THE CHINESE ANTI-INFLAMMATORY AND IMMUNOSUPPRESSIVE HERBAL REMEDY TRIPTERYGIUM WILFORDII HOOK F

COMPLEMENTARY AND ALTERNATIVE THERAPIES FOR RHEUMATIC DISEASES I1

0889-857X/OO $8.00

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THE CHINESE ANTIINFLAMMATORY AND IMMUNOSUPPRESSIVE HERBAL REMEDY TRIPTERYGIUM WILFORDII HOOK F Xuelian Tao, MD, and Peter E. Lipsky, MD

Tripterygium wilfordii Hook F (TwHF) is a member of Celastraceae family of perennial vine-like plants that grow in the wild in southern China along the Yongzhi River and in Fujian Province and Taiwan. TwHF and three additional members of the Celastraceae family, T. hypoglaucum Hutch, T. regeli Spraque et Takeda (TrST) and T. forresti Dicls (TfD), have been used as remedies to treat arthritis, muscle and skeletal injury, and skin diseases for several centuries. Because of the associated toxicity, the major use of the agents in China outside of traditional medicine has been as agricultural insecticides until recently. Despite their broad use in Chinese traditional medicine, they have only been used in Chinese allopathic medicine for the past 30 years.49In the early 1960s, TwHF was successfully used to treat patients with inflammatory lesions of leprosy, and patients with rheumatoid arthritis were treated in three county hospitals in Fujian Province.22, 62 About the same time, T. hypoglaucum was reported to be effective in the treatment of rheumatoid arthritis (RA) patients in Yunan Province, where this plant grows abundantly.ll

From the Harold C. Simmons Arthritis Center, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

RHEUMATIC DISEASE CLINICS OF NORTH AMERICA VOLUME 26 * NUMBER 1 * FEBRUARY 2000

29

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TAO & LIPSKY

Originally, a decoction of TwHF, or hot water extract, which is one of the most common preparations in traditional Chinese medicine, was employed to treat patients. Results from these uncontrolled trials reported excellent therapeutic effects but noted a large number of adverse effects.22,62 This stimulated pharmaceutical development with the goal of maintaining efficacy but diminishing toxicity of the TwHF preparations. As a result of this activity, two new preparations of TwHF were developed in China in the 1970s. One was an ethyl acetate (EA) extract, and the second was a chloroform-methanol extract known as T2. Both of these extracts of TwHF claimed to have better therapeutic benefit with reduced adverse effects.l0C'lo Both of these preparations have become commercially available in China and have been used extensively. The availability of these preparations has made it possible to carry out numerous clinical trials of TwHF in a variety of autoimmune and inflammatory diseases.'" In parallel with clinical trials, there has been extensive pharmacologic, toxicologic, chemical, and pharmaceutic analysis of TwHF. Most investigators have agreed that the diterpenoid components with epoxide structures are responsible for the therapeutic effect of TwHF, although many other components of this plant have also been shown to have 26, 72, lo8 Recertain anti-inflammatory or immunosuppressive acti~ities.'~, sults from studies on the effects of TwHF on in vitro and in vivo inflammatory and immune responses suggest that the anti-inflammatory and immunosuppressive effects are related to inhibition of the production of a series of pro-inflammatory cytokines, including interleukin-2 (IL-2) and interferon-? (IFN-y),12,56, 58, 71, 73 and other inflammatory media70, 75, 77 More tors, including prostaglandin E, (PGE,) and nitric oxide.50* detailed analysis of the impact of TwHF on the signaling pathways leading to the production of IL-2 and cyclooxygenase-2 (COX-2) further documented that suppression of the upregulation of expression of proinflammatory and immunologically active molecules is one of the major mechanisms by which TwHF exerts its immunosuppressive and antiinflammatory effects.73,75, 77 CHEMICAL STUDIES

As uncontrolled trials suggested that TwHF was effective in the treatment of a variety of autoimmune and inflammatory diseases, efforts to isolate and characterize the active components were undertaken. Clinical trials have shown that extracts of the roots of the plant are therapeutically active and appear to induce fewer side effects. Therefore, much of the medicinal chemistry has focused on the roots. More than 70 components have been isolated from the roots of TwHF, including

TRlPTERYGlUM WILFORDII HOOK F

31

diterpenoids, triterpenoids, sesquiterpenoids, alkaloids, p-sitosterol, dulcitol, and glycoside~.~~, 69, 79, lol, Similar components have also been isolated from the leaves and stem of TwHF, suggesting that these components might also be a useful source of pharmacologically active component^.^^, loo Besides diterpenoids,l7-I9,87, lo2,lo9 some of the components isolated from TwHF, including triterpenoidss7,lo* and alkal0ids,4~also showed anti-inflammatory and immunosuppressive effects in vitro and in vivo (Fig. 1). Clinical and animal studies employing fractions of the EA extract and T2 indicated that the diterpenoid components accounted for

