White feet, maybe treat? Testing for the MDR-1 gene in collie breeds (Proceedings)

What is pharmacogenomics?

Pharmacogenomics is an emerging field studying how genetic differences within a population can cause differences in pharmacology. Usually the genetic change will influence the pharmacokinetics of the drug, through altered drug absorption, distribution, metabolism, or excretion. Although there is always variation within a population when it comes to drug pharmacokinetics, differences in genetic traits can cause results that lie far outside the "normal" distribution, creating a bi-modal distribution. These genetic differences are responsible for some types of breed-specific drug toxicity that veterinarians encounter. Probably the most common example is the susceptibility of some types of herding (collie) breeds to iveremctin and other macrocyclic lactones.

P-glycoprotein, MDR, ABC...more than alphabet soup!

Some collie breeds are more susceptible to ivermectin because of a dysfunctional P-glycoprotein (P-gp). This protein spans the membrane of cells in the intestinal mucosa, liver canaliculi, renal proximal tubules, and the blood-brain barrier (among others). P-gp acts primarily as an efflux pump, moving xenobiotic substances from inside the cell to the outside in an effort to keep the body safe. On intestinal cells, P-gp acts like a bouncer at the front door of an exclusive club (the body). It screens the molecules that want entry into the club, and removes the unknown (xenobiotic) particles before they can get inside. In this way oral drug absorption is minimized. In the kidney and liver, P-gp transports foreign molecules so they can be excreted in the urine or bile, respectively (sort of like the bouncer inside the club, throwing troublemakers out the back door). In PK terms, this action will increase drug clearance. Finally, P-gp on endothelial cells at the blood-brain barrier (BBB) keeps xenobiotic particules from entry into the CNS. Think of these transporters as the bouncers in the body's VIP room...the brain.

P-gp is a large protein with a complex structure. Like all proteins, P-gp is encoded by certain genes. In this case the gene is MDR-1 (multi-drug resistant-1), also called the ABCB1 (ATP-binding cassette B1) gene. It is genetic abnormalities in this gene that causes a phenotypic change in P-gp function. Some herding breed populations have a 4 base-pair deletion in the ABCB-1 gene which causes a premature stop codon. The truncated P-gp protein that is produced in these cases is not functional; therefore drugs which gain entry into cells guarded by P-gp are not removed as easily.

Ivermectin effects in collie breeds

Because of their broad-spectrum endo- and ecto-parasiticide effects and low cost, macrocyclic lactones (ivermectin, doramectin, abamectin, etc.) are among the most commonly used veterinary drugs for many different species. They are generally extremely safe in mammals, even at doses higher than recommended (0.2 mg/kg). For example, a common ivermectin dose for treatment of demodectic mange is 0.4 - 0.6 mg /kg for 2 – 4 months. However, some collie breed dogs show signs of neurotoxicity (such as ataxia, weakness, hypersalivation) at much lower doses (< 0.1 mg/kg). This is because the P-gp at the blood-brain barrier which normally keeps ivermectin out of the brain is defective. Other drugs within this class (such as selamectin and moxidectin) do not appear to be as toxic to collie breeds as ivermectin, possibly because there are other mechanisms for their elimination from the BBB. However, a cautious approach is required before any drugs of this class are used in collie breeds.

There are also differences in ivermectin toxicity with the collie breeds. Veterinarians have long known that not all collies were ivermectin-sensitive, but enough were that is wasn't worth taking the risk of treating them. In many cases there are appropriate alternatives to using ivermectin, but in some cases these alternatives are not feasible. Cases of demodectic mange are one example. Due to the long treatment time and high doses required, the low cost and safety of oral ivermectin make it an excellent choice...except in collies! What to do in a case like this?

Testing for the ABCB-1 gene

Obviously employing a "trial and error" approach to ivermectin toxicity is not a good idea. However, a better option has emerged—genetic testing to determine if the ABCB-1 gene is producing functional P-gp. The clinical pharmacology lab at Washington State University (led by Dr. Katrina Mealey, who did some of the decisive research in this area) can now perform an inexpensive genetic test which identifies mutant ABCB-1 genes in dogs. The genotypic expression (homozygous wild-type or normal, homozygous mutant, or heterozygous) allows the veterinarian to know in advance if a phenotypic response (such as toxicity to ivermectin or other P-gp substrates) is likely. Heterozygous dogs typically have a phenotypic expression intermediate between the homozygous animals, in that they are more susceptible to ivermectin toxicity at lower doses than homozygous wild-type dogs, but not as susceptible as homozygous mutant carriers. The ABCB-1 mutation has currently been identified in 10 different dog breeds, including a few non-collie breeds. The frequency of the ABCB-1 mutation is as follows:

How to test for the ABCB-1 mutant gene

Test kits can be ordered from the WSU Clinical Pharmacology lab (509-335-3745, http://www.vetmed.wsu.edu/depts-VCPL). Test prices (as of July 2008) are $70/test for 1 - 4 tests or $60/test for 5 or more. The test requires some cheek or gum cells which are taken by gently scrubbing the mouth with a small brush for 20 – 30 seconds. Results are usually available within one or two weeks.

Other P-gp substrates

Macrocyclic lactones aren't the only drugs which are subtrates for P-gp. Other commonly used veterinary drugs are also transported by P-gp, including:

     • Loperamide (Immodium®): This anti-diarrheal drug is an opioid similar to morphine. Unlike morphine, loperamide usually does not show CNS activity because it is kept out of the CNS by P-gp at the BBB. Dogs with the ABCB-1 mutation can develop neurologic signs like ataxia, disorientation, and ptyalism when treated with loperamide at normal doses (0.2 mg/kg).

     • Vincristine, doxorubicin: These chemotherapy drugs are also P-gp substrates. Clearance of these drugs by the biliary system is decreased in mutant ABCB-1 dogs, leading to an increased risk of myelosuppression and GI toxicity.

     • Digoxin: Oral bioavailability of digoxin is normally low, but ABCB-1 mutants may have increased absorption from the intestines because of decreased P-gp function. It does not appear that ABCB-1 mutant dogs are at increased risk of digoxin toxicity, however, therapeutic drug monitoring is even more important in these animals.

References

Mealey KL. Therapeutic implications of the MDR-1 gene. J. vet. Pharmacol. Therap. 2004; 27:257-264.

Schrickx JA, Fin-Gremmels J. Implications of ABC transporters on the disposition of typical veterinary medicinal products. Eur. J. Pharm. 2008; 585:510-519.

Martinez M, Modric S, Sharkey M, et al. The pharmacogenomics of P-glycoprotein and its role in veterinary medicine. J. vet. Pharmacol. Therap. 2008; 31:285-300.

Mealey KL. Case presentations: Adverse drug reactions involving P-glycoprotein. 26th ACVIM Forum Proceedings; 2008; 64-66.

Dowling PM. Pharmacogenetics: It's not just about ivermectin in collies. Can. Vet. J. 2006; 47:1165-1168.

Barbet JL, et al. ABC�-1 Delta (MDR1-1 Delta) genotype is associated with adverse reactions in dogs treated with milbemycin oxime for generalized demodicosis. Vet Dermatol. 2009; 20(2):111-114.

Sherman JG, Paul AJ, Firkins LD. Evaluation of the safety of spinosad and milbemycin 5-oxime orally administered to Collies with the MDR1 gene mutation. Am J Vet Res. 2010;71(1):115-9.

Mealey, K. Canine ABC� and macrocyclic lactones: heartworm prevention and pharmacogenetics. Vet Parasitol/2008; 158(3): 215-22.