Pharmacokinetics of Oral Contraceptive Steroids and Drug Interaction: Salzburg, Austria, September 14-16, 1989

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Decemb r 1 990 in two p11,11,, ptut 2 volume 163, numbt•r 6

SUPPLEMENT TO

� OBSTETRICS AND GYNECOLOGY Copyright© 1990 by Mo,by-Year Book, Inc.

PHARMACOKINETICS OF ORAL CONTRACEPTIVE STEROIDS AND DRUG INTERACTION Salzburg, Austria September 14-16, 1989 Editor

Prof. Dr. phil. nat. Herbert Kuhl Department of Obstetrics and Gynecology J. W. Goethe University FranHurt am Main, Germany

PubU,hed by MOSBY-YEAR BOOK, INC.

St. Louis, Missouri 63146-3318

ISSN OOM-9378

American Journal

oJOBSTETRICS AND GYNECOLOGY Copyright © 1990 by Mosby-Ye11r Book, J11c.

PHARMACOKINETICS OF ORAL CONTRACEPTIVE STEROIDS AND DRUG INTERACTION Salzburg, Austria September 14-16, 1989

Editor Prof. Dr. phil. oat. Herbert Kuhl Department of Obstetrics and Gynecology J. W. Goethe University Frankfurt am Main, Germany

~T..41 Mosby rl n Year Book

American Journal of Obstetrics

and Gynecology Founded in I 920

Copyright© 1990 by Mosby-Year Book, Inc.

December Part 2 1990

Introduction

2113

Prof. Dr. Herbert Kuhl, PhD, Guest Editor

SESSION I. PHARMACOKINETICS OF ORAL CONTRACEPTIVE STEROIDS Chairman: K. Fotherby Pharmacokinetics of ethinyl estradiol and mestranol

2114

Joseph W. Goldzieher, MD, and Steven A. Brody, MD Houston, Texas Interindividual and intraindividual variations as well as ethnic factors affect the pharmacokinetics of ethinyl estrogens.

Pharmacokinetics of the contraceptive steroids levonorgestrel and gestodene after single and multiple oral administration to women

2120

Wilhelm Kuhnz, PhD Berlin, West Germany During the long-term administration of the synthetic progestins levonorgestrel and gestodene, the observed steady-state concentrations in serum were higher than anticipated from single-dose pharmacokinetics.

Chairman: J. W. Goldzieher Pharmacologic and pharmacokinetic characteristics of norgestimate and its metabolites

2127

John L. McGuire, PhD, Audrey Phillips, PhD, DoWon Hahn, PhD, Edward L. Tolman, PhD, Soledad Flor, PhD, and Michael Edwin Karfrissen, MD, MSPH Raritan, Ne·rv Jersey Data suggest that 17-deacetyl norgestimate contributes to the pharmacology of its parent compound, norgestimate.

(Contents continued on page 4A) Vol. 163, No. 6, Part 2, December 1990. The American Journal of Obstetrics and Gynecology (ISSN 0002-9378) is published monthly (six issues per volume, two volumes per year) by Mosby-Year Book, Inc., 11830 Westline Industrial Drive, St. Louis, Missouri 63146-3318. Second-class postage paid at St. Louis, Missouri, and additional mailing offices. POSTMASTER: Send change of address to American Journal of Obstetrics and Gynecology, Mosby-Year Book, Inc., 11830 Westline Industrial Drive, St. Louis, Missouri 63146-3318, (314) 872-8370, ext. 4351. Annual subscription rates for 1991: domestic, $86.00 for individuals and $150.00 for institutions. Printed in the U.S.A. Copyright © 1990 by Mosby-Year Book, Inc.

3A

Contents

continued from page 3A 2132

Serum pharmacokinetics of orally administered desogestrel and binding of contraceptive progestogens to sex hormone-binding globulin Willem Bergink, PhD, Roel Assendorp, MSc, Lenus Kloosterboer, PhD, Wim van Lier, MSc, Gerrit Voortman, MSc, and lngelise Qvist, MD Oss. The Netherlands, and Horsens. Denmark The pharmacokinetics of desogestrel (0.150 mg) and ethinyl estradiol (0.030 mg) and t he binding affinities of progestogens derived from norethisterone for sex hormone-binding globulin were estimated.

SESSION II. GASTROINTESTINAL METABOLISM OF ORAL CONTRACEPTIVE STEROIDS

Chairman: J. Hammerstein Gastrointestinal metabolism of contraceptive steroids

2138

David J. Back, PhD, Steven Madden, BSc, and Michael L'E. Orme, MD Liverpool, England The article summarizes both in vitro and in vivo studies on gut wall metabolism of the contraceptive steroids ethinyl estradiol, desogestrel, and norgestimate.

Factors affecting the enterohepatic circulation of oral contraceptive steroids

2146

Michael L'E. Orme, MD, and David J. Back, PhD Liverpool, England The enterohepatic circulation of contraceptive steroids applies only to the estrogens, particularly ethinyl estradiol, and clinical and laboratory studies have not fou nd convi ncing evidence of its importance.

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4A

Contents SESSION III. HEPATIC METABOLISM OF ORAL CONTRACEPTIVE STEROIDS Chairman: M. Orme Interactions with oral contraceptives

2153

Kenneth Fotherby, PhD London, England The many factors that may affect the pharmacokinetics and pharmacodynamics of oral contraceptives and the interaction of the estrogen and gestagen components are considered.

Inhibition of oral contraceptive steroid-metabolizing enzymes by steroids and drugs

2159

F. Peter Guengerich, PhD Nashville, Tennessee The major oxidative pathway of l 7a-ethinyl estradiol metabolism, 2-hydroxylation, is inhibited by several chemicals; gestodene is an inactivator in the human liver.

Formation, metabolism, and physiologic importance of catecholestrogens

2163

Peter Ball, Prof. Dr. med, and Rudolf Knuppen, Prof. Dr. Rer. Nat. Lubeck, West Germany Aromatic orthohydroxylation of primary estrogens, such as estradiol and ethinyl estradiol, is an important reaction in mammals.

SESSION IV. HORMONAL EFFECTIVENESS OF ORAL CONTRACEPTIVE STEROIDS Chairman: M. Breckwoldt Binding of oral contraceptive progestogens to serum proteins and cytoplasmic receptor

2171

Michael Juchem and Kunhard Pollow, MD, PhD Mainz , West Germany Progestogens that are used in oral contraceptives are characterized at the level of receptors as well as at the level of high-affinity steroid-binding serum proteins.

(Contents continued on page 6A)

5A

Contents

continued from page SA

Pharmacokinetics and pharmacodynamics of oral contraceptive steroids: Factors Influencing the steroid metabolism

2183

Claudia Jung-Hoffmann, MD, and Prof. Dr. Herbert Kuhl, PhD Frankfurt am Main, Federal Republic of Germany The pharmacokinetics of contraceptive steroids during treatment with low-dose oral contraceptives indicate that ethinyl estradiol and nortestosterone derivatives may inhibit steroid metabolism.

2198

Prodrugs: Advantage or disadvantage? Jiirgen Hammerstein, MD Berlin, West Germany The use of hormonal prodrugs instead of drugs results in minor pharmacokinetic, pharmacodynamic, and potency changes with limited clinical impact, if any.

SESSION V. CLINICAL IMPLICATIONS Chairman: H. D. Taubert Gastrointestinal disease and oral contraception

2204

Jurgen P. Hanker, MD Munst~r. West Germany Gastrointestinal disease may impair contraceptive protection by reducing the bioavailability of oral contraceptive steroids.

Influence of oral contraceptives on drug therapy

2208

Alexander T . Teichmann, Prof. Dr. med. Gottingen, West Germany Oral contraceptives can modify pharmacokinetics of various drugs mainly by inhibition of oxidative metabolism and stimulation of conjugation with acids and may also interfere with pharmacodynamics.

2213

Oral contraception in disease states M. Breckwoldt, P. Wieacker, and F. Geisthovel Freiburg im Breisgau, Germany Absolute and relative contraindications to the use of oral contraceptives are p;-esented and discussed, with special reference to long-term diseases.

2216

Conclusion Prof. Dr. Herbert Kuhl, PhD Frankfurt am Main , Federal Republic of Germany

6A

American Journal of Obstetrics

and Gynecology Founded in 1920

volume 163

number 6 part 2

DECEMBER

1990

Introduction The purpose of an oral contraceptive is to inhibit ovulation reliably, to maintain good cycle control, and to cause minimal adverse effects. The response of individual women to a given formulation differs to a large degree. Although the pill producers and the medical boards and authorities are, in the first place, interested in average figures of efficacy, adverse effects, and mortality, women who take the pill and the practitioner who prescribes it, are faced with individual susceptibilities, responses, and side effects. For the past 30 years ago, choosing the most suitable formulation has remained for each woman a matter of experience. Undoubtedly all formulations, including low-dose preparations, are overdosed for most women, because they must suppress ovulation in all women. The pill is a combination of two hormones that, as far as the dose is concerned, belong to the most potent drugs known. Although it has a large therapeutic margin and serious adverse effects are rare, liver damage, stroke, or thromboembolic disease during intake of an oral contraceptive is a vital question for the individual. Any attempt to individualize the prescription of oral contraceptives on a scientific basis is dependent on the pharmacokinetics of the estrogen and progestogen components, which show large variations from woman to woman and from day to day. Therefore one of the most important aims of research must be to investigate the factors that influence the pharmacokinetic properties of contraceptive steroids. The history of the pill since the days of Pincus is characterized by not only empiricism but also by errors. The suspicion that the estrogen component is involved in the etiology of cardiovascular diseases has led to a continuous reduction in the dose, which was hampered from time to time by the assumption that there must be a threshold dose of ethinyl estradiol with respect to good cycle control (e.g., 50 J.Lg 20 years ago or 30 J.Lg today). In both cases reality proved it to be wrong, provided that the progestogen was chosen properly. We are still waiting for a new estrogen that has no toxic effects on the liver. The development of the progestogens was decisively influenced by the "beagle story," when derivatives of progesterone were unfoundedly 6/0/23620

suspected of causing breast cancer. Although the hepatic effects of this type of progestogen are admittedly much less than those of ethinylated nortestosterone derivatives, new courses were set for the development of the so-called "gonanes." By the same measure, as potency was increased, the dose was reduced, which was claimed to be progress because of a pretendedly less burden of hepatic metabolism. However, a comparison of the pharmacokinetics would easily demonstrate that this is very questionable. The gonanes are orally more potent because their elimination is slowed down, and the serum concentrations of new and very low-dose progestogens may reach very high values. There are some indications that the slow inactivation rate of nortestosterone derivatives is not primarily the result of a steric hindrance of enzymatic action by the ethinyl group, but rather of a direct inhibition of enzymes by the chemically activated ethinyl group. Even though there is no doubt that contraceptive steroids in the blood are of much higher importance for the assessment of the effects and risks than the dose ingested, pharmacokinetics are of limited interest for most gynecologists and endocrinologists. One of the aims of the symposium on "Pharmacokinetics of Oral Contraceptive Steroids and Drug Interactions" was to focus attention on this topic. More than 10 years have passed since a similar workshop conference was held in Igls, Austria, in 1978, and among the 18 experts assembling in Salzburg on September 14 to 16, 1989, some participated in the meeting held 1 decade ago. Obviously, the questions and problems discussed in Salzburg in 1989 have not changed since 1978. As with the workshop conference in Igls, Austria, in 1978, the symposium held in Salzburg in 1989 was sponsored by Organon, Munich. It was because of Mrs. Franz and Dr. Geissler (Organon GmbH, Munich) that the organizational setting and the preconditions for a successful meeting have been optimal. Moreover, the generous support from Organon GmbH (Munich) and Organon International bv (Oss, Netherlands) enabled the publication of all the papers presented at the symposium in this supplement to the AMERICAN JOURNAL OF OBSTETRICS Al\'D GYNECOLOGY. Prof Dr. Herbert Kuhl, PhD Guest Editor 2113

SESSION I. PHARMACOKINETICS OF ORAL CONTRACEPTIVE STEROIDS Chairman: K. Fotherby

Pharmacokinetics of ethinyl estradiol and mestranol Joseph W. Goldzieher, MD, and Steven A. Brody, MD Houston, Texas Pharmacokinetally, a 50 f,Lg oral dose of mestranol (which itself is inactive) is bioequivalent to a 35 f,Lg dose of ethinyl estradiol. Physiologically, mestranol ranges from 50% to 100% of the activity of ethinyl estradiol, depending on the endpoint chosen. Compounds such as these, which are metabolized with a first-pass effect and are enterohepatically recirculated, demonstrate large interindividual and intraindividual variability in their pharmacokinetics. Thus a given dose of ethinyl estradiol in one person may produce an effect equivalent to a substantially larger (or smaller) dose in another person. This wide variability confounds efforts to establish tight dose-response relationships, a pOint rarely considered in clinical or epidemiologic studies of these compounds. The circulating levels of ethinyl estradiol sulfates may be higher than those of free ethinyl estradiol itself. It has been thought that these sulfates represent a "reservoir" of ethinyl estradiol. Our studies show that this idea is untenable because the half-life of the sulfates is not long enough for such an effect. Differences in the pharmacokinetics of ethinyl estradiol and mestranol have been observed in studies of various populations. The reality of these group differences is affirmed by analyses of urinary metabolite patterns. (AM J OSSTET GVNECOL 1990;163:2114-9.)

Key words: Ethinyl estradiol, mestranol, pharmacokinetics All oral contraceptives currently in use rely on ethinyl estrogens for their estrogenic component. The reason for this is the greatly increased pituitary-inhibiting activity relative to other estrogenic effects, that is imparted by the ethinyl group. In addition, studies have shown that the ethinyl estrogens act synergistically with the 19-nor progestins with respect to gonadotropin suppression. Whether a synergism of non-ethinyl estrogens with these progestins exists is uncertain. This amplification is the basis on which the contraceptive effectiveness of the very low-dose formulations rests, and the reason why the dosage in the original "sequential" regimens could not be lowered to currently desired levels. Some of the adverse symptomatic effects of the estrogens are clearly dose related. Therefore it becomes important to have an in-depth understanding of their pharmacokinetics.

Mestranol In vitro receptor studies l have demonstrated that mestranol is an inactive compound that becomes biologically active on conversion to ethinyl estradiol (EE). The degree of conversion varies with different species. From the Department of Obstetrics and Gynecology, Baylor College of Medicine. Reprint requests: Joseph W. Goldzieher, MD, Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. 6/0/23775

2114

Published human studies of the pharmacokinetics of EE derived from mestranol generally suffer from the small number of subjects used (usually < 10) and from insufficient sampling frequency. Thus the general impression of a 50% conversion rate of mestranol to EE is based on rather uncertain grounds. In view of what is known today about the magnitude of interindividual and intraindividual variation with these compounds, a reinvestigation is clearly needed. We 2 have recently examined the pharmacokinetic parameters derived from analysis of plasma EE levels after administration of a single dose of three bioequivalent norethindrone (1 mg)/EE (35 fLg) formulations from different manufacturers, as well as three norethindrone (1 mg)/mestranol (50 fLg) formulations from the same manufacturers. The protocol was designed as an open-label, three-way crossover study. Each subject received a two-tablet dose of a drug within the first 6 days of three consecutive menstrual cycles. This double dose was used to improve assay reliability. The drugs were assigned in a randomized sequence so that all three drugs were tested in each subject at a giveh estrogen dose. Twenty-four women took each of these EE-35 formulations; another 27 took the three mestranol-50 formulations. Some of the derived phar~ macokinetic parameters are shown in Table I. The data clearly show the delay in achieving maximum plasma EE levels derived from mestranol compared with EE as expected. Interestingly, the peak plasma concentration (e max ) values for EE derived from

Pharmacokinetics of ethinyl estrogens

Volume 163 Number 6, Part 2

2115

Table I. Pharmacokinetic parameters for EE after oral administraion of single two-tablet doses of each oral contraceptive Formulation Variable

Dose

AVC (pg-hr/ml)

1I35EE 1/50ME 1I35EE 1/50ME 1/35EE ]/50ME

Cmax.

