TWI355933B - Andrographolide and its derivatives as tnf-α antag - Google Patents

Description

1355933 Ο) Description of the Invention [Technical Field] The present invention relates to a novel pharmaceutical composition comprising andrographolide which is a tumor necrosis factor (TNFa) antagonist or inhibitor and/or derivative. Prior art

Andrographolide has the formula (I R,0

0 where Ri=R2=R3 = R4 = H. Andrographolide is a major component of Andrographis paniculata, which was originally isolated by Gorter1. Andrographolide is classified as a diterpene lactone, which has been shown to protect liver i, stimulate immunity 2, anti-inflammatory 3, anti-oxidation 4, anti-cancer 5, anti-HIV infection 6, antibacterial 7, antipyretic 8, anti-infective 9, analgesic 1Q, antithrombotic 11, anti-cardiovascular disease 12, antihypertensive 13, anti-allergic 14, anti-diarrhea 15 and anti-high cholesterol 16 activity. Regarding anti-inflammatory activity, it has been reported that andrographolide has a synergistic inhibitory effect on cyclic oxidase-2, and manufacture and adhesion of neutrophilic oxygen-containing substances to rats -6 - (2) (2) 1355933 The inhibitory effect of action 4b, the inhibitory effect on the expression of nitric oxide synthase which can be triggered 4d, and the up-regulation of ICAM-1 (intercellular adhesion molecule-1) induced by TNFot and endothelial cell mononuclear ball The inhibitory effect of adhesion is 18. However, the anti-inflammatory machine of andrographolide has not yet been established. Among some of the currently known inflammatory modulators, TNFa is one of the most potent and defined cytokines available today, and was selected for testing in cell proliferation/cytotoxicity assays, whether andrographolide inhibits TNFa binding to The role of TNFa-Rl. TNFa is an important factor in host defense, which is resistant to many pathogenic microorganisms and some viruses. Even though TNFa undoubtedly has a beneficial function (mainly at a systemic level), it may lead to pathological results. TNFa is an important factor in the pathogenesis of septic shock, which is characterized by hypotension and multiple organ failure. TNFa is a major regulator of cachexia, with cachexia characterized by abnormal body weight loss in cancer patients. TNFa is often detected in the joint fluid of patients with arthritis. TNFa may be an important factor in a wide range of diseases, and therefore, compounds that bind to the TNFa receptor may be used to treat a variety of diseases or conditions involving TNFa, such as rheumatoid arthritis, Crohn's disease, Plaque sclerosis, septic shock, or cancer or cachexia associated with immunodeficiency. SUMMARY OF THE INVENTION The present invention relates to a novel T N F a inhibitor or inhibitor, the stereoisomer of (3) 1355933, and pharmaceutically acceptable salts thereof. In particular, the present invention relates to novel andrographolide derivatives, stereoisomers thereof, and salts. The novel andrographolide derivative has the general formula (1):

Wherein h, R2 and r3 - - π are independent of AL or unsubstituted groups, for example: alkyl, aryl, heteroaryl, male, heteroaryl, (tetra), enoyl , aromatic group, heteroaryl, sulfhydryl, sulfhydryl, aryl, aryl or aryl, _ or group -(C0)- W-r5, where 1 is 〇3 or 6 , 眞

R6 is hydrogen or (C丨-Cdei is a ratio, " base, R is substituted or ungrouped', for example: alkyl, aryl~^, aryl, aryl, 〇R2, 〇 Or substituted or unsubstituted q ^ 7 7a a has a cyclic structure of carbon and oxygen atoms, R 4 is halogen or ^7, (4) take W, medium X is 〇, S or NH, and 4 1 work Substituted or unsubstituted group, for example: alkyl, aryl, aryl 'cylalkyl' olefinic group, 垸_π% alkyl fluorenyl group, aryl fluorenyl group, heteroaryl fluorenyl group, aryl group: a dimercapto group, a fluorenyl group, or a group - (the TM group is a substituted or unsubstituted aralkyl group. The penetrazine is known to be a compound of the above formula (1), but -8 - (4) (4)1355933

