tacrolimus and triptolide

Fujisawa is committed to improving the outcomes of transplant patients worldwide. Research and development programs are underway for a new modified release dosage form of tacrolimus (MR-4), a new analog of leflunomide (FK 778), and several novel compounds (PG 490-88, AGI 1096) in collaboration with other companies. These programs are targeted to address many of the unmet medical needs in transplantation including (1) improving compliance, (2) reducing chronic rejection, and (3) improving long-term safety by reducing infectious and cardiovascular risk.

Topics: Alkynes; Animals; Delayed-Action Preparations; Diterpenes; Epoxy Compounds; Graft Rejection; Humans; Immunosuppressive Agents; Isoxazoles; Nitriles; Phenanthrenes; Probucol; Tacrolimus

Induction of tolerance and minimizing the toxicity of immunosuppression are two fundamental goals in vascularized composite allotransplantation. Accumulating data indicate that triptolide is an agent that may have the capacity to suppress inflammation and immunologic rejection.. A heterotopic hindlimb allotransplantation model from Brown Norway to Lewis rats was established and treated with different doses of tacrolimus combined with or without triptolide. Mean survival time of the transplants was monitored, and histopathologic examination of the skin was performed. The level of inflammatory cytokine interleukin-1β, interleukin-6, and tumor necrosis factor-á in peripheral blood was assayed. The percentage of T lymphocytes and its subsets was measured using flow cytometry. The level of recipient peripheral chimerism and the apoptosis of donor bone marrow cells were evaluated. The apoptotic related genes bcl-2 and Bax were detected by real-time polymerase chain reaction.. The authors' results showed that triptolide not only reduces the dose of tacrolimus required for immunosuppression, but also decreased drug side effects in terms of weight gain and diarrhea. Triptolide had an obvious effect on proinflammatory cytokine expression and T-lymphocyte proliferation in the peripheral blood. Interestingly, triptolide could increase the mixed chimerism level of recipients, possibly by inhibiting the apoptosis of transplanted bone marrow cells by means of regulation of the apoptotic genes bcl-2 and Bax.. Triptolide reduces the dose of tacrolimus required for immunosuppression by attenuating inflammation and by T-cell suppression. Furthermore, triptolide increases the chimerism level, which might contribute to acceptance of the allografts.

Topics: Animals; Apoptosis; Biomarkers; Chimerism; Cytokines; Diterpenes; Dose-Response Relationship, Drug; Drug Therapy, Combination; Epoxy Compounds; Graft Rejection; Graft Survival; Hindlimb; Immune Tolerance; Immunosuppressive Agents; Male; Phenanthrenes; Random Allocation; Rats; Rats, Inbred Lew; T-Lymphocytes; Tacrolimus; Vascularized Composite Allotransplantation

Membranous nephropathy is a major cause of nephrotic syndrome in adults where podocyte injuries were found to mediate the development of proteinuria. Triptolide, a major active component of Tripterygium wilfordii Hook F, has potent immunosuppressive, anti-inflammatory and antiproteinuric effects. To study its antiproteinuric properties, we established an experimental rat model of passive Heymann nephritis and a C5b-9 injury model of podocytes in vitro. Treatment or pretreatment with triptolide markedly reduced established proteinuria as well as the titer of circulating rat anti-rabbit IgG antibodies in these nephritic rats, accompanied by a reduction in glomerular C5b-9 deposits. Expression of desmin, a marker of podocyte injury, diminished after triptolide treatment, whereas quantitative analysis of mean foot process width showed that effacement of foot processes was substantially reversed. In in vitro studies we found that triptolide deactivated NADPH oxidase, suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase, and restored RhoA signaling activity. Triptolide did not interfere with the formation of C5b-9 on the membrane of podocytes. Thus, triptolide reduces established heavy proteinuria and podocyte injuries in rats with passive Heymann nephritis, and protects podocytes from C5b-9-mediated injury.

Topics: Administration, Oral; Animals; Cell Line; Complement Membrane Attack Complex; Cytoprotection; Desmin; Disease Models, Animal; Diterpenes; Epoxy Compounds; Female; Glomerulonephritis, Membranous; Heymann Nephritis Antigenic Complex; Immunoglobulin G; Immunosuppressive Agents; Mice; NADPH Oxidases; p38 Mitogen-Activated Protein Kinases; Phenanthrenes; Podocytes; Proteinuria; Rabbits; Rats; Rats, Sprague-Dawley; Rats, Wistar; Reactive Oxygen Species; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; Signal Transduction; Tacrolimus; Time Factors

