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The cytochrome P450 (CYP) 3A family is often considered the most important CYP subfamily with regard to drug metabolism in people. Certainly, CYP3A4 contributes to poor oral bioavailability of a number of drugs, and a tremendous amount of effort has been made in attempting to find an appropriate model system to predict the oral bioavailability of candidate drugs. The dog is a species widely used as a preclinical model for the evaluation of drug safety and pharmacokinetics. Compared with other species, little information is available on the tissue distribution of CYP enzymes. The purpose of this study was to determine the level of messenger RNA (mRNA) expression of the canine CYP3A subfamily (CYP3A12 and CYP3A26) in the liver and duodenum. Overall, expression of CYP3A mRNA was greater in the liver than in the duodenum. Hepatic expression of CYP3A26 was greater than CYP3A12 in all dogs, with CYP3A26 comprising 75.2% of the hepatic mRNA CYP3A pool. Conversely, duodenal expression of CYP3A12 was greater than CYP3A26 in all dogs, with CYP3A12 comprising 99.8% of the duodenal mRNA CYP3A pool. In summary, these results represent an important step toward the systematic comparison of human and canine CYP3A enzymes, particularly in relation to oral bioavailability of substrate drugs.

Despite advancements in pharmacogenetics in human medicine, the incorporation of pharmacogenetics into veterinary medicine is still in its early stages of development. To date, efforts to understand the pharmacologic impact of genetic variation in veterinary species have largely focused on genes encoding for the membrane transporter, P-glycoprotein (P-gp). The emphasis on the role of P-gp is largely because of safety concerns associated with the use of some macrocyclic lactones in dogs. Because of the body of information available on this topic, we use P-gp as a platform for understanding the importance of population diversity in veterinary medicine. The impact of P-gp on drug pharmacokinetics and pharmacodynamics is considered, along with endogenous and exogenous factors that can modulate P-gp activity. The review includes discussion of how population diversity in P-gp activity can lead to susceptibility to certain diseases or alter patient response to environmental stress or pharmaceutical intervention. In addition, phenotypic diversity also needs to be considered, as demonstrated by the impact of P-gp up-regulation and drug resistance. The aim of this review was to set the stage for further exploration into the impact of genetic and phenotypic variability on drug pharmacokinetics, disease propensity, product formulation and drug response in both companion and food-producing animals.

Hypertrophic cardiomyopathy (HCM) is a familial myocardial disease with a prevalence of 1 in 500. More than 400 causative mutations have been identified in 13 sarcomeric and myofilament related genes, 350 of these are substitution mutations within eight sarcomeric genes. Within a population, examples of recurring identical disease causing mutations that appear to have arisen independently have been noted as well as those that appear to have been inherited from a common ancestor. The large number of novel HCM mutations could suggest a mechanism of increased mutability within the sarcomeric genes. The objective of this study was to evaluate the most commonly reported HCM genes, beta myosin heavy chain (MYH7), myosin binding protein C, troponin I, troponin T, cardiac regulatory myosin light chain, cardiac essential myosin light chain, alpha tropomyosin and cardiac alpha-actin for sequence patterns surrounding the substitution mutations that may suggest a mechanism of increased mutability. The mutations as well as the 10 flanking nucleotides were evaluated for frequency of di-, tri- and tetranucleotides containing the mutation as well as for the presence of certain tri- and tetranculeotide motifs. The most common substitutions were guanine (G) to adenine (A) and cytosine (C) to thymidine (T). The CG dinucleotide had a significantly higher relative mutability than any other dinucleotide (p < 0.05). The relative mutability of each possible trinucleotide and tetranucleotide sequence containing the mutation was calculated; none were at a statistically higher frequency than the others. The large number of G to A and C to T mutations as well as the relative mutability of CG may suggest that deamination of methylated CpG is an important mechanism for mutation development in at least some of these cardiac genes.

Objective—To determine the pharmacokinetics of gallium maltolate (GaM) after intragastric administration in healthy foals. Animals—6 healthy neonatal foals. Procedures—Each foal received GaM (20 mg/kg) by intragastric administration. Blood samples were obtained before (time 0) and at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 hours after GaM administration for determination of serum gallium concentrations by use of inductively coupled plasma mass spectroscopy. Results—Mean ± SD pharmacokinetic variables were as follows: peak serum gallium concentration, 1,079 ± 311 ng/mL; time to peak serum concentration, 4.3 ± 2.0 hours; area under the serum concentration versus time curve, 40,215 ± 8,420 ng/mL/h; mean residence time, 39.5 ± 17.2 hours; area under the moment curve, 1,636,554 ± 931,458 ng([h]2/mL); and terminal half-life, 26.6 ± 11.6 hours. The mean serum concentration of gallium at 12 hours was 756 ± 195 ng/mL. Conclusions and Clinical Relevance—Gallium maltolate administered via nasogastric tube at a dose of 20 mg/kg to neonatal foals resulted in gallium serum concentrations considered sufficient to suppress growth or kill Rhodococcus equi in macrophages and other infected tissues.

Objective: To evaluate the hypothalamic–pituitary–adrenal (HPA) axis in MDR1-1D (dogs with the MDR1 mutation associated with ivermectin sensitivity) and MDR1 wildtype dogs. Design: Prospective study. Setting: Institutional vivarium. Animals: Seven healthy Collie dogs. Measurements: MDR1 genotyping was used for allocation of dogs to 1 of 2 groups: dogs homozygous for the wildtype MDR1 allele (MDR1 wildtype) and those homozygous for the MDR1-1D mutation (MDR1 mutant). Blood samples were obtained for determination of cortisol and adrenocorticotropin hormone (ACTH) concentrations under basal conditions, before and after ACTH administration, and before and after dexamethasone administration. Main results: Significant differences were identified between the MDR1 mutant and MDR1 wildtype groups. Basal plasma cortisol concentrations and cortisol concentrations after ACTH administration were significantly lower in MDR1 mutant dogs as compared with MDR1 wildtype dogs. Plasma ACTH concentrations after dexamethasone administration were significantly lower in MDR1 mutant dogs as compared with MDR1 wildtype dogs. Conclusions: Results suggest that P-glycoprotein (P-gp) plays a role in regulation of the HPA axis. Furthermore, it appears that the HPA axis in MDR1 mutant dogs that lack P-gp is suppressed compared with MDR1 wildtype dogs. This finding may explain some clinical observations in breeds known to harbor the MDR1 mutation including Collies, Shelties, Australian Shepherds, and others. There is a clinical impression that many of these dogs have worse outcomes in response to stress and, at times, respond poorly to appropriate therapy. HPA axis suppression, secondary to the MDR1 mutation, could result in a relative adrenal insufficiency (RAI) state during times of stress or illness. Further studies are required to determine the relationship between the MDR1 genotype and RAI.

