PCR 500 bp CYP2C fragment (436 bp )

1. GUINEA PIG IS A GOOD ANIMAL MODEL FOR STUDYING CARDIOVASCULAR DRUG-DRUG INTERACTIONS Ibrahim Hasibu1,2 B.Sc., Sylvie Pilote 1 M.Sc., Dany Patoine1,M.Sc., Benoît Drolet 1, 2, B. Pharm., Ph.D., Chantale Simard 1, 2, B. Pharm., Ph.D.1. Centre de recherche de l’Institut universitaire de cardiologie et pneumologie de Québec, 2. Faculté de pharmacie, Université Laval introduction Methods (cont.) Abstract Results Context As opposed to other rodents, the guinea pig was shown to be an excellent model for studying drug-induced QT prolongation, as it expresses the same potassium channels as humans. Interestingly, guinea pig was also shown to express drug-metabolizing enzymes such as CYP1A, CYP2D and CYP3A subfamilies. However, CYP2C subfamily, which metabolizes drugs such as warfarin and angiotensin II receptor antagonists, has not been characterized. As cardiovascular deleterious side effects are often the result of drug interactions, the objective of our study was to demonstrate the presence of CYP2C subfamily and the pertinence of using this animal for cardiovascular drug-drug interaction studies. Methods Sequence alignments of few members of CYP2C subfamily DNA transcripts in different species (human, bovine, mouse, rat, and boar) have been performed. A part of sequence with 100% homology has been identified. A « BLAST » of these short sequences has been performed with the guinea pig total genomic sequence (www.ensembl.org). Then, coding regions (cDNA) of guinea pig CYP2C were determined. RNA extraction was performed with the Trizol® method and treated with DNase. The RT-PCR was performed with the iScriptcDNA synthesis kit (Bio-Rad Laboratories) using primers specific to the CYP2C enzymes. Western blot analyses with Abcamanti-CYP2C9 antibody staining was performed on guinea pig liver proteins extracts. Ex vivo standard incubations were performed using hepatic microsomes from guinea pig and a CYP2C9 probe drug (diclofenac) was used. Concentration of diclofenac and its metabolites were analyzed by an HPLC method using ultraviolet detection (absorbance at 280 nm). The mobile phase is 5% acetonitrile, 15% methanol in 30 mM phosphate buffer changing to 100% methanol over 30 minutes and at a flow rate of 1.0 mL/min. Results Specifically designed primers for guinea pig CYP2C allowed us to obtain DNA amplification. This is strongly suggesting the presence of CYP2C in this animal. Western blot allowed us to identify a 55 kDa DNA band, highly suggesting CYP2C signatures. Experiments performed with incubations of hepatic microsomes obtained from animals revealed formation of diclofenac specific CYP2C metabolites, suggesting that guinea pig has active CYP2C drug-metabolizing enzymes. Conclusion We have shown that the guinea pig expresses hepatic CYP2C subfamily of drug-metabolizing enzymes. Moreover, we have shown that guinea pig hepatic microsomes generated a specific CYP2C-formed metabolite of diclofenac, a probe substrate of CYP2C9. When added to the confirmed expression of CYP1A, CYP2D and CYP3A subfamilies and considering the number of drugs metabolized by these hepatic enzymes and the well established use of this animal in cardiac electrophysiology studies, this is reinforcing the pertinence of using the guinea pig as model for studying cardiovascular drug-drug interactions. • PCR • 500 bp • CYP2C fragment (436 bp) • The sequence of this fragment presents 100% homology with the predicted guinea pig CYP2C. • Western blot • Band approximativeweight: ~ 55 kDa • HPLC Analyses • Fig. 2. Chromatograms show diclofenac metabolites produced by CYP2C9 (A), CYP3A4 (B) and guinea pig hepatic microsomes (C) • Retention times for 4’-OH diclofenac, 5-OH diclofenac and diclofenac are 17.1, 18.8 and 22 min respectively. • We have shown that the guinea pig expresses hepatic CYP2C subfamily of drug-metabolizing enzymes. Moreover, we have shown that guinea pig hepatic microsomes generated a specific CYP2C metabolite of diclofenac, a probe substrate of CYP2C9. When added to the confirmed expression of CYP1A, CYP2D and CYP3A subfamilies and considering the number of drugs metabolized by these hepatic enzymes and the well established use of this animal in cardiac electrophysiological studies, this is reinforcing the pertinence of using the guinea pig as model for studying cardiovascular drug-drug interactions. • Danielson, P.B. (2002) Curr Drug Metab, 3(6), 561-597. • Hanatani, T., Fukuda ,T., Ikeda, M., Imaoka, S., Hiroi, T., Funae, Y., Azuma, J. (2001) Pharmacogenomics J, 1(4), 288-292. • Tang, W. (2003) Curr Drug Metab, 4(4), 319-329 • Gallelli, L., Ferraro, M., Spagnuolo, V., Rende, P., Mauro, G.F., De Sarro, G. (2009) Drug metabol drug Interact, 24(1), 