Diterpenoid components

Triptolide

Triterpenoid components

Triptolide-related Diterpenoid Components Isolated from TwHF Chemical name

Structure formula

Tripdiolide

C2-OH

Triptonide

C14=0

16-hydroxytriptolide

C16-OH

Tripchlorolide

c12-CI C13-OH

Triptriolode

a-C1Z-OH C13-OH

Epitriptriolide

C12-OH C13-OH

Al kyloid

/OAc I

Celastrol (Tripterine)

j

'

OH

'b

\

Wilfortrine

Figure 1. Immunosuppressive anti-inflammatory components isolated from Tripferygium wilfordii Hook F (TwHF).

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TAQ & LIPSKY

most of the immunosuppressive and anti-inflammatory activities.", lo8 Only small quantities of two active diterpenoids, triptolide and tripdiolide, were found in the EA extract and T2 (Table 1).Quantitative analysis by high-performance liquid chromatography documented that triptolide and tripdiolide could fully account for the in vitro immunosuppressive and anti-inflammatory activities of the EA extract, whereas the combination of triptolide and tripdiolide and perhaps some additional unknown diterpenoid component explained the activity of T2". 75 (Table 2). In agreement with this conclusion, Gu et a P Oanalyzed the components accounting for the therapeutic activity of an aqueous extract of TwHF and reported that triptolide and tripdiolide accounted for the capacity of this material to suppress collagen-induced arthritis in rodents and autoimmune disease in MLR-Zpr/lpr mice. A series of sesquisterpenoids was isolated from TwHF and reported to be inhibitory of herpes simplex virus type 1 replication in v i t r ~ . ~ ~ Recently, another diterpene lactone, tripterifordin, was also isolated from the roots of TwHF and reported to exert antireplicative activity on human immunodeficiency virus in H9 lymphocytes.6There is no information available on whether these components also possess immunosuppressive or anti-inflammatory effects. Active diterpenoids isolated from TwHF all have three epoxide moieties (see Fig. 1).Efforts have been made to reduce the toxic effects of the triepoxides of TwHF by structural modification of triptolide, the immunosuppressive and anti-inflammatory diterpenoid prototype. Nine compounds have been produced by modifying the 12,13-epoxide group.51,94 Among these derivatives, tripchlorolide and tripbromolide were reported to exert inhibitory activities on antibody formation in vivo in mice and on in vitro proliferation of murine lymphocytes. The inhibitory activities of the two compounds were comparable to that of t r i ~ t o l i d ewhereas ,~~ the remaining products of the modification showed

Table 1. DITERPENOID CONTENT OF THE ETHYLACETATE EXTRKT AND T2 Diterpenoid Content of Tripterygium wilfordii Hook F Preparation (pg/mg of extract) Preparations

Triptolide

Tripdiolide

Triptonide

Triptophenolide

Ethyl acetate T2

1.08 2 0.07

0.31 2 0.01 0.68 2 0.02

0.79 t 0.03 0.03 k 0.01

4.09 & 0.16 0.04 2 0.01

0.36

* 0.04

From Tao XL, Ca JJ, Lipsky PE: The identity of immunosuppressive components of the ethyl acetate extract and chloroform methanol extract (TJ of Tripfevygium wilfordii Hook f. J Pharmacol Exp Ther 272:1305-1312, 1995; with permission.

W W

946.67 i 141.89 0.61 ? 0.11 0.86 i 0.13 2.46 i 0.21 830.11 ? 102.23

T2

Triptolide Tripdiolide Triptonde Triptophenolide

102.23

+ 51.93

?

i 0.21

* 0.13

5 61.10 ? 0.11

i 72.34

EC5Ot (ng/mL)

806.67 983.33 0.61 0.86 2.46 830.11 497.67

Preparation

Ethyl acetate extract T2 Triptolide Tripdiolide Triptonide Triptophenolide Ethyl acetate extract 0.34 i 0.05

0.54 f 0.06

0.87 i 0.07 0.35 5 0.02

Triptolide

Triptonide

0.39 i 0.02 0.03 i 0.01

0.64 i 0.10

0.64 ? 0.02 0.03 i 0.01

0.15 i 0.02

0.25 i 0.02 0.67 ? 0.09

Tripdiolide

0.04 i 0.01

2.04 i 0.01

3.30 i 0.13 0.04 i 0.13

Triptophenolide

Amount of Component in Extract at EC5Ot (ng/mL)

