4-pregnene-3-one-20 beta-carboxaldehyde: a potent inhibitor of 17 alpha-hydroxylase/C17,20-lyase and of 5 alpha-reductase.

The pregnene derivative, 4-pregnene-3-one-20 beta-carboxaldehyde (22-A) was evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and of 5 alpha-reductase in human prostatic homogenates. The effect of the compound in vivo was studied in adult male rats. The 22-A demonstrated potent and competitive inhibition of 17 alpha-hydroxylase and C17,20-lyase with Ki values 8.48 and 0.41 microM, respectively, significantly below the Km values for these two enzymes (33.75 and 4.55 microM). This compound also showed potent inhibition of 5 alpha-reductase with a Ki value of 15.6 nM (Km for this enzyme is 50 nM). By comparison, ketoconazole, a currently studied 17 alpha-hydroxylase/C17,20-lyase inhibitor for the treatment of prostatic cancer, showed less potent inhibition of 17 alpha-hydroxylase (Ki 39.5 microM) and C17,20-lyase (Ki 3.6 microM) and did not inhibit 5 alpha-reductase. Progesterone which has been reported to inhibit the 17 alpha-hydroxylase/C17,20-lyase, did not significantly reduce the production of testosterone by rat testes in vitro in comparison to controls, while the same concentration of 22-A demonstrated a 42% reduction of testosterone biosynthesis. When the adult male rats were injected s.c. with 22-A at 50 mg/day/kg for a 2 week period, the testosterone concentrations in the rat sera were significantly lower than control values (P less than 0.05), whereas serum corticosterone levels did not change. These results suggest that 22-A is a selective potent inhibitor for 17 alpha-hydroxylase and C17,20-lyase, but is more potent for the C17,20-lyase. The compound also inhibits 5 alpha-reductase, and therefore may reduce biosynthesis of testosterone and dihydrotestosterone effectively. Thus, 22-A may be useful in the treatment of problems associated with the androgen excess and prostatic cancer.     

C(17,20)-lyase inhibitors. Part 2: design, synthesis and structure-activity relationships of (2-naphthylmethyl)-1H-imidazoles as novel C(17,20)-lyase inhibitors

A series of 1- and 4-(2-naphthylmethyl)-1H-imidazoles (3 and 4) has been synthesized and evaluated as C(17,20)-lyase inhibitors. Several 6-methoxynaphthyl derivatives showed potent C(17,20)-lyase inhibition, suppression of testosterone biosynthesis in rats and reduction in the weight of prostate and seminal vesicles in rats, whereas most of these compounds increased the liver weight after consecutive administrations. The effect on the liver weight was removed by incorporation of a hydroxy group and an isopropyl group at the methylene bridge, as seen in (S)-28d and (S)-42. Selectivity for C(17,20)-lyase over 11beta-hydroxylase is also discussed, and (S)-42 was found to be a more than 260-fold selective inhibitor. Furthermore, (S)-42 showed a potent suppression of testosterone biosynthesis after a single oral administration in monkeys. These data suggest that (S)-42 may be a promising agent for the treatment of androgen-dependent prostate cancer.     

Orteronel (TAK-700), a novel non-steroidal 17,20-lyase inhibitor: effects on steroid synthesis in human and monkey adrenal cells and serum steroid levels in cynomolgus monkeys

