Principles of General and Autonomic Pharmacology [2 ed.] 1621311481, 9781621311485

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REVISED EDITION

By Robert Rodgers University of Rhode Island

Bassim Hamadeh, CEO and Publisher Christopher Foster, General Vice President Michael Simpson, Vice President of Acquisitions Jessica Knott, Managing Editor Kevin Fahey, Cognella Marketing Manager Jess Busch, Senior Graphic Designer John Remington, Acquisitions Editor Jamie Giganti, Project Editor Brian Fahey, Licensing Associate Copyright © 2013 by Cognella, Inc. All rights reserved. No part of this publication may be reprinted, reproduced, transmitted, or utilized in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information retrieval system without the written permission of Cognella, Inc. First published in the United States of America in 2013 by Cognella, Inc. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Printed in the United States of America ISBN: 978-1-62131-148-5 (pbk)

Contents

Chapter 1

1

The Processing of Drugs By The Body: Drug Absorption and Disposition

Chapter 2

39

Assessing Drug Action

Chapter 3

79

Drugs That Interact With the Autonomic Nervous System

Glossary

123

Bibliography

127

Acknowledgements

The author is profoundly grateful to the following people for their generous and expert assistance in the preparation of this book: Sara Rosenbaum, Ph.D., for her patient explanations of topics in drug metabolism; Abraham Kovoor, Ph.D., for his insights in receptor pharmacology; Mikko Niemi, M.D., for his expertise in the effects of genetic polymorphism on simvastatin metabolism and toxicity; and John Babson, Ph.D., friend and colleague, who taught the course with the author for many years.

Chapter 1

The Processing of Drugs By The Body: Drug Absorption and Disposition

Introduction

W

hat is a drug? It is a chemical that is not a nutrient but nevertheless alters biological functions of organisms, including human beings. A common misconception is that the term “drug”* applies only to powdered chemicals that are often illegal and used or abused recreationally (cocaine, heroin, amphetamines, etc.). That erroneous concept, for example, is implicit in the unfortunate but ubiquitous phrases “drugs and alcohol” and, worse, “drugs or alcohol.” Perhaps because alcohol is a liquid at room temperature, is legal, and is so widely accepted as a social medium of exchange, it seems to have been segregated from other drugs in the collective consciousness. More accurate phrases in this context would be “alcohol and other drugs” or “drugs, including alcohol.” Of course, relatively few drugs are used or abused recreationally. There are literally hundreds of drugs, the vast majority of which are used for therapeutic purposes; i.e., to treat diseases. In addition, drugs come in multiple physical forms prepared in liquids, capsules, tablets, aerosols, skin patches, and other dosage forms. One characteristic that all seemingly disparate drugs share is that they each produce some specific measurable response or set of responses in living organisms, some of which are beneficial, and some adverse. Drugs can, for instance, induce or delay sleep, excite or calm, fight or exacerbate diseases, improve or impair health. Another important principle to keep in mind is that no drug is truly clean. All drugs—whether therapeutic or recreational—can produce adverse effects in sufficient quantities, many even at recommended doses. Once a drug is taken by someone, what happens to it? Where does it go within the body? What, if anything, does the body do to the drug? How does the body get rid of it? These are among the fundamental questions that are addressed by the broad discipline of pharmacokinetics. It is useful to approach this extensive subject from the following point of view: The human body really does not like to be invaded by most drugs. It will use a variety of tools in its box to reject, destroy, and dispose of the drug as fast and as thoroughly as it can. The body strives to preserve the status quo that existed before the drug was administered, even if it is diseased or in ill health. It rightly considers most drugs to be xenobiotics, or foreign substances, and reacts accordingly. One of the most challenging aspects of drug development is to design drugs and their formulations to withstand and overcome the body’s counterattack. One of the central aims of pharmacotherapy is to maximize drug bioavailability. The term “bioavailability” can be defined as the fraction of the drug’s dose that ends up in the bloodstream. Only the free drug dissolved in blood or extracellular fluid can interact with its molecular target: receptor, transporter, or enzyme. Therefore, other factors beyond the rate of absorption can influence the free drug concentration up or down, and thus will have an impact on its therapeutic effectiveness. An important objective is to keep blood levels of the drug within defined limits, known as the therapeutic window. If levels are

* Glossary terms are printed in bold type when they first appear in the text.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 3

Toxic Levels

A Blood Levels Of Drug

C

B

Therapeutic Window

Sub-Therapeutic Levels

Time

Figure 1. Time course of appearance and disappearance of an orally administered drug in the bloodstream. A major objective of pharmacotherapy is to maintain blood concentrations of drugs within the therapeutic window (the range of therapeutically effective concentrations). Total blood concentrations are depicted. Free concentrations are lower, and can vary depending on how much of the drug is bound to plasma proteins. Excessive levels (A), i.e., increased area under the curve (AUC), would increase the risk of adverse events and toxicity. Conversely, insufficient levels or a decreased AUC (B) would compromise therapeutic effectiveness. Repeated oral dosing (arrows) should be timed to maintain drug levels within the therapeutic window (C). too low, then the drug’s effectiveness is restricted. But if they are too high, then the risk of adverse effects and toxicity is increased (Figure 1). The challenge to maintain a drug’s blood levels within the therapeutic window is daunting because there are many obstacles to overcome (Figure 2). Mainly because of convenience and relative safety, the most common route of drug administration is oral, and thus the most common dosage forms are tablets, capsules, or liquids. After oral administration, drugs must then traverse one or more barriers to absorption in the gastrointestinal (g.i.) tract in order to get into the bloodstream (Figure 2, sites 2–4). But the g.i. tract can be a formidable barrier to absorption of many drug molecules. For instance, it is useless to orally administer drugs that are naked peptides or proteins. The gut will digest and inactivate them very quickly. Such agents must be administered parenterally. Also, even if a drug molecule is not a protein or peptide, it may not be absorbed very well anyway. If the molecule is too big and dissolves too easily in water, i.e., if it is too hydrophilic, then its intestinal absorption will be severely restricted. The anticoagulant heparin would be a drug of this type. The intestine, where the greatest fraction of most orally administered drugs is absorbed, will not allow molecules above a certain size to cross cell membranes and cellular barriers to enter the bloodstream unless they are sufficiently lipophilic. As if that were not enough, the cells of the intestine (enterocytes) have enzymes in them that can inactivate drug molecules even before they are absorbed into the bloodstream. They also have transport proteins in their membranes that can kick the drug molecules back into the intestinal lumen for ultimate excretion. If the drug molecule manages to survive all this, successfully passes the intestinal barrier, and is absorbed into the bloodstream, its challenges are only beginning. The next major barrier is a serious one. Remember that a drug is a xenobiotic, and as such the body considers it to be a foreign substance. Over the millennia, organisms including humans have evolved a

4 | Principles of Pharmacology and Autonomics

X

1 M

D

-Intestinal Lumen-

2 D

3

-Enterocyte-

M

4

Hepatic Portal System

5 D

6

M

7

-Hepatocyte-

D,M

Excretion

-Bile8

-systemic circulationD,M

11

9

D

M

D,M

10 -Non-target tissues-

PP 12

-Kidney-

Target tissue (Site of action)

Figure 2. Major routes of absorption and disposition of drugs. After oral administration, the unbound drug molecule (1) is available for intestinal metabolism and absorption (sites 2, 3, and 4) followed by hepatic processing (5, 6). Either the parent drug (D) or metabolites (M) can be excreted into the bile (7) or returned to the systemic circulation (8). The unchanged parent drug or its metabolites can then be distributed to non-target tissues (9), bound to plasma proteins (PP, 10), or can be excreted by the kidneys (11). The remaining free drug molecules in the extracellular fluid are available to interact with their receptors at the target tissue (12).

variety of mechanisms to minimize harmful effects of xenobiotics of many types. Important among these is a collection of blood vessels called the hepatic portal system (HPS). The HPS basically is an express route from the intestines to the liver, which stands as a sentinel to protect the rest of the body against invasion by toxic substances. The liver in turn has evolved into an effective discriminator, allowing nutrients and other essential molecules to pass through, while destroying or altering many non-nutrient substances that the liver has determined to be potentially detrimental to the organism’s health (Figure 2, sites 5 and 6). To accomplish this, the liver relies on an impressive array of membrane transporter proteins and intracellular enzymes to process xenobiotics in such a way that they usually become more soluble in water (more hydrophilic), less soluble in oil (less lipophilic), and less biologically active (or less toxic). With regard to drugs specifically, biotransformation of parent drugs produces metabolites that usually have lower affinity for the drug’s receptor, or ultimate molecular target (see Chapter 2).

The Processing of Drugs By The Body: Drug Absorption and Disposition | 5

Accordingly, biotransformation usually destroys the therapeutic utility of the drug. It also increases the ease of its elimination from the body, mainly by the kidneys (Figure 2, site 7). The major site of biotransformation is the liver, but additional sites include the intestinal enterocyte, tubular epithelial cells in the kidney, occasionally the lung, and some other tissues. The liver usually gets better at metabolizing xenobiotics as time goes on. The invading agent, upon first exposure, will very likely serve as a substrate for at least one member of an impressive array of families and subfamilies of hepatic drug-metabolizing enzymes (DME). With repeated exposure to the drug, the liver often increases its expression of specific DMEs, which in turn enhances its ability to destroy that drug, and perhaps others that can be substrates for that enzyme isoform as well. As a result, this induction of hepatic DMEs increases the doses required to maintain therapeutic effects. This is the major mechanism of pharmacokinetic tolerance and cross-tolerance. The blood itself can impede a drug molecule’s journey to its objective. Blood is composed basically of water, salts, and proteins, and includes erythrocytes, leukocytes, and platelets. Blood proteins, especially globulins and albumins, are fairly large and have many pockets in their tertiary structures into which drug molecules can nestle. Both lipophilic and hydrophilic drugs can bind nonspecifically and extensively to large plasma proteins (Figure 2, site 10). Drugs can also be taken up by blood cells and platelets. Nonselective binding and cellular uptake (Figure 2, site 9) decrease the concentration of the drug that is dissolved freely in the aqueous phase (salt water). Only the drug molecules that are freely dissolved in the extracellular fluid can interact with their target receptors on or in cells. Extensive plasma protein binding is therefore a two-edged sword. On the one hand, binding to plasma proteins tends to retard extensive extra-vascular distribution, keeping the absorbed drug in the blood longer. This would maintain a repleting reserve of freely dissolved drug molecules that are available for interaction with their target receptors or enzymes. On the other hand, protein-bound drug molecules are subject to displacement by other drugs that might share an affinity for the same binding sites, one category of potentially adverse drug interactions. Even if a drug molecule survives hepatic biotransformation and exits the liver unscathed into the systemic circulation, it still faces more obstacles to achieving its ultimate therapeutic destination. Let’s say the patient takes a drug to treat depression. Of course, in order for that drug to exert its therapeutic effects, it must be delivered by the circulation to the brain. But the circulation delivers blood everywhere, so the drug will usually be distributed widely, with only a fraction of it going to the brain. To compound the problem, the brain—with the heart, the body’s most vital organ—has evolved its own unique protective mechanism called the blood–brain barrier (BBB). The BBB is a system of capillaries whose walls are atypically resistant to crossing by intermediate-to-large molecules, unless those molecules are sufficiently lipid soluble. As expected, clinically useful antidepressant drugs generally dissolve in lipids well enough to pass through the BBB fairly easily. But other drugs, such as many anticancer drugs used to treat brain tumors, either penetrate the BBB with difficulty or are actively extruded back into the blood by specific membrane-bound transporters, presenting potentially serious therapeutic problems. Once the drug finally reaches its molecular target (Figure 2, site 12), it would appear that it has survived the gauntlet, and that the body has run out of options to fight the drug’s effects. It hasn’t. The scope of the body’s resources to resist actions of drugs is impressive, extending all the way down to the level of the target cell. The cell has evolved mechanisms to remove and replace receptors on the cell membranes. Removal and replacement of receptors remains in balance as long as the levels of neurotransmitters or hormones in the vicinity of the receptors are fairly constant. A sudden change in the amounts of endogenous ligands or drugs can influence this process, which can alter the number (i.e., the density) of receptors. The end result can be either an increase or a decrease

6 | Principles of Pharmacology and Autonomics

in the sensitivity of the cells for the hormones or drugs that bind to those receptors. In response to continual interaction with its receptors by either agonists or antagonists, the cell changes the rate at which it expresses and elaborates those receptors. Against agonists (drugs that activate their receptors), the cell decreases the sensitivity, expression, and density of its receptors to minimize the agonist’s effects. This is called receptor down-regulation. The normal physiological function of receptor down-regulation is to maintain cellular homeostasis within narrow limits in the face of fluctuations in receptor activation by endogenous agonists such as neurotransmitters. Conversely, against antagonists (drugs that bind to, but do not activate, receptors), the cell can change the balance, increasing the density of receptors on the membrane. This is called receptor up-regulation or, perhaps more accurately, reversal or inhibition of down-regulation. Because most therapeutic drugs act as antagonists, rather than agonists (Chapter 2), receptor up-regulation is an important mechanism of pharmacodynamic tolerance to many antagonist drugs. For example, the drug prazosin is used to decrease blood pressure in people who have hypertension. It works by blocking alpha receptors on vascular smooth muscle, interfering with the ability of the neurotransmitter norepinephrine to constrict small arteries. In many patients, prazosin’s antihypertensive effectiveness declines over time. This is a form of tolerance. A contributing factor may be up-regulation of alpha receptors on vascular smooth muscle. This would enhance the ability of the neurotransmitter norepinephrine to overcome prazosin’s blockade of the receptors and oppose the vasodilation. Receptor up-regulation may also render the tissue, and thus the patient, supersensitive to the actions of the competing endogenous agonist upon withdrawal of the antagonist drug. For example, chronic treatment with a beta blocker can elicit up-regulation of beta receptors in the target organ. Sudden discontinuation of the beta-blocking drug will leave a larger pool of functional beta receptors exposed to endogenous agonists such as epinephrine or norepinephrine. This is one mechanism, for example, of rebound tachycardia (fast heart rate) associated with interruption of beta-blocker therapy. Even if they reach their sites of action, the majority of drugs can still produce adverse effects, some of which can be serious or life-threatening. Pharmacotherapy commonly requires multiple drug treatments. If a patient is taking two or more drugs at the same time, the combination may produce new adverse effects that none of the drugs individually would have elicited by themselves. This is another sub-discipline of pharmacotherapy called adverse drug interactions. Unfortunately, the complex interdependent processes of drug absorption, distribution, metabolism, action, and elimination provide many opportunities for drugs to interact with each other in different ways. Each time a new drug is added to the list, the odds of adverse events go up accordingly. For example, one drug may interfere with the hepatic uptake or metabolism of another drug, increasing its blood levels and the risk of adverse reactions. Most or all of the sites depicted in Figure 2 can be points of drug–drug interactions. One drug might restrict absorption of another by binding to it in the intestinal lumen (Figure 2, site 1), by inhibiting its influx into intestinal enterocytes, or promoting its efflux back into the lumen (Figure 2, sites 1 and 2). This would reduce bioavailability and therapeutic effectiveness. In the liver, one drug may either interfere with or induce (increase levels of) hepatic DMEs, altering the rate of metabolism of itself or of a second drug. So not only do we need to be concerned about how well a drug is producing its desired (therapeutic) effects, but we also need to worry about how toxic it or its metabolites may be, and how they might interact adversely with other drugs. While all of this is going on, the body is busily engaged in getting rid of the drug and its metabolites entirely. The most important organs of drug elimination are the kidneys. To accomplish this objective, the liver and kidneys work in tandem (Figure 2, sites 5–7). The liver transforms the parent drugs into more water-soluble metabolites, enabling the kidneys to then filter or secrete those metabolites into the urine in order to excrete them. All of these processes—absorption, metabolism,

The Processing of Drugs By The Body: Drug Absorption and Disposition | 7

and excretion—are critically dependent on membrane-bound proteins called transporters. In the intestinal enterocytes, transporters allow hydrophilic drugs or metabolites to be absorbed. In the hepatocytes, transporters facilitate the import of drugs for metabolism by DMEs and the export of drugs or metabolites into the bile or systemic circulation. Renal transporters, in turn, often facilitate the secretion of drugs or their metabolites into the filtrate and ultimately into the urine. All of these processes can be disrupted if the functions of DMEs or transporters are altered by other drugs or by genetic polymorphisms (usually, altered transporter structure and function due to changes in the coding of their amino acid sequences). That effective and safe drugs can be developed at all, considering the formidable opposition, is a testament to scientific persistence, creativity, and expertise. Successful addressing of many of these issues can be accomplished by using rather clever strategies. For example, the tendency of the body to metabolically destroy drugs can be exploited for therapeutic purposes. Some drug molecules are synthesized in such a way that they are transformed in the intestine or liver to more, not less, therapeutically effective metabolites. A list of such pro-drugs includes the antihypertensive drug enalapril and the cholesterol-lowering drug simvastatin. Enalapril is hydrolyzed in the intestine to the diacid enalaprilat, which facilitates the transport of enalapril into the bloodstream without reducing its therapeutic effect. The parent simvastatin molecule is the inactive lactone form, which is converted to the active hydroxyl-acid form in the liver. In the following sections, important pharmacokinetic topics—including administration, absorption, biotransformation, distribution, elimination, tolerance, adverse effects, adverse interactions, and influences of genetic variability on drug responses—are presented in more detail. It is hoped that acquisition of a basic understanding of these pharmacokinetic principles will impart a greater appreciation of the complex challenges implicit in both the development and the delivery of safe and effective therapeutic agents.

Drug Administration Drugs can be taken by patients, or administered to patients by health care providers, by various routes. Each route has its own set of advantages and disadvantages (Table 1). The most common route of administration is oral, because it is the most convenient and, relatively, the safest. However, oral administration is not always possible, and an alternative route must be used. For example, patients with angina pectoris (tightness or pain in the chest secondary to coronary ischemia) may be prescribed the coronary vasodilator nitroglycerin. If nitroglycerin is administered orally, most or all of it will be destroyed in the gut and liver before it gets to the heart. Formerly, this problem was addressed by nitroglycerin formulations that allowed absorption of the drug into the bloodstream upon placement under the tongue (sublingual administration). This is possible because nitroglycerin is a small molecule and is very lipophilic, so that it can pass through the barriers of the oral mucosa and its capillary walls to enter the bloodstream. A significant fraction of the drug would then go directly to the heart without having to pass through the liver first. More recently, transdermal nitroglycerin (skin patch) is the preferred route to minimize hepatic biotransformation. Administering drugs intravenously bypasses absorption and decreases the extent of first pass hepatic metabolism, but is more risky because of the possibility of infection or extravasation.

8 | Principles of Pharmacology and Autonomics

Table 1. Various Routes of Drug Administration. Route

Advantages

Disadvantages

Oral

Convenient, usually safer

Subject to first pass hepatic metabolism, risk of systemic toxicity, slower absorption, dependent on patient compliance

Intravenous

Bypasses first pass metabolism, rapid absorption and response

Inconvenient, subject to infection or extravasation, risk of systemic toxicity

Intramuscular Favors slow and steady absorption, depot preparations

Subject to irritation and discomfort, infection possible

Transdermal

Bypasses first-pass metabolism, lower risk of systemic toxicity

Less convenient, subject to contact dermatitis

Topical

Suitable for skin or mucus membrane applications, low risk of systemic toxicity

Risk of contact dermatitis, some systemic absorption and toxicity possible with lipid-soluble drugs

Inhalational

Rapid response

Risk of bronchial irritation or infection, restricted applications (e.g., asthma, general anesthesia)

Rectal

Less first-pass metabolism than oral

Restricted applications, inconvenient

Movement of Drugs Across Cell Membranes: Transporters In order for orally administered drugs to be absorbed in the intestinal tract, metabolized in the liver, distributed to various tissues, or eliminated by the kidneys, they must cross cellular barriers from one compartment to the other (Figure 2, sites 1, 2, 4, 5, and 7–9). In the intestine, the barrier is the epithelium (enterocytes) lining the villi and microvilli, and the compartments are the intestinal lumen on one side and extracellular fluid on the other. In the liver, drugs must pass into hepatocytes from the portal blood and then they or their metabolites must exit the hepatocytes, either into the bile or back into the bloodstream. In the kidneys, drugs or their metabolites can be filtered (in the glomeruli) or secreted (across the tubular epithelium) from the blood into the lumen of the nephron and ultimately into the urine. Regardless of the cellular barrier, the principles are the same. If the drug molecules are lipid-soluble, they may cross the barrier by trans-cellular diffusion with their concentration gradients. If they are relatively small and water-soluble, they may pass between cells through tight junctions via para-cellular diffusion. However, many drugs are too large and water-soluble to easily cross cellular barriers by these routes, and thus require assistance in the form of membrane transporter proteins in order for trans-cellular transport to succeed (Figure 3). Carrier or transporter proteins exist because they are involved in the transport of nutrients or endogenous substances such as products of cellular metabolism. Many of the same transporters can carry xenobiotic substances as well. In general, transporters belong to one of two groups: 1) Solute-carrier proteins (SLC) that transport substrates, usually with their concentration gradients, without concomitant hydrolysis of ATP; and 2) ATP-binding cassette (ABC) proteins that require ATP hydrolysis, usually

The Processing of Drugs By The Body: Drug Absorption and Disposition | 9

DHP 1

D+ HC

DLP 2

3

D–HA 4

OCT 1

H+

DP 5

OAT 1

PEPT-1

αKG

D-GL ATP 6

ADP MRP 1

Figure 3. Transport of drug molecules across cellular barriers and examples of transporters. 1) Para-cellular diffusion of small, hydrophilic molecules (DHP); 2) Trans-cellular diffusion of lipophilic molecules (DLP); 3) Facilitated diffusion of hydrophilic cationic drug molecules (D+HC) by the solute carrier protein (SLC) organic cation transporter 1 (OCT 1); 4) Facilitated diffusion of a hydrophilic anionic drug molecule by the SLC organic anion antiporter (OAT1) coupled to alpha ketoglutarate (αKG) efflux; 5) Facilitated diffusion of peptide-like drugs (DP) via the SLC peptide symporter 1 (PEPT-1) coupled to hydrogen ion influx; 6) Efflux of a glucuronide-conjugated drug metabolite (D-GL) mediated by an ATP-binding cassette (ABC) protein, the multidrug resistanceassociated protein 1 (MRP 1). Most or all of these transporters also carry endogenous substrates (nutrients or byproducts of nutrient metabolism). Adapted from Zamek-Gliszczynski et al., 2006; Kusuhara and Suguyama, 2009; Shugarts and Benet, 2009; Masereeuw and Russel, 2010; Romaine et al., 2010.

transporting their substrates against a concentration gradient. In the intestine and liver, SLC transporters generally carry their substrates into the cell (influx), while ABC transporters mediate export (efflux) of their substrates. Transporters can be further subdivided into simple carriers, symporters, and antiporters. Simple carriers transport their substrates alone across the membrane with their concentration gradients. Symporters and antiporters couple the transport of their substrates with one or more co-transported substance, such as inorganic or organic ions. Usually, the cotransported substrate is moved with its concentration gradient either in the same direction (symporter) or in the opposite direction (antiporter) as that of the transported substrate in a process called facilitated diffusion. ABC proteins have binding sites for both ATP and their specific substrates. The hydrolysis of ATP results in a

10 | Principles of Pharmacology and Autonomics

Basolateral (Blood)

Apical (Lumen) 1

Dhp 2

D

Dlp

DMEs Phase 1 OCT N1,2 3

(CYPs) [SLC] M1

OAT P2B1

Phase 2

ADP

ADP (UGTs, GSTs, SULTs)

MDR 1

ENT 1,2 ATP

ATP 4

ADP [ABC]

[ABC] ADP M2

MRP 2 ATP

5

MRP 3 ATP

Absorption Intestinal Excretion

Figure 4. Drug transport and drug metabolism in the intestinal enterocyte. Small, water-soluble drugs may be absorbed para-cellularly (1), while more lipid-soluble drugs may diffuse into the bloodstream trans-cellularly (2). Solute-carrier transporters (SLC) can mediate the diffusional influx of larger, more hydrophilic drugs (3), while ATP-binding cassette (ABC) transporters mediate efflux of drugs or their metabolites back into the lumen (4) or into the bloodstream (5). As in the liver (see Figure 5), the intestinal enterocyte contains phase 1 and phase 2 drug-metabolizing enzymes (DMEs). For enzyme abbreviations, see Table 4. For transporter abbreviations, see Table 2. Adapted from Huang et al., 2009. conformational change in the carrier protein and either allows the transport of the substrate against its concentration gradient (active transport) or accelerates the transport of substances at a faster rate than that mediated by carrier-mediated simple diffusion. Specific examples of SLC and ABC transporters are depicted in Figure 3. Transporters of various types that belong to all of these classes are involved in intestinal absorption, hepatic metabolism, systemic distribution, and renal excretion of nearly all therapeutic drugs.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 11

Table 2. Intestinal Transporters and Representative Xenobiotic Substrates. Transporter

Location

Representative xenobiotic substrates

PEPT 1,2

AM

captopril

OAT P1A2

AM

rifampin

OAT P2B1

AM

rifampin

OCT 3

AM

cimetidine

OCT N1,2

AM

cimetidine, pyrilamine

CNT 1,2

AM

adenosine, thymidine

MDR 1 (p-GP)

AM

verapamil

MRP 2

AM

cyclosporine

MRP 1

BM

probenicid

MRP 3

BM

indomethacin

MRP 5

BM

sildenafil

ENT 1,2

BM

dipyridamole

SLC

ABC

Abbreviations: SLC, solute-carrier protein; ABC, ATP-binding cassette protein; PEPT, peptide transporter; OAT, organic anion transporter; OCT, organic cation transporter; CNT, concentrative nucleotide transporter; MDR, multidrug resistance protein; p-GP, p-glycoprotein; MRP, multidrug resistance-associated protein; ENT, equilibrative nucleoside transporter; AM, apical (luminal) membrane; BM, basolateral (blood-side) membrane. Adapted from Shugarts and Benet (2009), Huang et al. (2009), Fahrmeyer et al. (2010), Niemi et al. (2011).

Intestinal Absorption and Processing of Drugs After oral administration, drugs must first be absorbed into the bloodstream from the gastrointestinal tract. Absorption of most drugs takes place in the small intestine rather than in the stomach, mainly because the intestine has more surface area and the transit time in the intestine is longer. The intestinal wall therefore constitutes the first practical barrier to the absorption of orally administered drugs (Figure 2, sites 2–4). Transport of drugs around or across intestinal enterocytes is illustrated in Figure 4. As discussed above, lipid-soluble drugs can cross the cellular barrier by trans-cellular diffusion with their concentration gradients. Small hydrophilic drug molecules may be able to diffuse via the para-cellular route. However, many drug molecules are larger and hydrophilic, and thus must cross with the assistance of selective transporter or carrier proteins in the cell membrane. In the intestinal enterocyte, membrane

12 | Principles of Pharmacology and Autonomics

Basolateral (Blood)

Apical (Bile) 1

Dhp D

Dlp

2 DMEs ADP

OAT P1B1

Phase 1 3

[SLC] (CYPs, FMOs, EHs)

MDR 1

OAT P2B1

ATP 4

[ABC]

M1

OCT 1

ADP Phase 2

MRP 2 ATP

ADP

(UGTs, SULTs, NATs GSTs MTs)

MRP 3 ATP [ABC]

ADP

M2

5

MRP 5 ATP

Biliary Excretion Efflux into Circulation

Figure 5. Drug transporters and drug-metabolizing enzymes in the hepatocyte. Hydrophilic drug or metabolite molecules (Dhp) can enter the bile from the blood via para-cellular diffusion (1), or lipophilic parent drug molecules (Dlp) can enter the hepatocyte by trans-cellular diffusion (2). Hydrophilic drugs and metabolites can also enter the hepatocyte via solute-carrier proteins (3). Once in the hepatocyte, drug molecules can be transformed by phase 1 and phase 2 drug-metabolizing enzymes (DMEs) into one or more metabolites (M1, M2). Finally, either the unmetabolized drug molecule or its metabolites can be transported from the hepatocyte into the bile (4) or back into the blood (5) via ABC transporters. For transporter abbreviations, see Table 3. For enzyme abbreviations, see Table 4. Adapted from Xu et al., 2005; Sugarts and Benet, 2009.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 13

Table 3. Hepatic Transporters and Representative Xenobiotic Substrates. SLC

Location

Representative xenobiotic substrates

OAT P1B1

BM

many drugs, including statins, antibacterials, anticancer drugs, diuretics, antihypertensives

OAT P1B3

BM

digoxin, methotrexate, fexofenadine, bosentan

OAT P2B1

BM

statins, benzylpenicillin, fexofenidine

OAT P1A2

BM

rifampin

OCT 1

BM

acyclovir, metformin, cimetidine, desipramine quinidine, cisplatin

ABC

Location

Representative substrates

MRP 3

BM

etoposide, glucuronide conjugates, methotrexate

MRP 4

BM

cephazolin, topotecan

MRP 5

BM

mercaptopurine

MDR 1 (p-GP)

AM

colchicine, paclitaxel, vincristine

MDR 3

AM

digoxin, vinblastine

MRP 2

AM

cisplatin, glucuronide and sulfate conjugates

BSEP

AM

vinblastine

Abbreviations: SLC, solute carrier proteins; ABC, ATP-binding cassette proteins; OAT, organic anion transporter; OCT, organic cation transporter; MRP, multidrug resistance-associated protein; MDR, multidrug resistance protein; p-GP, p-glycoprotein; BSEP, bile salt export pump; BM, basolateral (blood-side) membrane; AM, apical (bile side) membrane. Adapted from Neuvonen et al., 2006; Kusuhara and Sugiyama, 2009; Shugarts and Benet, 2009; Fahrmeyer et al., 2010; Niemi et al., 2011. transporters on both the luminal (apical) and basolateral (blood) side are involved with the absorption of nutrients or the efflux of bile acids or other metabolites. They can also facilitate the transport of drug molecules in either direction: influx into the enterocyte, or efflux back out into the intestinal lumen on the apical side or into the bloodstream on the basolateral side of the cell. Note that, in general, SLC carriers mediate uptake, while ABC proteins mediate efflux of xenobiotics in the intestinal enterocyte. Important SLC carriers involved in drug transport can be found on the apical (luminal) side, while important ABC carriers are located on both the apical and basolateral sides of the cell. Intestinal absorption is net movement of nutrients or xenobiotics from the lumen ultimately into the blood, while intestinal excretion is net movement of substrates, usually metabolites, in the opposite direction, from the cytoplasm or blood into the lumen. Examples of specific intestinal SLC and ABC transporters and their xenobiotic substrates are listed in Table 2.

14 | Principles of Pharmacology and Autonomics

Table 4. Hepatic Drug-Metabolizing Enzymes. Enzymes

Representative reactions

Phase 1 (oxidations) Cytochrome P450 (CYP)

CYP D-H + O2 + 2e- → D-OH + H2O [NADPH, FAD, FMN]

Flavin-containing monooxygenases (FMOs)

Addition of oxygen atoms to S, P, or N atoms

Epoxide hydrolases (EHs)

Conversion of epoxides to dihydroxyls

Phase 2 (conjugations) UDP-glucuronosyltransferases (UGTs)

UGT UDP-glucuronic acid + D-OH → D-glucuronide

Glutathione-S-transferase (GST)

Addition of a glutathione group

Sulfotransferases (SULT)

Addition of sulfonyl group

N-acetyltransferase (NAT)

Addition of an acetyl group

Methyltransferases (MT)

Addition of a methyl group

Abbreviations: D, drug molecule; NADPH, nicotinamide adenine dinucleotide phosphate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide. Adapted from Xu et al. 2005, Coleman 2005.

In addition to transporters, enzymes that catalyze the biotransformation (metabolism) of xenobiotics can be found in intestinal enterocytes (Figure 2, site 3; Figure 4). Drug-metabolizing enzymes (DMEs) are also found in the liver—the main site of drug biotransformation—as well as in other tissues including the kidneys and the lungs. Most DMEs belong to one of two classes: phase 1 oxidizing enzymes and phase 2 conjugating enzymes. Processing of xenobiotics in phases 1 and 2 usually converts a more lipid-soluble parent drug molecule into a more water-soluble one, which is more easily excreted by the kidneys. In addition, DMEs usually inactivate the parent drug molecule, reducing its therapeutic effectiveness. The intestine, therefore, is a formidable barrier to the absorption of hydrophilic drugs. Intestinal DMEs can inactivate the parent drug, and its transporters can reject either the parent molecules or their metabolites by mediating their export back into the intestinal lumen. Both phase 1 and phase 2 DMEs are discussed in more detail in the following section on hepatic biotransformation.

Hepatic Biotransformation of Drugs Some orally administered drugs may be metabolized to a significant extent in the intestine. However, if a parent drug molecule is absorbed unchanged into the bloodstream from the intestine, it will likely be metabolized in the liver, to which it is delivered via the hepatic portal system (Figure 2, site 5). The

The Processing of Drugs By The Body: Drug Absorption and Disposition | 15

liver serves as the main sentinel to guard against invasion of the whole body by xenobiotic substances. Hepatocytes, like intestinal enterocytes, have both SLC and ABC transporters that mediate transport of xenobiotics across its outer membranes (Figure 5 and Table 3). Some of these transporters are found in both the enterocyte and hepatocyte, but some are unique to each cell type. SLC transporters can be specific for organic anions (e.g., OAT transporters) or organic cations (e.g., OCT transporters). As in the intestine, SLC transporters in the hepatocyte generally mediate diffusion of drugs into the cell (influx), where the parent drug is subject to biotransformation catalyzed by drug-metabolizing enzymes (DMEs). Efflux of metabolites from hepatocytes into the bile or blood is generally mediated by ABC carrier proteins. As discussed above for intestinal enterocytes, once the parent compound has entered the hepatocyte from the blood, its biotransformation often occurs in two phases (Table 4), with each phase comprised of different families and subfamilies of enzymes. In phase 1, xenobiotics are converted to less active, and sometimes less lipid-soluble, metabolites, usually by oxidation to hydroxyl or oxide metabolites. Phase 1 reactions catalyzed by cytochrome p450 (CYP) enzymes usually form monohydroxylated metabolites. Similarly, oxygen atoms can be added to sulfur, phosphorus, and nitrogen atoms in drug molecules to form their respective oxides via flavin monooxygenase (FMO) enzymes. The reactions catalyzed by FMOs are similar to those catalyzed by CYP enzymes. Occasionally, CYP reactions yield epoxide intermediates that can be converted to dihydroxy metabolites by membrane-bound or cytosolic epoxide hydrolases (EH) that are proximal to CYP enzymes in the hepatocyte. Chemical reactions catalyzed by phase 2 enzymes often involve the attachment of a glucuronide, sulfate, or other larger hydrophilic group to the hydroxyl that had been added to the molecule in phase 1. Phase 2 enzymes include a family of UDP-glucuronosyltransferases (UGTs) that catalyze the addition of a glucuronide group to a hydroxylated parent drug or metabolite. Other phase 2 enzyme families catalyze the additions of glutathione, sulfonyl, acetyl, and methyl groups (Table 4). Thus, hydroxylation and other changes in the parent compound in phase 1 often prepare the molecule for additional processing by phase 2 enzymes into larger, more hydrophilic molecules that are more easily excreted by the kidneys. Of course, without assistance in crossing the cell membranes, the larger, water-soluble metabolites would be effectively trapped inside the cell. They might eventually escape the cell, but the process would be much slower without the assistance of membrane carrier proteins. Efflux of metabolites is mediated by ABC transporters on either the apical (bile side) or basolateral (blood side) membrane of the hepatocyte. Those on the apical side promote biliary excretion, while those on the basolateral side send metabolites or, in some cases, the parent compound back into the systemic circulation. Collectively, the three phases of drug metabolism are often categorized as Phase 1 oxidative reactions, Phase 2 conjugations, and Phase 3 carrier-mediated transport of parent drugs or their metabolites.

