ASP2409, A Next-Generation CTLA4-Ig, Versus Belatacept in Renal Allograft Survival in Cynomolgus Monkeys.

Belatacept is the first costimulatory blockade agent approved for maintenance immunosuppression in kidney transplant recipients. Clinical results have indicated that belatacept is associated with superior renal function and improved metabolic profile; however, higher incidence of acute rejection and posttransplant lymphoproliferative disorder are the shortcomings of this agent. In this study, ASP2409, a new cytotoxic T-lymphocyte associated protein 4-immunoglobulin possessing 14-fold higher in vitro CD86 binding affinity than belatacept, was tested for renal allograft survival in cynomolgus monkeys. ASP2409 monotherapy dose-dependently prolonged renal allograft survival. Low-dose ASP2409 in combination with a subtherapeutic dose of tacrolimus showed much longer median survival time than monotherapy. Similar allograft survival results were observed in regimens based on high-dose ASP2409, belatacept, and therapeutic-dose tacrolimus. The results of renal allograft histopathology with high-dose ASP2409-based regimens were not inferior to the belatacept-based regimen. Moreover, higher frequencies of FoxP3-positive regulatory T cells in renal allografts were observed in ASP2409- and belatacept-based regimens compared with tacrolimus-based regimens. No serious side effects related to ASP2409 administration were found during the study. These data suggest that ASP2409 is a promising candidate for calcineurin inhibitor-sparing or -avoidance regimens.

Calcineurin inhibitor minimization in the Symphony study: observational results 3 years after transplantation.

The Symphony study showed that at 1 year posttransplant, a regimen based on daclizumab induction, 2 g mycophenolate mofetil (MMF), low-dose tacrolimus and steroids resulted in better renal function and lower acute rejection and graft loss rates compared with three other regimens: two with low-doses of cyclosporine or sirolimus instead of tacrolimus and one with no induction and standard cyclosporine dosage. This is an observational follow-up for 2 additional years with the same endpoints as the core study. Overall, 958 patients participated in the follow-up. During the study, many patients changed their immunosuppressive regimen (e.g. switched from sirolimus to tacrolimus), but the vast majority (95%) remained on MMF. During the follow-up, renal function remained stable (mean change: -0.6 ml/min), and rates of death, graft loss and acute rejection were low (all about 1% per year). The MMF and low-dose tacrolimus arm continued to have the highest GFR (68.6 +/- 23.8 ml/min vs. 65.9 +/- 26.2 ml/min in the standard-dose cyclosporine, 64.0 +/- 23.1 ml/min in the low-dose cyclosporine and 65.3 +/- 26.2 ml/min in the low-dose sirolimus arm), but the difference with the other arms was not significant (p = 0.17 in an overall test and 0.077, 0.039 and 0.11, respectively, in pair-wise tests). The MMF and low-dose tacrolimus arm also had the highest graft survival rate, but with reduced differences between groups over time, and the least acute rejection rate. In the Symphony study, the largest ever prospective study in de novo kidney transplantation, over 3 years, daclizumab induction, MMF, steroids and low-dose tacrolimus proved highly efficacious, without the negative effects on renal function commonly reported for standard CNI regimens.

Immunological evaluation of combination therapy with tacrolimus and sirolimus on long-term allograft survival in nonhuman primates.

UNLABELLED: We reported that a 60-day course of combination therapy with tacrolimus and sirolimus induced long-term survival of renal allograft after withdrawal of immunosuppressants in Vervet monkeys. In the present study, the mechanism of drug-induced allograft survival was evaluated via Th1/Th2 cytokines, apoptosis and MLC activity in primates. MATERIALS AND METHODS: Cytokines were evaluated by ELISA. MLR and CTL assays were performed by incorporation of 72 hours (3)H-TdR and 4 hours (51)Cr release assay. RESULTS: A 60-day course of tacrolimus with sirolimus resulted in long-term survival of kidney allografts. (67% > 100 days) without intermittent acute rejection. Low sensitivity to MLR was seen in long-term renal allograft survival among monkeys treated with tacrolimus and sirolimus. Increased levels of CD3(+)CD8(+), CD3(+)/CD56(+) NKT cells and CD86(+)CD8(-)CD11(+) dendritic cells were observed. A population of high expression of CD4(+)FasL(+) was detected. In addition, the concentrations of IL-2 and IFN-gamma from long-term allograft surviving monkeys was not significantly increased, rather a late phase dominance of Th2, IL-4, IL-10, and TGF-beta was found correlated with long-term survival of recipients. In conclusion, the mechanism of tacrolimus and sirolimus induced long-term allograft survival in primates relates to up-regulated FasL expression, NKT cells and dendritic cells, with downregulation of MLR sensitivity. It is also associated with late-dominant expression of Th2 cytokines.

