ARIMIDEX: a potent and selective aromatase inhibitor for the treatment of advanced breast cancer

Aromatase inhibitors have been available for a number of years and their ability to reduce circulating estradiol levels has been shown to produce clinical benefit in women with advanced breast cancer. Until recently, the only commercially available aromatase inhibitor was aminoglutethimide. Although aminoglutethimide has been shown to be efficacious in the treatment of advanced breast cancer, it does cause significant toxicity and requires the use of concomitant hydrocortisone therapy. Anastrozole is one of a new class of potent aromatase inhibitors able to suppress estradiol to the limit of detection of sensitive assays without suppressing adrenal steroidal synthesis. Two large clinical trials (n = 764) conducted in the U.S.A. and in Europe evaluated two doses of anastrozole, 1 and 10 mg a day, compared to megesterol acetate, 40 mg four times a day, in postmenopausal women who had progressed while on tamoxifen. Response rates and time to progression with anastrozole were similar to those of megesterol acetate. Objective responses (CR + PR) were 10.3%, 8.9% and 7.9% in the 1 and 10 mg of anastrozole and megesterol acetate treatment groups, respectively. Another 25.2%, 22.6% and 26.1% had stable disease for over 24 weeks on 1, 10 mg anastrozole and megesterol acetate, respectively. Anastrozole and megesterol acetate were well tolerated; however, more patients had significant weight gain on megesterol acetate than with anastrozole treatment. The weight gain seen with megesterol acetate continued to increase over time. Anastrozole has a better therapeutic index (fewer side-effects) and has recently been approved by the FDA and a number of other regulatory agencies around the world for the treatment of advanced breast cancer.     

ORG 33201: A new highly selective orally active aromatase inhibitor

Org 33201 has been selected as a very potent aromatase inhibitor. The compound is an enantiomer of a SC₂H₅ substituted imidazoylethylphenalene. Org 33201 inhibited human aromatase activity for 50% at a concentration of 2.2 × 10⁹ mol/l. More than 200-fold higher concentrations were needed for the inhibition of other cytochrome P-450 enzymes. In vivo the compound was active in rats (ED₅₀ = 0.035 mg/kg) and dogs (1 mg/kg gave 70% inhibition) after oral administration. It can be concluded that Org 33201 is a potent and highly selective orally active aromatase inhibitor.     

R 76713, a new specific non-steroidal aromatase inhibitor

The effects of R 76713, a new triazole derivative, on rat ovarian, testicular and adrenal steroidogenesis were investigated both in vitro and in vivo. In vitro R 76713 is a very potent inhibitor of the aromatase enzyme in rat granulosa cells, showing an IC50-value of 3.0 +/- 0.2 nM. The compound is about 1000 times more active than aminoglutethimide which shows an IC50-value of 3900 +/- 2800 nM in the same system. R 76713 is also a highly selective aromatase inhibitor. In cultures of ovarian, testicular and adrenal cells, formation of progesterone, androgens and glucocorticoids was only affected by drug concentrations higher than 1 microM. In vivo, single oral drug doses of 0.05 mg/kg lowered plasma estradiol levels of PMSG-primed female rats by more than 90%. An ED50-value of 0.005 mg/kg could be calculated. A single oral dose of 1 mg/kg suppressed plasma estradiol levels almost completely for 24 h. A dose of 0.1 mg/kg lowered plasma estradiol by more than 90% for 8 h. In vivo, R 76713 also showed a highly selective profile. In LHRH/ACTH-injected rats, plasma levels of testicular and adrenal steroids remained unchanged after administration of a drug dose of 20 mg/kg. R 76713 at drug concentrations of 10 microM, showed no interaction in vitro with estrogen-, progestin-, androgen- and glucocorticoid-receptors. Given orally at 20 mg/kg for 3 days the compound also showed no estrogen or androgen agonistic or antagonistic effects.     

