WO1995015174A1

WO1995015174A1 - Coronary arterial restenosis treatment method

Info

Publication number: WO1995015174A1
Application number: PCT/US1994/013785
Authority: WO
Inventors: Tien Wen Tao Wiedmann; Jian Wang
Original assignee: Pharmagenesis, Inc.
Priority date: 1993-12-02
Filing date: 1994-12-01
Publication date: 1995-06-08

Abstract

A method of preventing restenosis in a subject treated for coronary artery stenosis is disclosed. The method includes administering to the subject, a pharmaceutically effective amount of an ethanol extract from the root xylem of Tripterygium wilfordii.

Description

CORONARY ARTERIAL RESTENOSIS TREATMENT METHOD

Field of the Invention

The present invention relates to a method of preventing restenosis in a mammal treated for coronary artery stenosis.

References

Califf, R. , et al., J. Am. Col. Cardiol., 12(6) :2B (1991) .

Clowes, A., et al . , Laboratory Invest., 49: 208 (1983) .

Evans, R.G., JAMA 265.: 2382 (1991) . Hardoff, R. , J. Am. Coll. Cardiol., 15:1486 (1990).

Holmes, D. , et al . , J. Am. Col. Cardiol. , 53 :77C (1984) .

Jang, G.C., et al ., J. Am. Col. Cardiol., 53 :52C (1984) . Muller, D., et al . , J^". Am. Col. Cardiol.,

19(2) :418 (1992) .

Pepine, C. , Circulation 81:1753 (1990). Sahni, R. , et al., Am. Heart J. Jun. 121(6 Pt 1) :1600-1608 (1991) . Setsuda, M. , et al . , Clin. Ther. Mar-Apr. 15(2) :

374-382 (1993). Bacher, P., et al . , Thromb. Res. 7_0 (4) :295-306 (1993) .

Gapinski, J. P., et al . , Arch. Intern. Med. 152(13): 1595-1601 (1993). Gell an, J. , J. Am. Coll. Cardiol. 17:251

(1991) .

Yabe, Y., Circulation j30_. (Suppl.) :260 (1989). Nye, E. , Aust. N.Z. J. Med. 2JD:549 (1990). Demke, D., Brit. J. Haematol 7_6_ (Suppl.):20 (1990).

Darius, H. , Eur. Heart J. 12 (Suppl.):26 (1991) Okamoto, S., Circulation 82_ (Suppl.):428 (1990) Gottlieb, N., J. Am. Coll. Cardiol. 12 (Suppl. A) : 181A (1991) . Gorman, R.R., et al . , Prostaglandins 26 (2) :325-

342 (1983). de Vries, C. , Eur. Heart J. 2 (Suppl.):386 (1991) .

Graham, L.M. , et al . , J. Surg. Res. 46: 611-615 (1989) .

Payne, J.E., et al . , Aust. N.Z. J. Surg. 61: 619- 625 (1991) .

Wynalda, M.A. , et al . , Prostaglandins 26(2) :3ll- 324 (1983). O'Gara, A. and Defrance, T. : "Bioassays for interleukins", In Laboratory Methods in Immunoloαv. H. Zola, Ed. CRC Press (1990) .

Background of the Invention Atherosclerotic coronary artery disease is the most common cause of death in the Western world and accounts for a large portion of health care expenditures. The disease causes progressive narrowing of the coronary arteries, resulting in inadequate perfusion of the myocardium and the clinical syndromes of anginal chest pain, myocardial infarction, potentially fatal arrhythmias and congestive heart failure. The burden of the morbidity, mortality and cost of utilized health-care resources associated with coronary artery stenosis and occlusion is very high.

During the past 12 years, percutaneous coronary artery revascularization by mechanical means has been developed and greatly refined (Califf, 1991) . Most coronary stenoses can now be treated successfully with balloon dilatation, directional atherectomy, laser ablation or endoluminal stenting procedures (Holmes, 1984) . Such treatment avoids the morbidity and cost associated with open heart surgery as a means for coronary revascularization (Jang, 1984) . However, the long term efficacy of each of these procedures is limited by recurrent stenosis (re¬ stenosis) at the site of intervention, seen in 25-35% of treated stenoses. This condition is characterized by proliferation of the smooth muscle cells of the dilated segment of the arterial wall, resulting in luminal narrowing.