*The inhibitory effect of the ethyl acetate extract, T2, or the individual diterpenoids on interleukin-2 production and DNA synthesis by phytohemagglutinin (PHA)stimulated T cells was examined. Data represent the mean 2 SE of three separate experiments. SThe concentration of the extract causing 50% inhibition. Aduptedfrom Tao XL, Cai JJ,Lipsky PE: The identity of immunosuppressive components of the ethyl acetate extract and chloroform methanol extract (TJ of Tripterygium wilfordii Hook f. J Pharmacol Exp Ther 272:1305-1312, 1995; with permission.

Interleukin-2 production

Proliferation

T-cell Function

Table 2. COMPARISON OF THE CONCENTRATIONS OF DITERPENOIDS IN THE ETHYL ACETATE EXTRACT AND T2 WITH THEIR IMMUNOSUPPRESSIVE ACTIVITY

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TAO & LIPSKY

low inhibitory effects on the immune functions tested. Because tripchlorolide and tripbromolide could be easily converted to triptolide under alkaline conditions and triptolide could be converted to tripchlorolide in acetone-hydrogen chloride, it is presumed that tripchlorolide and tripbromolide exert immunosuppressive effects in vivo by conversion to t r i p t ~ l i d e .Other ~ ~ compounds with stable modifications of the 12,13epoxide group were inactive, indicating the importance of this component of triptolide for its immunosuppressive and anti-inflammatory activity. The hydroxyl group at 14-C of triptolide (14-OH) might also be important for activity, because replacements of the 14-OH of triptolide or tripchlorolide significantly reduced immunosuppressive activitie~.~~ The 12,13-epoxide and the 14-OH of the diterpenoid structure appear to be critical for immunosuppressive and anti-inflammatory activity.

CLINICAL STUDIES Efficacy During the past three decades, thousands of patients with a variety of autoimmune and inflammatory diseases have been reported to be successfully treated with various preparations of TwHF in China (Table 3). The diseases reported to be successfully treated include RAZ5,60, 66, 74, 95 systemic lupus erythematosus (SLE),27, 47, 59, 89 ankylosing spondyliti~,~, 21 p~oriasis'~, and psoriatic arthriti~,'~, 64* Behcets' disease,86, Io7 HenochSchonlein p u r p ~ r a ,52, ~~ 55 , reactive status of leprosy,14,32, 84 chronic nephrotic syndrome,28,41 IgA nephr0pathy,3~~ 81 adult and childhood nephritis?O. Io3 chronic lymphocytic thyroiditis,8 and a variety of skin diseases such as diffuse eczema, contact dermatitis, polymorphous light eruption, erythema multiforme,68 and diffuse neur~dermatitis.~~ More recently, clinical trials of T2 in the treatment of systemic ~cleroderma~~ and polymyo~itis~~ and in the prevention of graft rejection after kidney transplantation' have been reported. It is important to emphasize that although efficacy has been claimed in these various conditions, few of these claims have been substantiated by randomized controlled trials. The extracts of TwHF have been most frequently used in the treatment of RA. It should be noted that most of this clinical information was derived from uncontrolled clinical trials or retrospective reports. Nevertheless, the reports include observations from multiple medical centers and involve thousands of patients. In some of the reports, patients have been followed for more than 10 years.96One prospective, randomized, double-blind, crossover study of T2 in the treatment of RA has been reported.74In this trial, 70 patients were randomly divided into two groups and treated with either T2 at 20 mg three times daily (group

