Everolimus (Certican) 12-month safety and efficacy versus mycophenolate mofetil in de novo renal transplant recipients

BACKGROUND: Everolimus is a proliferation inhibitor designed to target chronic rejection, including prevention of acute rejection. Everolimus blocks growth factor-mediated transduction signals, preventing organ rejection by a mechanism different than that of calcineurin inhibitors and of mycophenolate mofetil (MMF). METHODS.: Everolimus (1.5 mg or 3 mg daily) was compared with MMF (2 g daily) in a randomized, multicenter, multinational, 12-month double-blind, double-dummy and 2-year open-label, phase 3 trial in de novo renal allograft recipients (n = 588) who also received cyclosporine and corticosteroids as part of a triple immunosuppressive regimen. RESULTS: At 12 months, there were no statistically significant differences between doses of 1.5 and 3 mg/day everolimus and MMF (2 g/day) in incidence of biopsy-proven acute rejection (23.2%, 19.7%, and 24.0%, respectively), graft loss (4.6%, 10.6%, and 9.2%), or death (5.2%, 4.0%, and 2.6%), respectively. Everolimus 1.5 mg/day and MMF were generally equally well tolerated. Both were better tolerated than everolimus 3 mg/day. The incidence of cytomegalovirus infection was significantly lower in patients receiving either 1.5 or 3 mg/day everolimus than in those receiving MMF (5.2% and 7.6% vs. 19.4%, respectively) (P = .001). CONCLUSIONS: Everolimus is effective in preventing acute rejection and graft loss in de novo renal allograft recipients receiving a triple immunosuppressive regimen. Prevention of acute rejection, along with reduction in cytomegalovirus infection, addresses two factors known to contribute to chronic rejection in such patients.     

Three-Year Efficacy and Safety Results from a Study of Everolimus Versus Mycophenolate Mofetil in de novo Renal Transplant Patients

Everolimus 1.5 or 3 mg/day was compared with mycophenolate mofetil (MMF) 2 g/day in a randomized, multicenter 36-month trial in de novo renal allograft recipients (n = 588) receiving cyclosporine microemulsion (CsA) and corticosteroids. The study was double-blind until all patients had completed 12 months, then open-label. By 36 months, graft loss occurred in 7.2, 16.7 and 10.7% of patients in the everolimus 1.5, 3 mg/day, and MMF groups, respectively (p = 0.0048 for everolimus 1.5 mg/day vs. 3 mg/day); efficacy failure (biopsy-proven acute rejection (BPAR), graft loss, death or lost to follow-up) occurred in 33.0, 38.9 and 37.2% of patients (p = 0.455 overall), respectively. Mortality and incidence of BPAR were comparable in all groups. Creatinine values were higher in everolimus groups, requiring a protocol amendment that recommended lower CsA exposure. Diarrhea, lymphocele, peripheral edema and hyperlipidemia were more common among everolimus-treated patients, whereas viral infections, particularly cytomegalovirus infection, increased in the MMF group. Overall safety and tolerability were better with MMF and everolimus 1.5 mg/day than with everolimus 3 mg/day. In conclusion, at 36 months, an immunosuppressive regimen containing everolimus 1.5 mg/day had equivalent patient, and graft survival and rejection rates compared with MMF in de novo renal transplant recipients, whereas everolimus 3 mg/day had inferior graft survival. Renal dysfunction in everolimus cohorts necessitates close monitoring.     

Everolimus Versus Mycophenolate Mofetil in Heart Transplantation: A Randomized,Multicenter Trial