The restenosis phenomenon has been refractory to a variety of mechanical and pharmacologic interventions. Given the number of angioplasty procedures performed in the US (-250-3000,000 per year) , the cost of restenosis in resources and utilized in patient morbidity is very great. A pharmacologic agent able to decrease the rate of restenosis could be expected to impact both the human and the economic costs of restenosis. To date there is no effective means to reduce the rate of restenosis in the large number of patients who are treated with angioplasty. Summary of the Invention

The present invention includes a method of preventing restenosis in a subject treated for coronary artery stenosis. The method includes administering to the subject, a pharmaceutically effective amount of an ethanol extract from the root xylem of Tripterygium wilfordii (TW) .

In one embodiment, the water-soluble components have been removed from the TW ethanol extract. In other embodiments, the TW extract is a partially purified fraction prepared by treating the above extract to remove components which elute from silica gel in acetone, and which are retained on silica gel in the presence of methylene chloride:methanol 97:3. Further, the invention includes the oral administration of the TW extract, preferably at a dose of 1-50 mg extract/kg body weight per day, and preferably over a period from 1-3 days preceding the stenosis treatment to several months following the treatment. In another embodiment, the extract is administered parenterally, preferably at a dose of 0.5 to 10 mg extract/kg body weight per day, also preferably over a period from 1-3 days preceding the stenosis treatment to several months following the treatment.

The present invention further includes, in one aspect, a purified compound for use in preventing restenosis in a subject treated for coronary artery stenosis, the compound referred to herein as tripterygin, having the structural formula:

In another aspect, the invention includes a pharmaceutical composition for use as an anti-hyper- proliferative agent to prevent hyperproli eration of vascular endothelium. The composition includes purified tripterygin in a pharmaceutically acceptable delivery vehicle.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

Brief Description of the Drawings

Fig. 1A is a flow diagram of a method for preparing Tripterygium wilfordii (TW) ethanol extracts. Fig. IB is a flow diagram of a method of further purification for preparing TW extracts useful in the method of the invention;

Fig. 2 is a thin layer chro atogram of various T . wilfordii ethanol extracts useful in the invention; Figs. 3A and 3B are 100x and 400x photomicrographs of a cross-section of a normal rat carotid artery;

Figs. 4A and 4B are lOOx and 40θx photomicrographs of a cross-section of a stenotic carotid artery 14 days following balloon dilatation; and

Figs. 5A and 5B are lOOx and 400x photomicrographs of a cross-section of a rat carotid artery 14 days following balloon dilatation, in an animal treated in accordance with the method of the present invention.

Fig. 6 shows a proposed structure of tripterygin.

Detailed Description of the Invention

1. Definitions

The terms below have the following meanings, unless specified otherwise: "Coronary arterial stenosis" refers to a progressive narrowing of a coronary artery, typically by neoendothelial cell proliferation.

"Coronary stenosis treatment" refers to any treatment designed to restore the normal cross sectional area of a stenosed artery. The treatment method may involve mechanical methods of stretching the lumen, such as involving balloon angioplasty, or other methods such as directional atherectomy, laser ablation, or endoluminal stenting procedures. An "ethanol extract of the root xylem from T. wilfordii " refers to a composition containing ethanol-soluble components extracted from the root xylem of T. wilfordii ; such a composition may include water soluble components that are also soluble in ethanol. The extract may be relatively unpurified (e.g., Fig. 1A) , or may exist in progressively more purified forms (e.g., Fig. IB), as described below. For example, water-soluble components and components which bind to silica gel in the presence of chloroform may be removed from the extract. II. Tripterγσium wilfordii (TW) Extracts

A. Preparation of a TW Ethanol Extract

The extract of the invention is obtained from the root xylem of Tripterygiu wilfordii (TW) , a medicinal plant which is grown in the Fujiang

Province and other southern provinces of China. Plant material can be obtained easily in China. One method of preparation of a TW ethanol extract is illustrated in Fig. 1A, and detailed in Example 1. Briefly, dried plant material is ground into a crude powder and then extracted by boiling in 95% ethanol with refluxing. The ethanol is removed and the extraction typically repeated twice. The resulting extracts are then combined and the ethanol removed (for example, by evaporation or heat-assisted evaporation, such as in a "ROTOVAP") . About 10 g dry ethanol extract is usually recovered per kg dry weight of plant material. This simple ethanol extract represents one preparation useful in the practice of the treatment method of the present invention.

