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.


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. Dogs were administered each drug 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. Drug concentrations in blood 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.


| INTRODUCTION
Immune-mediated disorders of dogs are commonly treated with 1 or more immunosuppressive agents, often a glucocorticoid in addition to a second immunosuppressive agent such as cyclosporine, azathioprine, mycophenolate mofetil, or leflunomide. 1 Glucocorticoids such as prednisone are a mainstay in the treatment of immune-mediated disorders, with multiple mechanisms of action involving both the humoral and cellmediated arms of immunity. 1 Cyclosporine has a specific mechanism of action, which is inhibition of the enzyme calcineurin in lymphocytes, leading to impaired lymphocyte function by suppression of nuclear factor of activated T-cell-regulated cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-γ). 1  In dogs receiving immunosuppressive treatment, therapeutic drug monitoring through the measurement of blood concentrations, the use of pharmacodynamic assays, or both can be used to ensure appropriate drug doses, particularly when there is individual dog-to-dog variability in drug metabolism and a narrow therapeutic window between ineffective and toxic doses. Currently, there are no commercially available assays for measuring blood concentrations of prednisone, mycophenolate mofetil, or azathioprine in dogs, although concentrations of mycophenolic acid (the active metabolite of mycophenolate mofetil) have been measured in an experimental setting. In North America, there are currently commercially available assays for measuring cyclosporine and teriflunomide (the active metabolite of leflunomide) blood concentrations. There is also a commercially available pharmacodynamic assay to evaluate the immunosuppressive effects of cyclosporine in dog via measurement of activated T-cell production of IL-2 and IFN-γ. This assay was developed and validated specifically for cyclosporine 5  The goal of this study was to identify the effects of the oral administration, at immunosuppressive doses, of 5 common immunosuppressive medications (prednisone, cyclosporine, azathioprine, mycophenolate mofetil, and leflunomide) on the expression of the T-cell cytokines IL-2 and IFN-γ in dogs. Our hypotheses were that cyclosporine and prednisone would suppress cytokine gene expression because they are known to affect transcription factors, with cyclosporine having the greatest effect, and that the proliferation inhibitors azathioprine, mycophenolate mofetil, and leflunomide would not have a significant effect.

| Animals
Eight healthy Walker hound dogs were used in this study. Four dogs were intact females and 4 were neutered males, with a median age of 2 years (range, 1.3-7.3 years). Typically, 6-8 dogs are used for standard pharmacokinetic and pharmacodynamic studies, as described by Riviere,6 and published by others. 2,3,7-9 Dogs were deemed healthy by detection of no abnormalities on physical examination and a minimum data base (including a complete blood count, serum biochemistry, urinalysis) as well as heartworm testing. The dogs were not exposed to any medications or vaccines for at least 1 month before the initiation of the study. Animal use was approved by the Mississippi State University College of Veterinary Medicine Institutional Animal Care and Use Committee and followed the requirements of a facility accredited by the American Association for Accreditation of Laboratory Animal Care.

| Drug administration and blood collection
In a 5-way, randomized, cross-over study, the dogs were administered either prednisone (West-Ward Pharmaceutical Corp., Eatontown, New Jersey) (2.0 ± 0.1 mg/kg, mean ± SD, PO, q24h), azathioprine (Mylan (4.0 ± 0.2 mg/kg, PO, q24h). With each medication, drugs were administered for 7 days, followed by a minimum recovery period of 21 days between dosing with another medication. In previous studies of cyclosporine, maximal changes in cytokine expression were seen within 1 week of commencing the drug, and cytokine expression had returned to values before treatment within 2 weeks of discontinuing the medications. 2,4,9 After each recovery period, the dogs switched groups, and the study was continued until all dogs had received all medications.
Before drug administration, 3 mL of blood was collected from each dog in a heparinized tube for baseline quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis for the assessment of IL-2 and INF-γ. At the end of each treatment period, blood was again collected in a heparinized tube from each dog, 2 hours after the morning drug dose was administered, for repeat RT-qPCR analysis. At the same time point (2 hours after the final morning pill), blood was collected to measure peak blood cyclosporine concentrations.
Additionally, within minutes before administration of the final morning drug dose, blood was collected to measure trough blood concentrations of the active metabolites of mycophenolate mofetil and leflunomide (mycophenolic acid and teriflunomide, respectively).

