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.

The use of inactivated brain homogenate to determine the in vitro fraction unbound in brain for unstable compounds.

The use of IBH-5 decreased the kdeg values and increased the half-life of the compounds PNZ, TCP, Cpd I and Cpd II with kdeg values of 1.10 × 10-4 s- 1 (t1/2 = 115 min), 4 × 10-5 s-1 (t1/2 = 289 min), 4 × 10-5 s-1 (t1/2 = 289 min), and 3 × 10-5 s-1 (t1/2 = 385 min) respectively, compared to kdeg values of 1.25 × 10-2 s-1 (t1/2 = 0.9 min), 1.1 × 10-4 s-1 (t1/2 = 105 min), 1.0 × 10-3 s-1 (t1/2 = 11.5 min) and 4.5 × 10-4 s-1 (t1/2 = 26 min) in FBHThe use of lower temperature (4 °C) for the determination of fu,brain in this study is not successful due to the instability of the compounds during longer equilibration times required at lower temperatures.The fu,brain values for a set of 15 CNS drugs determined in FBH and IBH-5 using HT-dialysis were similar and are consistent with the literature values. The use of IBH-5 led to the determination of fu,brain for unstable compounds that could not be determined by other methods.The use of IBH-5 is an easy and convenient method to determine the fu,brain of unstable compounds in FBH during drug discovery and development.

Absorption, distribution, metabolism, excretion (ADME), drug-drug interaction potential and prediction of human pharmacokinetics of SUVN-G3031, a novel histamine 3 receptor (H3R) inverse agonist in clinical development for the treatment of narcolepsy.

SUVN-G3031 is a potent and selective inverse agonist of Histmine-3 (H3) receptor that is being investigated for the treatment of narcolepsy. SUVN-G3031 has high passive permeability, not a substrate for P-glycoprotein, has high plasma unbound fractions and was equally distributed between blood and plasma. Major routes of metabolism in vitro were cyclization (Metabolite A) in microsomes and dealkylation (Metabolite D) in hepatocytes. Intrinsic clearance in liver microsomes and hepatocytes was low as monitored by metabolite formation approach. CYP3A4 and MAO-A were the major enzymes involved in the formation of metabolite A and metabolite D respectively. The human hepatic clearance estimated by well-stirred model from hepatocytes was low (2.7 L.h -1) illustrating the importance of metabolite formation kinetics for prediction of human clearance for SUVN-G3031. Renal clearance in humans (9.7 L.h -1) was predicted from dog renal clearance and accounts for ~78% of the total clearance. SUVN-G3031 was neither an inhibitor nor inducer of the P450 enzymes at clinically relevant concentrations. SUVN-G3031 did not inhibit the major uptake transporters and was not a substrate for the uptake transporters. The potential of SUVN-G3031 as a victim and perpetrator of drug-drug interactions is remote. The predicted human pharmacokinetic parameters were consistent with those observed in the first-in-human study.

Assessment of sigma-1 receptor occupancy in mice with non-radiolabelled FTC-146 as a tracer.

The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) is advantageous in in-vivo receptor occupancy assays at pre-clinical drug developmental stages. Relatively, its application is effective in terms of high throughput, data reproducibility, sensitivity, and sample processing. In this perspective, we have evaluated the use of FTC-146 as a non-radiolabelled tracer to determine the sigma-1 receptor occupancy of test drugs in mice brain. Further, the brain and plasma exposures of test drug were determined at their corresponding occupancies. In this occupancy method, the optimized tracer treatment (sacrification) time after intravenous administration was 30 min. The tracer dose was 3 µg/kg and specific brain regions of interest were frontal cortex, pons and midbrain. Mice were pretreated orally with SA4503, fluspidine, haloperidol, and donepezil followed by tracer treatment. Among the test drugs, SA4503 was used as positive control group at its highest test dose (7 mg/kg, intraperitoneal). There was a dose-dependent decrease in brain regional FTC-146 binding in pretreated mice. From the occupancy curves of SA4503, fluspidine, haloperidol, and donepezil the effective dose (ED50) value ranges are 0.74-1.45, 0.09-0.11, 0.11-0.12, and 0.07-0.09 mg/kg, respectively. Their corresponding brain effective concentration (EC50) values are 74.3-132.5, 3.4-3.7, 122.5-139.5, and 8.8-11.0 ng/g and plasma EC50 values are 34.3-53.7, 0.08-0.10, 7.8-9.5, and 0.6-0.7 ng/mL. Brain regional distribution and binding inhibition upon pretreatment were comparable with data reported with labeled [18F]FTC-146. Drug exposures were simultaneously determined and correlated with sigma-1 occupancy from the same experiment. Wide category drugs can be assayed for sigma-1 receptor engagement and their correlation with exposures aid in clinical development.

Incurred sample reanalysis of fingolimod and fingolimod phosphate in blood: stability evaluation and application to a rat pharmacokinetic study.

BACKGROUND: Incurred sample reanalysis (ISR) is an in-study validation parameter, which reinforces that the validated bioanalytical methods are reproducible. ISR of whole blood samples is complex when the test compounds can interconvert, ex vivo. Fingolimod and fingolimod phosphate are highly distributed in the blood cellular components and undergo rapid interconversion, both in vivo and ex vivo. An LC-MS/MS method capable of simultaneous quantification of fingolimod and fingolimod phosphate with the controlled sample preparation procedure is essential. RESULTS: The ex vivo analyte interconversion in blood was controlled by lysing the blood cells. CONCLUSION: Lysis of blood samples not only controlled the interconversion but also rendered homogeneity to the sample, which led to acceptable ISR results from the study.

Inhibition of cytochrome P450 enzymes by saturated and unsaturated fatty acids in human liver microsomes, characterization of enzyme kinetics in the presence of bovine serum albumin (0.1 and 1.0% w/v) and in vitro - in vivo extrapolation of hepatic clearance.

The objective of the study was to determine the effect of fatty acids on CYP enzymes and the effect of BSA on intrinsic clearance of probe substrates. The inhibitory effect of thirteen fatty acids including saturated, mono-unsaturated and polyunsaturated fatty acids on CYP enzymes, kinetic parameters and intrinsic clearance values of nine CYP marker probe substrate reactions in the absence and presence of BSA (0.1 and 1.0% w/v) were characterized in human liver microsomes. The results demonstrate that most of the unsaturated fatty acids showed marked inhibition towards CYP2C8 mediated amodiaquine N-deethylation followed by inhibition of CYP2C9 and CYP2B6 mediated activities. The addition of 0.1% BSA in the incubation markedly improved the unbound intrinsic clearance values of probe substrates by reducing the Km values with little or no effect on maximal velocity. The addition of BSA (0.1 and 1.0% w/v) did not influence the unbound intrinsic clearance of marker reactions for CYP2A6, and CYP3A4 enzymes. The addition of 0.1% w/v BSA is sufficient to determine the intrinsic clearance of marker probe reactions by metabolite formation approach. The predicted hepatic clearance values for the substrates using the well-stirred model, in the presence of BSA (0.1% BSA), are comparable to the in vivo hepatic clearance values.