ABCG2 (ATP binding cassette subfamily G, member 2) mediates resistance to a variety of cytotoxic agents. Although human ABCG2 is well characterized, the function of canine ABCG2 has not been studied previously. Feline ABCG2 has an amino acid substitution in the adenosine triphosphate-binding domain that decreases its transport capacity relative to human ABCG2. Our goal was to compare canine ABCG2-mediated chemotherapeutic drug resistance to feline ABCG2-mediated chemotherapeutic drug resistance. HEK-293 cells stably transfected with plasmid containing canine ABCG2, feline ABCG2 or no ABCG2 were exposed to carboplatin, doxorubicin, mitoxantrone, toceranib or vincristine, and cell survival was subsequently determined. Canine ABCG2 conferred a greater degree of chemotherapy resistance than feline ABCG2 for mitoxantrone. Neither canine nor feline ABCG2 conferred resistance to doxorubicin, vincristine or toceranib. Canine, but not feline, ABCG2 conferred resistance to carboplatin, a drug that is not reported to be a substrate for ABCG2 in other species.

Background Lactate dehydrogenase (LDH) exists as 5 isoenzymes in both serum and cerebrospinal fluid (CSF). Human studies have demonstrated that changes in LDH activity can be correlated with a particular disease. Objectives Conflicting reports regarding the stability of LDH made it necessary to determine storage conditions before further study of the diagnostic power of this enzyme's activity can be pursued in dogs. The purpose of this study was to optimize measurement of LDH activity and analysis of its isoenzyme profile in canine serum and CSF through proper storage. Methods Serum and CSF were collected from 5 healthy dogs. Samples were stored at 22°C, 4°C, or −20°C for up to 2 months. Total LDH activity was measured spectrophotometrically. Isoenzyme profiles were determined using the QuickGel LDH Isoenzyme technique and densitometric scanning. Retention of > 70% LDH activity in stored samples was considered clinically acceptable. Results Serum and CSF stored at −20°C retained > 85% of the total LDH activity for 4 weeks, although CSF total LDH activity degraded by > 10% within 24 hours of storage. All serum LDH isoenzymes retained > 85% activity for up to 4 weeks at −20°C. CSF LDH isoenzyme activity degraded rapidly, therefore CSF LDH should be evaluated within 72 hours to assure > 75% of LDH isoenzyme activity. Conclusions Proper storage at −20°C can optimize detection of total LDH activity and the LDH isoenzyme profile in canine serum and CSF. This information is important for evaluating the potential usefulness of LDH in veterinary medicine.

Pharmacogenetics, the study of genetic determinants of response to drug therapy, is likely the ultimate way to establish the right drug and dose for each patient, thereby optimizing efficacy and minimizing toxicity. Despite the fact that this branch of pharmacology is still in its infancy as a science, a number of important discoveries have already contributed to improved pharmacotherapy in human and veterinary patients.

Objective—To determine the disposition of a bolus of meloxicam (administered IV) in horses and donkeys (Equus asinus) and compare the relative pharmacokinetic variables between the species. Animals—5 clinically normal horses and 5 clinically normal donkeys. Procedures—Blood samples were collected before and after IV administration of a bolus of meloxicam (0.6 mg/kg). Serum meloxicam concentrations were determined in triplicate via high-performance liquid chromatography. The serum concentration-time curve for each horse and donkey was analyzed separately to estimate standard noncompartmental pharmacokinetic variables. Results—In horses and donkeys, mean ± SD area under the curve was 18.8 ± 7.31 μg/mL/h and 4.6 ± 2.55 μg/mL/h, respectively; mean residence time (MRT) was 9.6 ± 9.24 hours and 0.6 ± 0.36 hours, respectively. Total body clearance (CLT) was 34.7 ± 9.21 mL/kg/h in horses and 187.9 ± 147.26 mL/kg/h in donkeys. Volume of distribution at steady state (VDSS) was 270 ± 160.5 mL/kg in horses and 93.2 ± 33.74 mL/kg in donkeys. All values, except VDSS, were significantly different between donkeys and horses. Conclusions and Clinical Relevance—The small VDSS of meloxicam in horses and donkeys (attributed to high protein binding) was similar to values determined for other nonsteroidal anti-inflammatory drugs. Compared with other species, horses had a much shorter MRT and greater CLT for meloxicam, indicating a rapid elimination of the drug from plasma; the even shorter MRT and greater CLT of meloxicam in donkeys, compared with horses, may make the use of the drug in this species impractical.

Methadone is an opioid, which has a high oral bioavailability (>70%) and a long elimination half-life (>20 h) in human beings. The purpose of this study was to evaluate the effects of ketoconazole [a CYP3A and p-glycoprotein (p-gp) inhibitor] and omeprazole (an H+,K(+)-ATPase proton-pump inhibitor) on oral methadone bioavailability in dogs. Six healthy dogs were used in a crossover design. Methadone was administered i.v. (1 mg/kg), orally (2 mg/kg), again orally following oral ketoconazole (10 mg/kg q12 h for two doses), and following omeprazole (1 mg/kg p.o. q12 h for five doses). Plasma concentrations of methadone were analyzed by high-pressure liquid chromatography or fluorescence polarization immunoassay. The mean +/- SD for the elimination half-life, volume of distribution, and clearance were 1.75 +/- 0.25 h, 3.46 +/- 1.09 L/kg, and 25.14 +/- 9.79 mL/min.kg, respectively following i.v. administration. Methadone was not detected in any sample following oral administration alone or following oral administration with omeprazole. Following administration with ketoconazole, detectable concentrations of methadone were present in one dog with a 29% bioavailability. MDR-1 genotyping, encoding p-gp, was normal in all dogs. In contrast to its pharmacokinetics humans, methadone has a short elimination half-life, rapid clearance, and low oral bioavailability in dogs and the extent of absorption is not affected by inhibition of CYP3A, p-gp, and gastric acid secretion.

Dogs may be infected by Mycobacterium (M.) tuberculosis, M. bovis, and M. avium complex, and the clinical signs associated with each of these infections may be indistinguishable. Rapid speciation of the infecting organism is desirable because of the public health concerns associated with M. bovis and M. tuberculosis infections. A mycobacterial infection was suspected in the dog of this report based on acid-fast staining of organisms in macrophages obtained from liver aspirates and buffy-coat preparations. Polymerase chain reaction (PCR) analysis of a buffy-coat preparation identified M. avium.

