Cloning of canine cytochrome P450 2E1 cDNA: identification and characterization of two variant alleles.

Cytochrome P450 (CYP) 2E1 is a toxicologically important enzyme that inactivates a number of drugs and xenobiotics and also bioactivates many xenobiotic substrates to their hepatotoxic or carcinogenic forms. Although cDNAs for the human, rodent, and rabbit forms of CYP2E1 have been isolated and studied extensively, there is an absence of information about canine CYP2E1, despite the fact that the dog is routinely used in drug safety studies. In this study, we isolated and sequenced a full-length CYP2E1 cDNA from a beagle liver cDNA library. The deduced canine CYP2E1 amino acid sequence exhibited 75 to 76% identity with rat, mouse, and rabbit CYP2E1 sequences, and 77% identity with human CYP2E1. Two populations of clones, differing at a single nucleotide, were isolated from the unamplified library. The T1453C base change results in a Tyr485His amino acid substitution, which is well beyond the heme binding region but is possibly part of a beta-sheet structure. An allele-specific polymerase chain reaction-based restriction enzyme test was developed for genotyping individual dogs from genomic DNA samples. One hundred mixed breed dogs were genotyped, and the frequencies of the Tyr485 and His485 alleles were found to be 0. 85 and 0.15, respectively. The canine Tyr485 and His485 alleles and human CYP2E1 were expressed in Escherichia coli cells, and catalytic activities of the proteins were assessed using the substrate chlorzoxazone. Although the two canine enzymes had similar catalytic activity; significant kinetic differences were seen between canine and human CYP2E1s.

Pharmacokinetic-pharmacodynamic relations of losartan and EXP3174 in a porcine animal model.

The pharmacokinetics of losartan and EXP3174, an active metabolite of losartan, were evaluated in the anesthetized pig after both a single intravenous dose (3 mg/kg) and during constant intravenous infusion. The pharmacodynamic activities of losartan and EXP3174 were determined during constant intravenous infusion as the degree of inhibition of angiotensin II-induced increase in the diastolic pressure. The systemic plasma clearance of losartan was 22.1 +/- 4.4 ml/min/kg (mean +/- SEM) and had an apparent volume of distribution at steady state of 0.56 +/- 0.16 L/kg after a 3-mg/kg intravenous dose. The elimination half-life of losartan was 40 +/- 6 min. Less than 2% of the intravenous losartan doses was estimated to be present as unconjugated EXP3174. The plasma clearance of EXP3174 was approximately 50% that of losartan, 11.8 +/- 1.5 ml/min/kg, and had a smaller steady-state apparent volume of distribution, 0.18 +/- 0.04 L/kg. The elimination half-life for EXP3174 was slightly longer than that of losartan (52 min). The time course of the pharmacodynamic effects of losartan and EXP3174 closely followed their respective plasma concentrations. The apparent dissociation constant of EXP3174 to the angiotensin II receptor was estimated, based on the total plasma concentrations, to be approximately 5 times lower than that for losartan.

The influence of cimetidine on the pharmacokinetics of the enantiomers of verapamil in the dog during multiple oral dosing.

The disposition of intravenously (0.5 mg/kg) and orally (5 mg/kg) administered verapamil was studied in six dogs after 3 days' pre-treatment with verapamil alone (5 mg/kg, every 8 h) and during concomitant oral administration of cimetidine (16 mg/kg, every 8 h). Racemic verapamil and norverapamil, an active metabolite of verapamil, were measured by fluorescence high performance liquid chromatography using an achiral phenyl column. The isolated racemic verapamil was rechromatographed on an Ultron-OVM chiral column, which separated the two verapamil enantiomers. Cimetidine co-administration significantly reduced the systemic clearance of racemic verapamil as well as that of its enantiomers by 25-29%. The clearance of racemic verapamil administered orally as well as that of its enantiomers was also reduced by 28% during cimetidine coadministration. The decrease in verapamil metabolism by cimetidine appeared to be non-stereoselective. On the other hand, cimetidine co-administration had no significant effect on the apparent volume of distribution of racemic verapamil and its enantiomers or the plasma protein binding or the blood to plasma concentration ratio of racemic verapamil. In addition, the ratio of the area under the plasma concentration-time curve for norverapamil to that of verapamil was unaffected by cimetidine co-administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the stereochemical composition of the major metabolites of verapamil in dog urine with enantioselective liquid chromatographic techniques.

The stereochemical composition of verapamil and seven of its basic-extractable metabolites, isolated from the urine of dogs administered oral racemic verapamil, was determined by HPLC, using an Ultron OVM (ovomucoid) column. One dog was given oral (R)-verapamil alone in order to discriminate the (R)- and (S)-enantiomers of the metabolites. Structure identification of the isolated verapamil metabolites was accomplished using a combination of HPLC-MS and FAB-MS-MS techniques. Six of the urinary verapamil metabolites, including verapamil, were predominantly of the (R)-configuration, whereas one of the metabolites was predominantly in the (S)-form. The remaining isolated metabolite was comprised of approximately equal amounts of the two forms.