Renal elimination of drugs The most likely fate of blood-borne drug metabolites, originating in either the intestine or the liver, is renal excretion. The kidney is set up to excrete both unmetabolized parent drugs and their metabolites by the processes of filtration and secretion (Figure 6). The glomerulus filters most molecules whose molecular weights are below around 10,000 Daltons; thus, most drug metabolites would be subject to renal filtration into the proximal tubules. The kidneys do not filter all of the blood that enters them, however. The filtration fraction of the normal kidney is about 20%. In other words, about a fifth of the

16 | Principles of Pharmacology and Autonomics

A

D,M

G

1 PT D, M

D,M

B -Apical(Urine)

-Basolateral(Blood) 2

Dhp, Mhp

1 D,M

Dlp

3

ATP MRP 2,4

OAT 1,3 ADP ATP

BCRP

OAT P4C1 ADP ATP

MDR 1 (p-GP)

OCT 2 ADP

MATE 1,2

OCT 1,2

PEPT 1,2 4 D,M Secretion Reabsorption

Figure 6. Renal drug transporters. A: Both unmetabolized parent drugs (D) and their metabolites (M) are filtered (1) in the glomerulus (G) into the proximal tubule (PT). B: Unfiltered hydrophilic drugs (Dhp) and metabolites (Mhp) can be secreted para-cellularly (2). Unfiltered lipophilic drugs (Dlp) can be secreted by trans-cellular diffusion (3). Note that the majority of transporters mediate the secretion of xenobiotics, rather than their reabsorption. Both filtered and secreted D and M molecules are excreted into the urine (4) unless they are further processed by distal sections of the nephron before entering the collecting duct (not shown). For transporter abbreviations, see Table 5. Adapted from Kusuhara and Sugiyama, 2009; Feng et al., 2010; Masereeuw and Russel, 2010. A more complete list of renal transporters and representative substrates is provided in Table 5.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 17

Table 5. Renal Drug Transporters. SLC

Location

Representative xenobiotic substrates

PEPT 1

AM

beta-lactam antibiotics, valcyclovir

PEPT 2

AM

cephalexin

NPT 1

AM

indomethacin, mevalonate

OAT 1

BLM

loop diuretics, antiviral drugs, pravastatin

OAT 2

BLM

anti-inflammatory drugs

OAT 3

BLM

ranitidine, pravastatin

OAT 4

AM

cimetidine, zidovudine

OCT 2

BLM

procainamide, metformin

URAT 1

AM

diuretics

OATP 4C1

BLM

cardiac glycosides

MATE1,2

AM

metformin, procainamide, cimetidine

ABC

Location

Representative xenobiotic substrates

MDR 1 (p-GP)

AM

doxorubicin, verapamil

MRP 1

BM*

etoposide glucuronide, ethacrynic acid, glutathione

MRP 2

AM

statins, vinblastine

MRP 3

BLM**

dinitrophenol, glutathione

MRP 4

AM

methotrexate, cephalosporins

BCR P

AM

daunorubicin, statins

Most of the listed transporters have been reported to be in the proximal tubule of the nephron. Abbreviations: SLC, solute carrier proteins; ABC, ATP-binding cassette proteins; AM, apical or luminal (brush-border) membrane; BM, basolateral (blood side) membrane; PEPT, oligopeptide transporter; OAT, organic anion transporter; OCT, organic cation transporter; URAT, urate transporter; OATP, organic anion transport protein; MDR, multidrug resistance protein; MATE, multidrug and toxin extrusion protein; MRP, multidrug resistance-associated protein; BCRP, breast cancer resistance protein. *Collecting duct; **Distal tubule. Adapted from Kusuhara and Sugiyama, 2009; Feng et al., 2010; Masereeuw and Russel, 2010.

18 | Principles of Pharmacology and Autonomics

blood entering each kidney is filtered. The rate of renal excretion of metabolites may exceed filtration through the process of secretion, or the transport of molecules from renal blood into the tubules of the nephron, usually the proximal tubule. Water-soluble metabolites would be excreted more easily by the kidneys than lipid-soluble molecules would be, for at least two reasons: 1) The filtrate is essentially deproteinized plasma, which is a highly aqueous environment; and 2) secretion is a process that is mediated by SLC and ABC transporters—in membranes of tubular epithelial cells—that are selective for anionic or cationic, rather than electroneutral, metabolites.

Distribution of drugs After intestinal absorption (Figure 2, sites 2–4) and hepatic metabolism (Figure 2, sites 5–8), but before renal excretion (Figure 2, site 11), drugs and their metabolites can be distributed to various nontarget tissues (Figure 2, site 9), or to the drug’s site of action (Figure 2, site 12). In addition, drugs can bind to lesser or greater extents to plasma proteins (Figure 2, site 10). Blood flow is an important determinant. Initially, drugs will tend to be distributed more rapidly and extensively to organs whose perfusion rates are relatively high, such as the brain, liver, heart, and kidneys. Tissue fat content also significantly affects distribution. Highly lipophilic drugs that escape intestinal or hepatic biotransformation will tend to accumulate more in adipose tissue or the brain and less in lean muscle or other tissues. Later on, parent drugs or metabolites may redistribute, most commonly from high-lipid tissues such as brain or adipose, back into the bloodstream. Extensive redistribution might be detected as a secondary and later increase in blood levels, usually lower than the initial peak after administration. Extensive plasma protein binding would restrict metabolism and distribution because only the drug molecules that are freely dissolved in plasma can be metabolized or can interact with their molecular targets.

Influence of pH on drug distribution Many drugs are weak electrolytes. As such, their rates of absorption and distribution—either by transcellular or carrier-mediated processes—might be influenced by the pH of the solution in which they are dissolved. Remember that ionization enhances water solubility and retards carrier-independent transmembrane diffusion, while electrical neutralization enhances lipid solubility and promotes transmembrane diffusional transport. The lipid- vs. water-solubility of many ionizable drugs can be influenced by the pH, or the hydrogen ion (proton) concentration. Therefore, pH can influence the rates of transmembrane diffusion, and thus the absorption and distribution of many drugs. The electrolytic nature of most drugs is often conferred by either of two functional groups with dissociable hydrogen ions in the form of amines (-NH2) or carboxylic acids (COOH): D1-NH3+ D1-NH2 + H+

D2-COOH  D2-COO- + H+

As summarized in the Henderson-Hasselbalch equation below, the pKa of a weak electrolyte is the pH at which the molecule is half-dissociated; i.e., the concentrations of the unprotonated and protonated forms are equal (the concentration ratio is 1, and log 1 = 0):

(

pKa = pH – log un-protonated form protonated form

)

The Processing of Drugs By The Body: Drug Absorption and Disposition | 19

-ECF-Blood-

Buffers O D1

NH3+

D1

NH2

[pka ≅ 10]

+

H+

O C

+

D2

–O

pH ≅ 7

C HO

D2 [pka ≅ 4]

Figure 7. The influence of pH on the distribution of weak electrolytes. A solution buffered at a pH of around 7 (typical of blood) will tend to retard transmembrane diffusion of electrically charged weak electrolytes. Drug molecules with an amine group (D1) might have a pKa of around 10, while drugs with a carboxyl group (D2) might have a pKa of around 4. The Henderson-Hasselbalch equation predicts that both would be extensively ionized at pH 7; the amine by retaining H+ and becoming positively charged, the carboxyl by losing H+ and becoming negatively charged. ECF: extracellular fluid.

[rearranging]: log

[at pH = 7]:

log

[protonated form] = pKa – pH ( [un-protonated form] ) [protonated form] ( [un-protonated form] )

= pKa – 7

For amines (D1 above), the protonated form is ionized (positively charged), and a representative pKa value could be around 10. For carboxylic acids (D2 above), the un-protonated form is ionized (negatively charged), and the pKa value might be around 4. So, as the Henderson-Hasselbalch equation predicts, a buffered pH of around 7 (typical of blood) would favor the charged form of either the amine or the carboxylic acid. In the case of amines, (pKa – 7) would be (10 – 7) = 3. The antilog = 103 = 1,000. Thus, the concentration of the protonated (ionized) form (numerator) would be 1,000-fold greater than that of the un-ionized form (denominator). For the carboxylic acids, (pKa – pH) would be (4 – 7) = -3. The antilog = 10-3, and again the concentration of the ionized form (this time in the denominator) would be 1,000-fold greater than that of the un-ionized form (in the numerator). As shown in Figure 7, either the amine or the carboxylic acid would tend to be trapped in a medium of pH 7 because both would exist mainly in their more water-soluble (ionized) forms, and consequently would be less able to traverse biological membranes without the help of membrane carrier proteins. Recall that water solubility favors renal excretion. So the pH of the blood relative to the pKa of a weak electrolyte parent drug or metabolite can influence their water solubility, and thus indirectly influence their rate and extent of elimination by the kidneys.

20 | Principles of Pharmacology and Autonomics

-ECF-

-BBB EC-

OAT P1A4

OAT P1A4 ADP

BCRP

-BloodDHP OAT 3

ATP

ADP

DLP MRP 4

(TJ) ADP

ATP

MDR 1 (p-GP)

MRP 1 ADP

ATP

ATP

Figure 8. Transport across blood–brain barrier. Capillaries of the blood–brain barrier (BBB) have very tight junctions (TJ) between endothelial cells (EC), restricting the entry of hydrophilic drugs (Dhp) into the brain. Lipophilic drugs (Dlp) can enter the brain via trans-cellular diffusion. Both organic anion SLC transporters (OAT) and ABC transporters (MRP, MDR, and BCRP) have been found in EC of the BBB. Note that most carriers so far identified mediate efflux of xenobiotics from the extracellular fluid (ECF) into the blood. Abbreviations: OAT, organic anion transporter; MRP, multidrug resistance-associated protein; MDR, multidrug resistance protein; p-GP, p-glycoprotein; BCRP, breast cancer resistance protein. Adapted from Kushuhara and Sugiyama, 2009; Colabuto et al., 2009. Blood–brain barrier The blood–brain barrier (BBB) is a system of capillaries in the brain. Their walls are composed of specialized endothelial cells that are very tightly connected. Junctions between BBB endothelial cells are atypically tight compared to those of systemic capillaries. Lipid-soluble drugs can gain entry into the brain across the BBB via trans-cellular diffusion, but hydrophilic drug molecules are restricted. Like

The Processing of Drugs By The Body: Drug Absorption and Disposition | 21

Table 6. Categories of Adverse Reactions to Drugs. Category

Example

1. Toxic reactions Pharmacological toxic reaction Cytotoxic pathological effects Genotoxic reactions Teratogenic effects

Impaired breathing caused by opioids Hepatotoxicity caused by acetaminophen Mutations caused by anticancer drugs Birth defects caused by diphenylhydantoin

2.

Allergic (hypersensitivity) reactions

Skin rash produced by penicillin antibiotics

3.

Idiosyncratic reactions

Lupus-like syndrome caused by procainamide

4.

Immunosuppression

Increased risk of infection caused by steroid antiinflammatory drugs

the intestinal enterocyte, hepatocyte, and renal tubular epithelial cells discussed above, the endothelial cells of the BBB have membrane transporters to allow the movement of hydrophilic xenobiotics and nutrients across the barrier (Figure 8). However, most of the BBB carriers that have been identified so far, particularly the ABC carriers, mediate efflux of substrates from the extracellular fluid within the brain to the blood, rather than influx from the blood into the brain. Thus, the BBB stands as a formidable obstacle to the distribution of drugs to the brain. This can pose a significant therapeutic problem, particularly for the delivery of hydrophilic drugs to treat brain cancers. For example, MDR1 (p-GP) mediates the efflux of colchicine, daunorubicin, and fluoropaclitaxel (Kusuhara and Sugiyama, 2009), all anticancer drugs.

Adverse reactions and toxicity A principle of pharmacology is that all drugs can have adverse effects on the body; i.e., no drug is absolutely clean. The spectrum of adverse effects can range from mild or tolerable (“side effects”) all the way to severe toxic reactions that can cause physical damage and, in some cases, can be life-threatening. Adverse reactions to drugs can be classified into four categories: toxic reactions, allergic reactions, idiosyncratic reactions, and immunosuppression (Table 6). Some of these responses are manifested at high doses or blood levels that exceed the therapeutic window, while others can occur within the therapeutic window (Figure 1, A). A single drug may produce multiple adverse effects in more than one of these categories. Toxic reactions are the most common. Pharmacological toxic reactions can be extensions of the therapeutic effect, and are usually produced at excessive blood concentrations. Examples are widespread and varied, from opioid- or barbiturate-induced respiratory suppression, to severe hypoglycemia produced by an overdose of insulin in diabetes, to excessive bleeding caused by high doses of warfarin or other anticoagulants. However, pharmacological toxic reactions might also be produced by drugs that act on more than one receptor or receptor subtype (nonselective drugs) at blood levels that are within the therapeutic window. Classic examples include nonselective beta blockers such as propranolol. Therapeutic effects of propranolol, such as suppression of renin secretion, are generally mediated by inhibition of beta-1 receptor isoforms. By contrast, adverse effects are usually the result of blockade of

22 | Principles of Pharmacology and Autonomics

Table 7. Phase 1 Cytochrome p-450 Drug-Metabolizing Enzymes Linked to Drug-Induced Hepatotoxicity. Cyp isoform

Representative drug

Proposed mechanism

CYP 1A2

tacrine (anticholinesterase)

formation of protein-reactive metabolites

CYP 2E9

diclofenac (anti-inflammatory)

formation of protein adducts with glucuronide metabolite

CYP 2E1

acetaminophen (analgesic)

formation of reactive metabolite NAPQI

CYP 3A

valproic acid (antiepileptic)

formation of an unstable free radical intermediate

Adapted from Madden et al. 1993, Kretz-Rommel et al. 1994, Rettie et al. 1997, Villeneuve and Pichette 2004.

beta-2 receptor subtypes. One example is bronchoconstriction produced by blockade of beta-2 receptors on bronchial smooth muscle cells. Antihistamines target H-1 receptors to decrease the severity of mild allergy symptoms, but can also simultaneously inhibit muscarinic cholinergic receptors, producing adverse effects such as tachycardia and dry mouth. Cytotoxic reactions refer to drug-induced structural cell damage. Drugs may produce cytotoxic effects directly. A classic example is acetaminophen-induced hepatotoxicity. In the hepatocyte, acetaminophen is subject to phase 1 and phase 2 metabolism, forming sulfonyl and glucuronide conjugates, which are not considered to be toxic. At high levels, however, acetaminophen may undergo additional metabollism by cyp2E1-mediated conversion to the reactive metabolite NAPQI (N-acetyl-p-quinoneimine). NAPQI then conjugates with the cytoprotective molecule ubiquinone. If that pathway becomes saturated—as may also occur with higher doses—then NAPQI may react with other proteins in the cell, producing structural damage and hepatic dysfunction. Conversion of parent compounds to hepatotoxic metabolites by hepatic drug-metabolizing enzymes is a clinically important mechanism of drug-induced hepatotoxicity (Table 7).

Assessment of drug toxicity The safety of a drug can be quantified by administering a wide range of doses, usually to experimental animals, and then measuring both desired (therapeutic) and adverse (toxic or lethal) responses (Figure 9). Two dose-response curves are usually generated (see Chapter 2), one over the therapeutic dose range, and the other over the toxic or lethal dose range. The index of potency for each curve is the dose that, on average, produces 50% of the maximum therapeutic (ED50), toxic (TD50), or lethal response (LD50). One way of quantifying a drug’s safety is to simply obtain the ratio of either the TD50 or the LD50 divided by the ED50. This is called the therapeutic index (TI). A low TI value reflects a short distance (dose range) between the curves. This is referred to as a narrow TI. Digoxin is a classic example of a drug with a narrow TI. The molecular target of digoxin is the ubiquitous sodium-potassium ATPase (sodium pump) enzyme. By inhibiting the enzyme on cardiac muscle cells, digoxin indirectly increases the intracellular

The Processing of Drugs By The Body: Drug Absorption and Disposition | 23

100 Toxic or lethal effect

Therapeutic Effect

Response (% Maximum) 50 TI =

TD50 or LD50 ED50

MS

0 ED50

TD50 or LD50 Log Dose

Figure 9. Therapeutic index and margin of safety. Two approaches to measuring a drug’s safety in human beings or whole animals are illustrated. The therapeutic index (TI) is the ratio of a drug’s index of toxic (TD50) or lethal potency (LD50) divided by its index of therapeutic potency (ED50). The shaded area represents the margin of safety (MS), or the dose range that drug produces no observable toxic or lethal effects. Note that the slopes of the two curves are different, suggesting that the mechanisms of the drug’s therapeutic and toxic effects are probably different as well. For more complete descriptions of log dose-response curves and the concept of drug potency, see Chapter 2.

concentration of free calcium ions, thus strengthening cardiac contraction. Not surprisingly, one therapeutic application for digoxin is the treatment of congestive heart failure. But inhibition of the sodium pump also disturbs the normal transmembrane distribution of sodium and potassium ions. This can lead to electrical instability and potentially life-threatening cardiac arrhythmias. The two curves, one for strengthening the heart and one for generating arrhythmias, are uncomfortably close, reflecting a very narrow TI. This has been repeatedly validated both experimentally and clinically. Accordingly, patient compliance and close monitoring of the dose are of critical importance during digoxin therapy. The TI alone, however, does not take into account the respective slopes of the two curves. If they are parallel, steep, and widely separated, then the TI can be a useful first estimate of a drug’s safety. But if the curve for the toxic or lethal effects is shallower than that of the curve for the therapeutic effect and the curves are close together, then the two curves could significantly overlap in spite of a fairly high numerical value of the TI (Figure 9). In that situation, a useful additional index is the margin of safety (MS). The MS describes the dose range that produces only therapeutic responses, with no observable evidence of toxic or lethal effects. Of course, the ideal situation would be no overlap at all. Unfortunately, many drugs are not that safe. In those cases where the MS is lower than the full therapeutic dose range

24 | Principles of Pharmacology and Autonomics

Table 8. Examples of Drug–Drug Interactions. Type of Interaction

Representative Mechanism

Pharmacokinetic antagonism

Induction of hepatic DMEs by one drug enhances the rate of hepatic metabolism of itself or other drugs that are metabolized by the same DME.

Pharmacokinetic potentiation

Inhibition of hepatic or intestinal DMEs or transporters increases the AUC of other drugs that are metabolized by the same DMEs or carried by the same transporter.

Functional antagonism

The pharmacological effects of two agonist drugs, that act on different receptors, have opposing effects on the same cell or tissue.

Pharmacological antagonism

Two drugs have opposing effects on the same receptor.

and the toxic effects are severe, it must be determined whether sub-maximal doses would be sufficient to produce acceptable therapeutic responses.

Drug-drug interactions It is quite common that the disease or diseases afflicting an individual will call for multiple drug therapy. Two or more drugs, taken simultaneously, can interact with each other in beneficial ways. One drug can enhance the therapeutic effectiveness of the other, reduce the severity of the other’s adverse effects, or both. This type of mutually beneficial interaction is often exploited therapeutically, and is referred to as “rational drug co-administration.” Diuretic therapy provides an illustrative example. A potentially serious adverse effect of thiazide or loop diuretics is a reduction in blood potassium ion concentration (hypokalemia). In contrast, anti-aldosterone diuretics such as spironolactone lack this adverse effect, and can actually increase blood potassium levels during therapy; thus, spironolactone and similar drugs are called “potassium-sparing” diuretics. If thiazide diuretics are given in combination with potassiumsparing diuretics, their opposing actions on blood potassium levels usually offset, and normokalemia is preserved. This combination is so rational that thiazide or loop diuretics are often combined with potassium-sparing diuretics in the same formulation (e.g., Dyazide®, a combination of hydrochlorothiazide and triamterene). Unfortunately, combinations of two or more drugs can also work against each other, producing adverse interactions that can be anywhere from mild to life-threatening. Among adverse drug–drug interactions that are of serious concern are those that increase or decrease the AUC of the affected drug, either increasing the risk of toxicity or decreasing therapeutic effectiveness (Figure 1). A summary of different types of drug–drug interactions, and examples of mechanisms for each, is provided in Table 8. Of the types of interactions included in Table 8, the first two—pharmacokinetic antagonism and potentiation—are the most common and thus of most potential clinical concern. They are discussed in the following sections.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 25

An example of functional antagonism would be the opposing actions of digoxin and terbutaline on heart rate. Digoxin, used to treat certain types of cardiac arrhythmias, slows heart rate by intensifying the actions of the vagus nerve, indirectly activating cardiac muscarinic receptors (Chapter 3). Terbutaline, used to treat asthma, accelerates heart rate by activating cardiac beta adrenergic receptors. The opposing actions of the two drugs could exacerbate the arrhythmia. Incidents of pharmacological antagonism are rarely encountered in practice because they are so irrational. For example, a patient with hypertension and asthma would typically require at least two drugs to treat the two conditions. Terbutaline is a drug that is used to treat asthma, and propranolol is an antihypertensive drug. However, it would be quite evident that administering propranolol to an asthmatic patient taking terbutaline would be a big mistake. Terbutaline dilates airways by activating beta-2 adrenoceptors on bronchiolar smooth muscle. Propranolol blocks beta-2 receptors, so its effects would be obviously counter-therapeutic in that situation. An antihypertensive drug that reduces blood pressure by a different mechanism would be a much better choice.

Pharmacokinetic tolerance Continual or repeated exposure often leads to diminishing responses of the target tissues or organs to drugs. This phenomenon is referred to as drug tolerance. Specifically, tolerance can be described as the need to increase the dose over time in order to maintain the response. Alternatively, tolerance can manifest itself as a diminishing response to a fixed dose over time. There are two important mechanisms of drug tolerance: 1) Pharmacodynamic tolerance, which is a progressive change in either the sensitivity or the numbers (density) of the drug’s receptors on or in the target cell (see Chapter 2); and 2) Pharmacokinetic tolerance, which is most often manifested by a progressive decline in a drug’s bioavailability due to acceleration of its metabolism by DMEs. An important cause of pharmacokinetic tolerance is the induction of hepatic DMEs by drugs and other xenobiotics. Induction refers to receptor-mediated increases in the expression of DMEs through the interaction of drug molecules with nuclear steroid-like receptors (Figure 10). The two steroid-like receptors that are most often involved in drug-induced DME expression are the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Either of these can bind to drug molecules in the hepatocyte and form complexes with another constitutive regulatory nuclear receptor, the retinoid X receptor (RXR). The drug-bound heterodimeric complex then binds to specific loci (response elements) in target genes to increase the transcription and translation of a variety of DMEs and other proteins. Examples of phase 1 and phase 2 DMEs and drug transporters that can be induced by ligand interactions with PXR or CAR are listed in Table 10. Because hepatic DMEs, especially CYP3A4, are involved in the biotransformation of many drugs, the induction of phase 1 or phase 2 enzymes by one drug may very well accelerate the metabolism not only of itself but also of other co-administered drugs (Figure 9). This is called pharmacokinetic cross-tolerance.

Influences of genetic polymorphism Not everyone responds to drugs equally. The same dose may be effective in one patient, ineffective in another, or toxic in a third. Often, the explanation for this type of variability in drug responses lies in the patient’s genetic profile. The study of genetic variability and its effects on the kinetics and dynamics of drug responses is the focus of the emerging discipline of pharmacogenomics (formerly termed

26 | Principles of Pharmacology and Autonomics

D1

PXR RXR

D1 RXR

PXR CYP 3A4 gene PXRE Transcription

D1 or D2

D1 – OH or D2 – OH

CYP 3A4 mRNA

CYP 3A4

Figure 10. Steroid-like receptors and induction of CYP3A4.In hepatocytes, drug molecules (D1) form ternary complexes with constitutive nuclear receptors RXR and PXR. The complex binds to a PXR response element on the CYP3A4 gene, inducing transcription of CYP3A4 RNA and increased synthesis of the enzyme. Induction can influence the metabolism not only of the inducing drug (D1) but also other drugs (D2) that are substrates of the same enzyme. Abbreviations: RXR, retinoid X receptor; PXR, pregnane X receptor; PXRE, PXR response element; CYP3A4, cytochrome p450 3A4. Adapted from Xu et al., 2005; Pan et al., 2009.

Table 9. Induction of DMEs and Transporters by PXR and RXR.

Receptor Class

Examples of induced DMEs and transporters

PXR/RXR

CYP3A4, UGT1A, MDR1

rifampicin, phenobarbital, clotrimazole, nifedipine, paclitaxel

CAR/RXR

CYP2B6, SULT2A1, MRP2

clotrimazole

Representative inducing ligands

Abbreviations: PXR, pregnane X receptor; CAR, constitutive androstane receptor; RXR, retinoid X receptor; CYP3A4, cytochrome P4503A4; UGT1A, UDP-glucuronosyl transferase 1A; MDR1, multidrug resistance protein 1; SULT2A1, sulfotransferase 2A1; MRP2, multidrug resistance-associated protein 2. Selected from Willson and Kliewer 2002, Klaassen and Slitt 2005, Xu et al. 2005, Zayek-Gliszcynski et al 2006, Shugarts and Benet 2009.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 27

Table 10. Polymorphisms and Drug Responses. Isoform

Drug

Affected response

CYP2C9

warfarin

altered warfarin metabolism and variable blood levels

UGT287

morphine

increased morphine levels

beta-2 receptor

albuterol

altered extent of bronchodilation and tachycardia

From Roden 2003, Daly 2010.

pharmacogenetics). Variations in genetic makeup can markedly influence bioavailability, effectiveness, and toxicity of many drugs. It is becoming increasingly clear, for example, that genetic differences in DME isoforms or expression can explain why some people metabolize certain drugs much faster than others do. Of course, variations in the processing of and responses to specific drugs can run in families, in ethnic populations, or in whole populations of unrelated individuals. They can involve single (monogenic) or multiple (multigenic) traits. Most polymorphisms are of the single nucleotide variety (SNP), in which only one base pair in a sequence is affected. SNPs can result in missense or nonsense sequences and dysfunctional or nonfunctional gene products. Less often, polymorphisms are due to insertions or deletions, which can result in over- or under-expression of the affected protein. Defining the precise alteration—in a gene, promoter region, exon, or other component of the transcriptional machinery—can be a very complex undertaking. What is known for certain is that genetic polymorphisms can render some individuals resistant and others hypersensitive to drug effects. A classic example is the anticoagulant drug warfarin. Warfarin is famous for its defiance of attempts to maintain stable blood levels in all patients. Variations in blood levels up or down increase the risk of bleeding or thrombotic episodes. The molecular target for warfarin is the hepatic enzyme vitamin K epoxide reductase (VKOR). Inhibition of VKOR depletes the blood of active vitamin K-dependent clotting factors. Warfarin is metabolized by hepatic CYP2C9. Inter-individual differences in VKOR and CYP2C9 isoforms contribute significantly to the wide range of doses required to achieve adequate anticoagulant control in large populations. Other examples of polymorphisms in DMEs and drug receptors, and their effects on drug responses, are summarized in Table 10.

Implications: The absorption and disposition of simvastatin In order to illustrate many of the principles discussed in this chapter, it might be helpful to consider the absorption and disposition of a model drug. The fundamentals of bioavailability, transport, metabolism, drug–drug interactions, adverse effects, and influences of genetic polymorphism can be illustrated very well by following the drug simvastatin through its journey of intestinal absorption and hepatic metabolism. Simvastatin would be a useful choice for several reasons: 1) it belongs to a class of drugs called “statins,” which are widely used in the treatment of specific cardiovascular diseases; 2) quite a bit of information has been collected about the absorption and metabolism of this drug; 3) it is an example of a pro-drug, which is a drug that must be transformed from the parent (administered) molecule into a metabolite in order to be therapeutically effective; 4) the molecular target of simvastatin is within the

28 | Principles of Pharmacology and Autonomics

SVL

SVA

HO EA

O SVL

SVL

O

HO O

PON

O

COO– OH

O

PON

MCT 1

O

EA

SVA

CH3 H3C

H3C Hepatic Metabolism

CYP 3A4 O

HO O

COOH OH

O O

CH3

H3C

OH

UGT 1A 1 O

HO ADP

O O

SVG

MRP 2 ATP

H3C

COO OH

O

ADP

CH3

MRP 3 O

O

O–

SVG

ATP

O Fecal Excretion

HO

OH

HO

Renal Excretion

Figure 11. Processing of simvastatin by the intestinal enterocyte. Abbreviations: SVL, simvastatin lactone; SVA, simvastatin acid; SG, simvastatin glucuronide; OAT P1B1, organic anion transport protein 1B1; MRP 2 or 3, multidrug resistance-associated protein 2 or 3; MCT1, monocarboxylate tranporter protein 1; CYP3A4, cytochrome P-450 3A4; UGT1A1, uridine diphosphate glucuronosyltransferase 1A1; EA, esterase; PON, para-oxonase. Compiled from Draganov et al., 2004; Prueksaritanont et al., 2005; Bottorff, 2006; Neuvonen et al., 2006; Shugarts and Benet, 2009; Huang et al., 2009; Fahrmeyer et al., 2010; Mackness et al., 2010.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 29

hepatocyte, providing an opportunity to examine relationships between its transport, metabolism, and therapeutic effectiveness in the same tissue; and 5) clinically important adverse interactions between simvastatin and other drugs, as well as influences of genetic polymorphisms on adverse effects of simvastatin, are well described.

Intestinal absorption of simvastatin Simvastatin is a drug used to treat dyslipidemias (disorders of blood cholesterol metabolism). It is administered in the lactone form as an inactive pro-drug (Figure 11). Because it is fairly lipid-soluble, the lactone can diffuse into the intestinal enterocyte by simple transcellular diffusion. Once in the enterocyte, the lactone can be absorbed into the bloodstream unchanged (again by simple diffusion), or it can be converted to the more biologically active simvastatin acid by esterases or paraoxonases. The acid form is much more water-soluble than the lactone, so it requires assistance by membrane transporters (e.g., MCT-1 on the basolateral side) for its absorption. Either the acid or the lactone can also be transformed by intestinal phase 1 CYP3A4 and phase 2 UGT1A1 to the glucuronide, which may then be exported via the ABC carriers MRP2 back into the intestinal lumen or MRP3 into the blood. The unmetabolized lactone and acid forms of simvastatin are then subject to further metabolism in the liver, while the blood-borne glucuronides are excreted by the kidneys. The target organ for simvastatin is the liver, where the drug is also further metabolized. The bioavailability of simvastatin is only around 5%. This implies that the intestine is a formidable barrier to the absorption of the active acid, and that the drug undergoes extensive hepatic metabolism.

Hepatic metabolism of simvastatin The active simvastatin acid form is taken up into the liver by the SLC transporter OATP1B1 on the basolateral side of the hepatocyte (Figure 12). OATP1B1 is the primary, if not exclusive, carrier for hepatic uptake of SA. The molecular target for simvastatin acid inside the hepatocyte is hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in the cholesterol-biosynthetic pathway. Simvastatin acid, but not the lactone, competitively inhibits the enzyme, decreasing the rate of hepatic cholesterol biosynthesis. One consequence of this action is increased expression of hepatocyte membrane receptors for low-density lipoproteins (LDL). This increases the clearance of LDL particles from the blood, decreasing LDL blood levels, thereby reducing the risk of atherosclerosis. Also, simvastatin acid can be exported into the bile for excretion via the ABC transporter MDR1 on the apical side of the cell. It can also undergo phase 1 and phase 2 metabolism, primarily by CYP3A4 and UGT1A1, respectively. A predominant metabolite is simvastatin acid glucuronide, which is either transported into the bile by the ABC transporter MRP2 for biliary excretion or transported back into the blood by the ABC transporter MRP3 for ultimate excretion by the kidneys.

Simvastatin interactions Hepatic uptake and metabolism of simvastatin can be affected by other drugs or by genetic polymorphisms that affect the activities of DMEs or transporters (Figure 12). For example, the antibiotic clarithromycin or the oral hypoglycemic drug gemfibrozil can interfere with the phase 1 and phase 2 metabolism of simvastatin by inhibiting CYP3A4 or UGT 1A1, respectively. Impaired hepatic metabolism would increase the intracellular levels of simvastatin acid, increasing the extent of its inhibitory action on HMG CoA reductase. Thus, clarithromycin, ritonavir, and other drugs that can inhibit phase

30 | Principles of Pharmacology and Autonomics

Apical (Bile)

Basolateral (Blood) HMG-CoA Reductase (–)

SA

Genetic polymorphism

HO O

MDR1

(–)

COO– OH

Cyclosporine (–)

OATP1B1

SA

O CH3

EA

H3C

SL

CYP3A4 (–)

HO

Clarithromycin

PON

COO OH

O O

CH3 H3 C

OH

UGT1A1 (–) Ritonavir

HO O

COO OH

O CH3 ADP SVG

H3C

ADP

O

O

O

O

MRP2

MRP3 HO

ATP

SG

OH

HO

ATP Renal Excretion

Biliary Excretion

Figure 12. Processing of simvastatin acid by the hepatocyte. Abbreviations: SL, simvastatin lactone; SA, simvastatin acid; SVG, simvastatin acid glucuronide; OAT P1B1, organic acid transport protein 1B1; MDR1, multidrug resistance protein 1 (also known as p-GP, or p-glycoprotein); MRP 2 or 3, multidrug resistance-associated protein 2 or 3; cyp 3A4, cytochrome P-450 3A4; UGT 1A1, uridine diphosphate glucuronosyltransferase 1A1. Compiled from Prueksaritanont et al., 2002; Jacobson, 2004; Bottorff, 2006; Jacobson et al., 2006; Pasanen et al., 2006; Neuvonen et al., 2008; Fahrmayer et al., 2010; Romaine et al., 2010; Niemi, 2010; Zhou et al., 2011.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 31

7 CC

Simvastatin acid (ng/ml)

6 5 4 TC 3 2 1

TT

0 0

1

2

3

4

5 7 Time (h)

9

12

20

Cumulative percentage of patients with myopathy

CC

15

10

5 TC TT 0 0

1 2 3 4 5 6 Years since starting simvastatin (80 mg/day)

Figure 13. Effect of genetic polymorphism on simvastatin toxicity. Simvastatin was administered to volunteers who were polymorphic with respect to the expression of the hepatic SLC transporter OATP1B1. Those who lacked functional transporters (CC) had much higher blood levels (top) and incidence of myopathy (bottom) than those who were heterozygous (TC) or homozygous (TT) for OATP1B1 expression. From Pasanen et al., 2006; Link et al., 2008; Niemi, 2010.

