RU2206326C2 - Application of sodium nucleospermate for treatment of hiv-infection and method of treatment - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: invention relates to application of sodium nucleospermate as medicinal agent for treatment of HIV-infection. EFFECT: enhanced effectiveness of treatment of HIV-infection. 18 cl, 10 tbl

Description

 The present invention relates to medicine and can be used to create drugs (drugs) and treatment of HIV infection.

State of the art
Approaches to chemotherapy for HIV infection are known to include inhibition of various stages of HIV reproduction (Fundamental and Applied Issues of AIDS, M, 1988).

 The most successfully developing of these strategic areas of chemotherapy are associated with the suppression of the functioning of the reverse transcriptase of the virus by abnormal nucleosides, i.e. modified nucleic acid precursors. In this regard, it is no coincidence that at present, both drugs approved for medical practice - azidothymidine (thymazide) and dideoxyinosine belong to the class of nucleic acid precursors. At the same time, this direction has several limitations, among which the first place is the correlation of the antiviral effect of drugs of this class with their toxicity, which significantly limits the treatment with chemotherapeutic effects. Relatively safe are compositions based on serine protease inhibitors that inhibit virus replication in infected cells, in particular monocytes (RF Patent 2126266, 1999). However, their clinical efficacy has not been confirmed.

 In recent years, studies on the development of drugs based on natural deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) have become widespread. Many of them, as shown by preliminary tests, have antiviral and immunotropic activity (RF Patents 2136286, 1996, and 2137477, 1995). A number of drugs containing DNA metal complexes also found activity against the human immunodeficiency virus (RF Patents 2108797, 1998 2158592, 2000). At the same time, the activity of the above drugs was studied only in "in vitro" experiments and there is currently no encouraging information about what activity certain DNA-based drugs will exhibit under the conditions of the body's life.

 A number of studies on drugs containing modified RNA have also revealed their antiviral orientation, including some drugs against human immunodeficiency viruses (EP 0213921, 1987). So, this activity was established for a DNA-RNA hybrid obtained by the method described in RF patent 2108796 of 1998.

 At the same time, there is evidence that the toxicity of drugs based on nucleic acids is largely determined by the content of RNA in them (V.I. Masycheva V.I. et al. Antibiotics Journal, 1986, 5, p. 374- 378). This, perhaps, explains the fact that until recently there was no drug based on DNA or RNA, with the exception of the first DNA vaccines admitted to clinical trials in the treatment of AIDS (Medical News, 1998, 5, p. 30-31) .

 We also have to admit that there is no definite point of view on how these or other physicochemical properties of drugs based on nucleic acids are specifically related to their biological, in particular, antiviral, especially selective viral activity, with the introduction of the drug "in vivo" and this difficult targeted synthesis of drugs.

 Thus, at present, there are extremely few drugs and methods for effective treatment of HIV infection. The currently used drugs and methods of treatment for HIV infection have the main drawback - the low effectiveness of the treatment of HIV infection.

 As a prototype of the claimed method, a method of treating HIV infection can be adopted, which consists of at least administering to the human body a drug having a virus-suppressing effect, for example thymazide (Brief Medical Encyclopedia: 2 tons / Ed., Academician RAMS V.I. Pokrovsky. - M.: Scientific-Production Medical Encyclopedia NGO, 1999.- T. 1, A - Mechanotherapy, 608 p.).

 The main disadvantage of the prototype is the low effectiveness of the treatment of HIV infection and high toxicity.

SUMMARY OF THE INVENTION
It is known that a medicinal product (s) of sodium nucleospermate is allowed to be used (see the Temporary Pharmacopoeial Article VFS 42-2566-95, effective from October 27, 1995). The drug (drug) has a complex biologically active base, which is a mixture of sodium salts of polychlorohydrates of deoxyribonucleic and ribonucleic acid derivatives from sturgeon milk with a nitrogen to phosphorus ratio of 1.3 to 1.8 with a molecular weight of 66000-400000 Daltons, more than 90% of the active principle the drug is DNA, and among the rest, pharmacologically significant amounts (up to 6%) of RNA are present. Sodium nucleospermatum is used as a hemostatic stimulator for the treatment of leukopenia that occurs after standard chemotherapy or to prevent deep leukopenia when using high doses of cytostatics. At the same time, low toxicity of the drug and the absence of side effects are noted.

 In addition, it is known, approved for use and has proven itself in clinical practice as a drug - sodium nucleospermate solution (see VFS Temporary Pharmacopoeial Article 42-2565-95, effective from October 27, 1995), consisting of sodium nucleospermate (1.5 g ), sodium chloride (0.1 g) and water for injection (up to 100 ml). The sodium nucleospermate solution is designed to correct the effects of chemoradiotherapy, has the property of stimulating leukopoiesis. For these purposes, it is administered 75-150 mg in 5-10 ml of a 1.5% solution subcutaneously (RF Patent 2110995, 1998).

 As it turned out later, sodium nucleospermate has the property of reparative tissue regeneration, which allowed it to be used to create a means for the regeneration of various tissues of the human body (RF Patent 2115424, 1999) in patients with long-term healing wounds or ulcers.

 Known plasma substituting solution for intravenous administration, having immunomodulating and hemocorrectional properties (RF Patent 2166944, 2001). In the described plasma-substituting solution, low molecular weight DNA is used as the active ingredient causing the stimulation of hematopoiesis, which is characterized by physicochemical parameters that absorb the physicochemical parameters of sodium nucleospermate. Those. the plasma-replacing solution described above is, in particular, one of the dosage forms of sodium nucleospermate used to stimulate human hematopoiesis.

