TW200944223A - Medicine composition containing Korean ginseng for making HIV-1 have 5'LTR and/or gag defects - Google Patents

Abstract

The present invention relates to using Korean ginseng as active ingredients of medicine composition that is able to make HIV-1 with 5'LTR and/or gag gene defects. By giving the pharmaceutical compositions of this invention to an HIV infected individual, 5'LTR and/or gag gene defects in HIV-1 can be induced. Therefore, this invention can be used for AIDS prevention and treatment.

Description

[Technical Field] The present invention relates to a pharmaceutical composition containing Korean ginseng as an active ingredient for causing HIV-1 to have a 5'LTR and/or gag gene defect. [Prior Art] HIV-1 (Human Immunodeficiency Virus-1) is the cause of Acquired Immunodeficiency Syndrome (hereinafter referred to as "AIDS"). HIV-1 and HI V-2 belong to the lentiviral family of the retroviridae, which has an RNA (+) chromosome formed by two identical single strands. When HIV-1 infects a cell, the RNA is converted into double-stranded DNA by the reverse transcriptase encoded by the above RNA (+), and the DNA is inserted into the chromosome of the host cell to form a provirus. In the proviral state, the virus is proliferated by transcription of the mRNA of the protein necessary for the production of the viral gene in replication and assembly. The HI V-1 genome (9.2 kb) consists of the structural genes of gag, pol, env, and the accessory genes of tat, rev, nef, vif, vpu, vpr, and the long terminal repeats at both ends. (long terminal repeat sequence (LTRs)) configuration (refer to Fig. 8). The gag and pol proteins decompose to form a precursor protein, and the env protein becomes a gpl60 precursor protein, which is decomposed into gpl20 and gp41 during assembly. Initially infected with HIV, there is no symptom, or it is manifested as a cold, and the virus is latent infection in CD4 T cells, giant sputum cells, small sputum cells, etc., and is in a latent state for a long time. Antibodies to gpl 20 appear 3 to 20 weeks after infection, after which 01343-TW / OTW 9869 JJP 5 200944223 can be diagnosed for infection. After an incubation period of 2-1 years, the immune system induces changes. 'Before the symptoms of immunodeficiency, it shows prodromal symptoms (AIDS-related syndrome (ARC) - fatigue, fever, weight loss, abdominal cleansing) ° due to HIV Infection causes the number of CD4 T cells to decrease to less than 200/ul. At this time, symptoms of immunodeficiency are manifested, which is clinically defined as AIDS - Panax ginseng C. A· Meyer ( Panax schinsengNees) belongs to Wu Jia Araliaceae ginseng's multi-year-old perennial herb has been used in East Asian countries for the treatment of various diseases for thousands of years. Many pharmacological experiments to date have shown that Korean ginseng has the following activities: lowering cholesterol, anti-lipid peroxidation, lowering blood pressure, increasing blood flow, dilating cerebral blood vessels, improving heart function, anti-arrhythmia, anti-blood test, and treatment of chronic renal failure. Effects, immune regulation, memory enhancement, brain metabolism, anti-stress, anti-oxidation, anti-aging, anti-ulcer and inhibition of gastric secretion, anti-diabetes, detoxification, increase of hepatocyte enzymes, treatment of asthma, anti-inflammatory, analgesic effect, Treating anemia, improving fertility, lowering blood sputum concentration, anti-allergic, anti-cancer, etc. In addition, Korean ginseng and red ginseng (Red Ginseng) are distinguished according to processing methods. The so-called Korean ginseng is not processed. 'The dried Korean ginseng is called dry ginseng, and the undried Korean ginseng is called ginseng. Red ginseng is steamed and dried to make the moisture content 14% or less. During the preparation process, the browning reaction is promoted to form a hard form which is a dark brown color and is kept in a round shape of Korean ginseng. Therefore, 'Koryo ginseng is difficult to store for a long time in the state of ginseng containing about 75% of water, and it is caused by microbial contamination in the circulation state. 01343-TW / OTW 9869 JJP 6 200944223 Corruption, or various enzymes contained in Korean ginseng itself The Korean ginseng ingredient occurs as a solution, and the red ginseng has the advantage of preventing bacterial, moldy, and microbial contamination by reducing moisture after processing and drying, and has the advantages of reduced volume and weight, and easy storage and transportation. Due to the development of the quantitative method of saponin and the development of the product, which is a representative effective component of Korean ginseng, I can minimize the decomposition of the sputum during processing. In addition to the bitterness, it is possible to further produce active ingredients such as maltitol and ginseng Rh2. That is, the main component of Korean ginseng is 41, and red ginseng is a high-grade ginseng that has not changed its function after processing. Red ginseng has sedative and excitatory effects especially for the middle sacral nerve. It acts on the circulatory system to prevent hypertension and arteriosclerosis. In addition, it can reduce hematopoiesis and blood sugar levels, protect the liver, and act indirectly on the endocrine system. It is effective in sexual and reproductive functions, has anti-inflammatory and anti-tumor effects, has anti-radiation effects, protects and smoothes skin. In addition, it is important in the effect of red ginseng to increase the defensive ability of various tears and various types of stresses from the surrounding environment as an adaptor effect, making the body more adaptable. Effect. So far, there have been many studies on the ingredients and pharmacological effects of red ginseng in Korea and abroad. In particular, it has been reported that red ginseng has antioxidant activity (Xi Cai > • 3 y 7 > 々g, Korean ginseng society, 24(1), 29_34, 2〇〇〇). In addition, the present inventors have previously studied the treatment of HIV-1 infected patients with separate Korean ginseng (KRG, Korean red ginseng) (see references 1-4), and after 1991, using Koreans to participate in azidothymidine ( Treatment of ZDV, zidovudine) (see Reference 5), and treatment of highly active antiretroviral drugs 01343-TW / OTW 9869 JJP 7 200944223 with Koreans after 1997 (HAART, highly active antiretroviral drug therapy) (See Reference 6) e During clinical application (see References 1-7) 'There is a significant inverse correlation between KRG treatment and progression rate of acquired immunodeficiency syndrome (AIDS) (See Reference 8) The coefficient of variation of the β ίπν gene is inversely related to 〇G treatment. Moreover, KRG treatment can delay the development of resistance to azidothymidine. According to the findings of the present inventors, EG treatment can reduce the decrease of CD4 τ cells by irrespective of other characteristics such as treatment or patients. Analysis of the combination therapy of KRG and HARRT revealed that no drug-resistant mutations were present, and consistent viral regulation was achieved by this combination therapy (see Reference 7). The cohorts of the present inventors herein describe acquired immunodeficiency syndromes that are visible from long-term non-progressors (LTNPs) or long-term survivors (LTSs). The reason for the slow progress. Recently, the present inventors reported on the correlation between the ratio of & large-scale defects of the nef gene of 10 LTSs which were continuously treated with KRG and the KRG uptake time (see Reference 9). The amplified products in the nef gene of these 10 long-term survivors were compared, and as a result, in the products amplified in the gag region and the 5'LTR region, the full-length domain sequence of the provirus exhibited two bands at a high ratio (band). ) (wild type and unshortened band). A number of studies have been conducted on large-scale defects in the nef gene in LTNP (see References 1〇-18). However, the correlation of large-scale defects in the gag region and the 5'LTR region has been reported so far in only a few occasions. . More specifically, 'one that has been infected with HIV subtype C has been reported

