Mycophenolate Pharmacokinetics and Association with Response to Acute Graft-Versus-Host Disease Treatment from the Blood and Marrow Transplant Clinical Trials Network

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Mycophenolate Pharmacokinetics and Association with Response to Acute Graft-versus-Host Disease Treatment from the Blood and Marrow Transplant Clinical Trials Network

Pamala A. Jacobson,1 Jiayin Huang,1,2 Juan Wu,3 Miae Kim,1 Brent Logan,4 Amin Alousi,5 Michael Grimley,6 Javier Bolan˜os-Meade,7 Vincent Ho,8 John E. Levine,9 Daniel Weisdorf10

There are limited data as to the effectiveness of mycophenolate mofetil (MMF) plus high-dose corticosteroids for the treatment of acute graft-versus-host disease (aGVHD), and even less data regarding the pharmacokinetic disposition and exposure–response relationship of MMF in individuals with GVHD. MMF pharmacokinetics were studied in a multicenter Blood and Marrow Transplant Clinical Trials Network randomized phase II trial evaluating the effectiveness of MMF as one of 4 agents added to corticosteroids as treatment of aGVHD. Thirty-two of the patients randomized to receive MMF underwent pharmacokinetic sampling in weeks 1 and 2 were studied. Mean age was 41 6 13.6 years. Twenty one (65.6%), 5 (15.6%), 6 (18.8%) patients had a complete response (CR), partial response (PR) or lesser response by day 28, respectively. Twenty-five (78.1%), 2 (6.3%), 5 (15.6%) patients had a CR, PR, or other response by day 56 to treatment, respectively. Mycophenolic acid (MPA) pharmacokinetic measurements from weeks 1 and 2 did not correlate with CR at either day 28 or day 56 (P ..07); however, if the mean of weeks 1 and 2 total MPA troughs was .0.5 mg/mL or that of an unbound trough was .0.015 mg/mL, then a significantly greater proportion achieved CR 1 PR at days 28 and 56. CR 1 PR at day 28 was observed in 19 of 19 patients (100%) with a mean total trough .0.5 mg/mL, but in only 7 of 13 (54%) with a mean total trough #0.5 mg/mL (P 5 .002). Similarly, CR 1 PR at day 28 was seen in 15 of 15 patients (100%) with an unbound trough concentration .0.015 mg/mL, but in only 11 of 17 (65%) with an unbound trough concentration #0.015 mg/mL (P 5 .02). There was no association between the pharmacokinetic measures and risk of infection by day 90 or overall survival (OS) at day 180 postrandomization. About one-half of subjects did not achieve the favorable MPA total and unbound trough concentrations. The current practice of MMF 1 gm twice daily dosing provides low plasma concentrations in many patients. Higher doses may improve the efficacy of MMF as aGVHD therapy. Biol Blood Marrow Transplant 16: 421-429 (2010) Ó 2010 American Society for Blood and Marrow Transplantation KEY WORDS: Mycophenolate mofetil, Mycophenolic acid, Pharmacokinetics, acute GVHD

INTRODUCTION resulting in greater treatment-related mortality (TRM) and delayed patient recovery after transplanta- Acute graft-versus-host disease (aGVHD) is a com- tion. Standard initial therapy for aGVHD is methyl- mon complication, occurring in 35%-50% of recipi- prednisolone 1-2.5 mg/kg/day; however, only 50% of ents of hematopoietic cell transplantation (HCT), patients achieve a durable response [1]. Numerous

From 1Experimental and Clinical Pharmacology, College of Phar- Arbor, Michigan; and 10Division of Hematology, Oncology macy, University of Minnesota, Minneapolis, Minnesota; 2Clin- and Transplantation, University of Minnesota, School of Med- ical Pharmacology, Vertex Pharmaceuticals, Cambridge, icine, Minneapolis, Minnesota. Massachusetts; 3The EMMES Corporation, Rockville, Mary- This trial is registered at http://www.clinicaltrials.gov as NCT00224874. land; 4Department of Population Health, Medical College of Financial disclosure: See Acknowledgments on page 428. Wisconsin, Milwaukee, Wisconsin; 5Department of Stem Cell Correspondence and reprint requests: Pamala Jacobson, PharmD, Transplantation and Cellular Therapy, University of Texas University of Minnesota, College of Pharmacy, Experimental MD Anderson Cancer Center, Houston, Texas; 6Texas Trans- and Clinical Pharmacology, WDH 7-151, 308 Harvard Street plant Institute, San Antonio, Texas; 7Department of Oncology, SE, Minneapolis, MN 55455 (e-mail: [email protected]). Johns Hopkins University, Baltimore, Maryland; 8Department Received October 16, 2009; accepted November 10, 2009 of Medical Oncology/Hematologic Oncology, Dana-Farber Ó 2010 American Society for Blood and Marrow Transplantation Cancer Institute, Boston, Massachusetts; 9Department of Pedi- 1083-8791/10/163-0015$36.00/0 atrics and Internal Medicine, University of Michigan, Ann doi:10.1016/j.bbmt.2009.11.010

