Metabolism of steroid-modifying anticancer agents

Pharmac. Ther. Vol. 36, pp. 41 to 103, 1988

0163-7258/88 $0.00+0.50 Copyright © 1987 Pergamon Journals Ltd

Printed in Great Britain. All rights reserved

,Specialist Subject Editor: GARTFI POWIS

METABOLISM OF STEROID-MODIFYING A N T I C A N C E R AGENTS S. P. ROBINSONand V. C. JORDAN Department of Human Oncology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792, U.S.A.

1. INTRODUCTION Three decades before the first steroid hormone, estrone, had been isolated and the steroid nucleus identified (Marrion, 1930), a supportive role had been indicated for an ovarian product in breast cancer growth (Beatson, 1896). Studies on ovarian function started at the turn of the century when transplanted rabbit ovaries were demonstrated to be capable of stimulating uterine growth (Halban, 1900; Knauer, 1900). In 1905, Marshal and Jolly proposed that ovarian cells secrete a compound that induces estrus when administered to castrated rodents. The graafian follicle was later identified as a rich source of estrogenic activity (Allen and Doisy, 1923). Following the isolation of estrone from the urine of pregnant women by Marrion (1930), an extensive series of studies were performed by many laboratories on steroid metabolism. These studies often involved the administration of large doses of known crystalline steroids with the subsequent isolation and analysis of crystalline metabolites from urine or other body fluids. In this way, the conversion of pregnanediol to progesterone (Callow, 1939; Dorfman and Hamilton, 1939), estradiol-17fl to estrone (Fish and Dorfman, 1941) and deoxycorticosterone to pregnanediol (Westphal, 1942) was established. The application of new and improved techniques to separate steroid hormones (i.e. adsorption column chromatography, paper chromatography, partition colunms, thin layer chromatography and countercurrent distribution) and the development of new physical means to identify compounds (i.e. X-ray crystallography, optical rotationary dispersion, nuclear magnetic resonance, mass spectroscopy, gas chromatography and infrared spectroscopy) increased the sensitivity of the determination and allowed the elucidation of the steps in steroid biosynthesis (for detailed reviews, see Dorfman and Unger, 1965; Samuels and Eik-Nes, 1968; Briggs and Brotherton, 1970). However, one of the crucial advances in the study of hormone biosynthesis and action came with the use of radiolabeled compounds (deuterium, 14C or tritium). The use of labeled compounds in vivo and the perfusion of isolated glands with media-containing labeled precursors enabled the identification of specific biosynthetic pathways (see Doffman and Unger, 1965). With the information gathered from these types of investigations, biosynthetic pathways have been found for the steroid hormones in the ovary, testis and adrenal gland. These pathways have a common root and can be unified (Ryan, 1972) into a single model (see Fig. 1). During the years when these pathways were being elucidated, the treatment of cancer by ablative and additive therapies was being developed. Beatson, in 1896, was the first to show that oophorectomy could inhibit the growth of advanced breast cancer in some women. This effect was reproduced by many workers during the next five years. In 1940, Zondek attempted to inhibit the growth of breast cancer in a 26-year-old woman by the administration of 10 mg estradiol benzoate. The rationale for his investigation was that large doses of the estrogenic hormone produced dwarfing by inhibiting the release of 41

42

S . P . ROBINSON and V. C. JORDAN

growth hormone from the anterior pituitary and that estrogens inhibit gonadotrophic hormone secretion. Zondek thus attempted to suppress ovarian function by hormone castration. Haddow (1938) and colleagues (Haddow et aL, 1944) showed that large doses of different estrogens retard tumor growth and they considered that suppression of pituitary function was partially responsible for this general growth inhibiting effect. Gardner (1941) demonstrated that very large doses of estrogenic material administered to animals resulted in little or no breast development and suggested that this was caused by suppression of pituitary function by the estrogenic hormone. In 1952, Huggins and Bergenstal described the use of adrenalectomy as a further ablative therapy after castration for the treatment of both breast and prostate cancer. Adrenalectomy was made possible by replacement therapy using cortisone acetate to maintain salt balance. The authors speculated that the mechanism behind the positive results involved estrogenic and/or androgenic changes; however, at the time, details of hormone biosynthesis in the adrenals had not been established. Later, hypophysectomy was used to produce 'super castration'; this extreme ablative procedure achieved some further remission (Luft and Olivecrona, 1953). The elucidation of the early events involved in the growth of hormone-dependent tumors was enhanced by the development of tritium-labeled estrogens and androgens. In 1959, Glascock and Hoekstra demonstrated the selective accumulation of tritiated hexestrol, a synthetic estrogen, by the reproductive organs of female goats and sheep, and, in 1962, Jensen and Jacobson reported the specific uptake and retention of [6-73H]estradiol 17fl by the uterus, vagina and pituitary of immature rats. The finding that these target tissues could retain significant quantities of the hormone against a marked concentration gradient, whereas nontarget tissues such as diaphragm, muscle and kidney could not, prompted the assumption that a specific binding site was present in some organs. Toft and Gorski (1966) demonstrated the presence of a macromolecule capable of binding estradiol in the cytosol fraction of rat uterus using sucrose-density gradient centrifugation. The model proposed simultaneously by both Gorski's 0968) and Jensen's 0968) laboratories that estrogen action is mediated through receptor binding in target tissue prompted the examination of breast cancers for receptors. The determination of estrogen receptor in tumors is of more than academic interest to the oncologist. Use of additive or ablative endocrine procedures to treat advanced breast cancer achieves an overall objective response rate of only about 30%; however, since 70% of patients will not respond to oophorectomy or adrenalectomy, a predictive test for success is clearly of value. The use of estrogen receptor measurements to provide a predictive marker for the selection of therapy has been shown to be partially successful (McGuire et al., 1978). Very few tumors shown to have no estrogen receptors respond to endocrine therapy whereas 55-60% of estrogen receptor-containing tumors show objective tumor regression following similar therapy. Furthermore, the detection of estrogen receptors in primary tumors has been correlated with an extended disease-free interval compared with receptor-poor tumors.

CH s C O O H ACETATE

>

°

°

PREGNENOLONE

PROGESTERONE

HO

CHOLESTEROL

J

,k ADRENALS

TESTIS o

OVARY OH

OH

t5 CORTISOL

ANDROSTENEDIONE

TESTOSTERONE

ESTRADIOL 17/r

Fit;. 1. A general scheme to illustrate the common root of steroid hormone synthesis from cholesterol in different organs.

Metabolism of steroid-modifyinganticanceragents

43

The fact that about 45% of advanced estrogen receptor-positive breast tumors fail to respond to endocrine therapy has prompted further refinements of the test. It is known that progesterone receptor production is stimulated by estrogen via estrogen receptor activation (Freifeld et aL, 1974). Therefore, tumors with both estrogen and progesterone receptors would be expected to have a functioning estrogen receptor system and be hormone responsive. Studies by McGuire's group (McGuire, 1980) have shown that 77% of tumors with both measurable progesterone and estrogen cytosolic receptors will undergo objective remission to endocrine therapy, whereas only 11% of tumors with no cytosolic progesterone or estrogen receptors will respond to the same therapy. The 60-80% response rate of prostate tumors to hormonal therapy (Bailar et al., 1970; Barnes and Ninan, 1972) makes the development of predictive tests for the hormone dependency of this cancer unnecessary. However, androgen receptors have been measured in the male reproductive tract (Mainwaring, 1975; Mobbs et al., 1975) and in some prostate tumors (Mobbs et al., 1978) using tritium-labeled androgens. This brief historical outline describes how the general ideas behind endocrine therapy have developed. However, with the increased understanding of hormone-dependent cancers has come the development of new pharmacological agents to treat the disease. The treatment strategies that are commonly employed or that are attracting interest with new agents have been briefly surveyed in order to facilitate an understanding of the various (]rug groups that will be considered in detail later in the review.

2. TREATMENT STRATEGIES FOR HORMONE-DEPENDENT CANCERS The treatment of cancer is dependent upon the extent of spread of the disease. However, even with disease that apparently has no detectable spread, surgery alone is unable to cure

Hypothalamus Vx !

od

(3

"~O O~/D"

FIG. 2. Illustrationof the feedbackmechanismsbetween the hypothalamo-pituitaryaxis and the ovary or adrenal gland whichcontrol steroidogenesis.

44

S.P. ROBINSONand V. C. JORDAN

the patient. We have presented an historical overview to demonstrate the hormone dependence of breast and prostate cancer, but the surgical approaches of endocrine ablation to control breast or prostate cancer are not successful in all cases. The majority of breast cancer cases do not respond to oophorectomy or adrenalectomy, and adrenalectomy requires continuous treatment of patients with corticoid supplements. Unfortunately, this replacement therapy also has troublesome side-effects of its own. A significant disadvantage of the surgical approach to endocrine ablation is the fact that the surgical procedures are irreversible, whether they effect the patient's disease or not. This has prompted the development of a medical strategy which has the advantage of reversibility should the therapy be found to be ineffective. Furthermore, the specificity of the treatment often has a low attendant incidence of side-effects. The development of drugs to affect the regulatory mechanism of steroid synthesis, the enzymes directly involved in steroid biosynthesis or steroid receptor-mediated cell responses have all been used as treatment approaches to prevent tumor growth. For convenience, each target will be considered separately; however, some drugs interact through more than one target and sometimes an integrated approach to therapy which employs more than one agent is used. 2.1. REGULATIONOF STEROIDOGENESISTHROUGH THE HYPOTHALAMOPITUITARYAXIS In man and the premenopausal woman, the regulation of sex steroid synthesis is via the gonadotrophins secreted by the pituitary gland. In women, estrogen synthesis in the ovary is dependent on follicular development which is regulated by follicle stimulating hormone (FSH) and luteinizing hormone (LH) (see Fig. 2). The gonadotrophins act on the primary follicle causing maturation and the development of enzymes needed for estrogen synthesis.

HYPOTHALAMO-

PITUI

~ L H R H / ~ ~ AGONIST/ I vo , . \ ANTAGONIST

/11 I

AMINO- I GLUTETHIMIDE ANTI-

/

,~ ADRENAL

I

/

A~DROGEN PROSTATIC I

,MP"___.~.~CARClNOMA/

ANDROGEN~ " - ~

~//

TEiOSTER'~

TESTES

FIG. 3. Treatmentstrategies for prostratecancer: inhibitionof gonadotrophin release (represented by LHRH agonist/antagonist) or adrenal enzymes (representedby aminoglutethimide)is aimed at inhibiting steroidogenesis and reducing circulating androgen levels. Antiandrogens are used to block androgen action at the cellular level.

45

Metabolism of steroid-modifying anticancer agents

CHOLESTEROL

C=O LUTEAL PHASE

FOLLICULARPHASE HO CH I 3 C=O

CH 3 I

PREGNENOLONE

C=O

HO -~)H 17a-HYDROXYPREGNENOLONE

PROGESTERONE CH 3 I

O

~

II

DEHYDROEPIANDROSTERONE ~

0~ * ' ~ 7 a

- HYDROXYPROGESTHERONE

TESTOSTERONE

ANDROSTENEDIONE

I HO~

° K

ESTRONE

C=O j~ , ~i~-OH

>HO"

~ ~ ESTRADIOL

FIG.4. Biosynthetic pathway of ovarian steroidogenesis. LH causes ovulation of the mature follicle and the subsequent synthesis of large quantities of progesterone by the corpus luteum. In men, testosterone production is predominantly by the Leydig cells of the testis and is regulated by LH (see Fig. 3). Regulation of gonadotrophin release is via a sex steroid feedback mechanism on the pituitary gland and hypothalamus. Hypothalamic stimulation of gonadotrophin production and release by the pituitary gland is by luteinizing hormone releasing hormone (LHRH) (see Figs 2 and 3). A strategy used to produce medical castration is to prevent the release of the gonadotrophins by the pituitary. One approach in men is to administer pharmacological doses of estrogens (diethylstilbestrol) which causes a rapid decrease in the circulating levels of LH and a decrease in circulating testosterone to castrate levels (Leuprolide Group, 1984). Unfortunately, long-term high-dose estrogen therapy is associated with severe side-effects (breast enlargement, nipple pain and thromboembolic disorders). These side-effects have focused current attention on the use of continuous administration of synthetic LHRH agonists to regulate LH secretion (Fig. 3). These drugs, unlike estrogens, cause an initial elevation of circulating LH and testosterone levels, but eventually (within 2 weeks) the pituitary becomes desensitized to the constant stimulation and LH release is inhibited

46

S.P. ROBINSON and V. C. JORDAN

(Leuprolide Group, 1984). Circulating testosterone ultimately drops to castrate levels. Although most research has been conducted in patients with prostate carcinoma, this type of medical castration has also been found to be effective at lowering estrogen levels in the premenopausal breast cancer patient (Nicholson et al., 1984). 2.2.

STEROIDOGENESIS AND ITS BLOCKADE

The biosynthetic pathways of the sex steroids for the ovary, testes and adrenal glands are shown in Figs 4, 5 and 6. In postmenopausal women, the biosynthesis of estrogens by the ovary has stopped; however, the synthesis of androgens still occurs in the adrenal gland and peripheral aromatizing enzymes are capable of producing significant circulating levels of estrogens from androstenedione. There is current interest in the development of drugs to regulate adrenal steroidogenesis (Fig. 7). Aminoglutethimide (with hydrocortisone to inhibit ACTH production) blocks the synthesis of pregnenolone in the adrenals and the aromatization of androstenedione to estrone in the peripheral tissues (Stuart-Harris and Smith, 1984; Santen and Brodie, 1982). The drug has, however, significant side-effects (e.g. drowsiness and rash) and hydrocortisone must be administered to replace essential corticoids. Hydrocortisone is also essential to prevent a reflex increase in ACTH from occurring which would overwhelm the aminoglutethimide blockade in the adrenals (Santen and Brodie, 1982). The alteration of adrenal physiology is clearly a therapeutic disadvantage and this

CHOLESTEROL

I

oEL-rA-,

CH3

I

CH3

- ~oo |.

cl=o HO"~,,"'%J

PROGESTERONE

,

oE~,..~

| PREGNENOLONE CH3

]

Cl=O

17el-HYDROXYPREGNENOLONE

lo

17=-HYDROXYPROGESTER O N E

lo

A4-ANDROSTENEDIONE OH

DEHYDROEPIANDROSTERONE

ANDROSTENE DIOL

O~TERONE FIO, 5. Biosynthetic pathway of tcsticular androgen stcroidogcnesis.

Metabolism of steroid-modifyinganticanceragents

47

has stimulated a search for agents that block only peripheral aromatization enzyme systems without affecting adrenal steroidogenesis (Fig. 7). There are several 'suicide inhibitors' of the aromatization enzyme system but only one compound, 4hydroxyandrostenedione, has entered clinical trial (Coombs et al., 1984). It should be pointed out that these studies all pertain to postmenopausal women. Aminoglutethimide is ineffective in inhibiting estrogen production by the ovary of the premenopausal woman (Santen et al., 1980). Finally, it should be mentioned that there is also current interest in the antifungal agent, ketoconazole. Studies are still preliminary, but the compound affects testicular and adrenal steroidogenesis which indicates a potential role in the treatment of prostate cancer (Trachtenberg, 1984). 2.3. HORMONEANTAGONISTS A model for the cellular interaction of estradiol with an estrogen-dependent tumor cell is shown in Fig. 8. Circulating estradiol dissociates from plasma proteins and diffuses into

Cholesterol

17~ Hydroxypregnenolone

Pregnenolone

Dehydroepiondrosterone

$

$ 0

>o

O~

>o

17K Hydroxyprooesterone

Progesterone

Androstenedione

O@OH

OH 0

0

II Deoxycortisol

21Hydroxyprogesterone (11Deoxycorticosterone)

OH Testosterone

$

$

O~ •

Corticosterone

OH

..OH

Cortisol

(Hydrocortisone)

FIG. 6. Biosyntheticpathwayof adrenal steroidogenesis.

48

S.P. ROBINSONand V. C. JORDAN

f ANTIESTROGEN

E/STROGEN IDROSTENE-~ ~ DIONE ~

AMINOGLUTETHIMIDE

INHIBITOR

FIG. 7. Treatment strategies for breast cancer in postmenopausal women: inhibition of adrenal enzymes (represented by aminogiutethimide) or peripheral aromatization (shown as aromatase inhibitor or aminoglutethimide) are aimed at reducing circulating estrogens. Antiestrogens are used to block estrogen action at the cellular level. I

the cell where it binds to the estrogen receptor. The localization of the unoccupied estrogen receptor has recently become the subject of some debate. However, several lines of evidence suggest that the receptor is located in the nucleus rather than the cytoplasm (Welshons et al., 1984; King and Greene, 1984). Be that as it may, the resulting estradiol-estrogen receptor complex apparently becomes activated and binds to, as yet unidentified, nuclear acceptor sites. This interaction results in a cascade of subcellular events and the expression of an estrogenic response. Antiestrogens such as tamoxifen block the binding of estrogen to estrogen receptors and inhibit estrogen stimulation. There are currently several strategies for the endocrine treatment of breast cancer in postmenopausal patients. High-dose estrogen, androgen or progestin therapy appear to have a direct effect upon the replication of breast cancer cells, but the exact mechanism of action is unknown. Antiestrogen (tamoxifen) therapy is now the treatment of choice in postmenopausal patients because it has a low incidence of side-effects (Furr and Jordan, 1984) and has largely replaced adrenalectomy. Tamoxifen effectively blocks the binding of the small, but significant, quantities of estrogen that are produced by the peripheral aromatization of androstenedione produced by the adrenals (Fig. 7). An antiestrogen also blocks the action of any exogenous estrogens such as phytoestrogens from the patient's diet (Jordan et al., 1985). The use of antiestrogens for breast cancer therapy in the premenopausal patient has also been examined (Sawka et al., 1986). However, in concert with an initial tumor response is a stimulation of the ovary to increase estrogen production. This stimulation is apparently without a significant rise in circulating gonadotropins (Sherman et al., 1979). After the initial response, tumors regrow, and many of these patients subsequently respond to oophorectomy (Sawka et al., 1986). This suggests that the failure of tamoxifen results from an overproduction of ovarian estrogen which reverses the blockade of estrogen receptors by antiestrogens in the tumor. A model similar to the estrogen receptor in estrogen target tissues can be used to describe the androgen receptor in androgen target tissue. In this case though, there is first

Metabolism of steroid-modifying anticancer agents

/-Cytoplasm

~\ ~\

49

Protein Synthesis mRN¢ 1 progesterone

~,

\~

Receptor

R'°°'7°' Resy.the$is

PE~--~ P+E.

" EiR,E. . . . . . .

i Proliferation • Cell

I Traneformotion Troneloeetion Model

E f

Cytoplasm

~ ~,

Protein '~ Synthesis J

,, ~

/

P+E~-

/

~

~

=

tuRN P,og,,,.....