B. Exemplary Method for Further Purification of the TW Ethanol Extract The ethanol extract may be further purified as desired. One method of further purification of the

TW ethanol extract is illustrated in Fig. IB, and detailed in Example 2. Briefly, the ethanol extract is filtered and volume is reduced under vacuum. The resulting syrup is diluted with water.

Chloroform-soluble components are extracted by the addition of chloroform, separation of the non-aqueous-phase material, and discarding the aqueous-phase components. The chloroform extract is concentrated (e.g., by evaporation) and applied to a δ silica gel column. The TW extract is then eluted successively with chloroform and chloroformrmethanol (95r5) and with chloroformrmethanol (90rl0), at a yield of about 1 g extract material per 20 g of original ethanol extract, corresponding to about 100 g dry weight starting plant material. This extract is referred to as a lrlOOO TW extract.

The l lOOO extract can be further purified (Example 2) by application to a silica gel column and elution with methylenechloridermethanol (97r3) .

Typically, six fractions are collected with the first and last of the six fractions to be discarded. The four intermediate eluted fractions are combined, with a yield of about 20 g material per 100 gram lrlOOO extract. The resulting extract is referred to herein as a l 5000 extract.

The lr500 extract is further purified by the same procedure, i.e., by elution on silica gel with methylene chloridermethanol (97r3) and collection of the intermediate fractions, with a final yield of about 1 g material per 2 gram lr5000 extract. This final purified extract is referred to herein as a lrl0,000 and also as a purified TW extract.

C. Characterization of the TW Extracts

The physical and chemical properties of TW extracts have been evaluated as follows. Fig. 2 shows a thin layer chromatogram (TLC) of the following TW extractsr lr1,000, lr5,000 and lrl0,000. Comparison of the results of analysis of the TLC with different reagents indicates that a number of major plant components have been removed from these extractsr for example, no alkaloids appear to be present (Example 3) . III. Purified Tripterygin.

In one aspect, the invention includes a purified compound isolated from Tripterygium wilfordii . The compound, designated herein as "tripterygin", is believed to have the structural formula shown in Figure 6.

Tripterygin can be purified from the root xylem of Tripterygium wilfordii (Example 4) . The presence of tripterygin in the fractions generated at various stages of purification can be monitored by use of one or more assays, including the inhibition of IL-1 action on the proliferation of mouse thymoσytes (Example 4) . However, other compounds present in the mixture may also be detectable in the assays, thereby precluding unambiguous distinction of tripterygin from such other compounds.

A protocol for isolating purified tripterygin is described in Example 4. Figure 6 shows a structure and carbon atom numbering scheme for the tripterygin compound of the invention.

IV. Treatment Method

In the treatment method of the invention, TW extracts are administered to a mammal prior to and following coronary artery angioplasty, in order to prevent restenosis. Initial treatment is administered three days or more prior to the angioplasty procedure and continued for a period of several months. A TW extract may also be administered at later times following angioplasty, in order to limit the development of late-occurring restenosis.

The TW ethanol extract may be administered by oral or parenteral administration. For oral administration, the extract may be given in tablet form, at a preferred dose of 1 to 50 mg/kg patient body weight per day. A parenteral suspension can be administered by injection, e.g., intravenously, intramuscularly, subcutaneously, or intraperitoneally. A dose of preferably between about 0.5 to 10 mg extract/kg body weight per day is anticipated. Other pharmaceutical formulations of TW extract are described in Section V.

Treatment results can be monitored, if indicated, by coronary angiography, or other non- invasive methods that may become available in the future.