 A known dosage form in the form of a liposome pharmaceutical preparation (Application for the grant of a patent 95102712/14 from 23/02/95, a positive decision 139 NK / 97 from 21/10/97). In this preparation, low molecular weight DNA is used as the active component of the liposome, which is characterized by physicochemical parameters that absorb the physicochemical parameters of sodium nucleospermate. That is, the liposome pharmaceutical preparation described above is, in particular, one of the dosage forms of sodium nucleospermate used to stimulate human hematopoiesis.

 Enteral dosage forms are known, in particular nasal, which is a 0.25% solution of the sodium salt of DNA mentioned above, as well as soft ointment and suppository dosage forms, including the sodium salt of DNA with the above physicochemical characteristics as the active ingredient (US Patent # 5547684 August, 20, 1996).

 In connection with the identification of new properties of sodium nucleospermate, its study was continued, while it was found that sodium nucleospermate in its pure form has the ability to treat (suppress) HIV infection both in in vitro experiments and in an HIV-infected organism (Article " Hope Earth’s compass ", Moscow News weekly March 10, 12-12, 2001), while there is a correlation between the activity of the drug and its molecular weight.

The objectives of the invention are:
A. Improving the effectiveness of the treatment of HIV infection through the use of the known drug sodium nucleospermate as a drug (s) for treating HIV infection.

 B. Reduction of costs and time for creating a new drug for treating HIV infection and expanding the range of drugs for treating HIV infection for their further joint use.

The invention consists in:
1. The use of sodium nucleosperm as an agent (drug) for the treatment of HIV infection or as an active principle (component) of a drug for the treatment of HIV infection.

This will provide:
A. Improving the effectiveness of the treatment of HIV infection through the use of the known drug sodium nucleospermate as a drug (s) for treating HIV infection.

 B. Reduction of costs and time for creating a new drug for treating HIV infection and expanding the range of drugs for treating HIV infection for their further joint use.

 2. In the development of a method of treating HIV infection, comprising administering a drug (preparation) into the human body and using at least sodium nucleosperm as the drug or as the active principle (component) of the drug.

This will provide:
A. Improving the effectiveness of treating HIV infection through the use of the known drug sodium nucleospermate as a drug (drug) for treating HIV infection and using a new technique for its use.

 B. Reduction of costs and time for the creation of a new drug for the treatment of HIV infection and the expansion of the range of therapeutic drugs and methods for treating HIV infection for their further joint use.

 As is known, sodium nucleospermate during traditional parenteral administration can cause tissue and temperature reactions. These reactions, taking into account the clinical features of AIDS, are especially undesirable in such patients. A study of the pharmacokinetics of the drug made it possible to suggest a rectal route of administration. It is also possible to use its traditional 1.5% solution, but a single dose of the drug should be increased, for example, to 20-50 ml. Such a dosage form and method of administration provides the convenience, simplicity and painlessness of use with the same dosage accuracy. The drug is delivered to the places of the necessary effects by endolymphatic transport by transporting it into the large circle of blood circulation.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, sodium nucleospermate is used as an agent (drug) for treating HIV infection or as an active principle (component) of a drug for treating HIV infection .

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods of treating HIV infection, sodium nucleospermate with a molecular weight of 66,000 Daltons to 320,000 Daltons is used.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods of treating HIV infection, sodium nucleospermate with a molecular weight of 70,000 Daltons to 270,000 Daltons is used.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, sodium nucleospermate includes a mixture of sodium salts of deoxyribonucleic and ribonucleic acids, in which the ratio of the percentage by weight of nitrogen to the percentage by weight of phosphorus in terms of dry matter is from 1.3 to 1.8.

In order to increase the effectiveness of treating HIV infection, expanding the range of drugs and methods of treating HIV infection, sodium nucleospermate is used, including a mixture of sodium salts of polychlorohydrates of deoxyribonucleic and ribonucleic acid derivatives, with a hyperchromic effect of 33 to 47%, and an intrinsic viscosity of 1.55 • 10 ² to 3.25 • 10 ² ml / g, the content of chloride ion in terms of dry matter from 7 to 13.5%, the content of sulfate ash from 0.001 to 30% by weight (by weight), the content of heavy metals in sulphated ash from 0.000001 d about 0.001%, protein content from 0.001 to 1.5% in terms of dry matter, nucleic acids in terms of sodium deoxyribonucleate in the range from 90 to 105% in terms of dry matter, derivatives of ribonucleic acids in terms of sodium ribonucleinate 5% in terms of dry matter, the content of ethyl alcohol from 0.00001 to 0.3% in terms of dry matter.

 In order to increase the effectiveness of treating HIV infection, expanding the range of drugs and methods of treating HIV infection, sodium nucleospermate is used in conjunction with at least one antiviral drug, in particular thymazide.

 In order to increase the effectiveness of treating HIV infection and expanding the range of drugs and methods of treating HIV infection, sodium nucleospermate is used together with at least one drug that suppresses at least one AIDS-associated or opportunistic infection, for example, herpetic, in particular with ridostin or zovirax.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, sodium nucleospermate is used in combination or with a mixture of at least one plasma-substituting solution, in particular polyglucin, or macrodex, or dextran, or intradex, or plasmodex or oncovertin, or reopolyglucin, or reomacrodex, or lomodex, or dextran-40, or hemodex.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, sodium nucleospermate is used in the form of a soft dosage form or as a component (active ingredient) of a soft dosage form, in particular ointment, paste, liniment, suppository , for example rectal suppositories.

 In order to increase the effectiveness of treating HIV infection, expanding the range of drugs and methods of treating HIV infection, sodium nucleospermate is used in the form of a liquid dosage form or as a component (active ingredient) of a liquid dosage form, in particular a solution, suspension, for example, a stabilized suspension nanoparticles.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, sodium nucleospermate is used in the form of a solution, in particular aqueous, including from 0.6 to 2 g of sodium nucleosperm per 100 ml of solvent.

