P82 THE USE OF BOVINE SERUM ALBUMIN IN PHENOTYPING EXPERIMENTS BY SUBSTRATE DEPLETION APPROACH TO DETERMINE fm, cyp: APPLICATION TO CYP2B6 SUBSTRATES WITH EMPHASIS ON BUPROPION

Raghava Choudary Palacharla , Drug Metabolism and Pharmacokinetics, Suven Life Sciences Ltd, Hyderabad, India
Venkatesham Uthukam , Drug Metabolism and Pharmacokinetics, Suven Life Sciences Ltd, Hyderabad, India
Arun Kumar Manoharan , Drug Metabolism and Pharmacokinetics, Suven Life Sciences Ltd, Hyderabad, India
Ilayaraja Kalaikadhiban , Suven Life Sciences Ltd., Hyderabad, India
Rajesh Kumar Boggavarapu , Bioanalytical Research, Suven Life Sciences Ltd, Hyderabad, India
Nagasurya Prakash Padala , Dmpk, Suven Life Sciences Ltd, Hyderabad, India
Ramakrishna Nirogi , Dmpk, Suven Life Sciences Ltd, Hyderabad, India
Determination of accurate fm,CYP values, also known as phenotyping, is necessary for the prediction of victim potential of new molecular entities during drug discovery and development. Metabolite formation and substrate depletion approaches are routinely employed in phenotyping studies. Authentic standards of metabolites are not available during discovery stage of the molecule and hence substrate depletion is the preferred approach for phenotyping. It is well established that the CYP1A2, CYP2B6, CYP2C9, CYP2C19 and CYP2E1 mediated activities are inhibited by fatty acids that are released during the course of the incubation1. The inhibition effects are abolished by using bovine serum albumin in metabolite formation experiments. The purpose of the present study was to evaluate the effect of BSA in phenotyping CYP2B6 substrates using substrate depletion approach. Test compounds were incubated with 0.5 mg.mL-1 of HLM (1.0 mg.mL-1 for bupropion and sertraline), in the presence and absence of specific chemical inhibitors, in the absence and presence of BSA (0.5% w/v) in phosphate buffer (100 mM, pH 7.4) in a final incubation volume of 200 uL at 37 °C. The final concentration of test compounds in the incubation mixture was 1 uM. In chemical inhibition studies, the specific chemical inhibitors, furafylline (50 µM), tranylcypromine (0.5 µM), Phenyl piperidinyl propane (PPP) (30 µM), montelukast (5 µM), sulfaphenazole (5 µM), N-benzyl nirvanol (4.5 µM), quinidine (1 µM), 4­-methyl pyrazole (10 µM), and azamulin (6 µM), were used against CYP1A2, 2A6, 2B6 , 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 enzymes respectively. All the incubations were initiated by the addition of NADPH and were performed in triplicates. An aliquot of the incubation mixture was removed and added to three volumes of ice-cold acetonitrile containing an internal standard. A significant difference was observed in fm, CYP2B6 values of test compounds in the absence and presence of BSA (0.5% w/v). Bupropion is significantly metabolized by CYP2C19 in the presence of BSA. The involvement of CYP2C19 (40%) and CYP2B (17%) in the metabolism of bupropion could explain the fold change in AUC of bupropion when co-administered with ticlopidine.

References

  1. Palacharla RC, Uthukam V, Manoharan A, Ponnamaneni RK, Padala NP, Boggavarapu RK, Bhyrapuneni G, Ajjala DR, Nirogi R. (2017) 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. Eur J Pharm Sci. 1(101):80-89