Pharmacokinetics of Oral Mycophenolate Mofetil in Dog: Bioavailability Studies and the Impact of Antibiotic Therapy

Abstract

An improved understanding of the pharmacokinetics of mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is needed because of the increasing use of MMF in patients undergoing solid-organ transplantation [1Shaw L.M. Korecka M. Venkataramanan R. Goldberg L. Bloom R. Brayman K.L. Mycophenolic acid pharmacodynamics and pharmacokinetics provide a basis for rational monitoring strategies (review).Am J Transplant. 2003; 3: 534-542Crossref PubMed Scopus (219) Google Scholar] or hematopoietic cell transplantation (HCT) [2Nash R.A. Johnston L. Parker P. et al.A phase I/II study of mycophenolate mofetil in combination with cyclosporine for prophylaxis of acute graft-versus-host disease after myeloablative conditioning and allogeneic hematopoietic cell transplantation.Biol Blood Marrow Transplant. 2005; 11: 495-505Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar, 4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar]. Plasma concentrations of MPA have been correlated with various clinical outcomes in solid-organ transplant recipients [1Shaw L.M. Korecka M. Venkataramanan R. Goldberg L. Bloom R. Brayman K.L. Mycophenolic acid pharmacodynamics and pharmacokinetics provide a basis for rational monitoring strategies (review).Am J Transplant. 2003; 3: 534-542Crossref PubMed Scopus (219) Google Scholar]. However, only recently have pharmacodynamic associations been described in patients undergoing HCT, specifically the correlation of increased MPA concentrations at steady state [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar] and the unbound MPA area under the concentration-time curve (AUC) [4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar], with higher degrees of donor T cell chimerism [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar] and lower incidences of graft rejection [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar, 4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar], respectively.Recent studies in a well-established preclinical canine model of HCT have identified effective immunosuppressive drug combinations that include MMF for the prevention of acute allograft rejection and graft-versus-host disease [5Yu C. Seidel K. Nash R.A. et al.Synergism between mycophenolate mofetil and cyclosporine in preventing graft-versus-host disease among lethally irradiated dogs given DLA-nonidentical unrelated marrow grafts.Blood. 1998; 91: 2581-2587PubMed Google Scholar, 6Storb R. Yu C. Wagner J.L. et al.Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation.Blood. 1997; 89: 3048-3054PubMed Google Scholar]. In these studies, MMF was administered subcutaneously at a dose of 10 mg/kg twice daily. We sought to determine in a healthy 25-month-old male beagle the bioavailability and interdose variability of oral MMF to develop an understanding of drug dosing required for substituting oral for subcutaneous MMF. Because enterohepatic circulation mediated through gram-negative intestinal bacteria plays a major role in MPA pharmacokinetics [7Bullingham R.E. Nicholls A.J. Kamm B.R. Clinical pharmacokinetics of mycophenolate mofetil (review).Clin Pharmacokinet. 1998; 34: 429-455Crossref PubMed Scopus (716) Google Scholar], we also addressed the question of whether gut decontamination with nonabsorbable antibiotics affects the enterohepatic circulation in the dog, which could have potential clinical implications for canine marrow graft recipients. MPA bioavailability was investigated on 3 occasions, each separated from the others by 1 month, after single doses (ie, 10, 15, and 20 mg/kg) of oral MMF (CellCept Oral Suspension, Roche Laboratories. Nutley, NJ; 200 mg/mL) and compared with that of a single subcutaneous dose of 10 mg/kg MMF (CellCept Intravenous, Roche Laboratories; 25 mg/mL); each oral dose was separated from the baseline subcutaneous dose by a 3-day rest period. The effect of nonabsorbable antibiotics on oral MMF pharmacokinetics was evaluated on 4 occasions, 1 month apart. At baseline, the dog was given a single dose of 15 mg/kg oral MMF, followed by 3 days of rest, and then received a nonabsorbable antibiotic mixture consisting of a 1:1 ratio of neomycin sulfate (Biosol, The Upjohn Company, Kalamazoo, MI) and polymyxin B sulfate (Polymyxin B, Bedford Laboratories, Bedford, OH), each administered at a dose of 1 g orally per total body weight, 3 times daily, 10 days before and concomitant with another dose of 15 mg/kg oral MMF. Plasma samples were obtained before and 1, 2, 4, 6, 8, 10, and 12 hours, respectively, after each MMF dose. Pharmacokinetic parameters were quantified and analyzed as previously reported [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar]. The interdose variability was assessed by comparing the variances of the various measurements of the pharmacokinetic parameters using the F test; P < 0.05 was considered statistically significant.The pharmacokinetic data are presented in Table 1. Bioavailabilities were 54%, 65%, and 87% after 10, 15, and 20 mg/kg oral MMF, respectively. The plasma concentration-time profile after oral MMF was characterized by secondary maxima induced by the enterohepatic circulation within 4-12 hours of dosing. There were statistically significant differences in interdose variabilities in the MPA clearance and total MPA maximum plasma concentration (Cmax; P = .02 and .006, respectively), but not in the MPA-free fraction and AUC from 0 to 12 hours (AUC0-12h; P = .16 and .07, respectively) after oral versus subcutaneous MMF administrations (Table 1). Administration of preceding and concomitant nonabsorbable antibiotics with a single oral MMF dose did not appear to reduce the MPA-free fraction, AUC0-12h, clearance, and Cmax when compared with oral MMF given without antibiotics (Table 1); however, the average of the secondary MPA peaks to the AUC0-12h was reduced by 17.2% (P = .64). The interdose variabilities in the MPA-free fraction, AUC0-12h, clearance, and Cmax were not statistically significantly different after oral MMF given with or without nonabsorbable antibiotics (P = .30, .39, .42, and .21, respectively; Table 1); high interdose variabilities, although not statistically significantly different, were seen in the amplitude of the secondary MPA peaks to the AUC0-12h after oral MMF given with preceding and concomitant nonabsorbable antibiotics compared with oral MMF given alone (P = .06; Table 1).Table 1Pharmacokinetic Parameters in a Healthy Dog Following Administration of Single Doses of Subcutaneous and Oral MMFMMF Route/Dose (mg/kg)NUnbound MPA (%)MPA AUC0-12h (mg⁎Average ± standard deviation.hr/ml)MPA AUC Secondary Peak (%)MPA Clearance (l/hr⁎Average ± standard deviation.kg)MPA Cmax (μg/ml)SC 1032.09 ± 0.4⁎Average ± standard deviation.20.89 ± 2.97⁎Average ± standard deviation.NA0.485 ± 0.064⁎Average ± standard deviation.5.38 ± 0.7⁎Average ± standard deviation.PO 1011.9313.1748.40.7594.66 1551.95 ± 0.23⁎Average ± standard deviation.20.77 ± 10.8⁎Average ± standard deviation.39.3 ± 11.66⁎Average ± standard deviation.0.859 ± 0.390⁎Average ± standard deviation.9.87 ± 9.15⁎Average ± standard deviation. 2011.8233.733.70.59317.95PO + nAb 1541.75 ± 0.16⁎Average ± standard deviation.21.94 ± 12.4⁎Average ± standard deviation.32.57 ± 28.81⁎Average ± standard deviation.0.874 ± 0.502⁎Average ± standard deviation.10 ± 5.48⁎Average ± standard deviation.Abbreviations: AUC0-12h = area under the concentration-time curve from 0 to 12 hours; Cmax = maximum plasma concentration; MMF = mycophenolate mofetil; MPA = mycophenolic acid; N = number; NA = not applicable; nAb = non-absorbable antibiotics. Average ± standard deviation. Open table in a new tab Our data suggested that interdose variability after oral MMF administration occurred even under well-controlled environmental conditions. Using oral MMF after HCT would expose the dogs to unwarranted risks due to excessive or suboptimal MPA concentrations that could lessen the potency of immunosuppression necessary for successful HCT; therefore, oral MMF would not be a good substitute for subcutaneous MMF in the preclinical canine HCT model, where MPA plasma levels are not routinely monitored and adequate levels of immunosuppression are essential for preclinical validation of the HCT protocols. Our findings support the need for rational individual therapeutic monitoring of MPA levels in patients undergoing HCT, where the use of pretransplantation test doses did not accurately predict total or unbound MPA AUC after HCT [4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar].On average, the effects of nonabsorbable antibiotics on MPA pharmacokinetics were not significant relative to the interdose variability seen. It has been recently reported that patients after HCT had lower plasma MPA levels and a shorter MPA half-life after oral MMF administration compared with healthy volunteers or solid-organ transplant recipients [2Nash R.A. Johnston L. Parker P. et al.A phase I/II study of mycophenolate mofetil in combination with cyclosporine for prophylaxis of acute graft-versus-host disease after myeloablative conditioning and allogeneic hematopoietic cell transplantation.Biol Blood Marrow Transplant. 2005; 11: 495-505Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. That might be attributed to the decreased bacterial flora in the gastrointestinal tract from broad-spectrum prophylactic antibiotic use. Further, it has been suggested that a significant fraction of the interdose variability in MPA pharmacokinetics was caused by day-to-day fluctuations in the contribution of MPA enterohepatic circulation to the MPA AUC [8Shaw L.M. Nawrocki A. Korecka M. Solari S. Kang J. Using established immunosuppressant therapy effectively: lessons from the measurement of mycophenolic acid plasma concentrations (review).Ther Drug Monit. 