The AusD Study: a population-based study of the determinants of serum 25-hydroxyvitamin D concentration across a broad latitude range.

Observational studies suggest that people with a high serum 25-hydroxyvitamin D (25(OH)D) concentration may have reduced risk of chronic diseases such as osteoporosis, multiple sclerosis, type 1 diabetes, cardiovascular disease, and some cancers. The AusD Study (A Quantitative Assessment of Solar UV Exposure for Vitamin D Synthesis in Australian Adults) was conducted to clarify the relationships between ultraviolet (UV) radiation exposure, dietary intake of vitamin D, and serum 25(OH)D concentration among Australian adults residing in Townsville (19.3°S), Brisbane (27.5°S), Canberra (35.3°S), and Hobart (42.8°S). Participants aged 18-75 years were recruited from the Australian Electoral Roll between 2009 and 2010. Measurements were made of height, weight, waist:hip ratio, skin, hair, and eye color, blood pressure, and grip strength. Participants completed a questionnaire on sun exposure and vitamin D intake, together with 10 days of personal UV dosimetry and an associated sun-exposure and physical-activity diary that was temporally linked to a blood test for measurement of 25(OH)D concentration. Ambient solar UV radiation was also monitored at all study sites. We collected comprehensive, high-quality data from 1,002 participants (459 males, 543 females) assessed simultaneously across a range of latitudes and through all seasons. Here we describe the scientific and methodological issues considered in designing the AusD Study.

Prolonging hormone sensitivity in prostate cancer xenografts through dual inhibition of AR and mTOR.

BACKGROUND: To determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer (PCa), we investigated the relationship between the androgen receptor (AR) and mTOR pathways and the impact of inhibiting both pathways in androgen-dependent and castration-resistant PCa models. EXPERIMENTAL DESIGN: Androgen-dependent (LNCaP) and castration-resistant PCa (HP-LNCaP) cells were grown as tumours in SCID mice. Once tumours reached 500 mm(3), animals were grouped and injected subcutaneous with vehicle, our novel anti-androgen/androgen synthesis inhibitor, VN/124-1, bicalutamide, and everolimus. Tumour volumes were measured biweekly. The PSA and protein analyses were performed after completion of the treatment. RESULTS: The addition of everolimus to bicalutamide treatment of resistant tumours significantly reduced tumour growth rates and tumour volumes. Anti-androgen treatment also increased protein expression of multiple signal transduction pathways earlier than vehicle-treated control xenografts. VN/124-1 plus everolimus acted in concert to reduce tumour growth rates in our castration-resistant xenograft model. CONCLUSIONS: This study suggests that dual inhibition of AR and mTOR in castration-resistant xenograft models can restore sensitivity of tumours to anti-androgen therapy. Furthermore, after bicalutamide failure, dual inhibition with VN/124-1 and everolimus was the most effective treatment.

Novel retinoic acid metabolism blocking agents have potent inhibitory activities on human breast cancer cells and tumour growth.

Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER -ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumour xenografts. The ER +ve cell lines were more sensitive (IC(50) values between 3.0 and 609 nM) to the RAMBAs than the ER -ve MDA-MB-231 cell line (IC(50)=5.6-24.0 microM). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-alpha (ER-alpha). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer.

The effect of tamoxifen on the pharmacokinetics of letrozole in female rats.