Surgical or pharmacologic methods to control gonadal androgen biosynthesis are effective approaches in the treatment of a variety of non-neoplastic and neoplastic diseases. For example, androgen ablation and its consequent reduction in circulating levels of testosterone is an effective therapy for advanced prostate cancers. Unfortunately, the therapeutic effectiveness of this approach is often temporary because of disease progression to the 'castration resistant' (CRPC) state, a situation for which there are limited treatment options. One mechanism thought to be responsible for the development of CRPC is extra-gonadal androgen synthesis and the resulting impact of these residual extra-gonadal androgens on prostate tumor cell proliferation. An important enzyme responsible for the synthesis of extra-gonadal androgens is CYP17A1 which possesses both 17,20-lyase and 17-hydroxylase catalytic activities with the 17,20-lyase activity being key in the androgen biosynthetic process. Orteronel (TAK-700), a novel, selective, and potent inhibitor of 17,20-lyase is under development as a drug to inhibit androgen synthesis. In this study, we quantified the inhibitory activity and specificity of orteronel for testicular and adrenal androgen production by evaluating its effects on CYP17A1 enzymatic activity, steroid production in monkey adrenal cells and human adrenal tumor cells, and serum levels of dehydroepiandrosterone (DHEA), cortisol, and testosterone after oral dosing in castrated and intact male cynomolgus monkeys. We report that orteronel potently suppresses androgen production in monkey adrenal cells but only weakly suppresses corticosterone and aldosterone production; the IC(50) value of orteronel for cortisol was ~3-fold higher than that for DHEA. After single oral dosing, serum levels of DHEA, cortisol, and testosterone were rapidly suppressed in intact cynomolgus monkeys. In castrated monkeys treated twice daily with orteronel, suppression of DHEA and testosterone persisted throughout the treatment period. In both in vivo models and in agreement with our in vitro data, suppression of serum cortisol levels following oral dosing was less than that seen for DHEA. In terms of human CYP17A1 and human adrenal tumor cells, orteronel inhibited 17,20-lyase activity 5.4 times more potently than 17-hydroxylase activity in cell-free enzyme assays and DHEA production 27 times more potently than cortisol production in human adrenal tumor cells, suggesting greater specificity of inhibition between 17,20-lyase and 17-hydroxylase activities in humans vs monkeys. In summary, orteronel potently inhibited the 17,20-lyase activity of monkey and human CYP17A1 and reduced serum androgen levels in vivo in monkeys. These findings suggest that orteronel may be an effective therapeutic option for diseases where androgen suppression is critical, such as androgen sensitive and CRPC.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

CYP17 inhibitors for prostate cancer therapy

Prostate cancer (PC) is now the second most prevalent cause of death in men in the USA and Europe. At present, the major treatment options include surgical or medical castration. These strategies cause ablation of the production of testosterone (T), dihydrotestosterone (DHT) and related androgens by the testes. However, because these procedures do not affect adrenal, prostate and other tissues' androgen production, they are often combined with androgen receptor antagonists to block their action. Indeed, recent studies have unequivocally established that in castration-resistant prostate cancer (CRPC) many androgen-regulated genes become re-expressed and tissue androgen levels increase despite low serum levels. Clearly, inhibition of the key enzyme which catalyzes the biosynthesis of androgens from pregnane precursors, 17α-hydroxy/17,20-lyase (hereafter referred to as CYP17) could prevent androgen production from all sources. Thus, total ablation of androgen production by potent CYP17 inhibitors may provide effective treatment of prostate cancer patients. This review highlights the role of androgen biosynthesis in the progression of prostate cancer and the impact of CYP17 inhibitors, such as ketoconazole, abiraterone acetate, VN/124-1 (TOK-001) and TAK-700 in the clinic and in clinical development. Article from the special issue on Targeted Inhibitors.     

A novel molecular modelling study of inhibitors of the 17alpha-hydroxylase component of the enzyme system 17alpha-hydroxylase/17,20-lyase (P-450(17alpha)).

The enzyme 17alpha-hydroxylase/17,20-lyase (P-450(17alpha) has recently become the focus of research into the fight against hormone dependent prostate cancer. However, the specific nature of this enzyme, in particular, the dual role of its active site, remains unknown. In our drive to elucidate further information regarding P-450(17alpha), and in light of our experience of other cytochrome P-450 enzymes, we chose to consider each part of this complex enzyme separately (i.e. the 17alpha-hydroxylase (17alpha-OHase) and the 17,20-lyase components). We therefore initiated a series of molecular modelling studies involving the construction of a 'substrate heme complex' for each of the two components. Here, we consider the construction and use of the complex for the 17alpha-OHase component of this enzyme. Using this approach, we have successfully considered: the binding of steroidal and non-steroidal reversible inhibitors: the structural features necessary for potent inhibition: and, rationalised the mode of action of a number of compounds whose inhibitory activity has not been previously explained, for example aminoglutethimide (an inhibitor of another related cytochrome P-450 enzyme, aromatase AR). The study concludes that the ability of the inhibitors of 17alpha-OHase to undergo polar polar interaction with the active site and for the compounds to closely mimic the substrate plane is a major factor in determining potency. Factors such as log P (log of the partition coefficient value for the distribution of a compound between octanol and water) would then appear to determine the extent of overall inhibitory activity. Overall, the study suggests that the novel substrate-heme complex approach has provided a good approximation of the 17alpha-OHase active site and has proved to be a useful tool in drug design and discovery.     