Rl=R2 = R3 = R4 = H. The andrographolide and its derivative represented by the general formula (I) as defined above can be used for inhibiting the release of TNFcx, and can be used for treating various diseases involving TNFoc, such as rheumatoid arthritis. , Crohn's disease, plaque sclerosis, septic shock, or cancer or cachexia associated with immunodeficiency. The present invention also relates to a pharmaceutical composition for inhibiting or anti-TNFot in mammals, including humans, comprising a compound of the formula (I), or a stereoisomer thereof, in an amount effective to inhibit or antagonize TN Fa, Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The invention also relates to a pharmaceutical composition for treating a condition or condition involving TNFoc, such as rheumatoid arthritis, Crohn's disease, plaque sclerosis, septic shock, or cancer or cachexia associated with immunodeficiency, The pharmaceutical composition comprises a compound of the formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is effective for inhibiting or antagonizing TNFa, and a pharmaceutically acceptable carrier. The present invention also relates to andrographolide or a compound of the formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is used in the manufacture to inhibit or antagonize in mammals, including humans. The use of TNFa for drugs. The invention also relates to andrographolide or a compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a condition or condition involving TNFa, such as a class Use of rheumatoid arthritis 'clonal disease, plaque sclerosis, septic shock, or a drug associated with cancer or cachexia associated with immunodeficiency-9-(5) 1355933. DETAILED DESCRIPTION OF THE INVENTION AND GENERAL SYSTEM: The compound of formula (I) of the present invention or a pharmaceutical thereof can be administered to mammals (e.g., via subcutaneous, intravenous, intramuscular, j), transrectal via any of the following means (e.g., via subcutaneous, intravenous, intramuscular, j) , through the nose, through the local and through the penetration of A using the patch). The pharmaceutically acceptable salts of the formula (I) of the present invention can be administered alone or together with a pharmaceutically acceptable agent, as described above, and can be administered in an amount as described above. Suitable pharmaceutical carriers include solid diluents or graves and various non-toxic organic solvents and the like. [Examples] Experimental procedure 1 · Purification of rhTNFa from E. coli and biotinylation of IPTNF (isopropyl-β-D-L. thiobacillus BL2 1 (DE3) in rhTNFa (recombinant sense, and different The concentration of imidazole is pure under natural conditions. 2. Preparation of methanol extract of Andrographis paniculata 50 grams of Andrographis paniculata is washed and dried, followed by Rhododendron (40/1, volume/weight), in the room for three days. Filtration to allow acceptable salts for filtration: oral, parenteral-intra-osseous and infusion-based skin (for example, via a pharmaceutically acceptable carrier or a diluted single or multiple dose, sterile aqueous rhTNFa) Glycoside) induces the expression of the large intestine, TNFtx-like. The methanol is added to the enthalpy, and the extract of Andrographis paniculata is concentrated in a rotary evaporator-10-(6)(6)1355933 (Heidolph Laborota 40 00) until the final volume is reduced to about 50 ml. Figure 2 shows a chromatogram of the crude methanol extract of Andrographis paniculata on a TSK Gel 0DS 80TM (TOSOH) reverse phase chromatography column (250 x 4.6 mm) filled with 5 micron micelles. 