John Wilton

Sequential Use of the Androgen Synthesis Inhibitors Ketoconazole and Abiraterone Acetate in Castration-Resistant Prostate Cancer and the Predictive Value of Circulating Androgens

Purpose: Patients previously treated with ketoconazole were excluded from phase III trials of abiraterone acetate due to potential overlapping mechanism of action. The purpose of this study was to determine the clinical utility of abiraterone and its impact on circulating androgens following ketoconazole. Experimental Design: Chemotherapy-naïve patients with progressive metastatic castration-resistant prostate cancer (mCRPC) and prior ketoconazole therapy ≥28 days received abiraterone acetate 1,000 mg daily and prednisone 5 mg twice daily. The primary endpoint was the proportion of patients with PSA response, defined as ≥30% PSA decline at 12 weeks. H0 = 0.30 versus H1 = 0.50 (α = 0.05, power = 0.83). Circulating androgen levels were measured using liquid chromatography tandem mass spectrometry. Results: Thirty-nine patients were included in the final analysis. Twenty (51%; 95% confidence interval, 36%–66%) patients had ≥30% PSA decline; the null hypothesis was rejected. Sixteen (41%) had ≥50% PSA decline. Median PFS (progression-free survival) was 16 weeks; median radiographic PFS (rPFS) was 36 weeks. Samples for measurement of baseline androgens were available in 37 patients. The PSA response proportion was 59% in 29 patients with DHEA ≥ limit of quantitation (LOQ), compared with 13% in 8 patients with DHEA < LOQ (P = 0.042). Median PFS was 6 and 16 weeks in DHEA < LOQ and DHEA ≥ LOQ patients, respectively (P = 0.017); median rPFS was 14 and 36 weeks in DHEA < LOQ and DHEA ≥ LOQ patients, respectively (P < 0.001). Conclusions: Abiraterone demonstrates modest clinical efficacy in mCRPC patients previously treated with ketoconazole. Patients with DHEA ≥ LOQ were more likely to demonstrate PSA responses and longer PFS. Analysis of circulating androgens merits further investigation as a biomarker for response to androgen synthesis inhibitor therapy. Clin Cancer Res; 20(24); 6269–76. ©2014 AACR.

Prostate cancer cells differ in testosterone accumulation, dihydrotestosterone conversion, and androgen receptor signaling response to steroid 5α-reductase inhibitors†‡

Blocking 5α‐reductase‐mediated testosterone conversion to dihydrotestosterone (DHT) with finasteride or dutasteride is the driving hypothesis behind two prostate cancer prevention trials. Factors affecting intracellular androgen levels and the androgen receptor (AR) signaling axis need to be examined systematically in order to fully understand the outcome of interventions using these drugs.

Roles for the Backdoor Pathway of Androgen Metabolism in Prostate Cancer Response to Castration and Drug Treatment

Almost all men who present with advanced prostate cancer (CaP) and many men who fail potentially curative therapy are treated with androgen deprivation therapy (ADT). ADT is not curative and CaP recurs as the lethal phenotype. The goal of this review is to describe the evolution of adrenal androgen blockade, how new androgen measurement methods have furthered understanding of androgen metabolism, and how further understanding of the backdoor pathway of androgen metabolism may lead to interventions that extend survival even more.

Androgenic biomarker profiling in human matrices and cell culture samples using high throughput, electrospray tandem mass spectrometry

A high throughput, high pressure liquid chromatographic (HPLC) method with triple quadrupole mass spectral detection (LC/MS/MS) was validated for the measurement of 5 endogenous androgens in human plasma and serum and applied to various in vivo and in vitro study samples to pursue a better understanding of the interrelationship of the androgen axis, intracrine metabolism, and castration‐recurrent prostate cancer (CaP).

A Pilot Study Comparing the Effect of Flaxseed, Aromatase Inhibitor, and the Combination on Breast Tumor Biomarkers

Use of complementary approaches is common among breast cancer survivors. Potential interactions between aromatase inhibitors (AI) and high phytoestrogen foods, such as flaxseed (FS), are not often described. We conducted a pilot 2 × 2 factorial, randomized intervention study between tumor biopsy and resection, in 24 postmenopausal women with estrogen receptor positive (ER+) breast cancer, to assess the effects of FS and anastrozole, and possible interactions between them, on serum steroid hormone and tumor-related characteristics associated with long-term survival (Roswell Park Cancer Institute, 2007–2010). The effect of each treatment vs. placebo on outcomes was determined by linear regression adjusting for pretreatment measure, stage, and grade. Although not statistically significant, mean ERβ expression was approximately 40% lower from pre- to postintervention in the FS + AI group only. We observed a statistically significant negative association (β ± SE −0.3 ± 0.1; P = 0.03) for androstenedione in the FS + AI group vs. placebo and for DHEA with AI treatment (β ± SE −1.6 ± 0.6; P = 0.009). Enterolactone excretion was much lower in the FS + AI group compared to the FS group. Our results do not support strong effects of FS on AI activity for selected breast tumor characteristics or serum steroid hormone levels but suggest AI therapy might reduce the production of circulating mammalian lignans from FS.