(pg/ml) Tmax

(hr)

Kel

I

1I35EE ]/50ME

1089 996 171 175 1.3

± ± ± ± ± 1.7 ±

OrthoNovum

I

NOYr£pt 570 454 57 57 0.5 0.7 P = 0.008 0.08 ± 0.07 0.13 ± 0.10

993 940 178 175 1.3 1.8

± ± ± ± ± ±

P=

0.09 ± 0.18 ±

P=

270 494 45 69 0.6 0.8 0.02 0.07 0.13 0.04

I

Norinyl 1024 983 174 175 1.4 2.0

± ± ± ± ± ±

454 429 59 50 0.6 0.8 P = 0.003 0.11 ± 0.07 0.13 ± 0.10

Total 1036 963 174 175 1.3 1.9

± ± ± ± ± ±

483* 544t 67 72 0.5 0.8 P = < 0.0001 0.09 ± 0.09 0.15 ± 0.12

Reproduced with permission from Brody]A, Turkes A, Goldzieher ]W. Contraception 1989;40:269-84. Values shown are mean ± SD for area under the plasma concentration/time curve (AVC o.,4) by the trapezoidal rule; peak plasma concentration (C max ); time to peak concentration (T m ,,); and the elimination rate constant (1(,,1). *n = 24 x three trials. tn = 27 x three trials.

Table II. Interindividual variability of EE plasma concentrations after administration of two 35 Il-g EE and two 50 Il-g mestranol pills Parameter EE (35 fLg EE pills) AVC o/24 (pg-hr/ml) C max (pg/ml) EE from mestranol (50 fLg mestranol pills) AVC o.2 • (pg-hr/ml) Cm" (pg/ml)

Mean ± SD

Range

CV (%)

1036 ± 483 174 ± 67

284-2498 55-311

47 39

963 ± 544 175 ± 72

215-2122 67-391

57 41

Data from Brody]A, Turkes A, Goldzieher ]W. Contraception 1989;40:269-84. CV, Coefficient variation.

Table III. Range of pharmacokinetic values for EE reported in the literature tl;2 (at) tl;, ([3) tl;2 (Ka) Bioavailability K!2 K2! KIO Tmax

0.5-2.4 hr 13.1-27.0 hr 0.2-0.4 hr 0.38-0.48 0.182-0.249/hr 0.101-0.245/hr 0.193-0.309/hr 1-2 hr

Estimate of tl;, (u) has a wide range and needs further documentation. Ranges of values indicated are derived from Newburger and Goldzieher.!6 tl;2 (u) is the half-life of drug disposition from the central to the peripheral compartment. tl;2 ([3) is the elimination half-life. tl;2 (Ka) is the half-life of drug delivery to the central compartment, where Ka is the transfer rate constant in this direction. K!2 is the transfer rate constant from the central to the peripheral compartment; K2! is the transfer rate constant in the reverse direction. KIO is the same as the elimination rate constant (KeI)' Bioavailability is calculated by dividing AVC after oral administration by AVC after intravenous administration with the same dose of drug. T m.x is the time of maximum plasma concentration of drug. The theoretic model for these pharmacokinetic parameters is described in Goldzieher et al. 1I

2116

Goldzieher and Brody

December 1990

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Fig. 1. Plasma EE contrations after administration of N orceptE1/35. A single dose of two tablets consisting of EE 35 fJ.g/norethindrone I mg was given by mouth at time = O. The mean ± SD is depicted along with highest and lowest individual responses. (Reproduced with permission from Brody SA, Turkes A, Goldzieher jW. Contraception 1989;40:26984.)

50 /Lg of mestranol are identical with those derived from 35 /Lg of EE, both of which were given along with 1 mg of norethindrone. The mean area under the plasma concentration/time curve from 0 to 24 hours (AUC n. e,) values for EE derived from 50 /Lg of mestranol are clearly lower than those derived from 35 /Lg of EE as shown, but the very large intersubject variability keeps this difference from being statistically significant. From these data it may be concluded that EE from mestranol would yield about 70% of the C max value of a similar dose of EE. Based on AUC o. e4 , mestranol is somewhat less than 70% as bioavailable as EE. It appears that the usual estimate of 50% is probably low in terms of AUC, The interindividual variation of plasma EE levels derived from EE or mestranol was similar; the coefficient of variation of AUC for EE was 47%; that for EE from mestranol was 57%. The coefficient of variation of C max for EE was 39%; for EE derived from mestranol it was 41 % (Table II), These pharmacokinetic data are not necessarily concordant with pharmacodynamic observations. Studies of human endometrial response and measurements of hepatic synthesis of certain proteins suggest a potency of about 50% for mestranol compared with EE."·' However, other studies of human endometrial response have found that the two compounds are bioequivalent over the range of 50 to 100 /Lg/day. Furthermore, studies of the effects on plasma gonadotropins, cortisol, testosterone, and androstenedione and their binding globulins and the effects on carbohydrate and lipid metabolism also found bioequivalence. These studies were conducted with estrogens alone rather than with commercial oral contraceptive formulations. It must be added, however, that these studies were not carried out on the numbers of patients we require in light of con-

EE (35 fJ.g EE pills) EE from mestranol (50 fJ.g mestranol pills)

(%J

41 42

Range*

-66%-+71% -70%-+92%

Data from Brody .lA, Turkes A, Goldzieher .JW. Contraception 1989;40:269-84. CV, Coefficient of variation. *The percent difference of high and low values from the mean of three trials per subject.

temporary knowledge of interindividual variability.n.1O These findings are highly relevant to epidemiologic studies, which frequently analyze data sets in terms of the quantity of estrogen in contraceptive formulations without regard to the chemical nature of the estrogen. Thus some "high-dose" mestranol pills have a lower biologically active estrogen content than do some "lowdose" EE pills. 2 EE

The literature on the pharmacokinetics of EE is substantial. There are large discrepancies in the values reported for the various parameters. These are probably because of limitations in sampling frequency, difficulties in measuring of plasma EE levels at clinically relevant dosages, and the effects of enterohepatic recirculation. The large interindividual variation seen with these drugs is a major confounding factor, accounting for the discrepant findings reported. Analysis of these difficulties has been presented elsewhere." Our best estimate of the relevant pharmacokinetic parameters is shown in Table III. As Table III indicates, the half-lives for EE (tI;2-J 0-

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Fig. 2. Pattern of reference EE and non-ethinyl estrogens on a high-performance liquid chromatography. Chromegaprep Diol column, 9.6 mm by 50 cm, 10 f1; flow-7.5 mUmin; pressure, 1000 psi; linear gradient, 2.25 f1 to 15% isopropanol in heptane in 158 minutes; detection by ultraviolet absorption at 280 nm. (Reproduced with permission from Butterworth-Heinemann. Williams MC, Goldzieher, Jw. Steroids 1980;36:255-82.)

Table V. Representative plasma AUC o24 values (pg-hr/ml) for EE and norethindrone after repeated trials in the same persons Ethynyl Estradiol Subject No.

Norcept

I

Ortho-Novum

2 9 12 13 20

1002 252 1892 1563 657

431 499 637 539 1594

104 106 3 16 25

215 1355 768 1033 433

1230 854 963 839 289

1

Norethindrone Nonnyl

Norcept

I + 35 EE formulations 855 29.2 569 32.2 1390 79.7 98.9 934 2122 90.3 I + 50 ME formulations 658 30.3 1080 175.0 696 109.8 818 58.5 331 68.1

1

Ortho-Novum

I

Nonnyl

23.1 55.7 77.2 169.9 86.3

49.3 46.8 91.7 127.2 88.0

73.7 33.9 86.6 38.4 62.1

120.5 15.6 31.6 109.0 62.5

Reproduced with permission from Brody JA, Turkes A, Goldzieher JW. Contraception 1989;40:269-84.

both the 35 f.Lg EE and the 50 f.Lg mestranol preparations, there was a large intraindividual coefficient of variation: 41% to 42%. Within individual subjects the range of AUC varied enormously, from -70% to + 92% of the mean of the three trials. Typical examples of this variability are shown in Table V. The unexpectedly large intraindividual variance, which showed

no product-specific tendency, is apparent from the table. Moreover, the rank order of values for AUC r[ in the three trials per individual does not correlate with the rank order of the three results for AUC"n (norethindrone), a point that has already been made by Fotherby.13 A prevailing opinion has been that the conjugates of

2118

Goldzieher and Brody

December 1990 Am J Obstel Gynecol

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FRACTION NUMBER

Fig. 3. Representative patterns of urinary mestranol and EE metabolites in women from Sri Lanka, Nigeria and the United States. High-performance liquid chromatography Chromegaprep Diol column, 9.6 mm by 50 cm; 10 flo; flow-7.5 mllmin; pressure, 1000 psi; linear gradient, 2.25% to 15% isopropanol in heptane in 158 minutes, scintillation counting of I-minute fractions. Results from the two U.S. women illustrate the wide range of oxidative metabolism seen in this population. (Reproduced with permission from Butterworth-Heinemann. Williams MC, Goldzieher ]W. Steroids 1980;36:255-82.)

EE, specifically the 3-sulfates, have relatively long halflives and through enterohepatic recirculation might be deconjugated to provide a slow-release reservoir of EE. We have synthesized the various sulfates and have carried out clinical trials with these substances. A detailed analysis of the pharmacokinetic parameters has been published. 14 In summary, only 3.4% of intravenously administered and 11.4% of orally administered 17sulfate appeared in the blood as free EE; with the 3sulfate the conversion rates were 13.7% and 20.7%, respectively, which suggests that the sulfates are not important reservoirs. Moreover, the half-lives of free and sulfoconjugated EE are similar, ranging from 8.8 to 11.2 hours. Finally, the tII2 of the 17-sulfates after intravenous administration was similar to that of free EE. As expected, much more individual variation was encountered with oral administration than with the intravenous route. This phenomenon was apparent for both the 3- and 17 -sulfates, as reflected in measurement of the total circulating EE sulfates.

Ethnic differences

Several investigators 15- l7 have studied the pharmacokinetics of contraceptive steroids in various populations. Substantial differences in bioavailability have been noted. In comtemporary perspectives, the sample sizes studies and the blood sampling schedules used raise questions about the impact of interindividual variability as a possible confounding factor in the interpretation of these data. Although evidence of quantitative differences between populations is perhaps now less certain than was formerly thought, qualitative studies unequivocally support the conclusion that these population differences are real. In published studies l8 we have analyzed the pattern of urinary conjugates (glucuronides and sulfates) of EE after oral administration of radioactively labeled material to women in the United States, Sri Lanka, and Nigeria. In the three populations studied, the proportions of glucuronides and sulfoconjugates were similar: about 70% and 18%, respectively. However, the patterns of glucuronide con-

Pharmacokinetics of ethinyl estrogens

Volume 163 Number 6, Part 2

jugates seemed to differ. Further evidence of metabolic differences between populations can be demonstrated by examination of the oxidative metabolites of EE itself. High-performance liquid chromatography profiling permits high-resolution identification of the various metabolites of EE (Fig. 2). There was a consistent difference, with Nigerian women (n = 10) displaying the least, Sri Lanka women (n = 10) intermediate, and American women (n = 6) the highest degree of oxidative metabolism, as evidenced by multiple chromatographic peaks identifying distinct metabolic products (Fig. 3). Such findings rule out the possibility that the observed pharmacokinetic differences between ethnic groups were from the confounding effects of interindividual variation. More important, they raise interesting questions regarding ethnic differences in the enterohepatic metabolism of EE. Further studies will be needed to address the role of nutritional factors, intestinal bacterial flora, genetic steroid-metabolic differences, and other as yet undiscovered influences on the metabolism of these compounds. REFERENCES I. Kappus H, Bolt HM, Remmer H. Affinity of ethynylestradiol and mestranol for the uterine estrogen receptor and for the microsomal mixed function oxidase of the liver.] Steroid Biochem 1973;4:121-8. 2. Brody SA, Turkes A, Goldzieher ]W. Pharmacokinetics of three bioequivalent norethindrone I mestranol-50f.\-g and three norethindrone/ethynyl estradiol-35f.\-g OC formulations: are "low-dose" pills really lower? Contraception 1989;40:269-84. 3. Delforge ]P, FerinJ. A histometric study of two estrogens: ethinylestradiol and its 3-methyl-ester derivative (mestranol); their comparative effect upon the growth of the human endometrium. Contraception 1970;1:57-63. 4. Teter ], Stupnicki R. A comparative study of the estrogenic potential of two synthetic estrogens (mestranol and ethinylestradiol). Acta Cytol 1971; 15: 167-70. 5. Goldzieher ]W, Maqueo M, Chenault CB, Woutersz TB. Comparative studies of the ethynyl estrogens used in oral contraceptives. I. Endometrial response. AM] OBSTET GvNECOL 1975; 122:615-8. 6. Goldzieher ]W, de la Pena A, Chenault CB, Cervantes A.

7.

8.

9.

10.

II. 12.

13. 14. 15. 16. 17. 18.

2119

Comparative studies of the ethynyl estrogens used in oral contraceptives. III. Effect on plasma gonadotropins. AM ] OB5TE1' GVNECOL 1975;122:625-36. Goldzieher ]W, Chenault CB, de la Pena A, Dozier TS, Kraemer DC. Comparative studies of the ethynyl estrogens used in oral contraceptives: effects with and without progestational agents on plasma cortisol and cortisol binding in humans, baboons, and beagles. Ferti! Steril 1977;28:1182-90. Goldzieher ]W, Chenault CB, de la Pena A, Dozier TS, Kraemer DC. Comparative studies of the ethynyl estrogens used in oral contraceptives: effects with and without progestational agents on plasma androstenedione, testosterone, and testosterone binding in humans, baboons, and beagles. Fertil Steril 1978;29:388-96. Goldzieher ]W, Chenault CB, de la pena A, Dozier TS, Kraemer DC. Comparative studies of the ethynyl estrogens used in oral contraceptives. VI. Effects with and without progestational agents on carbohydrate metabolism in humans, baboons, and beagles. Fertil Steril 1978;30: 146-53. Goldzieher ]W, Chenault CB, de la Pena A, Dozier TS, Kraemer DC. Comparative studies of ethynyl estrogens used in oral contraceptives. VII. Effects with and without progestational agents on ultracentrifugally fractionated plasma lipoproteins in humans, baboons, and beagles. Fertil Steril 1978;30:522-33. Newburger], Goldzieher ]W. Pharmacokinetics of ethynyl estradiol: a current view. Contraception 1985;32:3344. Back D], Breckenridge AM, Crawford FE, Maciver M, Orme ML'E, Rowe PH. Interindividual variation and drug interactions with hormonal steroid contraceptives. Drugs 1981;21:46-61. Fotherby K. Pharmacokinetics of ethynyloestradiol in humans. Methods Find Exp Clin Pharmacol 1982;4:13141. Goldzieher ]W, Mileikowsky G, Newburger ], Dorantes A, Stavchansky SA. Human pharmacokinetics of ethynyl estradiol3-sulfate and 17-sulfate. Steroids 1988;51 :63-79. Goldzieher ]W, Dozier TS, de la Pena A. Plasma levels and pharmacokinetics of ethynyl estrogens in various populations. I. Ethynylestradiol. Contraception 1980;21:1-16. Fotherby K, Akpoviroro ], Abdel-Rahman HA, et al. Pharmacokinetics of ethynyloestradiol in women from different populations. Contraception 1981 ;23:487-96. Fotherby K. Variability of pharmacokinetic parameters for contraceptive steroids. ] Steroid Biochem 1983; 19:817-20. Williams MC, Goldzieher ]W. Chromatographic patterns of urinary ethynyl estrogen metabolites in various populations. Steroids 1980;36:255-82.

Pharmacokinetics of the contraceptive steroids levonorgestrel and gestodene after single and multiple oral administration to women Wilhelm Kuhnz, PhD Berlin, West Germany Little is known about the pharmacokinetics of the two progestins levonorgestrel and gestodene during long-term administration compared with single-dose pharmacokinetics. The predictive value of single-dose administration for the pharmacokinetic behavior of a progestin during long-term treatment was investigated for two triphasic oral contraceptives. One contained levonorgestrel and the other gestodene, each in combination with ethinyl estradiol. In eight Japanese women who received the levonorgestrel-containing formulation over a treatment cycle, steady-state trough levels of levonorgestrel were higher than those obtained by computer simulation based on single-dose administration. An analogous observation was made in a group of 10 white women who received the gestodene-containing formulation. A close correlation between gestodene and sex hormone-binding globulin concentrations was demonstrated for eight subjects; the other two patients already had initially high sex hormone-binding globulin levels. Ethinyl estradiol-induced production of sex hormone-binding globulin seems to be a major factor that contributes to the accumulation of the two progestins in the plasma. Computer simulation, based on single-dose pharmacokinetics, allows an estimation of this contribution. (AM J OSSTET GVNECOL 1990;163:2120-7.)

