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The Beauty and the Beast of Abiraterone Metabolism Mohammad Alyamani1, Zhenfei Li1, Sunil K. Upadhyay2, Steven P. Balk3, Mary-Ellen Taplin3, Richard J.

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Presentation on theme: "The Beauty and the Beast of Abiraterone Metabolism Mohammad Alyamani1, Zhenfei Li1, Sunil K. Upadhyay2, Steven P. Balk3, Mary-Ellen Taplin3, Richard J."— Presentation transcript:

1 The Beauty and the Beast of Abiraterone Metabolism Mohammad Alyamani1, Zhenfei Li1, Sunil K. Upadhyay2, Steven P. Balk3, Mary-Ellen Taplin3, Richard J. Auchus2 & Nima Sharifi1,4 1Cleveland Clinic, Cleveland, OH; 2University of Michigan Medical School, Ann Arbor, MI; 3Harvard Medical School, Boston, MA.4Case Comprehensive Cancer Center, Cleveland, OH Abstract D4A Inhibits Steroidogenic Enzymes Results Abiraterone acetate (AA, the prodrug of abiraterone) is FDA-approved for the treatment of castration-resistant prostate cancer. Abiraterone (Abi) is metabolized in patients to a more potent analogue, D4A, which can block steroidogenic enzymes and the androgen receptor (AR) directly, and drive the anti-tumor activity of AA in prostate cancer1. In addition, similar to testosterone and androstenedione, D4A has a Δ4, 3-keto structure, which allows D4A to be further metabolized by steroidogenic enzymes. We determined that 6 structurally related metabolites were generated from D4A metabolism; 3 resulted from 5β-reductase and 3 from 5α-reductase, one of which can promote tumor progression and activate AR-regulated genes2. Therefore it may be important to block D4A metabolism to achieve the maximum efficacy for treatment. Analysis of serum samples obtained from CRPC patients treated with AA shows that Abi and all its 7 steroidal metabolites are present in the samples. In order to test the effect of dutasteride (a 5α-reductase inhibitor) on abiraterone metabolism, blood samples from patients treated on a phase 2 clinical trial with AA followed by AA plus dutasteride were collected. The results show depleted levels of 5α-reduced Abi metabolites, increases in Abi and D4A, and no change in 5β-reduced Abi metabolites. To analyze the patient samples, we developed a liquid chromatography tandem mass spectrometry method that allows not just determination of the metabolites but also accurate quantification of their concentrations in patients with castration-resistant prostate cancer treated with AA. In the method, an AB Sciex 5500 Qtrap mass analyzer with electrospray ionization in positive mode was applied, the mass analyzer was coupled with Shimadzu Nexera X2 UPLC station, and the analytes were separated using a Zorbax Eclipse Plus C x 2.1 mm, 3.5 µm column at 40°C. The method was validated according to US FDA guidelines for bioanalytical method validation3. The validated LC–MS/MS method with reversed-phase chromatographic conditions resolved and quantitated all the metabolites despite the similarity in their structures and mass transition. Test Min Max Acceptance Criteria Linearity (R2) 0.9990 0.9996 > Intra day Accuracy 91.7 107.4 % Intra day Precision 0.99 10.75 ≤ 15 % Inter day Accuracy 93.8 103.8 Inter day Precision 2.84 12.18 Sensitivity Precision 2.63 16.89 ≤ 20 % Sensitivity Accuracy 85.3 111.2 Selectivity 6.7 592.6 > 5 Recovery 85.1 105 - Matrix Effect 88.5 114.7 Solution Stability (9 days) 83.2 109.5 % Post preparative (42 h) 96.1 103.3 Short term (21 h) 88.2 114.8 Freeze & Thaw ( 3 cycles) 86.3 96.7 Long term (6 months) 86.7 95.6 Abiraterone Metabolites Table 1. Validation tests results. Spiked serum with known concentration were used to study the validation of the LC-MS/MS method. n=5 and n=15 samples were used to study the intra-day and inter-day accuracy and precision. N = 5 LLOQ samples were prepared to study the sensitivity. N = 6 LLOQ samples, each prepared in different serum batches, were compared to the blank samples to test the selectivity. The ratio between samples spiked before extraction to samples spiked after extraction was used to calculate the recovery. Matrix effect is quantified by calculating the area ratio of analyte/internal standard of a QC low (post-extraction addition of analyte) over the area ratio of analyte/internal standard in a methanol/water solution. Analytes were stable in solution and in serum. Even though all analytes did show a decrease in concentration when subjected to three freeze-thaw cycles or stored for 6 months, the results were within the criterion range for accuracy. Fig 3. D4A inhibits,CYP17A1, 3βHSD and SRD5A enzymatic activity in vitro. A. HEK293 cells, B. LNCaP cells, and C. LAPC4 cells were treated with either D4A or Abi at the indicated concentrations and