3. Preparation of Andrographis water extract will be 50 grams The andrographis panicula is washed and dried, then water is added to the weighed andrographis (10/1, volume/weight), and the penguin is extracted twice at 95. (:: the extract is filtered for 2 hours. The extract is filtered to make the filtrate rotate. Concentrate in an evaporator (Heidolph Laborota 4000) until the final volume is reduced to approximately 100 mL. Figure 3 shows a TSK Gel 0DS 80TM (TOSOH) reverse phase chromatography column (250x4.) filled with 5 micron colloids. On the 6 mm), the chromatogram of the crude extract of Andrographis paniculata 4. Purification of andrographolide 50 g of Andrographis paniculata leaves were extracted twice with 95% absolute ethanol at the boiling point for 2 hours. The extract was filtered in vacuo. The solvent is removed from the filtrate. The resulting dark green crystals are washed several times with toluene until most of the colored material is removed from the residue. The toluene is then completely removed from the residue. The remaining crystalline material is dissolved in hot methanol. Then, it is cooled in a refrigerator to crystallize it. This step is repeated several times until colorless crystals are obtained. 5. Identification of andrographolide (1) Thin layer chromatography (TLC) -11 - (7) (7) 1355933 In order to carry out TLC experiments, use some Pre-coated with silicone disc 60F254 (E. Merck) and spotted with a capillary. Scan the discs on a UV observation box (Gam ag). The solvent system for pure andrographolide is chloroform: ethanol A mixture of 9: 1. The TLC of the separated andrographolide showed a single point, and the enthalpy in the solvent system was 0.46. (2) UV/visible absorption spectrum φ The UV absorption spectrum of andrographolide and the reference standard in methanol was recorded on a UV/visible spectrophotometer (Beckman). The maximum absorption band (human max) of andrographolide in H20 is 230 nm' absorption coefficient is 13,000. In methanol, it shows a max = 222 nm and an absorption coefficient of 13200. (3) LC/MS spectrum Obtained on LC/MS (Liquid Chromatography Mass Spectrometry) (Varian) · ESI mass spectrum of molecular ion of atmospheric pressure ionization. The mobile phase was MeOH + 2 mM NH4OAc. Andrographolide mass spectrometry data: 351 (M + H) +, 3 68 (M + NH4) +, 3 73 (M + Na)+. (4) HPLC spectroscopy HPLC spectra of andrographolide and reference standards were obtained on a TSK Gel ODS 80 ΤΜ (5 μιη) TOSOH reverse phase chromatography column (4.6 x 250 mm) using a Shimadzu HPLC system with mobile phase containing acetonitrile and water. -12- (8) (8)1355933 (1:1), with a flow rate of 0.75 ml/min and a gradient of 0-100, and the eluted compound was detected at 230 nm. The mobile phase used was a mixture of acetonitrile (B) and hydrazine 20 (Α) at a flow rate of 0.75 ml/min. The column is eluted in the following manner: 0%~30% B in the first 5 minutes; 30%~50% B-shaped gradient in 5~25 minutes; 50~70% in 25~30 minutes B; 30~ 35 minutes is 70~100% B; 35~45 minutes is 100% B. Detection was performed at a wavelength of 23 0 nm with a detection sensitivity of 0.01 AUFS. HPLC analysis of andrographolide showed a single peak with a residence time of 20.5 minutes, the same as the andrographolide standard. Figure 4 shows a chromatogram of andrographolide on a TSK Gel ODS 80tm (TOSOH) reverse phase chromatography column (250 x 4.6 mm) filled with 5 micron particles. 6. Inhibitory effect of andrographolide on TNFa binding to TNFot-Rl Different ratios of andrographolide and biotinylated rhTNFa mixture were preincubated for 30 minutes in 37.0. The mixture was then added to a 96-well microtiter plate which was previously pre-coated with rhTNFa receptor (rhTNFoc-R1). After 2 hours of incubation at 37 °C, the plate was washed with TBST (Tris buffered saline/Tween 20). Avidin (acedin) conjugated with alkaline phosphatase was used for detection. Measured at a wavelength density of 405 nm, the inhibitory ability of andrographolide was calculated as follows: % inhibition (%) = 100 - OD (optical density) of andrographolide / positive control group - 13 - 1355933 (9) 7. In vitro L929 cell proliferation/cytotoxicity assay assays Antagonistic activity against TNFtx In vitro L929 cell proliferation/cytotoxicity assays were performed on microtiter plates. L929 cells were cultured in Eagle's minimal essential medium (EMEM) containing 10% bovine blood sputum, 1% P/S and 1% non-essential amino acids. The pooled L929 cells were washed with 2 ml of PBS (phosphate buffer) solution, then trypsinized, and then resuspended in complete medium. Two hundred microliters of the cell suspension was taken for calculation of cell