Assessment of cytochrome P450 induction in canine intestinal organoid models.

Understanding cytochrome P450 (CYP) enzymes in the canine intestine is vital for predicting drug metabolism and developing safer oral medications. This study evaluates canine colonoids as a model to assess the expression and induction of essential intestinal CYP enzymes.Canine colonoids were cultured in expansion medium (EM) with Wnt-3A and in differentiation medium (DM) without Wnt-3A. We assessed the mRNA expression of CYP2B11, CYP2C21, CYP3A12, and CYP3A98 using qPCR and examined the effects of rifampicin and phenobarbital as inducers.Our findings show that DM significantly increased the mRNA expression of CYP3A98 and CYP2B11, but not CYP3A12, compared to EM. CYP2C21, not typically expressed in the intestine, remained unexpressed in colonoids. Rifampicin induced CYP3A98, aligning with pregnane x receptor (PXR) regulation, while phenobarbital did not, suggesting no constitutive androstane receptor (CAR) involvement. CYP2B11 did not respond to either inducer, suggesting alternative regulatory pathways in canine colonoids.This study is a pioneering effort to establish conditions for studying P450 expression in canine colonoids, confirming significant CYP3A98 expression in the canine intestine. It demonstrated colonoids can induce CYP activity post drug treatments. Further research is needed to enhance species-specific drug metabolism understanding and validate this model for broader applications.

Cannabidiol and cannabidiolic acid: Preliminary in vitro evaluation of metabolism and drug-drug interactions involving canine cytochrome P-450, UDP-glucuronosyltransferase, and P-glycoprotein.

Phytocannabinoid-rich hemp extracts containing cannabidiol (CBD) and cannabidiolic acid (CBDA) are increasingly being used to treat various disorders in dogs. The objectives of this study were to obtain preliminary information regarding the in vitro metabolism of these compounds and their capacity to inhibit canine cytochrome P450 (CYP)-mediated drug metabolism and canine P-glycoprotein-mediated transport. Pure CBD and CBDA, and hemp extracts enriched for CBD and for CBDA were evaluated. Substrate depletion assays using pooled dog liver microsomes showed CYP cofactor-dependent depletion of CBD (but not CBDA) and UDP-glucuronosytransferase cofactor-dependent depletion of CBDA (but not CBD) indicating major roles for CYP and UDP-glucuronosytransferase in the metabolism of these phytocannabinoids, respectively. Further studies using recombinant canine CYPs demonstrated substantial CBD depletion by the major hepatic P450 enzymes CYP1A2 and CYP2C21. These results were confirmed by showing increased CBD depletion by liver microsomes from dogs treated with a known CYP1A2 inducer (ß-naphthoflavone) and with a known CYP2C21 inducer (phenobarbital). Cannabinoid-drug inhibition experiments showed inhibition (IC50 = 4.6-8.1 µM) of tramadol metabolism via CYP2B11-mediated N-demethylation (CBD and CBDA) and CYP2D15-mediated O-demethylation (CBDA only) by dog liver microsomes. CBD and CBDA did not inhibit CYP3A12-mediated midazolam 1'-hydroxylation (IC50 > 10 µM). CBD and CBDA were not substrates or competitive inhibitors of canine P-glycoprotein. Results for cannabinoid-enriched hemp extracts were identical to those for pure cannabinoids. These in vitro studies indicate the potential for cannabinoid-drug interactions involving certain CYPs (but not P-glycoprotein). Confirmatory in vivo studies are warranted.

Application of eprinomectin-containing parasiticides at label doses causes neurological toxicosis in cats homozygous for ABCB11930_1931del TC.