RNA~

Receptor

==E~/E +R~-~ P°l;~eArase~ / /

Proc;ssing P

/ ER ~ '

/

Ace'tp'°' ) P o l y r n e r ~

\\%.\

"Proliferation

ase/

Model 2

Protein _ Synthesis ''~

/

~ /

R

X~~

r

N

~ 1

A

Autocrine GrowthFactors (GF)

~ i= ~

~7"

"Pi'O'lileroti°n •

II

Cytoplasm J Model 5 i

FIG. 8. Models to describe the subcel]ular mechanisms of estrogen (E) action. E dissociates from plasma sex hormone binding globulin (P) and diffuses into target tissue cells. E binds to estrogen receptors (R) in the cytoplasm (Model I) (Gorski et el., 1968; Jcnsen et el., 1968) or the nucleus (Model 2) (King and Greene, 1984; Welshons et el., ]984). The estrogen receptor complex (ER) is activated (*) (transformation) before initiating the events associated with estrogen action. RNA polymerase is activated to synthesize proteins, e.g. progesterone receptor. DNA polymcrasc is activated to cause cell division• Current research (Lippman et el., 1986) suggests that cell division occurs through an autocrine mechanism and the activation of growth factor receptors (GFR) (Model 3). JPT 36/1--D

50

S.P. ROBINSONand V. C. ,JORDAN

a metabolic activation of testosterone in the target cell cytoplasm to a high affinity ligand (dihydrotestosterone) before binding occurs to the receptor (see Fig. 9) (Mainwaring, 1975). The localization of unoccupied androgen receptors has not been studied in detail so their subcellular compartmentalization must remain in question. The direct effects of androgens to stimulate prostate tumor growth can be inhibited with antiandrogens that block the androgen receptor. There is also the potential of using drugs that prevent the conversion of testosterone to dihydrotestosterone. Compounds have been identified that block 5~-reductase activity but none is available for clinical use (Petrow et al., 1983, 1984). There are, however, clinically available steroidal and nonsteroidal antiandrogens. Unfortunately, this approach to antihormonal therapy in endocrinologically normal men may inadvertently cause a rise in circulating androgens by an inhibition of the negative feedback mechanisms by androgens in the hypothalamo-pituitary axis (Neumann and Jacobi, 1982). As a result, the pituitary increases LH output to stimulate additional steroidogenesis in the Leydig cells of the testes. The increase in circulating testosterone can then reverse the effects of the antiandrogen in the tumor. One approach to avoid the problem is to administer LHRH agonists and an antiandrogen (Labrie et al., 1983a). The continuous administration of the L H R H agonist causes a decrease in testicular androgen production and the antiandrogen blocks the binding of adrenal, or residual testicular, androgen to androgen receptors in the prostatic carcinoma cells. The role of steroid-modifying agents in the treatment of cancer has increased during the past decade with the success of the antiestrogen, tamoxifen. This success together with the finding that combination chemotherapy is unable to cure breast and prostate cancer has resulted in a re-evaluation of antihormonal strategies. As a consequence, the medicinal chemist is now being asked to design a new generation of drugs to inhibit steroidogenesis selectively or to block the actions of steroid hormones. Development of these agents for unrestricted clinical use requires an investigation of their pharmacokinetics and metabolism. The importance of these studies is increased with the realization that treatment may be required for prolonged periods or indefinitely if tumor relapse is to be avoided (Jordan, 1983). It is the aim of this chapter to review the metabolism of some of the agents used as therapeutic agents in the treatment of hormone-dependent disease. Where possible, every effort has been made to point out the relevance of potential metabolic routes on the overall pharmacology of the parent compound.

1

Cytoplasm

~

Protein Synt~sis

~DHT÷AR RNA~ ,/~//~\ {. Polymerose ] / \ ~ DNA J /'~ \ DH T - A R ~ P o l y m D N A J / \\

P T ~ P*T;~--- ~

[

.

Ch/ o--Testosterone(T)

[ LOWAffinity fOr._AR

l•ce. Proliferation

-;o/-M, Olhydrotestolterone High AlfiDnHtT~}for AR

I

FIG. 9. Model to describe the subcellular mechanism of androgen (T) action. T dissociates from plasma proteins and is converted to dihydrotestosterone (DHT) by the enzyme 5~-reductas¢. DHT has a high affinity for the androgen r ~ p t o r (AR). (The subccllular location of the unoccupied receptor has not been established.) The DHT-AR complex then initiates the events associated with androgen action (Mainwaring, 1975).

Metabolism of steroid-modifying anticancer agents

51

3. INHIBITORS OF GONADOTROPHIN RELEASE 3.1. LUTEINIZING HORMONE RELEASING HORMONE ANALOGS Luteinizing hormone releasing hormone (LHRH) is a decapeptide (see Fig. 10) found in the hypothalamus of mammals (e.g. sheep, pigs, baboons, dogs and man). LHRH acts on the pituitary gland via an adenylate cyclase-linked receptor system to stimulate the production and release of both LH and FSH. Analogs of LHRH were initially investigated with the intent of developing an antagonist as an antifertility agent. The finding that potent agonist analogs of LHRH would prevent puberty when given chronically to immature female rats and would cause ovarian and uterine atrophy in mature rats (Johnson et al., 1976) prompted the examination of the effects of these agents on hormone-dependent tumors. Administration of 5 or 20/~g/day of the agonist analog leuprolide (see Fig. 10) to rats bearing DMBA-induced mammary tumors was essentially as effective in causing tumor regression as surgical ovariectomy (DeSombre et al., 1976). The treatment of metastatic breast cancer in premenopausal women with L H R H analog agonists has also had some success. Klijn and DeJong 0982) reported that two out of four patients showed improvement after 7 weeks of treatment with the L H R H analog agonist Buserelin (see Fig. 10). However, the ovarian function did not decrease until 1-2 weeks of treatment though subsequent plasma estradiol concentrations were reduced to postmenopausal levels. Gonadotrophin levels were initially increased following the administration of Buserelin and gradually fell to below pretreatment levels after 2 weeks. Very low plasma levels (1 or 2 IU/1) were, however, not reached. More recently, the same authors have reported the treatment of 31 premenopausal patients with metastatic breast cancer with Buserelin. They found Buserelin therapy as effective as surgical castration: 9 out of 22 patients (41%) treated with Buserelin alone showed an objective response (Klijn and DeJong, 1984). Similar findings for hormonal changes seen with Buserelin treatment have been observed using the L H R H analogs, Zoladex (Fig. 10) (Nicholson et al., 1984) and leuprolide (Fig. 10) (Harvey et al., 1984). Prolonged treatment with L H R H analogs in the male results in a chemical castration. Following initial therapy with L H R H analogs, such as Zoladex, Buserelin or leuprolide, an initial rise in LH has been observed with a corresponding increase in testosterone levels. This rise in testosterone may result in a 'flare' of the disease. After 2-4 weeks of treatment, LH levels are markedly lower than control, and testosterone levels are down to those observed in castrate patients (see Schally et al., 1984; Tolis et al., 1983, Nicholson et al., 1984).

I

Z

3

4

5

6

Pyro Glu - His- T r p - S e r - Tyr -

Gly

7

8

9

iO

- L e u - A r g - P r o - G l y NH=

LHRH Pyro Glu- His- T r p - S e r - Tyr - D-Ser(But)-Leu - A r g - P r o - N H

ET

Hoe 766 (Buserelin) Pyro

G I u - H i s - T r p - S e r - Tyr -

D-Leu

- L e u - A r g - P r o - NH ET

A 43818 (Leuprolide) Pyro G l u - His- T r p - S e r - T y r - D-Ser (Bu t ) - Leu - A r g - P r o - A z G l y N H 2

IC1118630 (Zolodex) PyroGlu-His-Ttp-Ser-Tyr-

D-NoI(2) - L e u - A r g - P r o - G l y N H =

Nafarelin Fro. 10. The amino acid sequence of luteinizing hormone releasing hormone (LHRH) and synthetic LHRH agonists.

52

S.P. ROBINSONand V. C. JORDAN

The response of prostate cancer to treatment with LHRH analogs is similar to those observed after castration or estrogen therapy but without the serious side-effects (Schally et al., 1984; Tolis and Koutsilieris, 1984). The mechanism by which LHRH analogs produce their antitumor action in humans is uncertain. Reduced estradiol or testosterone levels almost certainly play an important role in this action. In the rat, evidence has been obtained to suggest LHRH is not only produced in the hypothalamus but also in the ovary and testes and has a regulating role in these organs (see Sharpe, 1982, for review). Inhibition of steroidogenesis in this species may thus be via direct effects on the ovary or testes. However, in the human, this does not appear to be the case (Casper et al., 1984; Richardson et al., 1984), and evidence suggests that neither LHRH nor its analogs directly affect granulosa or luteal cell function. Studies in rhesus monkeys (Belchetz et al., 1978) and rat pituitary cell cultures (Smith and Vale, 1981) indicate continuous exposure to LHRH is unable to maintain the release of FSH and LH. This pituitary desensitization can explain the reduced circulating LH and FSH levels following long-term treatment with LHRH agonist analogs and the resulting reduction in steroidogenesis which occurs during the treatment of metastatic breast and prostate cancer (Klinj and DeJong, 1982; Nicholson et al., 1984). A sustained circulating level of active agent must therefore be maintained for effective antitumor activity. LHRH itself has limited usefulness as a therapeutic agent because of its short half-life (Bennett and McMartin, 1979). Through studies of the routes of inactivation and structure-activity relationships, agents with considerably greater biological potency have been developed. 3.1.1. Development of L H R H Analogs: Structure-Function Relationships

The development of LHRH analogs with increased biological activity has been a progressive process involving the synthesis of many hundreds of derivatives. Systematic studies have allowed optimization of various characteristics. For example, derivatives with amide substitutions of the C-terminal of LHRH are only weakly active whereas maintenance of a peptide length similar to that of LHRH by synthesis of alkyl amides of the LHRH nonapeptide produces the ethylamide derivative which is six times as potent as LHRH itself (see Nestor, 1984). Substitution for Gly6 of LHRH with o-amino acids increases the biological activity. These structural changes correspond with an increased receptor binding affinity and decreased susceptibility to enzyme degradation when tested with brain preparations (Griffiths and Hopkinson, 1979; Horsthemke et al., 1981). The first anticancer studies in vivo were performed using an LHRH analog which had the Gly6 of LHRH substituted with o-Leu and the C-terminal Gly substituted with ethylamide (leuprolide, see Fig. 10) (DeSombre et al., 1976). This agent is now in clinical use for the treatment of prostate cancer. Modification at the Gly6 position of LHRH by incorporation of o-Trp produces one of the most active analogs with a natural amino acid substitution (Nestor et al., 1984a). Substitution of the Gly 6 of LHRH by incorporation of the tertiary butyl ester of D-serine ([o-Ser(tBu)]) combined with the ethylamide modification gave the highly potent analog, Buserelin (see Fig. 10). This derivative is 140 times more potent than LHRH for induction of ovulation in the rat compared with 80 times for leuprolide (Nestor, 1984). Buserelin is undergoing clinical trials for the treatment of hormone-dependent breast cancer and cancer of the prostate (Klijn and DeJong, 1984; Wenderoth and Jacobi, 1984). Incorporation of aza-Gly in Buserelin instead of the ethylamide moiety produces the analog Zoladex ([o-Ser(tBu)6,aza-Gly~°]LHRH) (Fig. 10). This agent is seven times more potent than Buserelin in the ovulation induction assay (Nestor et al., 1984a). Zoladex is undergoing clinical trials for the treatment of both breast and prostate cancer (Nicholson et al., 1984). Further structure-activity studies of LHRH by substitutions in the 6 position with novel hydrophobic 'amino acids' has been examined. This alteration increases binding to plasma membranes and hydrophobic carrier proteins and results in reduction of renal

Metabolism of steroid-modifyinganticancer agents

53

clearance. For example, introduction of the strongly hydrophobic 3-(2-naphthyl)-D-alanine([Nal(2)]) in place of Gly 6 produces the very potent analog, Nafarelin (see Fig. 10). Nafarelin ([NaI(2)]6LHRH) is currently being evaluated in clinical trials for the treatment of breast and prostate cancer. Substitution with azo-Gly l° in Nafarelin produces one of the most potent analogs developed to date (Nestor et al., 1984b). 3 1.2. Routes of Administration

Enzymatic degradations and/or lack of absorption from the gastrointestinal tract makes oral administration of L H R H analogs inappropriate. As a result, various alternative routes of administration have been examined. These include nasal, vaginal, rectal, intramuscular and subcutaneous injection (see Anik et al., 1984). The analogs so far developed for clinical use in the treatment of hormone-dependent cancers are administered by injection. Luprolide acetate has been marketed as Lupron ® for use by subcutaneous injection in the treatment of prostate cancer. To avoid the discomfort of daily injection, sustained-release preparations are being investigated. Clinical trials are being performed with Zoladex in a copolymer depot preparation which allows sustained release of the L H R H analog over a 28-day period following subcutaneous implantation (Ahmann et al., 1986). Apart from the convenience of sustained-release preparations for the patient, the formulation also ensures compliance with the treatment regimen. Several studies have used the nasal route of administration; however, the variable absorption rate may make long-term therapy unreliable. The lack of oral activity limits the usefulness of these peptides; however, the development of orally active agents such as CGP 19986 (see nonsteroidal (nonpeptide) inhibitors of gonadotrophin release) may open up a new line of investigation for the medicinal chemist. 3.1.3. Clearance and Distribution of L H R H and Analogs

Studies in the rat, dog, pig and human have shown L H R H to be rapidly cleared from the circulation. The clearance, estimated after a single intravenous injection, has been found to have a two-component half-life: the initial component with a time of 2-12 min and the second component of about 25-60 min. However, the bolus injection of compound used to estimate the clearance in many of the reports may result in misleading estimates of half-lives because rapid distribution outside the vascular circulation is still occurring during early measurements (for reviews see Bennett and McMartin, 1979; Griffiths, 1976). The removal of the GIy--NH2 terminal of L H R H and introduction of ethylamide combined with substitution of Gly 6 with D-Ser(tBU) (Buserelin) or D-ser produces analogs with resistance to enzyme degradation; however, these alterations have not been found to prolong the circulating half-life following i.v. injection to sheep (Swift and Crighton, 1979). In contrast, metabolic clearance studies in humans using an infusion of the highly active analog [D-Trp6]LHRH show a significantly longer tl/2 for this analog than L H R H (Barron et al., 1982). Studies using Nafarelin, which has a strongly hydrophobic substitution at position 6, have demonstrated a longer half-life than L H R H in the rat (33.6 min vs 6.7-7.5 min), monkey (120 min vs 33 min) and human (120 min vs 20-450 min) (Chan and Chaplin, 1985a). The ratio of bound to free drug determined in plasma for Nafarelin by equilibrium dialysis is about 80:20 whereas for L H R H it is only about 25:75 (Chan and Chaplin, 1985a; Nestor et al., 1984b). The binding to albumin has been shown to play an important role in the high plasma binding of Nafarelin, presumably as a consequence of the hydrophobicity of this compound. This property probably contributes to the decreased clearance of the analog (Chan and Chaplin, 1985a). Buserelin and leuprolide on the other hand are bound to about the same extent as L H R H in rat and human serum (Sandow et al.,

54

S.P. ROBINSONand V. C. JORDAN

1984; Tharandt et al., 1979) and have similar plasma half-lives to LHRH (Okada et al., 1984; Swift and Crighton, 1979). Plasma elimination of immunoreactive Buserelin in the beagle after i.v. injection of 5 mg has been reported to show a multi-exponential decline, with an initial half-life of 3.1-3.7 min followed by an increasing half-life of between 60 and 180 min. Biological activity in the plasma on the other hand decreased more rapidly (Sandow et al., 1984). Studies examining the distribution of tritium-labeled LHRH in rats and mice show an accumulation of radioactivity in the anterior and posterior pituitary, pineal gland, kidney and liver (Redding and Schally, 1973; DuPont et al., 1974). Sandow and Konig (1979) reported accumulation of 125I-labeled Buserelin in the anterior pituitary, kidney and liver of 400 g male rats following i.v. administration, and Chu et al., (1985) observed high concentrations of radioactivity in the kidney, liver and intestine of male rats treated i.v. with [14C]Nafarelin. These studies indicate a similar distribution between LHRH and its analogs. Analysis of tissue samples from male rats following injection of [125I]Buserelin showed that metabolism in the liver and kidney was to the 4-9 hexapeptide (Sandow et al., 1980). Urinary excretion of immunoreactive Buserelin in the rat has been reported to be 30% of the dose administered, whereas the excretion of immunoreactive LHRH averaged only 0.23% (Sandow et al., 1984). This may be a consequence of the greater stability of Buserelin which prevents metabolism in the kidney to nonimmunoreactive components. Urinary excretion of Buserelin metabolites in the dog, on the other hand, reaches 20.7% of the dose administered (5 mg i.v.) by 23 hr, but the concentration of the agent corresponding to Buserelin in HPLC fractions represents only 2.4% of the dose administered (Sandow et al., 1984). Species differences in the handling of LHRH analogs are also indicated by the finding that most ( > 80%) of the radioactivity from [~4C]-labeledNafarelin acetate is eliminated in the urine following administration to monkeys whereas only about one-third is eliminated in the urine in rats (Chu et al., 1985). These authors reported that remaining radioactivity is excreted in the bile. The rapid accumulation of LHRH and its analogs in the kidney and urine indicates a dominant role for this organ in the clearance of these peptides. This is further supported by an increased half-life of LHRH observed in patients with renal failure (Pimstone et al., 1977). Detailed studies of renal metabolism of LHRH have been performed primarily in the rat and rabbit. Metabolism in kidney proximal tubule brush border has been demonstrated to result in the formation of Pyro Glu--His, Pyro Glu--His--Trp and Pyro Glu--His--Trp--Ser from LHRH and the additional intracellular metabolism to produce Pyro Glu (Carone et al., 1982; Stetler-Stevenson et al., 1981, 1983). Analysis of urine from female rhesus monkeys after a single i.v. injection of [14C]Nafarelin acetate has identified five major metabolites. Four of these metabolites were identified as short peptides: 5-10 hexapeptide amide, 6-10 pentapeptide, 5-7 tripeptide and the 6-7 dipeptide. The fifth metabolite, which accounted for 15% of the radioactivity, was identified by NMR and mass spectroscopy to be 2-naphthylacetic acid (Chan and Chaplin, 1985b). An important step in the inactivation of Nafarelin acetate in the rhesus monkey involves the cleavage of the Ser4 Tyr 5 bond. Chan and Chaplin (1985b) suggested this step may be performed by the same enzyme which cleaves the native LHRH in the kidney. Further breakdown may then follow by aminopeptidase activity. It should be pointed out that some of the Nafarelin acetate metabolites identified in the urine are also found in the plasma (Chan and Chaplin, 1985b). In patients receiving high-dose Buserelin to treat prostate cancer, both the parent compound and a second nonbiologically active metabolite have been observed in urine samples. The metabolite has not, however, been identified. Only the parent compound is found in the plasma (Sandow et al., 1984).