Key words: Pharmacokinetics, synthetic progestins, long-term administration, steady-state concentrations, sex hormone-binding globulin Levonorgestrel (LNG) and gestodene (GEST) are two synthetic progestins that are used in combination with ethinyl estradiol (EE t ) as oral contraceptives. Whereas LNG has been used for many years, GEST was introduced only a few years ago, and accordingly, a fairly large number of clinical pharmacokinetic studies have been performed with LNG-containing oral contraceptives, whereas a comparatively smaller number of analogous studies have been reported for GEST. Consequently, the pharmacokinetic characteristics of LNG after single-dose administration (intravenous and oral) are well known, I whereas analogous information on the pharmacokinetics of GEST has become available more recently.t." Compared with single-dose pharmacokinetics, much less published data are available on the administration of both progestins throughout a whole treatment cycle in healthy women."!' 9 Because single-dose pharmacokinetics of a particular progestin hardly reflects the real situation of women taking an oral contraceptive for long durations, usually for many years, at least one important question must be addressed: Is the pharmacokinetic profile of a particular progestin the same after single- and multipleFrom the Research Laboratories, Schering AG. Reprint requests: Wilhelm Kuhnz, PhD, Department of Pharmacokinetics, Schering AG, MullerstraJ3e 170-178, D-JOOO Berlin 65, West Germany. 610123263

2120

dose administration or, in others words, is it possible to simply predict the pharmacokinetic behavior of a progestin during long-term treatment from the characteristics obtained after the administration of a single dose? In this article this issue will be discussed for two triphasic oral contraceptives, one containing LNG and the other GEST, in combination with EE t as the estrogenic component. Material and methods

Study design: Study I. This study was made up of two parts. The first included a group of seven healthy Japanese women between 22 and 34 years old, who received a single-coated tablet of each of the three dosage forms of the triphasic formulation (Triquilar) at the adequate time of the cycle (days 1,7, and 13). Blood samples were taken at the following times: before administration and 0.5, 1,2,3,4,8, and 24 hours after drug intake. The second part of the study included eight healthy Japanese women between 21 and 35 years, who received the triphasic formulation during a whole cycle according to the following schedule: days 1 through 6: 0.05 mg LNG + 0.03 mg EE 2 ; days 7 through 11: 0.075 mg LNG + 0.04 mg EE 2 ; and days 12 through 21: 0.125 mg LNG + 0.03 mg EE 2 • Blood samples were taken during this period at the

Pharm"C'okinetics of levonorgestrel and gestodene

Volume 163 l\ umber 6, Part 2

Table I. Pharmacokinetic parameters (mean values) of LNG obtained from seven Japanese women who received three single-coated tablets of the triphasic oral contraceptive at adequate times during one cycle; parameters were calculated on the basis of an open two-compartment model; concentration values of LNG obtained after single administration of 0.05 mg LNG + 0.03 mg EE, were not suitable for model-fitting and therefore were not included

..

LNG (ng/mll

3 005 mg ______ 0.075 mg

--4--

- 0 . 1 2 5 mg

2

o

o

Dose of LNG (mg) ..

Parameter

0.075

(j.125

em", (ng/ml) t",,, (hr) Aue (0-24 hr) (ng . ml- I . hr) AUe (ng' ml- I . hr) t*" (hr) V" (I) Cl (mi· min' I. kg-I)

1.6 1.0 8.5 11.2 13.1 90.6 2.0

2.6 1.2 17.1 20.0 13.1 85.0 1.9

10 15 time p admln (hi

20

following times: days 2, 4, 6, 8, 10, 12, 16, 18, and 20 before drug administration, and day 21 before drug administration and 0,5, 1, 2, 4, 8, 24, and 48 hours after drug intake. All blood samples were collected in heparinized tubes and centrifuged, and plasma samples were frozen at - 20° C until analysis. Study design: Study II. This study was designed as an open investigation with 10 healthy white women between 23 and 39 years, who received a single-coated tablet of the triphasic preparation (Milvane) that contained 0.1 mg of GEST together with 0.03 mg of EE, on day 21 of a pretreatment cycle. Blood samples were taken at the following times: before administration and 0.5,1,1.5,2,4,6,8,10,12, 24, 48, and 72 hours after drug intake. After a wash-out phase of 7 days, the same women received, in the second part of the study, the triphasic formulation during a whole cycle: days 1 through 6: 0.05 mg GEST + 0.03 mg EE,; days 7 through 11: 0.07 mg GEST + 0.04 mg EE,; and days 12 through 21: 0.1 mg GEST + 0.03 mg EE,. Blood samples were taken at the following times': days 2 and 7: before and 0.5, 1, 1.5,2,4,6,8, and 12 hours after drug administration, and day 21: before and 0.5, 1, 1.5,2,4,6,8, 12,24,48, 72, and 96 hours after drug intake. Additional blood samples were obtained on days 3, 5, 8, 10, 12, 14, 16, 18, and 20 before drug ad-

25

LNG (ng/mll

_

V,\, Apparent volume of distribution at steady state; C/, clearance. *Terminal half-life calculation was based on two concenu'ation values only.

2121

0.125 mg

- - 0075 mg

o

o

15 10 time p. admln (hi

20

25

Fig. 1. Mean concentrations of LNG in plasma of seven Japanese women who received single doses of 0.05, 0.075, and 0.125 mg of LNG as coated tablets of a triphasic oral contraceptive at adequate times (days 1, 7, and 13) of the cycle. Directly obtained LNG concentration/time curves (top) are presented along with curves fitted on the basis of an open twocompartment model (bottom).

ministration. All blood samples were kept at 4° C until coagulation; the serum was separated and stored at - 20° C until analysis. Analytic determinations. LNG was determined in all plasma samples of study I by a specific radioimmunoassay.' The serum samples of study II were analyzed for total concentration of GEST by a specific radioimmunoassay." The free fraction of GEST, as well as the distribution of GEST over the serum-binding proteins albumin and sex hormone-binding globulin (SHBG), was determined by ultrafiltration 7 with and without heat treatment of the serum samples. 8 In study II SHBG and cortisol-binding globulin (CBG) concentrations were measured by radioimmunologic means with two commercially available assay kits (SHBG: Diagnostic Products Corp., Los Angeles, Calif.; CBG: IRE Medgenix, Fleurus, Belgium). Pharmacokinetic evaluation. Plasma concentrations of LNG, which were obtained during study I, were used for the model-free evaluation of maximum drug concentration (c max ), time of maximum concentration (t ma ,), and area under the curve (AUC). In addition, the LNG

2122 Kuhnz

December 1990 Am J Obstet Gynccol

"4

LNG (ng/ml) 0.125 mg

0.075 mg

0.05 mg

LNG

3

2

~ _.-----+-----+---+-+ ----_ ..... ----- ... ----_ ... -----...----- .... -----

1

0

0

2

4

_ _ treatment

6

8

10 12 day of cycle

14

16

18

20

---+--- simulation

Fig. 2. Mean trough concentrations of LNG in plasma of eight Japanese women who received the triphasic oral contraceptive during one cycle. Experimentally measured and computer-simulated values are presented.

concentrations in plasma obtained after single-dose administration were evaluated by model-fitting (TOPFIT, Thomae GmbH, Biberach, West Germany) on the basis of an open two-compartment model. The serum concentrations of GEST obtained in study II were evaluated by model-fitting only. Apparent oral clearances and volumes of distribution were calculated for both progestins assuming 100% bioavailability.2.5 10 The data obtained by model-fitting were used for the simulation of progestin concentrations in plasma (serum) during treatment with the respective triphasic formulations over a whole treatment cycle. Accumulation factors were calculated as follows:

R = ------------- e ( "'" ,) -

R*

I"

C:~in

C~"in

(TC) (SC)

where tV2 = terminal half-life; T = dosing interval; C:~in = minimum concentration at steady state; TC = treatment cycle; and SC = simulated treatment cycle. AUC (0-24 hr) TC AUC sD single dose. R**

where SD

=

Results

Study I: Pharmacokinetics of LNG after single-dose administration to Japanese women. The maximum concentration values of LNG, which were observed in the plasma of the seven women after single oral administration of the three different dosages (0.05, 0.075,

and 0.125 mg of LNG), were 0.72 ± 0.34 ng/ml, 1.83 ± 0.61 ng/ml, and 2.74 ± 0.96 ng/ml, respectively. The corresponding t max values were 2 ± 1 hour, 1.1 ± 0.7 hour, and 1.1 ± 0.6 hour, respectively. The observed AUC (0 to 24 hours) - values were 5.0 ± 4.1 ng*ml-I*hr, 8.8 ± 5.3 ng*ml-I*hr, and 17.1 ± 6.4 ng * ml- ' * hr, respectively. The time course of LNG levels in the plasma is shown in Fig.!. The mean LNG concentration values in plasma obtained after the administration of a single dose of 0.075 and 0.125 mg of LNG, respectively, were fitted on the basis of an open two-compartment model (Fig. 1). The derived pharmacokinetic parameters were identical to those determined without model-fitting (Table I). A computer simulation of LNG trough levels in plasma during a 21-day treatment period was performed on the basis of those pharmacokinetic parameters that were obtained with high-dose LNG (0.125 mg) except that the terminal half-life was set to a value of 22 hours, which represented the experimentally observed value at the end of one treatment cycle in the second part of study 1. The result of this simulation is presented in Fig. 2, along with the experimentally obtained concentration values. The comparison of both curves demonstrated the following: (1) that simulated trough levels of LNG increased during treatment, reaching steady state on day 16 of treatment, and (2) that experimentally measured LNG trough levels closely followed simulated ones; however, they increased more markedly and reached a considerably higher plateau during the last week of treatment. Both curves demonstrate an increase in LNG plasma levels during 1 month of treatment.

Pharmacokinetics of levonorgestrel and gestodene

Volume 163

Number 6, Part 2

T

2123

GEST (ng/ml)

10 0,1 mg

0,07 mg

0,05 mg

8

GEST

6

4

_...... -..... -.-.- .... -.- .... -

2

.... -. - .... -+ - .... -.- .... -.

O,,----r---,----,---,----r---,----,---,----.---.-~

a

2 _

4

treatment

10 12 day of cycle - .... - simulation

6

8

14

16

18

20

Fig. 3. Mean trough concentrations of GEST in the serum of 10 white women who received a GESTcontaining triphasic oral contraceptive during one cycle, Experimentally measured and computersimulated values are presented,

Table II. Pharmacokinetic parameters of LNG (mean ± SD) of eight Japanese women who received the triphasic formulation during one treatment cycle; data were calculated without model-fitting Parameter Cm" (ng/ml)

t mox (hr)

AUC (0-24 hr) (ng , ml- I . hr) AUC (ng' ml- I . hr) t"2 (hr)

Day 21 of treatment 6.2 1.3 80.7 154.2 22.1

± 2.1

± 0.5 ± 34.8

± 69.7 ± 5.7

An accumulation factor (R) of 1.9 can be calculated for LNG from a terminal half-life of 22 hours and a dosing interval of 24 hours. However, this only applies if accumulation is determined only by the terminal halflife of the drug and the dose interval chosen. If this was the case, the ratio (R*) of experimentally determined and simulated steady-state trough levels should be unity. Likewise, the ratio (R**) of Aue obtained on day 21 of the pretreatment cycle and Aue (0 to 24 hours) obtained on day 21 of the treatment cycle should also be unity. However, a value of 2.9 was calculated for the former and a value of 4.0 for the latter ratio. Study I: Pharmacokinetics of LNG after multiple dose administration to Japanese women. Mean trough concentrations of LNG, which were measured during the treatment cycle, reached a steady state on about day 16 (Fig. 2). On the last day of treatment (day 21), maximum concentrations of LNG in the plasma were observed 1.3 ± 0.5 hour after administration and amounted to 6.2 ± 2.1 ng/ml. Postmaximum drug concentrations in plasma de-

Table III. Pharmacokinetic parameters of GEST (mean ± SD) obtained by model-fitting based on total and free concentrations in serum after single oral administration of a coated tablet containing 0.1 mg of GEST together with 0.03 mg of EE2 on day 21 of a pretreatment cycle to 10 women Parameter Cm" (ng/ml) t m " (hr) AUC (0-24 hr) (ng . ml- I . hr) AUC (ng' ml- I . hr) tl12 (hr) Vss (I)

CI (mi· min-I. kg-I)

Total GEST

7.2 1.3 50.6 74.3 15.8 32.2 0.48

3.2 1.1 22.9 37.5 4.9 ± 19.4 ± 0.25

± ± ± ± ±

I

Free GEST 0.09 1.3 0.65 0.97 15.9 2242 34.7

± 0.04

± 1.1 ± 0.25 ± 0.46 ± 5.4 ± 974 ± 15.8

V,S, Apparent volume of distribution at steady state; clearance.

ct,

clined, with a mean half-life of disposition of22.1 ± 5.7 hours. The AUe (0 to 24 hours) was 80.7 ± 34.8 ng * ml- I * hr (Table II). Study II: Pharmacokinetics of GEST after singledose administration to white women. The maximum concentration values of GEST, which were observed in the serum of the 10 women after a single oral administration of 0.1 mg of GEST together with 0.03 mg of EE 2 , were observed after 1.3 ± 1.1 hour and amounted to 7.2 ± 3.2 ng/ml. The terminal half-life of disposition was 15.8 ± 4.9 hours. The AUe was 74.3 ± 37.5 ng * hr * ml- I • Total serum clearance was calculated to 0.48 ± 0.25 ml * min- I * kg- I (Table III). Protein binding of GEST and drug distribution over the binding proteins were determined before drug administration and 1 and 10 hours after drug intake, and they were identical in all cases. The free fraction of GEST

2124 Kuhnz

December 1990 Am J Obstet Gynecol

Table IV. Protein binding of GEST and concentration of SHBG and CBG (mean ± SD) in the serum of 10 women on day 21 of a pretreatment cycle and on day 21 of treatment Day 21 pretreatment cycle

Parameter

GEST Free fraction (%) SHBG-bound (%) Albumin-bound (%) SHBG (nmollL) CBG (l1g/ml)

1.37 66.5 32.2 90.3 37.4

± 0.31 ± 6.9 ± 6.6 ± 36.0 ± 5.2

Day 21 treatment cycle

0.81 76.6 22.6 198.8 84.9

± 0.15

± 5.8 ± 5.8 ± 52.0

± 11.4

Table V. Pharmacokinetic parameters of GEST (mean ± SD) obtained by model-fitting based on total and free concentrations in serum of 10 women (on day 21 of treatment) who received the triphasic preparation during one cycle Total GEST

Parameter

C m " (ng/ml) t max

(hr)

AVC (0-24 hr) (ng . ml- I AVC (ng . ml- I . hr)

t"2 (hr)

•

18.2 0.8 hr) 226.6 422.6 22.0

± 4.5 ± 0.4

I

Free GEST

0.15 0.9 1.8 ± 77.1 ± 178.5 3.5 22.9 ± 4.1

± ± ± ± ±

0.04 0.4 0.6 1.5 5.4

was determined to 1.37% ± 0.31% and the fraction bound to SHBG and albumin was 66.5% ± 6.9% and 32.2% ± 6.6%, respectively. CBG concentrations in the serum were 37.4 ± 5.2 /-lg/ml; SHBG concentrations were 90.3 ± 36.0 nmoliL (Table IV). The free concentrations of GEST were used to calculate the pharmacokinetic parameters of free GEST by model-fitting (Table III). A computer simulation of GEST trough levels in the serum during a complete treatment cycle of 21 days was performed on the basis of the data obtained after single-dose administration on day 21 of the pretreatment cycle (Fig. 3) except that the terminal half-life was set to a value of 22 hours, which represented the experimentally observed value at the end of one treatment cycle in the second part of study II. Both curves showed similar time course except that the experimentally determined trough levels of GEST, which reached a steady state on approximately day 16, increased more markedly and reached a considerably higher plateau in the last week of treatment compared with the simulated trough levels. An accumulation factor (R) of 1.9 can be calculated from a terminal half-life of 22 hours and a dosing interval of 24 hours. Values calculated for R* and R** were 2.3 and 3.4, respectively. Corresponding values of R* and R** for the free GEST were 1.3 and 1.9, respectively.