17,20-lyase inhibitors. Part 4: design, synthesis and structure-activity relationships of naphthylmethylimidazole derivatives as novel 17,20-lyase inhibitors

A novel series of naphthylmethylimidazole derivatives and related compounds have been investigated as selective 17,20-lyase inhibitors. Optimization of the substituent at the 6-position on the naphthalene ring was performed to yield a methylcarbamoyl derivative, which exhibited potent inhibitory activity against human 17,20-lyase and promising selectivity (>200-fold) for 17,20-lyase over CYP3A4. Further modifications of the methylcarbamoyl derivative led to the discovery of the corresponding tricyclic compound, which showed highly potent activity against human 17,20-lyase (IC(50) 19 nM) and good selectivity (>1000-fold) for inhibition of 17,20-lyase over CYP3A4. Additional biological evaluation revealed that the tricyclic compound had potent in vivo efficacy in monkeys and favorable pharmacokinetic profiles when administered in rats. Asymmetric synthesis of the selective tricyclic inhibitor was also achieved using a chiral α-hydroxy ketone.     

Imidazole substituted biphenyls: a new class of highly potent and in vivo active inhibitors of P450 17 as potential therapeutics for treatment of prostate cancer.

3- And 4-imidazol-1-yl-methyl substituted biphenyl compounds (named as meta- and para-substituted compounds) were synthesized bearing additional substituents in 3'-/4'-position as inhibitors of P450 17 (17alpha-hydroxylase-C17,20-lyase). P450 17 is the key enzyme of androgen biosynthesis. Its inhibition is a novel therapeutic strategy for treatment of prostate cancer (PC). Twenty-nine compounds were synthesized by Ar-Mg-Br, Negishi or Suzuki aryl-aryl cross coupling and tested toward human and rat enzyme. Most of the compounds showed moderate to excellent activity against one of the enzymes (0.087 microM < or = IC50 < or = 7.7 microM (ketoconazole: 0.74 microM) for the human enzyme, 0.63 microM < or = IC50 < or = 32 microM (ketoconazole: 67 microM) for the rat enzyme). Interestingly, strong species differences were observed. In addition compounds were tested for inhibition toward P450 arom. The 3-imidazol-1-yl-methyl substituted compounds showed good inhibitory activity of P450 arom, while for the 4-substituted compounds negligible inhibition was found. For the most active group of P450 17 inhibitors, (i.e. the 4-imidazol-1-yl-methyl substituted compounds) a QSAR study was performed for inhibition of the human enzyme leading to the result that a hydrophilic substituent in 3'-/4'-position is very important. The most promising compounds (with respect to activity toward both enzymes) were tested in vivo using SD-rats for reduction of plasma testosterone concentrations 2 and 6 h after single i.p. application. The fluorine substituted compound 8c decreased the testosterone plasma concentration to castration level (after 2 h; 5 mg/kg) showing a biological half live of about 6 h.     

17,20-Lyase inhibitors. Part 3: Design, synthesis, and structure-activity relationships of biphenylylmethylimidazole derivatives as novel 17,20-lyase inhibitors

A novel series of biphenylylmethylimidazole derivatives and related compounds were synthesized as inhibitors of 17,20-lyase, a key enzyme in the production of steroid hormones, and their biological activities were evaluated. In an attempt to identify potent and selective inhibitors of 17,20-lyase over the related CYP3A4 enzyme, a homology model for human 17,20-lyase was developed using the X-ray crystallographic structure of the mammalian CYP2C5 enzyme. With the aid of molecular modeling, optimization of the biphenyl moiety was performed to give an acetamide derivative, which was resolved by HPLC to give the active (-)-enantiomer. The obtained active enantiomer showed not only potent inhibition of both rat and human 17,20-lyase,with IC(50) values of 14 and 26 nM, respectively, but also excellent selectivity (>300-fold) for inhibition of 17,20-lyase over CYP3A4. Moreover, the active enantiomer significantly reduced both serum testosterone and DHEA concentrations in a monkey model after single oral administration. Asymmetric synthesis of the active enantiomer was also developed via a chiral intermediate using a diastereoselective Grignard reaction.     

Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo

Aiming at the development of new drugs for the treatment of prostate cancer, the effects of steroidal compounds and one non-steroidal substance on androgen biosynthesis were evaluated in vitro and in vivo. Sa 40 [17-(5-pyrimidyl)androsta-5,16-diene-3beta-ol], its 3-acetyl derivate Sa 41 and BW 19 [3,4-dihydro-2-(4-imidazolylmethyl)-6-methoxy-1-methyl-naphthalene] are compounds from our group, which have been developed as inhibitors of CYP 17 (17alpha-hydroxylase-C17, 20-lyase, the key enzyme in androgen biosynthesis). They have been compared with CB 7598 [abiraterone: 17-(3-pyridyl)androsta-5,16-diene-3beta-ol], its 3-acetyl compound CB 7630 and ketoconazole, compounds which already have been used clinically. The most potent compound toward human CYP 17 (testicular microsomes) was Sa 40 (IC(50) value of 24 nM), followed by Sa 41, CB 7598, BW 19, CB 7630 and ketoconazole. Sa 40 shows a type II difference spectrum and a non-competitive type of inhibition (K(i) value of 16 nM). No recovery of enzyme activity was observed after preincubation of CYP 17 with Sa 40 and subsequent charcoal treatment. In Escherichia coli cells coexpressing human CYP 17 and NADPH-P450 reductase, Sa 40 was more active than CB 7598 and BW 19, whereas the acetyl compounds were not active. The latter three compounds were equally active towards rat CYP 17. Male Sprague-Dawley (SD) rats were administered daily for 14 days BW 19 and the acetyl derivatives Sa 41 and CB 7630 as prodrugs (0.1 mmol/kg intraperitoneally). The test compounds strongly reduced plasma testosterone concentration, as well as prostate and seminal vesicles weights. They showed moderate inhibitory effects on the weights of levator ani, bulbocavernosus and testes, whereas they led to an increase in adrenal and pituitary weights. The only exception was BW 19 which did not change pituitary weights. Based on its superiority on the human enzyme, it was concluded that Sa 40 in its 3beta-acetate form (Sa 41) could be a promising candidate for clinical evaluation.     

Discovery of orteronel (TAK-700), a naphthylmethylimidazole derivative, as a highly selective 17,20-lyase inhibitor with potential utility in the treatment of prostate cancer

A novel naphthylmethylimidazole derivative 1 and its related compounds were identified as 17,20-lyase inhibitors. Based on the structure-activity relationship around the naphthalene scaffold and the results of a docking study of 1a in the homology model of 17,20-lyase, the 6,7-dihydro-5H-pyrrolo[1,2-c]imidazole derivative (+)-3c was synthesized and identified as a potent and highly selective 17,20-lyase inhibitor. Biological evaluation of (+)-3c at a dose of 1mg/kg in a male monkey model revealed marked reductions in both serum testosterone and dehydroepiandrosterone concentrations. Therefore, (+)-3c (termed orteronel [TAK-700]) was selected as a candidate for clinical evaluation and is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer.     

Cytochrome P450 17alpha hydroxylase/17,20 lyase (CYP17) function in cholesterol biosynthesis: identification of squalene monooxygenase (epoxidase) activity associated with CYP17 in Leydig cells

Cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17) is a microsomal enzyme catalyzing two distinct activities, 17alpha-hydroxylase and 17,20-lyase, essential for the biosynthesis of adrenal and gonadal steroids. CYP17 is a potent oxidant, it is present in liver and nonsteroidogenic tissues, and it has been suggested to have catalytic properties distinct to its function in steroid metabolism. To identify CYP17 functions distinct of its 17alpha-hydroxylase/17,20-lyase activity, we used MA-10 mouse tumor Leydig cells known to be defective in 17alpha-hydroxylase/17,20-lyase activity. A CYP17 knocked down MA-10 clone (MA-10(CYP17KD)) was generated by homologous recombination and its steroidogenic capacity was compared with wild-type cells (MA-10(wt)). Although no differences in cell morphology and proliferation rates were observed between these cells, the human chorionic gonadotropin-induced progesterone formation and de novo synthesis of steroids were dramatically reduced in MA-10(CYP17KD) cells; their steroidogenic ability could be rescued in part by transfecting CYP17 DNA into the cells. Knocking down CYP17 mRNA by RNA interference yielded similar results. However, no significant difference was observed in the steroidogenic ability of cells treated with 22R-hydroxycholesterol, which suggested a defect in cholesterol biosynthesis. Incubation of MA-10(CYP17KD) cells with (14)C-labeled squalene resulted in the formation of reduced amounts of radiolabeled cholesterol compared with MA-10(wt) cells. In addition, treatment of MA-10(CYP17KD) cells with various cholesterol substrates indicated that unlike squalene, addition of squalene epoxide, lanosterol, zymosterol, and desmosterol could rescue the hormone-induced progesterone formation. Further in vitro studies demonstrated that expression of mouse CYP17 in bacteria resulted in the expression of squalene monooxygenase activity. In conclusion, these studies suggest that CYP17, in addition to its 17alpha-hydroxylase/17,20-lyase activity, critical in androgen formation, also expresses a secondary activity, squalene monooxygenase (epoxidase), of a well-established enzyme involved in cholesterol biosynthesis, which may become critical under certain conditions.     