A mutation in the canine multidrug resistance gene, MDR1 , has previously been associated with drug sensitivities in two breeds from the collie lineage. We exploited breed phylogeny and reports of drug sensitivity to survey other purebred populations that might be genetically at risk. We found that the same allele, mdr1-1 Δ, segregated in seven additional breeds, including two sighthounds that were not expected to share collie ancestry. A mutant haplotype that was conserved among affected breeds indicated that the allele was identical by descent. Based on breed histories and the extent of linkage disequilibrium, we conclude that all dogs carrying mdr1-1 Δ are descendants of a dog that lived in Great Britain before the genetic isolation of breeds by registry ( ca. 1873). The breed distribution and frequency of mdr1-1 Δ have applications in veterinary medicine and selective breeding, whereas the allele's history recounts the emergence of formally recognized breeds from an admixed population of working sheepdogs.

Objective—To evaluate survival time of dogs with idiopathic Fanconi syndrome. Design—Case series. Animals—60 dogs with idiopathic Fanconi syndrome. Procedure—Data were collected by means of questionnaires distributed to owners and veterinarians of dogs with idiopathic Fanconi syndrome and by examination of medical records when accessible. Questionnaires and records were reviewed for criteria used in diagnosis, treatments administered, survival time, and subjective owner perceptions regarding their dogs' general condition. Results—58 of the dogs were Basenjis. Fifty-seven dogs (95%) were reportedly managed by use of a single therapeutic regimen. Median survival time after diagnosis of Fanconi syndrome was 5.25 years; median estimated lifespan was calculated to be between 11.3 and 12.1 years. Owners of 28 of 29 (97%) dogs still alive at the time of the study subjectively assessed their dogs' general condition as good to excellent. Seizures or other neurologic dysfunction was reported for 11 dogs. Conclusions and Clinical Relevance—Results suggest that expected lifespan for dogs with idiopathic Fanconi syndrome was not substantially reduced, compared with expected lifespan for unaffected dogs, and that affected dogs generally had a good to excellent quality of life, as subjectively assessed by their owners. What effect the treatment regimen had on survival time or lifespan could not be determined, given the small number of dogs managed with other methods. The high percentage of dogs with neurologic abnormalities was a concern, but whether this was related to Fanconi syndrome or represented a breed-related predisposition to neurologic disease could not be determined. ( J Am Vet Med Assoc 2004;225:377–383)

The product of the canine mdr1 gene, P-glycoprotein (P-gp), plays an important role in chemotherapeutic drug resistance of several canine tumours. Increased expression of P-gp by tumour cells is associated with the multidrug-resistant phenotype. Because of its importance in cancer chemotherapy, a great deal is known about the regulation of mdr1 gene expression in human cancer patients and rodent cancer models. In contrast, there is no information regarding the regulation of P-gp expression in dogs. Initial information regarding the regulation of mdr1 gene expression can be gained by evaluating the mdr1 promoter. The downstream promoter of the canine mdr1 gene was sequenced. Several regulatory elements were identified, including an AP-1 site, AP-2 site and SP-1 site. The presumed canine mdr1 promoter was similar to that of other species; however, low overall sequence homology may suggest that aspects of P-gp regulation are distinctive in dogs.

To determine plasma disposition after dermal application of a liposome-encapsulated formulation of lidocaine in cats.6 healthy adult cats with a mean (+/- SD) body weight of 4.1 +/- 0.44 kg.CBC determination and biochemical analysis of blood samples were performed for all cats. Cats were anesthetized by use of isoflurane, and catheters were placed IV in a central vein. The next day, blood samples were obtained from the catheters before and 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours after applying a 4% liposome-encapsulated lidocaine cream (15 mg/kg) to a clipped area over the cephalic vein. Plasma concentrations of lidocaine were analyzed with a high-performance liquid chromatography assay. Results-Two cats had minimal transdermal absorption of lidocaine, with lidocaine concentrations below the sensitivity of the assay at all but 1 or 2 time points. In the other 4 cats, the median maximum plasma concentration was 149.5 ng/ml, the median time to maximum plasma concentration was 2 hours, and the median area under the concentration versus time curve from zero to infinity was 1014.5 ng.h/ml.Maximum plasma concentrations of lidocaine remained substantially below toxic plasma concentrations for cats. On the basis of these data, topical administration of a liposome-encapsulated lidocaine formulation at a dose of 15 mg/kg appears to be safe for use in healthy adult cats.

Questions about pathogenesis and therapy for Helicobacter infections in dogs could be answered with a simple, noninvasive, sensitive, and specific diagnostic test. We hypothesized that a fecal polymerase chain reaction (PCR) assay would detect Helicobacter and could be useful for assessing therapeutic responses. Paired gastric biopsies and fecal samples were obtained from 39 random source dogs (group 1). Gastric biopsies from each of these dogs had histologic evidence of gastric spiral bacteria, and paired gastric tissue and fecal samples produced a 375-base pair (bp) product when amplified by PCR with Helicobacter-specific primers. Specificity of the PCR product was confirmed by detection of expected 60-, 119-, and 196-bp products following Hinfl digestion. Direct sequencing of amplicons from paired PCR products from gastric biopsy and fecal samples from 8 group I dogs showed that gastric products had the highest homologies with known gastric Helicobacter species, whereas fecal products had the highest homologies with intestinal species. Healthy mixed-breed dogs (group II; n = 8) with histologically confirmed spiral bacteria infection were treated with a 21-day course of metronidazole, amoxicillin, and famotidine. Fecal samples were collected from group II dogs twice before and within 3 days of completion of treatment. The PCR results correctly identified 15/16 pretreatment samples as positive: 1 pretreatment sample was negative. PCR results identified 8/8 posttreatment samples as Helicobacter negative. Fecal PCR is a useful test for detecting Helicobacter infection in dogs. This assay may be useful as a screening test for infection and could be used to address questions relevant to pathogenesis and therapy.

Objective—To compare serum disposition of sulfamethoxazole and trimethoprim after IV administration to donkeys, mules, and horses. Animals—5 donkeys, 5 mules, and 3 horses. Procedure—Blood samples were collected before (time 0) and 5, 15, 30, and 45 minutes and 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, and 24 hours after IV administration of sulfamethoxazole (12.5 mg/kg) and trimethoprim (2.5 mg/kg). Serum was analyzed in triplicate with high-performance liquid chromatography for determination of sulfamethoxazole and trimethoprim concentrations. Serum concentration-time curve for each animal was analyzed separately to estimate noncompartmental pharmacokinetic variables. Results—Clearance of trimethoprim and sulfamethoxazole in donkeys was significantly faster than in mules or horses. In donkeys, mean residence time (MRT) of sulfamethoxazole (2.5 hours) was less than half the MRT in mules (6.2 hours); MRT of trimethoprim in donkeys (0.8 hours) was half that in horses (1.5 hours). Volume of distribution at steady state (Vdss) for sulfamethoxazole did not differ, but Vdss of trimethoprim was significantly greater in horses than mules or donkeys. Area under the curve for sulfamethoxazole and trimethoprim was higher in mules than in horses or donkeys. Conclusions and Clinical Relevance—Dosing intervals for IV administration of trimethoprim-sulfamethoxazole in horses may not be appropriate for use in donkeys or mules. Donkeys eliminate the drugs rapidly, compared with horses. Ratios of trimethoprim and sulfamethoxazole optimum for antibacterial activity are maintained for only a short duration in horses, donkeys, and mules. (Am J Vet Res 2002;63:349–353)

