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Polymorphisms in genes encoding CYP enzymes could explain adverse drug effects or therapeutic failure in canine patients.
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A premature stop codon mutation in CYP1A2 is commonly found in certain dog breeds, including Beagle and Irish wolfhound.
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Although the CYP1A2 premature stop codon has shown large effects on the pharmacokinetics of some experimental compounds, effects on commonly used clinical drugs is currently unknown.
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Polymorphisms also exist in genes encoding canine CYP2C41, CYP2E1, CYP2D15, and CYP3A12 that have the potential to impact the metabolism of a large number of different drugs.
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Anesthetic drug hypersensitivity in Greyhounds may be the result of a genetic variant affecting canine CYP2B11 expression or function.
Introduction
The cytochrome P-450 (CYP) drug-metabolizing enzymes are critical to the efficient elimination of many drugs used in clinical practice. Unfortunately in humans, and probably in all species of veterinary importance, there is considerable interindividual variability in the activity of these enzymes.
Consequently, for a given drug dosage the effect can range from undetectable or suboptimal (with high enzyme activity, high drug clearance, and low plasma levels) to excessive or toxic (with low enzyme activity, low drug clearance, and high plasma levels). Causes of this variability can include concurrent exposure to CYP enzyme inhibitors or inducers in the diet or from coadministered medications (previously reviewed
). Genetic variation is also a well-established cause of CYP activity variability in humans, and current evidence suggests that it may be equally important in veterinary species, including dogs.
Consequently, clinical assays for CYP gene variants that significantly impact drug disposition could be a useful tool to enable rational drug selection and dosage for the individual patient. This article reviews the current state of knowledge regarding the dog CYPs focusing on potentially clinically important genetic variants that could influence drug efficacy and toxicity.
Dog-human CYP similarities and differences
Much of the available published data on the CYPs so far concern the human CYPs. Indeed, the US Food and Drug Administration requires detailed label information regarding the involvement of specific human CYPs in the metabolism of all newly approved drugs intended for use in humans. Although much of the information can be applied in a general fashion to the dog CYPs, it is becoming increasingly apparent that there are important differences in the metabolism of drugs by human and dog CYPs, much of which have yet to be determined. Specific examples of some of the known similarities and differences are discussed next.
CYP Substrate Specificity
Table 1 lists common CYP drug substrates in humans compared with dogs. The CYPs are named according to gene sequence similarity and grouped according to family (number), subfamily (letter), and unique gene product (number), as in the canine CYP2B11 gene (family, 2; subfamily, B; 11th gene identified). Because of significant species differences in gene sequence of these enzymes, each species tends to have their unique CYP names, although orthologs (genes derived from the same ancestral gene that diverged after speciation) are found in most species. For example, CYP2B11 is considered to be the canine ortholog of human CYP2B6.
However, significant species differences exist. For example, midazolam is metabolized exclusively by human CYP3A4 and CYP3A5 (but not by human CYP2B6), whereas dog CYP2B11 (and not CYP3A12) primarily metabolizes midazolam.
whereas only human CYP1A2 (and not human CYP2A6) metabolizes this drug. Although most dog CYPs have unique names, three of the drug-metabolizing CYPs (CYP1A1, CYP1A2, and CYP2E1) have identical names to those found in other mammalian species, in part because they have relatively conserved gene sequences between species, and in part because their naming preceded the convention to give unique names to the drug-metabolizing CYPs in different species.
Table 1Examples of drugs that are known substrates for specific human
Data from P450 Drug Interaction Table. Indiana University Department of Medicine. 2009. Available at: http://medicine.iupui.edu/clinpharm/ddis/table.aspx. Accessed April 20, 2013.
Data from Martinez MN, Antonovic L, Court M, et al. Challenges in exploring the cytochrome P450 system as a source of variation in canine drug pharmacokinetics. L Drug Metab Rev 2013;45(2):218–30.
b Data from Martinez MN, Antonovic L, Court M, et al. Challenges in exploring the cytochrome P450 system as a source of variation in canine drug pharmacokinetics. L Drug Metab Rev 2013;45(2):218–30.
Apart from differences in catalytic properties between dog and human CYPs, these enzymes also differ in the relative amount of each family and subfamily between dogs and humans. Fig. 1 shows the distribution of the different CYPs in liver and small intestinal mucosa of human
Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians.
Mass spectrometry-based quantification of CYP enzymes to establish in vitro/in vivo scaling factors for intestinal and hepatic metabolism in beagle dog.