 In order to increase the effectiveness of treating HIV infection, expanding the range of drugs and methods for treating HIV infection, sodium nucleospermate is used as the active ingredient associated with the molecular carrier through the biological membrane and an element of the sustained release dosage form, in the form, for example, of free or liposomes polymerized, in particular liposomes with encapsulated sodium nucleospermate, surrounded by a phospholipid layer (s) and with an outer shell of polyethylene glycol and / or achestve element of conventional liposomes obtained, in particular from glyceryl phospholipids with substitution of glycerol, e.g., ribose, and the skeleton which is the basis carboxylate; and / or as an element associated with a peptide or peptide-like (peptide derivative or monoclonal antibody repeating the peptide by properties) delivery system according to the principle of receptor-mediated transcytosis.

 In order to increase the effectiveness of treating HIV infection, expanding the range of drugs and methods of treating HIV infection, a method of treating HIV infection involves administering to the human body a drug, which, at least, is used as an active principle (component) sodium nucleospermate.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, a method of treating HIV infection involves administering a drug into the human body by the enteral route, in particular rectally and / or nasally; and / or parenteral route, in particular subcutaneously and / or intramuscularly and / or intravenously and / or transdermally.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, the treatment method consists in introducing the drug into the human body at least in the form of a soft dosage form or as an integral part (active ingredient) soft dosage forms, in particular ointments, pastes, laminants, suppositories, for example rectal suppositories; and / or administered as a liquid dosage form or as an integral part of a liquid dosage form, in particular a solution, suspension, for example, a stabilized suspension of nanoparticles.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, the treatment method consists in injecting the drug into the human body in a single dose, including 10-50 ml of a 0.6-2% sodium solution nucleospermate in water (for example, one and a half percent sodium nucleospermate solution) or another solvent pharmaceutically acceptable to the human body.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, the treatment method consists in injecting sodium nucleospermatum into the human body rectally 2-3 times a week, two single doses per day, for example in the morning and in the evening, or with a break between administration at 5 - 12 hours.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, the treatment method consists in adding at least one drug, for example, thymazide, to the human body.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, the method of treatment is that sodium nucleosperm is introduced into the human body in the form of an active ingredient associated with a molecular carrier through a biological membrane and an element of a delayed dosage form release, for example, of free or polymerized liposomes, in particular liposomes with encapsulated sodium nucleospermate surrounded by a phospholipid layer (s) and with an outer shell of polyethylene glycol and / or as an element of the conventional liposome, obtained, in particular, from glyceryl phospholipids with glycerol substitution, for example, ribose, and which is the basis of the carboxylate skeleton; and / or as an element associated with a peptide or peptide-like (peptide derivative or monoclonal antibody repeating the peptide by properties) delivery system according to the principle of receptor-mediated transcytosis.

 In order to increase the effectiveness of treating HIV infection, expanding the range of therapeutic drugs and methods for treating HIV infection, the treatment method consists in injecting sodium nucleospermate in a mixture with one or more chemicals that are not part of the sodium nucleospermate into the amount not exceeding the highest single therapeutic dose for these substances, for example sodium phosphate monosubstituted 2-aqueous in an amount of 1.5-3.5% by weight of the amount of sodium nucleospermate and / or sodium phosphate d substituted 12-aqueous in an amount of 2-4% by weight of the amount of sodium nucleospermate.

The term WATER refers to a liquid, the main part of which is hydrogen oxide - H ₂ O. Water is an essential component of almost all technological processes in both industrial and agricultural production. High purity water is used in food production, as well as in medicine, the latest industries
The term “TREATMENT” means a set of measures aimed at eliminating the patient’s suffering and restoring his health (See page 542, Brief Medical Encyclopedia: 2 vol. / Ed. By the Academician of the RAMS V.I. Pokrovsky. - M .: Medical Encyclopedia ", 1999.- T. 1, A - Mechanotherapy, 608 p.). There are etiotropic, pathogenetic and symptomatic treatment. Etiotropic treatment is used in cases where the cause of the disease is established, and it is possible to act on it. An example of this is the use of antibiotics for pneumonia, specific serum for diphtheria or quinine for malaria, removal of a foreign body in case of injury, etc. (See page 542, Brief Medical Encyclopedia: 2 tons / Ed., Academician RAMS V.I. Pokrovsky. - M.: NPO Medical Encyclopedia, 1999.- T. 1, A-Mechanotherapy, 608 p.). Etiotropic therapy aimed at eliminating the cause of the disease is carried out mainly with the help of chemotherapeutic drugs that suppress the vital activity of infectious agents (See p. 534, Brief Medical Encyclopedia: 2 vol. / Ed. By the Academician of the RAMS V.I. Pokrovsky. - M .: NPO "Medical Encyclopedia", 1999. - T. 1, A - Mechanotherapy, 608 p.). Pathogenetic treatment - therapeutic methods that affect the pathogenesis of the disease as a whole or its individual links. Pathogenetic treatment can be aimed at restoring the organs and systems functions impaired as a result of the disease (See p. 542, Brief Medical Encyclopedia: 2 vol. / Ed. By Academician RAMS V.I. Pokrovsky. - M.: Medical Encyclopedia NGO ", 1999.- T. 1, A-Mechanotherapy, 608 p.).

 The term MEDICINES refers to chemicals of natural or synthetic origin, or combinations of substances used for the treatment, prevention, and diagnosis of human and animal diseases (See page 544. Brief Medical Encyclopedia: 2 vol. / Ed. By Academician RAMS V. I. Pokrovsky. - M .: NPO Medical Encyclopedia, 1999. - T. 1, A - Mechanotherapy, 608 p.).

