Introduction
Autoimmune diseases, such as hemolytic anemia, thrombocytopenia, pemphigus, and vasculitis, in horses can be challenging with regard to treatment.1–5 Corticosteroids, injectable preparations of gold salts, and, to a lesser extent, azathioprine are the only immunosuppressive treatments that have been used somewhat frequently in horses.1–3,6 These agents are often effective. However, the high doses of corticosteroids necessary for successful treatment are associated with an increased risk of adverse effects, including corticosteroid hepatopathy, laminitis, and iatrogenic hyperadrenocorticism. Injectable preparations of gold salts are not always readily available.1,4,7,8 Therefore, investigations of other immunosuppressive agents for treatment of autoimmune diseases in horses are needed.
Mycophenolate mofetil is the prodrug of MPA. Mycophenolic acid is a potent and reversible inhibitor of de novo purine biosynthesis required for T- and B-lymphocyte proliferation9,10; thus, MPA reduces the production of antibodies. Although MMF was originally developed to suppress organ rejection in human transplant recipients, more recently it has been used as a treatment for immune-mediated diseases in people and (as the oral form) small animals.11–16 Safety studies of cats17–19 and pharmacokinetic and pharmacodynamic studies20,21 of dogs have provided evidence of the safety (although not necessarily the efficacy) of MMF administered at a dose of 10 mg/kg twice daily in cats and a dose of 8 to 15 mg/kg twice daily in dogs. Adverse effects of MMF treatment generally involve the gastrointestinal tract (eg, vomiting and diarrhea).12,13,19
The objective of the study reported here was to characterize the pharmacokinetics of MMF following single-dose IV or PO administration, characterize the pharmacokinetics of MMF following multiple-dose PO administration, and describe the clinicopathologic effects of long-term PO MMF administration in horses.
Materials and Methods
Animals
For the single-dose assessment, 6 university-owned horses were used. The horses included 1 Quarter Horse mare and 5 Thoroughbred geldings. The mean ± SD age and weight of the horses were 10.8 ± 3.4 years (range, 7 to 15 years) and 540.0 ± 39.8 kg (range, 472 to 572 kg), respectively. For the multiple-dose study, 6 other university-owned horses were used. The horses included 1 Standardbred mare, 1 Thoroughbred mare, and 4 Thoroughbred geldings. The mean ± SD age and weight of these horses were 11.3 ± 7.8 years (range, 3 to 23 years) and 521.1 ± 61.1 kg (range, 449.4 to 615.4 kg), respectively.
Prior to commencement of the study, all horses were considered healthy on the basis of physical examination findings and results of a CBC; assessments of serum activities of aspartate aminotransferase, creatine kinase, alkaline phosphatase, and sorbitol dehydrogenase; and concentrations of total bilirubin, BUN, and creatinine. Throughout the study, all clinicopathologic analyses were performed with standard protocols at 1 clinical pathology laboratory.a Horses did not receive any other medications for at least 2 weeks prior to participation in the study. The study was approved by the Institutional Animal Care and Use Committee of the University of California-Davis (protocol 20319).
Study design, catheter placement, and drug administration
The study was conducted in 2 phases. Phase 1 was conducted in a randomized (with respect to treatment order) balanced crossover design. In phase 1, 6 horses received a single IV (2.5 mg/kg) or PO (5 mg/kg) dose of MMF; after a minimum washout period of 2 weeks, each horse received a second single dose by the other route of administration. Each horse was weighed immediately prior to IV or PO drug administration. For IV administration of MMF, a 14-gauge catheter was aseptically placed in the external jugular vein of each horse immediately prior to drug administration. One catheter was used for drug administration, and the other catheter was used for blood sample collection. The dosing catheter was removed following drug administration. For PO administration of MMF, a 14-gauge catheter was aseptically placed in 1 external jugular vein of each horse immediately prior to drug administration. This catheter was used for blood sample collection. For IV administration, MMFb was diluted in 250 mL of fluids (5% dextrose and saline [(0.9% NaCl] solution) and administered through the dosing catheter over a period of 2 hours. For PO administration, MMF tabletsc were dissolved in water and administered with a dosing syringe directly into the oral cavity of each horse. Food was withheld from each horse for approximately 8 hours before and 2 hours after drug administration; water was available ad libitum.