In an open‐label, 24‐month trial, 721 de novo heart transplant recipients were randomized to everolimus 1.5 mg or 3.0 mg with reduced‐dose cyclosporine, or mycophenolate mofetil (MMF) 3 g/day with standard‐dose cyclosporine (plus corticosteroids ± induction). Primary efficacy endpoint was the 12‐month composite incidence of biopsy‐proven acute rejection, acute rejection associated with hemodynamic compromise, graft loss/retransplant, death or loss to follow‐up. Everolimus 1.5 mg was noninferior to MMF for this endpoint at month 12 (35.1% vs. 33.6%; difference 1.5% [97.5% CI: –7.5%, 10.6%]) and month 24. Mortality to month 3 was higher with everolimus 1.5 mg versus MMF in patients receiving rabbit antithymocyte globulin (rATG) induction, mainly due to infection, but 24‐month mortality was similar (everolimus 1.5 mg 10.6% [30/282], MMF 9.2% [25/271]). Everolimus 3.0 mg was terminated prematurely due to higher mortality. The mean (SD) 12‐month increase in maximal intimal thickness was 0.03 (0.05) mm with everolimus 1.5 mg versus 0.07 (0.11) mm with MMF (p < 0.001). Everolimus 1.5 mg was inferior to MMF for renal function but comparable in patients achieving predefined reduced cyclosporine trough concentrations. Nonfatal serious adverse events were more frequent with everolimus 1.5 mg versus MMF. Everolimus 1.5 mg with reduced‐dose cyclosporine offers similar efficacy to MMF with standard‐dose cyclosporine and reduces intimal proliferation at 12 months in de novo heart transplant recipients.     

A proteasome inhibitor effectively prevents mouse heart allograft rejection

BACKGROUND: We have previously demonstrated in vitro that proteasome inhibitors could suppress proliferation and induce apoptosis of activated T cells. This finding suggests that such inhibitors could be used as a novel category of immunosuppressants in blocking allograft rejection. METHODS: The proteasome inhibitor dipeptide boronic acid (DPBA) was tested in vitro for its inhibitory effect on mouse T-cell proliferation and lymphokine secretion. DPBA was also used in vivo to treat mouse heterotopic heart allograft rejection. Possible side effects of this compound were examined according to blood chemistry of mice treated with DPBA. RESULTS: DPBA suppressed the T-cell proliferation and potently inhibited interleukin (IL)-2, IL-6, IL-10, IL-13, and IFN-gamma produced by anti-CD3-activated T cells. Given i.p. starting 1 day after transplantation at 0.66 mg/kg per day for 16 days, or at 1 mg/kg per day for 4 days followed by 0.5 mg/kg per day for 12 days, DPBA could prolong heart allograft survival to 35.5 days (mean survival time, MST) and to 36.2 days, respectively. The control group had MST of 7.3 days. When administrated 72 hr post operation at 1 mg/kg per day for 4 days, DPBA could prolong the graft survival to 19.8 days. During the course of these effective dosages, DPBA had no apparent toxicity in the liver, kidney, pancreas, or heart, according to analysis of blood chemistry. CONCLUSIONS: The proteasome inhibitor could repress allograft rejection in mice without apparent side-effects at the effective dosages. This finding has opened a new dimension in development of novel immunosuppressants for organ transplantation.     

Everolimus Versus Azathioprine in Maintenance Lung Transplant Recipients: An International, Randomized, Double-Blind Clinical Trial

Everolimus is a proliferation signal inhibitor with immunosuppressive activity that may reduce the rate of progression of chronic rejection, bronchiolitis obliterans syndrome (BOS), after lung transplantation. In a randomized, double-blind clinical trial, 213 BOS-free maintenance patients received everolimus (3 mg/day) or azathioprine (AZA, 1-3 mg/kg/day) in combination with cyclosporine and corticosteroids. The prospectively defined primary endpoint was the incidence of efficacy failure (decline in FEV1 >15%[deltaFEV1 >15%], graft loss, death or loss to follow-up) at 12 months. Incidence of efficacy failure at 12 months was significantly lower in the everolimus group than AZA (21.8% vs. 33.9%; p = 0.046); at 24 months, rates of efficacy failure became similar between the groups. At 12 months, the everolimus group had significantly reduced incidences of deltaFEV1 >15%, deltaFEV1 >15% with BOS, and acute rejection. At 24 months, only incidence of acute rejection remained significantly less in the everolimus group. Treatment discontinuations (particularly due to adverse events), serious adverse events and high serum creatinine values were more common with everolimus. For the first time, a drug has demonstrated significant slowing of loss in lung function, suggesting that patients kept on prolonged maintenance treatment with everolimus may benefit from replacing AZA with everolimus 3 months after lung transplantation.     