The treatment of restenosis, in accordance with the invention, is illustrated in Example 5. A established animal model system for restenosis was employed. The left distal common carotid and external carotid arteries of male Sprague-Dawley rats were exposed under aseptic conditions. A #2 Fogarty arterial embolectomy catheter was introduced into the external carotid artery and passed proximally into the common carotid artery. The catheter balloon was then distended with 0.25 ml of room air, using a 1 ml syringe, and the inflated balloon was passed three times through the common carotid, dilating the vessel, in a manner analogous to balloon coronary angioplasty. The balloon was then deflated and the catheter withdrawn. The external carotid was ligated using absorbable 4-0 suture. After fourteen days, animals were anesthetized, sacrificed, and the left common carotid arteries were removed, stained, imbedded in paraffin and sectioned for microscopy. Animals were treated with 30 or 50 mg/kg body weight of TW extract or vehicle control, administered orally, from three days prior to balloon dilatation to 13 days following balloon dilatation (one day prior to the conclusion of the experiment) . The dried extract was prepared in olive oil vehicle.

Arterial specimens were examined using light microscopy. The effect of the plant extract on neointimal proliferation was determined by estimating the percentage of the interluminal area (defined as the area within the internal elastic lamina of the excised arterial segment that was occupied by proliferative smooth muscle cells. Figures 3A and 3B show exemplary results of lOOx and 400x magnification photomicrographs of a cross- section of a normal rat carotid artery. Figure 4A and 4B (lOOx and 400x magnification, respectively) show exemplary photomicrographs of a cross-section of a stenotic carotid artery 14 days following balloon dilatation. Comparison of these two sets of figures show the result of neointimal thickening in the stenotic artery.

Figures 5A and 5B (lOOx and 400x magnifications, respectively) show exemplary photomicrographs of a cross-section of a rat carotid artery 14 days following balloon dilatation, in an animal treated with TW extract as described above. These results demonstrate the inhibition of neointimal thickening in the carotid artery of a mammal treated with TW extract.

These results indicate that in vivo TW extract was effective in preventing restenosis that develops following percutaneous transluminal coronary angioplasty. Specifically, TV? extract is useful in preventing or treating intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion in a mammal, particularly following mechanically mediated vascular injury, or under conditions that would predispose a mammal to suffering such a vascular injury.

V. Pharmaceutical Preparations of Tripterygium wilfordii Ethanol Extract

Tripterygium wilfordii extract, when employed in the prevention or treatment of restenosis, can be formulated alone or with the addition of a pharmaceutical carrier. Such pharmaceutical carriers may be solid or liquid. Pharmaceutical preparations containing Tripterygium wilfordii extract are administered to mammals, in need of treatment, at a therapeutically effective dose.

TW extract-containing compositions of the present invention can also be administered orally either in solid composition or liquid form.

A solid carrier for TW extracts can include one or more substances. A carrier can also provide suspending agents, flavoring agents, binders, lubricants, glidants, compression aids, solubilizers or tablet-disintegrating agents. Further, a carrier may also function as an encapsulating material.

When the extract is employed in the form of solid preparations for oral administration, the preparations may be tablets, granules, powders, capsules or the like. In a tablet formulation, the extract is typically formulated with additives, for example, an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator and so on, all being ones usually used in the manufacture of medical preparations.

Powder formulations are typically a mixture of a finely rendered solid carrier which is in a mixture with the finely rendered TW extract. In tablet form, a carrier having the necessary compression properties is mixed, in appropriate proportions with the TW extract. The mixture is compacted into the desired shape. Powders and tablets can contain up to about 98% of the active ingredient. A number of suitable solid carriers are available, including, but not limited to, the following sugars, lactose, dextrin, starch, gelatin, calcium phosphate, magnesium stearate, talc, polyvinylpyrrolidone, low melting waxes, ion exchange resins, cellulose, methyl cellulose, and sodium carboxymethyl cellulose.

For use in oral liquid preparation, the ethanol extract may be prepared as a liquid suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.

