Pharmacokinetics and pharmacodynamics of mycophenolic acid in healthy cats after twice-daily intravenous infusion of mycophenolate mofetil for three days

Jennifer E. Slovak Program in Individualized Medicine, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164.

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 DVM, MS
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Sol M. Rivera-Velez Program in Individualized Medicine, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164.

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Julianne K. Hwang Program in Individualized Medicine, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164.

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Nicolas F. Villarino Program in Individualized Medicine, Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164.

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 Med Vet, DVSc, PhD

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Abstract

OBJECTIVE To evaluate the plasma disposition of mycophenolic acid (MPA) and its derivatives MPA glucuronide and MPA glucoside after twice-daily infusions of mycophenolate mofetil (MMF) in healthy cats for 3 days and to assess the effect of MMF administration on peripheral blood mononuclear cell (PBMC) counts and CD4+-to-CD8+ ratios.

ANIMALS 5 healthy adult cats.

PROCEDURES MMF was administered to each cat (10 mg/kg, IV, q 12 h for 3 days). Each dose of MMF was diluted with 5% dextrose in water and then administered over a 2-hour period with a syringe pump. Blood samples were collected for analysis. A chromatographic method was used to quantitate concentrations of MPA and its metabolites. Effects of MMF on PBMC counts and CD4+-to-CD8+ ratios were assessed by use of flow cytometry.

RESULTS All cats biotransformed MMF into MPA. The MPA area under the plasma concentration–time curve from 0 to 14 hours ranged from 14.6 to 37.6 mg·h/L and from 14.4 to 22.3 mg·h/L after the first and last infusion, respectively. Total number of PBMCs was reduced in 4 of 5 cats (mean ± SD reduction, 25.9 ± 15.8% and 26.7 ± 19.3%) at 24 and 48 hours after the end of the first infusion of MMF, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE Plasma disposition of MPA after twice-daily IV infusions for 3 days was variable in all cats. There were no remarkable changes in PBMC counts and CD4+-to-CD8+ ratios.

Abstract

OBJECTIVE To evaluate the plasma disposition of mycophenolic acid (MPA) and its derivatives MPA glucuronide and MPA glucoside after twice-daily infusions of mycophenolate mofetil (MMF) in healthy cats for 3 days and to assess the effect of MMF administration on peripheral blood mononuclear cell (PBMC) counts and CD4+-to-CD8+ ratios.

ANIMALS 5 healthy adult cats.

PROCEDURES MMF was administered to each cat (10 mg/kg, IV, q 12 h for 3 days). Each dose of MMF was diluted with 5% dextrose in water and then administered over a 2-hour period with a syringe pump. Blood samples were collected for analysis. A chromatographic method was used to quantitate concentrations of MPA and its metabolites. Effects of MMF on PBMC counts and CD4+-to-CD8+ ratios were assessed by use of flow cytometry.

RESULTS All cats biotransformed MMF into MPA. The MPA area under the plasma concentration–time curve from 0 to 14 hours ranged from 14.6 to 37.6 mg·h/L and from 14.4 to 22.3 mg·h/L after the first and last infusion, respectively. Total number of PBMCs was reduced in 4 of 5 cats (mean ± SD reduction, 25.9 ± 15.8% and 26.7 ± 19.3%) at 24 and 48 hours after the end of the first infusion of MMF, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE Plasma disposition of MPA after twice-daily IV infusions for 3 days was variable in all cats. There were no remarkable changes in PBMC counts and CD4+-to-CD8+ ratios.

Few immunosuppressants have been evaluated for use in cats, which has resulted in limited options when corticosteroids are insufficient.1 This is unfortunate because cats are susceptible to multiple immune-mediated diseases (eg, immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, inflammatory bowel disease, and various dermatologic conditions) that require immunosuppressive treatments.1 The prodrug MMF, which is used in human organ transplant recipients, has become increasingly popular in clinical veterinary medicine as an alternative or adjuvant treatment.2–6 Mycophenolate mofetil is commercially available as a solution for IV administration as well as a liquid suspension and tablets for oral administration, which is desirable when medicating ill veterinary patients.