The liver has the highest content of drug-metabolizing CYPs and is the most important organ for CYP-mediated drug elimination. The small intestine also has a high specific content of certain CYPs located within the mucosa and serves to decrease absorption of intact (unmetabolized) drugs thereby limiting systemic availability of orally administered drugs. Similarities between dog and human are apparent in that the CYP3A subfamily enzymes are the predominant isoforms in liver and intestines of both species. However, the CYP2D subfamily enzyme CYP2D15 is more highly expressed (as a percentage of total CYPs) in the livers of dogs versus CYP2D6 in humans. Furthermore, the CYP2B subfamily enzyme CYP2B11 is more highly expressed in both livers and intestines of dogs than CYP2B6 in human livers and intestines. This difference could be a consequence of the many genetic mutations that have been associated with the CYP2D6 and CYP2B6 genes in humans. A clinical consequence is that drugs metabolized by CYP2D or CYP2B may have lower systemic levels in dogs than in humans.
Fig. 1Comparison of relative CYP isoform protein levels in dog and human liver and small intestines. Protein amounts were determined by quantitative immunoblotting for human liver
Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians.
Mass spectrometry-based quantification of CYP enzymes to establish in vitro/in vivo scaling factors for intestinal and hepatic metabolism in beagle dog.
Table 2 summarizes published data regarding known genetic polymorphisms in the canine drug-metabolizing CYP enzymes including variant description, allele frequencies, and effects of the variant on enzyme function in vitro and in vivo.
Table 2Summary of known canine CYP genetic polymorphisms
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
Isolation of a new canine cytochrome P450 CDNA from the cytochrome P450 2C subfamily (CYP2C41) and evidence for polymorphic differences in its expression.
The most comprehensively studied canine CYP genetic polymorphism is the premature stop codon mutation (c.1117C>T; R373X) located in the coding region of the CYP1A2 gene that results in complete loss of hepatic CYP1A2 protein and associated enzyme activity.
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
This mutation was discovered independently by two Japanese pharmaceutical companies during preclinical testing of two unrelated investigational compounds (YM-64227
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
) that showed highly polymorphic pharmacokinetics of these compounds in their Beagle dog colonies. Both groups used genetic testing to screen a large number of their Beagle dog colonies and found that from 11% to 17% of their dogs had the homozygous mutant genotype and consequently did not express functional CYP1A2.
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
Elucidation of the effects of the CYP1A2 deficiency polymorphism in the metabolism of 4-cyclohexyl-1-ethyl-7-methylpyrido 2,3-d pyrimidine-2-(1h)-one (YM-64227), a phosphodiesterase type 4 inhibitor, and its metabolites in dogs.
The canine CYP1A2 deficiency polymorphism dramatically affects the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido 2,3-D -pyrimidine-2-(1H)-one (YM-64227), a phosphodiesterase type 4 inhibitor.
However, animals that had at least one normal CYP1A2 copy (heterozygotes) did not have substantially different drug levels from the wild-type dogs. Several other investigational compounds including GTS-21
have also been associated with variable CYP1A2 metabolism in Beagles.
Fig. 2Effect of the CYP1A2 premature stop codon mutation (R373X) on the pharmacokinetics of the investigational compound YM-64227 (left) and the analgesic drug phenacetin (right) after oral administration to genotyped Beagle dogs. The effect of the CYP1A2 premature stop codon on YM-64227 (about 25 times greater mean plasma area under the curve) was much greater than on phenacetin (about two times greater area under the curve).
(YM-64227 Data from Tenmizu D, Noguchi K, Kamimura H, et al. The canine CYP1A2 deficiency polymorphism dramatically affects the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido 2,3-D -pyrimidine-2-(1H)-one (YM-64227), a phosphodiesterase type 4 inhibitor. Drug Metab Dispos 2006;34(5):800; Phenacetin Data from Whiterock VJ, Morgan DG, Lentz KA, et al. Phenacetin pharmacokinetics in CYP1A2-deficient beagle dogs. Drug Metab Dispos 2012;40(2):228–31.)
using liver microsomes from CYP1A2-deficient and -expressing dogs indicated that phenacetin and tacrine (both selectively metabolized by human CYP1A2) were more slowly metabolized in deficient livers. However, other (human selective) CYP1A2 substrates including caffeine and melatonin were unaffected by the deficiency, implying that dog CYP1A2 does not selectively metabolize these latter drugs (unlike human CYP1A2). A recent study of phenacetin pharmacokinetics following oral and intravenous administration to CYP1A2 genotyped Beagles
showed about twofold higher phenacetin exposure (based on area under the plasma concentration time curve) after oral administration in CYP1A2-deficient dogs, but there were much smaller (nonsignificant) differences in phenacetin levels after intravenous exposure. The authors concluded that phenacetin was not a selective or robust in vivo probe for CYP1A2 probably because of metabolism of phenacetin by other enzymes (eg, canine CYP1A1 or canine CYP2A13). These findings indicate that the effect of CYP1A2stop on a particular drug depends on the degree of importance of canine CYP1A2 in clearance and cannot be extrapolated directly from human data.