 The term “MEDICINAL FORMS” means the conditions of medicinal products or herbal raw materials that are convenient for use and storage, which provide the optimal therapeutic effect (See page 535, Brief Medical Encyclopedia: 2 tons / Ed. By the Academician of the Russian Academy of Medical Sciences V.I. Pokrovsky. - M .: Scientific-medical encyclopedia NGO, 1999.- T. 1, A - Mechanotherapy, 608 s).

 The term INFECTION (late late. Infectio infection) refers to the introduction into the body and the multiplication of microbes in it with the subsequent development of various forms of their interaction - from carriage of pathogens to a clinically expressed disease. The process that develops in the body as a result of the introduction of a pathogenic pathogen into it is commonly called the infectious process (See p. 422, Brief Medical Encyclopedia: 2 vol. / Under the editorship of the Academician of the RAMS V.I. Pokrovsky. - M .: NGO " Medical Encyclopedia ", 1999. - T. 1, A - Mechanotherapy, 608 p.).

 The term HIV infection is understood as an infectious disease that develops as a result of long-term persistence in lymphocytes, macrophages and cells of the nervous tissue of the human immunodeficiency virus (HIV) and is characterized by a slowly progressing defect of the immune system, which leads to the death of the patient from secondary lesions, described as acquired immunodeficiency syndrome (AIDS), or from subacute encephalitis (See p. 137, Brief Medical Encyclopedia: В 2т. / Ed. By the Academician of the Russian Academy of Medical Sciences V.I. Pokrovsky. - M .: Medical Encyclopedia opedia ", 1999.- T. 1, A - Mechanotherapy, 608 p.).

 The term DOSE (Greek: Dosis portion) refers to the dose assigned to one dose, called a single dose, and taken during the day - the daily. In some cases, doses are established for the entire course of treatment - course doses. The minimum dose in which the drug has a therapeutic effect is indicated as the minimum therapeutic dose, and the highest dose is the highest therapeutic dose. Distinguish the highest single and higher daily doses. In practice, the average therapeutic doses are most often used (See p. 338, Brief Medical Encyclopedia: 2 vol. / Under the editorship of the Academician of the Russian Academy of Medical Sciences V. Pokrovsky. - M.: Medical Encyclopedia NGO, 1999.-T. 1, A - Mechanotherapy, 608 p.).

 Thus, the goal of the invention is achieved.

 The patentability of inventions was evaluated in accordance with the requirements of the "Rules for the Compilation, Filing and Consideration of an Application for a Patent for an Invention". When developing inventions, the author did not find information on the use and methods of using sodium nucleospermate for the treatment of HIV infection.

 Information confirming the possibility of carrying out the invention.

 The test of "sodium nucleospermate solution" (hereinafter referred to as HH solution) as a drug for the treatment of HIV infection was carried out in two stages. At the first stage, the cytotoxic activity of the drug was studied, at the second stage, specific antiviral activity. The study used the transplantable line of human T cells MT-4, characterized by high sensitivity to HIV-1 and the laboratory strain of HIV-1 IIIB. At all stages of the work, the effects of the drug and azidothymidine were compared.

The study of the cytotoxic activity of the drug NN in relation to HIV infection
In the study of the cytotoxic activity of the drug, 24-well culture plates were used. The first row of dies was used to prepare five- or two-fold dilutions of the drug. Dilutions of the preparations were carried out in culture medium (CS) with 5% serum of cow embryos (CEC) without antibiotic. A new pipette was used for each dilution. The remaining wells were added 0.8 ml of cell suspension with a concentration of 150-300,000 cells / ml. Cells were pre-washed with a 100-fold volume of medium without garamycin, diluted with KS without antibiotic, the concentration in the Goryaev chamber was calculated, and 0.8 ml of suspension was poured into the wells of 24-well plates. Then 0.2 ml of the drug was added to the cells. Three dilutions were used for each dilution. The culture medium was added to three wells. Dies were placed in a thermostat.

After 3-4 days, living cells were counted in a Goryaev chamber in the presence of trypan blue. Tests for each sample were performed in triplicate. For each dilution of the drug in each experiment, the average value of the percentage of cell viability was determined. For 100 percent, the number of cells in the sample without the drug was taken. Using the points obtained in three independent experiments, a curve was built of the dependence of cell viability on the concentration of the drug in a semi-log scale, and a 50% toxic dose of the drug was determined from this curve. The number of cells in the samples on the day of the experiment was taken as the background value. The results of 3 independent experiments are presented in table. 1. Based on the results, a 50% cytotoxic dose (TD50) was determined for this drug,
corresponding to its concentration, which reduces the growth properties of cells by 50%.

Based on the results obtained, the following conclusions can be drawn:
1. The drug NN is characterized by low cytotoxic activity on the MT-4 cell line.

 2. NN is more than 1000 times less cytotoxic than azidothymidine.

Studies of the specific antiviral activity of the drug NN in relation to HIV infection
At the second stage of the studies, the antiviral activity of HH was studied. We used the MT-4 transplantable T-cell line obtained from Prof. Robin Weiss, Chester Beatty Laboratories, The Institute of Cancer Research, London, United Kingdom). It was previously shown that this cell line is highly sensitive to HIV-1 and can be used to test antiviral drugs (Pauwels R., De Clercq E. et. Al. Sensitive and rapid assay on MT-4 cells for detection of antiviral compaunds against the AIDS virus. J. Virol Meth. 1987; 16: 171-185).

 For infection of cells, a standard HIV-1 laboratory strain IIIB (Gallo RC et al. Isolation of human T-cell leukemia virus in aquired immune deficiency syndrome (ADDS). Science 1983; 220: 865-867) from the Museum of Viral Cultures of the Research Institute of Virology was used named after D.I. Ivanovsky RAMS.