Therapeutic Drug Monitoring for Everolimus in Heart Transplant Recipients Based on Exposure–Effect Modeling

Everolimus, a proliferation signal inhibitor, is an immunosuppressant that targets the primary causes of progressive allograft dysfunction, thus improving the long-term outcome after heart transplantation. The present study investigated whether therapeutic drug monitoring (TDM) of everolimus would benefit heart transplant patients. Data from a twelve-month phase III trial comparing everolimus (1.5 or 3 mg daily) with azathioprine were used to evaluate everolimus pharmacokinetics, exposure-efficacy/safety and TDM prognostic simulations. Everolimus trough levels were stable in the first year post-transplant and averaged 5.2 +/- 3.8 and 9.4 +/- 6.3 ng/mL in patients treated with 1.5 and 3 mg/day, respectively. Cyclosporine trough levels were similar in all treatment groups. Biopsy-proven acute rejection (BPAR) was reduced with everolimus trough levels > or =3 ng/mL. Intravascular ultrasound (IVUS) analysis showed evidence of reduced vasculopathy at 12 months with increasing everolimus exposure. Unlike cyclosporine, increasing everolimus exposure was not related to a higher rate of renal dysfunction. The TDM simulation, which was based on two everolimus dose adjustments and an initial starting dose of 1.5 mg/day, showed that the simulated BPAR rate (with TDM) was 21% versus 26% in the group with fixed dosing. Therefore, TDM in heart transplantation could optimize immunosuppressive efficacy and reduce treatment-related toxicity.     

Late acute cardiac allograft rejection: new therapeutic options?

BACKGROUND: Late acute cellular rejection is associated with decreased survival and the development of CAV. Among new immunosuppressive drugs introduced into clinical practice, everolimus, has been shown to be safe in cardiac transplantation. We report our experience with everolimus in heart transplant recipients who developed late acute cellular cardiac rejection. METHODS: Patients with a history of previous rejection episodes who experienced cardiac rejection were switched to an everolimus, cyclosporine, and steroid immunosuppressive regimen. All patients had already received statins and antihypertensive medications. Everolimus, cyclosporine trough levels, and laboratory values were controlled monthly. Drug administration was adapted to an everolimus trough level between 3 and 8 ng/mL, mean maintenance dosage was 0.25 to 1.5 mg twice a day. Death, safety, side effects, biopsy-proven acute rejection episodes, laboratory values, and blood levels were evaluated retrospectively. RESULTS: Four cardiac allograft recipients (two male, two female), at a median of 1473.25 days post-orthotopic heart transplantation (oHTx) (range = 65 to 3045), received 1 to 1.5 mg everolimus per day. Over a follow-up period of at least 2 month (range = 2 to 10) the mortality was 0%. The drug was well tolerated; no acute cellular rejection greater than grade 1a (ISHLT grading) was observed after 2 months. In one patient increased cholesterol values and in two others, elevated triglyceride levels were seen, but were controlled with increased statin therapy. No obvious increased creatinine values were seen with everolimus. CONCLUSION: In conclusion, conversion to an everolimus-based immunosuppressive regimen after late cardiac rejection is safe and effective; no major side effects were observed.     

Role for primary immunosuppression with everolimus after pulmonary transplantation

PurposeEverolimus is a proliferation signal inhibitor used for triple immunosuppressive therapy following solid organ transplantation. Its positive benefits, such as reduction of acute rejection episodes and reduced nephrotoxicity have been shown in kidney- as well as heart transplantation. However the role of everolimus is less well defined in lung transplantation. We thus wished to study the effect of primary immunosuppression with everolimus in a preclinical large animal lung transplantation model.MethodsLeft-sided single lung transplantation from MHC-mismatched donors was performed in 11 adult minipigs. Intravenous pharmacologic immunosuppression was maintained for 28 days with 1.5 mg/kg/d methylprednisolone, 1.0 mg/kg/d azathioprine and cyclosporine A (blood levels 300-500 ng/ml; CsA group; n = 5). A further group (CsA + Ev; n = 6) received methylprednisolone, CsA (200–300 ng/ml) and Everolimus (5–10 ng/ml). Immunosuppression was discontinued on postoperative day (POD) 28. Graft survival was monitored by sequential chest X-rays, bronchoscopies and transbronchial biopsy histology.ResultsAll animals survived the 28 day course of immunosuppressive therapy and showed healthy grafts on POD 28. Median allograft survival in the CsA group was 55 ± 15 days. CsA + Ev grafts showed median survival of 49 ± 86 days (p = 0.37).ConclusionWhereas everolimus might be advantageous as maintenance immunosuppressive agent, this data does not support an important role for everolimus in the immunosuppressive induction phase following lung transplantation.     