Corticosteroids are one of the most extensively used class of therapeutic agents in dogs. In human patients, response to corticosteroid therapy has been correlated with the presence of certain polymorphisms of the glucocorticoid receptor gene (NR3C1). Depending on the polymorphism present, patients may show either increased sensitivity to glucocorticoid-induced adverse effects or resistance to their therapeutic effects. Because response to corticosteroid therapy in dogs can also be variable and unpredictable, we hypothesized that genetic variability exists in the canine NR3C1 gene. The aim of this study was to sequence the coding regions of the canine NR3C1 gene in a representative sample of dogs. Samples from 97 dogs from four previously identified genetic groupings of domestic breeds (Asian/Ancient, Herding, Hunting, and Mastiff) were sequenced and evaluated. Four exons contained polymorphisms and four exons showed no variation from the reference sequence. A total of six single nucleotide polymorphisms (SNPs) were identified including four synonymous SNPs and two nonsynonymous SNPs (c.811A>T and c.2111T>C). No dogs were homozygous for either variant allele, while 23 dogs were heterozygous for the c.811A>T allele and 2 were heterozygous for c.2111T>C allele. The amino acid changes caused by c.811A>T (serine to cysteine) and c.2111T>C (isoleucine to threonine) were both predicted by in silico analysis to be ‘probably damaging’ to structure and function of the resulting protein. We conclude that NR3C1 polymorphisms occur in dogs and may cause individual variation in response to corticosteroid therapy.

Drug–drug interactions can cause unanticipated patient morbidity and mortality. The consequences of drug–drug interactions can be especially severe when anticancer drugs are involved because of their narrow therapeutic index. Veterinary clinicians have traditionally been taught that drug–drug interactions result from alterations in drug metabolism, renal excretion or protein binding. More recently, drug–drug interactions resulting from inhibition of P-glycoprotein-mediated drug transport have been identified in both human and veterinary patients. Many drugs commonly used in veterinary patients are capable of inhibiting P-glycoprotein function and thereby causing an interaction that results in severe chemotherapeutic drug toxicity. The intent of this review is to describe the mechanism and clinical implications of drug–drug interactions involving P-glycoprotein and anticancer drugs. Equipped with this information, veterinarians can prevent serious drug–drug interactions by selecting alternate drugs or adjusting the dose of interacting drugs.

A 9-year-old cat with hyperthyroidism was referred for radioactive iodine treatment. The cat also had a ventral cervical mass that the owners reported had been present for several years and had increased in size during the past few weeks. On physical examination, the mass was found to have caused lateral displacement of the trachea, esophagus, jugular vein, and common carotid artery. The mass was aspirated and was determined to be cystic in nature. Concentrations of thyroid hormones in the cystic fluid were similar to serum concentrations, and nuclear scintigraphy revealed thyroactive tissue lining the cyst wall. Magnetic resonance imaging suggested that the cyst originated from the right lobe of the thyroid gland. The cat was treated with sodium iodide I 131 but died 4 days later, presumably as a result of aspiration of gastric or esophageal contents. A necropsy was not performed, but histologic examination of a biopsy specimen of the mass indicated that it was a cystic thyroid adenoma.

beta-Lactamase production by bacteria continues to be one of the main mechanisms of bacterial resistance to beta-lactam antibiotics, and it seems likely to remain so. beta-Lactamase inhibitors provide 1 strategy to overcome this mechanism of bacterial resistance. Although 3 beta-lactamase inhibitor/antibiotic combinations are currently available, only 1 is approved for veterinary use. Because the beta-lactamase inhibitor must be present concurrently with the antibiotic for synergistic activity, it is important to consider the pharmacokinetic profile of these drugs in combination. These combinations were developed and optimized for human patients, so it is unlikely that they would achieve the ideal plasma and tissue concentrations and ratios in veterinary patients. Indeed, several differences in pharmacokinetic variables of beta-lactam antibiotic/beta-lactamase inhibitor agents have been described in dogs, compared with people. Such pharmacokinetic differences should be considered when interpreting in vitro susceptibility results in veterinary species, because these tests use ratios of drug that were established for humans. The beta-lactamase inhibitors represent a successful example of targeted drug development. However, the currently available inhibitors are active primarily against class-A beta-lactamases. Because the frequency with which class-C beta-lactamases are recognized is rapidly increasing in human isolates, and because beta-lactamase enzymes continue to evolve, new beta-lactamase inhibitors will need to be developed to target these enzymes.