TRIPTERYGIUM WILFORDII HOOK F

35

A) or placebo (group B). The first treatment course was 12 weeks, which was followed by a 4-week crossover treatment period. In comparison with patients in group B, patients in group A showed significant improvement in all parameters of clinical disease activity and laboratory abnormalities. Approximately 90% of patients in group A and group B demonstrated significant improvement after the first and second treatment courses, respectively. Peripheral blood mononuclear cells obtained from patients treated with TwHF produced significantly less IgM-rheumatoid factor than those of placebo-treated patients.76These results suggest that the extract of TwHF exerted potent immunosuppressive and anti-inflammatory effects in RA patients. A single trial of triptolide, one of the active components of the extracts of TwHF, in the treatment of RA has been In this trial, two groups of 15 patients were treated with either the EA extract (120 mg/d) of TwHF or triptolide (0.5 mg/d to 0.75 mg/d) for 30 days. Significant improvement was observed in both groups after the treatment; however, adverse reactions developed more often and were more severe in patients treated with triptolide than in those treated with the EA extract. The authors concluded that triptolide might be one of the major components that accounts for both the therapeutic benefits and side effects of the EA extract. They also suggested that besides triptolide, other components might play a role in the therapeutic effect of the EA extract of TwHF, because the amount of triptolide contained in the beneficial dose of the EA extract was only half of that employed in the triptolide-treated patients. Therapeutic benefits of TwHF preparations in the treatment of a total number of 249 patients with SLE have been reported from five open trials.27,45, 47, 59, 89 Treatment with TwHF improved clinical manifestations of SLE, including fatigue, arthralgia, fever, skin rash, lymphadenopathy, hepatomegaly, and abnormalities in laboratory findings such as proteinuria, thrombocytopenia, antinuclear antibodies, and renal function. In one trial of 103 SLE patients, 12 patients were able to withdraw from glucocorticoids after TwHF treatment and 42 patients significantly reduced their dose of prednisone. Average daily doses of prednisone before and after the TwHF treatment course were 19.5 mg and 6.5 mg, re~pectively.~~ One report described the results of treatment in 92 SLE patients with a combination of a decoction of TwHF and prednisone for a mean of 19.4 months. After treatment, 18% of the patients experienced remission, 56% improved, and 26% were not affected. Of the patients who had repeat renal biopsies, 3 of 10 were improved, whereas 7 of 10 were not. These results suggest that TwHF might be an alternative or additional drug for those SLE patients in whom steroid therapy is insufficiently effective or contraindicated. A preliminary study on the therapeutic effect of T2 in comparison

Psoriatic arthritis Chronic follicular thyroiditis

Ankylosing spondylitis Systemic sclerosis Polymyositis Behcet‘s disease

Systemic lupus erythematosus

Rheumatic disease Rheumatoid arthritis Tincture60 ~295 ~274‘ Ethyl acetate extractt“ T225 Decoction59 Ethyl a ~ e t a t e ~ ” ~ ~ ~245 Decoction” Decoctionz7 Tin~ture~,~~ T67t7 Decoctiona DecoctionIw T2ffi T2a ~ 2 3

Tripterygium wilfordii Hook F Preparation

31 144 70 270 34 103 18 15 86 92 13 40 23 47 13 18 12

Number of Patients

43 79 38

29 55 7 32 15 54 44 33 55 25 23

Significantly Improved

Table 3. THERAPEUTIC EFFECT OF VARIOUS PREPARATIONS OF TRlfTERYGlUM WILFORDII HOOK F

64 38 93 57 71 37 50 66 13 51 38 36-76$ 39 21 54 89 100

Improved

Therapeutic Effect (%)

18 0 8

7 7 0 11 14 9 6 34 32 24 39

Ineffective

u

W

5 62 27 36 284 51 50 71 8 26 66 30 20 119 87

Decoctions1+ T2% Ethyl acetate extracts1 T241.103 T2" ~21~3 T242.55 T2It

44 303 93

T288 Triptolide ointmenpt De~oction~~t T288 T288 T2@ T288 Decocti~n~~t T234.81 T288 39 42 31 15 60 10 10

88 41 20 0 97 37 11 8 16 80

10 8

27 8 13 9

7 6 2 2

15 13 0

Compared with control group, shorter normalization time of graft function and longer graft survival time were seen in T2-treated patients

80 82

34 50 56 76

56 83 90 82

5

44 67

*Double-blind controlled trial. tControlled studies. SThirty-eight of 40 patients who completed a 1-year treatment with T2 exhibited the most improvement in skin sclerosis (76%) and the least improvement in extremity ulcers (36%). Thirty-seven of the patients who were treated with colchicine also obtained the most improvement in skin sclerosis (35%) and the least improvement in Raynaud's phenomenon (16%).

Henoch-Schonleinpurpura nephritis Transplantation Kidney graft rejection

Childhood nephropathy Adult nephritis

Discoid lupus Kidney disease Idiopathic IgA nephropathy

Skin disease Pustular psoriasis Diffuse neurodermatitis Allergic and contact dermatitis Erythema multiforme Diffuse eczema SweeCs syndrome Reactive state of leprosy

