Ivo P. Nnane

Effects of new 17α-hydroxylase/C17,20-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 17α-hydroxylase/C17,20-lyase (P450c17), with the aim of improving prostate cancer treatment. We designed and evaluated two groups of azolyl steroids: Δ5-non-competitive inhibitors (Δ5NCIs), VN/63-1, VN/85-1, VN/87-1 and their corresponding Δ4 derivatives (Δ4NCIs), 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 Δ5NCIs and VN/108-1 blocked the growth-stimulating effects of androgens. In steroid-free media, the Δ5NCIs decreased the proliferation of LNCaP cells by 35–40%, while all of the Δ4NCIs 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 Δ4NCIs (5 μM) displaced 77–82% of synthetic androgen R1881 (5 nM) from the LNCaP AR. The anti-androgen flutamide and the Δ5NCIs displaced 53% and 32–51% of R1881 bound to AR respectively. These results suggested that the Δ5NCIs may also be acting as anti-androgens. We further evaluated our inhibitors in male severe combined immuno- deficient 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.

Effects of novel 17-azolyl compounds on androgen synthesis in vitro and in vivo.

17-Azolyl steroids were synthesized and evaluated as inhibitors of androgen synthesis in vitro and in vivo. Several of the novel compounds exhibit potent noncompetitive inhibition of human 17alpha-hydroxylase/C17,20-lyase with IC50 values ranging from 7 to 90 nM, and Ki values from 1.2 to 41 nM. VN/85-1 and VN/108-1 were the most potent inhibitors against this enzyme with IC50 value of 8 nM (Ki of 1.2 nM) and 7 nM (Ki of 1.9 nM), respectively. VN/107-1, VN/108-1 and VN/109-1 also showed moderate inhibition of 5alpha-reductase in human prostatic microsomes. Normal adult male rats were treated with these novel 17-azolyl steroidal compounds at a dose level of 50 mg/kg, s.c., for 14 consecutive days, sacrificed 1-2 h after the last administered dose and blood, prostate and other tissues were collected. The organs were weighed and tissue concentrations of testosterone (T) and dihydrotestosterone (DHT) were measured. Tissue T levels were significantly (p<0.05) lower in rats treated with the novel 17-azolyl steroids by more than 50% compared to the control group. Similarly, the concentration of DHT in the serum and prostates was significantly (p<0.05) diminished in rats treated with the 17-azolyl steroids by 39-80% compared to the control group. Furthermore, the wet weights of the prostates and seminal vesicles were significantly (p<0.05) reduced by several of the novel steroids. Although only one dose was evaluated in these studies, VN/85-1 was the most effective compound and reduced prostatic androgen levels by more than 80% and the wet weights of the prostate and seminal vesicles in rats by about 50%. These findings suggest that these novel compounds may provide useful leads for the research and development of suitable agents for the treatment of androgen dependent prostate cancer.

Potent CYP17 inhibitors: Improved syntheses, pharmacokinetics and anti-tumor activity in the LNCaP human prostate cancer model

A facile preparation of azolyl steroids, VN/85-1 and VN/87-1 (potent inhibitors of CYP17) has been developed. This process without tedious chromatographic separations improved the overall yields from 55 and 45% to 70 and 65% for VN/85-1 and VN/87-1, respectively. Pharmacokinetic studies of VN/85-1 were conducted in male SCID mice. Following subcutaneous (s.c.) administration of 100mg/kg of VN/85-1, peak plasma level of 16.73 microg/ml occurred after 45 min, and the compound was cleared rapidly with a t(1/2) of 52.34 min. The bioavailability of VN/85-1 after s.c. administration was 83.0%. VN/85-1 was also rapidly metabolized to the corresponding 3-oxo-4-ene analog, 17-(1H-imidazol-1-yl)androsta-4,16-diene-3-one (VN/108-1). In our attempt to optimize the anti-tumor efficacy of these two CYP17 inhibitors, we studied their anti-tumor efficacies in male SCID mice bearing LNCaP tumor xenografts, utilizing various drug doses and drug scheduling. Three times a day dose regimen (3 x dose regimen) of VN/85-1 was more effective than a once daily dose. In contrast, 3 x dose regimen doses of VN/87-1 were less effective than the once daily dose. However, at their effective dosage regimes, VN/85-1 and VN/87-1 were each as effective as castration and more effective than finasteride or casodex, an anti-androgen used for prostate cancer (PC) therapy. For all of the treatments, there was a strong correlation between the tumor volumes and other associated parameters, such as, tumor weights, and serum testosterone (T) and PSA levels. These results indicate that VN/85-1 or VN/87-1 may be useful in the treatment of hormone-dependent prostate cancer.

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 17α-hydroxylase/C17,20-lyase with an IC50 value of 59 nM and Ki of 22 nM. L-39 also showed potent and competitive inhibition of 5α-reductase in human prostatic microsomes with IC50and Ki values of 33 and 28 nM respectively. L-39 (5 μM) 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 próstate cancer xenografts (PC-82) were grown in nude mice and the effects of L-39 (50 mg kg–1day–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 t1/2 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.