32 | Principles of Pharmacology and Autonomics

1 or phase 2 DMEs might increase the apparent therapeutic potency of a given dose of simvastatin. DME inhibitors can also enhance hepatotoxic or systemic toxic effects of many drugs by increasing their intracellular concentrations or AUC. As another example, the antibiotic cyclosporine can bind to OATP1B1, competitively inhibiting the hepatic uptake of simvastatin acid. The resulting accumulation (“exposure”) of simvastatin in the blood (i.e., increased AUC) might exceed the upper limits of the therapeutic window (Figure 1), increasing the risk of systemic toxicity. Similarly, genetic polymorphisms of the functional OATP1B1 transporter may inhibit transport function, enhance exposure, and increase the risk of systemic toxicity. Effects of genetic variability on the pharmacokinetics of simvastatin are illustrated in Figure 13. The study involved 32 patients who were polymorphic for SLC01B, the gene that encodes the OAT P1B1 transporter. Recall that the OATP1B1 SLC is the major, if not the exclusive, transporter that mediates the uptake of simvastatin acid into the hepatocyte (Figure 12). The three groups of volunteers were 1) homozygous for the complete reference genotype (TT, n =16); 2) heterozygous for the gene (TC, n = 12); and 3) those for whom a critical component of the gene was missing and thus did not express functional OATP1B1 transporters (CC, n = 4). All were administered the same dose of simvastatin. As shown in Figure 13 (top), the AUC for the CC group after a single dose was much greater than the AUCs for either the TC or TT groups. The likely mechanism for this observation was that hepatic uptake of simvastatin acid in the CC group was blocked, resulting in a marked accumulation in the blood. A known adverse effect of the statins, including simvastatin, is myopathy (damage to the skeletal muscles). Presumably, absence of hepatic OATP1B1 transporter interfered with hepatic uptake of simvastatin acid, markedly increasing blood levels above the therapeutic window (Figure 1A), thus significantly increasing the incidence of myopathy (Figure 13, bottom). This is an excellent example of how genetic polymorphism can substantially influence drug bioavailability and toxicity.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 33

Chapter 1: Bottom Lines • A major objective of pharmacotherapy is to maintain blood concentrations (AUC) within the therapeutic window. • The bioavailability of a drug can be influenced by its absorption, metabolism, distribution, and excretion, by interactions with other drugs, and by genetic polymorphisms. • Metabolism (biotransformation) of drugs tends to increase water solubility, decrease lipid solubility, decrease both therapeutic effectiveness and toxicity, and facilitate excretion of parent drug molecules. • The three phases of drug metabolism and disposition are Phase 1 oxidative metabolism, Phase 2 conjugating reactions, and Phase 3 carrier-mediated transport. • The two major sites of drug metabolism are the liver (primary importance) and the intestine (secondary importance). • The major route of drug administration is oral; other routes include intravenous, topical, transdermal, and intramuscular. • Major drug transporters in the intestinal enterocyte are the SLC carrier protein OAT P2B1 and the ABC transporter MDR1 (p-glycoprotein). • Major drug transporters in the hepatocyte are the SLC carrier protein OAT P1B1 and the ABC transporter MDR1 (p-glycoprotein). • In both the liver and intestine, the main phase 1 drug metabolizing enzyme is cytochrome P 450 (CYP) 3A4, and the major phase 2 enzymes are UDP glucuronosyl transferases. • The kidneys eliminate parent drugs and metabolites by a combination of filtration and secretion; the latter often requires carrier-assisted transport across tubular epithelium from the blood to the filtrate (urine). • Important determinants of drug distribution are blood flow and lipid vs. water solubility of the drug molecule. • In blood (pH buffered at around 7), both amine- and carboxylate-containing drug molecules are largely ionized, and thus require carrier assistance for trans-membrane diffusion. • In the blood–brain barrier, drugs are actively extruded from the brain to the blood by ABC transporters such as MDR-1 (p-glycoprotein). • Four major categories of adverse drug reactions are toxic reactions, allergic reactions, idiosynchratic reactions, and immunosuppression. • Toxic reactions can be subdivided into pharmacological toxic reactions, cytotoxic effects, genotoxic effects, and teratogenic effects. • Adverse effects of drugs can be quantified by the therapeutic index and margin of safety. • Four major examples of potentially adverse drug–drug interactions are pharmacokinetic antagonism, pharmacokinetic potentiation, functional antagonism, and pharmacological antagonism. • Induction of drug metabolizing enzymes is an important example of pharmacokinetic tolerance. • Genetic polymorphism can contribute significantly to variations in both therapeutic effectiveness and toxicity of drugs. • Genetic polymorphisms in the hepatic transporter OAT P1B1 increase the AUC of simvastatin, and thus increase simvastatin-induced myopathy while decreasing therapeutic effectiveness.

34 | Principles of Pharmacology and Autonomics

Glossary Absorption: The movement of drug molecules from the site of administration to the bloodstream. Absorption is bypassed by intravenous injection. Active transport: The movement of drug molecules across barriers (usually membranes of individual cells) by specialized transport proteins, most often against the concentration gradient with the application of external chemical energy. Additive effects: Combined effects of two or more drugs administered together are the sum of their individual effects. Administration: Route by which a drug is taken by or given to a person or an experimental animal. Adverse effects: Effects of drugs that are neither therapeutic nor desirable; also called adverse reactions. Allergic reactions: Broad category of adverse effects resulting from reactions of the immune system to drugs. Antagonism: The presence of one drug diminishes the effect of another drug. Antiporter: Transport protein that carries more than one molecule across cell membranes, at least two in the opposite direction. Bioavailability: The fraction of an administered drug that is measured in the bloodstream. Cross-tolerance: The secondary development of tolerance to additional drugs associated with, or in response to, the development of tolerance to the primary drug. Cytochrome p450: A large group of mixed function oxidase enzymes, found mainly in the liver but also in the intestine and some other tissues, that constitutes the most important drug-metabolizing enzyme system. Cytotoxic reactions: Cell damage caused by adverse actions of drugs on either target or nontarget tissues. Distribution: The passage of drugs in circulating fluids from the site of administration to various tissues or binding sites in the body. Drug: A chemical that is not a nutrient but nevertheless alters biological functions of organisms, including human beings. Drug interactions: Two or more drugs, when taken together during a course of therapy, may interact either positively (one drug enhancing therapeutic effects or decreasing adverse effects of the other) or negatively (one drug decreasing therapeutic effects or enhancing adverse effects of the other). Drug metabolism: The enzymatic conversion of drug molecules, usually by the liver, from the parent compound to one or more metabolite compounds; also called biotransformation. Drug tolerance: Tolerance to drugs can be expressed in either of two ways: 1) The progressive decline of a drug’s activity with repeated administrations; or 2) The progressive requirement of increasing doses over time in order to maintain a drug’s effect. ED50: The dose of a drug that produces an effect in 50% of the animals to which it is administered (index of potency in whole animals). Endogenous: Produced inside the body. Enteral: Administration of a drug either orally (by mouth) or rectally. Excretion: The passage of drugs or their metabolites from the circulation into the urine (or, in minor cases, the bile, feces, sweat, lung exhalations, or breast milk). Exogenous: Produced outside the body. Extravasation: Deposition of an administered drug, usually intravenous, in the tissue surrounding a blood vessel instead of the intended intravascular compartment. Facilitated diffusion: The movement of drug molecules across barriers mediated by specialized transport proteins, with the concentration gradient and without the application of external chemical energy. First-pass metabolism: Significant destruction of an orally administered drug upon its initial contact with and passage through the liver.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 35

Functional antagonism: Opposing effects of two agonist drugs on the same cell or tissue that are mediated by activation of different receptors. Genotoxic reactions: Damage to DNA caused by nonselective effects of drugs. Hydrophilic: Tendency to dissolve in aqueous media (e.g., blood plasma, extracellular fluid, or urine). Idiosyncratic reactions: Unexpected and usually adverse effects of drugs, other than allergic responses, that bear no obvious relation to the drug’s known mechanism of action. Immunosuppression: A suppression of the immune system and immune responses produced by drug therapy. Can be either intentional or unintentional (i.e., either therapeutic or adverse), depending on the therapeutic setting. Induction: Drug-induced increases in the expression and activities of proteins; most often referring to hepatic drug-metabolizing enzymes. Inhalational: Administration of a drug into the lungs or upper respiratory tract, usually by an inhaler. Intra-arterial: Administration of a drug directly into an artery, usually by injection or catheterization. Intradermal: Administration of a drug, usually by injection, just below the surface of the skin. Intramuscular: Administration of a drug, usually by injection, into the muscles. Intravenous: Administration of a drug directly into a vein, usually by injection or catheterization, bypassing absorption. LD50: The dose of a drug that results in the death of 50% of the group of organisms to which the drug is administered (index of lethality). Lipophilic: Pertaining to a molecule that dissolves readily in oil or fat but not readily in water (“fat-loving”). Also called “hydrophobic” (or “water-fearing”). Metabolism: Biotransformation of drug molecules by enzymatic reactions in the liver and other tissues. Usually, metabolism of a drug produces a more water-soluble (hydrophilic) and less biologically active or toxic form of the molecule. Metabolite: A product of a drug’s biotransformation, usually by the liver. Metabolites are generally less active, and more hydrophilic, than the parent compound. Paracellular: Between (around) cells within a layer. Parenteral: Any route of drug administration other than enteral (into the intestinal tract); the term is most commonly applied to intravenous administration. Passive diffusion: The movement of drug molecules across barriers, usually cell membranes or single layers of cells, with the concentration gradient and without the application of external chemical energy. Permissive effect: Two or more drugs without effects of their own produce effects when administered in combination. Pharmacodynamic tolerance: Tolerance to drugs caused by reductions in the responsiveness to drugs at the receptor level. Pharmacogenetics: The study of the influence of genetic factors on overall reactions of patients or experimental animals to drugs; similiar to Pharmacogenomics. Pharmacogenomics: The study of the influences of specific genetic variations on the processes of absorption, distribution, actions, metabolism, adverse effects, or excretion of drugs. Pharmacokinetic antagonism: Most often, one drug enhances the metabolic inactivation of another by the liver. Pharmacokinetic tolerance: Tolerance to drugs caused by changes in the rates of their biotransformation, usually by changes in the expressions or activities of hepatic drug-metabolizing enzymes. Pharmacokinetics: The study of drug absorption, distribution, metabolism, and excretion. Pharmacologic adverse reactions: Inappropriately severe extensions of the therapeutic effect (e.g., coma produced by overdose of a barbiturate).

36 | Principles of Pharmacology and Autonomics

Pharmacologic antagonism: Antagonism of the effects of agonists on receptors by reversible or irreversible antagonists. Potentiation: The combined effects of two drugs administered together exceed the sum of individual effects of each drug alone; also called synergism. Receptor down-regulation: The decrease of cellular receptor expression and density in response to continual agonist activation. Redistribution: A secondary and delayed reappearance of drugs in the bloodstream due to the movement of drugs from previous distribution sites (often adipose tissue). Pro-drug: A drug whose biological activity is increased by metabolism. Side effects: Tolerable adverse reactions that do not lead to damage to the organism. Subcutaneous: Administration of a drug, usually by injection, just below the skin; also called subdermal. Sublingual: Administration of a drug topically under the tongue. Symporter: Transport protein that carries more than one molecule across cell membranes in the same direction. Synergistic effects: Combined effects of two or more drugs are more than the sum of the individual effects, also called potentiation. TD50: The dose of a drug that that produces 50% of the maximal toxic effect produced by the highest toxic dose. Teratogenic effects: Damage to fetal development (“monster-producing”) caused by cytotoxic and genotoxic effects of drugs administered in pregnancy. Therapeutic index: The ratio of the index of toxicity to the index of therapeutic potency of a drug, usually the ratio of LD50/ED50 or the TD50/ED50. Therapeutic window: Range of concentrations of a drug in the bloodstream sufficient to produce desired effects without exerting intolerable adverse effects. Tolerance: Progressive decline in the response to a single drug dose; alternatively, a progressive increase in the dose required to maintain a drug’s effect. Topical: Administration of a drug on any exposed surface such as the skin, eye, or visible surfaces of the mouth. Transcellular: Across a layer of cells. Transdermal: Administration of a drug across the skin, most commonly by skin patch. Toxic reactions: Severe adverse reactions to drugs, usually associated with structural damage to cells, tissues, or organisms. Volume of distribution: A virtual term, describing the volume of liquid in which the total drug dose must be dissolved in order to equal the observed concentration in blood. Xenobiotic: An exogenous bioactive substance, such as a drug or toxin, that is treated by the body as a foreign substance.

The Processing of Drugs By The Body: Drug Absorption and Disposition | 37

Chapter 2

Assessing Drug Action

Introduction The hazardous journey a drug molecule must take to reach its site of action was described in Chapter 1. Here, we will consider how a drug produces its effects once it arrives at its destination. A good place to start might be to consider the following axiom: Drugs do not act unless they bind to receptors. What is a receptor? Nearly every drug in existence produces its effects by interacting selectively with specific receptors throughout the body. With very few exceptions, receptors are proteins. Most reside in outer membranes of various types of cells, but some are located inside the cell. Receptors for drugs can be selective binding sites for biological substances, or they can be enzymes, transporters, or regulatory proteins. Receptors come in many sizes and shapes, and participate in the regulation of a variety of physiological functions, from muscle contraction to nerve transmission to glandular secretion. The normal function of most drug receptors is to mediate the actions of hormones, neurotransmitters, and other biological substances on cells, tissues, and organs. Drugs can activate receptors or inhibit them, and thus can imitate or interfere with the effects of natural biological substances. The discipline of pharmacology is, fundamentally, the study of drug effects on cells, tissues, organs, and organisms in terms of their interactions with their receptors. This chapter is devoted to an important component of pharmacology called pharmacodynamics. It starts with a discussion of different types of receptors and examples of their intracellular effectors or signals that mediate their responses to agonists. It then progresses to mechanisms involved in receptor-mediated cellular regulation, including receptor desensitization and downregulation. Another section is devoted to drug-receptor interactions and methods used to quantify affinity, efficacy, and potency of agonist and antagonist drugs. The chapter concludes with brief descriptions of theoretical topics including the concepts of spare receptors and full versus partial agonism.

Molecular Targets of Drugs: Receptors and Their Cellular Signals As stated above, in order to exert their effects in the body, almost all drugs must interact with specific proteins called receptors:

DRUG

RECEPTOR

BIOLOGICAL EFFECT

Receptors are usually located on the surface of cell membranes, but can also reside within the cytoplasm, on intracellular membranes, or in the nucleus of virtually every cell type in the body. Receptors exist because they mediate effects of natural (endogenous) ligands such as hormones, neurotransmitters, or other biologically active substances such as growth factors or cytokines. Activation of receptors by these ligands generates or alters the activities of signal pathways and effectors. Thus, the interface

Assessing Drug Action | 41

Table 1. Five Major Categories of Receptors in Humans. Receptor class

Representative Effectors or Signals

Representative Ligands

GPCR1

AC/cAMP; PKC/DAG/IP3, receptor-activated ion channels

Most neurotransmitters and many hormones and drugs

Ion transporters2

GABA receptor (chloride channel)

GABA, benzodiazepines

Growth factor3

MAPKs, PI-3Ks

Insulin, growth factors, cytokines, interleukins, atrial natriuretic peptides, peptide drugs

Intracellular4

PXRs, CARs, RXRs

Steroids, many drugs

Enzymes as receptors5

Phosphodiesterases

Theophylline, sildenafil

1

Figures 1 and 2

2

Figure 3

Figure 4; these are also known as “receptors as enzymes.” They include tyrosine kinase receptors, tyrosine kinase-associated receptors, and receptor serine-threonine kinases. 3

4

For a discussion of intracellular receptors and their involvement in the induction of drug-metabolizing enzymes, see Chapter 1, Figure 10.

5

Figure 1

Abbreviations: GPCR, G-protein-coupled receptor; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; PKC, protein kinase C; DAG, diacylglycerol; IP3, inositol triphosphate; GABA, gamma aminobutyric acid; MAPK, mitogen-activated protein kinase; PI-3K, phosphoinositol-3 kinase; PXR, pregnane X receptor; CAR, constitutive androstane receptor; RXR, retinoid X receptor.

between neurotransmitters or hormones and their receptors are the focal points of homeostatic regulation by endocrine and nervous systems. Intracellular signal pathways translate the binding of receptors to their cognate ligands into cellular responses. A signal is a series of enzyme-mediated intracellular chemical reactions that culminate in one or more observable changes in the activity of the cell. Effector proteins are ultimate intracellular targets of signal pathways. There are three major types of effectors: 1) Metabolic enzymes, whose activation or inhibition by the signal alters cellular metabolism; 2) Gene regulatory proteins, whose activation or inhibition alters gene expression and protein synthesis and expression; and 3) cytoskeletal proteins, whose alteration by signal pathways affects cellular movement, shape, or—in the case of neurons or gland cells—secretion. There are five major categories of receptors (Table 1): G-protein coupled receptors (GPCR), receptors that are also ion channels, growth factor receptors, intracellular receptors, and enzymes whose activities can be directly influenced by drug molecules. By far the most common receptor type is the GPCR, but the other receptor classes can also be very important molecular targets of drugs. Nearly all receptor classes can be further sub-divided into subtypes or isoforms, which are slightly different forms of the same general type of receptor. Usually, isoforms can be distinguished on the basis

42 | Principles of Pharmacology and Autonomics

Table 2. Examples of G protein Subunit Isoforms and Representative Effectors or Signals. G protein subunit

Representative Signal or Effector

Gαs

Stimulation of adenylate cyclase

Gαi

Inhibition of adenylate cyclase

Gαq

Stimulation of phospholipase C

Gβγ

Altered conductance of G-protein-regulated potassium or other ion channels

Adapted from Pierce et al., 2002.

of their amino acid sequences. Changes in one or a few amino acids can subtly affect the shape (tertiary structure), secondarily influencing characteristics such as how the receptor is situated in the membrane, how it interacts with other proteins, how it binds to its ligand, or with which signal it is most commonly associated. For example, two isoforms of the beta (β) GPCR, β1 and β2, are both linked to the adenylate cyclase (AC) signal pathway (Table 1). Nevertheless, the two isoforms can be distinguished by their respective affinities for the neurotransmitter norepinephrine (NE). NE binds with fairly high affinity to β1 receptors, but its affinity for β2 receptors is so low that it would not be expected to bind to those receptors at physiological concentrations. This selectivity for isoforms of β receptors explains, for instance, the ability of NE to increase heart rate (a β1-mediated, AC-linked response in cardiac pacemaker cells) and its inability to produce vasodilation (a β2-mediated, AC-linked response in vascular smooth muscle cells). In contrast, the neurohormone epinephrine (EPI), chemically similar to NE, binds with roughly equal and high affinity to β1 and β2 receptors. As a result, EPI can increase heart rate and dilate blood vessels with roughly equal proficiency at physiologically relevant concentrations (see Chapter 3).

G-protein-coupled receptors (GPCR) and their major signal pathways G-protein-coupled receptors (GPCR) reside in outer membranes of nearly all cell types (Figures 1–3). The GPCR superfamily is by far the most prevalent of the five major receptor categories. In general, GPCRs are made up of three basic components: 1) the receptor protein itself that contains binding sites for the endogenous ligand and for G proteins. It is composed of seven transmembrane domains; 2) a G protein transducer that binds to, and is regulated by, either guanosine triphosphate (GTP) or guanosine diphosphate (GDP); and 3) a signal pathway and effector proteins. GPCRs, like most receptor classes, can be named according to the endogenous ligand that interacts with the ligand-binding subunit. Many of those are named for neurotransmitters found in the central or peripheral nervous systems. For example, the broad category of GPCRs include those that are selective for GABA (gamma aminobutyric acid), serotonin (5-hydroxytryptamine or 5-HT), endogenous opioids, and so forth. Receptors are also assigned letters, such as kappa (κ) or mu (μ) for opioids (e.g., morphine), alpha (α) or beta (β) for NE and EPI, D for dopamine, or M (muscarinic) for acetylcholine (see Chapter 3). Receptor isoforms, or subcategories based on small differences in the amino acid sequences of their binding sites, are usually assigned numbers: α1, α2, β1, β2, D-1, D-2, and so forth. Isoforms of the binding site may bind with greater or lesser affinity for their ligands. There are also isoforms for the associated G proteins. They are

Assessing Drug Action | 43

usually named with Greek and Arabic letters, such as Gαq. Specific isoforms of G protein subunits are associated with different effectors or signals (Table 2). A generalized GPCR associated with the adenylate cyclase/cyclic AMP signal pathway is shown in Figure 1A. The receptor is in its basal (low activity) state when guanosine diphosphate (GDP) is bound to the alpha subunit of the G protein. Binding of an agonist (e.g., endogenous neurotransmitter or hormone, or exogenous agonist drug) to the ligand binding site activates the receptor. Upon activation, the alpha subunit of the G protein exchanges bound guanosine diphosphate (GDP) for guanosine triphosphate (GTP). The activated alpha subunit can then in turn activate the enzyme adenylate cyclase, which then catalyzes the conversion of ATP to cyclic adenosine monophosphate (cAMP), the primary signal molecule. Binding of cAMP to regulatory subunits of its primary target, cyclic AMP-dependent protein kinase, disinhibits the enzyme, leading to the subsequent phosphorylation and activation of downstream protein kinases and other signal enzymes, ultimately generating cellular responses. In addition, the beta and gamma subunits of the G protein can dissociate from the alpha subunit and participate in the generation of a distinct cellular response. Cyclic AMP-dependent protein kinases (PKa) can mediate either rapid (acute) or delayed responses. Within seconds, the signal can begin to mediate increases in such processes as liver glycogenolysis or cardiac contraction produced by EPI (Chapter 3). Under some conditions and in some cells, however, activation of the AC/cAMP pathway can result in a change in the rate of gene expression and protein synthesis, processes that take longer to develop. Activated protein kinases can enter the nucleus, where they are associated with regulatory proteins and certain regions of the genome called response elements, altering the rate of mRNA synthesis (transcription) and ultimately the synthesis of proteins in the cytoplasm (translation). An example of a delayed response mediated by the AC/cAMP signal would be a decrease in the expression of the hepatic metabolic enzyme pyruvate kinase produced by high concentrations of glucagon (Ichai et al., 2001). The process is reversible. After a short time, GTP is hydrolyzed back to GDP and the G-protein subunits reassemble, returning the GPCR back to its basal state. cAMP itself is inactivated by phosphodiesterase enzymes, and protein kinases and other phosphorylated regulatory proteins are inactivated by phosphatase-catalyzed dephosphorylation. A balance between protein phosphorylation by protein kinases and dephosphorylation by phosphatases is a common mechanism of signal activity regulation. Like the AC/cAMP signal illustrated in Figure 1A, many other signal pathways consist of a family of protein kinases and protein phosphatases. A kinase is an enzyme that catalyzes the addition of a phosphate group to substrate proteins. Kinase substrates may include other kinases in the signal pathway, or may be other enzymes or proteins with different functions. In any case, the phosphate donor is usually ATP and the phosphorylated amino acid on the substrate protein is commonly serine or threonine. Phosphate groups are highly negatively charged in the intracellular environment. Adding those negative charges to the substrate protein alters the local charge density around the bonding site, changing the shape (tertiary structure) and thus the function of the phosphorylated protein. The process of phosphorylation can either enhance or inhibit the activity and function of a target enzyme or regulatory protein. The original (pre-phosphorylation) functional state of the target protein is restored by dephosphorylation, or removal of the phosphate. Dephosphorylation is usually catalyzed by a phosphatase enzyme. Variations in the strength of most cellular signals, and thus of the magnitude of the cellular response, depend to a great extent on the degree of phosphorylation of component signal proteins. Another important component of most signal-generated responses is an alteration in the concentration of free intracellular calcium ion in the cytoplasm. Both the structure and function of many intracellular proteins are affected by the extent of their binding to calcium. Many receptor-linked signals, frequently linked to a GPCR, are designed to control the levels of free intracellular calcium in the cytosol,

44 | Principles of Pharmacology and Autonomics

and thus the functional state of calcium-sensitive enzymes and other cellular proteins. Not surprisingly, most cellular functions are calcium-dependent, from glandular secretion to neurotransmission to muscle contraction. For example, β GPCR are almost always associated with the AC/cAMP signal. In the heart, activation of β1 receptors on cardiac muscle cells increases their force of contraction. Certain protein kinases in the AC/cAMP signal pathway interact with regulatory proteins in cardiac myocytes to enhance the peak levels of free calcium during the contraction phase of the cardiac cycle. In turn, the elevated calcium levels increase the extent of interaction between contractile proteins, strengthening the contraction of the affected cells and thereby of the whole atrium or ventricle. Activation of the same signal in smooth muscle cells, however, can decrease intracellular calcium levels and reduce the strength of tonic contraction. One example of a GPCR that is a molecular target of drug therapy is the beta-2 (β2) receptor (Figure 1B). EPI (adrenaline) is the main endogenous agonist for β2 receptors. Agonist drugs such as albuterol also, like EPI, bind to and activate β2 GPCRs. An important therapeutic use of albuterol is the treatment of asthma. One of the defects in asthma is constriction of airways (bronchioles), which are lined by smooth muscle cells. Binding of albuterol to β2 receptors on the outer membranes of bronchiolar smooth muscle cells activates the AC/cAMP pathway inside the cells. The cellular response in this case is a reduction in intracellular free calcium levels, relaxation of the smooth muscle cells lining the bronchioles, and opening of the airways. By the same mechanism, EPI also activates bronchiolar smooth muscle β2 receptors, increases intracellular cAMP levels, and opens lung airways. This is the basis for one of EPI’s beneficial effects in treating acute anaphylactic (hypersensitivity) reactions, for people who are very allergic to bee stings, peanuts, cat hair, or other allergens. EPI is not suitable to treat asthma because it has too many adverse effects (see Chapter 3). Activating a receptor on the cell surface is not the only way to alter a cellular signal. Like albuterol, theophylline can also increase cAMP levels in lung smooth muscle cells. Also like albuterol, theophylline can be used to treat asthma. But theophylline does not target the β2 receptor. Instead, theophylline binds to and inhibits the enzyme cAMP-dependent phosphodiesterase (PDIEST; Table 1 and Figure 1A), a downstream regulator of the cAMP signal pathway. PDIEST is an example of an enzyme as a drug receptor. Recall that PDIEST catalyzes the breakdown of cAMP into the inactive AMP (Figure 1A). Inhibition of the enzyme by theophylline thus retards the breakdown of cAMP, increasing its intracellular levels, amplifying the signal, decreasing cellular calcium, and promoting relaxation of the smooth muscle and the opening of the airways. This is a good illustration of two drugs that are used to treat the same disease by amplifying the same signal in the same cell, one (albuterol) by increasing the production of the primary signal molecule, and the other (theophylline) by inhibiting its breakdown. Another major signal pathway associated with GPCRs, in addition to the AC/cAMP signal, is the PLC/IP3/DAG pathway (Figure 2). Multiple GPCRs are associated with this pathway, including α adrenergic and muscarinic (M) receptors (Chapter 3). When the appropriate agonist binds to the receptor subunit, the associated G protein is induced to activate phospholipase Cβ (PLCβ), which catalyzes the conversion of specific membrane phospholipids, mainly phosphoinositol -4,5–bisphosphate (PIP2), into two products: 1) inositol 1,4,5-triphosphate (IP3); and 2) diacylglycerol (DAG). The two products have different effectors. The phospholipid IP3 acts to mobilize Ca2+ from storage sites, which in turn activates various intracellular calcium-sensitive proteins. The ubiquitous calcium-activated protein calmodulin (CAM) is highlighted in Figure 2. Together with Ca2+, DAG activates the enzyme protein kinase C, which phosphorylates target effector proteins whose identities vary with cell type. As is true for the AC/ cAMP signal pathway, PLC/IP3/DAG signal components can be organized by docking or scaffolding proteins, promoting intracellular target specificity. Also like the AC/cAMP signal, the PLC/IP3/DAG signal is turned off by calcium resequestration combined with phosphatase-mediated dephosphorylation of component protein kinases.

Assessing Drug Action | 45

A AG Theo

AG GPCR GDP Basal

(–)

AC

GPCR

Pdiest

Gα2 b

g

ATP

Active GTP

AMP

CAMP

Gα2 b

b

g PKA

(Inactive)

PKA

(Active)

g

ATP Cellular Response

PK

PK

(Inactive)

PO4 Pase (Active)

B

Figure 1. GPCR associated with the adenylate cyclase signal pathway. A: Two signal components mediating the response are illustrated: cyclic AMP-dependent and downstream protein kinases; and beta-gamma subunits dissociated from the G protein. B: Crystallized β2 receptor showing the seven transmembrane alpha-helical domains (typical of GPCRs in general) and the binding site for the inverse agonist carazolol. Abbreviations: GPCR, G protein-coupled receptor; Gαs, stimulatory alpha subunit of the G protein; β, γ, beta and gamma subunits of the G protein; AC, adenylate cyclase; AG, agonist ligand; Theo, theophylline; Pdiest, phosphodiesterase; GDP, guanosine diphosphate; GTP, guanosine triphosphate; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; PKA, cyclic AMP-dependent protein kinase; PK, protein kinase; Pase, phosphatase. Modified from Pierce et al., 2002.

46 | Principles of Pharmacology and Autonomics

AG K+

AG PLC

GPCR

PIP2

PKC

DAG

GAK

b

Gαq

g

CaCh

ER

ADP

E – PO4 (Active)

IP3

ATP

E (Inactive)

Ca2+ CAM ATP

ADP

Pase

Ca2+

Ca ATPase

4Ca2+ E (Inactive)

CAM Cellular Response

E

CAM (Active)

Figure 2. GPCR associated with the PLC/IP3/DAG signal pathway and a potassium channel. Abbreviations (see legend, Figure 1): GAK, G-protein-activated (inwardly rectifying) potassium channel; PLC, phospholipase C; PIP2, phosphoinositol-4,5-bisphosphate; IP3, inositol triphosphate; DAG, diacylglycerol; PKC, protein kinase C; E, enzyme; ER, endoplasmic (or sarcoplasmic) reticulum; CaATPase, calcium ATPase. Three effector molecules are illustrated: 1) calmodulin (CAM), a cytosolic protein involved in the regulation of cell movement, shape, or contraction; 2) an endoplasmic reticular calcium channel (CaCh); and 3) an endoplasmic reticular calcium pump (Ca ATPase).

When considering GPCRs, think in terms of “options.” Perhaps more than any other receptor type, GPCRs and their associated signals exert profoundly complex regulatory control over cellular processes. These are not straightforward one ligand, one GPCR, one signal, one effector, one response systems. Instead, control points can be found at multiple levels, including the cell membrane, individual receptors, and intracellular signals.

Assessing Drug Action | 47

A

Modulator

Cl– GABA

Membrane Hyperpolarization GABA Cl– Inhibitory Response

B

GABA

GABA + enhancing drug

GABA α

β

benzodiazepine α

γ

ethanol Cl–

β

neurosteroids barbiturates

Figure 3. The GABAA receptor: An example of an ion channel as a drug receptor. A: GABA (gamma aminobutyric acid) is an inhibitory neurotransmitter in the central nervous system. One of its receptors, the GABAA isoform, is a chloride channel in neuronal membranes. B: A number of drugs can bind to the GABAA receptor at sites other than those for GABA, acting as “allosteric modulators,” enhancing the inhibitory action of the neurotransmitter. Adapted from Uusi-Oukari and Korpi, 2010.

For example, the surface of a single cell can display dozens of receptor categories including GPCRs, each consisting of many individual receptors. Receptors can congregate on the cell surface, or their signal proteins can coalesce inside the cell. Clusters of receptors on the membrane are called “lipid rafts” (Luttrell, 2006). These gatherings of receptors may promote signal amplification or may partially account for the specificity of agonist-induced receptor down-regulation (see "Desensitization and downregulation" below). Individual protein molecules within a signal pathway can be physically connected to each other by other proteins called “scaffold” or “docking” proteins (e.g., IRS1). Further, two or more signal pathways may come together to ultimately share a common pathway. “Signal convergence” and

48 | Principles of Pharmacology and Autonomics

IGF-1

IGFR

IRS-1 P

P

P1-3K

Raf

Akt

MEK

mTOR

ERK Cell Growth and Proliferation

Figure 4. The IGF-1 receptor: An example of a tyrosine kinase growth factor receptor. Two important signaling pathways associated with IGF-like receptors are illustrated: 1) Raf-MEKERK; and 2) PI-3K-Akt-mTOR. Abbreviations: IGF-1, insulin-like growth factor 1; IGFR, insulinlike growth factor receptor 1; IRS-1, insulin receptor substrate 1; PI-3K, phosphoinositol 3-kinase; mTOR, mammalian target of rapamycin; MEK, mitogen-activated protein kinase; ERK, extracellular-regulated protein kinase. Adapted from Ryan and Goss, 2008.

scaffolding may be two mechanisms, among others, of maintaining selectivity and site-direction of signal-mediated responses within the cell. With regard to GPCRs specifically, multiple types, subtypes, or isoforms can exist on one cell. While different categories of GPCR may often be coupled to the same signal, isoforms of GPCR subtypes may be coupled to distinct signals depending in part on the associated G protein. Alpha (α) adrenoceptor subtypes (Chapter 3) can serve as an illustration here. Activation of α1 receptor subtypes on heart muscle by the α-agonist phenylephrine both increases and decreases levels of calcium inside the cell (O-Uchi et al., 2008). This apparent paradox is explained by the observation that the opposing responses are mediated by two isoforms of the α1 receptor, α1A and α1B. .The 1A isoform is coupled to Gq protein and the PLC/DAG/IP3 cellular signal that ultimately opens a membrane calcium channel, increasing intracellular calcium levels. In contrast, the 1B isoform

Assessing Drug Action | 49

is coupled to a Go protein, whose βγ subunit complex inhibits the same calcium channel, decreasing calcium concentrations. Isoform-specific signals of this type have been called “signaling bias.” As another example, two ligand molecules of similar structure may induce a given GPCR subtype to generate different signals. This is referred to as “functional ligand selectivity” (Evans et al., 2010). In addition, one pathway, activated by one GPCR type, may subsequently activate or inhibit other signal pathways within the cell. Interactions of this type are often called signal “crosstalk.” Clearly, GPCRs, perhaps more than any other receptor category, confer a degree of regulatory control that is nothing short of astounding. It seems likely that the emerging picture of GPCR function and its varied roles in cellular regulation will have a significant influence on drug development and therapeutic strategies in the future.