Cardioprotective effects of the lazaroid U74389G following cold preservation and transplantation of rat hearts.

Limited recovery of contractile function and loss of coronary reactivity have been observed following prolonged hypothermic storage and transplantation of the heart. Since lipid peroxidation has a significant role in these deficits, we investigated the cardioprotective effects of a 21-aminosteroid. U74389G, 3 mg/kg i.v., was given daily for 2 consecutive days to donor Lewis rats before the hearts were harvested and to recipient Lewis rats for 3 consecutive days after heart transplantation. Donor hearts were preserved for 4 hr in cold saline (4 degree C) before transplantation. Left ventricular developed pressure (LVP), basal coronary perfusion, and coronary reactivity to endothelium-dependent dilation (bradykinin, 0.1 microM) or endothelium-dependent dilation (sodium nitroprusside, 0.5 microM) were studied in isolated, buffer-perfused heart, using a modified Langendorff model. Cold preservation alone significantly reduced LVP and coronary perfusion. Coronary reactivity to bradykinin and sodium nitroprusside was also significantly impaired. In U74389G-treated donor hearts, 4 hr of cold ischemia did not alter contractile function, coronary perfusion or endothelial reactivity, although the response to sodium nitroprusside did not fully recover. In untreated recipients, in vivo reperfusion (transplantation) resulted in reduced LVP and perfusion deficits. Treating donors and recipients with U74389G improved left ventricular contractibility and coronary perfusion, although endothelium-dependent and -independent coronary reactivity remained affected. These results indicate that the lazaroid U74389G exerts significant cardioprotection during both preservation and transplantation of the heart. Donor and recipient pretreatment is mandatory for maximal efficacy with U74389G.

Immunosuppressive properties of the benzothiazepine calcium antagonists diltiazem and clentiazem, with and without cyclosporine, in heterotopic rat heart transplantation.

In vitro studies have shown that calcium channel blockers (CCB) exert inhibitory effects on immunocompetent cells but conflicting results have been reported as to the translation of these effects into significant in vivo immunosuppression. In this study we evaluated the effects of the benzothiazepine-like calcium blockers diltiazem and clentiazem, given alone or associated with cyclosporine on survival improvement of heterotopic rat heart transplants. Inbred male rats of the Lewis strain were used as recipients and Wistar-Furth as donors. Following abdominal implantation of the graft, recipients were randomly divided into 9 groups (n = 5). Group 1 were control isografts (Lew-Lew); group 2 were control allografts (WFu-Lew), and group 3 were allografts treated with low-dose oral cyclosporine 2 mg/kg/day. Groups 4 and 5 were allografts treated with oral diltiazem 0.25 and 2.50 mg/kg/day. Groups 6 and 7 were treated with oral clentiazem, 0.25 and 2.50 mg/kg/day. Groups 8 and 9 consisted of allografts receiving low-dose cyclosporine with either diltiazem or clentiazem 2.50 mg/kg/day, all drugs were administered daily by gavage. Graft function was monitored by transabdominal palpation, and rejection was considered to be complete when no contraction of the graft could be detected. Mean survival time of untreated allografts was 6.4 +/- 0.5 days. Cyclosporine alone increased the mean survival time to 10.6 +/- 2.7 days (P < 0.05 vs. group 2). At all doses studied, diltiazem and clentiazem significantly increased mean survival time of allografts, clentiazem being slightly more potent than diltiazem. In addition, the observed beneficial effects of the benzothiazepine-like calcium channel blockers were dose-dependent. When combined with cyclosporine, diltiazem and clentiazem interacted synergistically (mean survival time increased to 16.8 +/- 3.4 days for diltiazem and 15.8 +/- 1.4 days for clentiazem). These results demonstrate that the benzothiazepine-like calcium channel blockers, as opposed to phenylalkylamines or dihydropyridines, afford significant immunosuppression when used alone. These observations, and the fact that they beneficially interact with cyclosporine, suggest that the benzothiazepine-like calcium antagonists should be considered the drugs of choice when CCBs are to be selected in transplanted patients.

