1). Background leading to the current reporting period a). We have established the techniques to produce and identify the ethyl acetate (EA) extract of Chinese herbal medicine, Tripterygium wilfordii hook F TwHF for clinical trials and obtained and identified the usefulness of large quantity of the TwHF materials for further clinical trials. We have shown the benefit of the EA extract of (TwHF), to the patients with rheumatoid arthritis by a double blind trial; b). In 2004, we have started the preclinical trail of the EA extract of TwHF on NZB/WF1 mice, the human SLE model. We have found that most animals developed varying degrees of kidney disease at 25 week of age. Therefore, the animals were treated from the age of 25 weeks with the EA extract at the doses equivalent to 1/20 or 1/10 of the LD50 or vehicle only. c). From the pharmacokinetic study on the EA extract and its active component, triptolide, in rats, we have found that most of the administered triptolide was eliminated as a single peak that processes similar HPLC pattern and bioactivities to another active diterpenoid of TwHF, tripdiolide. Therefore, we separated and purified the metabolites from urine obtained from the rats treated with triptolide. Three metabolite components were separated from the urine, two (Metabolite A and B) of the three showned suppressive activity on cell proliferation and production of IL-1, IL-2 and TNF by human peripheral mononuclear cells. Metabolite A also inhibits mitogen stimulated NFkB and AP-1 transcription by jurkat cells. These studies delineated the mechanism of the anti-inflammatory and immunossupressive action of the extract of TwHF and its active components that is relevant to inhibited transcriptional activation of NFkB, AP-1 and NFAT; d). Though, preclinical and clinical trials have been conducted, the toxicology of the EA extract or its active components has never been examined. ? ? 2). Objectives of the current work: ? a). Support the phaseII/III clinical trial on RA conducted at the Clinical ? Center of NIH; b). complete the preclinical trial of the EA extract on glomerulonephritis developed in NZB/W F1 mice. Further explore the therapeutic efficacy of triptolide and ? tripdiolide, the active components of the EA extract, on lupus nephritis in NZB/W F1 ? mice. c). examine and identify the chemical structures of the metabolites of triptolide ? d). Examine the sub-chronic and chronic toxic effects of triptolide, the major active ? component of the EA extract; and e). further elucidate the mechanism by which the EA ? extract and its active components exert their immunosuppressive and anti-inflammatory ? action. ? ? 3). Results: ? a). There have been 116 patients enrolled in the clinical trail of the EA extract of TwHF on RA conducted by clinical stuff of NIAMS. An interim analysis has been done showing significant differences between groups in disease improvement. There has been no severe adverse effect observed in treated patients. ? b). The preclinical trial of the EA extract on NZB/W F1 mice has been completed. Methods for determination of serum anti-DNA antibody titers, glomerular deposition of IgG and complement lymphocyte subpopulation in kidneys and spleens by immunohistochemical staining, lymphocyte subpopulations by flow cytometry were established. Results from the studies show that at 28 weeks of age, proteinuria (> 30mg/dL) and anti-ds-DNA antibody were found in all mice of the 3 groups. Fourteen, 16 and 15 mice in the vehicle, EAlow and EAhigh group, respectively, completed at least 4 weeks of treatment. At the end of treatment, the mean proteinuria of the EAlow and EAhigh groups was significantly less than that of the vehicle group and no different than proteinuria at the onset of treatment. Histological evidence of glomerulonephritis, glomerular deposition of IgG and complement and cellular infiltration in the interstitium and perivascular regions were significantly less severe in the EA extract- treated mice than in vehicle-treated mice. Treatment had no significant effect on the levels of anti-dsDNA antibody. The conclusion is that treatment with the EA extract significantly inhibited the progression of kidney disease in NZB/W F1 mice. A paper has been completed and submitted to !?Arthritis & Rheumatism!? for publication. ? To further examine whether the active components, triptolide and tripdiolide, also exert similar effect as the EA extract on lupus nephritis, we have conducted a preclinical trial where the NZB/W F1 mice, 17 in each group, were treated with vehicle only or triptolide or tripdiolide for 16 weeks at a dose equivalent to 1/7 of the dose that caused death in 50% of treated mice. At the end of study, the incidence of severe kidney disease evidenced by excessive proteinuria and significantly elevated BUN were 88%, 29% and 29% for the groups treated with vehicle only, triptolide and tripdiolide, respectively. During the treatment course, 12, 3 and 2 mice were dead in the groups treated with vehicle only, triptolide and tripdiolide, respectively. These primary data confirmed what we have found in our previous in vitro and in clinical studies that triptolide and tripdiolide are the two major components responsible for most of the immunosuppressive and anti-inflammatory effect of the EA extract. Histological and immunopathologic studies are currently being performed for further evaluation of the results of the trial.? c). Because of the extremely low content of the metabolites of triptolide in the urine, we have had to separate and purify the metabolites from multiple groups of rats to obtain enough of these compounds for structural identification. So far, we have purified three unknown compounds A( about 250 ?Yg ),B ( about 40 ?Yg ), and C( about 150 ?Yg ), from the urine. We have identified their molecular weight by MS. Based on the MW and HPLC pattern, metabolite A, most likely, is tripdiolide. We have sent metabolite A, B and C to Dr. Kirk Gustafson of NCI for confirmation of their chemical structure by cellular NMR. Results are pending. ? d). The toxicity studies were designed to have 4 groups of rats. Each group includes 8 rats for triptolide treatment and 4 for control. Triptolide was administered orally 5 days a week for 16 weeks at 1/7 of the dose that caused death in 50% of treated rats. The rats were sacrificed 0, 3, 9 and 12 months, respectively, after completion of the treatment course. Up to now, we have completed 3 groups for the studies. The last group that is going to be observed for 3 months is currently under treatment course.? There was no significant difference in bodyweight between the treated or the control animals at the end of treatment course. Organs from all animals of the 3 groups have been sent to Histoserv and detailed histological findings are pending.? e). In terms of the mechanism by which triptolide exerts its immunosuppressive and anti-inflammatory action, we have found that triptolide inhibited expression of reporter gene driven by varying promoters, including NFkB, AP-1, NFAT, iNOs as well as COX 2. Previous results from binding tests using multiple methods show that triptolide could not compete with Dex to bind the glucocorticoid receptor (GR) either in a whole cell or in GR-labeled particles. However, recently, we have further confirmed that triptolide strongly inhibited mitogen stimulated reporter gene expression driven by p-MMTV promoter that was greatly enhanced by dexamethasone (Dex). This inhibition was in a concentration-dependent manner suggesting that triptolide interfered with the complex of Dex and its receptor (GR) to bind to the DNA element for glucocorticoid receptor (GRE) activation.
| Tao, Xuelian; Fan, Fred; Hoffmann, Victoria et al. (2006) Therapeutic impact of the ethyl acetate extract of Tripterygium wilfordii Hook F on nephritis in NZB/W F1 mice. Arthritis Res Ther 8:R24 |
| Tao, Xuelian; Younger, Jean; Fan, Fred Z et al. (2002) Benefit of an extract of Tripterygium Wilfordii Hook F in patients with rheumatoid arthritis: a double-blind, placebo-controlled study. Arthritis Rheum 46:1735-43 |
