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U.S. Department of Health and Human Services

Animal & Veterinary

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Understanding Genetic Variability in Dogs and Its Potential Role in Drug Development

by Michele Sharkey, D.V.M.; Marilyn Martinez, Ph.D.; Sanja Modric, D.V.M., Ph.D.; Lisa Troutman, D.V.M., MS; and Lynn Walker, D.V.M.; Office of New Animal Drug Evaluatio
FDA Veterinarian Newsletter January / February 2008 Volume XXIII, No I

Veterinary medicine is in the early stages of understanding how genetic differences in animals can affect the way drugs work. This field of pharmacogenomics offers promise in veterinary medicine, as it does in human medicine. Researchers at the Center for Veterinary Medicine’s Office of Research have begun studies in pharmacogenomics to determine when genetic tests can be used to determine drug safety in specific breeds of dogs. The Office of Research’s work is being car-ried out under the Food and Drug Administration’s Critical Path Initiative, which is designed to help move appropriate medical innovations that are safe and effective out of the laboratories to where they can help human and animal patients.

Background

In March 2004, FDA launched the Critical Path Initiative in an effort to stimulate and modernize the processes through which FDA-regulated products are developed, evaluated, and manufactured. To meet the Critical Path objectives, FDA plans to apply relevant disciplines (e.g., physiology, pharmacology, clinical pharmacology, and pharmacogenomics) and to identify ways to better correlate laboratory-generated data to clinical outcomes when the drug is administered to the broader population.

As part of the Critical Path Initiative, novel biomarkers may be identified that will serve as tools for ensuring the safe and effective use of products in either human or veterinary patients. Pharmacogenomics can be used as a tool to help identify novel biomarkers or physiological characteristics that impact a patient’s drug response, both in human and veteri-nary medicine. Knowledge and understanding of genetic variability in drug response is critical because clinical testing may not always detect rare but important safety problems or the sample size is too small to detect trends that can occur in the broader population.

The Critical Path Initiative evolving

The Critical Path Initiative is one of FDA’s top priorities. “It is fostering strong, sustained scientific advances that will en-hance the health and well-being of all Americans,” said Dr. Andrew von Eschenbach, Commissioner of Food and Drugs.

As part of its effort under the Critical Path Initiative, FDA is striving to obtain better information throughout the entire drug development process in an effort to improve the predictability of product clinical performance. A component of that effort is the identification of patient characteristics for which a drug might pose an unacceptable risk.

For example, concentrations of a drug in blood may be markedly affected by body condition, such as obesity. The de-gree of obesity was recently suggested as a major determinant of moxidectin kinetics in dogs, because obesity modulates the volume of distribution and, therefore, terminal half-life1 of the drug. Similar results were reported with moxidectin in swine with different body condition2.

With the objective of obtaining better information, CVM has launched two initiatives to address some of these scientific hurdles as they pertain to the veterinary profession. Those two initiatives—CVM’s recent collaborative research on drug transporters, specifically P-glycoprotein (P-gp), and breed effects as they influence product safety and effectiveness in certain breeds of dogs—are discussed in this article. Both of these efforts have resulted in recent publications3,4.

Advancing veterinary drug development

Pharmacogenomics is the study of how genetic variation in animals influences the safety and effectiveness of drug products administered to those animals. (This science is a rapidly evolving tool within human medicine, supporting efforts to improve the risk/benefit relationship of pharmacotherapy within the individual patient5.)

Although there is a lack of genetic information in veterinary medicine, breed specific differences in response to endoge-nous substances (produced by the animal) and exogenous substances (from external sources) have been reported across a range of species, including cattle6, sheep7, chickens8, and pigs9.

With regard to dogs, there are more than 400 breeds recognized worldwide and 156 breeds recognized by the Ameri-can Kennel Club. A consequence of the genetic selection associated with the propagation of breed-specific characteristics is a host of breed-related medical issues, which are recognized by the veterinary profession.

