P41 EVALUATION OF METHOXSALEN AS A PAN-CYP450 DIRECT AND TIME DEPENDENT INHIBITOR IN HUMAN LIVER MICROSOMES: COMPARISON WITH ABT AS A TOOL FOR IN VITRO PHENOTYPING STUDIES

Raghava Choudary Palacharla , Drug Metabolism and Pharmacokinetics, Suven Life Sciences Ltd, Hyderabad, India
Parusharamulu Molgara , 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
Ranjith kumar Ponnamaneni , Drug Metabolism and Pharmacokinetics, Suven Life Sciences Ltd, Hyderabad, India
Gopinadh Bhyrapuneni , Discovery Research, Suven Life Sciences Ltd, Hyderabad, India
Ramakrishna Nirogi , Dmpk, Suven Life Sciences Ltd, Hyderabad, India
Abstract

Methoxsalen (8-Methoxypsoralen, Ammoidin, 8-MOP, Xanthotoxin) is routinely used as CYP2A6 inhibitor and is also a non-selective inhibitor of other CYP isoforms1. The purpose of the study is to evaluate 8-methoxypsoralen as a direct and time dependent inhibitor of CYP450 enzymes and to determine the optimal concentration that can be used in reaction phenotyping studies. The inhibition potential of Methoxsalen is compared with Aminobenzotriazole (ABT) which is used as a pan-CYP inhibitor in phenotyping studies. Inhibition potential towards major CYP isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) was determined for both compounds using marker reactions in human liver microsomes. For measuring direct inhibition potential, compounds were incubated with HLM and probe substrates in the presence of NADPH followed by termination of reactions with acetonitrile. Time dependent inhibition potential was measured by incubating the compound with HLM and NADPH in the presence and absence of pre-incubation for 30min followed by dilution in to an activity assay containing probe substrate. IC50 values of 8-Methoxypsoralen are ranging from 1.4µM - 100µM and that of 1-aminobenzotriazole are 90µM - 3000µM. Percent time dependent inhibition of CYP450 activities are in the range of 50.0 - 90.0 and 27.0 - 98.0 for 8-methoxypsoralen and 1-aminobenzotriazole respectively for tested CYP isoforms. 8-Methoxypsoralen showed higher inhibition (80%) compared with 1-aminobenzotriazole (27%) towards CYP2C9 isoform. No inhibitor showed complete inhibition in time dependent inhibition mode against all P450 isoforms. In direct inhibition assay, 80-90% inhibition of all CYP mediated activities was observed at 330µM for 8-methoxypsoralen and at 10000µM for 1-aminobenzotriazole indicating high potency of 8-methoxypsoralen than 1-aminobenzotriazole. 8-Methoxypsoralen can be used as pan-CYP inhibitor in reaction phenotyping studies. 8-Methoxypsoralen is 50 fold more cost effective than ABT.

References

  1. Nirogi R, Palacharla RC, Uthukam V, Manoharan A, Srikakolapu SR, Kalaikadhiban I, Boggavarapu RK, Ponnamaneni RK, Ajjala DR, Bhyrapuneni G. Chemical inhibitors of CYP450 enzymes in liver microsomes: combining selectivity and unbound fractions to guide selection of appropriate concentration in phenotyping assays. Xenobiotica. 2015 Feb; 45(2):95-106.