JP2009263327A - PHARMACEUTICAL COMPOSITION FOR 5'LTR/gag GENE DEFICIENCY OF HIV-1, CONTAINING PANAX GINSENG AS EFFECTIVE INGREDIENT - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for 5'LTR (long terminal repeat sequence) and/or gag (group-specific antigen) gene deficiencies, containing Panax ginseng as an effective ingredient. <P>SOLUTION: The invention relates to a pharmaceutical composition for HIV-1 5'LTR and/or gag gene deficiencies, containing Panax ginseng as an effective ingredient. The deficiencies of HIV-1 5'LTR and/or gag gene can be induced by administration of the pharmaceutical composition to the HIV-infected individuals. Therefore, the present invention can be applied for the prevention and treatment of AIDS. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a pharmaceutical composition for HIV-1 5'LTR and / or gag gene deficiency comprising ginseng as an active ingredient.

  Human immunodeficiency virus type 1 (HIV-1) is the etiology of acquired immunodeficiency syndrome (AIDS). HIV-1 belongs to the retroviral lentiviridae together with HIV-2, and infectious virions have an RNA (+) genome consisting of two identical single strands. When HIV-1 infects cells, RNA is converted to double-stranded DNA by the reverse transcriptase encoded by the RNA (+) genome, and this DNA is inserted into the host cell chromosome to form a provirus. And, in the provirus state, transcription of the viral gene produces mRNAs of proteins necessary for replication and construction, and the virus is propagated.

  The HIV-1 genome (9.2 kb) is composed of gag, pol, env structural genes, accessory genes such as tat, rev, nef, vif, vpu, vpr, and long terminal repeat sequences (LTR). ) And.

  The gag protein and the pol protein are produced by degrading the precursor protein, and the env protein becomes the gp160 precursor protein, which is degraded by gp120 and gp41 during the construction process. When HIV is first infected, there are no symptoms or symptoms such as colds, and the virus is latently infected with CD4 T cells, macrophages, microglia cells, etc., and enters a long-term latent state. An antibody against gp120 appears 3 to 20 weeks after infection, and the presence or absence of infection can be diagnosed after this time. Changes in the immune system are induced during the incubation period of about 2 to 10 years, and prodromal symptoms (AIDS-related complex (ARC): fatigue, fever, weight loss) before immune deficiency symptoms appear Diarrhea) appears. If the number of CD4 T cells is reduced to 200 / μl or less due to HIV infection, symptoms of immune deficiency appear and are clinically defined as AIDS.

  Ginseng (Panax ginseng CA Meyer (Panax schinseng Nees)) is a perennial perennial plant belonging to the genus Panax of the Araliaceae family, and has been supporting the treatment of various diseases in the Oriental region for thousands of years. Have been used. Ginseng has undergone many pharmacological experiments to lower cholesterol, inhibit lipid peroxidation, lower blood pressure, increase blood flow, cerebral vasodilation, cardiac dysfunction, antiarrhythmia, antithrombosis, chronic renal failure treatment effect, immune regulation Action, increased memory, increased brain metabolism, anti-stress, anti-oxidation, anti-aging, anti-ulcer and gastric secretion inhibition, anti-diabetes, detoxification, hepatocyte enzyme increase, asthma treatment, anti-inflammatory, analgesic, anemia treatment, It is known to have the effects of promoting fertility, lowering alcohol blood levels, antiallergy, and anticancer agents.

  In addition, ginseng and red ginseng are classified according to the processing method. Ginseng refers to unprocessed one, dried one is called dry ginseng, and undried one is called ginseng. Red Ginseng refers to a ginseng steamed and dried to a moisture content of 14% or less. The browning reaction is promoted during the manufacturing process, and it has a strong brown color. Is a ginseng with a round shape.

  Therefore, it is difficult to preserve for a long time in the state of ginseng containing about 75% water, and ginseng components are decomposed by various enzymes contained in the ginseng itself due to spoilage due to microbial contamination during distribution. Red ginseng has the advantage that it is easy to store and transport because it reduces moisture by processing and drying, prevents contamination by bacteria, sputum and microorganisms, and reduces volume and weight. Quantification of saponin, a representative active ingredient of ginseng, and the development of quality control make it possible to minimize the degradation of saponin during the processing process. In addition to saponin, maltol and ginsenoside Rh2 Further active ingredients such as were generated. That is, the main ingredient of ginseng is saponin, and red ginseng is processed so that this effect is not altered.

  Red ginseng in particular has sedative and excitatory effects on the central nervous system and acts on the circulatory system to prevent hypertension and arteriosclerosis, while lowering hematopoiesis and blood glucose levels to protect the liver. It acts on the endocrine system and acts indirectly on sexual behavior or reproductive effects, has anti-inflammatory and anti-tumor effects, has a protective effect against radiation, and has an action of softening while protecting the skin. In addition, the important effect of red ginseng is that it adapts more easily by increasing the protective ability against various adverse effects such as epiphora and various stress caused by the surrounding environment as an adaptogen effect. Has the effect of So far, many studies have been conducted on the ingredients and pharmacological effects of red ginseng, both in Korea and abroad, and it has been reported that red ginseng has anti-oxidative activity (Kwon Yong Hoon et al., Korea). Ginseng Journal, 24 (1), 29-34, 2000).

