Mohammad Alyamani

Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer

Prostate cancer resistance to castration occurs because tumours acquire the metabolic capability of converting precursor steroids to 5α-dihydrotestosterone (DHT), promoting signalling by the androgen receptor and the development of castration-resistant prostate cancer. Essential for resistance, DHT synthesis from adrenal precursor steroids or possibly from de novo synthesis from cholesterol commonly requires enzymatic reactions by 3β-hydroxysteroid dehydrogenase (3βHSD), steroid-5α-reductase (SRD5A) and 17β-hydroxysteroid dehydrogenase (17βHSD) isoenzymes. Abiraterone, a steroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitor, blocks this synthetic process and prolongs survival. We hypothesized that abiraterone is converted by an enzyme to the more active Δ4-abiraterone (D4A), which blocks multiple steroidogenic enzymes and antagonizes the androgen receptor, providing an additional explanation for abiraterone’s clinical activity. Here we show that abiraterone is converted to D4A in mice and patients with prostate cancer. D4A inhibits CYP17A1, 3βHSD and SRD5A, which are required for DHT synthesis. Furthermore, competitive androgen receptor antagonism by D4A is comparable to the potent antagonist enzalutamide. D4A also has more potent anti-tumour activity against xenograft tumours than abiraterone. Our findings suggest an additional explanation—conversion to a more active agent—for abiraterone’s survival extension. We propose that direct treatment with D4A would be more clinically effective than abiraterone treatment.

Redirecting abiraterone metabolism to fine-tune prostate cancer anti-androgen therapy

Abiraterone blocks androgen synthesis and prolongs survival in patients with castration-resistant prostate cancer, which is otherwise driven by intratumoral androgen synthesis. Abiraterone is metabolized in patients to Δ4-abiraterone (D4A), which has even greater anti-tumour activity and is structurally similar to endogenous steroidal 5α-reductase substrates, such as testosterone. Here, we show that D4A is converted to at least three 5α-reduced and three 5β-reduced metabolites in human serum. The initial 5α-reduced metabolite, 3-keto-5α-abiraterone, is present at higher concentrations than D4A in patients with prostate cancer taking abiraterone, and is an androgen receptor agonist, which promotes prostate cancer progression. In a clinical trial of abiraterone alone, followed by abiraterone plus dutasteride (a 5α-reductase inhibitor), 3-keto-5α-abiraterone and downstream metabolites were depleted by the addition of dutasteride, while D4A concentrations rose, showing that dutasteride effectively blocks production of a tumour-promoting metabolite and permits D4A accumulation. Furthermore, dutasteride did not deplete the three 5β-reduced metabolites, which were also clinically detectable, demonstrating the specific biochemical effects of pharmacological 5α-reductase inhibition on abiraterone metabolism. Our findings suggest a previously unappreciated and biochemically specific method of clinically fine-tuning abiraterone metabolism to optimize therapy.

Aberrant corticosteroid metabolism in tumor cells enables GR takeover in enzalutamide resistant prostate cancer

Prostate cancer is driven by androgen stimulation of the androgen receptor (AR). The next-generation AR antagonist, enzalutamide, prolongs survival, but resistance and lethal disease eventually prevail. Emerging data suggest that the glucocorticoid receptor (GR) is upregulated in this context, stimulating expression of AR-target genes that permit continued growth despite AR blockade. However, countering this mechanism by administration of GR antagonists is problematic because GR is essential for life. We show that enzalutamide treatment in human models of prostate cancer and patient tissues is accompanied by a ubiquitin E3-ligase, AMFR, mediating loss of 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2), which otherwise inactivates cortisol, sustaining tumor cortisol concentrations to stimulate GR and enzalutamide resistance. Remarkably, reinstatement of 11β-HSD2 expression, or AMFR loss, reverses enzalutamide resistance in mouse xenograft tumors. Together, these findings reveal a surprising metabolic mechanism of enzalutamide resistance that may be targeted with a strategy that circumvents a requirement for systemic GR ablation. DOI: http://dx.doi.org/10.7554/eLife.20183.001