The feline MDR1 mutation (ABCB11930_1931delTC) has been associated with neurological toxicosis after topical application of eprinomectin products labeled for feline use. Information was collected from veterinarians who submitted samples for ABCB11930_1931delTC genotyping. In most cases, the submission form indicated an adverse event involving eprinomectin, in other cases submitting veterinarians were contacted to determine whether the patient had experienced an adverse drug event involving eprinomectin. If so, additional information was obtained to determine whether the case met inclusion criteria. 14 cases were highly consistent with eprinomectin toxicosis. Eight cats were homozygous for ABCB11930_1931del TC (3 died; 5 recovered). Six cats were homozygous wildtype (2 died; 4 recovered). The observed ABCB11930_1931delTC frequency (57%) was higher than the expected frequency (≤1%) in the feline population (Fisher Exact test, p < 0.01). Among wildtype cats, four were concurrently treated with potential competitive inhibitors of P-glycoprotein. Results indicate that topical eprinomectin products, should be avoided in cats homozygous for ABCB11930_1931delTC. This is a serious, preventable adverse event occurring in an identifiable subpopulation treated with FDA-approved products in accordance with label directions. Acquired P-glycoprotein deficiency resulting from drug interactions may enhance susceptibility to eprinomectin-induced neurological toxicosis in any cat, regardless of ABCB1 genotype.

Association of cytochrome P450 2D15 (CYP2D15) nonsynonymous polymorphisms and exon 3 deleted RNA splice variant with CYP2D15 protein content and enzyme function in dog liver microsomes.

CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3-delta) was associated with differences in CYP2D15-selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15-selective activities measured included dextromethorphan o-demethylation and tramadol o-demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3-delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3-delta was not correlated with CYP2D15 activities or protein content. Compared with wild-type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p < .05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O-demethylation activities versus wild-type livers (p < .05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver.

Canine orosomucoid (alpha-1 acid glycoprotein) variants and their influence on drug plasma protein binding.

Orosomucoid polymorphisms influence plasma drug binding in humans; however, canine variants and their effect on drug plasma protein binding have not yet been reported. In this study, the orosomucoid gene (ORM1) was sequenced in 100 dogs to identify the most common variant and its allele frequency determined in 1,464 dogs (from 64 breeds and mixed-breed dogs). Plasma protein binding extent of amitriptyline, indinavir, verapamil, and lidocaine were evaluated by equilibrium dialysis using plasma from ORM1 genotyped dogs (n = 12). Free and total drug plasma concentrations were quantified by liquid chromatography-mass spectrometry. From the five polymorphisms identified in canine ORM1, two were nonsynonymous. The most common was c.70G>A (p.Ala24Thr) with an allele frequency of 11.2% (n = 1464). Variant allele frequencies varied by breed, reaching 74% in Shetland Sheepdogs (n = 21). Free drug fractions did not differ significantly (p > .05; Mann-Whitney U) between plasma collected from dogs with c.70AA (n = 4) and those with c.70GG (n = 8) genotypes. While c.70G>A did not affect the extent of plasma protein binding in our study, the potential biological and pharmacological implication of this newly discovered ORM1 variant in dogs should be further investigated.

Effect of Rifampin-Isoniazid-Containing Antituberculosis Therapy on Efavirenz Pharmacokinetics in HIV-Infected Children 3 to 14 Years Old.