Exemestane,a new steroidal aromatase inhibitor of clinical relevance

Breast cancer is the leading cause of death among women and the contribution of circulating oestrogens to the growth of some mammary tumours has been recognized. Consequently, suppression of oestrogen action by inhibition of their biosynthesis at the androstenedione-oestrone aromatization step, by means of selective inhibitors of the enzyme aromatase, has become an effective therapeutic option for the treatment of hormone-dependent breast cancer. Exemestane (6-methylenandrosta-1,4-diene-3,17-dione) is a novel steroidal irreversible aromatase inhibitor recently approved and introduced into the global market under the name Aromasin. The design, laboratory and viable syntheses of exemestane, starting from a variety of steroidal precursors, are presented and discussed. Data from biochemical and pharmacological studies as well as the clinical impact of the compound are briefly reviewed. The drug is an orally active and well-tolerated hormonal therapy for postmenopausal patients with advanced breast cancer that has become refractory to standard current hormonal therapies.     

A photoaffinity inhibitor of aromatase

Several 7-substituted 4-androstene-3, 17-diones are potent inhibitors of the biosynthesis of estrogens, with the most effective being 7-(4'-amino)phenylthio-4-androstene-3, 17-dione. An azide derivative of this 7-thioether compound has been prepared as a potential photoaffinity inhibitor. The enzyme kinetics of the azide analog were examined under both dark conditions and UV irradiation. In the dark, the azide was a very potent competitive inhibitor, with an apparent K_i of 1.3 nM. Under UV-irradiation, a time-dependent loss of aromatase activity was also observed. These studies indicate that the 7-substituent enhances the affinity of the steroidal analogs for the enzyme site.     

Growth suppression of MCF-7 human breast cancer cells by aromatase inhibitors: A new system for aromatase inhibitor screening

In our previous study we found that MCF-7 cells possess aromatase activity and stimulate estrogen receptor-mediated growth. The pathways through which androgens are converted to estrogens by aromatase and estrogens interact with estrogen receptors contribute significantly to growth stimulation. The administration of aromatase inhibitor results in suppression of growth stimulation by androgens. This system enabled us to assess directly the biological activities of aromatase inhibitors. Aromatase activity was inhibited in a dose-dependent manner by the addition of aminoglutethimide and CGS 16949A, competitive inhibitors, and of 14-hydroxy-4-androstene-3,6,17-trione and 4-hydroxy-androstenedione, mechanism-based inhibitors. After preincubation with mechanism-based inhibitors, aromatase activity was significantly suppressed, whereas after preincubation with competitive inhibitors, it was adversely increased. These effects were concentration- and time-dependent. Preincubation with competitive inhibitors resulted in augmentation of subsequent androgen stimulation of thymidine incorporation, while preincubation with mechanism-based inhibitors resulted in diminished stimulation by subsequent androgen administration. These results suggest that in MCF-7 cells competitive inhibitors adversely induce aromatase and accelerate the subsequent androgen stimulation of DNA synthesis. Suicide inhibitors are more effective than competitive inhibitors. This system will be useful for aromatase inhibitor screening.     

Exemestane (FCE 24304), a new steroidal aromatase inhibitor.

