ZY12201, A Potent TGR5 Agonist: Identification of a Novel Pan CYP450 Inhibitor Tool Compound for In-Vitro Assessment.

BACKGROUND: Identification of clinical drug-drug interaction (DDI) risk is an important aspect of drug discovery and development owing to poly-pharmacy in present-day clinical therapy. Drug metabolizing enzymes (DME) plays important role in the efficacy and safety of drug candidates. Hence evaluation of a New Chemical Entity (NCE) as a victim or perpetrator is very crucial for DDI risk mitigation. ZY12201 (2-((2-(4-(1H-imidazol-1-yl) phenoxy) ethyl) thio)-5-(2-(3, 4- dimethoxy phenyl) propane-2-yl)-1-(4-fluorophenyl)-1H-imidazole) is a novel and potent Takeda-G-protein-receptor-5 (TGR-5) agonist. ZY12201 was evaluated in-vitro to investigate the DDI liabilities. OBJECTIVE: The key objective was to evaluate the CYP inhibition potential of ZY12201 for an opportunity to use it as a tool compound for pan CYP inhibition activities. METHOD: In-vitro drug metabolizing enzymes (DME) inhibition potential of ZY12201 was evaluated against major CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4/5), aldehyde oxidase (AO), monoamine oxidase (MAO), and flavin-containing monooxygenase (FMO in human liver cytosol/mitochondrial preparation/ microsomes using probe substrates and Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) method. RESULTS: The study conducted on ZY12201 at 100 µM ZY12201 was found to reduce the metabolism of vanillin (AO probe substrate), tryptamine (MAO probe substrate), and benzydamine (FMO probe substrate) by 49.2%, 14.7%, and 34.9%, respectively. ZY12201 Ki values were 0.38, 0.25, 0.07, 0.01, 0.06, 0.02, 7.13, 0.03 and 0.003 µM for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5 (substrate: testosterone) and CYP3A4/5 (substrate: midazolam), respectively. Time-dependant CYP inhibition potential of ZY12201 was assessed against CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 and no apparent IC50 shift was observed. CONCLUSIONS: ZY12201, at 100 µM concentration showed low inhibition potential of AO, MAO, and FMO. ZY12201 was found as a potent inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 while moderately inhibits to CYP2E1. Inhibition of CYP1A2, CYP2B6, CYP2C19, and CYP2E1 by ZY12201 was competitive, while inhibition of CYP2C8, CYP2C9, CYP2D6, and CYP3A4/5 was of mixed-mode. ZY12201 is a non-time-dependent inhibitor of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5. In summary, the reported Ki values unequivocally support that ZY12201 has a high potential to inhibit all major CYP isoforms. ZY12201 can be effectively used as a tool compound for in-vitro evaluation of CYP-based metabolic contribution to total drug clearance in the lead optimization stage of Drug Discovery Research.

Simultaneous determination of sacubitrilat and fimasartan in rat plasma by a triple quad liquid chromatography-tandem mass spectrometry method utilizing electrospray ionization in positive mode.

An LC-tandem mass spectrometry method was developed and validated for the simultaneous quantitation of fimasartan and sacubitrilat using positive ion mode. The protein precipitation method was employed for the extraction of fimasartan, sacubitrilat and alprazolam (internal standard) from rat heparinized plasma. Baseline separation of the analytes was accomplished using an ACE-5, C18 (4.6 × 50 mm) column and gradient elution of mobile phase A (5 mm ammonium formate and 0.1% formic acid in purified water) and B (acetonitrile:methanol, 80:20; v/v). All peaks of interest were eluted within a 5-min runtime. The quantitation was achieved in the selected reaction monitoring mode. The developed method was validated as per US Food and Drug Administration guidelines and met the pre-defined acceptance criteria. The method showed linearity from 5 to 10,000 ng/mL. The accuracy/precision of intra- and inter-batch assays was 96.64%/2.05% to 109.17%/13.70% and 100.74%/3.76% to 106.39%/9.75% for fimasartan and 100.02%/1.49% to 113.80%/9.38% and 100.75%/2.31% to 108.40%/7.74% for sacubitrilat, respectively, in rat plasma. Fimasartan and sacubitrilat remained stable in rat plasma at different experimental conditions up to 21 days. The developed method was sensitive, selective and applied successfully to monitor plasma concentrations of fimasartan and sacubitrilat in an oral rat pharmacokinetic study.