Liquid carriers can be used to prepare syrups, suspensions, elixirs, solutions and emulsions. The TW extract is dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, organic solvent, a mixture of water and one or more organic solvent, or pharmaceutically accepted oils or fats. The liquid carrier can contain other suitable pharmaceutical additives including, but not limited to, viscosity regulators, preservatives, sweeteners, solubilizers, emulsifiers, buffers, thickening agents, flavoring agents, suspending agents, colors, stabilizers and osmolarity regulators. Suitable examples of liquid carriers for oral and parenteral administration of TW extract preparations include water (partially containing additives as above, e.g., cellulose derivatives, preferably sodium carboxymethyl cellulose solution) and alcohols (including ethanol/water mixtures) . The ethanol extract of the present invention may be injected in the form of aqueous solutions, suspensions or oily or aqueous emulsions, such as liposome suspensions. For parenteral administration of TW extract the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Typically, for parenteral administration, the extract is formulated as a lipid, e.g., triglyceride, or phospholipid suspension, with the extract components being dissolved in the lipid phase of the suspension. Sterile carriers are used to form sterile liquid compositions for parenteral administration. Sterile liquid pharmaceutical compositions, solutions or suspensions can be utilized by, for example, intraperitoneal injection, intramuscular, subcutaneous or intravenous injection. TW extract can be also be administered intravascularly or via a vascular stent impregnated with TW extract, for example, during balloon catheterization to provide localized effects immediately following injury.

TW extract can be rectally administered in the form of a conventional suppository. Alternatively, the drug may be administered transdermally through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non-toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin. Carriers for transdermal absorption may include pastes, e.g., absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient with or without a carrier, or a matrix containing the active ingredient; creams and ointments, e.g., viscous liquid or semi-solid emulsions of either the oil-in-water or the water-in- oil type; gels and occlusive devices. Preparations of TW extract, may be administered topically as a solution, cream, or lotion, by formulation with pharmaceutically acceptable vehicles containing the active compound.

The dosage requirements for treatment with TW extract vary with the form of TW extract used (for example, level of purification) , particular compositions employed (e.g., amount and type of carrier) , the route of administration, the severity of the disease presented, and the particular subject being treated. Precise dosages for oral, parenteral, intravascular, transdermal, or rectal administration are determined by the administering physician based on experience with the individual subject treated. TW extract is generally administered at a concentration that provides effective results without causing any harmful or deleterious side effects. Such a concentration can be delivered by administration of either (i) a single unit dose, or (ii) the dose divided into convenient subunits at suitable intervals throughout the day.

VI. Utility Mechanical injuries leading to intimal thickening follow typically as the result of invasive processes that disrupt vascular integrity, such as, balloon angioplasty, vascular surgery, and transplantation surgery. Injury to the smooth muscle cells of the tunica media is followed by a proliferation of the medial smooth muscle cells. These smooth muscle cells then migrate into the intima through fenestrations in the internal elastic lamina and continue to proliferate there to produce neointimal thickening. Such lesions result in the partial or complete blockage of blood vessels.

Coronary arterial stenosis is typically detected and evaluated using angiographic or sonographic imaging techniques (Evans) . Stenosis is now commonly treated by percutaneous transluminal coronary angioplasty (balloon catheterization and dilation) . However, within a few months following angioplasty, stenosis recurs in approximately 30-40 percent of treated patients. Restenosis is caused by the proliferative response of the arterial wall cells to the mechanical injury induced by the angioplasty procedure (Pepine; Hardoff) itself.

Numerous pharmaceutical agents have been employed clinically, concurrent with or following angioplasty, to try to (i) prevent restenosis, or (ii) or by reducing intimal smooth muscle cell proliferation following angioplasty; most such pharmaceutical agents have been unsuccessful. The experiments performed in support of the present invention indicate that TW extract is nontoxic and an effective in vivo agent for preventing intimal smooth muscle thickening following arterial injury. The use of TW extract may provide new therapeutic strategies for the control of restenosis following invasive arterial invasive widening. The present invention generally relates to the use of TW extract for the treatment of coronary arterial restenosis in a mammal. Further, the present invention contemplates the use of compounds isolated from TW extracts for the treatment of restenosis, e.g., the compound tripterygin, described above, having the structural formula presented in Figure 6. Such isolated compounds can be formulated in pharamceutical compositions, essentially as described above for TW extract. Typically, the composition includes purified tripterygin in a pharmaceutically acceptable delivery vehicle. The composition may further include other pharmaceutical agents employed to prevent or reduce the extent of restenosis. Several such agents have been identified for which favorable preliminary clinical results have been reported, including the followingr lovastatin (Sahni; Gellman) ; thromboxane A₂ synthetase inhibitors such as DP-1904 (Yabe) ; eicosapentanoic acid (Nye) ; ciprostene (a prostacyclin analog) (Demke; Darius) ; trapidil (Okamoto) ; angiotensin converting enzyme inhibitors (Gottlieb) ; low molecular weight heparin (de Vries; Bacher, et al . ) ; 5-(3'-pyridinylmethyl)benzofuran-2- carboxylate (Gorman, et al . ; Payne, et al . ; Wynalda, et al . ; Graham, et al . ) ; "PROBUCOL" (Setsuda, et al . ) ; and fish oils - omega-3 fatty acids (Gapinski, et al . ) .