The effects of single doses of tamoxifen (TAM; 0.5-5 mg/kg, i.v.) and chronic pretreatment with TAM (0.1-5.0 mg/kg/day, i.p. for 7 consecutive days) on letrozole (0.5 mg/kg, i.v.) pharmacokinetics were evaluated in female Sprague-Dawley rats. The plasma concentration-time profiles of letrozole (0.1-2.0 mg/kg) after single i.v. doses were analysed by the non-compartment model with terminal half-lives (t(1/2,lambdaz)) ranging from 34.3 to 37.5 h. The volume of distribution at the terminal phases (Vd(lambdaz)) ranged from 1.9 to 2.1 l/kg and clearance (CL) varied from 0.036 to 0.042 l/(h.kg). After co-administration of TAM and letrozole intravenously, the t1/2, Vd(lambdaz) and CL of letrozole were not significantly altered. Chronic pretreatment with TAM significantly decreased the t1/2 of letrozole by about 33%, and increased its clearance by an average of 40%. However, TAM pretreatment did not significantly affect the Vd(lambdaz) of letrozole in female rats. Co-administration of letrozole and TAM orally increased the absorption half-life of letrozole threefold although the absolute bioavailability remained unchanged. These observations suggest that single oral doses of TAM delay the absorption of letrozole while chronic pretreatment with TAM accelerates the elimination of letrozole, probably due to induction of cytochrome P450 enzymes in rats.

A versatile synthesis of 17-heteroaryl androstenes via palladium-mediated Suzuki cross-coupling with heteroaryl boronic acids.

Suzuki coupling of 17-iodoandrosta-5,16-dien-3beta-ol (1) and 17-iodoandrosta-4,16-dien-3-one (2) with nine heteroaryl boronic acids (mainly 2- or 3-furanyl, thienyl, benzofuranyl and benzothienyl boronic acid derivatives) were carried out under normal Suzuki condition (Pd(PPh(3))(4), 2M Na(2)CO(3) and MeOH), generally yielded C(17)-heteroaryl steroids in moderate (10-60%) yields, but furanyl-2- and 5-chlorothienyl-2-boronic acid did not give any coupling product.

Inhibitory effects of retinoic acid metabolism blocking agents (RAMBAs) on the growth of human prostate cancer cells and LNCaP prostate tumour xenografts in SCID mice.

In recent studies, we have identified several highly potent all-trans-retinoic acid (ATRA) metabolism blocking agents (RAMBAs). On the basis of previous effects of liarozole (a first-generation RAMBA) on the catabolism of ATRA and on growth of rat Dunning R3227G prostate tumours, we assessed the effects of our novel RAMBAs on human prostate tumour (PCA) cell lines. We examined three different PCA cell lines to determine their capacity to induce P450-mediated oxidation of ATRA. Among the three different cell lines, enhanced catabolism was detected in LNCaP, whereas it was not found in PC-3 and DU-145. This catabolism was strongly inhibited by our RAMBAs, the most potent being VN/14-1, VN/50-1, VN/66-1, and VN/69-1 with IC50 values of 6.5, 90.0, 62.5, and 90.0 nM, respectively. The RAMBAs inhibited the growth of LNCaP cells with IC50 values in the microM-range. In LNCaP cell proliferation assays, VN/14-1, VN/50-1, VN/66-1, and VN/69-1 also enhanced by 47-, 60-, 70-, and 65-fold, respectively, the ATRA-mediated antiproliferative activity. We then examined the molecular mechanism underlying the growth inhibitory properties of ATRA alone and in combination with RAMBAs. The mechanism appeared to involve the induction of differentiation, cell-cycle arrest, and induction of apoptosis (TUNEL), involving increase in Bad expression and decrease in Bcl-2 expression. Treatment of LNCaP tumours growing in SCID mice with VN/66-1 and VN/69-1 resulted in modest but statistically significant tumour growth inhibition of 44 and 47%, respectively, while treatment with VN/14-1 was unexpectedly ineffective. These results suggest that some of our novel RAMBAs may be useful agents for the treatment of prostate cancer.

Biochemical assessment of limits to estrogen synthesis in porcine follicles.

Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17 alpha-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

Regulation of microsomal P450, redox partner proteins, and steroidogenesis in the developing testes of the neonatal pig.

