Saving Sighthounds from Anesthetic Drug Death
The locomotion of the long, lean muscles of running Greyhounds is simultaneously elegant and breathtaking. Like other ancient sighthound breeds hardwired to pursue prey by speed and sight, the Greyhound has idiosyncrasies that include having a potentially life-threatening, slow recovery from anesthetic drugs.
Identifying gene mutations responsible for breaking down, or metabolizing, commonly used drugs in Greyhounds and other sighthound breeds is the focus of research underway at Washington State University. Lead investigator Michael Court, BVSc, PhD, professor and the William R. Jones Endowed Chair, says the goal is to develop a drug sensitivity test from saliva, urine or blood that will identify dogs with a slow drug metabolism of certain drugs.
“This would allow a veterinarian to make dose adjustments or select an alternative drug for optimal drug treatment,” he says. “A personalized or individual approach to drug selection would be the ideal outcome for affected dogs.”
A veterinary anesthesiologist who has worked in research since 1983, Dr. Court is revolutionizing a cocktail drug phenotyping approach to use in dogs that will enable several drugs to be tested at the same time to determine their metabolism ratio in the body. The test would produce results classifying a dog as a slow, normal or fast metabolizer for a specific group of drugs.
“The original problem drug in sighthounds was a barbiturate, thiopental, which is used to induce anesthesia,” Dr. Court says. “Greyhound owners in Australia and the U.S. reported that it took their dogs 48 hours to stand unassisted after having thiopental anesthesia. Although thiopental is no longer used in the U.S., its use continues in most of the world. The replacement drug in the U.S. is propofol, though even with this drug, recovery in Greyhounds can be double what is normal, taking 40 minutes instead of 20 minutes.”
Since all of the problem anesthetic drugs are highly fat-soluble, the sensitivity of Greyhounds and other lean-bodied sighthounds to these drugs was originally thought to result from a reduced ability of their tissues to “soak up” the drug, keeping it away from the brain. However, more recent work has implicated a reduced ability of the dog’s liver to break down and remove the drug from the body. Specifically, there appears to be deficiency of an important drug metabolism enzyme, called cytochrome P450 (CYP).
“Using a candidate gene approach to identify gene mutations in Greyhounds that decrease anesthetic drug metabolism, we found two mutations that may explain the slow metabolism,” says Dr. Court. “One is a mutation in one of the CYP (CYP2B11) genes, and the other is in a gene for an enzyme needed for CYP to work efficiently, called P450 oxidoreductase (POR). Together, these drug-metabolizing enzymes make the drugs more water soluble, so they can be easily flushed out through the kidneys.
“Both mutations are found primarily in Greyhounds and some sighthound breeds. Rarely in non-sighthound breeds.”
Developing A Novel Drug Sensitivity Test
The sighthound adverse drug reaction research, now in phase two, focuses on developing a cocktail drug phenotyping test. Funding of $172,765 for the two-year study that began in June 2018 is provided by the AKC (American Kennel Club) Canine Health Foundation. Phase one of the research, which ran from 2016 to 2018, received funding of $150,000 from the AKC Canine Health Foundation. This work involved functional studies of the mutations in the laboratory.
“The AKC Canine Health Foundation is committed to advancing a future for personalized medicine for canine patients,” says Dr. Diane Brown, CEO of the AKC Canine Health Foundation. “We consider Dr. Court’s work in this area as an important series of first steps toward that future, and have dedicated funding to these efforts since 2016.”
Although the adverse drug reactions in sighthounds is similar to the MDR1 gene mutation in herding breeds that causes a neurotoxic reaction to ivermectin and other drugs (see below), there are differences. While the mutations in sighthounds dramatically reduce the function of the enzymes, they do not wipe them out, as occurs with dysfunction of the P-gp transporter protein in herding breeds.
“These are two different processes with the same goal, essentially trying to limit the amount of a drug that gets into the body,” Dr. Court explains. “P-gp keeps drugs out of the body, and especially the brain, by pumping the drug out. Drug-metabolizing enzymes like CYP2B11 make drugs in the body water soluble, so they can be easily flushed out, usually through the kidneys. The slow metabolism also changes the drugs chemically so they often lose their effect.”
