Characterization of Equine CYP3A94, CYP3A95 and CYP3A97 Isoenzymes

Vimercati, Sara (2018). Characterization of Equine CYP3A94, CYP3A95 and CYP3A97 Isoenzymes (Unpublished). (Dissertation)

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Introduction: CYP3A is a human cytochrome P450 (CYPs) subfamily that catalyzes drug metabolism of more than 50% of on the marketed drugs. In equines, this subfamily is known to share more than 75% sequence identity with the human orthologue CYP3A4. Nevertheless, knowledge about drug metabolism in equines is still based on in vivo studies in contrast to humans, where drug metabolism is studied using CYP test batteries. Certain drugs might cause CYP induction or CYP inhibition in horses and may therefore lead to failure of therapy or toxicity, respectively. Furthermore, drug-drug interactions may be anticipated, as polypharmacy is common practice in equine medicine.
CYPs have been reported to be highly polymorphic and therefore, the drug metabolism is likely to be changed. In equines, little information about genetic variants is available, especially, the functional impact in drug metabolism of such variants. Hence, characterizing equine CYPs involved in drug metabolism and their genetic variants need to be studied to be able to predict unwanted effects and achieve a higher level of safety.

Aims: The goals of this thesis were to characterize three members of the equine CYP3A subfamily in comparison to the human orthologue CYP3A4 (I), to investigate possibledrug-drug interactions using ketamine and drugs frequently used in combination with ketamine in equine anesthesia (II), andtoidentify and study CYP3A genetic variants invitroand insilico(III).

Methods: First, three equine CYP3A subfamily members (CYP3A94, CYP3A95 and CYP3A97) were heterologously expressed in Sf9 cells. Microsomes containing these single CYPs were used to functionally characterize these CYPs using testosterone with HPLC (high performance liquid chromatography) and CE (capillary electrophoresis). Michaelis-Menten kinetic studies were performed. 3D-models of these CYP isoenzymes were built to predict possible changes in drug metabolism of these genetic variants. Based on the allele frequency, five of these variants were investigated in silico and in vitro.

Results: First, the basic characterization revealed that the main metabolite produced by the human CYP3A4, namely 6β-hydroxy-testosterone (6β-OH-TES), was only produced by CYP3A94, whereas 2-hydroxy-testosterone (2-OH-TES) was formed by all three equine CYPs. Androstenedione was also formed by all three equine CYPs. However, quantification was hampered as the Sf9 cells also produced this metabolite. Second, Michaelis-Menten kinetics 9 revealed a higher testosterone 2β-hydroxylation formation rate for CYP3A95 compared to CYP3A94. Similar to CYP3A4, ketoconazole inhibited the testosterone metabolism in the equine CYPs with the effect differing regarding the CYP isoenzyme. Third, ketamine N-demethylation was found in all three CYPs with intra-species differences. In contrast to diazepam and methadone, the α2-receptor agonist, medetomidine decreased S- and R-norketamine (S-NK, R-NK) formation. Five equine CYP3A genetic variants with a minor allele frequency (MAF) ranging from 0.2 to 0.4 were obtained. Based on the equine 3D-models built, CYP3A94:p.Asp217Asn and CYP3A95:p.His214Asp, were located on the F-α-helix, where the most prominent conformational changes were reported for CYP3A4. For CYP3A94:p.Asp217Asn, the 217Asn-allele resulted in decreased in testosterone 2β- and 6β-hydroxylation compared to the reference 217Asp-allele. For CYP3A95:p.His214Asp, a significant increase in the 2β-OH-TES formation rate was obtained for the 214His-allele.At the variant CYP3A95:p.Thr392Ser, the reference 392Thr-allele demonstrated a lower 2β-hydroxylation activity compared to the alternate allele.

Conclusions: The three members of the equine CYP3A subfamily investigated revealed intra-species differences and alterations in comparison to the human CYP3A4. Besides differences in the number of CYPs of the 3A subfamily, the activity differs between equines and humans, suggesting differences in the dosage regime. Although the in vitroapproach may not completely reflect the in vivo situation, single CYPs can be used to investigate the metabolism of drugs under development, drugs currently used in equine medicine and drug-drug interactions. The combination of in silico and in vitro analyses revealed that the investigated genetic variants differed in their activity when the reference variant was compared to the alternate variant. Hence, in silico modeling helps to predict differences in biotransformation in genetic variants and therefore it may identify variants for subsequent in vitro investigations. Knowledge about these differences might be used in the future when personalized medicine will be applied in equine medicine.

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