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TAO & LIPSKY

to colchicine in the treatment of systemic sclerosis has been In this study, groups of 40 patients were treated with either T2 at 40 to 60 mg/d or colchicine at 1 mg/d for 5 days per week for a total course of 1 year. In comparison with colchicine, patients treated with T2 experienced significant improvement in skin thickness, Raynaud’s phenomenon, arthritis, grip strength, and dysphagia as well as improvements in the erythrocyte sedimentation rate (ESR) and serum IgG. Various types of nephritis have also been claimed to be successfully treated with TwHF. In a randomized open trial, 52 patients with biopsydocumented idiopathic IgA nephropathy were randomly divided into two groups and treated with T2 at 20 mg/d to 60 mg/d or prednisone at 20 mg/ d to 30 mg/d, respectively, for at least 6 Effectiveness of treatment was defined as disappearance of edema and normalization of blood pressure, renal function, and routine urine analysis. After treatment, the percentages of patients fulfilling these criteria were 76.9% and 38.4% for the T2 group and the prednisone group, respectively ( P < 0.05). The first clinical trial of the extract of TwHF in cadavaric kidney transplantation was reported recent1y.l In this randomized controlled trial, 87 patients were treated with T2 in combination with prednisone and cyclosporine. The control group included 85 patients who were treated with a combination of azathioprine with cyclosporine and prednisone. In comparison to the control group, significant therapeutic benefits indicated by shorter time to normalization of graft function and higher graft survival rates were observed in TwHF-treated patients. Adverse Effects

The major side effects of the different preparations of TwHF have been reported to be similar (Table 4). These include gastrointestinal (GI) tract disturbances, especially diarrhea, skin rash and pigmentation, decrease in blood components, and malfunction of the male and female reproductive system.34,40, 52, 74, 95 Skin rash and pigmentation occurred most often but rarely necessitated cessation of treatment. GI tract adverse events usually developed earlier and were dose dependent. A small percentage of patients (OYO to 6%) developed leukopenia in different trials employing different batches of the same preparations or various preparations of TwHF. Most of these adverse reactions ceased either spontaneously or after dose adjustment. The side effect-related withdrawal rate for T2 in a 3-month trial was 2.9%.74GI tract disturbances were the most frequent adverse reactions causing early withdrawals in patients treated with the EA extract of TwHF.~~ Attempting to reduce the adverse effects of TwHF on the GI tract, sustained release tablets (TW-SR) of the EA extract have been prepared.

TRIPTERYGILIM WILFORDII HOOK F

39

Table 4. ADVERSE EFFECTS RELATED TO T2 TREATMENT IN 870 PATIENTS* Clinical Manifestation

Dryness of mouth Loss of appetite Nausea or vomiting Abdominal pain Diarrhea Leukopenia Thrombocytopenia Skin rash Skin pigmentation Amenorrhea

Number of Cases

Incidence (%)

87 63 21 13 12 44 5 71 80 23

9.1 (0-18.7) 6.6 (5.7-20.8) 2.2 (0.7-7.6) 1.3 (2.0-7.4) 1.2 (0-8.5) 4.6 (0-6.3) 0.5 (0-1.3) 7.4 (5.4-55.5) 8.4 (7.1-13.2) 2.4 (1.8-8.7)

*Numbers are from 214 patients with rheumatoid arthritis, 53 with Henoch-Shonlein purpura, 40 with systemic sclerosis, 26 with IgA with nephropathy, and 537 with other diseases. Data from references 52, 67, 74, 81, 88, and 95.

A prospective, randomized, single-blind trial to compare the therapeutic effects of TW-SR with those of the standard EA extract in the treatment of RA was carried out.80The results of this trial indicated that patients receiving either treatment regimen obtained comparable therapeutic benefit, whereas the incidence of side effects in TW-SR-treated patients was less than in the standard EA extract-treated patients.39 Yu96described 101 RA patients treated with T2 for 3 to 13 years, with a mean treatment duration of 5.8 years. Most side effects of longterm TwHF treatment could be resolved by dose adjustment, except for amenorrhea. Development and reversibility of amenorrhea was correlated to the age of the patient and the accumulated quantity of the drug taken. Amenorrhea was reversed in most patients younger than 40 years old who experienced amenorrhea for less than 2 years. In contrast, perimenopausal patients tended to develop irreversible amenorrhea within several months of the initiation of TwHF treatment.30Symptomatic treatment of this side effect with supplemental low-dose estrogen appeared to be helpful, and as a result, treatment with the extracts of 96 TwHF could be continued.30, The incidence of side effects of T2 were higher in patients with intrinsic renal disease. One report of 106 patients with various types of chronic nephritis and nephropathy who were treated with 1 mg/kg/d to 1.5 mg/kg/d of T2 for at least 1month noted incidences of leukocytopenia, anorexia, and general edema of 57%, 4%, and 9%, respectively, which were higher than those developing in the patients with normal renal function who received the same treatment. The results suggest that special care, including appropriate dosing adjustment, should be given to patients with abnormal kidney function who receive TwHF treatment .83