Metabolism of steroid-modifyinganticancer agents

55

3.1.4. Tissues That M e t a b o l i z e L H R H

L H R H has blocked N(pyro--Glu l) and C(GIy--NH2 l°) terminals making the peptide resistant to conventional amino or carboxy-peptidase degradation. However, degradation of L H R H by several tissues including kidney, liver, hypothalamus, pituitary, lung and skeletal muscle has been observed. Reports suggest that if plasma can cause the degradation of LHRH, then this occurs only at a very slow rate (for review see Bennett and McMartin, 1979; Griffiths, 1976). Rapid enzymatic degradation of synthetic L H R H has been reported in both the supernatant and particulate fractions of hypothalamic and pituitary homogenates. The enzymes found in these different tissues have been demonstrated to have similar characteristics (for reviews see Griffiths and Kelly, 1979; Griffiths and McDermott, 1983). Early studies demonstrated the LHRH-degrading activity in the hypothalamus was altered by hormonal manipulation such as castration or injection of gonadal steroids (Griffiths et al., 1975b; Kuhl et al., 1978) and that there was an uneven distribution of peptidase activity from different areas (Advis et al., 1982a). This suggests a potential functional role for the enzyme system in regulating L H R H in the hypothalamus. Studies examining the subcellular distribution of LHRH-degrading enzymes indicate substantial activity to be found in nerve endings. This activity is mainly found in the soluble fraction of the cells rather than the membrane fraction (Parker et al., 1979; Joseph-Bravo et al., 1979; McDermott et al., 1983). Investigation into the fate of the radiolabeled L H R H agonist, [D-AIa6]des-GlyI°LHRH ethylamide, taken up in rat pituitary gonadotrophs in vivo indicated accumulation and degradation by lysosomes and Golgi apparatus. It was speculated this may be a consequence of binding to the receptor then receptor-mediated endocytosis followed by transportation to the Golgi apparatus or lysosomes (Duello et al., 1983) Studies on the degrading enzymes have identified an endopeptidase which acts on the central region of the decapeptide (Griffiths et al., 1975a). This neutral endopeptidase was initially believed to cleave at the Gly6~Leu 7 bond of LHRH (see Griffiths and Kelly, 1979). However, more recent studies indicate the TryS--Gly 6 bond is more likely the site of deactivation, although some evidence still supports the Glyr---Leu 7 bond as the site of cleavage for the enzymes in the pituitary gland (see Griffiths and McDermott, 1983, for review). A postproline cleaving enzyme that is thiol dependent has been identified which acts on the Prog~Gly--NH2 ~°bond. This enzyme has a high affinity (Kin---2.3 #M) for L H R H and may be the main enzyme responsible for L H R H degradation in the pituitary (see Griffiths and McDermott, 1983). The enzyme, pyroglutamate aminopeptidase, found in the brain, has been shown to degrade L H R H by removal of the N-terminal pyroglutamate (Horsthemke et al., 1981) Studies using analogs of L H R H have indicated the importance of neutral endopeptidase and the postproline cleaving enzymes in the deactivation of LHRH. Analogs with D-amino acid substitution for Gly 6, such as D-Ala or D-Ser(tBu), and the C-terminal Gly--NH2 l° replaced by ethylamide make the agent more resistant to enzyme degradation than L H R H (Griffiths and Hopkinson, 1979; Horsthemke et al., 1981). However, the reistance to hypothalamic or pituitary degradation or increased binding affinity for the L H R H receptor has not necessarily resulted in an increase in biological activity in vivo (Swift and Crighton, 1979). Since only a small fraction of the circulation passes through the pituitary and hypothalamus, there would be little effect of these tissues on the blood levels of L H R H or its analogs. However, the enzymes may influence intra-tissue concentrations and play a role in the control of hormone binding to specific receptors in vivo (Clayton et al., 1979). Analysis of anterior pituitary tissue following the administration of ~25I-labeled Buserelin showed the presence of an unknown metabolite, possibly the 2-9 octapeptide (Sandow et al., 1980). This demonstrates that L H R H analog deactivation does occur even though these agents are less susceptible to degradation.

56

S.P. Rom~soN and V. C. JORDAN

3.1.5. Activity of Peptide Fragments

The replacement of the C-terminal glycinamide of LHRH with ethylamide stabilizes the peptide to degradation without loss of biological activity (Grifliths and Hopkinson, 1979; Horsthemke et al., 1981). However, removal of the C-terminal glycinamide without substitution substantially reduces the ability of LHRH to cause LH release in vivo (Griffiths et al., 1975a). Removal of Prog---GIy--NH2 l° leaving the LHRH (1-8) octapeptide decreases the biological activity to 0.01% of the complete LHRH decapeptide (Mohaham et al., 1972). Studies performed on Buserelin indicate not only a reduced susceptibility to metabolism but also the retention of biological activity of some of the metabolites. Buserelin has been demonstrated to be metabolized to the 4-9 hexapeptide and perhaps the 2-9 octapeptide (Sandow et al., 1980). Sandow and Konig (1979) showed the induction of ovulation in phenobarbitone-blocked rats can be achieved by Buserelin fragments 3-9, 4-9, 5-9 and even 6--9, although more compound is required as the peptide is shortened. In contrast, the removal of the comparable N-terminal amino acids of LHRH to produce the 3-10 octapeptide results in an almost complete loss of activity (Sandow et al., 1976). These differences between LHRH and Buserelin fragments may result from the resistance of the Buserelin fragment to further degradation. The relevance of this to the greater activity of the analogs of LHRH is uncertain. 3.1.6. Assay Procedures Determination of LHRH degradation was first assessed using bioassay procedures such as stimulation of ovulation in androgen-sterilized rats (Griffiths et al., 1973) or stimulation of LH production in vivo determined by radioimmunoassay (Griffiths et al., 1973; Griffiths et al., 1975a). Unfortunately, these procedures require large quantities ( > 100 ng) of compound and, like all procedures in vivo, cannot quantitate the contributions of active metabolites. Radiolabeled LHRH or its analogs has permitted detection of low concentrations of these agents in body tissues and urine (Redding and Schally, 1973; Sandow et al., 1980; Chu et al., 1985). Furthermore, chromatographic or column separation of tissue extracts following administration of radiolabeled compounds has allowed separation of metabolites (Redding et al., 1973; Berger et al., 1982; Stetler-Stevenson et al., 1983; Sandow et al., 1980; Chan and Chaplin, 1985b). Production of antibodies to LHRH or LHRH analogs provides a sensitive assay (picograms) to measure circulating levels (Nett et al., 1973; Jeffcoate et al., 1974; Arimura et al., 1973; Sandow et al., 1984). However, cross-reactivity of antibodies between parent compound and fragments (Berger et al., 1982; Griffiths and McDermott, 1983) makes experiments to study metabolism using this method difficult to interpret unless antibodies to different regions are used together or antibodies that have demonstrated specificity for only the parent compound are used (Barron et al., 1982). The use of HPLC procedures to separate and to identify metabolites has resolved many of the difficulties in metabolite determination (Advis et al., 1982b; Krause and McKelvy, 1983). The addition of amino acid analysis of peaks eluted from the HPLC has allowed full determination of degradation products (Krause et al., 1982; McDermott et al., 1982). 3.2. NONSTEROIDAL (NONPEPTIDE) INHIBITORS OF GONADOTROPHIN RELEASE

Paget and coworkers (1961) reported that some derivatives of dithiocarbamoylhydrazine inhibit pituitary gonadotrophin function in rats, dogs and rhesus monkeys. The most active compound was methallibure (ICI 33,828) (Fig. 11). Subsequent studies demonstrated that methallibure (50 mg b.i.d.) depressed ovarian activity and inhibited ovulation in premenopausal women (Bell et al., 1962), but larger doses

Metabolism of steroid-modifyinganticanceragents

57

(125-660 mg) depressed gonadotrophin output in postmenopausal women. Methallibure was found to inhibit gonadotrophin activity, notably depressing pituitary FSH levels in rats (Brown, P. S., 1963). Although the initial application of methallibure was for the regulation of estrous cycles in farm animals (Polge, 1965), the drug was also shown to inhibit rat mammary cancer induced by 3-methylcholanthrene (Shay et al., 1964). Clinical trials were initiated in England for the treatment of patients with breast and prostate cancer. Unfortunately, these had to be discontinued because of severe side-effects (nausea, drowsiness and lassitude). Ten years later a structurally related agent, GP 48989, (Fig. 11) was shown to have antitumor activity in the DMBA-induced rat mammary carcinoma model. Interestingly enough the compound apparently was active against both hormone-dependent and 'independent' mammary tumors (Schmidt-Ruppin et al., 1973, 1976). Unfortunately, GP 48989 was not active against a transplantable hormone-independent rat mammary tumor (Jordan et al., 1979). However, the same study showed that circulating LH levels were reduced in ovariectomized rats treated with GP48989. An interesting feature of the drug, GP 48989, is the low toxicity in laboratory animals. This fact, as well as oral activity, prompted further investigation of structure-activity relationships. A successor drug, CGP 19984 (Fig. 11), with greater solubility than GP48989, has recently been shown to be active against the androgen-dependent Dunning R3327 rat prostate adenocarcinoma and the MTW-9B rat mammary tumor (Ip et al., 1986). The study showed that CGP 19984 inhibits gonadal function and reduces LH release. Since the drug is orally active, there is the potential of clinical applications for the treatment of prostate and possibly breast cancer.

TABLE l. Methallibure and Principal Metabolites Isolated from Cow and Gilt Urine

Following the Oral Administration of Radiolabeled Compound Reference No.

Structure

Parent

CH~ S S I II II CH- CHNH- CNH NH C NHCH3 II CHa

1

N--N " /-NHCHa II CH2:CH-CH-NH-C~ CHa S

% Total '4C in urine

Cow 1-2

Gilt 12-20

20-25

7-10

9-12

12-20

2

N--N II II H2N-C sC-NHCH 3

3

H2N-CxsC-NHa_

9-12

8-15

N--N II II CHa=CH-CHNHC" C'SCH3 t -N ~ CH~

<1

<1

5

N--N CH2=CH'CH-NH-~,, #-SH N CH~ CH~


6

N--N il II CHa=CH_.CH_NH_C,,sC_NH 2 " CH3

< 1

N--N If

4

II

CH3

S.P. ROBINSONand V. C. JORDAN

58

3.2.1. Metabolism of Methallibure Only the metabolism of methallibure has been reported. The principal metabolites isolated from cow or gilt urine following the oral administration of 14C-methallibure are shown in Table 1. It is interesting to point out that methallibure is not stable in cow urine. If ~4C-methallibure is added to urine, stored overnight at 5°C, followed by frozen storage at - 2 0 ° C for 7 days, 82% of a chloroform extract is converted to Metabolite 1 (2-methylamino-5(~-methylallylamino)-l,3,4-thiadiazole. This apparently occurs only to a small extent in gilt urine. In the main, there is no major difference in the metabolites observed in cow or gilt urine (Aschbacher et al., 1971, 1972), and the low amounts of methallibure observed in cow urine may be an artifact because of conversion to Metabolite 1. No detailed biological investigation of the metabolites has been reported. 3.3. STEROIDAL INHIBITORS OF GONADOTROPHIN RELEASE Danazol is a novel steroid structurally related to both testosterone and ethisterone. Clinically, danazol has been used to treat endometriosis, benign breast disease and some preliminary studies have reported its use in the treatment of breast cancer. Coombes and coworkers (1980a) reported a positive response in 7 of 41 patients with advanced breast cancer treated with danazol. The original studies of the pharmacology of this agent concluded danazol is an antigonadotrophin with mild androgenic activity and no other hormonal properties (Potts et al., 1974). However, recently, a more complex series of interactions has been identified. Danazol has been demonstrated to prevent the midcycle surge of LH and FSH but does TABL~2. Metabolites o f Danazol Unequivocally Identified in Human or Monkey Urine or Monkey Feces Structure

Compound

Source

Reference

CmCH

Danazol

CNCH

CH~ OH

Ethisterone

CH3

CinCH OH

C~"

CH3 HOCH2--

Monkey urine Human urine Monkey urine and feces

Rosi et al., 1977 Davison et al., 1976

2-Hydroxymethyl ethisterone (major metabolite in human, rat and monkey)

Human urine

Rosi et aL, 1977

Monkey urine, plasma and feces

Davison et al., 1976

A'-2-Hydroxymethylethisterone

Human urine

Rosi et al., 1977

Monkey urine

Davison et al., 1976

Human urine

Rosi et al., 1977

Monkey urine and feces

Davison et al., 1976

Monkey feces (after chronic dosing)

Davison et al., 1976

CINCH

HOCH2 0 ~ , ~ CINCH ~o.

6fl Hydroxy-A'-2-hydroxymethylethisterone

CH 3

CmCH

'

CH3 I

I

I

2-Ketoethisterone

Metabolism of steroid-modifying anticancer agents

59

not suppress basal LH and FSH levels in gonadally intact humans. Danazol binds to androgen, progesterone and glucocorticoid receptors and can cause androgen receptor nuclear occupancy and initiate androgen-dependent RNA synthesis. Multiple enzymes involved in steroidogenesis have been shown to be inhibited by danazol and an increase in the clearance rate of progesterone has been demonstrated (see Barbieri and Ryan, 1981, for review). 3.3.1. M e t a b o l i s m o f D a n a z o l

Pharmacokinetic studies of danazol have been performed in the rat, monkey and human (Davison et al., 1976; Rosi, et al., 1977). Danazol is well absorbed following oral administration and rapidly metabolized. About 60 different metabolites have been seen in monkey urine (Davison et al., 1976). Tissue distribution studies in monkeys and rats show greater concentrations in the liver, adrenal glands and kidneys than is present in the plasma; however, the hypothalamus-midbrain area does not show accumulation of this compound (Davison et al., 1976). The majority of an administered dose of danazol is excreted in the rat via the feces (77%) in preference io the urine (8.9%), whereas in the monkey equal amounts are excreted in the urine (35.9%) and feces (47.9%) (Davison et al., 1976). Experiments on animals with cannulated bile ducts demonstrate a rapid accumulation of the administered dose in the bile of the rat (70% in 11.5 hr) and monkey (40% in 8.5 hr). However, even though excretion is rapid, considerable levels of radioactivity in monkey plasma persist for at least 2 weeks following administration of radiolabeled danazol. This is probably a consequence of enterohepatic circulation (Davison et al., 1976). Steady state plasma concentrations of danazol is reached within 7 days at low doses (100 mg danazol bid) or 14 days at higher doses (200 mg danazol bid) (Williams et al., 1978). Tentative metabolic pathways for danazol have been proposed based upon metabolites identified in monkey urine and feces by Davison et al., (1976) and in human urine by Rosi et al. (1977) (see Table 2). Rosi and coworkers (1977) reported that none of the five metabolites they identified in human urine exhibited pituitary-inhibiting activity comparable to danazol; however, 17~-ethyltestosterone (ethisterone), one of the major metabolites on this agent, is known to have progestational and androgenic activity. This metabolite may play a role in the progestational activity seen in some studies of danazol (Barbieri and Ryan, 1981). Danazol and some of its principal metabolites have been reported to interfere with the binding of endogenous steroids to plasma proteins (Haning et al., 1982; Nilsson et al., 1982). /CH3 HN I

s Soc~NH~NH~C//I NH

C.;H-CH¢CH

Me~hallibure (ZCI 33,828)

0,,~

N~'CH3

f_ sc.,

0 0,~

oc..

GP 48,989

N/CH3 --o

CHz-c=CH2 I CH3 CGP 19,984

FIG. 11. Structures of nonsteroid (nonpeptide) inhibitors of gonadotrophin release.

60

S.P. ROBINSONand V. C. JORDAN 4. INHIBITORS OF STEROID HORMONE BIOSYNTHESIS 4.1. HISTORICALDEVELOPMENT

The report by Huggins and Bergenstal (1952) that adrenalectomy could produce remission in some breast cancer patients, even after castration had failed, prompted an examination of agents which would interfere with adrenal steroidogenesis. In an attempt to treat advanced breast cancer, high-dose cortisone or prednisone treatment has been used to inhibit ACTH release and produce adrenal atrophy (Segaloff, et al., 1954; West et al., 1954; Pearson et al., 1955). However, the limited success with low-dose treatment and the severe side-effects of the high-dose therapies have limited the usefulness of this approach. Amphenone was one of the first nonsteroidal agents used to treat breast and prostate cancer (Hertz et al., 1956). This diphenylmethane (see Fig. 12) has been shown to interfere with adrenal steroidogenesis (reviewed by Hertz et al., 1955). Unfortunately, the drug's marked side-effects (sedation, nausea, methemoglobinemia and impaired liver function), resulting, in part, from the blockade of 17-hydroxycorticosteroid production and thyroid function, limited its general clinical usefulness. In the search for an agent with the ability to inhibit adrenal steroidogenesis without the unpleasant side-effects, Bencze and Allen (1959) synthesized the pyridine derivative, metyrapone (see Fig. 12). This agent was shown to reduce the production of 17-hydroxycorticosteroid by inhibition of l l/~-hydroxylase, a mitochondrial enzyme found in all zones of the adrenal cortex (Liddle et al., 1958; Milewich and Azelrod, 1972). However, the drug fails to inhibit the side-chain cleavage of l%t-hydroxYprogesterone or 17-hydr0xyprogesterone (Neher and Kahnt, 1965). Although metyrapone has not become useful as an endocrine therapy for cancer, the compound is clinically useful in testing for the functional ability of the pituitary to produce ACTH by blocking the synthesis of cortisol in the adrenal (Metcalf and Beavan, 1968; Jubiz et al., 1970; Donald et al., 1972). Aminoglutethimide (Fig. 12) was the first compound, which inhibits adrenal steroidogenesis, to be used routinely for breast cancer therapy in postmenopausal women (see Stuart-Harris and Smith, 1984, for review). 4.2. AMINOGLUTETHIMIDE 4.2.1. General Introduction Aminoglutethimide was initially marketed as an anticonvulsant; however, the low potency (Council on Drugs, 1962) and side-effects (the drowsiness, dizziness and ataxia) (Hughes and Burley, 1970) limited the usefulness of the drug. In 1965, a case of pseudohermaphroditism was reported in a baby born to a mother taking aminoglutethimide (Iffy et al., 1965) and, in 1966, adrenal insufficiency was noted in two children treated with this drug (Camacho et al., 1966). Aminoglutethimide was withdrawn from the market in 1966 as a result of the severe side-effects, but studies continued into its mode of action as an inhibitor of adrenal steroidogenesis. Aminoglutethimide was subsequently reintroduced as an experimental treatment for advanced breast cancer (Cash et al., 1967). Dexamethasone was added to the therapy to prevent the elevated levels of ACTH from reversing the adrenal blockade (Hall et al., @ H2N

C2H5

CH3 C I C H 5,,"C "~'0

ONH2

Arnphenone B

O~~'-NH2 H

p-arninoglutethirnide

Metyrapone

FIG. 12. Structure of agents examined in the search for an effective inhibitor of adrenal steroidogenesis.