The study of the virucidal effect of the drug NN
The initial concentration of the drug was 15 mg / ml.

0.1 ml of virus-containing culture fluid with a titer of 10 ⁷ ID50 / ml (this corresponds to 10 mg of the drug and 3 • l0 ⁶ ID50 in 1 ml of solution) was added to 0.2 ml of a sample of the drug, the samples were incubated for 15 min at room temperature, then added 0.7 ml of culture medium and 0.1 ml of solution were added to 0.9 ml of MT-4 cell suspension with a concentration of 250,000 cells / ml. Thus, the multiplicity of infection was 0.5 ID50 per cell.

Cells were cultured in 24-well culture plates at 37 ^{° C.} in the presence of 3% carbon dioxide for 72 hours.

 To evaluate the results used the method proposed by Pauwels R., De Clercq E. et. al. (Sensitive and rapid assay on MT-4 cells for detection of antiviral compaunds against the AIDS virus. J. Virol Meth 1987; 16: 171-185). The essence of the method is that infection of MT-4 cells with HIV-1 leads to their death. If the test compound has an antiviral effect, then it prevents their death. Therefore, the antiviral effect is evaluated by increasing the number of living cells in cultures infected with the virus treated with the test compound. In this case, the number of living cells in the samples is counted (each sample includes three parallel samples) in the Goryaev’s chamber in the presence of trypan blue and then the percentage of living cells is determined taking the number of cells in the virus-free sample as 100%.

 The results of this test are presented in table. 2.

The study of the anti-replicative effect of the drug NN
Cells (500,000 cells / ml) of 1 ml were added to the wells of 24-well culture plates, 0.1 ml of virus-containing culture fluid was added. In this case, infectious doses were 0.01 ID50 / cell or 0.02 ID50 / cell. Then the samples were incubated in a thermostat under standard conditions for 2 hours and then 0.3 ml of the drug was added to a final concentration of 3 mg / ml. The plates were placed in a thermostat and the results were taken into account after 96 hours according to the procedure indicated previously. The results of the experiment are presented in table. 3.

 From the table. Figure 3 shows that under these experimental conditions, the NN preparation exhibits antiviral activity comparable to the activity of azidothymidine at a concentration of 0.1 μg / ml.

Thus, the preliminary stage of testing the samples of the drug NN allowed to draw the following conclusions:
1. The drug NN has antiviral activity, in particular, at a concentration of 3 mg / ml.

 2. The greatest antiviral effect is observed when the drug is added only after at least the early stages of the infection process have passed - the adsorption and penetration of viral particles into cells.

 Based on the results obtained at the preliminary stage of the tests, the antiviral activity of HH was studied using commercial test systems to determine the content of p24 protein of HIV-1 and RNA of HIV-1.

- VIRONOSTIKA HIV-1 ANTIGEN, microelisa system - ELISA (Organon Teknika, USA);
- AMPLICOR HIV-1 MONITOR Test Kit, version 1.5, ART: 11 1048 9, US: 87674, 24 tests, Roche, USA.

MT-4 cells (80-100 ml) 24 or 48 hours after reseeding were besieged at 500 rpm for 7 minutes, the pellet was washed three times with growth culture medium without antibiotics. The cell pellet was suspended in 5-7.5 ml of medium depending on the initial concentration of the cells, but so that their concentration corresponded to 10 ⁷ cells / ml, then 1/10 of the standard virus-containing culture liquid was diluted at a dilution of 1:10. Thus, the multiplicity of infection was 0.01 ID50 / cell, and then one of the following options for setting up the experiment was used:
1. Cells with the virus were placed in a thermostat and incubated, stirring every 15-20 minutes. After 90 minutes, the cells were washed three times with a 100-fold volume of fresh medium to get rid of the free virus that did not penetrate into the cells, then the cells were poured into separate disposable culture tubes, the preparations were applied according to the test schedule, and they were further cultured in an incubator under standard conditions.

 2. Cells with the virus were poured 1 ml into culture tubes with a volume of 15 ml, the preparations were added according to the experimental scheme and placed in an incubator for incubation under standard conditions. After 18 hours, the cells of each sample were washed three times with a 100-fold volume of culture medium, then they were suspended in fresh medium at the appropriate concentration of the preparation according to the experimental scheme and placed in a thermostat. 96 hours after infection, in both cases, the cells were besieged at 1000 rpm for 10 minutes, the supernatant was taken and then used to evaluate the accumulation of HIV-1 p24 protein in it. Some of the samples were used for additional analysis of the accumulation of HIV-1 RNA in them. Each test point included three parallel samples.

 In the first experiment, sample 1 of the NN preparation at a final concentration of 3 mg / ml was introduced 0, 1, 4, and 18 hours after infection of the cells. When determining the semiquantitative concentration of p24 protein accumulation in samples, their serial ten-fold dilutions were prepared. For analysis used 0.1 ml of each of the obtained dilutions. The results are presented in table. 4. For comparison, in table. 5 presents the results of an experiment with azidothymidine.

 Based on the results obtained, it can be concluded that the best antiviral effect is observed when the drug is added after such stages of the infection process as the adsorption of the virus on the cell surface and the fusion of the cell and viral membranes take place. A similar antiviral effect is found when the virus is added to the later stages of the first cycle of the infectious process and at the stage of the second cycle of the infectious process (making the drug 18 hours after infection).

 The culture fluid of the samples from the above experiments, in which the best antiviral activity was observed according to p24 of the HIV-1 protein, was examined for the presence of the number of RNA copies in them using the AMPLICOR HIV-1 MONITOR test system. The results of the analysis are presented in table. 6.