Reversal of ongoing heart, kidney, and pancreas allograft rejection and suppression of accelerated heart allograft rejection in the rat by rapamycin.

Rapamycin was examined for its effects on reversal of ongoing rejection of heart, kidney, and pancreas allografts and on suppression of accelerated heart allograft rejection in the rat. A 14-day continuous intravenous infusion of RAPA by an osmotic pump at 0.02, 0.08, and 0.8 mg/kg/day to WFu recipients, starting 4 days postoperation, significantly protected the BUF heart allografts with a mean survival time (MST) +/- 1 SD of 33.2 +/- 19.8 (p < 0.001), 48.2 +/- 14.8 (p < 0.001), and 107.0 +/- 86.3 (p < 0.001) days, respectively, as compared with 7.2 +/- 0.8 days in vehicle-treated controls. Combination of low dose RAPA (0.02 mg/kg or 0.08 mg/kg) and low dose CsA (2 mg/kg) achieved significantly longer cardiac allograft survival than RAPA or CsA alone. RAPA's effect in reversing ongoing rejection of renal and pancreatic allografts was also significant. The BUF kidney and pancreas in WFu recipients treated with a 14-day course of RAPA (0.8 mg/kg/day starting 4 days postoperation) had an MST of 44.7 +/- 15.9 (p < 0.001) and 46.4 +/- 12.5 (p < 0.001), while in vehicle-treated controls, the grafts were rejected within 10 days. RAPA could also suppress accelerated cardiac allograft rejection. Hyperimmunized WFu recipients were treated with two 14-day courses of continuous i.v. RAPA at 0.8 mg/kg/day before and after BUF heart allografting. Significantly longer survival of the grafts (25.5 +/- 3.7 days, p < 0.001) was achieved compared with that of the vehicle-treated controls (3.8 +/- 1.0 days). One-course RAPA treatment before or after heart transplantation was considerably less effective. RAPA was also shown to prevent the increase of serum IgG levels and to inhibit the production of specific cytotoxic Ab in the rat receiving repetitive immunizations. Such effects presumably contribute to the inhibition of the accelerated rejection. The results of this study suggest that RAPA is potentially useful in treatment of ongoing as well as accelerated allograft rejection.

Rapamycin, a potent immunosuppressive drug for vascularized heart, kidney, and small bowel transplantation in the rat.

The effectiveness of rapamycin (RAPA) was examined for heart, kidney, and small bowel allografts in rats. Untreated or vehicle only-infused Wistar Furth (RT1u) recipients rejected Buffalo (RT1b) heart allografts within a mean survival time (MST) of 6.5 +/- 0.5 and 6.3 +/- 0.5 days, respectively. In contrast, a 14-day continuous intravenous (i.v.) infusion by an osmotic pump of 0.08 mg/kg/day RAPA to WFu recipients prolonged BUF heart allograft survival to an MST of 34.4 +/- 12.1 days (P = 0.0001). There was a graded dose-response to 0.16 mg/kg (39.0 +/- 8.7 days; P = 0.0001), 0.32 mg/kg (55.7 +/- 3.3 days; P = 0.0001) and 0.8 mg/kg (48.0 +/- 3.6; P = 0.0001). Furthermore, intraarterial/intragraft but not i.v. infusion of 0.02 mg/kg/day prolonged BUF heart allografts--namely, an MST of 14.6 +/- 1.4 days versus 8.6 +/- 2.6 days (P = 0.0001), respectively. Local delivery doses of RAPA were about as effective as the same dose delivered i.v.: 0.08 mg/kg MST 37.0 +/- 18.3 days (P = 0.0001); 0.32 mg/kg, 40.0 +/- 3.9 days (P = 0.0001); and 0.8 mg/kg, 54.8 +/- 8.2 days (P = 0.0001). Systemic i.v. RAPA therapy with 0.08 or 0.8 mg/kg/day prolonged the survival of BUF kidney grafts in WFu recipients--namely, an MST of 52.7 +/- 42.7 (NS) and 90.2 +/- 62.4 (P = 0.001) days, respectively, versus an MST of 11.6 +/- 1.5 days in control WFu recipients only infused with vehicle. While normal WFu rats reject heterotopic BUF small bowel allografts within an MST of 10.0 days, a 14-day course of i.v. RAPA treatment significantly (P = 0.0001) prolonged small bowel allograft survival to an MST of 26.8 +/- 3.7 days.

