Genetic predisposition to adverse drug events in dogs. in: veterinary focus 17(2); 2007

Published in IVIS with the permission of the editor Genetic predisposition
to adverse drug events in dogs
KEY POINTS
➧ There is growing evidence that clear genetic links exist between the phenotypic characteristics of pharmacogenomics are rapidly expanding the promise of the clinical application of genetic data to Margo Karriker, PharmD
help prevent adverse events, predict drug behavior, WALTHAM UCVMC-SD Clinical Nutrition Program,
University of California Veterinary Medical Center,
➧ Among the most widely studied genetic variances San Diego, USA
observed in dogs is the multi-drug resistance Dr. Karriker graduated with a doctorate from the School of (MDR) gene deletion mutation resulting in altered Pharmacy at the University of North Carolina, Chapel Hill in p-glycoprotein expression and altered drug 2003. She completed a two-year Veterinary Clinical Pharmacy residency at the University of California, Davis Veterinary Teaching Hospital in 2005 and began her position ➧ The future of pharmacogenetics and its clinical as the WALTHAM Specialist in Clinical Pharmacy for the applicability will have a lasting impact on the WALTHAM UCVMC-SD Clinical Nutrition Program in San Diego the same year. While continuing to serve in this position, Dr. Karriker is also clinical faculty at the UC San Diego Skaggs School of Pharmacy and pharmacist for a federal Veterinary mental influence and other factors, we have never been able to map these events to one geneticlocation or associate them unequivocally with onesite in the genome. Introduction
The goal of drug treatment is to maximize the In recent years, there has been much focus on the therapeutic effect while minimizing adverse drug genetic basis of the pharmacokinetic (absorption, events and drug interactions. Seldom is drug distribution, metabolism, excretion) and pharmaco- therapy this simplistic. Complex patients, variable dynamic (drug interactions with targets such as disease processes, and intricate medication regimens receptors and transporters) interactions drugs have complicate most clinical scenarios. There are times in the body. This field of study has been termed when our best effort at predicting, avoiding, and pharmacogenetics and is designed to provide an managing adverse drug events is not enough. understanding of the genetic variation in populationsthat predict an individual’s response to drug therapy.
While we have long noted trends in medication This field of study promises us the ability to events seemingly related to breed, age, environ- effectively and efficiently predict a sound outcome Vol 17 No 2 / / 2007 / / Veterinary Focus / / 11 in almost every patient as it relates to identified events. Until recently, these insights were based primarily on the phenotypic, or visually expressed,traits such as hair coat and eye color. A pharmaco- With the completion of the human genome project genetic approach to adverse drug events stands to and continued focus on mapping the genetic profile provide us with drug therapy that is unique to of other species, pharmacogenetic data and, in each patient and allows for an individualized turn, clinical predictors of drug response, are likely to become more commonplace in veterinarypractice. As our clinical picture progresses toward As we currently stand, a population of seemingly continued treatment of complex disease states, similar animals may experience a variety of clinical management of chronic conditions, and diagnosis responses to one common drug treatment. The of less common disease processes, the ability to variable clinical response we see may be genetically predict a patient’s interaction with their medication based in some cases and the insight pharmaco- regimen, and perhaps a clinical success probability, genetics gives us will change our practice on a real- should prove to be an invaluable part of compre- time level. Using focused screening tests, we may be able to predict which animals express the alteredprotein or gene sequence responsible for the adverse Currently, we have a small amount of clinical and event associated with a specific drug or class of anecdotal evidence that allows us to have some drugs. With the tools that pharmacogenetics offers, insight into breed or population characteristics we will likely be able to avoid or alter this event by that appear to predispose animals to adverse drug Table 1.
Examples of inherited and acquired variations in enzymes, receptors and drug transporters found
to be clinically relevant in human medicine (2)
Variant phenotypes
Drug affected
Response modification
Transporters
Many - See Table 2
Receptors
12 / / Veterinary Focus / / Vol 17 No 2 / / 2007 GENETIC PREDISPOSITION TO ADVERSE DRUG EVENTS IN DOGS
Current evolution of pharmacogenetics
ideal settings to explore the genetics of a population.