A 9-year-old, 29.6-kg, female spayed Collie was referred to the University of Wisconsin–Madison Veterinary Medical Teaching Hospital (VMTH) for evaluation of tachycardia. Past history included surgery for patent ductus arteriosus (PDA) at 9 months of age. The ductus remained patent after surgery, although the murmur had changed in quality. The owner elected not to pursue a second surgery or the use of thrombotic coils, and the dog remained asymptomatic until 9 years of age. An ECG was performed at the VMTH; results were consistent with paroxysmal ventricular tachycardia, with a heart rate of 180 beats/min. Lidocaine (2 mg/kg IV) was administered as a bolus to suppress the arrhythmia, and continued as a constant rate infusion at 60 mg/kg/min. Radiographs of the thorax revealed pulmonary edema and pleural effusion with cardiomegaly, aneurysmal dilatation of the descending aorta, enlarged pulmonary vessels, and a bulge of the main pulmonary artery, findings consistent with PDA and biventricular congestive heart failure. Serum biochemistry values and total T4 concentration reported by the referring veterinarian were within the reference range with the exception of total protein (4.8 g/dL, reference range 5.0–7.4 g/dL), consistent with movement of protein into the pleural and alveolar spaces. Additional medications given on the day of admission included furosemide (2.7 mg/kg) IV once, and then 1.7 mg/kg PO q8h, digoxin 0.002 mg/kg PO in the morning and 0.004 mg/kg PO in the evening, and enalapril 0.51 mg/kg PO q12h. Doppler echocardiography, performed after the arrhythmia was controlled, documented continuous flow in the pulmonary artery, moderate to severe mitral valve regurgitation with mitral valve thickening, left atrial and left ventricular dilation, and myocardial failure (fractional shortening 12%, reference range 25– 35%). Lidocaine was tapered and discontinued on the 2nd hospital day, and oral mexiletine was administered at a dosage of 5.1 mg/kg PO q8h. The dog had no interest in eating and vomited immediately after the 2nd mexiletine dose. Oral famotidine and metoclopramide were administered with the cardiac medications, and the dog was discharged on the 3rd hospital day. Seven days after the initial presentation for tachycardia, the dog re-presented to the VMTH for anorexia and abnormal mentation. The owner reported that the dog was being force-fed, and a 3-hour episode of circling had occurred at home. In addition, the dog had not reacted normally to stimuli in the home environment since beginning the medications. On physical examination, the cardiac rhythm was regular and the dog appeared dehydrated. A serum digoxin concentration was obtained 8 hours after dosing, and the dog was administered 500 mL lactated Ringer’s solution SC. The owner was advised to not administer the next dose of furosemide and to discontinue administration of digoxin. The serum digoxin concentration, reported the next day, was 4.5 ng/mL (reference range, 0.8–1.2 ng/ mL). The dog presented again to the VMTH on an emergency basis 3 days later with a history of repeated vomiting and continued anorexia. Current medications included enalapril and mexiletine at the previously prescribed dosages. Subcutaneous fluid administration was repeated, and treatment with metoclopramide and famotidine was again recommended with continuation of the enalapril (0.51 mg/kg q12h) and mexiletine (5.1 mg/kg q8h). One week later, the dog had another episode of confusion and anxiety, characterized by walking into walls, uncontrollable pacing, and attempting to crawl under furniture. The dosage of mexiletine was reduced to q12h on the basis of suspicion of mexiletine toxicosis, and further reduced by the owner to q24h administration after 2 days. The dog was re-examined 20 days after the 1st presentation. The dog had lost 3.4 kg, which was 11.5% of her initial body weight, over 20 days. The owner reported that the dog was no longer anxious or excited since decreasing administration of mexiletine to once daily, and that appetite was returning. An ECG revealed a heart rate of 120 beats/min and sinus rhythm without ventricular arrhythmias. Mexiletine neurotoxicosis was suspected on the basis of the history, and administration of the drug was discontinued. A buccal swab DNA sample was obtained to determine if the dog had a functional defect in Pglycoprotein (P-gp) on the basis of adverse reactions to digoxin and mexiletine. Swabs were submitted for MDR1 genotyping. DNA was extracted using previously published methods and used as a template for polymerase chain reaction amplification. Labeled amplicons were detected using amplified fragment length polymorphism and analyzed. Primers were designed such that they spanned the mutation area, base pairs 294–297 of the canine MDR1 gene (GenBank From the Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI (Henik, Kellum); and the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA (Bentjen, Mealey).

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.

Beta-adrenergic receptor antagonists are utilized for the management of several cardiac diseases in the dog. In humans the beneficial effects of beta-adrenergic receptor antagonists are variable and are associated with a genetic variability in the beta one adrenergic receptor gene (ADRB1). To determine if DNA variants were present in the canine ADRB1 gene, DNA from five breeds of dogs was evaluated. Two deletions were identified within the region of the gene that encodes the cytoplasmic tail of ADRB1. The functions of this region are not well understood although it is important in differentiating subtypes of adrenergic receptors and may be associated with control of receptor downregulation. The functional consequences of these identified variants deserve further study.

Objective—To determine whether the reported drug-drug interaction between the flea medication spinosad and ivermectin is attributable to inhibition of P-glycoprotein by spinosad. Animals—6 healthy adult dogs with the ABCB1 wildtype genotype. Procedures—The study was conducted as a prospective, masked, randomized crossover design. Six dogs were allocated to 2 groups; each dog served as its own control animal. Dogs in one of the groups received spinosad at the manufacturer's recommended dose; the other group received no treatment. Forty-eight hours later, scintigraphic imaging of the head and abdomen were performed with the radiolabeled P-glycoprotein substrate methoxy-isobutyl-isonitrile (sestamibi) in both groups of dogs. After a washout period of 60 days, the dogs in each group received the alternate treatment, and scintigraphic imaging again was performed 48 hours later. Gallbladder-to-liver and brain-to-neck musculature ratios of technetium Tc 99m sestamibi were calculated for each dog and compared between treatments. Results—No significant differences in gallbladder-to-liver or brain-to-neck musculature ratios were found between treatments. Conclusions and Clinical Relevance—Results provided evidence that spinosad did not inhibit P-glycoprotein function 48 hours after spinosad was administered at the manufacturer's recommended dose. Further investigations will be necessary to elucidate the mechanism of the reported toxic interaction between spinosad and ivermectin.

To describe the outcome of 3 cases of ivermectin toxicosis in dogs homozygous for the ABCB1-1Δ gene mutation treated with intravenous fat emulsion (IFE).One Australian Shepherd and 2 Miniature Australian Shepherds were treated for naturally occurring ivermectin toxicosis with IFE. All 3 dogs were homozygous for the ABCB1-1Δ gene mutation. Serum ivermectin concentrations confirmed ivermectin exposure in each case. All 3 dogs exhibited tremors, ptyalism, and central nervous system depression, which progressed over several hours to stupor in 2 dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. A 20% formulation of IFE(a) was administered as an IV bolus (1.5 mL/kg) followed by a slow IV infusion (7.5-15 mL/kg [0.25-0.5 mL/kg/m], over 30 minutes). No change was observed in the neurologic status of any patient. Lipemia visible upon blood sampling persisted for 36 hours in 1 dog however, no other adverse effects were noted. Flumazenil (0.01 mg/kg IV), followed by a constant rate infusion(CRI) of 0.01 mg/kg/h IV was administered in 1 case, without any apparent clinical benefit or adverse effect.IFE was ineffective in the treatment of ivermectin toxicosis in these ABCB1-1Δ homozygous mutant dogs. Further investigation is necessary to determine why IFE treatment was unsuccessful in these cases and whether its use can be optimized to yield better results.