The immunosuppressive effect of rapamycin on mouse small bowel transplantation.

The efficacy of rapamycin (RAPA) was tested on small bowel transplantation in the mouse and compared with cyclosporine (CsA). Four groups were involved, each one included three combinations (n > or = 6) for evaluation of host-versus-graft (HVG, C57BL/6 X BALB/c F1 (CB6F1)-to-BALB/c), graft-versus-host (GVH, BALB/c-to-CB6F1), and combined HVG and GVH responses (C57BL/6-to-BALB/c). Grafts were transplanted to recipients heterotopically. Groups were as follows: group 1: naive controls; groups 2 and 3: recipient mice treated with RAPA 2 mg/kg/day and 4 mg/kg/day orally for 14 days, respectively; group 4: recipient mouse treated with CsA 4 mg/kg/day orally for 14 days. In the HVG model, the mean survival time (MST) of recipients was significantly longer in group 2 (32.9 +/- 17.7 days, P=0.006), group 3 (32.7 +/- 10.4 days, P=0.0001), and group 4 (37.9 +/- 11.8 days, P=0.0001), compared with naive controls in group 1 (8.5 +/- 1.6 days). In the GHV model, the MST of recipients in group 2 (41.8 +/- 19.9 days, P=0.002), group 3 (48.2 +/- 21.4 days, P=0.001) and group 4 (56.5 +/- 30.6 days, P=0.003) were significantly prolonged compared with control group 1 (8.5 +/- 1.6 days). In combined HVG and GVH responses, MST of recipient in group 2 (20.9 +/- 4.9 days, P=0.0001), group 3 (27.0 +/- 4.3 days, P=0.008), and group 4 (35.2 +/- 23.9 days, P=0.0001) were also significantly longer than that in controls (6.9 +/- 1.4 days), but in all three combinations, there were no statistically significant differences between groups 2 and 3, groups 2 and 4, or groups 3 and 4 (P>0.05). RAPA is a potent immunosuppressant able to significantly prolong small bowel allograft survival in mice using a short-term treatment. There is no statistically significant difference in recipient survival between low and high doses of RAPA treatment and the CsA standard dose used in this study.

Inhibition of in vitro immunoglobulin production by rapamycin.

Like FK506, rapamycin, a structural analog of FK506, is a strong immunosuppressant. The immunosuppressive effect of Rapa in in vitro IgG, IgM, and IgA production by human lymphocytes was examined in this study. To inhibit spontaneous or pokeweed mitogen-stimulated production of Ig by human peripheral blood lymphocytes, about one thousandfold lower concentrations of Rapa (IC50 = 0.3 nM-2 nM) were required than of cyclosporine (IC50 = 0.3 microM-2 microM). T cells were the direct targets of Rapa, because preincubation of T cells with Rapa abolished the T cells helper effect to T-dependent Ig production. Rapa also had direct suppressive effect on B cells, since Rapa suppressed IgG production by pure B cells stimulated with IL2 and Staphylococcus aureus Cowan I. Kinetic studies measuring IgG production and cell proliferation revealed that Rapa acted at the activation stage of T and B cells. Exogenous IL2 substantially reversed the inhibitory effect of CsA but not that of Rapa in Ig production. This study is the first report on the strong suppressive effect of Rapa on human humoral immune response with a quantitative comparison with that of CsA. The underlying mechanisms are also explored. The results indicate the potential usefulness of this drug in treatment of presensitized transplantation patients, with whom cytotoxic Ab is a major obstacle to a successful transplantation.

Inhibition of host-versus-graft and graft-versus-host responses after small bowel transplantation in rats by rapamycin.

