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INVENTION
Russian Federation Patent RU2043767

Method of inhibiting HIV infection

Method of inhibiting HIV infection

Name of the inventor: AV Asaph .; Bezyulev VV .; Anosova IG
The name of the patentee: Research and Production Enterprise "Farmek"
Address for correspondence:
Starting date of the patent: 1993.05.24

The invention relates to medicine and can be used to inhibit HIV infection that causes AIDS in humans. The essence of the invention consists in that for the suppression of HIV infection using exogenous DNA complexes with polyvalent metals. The method has a high antiviral activity with low toxicity.

DESCRIPTION OF THE INVENTION

The invention relates to medicine, namely to virology, and can be used to inhibit HIV infection that causes AIDS in humans.

The increase in morbidity and mortality from AIDS increasing at impossibility of effective treatment makes it relevant to the search for new drugs to combat HIV.

Known methods of inhibiting HIV infection comprising administering nucleotide analogues, in particular 3-asu-3-deoxythymidine-AZT. However, this drug is effective in the early stages of the disease and even in therapeutic doses is highly toxic, giving a clinical complications (headache, anemia, disorders of the gastrointestinal tract), limiting its use. Known methods comprising administering diftorirovannyh nucleosides, nucleoside analogues liposome, but their efficiency is low.

The purpose of the invention to develop an effective method of inhibiting HIV infection, which combines high efficiency with low toxicity.

The proposed method lies in the fact that they use complex of sodium salt of deoxyribonucleic acid (DNA) with polyvalent metals (zinc, nickel, cobalt, iron) at the ratio of 1: 1 to 1: 1000, and administration of the preparation is carried out subcutaneously, intraperitoneally, intranasally or spinal channel depending on the form of the disease in a therapeutically equivalent doses.

The essential features of the invention should be considered within the process using the sodium salt of DNA complexed with polyvalent metals, according to indications previously unused, their proportions and dose and administration regimens and formulations. For the first time the possibility of obtaining an antiviral effect at different modes of administration of the drug, which is very important in different clinical forms of AIDS, pulmonary, gastrointestinal, CNS lesions.

DNA sodium salt complex metal compounds consists of low molecular weight sodium salt of DNA, nizkopolimernoy contains at least 80% of the sodium salt of the native DNA derived from sturgeon milt, and has the following characteristics: Mol. 270-500 m 10 3 Dalton hyperchromic effect: 37% less than 1% protein by weight, not more than 17% moisture by weight, molar ratios of adenine nucleotides thymine 29.0 27.0 22.0 guanine, cytosine and 20.0 polyvalent metal namely zinc (Zn), cobalt (Co), nickel (Ni), iron (Fe).

To suppress HIV in in vitro systems using Na-DNA complexes with zinc, cobalt, iron and nickel at different concentrations of components. "Wellcome" companies have used AZT production comparison. The antiviral activity of the preparations was evaluated using the methods recommended by the WHO for this purpose. culture were used continuous cell lines CEM-SS and MT-4. Cells were cultured at a concentration of (0.03-0.05) x 10 5 cells per 1 mL medium RRMI 1640 with 10% fetal calf serum, 300 mg / ml L-glutamine, 100 ug / ml gentamycin and grown in suspension. Cell viability was checked by staining them a 0.4% trepanovogo blue solution. As the source of the virus strains used HIV / IVS and HIV-1, HTLV / IIIB.

The cell suspension was placed in a 24 well panels were treated with different doses of the drug and the HIV infected. The multiplicity of infection was 0.01 TPA 50 per cell. The cultures were incubated at 37 ° C in an atmosphere containing 5% CO 2 at 98% humidity for 5-7 days until cytopathic effect determination virus in cell culture.

To assess the formation of viral antigen enzyme immunoassay used.

The results are shown in Table 1.

From the above table shows that, when introduced into the culture medium of the DNA-metal marked suppression of HIV infection in vitro. Moreover, the optimal ratio of DNA-metal is in the range 1: 1-1: 1000. This viralstatic effect combined with low cytotoxicity. When introduced into the culture medium of the drug AZT have a high cytotoxicity with moderate antiviral effect.

Subsequently, the toxicity of drugs by using the proposed method was tested in experimental animals in the system in vivo.

The tests were conducted in parallel on all four coded preparations compared with AZT at doses of 5, 10, 20, 35, and 50 mg / kg. Toxicity studies and four drugs azidothymidine nonlinear conducted on albino mice weighing 6-7 g using different concentrations of drug per kilogram body weight of animals in a volume of 0.2 ml in the form of subcutaneous, intranasal, and intraperitoneal injection and 0.03 ml in volume in the form of intracerebral injection . The animals were kept under observation for 2 weeks, then LD 50 was calculated by the method Curber. For a dose of LD 50 took a lethal dose of the drug that caused the death of 50% of the animals.