421 422 P. A. Jacobson et al. Biol Blood Marrow Transplant 16:421-429, 2010 immunosuppressants have been investigated for treat- that exposure may be altered in the setting of aGVHD ing aGVHD, either in combination with steroid ther- [34], pharmacokinetic analyses were performed in apy or as single agents [2-8]. Several newer agents, subjects on the MMF arm of BMT CTN 0302, and including mycophenolate mofetil (MMF), have the data are reported here. Our primary objective emerged as potentially effective for aGVHD; however, was to evaluate the association between mycopheno- well-controlled trials defining the most effective agent late pharmacokinetics and aGVHD CR and partial are lacking [9-15]. response (PR) at days 28 and 56 posttreatment. Recently, the Blood and Marrow Transplant Clin- Secondary objectives were to assess the relationships ical Trials Network (BMT CTN) conducted a multi- between pharmacokinetics and the incidence of severe center randomized phase 2 trial (BMT CTN 0302) infections and survival. comparing corticosteroids combined with either etanercept, MMF, denileukin diftitox, or pentostatin for treating newly diagnosed aGVHD in 180 patients METHODS who had undergone allogeneic HCT [16]. Compared with the other arms, the MMF arm had a greater rate Study Design and Patients of complete response (CR) at day 28 (60%; 95% con- This was a prespecified analysis in patients ran- 5 fidence interval [CI] 46%-74%) and day 56 (73%; domized to the MMF arm of CTN 0302. Forty-five 5 95% CI 60%-86%), and fewer treatment failures patients aged $6 years with newly diagnosed 5 at day 56 (9%; 95% CI 0.6%-17%), although no aGVHD requiring systemic treatment were treated formal statistical comparisons were done because of with MMF; pharmacokinetics were studied in 32 of the small numbers in the trial. These data suggest these patients. The patients received treatment with that MMF may have significant activity against MMF plus methylprednisolone 2 mg/kg/day i.v. aGVHD, and a follow-up phase III trial is planned divided into 2-3 daily doses (or prednisone 2.4 mg/ to confirm this. Given these findings, understanding kg/day orally) for a minimum of 7 days. A patient could MMF exposure–response relationships and defining not have received previous systemic therapy for the the optimal dose for GVHD treatment is critical and treatment of aGVHD, except for a maximum of 48 may further improve the efficacy of MMF. hours of high-dose steroid therapy (methylpredniso- MMF is a prodrug designed to enhance the bioavail- lone $1 mg/kg/day). All patients had undergone ability of the active form, mycophenolic acid (MPA). related or unrelated allogeneic HCT. Patients with MMF is extensively hydrolyzed to MPA by esterases an uncontrolled infection, absolute neutrophil count located in the systemic circulation, gut wall, liver, and (ANC) \500 cells/mL, or creatinine clearance #30 tissues [17,18]. Glucuronidation of MPA occurs in the mL/min were ineligible. Patients also were ineligible kidney, liver, and intestine by uridine 5’-diphospho- if they had received MMF for GVHD prophylaxis glucuronosyltransferases (UGTs) to the main inactive within the 7 days before study enrollment. The study metabolite, mycophenolic acid glucuronide (MPAG) was approved by each participating institution’s insti- [19,20]. MPAG is then transported in the bile to the tutional review board (IRB) and the IRB of the BMT gastrointestinal (GI) tract by multidrug-resistance CTN. All patients provided written informed consent. protein 2 or excreted into the urine by active tubular secretion and eliminated [18]. MMF also may undergo enterohepatic recirculation, in which biliary MPAG is MMF Treatment deglucuronidated to MPA by gut bacteria and then MMF (Cellcept; Roche, Nutley, NJ) was given at reabsorbed into the systemic circulation [18,21,22]. a dose of 20 mg/kg twice daily orally or i.v. in patients MPA pharmacokinetic disposition and exposure– with a body surface area (BSA) .1.5 m2 (maximum 1 g response relationships in the setting of aGVHD treat- twice daily) and 750 mg twice daily orally or i.v. ment are poorly understood. It is possible that more in those with a BSA \1.5 m2 at time of GVHD intensive immunosuppression is required during diagnosis. The choice of oral or i.v. administration GVHD treatment, and that different MPA plasma tar- was determined clinically at each site. Patients with gets may be required relative to GVHD prophylaxis. GI GVHD who were unable to tolerate more than In addition, MPA is highly dependent on metabolism 500 mL of oral fluids/day received i.v. MMF for at least in the intestine and liver, and given that GVHD may the first 3 days of therapy. In patients with suspected involve these sites, it is reasonable to speculate that mycophenolate-related GI toxicity, the MMF dose glucuronidation, enterohepatic recirculation, or bio- was reduced by 50%. MMF was discontinued in those availability might be altered. Although controversial, with persistent or severe GI symptoms. Likewise, the data from organ transplantation and HCT suggest MMF dose was reduced by 50% in patients with an that a minimal required plasma exposure of mycophe- ANC of 500-1000 cells/mL and discontinued in those nolate is required for optimal immunosuppression in with an