Testicular growth and plasma androgen concentrations increase markedly in the first weeks of neonatal life of pigs. The regulation of steroidogenesis through this period was examined by measuring total microsomal cytochromes P450 (P450), 17alpha-hydroxylase/17,20-lyase P450 (P450c17) and aromatase P450 (P450arom) enzyme activities, and the redox partner proteins nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome P450 reductase (reductase) and cytochrome b(5) in testicular microsomes. Testes were collected from 1-24 d of age, and testicular development was suppressed by a GnRH antagonist in some animals from d 1-14. Both 17/20-lyase and aromatase activities increased from d 1-7 but not thereafter, and 17-20-lyase activity was always at least 200-fold higher than aromatase activity. Reductase decreased in wk 1, then increased to d 24. No changes were seen in cytochrome b(5) expression. GnRH antagonist treatment suppressed plasma LH, testosterone and testes growth to d 14. 17,20-Lyase and aromatase activities in testicular microsomes were reduced by 20% and 50%, respectively. Total microsomal P450 concentration was reduced by 50% on d 7, but there was no effect of treatment on reductase or cytochrome b(5) expression. These data support the hypothesis that the rise in neonatal testicular androgen secretion is more likely due to gonadotropin-stimulated gonadal growth, rather than specific P450c17 expression. Neither P450c17 nor P450arom can account for the decline in total microsomal P450. Reductase and cytochrome b(5) expression appears to be constitutive, but reductase levels saturate both P450c17 and P450arom.

Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors.

An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10-30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress.

Aromatase and COX-2 expression in human breast cancers.

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E2 is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE2 produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.

Antiandrogenic effects of novel androgen synthesis inhibitors on hormone-dependent prostate cancer.

We have found that in addition to being potent inhibitors of 17alpha-hydroxylase/C17,20-lyase and/or 5alpha-reductase, some of our novel androgen synthesis inhibitors also interact with the mutated androgen receptor (AR) expressed in LNCaP prostate cancer cells and the wild-type AR expressed in hormone-dependent prostatic carcinomas. The effects of these compounds on the proliferation of hormone-dependent human prostatic cancer cells were determined in vitro and in vivo. L-2 and L-10 are delta4-3-one-pregnane derivatives. L-35 and L-37 are delta5-3beta-ol-androstane derivatives, and L-36 and L-39 are delta4-3-one-androstane-derived compounds. L-2, L-10, and L-36 (L-36 at low concentrations) stimulated the growth of LNCaP cells, indicating that they were interacting agonistically with the mutated AR expressed in LNCaP cells. L-35, L-37, and L-39 acted as LNCaP AR antagonists. To determine whether the growth modulatory effects of our novel compounds were specific for the mutated LNCaP AR, competitive binding studies were performed with LNCaP cells and PC-3 cells stably transfected with the wild-type AR (designated PC-3AR). Regardless of AR receptor type, all of our novel compounds were effective at preventing binding of the synthetic androgen methyl-trienolone[17alpha-methyl-(3H)-R1881 to both the LNCaP AR and the wildtype AR. L-36, L-37, and L-39 (5.0 microM) prevented binding by >90%, whereas L-35 inhibited binding by 30%. To determine whether the compounds were acting as agonists or antagonists, LNCaP cells and PC-3AR cells were transfected with the pMAMneoLUC reporter gene. When luciferase activity was induced by dihydrotestosterone, all of the compounds were found to be potent inhibitors of transcriptional activity, and the pattern of inhibition was similar for both receptor types. However, L-2, L-10, and L-36 were determined to be AR agonists, and L-35, L-37, and L-39 were wild-type AR antagonists. When tested in vivo, L-39 was the only AR antagonist that proved to be effective at inhibiting the growth of LNCaP prostate tumor growth. L-39 slowed tumor growth rate in LNCaP tumors grown in male SCID mice to the same level as orchidectomy, significantly reduced tumor weights (P < 0.05), significantly lowered serum levels of prostate-specific antigen (P < 0.02), and significanty lowered serum levels of testosterone (P < 0.05). L-39 also proved to be effective when tested against the PC-82 prostate cancer xenograft that expresses wild-type AR. These results show that some of our compounds initially developed to be inhibitors of androgen synthesis also interact with the human AR and modulate the proliferation of hormone-dependent prostatic cancer cells. Therefore, compounds such as L-39, which have multifunctional activities, hold promise for the treatment of androgen-dependent prostate tumors.