The following examples illustrate the method for obtaining ethanol extracts of T. wilfordii , show the physical properties of the extract, and demonstrate the use of an ethanol extract for preventing restenosis in an animal model system. The examples are intended to illustrate, but in no way limit the scope of the invention.

Example 1 Preparing Tripterygium wilfordii

Ethanol Extract

Tripterygium wilfordii plants were obtained in

Fujiang Province, China. Plants were air dried in sunlight. The root xylem of the plants (300 g) was ground into a crude powder and extracted with 5 volumes (1.5 1) of 95% ethanol, under reflux at 85°C for 4 hours. The filtered xylem powder was then extracted two more times in 3 volumes (900 ml total) of 95% ethanol. The three extracts (total of about 3.3 1) were combined and the resulting mixture was concentrated at 50°C under vacuum, to about 2% of the original volume, i.e., about 66 ml.

Example 2 Further Purifications of the

TW Ethanol Extract

A. The CH₂C1, TW Extract

The ethanol extract syrup obtained in Example 1 was then diluted with 33 ml water, filtered through Whatman # 1 filter paper. The filtrate was extracted 4 times (50 ml/extraction) with methylene chloride (CH₂C1₂) .

B. The 1r 1 , 000 TW Extract The combined, CH₂Cl₂-extracted filtrate (about

200 ml) was concentrated, and applied to a 1 cm (diameter) x 5 cm column containing silica gel (1.5 kg; 60-200 mesh) . The column was washed successively with 100 ml methylene chloride, and 100 ml methylene chloride methanol (95 5) . The fraction which eluted in 95 5 solvent contained about 0.3 g material, and is referred to herein as a lrlOOO extract.

C. The l 5,000 TW Extract Forty grams of lr 1,000 prepared as described above (in scale-up) was concentrated to a small volume in 20 ml acetone. The solution was applied to a 13 cm x 14 cm column containing silica gel (800 gm; 60-200 mesh) and eluted with methylene chloridermethanol 97r 3 to produce six 1 liter ml fractions. The yield of each fraction was about 5% or 2 grams. Fractions 2-5 were combined and the resulting 8 grams of material are referred to herein as the lr5000 TW extract.

D. The lrlO-000 TW Extract

Fractions 2-5 were combined and the resulting 8 grams of material, the lr5000 TW extract, was then applied to a 8 cm x 40 cm column containing silica gel (320 gm; 260-400 mesh) and eluted with methylene chloridermethanol (97r3) to produce five 300 ml fractions. Fractions 2-4, which were yellowish in color, were combined. The solvent was removed by evaporation under vacuum to yield 4 grams of light brown powder, referred to herein as the purified (lr 10,000) TW extract.

Example 3

Physical Characteristics of the Ethanol Extract and of Further Purified Mixtures

One microgram samples of extracts were applied to a silica gel coated aluminum thin layer chromatography plate (Whatman, catalog # 4420 222).

The development solvent was hexanermethylene chloridermethanol in volume ratios of lrlrθ.15.

Following separation, samples were visualized using an ultraviolet lamp and by application of an aerosol of 0.5% vanillin in H₂S0₄-ethanol (4rl).

TLC profiles of the various TW extracts are shown in Figure 2. Lane A shows the lr1,000 extract, lane B shows the lr5,000 extract and lane C the lrl0,000 extract, herein called the purified TW extract. It can be seen that purification between the lrlOOO, lr5000 and lr10000 extracts has removed a number of major plant components. For these TW extracts, the thin layer chromatographic profile showed no alkaloid in the extract, as determined by application of the Dragendorff reagent.

Example 4

Preparation of Tripterygin A. Bioassay.

Fractions produced at various stages in the purification procedure below were assayed to identify fractions containing tripterygin by one or more assays, including suppression of PBL proliferation in vitro, inhibition of IL-2 action, evaluating the effects of tripterygin on cytokine production.