Study II: Pharmacokinetics of GEST after multipledose administration to white women. Mean trough concentrations of GEST increased during the treatment cycle and reached a steady state on about day 16 (Fig. 3). On the last day of treatment (day 21), maximum concentrations of GEST in the serum were observed 0.8 ± 0.4 hour after administration and amounted to 18.2 ± 4.5 ng/m!. The terminal half-life of disposition was 22.0 ± 4.1 hours. The AVC (0 to 24 hours) was 226.6 ± 77.1 ng * hr * ml- I ; corresponding AVC value was 422.6 ± 178.5 ng * hr * ml- I • The corresponding pharmacokinetic parameters calculated from the free GEST concentrations in serum are also presented (Table V). The free fraction of GEST was determined in the serum samples obtained throughout the treatment cycle. During treatment free GEST fractions decreased from a pretreatment value of 1.37% ± 0.31 % to a value of 0.81% ± 0.15% on day 21 of treatment. The fractions of GEST bound to SHBG and albumin were 76.6% ± 5.8% and 22.6% ± 5.8%. The mean SHBG and CBG levels on day 21 were 198.8 ± 52.0 nmoliL and 84.9 ± 11.4 /-lg/ml, respectively (Table IV). SHBG concentrations showed a steady increase in 8 of 10 women throughout treatment and were correlated significantly with the corresponding GEST concentrations in serum (Fig. 4). In two women relatively high SHBG concentrations were already observed when treatment started, and they remained nearly unchanged throughout treatment. The correlation coefficients for total and free GEST concentrations, respectively, and the corresponding SHBG levels were calculated individually, and for eight women were in the range of 0.7101 and 0.9112 (mean, 0.8195) and 0.5553 and 0.9126 (mean, 0.7914), respectively. In two women there was no correlation between both parameters. Comment

Two different triphasic oral contraceptives were investigated, one containing LNG and the other GEST as progestagenic compound. However, the studies were carried out in two different ethnic populations and with slightly different design, since they were performed independent of each other and not particularly for comparative purposes. Nevertheless, it became obvious that both progestins, when administered in combination with EE2 as triphasic formulations, showed very similar concentration profiles during a treatment cycle. Pharmacokinetic parameters of LNG obtained after the administration of a single oral dose to Japanese women were in good accordance with published data on single dose administration in white subjects, I although somewhat higher drug levels were found in the plasma of Japanese women because of their comparatively lower body weight. Because terminal half-life

Volume 163 Number 6, Part 2

Pharmacokinetics of levonorgestrel and gestodene

T

SHBG (nmol/I)

GEST (ng/ml)

250

T

10

200

150

.-..-

100

,, ,,

.---.-

.- -

-_ .... - - - .... - - - +- - - - .... - - - ... -. --_.- -

8

6

4

2

50

0

2125

0

2

4

6

8

10

12

14

16

18

20

22

0

day of cycle ---- GEST

--+-

SHBG

Fig. 4. Mean serum levels of SHBG and GEST in 10 white women during one treatment cycle.

estimation was based on 8 and 24 hour values only, the half-life of LNG was somewhat shorter than reported previously,5 The pharmacokinetic parameters of LNG obtained with and without the use of model-fitting were identical, thus proving the validity of the model. During 1 month of treatment with the triphasic preparation, plasma trough levels of LNG increased concomitantly with the increasing dose of LNG administered and reached a steady state on about day 16 of treatment. Simulated trough levels of LNG showed a similar time course, although the rise was less steep and the steady state reached was considerably lower compared with experimentally determined values, Computer simulation assumed linear pharmacokinetics of LNG during long-term administration and any deviation of experimentally determined trough levels from the simulated ones indicated additional influences on the drug's disposition, which were due to long-term treatment and did not occur during single-dose administration. At least two different factors, which may affect the drug's distribution and disposition from plasma, have been reported for contraceptive steroids; one is an impairment of hepatic metabolic capacity, whereas the other relates to changes in protein binding. There have been numerous reports on impairment of hepatic drugmetabolizing enzymes in women under oral contraceptive therapy.II-13 This was true for formulations containing LNG and other progestins, and although GEST in particular was not yet investigated, it can be assumed that it also follows the general line. For steroids such as LNG and GEST, which are characterized by a restrictive hepatic elimination, any impairment of the metabolic capacity during long-term treatment could lead to higher drug levels in the plasma than anticipated from single-dose pharmacokinetics. On the other hand, it is well known that concomitant administration of EE2 can increase SHBG concentration throughout treatment, and be-

cause LNG and GEST bind with a high affinity to SHBG, an increase in total drug levels in the serum is the result. Although SHBG was not determined in the present study with Triquilar, most published information on this particular triphasic formulation indicates about a 100% increase in SHBG levels during a treatment cycle,14-16 although a smaller increase has been reported as well. 17 Because computer simulation did not account for any changes in hepatic metabolic capacity or in plasma protein binding during treatment, a comparison of simulated and experimentally obtained trough level curves allowed an estimation of the relative contribution of both factors to the observed increase in LNG concentrations in plasma. Therefore a ratio of 2.9 for the two steady-state concentrations indicated about threefold higher LNG concentrations during treatment than anticipated on the basis of single-dose pharmacokinetics alone. Similarly, when the ratio of the AUC obtained after single-dose administration and the AUC of one dosing interval on day 21 of the treatment cycle was examined, fourfold higher drug levels were found during treatment. Thus the relative contribution of factors other than the relationship of terminal half-life and dosing interval to the total accumulation of LNG in the plasma was about 75%. A very similar observation was made with GEST. Data obtained from single-dose administration to white women were in good agreement with those observed in previous studies (Kuhnz W, et al. Unpublished data). However, compared with a previously performed study on single-dose administration of a GEST-containing formulation, a slightly different protein binding pattern of GEST was observed in the present study. Compared with the former study, in the present study, higher SHBG concentrations and a higher proportion of GEST bound to SHBG were found in the serum,

2126 Kuhnz

and a smaller fraction of GEST unbound was observed. The most likely explanation for this phenomenon is that only women who had not previously taken any oral contraceptives were included in the previously performed study, whereas in the present study most women were regularly taking oral contraceptives and only stopped taking the pill between 2 and 6 months before the study started. The administration of GEST over a whole treatment cycle resulted in increasing trough levels, which reflected the triphasic pattern of the GEST doses administered. The increasing trough levels were paralleled by a 2.2-fold increase in SHBG concentrations in serum, and a close correlation between both parameters was found in 8 of 10 women. However, two women showed no such correlation; they already had high pretreatment levels of SHBG that decreased slightly during treatment, although GEST concentrations increased in a similar way as in the other women. A very similar observation has been reported previously on LNG, IS in which seven of nine subjects had a close positive correlation between SHBG and LNG levels, whereas two other women had no such correlation. Both women already started with high SHBG levels, which decreased during treatment. In the present study a comparison with experimentally observed steady-state concentrations revealed 2.3-fold higher GEST concentrations during treatment than those calculated on the basis of single dose pharmacokinetics. Likewise, an evaluation of the AUCs revealed 3.4fold higher drug levels during treatment. Therefore the relative contribution of serum protein binding and hepatic metabolic impairment to the total concentration of GEST in serum at steady state was about 71 %. When free GEST concentrations were considered in the same way, only 1.3-fold and 1.9-fold higher drug levels were calculated. Although this was still somewhat higher than unity, it clearly demonstrated the influence of protein binding for the observed increase in GEST trough levels in serum. For restrictively cleared drugs such as LNG and GEST, an increase in serum protein binding affects not only volume of distribution and thus drug levels in serum, but also the progestin's terminal half-life. Because of the relative increase in the SHBGbound fraction of the progestin, which is not readily metabolized by the liver, the drug'S terminal half-life will be prolonged, which in fact was observed with GEST. Both increasing progestin levels and prolonged terminal half-lives could also be explained by an impairment of hepatic metabolism. However, where there is evidence for the contribution of protein binding, it remains unclear whether metabolic impairment plays an important role.

December 1990 Am.J Obstet Gyneco1

In conclusion, it became apparent that the pharmacokinetic parameters obtained after single-dose administration of either of the two progestins cannot be used to predict their pharmacokinetic behavior completely during a treatment cycle; the main reason is the continuously altered protein binding of the progestins because of EE2-induced SHBG synthesis. However, single-dose pharmacokinetics of a progestin, together with computer simulation based on pharmacokinetic modeling, can be a useful tool to detect any deviations from linear pharmacokinetics during repeated administration. Along with additional information, for example, on altered binding capacity of SHBG, the interpretation of experimentally obtained results is greatly facilitated.

REFERENCES 1. Orme ML'E, Back D], Breckenridge AM. Clinical pharmacokinetics of oral contraceptive steroids. Clin Pharmacokinet 1983;8:95-136. 2. Tauber U, Tack ]W, Matthes H. Single dose pharmacokinetics of gestodene in women after intravenous and oral administration. Contraception 1989;40:461-79. 3. Diisterberg B, Tack ]W, Krause W, Hiimpel M. Pharmacokinetics and biotransformation of gestodene in man. In: Elstein M, ed. Gestodene. Carnforth, England: Parthenon Publishing, 1987:35-44. 4. Kuhl H, lung-Hoffmann C, Heidt F. Alterations in the serum levels of gestodene and SHBG during 12 cycles of treatment with 30 flog ethinylestradiol and 75 flog gestodene. Contraception 1988;38:477-86. 5. Hiimpel M, Wendt H, Pommerenke G, Weiss C, Speck W. Investigations of pharmacokinetics of levonorgestrel to specific consideration of a possible first pass effect in women. Contraception 1978; 17:207-20. 6. Nieuweboer B, Tack], Tauber U, Hiimpel M, Wendt H. Development and application of a radioimmunoassay of the new progestogen gestodene. Contraception 1989;40: 313-23. 7. Kuhnz W, Pfeffer M, AI-Yacoub G. Protein binding of the contraceptive steroids gestodene, 3-keto-desogestrel and ethinylestradiol in human serum. ] Steroid Biochem 1990;35:313-8. 8. Hammond GL, Lahteenmaki PLA, Lahteenmaki P, Luukkainen T. Distribution and percentages of non-protein bound contraceptive steroids in human serum.] Steroid Biochem 1982;17:375-80. 9. Jenkins N, Limpongsanurak S, Fotherby K. Circulating levels of synthetic steroids in women using a triphasic formulation: a comparison with different ethinylestradiol doses.] Obstet Gynaecol 1981;2:37-40. 10. Back D], Bates M, Breckenridge AM, et al. The pharmacokinetics of levonorgestrel and ethinylestradiol in women-studies with Ovran and Ovranette. Contraception 1981;23:229-39. II. O'Malley K, Stevenson I, Crooks]. Impairment of human drug metabolism by oral contraceptive steroids. Clin Pharmacol Ther 1972;13:552-7. 12. Herz R, Koelz HR, Haemmerli UP, Benes I, Blum AL. Inhibition of hepatic de methylation of aminopyrine by oral contraceptive steroids in humans. Eur] Clin Invest 1978;8:27-30. 13. Tornatore KM, Kanarkowski R, McCarthy TL, Gardner M], Yurchak AM, ]usko WJ. Effect of chronic oral contraceptive steroids on theophylline disposition. Eur] Clin PharmacoI1982;23:129-34.

Pharmacokinetics of levonorgestrel and gestodene

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14. Larsson-Cohn U, Fahraeus L, Wallentin L, Zador G. Ef~ fects of the estrogenicity of levonorgestrelf ethinylestradiol combinations on the lipoprotein status. Acta Obstet Gynecol Scand Suppl 1982;105:37-40. 15. Gaspard UJ, Romus MA, Gillain D, Duvivier J, DemeyPonsart E, Franchimont P. Plasma hormone levels in women receiving new oral contraceptives containing ethinyl estradiol plus levonorgestrel or desogestrel. Contraception 1983;27:577-90. 16. Lepot MR, Gaspard UJ. Metabolic effects of two low-dose

triphasic oral contraceptives containing ethinylestradiol and levonorgestrel or gestodene. lnt J Ferti! 1987;32: 1520. 17. Limpongsanurak S, Fotherby K. Effect of contraceptive steroids on serum levels of sex hormone binding globulin and ceruloplasmin. Curr Med Res Opinion 1981;7:18591.

18. Victor A, Weiner E, Johannson EDB. Relation between sex hormone binding globulin and d-norgestrel levels in plasma. Acta Endocrinol 1977;86:430-6.

Chairman: J. W. Goldzieher

Pharmacologic and pharmacokinetic characteristics of norgestimate and its metabolites John L. McGuire, PhD, Audrey Phillips, PhD, DoWon Hahn, PhD, Edward L. Tolman, PhD, Soledad Flor, PhD, and Michael Edwin Kafrissen, MD, MSPH Raritan, New Jersey Biotransformation, pharmacologic, and pharmacokinetic studies of norgestimate and its metabolites indicate that 17-deacetyl norgestimate, along with the parent drug, contributes to the biologic response. The postulated metabolic pathway, which is based on the identification of urinary products had indicated that three metabolites of norgestimate, 17-deacetyl norgestimate, 3-keto norgestimate, and levonorgestrel, might participate in the response. The pharmacologic evaluation of these metabolites demonstrates that only 17-deacetyl norgestimate has a pharmacologic profile consistent with that of norgestimate, and significant concentrations of this metabolite have been measured in the serum of women after the administration of norgestimate. These studies indicate that 17-deacetyl norgestimate contributes to the pharmacologic response to norgestimate. (AM J OSSTET GVNECOL 1990;163:2127-31.)

Key words: Norgestimate, norgestimate metabolites, 17-deacetyl norgestimate

Norgestimate is a progestin used in combination with ethinyl estradiol as an oral contraceptive. Because some steroids exert their pharmacologic effects through active metabolites, in addition to the parent drug, we have investigated whether the pharmacologic activity of norgestimate (Fig. I) might be mediated, at least in part, through any of its metabolites. Biotransformation studies have been conducted to examine the metabolism of norgestimate; the pharmacologic profiles of three metabolites have been compared with that of norgestimate, and the pharmacokinetics of norgestimate and 17-deacetyl norgestimate, a metabolite believed to con-

From the R. W. Johnson Pharmaceutical Research Institute. Reprint requests: Audrey Phillips, PhD, R. W. Johnson Pharmaceutical Research Institute, Route 202, Box300, Raritan, NJ 08876. 6/0/23622

tribute significantly to the biologic response, have been examined. In this article the results of these studies are reviewed, with emphasis placed on how they contribute to our further understanding of norgestimate's action.

Pharmacokinetics and biotransformation The pharmacokinetics of 14C-norgestimate have been studied in several animal species and appear to be consistent with those of other contraceptive progestins. 1 In rats, dogs, and monkeys, orally administered 14C_ norgestimate is rapidly absorbed, with peak levels of radioactivity achieved within 4 hours of administration, and it is eliminated with a half-life that ranges from 30 to 67 hours. After oral administration to women, 14C_ norgestimate is rapidly absorbed, with peak levels of radioactivity ranging from 30 minutes to 2 hours, and

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18

16

2

o /I

O-C- CH 3

3 4

6

HON Fig. 1. Structure of norgestimate and steroid nucleus.

it is eliminated with a half-life ranging from 45 to 71 hours! The biotransformation of norgestimate in women has been investigated by identifying urinary metabolites after the oral administration of 14C-norgestimate.' As in studies of other steroids, the identification and quantification of terminal norgestimate metabolites represent a sampling, depending on the availability of reference standards. Although these metabolites and their amounts do not necessarily reflect the total metabolite profile or their contribution to the biologic response, the chemical structures of identified urinary metabolites provide a basis to postulate the major metabolic pathways. After extraction and chromatographic separation, five 17a-ethinyl metabolites or norgestimate have been identified by gas chromatography-mass spectrometry and by comparison with several available reference standards.' The chemical structures of these terminal metabolites indicate that in women, norgestimate undergoes metabolism involving hydrolysis at position 17, cleavage of the oxime and subsequent reduction of the ketone at position 3, hydroxylation in the A and D rings, reduction of the double bond at C 4 C,' and subsequent conjugation to a glucuronide or sulfate. Urinary metabolites identified in analogous studies that use 14C-norgestimate in rats, dogs, and monkeys suggest that metabolic pathways in these species are similar to those in women. These studies indicate the possibility that 17 -deacetyl norgestimate, 3keto norgestimate, and 17-deacetyl-3-keto norgestimate (levonorgestrel) might contribute to the pharmacologic response observed after oral administration of norgestimate to women. To examine this possibility further, we have investigated the pharmacologic profiles of these metabolites and have compared them with that of norgestimate.