2004; 26: 347-351Crossref PubMed Scopus (46) Google Scholar]. In addition to generating a hypothesis, our study provided preliminary data regarding the effects of nonabsorbable antibiotics on the enterohepatic circulation of MPA, which could be further investigated in patients after HCT. An improved understanding of the pharmacokinetics of mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is needed because of the increasing use of MMF in patients undergoing solid-organ transplantation [1Shaw L.M. Korecka M. Venkataramanan R. Goldberg L. Bloom R. Brayman K.L. Mycophenolic acid pharmacodynamics and pharmacokinetics provide a basis for rational monitoring strategies (review).Am J Transplant. 2003; 3: 534-542Crossref PubMed Scopus (219) Google Scholar] or hematopoietic cell transplantation (HCT) [2Nash R.A. Johnston L. Parker P. et al.A phase I/II study of mycophenolate mofetil in combination with cyclosporine for prophylaxis of acute graft-versus-host disease after myeloablative conditioning and allogeneic hematopoietic cell transplantation.Biol Blood Marrow Transplant. 2005; 11: 495-505Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar, 4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar]. Plasma concentrations of MPA have been correlated with various clinical outcomes in solid-organ transplant recipients [1Shaw L.M. Korecka M. Venkataramanan R. Goldberg L. Bloom R. Brayman K.L. Mycophenolic acid pharmacodynamics and pharmacokinetics provide a basis for rational monitoring strategies (review).Am J Transplant. 2003; 3: 534-542Crossref PubMed Scopus (219) Google Scholar]. However, only recently have pharmacodynamic associations been described in patients undergoing HCT, specifically the correlation of increased MPA concentrations at steady state [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar] and the unbound MPA area under the concentration-time curve (AUC) [4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar], with higher degrees of donor T cell chimerism [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar] and lower incidences of graft rejection [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar, 4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar], respectively. Recent studies in a well-established preclinical canine model of HCT have identified effective immunosuppressive drug combinations that include MMF for the prevention of acute allograft rejection and graft-versus-host disease [5Yu C. Seidel K. Nash R.A. et al.Synergism between mycophenolate mofetil and cyclosporine in preventing graft-versus-host disease among lethally irradiated dogs given DLA-nonidentical unrelated marrow grafts.Blood. 1998; 91: 2581-2587PubMed Google Scholar, 6Storb R. Yu C. Wagner J.L. et al.Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation.Blood. 1997; 89: 3048-3054PubMed Google Scholar]. In these studies, MMF was administered subcutaneously at a dose of 10 mg/kg twice daily. We sought to determine in a healthy 25-month-old male beagle the bioavailability and interdose variability of oral MMF to develop an understanding of drug dosing required for substituting oral for subcutaneous MMF. Because enterohepatic circulation mediated through gram-negative intestinal bacteria plays a major role in MPA pharmacokinetics [7Bullingham R.E. Nicholls A.J. Kamm B.R. Clinical pharmacokinetics of mycophenolate mofetil (review).Clin Pharmacokinet. 1998; 34: 429-455Crossref PubMed Scopus (716) Google Scholar], we also addressed the question of whether gut decontamination with nonabsorbable antibiotics affects the enterohepatic circulation in the dog, which could have potential clinical implications for canine marrow graft recipients. MPA bioavailability was investigated on 3 occasions, each separated from the others by 1 month, after single doses (ie, 10, 15, and 20 mg/kg) of oral MMF (CellCept Oral Suspension, Roche Laboratories. Nutley, NJ; 200 mg/mL) and compared with that of a single subcutaneous dose of 10 mg/kg MMF (CellCept Intravenous, Roche Laboratories; 25 mg/mL); each oral dose was separated from the baseline subcutaneous dose by a 3-day rest period. The effect of nonabsorbable antibiotics on oral MMF pharmacokinetics was evaluated on 4 occasions, 1 month apart. At baseline, the dog was given a single dose of 15 mg/kg oral MMF, followed by 3 days of rest, and then received a nonabsorbable antibiotic mixture consisting of a 1:1 ratio of neomycin sulfate (Biosol, The Upjohn Company, Kalamazoo, MI) and polymyxin B sulfate (Polymyxin B, Bedford Laboratories, Bedford, OH), each administered at a dose of 1 g orally per total body weight, 3 times daily, 10 days before and concomitant with another dose of 15 mg/kg oral MMF. Plasma samples were obtained before and 1, 2, 4, 6, 8, 10, and 12 hours, respectively, after each MMF dose. Pharmacokinetic parameters were quantified and analyzed as previously reported [3Giaccone L. McCune J.S. Maris M.B. et al.Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.Blood. 2005; 106: 4381-4388Crossref PubMed Scopus (65) Google Scholar]. The interdose variability was assessed by comparing the variances of the various measurements of the pharmacokinetic parameters using the F test; P < 0.05 was considered statistically significant. The pharmacokinetic data are presented in Table 1. Bioavailabilities were 54%, 65%, and 87% after 10, 15, and 20 mg/kg oral MMF, respectively. The plasma concentration-time profile after oral MMF was characterized by secondary maxima induced by the enterohepatic circulation within 4-12 hours of dosing. There were statistically significant differences in interdose variabilities in the MPA clearance and total MPA maximum plasma concentration (Cmax; P = .02 and .006, respectively), but not in the MPA-free fraction and AUC from 0 to 12 hours (AUC0-12h; P = .16 and .07, respectively) after oral versus subcutaneous MMF administrations (Table 1). Administration of preceding and concomitant nonabsorbable antibiotics with a single oral MMF dose did not appear to reduce the MPA-free fraction, AUC0-12h, clearance, and Cmax when compared with oral MMF given without antibiotics (Table 1); however, the average of the secondary MPA peaks to the AUC0-12h was reduced by 17.2% (P = .64). The interdose variabilities in the MPA-free fraction, AUC0-12h, clearance, and Cmax were not statistically significantly different after oral MMF given with or without nonabsorbable antibiotics (P = .30, .39, .42, and .21, respectively; Table 1); high interdose variabilities, although not statistically significantly different, were seen in the amplitude of the secondary MPA peaks to the AUC0-12h after oral MMF given with preceding and concomitant nonabsorbable antibiotics compared with oral MMF given alone (P = .06; Table 1). Abbreviations: AUC0-12h = area under the concentration-time curve from 0 to 12 hours; Cmax = maximum plasma concentration; MMF = mycophenolate mofetil; MPA = mycophenolic acid; N = number; NA = not applicable; nAb = non-absorbable antibiotics. Our data suggested that interdose variability after oral MMF administration occurred even under well-controlled environmental conditions. Using oral MMF after HCT would expose the dogs to unwarranted risks due to excessive or suboptimal MPA concentrations that could lessen the potency of immunosuppression necessary for successful HCT; therefore, oral MMF would not be a good substitute for subcutaneous MMF in the preclinical canine HCT model, where MPA plasma levels are not routinely monitored and adequate levels of immunosuppression are essential for preclinical validation of the HCT protocols. Our findings support the need for rational individual therapeutic monitoring of MPA levels in patients undergoing HCT, where the use of pretransplantation test doses did not accurately predict total or unbound MPA AUC after HCT [4Jacobson P. Rogosheske J. Barker J.N. et al.Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation.Clin Pharmacol Ther. 2005; 78: 486-500Crossref PubMed Scopus (70) Google Scholar]. On average, the effects of nonabsorbable antibiotics on MPA pharmacokinetics were not significant relative to the interdose variability seen. It has been recently reported that patients after HCT had lower plasma MPA levels and a shorter MPA half-life after oral MMF administration compared with healthy volunteers or solid-organ transplant recipients [2Nash R.A. Johnston L. Parker P. et al.A phase I/II study of mycophenolate mofetil in combination with cyclosporine for prophylaxis of acute graft-versus-host disease after myeloablative conditioning and allogeneic hematopoietic cell transplantation.Biol Blood Marrow Transplant. 2005; 11: 495-505Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar]. That might be attributed to the decreased bacterial flora in the gastrointestinal tract from broad-spectrum prophylactic antibiotic use. Further, it has been suggested that a significant fraction of the interdose variability in MPA pharmacokinetics was caused by day-to-day fluctuations in the contribution of MPA enterohepatic circulation to the MPA AUC [8Shaw L.M. Nawrocki A. Korecka M. Solari S. Kang J. Using established immunosuppressant therapy effectively: lessons from the measurement of mycophenolic acid plasma concentrations (review).Ther Drug Monit. 2004; 26: 347-351Crossref PubMed Scopus (46) Google Scholar]. In addition to generating a hypothesis, our study provided preliminary data regarding the effects of nonabsorbable antibiotics on the enterohepatic circulation of MPA, which could be further investigated in patients after HCT. This work was supported in part by grants CA 78902, HL 36444, CA 15704, and CA 18029 from the National Institutes of Health, Bethesda, MD; ML was supported by a fellowship from the Exchange Scientist Program of the Office of International Affairs, National Institutes of Health.