Abstract
Objective
To determine the population variability in dextromethorphan metabolism by cytochrome (CY) P450 2D15 (CYP2D15) in dogs.
Methods
Healthy pet dogs were recruited from 2018 through 2024 from the Inland Pacific Northwest and phenotyped by orally administering the Program in Individualized Medicine cocktail, which contains dextromethorphan, a CYP2D15-specific probe drug. Glucuronidase-treated urine samples collected 6 hours after dosing were assayed for dextromethorphan and dextrorphan concentrations. Log-transformed metabolic ratios of dextrorphan divided by dextromethorphan (DOR/DXM Log MRs) were calculated. Dogs were genotyped for 5 missense CYP2D15 variants. Univariate and multivariate statistical approaches were used to evaluate associations between DOR/DXM Log MRs and demographic variables.
Results
105 dogs, including 34 mixed breeds and 71 dogs from 20 different owner-designated breeds, were enrolled and completed the study. There was a wide distribution of DOR/DXM Log MRs, from 0.97 to 2.76, representing a log unit range of 1.8 (63-fold variation DOR/DXM Log MRs). Log-transformed metabolic ratios of dextrorphan divided by dextromethorphan were normally distributed and unimodal. The mean (± SD) DOR/DXM Log MR was 2.04 ± 0.37. Multiple linear regression analysis showed significant association (R2 = 0.16) between DOR/DXM Log MRs and dog breed for Golden Retrievers (2.26 ± 0.29; N = 23) and Pugs (1.47 ± 0.29; N = 3). Log-transformed metabolic ratios of dextrorphan divided by dextromethorphan were not associated with dog sex, age, weight, or genotype.
Conclusions
There is substantial variability in DOR/DXM Log MR values among individuals, which can be partially attributed to differences between breeds.
Clinical Relevance
These findings predict high variability in the metabolism of drugs by CYP2D15 associated with differences between dog breeds.
Cytochrome (CY) P450 2D15 (CYP2D15) is one of the most abundant drug-metabolizing CYP450 enzymes in canine liver.1 Cytochrome P450 2D15 is known to metabolize a range of drugs in dogs, including dextromethorphan (DXM),2–4 propranolol,5 maropitant,6 and celecoxib.7 Although the full range of drugs that are metabolized by CYP2D15 in dogs is unknown, the human ortholog of CYP2D15 (CYP2D6) is involved in the metabolism of about 20% of commonly used drugs in people.8,9 Importantly, the metabolism of drugs by CYP2D6 demonstrates high interindividual variability, which is largely explained by a range of CYP2D6 gene variants that affect gene expression and enzyme activity. The pharmacokinetics of CYP2D6 substrates also varies between human populations according to race, ethnicity, and/or geographic origin. These differences result from population-level variation in the prevalence of CYP2D6 gene variants.
Interindividual and population-level (ie, breed) differences in the metabolism of drugs by CYP2D15 in dogs may also be explained by CYP2D15 genetic variation. Like humans, CYP2D15 is not inducible,3 meaning that genetic factors are likely to contribute to interindividual variation in enzyme activity more than environmental factors.9 Such differences may explain why some individual dogs or breeds of dogs are predisposed to adverse drug reactions resulting from the slow or fast metabolism of CYP2D15 substrate drugs.
A recent in vitro study10 evaluated whether 5 common CYP2D15 missense variants, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe, and p.Ile307Val, were associated with differences in CYP2D15 protein content or enzyme activities (tramadol O-demethylation or DXM O-demethylation) using a bank of genotyped livers from 59 dogs. The results of that study showed significant associations between genotypes for 2 of the CYP2D15 variants and reduced CYP2D15 expression and enzyme activity. However, additional in vivo evidence is needed to confirm and expand on those in vitro model findings.