Metabolism of steroid-modifying anticancer agents

61

1969). The administration of dexamethasone at 0.75 mg/day is initially effective as a replacement dose of glucocorticoid; unfortunately, aminoglutethimide stimulates the metabolism of dexamethasone (Santen et al., 1974). This effect can be avoided by either a stepwise increase of the dose of dexamethasone or by using a glucocorticoid which is not susceptible to drug-induced degradation, e.g. hydrocortisone (Santen et al., 1977). 4.2.2. Structural Features Aminoglutethimide (Orimeten®) is the p-amino derivative of the hypnotic glutethimide (see Fig. 13). p-Aminoglutethimide blocks the cholesterol side-chain cleaving enzyme desmolase, which converts cholesterol to pregnenolone, and the aromatase enzyme which converts androstenedione and testosterone to esterone and estradiol, respectively (for review see Stuart-Harris and Smith, 1984). Structure-activity relationship studies have shown the position of the amino group will markedly influence the degree and type of enzyme-inhibiting activity. The parent compound glutethimide, for example, has little desmolase or aromatase inhibitory action. On the other hand, the m-amino or the Namino derivatives (see Fig. 13) have equal or more desmolase inhibitory activities, respectively, than p-aminoglutethimide itself but only weak or no inhibitory activity on the aromatase enzyme (Foster et al., 1983). Substitution of pyridine for the aniline constituent of glutethimide to give 4-pyridylglutethimide (see Fig. 13) produces an agent with strong inhibitory action on the aromatase system but devoid of desmolase inhibitory activity. The 2- and 3-pyridyl derivatives, however, inhibit neither enzyme system (Foster et al., 1985). The primary aromatic amine makes aminoglutethimide metabolism substantially different to that of glutethimide by making the agent susceptable to N-acetylation. 4.2.3. Absorption, Clearance, Distribution and Metabolism 4.2.3.1. Laboratory animals. Studies in the rat have shown that aminoglutethimide is mainly (65%) absorbed from the lower intestine following oral administration. The maximum plasma concentration is reached 1 hr after dosing and an even distribution throughout the tissues of the body occurs (Douglas and Nicholls, 1965). By 48 hr after oral dosing of either 5 or 50 mg/kg of [~4C]-labeled aminoglutethimide to rats, almost complete elimination has occurred into the urine and feces (Egger et al., 1982). Nicholls (1982) reported that oral administration of [~4C]aminoglutethimide (60 mg/kg) to rats resulted in excretion of 10% of the dose in feces, 1% in expired air and the remainder in the urine. With a lower oral dose of 50 mg/kg of [~4C]aminoglutethimide administered to rats, Egger et al. (1982) measured an overall excretion of 25.1% in the feces and 60.1% in the urine by 96 hr. In rats receiving the same oral dose (50 mg/kg) of radiolabeled aminoglutethimide, about half the radioactivity was found in the bile and about half in the urine. The fact that bilary excretion is higher than the final amount observed in the feces indicates that some enterohepatic recirculation occurs (Egger et al., 1982). Bilary excretion in the rat has also been demonstrated following an intravenous R2

RI

I R3

glutethimide --no substitutions p-aminoglutethimide = NH 2 ot R I m-arninoglutethimide = NH z ot R~ N-ominoglutethirnide -- NH2 at R 3

N

H

4-pyridyl onalocjue

FIG. 13. Derivatives of glutethimide and a structural analog examined for aromatase and desmolase inhibitory activity.

62

S.P. ROBINSONand V. C. JORDAN

dose of []4C]aminoglutethimide; 72% and 24% of the dose was eliminated in the urine and the feces, respectively, over 48 hr (Nicholls, 1982). Results using rats with bile duct catheters confirmed bilary excretion and indicated enterohepatic circulation. Variation in these results has been noted in other species. Administration of [~4C]aminoglutethimide (60 mg/kg) orally to guinea-pigs results in 51% of the dose being eliminated in the urine and 40% in the feces whereas the rabbit excretes all the drug via the urine (Nicholls, 1982). Acetylaminoglutethimide has been shown to be the major urinary product in the rat constituting 37% of the radioactivity in the urine after oral administration of [~4C]aminoglutethimide (Egger et al., 1982). Twenty-four hours after the administration of 60 mg/kg of aminoglutethimide to rats, guinea-pigs or rabbits 24.3%, 4.2% and 8.0% of the dose, respectively, is identified in the urine as acetylaminoglutethimide. Aminoglutethimide can be detected in the urine as 1.8% (rat), 8.3% (guinea-pig) or 9.9% (rabbit) of the administered dose (Kamblawi et al., 1984). Detailed analysis of rat urine following oral administration of [t4C]aminoglutethimide has identified nine metabolites, of which seven have acetyl groups (see Fig. 14). The main metabolic transformations are acetylation of the aniline moiety, hydroxylation of the glutarimide ring at positions 3 and 4, and oxidative elimination of the ethyl side-chain (Egger et al., 1982). In this study, only 4% of the rat urinary excretion was in the form of [~4C]aminoglutethimide. A small amount of nitroglutethimide (see Fig. 15) has been detected; however, this may be the result of an impurity in the original agent because aminoglutethimide is synthesized from nitroglutethimide. Egger and coworkers (1982) reported that the bilary excretion of [14C]aminoglutethimide is much less complex with 22% occurring as aminoglutethimide and 8% as N-acetylaminoglutethimide. However, the bulk of radioactivity (45%) from bile extracts was not separated by the TLC system used.

'

,/

f ~ ~ ~ O - ' ' N " "0

~CONH2

CIa

"

~

~

CzH6

~'-NHCOCH3 0 t 3 OH I / C_H2,~-.-,

O,3F"~N,,,"~O

# "

' !

'-'

.,\ /o.

Acetylaminoglutethirnide

_ CzHsr./°s% O" - N - -0

IH

l

o --o

~.~ CzH~/"-~

~ Cz~F-~

0 ~ "~" " 0

OCi

Aminoglutethirnide

~

[~ ",[~==~ NHCOCHs

X

COl Y o. r X=OH, Y=NH2 X= NHz,Y=OH

1 NO2

F]o. 14. Metabolitesof aminoglutethimideidentifiedin rat urine (Eggeret al., 1982).

Metabolism of steroid-modifying anticancer agents

,-NHOH 0 ~ o

63

0 ~ 0

NH2

NHCOCH5

)

H

H

hydroxylaminoolutethimide (major metobolite)

H

aminoolutethimide

N-acetylaminoglutethimide (major metabolite)

D

~

N02

0-~~ "0

~

NHCHO

0"~ ~-~'0

nitroglutethimide

N- for mylominoglufethirnide

minor metabolites FIG. 15. Metabolites of aminoglutethimide identified in human urine.

4.2.3.2. H u m a n s . Aminoglutethimide is rapidly absorbed by humans following oral administration. Thompson et al. (1981) reported that maximum circulating levels of 5.9/tg/ml of aminoglutethimide were reached 1.5 hr after oral dosing of 500 mg in tablet form to six subjects. Similar values were reported by Nicholls (1982). In line with these observations, a plasma level of 2.4/~g/ml was measured 2 hr after oral administration of a 250-rag tablet, but this level dropped to 0.5/~g/ml within 24 hr (Menge and Dubois, 1984). Based on their pharmacokinetic data, a plasma half-life in humans of 10-15 hr has been determined for aminoglutethimide by Thompson et al. (1981). This agrees with the value of 11.8 hr reported by Nicholls (1982), 10.4 hr reported by Menge and Dubois (1984) and 13.3 hr reported by Murray et al. (1979). The latter authors observed a reduction in the half-life of aminoglutethimide to 7.3 hr after chronic treatment which matched an increase in the clearance rate from an initial value of 2.48 1/hr to 5.29 1/hr. Aminoglutethimide and its metabolites are not entirely soluble and distribute into various compartments in the blood. Human blood cells in vitro have been found to contain 1.4-1.7 times the concentration of aminoglutethimide found in plasma, and plasma proteins bind 21.3-25 % of available drug (Thompson, et al., 1981). Nicholls (1982) reported a slightly higher value of 32% plasma-binding at a concentration of 5#g/ml aminoglutethimide. This is in good agreement with the value of 31-36% plasma-binding observed for aminoglutethimide by Adams and Rogers (1984). Adams and Rogers (1984) determined a time cure for aminoglutethimide (plasma) and acetylaminoglutethimide (plasma and saliva) following oral administration of 500 mg of aminoglutethimide to human subjects. Menge and Dubois (1984) reported that the circulating plasma level of aminoglutethimide plateaus at 0.4 pg/g between 2 and 4 hr but then declines to 0.14/~g/g by 24 hr. Douglas and Nicholls (1972) reported 34-50% of the dose (0.1-1.0 g) of aminoglutethimide administered to humans was excreted in the urine as unchanged compound and 4-15% as acetylaminoglutethimide (Fig. 15). Subsequent studies have examined the influence of acetylator phenotype on the ability to metabolize aminoglutethimide. In one study, subjects characterized as slow acetylators excreted more aminoglutethimide (28% of 250-mg dose) than subjects characterized as rapid acetylators (12%) whereas rapid acetylators excrete more acetylaminoglutethimide (8.8% of 250-mg dose) than slow acetylators (3.9%) (Coombes et aL, 1982). These findings were supported by similar studies performed by Adam and coworkers (1984). Paradoxically, the later authors found the half-life of aminoglutethimide is longer in fast acetylators (19.5 hr) than slow acetylators (12.6 hr). Analyses of urine have, as in the rat, identified the presence at low levels of nitroglutethimide (Coombs et al., 1980b; Baker et al., 1981). These findings do not exclude

S.P. RoaiNso~ and V. C. JORDAr4

64

TABLE 3. Assay Methods Available to Measure the Concentration of Aminoglutethimide and Metabolites

Assay procedure Chromatographic and colorimetric Colorimetric Colorimetric

Compound measured Aminoglutethimide Acetylaminoglutethimide Aminoglutethimide Aminoglutethimide

HPLC

Aminoglutethimide Acetyl aminoglutethimide NFormulaminoglutethimide Nitroglutethimide Aminoglutethimide Acetylaminoglutethimide Aminoglutethimide Acetylaminoglutethimide Aminoglutethimide Acetylaminoglutethimide Hydroxylaminoglutethimide Hydroxylaminoglutethimide Aminoglutethimide

HPLC HPLC HPLC Colorimetric HPLC Gas-liquid chromatography HPLC HPLC

Aminoglutethimide

Sample Urine Serum Serum Urine Saliva Urine

Sensitivity --

Reference Douglasand Nicholls, 1972 1.8/zg/ml Murray et al., 1979 -Thompsonet al., 1981 --

Urine Plasma Urine

Plasma

Plasma Saliva Urine Acetylaminoglutethimide Plasma Saliva Urine Aminoglutethimide Plasma Saliva Urine Acetylaminoglutethimide Plasma Saliva Urine

Baker et al., 1981

Kamblawi et al., 1984 0.2/~g 0.2/tg/g ---

Mengeand Dubois, 1984 Goss et al., 1985

1/~mol/l Robinsonand Cornell, 1983 100 ng/ml Adamsand Rogers, 1984 10 ng/ml 250 ng/ml 250 ng/ml 5 #g/ml 100 ng/ml 100 ng/ml 2/zg/ml

Adamset al., 1985

the possibility that nitroglutethimide is not a contaminant of aminoglutethimide. However, the differences in levels of this compound in urine between slow (0.1% of dose) and fast (0.047% of dose) acetylators suggests metabolism has an influence on its abundance (Coombes et al., 1982). N-Formylaminoglutethimide (see Fig. 15) has also been identified as a further minor metabolite (Baker et al., 1981). This metabolite, however, was not influenced by acetylator type (Coombes et al., 1982). Following prolonged treatment with aminoglutethimide, its half-life is significantly reduced (Murray et al., 1979). This is consistent with self-induced metabolism. Analysis of urine samples from patients after multiple doses of aminoglutethimide has identified a further metabolite, hydroxylaminoglutethimide (see Fig. 15). This metabolite is not observed after a single dose and its appearance is consistent with the induction of an oxidation pathway (Jarman et al., 1983). The profile of metabolites measured in one patient was consistent with the formation of the hydroxylaminoglutethimide metabolite being at the expense of acetylaminoglutethimide metabolite formation. This was confirmed in a later study which demonstrated hydroxylaminoglutethimide as a product of an aminoglutethimide-induced pathway: the hydroxylaminoglutethimide:aminoglutethimide ratio increased with time whereas the acetylaminoglutethimide:aminoglutethimide ratio decreased (Goss et al., 1985). The production of the hydroxylaminoglutethimide metabolite has not been observed in the rat even after 23 days of treatment (Jarman et al., 1983). This demonstrates a difference in metabolite capacity between the human and rat and questions the use o f the rat as a model in long-term aminoglutethimide studies. Detailed analysis of urine from patients undergoing chronic treatment with amino-

Metabolism of steroid-modifyinganticancer agents

65

glutethimide has identified further minor metabolites (Foster et al., 1984). These recently identified metabolites are all products of hydroxylation (see Fig. 16). There appears to be a marked species difference between the metabolites of aminoglutethimide identified in the rat (Egger et al., 1982) (see Fig. 14) and the human (Foster et al., 1984; Douglas and Nicholls, 1972; Baker et al., 1981; Jarman et al., 1983) (see Figs 15 and 16). Almost all the metabolites in the rat are products of N-acetylation whereas most of the human metabolites (after chronic treatment) are products of hydroxylation.

4.2.4. Acth;ity of Aminoglutethimide Metabolites Patients receiving aminoglutethimide initially experience many side-effects (e.g. rash, fever, lethargy, dizziness and ataxia), but these diminish after the first few weeks of treatment. During the initial period of medication, the half-life of aminoglutethimide is markedly reduced (Murray et al., 1979) and the major metabolite changes from acetylaminoglutethimide to hydroxylaminoglutethimide (Goss et al., 1985). However, the severity of the side-effects has not been related to metabolite formation. The metabolites detected in human urine following single doses of aminoglutethimide do not appear to play a positive role in preventing adrenal steroidogenesis. The major metabolite, acetylaminoglutethimide, has no aromatase or desmolase inhibitory action and the minor metabolites, formylaminoglutethimide and nitroglutethimide, are only weakly active (Chohan et al., 1982; Foster et al., 1983). Hydroxylaminoglutethimide is pharmacologically less active than the parent compound in inhibiting aromatase and desmolase activity (Chohan et al., 1982; Goss et al., 1985) but is more active than acetylaminoglutethimide. The minor metabolite identified in human urine, cis p-amino-5-hydroxyglutethimide, does not inhibit either desmolase or aromatase activity in vitro (Foster et al., 1984). The oxidative enzymes induced by chronic aminoglutethimide treatment not only influence its own half-life (Murray et al., 1979) but also those of other drugs. This is illustrated by a decrease in the biological half-life of dexamethasone and warfarin during therapy with aminoglutethimide (see Murray et al., 1979).

4.2.5. Detection, Identification and Measurement of Aminoglutethimide and Metabolites The detection of aminoglutethimide was initially performed by a colorimetric reaction with Ehrlich's reagent forming a yellow Schiff's base (Douglas and Nicholls, 1965). Since the assay has low sensitivity and a number of other drugs can react with this reagent, alternative assay methods were developed.

:2.L

v

H

p-amino- 5-hydroxyglutethimide

HOCHC3H ~'~NH2 0 " "~" "0 p-amino-l- hydroxyglutethimide

o"

L v H

p- ocetylamino-5- hydroxyglutethimide

CH2CH2CON 2H ~NH2 "u~ "(3

Lactone from p- amino- 2'-hydroxyglutethimide

FIG. 16. Hydroxylatedmetabolitesof aminoglutethimide identified in human urine (Foster et al., 1984). JFT 36/1--E

66

S.P. ROBINSONand V. C. JORDAN

Absorption, distribution and metabolism studies of amin0glutethimide in animals have used the [t4C]-labeled compound (Nicholls, 1982; Egger et al., 1982), whereas clinical samples are measured by HPLC with u.v. detection. A list of assay methods, their sensitivities, and their ability to detect metabolites is shown in Table 3. Unknown metabolites have been identified by mass spectroscopy (Foster et al., 1984; Baker et al., 1981) and nuclear magnetic resonance spectroscopy (Egger et al., 1982).

4.3. NEW AGENTS TO INHIBIT STEROID HORMONE BIOSYNTHESIS

4.3.1. General Introduction The promising clinical results with aminoglutethimide have increased interest to find a new treatment regimen to reduce the side-effects or new therapeutic agents which produce fewer side-effects but are equally or more effective in suppressing estrogen production. Trials are being performed using low-dose (125 mg twice daily) aminoglutethimide therapy (Stuart-Harris et al., 1984) in the hope of maintaining efficacy but reducing sideeffects. Although the dose is lower than usually used to cause adrenal inhibition through desmolase blockade, effective aromatase inhibition is still achieved and a suppression of estrogen levels can be produced (Harris et al., 1983). In these recent studies, objective responses have been reported with this regimen (Stuart-Harris and Smith, 1984), and the number and degree of severity of side-effects have been reduced (Cantwell et al., 1984). However, it is important to ensure that autoinduction of metabolic enzymes does not reduce circulating levels of aminoglutethimide to those that are ineffective. Studies with new agents are focused on the novel compounds, ketoconazole and trilostane (Fig. 17), which inhibit steroid biosynthetic pathways. In the case of ketoconazole, an interaction with the cytochrome P-450 enzyme complex involved with CtT_20lyase has been indicated (Van den Bossche et al., 1985), and studies are concentrated on the inhibition of testosterone synthesis (Pont et al., 1982). Prolonged treatment with low doses of ketoconazole in intact men results in a compensatory rise in LH levels which ultimately overcomes the blockade of steroid synthesis. However, high-dose therapy may not be reversed (Santen et al., 1983), but some suppression of cortisol synthesis has also been reported which may lead to troublesome side-effects. Trilostane has been shown to inhibit adrenal steroid biosynthesis and has been successfully used in the treatment of some cases of Cushing's syndrome (Komanicky et al., 1978). The use of this agent in breast cancer therapy without additional corticosteroids has been reported to be ineffective (Coombes et al., 1985), whereas a more favorable response was observed in combination with corticosteroid replacement (Beardwell et al., 1983). Another approach is to study compounds which specifically inhibit the peripheral conversion of androstenedione to estrone without interrupting adrenal steroidogenesis. A'Testolactone (Fig. 17) was first used successfully in breast cancer therapy in the 1960s (Segaloff et al., 1962). The rationale was originally that this derivative of testosterone might be active as an androgen-like molecule. However, it was subsequently shown that A'testolactone is an inhibitor of the aromatase enzyme systems which could account for its pharmacological activity (Barone et al., 1979). While less effective than ablative surgery, A'testolactone has been shown to produce objective tumor regression in some breast cancer patients (Segaloff et al., 1962). This agent is well tolerated and produces few sideeffects. More recently, a group of androstenedione derivatives have been studied which are the most effective aromatase inhibitors found to date (Santen and Brodie, 1982). 4-Hydroxyandrostene-3,17-dione (Fig. 17) has been used clinically to treat hormone-dependent breast cancer in postmenopausal women (Brodie et al., 1986). Complete or partial regression was observed in 30% of 60 postmenopausal patients with advanced metastatic breast cancer even though all these patients had relapsed from previous therapy.