83-87. • Cytochromes P450 (CYP) constitute a superfamily of genes coding for enzymes metabolizing a large number of drugs and endogenous substrates such as steroids, eicosanoids, vitamins etc. These enzymes play a crucial role in detoxifying and protecting the organism. • The human genome encodes at least 57 CYP450s organized into 18 families and 43 subfamilies. The most significant in terms of drug metabolism are the members of CYP1, CYP2 and CYP3 families. • The human CYP2C subfamily includes four enzymes: CYP2C8, CYP2C9, CYP2C18 and CYP2C19. They are all over 80% homologous and their catalytic specificity overlaps. • This subfamily is generally accepted as the second most pharmacologically important group of CYP450s in human, after the CYP3A subfamily as it represents about 20% of CYPs in the liver and is involved in the metabolism of about 25% of all drugs. • Its members metabolize some clinically important cardiovascular drugs such as warfarin, angiotensin II receptor antagonists (Losartan, Candesartan, Irbesartan) and some HMG-CoAreductase inhibitors (Fluvastatin, Rosuvastatin). • However, the presence of this subfamily in the guinea pig has not been demonstrated. • To demonstrate the presence of the CYP2C subfamily in the guinea pig • To demonstrate the usefulness of the guinea pig in cardiovascular drug-drug interaction studies. • Sequence alignments of few members of CYP2C subfamily DNA transcripts in different species (human, bovine, mouse, rat, and boar) have been performed. A part of sequence with 100% homology has been identified. • A « BLAST » of these short sequences has been performed with the guinea pig total genomic sequence (www.ensembl.org). Then, coding regions (cDNA) of guinea pig predicted CYP2C were determined. • Male Hartley guinea pigs (weight 300-350 g; Charles River Laboratories, Montreal, Quebec, Canada). • Following cervical dislocation of the animals, liver was collected, washed in cold PBS and put in liquid nitrogen. • Microsomes were collected according to the Von Moltke method; 10 g of tissue has been homogenized and after several washes and centrifugations, the microsomal portion of the tissue was collected. • Protein concentration was determined by the 660 nm Protein assay kit (Thermo Scientific). • RNA extraction was performed with the Trizol® method (Invitrogen) and treated with Dnase (Ambion). • The RT-PCR was performed with the iScriptcDNA synthesis kit (Bio-Rad Laboratories). • PCR • The amplicon was sequenced at the CHUL Research Center sequencing service and the result was aligned with the guinea pig CYP2C hypothetical sequence using Vector NTI software (Invitrogen). • Western blot • Liver microsomal proteins (0.3 μg) were loaded on a 10% acrylamide gel, then blotted on a nitrocellulose membrane. Immunochemical staining with rabbit anti-CYP2C9 antibody (Abcam) was performed as described in the table 4. • Ex vivo incubations • Standard incubations were performed using either guinea pig hepatic microsomes (0.8 mg of protein) or purified CYP2C9 and CYP3A4 (Gentest) in the presence of diclofenac (100 μM), a CYP2C9 probe, during 60 min at 37 °C. • Diclofenac and its metabolites were analyzed by HPLC, according to Gentest method with few modifications, on a Prominence System (SHIMADZU) with an Ultrasphere ODS 5 μ 250 mm x 4,6 mm column (Beckmann) and a μ-Bondapak C18 precolumn (Waters). • The mobile phase consists of 5% acetonitrile, 15% methanol in 30 mM phosphate buffer changing to 100% methanol over 30 minutes at a flow rate of 1.0 mL/min. • Ultraviolet detection was performed at 280 nm CYP2C A: CYP2C9 B: CYP3A4 Diclofenac Diclofenac 4’-Hydroxydiclofenac 5-Hydroxydiclofenac C: Guinea pig hepatic microsomes objectives Diclofenac 5-Hydroxydiclofenac 4’-Hydroxydiclofenac background methods • In clinical settings, drug-drug interactions are often responsible for cardiovascular deleterious side effects such as prolongation of QT interval on the ECG, which may lead to a malignant ventricular arrhythmia called torsades de pointes. • As opposed to other rodents, the guinea pig (caviaporcellus) was shown to be an excellent animal model for studying drug-induced QT prolongation, as it expresses the same potassium channels as in humans, namely IKr and IKs, which are responsible for the repolarization process of the heart. • Interestingly, the guinea pig was also shown to express drug-metabolizing enzymes such as CYP1A, CYP2D and CYP3A subfamilies. • In a previous study, we have also shown that this animal expresses CYP2E subfamily. Conclusion Table 1. Guinea pig CYP450s in Uniprot website Figure 1. Proposed mechanism for diclofenacmetabolism (3) references acknowledgements Fonds d’enseignement et de recherche de la Faculté de pharmacie