01343-TW / OTW 9Β69 JJP 200944223 Defects in one of the three amino acid codons in the gag gene of LTS (see reference 19). In other studies, two of the 24 untreated patients showed i6 bp defects and 9 bp minor defects in the 5' LTR (see reference 20). However, there is no report on the possible link between maintaining a state of non-deterioration over a long period of time or a slow progression of HIV infection and a genetic defect in the 5' LTR and gag regions. References: 1. Cho YK, Kira YK, Lee I, Choi MH, and Shin Y0: The ❿ effect of Korean red ginseng (KRG), zidovudine (ZDV), and the combination of KRG and ZDV on HIV-infected patients. J Korean Soc Microbiol 1996; 31:353-360.

2. Cho YK, Lee HJ, Kim YB, Oh WI, and Kim YK: Sequence analysis of C2/V3 region of human immunodeficiency virus type 1 gpl20 and its correlation with clinical significance; the effect of long-term intake of KRG on env Gene variation. J ❹

Korean Soc Microbiol 1997; 32:611-623. 3. Cho YK, Lee HJ, and Desrosiers RC: Complete sequences of HIV-1 in a Korean longterm nonprogressor * with HIV-1 infection. J Korean Soc Microbiol 1999; 29:107 -118. 4. Cho YK, Sung H, Kim TK, Lim J, Jung YS, and Kang SM: KRG significant slows CD4 T cell depletion over 10 years in HIV-1 infected patients: association with 01343-TW / OTW 9869 JJP 9 200944223 HLA. J Ginseng Res 2004; 28:173-182. 5. Cho YK, Sung H, Ahn SH, Bae IG, Woo JH, Won YH, et al. : High frequency of mutations conferring resistance to nucleoside reverse transcriptase inhibitors In human immunodeficiency virus type-1 infected patients in Korea. J Clin Microbiol 2002;40:1319-1325.

6. Cho YK, Sung H, Lee HJ, Joo CH, and Cho GJ: Long-term intake of KRG in HIV-l-infected patients: development of resistance mutation to zidovudine is delayed. Int Immunopharmacol 2001; 1:1295-1305 7. Sung H, Jung YS, Kang MW, Bae IG, Chang HH, Chang MS, et al. : High frequency of drug resistance mutations in human immunodeficiency virus type 1-infected Korean patients treated with MART. (In press), AIDS Res Hum Retroviruses. 8. Sung H, Kang SM, Lee MS, Kim TG, and Cho YK: KRG slows depletion of CD4 T cells in human immunodeficiency virus type 1-infected patients. Clin Diagn Lab Immunol 2005; 12:497- 501. 9. Cho YK, Lim JY, Jung YS, Oh SK, Lee HJ, and Sung H: High frequency of grossly deleted nef genes in HIV-1 infected longterm survivor streated with Korean red ginseng. Curr HIV Res 2006; 4: 457-467. 01343-TW/OTW9869JJP 10 200944223 10. Kirchhoff F, Greenough TC, Brettler DB, Sul 1ivan JL, and Desrosiers RC: Brief report: absence of intact nef sequences in a long-term survivor with non Progressive HIV-1 infection. N Engl J Med 1995; 332:228-232.

11. Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, et a 1. : Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 1995; 270 : 988-991. 12. Mariani R, Kirchhoff F, Greenough TC, Sullivan JL, Desrosiers RC, and Skowronski J: High frequency of defective nef alleles in a long-term survivor with nonprogressive human immunodeficiency virus type 1 infection. J Virol 1996 70:7752-7764. 13. Salvi R, Garbuglia AR, Di Caro A, Pulciani S, Montel1 a F, and Benedetto A: Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor. J Virol 1998;, 72:3646-3657. 14. Brambilla A, Turchetto L, Gatti A, Bovolenta C, Veglia F, Santagostino E, et al. : Defective nef alleles in a cohort of hemophiliacs with progressing and nonprogressing HIV-1 infection Virology 1999; 01343-TW / OTW 9869 JJP 11 200944223 259:349-368. 15. Rhodes DI, Ashton L, Solomon A, Carr A, Cooper D, K Aldor J, et al. : Characterization of three nef-defective human immunodeficiency virus type 1 strains associated with long-term nonprogression. Australian Long-Term Nonprogressor Study Group. J Virol 2000: 74:10581-10588. 16. Foster JL, Molina RP, Luo T, Arora VK, Huang Y, Ho DD, et al. : Genetic and functional diversity of human immunodeficiency virus type 1 subtype B Nef primary isolates. J Virol 2001; 75:1672-1680. 17. Casartelli N, Di Matteo G, Argentini C, Cancrini C, Bernardi S, Caste 11i G, et al. : Structural defects and variations in the HIV-1 nef gene from rapid, slow and nonprogressor children. AIDS 2003; 17:1291-301. Rodes B, Toro C, Paxinos E, Poveda E, Martinez-Padial M, Benito JM, et al. : Differences in disease progression in a cohort of long-term non-progressors after more than 16 years of HIV-1 infection. AIDS 2004; 18:1109-1115.