Objective—To determine the frequency of the MDR1 gene mutation (polymorphism) associated with ivermectin sensitivity in a sample population of Collies in Washington and Idaho. Animals—40 healthy client-owned Collies. Procedure—A blood sample (8 ml) was collected from each dog and used for RNA extraction. Reverse transcriptase was used to generate MDR1 cDNA. Polymerase chain reaction (PCR) primers were designed to amplify a 1,061-base pair region of the MDR1 gene. The PCR products were sequenced to determine whether the Collies had 0, 1, or 2 mutant alleles. Pedigrees of some dogs were available for analysis to determine relatedness of affected dogs. Results—Of the 40 Collies, 9 (22%) were homozygous for the normal allele (normal), 17 (42%) were heterozygous (carrier), and 14 (35%) were homozygous for the mutant allele (affected). Pedigree analysis revealed that some, but not all, affected dogs were related to each other within the 4 most recent generations. Conclusions and Clinical Relevance—A high percentage of a sample population of Collies in Washington and Idaho are affected or carriers of the mutant MDR1 allele associated with ivermectin sensitivity. A similar frequency of this mutation may be detected in dogs from other geographic areas. Pharmacologic treatment with ivermectin, loperamide, vincristine, and other drugs that are substrates of P-glycoprotein, the MDR1 gene product, may result in neurologic toxicosis in a high percentage of Collies. (Am J Vet Res 2002;63:479–481)

A six-year-old, neutered, female collie was presented to an oncology specialty service after developing tetraparesis and self-mutilation that progressively worsened while receiving chemotherapy for lymphoma. Neurologic examination revealed ataxia, paresis and diminished conscious proprioception in all limbs with entire spinal reflexes. Magnetic resonance imaging of the brain and spinal cord was normal. Electromyography of the limbs ruled out a vincristine-induced peripheral neuropathy. Cerebrospinal fluid analysis and cerebrospinal fluid and serum testing for Neospora and Toxoplasma were normal. Results of MDR1 genotyping revealed that the dog was homozygous for the ABCB1-1Δ (MDR1) mutation. This clinical presentation strongly resembled the effects seen from inadvertent intrathecal administration of vincristine in humans. Dogs that are homozygous for the ABCB1-1Δ (MDR1) mutation should not receive standard dosages of chemotherapy drugs known to be eliminated by P-glycoprotein, the gene product of ABCB1. Testing for this mutation is strongly recommended before chemotherapy initiation for at-risk breeds.

Background In people, serum gastrin concentrations increase in response to administration of H2 receptor antagonists, but the effect of famotidine administration on serum gastrin concentrations has not been evaluated in dogs. Objectives To determine if serum gastrin concentrations increase in response to 14 days of famotidine treatment and the time needed to return to baseline after discontinuation of famotidine; define stability of gastrin in samples held at room temperature. Animals Eleven healthy dogs were included in part A (famotidine treatment) and 7 healthy dogs in Part B (serum gastrin stability). In part A, famotidine (0.5 mg/kg PO q12h) was administered for 14 days. Fasting blood samples were collected on days 0, 3, 7, 11, 14, 16, 18, 20, and 22. In part B, blood was collected after a 12-hour fast. Gastrin concentrations in serum samples held at room temperature for ≤30 minutes after sampling were compared to concentrations in samples held at room temperature for 150 minutes after sampling. Results Serum gastrin concentrations increased by day 3 of famotidine administration and returned to baseline concentrations in all dogs by day 14 despite continued famotidine administration. Serum gastrin concentrations were lower (20% mean decrease; P = .0005) in samples held at room temperature for 150 minutes. Conclusions and Clinical Importance After 14 days of famotidine administration, clinically healthy dogs have normal serum gastrin concentrations. In a dog with clinical features consistent with gastrinoma, chronic famotidine administration is unlikely to contribute to increases in serum gastrin concentrations.

Background ABCB4 functions as a phosphatidylcholine translocater, flipping phosphatidylcholine across hepatocyte canalicular membranes into biliary canaliculi. In people, ABCB4 gene mutations are associated with several disease syndromes including intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis (type 3), primary biliary cirrhosis, and cholelithiasis. Hepatobiliary disease, specifically gallbladder mucocele formation, has been recognized with increased frequency in dogs during the past decade. Because Shetland Sheepdogs are considered to be predisposed to gallbladder mucoceles, we initially investigated ABCB4 as a candidate gene for gallbladder mucocele formation in that breed, but included affected dogs of other breeds as well. Results An insertion (G) mutation in exon 12 of canine ABCB4 (ABCB4 1583_1584G) was found to be significantly associated with hepatobiliary disease in Shetland Sheepdogs specifically (P < 0.0001) as well as other breeds (P < 0.0006). ABCB4 1583_1584G results in a frame shift generating four stop codons that prematurely terminate ABCB4 protein synthesis within exon 12, abolishing over half of the protein including critical ATP and a putative substrate binding site. Conclusions The finding of a significant association of ABCB4 1583_1584G with gallbladder mucoceles in dogs suggests that this phospholipid flippase may play a role in the pathophysiology of this disorder. Affected dogs may provide a useful model for identifying novel treatment strategies for ABCB4-associated hepatobiliary disease in people.

Twenty-two dogs diagnosed with generalized demodicosis were treated with milbemycin oxime (MO) because of poor response to previous therapies or because the dog was a breed known to be susceptible to ivermectin toxicosis. Fifteen of the 22 dogs were herding breeds. Doses of MO ranged from 1.0 to 2.2 mg kg(-1) day(-1) per os. Cheek swab samples were obtained in order to determine each dog's ABCB1 genotype. Adverse drug reactions were recorded for each dog by the owners and/or veterinarians. The ABCB1-1 Delta genotype was significantly associated with the development of an adverse reaction (neurological toxicity) after treatment with MO. None of the 19 dogs with the wild-type ABCB1 allele experienced adverse reactions, whereas two dogs homozygous for the ABCB1-1 Delta mutation developed ataxia. Assessing the ABCB1-1 Delta genotype prior to MO administration may prevent neurological toxicity in these patients.

The impact of drug transporters on drug pharmacokinetics and pharmacodynamics has been increasingly recognized in recent years. P-glycoprotein (P-gp), the product of the ABCB1 (formerly MDR1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. P-gp is expressed by a variety of normal tissues, including the intestines, brain capillary endothelial cells, renal tubular cells, and biliary canalicular cells, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs and enhances their excretion from the body. Many drugs used in veterinary medicine are substrates for P-gp, including many chemotherapeutic agents and macrocyclic lactones (avermectins and milbemycin). A 4-base pair deletion mutation in the ABCB1 gene occurs in many herding breed dogs, including collies, Australian shepherds, and Shetland sheepdogs. The mutation (ABCB1-1Delta) renders affected animals extremely susceptible to toxicosis induced by substrate drugs, such as the macrocyclic lactones at doses well below those tolerated by dogs with the wild-type ABCB1 gene. However, at the manufacturer's recommended dose, all FDA-approved heartworm preventive products marketed in the United States are safe, even for dogs with the ABCB1 mutant/mutant genotype.

