Jochen Klupp

Comparison of the effects of tacrolimus and cyclosporine on the pharmacokinetics of mycophenolic acid.

Mycophenolate mofetil (MMF) is almost completely absorbed from the gut and is rapidly de-esterified into its active drug, mycophenolic acid (MPA). The main metabolite is glucuronidated MPA (MPAG), which is excreted into bile and undergoes enterohepatic recirculation. Studies in healthy volunteers treated with cholestyramine show that interruption of the enterohepatic recirculation decreases MPA exposure by approximately 40%. Published data show a difference in mycophenolic acid plasma concentrations between kidney transplant recipients treated with MMF plus cyclosporine (CsA) and those treated with MMF plus tacrolimus (TRL). However, the interpretation of these data is complicated by interpatient differences in variables that may influence MMF pharmacokinetics (e.g., underlying disease, co-medication, and time since transplantation). To understand the influence of TRL and CsA on MMF pharmacokinetics (PK) more completely, the authors eliminated confounding variables in clinical studies by performing drug interaction studies in inbred rats. To achieve a steady state, 3 groups of Lewis rats (n = 8 per group) were treated once daily with oral CsA (8 mg/kg), TRL (4 mg/kg), or placebo on days 0–6 before all rats began once-daily oral treatment with MMF (20 mg/kg) on day 7. Combined treatment with either MMF + CsA, MMF + TRL, or MMF + placebo was continued for 1 week (days 8–14). Thereafter, CsA and TRL treatments were stopped but MMF treatment was continued on days 14–21. Blood was sampled during the 24 hours subsequent to dosing on day 7 (after the first MMF dose), on day 14 (after multiple MMF doses) and on day 21 (after CsA/TRL washout). Rats in the MMF + TRL group and in the MMF + placebo group showed a second peak in the MPA-PK profiles consistent with enterohepatic recirculation of MPA. The MPA-PK profiles for the MMF + CsA–treated animals did not show a second MPA peak. On Day 14, the mean plasma MPA-AUC0–24 hours for the CsA-treated animals was significantly less than MPA exposures for rats in the MMF + TRL– and the MMF + placebo–treated groups. Furthermore, in contrast to results from other investigators, co-administration of CsA and MMF significantly increased MPAG-AUC0–24 hours. Serum creatinines did not differ among rats in the three groups. CsA but not TRL decreased MPA plasma levels and increased MPAG-AUC0–24 hours. These data suggest that CsA inhibits MPAG excretion into bile and offer an explanation for the well-known increased MPA exposure in organ transplant patients caused by conversion from CsA-to TRL-based immunosuppression.

Mycophenolatemofetil for immunosuppression after liver transplantation: A follow-up study of 191 patients

Background. Mycophenolatemofetil (MMF) combined with calcineurin inhibitors (CNIs) as immunosuppression after orthotopic liver transplantation (OLT) is still under discussion. We retrospectively investigated the immunosuppressive potency of MMF for treatment of steroid‐resistant acute rejection (AR) or chronic rejection (CR), chronic graft dysfunction, and CNI‐induced toxicity in patients after OLT. Methods. Between 1988 and 2001 we performed 1,386 OLTs in 1,258 patients. Since 1995, 191 patients have received MMF after OLT for steroid‐resistant AR or CR, chronic graft dysfunction (115 patients), and CNIinduced toxicity (76 patients). The mean follow‐up time was 56 months. Results. Of 47 patients with steroid‐resistant AR, 12 had been treated with OKT3, without resolving the rejection. Overall, bilirubin and transaminases decreased significantly within 2 weeks after the addition of MMF, and liver function normalized in 38 patients. Five of eight patients with CR demonstrated stable liver function after a follow‐up of 55±8 months; 52 of 60 patients with chronic graft dysfunction improved within 3 months; and 46 of 59 patients with CNI‐induced nephrotoxicity improved after MMF treatment and a reduction of CNIs (with a significant decrease in serum creatinine within 2 weeks and an increase of creatinine clearance within 3 months). Clinical symptoms improved in 10 of 12 patients with neurotoxicity and four of five patients with hepatotoxicity. Side effects of MMF, such as gastrointestinal disorders or bone marrow toxicity, occurred in 60 patients (31.4%). The incidence of infections did not increase. Patient survival was 93%, and graft survival was 88.2%. Conclusions. MMF is a potent and safe immunosuppressive agent in OLT recipients for rescue therapy in AR, CR, or chronic graft dysfunction and helps to reduce the serious toxic side effects of CNIs.

Coadministration of either cyclosporine or steroids with humanized monoclonal antibodies against CD80 and CD86 successfully prolong allograft survival after life supporting renal transplantation in cynomolgus monkeys.