12-month safety and efficacy of everolimus with reduced exposure cyclosporine in de novo renal transplant recipients

The proliferation signal inhibitor everolimus (Certican), has demonstrated efficacy with full-dose cyclosporine (CsA) (Neoral). Two multicenter randomized controlled studies were performed to compare 12-month efficacy and safety of everolimus 1.5 and 3.0 mg/day with reduced-dose CsA. Study 1 enrolled 237 de novo renal allograft recipients, randomizing 222 nonblack patients to either everolimus 1.5 or 3.0 mg/day, with the Neoral) dose guided by C(2) (monitoring of CsA concentration 2 h after dosing). Study 2 had a similar protocol, with basiliximab included, enrolling 256 recipients and randomizing 243 nonblack patients. In Study 1, there was a lower incidence of acute rejection in nonblack patients on 3 mg/day (16.4%) compared with 1.5 mg/day (25.9%), P = 0.08. In Study 2, the inclusion of basiliximab lowered the overall incidence of acute rejection; 14.3% of nonblack patients (3 mg/day) and 13.6% of nonblack patients (1.5 mg/day) had acute rejection by 12 months (P =0.891). Renal function was preserved throughout the study, with no differences observed between groups within studies. Everolimus was well tolerated with no significant differences between doses. Everolimus, in combination with reduced-dose Neoral), demonstrated efficacy and was well tolerated. Basiliximab allows for utilization of lower doses of everolimus with reduced dosing of Neoral).     

Everolimus suppresses cancellous bone loss, bone resorption, and cathepsin K expression by osteoclasts

The proliferation inhibitor of the macrolide class, everolimus, is a drug shown to be effective in the prevention of organ transplant rejection and to have a potential in the treatment of rheumatoid arthritis and certain cancers. As these diseases or their current treatments are associated with bone loss, we examined the effect of everolimus on mouse and human bone cells in vitro and on bone in an ovariectomized (OVX) rat model. Everolimus potently inhibited primary mouse and human osteoclast activity in the pit assay (IC50 values of 0.6-4.0 nM), as well as osteoclast formation, measured as the number of tartrate-resistant acid phosphatase (TRAP) multinucleated cells (IC50 values of 7.7-10.5 nM). Inhibition of osteoblastic differentiation was also observed (IC50 value of 13.5 nM). As expected, everolimus inhibited proliferation of osteoclast precursors and stimulated apoptosis, albeit with insufficient potency and efficacy to explain inhibition of osteoclast activity. Thus, everolimus appeared to directly inhibit bone resorption, which is in accord with the detected inhibition of mRNA and protein expression of cathepsin K; the main collagen-degrading protease in osteoclasts. Despite the in vitro antiproliferative activity of everolimus and the observed inhibition of osteoblast differentiation, no detrimental effects were detected at different skeletal sites in mature OVX rats at doses up to 3 mg/kg/day. This everolimus dose also prevented the OVX-induced loss of cancellous bone by 60%, an effect predominantly associated with decreased osteoclast-mediated bone resorption, resulting in a partial preservation of the cancellous bone network. Everolimus inhibited S6 kinase 1 activity in rat blood cells, skin, and bone, at doses equivalent to those used for efficacy experiments in the OVX rat model, which demonstrated in vivo targeting of the expected molecular pathway. In conclusion, everolimus directly inhibits bone resorption by osteoclasts and thus could at least be neutral or protective for bone in vivo, which would favor its use in disease indications associated with bone loss.     

Assessment of the in vivo immunosuppressive activity of the major cyclosporine metabolite by leukemia allograft rejection.

