Pharmacokinetics of mycophenolic acid and its effect on CD4+ and CD8+ T cells after oral administration of mycophenolate mofetil to healthy cats

Abstract Background Mycophenolate mofetil (MMF) is an immunosuppressant used in human and veterinary medicine. Little pharmacokinetic and pharmacodynamic information on MMF is available in cats. Objective To evaluate the plasma disposition of mycophenolic acid (MPA) and assess its effect on total peripheral blood mononuclear cells and CD4+/CD8+ ratios after PO administration of MMF. Animals Healthy cats (n = 10). Methods Mycophenolate mofetil was administered at a dosage of 10 mg/kg q12h (n = 3), 15 mg/kg q12h (n = 3), and 15 mg/kg q8h (n = 4) for 7 days. Concentrations of MPA and derivatives were determined using ultra‐high‐performance liquid chromatography. Flow cytometry was used to assess CD4+/CD8+ T‐cell ratios. Results All cats biotransformed MMF into MPA. Half of the cats (5/10) had adverse effects within 1 week of MMF administration. Area under the curve limit of quantification (AUC0‐LOQh) of MPA ranged from 1.27 to 2.03 hours·μg/mL and from 1.77 to 8.54 hours·μg/mL after the first and last PO dose of 10 mg/kg. The AUC0‐loqh of MPA ranged from 2.18 to 31 hours·μg/mL after the first dose of 15 mg/kg of MMF. Before the first dose of MMF, the average total number of PBMC ranged from 1.2 to 9.3 million/mL. At the last dose of MMF, the average total number of PBMC ranged from 3 to 5 million/mL. Conclusion Mycophenolic acid was detected in all cats. The dose 10 mg/kg given q12h for 1 week was tolerated (n = 3). The efficacy of MMF as an immunosuppressant and long‐term safety in cats of this dosage regimen is unknown.


| INTRODUCTION
Few orally administered immune suppressants are available in clinical veterinary medicine despite many conditions requiring their use. Many immune-mediated diseases that occur in veterinary medicine are unpredictable and impact the patient's quality of life. [1][2][3] Few investigations into the use of alternative medications in cats have been performed. Therefore, further research is warranted for clinically relevant and easily administered PO immunosuppressant drug options for cats.
Mycophenolate mofetil is an immunosuppressant used in human medicine in organ transplantation patients. [4][5][6][7] It has been used in veterinary medicine [8][9][10][11][12] as a secondary immunosuppressant with little published research in cats. 11,13,14 To date, safe and effective dosage regimens remain to be established. Mycophenolate mofetil is a prodrug of the active moiety mycophenolic acid (MPA), a fermentation product of Penicillium. 7,15 After PO administration, MMF undergoes rapid presystemic tissue de-esterification [15][16][17] and is converted to the active metabolite MPA. 15 In cats, MPA is highly bound to plasma proteins, 18 and is eliminated from the body rapidly likely by hepatic biotransformation into at least 2 metabolites: MPA phenol glucoside (MPAGls) and MPA phenol glucuronide (MPAG). 19,20 The primary action of MPA involves decreasing T and B lymphocyte proliferation by a specific and noncompetitive mechanism of action that decreases production of antibodies, decreases proliferation of CD4 + and CD8 + lymphocytes, and inhibits adhesion of glycoproteins to endothelial cells. 15,21 These effects occur by inhibiting inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo guanosine synthesis. 15,21 The disposition of MPA in cats has been studied after IV infusion of MMF, 19 suggesting that the disposition of MPA is highly variable. In addition, MMF administered IV at a dosage of 10 mg/kg q12h for 3 days in healthy cats resulted in little change in total peripheral blood mononuclear cell (PBMC) counts after MMF administration. 20 8,9,12 and cats. 19,20,23 Food was withheld 2 hours before and after drug administration. Water was available ad libitum to the cats. A repeat CBC and serum biochemistry profile were performed in all study cats within 24 hours of the last PO MMF administration.
The tubes were centrifuged at 1800g for 8 minutes. Plasma was separated and divided into 200 μL aliquots in Eppendorf (Eppendorf AG, Hamburg, Germany) tubes and stored at −80 C until analysis. Samples were analyzed in a single batch. For PBMC isolation, 1.5 mL of blood was collected and placed into glass tubes with lithium heparin before dosing, and 24, 144, and 168 hours after the initial MMF PO dose.
A volume <5% of the circulating blood volume of the cats was obtained for analysis during the course of the study.   Abbreviations: BLLOQ, below lower limit of quantification (0.3 μg/mL); NA, not applicable. For 10 mg/kg dosage regimen protocol, the median observed maximum plasma concentration and AUC 0-loq after the first and last dose were not different (P = .2).
WinNonlin v. 7 (Phoenix, version 7.1, Pharsight Corp Mountain View, California) where t is sampling time and Y is the observed outcome: The PK parameters were reported as mean and range unless otherwise noted. The AUC 0-loq of MPA after the first and last administration of 10 mg/kg of MMF was compared statistically using a Mann-Whitney test in GraphPad Prism v. 8. Significance level was P < .05.