Many years ago, the idea of organisms possessing the With traceable breed divergence, intensive in- capacity to adapt to influences placed on them by breeding programs, and short time periods between environmental or therapeutic factors was thought to generations, the process of finding the genetic link be based in inheritance. In the early 1900’s the to a given drug event is quite realistic. Several studies of several investigators provided proof that papers have already reviewed pharmacogenetics in there was indeed a connection between the bio- both human and veterinary medicine (2,5). This chemical processes that dictate drug metabolism article will ser ve to summarize the genetic and genetics (2). One of the first reports of an predictors of adverse drug events of clinical inherited difference in response to a foreign chemical significance that have been identified in dogs. was published by Snyder in 1932. Based on previouswork by Fox, Blakeslee and Salmon, “taste blindness” Multi-drug resistance (MDR-1)
to phenylthiocarbamide (PTC) was described and mutation and p-glycoprotein
linked to an autosomal recessive trait in humans (3).
Multiple sources and clinical case reports have now Later, during World War II, Archibald Garrod noticed reported the linkage between certain herding breeds a link between the development of a primiquine and adverse drug events related to antiparasitics and hemolysis reaction and African-American soldiers.
other drugs. Avermectins are a widely used class of Further study showed that this hemolysis was the drugs used in veterinary medicine to treat internal product of a genetic deficiency of glucose-6- and external parasites. One specific compound in phosphate dehydrogenase (G-6-PD) (4).
this class is ivermectin, which works to paralyzeinvertebrate organisms by activating the GABA As time progressed, observations of succinyl- (gamma amino butyric) or glutamate gated chloride choline, isonizid, and debrisoquine helped relate channels of the peripheral ner vous system.
individual patients’ responses to a genetic pattern.
Mammals generally have GABA expression within These early reports and case studies laid the the central nervous system protected by the blood- ground work for modern pharmacogenetics. Since those first advances, contemporary technology Clinically, it has been observed that Collies (Figure 1)
has allowed the study of pharmacogenetics to move and other related breeds are seen to be more forward at a heightened pace. Today, there is a focus sensitive to the CNS effects of ivermectins revealing on both pharmacogenetics (a collaboration of clinical signs such as tremors, hypersalivation, coma, biochemistry and pharmacology to correlate depression and ataxia. First described in the 1980’s, phenotypic markers to specific genetic links) and on very small doses (1/100-1/200th of standard) were pharmacogenomics. Pharmacogenomics differs shown to elicit these profound and acute adverse from pharmacogenetics in its more global focus.
events in some, but not all, Collies and related Using advanced technology such as high-throughput breeds. While exploring several possibilities, such DNA sequencing, gene mapping and bioinformatics, as altered protein binding, it was seen that Collies studies can focus on interpatient variables to predict demonstrating adverse events had a higher brain differences in drug behavior and response. The concentration of ivermectin relative to non- genomic approach will allow us to study the basis of observed drug response, and predict responses,identify new drug targets, and individualize The basis of this observation has been shown to be therapy in a way that reduces both drug cost and linked to a deletion mutation in the MDR-1 (Multi prevents unwanted effects. Some of the inherited or Drug Resistance 1) gene sequence that results in a acquired variations in drug enzymes, transporters series of premature stop codons to be expressed and receptors seen to be clinically relevant in human halting formation of almost 90% of the resulting medicine are outlined in Table 1.
p-glycoprotein amino acid sequence (6). P-glyco-protein, first identified in the mid-1970’s, is a Several recent pharmacogenetic discoveries have 170kDa glycosylated membrane protein that proven to be clinically relevant to veterinary functions to infer intrinsic resistance to a wide patients. The canine population is one of the more variety of drug substances by exporting these Vol 17 No 2 / / 2007 / / Veterinary Focus / / 13 substances out of the body. P-glycoprotein expression Many drugs, of veterinary clinical significance, have can be found in several tissues including the brain, been shown to be substrates of p-glycoprotein where it works to maintain an integral portion of (Table 2). During drug absorption, p-glycoprotein
the blood-brain barrier; the gut, where its location may significantly reduce the oral bioavailability on the brush border of enterocytes, limits the of its substrates. As shown in MDR-1 knockout absorption and bioavailability of substrates; on mice, p-glycoprotein substrate bioavailability is the surface of tumor cells, where it infers multi- considerably greater than wild-type mice. Investig- drug resistance; and the renal proximal tubules, ations to take advantage of this process showed that where its presence accelerates the secretion of the oral bioavailability of docetaxel, a substrate, was increased almost 20-fold when administered with ap-glycoprotein inhibitor. As drugs are distributed in P-glycoprotein’s role in the blood-brain barrier was the body, p-glycoprotein again plays a role. For first shown in MDR-1 knockout mice. Investigations those substrates that may cause adverse effects with ivermectin showed that the knockout popul- if given entry into the central nervous system, ation was 50-100 times more sensitive to the testes, or placenta, breeds suffering from the MDR-1 neurologic effects relative to the wild-type mice.