| Blood incubation, T-cell activation, and RNA extraction
All whole blood samples were activated before RNA extraction with 12.5 ng/mL of phorbol myristate acetate (Sigma, St. Louis, Missouri) and 0.8 μM of ionomycin (Sigma), as previously described. 5 All samples were then incubated for 5 hours at 37 C and 5% CO 2 .
RNA was extracted by using a previously published protocol. 5 Total RNA was isolated from 1 mL of heparinized whole blood by No. 79254). Samples were then stored at −80 C until further analysis.  3 with the flow rate set to 1 mL/min. 12 The standard curve ranged from 2.5 to 200 μg/mL, and was generated by spiking canine plasma with known amounts of teriflunomide (VWR, Radnor, PA) reference standard and accepted if the coefficient of determination (r 2 ) was at least .999 and the predicted concentrations were within 10% of the actual concentrations. Briefly, 500 μL of acetonitrile were added to tubes containing 250 μL of plasma sample. 14 The contents of each tube were mixed vigorously through vortexing, then subjected to centrifugation for 10 minutes at 3000 rpm (1900g). 12,14 The volume of clear supernatant injected into the column was 20 μL. 14 The retention time for teriflunomide was 4.5 minutes and UV absorbance was monitored at 302 nm 2 . The linear correlation coefficient for teriflunomide was .9995.

| Cytokine gene expression quantification
The limit of detection and the limit of quantification (LOQ) for teriflunomide were 1.0 and 2.5 μg/mL, respectively. The accuracy (% recovery) for teriflunomide plasma controls samples with concentra- Plasma mycophenolic acid levels were measured by using a homogeneous particle-enhanced turbidimetric inhibition immunoassay, automated assay, on the Siemens Dimension Xpand Plus system (Siemens Healthcare Diagnostics Inc). The assay used a synthetic particle-mycophenolic acid conjugate and monoclonal antibody specific to mycophenolic acid. Competition for antibody between the particle-conjugated mycophenolic acid and the mycophenolic acid in    Cytokine expression, presented as a percentage of baselineactivated samples before treatment, is represented in Figure 2.
Baseline-activated samples before treatment represents 100% cytokine production. Samples exposed to cyclosporine showed a marked suppression of cytokine expression after 7 days of drug administration.

| DISCUSSION
In this study, we utilized a RT-qPCR assay, validated in dogs, to explore the effects of the administration of 5 common immunosuppressive medications on T-cell cytokine production. This study dem- Immune-mediated and inflammatory disorders of dogs are commonly treated with ≥1 immunosuppressive agents. In dogs, this often includes a glucocorticoid in addition to a second immunosuppressive agent, not only to target the immune system with 2 medications with differing mechanisms of action, but also to enable earlier tapering of the glucocorticoid to minimize steroid-related adverse effects. 1 Prednisone, cyclosporine, mycophenolate mofetil, azathioprine, and leflunomide can all be used in dogs to induce immune suppression. 1,15 For all these medications, there is a range of recommended dosages for use in dogs, and this study used common starting dosages that are believed to be immunosuppressive in dogs. 1,15 The cyclosporine dosage chosen for this study was 10 mg/kg PO twice daily, and although this is within the accepted dosage range for initial treatment, it is at the high end of the dosage range. This dose was chosen to reliably induce immune suppression in all dogs. This study revealed that dogs administered cyclosporine at this high dose exhibited marked suppression of cytokine expression after 7 days of drug administration, demonstrating that this assay worked in a manner consistent with previous studies, and confirming that the drug dose used was reliably immunosuppressive. 2  Clinical Pharmacology Laboratory, a concentration greater than differences in both IL-2 and IFN-γ expression before and after treatment with cyclosporine or prednisone, as well as detecting differences in expression between cyclosporine and prednisone treated dogs, it is possible that the sample size of 8 dogs did not provide sufficient power to detect an effect by the other drugs resulting in Type II errors.
In conclusion, cyclosporine and prednisone had a significant effect on expression of IL-2 and IFN-γ, with cyclosporine having a more pronounced effect than prednisone. Azathioprine, mycophenolate mofetil, and leflunomide did not have a significant effect on these 2 cytokines.
At a cyclosporine dosage of 10 mg/kg PO every 12 hours, a significant and profound effect was seen in every dog. Prednisone had less pronounced and more variable effect on these cytokines. Blood drug concentrations were highly variable when dosing cyclosporine, mycophenolate mofetil, and leflunomide.

OFF-LABEL ANTIMICROBIAL DECLARATION
The authors declare no off-label use of antimicrobials.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.