Sirolimus-mediated prolongation of rat cardiac allograft survival is enhanced by beta1 integrin very late antigen-4 blockade.

The beta1 integrin very late antigen-4 (VLA-4) plays a key role in lymphocyte rolling and adhesion to endothelium, and in lymphocyte migration through fibronectin. Thus, VLA-4 blockade may modulate allograft rejection. Here, we examined the effect of WAY-279, a small molecule VLA-4 antagonist, combined with sirolimus in a model of vascularized heart allograft (BN --> LEW) in the rat. Recipients were treated with low doses of WAY-279 (10-50 mg/kg, bid) and/or sirolimus (0.04 mg/kg) for 14 days, starting on the day of transplantation. The median-effect principle and the combination index (CI) were used to assess the combined effect of WAY-279 and sirolimus (CI < 1: synergism; CI = 1: summation; CI > 1 antagonism). Low doses of WAY-279 or sirolimus alone slightly prolonged allograft survival as compared to control group (MST = 7 days). When recipients were treated with WAY-279 and sirolimus, the cardiac allograft survival was synergistically prolonged for up to 45 days (P < .001; CI = 0.174-0.970). We showed that a concomitant treatment of WAY-279 with sirolimus produced a synergistic effect in prolonging cardiac allograft survival in the rat.

Sirolimus: its discovery, biological properties, and mechanism of action.

Sirolimus is the USAN-assigned generic name for the natural product rapamycin. Sirolimus is produced by a strain of Streptomyces hygroscopicus, isolated from a soil sample collected from Rapa Nui commonly known as Easter Island. Although sirolimus was isolated as an antifungal agent with potent anticandida activity, subsequent studies revealed impressive antitumor and immunosuppressive activities. Sirolimus demonstrates activity against several murine tumors, such as B16 43 melanocarcinoma, Colon 26 tumor, EM ependymoblastoma, and mammary and colon 38 solid tumors. Sirolimus is a potent inhibitor of antigen-induced proliferation of T cells, B cells, and antibody production. Demonstration of the potent immunosuppressive activity of sirolimus in animal models of organ transplantation led to clinical trials and subsequent approval by regulatory authorities for prophylaxis of renal graft rejection. Interest in sirolimus as an immunosuppressive therapy in organ transplantation derives from its unique mechanism of action, its unique side-effect profile, and its ability to synergize with other immunosuppressive agents. The molecular mechanism underlying the antifungal, antiproliferative, and immunosuppressive activities of sirolimus is the same. Sirolimus forms an immunosuppressive complex with intracellular protein, FKBP12. This complex blocks the activation of the cell-cycle-specific kinase, TOR. The downstream events that follow the inactivation of TOR result in the blockage of cell-cycle progression at the juncture of G1 and S phase.

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.

Manipulation of the C(22)-C(27) region of rapamycin: stability issues and biological implications.

A novel series of rapamycin derivatives with modifications in the C(22)-C(27) region has been prepared. These compounds are evaluated for their ability to prevent ring fragmentation while still retaining immunosuppressive capabilities.

Rapamune (RAPA, rapamycin, sirolimus): mechanism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression.

OBJECTIVE: Rapamune is a novel immunosuppressive agent in Phase III clinical trial in renal transplantation. Its unique mechanism of action has created great interest in its use as a biochemical probe of signal transduction pathways that has provided insight into its molecular mechanism of action. This article reviews the current state of our understanding of the mechanism of action of rapamune.

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.

Therapeutic blood levels of sirolimus (rapamycin) in the allografted rat.

A study was conducted to determine the relationship among oral dose, trough whole blood levels, graft survival, and side effects in sirolimus-treated allografted rats. The heterotopic heart allograft model using Brown Norway donors and Lewis rat recipients was used. Rats were dosed daily with sirolimus or vehicle until graft failure or up to a maximum of 28 days. Upon graft failure, rats were bled for measurement of trough blood levels of drug and tissues sent for histopathologic analysis. Sirolimus blood concentration correlated positively with dose and graft survival. Significant graft survival occurred at whole blood trough levels of 0.5 ng/ml achieved at the 0.3 mg/kg/day dose. Analysis of the concentration-effect data using a sigmoidal Emax model calculated a whole blood EC50 of 2.0 ng/ml for graft survival. With mean trough concentrations of 7 ng/ml and higher, grafts survived after cessation of drug treatment. At the 0.8 mg/kg/day dose, there was a significant decrease in body weight gain in the rats. Histopathologic examination of sirolimus-treated animals detected thymic and lymphoid atrophy, both considered pharmacologic extensions of sirolimus's immunosuppressive activity and focal myocardial degeneration, an exacerbation of a spontaneous occurring lesion. These results demonstrate that sirolimus prolongs graft survival in rat in a concentration dependent manner with therapeutic whole blood levels of about 10 ng/ml.

Rapamune (Sirolimus, rapamycin): an overview and mechanism of action.