Mycophenolic acid, the active form of MMF, reduces lymphocyte proliferation by inhibiting inosine monophosphate dehydrogenase. Inosine monophosphate dehydrogenase is the rate-limiting enzyme in de novo guanosine (purine) synthesis.7,8 The specific and noncompetitive mechanism of action diminishes the production of antibodies, reduces proliferation of CD4+ and CD8+ T lymphocytes, and reduces adhesion of glycoproteins to endothelial cells.7,8 The specialized mechanism of action is especially helpful in patients with immune-mediated diseases because T cells contribute greatly to autoimmune disease through various activation, stimulatory, and cytotoxic mechanisms.9,10

Cats have inherent limitations in drug metabolism, which makes the selection and administration of medications challenging.11 Fortunately, some of the currently available immune suppressants for use in humans (eg, MMF) may be promising alternatives for use in cats. In vitro, MPA inhibits mitogen-stimulated proliferation of feline lymphocytes in a dose-dependent manner, thereby reducing CD4+ and CD8+ counts, compared with results for control samples.3 However, little is known about the effect of MPA on the immune system of cats.

A recent in vitro study12 of feline, canine, and human liver microsomes revealed that MPA was metabolized into phenol glucuronide, which is a stable and pharmacologically nonactive metabolite, and MPA phenol glucoside. Additionally, a pharmacokinetic study13 involving 6 healthy cats treated by use of a 2-hour constant rate infusion of MMF at a rate of 20 mg/kg every 12 hours for 1 day revealed that all the cats biotransformed MMF into the active moiety MPA. The disposition of MPA in cats is highly variable, which may impact the safety and efficacy of MMF treatment in cats.13,14 The authors of that study13 also suspected that there was variability in the disposition of MPA within some cats, as is seen in human patients. However, they did not know whether those observations were dose dependent and consistent for cats treated for long periods.7,8,13

The dosage regimen in that study13 of cats resulted in MPA exposure comparable to that reported necessary for treatment of human patients with autoimmune diseases.9 Unfortunately, not all of the cats tolerated the dosage regimen, which suggests that some cats will need to be treated with lower doses of MMF. It is highly likely that cats requiring intensive critical care and hospitalization would require IV administration of MMF for > 1 day, but it is unknown whether lower MMF doses administered for longer periods would result in safe and effective MPA exposure.

The disposition of MPA in cats and its effects on the immune system of cats are not fully known. Therefore, the primary objective of the study reported here was to evaluate the plasma disposition of MPA and its derivatives MPA glucuronide and MPA glucoside in healthy cats treated with MMF twice daily for multiple days. Furthermore, the secondary objective of the study was to obtain pharmacodynamic data by assessing the effect of the MMF dosage regimen on PBMC counts and CD4+-to-CD8+ ratios.

Materials and Methods

Animals

Five healthy adult client-owned cats were enrolled in the study. All the cats were indoor cats. All cats were neutered (3 females and 2 males). Mean age was 2.8 years (range, 1 to 5 years), and mean body weight was 4.6 kg (range, 3.5 to 5.7 kg). A signed consent form was obtained from each owner before cats were enrolled in the study. Each cat underwent a physical examination and hematologic and biochemical analyses (which included serum biochemical analysis, a CBC, urinalysis, and testing for FeLV and FIV) prior to enrollment. Cats were housed in and allowed to acclimate to feline-friendly rooms for 1 week before initiation of the study. A maximum of 2 cats was housed in each room at any time. The study was approved by the Washington State University Institutional Animal Care and Use Committee (ASAF No. 04665-005).

Experimental procedures

Twenty-four hours before the study commenced, cats were sedated by IM administration of butorphanol tartratea (0.4 mg/kg), ketamine hydrochlorideb (5 mg/kg), and acepromazine maleatec (0.02 mg/kg). A dual-port sampling catheter then was aseptically placed in a jugular vein. Catheters were bandaged; they were flushed every 6 to 8 hours with 1 mL of heparinized saline (0.9% NaCl) solution.

Study design

Each cat received 6 doses of MMFd (10 mg/kg, IV, q 12 h for 3 days). The MMF was diluted with 5% dextrose in water to achieve a final concentration of 6 mg/mL, which was then administered as a constant rate infusion over a 2-hour period via a syringe pump. The dose was selected on the basis of data for MPA in another study13 and a report5 of oral administration of MMF to 2 cats. The dosage regimen was selected in an effort to avoid phlebitis, thrombosis, and potential allergic reactions to the drug or components of the formulation.4,7,15 We also attempted to mimic a clinically relevant dosing regimen for critically ill patients that require IV medication before being transitioned to oral medication.