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MECHANISTIC STUDIES Effects on the Immune System and Inflammatory Mediators

Cellular Immune Responses

Inhibition of cellular immunity was initially documented by the findings that treatment of rodents with extracts of TwHF suppressed 113 as well as experimental allergic and delayed type hyper~ensitivity~~, contact d e r m a t i t i ~ Later, . ~ ~ results from different sources reported that T2, the EA extract, and a decoction of TwHF inhibited in vitro proliferation and IL-2 production by T cells in response to antigen and mitogen s t i m u l a t i ~ n58,. ~71,~73,~ 98 Similar to the impact on IL-2 production, in vitro production of IFN-7 was also inhibited by T2.". 73 In agreement with the results of in vitro studies, IL-2 production by spleen cells of mice treated with triptolide was lower than that from control animals? Inhibition of IL-2 production was correlated with significant downregulation of IL-2 mRNA expression. Furthermore, it was found that T2 inhibited mitogeninduced CAT reporter gene expression driven by the IL-2 promoter, clearly indicating that T2 inhibited IL-2 gene transcription. The responsiveness of T cells to exogenous IL-2 and IL-2 receptor expression was less sensitive to the inhibitory effect of T2 or the EA 72 This result was confirmed by the demonstration of the lack e~tract.~', of inhibition by T2 on mitogen-induced IL-2R mRNA expression." In other studies, inhibition of IL-2R expression was reported, although this contention is controversial and likely to be explained by different experimental methodology.'* The inhibitory effects of T2 on the signaling pathway leading to IL-2 production have been investigated in Results from these studies revealed that T-cell activation initiated by T-cell receptor (TCR)/ CD3 occupancy was inhibited by T2. In contrast, T-cell activation stimulated with anti-CD28 plus a phorbol ester or phorbol ester plus ionomycin was more resistant to the inhibitory effect. Of note, T2 did not inhibit early signaling events, including tyrosine phosphorylation of multiple proteins, generation of diacylglycerol, and inositol phosphates, and the translocation of protein kinase C. The inhibitory action of T2 could be localized to a step in the T-cell activation cascade resulting specifically from T-cell receptor occupancy and occurring after initial second messenger generation but before transcription of the IL-2 gene." Humoral Responses

Treatment in vivo with T2,"' the EA extract,37the decoction of TwHF,l13 and triptolideTS6 or celastrol (tripterine)lo2as well as with an

TRIPTERYGIUM WILFORDll HOOK F

41

alkyloid component, isowilfortrine,48has been reported to inhibit formation of antibody against sheep red blood cells (SRBC) in mice. Similarly, treatment of RA patients with T2 for 12 weeks significantly reduced the production of IgM and IgM-rheumatoid factor by nonstimulated or pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells separated from these patients.76In vitro, T2 inhibited proliferation and production of immunoglobulins by pokeweed mitogen (PWM)stimulated peripheral blood mononuclear cells93or purified human B cells in response to stimulation with Staphylococcus u u ~ e u s indicating ,~~ that T2 directly affected the function of B cells as potently as that of T cells. Proinflammatory Media tors

It has been reported that T2 inhibited in vitro PGE, production by PWM-stimulated human peripheral blood mononuclear cells.70In addition, one of the triterpenoid components of TwHF, celastrol (tripterine), has been reported to inhibit PGE, production by rat synovial cells in response to the calcium ionophore A23Xg7 More recently, the impact of T2, the EA extract of TwHF, and triptolide on in vitro production of PGE, and mRNA expression of COX2 by a variety of human cells was examined.75Results from these studies indicated that the extracts of TwHF or triptolide exerted an inhibitory effect on lipopolysaccharide-induced PGE, production comparable to the effect of dexamethasone. T2 and triptolide suppressed PGE, production and inhibited COX-2 mRNA expression by RA synovial fibroblasts, human monocytes, and human foreskin fibroblasts. Further studies revealed that components of TwHF inhibited PGE, production only by the cells that increased COX-2 mRNA in response to the stimuli, indicating that inhibition of production of this inflammatory mediator could be explained by a suppressive effect on the upregulation of COX-2. The direct anti-inflammatory effects of TwHF demonstrated in vitro were also observed in the air pouch model of carrageenan-stimulated acute inflammation in rats.77 Significantly lower volumes of the air pouch exudate, lower white blood cell counts with lower percentages of neutrophils, and lower concentrations of inflammatory mediators, including PGE, tumor necrosis factor-a, and nitrite in the exudate, were found in the animals treated orally with the EA extract. Correspondingly, spontaneous ex vivo production of PGE, tumor necrosis factor-a, and nitrite by exudate cells was significantly reduced in TwHF-treated animals. PGE, content and COX-2 mRNA expression in the air pouch lining tissue of the TwHF-treated rats was significantly less than that in the vehicle-treated animals. In contrast, PGE, content and mRNA expression of either COX-1 or COX-2 in the kidney or the stomach were not

