| dc.description.abstract | Cytochrome P450 (CYP) is an important enzyme superfamily, estimated to metabolize 70-90% of pharmaceuticals. Use of pharmaceuticals with at least 30% metabolism by CYP in humans require phenotyping to determine the CYP isoforms involved and the impact common polymorphisms may have. These same drugs are used off-label in canine and feline patients, yet knowledge regarding the CYP isoforms present in the liver and their enzymatic efficacy is unknown. Using human probes and substrates, differences in feline and canine CYP metabolism have been reported, and reflected in the limited pharmacokinetic (PK) studies of both species. The aim of this dissertation was to characterize canine and feline CYP transcriptome in canine and feline, compare and contrast the predicted hepatic clearance (CLhep) between the two species, and carry out reaction phenotyping of selected pharmaceuticals in canine recombinant CYP (rCYP). The first study determined the physiologic expression of CYP mRNA transcripts in whole blood, kidney, duodenum, liver and lung in healthy, adult male (n=4) and female (n=4) beagles via RNA-sequencing (RNA-seq). A total of 45 canine CYPs were identified, with liver, duodenum and lung expressing a high number of xenobiotic metabolizing CYPs, and expressing prominent endogenous metabolizing CYPs expression present in blood and kidney. In the second study, transcriptomes from the 99 Lives Cat Genome Sequencing Initiative databank combined with experimentally acquired whole transcriptome sequencing of healthy, adult male (n=2) and female (n=2) domestic felines was used to characterize CYP expression across a wide variety of tissues. A total of 20 tissues were analyzed and 47 CYP isoforms identified. Depending on the tissue, 9 to 33 CYP isoform transcripts were expressed. This study was the first to describe feline CYP transcriptome across a wide variety of tissues. Based on the differences in the transcriptome between the two species, the third study compared canine and feline CYP metabolism via in vitro liver microsomes. In canine liver microsomes, 3/30 substrates did not have quantifiable intrinsic clearance (CLint), while midazolam and amitriptyline CLint was too rapid for accurate determination. A predicted hepatic clearance (CLhep) was calculated for 29/30 substrates in feline microsomes. Overall, canine CLhep was faster compared to the feline, with fold differences ranging from 2 to 20 fold. A comparison between the well-stirred (CLhep,ws) and parallel tube model (CLhep,pt) indicates that the CLhep,pt model reports a slightly higher CLhep in both species. With evidence of the variation between the species, reaction phenotyping was applied to identify the CYP isoform pattern for targeted substrates. While the recombinant CYP (rCYP) isoforms are routinely used to screen novel human pharmaceuticals prior to approval, whether the canine isoforms metabolize these drugs in the same pattern is unknown. Utilizing an rCYP metabolic stability assay, 22 drugs used in veterinary medicine were phenotyped using canine rCYP1A1, 1A2, 2B6, 2C21, 2C41, 2D15, 3A12, and 3A26. Four of the 22 substrates required a two or four-fold rCYP dilution in order to achieve the three time points necessary to calculate the CLrCYP. The tricyclic antidepressants, amitriptyline and clomipramine, required a two or four-fold dilution of both rCYP2C41 and rCYP2D15. An isoform reported in only 11% of tested beagles, rCYP2C41, was involved in the metabolism of 9/22 substrates. The contribution of rCYP2B11 in canine drug metabolism altered the rCYP metabolism pattern for 8/22 substrates compared to the CYP metabolism pattern reported for humans. This body of research identified differences in the CYP transcriptome and CYP substrate depletion profile between the two species that suggests the need for species-specific pharmacokinetic studies as a basis for design of dosing regimens. In addition, canine CYP2B11 metabolism does not follow the reported human profile, highlighting the need for canine- and feline-specific reaction phenotyping. | en_US |
