2017 ACVIM Forum Research Abstract Program

Our study confirmed that oral MMF at maximally tolerated dose rates significantly inhibits both B and T cell proliferation in dogs. This effect, however, appears to be delayed for several weeks, suggesting that MMF may not be the ideal treatment choice for dogs with severe or emergent immune-mediated diseases that require rapid immunosuppression. The mechanism for this delay in the immunosuppressive effects of MMF has not been elucidated, although another purine synthesis inhibitor, azathioprine, is also reported to have a delayed effect, the cause of which has also not yet been determined. Diarrhea was, as has been previously reported, the most common dose-limiting side effect in our study. The exact mechanism of MMF-associated diarrhea has not been determined in either humans or dogs. Interestingly, in some dogs, drug-induced diarrhea, as with drug effects on lymphocyte proliferation, appears to be delayed until after the first week of therapy. Since a lower oral dose rate (10 mg/kg bid) of MMF still causes significant suppression of lymphocyte proliferation in some dogs, without associated diarrhea, this reduced dose rate warrants exploration in future pharmacodynamic studies. The enzyme cytochrome b 5 reductase (encoded by CYB5R3) detoxifies reactive metabolites of sulfonamide antibiotics. The single nucleotide polymorphism (SNP) CYB5R3 729A>G was recently associated with sulfonamide hypersensitivity in dogs. This synonymous coding SNP may partially explain risk for this idiosyncratic drug reaction, however, the functional significance of this variant is unknown. Based on in silico predictions, we hypothesized that the 729A>G SNP would decrease protein synthesis and total cellular cytochrome b 5 reductase activity without decreasing mRNA expression in an in vitro model. HepG2 cells were transfected with an expression vector containing either wildtype (A) or variant (G) CYB5R3 cDNA and incubated for 48 hours. Cells were harvested and CYB5R3 mRNA, protein, and function were measured with qRT-PCR, immunoblots, and cytochrome c reduction assay, respectively. There was no significant difference in protein expression between the A and G alleles. mRNA quantification and cytochrome b 5 reductase activity assays are underway. Preliminary results do not support CYB5R3 729A>G as a functional variant leading to decreased cytochrome b 5 reductase expression. However, immunoreactivity is only a semi-quantitative measure of protein expression; results of mRNA and enzyme activity assays may better inform the mechanistic significance of 729A>G SNP in sulfonamide hypersensitivity in dogs. Cyclosporine is a potent immunosuppressive agent commonly used to treat immune-mediated disorders in dogs. Cyclosporine targets T lymphocytes by decreasing cytokine expression (IL-2, IL-4 and IFN-c) of activated T cells. Unfortunately, significant complications, such as life-threatening secondary infections, can sometimes arise as a result of cyclosporine administration, necessitating cessation of therapy. Once cyclosporine is stopped, the expectation is that the patient's immune system will completely recover, and commence functioning appropriately. Currently, it is unknown how quickly the immune system and T-cell function recover following the cessation of cyclosporine therapy. The objective of this study was to utilize a pharmacodynamic bioassay to quantify the average time it takes for T-cell function to return to pre-treatment levels in healthy dogs after cessa