Potent inhibition of retinoic acid metabolism enzyme(s) by novel azolyl retinoids.

Novel (+/-)-4-azolyl retinoic acid analogues 4, 5, 7 and 8 have been designed and synthesized and have been shown to be powerful inhibitors of hamster microsomal all-trans-retinoic acid 4-hydroxylase enzyme(s). (+/-)-4-(1H-Imidazol-1-yl)retinoic acid (4) is the most potent inhibitor of this enzyme reported to date.

Anti-tumour effects and pharmacokinetic profile of 17-(5'-isoxazolyl)androsta-4,16-dien-3-one (L-39) in mice: an inhibitor of androgen synthesis.

17-(5'-Isoxazolyl)androsta-4,16-dien-3-one (L-39), a novel androstene derivative, was synthesized and evaluated in vitro and in vivo. L-39 showed potent and non-competitive inhibition of human testicular microsomal 17alpha-hydroxylase/C(17,20)-lyase with an IC50 value of 59 nM and Ki of 22 nM. L-39 also showed potent and competitive inhibition of 5alpha-reductase in human prostatic microsomes with IC50 and Ki values of 33 and 28 nM respectively. L-39 (5 microM) has also been shown to manifest anti-androgenic activity in cultures of human prostate cancer cell lines (LNCaP) by preventing the labelled synthetic androgen R1881 (5 nM) from binding to the androgen receptors. Androgen-dependent human prostate cancer xenografts (PC-82) were grown in nude mice and the effects of L-39 (50 mg kg(-1) day(-1)) on tumour growth and prostate-specific antigen (PSA) levels were determined after 28 days. L-39 significantly (P < 0.01) diminished tumour growth and wet weights to a similar extent as castration or flutamide treatment. L-39 also significantly (P < 0.01) reduced serum PSA levels by more than 80% in the mice bearing human prostate cancer xenografts. Pharmacokinetic studies were also conducted in male Balb/c mice. After subcutaneous administration of a single bolus dose, L-39 was rapidly absorbed into the systemic circulation. Peak plasma levels occurred at 0.75 h and then declined with a t(1/2) of 1.51 h. The bioavailability of L-39 after subcutaneous administration was 28.5%. These results demonstrate that L-39 is a potent inhibitor of androgen synthesis and is effective in reducing the growth of human prostate cancer xenografts in nude mice. Although improvements in the bioavailability are necessary, L-39 is a potential lead compound with this profile as an inhibitor of prostate cancer growth.

Aromatase inhibitors and their application in breast cancer treatment*.

Estrogens are known to be important in the growth of breast cancers in both pre- and postmenopausal women. The number of breast cancer patients with hormone-dependent disease increases with age, as does the incidence of breast cancer. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. Because aromatase catalyzes the rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme have been developed in the last few years as a logical treatment strategy. Two classes of aromatase inhibitors, steroidal and nonsteroidal compounds, are now in use. Among the steroid substrate analogs, formestane and examestane have been shown to be effective in breast cancer patients with advanced disease. Highly potent and selective nonsteroidal inhibitors have recently been found to suppress plasma and urinary estrogens by more than 95% in breast cancer patients. Two of these compounds recently were approved in the United States and have been shown to be more effective than other second-line agents in terms of overall response rates and treatment failure, as well as better tolerated. Although studies of the efficacy of these agents in earlier stage disease are awaited, it is evident that aromatase inhibitors can extend the duration of treatment in breast cancer patients.

Effects of new 17alpha-hydroxylase/C(17,20)-lyase inhibitors on LNCaP prostate cancer cell growth in vitro and in vivo.