Ion channels or ion transporters as receptors Ion channels are proteins, usually made of multiple subunits, that reside on cell membranes and mediate trans-membrane movements of ions such as sodium, potassium, chloride, calcium, and so on. Many ion channels are subject to regulation by neurotransmitters or hormones. The channels themselves can bind directly and with high affinity to endogenous ligands, and thus can also act as receptors for drugs with high enough affinities for binding sites on the channel (Figure 3). This category of receptor is not normally associated with a separate and distinct signal pathway; it is its own effector. The result of direct ligand binding would be a change in the conductance to the specific ion, usually affecting transmembrane voltage. The channel depicted in Figure 3 is both a chloride channel and a receptor for the neurotransmitter gamma aminobutyic acid (GABA). Activation of the channel by GABA opens the channel (increases its conductance to chloride), increasing the resting transmembrane voltage and thus hyperpolarizing the affected neuron. Hyperpolarization makes the neuron more difficult to excite by other neurotransmitters. Accordingly, GABA is considered to be an inhibitory neurotransmitter. Agonist drugs can also bind to the GABA receptor and act as modulators, increasing the effects of GABA to open the chloride channel and hyperpolarize the neuronal membrane. These include benzodiazepines (e.g., diazepam or Valium®), ethanol, and barbiturates such as phenobarbital. Not surprisingly, all of these drugs act as central nervous system depressants. Ion transporters can be molecular targets for drugs even if they are not under the control of endogenous ligands. For example, diuretic drugs can interact with specific ion channels or transporters on membranes of renal tubular epithelial cells. “Loop” diuretics such as bumetanide bind to, and inhibit, transporters for sodium, chloride, and potassium in the ascending limb of the Loop of Henle. Thiazide diuretics inhibit transporters for sodium and chloride that reside in the early distal tubule. Certain anti-aldosterone diuretics such as ameloride bind to and inhibit sodium channels in the late distal tubule and early collecting duct, functionally inhibiting the action of aldosterone at this site. The ability of these drugs to alter renal handling of ions and water underlies their therapeutic utility in the treatment of specific cardiovascular diseases.

Growth factor receptors (“receptors as enzymes”) Growth factor receptors (GFR), like GPCRs, are located on the surface membranes of responding cells. They are more precisely called “receptors as enzymes,” because most, but not all, GFRs mediate the effects of true growth factors. Also like GPCRs, GFRs may also be associated with AC/cAMP or

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PLC/IP3/DAG signal pathways. However, unlike GPCRs, GFRs are usually made up of single transmembrane components and are not coupled to G proteins. Instead, binding of GFRs to their ligands activate receptor-linked enzymes, including tyrosine kinases, serine/threonine kinases, or histidine kinases. GFR receptors mediate the effects of a variety of growth factors and hormones, including insulin, insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). The IGF-1 receptor (IGRF) is diagrammatically illustrated in Figure 4. Binding of IGF-1 to the receptor causes single-chain receptor components, which are latent protein kinases, to form dimers. The dimerization enhances the protein kinase activity, with the result that the enzymes phosphorylate each other (“autophosphorylation” or “cross-phosphorylation”). The phosphorylated enzymes can then interact with docking or signaling proteins, initiating signal pathways downstream. Two common pathways associated with the IGF-1 receptor are shown in Figure 4: 1) RafMEK-ERK; and 2) PI3K-akt-mTOR. Either pathway can promote delayed (minutes to hours) growth and proliferation responses by affecting gene transcription. In addition, PI3-K-associated pathways can be involved in early responses to hormones or growth factors. For example, a PI3-K pathway mediates the rapid (within seconds) increases in glucose uptake and glycolysis produced by the binding of insulin to its receptors on heart muscle.

Implications It is clear from the foregoing discussion that there are many different types of receptors and receptor isoforms—with a variety of effectors and signals—that respond to multiple hormones, neurotransmitters, growth factors, and other biologically active ligands. A point to emphasize here is that a single cell can simultaneously express many categories of receptors and their isoforms. Hundreds or even thousands of individual receptors, belonging to different classes and subclasses, can coexist on outer or inner membranes of individual cells. At any instant in time, many of these different types of receptors are engaged with their cognate ligands in vivo. Cellular regulation is thus a continual and cooperative interplay between disparate regulatory influences acting at the receptor level. Further, each receptor subtype or isoform might be linked to one or more signals, both stimulatory and inhibitory. To add even more complexity, signals do not exist in isolation either. They can interact inside the cell, phosphorylating each other’s signal components or target proteins. This endless chatter, or cross-talk, between cellular signals is another critical determinant of cellular homeostasis. Remember, in physiology it is never “all-or-nothing,” but rather it is always “more or less.” Endocrine and nervous systems are designed to produce subtle and continual adjustments at the cellular (receptor) level in order to maintain the function of organs and organ systems within appropriate homeostatic limits. Similarly, when drugs intervene, they neither totally block nor completely maximize collective receptor activity. In most cases they merely alter the direction or magnitude of hormonal or neural control. The following sections will focus on the quantification of interactions between drugs and their receptors.

Desensitization and down-regulation of receptors The ability of receptors to respond to agonists, i.e., agonist intrinsic activities, can diminish with time. Loss of responsiveness to agonists can occur by at least two interrelated mechanisms: 1) Receptor desensitization; and 2) Receptor downregulation. The essential difference between these two processes is that the first does not involve a change in the numbers of receptors on the cell surface, while the second is

Assessing Drug Action | 51

(H or DAG)

A

E

GPCR-1 (i)

GPCR-1 (a) β γ

β γ

Gαs

GPRK-2

P

GPCR-1 (i) β-ARR

P

1

P β γ

β-ARR

GPRK-2

P

GRK2 2

PK β

β γ

P

Gα

P

γ

Gα

GPCR-2 (i)

GPCR-2 (a)

B Dhom Tension

Dhet

1

1

2

Time

Figure 5. Homologous and heterologous desensitization of GPCRs. A: Upon binding to its agonist, an activated GPCR (GPCR-1) can rapidly become inactivated through phosphorylation mediated by G-protein receptor kinase 2 (GPRK-2) and subsequent binding to the modulating protein beta arrestin (β-ARR). This is a form of homologous desensitization (1). Activation of GPCR-1 by its cognate ligand can also desensitize another type of GPCR (GPCR2), through signal-mediated phosphorylation by protein kinases (PK). This is a form of heterologous desensitization (2). E, effector or signal protein. B: Tension responses to two agonist drugs demonstrating homologous and heterologous desensitization. When the desensitization is homologous, tension responses (e.g., of an isolated blood vessel) to the second administration of drug 1, but not of the first response to drug 2, is diminished. When the desensitization is heterologous, tension responses to the second administration of drug 1 or the first response to drug 2 are diminished. It is assumed that drugs 1 and 2 produce their respective responses by activating different GPCRs. Other abbreviations: H, hormone or neurotransmitter; DAG, agonist drug; S, signal (such as adenylate cyclase or protein kinase C); P, phosphate group; a, active; i, inactive; Dhom, homologous desensitization; Dhet, heterologous desensitization. Adapted from Luttrell and Lefkowitz, 2002; Perry and Lefkowitz, 2002.

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associated with a reduction in receptor density. In other words, desensitization occurs while the receptors are still in the membrane, while down-regulation usually involves internalization of the receptors for subsequent reassembly and recycling or destruction. Rapid loss of intrinsic activity (within seconds or minutes), by either mechanism, is called tachyphylaxis. Delayed loss of activity (after hours or days) is a form of longer-term pharmacodynamic tolerance. Desensitization of GPCRs has been well characterized (Figure 5). Binding of an agonist—such as a hormone, neurotransmitter, or agonist drug—to its GPCR initially activates the receptor and increases the activity of its signal or signals inside the cell. However, agonist binding can simultaneously initiate the rapid inactivation of the same receptor by promoting its phosphorylation on the inner side of the membrane (Ferguson et al., 2001), initiating the process of homologous desensitization (desensitization to the stimulating agonist). In Figure 5, the release of the beta-gamma subunit of the G protein, induced by ligand binding, recruits the enzyme G-protein receptor kinase 2 (GPRK-2), which in turn catalyzes the phosphorylation of serines and threonines on one or more of the seven transmembrane domains that are characteristic of most or all GPCRs (1 in Figure 5A). This promotes the binding of a modulating protein, beta arrestin, to the receptor, extending the duration of homologous desensitization. Reestablishment of receptor function follows dissociation of the arrestin proteins, and subsequent removal of the phosphates from the receptor protein mediated by phosphatase enzymes (not shown in the figure). Agonist ligands can desensitize their own receptors (homologous desensitization) as well as GPCRs for other ligands (heterologous desensitization). Unlike homologous desensitization, which is mediated by GPK-2 and beta-arrestin, heterologous desensitization of GPCRs (reduced sensitivity to a ligand other than the initiating agonist) is promoted by signal-associated protein kinases (2 in Figure 5A). Activation of a signal associated with one class of GPCR—and one or more of its constituent protein kinases—can also phosphorylate and inactivate another type of GPCR on the same cell (Figure 5 B). With regard to GPCRs at least, the processes of desensitization and down-regulation are interrelated. Beta arrestins are not only involved with desensitization of GPCRs, they are also central players in their down-regulation (Figure 6). The inactivated receptors bound to beta arrestin recruit additional regulatory proteins to form invaginations in the membrane called “coated pits.” These regulatory proteins include dynamin, clathrin, and beta-2-adaptin. Receptor-containing vesicles formed from the coated pits are then internalized into the cytoplasm, rendering them inaccessible to ligands. Some GPCRs are then degraded, while others may be recycled and returned to the membrane. To be recycled, the regulatory proteins and ligand are released from the vesicle-bound receptor. Still inactive, the receptor then releases its ligand and becomes de-phosphorylated via G protein-specific phosphatase-2. The vesicle, with restored GPCRs, merges with the outer membrane. The receptors are then readied for ligand activation by reassembly with G protein subunits and GDP. An illustrative example is the development of pharmacodynamic tolerance to morphine (Zhang et al., 1998).

Receptor supersensitivity Recall that nearly all endogenous ligands are agonists, but most therapeutic drugs are competitive antagonists. A potential hazard of chronic treatment of patients with antagonist drugs is rebound supersensitivity. Sudden discontinuation of antagonist therapy may produce adverse effects that can be attributed to exaggerated activation of affected receptors by endogenous agonists. An important underlying mechanism may be an interruption, by antagonists, of the ongoing processes of endogenous agonist-induced desensitization and down-regulation (Hendriks-Balk et al., 2008).

Assessing Drug Action | 53

H GPCR (d) -ARR

DYN DYN DYN

1 P P

H

AP2 DYN

R

R -A AP2 3

P P

DYN

CLATHRIN AP2

2

CLATHRIN -ARR H 4 LYSOSOMAL DEGRADATION PP PP2A

P

P

Figure 6. Down-regulation of GPCRs. Desensitized (d) GPCR bound to beta-arrestin (β) become incorporated into emerging membrane vesicles (coated pits) by interacting with and recruiting regulatory proteins (1) including clathrin, β-2 adaptin (AP2), and dynamin (DYN). The coated pits then internalize in the form of vesicles (2). Release of regulatory proteins, followed by dephosphorylation of the receptor via a specific phosphatase isoform, G-protein-specific phosphatase-2 (PP2A), prepares the vesicle for recycling (3) and subsequent reassembly and restoration of responsiveness to ligand (H) activation. Alternatively, internalized receptors can be destroyed by lysosomal degradation (4). Adapted from Penn et al., 2000; Luttrell and Lefkowitz, 2002.

The key assumption is that, physiologically, the overall sensitivity of the cell to endogenous ligands (such as neurotransmitters or hormones) over time is a balance between the concentrations of ligands on the one hand and availability of functional receptors on the other. The first is largely a function of the combined rates of secretion, delivery, removal, and destruction of the ligand (Chapter 3). The second is mainly determined by the rates of new receptor synthesis and existing receptor desensitization, downregulation, and lysosomal destruction. In a sense, the system works like a thermostat, but instead of a furnace going on and off to maintain a fairly constant temperature, the cell continually adjusts the sensitivity and density of its receptors to maintain a fairly constant level of function in the face of moderate variations in ligand levels. This is yet another form of cellular homeostasis.

54 | Principles of Pharmacology and Autonomics

Hypothetically, introduction of an exogenous antagonist would alter the balance. Initially, binding of antagonists to their GPCRs would interrupt signal activation by the endogenous agonists (the intended therapeutic effect). However, continual interruption of agonist-induced receptor desensitization and downregulation with prolonged therapy would diminish the extent of receptor desensitization and downregulation, resulting in an increase in the densities of functional receptors. This might manifest itself as a progressive decline in the antagonist’s effects during therapy (pharmacodynamic tolerance). Then, upon sudden discontinuation of antagonist treatment, the new balance would reveal itself, temporarily at least, as heightened responses—supersensitivity—to the endogenous agonist. After a time, the supersensitive state would decline as the original balance is restored. If this supersensitive state is associated with receptor densities that are above normal, the effect is sometimes referred to as “receptor upregulation”.* For example, beta blockers such as propranolol might be given to patients who suffer from cardiovascular diseases including hypertension, congestive heart failure, angina, and certain cardiac arrhythmias. In all of these conditions, an essential component of propranolol’s therapeutic effect is the interruption of the continual activation of cardiac β receptors by NE released from neurons or by blood-borne EPI. This would be manifested by, among other responses, a reduced and more stable heart rate in patients treated for congestive heart failure, angina, or cardiac arrrhythmias, or a reduction in blood pressure in hypertensive patients (see Chapter 3). However, if the patient suddenly discontinues beta blocker therapy after a prolonged treatment period, then his or her condition could be worse than before the patient took the drug in the first place. At least temporarily, heart rate could go up dramatically (tachycardia), the heart may beat erratically (arrhythmias), and blood pressure could rise to dangerous levels (rebound hypertension). Pre-treatment conditions will probably return eventually as the ligand-receptor balance at the level of the heart cell is restored. This “withdrawal syndrome,” as it applies to beta blockers specifically, can be a significant clinical problem. Analagous withdrawal phenomena, associated with other signs and symptoms but also attributable to changes in receptor sensitivity, have been observed in response to other drugs such as opioid analgesics and their actions on neurons in the brain.

The Interaction of Receptors with Drugs Drug potency and its quantification Also as discussed above, drugs cannot act unless they bind to receptors and either activate them (agonist drugs) or inhibit their activation by endogenous ligands (antagonist drugs). One of the first questions often asked about a drug is “How potent is it?” In order to communicate effectively about drug potency, it is of course necessary to understand exactly what the term means. A common misconception is that potency refers to a drug’s “strength,” or how intense its maximum effect can be at the highest effective doses. That is not what potency means. Potency specifically refers to the amount of drug required to produce a response of a defined magnitude. The lower the required dose or concentration, the greater the potency. The term is often used to compare two or more drugs that belong to the same class; i.e., drugs within a therapeutic class can be categorized according to their relative potencies, using one of the drugs in the class as a reference compound. As discussed above, any individual drug will belong to one of two broad categories: agonist or antagonist. Both types bind to receptors. If the drug is an agonist, it will activate the receptor and produce a response in the cell. By doing so, agonists imitate the effects of endogenous ligands (hormones,

* The term "upregulation" can have different meanings depending on the context. For example, it can be used to describe the effect of hormones to increase the synthesis of receptor and other proteins.

Assessing Drug Action | 55

neurotransmitters, and other biologically active substances) on those receptors. If the drug is an antagonist, it will bind to the receptor but will not produce a response by itself. Instead, it will interfere with the ability of endogenous agonists to activate the receptor and produce their effects on that cell. Both agonists and antagonists possess the property of potency, but for different reasons. The potency of either an agonist or an antagonist in vivo describes the relationship between its dose or concentration in blood and the magnitude of the ensuing response in the whole animal or patient. An agonist drug produces its effects directly by binding to its molecular target (receptor) and activating it. In contrast, an antagonist produces its effects by interfering with the continual activation of receptors by endogenous agonists such as neurotransmitters, hormones, or other biologically active substances. For example, phenylephrine is an agonist, and prazosin is an antagonist, on α GPCRs. By activating α receptors on smooth muscle surrounding blood vessels, phenylephrine imitates the endogenous agonist neurotransmitter NE to increase or maintain the state of contraction of those smooth muscles, producing vasoconstriction (narrowing of the lumen diameter of blood vessels). This effect can be measured as a dose-dependent reduction in blood flow locally or an increase in blood pressure systemically in vivo, or as a concentration-dependent increase in the contraction of isolated blood vessels ex vivo (outside the animal). The potency of phenylephrine can be determined directly by measuring the relationship between its concentration and the magnitude of the resulting vasoconstriction. By contrast, prazosin’s potency cannot be measured this way, because it does not activate receptors. Instead, the potency of prazosin on vascular smooth muscle α receptors can be measured indirectly by quantifying its ability to oppose the constricting actions of phenylephrine or of the endogenous transmitter NE. Thus, the potency of prazosin can be assessed in vivo by the relationship between its dose and its ability to oppose the blood pressure-maintaining effects of the endogenous α agonist NE, i.e., by its ability to reduce blood pressure. Ex vivo, the potency of prazosin can be assessed as its ability to oppose the contractile actions of an alpha agonist such as NE or phenylephrine on isolated vascular smooth muscle preparations. Because agonists such as phenylephrine or NE activate receptors upon binding to them, they are characterized as possessing the property of intrinsic activity on those receptors. By contrast, antagonists such as prazosin do not activate receptors after they bind to them, and thus lack the property of intrinsic activity. So both agonists and antagonists bind to receptors at physiological or pharmacological concentrations, and thus have appropriate affinity for them, but only agonists possess the property of intrinsic activity. Nevertheless, both agonists and antagonists produce observable and measurable effects in experimental animals and patients, and therefore both have the property of potency.

Drug potency assessed in patients or experimental animals (in vivo) Relative potencies of drugs within a single pharmacological or therapeutic class can be determined in patients or experimental animals. This can be done by measuring the responses to either single or multiple effective doses of the drugs. To illustrate this concept, we can focus on the thiazide and thiazide-like drugs, an important class of diuretics (drugs that enhance urine production). There are numerous members of this class, but three illustrative examples would be indapamide (Lozol®), hydrochlorothiazide (Hydrodiuril®), and chlorothiazide (Diuril®). All work within the kidney on the same molecular target, an ion transporter, to increase the production of urine. At roughly equivalent oral doses, all three would produce responses (increase in the rate of urine production) of approximately the same intensity (usually at or near the maximum in most patients). However, the equivalent doses of the three drugs are not the same: indapamide, 1.25 mg; hydrochlorothiazide, 25 mg; and chlorothiazide, 250 mg. Clearly, the rank order of the amounts of each drug required to produce similar diuretic responses are indapamide (lowest), hydrochlorothiazide (intermediate), and chlorothiazide (highest). Relative

56 | Principles of Pharmacology and Autonomics

potencies of these diuretics can then be quantified by assigning a reference drug in the group a potency value of 1.0, and ranking the others according to the ratio of their effective doses to that of the reference compound. Here, we can assign hydrochlorothiazide as the reference (of course, compared to itself, its relative potency value is 25/25 = 1.0). By this single-dose approach, the relative potency of indapamide vs. hydrochlorothiazide would then be 25/1.25 = 20, while that of chlorothiazide would be 25/250 = 0.1. In other words, indapamide is found to be 20× more potent than hydrochlorothiazide, but chlorothiazide is 10× less potent than hydrochlorothiazide, even though all three produce about the same diuretic effect in patients at their respective therapeutic doses. Absolute potencies of individual drugs can also be assessed quantitatively in vivo by administering multiple doses of one drug and recording the responses to each dose. Graphical representations of the relationship between doses of a drug and the responses to each dose are called log dose-response curves. The ultimate aim is to obtain a numerical index of potency in vivo for that drug, called the ED50. The ED50 in experimental animals or humans is defined as the average dose that is sufficient to generate 50% of the drug’s maximum effect. As an example, log dose-response curves for the loop diuretic bumetanide and the thiazide-like diuretic clopamide are compared in Figure 7. It is clear from the curves that bumetanide is nearly 20× more potent than clopamide is. Its ED50 is shown to be around 0.2 mg, while that of clopamide is around 3.8 mg, and 3.8/.2 = 19. In addition, bumetanide has around twice the clinical efficacy that clopamide does. At near-maximal effective doses, clopamide increases urine volume by around 2.5 mL/min., but the maximal effect of bumetanide is twice that, at around 5 mL/min. It is not surprising that, of the two, bumetanide is currently used far more often as a diuretic.

Drug potency assessed experimentally Just as drug potencies can be estimated at the clinical (therapeutic) level in patients, drug potencies can be quantified in more detail, and with more accuracy, under experimental (laboratory) conditions. The most common approach is to select a cell type or organ that is known to respond to the drug (responding system). The next step is to select a suitable experimental preparation (cultured cells, isolated organs or tissues, etc.), and a reliable, accurate, and consistent method of quantifying the response of the cells or organs to the drug of interest. Finally, the cells or tissues are exposed to several concentrations of the drug in order to quantify the relationships between multiple concentrations of the drug and the magnitude of the associated responses to each concentration. The ultimate goal is to obtain a single numerical index of potency for the drug of interest.

Experimental assessment of agonist potency One older but still illustrative example of a preparation that can be used to assess the potencies of both agonists and antagonists would be an isolated arterial preparation (Figure 8A). In this example, a section of aorta is isolated from a rat, rabbit, or other animal, and suspended under tension in an immersion bath. The aortic preparation can be in the form of a spiral strip or a tubular section (a rat aortic ring is illustrated). Again, a useful agonist is phenylephrine. Recall that phenylephrine activates α-1 GPCRs on vascular smooth muscle and therefore constricts the vessel. The measured responses are changes in tension (tonic contraction of smooth muscle) of the arterial ring induced by the addition of phenylephrine to the bathing solution. A resting tension is first imposed mechanically (in this case about three grams), which is necessary to optimize responsiveness of this preparation to phenylephrine. The α receptors on rat aortic smooth muscle seem to be exclusively α1d isoforms (Buckner et al., 1996), for which phenylephrine, like the endogenous neurotransmitter NE, has high affinity.

Assessing Drug Action | 57

6 - Bumetanide (B) - Clopamide (C)

Urine Volume 3 (mL/min)

ED50 (B) ≅ 0.2

0

0 .125 .25

ED50 (C) ≅ 3.8

.5 1 2 4 8 Dose (mg, log scale)

16

32

64 128

Figure 7. Log dose-response curves for bumetanide and clopamide in human volunteers. Adapted from Campbell and Moore, 1981; McNeil et al., 1987. After a suitable stabilization period, phenylephrine is added to the bath at increasing concentrations. After each addition, the new stable tension is recorded. The agonist is not washed out, but instead higher concentrations are added and further increases in tension are recorded. This method is called generation of a “cumulative dose-response curve,” referring to the fact that the increase in agonist concentration is cumulative (one added on top of the other). The process is repeated by adding higher and higher concentrations until the artery no longer responds because it has reached its maximum response capability. The index of agonist potency, the EC50, is derived from the incremental changes in tension produced by the multiple agonist concentrations. Recall that the EC50 is defined as the concentration of agonist necessary to generate 50% of the ultimate maximum response to the highest effective concentration. A quick scan of the data depicted in Figure 8A would indicate that the EC50 in this experiment should be somewhere around 30 × 10-8 M (3.0 × 10-7 M). In order to obtain a more accurate estimate of the EC50, the results are represented graphically (Figure 8B). The logarithmic value of the agonist’s concentration (molar) is plotted on the X axis and the response to each agonist concentration on the Y axis. The data then yield the sigmoidal log dose-response curve (or, more accurately, the log concentration-effect curve). If the concentrations are selected appropriately, they will be equally spaced on a logarithmic scale. Note that if the agonist concentration is plotted linearly instead of logarithmically on the X axis, the curve becomes compressed on the left side, obscuring the individual effects of lower concentrations (inset, Figure 8B). As in Figure 7 for in vivo data, the index of phenylephrine’s potency in this ex vivo preparation can then be derived directly from the dose-response curve shown in Figure 8B. The pD2 (the logarithmic form of the EC50) can be obtained by drawing a horizontal line from the 50% mark on the normalized Y axis to the curve, and then from the curve straight down to the corresponding concentration on the X axis.

58 | Principles of Pharmacology and Autonomics

A 2 Tb

t ( min)

T 0

4

15

30

45

PE 6 3 T(g) -37oC3 1 1

3

10

30

100

300

1000

PE (×10–8 M) O2/CO2

B

[PE] < 10–6 M 3 100 ΔT

ΔT (g)

[Linear Scale] 50

ΔT (% Max)

PD2 ≅ 6.7

0

0 B

8

7 – log [PE] (M)

6

5

Figure 8. Generation of a log-dose response curve ex vivo. A: Isolated rat aorta preparation. Rat aortic rings (1) are immersed in a tissue bath, and a basal tension (Tb) of 3 grams is imposed. Tensions are recorded by a transducer (2) connected to a recording device (3). Cumulative additions of phenylephrine (PE) to the bath (4) to yield final concentrations ranging between 10-8 and 10-5 molar, progressively increase aortic tension above basal, until a maximum is reached. B: The data generated in A can then be plotted, with the log of the PE concentration on the X axis and the change in tension above baseline on the Y axis. The index of potency for PE (PD2) can then be obtained directly from the resulting log dose-response (concentration-effect) curve. Inset: the shape of the curve if the PE concentrations were plotted linearly instead of logarithmically. Note the compression on the left side of the curve, making it difficult to resolve the EC50.

Assessing Drug Action | 59

This procedure is repeated several times on multiple curves (obtained from different aortic rings) until a statistical sample is achieved (mean pD2 value ± standard deviation or standard error). The pD2 can then be easily converted to its antilog, the EC50 (EC50 = 10-pD2). In the illustrated example, the pD2 value for phenylephrine, in enhancing the contraction of the rat aorta, is depicted to be about 6.7, which corresponds to an EC50 concentration of 2 × 10-7 molar (10-6.7 M), a representative potency for this drug on the vascular system.

Experimental assessment of competitive antagonist potency Most antagonists can be categorized as either reversible (competitive) or irreversible (non-competitive). Reversible antagonists do not form covalent or other strong bonds with their target receptors, and thus can be displaced from their binding sites by sufficient concentrations of agonists that compete for occupancy of the same receptor, usually at the same binding site. Irreversible blockers often interact covalently with specific amino acids at a site other than the agonist binding site on the receptor protein. Irreversible binding effectively takes the receptor out of action, preventing agonists from interacting appropriately with their binding sites. This renders the receptor incapable of activation by endogenous agonists such as neurotransmitters or hormones. As noted above, most therapeutically relevant drugs are antagonists instead of agonists because, in the treatment of most diseases, the goals and strategies of pharmacotherapy are to inhibit, rather than to directly activate or enhance, specific cellular processes. Further, most antagonists used in therapy are reversible or competitive, not irreversible, antagonists. Adverse effects of overaggressive therapy with competitive antagonists can usually be diminished or eliminated relatively quickly by simply reducing the dose. In contrast, overdose of irreversible antagonists would be more difficult to reverse quickly. Restoration of full receptor function in the face of irreversible blockade takes longer, because it usually requires cellular synthesis and elaboration of new receptors. For that reason, irreversible antagonists are, in most clinical settings, more dangerous and less useful than reversible ones, and are therefore used much less frequently in practice. Accordingly, the focus here will be on assessment of potency of reversible (competitive) antagonists. Recall that quantification of antagonist and agonist potencies require different approaches. Although both agonists and antagonists interact with receptors, antagonists do not subsequently generate or influence a cellular signal, because they lack intrinsic activity, which is the ability to generate or alter an intracellular signal upon binding to the receptor. Therefore, if an antagonist is added to an isolated tissue or organ, it will not produce an observable dose-response curve by itself. So the standard method of quantifying indexes of potency ex vivo for agonists (obtaining a pD2 value directly from a log doseresponse curve) cannot apply to antagonists. Instead, the potencies of antagonists are assessed according to their effectiveness in antagonizing responses to agonists. The isolated rat aorta preparation can serve again as an illustration (Figure 9). In quantifying competitive antagonist potency, the ultimate aim is to generate a graph known as a Schild plot (Arunlakshana and Schild, 1959), from which the index of competitive antagonist potency, the pA2, can be derived. The procedure is the same as that described above for obtaining the potency of the agonist phenylephrine, except that multiple dose-response curves are generated, one for phenylephrine alone, and several in the presence of increasing concentrations of prazosin, a competitive antagonist of phenylephrine on the α GPCR. It is clear from the figure that the presence of prazosin in the bath increases the concentrations of phenylephrine necessary to produce equivalent increases in tension, when compared to the curve generated by phenylephrine alone; i.e., prazosin shifts the phenylephrine log dose-response curve to the right. It is also clear that the higher the concentration of prazosin in the bath, the greater is the resulting curve shift. This effect can be attributed to incrementally increasing occupancy of α receptors by prazosin prior to exposure to phenylephrine. Note, however, that if the concentration of phenylephrine is high enough, prazosin can

60 | Principles of Pharmacology and Autonomics

be displaced by phenyleprhine from the α receptors. Receptor blockade by prazosin can then be overcome, and the maximum increase in tension produced by phenylephrine can ultimately be achieved. The logarithmic index of competitive antagonist potency, the pA2 value, is analagous to the pD2 value for agonists. Both are logarithmic expressions of molar concentrations. In order to obtain the antagonist pA2 value, data derived from multiple agonist dose-response curves, produced at increasingly higher antagonist concentrations (Figure 9A), are expressed in a Schild plot (Figure 9B). Note that the label for the Y axis of the Schild plot is log (DR – 1). The DR (dose ratio) is the ratio of two agonist EC50 values (EC50' / EC50) in the absence and presence of one concentration of the competitive antagonist. The denominator of the DR is the EC50 value obtained for phenylephrine in the absence of prazosin, and the numerator is the EC50' value for phenylephrine after the curve is shifted by a given concentration of prazosin. Then the integer 1 is subtracted from the DR value to yield (DR – 1), and finally the logarithmic value for (DR – 1) is obtained. In this example, a log (DR – 1) value is calculated for each of three antagonist concentrations to yield three points on the Schild plot. The X axis is simply the negative log value of the antagonist concentration (molar). As illustrated, three prazosin concentrations were used: 10-9.2 M, 10-8.6 M, and 10-8.2 M. These exponentially expressed concentrations correspond, respectively, to 6.3 × 10-10 M, 2.5 × 10-9 M, and 6.3 × 10-9 M. Note that the higher the prazosin concentration, the greater the curve shift and thus the higher the value of log (DR – 1) on the Schild plot. A best-fit line is then drawn through the three points. The resulting X intercept is the pA2 value, or the index of potency for the competitive antagonist. The pA2 is defined as the negative log of the molar concentration of the competitive antagonist that is necessary to double the EC50 of the competing agonist. As depicted in Figure 9B, the pA2 for prazosin against phenylephrine on the rat aorta was found to be 9.39. The corresponding antilog value is usually designated as the Ka (analogous to the EC50 value for an agonist). Since the Ka = 10-pA2, the Ka molar concentration is 10-9.39, or about 4 × 10-10 M, which is a representative potency index for prazosin in vascular α receptor systems (Asbun-Bojalil et al., 2002). We can verify the definition of pA2 by looking at the Schild plot in Figure 9. If the EC50 is doubled at the Ka concentration of prazosin, then the numerical value of the DR (ratio of the EC50 with and without the antagonist) would be 2. It follows that (DR – 1) is 1, and that the log (DR -1) = log 1, which is 0. Thus, by convention, the pA2 is defined as the concentration of the competitive antagonist at the X intercept of the Schild plot. At that point, the value of the Y axis is 0, corresponding to a DR of 2, where the agonist EC50' is double the EC50.

Implications Indexes of drug potency are more than mere numbers attached to drugs. Together with pharmacokinetic considerations (Chapter 1), they provide a guide, or basis, for predicting effective blood concentrations, and thus drug dosing, in patients. In addition, the indexes can be useful in gaining a more meaningful appreciation for just how extremely potent some drugs can be. For instance, the pA2 for prazosin of 9.39 in Figure 9 is a good predictor of its potency in hypertensive patients. Recall that the Ka equivalent is about 4 × 10-10 M, or 0.4 nanomolar. The molecular weight of prazosin is about 420 Daltons. Thus, its Ka concentration in grams per liter is 420 g/mole · 4 × 10-10 moles/liter = 1680 × 10-10 g/L, or 1.68 × 10-7 g/L. We can invert that last number to determine what volume in which 1g (less than a teaspoon full) of prazosin must be dissolved in order to achieve its Ka concentration: 1/1.68 × 10-7 = 0.6 × 107 L/g, or about 6 million liters. This impressive volume is about how much beer is consumed at the annual Oktoberfest in Munich. So that extreme dilution of prazosin in the blood would presumably be enough to begin to reduce blood pressure in hypertensive human beings. Now that is potency.

Assessing Drug Action | 61

A

100

Response (% Max)

Concentration of Prazosin in Bath (M) –8.6 10–9.2 10 0

1

50

2

10–8.2

3

0 B

8.0

6.5 6.0 7.0 7.5 - log [phenylephrine] (M)

5.5

5.0

4.5

B

2

3 1

2

log (DR-1)

1 0

pA2 = 9.39 9.5

8.5 8.0 9.0 - log [prazosin] (M)

7.5

Figure 9. Competitive antagonism of phenylephrine by prazosin. A. Log dose-response (concentration-effect) curves for phenylephrine on isolated rat aorta, as shown in Figure 8. The presence of the competitive alpha-1 receptor antagonist prazosin, at incrementally increasing concentrations, progressively shifts the phenylephrine dose-response curves to the right. B: The data obtained in A are recalculated and replotted in the form of a Schild plot. The X intercept of the Schild plot is the pA2, or the index of prazosin potency.

62 | Principles of Pharmacology and Autonomics

Affinity, intrinsic activity, and efficacy As stated previously, in the study of drug actions, a fundamental assumption is that drugs do not produce biological effects unless they bind to receptors (i.e., molecular targets of drugs in vivo):

DRUG

RECEPTOR

BIOLOGICAL EFFECT

The term “affinity” describes the ability of a drug molecule to bind with its cognate receptor (for example, the interaction of either the agonist phenylephrine or the antagonist prazosin with α GPCRs). Affinity is the relationship between a drug’s concentration in the vicinity of the receptors and the fraction of receptors occupied at that concentration. Both agonists and antagonists possess the property of affinity. Further, as discussed above, whether the binding of the drug molecule to the receptor subsequently generates or alters a cellular signal linked to that receptor determines whether the drug also possesses the property of intrinsic activity. Only agonists, by definition, possess intrinsic activity. Because agonists have intrinsic activity, they also have efficacy. The two properties are related but not identical. Intrinsic activity is qualitative and efficacy is quantitative. Specifically, efficacy is the relationship between the fraction of available receptors bound by an agonist and the resulting magnitude of the cellular response. In other words, if an agonist only needs to bind to a small fraction of the receptors to generate a maximal response, with all of the others unoccupied, then that agonist has high efficacy for that receptor system. Potency of antagonists is related only to affinity, because antagonists lack the property of intrinsic activity. Potency of agonists, by contrast, is a combination of affinity and efficacy (Figure 10). Relative efficacy values can range between maximum for full agonists and zero for full antagonists. Drugs whose efficacies fall between these extremes are termed “partial agonists.” Compared to the dose-response curves for full agonists on a system, those for partial agonists on the same system are depressed (see “Full vs. partial agonism” below).