In phase 2, the other 6 horses received MMF PO according to a long-term dosing protocol for a total of 60 days. Mycophenolate mofetil was administered PO as described for phase 1 at a dosage of 5 mg/kg every 24 hours for 30 days and then every 48 hours for an additional 30 days. The long-term dosing protocol was based, in part, on a protocol used in a previous study22 of azathioprine (another immunosuppressive prodrug in which the active metabolite also suppresses antibody production and lymphocyte function) in horses. The long-term dosing protocol was also selected in an attempt to avoid MMF-related adverse gastrointestinal effects.
Sample collection
In phase 1, when the horses received IV administration of MMF, a blood sample (10 mL) was collected at 0 minutes (immediately prior to the start of the IV infusion) and 5, 10, 15, 20, 30, and 45 minutes and 1 (during the infusion), 1.5 (during the infusion), 2 (at the time the infusion was ended), 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48, 72, and 96 hours after commencement of the IV infusion. In phase 1, when the horses received PO administration of MMF, a blood sample (10 mL) was collected at 0 minutes (immediately prior to drug administration) and at 15, 30, and 45 minutes and 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48, 72, and 96 hours after PO administration.
In phase 2, a blood sample (10 mL) was collected at 0 minutes (immediately prior to the initial drug administration) and 15, 30, and 45 minutes and 1, 2, 3, 4, 6, 8, 12, and 24 hours after the initial drug administration. The 24-hour blood sample was collected immediately prior to the second drug administration. Additional blood samples were collected on days 3, 5, 7, 12, 14, 17, 19, 21, 24, 26, 28, and 30 immediately prior to and 1 hour after drug administration. The 1-hour sample collection time was based on Cmax values derived from phase 1 data. Following drug administration on day 30, a blood sample was collected at 15, 30, and 45 minutes and 1, 2, 3, 4, 6, 8, 12, and 24 hours. Subsequent blood samples were collected on days 35, 41, 47, 55, and 60 immediately prior to and 1 hour after drug administration. Following the final dose administration on day 60, blood samples were collected as described for day 30, with additional samples collected 36, 48, 72, and 96 hours after drug administration. Blood samples were collected by direct venipuncture into tubes containing EDTAd and were centrifuged at 3,000 × g for 10 minutes. Plasma was immediately transferred into storage cryovialsd and stored at–20°C until analysis (approx 2 weeks after collection of the final sample in phases 1 and 2).
Assessment of clinicopathologic variables
During phase 2, a blood sample (10 mL) was collected from each horse for a CBC and serum biochemical analysis immediately prior to the initial drug administration and once every 7 days for the first 30 days of drug administration. A blood sample was also collected for a CBC and serum biochemical analysis immediately prior to drug administration on days 30, 35, 41, 47, 55, and 60.
Determination of MMF and MPA concentrations in plasma samples
Mycophenolate mofetile and MPAe were combined into 1 working solution. Plasma calibrators were prepared by dilution of the working standard solution with drug-free equine plasma to concentrations between 0.1 and 4,000 ng/mL. Calibration curves and negative control samples were prepared fresh for each quantitative assay. In addition, quality control samples (equine plasma fortified with analytes at 1 of 3 concentrations within the standard curve) were included with each sample set as an additional check of accuracy.
Prior to analysis, 500 µL of plasma was diluted with 100 µL of water containing 0.01/ng mL of a deuterated standard (d4-MMFe or d3-MPAe) and 2 mL of 0.1M phosphate buffer (pH 7). The samples were vortexed briefly to mix and underwent solid-phase extraction in extraction columns.f Samples were loaded onto the columns and then washed with 3 mL of water each before elution with 3 mL of methanol. Samples were dried under nitrogen and dissolved in 150 µL of 5% acetonitrile in water with 0.2% formic acid; 40 µL of the resultant solution was injected into an LC-MS/MS system.