19. McCormack GP, Glynn JR, Clewley JP, Crampin AC, Travers SA, Redmond N, et al. : Emergence of a three codon deletion in gag pi 7 in HIV type 1 subtype C 01343-TW / OTW 9869 JJP 12 200944223 long -term survivors, and general population spread. AIDS Res Hum Retroviruses 2006; 22:195-201. 20. Copeland KF, Chen Z, Fiebig M, Ni L, Savoy .S, Smai11 FM, et al.: Identification of mutations in Proviral long terminal repeats of HIV type 1-infected subjects naive to drug therapy. AIDS Res Hum Retroviruses 2004; 20:1019-1021.

21. Cho YK, Foley BT, Sung H, Kim YB, and Kim JH: Molecular epidemiologic study of an HIV-1 outbreak in hemophiliacs B infected through clotting factor 9 after 1990. Vox Sanguinis. 2007; 42:113-120, 22 Alexander L, Weiskopf E, Greenough TC, Gaddis NC, Auerbach MR, Malim Μ H, et al. : Unusual polymorphisms in human immunodeficiency virus type 1 associated with nonprogressive infection. J Virol 2000; 74:4361-4376. 23. Zhang L, Huang Y, Yuan H, Chen BK, Ip J, and Ho DD: Genotypic and phenotypic characterization of long terminal repeat sequences from long-term survivors of human immunodeficiency virus type 1 infection. J Virol 1997;71:5608-5613. 24. Huang Y, Zhang L, and Ho DD: Characterization of gag and pol sequences from long term survivors of human immunodeficiency virus type 1 infection. 01343-TW / OTW 9869 JJP 13 200944223

Virology 1998;240:36-49. 25. Calugi G, Montella F, Favalli C, and Benedetto A: Entire genome of a strain of human immunodeficiency virus type 1 with a deletion of nef that was recovered 20 years after primary infection:large Pool of proviruses with deletions of env. J Virol. 2006;80:11892-11896.

26. Blankson JN, Bailey JR, Thayil S, Yang HC, Lassen K, Lai J, et al. : Isolation and characterization of replication-competent human immunodeficiency virus type 1 from a subset of elite suppressors. J Virol. 2007; 81: 2508-18. 27. Jones KA, Kadonaga JT, Luciw PA, and T j ian R:

Activation of the AIDS retrovirus promoter by the cellular transcription factor, Spl. Science. 1986; 232:755-59. φ 28. Keum YS, Han SS, Chun KS, Park KK, Park JH, Lee SK, et al. : Inhibitory Effects of the ginsenoside Rg3 on phorbol ester-induced cyclooxygenase-2 expression, NF-kappaB activation and tumor promotion. Mutation Res. 2003;523-524:75-85. 29. Kira KH, Lee YS, Jung IS, Park SY, Chung HY, Lee IR, and Yun YS. Acidic polysaccharide from Panax ginseng, ginsan, induces Thl cell and macrophage 01343-TW / OTW 9869 JJP 14 200944223 cytokines and generates LAK cells in synergy with rIL-2. Planta Med. 1998;64 :110-115. 30. Han SK, Song JY, Yun YS, Yi SY. Ginsan improved Thl immune response inhibited by gamma radiation. Arch Pharm Res. 2005;28:343-50. 31. Larsen MW, Moser C, Hoiby N, Song Z, Kharazrai A. Ginseng modulates the immune response by induction of interleukin-12 production. APMIS. ® 2004;112:369-73. 32. Lee EJ, Ko E, Lee J, Rho S, Ko S, Shin MK, et al. Ginsenoside Rgl enhanc Es CD4(+) T cell activities and modulates Thl/Th2 differentiation. Int Imraunopharmacol. 2004; 4: 235-44. [Summary of the Invention] The present inventors studied AIDS treatment by licking HIV-1 The patient has long been administered red ginseng to confirm the defects of the 5, LTR and gag genes in Hiv-1, thereby completing the present invention. SUMMARY OF THE INVENTION An object of the present invention is to provide a pharmaceutical composition comprising Korean ginseng as an active ingredient for causing HIV-1 to have a 5, LTR and/or gag gene defect. In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for causing HIV-1 to have a 5,ltr and/or gag gene defect containing Korean ginseng as an active ingredient. 01343-TW / OTW 9869 JJP 15 200944223 The invention will be described in detail below. The present invention is characterized in that it is found that the uptake of Korean ginseng makes it possible to produce a defect in the LTR/gag gene which is closely related to the growth of the virus and the cause of AIDS. Preferably, the invention is characterized in that the Korean ginseng is red ginseng. In one embodiment of the present invention, in order to determine whether there is a correlation between the occurrence of large-scale defects (2Δ) in the LTR and gag regions and KRG uptake, the inventors have used KRG (total 13, 364). ±5, 364g) 10 long-term survivors who treated more than 12 years of long-term survival (l〇ng_term survivi〇rs; Ltss)' and no uptake or small intake of Korean ginseng (total l, 436±l, 027g) The 1,125 domain-based sequences surrounding the 5, LTR and gag regions were examined (control). Of the above 1 LTS, 189 PCR products were obtained in 80 peripheral blood mononuclear cell (PBMC) samples, 44 out of 8 PBMCs (55%) and 71 out of 189 PCR products (37 6 %) shows a large-scale defect (g^). The 5% of the LTS were found to be 55% 0 of the PBMC and 37.6% of the PCR products, but the LTS values of the eight control groups were 30.3% and 14.8%, respectively. Such differences are statistically significant (P < 0.05, and P can be 〇). Further, the proportion of defective genes in 28 general control patients who did not take red ginseng was 13 · 3 % and 8 _ 3 % based on PBMC and PCR products, respectively (refer to Example 3' Fig. 1 to Fig. 3). Therefore, by giving red ginseng to an individual, it is possible to induce a defect in the 5'LTR/gag gene in HI V-1, thereby contributing to prevention and treatment of AIDS. The red ginseng used as an active ingredient of the pharmaceutical composition of the present invention can be produced by a method known from 01343-TW / OTW 9869 JJP 16 200944223. In general, red ginseng is subjected to water at a certain temperature (about water) 75%) is obtained by a steaming stage of steaming and a drying stage of drying the steamed ginseng. The red ginseng thus obtained is subjected to heat extraction to obtain a red ginseng extract, and the red ginseng extract is concentrated to obtain a red ginseng concentrate. Further, in the present invention, the above red ginseng extract or concentrate may be used alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents, according to a conventional method. The above "medicine permit" {refers to physiologically acceptable means that when administered to a human body: - an allergic reaction which does not cause gastrointestinal disorders, dizziness, or the like, or a reaction similar thereto. Further, the red ginseng of the present invention formulated as described above can be administered by an appropriate administration route. Suitable administration routes may be oral administration, preferably in the form of powders, granules, tablets, pills, sugar-coated tablets, sputum liquid preparations, gels, syrups, suspensions, etc. for oral administration. A known method is used for formulation. For example, the preparation is prepared by mixing the active ingredient with a solid excipient, adding it to February, and adding a suitable auxiliary agent to form a mixture of granules, bismuth tablets or sugar-coated tablets. Suitable excipients may, for example, be glucose, sucrose, sorbitol, mannitol, xylitol::=:, and maltitol in (iv) sugars, including corn (four), small starch, and potato starch. Astringent class, not