Quantification of affinity The assessment of affinity is the measurement of the relationship between a drug’s concentration in the vicinity of its receptors and the fraction of the available receptors occupied by (bound to) the drug molecule at any instant in time. Quantification of affinity—of either agonists or antagonists—is based on principles behind Michaelis-Menten enzyme kinetics (Nelson and Cox, 2009). The equation below describes the relationship between an agonist’s free concentration—in the extracellular fluid surrounding the receptors—and the fraction of those drug molecules that are bound to receptors:

DR e =

D x Rt D + Kd

where DRe = number of drug-receptor complexes at equilibrium D = number of drug molecules Rt = total number of receptors Kd = affinity constant of the drug-receptor complex at equilibrium

Assessing Drug Action | 63

Potency Agonists (EC50) Antagonists (Ka) Affinity Receptor Occupancy

Efficacy Response Magnitude

Concentration

Receptor Occupancy

Kd

Receptor

Ligand

Binding

Signal

Response

Intrinsic Activity

Figure 10. Definitions of agonist and antagonist potencies. Affinity is defined as the fraction of receptors occupied at a defined ligand concentration. Intrinsic activity is the ability to generate a signal and response upon binding to the receptor. The magnitude of intrinsic activity, efficacy, can be quantified as the magnitude of the response at a defined fraction of receptors occupied. Both agonists and antagonists possess the property of affinity, but only agonists possess the property of intrinsic activity. The potency of antagonists is equal to their affinity. The potency of agonists is equal to their affinity and efficacy.

The derivation of the equation is based on three assumptions: Assumption 1: A drug binds reversibly to its receptor.

k1 D + R

k2

DR

where k1 and k2 are constants describing reversible rates of formation (k1) and dissociation (k2) of the drug-receptor complex. Assumption 2: Formation and dissociation of the drug-receptor complex will follow the Law of Mass Action: Rate of association: Rate of dissociation:

Vassoc. = k1 (D – DR) (Rt – DR) Vdiss. = k2 (DR)

64 | Principles of Pharmacology and Autonomics

The net rate of formation of the DR complex equals the difference of the two rates over time: dDR / dt = Vassoc. – Vdiss. or dDR / dt = k1 (D – DR) (Rt – DR) – k2 (DR) Under equilibrium conditions, the rates are equal: dDR / dt = 0. therefore: k1 (D – DRe) (Rt – DRe) = k2 (DRe) Assumption 3: DRe is a small value compared to value of D. In other words, the number of receptors bound to the drug (ligand) is small compared to the total number of free drug molecules in the vicinity of the receptors. therefore: (D – DRe) ≈ D substitute and simplify: k1 (D) (Rt – DRe) = k2 (DRe) rearrange the equation: (D x Rt) – (D x DRe) = k2 / k1 (DRe) A new term has evolved: k / k1 = Kd [the affinity constant is the ratio of the dissociation and association constants] substitute Kd: (D x Rt) – (D x DRe) = Kd (DRe) solve for DRe: D x Rt = Kd (DRe) + (D x DRe) D x Rt = DRe (Kd + D) By rearrangement, we return to the original equation:

DR e =

D x Rt D + Kd

Assessing Drug Action | 65

This equation describes the presumption that, statistically, the number of total available receptors bound to drug molecules at any instant in time is directly proportional to the product of the free concentration of the drug in the vicinity of the receptors and the total number of available receptors, and inversely proportional to the sum of the free drug concentration and the concentration representing the affinity of the receptors for the drug molecules. It is clear from this equation that the higher the free concentration of the drug (D), the more of the available receptors will be occupied (DRe). Note also: When the drug concentration (D) equals the Kd, then the denominator equals 2D and the value of DRe is D/2D × Rt, or 0.5 Rt. In other words, when the drug concentration is equal to the Kd, 50% of the receptors are occupied according to this equation. The Kd is thus the affinity constant of the drug for its receptor, equal or close to the potencies of antagonists but not usually equal to the potencies of agonists (see below). Like logarithmic and antilogarithmic forms of the indexes of potency, the Kd can be also expressed as the negative log of the concentration, or pKd: Kd = 10-pKd (i.e., pKd is the negative log of the Kd). Example: a Kd concentration of 3 × 10-6 M can be alternatively expressed as a pKd of 5.5 (10-5.5 = 3 × 10-6).

Experimental quantification of drug affinity (Kd) A variety of experimental techniques can be used to quantify Kd, or the affinity of ligands for their receptors. These techniques can determine the affinities for either agonist or antagonist drugs. All of the methods yield quantitative relationships between the ligand concentration and the fraction of receptors bound to the ligand at that concentration. An example of one of those techniques is shown in Figure 11. In that approach, a tissue sample is homogenized and the membrane fraction is isolated, usually by centrifugation. Samples containing suspensions of membrane fraction are incubated with various concentrations of nonradioactive ligand molecules containing tracer amounts of radiolabeled ligands (such as those labeled with tritium or carbon-14). The incubation duration and temperature are assumed to yield equilibrium binding of ligands to their membrane-bound receptors. It is also assumed that the amount of ligand that is bound to the membrane at equilibrium does not significantly decrease the free ligand concentration. Separation of receptor-bound from free ligand molecules (with suitable corrections for nonspecific binding) is achieved by filtration (Figure 11 A). The radioactivity on the filters is counted to determine the extent of ligand binding at each concentration. The data are used to create ligand binding curves (Figure 11 B). Receptor binding is commonly represented by Scatchard plots, which are recalculated representations of the ligand binding curve (Figure 11 C). The slope of the line in the Scatchard plot is the inverse of the affinity constant Kd. Scatchard analysis yields more accurate statistical estimates of the Kd than raw binding curves do.

The difference between potency and affinity of agonists: Spare receptors Recall that the potency of an agonist is a combination of its affinity and efficacy. Therefore, an agonist’s affinity constant (Kd) is related to, but rarely identical with, its index of potency (EC50). Recall also that the Kd is the molar concentration necessary to occupy 50% of the available receptors, while the EC50 is the

66 | Principles of Pharmacology and Autonomics

A 1

L+ *L

F

2 *L Filter

L

B

*L M ↓P Free L + *L

B

100

L

cpm

Bmax

B 3

C Scatchard Plot

%L Bound 50 (B)

–1

Kd

B/F Kd Bmax 0

Free L Concentration (F)

B

Figure 11. Affinity constants (Kd) of agonists or antagonists quantified by a receptor-binding technique. A. Solutions of varying concentrations of ligand (agonist or antagonist drug) with tracer amounts of radiolabeled ligand are incubated with membrane fractions from the tissue of interest (1). The membranes bound to the ligand (nonlabeled and labeled) are separated from the suspension by filtration (2). The filter paper is then placed in a scintillation vial and counted for radioactivity (3). B: The amount of radioactivity representing the extent of bound ligand is then plotted against the ligand concentration. C: The data in B are recalculated and re-plotted in the form of a Scatchard plot whose slope is the inverse of Kd, the affinity constant of the ligand.

concentration of agonist required to produce 50% of the maximal response. If it were necessary for an agonist to bind to 100% of the available receptors in order to generate 100% of the maximum response, then its EC50 and Kd would be the same molar concentration, as predicted by the equation describing

Assessing Drug Action | 67

the relationship of DRe, D, Rt, and Kd derived above. But for many receptors and cells, particularly in the central nervous system, the Kd of an agonist is a higher concentration than the EC50. The reason for the discrepancy is that most systems contain spare receptors (Zhu, 1993); that is, for many agonist drugs and endogenous neurotransmitters or hormones, only a fraction of the available receptors need to be occupied by agonists in order to produce maximal responses. One way to experimentally reveal the presence of spare receptors in a responding system is to compare agonist dose-response curves in the absence and in the presence of increasing concentrations of an irreversible antagonist (Figure 12). Without spare receptors, agonists need to bind to 100% of the available receptors in order to generate 100% of the maximum response. Irreversible binding of an antagonist to any of those receptors, even at very low antagonist concentrations, would diminish the agonist’s effects. This would translate as a rightward and downward shift of the agonist dose-response curve (Figure 12A). However, if the system contains spare receptors, then even if some of those receptors could be eliminated by covalently binding to the irreversible antagonist, the maximum effect of the agonist could still be achieved. At appropriately low concentrations of an irreversible antagonist in a spare-receptor system, the agonist dose-response curve could be shifted to the right but the maximum response could still be attained (Figure 12B). Higher concentrations of irreversible antagonist would be required to eliminate enough spare receptors to decrease the maximum agonist response. The concept of spare receptors is further illustrated by the data in Table 2. Recall that phenylephrine is an alpha (α) receptor agonist that produces vasoconstriction. The table compares the potency of phenylephrine to constrict isolated rat caudal arteries with its binding affinity for α adrenoceptors in the same preparation. It can be seen that the concentration of phenylephrine necessary to produce a half-maximal contractile response (EC50), by activating α1 adrenoceptors (see Figure 8), is about 30 fold less than the concentration required to half-saturate α adrenoceptors (Kd) in the same preparation (Kd, Figure 11). These findings are good evidence that the smooth muscle cells surrounding the rat caudal artery contain substantial numbers of spare α adrenoceptors. The story is different for competitive antagonists. Recall that the potency of competitive antagonists is related only to affinity. Efficacy is not a component because antagonists lack intrinsic activity. All an antagonist has to do is bind to the receptor and thus inhibit agonist binding. So the presence of spare receptors will not alter the potency of antagonists, but will affect the potency of agonists because spare receptors will affect agonist efficacy (relationship between the fraction of receptor binding and magnitude of the response). In other words, in the presence of spare receptors, the EC50 for an agonist will be a lower concentration than the Kd (Table 2), but the Ka (index of potency derived from the Schild plot) for competitive antagonists should be the same as, or very nearly identical to, the Kd derived from the Scatchard plot (Table 3).

Implications Why do spare receptors exist? Why do so many cells seem to require redundant receptors for biologically active agents? The presence of spare receptors on (or in) cells may have evolutionary, pathologic, and therapeutic implications. It was proposed early on that spare receptors might have evolved as a mechanism to preserve maximal sensitivity of cells to hormones and neurotransmitters under variable environmental conditions (Abbott and Nelstestuen, 1988). As we saw, down-regulation is one

68 | Principles of Pharmacology and Autonomics

A - No Spare Receptors

1

2 Response

3

0 Log agonist concentration B - Spare Receptors

1

Response

2

3

Log agonist concentration

Figure 12. Influence of spare receptors on responses to agonists in the absence and presence of irreversible antagonists. The figures show responses to a full agonist alone (1), and to the agonist in the presence of a lower (2) and higher (3) concentration of an irreversible antagonist. A: In the absence of spare receptors, the agonist curve is progressively depressed and shifted to the right by increasing concentrations of the irreversible antagonist. B: In the presence of spare receptors, very low blocking concentrations of the irreversible antagonist might shift the agonist curve to the right without affecting the maximum response. Depression of the curve would then require higher concentrations of the irreversible antagonist.

Assessing Drug Action | 69

Table 2. Indexes of Potency and Affinity of the Agonist Phenylephrine on Vascular Alpha Adrenoceptorsa . Logarithmic value Phenylephrine potency

Phenylephrine affinity

Antilog valueb

pD2 b

EC50

6.44

3.6 × 10-7 M

pKd

Kd

4.89

1.3 × 10-5 M

a

Data are from Abel and Minneman, 1986.

b

EC50 = 10-pD2, Kd = 10-pKd; ratio of Kd to EC50 = 36.

Method Obtained from the agonist log doseresponse curve

Obtained from the Scatchard plot

Note that the potency of phenylephrine is a lower concentration than its affinity; therefore, this is a spare receptor system.

Table 3. Indexes of Potency and Affinity of the Competitive Antagonist Prazosin on Vascular Alpha Adrenoceptorsa . Logarithmic value Prazosin potency

Prazosin affinity

Antilog valueb

pA2 b

Ka

10.1

7.9 × 10-11 M

pKd

Kd

10.0

1.0 × 10-10 M

a

Data are from Schulingkamp et al., 2005. Competing agonist: phenylephrine.

b

Ka = 10-pA2, Kd = 10-pKd; ratio of Kd to Ka = 1.3.

Method Obtained from the Schild plot

Obtained from the Scatchard plot

Note that the pA2 and pKd values are not significantly different. Thus, even though this is a spare-receptor system for agonists (Table 2), the potency and affinity of the competitive antagonist prazosin on vascular alpha receptors are identical.

mechanism that cells evoke to maintain homeostasis. If there were no spare receptors, then any stressinduced decrease in receptor density might acutely compromise cellular responsiveness and sensitivity. This might interfere with the ability of the organism to adapt to variable selection pressures such as predation or climate fluctuations. Further, recall that receptors of different classes can share common intracellular signals (Table 1). In this context, spare receptors might confer adaptive receptor-signal redundancy. This may in turn preserve cellular function in the face of variations in the relative contributions of various hormones or neurotransmitters to the regulation of cellular homeostasis. Spare receptors might also affect the progression of certain diseases. For example, without spare nicotinic receptors at

70 | Principles of Pharmacology and Autonomics

the neuromuscular junction (NMJ), the progression of the muscle weakening disease myasthenia gravis might be more rapid. Fortuitously, spare receptors throughout the organism may provide a buffer against adverse effects of wide fluctuations in blood concentrations of drugs, as might occur as a consequence of poor patient compliance. The density of spare receptors may also influence, for example, the extent and rate of the development of tolerance to specific therapeutic and recreational drugs, particularly in the central nervous system (Berger and Whistler, 2010).

Drug Efficacy and the Concept of Full, Partial, and Inverse Agonism The concept of drug efficacy, like potency, is often misunderstood. In the clinical literature and elsewhere, “efficacy” is often used to simply describe the magnitude or intensity of the maximal response to a drug at its highest effective dose or concentration when administered to patients (Figure 7). In the clinical sense, the term “efficacy” can apply to either agonists or antagonists, because both produce observable responses in patients. However, in pharmacology, “efficacy” has a more specific meaning, applying only to agonists.

Full vs. partial agonism As we have seen, “efficacy” in the stricter pharmacological sense describes the relationship between the fraction of receptors occupied by an agonist and the magnitude of the resulting cellular response. So pharmacological efficacy applies only to agonists, while clinical efficacy can apply to either agonists or antagonists. The term “pharmacological efficacy” includes the concepts of full vs. partial agonism. Different agonists can have different efficacies on the same receptor and responding system. Relative efficacies of different agonists that act on the same receptor can be compared using standard methods described above. For example, two α agonists, the full agonist phenylephrine and the partial agonist oxymetazoline, increase tension of isolated rat aorta preparations (Figure 13). However, their concentration-effect curves are somewhat different. Compared to the curve for phenylephrine, the one for oxymetazoline is displaced to the right and downward; i.e., its maximal response is lower than that produced by phenylephrine. By these criteria, on α receptors in rat aortic smooth muscle, phenylephrine would be considered to be a full agonist, while oxymetazoline is a partial agonist. Here is where “efficacy” comes in. Remember that vascular smooth muscle was shown above to contain spare α receptors (Table 2). Accordingly, phenylephrine produces its maximum response (Figure 13) at a concentration that binds to fewer than 100% of the available alpha receptors (Table 2). By contrast, oxymetazoline generates a lower maximal effect, even at concentrations that bind to a greater fraction of the same receptors. Because it must bind to more α receptors in order to generate a weaker overall response, the partial agonist oxymetazoline is considered to have lower efficacy than the full agonist phenylephrine. Differences in efficacy between agonists can be explained, at least in part, by differences in the efficiency of coupling between receptor binding and the generation of the intracellular signal. In pharmacotherapy, there are a variety of full and partial agonists that vary in their efficacies on many different receptor types, such as those for histamine, EPI, endorphins, or steroid hormones. Both types of agonists, full or partial, can have therapeutic utility. For example, EPI, a full agonist on both α1 and β2 GPCRs, is the drug of choice for treating anaphylactic shock (extremely low blood pressure associated with acute hypersensitivity reactions). Clonidine, a partial agonist on α2 receptors on neurons in the brain, can be used to treat essential hypertension (see Chapter 3).

Assessing Drug Action | 71

100

- Phenylephrine - Oxymetazoline

Response (% PE 50 max)

0 9

8 7 6 5 -log agonist concentration (M)

5

Figure 13. Comparison of dose-response curves for the full agonist phenylephrine and the partial agonist oxymetazoline on rat aorta. Adapted from Ruffolo and Waddell , 1982; Chang and Stevens, 1992.

Inverse agonism and constitutive activity Until fairly recently, cellular signals were viewed as being created de novo by the interaction of agonists with their receptors. The agonist binds, signals are generated where none existed before, cellular function is altered, and responses are observed. However, it is now established that substantial signal activity can be present even in the complete absence of agonists. The basal cellular signal is called “constitutive activity.” Think of constitutive activity as an idling engine, and agonist-induced increases in signal activity as the foot on the accelerator. The newer view is that many agonists, rather than create signals where little or none had existed before, actually alter the magnitude of signals that are already there. Consititutive activity is now considered to be a component of basal cellular function. It can be conceptualized as just one set of the vast and complex variety of biochemical reactions that sustain cellular function. Agonists do not create signals, they modify them. The basal rates of many of these signal pathway reactions can be altered by the interactions of neurotransmitters, hormones, or agonist drugs on certain receptors, affecting cellular function in specific ways. Interestingly, another recent finding is that not all agonists intensify signal activity (Figure 14). Some agonists, when they bind to their receptors, actually decrease constitutive activity, actively suppressing the signal (Cotecchia, 2007; Dickey et al., 2010). Their effects on the cell and tissue would thus be opposite to those of full or partial agonists that may act on the same receptor. Accordingly, they are called inverse agonists. Extending the above car analogy, inverse agonists would turn down the engine idling speed (maybe by adjusting the fuel injector). For example, either α antagonists or reverse α agonists can relax vascular smooth muscle by different mechanisms. An α receptor antagonist, that lacks intrinsic activity, can relax the muscle by binding to

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+

Full agonist (Maximum efficacy)

Direction and magnitude of signal

Increase (Stimulatory)

Partial agonist (Intermediate efficacy) Antagonist (Zero efficacy)

(Constitutive signal activity)

Observable response

0

–

Inverse agonist (Negative efficacy)

Decrease (Inhibitory)

0 0 Drug Concentration

Figure 14. Conceptual dose-response curves for full, partial, and inverse agonists.

the receptor and inhibiting the constricting effect of an α receptor agonist such as NE. An inverse α agonist, with negative efficacy, can relax the muscle by suppressing a constitutive signal, associated with the α receptor, that continually contributes to the maintenance of a basal contractile state (tone). In one case, the neutral antagonist drug is working against an endogenous agonist; in the other, an inverse agonist is working against a constitutive signal. But in both cases, the observable response is the same: an inhibition of the signal-related cellular activity. So full and partial agonists have positive efficacy (intrinsic activity), neutral antagonists have zero efficacy, and inverse agonists have negative efficacy (Figure 14).

Implications Constitutive signal activity and inverse agonism have been linked mainly with GPCRs—including α, β, and M receptors that respond to neurotransmitters, and much less with other receptor classes such as those for growth factors or insulin (Bridge, 2010). The list of inverse agonists is growing, and includes endogenous hormones or neurotransmitters as well as exogenous agents. The properties of negative efficacy and intrinsic activity is being increasingly considered as a potentially beneficial characteristic of therapeutic agents. For example, inverse β2 receptor agonists have been proposed to be of unique therapeutic advantage in the treatment of bronchial asthma (Dickey et al., 2010). The binding of carazolol, an inverse agonist, to its target (the beta-2 receptor) is shown in Figure 1B.

Assessing Drug Action | 73

Synthesis of Chapter 1 and Chapter 2: The Bioavailability of Prazosin An important concept discussed in Chapter 1 was bioavailability, or the amount of a drug’s dose that ends up dissolved freely in the blood. For a drug to be therapeutically effective, this concentration should be sufficient to activate or block its target receptors; i.e., should be within the therapeutic window. Pharmacokinetic data can be useful to predict effective doses of specific drugs. We can select prazosin as an example. Prazosin is a very potent competitive antagonist on alpha adrenoreceptors. It relaxes vascular smooth muscle, and thus is used therapeutically in the treatment of hypertension. Its mechanism of action is to antagonize the vasoconstricting effects of the α agonist NE, a neurotransmitter in the autonomic nervous system (Chapter 3). A good rule of thumb is that, in order to be effective, the free concentration of a competitive antagonist drug in plasma should be, on average, at least 10 times its Ka (antilog of the pA2). Most drugs have a wide enough margin of safety so that this concentration should be within the therapeutic window but below the toxic range. Recall that the Ka (or logarithmically, its pA2) is the molar concentration of a competitive antagonist required to double the EC50 of the competing agonist. The Ka concentration is a useful index of potency, but it is not sufficient to be therapeutically active. Merely doubling an agonist’s EC50 would barely shift the agonist's dose-response curve, and would not produce much of a response in vivo. If, however, the concentration of the competitive antagonist were 10× greater than its Ka concentration, then the agonist dose-response curve would be shifted nearly one log unit (nearly 10 fold), a distance that should represent significant inhibition of the agonist’s actions in vivo. In this case, if prazosin were administered to hypertensive patients at a dose that would achieve at least 10× its Ka in plasma, then it would be expected to produce substantial inhibition of norepinephrine-induced vasoconstriction, and consequently produce measurable reductions in blood pressure.

Question: Are representative clinical doses of prazosin sufficient to achieve free concentrations of at least 10× its Ka in plasma?

Answer: To answer this question, the following information—from Appendix II in Brunton LL et al., (eds.) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 11th edition—is required: Representative dose (D): 5 mg (0.005g) Plasma protein binding (Bp): 95% (0.95) Volume of distribution (Vd): 0.60 L/Kg Molecular weight of prazosin (MW): 383 g/mole Body weight (BW): 70 Kg. pA2: 9.39 (From Figure 9B and Asbun-Bojalil et al., 2002.) We can use the above information to yield an estimate of the peak free concentration of prazosin in plasma: Amount of drug in the body (A) = D/BW = 0.005 g / 70 Kg = 7.14 × 10-5 g/Kg.

74 | Principles of Pharmacology and Autonomics

Total blood concentration (Ct) = A/Vd = 7.14 × 10-5 g/Kg ÷ 0.6 L/Kg = 1.19 × 10-4 g/L. Free blood concentration (Cf) in g/L = (1 – Bp) × Ct = 0.05 × 1.19 × 10-4 g/L = 5.95 × 10-6 g/L Free blood concentration (Cfm) in moles/L = Cf / MW = 5.95 × 10-6 g/L ÷ 383 g/mole = 1.16 × 10-8 M

A pA2 value of 9.39 represents a Ka concentration of 10-9.39, or 4.07 × 10-10 M. So, the ratio of the calculated free prazosin concentration in plasma—at a 5 mg dose—to its Ka concentration is: Cfm/Ka = 1.16 × 10-8 M / 4.07 × 10-10 M = 116/4.07 = 28.5. Therefore, pharmacokinetic data predicts that a prazosin dose of only 5 mg is sufficient to yield a free plasma concentration of nearly 30× its Ka, which, as clinical experience demonstrates, is more than sufficient to lower blood pressure in hypertensive patients.

Assessing Drug Action | 75

Chapter 2: Bottom Lines • The vast majority of drugs cannot act unless they bind to receptors on or in cells. • There are five major categories of receptors: G protein-coupled receptors (GPCR), ion transporters, growth factor receptors (also known as “receptors as enzymes”), intracellular receptors, and enzymes as receptors. • The most prevalent and ubiquitous receptors are GPCR. • All drugs are either agonists or antagonists. • Both agonists and antagonists have affinity for receptors. • Only agonists possess the property of intrinsic activity; i.e., only agonists activate receptors and subsequently alter the rates of receptor-coupled intracellular signal reactions. • Agonists can be subdivided into three categories: full agonists, partial agonists, and inverse agonists. • Upon binding to receptors, full or partial agonists intensify constitutive signal activity, but inverse agonists suppress it. • Inverse agonists produce effects in cells that are opposite to those of full or partial agonists. • Efficacy of agonists is described as the relationship between the fraction of receptors bound and the magnitude of the subsequent response. • Full agonists display greater efficacy than partial agonists do, because full agonists can generate responses by binding to fewer receptors. • Antagonists can be subdivided into competitive (reversible) and irreversible categories, depending on the strength of the chemical bonds between the drug molecule and its receptor protein. • Receptor blockade by competitive antagonists can be overcome by sufficient concentrations of agonists, but blockade by irreversible antagonists cannot. • Potencies of agonists are molar concentrations (EC50 or pD2) that are sufficient to produce 50% of the maximum response in target cells. • Agonist potencies can be obtained directly from the log dose-response curve. • Potencies of competitive antagonists are molar concentrations (Ka or pA2) that are sufficient to double the EC50 of the competing agonist. • Competitive antagonist potencies can be quantified as X intercepts of Schild plots. • Both agonists and antagonists have affinity for their receptors, but only agonists have intrinsic activity and efficacy. • Affinity (Kd) can be quantified from Scatchard plots derived from receptor binding curves. • Potencies of agonists are related to their affinities and efficacies, but potencies of competitive antagonists are related only to their affinities for their receptors. • Both agonists and antagonists have effects in whole organisms, including patients. • Agonist drugs generate responses in patients by imitating the actions of endogenous agonists such as neurotransmitters or hormones. • Antagonists generate responses in patients because they interrupt continual receptor activation by endogenous agonists. • The majority of therapeutic drugs are competitive antagonists.

76 | Principles of Pharmacology and Autonomics

Glossary Adenylate cyclase: A signal enzyme associated with GPCRs that converts ATP to cyclic adenosine monophosphate (cAMP), a downstream signal molecule. Affinity: Relationship between the free concentration of a ligand in the vicinity of its receptors and the fraction of the total number of accessible receptors that are bound to the ligand. Both agonists and antagonists have affinity for their receptors. Agonist: A ligand that activates a receptor upon binding to it. Agonists possess the property of intrinsic activity. Agonist potency: At the cellular level, the relationship between the concentration of an agonist in the vicinity of its receptors and the magnitude of the ensuing response of the cell, tissue, or organ; at the whole-animal or patient level, the relationship between the dose of an agonist drug and the magnitude of the observable response. Antagonist: A ligand that binds to a receptor but does not activate it. Antagonists lack intrinsic activity. Antagonist potency: At the cellular level, the relationship between the concentration of an antagonist in the vicinity of its receptors and the magnitude of inhibition of agonist effects on those receptors; at the whole-animal or patient level, the relationship between the dose of an antagonist drug and the magnitude of the observable response. Constitutive signal: Basal signal activity in a cell that is present even in the absence of receptor activation by an agonist. Drug: Any non-nutrient chemical substance that, in sufficient quantities, alters the activities of biological systems. Drug concentration: Amount of a drug dissolved in blood or other solutions in patients or whole animals, or in perfusing or bathing solutions in isolated cells or organs (expressed in units such as moles per liter [M] or micrograms per deciliter [μg/dL]). Drug dose: Amount of drug administered to a patient or whole animal (expressed in units such as milligrams, milligrams per Kg body weight or per square meters of surface area, etc.). EC50: Index of agonist potency ex vivo. It is the antilog of the pD2. ED50: Index of potency in vivo (in whole animals or patients) of an agonist or antagonist drug. Ex vivo: Outside the body or isolated from the body; usually isolated cells, tissues, or organs. Effect (response): Specific and measurable (observable) change in activity or function of a cell, tissue, organ, or whole animal produced by a drug (agonist or antagonist), hormone, or neurotransmitter. Efficacy: Relationship between the fraction of the total number of receptors bound to an agonist and the magnitude of the change in cellular signal or observable response to the agonist. Effector: As used in this text, a protein or other macromolecule that is an ultimate target of a signal pathway (e.g., an ATPase on an intracellular membrane whose activity is altered by the ultimate signal component). Endogenous: Originating from, and acting, inside the body (e.g., hormones or neurotransmitters). Exogenous: Produced, or administered from, outside the body. All drugs, even hormones or neurotransmitters used as drugs, are exogenous. Full agonist: An agonist whose intrinsic activity is sufficient to produce maximum achievable responses in cells, organs, or whole animals or patients. GPCR: G protein-coupled receptors; A broad category of receptors that are associated with regulatory protein complexes called G proteins because they bind to guanosine triphosphate (GTP) and guanosine diphosphate (GDP). Graded response: A response to a drug that is, more or less, on a continuum; e.g., changes in urine production or blood pressure. See “quantal response.” Inhibitory effect: A decrease in the activities of cells, tissues, or organs in response to binding of ligands to their receptors; also called inhibitory responses.

Assessing Drug Action | 77

Inhibitory receptor: A receptor whose activation decreases the activities of the cell, tissue, or organ. Intrinsic activity: Property of agonist drugs; ability to affect (alter or influence) signal activity in target cells; related to, but not identical with, efficacy. Inverse agonist: An agonist that produces cellular responses by decreasing constitutive signal activity. In vivo: Inside the intact living organism; pertaining to the whole animal or patient. Ka: Index of antagonist potency ex vivo, i.e., at the cellular, tissue, or organ level; the average free concentration (in moles per liter) of a competitive antagonist sufficient to double the EC50 of an agonist acting on the same receptor. It is the antilog of the pA2. Ligand: Collective term for any drug, neurotransmitter, or hormone that binds with defined affinity to specific receptors. pA2: Logarithmic form of the index of competitive antagonist potency; the molar concentration of competitive antagonist required to double the EC50 of the agonist. It is the negative log (base 10) of the Ka. Partial agonist: An agonist whose efficacy is not sufficient to produce maximum achievable responses in cells, organs, or tissues. Pharmacology: The study of drugs, their actions, and their mechanisms of action. Pharmacokinetics: The study of the processing of drugs or toxins by the body, including absorption, distribution, metabolism, and excretion of drugs. pD2: Logarithmic form of the index of agonist potency; the molar concentration of agonist required to produce 50% of the maximal response of a cell, tissue, or organ to antagonist. It is the negative log (base 10) of the EC50. PKC: Protein kinase C, a signal enzyme protein that is activated by diacylglycerol. PLC: Phospholipase C, an enzyme associated with receptors and that catalyzes the conversion of membrane lipids into downstream signal molecules. Pharmacodynamics: The study of interactions between drugs—either agonists or antagonists—and their receptors. Potency: At the cellular level, relationship between the concentration of a drug (agonist or antagonist) accessible to cellular receptors and the magnitude of the response of the cell, tissue, or organ to that drug; In the whole animal or patient (in vivo), relationship between the dose of a drug and the magnitude of the response. Quantal response: A response to a drug that is either present or absent, e.g., induction of sleep. See “graded response.” Receptor: A molecule—most commonly a protein on or in a cell—that binds with defined affinity to a ligand and mediates its biological effects. Signal: A series of enzymatic reactions or molecular interactions that are functionally linked to receptors and that mediate and account for the cellular response to agonists on those receptors. Stimulatory effect: An increase in the activities of cells, tissues, or organs in response to binding of ligands to their receptors. Also called a stimulatory response. Stimulatory receptor: A receptor whose activation ultimately results in an acceleration or intensification of the activities of the cell, tissue, or organ. Toxicology: The study of toxins, their actions, and their mechanisms of action. Toxin: Any chemical substance that, in sufficient quantities, exerts harmful effects on biological systems.

78 | Principles of Pharmacology and Autonomics

Chapter 3

Drugs That Interact With the Autonomic Nervous System

Introduction The autonomic nervous system (ANS) controls a variety of physiological processes, from blood pressure to gastrointestinal (g.i.) function to glandular secretion. It is emphasized here because many drugs interact with the ANS to produce either therapeutic or adverse effects. In addition, the ANS serves as a useful transitional subject, situated between the fundamentals of drug action on the one hand (Chapters 1 and 2) and the study of specialized categories of therapeutic drugs—cardiovascular, antidepressant, gastrointestinal, and so forth—on the other (beyond the scope of this text). A good basic understanding of the ANS—what it does and how it works—goes a long way toward understanding how a broad spectrum of drugs acts, particularly on the cardiovascular and central nervous systems. The ANS is an important target system for therapeutic actions of many drugs. Prazosin, highlighted in previous chapters, would serve as an excellent example here as well. The drug lowers blood pressure of people with hypertension primarily, if not exclusively, by interfering with specific actions of the ANS. In addition, many drugs exert adverse effects by directly or indirectly influencing ANS activity. For instance, tachycardia (fast heart rate) is an important adverse effect common to a variety of drugs with diverse mechanisms of action. Tachycardia at rest is increasingly recognized as a drug effect that should be avoided or minimized, because it increases the risk of adverse coronary events (oxygen supply–demand imbalances in the heart). Understanding of the workings of the ANS explains the tachycardic effects of such disparate drugs as prazosin (antihypertensive), scopolamine (anti-nausea), terbutaline (anti-asthma), and nitroglycerin (coronary vasodilator). In summary, a firm grasp of how drugs interact with the ANS often allows educated prediction, rather than mere memorization, of their therapeutic and adverse effects. It will become clear early on that reading the following discussion of the ANS is like learning a new language. It is replete with terminology that is not familiar to most people. In order to facilitate the process, a glossary is provided at the end of the chapter. As in previous chapters, glossary terms are highlighted in bold type as they first appear in the text. Initially, at least, it may be useful to flip back and forth between the text and the glossary in order to keep up with the terminology. With time, though, this process should become less necessary. In an effort to facilitate the process of learning the ANS and drugs that interact with it, all figures in this chapter are color-coded. A summary chart is presented at the end of the chapter.