Pharmacologic evaluation of norgestimate, 17-deacetyl norgestimate, 3-keto norgestimate, and levonorgestrel

Laboratory studies. The pharmacology of norgestimate, which has been studied extensively and reported previously,'·s is summarized here briefly. The affinity of norgestimate for rabbit uterine cytosolic progestin receptors in vitro is similar to that of progesterone. The stability of norgestimate throughout the incubation period in the receptor-binding studies has been monitored, confirming the affinity of unchanged norgestimate for progestin receptors. 6 Additional evidence of norgestimate's inherent progestational activity is provided by experiments that show its local stimulation of the rabbit endometrium when injected directly into the uterine lumen 6 and its ability to directly inhibit luteinizing hormone-releasing hormone-stimulated luteinizing hormone release from rat pituitary cells in vitro." In conventional endocrine bioassays, norgestimate exhibits activity characteristic of a progestin. It stimulates the endometrium after oral administration to estrogen-primed immature rabbits.' In this test system the potency of norgestimate is maintained after subcutaneous administration (0.24 and 0.34 times that of levonorgestrel after oral and subcutaneous administration, respectively) which shows that the progestational action of norgestimate is not dependent on first-pass oral metabolism. Norgestimate, like progesterone, also maintains pregnancy in ovariectomized rats and effectively inhibits ovulation in several species. The relative lack of androgenicity of norgestimate is shown by its very weak affinity for rat prostatic androgen receptors (relative binding affinity of 0.003 times that of dihydrotestosterone), its ability to stimulate ventral prostate growth in immature castrated rats only to the same degree as progesterone (relative potency of

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Norgestimate and its metabolites

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......... Norgestimate (0.01) 100

Ci

,S

0····-0

90

. . . - . . 3-Keto-Norgestimate (0.09)

80

....... Levonorgestrel (0.1)

~ 70

~

a: 0

17-DeacetylatedNorgestimate (0.007)

o--a Testosterone

60

Propionate (1.0)

50

~ E 40 Q)

>

c:

CO

Q)

~

30 20 10

0.0

0.10

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Total Dose, mg

Fig. 2. Stimulation of ventral prostate growth after administration to immature castrated rats. (Relative potency estimates from Phillips et a1. 6 )

0.003 to 0.03 times that of testosterone) ,6 and its virtual lack of affinity for human sex hormone-binding globulin (SHBG) in vitro (inhibiting concentration of 50% [IC oo ] > 10,000 nmoIlL).7 To determine whether 17-deacetyl norgestimate, 3keto-norgestimate, or levonorgestrel contributes to the pharmacologic activity of the parent drug, these steroids were tested in the same assays described above. If any of these metabolites contribute significantly to the pharmacologic activity of norgestimate, their pharmacologic activity profile would be consistent with that observed after the administration of norgestimate. 17-Deacetyl norgestimate, 3-keto norgestimate, and levonorgestrel demonstrate affinity for progestin receptors by displacing 3H-R5020 from rabbit uterine receptors with relative binding affinities of 0.94, 5.21, and 5.41 times that of progesterone, respectively.6 These steroids also stimulate the endometrium of estrogenprimed immature rabbits with relative potencies of 1.4, 3.4, and 4.9 times that of norgestimate, respectively.6 Therefore each of these three steroids show progestational activity. However, to evaluate whether they might act as biologically active metabolites of norgestimate, it was necessary to also assess their androgenicity, because a lack of androgen-related pharmacology is a characteristic or norgestimate. Comparative laboratory assays show that levonorgestrel and 3-keto norgestimate have a pharmacologic profile different from that of norgestimate. Therefore it appears unlikely that either of these steroids contributes significantly to the pharmacologic response of norgestimate. In contrast to norgestimate, levonorgestrel has significant affinity for androgen receptors in vitro (relative binding affinity of 0.22 times that of dihydrotestosterone),6 significant stimulation of prostate growth in rats (relative potency of about 0.1 times that of testosterone; Fig. 2),6 and significant affinity for hu-

man SHBG in vitro (IC oo = 53.4 nmoIlL).7 3-Keto norgestimate also appears to be significantly androgenic, though less so than levonorgestrel. The relative binding affinity of 3-keto norgestimate for rat androgenic receptors is 0.025 times that of dihydrotestosterone; its potency in stimulating prostate growth in rats is equal to that of levonorgestrel (Fig. 2), but this progestin exhibits no affinity for human SHBG in vitro (lC 50 > 10,000 nmoIlL). In contrast to levonorgestrel and 3-keto norgestimate, 17-deacetyl norgestimate, like norgestimate, has little or no activity in each of the three androgenic test models. Its relative binding affinity for rat androgen receptors is only 0.013 times that of dihydrotestosterone; whereas its ability to stimulate prostate growth in rats is similar to that of norgestimate (Fig. 2). Finally, it shows a lack of affinity for human SHBG in vitro (IC oo > 10,000 nmoIlL). These data suggest that it is unlikely that either levonorgestrel or 3-keto norgestimate contributes significantly to the pharmacologic activity of norgestimate, since the androgenicity of these compounds is inconsistent with the pharmacologic profile of norgestimate. However, the pharmacologic profile of 17 -deacetyl norgestimate is similar to that of norgestimate, which suggests that it may be an important contributing metabolite. Clinical studies. Based on the results of preclinical studies, norgestimate and 17 -deacetyl norgestimate should have minimal effect on ethinyl estradiolinduced elevations in circulating levels of SHBG and high-density lipoprotein (HDL), whereas levonorgestrel and 3-keto norgestimate should significantly reduce these levels. This prediction has been based on the sensitivity of these levels to androgenic stimulation. SHBG levels in women during combined oral contraceptive use are considered a measurement of the relative es-

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4200

~~-.--.~,. -- .. ---.-----+--------. ..-------------- .. • ····4

Norge!!timate - Single Dose

. - . . . Norgestimate - Steady-State

1

o

.......

17-Deacetyl

Norgestimate - Single Dose

........

17-Deacetyl

Norgestimate - Steady-State

20

10

30

40

Hours Post Drug Administration

Fig. 3. Mean serum levels of norgestimate and 17-deacetyl norgestimate after single- and multipledose administration of 360 fLg of gestimate to 10 human female subjects.

trogen and androgen activities of the combined product, since estrogens elevate and androgens lower SHBG levels. 9 ,IO Another androgen-sensitive parameter is lipid levels. It has been shown that estrogens elevate HDL levels whereas androgens have the opposite effect."- l3 Two of the progestins, norgestimate and levonorgestrel, are used in combination with ethinyl estradiol as oral contraceptives and have been compared for their effects of SHBG and HDL levels. In a study of 40 women after four treatment cycles, norgestimate does not appreciably inhibit estrogen-induced elevations in SHBG levels (161.2% change from pretreatment levels after norgestimate: ethinyl estradiol, 250 : 35 /Lg).l4 However, the relatively lower SHBG levels in women treated with the levonorgestrel product (26.2% change from pretreatment after levonorgestrel : ethinyl estradiol, 150 : 30 /Lg) indicate that levonorgestrel has markedly reduced the elevation in SHBG levels resulting from ethinyl estradiol. In a large multicenter study of these same two oral contraceptives in more than 1000 women, significant elevations in HDL levels were observed after 3, 6, 12, and 24 cycles of treatment with the norgestimate-ethinyl estradiol product (S.S% maximum increase), which is in contrast to the significant decrease in HDL levels observed during these same cycles of treatment with the levonorgestrel product (7.6% maximum decrease).l4 These clinical data and the laboratory data demonstrate very different profiles for norgestimate and levonorgestrel, suggesting that levonorgestrel does not appear to make an important contribution to the pharmacologic activity of norgestimate. Pharmacokinetics of norgestimate and 17-deacetyl norgestimate

The pharmacologic evaluation of norgestimate's metabolites has suggested that 17 -deacetyl norgestimate might contribute to the response. Studies by Madden

and Back (see this Supplement) demonstrate that 17deacetyl norgestimate is the major metabolic product of norgestimate in human intestinal and hepatic tissues in vitro. A recent study in our laboratory that characterized the pharmacokinetics of norgestimate and 17 -deacetyl norgestimate in the serum of women after norgestimate administration is consistent with the significant contribution of this metabolite to the response to norgestimate. In this study, 10 healthy women have received single (day 1) and multiple doses (days 4 to 10) of two identical tablets, each containing 0.IS0 mg of norgestimate and 0.035 mg of ethinyl estradiol (the norgestimate clinical dose is 0.250 mg daily). Intensive blood sampling has been performed after the first and last doses. Serum concentrations of norgestimate and 17deacetyl norgestimate have been measured by highpressure liquid chromatography-radioimmunoassay procedures (unpublished data). Norgestimate is rapidly absorbed from the tablets. Mean peak norgestimate concentrations of 100 pg / ml are observed within 1 hour of administration, both after single dose and at steady state (Fig. 3), followed by a rapid decline caused by distribution and elimination. Norgestimate concentrations after single- or multiple-dose administration are generally below assay detection within 5 hours after dosing, which precludes an accurate determination of its elimination half-life. Consistent with the in vitro studies in human intestinal and hepatic tissues, the metabolite 17-deacetyl norgestimate appears rapidly in serum after single and multiple norgestimate doses, with concentrations greatly exceeding those of norgestimate (Fig. 3). Mean peak concentrations of 3597 and 4436 pg / ml are observed at 1.5 and 1.4 hours after single dose and at steady state, respectively, which indicates that there is a rapid and substantial conversion of norgestimate to 17 -deacetyl norgestimate after oral administration of

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norgestimate. A certain degree of accumulation of the 17-deacetyl norgestimate is observed after multiple dosing, as would be anticipated from its estimated halflife of 16 to 17 hours.

Comment Significant progress has been made in understanding the contribution of metabolites to the response to norgestimate. However, additional studies are needed. Other metabolites need to be identified, but it is important to point out that relatively few metabolites have been identified for any steroid to date and that such work is painstakingly difficult and time consuming. Blood levels of other metabolites also need to be determined. However, because some of these metabolites bind strongly to SHBG in serum and SHBG is significantly elevated in women using norgestimate-ethinyl estradiol, the measurements of these metabolites and the relationship of such measurements to their contribution to the response are complicated. The studies reported here indicate that 17-deacetyl norgestimate, together with the parent drug, contributes significantly to the response to norgestimate. Significant blood levels of this metabolite have been demonstrated in women after norgestimate administration, and its pharmacology is consistent with that of norgestimate. REFERENCES 1. Orme MLE, Back DJ, Breckenridge AM. Clinical pharmacokinetics of oral contraceptive steroids. Clin Pharmacokin 1983;8:95-136. 2. Weintraub HS, Abrams LS, Patrick JE, McGuire JL. Dis-

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position of norgestimate in the presence and absence of ethinyl estradiol after oral administration to humans. J Pharm Sci 1978;67:1406-8. 3. Alton KB, Hetyei NS, Shaw C, Patrick JE. Biotransformation of norgestimate in women. Contraception 1984; 29:19-29. 4. Killinger J, Hahn DW, Phillips A, Hetyei NS, McGuire JL. The affinity of norgestimate for uterine progestogen receptors and its direct action on the uterus. Contraception 1985;32:325-32. 5. Phillips A, Hahn DW, Klimek S, McGuire JL. A comparison of the potencies and activities of progestins used in oral contraceptives. Contraception 1987;36: 181-92. 6. Phillips A, Demarest K, Hahn DW, Wong F, McGuire JL. Progestational and androgenic receptor binding affinities and in vivo activity of norgestimate and other progestins. Contraception 1990;41:399-410. 7. Phillips A, Hahn DW, McGuire JL. Relative binding affinity of norgestimate and other progestins for sex hormone binding globulin. Steriods 1990;55:373-5. 8. Phillips A. The selectivity of a new progestin. Acta Obstet Gynaecol Scand 1990;152:21-4. 9. El Makhzangy MN, Wynn V, Lawrence DM. Sex hormone binding globulin capacity as an index of estrogenicity or androgenicity in women on oral contraceptive steroids. Clin Endocrinol 1979; 10:39-45. 10. Victor A, Weiner E, Johansson EDB. Relationship between sex hormone binding globulin and d-norgestrel levels in plasma. Acta Endocrinol 1977;86:430-6. 11. Alen M, Rahkila P. Reduced high density lipoprotein cholesterol in power athletes. Use of male sex hormone derivatives, an atherogenic factor. Int J Sports Med 1984; 5:341-2. 12. Bradley DD, Wingerd J, Pettiti DB, Serum high density lipoprotein cholesterol in women using oral contraceptives, estrogens and progestins. N Engl J Med 1978; 299: 17-20. 13. Fotherby K. Oral contraceptives, lipids, and cardiovascular disease. Contraception 1985;31:367-94. 14. Chapdelaine A, Desmarais JL, Derman RJ. Clinical evidence of the minimal androgenicity of norgestimate. Int J Fertil 1989;34:347-52.

Serum pharmacokinetics of orally administered desogestrel and binding of contraceptive progestogens to sex hormone -binding globulin Willem Bergink, PhD," Roel Assendorp, MSc," Lenus Kloosterboer, PhD," Wim van Lier, MSc," Gerrit Voortman, MSc," and Ingelise Qvist, MDb Oss, The Netherlands, and Horsens, Denmark Serum levels of 3-ketodesogestrel and ethinyl estradiol were analyzed by radioimmunoassay in a balanced crossover study with two tablet formulations containing desogestrel (0.150 mg) and ethinyl estradiol (0.030 mg) in 25 women under steady-state conditions after 21 days of treatment. The pharmacokinetic properties of desogestrel were characterized by the following parameters: (1) maximum serum concentration, (2) time to maximum serum concentration, (3) total area under the serum concentration versus time curve, and (4) serum half-life of elimination. The interindividual variation in these parameters was comparable with that observed with other contraceptive combinations containing ethinyl estradiol and norethisterone, levonorgestrel, or gestodene. The serum distribution of contraceptive progestogens is known to be determined by their affinity to sex hormone-binding globulin and the concentration of sex hormone-binding globulin. We analyzed the structural features that determine binding to sex hormone-binding globulin. The .18-methyl group increased and the 11-methylene group weakened the binding to sex hormone-binding globulin. The double bond at C-15 reinforced the binding only when combined with an 18-methyl group. Therefore, the binding of levonorgestrel (the 18-methyl derivative of norethisterqne) and gestodene (the 8-15,18 methyl derivative of norethisterone) to sex hormone-binding globulin was much stronger than that of 3-keto-desogestrel and norethisterone. (AM J OSSTET GVNECOL 1990;163:2132-7.)

Key words: Desogestrel, oral contraceptive, pharmacokinetics, sex hormone-binding globulin

The pharmacokinetics and metabolism of contraceptive progestogens and ethinyl estradiol (EE) have been analyzed in several studies,I-7 and considerable variation was observed in the pharmacokinetic parameters. Only a few studies are adequate for a meaningful comparison of the rate and the extent of absorption and the rate of elimination, because in many studies data have been obtained with only a limited number of volunteers. Parameters that are characteristic for the pharmacokinetic properties of the hormonal components in an oral contraceptive tablet, namely, maximal serum concentration (e max) , time to maximum serum concentration (T rna.), area under the serum concentration versus time curve (AUC), and serum elimination half life (T !l2el), are subject to several factors, which influence the variability. These factors result not only in considerable interindividual variation;' 7 but also in intercenter variation in the pharmacokinetic parameters. From the Scientific Develapment Group, Organon International B. V.,' and the Department of Obstetrics and Gynaecology, Horsens Hospital.' Reprint requests: Willem Bergink, PhD, Scientific Development Group, Organon International B.V., P. O. Box 20,5340 BH Oss, The Netherlands. 6/0/24389

2132

Relevant to this aspect may be variations in environmental factors, patient characteristics, assay methods, and the first-pass effect. The latter was reported as specifically relevant to plasma level variability.s It has also been concluded that levonorgestrel and gestodene are 80% to 100% bioavailable and therefore have no or a limited first-pass effect. g • 10 The bioavailability of other compounds (e.g., norethisterone, EE, and desogestrel) has been reported to be less. Therefore in the present study we analyzed the interindividual variability of the mean values of the pharmacokinetic parameters (emax> tmax> AUe, and t!l2el) for orally administered desogestrel in comparison with the values found in other studies with oral contraceptives containing EE and desogestrel, EE and levonorgestrel, EE and norethisterone, and EE and gestodene. In addition to pre systemic metabolism (i.e., absorption by the gut wall and first-pass metabolism in the liver), the plasma levels of contraceptive steroids strongly depend on binding to plasma proteins. A close correlation between plasma concentrations of levonorgestrel or norethisterone and sex hormone-binding globulin (SHBG) binding capacity as found by Victor and Johansson ll and Back et al. 12 indicates the importance of SHBG binding for the pharmacokinetics of

Volume 163 Number 6, Part 2

contraceptive progestogens. Therefore we have also analzyed the binding affinity of contraceptive progestogens to SHBG. Material and methods

