John M. Fidler

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. I. Prolongation of rat cardiac and renal allograft survival by the PG27 extract and immunosuppressive synergy in combination therapy with cyclosporine.

BACKGROUND PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant, Tripterygium wilfordii. We tested PG27 in rat cardiac and renal allotransplantation, and we examined the immunosuppressive interaction with cyclosporine (CsA). METHODS Brown Norway (BN) rat heart or kidney allografts were transplanted into the abdomen of Lewis rats, which were treated by the intraperitoneal or oral route with PG27, CsA, or both. RESULTS PG27 administered intraperitoneally to Lewis recipients for 16 days at 10-30 mg/kg/day significantly increased the median survival time of BN heart allografts from 7 to 18-22 days. Oral administration was effective, with cardiac allograft survival prolonged to > 100 days with 52 days of treatment. PG27 at 20-30 mg/kg/day significantly extended the median survival time of BN kidney allograft recipients from 9 to 36.5-77 days, and 30 mg/kg/day for 52 days extended survival beyond 200 days. PG27 combined with CsA significantly enhanced heart and kidney allograft survival, even at doses of CsA ineffective when administered alone. The addition of 5 or 10 mg/kg/day PG27 reduced by 50-75% the CsA dose needed for 100% kidney allograft survival. The combination index was less than 1.0, indicating synergy of PG27 with CsA in prolonging cardiac and renal allograft survival. Furthermore, the PG27/CsA combination exerted a positive influence on renal allograft function. PG490 (triptolide, a constituent of PG27) and PG490-88 (a semisynthetic derivative of PG490) suppressed rejection of cardiac and renal allografts. CONCLUSIONS The PG27 herbal extract demonstrated immunosuppressive activity by prolonging heart and kidney allograft survival, displaying synergy in the immunosuppressive interaction with CsA, and improving renal allograft function in combination with CsA. PG490 and PG490-88 compounds were also effective.

Prevention of graft-versus-host disease by a novel immunosuppressant, PG490-88, through inhibition of alloreactive T cell expansion.

Background. PG490–88 is a water soluble, semisynthetic derivative of a novel compound PG490 (triptolide) purified from the Chinese herb Tripterygium Wilfordii Hook F. Methods. PG490–88 was administrated into recipient mice in a model (B10.D2→BALB/c) of lethal graft-versus-host disease (GVHD) to study the effects of PG490–88 on GVHD and on the various steps involved in the pathological course of GVHD. Results. Injection of PG490–88 i.p. at a dose of 0.535 mg/kg/day for the first 3 weeks after transplantation protected all the recipients from developing GVHD up to 100 days after transplantation. PG490–88 inhibited in vivo both CD4+V&bgr;3+ and CD8+V&bgr;3+ T cell (alloreactive T cells in this model) expansion in the spleen by 64.09 and 34.02%, respectively, at the time when V&bgr;3+ cell expansion was in the logarithmic phase (day 3 after transplantation). Intracellular cytokine staining without further in vitro activation demonstrated 47.42% inhibition of IL-2 production among CD4+ spleen cells in PG490–88-treated mice as compared to GVHD control on day 3 after transplantation. In contrast, CD25 (&agr; chain of interleukin-2 receptor) expression did not differ. Conclusions. PG490–88 is highly effective in prevention of murine GVHD. The immunosuppressive effect of PG490–88 is mediated by inhibition of alloreactive T cell expansion through interleukin-2 production.

Mechanisms of tolerance induced by PG490-88 in a bone marrow transplantation model.

Background. PG490‐88, a semisynthetic derivative of a novel compound PG490 (triptolide) purified from a Chinese herb (Tripterygium wilfordii Hook F), is effective in prevention of murine graft‐versus‐host disease (GVHD). Methods. PG490‐88 was administrated into recipients in a model (B10.D2 [H2d, Mls‐2b, Mls‐3b]→BALB/c [H2d, Mls‐2a, Mls‐3a]) of lethal GVHD. Tolerance was evaluated by transplantation of neonatal hearts. The mechanisms of tolerance were studied. Results. Host‐specific tolerance was established in PG490‐88‐treated BALB/c recipients. Significant numbers of host reactive V&bgr;3+ T cells (3.56±1.66% among CD4, 4.06±1.62% among CD8, P<0.0001 vs. normal BALB/c mice, P>0.05 vs. normal B10.D2 mice) were present in PG490‐88‐treated mice, suggesting that clonal deletion was not responsible for the observed tolerance. Spleen cells from PG490‐88‐treated mice could not respond to the host antigens measured by a popliteal lymph node weight gain assay. The unresponsiveness was unable to be overcome by supplementation of exogenous interleukin (IL)‐2. Tolerant V&bgr;3+ T cells obtained from PG490‐88‐treated mice proliferated normally to nonantigen‐specific T cell receptor cross‐linking. Neither antigen‐specific nor nonantigen‐specific suppressor cells were found in PG490‐88‐treated mice. The tolerant mice produced IL‐4 rather than IL‐2 and interferon (IFN)‐&ggr;. Conclusions. Host‐specific tolerance induced by PG490‐88 in a murine bone marrow transplantation model is not due to deletion of alloreactive cells. Moreover, suppressor cells are not involved in the maintenance of tolerance. Rather, PG490‐88 seems to lead to allogeneic tolerance either through the induction of a state of antigen‐specific anergy of the responding T cells or through the induction of T‐helper cell, type II (TH2) responses.