Functional Anatomy of the ANS Using either anatomical or functional criteria, the ANS can be divided into two main branches (Figure 1): 1) The sympathetic branch or system (SNS); and 2) The parasympathetic (around the sympathetic) branch or system (PNS). Anatomically, sympathetic efferent neurons leave the CNS from the two middle regions of the spinal cord: the thoracic (chest) region and the lumbar (loins) region. Parasympathetic

Drugs That Interact With the Autonomic Nervous System | 81

SMNS

-CNS-

-CNSNMJ ACh

PSG

NEJ

PSN

CRA

ACh

ACh SG SN

ACh

THOR/ LUMB

SN SN SG ACh

NE AM

ACh

EPI

NE

Renin

DA

Effector Organs ACh ACh

PSG

NE Art

SG ACh

SN

SG ACh

SCN

Sweat

ACh SWG PSN

SAC

Figure 1. Major components of the autonomic nervous system. Abbreviations: CNS, central nervous system; CRA, cranial region of the spinal cord and base of the brain; THOR/LUM, thoracolumbar region of the spinal cord; SAC, sacral region of the spinal cord; PSN, parasympathetic nerve; SCN, sympathetic cholinergic nerve; PSG, parasympathetic ganglion; SN, sympathetic nerve; AM, adrenal medulla; ART, arteriole; ACh, acetylcholine; EPI, epinephrine; NE, norepinephrine; DA, dopamine; SWG, sweat gland; NEJ, neuroeffector junction; NMJ, neuromuscular junction. Inset: motor nerve of the somatomotor nervous system (not part of the ANS) innervating skeletal muscles. efferent neurons leave the CNS from the cranial (head) and sacral (sacred) regions of the brain and spinal cord. The efferent neurons of each branch, whether sympathetic or parasympathetic, are interrupted by ganglia. Autonomic ganglia are collections of neuronal synapses, which are connections between neurons. Groups of neurons that terminate at ganglia (i.e., carry efferent impulses from the spinal cord or brain to the ganglion) are called preganglionic (presynaptic) neurons, while groups of neurons that

82 | Principles of Pharmacology and Autonomics

Table 1. Cholinergic and adrenergic components of the ANS. Component Cholinergic

Neurotransmitter or Neurohormone Acetylcholine (ACh)

Cells of Origin

Preganglionic neurons of the sympathetic and parasympathetic nervous systems Postganglionic neurons of the parasympathetic nervous system Postganglionic neurons of the sympathetic nervous system innervating the sweat glands Motor neurons innervating skeletal muscle (somatomotor nervous system, not the ANS)

Adrenergic

Norepinephrine (NE)

Most postganglionic neurons of the sympathetic nervous system*

Epinephrine (EPI)

Chromaffin cells of the adrenal medulla

* Some sympathetic postganglionic neurons innervating the kidneys are dopaminergic (release dopamine as the neurotransmitter; see Figure 1).

leave ganglia and carry efferent impulses to their effector cells at the neuroeffector junction (NEJ) are called postganglionic (prejunctional) neurons. Functionally, the two branches of the ANS can also be distinguished by the neurotransmitter that is released by the postganglionic neuron at the NEJ (Figure 2). As shown in Figure 1, the neurotransmitter of the sympathetic postganglionic neuron is almost always noradrenaline, also known as norepinephrine (NE), while that of the parasympathetic postganglionic neuron is acetylcholine (ACh). Thus, common adjectives describing the two branches are “adrenergic” for the sympathetic branch, and “cholinergic” for the parasympathetic branch. The preganglionic neurons of both branches—sympathetic and parasympathetic—release ACh, and are thus described as cholinergic. So overall, the ANS is predominantly a cholinergic system, except that the postganglionic portion of the SNS is, with some exceptions, adrenergic (Figure 1 and Table 1). Note from Figure 1 similarities and differences between the two branches of the ANS, the PNS and SNS, with regard to the mechanisms of their control of effector organs. The post-synaptic (postganglionic) neuron of each branch delivers its neurotransmitters directly to distinct points or zones on target tissues or organs. This allows local control in restricted areas. For example, each branch of the ANS sends neurons to the sinoatrial node (pacemaker) of the heart, exerting site-directed and opposing control of heart rate. However, regulation by the sympathetic branch of the ANS is supplemented by an endocrine component. The neurohormone EPI is delivered diffusely, via the bloodstream, to multiple effector organs at the same time. At the effector organ, EPI exerts a broader influence over a wider area, limited mainly by the density, location, and arrangement of tissue capillaries. The site-directed influence of the sympathetic neurons on heart rate, mediated by the neurotransmitter NE, is thus supplemented by the more diffuse delivery of circulating EPI to the same site and other sites. Both the transmitter (NE) and the neurohormone (EPI) increase heart rate by acting on the same receptor. Not surprisingly, general systemic processes—such as hepatic glucose metabolism or systemic blood pressure—are

Drugs That Interact With the Autonomic Nervous System | 83

Acetylcholine H 3C + N

O H3C

Norepinephrine

O

CH3

Parasympathetic neuroeffector junctions, autonomic ganglia

CH3

HO

H

HO N

HO

H

Sympathetic neuroeffector junctions

Dopamine

Epinephrine H

HO

HO

N HO Sympathetic neuroeffector junctions in kidney

H

HO N

H

HO

CH3

Bloodstream from adrenal medulla

Figure 2. Chemical structures and major locations of ANS neurotransmitters and the neurohormone epinephrine. The “nor” in norepinephrine stands for the German “N ohne Radikal,” meaning the methyl group on the nitrogen in epinephrine is absent in norepinephrine.

regulated much more extensively by the SNS than they are by the PNS. Note also in Figure 1 that a separate but similar nervous system is depicted in the inset. Neural control of skeletal muscle activity is accomplished by the somatomotor nervous system (SMNS) rather than the ANS. Unlike neurons of the ANS, those of the SMNS are not interrupted by ganglia. The distinct neuroeffector junction (NEJ) of the SMNS is called the neuromuscular junction (NMJ). The neurotransmitter at the NMJ is ACh, like that of the PNS at its NEJ. However, the receptors activated by ACh at the two junctions are different (Table 2). The general rule is that postganglionic fibers of the PNS and SNS release ACh and NE, respectively, at their effector cells (Table 1). However, there are exceptions to this rule. Some specialized sympathetic postganglionic neurons release ACh instead of NE; those neurons innervate the sweat glands, and are termed the “sympathetic cholinergic” system (Table 1 and Figure 1). Other sympathetic neurons to the kidneys release dopamine (DA) instead of NE. Their main function is to influence renal blood flow (Figure 1). Still other neurons and neurotransmitters are part of a recently recognized group called the nonadrenergic, noncholinergic (NANC) system (Witticombe, 1998; Said and Rattan, 2004; Brain and Cox, 2006; Jobling, 2010). Whether all NANC neurons are part of the ANS has not been definitively established. In the SNS, NANC neurotransmitters include ATP and neuropeptide Y. They are released with NE by sympathetic postganglionic neurons, and are thought to contribute to the overall effects of sympathetic innervation on effector cells such as vascular smooth muscle and exocrine glands. Other NANC transmitters include vasoactive intestinal peptide (VIP), nitric oxide (NO), and carbon monoxide (CO). All three are involved in relaxing smooth muscle surrounding such structures as airways, intestine, genitourinary tract, and blood vessels. NO is an important regulator of penile erection through the activation of guanylate cyclase in the corpus cavernosum and the production of cyclic guanosine monophosphate (cGMP). Cyclic GMP-dependent phosphodiesterase type 5 is a target of male performance-enhancing drugs such as sildenafil (Viagra®).

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Table 2. Autonomic receptors and their signals at the NEJ. Receptor Type*

Agonist

Predominant G protein

Representative Signals

Parasympathetic muscarinic (M1, M3)

ACh

Gαq

PLC/DAG/IP3

muscarinic (M2, M4)

ACh

Gαi

adenylate cyclase/cAMP

nicotinic (Nn, Nm)

ACh

NA

NA (ion channels, not GPCR)

alpha (α-1)

NE, EPI

Gαq

PLC/DAG/IP3

alpha (α-2)

NE

Gαi (βγ subunits)

adenylate cyclase/cAMP K+ channels

beta (β-1)

NE, EPI

Gαs

adenylate cyclase/cAMP

beta (β-2)

EPI

Gαs

adenylate cyclase/cAMP

Sympathetic

* Indicated agonists are known to activate the corresponding receptors in vivo. The affinities of ACh for all listed M receptor isoforms are roughly equal. NE has a greater affinity for α2 receptors than EPI does, but EPI has a greater affinity for β2 receptors than NE does. Their relative affinities for α1 and β1 receptors are about the same (see Table 2 and Figure 4B).

Abbreviations: PLC, phospholipase C; DAG, diacylglycerol; IP3, inositol triphosphate; cAMP, cyclic adenosine monophosphate; NA, not applicable. Other receptor isoforms are not listed because they are not prominent drug targets.

How the ANS Controls Physiological Processes The ANS regulates involuntary physiologic processes such as blood pressure, gastrointestinal motility and secretions, airway resistance, glandular secretions, and so forth. It does this by varying the rate at which its neurons release their neurotransmitters, and consequently activate receptors on the surface of effector cells of tissues or organs that are innervated by the ANS. All of this is controlled by the central nervous system (CNS), from areas inside both the brain and the spinal cord. These areas may receive input from afferent sensory nerves (such as touch, pain, temperature, olfactory, or visual) or afferent reflex neurons (e.g., reflexes that respond to changes in blood pressure, muscle stretch, and so forth). There are also independent rhythmic control centers within the CNS such as those that determine diurnal rhythms, hormonal cycles, sleep–wake cycles, and other automatic processes. There is some limited conscious control of the ANS as well. We can exert some influence over our own heart rates, for example, through relaxation and concentration. In response to sensory, reflex, and to a minor extent conscious input, the CNS in turn regulates the frequency of action potential generation by the efferent (preganglionic) neurons leaving the brain or spinal cord, terminating at ganglia. A secondary routing of impulses to different destinations occurs in ganglia. The frequency of impulse generation in

Drugs That Interact With the Autonomic Nervous System | 85

Table 3. Locations of autonomic receptors. Neurotransmitter or Neurohormone ACh

Receptor Nicotinic (Nn)

Locations Postsynaptic neurons in sympathetic and parasympathetic ganglia* Chromaffin cells of the adrenal medulla

Nicotinic (Nm)

Skeletal muscle cells**

Muscarinic (M)

Cells of effectors innervated by cholinergic neurons at neuroeffector junctions (NEJ) Endothelial cells of blood vessels (sparsely innervated)

NE

EPI

Alpha (α) 1 or 2

Cells of effectors innervated by adrenergic neurons at neuroeffector junctions (NEJ); terminal regions of sympathetic postganglionic neurons (α-2)***

Beta (β) 1

Cells of effectors innervated by adrenergic neurons at neuroeffector junctions (NEJ)

Alpha (α) 1

Many effector cells

Beta (β) 1 or 2

Many effector cells

*In some ganglia, M receptors contribute to neurotransmission. **Somatomotor nervous system, not the ANS (see Appendix). ***Presynaptic (more correctly, prejunctional) α2 receptors mediate feedback inhibition of NE release by sympathetic postganglionic neurons. Neuronal β (SNS) and M receptors (PNS) can also mediate feedback regulation of transmitter release at various sites.

the postganglionic fibers then determines the amounts of neurotransmitter that are released at the effector cells. As action potential frequencies go up, more neurotransmitter is released, and consequently a greater fraction of receptors on the recipient cells is bound to, and activated by, the neurotransmitter. The opposite occurs as action potential frequencies decline. The amount of neurotransmitter that is accessible to the receptors at the synapse or NEJ at any given time is a balance between the rate of release from the incoming neurons and the rate of inactivation by either reuptake into the neuron (adrenergic) or enzymatic destruction (adrenergic and cholinergic).

Cholinergic innervation and control As an illustration, we can focus on the PNS, because the same neurotransmitter, ACh, is released at the ganglia and at the NEJ. Eight major steps in the process can be summarized as follows (Figures 1 and 3): 1) Neurons originating in specific regions of the brain and spinal cord communicate via synaptic transmission with other neurons that ultimately emerge from the spinal cord, becoming preganglionic neurons that terminate in ganglia; 2) In the ganglia, axons of multiple presynaptic cholinergic neurons

86 | Principles of Pharmacology and Autonomics

form synapses with dendrites and cell bodies of postsynaptic neurons; 3) Variations in action potential frequency in the preganglionic cholinergic neurons—controlled in the CNS—determine the rate of release of ACh by these neurons in the ganglionic synaptic clefts; 4) ACh in ganglionic synapses activates receptors—predominantly nicotinic cholinergic receptors—on the surface of postsynaptic (postganglionic) neurons; 5) Receptor activation in ganglia determines the frequency of action potential generation (increase or decrease) in the postsynaptic neurons; 6) The frequency of postsynaptic neuronal action potential generation determines the extent of release of its transmitter at the neuroeffector junction (NEJ); 7) ACh released at the NEJ activates muscarinic (M) receptors on the effector cell surface; and 8) Activation of target-cell M receptors and their associated signals alters the activity of the effector cells and organs, ultimately regulating their function. Recall from Figure 1 and Tables 1 and 2 that somatomotor nerves are also cholinergic but are not interrupted by ganglia. In that case, control of ACh release at the NMJ originates entirely in the brain and spinal cord, with no intervening ganglionic regulation, and the receptors at those targets would be nicotinic, not muscarinic (see Appendix). A good grasp of the principles of cholinergic innervation can be obtained by comparing Figures 1 and 3. Figure 1 includes macroscopic arrangements of cholinergic nerves in the ANS and SMNS, showing their origins in the CNS and their terminals at ganglia or NEJs. Figure 3 focuses more closely on the cholinergic synapses (ganglia) or NEJs (effector cells) depicted in Figure 1. In all cholinergic neurons, ACh is synthesized from acetyl CoA and choline by choline acetyltransferase (Figure 3A). The acetyl component is derived from cytosolic acetyl CoA, which in turn comes mainly from mitochondrial metabolism of pyruvate or fatty acids. The choline is largely recycled by reuptake from the synaptic or junctional cleft via a specific reuptake transporter (CRT). ACh is packaged into vacuoles in the cytoplasm for ultimate exocytotic release into the synaptic or junctional cleft. The rate of ACh release is proportional to the rate of action potential generation in the releasing neuron. Action potentials promote the influx of calcium ions into the neuron through voltage-sensitive calcium channels in the membrane. The processes of ACh packaging and release are calcium-dependent. So the greater the frequency of action potential generation, the greater the levels of intracellular calcium levels, and the more ACh is released into the synapse or neuroeffector junction. Levels of intracellular calcium are a balance primarily between action potential-induced influx on the one hand, and removal (efflux) by membrane ABC transporters on the other (see Chapter 1). Once the ACh is released, it either binds to and activates receptors on postsynaptic neurons or postjunctional cells, or is destroyed by acetylcholinesterase (AChE)-mediated hydrolysis of ACh to choline and acetate. The amount of ACh in the cleft at any moment is thus a balance between the rate of neuronal ACh release and extracellular enzymatic destruction (in this case, AChE). It follows that ACh levels in the synaptic or junctional gap, and thus the extent of receptor activation, can be increased in at least two ways: 1) increasing the rate of transmitter release by increasing the activity of the releasing neuron; or 2) decreasing the rate of transmitter destruction by inhibiting the AChE enzyme. The latter is the mechanism of action of indirect-acting cholinergic stimulating drugs (see below). Figure 3B depicts the cholinergic receptor at autonomic neuroeffector junctions. These might include the point of contact between parasympathetic postganglionic neurons and their effector cells (secretory glands, bronchial smooth muscle, etc.). Or it might be the junction between sympathetic cholinergic neurons and sweat glands (again, refer to Figure 1 for the bigger picture). Receptors at those sites are M (muscarinic) GPCRs. In the ANS, M receptors are usually associated with either the PLC-IP3-DAG signal or membrane potassium channels, depending on the isoforms of the receptor and their associated G proteins (see Chapter 2). The response to M receptor activation depends on the type of cell that is being innervated by the cholinergic neuron. For example, more extensive M receptor activation by ACh in bronchiolar smooth muscle would increase airway constriction, while activation of similar receptors

Drugs That Interact With the Autonomic Nervous System | 87

A. Ganglionic synapses or cholinergic neuroeffector junctions (NEJs) Ca2+ (+)

CAT AcCoA + Chol.

ACh

ACh

Na+ Chol. + Acetate

R (in A. above) is a muscarinic receptor on postjunctional cells at the NEJ ACh +

R

K+ M

Gα β γ

1

Δ Em 2 Response (e.g. decrease in heart rate)

Signal

R (in A. above) is a nicotinic (Nn) receptor on postsynaptic neurons or adrenomedullary chromaffin cells

C.

ACh +

Na+ Na+

Δ Em Na+

Response (i.e., increase in NE, ACh, or EPI release)

88 | Principles of Pharmacology and Autonomics

R AChE

CRT

B.

(+)

⇑ Frequency of action potential generation

(–) Physostigmine

Figure 3. Cholinergic neurotransmission (previous page). A. Cholinergic synapse or neuroeffector junction (NEJ) (see Figure 1): The diagram depicts either a synapse in autonomic ganglia (sympathetic or parasympathetic) or a cholinergic NEJ. The latter can be an NEJ in the parasympathetic nervous system (PNS), the junction between a sympathetic preganglionic neuron and an adrenomedullary chromaffin cell, or a junction between a sympathetic postganglionic neuron and a sweat gland cell. Acetylcholine in the preganglionic (prejunctional) neuron is synthesized from acetyl CoA and choline recycled via choline acetyltransferase (CAT). Action potentials promote uptake of calcium ions through voltage-operated calcium channels, which in turn stimulates the release of ACh into the synapse or neuroeffector junction. ACh is broken down and inactivated by acetylcholinesterase (AChE) to choline and acetate. The choline is then taken back into the neuron through a reuptake transporter (CRT). B. In cholinergic neuroeffector junctions, the receptors on post-junctional cells are muscarinic (M) GPCRs. Depending largely on the isoforms of the M receptor and the G protein (see Chapter 2), activation of the receptor by ACh usually either: 1) activates one or more signal pathways in the cell producing a signal-mediated response; or 2) changes the conductance of a potassium channel on the membrane, hyperpolarizing the cell (ΔEm). The nature of the response depends on cell type (see Table 3). C. In autonomic ganglia (SNS or PNS), or at the neuromuscular junction (NMJ) in the somatomotor nervous system (SMNS), the predominant or exclusive receptor on the postsynaptic neuron or the skeletal muscle cell is a nicotinic (N) receptor. There are two dominant nicotinic receptor isoforms in the two tissue types: 1) Nn receptors in ganglionic neurons; and 2) Nm receptors in skeletal muscle cells. The isoforms are determined by the combination of α and β subunits making up the 5-subunit receptor. In the ANS or SMNS, N receptors are mainly sodium channels. Activation of either isoform increases its conductance to sodium ions, partially depolarizing the membrane (ΔEm). This in turn activates voltage-sensitive fast sodium channels in the membrane, increasing the frequency of action potential generation. In neurons, this leads to enhanced release of their transmitters downstream. In muscle cells, this leads to a greater twitch frequency (skeletal muscle) or sustained tonic contraction (smooth muscle).

in secretory glands would increase the rate of secretion of their products. For a more complete list of responses to cholinergic innervation at neuroeffector junctions, see Table 4. Figure 3C depicts the cholinergic receptor at autonomic ganglia (sympathetic or parasympathetic) or at the neuromuscular junction in the somatomotor system. At both sites, the receptor is nicotinic (N), not muscarinic (M). The N receptor is an ion channel, reminiscent of the GABA chloride channel (Chapter 2), consisting of five subunits (α and β). In ganglia, the predominant isoform is Nn, but at the neuromuscular junction, it is Nm. The two isoforms are distinguished by the ratio of α and β subunits. They both bind to ACh with similar affinities. However, they can also be pharmacologically characterized by their respective affinities for antagonist drugs (see below). In the ANS and somatomotor nervous systems, N receptors are predominantly sodium channels. As an aside, the brain also has N receptors that can be channels for either sodium or calcium. Activation of N receptors by ACh in ganglia or at the NMJ increases their conductances mainly for sodium ions, slowly depolarizing the cell membrane (decreasing transmembrane voltage). If the change in voltage is appropriate, in both duration and magnitude, then voltage-sensitive fast sodium channels will be activated and trigger action potentials in the recipient cells. In ganglia, activation of Nn receptors in postganglionic neurons translates into increased

Drugs That Interact With the Autonomic Nervous System | 89

frequency of firing and correspondingly increased release of their neurotransmitters downstream at their neuroeffector junctions. At the NMJ, activation of Nm receptors also increases the frequency of action potential generation in skeletal muscle cells, but in those cells the response in increased frequency of muscle twitches or, at higher frequencies, increased tonic tension of the muscle.

Adrenergic innervation and control Compared to cholinergic innervation, adrenergic innervation is relatively straightforward. It applies only to the SNS, and then only to postganglionic neurons (Table 1, Figure 4). By far the most prevalent neurotransmitter at the sympathetic NEJ is norepinephrine (NE), also called noradrenaline. However, the SNS also includes chromaffin cells in the adrenal medulla that in humans secrete predominantly epinephrine (EPI), also called adrenaline. Sympathetic neurons that go to the sweat glands release ACh (activating M receptors on the gland) instead of NE (activating alpha or beta receptors on the target cells). In addition, some neurons in the SNS—innervating the kidney for example—secrete dopamine (DA) instead of NE (see Figure 1). Cellular synthesis of DA, EPI, or NE starts with the amino acid tyrosine (Figure 4A). In the cytoplasm, tyrosine is first converted to dopa and then to dopamine by the enzymes tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC), respectively. Dopamine then enters secretory granules via carrier-mediated transport. In dopaminergic sympathetic neurons, there is no further conversion and the dopamine is secreted into the junctional cleft. In noradrenergic sympathetic neurons, DA is further converted to NE by the intragranular enzyme dopamine beta hydroxylase (DβH), and it is the NE that is secreted by the neuron. Recall that the major catecholamine secreted by the adrenal medulla is EPI, not NE. EPI is essentially methylated NE. In the chromaffin cells, one additional synthetic step takes place. NE exits the granule and is converted to EPI by the cytosolic enzyme phenyethanolamine-Nmethyltransferase (PNMT). Epinephrine then reenters the granule and is secreted into the bloodstream along with some unconverted NE. In both sympathetic neurons and adrenomedullary chromaffin cells, the secretory process is calcium-dependent. Other substances in sympathetic neuronal granules, including ATP and neuropeptide Y, are secreted with NE and exert biological effects by activating their own cognate receptors in certain effector cells. However, neither the physiological roles of these co-released substances nor the full potential of their receptors as drug targets has been clearly established. Therefore, they have been omitted from Figure 4. Once in the junctional cleft, NE can stimulate isoforms of either α or β GPCR (Figure 4 A and B). Circulating EPI can also stimulate α or β receptors. However, the two neurotransmitters have different affinities for different isoforms. Both have similar affinities for α1 and β1 receptors; i.e., both can activate these receptors on various target organs in vivo. In contrast, NE has much higher affinity than epinephrine for α2 receptors, while epinephrine has much higher affinity than NE for β2 receptors. In vivo, therefore, both EPI and NE activate α1 or β1 receptors on effector cells, but only EPI activates β2 receptors and mainly NE activates α2 receptors. Predominant G proteins and signals associated with autonomic α and β GPCRs are listed in Table 2. There are several redundant mechanisms for inactivation of NE. It can be broken down to normetanephrine in the junctional cleft by the enzyme catechol-O-methyl transferase (COMT) on the effector cell. Or, it can be taken back up into the neuron by a selective carrier protein, the NE reuptake transporter (NRT). This is the predominant mechanism of inactivation. Also, either synthesized or recycled NE can be metabolized within the neuron to 3,4,-dihydroxyphenylglycolaldehyde (DOPGAL) by the mitochondrial enzyme monoamine oxidase (MAO). Finally, NE can inhibit its own secretion (feedback inhibition) by activating prejunctional (erroneously termed “presynaptic” at the NEJ) α2 receptors on

90 | Principles of Pharmacology and Autonomics

A +

Ca2+

NME

TYR α2 TH TYR

(NE)

AADC D

DbH

DA

DA VMAT

COMT

PNMT +

NE (EPI)

+

NE

(–) [Reserpine]

Effector Cell-

(–)

(EPI)

R

DOPGAL [MAO]

+

(–) [Cocaine]

(EPI)

NRT

B NE

+

+

+

α2

+

EPI

+

α1

+

β1

β2

Gαi β γ

Gαq β γ

Gαs β γ

Gαs β γ

Signal

Signal

Signal

Signal

Response

Response

Response

Response

(e.g., decrease in NE release)

(e.g., increase (e.g., increase in smooth muscle in heart rate) contraction)

(e.g., decrease in smooth muscle contraction)

Figure 4. Adrenergic (sympathetic) neurotransmission. A. Adrenergic neuroeffector junctions. In sympathetic neurons, NE is synthesized from the amino acid tyrosine in three steps. In the cytoplasm, tyrosine is converted to dopa (D) by tyrosine hydroxylase (TH) and dopa is then converted to dopamine (DA) by aromatic L-amino acid decarboxylase (AADC). DA is then taken up into secretory granules through selective membrane transporters, where it undergoes further conversion to norepinephrine (NE) by dopamine beta hydroxylase (DβH). The last step does not take place in dopaminergic neurons. In adrenomedullary chromaffin cells, but not in sympathetic adrenergic neurons, cytosolic NE is further converted to epinephrine (EPI) by phenylethanolamine-N-methyltransferase (PNMT). NE leaves the granule and is converted to EPI in the cytosol. The EPI is then taken up by the granule and secreted into the bloodstream. Neuronal NE, released into the junctional cleft, can activate adrenergic receptors on the post-junctional effector cell, can activate prejunctional (“presynaptic”) α2 receptors on the neuron to inhibit its own release, can be inactivated by catechol-O-methyltransferase (COMT) on the postjunctional cell, or can be taken back up into the releasing neuron by selective reuptake transporters (NRT). Inside the neuron, either synthesized or recycled NE can be metabolized and inactivated by the mitochondrial enzyme monoamine oxidase (MAO) to 3,4-dihydroxyphenylglycolaldehyde (DOPGAL). B. In vivo, NE from neurons and EPI from the adrenal do not activate all adrenergic receptors identically. Both activate α1 and β1 receptors similarly, but NE also activates α2 receptors, while EPI also activates β2 receptors.

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Table 4. ANS control of effector cell (target organ) function. 1. Dominant or exclusive control by the parasympathetic branch of the ANS (ACh) Effector

Response

Receptor Subtypes*

Exocrine glands**

Increased secretion

M1, M2, M3

Circular muscle of the pupil

Contraction (miosis)

M2, M3

Ciliary muscle of the lens

Contraction (accommodation)

M2, M3

Intestinal smooth muscle

Increased contraction

M2, M3

Urinary bladder detrusor muscle

Contraction

M2, M3

G.I. sphincter smooth muscle

Relaxation

M2, M3

*M2 and M3 receptor subtypes are activated by ACh with approximately equal affinity. **Includes salivary, bronchial, nasopharyngeal, and gastrointestinal digestive glands and gastric acid-secreting cells.

2. Dominant or exclusive control by the sympathetic branch of the ANS (NE or EPI)* Effector

Response

Major Receptor Subtype

Heart ventricles**

Increased force of contraction

β1

Heart ventricles**

Increased conduction velocity

β1

Kidney juxtaglomerular cells**

Increased renin secretion

β1

Arteriolar smooth muscle

Decreased tonic contraction (vasodilation)

β2

Uterine smooth muscle

Decreased tonic contraction

β2

Liver

Increased glycogenolysis

β2

Skeletal muscle

Increased potassium uptake

β2

Pancreatic islet beta cells

Increased insulin secretion

β2

Pancreatic islet beta cells

Decreased insulin secretion

α2

Arteriolar smooth muscle**

Increased tonic contraction (vasoconstriction)

α1***

Increased tonic contraction (mydriasis)

α1

Increased tonic contraction

α1

Radial muscle of the pupil Genitourinary smooth muscle

*Responses mediated by α1 and β1 receptors are generated by either NE or EPI in vivo; responses mediated by β2 receptors are generated exclusively by blood-borne EPI in vivo; responses mediated by α2 receptors are generated nearly exclusively by NE in vivo. **Innervated predominantly or exclusively by the SNS (little to no PNS innervation) ***Arteriolar smooth muscle generally contains a mixture of alpha-1 and alpha-2 receptors, with alpha-1 predominant.

92 | Principles of Pharmacology and Autonomics

3. Significant control by both the parasympathetic and sympathetic branches of the ANS Effector Heart atrial muscle*

Heart sinus node

Heart atrioventricular node

Bronchiolar smooth muscle

Response

Major Receptor Subtypes

Increased force of contraction

β1

Decreased force of contraction

M2

Increased heart rate

β1

Decreased heart rate

M2

Increased conduction velocity

β1

Decreased conduction velocity

M2

Decreased contraction (bronchodilation)

β2

Increased contraction (bronchoconstriction)

M2, M3

* Cardiac tissue also expresses beta-2 receptors; most areas of the heart express a mixture of muscarinic subtypes 2 and 3.

4. Special cases Effector

Response

Major Receptor Subtype

Intestinal smooth muscle

Relaxation

α2*

Sweat glands

Increased secretion

M1**

Adrenal medulla

Increased secretion of EPI (and some NE)

Nn***

*Control of intestinal tone by the sympathetic branch of the ANS is complex (see text). **Sympathetic cholinergic system (see text). ***Innervated by sympathetic preganglionic neurons (see text).

the neuronal membrane. Drugs can indirectly amplify the effects of NE by inhibiting COMT or MAO, by inhibiting the reuptake transporter, or by inhibiting prejunctional α2 receptors (see “Indirect-acting sympathomimetics and sympatholytics” below). Keep in mind that autonomic control of organ function is a dynamic process. Efferent autonomic nerve activities go up and down, and the responses of the affected target organs fluctuate accordingly. But also remember that it is a continual process, always more or less, but never all or nothing. Using a stereo as an illustration, the CNS and ANS are not “on–off ” switches, but rather control units that produce moderate but uninterrupted fluctuations in speaker volume (responses of the effector cells). Thus, when drugs affect ANS activity, they don’t act as “on–off ” switches either, but rather enhance or reduce the average volume (cell and organ activity) levels. For example, variations of sympathetic nerve activity, and of EPI in the blood, act on the heart to produce greater or lesser enhancements of heart rate during the day. A drug such as the β receptor antagonist propranolol will suppress this activity but, at therapeutic doses, will not shut it down. Resting heart rate of an untreated patient, for instance, might

Drugs That Interact With the Autonomic Nervous System | 93

vary during the day between a low of 60 and a high of 80 beats per minute, in part because of fluctuations in the activity of the cardiac sympathetic nerve. If that patient were hypertensive, then he or she might be taking propranolol. At normal doses, propranolol might decrease both upper and lower limits of heart rate to, say, 75 and 50, but would certainly not reduce it to zero. That would kill the patient. During propranolol therapy, heart rate still varies up and down, but the daily average is lower.

Responses of Effector Cells and Organs to Autonomic Innervation Most effector organs are innervated by both branches of the ANS, but some are innervated by only one. When an effector is dually innervated, the influences of the two branches may be roughly equal or the influence of one branch may be greater than the other. In most cases of dual innervation, the effects of the two branches are opposite. For example, the cardiac sympathetic nerve acts to increase heart rate, while the vagus (cranial nerve X, a predominant parasympathetic nerve) acts to decrease heart rate. In all cases, the actions of the nerves are mediated by their transmitters and the receptors on the surfaces of the effector cells that respond to those transmitters. Important effectors, their responses to sympathetic or parasympathetic innervation, and the predominant receptors that mediate those responses are summarized in Table 4. The information summarized in Table 4 is central to understanding autonomic pharmacology. A number of critical concepts and principles are implicit in that table. First, note that the influence of the two branches of the ANS may not be equal on some effectors (target cells, tissues, or organs). A given effector may be innervated by both branches (dually innervated), but one of the branches may exert dominant control. Glandular secretion is an example. Although both branches may innervate salivary or other exocrine glands, the parasympathetic branch has the most influence in regulating their secretion. Another reason for unequal influence by the two branches is anatomical. Some effectors are innervated by only one of the two branches (singly innervated) and of course would be under exclusive control by that branch. An example of such an effector would be systemic arterioles (the smallest of the arteries), which control blood pressure. They are innervated nearly exclusively by the SNS; parasympathetic nerves, for the most part, do not even go to arterioles. Therefore, parasympathetic innervation has little or no direct influence on blood pressure or on bloodflow, except weakly in the heart and a few other vascular beds. Similarly, only sympathetic nerves go to renal juxtaglomerular cells and regulate their secretion of the enzyme renin into the bloodstream. Further, heart ventricular tissue is richly innervated by sympathetic neurons but only sparsely by parasympathetic neurons. As a result, ANS regulation of the force of ventricular muscle contraction is carried out exclusively by the SNS. Heart atrial and nodal tissues, on the other hand, are representative of effectors that are under roughly equal and opposite control by the two branches of the ANS. Heart rate at rest, for example, is a balance between the tendency of the parasympathetic (vagus) nerve to suppress sinoatrial (SA) nodal activity and thus reduce heart rate, and that of the sympathetic branch (cardiac sympathetic nerve) to stimulate SA nodal activity and thus increase heart rate. Similarly, conduction of impulses (action potentials) through the atrioventricular (AV) node, from the atria to the ventricles, is slowed by parasympathetic activity and accelerated by sympathetic activity. The lungs are another example. Resistance to air flow in the lungs is determined primarily by the state of contraction of smooth muscles surrounding bronchioles, the smallest of the airways. Parasympathetic nerves innervate bronchiolar smooth muscles, but sympathetic nerves effectively do not. Nevertheless, the sympathetic nervous system does influence bronchiolar diameter by regulating the secretion of EPI from the adrenal medulla. Bronchiolar luminal

94 | Principles of Pharmacology and Autonomics

diameter is decreased by parasympathetic innervation (M receptors) but increased by actions of EPI (β2 receptors) delivered to the lungs via the bloodstream. The “special cases,” listed under section 4 of Table 4, have been segregated because they are atypical. First, activation of α receptors on smooth muscle (e.g., vascular) in general, by NE or EPI, would normally enhance their tonic contraction. But in the intestine, sympathetic activation of α adrenoceptors ultimately results in relaxation of the smooth muscles lining the intestinal walls, inhibiting propulsive movements. The likely reason for this paradoxical effect is that the control of intestinal tone involves a complex interaction of local hormones and transmitters arising from an intrinsic plexus of neuron-like cells within the intestinal submucosal layer. Enhanced sympathetic nerve activity may indirectly relax the intestine by promoting the release of local inhibitory regulators (Gournine et al., 2009). Second, recall that the sympathetic nerves going to the sweat glands are cholinergic, not adrenergic. Even though these neurons are anatomically sympathetic (arising from paravertebral ganglia in the thoracolumbar regions of the spinal cord), they are functionally cholinergic instead of adrenergic (releasing ACh instead of NE at the neuroeffector junction). Therefore, the sympathetic cholinergic neurons activate M receptors, not α or β receptors, on cells of the sweat glands. Finally, the adrenal medulla is an unusual effector: A) it is innervated by preganglionic, not postganglionic, neurons; B) it is singly innervated by sympathetic neurons that are cholinergic because they are preganglionic; and C) the receptors on the surface of the EPI-secreting chromaffin cells responding to ACh are nicotinic (Nn), not muscarinic (M).

Major Categories of Drugs that Interact with the ANS at the NEJ: Direct-Acting Autonomic Receptor Agonists and Antagonists The focal point of autonomic regulation, the NEJ, is also the most common site of action of drugs that influence ANS activity. ANS drugs are categorized according to the receptors that they either activate or block at the NEJ. Most drugs that influence the activity of the PNS fall into one of two categories: they are either agonists or antagonists on M receptors. Drugs that interact with the SNS at the NEJ, however, generally fall into one of four categories: α receptor agonists, α receptor antagonists, β receptor agonists, or β receptor antagonists. In the majority of cases, drugs that influence the ANS act directly on receptors residing on the surface of effector cells in the NEJ; these are called direct-acting agents (Table 5). Direct-acting agents used clinically are usually competitive (reversible) antagonists (see Chapter 2). They can be further subdivided according to their selectivity for receptor subtypes. For example, α blockers can be nonselective (e.g., phentolamine), with approximately equal affinities for α1 and α2 adrenoceptor subtypes. Most α blockers used clinically, however, have greater affinity for α1 than for α2 adrenoceptors (e.g., prazosin). The rationale for developing subtype-selective drugs is usually to narrow the target in order to maximize therapeutic efficacy and minimize adverse effects related to nonselectivity. Similarly, the prototype M receptor blocker, atropine, is not selective for M receptor isoforms. Pirenzipine, on the other hand, is an M1-selective blocking agent, and can be used therapeutically to treat peptic ulcers. The chemical structures of representative direct-acting ANS agents are shown in Figure 5. For an expanded list of drugs in each category, see Table 5.