Objective—To evaluate the breed distribution of the ABCB1-1Δ polymorphism in a large number of dogs in North America, including dogs of several herding breeds in which this polymorphism has been detected and other breeds in which this polymorphism has not yet been identified. Design—Cross-sectional study. Animals—5,368 dogs from which buccal swab samples were collected for purposes of ABCB1 genotyping. Procedures—From May 1, 2004, to September 30, 2007, DNA specimens derived from buccal swab samples collected from 5,368 dogs underwent ABCB1 genotyping. These data were reviewed, and results for each dog were recorded in a spreadsheet, along with the dog's breed. The genotypes for each breed were tallied by use of a sorting function. Results—The ABCB1-1Δ allele was identified in 9 breeds of dogs and in many mixed-breed dogs. Breeds that had the ABCB1-1Δ allele included Collie, Longhaired Whippet, Australian Shepherd (standard and miniature), Shetland Sheepdog, Old English Sheepdog, Border Collie, Silken Windhound, and German Shepherd Dog (a breed in which this mutation had not been detected previously). Conclusions and Clinical Relevance—The ABCB1-1Δ polymorphism is associated with increased susceptibility to many adverse drug reactions and with suppression of the hypothalamic-pituitary-adrenal axis and is present in many herding breeds of dog. Veterinarians should be familiar with the breeds that have the ABCB1-1Δ polymorphism to make appropriate pharmacologic choices for these patients.

P-glycoprotein is considered to be a major factor impeding effective drug therapy for many diseases of the central nervous system (CNS). Thus, efforts are being made to gain a better understanding of P-glycoprotein's role in drug distribution to brain parenchyma and cerebrospinal fluid (CSF). The goal of this study was to validate and introduce a novel P-glycoprotein-deficient (ABCB1-1Delta) canine model for studying P-glycoprotein-mediated effects of drug distribution to brain tissue and CSF. CSF concentrations of drug are often used to correlate efficacy of CNS drug therapy as a surrogate for determining drug concentration in brain tissue. A secondary goal of this study was to investigate the validity of using CSF concentrations of P-glycoprotein substrates to predict brain tissue concentrations. Loperamide, an opioid that is excluded from the brain by P-glycoprotein, was used to confirm a P-glycoprotein-null phenotype in the dog model. ABCB1-1Delta dogs experienced CNS depression following loperamide administration, whereas ABCB1 wild-type dogs experienced no CNS depression. In summary, we have validated a novel P-glycoprotein-deficient canine model and have used the model to investigate transport of the P-glycoprotein substrate (99m)Tc-sestamibi at the blood-brain barrier and blood-CSF barrier.

Objective—To determine whether antiepileptic drugs (AEDs) are substrates for canine P-glycoprotein (P-gp). Sample Population—OS2.4/Doxo cells (canine osteosarcoma cells induced via exposure to doxorubicin to highly express P-gp). Procedures—Competitive inhibition of rhodamine 123 efflux from OS2.4/Doxo cells was used to determine whether AEDs were substrates for canine P-gp. Flow cytometry was used to quantify mean fluorescence intensity of cells treated with rhodamine alone and in combination with each experimental drug. Results—Known P-gp substrate drugs ivermectin and cyclosporin A altered rhodamine efflux by 90% and 95%, respectively. Experimental drugs altered rhodamine efflux weakly (diazepam, gabapentin, lamotrigine, levetiracetam, and phenobarbital) or not at all (carbamazepine, felbamate, phenytoin, topirimate, and zonisamide). Conclusions and Clinical Relevance—At clinically relevant doses, it appeared that AEDs were weak substrates (diazepam, gabapentin, lamotrigine, levetiracetam, and phenobarbital) or were not substrates (carbamazepine, felbamate, phenytoin, topirimate, and zonisamide) for canine P-gp. Therefore, it seems unlikely that efficacy of these AEDs is affected by P-gp expression at the blood-brain barrier in dogs.

Objective—To determine the lowest of 5 doses of cosyntropin (1.0, 0.5, 0.1, 0.05, or 0.01 μg/kg) administered IV that stimulates maximal cortisol secretion in clinically normal dogs. Animals—10 clinically normal dogs. Procedures—5 dose-response experiments were performed in each of the dogs. Each dog received 5 doses of cosyntropin (1.0, 0.5, 0.1, 0.05, and 0.01 μg/kg) IV in random order (2-week interval between each dose). Serum samples for determination of cortisol concentrations were obtained before (baseline) and at 10, 20, 30, 40, 50, 60, 120, and 240 minutes after cosyntropin administration. Results—Compared with baseline values, mean serum cortisol concentration in the study dogs increased significantly after administration of each of the 5 cosyntropin doses. Mean peak serum cortisol concentration was significantly lower after administration of 0.01, 0.05, and 0.1 μg of cosyntropin/kg, compared with findings after administration of 0.5 and 1.0 μg of cosyntropin/kg. After administration of 0.5 and 1.0 μg of cosyntropin/kg, mean peak serum cortisol concentration did not differ significantly; higher doses of cosyntropin resulted in more sustained increases in serum cortisol concentration, and peak response developed after a longer interval. Conclusions and Clinical Relevance—Administration of cosyntropin IV at a dose of 0.5 μg/kg induced maximal cortisol secretion in healthy dogs. Serum cortisol concentration was reliably increased in all dogs after the administration of each of the 5 doses of cosyntropin. These data should be useful in subsequent studies to evaluate the hypothalamic-pituitary-adrenal axis in healthy and critically ill dogs.

A study was performed to determine the frequency of the mutant MDR1 allele associated with ivermectin sensitivity in a sample of Collies and other herding breeds living in Australia. Buccal swab samples were collected from 33 Collies, 17 Australian Shepherds, 7 Border Collies and 7 Shelties for determination of MDR1 genotype. DNA was extracted and the polymerase chain reaction was performed to amplify a 148 base pair (wildtype MDR1 genotype or 144 base pair (mutant MDR1 genotype) amplicon containing the MDR1 mutation. Sequence analysis was performed to determine the genotype of each dog. Adequate quantities of DNA for unequivocal genotyping were obtained from 61 of 64 samples. The previously described MDR1 mutation was identified in Collies, Australian Shepherds and Shelties living in Australia, but not in Border Collies (although sample numbers were low). Twelve percent (4/33) of the Collies studied were homozygous for the normal allele (normal), 64% (21/33) were heterozygous (carrier) and 24% (8/33) were homozygous for the mutant allele (affected). Results of this study indicate that a high percentage of herding breeds presenting to veterinarians in Australia harbor the MDR1 mutation, thus impacting some therapeutic decisions.