Foode was withheld for 2 hours before and after drug administration. Water was always freely available to the cats. A repeated CBC and serum biochemical analysis were performed within 24 hours after the last MMF infusion.

Collection of blood samples

Blood samples for determination of plasma concentrations of MPA were collected immediately before (time 0) and at the end of the first MMF infusion and 0.75, 1.5, 3, 6, 9, 12, 24, 36, and 48 hours after the end of the first MMF infusion. Additionally, blood samples were collected at 59, 60, 61, 62, 62.5, 62.75, 63, 63.5, 64, 66, 68, 70, and 72 hours (which corresponded to 1 hour after starting the last MMF infusion and 0, 1, 2, 2.5, 2.75, 3, 3.5, 4, 6, 8, 10, and 12 hours after the end of the last MMF infusion, respectively). Blood samples (1.1 mL/sample) were collected, transferred to glass tubes containing citrate, and centrifuged at 1,800 × g for 8 minutes. Plasma was harvested, placed as 200-μL aliquots in plastic Eppendorf tubes, and stored at −80°C until analysis. All samples for the study were analyzed in 1 batch.

Blood samples (2 mL/sample) for PBMC isolation were collected from each cat and placed into glass tubes containing lithium heparin before the start of the first infusion of MMF and 24, 48, and 72 hours after the end of the first infusion. A total of < 5% of the circulating blood volume of each cat was obtained for analysis.

Measurement of MPA, MPA glucuronide, and MPA glucoside concentrations

Mycophenolic acid and its derivatives MPA glucuronide and MPA glucoside were extracted from plasma and quantified by use of a chromatographic method developed by our laboratory group.16,17 The method was validated in accordance with guidelines published by the FDA.18

Quantitation of MPA, MPA glucuronide, and MPA glucoside concentrations

Briefly, MPA and its derivatives were extracted by use of a protein-precipitation method with a mixture of methanolf and acetonitrilef (1:1 [vol/vol]) as the precipitating agent. Quantitation of MPA was performed on an ultra–high-performance liquid chromatography C18 columng (1.5 μm; 2.1 × 100 mm). The mobile phase consisted of solvent A (0.05% mixture of 85% phosphoric acidf in water) and solvent B (100% acetonitrile); elution was with a linear gradient. Flow rate was 0.25 mL/min. Column and autosampler temperatures were 32° and 25°C, respectively. Injection volume was 1 μL. Analytes were detected at 215 nm.

Quality control samples and calibration standards (MPA,h MPA glucuronide,i and MPA glucosidei) were prepared in feline plasma and assayed with the study samples; phenolphthalein β-d-glucuronideh was included as an internal standard. The calibration curve for MPA ranged from 0.3 to 20 μg/mL, whereas the calibration curve for MPA glucuronide ranged from 0.3 to 3 μg/mL, and the calibration curve for MPA glucoside ranged from 0.5 to 3 μg/mL. As recommended in the FDA guidelines,18 precision and accuracy of the method were assessed by use of quality control samples for each analyte. We included quality control samples with 3 concentrations within the calibration curve for each analyte. The calibration curve was linear (R2 > 0.99), and the method was highly precise (coefficient of variation, ≤ 15%) and accurate (error, ≤ 7.6%).

Pharmacokinetic analysis

Pharmacokinetic parameters were determined with noncompartmental analyses.j Values for AUC were estimated by use of the linear trapezoidal rule. The maximum plasma concentration and time of maximum plasma concentration of MPA corresponded to the maximal concentration and time of maximum plasma concentration observed for each cat.

PBMC preparation and analysis

The PBMCs were isolated from blood samples that were collected from the indwelling jugular catheter into tubes containing lithium heparin as an anticoagulant. Blood samples were allowed to sit undisturbed for 20 minutes at room temperature (21°C). Each blood sample then was diluted with an equal volume of PBS solution. Diluted blood samples were carefully layered over a commercially available gradient solutionk in 15-mL conical tubes at room temperature. Samples were centrifuged at 700 × g for 30 minutes at room temperature without braking during deceleration. The top layer was discarded, and the PBMC layer was collected from the gradient solutionk interface. The collected sample was washed with 12 mL of PBS solution via centrifugation at 300 × g for 10 minutes. The RBCs were removed by adding 3 mL of RBC lysis buffer (150mM NH4Cl, 10mM KHCO3, and 0.1mM disodium EDTA; pH, 7.3) and incubating the mixture at room temperature for 10 minutes. Samples were centrifuged at 300 × g for 8 minutes, and PBMCs were collected and washed again with 10 mL of PBS solution. The number of cells was counted by use of an automated thin-film sensor cell counterl with cell count cassettes.m The healthy cell population and viability were calculated by use of the internal curve-fitting algorithm of the automated cell counter software.