 From the data table. 6 that the drug tested using the AMPLICOR HIV-1 MONITOR test system shows a certain antiviral activity. The data obtained are consistent with the results of the determination of p24 HIV-1 and the number of viable cells presented above.

 Thus, our in vitro studies on HIV showed the presence of a certain antiviral activity of the drug proposed for testing. Summarizing the data on the determination of the number of viable cells, HIV-1 p24 protein in the culture fluid, as well as the number of copies of virus-specific RNA in the culture medium, it can be concluded that the HH preparation is able to suppress the reproduction of HIV-1 virus of strain IIIB in MT-sensitive cells 4. At the same time, a 50% effective dose of the drug was in the range of 0.9-2.1 mg / ml. The drug has low cytotoxicity. The calculated value of TD50 was 15.85 mg / ml. However, the selectivity index of HH is two orders of magnitude smaller than the selectivity index of azidothymidine. It should also be noted that the greatest antiviral effect was achieved with the introduction of the drug only some time after infection of sensitive cells. In this regard, it can be assumed that its antiviral effect can occur by binding to certain signaling receptors on the cell surface, which are involved in the negative regulation of virus reproduction. It is also important to emphasize the relative instability of this drug during incubation in the culture fluid, however, this disadvantage does not seem significant to us, since the low cytotoxicity of the drug, apparently, will allow it to maintain sufficiently high concentrations in the body.

The results of experiments with a rectal method of introducing NN
The pharmacokinetics of tritium-labeled sodium nucleospermate ( ³ H-HH) in the blood of mice after a single rectal administration of the drug was studied.

 Given the specific nature and chemical structure of the studied drug NN, the development of a method for identifying it in biological substrates was carried out in the only possible direction: obtaining labeled NN by introducing a radioisotope tag into its structure.

Obtaining ³ H-HH was carried out using the method of thermal activation of tritium. Two types of targets were used for labeling. First, the target was prepared by applying a suspension of HH powder in ethanol to the walls of a cylindrical reaction vessel with a diameter of 7 cm with a tungsten helix located along the reaction vessel. After freezing with liquid nitrogen, ethanol was removed in vacuo. The resulting target was treated with atomic tritium and dissolved in water. The HH solution was frozen and the treatment with atomic tritium was repeated.

 To obtain atomic tritium, the atomizer — the tungsten wire — was heated to 1900 K. The pressure of molecular tritium in the system was 0.5 Pa, which ensured the free path of tritium atoms from the atomizer to the target. Every 10 s, the gas phase was updated, evacuating the reactor and letting in a new portion of tritium. During the reaction, the walls of the vessel with the target were cooled with liquid nitrogen.

 Three portions of NN of 30 g each with different molecular weights were tagged.

The initial radioactivity of the solution was 50 mCi. The solution was dialyzed for 2 days in the refrigerator. Then the preparation was purified: ³ H-HH was precipitated by adding ammonium acetate to 2M concentration and 2 volumes of ethanol. Maintained for 2 hours at -20 ^o C, then after centrifugation, the resulting precipitate was dried and dissolved in 3 ml of water. Conducted 3 extraction with equal volumes of a mixture of phenol-chloroform (1: 1) and one extraction with a mixture of chloroform-isoamyl alcohol (24: 1) or repeated extraction with a mixture of phenol-chloroform. Ammonium acetate was added to the aqueous phase to a 2M concentration and 2 volumes of ethanol, held for 2 minutes and precipitated with ³ H-HH by centrifugation. The precipitate was washed with 70% ethanol, dried, weighed and dissolved in water. The drug ³ H-HH was analyzed using analytical agarose gel electrophoresis with ethidium bromide in comparison with the original HH and the marker λP _S t _I. Labeled with tritium NN was similar to the initial preparation for a set of DNA fragments with a length from -300 to -500 bp with a small admixture of -1200 bp up to -3000 bp

 As a result of purification, the total radioactivity of the preparative mixture decreased from ~ 50 to 7 mCi and the weight of the purified HH for each of the samples was 2 g. The radioactivity of the drug at certain stages of purification was determined on a Mark (Holland) liquid scintillation counter.

The experiments were performed on male mice of VDF ₁ line weighing 25 g. To confirm the dependence of the absorption rate (bioavailability) of the NN preparation on its average molecular weight, the experiments with the use of three samples of the drug with an average MM of 70 KD, 150 KD, and 270 KD were most demonstrative.

Upon rectal administration, each of the 3 samples of the NN preparation was administered once to groups of mice, three animals per group at a dose of 150 mg (1.83 x ^{10 9} dpm) / Kg or 3.7 (4.57 • 10 ⁷ dpm) per mouse as 3% 0.125 ml aqueous solution.

With the rectal route of administration of the drug, a clamp was applied to the anus of animals, which was removed after 1 hour. The content of ³ H-HH in the cecum and colon was determined immediately after removal of the clamp in each individual separately in a group of 3 animals. Blood samples were taken individually from each mouse from the intraorbital sinus in a volume of 50 μl in a group of 3 animals at 0, 25; 0.5; 1, 3, 5 and 24 hours after administration of the drug.

 The total radioactivity in biological samples (blood, serum, blood cells, tissues, urine and feces) was determined by the radioisotope method after burning in nitric acid in a ratio of 1: 5 in a boiling water bath for 10-20 minutes. 0.1 ml of the resulting solution was added to vials containing 15 ml of ZhS-8 scintillation fluid, and radiometric measurements were carried out on a Wallac β-counter LKB (Sweden).

In the table. 7 shows, for example, the pharmacokinetics of ³ H-HH in the blood of the 2nd group of BDF ₁ mice after a single rectal administration of the drug at a dose of 150 mg (1.83 • 10 ⁹ dpm) / kg.