Dietary folate levels alter the kinetics and molecular mechanism of prostate cancer recurrence in the CWR22 model

Folate impacts the genome and epigenome by feeding into one-carbon metabolism to produce critical metabolites, deoxythymidine monophosphate and s-adenosylmethionine. The impact of folate exposure and intervention timing on cancer progression remains controversial. Due to polyamine metabolism’s extraordinary biosynthetic flux in prostate cancer (CaP) we demonstrated androgen stimulated CaP is susceptible to dietary folate deficiency. We hypothesized dietary folate levels may also affect castration recurrent CaP. We used the CWR22 human xenograft model which recurs following androgen withdrawal. Engrafted mice were fed a folate depleted or supplemented diet beginning at androgen withdrawal, or prior to xenograft implantation. Both folate depletion and supplementation at the time of withdrawal significantly decreased recurrence incidence. Folate supplementation prior to xenograft implantation increased time to recurrence, suggesting a protective role. By contrast, folate depleted recurrent tumors exhibited transcriptional adaptive responses that maintained high polyamine levels at the expense of increased DNA damage and DNA methylation alterations. Mining of publically available data demonstrated folate related pathways are exceptionally dysregulated in human CaP, which correlated with decreased time to biochemical recurrence. These findings highlight the potential for novel therapeutic interventions that target these metabolic pathways in CaP and provide a rationale to apply such strategies alongside androgen withdrawal.

Serum-free complete medium, an alternative medium to mimic androgen deprivation in human prostate cancer cell line models

Almost all men who present with advanced prostate cancer (CaP) and some men who fail therapy for clinically localized CaP are treated with androgen deprivation therapy (ADT). CaP cell lines are used to identify and characterize new agents for ADT or investigate mechanisms of ADT resistance. CaP cell lines are maintained in culture medium that contains fetal bovine serum, which contains testosterone (T). Androgen deprivation experiments are performed using media supplemented with androgen‐free serum, such as charcoal stripped fetal bovine serum (CS‐FBS). However, CS‐FBS composition varies from batch‐to‐batch and variations may impact experimental reproducibility. Serum free media (SFM) may provide a better defined alternative to media supplemented with CS‐FBS (CSM).

Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade

Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert 5α-androstane-3α,17β-diol (DIOL) to dihydrotestosterone (DHT). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to DHT. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the 5α-reductase inhibitor, dutasteride, decreased DHT levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of DHT synthesis provides a therapeutic strategy to inhibit CRPC development and growth.

MP87-12 ADRENAL ANDROGENS FACILITATE PROSTATE CANCER CELL RESISTANCE TO ANDROGEN DEPRIVATION THERAPY

INTRODUCTION AND OBJECTIVES: We recently identified and validated 3 intrinsic prostate cancer subtypes (PCS) based on a 37gene expression signature. To evaluate the PCS system as a prognostic tool, we determined the frequencies of PC subtypes in diagnostic prostate needle biopsies (PNBX) collected from men with high-grade localized PC (clinical stage M0) who remained disease-free or progressed to castration-resistant prostate cancer (CRPC) after definitive treatment as well as in PNBX of men who were newly diagnosed with metastatic disease (clinical stage M1). METHODS: PNBX cases were selected from a cohort of 486 patients with high-grade localized or metastatic prostate cancer (mPC), diagnosed and treated in the Greater Los Angeles VA Healthcare System between 2000 and 2015. RNA sequencing (RNAseq) was performed on 86 tumor foci from 68 formalin-fixed, paraffin-embedded (FFPE) PNBXs: thirty nine cases with de novo metastases (M1), 6 cases who progressed to metastasis (M0-P), and 23 PC cases without progression (M0-NP). Computation of pathway activation profiles, principal component analysis, and application of the 37-gene PCS classifier were performed for assignment of subgroups. The results of this analysis were compared to publically available datasets (PCF and SU2C datasets) of primary PC and CRPC metastases. RESULTS: Importantly, analysis of RNAseq data revealed adequate levels of transcriptome coverage (>18,000 genes) in all 68 cases. Significant differences in survival were observed in M1 cases compared to M0-P and M0-NP. The frequency of PCS groups in PNBX specimens of patients with M1 stage (36% PCS1; 21% PCS2; 44% PCS3) was similar to that of biopsies from metastatic CRPC in the PCF and SU2C cohort (35% PCS1; 20% PCS2; 45% PCS3). A high proportion of the poor prognosis PCS1 (n1⁄414 of 15 cases) was identified in PNBX of patients with M1 stage compared to M0 stage. CONCLUSIONS: Although the subtyping of PNBX and derived staging and prognostic information warrants further confirmation in a larger cohort, the data demonstrate a promising potential for a footprint of concurrent or future metastatic disease in PNBXs obtained at the time of PC diagnosis.