Relevance of preclinical rodent pharmacokinetics in the selection of a companion antibiotic for combining with beta-lactamase inhibitor.

Recent approvals of beta-lactamase inhibitor (BLI) drug in combination with cephalosporins/penems have provided the right impetus for novel BLIs. One important research question, hitherto not addressed, is pertaining to the relevance of preclinical pharmacokinetics for pairing the antibiotic with existing/novel BLI.Two BLI combination drugs: (a) approved (i.e. ceftazidime/avibactam); (b) clinical development (i.e. cefepime/zidebactam) were explored to provide insights to address the research question.Individual intravenous dosing of ceftazidime, avibactam, cefepime and zidebactam was done at 1 mg/kg by intravenous route in Balb/c mice and Wistar rats. Serial blood samples were collected and analysed by LC-MS/MS method.Examination of the ratios of pharmacokinetic parameters (CL, VSS and T1/2) for individual drugs in combinations (for instance, CL (ceftazidime)/CL (avibactam); CL (cefepime)/CL (zidebactam)) suggested that the pharmacokinetic data gathered in rats were generally within 0.5- to 2-fold; but mouse data revealed larger disparity for VSS (0.11- to 8.25-fold) or CL (0.49- to 4.03-fold).The observed ratio for CL/VSS observed in rats agreed with corresponding human ratios for the pairwise comparison of the individual drugs in the combinations.Retrospectively, current pharmacokinetic findings suggest rat pharmacokinetic data may aid the combination of BLI with an appropriate antibiotic.

Lack of inhibition of CYP2C8 by saroglitazar magnesium: In vivo assessment using montelukast, rosiglitazone, pioglitazone, repaglinide and paclitaxel as victim drugs in Wistar rats.

Saroglitazar, a PPAR αÒ® agonist, is currently undergoing global development for the treatment of NASH and other indications. Saroglitazar showed CYP2C8 inhibition in human liver microsomes (IC50: 2.9 µM). The aim was to carry out drug-drug interaction (DDI) studies in Wistar rats using saroglitazar (perpetrator drug) with five CYP2C8 substrates. Also, the in vitro CYP2C8 inhibitory potential of saroglitazar in rat liver microsomes (RLM) was evaluated to justify use of preclinical model. The oral pharmacokinetics of various CYP2C8 substrates; montelukast, rosiglitazone, pioglitazone, repaglinide and intravenous pharmacokinetics of paclitaxel was assessed in the presence/absence of oral saroglitazar (4 mg/kg) in Wistar rats. A separate study was performed to assess the oral pharmacokinetics of saroglitazar. Serial blood samples were collected from all studies and the harvested plasma were stored frozen until bioanalysis. LC-MS/MS was used for the analysis of various analytes; concentration data was subjected to noncompartmental pharmacokinetic analysis. Statistical tests (unpaired t-test) were employed to judge the level of DDI. Generally, the pharmacokinetics of CYP2C8 substrates was not affected by the concomitant intake of saroglitazar as judged by the overall exposure (AUC0-last and AUC0-inf) and elimination half-life. The CYP2C8 IC50 of 4.5 µM in RLM for saroglitazar, supported the use of rats for this DDI study. In conclusion, pharmacokinetic data of diverse CYP2C8 substrates suggested that coadministration of saroglitazar did not cause clinically relevant DDI.

Incurred sample reanalysis in drug discovery bioanalysis.