For example, specific metabolic diseases appear to follow breed lines. Human inborn errors of metabolism are gener-ally attributable to several different mutations in a particular gene across a population of individuals, whereas in dogs (and cats) the same mutation is generally responsible for the specific disease within a breed10. While only 5 percent to 10 per-cent of human genetic variation has been shown to be associated with populations or races, 27 percent of genetic varia-tion in dogs is associated with differences in breed11.

Currently, there are more than 100 DNA-based tests for inherited diseases and traits in dogs. For example, a test is available to determine the presence of a multi-drug resistance gene 1 (MDR-1) mutation in dogs. The mutation results in nonfunctional P-gp. Dogs with nonfunctional P-gp show an increase in toxicity when administered certain P-gp substrates, such as ivermectin.12,13 Considering the extensive research already generated by Dr. Katrina Mealey et al.14 on the con-sequence of genetic mutations of the MDR-1 gene, CVM elected to focus on P-gp as a biomarker to enhance the evalua-tion of safe and effective new animal drugs.

P-gp and its role in therapeutics

P-gp is a transmembrane efflux (able to pump substances out of a cell) protein that affects the absorption, distribution, and elimination of certain drugs. It is part of a family of efflux transporters found in a variety of organs, including the intes-tine, the kidneys, the biliary system, and the central nervous system. P-gp provides the body with a mechanism to protect itself from potentially harmful substances by transporting substrates (e.g., across the intestinal tract [influx—pumping a substance in—and efflux], out of the brain, into the urine, and into the bile).

In 2001, Dr. Katrina Mealey reported a mutation in the MDR-1 gene that encodes for P-gp in dogs. The genetic muta-tion, believed to have first evolved in England in the late 1800s, creates a nonfunctional fragment of the original P-gp pro-tein molecule. While the genetic defect has been commonly seen in herding breed dogs, it has also been found in some hounds.

Ivermectin sensitivity, a result of the nonfunctioning P-gp protein molecule, is most commonly reported in Collies; the MDR-1 mutation is postulated to affect 30 percent to 50 percent of the Collie population15,16. Sporadic descriptions of iv-ermectin sensitivity have been reported in other breeds, including Shetland Sheepdogs, Australian Shepherds, and Old English Sheepdogs.

Dogs (like humans) have two alleles for each trait. These alleles can be dominant or recessive.

Dogs can have one of three possibilities for the MDR-1 mutation. They can be homozygous recessive (in which case the MDR-1 alleles are mutant/mutant), heterozygous (with normal/mutant alleles), or wild-types (normal/normal alleles).

Because P-gp is an important efflux transporter of a wide range of compounds, dogs homozygous recessive for the MDR-1 mutation (mutant/mutant) have nonfunctioning P-gp, and therefore may have altered pharmacokinetic and toxicity profiles for P-gp substrates, including avermectins. In that case, avermectins accumulate in the brain, resulting in the dogs exhibiting clinical signs of neurotoxicity, such as ataxia, convulsions, vomiting, salivation, and tremors. The resulting neu-rotoxicity is dose-dependent and can be fatal.

In addition to neurotoxicity due to the macrocyclic lactones (ivermectin, moxidectin, milbemycin, and selamectin), dogs homozyogous for the MDR-1 defect have been reported to exhibit signs of toxicity from other drugs at doses used without side effects in MDR-1 wild-type (normal/normal) dogs. For example, MDR-1 (mutant/mutant) dogs have exhibited neuro-toxicity with standard doses of loperamide, a drug that is normally excluded from the brain by P-gp17. Dogs homozygous for the MDR-1 mutation have also been reported to have increased sensitivity (pronounced and protracted central nervous system depression) to acepromazine and butorphanol18. Altered biliary and/or renal excretion of vincristine and doxorubi-cin was proposed to cause myelosuppression and gastrointestinal tract toxicosis in a MDR-1 (mutant/mutant) Collie19. Similarly, digoxin toxicity was also documented in an MDR-1 (mutant/mutant) Collie20.