To explore the effects of FK506 on the inhibition of cell proliferation and the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their products PGE subset2 and NO in TNF-alpha-stimulated human rheumatoid arthritis synovial fibroblasts (RASF) treated with triptolide (TP), and to study the mechanisms involved when combining FK506 and TP in RA therapy.. RASF used in the experiments were obtained from the synovial tissue of patients with RA before being cultured. RASF were pretreated with FK506 (10 approximately 1000 nM) for 2 hours before being stimulated with TNF-alpha (20 ng/ml) in the presence or absence of TP (10 ng/ml) . RASF proliferation was determined by [3 supersetH]-TdR incorporation. Production of PGE subset2 and NO in culture supernatants of RASF was detected by competitive ELISA and enzymatic reduction of nitrate, respectively. Expressions of COX-2 and iNOS mRNA in RASF were analyzed by semi-quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western-blot and a cellular enzyme immunoassay. NFkappaB activity in whole-cell extract of treated RASF was also measured using an ELISA-based method.. Neither FK506 nor TP at a lower concentration (10 ng/ml) affected TNF-alpha-induced COX-2 and iNOS expressions or PGE subset2 and NO productions in synovial cells. Combined treatment of FK506 and a lower concentration of TP (10 ng/ml) reduced both COX-2 and iNOS mRNA and protein expression, and correspondingly reduced PGE subset2 and NO produced by synovial fibroblasts. This effect was highly correlated with FK506 concentration (10 approximately 1000 nM). NFkappaB activity in TNF-alpha-stimulated synovial cells was suppressed more profoundly by FK506 plus TP (10 ng/ml) than by TP (10 ng/ml) alone. However, no change was observed regarding the inhibition of synovial cell proliferation after combined treatment of FK506 and TP.. FK506 enhanced TP-mediated down-regulation of COX-2 and iNOS as well as their products PGE subset2 and NO in human TNF-alpha-stimulated RASF by more profoundly suppressing the activity of NFkappaB.

Topics: Arthritis, Rheumatoid; Base Sequence; Cell Line; Cyclooxygenase 2; Dinoprostone; Diterpenes; DNA Primers; Down-Regulation; Drug Synergism; Epoxy Compounds; Fibroblasts; Humans; Immunosuppressive Agents; Membrane Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenanthrenes; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; Synovial Membrane; Tacrolimus; Tumor Necrosis Factor-alpha

Recent studies have shown that triptolide inhibits T cell activation through mechanisms different from those of cyclosporine A and tacrolimus and we postulated that triptolide might have a synergistic effect with tacrolimus to enhance immunosuppression. Using a F344 donor-to-Lewis recipient rat combination, we investigated the immunosuppressive effects of triptolide alone or in combination with tacrolimus on the survival of cardiac allografts. Recipients were treated with placebo, triptolide, tacrolimus, and triptolide in combination with tacrolimus at different doses. The median survival time (MST) was 8 days for placebo; 9.5, 11, 14 and 19 days for triptolide monotherapy at doses of 0.04, 0.08, 0.16, and 0.32 mg/kg/day, respectively, and 11, 13.5, and 52 days for tacrolimus monotherapy at doses of 0.025, 0.05, and 0.1 mg/kg/day, respectively. Tacrolimus 0.025 mg/kg/day combined with triptolide 0.08 and 0.16 mg/kg/day prolonged the MST to 17.5 and 20 days, respectively; while tacrolimus 0.05 mg/kg/day combined with triptolide 0.04, 0.08, and 0.16 mg/kg/day prolonged the MST to 21, 23, and 23 days, respectively. These results suggest that triptolide is a moderately effective immunosuppressive agent. Triptolide combined with a subtherapeutic dose of tacrolimus produced a synergistic effect in prolonging rat cardiac allograft survival.

Topics: Animals; Diterpenes; Drug Synergism; Epoxy Compounds; Graft Survival; Heart Transplantation; Immunosuppressive Agents; Male; Phenanthrenes; Rats; Rats, Inbred Lew; Tacrolimus; Transplantation, Homologous