Exemestane (FCE 24304; 6-methylenandrosta-1,4-diene-3,17-dione) is a novel orally active irreversible aromatase inhibitor. Its in vitro and in vivo pharmacological properties have been compared to 4-hydroxyandrostenedione (4-OHA). In preincubation studies with human placental aromatase, exemestane, like 4-OHA, showed enzyme inactivating properties with a similar affinity (Ki 26 vs 29 nM) and a lower rate of inactivation (t1/2 13.9 vs 2.1 min). Conversely, when tested in pregnant mares' serum gonadotropin-treated rats, exemestane was more potent in reducing microsomal ovarian aromatase activity than 4-OHA, after both subcutaneous (ED50 1.8 vs 3.1 mg/kg) and oral dosing (ED50 3.7 vs greater than 100 mg/kg). No interference of exemestane on desmolase or 5 alpha-reductase activity was found. The compound did not show any relevant binding affinity to steroidal receptors, but slight binding to the androgen receptor (approximately 0.2% of dihydrotestosterone), like 4-OHA. In the first phase I trial, healthy postmenopausal volunteers were given single oral doses of exemestane, ranging from 0.5 to 800 mg, and plasma [estrone (E1), estradiol (E2) and estrone sulphate (E1S)] and urinary estrogens (E1 and E2) were measured up to 5-8 days. The minimal effective dose in decreasing estrogens was 5 mg. At 25 mg the maximal suppression was observed at day 3: plasma estrogens fell to 35 (E1), 39 (E2) and 28% (E1S), and urinary estrogens fell to 20 (E1) and 25% (E2) of basal values, these effects still persisting on day 5. No effects on plasma levels of cortisol, aldosterone, 17-hydroxyprogesterone, DHEAS, LH and FSH, and no significant adverse events were observed up to the highest tested dose of 800 mg exemestane.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.     

Determination of exemestane, a new aromatase inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection

A sensitive and selective high-performance liquid chromatographic method for the determination of 6-methylen-androsta-1,4-diene-3,17-dione (exemestane) and its 17-dihydro metabolite in human plasma has been developed. The analytes and internal standard (Norgestrel) were extracted from plasma samples with a methylene chloride—iso—octane mixture; the organic phase was dried and the residue was reconstituted with an acetonitrile—water mixture, then analyzed by reversed-phase liquid chromatography. Quantification was achieved by ultraviolet detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human blank plasma was observed. The lower limit of quantification was 10 ng/ml plasma. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from healthy male volunteers who had received a 200-mg single oral dose of the test compound.     

6-Methylenandrosta-1,4-diene-3,17-dione (FCE 24304): a new irreversible aromatase inhibitor

FCE 24304 (6-methylenandrosta-1,4-diene-3,17-dione), a new irreversible aromatase inhibitor, has been identified and characterized in vitro and in vivo. The compound caused time-dependent inactivation of human placental aromatase with a t1/2 of 13.9 min and ki of 26 nM. When tested in PMSG-treated rats, ovarian aromatase activity was reduced 24 h after dosing by both the s.c. (ED50 1.8 mg/kg) and the oral (ED50 3.7 mg/kg) routes. No interference with 5 alpha-reductase activity nor any significant binding affinity for estrogen receptor was found. Slight binding affinity for the androgen receptor (RBA 0.2% of DHT) was observed.     

Behavioral demasculinization of female quail is induced by estrogens: studies with the new aromatase inhibitor, R76713.

The injection before Day 12 of incubation of estradiol benzoate (EB) into Japanese quail eggs produces a complete behavioral demasculinization of adult males that will hatch from these eggs. These males never show copulatory behavior even after administration of high levels of exogenous testosterone (T). It is usually assumed that such a demasculinization normally takes place in female embryos under the influence of endogenous estrogens but few experimental data are available to confirm the validity of this model. A series of four experiments was performed during which R76713, a triazole derivative that specifically inhibits aromatase (estrogen synthetase) activity, was injected into quail eggs at different stages of incubation to prevent the production of endogenous estrogens. The consequences of these embryonic treatments on the T-activated sexual behavior in adults were then quantified. When injected before Day 12 of incubation, R76713 completely blocked the behavioral demasculinization of females without affecting the behavior of the males. After a treatment with T, almost all R76713-treated females showed as adults a masculine copulatory behavior that was undistinguishable from the behavior of intact males. This effect was fully reversed by the injection in egg of EB demonstrating that the effects of R76713 were specifically due to the suppression of endogenous estrogens. Injection of R76713 during the late phase of the incubation (Day 12 or Day 15) only maintained weak copulatory behavior in females which confirmed that the behavioral demasculinization in quail takes place mainly though not exclusively during the early stages of ontogeny. In a last experiment, we combined an early R76713 treatment with an injection of EB either on Day 9 or on Day 14 of incubation. This showed that the sensitivity to differentiating effects of estrogens varies with age in a sexually differentiated manner. The EB injection on Day 9 demasculinized both male and female embryos. If this injection was delayed until Day 14, it was no longer effective in males but still caused a partial demasculinization of females. This demonstrates that even if females are not yet behaviorally demasculinized on Day 9 of incubation (suppression of aromatase activity at that age will maintain the behavior), their sensitivity to estrogens is already different from that of males.     