MRx102, a triptolide derivative, has potent antileukemic activity in vitro and in a murine model of AML

Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently induce apoptosis in various malignant cells by inhibiting RNA synthesis and nuclear factor-κB activity. Previously, we showed that triptolide promotes apoptosis in acute myeloid leukemia (AML) cells via the mitochondria-mediated pathway, in part, by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a triptolide derivative, currently in preclinical development. Here we show that MRx102 potently promoted apoptosis in AML cell lines, with EC50 values of 14.5±0.6 nM and 37.0±0.9 nM at 48 h for OCI-AML3 and MV4–11 cells, respectively. MRx102, at low nanomolar concentrations, also induced apoptosis in bulk, CD34+ progenitor, and more importantly, CD34+CD38− stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. In vivo, MRx102 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Collectively, we demonstrated that MRx102 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.

Immunosuppressive activity of the Chinese medicinal plant Tripterygium wilfordii. III. Suppression of graft-versus-host disease in murine allogeneic bone marrow transplantation by the PG27 extract.

Background. PG27 is an active fraction purified from an extract of a Chinese medicinal plant, Tripterygium wilfordii Hook f. We tested PG27 in murine allogeneic bone marrow transplantation (BMT) and investigated the mechanism of graft-versus-host disease (GVHD) suppression. Methods. Recipients in the C57BL/6 → BDF1 murine BMT model received oral or intraperitoneal PG27. Results. Fourteen days of PG27 given orally or intraperitoneally prevented GVHD development and produced extended disease-free survival (more than 300 days) for many animals. PG490–88, a semisynthetic derivative of PG490 (triptolide, present in PG27), was also efficacious. PG27 reduced day 7 splenic allospecific cytotoxic T lymphocyte levels by more than 99% compared with vehicle-treated mice. Compared with normals, spleens from control allogeneic BMT mice displayed significantly reduced mononuclear cell content, an increased percentage of CD8+ cells, fewer CD4+ cells, and more activated ([interleukin-2 receptor+], IL-2R+) CD8+ T cells. PG27 increased mononuclear cell recovery, and significantly reduced the day-14 percentages of CD3+ and IL-2R+ cells in allogeneic BMT mice, producing results similar to those for syngeneic BMT mice. PG27 significantly increased concanavalin A-stimulated in vitro IL-4 production by day-14 splenocytes, with a 7- to 8-fold higher level than that produced by control cells. Conclusions. PG27 treatment for only 14 days prevented GVHD induction and development and produced long-term survival. PG27 largely normalized splenic T lymphocyte subsets, reduced allospecific cytotoxic T lymphocyte activity, and increased IL-4 production capability. PG27 may suppress GVHD by the induction of anergy and a deviation away from a proinflammatory phenotype, which could be reflected in the increased potential for IL-4 production.

Immunosuppressive activity of the chinese medicinal plant Tripterygium wilfordii. II. Prolongation of hamster-to-rat cardiac xenograft survival by combination therapy with the PG27 extract and cyclosporine

BACKGROUND PG27 is an immunosuppressive fraction purified from an extract of a Chinese medicinal plant Tripterygium wilfordii, which we investigated alone and in combination with cyclosporine (CsA) in a concordant, hamster-to-rat cardiac xenotransplantation model. METHODS Golden Syrian hamster hearts were heterotopically transplanted into the abdomen of Lewis rat recipients, which were treated intraperitoneally or orally with PG27, CsA, or both. RESULTS Combination therapy with 30 mg/kg(day of PG27 and CsA at 10 mg/kg/day successfully suppressed acute hamster-to-rat cardiac xenograft rejection. Treatment with PG27 or CsA alone was ineffective. Among several effective combinations, the best regimen involved PG27 at 30 mg/kg/day and CsA at 5 mg/ kg/day from days 8 to 35 and then CsA at 5 mg/kg/day from days 36 to 100, which produced 100% survival beyond 100 days. CsA suppressed the heterospecific lymphocytotoxic antibody response and inhibited IgG but not IgM xenoantibody production (which led to xenograft rejection), whereas PG27 alone did not prevent antibody production. The PG27/CsA combination blocked the lymphocytotoxic antibody response and IgG and IgM xenoantibody production induced by cardiac xenotransplantation. CONCLUSIONS PG27 combined with CsA substantially prolonged hamster-to-rat cardiac xenograft survival, as well as completely inhibiting xenoantibody production.

