Cannabinoid Receptor Antagonist-Induced Striated Muscle Toxicity and Ethylmalonic-Adipic Aciduria in Beagle Dogs

Abstract

Ibipinabant (IBI), a potent cannabinoid-1 receptor (CB1R) antagonist, previously in development for the treatment of obesity, causes skeletal and cardiac myopathy in beagle dogs. This toxicity was characterized by increases in muscle-derived enzyme activity in serum and microscopic striated muscle degeneration and accumulation of lipid droplets in myofibers. Additional changes in serum chemistry included decreases in glucose and increases in non-esterified fatty acids and cholesterol, and metabolic acidosis, consistent with disturbances in lipid and carbohydrate metabolism. No evidence of CB1R expression was detected in dog striated muscle as assessed by polymerase chain reaction, immunohistochemistry, Western blot analysis, and competitive radioligand binding. Investigative studies utilized metabonomic technology and demonstrated changes in several intermediates and metabolites of fatty acid metabolism including plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate. These results indicated that the toxic effect of IBI on striated muscle in beagle dogs is consistent with an inhibition of the mitochondrial flavin-containing enzymes including dimethyl glycine, sarcosine, isovaleryl-CoA, 2-hydroxyglutarate, and multiple acyl-CoA (short, medium, long, and very long chain) dehydrogenases. All of these enzymes converge at the level of electron transfer flavoprotein (ETF) and ETF oxidoreductase. Urinary ethylmalonate was shown to be a biomarker of IBI-induced striated muscle toxicity in dogs and could provide the ability to monitor potential IBI-induced toxic myopathy in humans. We propose that IBI-induced toxic myopathy in beagle dogs is not caused by direct antagonism of CB1R and could represent a model of ethylmalonic-adipic aciduria in humans.

Spatial distribution of cannabinoid receptor type 1 (CB1) in normal canine central and peripheral nervous system

“…A commercially available antibody against human CB 1 was used, corresponding to C terminal amino acids 461–472 of human cannabinoid receptor type 1. The CB 1 protein sequence is highly conserved across mammalian species [33], moreover, crossreactivity of this specific CB 1 antibody with canine tissue has been previously demonstrated in peripheral tissues, hippocampus and cerebellum of adult dogs [26, 34] and in canine embryos [35]. …”

Plasma 2-hydroxyglutarate and hexanoylcarnitine levels are potential biomarkers for skeletal muscle toxicity in male Fischer 344 rats

“…It was reported that the off-target mechanism of myotoxicity accompanied by increased plasma levels of acylcarnitines in dogs that is induced by ibipinabant, which is a cannabinoid-1 receptor antagonist, involved the inhibition of mitochondrial ADP/ATP exchange (Schirris et al, 2015). It was suggested that increases in the plasma levels of acylcarnitines are due to impaired fatty acid oxidation in skeletal muscle (Tomlinson et al, 2012). It is well Table 2.…”

“…Our present data, which show that the levels of 2HG and hexanoylcarnitine were increased in plasma and muscle tissues, suggest that CER-and TMPD-induced skeletal muscular injury can be monitored in the plasma, which would be useful in routine animal studies. In addition, 2HG and hexanoylcarnitine may be potential biomarkers for muscle toxicity in dogs, because Tomlinson et al (2012) reported that 2HG and hexanoylcarnitine were increased in dogs with skeletal and cardiac myopathies induced by cannabinoid-1 receptor antagonist. Fig.…”

“…Compared to classical diagnostic approaches and conventional clinical biomarkers, metabolomics offers potential advantages owing to its sensitivity and specificity (Monteiro et al, 2013). Metabolomic approaches have been used to show changes in plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate (2HG) in cannabinoid-1 receptor antagonist-induced skeletal and cardiac myopathy in dogs (Tomlinson et al, 2012).…”

Metabolomics in the pharmaceutical industry

“…In preclinical studies evaluating a cannabanoid-1 receptor antagonist, dogs developed a drugrelated, microscopically observable lipid accumulation in muscle tissue. Urinary metabolomics revealed a coincident ethylmalonic acid accumulation in the urine, which was subsequently carried into phase 1 clinical trials as a safety biomarker [41]. Xenobiotic metabolites are also part of the metabolome, and these can also be characterized through metabolomic studies.…”