The concentration of MMF and MPA was measured in plasma by LC-MS/MS with positive electrospray ionization. Quantitative analysis of plasma was performed on a triple quadrupole mass spectrometerg that had a liquid chromatography system.h The spray voltage was 4,500 V, vaporizer temperature was 217°C, and sheath and auxiliary gas were 40 and 25 arbitrary units, respectively. Product masses and collision energies of each analyte were optimized by infusion of the standards into the mass spectrometer. Chromatography involved a 10 cm × 2.1 mm × 3-µm columni and a linear gradient of acetonitrile in water, both with 0.2% formic acid, at a flow rate of 0.4 mL/min. The initial acetonitrile concentration was held at 10% for 0.4 minutes and then ramped to a concentration of 99% over 4.5 minutes and held at that concentration for 0.3 minutes before re-equilibration for 4.3 minutes at initial conditions.
Detection and quantification were conducted with selective reaction monitoring of the initial precursor ion for MMF (m/z, 434.2), MPA (m/z, 321.1), and internal standards d4-MMF (m/z, 438.2) and d3-MPA (m/z, 324.1). The instrument response of the product ions for MMF (m/z, 159), MPA (m/z, 207 and 159), and internal standards d4-MMF (m/z, 118) and d3-MPA (m/z, 210) were plotted, and peaks at the proper retention time were integrated with softwarej to generate calibration curves and quantitate MMF and MPA concentrations in all samples by linear regression analysis. A weighting factor of 1/X was used for all calibration curves.
Determination of MPAG and MPAAG concentrations in plasma samples
Mycophenolic acid glucuronidek and MPAAGl stock solutions and working solutions were prepared as described for assessment of MMF and MPA plasma concentrations. Plasma calibrators were prepared, as described, to concentrations between 0.5 and 2,000 ng/mL. Solid-phase extraction was performed as that described for assessment of MMF and MPA plasma concentrations, with the exception that the internal standards d3-MPAG and d3-MPA were used each at a concentration of 0.2 ng/µL.
The concentrations of MPAG and MPAAG were measured in plasma samples by LC-MS/MS with negative electrospray ionization. Quantitative analysis of plasma samples was performed on a triple quadrupole mass spectrometerm coupled with a liquid chromatography system.n The spray voltage was 2,900 V, vaporizer temperature was 400°C, and sheath and auxiliary gas were 50 and 15 arbitrary units, respectively. Chromatography was performed as described for assessment of MMF and MPA plasma concentrations, except the acetonitrile concentration was held at 5% for 0.2 minutes, ramped to a concentration of 60% over 4.2 minutes, and then increased to a concentration of 95% over 0.1 minutes and held at that concentration for 0.1 minutes before re-equilibration for 3.4 minutes at initial conditions.
Detection and quantification were performed as described for assessment of plasma MMF and MPA concentrations with MPAG and MPAAG precursors (m/z, 495.1 each) and the internal standards d3-MPAG (m/z, 498.1) and d3-MPA (m/z, 324.1). The instrument response of the product ions for MPAG (m/z, 191.0, 205.1, and 287.0), MPAAG (m/z, 305.0), and internal standards d3-MPAG (m/z, 322), and d3-MPA (m/z, 210) were plotted with software.j
Pharmacokinetic parameter calculations
By use of commercially available software,o noncompartmental analysis was used to determine pharmacokinetic values for MPA. The time to maximal plasma concentration and Cmax were determined on the basis of visual inspection of the plasma concentration versus time plot. The terminal-phase half-life, AUC from time 0 hours to the last measured concentration, AUC from time 0 hours to infinity, and extrapolated percentage of the AUC were determined. The AUC was calculated with the log-linear trapezoidal rule and was extrapolated to infinity by dividing the last measured plasma concentration by the terminal slope. Fluctuation, or the percentage difference between peak and trough concentrations, within a dosing interval and the accumulation at steady state for both the 30- and 60-day time points were determined. Pharmacokinetic parameters for MMF and MPA are reported as mean ± SD, median, and range.