Our laboratory has been developing new inhibitors of a key regulatory enzyme of testicular and adrenal androgen synthesis 17alpha-hydroxylase/C(17,20)-lyase (P450c17), with the aim of improving prostate cancer treatment. We designed and evaluated two groups of azolyl steroids: delta5-non-competitive inhibitors (delta5NCIs), VN/63-1, VN/85-1, VN/87-1 and their corresponding delta4 derivatives (delta4NCIs), VN/107-1, VN/108-1 and VN/109-1. The human P450c17 gene was transfected into LNCaP human prostate cancer cells, and the resultant LNCaP-CYP17 cells were utilized to evaluate the inhibitory potency of the new azolyl steroids. VN/85-1 and VN/108-1 had the lowest IC50 values of 1.25 +/- 0.44 nM and 2.96 +/- 0.78 nM respectively, which are much lower than that of the known P450 inhibitor ketoconazole (80.7 +/- 1.8 nM). To determine whether the compounds had direct actions on proliferation of wild-type LNCaP cells, cell growth studies were performed. All of the delta5NCIs and VN/108-1 blocked the growth-stimulating effects of androgens. In steroid-free media, the delta5NCIs decreased the proliferation of LNCaP cells by 35-40%, while all of the delta4NCIs stimulated LNCaP cells growth 1.5- to 2-fold. In androgen receptor (AR) binding studies, carried out to determine the mechanism of this effect, all of the delta4NCIs (5 microM) displaced 77-82% of synthetic androgen R1881 (5 nM) from the LNCaP AR. The anti-androgen flutamide and the delta5NCIs displaced 53% and 32-51% of R1881 bound to AR respectively. These results suggested that the delta5NCIs may also be acting as anti-androgens. We further evaluated our inhibitors in male severe combined immunodeficient mice bearing LNCaP tumour xenografts. In this model VN/85-1 was as effective as finasteride at inhibiting tumor growth (26% and 28% inhibition, respectively) and the inhibitory effect of VN/87-1 was similar to that of castration (33% and 36% inhibition respectively). These results suggest that VN/85-1 and VN/87-1 may be potential candidates for treatment of prostate cancer.

Comprehensive pharmacology and clinical efficacy of aromatase inhibitors.

The goal of hormone therapy is to deprive breast tumours of estrogens, since estrogens have been implicated in the development or progression of tumours. This can be accomplished by the use of antiestrogens that block estrogen action or by inhibiting aromatase, the enzyme that catalyses the final and rate-limiting step in estrogen biosynthesis. A number of steroidal and nonsteroidal compounds have been developed as aromatase inhibitors. This review highlights the valuable role that a few of these aromatase inhibitors have played, and continue to play, in the treatment of breast cancer. Following background information regarding the biochemistry of aromatase, the rationale for its inhibition, and an outline of the test systems for evaluating and characterising aromatase inhibitors, the discussion focuses on the new generation of aromatase inhibitors that are in clinical trials or clinically available. Specifically, it discusses the pharmacology and clinical efficacy of formestane, exemestane, rogletimide, fadrozole, vorozole, anastrozole and letrozole. The role of these agents as the optimal second-line agents (after tamoxifen) for the treatment of advanced breast cancer has been established; their prospects in other clinical settings and as potential breast cancer chemopreventives are warranted but are yet to be fully determined.

The aromatase inactivator 4-hydroxyandrostenedione (4-OH-A) inhibits tamoxifen metabolism by rat hepatic cytochrome P-450 3A: potential for drug-drug interaction of tamoxifen and 4-OH-A in combined anti-breast cancer therapy.