density, while the other suspensions were centrifuged at 1,500 rpm for 5 minutes. Remove the upper sputum and add the complete medium to dilute the cells to a final concentration of 1.5 x 105 cells/ml. One microliter of the cell suspension was added to each well of a 96-well flat-bottom microtiter plate and incubated for 24 hours at 5% CO 2 gas at 37 °C. The resulting chemical was resuspended in lxPBS and sterilized with a 0.22 micron filter. Chemicals of various concentrations were incubated for 1 hour in an equal volume of commercially available TNFoc 0.2 ng/ml. Prior to the end of the 1-hour pre-incubation process, the culture broth was removed from the 24-well 96-well plate, and 50 μl of fresh culture medium containing 4 μg/ml Actinomycin D was added. The 50 microliter pre-incubated chemical and TNFa mixture was transferred to a 96-well dish filled with actinomycin sputum to achieve a final concentration: actinomycin D at 2 μg/ml, TNFa It is 0·1 ng/ml. Actinomycin d 2 μg/ml with TNFa 0.1 ng/ml was added as a positive control -14 - (10) 1355933 group, actinomycin D 2 μg/ml was added as a negative control group. After gentle shaking to mixing, the 96-well plate was incubated for 24 hours at 5% CO 2 gas and 37 ° C. The cytotoxicity was the same as that used to test TNFcx activity, and was added to the culture containing actinomycin D. The liquid was centrifuged in a 96-well dish to achieve a final concentration of actinomycin D 2 μg/ml. The 96-well plate was gently shaken with 5% CO 2 gas and 37. (: 24 hours incubation. 50 μl of XTT (ie 2,3-bis(2-methoxy-4-nitro-5-sulfonic acid phenyl)-5-[(anilino)carbonyl)] A mixture of 2H-tetrazolium hydroxide was added to each well and incubated for 4 hours. The EIS A was read at OD 490/ 630 nm.

Inhibition of TNF TNFot inhibition and cytotoxicity % = Pregnancy +77\yg + Actinomycin £> x 1 〇〇% - uuTNFa + actinomycin d 1 0 0 % cytotoxicity = liquid + actinomycetes abandoned. Efficacy of Actinomycin d 8. Andrographis chinensis extract in L929 cell proliferation/cytotoxicity assay In vitro L92 9 cell proliferation/cytotoxicity assay performed on a microtiter plate as described above to determine Andrographis paniculata The inhibitory effect of methanol extract on TNFa. The results showed that the methanol extract of Andrographis paniculata was an antagonist of TNFot, and the methanol extract having a concentration of 500 mg/ml had a 40% inhibitory effect on -15-(11) (11)1355933, and no cytotoxicity was observed. 9. Erythrocyte water extract in L929 cell proliferation/cytotoxicity test The same procedure as above was used to determine the inhibitory activity of the aqueous extract of Andrographis paniculata on TNFot. The results showed that the water extract of Andrographis paniculata was an antagonist of TNFoe, the water extract with a concentration of 500 mg/ml had a 34% inhibitory effect, and no cytotoxicity was observed. 10. The L929 cell proliferation/cytotoxicity test was performed by Andrographis paniculata. The water extract was found to be an inhibitor of TNFa in the plant components isolated from the aqueous extract of Andrographis chinensis by HPLC to identify possible inhibitors of TNFa. After the collected plants were washed and dried, water was added to the weighed plants (10 / 1, volume / weight) to extract the plant components. The extraction step consisted of blending the mixture and collecting the mash after centrifugation at 8 000 rpm for 30 minutes. The procedure was repeated twice and all of the above mash was concentrated and concentrated on a rotary evaporator (Heidolph Laborota 4000) until the final volume was reduced to approximately 50 ml. Following the separation step, 100 microliters of concentrated sputum (i.e., plant extract) was applied to a pre-equilibrated HPLC system (Shimadzu). Separation was performed using a TSK Gel 80TM reverse phase chromatography column (TOSOH). The solvent used for the separation was double distilled water and anhydrous ethanol, and the gradient was 〇~1〇〇% for 96 minutes, and the flow rate was 0.75 ml/min. -16- (12) (12)1355933 Collect the fractions at 1 