Objective—To compare plasma disposition of the R(–) and S(+) enantiomers of carprofen after IV administration of a bolus dose to donkeys and horses. Animals—5 clinically normal donkeys and 3 clinically normal horses. Procedure—Blood samples were collected from all animals at time 0 (before) and at 10, 15, 20, 30, and 45 minutes and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 24, 28, 32, and 48 hours after IV administration of a bolus of carprofen (0.7 mg/kg). Plasma was analyzed in triplicate via high-performance liquid chromatography to determine the concentrations of the carprofen enantiomers. A plasma concentration-time curve for each donkey and horse was analyzed separately to estimate noncompartmental pharmacokinetic variables. Results—In donkeys and horses, the area under the plasma concentration versus time curve (AUC) was greater for the R(–) carprofen enantiomer than it was for the S(+) carprofen enantiomer. For the R(–) carprofen enantiomer, the AUC and mean residence time (MRT) were significantly less and total body clearance (ClT) was significantly greater in horses, compared with donkeys. For the S(+) carprofen enantiomer, AUC and MRT were significantly less and ClT and apparent volume of distribution at steady state were significantly greater in horses, compared with donkeys. Conclusions and Clinical Relevance—Results have suggested that the dosing intervals for carprofen that are used in horses may not be appropriate for use in donkeys. (Am J Vet Res 2004;65:1479–1482)

Objective—To compare plasma disposition of alkaloids after lupine challenge in cattle that had given birth to calves with lupine-induced arthrogryposis and cattle that had given birth to clinically normal calves and determine whether the difference in outcome was associated with differences in plasma disposition of anagyrine. Animals—6 cows that had given birth to calves with arthrogryposis and 6 cows that had given birth to clinically normal calves after being similarly exposed to lupine during pregnancy. Procedure—Dried lupine (2 g/kg) was administered via gavage. Blood samples were collected before and at various time points for 48 hours after lupine administration. Anagyrine, 5,6-dehydrolupanine, and lupanine concentrations in plasma were measured by use of gas chromatography. Plasma alkaloid concentration versus time curves were generated for each alkaloid, and pharmacokinetic parameters were determined for each cow. Results—No significant differences in area under the plasma concentration versus time curve, maximum plasma concentration, time to reach maximum plasma concentration, and mean residence time for the 3 alkaloids were found between groups. Conclusions and Clinical Relevance—Because no differences were found in plasma disposition of anagyrine following lupine challenge between cattle that had given birth to calves with arthrogryposis and those that had not, our findings do not support the hypothesis that between-cow differences in plasma disposition of anagyrine account for within-herd differences in risk for lupine-induced arthrogryposis. (Am J Vet Res 2004;65:1580–1583)

Drug transporters significantly influence drug pharmacokinetics and pharmacodynamics. P-glycoprotein (P-gp), the product of the MDR1 (ABCB1) gene, is among the most well-characterized drug transporters, particularly in veterinary medicine. A number of clinically relevant, structurally and functionally unrelated drugs are substrates for P-gp. P-gp is expressed by a variety of normal tissues including the intestines, renal tubular cells, brain capillary endothelial cells, biliary canalicular cells, and others, where it functions to actively extrude substrate drugs. In this capacity, P-gp limits oral absorption and central nervous system entry of many substrate drugs. A number of MDR1 polymorphisms have been described in human patients, some of which result in altered drug pharmacokinetics and susceptibility to diseases such as Parkinson's disease, inflammatory bowel disease, refractory seizures, and others. An MDR1 polymorphism in herding breed dogs, including collies and Australian shepherds, has been demonstrated to be the cause of ivermectin sensitivity in these breeds. Recent evidence suggests that this polymorphism, a 4-bp deletion mutation, results in increased susceptibility to the toxicity of several drugs in addition to ivermectin. Furthermore, data in rodent models suggest that P-gp may play an important role in regulating the hypothalamic-pituitary-adrenal axis.

A study was performed to determine the frequency of the mutant MDR1 allele associated with ivermectin sensitivity in a sample of collies living in France. Buccal swab samples were collected from approximately 83 collies for determination of MDR1 genotype. DNA was extracted and the polymerase chain reaction was performed to amplify a 148 bp (wildtype MDR1 genotype) or 144 bp (mutant MDR1 genotype) amplicon containing the MDR1 mutation. Sequence analysis was performed to determine the genotype of each dog. Adequate quantities of DNA for unequivocal genotyping were obtained from only 25 of 83 swabs. Twenty percent (5/25) of the collies studied were homozygous for the normal allele (normal), 32% (8/25) were heterozygous (carrier), and 48% (12/25) were homozygous for the mutant allele (affected). The results of this study indicate that a high percentage of collies presenting to veterinarians in France harbor the MDR1 mutation, thus impacting some therapeutic decisions.

Lymphoma was diagnosed in a 4-year-old spayed female Collie, and treatment with a combination chemotherapy protocol incorporating prednisone, L-asparaginase, vincristine, vinblastine, doxorubicin, and cyclophosphamide was initiated. The dog had signs of gastrointestinal tract toxicosis and myelosuppression after treatment with P-glycoprotein-substrate drugs (vincristine, vinblastine, and doxorubicin) even when dosages were reduced, but did not have signs of toxicosis after treatment with cyclophosphamide, a non-P-glycoprotein-substrate drug, even when administered at the full dosage. It was postulated that a deletion mutation in the canine MDR1 gene (deltaMDR1 295-298) could be responsible for the drug toxicoses in this dog. This mutation has been identified as the cause of a functional P-glycoprotein defect in Collies susceptible to the toxic effects of ivermectin, another P-glycoprotein-substrate drug. The MDR1 genotype of this dog consisted of 1 normal and 1 mutant MDR1 allele. Because P-glycoprotein contributes to renal, biliary, and intestinal excretion of P-glycoprotein-substrate drugs, it is possible that drug excretion was delayed in this patient, resulting in clinical signs of toxicosis.

A 6-year-old, 43-kg, spayed female rottweiler was presented for a 1-month history of progressive, left hind-limb lameness. Upon physical examination, a cranial drawer sign and joint distention were present in the left stifle. Radiographically, the stifle had evidence of effusion, remodeling of the patella, and an enlarged popliteal lymph node. Marked synovial thickening and an intact cranial cruciate ligament were noted during surgery. Despite finding a nonspecific, mixed inflammatory response on joint fluid cytopathology, histopathology demonstrated T-cell lymphoma of the synovium. Lameness may be the sole presenting clinical sign in canine lymphoma.

A subpopulation of collie dogs is extremely sensitive to neurotoxicity induced by ivermectin. The aim of this study was to determine the mechanistic basis for this phenomenon. The multi-drug-resistance gene (mdr1) encodes a large transmembrane protein, P-glycoprotein (P-gp), that is an integral part of the blood-brain barrier. P-gp functions as a drug-transport pump at the blood-brain barrier, transporting a variety of drugs from the brain back into the blood. Since ivermectin is a substrate for P-gp, we hypothesized that ivermectin-sensitive collies had altered mdr1 expression compared with unaffected collies. We report a deletion mutation of the mdr1 gene that is associated with ivermectin sensitivity. The 4-bp deletion results in a frame shift, generating several stop codons that prematurely terminate P-gp synthesis. Dogs that are homozygous for the deletion mutation display the ivermectin-sensitive phenotype, while those that are homozygous normal or heterozygous do not display increased sensitivity to ivermectin.