BACKGROUND Recent studies have shown some efficacy using monotherapy with monoclonal antibodies (mAb) against CD80 and CD86 receptors after life-supporting renal transplantation in non-human primates. Our study was designed to evaluate the efficacy of combinations of the same mAbs with either microemulsion cyclosporine (CsA) or steroids. METHODS Unilateral renal transplantation was performed in 16 blood group-matched and MLR-mismatched cynomolgus monkeys that were assigned to four different treatment groups. All monkeys in groups I, II, and IV were treated with the combination of a CD80 (h1F1) and CD86 (h3D1) mAb given at 20 mg/kg each preoperatively, then 5 mg/kg at weekly intervals starting postoperative (po) day 0 until poday 56 (9 doses). In group I the animals (n=4) were treated with mAbs only. In group II (n=4) mAbs were combined with a CsA regimen adjusted daily to maintain target 24 hr trough levels of 150-300 ng/ml CsA for poday 0 to poday 56. In group III (n=4) the animals received CsA monotherapy according to the same regimen as group II. In group IV methylprednisone was administered at 2 mg/kg IV on poday 0-2, then at 0.5 mg/kg/day prednisone per gavage that was and tapered to 0.2 mg/kg/day on which they were maintained until poday 56. All animals were off all immunosuppressive treatment after poday 56 and were then followed until poday 119. RESULTS The mean survival of groups I-IV was 74 (range 9-119 days), 113 (96-119 days), 39 (22-71 days), and 79 days (6 to 119), respectively. All animals in group I showed clinical evidence of acute severe rejection (fever, creatinine increase, anuria) within the first week posttransplant, including those that retained renal function until poday 119. Only one animal in group II had a moderate clinical rejection during the treatment period and three of four animals survived the intended follow-up period. All animals in group III had multiple biopsy proven or severe clinical rejection episodes within the first 21 days and only one animal survived beyond poday 40. Moderate or severe acute rejection was diagnosed in three of four animals of group IV within the first 28 days post transplant and only one animal survived until poday 119. CONCLUSION Our data show that combining a calcineurin inhibitor or prednisone with mAbs designed to block costimulatory signals does not antagonize the immunosuppressive efficacy of these mAbs. In addition, combining CsA with mAbs directed against the CD80 and CD86 receptors significantly prolongs graft survival when compared to CsA monotherapy. Therefore clinical trials of humanized mAbs to CD80 and CD86 used in combination with conventional immunosuppression can be considered.

Successful infliximab treatment of steroid and OKT3 refractory acute cellular rejection in two patients after intestinal transplantation

Acute rejection resistant to established immunosuppressive rescue protocols remains the most prominent risk factor after intestinal transplantation. In two patients presenting with steroid-resistant severe acute cellular rejection 9 months and 2 years after intestinal transplantation, complete resolution was not achieved despite 5 and 10 days of OKT3 treatment, respectively, and high-dose triple baseline immunosuppression with tacrolimus, rapamycin, and steroids. There was a dissociated course of rejection with persistent moderate to severe rejection in the terminal portion of the graft despite complete recovery from rejection in the proximal parts. Both patients were treated with four subsequent infusions of infliximab (3 mg/kg body weight), a chimeric anti-tumor necrosis factor-&agr; antibody. There was an immediate response regarding macroscopic appearance, graft histology, and clinical symptoms. Both patients recovered. In conclusion, infliximab has proven to be an effective rescue therapy in a selected group of patients with steroid and OKT3 refractory severe acute rejection after intestinal transplantation.

Treatment with humanized monoclonal antibodies against CD80 and CD86 combined with sirolimus prolongs renal allograft survival in cynomolgus monkeys.

Background. Co-stimulatory blockade has been shown to prolong allograft survival in different transplant models. We investigated the effect of combining humanized anti-CD80 and anti-CD86 monoclonal antibodies (mAb) with sirolimus in cynomolgus monkey renal transplant recipients. Methods. After renal transplantation, groups of four animals were treated daily with sirolimus, sirolimus and nine weekly doses of mAb, two weekly doses of mAb, or sirolimus and two weekly doses of mAb. Results. Survival was significantly better in monkeys treated with the combination of sirolimus and mAb when compared with treatment with either agent alone (P =0.0067 by log-rank analysis). When combined with sirolimus, nine weekly doses of mAb did not result in an additional survival benefit compared with only two mAb doses (P =0.74). None of the treatment regimens used in this study resulted in development of transplantation tolerance. Conclusions. Sirolimus can be successfully combined with humanized mAb against CD80 and CD86. Induction with a short course of mAb is effective in prolonging allograft survival in combination with sirolimus.

Extended indications in living-donor liver transplantation: bile duct cancer.

The advantages of living donor liver transplantation are an individually available graft and a tremendously reduced waiting time until transplantation. One consequence is that many centers have extended the pretransplant selection criteria, especially for potential recipients suffering from hepatocellular carcinoma. In contrast, reports on living donor liver transplantation for cholangiocarcinoma are restricted to few case reports. We have analyzed our experience with seven patients suffering from cholangiocarcinoma (Klatskin tumors, n=5; intrahepatic cholangiocarcinoma, n=2). During a median follow-up of 20 months (range 2–46 months), all patients are alive except for one posttransplant death. Four patients suffering from Klatskin tumors are alive without recurrence; both patients suffering from intrahepatic cholangiocarcinoma are alive with bone and peritoneal metastases. Living donor liver transplantation may be beneficial in selected patients suffering from Klatskin tumors, whereas caution should prevail when considering intrahepatic cholangiocarcinoma.