An in vivo approach to quantify the rate of drug metabolism noninvasively, selectively, and simultaneously by 3 of the major canine drug-metabolizing enzymes (CYP2D15, CYP2B11, and CYP3A12) has recently been developed and validated for use in dogs.2 The Program in Individualized Medicine (PrIMe) cocktail drug metabolism phenotyping method involves orally administering clinically accepted dosages of the probe drugs DXM (CYP2D15), bupropion (CYP2B11), and omeprazole (CYP3A12) and measuring concentrations of each of these drugs and the associated metabolites formed by the respective CYP450 in urine and plasma samples. The calculated metabolite-to-parent-drug concentration ratio (MR) value provides a quantitative index of the rate of in vivo drug metabolism for each CYP450. This approach is commonly used in human pharmacology research to identify genetic and environmental factors influencing CYP450 metabolism and to evaluate the potential for drug-drug interactions.11,12
The aim of this study was to use the PrIMe cocktail to determine the distribution of CYP2D15 MR values in a population of healthy owned pet dogs. The association of these MR values with demographic and genetic factors, including dog sex, age, weight, breed, and CYP2D15 missense variant genotype, were then evaluated.
Methods
Study subjects
This study was approved by the IACUC at Washington State University (protocol #6115). All owners gave verbal and written informed consent for their dogs to participate prior to commencement of the study.
Owned healthy dogs were recruited from the Inland Pacific Northwest region (Eastern Washington and Northern Idaho) from 2018 through 2024. Health status was determined by an initial phone screen for health history and medication use followed by an in-person physical examination. To be included in the study, dogs needed to be healthy, weigh ≥ 5 kg, and not have any medications or supplements being administered except for lokivetmab injections or monthly flea, tick, and parasite preventatives. For dogs receiving these medications, the study did not commence until at least 2 weeks after their last dose.
Study design
On the day prior to the trial, food (but not water) was withheld after the dog’s normal evening meal. Dogs arrived at the study site prior to 9 am, received a brief physical examination, and were assigned a kennel for the day. Owners supplied their dog’s normal morning ration of food. Two hours prior to drug administration, dogs were offered half of their morning food ration, with the remainder given at the end of the study. All dogs had free access to water throughout the study.
Each dog was administered commercially available DXM (0.46 to 1.5 mg/kg), bupropion (1.2 to 3.8 mg/kg), and omeprazole (0.6 to 2 mg/kg) dosed according to their weight range (5 to 10 kg, 10 to 20 kg, and 20 to 65 kg). Details regarding the drug formulations and the doses given to dogs in each weight range are given in Supplementary Table S1. Tablets and capsules were administered in pill pockets (Canine Greenies; The Nutro Company), whereas DXM syrup was directly administered into the back of the mouth using a plastic syringe. Six hours after the drugs were administered, the dogs were returned to their owners. All owners were contacted the day after the study to inquire about possible drug side effects.
Heparinized blood samples (2 to 3 mL) were collected aseptically from a peripheral vein before (time 0) and at 4 hours after drug administration. The blood samples were centrifuged at 2,000 X g for 15 minutes, and the plasma (clear supernatant) for drug analysis and buffy coat (top layer of the cellular fraction) for DNA extraction were transferred to separate 2-mL polypropylene tubes and stored at −80 °C until analysis. Plasma samples were collected for the measurement of omeprazole, omeprazole sulfone, bupropion, and hydroxybupropion concentrations as phenotypic measures of CYP3A12- and CYP2B11-mediated drug metabolism, which will be reported elsewhere.
Urine samples taken by free catch prior to drug administration (time 0) and 6 hours after drug administration were transferred to separate 2-mL polypropylene tubes and stored at −80 °C until analysis.
Dextromethorphan and dextrorphan assay
Concentrations of DXM and dextrorphan (DOR) in urine samples were determined by HPLC-MS-MS using a previously described and validated method with minor modifications.2 Briefly, 100 µL internal standard solution containing 10 ng of DXM-d3 and 250 ng of DOR-d3 (Cayman Chemical) were added to 1.5 mL polypropylene tubes and evaporated to dryness in a vacuum centrifuge. Methanolic solutions of DXM (1 to 75 ng) and DOR (10 to 2,000 ng; GlaxoSmithKline) were also added to calibrator and quality control samples before drying. Next, 25 µL of urine test sample (or 25 µL of pooled blank urine for calibrator samples) and 75 µL of 200 mM aqueous sodium acetate buffer (pH 5.0) containing 375 U of Type HP2 β-Glucuronidase from Helix pomatia (Sigma G7017) were added to each tube, capped, and incubated overnight at 37 °C in a shaking incubator. Then, 100 µL of acetonitrile containing 1% formic acid was added to each tube, vortexed, and centrifuged at 15,000 X g for 10 minutes. A 100-µL aliquot of the supernatant was then diluted with 100 µL of ultrapure water, and 20 µL of that was injected into the HPLC-MS-MS apparatus.