Metabolism of steroid-modifying anticancer agents

N-C-~o

67

NJ]I

OH

0 CH3C--

CH2 Cl

" C"-O-

TRILOSTANE

0,.~.'~0--. ~ 0 CHz m,=,I H

CI

KETOCONAZOLE

0 0

0

HO 4,HYDROXYANDROST-4- ENE3,17- DIONE

A' TESTOL ACTONE

FIG. 17. Additional inhibitors of steroid hormone biosynthesis presently being studied in clinical trials.

A few studies have reported the pharmacokinetics of trilostane, ketoconazole, A'testolactone and 4-hydroxyandrostene-3,17-dione and these will be reviewed. 4.3.2. Trilostane The disposition of radiolabeled trilostane has been examined in the monkey and rat (Baker et al., 1980). In the monkey, a peak plasma concentration of 15/~g/ml was observed 2 hr after oral administration of [~4C]trilostane (20 mg/kg). The elimination of radioactivity from the plasma compartment was triphasic with half times of 10 min, 1.1 hr and 34 hr (estimated from i.v. injection). The percentage of total plasma radioactivity accounted for by intact trilostane decreased from 100% at 5 min to 50% by 1 hr to < 10% at 7 hr, indicating the rapid metabolism (Baker et al., 1980). Baker et al. (1980) also reported peak blood levels of radioactivity equivalent to 2 #g/ml of trilostane were reached 0.5-1 hr after oral administration of 25 mg/kg of [~4C]trilostane to rats. Using the higher dose of 30 mg/kg of trilostane, a peak level of about 2 #g/ml was measured in rat serum by gas chromatography-mass spectrometry 2 hr after administration (Mori et al., 1981a). One hour after oral administration of [~4C]trilostane to rats, Baker et al., (1980) found most of the radioactivity in the grastrointestinal tract; however, from the area under blood-time curves they estimated 8~% of the dose was subsequently absorbed. Examination of tissues at 1 hr after oral administration showed the highest levels of radioactivity in the liver and adrenals with the lungs and kidneys possessing significantly higher levels than blood. Elimination of [14C]trilostane from the blood following i.v. administration to rats gives a triphasic clearance pattern with half times of 26 min, 2 hr and 50 hr (Baker eta/., 1980). Twenty-four hours after oral administration of [14C]trilostane (20 mg/kg), 83% of the dose is eliminated primarily via the feces. However, significant levels of radioactivity are still observed in the adrenals and gastrointestinal tract (Baker et al., 1980). Gas chromatography-mass spectrometry analysis of urine from rats given trilostane 1130mg/kg) orally show that only about 3% of the dose is excreted via this route over

68

S.P. ROaINSONand V. C. JORDAN

24hr, the majority (2.7%) as the parent compound (Mori et al., 1981a). In contrast, higher levels of urinary excretion are measured when radiolabeled trilostane is administered either orally (13.7% of 20 mg/kg) or i.v. (18.4% of 5 mg/kg) and samples determined over a 5-day period (Baker et al., 1980). The predominant route of excretion is via the feces. Baker et al., (1980) measured 71% of an i.v. administered dose (5 mg/kg) and 81% of an orally administered dose (20 mg/kg) in the feces. Mori et al. (1981a) reported that in rats with ligated bile ducts 30.2% of a 30-mg oral dose was excreted into the bile. These results contrast with those obtained using monkeys when 54-64% of a dose of trilostane is eliminated in the urine and only 27-39% in the feces (Baker et al., 1980). Analysis of rat urine and bile by gas chromatography, nuclear magnetic resonance spectroscopy and infrared spectroscopy has positively identified trilostane and three metabolites and tentatively assigned structures to two other metabolites (Mori et al., 1981a,b). Urine from rats given trilostane orally contains unconjugated metabolites; whereas bile contains mainly conjugated ones. From these data, a metabolic pathway has been proposed (see Fig. 18; Mori et al., 1981a). The major metabolite identified is 17ketotrilostane. This metabolite can be measured at substantially higher plasma levels than trilostane following an oral dose of 30 mg/kg (Mori et al., 1981a). Baker et al. (1980) have identified five major metabolites of trilostane in monkey urine and have partially characterized them. The primary metabolic pathways involve hydroxylation and glucuronide formation. The development of new HPLC methodology has allowed the analysis of circulating trilostane levels in human subjects (Powles et al., 1984; Robinson et al., 1984; Brown et al., 1985b). 17-Ketotrilostane has been identified in human plasma as the major metabolite (Powles et al., 1984). From preliminary measurements of plasma from a subject who was OH

N s NE ,

OH OH

. I N ' C ~

=~ N- C

I%

~~ O H ~ ~

' HO"W'~gV~ "OH FIG. 18. Metabolicpathwayof trilostane proposedby Mori et GL(1981a).[ ] not detected;*structure only tentatively assigned.

Metabolismof steroid-modifyinganticanceragents

69

given 120 mg trilostane orally, levels of parent compound were in the range 0--1 #g/ml and 17-ketotrilostane in the range 0-2.5/~g/ml (Powles et al., 1984). These estimates are in agreement with the values reported by Brown et al. (1985b) who measured plasma levels over 6 hr. Maximum levels (0.48/~g/ml) of the parent compound were reached by 1 hr and the metabolite, 17-ketotrilostane, (1.3/~g/ml) within 2-3 hr. Robinson et al. (1984) analyzed plasma taken over 8 hr from 10 male subjects following the administration of 120 mg trilostane orally. The metabolite 17-ketotrilostane was identified in plasma at approximately three times the concentration of the parent drug in most samples analyzed. Blood levels varied markedly from subject to subject; however, levels were generally >2 X 10-TM by 1 hr, and there was complete clearance from the blood by 6--8 hr. A comparison of the relative potencies of trilostane and its 17-keto metabolite by bioassay (inhibition of 3fl-hydroxysteroid dehydrogenase in the rat ovary) (Earnshaw et al., 1984) demonstrates that the metabolite is almost twice as effective as an enzyme inhibitor than the parent compound (Robinson et al., 1984). Since the concentration of metabolite required in vitro is achieved in vivo and the concentration of the metabolite in plasma is greater than the parent compound, then the metabolite, 17-ketotrilostane, is clearly very important for the activity of the drug during therapy (Robinson et al., 1984). 4.3.3. Ketoconazole 4.3.3.1. M e t a b o l i s m in laboratory animals. Circulating levels of ketoconazole have been determined in the rat (Pont et al., 1982; Bhasin et al., 1986; Heel, 1982), mouse (Harvey et al., 1980; Levine and Cobb, 1978) and human (Pont et al., 1982; Santen et al., 1983; Badcock, 1984; Alton 1980; Swezey et al., 1982) following administration. The absorption of radiolabeled ketoconazole from the gastrointestinal tract is more rapid in rats and guinea-pigs (maximum plasma concentrations at 0.25-1 hr) than in rabbits and dogs (maximum plasma concentrations at 1-2 hr). Following a dose of 10 mg/kg, peak plasma concentrations were 1, 3.7, 8.9 and 16.5/~g/ml in rabbits, guineapigs, dogs and rats, respectively (Heel, 1982). Heel (1982) reported absorption of ketoconazole in fasted and nonfasted rabbits is similar. Peak circulating levels following oral administration to mice occurs within 1 hr of dosing (Levine and Cobb, 1978; Harvey et al., 1980). Harvey and coworkes (1980) observed relatively greater increases in peak serum levels in mice for each increase in dose and suggested this may be a function of the first pass through the liver. A large fraction of low doses may be absorbed and metabolized by the liver, but the capacity may be saturated at higher doses (Harvey et al., 1980). An interaction with the rat liver enzyme systems by ketoconazole has been demonstrated by the reduced metabolism of chlordiazepoxide when administered together (Brown et al., 1985a). Indeed the highest tissue concentration of tritiated ketoconazole occurs in the liver (125 #g/kg) following oral administration (20 mg/kg) (Heel, 1982). Radioactivity is also found in high levels in the adrenals and moderate levels in the lungs, kidney, bladder, bone marrow, teeth, myocardium and various glandular tissues. The lowest tissue levels occur in the testis and brain, peak brain levels being about one-tenth of the plasma concentration (Heel, 1982). Ketoconazole is also reported by Heel (1982) to be poorly absorbed across the placental barrier. Excretion of tritiated ketoconazole and metabolites occurs via the feces in both the rat and the dog. However, a higher proportion of unchanged drug is recovered from the feces of dogs (55%) than from rats (4 to 6%) (Heel, 1982). The major metabolic pathways in these species involves oxidation and subsequent scission and degradation of the imidazole ring, scission and degradation of the piperazine ring, scission of the dioxolane ring and oxidative-O-dealkylation (Heel, 1982). 4.3.3.2. M e t a b o l i s m in humans. In the human, ketoconazole is rapidly absorbed following oral administration, peak plasma levels being reached 2 hr after a 200-mg dose (Santen et al., 1983; Swezey et al., 1982; Heel, 1982). Swezey et al. (1982) compared measurements using an HPLC method with concentrations estimated with a bioassay (Harvey et al.,

70

S.P. ROaINSONand V. C. JORDAN TABLE4.Metabolites of 4-Hydroxyandrostene-3,17-dione Identified In Vitro and In Vivo in the Rat o 4-Hydroxyandrostene-3,17-dione

HO

OH

4-Hydroxytestosterone

HO

O 4-Hydroxyestrone

HO

0

3fl-hydroxy-5~-androstan-4,17dione

o 3a-hydroxy-5fl-androstan-4,17dione o 3fl,4fl dihydroxyandrostan-17-one

HO

0

2,4-dihydroxyandrost-4-ene-3,17dione HO

O 2,4-dihydroxyandrostan-3,17-dione

4fl,5~-dihydroxyandrostan-3,17dione

2,4,17fl-trihydroxyandrostan-3-one

1980) and found similar results. Peak levels determined by HPLC were about 7 #g ketoconazole/ml of plasma 2 hr after the oral administration of a 200 mg dose. This level is higher than the value of 2.65 #g/ml reported for the same time period and dose by Santen et al. (1983) and the 3-4.5 #g/ml reported by Heel (1982) and is closer to the level determined by Pont et al., (1982) of 7.9 #g/ml 2 hr after a 600-rag oral dose. The circulating levels of ketoconazole are reduced to low (Pont et al., 1982) or nondetectable (Santen et al., 1983) at 24 hr after an oral dose. The fall in ketoconazole blood levels is exponential in nature (Santen et al., 1983). Studies by Santen et al. (1983) indicate higher circulating levels occur after 12 months of treatment than after 3 months but indicate the time of sampling may have influenced the observed values. Data cited by these authors supported no change in clearance occurring

Metabolism of steroid-modifyinganticancer agents

71

following chronic treatment. In contrast, it has been suggested that higher doses (200 mg, 4 times daily) may accumulate over several weeks of treatment (Maksymink et al., 1980). The absorption of ketoconazole is markedly reduced by alteration in the gastric acidity by agents such as cimetidine or antacids (Van der Meer et aL, 1980). Since ketoconazole is a dibasic compound, sufficient gastric acid is required for dissolution and absorption. Although early data suggested the administration of ketoconazole with food might improve adsorption (Heel, 1982), a later study indicates administration to fasted subjects results in higher serum levels (Heel et al., 1982). Distribution studies in the human indicate ketoconazole passes into urine, saliva, sebum cerumen and cerebrospinal fluid (Heel et al., 1982; Heel, 1982). However, reports of the extent of penetration into the cerebrospinal fluid have been somewhat variable (see Heel et al., 1982, for review). Ketoconazole is about 99% bound by human plasma protein, primarily to albumin (Heel et al., 1982). In whole blood, 1% was present as free drug with 84% protein bound and 15% associated with blood cells (Heel et al., 1982). Excretion of ketoconazole in the human is predominantly via the feces with some in the urine (Heel, 1982). Following adsorption, ketoconazole is extensively metabolized. Heel (1982) reported unchanged drug represented 20-65% of the fecal excretion and only 2--6% in the urine. The main identified metabolic pathways are oxidation of the imidazole ring, degradation of the oxidized imidazole, oxidative O-deakylation, oxidative degradation of the piperazine ring and aromatic hydroxylation (Heel, 1982; Heel et al., 1982). The half-life of elimination of ketoconazole has been determined in a few studies; these estimates range from 2.7 to 12 hr (see Heel et al., 1982, for review). Studies of the influence of renal or hepatic impairment are inconclusive and further investigation is needed (see Heel et al., 1982; Heel, 1982). 4.3.4. A'Testolactone Segaloff et al. (1966a) reported the identification of nonconjugated A'testolactone and 3-keto-4,5fl-dihydro-A'testolactone in urine following the oral administration of 14Clabeled A'testolactone to breast cancer patients (see Fig. 19). In addition, 3g,5fltetrahydro-A'testolactone metabolite was isolated following treatment of urine with flglucuronidase (see Fig. 19). No metabolites were found in the feces. The majority (96%) of the administered dose was collected in the urine by 72 hr. This differs from the excretion of testolactone which is complete within 36 hr and has both 5g and 5fl metabolites (Segaloff et al., 1966b). The difference in excretion rates suggests the double bond at C-1 has a profound effect on the rate of metabolism. Recently the 4,5-dihydro-A'testolactone metabolite has been identified in plasma and urine of male patients given A'testolactone (Yeager et al., 1985; Pascucci et al., 1983). Studies of sequential samples taken over 12 hr from patients who received 500 mg of °

A'- testolactone

°

4, 5- dihydro ~ - testolactone (Major metabolite )

HO.~~ 0 3a, 5,8- tetrahydro- A' testolactone

FIG. 19. The structure of A'testolactoneand metabolites.

72

S.P. ROBINSON and V. C. JORDAN

A'testolactone 4 times per day for 3 months indicated that peak plasma levels of about 1/~g/ml occurred for both A'testolactone and 4,5-fl-dihydro-A'testolactone about 3 hr after administration (Pascucci et al., 1983). The activity of the metabolites of A'testolactone has not been reported. 4.3.5. 4-Hydroxyandrostene-3,17-dione A study by Brodie et al. (1981) of the metabolism of radiolabeled 4-hydroxyandrostene3,17-dione in the rhesus monkey identified the unconjugated parent compound and the metabolite, 4-hydroxytestosterone, in the blood 1-180 min after i.v. injection. The clearance of both these agents was reported as being very rapid (Brodie et al., 1981). In rats injected with [6,73H]4-hydroxyandrostene-3,17-dione, the metabolite, 4-hydroxytestosterone, was also identified in the free fraction of blood; however, it represented only 0.5% of the total extract (Marsh et al., 1982). A further metabolite, 3fl-hydroxy-5ctandrostane-4,17-dione accounted for the largest proportion of the free extract (20%). In a review article, Santen and Brodie (1982) reported that glucuronide conjugates of 4hydroxyandrostene-3,17-dione, 4-hydroxytestosterone and 3fl-hydroxy-5~-androstene4,17-dione, were found in the blood of rats collected 30, 60 and 120 min after injection of radiolabeled parent compound (see Table 4). The glucuronide of 4-hydroxyandrostene-3,17-dione has been found in the bile, but not in the urine, of rats given the drug orally. This is in contrast to patients given 4-hydroxyandrostene-3,17-dione orally, where the glucuronide is the major urinary metabolite (Goss et al., 1986). Distribution studies in the rat using [6,7-3H]-labeled 4-hydroxyandrostene-3,17-dione showed that, of the total radioactivity/mg tissue recovered at 1 hr after injection, 42% was associated with the ovaries, 3 0 0 with the liver, 10% with the kidneys, 10% with the adrenals and 3% with the pituitary. This indicates preferential binding of the aromatase inhibitor in the ovaries rather than other tissues (Santen and Brodie, 1982). The high levels of radioactivity in the liver may be the consequence of metabolism in this organ. Studies in vitro have shown rat ovarian microsomes convert 4-hydroxyandrostene-3,17dione to the major metabolite, 4-hydroxytestosterone, and to a very small amount of 4hydroxyestrone (Marsh et al., 1982). Rat hepatocytes have been reported by Foster and coworkers (1986) to metabolize 99% of [~4C]-labeled 4-hydroxyandrostene-3,17-dione within 5 min. Conversion is to an array of Phase I metabolites formed by reduction, hydroxylation and hydration reactions (see Table 4). After the incubation of radiolabeled parent drug with hepatocytes for 10min, 4-hydroxyandrostene-3,17-dione glucuronide represented 30% of the radioactivity and by 15 min the glucuronide fraction represents 60%. On further investigation, the glucuronide fractions were found to contain several compounds (Foster et al., 1986). Marsh and coworkers (1982) reported that none of the metabolites of 4-hydroxyandrostene-3,17-dione had greater aromatase inhibitory action than the parent compound itself. Using rat ovarian microsomes, 3fl-hydroxy-50~-androstane-4,17-dione had little inhibitory effect (10% of parent drug) while 4-hydroxytestosterone had about 65% of the inhibitory activity of 4-hydroxyandrostene-3,17-dione. Using human placental microsomes, the 30thydroxy-5fl-androstane-4,17-dione, 3fl-hydroxy-5~t-androstane-4,17-dione and 4fl-5ctdihydroxyandrostane-3,17-dione metabolites had little or no aromatase inhibiting activity when compared with 4-hydroxyandrostene-3,17-dione whereas 2,4-dihydroxyandrost-4ene-3,17-dione was about 46% as active as the parent drug (Foster et al., 1986). 5. ANTIESTROGENS 5.1. HISTORICALDEVELOPMENT Lerner and coworkers (1958) reported the pharmacological properties of the first nonsteroidal antiestrogen, ethamoxytriphetol (MER 25) (Fig. 20). The compound inhibits estrogen action in all species tested (rats, mice, chickens, rabbits and monkeys) and exhib-

Metabolism of steroid-modifyinganticancer agents

73

its no other hormonal or antihormonal properties. The most important action of MER 25 (which stimulated further study) is its ability to prevent implantation of blastocysts in the rat (Segal and Nelson, 1958). These observations heralded a new era for the potential clinical application of antiestrogens as contraceptives. Initial reports (Kistner and Smith, 1959; 1961) demonstrated the efficacy of MER 25 in clinical studies to evaluate the antiestrogenic properties, but eventually the drug was withdrawn because of its low potency and toxic side-effects (Lerner, 1981). A search for other nonsteroidal antiestrogens for use as postcoital contraceptives yielded the derivative of triphenylethylene, clomiphene (MRL 41) (Holtkamp et al., 1960). 'The drug is more potent than MER 25, but this is at the expense of increased estrogenic activity. Furthermore, the drug that was subsequently tested in clinical trials is a mixture of cis (zuclomiphene) and trans (enclomiphene) geometric isomers (Fig. 20). Zuclomiphene is a weak estrogen, whereas enclomiphene is a partial estrogen agonist in the rat with antiestrogenic properties (Jordan et al., 1981). Clomiphene is an effective OCHICH=N/C=H= ~'C=Hs

MER 25

OCH2CH=N/C=H5

/NCH =CH20 CIHs ENCLOMIPHENE

ZUCLOMIPHENE

OCH2CH2N~CH= CH3

OCH2CH2N~CH3 CH;

C TAMOXIFEN

4 - HYDROXYTAMOXIFEN

] OCH2CH2N

LY 117018

FIG. 20. The structure of the first nonsteroidal anticstrogcn (MER25) and later developments. Tamoxifen has become the front-line endocrine therapy for breast cancer.