deletion mutation will be more likely to experience By showing that ivermectin is a substrate for adverse events even at low clinical doses. Animals p-glycoprotein, the link between the absence of that are heterozygous for the deletion may not this protein and the reported adverse events was experience adverse events initially or with one clear (8). The consequence of alterations in the dose, but at high or chronic doses, toxicity may expression of this protein stands to have great clinical significance due to its wide tissue expressionand relative lack of substrate specificity. During drug excretion, p-glycoprotein expressionon the renal tubules has altered the clearance of The distribution of the MDR-1 mutation among the some substrates, particularly chemotherapeutic canine population has been described. Overall, it medications. The concurrent administration of a has been reported that almost 75% of Collies in the p-glycoprotein inhibitor to rats decreases the billiary US, France and Australia have one mutant allele for the expression of altered p-glycoprotein. Affectedbreeds are suspected to have similar lineage and Clearly the implications of the MDR-1 deletion mutation include other herding breeds such as Old English and subsequent expression of p-glycoprotein will Sheepdogs, Australian Shepherds, Shelties, English stand to gain even more clinical relevance as we Shepherds, Border Collies, German Shepherds, are able to understand and test for those animals that Long-haired Whippets, and Silken Windhounds.
have altered expression. Currently, at least one com- Case reports in other non-related breeds are less mercial laboratory will analyze samples for canines to provide genotyping information (Washington St ateUniversity, www.vetmed.wsu.edu/vcpl).
Figure 1.
Standard American
Collie.
Cytochrome P450 enzymes
Drug metabolism is mediated by several complexsystems. While better understood in humans, theCYP450 (cytochrome P450) enzyme system isbecoming better delineated in canines. This classof enzymes is responsible for the metabolism of awide variety of drugs and can be expressed in severallocations within the body. It has been shown thatthese enzymes may be induced or inhibited by certaindrugs; they may be over or underexpressed in certainpopulations; and they may vary as to their expressionin certain individuals within a population.
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CYP1A2 has been shown to be deficient in 10% of one small population of Beagles (9). While few Substrates of p-glycoprotein (7)
drugs used clinically in veterinary medicine havebeen identified to be substrate of this enzyme, Cytotoxic drugs
future directions may provide greater clinical CYP2B11 has up to a 14-fold variation in its activity Cardiac drugs
in dogs of mixed breed pedigree, Greyhounds were shown to have a particularly low activity within the Immunosuppressives
larger canine population. Several drugs including propofol are substrates of CYP2B11 and some Antiemetic drugs
evidence that this enzyme may be expresseddifferently in male and female dogs (10).
Antidiarrheal agents
There is also some evidence that CYP2D15 may exhibit polymorphism in dogs. In Beagles, approxi- Antibiotics
mately half of the population appears to metabolize well celecoxib (a substrate of CYP2D15), while the Steroids
other half appears to be poor metabolizers. This effect has been shown to increase the half-life of elimination of this drug up to 5-fold (11). While the H2-blockers
extrapolation of this finding to other non-steroidals of similar structure, such as deracoxib, has not been shown, further clinical evidence and study may produce more information. Other drugs are shown to be CYP2D15 substrates in humans, although theseare used less frequently in veterinary medicine.
As the exploration of the metabolic process of drug metabolism in dogs continues to grow, we may have evidence to show that there are more variants in the metabolic enzymes. As we know more about the source of these mutations, we will likely find that other breeds may be affected and expandeddrug classes are involved. Further evidence willlikely help us avoid dangerous drug-drug, drug- metabolizing enzyme has greatly varying levels, even in seemingly similar breed populations,which is also similar to the situation in humans.
Thiopurine S-methyl transferase (TPMT)
Their study included 56 different dog breeds and Thiopurine S-methyl transferase has been studied mixed breed subjects. The study first observed the in humans and is shown to catalyze the methyla- TPMT levels occurring in 145 samples and found tion of drugs such as 6-mercaptopurine and the level of activity varied over a 9-fold range.