The HPLC-MS-MS apparatus consisted of an Agilent 1100 HPLC coupled to an AB Sciex API 4000 triple quadrupole mass spectrometry detector run in positive ion mode. Chromatographic separation was achieved using an Agilent Zorbax Eclipse XDB-C18 2.1 X 50 mm 5-μm column (Agilent Technologies, Santa Clara, CA). The mobile phase pumped at 1 mL/min consisted of 0.1 % formic acid in water (A) variably mixed with acetonitrile (B) as a gradient starting at 95:5 (A:B volume per volume), linearly increasing from 95:5 at 1 minute run time to 5:95 at 2.5 minutes, and returning from 5:95 at 3 minutes to 95:5 at 3.5 minutes. The total run time was 6 minutes. Ion transitions (m/z+) monitored by the mass detector included 272.4 → 215, 275.4 → 215, 258.2 → 201, and 261.2 → 201 for DXM, DXM-d3, DOR, and DOR-d3, respectively. Analyte concentrations were calculated from standard curves using Analyst, version 16.3 (AB Sciex LLC, Framingham, MA). Interassay accuracy (determined as the percentage of bias) for quality control samples averaged 8% for DXM and 6% for DOR, whereas interassay precision (determined as the percentage of the coefficient of variation) averaged 13% for DXM and 5% for DOR. The lower limit of quantitation was 10 ng/mL for DXM and 100 ng/mL for DOR. All samples were assayed 3 times (on separate days), and the results were averaged. The results for each dog were expressed as a log-transformed metabolic ratio (Log MR) by dividing the urinary molar concentration of DOR by the molar concentration of DXM and log transforming (DOR/DXM Log MR).
Cytochrome P450 2D15 genotyping
Custom allele discrimination assays (Applied Biosystems TaqMan SNP Genotyping Assay; Thermo Fisher Scientific) were used to genotype DNA samples obtained from blood from 106 dogs for the following 5 known CYP2D15 missense variants: c.325 A>G (p.Ile109Val), c.345 A>G (p.Leu115Phe), c.556 G>A (p. Ser186Gly), c.748 A>T (p.Ile250Phe), and c.919 A>G (p.Ile307Val). Primer/probe sequences are given in Supplementary Table S2. Assays were performed using a real-time PCR instrument (CFX96 Touch; Bio-Rad). Assay accuracy was confirmed by sequencing representative DNA samples. Genotypes for each variant were classified as either homozygous wild type (WT), heterozygous (HET), or homozygous variant.
Statistical analysis
Statistical analyses were performed with Sigma Plot, version 14.5 (Grafiti LLC). The frequency histogram and probit plots were created using Excel, version 2410 (Microsoft Corp). For probit plots, sorted DOR/DXM Log MR data were used to calculate the cumulative probability, which was transformed to the z value for the standard normal distribution using the Excel function NORMSINV. Prior to statistical hypothesis testing, DOR/DXM Log MR data were evaluated for distribution normality (Shapiro-Wilk test) and constant variance to ensure valid use of the proposed parametric test. If either test had failed (nonnormality or unequal variance), an equivalent nonparametric test would have been used. The associations of DOR/DXM Log MR with dog age and body weight were evaluated by linear regression, whereas a Student t test analyzed the association of DOR/DXM Log MR with the sex of the dog. Analysis of variance was performed to compare DOR/DXM Log MR values between dog groups classified according to genotype (WT, HET, and homozygous variant) or by breed. Pairwise multiple comparisons testing was performed using the Holm-Sidak method to identify specific breed and genotype groups that were different. For phenotype-genotype association analysis, the Jonckheere-Terpstra trend test13 was also used to evaluate the association between DOR/DXM Log MR values and variant allele number when the ANOVA P value was less than .1. The Student t test was used when only 2 genotype groups were identified. Stepwise forward and backward multiple linear regression analysis was also used to simultaneously evaluate associations between independent variables (sex, age, weight, breed, and genotype) and DOR/DXM Log MR values. For all statistical tests, a P value of less than .05 was considered statistically significant.