Pharmacologic, toxicologic, and marrow transplantation studies in dogs given succinyl acetone

A novel immunosuppressant, succinyl acetone (4,6-dioxoheptanoic acid), was studied in dogs. Results with bolus intravenous injections at doses ranging from 50 to 1600 mg/kg showed dose-dependent α and β half-lives, ranging from 30 to 80 min and 7 to 27 hr, respectively. Results suggested that continuous i.v. infusion was necessary to maintain constant plasma levels. Four dogs were given 9.2 Gy total-body irradiation and autologous marrow transplants along with continuous i.v. infusion of succinyl acetone at 50, 100, 200, or 400 mg/kg/day for 21 days, and all four had rapid, sustained hematopoietic engraftment. However, two of the four dogs receiving 200 and 400 mg succinyl acetone/kg/day, respectively, developed bilateral hind-limb ataxia, with histologically confirmed cerebellar lesions in the dog given the higher dose, thus establishing a potential doselimiting neurotoxicity. Prevention of graft-versus-host disease was studied in recipients of allogeneic marrow. Dogs were given 9.2 Gy TBI, followed by hematopoietic grafts from unrelated DLA-nonidentical or DLA-haploidentical littermate dogs. Succinyl acetone was given as continuous infusion for 21 days after transplant at doses of 100–300 mg/kg/day. Starting succinyl acetone on the day of marrow infusion in four dogs failed to prevent rapid onset of acute GVHD, and dogs survived no longer than controls. Starting succinyl acetone 3 days before transplant delayed the onset of acute GVHD and prolonged survival significantly compared with that of dogs not given postgrafting immunosuppression (P=0.008); survival was comparable to that in previously reported dogs given either methotrexate or cyclosporine as postgrafting immunosuppression (P=0.88 and 0.99, respectively). Seven of the sixteen allogeneic recipients developed evidence of neurotoxicity during succinyl-acetone infusion. Neurological dysfunctions were manifested by hind-limb ataxia and posterior paresis. In conclusion, succinyl acetone significantly delayed the onset of GVHD and prolonged survival of DLA-nonidentical marrow graft recipients but did not induce graft-host tolerance and was associated with dose-limiting neurotoxicity.

Suppression of graft-versus-host disease by succinyl acetone in a rat allogeneic bone marrow transplantation model.

The efficacy of succinyl acetone (SA, 4,6-dioxoheptanoic acid) was explored in the allogeneic rat bone marrow transplant model of graft-vs.-host disease. Lethally irradiated Wistar Furth rats receiving Fischer 344 allogeneic bone marrow and spleen cells developed severe GVHD, resulting in mortality at 25–45 days posttransplant. Treatment for 14 days with 250 mg/kg/ day of SA by Alzet osmotic pumps implanted subcuta-neously 3 days before cell transfer prevented GVHD and produced long-term survivors that were allogeneic he-matopoietic chimeras. SA doses below 250 mg/kg/day and treatment for less than 14 days were less efficacious. Initiation of SA therapy could be effectively delayed up to 7 days after BMT. Pharmacokinetic studies with i.v. bolus administration in normal CD rats revealed a plasma mean residence time that increased with dose and a systemic clearance that decreased with dose. Three dose-dependent half lives were apparent (ca. 7–18 min, 0.8–3 hr, and 12 hr). The s.c. bioavailability was ca. 82%. Relatively constant plasma SA levels were obtained with s.c. Alzet osmotic pumps, indicating no change in clearance with continuous exposure. Allogeneic BMT exerted no major influence upon SA clearance. These studies show that SA is a robust therapeutic agent that suppressed GVHD in the allogeneic rat BMT model under a variety of circumstances.

Differential development of murine immunocompetence to transplantation antigens.

SUMMARY The graft-versus-host (GVH) reaction, which is abrogated by pretreatment of the inoculated cells with goat antibrain-associated &thgr; (BA&thgr;) serum plus complement, was used to determine the presence of competent T cells. The spleen: thymus GVH ratio of competence changes from 2.2 at day 8 after birth to 16 in the adult. This increase is not caused by a change in the cells of the thymus, the reactivity of which remains constant from birth through adulthood. However, the reactivity in the spleen changes, rising rapidly from birth to 8 days thereafter and at a decreased rate to the adult level at 35 days of age. Competent T cells are present in the thymus at birth, while they appear first in significant numbers in the spleen at 2-3 days of age. The implications of this differential development of GVH immunocompetence in terms of cell migration patterns and the development of immunocompetence in various organs are discussed.