We obtained individual, for 3 animals, pharmacokinetic curves describing the change in the concentration of ³ H-compounds in the blood for each of the samples of the NN preparation. Blood samples from mice were taken from the intraorbital sinus in a volume of 50 μl. As follows from the experimental data, three individual curves for one sample of the drug are both qualitatively and quantitatively similar in their parameters. At the same time, as can be seen from the table. 8, the rate of absorption decreases with increasing molecular weight of the sample of the NN preparation.

In the time interval of 0-5 hours, a fairly rapid increase in the concentration of ³ H-HH in the blood occurs when the rate of the absorption process exceeds the rate of elimination. Then, by 24 hours, there is a phase of a decrease in the level of ³ H-compounds, associated with the prevalence of the distribution of the drug in organs and tissues and the removal of ³ H-compounds from the animal organism. Thus, the concentration of the drug in the blood of animals at maximum points and one day after administration are close in value.

 Due to the fact that the sphincter is absent in mice, the administration of the drug to the animals was rectally accompanied by the application of a clamp on the anus. 1 hour after administration, the clamps were removed, and in the cecum and colon (together with the contents), the amount of the drug that had not absorbed by that time was determined.

From the data obtained it turned out that in the cecum a small amount of ³ H-HH is determined, and in the colon - on average up to 80% of the administered dose.

Data on the excretion of ³ H-compounds with animal urine and feces after a single rectal administration of ³ H-HH show that about 64% of the administered dose, with urine 2.5%, with feces 61.5, are excreted in 24 hours with urine and feces %

Thus, analyzing in a complex all the data obtained, we can conclude that the drug ³ H-HH has the ability to be absorbed in the large intestine of animals. Despite the fact that the clamp from the anus in mice was removed after 1 hour, part of the drug is absorbed in the rectum of animals and is not excreted simultaneously with feces. The absorption process continues for a long time: within 5 hours, the rate of ³ H-HH entry into the blood of mice exceeds the elimination rate, which reflects the distribution of ³ H-HH in organs and tissues and the excretion of the drug and metabolites. Then there is a transition of the pharmacokinetic curve from the absorption phase to the elimination phase.

Studies have led to the conclusion that the drug ³ H-HH, administered rectally, has bioavailability for the body. Rectal administration of mice received from 40% of the administered dose (for a sample with MM 70 KD) to 9.2% (for a sample with MM 270 KD). From the obtained experimental data, it follows that for rectal administration, it is preferable to use an NN preparation with a molecular weight of up to 270 KD, a further increase in the molecular weight of the drug leads to a sharp decrease in its bioavailability, which in turn makes its use ineffective.

Clinical studies of virus-suppressing activity of NN in a group of HIV-infected volunteers
Clinical studies were conducted in accordance with the permission of the Department of State Control of Quality, Efficiency, Safety of Medicines and Medical Equipment of the Ministry of Health of the Russian Federation.

According to the protocol, 20 HIV-infected patients in 2 groups participated in the study:
Group P - 10 patients (main group):
- the average age of the patients was 29.9 years, 2 women and 8 men were in the group;
- the term of infection (the limitation of diagnosis) is from 2 to 14 years, in 4 - more than 9 years, in 6 - more than 2 years;
- route of infection: 5 - intravenous, 3 - heterosexual, 1 - nosocomial, 1 - homosexual.

Group K - 10 patients (comparison group):
- the average age of the patients was 22.8 years, the group consisted of 4 women and 6 men;
- the infection period is from 1 to 10 years, in 2 more than 3 years, in 7 more than 2 years, in 1 more than 10 years;
infection route: 8 - intravenous route (drug addicts), 1 - heterosexual route, 1 - nosocomial.

 This test was conducted on HIV-infected patients at stages A3, B1, B2, B3 with an established diagnosis of HIV infection by ELISA and confirmed WESTERN BLOT.

At the beginning of this test:
In group P were observed: generalized lymphadenopathy in 8 patients, oropharyngeal candidiasis in 4, dysbiosis in 2, proteinuria in 2, increased alkaline phosphatase in 8, increased ALT / AST in 2, monocytosis in 3, eosinophilia in 2, erythropenia in 1, thrombocytopenia in 1 patient.

 In group K were observed: generalized lymphadenopathy in 7 patients, oropharyngeal candidiasis in 5, proteinuria in 3, increased alkaline phosphatase in 8, increased ALT / AST in 2, eosinophilia in 2, erythropenia in 1 patient.

According to the approved protocol, the following treatment regimen was used to treat HIV-infected patients:
In group P (main group) for 25 weeks:
NN in a 1.5% solution 2-3 times a week in a daily dose of up to 50 ml rectally (2 times 25 ml per day) + thymazide (200 mg 3 times a day, orally, daily. The first two months they used NN with a molecular weight of 70-100 KD, NN with MM 120-170 KD was used for the next 2 months, NN with MM 220-320 KD were used for the last two months.The molecular weight of all samples of the NN preparation used in the treatment process was within the limits established by the pharmacopeia article.

In group K (comparison group) for 25 weeks:
Timazide 200 mg 3 times a day, orally, daily.

 The results of the study of group P are given in table. 9, groups K - in table. 10.

 The results of the 1st month of therapy showed that 9 patients of the main group out of 10 showed a significant decrease in viral load (the maximum decrease in patient 8 was 17.3 times), in one patient 9 there was a slight increase in the number of viral copies of RNA per ml (0, 6 times). With a thorough history of patient 9 (with the participation of his close relatives), it turned out that at the moment the patient occasionally continued to take intravenous drugs.