We compared efavirenz pharmacokinetic (PK) parameters in children with tuberculosis (TB)/human immunodeficiency virus (HIV) coinfection on and off first-line antituberculosis therapy to that in HIV-infected children. Children 3 to 14 years old with HIV infection, with and without TB, were treated with standard efavirenz-based antiretroviral therapy without any efavirenz dose adjustments. The new World Health Organization-recommended antituberculosis drug dosages were used in the coinfected participants. Steady-state efavirenz concentrations after 4 weeks of antiretroviral therapy were measured using validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays. Pharmacokinetic parameters were calculated using noncompartmental analysis. Between groups, PK parameters were compared by Wilcoxon rank-sum test and within group by signed-rank test. Of the 105 participants, 43 (41.0%) had TB coinfection. Children with TB/HIV coinfection compared to those with HIV infection were younger, had lower median weight-for-age Z score, and received a higher median efavirenz weight-adjusted dose. Geometric mean (GM) efavirenz peak concentration (Cmax), concentration at 12 h (C12h), Cmin, and total area under the curve from time 0 to 24 h (AUC0-24h) values were similar in children with HIV infection and those with TB/HIV coinfection during anti-TB therapy. Geometric mean efavirenz C12h, Cmin, and AUC0-24h values were lower in TB/HIV-coinfected patients off anti-TB therapy than in the children with HIV infection or TB/HIV coinfection on anti-TB therapy. Efavirenz clearance was lower and AUC0-24h was higher on than in patients off anti-TB therapy. Reduced efavirenz clearance by first-line anti-TB therapy at the population level led to similar PK parameters in HIV-infected children with and without TB coinfection. Our findings do not support modification of efavirenz weight-band dosing guidelines based on TB coinfection status in children. (The study was registered with ClinicalTrials.gov under registration number NCT01704144.).

Effect of First-Line Antituberculosis Therapy on Nevirapine Pharmacokinetics in Children Younger than Three Years Old.

Nevirapine-based antiretroviral therapy (ART) is one of the limited options in HIV-infected children younger than 3 years old (young children) with tuberculosis (TB) coinfection. To date, there are insufficient data to recommend nevirapine-based therapy during first-line antituberculosis (anti-TB) therapy in young children. We compared nevirapine pharmacokinetics (PK) in HIV-infected young children with and without TB coinfection. In the coinfected group, nevirapine PK was evaluated while on anti-TB therapy and after completing an anti-TB therapy regimen. Of 53 participants, 23 (43%) had TB-HIV coinfection. While the mean difference in nevirapine PK parameters between the two groups was not significant (P > 0.05), 14/23 (61%) of the children with TB-HIV coinfection and 9/30 (30%) with HIV infection had a nevirapine minimum concentration (Cmin) below the proposed target of 3.0 mg/liter (P = 0.03). In multivariate analysis, anti-TB therapy and the CYP2B6 516G>T genotype were joint predictors of nevirapine PK parameters. Differences in nevirapine PK parameters between the two groups were significant in children with CYP2B6 516GG but not the GT or TT genotype. Among 14 TB-HIV-coinfected participants with paired data, the geometric mean Cmin and area under the drug concentration-time curve from time zero to 12 h (AUC0-12) were about 34% lower when patients were taking anti-TB therapy, while the nevirapine apparent oral clearance (CL/F) was about 45% higher. While the induction effect of anti-TB therapy on nevirapine PK in our study was modest, the CYP2B6 genotype-dependent variability in the TB drug regimen effect would complicate any dose adjustment strategy in young children with TB-HIV coinfection. Alternate ART regimens that are more compatible with TB treatment in this age group are needed. (This study has been registered at ClinicalTrials.gov under identifier NCT01699633.).

Personalized medicine: going to the dogs?

Interindividual variation in drug response occurs in canine patients just as it does in human patients. Although canine pharmacogenetics still lags behind human pharmacogenetics, significant life-saving discoveries in the field have been made over the last 20 years, but much remains to be done. This article summarizes the available published data about the presence and impact of genetic polymorphisms on canine drug transporters, drug-metabolizing enzymes, drug receptors/targets, and plasma protein binding while comparing them to their human counterparts when applicable. In addition, precision medicine in cancer treatment as an application of canine pharmacogenetics and pertinent considerations for canine pharmacogenetics testing is reviewed. The field is poised to transition from single pharmacogene-based studies, pharmacogenetics, to pharmacogenomic-based studies to enhance our understanding of interindividual variation of drug response in dogs. Advances made in the field of canine pharmacogenetics will not only improve the health and well-being of dogs and dog breeds, but may provide insight into individual drug efficacy and toxicity in human patients as well.