AM1 (M17) is the major metabolite of cyclosporine found in the blood of human transplant recipients, and trough levels of this derivative exceed those of the parent compound approximately two-fold. Studies performed in vitro indicate that AM1 retains only 10-20% of the biological activity of the parent compound, but very little is known about its in vivo immunosuppressive effects. We therefore developed a rapid and sensitive method, based on the rejection of allogeneic L1210 (H-2d) leukemia cells by C57BL/6 (H-2b) mice, to assess the immunosuppressive activity of AM1 in vivo. Rejection of the leukemia allograft was determined by analyzing the spleens from mice injected intravenously with 10(5) L1210 cells for the presence of H-2Kd-positive cells by flow cytometry using an FITC-conjugated monoclonal anti-H-2Kd antibody. Nonimmunosuppressed mice rejected the allogeneic cells and survived indefinitely. Spleens from these mice were virtually free of H-2Kd-positive cells (0.51 +/- 0.21%) by day 7. In contrast, C57BL/6 mice treated with 10 mg/kg/day s.c. of CsA all died from the L1210 challenge (mean survival time of 9 +/- 1 days). Spleens from mice treated in this manner contained 11.02 +/- 3.31% H-2Kd-positive cells on day 7. There was a direct correlation between the dose of CsA administered (7.5-50 mg/kg/day) and the percentage of H-2Kd-positive cells in the spleen. We then compared the immunosuppressive activity of AM1 and CsA in this model. AM1 was purified from the urine of CsA-treated renal allograft recipients by a combination of preparative adsorption-desorption chromatography and preparative elution high-performance liquid chromatography. AM1 at a dose of 10 mg/kg/day exhibited no demonstrable immunosuppressive effect, and trough levels of AM1 on day 7 were only 36 +/- 4 ng/ml. Increasing the dose of AM1 to 50 mg/kg/day resulted in only 1.05 +/- 0.16% H-2Kd-positive cells in the spleens (P = NS) and a mean trough level of 221 +/- 27 ng/ml. In contrast, mice treated with 50 mg/kg/day of CsA exhibited 17.7 +/- 2.9% H-2Kd-positive cells in their spleens and a mean trough CsA level of 3036 +/- 277 ng/ml. The half-life of a single subcutaneous dose of 10 mg/kg of AM1 (4.6 hr) was significantly shorter than that of CsA (9.7 hr) in mice. Compared with CsA, the lack of immunosuppressive effect of AM1 in vivo therefore appears to be due to a combination of decreased immunosuppressive activity and increased rate of clearance in mice.     

Safety, tolerability, and efficacy of everolimus in de novo liver transplant recipients: 12- and 36-month results.

Everolimus is a macrolide immunosuppressive agent with known consistent absorption. In this double-blind study, we examined the safety and tolerability of everolimus vs. placebo in de novo liver transplant recipients. One hundred and nineteen liver allograft recipients were randomized to 1 of 4 groups: everolimus 0.5 mg bid, everolimus 1.0 mg bid, everolimus 2 mg bid, or placebo. Patients received oral cyclosporine to achieve a target trough level of 150-400 ng/mL in combination with prednisone. Primary and secondary endpoints of safety, tolerability, and efficacy were determined at 12 months, and patients were followed through 36 months. There was a trend toward fewer treated acute rejections in the everolimus group than in the placebo group: everolimus 0.5 mg: 39.3%; everolimus 1.0 mg: 30.0%; everolimus 2 mg: 29.0%; placebo: 40.0% (P = not significant). Adverse events were higher in everolimus-treated patients especially at the 4-mg/day dose, but there was no difference in the incidence of thrombocytopenia or leukopenia between all groups and renal function as determined by serum creatinine, and creatinine clearance remained stable to 36 months in everolimus-treated patients. Mean cholesterol and triglycerides increased from baseline in all treatment groups, and maximum levels were seen at 6 months. In conclusion, this study demonstrates that everolimus in combination with oral cyclosporine had an acceptable safety and tolerability profile, paving the way for additional studies in this transplant indication.     