PBMC cryopreservation and thawing

The PBMCs were suspended at a concentration of 1 × 107 cells/mL in 0.5 mL of PBS solution at room temperature. Freezing medium (50% RPMI 1640, 40% fetal bovine serum, and 10% dimethyl sulfoxide) at room temperature was added, and the suspension was gently mixed. The resulting cell suspension was divided into two 1-mL aliquots, which were placed in cryogenic polypropylene vials. The vials were placed into freezing containersn with an isopropyl alcohol medium; containers were placed into a freezer (–80°C) to decrease the temperature by approximately 1°C/min. Twenty-four hours later, samples were transferred quickly into a liquid nitrogen tank (−195°C) and stored until testing.

Thawing of samples was accomplished by placing cryovials in a 37°C water bath. As soon as samples were thawed, cells were pipetted into a 15-mL conical tube containing 10-fold amounts of warm (37°C) complete RPMI medium (96.4% RPMI 1640, 2.5% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin-glutamine, and 0.1% 2-mercaptoethanol). Cells were washed by centrifugation at 300 × g for 10 minutes at room temperature. Cell counting and viability were assessed by use of the trypan blue dye exclusion test.19

Cell staining and flow cytometry

Cells were washed with complete RPMI medium and diluted to a concentration of 1 × 106 cells/100 μL. Cells in the 2 aliquots (100 μL/aliquot) were pelleted by centrifugation at 400 × g for 5 minutes and then incubated with 100 μL of PBS solution and optimal concentrations of fluorescein isothiocyanate–conjugated CD4+o and phycoerythrin-conjugated CD8+p for 15 minutes at 4°C in the dark. Concentration of the antibodies was 1 μg of IgG/10 μL of PBS solution. Cells were washed twice with 200 μL of flow cytometry buffer (97% PBS solution and 3% heat-inactivated fetal bovine serum) followed by centrifugation at 400 × g for 4 minutes. Cells were resuspended in flow cytometry buffer, and lymphocytes were gated for characteristic forward- and side-scatter profiles. The percentage of cells stained with antibody against CD4+ and CD8+ was determined as 2 color-flow cytometry profiles.q Percentages of stained cells were calculated by use of flow cytometer software.r

Statistical analysis

Estimated pharmacokinetic parameters and T-cell and PBMC response to treatments were evaluated. The AUC0–14h after the first and last infusion were compared. Distribution of the data was tested for normality by use of the Kolmogorov-Smirnov test. The AUC0–14h after the first and last infusion was compared by use of a paired Student t test. Median number of PBMCs per milliliter at 0 and 72 hours was compared by use of the Mann-Whitney U test. Median values for CD4+-to-CD8+ ratios were compared by use of the Friedman test. Significance for all statistical comparisons was set at P < 0.05. All analyses were performed with statistical software.s

RESULTS

Hyporexia, vomiting, or reactions to MMF infusion were not detected in any of the cats. Serum biochemical variables remained within reference limits after the MMF infusions; PCV, number of platelets, and total protein concentration of all cats decreased slightly, although values were still within respective reference limits. One cat had transient large bowel diarrhea once within 12 hours after the last MMF infusion; the diarrhea resolved without treatment within 24 hours.

All cats biotransformed MMF into MPA (Figure 1). Mean ± SD plasma concentration for all 5 cats was calculated (Figure 2). Pharmacokinetic parameters were calculated (Table 1). The AUC0–14h after the first and last infusion did not differ significantly (P = 0.199).

Figure 1—
Figure 1—

Plasma concentrations of MPA (black circles), MPA glucuronide (black triangles), and MPA glucoside (white circles) in each of 5 cats (A through E) after 6 infusions of MMF. The MMF was administered to each cat (10 mg/kg, IV, q 12 h) for 3 days, with each dose of MMF diluted with 5% dextrose in water and then administered over a 2-hour period. Time 0 was immediately before the start of the first MMF infusion.

Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1093

Figure 2—
Figure 2—

Mean ± SD plasma concentration of MPA for 5 cats after 6 infusions of MMF. See Figure 1 for key.

Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1093

Table 1—

Pharmacokinetic parameters of MPA in plasma from 5 cats after twice-daily IV infusion of MMF for 3 days.