minute and dry them with SpeedVac (Savant). Each fraction was redissolved in 1 ml of H20 to screen for TNFoc inhibitors. Fractions with TNFa inhibitor activity were purified by HPLC until the purity was above 95%. The procedure as described above was used to find a compound having TNFct inhibitor activity in the methanol extract of Andrographis paniculata. The crude methanol extract of Andrographis paniculata was isolated by using a TSK Gel 80tm (TOSOH) reverse phase chromatography column. The column is filled with a 5 μm diameter and a column size of 250 x 4.6 mm. The mobile phase used was a mixture of acetonitrile (B) and hydrazine 20 (Α) at a flow rate of 0.75 ml/min. The column was washed as follows in the following manner: 0 ° / for the first 5 minutes. ~30% B; 5~25 minutes is a linear gradient of 30%~50% B; 50~70°/ 25~30 minutes. B; 30~35 minutes is 70~100% B; 35~45 minutes is 100% B. Detection is performed at a wavelength of 230 nm with a detection sensitivity of 〇·〇1 AUFS. The fraction with TNFa inhibitor activity was purified from the collected solution by a TSK Gel ODS 80tm (TOSOH) reverse phase chromatography column with a residence time of 20 to 21 minutes. Fractions were identified by LC/MS and their residence time was the same as the andrographolide standard analyzed using HPLC. 1 1. Evaluation of the effect of andrographolide as a TNFa inhibitor by L929 cell proliferation/cytotoxicity test The inhibitory effect of andrographolide on TNFa was determined by the same L929 cell proliferation/cytotoxicity test as described above on a microtiter plate. . The inhibitory effect and cytotoxicity of andrographolide on TNFa of L929 cell line is shown in Figure -17-(13) 1355933. The results in Fig. 5 show that when the andrographolide concentration is high, the percentage of inhibition is increased, and it is found that 値 as a TNFa inhibitor is 60 μΜ, and no cytotoxicity is found at this concentration. 12. The effect of andrographolide on the inflammation of large rats with collagen-induced arthritis

SPF-class SD large mice are supplied by BioLasco. Animals were acclimated for 4 days after receiving the animals before proceeding. The weight of each animal, blood test, paw volume and other related records were measured. Each mouse was injected with the original protein II-IFA (incomplete Freund's adjuvant, Sigma for immunization and subsequent injection to induce arthritis (CIA). CIA was divided into 6 groups, and the drug candidate, ie, andrographolide, was injected daily. Dexamethasone (0.2 mg) was used as the positive group, and 5% ethanol was used as the negative control group. The treatment period was 7 days. Body weight and paw volume, the blood test at 0' 3, 6, 10 and 14 days last time Six days after the administration of the drug, all animals were sacrificed. The hind paws were taken for tissue analysis. Body weight and paw volume before and after treatment with the drug candidate were measured and compared. On the day, it was found that inflammation was induced by collagen, and the hind paw volume was swollen to 2 to 2.5 times of the normal hind paw (Fig. 6A!). The group treated with andrographolide showed a small edema volume of the hind paw 4 8 · 1 On the 3rd and 7th day after treatment was stopped, the edema was reduced by 6 1.4 3% and 5 0.24%, respectively (Figures 7A and 7B). The dexamethasone group was used as a positive control group during the treatment period and the non-treatment group. For comparison, increase IC5, anti-hair test, scale cattle Glue should be) rat. Make a control measurement. During the infection period, a 6B had a reduced treatment, and the percentage of edema volume decreased by 28.1 1% and 29.97% on days 3 and 7 after -18-(14) (14)1355933 stopped taking. Figures 8A and 8B). Among all arthritis samples, significant histopathological changes, loose connective tissue, lymphocytosis around the joints, edema around the joints, and proliferation of the lining of the joints were observed (Figures 10-15). ), but in normal samples there is no such situation (Figure 9). Synthesis Example: Preparation of 3-ethoxylated