Therapeutic drug monitoring for everolimus in kidney transplantation using 12-month exposure, efficacy, and safety data

The aims of the current study were to determine whether therapeutic drug monitoring (TDM) might benefit kidney transplant recipients receiving everolimus, and to establish dosage recommendations when everolimus is used in combination with cyclosporine and corticosteroids. The analysis was based on data from 779 patients enrolled in two 12-month trials. Everolimus trough concentrations >/=3 ng/mL were associated with a reduced incidence in biopsy-proven acute rejection (BPAR) in the first month (p = 0.0001) and the first 6 months (p = 0.0001), and reduced graft loss compared with lower concentrations (4% vs. 20%, respectively). By contrast, cyclosporine in the standard concentration range had no impact on BPAR within the same timeframes. Most patients receiving everolimus 1.5 or 3 mg/d achieved trough concentrations above the therapeutic threshold of 3 ng/mL, regardless of reductions in cyclosporine dose. TDM simulation showed that just two dose adjustments would achieve median everolimus trough values >/=3 ng/mL in 95% of patients during the first 6 months. This investigation indicates that improved efficacy is likely when TDM is considered as an integral component of the immunosuppressive strategy of everolimus.     

Long-term results of kidney transplantation with cyclosporine- and everolimus-based immunosuppression

The aim of the study was to evaluate safety and efficacy of everolimus with cyclosporine (CsA) in de novo renal transplant recipients. The immunosuppressive regimen, including basiliximab, everolimus (3 mg), and low-dose CsA, was administered to 17 patients, of whom 15 were part of a multicenter randomized study that stipulated cessation of steroids at 7 days posttransplantation in 5 recipients. Five patients underwent dialysis after transplantation for delayed graft function (DGF; 29%), all of whom showed a good recovery within 3 weeks. The mean follow-up was 45.7 months (SD +/- 13). The 1-year graft survival was 100%. We observed one acute rejection episode. No patient experienced a cytomegalovirus infection. Increased cholesterol and triglyceride levels were reported in almost all patients. Severe arthralgia (n = 3) was treated by everolimus dose reduction to maintain trough levels at 3 ng/mL. We noted a high rate of switch to mycophenolate mofetil (MMF) throughout follow-up (n = 7), due to everolimus-induced side effects. However, we did not observe normalization of lipids after the switch: patients always required stain treatment, resulting in slightly lower serum cholesterol and triglycerides. Everolimus plus CsA was effective to prevent acute rejection after kidney transplantation. To manage the induced side effects of the drugs C(2) monitoring is mandatory, targeting 350 ng/mL during 1 year and 200 to 250 ng/mL thereafter. Careful reduction of everolimus trough levels to 3 ng/mL is recommended for patients with arthralgia.     

Immune cells in a heterotopic lamb-to-pig bronchial xenograft model.

We developed our porcine model to elucidate the cellular rejection mechanisms of xenografts. Bronchial segments from a donor lamb were implanted into domestic pigs. The immunosuppressive regimens consisted of no immusuppression, or of daily oral cyclosporine A (CsA) 15 mg/kg, or of everolimus, 1.5 mg/kg, or of both. Implants were serially harvested during 17 days. Epithelial damage and obliteration were graded histologically, followed by a count of CD4+, CD8+, MHC class II-expressing cells, and macrophages. Furthermore, we studied the pharmocokinetics of everolismus. Epithelial damage preceded luminal obliteration, which was eventually total, except when both drugs had been given. In xenografts, an influx of cells with CD8+ cells dominating peaked on day 9, thereafter declining, except in the combination drug group. There, the immunological reaction was delayed and blunted, with CD4+ cells dominating. More macrophages appeared in xenografts than in allografts except with the combination CsA and everolimus. A dose of 1.5 mg/kg everolimus yields adequate blood concentrations for porcine studies. In this xenograft model, chronic rejection appears to be caused by an immune response to the graft, but it is more short-lived than the response in allografts. The combination of CsA and everolimus was able to blunt the response and delay the subsequent obliteration.     

A highly selective inhibitor of IkappaB kinase, BMS-345541, augments graft survival mediated by suboptimal immunosuppression in a murine model of cardiac graft rejection