Development and validation of a novel LC-MS/MS method for simultaneous determination of abiraterone and its seven steroidal metabolites in human serum: Innovation in separation of diastereoisomers without use of a chiral column

Abiraterone acetate (AA), the prodrug of abiraterone, is FDA-approved for the treatment of castration-resistant prostate cancer. Abiraterone is metabolized in patients to a more potent analogue, D4A. However, we have recently reported that this analogue is further metabolized to additional metabolites in patients treated with AA. Here, we present a liquid chromatography-tandem mass spectrometry method developed to resolve and detect abiraterone and its seven metabolites in human serum using an AB Sciex Qtrap 5500 mass analyzer coupled with a Shimadzu Nexera UPLC station. Analytes and the internal standard (abiraterone-d4) were extracted from human serum using the liquid-liquid extraction procedure. The analytes were separated using a Zorbax Eclipse Plus C18 150×2.1mm, 3.5μm column at 40°C and an isocratic mobile phase 35% A (0.1% formic acid in water), 65% B (0.1% formic acid in methanol:acetonitrile; 60:40). Electrospray ionization in positive mode was applied with multiple reaction monitoring in a total run time of 13min. Abiraterone detection was linear in the range 2-400ng/mL and all metabolites from 0.1-20ng/mL. The method was validated following US FDA guidelines for bioanalytical method validation, and all the metabolite results were within the acceptance limits. Despite the similarity in structure and mass transition between the metabolites, the validated method separated all the metabolites, including diastereomers, to allow accurate identification and quantitation of each compound.

Association of tissue abiraterone levels and SLCO genotype with intraprostatic steroids and pathologic response in men with high-risk localized prostate cancer

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes. Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes. Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05). Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592–601. ©2017 AACR.

HSD3B1(1245A>C) variant regulates dueling abiraterone metabolite effects in prostate cancer

BACKGROUND. A common germline variant in HSD3B1(1245A>C) encodes for a hyperactive 3&bgr;-hydroxysteroid dehydrogenase 1 (3&bgr;HSD1) missense that increases metabolic flux from extragonadal precursor steroids to DHT synthesis in prostate cancer. Enabling of extragonadal DHT synthesis by HSD3B1(1245C) predicts for more rapid clinical resistance to castration and sensitivity to extragonadal androgen synthesis inhibition. HSD3B1(1245C) thus appears to define a subgroup of patients who benefit from blocking extragonadal androgens. However, abiraterone, which is administered to block extragonadal androgens, is a steroidal drug that is metabolized by 3&bgr;HSD1 to multiple steroidal metabolites, including 3-keto-5&agr;-abiraterone, which stimulates the androgen receptor. Our objective was to determine if HSD3B1(1245C) inheritance is associated with increased 3-keto-5&agr;-abiraterone synthesis in patients. METHODS. First, we characterized the pharmacokinetics of 7 steroidal abiraterone metabolites in 15 healthy volunteers. Second, we determined the association between serum 3-keto-5&agr;-abiraterone levels and HSD3B1 genotype in 30 patients treated with abiraterone acetate (AA) after correcting for the determined pharmacokinetics. RESULTS. Patients who inherit 0, 1, and 2 copies of HSD3B1(1245C) have a stepwise increase in normalized 3-keto-5&agr;-abiraterone (0.04 ng/ml, 2.60 ng/ml, and 2.70 ng/ml, respectively; P = 0.002). CONCLUSION. Increased generation of 3-keto-5&agr;-abiraterone in patients with HSD3B1(1245C) might partially negate abiraterone benefits in these patients who are otherwise more likely to benefit from CYP17A1 inhibition. FUNDING. Prostate Cancer Foundation Challenge Award, National Cancer Institute.

