Involvement of CYP2C8 and UGT1A9 in the metabolism of a novel gastroprokinetic agent, Z-338

Transcription

1 Involvement of CYP2C8 and UGT1A9 in the metabolism of a novel gastroprokinetic agent, Z-338 S. Furuta 1, E. Kamada 1, T. Sugimoto 1, Y. Kawabata 1, X. C. Wu 2, J. Skibbe 3, E. Usuki 3, A. Parkinson 3 and T. Kurimoto 1 1 Central Research Laboratories, ZERIA Pharmaceutical Co., Ltd., Saitama, Japan 2 Cerep Inc., WA, USA 3 XETEC, L.L.C., KS, USA

2 Introduction Z-338 is a novel gastroprokinetic agent, which is synthesized by Zeria Pharmaceutical Co., Ltd (Fig. 1). Z-338 is under development for the treatment of functional dyspepsia, and is now in clinical trials. The purpose of the study was to evaluate possible involvement of cytochrome P45 (CYP) enzyme(s) and UDP-glucuronosyltransferase (UGT) enzyme(s) responsible for metabolizing Z-338. Potential interactions of Z-338 with CYP enzymes were also investigated, and we compared its inhibitory potentials with cisapride and Z-338 analog,.

3 CYP2C8 (>>CYP3A4 or 1A1) Me S Me S Me Z-338 deisopropyl metabolite Me Me Me Me Z-338 S UGT1A9 (>>UGT1A8) Me Me CYP2C8 (>CYP3A4 or 1A1) Me Me Glu Me S S Z-338 glucuronide deisopropyl metabolite C 3 3 C CYP3A4 C 3 3 C F (C 2 ) 3 C 2 C 2 Cisapride Cl -dealkylcisapride Cl Fig.1 Metabolic Scheme of Z-338, and Cisapride

4 Method: In vitro metabolism study PLC methods that were validated for the quantification of Z-338, Z-338 deisopropyl metabolite and were employed. Z-338 and were incubated with a panel of recombinant human enzymes to investigate possible involvement of CYP and UGT enzymes responsible for the metabolism of Z-338 and. rcyp and rugt enzymes were obtained from Gentest Corporation (Woburn, MA) or Cypex Ltd, (Scotland, UK). nce initial rate conditions were established, Km and Vmax were determined for the dealkylation of Z-338 by human liver microsomes (LM), recombinant CYP2C8 and recombinant CYP3A4. LM was prepared by XenoTech (Lenexa, KS). An antibody inhibition experiment was performed to determine the relative contribution of CYP2C8 and CYP3A4 to the metabolism of Z-338 and by LM. Polyclonal antibodies and intact rabbit serum were obtained from osan Corporation (Yokohama, Japan).

5 Method: In vitro inhibition study The following marker substrate activities were used to evaluate the potential inhibitory effects on CYP isoforms: 7-ethoxyresorufin deethylation activity (CYP1A1/2), coumarin hydroxylation activity (CYP2A6), 7-benzyloxyresorufin debenzylation activity (CYP2B6), dibenzylfluorescein debenzylation activity (CYP2C8), tolbutamide hydroxylation activity (CYP2C9), (S)-mephenytoin hydroxylation activity (CYP2C19), bufuralol hydroxylation activity (CYP2D6), chlorzoxazone hydroxylation activity (CYP2E1), testosterone 6βhydroxylation activity and terfenadine hydroxylation activity (CYP3A4). LM was obtained from the International Institute for the Advancement of Medicine (Exton, P.A., USA), and rcyp enzymes were obtained from Gentest Corporation (Woburn, MA) or Cypex Ltd, (Scotland, UK). The Ki values were calculated from Dixon plots based on a competitive inhibition model.

6 mvolts 4 2 a b c 4 2 mvolts Z µm 6-min incubation rcyp2c8 (4 pmol P45/incubation) a: Z-338 deisopropyl metabolite b: I.S. () c: Z Minutes mvolts 4 2 a b c 1 µm 5-min incubation rcyp2c8 (4 pmol P45/incubation) Minutes a: deisopropyl metabolite b: c: I.S. (Z-338) Fig. 2 PLC Chromatograms of In Vitro Metabolism Study

7 CYP1A1 CYP1A2 CYP2A6 CYP2B6 CYP2C8 CYP2C9 CYP2C18 CYP2C19 CYP2D6 CYP2E1 CYP3A4 CYP3A5 CYP4A11 Z µm 6-min incubation 4 pmol P45/incubation (mean±sd, n=3) Metabolite formation (nm) CYP1A1 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP3A4 1 µm 5-min incubation 4 pmol P45/incubation (mean, n=2) Metabolite formation (nm) Fig. 3 Metabolite Formation from Z-338 and incubation with a Panel of Recombinant CYP Enzymes