In addition, the present inventor found that in previous studies, HIV-1 infected patients were treated with ginseng (hereinafter referred to as KRG) alone (references 1 to 4), or after 1991, zidovudine (ZDV). Along with (Reference 5), after 1997, the patient was treated with highly active antiretroviral drug therapy (HAART) (Reference 6). During clinical application (Refs. 1-7), a meaningful inverse correlation was shown between KRG treatment and the rate of progression of AIDS (Ref. 8). The HIV-1 gene shift rate is inversely related to treatment with KRG. Furthermore, treatment with KRG delays the development of resistance to zidovudine. According to the inventor's results, treatment with KRG reduced the rate at which CD4 T cells were depleted regardless of the therapy or other characteristics of the patient. Recent results analyzing mixed treatments with KRG and HAART showed that consistent virus regulation is possible with these combinations without the occurrence of drug resistance shifts (Ref. 7) .

The reason why the progression of AIDS in the long-term nonprogressor (LTNP) or the long-term surviviors (LTS) in the inventor's cohort is delayed will be described here. Recently, the present inventor has reported an association between the ratio of large-scale deletion of the nef gene in 10 LTS treated with continuous KRG and the duration of KRG intake (Ref. 9). . Comparing the products amplified with the nef gene from these 10 LTSs, the product amplified in the gag region and the 5 ′ LTR region has two bands (wild Molds and short bands) appeared at a high rate.

  Many studies have been conducted on large-scale deletions of the nef gene in LTNP, but only a few cases have reported the association of large-scale deletions in the gag region and the 5 ′ LTR region to date (references 10-10). 18). More specifically, one LTS infected with HIV subtype C was reported to have one deletion in 3 amino acid codons in the gag gene (Ref. 19). In other studies, 2 of 24 people without treatment experience showed a 16 bp deficiency and a small 9 bp deficiency in the 5 'LTR (Ref. 20). However, there has been no report of an association between 5 'LTR region and gag region genetic defects, which is caused by maintaining the state where there is no long-term progression or slowing the progression of HIV infection.

  The present inventor completed the present invention by confirming the deficiency of 5 'LTR and gag gene of HIV-1 when red ginseng was administered to a patient infected with HIV-1 for a long time while studying the treatment method of AIDS. did.

  An object of the present invention is to provide a pharmaceutical composition for HIV-1 5 'LTR and / or gag gene deficiency comprising ginseng as an active ingredient.

  In order to achieve the above object of the present invention, the present invention provides a pharmaceutical composition for HIV-1 5 'LTR / gag gene deficiency containing ginseng as an active ingredient. The present invention will be described in detail below.

  The present invention is characterized by the possibility of deficiency of HIV-1 5'LTR / gag gene, which is closely related to viral growth and AIDS etiology, by ingestion of ginseng.

  Preferably, the ginseng is red ginseng.

  In one embodiment of the present invention, in order to investigate whether there is a relationship between the occurrence of large-scale deletions (gΔ) in the 5 ′ LTR region and the gag region and the intake of KRG (total amount 13,364 ± 5 out of 10 LTSs treated for 12 years or more and 8 LTSs (control group) who did not take KRG or took a small amount (total 1,436 ± 1,027 g) (partial 5) The nucleotide sequence of the 1,125 bp PCR product representing the 'LTR part and the gag gene was examined. In the 10 LTSs, 189 PCR products were obtained in 80 peripheral blood mononuclear cell (PBMC) samples, 44 (55%) in a total of 80 PBMC and 189 in PCR products. 71 (37.6%) showed large-scale defects (gΔ). 55% PBMC and 37.6% PCR products were found in 10 LTS, while the 8 control group LTS values were 30.3% and 14.8%, respectively. Such differences are statistically significant (P <0.05 and P = 0). Furthermore, the ratio of the defective genes in 28 patients in the general control group who did not take red ginseng was 13.3% and 8.3%, respectively, based on PBMC and PCR products (Example 3, FIGS. 1 to 3). reference).

  Therefore, administration of red ginseng to an individual can induce HIV-1 5 'LTR / gag gene deficiency, which can be used for the prevention and treatment of AIDS.

  Red ginseng used as an active ingredient of the pharmaceutical composition of the present invention can be produced by a known method.

  In general, red ginseng is produced through a steaming step in which steamed ginseng (water content: about 75%) is steamed under a certain temperature condition and a drying step in which steamed water ginseng is dried. The red ginseng extract can be produced by heating and extracting the red ginseng produced in this way and then concentrated to produce a red ginseng concentrate.

  On the other hand, in the present invention, the above red ginseng extract or concentrate is used alone, or one or more pharmaceutically acceptable carriers, excipients or diluents are additionally contained, and the agent is prepared in a usual manner. Can be used after shaping. The term “pharmaceutically acceptable” means a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Say. In addition, the red ginseng of the present invention formulated as described above can be administered through an appropriate administration method. A suitable method of administration may be oral administration.