Vitamins-based fibers-propylmethylcellulose Si II 01343-TW / OTW 9869 JJP 17 200944223 Vitamins, including fillers such as gelatin and polyvinylpyrrolidone. Further, a disintegrating agent including crosslinked polyvinylpyrrolidone, bad fat, alginic acid, or sodium alginate may be added depending on the case. Further, the pharmaceutical composition of the present invention may further contain a deflocculant, a lubricant, a wetting agent, a perfume, an emulsifier, a preservative, and the like. Further, red ginseng which is an active ingredient of the pharmaceutical composition of the present invention is commercially available. In one embodiment of the present invention, a red ginseng powder capsule sold by the Korean Ginseng Corporation is used. The pharmaceutical composition of the present invention can be used for administration to mammals, particularly animals including humans. It is preferred to administer a patient infected with Ηΐν-丨 in an effective amount. The above "effective amount" means an effective amount for causing a defect in the nef gene in HIV-1 in vitro or in vivo. The effective amount of the pharmaceutical composition of the present invention needs to take into consideration various factors such as the route of administration, the number of treatments, the age, body weight, health status, sex, severity of disease, diet, and excretion rate of the individual necessary for treatment, and those skilled in the art can The appropriate effective dose is determined according to the particular use. Preferably, the effective amount of the red ginseng-containing pharmaceutical composition of the present invention is 50-1 00 mg/kg/day for men and 2〇-7 mg/kg/day for women. The pharmaceutical composition of the present invention can be administered continuously after the diagnosis of the individual to delay the development of the AIDS disease. In addition, in the present invention, the defect of the above 5'LTR and/or gag gene can be defined as having a premature stop (premature stop). Codon ), lack of initiation codon (initiati〇n codon) e In one embodiment of the invention, amplification products generated from 10 LTS

01343-TW / OTW 9869 JJP 18 200944223 A pre-stop codon caused by a non-methionine initiation codon, a G to A hypermutation, and a small defect gene defect (Example 4) were observed. Defects, duplications or insertions are not exactly the same size in each patient and in 10 LTS. Even in the eight control LTSs, gene defects caused by premature stop codons and non-methionine start codons were observed (Fig. 4). Preferably, the defective 5'LTR and/or gag gene produced by the method of the present invention can be expressed as the following sequence: GenBank accession numbers EF 〇370172, EF 370173, EF 370175, EU 047612, EF 370184, EF 3 701 86, EF 370187, EF 370188, EF 370191, EF 3701 92, EF 3701 93, EF 3701 96, EF 3701 97, EF 3701 99, EF 370201, • EF 370 200, EF 37020 2, EF 370 205, EF 3 7020 7, EF 3702U, EF 370213, EF 37021 1 5, EF 3702116, EF 3702118, EF 370219, EF 37022, EF 370223, EF 370224, EF 370225, EF 370226, EF 370227, EF 370228, EF 370231, EF 370235, EF 370237, EF 370239, EU 047647, EF 370245, EF 370250, 〇EF 37025 EF 370252, EF 370253, EF 370256, EF 370259, EF 370262, EF 37026 EF 370265, DQ 2 951 96, EF 370275, EF 3 70276, EF 370277, EF 3702778, EF 37027 9, EF 370 282, EU 047673, EF 370283, EF 370285, EF 370286, EF 370287, EF 370288, EF 370292. As described above, the pharmaceutical composition of the present invention can induce HIV-1 to have a 5'LTR and/or gag gene defect by administering to an HIV-infected individual. Therefore, the present invention can be used for the prevention and treatment of AIDS. 01343-TW / OTW 9869 JJP 19 200944223 [Embodiment] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to exemplify the invention, and the present invention is not limited by the following examples, and various modifications and changes can be made. Example 1 Experimental items and KRG treatment ® The study group included: 1 long-term survivors (LTSs; l〇ng-term surviv〇rs) who ingested the greatest amount of KRG, and 8 control groups of HIV-infected patients with KRG. In this study, an LTS has been described in detail in the inventors' previous studies (see Reference 9). All of the above 10 LTS were diagnosed between 1987 and March 1996. In this study, no patients were given any antiretroviral treatment except one patient (93-04). The patient (93-04) discontinued ZDV (daily dose 200 mg or 3 mg) from April 1993 to August 1997. Eight control group LTSs were selected by the inventors' research group (c〇h〇rts). The patients assigned to the above-mentioned control group LTS were infected with HIV-1 in 1996, and they used the minimum dose (L526H, 183g) of krg without strong antiretroviral therapy. No use of KRG at all (patient 89_〇5 completely unused KRG eight only one patient 91-23 from April 993 to August 999 using ZDVe patient 89-05 went to other countries in 1997, therefore Removed from the study sample of the present invention. In 8 control group LTS, including long-term survival

01343-TW / OTW 9869 JJP 200944223 The two recipients of the 91-20 blood transfusion (recipients, patients 9 Bu 22 and 9 Bu 23). Patients 89-26 and 92-23 resisted uptake of KRG. Patient 90-01 was performed on 83 plasma donors who provided plasma for a fee. This patient 90-01 is referred to as the provider 〇 (see reference 21). Therefore, the baseline characteristics are the same when comparing one high KRG uptake LTS with eight control lts.