The prevalence of CYP1A2stop seems to vary considerably between and within dog breeds. Apart from research colony Beagle dogs in Japan,
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
The Irish Wolfhound had the highest allele frequency (42%) followed by the Japanese Beagles (37%–39%) and Berger Blanc Suisse (28%). Interestingly, the beagles studied in Germany
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
possibly reflecting colony founder effect differences. The remaining breeds studied had allele frequencies of 10% or less indicating that the likely frequency of the enzyme-deficient homozygous variant dogs would be 1% or less in the population (ie, relatively rare). Interestingly, many of the remaining affected breeds were herding dogs including Australian Shepherd, Collie, Shetland Sheepdog, Bearded Collie, Border Collie, and Old English Sheepdog.
Although this could be sampling bias, it might also indicate a common (although perhaps more recent) ancestry of CYP1A2stop with the MDR1 gene deletion (MDR1del) mutation, which is commonly found in herding breed dogs.
The latter results in drug sensitivity from deficiency of the P-glycoprotein transporter encoded by MDR1 (discussed elsewhere in this issue). Regardless, the clinical consequence is that herding breed dogs could be affected by multiple genetic defects (MDR1del and CYP1A2stop) influencing drug disposition and response.
Fig. 3Allele frequency (number of variant alleles as percent of total alleles) of the CYP1A2 stop codon mutation (R373X) in different dog breeds. Shown after each breed are the numbers of individual dogs that were sampled. Data are from dogs located in Brazil (*Scherr and colleagues
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
). Only data from breeds in which DNA from at least 10 different dogs in that breed were sampled are shown. Other breeds in which at least one dog had the deficient allele included Whippet, Deerhound, Dalmatian, and Jack Russell Terrier (Aretz and Geyer
Isolation of a new canine cytochrome P450 CDNA from the cytochrome P450 2C subfamily (CYP2C41) and evidence for polymorphic differences in its expression.
This latter isoform was found to be present at the RNA and genomic DNA level in only about 16% (4 of 25) of dogs (2 of 10 mixed breeds and 2 of 18 Beagles). This contrasted with CYP2C21 that was found to be expressed in all dogs examined. This finding suggests the presence of a partial or complete deletion of the CYP2C41 gene in many dogs. This was confirmed (albeit with a lower deletion frequency) by a study in another laboratory that showed detectable CYP2C41 mRNA in 6 of 11 Beagle dogs.
In vitro studies of recombinant canine CYPs indicate that CYP2C41 metabolizes many of the same substrates as CYP2C21 (including diclofenac and S-mephenytoin), although with much less efficiency.
Consequently, the impact of the CYP2C41 deletion on canine drug metabolism or pharmacokinetics may be somewhat limited.
CYP2D15 Amino Acid Variants
Several studies have identified different CYP2D15 mRNA forms expressed in liver that vary in predicted amino acid coding sequence at three to five different residues (see Table 2). Although it is presumed that these changes are the result of single nucleotide polymorphisms (SNP), this has not yet been established, such as through genotyping of multiple dogs. In vitro studies of expressed amino acid variants suggest that the impact of these coding changes on enzyme function may be somewhat limited. The only exception was the WT2 form identified by Roussel and colleagues,
originally undertook their study of CYP2D15 variants to explain polymorphic celecoxib clearance in vivo and celecoxib hydroxylation in vitro in a research colony of Beagle dogs. However, they did not directly address this hypothesis, such as through CYP2D15 genotyping of phenotyped dogs. Furthermore, although CYP2D15 was shown to be capable of hydroxylating celecoxib, a significant role for other CYPs was not excluded. Consequently, the mechanism underlying the celecoxib pharmacokinetic polymorphism remains unexplained.
CYP2E1 Amino Acid Variant
An SNP (1453T>C) resulting in a tyrosine for histidine substitution at amino acid position 485 (H485Y) was discovered during initial cloning of the CYP2E1 cDNA from canine liver RNA.
Survey genotyping found an allele frequency of 15% in 100 mixed-breed dogs, and 19% in 13 Beagles. An in vitro study comparing expressed wild-type (485H) and variant (485Y) CYP2E1 isoforms showed no difference in chlorzoxazone hydroxylation activity. However, because only one substrate was evaluated, substrate-dependent effects cannot be excluded. In vivo effects of this SNP on drug pharmacokinetics have not been reported.