Oral Coadministration of Fluconazole with Tramadol Markedly Increases Plasma and Urine Concentrations of Tramadol and the O-Desmethyltramadol Metabolite in Healthy Dogs.

Tramadol is used frequently in the management of mild to moderate pain conditions in dogs. This use is controversial because multiple reports in treated dogs demonstrate very low plasma concentrations of O-desmethyltramadol (M1), the active metabolite. The objective of this study was to identify a drug that could be coadministered with tramadol to increase plasma M1 concentrations, thereby enhancing analgesic efficacy. In vitro studies were initially conducted to identify a compound that inhibited tramadol metabolism to N-desmethyltramadol (M2) and M1 metabolism to N,O-didesmethyltramadol (M5) without reducing tramadol metabolism to M1. A randomized crossover drug-drug interaction study was then conducted by administering this inhibitor or placebo with tramadol to 12 dogs. Blood and urine samples were collected to measure tramadol, tramadol metabolites, and inhibitor concentrations. After screening 86 compounds, fluconazole was the only drug found to inhibit M2 and M5 formation potently without reducing M1 formation. Four hours after tramadol administration to fluconazole-treated dogs, there were marked statistically significant (P < 0.001; Wilcoxon signed-rank test) increases in plasma tramadol (31-fold higher) and M1 (39-fold higher) concentrations when compared with placebo-treated dogs. Conversely, plasma M2 and M5 concentrations were significantly lower (11-fold and 3-fold, respectively; P < 0.01) in fluconazole-treated dogs. Metabolite concentrations in urine followed a similar pattern. This is the first study to demonstrate a potentially beneficial drug-drug interaction in dogs through enhancing plasma tramadol and M1 concentrations. Future studies are needed to determine whether adding fluconazole can enhance the analgesic efficacy of tramadol in healthy dogs and clinical patients experiencing pain.

Absolute Quantitation of Drug-Metabolizing Cytochrome P450 Enzymes and Accessory Proteins in Dog Liver Microsomes Using Label-Free Standard-Free Analysis Reveals Interbreed Variability.

Dogs are commonly used in human and veterinary pharmaceutical development. Physiologically based pharmacokinetic modeling using recombinant cytochrome P450 (CYP) enzymes requires accurate estimates of CYP abundance, particularly in liver. However, such estimates are currently available for only seven CYPs, which were determined in a limited number of livers from one dog breed (beagle). In this study, we used a label-free shotgun proteomics method to quantitate 11 CYPs (including four CYPs not previously measured), cytochrome P450 oxidoreductase, and cytochrome b5 in liver microsomes from 59 dogs representing four different breeds and mixed-breed dogs. Validation included showing correlation with CYP marker activities, immunoquantified protein, as well as CYP1A2 and CYP2C41 null allele genotypes. Abundance values largely agreed with those previously published. Average CYP abundance was highest (>120 pmol/mg protein) for CYP2D15 and CYP3A12; intermediate (40-89 pmol/mg) for CYP1A2, CYP2B11, CYP2E1, and CYP2C21; and lowest (<12 pmol/mg) for CYP2A13, CYP2A25, CYP2C41, CYP3A26, and CYP1A1. The CYP2C41 gene was detected in 12 of 58 (21%) livers. CYP2C41 protein abundance averaged 8.2 pmol/mg in those livers, and was highest (19 pmol/mg) in the only liver with two CYP2C41 gene copies. CYP1A2 protein was not detected in the only liver homozygous for the CYP1A2 stop codon mutation. Large breed-associated differences were observed for CYP2B11 (P < 0.0001; ANOVA) but not for other CYPs. Research hounds and Beagles had the highest CYP2B11 abundance; mixed-breed dogs and Chihuahua were intermediate; whereas greyhounds had the lowest abundance. These results provide the most comprehensive estimates to date of CYP abundance and variability in canine liver. SIGNIFICANCE STATEMENT: This work provides the most comprehensive quantitative analysis to date of the drug-metabolizing cytochrome P450 proteome in dogs that will serve as a valuable reference for physiologically based scaling and modeling used in drug development and research. This study also revealed high interindividual variation and dog breed-associated differences in drug-metabolizing cytochrome P450 expression that may be important for predicting drug disposition variability among a genetically diverse canine population.

