A selective and accurate liquid chromatography-tandem mass spectrometry method for the quantitation of the novel 5-HT4 receptor partial agonist SUVN-D4010 (Usmarapride) in human plasma and urine.

Liquid chromatography and the tandem mass spectrometry method to quantitate SUVN-D4010 (Usmarapride) in human plasma and urine have been developed and fully validated in compliance with regulatory guidelines. The sample preparation technique is simple and rapid consisting of acetonitrile precipitation followed by dilution of supernatant with a compatible solvent. Chromatographic separation was achieved on an X-Bridge C18 (2.1×50 mm, 3.5 µm) column using 0.1% v/v ammonium hydroxide and acetonitrile as mobile phase components, delivered at a flow rate of 0.75 mL min-1. Electrospray Ionization technique in positive mode was used for mass spectrometric detection. Selective reaction monitoring (SRM) transitions of m/z 384.2 → 352.1 for SUVN-D4010 and m/z 388.2 → 356.1 for SUVN-D4010-d4 were used for quantitation. Calibration curves for SUVN-D4010 were linear across the concentration range of 0.3-300 ng mL-1 in human plasma and 5.00-5000 ng mL-1 in human urine. The method generated results with acceptable accuracy (± 9.0%), precision (%CV, ≤8.7), and mean extraction recovery (≥93.4%) with negligible matrix effect in both plasma and urine. SUVN-D4010 was found to be stable in human plasma and urine at the defined storage conditions. The validated method was successfully applied to quantitate SUVN-D4010 in human plasma and urine from a clinical first-in-human study conducted to evaluate its safety, tolerability, and pharmacokinetics in healthy adults.

Evaluation of monoamine oxidase A and B type enzyme occupancy using non-radiolabelled tracers in rat brain.

Monoamine oxidase (MAO) enzymes, type A and B metabolise the amine neurotransmitters of the body. Selective inhibition of either enzyme is an approach for treating neurodegenerative and stress-induced disorders, and inhibition of an enzyme is proportional to the binding of the MAO inhibitor. Conventionally, the binding of test compounds to enzymes is assessed by radiolabelled ligands in ex vivo and in vivo occupancy assays. Regulatory restrictions and turnaround time are the limitations of the methods that use radiolabelled ligands. But the use of non-radiolabelled tracers and sensitive mass spectrometry (LC-MS/MS) based assays accelerated the determination of target occupancy in pre-clinical species. A report on use of non-radiolabelled ligand in in vivo MAO occupancy assay is not available. The objectives of the present study were to optimise non-radiolabelled harmine and deprenyl as selective tracers in MAO-A and MAO-B occupancy assays and evaluate MAO occupancy of test compounds in rat brain. Tracer optimisation resulted in a detectable, stable, and low ratio (<3.0) of tracer concentrations between any two brain tissues. In occupancy assay, tracer was intravenously administered (10 µg/kg, harmine or 60 µg/kg, L-deprenyl) after the treatment with test compound (clorgyline or tranylcypromine or pargyline or phenelzine or thioperamide). Specific brain tissues were isolated at a defined interval and tracer concentrations were quantified using LC-MS/MS method. Pre-treatment with MAO inhibitors resulted in a decrease (maximum, 80-85%) in harmine or an increase (maximum, 85-300%) in L-deprenyl concentrations. But we considered the change in tracer concentration, relative to the vehicle and positive control groups to calculate MAO occupancy. The observed selectivity and ratio of occupancies (ED50) of test compound towards MAO-A and MAO-B are comparable with the results from in vitro radiolabelled ligand-based inhibition assay. The results demonstrated the application of these non-radiolabelled tracers as suitable pre-clinical tools to determine MAO occupancy.

LC-MS/MS method for the quantification of SUVN-G3031, a novel H3 receptor inverse agonist for narcolepsy treatment.

Background: A LC-MS/MS method was validated for the quantification of SUVN-G3031, a novel H3 receptor inverse agonist in clinical development for the treatment of patients with narcolepsy, with and without cataplexy. Methodology: SUVN-G3031 was extracted from plasma following acetonitrile protein precipitation, separated by Ultra HPLC and quantified using positive ESI-MS/MS. Results: The method was linear across the range of 0.1-100 ng ml-1 in plasma. Results for intra and inter-day accuracy were from 99.8 to 104% and precision (%CV) was ≤10.6%. Conclusion: The method was applied to a first-in-human study in healthy volunteers. The method is precise, accurate and highly selective for the quantification of SUVN-G3031 in human plasma.

Development and Validation of a Higher-Throughput Cytochrome P450 Inhibition Assay with the Novel Cofactor-Supplemented Permeabilized Cryopreserved Human Hepatocytes (MetMax Human Hepatocytes).

Here, we report the application of a novel hepatocyte system, the cofactor-supplemented permeabilized cryopreserved human hepatocytes [MetMax human hepatocytes (MMHHs)] in a higher-throughput 384-well plate assay for the evaluation of cytochrome P450 (P450) inhibition. The assay was created to develop physiologically relevant P450 inhibition information, taking advantage of the complete organelle composition and their associated drug-metabolizing enzymes of the MMHH but with the ease of use of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and thaw and use without centrifugation and microscopic evaluation as required for intact hepatocytes. Nine key P450 isoforms for drug metabolism (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were evaluated using multiple isoform-selective inhibitors. Results with MMHH were found to be comparable to those obtained with intact cryopreserved human hepatocytes (CHHs). Isoform-selective drug-metabolizing enzyme pathways evaluated were phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine N-deethylation (CYP2C8), diclofenac 4-hydroxylation (CYP2C9), s-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation and testosterone 6ß-hydroxylation (CYP3A4). The Km values obtained with MMHHs were comparable with those reported in the literature for CHHs. Using substrate concentrations at or near Km values, the IC50 values for the standard inhibitors against the P450 activities were found to be comparable between MMHHs and CHHs, with 73% and 84% of values falling within 2-fold and 3-fold, respectively, from the line of unity. The results indicate that MMHHs can be an efficient experimental system for the evaluation of P450 inhibition in hepatocytes. SIGNIFICANCE STATEMENT: MetMax human hepatocytes (MMHHs) are cofactor-supplemented cryopreserved human hepatocytes with the complete drug-metabolizing enzyme pathways of the conventional hepatocytes but with the convenience of human liver microsomes, including storage at -80°C instead of in liquid nitrogen, and direct thaw and use without a need for centrifugation and microscopic examination. Here, we report the application of MMHH in a high-throughput assay in a 384-well plate format for the evaluation of cytochrome P450 (P450) inhibition. Our results show that data obtained with MMHH are similar to those with conventional hepatocytes, suggesting that the MMHH 384-well P450 inhibition assay can be used routinely for the evaluation of drug-drug interaction potential of new chemical entities in drug development.