The effect of rapamycin (RAPA) on both host-versus-graft (HVG) and graft-versus-host (GVH) immune responses was examined in small bowel transplant models using strongly histoincompatible donor-recipient combinations. Normal Wistar Furth (WFu; RT-1u) recipients rejected Buffalo (BUF; RT-1b) small bowel allografts within a mean survival time (MST) of 10.5 +/- 0.5 days. Administration of RAPA (0.8 mg/kg) by continuous intravenous infusion for 14 days via an osmotic pump prolonged graft survival to 25.0 +/- 4.6 days (P = 0.01). In a second strain combination, the 12.5 +/- 2.2 day survival of Brown Norway (BN; RT-1n) small bowel allografts in Lewis (RT-1l) recipients was prolonged to 21.6 +/- 2.0 and 28.5 +/- 2.8 days by 14 days of i.v. RAPA at doses of 0.8 and 1.6 mg/kg, respectively. In this model RAPA is five times more effective than cyclosporine, which at 4.0 mg/kg prolongs BN small bowel allografts in Lewis recipients to 21.6 +/- 6.3. To isolate HVG and GVH immune responses, (BN x Lewis)F1 hybrid rats served as the graft donor or host, respectively. In the HVG model, (BN x Lewis)F1 small bowel allografts, which were rejected by normal Lewis recipients at 12.2 +/- 3.6 days, were prolonged to 40.8 +/- 5.8 days (P = 0.001) by RAPA (0.8 mg/kg x 14 days). In the GVH model, the ability of Lewis small bowel allografts to produce severe GVH disease in untreated (BN x Lewis)F1 recipients at 12.3 +/- 2.8 days was delayed to 21.3 +/- 5.2 days by 0.8 mg/kg RAPA (P = 0.025). Thus, RAPA protects small bowel allografts more effectively against HVG than GVH immune responses.

Rapamycin graft pretreatment in small bowel and kidney transplantation in the rat.

The effect of rapamycin (RAPA) as graft pretreatment was evaluated in orthotopic small bowel and kidney allotransplantation (Tx) in the rat. In the small bowel Tx model, six groups were involved, each including three combinations for evaluation of host-versus-graft (HVG) [Lewis (LEW) x Brown Norway (BN) (LBN)-F1-->Lewis], graft-versus-host (GVH) (LEW-->F1), and combined HVG and GVH immune responses (BN-->LEW). RAPA graft pretreatment alone (16 micrograms/ml x 3 ml) was able to induce a modest but significant prolongation of survival in all three combination models compared with controls (P < 0.05). The same was observed for low dose CsA treatment (2 mg/kg/day x 14 days) of the recipient only (P < 0.05). Combination of graft pretreatment with RAPA and CsA recipient treatment produced a marked prolongation of survival especially in HVG response. Recipients treatment with one 48-microgram bolus of RAPA i.v. immediately after graft revascularization failed to achieve any prolongation of survival for the GVH or combined HVG and GVH responses. This seems to exclude a "carry-over" effect of RAPA from graft to recipient. RAPA efficacy was also clearly confirmed in the kidney graft pretreatment model as compared to recipient treatment with an equivalent RAPA dose. These results demonstrate that graft RAPA pretreatment prolongs SB survival after Tx in the rat for HVG, GVH, and bidirectional immune responses. Intragraft interaction with passenger leukocytes or APC function appears as one of the possible mechanisms. RAPA graft pretreatment potentiates low dose CsA recipient treatment suggesting a possible use in clinical organ Tx.

Effect of tacrolimus (FK506) and sirolimus (rapamycin) mono- and combination therapy in prolongation of renal allograft survival in the monkey.

BACKGROUND: Our previous studies confirmed that tacrolimus (FK506) and sirolimus [rapamycin (RAPA)], in combination, are not antagonistic but are synergistic in the prolongation of heart and small bowel grafts in the rodent. The aim of this study was to confirm further the synergistic effect of combined FK506 and RAPA in the more clinically relevant model, kidney transplantation in monkeys. METHODS: A total of 60 male Vervet monkeys were randomly assigned to 10 groups (n> or =5). Monkeys with renal allografts were treated with different doses of FK506 and/or RAPA orally for 60 days. Graft survival, body weight, clinical biochemistry determinations, oral glucose tolerance test, trough levels of the two drugs, and histopathology were investigated. RESULTS: Low doses of FK506 (1 or 4 mg/kg) combined with RAPA (0.5 mg/kg) produced synergistic effect in the prolongation of renal graft survival [combination index (CI) = 0.292, 0.565]. There were no additive or synergistic drug-associated toxicities such as hyperglycemia, nephrotoxicity, and hyperlipidemia. There also was no pharmacological antagonism. CONCLUSION: Concomitant therapy of low-dose (drug-optimal) FK506 and RAPA produced a synergistic effect in the prolongation of kidney allograft survival in Vervet monkeys without additive drug-associated toxicities.

Blocking of CD44-hyaluronic acid interaction prolongs rat allograft survival.

