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.

Assessment of verdinexor as a canine P-glycoprotein substrate.

The P-glycoprotein (P-gp) substrate status of antineoplastic drugs intended for veterinary patients is an important characteristic to define for two reasons. First, neoplastic cells expressing P-gp can actively efflux drugs that are P-gp substrates curtailing their efficacy. Second, antineoplastic drugs tend to have a narrow therapeutic index. Antineoplastic drugs that are P-gp substrates can cause severe adverse reactions in animals with P-gp dysfunction such as dogs with ABCB1-1Δ and cats with ABCB11930_1931del TC. Animals with P-gp dysfunction experience greater overall exposure to P-gp substrate drugs due to mechanisms such as increased intestinal absorption, decreased biliary clearance and greater central nervous system penetration compared with animals with normal P-gp function. Accordingly, knowing the P-gp substrate status of antineoplastic drugs is an important safety consideration prior to use in canine or feline cancer patients. This study used a cell line overexpressing canine P-gp to assess the P-gp substrate status of verdinexor. Based on both a cytotoxicity assay and a competitive flow cytometry assay verdinexor is not a substrate for canine P-gp.

Canine and feline P-glycoprotein deficiency: What we know and where we need to go.

In 2001 the molecular genetic basis of so-called "ivermectin sensitivity" in herding breed dogs was determined to be a P-glycoprotein deficiency caused by a genetic variant of the MDR1 (ABCB1) gene often called "the MDR1 mutation." We have learned a great deal about P-glycoprotein's role in drug disposition since that discovery, namely that P-glycoprotein transports many more drugs than just macrocyclic lactones that P-glycoprotein mediated drug transport is present in more places than just the blood brain barrier, that some cats have a genetic variant of MDR1 that results in P-glycoprotein deficiency, that P-glycoprotein dysfunction can occur as a result of drug-drug interactions in any dog or cat, and that the concept of P-glycoprotein "inhibitors" versus P-glycoprotein substrates is somewhat arbitrary and artificial. This paper will review these discoveries and discuss how they impact drug selection and dosing in dogs and cats with genetically mediated P-glycoprotein deficiency or P-glycoprotein dysfunction resulting from drug-drug interactions.

Tolerance and Pharmacokinetics of Galliprant™ Administered Orally to Collies Homozygous for MDR1-1Δ.

The objectives of the study were to evaluate the pharmacokinetics and tolerance of grapiprant, a substrate of the human P-gp transporter, in collies homozygous for MDR1-1Δ when administered at the labeled dosage of 2 mg/kg once daily for 28 days. Twelve collie dogs with homozygous for MDR1-1Δ genotype from a commercial colony were used in the study, eight in the treated group and four as placebo-treated controls. The only treatment-related clinical sign was self-limiting vomiting (in 2/8 treated animals) and the only treatment-related clinical pathological changes seen were a slight decrease in serum albumin in one dog (2.6 g/dL; reference 2.7 to 3.9 g/dL) and total protein (5.1 g/dL; reference 5.5 to 7.7 g/dL). Absorption of grapiprant was rapid with a median Tmax of 1 h, Cmax of 5.2 µg/mL, AUC0-24 of 17.3 ± 7.1 h* µg/mL and median terminal t½ of 4.3 h after the first dose. To determine whether MDR1-1Δ animals handle grapiprant differently from normal dogs, a population pharmacokinetic analysis was performed utilizing data from the collies and historical beagle data. Volume of the peripheral compartment of collies was estimated to be 45% that of beagles, and clearance from the central compartment was 71% less in collies than in beagles. Self-liming vomiting events occurred at a numerically higher rate (2/8; 25%) in this group of P-gp-deficient dogs than seen in a clinical study (17%) composed of various dog breeds but limited numbers in this PK study make comparisons difficult. Grapiprant was otherwise well tolerated in collies homozygous for MDR1-1Δ despite increased drug exposure compared to dogs without this mutation.

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.

