Walter Schuler

Sdz Rad, A New Rapamycin Derivative: Pharmacological Properties In Vitro and In Vivo

BACKGROUND This report describes the preclinical pharmacological profile of the new rapamycin analog, SDZ RAD, i.e., 40-O-(2-hydroxyethyl)-rapamycin. METHODS The pharmacological effects of SDZ RAD were assessed in a variety of in vitro and in vivo models, which included an autoimmune disease model as well as kidney and heart allotransplantation models using different rat strain combinations. RESULTS SDZ RAD has a mode of action that is different from that of cyclosporine or FK506. In contrast to the latter, SDZ RAD inhibits growth factor-driven cell proliferation in general, as demonstrated for the in vitro cell proliferation of a lymphoid cell line and of vascular smooth muscle cells. SDZ RAD is immunosuppressive in vitro as demonstrated by the inhibition of mouse and human mixed lymphocyte reactions and the inhibition of antigen-driven proliferation of human T-cell clones. The concentrations needed to achieve 50% inhibition in all of these assays fall into the subnanomolar range. SDZ RAD is effective in the in vivo models when given by the oral route in doses ranging between 1 mg/kg/day and 5 mg/kg/day. When compared with rapamycin, the in vitro activity of SDZ RAD is generally about two to three times lower; however, when administered orally, SDZ RAD is at least as active in vivo as rapamycin. CONCLUSIONS In conclusion, SDZ RAD is a new, orally active rapamycin-derivative that is immunosuppressive and that efficiently prevents graft rejection in rat models of allotransplantation. SDZ RAD has therefore been selected for development for use in combination with cyclosporine A to prevent acute and chronic rejection after solid organ allotransplantation.

SDZ RAD, a new rapamycin derivative: synergism with cyclosporine.

BACKGROUND SDZ RAD is a new rapamycin analog with potent immunosuppressive activity. Compounds of the rapamycin class differ in their mode of action from cyclosporine, thus providing a rationale for potential synergism of these two potent immunosuppressants. METHODS The two-way mouse mixed lymphocyte reaction (BALB/c-CBA strain combination) was applied. Orthotopic kidney and heterotopic heart allografting was performed in the stringent DA-to-Lewis rat strain combination, with administration of compounds orally as microemulsion preconcentrate (i.e., Neoral in the case of cyclosporine). RESULTS Isobologram analysis of checkerboard titrations of SDZ RAD and cyclosporine in two-way mouse mixed lymphocyte reactions indicates a synergistic interaction in vitro. In vivo, the minimal effective dose of microemulsion cyclosporine giving long-term graft survival was 5.0 mg/kg/day; for SDZ RAD, the minimal effective dose was 5.0 mg/kg/day in kidney transplantation and >5.0 mg/kg/day in heart transplantation. Long-term allograft survival was noted for combinations of microemulsion cyclosporine administered at 1.0 or 2.0 mg/kg/day and SDZ RAD given at between 0.5 and 2.0 mg/kg/day. The index of synergy in different combinations ranged between 0.3 and 0.7. CONCLUSIONS SDZ RAD and cyclosporine show synergism in immunosuppression, both in vitro and in vitro. They form a promising synergistic drug combination in allotransplantation.

Immunosuppressive tor kinase inhibitor everolimus (RAD) suppresses growth of cells derived from posttransplant lymphoproliferative disorder at allograft-protecting doses

Background. Posttransplant lymphoproliferative disorders (PTLDs) represent a life-threatening complication of standard immunosuppressive therapy. The impact of novel, rapamycin-related immunosuppressive drugs on the pathogenesis of PTLDs remains undefined. Methods. We tested the effect of everolimus (RAD, Novartis Pharma AG, Basel, Switzerland) on human PTLD-derived cells using in vitro assays and an in vivo severe combined immunodeficiency disease mouse xenotransplant model. Results. Everolimus profoundly inhibited the proliferation, cell-cycle progression, and survival of the PTLD-1 cell line established from a pulmonary PTLD. Equally profound inhibition of PTLD-1 growth was achieved in vivo at well-tolerated everolimus doses of 0.5 to 5 mg/kg per day. Five mg/kg per day of everolimus, given once per day, inhibited PTLD-1 tumor volume gain by more than 10-fold in treated mice compared with untreated mice. Because the subsequent pharmacokinetic analysis indicated rapid everolimus absorption, distribution, and clearance in mice (with a half-life of 3 to 6 hr and maximum drug blood concentration reached after 0.5 to 1 hr), treatment was changed to a twice-daily regimen. Everolimus given twice daily at 0.5 mg/kg per dose inhibited tumor-volume gain by more than 60-fold and at 0.25 mg/kg per dose by more than 10-fold. Similar everolimus doses were required to prevent graft rejection in a mouse heart allotransplantation model; the highest dose tested (1.5 mg/kg twice daily) resulted in long-term graft survival in all mice that underwent transplantation. Conclusions. Everolimus displays a potent inhibitory effect on PTLD-derived cells in vitro and in vivo in a dose range leading to prevention of allograft rejection and may prove effective in both the prevention and treatment of PTLDs in transplant patients.

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Protein Kinase C Inhibitor Sotrastaurin Selectively Inhibits the Growth of CD79 Mutant Diffuse Large B-Cell Lymphomas

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G₁-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.

Monotherapy with the novel human anti-CD154 monoclonal antibody ABI793 in rhesus monkey renal transplantation model.