Stephanie Martinez, PhD, a postdoctoral research associate in the Veterinary Clinical Pharmacogenetics Laboratory at Washington State University, is working with Dr. Court on the sighthound research. “In the first two-year study, we saw a significant loss of enzyme function from the mutations when we tested them in the lab,” she says.
In May 2018, Dr. Martinez adopted two retired Greyhound racers, “Seamus” and “Otis.” As a new Greyhound owner, she relates to concerns about having her dogs anesthetized for even a dental cleaning procedure. “To be honest, I am a little nervous to put them under anesthesia,” she says. “I had them genotyped right away. Both are heterozygous for one of the anesthesia adverse reaction mutations, which means they have one copy of the mutated allele, though I do not know how significantly they are affected.
“In addition to the delayed anesthesia recovery issues, some sighthound breeds like Scottish Deerhounds and Greyhounds experience bleeding issues that occur 24 to 72 hours after surgical procedures. We think it is caused by a genetic mutation that causes the blood clot to break down prematurely. Otis is homozygous for a candidate gene we’re currently investigating, which means he may experience postoperative bleeding problems.”
A separate study of delayed postoperative bleeding, funded in part by the Scottish Deerhound Club of America, is underway in conjunction with the anesthesia research. The team is seeking DNA samples from sighthounds that experienced delayed bleeding 24 to 72 hours after surgery, as well as DNA from littermates of dogs that died from this condition.
The cocktail drug phenotyping research involves testing 12 dogs with three drugs to evaluate their rate of metabolism. “The drug cocktail includes an antidepressant, which we believe will be metabolized slowly in dogs with the mutation, because it is only metabolized by CYP2B11,” Dr. Court explains. “The other drugs are an over-the-counter acid reducer and a cough suppressor. These latter two should be metabolized normally in dogs with the mutation because they are metabolized via other CYP enzymes.”
Currently, the research team is working to ensure that the three drugs can be safely given at low doses together without interfering with one another. They also want to be sure the saliva concentrations of the drugs and metabolites match the blood and urine concentrations so that saliva can be used for the test.
“The power of this approach is that we can readily test hundreds of dogs,” says Dr. Court. “Once we have this cocktail phenotyping test worked out, then we plan to use it in dogs with and without the mutations to show that the metabolism of the CYP2B11 enzyme is slow, but metabolism of the other CYP probes is normal.
“Ultimately, owners could perform a saliva test at home that would determine the ability of individual dogs to handle drugs that could be affected by rare mutations, even those not yet discovered,” he says. “The impact of non-genetic factors, such as age, disease, diet, and environment, on drug metabolism would also be detectable by this novel drug sensitivity test.”
“I hope that owners of sighthounds will not have to worry when their dogs need to undergo necessary medical procedures,” Dr. Martinez says. “The potential of being able to proactively customize drugs used for anesthesia for individual dogs to reduce the risk of slow recovery or identify a dog that is prone to a fatal bleeding problem and ensure they get lifesaving drugs is exciting.” n
Sighthound Owners Can Contribute to Research
Researchers at the Program in Individualized Medicine (PrIMe) at Washington State University are seeking DNA samples from sighthounds for two separate ongoing studies. One study is examining adverse drug reactions to anesthesia, and the other is investigating delayed postoperative bleeding. For information, go to https://prime.vetmed.wsu.edu or send an email to [email protected]
To read the abstract of the adverse drug reaction grant that is funded by the AKC Canine Health Foundation, go to: https://bit.ly/2FG1vjh. You also may donate to this research via the link.
MDR1 GENE MUTATION IN HERDING BREEDS TIED TO ADVERSE DRUG REACTIONS
The discovery in 2001 of a mutation in the MDR1 (multidrug resistance) gene in Collies and other herding breeds that causes a neurotoxic reaction to ivermectin, a parasitic preventive, provided insights into treating affected dogs. It also initiated the use of pharmacogenetics to determine how an individual dog’s genetic makeup impacts its response to drugs.