Tamoxifen (tam), an anti-breast cancer agent, is metabolized into tam-N-oxide by the hepatic flavin-containing monooxygenase and into N-desmethyl- and 4-hydroxy-tam by cytochrome P-450s (CYPs). Additionally, tam is metabolically activated by hepatic CYP3A, forming a reactive intermediate that binds covalently to proteins. Tam and 4-hydroxyandrostenedione (4-OH-A) are currently used to treat breast cancer, and it has been contemplated that 4-OH-A be given concurrently with tam to contravene potential tumor resistance to tam. Because alterations in tam metabolism may influence its therapeutic efficacy, the effect of 4-OH-A on tam metabolism was examined. Incubation of tam with liver microsomes from phenobarbital-treated rats, in the presence of 4-OH-A (10-100 microM), resulted in marked inhibition of tam-N-demethylation and tam covalent binding and in decreased tam-N-oxide accumulation; however, there was no inhibition of the formation of 4-hydroxy-tam and of 3,4-dihydroxytamoxifen. These findings indicate that 4-OH-A inhibits CYP3A, but not P-450(s) that catalyze tam 4-hydroxylation. The diminished tam-N-oxide accumulation could be due to decreased N-oxide formation and/or due to increased N-oxide reduction. Incubation of tam-N-oxide with liver microsomes containing heat-inactivated flavin-containing monooxygenase demonstrated that 4-OH-A increases the accumulation of tam, possibly by diminishing its P-450-mediated metabolism. Kinetic studies indicate that 4-OH-A is a competitive inhibitor of CYP3A, but not a time-dependent inactivator. Consequently, the concurrent treatment of tam and 4-OH-A may result in increased tam half-life and thus could potentiate the therapeutic efficacy of tam and diminish the potential side effects of tam by inhibiting its covalent binding to proteins and possibly to DNA.

Inhibitors of 17alpha-hydroxylase/17,20-lyase (CYP17): potential agents for the treatment of prostate cancer.

Prostate cancer (PCa) is now the most prevalent cancer in men in the U.S.A. and Europe. At present the major treatment options include surgical or medical castration. These strategies depend on the abolition of the production of testosterone by the testes. However, as these procedures do not affect adrenal androgen production, they are frequently combined with androgen receptor antagonist to block their action. Inhibition of the key enzyme which catalyzes the biosynthesis of androgens from pregnane precursors, 17alpha-hydroxylase/17,20-lyase (hereafter referred to as CYP17 ) could prevent androgen biosynthesis from both sources. Thus total blockade of androgen production by CYP17 inhibitors may provide effective treatment of prostate cancer patients. Indeed, this strategy is now an area of intense interest within research institutions and the pharmaceutical industry. This review highlights development in the design and evaluation of both steroidal and non-steroidal CYP17 inhibitors since 1965. Major emphasis is given to the potent CYP17 inhibitors and those which may show clinical promise. The review could function as a comprehensive working reference of research accomplishment in the field and what problems remain to be tackled in the future.

Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17alpha-hydroxylase-C17,20-lyase inhibitors.

We have designed and synthesized a number of cytochrome P450 17alpha-hydroxylase-C17,20-lyase (P450c17) inhibitors with the aim of inhibiting androgen synthesis. To select the most potent inhibitors, we initially used human testicular microsomes, which have a high level of expression of this enzyme. However, due to lack of availability of human tissue and variability among the samples, we utilized recombinant human enzyme expressed in Escherichia coli. We designed a simple and economical protocol based on the report that recombinant bovine P450c17 can be functionally active in live bacteria. In the assay we report here, we substituted high-performance liquid chromatography product isolation with a rapid biochemical acetic acid releasing assay and utilized intact P450c17-expressing E. coli for the source of the enzyme. Enzymatic parameters of the bacterial system (Km = 5.1 x 10(-7) M, Vmax = 15.0 pmol/min/mg) were similar to those of human testicular microsomes (Km = 4.8 x 10(-7) M, Vmax = 40.0 pmol/min/mg), and our compounds displayed a similar pattern of inhibition in both systems. This new system is a fast, reliable, and reproducible method for screening P450c17 inhibitors. Furthermore, it eliminates our dependence on human tissue and potential data fluctuations caused by variations in enzymatic activity between donors.