Results
The instrument responses for MMF, MPA, MPAG, and MPAAG were linearly correlated with concentration (correlation coefficients of ≥ 0.99). The intraday, interday, and analyst-to-analyst precision and accuracy of the assay were determined by assaying quality control samples in replicates (n = 6) for all analytes (Table 1). Accuracy was reported as the percentage of nominal concentration, and precision was reported as percentage relative SD. The technique was optimized to provide limits of quantitation of 0.1 ng/mL for MMF and 0.5 ng/mL for MPA, MPAG, and MPAAG. The limits of detection were approximately 0.05 ng/mL for MMF and 0.25 ng/mL for MPA, MPAG, and MPAAG. Following single-dose IV, single-dose PO, and long-term PO administrations, MMF was rapidly converted to MPA. The mean plasma MMF and MPA concentrations versus time curves (Figure 1) and the mean plasma MPAG and MPAAG concentrations versus time curves (Figure 2) following single-dose IV or PO administration were plotted. The MMF concentrations were below the limit of quantitation in all but 1 horse by 4 hours after commencement (ie, 2 hours after termination) of the IV infusion. Following PO administration of a single-dose, MMF concentration was low at all time points; the concentration was below the limit of quantitation in 4 of 6 horses by 12 hours after drug administration. The mean plasma MPA concentration versus time curve for horses receiving long-term PO administration of MMF was plotted (Figure 3).
Accuracy and precision values for LC-MS/MS analysis of MMF, MPA, MPAG, and MPAAG in equine plasma samples.
| Analyte | Concentration (ng/mL) | Intraday accuracy (% nominal concentration) | Intraday precision (% relative SD) | Interday accuracy (% nominal concentration) | Interday precision (% relative SD) |
|---|---|---|---|---|---|
| MMF | 0.75 | 97.0 | 11.0 | 104 | 6.0 |
| 5.0 | 95.0 | 6.0 | 105 | 3.0 | |
| 2,500 | 88.0 | 3.0 | 101 | 3.0 | |
| MPA | 0.75 | 95.0 | 19.0 | 100 | 14.0 |
| 5.0 | 87.0 | 3.0 | 98.0 | 7.0 | |
| 2,500 | 84.0 | 6.0 | 94.0 | 5.0 | |
| MPAG | 1.5 | 107 | 5.0 | 104 | 6.0 |
| 50.0 | 112 | 3.0 | 108 | 4.0 | |
| 1,000 | 101 | 2.0 | 102 | 2.0 | |
| MPAAG | 1.5 | 104 | 6.0 | 102 | 5.0 |
| 50 | 110 | 3.0 | 107 | 4.0 | |
| 1,000 | 100 | 4.0 | 101 | 2.0 |
Quality control samples (drug-free equine plasma fortified with analytes at 1 of 3 concentrations within the standard curve) were included with each sample set as an additional check of accuracy. Accuracy is reported as percentage nominal concentration and precision is reported as percentage relative SD. Values represent the mean of 6 replicates.
Mean ± SD plasma MMF (white circles) and MPA (black circles) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (A) or following a single dose of MMF (5 mg/kg) administered PO (B) to 6 horses (phase 1). Phase 1 had a randomized (with respect to treatment order) balanced crossover design. Horses received a single dose of MMF by either route of administration; after a minimum washout period of 2 weeks, each horse received a single dose of MMF by the other route of administration.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MMF (white circles) and MPA (black circles) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (A) or following a single dose of MMF (5 mg/kg) administered PO (B) to 6 horses (phase 1). Phase 1 had a randomized (with respect to treatment order) balanced crossover design. Horses received a single dose of MMF by either route of administration; after a minimum washout period of 2 weeks, each horse received a single dose of MMF by the other route of administration.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MMF (white circles) and MPA (black circles) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (A) or following a single dose of MMF (5 mg/kg) administered PO (B) to 6 horses (phase 1). Phase 1 had a randomized (with respect to treatment order) balanced crossover design. Horses received a single dose of MMF by either route of administration; after a minimum washout period of 2 weeks, each horse received a single dose of MMF by the other route of administration.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MMF (white circles) and MPA (black circles) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (A) or following a single dose of MMF (5 mg/kg) administered PO (B) to 6 horses (phase 1). Phase 1 had a randomized (with respect to treatment order) balanced crossover design. Horses received a single dose of MMF by either route of administration; after a minimum washout period of 2 weeks, each horse received a single dose of MMF by the other route of administration.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MMF (white circles) and MPA (black circles) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (A) or following a single dose of MMF (5 mg/kg) administered PO (B) to 6 horses (phase 1). Phase 1 had a randomized (with respect to treatment order) balanced crossover design. Horses received a single dose of MMF by either route of administration; after a minimum washout period of 2 weeks, each horse received a single dose of MMF by the other route of administration.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG (A) and MPAAG (B) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (black circles) or following a single dose of MMF (5 mg/kg) administered PO (white circles) to the same 6 horses in Figure 1 (phase 1).