One exemplary assay uses the inhibition of IL-1 action on the proliferation of mouse thymocytes to identify tripterygin containing fractions. In this assay, mouse thymocytes were prepared, and the action of IL-1, which stimulates thymocyte proliferation, was measured using standard techniques (0^,Gara) . Briefly, untreated cells showed minimal DNA synthesis (thymidine incorporation 80 cpm/well) . Phytohemagglutinin (PHA) alone typically stimulated thymidine incorporation 2-3 fold. Treatment with 0.1 ng/ml IL-1 in the presence of PHA typically resulted in a 60-fold increase. Addition of the tripterygin resulted in a dose-dependent inhibition of IL-1 stimulation. Inhibition was typically measured over a range of about 10"⁹ M to 2 x 10"⁶ M- Half-maximal inhibition occurred at 2 x 10^*9 M.

B. Small-Scale Preparation of Crude Extract.

Tripterygium wilfordii plants were obtained in Taiwan or in Fujiang Province, China and were air- dried in sunlight. The root xylem of the plant (300 g) was ground into a crude powder and extracted with 1.5 1 of 95% ethanol under reflux at 85°C for 4 hours. Following reflux, the residual crude powder was collected by filtration and extracted two more times as above using 900 ml 95% ethanol each time.

The three ethanol extracts (total volume of about 3.3 1) were combined and the resulting mixture was concentrated at 50^*C under vacuum, to about 2% of the original volume, i.e., about 66 ml. The concentrated mixture was then diluted with

33 ml water and filtered through Whatman # 1 filter paper. The filtrate was extracted 4 times (50 ml/extraction) with methylene chloride. The combined filtrate (about 200 ml) was concentrated, and applied to a 6.5 cm (diameter) x 12 cm column of silica gel (-60-200 mesh) . The column was washed successively with 600 ml methylene chloride, and 1500 ml methylene chloridermethanol (95r5) . The fractions which eluted with the methylene chloridermethanol solvent were then concentrated under reduced pressure, yielding about 0.9 g of crude product (concentrate #1) .

C. Further Purification of Extract.

Forty grams of concentrate #1, prepared by scale-up of the procedure above, was applied to a 9 cm x 25 cm column of silica gel and eluted with the following series of methylene chloridermethanol mixtures (vrv) lOOrO (3 liters), 97r3 (4 liters), 95r5 (4 liters), and 90rl0 (3 liters). The fractions which were eluted by the 95r5 mixture were concentrated under reduced pressure, yielding 12.9 g of concentrate #2.

Concentrate #2 was loaded on a 5.5 x 49 cm silica gel column and eluted with the following series of mixtures of hexanerCH₂Cl₂ methanol as followsr Ir2r5 (2 liters), lr2riθ (2 liters), Ir2rl5 (2 liters), and Ir2r20 (2 liters). Fractions which were eluted by the Ir2rl5 mixture were concentrated under reduced pressure, yielding 2.26 g of a concentrate #3.

Concentrate #3 was purified by silica gel chromatography (2 cm x 42 cm) using the following series of hexaneracetone mixturesr 9rl (500 ml), 8r2 (1000 ml), 7r3 (lOOO ml), and 6r4 (300 ml).

Fractions which eluted with the 7 3 mixture were combined and concentrated, yielding 271 mg of a concentrate #4.

Concentrate #4 was purified by reversed phase HPLC on a 20 x 250 mm ODS column (Japanese

Analytical, Inc., distributed by DyChrom, Santa Clara, CA) . The mobile phase was 70% methanol in water, at a flow rate of 3 ml/min (100 mg/run) . The elution time of tripterygin was 3.6 min. Tripterygin-containing fractions were concentrated under reduced pressure (75°C) and further purified by another ODS HPLC column (10 x 250 mm) using a mobile phase of 70% methanol in water, at a flow rate of 1 ml/min (20 mg/run) . The elution time of tripterygin was 2.5 min.

As a final step, purified tripterygin was obtained by crystallization from methanol, yielding 13 mg of purified product.