Rapamycin (Sirolimus, Rapamune), a potent immunosuppressive agent, has been demonstrated to have remarkable activity in inhibiting allograft rejection in animal models of transplantation. It is currently in phase II clinical trials. Rapamycin belongs to the class of macrocyclic immunosuppressive drugs that are bioactive only when bound to immunophilins. Cyclosporin A and FK506, two other members of this class, selectively block the transcriptional activation of several cytokine genes, thereby inhibiting cytokine production. Although rapamycin and its structural analog FK506 bind to the same immunophilin (FKBP), rapamycin acts at a later stage in T-cell cycle progression by blocking cytokine-mediated signal transduction pathways. This inhibition is the consequence of modulation of activity of a target protein by the rapamycin: FKBP complex [sirolimus effector protein (SEP)]. Although the identification of SEP has recently been reported, its function in cell-cycle progression is not known. The biochemical events that rapamycin has been shown to inhibit are (a) activation of p70S6 kinase, (b) activation of cdk2/cyclin E complex, (c) phosphorylation of retinoblastoma protein, and (d) suppression of cdc2 and cyclin A transcription.

Rapamycin (sirolimus, rapamune).

Rapamycin is a novel immunosuppressive agent that is undergoing clinical trials for use in allograft rejection therapy. This paper reviews its in-vitro biological properties, the current state of knowledge concerning its mechanism of action, and its therapeutic applications.

Prolongation of renal allograft survival in a large animal model by oral rapamycin monotherapy.

We assessed the efficacy of 5 dose levels of oral rapamycin for prolonging renal allograft survival in pigs. Untreated and triple therapy groups (cyclosporine, azathioprine, and prednisone) served as controls. Immunosuppression was administered for 28 days posttransplant and then stopped. Rapamycin whole-blood concentrations were followed weekly. Chemistry, hematology, and lipid values were monitored post-transplant. For rapamycin-treated pigs, median survival time (MST) correlated with both dose and trough levels (ng/ml). All kidneys had some degree of rejection seen on necropsy. After rejection, pneumonia was the most common cause of death. No specific end-organ toxicity was noted on histopathologic examination. Triglyceride and cholesterol levels increased in all treated pigs (both rapamycin and triple therapy) vs. untreated controls--however, all values were within normal limits. Mean ALT levels increased in weeks 2 to 4 in the higher-dose rapamycin groups but returned to baseline in pigs surviving after the drug was stopped. ALT levels did not increase above twice normal in any group. Creatinine levels correlated with the degree of rejection seen on biopsy. We noted no other toxicities. We conclude that rapamycin, given as oral monotherapy, is an effective and safe immunosuppressant in our large animal renal allograft model. Outcome correlated with dose and whole-blood levels.

Rapamycin prolongs survival and arrests pathophysiologic changes in murine systemic lupus erythematosus.

OBJECTIVE: To evaluate the effects of oral rapamycin (RAPA), a macrolide immunosuppressant that has been shown to interfere with T cell activation events, on the course of spontaneous disease progression in the MRL/MpJ/lpr/lpr (MRL/l) mouse model of lupus. METHODS: RAPA treatment (6, 12, or 25 mg/kg 3 times per week) was evaluated by monitoring survival rates, autoantibody levels, and urinary albumin levels. Additionally, concanavalin A responsiveness, interleukin-2 (IL-2) production, lymphoid organ size, and histopathology were evaluated ex vivo. RESULTS: RAPA prevented the typical rise in anti-double-stranded DNA antibody and urinary albumin levels and prolonged survival. Spleen and lymph node sizes were significantly decreased, inflammatory changes in the lung, liver, kidney, spleen, lymph node, and thymus were significantly reduced, and T cell mitogen-stimulated splenocyte proliferation and IL-2 production were restored. CONCLUSION: Data from 3 independent experiments demonstrated that RAPA significantly reduced or prevented many pathologic features of lupus normally seen in the MRL/l mouse, and suggest that RAPA may be useful as a therapeutic agent in SLE in humans.

Renal effects of rapamycin in the spontaneously hypertensive rat.

The effects of rapamycin (RAPA), administered at therapeutic doses, were investigated in the spontaneously hypertensive rat (SHR). Additionally, the reversibility of RAPA's renal effects was investigated at a supratherapeutic dose. At doses that were active in preventing heart and kidney allograft rejection in the rat (0.01-0.08 mg/kg i.v.), RAPA had no effect on kidney function or rat body weight gain. At higher doses (0.8 mg/kg), RAPA produced significant changes in kidney function parameters and caused a loss in body weight. Histopathologic changes, including necrotizing vasculopathy and tubular atrophy, were noted at therapeutic doses. The effects of RAPA on kidney function were completely reversible after a 2-week washout period, though the histopathologic changes were still evident. These studies demonstrate that RAPA does not impair kidney function at therapeutic doses when administered for 2 weeks but does appear to accelerate the naturally occurring renal lesions of the SHR.