  Preferably, it is prepared using methods known in the art in the form of preparations for oral administration such as powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, slurries, suspensions, etc. Can be shaped. For example, an oral preparation can be obtained by blending an active ingredient with a solid excipient, pulverizing it, adding an appropriate adjuvant, and then processing into a granule mixture to obtain a tablet or a sugar-coated tablet.

  Suitable excipients such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol and other sugars, corn starch, wheat starch, rice starch and potato starch, cellulose, methylcellulose Fillers such as celluloses including sodium carboxymethylcellulose and hydroxypropylmethylcellulose, gelatin, polyvinylpyrrolidone and the like may be included. In some cases, cross-linked polyvinyl pyrrolidone, agar, alginic acid or sodium alginate can be added as a disintegrant. The pharmaceutical composition of the present invention may further contain an anti-aggregating agent, a lubricant, a wetting agent, a fragrance, an emulsifier, an antiseptic and the like.

  Moreover, the red ginseng which is an active ingredient of the pharmaceutical composition of this invention can purchase and use a commercially available thing. In one embodiment of the present invention, red ginseng powder capsules sold by Korea Ginseng Corporation were used.

  The pharmaceutical composition of the present invention can be administered to mammals, particularly animals including humans. Desirably, it can be administered in an effective amount to patients infected with HIV-1. The “effective amount” refers to an amount effective to delete the nef gene of HIV-1 in vitro or in vivo.

  The effective amount of the pharmaceutical composition according to the present invention takes into consideration various factors such as the route of administration, the number of treatments, the age, weight, health status, sex, severity of the disease, diet and excretion rate of the individual in need of treatment, One skilled in the art can determine the appropriate effective dosage for a particular application. Desirably, an effective amount of a pharmaceutical composition comprising red ginseng of the present invention is 50-100 mg / kg / day for men and 20-70 mg / kg / day for women.

  The pharmaceutical composition of the invention can be administered continuously to delay the progression of AIDS disease after diagnosis of the individual.

  On the other hand, in the present invention, the 5 'LTR and / or gag gene deficiency can be defined by having an early maturation stop codon and lacking a start codon.

  In one embodiment of the present invention, genetic defects such as a non-methionine start codon, an early maturation stop codon due to GA hypermutation, and a small deletion are generated from amplification products generated from 10 LTSs. Observed (Example 4). The size of the defect, duplication, or insertion all appeared differently in each patient and 10 LTS.

  A genetic defect due to the early maturation stop codon and non-methionine start codon was also observed in the 8 control group LTS (see FIG. 4).

  Desirably, the 5 'LTR and / or gag gene deleted by the method of the present invention is represented by the following sequence. Genbank registration number EF370172, EF370173, EF370175, EU047612, EF370184, EF370186, EF370187, EF370188, EF370191, EF370192, EF370193, EF3700197, EF37020, E3700201, EF37002, EF37021118, EF370219, EF370221, EF370223, EF370224, EF370225, EF370226, EF370227, EF370228, EF370235, EF370235, EF370237, EF 370239, EU047647, EF370245, EF370250, EF370251, EF370252, EF370253, EF370256, EF370259, EF370262, EF370261, EF370265, DQ295196, EF370275, EF370276, EF370277, EF3702778, EF370279, EF370282, EU047673, EF370283, EF370285, EF370286, EF370287, EF370288, EF370292

  As described above, the pharmaceutical composition of the present invention can cause a deficiency in the 5 'LTR and / or gag gene of HIV-1 when administered to an HIV-infected individual. Therefore, the present invention can be used for the prevention and treatment of AIDS.

  Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are intended to illustrate the present invention, and the present invention can be variously modified and changed without being limited by the following examples.

  FIG. 1 shows administration of a minimum of ginseng without antiretroviral treatment, or 8 control groups LTS (FIGS. 1k to 1r) that did not receive ginseng and a maximum of ginseng. Figure 3 shows the change in CD4 T cell counts relative to human LTS (Figures 1a-1j). The administration period of red ginseng is shown by a straight line. As the three PCR reactions amplify one sample, samples used for 5 'LTR and gag gene sequences and samples showing large-scale deletions of 5' LTR and gag genes are below each Indicated by the lower and upper arrows. In both groups, the number of CD4 T cells decreased over a decade. There was no significant difference between the two groups except for the dose of ginseng.

  FIG. 2 shows analysis of 5 'LTR and gag sequences in peripheral blood mononuclear cells obtained from the three LTS diagnosed earliest. The amplified products were separated by electrophoresis on a 1% agarose gel. PCR products from patients 87-05, 89-17, 90-05 were expressed in lanes 1-10, 11-18, 19-31, respectively. Patient 87-05 appears to have 4 wild-types (lanes 1, 3, 6, 9) with 3 overlapping bands (lanes 2, 7, 10) and 2 single short bands (lanes 4, 8) Had five short bands. Patient 89-17 showed 7 short bands (lanes 11, 12, 13, 14, 16-18), of which lane 14 was one short band (14%). Patient 90-05 showed 7 short bands (lanes 19, 22, 23, 24, 26, 29, 30). Among them, lanes 22, 24, 26, 29 and 30 showed one short band (71%) (M: marker, WT: wild type band).