Canine Albumin Polymorphisms and Their Impact on Drug Plasma Protein Binding.

Drug binding to plasma proteins is routinely determined during drug development. Albumin polymorphisms c.1075G>T (p.Ala359Ser) and c.1422A>T (p.Glu474Asp) were previously shown to alter plasma protein binding of a drug candidate (D01-4582, 4-[1-[3-chloro-4-[N'-(2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidine-2-yl]methoxybenzoic acid) in a colony of Beagles. Our study investigated the hypothesis that drug-protein binding in plasma from dogs with the albumin H1 (reference) allele would be greater than in plasma from dogs with the albumin H2 allele (c.1075G>T and c.1422A>T) (n = 6 per group). The plasma protein binding extent of four drugs (D01-4582, celecoxib, mycophenolic acid, and meloxicam) was evaluated using ultracentrifugation or equilibrium dialysis. Free and total drug concentrations were analyzed by liquid chromatography-mass spectrometry. The albumin gene coding region was sequenced in 100 dogs to detect novel gene variants, and H1/H2 allele frequency was determined in a large and varied population (n = 1446 from 61 breeds and mixed-breed dogs). For meloxicam, H1 allele plasma had statistically significant higher free drug fractions (P = 0.041) than H2 allele plasma. No significant difference was identified for plasma protein binding of D01-4582, celecoxib, or mycophenolic acid. c.1075G>T and c.1422A>T were the most common single nucleotide polymorphisms in canine albumin, present concurrently in most study dogs and occasionally identified independently. Our findings suggest a potential influence of c.1075G>T and c.1422A>T on plasma protein binding. This influence should be confirmed in vivo and for additional drugs. Based on our results, albumin genotyping should be considered for canine research subjects to improve interpretation of pharmacokinetic data generated during the drug development process for humans and dogs.

High interindividual variability in plasma clopidogrel active metabolite concentrations in healthy cats is associated with sex and cytochrome P450 2C genetic polymorphism.

Clopidogrel response variability has been identified in cats. In humans, evidence suggests that variable clopidogrel active metabolite (CAM) generation is the primary explanation for clopidogrel response variability with differences in body weight, sex, and variable metabolism of clopidogrel primarily due to polymorphisms of the gene encoding cytochrome P450 (CYP) 2C19 as some proposed mechanisms. The aim of this study was to evaluate whether variation in CAM concentrations exists in healthy cats and what the cause of such variation might be. Nineteen healthy cats were given 18.75 mg clopidogrel by mouth. Blood was collected 2 hr later. Plasma CAM concentrations were measured using high performance liquid chromatography and tandem mass spectrometry. Clopidogrel metabolism was estimated by calculating CAM metabolic ratio. DNA was collected, and feline CYP2C genotyping was performed. The cats demonstrated high interindividual variation of plasma CAM concentrations. Approximately 69% of this interindividual variation was primarily explained by differences in clopidogrel metabolism as measured by CAM metabolic ratio with some influence by sex but not by weight. A single nucleotide polymorphism was identified in the feline CYP2C gene that explained in part individual differences in CAM metabolic ratio and CAM plasma concentrations.

Investigation into the causes of aspirin resistance in healthy dogs.