Results
A total of 105 dogs were enrolled in the study, including 48 females (8 intact and 40 spayed) and 57 males (9 intact and 48 neutered). The median age was 5 years (range, 1 to 11 years), and the median weight was 25.5 kg (range, 6.1 to 65.2 kg). Twenty different owner-designated dog breeds were phenotyped, including Golden Retrievers (N = 24), Labrador Retrievers (N = 10), Border Collies (N = 4), Coonhounds (N = 5), Bernese Mountain Dogs (N = 3), Pugs (N = 3), Cardigan Welsh Corgis (N = 3), Australian Cattle Dogs (N = 3), Miniature Australian Shepherds (N = 2), Wire Fox Terriers (N = 2), Wirehaired Pointing Griffons (N = 2), English Springer Spaniels (N = 2), Bassett Hounds (N = 2), and 1 of each of the following breeds: German Shepherd, Siberian Husky, Shiba Inu, Dalmatian, Anatolian Shepherd, Boxer, and American Pitbull Terrier. There were also 34 mixed-breed dogs.
All enrolled dogs completed the study. Of those 105 dogs, 10 dogs experienced mild gastrointestinal side effects (vomiting or diarrhea) associated with administration of the PrIMe cocktail. These signs resolved completely within 24 hours of the study’s conclusion.
The DOR/DXM Log MR values were obtained from all 105 dogs enrolled. The mean (± SD) DOR/DXM Log MR was 2.04 ± 0.37, and the median (IQR) MR was 2.07 (1.78 to 2.32). There was a wide distribution of DOR/DXM Log MRs, ranging from 0.97 to 2.76. This difference (a log unit range of 1.8) represents a 63-fold variation in (untransformed) DOR/DXM MRs.
A frequency histogram plot of all DOR/DXM Log MR values is shown in Figure 1. The distribution of values was normal based on the Shapiro-Wilk test (W = 0.99; P = .33), and there was no clear visual evidence for multimodality (ie, bi- or trimodal distribution). This was confirmed by visual inspection of the probit plot, which was generally linear (R2 = 0.99; P < .001), except for the phenotypic extremes, without any clear antimode breakpoints.14
Distribution of log-transformed dextrorphan/dextromethorphan metabolic ratios (DOR/DXM Log MRs) measured in a population (N = 105) of healthy owned dogs. A—Frequency histogram. B—Probit plot.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0377
Next, possible associations of DOR/DXM Log MRs with demographic variables, including sex, age, weight, breed, and CYP2D15 genotype, were evaluated by univariate and multivariate analysis. As shown in Figure 2, DOR/DXM Log MR values were not statistically different (P = .34; t test) between male (2.07 ± 0.41; mean ± SD; N = 57) and female (2.00 ± 0.33; N = 48) dogs. Additionally, when females and males were grouped and compared according to whether they were intact (females: 2.13 ± 0.20, N = 8; males: 2.00 ± 0.30, N = 9) or desexed (spayed females: 1.985 ± 0.35, N = 40; neutered males: 2.088 ± 0.42, N = 48), there were no significant differences between these groups (P = .53; ANOVA). Furthermore, there was no association (R2 = 0.017; P = .18; linear regression) between DOR/DXM Log MR values and dog age (Figure 1). However, DOR/DXM Log MR values were weakly positively correlated (R2 = 0.089; P = .002; linear regression) with increasing body weight (Figure 1).
Association between DOR/DXM Log MRs measured in a population (N = 105) of healthy owned dogs and subject sex (A), age (B), and body weight (C). A—Box plot showing the median, mean, IQR, minimum, maximum, and outlier DOR/DXM Log MR values for female (N = 48) and male dogs (N = 57). Also shown is the result (P value) for statistical comparison between these groups. B and C—Scatter plot showing the relationship between age (B) or weight (C) and DOR/DXM Log MR values for each dog. Also shown in these plots are the fitted regression lines and associated R2 and P values.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0377
The DOR/DXM Log MR values showed substantial variation between breeds (P < .001; ANOVA; Figure 3; Supplementary Table S3). However, 12 of the 20 breeds studied were represented by only 1 or 2 individuals, limiting the power to identify individual breed groups that were different. When the ANOVA was limited to the remaining 7 dog breed groups and mixed-breed dogs that contained at least 3 tested individuals, Golden Retrievers (2.26 ± 0.29; N = 23) showed significantly higher DOR/DXM Log MR values compared with Pugs (1.47 ± 0.29; N = 3; P = .008; Holm-Sidak). Furthermore, when the ANOVA was limited to the 3 dog groups containing at least 10 individuals (Golden Retrievers, Labrador Retrievers, and mixed-breed dogs), Golden Retrievers (2.26 ± 0.29; N = 23) also showed significantly higher DOR/DXM Log MR values compared with mixed-breed dogs (1.99 ± 0.36; N = 28; P = .01; Holm-Sidak). No other significant differences between breed groups were revealed.