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG (A) and MPAAG (B) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (black circles) or following a single dose of MMF (5 mg/kg) administered PO (white circles) to the same 6 horses in Figure 1 (phase 1).
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG (A) and MPAAG (B) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (black circles) or following a single dose of MMF (5 mg/kg) administered PO (white circles) to the same 6 horses in Figure 1 (phase 1).
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG (A) and MPAAG (B) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (black circles) or following a single dose of MMF (5 mg/kg) administered PO (white circles) to the same 6 horses in Figure 1 (phase 1).
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG (A) and MPAAG (B) concentrations versus time curves following a single dose of MMF (2.5 mg/kg) administered IV (black circles) or following a single dose of MMF (5 mg/kg) administered PO (white circles) to the same 6 horses in Figure 1 (phase 1).
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG concentrations versus time curve following PO administration of 5 mg of MMF/kg every 24 hours for 30 days, followed by administration of that dose every 48 hours for an additional 30 days (total of 60 days [phase 2]) in 6 additional horses.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG concentrations versus time curve following PO administration of 5 mg of MMF/kg every 24 hours for 30 days, followed by administration of that dose every 48 hours for an additional 30 days (total of 60 days [phase 2]) in 6 additional horses.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Mean ± SD plasma MPAG concentrations versus time curve following PO administration of 5 mg of MMF/kg every 24 hours for 30 days, followed by administration of that dose every 48 hours for an additional 30 days (total of 60 days [phase 2]) in 6 additional horses.
Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.502
Because of the low plasma MMF concentrations, it was not possible to calculate pharmacokinetic parameters for this compound given by either route of administration. Pharmacokinetic parameters for MPA following single-dose IV or PO administration (Table 2) and following multiple-dose PO administration (Table 3) were calculated. Pharmacokinetic parameters for MPAG and MPAAG following single-dose IV or PO administration were also calculated (Table 4).
Mean ± SD pharmacokinetic parameters for MPA following a single dose of MMF (2.5 mg/kg) administered IV or following a single dose of MMF (5 mg/kg) administered PO to 6 horses (phase 1).
| Parameter | IV administration | PO administration |
|---|---|---|
| Cmax (ng/mL) | 2,585 ± 264.3 | 1,778.3 ± 441.5 |
| Tmax (h) | NA | 0.71 ± 0.29 |
| λz (1/h) | 0.180 ± 0.04 | 0.181 ± 0.04 |
| t1/2λ (h)* | 3.99 ± 0.865 | 4.02 ± 1.01 |
| AUC0–∞ (h•ng/mL) | 3,819 ± 848 | 6,091 ± 1520 |
| CL (L/h/kg) | 0.689 ± 0.194 | NA |
| Vdss (L/kg) | 1.57 ± 0.626 | NA |
Phase 1 had a randomized (with respect to treatment order) balanced crossover design. Horses received a single dose of MMF by either route of administration; after a minimum washout period of 2 weeks, each horse received a single dose of MMF by the other route of administration. All values were generated by noncompartmental analysis.
Harmonic mean.
AUC0–∞ = AUC from time 0 to infinity. CL = Systemic clearance. λz = Terminal rate constant. NA = Not applicable. t1/2λ = Terminal half-life. Tmax = Time to maximal plasma concentration after dose administration. Vdss = Volume of distribution at steady state.
Mean ± SD pharmacokinetic parameters for MPA following long-term PO administration of 5 mg of MMF/kg every 24 hours for 30 days followed by administration of that dose every 48 hours for an additional 30 days (total of 60 days [phase 2]) in 6 additional horses.