74

S.P. ROBINSONand V. C. JORDAN

postcoital antifertility agent in the rat (Segal and Nelson, 1961), but ironically the first clinical studies demonstrated that the drug induced ovulation (Greenblatt et al., 1961, 1962). Clomid ® (a cis/trans mixture of clomiphene) is currently available for the induction of ovulation in subfertile women with a functional ovarian-hypothalamo-pituitary axis (Huppert, 1979). The decline in interest in postcoital contraception was followed by the application of antiestrogens to treat advanced breast cancer. Several antiestrogens were tested in Phase I and Phase II clinical trials in postmenopausal patients with advanced disease (Legha and Carter, 1976), but only tamoxifen (Fig. 20) was found to be of value for further development. The drug has a low incidence of side-effects and is the front-line endocrine therapy for breast cancer in more than 70 countries around the world (Furr and Jordan, 1984). The enormous success of antiestrogen therapy for advanced disease, the application of tamoxifen as an adjuvant therapy following surgery (Jordan et al., 1987) and the potential application as a preventive therapy for breast cancer (Jordan, 1986) have focused interest on the development of additional agents to complement or replace tamoxifen. The finding that tamoxifen is metabolized to 4-hydroxytamoxifen (Fig. 20) (Fromson et aL, 1973b), which has a very high binding affinity for the estrogen receptor (Jordan et al., 1977), suggested that the metabolite might be a more potent antiestrogen and antitumor agent. There is some evidence that this is the case in vitro (Coezy et al., 1982), but 4hydroxytamoxifen has not proved to be more potent than tamoxifen in vivo (Jordan and Allen, 1980). The antiestrogen, LY 117018 (Fig. 20), was developed based upon the concept that compounds with a high binding affinity for the estrogen receptor might be potent agents in vivo. LY 117018 and structurally related antiestrogens have a high affinity for the receptor (Black et al., 1981), potent antitumor activity in vitro (Scholl et al., 1983), low estrogenic activity in vivo (Black and Goode, 1980; Jordan and Gosden, 1983a) but only weak antitumor activity when compared with tamoxifen (Clemens et al., 1983). No antiestrogen with novel properties has yet been approved for clinical use that could be used as an alternative to tamoxifen. For this reason, we will focus attention on the structure-activity relationships and metabolism of tamoxifen in animals and man. However, the metabolism of the earlier antiestrogens has recently been reviewed (Jordan, 1984), and the reader is referred to this source for additional information. After this section on the metabolism of tamoxifen, we will briefly discuss several new antiestrogens that have recently entered clinical trials around the world. Very little information is available about these drugs, but some speculation about their possible metabolism will be offered based upon their structures. 5.2. TAMOXIFEN:A NONSTEROIDAL ANTIESTROGEN

5.2.1. Structural Features The structure-activity relationships of nonsteroidal estrogens (Jordan et al., 1985) and antiestrogens (Jordan, 1984) have been extensively reviewed. It is, therefore, the aim of this section to point out the salient features of the drug molecule that will affect the pharmacology. The important structural features of the tamoxifen molecule (Fig. 21) can be divided into two main categories: substitution in the phenyl equivalent to the A ring of the steroid nucleus, and alteration or removal of the dimethylaminoethoxy side-chain. Most of the important changes are observed with tamoxifen's different metabolites. Introduction of a single para hydroxyl into tamoxifen to produce Metabolite B (4hydroxytamoxifen) markedly increases the affinity of the ligand for the estrogen receptor (Jordan et al., 1977) and the antiestrogenic potency. The introduction of two hydroxyls, one in the para and one in the meta position of tamoxifen, to produce Metabolite D again increases ligand affinity when compared with tamoxifen but the antiestrogenic activity in vivo is low (Jordan et al., 1977). This may be becuse the catechol derivative of tamoxifen is very unstable (Jordan et al., 1984).

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75

TABLE 5 Assay M e t h o d s Available to Measure the Concentration o f Tamoxifen and Metabolites Assay procedure Thin layer chromatography High performance liquid chromatography

Gas chromatography Mass spectrometry

Gas chromatography

Sensitivity

Reference

Tamoxifen N-Desmethyltamoxifen Tamoxifen

Compound measured

2.5 ng 2.5 ng 1 ng

Adam et al, (1980a) Adam et al. (1980b) Mendenhall et al. (1978)

4-Hydroxytamoxifen Tamoxifen N-Desmethyltamoxifen 4-Hydroxytamoxifen Tamoxifen N-Desmethyltamoxifen Metabolite Y Tamoxifen N-Desmethyltamoxifen 4-Hydroxytamoxifen Tamoxifen N-Desmethyltamoxifen 4-Hydroxytamoxifen Tamoxifen Tamoxifen 4-Hydroxytamoxifen Tamoxifen

I ng 100 pg 100 pg 100 pg 2 ng 2 ng 1 ng 100 pg 100 pg 100 pg 8 ng 8 ng 8 ng -1 ng 1 ng -Daniel et al. (1981) ---

N-Desmethyltamoxifen 4-Hydroxytamoxifen Tamoxifen

Mendenhall et al. (1978) Golander and Sternson (1980) Golander and Sternson (1980) Golander and Sternson (1980) Brown et al. (1983) Brown et al. (1983) Brown et al. (1983) Camaggi et al. (1983) Camaggi et al. (1983) Camaggi et al. (1983) Wilbur et al. (1985) Wilbur et al. (1985) Wilbur et al. (1985) Gaskell et al. (1978) Daniel et al. (1979) Daniel et al. (1975) Daniel et al. (1981) Daniel et al. (1981) Sane et al. (1985)

Modification of the side-chain of tamoxifen by removal of one methyl group (Metabolite X), two methyl groups (Metabolite Z) or replacement of the dimethylamine by hydroxyl (Metabolite Y) produces metabolites with low binding affinity for the estrogen receptor and weak antiestrogenic activity (Jordan et al., 1983; Kemp et al., 1983). In contrast, removal of the dimethylaminoethane side-chain to form Metabolite E produces a metabolite with a low binding affinity for the estrogen receptor but estrogenic properties (Jordan and Gosden, 1982). 5.2.2. Metabolic P a t h w a y s The metabolism of tamoxifen has been described in the mouse (Fromson et al., 1973a; Wilking et al., 1981, 1982; Lyman and Jordan, 1985a), rat, dog, rhesus monkey (Fromson et al., 1973a) and man (Fromson et al., 1973b; Adam et al., 1979, 1980b; Kemp et al., 1983). Using [14C]tamoxifen, it has been shown that, in these species, the drug is well X

R2

X

Ri

R2

Tamoxifen

H

H

Metabolite B-

OH

H

OCH2CHzN (CH3) 2

Metabolite D

OH

OH

OCHzCH2N (CH~) z

OCH2CH2N(CHz) 2

Metabolite X

H

H

OCH2CH2 NHCH3

Metabolite Z

H

H

OCHzCH2NH 2

Metabolite Y Metabolite E

H H

H H

OCH2CH20H OH

FIG. 21. Metabolites of the antiestrogen, tamoxifen.

76

S.P. ROBINSONand V. C. JORDAN TABLE 6. Concentration o f Tamoxifen, N-Desmethyltamoxifen, 4-Hydroxytamoxifen and Metabolite Y in Blood from Patients with Breast Cancer. The Values Shown are Mean with the Range in Parentheses. Drug Administration in These Studies was Chronic

Dose of tamoxifen 10 nag b.i.d. 10 mg/m b.i.d. 20 mg b.i.d. 10 mg b.i.d. 20 mg b.i.d.

20 mg/m a f t e r loading doses 10 mg b.i.d. 10 mg b.i.d.

20 mg b.i.d.

Compound measured Tamoxifen 4-HO-Tam Tamoxifen Tamoxifen N-Desmethyl-Tam Tamoxifen N-Desmethyl-Tam Tamoxifen N-Desmethyl-Tam 4-HO-Tam Tamoxifen N-Desmethyl-Tam 4-HO-Tam Tamoxifen N-Desmethyl-Tam Tamoxifen N-Desmethyl-Tam Metabolite Y Tamoxifen N-Desmethyl-Tam Metabolite Y

Concentration (ng/ml) 167 (143-197) 3,4 (2-5) 260 285 (153-494) 477 (189-202) 125 160 300 (270-520) 462 (210-761) 6.7 (2.8-11.4) 163 (95-240) 289 (187-325) 10 (4-21) 165 (89-274) 294 (208-452) 113 (77-189) 214 (163-265) 18 (5-49) 310 (164-494) 481 (300-851) 49 (22-136)

Reference Daniel et al. (1979) Fabian et al. (1980) Patterson et al. (1980) Wilkinson et al. (1980) Daniel et al. (1981)

Fabian et al. (1981); Fabian and Sternson (1981) Jordan (1982b) Jordan et al. (1983)

Kemp et al. (1983)

4-HO-Tam = 4-hydroxytamoxifen; N-Desmethyl-Tam = N-desmethyltamoxifen.

absorbed and extensively metabolized after oral administration. The major metabolites are shown in Fig. 21. Metabolites are excreted chiefly as conjugates in the bile and little or no tamoxifen is eliminated as unchanged drug. Urinary excretion appears to be greater in primates, including man, than in rodents but is still only a minor route of elimination of the drug. A similar pattern of metabolites was found in animals and patient serum by thin layer chromatography which led Fromson et al. (1973b) to conclude, erroneously, that 4hydroxytamoxifen was present in human serum at higher concentrations than the parent drug. It is now clear, however, that the chromatographic peak designated as 4-hydroxytamoxifen is, in fact, Metabolite X (ICI 55548; N-desmethyltamoxifen). In serum from women treated with tamoxifen, 4-hydroxytamoxifen is present only at very low concentrations (Daniel et al., 1979). Recently, Metabolite Y, a side-chain primary alcohol, has been identified in serum from women treated with tamoxifen (Bain and Jordan, 1983) and Metabolite Z (Ndesdimethyltamoxifen) has been tentatively identified (Kemp et al., 1983). In addition using a combination of thin layer chromatography and a high-pressure liquid chromatography system (Brown et al., 1983), Metabolite Y has been measured in patients on highdose (150 mg b.i.d.) tamoxifen therapy (Jordan et al., 1983). 5.2.3. Assay Procedures

Assay methods have been developed for tamoxifen, 4-hydroxytamoxifen and Ndesmethyltamoxifen, using either thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS). The assays used and their sensitivities are shown in Table 5. The compounds are estimated in TLC or HPLC assays by fluorescence detection after u.v. conversion to the phenanthrene derivatives (Fig. 22). Alternatively, the ratio of peak heights of the molecular ion of a tamoxifen derivative and an internal standard are used to determine tamoxifen concentrations by high resolution mass spectrometry after gas-liquid chromatography.

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77

UV

Triphenylethylene

Phenanthrene

FIG. 22. The u.v.-activatedconversionof triphenylethyleneto phenanthrene used to allowfluorescence detection of antiestrogens such as tamoxifen.

5.2.4. Distribution After i.v. injection of spayed female mice with [lac]tamoxifen, concentrations are higher after 5 min and 4 hr in lung, liver, adrenals, kidney, pancreas, uterus, salivary glands, and mammary tissue than in blood (Wilking et al., 1981, 1982). Although high concentrations of drug would be predictable in the mammary glands and in the organs of metabolism and excretion, the high concentrations of tamoxifen in the pancreas and lung were not to be expected. However, estrogen receptors have been shown to be present in rat lung (Morishige and Uetake, 1978) and rat and human pancreas (Greenway et al., 1981; Satake et al., 1982) so these findings may indicate a genuine concentration of [laC]drug by the pancreas and lungs, rather than intravascular clotting following the i.v. injection as suggested by Wilking et al., (1981). There is also the possibility of binding to antiestrogen binding components in tissues which are not true estrogen target tissues (Sutherland et al., 1980; Sudo et al., 1983). Using [3H]tamoxifen, Major et al. (1976) showed that radioactivity was concentrated and retained by the reproductive tract and pituitary gland of female rats. This was later confirmed by direct measurement when higher uterine, compared with blood, concentrations of both tamoxifen and N-desmethyltamoxifen were found (Adam, 1981b). It was also shown in this study that tamoxifen was the predominant species in subcellular fractions of rat uterus. After administration of [lac]tamoxifen to patients before hysterectomy, Fromson and Sharp (1974) found, perhaps predictably, radioactivity to be 2-3 times higher in uterus than in blood. The distribution of [3H]4-hydroxytamoxifen has been reported in the immature rat (Jordan and Bowser-Finn, 1982). This antiestrogen has a longer biological half-life when compared with [3H]estradiol. However, like estradiol, the 4-hydroxytamoxifen binds (with estrogen specificity) in the uterus and vagina. 4-Hydroxytamoxifen also accumulates in the liver but this binding is not estrogen specific. The binding of 4-hydroxytamoxifen in the liver is, however, inhibited by the co-administration of antiestrogens. This result demonstrates the ability of antiestrogens to bind to sites other than the estrogen receptor. 5.2.5. Pharmacokinetics 5.2.5.1. Blood concentrations o f tamoxifen and metabolites. Gaskell et al. (1978) studied the plasma concentrations of tamoxifen in ovariectomized rats after i.m. doses of tamoxifen of 0.9, 8 and 29 mg/kg. The concentrations they found were 10.6, 97.3 and 244 ng/ml, respectively, suggesting approximate linearity of drug concentrations over the dosage range. In female patients, Mendenhall et al. (1978) showed that after a single oral dose of 10 mg/m 2 a peak serum value of 30 ng/ml was obtained after 3 hr. This preliminary work was extended by Fabian and Sternson (1979) who found a median blood concentration of 16 ng/ml between 3 and 6 hr after administration of the drug. After a single dose to patients with breast cancer, there is, however, a wide spread in serum concentrations; the differences between the highest and lowest values are around 20-fold at 3 hr and 7-fold at 6 hr after dosing (Fabian et al., 1980). Similarly, Wilkinson et al., (1980) found mean peak serum concentrations of 17.4 ng/ml, ranging between 15 and 25 ng/ml at 3 hr after a single

78

S.P. ROBINSONand V. C. JORDAN

oral dose of 10mg tamoxifen, which is roughly equivalent to 15mg/m 2. NDesmethyltamoxifen was undetectable (<2.5 ng/ml) in this study. In a detailed study in healthy male volunteers given a single oral dose of 20 mg tamoxifen, mean peak serum concentrations of tamoxifen and N-desmethyltamoxifen were 42 ng/ml (range 35-45) and 12 (range 10-20)ng/ml, respectively (Adam et al., 1980a). A number of studies have described the serum concentrations of tamoxifen, Ndesmethyltamoxifen, 4-hydroxytamoxifen and Metabolite Y under steady state conditions in women with breast cancer. The results of these studies, which show excellent agreement, are summarized in Table 6. Tamoxifen concentrations in blood are approximately 300 ng/ml after chronic doses of 20 mg b.i.d. In all of the studies, concentrations of Ndesmethyltamoxifen are about 50% higher than those of tamoxifen while those of 4hydroxytamoxifen are very low (approximately 3 ng/ml at 10 mg b.i.d, and approximately 6 ng/ml at 20 mg b.i.d.). 5.2.5.2. Tumor concentrations of tamoxifen and metabolites. After daily doses of 40 mg tamoxifen, tumor biopsy samples from women contained mean concentrations of tamoxifen of 25.1 ng/mg protein (range 5.4-117), N-desmethyltamoxifen of 52ng/mg protein (range 7.8-210) and 4-hydroxytamoxifen of 0.53 ng/mg protein (range 0.29-1.13) (Daniel et al., 1981). These values are roughly in proportion to the concentrations in plasma but there is no information on the subcellular concentrations of these compounds. 5.2.5.3. Half-life. The administration of [14C]tamoxifen to rats, mice, rhesus monkeys, dogs and women has shown that the drug has a long half-life (Fromson et al., 1973a,b). Excretion of radioactivity is apparently biphasic; in rats, the initial half-life is 53 hr with a terminal half-life of 10 days. In women, peak [14C]-values in serum were found 4-7 hr after a single oral dose, but at the time only 20-30% of the radioactive material was tamoxifen. After an initial blood half-life of 7-14 hr, serum concentrations of total radioactivity decayed with a secondary half-life of > 7 days. Using direct assay methods, initial reports described the elimination half-life of tamoxifen in patients to be between 5 and 44 hr (Fabian and Sternson, 1979; Wilkinson et al., 1980). However, when the duration of the study was extended, it became clear that the true half-life was around 7 days for tamoxifen (Adam, 1981b). This concurs with the results of Patterson et al. (1980) who showed serum tamoxifen concentrations to be maximal 3 hr after oral administration and that steady state serum concentrations are achieved only after four weeks' treatment. Steady state serum N-desmethyltamoxifen concentrations are reached after 8 weeks indicating a likely biological half-life for the metabolite of 14 days (Patterson et al., 1980). In healthy males, half-lives of 4 and 7 days for tamoxifen and N-desmethyltamoxifen, respectively, have been recorded (Adam, 1981a). The long biological half-life of the drug reflects not only a high level of plasma binding (Adam, 1981c), most probably to sites on albumin (Sjoholm et al., 1979), but also enterohepatic recirculation, which has been shown to occur in the rat and dog (Fromson et al., 1973a). It is also important to appreciate that tamoxifen has a high affinity (Kd approximately 1 riM) for sites ('antiestrogen binding sites') in most tissues (Sutherland et al., 1980; Kon, 1983; Sudo et al., 1983). The liver has a high concentration of these sites, primarily located in the microsomal fraction of the cells (Sudo et al., 1983). The function of the binding sites is unknown, however, a variety of pharmacological agents will compete for [3H]tamoxifen binding (Lyman and Jordan 1985b,c). In fact, several compounds have been described that do not bind to the estrogen receptor but do inhibit the binding of [3H]tamoxifen to microsomal binding sites (Lyman and Jordan, 1985b). These compounds do not inhibit estrogen action. 5.2.6. Metabolism of Tamoxifen in Laboratory Animals In Vivo The metabolism of ~4C-labeled tamoxifen in rat, mouse, rhesus monkey and dog was first reported by Fromson and coworkers (1973a). Conversion of tamoxifen to 4-hydroxytamoxifen was observed in all species. However, several other metabolites have been