BACKGROUND: Lymphocyte activation and infiltration into a transplanted organ is an integral component of the rejection process. Graft infiltration of lymphocytes requires adhesion of leukocytes to the endothelium, diapedesis, and transmigration. One of several proteins involved in this process is CD44, which is known to interact with endothelial hyaluronan (HA). Blockade of cell-matrix and cell-cell interactions have been used extensively for modulation of immune responses and graft rejection. Based on these observations, we evaluated the effects of blocking CD44-HA interactions in a transplantation model. METHODS: We used a low molecular weight hyaluronic acid formulation (LMWHA) for the treatment of rat renal and cardiac allograft recipients. LMWHA was administered intraperitoneally at 0.5-5 mg/kg for 5-10 days after transplantation with or without a subtherapeutic dose of cyclosporine. RESULTS: LMWHA monotherapy prolonged allograft survival significantly, but only for a few days. In combination with low-dose cyclosporine, long-term survival of allografts was observed in some of recipients. CONCLUSION: Further definition of the underlying mechanism of LMWHA therapy may provide a rationale for the development of novel, nontoxic, nonimmunogenic immunotherapies.

Tacrolimus (FK506) and sirolimus (rapamycin) in combination are not antagonistic but produce extended graft survival in cardiac transplantation in the rat.

Combined use of tacrolimus (FK506) with sirolimus (rapamycin [RAPA]) was examined in a model of vascularized heart allograft in the rat. For prevention of acute rejection, three different combinations of low doses of FK506 and RAPA from day 1 up to day 14 after transplantation produced significantly longer cardiac allograft survival than each agent alone (P<0.05). Identical results were observed in a model of reversal of ongoing acute rejection, where two combinations of low doses of FK506 and RAPA from day 4 up to day 18 after surgery also demonstrated significantly longer graft survival than each immunosuppressant alone (P<0.05). All the low-dose-treated groups in these two models presented significantly longer heart graft survival than naive controls (P<0.05), confirming that both agents are potent immunosuppressants in the models chosen. These results also indicate that, in contrast with in vitro studies, the combined use of FK506 and RAPA in vivo did not produce antagonism, but rather had synergistic effect in prolonging the allograft survival as compared with each agent alone. It appears likely that the abundance of FKBP-12 available for binding in vivo prevents inhibitive competition of the two agents for their receptor.

Synergistic effects of mycophenolate mofetil and sirolimus in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat.

BACKGROUND: The effect of mycophenolate mofetil (MMF) and sirolimus (rapamycin, RAPA) mono- and combination-therapy was examined in prevention of acute heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat. METHODS: Both drugs were administered orally for up to 30 days. Eleven groups (n=6) were involved in the first part of the heart allografting model. Brown Norway (RT1n) to Lewis (RT1(1)) combination was used in the heart and pancreas transplantation models, whereas Buffalo (RT1b) to Wistar Furth (RT1u) was used in the kidney transplantation model. RESULTS: The naive control group showed a mean survival time of 6.5+/-0.6 days. There were graded dose-responses to monotherapy of MMF 10 and 20 mg(kg/ day (12.5+/-2.6 days; 19.3+/-9.0 days) and RAPA 0.2, 0.4, 0.8, and 1.8 mg/kg/day (19.2+/-2.0 days; 30.0+/-7.3 days; 50.8+/-12.5 days; 51.2+/-2.6 days), respectively (P=0.001). Results with the combined use of drugs indicate that a synergistic or very strong synergistic interaction was produced when compared with monotherapy of MMF or RAPA: MMF 10 mg(kg/day+RAPA 0.2 mg/kg(day (52.7+/-5.7 days, combination index [CI] =0.189), MMF 20 mg(kg/day+RAPA 0.2 mg/kg/day (57.7+/-5.7 days, CI=0.084), MMF 10 mg/kg/day+RAPA 0.4 mg(kg/day (50.2+/-13.5 days, CI=0.453), and MMF 20 mg/kg(day+ RAPA 0.4 mg/kg(day (51.5+/-6.8 days, CI=0.439), respectively. These results were repeatable in the prevention of acute pancreas and kidney allograft rejection in the rat. In the second part of the study of reversal of ongoing acute heart allograft rejection model, the combined treatment of MMF 10 mg/kg(day+RAPA 0.2 mg/ kg(day (35.5+/-16.0 days, CI=0.794) and MMF 20 mg/kg day+RAPA 0.2 mg(kg/day (57.2+/-4.7 days, CI=0.310) represented synergistic interaction compared with monotherapy of MMF or RAPA. CONCLUSIONS: Concomitant therapy of MMF and RAPA produces a synergistic effect in prevention of heart, pancreas, and kidney allograft rejection and in reversal of ongoing heart allograft rejection in the rat.