| Statistical analysis
Estimated PK parameters and T-cell and total PBMC response to treatments were evaluated using descriptive statistics (GraphPad Prism, version 7, GraphPad Software Inc, San Diego, California).

| RESULTS
All cats (4/4) had gastrointestinal signs (self-limiting diarrhea and hyporexia) in the 15 mg/kg q8h group; no cats completed the trial. One cat in the 15 mg/kg q12h group had self-limiting diarrhea; 2 of 3 cats completed the trial. No cats in the 10 mg/kg q12h group had diarrhea or hyporexia; 3 of 3 cats completed the trial. Once adverse effects were seen in affected cats, MMF was discontinued immediately. Serum biochemical results such as alanine transferase activity remained similar pre-and post-MMF treatment. Platelet counts and PCV decreased in 9 of 10 cats post-MMF treatment but remained adequate, based on blood smear slide evaluation by a clinical pathologist, as previously reported. 13 No housing or food intake was altered in any of the cats during the study period.

| Pharmacokinetic results
The disposition of MPA was evaluated after PO administration of MMF. After PO administration of MMF at 10 and 15 mg/kg, all cats biotransformed MMF to MPA (Table 1 and Figure 1). Pharmacokinetic parameters are presented in Table 1.
For all treatment groups, MPAG and MPAGls were detected in all cats, but the concentrations were below the validated LLOQ in some cats. After the last administration of 10 mg/kg q12h MMF,  (Figures 2 and 3).

| Determination of the effect of the repeated administration of MMF
Cell counting was assessed by use of the trypan blue dye exclusion test. 27 We assessed cell viability using the Moxi population index; all tested samples had an average of 95%-98% viability.
F I G U R E 2 MPA concentrations for all treatment groups. *Orange line reflects that many values shown were below the lower limit of quantification but above the lower limit of detection F I G U R E 3 A, MPAGls concentrations for all treatment groups. *Orange line reflects that many values shown were below the lower limit of quantification but above the lower limit of detection. B, MPAG concentrations for all treatment groups. *Many values shown were below the lower limit of quantification but above the lower limit of detection

| DISCUSSION
We report the disposition of MPA in plasma after PO administration These observations are consistent with those of previous studies. 19,20,23 The plasma disposition of MPA was variable in the cats as was the peak concentration of MPA ( Figure 1 and Table 1  Additionally, MMF may not have been given for a long enough time period, particularly considering that MPA has a cytostatic effect. 5,28 Recent studies performed in healthy dogs suggest that MMF may need to be given for at least 2 weeks before decreased lymphocyte proliferation is observed. 31 Interestingly, there is evidence that over time, a cumulative effect may occur after MMF administration, or initially other inhibitory actions on the immune system may occur, such as targeting key functions of dendritic cells. 15,32 In human medicine, conflicting evidence exists regarding the best pharmacodynamic marker after MMF administration, including, assessing disease activity scores, quantifying MPA F I G U R E 5 Total PBMC (mean ± standard deviation) for all cats (n = 10) given different doses of MMF on days 1, 2, 7, 8 plasma concentrations, and measuring inhibition of IMPDH activity or the concentrations of MPA in PBMC. 21,33 Currently, a validated method to measure IMPDH activity in cats is not available, and little research has been done to evaluate the effect of MPA on PBMC in cats. Previous studies have documented an effect of MMF on feline lymphocyte proliferation in vitro. 28 Further studies assessing lymphocyte proliferation after PO dosing may help clarify MMF's effects in cats.
Another factor that could have contributed to the lack of effect on the lymphocyte counts is variability in the MPA target of IMPDH in certain cats.
Recently, investigations in human medicine suggest that single nucleotide polymorphisms (SNPs) in genes encoding IMPDH may influence the inhibitory activity of MPA. 34 Unfortunately, we did not assess the genetic background of our cats or whether or not genetic polymorphisms in genes encoding for IMPDH could have contributed to our findings. However, SNPs in genes encoding IMPDH should be considered in future studies.
In conclusion, we obtained novel information regarding the disposition of MPA and its effects on total PBMC and CD4 + /CD8 + T-cell ratios after PO MMF administration in healthy cats. Because of variability in tolerance to MMF and current information on its effect on lymphocytes in vitro, MMF cannot be recommended for the routine treatment of immune-mediated disorders in cats.