Hydrazinobenzoylcurcumin inhibits androgen receptor activity and growth of castration-resistant prostate cancer in mice.

There is a critical need for therapeutic agents that can target the amino-terminal domain (NTD) of androgen receptor (AR) for the treatment of castration-resistant prostate cancer (CRPC). Calmodulin (CaM) binds to the AR NTD and regulates AR activity. We discovered that Hydrazinobenzoylcurcumin (HBC), which binds exclusively to CaM, inhibited AR activity. HBC abrogated AR interaction with CaM, suppressed phosphorylation of AR Serine81, and blocked the binding of AR to androgen-response elements. RNA-Seq analysis identified 57 androgen-regulated genes whose expression was significantly (p ≤ 0.002) altered in HBC treated cells as compared to controls. Oncomine analysis revealed that genes repressed by HBC are those that are usually overexpressed in prostate cancer (PCa) and genes stimulated by HBC are those that are often down-regulated in PCa, suggesting a reversing effect of HBC on androgen-regulated gene expression associated with PCa. Ingenuity Pathway Analysis revealed a role of HBC affected genes in cellular functions associated with proliferation and survival. HBC was readily absorbed into the systemic circulation and inhibited the growth of xenografted CRPC tumors in nude mice. These observations demonstrate that HBC inhibits AR activity by targeting the AR NTD and suggest potential usefulness of HBC for effective treatment of CRPC.

using a high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method as described below. The tissue samples were homogenized in 4 volumes of human plasma. A 250 μl aliquot of tissue homogenate or mouse plasma was spiked with 1 ml ethyl acetate. The mixture was vortex-mixed, centrifuged at 14,000 rpm for 5 min, and the top layer was transferred and evaporated to dryness under a stream of nitrogen in a water bath at 50 0 C ± 5 0 C. The residue was reconstituted in 100 μl of methanol/water containing 0.45% formic acid (70:30, v/v), and centrifuged at 14,000 rpm for 5 min.
Ten microliters of the supernatant was injected into the LC-MS/MS. The chromatographic separation was achieved on a Waters XTerra MS column (2.1 × 50 mm, 3.5 μm i.d.) with a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) at a flow rate of 0.2 ml/min. The column effluent was monitored using a Waters Quattro Micro TM triple quadruple mass-spectrometric detector equipped with electrospray ionization source (Milford, MA, USA). HBC was monitored in the positive ionization mode at the mass transition of 485.3→469.4. The calibration curve was constructed in human plasma over the concentration range of 2 to 10,000 ng/ml. The within-and between-day precision and accuracy of the assay were <15%.
Pharmacokinetic data analysis: The plasma and tissue pharmacokinetic parameters of HBC were estimated using noncompartmental analysis with the WinNonlin software (Pharsight). The maximum concentration (C max ) and the time of occurrence for maximum concentration (T max ) were obtained by visual inspection of the plasma/tissue concentration-time curves after the drug administration.
The total area under the concentration-time curve from time zero to the last measurable time point (AUC last ) was calculated using the linear and logarithmic trapezoidal method for the ascending and descending plasma concentrations, respectively. The total area under the concentration-time curve from time zero to infinity (AUC 0- ) was calculated as the sum of AUC last and the extrapolated area, which was calculated by the last observed concentration divided by the terminal rate constant ( z ), where  z was estimated by terminal log-linear phase of the concentration-time curve. The terminal elimination half-life (T 1/2, ) was calculated as 0.693/ z . The apparent systemic clearance (CL/F) was calculated as Dose/AUC 0- . The apparent volume of distribution (V z /F) was estimated as (CL/F)/ z .    Table 2.

Figure S4: A) Body weight of mice is unaffected by HBC. Nude mice bearing C4-2B tumors
were given a daily (5 days/week) i.p. injection of 100 mg/Kg and body weight was measured twice a week. Data points, mean body weight (n=5); bars, SE. B) HBC has no noticeable effect on histology of major organs, viz., kidney, liver, lung and heart. Paraffin-embedded tissue sections were deparaffinized, hydrated, and stained with hematoxylin for 1 min. After rinsing, the slides were stained with eosin for 1 min, rinsed thoroughly, and mounted with Permount.    CL/F (L/h/kg) 5.45 n/a n/a n/a n/a Vz/F (L/kg) 3.76 n/a n/a n/a n/a a Pharmacokinetic parameters were estimated using noncompartmental analysis of the mean concentration time profiles from two mice.
n/a, not estimated