换 There was no significant difference in the rate of basic disease progression (pr〇gressi〇n rate) or CD4 T cell between the 10 LTS and the 8 control LTS except for the intake of KRG. In addition, 28 other control patients were also investigated. These 28 people also include 87-05 and 90-50 wife. Some of these 28 patients are long-term survivors. However, they were diagnosed after 1997 and therefore are not included in the long-term survivor group. Each participant provided a consent form. KRG treatment for patients infected with HIV-1 began in the second half of 1991 at the NIH in Korea. The KRG used in the experiment was a red ginseng powder capsule sold by the Korean Ginseng Corporation. The amount of KRG per day for each patient was 5. 4 g (- three times a day, three capsules per day), and 1.8 g for women. The KRG administration time for each patient is shown in Figure 。. ^ , The average dose of red ginseng taken by 10 LTS was 13, 364 ± 5,364 g' in the 155 ± 28 month period! The sputum a of the red ginseng taken was during the period of 132 ± 29 months (Fig. 5). In the _ 1 LTS and the 8 control LTS, the annual dose of KRG was ίο// 131 g. In the KRG intake level, 7.9 times has a statistical H (P < 0.001) 〇, 01343-TW / OTW 9869 JJP 21 200944223 Example 2 CD4 T cell number and plasma HIV-1 RNA replication level (C0py ievei) After 3 months to 6 months, the patients in the sputum of the example were subjected to blood collection, and the peripheral mononuclear cells in each sample were combined with phycoerythrin' PE and luciferin isothiocyanate. (The antibody corresponding to the CD4 antigen of FIT (7) was cultured (Siraultest reagent; Becton Dickinson, San Jose, CA, USA). The level of CD4 T cells was measured by FACScan (Becton-Dickinson) flow cytometry (fjow cytometry). Plasma HIV-1 RNA concentrations were determined by the Amp licor HIV-1 Monitor kit (Roche Diagnostics, Branchburg, NJ) (see Reference 7) « Every 6 months. Visually ingested 1 LTS and 8 of red ginseng The number of CD4 T cells in the control group LTS was the same as that shown in Fig. 1. The number of CD4 T cells receiving 10 LTS of the maximum red ginseng treatment dose was significantly reduced from the initial measurement of 555 ± 208 / ul to 245±160/ul (-year reduction) measured after 168 soil for 29 months 22/id, Figure la to Figure 1 j). In addition, the number of CD 4 T cells in 8 control LTS decreased from the initial measured 5 7 4 ± 2 2 2 / u 1 to 132 ± 29 months. 353 ± 297 / ul (-year reduction of 20 / ul, Figure lk to Figure lr). It can be confirmed that the number of CD4 T cells in both groups decreased in a similar manner in 10 years. 01343-TW / OTW 9869 JJP 22 200944223 Example 3 Partial 5' LTR region and pre-amplification viral DNA of the gag gene were extracted from the uncultured PBMCs obtained in Example 2. By using the first round primer CE23 (5 '-tgtggatctaccacacacaaggctactt-3'; 46 to 73; SEQ ID NO: 1), 514 (5,-tccagaatgctggtagggtatac-3'; 1,617 to 1,640; SEQ ID NO: 2), second round of primer CE1 (5'-cgagagctgcatccggagtacta- 3'; 297 to 319; SEQ ID NO: 3), 512 (5'-ctgcagcttcctcattgatggtc-3'; 1,398 to 1.420; SEQ ID NO: 4), third round of primer 501 (5'-gtgtggcctgggcgggactg-3,; 380 to 399 ; SEQ ID NO: 5) and 510 (5'-gatgtaccatttgcccctgga-3, ; 1,202 to 1,222; SEQ ID NO: 6) Heavy (very few triple) nested polymerase chain reaction (nested PCR), from i. 297 to 1,421 (here and below, numbered from the sequence of HIV NL4-3), i25 bp PCR product Sample amplification (the above positions from 297 to 1,421 are representative of a portion of the 5' LTR portion and a portion of the gag gene). The maximum pcR response was performed once for each sample containing the negative control group, and the maximum PCR reaction was performed once for each sample containing the negative control group in order to amplify the nef gene. Refine the PCR products and sort them directly. The presence or absence of contamination of the pcR was monitored by physical separation of the PCR product, and the physical separation of the pcR product was performed by 35BLAST search, amino acid codon comparison by manual crypto-alignment, and systematic analysis.

01343-TW / OTW 9869 JJP 23 200944223 The experimental results are expressed as mean ± standard deviation. Statistical significance was confirmed by SPSS package version 12. 0 by Student's two-tailed t-test and Chi-square test. As a result of the experiment, 80 PMBC samples obtained from 1 LTS at different time points were amplified into 189 PCR products (Fig. 1a to Fig. 1j). At most time points, all of the 1 LTSs showed a gΔ at the 5, LTR/gag gene region (Fig. 1). Defects were observed in 44 (55%) of the 80 samples (Fig. 2). Of the 189 pCR products, 71 (37.6%) showed gA5'LTR/gag. Among them, 23 (32.4%) amplifications showed only a short band and no wild type gene. The characteristics of the 71 LTR/gag are summarized in Figure 7. 5 PCR products (from patient 87-05 in January 2007, patient 89-17 in July 2001, patient 90-05 in October 1993, and patient 92- in February 2006 and May 2004) 13 obtained) and 2 PCR products (Δ86 bp obtained from patient 90-05 in 1993 and Δ420 bp obtained from patient 90-50 2006 in 2006) contained gΔ only in the 5, LTR region or gag gene, respectively. (table 3). The defects in the remaining PCR products were located at 5, the two-terminal portion of the LTR (containing at least the 753 position; A37 bp) and the initial portion of the gag gene (Δ 29 bp at position 818 in HIV-1 NL43). In other words, the defect is 66 bp or more in the vicinity of the start codon of the gag gene. Therefore, the ratio of g Δ in the 5' LTR region was 36 5% (69/189), and the ratio of g Δ based on the gag gene was 34.9% (66/1). This indicates that the effect of KRG administration on the 5' LTR is greater than that on the gag gene.