InfusionPharmacokinetic parameterMean ± SDMinimumMaximum
FirstTime to maximum concentration after start of infusion (h)2.65 ± 0.8224
 Maximum concentration (μg/mL)6.27 ± 2.103.739.21
 Concentration at the end of the dosing interval (μg/mL)1.51 ± 1.090.982.96
 AUC0–14h (mg·h/L)23.70 ± 9.4414.6037.60
LastMaximum concentration (μg/mL)3.65 ± 1.561.675.52
 Concentration at the end of the dosing interval (μg/mL)1.68 ± 2.480.426.10
 AUC0–14h (mg·h/L)18.50 ± 3.4114.4022.30

Each cat received 6 doses of MMF (10 mg/kg, IV, q 12 h for 3 days). The MMF was diluted with 5% dextrose in water and administered as a constant rate infusion over a 2-hour period. Time 0 was immediately before the start of the first infusion.

— = Not applicable.

The metabolites MPA glucuronide and MPA glucoside were detected at quantifiable concentrations in only 4 of 5 cats (Figure 1). The concentration of MPA glucuronide ranged from 0.32 to 0.38 μg/mL, and the concentration of MPA glucoside ranged from 0.50 to 0.82 μg/mL.

Counts of the total number of PBMCs isolated were variable among all cats in samples obtained before and 24, 48, and 72 hours after the first MMF infusion (Figure 3). The total number of PBMCs was reduced in 4 of 5 cats at 24 (mean ± SD reduction, 25.9 ± 15.8%) and 48 (mean ± SD reduction, 26.7 ± 19.3%) hours after the first infusion of MMF. The median number of PBMCs per milliliter at 72 hours was not significantly different from the value before the first infusion. However, at 72 hours (12 hours after the end of the last infusion), the total number of lymphocytes was slightly reduced by 14.7 ± 8.4%. One cat had an increase of 11% to 67% in the number of lymphocytes. The CD4+-to-CD8+ ratios did not differ significantly (P = 0.29) over time (Table 2).

Figure 3—
Figure 3—

The CD4+-to-CD8+ ratios (A) and PBMC counts (B) for 5 cats before and after IV infusion of MMF. For panels A and B, each symbol represents results for 1 cat.

Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1093

Table 2—

The CD4+-to-CD8+ ratios of 5 cats before and after twice-daily IV infusion of MMF for 3 days.

Time (h)MedianMinimumMaximum
05.83 × 1064.88 × 10613.86 × 106
246.10 × 1063.00 × 10611.59 × 106
486.80 × 1063.34 × 10612.16 × 106
728.98 × 1064.48 × 10612.01 × 106

See Table 1 for key.

Discussion

In the study reported here, pharmacokinetics of multiple twice-daily IV infusion of MMF in cats was evaluated. The study provided novel information about short-term in vivo effects of MMF on PBMC counts and CD4+ and CD8+ lymphocytes in cats during and after MMF infusions.

All cats metabolized MMF into the active moiety MPA. Shape of the plasma concentration–time profile of MPA in plasma reflected a complex disposition of MPA, which was characterized by multiple maximum concentrations, as has been reported for cats and other species.4,7,8,13,20 The complexity of the disposition of MPA in plasma could have been the result of several factors, including the rate of infusion and elimination of MMF and the rate of formation, elimination, and distribution of MPA in plasma. In addition, enterohepatic recycling of MPA could also explain the multiple peaks of MPA.6,9

Similar to results of another study13 of cats, MPA was eliminated rapidly from the cats of the present study. Trough concentrations were relatively constant over time, which suggested that there was not a noticeable accumulation of MPA over time. This was also reflected in comparable AUC0–14h values after the first and last infusion of MMF.

Similar to results of a recently published in vitro study,12 MPA was metabolized by the cats of the present study into at least 2 metabolites (MPA glucuronide and MPA glucoside), likely by hepatic biotransformation. In another in vivo study13 of cats, glucosidation, but not glucuronidation, of MPA appeared to be the primary route for MPA elimination. However, in the study reported here, glucuronidation of MPA appeared to be more relevant than glucosidation for the elimination of MPA in 2 cats, which suggested that there is interanimal variability in biotransformation of MPA. Interestingly, the apparent interindividual differences in the metabolic routes used to biotransform MPA did not have a significant impact on the plasma concentrations of MPA. However, this observation would need to be confirmed in a larger study.