Antiplatelet effects of acetylsalicylic acid (ASA, aspirin) may be poor in some individuals. Additionally, no method exists for predicting poor ASA response (resistance) in individual dogs. This study's main objective was to determine whether poor ASA response results from pharmacodynamic or pharmacokinetic causes. ASA concentrations causing 50% inhibition of platelet aggregation (in vitro IC50) were determined using whole blood collected from 21 drug-free healthy dogs to evaluate intrinsic sensitivity of platelets to ASA. Dogs were then administered ASA at 4 mg/kg once orally. Percent decrease in platelet aggregation from baseline, and plasma ASA and salicylic acid (SA) concentrations (expressed as AUC values) were measured for up to 3 hr. By 3 hr, 13/21 (62%) dogs showed >50% aggregation inhibition, while 8/21 (38%) dogs showed <50% inhibition. Aggregation inhibition values were negatively correlated with in vitro IC50 values (Rs = -0.49; p = 0.028) and positively correlated with ASA concentrations (Rs = 0.48; p = 0.03). Furthermore, ASA concentrations were strongly negatively correlated (Rs = -0.88; p < 0.001) with SA/ASA concentration ratios, an index of ASA metabolism to SA by esterase enzymes. Multiple linear regression analysis indicated that 59% (p < 0.001) of interindividual variability in aggregation inhibition was explained by in vitro IC50 values (29% of variability) and ASA concentrations (29% of variability). Consequently, poor in vivo ASA response in these dogs resulted from both pharmacodynamic (decreased platelet sensitivity) and pharmacokinetic (lower ASA concentrations) causes. Lower ASA concentrations may be explained by reduced bioavailability associated with higher esterase activities.

Effect of Genetic Variation of NAT2 on Isoniazid and SLCO1B1 and CES2 on Rifampin Pharmacokinetics in Ghanaian Children with Tuberculosis.

Isoniazid and rifampin are essential components of first-line antituberculosis (anti-TB) therapy. Understanding the relationship between genetic factors and the pharmacokinetics of these drugs could be useful in optimizing treatment outcomes, but this is understudied in children. We investigated the relationship between N-acetyltransferase type 2 (NAT2) genotypes and isoniazid pharmacokinetics, as well as that between the solute carrier organic anion transporter family member 1B1 (encoded by SLCO1B1) and carboxylesterase 2 (CES2) single nucleotide polymorphisms (SNPs) and rifampin pharmacokinetics in Ghanaian children. Blood samples were collected at times 0, 1, 2, 4, and 8 h postdose in children with tuberculosis on standard first-line therapy for at least 4 weeks. Isoniazid and rifampin concentrations were determined by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and pharmacokinetic parameters were calculated using noncompartmental analysis. Genotyping of NAT2, SLCO1B1, and CES2 SNPs were performed using validated TaqMan genotyping assays. The Kruskal-Wallis test was used to compare pharmacokinetic parameters among the three genotypic groups and was followed by the Wilcoxon rank sum test for pairwise group comparisons. Genotype status inferred by the NAT2 4-SNP and 7-SNP genotyping panels identified children with a slow acetylator phenotype but not the rapid genotype. For rifampin, only the rare SLCO1B1*1b homozygous variant was associated with rifampin pharmacokinetics. Our findings suggest that NAT2 and SCLCO1B1*1b genotyping may have minimal clinical utility in dosing decisions at the population level in Ghanaian children, but it could be useful at the individual level or in populations that have a high frequency of implicated genotypes. Further studies in other populations are warranted.

Population variability in animal health: Influence on dose-exposure-response relationships: Part I: Drug metabolism and transporter systems.

There is an increasing effort to understand the many sources of population variability that can influence drug absorption, metabolism, disposition, and clearance in veterinary species. This growing interest reflects the recognition that this diversity can influence dose-exposure-response relationships and can affect the drug residues present in the edible tissues of food-producing animals. To appreciate the pharmacokinetic diversity that may exist across a population of potential drug product recipients, both endogenous and exogenous variables need to be considered. The American Academy of Veterinary Pharmacology and Therapeutics hosted a 1-day session during the 2017 Biennial meeting to explore the sources of population variability recognized to impact veterinary medicine. The following review highlights the information shared during that session. In Part I of this workshop report, we consider sources of population variability associated with drug metabolism and membrane transport. Part II of this report highlights the use of modeling and simulation to support an appreciation of the variability in dose-exposure-response relationships.