Direct-acting M receptor agonist and antagonist drugs: Actions and adverse effects As discussed above, drugs that affect the function of the ANS nearly always work at the junction between an autonomic neuron and a non-neural cell (NEJ) or (less often) at the junction between one neuron and

Drugs That Interact With the Autonomic Nervous System | 95

Table 5. Direct-acting drugs that influence ANS activity at the NEJ. Drug category

Representatives

I. Parasympathetic (cholinergic) system* A. M receptor agonists (nonselective) B. M receptor antagonists nonselective M1-selective

methacholine, carbamylcholine (carbachol), bethanechol, pilocarpine, muscarine, arecoline atropine, homatropine, glycopyrrolate, dicyclomine, methscopolomine pirenzepine, telenzepine, mepenzolate

II. Sympathetic (adrenergic) system A. β adrenergic agonists nonselective

isoproterenol

nonselective with some α agonist activity

dobutamine, ephedrine, pseudoephedrine, phenylpropanolamine

β2-selective

terbutaline, albuterol, metaproterenol, isoetharine, pirbuterol, fenoterol, salmeterol, ritodrine

B. β adrenergic antagonists nonselective

propranolol, timolol, nadolol

β1-selective

metoprolol, atenolol, esmolol

β1-selective + ISA**

pindolol, acebutolol

β antagonists + α1 blockade

labetalol (+ β2 agonism), carvedilol

C. Alpha adrenoceptor agonists nonselective (some β agonist activity)

α1-selective agonists: D. Alpha adrenoceptor antagonists nonselective α1-selective

phenylephrine, dobutamine, ephedrine, pseudoephedrine, phenylpropanolamine, methoxamine phentermine, metaraminol, tetrahydrozoline, naphazoline, mitodrine phenoxybenzamine, phentolamine, tolazoline prazosin, terazosin, doxazosin, trimazosin, indoramin

*Also includes sympathetic cholinergic system innervating the sweat glands. **ISA: Intrinsic sympathomimetic activity (i.e., partial agonists on β receptors).

96 | Principles of Pharmacology and Autonomics

H3C

CH3 + CH3 N

CH3

O O

CH3 HO

CH3

N

O

O

O C O

Methacholine (M receptor agonist)

N

H N

CH3 CH3

HO

H3C

H OH O

OH

C

O

H H N

N N

CH3

C Metoprolol (β1-selective antagonist)

HO

CH3

CH3 O

CH3 Phentolamine (α receptor antagonist)

O

N

O

N N

N Phenylephrine (α receptor agonist)

CH3

N

CH3

H3C HO

H OH

CH3 Propranolol (β receptor antagonist)

Isoproterenol (β receptor agonist)

CH3

O

CH3

N

N

N Pirenzepine (M1-selective antagonist)

Atropine (M receptor antagonist) OH

N

N

N

C O

NH2 Prazosin (α1-selective antagonist)

Figure 3B. Chemical structures of direct-acting ANS agonists and antagonists.

another (ganglionic synapse). For efferent cholinergic neurons emerging from the spinal cord (Figure 1), the targets for ACh are Nn receptors in ganglia or at the adrenal medulla, or Nm receptors on skeletal muscle. At all other cholinergic junctions, i.e., between postganglionic cholinergic neurons and their cellular targets (effectors), the predominant receptors on the postjunctional cells responding to ACh are muscarinic (M) (Figure 3B). Most therapeutically relevant cholinergic drugs fall under the last category, and thus alter the effects of the PNS at the target cells and tissues. Therefore, the emphasis here is on drugs that directly imitate or inhibit the actions of ACh at the parasympathetic NEJ. Subsequent sections are reserved for actions and adverse effects of indirect-acting cholinergic drugs—those that end up indirectly activating both M and N receptors—as well as direct-acting nicotinic receptor agonists and blockers. Either promoting or inhibiting the actions of parasympathetic nerves with drugs can have therapeutic implications. Drugs that activate muscarinic receptors (muscarinic agonists)—such as methacholine (Figure 5)—tend to be used to treat conditions that could benefit from increases in smooth muscle tone or enhanced glandular secretion (Table 6). Representative therapeutic applications thus include gastric atony (to increase activity of a hypo-motile stomach), urinary retention (to reverse low activity of smooth muscle lining the urinary tract), certain types of glaucoma (to produce miosis and promote fluid drainage), and xerostomia (to stimulate salivary secretion in dry mouth). These drugs generally do not discriminate between muscarinic receptor subtypes. Not surprisingly, adverse effects of muscarinic agonists tend to reflect overstimulation of smooth muscle contraction or glandular secretion (Table 7). Two exceptions are hypotension and bradycardia. The first is due to activation of M receptors on

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vascular endothelial cells, promoting nitric oxide-induced vasodilation. The second, a low heart rate, is the result of activating M receptors on the sinoatrial (SA) node in the heart.

Table 6. Therapeutic uses of M receptor agonist drugs (e.g., bethanechol) and their rationales. Condition

Rationale

Gastric atony

Stimulation of M receptors on gastric smooth muscle stimulates tonic movements [e.g., bethanechol].

Urinary retention

Stimulation of M receptors on smooth muscle of the genitourinary tract also stimulates contractile activity and urinary excretion [e.g., bethanechol].

Xerostomia

Stimulation of M receptors on salivary glands activates secretion of saliva, correcting dry mouth (xerostomia) [e.g., pilocarpine].

Glaucoma

In acute narrow angle glaucoma, elevated intraocular pressure is caused by impaired fluid drainage through the canal of Schlemm. By producing miosis (smaller pupil) and ciliary body constriction, cholinergic agonists facilitate fluid drainage through the canal [e.g., pilocarpine].

Table 7. Adverse effects of M receptor agonist drugs (e.g., bethanechol, pilocarpine) and their explanations.* Adverse Effect

Explanation

Marked miosis

Overstimulation of M receptors on circular muscles surrounding the pupil.

Respiratory distress

Overstimulation of M receptors on smooth muscles and glands of the airways, producing bronchospasm and hypersecretion.

Hypersalivation

Overstimulation of M receptors on salivary glands.

Sweating

Overstimulation of M receptors on the sweat glands (sympathetic cholinergic system).

Intestinal hyperactivity

Overstimulation of M receptors on smooth muscle and glands of the intestinal tract leading to abdominal cramps and diarrhea.

Hypotension

Overstimulation of M receptors on vascular endothelial cells produces NO-dependent vasodilation and potential hypotension (low blood pressure).

Bradycardia

Overstimulation of M receptors on the SA node and other cardiac cells can produce severe bradycardia and cardiac arrhythmias.

*The actions listed in the table apply to both direct-acting and indirect-acting cholinergic agents. See Table 20 for adverse effects attributable only to indirect-acting agents (anticholinesterases), but not to direct-acting agents (M agonists).

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Table 8. Therapeutic uses of M receptor antagonists and their rationales. Therapeutic Use

Rationale

Ulcer

Selective blockade of M1 receptors in acid-secreting cells of the stomach inhibits acid secretion (e.g., pirenzipine, telenzepine, mepenzolate).

Asthma

Blockade of M receptors on smooth muscle surrounding the bronchioles inhibits the constricting effect of parasympathetic innervation. Use of quaternary ammonium derivatives retards systemic absorption (e.g., ipratropium, tiotropium).

First-degree AV block

Blockade of M receptors on the AV node antagonizes the conduction-slowing effects of the vagus, improving minor conduction block in this structure (usually atropine).

Pre-surgery

Treatment with an M receptor blocker prior to surgery inhibits secretions in the upper airway and inhibits reflex-induced bradycardia or cardiac arrest during surgery (atropine, glycopyrrolate, others).

Irritable bowel

Blockade of M receptors on intestinal smooth muscle and secretory cells inhibits propulsive contractions and hypersecretion induced by parasympathetic innervation; use of quaternary ammonium derivatives retards absorption and limits systemic adverse effects (glycopyrrolate, dicyclomine, methscopolomine, others).

Hyperhydrosis

Blockade of M receptors on sweat glands inhibits the sweating response to sympathetic cholinergic innervation.

Ocular exams

Blockade of M receptors on circular muscle surrounding the pupil inhibits constricting effect of cholinergic innervation, resulting in mydriasis for eye exams; use of hydrophilic derivatives minimizes lipid solubility and systemic absorption (homatropine, cyclopentolate, others).

Mushroom poisoning

Some mushrooms (e.g., Inocybe genus) contain M receptor agonists (e.g., muscarine or pilocarpine), which are toxic at high levels (e.g., hypersalivation, bradycardia, bronchospasm, hallucinations); since most of the toxicity is attributable to overstimulation of M receptors, atropine and similar agents are used as antidotes.

Anticholinesterase poisoning

Overactivation of M receptors is an important component of reactions to overdoses of anticholinesterases (insecticides, nerve gases); atropine is the M receptor blocker of choice.

Motion sickness

Lipid-soluble M receptor blockers such as scopolamine have central effects that include prophylactic effectiveness against motion sickness.

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Table 9. Adverse effects of M receptor antagonists and their explanations. Adverse effect

Explanation

Tachycardia

Blockade of M receptors in the SA node of the heart inhibits the bradycardic effects of the vagus.

Xerostomia

Blockade of M receptors in the salivary glands inhibits the salivation-promoting effects of cholinergic innervation.

Blurred vision

Blockade of M receptors on the ciliary muscle to the lens inhibits cholinergic neural control of lens shape, and thus promotes blurred vision.

Sedation

Lipid-soluble anticholinergic agents more easily enter the brain and have sedative effects.

Therepeutic uses of muscarinic receptor antagonists (anticholinergic agents)—such as atropine (Figure 5)—rely heavily on their ability to either relax smooth muscle or inhibit glandular secretion (Table 8). For example, by suppressing the actions of parasympathetic innervation on the stomach, lung, and intestinal tract, anticholinergic agents can suppress acid secretion, dilate bronchioles, and counteract symptoms of irritable bowel syndrome. Their use prior to general surgery is based largely on their ability to decrease secretions of the airways and to antagonize reflex-induced activation of the vagus nerve and resulting bradycardia during surgery (the last as might occur after mechanical stretching of large blood vessels). The antimuscarinic drugs, with few exceptions, are nonselective (i.e., they do not discriminate between muscarinic receptor subtypes). Of course, anticholinergic agents are useful as antidotes against poisoning by muscarinic agonists such as those found in mushrooms (muscarine or pilocarpine) or as partial antidotes to anticholinesterases like insecticides such as Sevin or nerve gases such as Sarin (see section on anticholinesterases below). Special case: paradoxical bradycardia caused by atropine As shown in Table 9, tachycardia is an important adverse effect of atropine or other M receptor antagonists. However, sometimes low doses of atropine can produce a brief bradycardia instead. Release of ACh at the SA node of the heart is under partial control by prejunctional M receptors on the parasympathetic neurons. Activation of these receptors by ACh decreases its own release through feedback inhibition. (This is analogous to feedback inhibition of NE released from sympathetic neurons through activation of prejunctional α2 receptors.) Atropine, by blocking prejunctional M receptors at this site, would interfere with feedback inhibition of ACh release, briefly increasing the levels of ACh in the cleft, enhancing activation of (presumably less extensively blocked) M receptors on the SA nodal cells, and decreasing heart rate. In a short time, however, nodal cell M receptors are also blocked by atropine and the bradycardia is reversed to tachycardia. Special case: Effects of exogenous acetylcholine on blood flow As mentioned above, most blood vessels are not innervated by parasympathetic neurons. In those few that are (some in skeletal muscle blood vessels, coronary arteries, and a few others), the effect of cholinergic innervation is vasodilation. The ACh released from cholinergic neurons at these few sites activate

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M receptors on vascular endothelial cells. This increases the synthesis and release of nitric oxide (NO). The NO acts on the nearby smooth muscle layer to promote the synthesis of cyclic GMP and relax the muscle, dilating the vessel. For some reason, even non-innervated vascular endothelium expresses M receptors. Injections of exogenous ACh (or other M agonist drugs such as methacholine) can cause regional or systemic vasodilation by activating endothelial M receptors and enhancing the synthesis of NO. If, however, the endothelium is first removed or damaged, the effect of ACh becomes constriction instead of dilation. Without the intervention of functional endothelium, activation of M receptors on naked vascular smooth muscle will increase their tonic contraction and promote vasoconstriction instead of dilation. One of the first casualties of an unhealthy lifestyle (poor diet, obesity, diabetes, smoking, etc.) is damaged endothelium. The health of vascular endothelium can be assessed by a diagnostic procedure called the forearm blood flow test. A cuff or other device is placed around the forearm to record changes in forearm size, which in turn is related to the extent of blood flow in that vascular bed. ACh is then injected into an artery feeding the forearm (less invasive procedures are now being used). Healthy endothelial cells lining the blood vessels will release NO, dilate the vessels, and increase blood flow. This response is blunted if the endothelium is not healthy.

Direct-acting adrenergic (α and β) receptor agonist and antagonist drugs: Actions and adverse effects Direct-acting adrenergic agonists include drugs that interact with both β and α receptors. Therapeutic uses of beta agonist drugs are somewhat restricted (Table 10). Activation of β2 receptors generally relaxes smooth muscle, including those around the airways. Not surprisingly, then, a major therapeutic application of β2-selective agonists is asthma. Agonist drugs that are selective for β2 receptors include terbutaline and albuterol. Activation of β1 receptors, on the other hand, produces effects that are generally regarded as adverse, such as tachycardia, increased renin secretion, and so forth (Table 11). Often, drugs such as terbutaline, which are described as being selective for beta subtype 2 receptors, can nonetheless produce significant beta 1 agonist effects during therapy. Thus, some of their adverse effects are attributable to beta-1 receptor activation. Beta blocking drugs, by contrast, are widely used therapeutic agents, especially for cardiovascular diseases (Table 12). They slow the heart down, which is very desirable in congestive heart failure and angina (chest discomfort associated with coronary artery disease) primarily because of the associated decrease in oxygen demand. Another common therapeutic application of beta blockers is hypertension. Beta blockers can reduce elevated blood pressure mainly by decreasing the SNS-induced release of renin from juxtaglomerular cells in the kidney. Adverse effects of beta blockers generally fall into two categories: 1) bronchoconstriction due to blockade of β2 receptors on bronchiolar smooth muscle (recent evidence suggests, however, that long term beta-blocker therapy may not be as detrimental to chronic obstructive pulmonary disease as previously assumed (Cochrane et al., 2011)); and 2) Severe bradycardia and heart block due to over-blockade of cardiac β1 receptors. Again, even if a beta blocking drug is characterized as being selective for beta subtype 1 receptors, it can produce adverse effects—such as bronchoconstriction—attributable to blockade of beta subtype 2 receptors. The therapeutic applications of α agonists usually require topical applications or local injections (Table 14). Local constriction of blood vessels is involved, for example, in the ability of α agonists to relieve nasal congestion or restrict the delivery and distribution of local anesthetics. In most cases, adverse effects appear when the α agonist drug finds its way into the systemic circulation (Table 15).

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Table 10. Therapeutic uses of beta receptor agonists and their rationales. Therapeutic Use

Rationale

Asthma and other COPD

β2-selective agonists stimulate β2 receptors on bronchiolar smooth muscle, relaxing the smooth muscle and opening the airways (terbutaline, albuterol, fenoterol, others).

Restricted cardiac applications

The nonselective β receptor agonist isoproterenol can be used as a cardiac stimulant drug in attempts to overcome bradycardic conditions and certain specific cardiac arrhythmias. Dobutamine is a nonselective β agonist with some α agonist activity. It can be used to stimulate the heart in some patients with acute congestive heart failure or myocardial infarction.

Table 11. Adverse effects of beta receptor agonists (e.g., terbutaline, albuterol, others) and their explanations.* Adverse Effect

Explanation

Severe tachycardia

Overstimulation of β receptors on SA nodal (pacemaker) cells and other conductive tissue in the heart increases heart rate.

Cardiac arrhythmias

Overstimulation of β receptors on conducting tissue and cardiomyocytes decreases their electrical stability.

Angina pectoris

Overactivation of β receptors on cardiomyocytes increases contractile force and oxygen demand; attendant tachycardia adds to the oxygen demand, increasing the risk of angina, necrosis (tissue damage), and myocardial infarction (heart attack) in vulnerable patients.

Skeletal muscle weakness

Stimulation of β2 receptors on skeletal muscle promotes the movement of potassium ions from the blood into the muscle cells, producing low blood potassium levels (hypokalemia); this in turn can lead to hyperpolarization and muscle weakness.

* Note: Although most beta agonists in clinical use are beta-2 selective, they can produce beta-1 effects such as tachycardia and cardiac arrhythmias.

Hypertension, coronary ischemia, necrosis at the site of injection, or cerebral ischemia are all potential adverse effects of α agonists, and all can be attributed to severe local or systemic vasoconstriction. It should not be surprising, then, that many of the therapeutic effects of α blocking drugs are related to their ability to relax vascular smooth muscle and thus to dilate blood vessels (Table 16). For this purpose, α1-selective agents such as prazosin are the most useful. Dilation of systemic arterioles decreases total peripheral vascular resistance. This effect benefits hypertension because it reduces blood pressure, and benefits congestive heart failure because it improves cardiac output and relieves elevated pressure loading on the heart. Alpha blockers can also be used to treat conditions of severe hypertension

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Table 12. Therapeutic uses of beta receptor antagonists (e.g., propranolol, carvedilol, others) and their rationales. Therapeutic Use

Rationale

Hypertension

Blockade of β1 receptors: 1) inhibits the secretion of renin by juxtaglomerular cells in the kidney, reducing blood levels of the vasoconstrictor angiotensin II and the sodium-retaining hormone aldosterone; and 2) moderately decreases the force of cardiac contraction, correspondingly reducing blood pressure.

Congestive heart failure

Inhibition of renin secretion (see above) mitigates fluid retention (decreased aldosterone levels) and reduces vascular resistance (decreased angiotensin levels) improving cardiac output; blockade of β1 receptors in the sinus node decreases heart rate and oxygen demand.

Angina pectoris

Bradycardic effect (see above) decreases oxygen demand in the heart and indirectly promotes coronary flow by extending the duration of diastole (time between beats).

Cardiac arrhythmias

Some disorders of heart rhythm are caused or exacerbated by β1 receptor-mediated electrophysiological actions of the SNS on the heart; β blockers can be effective.

Table 13. Adverse effects of beta receptor antagonists and their explanations. Adverse Effect

Explanation

Bronchoconstriction

Blockade of β2 receptors in the lung antagonizes the bronchodilating effects of EPI (or of β2 agonist drugs such as terbutaline).

Vasoconstriction

Blockade of vascular β2 receptors antagonizes vasodilating actions of EPI.

Severe bradycardia

Overdosing with β blockers interferes with cardioaccelerating actions of the SNS and lifts its opposition to the bradycardic effects of the vagus.

AV block

Overdosing with β blockers interferes with the positive dromotropic (conduction-accelerating) effects of the SNS at the AV node and lifts opposition to the negative dromotropic effects of the vagus on this structure.

Rebound hypertension, tachycardia, or angina

During chronic therapy, β receptors may upregulate (i.e., endogenous agonist-induced β-receptor downregulation may be inhibited in heart and other tissues); sudden discontinuation can then result in supersensitivity to β-activating effects of NE and EPI (see Chapter 2).

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Table 14. Therapeutic uses of alpha receptor agonists (e.g., dobutamine, pseudoephedrine, others) and their rationales. Therapeutic Use

Rationale

Nasal congestion

Constriction of blood vessels in the nasal mucosa counteracts swelling and congestion of the nasal passages.

In local anesthetics

Addition of α agonists to local anesthetic preparations constricts blood vessels at the site of injection, retarding systemic absorption and thus prolonging the anesthetic effect while decreasing the risk of systemic toxicity.

In ocular preparations

Constriction of blood vessels on the surface of the eyes decreases redness.

Anaphylactic shock

Epinephrine (drug of choice) functionally antagonizes histamineinduced low blood pressure (α1-mediated vasoconstriction) and bronchoconstriction (β2-mediated bronchodilation), and inhibits histamine release from mast cells (β2-mediated).

associated with overabundance of catecholamines (EPI and NE) in the bloodstream, as would occur pheochromocytoma, a hypersecreting tumor of the adrenal medulla. Adverse effects of α blockers are commonly related to reflex responses to their vasodilating effects (Table 17, Figure 6). Indirect (reflex) activation of the SNS induced by vasodilating drugs in general, including α blockers, enhance the production of NE and EPI, with consequences ranging from tachycardia to fluid retention (related to increased production of renin by the kidneys and subsequent release of aldosterone from the adrenal medulla). Reflex-induced tachycardia and fluid retention are clinically important adverse effects of vasodilating drugs. Extreme vasodilation can also lead to acute orthostatic (postural) hypotension, manifested in part by dizziness or fainting upon a sudden transition from a prone to a standing position. Special case: Cardiovascular effects of epinephrine. Epinephrine (EPI) is a mixed agonist, with roughly equal potencies on β1, β2, and α1 adrenoceptors. It has much lower affinity and potency on α2 receptors (e.g., those on prejunctional sympathetic neurons), and probably does not substantially activate them at blood concentrations achieved in vivo. Its actions on the heart are fairly straightforward. Heart muscle and conducting tissues are rich in functional β1 receptors, somewhat poorer in β2 receptors, and even poorer in functional α1 receptors. By stimulating β1 receptors, EPI is very active as a cardiac stimulant drug, increasing heart rate, conduction velocity, and force of ventricular muscle contraction. Vascular smooth muscle, by contrast, is relatively rich in α1 and β2 receptors. The ability of EPI to activate both α1 and β2 receptors, with approximately equal potency, on vascular smooth muscle makes EPI a bit schizophrenic on this tissue. On the one hand, as an α1 agonist, EPI would produce vasoconstriction, which would decrease blood flow locally and, if widespread, increase blood pressure systemically. On the other hand, as a β2 agonist, EPI would produce vasodilation, which would increase blood flow locally and decrease blood pressure systemically. If EPI is injected into patients, the net effect would be dependent on a number of factors, including the dose of EPI and its route of administration (e.g., intramuscular, intravenous, or intra-arterial), whether a given

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- CNS -

VMC

2

(–) 3

(+)

1 BP (–) ↑ HR ↑ CF

Vasodilating Drug

6 (+)

↑ VC 4

Art

(+)

EPI (+)

Aldost.

A2

(+)

Renin A0

Sodium and Fluid Retention

ACE A1

5

Figure 6. Baroreceptor reflex response to rapid systemic vasodilation. Administration of a vasodilating drug decreases blood pressure (1), which is sensed by specialized nerve endings—in the walls of large arteries including the carotid—called baroreceptors (2). The frequency of afferent nerve impulses to the vasomotor center (VMC) is altered (2), decreasing efferent PNS activity while increasing efferent SNS activity to the heart (3) and other organs. Secretion of both EPI from the adrenal medulla (4) and renin from juxtaglomerular cells in the kidney (5) are increased. Secondary effects of the enhanced renin secretion include elevations in aldosterone secretion from the adrenal cortex. Overall reflex responses to a sudden fall in blood pressure include increased heart rate (HR) and cardiac contractile force (CF), vasoconstriction (VC), and sodium and fluid retention. All three reflex responses, especially the vasoconstriction (6), work against the vasodilating actions of the drug. Other abbreviations: Art, arteriole; ACE, angiotensin-converting enzyme; A0, angiotensinogen; A1, angiotensin 1; A2, angiotensin 2.

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Table 15. Adverse effects of alpha receptor agonists and their explanations. Adverse Effect

Explanation

Hypertension

Overdose-induced systemic vasoconstriction by activation of α1 receptors on arteriolar smooth muscle.

Myocardial necrosis/infarction

Extreme α-mediated vasoconstriction of coronary arteries.

Cerebrovascular accident

Alpha-mediated constriction of cerebral blood vessels.

Table 16. Therapeutic uses of alpha receptor antagonists (e.g., prazosin, doxazosin, others) and their rationales. Therapeutic Use

Rationale

Hypertension

Blockade of α1 receptors on systemic arteriolar smooth muscle produces vasodilation and a reduction in total peripheral vascular resistance, decreasing blood pressure.

Congestive heart failure

Reduction of total peripheral vascular resistance improves cardiac output and relieves pressure loading on the heart.

Pheochromocytoma

Hypersecretion of NE and EPI from adrenal tumor produces profound vasoconstriction and increases in blood pressure that are antagonized by α receptor blockade on vascular smooth muscle.

Benign prostatic hyperplasia

Blockade of α1 receptors in urethral smooth muscle dilates the urethra within an enlarged prostate and improves urine flow.

Miotic agent

Blocking α1 receptors on radial muscles controlling pupil size would interfere with the ability of sympathetic nerves to contract the muscles; the resulting relaxation of radial muscles would decrease pupil size (promote miosis).

vascular bed is relatively rich in functional α or β receptors, and so forth. So mean blood pressure, for example, might go up, down, or sideways, depending on which and how many of the multiple variables predominate in a given situation. Generally, systemic arteriolar vasoconstriction (α) increases mean, systolic, and diastolic blood pressures, while vasodilation (β2) would produce opposite effects. Increased cardiac contractile force (β1) tends to increase systolic and mean pressures more than diastolic pressure. In practice, the receptor with greatest overall influence can be inferred from the observed response: if mean, systolic, and diastolic blood pressures go up, then “it must have been vascular α with cardiac β.” If systolic pressure goes up but mean and diastolic pressures go down, then “it must have been vascular β2 and cardiac β1.”

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Table 17. Adverse effects of alpha receptor antagonists and their explanations. Adverse Effect

Explanation

Tachycardia

Rapid reduction in blood pressure activates the baroreceptor reflex (Figure 6) that stimulates the SNS while inhibiting the PNS, promoting tachycardia (increased β-receptor activation by the SNS and decreased M receptor activation by the PNS at the cardiac SA node).

Fluid retention

Another effect of activating the baroreceptor reflex (increased SNS-mediated activation of the endocrine renin-angiotensinaldosterone system).

Angina

Tachycardia increases myocardial oxygen demand, which can precipitate angina in the presence of coronary artery disease or congestive heart failure.

Syncope and orthostatic hypotension

Peripheral vasodilation can interfere with ability to regulate and maintain blood flow to the brain during changes in body position.

Characteristics of beta-blocking drugs Beta receptor antagonists (β blockers) are among the most widely used ANS drugs. It is clear from Table 12 that they have broad therapeutic applications, particularly for diseases of the cardiovascular system. Older β blocking drugs, such as the prototype propranolol, are not selective for β receptor subtypes; i.e., they have approximately equal binding affinities for β1 and β2 receptors. A comparison of Tables 12 and 13 reveals that the therapeutic benefits of β blockers are largely or exclusively attributable to blockade of β1 receptors, while adverse effects (such as broncho- or vasoconstriction) result mainly from inhibition of β2 receptor subtypes. In attempts to maximize the former and minimize the latter, β1-selective agents have been more recently developed (Table 5). Another property of some β blockers is intrinsic sympathomimetic activity (ISA), or partial agonism on β receptors. Among presumed therapeutic advantages of ISA on β1 receptors is the reduced risk of rebound hypertension or angina upon sudden discontinuation of therapy by minimizing the extent of receptor upregulation during therapy (Table 13). Still other β blockers exhibit some α receptor blockade as well. Pure β blockers do not directly relax vascular smooth muscle. The additional property of α blockade would confer vasodilating activity and fill a therapeutic gap in the treatment of hypertension or congestive heart failure. Dopamine and fenoldopam Recall from Figure 1 that some sympathetic neurons innervating the kidney are dopaminergic. Activation of D-1 receptors in the kidney produces dilation of selected blood vessels, decreasing their resistance to blood flow. Exogenous dopamine stimulates a variety of receptors, including β1, α1, and D-1 receptors. Fenoldopam is a selective D-1 receptor agonist. The ability of either of these drugs to dilate renal blood vessels and increase renal blood flow makes them potentially useful for the treatment of cardiogenic or hypovolemic shock (severely reduced blood pressure and cardiac output). The renal vasodilation

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helps to maintain perfusion of the kidneys, when systemic blood pressure or cardiac output is very low, decreasing the risk of renal failure.

Implications: Predicting Therapeutic and Adverse Effects of Drugs that Directly Interact with ANS Receptors As emphasized earlier, the majority of therapeutic drugs that target the ANS interact with autonomic receptors at the NEJ. Of those, some are agonists but most are competitive antagonists. Information contained in Tables 4–17 can be used to predict effects of drugs that either activate or block cholinergic or adrenergic receptors at the NEJ. Table 4 summarizes effects of the neurotransmitters themselves at different target organs, and which receptors mediate the responses. Table 5 is a list of drugs that either imitate (agonists) or inhibit (antagonists) the actions of neurotransmitters at their NEJ receptors. To predict the effects of these drugs in a step-by-step way, a useful sequence would be 1) select a drug of interest; 2) from Table 5, determine its receptor and whether the drug is an agonist or antagonist on that receptor; 3) from Table 4, determine effects that the ANS would produce by activating the same receptor on various target tissues; and 4) from that information predict potential therapeutic or adverse effects that the drug might produce. If the drug is an agonist, then it should imitate the effects of the ANS on the receptor. If the drug is an antagonist, then its effects should be opposite to those of the ANS on that receptor. Then check your predictions by referring to the two appropriate tables among Tables 6 to 17. As an illustrative example, we can select our old friend prazosin as the drug of interest. From Table 5, prazosin is shown to be an α1-selective antagonist. From Table 4, it is predicted that activation of α1 receptors by the SNS (i.e., by NE or EPI) would promote vasoconstriction, promote the contraction of genitourinary smooth muscle such as the urethra, and promote contraction of the radial muscle around the pupil of the eye. Since prazosin is an antagonist on α1 receptors, then it would not be surprising if that drug, at therapeutic blood levels, would oppose the actions of the SNS at each site. Therefore, prazosin would be expected to dilate blood vessels, relax the urethra, and relax radial muscles around the pupil of the eye. All three might be judged to have therapeutic potential. Prazosin would not be expected to substantially alter insulin secretion, because the alpha receptor subtype involved is α2, not α1, and the predominant endogenous regulator of insulin secretion is blood glucose, not the ANS. As it turns out (Table 16), the most therapeutically relevant effect is the vasodilation (specifically, dilation of arterioles), which is the rationale for prazosin’s use as an antihypertensive drug. Prazosin or similar α1 antagonists might also be used to treat benign prostatic hyperplasia (BPH) by dilating a urethra that is partially restricted by an enlarged prostate gland. Alpha blocking drugs might also produce miosis (small pupils) if applied topically in the eye. Miotic agents can be used to treat certain kinds of glaucoma. Alpha blockers would not be very effective for this purpose, however, because the predominant control of pupil size is cholinergic (M receptors). Two other therapeutic applications of alpha blockers listed in Table 16, congestive heart failure (CHF) and pheochromocytoma, make sense after some thought. Remember that prazosin is an effective systemic vasodilator. Systemic vasoconstriction contributes to the pathology of both CHF and pheochromocytoma. In CHF, prazosin-induced arteriolar dilation would reduce peripheral vascular resistance, decreasing workload (pressure load) on the heart and improving cardiac output. In pheochromocytoma, oversecretion of the adrenomedullary alpha agonist catecholamines NE and EPI produces profound vasoconstriction and hypertension. By blocking vascular α receptors, prazosin or other α blocking drugs would act to decrease the elevated blood pressure and thus reduce the risk of hypertensive crisis.

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Four potential adverse effects of alpha blockers listed in Table 17 are tachycardia, fluid retention, angina, and orthostatic hypotension and syncope. Prediction of those effects requires additional information. Recall that the major therapeutic effect of prazosin is vasodilation, which is the basis for the drug’s utility in treating hypertension, CHF, and pheochromocytoma. Unfortunately, vasodilating drugs in general can trigger the baroreceptor reflex, which can restrict their therapeutic effectiveness (Figure 6), particularly if they produce a rapid and marked reduction in pressure. Remember from Chapter 1 that the body usually fights effects of xenobiotics. This is another example of that principle. Vasodilating drugs including prazosin reduce blood pressure. Sensory nerve endings (baroreceptors) embedded in the walls of large arteries, notably in the carotid sinus, can sense changes in pulsatile activity resulting from the fall in pressure. This in turn alters afferent nerve activity delivered to the vasomotor center in the medulla of the brain. The result is an increase in efferent sympathetic nerve activity together with a reduction in efferent parasympathetic nerve activity. The combination of increased activation of β1 receptors by NE and diminished activation of M receptors by ACh in the SA node of the heart would increase heart rate. Tachycardia in turn increases myocardial oxygen demand, increasing the risk of angina in vulnerable patients. Fluid retention is promoted by enhanced activation of β1 receptors by NE on renal juxtaglomerular cells, promoting renin excretion. Downstream, elevations in angiotensin 2 would enhance the secretion of aldosterone from the adrenal cortex, promoting sodium and fluid retention through its actions on the kidney. In addition, reflex activation of sympathetic efferents to systemic arterioles would competitively oppose the vasodilatory effects of prazosin or functionally oppose effects of other vasodilating drugs that work by different mechanisms. Reflex activation of the SNS is a common complication of vasodilator therapy. Related effects of vasodilatory drugs in general, including prazosin, are syncope (fainting) and orthostatic hypotension (e.g., dizziness or fainting upon abruptly standing up from a prone position).

The Concept of Indirect-Acting ANS Drugs Some drugs can influence ANS activity without directly binding to ANS receptors on the effector cells at the NEJ. Instead, they exert their effects indirectly by altering the levels of the endogenous agonists ACh, NE, or EPI at the NEJ. Accordingly, such drugs are called indirect-acting agents. Like the direct-acting drugs discussed above, indirect-acting drugs also alter the extent of activation of M, α, or β receptors on the effector cells. However, they do this not by binding to the receptors themselves, but by making more or less of the endogenous neurotransmitter or neurohormone available for binding to, and activating, their receptors. The overall effects of indirect-acting agents that increase neurotransmitter production are thus similar, but not identical, to those of direct-acting agonists. Indirect-acting ANS drugs generally fall into one of two categories: 1) Indirect-acting sympathomimetics; and 2) Anticholinesterases. The first is discussed here. Pharmacological effects of anticholinesterases are included with those of ganglionic agonist and blocking drugs in the following section.

Indirect-acting sympathomimetics and sympatholytics Indirect-acting sympathomimetic or sympatholytic drugs enhance or decrease SNS activity by increasing or reducing the secretion of NE from sympathetic or CNS neurons, or of EPI from the adrenal medulla (Table 18). Some of these drugs also alter levels of dopamine in ANS and CNS neurons. Cocaine is a classic example. It does not activate α or β adrenoceptors directly. Instead, it makes more NE available to activate the receptors on effector cells such as heart, blood vessels, etc. Cocaine does that

Drugs That Interact With the Autonomic Nervous System | 109

Table 18. Drugs that indirectly influence the actions of adrenergic neurotransmitters. Drug

Predominant Action

Effects

Cocaine

NE reuptake transporter

Increased NE at NEJ, increasing activation of α and β receptors on effector cells.

Amphetamine

NE or DA release

Increased NE at NEJ, increased activation of α or β receptors on effector cells.

Tranylcypromine

MAO

Increased levels of NE, DA, or EPI inside neurons or chromaffin cells, increasing their secretion and effects.