As previously mentioned, the disposition of MPA in plasma after the administration of MMF was highly variable. This is not a novel finding. In fact, one of the biggest concerns regarding the disposition of MPA in cats and other species is the relatively high interindividual variability of plasma concentrations of MPA.2,4,8,13,21,22 In clinical settings, high interindividual variability and unpredictable exposure to MPA could result in overexposure or underexposure to MPA.

In another study13 conducted by our research group, the disposition of MPA after a constant rate infusion of a 20-mg/kg dose of MMF was evaluated. That dosage regimen resulted in a mean exposure to MPA (mean AUC0–14h, 32 μg·h/mL) comparable to the exposure that reportedly8 is effective in human patients with autoimmune diseases (AUC0–12h, > 35 μg·h/mL). Authors of another study23 set the therapeutic target for AUC0–12h at 30 to 60 mg·h/L. However, in that study,23 not all of the cats tolerated the dose of MMF (20 mg/kg, IV). Therefore, for the study reported here, we decided to evaluate the disposition of MPA after a constant rate infusion of 10 mg of MMF/kg. For this lower dose, the mean AUC0–14h was 23.7 and 18.5 mg·h/L after the first and last infusion, respectively. It should be mentioned that variability of AUC0–14h obtained in the present study was relatively lower than that reported13 for cats treated twice with an infusion of MMF at 20 mg/kg. It has been suggested that optimal immune suppression of human patients is achieved when trough concentrations range from 1.0 to 5.0 μg/mL, depending on the condition treated.24,25 In the study reported here, trough concentrations of MPA ranged from 0.29 to 6.10 μg/mL, which suggested that the infusion of MMF at a dose of 10 mg/kg may result in immunosuppressive concentrations in only some cats. Interestingly, the plasma concentrations of MPA detected in the cats for the present study are comparable to those (range, 0.2 to 3.2 μg/mL) reported13 in cats after 2 infusions of MMF at a dose of 20 mg/kg, which may suggest that there is a lack of dose proportionality and nonlinear kinetics of MMF or MPA (or both) when MMF is administered at doses between 10 and 20 mg/kg. However, this would need to be evaluated in studies designed to assess dose proportionality.

Antiproliferative effects of MPA on T and B lymphocytes appears to be a concentration-dependent phenomenon.3,21 In the study reported here, infusion of MMF resulted in fluctuations in total lymphocyte numbers in the cats (PBMC counts were reduced in 4/5 cats), and the CD4+-to-CD8+ ratios remained unaltered. Many factors may have contributed to the minimal changes in PBMC counts and their relatively inert response for the CD4+-to-CD8+ ratio. One factor may have been that the dose of MMF used was too low. However, it would be premature to conclude that administration of MMF at a dose of 10 mg/kg every 12 hours for 3 days is ineffective because the MMF may not have been given for a long enough period to enable us to detect an appreciable change in PBMC counts. These cats were medicated for only 3 days. The dosage regimen was chosen on the basis of the authors' clinical experience with regard to the typical hospitalized immune-mediated patient that requires IV treatment. A recent in vitro study22 involving the effect of MMF on cytokine-induced killer cells revealed that treatment with MMF for an intermediate amount of time (3 days) induced mixed or mild changes in proliferation and viability of T cells, but long-term (7 days) treatment caused alterations in the CD4-to-CD8 ratio. In fact, an in vivo study23 of humans found that MMF may have a cumulative effect over time that leads to a progressive reduction in PBMC counts, including those of CD4+ and CD8+ T lymphocytes, which suggests that a longer course of treatment might be necessary to enable investigators to detect an effect on PBMCs.

Finally, it is worth mentioning that the present study included only healthy cats with presumably un-activated lymphocytes, which would be in contrast to lymphocytes of diseased cats. This is important because MPA has a cytostatic effect.2,4,7,8 The MPA selectively inhibits T-lymphocyte proliferation at the S phase in the thymus and spleen.26 Also, MPA induces apoptosis of activated T lymphocytes, which may eliminate clones of cells responding to antigenic stimulation.26 These 2 mechanisms of action suggest that the effect of MMF would be detectable in cats with a more rapid or abnormal rate of proliferation of lymphocytes, as is seen in patients with immune-mediated conditions.21

The extent to which MPA binds to plasma proteins in cats may have a relevant impact on its plasma disposition, safety, and efficacy. In humans, MPA is highly bound to albumin (> 98%).27 Whether MPA is highly bound to plasma proteins in cats remains to be determined.