Effects of diltiazem on the disposition and metabolism of the enantiomers of propranolol in the dog during multiple oral dosing.

The intravenous and oral dose kinetics and metabolism of the enantiomers of propranolol were investigated in five dogs during steady-state oral racemic propranolol dosing (5 mg/kg, every 8 hr for 3 days). These results were compared with those obtained during concomitant administration of oral diltiazem (2.5 mg/kg, every 8 hr for 3 days) in the same animals. The oral and intravenous propranolol test doses consisted of a pseudoracemic mixture of equal amounts of hexadeuterated-(R-(+))- and dideuterated-(S-(-))-propranolol. Propranolol metabolism in the urine was evaluated by coadministering 150 muCi of [4'-3H]racemic propranolol HCl, along with the deuterium-labeled compounds. Plasma concentrations of the deuterated enantiomers were measured by HPLC-thermospray MS, using undecadeuterated racemic propranolol as the internal standard. Diltiazem coadministration had no significant effects on either the systemic clearance, renal clearance, the apparent volume of distribution, or the elimination half-lives of either enantiomer. On the other hand, concomitant diltiazem treatment significantly reduced the oral clearance of S-(-)- and R-(+)-propranolol by 58 and 61%, respectively. These reductions resulted in an increase in their respective apparent steady-state oral availabilities of 129 and 106%. The S/R enantiomeric ratio of the oral availability of propranolol was not significantly changed from control. The urinary propranolol metabolites were isolated and purified by solvent extraction and HPLC and quantitated by radioactivity. Twelve metabolites, including propranolol, were isolated and quantitated in the urine. A significant reduction in the percentage of ring oxidation products and a significant increase in the percentage of naphthoxylactic acid and propranolol glucuronide excreted in the urine occurred in the diltiazem-treated animals. The S/R enantiomeric ratios of urinary excreted propranolol, propranolol glucuronide, 4'-hydroxypropranolol glucuronide, and its sulfate were not altered by diltiazem. These results suggest that the decreased oral clearances of the enantiomers of propranolol by diltiazem is caused by a selective decrease in the formation of ring-oxidized products.

Pharmacokinetics of the enantiomers of verapamil in the dog.

The intravenous (0.5 mg/kg) and oral (5 mg/kg) dose kinetics of verapamil were studied in 6 dogs during steady-state oral verapamil dosing (5 mg/kg every 8 h for 3 days). Racemic verapamil and norverapamil, a metabolite of verapamil, were quantitated in plasma by HPLC-fluorescence detection. The verapamil peaks eluting off the column were collected and rechromatographed on an Ultron-OVM column, which resolved the two verapamil enantiomers. After intravenous administration, the systemic clearance and apparent volume of distribution of (-)-(S)-verapamil were nearly twice that of the (+)-(R)-isomer. There was no difference in the elimination half-lives between the two isomers. After oral administration, the oral clearance of (-)-(S)-verapamil was 20 times that of the (+)-(R)-isomer. The apparent bioavailability of (+)-(R)-verapamil was over 14 times that of (-)-(S)-verapamil. The plasma protein binding of the (+)-(R)-isomer was slightly higher by 5% than (-)-(S)-verapamil; however, this effect was not enough to account for the difference between the apparent volume of distribution of the enantiomers, indicating that the tissue binding of (-)-(S)-verapamil was greater than that of the (+)-(R)-isomer. This data on the disposition of the enantiomers of verapamil in the dog is similar to that reported for man and demonstrates that the dog may be an appropriate animal model for man in future studies on the disposition of the enantiomers of verapamil.

Effects of propranolol on the disposition and negative dromotropic activity of diltiazem in the dog during multiple dosing.

The effects of propranolol coadministration on the disposition and negative dromotropic action of intravenous and oral diltiazem were studied in six dogs after 3 days pretreatment with diltiazem alone (2.5 mg/kg, every 8 hr) and with coadministration of oral propranolol (5 mg/kg, every 8 hr). Diltiazem and two of its metabolites, desacetyldiltiazem and demethyldiltiazem, were measured by HPLC. Propranolol coadministration had no significant effects on either the systemic clearance, the apparent volume of distribution, elimination half-life, or the blood binding of diltiazem. On the other hand, the oral clearance of diltiazem was significantly reduced by 51%, and its oral bioavailability was significantly increased by 48% during propranolol coadministration. The area under the plasma demethyldiltiazem concentration-time curve after oral diltiazem increased significantly during propranolol coadministration. This increase was in proportion to the increase in the plasma diltiazem area under the concentration-time curve, such that the ratio of the areas of demethyldiltiazem to that of diltiazem remained the same between control and propranolol coadministration. Propranolol coadministration increased the area under the negative dromotropic activity-time curve after both intravenous and oral diltiazem by 37 and 48%, respectively. Using a log-linear pharmacodynamic model to analyze the data, there were no significant effects on either the slope, y-intercept, or the estimated diltiazem concentration needed to increase the PR interval by 20% of either intravenous or oral diltiazem with propranolol coadministration. These data suggest that propranolol coadministration can result in an increase in the pharmacological activity of diltiazem due to a kinetic drug interaction by increasing its oral bioavailability.