Estimation of SHBG binding capacity. The SHBG binding capacity has been estimated with the dextrancoated charcoal method for the separation of bound and free steroid as described by Hammond and Lahteenmaki. 13 The binding was determined in the presence of cortisol (200 nmollL) to prevent binding to transcortin-binding sites. Human serum was diluted (dilution factor 26) with a 0.25% dextran-coated charcoal, 0.025% dextran T70 suspension in buffer (10 mmollL TRIS, 5 mmollL CaCI 2 , glycerol 0.3 mollL, pH 7.4) and incubated for 30 minutes at room temperature to remove endogenous steroids. The mixture was centrifuged (15,000 N Ikg for 10 minutes), and the stripped serum was used for the determination of the relative binding affinities of progestogens to SHBG. The relative binding affinities were estimated with a 3-point parallel line assay.14 To determine relative affinity, 0.100 ml of stripped serum and 0.100 ml of a saturating concentration of labeled 5a-dihydrotestosterone and increasing concentrations of unlabeled progestogens were incubated for 30 minutes at 37° C. Nonspecific binding was measured in the presence of 100 nmollL of unlabeled 5a-dihydrotestosterone. The results of several tests were combined according to the procedure described in the United States Pharmacopeia. 15 Estimation of pharmacokinetic parameters for EE and desogestrel. The preparations were as follows: (1) a tablet with a diameter of 6 mm, containing 0.150 mg of desogestrel + 0.030 mg ofEE and (2) a smaller tablet with a diameter of 5 mm, which also contained 0.150 mg of desogestrel + 0.030 mg of EE. Volunteers. Twenty-five healthy female volunteers, ages 20 to 29 years, who voluntarily gave written informed consent, participated in this study. The subjects were accurate with respect to tablet intake, had a cycle length around 28 days (28 ± 2 days), and had less than three abnormal-linked laboratory values during biochemical and hematologic screening before treatment. The subjects also fulfilled the following criteria: (1) women were excluded during 3 months after a partus, an abortion, or stopping breast-feeding; (2) women should not be heavy smokers (> four cigarettes daily), heavy drinkers (not more than five glasses per week and no alcohol was taken within 3 days before an assessment day), or overweight « ideal weight + 20%); (3) women should not have been treated with oral contraceptives or other hormonal preparations within a 2month period before the start of the study or treated with an implant or injectable contraceptive within the previous 6 months were excluded. Moreover, women with a poor physical condition, abnormal fasting glu-

Serum pharmacokinetics of desogestrel

2133

Table I. Relative affinities of norethisterone derivatives of norethisterone for SHBG

Compound

Testosterone (reference) NET .1.-lS-NET II-methylene-NET 18-methyl-NET (levonorgestrel) II-methylene,18-methyl-NET (3-ketodesogestrel) II-methylene,.1.-IS-NET .1.-lS,18-methyl-NET (gestodene) II-methylene,.1.-lS,18-methyl-NET

Relative affinity to 5a-DHT* (with 95% confidence limits)

22.0 2.3 1.3 2.6 11.S 3.9

(20.0-24.2) (2.1-2.6) (1.2-1.4) (2.4-2.9) (10.2-13.0) (3.S-4.2)

1.S 17.3 4.7

(1.3-1.6) (IS.8-18.9) (4.3-S.1)

5a-DHT, Sa-Dihydrotestosterone; NET, norethisterone. *Results of three tests were combined.

cose levels, a history of gestational diabetes, a history or a family history of diabetes mellitus, and diseases known to be associated with lipid disorders were also excluded. Special attention was also given to the general exclusion criteria (thromboembolic disease, known or suspected estrogen-dependent tumors, etc.). Finally, subjects did not take rifampicin, tetracycline, phenylhydantoin, and phenobarbital during the 2 months before the start of treatment and during treatment. Design. The volunteers were randomly divided over two groups. Group I consisted of 11 volunteers and group II consisted of 14 volunteers. Group I started with the 6 mm tablet and group II started with the 5 mm tablet in the first treatment cycle. After 21 days of treatment a tablet-free period of 7 days followed, after which the treatments were reversed. Group I then received the 5 mm tablet for 21 days and group II received the 6 mm tablet for 21 days. Blood sampling scheme. Just before and after the twenty-first dose, blood samples were taken for 3ketodesogestrel and EE determinations. Sampling on day 21 started at 8:00 AM, just before the intake of tablet 21. The day before sampling, no food was taken after 8:00 PM. The samples were taken at the following times: 0 Gust before intake), 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 16, 24, 48 and 72 hours after intake of the tablet. Blood was allowed to clot at room temperature for 30 minutes and then centrifuged at 2.5 X 104 N Ikg for 10 minutes at 4° C. Serum was collected, frozen, and stored at - 20° C until analysis. EE and 3-ketodesogestrel assay. EE concentrations were estimated by a specific radioimmunoassay using 17a-[6,7-3H]-EE (New England Nuclear) as tracer, specific activity 2200 gigabecquerel (GBq)/mmol, and anti-serum raised in rabbits against EE-3-succinylbovine serum albumin. The working dilution of the antiserum was 1I 16000. The detection was accurate

2134

8ergink et al.

December 1990 Am J Obstet Gynecol

NET (norelhislerone) RBA: 2.3 %

I .1-15-NET

18-CH3-NET (Ievonorgeslrel) RBA: 11.5 %

RBA: 1.3 %

RBA: 2.6 %

.1-15

11 =CH 2 • .1-15-NET

II=CH2·18-CH3-NET (3-kelodesogeslrel) RBA: 3.9 %

RBA: 1.5 %

I

.1-15

.:\-15. 18-CH3-NET (geslodene) RBA: 17.3 %

I II=CH 2 ·.1-15.18-CH 3-NET

RBA: 4.7 %

Fig. 1. Relative binding affinities

(RBAs)

of progestogens derived from norethisterone

with variation coefficients of 90% of both the nifedipine oxidase and ethinyl estradiol 2hydroxylase activities in human liver micro somes containing either high- or low-catalytic activity.12 Rates of ethinyl estradiol 2-hydroxylation were well correlated with both rates of nifedipine oxidation and immunochenm:ally determined P-4S0NF in liver samples obtained trom different persons. Furthermore, troleandomycin (which has been shown to selectively inhibit cytochrome P-4S0 enzymes in the P-4S0 IlIA family)13 was converted to a (nitroso) metabolite that blocked both nifedipine oxidation and ethinyl estradiol 2hydroxylation. Several lines of evidence indicate that other cytochrome P-4S0 enzymes do not catalyze the reaction, including P-450DB (IID6), P-4S0PA (IA2), P-4S0 (lIE I), and the enzymes in the P-4S0 lIe famil}.I" It saould be pointed out that P-4S0NF (I1IA4) is a member of a complex multigene family, with at least three proteins having >80% sequence identity.14.16 The full clt:ails of specific expression and regulation of the gene, are not understood at this time, and the possibility exists that more than one of these related proteins might contribute to ethinyl estradiol 2-hydroxylation and Ix: recognized by the antibodies. Another point is that specific forms of cytochrome P-4S0 involved in ethinyl estradiol2-hydroxylation in animal models have not been identified. If the 2-hydroxylation of estradiol can be used as a guide, then the rat model may be more complex. We previously reported that in contrast to the human liver, in which P-4S0NF is the principal catalyst of 2-hydroxylation, at least three cytochrome P-4S0 en-

zymes (the products of three different gene families) contribute to the reaction in the rat liver.17 Inhibitors of ethinyl estradiol hydroxylation

Purba et al. 18 examined several drugs and steroids for their ability to inhibit ethinyl estradiol 2hydroxylation in vitro (in human microsomes, Km 9 fJ.moIlL). Several steroids were found to be competitive inhibitors, including estradiol (K, 12 fJ.moIlL), progesterone (K, 69 fJ.moIlL), and norgestrel (K, 69 fJ.moI/L). Primaquine and tolbutamide were noncompetitive inhibitors (K, 69 fJ.mollL and 81 fJ.moI/L, respectively). Several other compounds were tested and found to be rather poor inhibitors, including chloroquine, 1methylimidazole, cholesterol, cortisol, diethylstilbesterol, antipyrine, mephenytoin, and metronidazole. Bolt and KasseP9 reported that the insecticide synergist, l-naphthyl-4(S)-imidazole, was a competitive inhibitor of microsomal ethinyl estradiol 2-hydroxylation (K, 3 fJ.moI/L) on the rat liver. Troleandomycin has already been shown to be an inhibitor of microsomal ethinyl estradiol 2-hydroxylation in vitro in the human liver. 12 Such macrolide antibiotics are known to be effective inhibitors in vivo l3 and one would also expect erythromycin to be a selective inhibitor of ethinyl estradiol 2-hydroxylation. Mechanism-based inactivation by acetylenic steroids

In addition to the types of inhibition previously mentioned, mechanism-based or "suicide" inactivation of cytochrome P-4S0 is seen with the acetylenic steroids. White and Muller-Eberhard 20 reported that treatment of rats with ethinyl estradiol or norethindrone decreased the level of cytochrome P-450, and White 21 demonstrated the formation of green pigments formed from the heme of cytochrome P-450 in rats treated with ethinyl testosterone. Subsequently, the heme adducts formed by acetylenic steroids have been interpreted in terms of mechanism-based inactivation. 22.23 Mechanismbased enzyme inactivation is of significance in that only the enzyme involved in the biotransformation of the compound becomes inactivated, and the damage is therefore targeted. 24 Many of the acetylenic steroids appear to behave as inhibitors in this way.25 Although the chemistry of the green Nalkylporphyrins resulting from heme destruction has been deduced by Ortiz de Montellano et aI., Davies et aP6 have subsequently found that during the oxidation of norethindrone by rat liver microsomes, a considerable portion of the heme destroyed becomes irreversibly attached to the cytochrome P-4S0 apoprotein. The 2-hydroxy ethinyl estradiol that is formed by metabolism is readily oxidized in vitro to an ortho quinone that can covalently bind to protein,9 but the significance of this reaction is unclear.

Enzyme inhibition

Volume IG3 1\ umber G, Part 2

Ethinyl estradiol has been found to be a mechanismbased inactivator of human liver microsomal cytochrome P-450 and P-450KF (in vitro). The partition ratio, or number of times that an enzyme turns over a substrate before it is inactivated/' was estimated at -120. ' "

2161

rP>

'1

HO~

Desogeslrel

17a-Elhynyleslradiol

(75175)

(66/81)

Inhibition of cytochrome P-450 by acetylenic progestogens

Since progestogens are commonly used with ethinyl estradiol in contraceptive formulations, their interactions with estrogens are of great importance."7 Kuhl et al."H reported that gestodene levels rise during therapy, and they considered the possibility that the increase was due to decreased metabolism. Furthermore, Kuhl et aU" found that in comparison with desogestrel, gestodene had a marked effect in increasing plasma levels of ethinyl estradiol, and they also showed that the cause was decreased clearance of the drug. In addition, cortisol levels were elevated in patients treated with gestodene, which might be expected if cytochrome P-450I\F, the cortisol 6f)-hydroxylase, was inhibited (Table I). A series of 17a-acetylenic steroids was examined for the ability to inactivate human liver microsomal nifedipine oxidase and ethinyl estradiol 2-hydroxylase, activities of cytochrome P-450KF (Fig. 1). All the steroids proved to be inactivators, but clearly gestodene was the most effective when tested at the single concentration of 100 jJ-moll L. Indeed, the extent of inhibition seen with gestodene under these conditions was more marked than that found with a 20 jJ-moll L concentration of the classic inhibitor troleandomycin, which reduced the activity of nifedipine oxidase to 21% and that of ethinyl estradiol 2-hydroxylase to 65% under these conditions. The effects of gestodene were examined in more detail, the inhibition pattern is classic for mechanism-based inactivators 24 in that inactivation is first order in time and the rate increases with inhibitor concentration. The data can be used to derive the parameters K; = 46 jJ-nlOlI Land konan;,.a'inn = 0.39 minute. - I The kina"i'."ion value is one of the highest ever measured for a microsomal cytochrome P-450 enzyme. 2 " In other studies it was possible to demonstrate the loss of spectrally detectable cytochrome P-450 in human liver microsomes during the oxidation of gestodene. In this particular sample, which is enriched with P-450I\F, the level of cytochrome P-450 was decreased to 40% and thereafter remained constant. The difference in the rates of destruction of cytochrome P-450 by gestodene and ethinyl estradiol is quite dramatic, and logarithmic plots indicate that the rate is approximately 50-fold greater in the case of gestodene. Gestodene was effective in destroying microsomal cytochrome P-450, even at the lower concentration of 10

Norethisterone

Gestodene

(91181)

(15/16)

osnOH. 3-Kelo-desogeslrel

Levonorgestrel

(49/54)

(77/63)

osn

O~.",,===

a

.#

l1-CH 2 -Norethisterone (92168)

",,===

;)

0

.#

15

•

l1-CH 1 -d. -Norethlsterone (82168)

Fig, 1. Structures of synthetic 17a-ethynyl and 1713-hydroxy steroids examined as mechanism-based inactivators of human liver microsomal cytochrome P-450. Each steroid (100 fLmollL) was incubated with 250 pmol of human liver cytochrome P-450 (liver sample HL 110) and an NADPHgenerating system in a reaction volume of 50 fLl at 37° C for 30 minutes. At that time the reaction was diluted to a volume of 1.0 ml with the NADPH-generating system and either 200 fLmollL nifedipine or 100 fLmollL 17a-ethinyl estradiol, and these incubations proceeded for an additional 10 minutes. Rates of nifedipine oxidation and l7a-ethinyl estradiol 2hydroxylation were determined by high-performance liquid chromotagraphy and rates of the activities relative to those measured in similar experiments without steroids are presented (as percentages of uninhibited values). The first of each pair of numbers (in parentheses) indicates the relative rate of nifedipine oxidation, and the second indicates the relative rate of ethinyl estradiol 2-hydroxylation. NADPH, Nicotinamide adenine dinucleotide phosphate.

jJ-mollL (57% ± 4% cytochrome P-450 remaining after 10 minutes). The destruction could be partially blocked by high concentrations of the P-450NF substrates quinidine (l00 jJ-moIlL, 76% ± 1% original cytochrome P450 after 10 minutes) and nifedipine (200 jJ-moIlL, 82% ± 4% original cytochrome P-450 remaining after 10 minutes). The Km values for quinidine and nifedipine are 4 and 19 jJ-moIlL, respectively.!o.!! Comparison of the rates of nifedipine oxidase inactivation and gestodene disappearance (at a single concentration of gestodene) yields an estimated partition ratio of 9 gestodene, which can be compared with the value of 120 for ethinyl estradiol.!2 Therefore a given dose of gestodene

2162

Guengerich

would be expected to inactivate 15 times as much cytochrome P-450KF as does ethinyl estradiol. More than 10 gestodene metabolites are known,27 with biotransformation resulting from the formation of the 3u- and 313-hydroxyl compounds, 4, 5hydrogenation, hydroxylation at the 1, 6u, 11 and other unidentified positions, and homoannulation (conversion of the D ring to a cyclohexanone with release of CO 2 ).,o It is somewhat surprising that a 613-hydroxy product has not been identified, since 613-hydroxylation is observed in the cytochrome P-450NF oxidations of the .:1 4 3-keto steroids testosterone, androstenedione, cortisol, and progesterone. Conclusions

P-450NF (and possibly closely-related enzymes) is the major catalyst involved in the 2-hydroxylation of ethinyl estradiol, the major step in the clearance of the compound in the human liver. The enzyme appears to be induced by the administration of rifampicin, barbiturates, or certain steroids such as dexamethasone. The enzyme catalyzes the oxidation of several steroids, drugs, and carcinogens (Table I), and variations in the activity of the enzyme would alter the rates of biotransformation of all these compounds. Most of the inhibitors of the enzyme require metabolism to exert their effects. All 17u-acetylenic steroids examined are mechanism-based in activators of P-450NF and its activities, but gestodene shows a remarkably high activity in this regard. This property ofthe compound can explain its ability to elevate plasma levels of ethinyl estradiol and cortisol. Examination of the effects of the different steroids tested does not permit elucidation of the structure I function relationships related to mechanismbased inactivation of P-450NF. Because the inhibition seen with gestodene is mechanism based, only those cytochrome P-450-mediated activities associated with P-450NF (Table I) would expected to be compromised in patients receiving the drug. Finally, gestodene appears to be an excellent compound to be used in the further in vitro and possibly in vivo examination of which compounds are oxidized by cytochrome P450NF. REFERENCES I. Innhoffen HH, Hohlweg W. Neue per os-wirksame weibliche Keimdrusenhormon-Derivate: 17-Athinylostradiol und Pregnen-in-on-3-01-17. Naturwissenschaften 1938; 26:96. 2. de la Pena A, Chenault CB, Goldzieher Jw. Radioimmunoassay of unconjugated plasma ethynylestradiol in women given a single dose of ethynylestradiol or mestranol. Steroids 1975;25:773-80. 3. Stadel BC, Stern thai PM, SchlesselmanJJ, et al. Variation of ethinylestradiol blood levels among healthy women using oral contraceptives. Ferti! Steril 1980;33:257-60. 4. Jung-Hoffman C, Kuhl H. Interaction with the pharmacokinetics of ethinyl estradiol of progestogens contained in oral contraceptives. Contraception 1989;40:299312.