| Parameter | Time point | ||
|---|---|---|---|
| After first dose | After day 30 dose | After day 60 dose | |
| Tmax (h) | 0.75 ± 0.27 | 1.02 ± 1.46 | 0.72 ± 0.0 |
| Cmax (ng/mL) | 1,377 ± 510.6 | 1,557 ± 511 | 1,489 ± 242 |
| Cmin (ng/mL) | — | 13.2 ± 7.09 | 7.57 ± 3.84 |
| Css mean (ng/mL) | — | 255.7 ± 40.8 | 120.7 ± 33.8 |
| λz (1/h) | 0.188 ± 0.040 | 0.200 ± 0.049 | 0.150 ± 0.047 |
| t1/2λ (h)* | 3.68 ± 0.80 | 3.62 ± 0.89 | 4.59 ± 1.25 |
| AUCτ (h•ng/mL) | 5,553 ± 1433 | 6,136 ± 979 | 5,796 ± 1624 |
| Fluctuation (%) | — | 615 ± 242 | 1,280 ± 287 |
| Accumulation index | — | 1.02 ± 0.01 | 1.00 ± 0.002 |
All values were generated with noncompartmental analysis. Fluctuation represents the percentage difference between peak and trough concentrations within a dosing interval. Accumulation index was determined at steady state by use of the equation (AUC [(30 days or 60 days), τ])/(AUC [1, τ]), where 30 and 60 represent doses on day 30 and 60 and 1 represents the first dose.
— = Not determined. Cmin = Minimum plasma concentration. Css mean = Mean plasma concentration at steady state. AUCτ = AUC during a dosing interval.
See Table 2 for remainder of key.
Mean ± SD pharmacokinetic parameters for MPA and MPAAG following a single dose of MMF (2.5 mg/kg) administered IV or following a single dose of MMF (5 mg/kg) administered PO to 6 horses (phase 1).
| Parameter | MPAG | MPAAG | ||
|---|---|---|---|---|
| IV administration | PO administration | IV administration | PO administration | |
| Cmax (ng/mL) | 673.5 ± 292.2 | 1,002.3 ± 337.5 | 281.0 ± 214.5 | 290.8 ± 165.8 |
| Tmax (h) | 1.85 ± 0.742 | 3.67 ± 0.52 | 1.55 ± 0.622 | 2.17 ± 2.89 |
| λz (1/h) | 0.145 ± 0.024 | 0.128 ± 0.031 | 0.200 ± 0.064 | 0.152 ± 0.05 |
| t1/2λ (h)* | 4.78 ± 0.763 | 5.40 ± 1.50 | 3.46 ± 0.898 | 4.57 ± 1.69 |
| AUC0-Ȟ (h•ng/mL) | 2,805 ± 1262 | 7,642 ± 3097 | 721.6 ± 286.6 | 1,608 ± 487 |
See Table 2 for key.
Mycophenolate mofetil administered as a single dose IV or PO in phase 1 of the study or administered PO long-term (multiple doses) in phase 2 of the study was well tolerated by the horses. No clinical adverse effects were noted for any horse, and no horse had any consistently abnormal clinicopathologic findings (Supplementary Tables S1 and S2, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.6.502). Results of the CBC and serum biochemical analysis were reported for only 5 horses at week 3 because the blood sample for 1 horse clotted following collection and its analysis was not possible.
Discussion
The present study investigated the pharmacokinetics of MPA following single-dose IV or PO administration of MMF as well as following long-term administration to horses. To the authors' knowledge, the pharmacokinetics of MPA in horses has not been previously described. Similar to reports of other species,18 plasma concentrations of MMF quickly decreased to less than the limit of quantitation of the analytic assay whereas plasma MPA concentrations increased rapidly, indicating that MMF is rapidly converted to MPA. Therefore, as in previous investigations describing the pharmacokinetics of MMF, pharmacokinetic parameters were determined for MPA and not MMF in the present study.
The pharmacokinetics of MPA following administration of MMF by IV infusion in cats, rabbits, and humans has been reported.18,23,24 In the present study, samples were collected throughout the infusion as well as for a period following completion of the infusion. Although plasma MMF concentrations were low throughout the infusion, plasma MPA concentrations remained high. Interestingly, the time when MPA appeared in the plasma varied among horses (15 minutes to 1.5 hours), which suggested individual variation with respect to the rate of conversion. In the present study, the horses' plasma MPA concentrations peaked during the infusion; however, in humans, peak concentrations are not achieved until the end of the 60-minute infusion.23 Because blood samples were not collected during the infusion in other studies,18,24 it was not possible to compare the data for horses with those for cats or rabbits.