ANC \500 cells/mL. Patients with GVHD the prophylactic setting [23-33]. Because of concerns in CR at day 28 continued to receive MMF through Biol Blood Marrow Transplant 16:421-429, 2010 MMF Pharmacokinetics in Acute GVHD 423 the completion of the steroid taper, with MMF then Intravenous MMF was administered at a constant rate tapered over the subsequent 4 weeks. over 2 hours. Blood samples were spun, and plasma was removed and aliquoted into two 1.5-mL Eppen- aGVHD Response and Toxicity Assessment dorf screw cap vials, and then frozen at 220C. All samples were shipped to a central laboratory at the aGVHD was clinically diagnosed or biopsy- University of Minnesota for analysis. For each patient, proven in all patients. GVHD was scored and overall a total and unbound MPA area under the curve grade was assigned as defined by the Consensus Crite- (AUC) was determined from a limited sampling ria [35] at day 28, day 56, and then monthly for 9 0-12 model developed for HCT recipients [38]. For oral months following the initiation of treatment. CR in administration, total AUC ,inmg hour/mL, was GVHD was defined as complete resolution of all signs 0-12 4.43 1 2.76*C0 1 0.51*C1 1 1.97*C2 1 4.27*C6, and symptoms of GVHD in all evaluable organs with- and unbound AUC , in ng hr/mL, was 63.92 1 out additional therapy. PR was defined as improve- 0-12 2.01*C0 1 0.67*C1 1 2.05*C2 1 4.26*C6. For i.v. ment by at least one stage in one or more involved administration, total AUC ,inmg hour/mL, was organs without progression. A mixed response (MR) 0-12 -0.49 1 1.58*C2 1 0.41*C4 1 13.88*C6, and unbound was defined as improvement in at least one involved AUC , in ng hour/mL, was 7.99 1 1.40*C2 1 organ with progression or newly developed GVHD 0-12 2.47*C4 1 9.54*C6. Total and unbound MPA in one or more organs. No response (NR) was defined AUC and MPAG AUC were calculated by non- as a lack of improvement or worsening in any organ 0-6 0-6 compartmental analysis using WinNonLin version within 14 days of the start of therapy. Progression 5.2 (Pharsight Corp, Mountain View, CA). Steady- was defined as worsening by one or more stages in state trough was the concentration at time 0 (C0). one or more organs without improvement in any Cmax was the highest observed concentration. The organ. Failure of therapy was defined as PR, MR, free fraction of MPA was determined by calculating NR, progression, death, or development of chronic the ratio of unbound MPA AUC to total MPA GVHD (cGVHD). Severe, fatal, or life-threatening 0-6 AUC . The MPAG:MPA ratio was determined infections were recorded through day 90. Infections 0-6 from the trough concentration. were reported regardless of attribution to study treatment and were graded according to the National Can- cer Institute’s Common Terminology Criteria for Statistical Analysis Adverse Events, version 3.0. Death from any cause The distribution of pharmacokinetic measures (to- was considered an event in the survival analysis. tal and unbound trough, AUC0-6 and AUC0-12,Cmax) was evaluated. Week 1 and week 2 pharmacokinetic Mycophenolate Bioanalysis and measurements were compared using Wilcoxon’s Pharmacokinetics signed-rank test or McNemar’s test in patients with Total and unbound MPA and total MPAG in the both measurements. Comparisons of i.v. and oral plasma were measured in each sample by high-perfor- pharmacokinetics were conducted using Wilcoxon’s mance liquid chromatography (Series 1100 system; rank-sum test or Fisher’s exact test. The association Agilent Technologies, Wilmington, DE) with UV among various pharmacokinetic measurements was as- detection as described previously [36,37]. The validated sessed using Spearman’s r. The association between assays were linear in the range of 0.025-20 mg/mL for pharmacokinetic measurements (total and unbound total MPA, 1-100 mg/mL for total MPAG, and 0.001- AUC0-6, AUC0-12 and troughs) and clinical outcomes 0.5 mg/mL for unbound MPA. Assay accuracy and intra- (CR, PR, CR 1 PR, infection, survival) was conducted day and interday variability (as percent relative standard by dichotomizing the outcome at select time points. deviation) were 97%-111%, 0.6%-4.4%, and 1.0%- Pharmacokinetic measures were explored as continu- 6.0%, respectively, for total MPA; 97%-101%, 0.9%- ous variables using nonparametric tests to test the 1.4%, and 0.7%-4.8% for MPAG; and 96%-118%, association between outcomes and the MPA pharma- 0.7%-5.4%, and 0.5%-7.8% for unbound MPA. cokinetic measurements. We postulated dichotomized Patients were evaluated at steady state with phar- values using published data for each pharmacokinetic macokinetics in weeks 1 and 2 after initiation of measurement and performed chi-square tests or MMF treatment. Steady state was defined as .5 half- Fisher’s exact tests to test the association between lives from initiation of MMF. A limited sampling strat- dichotomized values and clinical endpoints. We ana- egy was used, in which 4 blood samples were obtained lyzed the pharmacokinetic measurements at week 1 over 6 hours after MMF administration. Blood and week 2 separately, as well as the mean of weeks 1 samples (5 mL) were collected in purple-top tubes and 2 to stabilize the measurement; the results were containing EDTA at 0 (C0), 1 (C1), 2 (C2), and 6 generally