BACKGROUND: We previously demonstrated in vitro and in vivo that an IkappaB kinase (IKK) inhibitor blocks cytokine production and suppresses immune responses. These results indicate that a potent IKK inhibitor may have the potential of being a novel therapeutic agent for the prevention of graft rejection. METHODS: The IKK inhibitor BMS-345541 was tested in mice for its ability to inhibit anti-CD3-induced interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha production and T-cell proliferation in an in vivo mixed lymphocyte reaction. BMS-345541 was further tested for its ability to suppress graft rejection in a murine nonvascularized heterotopic cardiac allograft model. BMS-345541 was tested as a single agent and in combination with other immunomodulators for inhibition of T-cell proliferation and graft rejection in vivo. RESULTS: BMS-345541 suppressed, in a dose-dependent manner, the production of both IL-2 and TNF-alpha in mice stimulated with an injection of anti-CD3 antibody. Approximately 70% inhibition of both IL-2 and TNF were observed at a dose of 100 mg/kg. When BMS-345541 was administered at 100 mg/kg as a single agent, in vivo T-cell proliferation was not inhibited. However, when combined with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin (200 microg), a synergistic antiproliferative effect was observed, resulting in 77% inhibition of CD4+ T-cell proliferation. In the murine heterotopic heart transplant model, BMS-345541 did not prolong graft survival when administered at 50 mg/kg as a single agent. However, when administered with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin or cyclosporine A (15 mg/kg), graft survival was significantly increased compared with either agent alone. CONCLUSIONS: These results indicate that inhibition of IKK may serve as novel adjunctive therapy for the prevention of graft rejection.     

FK778 and FK506 combination therapy to control acute rejection after rat liver allotransplantation

BACKGROUND: In organ transplantation, several immunosuppressants are currently used to control graft rejection. Clinically, no single immunosuppressive agent can completely prevent posttransplantation immunoreaction; thus, combination therapy is usually performed. Novel agents with more powerful immunosuppressive activity and less toxicity need to be developed. METHODS: Lewis rat livers were orthotopically transplanted into Brown-Norway recipients. FK778 was administered orally from day 0 to day 6 to prevent acute rejection and from day 7 to day 13 to rescue ongoing rejection. To assess the combined effects, recipients were treated with intramuscular injection of FK506 and oral administration of FK778 from day 0 to day 6. Blood chemistry and histopathologic findings were measured to determine the patient's general condition and the graft condition. Allospecific antibodies were measured using enzyme-linked immunosorbent assays. The FK778 trough concentration was examined by using high-performance liquid chromatography. RESULTS: The acute immune response was suppressed by FK778 alone in a dose-dependent manner. The optimal FK778 dosage was determined to be 20 mg/kg per day, because adverse effects (weight loss and intestinal bleeding) occurred at 30 mg/kg per day. FK778 treatment from day 7 to day 13 rescued liver grafts from ongoing rejection. The intramuscular FK506 (0.125 mg/kg per day) injection and the oral FK778 (20 mg/kg per day) gavages suppressed acute liver graft rejection effectively and maintained better graft condition compared with monotherapy. CONCLUSIONS.: FK778 treatment effectively prevented acute rejection and rescued ongoing rejection after liver transplantation. The optimal dosage was determined to be 20 mg/kg per day. Combination therapy with FK506 was more beneficial than FK778 monotherapy.     

Combined immunosuppression with cyclosporine (neoral) and SDZ RAD in non-human primate lung transplantation: systematic pharmacokinetic-based trials to improve efficacy and tolerability

BACKGROUND: We studied the efficacy and tolerability of combined immunosuppressive therapy with cyclosporine A microemulsion (Neoral) plus the macrolide SDZ RAD 40-0 (2-hydroxyethyl) rapamycin (RAD) in a stringent cynomolgus monkey lung graft model in comparison with cyclosporine or SDZ RAD monotherapy. METHODS: Thirty-nine cynomolgus monkeys received mixed lymphocyte reaction (MLR) mismatched unilateral lung transplants. Immunosuppressants were administered orally as single daily doses. The observation period was 28 days and follow-up included serial trough blood drug concentrations measured by high performance liquid chromatography/mass spectrometry, blood analyses, chest radiographs, open lung biopsies, as well as tissue drug concentrations and graft histology at necropsy. RESULTS: Graft biopsies in monkeys treated with vehicle (n=4), Neoral (day 1-7: 150 mg/kg/day; day 8-28: 100 mg/kg/day; n=6; mean +/- SE trough level (MTL): 292+/-17 ng/ml) or SDZ RAD monotherapy (1.5 mg/kg/day; n=6; MTL: 15+/-1 ng/ml) showed severe rejection. Coadministration in two transplant monkeys of Neoral (150/100 mg/kg/day) and SDZ RAD (1.5 mg/kg/day) caused their early death. In both animals, SDZ RAD blood levels were more than 5-fold higher than under monotherapy (MTL: 82+/-18 ng/ml). Simultaneous administration (n=6) of Neoral (150/100 mg/kg/day; MTL: 217+/-16 ng/ml) and SDZ RAD (0.3 mg/kg/day; MTL: 24+/-2 ng/ml) improved graft outcome (mild rejection). Side effects included renal failure (n=2) and seizures (n=1). Three monkeys survived to day 28. In this group the MTL for cyclosporin was 143+/-13 and for RAD 38+/-3.Staggered treatment completely prevented rejection in four of six grafts. However, five of six monkeys had moderate to severe diarrhea. In a concentration-controlled trial of simultaneously administered Neoral and SDZ RAD in transplant monkeys (target SDZ RAD MTL: 20-40 ng/ml; cyclosporine MTL: 100-200 ng/ml) all six monkeys survived with improved drug tolerability and an average biopsy score of mild rejection. CONCLUSION: Combination of orally administered SDZ RAD and Neoral showed excellent immunosuppressive efficacy in a stringent lung transplant model. The drug interaction and the narrow therapeutic index of this drug combination required careful dose adjustments to optimize tolerability and efficacy.     