To explore the effects of FK506 on the inhibition by triptolide (TP) of cell proliferation and expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and their inducing products PGE2, NO in human rheumatoid arthritis synovial fibroblasts (RASF), and to study the mechanisms of combination of FK506 and TP in RA therapy, RASF used in the experiments were obtained from synovial tissue of patients with RA and were cultured. RASF were pretreated with FK506(10-1000 nmol/L)for 2 h, then the cells were stimulated with TNF alpha(20 microg/L) in the presence or absence of TP (10 microg/L). The RASF proliferation was determined by [(3)H]-TdR incorporation, and the productions of PGE2 and NO in culture supernatants of RASF were detected with competitive ELISA and enzyme reduction of nitrate. Expression of COX-2 and iNOS mRNA in RASF were analyzed by semi quantitative RT-PCR. Expressions of COX-2 and iNOS protein were estimated by Western blot method and cellular enzyme immunoassay in synovial fibroblasts. NF-kappa B activity in whole-cell extract of RASF was also measured by an ELISA-based method. Results showed that neither FK506 nor TP at lower concentration (10 microg/L) alone affected TNF alpha-induced COX-2, iNOS expression and production of PGE2, NO in synovial cells. Combined treatment of FK506 and a lower concentration of TP (10 microg/L) down-regulated COX-2 and iNOS mRNA and protein expression, and their inducing products PGE2 and NO of synovial fibroblasts. This effect was positively correlated with FK506 concentrations (10-1000 nmol/L). NF-kappa B activity in TNF alpha-stimulated synovial cells was suppressed more profoundly by FK506 plus TP (10 microg/L) treatment than those with TP (10 microg/L) alone. No change was observed in inhibition of proliferation of synovial cells after combined treatment of FK506 and TP. In conclusion, FK506 enhanced TP-mediated down-regulation of COX-2, iNOS and their inducing products PGE2, NO in human RASF by suppressing the activity of NF-kappa B.

Topics: Arthritis, Rheumatoid; Blotting, Western; Cell Division; Cells, Cultured; Cyclooxygenase 2; Dinoprost; Diterpenes; Enzyme-Linked Immunosorbent Assay; Epoxy Compounds; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Immunosuppressive Agents; Isoenzymes; Membrane Proteins; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenanthrenes; Prostaglandin-Endoperoxide Synthases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Synovial Membrane; Tacrolimus; Tumor Necrosis Factor-alpha

Previous studies have shown that triptolide possesses potent immunosuppressive and anti-inflammatory properties. Increasing recognition of the importance of the proximal tubular epithelial cells (PTEC) in renal disease and renal transplantation raises the question of whether triptolide suppresses the pro-inflammatory activity of PTEC.. Cultured human PTEC were exposed to tumor necrosis factor-alpha (TNF-alpha) and immunosuppressant (triptolide or CsA or FK506) for 24 hours, followed by RT-PCR, ELISA, flow cytometry and Western blotting analysis for complement C3, CD40, B7h expression.. TNF-alpha up-regulated C3, CD40 and B7h production by PTEC. This up-regulation was inhibited by all three immunosuppressants with different intensity. Firstly, triptolide (4 to 8 ng/mL), CsA (4000 to 6000 ng/mL) and FK506 (2000 ng/mL) inhibited up-regulation of C3 mRNA, but CsA and FK506 had less of an effect than triptolide. Secondly, triptolide (4 to 8 ng/mL) completely inhibited C3 expression at both mRNA and protein levels. In contrast, CsA and FK506 had only slight effects on C3 expression at the protein level. Thirdly, triptolide (4 to 8 ng/mL), CsA (500 to 2500 ng/mL) and FK506 (1250 ng/mL) inhibited up-regulation of CD40 and B7h mRNA, the effect on B7h and CD40 mRNA expression by CsA and FK506 being greater than that on C3 mRNA expression.. Triptolide effectively inhibited up-regulation of C3, CD40 and B7h on PTEC. Triptolide was more effective than CsA and FK506 at inhibiting C3 expression. This suggests that triptolide, at non-cytotoxic concentrations, has the potential to reduce the inflammatory and immunostimulatory properties of PTEC, in addition to any of the previously reported actions on T cell or B cell function.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD; Antineoplastic Agents; CD40 Antigens; Complement C3; Cyclosporine; Diterpenes; Epoxy Compounds; Gene Expression; Humans; Immunosuppressive Agents; Inducible T-Cell Co-Stimulator Ligand; Kidney Tubules, Proximal; Phenanthrenes; Proteins; RNA, Messenger; Tacrolimus; Tumor Necrosis Factor-alpha

The Chinese traditional herb Tripterygium wilfordii Hook. F (TWHF) has been reported to be effective in the treatment of a variety of autoimmune disorders. The major active component of this herb is triptolide and most of the efficacy of this herb immunosuppression is attributed to triptolide. FK506 is also a potent immunosuppressive agent and is currently being used clinically. The present studies compare the effectiveness of triptolide and FK506 to suppress certain human T cell functions. Specifically human T cell proliferation, IL-2 and IFNgamma were compared. The results show that, overall, triptolide is more effective at inhibiting T cell proliferation and IFNgamma production than FK506 and the two compounds inhibit IL-2 production in an equivalent manner.

Topics: Cells, Cultured; Diterpenes; Epoxy Compounds; Humans; Immunosuppressive Agents; Interferon-gamma; Lymphocyte Activation; Medicine, Chinese Traditional; Phenanthrenes; Phytotherapy; T-Lymphocytes; Tacrolimus; Tetradecanoylphorbol Acetate