P-gp can also impact canine medicine in ways unrelated to the MDR-1 mutation. For example, the failure of predniso-lone to successfully treat naturally occurring chronic canine enteropathies in various dog breeds could be predicted by the over-expression of P-gp in the dog’s lamina propria lymphocytes during steroid exposure21. In these animals, the diseased tissues effectively became resistant to steroid therapy. Accordingly, the question may not only be related to the integrity of a patient’s P-gp function, but it could also have to do with whether a drug is a potential P-gp substrate or inhibitor.

Considering the importance of P-gp in modulating drug transport, CVM has safety concerns for other P-gp substrates. Therefore, CVM is examining some of the available technologies that can be used to screen for P-gp substrates, particularly with respect to predicting drug toxicity in P-gp deficient dogs.

Ongoing research efforts

  • CANINE GENETIC TESTING: A DNA test for the presence of the MDR-1 mutation is commercially available through Wash-ington State University. Studies have yet to confirm that this test is sensitive or specific for ivermectin sensitivity.
    Nevertheless, the test does allow veterinarians to screen dogs for the MDR-1 mutation. Dogs heterozygous for this mu-tation may also be at risk. Initial information suggests that for some substrates, there may be compromised P-gp func-tion in the heterozygous animal22. An understanding of the potential consequences of the MDR-1 genetic defect on drug pharmacokinetics should improve the ability to predict potential safety and effectiveness concerns in dogs carrying this mutation.
  • FDA/CVM’S CRITICAL PATH INITIATIVE: FDA has recently approved a research proposal submitted by CVM’s Office of Research under FDA’s Critical Path Initiative to explore the potential impact of the MDR-1 gene mutation on drug safety and effectiveness. The research project will also explore methods for determining which drugs have safety and/or effec-tiveness profiles that may necessitate studies in dogs known to carry the mutation.

    Involving review scientists within CVM’s Office of New Animal Drug Evaluation, the research program will initially ad-dress the potential differences in the pharmacokinetics of several known P-gp substrates when administered to dogs that are homozygous recessive, heterozygous, or wild-types for the MDR-1 mutation.

    Reliable screening procedures for identifying potential P-gp substrates, particularly those molecules at risk of crossing the blood-brain barrier in P-gp deficient dogs, are needed to help evaluate the safety of new drugs for use in dogs. Therefore, identifying alternative in vitro and in vivo tests that can serve as screening methods for detecting such P-gp substrates is needed. The Office of Research will also work on the development of an in vitro method for determining whether a compound is a P-gp substrate and if an in vivo transgenic knockout mouse model can serve as a reliable testing method.

Conclusion

Despite the current focus on P-gp, it is important to consider the overall objectives of the Critical Path Initiative to iden-tify physiological variables, genetic screens, or novel biomarkers that can be used to improve the safety and effectiveness data generated from small studies.

Similar to human medicine, the genetic variations present within veterinary species can affect drug safety and effec-tiveness. However, veterinary medicine is in the early stages of understanding the role of pharmacogenomics in drug re-sponse. Information relating to P-gp deficiencies in dogs can serve as a starting point upon which veterinary scientists build a pharmacogenomic database. Ultimately, the goal is to utilize information derived not only in dogs but across all species, to better understand population variability, test for sources of this variability, and minimize the risk of adverse drug reactions in veterinary species.

Endnotes

1 Lallemand E, Lespine A, Alvinerie M, Bousquet-Melou A, Toutain PL. Estimation of absolute oral bioavailability of mox-idectin in dogs using a semi-simultaneous method: influence of lipid co-administration. J Vet Pharmacol Ther. 2007 Oct; 30(5):375-80.

2 Craven J, Bjørn H, Hennessy DR, Friis C. The effects of body composition on the pharmacokinetics of subcutaneously injected ivermectin and moxidectin in pigs. J Vet Pharmacol Ther. 2002 Jun;25(3):227-32.

