Orteronel (TAK-700) is a novel and selective inhibitor of CYP17A1, which is expressed in testicular, adrenal and prostate tumor tissues. Orteronel is currently in Phase-III clinical development for metastatic castration-resistant prostate patients. The objective of the study is to assess the permeability, metabolic stability (in various preclinical and human liver microsomes), identify the major CYPs involved in the metabolism of Orteronel. We have also studied the pharmacokinetics and excretion of Orteronel in Sprague-Dawley rats. Orteronel was found to be stable in various liver microsomes tested. The half-life (t ½) of Orteronel with intravenous (i.v.) route was found to be 1.65 ± 0.22 h. The clearance and volume of distribution by i.v. route for Orteronel were found to be 27.5 ± 3.09 mL/min/kg and 3.94 ± 0.85 L/kg, respectively. The absorption of Orteronel was rapid, with maximum concentrations of drug in plasma of 614 ± 76.4, 1,764 ± 166, 4,652 ± 300 and 17,518 ± 3,178 ng/mL attained at 0.38, 0.75, 0.50 and 0.83 h, respectively, after oral administration of Orteronel at 5, 10, 30 and 100 mg/kg as a suspension. In the dose proportional oral pharmacokinetic study, the mean t ½ by oral route was found to be ~3.5 h and bioavailability ranged between 69 and 89 %. The primary route of elimination for Orteronel is urine.

The derived pharmacokinetic parameters (AUC and C at 600 mg dose) and simulated plasma concentration-time profiles of ulixertinib in humans were predicted with good confidence by allometric approach.

Aim and Background:A simple, sensitive and rapid method was developed for quantitation of theophylline in rabbit plasma utilizing Triple Quadrupole LC/MS.Materials and Methods:An aliquot of 0.1 mL of plasma sample was extracted with ethyl acetate using Heidolph Vortex. The chromatographic separation was performed by using HyPURITY ADVANCE™ C18 Column (3 × 50 mm) with a mobile phase of 80% methanol and 20% 2 mM ammonium acetate buffer followed by MS/MS detection. The analyte was quantitated in positive ionization mode. Multiple reaction monitoring (MRM) using the transition m/z 181.1→124.2 and m/z 180.2→110.1 was performed to quantify theophylline with internal standard (IS, Phenacetin), respectively. The method had a total chromatographic runtime of 3 min and linear calibration curves over the concentration range of 50.418–5062.063 ng/mL. The lower limit of quantification (LLOQ) was 50.418 ng/mL Sodium heparin (3.50%) used as an anticoagulant to prepare rabbit plasma and samples were maintained at 10°C in the auto sampler during the assay period. Inter and intraday batch precision and accuracy of the method were determined by using six quality control samples.Results:Average accuracies for the assay were 89 to 106%, inter and intra-day coefficients variation (CV) of <9% and the recovery is 39.30% for theophylline and 57.00% for Phenacetin.Conclusion:Currently we are extensively using this method in our laboratory for quantitative analysis of theophylline in rabbit plasma samples and proved to be simple, accurate and precise.

A simple, sensitive and rapid method was developed for quantitation of tacrolimus in rat whole blood utilizing triple Quadrupole LC/MS. An aliquot of 0.1 mL of whole blood sample was extracted with t-butyl methyl ether using a Heidolph vortexer. The chromatographic separation was performed by using chromolith fast gradient HPLC RP 18e (2mmX50mm.i.d) column with a mobile phase of 90% methanol and 10% 2mM ammonium acetate buffer followed by MS/MS detection. The analyte was quantitated in negative ionization mode. Multiple reaction monitoring (MRM) using the transition m/z 802.4→560.2 and m/z 808.4→548.6 was performed to quantify tacrolimus with IS (pimecrolimus), respectively. The method had a total chromatographic run time of 2.5 min; and linear calibration curves over the concentration range of 20.931 -1000.703 ng/mL.The lower limit of quantification (LLOQ) was 20.931 ng/mL. Use of sodium citrate (3.85%) as an anticoagulant in rat whole blood and samples were stable for at least the time required to assay the number of samples that could be placed in the auto sampler which is maintaining temperature of 10°C. The between and within batch precision and accuracy of the method were determined by using 6 quality control samples. The highest %CV 478.908 ng/mL (8.01% within run & 3.07 between run), with other %CV<5%. The recovery ranged 23.92% for tacrolimus over range of 50.285 to 798.179 ng/mL and was 18.52% for pimecrolimus respectively. The validated method was successfully applied to the quantification of tacrolimus concentration in rat whole blood.

Introduction: Lysine Specific Demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that has been reported to be over-expressed in many malignant tumors. Down-regulation of LSD1 has been shown to effectively treat cancers by inducing re-expression of aberrantly silenced genes. Studies have shown that LSD1 may contribute to acute myelogenous leukemia pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML. Similarly, HDAC isoform selective inhibitors are beginning to be explored as less toxic alternatives to panHDAC inhibitors in select cancers. Further, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting multiple cancers. Here, we show that JBI-295, a dual inhibitor targeting both LSD1 and HDAC6/8 shows stronger efficacy without enhancing systemic toxicity, in a subset of AML and JAK-dependent myeloproliferative cancer. Methods: Computational chemistry approaches were used to design LSD1 specific and LSD1-HDAC dual inhibitors. To assess in vitro LSD1 potency, TR-FRET assay was used. For assessing in vitro HDAC activity fluorescence based HDAC6 activity assay was performed. Western blotting was used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Results: Several compounds from this series show strong in vitro potency against LSD1 with more than excellent selectivity against MAOs. JBI-295 one of the lead dual molecules, showed strong LSD1 potency (IC50 of 0.07 μM) and isoform selective HDAC6/8 activity (IC50 of 0.006 and 0.08 µM on HDAC6 and HDAC8, respectively), with about 100 fold selectivity against other HDAC isoforms. JBI-295 showed strong anti-proliferative activity on leukaemia and multiple myeloma cell lines. In cell based and in vivo target engagement studies there was a concomitant increase in CD11b, CD86 and GFI1b and tubulin acetylation levels. JBI-295 was more efficacious in inhibiting the growth of leukemia HEL92.1.7 xenograft by oral administration when compared to IP administration of ACY-1215, a HDAC6 selective inhibitor. Stronger tumor growth inhibition was also observed in melanoma A375 xenograft model as compared to inhibitors with single activity. In addition, JBI-295 showed single agent activity in a syngeneic murine colon cancer model CT26 and also resulted in stronger tumor growth inhibition when combined with anti-PDL1 antibody. Conclusion: The data obtained demonstrate that dual LSD1-HDAC6/8 inhibitors could serve as novel, effective therapeutic agents for treatment of select subset of cancer. Advanced efficacy and toxicology studies with JBI-295 are in progress. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Sreekala Nair, Chandru Gajendran, Dimpy Ghosh, P Nagaraj, Subramanyam J. Tantry, Purushottam Dewang, Mahanandeesha S. Hallur, Kannan Murugan, Srinatha K. C, Damodara Kuntrapaku, M Dilipkumar, R Sharma, S Meghashree, Durga Prasanna Kumar, Suraj P. Ingle, Mohd Zainuddin, A B. Vinod, Sriram Rajagopal. Novel dual inhibitors of LSD1-HDAC6/8 for treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 11.