01343-TW/OTW9869JJP 24 200944223 The initial observation of gΔ in the 5' LTR and gag regions after the start of KRG uptake was earlier than the initial observation of nef gene defects, except for patients with no samples at 9 and 20 and 93-60. . The intermediate time from the onset of KRG to the initial gΔ observation was 26 months (19 months to 101 months). This is shorter than the time required for nef gene defect observation. Patient 87-05 was simultaneously infected with HIV-I subtype B and HCV from a contaminated corporate fluid coagulation factor of 9 produced abroad. The patient started KRG administration on June 3, 1994. Although there were small defects in _ in all of the amplifications, in 10 LTS towels, only the patient did not express heart (4) (see Reference 9). Surprisingly, the inventors observed gΔ in the initially useful samples obtained in April 1997, and a total of 7 gΔ were observed from 36 PCR products obtained from 4 out of 12 samples (Fig. Up to the figure 'Figure 5'. The patient did not show any symptoms associated with Acquired Immunodeficiency Syndrome since April, which was the first patient in Korea to survive for more than 2 years without any antiretroviral therapy. Patient 89 17 began KRG administration in December 1991. However, compliance with administration is not consistent. An exception was reported for a gA reported in July 1993. The reason is that △ 917 bp is replaced by the 474 bp of the p〇1 gene (in the opposite direction NL4-3, No. 4,296 to No. 4,037) and the termination portion of the (iv) gene (2,029 bp to 1,838 bp). The patient started HARRT treatment in February 2002. The number of CD4 T cells was 513/ul in April and April. Patient 90-05 was diagnosed with invy infection in February 1990 and KRG was started in December 1991. This patient in the study for the krg treatment table

01343-TW / OTW 9869 JJP 25 200944223 The best compliance is achieved. In addition to the 21 522 g of KRG supplied, the patient also ingested his own KRG. The dose of KRG in the 95% compliance is 6.0 g (- twice a day, 1 capsule per capsule). The initial observation of gΔ was 87 months earlier than the initial observation of gAnef in January 2001 (KR (KRG12, 012g in total for the first month after the start of J administration) (Fig. 6a, 6b, 6c) The details of gΔ and the details of the remaining 7 LTSs are shown in Figure i and Figure 6. In addition, 88 PCR products of 33 pBMC samples obtained from the additional 8 control LTSs were amplified. 13 g Δ (1. 8% of the amplified pcR product) were present in 1 PBMC sample (total 3 〇 3%). Compared to L0 LTS on PBMC samples and PCR products Eight control groups LTS' contained a significant south ratio of gΔ (previously p<〇. 〇5, latter Ρ&lt;〇·0001) (Fig. 3). In addition, KRG was never given 28 in the clinical stage. Sixty PCR products from 30 PBMC samples obtained from each patient were amplified. Five gΔ (8.3% of the amplified PCR products) were present in four pMC ® (13.3% in total). Example 4 Genetic Defects and Small Defects In addition to large-scale defects, the inventors observed genetic defects such as non-methionine initiation codon and G to a hypermutation. Early termination codon and small defects. In 10 LTS, patient 87-05 is shown in 4 sequences (EU〇476〇1, eu〇47605, 01343-TW / OTW 9869 JJP 26 200944223 EU047607, EU047609) Premature stop codon, patient 89-17 also contains isoleucine as a premature stop codon and start codon (GenBank EF370193). Patient 90-18 has a 6 bp insertion in the gag gene 'patient 92-13 has L〇2bp (GenBank EF370252) two duplications, 159bp defect in the 5' LTR region, and G to A hypermutation (Genbank EF370256), and the patient has regions 327 and 328 (GenBank EF370258) 6 bp insertion (Fig. 4). ® Patient 9b 22 in the control group LTS has an isoleucine as a start codon in the premature stop codon or 3 sequences (GenBank EF370314, EU047654, EU047655) Patient 89-26 has two premature stop codons in one sequence ' (EU047635). Example 5 Frequency of large-scale defects that vary with the number of CD4 T cells 〇 The inventors analyzed the number of CD4 T cells. Different big Defect mode frequency. Among them, it is preferable to group 10 LTS PCR amplification products in accordance with the CD4 T cell number of 200 / ul or less, 200 - 400 / ul, and 400 / ul or more. The frequency of large-scale defects in the 10 LTSs given KRG was 47.4% (9/19), 51.8% (14/27), and 61.8% (21/34), indicating the CD4 T Several dependencies on the number of cells. However, in the 8 control group LTS, the proportion of large-scale defects was 44.4% (4/9), 20% (4/20), 50% (2/4), and 28 HIV-not given KRG- 1 positive patient was 18. 2% ( 2/11 ), 14. 3% 01343-TW / OTW 9869 JJP 27 200944223 (1/7) ' 8. 3% ( 1/12) » Discussion

The present inventors have long been concerned about the health of LTS or LTNP in Korea, and as a result, even if there is no beneficial host factor such as a 32 bp defect in CCR5 or a Korean HLA-B57 'the present inventor can Most LTS of the 323 HI V-1 infected individuals diagnosed on December 31 were observed. More specifically, nine HI V-1 infected patients were diagnosed in Korea in 1987. Three of the patients were still alive, and all three were treated with KRG alone or with HARRT. Long-term survivor 87-05, one of these patients, began to take KRG after June 1996 (Fig. la). The other two of these patients received both HARRT and KRG treatment from 1998 to 1999. Their CD4 T cell count increased from 231/ul in 1998 to 6/ul in 1999 to 1 in December 2004. , 〇52/ul, 1,127/ul in 2006. In addition, 22 patients with HI V-1 were diagnosed in 1988, and only two patients (9%) who underwent gRG treatment were still alive. The patient's H丨v_〗 subtype is CRF02_AG, so this patient is not included in the study of the present invention, but the patient was assigned to the K4 treatment of most patients diagnosed after 1997 with the number of CD4T cells above 500/ul. And with HARRT treatment, they do not have drug resistance mutations (drug resistance mutati〇ns)' these patients showed a good response to the parallel treatment. The present inventors have reported HIV-1 infected patients through previous studies.