42

TAO & LIPSKY

significantly different between TwHF- and vehicle-treated animals. These results are consistent with the conclusion that the EA extract inhibited PGE, production by downregulating expression of the COX-2 gene at the inflammatory site without interfering with the COX-1-regulated PGE, production in the noninflammatory organs. Effects on Animal Models

Extracts of TwHF inhibited various inflammatory and autoimmune diseases in animal models (Table 5). A decoction of TwHF has been reported to be inhibitory of type I1 collagen-induced arthritis (CIA) in both rats and mice.17,l8 CIA in the rat was significantly suppressed even when the TwHF treatment was initiated 3 weeks after immunization with collagen I1 (CII). More recently, a chloroform extract of TwHF was reported to prevent the development of CIA and reduce the production of anticollagen antibody in CII-immunized mice.3 TwHF treatment also inhibited proliferative responses and the production of IL-2 and IFN-y by ex vivo CII-rechallenged lymph node cells from these animals. Neither the clinical course of arthritis nor the immune responses to CII were changed when the TwHF treatment was initiated 3 weeks after the

Table 5. ANTI-INFLAMMATORY AND IMMUNOSUPPRESSIVE EFFECTS OF EXTRACTS OF TRlPTERYGlUM WILFORDII HOOK F IN ANIMAL MODELS Model

Animal

Allergic encephalomyelitis Bovine serum albumin-induced serum sickness Masugi nephritis

Rat Rat Mouse Mouse Rat Mouse Mouse Guinea pig Rabbit Rabbit

Adriamycin-induced nephrotic syndrome Nephrotoxic nephritis Allergic and contact dermatitis Syngeneic heart transplantation Heart transplantation Intestinal transplantation Cornea allograft Skin graft Chronic graft-versus-host disease

Rat Rabbit Guinea pig Mouse Rat Pig Rabbit Mouse Mouse

Adjuvant arthritis Collagen arthritis Collagen arthritis MRL-lpr /lpr autoimmunity

Extract

Ethyl a ~ e t a t e ~ ~ , " ~ T2111 Chloroform3 Decoctionls Decoction17 Decoction20 T2Io4 ~27 TZU Decoction24 Water-ethano12* Polyglycosidesz4 Ethyl acetatez4 Ethyl acetate44 ~224 Decoction57 Decoctionlo6 T238. Polyglycosidego 46.116 ~ 2 4 3

~ 2 3 3

Chloroform2

TRIPTERYGIUM WlLFORDll HOOK F

43

onset of arthritis, suggesting that the extract of TwHF could exert its suppressive effect on the inductive phase of the d i ~ e a s eThese .~ findings are somewhat different from those reported by Gu et a1,18 who found that CIA was suppressed even when treatment with TwHF was initiated after disease onset. This conflict might be related to differences between the aqueous and chloroform extracts employed in these two studies. Another chronic arthritis model, adjuvant arthritis in rats, was also effectively treated with the EA extract.37, Arthritis spontaneously developing in HLA-B27 transgenic rats was also significantly improved by oral treatment with the EA extract.78Correlating with suppression of joint inflammation, decreases were found in mitogen-stimulated proliferation and IL-2 production by spleen cells separated from the animals, indicating the immunosuppressive effect of the EA extract in HLA-B27 transgenic rats. Suppressive effects of extracts of TwHF have also been found in the autoimmune disease developed in MRL-Zprtlpr micezo,lo*; rejection of skin, cornea, and cardiac grafts in rodents33,43, 90, Io6; small bowel allografts in pigs38, 46, 116,. and chronic graft-versus-host disease in DBA/ 2 mice.2 Experimental allergic encephalomyelitisin the guinea pig7bovine serum albumin-induced acute serum sickness,1o6and antimouse antiseruminduced nephritis in rats" were also inhibited by T2. These findings indicate that extracts of TwHF exerted potent immunosuppressive effects in a number of animal models of immunologically mediated tissue damage. A direct anti-inflammatory effect of extracts of TwHF has also been indicated by the findings that treatment with the extract of TwHF improved adriamycin-induced nephr~pathy,'~ joint swelling induced by agar or f~rmaldehyde,~~, 37, lo8, lo9 and carrageenan-induced air pouch inflammation." Effects on Pituitary-Adrenal Axis