andrographolide A mixture of andrographolide (5 g) and triphenylmethane chloride (10 g) in anhydrous pyridine (30 ml) at 60 °C Heat for 6 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled and diluted with diethyl ether. The organic layer was washed with an aqueous solution of copper sulfate, then washed with water and dried over Na 2 SO 4 . After the solvent was removed, the chromatographic method was carried out on a silica gel column (23 0 to 400 mesh size, using a mixture of light gasoline·ethyl acetate=6:4 as an eluent), and 19-trityl andrographolide (5 g) was obtained. The 19-trityl andrographolide (1 g) prepared in the above procedure was retained in distilled acetic anhydride (40 ml) for 5 minutes. After completion of the reaction (monitored by TLC), the contents were cooled to room temperature, diluted with water and then extracted with dichloromethane. The organic layer was separated, dried over Na 2 SO 4 and concentrated. The crude material was purified by flash chromatography (purified column: 230 to 400 mesh size, elution system was a mixture of chloroform and acetone 95: 5) to obtain pure 3-ethoxycarbonyl-1 9-triphenyl. Methyl andrographolide (300 mg). -19- (15) (15)1355933 3-Ethyloxy-19-trityl andrographolide (300 mg) prepared above with a mixture of formic acid and dichloromethane (1:1) (10 ML) was treated at room temperature for 10 minutes. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was diluted with EtOAc EtOAc. After the solvent is removed, the chromatographic method is carried out on a ruthenium tube column (23 0 to 400 mesh size, and the elution system is a mixture of chloroform and acetone 92:8) to obtain 3-acetonitrile. Oxyandrographolide (100 mg), which is a colorless solid, mp 205 0 C, m/z 3 92. • H-NMRiCDCh): <5 6.95 (t, 1H, H-12), 5.0 (d, 1H, H-14), 4.9 (s, 1H, H-17a), 4.65 (m), 4.6 (s , 1H, H-17b), 4.45 (dd, 1H, H-3), 4.25 (d), 4.15 (d, 1H, H-19a), 3.4 (d, 1H, H-19b), 2.5 (m) , 2.1 (s, 3H), 1.2-2.0 (m), 1.1 (s, 3H), 0.7 (s, 3H). BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows SDS-P AGE (sodium dodecyl sulfate polypropene) purified rhTNFa (recombinant human TNFa) (column 2) and biotinylated rhTNFcc (column 3) Amine gel electrophoresis). Figure 2 shows a chromatogram of the crude methanol extract of Andrographis paniculata on a TSK Gel ODS 80TM (TOSOH) reverse phase chromatography column (250 x 4.6 mm) filled with 5 micron micelles. Figure 3 shows the water thickening of Andrographis ternary -20-(16) (16)1355933 on a TSK Gel 0DS 80TM (TOSOH) reverse phase chromatography column (250 x 4.6 mm) filled with 5 micron colloidal particles. Chromatogram of the extract. Figure 4 shows a chromatogram of andrographolide on a TSK Gel ODS 80TM (TOSOH) reverse phase chromatography column (250 x 4.6 mm) filled with 5 micron micelles. Figure 5 shows the percentage inhibition and cytotoxicity of the aqueous andrographolide solution on the microtiter plate in the proliferation/cytotoxicity test of L929 cells in vitro. Fig. 6A shows the hind paw of a large mouse that has been induced by collagen, and edema and erythema are observed. Figure 6B shows the hind paw of a large mouse before it is induced by collagen. Fig. 7A shows the left hind paw volume change (percent reduction) of the test group treated with andrographolide. The volume T0 is before the injection of CII-INF, T1 is before treatment, and T3 is on the 6th day of treatment. 'T4 and T5 are the 3rd and 7th days of administration of andrographolide, respectively. Figure 7B shows the percentage of paw enlargement calculated as volume change when the treatment group was compared to the untreated group at different time points. T3 is 1 -(T3-T1/T1-T0)%' T4 is 1-(T1-T4/T1-T0). /. , and T5 is 卜 (Τ 1 - Τ 5 / Τ 1 - Τ 0) %. Figure 8 shows the left hind paw volume treated with dexamethasone. The volume T0 is before the injection of CII-IFA, T1 is before treatment, and T3 is on the third day of treatment, 'T4 and T5 are on the 3rd and 7th days of taking dexamethasone, respectively. Figure 8 shows the percentage of paw enlargement calculated by volume change when the