Metabolism of steroid-modifying anticancer agents

79

identified (Fig. 21). The catechol, Metabolite D, is present as a glucuronide in the feces of all species studied and Metabolite E, tamoxifen without the amino ether side-chain, is tbund as a minor metabolite in dog bile. The major metabolite of tamoxifen in the chicken (Borgna and Rochefort, 1981a; Lazier and Jordan, 1982; Lyman and Jordan 1985a) and frog (Reigel et al., 1986) has been shown to be 4-hydroxytamoxifen. Similarly, large doses of tamoxifen have been used to demonstrate the metabolism to 4-hydroxytamoxifen in the ovariectomized rat (Bowman et al., 1982). Nevertheless, several different polar metabolites have been observed but not characterized. Borgna and Rochefort (1981b) described a polar metabolite, M2, which accumulates in the nucleus of rat uteri following [3H]tamoxifen administration. Robertson and coworkers (1982) have observed several polar metabolites of tamoxifen in rat uterus. One goal of the study of tamoxifen metabolism in different species is to identify potentially estrogenic metabolites which might explain the disparate pharmacology of the drug. Tamoxifen is a full estrogen in the mouse, a partial estrogen/antiestrogen in rats and humans and an antiestrogen in chicks. The pharmacology of tamoxifen has recently been reviewed (Furr and Jordan, 1984; Jordan, 1984). As yet, however, no significant differences in the metabolism of tamoxifen in different species have been noted (Lyman and Jordan, 1985a). In fact, there are some interesting biological models that argue against the idea that differential metabolism or the production of estrogenic metabolites from tamoxifen is responsible for estrogenic actions in the mouse. The breast cancer cell line MCF7 will grow into solid tumors in athymic mice only if the animals are co-transplanted with slow-release estrogen pellets. Growth is dependent upon estrogen stimulation. In contrast, tamoxifen causes an increase in the uterine weight of athymic mice but does not cause growth of implanted MCF-7 breast cancer cells (Gottardis et al., 1985). Tamoxifen inhibits estrogen-stimulated growth of MCF-7 breast cancer cells in vivo (Osborne et al., 1985). The metabolism of tamoxifen in the athymic mouse is currently under investigation and 4hydroxytamoxifen has been identified as the primary metabolite which binds to both the uterine and human tumor estrogen receptor (Robinson and Joroan, unpublished observations). Since the same metabolites are sequestered in different tissues, the ligand-receptor complex must be interpreted differentially to act as either a growth promoter or a growth inhibitor. 5.2.7. Metabolism o f Tamoxifen In Vitro Borgna and Rochefort (1981 b) found that tamoxifen is converted to 4-hydroxytamoxifen by liver, chicken oviduct and lamb uterus but not by rat uterus and MCF-7 breast ca~,~cer cells. Indeed, chick liver was used to produce the first quantity of [3H]4-hydroxytamoxifen from [3H]tamoxifen (Borgna and Rochefort, 1980). Rat liver microsomes convert tamoxifen to 4-hydroxytamoxifen and N-desmethyltamoxifen (Foster et al., 1980). The N-oxide of tamoxifen has been identified as a metabolite of tamoxifen if microsomes from phenobarbital treated rats are used (Foster et al., 1980). Ruenitz and coworkers (1984) have studied the effects of phenobarbital pretreatment on tamoxifen metabolism by rat liver microsomes. Barbiturate treatment caused an increase in N-demethylation and a decrease in 4-hydroxylation. The enzyme inhibitor metyrapone had no effect on tamoxifen metabolism by rabbit microsomes but decreased N-demethylation by rat microsomes. In contrast, SKF525A inhibited microsomal metabolism in both species although N-oxidation of tamoxifen was unaffected in either species. Two novel metabolites have been observed: 4'-hydroxytamoxifen and tamoxifen epoxide (Ruenitz et al., 1984). The epoxide may be a precursor of Metabolite A, a triphenyl ethanol reported originally by Fromson et al., (1973a) (Fig. 23). There are no biological data about Metabolite A but the structurally related compound MER 25 is an antiestrogen in all species (I,erner et al., 1958; Lyman and Jordan, 1985b). The metabolite 4'-hydroxytamoxifen has a higher binding affinity for the estrogen receptor than tamoxifen and is an antiestrogen in vitro (Jordan et al., 1984) and in vivo (Ruenitz et al., 1982). Conversely, tamoxifen and its metabolites might affect the metabolism of other agents.

80

S.P. ROBINSONand V. C. JORDAN

/CH3 2CH2N\cH3

Tamoxifen epoxide

OCH2CH2N/cH3

Metabolite A

OH

4' hydroxytomoxifen FIG. 23. Putative metabolites of tamoxifen.

Meltzer and coworkers (1984) have demonstrated that tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen are potent inhibitors of hepatic cytochrome P-450-dependent mixed function oxidases. This finding raises the question of whether tamoxifen can adversely affect the metabolism and excretion of cytotoxic cancer chemotherapeutic agents. 5.2.8. Pharmacological Activity of Metabolites 5.2.8.1. Metabolite B: 4-hydroxytamoxifen (ICI 79280). The pharmacological properties of the known metabolites of tamoxifen have been studied mainly in the rat. There is general agreement that 4-hydroxytamoxifen binds to the estrogen receptor with much higher affinity than tamoxifen (Jordan et al., 1977, 1978; Binart et al., 1979; Nicholson et al., 1979; Rochefort et al., 1979; Fabian et al., 1981; Jordan et al., 1980; Borgna and Rochefort, 1981b; Rochefort and Borgna, 1981; Wakeling and Slater, 1981a,b; Coezy et al., 1982; Jordan, 1982b; Jordan and Bowser-Finn, 1982). In fact, the affinity of 4hydroxytamoxifen is at least as great as that of estradiol for estrogen receptor. Like estradiol, and in contrast to tamoxifen, 4-hydroxytamoxifen shows a very slow dissociation rate from the receptor (Rochefort et al., 1979). 4-Hydroxytamoxifen localizes estrogen receptor complexes in the nucleus (Dix and Jordan, 1980a,b; Wakeling and Slater, 1981a,b). After administration of [3H]4-hydroxytamoxifen to ovariectomized or immature female rats the compound accumulates in uterus, vagina and pituitary gland (Jordan, 1982a; Jordan and Bowser-Finn, 1982). 4-Hydroxytamoxifen is about 100 times as potent as tamoxifen in inhibiting growth of human mammary cancer, MCF-7, cells in culture which is in accord with its higher affinity for the estrogen receptor (Coezy et al., 1982). Like tamoxifen, it behaves as a pure antiestrogen and inhibits the secretion of the estrogen-induced 46K protein by MCF-7 cells (Westley and Rochefort, 1980). The very high potency in vitro is not paralleled in a number of tests in vivo. Several groups have shown that 4-hydroxytamoxifen will inhibit the increase in uterine weight induced in rats by estradiol (Jordan et al., 1977, 1978; Rochefort et al., 1979; Nicholson and Griffiths, 1980; Wakeling and Slater, 1981a,b; Kemp et al., 1983) but there is no agreement about the relative potency compared with tamoxifen. Jordan et al. (1977) found it to be eight times as potent as tamoxifen whereas Wakeling and Slater (1981a) and Kemp et al. (1983) suggested that the compounds were equipotent. Although the reason for this discrepancy is unclear, it may be related to

Metabolism of steroid-modifyinganticancer agents

81

differences in the route of administration and to different rates of metabolism of the compounds in the strains of rats used. Certainly, the duration of action of 4-hydroxytamoxifen is much shorter than tamoxifen in vivo (Jordan and Allen, 1980), probably because 4-hydroxytamoxifen can be more readily conjugated and excreted. There is also agreement that 4-hydroxytamoxifen is less potent than tamoxifen on chronic dosing in the DMBA-induced rat mammary tumor model (Jordan and Allen, 1980; Nichoison and Griffiths, 1980) again probably because it has a shorter half-life. Like tamoxifen, 4-hydroxytamoxifen is a partial agonist and will stimulate uterine weight increases in immature rats (Joran et al., 1977; Dix and Jordan, 1980a,b; Wakeling and Slater, 1981a,b; Jordan 1982a; Vass and Green, 1982) and progesterone receptor synthesis. It is a full agonist in the ovariectomized mouse (Jordan et al., 1978). 5.2.8.2. Metabolite X: N-desmethyltamoxifen (ICI 55548). N-Desmethyltamoxifen has a low binding affinity for the estrogen receptor from rat and lamb uterus and human mammary cancer (Nicholson and Griffiths, 1980; Wakeling and Slater, 1980, 1981b; Fabian et al., 1981; Coezy et al., 1982; Kemp et al., 1983). N-Desmethyltamoxifen has similar biological properties to tamoxifen because it will antagonize the estrogen-induced increase in uterine weight in immature rats (Nicholson and Griffiths, 1980; Wakeling and Slater, 1980, 1981b; Kemp et al., 1983); however, it is less potent than tamoxifen. NDesmethyltamoxifen is a partial agonist and will not suppress completely the estrogeninduced increase in uterine weight (Jordan et al., 1983), but inhibits the growth of DMBAinduced rat mammary tumors in vivo (Nicholson and Griffiths, 1980) and MCF-7 cells in vitro (Coezy et al., 1982). 5.2.8.3. Metabolite D: 3,4-dihydroxytamoxifen (ICI 77307). Metabolite D has a high affinity for the rat uterine estrogen receptor, comparable to that of estradiol (Jordan et al., 1977). Interestingly, Metabolite D has no detectable estrogenic activity in the immature rat uterine weight test but is antiestrogenic at high doses (Jordan et al., 1977). Since the catechol structure can be readily oxidized and conjugated, it is likely that the low potency m vivo results from a very rapid clearance of the compound. In the ovariectomized mouse, where both tamoxifen and 4-hydroxytamoxifen are full agonists, Metabolite D is a partial agonist and has antiestrogenic activity (Jordan et al., 1978). Metabolite D is unstable in vitro but does inhibit estradiol-stimulated prolactin by primary cultures of rat pituitary cells. The potency of Metabolite D is increased by the antioxidant ascorbic acid and the inhibitor of catechol-O-methyl transferase U-0521 (Jordan et al., 1984). The antiestrogenic potency of the stabilized catechol metabolite is consistent with its high binding affinity for the estrogen receptor. 5.2.8.4. Metabolite E (ICI 141389). Removal of the basic side-chain from tamoxifen converts the compound to a full estrogen agonist (Jordan and Gosden, 1982). Metabolite E has a lower affinity than tamoxifen for estrogen receptors in the rat (Jordan and Gosden, 1982) and lamb (Coezy et al., 1982) uterus and is less effective in inhibiting the binding of [3H]estradiol to human breast cancer estrogen receptors (Jordan et al., 1983). Metabolite E will cause a full agonist response on the immature rat uterus, both on uterine weight and histology, and will induce progesterone receptor synthesis (Jordan and Gosden, 1982). Metabolite E has little inhibitory effect on growth of MCF-7 mammary tumor cells in culture (Coezy et al., 1982). Indeed, Metabolite E is a full agonist for the stimulation of prolactin synthesis by primary cultures of rat pituitary cells (Lieberman et al., 1983a; Jordan et al., 1984). This is consistent with the general structural features of the triphenylethylenes (Jordan et al., 1985). 5.2.8.5. Metabolite Y (ICI 142269). Metabolite Y has less affinity for the rat uterine estrogen receptor than tamoxifen (Jordan, 1982b; Kemp et al., 1983). Metabolite Y will inhibit implantation if given on day 4 of pregnancy in rats and partially prevent the JPT 36/I--F

82

s.p. Romr~soNand V. C. JORDAN

estrogen-induced increase in uterine weight in immature rats (Kemp et al., 1983) but is less potent than tamoxifen. It is also a partial agonist (Jordan et al., 1983). 5.2.8.6. Metabolite Z: N-desdimethyltamoxifen (ICI 142268). N-Desdimethyltamoxifen has similar properties to Metabolite Y and will bind to the rat uterine estrogen receptor and inhibit implantation in pregnant rats (Kemp et al., 1983). 5.2.8.7. Tamoxifen N-oxide. This metabolite has been identified from studies in vitro (Foster et al., 1980) and shown to have weak antiproliferative activity against MCF-7 breast cancer cells in culture (Bates et al., 1982). 5.2.9. Metabolic Activation and Antiestrogen Action The administration of [3H]tamoxifen to immature rats results in the binding of polar metabolites in the uterus (Borgna and Rochefort, 1981a). However, this should not imply that metabolic activation is required for the antiestrogen action of tamoxifen because the pharmacokinetics of a single injection do not correspond to the steady state conditions observed during continuous therapy. An extremely complex interaction of parent compound and metabolites with the estrogen receptor will occur during the therapeutic use of antiestrogens. If tamoxifen has to be converted to 4-hydroxytamoxifen before receptor binding in target tissues in vivo, then perhaps an inhibition of this metabolic step would yield compounds without activity. A series of 4 fluor-, chloro- and methyl-derivatives of tamoxifen that cannot be converted to 4-hydroxytamoxifen have been shown to be weakly antiestrogenic in immature rat uterine weight tests (Allen et al., 1980). Similarly, all the substituted derivatives of tamoxifen are partial estrogen agonists in the rat uterus, but are less potent than tamoxifen. 4-Hydroxytamoxifen is, however, at least at potent as tamoxifen, suggesting that the effect of tamoxifen is probably the combined effect of the parent compound and this hydroxylated metabolite. In man, where N-desmethyltamoxifen is present at higher concentrations than tamoxifen and there are negligible concentrations of 4-hydroxytamoxifen present under steady state conditions, N-desmethyltamoxifen may also contribute to the pharmacological activity observed (Reddel et al., 1983). Overall, these results suggest that conversion of tamoxifen to 4-hydroxytamoxifen and Ndesmethyltamoxifen may be an advantage but is not a requirement for the inhibition of estrogen action. This conclusion is consistent with the observed activity of tamoxifen in vitro where metabolism cannot be detected (Horwitz et al., 1978). Furthermore, tamoxifen and 4-methyltamoxifen have a similar ability to inhibit the binding of [3H]estradiol to estrogen receptors from rat pituitary glands and to inhibit estradiol-stimulated increases in prolactin synthesis in culture (Lieberman et al., 1983b). 5.3. NEW ANTIESTROGENSFOR FUTURECLINICAL USE Several new antiestrogens have recently been targeted for future clinical development in the treatment of advanced breast cancer. Unfortunately, there is, as yet, not very much data available to evaluate their efficacy in clinical trials. In this section, we will discuss these new compounds and comment upon their metabolic transformations. 5.3.1. Toremifene (Fc 1157a) The hormonal (Kallio et al., 1986) and antitumor properties (Kangas et al., 1986) of toremifene have recently been described. As might be expected from its structure (Fig. 24), the pharmacological properties are very similar to those reported for tamoxifen. The information concerning the metabolism of toremifene in rats and patients is available from the manufacturer (Farmos-Group Research Center, Cancer Research Laboratory, P.O. Box 425, 20101 Turku, Finland). The half-life of distribution of toremifene in the rat

Metabolism of steroid-modifying anticancer agents

83

is 4 hr and the t~/2 for elimination is approximately 24 hr. Very little toremifene is eliminated via the urine. The major route of elimination for toremifene (almost completely as metabolites) is in the feces via biliary excretion. The main metabolites found in the rat are shown in Fig. 24. Deamination of the side-chain also occurs to produce the glycol and carboxylic acid derivatives. Apparently, the chlorine atom is stable. In humans, the distribution and elimination t~/2 of toremifene are 4 hr and 5 days, respectively. Apparently, the pharmacokinetics are not dose dependent over the range of 3-680 mg. At steady state, a serum concentration of 600 ng/ml is obtained for toremifene if given at a daily dose of 60mg. The principal metabolite in humans is Ndesmethyltoremifene which has similar pharmacological properties to the parent drug. At present, there is no published evidence to suggest that toremifene offers an advantage over tamoxifen. However, it is interesting to point out that current clinical studies with toremifene are using much higher doses (60-100 mg daily) than those that are recommended for tamoxifen (20-40 mg daily). Whether this strategy reflects a belief that toremifene is less potent than tamoxifen or that greater therapeutic benefit can be obtained with higher doses of antiestrogen must await critical evaluation of published laboratory and clinical studies. 5.3.2. Droloxifene (KO6OE) Droloxifene, or 3-hydroxytamoxifen (Fig. 25), has a higher binding affinity for the estrogen receptor than tamoxifen (Ruenitz et al., 1982; Roos et al., 1983; Jordan et al., 1984) and in some assays in vitro this translates into a higher biological activity when compared with tamoxifen. Short-term assays of antitumor activity have demonstrated efficacy against the transplantable rat mammary tumor, R3230AC (L6ser et al., 1985a). -CH 3 OCH2CH 2 N ~ , c H 3

TOREMIFENE (RBA=9%)

\

~

/,CH 3 2CH 2N~,~ CH 3

4-HYDROXYTOREMIFENE (RBA=158%)

OCH2CH 2 NH CH 3

N-DESMETHYLTOREMIFENE (RBA=3%) FIG. 24. The structure of toremifene and main metabolites.

84

S.P. ROBINSONand V. C. JORDAN

This result is interesting because this particular tumor model is ovarian independent. However, tamoxifen was also active in these experiments. The major metabolite in rats and marmosets is N-desmethyldroloxifene (Fig. 25) (Huber and Stanislaus, 1985) which has very similar pharmacological properties to the parent compound (L6ser et al., 1985b). As yet, no clinical studies have been reported for droloxifene. It is perhaps important to point out that the hydroxylated antiestrogens, 4hydroxytamoxifen and LY 117018, which both have a high binding affinity for the estrogen receptor (Jordan et al., 1977; Black et al., 1981) and potent antitumor activity in vitro (Coezy et al., 1982; Scholl et al., 1983) were found to exhibit poor antitumor properties in vivo (Jordan and Allen, 1980; Wakeling and Valcaccia, 1983). This has been explained by the fact that the hydroxylated antiestrogens have short biological half-lives (Jordan and Gosden, 1983b) and may be unable to maintain the high blood levels that may be required for antitumor activity. Certainly, hydroxylated antiestrogens will be more susceptible to Phase II metabolism on first pass through the liver after absorption from the gastrointestinal tract. There are no published studies that address this question with the hydroxylated antiestrogen, droloxifene.

5.3.3. Zindoxifene (D16726) Zindoxifene (Fig. 26) has been extensively evaluated as an antitumor agent in laboratory models both in vitro and in vivo (von Angerer et al., 1985a). The drug is a weak partial agonist in mouse uterine weight tests (von Angerer, 1984) and effective in inhibiting the growth of DMBA-induced rat mammary tumors (von Angerer et al., 1985b). These biological effects are particularly interesting because the deacetylated derivative, D15414 (Fig. 26), is probably the active agent at the receptor level. We have evaluated the biological properties of D15414 in assays in vitro (prolactin synthesis and progesterone receptor synthesis) and found that D15414 is a weak estrogen with no antiestrogenic properties (Robinson and Jordan, unpublished observations).

/CH3 OCHzCH2N~CH3

OCH2CH2NHCH3

OH

OH

DROLOXlFENE (KO6OE) (3-hydroxytamoxifen)

N-DESMETHYLDROLOXIFENE

FIG. 25. The structure of droloxifene and main metabolite.

qHs C H s C O ' ~ I

CH2CH~ ZINDOXIFENE (D16726)

OCCHs

H

qH~ O ~ I

CH2CHs D15414

FIG. 26. The structure of zindoxifene and the deacetylated derivative (D15414).