CYP3A12 Amino Acid Variants
A variant (called CYP3A12*2) that included five different nucleotide differences from the initial cloned sequence, and predicted to cause five unique amino acid changes, was also discovered during cloning of the CYP3A12 cDNA from canine liver RNA.
In vitro experiments showed no effect of these amino acid changes on testosterone 6-β-hydroxylation by recombinant enzymes. Genotype frequencies or any association of genotype with drug metabolism phenotype measured in vivo have not been reported.
Other dog CYPs associated with phenotypic variability
CYP2B11 and Breed-related Anesthetic Drug Hypersensitivity
Severely delayed recovery has been reported for certain dog breeds (primarily Greyhounds and possibly other sighthounds) after use of injectable anesthetic agents, including thiopental and thiamylal.
Comparative pharmacokinetics and anesthetic effects of methohexital, pentobarbital, thiamylal, and thiopental in Greyhound dogs and non-Greyhound, mixed-breed dogs.
Although initially attributed to decreased drug redistribution from the central compartment resulting from reduced body fat in Greyhounds, a series of studies demonstrated that the effect could be attributed to decreased drug clearance in Greyhounds compared with mixed-breed dogs, and was also prevented by pretreatment with a microsomal enzyme inducer (phenobarbital).
Comparative pharmacokinetics and anesthetic effects of methohexital, pentobarbital, thiamylal, and thiopental in Greyhound dogs and non-Greyhound, mixed-breed dogs.
The molecular genetic basis for this breed-dependent difference in drug metabolism has not been reported.
Fig. 4Greyhounds show slower clearance (left) and lower liver metabolism (right) of propofol compared with mixed-breed and Beagle dogs. (Left) Mean propofol concentrations in 10 Greyhounds and 10 mixed-breed dogs given propofol intravenously at a dose of 5 mg/kg body weight. Time of return of the righting reflex (solid arrow) and ability to stand (open arrow). (Right) Propofol hydroxylation rates measured by high-performance liquid chromatography with in vitro incubations of liver microsomes prepared from male Greyhounds, Beagles, and mixed-breed dogs (five each). The circles represent data from individual dog livers. The horizontal lines indicate the mean values of each group.
([Left panel] Data from Zoran DL, Riedesel DH, Dyer DC. Pharmacokinetics of propofol in mixed-breed dogs and greyhounds. Am J Vet Res 1993;54(5):755; [Right panel] Data from Court MH, Hay-Kraus BL, Hill DW, et al. Propofol hydroxylation by dog liver microsomes: assay development and dog breed differences. Drug Metab Dispos 1999;27(11):1293.)
Published evidence indicates that variability in drug metabolism by CYP in dogs is likely to be considerable and is explained in part by the presence of genetic polymorphisms that vary between dog breeds. However, few CYPs (mainly CYP1A2) have been systematically investigated, and the influence of the discovered genetic variants on the pharmacokinetics of clinically used drugs and their effects is unclear. Predictions of genetic effects on particular drugs (eg, the effect of CYP1A2 stop codon mutation on phenacetin pharmacokinetics) from human data are complicated by human-dog differences in CYP substrate specificity and abundance. Consequently, clinical studies confirming the impact of discovered variants on drug response in canine patients are essential.
Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians.
Mass spectrometry-based quantification of CYP enzymes to establish in vitro/in vivo scaling factors for intestinal and hepatic metabolism in beagle dog.
Polymorphic expression of CYP1A2 leading to interindividual variability in metabolism of a novel benzodiazepine receptor partial inverse agonist in dogs.
Isolation of a new canine cytochrome P450 CDNA from the cytochrome P450 2C subfamily (CYP2C41) and evidence for polymorphic differences in its expression.
Elucidation of the effects of the CYP1A2 deficiency polymorphism in the metabolism of 4-cyclohexyl-1-ethyl-7-methylpyrido 2,3-d pyrimidine-2-(1h)-one (YM-64227), a phosphodiesterase type 4 inhibitor, and its metabolites in dogs.
The canine CYP1A2 deficiency polymorphism dramatically affects the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido 2,3-D -pyrimidine-2-(1H)-one (YM-64227), a phosphodiesterase type 4 inhibitor.
Comparative pharmacokinetics and anesthetic effects of methohexital, pentobarbital, thiamylal, and thiopental in Greyhound dogs and non-Greyhound, mixed-breed dogs.
Disclosures: This work was supported by funds provided by the William R. Jones Endowed Chair in Veterinary Medicine at Washington State University. There are no conflicts of interest to report.
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