01343-TW / OTW 9869 JJP 28 200944223 There is a link between KRG treatment and the high frequency of large defects in the nef gene (see Reference 9), and KRG treatment can slow the progression of the disease (see Reference 18). In the present invention, 'three PCR products from each sample are amplified. Although this is less than four PCR products amplified by the nef gene (see Reference 9), the inventors are in the same LTS. It was observed that compared with the nef defect (34.3% of the sample and 18.8% of the PCR product) (see Reference 9), large-scale defects (55% of the sample and 37 6 © % of the pcR product) The frequency is more (Ρ&lt;〇·〇〇〇1)°5' The defect frequency of LTR/gag is 1. 6 times / 2. times higher than the frequency confirmed in the nef gene defect. Amplification of the nef gene was previously performed in 61 of the total of 8 samples. Thirteen of the 61 samples (21.3%) had large defects in both genes. In particular, patients 87-05, 89-17, 90-05, 90-50, 96-51 showed a significantly higher frequency of large-scale defects in the LTR/gag gene than in the nef gene (P&lt;0. 05) (Figure 5). In addition, the proportion of samples showing large-scale defects was confirmed to be 37.6% in 1 LTS treated with KRG, which was better than 8 对照组 control group LTS (1.48%) and 28 ΗIV-1 infected patients. (8.3%) is obviously higher. Moreover, in the present invention, it can be confirmed that there is no relationship between the large-scale defect frequency and the number of CD4 T cells in any of the eight control group LTS or other 28 patients (Example 5), according to which it can be proved The relationship between large-scale defects and KRG intake. The above findings show a good comparison with the appropriate dependence (m〇dest dependency) on the number of CD4 Τ cells in 1 LTS. The number of CD4 T cells was maintained in 87-05, 90-05, 92-13, and 96-51 patients who had been consistently treated with KRG (Figures la, ic, 01343-TW / OTW 9869 JJP 29 200944223 lg , 1 j ), but on the contrary, the number of CD4 T cells of patients 90-50, 91-20, 93-60 that were not adhered to in the last 1-2 years was rapidly reduced (Fig.ie, 1 f, ! i ). As mentioned above, the reduction in the number of CD4 T cells caused by attitude has already occurred in patients 89-17 and 90-18 (Fig. lb, Id). There are no AIDS-related symptoms for a long period of more than one year, so most patients have a tendency to ignore their health or ignore the intake of KRG. Several recent papers have published the genetic characteristics of full-length viral sequences in LTNP. Alexander et al. (see Reference 22) found no premature termination of codons, out-of frame deletions, and overall defects, except for defects found in other genes in the nef gene. Uncommon p〇lymorphism and nef gene large-scale defects were found in 8 LTNPs. In addition, Huang et al. (see references 23, 24) reported genetic defects in the 5, LTR and gag genes in eight LTSs. Full-length viral sequences were obtained in this study, and G to a hypermutations were found only in the 5'LTR and gag genes of one patient. Recently, Cal ugi et al. identified large-scale defects in the env gene (a gene adjacent to the nef © gene) (see references)

25) Blankson et al. (see Reference 26) are not in the domain-based sequence of HIV-1 full-length in the inhibitory factor (SUpressor) at a viral load (virai i〇ads) of 50 copies/mL:&gt; Discover any large-scale defects.

HIV-1 gene expression is regulated by several South-preserved LTSs in the binding region containing the Tata box, transcription factor NF-kB, and Spl (see Reference 27); cis acung elements. Certain components of ginseng inhibit human cell lines

NF-kB and Ap-丨 transcription factor activity in 01343-TW/OTW9869JJP 30 200944223 (see Reference 28). The LTR fraction is more likely to be affected by 〇G uptake than other parts of HIV-1. In the present invention, a large-scale defect of 5' LTR was observed in 10 LTS including patient 87-05 (not showing a large-scale defect of the nef gene). Moreover, the large scale defects observed are larger in size, but only in a single location within the gene. The present inventors confirmed large-scale defects of the env gene of six LTSs (patients 90-05, 90-18, 90-50, 9b 20, 92-13 ® and 96_51) when obtaining the Hiv-i full-length domain-based sequence ( &gt; 1 000 bp) (data not shown). Therefore, 'the relationship between KRG uptake and gene defects is not considered, and although the observation of the present invention is only one of various beneficial factors obtained in the study of the present invention, the results of the present invention provide preliminary information, which preliminary Data indicate that large-scale defects in the 5' LTR and gag portions are significantly associated with the rate of progression of jjiv disease (see References 9). Regarding the possible mechanism by which viral gene defects affect LTS or LNTP, the inventors hypothesized that gene fragmentation is the result of indirect immunomodulation caused by uptake of KRG. It is well known that Korean ginseng mediates the general immune activity state in HIV-1 infection by mediating the cell mediated immune response of Thl cytokines (see Reference 2 9-32). Immune activation state). However, LTS without KRG still has a more aberrant 5&apos; LTR/gag deficiency compared to the other 28 patients infected with HIV&apos; so KRG uptake may not be the only factor. This finding demonstrates the importance of gene defects caused by krg uptake in the life extension of LTS or LNTPs 01343-TW / OTW 9869 JJP 31 200944223. <Sequence DATA>&gt; A total of 178 of the 336 domain-based sequences have been submitted to GenBank, and GenBank accession numbers are given: EF370172 to EF370349, EU047600 to EU047667, EU047670 to EU047676, And EU047681 to EU047693. Industrial Applicability Specifically, the pharmaceutical composition of the present invention can induce HIV-1 to have a 5'LTR and/or gag gene defect by administering to an HIV-infected individual. Therefore, the pharmaceutical composition provided by the present invention can be used for the prevention and treatment of AIDS. 01343-TW/OTW9869JJP 32 200944223 [Simplified Schematic] Figure 1 shows 8 control groups of 8 control groups without the presence of sputum-retroviral therapy, administration of the most + KRG or no KRG. Lk~r) and a large number of KRG-administered sputum and the most LTS (Fig. la~j) CD4 T cell number changes. The period in which the number of red ginseng periods is given is represented by a bar (bar). = PCR reaction accompanied by a sample - phantom increase ' 5 LTR and gag gene used in the sample of 戾 and 5, τ Τ 1 Λ 序列 序列 sequence 5 LTR and gag gene large-scale defects in the lower part of the downward arrow Buckle au @ +

❹ Head Xin said upward arrow. In both groups, the number of c T cells decreased after 1 year. Accounted for lL ^ 4 dry silly for V. Thus, there was no significant difference between the two except for the amount of KRG administered. Fig. 2 is a view showing sequence analysis of 5 LTR and gag in peripheral blood mononuclear cells obtained from three LTSs initially diagnosed. The product amplified by electrophoresis of an agarose gel was isolated. The PCR products from patients 87_〇5, 8917, 9〇 〇5 are represented by lanes uo, 1828, 19 31, respectively. Patient 87-05 has 4 wild types (wild type, 3, 6, 9), with 3 repeat bands (lanes 2, 7, i〇) and two single short bands (lanes 4, 8). Five short belts. Patient 894 7 represents 7 short bands (lanes, 12, 13, 14, 16-18), of which track 14 is a short band (14%) » patient 90-05 has 7 short bands (channel 19, 22, 23, 24, 26, 29, 30), wherein the tracks 22, 24, 26, 29, 30 appear as a short band (71%). (M: mark 'WT: wild type band)