AR Signaling in Prostate Cancer Regulates a Feed-Forward Mechanism of Androgen Synthesis by Way of HSD3B1 Upregulation

3βHSD1 enzymatic activity is essential for synthesis of potent androgens from adrenal precursor steroids in prostate cancer. A germline variant in HSD3B1, the gene that encodes 3βHSD1, encodes for a stable enzyme, regulates adrenal androgen dependence, and is a predictive biomarker of poor clinical outcomes after gonadal testosterone deprivation therapy. However, little is known about HSD3B1 transcriptional regulation. Generally, it is thought that intratumoral androgen synthesis is upregulated after gonadal testosterone deprivation, enabling development of castration-resistant prostate cancer. Given its critical role in extragonadal androgen synthesis, we sought to directly interrogate the transcriptional regulation of HSD3B1 in multiple metastatic prostate cancer cell models. Surprisingly, we found that VCaP, CWR22Rv1, LNCaP, and LAPC4 models demonstrate induction of HSD3B1 upon androgen stimulation for approximately 72 hours, followed by attenuation around 120 hours. 3βHSD1 protein levels mirrored transcriptional changes in models harboring variant (LNCaP) and wild-type (LAPC4) HSD3B1, and in these models androgen induction of HSD3B1 is abrogated via enzalutamide treatment. Androgen treatment increased flux from [3H]-dehydroepiandrosterone to androstenedione and other downstream metabolites. HSD3B1 expression was reduced 72 hours after castration in the VCaP xenograft mouse model, suggesting androgen receptor (AR) regulation of HSD3B1 also occurs in vivo. Overall, these data suggest that HSD3B1 is unexpectedly positively regulated by androgens and ARs. These data may have implications for the development of treatment strategies tailored to HSD3B1 genotype status.

High-Dose Abiraterone Acetate in Men With Castration Resistant Prostate Cancer

Micro‐Abstract Abiraterone acetate with prednisone prolongs progression‐free and overall survival in men with advanced prostate cancer, but most eventually acquire resistance to treatment. In this study we evaluated the clinical benefit of increasing the dose of abiraterone acetate in patients who develop acquired resistance to standard‐dose therapy while exploring the pharmacokinetics and pharmacodynamics of resistance. Background: Abiraterone acetate (AA) inhibits androgen biosynthesis and prolongs survival in men with metastatic castration‐resistant prostate cancer (mCRPC) when combined with prednisone (P). Resistance to therapy remains incompletely understood. In this open‐label, single‐arm, multicenter phase II study we investigated the clinical benefit of increasing the dose of AA at the time of resistance to standard‐dose therapy. Patients and Methods: Eligible patients had progressive mCRPC and started AA 1000 mg daily and P 5 mg twice daily. Patients who achieved any prostate‐specific antigen (PSA) decline after 12 weeks of therapy continued AA with P until PSA or radiographic progression. At progression, AA was increased to 1000 mg twice daily with unchanged P dosing. Patients were monitored for response to therapy for a minimum of 12 weeks or until PSA or radiographic progression. The primary end point was PSA decline of at least 30% after 12 weeks of therapy at the increased dose of AA. Results: Forty‐one patients were enrolled from March 2013 through March 2014. Thirteen men experienced disease progression during standard‐dose therapy and were subsequently treated with AA 1000 mg twice per day. Therapy was well tolerated. No PSA declines ≥ 30% nor radiographic responses were observed after 12 weeks of dose‐escalated therapy. Higher baseline dehydroepiandrosterone levels, lower circulating tumor cell burden, and higher pharmacokinetic levels of abiraterone and abiraterone metabolites were associated with response to standard‐dose therapy. Conclusion: Increasing the dose of abiraterone at the time of resistance has limited clinical utility and cannot be recommended. Lower baseline circulating androgen levels and interpatient pharmacokinetic variance appear to be associated with primary resistance to AA with P.

