The guinea-pig expresses functional CYP2C and P-glycoprotein: further validation of its usefulness in drug biotransformation/transport studies.

BACKGROUND: The guinea-pig is an excellent animal model for studying cardiopulmonary physiology/pharmacology. Interestingly, it also possesses a number of drug-metabolizing enzymes found in humans, such as CYP1A, CYP2D and CYP3A. OBJECTIVE: To evaluate the hypothesis that the guinea-pig also expresses a functional CYP2C drug-metabolizing enzyme and the P-glycoprotein (P-gp) drug transporter in various tissues. METHODS: cDNAs encoding CYP2C and P-gp were obtained from guinea-pig liver or small intestine and sequenced. Western blotting was performed to confirm the expression of CYP2C and P-gp. The functional enzymatic activity of guinea-pig CYP2C was evaluated with microsomal preparations using diclofenac and tolbutamide as specific drug substrates in HPLC analyses. To further study both P-gp and CYP2C functional activities, the guinea-pig ABCB1/MDR1 and CYP2C genes were cloned. The recombinant plasmids were then transfected in HEK293 (human embryonic kidney) cells and either calcein-acetoxymethyl ester (AM) accumulation assays or 14,15-EET/DHET formation experiments were performed to evaluate either P-gp transport activity or CYP2C epoxygenase activity, respectively. The guinea-pig tissue distribution of P-gp was studied by Western blotting. RESULTS: Functional expression of CYP2C was demonstrated in guinea-pig liver microsomal preparations. CYP2C-mediated biotransformation of diclofenac and tolbutamide were shown. Expression of P-gp protein was detected in guinea-pig liver and small intestine. Functional activity of guinea-pig P-gp was demonstrated in ABCB1/MDR1-transfected cells. GP-CYP2C-transfected cells also showed functional epoxygenase activity. CONCLUSION: The guinea-pig expresses functional CYP2C and P-gp, thus suggesting its usefulness for further validating data obtained with other animal models in drug biotransformation/transport studies.

A novel KCNQ1 variant (L203P) associated with torsades de pointes-related syncope in a Steinert syndrome patient.

BACKGROUND: A 43-year-old woman suffering from Steinert syndrome was admitted after experiencing multiple episodes of torsades de pointes-related syncope. OBJECTIVES: To elucidate the pathophysiology of these arrhythmic events. METHODS AND RESULTS: We obtained DNA from the patient and sequenced the coding region of KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. A single nucleotide change was identified in the KCNQ1 gene at position 608 (T608C), resulting in a substitution from leucine to proline at position 203 (L203P). CHO cells were used to express either wild-type KCNQ1, wild-type KCNQ1+L203P KCNQ1 (50:50), or L203P KCNQ1, along with KCNE1 to recapitulate the slow cardiac delayed rectifier potassium current (I(Ks)). Patch-clamp experiments showed that the variant L203P causes a dominant negative effect on I(Ks). Coexpression of wild-type KCNQ1 and L203P KCNQ1 (50:50) caused a ~75% reduction in current amplitude when compared to wild-type KCNQ1 alone (131.40 ± 23.27 vs 567.25 ± 100.65 pA/pF, P < .001). Moreover, when compared with wild-type KCNQ1 alone, the coexpression of wild-type KCNQ1 and L203P KCNQ1 (50:50) caused a 7.5-mV positive shift of midpoints of activation (from 27.5 ± 2.4 to 35.1 ± 1.2 mV, P < .05). The wild-type KCNQ1 and L203P KCNQ1 (50:50) coexpression also caused alteration of I(Ks) kinetics. The activation kinetics of the L203P variant (50:50) were slowed compared with wild-type KCNQ1, while the deactivation kinetics of L203P (50:50) were accelerated compared with wild type, all these further contributing to the "loss-of-function" phenotype of I(Ks) associated with the variant L203P. CONCLUSION: Torsades de pointes and episodes of syncope are very likely to be due to the KCNQ1 variant L203P found in this patient.