Background. This study assesses the safety and efficacy of the novel human anti-human CD154 monoclonal antibody ABI793 in rhesus monkeys. Methods. Outbred rhesus monkeys were used for renal transplantation from major histocompatibility complex-mismatched donors. Seven recipients were treated with ABI793, and six untreated recipients were used as controls. Graft function was monitored by urine output, serum creatinine, and renal biopsy. Phenotypic analysis of peripheral blood lymphocytes and mixed lymphocyte reaction were performed before transplantation and periodically after transplantation. Anti-donor major histocompatibility complex class I antibody levels were measured at the time of sacrifice. Results. Monkeys in the treated group demonstrated prolonged graft survival compared with controls. One monkey was sacrificed because of a urine leak on postoperative day 13. Three monkeys were sacrificed because of acute rejection (days 44, 149, and 158). Two monkeys were sacrificed because of chronic active rejection (days 154 and 221). One monkey was sacrificed on day 139 without rejection to observe the effects of ABI793 in the absence of rejection. There were no obvious clinical side effects of ABI793, but microscopic thromboembolic changes were observed in two monkeys. Lymphocyte subsets remained unaltered in all monkeys. Mixed lymphocyte reaction showed nonspecific suppression 6 weeks after transplantation. The monkeys with chronic active rejection showed relatively strong alloantibody responses. Conclusions. ABI793 induces prolonged renal allograft survival in rhesus monkeys. Nevertheless, thromboembolic complications may occur and chronic allograft nephropathy may develop after anti-CD154 treatment is discontinued.

Oral Efficacy Of The Macrolide Immunosuppressant Sdz Rad And Of Cyclosporine Microemulsion In Cynomolgus Monkey Kidney Allotransplantation

BACKGROUND 40-O-(2-Hydroxyethyl)-rapamycin (SDZ RAD) is a novel, potent, macrolide immunosuppressant. Its efficacy in rodent transplantation models provided the rationale for us to evaluate the compound in a more relevant, large animal transplantation model. METHODS Life-supporting kidney allotransplantation was performed in cynomolgus monkeys: rejection was inferred from a rise in serum creatinine or urea and was subsequently confirmed by histopathology. This model was validated with the microemulsion formulation of cyclosporine (i.e., Neoral). Two studies with a microemulsion formulation of SDZ RAD were performed. First, in a dose-finding study, the SDZ RAD dose was reduced in a stepwise fashion until rejection occurred, either with SDZ RAD as monotherapy, or in combination with a fixed, suboptimal dose of cyclosporine. Second, an efficacy study was performed in which two fixed SDZ RAD doses (0.75 and 1.50 mg/kg/ day) were evaluated in monotherapy and compared with the same doses of rapamycin (sirolimus). All immunosuppressants were administered once daily by gastric gavage. RESULTS Untreated control animals rejected their grafts between 4 and 8 days after transplantation. Cyclosporine (initially at 150 mg/kg/day, reduced to 100 mg/kg/day 2 weeks after transplantation) yielded long-term (>100 days) rejection-free allograft survival in four of five animals. A 10 mg/kg/day dose of cyclosporine led to rejection between 10 and 27 days after transplantation and was considered suboptimal. In the dose-finding study with SDZ RAD monotherapy, rejection occurred in most of the cases (four of six animals) when a dose level of 0.63 mg/kg/day had been reached. Combined with suboptimal cyclosporine, this threshold SDZ RAD dose was about 2-fold lower. In the efficacy study, median graft survival with histologically proven rejection was 32 days (range 8-91 days, n=6) for 0.75 mg(kg/day SDZ RAD and 59 days (range 28-85 days, n=6) for 1.50 mg/kg/day SDZ RAD. For sirolimus, median graft survival was 43 days (range 5-103 days, n=7) for the 0.75 mg/kg/day dose and 56 days (range 8-103 days, n=8) for the 1.50 mg/kg/day dose. There was no statistically significant difference in efficacy between SDZ RAD and sirolimus. CONCLUSION SDZ RAD, in the absence of any other immunosuppressant and at doses that do not show any overt toxicity, considerably prolongs rejection-free survival of cynomolgus monkeys after life-supporting kidney allotransplantation.

2,6-Naphthyridines as potent and selective inhibitors of the novel protein kinase C isozymes.

The present study describes a novel series of ATP-competitive PKC inhibitors based on the 2,6-naphthyridine template. Example compounds potently inhibit the novel Protein Kinase C (PKC) isotypes δ, ε, η, θ (in particular PKCε/η, and display a 10-100-fold selectivity over the classical PKC isotypes. The prototype compound 11 was found to inhibit PKCθ-dependent pathways in vitro and in vivo. In vitro, a-CD3/a-CD28-induced lymphocyte proliferation could be effectively blocked in 10% rat whole blood. In mice, 11 dose-dependently inhibited Staphylococcus aureus enterotoxin B-triggered IL-2 serum levels after oral dosing.

Novel ROCK inhibitors for the treatment of pulmonary arterial hypertension.

A novel class of selective inhibitors of ROCK1 and ROCK2 has been identified by structural based drug design. PK/PD experiments using a set of highly selective Rho kinase inhibitors suggest that systemic Rho kinase inhibition is linked to a reversible reduction in lymphocyte counts. These results led to the consideration of topical delivery of these molecules, and to the identification of a lead molecule 7 which shows promising PK and PD in a murine model of pulmonary hypertension after intra-tracheal dosing.

Cleavage of the cyclohexyl-subunit of rapamycin results in loss of immunosuppressive activity

The cyclohexyl-subunit of rapamycin was cleaved by a sequence involving a Baeyer-Villiger reaction and acid hydrolysis of the resulting lactone-acetal as key steps. Binding of this new rapamycin derivative to FKBP12 was only slightly reduced by this modification, whereas the loss of antiproliferative and immunosuppressive activity was dramatic. These findings indicate that part of the cyclohexyl-subunit of rapamycin could belong to its effector domain.