Box plots showing the mean, median, IQR, minimum, maximum, and outlier DOR/DXM Log MR values for 105 dogs grouped by owner-designated breed. Shown are the results (P values) of pairwise statistical comparisons between breeds. Also shown are the numbers of dogs studied in each breed group.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0377
Cytochrome P450 2D15 missense variant genotype and allele frequencies for all dogs (mixed breeds, Golden Retrievers, Labrador Retrievers, and other breeds [combined]) are given in Supplementary Table S4. None of the dogs tested had the WT genotype for the p.Ile250Phe or p.Ile307Val variants. Only 5 dogs were HET for the p.Ile250Phe variant, and 3 dogs were HET for the p.Ile307Val variant, resulting in variant allele frequencies of 0.98 and 0.99, respectively. Allele frequencies (Figure 4) for all dogs were 0.16, 0.21, and 0.43 for the p.Ile109Val, p.Leu115Phe, and p.Gly186Ser variants, respectively. Interestingly, while mixed-breed dogs, Labrador Retrievers, and the other breeds group showed similar allele frequencies to all dogs combined, Golden Retrievers had much lower frequencies of 0.02, 0.11, and 0.17 for the p.Ile109Val, p.Leu115Phe, and p.Gly186Ser variants, respectively.
A—Cytochrome P450 2D15 allele frequencies (mean ± SD) for the entire population studied (all dogs; N = 105) and 4 population subgroups, including mixed breeds (N = 34), Golden Retrievers (N = 23), Labrador Retrievers (N = 10), and all remaining dogs combined (other breeds; N = 38). B—Box plots showing the median, mean, IQR, minimum, maximum, and outlier DOR/DXM Log MR values grouped by cytochrome P450 2D15 (CYP2D15) p.Gly16Ser genotype (homozygous wild type [no variant allele; WT], heterozygous [1 variant allele; HET], or homozygous variant [2 variant alleles; VAR]). Also shown are the results (P value) evaluating the relationship between DOR/DXM Log MR values and variant allele number.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.24.12.0377
The results of statistical evaluation of phenotype-genotype associations between DOR/DXM Log MR values and genotypes for each CYP2D15 missense variant are given in Table 1. Initial evaluation by ANOVA and the Student t test (for p.Ile250Phe or p.Ile307Val genotypes) found no statistically significant association (P > .05) between DOR/DXM Log MRs and any of the 5 variants evaluated (p.Ile109Val, P = 0.471; p.Leu115Phe, P = .38; p.Gly186Ser, P = .08; p.Ile250Phe, P = .139; p.Ile307Val, P = .065). However, using the Jonckheere-Terpstra trend test, a statistically significant decrease in DOR/DXM Log MR values (P = .016) was found in association with increasing variant allele number (Figure 4).
Comparison of dextrorphan/dextromethorphan log metabolic ratio values between 105 pet dogs genotyped as wild type (WT), heterozygous (HET), or variant (VAR) for 5 different missense cytochrome P450 2D15 gene variants.