Indications of mycophenolate mofetil in liver transplantation.

Mycophenolate mofetil (MMF) is approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation as well as for pediatric patients after kidney transplantation. MMF, a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH), blocks de novo purine synthesis which leads to an effective inhibition of proliferation selectively in T and B lymphocytes, smooth muscle cells, and fibroblasts. MMF shows additional effects with inhibition of the expression of activating and adhesion molecules on the surface of lymphocytes. The beneficial safety profile with distinct side effects compared to calcineurin inhibitors (CNI) enable efficacious combination with ciclosporin or tacrolimus as de novo therapy after liver transplantation. Furthermore, recent studies show the possibility to reduce CNI induced toxicities by adding MMF to primary immunosuppression. MMF is also used to enable early steroid withdrawal after liver transplantation. MMF can increase efficacy of immunosuppressive therapy and thereby support the treatment of steroid resistant acute rejections, chronic rejections and chronic graft dysfunction.

Sirolimus (Rapamycin) Monotherapy Prevents Graft Vascular Disease in Nonhuman Primate Recipients of Orthotopic Aortic Allografts

Background—Delayed treatment with sirolimus (SRL) halts progression of graft vascular disease (GVD) in nonhuman primate (NHP) aortic allograft recipients. In this study, we investigated whether SRL monotherapy prevents the development of GVD. Methods and Results—Pairs of 3-cm infrarenal aortic segments were exchanged between mixed lymphocyte reaction–mismatched, blood group–compatible NHPs (n=12). Six NHPs were untreated controls, and 6 were treated orally with SRL starting on the day of transplantation. Follow-up was 105 days. SRL doses were adjusted individually by assessing SRL blood concentrations, immune function, and clinical status. The severity of GVD was determined every 3 weeks by intravascular ultrasound, which quantified intimal area (IA) and intimal volume (IV) for the middle 1-cm graft segments. The mean±SEM SRL plasma levels were 14.5±9 ng/mL. In grafts from treated NHPs, IA and IV values on days 63, 84, and 105 were significantly lower than for controls (P <0.05 to P <0.001). On day 105, in the grafts from SRL-treated NHPs compared with grafts from controls, values (mean±SEM) were IA, 2.9±0.9 versus 5.5±0.7 mm2, P <0.001 and IV, 29.6±4.6 versus 55.2±2.8 mm3, P <0.001; IA and IV values for grafts from SRL-treated NHPs did not increase significantly between days 21 and 105. Conclusions—We show that SRL monotherapy prevented GVD in NHP aortic allograft recipients, suggesting the value of SRL for controlling GVD in clinical transplantation.

Effects of the malononitrilamide FK778 on immune functions in vitro in whole blood from non-human primates and healthy human volunteers.

BACKGROUND FK778, a malononitrilamide analog of leflunomide, is currently being investigated for use in clinical transplantation. METHODS Whole blood from cynomolgus monkeys (n=4) and healthy human volunteers (n=4) was incubated with different concentrations of FK778 and stimulated with mitogens in culture medium. Lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow-cytometric analysis of expression of proliferating cell nuclear antigen on cells in S/G(2)M phase. Flow cytometry was also used to assess expression of T and B lymphocyte activation surface antigens and production of intracellular cytokines by T cells. RESULTS Not only lymphocyte proliferation, but also expression of various T cell surface antigens (CD25, CD11a, CD95, CD154) was suppressed by FK778. Fifty percent effective concentration values for the different immune functions were lower in human blood than in blood from cynomolgus monkeys. CONCLUSIONS FK778 inhibits multiple immune functions. Their flow cytometric evaluation can be used to assess the effects of the drug in vivo.

Treatment by Mycophenolate Mofetil of Advanced Graft Vascular Disease in Non‐Human Primate Recipients of Orthotopic Aortic Allografts

Failure to control chronic graft dysfunction [e.g. graft vascular disease (GVD)] is the primary cause of immunologic graft failure. This is the first study of mycophenolate mofetil (MMF) for the treatment of GVD in non‐human primate recipients of aortic allografts. Abdominal aortic allografts were exchanged between mixed leukocyte reaction (MLR) ‐mismatched, blood‐group‐compatible cynomolgus monkeys. Six control recipients were untreated. Individualized treatment with frequent dose adjustments of MMF insured that treatment was close to the maximum tolerated dose (mean 99.2 mg/kg/day). Immune‐mediated injury proceeded unhindered until day 45, after which MMF treatment began. Changes in intimal volume (IV) were quantified by intravascular ultrasound (IVUS) and compared to histology on day 105. Serial IVUS measurements of IV (mm3) in controls showed progressive GVD. In four out of six animals, MMF was well tolerated, thus enabling optimum treatment; in all these animals, IV was significantly less than in the control animals (p = 0.02). In the two remaining animals, high doses were not tolerated; at day 105, there was no significant difference in IV between them and the controls. We found a significant correlation between the mean MMF tolerated dose and the inhibition of progression of IV (r = −0.88, p = 0.015). When high MMF doses were tolerated, MMF slowed progression of GVD.