Pharmacology of novel steroidal inhibitors of cytochrome P45017α (17α-hydroxylase/C17–20 lyase)

Medical or surgical castration for the treatment of prostatic cancers prevents androgen production by the testes, but not by the adrenals. Inhibition of the key enzyme for androgen biosynthesis, cytochrome P450_17α, could prevent androgen production from both sources. The in vivo effects of 17-(3-pyridyl)androsta-5,16-dien-3β-ol (CB7598) and 17-(3-pyridyl)androsta-5,16-dien-3-one (CB7627), novel potent steroidal inhibitors of this enzyme, on WHT mice were compared with those of castration and two clinically active compounds, ketoconazole and flutamide. Flutamide and surgical castration caused significant reductions in the weights of the ventral prostate and seminal vesicles. CB7598, in its 3β-O-acetate form (CB7630), and CB7627 caused significant reductions in the weights of the ventral prostate, seminal vesicles, kidneys and testes when administered once daily for 2 weeks. Ketoconazole, given on the same schedule, caused no reductions. Plasma testosterone was reduced to 0.1 nM by CB7630, despite a 3- to 4-fold increase in the plasma level of luteinizing hormone. Adrenal weights were unchanged following treatment with CB7630 or CB7627 but were markedly increased following ketoconazole, indicating no inhibition of corticosterone production by these steroidal compounds. These results indicate that CB7598, CB7630 or CB7627 may be useful in the treatment of hormone-dependent prostatic cancers.     

Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17

Cytochrome P450c17 (CYP17) catalyzes both the 17alpha-hydroxylase and 17,20-lyase reactions in human steroid biosynthesis. Cytochrome b5 (b5) stimulates the rate of the 17,20-lyase reaction 10-fold with little influence on 17alpha-hydroxylase activity. Studies with apo-b5 suggest that stimulation of 17,20-lyase activity results from an allosteric action on the hCYP17 x POR complex, rather than electron transfer by b5. We hypothesized that specific residues on b5 interact with the hCYP17 x POR complex and that targeted mutation of surface-exposed residues might identify b5 residues critical for stimulating 17,20-lyase activity. We constructed, expressed, and purified 14 single plus 3 double b5 mutations and assayed their ability to stimulate 17,20-lyase activity. Most mutations did not alter the capacity of b5 to stimulate 17,20-lyase activity or appeared to modestly alter the affinity of b5 for the hCYP17 x POR complex. In contrast, mutation of E48, E49, or R52 reduced the maximal stimulation of 17,20-lyase activity. In particular, b5 mutation E48G + E49G lost over 95% of the capacity to stimulate 17,20-lyase activity, yet this mutation retained normal electron transfer properties. In addition, mutation E48G + E49G did not impair stimulation of 17,20-lyase activity by wild-type b5, suggesting that the mutation binds poorly to the site of the hCYP17 x POR complex occupied by b5. These data suggest that a specific allosteric binding site on b5, which includes residues E48, E49, and possibly R52, mediates the stimulation of 17,20-lyase activity.     