December 1990 Am J Obstet Gynecol

5. Reimers D, Jezek A. Rifampicin und andere Antituberkulotika bei gleichzeitiger oraler Kontrazeption. Prax PneumoI1971;25:255-62. 6. Nocke-Finck L, Brewer H, Reimers D. Wirkung von Rifampicin auf den Menstruationszylkus und die Ostrogenausscheidung bei Einnahme oraler Kontrazeptiva. Dtsch Med Wochenschr 1973;98:1521-3. 7. Janz D, Schmidt D. Anti-epileptic drugs and failure of oral contraceptives. Lancet 1974; I: 1113. 8. Bolt HM, Kappas H, Remmer H. Studies on the metabolism of ethynylestradiol in vitro and in vivo: the significance of 2-hydroxylation and the formation of polar products. Xenobiotica 1973;3:773-85. 9. Bolt HM. Metabolism of estrogens-natural and synthetic. Pharmacol Ther 1979;4:155-81. 10. Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ. Characterization of the rat and human microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype of genetic polymorphism in oxidative drug metabolism.J Bioi Chern 1986;261:505160. 11. Guengerich FP, Muller-Enoch D, Blair IA. Oxidation of quinidine by human liver cytochrome P-450. Mol Pharmacol 1986;30:287-95. 12. Guengerich FP. Oxidation of 17Cl-ethynylestradiol by human liver cytochrome P-450. Mol Pharmacol 1988;33: 500-8. 13. Pessayre DV, Descatoire V, Konstantinova-Mitcheva M, et al. Self-induction by triacetyloleandomycin of its own transformation into a metabolite forming a stable 456 nmabsorbing complex with cytochrome P-450. Biochem PharmacoI1981;30:553-8. 14. Nebert DW, Nelson DR, Adesnik M, et al. The P450 superfamily: update on listing of all genes and recommended nomenclature of the chromosomal loci. DNA 1989;8: 1-13. 15. Beaune PH, Umbenhauer DR, Bork RW, Lloyd RS, Guengerich FP. Isolation and sequence determination of a human liver cytochrome P-450 clone related to human cytochrome P-450 nifedipine oxidase. Proc Nat! Acad Sci USA 1986;83:8064-8. 16. Bork RW, Muto T, Beaune PH, Srivastava PK, Lloyd RS, Guengerich FP. Characterization of mRNA species related to human liver cytochrome P-450 nifedipne oxidase and regulation of catalytic activity. J Bioi Chern 1989;264: 910-9. 17. Dannan GA, Porubek DJ, Nelson SD, Waxman DJ, Guengerich FP. 1713-EstradioI2- and 4-hydroxylation catalyzed by rat hepatic cytochrome P-450: roles of individual forms, inductive effects, developmental patterns, and alterations by gonadectomy and hormone replacement. Endocrinology 1986; 118: 1952-60. 18. Purba HS, Maggs JL, Orme M L'E, Back DJ, Park BK. The metabolism of 17Cl-ethinyloestradiol by human liver microsomes: formation of catechol and chemically reactive metabolites. Br J Clin Pharmacol 1987;23:447-53. 19. Bolt HM, Kasssel H. Effects of insecticide synergists on microsomal oxidation of estradiol and ethynylestradiol and on microsomal drug metabolism. Xenobiotica 1976; 6:33-8. 20. White INH, Muller-Eberhard U. Decreased liver cytochrome P-450 in rats caused by norethindrone or ethynyloestradiol. Biochem J 1977; 166:57-64. 21. White INH. Metabolic activation of acetylenic substituents to derivatives in the rat causing the loss of hepatic cytochrome P-450 and haem. BiochemJ 1978;174:853-61. 22. Ortiz de Montellano PR, Kunze KL, Yost GS, Mico EA. Self-catalyzed destruction of cytochrome P-450: covalent binding of ethynyl sterols to prosthetic heme. Proc Nat! Acad Sci USA 1979;76:746-9. 23. Ortiz de Montellano PR, Kunze KL. Self-catalyzed inactivation of hepatic cytochrome P-450 by ethynyl substrates. J Bioi Chern 1980;255:5578-85. 24. Silverman RB. Mechanism-based enzyme inactivation:

Enzyme inhibition

VoluIlle 163 1\ umber 6, Part 2

25. 26.

27.

28.

29.

30.

chemistry and enzymology. Vols I and 2. Boca Raton. Florida: CRC Press, 1988. Ortiz de Montellano PR, Correia MA. Suicidal destruction of cytochrome P-450 during oxidative drug metabolism. Annu Rev Pharamacol Toxicol 1983;23:481-503. Davies HW, Britt SG, Pohl LR. Inactivation of cytochrome P-450 by 2-isopropyl-4-pentenamide and other xenobiotics leads to heme-derived protein adducts. Chem-Biol Interact 1986;58:345-52. Diisterberg B, Tack J-W, Krause W, Hiimpel M. Pharmacokinetics and biotransformation of gestodene in man. In: Elstein M, ed. Gestodene: development of a new gestodene-containing low dose oral contraceptive. Carnforth: Parthenon Publishers, 1987:35-44. Kuhl H, Jung-Hoffman C, Heidt F. Serum levels of 3keto-desogestrel and SHBG during 12 cycles of treatment with 30 J.Lg ethinylestradiol and ISO J.Lg desogestrel. Contraception 1988;38:381-90. Hammons GJ, Alworth WL, Hopkins NE, Guengerich FP, Kadlubar FF. Mechanism-based inactivation of cytochrome P-450-dependent 2-naphthylamine N-oxidation activity of liver microsomes by 2-ethynylnaphthalene. Chem Res Roxicol 1989;2:367-374. Schmid SE, Au WYW, Hill DE, Kadlubar FF, Slikker WM Jr. Cytochrome P-450-dependent oxidation of the 17aethynyl group of synthetic steroids: D-homoannulation or enzyme inactivation. Drug Metab Dispos 1983; II :531-6.

31. Waxman DJ, Attisano C, Guengerich FP. Lapenson DP. Cytochrome P-450 steroid hormone metabolism catalyzed by human liver microsomes. Arch Biochem Biophys 1988;263:424-36. 32. Shimada T, Guengerich FP. Evidence for P-450:'-iF, the nifedipine oxidase, being the principal enzyme involved in the bioactivation of aflatoxins in human liver. Proc Natl Acad Sci USA 1989;86:462-5. 33. Shimada T, Iwasaki M, Martin MV, Guengerich FP. Human liver microsomal cytochrome P-450 enzymes involved in the bioactivation of procarcinogens detected by umu gene response in Salmonella typhimurium TA1535/pSKl002. Cancer Res 1989;49:3218-28. 34. Shimada T, Martin MV, Pruess-Schwartz D, Marnett LJ, Guengerich FP. Roles of individual forms of human cytochrome P-450 enzymes in the bioactivation ofbenzo(a)pyrene, 7,8-dihydroxy-7,8-dihydrobenzo (apyrene, and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons. Cancer Res 1989;49:6304-12. 35. Ged C, Rouillon JM, Pichard L, et al. Cortisol 613hydroxylase in human liver microsomes. Br J Clin Pharmacal 1989;28:373-87. 36. Guengerich FP. Biochemical characterization of human cytochrome P-450 enzymes. Annu Rev Pharmacol Toxicol 1989;29:241-64.

Formation, metabolism, and physiologic importance of catecholestrogens Peter Ball, Prof. Dr. med,' and Rudolf Knuppen, Prof. Dr. Rer. Nat. b Liibeck, West Germany The metabolism of natural and synthetic estrogens is governed primarily by hydroxylations, leading to polyhydroxylated derivatives of the steroid molecule. In mammals aromatic hydroxylation is most prominent quantitatively. The 2- and 4-hydroxyestrogens (catecholestrogens) formed are secreted not only in high amounts in urine but are also present in significant quantities in different organs, such as the liver, pituitary gland, and hypothalamus. This A ring hydroxylation of primary estrogens is affected by peroxidases, tyrosinases, and unspecific monooxygenases by mechanisms still not completely understood. The activity of the aromatic hydroxylases is regulated not only with respect to the overall extent but also to the relative rate of hydroxylation at C-atoms 2 and 4. The metabolism of catechol estrogens may be divided into reversible and irreversible reactions, of which the reaction with the catechol-O-methyltransferase, and thereby the interaction with catecholamines, the conjugation, and the thioether formation are the most prominent. Low- and high-affinity binding is operative in binding to plasma proteins and receptors. Finally, irreversible binding to cellular macromolecules, such as proteins and deoxyribonucleic acid, and the oncogenic potential of natural and synthetic catecholestrogens are discussed. (AM J OSSTET GVNECOL 1990;163:2163-70.)

Key words: Catecholestrogens, formation, metabolism, humans

From the Klinische Endokrinologie," and Biochemische Endokrinologie, Medizinische Universitat zu Lubeck." Reprint requests: Dr. P. Ball, Klinische Endokrinologie, Medizinische Universitiil zu Lubeck, D-2400 Lubeck West Germany.

610123623

Since the discovery of the "classic" estrogensestrone (El)' estradiol (Eel, and estriol (E,)-some 50 years ago, intense research work has been carried out on the metabolism of these hormones that, before and 2163

2164

Ball and Knuppen

December 1990 Am J Obstet Gyneco1

Table I. Concentrations of 2-substituted E, in plasma of humans (picograms per milliliter) Subject

2-0HEJ unconjugatedl conjugated

Children, 5-9 yr Men Women Periovulation Pregnant Children, newborn

2-0CE J unconjugatedl conjugated

20145/1000

6011130 8011320

65/2300 100/5000 500/3000

18011180 3980/5850 -1-

Table II. Excretion of 2-substituted E, in urine of humans (micrograms per 24 hours) Subject

Children Men Women Cycling Pregnant Menopausal

2-0HEJ

2-0CEJ

2-Substituted 1total E

1.0 9.0

2.4 12

1.3 1.6

30 700 5.0

20 450 9.0

0.7

in addition to co~ugation, are essentially transformed by hydroxylation reactions. In mammals these hydro xylations occur not only at the aliphatic C-atoms 18, 16, 15, 14, 11, 7, and 6, but also at aromatic positions 4 and 2. This aromatic ortho-hydroxylation of phenols, also well known in the biotransformation of many nonsteroidal compounds with physiologic and pharmacologic importance, has several interesting features that stimulated intense and steadily increasing investigations into 2- and 4-hydroxyestrogens in recent years. Because of the evident structural relationship between E 2, ethinyl estradiol (EE), and in part tyrosine, the reactions given here for the natural estrogens are, in general, the same for all phenols. Metabolism of E2 in vitro in rat and human liver and in vivo in humans',2

When [6,7 -3H]E 2 is incubated with rat liver slices for hour under O 2, approximately 50% of the radioactivity remains lipophilic, 30% becomes water soluble, and 4% is irreversibly bound to protein. In the water-soluble fraction containing the "classic" conjugates, co~ugates with sulfuric and glucuronic acid, and the "newer" co~ugates, mainly glutathione, the sulfate esters predominate. Within the more than 20 metabolites isolated from the lipophilic and, after hydrolysis, water"soluble fractions of rat liver, the main metabolites are 2-hydroxylated estrogens (25%), followed by their isomeric monomethyl ethers (15%), 4hydroxyestrogens are of minor importance (8%). Essentially the same distribution pattern of metabolites is found on incubation of human liver slices, although the turnover of the substrate is reduced. In the same patient whose in vitro metabolism in the liver was explored, [4-14C]E2 was administered orally,

1.7

and the excretion pattern of radioactive metabolites was monitored in urine. Again, 2-substituted estrogens were the main products (10%), although they did not exceed the sum of primary (E, + E2 = 8%) and 16uhydroxylated (16u-OHE, and E, = 6%) estrogens. Concentrations of endogenous 2-substituted estrogens in tissue, blood, and urine

Using a monoisotope-derivative method (radioenzymatic assay) and ether extracts of the tissues, preliminary values for the concentration of 2-hydroxyestrone (2-0HE,) in the female rat were 560 pg/gm for the liver, 770 pg/gm for the pituitary gland, and 150 pgl gm for hypothalamic tissue. In comparison, rat plasma contained about 520 pg/ml.' With the use of newly developed radioimmunoassays for all relevant catecholestrogens, the determination of plasma concentrations became feasible. The obtained normal values in humans are given in Table I.'.4 U nconjugated and conjugated steroids were determined separately. The 2-0HE, concentrations were in the range of those of E" and the 2-0HE, 2-methyl ether (2-0CE,) concentrations were even slightly higher than those of E, and 2-0HE, together. Determinations of the co~ugated 2-0HE, and 2-0CE, concentrations resulted in 10 to 20 times higher values than those obtained for the unco~ugated steroids. Using the same radioimmunoassays, urinary excretion rates of 2-substituted and 4-substituted estrogens were monitored.' The results for the 2-substituted estrogens are given in Table II. Of special interest were the molar ratios of 2-substituted to 2-nonsubstituted estrogens, the so-called total estrogens. With the exception of women in the reproductive phase, the amount of 2-substituted estrogens clearly surpassed the

Physiology of catecholestrogens

Volume 163 "umber 6, Part 2

2165

H2 0

perox idose

-----1

HoD --

~

HO

O~

lOW

oon Han H+

l-H2

---...

HO

~

o-Quinone

oQ.

Phenoxy- rod icol

Phenol

on

-D

H __ HOO-~O

oQH

:

I

HO

Semiquinone

~

Catechol

Fig. 1. Peroxidase-proposed mechanism of action. (From Kuss E. Estrogens and glutathione. In: Dolphin D, Voulson R, Avramovic 0, eds. Glutathione. New York: John Wiley & Sons, 1989:51249. Copyright© 1989 by John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.)

phenol o-hydrox.

catecho l oxidase

H0yY

~ HO~

HO~ Catechol

A1enol

o~o O~

h-

a-Quinone

Fig. 2. Tyrosinase-catalyzed reactions.

amount of all other estrogens together. For the excretion rates of 2-0HE, and 4-0HE, during the menstrual cycle of four healthy subjects, see ref. 4. Formation of catecholestrogens: Enzymatic hydroxylation of estrogens

Enzymatic A ring hydroxylation of primary estrogens is thought to be affected by three different hydroxylating systems: peroxidases, tyrosinases, and the "unspecific monooxygenases." The mechanism of action of these three enzymes is still poorly understood. It is strongly interconnected with glutathione metabolism and thus thioether formation.' It is believed that peroxidases, through the generation of hydroxyl radicals, initially oxidize phenols to phenoxy radicals, eventually leading to dihydroxybenzenes (catechols), partly in a semiquinone or o-quinone-reactive form (Fig. 1). These quinones show a high binding to deoxyribonucleic acid and protein, a reaction that may be competitively inhibited by

glutathione (GS-SG and thioether formation) and lipids (lipid peroxidation).3 Recently, the estrogen 2-/4hydroxylase activity of the soluble fraction 01 ;, ,man breast cancer tissue was found to behave sin,'Lrly to the peroxidase type from the rat uterus. 6 Tyrosinases that show both phenol o-hydro .ylase and catechol oxidase activity catalyze a two-,:kctron transfer to form o-quinones (Fig. 2). The quinones have the remarkable ability to conjugate with reactive groups on the tyrosinase molecule itself and thus have the potential to inhibit the further action of the t'l1zyme (product inhibition from the suicide type). The addition of glutathione enhances tyrosinase activit~·, presumably by protecting functional groups of the enzyme or reactive groups of the product, thereby inhibiting the suicide reaction by enhancing not only th;oether conjugation but also the formation of protein ;idducts. Similar reactions were observed with EE and ,hpa as substrates." However, the classic estrogen 2-/4-hydroxyla:..t' ,cems