Aminoglutethimide, an inhibitor of aromatase from chick embryo ovary

The gonads from 13-day-old female chick embryos were cultured in vitro on TC medium 199, and oestradiol production was measured by radioimmunoassay. In the presence of dehydroepiandrosterone as substrate, oestradiol synthesis was markedly increased, but when aminoglutethimide was also present, it was greatly reduced, depending on the concentration of the drug. This result demonstrates inhibition of the aromatizing enzyme system of the chick embryo ovary by aminoglutethimide in vitro. However, sex differentiation of the female gonads was not modified after in vivo treatment. Since it is not known whether their production is completely suppressed in vivo, the hypothesis cannot be dismissed that oestrogens play a role in ovarian differentiation.     

Mannostatin A, a new glycoprotein-processing inhibitor

Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal alpha-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward alpha- and beta-glucosidase and galactosidase as well as beta-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal alpha-mannosidases, with estimated IC50's of 70 nM, 450 nM, and 160 nM, respectively. The type of inhibition was competitive in nature. This compound also proved to be an effective competitive inhibitor of the glycoprotein-processing enzyme mannosidase II (IC50 of about 10-15 nM with p-nitrophenyl alpha-D-mannopyranoside as substrate, and about 90 nM with [3H]mannose-labeled GlcNAc-Man5GlcNAc as substrate). However, it was virtually inactive toward mannosidase I. The N-acetylated derivative of mannostatin A had no inhibitory activity. In cell culture studies, mannostatin A also proved to be a potent inhibitor of glycoprotein processing. Thus, in influenza virus infected Madin Darby canine kidney (MDCK) cells, mannostatin A blocked the normal formation of complex types of oligosaccharides on the viral glycoproteins and caused the accumulation of hybrid types of oligosaccharides. This observation is in keeping with other data which indicate that the site of action of mannostatin A is mannosidase II. Thus, mannostatin A represents the first nonalkaloidal processing inhibitor and adds to the growing list of chemical structures that can have important biological activity.     

Mechanism of human placental aromatase: A new active site model

Based on Akhtar's ferric peroxide mechanism and on recent studies in our own laboratory, we present a detailed proposal for aromatase action. This picture can account for the known stereochemical consequences at C-19 observed by others using isotopes of hydrogen and oxygen. The postulated process involves anchoring of the 19-hydroxymethyl and 19-oxo groups at the active site by a glutamate residue, which also serves to activate the 19-oxo group for attack by ferric peroxy species in the third oxidative step.     

Sexual differentiation of brain and behavior in quail and zebra finches: Studies with a new aromatase inhibitor, R76713