Bioanalysis plays a key role during the drug discovery process to generate the pharmacokinetic data to facilitate unbiased evaluation of leads, optimized leads and drug candidates. Such pharmacokinetic data are used to enable key decisions in the drug discovery process. The aim of the work is to put forward a new strategy of performing the incurred sample reanalysis for select small molecule novel chemical entities at different stages of drug discovery prior to candidate selection. Three discovery programs representing hits, leads and optimized lead candidates were selected for the incurred sample reanalysis (ISR) analysis. From each discovery program, two novel chemical entities were selected for the ISR analysis. The time points considered for ISR generally varied among the programs; however, samples coinciding with drug absorption, distribution and elimination were considered in the ISR assessment. With the exception of a single ISR value that gave a high deviation (about 63%), the observed ISR values supported the discovery bioanalytical assays. While the individual bioanalytical laboratory can draw an algorithm for selecting novel chemical entities and fixing the acceptance criteria for the ISR data, it is proposed that the percentage difference between ISR vs. original concentration for 67% of the repeat samples is contained within ±30% for discovery bioanalysis.

In Vitro Drug-Drug Interaction Potential of Sulfoxide and/or Sulfone Metabolites of Albendazole, Triclabendazole , Aldicarb, Methiocarb, Montelukast and Ziprasidone.

BACKGROUND: The use of polypharmacy in the present day clinical therapy has made the identification of clinical drug-drug interaction risk an important aspect of drug development process. Although many drugs can be metabolized to sulfoxide and/or sulfone metabolites, seldom is known on the CYP inhibition potential and/or the metabolic fate for such metabolites. OBJECTIVE: The key objectives were: a) to evaluate the in vitro CYP inhibition potential of selected parent drugs with sulfoxide/sulfone metabolites; b) to assess the in vitro metabolic fate of the same panel of parent drugs and metabolites. METHODS: In vitro drug-drug interaction potential of test compounds was investigated in two stages; 1) assessment of CYP450 inhibition potential of test compounds using human liver microsomes (HLM); and 2) assessment of test compounds as substrate of Phase I enzymes; including CYP450, FMO, AO and MAO using HLM, recombinant human CYP enzymes (rhCYP), Human Liver Cytosol (HLC) and Human Liver Mitochondrial (HLMit). All samples were analysed by LC-MS-MS method. RESULTS: CYP1A2 was inhibited by methiocarb, triclabendazole, triclabendazole sulfoxide, and ziprasidone sulfone with IC50 of 0.71 µM, 1.07 µM, 4.19 µM, and 17.14 µM, respectively. CYP2C8 was inhibited by montelukast, montelukast sulfoxide, montelukast sulfone, tribendazole, triclabendazole sulfoxide, and triclabendazole sulfone with IC50 of 0.08 µM, 0.05 µM, 0.02 µM, 3.31 µM, 8.95 µM, and 1.05 µM, respectively. CYP2C9 was inhibited by triclabendazole, triclabendazole sulfoxide, triclabendazole sulfone, montelukast, montelukast sulfoxide and montelukast sulfone with IC50 of 1.17 µM, 1.95 µM, 0.69 µM, 1.34 µM, 3.61 µM and 2.15 µM, respectively. CYP2C19 was inhibited by triclabendazole and triclabendazole sulfoxide with IC50 of 0.25 and 0.22, respectively. CYP3A4 was inhibited by montelukast sulfoxide and triclabendazole with IC50 of 9.33 and 15.11, respectively. Amongst the studied sulfoxide/sulfone substrates, the propensity of involvement of CY2C9 and CYP3A4 enzyme was high (approximately 56% of total) in the metabolic fate experiments. CONCLUSION: Based on the findings, a proper risk assessment strategy needs to be factored (i.e., perpetrator and/or victim drug) to overcome any imminent risk of potential clinical drug-drug interaction when sulfoxide/sulfone metabolite(s) generating drugs are coadministered in therapy.