  FIG. 3 is a bar graph comparing the frequency of large-scale defects in the three groups. Peripheral blood mononuclear cell samples representing large defects were further increased in LTS administered KRG from 26.7% of 9 control LTS as well as 28 other patients (13.3%) High (55%). Moreover, the ratio of positive PCR products for large defects is 37.6% in 10 LTSs administered ginseng, 13.9% in 8 control group LTSs, and It was significantly higher than the rate of 8.3% in the other 28 patients. Among the 28 patients were the wife of patients 87-05 and 90-50. Moreover, some of them had the potential to become LTS, although 10 years have not passed since the diagnosis of HIV-1.

  FIG. 4 shows the predicted amino acid sequence of a representative partial gag protein of 10 LTSs infected with HIV-1. The consensus amino acid sequence of gag proteins from non-progressive patients in the United States is shown in the top row (Ref. 23), and the predicted sequence for 10 LTSs is shown in the bottom row. The dots indicate that the sequences are identical, and the straight line indicates a partial or large deletion other than the mutated portion. The question mark means an early maturation stop codon. The amino acid sequence is indicated by one letter code. The origin of each sequence was confirmed by each patient code (the first two digits indicate the year of HIV diagnosis and the two digits after the hyphen indicate the unique number assigned to each patient in Korea) . The date of sampling for each Genbank registration number is shown in FIG. In FIG. 7, seven types of base sequences not containing the gag gene deletion were not shown.

  FIG. 5 is a table showing the clinical characteristics of 18 LTSs infected with HIV-1. Except for the last year, the compliance of patients 91-20 in LTS was the best with patients 90-05, and their actual ginseng dose was over 22,422 g by personal purchase. Patients 91-22 and 91-23 were infected with HIV-1 from blood provided by patient 91-20. With the exception of LTS87-05, all patients were infected with HIV-1 subtype B Korean subclade.

  FIG. 6 shows the characteristics of a large deletion of the 5 'LTR / gag gene for 18 LTS and 28 ginseng untreated patients.

  FIG. 7 shows the frequency of large-scale deletions of the 5'LTR / gag gene compared to large-scale deletions in the nef gene.

Study subjects and treatment with KRG The study group consisted of 10 LTSs that received the maximum KRG, 8 control LTSs, and 28 HIV-infected patients who did not receive KRG.

  The 10 LTSs in this study were described in detail in our previous study (Ref. 9). All 10 were diagnosed between 1987 and March 1996 and did not receive any antiretroviral treatment during the study period, with the exception of one patient (93-04). The patient was intermittently administered ZDV (daily dose 200 mg or 300 mg) from April 1993 to August 1997.

  The eight control group LTS was selected from our cohort. Patients classified into the above control group LTS were diagnosed with HIV-1 infection before 1996 and they had a minimal dose (1,526 ± 1, 1) in the absence of treatment with very potent antiretroviral drugs. 183g) or no KRG was administered (patients 89-05). Only patients 91-23 were administered ZDV from April 1993 to August 1999. Patient 89-05 went to another country in 1997 and was removed from the study sample of the present invention. Two recipients (patients 91-22 and 91-23) who received blood transfusions from LTS91-20 were added to the 8 control group LTS. Patients 89-26 and 92-23 hated taking KRG. Patient 90-01 was a plasma donor who sold blood plasma 83 times. The patient was described as provider O (Ref. 21). Therefore, comparing the 10 high KRG ingested LTSs with the 8 control LTSs, the most basic features were similar.

  In other words, there was no significant difference in basic disease progression rate or CD4 T cell count between 10 LTS and 8 control group LTS, only a difference in KRG intake.

  In addition, another 28 control group patients were investigated. Among these 28 were the wife of patients 87-05 and 90-50. Some of these 28 patients were LTS. However, they were diagnosed after 1997 and were not included in the LTS group. The consent form provided by each participant was accepted.

  Treatment with HIV-1 infected patients with KRG began in the late 1991 at the National Institutes of Health in Korea (Refs. 1, 2). The KRG used in the experiment was a red ginseng powder capsule sold by Korea Ginseng Corporation. The daily dose of KRG to each patient is 5.4 g (6 300 mg capsules 3 times a day) for males and 2.7 g (300 mg capsules for females). 3 doses 3 times a day). The administration period of KRG for each patient is shown in FIG.

  The average amount of red ginseng administered to 10 LTSs was 13,364 ± 5,364 g for 155 ± 28 months, and 1,436 ± 1 for 132 ± 29 months for the 8 control group LTS. , 027 g (see FIG. 5). The annual doses of KRG were 1035 g and 131 g in 10 LTS and 8 control group LTS, respectively. A 7.9-fold difference in the dose level of KRG is statistically significant (P <0.001).