consistent, so except as noted the mean of (C6) hours after administration of an oral dose and at weeks 1 and 2 data are presented. Although this is 0, 2, 4 (C4), and 6 hours after the start of i.v. infusion. an exploratory study, we considered adjustment for 424 P. A. Jacobson et al. Biol Blood Marrow Transplant 16:421-429, 2010 multiple comparisons based on the 6 pharmacokinetic Table 1. Patient Characteristics measurements tested for association with GVHD Age, years, mean ± standard deviation 41 ± 13.6 response, so that a p-value \ 0.05/6 5 0.0083 should Weight at time of study entry, kg, 82.6 ± 25.4 mean ± standard deviation be considered significant after Bonferroni adjustment. Primary disease, n (%) No further adjustment for the multiple PK time points Acute myelogenous leukemia 15 (46.9%) (week 1 vs. week 2 vs. average of weeks 1 and 2) or for Acute lymphoblastic leukemia 6 (18.8%) Chronic myelogenous leukemia 2 (6.3%) the multiple outcomes (day 28 CR1PR vs. day 56 Myelodysplastic syndrome 1 (3.1%) CR1PR) were done because the tests were highly Lymphoma 4 (12.5%) correlated and consistent. We also examined the asso- Other 4 (12.5%) Donor source, n (%) ciation between non-pharmacokinetic variables (con- Related 16 (50%) ditioning regimen, donor and graft source, GVHD Unrelated 16 (50%) prophylaxis at transplantation and grade of GVHD Stem cell source, n (%) Bone marrow 11 (34.3%) at enrollment) and outcome. Survival analysis was per- Peripheral blood 19 (59.4%) formed to evaluate the association between pharmaco- Cord blood 2 (6.3%) kinetic measures and overall survival. Dichotomized GVHD prophylaxis at time of transplantation, n (%) Tacrolimus/methotrexate 16 (50%) results are reported since they provide interpretations Cyclosporine/other 6 (19%) that can be used in clinical care. Multiple regression Cyclophosphamide 6 (19%) analyses were not performed due to limited sample Tacrolimus/other 4 (12%) Calcineurin Inhibitor at time of pharmacokinetics size. SAS 9.1 statistical software was used for data Tacrolimus 23 (72%) analysis. Cyclosporine 3 (9%) None 6 (19%) Conditioning regimen, n (%) Nonmyeloablative or reduced intensity 4 (12.5%) RESULTS Myeloablative 28 (87.5%) Acute GVHD grade at time of enrollment, n (%) Pharmacokinetics I 1 (3.1%) II 19 (59.4%) Mycophenolate pharmacokinetics were studied in III 12 (37.5%) 32 of the 45 patients randomized to MMF. Thirteen IV 0 of the patients were not studied because the patient GVHD indicates graft-versus-host disease. was unable or declined to undergo additional blood sampling for pharmacokinetics, or was taken off MMF therapy before sampling. Characteristics and (r 5 0.53; P 5 .003) and week 2 (r 5 0.75; P \ .001). response to MMF treatment did not differ between Correlations between unbound trough and unbound the 32 evaluable patients and the whole MMF group AUC0-12 in weeks 1 and 2 were r 5 0.49 (P 5 .006) (n=45, data not shown). Patient characteristics are and r 5 0.40 (P 5 .028), respectively. summarized in Table 1. Five patients were studied only once, resulting in a total of 59 pharmacokinetic GVHD Response and MPA Exposure profiles. Thirty patients received MMF 1 g twice daily, The majority of patients achieved either a CR (n 5 and 2 patients received 0.75 g twice daily. MMF was 21, 66%) or PR (n 5 5, 15%) by day 28 after initiation administered orally or i.v. based on clinical status. of therapy while the remaining 6 patients (19%) had ei- Twenty patients received oral MMF during both ther NR or MR or GVHD progression. At day 56 the sampling periods. Median MPA pharmacokinetic response rates were 78% (n 5 25) with CR, 6% (n 5 2) measures were similar in weeks 1 and 2 of treatment with PR, and 16% (n55) with NR, MR or progression. except for total troughs, which were higher in week 2 Pharmacokinetic measurements (total and unbound (Table 2). Oral administration yielded 4- to 6-fold AUC0-6, AUC0-12, trough) at either 1 or 2 weeks did higher trough concentrations than i.v. administration, not correlate with achieving CR at day 28 or 56 whereas i.v. administration provided higher AUC (p.0.07). However, higher mean trough concentra- values (Table 3). In all pharmacokinetic sets, the tions (taken by averaging the trough levels from both median unbound MPA fraction was 2.2% (range, weeks 1 and 2) were associated with a significantly 0.9%-5.6%) and was significantly higher with i.v. ad- better combined CR1PR at both days 28 and 56. ministration compared with oral administration. The As shown in Figure 1, patients whose mean total median MPAG:MPA ratio [range] was higher in trough of weeks 1 and 2 was .0.5 mcg/ml were week 1 than in week 2 (66.0 [17.6-447.1] vs 45.7 more likely to achieve CR1PR at day 28 than patients [14.9-576.7]). with lower mean total troughs (19/19, 100% vs 7/13, Total and unbound trough values were highly cor- 54%, p50.002). At day 56, a CR1PR was also higher related in weeks 1 and 2 (Spearman’s r 5 0.88 and in those with a mean week 1 and 2 total trough 0.87, respectively; P \ .001). Total trough and total .0.5 mcg/mL than those with lower troughs (19/19, AUC0-12 were only moderately correlated in week 1 100% vs 8/13, 62%, p = 0.006). These results were Biol Blood Marrow Transplant 16:421-429, 2010 MMF Pharmacokinetics in Acute GVHD 425