| Genotype | Holm-Sidak test P value | |||||||
|---|---|---|---|---|---|---|---|---|
| Missense variant | WT | HET | VAR | P value | WT vs HET | VAR vs HET | VAR vs WT | |
| c.325 A>G (p.Ile109Val) | Mean | 2.068 | 1.969 | 2.098 | .483 | .561 | .819 | .893 |
| SD | 0.378 | 0.368 | 0.401 | |||||
| N | 74 | 28 | 3 | |||||
| c.345 G>C (p.Leu115Phe) | Mean | 2.079 | 1.966 | 2.017 | .390 | .483 | .749 | .897 |
| SD | 0.362 | 0.408 | 0.349 | |||||
| N | 68 | 30 | 7 | |||||
| c.556 G>A (p.Gly186Ser) | Mean | 2.143 | 2.011 | 1.938 | .08 | .436 | .216 | .095 |
| SD | 0.351 | 0.373 | 0.384 | |||||
| N | 39 | 41 | 25 | |||||
| c.748 A>T (p.Ile250Phe) | Mean | — | 2.285 | 2.03 | .139 | |||
| SD | — | 0.406 | 0.371 | |||||
| N | 0 | 5 | 100 | |||||
| c.919 A>G (p.Ile307Val) | Mean | — | 2.434 | 2.031 | .066 | |||
| SD | — | 0.298 | 0.372 | |||||
| N | 0 | 3 | 102 | |||||
Additional phenotype-genotype association analyses were conducted within breed-designated groups containing at least 10 individuals/group, including mixed-breed dogs (Supplementary Table S5), Golden Retrievers (Supplementary Table S6), and Labrador Retrievers (Supplementary Table S7). No statistically significant association (P > .05) was found within these breed groups between DOR/DXM Log MRs and genotypes for any of the 5 CYP2D15 variants evaluated.
Multivariate analysis was then performed using stepwise forward and backward multiple linear regression analysis to determine the minimum set of independent variables (sex, age, weight, breed, and/or genotype) predictive of DOR/DXM Log MR. For this analysis, the breed and genotype variables were limited to those identified as being significantly associated with DOR/DXM Log MR in the univariate analysis, which included Golden Retriever, Pug, and mixed-breed designations (each coded as either 1 or 0) and the number of CYP2D15 p.Gly186Ser variant alleles (0, 1, or 2). Identical results were obtained with both forward and backward stepwise approaches. Sex (P = .57), age (P = .27), weight (P = .18), mixed-breed designation (P = .97), and p.Gly186Ser allele number (P = .895) were eliminated. The final multiple regression model accounted for 16% of the observed variability (R2 = 0.16; P < .001) and included Golden Retriever breed (standardized coefficient = 0.30; P = .001) and Pug breed (standardized coefficient = −0.24; P = .011). Finally, since the final multivariate model did not include weight as a predictive variable, a reanalysis of the association between DOR/DXM Log MR values and dog weight by linear regression was performed after exclusion of the golden retriever and Pug data. As shown in Supplementary Figure S1, there was no significant association between weight and DOR/DXM Log MR values (R2 = 0.0067; P = .47) in the remaining 79 dogs.
Discussion
This is the first study to report in vivo phenotyping of a large population of dogs using a CYP450-selective probe for a major drug-metabolizing enzyme (CYP2D15). The results showed high interindividual variability in DOR/DXM Log MR values that was explained in part by differences between dog breeds, with Golden Retrievers showing higher values (faster metabolism) and pugs showing lower values (slower metabolism) relative to the entire population studied.
This study also included 36 mixed-breed dogs, which are the most commonly owned dog type in US households.15 Interestingly, DOR/DXM Log MR values for mixed-breed dogs essentially spanned the entire range of MRs of the entire population. Furthermore, the mean (± SD) DOR/DXM Log MR of mixed-breed dogs (1.99 ± 0.36; N = 36) was essentially identical to the values for the non–mixed-breed dogs (2.04 ± 0.38; N = 69). This finding is likely a consequence of the genetic admixture of the mixed-breed dogs studied. Although there is little published on the breed composition of mixed-breed dogs in the US, genetic data from 200,000 mixed-breed dogs gathered by a commercial genetic testing company suggests that some breeds predominate, including German Shepherds, American Pit Bull Terriers, Labrador Retrievers, and Chihuahuas (https://embarkvet.com/resources/top-dog-breed-by-state/). Interestingly, consistent with the high variability observed for mixed-breed dogs, the dog with the highest DOR/DXM Log MR value was a German Shepherd, whereas the dog with 1 of the lowest DOR/DXM Log MR values was an American Pit Bull Terrier. However, these (and other) “outlier” breeds were represented by only 1 or a small number of individuals. Consequently, further studies are needed to sample additional individuals from the breeds tentatively located at the phenotypic extremes (ie, fast and slow metabolizers) to confirm these differences.