Compromised kidney graft rejection response in Vervet monkeys after withdrawal of immunosuppressants tacrolimus and sirolimus.

BACKGROUND: In nonprimates, organ allografts are often not rejected after withdrawal of immunosuppression. In this study, we examined whether such a phenomenon also occurs in primates. METHODS: Vervet monkeys were transplanted with renal allografts and treated for 60 days with tacrolimus, or tacrolimus plus sirolimus. The drugs were totally withdrawn on day 61. The survival of the monkeys was monitored, and their response to donor- or third party-derived alloantigens was examined in vivo and in vitro. RESULTS: The majority (80-100%) of the grafts survived for at least additional 30 days with no signs of acute rejection. The compromised rejection is donor-specific, because recipient monkeys failed to reject a donor-derived skin graft, but a third-party skin graft was rejected. In vitro mixed lymphocyte reaction and interleukin-2 production in the mixed lymphocyte reaction between the recipients and their donors or between the recipients and a third party had no discernable patterns, and thus did not reflect the in vivo status of the immune system. Although the recipients could not reject the graft acutely after drug withdrawal, the kidney grafts and the donor-derived skin grafts had pathological findings of chronic rejection. CONCLUSIONS: The rejection response of the monkeys to an established graft after withdrawal of immunosuppression is compromised. The compromised rejection is specific and is not due to a permanent alteration of the immune system by the initial drug treatment. The allografts are not inert but have low levels of interaction with the recipient immune system.

A randomized, prospective multicenter pharmacoepidemiologic study of cyclosporine microemulsion in stable renal graft recipients. Report of the Canadian Neoral Renal Transplantation Study Group.

BACKGROUND: The safety, tolerability, and pharmacokinetics of conventional cyclosporine (ConCsA) and cyclosporine microemulsion (MeCsA) were compared under conditions of normal clinical practice in a prospective, randomized, concentration-controlled, pharmacoepidemiologic study. METHODS: Between September 1994 and March 1995, 1097 stable renal transplant recipients in 14 Canadian centers were randomized 2:1 to treatment with MeCsA or ConCsA. Patients were commenced on each study drug at a dose equal to their previous therapy with ConCsA, and the dose was adjusted to maintain predose whole blood cyclosporine concentrations within the therapeutic range established for each center. Prednisone and azathioprine were continued unless dose adjustment was required for clinical reasons. RESULTS: The mean cyclosporine concentration was comparable in both treatment groups at all time points throughout the 6 months of follow-up. The mean dose of cyclosporine was 3.6 mg/kg/day in both treatment groups at entry to the study, and declined by 0.3% and by 2.8% in patients receiving ConCsA and MeCsA, respectively. The nature and severity of adverse events were similar in both treatment groups, but there was a transient increase in neurological and gastrointestinal complications in the group receiving MeCsA within the first month after conversion (P<0.05). Serum creatinine and creatinine clearance did not change in either treatment group throughout the study. Biopsy-proven acute rejection occurred in three patients (0.8%) receiving ConCsA and in seven patients (0.9%) receiving MeCsA, with non-histologically proven acute rejection in an additional three patients (0.8%) receiving ConCsA and five patients (0.6%) receiving MeCsA (P=NS). Serum creatinine rose transiently in 35 patients (9.8%) receiving ConCsA and 138 patients (18.7%) receiving MeCsA (P<0.05) and resolved either spontaneously or after a reduction in the cyclosporine dose. One graft was lost in the MeCsA group due to irreversible rejection, and seven patients died, three in the group receiving ConCsA and four of those receiving MeCsA (P=NS). Absorption of cyclosporine was more rapid and complete from MeCsA than from ConCsA during the first 4 hr of the dosing interval, resulting in almost 40% greater exposure to the drug (P<0.001). There was close correlation between area under the time-concentration curve (AUC) over the first 4 hr of the 12-hr dosage interval and AUC over the entire 12-hr dosage interval for both formulations, making AUC over the first 4 hr a good predictor of total cyclosporine exposure. Using this parameter, patients with low absorption randomized to receive MeCsA showed a marked increase in drug exposure by months 3 and 6, whereas there was no change in those who continued on ConCsA. A limited sampling strategy utilizing samples at the predose and postdose trough levels provided an excellent correlation with drug exposure, particularly for patients receiving MeCsA (r2=0.94 MeCsA vs. r2=0.89 ConCsA). CONCLUSIONS: MeCsA appears to be a safe and effective therapy in stable renal transplant patients and provides superior and more consistent absorption of cyclosporine when compared with ConCsA. Transient toxicity after conversion to MeCsA occurs in some patients, and may reflect the increased exposure to cyclosporine. Use of a limited sampling approach combining trough and 2-hr postdose concentrations may provide an effective way to monitor this exposure.