Table 2. MPA Pharmacokinetics in Weeks 1 and 2 after Initiation of MMF Treatment*

Week 1 (n 5 29) Week 2 (n 5 30) P† i.v./oral administration, n 11/18 7/23 .38‡ Day posttransplantation 36.0 (17-81) 46 (22-88) Dose 1000 mg/750 mg, n 27/2 28/2 Serum creatinine, mg/dL 0.90 (0.4-1.7) 0.90 (0.4-2.8) .12 Total bilirubin, mg/dL 0.70 (0.2-19.7) 0.70 (0.3-21.7) .35 Serum albumin, g/dL 2.95 (1.6-4.3) 3.30 (1.5-3.9) .21 MPA, total Trough, mg/mL 0.465 (0.036-2.54) 0.782 (0.0-5.10) .05 AUC0-6, mg*hour/mL 20.11 (5.86-44.1) 18.70 (3.8-53.7) .61 AUC0-12, mg*hour/mL 23.97 (7.9-44.4) 21.90 (11.6-76.2) .98 MPA, unbound Trough, mg/mL 0.006 (0.0-0.106) 0.015 (0.0-0.149) .14 AUC0-6, mg*hour/mL 0.445 (0.093-0.126) 0.384 (0.099-2.80) .80 AUC0-12, mg*hour/mL 0.467 (0.169-1.33) 0.444 (0.176-3.257) .28 MPAG trough, mg/mL 32.88 (3.89-163.2) 33.76 (5.65-209.3) .27 MPA unbound fraction 0.0195 (0.012-0.044) 0.023 (0.009-0.056) .44