3 Martinez, M, Modric S, Sharkey M, Troutman L, Walker L, Mealey K. The pharmacogenomics of P-glycoprotein (P-gp) and its role in veterinary medicine. Submitted, J Vet Pharmacol Therap. 2007.

4 Fleischer S, Sharkey M, Mealey K, Ostrander E, Martinez M. Pharmacogenetic and Metabolic Differences Between Dog Breeds: Their Impact on Canine Medicine and the Use of the Dog as a Preclinical Animal Model. AAPS Journ., Ac-cepted January 18, 2008.

5 Tomalik-Scharte D, Lazar Al Fuhr U, and Kirchheiner J. The clinical role of genetic polymorphisms in drug-metabolizing enzymes. Pharmacogenomics Journ, 2008 Feb;8(1): 4–15.

6 Sallovitz J, Lifschitz A, Imperiale F, et al. Breed differences on the plasma availability of moxidectin administered pour-on to calves. Vet J. 2002;164:47-53.

7 Ammoun I, Encinas T, Veiga-Lopez A, et al. Effects of breed on kinetics of ovine FSH and ovarian response in su-perovulated sheep. Theriogenology. 2006;66:896-905.

8 Opdycke JC, Menzer RE. Pharmacokinetics of diflubenzuron in two types of chickens. J Toxicol Environ Health. 1984;13:721-733.

9 Sutherland MA, Rodriguez-Zas SL, Ellis M, Salak-Johnson JL. Breed and age affect baseline immune traits, cortisol, and performance in growing pigs. J Anim Sci. 2005; 83:2087-2095.

10 Sewell AD, Haskins ME, Giger U. Inherited metabolic diseases in companion animals: searching for natures mistakes. Vet J. 2007;174:252-259.

11 Ibid.

12 Mealey, KL. Therapeutic implications of the MDR-1 gene. J Vet Pharmacol Ther. 2004; 27:257-264.

13 Mealey, KL. Adverse Drug Reactions in Herding-Breed Dogs: The Role of P-gp. Compendium, January 2006, 23-33.

14 Mealey KL, Bentjen SA, Gay JM, et al. Ivermectin sensitivity in Collies is associated with a deletion mutation of the MDR-1 gene. Pharmacogenetics 2001; 11(8):727-33.

15 Mealey KL, Bentjen SA, Waiting DK. Frequency of the mutant MDR-1 allele associated with ivermectin sensitivity in a sample population of Collies from the northwestern United States. American Journal of Veterinary Research, 2002a; 63:479-481.

16 Hugnet C, Bentjen SA, Mealey KL. Frequency of the mutant MDR-1 allele associated with multi-drug sensitivity in a sample of Collies from France. J Vet Pharmacol Ther. 2004; 27:227-229.

17 Sartor L, Bentjen SA, Trepanier L, Mealey KL. Loperamide Toxicity in a Collie with the MDR-1 Mutation Associated with Ivermectin Sensitivity. J Vet Intern Med 2004; 18:117-118.

18 Mealey KL. Adverse Drug Reactions in Herding Breed Dogs: The Role of P-glycoprotein. Compendium. Jan 2006; 28:23-33.

19 Mealey K, Northrup NC, Bentjen SA. Increased toxicity of P-glycoprotein-substrate chemotherapeutic agents in a dog with the MDR-1 deletion mutation associated with ivermectin sensitivity. J Am Vet Med Assoc. 2003 Nov 15; 223(10):1453-1455.

20 Henik RA, Kellum HB, Bentjen SA, Mealey KL. Digoxin and mexiletine sensitivity in a Collie with the MDR-1 mutation. J Vet Intern Med. 2006 Mar-April; 20(2):415-417.

21 Allenspach K, Rüfenacht S, Sauter S, Gröne A, Steffan J, Strehlau G, Gaschen F. Pharmacokinetics and clinical effi-cacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease. J Vet Intern Med. 2006; 20:239-44.

22 Mealey KL. Pharmacogenetics, Veterinary Clinics of North America Small Animal Practice. Sept 2006; 36:5, 965.