PREVALENCE OF THE ABCB1-1Δ GENE IN NONDOMESTIC SPECIES OF THE CANIDAE FAMILY.

The ABCB1 gene is responsible for encoding the P-glycoprotein (P-gp) efflux transporter that prevents accumulation of exogenous substances in the body by utilizing ATP hydrolysis to transport these substances against their concentration gradient. In dogs, homozygous or heterozygous mutations for the previously described ABCB1-1Δ mutation lead to ineffective P-gp efflux transport function and puts the animal at risk for potentially devastating adverse drug effects. The purpose of this study was to evaluate ABCB1-1Δ gene mutation status in species belonging to the Canidae family, including each of the following: maned wolf (Chrysocyon brachyurus), gray wolf (Canis lupus), red wolf (Canis rufus), coyote (Canis latrans), dingo (Canis lupus dingo), New Guinea singing dog (Canis lupus dingo), arctic fox (Vulpes lagopus), and fennec fox (Vulpes zerda). These species were chosen based on an evolutionary study conducted by Belyaev that noted foxes, bred for temperament, tended to have similar behaviors seen in the modern-day dog. Wolves, known predecessors to the modern dog, were also included. In the current study, a buccal swab was performed on each animal and then tested at Washington State University's Veterinary Clinical Pharmacology Lab, where they were tested according to previously published methods validating buccal swab samples and polymerase chain reaction (PCR) -based genetic analysis. Knowledge of Canidae species ABCB1-1Δ gene mutation status allows for safe and effective therapeutic treatment of nondomestic animals, ensuring any anticipated adverse drug events are prevented. All eight species were found to have the wild-type ABCB1 gene and thus, expected to have normally functioning P-gp efflux transporters. Although these data can be used to guide clinical decision making, because of a small sample size, a more robust study is necessary to assess Canidae ABCB1-1Δ mutation status comprehensively.

Development of prednisone resistance in naïve canine lymphoma: Longitudinal evaluation of NR3C1α, ABCB1, and 11ß-HSD mRNA expression.

Prednisone resistance develops rapidly and essentially universally when dogs with lymphoma are treated with corticosteroids. We investigated naturally occurring mechanisms of prednisone resistance in seven dogs with naïve multicentric lymphoma, treated with oral prednisone; four dogs were administered concurrent cytotoxic chemotherapy. Expression of NR3C1α, ABCB1 (formerly MDR1), 11ß-HSD1, and 11ß-HSD2 mRNA was evaluated in neoplastic lymph nodes by real-time RT-PCR. Changes of expression levels at diagnosis and at time of clinical resistance to prednisone were compared longitudinally using a Wilcoxon signed-rank test. Clinical resistance to prednisone was observed after a median of 68 days (range: 7-348 days) after initiation of treatment. Relative to pretreatment samples, prednisone resistance was associated with decreased NR3C1α expression in biopsies of all dogs with high-grade lymphoma (six dogs, p=.031); one dog with indolent T-zone lymphoma had increased expression of NR3C1α. Resistance was not consistently associated with changes in ABCB1, 11ß-HSD1, or 11ß-HSD2 expression. Decreased expression of the glucocorticoid receptor (NR3C1α) may play a role in conferring resistance to prednisone in dogs with lymphoma. Results do not indicate a broad role for changes in expression of ABCB1, 11ß-HSD1, and 11ß-HSD2 in the emergence of prednisone resistance in lymphoma-bearing dogs.

Excessive Cyclosporine-Associated Immunosuppression in a Dog Heterozygous for the MDR1 (ABCB1-1Δ) Mutation.