MMF indicates mycophenolate mofetil; MPA, mycophenolic acid; MPAG, mycophenolic acid glucuronide; PK, pharmacokinetics. *Data are median (range); P is a comparison of week 1 and week 2 pharmacokinetics for only those subjects with an assessment at both week 1 and week 2 (paired analysis; n 5 27). †Wilcoxon’s signed-rank test except where noted otherwise. ‡McNemar’s test (exact P value). consistent using week 1 (p = 0.02 for day 28, p = 0.05 week 1, 16 of 29 patients (55%) had a total trough for day 56) and week 2 (p = 0.003 for day 28, p = \0.5 mg/mL, and 20 of 29 (69%) had an unbound 0.003 for day 56) troughs separately. Not surprisingly, trough \0.015 mg/mL. At week 2, 11 of 30 (37%) higher mean unbound troughs also correlated with re- had a total trough \0.5 mg/mL, and 15 of 30 (50%) sponse to treatment, although these were not signiﬁ- had an unbound trough \0.015 mg/mL. Interest- cant after adjustment for multiple comparisons. As ingly, other variables, including donor type, graft shown in Figure 2, patients with a mean of weeks 1 source, conditioning regimen, GVHD prophylaxis, and 2 unbound troughs .0.015 mcg/ml were more and GVHD grade at enrollment, were not associated likely to achieve CR1PR at day 28 than patients with response (all P . .09). with lower mean unbound troughs (15/15, 100% vs 11/17, 65%, p 5 0.02). At day 56, a CR1PR was also higher in those with a mean week 1 and 2 unbound Site of GVHD and MPA Exposure trough .0.015 mcg/mL than those with a lower GVHD organ involvement did not predict MPA trough (15/15, 100% vs 12/17, 71%, p 5 0.04). The exposure, although the number of observations in our mean of weeks 1 and 2 total and unbound AUC0-6 study was limited. In patients with only skin GVHD and AUC0-12 were not associated with a better com- (n 5 10), the median MPA total and unbound trough bined CR1PR at both days 28 and 56 (p $ 0.33, concentrations were 1.18 mg/mL (range, 0.02-2.11 data not shown). mg/mL) and 0.017 mg/mL (0.002-0.032 mg/mL), Substantial numbers of patients had MPA expo- respectively. Patients with liver involvement (n 5 6) sure below these determined trough thresholds. At had total and unbound trough concentrations of