Example 5

Effect of the Ethanol Extract on Arterial Stenosis in a Rat Arterial Injury Model

A modification of an established arterial injury model (Clowes, 1983; Muller, 1992) was used. Male Sprague-Dawley rats, weighing 400-450 grams, were obtained from Simonsen Laboratories, Inc. (Gilroy, CA) . Animals were anesthetized using a mixture of ketamine (15 mg/kg) and xyloxine (9 mg/kg) , given by intraperitoneal injection, following initial inhalation of ethyl ether. The left distal common carotid and external carotid arteries were exposed under aseptic conditions, using a left paramidline cervical incision.

A #2F Fogarty arterial embolectomy catheter (Baxter Healthcare Corporation, Santa Ana, CA) was introduced into the external carotid and passed proximally into the common carotid artery. The catheter balloon was then distended with 0.25 ml of room air, using a 1 one ml syringe, and the inflated balloon was passed three times through the common carotid, dilating the vessel and denuding the endothelium (thus mimicking balloon coronary angioplasty) . The balloon was then deflated and the catheter withdrawn. The external carotid was ligated using absorbable 4-0 suture.

After fourteen days, animals were anesthetized, sacrificed, and the left common carotid arteries were removed. The arteries were stained, imbedded in paraffin and sectioned for microscopy. Animals were treated with varying doses of the plant extract or vehicle control, administered orally, from three days prior to balloon dilatation to day 13 following dilatation, one day prior to the conclusion of the experiment. The dried extract, typically the^' lrl0,000 extract, was prepared in ethanol (50 mg/ml) and diluted in sterile normal saline.

The effect of the plant extract on intimal proliferation was determined by measuring the percentage of the interluminal area (defined as the area within the internal elastic lamina) of the excised arterial segment that was occupied by proliferative smooth muscle cells.

Exemplary photomicrograph data are presented in the following figures. Figs. 3A and 3B show lOOx and 400x magnification photomicrographs of a cross- section of a normal rat carotid artery. These figures can be compared to Figs. 4A and 4B (lOOx and 400x magnification, respectively) show photomicrographs of a cross-section of a stenotic carotid artery 14 days following balloon dilatation. Comparing these two figures clearly shows the degree of neointimal proliferation that has occurred in the dilatated artery. Figs. 5A and 5B (lOOx and 400x magnification, respectively) show photomicrographs of a cross-section of a rat carotid artery 14 days following balloon dilatation, in an animal treated with TW extract as just described. Comparison of Figs. 5A/B to 3A/B and 4 A/B demonstrate the ability of TW extract to inhibit neointimal cell proliferation and restenosis.

Treatment with the olive oil vehicle alone did not affect endothelial proliferation. Treatment with oral TW extract at 30 and 50 mg/kg body weight reduced proliferation. Microscopic examination of arteries obtained from untreated animals showed marked proliferation while arteries obtained from the TW extract treated animals were nearly indistinguishable from the control (non-injured) vessels.

No toxicity was observed in the treated animals.

Although the invention has been described with respect to particular methods and applications, it will be appreciated that various changes and modifications may be made without departing from the spirit of the invention.

Claims

IT IS CLAIMEDr

1. A method of preventing restenosis in a mammal treated for coronary stenosis, comprising administering to a mammal in need of treatment in a pharmaceutically effective amount of an ethanol extract from the root xylem of Tripterygium wilfordii .

2. The method of claim 1, wherein said administering includes oral administration, at a dose of 1-50 mg extract/kg body weight per day.

3. The method of claim 2, wherein said administering includes giving the extract to the mammal prior to coronary stenosis treatment, and continuing said administering for a period of at least 1 month following stenosis treatment.

4. The method of c3aim 1, wherein said administering includes parenteral administration, at a dose of 0.5 to 10 mg extract/kg body weight per day.

5. The method of claim 4, wherein said administering includes giving the extract to the mammal prior to coronary stenosis treatment, and continuing said administering for a period of at least 1 month following stenosis treatment.

6. The method of claim 1, wherein said extract is an ethanol extract of Tripterygium wilfordii root xylem from which water-soluble components and components which bind to silica gel in the presence of chloroform have been removed.

7. The method of claim 6, wherein said extract is a partially purified extract prepared by treating the extract from claim 6 to remove components which elute from silica gel in acetone, and which are retained on silica gel in the presence of methylene chloridermethanol 97r3.