CD4 T cell count and plasma HIV-1 RNA copy level Blood was collected from each patient of Example 1 at intervals of 3 to 6 months, and peripheral blood mononuclear cells (PBMC) were phycoerythrin (PE) in each sample. -And Fluorescein Isothiocyanate (FITC)-each incubated with antibodies to conjugated CD4 antigen (Simultest reagent; Becton Dickinson, Becton Dickinson, San Jose, CA, USA). CD4 T cell levels were measured using a FACScan (Becton Dickinson) flow cytometer. Plasma HIV-1 RNA levels are measured with the Amplicor HIV-1 Monitor kit (Roche Diagnostics, Branchburg, NJ, USA). (Reference 7).

  The number of CD4 T cells was measured at the 6th month every month with 10 LTSs taking red ginseng and 8 control group LTSs, and the results are the same as those shown in FIG.

  The number of CD4 T cells in 10 LTS treated with maximal red ginseng administration decreased significantly from 555 ± 208 / μl to 245 ± 160 / μl in the first measurement after 168 ± 29 months (Decrease of 22 / μl per year, see FIGS. 1a-1j). Also, the number of CD4 T cells in the 8 control group LTS decreased from 574 ± 222 / μl to 353 ± 297 / μl in the first measurement after 132 ± 29 months (20 / μl decrease per year, FIG. 1k). To FIG. 1r).

  In both groups, CD4 T cells were confirmed to have decreased in a similar manner over a 10 year period.

Amplification of partial 5 ′ LTR sites and the gag gene Proviral DNA was extracted from unincubated PBMCs obtained in Example 2. First round primers CE23 (5′-tgtggatta cccacacaa gggctactt-3 ′; 46-73; SEQ ID NO: 1) and 514 (5′-tccagaatgc tggtaggggta tac-3 ′; 1,617-1,640; SEQ ID NO: 2), second round primer CE1 (5′-cgagagctgc atccggagta cta-3 ′; 297 to 319; SEQ ID NO: 3) and 512 (5′-ctgcaggtc ctcattgatg gtc-3 ′; 1,398 to 1,420 SEQ ID NO: 4) and third round primer 501 (5′-gtgtggccctg ggcgggactg-3 ′; 380-399; SEQ ID NO: 5) and 510 (5′-gagtgtcatt ttgcccc No. 297 to 1,421 (numbering here is HIV NL4) by double- (rarely triple-) nested PCR using gg a-3 ′; 1202-1, 222; SEQ ID NO: 6). 1,125 bp PCR product (derived from the sequence of -3) was amplified from each sample (positions 297 to 421 are the part representing the partial 5 ′ LTR region and the gag gene) .

  A maximum of 4 PCR reactions including the negative control group are performed at once on every sample, while a total of 5 PCR reactions (including the negative control group) are used to amplify the nef gene. The PCR product was purified and immediately sequenced. The presence or absence of PCR contamination was monitored by physical separation of PCR products by BLAST search, amino acid codon comparison by manual alignment, and phylogenetic analysis.

  The experimental results are shown as mean ± standard deviation. Statistical significance was evaluated by Student's two-tailed t-test and chi-square test using SPSS package version 12.0.

Experimental Results 189 PCR products were amplified from 80 PMBC samples collected from 10 LTS at different time points (see FIGS. 1a to 1j). At many time points all 10 LTSs showed gΔ in the 5 ′ LTR / gag gene region (see FIG. 1). Defects were observed in 44 out of 80 samples (55%) (see FIG. 2). Of 189 PCR products, 71 (37.6%) showed gΔ. Of these, 23 (32.4%) amplifications showed only one short band without the wild type gene. The characteristics of 71 gΔ are summarized in FIG.

  Five PCR products (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) and two PCR products (patient 90-05 to Δ86 bp in October 1993 and patient 90-50 to Δ420 bp in October 1993) simply contain gΔ in the 5 ′ LTR site or in the gag gene, respectively. (Table 3). The defect in the remaining PCR product is located in the bilateral terminal region of the 5 ′ LTR (including at least position 753; Δ37 bp) and the start of the gag gene (Δ29 bp at position 818 in HIV-1 NL43). In other words, the deletion was 66 bp or more in the vicinity of the gag gene start codon.

  Therefore, the ratio of gΔ in the 5 ′ LTR region was 36.5% (69/189), and the gΔ ratio based on the gag gene was 34.9% (66/189). This indicates that the administration of KRG affected the 5 'LTR rather than the gag gene.

  Aside from patients 91-20 and 93-60 for which samples were not available, the first detection of gΔ in the 5 ′ LT region and the gag region after the start of KRG ingestion is more likely than the first detection of a nef gene deficiency. Was also early (Reference 9). The median time from the start of KRG ingestion for the first detection of gΔ was 26 months (range 19-101 months). This was much shorter than the time required to detect the defect in the nef gene (Reference 9).

  Patient 87-05 was co-infected with hepatitis C virus (HCV) and HIV-1 subtype B derived from introduced and contaminated coagulation factor 9. This patient was started on KRG in June 1994. All amplifications had a small deletion of 9 bp, but did not show gΔnef only in 10 LTS (Ref. 9). Surprisingly, we observed gΔ in the earliest used sample obtained in April 1997 and found a total of 7 out of 36 PCR products obtained from 4 out of 12 samples. gΔ was observed (see FIGS. 1a to 1j and FIG. 5). The patient had no AIDS related symptoms since April 2007. This is the first case in Korea that has survived over 20 years without antiretroviral therapy.