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Ib

Ie

2b

2e

J Obstet Gynecol

Fig. 3. Hypothetic intermediates in the biosynthesis of 2- and 4-hydroxyestrogens. (From Kuss E. Estrogens and glutathione. In: Dolphin D, Voulson R. Avramovic 0, eds. Glutathione. New York: John Wiley & Sons, 1989:512-49. Copyright© 1989 by John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons, Inc.)

to be a cytochrome P-450-dependent microsomal enzyme system. The labile 1,2- and 4,5-arenoxides (dienol epoxides) (Fig. 3), in equilibrium with the more stable keto- and oxepin tautomers, are the postulated intermediates. Arenoxides are unlikely precursors of thioethers, which need an o-quinone, or more likely a semiquinone radical, as molecular species immediately preceding their formation.' Hydroxylase activity is strongly dependent on the nature of the substrate: 17a-EE is a far better substrate than are natural estrogens (EE > E2 > EI)' Enzymatic activity is increased by higher age and the male sex-androgens and castration7-and a widely varying array of substances such as phenobarbital,' EE," and rifampicin,g· IO and decreased by cytochrome P-450 inhibitors,' 2- and 4-haloestrogens," and growth hormone. 12 Finally, urinary excretion of 2hydroxyestrogens was increased by malnutrition, hyperthyroidism, and tobacco smoking and decreased by liver cirrhosis. 3 . , Because the C-2 and C-4 hydroxylations are strongy correlated (ratio of activity 10': 1 to 20: 1), the same mechanism seems to be operative in both reactions. Nevertheless, the relative C-2/C-4 reaction rates vary depending on both the substrate and the source of the enzyme. I. 1:1 Metabolism and binding of catecholestrogens

To elucidate the importance of catecholestrogens in physiology, the rate of formation is one important parameter; the metabolism and binding are others. Within the latter we must discriminate between enzymatic (nonenzymatic) metabolism of the catecholestrogens, both reversible and irreversible, and low- and high-affinity binding, with functional preservation of the catechol structure. One of the reversible interactions is the reaction

with enzymes such as the catechol-O-methyltransferase (COMT). In 1958 Axelrod and Tomchick 14 already showed that the COMT is one of the key enzymes in the metabolism of catecholamines. We purified the enzyme from the liver, characterized it, and proved Knuppen's hypothesis" that the enzymatic methylation of 2-hydroxyestrogens and catecholamines is accomplished by the same methyltransferase. 16 Moreover, we showed that under standard assay conditions, catecholestrogens were not only the preferred substrates (Table 111)17, but also the most potent inhibitors of the methylation of catecholamines in vitro (Fig. 4).18.20 We suggested that the inhibition of catecholamine metabolism by the catechol estrogens might cause prolongation and potentiation of the physiologic actions of the catecholamines, especially blood pressure and gonadotropin release. 21 As demonstrated already by Axelrod and Tomchick, COMT is an ubiquitous enzyme. Of special interest is its high activity in red blood cells,22 which leads to the high plasma metabolic clearance rate of catecholestrogens (Table IV?' and falsely low catecholestrogen concentrations if the blood is handled incorrectly.22 Catecholestrogens monomethyl ethers are prone to extensive demethylation. On incubation with rat liver slices, apparent Kms of 12 and 3 j.lmollL for 2-0CEI and 2-0CE2 were obtained. 17 Catecholestrogens are metabolized by two other reversible reactions: coqjugation with glucuronic and sulfuric acid on incubation of [4-14C]2-0HE2 or [4-14C]E2 with rat liver slices; the most prominent finding is the extensive conjugation of the catecholestrogens,24 a conjugation rate that is significantly higher (58%) than that of the primary estrogen (47%). Finally, two irreversible reactions of catecholestrogens should be mentioned. It has long been known that

Physiology of catecholestrogens

Volume 163 l'.'umber 6, Part 2

Table III. Substrate specificity of COMT from human liver Substrate

K", (iJ-moIlL)

2-0HE, 2-0HE, 4-0HE, Dopamine Epinephrine EE

15 20 20 250 300 IO

2167

.

0,35 '0 ~

c

"'c

0,3

E

a

E, > 2-0HE > 4-0HE NE = 2-0HE 2; E2 inactive 17a-EE > 2-0HE, > 2-0HE, E, > 2-0HE,

CA, Catecholamine; NE, norepinephrine.

REFERENCES 1. Ball P, Haupt M, Knuppen R. Comparative studies on the metabolism of oestradiol in the brain, the pituitary and the liver of the rat. Acta Endocrinol 1978;87:1-11. 2. Ball P, Farthmann E, Knuppen R. Comparative studies on the metabolism of oestradiol-I7(3 and 2-hydroxyoestradiol-17(3 in man in vitro and in vivo. J Steroid Biochern 1976;7:139-43. 3. Ball P, 'Knuppen R. Catecholoestrogens. Acta Endocrinol SuppI1980;232:1-127. 4. Emons G, Ball P, Knuppen R. Radioimmunoassays of catecholestrogens. In: Merriam GR, Lipsett MB, eds. Catecholestrogens. New York: Raven Press, 1983:71-81. 5. Kuss E. Estrogens and glutathione. In: Dolphin D, Voulson R, Avramovic 0, eds. Glutathione. New York: john Wiley & Sons, 1989:512-49. 6. Levin M, Weisz j, Bui QD, Santen RJ. Peroxidatic catecholestrogen production by human breast cancer tissue in vitro. j Steroid Biochem 1987;28:513-20. 7. Dannan GA, Porubek DJ, Nelson SD, Waxman DJ, Guengerich FP. 17(3-estradiol, 2- and 4-hydroxylation catalyzed by rat hepatic cytochrome P-450: roles of individual forms, inductive effects, developmental patterns, and alterations by gonadectomy and hormone replacement. Endocrinology 1986;118:1952-60. 8. Shiverick KT, Notelovitz M. Regulation of cytochrome P450-dependent catechol estrogen formation in rat liver microsomes. Biochem Pharmacol 1983;32:2399-403. 9. Nocke-Finck L, Breuer H, Reimers D. Wirkung von Rifampicin auf den Menstruationzyklus und die Ostrogenausscheidung bei Einnahme oraler Kontrazeptiva. Dtsch Med Wochenschr 1973;98:1521-3. 10. Bolt HM, Kappus H, Bolt M. Effect of rifampicin treatment on the metabolism of oestradiol and 17a-ethynyloestradiol by human liver microsomes. Eur J Clin PharmacoI1975;8:30l-7. 11. Brueggemeier RW, Kimball JG. Kinetics of inhibition of estrogen 2-hydoxylase by various haloestrogens. Steroids 1983;42:93-103. 12. Quail JA, Jellinck PH. Modulation of catechol estrogen synthesis by rat liver microsomes: effects of treatment with growth hormone or testosterone. Endocrinology 1987; 121:987-92. 13. Purdy RH, Moore PH, Williams MC, Goldzieher jW, Paul SM. Relative rates of 2- and 4-hydroxyestrogen synthesis are dependent on both substrate and tissue. FEBS Lett 1982;138:40-4.

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14. Axelrod J, Tomchick R. Enzymatic O-methylation of epinephrine and other catechols. ] Bioi Chern 1958;233: 702-5. 15. Knuppen R, Breuer H, Pangels G. Stoffwechsel von 2-Hydroxyostradiol-(l7J3) und 2-Methoxy-ostradiol-(l7J3) in Geweben des Menschen und der Ratte. Hoppe-Seyler's Z Physiol Chern 1961 ;324: 108-17. 16. Ball P, Knuppen R, Breuer H. Purification and properties of a catechol-O-methyltransferase of human liver. Eur J Biochem 1971;21:517-25. 17. Ball P, Haupt M, Knuppen R. Biogenesis and metabolism of catechol estrogens in vitro. In: Merriam GR, Lipsett MB, eds. Catecholestrogens. New York: Raven Press, 1983:91-103. 18. Ball P, Knuppen R, Haupt M, Breuer H. Kinetic properties of a soluble catechol O-methyltransferase of human liver. Eur r Biochem 1972;26:560-9. 19. Ball P, Knuppen R, Haupt M, Breuer H. Interactions between estrogens and catechol amines III. Studies on the methylation of catechol estrogens, catechol amines and other catechols by the catechol O-methyltransferase of human liver. J Clin Endocrinol Metab 1972;34:736-46. 20. Ball P, Gelbke HP, Haupt 0, Knuppen R. Metabolism of 17-0l-ethynyl-4 1•1C oestradiol and 4J IC mestranol in rat liver slices and interaction between 17 -0l-ethynyl-2hydroxyoestradiol and adrenaline. Hoppe-Seyler's Z Physiol Chern 1973;354:1567-75. 21. Ball P, Knuppen R, Wennrich W, Breuer H. Interactions between oestrogens and catecholamines: influence of oestrogens on the effect of catecholamines on blood pressure in rats. Acta Endocrinol Suppl 1972; 159:85. 22. Bates GW, Edman CD, Porter ]C, MacDonald PC. Metabolism of catechol estrogen by human erythrocytes. ] Clin Endocrinol Metabol 1977;45: 1120-3. 23. Lipsett MB, Merriam GR, Kono S, Brandon DD, Pfeiffer DG, Loriaux DL. Metabolic clearance of catechol estrogens. In: Merriam GR, Lipsett MB, eds. Catecholestrogens. New York: Raven Press, 1983:105-14. 24. Ball p, Hoppen H-O, Knuppen R. Metabolism of oestradiol-17J3 and 2-hydroxyoestradiol-17J3 in rat liver slices. Hoppe-Seyler's Z Physiol Chern 1974;355: 1451-62. 25. Riegel IL, Mueller GC. Formation of a protein-bound metabolite of estradiol-16-C 14 by rat liver homogenates. ] Bioi Chern 1954;210:249-57. 26. ]ellinck PH, Lewis], Boston F. Further evidence for the formation of an estrogen peptide conjugate by rat liver in vitro. Steroids 1967;10:329-46.

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27. Kuss E. Wasserlosliche Metabolite des 17J3-6stradiols. Hoppe-Seyler's Z Physiol Chern 1967;348: 1707-1708. 28. Metzler M. Metabolism of stilbene estrogens and steroidal estrogens in relation to carcinogenicity. Arch Toxicol 1984;55:104-9. 29. Rajan R, Daly M], Reddy VVR. Estrogen effects on NADH oxidase and superoxide dismutase in prepubertal female rats. Steroids 1982;40:651-60. 30. Vandewalle B, Peyrat ]-P, Bonneterre J, Lefebvre J. Catecholestrogen binding sites in breast cancer. ] Steroid Biochem 1985;23:603-10. 31. Liehr ]G, Ulubelen AA, Strobel HW. Cytochrome P-450mediated redox cycling of estrogens.] Bioi Chern 1986; 261: 16865-70. 32. Liehr ]G, Fang WF, Sirbasku DA, Ari-Ulubelen A. Carcinogenicity of catechol estrogens in Syrian hamsters. ] Steroid Biochem 1986;24:353-6. 33. Aswegen CH, Purdy RH, Wittliff JL. Binding of 2Hydroxyestradiol and 4-Hydroxyestradiol to estrogen receptors from human breast cancers. ] Steroid Biochem 1989;32:485-92. 34. Dunn ]F. Transport of estrogens in human plasma. In: Merriam GR, Lipsett MB, eds. Catechol estrogens. New York: Raven Press, 1983:167-76. 35. Martucci Ch, Fishman J. Uterine estrogen receptor binding of catecholestrogens and of estetrol (1,3,5(10)estratriene-3, 15a, 16a, 17J3-tetrol). Steroids 1976;27:32533. 36. MacLusky N], Barnea ER, Clark CR, Naftolin F. Catechol estrogens and estrogen receptors. In: Merriam GR, Lipsett MB, eds. Catechol estrogens. New York: Raven Press, 1983:151-65. 37. Ghraf R, Hiemke Ch. Interaction of catechol estrogens with catecholamine synthesis and metabolism. In: Merriam GR, Lipsett MB, eds. Catechol estrogens. New York: Raven Press, 1983:177-87. 38. Keizer HA, Kuipers H, Verstappen FT],]anssen E. Limitations of concentration measurements for evaluation of endocrine status of exercising women. Can] Appl Spt Sci 1982;7:79-84. 39. Purdy RH, Goldzieher ]W, Le Quesne PW, Abdel-Baky S, Durocher CK, Moore PH Jr. Active intermediates and carcinogenesis. In: Merriam GR, Lipsett MB, eds. Catechol estrogens. New York: Raven Press, 1983:123-40.

SESSION IV. HORMONAL EFFECTIVENESS OF ORAL CONTRACEPTIVE STEROIDS Chairman: M. Breckwoldt

Binding of oral contraceptive progestogens to serum proteins and cytoplasmic receptor Michael Juchem* and Kunhard Pollow, MD, PhD Mainz, West Germany Some progestogens widely used in oral contraceptives are characterized at the level of high-affinity receptor binding as well as binding to sex hormone-binding globulin and corticosteroid-binding globulin. With regard to binding to sex hormone-binding globulin, gestodene, levonorgestrel, and to a lesser extent 3-ketodesogestrel (which is only formed from the prodrug desogestrel in the body), show a behavior that is manifested in the relatively high affinity to sex hormone-binding globulin, whereas desogestrel and norgestimate do not display any measurable affinity for this specific steroid-binding serum protein. Furthermore, levonorgestrel and gestodene dissociate very much more slowly from the binding sites of sex hormone-binding globulin than 3-ketodesogestrel. A natural affinity of all these synthetic progestogens tested for corticosteroid-binding globulin could not be established. Gestodene, levonorgestrel, and 3-ketodesogestrel bind to the progesterone, glucocorticoid, and androgen receptor with high affinity, apart from slight differences, whereas estrogen receptor affinity could not be demonstrated in any of the progestogens investigated. In relation to aldosterone, the relative binding affinity values of gestodene, levonorgestrel, and the natural progestogen progesterone are relatively high, whereas 3-ketodesogestrel does not display any measurable affinity for this receptor species. (AM J OSSTET GVNECOL 1990;163: 2171-83.)

Key words: Sex hormone-binding globulin, competition studies, steroid receptors, gestodene, desogestrel, levonorgestrel, norgestimate

A generally accepted classical principle of therapy with pharmacologically active substances is that the greatest possible effectiveness is to be reached at the lowest dosage. The idea behind this approach is based on the assumption that the lower the therapeutic dose, the less the burden on the body as a whole, especially in terms of possible adverse drug effects. With regard to oral contraceptives, efforts have been made over many years to reduce the overall amount of estrogenically and progestogenically active steroid components applied over the menstrual cycle and to find synthetic steroids that offer not only efficacy but also a large measure of tolerance. The proportion of estrogen in the oral contraceptives of the most recent generation has been reduced; for example, it is down to 30 f.Lg per pill in the most widespread form of ethinyl estradiol, above all to lower markedly the incidence of adverse drug effects caused From the Department of Experimental Endocrinology, Johannes Gutenberg University. Reprint requests: Kunhard Pollow, MD, PhD, Department of Experimental Endocrinology, Johannes Gutenberg University, Langenbeckstrasse 1, 6500 Mainz 1, Federal Republic of Germany. *This work is part of the PhD thesis of M. Juchem. 610124390

by estrogens. Regarding the gestagen constituent in oral contraceptives, however, there is no uniformity with regard to the steroid used or to the dosage. The synthetic progestogens most frequently used are levonorgestrel, desogestrel, gestodene, cyproterone acetate, and norgestimate. Thus levonorgestrel, the classic orally active synthetic progestogen, presents the advantage of balancing overshoot and adverse effects of the estrogen component besides providing a markedly reduced dosage. I The antiandrogenically active cyproterone acetate had the advantage of processing a highly antiandrogenic activity component, in addition to its potent gestagenic effects, which allowed an additional effective treatment in androgenization manifestations of the skin in potential users of oral contraceptives!'; Desogestrel,6.9 gestodene,IO·13 and norgestimate,11 further agents from the 19-nortestosterone series, which are now available for clinical application in oral contraceptives, appeared to set new standards with regard to activities and hormonal profiles. The dosage of progestogens used in oral contraceptives shows a large range of variation. However, a dose reduction of a certain progestogen is limited by its hormonal effectiveness, because the component used in oral contraceptives has to suppress ovulation reliably.

2172 Juchem and Pollow

December 1990 Am J Obstet Gynecol

Table I. Relative binding affinities (%) of various progestogens in comparison with cortisol and DHT for binding to human CBG and SHBG Cortisol DHT Progesterone Medroxyprogesterone acetate Cyproterone acetate Org2058 R5020 Levonorgestrel Gestodene 3-Ketodesogestrel Desogestrel Norgestimate

CGB

SHBG

100