Table 3. Mycophenolate Pharmacokinetics During Intravenous and Oral Administration in Week 1 of Treatment*

Route of Administration i.v. (n 5 11) Oral (n 5 18) P†

MMF dose, 1000 mg twice daily/750 mg twice daily, n 9/2 18/0 .14‡ Day posttransplantation 35 (21-81) 38.5 (17-74) .67 Total MPA Trough (mg/mL) 0.22 (0.04-0.62) 0.96 (0.04-2.54) .0028 AUC0-6, (mg*hour/mL) 20.36 (9.1-37.0) 18.76 (5.86-44.06) .57 AUC0-12, (mg *hour/mL) 21.12 (7.9-32.57) 24.0 (11.4-44.41) .80 Unbound MPA Trough (mg/mL) 0.005 (0.0-0.027) 0.014 (0-0.106) .03 AUC0-6, (mg*hour/mL) 0.612 (0.262-1.262) 0.367 (0.093-1.15) .02 AUC0-12, (mg*hour/mL) 0.502 (0.235-1.018) 0.450 (0.17-1.33) .20 MPAG trough, mg/mL 39.29 (3.89-89.99) 32.80 (4.6-163.23) .51 MPA unbound fraction 0.029 (0.017-0.042) 0.017 (0.012-0.044) .005

MMF indicates mycophenolate mofetil; MPA, mycophenolic acid; MPAG, mycophenolic acid glucuronide. *Data are median (range). †P value is comparison of i.v. and oral pharmacokinetics by Wilcoxon’s rank-sum test, unless noted otherwise. ‡Fisher’s exact test. 426 P. A. Jacobson et al. Biol Blood Marrow Transplant 16:421-429, 2010

Figure 1. Total MPA trough values by response at days 28 and 56 posttreatment. Troughs are the mean of weeks 1 and 2 in each subject. Only subjects with both week 1 and 2 pharmacokinetics data are included. The box-and-whisker plots extend to the 5th and 95th percentiles.