Abstract 3372: Pharmacokinetic analysis of CFAK-C4 in dogs and estimation of first dose in man.

Purpose: Inhibition of the FAK-VEGFR-3 interaction by the small molecule inhibitor chloropyramine hydrochloride (CFAK-C4) has been shown to reduce tumor growth both alone and synergistically with doxorubicin and gemcitabine in an animal tumor model. A multiple dose study of CFAK-C4 in dogs was investigated to gain knowledge in estimating a first in man dose. Methods: CFAK-C4 was given to two female and two male dogs as an IV infusion at 1.25 or 2.5 mg/kg for five minutes, on three consecutive days, days 1, 2 and 3. Single- and multiple-dose PK samples were collected on days 1 and 3 of dosing. Plasma samples were collected at 0.5, 1, 2, 4, 8, and 24 hours post-dose. CFAK-C4 concentrations were determined using LC-MS/MS, with a LLOQ of 2.5 pg/ml. Noncompartmental PK analysis was performed using WinNonlin (Pharsight, version 5.3). Results: The mean CFAK-C4 plasma concentration-time profiles revealed a biexponential decline of drug following IV infusion, independent of day of dosing. The median (range) half-life was 3.42 hours (2.75-4.06 hours) and 3.79 (3.5-5.0) on days 1 and 3 following administration of 1.25 mg/kg CFAK-C4. At the 2.5 mg/kg dose level, the median half-life was similar. The median (range) Cmax of 1.25 mg/kg CFAK-C4 was determined to be 75 ng/ml (41-87 ng/ml) on day 1 and 99 ng/ml (84-102 ng/ml) on day 3. After the 2.5 mg/kg dose the Cmax was 185 ng/ml (165-244 ng/ml) and 171 ng/ml (153-181 ng/ml) on days 1 and 3 respectively. The AUC0-24 at the 1.25 mg/kg dose level was similar on both days 1 and 3 with a median (range) of 167 ng*hr/ml (117-231 ng*hr/ml) and 178 ng*hr/ml (170-194 ng*hr/ml), respectively. The AUC0-24 was also similar after the 2.5 mg/kg dose on days 1 and 3 with a median (range) of 447 ng*hr/ml (434-495 ng*hr/ml) and 406 ng*hr/ml (400-460 ng*hr/ml), respectively. The median (range) clearance for dose 1.25 mg/kg was 7.46 L/hr/kg (5.36-10.59 L/hr/kg) on day 1 and 6.47 L/hr/kg (6.07-6.91L/hr/kg) on day 3. For the 2.5 mg/kg dose level, the median (range) clearance was slightly lower compared with the 1.25 mg/kg dose, with values of 5.48 L/hr/kg (5.02-5.66 L/hr/kg) and 5.85 L/hr/kg (5.04-5.93 L/hr/kg) on days 1 and 3, respectively. Utilizing allometric scaling techniques of PK parameters from mice and dogs for CFAK-C4, the estimated human PK parameters for volume of distribution, clearance and half-life were 1695 L, 126 L/hr, and 9.34 hours, respectively. Subsequently, using a no observable adverse event level (NOAEL) of 44.5 mg/kg IP for mice and a NOAEL of 2.5 mg/kg IV for dog, the human starting dose (assuming a 70 kg human) would be a daily dose of 25 mg and 10 mg based on mouse or dog, respectively. Given that dogs are the most sensitive species, 10 mg daily is the suggested first dose in man for CFAK-C4. Conclusions: These results demonstrate that CFAK-C4 has linear kinetics and that no accumulation of drug occurs after multiple dosing. Allometric scaling of mouse and dog data allowed for the estimation of the first in man dose of CFAK-C4. Citation Format: Allison Gaudy, John Wilton, Leslie Curtin, Elena Kurenova, Sandra Buitrago, William Cance, Gerald Fetterly. Pharmacokinetic analysis of CFAK-C4 in dogs and estimation of first dose in man. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3372. doi:10.1158/1538-7445.AM2013-3372