Fig. 3 is a view showing comparison of three groups of large-scale defect frequencies. The proportion of peripheral blood mononuclear cells representing large-scale defects, KRG LTS (55%) was not only higher than that of the other 28 patients (13.3%), but also compared with 9 control groups LTS 01343-TW / OTW 9869 JJP 33 26.7% of 200944223 is high. Moreover, the proportion of PCR products positive for large-scale defects, one LTS (37.6%) given KRG was not only significantly higher than the other 28 patients (8.3%), but also compared with the eight control groups LTS Ip3 9% is significantly higher. The 28 patients also included 87-05 and 90-50 wives. Moreover, some of them may become LTS even if they have not been diagnosed with HIV-1 for more than a year. Fig. 4 is a diagram showing the predicted amino acid sequence of a representative partial gag protein of 10 LTS infected with HIV-1. The common amino acid sequence of the protein from non-progressive individuals in the United States is shown in the top (see Reference 2 3)' and the expected sequence of one LTS is as follows. The point (d〇t) indicates the same part of the sequence, and the bar (bar) indicates a part or a large defect other than the variant part. In addition, the 'question mark indicates the early termination of the codon. The amino acid sequence is represented by a text code. The source of each sequence is coded by each patient (the first two digits indicate the year of HIV diagnosis, and the two-digit digits after the character indicates the unique number assigned to each patient in Korea). The samples corresponding to the respective GenBank numbers are sampled. The date is shown in Figure 7. 7 Seven kinds of domain-based sequences lacking defects in the gag gene are not seen in Fig. 7. Fig. 5 is a table showing the clinical features of 18 LTSs infected with inv-i. In addition to the past year, the compliance of patients 91-20 in the LTS was the same as that of the 90-05' and his actual KRG intake reached more than 22,422g through personal purchases. Patients 91-22 and 91-23 were infected with Hiv-1 by receiving blood supplied by 9 Bu 20. Except for LTS 87-05, all patients were infected with the Korean branch of HIV-1 subtype B (subcUde). Figure 6 shows the 18 LTS and 28 non-KRG-administered patients. 01343-TW / OTW 9869 JJP 34 200944223 5 Characteristics of large-scale defects of the 'LTR/gag gene. Fig. 7 is a table showing the frequency of large-scale defects of the 5'LTR/gag gene as compared with the large-scale defect of the nef gene. [Main component symbol description]

01343-TW / OTW 9869 JJP 35 200944223 Sequence Listing &lt;110&gt; Korea Ginseng Corporation Ulsan University School of Industry and Research Association &lt;120&gt; Contains Korean ginseng for making HIV-1 5' LTR/gag gene defect Pharmaceutical composition &lt;150&gt; KR10-2008-0037555 &lt;151&gt; 2008-04-23 &lt;160&gt; 6 &lt;170&gt; Patentln version 3.3

&lt;210&gt; 1 &lt;211>28 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; First round primer CE23 &lt;400&gt; 1 tgtggatcta ccacacacaa ggctactt &lt;210&gt; 2 &lt;211&gt 23 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; First round primer 514 &lt;400>2 tccagaatgc tggtagggta tac &lt;210&gt; 3 &lt;211&gt; 23 &lt;212&gt; DNA &lt;;213&gt; Manual sequence &lt;220&gt; 01343-TW / OTW 9869 JJP 36 200944223 &lt;223&gt; Second round of primers CE1 &lt;400&gt; 3 cgagagctgc atccggagta eta &lt;210&gt; 4 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Second round of primer 512 &lt;400&gt; 4 ctgcagcttc ctcattgatg gtc &lt;210&gt; 5 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial sequence&lt;220&gt;&lt;223&gt; third round of primers 501 &lt;400&gt; 5 gtgtggcctg ggcgggactg

&lt;210&gt; 6 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Third round of primer 510 &lt;400&gt; 6 gatgtaccat ttgcccctgg a 01343-TW / OTW 9869 JJP 37

Claims (1)

200944223 X. Patent application scope: 1. A pharmaceutical composition for causing HIV-1 to have a 5'LTR and/or gag gene defect, characterized in that the composition contains Nanli ginseng as an active ingredient. 2. The composition of claim 1, wherein the Korean ginseng is red ginseng. 3. The composition of claim 1 or 2, wherein the composition is for administration to a patient infected with HIV-1. 4. The composition of claim 2, wherein the male is administered at a dose of 50-100 mg/kg/day, and administered at 20-70 mg/kg/day. The amount is administered to women. 5. The composition of claim 1 or 2, wherein the 5&apos; LTR and/or gag gene defect is selected from the group consisting of having an early termination codon and a lack of a start codon. 6. The composition of claim 2, wherein the 5'LTR and/or gag gene defects are expressed as sequences having the following GenBank accession numbers: EF 370172, EF 370173, EF 3701 75 , EU 047612, EF 370184, EF 370186, EF 370187, EF 3701 88, EF 370191, EF 370192, EF 370193, EF 3701 96, EF 370197, EF 370199 'EF 370201, EF 370200, EF 370202, EF 370205, EF 370207 , EF 370211, EF 370213, EF 3702115, EF 3702116, EF 3702118, EF 370219, EF 370221, EF 370223, EF 370224, EF 370225, EF 370226, EF 370227, EF 370228, EF 37023 EF 370 235, 01343-TW / OTW 9869 JJP 38 200944223 EF 370237 , EF 370239 , EU 047647 , EF 370250 , EF 370251 , EF 370252 , EF 370253 EF 370259 , EF 370262 , EF 370261 , EF 2951 96 , EF 370275 , EF 370276 , EF 370277 , EF 370279 , EF 370282, EU 047673, EF 370285, EF 370286, EF 370287, EF 370288 370245, EF, EF 370256, 370265, DQ EF 3702778, 370283, EF, EF 370292 . 01343-TW / OTW 9869 JJP 39