incubated with the designated steroids. Analysis of steroids was done by HPLC. DHEA: Dehydroepiandrosterone; AD: Androstenedione; 5α-dione: 5α-Androstanedione. LC-MS/MS Method Abiraterone Metabolites in CRPC Patients An AB Sciex Qtrap 5500 mass analyzer operated in positive ion mode with an electrospray ionization (ESI) source coupled with a Shimadzu Nexera UPLC station was used to detect Abi and its metabolites. The separation of the analytes was achieved using a Zorbax Eclipse Plus C18 150*2.1mm, 3.5-µm column at 40°C, and isocratic mobile phase 35% A (H2O), 65% B (methanol:acetonitrile; 60:40) with 0.1% (formic acid) in both and flow rate 0.2 ml/min. Analytes and the IS (Abiraterone-d4) were extracted from 100µl human serum (collected from CRPC patients treated with AA) with 2 ml methyl tert-butyl ether. After evaporation, the residue was reconstituted using 300 µl 50% methanol and 10 µl was injected. A 7-point calibration curve for all analytes was constructed, and 6 quality control samples; 2 (low, mid, and high) were injected with the samples. The analytes were quantified using multiple reaction monitoring (MRM). Data were processed using Analyst software (AB Sciex). All the analytes were successfully separated with the developed method despite their closely related structures (Fig 4). Analysis in human serum showed that Abi was linear in the range of ng/ml, and all the metabolites were linear in the range of ng/ml. n=36 Abiraterone D4A 3-keto-5α-abi 3α-OH-5α-abi 3β-OH-5α-abi 3-keto-5β-abi 3α-OH-5β-abi 3β-OH-5β-abi Mean 41.33 2.38 9.207 1.809 0.6742 11.59 11.48 16.26 Min 0.00 0.06 0.58 0.17 0.08 0.12 0.23 0.22 Max 213.3 7.19 65.22 8.085 4.137 46.64 32.81 41.56 Table 2. The mean with minimum and maximum concentrations (ng/ml) of abiraterone metabolites. N=36 patients received abiraterone acetate 1 gm/day and prednisone 5 mg twice/day. 3-keto-5α-abi (AR agonist) levels were higher than D4A (AR antagonist). Effect of Dutasteride on Abiraterone Metabolism Separation of Abi and its Metabolites Fig 1. Steroidogenic enzyme metabolism of abiraterone and structure of the resultant metabolites. Abi is a Δ5, 3β-hydroxysteroid, and therefore it may undergo enzymatic reaction with 3β-hydroxysteroid dehydrogenase (3βHSD) and be converted to its 3 keto Δ4 -metabolite (D4A). D4A can undergo metabolism by two possible pathways: via steroid 5α-reductase (SRD5A) or steroid 5β-reductase. The resultant metabolites can also be metabolized by two enzymes: 3α- hydroxysteroid dehydrogenase (3αHSD) or 3βHSD. D4A .vs. 3 Keto-5α-Abi Activity Fig 5. Fold change in the metabolite concentrations. N=16 patients received abiraterone acetate for 8 weeks then dutasteride (SRD5A inhibitor) was added to the treatment. Blood samples were collected when the patients were under AA alone (T0) , after 4 weeks of initiating dutasteride (T1) , and then after 16 weeks (T2). The levels were normalized to AA alone treatment to show the effect of adding dutasteride. Conclusions New abiraterone metabolites that result from steroidogenic enzymes were identified. Steroidogenic enzymes which converts abiraterone to the more potent analogue D4A (The Beauty), also converts D4A to a tumor progression promoter; 3-keto-5α-Abi (The Beast). Abiraterone metabolism can be altered by SRD5A inhibition. The validated LC-MS/MS method with reversed-phase chromatographic conditions resolved and quantitated all the metabolites despite the similarity in their structures. Fig 4. Chromatogram for abiraterone and its metabolites. The results were obtained by injecting 10 μL of 50 ng/mL standard solutions for A, Abi; B, D4A; C, 3-keto-5α-Abi; D, 3α-OH-5α-Abi; E, 3β-OH-5α-Abi; F, 3-keto-5β-Abi; G, 3α-OH-5β-Abi; and H, 3β-OH-5β-Abi. Validation References Fig 2. D4A (A) but not 3-Keto-5α-Abi (B) inhibits xenograft tumor. Male NSG mice (6-8 weeks) were castrated and implanted with DHEA and injected with 107 VcaP cells. (C) 3-Keto-5α-Abi stimulates AR responsive genes in LAPC4 Cells. (D) 3 Keto-5α-Abi levels are higher than D4A in humans treated with single dose of AA. Numbers in the circles represent the concentration in ng/ml. The method was validated according to US FDA guidelines for bioanalytical method validation. As shown in Table 1, all criteria of the guideline were met. The criteria for linearity, accuracy, and precision fell within the acceptance criteria. All the analytes were stable in solution and serum, and the method gave excellent recovery without matrix effects or interference. Li Z, Bishop A, Alyamani M et al (2015). Nature 523:347–351. Li Z, Alyamani M et al (2016). Nature 533:547–551. Alyamani M et al (2016). J. Steroid Biochem. Mol. Biol (In Press).


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