The study reported here provided novel information about the disposition of MPA after multiple infusions of MMF to cats. In all cats, MMF was transformed to the active metabolite MPA. Drug exposure was lower than that reported to be optimal in human patients requiring sustained and aggressive immunosuppressive treatment, and the shape of the plasma concentration–time profile of MPA was variable among the cats. However, the effect of MMF and MPA on the immune system of cats remains unclear and deserves further investigation. A study on the pharmacokinetics and pharmacodynamics for a population of cats would provide substantial evidence to aid in creating guidelines for the safe and effective use of MMF in cats that require novel and aggressive immunosuppressive treatments.

Acknowledgments

Funded by grants from the Washington State University Intramural Research Fund, Beardsley-Blanco Endowment Fund, and Dorothy Shea Brink Memorial Fund.

The authors declare that there were no conflicts of interest.

Presented in part as a poster at the 2016 European College of Veterinary Internal Medicine–Companion Animal Congress, Gothenburg, Sweden, September 2016.

The authors thank Kate Spies for assistance with care of the cats.

ABBREVIATIONS

AUC

Area under the plasma concentration–time curve

AUC0–12h

Area under the plasma concentration–time curve from time 0 to 12 hours

AUC0–14h

Area under the plasma concentration–time curve from time 0 to 14 hours

MMF

Mycophenolate mofetil

MPA

Mycophenolic acid

PBMC

Peripheral blood mononuclear cell

Footnotes

a.

Torbugesic injectable (100 mg/mL), Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo.

b.

Ketaset injectable (100 mg/mL), Zoetis Inc, Kalamazoo, Mich.

c.

Promace injectable (10 mg/mL), Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo.

d.

Cellcept IV MMF hydrochloride for injection (6 mg/mL), Genetech USA Inc, South San Francisco, Calif.

e.

Purina Cat Chow indoor formula, Purina Animal Nutrition LLC, Gray Summit, Mo.

f.

Fisher Scientific, Waltham, Mass.

g.

Accucore Vanquish UHPLC C18, Thermo Fisher Scientific, Waltham, Mass.

h.

Sigma-Aldrich Corp, St Louis, Mo.

i.

Toronto Research Chemicals Inc, Toronto, ON, Canada.

j.

Phoenix WinNonolin, version 7.1, Certara, Princeton, NJ.

k.

Histopaque 1077, MP Biomedicals LLC, Solon, Ohio.

l.

Orflo Moxi Z, Orlfo Technologies, Ketchum, Idaho.

m.

Type S, Orlfo Technologies, Ketchum, Idaho.

m.

Mr. Frosty freezing containers, Thermo Fisher Scientific, Rochester, NY.

o.

Mouse anti-cat CD4:FITC, Bio-Rad Laboratories Inc, Hercules, Calif.

p.

Mouse anti-cat CD8 alpha/beta:RPE, Bio-Rad Laboratories Inc, Hercules, Calif.

q.

BD FACSCalibur, BD Biosciences, San Jose, Calif.

r.

FCS Express 4, BD Biosciences, San Jose, Calif.

s.

GraphPad Prism, version 7, GraphPad Software Inc, San Diego, Calif.

References

  • 1. Viviano KR. Update on immunosuppressive therapies for dogs and cats. Vet Clin North Am Small Anim Pract 2013;43:11491170.

  • 2. Wagner M, Earley AK, Webster AC, et al. Mycophenolic acid versus azathioprine as primary immunosuppression for kidney transplant recipients. Cochrane Database Syst Rev 2015;12:CD007746.

    • Search Google Scholar
    • Export Citation
  • 3. Kyles AE, Gregory CR, Craigmill AL. Comparison of the in vitro antiproliferative effects of five immunosuppressive drugs on lymphocytes in whole blood from cats. Am J Vet Res 2000;61:906909.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Lange S, Mueller SC, Altmann S, et al. Pharmacokinetics of oral mycophenolate mofetil in combination with CsA in dogs after nonmyeloablative allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2008;41:667674.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Bacek LM, Macintire DK. Treatment of primary immune-mediated hemolytic anemia with mycophenolate mofetil in two cats. J Vet Emerg Crit Care (San Antonio) 2011;21:4549.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. West LD, Hart JR. Treatment of idiopathic immune-mediated hemolytic anemia with mycophenolate mofetil in five dogs. J Vet Emerg Crit Care (San Antonio) 2014;24:226231.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Abd Rahman AN, Tett SE, Staatz CE. Clinical pharmacokinetics and pharmacodynamics of mycophenolate in patients with autoimmune disease. Clin Pharmacokinet 2013;52:303331.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Bullingham RE, Nicholls AJ, Kamm BR. Clinical pharmacokinetics of mycophenolate mofetil. Clin Pharmacokinet 1998;34:429455.