Effect of ketoconazole on human ovarian C17,20-desmolase and aromatase

Ketoconazole, an imidazole antimycotic drug, inhibits steroid biosynthesis in adrenal and testicular tissue by blocking cytochrome P-450 dependent enzymes. To study the effect of ketoconazole on steroid biosynthesis in the human ovary we incubated human ovarian tissue (mainly theca cells) or granulosa cells with radiolabeled precursors and increasing concentrations of ketoconazole. After incubation, steroids were extracted and separated by thin layer chromatography (TLC). Activity of C17,20-desmolase and aromatase was estimated by measuring the amount of their radioactive products with liquid scintillation counting. After incubation of ovarian tissue with [3H]17-hydroxyprogesterone the production of [3H]androstenedione was reduced by increasing concentrations of ketoconazole (0-200 microM) to a minimum of 31% of basal production. This indicates a strong inhibition of ovarian C17,20-desmolase by ketoconazole with a 50% inhibiting concentration (IC50) of 23 microM. After incubation of human granulosa cells with ketoconazole (0-2000 microM) and [3H]androstenedione the production of [3H]estrone and [3H]estradiol was suppressed to minimally 37 and 35% of basal values, indicating a significant inhibition of ovarian aromatase. IC50-values were 105 microM ketoconazole for estradiol and 130 microM for estrone. In conclusion, ketoconazole was shown to inhibit human ovarian C17,20-desmolase and aromatase in vitro. As in human adrenals and testes ovarian C17,20-desmolase seems to be most sensitive to the inhibitory effect of ketoconazole.     

Synthesis and biochemical evaluation of a range of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P-450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. Here, we report the synthesis and biochemical evaluation of a range of benzyl imidazole-based compounds which have been targeted against the two components of this enzyme, that is, 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are good inhibitors, with N-4-iodobenzyl imidazole (5) (IC50=10.06 microM against 17alpha-OHase and IC50=1.58 microM against lyase) showing equipotent activity against lyase compared to the standard compound, ketoconazole (KTZ) (IC50=3.76+/-0.01 microM against 17alpha-OHase and IC50=1.66+/-0.15 microM against lyase). Furthermore, the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha).     

In vitro and in vivo models for the evaluation of new inhibitors of human steroid sulfatase,devoid of residual estrogenic activity

The goal of our research project is to develop a new class of orally active drugs, estrone sulfatase inhibitors, for the treatment of estrogen-dependent (receptor positive) breast cancer. Several compounds were synthesized and their pharmacological potencies explored. Based on encouraging preliminary results, three of them, TX 1299, TX 1492 and TX 1506 were further studied in vitro as well as in vivo. They proved to be strong inhibitors of estrone sulfatase when measured on the whole human JEG-3 choriocarcinoma and MCF-7 breast cancer cells and their IC(50)s found to be in the range of known standard inhibitors. Their residual estrogenic activity was checked as negative in the test of induction of alkaline phosphatase (APase) activity in whole human endometrial adenocarcinoma Ishikawa cells. In addition, their effect on aromatase activity in JEG-3 cells was also examined, since the goal of inhibiting both sulfatase and aromatase activities appears very attractive. However, it has been unsuccessful so far. Then, in vivo potencies of TX 1299, the lead compound in our chemical series, were evaluated in comparison with 6,6,7-COUMATE, a non-steroidal standard, in two different rat models and by oral route. First, the absence of any residual estrogenic activity for these compounds was checked in the uterotrophic model in prepubescent female rats. Second, antiuterotrophic activity in adult ovariectomized rat supplemented with estrone sulfate (E(1)S), showed that both compounds were potent inhibitors, the power of TX 1299 relative to 6,6,7-COUMATE being around 80%. This assay was combined with uterine sulfatase level determination and confirmed the complete inhibition of this enzyme within the target organ.Preliminary studies indicated that other non-steroid compounds in the Théramex series were potent in vitro and in vivo inhibitors of estrone sulfatase in rats and further studies are in progress.     

Chimerogenesis in Estimation of Specificity and Pathway Directions for Cytochrome P45017α Catalyzed Reactions