0.54 mg/mL (0.10-0.99 mg/mL) and 0.015 mg/mL generally higher, at 0.99 mg/mL (0.021-2.31 mg/mL) (0.004-0.021 mg/mL), respectively, whereas those with- and 0.015 mg/mL (0.0018-0.087 mg/mL). out liver involvement (n 5 26) had respective trough concentrations of 0.81 mg/mL (0.021-2.88 mg/mL) Infections, OS, and MPA Exposure and 0.012 mg/mL (0-0.088 mg/mL). In patients with Severe, life-threatening, or fatal infections (n 5 12; lower GI involvement (n 5 13), total and unbound 8 viral, 3 bacterial, and 1 fungal) occurred in 10 indi- MPA concentrations were somewhat lower, at 0.38 viduals by day 90 postrandomization. MPA exposure mg/mL (0.04-2.88 mg/mL) and 0.007 mg/mL (0.000- measurements were not signiﬁcantly different between 0.088 mg/mL), respectively, whereas in those without those with and without a serious infectious event (all lower GI involvement (n 5 19), concentrations were P . .19). There was no association between the

Figure 2. Unbound MPA trough values by response at days 28 and 56 posttreatment. Troughs are the mean of weeks 1 and 2 in each subject. Only subjects with both week 1 and 2 pharmacokinetics data are included. The box-and-whisker plots extend to the 5th and 95th percentiles. Biol Blood Marrow Transplant 16:421-429, 2010 MMF Pharmacokinetics in Acute GVHD 427 pharmacokinetic measurements and OS at day 180 subjects in week 1 of MMF treatment and in 37%- postrandomization (P $. 23). 50% of subjects in week 2. These data suggest that an MMF dose of .2 g/day may be required in a substantial number of patients. DISCUSSION Higher doses of MMF (3 g/day) are increasingly common in the prophylactic setting. Significantly The pharmacokinetics and pharmacodynamics of higher donor T cell chimerism was observed in a group MMF have been studied extensively in solid organ of patients receiving prophylactic MMF 3 g/day com- transplantation; however, relatively limited data are pared with those receiving 2 g/day [54]. Although available in HCT. Numerous studies have reported there was a slightly higher rate of infection in the a relationship between MMF pharmacokinetics and 3-g/day group, no difference in OS or progression- development of acute allograft rejection in organ trans- free survival (PFS) was observed. Studies in kidney plantation, and, although controversial, most support transplantation and HCT have shown a higher an association between higher MPA exposure and pro- incidence of leukopenia or cytomegalovirus (CMV) tection against acute rejection [33,39-42]. More recent reactivation in patients with high MPA exposure data in kidney transplantation indicate that prophylac- [28,30,31,55]. In our analysis, only a small number of tic calcineurin inhibitors can be safely minimized or patients achieved exposure levels potentially associated corticosteroids withdrawn if total MPA trough concen- with these events. Although higher MMF doses might trations are maintained at .1.2 or 1.6 mg/mL, or if result in more patients’ MPA exposure in the range as- AUC0-12 is $ 40 mg hour/mL [27,43]. To achieve these sociated with higher risk of infectious complications, targets, oral MMF doses of 2.5-3 g/day are typically re- no data in the context of GVHD treatment supports quired [27]. Higher starting doses of MMF in kidney this conjecture. The risk may be mitigated by better transplantation are currently under investigation [33]. GVHD control and more rapid discontinuation of im- The optimal dose of MMF in HCT remains a mat- munosuppression. In the current study, we found no ter of debate. Most centers have adopted the common association between higher MPA exposure and in- prophylactic dose for kidney transplantation of 1 g creased rate of infection. twice daily. However, at this dose, plasma concentra- Several factors that affect MMF pharmacokinetics tions are typically lower in HCT recipients relative to warrant discussion. First, the potential effect of intes- organ transplant recipients, especially when combined tinal and liver GVHD on MPA disposition is of con- with cyclosporine (CsA) [18,22,23,28,34,44-52].We cern. MPA is metabolized in the intestine and liver, have previously studied mycophenolate pharmacoki- common sites of GVHD involvement. A pilot study netics early posttransplantation after nonmyeloablative of 14 patients treated with MMF for aGVHD found conditioning and found that low total MPA trough lower total MPA concentrations in patients with intes- concentrations are associated with poorer engraftment tinal GVHD compared with those with skin or liver in- and low unbound MPA AUCs are associated with volvement [34]. This finding is supported by our a higher rate of acute GVHD [23]. Similarly, Giaccone analysis, which revealed lower trough concentrations et al. [28] reported an association between low total in patients with lower GI or liver GVHD compared MPA exposure and lower donor T cell chimerism after with those with skin- only involvement. Larger studies nonmyeloablative HCT. A dose of 3 g/day is typically with pharmacokinetic analyses are needed to deter- required to achieve the target concentrations defined mine whether the site of GVHD involvement affects in those studies when combined with CsA [37]. dosing recommendations. Second, steroids are potent The use of MMF to treat aGVHD is increasing, inducers of glucuronidation of many substrates in vitro and data from BMT CTN trial 0302 suggest that and might affect MPA metabolism, which is highly de- MMF plus corticosteroids may have significant activity pendent on glucuronidation to form its metabolites, against GVHD [16]. In the treatment setting, MPA including the major metabolite, MPAG [56]. MPAG pharmacokinetic disposition, exposure–response rela- is known to displace MPA from protein-binding sites, tionship, and optimal dosing are unclear. Two earlier thereby increasing unbound MPA. It is possible that studies evaluating the use of MMF to treat aGVHD steroids may induce glucuronidation, thereby enhanc- and cGVHD suggested a better GVHD response in ing MPAG formation and lowering MPA concentra- patients with higher MPA exposure; both studies tions. The effect of corticosteroids on MPA were small, however [34,53]. In the current pharmaco- metabolism has been studied in organ transplantation, kinetic analysis conducted with BMT CTN 0302, we with conflicting conclusions [57,58]. In our analysis, found that patients with a total MPA trough concen- the MPAG:MPA exposure ratio was high (45-66) com- tration .0.5 mg/mL or an unbound trough .0.015 pared with the ratios of around 30 reported in other mg/mL were more likely to achieve CR 1 PR at day HCT studies [23,59]. This suggests that MPAG for- 28 and day 56. MPA exposure was below these mation may be enhanced or MPAG excretion may be therapeutically favorable thresholds in 55%-69% of reduced. MPAG readily accumulates in renal 428 P. A. Jacobson et al. Biol Blood Marrow Transplant 16:421-429, 2010 dysfunction; however, the mean serum creatinine in prednisolone: a multicenter randomized trial from the Italian our subjects was 0.9 mg/dL; therefore it is unlikely Group for Bone Marrow Transplantation. Blood. 1998;92: 2288-2293. that MPAG accumulated as a result of poor renal clear- 4. Martin PJ, Nelson BJ, Appelbaum FR, et al. Evaluation of ance alone. Therefore, high dose corticosteroids may a CD5-specific immunotoxin for treatment of acute graft- affect the formation of MPAG and MPA concentra- versus-host disease after allogeneic marrow transplantation. tions; however, this requires confirmation in formal Blood. 1996;88:824-830. 5. Mayer J, Krejci M, Doubek M, et al. Pulse cyclophosphamide for drug interaction studies. Finally, MPA concentrations corticosteroid-refractory graft-versus-host disease. Bone Marrow are lower in patients receiving CsA compared to tacro- Transplant. 2005;35:699-705. limus; therefore; MMF dose requirements may vary 6. Carpenter PA, Lowder J, Johnston L, et al. A phase II multicen- depending on the underlying calcineurin inhibitor ter study of visilizumab, humanized anti-CD3 antibody, to treat steroid-refractory acute graft-versus-host disease. Biol Blood [60,61]. Although the MPA exposure target is likely Marrow Transplant. 2005;11:465-471. the same for either regimen. 7. Patriarca F, Sperotto A, Damiani D, et al. Infliximab treatment Interestingly, total MPA trough concentrations for steroid-refractory acute graft-versus-host disease. Haemato- logica. 2004;89:1352-1359. were higher in subjects receiving oral MMF than in 8. Lee SJ, Zahrieh D, Agura E, et al. Effect of up-front daclizumab those receiving i.v. MMF. 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