  • 9. Santamaria P. Effector lymphocytes in autoimmunity. Curr Opin Immunol 2001;13:663669.

  • 10. Devarajan P, Chen Z. Autoimmune effector memory T cells: the bad and the good. Immunol Res 2013;57:1222.

  • 11. Court MH. Feline drug metabolism and disposition: pharmacokinetic evidence for species differences and molecular mechanisms. Vet Clin North Am Small Anim Pract 2013;43:10391054.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Slovak JE, Mealey K, Court MH. Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human, dog, and cat liver microsomes. J Vet Pharmacol Ther 2017;40:123129.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Slovak JE, Rivera SM, Hwang JK, et al. Pharmacokinetics of mycophenolic acid after intravenous administration of mycophenolate mofetil to healthy cats. J Vet Intern Med 2017;31:18271832.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Slovak JE, Villarino NF. Safety of oral and intravenous mycophenolate mofetil in healthy cats. J Feline Med Surg 2018;20:184188.

  • 15. Wang A, Smith JR, Creevy KE. Treatment of canine idiopathic immune-mediated haemolytic anaemia with mycophenolate mofetil and glucocorticoids: 30 cases (2007 to 2011). J Small Anim Pract 2013;54:399404.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Rivera Vélez SM, Morassi A, Court MH, et al. Development and validation of an ultrafast chromatographic method for quantification of the immunosuppressant mycophenolic acid in canine, feline and human plasma. J Pharm Biomed Anal 2016;131:94102.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Rivera Vélez SM, Hwang JK, Slovak JE, et al. Simultaneous determination of mycophenolic acid and its glucuronide and glycoside derivatives in canine and feline plasma by UHPLC-UV. Biomed Chromatogr 2017;31:19.

    • Search Google Scholar
    • Export Citation
  • 18. US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Bioanalytical method validation: guidance for industry. May 2001. Available at: www.fda.gov/downloads/Drugs/Guidance. Accessed May 12, 2017.

    • Search Google Scholar
    • Export Citation
  • 19. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol 2015;111:A3.B.1A3.B.3.

  • 20. Yoshimura K, Yano I, Yamamoto T, et al. Population pharmacokinetics and pharmacodynamics of mycophenolic acid using the prospective data in patients undergoing hematopoietic stem cell transplantation. Bone Marrow Transplant 2018;53:4451.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21. Bravo Soto JA, Esteban de la Rosa RJ, Luna del Castillo JD, et al. Effect of mycophenolate mofetil regimen on peripheral blood lymphocyte subsets in kidney transplant recipients. Transplant Proc 2003;35:13551359.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Bremm M, Huenecke S, Zimmermann O, et al. In-vitro influence of mycophenolate mofetil (MMF) and ciclosporin A (CsA) on cytokine induced killer (CIK) cell immunotherapy. J Transl Med 2016;14:112.

    • Search Google Scholar
    • Export Citation
  • 23. Iwaszkiewicz-Grzes D, Cholewinski G, Kot-Wasik A, et al. Investigations on the immunosuppressive activity of derivatives of mycophenolic acid in immature dendritic cells. Int Immunopharmacol 2017;44:137142.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Dipchand AI, Pietra B, McCrindle BW, et al. Mycophenolic acid levels in pediatric heart transplant recipients receiving mycophenolate mofetil. J Heart Lung Transplant 2001;20:10351043.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Gajarski RJ, Crowley DC, Zamberlan MC, et al. Lack of correlation between MMF dose and MPA level in pediatric and young adult cardiac transplant patients: does the MPA level matter? Am J Transplant 2004;4:14951500.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Allison AC. Immunosuppressive drugs: the first 50 years and a glance forward. Immunopharmacology 2000;47:6383.

  • 27. Nowak I, Shaw LM. Mycophenolic acid binding to human serum albumin: characterization and relation to pharmacodynamics. Clin Chem 1995;41:10111017.

    • Crossref
    • Search Google Scholar
    • Export Citation
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