Cytochrome P45017alpha is a key enzyme in steroid hormone biosynthesis. It catalyzes the reaction of 17alpha-hydroxylation of progesterone (P4) and pregnenolone (P5) and the 17,20-lyase reaction resulting in side chain cleavage of C21 steroids to form C19 steroids. Depending on the activity of cytochrome P45017alpha, steroid hormone biosynthesis pathways are directed either for biosynthesis of mineralocorticoids and glucocorticoids or sex hormones. The formation of sex hormones starts from biosynthesis of androstenedione. Androstenedione formation is a result of two reactions: 17,20-lyase reaction of 17alpha-hydroxyprogesterone (Delta4-pathway) and 3beta-hydroxysteroid dehydrogenase/Delta4,Delta5-isomerase reaction using dehydroepiandrosterone as substrate (Delta5-pathway). In case of exclusive direction of the 17,20-lyase reaction either through the Delta4- or the Delta5-pathway, the formation of sex hormones depends more on specificity and activity of 3beta-hydroxysteroid-dehydrogenase/Delta4,Delta5-isomerase. Depending on species, the cytochromes P45017alpha can utilize as a substrate for 17,20-lyase activity Delta4-steroids, Delta5-steroids, or both types of steroids. To identify the structural elements of cytochrome P45017alpha responsible for substrate recognition, in the present work we used exchange of homologous fragments of cytochrome P45017alpha having different types of activities. We engineered more than 10 different types of chimeric cytochrome P45017alpha. Chimeric cytochromes P45017alpha have been expressed in E. coli and purified. The expression of chimeric cytochrome P45017alpha with the point of exchange between exons III and IV results in inability of the recombinant hemeprotein to properly bind heme. The determination of activity of chimeric cytochromes P45017alpha shows that the structural element responsible for switching activity between Delta4- or Delta5-pathway is located in the region of polypeptide chain coded by exons II-V of CYP17 gene.     

Synthesis and evaluation of novel 17-indazole androstene derivatives designed as CYP17 inhibitors

A series of novel 1H- and 2H-indazole derivatives of the commercially available dehydroepiandrosterone acetate have been synthesized and tested for inhibition of human cytochrome 17alpha-hydroxylase-C(17,20)-lyase (CYP17), androgen receptor (AR) binding affinity, and cytotoxic potential against three prostate cancer (PC) cell lines.     

P450-dependent enzymes as targets for prostate cancer therapy

Metastatic prostate adenocarcinoma is a leading cause of cancer-related deaths among men. First line treatment is primarily aimed at blocking the synthesis and action of androgens. As primary endocrine treatment, androgen deprivation is usually achieved by orchidectomy or LHRH analogues, frequently combined with androgen receptor antagonists in order to block the residual adrenal androgens. However, nearly all the patients will eventually relapse. Available or potential second line therapies include, among others, alternative endocrine manipulations and chemotherapy.

Cytochrome P450-dependent enzymes are involved in the synthesis and/or degradation of many endogenous compounds, such as steroids and retinoic acid. Some of these enzymes represent suitable targets for the treatment of prostate cancer.

In first line therapy, inhibitors of the P450-dependent 17,20-lyase may achieve a maximal androgen ablation with a single drug treatment. Ketoconazole at high dose blocks both testicular and adrenal androgen biosynthesis but its side-effects, mainly gastric discomfort, limit its widespread use. A series of newly synthesized, more selective, steroidal 17,20-lyase inhibitors related to 17-(3-pyridyl)androsta-5,16-dien-3β-ol, may open new perspectives in this field.

In prostate cancer patients who relapse after surgical or medical castration, therapies aiming at suppressing the remaining adrenal androgen biosynthesis (ketoconazole) or producing a medical adrenalectomy (aminoglutethimide + hydrocortisone) have been used, but are becoming obsolete with the generalization of maximal androgen blockade in first line treatment. The role of inhibition of aromatase in prostate cancer therapy, which was postulated for aminoglutethimide, could not be confirmed by the use of more selective aromatase inhibitors, such as formestane.

An alternative approach is represented by liarozole fumarate (LIA), a compound that blocks the P450-dependent catabolism of retinoic acid (RA). In vitro, it enhances the antiproliferative and differentiation effects of RA in cell lines that express RA metabolism, such as F9 teratocarcinoma and MCF-7 breast carcinoma cells. In vivo, monotherapy with LIA increases RA plasma levels and, to a greater extent, endogenous tissue RA levels leading to retinoid-mimetic effects. In the rat Dunning prostate cancer models, it inhibits the growth of androgen-independent as well as androgen-dependent carcinomas relapsing after castration. Concurrently, changes in the pattern of cytokeratins characteristic of increased differentiation were observed. Early clinical trials show that LIA, in second or third line therapy in metastatic prostate cancer, induces PSA responses in about 30% of unselected patients. In some patients regression of soft tissue metastasis has been observed. In a subgroup of patients, an important relief of metastatic bone pain was also noted.