Katrina Mealey, DVM, PhD, DACVIM, DACVCP, professor and the Richard L. Ott Endowed Chair in Small Animal Medicine and Research at Washington State University, identified a 4-base pair deletion mutation in the MDR1 gene that causes a stop codon prematurely terminating P-glycoprotein (P-gp) synthesis.
“The MDR1 gene encodes the P-gp transmembrane protein that functions in a protective capacity by transporting a variety of drugs from the blood-brain barrier into the body,” explains Dr. Mealey. “Dogs with the MDR1 mutation do not have this protective barrier. Thus, drugs like ivermectin can cause toxic reactions that result in debilitating illness and even death.”
In the affected dogs, high concentrations of ivermectin accumulated in their brain tissue after a single dose of the drug. Since ivermectin is processed from the brain through P-gp into the body, Dr. Mealey hypothesized that ivermectin-sensitive collies had altered MDR1 gene expression.
Her research led to the MDR1 gene mutation discovery and development of a genetic test that is available through the Veterinary Clinical Pharmacology Laboratory at Washington State University (www.vcpl.vetmed.wsu.edu). A DNA cheek swab test that identifies a dog’s phenotype for the mutation can be ordered and processed for $60.
The autosomal dominant gene mutation impacts homozygous and heterozygous affected dogs. Dogs that are homozygous inherit two copies of the mutant allele and pass one copy of the defective gene to their offspring. Homozygous dogs are more likely to have severe, life-threatening drug reactions.
Affected dogs that are heterozygous have one copy of the mutant allele and one copy of the normal allele, meaning they have a 50 percent chance of passing the defective gene to their offspring. These dogs have less severe adverse drug reactions compared with those that are homozygous.
The MDR1 mutation primarily occurs in herding breeds. Affected dogs are thought to be descendants of a single dog that lived in Great Britain in the early 1800s before breeds were selectively bred. The mutation also occurs in two sighthound breeds, the Longhaired Whippet and Silken Windhound, believed to share a collie ancestry. Mixed breeds — any dog with a herding breed in its ancestry — may be at risk for drug toxicity.
“As a pharmacologist, the most fascinating aspect of the MDR1 gene mutation is the sheer number of drugs that are affected by a single gene mutation,” Dr. Mealey says. “Not all drugs must be avoided in dogs with altered P-glycoprotein function. Many drugs can be used safely with no need to alter the dose.”
However, adverse drug reactions involve over a dozen different drugs. The most serious adverse drug reactions involve antiparasitic preventives, such as ivermectin and milbemycin, the antidiarrheal medication loperamide (Imodium®), and several anticancer drugs, including vincristine and doxorubicin.
Since the genetic test was first offered in 2004, there has been a reduction of the mutation in some breeds via selective breeding. Importantly, identifying dogs with the P-gp dysfunction allows veterinarians to decrease the drug dose or consider alternate drugs to avoid a potentially fatal drug reaction.
Breed Frequency of MDR1 Gene Mutations1
Breed Approximate Frequency
Collie (Rough- & Smooth-Coated) 70 Percent
Longhaired Whippet 65 Percent
Australian Shepherd 50 Percent
Miniature Australian Shepherd 50 Percent
McNab Shepherd 30 Percent
Silken Windhound 30 Percent
English Shepherd 15 Percent
Shetland Sheepdog 15 Percent
German Shepherd Dog 10 Percent
Herding Breed Cross 10 Percent
Mixed Breed 5 Percent
Old English Sheepdog 5 Percent
Border Collie <5%
1Veterinary Clinical Pharmacology Laboratory at Washington State University (www.vcpl.vetmed.wsu.edu)
Drugs Affected by the MDR1 Gene Mutation1
Drug Category Drug
Antidiarrheal Loperamide (Imodium®)
*Note that giving dogs FDA-approved heartworm preventive products according to the recommended doses is safe, even in dogs with the MDR1 mutation.
1Veterinary Clinical Pharmacology Laboratory at Washington State University (www.vcpl.vetmed.wsu.edu)