  Administration of KRG was started in December 1991 for patients 89-17. And compliance with dosing was not consistent. One gΔ reported in July 1993 was very exceptional. This is because the deletion of Δ917 bp was replaced with 474 bp of the pol gene (in the reverse direction, 4296 bp to 4037 bp of NL4-3) and the terminal part of the gag gene (2029 bp to 1838 bp). The patient started HAART in February 2002 and had a CD4 T cell count of 513 / μl in April 2007.

  Patient 90-05 was diagnosed with HIV-1 infection in February 1990 and started taking KRG in December 1991. The patients showed the best compliance for treatment with KRG in this study. Along with the supply of 21,522 g of KRG, the patient himself took the KRG he had personally. The daily dose of KRG was 6.0 g (10 300 mg capsules twice a day) with 95% compliance. The first detection of gΔ was 87 months earlier than the first detection of gΔnef in January 2001, 111 months after the start of KRG treatment (a total of 12,012 g was ingested). Detailed information on the gΔ example and the remaining seven LTSs is shown in FIGS.

  In addition, 88 PCR products were amplified from 33 PBMC samples collected from 8 control group LTS. Thirteen gΔ (14.8% of the amplified) were present in 10 PBMC samples (30.3% of the total). Ten LTSs contained significantly higher ratios of gΔ in the PBMC samples and all PCT products than the eight control LTSs (P <0.05 for the former, P <0.0001 for the latter) (FIG. 3).

  In addition, 60 PCR products were amplified from 30 PBMC samples obtained from 28 patients who did not receive KRG regardless of clinical stage. Five gΔ (8.3% of the amplified) were present in 4 PBMCs (13.3% of total).

Genetic defects and small defects In addition to large defects, we observed genetic defects such as non-methionine start codon, early mature stop codon due to GA hypermutation, and small defects. . Among the 10 LTSs, patient 87-05 has an early maturation stop codon with 4 sequences (EU047601, EU047605, EU047607, EU047609), and patient 89-17 also has an early maturation stop codon and an initiation codon. It had isoleucine as (Genbank registration number EF370193). Patient 90-18 has a 6 bp insertion in the gag gene, patient 92-13 has two duplications of 102 bp (Genbank EF370252), a 159 bp deletion in the 5 'LTR region, and G- It had an A hypermutation (Genbank EF370256), and also had a 6 bp insertion between 327 and 328 (Genbank EF370258) (see FIG. 4).

  Of the 8 control LTS patients 91-22 had an early mature stop codon or 3 sequences (Genbank EF370314, EU047654, and EU047655) with isoleucine as the start codon. Patient 89-26 had two early mature stop codons in one sequence (EU047635).

Frequency of large-scale defects according to the number of CD4 T cells The present inventor analyzed the frequency of large-scale defects according to the number of CD4 T cells. PCR amplification products of 10 LTSs were grouped at a CD4 T cell count of 200 / μl or less, 200 to 400 / μl, or 400 / μl or more. The frequency of large deficits in 10 LTSs administered KRG appeared in 47.4% (9/19), 51.8% (14/27) and 61.8% (21/34), Some dependence on the number of CD4 T cells can be shown. However, in the 8 control group LTS, the large deficiency ratio was 44.4% (4/9), 20% (4/20), 50% (2/4), and KRG was not administered. The 28 HIV-1 positive patients were 18.2% (2/11), 14.3% (1/7), and 8.3% (1/12).

DISCUSSION The present inventor has a persistent interest in the health of LTS or LTNP in Korea, and as a result, despite the absence of beneficial host factors such as CCR5 or Δ32bp in HLA-B57 in Korean people, The inventor was able to observe many LTSs in 323 HIV-1 infected patients diagnosed from December 31, 1993.

  More specifically, nine people were diagnosed with HIV-1 infection in Korea in 1987, and three of them were still alive, and all three of them received KRG as a treatment. Or it went in parallel with HAART. LTS87-05 has been taking KRG as one of these patients since June 1996 (see FIG. 1a). The other two of these patients received HAART with administration of KRG from 1998 to 1999, and their CD4 T cell count was 231 / μl in 1998, 6 / μl in 1999, December 2004 Increased to 1,052 / μl and in 2006 to 1,127 / μl.

  In 1988, 22 people were diagnosed as HIV-1 patients, and only 2 patients (9%) who had been treated with KRG were alive. Since the patient's HIV-1 subtype was CRF02_AG, this patient was not included in the study of the present invention, but the patient was classified as LTNP with a CD4 T cell count of 500 / μl or more. Many patients diagnosed since 1997 receive HAART with KRG administration, indicating a lack of drug resistance mutations. These patients responded surprisingly well to the combination therapy.

  Through previous studies, the present inventor has reported that treatment with HIV-1 infected patients with KRG is associated with a high frequency of large-scale deletions of the nef gene (Ref. 9) and slows disease progression (see References 1, 8).