Pharmacodynamic monitoring was used to titrate cyclosporine dosing in a dog with immune-mediated hemolytic anemia. Development of a suspected secondary infection, with subsequent discovery of an unexpectedly high level of T-cell suppression despite a relatively low cyclosporine dose, prompted an investigation into the cause of possible excessive immunosuppression. Blood cyclosporine concentrations were within expected target ranges, and the dog was determined to be heterozygous for the multidrug resistance protein 1 (MDR1; ATP-binding cassette sub family B member 1-1Δ) gene mutation. The MDR1 mutation was suspected to have contributed to the excessive immunosuppression experienced by this patient. This case highlights the need to monitor immunosuppressive therapy in the individual patient, especially when the patient is not responding to therapy at typical dosages or when secondary infections develop at dosages lower than expected to cause significant immunosuppression. Pharmacodynamic monitoring can be used to help identify unexpected excessive immunosuppression in dogs receiving cyclosporine, and MDR1 genotyping should be further explored as a potential method of predicting and preventing its occurrence.

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.

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.

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.

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.

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.

Tramadol metabolism to O-desmethyl tramadol (M1) and N-desmethyl tramadol (M2) by dog liver microsomes: Species comparison and identification of responsible canine cytochrome P-450s (CYPs).

Tramadol is widely used to manage mild to moderately painful conditions in dogs. However, this use is controversial since clinical efficacy studies in dogs showed conflicting results, while pharmacokinetic studies demonstrated relatively low circulating concentrations of O-desmethyltramadol (M1). Analgesia has been attributed to the opioid effects of M1, while tramadol and the other major metabolite (N-desmethyltramadol, M2) are considered inactive at opioid receptors. The aims of this study were to determine whether cytochrome P450 (CYP) dependent M1 formation by dog liver microsomes is slower compared with cat and human liver microsomes; and identify the CYPs responsible for M1 and M2 formation in canine liver. Since tramadol is used as a racemic mixture of (+)- and (-)-stereoisomers, both (+)-tramadol and (-)- tramadol were evaluated as substrates. M1 formation from tramadol by liver microsomes from dogs was slower than from cats (3.9-fold), but faster than humans (7-fold). However, M2 formation by liver microsomes from dogs was faster than from cats (4.8-fold) and humans (19-fold). Recombinant canine CYP activities indicated that M1 was formed by CYP2D15, while M2 was largely formed by CYP2B11 and CYP3A12. This was confirmed by dog liver microsomes studies that showed selective inhibition of M1 formation by quinidine and M2 formation by chloramphenicol and CYP2B11 antiserum, and induction of M2 formation by phenobarbital. Findings were similar for both (+)-tramadol and (-)-tramadol. In conclusion, low circulating M1 concentrations in dogs is explained in part by low M1 formation and high M2 formation, which are mediated by CYP2D15 and CYP2B11/CYP3A12, respectively.

Impact of the canine double-deletion ß1 adrenoreceptor polymorphisms on protein structure and heart rate response to atenolol, a ß1-selective ß-blocker.

OBJECTIVE: ß-Adrenergic receptor antagonists are widely utilized for the management of cardiac diseases in dogs. We have recently identified two deletion polymorphisms in the canine adrenoreceptor 1 (ADRB1) gene.We hypothesized that canine ADRB1 deletions would alter the structure of the protein, as well as the heart rate response to the ß-adrenergic receptor antagonist, atenolol. The objectives of this study were to predict the impact of these deletions on the predicted structure of the protein and on the heart rate response to atenolol in a population of healthy adult dogs. METHODS: Eighteen apparently healthy, mature dogs with (11) and without (seven) ADRB1 deletions were evaluated. The heart rate of the dogs was evaluated with a baseline ambulatory ECG before and 14-21 days after atenolol therapy (1 mg/kg orally q12 h). Minimum, average, and maximum heart rates were compared between groups of dogs (deletions, controls) using an unpaired t-test and within each group of dogs using a paired t-test. The protein structure of ADRB1 was predicted by computer modeling. RESULTS: Deletions were predicted to alter the structure of the ADRB1 protein. The heart rates of the dogs with deletions were lower than those of the control dogs (the average heart rates were significantly lower). CONCLUSION: ADRB1 deletions appear to have structural and functional consequences. Individual genome-based treatment recommendations could impact the management of dogs with heart disease.