Safety and tolerability of cyclosporine and cyclosporine microemulsion during 18 months of follow-up in stable renal transplant recipients: a report of the Canadian Neoral Renal Study Group.

BACKGROUND: There has been concern that the increased drug exposure associated with treatment with cyclosporine microemulsion (CsA-ME) would lead to an increase in adverse events. METHODS: The long-term safety and tolerability of conventional cyclosporine (CsA) and CsA-ME were compared in a randomized, multicenter, pharmacoepidemiologic study involving 1097 stable renal transplant patients after 18 months of follow-up. RESULTS: No significant difference was seen in change in serum creatinine or calculated creatinine clearance between the two groups. Episodes of deterioration in renal function (change in serum creatinine > or = 20%) were categorized with the following results for CsA-ME versus CsA, respectively: acute rejection, 4.5% vs. 4.5%; chronic rejection, 8% vs. 11%; CsA nephrotoxicity, 12% vs. 7% (P=0.008); transient changes, 17% vs. 12%; other causes, 4% vs. 6%. During the first 6 months of the study, a transient increase in the incidence of gastrointestinal and neurological adverse events was seen in the CsA-ME group compared with the CsA group. Up to 18 months, patients in the CsA group reported significantly fewer hearing and vestibular disorders, but more cardiovascular problems than those in the CsA-ME group (P=0.035). CONCLUSIONS: Tolerance to CsA and CsA-ME was similar. Renal function over 18 months was not adversely affected by the increased drug exposure with CsA-ME, although there was a transient increase in nephrotoxicity. The frequency of acute and chronic rejection did not change.

Replacement therapy for inherited enzyme deficiency: liver orthotopic transplantation in Niemann-Pick disease type A.

Liver homotransplantation was attempted as replacement therapy in a 2-year-old patient with near total absence of sphingomyelinase activity of Niemann-Pick disease type A. Satisfactory function of the graft was observed until the death of the recipient from respiratory complication 2 years after transplantation. The clinical stigmata of the disease became less severe during the first 6 months after transplantation, with no further improvement thereafter. Sphingomyelinase activity was restored to near normal levels in serum, was present in cerebrospinal fluid and was maintained in the graft at normal or supranormal levels. No accumulation of sphingomyelin was observed in the transplanted organ as evaluated by histopathological and chromatographic studies. These findings support the interest of organ transplantation for long-term enzyme replacement in Niemann-Pick disease type A and similar lysosomal deficiencies.

Long-term in vivo effects of rapamycin on humoral and cellular immune responses in the rat.

Rapamycin (RAPA) is a strong immunosuppressant and is able to prevent allograft rejection in animal models. We have demonstrated that RAPA could strongly inhibit in vitro immunoglobulin (Ig) production by human lymphocytes. The present study investigated the long-term in vivo effect of RAPA on humoral and cellular immune responses, and the effect of RAPA on accelerated rejection. It was shown that RAPA strongly inhibited antigen (Ag) specific antibody (AB) production (i.e. cytotoxic Ab to donor lymphocytes and Ab to tetanus toxoid) during the period of drug administration. The accelerated rejection of cardiac allografts in presensitized animals was alleviated by RAPA administration. These results suggest the potential application of RAPA in treatment of presensitized candidates for organ transplantation. A little more than two months after the drug withdrawal, the rats were basically competent in Ab response to further Ag challenges. When tested 4 months after the RAPA-treatment, the rats showed uncompromised cardiac allograft rejection, and the cellular immune response in vitro according to mixed lymphocyte reaction (MLR) and mitogen-stimulated proliferation were not hampered. Such results suggest that the immune system can return to normal status without sequelae after discontinuation of RAPA.