In many species of vertebrates, major sex differences affect reproductive behavior and endocrinology. Most of these differences do not result from a direct genomic action but develop following early exposure to a sexually differentiated endocrine milieu. In rodents, the female reproductive phenotype mostly develops in the absence of early steroid influence and male differentiation is imposed by the early action of testosterone, acting at least in part through its central conversion into estrogens or aromatization. This pattern of differentiation does not seem to be applicable to avian species. In Japanese quail (Coturnix japonica), injection of estrogens into male embryos causes a permanent loss of the capacity to display male-type copulatory behavior when exposed to testosterone in adulthood. Based on this experimental result, it was proposed that the male reproductive phenotype is “neutral” in birds (i.e. develops in the absence of endocrine influence) and that endogenous estradiol secreted by the ovary of the female embryo is responsible for the physiological demasculinization of females. This model could be recently confirmed. Females indeed display a higher level of circulating estrogens that males during the second part of their embryonic life. In addition, treatment of female embryos with the potent aromatase inhibitor, R76713 or racemic vorozole™ which suppresses the endogenous secretion of estrogens maintains in females the capacity to display the full range of male copulatory behaviors. The brain mechanisms that control this sexually differentiated behavior have not been identified so far but recent data suggest that they should primarily concern a sub-population of aromatase-immunoreactive neurons located in the lateral parts of the sexually dimorphic preoptic nucleus. The zebra finch (Taeniopygia guttata) exhibits a more complex, still partly unexplained, differentiation pattern. In this species, early treatment with exogenous estrogens produces a masculinization of singing behavior in females and a demasculinization of copulatory behavior in males. Since normal untreated males sing and copulate, while females never show these behaviors even when treated with testosterone, it is difficult to understand under which endocrine conditions these behaviors differentiate. In an attempt to resolve this paradox, we recently treated young zebra finches with R76713 in order to inhibit their endogenous estrogens secretion during ontogeny and we subsequently tested their behavior in adulthood. As expected, the aromatase inhibitor decreased the singing frequency in treated males but it did not affect the male-type copulatory behavior in females nor in males. In addition, the sexuality differentiated brain song control nuclei which are also masculinized in females by early treatment with estrogens, were not affected in either sex by the aromatase inhibitor. In conclusion, available data clearly show that sexual differentiation of reproductive behaviors in birds follows a pattern that is almost opposite to that of mammals. This difference may be related to the different mechanisms of sex determination in the two taxa. In quail, the ontogeny of behavioral differentiation is now well understood but we only have a very crude notion of the brain structures that are concerned. By contrast, in zebra finches, the brain mechanisms controlling the sexually differentiated singing behavior in adulthood have been well identified but we do not understand how these structures become sexually dimorphic during ontogeny.     

Fadrozole hydrochloride, a new nontoxic aromatase inhibitor for the treatment of patients with metastatic breast cancer.

Eighty previously treated postmenopausal women with metastatic breast cancer were randomized to receive fadrozole (CGS 16 949A), a new aromatase inhibitor, 1 or 4 mg orally per day. Seventy eight patients were evaluable for toxicity and response. Only mild to moderate toxicity, namely hot flushes (28%), nausea and vomiting (13%), fatigue (8%) and loss of appetite (5%) occurred. Complete response was documented in 10% and partial response in 13% of patients with 45% having a no change status for at least 2 months. The median time to treatment failure is 4.1 months. The median survival is 23.7 months. The median survival is 23.7 months. The response and survival in patients with estrogen receptor positive and estrogen receptor unknown disease were not significantly different. Neither response nor survival was significantly different between the patients receiving 1 or 4 mg of fadrozole per day. Fadrozole is a well tolerated, effective second line treatment for women with metastatic breast cancer.     

Summary of aromatase inhibitor trials: the past and future

Antagonizing estrogen by inhibition of aromatase has become a mainstay of adjuvant endocrine therapy in women with hormone receptor positive (ER+) breast cancer. Recent trials have shown an incremental gain for the AIs over tamoxifen when given as an up-front alternative to tamoxifen, but additionally added benefit is achieved by giving them in sequence with tamoxifen after either an early switch (2–3 years) or as a late switch (5 years). The true clinical implications of accelerated bone resorption from AIs is becoming better understood and its management defined. AI minimally effect quality of life. The chronic relapsing nature of ER+ breast cancer implies long term therapy will be of benefit in selected patients. Outstanding issues under investigation include optimal duration of endocrine therapy, optimal sequence, optimal agents and whether combining anti-estrogens will yield advantage. The role of AIs is also under investigation in premenopausal women in combination with ovarian function suppression. Identifying prognostic and predictive factors of endocrine therapy is important as is the identification and overcoming of resistance mechanisms. Both tumor and host signatures are being pursued to this end. Optimizing, expanding and extending endocrine therapy is likely to add further to patient outcome.