azathioprine. Recently, a genetic polymorphism Using information on the sequence and structure has been identified in dogs that leads to some of the TPMT gene, the researchers resequenced all considerable variation in the expression of this of the exons of the canine TPMT gene using DNA enzyme. Salavaggione, et al. (12) found that from 39 dogs selected based on different levels of TPMT levels in the average canine red blood cell RBC TPMT activity. Subsequently, nine poly- (RBC) were similar to those found in human morphisms were obser ved. Six of the nine studies. Furthermore, it was found that this drug- polymorphisms were associated with 67% of the Vol 17 No 2 / / 2007 / / Veterinary Focus / / 15 variation in level of the RBC TPMT activity in the other breeds lending them a greater risk for 39 samples. When those 6 single-nucleotide poly- adverse events from those more lipophilic drugs morphisms were assayed using DNA from all 145 such as barbiturates that are cleared from the brain dogs, 40% of the phenotypic variance could be to muscle and fat with subsequent elimination by the liver. While anecdotal evidence has shown thatnot all sighthounds have the same reaction to The clinical application of these obser ved barbiturates and propofol, these reactions are variations can not be fully elucidated at this time.
likely contributed to a combination of factors, We know that those patients with low TPMT including polymorphic gene expression, variable activity are at much higher risk of developing enzyme expression and environmental influences.
potentially lethal toxicity when administeredthiopurine drugs at standard doses. Likewise, Idiosyncratic sulfonamide toxicity
those patients with high enzyme activity are likely There is a growing body of evidence that supports to see clinical failure due to under-treatment. It has the theories that pharmacogenetic differences can been observed that Giant Schnauzers have lower also potentiate adverse drug reactions that are not TPMT activity, while Alaskan Malamutes had high related to a drug concentration and are not TPMT activity (13). While the clinical application predictable. These idiosyncratic reactions have been of this information may not be readily available characterized in humans for some time. The until standardized testing methods have been sulfonamide antibiotics (sulfamethoxazole, sulfadia- established, it is nonetheless important to consider zine, sulfadimethoxine) have been shown to cause that variations do occur that may predispose some numerous dose-dependant reactions in dogs such as breeds to greater risk of adverse events such as hematuria, non-regenerative anemia, and an inter- bone marrow suppression, and should be consider- ference with thyroid hormone synthesis. Other adverse reactions have also been seen at therapeuticdoses that tend to be more generalized and more Sighthounds and anesthesia
typical of delayed immunologic reactions and can The sighthound class has long been a highly domestic- even manifest once a short treatment (10 days or less) ated, strictly bred, group of dogs. Predisposing has been completed. These reactions tend to include these animals to a myriad of physiological and signs such as hepatotoxicity, skin eruptions, fever, anatomical anomalies, the physical makeup of these hemolytic anemia, uveitis, polyarthritis, proteinuria dogs has seemingly caused idiosyncratic reactions to and facial swelling. Work is currently underway in certain classes of drugs. As described above, some the United States to characterize dogs with these of the reactions we see related to sensitivity to reactions using ELISA for anti-drug antibodies, anesthetic agents, such as propofol, may likely be in vitro cytotoxicity assays and other methodologies related to a cytochrome P450 variance relative to other non-related breeds. We do see other reactionsthat are more likely the consequence of breeding Conclusions
patterns, exercise pressures and performance We have clear data that helps to verify, on a genetic demands placed on these animals by humans. basis, some of the adverse drug effects we have seenin certain breeds of dogs. The previous approaches Sighthounds are typically thought to include the of identifying a genetic predisposition to adverse Greyhound, Whippet, Borzoi, Irish Wolfhound, drug events based on phenotype and then searching Basenji, Saluki and Rhodesian Ridgeback and were for the genetic link will soon give way to identifying bred to hunt based on sight, as opposed to scent.
predisposing genetic factors and modifying drug They have been characteristically stream-lined to therapy in anticipation of the events. Several show similar characteristics of lean body mass, genetic factors that have clinical implication with prominent musculature, long limbs and a deep regard to drug therapy have already been described. thorax. These breeds tend to have highly stressed Pharmacogenetics provides us with the opportunity demeanors and can be more likely to have stress- to practice, on a clinical level, the best possible drug related complications when placed in the clinical therapy. We will be able to screen for those animals environment. They have much less body fat than who may be at risk; test to verify their genotypic 16 / / Veterinary Focus / / Vol 17 No 2 / / 2007 GENETIC PREDISPOSITION TO ADVERSE DRUG EVENTS IN DOGS
characteristics; and customize drug therapy for the adverse events and maximizes therapeutic benefit.
individual rather than the population. With the Academic and clinical support for the research advances that clear genetic answers provide us, we efforts in pharmacogenetics and pharmaco- will be able to identify new and more precise drug genomics stand to benefit the whole of veterinary targets to allow for drug therapy that minimizes REFERENCES
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