  In the present invention, three PCR products were amplified from each sample. Although this was less than four PCR products amplified from the nef gene (Reference 9), the present inventor In LTS, compared to nef deficiency (34.3% of the sample and 18.8% of the PCR product) (Ref. 9), there was a large deficiency (55% of the sample, 37.6% of the PCR product). A higher frequency could be observed (P <0.0001). The frequency of 5 ′ LTR / gag deletion was 1.6 and 2.0 times higher than the frequency confirmed for the nef gene deletion. Of the 80 samples in total, 61 have previously been applied to nef gene amplification, and 13 of the 61 samples (21.3%) are large in all 2 genes. Showed a deficiency. In particular, patients 87-05, 89-17, 90-05, 90-50, and 96-51 showed a large deletion frequency significantly higher in the 5 ′ LTR / gag gene than in the nef gene (P <0). .05) (see FIG. 5). Also, the proportion of samples that showed large deficits was confirmed in 37.6% in 10 LTS treated with KRG, which included 8 control LTS (14.8%) and 28 Significantly higher than in HIV-1 infected patients (8.3%). Furthermore, the present invention has confirmed that there is no association between the frequency of large defects and the number of CD4 T cells in any group of 8 control LTS or other 28 patients (Examples). 5). Such findings prove a relationship between large-scale deficits and KRG intake.

  Furthermore, the association between gΔ and KRG intake was supported by the discovery that there was no association between gΔs frequency and CD4 T cell count in 8 LTS control groups or another 28 patients. Is done. This finding shows a good contrast with the moderate dependence of 10 LTSs on CD4 T cells. Among 10 LTSs treated with KRG, patients 87-05, 90-05, 92-13, 96-51 with stable and good adhesion maintained the CD4T somatic cell count, while the past 1 Patients 90-50, 91-20, 93-60 who did not show good adherence during ~ 2 years showed a sharp decrease in the number of CD4 T cells. Based on this result, a decrease in CD4 T cells has already been shown in patients 89-18 and 90-18. After more than 10 years without any AIDS-related symptoms, many patients tend to fail to take KRG or pay attention to their health.

  Several papers have published features of the full-length viral sequence in LTNP. Alexander et al. (Reference 22) failed to find defects in other genes besides the nef gene, but found early maturation stop codons, out-of-frame deletions, and global defects. did. Unusual polymorphism and a large deficiency in the nef gene were expressed in 8 LTNPs. Huang et al. (References 23, 24) also reported genetic defects in the 5 'LTR and gag genes from 8 LTSs. This study yielded the entire viral sequence, and only one patient expressed GA hypermutation in the 5'LTR and gag genes. Recently, Calugi et al. Confirmed a large-scale deficiency in the env gene (a gene located beside the nef gene) (Reference 25). Blankson et al. (Ref. 26) did not observe any large-scale deletions in the full-length HIV-1 nucleotide sequence of the suppressor with viral load of plasma RNA of less than 50 copies / mL.

  The expression of the HIV-1 gene is expressed in several very highly conserved cis-acting regulatory elements in the LTR that contain the TATA box and the binding sites of transcription factors NF-κB and Sp1 (ref. 27). elements). Certain components are known to suppress NF-κB and Ap-1 transcription factor activity in human cell lines (Ref. 28). The LTR region can be affected by KRG intake from other parts of HIV-1.

  In the present invention, a large 5 'LTR deficiency was observed in 10 LTSs, including patient 87-05 (which did not show a large deficiency in the nef gene). In addition, the size of each of the large defects observed was large, but located only at a single site within the gene. We have obtained a large env gene deletion (> 1,000 bp) from 6 LTS (patients 90-05, 90-18, 90-50, 91-20, 92-13 and 96-51) HIV. -1 Confirmation was made when the entire length base sequence was obtained (data not shown).

  Thus, without a relationship between KRG intake and genetic defects, the observations of the present invention are not merely a discovery among the various beneficial elements observed in the present study, but the present invention. The results provide primary evidence that large-scale deletions in the 5 ′ LTR region and the gag region are significantly associated with the rate of progression of HIV disease (references 1-9). Regarding possible mechanisms for how viral gene deficiency contributes to LTS or LTNP, we consider that gene deficiency may be an indirect result of immune modulation by ingestion of KRG. It is well known that KRG mediates cell-mediated immune responses through Th1 cytokines (refs. 29-32) and suppresses the generalized immune activation state in HIV-1 infection. . Nonetheless, LTS without KRG still has a higher 5 'LTR / gag deficiency than the other 28 patients infected with HIV, which suggests that KRG intake is not the only factor It is done. Such findings support the importance of genetic defects induced by KRG for the survival of LTS or LNTP.

Base Sequence Data Of the total 336 base sequences, 178 were submitted to Genbank, and Genbank registration numbers EF370172 to EF370349, EU047600 to EU047667, EU047670 to EU047676 and EU047681 to EU047693 were assigned.

