Excessive Cyclosporine-Associated Immunosuppression in a Dog Heterozygous for the MDR1 (ABCB1-1Δ) Mutation.

Pharmacodynamic monitoring was used to titrate cyclosporine dosing in a dog with immune-mediated hemolytic anemia. Development of a suspected secondary infection, with subsequent discovery of an unexpectedly high level of T-cell suppression despite a relatively low cyclosporine dose, prompted an investigation into the cause of possible excessive immunosuppression. Blood cyclosporine concentrations were within expected target ranges, and the dog was determined to be heterozygous for the multidrug resistance protein 1 (MDR1; ATP-binding cassette sub family B member 1-1Δ) gene mutation. The MDR1 mutation was suspected to have contributed to the excessive immunosuppression experienced by this patient. This case highlights the need to monitor immunosuppressive therapy in the individual patient, especially when the patient is not responding to therapy at typical dosages or when secondary infections develop at dosages lower than expected to cause significant immunosuppression. Pharmacodynamic monitoring can be used to help identify unexpected excessive immunosuppression in dogs receiving cyclosporine, and MDR1 genotyping should be further explored as a potential method of predicting and preventing its occurrence.

Effects of oral administration of 5 immunosuppressive agents on activated T-cell cytokine expression in healthy dogs.

BACKGROUND: Dogs are often adminstered >1 immunosuppressive medication when treating immune-mediated diseases, and determining whether these different medications affect IL-2 expression would be useful when performing pharmacodynamic monitoring during cyclosporine therapy. HYPOTHESIS/OBJECTIVES: To determine the effects of 5 medications (prednisone, cyclosporine, azathioprine, mycophenolate mofetil, and leflunomide) on activated T-cell expression of the cytokines IL-2 and interferon-gamma (IFN-γ). ANIMALS: Eight healthy dogs. METHODS: Randomized, cross-over study comparing values before and after treatment, and comparing values after treatment among drugs. Dogs were administered each drug at standard oral doses for 1 week, with a washout of at least 21 days. Activated T-cell expression of IL-2 and IFN-γ mRNA was measured by quantitative reverse transcription polymerase chain reaction. Blood drug concentrations were measured for cyclosporine, mycophenolate, and leflunomide metabolites. RESULTS: Least squares means (with 95% confidence interval) before treatment for IL-2 (2.91 [2.32-3.50] ΔCt) and IFN-γ (2.33 [1.66-3.00 ΔCt]) values were significantly lower (both P < .001) than values after treatment (10.75 [10.16-11.34] and 10.79 [10.11-11.46] ΔCt, respectively) with cyclosporine. Similarly, least squares means before treatment for IL-2 (1.55 [1.07-2.02] ΔCt) and IFN-γ (2.62 [2.32-2.92] ΔCt) values were significantly lower (both P < .001) than values after treatment (3.55 [3.06-4.00] and 5.22 [4.92-5.52] ΔCt, respectively) with prednisone. Comparing delta cycle threshold values after treatment among drugs, cyclosporine was significantly different than prednisone (IL-2 and IFN-γ both P < .001), with cyclosporine more suppressive than prednisone. CONCLUSIONS AND CLINICAL IMPORTANCE: Prednisone and cyclosporine both affected expression of IL-2 and IFN-γ, suggesting that both have the ability to influence results when utilizing pharmacodynamic monitoring of cyclosporine treatment.

Effects of pentoxifylline on whole blood IL-2 and IFN-gamma gene expression in normal dogs.

BACKGROUND: Pentoxifylline (PTX) is a methylxanthine phosphodiesterase inhibitor that is used as a hemorrheologic and anti-inflammatory agent in veterinary and human medicine. In human studies, PTX has been shown to decrease T-cell production of cytokines such as IL-2 and IFN-γ. A RT-qPCR assay to measure activated T-cell gene expression of IL-2 and IFN-γ has been validated in dogs. OBJECTIVES: The goal of this study was to utilize this assay to investigate the effects of PTX on in vitro cytokine gene expression in canine whole blood. METHODS: Whole blood from seven healthy dogs was collected and incubated with various concentrations of PTX for 1 hr before activation. PTX concentrations spanned and exceeded blood concentrations achieved when administered at clinically relevant dosages (1, 2, 10, 50 and 200 µg/ml). Cyclosporine was used at a concentration of 500 ng/ml as a positive control. All blood samples, including untreated activated baseline samples, were then activated with phorbol myristate acetate and ionomycin for 5 hrs. RESULTS: Analysis of activated whole blood by RT-qPCR revealed that there was not a significant suppression of IL-2 or IFN-γ gene expression at any concentration of PTX when evaluating ΔCt values. All samples exposed to cyclosporine showed significant changes from untreated activated baseline samples, demonstrating marked suppression as the positive control. Cytokine expression, presented as a percentage of untreated activated baseline samples, was also evaluated. After exposure to the highest concentration of PTX (200 µg/ml), median percentage cytokine expression was suppressed to just below 50% of baseline values. This concentration, however, is much higher than blood concentrations reported to be achieved at standardly used pentoxifylline doses. CONCLUSIONS: PTX does not appear to significantly suppress T-cell cytokine production in samples from most dogs at clinically relevant drug concentrations. Further testing is needed to establish the full effects of PTX on the immune system in dogs.

Alterations in activated T-cell cytokine expression in healthy dogs over the initial 7 days of twice daily dosing with oral cyclosporine.

Cyclosporine is a powerful T-cell inhibitor used in the treatment of immune-mediated and inflammatory diseases in the dog. There is limited information on how to best monitor patients on cyclosporine therapy. Currently, pharmacokinetic and pharmacodynamic assays are available. Pharmacokinetic assays that measure the concentration of cyclosporine in the blood are used to assess if an appropriate drug concentration has been achieved; however, target blood drug concentrations have not been shown to reliably correlate with suppression of T-cell function in the dog. In human transplant recipients, therapeutic drug monitoring has shifted to include pharmacodynamic-based monitoring. Our laboratory has validated a RT-qPCR assay to measure the pharmacodynamic effects of cyclosporine in the dog. In this study, activated T-cell expression of IL-2 and IFN-γ was measured using RT-qPCR daily for 7 consecutive days in 8 healthy Walker hounds receiving oral cyclosporine at a dosage of 10 mg/kg every 12 hr. Cytokine production was found to be markedly decreased within 24 hr after the initiation of cyclosporine and remained significantly decreased for the duration of the project. Based on these results, cyclosporine causes a rapid drop in T-cell cytokine production that is sustained with continued dosing in healthy dogs. Although performed in healthy dogs, this study demonstrated a marked decrease in cytokine suppression within 24 hr of drug administration, suggesting that pharmacodynamic monitoring of cyclosporine's effects on T cells could be considered within several days of commencing therapy in dogs suffering from life-threatening immune-mediated disorders.