Prediction of pharmacokinetic clearance and potential Drug-Drug interactions for omeprazole in the horse using in vitro systems.

Horses are exposed to various kinds of medication, however, there are limited determinations of plasma clearance (CLp) for the drugs used due to the high cost of equine in vivo studies.Many of the CLp values generated come from the equine sports industry for determining drug plasma screening limits in the control of medications at the time of competition.The kinetics of omeprazole metabolism were investigated in freshly isolated and cryopreserved equine hepatocytes and hepatic microsomes (n = 3 horses).The Vmax, Km and intrinsic clearance (CLint) of omeprazole were determined via the substrate depletion method as well as Km values for the formation of three metabolites.The CLint values were extrapolated to in vivo hepatic plasma clearance (CLH) using the well stirred and parallel tube models.Clp for omeprazole was successfully predicted using freshly isolated or cryopreserved equine hepatocytes, while microsomes under-predicted.Equine microsomes were used to perform a drug-drug interaction (DDI) study between omeprazole and chloramphenicol. The average inhibitor constant Ki, assuming competitive inhibition, was 15.4 ± 5 µM.To the authors' knowledge, this is the first report showing the successful extrapolation of drug CLp in the horse using equine hepatocytes and the prediction of a DDI using microsomes.

The effects of aging on hepatic microsomal scaling factor and hepatocellularity number in the horse.

1. Scaling factor values for the in vitro-in vivo extrapolation of hepatic metabolic clearance for xenobiotics have not yet been determined in horses. Scaling factors were determined by comparing the total protein and or cytochrome (CYP) P450 content in microsomes and cryopreserved hepatocytes against the content in the liver. 2. Microsomal protein per gram of liver (MPPGL) and hepatocellularity number per gram of liver (HPGL) using CYP P450 content method ranged 41-73 mg/gram of liver (mean= 57 mg/gram of liver, n = 39) and 146-320 × 106 cells/g of liver (mean = 227× 106 cells/g of liver, n = 18), respectively and 156-352 × 106 cells/g of liver (mean = 232× 106 cells/g of liver) using total protein method. 3. A non-monotonic and inverse relationship between age and MPPGL and HPGL, respectively, was observed. Between one and 20 y of age, the liver cell size decreases as age increases. Subsequently, the cell size increases until the hepatocytes of the oldest horses approached the size found in the youngest horses. Hepatocyte density was inversely related to the size of the hepatocytes. 4. This study provides the first extensive and comprehensive data demonstrating the relationship between the size of hepatocytes and HPGL in any species.

Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies.

Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Clint) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two-step collagenase perfusion method, with an average cell yield of 2.47 ± 2.62 × 106 cells/g of perfused liver tissue and viability of 84.1 ± 2.62%. These cells were cryopreserved with William's medium E containing 10% fetal bovine serum with 10% DMSO. The viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. These purified cells were used to determine the in vitro Clint of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Clint compared to fresh cells for these three drugs as well as producing the same metabolites. This work paves the way for establishing a bank of cryopreserved equine hepatocytes that can be used for estimating pharmacokinetic parameters such as the hepatic metabolic in vivo clearance of a drug as well as producing horse-specific drug metabolites.