  As described in detail, the pharmaceutical composition of the present invention can induce a deficiency in HIV-1 5 'LTR and / or gag gene by administration to an HIV-infected individual. Thus, the present invention can be used to help prevent and treat AIDS.

References
1. Cho YK, Kim YK, Lee I, Choi MH, and Shin YO: The 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 gp120 and its correlation with clinical significance; the effect of long-term intake of KRG on env gene variation. J 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 significantly slows CD4 T cell depletion over 10 years in HIV-1 infected patients: association with 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-1-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 HAART. (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.
10. Kirchhoff F, Greenough TC, Brettler DB, Sullivan JL, and Desrosiers RC: Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive 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 al .: 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, Montella 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; 259: 349-368.
15. Rhodes DI, Ashton L, Solomon A, Carr A, Cooper D, Kaldor 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, Castelli G, et al .: Structural defects and variations in the HIV-1 nef gene from rapid, slow and nonprogressor children.AIDS 2003; 17: 1291 -301.
18. 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 p17 in HIV type 1 subtype C long-term survivors, and general population spread. Res Hum Retroviruses 2006; 22: 195-201.
20. Copeland KF, Chen Z, Fiebig M, Ni L, Savoy S, Smaill 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 MH, 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 longterm survivors of human immunodeficiency virus type 1 infection. 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. 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 Tjian R: Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. Science. 1986; 232: 755-59.
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. Kim KH, Lee YS, Jung IS, Park SY, Chung HY, Lee IR, and Yun YS. Acidic polysaccharide from Panax ginseng, ginsan, induces Th1 cell and macrophage cytokines and generates LAK cells in synergy with rIL-2. Med. 1998; 64: 110-115.
30. Han SK, Song JY, Yun YS, Yi SY.Ginsan improved Th1 immune response inhibited by gamma radiation. Arch Pharm Res. 2005; 28: 343-50.
31. Larsen MW, Moser C, Hoiby N, Song Z, Kharazmi 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 Rg1 enhances CD4 (+) T cell activities and modulates Th1 / Th2 differentiation. Int Immunopharmacol. 2004; 4: 235-44 .

The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4T cells with respect to 10 LTS which received administration of the maximum amount of KRG. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. The graph which shows the change of the number of CD4 T cells with respect to eight control group LTS which did not administer the minimum KRG without antiretroviral treatment. Figure 5 shows analysis of 5 'LTR and gag sequences in peripheral blood mononuclear cells obtained from 3 earliest diagnosed LTSs. It is a bar graph comparing the frequency of large-scale defects in three groups. A predicted amino acid sequence of a representative partial gag protein of 10 LTSs infected with HIV-1. A predicted amino acid sequence of a representative partial gag protein of 10 LTSs infected with HIV-1. A predicted amino acid sequence of a representative partial gag protein of 10 LTSs infected with HIV-1. A predicted amino acid sequence of a representative partial gag protein of 10 LTSs infected with HIV-1. A predicted amino acid sequence of a representative partial gag protein of 10 LTSs infected with HIV-1. A predicted amino acid sequence of a representative partial gag protein of 10 LTSs infected with HIV-1. 2 is a table showing the clinical characteristics of 18 LTSs infected with HIV-1. Figure 5 shows the characteristics of a large deletion of the 5 'LTR / gag gene for 18 LTS and 28 non-treated patients with KRG. 2 shows the characteristics of a large deletion of the 5 'LTR / gag gene for 18 LTS and 28 non-treated patients with KRG. Figure 5 shows the characteristics of a large deletion of the 5 'LTR / gag gene for 18 LTS and 28 non-treated patients with KRG. Shows the frequency of large-scale deletions in the 5'LTR / gag gene compared to large-scale deletions in the nef gene.

Claims (6)

  A pharmaceutical composition for HIV-1 5 'LTR and / or gag gene deficiency comprising ginseng as an active ingredient.   The composition according to claim 1, wherein the ginseng is red ginseng.   The composition according to claim 1 or 2, which is administered to a patient infected with HIV-1.   3. The composition according to claim 2, wherein the composition is administered at a dose of 50 to 100 mg / kg / day for men and 20 to 70 mg / kg / day for women.   The composition according to claim 1 or 2, wherein the 5 'LTR and / or gag gene deficiency is defined by having an early maturation stop codon and lacking a start codon. The composition according to claim 2, wherein the 5 ′ LTR and / or gag gene deficiency is represented by the following sequence.
GenBank registration numbers EF370172, EF370173, EF370175, EU047612, EF370184, EF370186, EF370187, EF370188, EF370191, EF370190, EF370701F, EF370701F, EF370701F, EF370701F, EF37070 , EF37021116, EF3702118, EF370219, EF370221, EF370223, EF370224, EF370225, EF370226, EF370227, EF370228, EF370235, EF370235, EF370237, E F370239, EU047647, EF370245, EF370250, EF370251, EF370252, EF370253, EF370256, EF370259, EF370262, EF370261, EF370265, DQ295196, EF370275, EF370276, EF370277, EF3702778, EF370279, EF370282, EU047673, EF370283, EF370285, EF370286, EF370287, EF370288, EF370292

Images