The results of toxicity studies of these preparations are given in Table 2.

The tables show that all five drugs, regardless of the dose were non-toxic to white mice weighing 5-7 g subcutaneous, intranasal, intra-abdominal administration (time of observation 2 weeks). At the same time, it recorded a 50% mortality in mice at doses exceeding the DNA-Na-Fe 50 mg / kg for intracerebral administration. Azidothymidine and caused the death of 50% of the mice at intracerebral administration in doses of 10 to 50 mg / kg of animal weight. Characteristically, the complexes, virtually any mode of administration, antiviral activity is retained, at the same time as that of AZT is better expressed in intracerebral, subcutaneous and intraperitoneal administration.

The proposed complexes of Na-DNA-metal possess a pronounced anti-HIV activity; their antiviral activity combined with low toxicity, virtually any method of administering a dose of 10-50 mg / kg and antiviral activity manifests virtually any route of administration.

EXAMPLE Example 1. The experiment used a cell culture of continuous lines CEM-SS and MT-4. Cells were cultured at a concentration of 0,03- 0,05 · 10 6 cells per 1 ml of medium RRMI 1640 with 10% fetal calf serum, 300 ug / ml L-glutamine, 100 ug / ml gentamycin and grown in suspension. Cell viability was verified by staining with a 0.4% solution trepanovogo blue. As the source of the virus strains used HIV / IVS and HIV-1 HT2V / IIIB. The cell suspension was placed in a 24 well panels were treated with various doses of a complex DNA-Na-Zn at a ratio of DNA and metal (M / M) 1: 1; 4: 9; 3:10; 0.5: 350; 1: 1000; 1: 1100 and infected with HIV. The multiplicity of infection was 0.01 TSD 50 per cell. The cultures were incubated at 37 ° C in an atmosphere containing 5% CO 2 at 98% humidity for 5-7 days until cytopathic effect determination virus in cell culture. To assess the formation of viral antigen enzyme immunoassay used.

In the experiment, it was shown that the number of syncytia in percent of control for virus DNA at a ratio of 3:10 metal was 25% with 69% of viable cells; at a ratio of 0.5: 350 to 35% at 81.2% of viable cells; at a ratio of 1: 1000 of 70% and 84.3% viable cells at a ratio of 1: 1100 to 100% and 86.2% viable cells.

EXAMPLE Example 2. Experimental Production as in Example 1.

The cell suspension was treated with various doses of a complex DNA-Na-nickel under the same component ratio as in Example 1. The experiment shows that the number of syncytia in percent of control by the virus at a DNA: nickel 0.5 3:10 350 equaled zero for cell viability 71 and 79% at a ratio of 1: 1000 with 68% 84.3% viable cells at a ratio of 1: 1100 to 87.5% at 100% viable cells.

EXAMPLE Example 3. Experimental Production as in Example 1.

The cell suspension was treated with various doses of a complex DNA-Na-Co under the same component ratio as in Example 1. The experiment shows that the number of syncytia in percent of control by the virus at a DNA-Na-Co 3:10 corresponds to 10% viability 78.4% cells at a ratio of 0.5: 350 to 15% cell viability at 87.1 at a ratio of 1: 1000 with 66% viability and 76.3% at 1: 1100 respectively viability was 82.1%

EXAMPLE 4. Example Formulation experiment as in Example 1.

The cell suspension was treated with various doses of a complex DNA-Na-iron, with the same proportions of components as in Example 1. The experiment shows that the number of syncytia in percent of control by the virus at a DNA-Fe 3:10, 0,5: 350 0 corresponds with cell viability, respectively 79.3 and 82.1% when the ratio of Fe DNA of 1: 1000 the number of syncytia in 61% cell viability to 81.7, at a ratio of 1: 1100 to 100% at 84.5% viability

Thus, these examples illustrate the high anti-HIV activity of the proposed method with low toxicity.

Such a method is compared with the known has the following advantages: high anti-HIV activity in combination with a low toxicity, effective in low doses for any method of administration that allows selecting an adequate route of administration of the drug in different clinical forms of AIDS. The method can be recommended for clinical study.

CLAIM

METHOD FOR SUPPRESSION OF HIV comprising administering a preparation of nucleic acids, characterized in that the preparation derived from sturgeon milt, containing at least 80% of the sodium salt of the native DNA molecular weight 270500 × 10 3 D at a molar ratio of 29.0 nucleotides adenine, thymine, 27.0, 22.0 guanine, cytosine 20.0, in combination with non-toxic polyvalent metal in a molar ratio 1 1 1 1000 which is administered subcutaneously, intraperitoneally or into the spinal canal, depending on the clinical form of the disease, choosing the appropriate therapeutic dose.

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Publication date 06.01.2007gg