Abstract A17: Abiraterone metabolism and a novel therapeutic strategy for castration resistant prostate cancer

Prostate cancer is the most common cancer in men in the United States. Abiraterone (abi) is approved by the FDA for treatment of castration resistant prostate cancer (CRPC). However, ~30% of patients have limited or no response to abi. The underlying mechanism of resistance is elusive in these cases, and the best treatment plan for such patients is not clear. Thus we sought to determine a potential mechanism of resistance by analyzing abi metabolism. We found that abi undergoes metabolism catalyzed by steroidogenic enzymes. Abi is converted sequentially to delta-4-abi (D4A), then undergoes C5 reduction to produce several metabolites, including 5α-abi and 5 other metabolites. D4A has more potent anti-tumor activity than abi itself because it inhibits multiple enzymes and antagonizes the androgen receptor (AR) directly. D4A inhibits cell proliferation and xenograft growth better than Abi. However, 5α-Abi has more limited effects on steroidogenic enzymes and is a modest AR agonist, which might promote abi resistance. We suggest that abi metabolism could be a biomarker for abi response, and patients with limited or no response to abi might have less D4A or more 5α-Abi. Furthermore, we demonstrate that dutasteride, a 5α-reductase inhibitor, suppresses the conversion from D4A to 5α-Abi in both prostate cancer cell lines and patients, which might enhance the clinical response to abi. Taken together, these data reveal a novel metabolic pathway of abi that can be exploited for treatment, specifically as a biomarker. It is achievable in clinic to fine tune abi metabolism to obtain greater concentrations of D4A, the most potent anti-tumor abi metabolite, in patients by supplementing abi treatment with dutasteride. Citation Format: Zhenfei Li, Mohammad Alyamani, Jianneng Li, Mary-Ellen Taplin, Nima Sharifi. Abiraterone metabolism and a novel therapeutic strategy for castration resistant prostate cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A17.

A PK/PD study of Delta-4 abiraterone metabolite in metastatic castration-resistant prostate cancer patients

ABSTRACT &Dgr;4‐abiraterone (&Dgr;4A) is an activemetabolite of abiraterone (ABI), which is approved in the treatment of metastatic castration resistant prostate cancer (mCRPC). The contribution of &Dgr;4A to the clinical antitumor activity of ABI remains unknown. The aim of this study was to explore the relationship between plasma &Dgr;4A concentration and survival in 36 mCRPC patients treated with abiraterone acetate (1000 mg/day) plus prednisone (10 mg/day). Plasma trough ABI and &Dgr;4A concentrations were monthly assayed using liquid chromatography during the first 3 months of treatment. ABI and &Dgr;4A Cmin were defined as the mean of trough concentrations measured for each patient. Predictive factors regarding progression‐free survival (PFS) and overall survival (OS) were explored using univariate Cox model. Mean plasma ABI and &Dgr;4A Cmin were 12.6 ± 6.8 ng/mL and 1.6 ± 1.3 ng/mL, respectively. The mean metabolic ratio &Dgr;4A/ABI was of 0.18 ± 0.25. In regard with in vitro pharmacodynamic data, effective plasma concentrations for ABI and &Dgr;4A were reached in 30 patients (83.3%) and only 2 patients (5.6%), respectively. Higher &Dgr;4A Cmin was associated with shorter OS (Hazard ratio, HR 1.54; CI95% 1.06–2.22; p = 0.022) but not with PFS. The HR associated with the metabolic &Dgr;4A/ABI ratio for PFS and OS were 7.80 (CI 95% 1.63–37.38; p = 0.010) and 12.52 (CI 95% 1.95–80.47, p = 0.0078), respectively. The present study shows &Dgr;4A is unlikely to have meaningful contribution to pharmacodynamic activity of ABI in mCPRC, rather that higher plasma &Dgr;4A concentration is associated with worse clinical outcomes. A high &Dgr;4A/ABI metabolic ratio could help to identify mCRPC patients with poorer survival.