Tyrosine kinase inhibitors enhance ciprofloxacin-induced phototoxicity by inhibiting ABCG2.

The tyrosine kinase inhibitor (TKI) class of anticancer agents inhibits ABCG2-mediated drug efflux. ABCG2 is an important component of the blood-retinal barrier, where it limits retinal exposure to phototoxic compounds such as fluoroquinolone antibiotics. Patients treated with TKIs would be expected to be at greater risk for retinal phototoxicity. Using an in vitro system, our results indicate that the TKIs gefitinib and imatinib abrogate the ability of ABCG2 to protect cells against ciprofloxacin-induced phototoxicity. We conclude that the concurrent administration of ABCG2 inhibitors with photoreactive fluoroquinolone antibiotics may result in retinal damage.

Challenges in exploring the cytochrome P450 system as a source of variation in canine drug pharmacokinetics.

The cytochrome P450 (CYP) superfamily constitutes a collection of enzymes responsible for the metabolism of a wide array of endo- and xenobiotic compounds. Much of the knowledge on substrate specificity and genetic identification of the various CYP isoforms is derived from research in rodents and humans and only limited information has been captured in the dog. Currently, there exist many gaps in our knowledge of canine CYP diversity as a result of the paucity of studies focusing on canine CYPs, canine CYP polymorphisms, and the therapeutic consequences of these genetic variants. Challenges engendered by this lack of information is further amplified by inter- and intraspecies differences in the specificity and affinity of substrates and inhibitors, prohibiting a simple extrapolation of probe substances used in human CYP research. This creates a need to develop and validate canine-specific CYP probes. Failure to understand this potential metabolic and pharmacogenomic diversity can also influence the interpretation of data generated in dogs to support human drug development. It is with these objectives in mind that we provide an overview of what is currently known about canine CYPs with the hope that it will encourage further exploration into this important area of research.

Molecular genetic basis for fluoroquinolone-induced retinal degeneration in cats.

OBJECTIVES: Distribution of fluoroquinolones to the retina is normally restricted by ABCG2 at the blood-retinal barrier. As the cat develops a species-specific adverse reaction to photoreactive fluoroquinolones, our goal was to investigate ABCG2 as a candidate gene for fluoroquinolone-induced retinal degeneration and blindness in cats. METHODS: Feline ABCG2 was sequenced and the consensus amino acid sequence was compared with that of 10 other mammalian species. Expression of ABCG2 in feline retina was assessed by immunoblot. cDNA constructs for feline and human ABCG2 were constructed in a pcDNA3 expression vector and expressed in HEK-293 cells, and ABCG2 expression was analyzed by western blot and immunofluorescence. Mitoxantrone and BODIPY-prazosin efflux measured by flow cytometry and a phototoxicity assay were used to assess feline and human ABCG2 function. RESULTS: Four feline-specific (compared with 10 other mammalian species) amino acid changes in conserved regions of ABCG2 were identified. Expression of ABCG2 on plasma membranes was confirmed in feline retina and in cells transfected with human and feline ABCG2, although some intracellular expression of feline ABCG2 was detected by immunofluorescence. Function of feline ABCG2, compared with human ABCG2, was found to be deficient as determined by flow cytometric measurement of mitoxantrone and BODIPY-prazosin efflux and enrofloxacin-induced phototoxicity assays. CONCLUSION: Feline-specific amino acid changes in ABCG2 cause a functional defect of the transport protein in cats. This functional defect may be owing, in part, to defective cellular localization of feline ABCG2. Regardless, dysfunction of ABCG2 at the blood-retinal barrier likely results in accumulation of photoreactive fluoroquinolones in feline retina. Exposure of the retina to light would then generate reactive oxygen species that would cause the characteristic retinal degeneration and blindness documented in some cats receiving high doses of some fluoroquinolones. Pharmacological inhibition of ABCG2 in other species might result in retinal damage if fluoroquinolones are concurrently administered.