The evaluation of DOR/DXM Log MR values for all dogs did not reveal clear evidence for a multimodal (bi- or trimodal) data distribution that would indicate a strong effect on phenotype from 1 or a small number of CYP2D15 genetic variants in the population studied. Over 130 different CYP2D6 variant alleles have been identified in humans with varying degrees of effect on enzyme function, ranging from no enzyme function (null allele) to substantially increased function (gene duplication).8 Differences in the prevalence of these variants between human populations vary greatly according to geographic and ethnic origin and account for differences in the distribution of phenotypes in those populations.16 Consequently, multiple CYP2D15 variants may occur in dogs with varying effects on enzyme function, resulting in unimodal data distribution as was observed in this study. Furthermore, differences in the prevalence of these variants across breeds may account for breed differences in CYP2D15 genotype observed in this study.
A relatively weak association was found between the number of CYP2D15 p.Gly186Ser variant alleles (0, 1, or 2) and decreased DOR/DXM Log MRs. A similar association was observed in a previous in vitro study10 that found decreased DXM O-demethylation, tramadol O-demethylation (another CYP2D15-selective activity), and CYP2D15 protein content in a bank of dog livers. This result provides additional validation of the in vitro liver microsomes phenotype-genotype model approach. However, in both instances, the effect was relatively small, indicating that genetic variants other than those studied here are more likely to explain the observed CYP2D15 phenotype variability.
Interestingly, a breed difference was observed for the p.Ile109Val, p.Leu115Phe, and p.Gly186Ser variants, with substantially lower frequencies for Golden Retrievers compared with mixed-breed dogs, Labrador Retrievers, and other breeds. Consequently, the higher DOR/DXM Log MRs observed in Golden Retrievers might be a consequence of the lower frequency of the p.Gly186Ser allele that was associated with lower DOR/DXM Log MRs in the univariate analysis. However, only breed (Golden Retriever and Pug), but not p.Gly186Ser allele number, was significantly associated with DOR/DXM Log MR, indicating that the genetic variants other than p.Gly186Ser explain the higher DOR/DXM Log MRs in Golden Retrievers.
There was no association between dog sex (including sexes subcategorized as desexed or intact) or age and DOR/DXM Log MR. No significant sex or age-dependent differences in CYP2D6-mediated DXM metabolism have been reported in humans and horses.17–19 A small, albeit statistically significant, increase in DOR/DXM Log MRs was observed with increasing body weight in the univariate analysis but not in the multivariate analysis. The cause of this discrepancy may be covariance between the weight and breed variables. Specifically, the faster-metabolizing Golden Retriever breed had higher body weight (33 ± 8 kg; N = 23) than the slower-metabolizing Pugs (6.6 ± 0.8 kg; N = 3). Consequently, this apparent effect of weight in the univariate analysis was not observed in the multivariate analysis after inclusion of Golden Retriever and Pug breeds as covariates.
This study had some limitations. Although a relatively large number of dogs were studied, most were mixed-breed dogs or individual dogs from multiple different breeds. Consequently, this limited the ability to discriminate against potential breed differences. Regardless, the study was able to show that Golden Retrievers are relatively fast CYP2D15 metabolizers. Furthermore, Pugs were identified as relatively slow metabolizers, although this finding was based on only 3 individual dogs, which needs to be confirmed by studying additional dogs in this breed. Finally, drug metabolism phenotype was determined with a single CYP2D15 probe using a limited sampling strategy (single 6-hour urine samples) that is appropriate for screening large populations. Consequently, any findings will need confirmation using complementary approaches, including determining complete plasma pharmacokinetic profiles with DXM and the evaluation of alternate CYP2D15 probes.
In conclusion, like human CYP2D6, canine CYP2D15 demonstrates high interindividual phenotypic variability in dogs. Also, like human CYP2D6, breed (population group) differences are evident. However, unlike human CYP2D6, the genetic causes of this variability are not yet understood.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
This work was supported by the American Kennel Club Canine Health Foundation (grant Nos. 2242 and 2529) and William R. Jones Endowment at Washington State University College of Veterinary Medicine and Morris Animal Foundation Grant Nos. D21CA-608 and D22CA-310.
ORCID
T. P. Jimenez https://orcid.org/0000-0002-5867-5276
S. Martinez https://orcid.org/0000-0001-8430-4973
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