Methoxsalen as an in vitro phenotyping tool in comparison with 1-aminobenzotriazole.

The objective is to evaluate methoxsalen as an in vitro phenotyping tool in comparison to ABT as a nonspecific inactivator of P450 mediated metabolism. The reversible inhibition of methoxsalen and ABT against the P450, FMO, AO, MAO-A and -B, enzymes were evaluated using standard marker probe reactions. The time-dependent inhibition of P450 enzymes was evaluated in human liver microsomes. CES1 activities were determined by monitoring the depletion of known substrate, the clopidogrel. The metabolism of P450 substrates in the presence and absence of methoxsalen or ABT was evaluated in human liver microsomes. Methoxsalen is a direct inhibitor and inhibited the activities (>90%) of all enzymes at a concentration of 300 µM except for CYP2C9. Methoxsalen is also a potent time-dependent inhibitor of all P450 enzymes except for CYP2C19 (moderate) at a concentration of 300 µM. Methoxsalen inhibited the metabolism of P450 substrates in the pre-incubation mode. ABT is a potent TDI of several P450 except for CYP2C19 (47%) and CYP2C9 (27%). The results indicate that methoxsalen is a potent pan P450 inhibitor than ABT and can be a better tool in distinguishing P450 mediated metabolism form non-P450 metabolism in human liver microsomes.

Quantitative in vitro phenotyping and prediction of drug interaction potential of CYP2B6 substrates as victims.

1. Determination of fm, CYP for a compound is critical to assess the potential risk of a drug candidate as a victim of DDI. Several compounds are identified as CYP2B6 substrates, but the fm, CYP2B6 values are not determined quantitatively. 2. Two methods of reaction phenotyping, the chemical inhibition method and metabolism in rCYP enzymes, were used to determine the relative contributions of the enzymes. Chemical inhibition method was also conducted in the presence of BSA (0.5% w/v). 3. The results confirm with the earlier studies concerning the identity of the CYP2B6 enzyme. The fm, CYP2B6 values for artemisinin, bupropion, clopidogrel, ketamine, selegiline, sertraline and ticlopidine were 0.24, 0.28, 0.15, 0.45, 0.46, 0.42 and 0.54, respectively, in HLM determined by chemical inhibition method. The fm, CYP2B6 values for artemisinin, bupropion, clopidogrel, ketamine, selegiline, sertraline and ticlopidine were 0.46, 0.17, 0.15, 0.60, 0.51, 0.66 and 0.77, respectively, in HLM determined by chemical inhibition method in the presence of BSA (0.5% w/v). 4. Bupropion metabolism is majorly mediated by CYP2C19 (0.41) with a minor contribution from CYP2B6 (0.16) in the presence of BSA. Ticlopidine is a time-dependent inhibitor of both CYP2B6 and CYP2C19 that can inhibit the bupropion metabolism by 50-60%.

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.

Evaluation of metabolism dependent inhibition of CYP2B6 mediated bupropion hydroxylation in human liver microsomes by monoamine oxidase inhibitors and prediction of potential as perpetrators of drug interaction.

The objective of the study was to evaluate the metabolism dependent inhibition of CYP2B6 catalyzed bupropion hydroxylation in human liver microsomes by monoamine oxidase (MAO) inhibitors and to predict the drug-drug interaction potential of monoamine oxidase inhibitors as perpetrators of drug interaction. Human liver microsomal CYP2B6 activities were investigated using bupropion hydroxylation as probe substrate marker. The results from single point time dependent inhibition and shift assays suggest that clorgyline, pargyline, phenelzine, and selegiline were metabolism based inhibitors of CYP2B6. In IC50 shift assays, clorgyline, pargyline, phenelzine and selegiline are metabolism based inhibitors of CYP2B6 with fold shit of 3.0-, 3.7-, 2.9-, and 11.4-fold respectively. The inactivation of clorgyline was characterized by KI value of 2.5 ± 0.3 and k(inact) value of 0.045 ± 0.001 min(-1). Phenelzine inactivated CYP2B6 with KI and k(inact) values of 44.9 ± 6.9 µM and 0.085 ± 0.003 min(-1) respectively. Inactivation of selegiline was characterized with KI and k(inact) values of 22.0 ± 3.3 and 0.074 ± 0.002 min(-1) respectively. The inactivation caused by these inhibitors was not reversed by dialysis indicating irreversible inhibition. Based on the mechanistic static model, selegiline showed an increase in the area under the curve (AUC) of efavirenz and bupropion by 1.01-fold. Phenelzine predicted to cause an increase in the AUC of efavirenz and bupropion by 9.4- and 2.4-fold respectively considering unbound hepatic inlet concentrations of phenelzine. In conclusion, the results from this study demonstrated that MAO inhibitors can inactivate human liver microsomal CYP2B6. The likelihood of drug interaction when selegiline co-administered with CYP2B6 substrates is remote. Caution is required while co-administering phenelzine with substrates that are exclusively metabolized by CYP2B6 enzyme and substrates that have narrow therapeutic index.

Chemical inhibitors of CYP450 enzymes in liver microsomes: combining selectivity and unbound fractions to guide selection of appropriate concentration in phenotyping assays.