OH

Metabolism of steroid-modifying anticancer agents

85

No clinical trials with zindoxifene have been reported. 5.3.4. S u m m a r y The antiestrogen, tamoxifen, has had an enormous impact upon the therapy of breast cancer. It is the most widely used antihormonal therapy. The success of tamoxifen has spurred the development of toremifene, droloxifene and zindoxifene; however, their clinical evaluation is not sufficently advanced to be able to provide any conclusions about their potential value. 6. ANTIANDROGENS 6.1. GENERALINTRODUCTION Surgical castration or treatment with estrogen therapy provides temporary objective and/or subject improvement in 60-70% of cases of prostatic cancer (Bailer et al., 1970; Barnes and Ninan, 1972; Byar, 1973). Both these procedures prevent testicular androgen secretion and lower circulatory testosterone levels. However, surgical castration is not always well accepted by patients and treatment with estrogens causes serious cardiovascular side-effects which often offset the benefits (Klein, 1979). The development of the antiandrogen, cyproterone acetate (Fig. 27), provided a new alternative for therapy (Scott and Schirmer, 1966). Cyproterone acetate was originally being developed as a synthetic progestin and indeed possesses substantial progestational activity (Neumann et al., 1979). The antiandrogenic action of this agent was identified by the feminization effect it induced in the offspring when it was administered to gestating female rats (Neumann et al., 1979). Subsequent studies have shown cyproterone acetate competitively binds to the androgen receptor and blocks androgen action (see Neff, 1976, for review). Studies have examined the potential use of cyproterone acetate to treat a variety of androgen-dependent conditions including acne, seborrhea, idiopathic hirsutism, androgenic alopecia and prostatic cancer (Neumann, 1977). The administration of cyproterone acetate will cause atrophy of the prostate and seminal vesicles in experimental animals and man (Neri, 1976). Clinical trials have demonstrated that the treatment of prostate cancer with this agent is as effective as estrogen therapy but with fewer side-effects (see Jacobi et al., 1980; Neumann and Jacobi, 1982, for reviews). The beneficial effects of cyproterone acetate are less pronounced in patients who have failed other endocrine therapies (estrogen or castration) compared with patients who received this agent as a first line of treatment. This may be because patients who failed one endocrine therapy now have hormone-independent disease (Jacob et al., 1980; Neumann and Jacob, 1982). Along with the antiandrogenic and progestational activity, cyproterone acetate also possesses antiestrogenic, antigonadotropic and ACTH-suppressing activity. In contrast, the development of the nonsteroidal agent, flutamide (Fig. 27), provided an agent with purely antiandrogenic activity (Neri et al., 1972). The use of this 'pure' antiandrogen in clinical trials has shown equal effectiveness as estrogen therapy (Neumann and Jacobi,

,,,,*OCCH5 0

CH2 P-,

,

I

I

I

0

O

, -'CH3

, ~

NH"'c'~CH"CH

Cyproterone

CF3 Acetate

. ~ . , , , , , / N . , ~ c.3

1. I1

OzN'~,,,,/

OzN CI

/t....

Flutamide

cF~ RU 2 3 9 0 8

FXG.27. The structure of the major steroidal and nonsteroidal antiandrogens.

86

S.P. ROBINSONand V. C. JORDAN

1982) in the treatment of prostate cancer. Side-effects are few and flutamide has been characterized as a 'safe antiandrogen' (Sogani and Whitmore, 1979). Flutamide is also devoid of the side-effects produced by the progestational activity of cyproterone acetate; however, the antigonadotropic activity produced as a consequence of this progestational activity is also lacking. Treatment with flutamide has been demonstrated to result in an increase in testosterone production (Prout et al., 1975) in noncastrate men. This results from an inhibition of the negative feedback of androgens at the pituitary gland which results in a rise in LH levels to stimulate androgen synthesis by the Leydig cells of the testis (Neumann and Schenck, 1976). This questions the wisdom of using pure antiandrogens alone for the prolonged treatment of prostate cancer. Following the development of superactive LHRH analogs and the use of these agents to block steroidogenesis in prostatic cancer patients (see section on LHRH analogs in this review), interest was directed at the combination of an LHRH analog and an antiandrogen (Labrie et al., 1983b). Initial treatment with LHRH analogs results in an increased androgen level and may produce tumor flare (Trachtenberg, 1983). Following prolonged treatment, androgen production is reduced to castrate levels. However, even in the castrate patient, androgen production from the adrenals will remain and circulating androgen levels may be sufficient to support tumor growth (Stanford et al., 1977). A combination of castration and antiandrogen therapy has been used in some clinical trials (Giuliani et al., 1980; Sander et al., 1982). Recently, combination of cyproterone acetate with buserelin (Klijn et al., 1985), flutamide with D-Trp 6 L H R H (Labrie et al., 1985) and RU 23908 with Buserelin (Labrie et al., 1984) have all been examined in clinical studies. RU 23908 is a pure antiandrogen derived from flutamide (Fig. 27). Labrie et al. (1984) have reported that the use of this agent in combination with Buserelin results in a positive response in all patients treated and an improved survival time. Further studies are required to confirm these promising results.

6.2. STRUCTURAL FEATURES OF STEROIDAL AND NONSTEROIDAL ANTIANDROGENS

The antiandrogens can be divided into two groups on the basis of structure. The first group to be developed was based on the steroid nucleus, and the most clinically useful agent has proved to be cyproterone acetate. This drug is a hydroxyprogesterone derivative (see Fig. 27) and was initially examined as a potential orally active progestogen (see Neumann et al., 1979). Studies have demonstrated a high affinity for the progesterone receptor and the production of progestational responses. In contrast, cyproterone (the free alcohol) has a much lower affinity for the progesterone receptor and little progestational activity (Grill et al., 1985). The second group of antiandrogens are nonsteroidal compounds. Until recently, the substituted anilide, flutamide, was accepted as the only clinically useful nonsteroidal antiandrogen (see Fig. 27). The drug appears to be specific for androgen receptors and does not interact with other steroid receptor systems. RU 23908 was developed from flutamide and has recently been introduced into clinical trials (see Fig. 27). This agent, like flutamide, is a pure antiandrogen (see Raynard et al., 1984, for review) and causes a rise in LH in men because it blocks the negative feedback mechanism by testosterone at the hypothalamo-pituitary axis. Although this is a rapidly expanding area of research, it should be pointed out that very little has been published about the structure-activity relationships of antiandrogens.

Metabolism of steroid-modifying anticancer agents

87

6.3. CYPROTERONE ACETATE: A STEROIDAL ANTIANDROGEN 6.3.1. A n i m a l Metabolism The systemic adsorption of cyproterone acetate is extensive following oral administration to rats, dogs and monkeys. The circulating half-life, as determined by radioimmunoassay of plasma following an intravenous injection of cyproterone acetate, is similar for the rat (26.3 hr) and rhesus monkey (25.3 hr) but considerably longer in the dog (109 hr) (Diisterberg et al., 1981). Schulz (1976) found, using radiolabeled cyproterone acetate, an average of 81% absorption of an oral dose in the monkey and a circulating half-life of 1.6-1.8 days. Radioactivity in the urine and feces was in the ratio of 0.7 : 1. Shortly (30 min) after intravenous administration of [3H]cyproterone acetate to male rats, a large amount of radioactivity accumulates in the liver. The radioactivity is, however, predominantly associated with a metabolite rather than the parent compound. Similarly, analysis of plasma demonstrates that the principal peak of radioactivity is also associated with a metabolite. Nevertheless, cyproterone acetate is the predominant compound identified in androgen target tissues such as the prostate, seminal vesicles, and bulbo-cavernosus and levator ani muscle (Szalay et al., 1975). Analysis of dog plasma following long-term (4 weeks) oral treatment (100 mg/kg/day) with cyproterone acetate has identified a derivative 15fl-hydroxycyproterone acetate (11 #g/ml) as well as the parent (4 #g/ml) (see Fig. 28). If plasma samples are analyzed from rhesus monkeys following long-term (12 weeks) oral treatment (40 mg/kg/day) with cyproterone acetate, only minimal amounts of unconjugated 15fl-hydroxycyproterone acetate and cyproterone acetate are detected. The major metabolite found in the urine of these monkeys is the 15fl-hydroxy derivative. However, additional undetermined minor metabolites are also observed (Bhargava et al., 1977). In the female rat, cyproterone acetate has been shown to cause considerable enlargement of the liver and to be a potent inducer of hepatic microsomal monoxygenase activity. This effect is less pronounced in the male rat and has been attributed to the inhibition of endogenous androgen which is known to induce certain pathways of drug metabolism (Schulte-Hermann and Parzefall, 1980; Schulte-Hermann et al., 1980).

6.3.2. H u m a n Metabolism Cyproterone acetate is almost completely absorbed following oral dosing of humans (2 mg), and plasma levels are maximum by 1-4 hr. The plasma half-life of cyproterone acetate is 1.5-2 days. About 30--35% of the total dose administered is excreted in the urine and about 50-60% in the feces within 8-10 days (Speck et al., 1976; Humpel et al., 1977). Repeated doses of cyproterone acetate (2 mg/day) result in increasing circulating levels of this agent. This accumulation reaches a steady-state level after 5-8 days of treatment which is 2-3 times that observed after a single administration. The apparent volume of distribution for cyproterone acetate is larger than that of body fluids indicating a distribution into body fat (Humpel et al., 1979). CH3 I

CH3 I

C-O

~,...~-ococH3 c82

r

T

"1

Cl

cH2

C,,O

~V~:OCOCHs F

•

1

Cl

15~-hydroxy cyproterone acetote FIG. 28. The structure of cyproteroneacetate and main metabolite.

cyproterone ocetate

88

S.P. ROBINSONand V. C. JORDAN

The major metabolite of cyproterone acetate in man is the 15fl-hydroxy derivative. Plasma levels of this metabolite reach about twice the levels of cyproterone acetate itself (Frith and Phillipou, 1985). The polar metabolite has a much smaller apparent volume of distribution than cyproterone acetate and is the predominant urinary product. Hence, overall, there appears to be a steady state reached between retransfer of cyproterone acetate from tissue, metabolism to 15fl-hydroxycyproterone acetate and/or elimination. The lipophilic nature of cyproterone acetate may be the determining factor in its long halflife. Free metabolite and parent drug are the predominant compounds excreted in the urine. About 30% of the metabolite in the urine and 50-60% in the bile are conjugated with glucuronic acid and 10% in both as sulfates (see Hiimpel et al., 1979, for detailed studies of pharmacokinetics). Cyproterone acetate has been shown to be extensively bound in serum (>93%) by a heat-stable component (predominantly albumin) whilst little of the drug is bound by sex hormone binding globulin (SHBG) (Hammond et al., 1982). The lack of binding to SHBG is supported by the reports of Geller et al. (1975), Mobbs et al. (1977) and Frolich et al. (1978), but conflict with the report of firm binding of cyproterone acetate to SHBG by Sammelwitz et al. (1973). The latter authors found cyproterone acetate did not displace [3H]corticosterone from corticosteroid binding globulin. 6.3.3. Activity o f Metabolites The 15fl-hydroxy derivative of cyproterone acetate has been observed to have a greater inhibitory action of sebacous gland function than the parent compound following systemic administration. However, the relative potency of this major metabolite in the classical target tissues is less than that of cyproterone acetate (Neumann et al., 1979). 15fl-Hydroxycyproterone acetate has a much lower binding affinity for the progesterone receptor than cyproterone acetate and has considerably less progestational activity (Frith and Phillipou, 1985; Grill et al., 1985). Studies in vitro indicate the formation of the 15fl-hydroxy derivative is not required for antiandrogen activity. 6.4. FLUTAMIDE AND R U 23908: NONSTEROIDAL ANTIANDROGENS

6.4.1. Animal Metabolism Few studies have been published on the metabolism of these agents in animals. From studies performed with flutamide on rat prostate tissue in vivo and on tissue in vitro a disparity is observed between the activities of the two systems. It is therefore hypothesized that a metabolite of flutamide may be the active agent. A study in humans has been used to identify the metabolic pathway (see following section) and a hydroxy derivative has been identified as the major plasma metabolite (Fig. 29). A similar metabolic pathway has been indicated for the rat (see review by Neri, 1976). The metabolism and excretion of [14C]RU 23908 in the rat has been studied and reported by Raynaud and coworkers (1985). Absorption following oral administration of [14C]RU 23908 (10 mg/kg) to rats is rapid and radioactivity in the plasma reaches a peak equivalent to 5-6 #g of drug per ml. The major proportion of plasma radioactivity observed is unchanged compound. The terminal half-life reported in the rat is 11 hr. The major route of excretion of [14C]RU23908 in the rat (two-thirds of the radioactivity) is via the kidneys after either oral or intravenous administration. The majority (94%) of urinary excretion takes place in the first 2 days. Analysis of rat urine following enzyme hydrolysis has identified the metabolites shown in Fig. 30 as the major contributors to the radioactivity found in urine. Only a very small quantity of the parent compound is excreted. The major metabolites are compounds resulting from partial (Metabolite A) and full (Metabolite B) reduction of the nitro group or the primary alcohol of Metabolite B (Metabolite C).

Metabolism of steroid-modifying anticancer agents

CH3~cH/CH3 I CO

CH3 /CH3 '~COH I CO

NH

NH

NH2 C

CF3

CF

NO2 FLUTAMIDE t

/

~

NH2 CF3

I\

NO2

NO2 MAJORPLASMA METABOLITE CH3~Io¢CH3

/%

89

T^ NO2

MAMJEOUoR TRALNTA~ Y

I \ I I~

?o

(c.,O<

,.pl/

NO2 FIG. 29. The metabolic pathway of flutamide in the human (proposed by Katchen and Buxbaum, 1975).

6.4.2. Human Metabolism Flutamide is rapidly and extensively absorbed following oral administration to humans and significant absorption has been reported following topical application (Katchen et al., 1976; Katchen and Buxbaum, 1975). Extensive metabolism of flutamide occurs. Just 1 hr after oral administration, levels of hydroxyflutamide (Fig. 29), the major plasma metabolite, have been reported to be 10 times that of flutamide itself. The rapid appearance of this metabolite suggests first-pass metabolism in the liver. The metabolic pathway for flutamide has been proposed as removal, by stepwise oxidation, of the isobutyryl sidechain followed by hydroxylation of the resulting nitro aniline of phenylenediamine (Fig. 29) (Katchen and Buxbaum, 1975). Excretion of tritium-labeled flutamide has been observed to be mainly through the kidney. Glucuronidase hydrolysis of the urine samples results in an increase of extractable tritium from 44.7% to 77.5%. The major urinary metabolite has been identified as ~tcttrifluoro-2-amino-5-nitro-p-cresol which constitutes 32% of the extractable radioactivity (Fig. 29) (Katchen and Buxbaum, 1975). 6.4.3. Activity of Metabolites Hydroxyflutamide is capable of inhibiting testosterone propionate stimulated growth of the prostate gland and seminal vesicles in castrated rats. This major metabolite is about 1.5 times as active as flutamide in this test. Both flutamide and its hydroxy derivative inhibit the uptake of radiolabeled androgen into rat prostate tissue in vivo. However,

90

S.P. ROBINSONand V. C. JORDAN

0

CIH3

#--C--CH3

O,N-W(~--.I ~ . l

#--NH 0 Ru 25908

CF3 II

A ~ o,c.~

/C-C-CH3 -

H

3

F

HO.

H2N~//

E

O, CH. <

o cm

-~ H2N-~/

C-c-ell. \~'-N/

c~',_~c.."°>~"_~cJ

> - = - , )c-.. CF3'

R

"C--C--CH 3 \~-'N( [

HOz H

I

°

C~

c-,.

°c_~_ ~

0

CF3

D

}-~. 0

o ~.3

.,.-{ }-,(

C-C--CH 3

I

#C--N~oH

CF 3

0

FIG. 30. The metabolicpathway of RU 23908in the rat (proposedby Raynaud et al., 1985).The major metabolitesare A, B and C. flutamide in vitro is considerably less effective than its metabolite in inhibiting the uptake of androgen (Neri et al., 1979). The higher binding affinity of hydroxyflutamide than flutamide for the androgen receptor supports the suggestion that this metabolite is the active form in vivo. Other metabolites of flutamide possess little or no antiandrogenic activity (Neri, 1976). The binding affinity of RU 23908 for the androgen receptor is similar to that of hydroxyflutamide (Wakeling et al., 1981). A study in vitro designed to examine possible activation of RU 23908 to a more potent agent found no evidence for this (Brown et al., 1981). Investigations using Metabolites A and B of RU 23908 (Fig. 30) indicate these compounds are unable to bind to the androgen receptor (Raynaud et al., 1985).

7. GENERAL SUMMARY AND CONCLUSIONS The application of steroid-modifying drugs as a strategy for the treatment of hormonedependent cancers has gained increasing popularity during the past decade. However, it is important to point out and emphasize that very few of the agents were originally designed for their current application. Most were designed for other purposes, predominantly fertility control (e.g. L H R H agonists and the antiestrogens). Nevertheless, now it is possible to integrate their actions to design rational therapies. There are many reasons for the current interest in antisteroidal drugs. The initial euphoria over the potential ability of combination chemotherapy to cure breast and prostatic carcinoma has proved to be premature. Combination chemotherapy has many severe side-

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effects which limits p a t i e n t acceptability, especially if the p a t i e n t realizes that the likelih o o d o f a cure is remote. In the main, a n t i s t e r o i d a l therapies d o n o t have m a n y sideeffects a n d those that d o , e.g. a m i n o g l u t e t h i m i d e , are the focus o f increased efforts in d r u g design to p r o d u c e increased d r u g specificity. F i n a l l y , there is a g r o w i n g realization t h a t h o r m o n e - d e p e n d e n t c a n c e r c o n t r o l with a nontoxic, a n t i s t e r o i d a l t h e r a p y m a y be the m o s t a c c e p t a b l e a p p r o a c h c u r r e n t l y available for early disease m a n a g e m e n t . C h e m o t h e r a p y w o u l d then be reserved as the final o p t i o n for treatment. T h e description o f d r u g m e t a b o l i s m has been central to the d e v e l o p m e n t o f synthetic L H R H a n a l o g s a n d a n u n d e r s t a n d i n g o f the m o d e o f action o f n o n s t e r o i d a l antiestrogens a n d a n t i a n d r o g e n s . The d i s c o v e r y o f steroid synthetic p a t h w a y s has been essential for the d e v e l o p m e n t o f the a r o m a t a s e inhibitors. This w h o l e area o f e n d e a v o r has n o w b e c o m e a m a j o r focus o f a t t e n t i o n for the m e d i c i n a l chemist. A new g e n e r a t i o n o f agents is entering clinical e v a l u a t i o n which will p r o v i d e a wealth o f valuable i n f o r m a t i o n a b o u t the successful (or unsuccessful?) m e t h o d s to c o n t r o l h o r m o n e - d e p e n d e n t disease. Since the success o r failure o f a d r u g c a n often d e p e n d u p o n f o r m u l a t i o n , p h a r m a c o k i n e t i c s , b i o a v a i l a b i l i t y o r m e t a b o l i s m , it is o u r h o p e t h a t this overview m i g h t help solve some o f the future problems. Acknowledgements--We are grateful for the support of the University of Wisconsin Clinical Cancer Center Anderson Fund. Some of the studies reported here were supported by grants P30-CAI4520, P01-CA20432 and R01-CA32713.

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