Based on the clinical and experimental studies, it is possible that TwHF exerted a steroid-like anti-inflammatory and immunosuppressive action. Studies on the influence of treatment with TwHF on the pituitaryadrenal axis revealed that urinary 17-hydroxycorticosteroid in the RA patients returned to normal after a month of treatment with the EA extract.l15The content of vitamin C and cholesterol in the adrenal gland was significantly decreased in the patients treated with the EA extract, implying increased synthesis of glucocorticoid.117Furthermore, it has been found that the EA extract of TwHF accelerated compensatory hypertrophy of the adrenal gland after unilateral adrenalectomy in This suggested that treatment with the EA extract could stimulate

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the pituitary gland and consequently increase the synthesis of glucocorticoid by the adrenal gland.l14No evidence of a steroid-like effect on the pituitary-adrenal axis was noted in these studies. Effects on the Reproductive System

It has been noted that treatment with either the decoction, the EA extract, or T2 for a long period of time (mostly 3-24 months) caused irregular menstruation or amenorrhea in women and gynecomastia in men.15,lh, 96 In the patients who developed amenorrhea after treatment with T2 or the decoction of TwHF, the serum levels of follicular stimulating hormone and lactogenic hormone increased, whereas estradiol de~reased,'~, l6 which presents a profile similar to that of postmenopausal changes. Semen examination was performed in nine male patients treated with T2 for a mean of 4 years and 8 months. Five of the nine men demonstrated no sperm, whereas three had few sperm with weak activity.96Examination of the effect of T2 and tripchlorolide on lactogenic cells of the pituitary glands in mice found a change similar to that seen in castrated rats.8O These results indicate that TwHF-associated amenorrhea and azoospermia resulted from a direct effect of TwHF on the reproductive system rather than through the pituitary gland. Screening the immunosuppressive and antifertility fractions of TwHF in experimental animals revealed that the fractions exerting antifertility effects could not be separated from those processing the immunosuppressive effects.'08

TOXICOLOGIC STUDIES

Treatment-related death was uncommon and mostly occurred in those patients who either received an overdose of the extract of TwHF, self-prepared decoctions, or tinctures of the TwHF plant. Direct causes of death included myocardial damage, renal failure, and low-volume shock secondary to severe intestinal tract disturbances.l17 Toxicity Testing of the Ethyl Acetate Extract

The dose of the EA extract that caused death in 50% of mice as a result of a single administration (LD50) was 608 to 858 mg/kg depending on the source of the material and the season of harvest.*' The most striking changes resulting from this administration were seen in the lymphatic tissue and were characterized by necrosis and loss of

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lymphocytes. The spermatogenetic cells of testes were decreased in number and degenerated with increased giant cell formation.'05Subacute toxicity testing was carried out by treatment of rats with the EA extract at doses equivalent to one sixteenth to one fourth of the LD50 for 6 months. Various pathologic changes were found in the lymph system, including decreased numbers of follicules and lymphocytes in the lymph nodes, spleen, and intestine. Decreased numbers of spermatozoa with various degrees of damage were found in the testes. No pathologic changes were noted in heart, liver, kidney, or ovaries.82These findings suggest that the major target organs of subacute toxicity were the lymphatic system and the reproductive tract. Toxicity Testing of T2

The LD50 of T2 was 159.7 mg/kg in mice.'O Rats treated with T2 at a daily dose of 30 mg/kg for 60 to 80 days manifested no changes in body weight or functions of the major organs. This suggested that compared with crude extracts, T2 exerted less toxicity. Common pathologic changes with various degrees of severity were found in the testes of mice, rats, and dogs treated with T2 at daily doses equivalent to one fourth to one twenty-fourth of the LD50 for 80 days, including damage of the seminiferous epidermis and reduction or absence of reproductive cells.'O

CONCLUSIONS

These results suggest that treatment with the extracts of TwHF at proper doses in most patients with rheumatic diseases is effective. To avoid severe intoxication, however, medical use of properly prepared preparations as well as medical monitoring is necessary, particularly for patients with diminished functions of the kidney. Long-term treatment may result in damage of the reproductive system. Despite several unknown aspects of this herbal remedy, currently available data have provided strong evidence indicating that TwHF is a safe and effective therapy for autoimmune and inflammatory diseases.

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Address reprint requests to Xuelian Tao, MD The University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Boulevard Dallas, TX 75235-8884