Immunosuppressive effects of FTY720 alone or in combination with cyclosporine and/or sirolimus.

BACKGROUND: We examined the ability of FTY720, a novel immunosuppressant that prolongs the survival of allografts in experimental animal models, to potentiate the immunosuppressive effects of cyclosporine (CsA) and/or sirolimus (SRL) in vitro and in vivo. METHODS: FTY720 alone (10-5000 ng/ml) or in combination with other drugs was added to human peripheral blood lymphocytes (PBLs) undergoing stimulation in vitro with phytohemagglutinin (PHA) or OKT3 monoclonal antibody. The combination index (CI) values were calculated to evaluate the nature of the interactions between FTY720 and CsA and/or SRL: CI values <1 reflect synergistic, CI=1, additive, and CI>1, antagonistic interactions. In addition, Wistar Furth (RT1u) rat recipients of Buffalo (RT1b) heart allografts were treated with FTY720 alone or in combination with other agents. FTY720 alone was also tested to block small bowel or liver allograft rejection in rats. RESULTS: FTY720 alone produced only modest inhibition of the proliferation of human PBL stimulated with PHA or OKT3 monoclonal antibody. In combination with CsA or SRL, however, FTY720 produced synergistic effects, namely, CI values of 0.58 and 0.36, respectively. A 14-day course of FTY720 (0.05-8.0 mg/kg/day) by oral gavage prolonged heart allograft survival in dose-dependent fashion. Although a 14-day oral course of CsA (1.0 mg/kg/day) alone was ineffective (mean survival time=7.0+/-0.7 vs. 6.4+/-0.6 days in treated vs. untreated hosts), treatment with a combination of 1.0 mg/kg/day CsA and 0.1 mg/kg/day FTY720 extended allograft survival to 62.4+/-15.6 days (P<0.001; CI=0.15). Similarly, a 14-day oral course of 0.08 mg(kg/day SRL alone was ineffective (6.8+/-0.6 days; NS), but the combination of SRL with 0.5 mg/kg/day FTY720 extended the mean survival time to 34.4+/-8.8 days (CI=0.28). The CsA/SRL (0.5/0.08 mg/kg/day) combination acted synergistically with FTY720 (0.1 mg/kg/day) to prolong heart survivals to >60 days (CI=0.18). CONCLUSIONS: FTY720 potentiates the immunosuppressive effects of CsA and/or SRL both in vitro (by inhibiting of T-cell proliferative response) and in vivo (by inhibiting allograft rejection).

15 AU81, a prostacyclin analog, potentiates immunosuppression and mitigates renal injury due to cyclosporine.

15 AU81, a synthetic prostacyclin analog, inhibits human mixed lymphocyte culture responses in a dose-dependent manner at concentrations within a range [0.1 to 10.0 micrograms/ml] similar to that of cyclosporine, methylprednisolone, antilymphocyte serum, and prostaglandin E1 and E2 analogs. Using the median-effect analysis to dissect immunosuppressive interactions, 15 AU81 and cyclosporine were shown to produce synergistic effects on human peripheral blood lymphocyte activation upon phytohemagglutinin or anti-CD3 monoclonal antibody stimulation. In vivo, 15 AU81 potentiated the effect of cyclosporine to prolong the survival of rabbit renal allografts using concentrations of each agent that were individually ineffective. Furthermore, addition of 15 AU81 mitigated functional damage of rabbit kidneys due to high-dose cyclosporine therapy. These observations suggest that the addition of 15 AU81 may broaden the therapeutic window of cyclosporine by potentiating immunosuppression and mitigating nephrotoxic effects.

The synergistic interactions in vitro and in vivo of brequinar sodium with cyclosporine or rapamycin alone and in triple combination.