8 UGTControl UGT1A1 UGT1A3 UGT1A4 UGT1A6 UGT1A8 UGT1A9 UGT1A1 UGT2B7 UGT2B15 Percent reduction of Z-338 by recombinant UGT isoforms The values show the percentage reduction from initial value (1nM)..25 mg microsomal protain/incubation UGT1A9: 3-incubation ther UGTs:12-min incubation Each value represents the mean±sd, (n=3) Metabolism(nM) Percent reduction from initial value v (nmol/min/mg protein of expressed microsome) 8 7 UGT1A Concentration ( M) v (nmol/min/mg protein of expressed microsome) UGT1A Concentration ( M) UGT1A8 Vmax :11.11 nmol/min/mg protain Km = µm Vmax/Km:.8 ml/min/mg protein UGT1A9 Vmax :15.24 nmol/min/mg protain Km = µm Vmax/Km:.353 ml/min/mg protein Fig. 4 Metabolism of Z-338 by uman Recombinant UGT Isoforms

9 Rate (pmol/min/mg protein) Rate (pmol/min/mg protein)/ [Z-338] (µm) Rate (pmol/min/pmol P45) Rate (pmol/min/pmol P45)/ [Z-338] (µm) Rate (pmol/min/pmol P45) Rate (pmol/min/pmol P45)/ [Z-338] (µm) uman Liver Microsomes rcyp2c8 rcyp3a4 Fig. 5 Eadie-ofstee Plots of Z-338 Metabolism Table 1 Kinetic Parameters of Z-338 by uman Liver Microsomes, Recombinant CYP2C8 and CYP3A4 enzymes Test System Km (µm) Vmax (pmol/ min/ mg protein or pmol P45) In vitro intrinsic clearance (µl/ min /mg protein or pmol P45) LM 318 ± ± rcyp2c8 152 ± ± rcyp3a ± ± Values are the mean ± standard error (n = 3). In vitro intrinsic clearance = Vmax/Km

10 Table 2 Inhibition of Z-338 and on the enzyme activities of rcyp isoforms Isoforms Inhibitors IC 5 ( mol/l) n rcyp2c8 (DBF substrate) Z-338 Quercetin > rcyp2c9 (7-MF substrate) Sulfaphenazole > rcyp3a4 (Testosterone substrate) Sulfaphenazole Effect of Z-338 on the CYP2C8 enzyme activity 1/v DBF.25 µm DBF.5 µm DBF 1 µm DBF 2 µm DBF 2.5 µm Z-338 ( M) Fig. 6 Inhibition of Z-338 on CYP2C8 activities by Dixon Plots Z-338: Ki = 121µM Quercetin : Ki = 5.8µM

11 Table 3 Inhibition constants (K i values, µm) on CYP isoforms of human liver microsomes by Z-338, cisapride and specific CYP inhibitors on several enzyme metabolic activities Z-338 Cisapride Inhibitor CYP1A1&1A2 7-Ethoxyresorufin deethylation activity (α-aphthoflavone ) 3.14 (Furafylline) CYP2A6 Coumarin hydroxylation activity CYP2B6 7-Benzyloxyresorufin debenzylation activity CYP2C9 Tolbutamide hydroxylation activity (8-Methoxypsoralen) ot inhibited ot inhibited (rphenadrine) (Sulfaphenazole) CYP2C19 S-mephenytoin hydroxylation activity >> (meprazole) 53.1 (Tranylcypromine) CYP2D6 Bufuralol hydroxylation activity (Quinidine) CYP2E1 Chlorzoxazone hydroxylation activity CYP3A (Anilline) 9.33 (Diethyldithiocarbamate) Testosterone 6β-hydroxylation activity >> (Ketoconazole) Terfenadine hydroxylation activity (Ketoconazole) The K i values were calculated from Dixon plots based on a competitive inhibition model.

12 Anti-CYP2C8 Anti-CYP3A4 Z-338 Table 4 Summary of the relative contribution of CYP2C8 and CYP3A4 to the metabolism of Z-338 by human liver microsomes Clearance Specific Content Adjusted Clearance Enzyme Relative (µl/min/pmol (pmol p-45/mg (µl/ min /mg System Contribution P45) protein) protein) rcyp2c % Anti-CYP2C8 Anti-CYP3A Inhibition (%) rcyp3a % r: recombinant Clearance = (Vmax Km) Specific content values are from Rodrigues, Adjusted Clearance (µl/mg protein/min) = Clearance (µl/min/pmol P45) x Specific Content (pmol/mg protein) Relative Contribution (%) = (Adjusted Clearance (µl/ min /mg protein) Total Adjusted Clearance (µl/min /mg protein)) x 1% Fig. 7 Percent inhibition for Z-338 and when incubated with human liver microsomes pretreated with polyclonal antibodies against CYP2C8 and CYP3A4

13 Conclusion Z-338 is predominantly metabolized to Z-338 glucuronide by UGT1A8 and UGT1A9 (probably UGT1A9>>UGT1A8). Z-338 is also catalyzed to deisopropyl metabolite by mainly CYP2C8. Z-338 showed little inhibition on CYP2C8 activities, which is significantly less than by its analog,. This may be based on the fact that its major metabolic pathway being glucuronidation rather than oxidation. Z-338 is considered unlikely to cause significant drugdrug interactions when coadministered with CYP substrates at clinically effective doses.