1. Chemical inhibition is the widely used method in reaction phenotyping assays for estimation of specific enzyme contribution to a given metabolic pathway. The results from phenotyping assays depend on the selectivity of chemical inhibitor and the concentration of inhibitor used in the incubation. 2. The higher protein concentrations used in the in vitro phenotyping assays will impact the inhibitory potency of chemical inhibitors. The objective of the study is to evaluate comprehensively the selectivity of chemical inhibitors and to guide in selecting appropriate concentration of the chemical inhibitors to be used in the phenotyping assays based on unbound fractions. 3. Selectivity of chemical inhibitors against nine major CYP450 isoforms was determined in liver microsomes using standard probe substrates. The unbound fractions of the selective inhibitors were determined in human liver microsomes using high-throughput equilibrium dialysis. Combining unbound inhibitor concentrations that are required to inhibit the CYP450 activities by 90% and unbound fractions of the chemical inhibitors in liver microsomes appropriate total concentrations of the inhibitors to be used in the phenotyping assays were reported. 4. The findings suggest that non-specific binding of the chemical inhibitors need to be taken into account while selecting concentrations for phenotyping assays.

Identification of a suitable and selective inhibitor towards aldehyde oxidase catalyzed reactions.

1. Aldehyde oxidase (AO) is a liver cytosolic molybdoflavoprotein enzyme whose importance in drug metabolism is gaining in the recent. The objective of this work is to find a potent and selective inhibitor for AO activity using phthalazine oxidation as a marker reaction. 2. Among organic solvents tested, it was identified that methanol was not a suitable choice for AO activity even at concentrations less than 0.2% v/v. Acetonitrile and DMSO did not show any effect till 0.5% v/v but thereafter activites tend to decrease. 3. For selectivity, 23 compounds were selected and evaluated for their effects on AO and nine CYP450 enzymes. Among the tested compounds chlorpromazine, estradiol, hydralazine, quetiapine and raloxifene were selected based on their potency of inhibition towards AO activity. 4. Raloxifene was found to be a non-specific inhibitor of all major tested CYP450 enzymes and was excluded as a selective inhibitor for AO. Quetiapine also showed a degree of inhibition towards the major CYP450 tested. Hydralazine used as a specific inhibitor during the past for AO activity demonstrated a stimulation of AO activity at high and low concentrations respectively and the inhibition noted to be time dependent while inhibiting other enzymes like monoamine oxidase. 5. Estradiol showed no inhibition towards the tested CYP450 enzymes and thus proved to be a selective and specific inhibitor for AO activity with an uncompetitive mode of inhibition.

In vivo receptor occupancy assay of histamine H3 receptor antagonist in rats using non-radiolabeled tracer.

INTRODUCTION: Rapid and reliable preclinical receptor occupancy measurement at the target organ in relevant species is critical in accelerating the drug hunting process. The aim of this study was to develop in vivo receptor occupancy assay for histamine H3 receptors (H3R) using the non-radiolabeled GSK189254 as a tracer and to correlate the occupancy-exposure relationship for H3R antagonists in the rats. METHODS: In vivo tracer characterization studies like brain regional distribution, dose and time dependent uptake were carried out for GSK189254 in the male Wistar rats after intravenous administration. The tracer specificity was validated by pretreatment with H3 antagonists like ciproxifan, thioperamide, and GSK334429. The brain regional tracer levels and H3R antagonist concentrations in plasma and brain were quantified using liquid chromatography tandem mass spectrometry. Receptor occupancy was calculated using the ratio of total binding (striatum or frontal cortex) to the nonspecific binding (cerebellum) of the tracer in animals pretreated with H3R antagonist. RESULTS: High degree of selective distribution of GSK189254 was found in striatum, frontal cortex, and low level in the cerebellum. Regional distribution of GSK189254 in the rat brain was consistent to that of H3R distribution mapped using ³H or ¹¹C-GSK189254 in human, porcine, and rat. The calculated occupancy ED50 values in the frontal cortex were 0.14, 1.58, and 0.14 mg/kg for ciproxifan, thioperamide, and GSK334429, respectively. The plasma EC50 values (ng/mL) were found to be 2.33, 292.2, and 3.54 for ciproxifan, thioperamide and GSK334429, respectively. DISCUSSION: Results from mass spectroscopy based approach to determine H3R occupancy in rat brain is comparable with reported radiolabeled method by scintillation spectroscopy. In conclusion, non-radiolabeled GSK189254 was successfully employed as a tracer for assessing the H3R occupancy in rats and it can be used as a preclinical tool for evaluation of novel H3R ligands in the drug discovery.

Effect of dimethyl sulfoxide on in vitro cytochrome P4501A2 mediated phenacetin O-deethylation in human liver microsomes.

In this study, we report the effect of dimethyl sulfoxide (DMSO), acetonitrile, and methanol on the CYP1A2-mediated metabolism of phenacetin in human liver microsomes. Phenacetin O-deethylation is the preferred probe reaction for CYP1A2, in which the metabolite, acetaminophen, is quantified using liquid chromatography-tandem mass spectrometry. DMSO was found to inhibit CYP1A2-mediated phenacetin O-deethylation even at low concentrations (0.1%). Acetonitrile did not significantly change the phenacetin O-deethylation activity at concentrations up to 2%. There was no effect on the phenacetin O-deethylation when methanol was present at levels up to 2%. We found that the DMSO level should be kept lower than 0.05% because a concentration of 0.1% strongly affected the metabolism of phenacetin. These findings should be taken into consideration when designing in vitro metabolism studies, especially studies in which metabolism of the investigational compound needs to be evaluated, which would confound the results. The findings from this study indicate that methanol is the suitable solvent and has no significant effects on CYP1A2-mediated phenacetin O-deethylation.