The rigorous median-effect analysis was used to assess the interactions between cyclosporine and drugs that inhibit nucleotide synthesis pathways. Using in vitro proliferation assays wherein human lymphocytes were triggered by phytohemagglutin, anti-CD3 monoclonal antibody, or mixed lymphocyte reactions, CsA was shown to display additive interactions with 6-mercaptopurine (6-MP), mizorbine (MZB), and mycophenolic acid (MPA), and a synergistic interaction with brequinar (BQR). In the in vitro assays, BQR contributed a further synergistic effect to the double-drug combination CsA/rapamycin (RAPA). Of the four inhibitors of nucleotide synthesis pathways, only BQR noncompetitively inhibited IL-2-stimulated proliferation of the CTLL-2 cell line. Using the in vivo assay of heterotopic Buffalo (BUF, RT-1b) cardiac allografts in Wistar-Furth (WFu, RT-1u) hosts, oral administration of BQR displayed about 100% bioavailability--which, like the bolus intravenous (i.v.) mode, was eight-fold more effective than continuous i.v. infusions. Furthermore median-effect analysis of serial amounts of orally administered BQR demonstrated that it contributes synergistically to the immunosuppressive effects of intravenously delivered CsA/RAPA (0.5/0.01 mg/kg/day). The degree of synergism was proportionate to the extent of the immunosuppression. These findings document the potency of the CsA/RAPA/BQR triple-drug combination and suggest that the synergistic effects may permit dose reductions of each component, thereby mitigating toxicities resulting from the large amounts of individual agents necessary to achieve allo-unresponsiveness.

Prospects for synergistic immunosuppressive drug therapy in the coming decade.

The major clinical strategy during the past three decades has been to discover a single drug that achieves immunosuppression with minimal toxic side effects. Regimens to optimize CyA immunosuppressive therapy have been limited by marked interindividual variations in drug pharmacokinetics and pharmacodynamics that cause simple drug administration routines to produce variable clinical outcomes. The approach presented herein seeks to employ multiple immunosuppressive agents that act synergistically to inhibit alloactivation. Using the rigorous median effect analysis, synergistic interactions have been discerned both using in vitro systems, as well as in vivo transplant situations. CyA has a profoundly synergistic interaction with RAPA, and a moderately synergistic effect with QCA. The next decade presents important opportunities to determine whether these immunologic effects are beclouded by synergistic drug toxicities. Studies of this type may allow us to achieve a uniformly effective, yet well-tolerated, immunosuppressive regimens in the future.

Synergistic interactions of cyclosporine and rapamycin to inhibit immune performances of normal human peripheral blood lymphocytes in vitro.

Rapamycin, an actinomycete macrolide lactone that inhibits cytokine-induced immunoactivation, and cyclosporine, an endecapeptide that prevents transcription of lymphokine messenger RNA, display mutually synergistic interactions in vitro and in vivo. Using the rigorous median-effect analysis to dissect the nature of immunosuppressive drug interactions, rapamycin significantly augmented the inhibitory effects of cyclosporine and/or dexamethasone upon human peripheral blood lymphocyte activation by phytohemagglutinin, anti-CD3 monoclonal antibody, and mixed lymphocyte reaction. Furthermore, the addition of rapamycin potentiated the activity of cyclosporine to reduce cytotoxic cell generation and precursor frequency during in vitro alloactivation, using cell-mediated lympholysis and limiting dilution analyses, respectively. Similarly, cyclosporine potentiated the inhibitory effects of rapamycin upon proliferation of IL-2 (CTLL-2) and IL-6 (MH60.BSF-2) lymphokine-dependent cell lines. Lineweaver-Burk plots of the Michaelis-Menton equation suggested rapamycin inhibits IL-2 signal transduction in competitive, and IL-6 signal transduction in noncompetitive fashion, suggesting distinctive components of the various cytokine-receptor mechanisms. In vivo the cyclosporine/rapamycin combination exerted synergistic immunosuppression of rejection reactions in rats toward heterotopic cardiac allografts, using concentrations at which drugs were individually ineffective. These observations suggest that cyclosporine and rapamycin may be combined at significantly reduced doses to achieve unprecedented levels of immunosuppressive efficacy.