treatment group was compared with the untreated group at -21 - (17) (17)1355933 at different time points. T3 is 1 -(Τ3-Τ1/Τ1-Τ0)%, Τ4 is 1-(Τ1·Τ4/Τ1-Τ0)%, and Τ5 is 1·(ΤΙ-Τ5/Τ1-TO)%. Figure 9 shows normal tissue sections of joints that were not immunized with collagen sputum. Figure 10 shows histopathological sections of rats suffering from arthritis induced by collagen 11 immunization and treated with andrographolide (intestinal administration). Proliferation of the inner lining cells and penetration of red blood cells and some lymphocytes were observed. Figure 11 shows histopathological sections of rats suffering from arthritis induced by collagen sputum immunization and treated with andrographolide (oral administration). Proliferation of the lining cells of the joint and penetration of the lymphocytes were observed. Fig. 1 2 shows histopathological sections of rats suffering from arthritis caused by collagen π immunoinjection and treated with andrographolide (external sputum). It exhibits proliferation of the lining cells of the joint and loose connective tissue. Figure 13 shows histopathological sections of rats suffering from arthritis induced by collagen II immunization and treated with dexamethasone (intestinal administration). Proliferation of the lining cells of the joints and penetration of some lymphocytes were observed. Figure 14 shows histopathological sections of rats suffering from arthritis induced by collagen II immunization and treated with 5% ethanol (intestinal administration). Proliferation of the lining cells of the joints and penetration of some lymphocytes were observed. Figure 15 shows histopathological sections of rats suffering from arthritis induced by collagen n immunotherapy and treated with dexamethasone (intestinal administration). Surgery around the joint and penetration of the lymphocytes were observed. -22- (18) (18)1355933 References (1) Rec. Trav. Chim. 1911,30,15 1-160. (2) (a) Toxicology in Vitro. 1999, 13(6), 897~903 (b) Fitoterpia. 1 99 5,66(5),415~420. (c) International Journal of Pharmacognosy. 1 995, 33(2), 135~138. (d) Asia Pacific Journal of Pharmacology. 1 994 , 9(2), 73~78. (e) Journal of Ethnopharmacology. 1993, 40(2), 131-136. (3) (a) Zhongguo Zhongyao, 2002, 27(2), 147-150. (b Journal of Natural Products. 1 9 9 3, 56(7), 995~999. (4) (a) Biological and Pharmaceutical Bulletin. 2002, 2 5(9), 1 1 69~1 174. (b) British Journal of Pharmacology. 2 002, 1 3 5(2), 399^406. (c) Planta Medica. 2000, 66(4), 3 1 4~3 1 7 . (d) British Journal of Pharmacology. 2000,129 (8), 1553-1560. (e) Phytotherapy Research. 1998, 12(1), 37-40. (f) Fitoterpia. 1 996, 67(5 ), 452~45 8. (5) (a) Life Science. 2000, 66(8), 709~723· -23- (19) (19)1355933 (b) Indian Journal of Pharmacology. 1994, 26(4), 296-300. (6) Journal of Experimental Therapeutics and O NCology. 2003,3 (3),1 4 7~ 1 5 8 . (7) Phytotherapy Research. 2000,14(5),3 3 3~3 3 8. (8) (a) BMC Pharmacology. 2003,3 (b) Drug Delivery. 2000, 7(4), 209~213. (9) Phytomedicine. 2 0 0 2, 9(7), 589~5 97. (10) CN 1293 955. CN 1 266699. ( 11) Phytomedicine. 1 999,6(1), 27-31. (12) Pharmacological Research. 1 99 8,3 8 (6),4 1 3~4 1 7 . (13) Phytotherapy Research. 1 999,13 (2), 157~159. (1 4) (a) Indian Journal of Pharmaceutical Sciences. 1 998,60(3),1 7 6- 1 7 8. (b) Pharmaceutical Biology. 1 998, 3 6( 1 ), 72-74. (15) International Journal of Crude Drug Research. 1 990, 28(4), 273-283 . (16) (a) Bangladesh Pharmaceutical Journal. 1 9 7 7, 6(2), 21~ 24. (b) Planta Medica. 2003, 69(1 2), 1 075~1 079. (17) WO 2003 03 5 007. WO 2003 00 7975 (18) Phytotherapy Research. 1998, 12(1), 37~ 40.

Claims (1)

1355933 Ο) / (4) Qing (Pseudo-Replacement Page) Patent Range 1. A pharmaceutical composition for inhibiting or antagonizing TNF-ct in a mammal, comprising an amount of orthoquinol which is effective for inhibiting or antagonizing TNF-ct Lactone 2. A pharmaceutical composition for treating a condition or condition involving TNF-α, comprising an amount of andrographolide effective to inhibit or antagonize TNF-α, wherein the condition involving TNF-α comprises Rheumatoid arthritis. -25-