Identification of canine cytochrome P-450s (CYPs) metabolizing the tramadol (+)-M1 and (+)-M2 metabolites to the tramadol (+)-M5 metabolite in dog liver microsomes.

We previously showed that (+)-tramadol is metabolized in dog liver to (+)-M1 exclusively by CYP2D15 and to (+)-M2 by multiple CYPs, but primarily CYP2B11. However, (+)-M1 and (+)-M2 are further metabolized in dogs to (+)-M5, which is the major metabolite found in dog plasma and urine. In this study, we identified canine CYPs involved in metabolizing (+)-M1 and (+)-M2 using recombinant enzymes, untreated dog liver microsomes (DLMs), inhibitor-treated DLMs, and DLMs from CYP inducer-treated dogs. A canine P-glycoprotein expressing cell line was also used to evaluate whether (+)-tramadol, (+)-M1, (+)-M2, or (+)-M5 are substrates of canine P-glycoprotein, thereby limiting their distribution into the central nervous system. (+)-M5 was largely formed from (+)-M1 by recombinant CYP2C21 with minor contributions from CYP2C41 and CYP2B11. (+)-M5 formation in DLMs from (+)-M1 was potently inhibited by sulfaphenazole (CYP2C inhibitor) and chloramphenicol (CYP2B11 inhibitor) and was greatly increased in DLMs from phenobarbital-treated dogs. (+)-M5 was formed from (+)-M2 predominantly by CYP2D15. (+)-M5 formation from (+)-M1 in DLMs was potently inhibited by quinidine (CYP2D inhibitor) but had only a minor impact from all CYP inducers tested. Intrinsic clearance estimates showed over 50 times higher values for (+)-M5 formation from (+)-M2 compared with (+)-M1 in DLMs. This was largely attributed to the higher enzyme affinity (lower Km) for (+)-M2 compared with (+)-M1 as substrate. (+)-tramadol, (+)-M1, (+)-M2, or (+)-M5 were not p-glycoprotein substrates. This study provides a clearer picture of the role of individual CYPs in the complex metabolism of tramadol in dogs.

Race, Gender, and Genetic Polymorphism Contribute to Variability in Acetaminophen Pharmacokinetics, Metabolism, and Protein-Adduct Concentrations in Healthy African-American and European-American Volunteers.

Over 30 years ago, black Africans from Kenya and Ghana were shown to metabolize acetaminophen faster by glucuronidation and slower by oxidation compared with white Scottish Europeans. The objectives of this study were to determine whether similar differences exist between African-Americans and European-Americans, and to identify genetic polymorphisms that could explain these potential differences. Acetaminophen plasma pharmacokinetics and partial urinary metabolite clearances via glucuronidation, sulfation, and oxidation were determined in healthy African-Americans (18 men, 23 women) and European-Americans (34 men, 20 women) following a 1-g oral dose. There were no differences in acetaminophen total plasma, glucuronidation, or sulfation clearance values between African-Americans and European-Americans. However, median oxidation clearance was 37% lower in African-Americans versus European-Americans (0.57 versus 0.90 ml/min per kilogram; P = 0.0001). Although acetaminophen total or metabolite clearance values were not different between genders, shorter plasma half-life values (by 11-14%; P < 0.01) were observed for acetaminophen, acetaminophen glucuronide, and acetaminophen sulfate in women versus men. The UGT2B15*2 polymorphism was associated with variant-allele-number proportional reductions in acetaminophen total clearance (by 15-27%; P < 0.001) and glucuronidation partial clearance (by 23-48%; P < 0.001). UGT2B15 *2/*2 genotype subjects also showed higher acetaminophen protein-adduct concentrations than *1/*2 (by 42%; P = 0.003) and *1/*1 (by 41%; P = 0.003) individuals. Finally, CYP2E1 *1D/*1D genotype African-Americans had lower oxidation clearance than *1C/*1D (by 42%; P = 0.041) and *1C/*1C (by 44%; P = 0.048) African-Americans. Consequently, African-Americans oxidize acetaminophen more slowly than European-Americans, which may be partially explained by the CYP2E1*1D polymorphism. UGT2B15*2 influences acetaminophen pharmacokinetics in both African-Americans and European-Americans.

Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms.

1. Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast. 2. Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes >80% of activity at bisphenol-A concentrations under 5 µM, while UGT1A9 contributes up to 50% of activity at higher concentrations. 3. Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p = 0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes. 4. Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from <0.2 to 56 fmoles/min/mg protein. Breast mRNA expression of UGTs capable of glucuronidating bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p = 0.006). 5. UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.

Simultaneous determination of mycophenolic acid and its glucuronide and glycoside derivatives in canine and feline plasma by UHPLC-UV.

Cats and dogs can suffer from multiple autoimmune diseases. Mycophenolic acid (MPA) is a potentially useful immunosuppressant drug in cats and dogs, because of its well-documented efficacy in controlling autoimmune disease in humans. However, the pharmacokinetics and pharmacodynamics in these species remain to be determined. We have developed and validated a sensitive, precise, accurate and reproducible method that provides consistent quantification of MPA and its major derivatives, MPA phenol glucoside and MPA phenol glucuronide, in canine and feline plasma using ultra-high-pressure liquid chromatography coupled to an ultraviolet detector. The main advantages of this novel method include a small sample volume, easy sample preparation, a short chromatographic analysis time and the option to select either phenolphthalein ß-d-glucuronide or mycophenolic acid carboxybutoxy ether as internal standard. Results of validation indicate that this analytical method is suitable to study the plasma disposition of MPA and its derivatives in dogs and cats.

Relationship between the melanocortin-1 receptor (MC1R) variant R306ter and physiological responses to mechanical or thermal stimuli in Labrador Retriever dogs.

OBJECTIVE: Variants in the MC1R gene have been associated with red hair color and sensitivity to pain in humans. The study objective was to determine if a relationship exists between MC1R genotype and physiological thermal or mechanical nociceptive thresholds in Labrador Retriever dogs. STUDY DESIGN: Prospective experimental study. ANIMALS: Thirty-four Labrador Retriever dogs were included in the study following public requests for volunteers. Owner consent was obtained and owners verified that their dog was apparently not experiencing pain and had not been treated for pain during the previous 14 days. The study was approved by the Institutional Animal Care and Use Committee. METHODS: Nociceptive thresholds were determined from a mean of three thermal and five mechanical replications using commercially available algometers. Each dog was genotyped for the previously described MC1R variant (R306ter). Data were analyzed using one-way anova with post hoc comparisons using Tukey's test (p < 0.05). RESULTS: Thirteen dogs were homozygous wild-type (WT/WT), nine were heterozygous (WT/R306ter), and eight were homozygous variant (R306ter/R306ter) genotype. Four dogs could not be genotyped. A significant difference (p = 0.04) in mechanical nociceptive thresholds was identified between dogs with the WT/WT genotype (12.1±2.1 N) and those with the WT/R306ter genotype (9.2±2.4 N). CONCLUSION: A difference in mechanical, but not thermal, nociceptive threshold was observed between wild-type and heterozygous MC1R variants. Differences in nociceptive thresholds between homozygous R306ter variants and other genotypes for MC1R were not observed. CLINICAL RELEVANCE: Compared with the wild-type MC1R genotype, nociceptive sensitivity to mechanical force in dogs with a single variant R306ter allele may be greater. However, in contrast to the reported association between homozygous MC1R variants (associated with red hair color) and nociception in humans, we found no evidence of a similar relationship in dogs with the homozygous variant genotype.