1). Background leading to the current reporting period, include a). we have shown the benefit of the ethyl acetate extract of the Chinese herbal medicine, Tripterygium wilfordii hook F (TwHF), to the patients with rheumatoid arthritis by a double blind trial; b). Established the techniques to produce and identify the EA extract of TwHF for clinical trials and obtained and identified the usefulness of large quantity of the TwHF materials for further clinical trials. c). Delineated the mechanism of the anti-inflammatory and immunossupressive action of the extract of TwHF and its active components that is related to inhibition of transcriptional activation of NFkB, AP-1 and NFAT ; d). previous year, we have completed the pharmacokinetic study on the new EA extract and its active component, triptolide, in rats. We have known from the studies that the bioavailability of the EA extract is about 60%. The absorbed triptolide is eliminated up to 90% through urinary tract within 24 hours after administration. Most of the administered triptolide was eliminated as a single metabolite that pocesses similar HPLS pattern and bioactivities to another active ditepenoid of TwHF, tripdiolide. 2). Objectives of the current work include: continually support the phaseII/III clinical trial on RA conducted by the clinical center of NIH, explore new clinical application of the EA extract of TwHF on systemic lupus ethethematosus (SLE), examine and identify the metabolites of triptolide and further elucidate the mechanism by which the EA extract and its active components suppress inflammation. 3). Results: a). Separated and purified the metabolites of triptolide from large amount urine obtained from the rats treated with triptolide. So far, we have purified three compounds from the urine, two (Metabolite A and B) of the three shown suppressive activity on cell proliferation and production by human peripheral mononuclear cells of IL-1, IL-2 and TNF-a. Metabolite A also inhibits mitogen stimulated NFkB and AP-1 transcription by jurkat cells. The molecular weights of the two compounds have been identified by two institutes (Dr. Kei Ma of NIAMS and Dr. Sonja Hess of NIDDK) with same results. Molecular weight for Metabolite A and B are 377. 1949 and 377.2001, respectively that is the same as tripdiolide (377.1949). However, retention time of metabolite A, but not metabolide B is the same as tripdiolide when analyzed by HPLC. Theae results suggest that metabolite A is tripdiolide while metabolite B is an isoform of tripdiolide. b). 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. The animals have been treated for three weeks so far without significant side effect. c). There are 86 patients have been enrolled in the clinical trail of the EA extract of TwHF on RA. Some impressive results have been seen with some patients. There has been no severe adverse effect observed on treated patients. An interim analysis is planned. d). From previous work, we have found that triptolide block DNA binding of AP-1, NFkB, NFAT as well as Oct-1; slightly stimulate IkB synthesis and reduce NFkB nuclear translocation. Triptolide also reduce the nuclear content of c-Jun and cellular abundance of phoaphorylated c-Jun suggesting one of the mechanism for triptolide inhibits transcription activation of AP-1, NFkB and NFAT may resulted from direct or indirect interfere with the DNA binding capacity of transcription factors. These natures of triptolide are very similar to dexamethasone (Dex). However, different from Dex, triptolide has no effect on phosphorylation of mitogen-activated protein kinases including JNK, ErK and p38 suggesting that triptolide shares similar mechanism with glucocorticoids for the anti-inflammatory and immunosuppressive effect at the cell activation process after kinase phosphorylation. Therefore, we examined whether the inhibitory effect of triptolide is mediated by glucocorticoid receptor or other nuclear receptors with negative regulatory effect such as PPAR-??. We have found that mitogen stimulated reporter gene expression driven by p-MMTV promoter was strongly enhanced by Dex that was significantly inhibited by addition of triptolide suggesting that triptolide may compete with Dex binding to glucocorticoid receptor (GR). However, results of binding tests by using multiple methods show that triptolide could not compete with Dex to bind to GR. In addition, triptolide inhibited reporter gene expression driven by p-MMTV promoter without Dex. Similarly, tritolide inhibited reporter gene expression driven by PPAR-?? promoter , however, inhibitory effect of triptolide on NFkB or AP-1 transription was not enhanced by transfection of hela cells with the PPAR-?? expression vector suggesting that the suppressive action of triptolide is not related to PPAR-???|?nFurther studies on other negative regulatory nuclear receptors are necessary. We can not exclude that inhibition of activation of multiple transcription factors may also be resulted from triptolide blocking signal transmission within the nuclei after DNA binding of transcription factors. 4). Publications: (a). Triptolide, an active component of the Chinese herbal remedy Tripterygium wilfordii Hook F, inhibits production of nitric oxide by decreasing inducible nitric oxide synthese gene transcription. 2004 Arthritis & Rheumatis 50 (9) 2995-3003 (b). Pharmacokinetics of Triptolide, the Major Active Component of the Chinese Herbal Medicine Tripterygium Wilfordii Hook F, in Rats Experimental Biology 2004 Annual Meeting. Abstract book B250, p159 5). Conclusions: Results obtained during the past year from both of clinical and preclinical studies suggest that the EA extract of TwHF is safe and applicable as a treatment strategy for autoimmune diseases. The major metabolites of triptolide are tripdiolide and its similar compounds. It is reasonable to expect that replace the EA extract of TwHF with these metabolites should provide similar therapeutic efficacy but less side effects. Therefore, it is worth of conducting preclinical trail of the metabolites on autoimmune diseases including SLE and psoriasis.
| 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 |