Reserpine

VMAT transporter

Increased MAO-mediated metabolism of NE or DA in neurons, or of EPI in adrenal chromaffin cells, depleting the cells of neurotransmitter or neurohormone, diminishing their effects.

Table 19. Therapeutic applications and adverse effects of indirect-acting sympathomimetic and sympatholytic drugs. Drug

Therapeutic Applications

Adverse Effects

Cocaine

Local anesthetic (Schedule II)

Systemic and coronary vasoconstriction, angina, tachycardia, cardiac arrhythmias (α and β).

Amphetamine

Hyperactivity syndromes (CNS)

Systemic and coronary vasoconstriction, angina, tachycardia, cardiac arrhythmias (α and β).

Tranylcypromine*

Depression (CNS)

Tachycardia, elevated blood pressure (α and β).

Reserpine

Pheochromocytoma

CNS depression, hypotension, fluid retention.

*And other MAO inhibitors

by inhibiting the NE-specific reuptake transporter, increasing the levels of NE in the junctional cleft. So cocaine would indirectly activate β1, α1, or α2 receptors at the NEJ, because those are the receptor subtypes that are targeted by NE. Similarly, drugs that inhibit the MAO enzyme (Figure 4) would retard the metabolic destruction of NE or EPI in sympathetic neurons or the adrenal medulla, increasing their accumulation and amplifying the effects of these substances on target cells. Other indirect-acting agents can interfere with sympathetic neurotransmission or adrenal function. For example, in the ANS and the CNS, reserpine depletes adrenergic neurons of NE, dopaminergic neurons of DA, and chromaffin cells of EPI, decreasing their release. Reserpine’s molecular target is the intracellular vesicular uptake transporter VMAT (Figure 4). Among reserpine’s restricted therapeutic applications is the treatment of pheochromocytoma. Depletion of neurotransmitters underlies both its ability to decrease blood

110 | Principles of Pharmacology and Autonomics

pressure peripherally (SNS) and its tendency to promote clinical depression centrally (neurons in the brain). Therapeutic actions (where applicable) and adverse effects of indirect-acting sympathomimetic and sympatholytic agents are summarized in Table 19.

Indirect-acting agents that influence the activities of both the PNS and SNS: Anticholinesterases and ganglionic agonists and antagonists Anticholinesterases Two additional groups of drugs can indirectly enhance the effects of both the SNS and PNS at their termini (the NEJ). One group consists of the anticholinesterases. Physostigmine is a prototype anticholinesterase (Figure 7). Other anticholinesterase drugs include neostigmine, pyridostigmine, rivastigmine, edrophonium, tacrine, donepezil, and galantamine. These drugs work by reversibly binding to and inhibiting the enzyme acetylcholinesterase (AChE) in ganglionic synapses and cholinergic NEJs (Figures 3, 8, 9). By inhibiting AChE, anticholinesterases interfere with the ability of the enzyme to hydrolyze and destroy ACh. Therefore, anticholinesterases would increase the amounts of ACh wherever it is released. These sites would include both SNS and PNS ganglia, as well as cholinergic NEJs including NEJs innervated by the PNS, synapses at the adrenal medulla, and the neuromuscular junction (NMJ). Note that the activation of cholinergic ANS neurons takes place at both the ganglionic and NEJ sites, but activation of adrenergic neurons is restricted to ganglia. At the adrenal medulla, anticholinesterases would also increase the release of EPI into the systemic circulation. Therefore, anticholinesterases would indirectly increase the activation of M receptors by ACh at cholinergic NEJs. In addition, by indirectly A. Indirect-Acting Adrenergic Agonists O O

NH2

NH O

O

H3C Tranylcypromine: Amplifies SNS by inhibiting monoamine oxidase (MAO)

Cocaine: Amplifies SNS by inhibiting neuronal NE reuptake.

B. Indirect-Acting Cholinergic Agonists with Indirect Adrenergic Agonist Activity

N

N N

H

3C

O

CH

3

Physostigmine: “reversible” anticholinesterase.

CH3

CH3 N

N

CH3 H DMPP: Nn receptor agonist.

Figure 7. Indirect-acting adrenergic and cholinergic agonists.

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Table 20. Effects of anticholinesterases. Representative Drug

Target

Predominant Effects

Physostigmine

Acetylcholinesterase

1. Increased ACh and activation of Nn receptors on postsynaptic neurons in parasympathetic ganglia (imitating actions of direct-acting nicotinic receptor agonists at parasympathetic ganglia), increasing release of ACh by postsynaptic neurons at the NEJ, increasing activation of M receptors on effector cells (imitating the actions of direct-acting M receptor agonists at the NEJ). 2. Increased ACh and activation of Nn receptors on postsynaptic neurons in sympathetic ganglia, increasing release of NE by postsynaptic neurons at the NEJ + increased release of ACh from preganglionic sympathetic neurons and subsequent activation of Nn receptors on adrenomedullary chromaffin cells (imitating actions of direct-acting nicotinic receptor agonists at sympathetic ganglia and at the adrenal medulla), enhancing the release of EPI into the bloodstream. Both actions increase stimulation of α and β adrenoceptors on effector cells (imitating the actions of direct-acting alpha and beta agonists at the NEJ). 3. Increased ACh and activation of Nm receptors at the neuromuscular junction (NMJ), enhancing muscle twitches and tonic contractions, imitating early effects of succinylcholine (somatomotor, not ANS, effect).

increasing the activation of N receptors in ganglia, anticholinesterases can secondarily enhance the activation of α and β receptors at adrenergic NEJs by NE or EPI downstream. Anticholinesterases can also stimulate muscle twitching and contraction at the NMJ (see Appendix). Finally, lipid-soluble anticholinesterases such as tacrine, metrifonate, and rivastigmine more readily cross the blood–brain barrier. Some have been tried in the treatment of Alzheimer’s disease or for prophylaxis against nerve gas poisoning (Table 21). It is evident that the net effects of anticholinesterases would be more difficult to predict than those of direct-acting receptor agonists, because actions of cholinergic and adrenergic neurons at dually innervated effectors often oppose each other. In general, anticholinesterases favor cholinergic rather than adrenergic effects, presumably because they enhance cholinergic neurotransmission at both the ganglionic and NEJ levels of the PNS, but only enhance adrenergic transmission of the SNS at the ganglionic level. Predominant actions and therapeutic and adverse effects of anticholinesterases, along with their rationales, are summarized in Tables 20–22.

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Table 21. Therapeutic uses of anticholinesterases and their rationales*. Condition

Rationale

Belladonna poisoning

Excessive M receptor blockade underlies atropine (belladonna) poisoning. Anticholinesterases increase ACh levels, competitively overriding the blockade.

Nerve gas poisoning

Prophylactic treatment with reversible anticholinesterases presumably protects against subsequent phosphorylation of the enzyme by organophosphates (Figure 7).

Alzheimer’s disease

Centrally acting anticholinesterases (e.g., tacrine, metrifonate) increase brain levels of ACh and presumably overcome deficits in Alzheimer’s.

Myasthenia gravis (MG)

MG is a muscle weakness associated with a deficit of Nm receptors at the NMJ. Anticholinesterases increase levels of ACh at the NMJ, activating a greater fraction of Nm receptors.

Miotic agents

Drugs such as demecarium have an extended duration of action to produced sustained miosis (narrowing of the pupil).

*Applies only to reversible cholinesterase inhibitors (physostigmine, neostigmine, etc.).

Special case: “irreversible” anticholinesterases Anticholinesterase drugs can be either reversible or irreversible, depending on how they inhibit the cholinesterase enzyme. Physostigmine and related drugs bind reversibly to the active site of the enzyme, preventing hydrolysis of the endogenous substrate ACh. The effects of these agents are temporary, with restoration of full enzyme activity determined substantially by the rate at which these drugs are metabolized and excreted. In contrast, another group of anticholinesterases can bind irreversibly, creating a permanent inactivation of the enzyme molecule. Organophosphates such as the insecticide Sevin® and the nerve gas Sarin® inactivate AChE by phosphorylating it. After a short time, the phosphate bond becomes permanent. Without pharmacological intervention, restoration of full activity in this case is dependent largely on the rate of replacement by newly synthesized enzyme. Poisoning with reversible and irreversible anticholinesterases are treated somewhat differently. The actions of reversible and irreversible anticholinesterases are compared in Figure 8. High concentrations of either non-organophosphate or organophosphate agents would elevate levels of ACh and correspondingly overactivate M receptors at the NEJ or NMJ, leading to toxic effects such as bronchoconstriction, increased secretions in the airways, severe bradycardia, skeletal muscle tremors, etc. (Table 22). The drug of choice to combat most of these effects is the competitive M receptor antagonist atropine. This treatment is sufficient for the non-organophosphates because their effects are temporary (Figure 8A). However, atropine is not sufficient for the organophosphates because their actions are relatively long-lasting and cannot be reversed by simple competitive inhibition of M receptors. For those agents, removal of the phosphate from the enzyme is necessary. “Cholinesterase reactivators” include pralidoxime (2-PAM), obidoxime, and HI-6, a more recently developed derivative of 2-PAM. These agents can reactivate AChE by removing the phosphate from the enzyme. They bind phosphates more avidly than the enzyme does. Cholinesterase reactivators should be administered as soon as possible after exposure

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M

A

5 Atropine + N

+

N

O

O

O

F

O

O

+

1

P

N ACh

O + N

+

4

O

OH

2-PAM

O

HO AC

N

N+

Chol

F Pyr O

2 3

OH GLU 334

P

O

O

Sarin

GLU 334 SER 203

SER 203

GLU 334

HIS 447

HIS 447 AChE

AChE

B

O + F R2

OH

C3H7

R1

R1

O

(Early)

R2

O

P

O O

AChE (SER 203)

R1 O

P

Sarin

Sarin-AChE

F (Later)

R2

O

P O

“Aged” Phosphorylated AChE

Figure 8. Treatment of poisoning by “reversible” and “irreversible” anticholinesterases. A. In the active site of the enzyme, ACh is hydrolyzed to choline and acetate (1). Important amino acids in the binding site are Glu334, His447, and Ser203. The “reversible” anticholinesterase pyridostigmine competitively inhibits ACh hydrolysis (2). The organophosphate nerve gas Sarin “irreversibly” blocks ACh hydrolysis by binding to, and phosphorylating, Ser203 (3). Restoration of functional enzyme is accomplished by a transfer of the phosphate from the enzyme to the reactivator 2-PAM (pralidoxime) (4). Atropine can mitigate the systemic effects of overstimulation of M receptors during AChE inhibition by either competitive (nonphosphorylating) or irreversible (phosphorylating) agents, but is not effective in restoring activity of a phosphorylated enzyme (5). B. “Aging” of the enzyme occurs with slow removal of C3H7 from the phosphate group of the bound sarin nerve gas. The aged enzyme is resistant to reactivation. Adapted from Taylor and Radic, 1994; Ekström et al., 2009.

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Table 22. Adverse effects of anticholinesterases and their explanations*. Adverse Effect

Explanation

Marked miosis

Overactivation of M receptors on the circular muscles surrounding the pupil.

Respiratory distress

Overstimulation of M receptors on smooth muscles surrounding bronchioles (bronchospasm) and on bronchial glands (hypersecretion).

Sweating

Overstimulation of M receptors on sweat glands (sympathetic cholinergic).

Hypersalivation

Overstimulation of M receptors on salivary glands.

Intestinal hyperactivity

Overstimulation of M receptors in the g.i. tract leads to abdominal cramps, diarrhea, and incontinence.

Cardiovascular

Inhibition of pseudocholinesterase in the bloodstream can increase circulating levels of ACh, activating M receptors on vascular endothelial cells, and producing NO-induced hypotension (severely low blood pressure); overstimulation of cardiac M receptors produces cardiac weakness, severe bradycardia, and arrhythmias.

CNS

Agents that enter the brain at high doses can cause confusion, convulsions, central respiratory compromise, and coma.

Muscle fasciculations

Overstimulation of Nm receptors leads to skeletal muscle hyperactivity (somatomotor system, not the ANS).

Paradoxical

Stimulation of Nn receptors in sympathetic ganglia and adrenal medulla can lead to paradoxical sympathetic effects (increased heart rate, increased blood pressure, tachycardia, etc.), which are opposite to expected actions.

*Many of these adverse effects are similar to those of direct-acting M receptor agonists (see Table 7).

to organophosphates. With time, the phosphorylated enzyme “ages” and becomes more resistant to reactivation (Figure 8B). Of course, reactivators would be inappropriate for non-organophosphate anticholinesterases. So the preferred treatment for poisoning with non-organophosphate anticholinesterases is atropine, but the treatment of organophosphate poisoning is atropine together with 2-PAM or a similar agent, HI-6. Nicotinic agonists The second group of indirect ANS activators is composed of drugs that stimulate neuronal nicotinic receptors (Nn receptors). These drugs are also known as short-term nicotinic agonists or ganglionic agonists. Tetramethylammonium (TMA) is a prototype Nn receptor agonist that lacks antagonist activity. Other Nn agonists include nicotine, lobeline, and dimethylpiperazinium (DMPP) (Figure A-1, Appendix). The Nn receptor agonist effects of nicotine and lobeline, however, are temporary. By activating Nn receptors, these agents generate small membrane depolarizations known as “excitatory

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Table 23. Effects of neuronal and adrenomedullary nicotinic receptor (Nn) agonists. Representative Drug

Target

Predominant Effects

Tetramethylammonium (TMA)

Parasympathetic ganglia

Increased release of ACh by postganglionic cholinergic neurons, increasing the activation of M receptors at the NEJ.

Sympathetic ganglia

Increased release of NE by postganglionic adrenergic neurons, increasing the activation of α and β receptors at the NEJ.

Adrenal medulla

Increased release of EPI by chromaffin cells, increasing the activation of α and β receptors on target cells.

Table 24. Adverse effects of Nn agonists and their explanations. Adverse Effect

Explanation

Tachycardia

Enhanced release of NE from sympathetic neurons and EPI from the adrenal activates cardiac β adrenoceptors.

Increased blood pressure

Enhanced release of NE from sympathetic neurons and EPI from the adrenal activates vascular α adrenoceptors.

Increased g.i. secretions and motility

Enhanced release of ACh from parasympathetic nerves to g.i. tract, increasing activation of M receptors on glands and smooth muscle cells.

Increased secretions in the upper respiratory tract.

Enhanced release of ACh from parasympathetic nerves to the lung.

Nicotine poisoning

Mainly CNS effects: nausea, vomiting, disturbed hearing and vision, dizziness, fainting, a fall in blood pressure, respiratory distress, and a weak pulse. Convulsions and death due to respiratory failure at high toxic doses.

post-synaptic potentials” or EPSPs. The small depolarizations are sensed by nearby voltage-sensitive fast sodium channels. Their activation by EPSPs increases the frequency of action potential generation, ultimately increasing the release of neurotransmitter downstream. However, sustained exposure of Nn receptors to these agonists reverses the stimulation to a “depolarizing blockade.” In that regard, these agonist/ depolarizing blockers in autonomic ganglia are analogous to depolarizing blockers of Nm receptors, such as succinylcholine, at the neuromuscular junction (Figure A-1, Appendix). While depolarizing Nn

116 | Principles of Pharmacology and Autonomics

blockers such as nicotine are acting as agonists, they activate both SNS and PNS ganglia and enhance the release of NE or ACh from postganglionic neurons, and EPI from the adrenal medulla, as anticholinesterases do. However, unlike anticholinesterases, Nn agonists do not supplement their ganglionic effects by enhancing neurotransmission at the cholinergic NEJ. Nevertheless, it should not be surprising that the early pharmacological actions of Nn agonists (Tables 23 and 24) are similar to those of anticholinesterases (Tables 20–22). The therapeutic use of nicotine is restricted largely to the treatment, usually in skin patches, of smoking addiction.

Ganglionic blocking agents Ganglionic blocking drugs are antagonists on Nn receptors without agonist activity. The alkaloids nicotine and lobeline are depolarizing Nn blockers (see above and Appendix Figure A-1) but hexamethonium, mecamylamine, pentolinium, and trimethaphan are competitive Nn antagonists that lack any short-lived agonist effects. All of these drugs can interfere with the activities of both the PNS and the SNS by blocking Nn receptors at parasympathetic and sympathetic ganglia and at the adrenal medulla. The net effect of these agents on target cell activity depends largely on which branch of the ANS is dominant on that effector (Table 25). For example, the actions of ganglionic blockers on blood pressure are easily predictable. Most systemic arterioles are innervated exclusively by the SNS without involvement of the PNS. Therefore, these drugs would be expected to relax arteriolar smooth muscle and decrease blood pressure by blocking sympathetic neurons at the ganglionic level and by inhibiting the release of EPI from the adrenal medulla. In that respect, the effects of mecamylamine at sympathetic ganglia, for instance, would be similar to those of the α1 receptor antagonist prazosin at vascular smooth muscle. In contrast, glandular secretion is controlled predominantly by the PNS with less influence of the SNS. By blocking Nn receptors in parasympathetic ganglia, these agents would act like atropine at the parasympathetic NEJ. That is, either Nn blockers or M blockers would produce xerostomia, diminished glandular secretion, and other anticholinergic effects, but would do so by acting at different sites. Where effectors are under dual ANS control, such as the cardiac SA node, the effect of Nn blockers on that specific target would be more difficult to predict. It depends on which branch is dominant on that effector. Resting heart rate in most people is influenced more by the PNS (vagus) than by the SNS (cardiac sympathetic nerve). So, in most patients, Nn blockers would act like atropine and produce some tachycardia, even though the drug is blocking both parasympathetic and sympathetic ganglia. However, this is only playing the odds. The Nn receptor blocker mecamylamine might instead produce bradycardia in some people whose heart rate is under dominant SNS control. In those patients, the drug would be mimicking the actions of the beta-blocking drug propranolol. Therapeutic applications of ganglionic blocking drugs are limited mainly to the treatment of acute, severe hypertensive crisis, to control arterial pressure during vascular surgery, and to treat autonomic hyperreflexia. Adverse effects of higher doses include marked hypotension, paralysis of the g.i. tract, fainting, and cycloplegia. Mecamylamine can enter the CNS and cause symptoms of both excitation and depression.

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Table 25. Most likely effects of ganglionic blocking agents (e.g. mecamylamine) on selected targets.* Effector

Predominant innervation

Effect of ganglionic blockade

Systemic arterioles

Sympathetic

Vasodilation and reduction of blood pressure

Cardiac ventricles

Sympathetic

Depressed contractility

Cardiac SA node

Parasympathetic

Tachycardia

Salivary glands

Parasympathetic

Xerostomia

Sweat glands

Sympathetic cholinergic

Anhydrosis

*Note that, in most people, the actions of ganglionic blocking drugs such as mecamylamine imitate those of α blockers such as prazosin on arterioles, of β blockers such as propranolol on ventricular muscle, and of M receptor blockers such as atropine on cardiac SA node and glandular secretion.

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Appendix Drugs That Inhibit Skeletal Muscle Contraction

Skeletal muscles are innervated by cholinergic motor nerves that comprise the somatomotor nervous system (SMNS), which are not part of the autonomic nervous system but share many of the characteristics of cholinergic autonomic nerves. The neurons of the somatomotor system emerge from the spinal cord but, unlike ANS nerves, somatomotor nerves are not interrupted by ganglia (inset, Figure 1). Motor nerves terminate at special NEJs called neuromuscular junctions (NMJs). In many ways, NMJs are similar to ganglionic synapses in the ANS. For instance, the predominant receptors on the innervated cells are nicotinic in both cases, but they are different isoforms, Nn and Nm. Both isoforms respond to ACh, but they differ with regard to which drugs selectively activate or block them. As discussed above, activation of Nn receptors (by, for instance, TMA) produces EPSPs. Activation of Nm receptors generates similar small changes in membrane voltage called miniature end-plate potentials (MEPPs). As in ganglia, there are depolarizing and nondepolarizing blocking drugs, but they are more selective for Nm than for Nn receptors. Nondepolarizing Nm blockers include d-tubocurarine and pancuronium. Depolarizing blockers include succinylcholine and decamethonium. Nicotinic neurotransmission at ganglia and the NMJ are compared in Figure A-1. Therapeutic and adverse effects of drugs that act at the NMJ are listed in Tables A-1 and A-2. A

- Presynaptic Neuron -

- Somatomotor Neuron -

SuccinylPhysostigmine ACh choline ACh Nicotine (+) (DB) (DB) (–) (–) Na+ d-tuboNa+ (+) MMA (–) (–) (–) + (NDB) Choline+ Na+ Na curarine Acetate (–) (NDB) Nm Nn AChE AChE AP

Na+ EPSP Na+ ↑ Neurotransmitter Release

- Postsynaptic Neuron -

- Muscle cell Na+

MEPP Na+

AP ↑ Muscle Twitch

Figure A-1. A. N receptors at the synapse in ANS ganglia (left) and at the NMJ of the somatomotor nervous system (right). The figure illustrates analogous effects of agonist and blocking drugs at the two sites. For example, the depolarizing Nn blocker nicotine acting at ganglia is analagous to the depolarizing blocker succinylcholine acting at the NMJ. Abbreviations: Nn, neuronal nicotinic receptor; Nm, muscle nicotinic receptor; NMJ, neuromuscular junction; DB, depolarizing blocker;

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NDB, nondepolarizing blocker; EPSP, excitatory postsynaptic potential; MEPP, miniature end plate potential; AChE, acetylcholinesterase; MMA, mecamylamine.

B

0

Em (mV)

–80 NDB

PS

DB

PS

Figure A-1. B. Nondepolarizing vs. depolarizing blockade at either ANS ganglia or the NMJ. Anticholinesterase drugs such as physostigmine (PS) can overcome blockade by nondepolarizing agents (left), but not of depolarizing blockers (right). Abbreviations: AP, action potential; NDB, nondepolarizing blocker; DB, depolarizing blocker.

Table A-1. Therapeutic uses of muscle-blocking drugs and their rationales. Therapeutic Use

Rationale

Adjuncts to surgical anesthesia

Blockade of diaphragm and intercostals facilitates artificial ventilation during surgery (patient does not fight the respirator)

Realignment of bone fractures

Blockade of muscles around fractures facilitates realignment

Facilitation of tracheal intubation

Blockade of skeletal muscles surrounding upper respiratory tract

Laryngoscopy and esophagoscopy

Removes reflex resistance to these procedures

Table A-2. Adverse effects of muscle-blocking drugs and their explanations. Adverse effect

Explanation

Malignant hyperthermia

Life-threatening condition precipitated by the combination of depolarizing muscle blockers and halothane anesthesia, characterized in part by contracture of all muscles. DOC is dantrolene, which interferes with calcium release in the muscle.

Hyperkalemia

Produced by depolarizing blockers. Widespread cellular depolarization promotes potassium movement from the cells into the bloodstream, resulting in hyperkalemia.

Histamine release

Curariform (nondepolarizing) blockers only. Curare promotes the release of histamine from mast cells, which may precipitate hypersensitivity reactions.

Ganglionic blockade

Although the Nm blocking drugs are designed to be specific to the nicotinic receptors on muscle, at higher doses they can also block Nn receptors in ganglia.

120 | Principles of Pharmacology and Autonomics

Chapter 3 Bottom Lines • The autonomic nervous system (ANS) is divided into two main branches: The sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS). • Efferent neurons of the ANS are divided into preganglionic and postganglionic fibers. • Acetylcholine (ACh) is the neurotransmitter of the preganglionic fibers of the SNS and PNS and of the postanglionic fibers of the PNS. • Norepinephrine (NE) is the major neurotransmitter of the postganglionic fibers of the SNS. • In the sympathetic cholinergic system, the SNS releases ACh and stimulates M receptors on sweat glands. • The predominant receptor for ACh in autonomic ganglia is the neural nicotinic receptor (Nn), mediating ganglionic neurotransmission. • The predominant receptor for ACh at the autonomic neuroeffector junction (NEJ) is the muscarinic (M) receptor. • The predominant receptors for NE at the NEJ are alpha (α) and beta (β) receptors. • In the SNS, epinephrine (EPI) is released into the bloodstream by chromaffin cells of the adrenal medulla. • EPI, like NE, stimulates both α and β receptors on target cells, but their subtype selectivities are different. • EPI and NE have approximately equal affinities for β1 and α1 receptors; EPI has greater affinity for β2 receptors, while NE has greater affinity for α2 receptors. • Prominent effects of the PSN (ACh) on M receptors at the NEJ include bradycardia, decreased cardiac AV nodal conduction, enhanced glandular secretion, increased g.i. tone and secretions, and miosis. • Prominent effects of M receptor blockers (e.g., atropine) thus include tachycardia, increased AV nodal conduction, decreased glandular secretion, decreased g.i. tone and secretions, and mydriasis (all anti-PSN effects), and anhydrosis (anti-sympathetic cholinergic effect). • Prominent effects of the SNS (NE, EPI, or both) on α receptors at the NEJ include vasoconstriction, mydriasis, and increased tonic contraction of genitourinary smooth muscle. • Prominent effects of α receptor blockers (e.g., prazosin) thus include vasodilation, miosis, and decreased tonic contraction of genitourinary smooth muscle. • Prominent effects of the SNS (NE, EPI, or both) on β receptors at the NEJ include tachycardia, increased force of cardiac contraction, increased AV nodal conduction, and increased renin secretion (all β1 by NE or EPI), and vasodilation, bronchodilation, increased hepatic glycogenolysis, and increased skeletal muscle potassium uptake (all β2 by EPI). • Prominent effects of nonselective β blocking drugs (e.g., propranolol) thus include bradycardia, decreased force of cardiac contraction, decreased AV nodal conduction, and decreased renin secretion (all β1 blockade), and vasoconstriction, bronchoconstriction, decreased hepatic glycogenolysis, and decreased skeletal muscle potassium intake (all β2 blockade). • Effects of anticholinesterases (e.g., physostigmine) include those of the PNS on M receptors plus possible ganglionic activation (both branches) plus skeletal muscle tremors. • Anticholinesterases can be used to treat belladonna poisoning or myasthenia gravis, and as miotic agents. • Nicotine and succinylcholine are depolarizing ganglionic blockers at autonomic ganglia and the neuromuscular junction, respectively. • Mecamylamine and d-tubocurarine are nondepolarizing (competitive) blockers at autonomic ganglia and the neuromuscular junction, respectively.

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Color Chart Red

NE, EPI, Beta agonists, indirect sympathomimetics

Pink

Beta antagonists, indirect sympatholytics

Orange

Alpha agonists

Yellow

Alpha antagonists

Green

Dopaminergic agonists

Blue

ACh, Muscarinic agonists

Light Blue

Muscarinic antagonists

Purple

Nicotinic drugs and anticholinesterases

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Glossary Acetylcholine: (ACh). Predominant or exclusive neurotransmitter of sympathetic and parasympathetic preganglionic and parasympathetic postganglionic efferent neurons, and minor neurotransmitter of selected sympathetic postganglionic neurons. Acetylcholinesterase: (AChE). Enzyme in or near the NEJ that breaks down acetylcholine into the biologically inactive products choline and acetate. Adrenergic: Describing the predominant efferent postganglionic neurons of the sympathetic branch of the ANS: Adren-, of the adrenal (EPI or NE); -ergic, pertaining to the predominant or exclusive neurotransmitter released by the neuron. Adrenoceptor: Alpha or beta autonomic receptor, located on membranes of effector cells at the neuroeffector junction (NEJ). Afferent: Direction of nerve transmission (action potential flow) toward the central nervous system (CNS); applicable to sensory neurons of the ANS. Alpha (α) receptor: Type of adrenoceptor on target cells, activated by epinephrine or NE, depending on the subtype. Anhydrosis: Impaired sweating. ANS: Autonomic nervous system. Anticholinergic: Narrowly but inaccurately, the term indicates interference with the parasympathetic nervous system by blocking muscarinic (M) receptors (synonyms: parasympatholytic, anti-M). More accurately, the term indicates interference with the effects of any cholinergic neuron, including parasympathetic pre- and post-ganglionic neurons as well as sympathetic preganglionic neurons, and thus would include both M and nicotinic (N) receptor blockers. Anticholinesterase: Drug that inhibits or inactivates cholinesterases, including acetylcholinesterase, thus increasing the amounts of acetylcholine. Anti-M: Drug that blocks M receptors. Antinicotinic: Drug that blocks nicotinic receptors. Antivagal: Interference with the actions of the vagus nerve. Arteriole: Smallest of arteries (pre-capillary); responsible for vascular resistance and the control of local blood flow or systemic blood pressure. Autonomic: Nervous system that continually regulates the activity of nearly every organ in the body. Autonomic receptor: Proteins found on the surfaces of cell membranes that bind selectively to ANS neurotransmitters. They are located on either postsynaptic neurons in ganglia or effector cells at the neuroeffector junction. AV node: Atrioventricular node; specialized structure between the atria and ventricles of the heart that is responsible for carrying electrical impulses from the atria to the ventricles. Belladonna: Plant source of anti-M alkaloids (literally “beautiful woman”). Beta (β) receptor: Type of adrenoceptor on target cells, activated by EPI or NE, depending on the subtype. Bradycardia: slow heart rate. Bronchiole: Small airway tubes in the lung, surrounded by smooth muscle; responsible for airway resistance. Catecholamine: Chemical class to which the sympathetic neurotransmitters and neurohormones (NE and EPI) belong.

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CHF: Congestive heart failure; the inability of the heart to maintain adequate cardiac output for systemic organ perfusion. Cholinergic: Describing all the predominant efferent neurons of the ANS except the vast majority of sympathetic postganglionic neurons: i.e., sympathetic and parasympathetic preganglionic neurons and postganglionic parasympathetic neurons: Cholin-, of acetylcholine; -ergic, pertaining to the predominant or exclusive neurotransmitter released by the neuron. Cholinoceptor: Autonomic receptor that binds and responds to acetylcholine (ACh) and related drugs; M or N receptors. Chromaffin cells: Cells of the adrenal medulla that secrete the neurohormone EPI and, to a lesser extent, NE, into the bloodstream (Chrom-, dye; -affin, affinity). Circular muscle: smooth muscle group in the eye that regulates the size of the pupil. Upon contraction, it produces miosis. Also called the sphincter muscle; see Radial muscle. Cycloplegia: Paralysis of accommodation in the eye (inability to focus). Effector: The term “effector” is used two ways in this book: 1) Target molecule of receptor-linked signal pathways (Chapters 1 and 2); and 2) Cells or tissues that are innervated and regulated by the autonomic nervous system (also called target cells or tissues), as used in this chapter. Effector cell: Non-neural target cell that is being innervated and influenced by a neurotransmitter released by postganglionic ANS neurons. Efferent: Direction of nerve transmission (action potential flow) away from the central nervous system (CNS) and terminating at the effector cell. Epinephrine (EPI): Predominant neurohormone released by chromaffin cells of the adrenal medulla. Also called adrenaline. Ganglia: Anatomical collections of neuronal synapses. Glycogenolysis: The breakdown of glycogen to glucose, mainly in liver and skeletal muscle. Hyperhydrosis: Excessive sweating. ISA: Intrinsic sympathomimetic activity; applies to partial beta adrenoceptor agonists, or beta blockers with some agonist activity. Ischemia: Diminished blood flow. Juxtaglomerular cells: Specialized cells near the glomeruli in the kidney that secrete the enzyme renin into the blood in response to sympathetic neural and other influences. Lacrimal: Type of gland that produces tears in the eye. Miosis: Constriction of the pupil. Muscarinic (M) receptor: Type of cholinoceptor on target cells, activated by acetylcholine. Mydriasis: Dilation of the pupil. NEJ: Neuroeffector junction; the interface between a postganglionic ANS neuron and a non-neural effector (target) cell. Neurohormone: Chemical released by specialized cells, upon stimulation by neurons, into the bloodstream to activate receptors on target cells; see Epinephrine. Neurotransmitter: Chemical released by neurons close to target cells that activates selective receptors on those cells; see Norepinephrine, Acetylcholine. Nicotinic (N) receptor: Type of cholinoceptor on neurons (Nn) or muscle (Nm) activated by ACh. Nonselective antagonist: Blocking agents with similar affinities for multiple receptor subtypes within a class. Norepinephrine (NE): Predominant neurotransmitter of sympathetic postganglionic efferent neurons of the ANS and a minor neurohormone secreted, with epinephrine, by chromaffin cells of the adrenal medulla. Also called noradrenaline.

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Organophosphate: One type of anticholinesterase (e.g., organophosphate insecticides) that inhibits the cholinesterase enzyme by phosphorylating it. Parasympathetic: Branch of the autonomic nervous system that emanates from the base of the brain and upper levels of the spinal cord (cranio) and the lower end of the spinal cord (sacral). Parasympathomimetic: Agonist drug that imitates effects of the parasympathetic nervous system and thus acts like ACh (also called cholinomimetics or cholinergic agonists). Pheochromocytoma: Tumor of the adrenal medulla resulting in very high blood levels of catecholamines including EPI and NE; produces severely high blood pressure. PNS: Parasympathetic nervous system. Preganglionic: Pertaining to ANS neurons that originate in the CNS and terminate in autonomic ganglia. Prejunctional: Pertaining to the end of a somatomotor neuron or an autonomic postganglionic neuron where it impinges on the effector cell at the neuroeffector junction. Presynaptic: Pertains to neurons that terminate at synapses and interact with postsynaptic neurons. Postganglionic: Pertaining to ANS neurons that originate in autonomic ganglia and terminate at the neuroeffector junction; also called “postsynaptic.” Postjunctional: Pertaining to the region of the effector cell that is innervated by a prejunctional neuron. Postsynaptic: Pertaining to neurons that are innervated by presynaptic neurons at synapses. Radial muscle: In the eye, the muscle group that radiates outward from the pupil, so that it produces mydriasis upon contraction. Also called dilator muscle; see Circular muscle. Renin: Enzyme released by the kidney into the bloodstream that converts angiotensinogen (renin substrate) to angiotensin I; involved in responses to hemorrhage, altered sodium balance, stress, and other adverse hemodynamic conditions. SA node: Sinoatrial node; specialized structure in the right atrium of the heart that is the major pacemaker, determining heart rate. Selective antagonist: Blocking agent that has a higher affinity for one receptor subtype over another within a class. SNS: Sympathetic nervous system. Sphincter muscle: In the eye, the muscle that surrounds the pupil (also called circular muscle) that, when it contracts, produces miosis. Sympathetic: Branch of the autonomic nervous system that emanates from the middle (thoracolumbar) regions of the spinal cord. Sympathoadrenal: The sympathetic branch of the ANS including innervations to the adrenal medulla and release of (predominantly) EPI. Sympathomimetic: Drug that imitates effects of the sympathetic nervous system and thus acts like either NE or EPI (also called adrenergic agonist). Sympatholytic agent: A drug that inhibits effects of the sympathetic nervous system; includes alpha blockers, beta blockers, and, technically, ganglionic blockers. Synapse: A connection, or small gap, between two neurons, where neurotransmitters are released by presynaptic neurons to exert their effects on postsynaptic neurons. Tachycardia: abnormally rapid heart rate. Vasoconstriction: Constriction of blood vessels (contraction of their smooth muscle). Vasodilation: Dilation of blood vessels (relaxation of their smooth muscle). Xerostomia: Dry mouth.

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