Among the horses of the present study, MMF was well tolerated following PO administration. Although it was not possible to directly compare Cmax determined in the present study with values determined in previous studies because of different dosing protocols, time to maximal plasma concentration was comparable among studies, with maximum concentrations achieved between 30 minutes and 1 hour in all species studied (humans and dogs).20,21,23,25
On the basis of the results of the present study, MPA was further metabolized to MPAG and MPAAG in horses, as described for other species. Both metabolites reportedly can undergo extensive enterohepatic recirculation as evidenced by a second peak on the plasma concentration curve occurring 6 to 12 hours after MMF administration.25,26 Such a second peak was observed at approximately 3 hours after MMF administration in the present study, which suggested that enterohepatic recirculation may occur in horses as well, albeit earlier than described for other species. If comparatively early enterohepatic recirculation occurs in horses, this could ultimately prolong the pharmacological effect of MMF because of prolonged residence time in the body. The mean terminal half-life following single-dose IV or PO administration to horses was similar to that previous reported for dogs (5.5 ± 3.8 hours).20
Long-term administration of MMF to humans and dogs is commonplace12,13,25,27,28; therefore, the pharmacokinetics of MMF following multiple-dose PO administration in horses was investigated to assess the potential for bioaccumulation and nonlinear elimination. The multiple-dose PO protocol of 5 mg/kg once daily for 30 days followed by once every 48 hours for 30 days was based on the protocol used in a previous study22 of the pharmacokinetics of azathioprine in horses. As evidenced by the accumulation ratio of 1.0 in the present study, MMF does not appear to bioaccumulate when administered long-term at the dosage used, regardless of administration daily or every 2 days. The mean terminal half-life after single-dose PO administration (4.02 ± 1.10 hours) was in agreement with the mean terminal half-life after 30 days (3.62 ± 0.89 hours) and 60 days (4.59 ± 1.25 hours) of drug administration. Thus, when MMF is administered with the long-term dosing protocol used in the present study, the drug follows linear elimination kinetics.
In cats, there is interanimal variability in glucuronidation or glucosidation regarding which is the major biotransformation pathway of MPA.18,19 We did not investigate the presence of MPA glucoside in the present study; whether the glucosidation pathway exists (and, if so, to what extent) in horses remains to be investigated.
The limitations of the present study were the small number of horses used and the restricted duration (60 days) of PO administration of MMF, which is a shorter period than the long-term dosing that is often required for treatment of autoimmune diseases. The dosage of 5 mg of MMF/kg given to horses PO once daily or every 2 days was not evaluated for efficacy in the treatment of disease. Results of a recent human study27 suggest an AUC of 30 to 60 mg•h/L provides better results in patients with renal transplants; this is much higher than the AUC achieved with either the IV or PO dosing in the horses of the present study. Future pharmacokinetic and clinical studies are needed to determine efficacious doses in horses with autoimmune diseases.
Abbreviations
| AUC | Area under the curve |
| Cmax | Maximum plasma concentration |
| d3-MPA | MPA with 3 deuterium atoms |
| d4-MMF | MMF with 4 deuterium atoms |
| LC-MS/MS | Liquid chromatography-tandem mass spectrometry |
| MMF | Mycophenolate mofetil |
| MPA | Mycophenolic acid |
| MPAAG | Mycophenolic acid acyl glucuronide |
| MPAG | Mycophenolic acid glucuronide |
Footnotes
Clinical Pathology Laboratory, William R. Pritchard Veterinary Medical Teaching Hospital, University of California, Davis, Calif.
Mycophenolate lypholized powder for injection, Cardinal Health, Dublin, Ohio.
Cardinal Health, Dublin, Ohio.
Phenix Research Products, Candler, NC.
Toronto Research Chemicals, Toronto, ON, Canada.
Cerex polycrom Clin II, Cera Inc, Baldwin Park, Calif.
TSQ Quantum Ultra, Thermo Scientific, San Jose, Calif.
1100 series, Agilent Technologies, Palo Alto, Calif.
ACE C18, Mac-Mod Analytical, Chadds Ford, Pa.
Quanbrowser, Thermo Scientific, San Jose, Calif.
Mycophenolic acid β-D-glucuronide disodium salt, Toronto Research Chemicals, Toronto, ON, Canada.
Toronto Research Chemicals, Toronto, ON, Canada.
TSQ Altis, Thermo Scientific, San Jose, Calif.
Vanquish, Thermo Scientific, San Jose, Calif.
Phoenix Winnonlin, version 8.0, Certera, Princeton, NJ.
References
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