FTY720 treatment prolongs skin graft survival in a completely incompatible strain combination

FTY720 has shown potent immunomodulatory activity in a variety of animal organ transplant models. However, the in vivo immunosuppressive mechanism of FTY720 is still not fully understood. It has been suggested that the marked decrease in the number of peripheral blood lymphocytes during FTY720 administration could be responsible for its immunosuppressive effects. Our aims were: (1) to study the effects of FTY720 treatment on skin graft survival using a fully mismatched strain combination and (2) to evaluate lymphocyte numbers in different sites at 5 days after skin transplant. C57BL/6 mice and BALB/c mice were the donors and recipients respectively. BALB/c mice received FTY720 (1 mg/kg/d) orally for 4 consecutive days. Drug administration started 1 day before skin transplants. A small segment of tail skin was affixed on the right dorsal side of the mouse via sutures. The administration of FTY720 (4 mg/kg) prolonged skin graft survival from 12.6 +/- 2.2 days (no treatment) to 16.6 +/- 4.2 days. The histologic findings of rejection were similar for all groups. Five days after transplant, lymphocyte numbers were significantly increased in lymph nodes compared with nontransplanted or isogenic graft mice. FTY720 decreased lymphocyte numbers only in the spleen. In conclusion, FTY720 prolonged skin graft survival in a fully mismatched strain combination when administered for 4 days (day -1 to day +2) at a dose of 1 mg/kg/d. The decreased number of lymphocytes in the spleen suggests that the spleen may be a target of FTY720 activity, during the early posttransplant period.     

Everolimus inhibits glomerular endothelial cell proliferation and VEGF, but not long-term recovery in experimental thrombotic microangiopathy.

BACKGROUND: Everolimus is a potent immunosuppressant used in renal transplant therapy, but its effects on renal endothelial cell regeneration after injury are unknown. The effects of an everolimus therapy were investigated in a model of renal thrombotic microangiopathy (TMA) with specific endothelial cell (EC) injury in the rat in vivo as well as in glomerular ECs in vitro. METHODS: During the early regenerative phase (day 3) of the renal microvascular injury model in vivo, everolimus inhibited glomerular EC proliferation by up to 60% compared with vehicle-treated rats, whereas apoptosis was not different in these groups. This decreased EC proliferation was associated with an enhanced deposition of fibrin in everolimus treated animals on day 3. In cultured glomerular endothelial cells, everolimus effectively and dose dependently inhibited cellular proliferation. This anti-proliferative effect was associated with a reduced phosphorylation of the p70S6 kinase and reduction of the pro-angiogenic factor VEGF in glomeruli in vivo and in cultured podocytes in vitro. RESULTS: Despite the prolonged EC repair and in contrast to the anti-Thy1 nephritis model, everolimus therapy did not disturb the long-term repair reaction in this thrombotic microangiopathy model. CONCLUSION: Everolimus is anti-proliferative for glomerular EC in vitro and in vivo and does not seem to have detrimental long-term effects in experimental renal TMA, when only the glomerular endothelium, but not the mesangium is severely injured.