 The results of the 1st month of thymazide therapy showed that 9 patients of the control group out of 10 showed an insignificant decrease in viral load, in one patient 4 there was an 8-fold decrease in the number of viral copies of RNA per ml.

 The results of the 3-month course showed that 9 patients of the main group out of 10 showed a further significant decrease in the number of viral copies of RNA per ml up to 9.7 times (7). One patient (9) showed a further increase in viral load by 9.7 times.

 The results of a 3-month course showed that in 9 patients of the control group out of 10, a slight increase in the number of viral copies of RNA per ml was observed up to 2.2 times (4). In one patient (7), the level of viral load did not change.

 According to the results of a 6-month course of treatment of the main group, 4 patients showed an increase in the number of RNA copies per ml from 3.1 (5) to 7.2 (8) times. 6 out of 10 patients showed a further decrease in viral load from 2 to 17.8 times (10). Patient 10 was included in the study program with the obviously lowest number of RNA copies per ml - 8.932. The viral load of patient 10 is reduced to a threshold of <500 copies per ml.

 According to the results of a 6-month course of treatment of the control group, the level of viral load slightly increased in 9 patients. Patient 1 showed a slight decrease in the number of RNA copies per ml by 3.1 times.

 According to the results of monthly monitoring, it is clear that the effectiveness of the drug in reducing the viral load in the first two months significantly exceeds its effectiveness in the last two months, and this decrease is correlated with an increase in the MM used to treat the drug during the entire course of treatment.

 Thus, the use of HH in combination with thymazide compared with the control group taking thymazide alone leads to a positive effect of suppressing the HIV virus in the patient and a significant reduction in the viral load after one month in 80% of patients, after a three-month course in 90% of patients. The use of the drug NN in combination with thymazide after a 6-month course is less effective and leads to a positive effect in 60% of cases. From the data presented, it can be concluded that the sodium nucleospermate preparation is quite effective in the field of molecular weight (MM) from 66,000 D (66,000 Daltons) to 320,000 D and the inappropriateness of using this drug to suppress HIV infection in the area of its MM is more than 320,000 D.

 All of the above, in our opinion, determines the prospects of using sodium nucleospermate in the complex treatment of HIV infection. Considering the fact that NN is an industrially produced medicinal product that has already passed all the necessary stages of control and testing, its use in a new quality has not only important medical value, but also provides a great economic effect.

Claims (18)

 1. The use of sodium nucleosperm as a medicine for treating HIV infection or as an active ingredient in a medicine for treating HIV infection.  2. The use according to claim 1 of sodium nucleospermate with a mol.m. 66000 - 320,000 daltons.  3. The use according to claim 1 of sodium nucleospermate with a mol.m. 70,000 - 270,000 daltons.  4. The use according to any one of claims 1 to 3, a sodium sperm containing a mixture of sodium salts of deoxyribonucleic and ribonucleic acids, in which the ratio of the percentage by weight of nitrogen to the percentage by weight of phosphorus in terms of dry matter is 1.3 - 1.8. 5. The use according to any one of claims 1 to 4, a sodium sperm comprising a mixture of sodium salts of polychlorohydrates of derivatives of deoxyribonucleic and ribonucleic acids, with a hyperchromic effect of 33 to 47%, a characteristic viscosity of 1.55 • 10 ² - 3.25 • 10 ² ml / g, the content of chloride ion in terms of dry matter is 7 - 13.5%, the content of sulfate ash is 0.001 - 30% by weight (by weight), the content of heavy metals in sulfate ash is 0.000001 - 0.001%, the protein content is 0.001 - 1.5% in terms of dry matter, the content of nucleic acids in terms of sodium deoxyribo ukleat within 90 - 105% calculated on the dry substance content of ribonucleic acids derivatives, based on the sodium ribonukleinat to 5% based on the dry substance content of ethyl alcohol 0.00001 - 0.3%, based on dry substance.  6. The use according to any one of claims 1 to 5 in combination with at least one antiviral drug, in particular thymazide.  7. The use according to any one of claims 1 to 6 in combination with at least one drug that suppresses at least one AIDS-associated or opportunistic infection, for example herpetic, in particular with zovirax.  8. The use according to any one of claims 1 to 7 in combination or mixture with at least one plasma substituting solution, in particular polyglucin, or macrodex, or dextran, or intradex, or plasmodex, or oncovertin, or reopolyglucin, or reomacrodex, or lomodex , or dextran-40, or hemodex.  9. The use according to any one of claims 1 to 7, sodium nucleospermate in the form of a soft dosage form or as an active ingredient in a soft dosage form, in particular a suppository, for example rectal suppositories.  10. The use according to any one of claims 1 to 8, sodium nucleospermate in the form of a liquid dosage form or as an active ingredient in a liquid dosage form, in particular a solution, suspension, for example, a stabilized suspension of nanoparticles.  11. The use according to any one of claims 1 to 8 and 10 sodium nucleospermate in the form of a solution, in particular aqueous, including 0.6 to 2 g of sodium nucleospermate per 100 ml of solvent.  12. A method of treating HIV infection, comprising administering a drug to the human body, characterized in that at least sodium nucleospermate is used as a drug or as an active ingredient of the drug.  13. The method according to p. 12, characterized in that the drug is administered rectally into the human body.  14. The treatment method according to claims 12 and 13, characterized in that the drug is administered to the human body in the form of a soft dosage form or at least as an active ingredient in a soft dosage form, in particular a suppository, for example rectal suppositories; and / or administered as a liquid dosage form, or at least as an active ingredient in a liquid dosage form, in particular a solution, suspension, for example, a stabilized suspension of nanoparticles.  15. The treatment method according to item 12 or 13, characterized in that the drug is administered into the human body in a single dose, including 10-50 ml of a 0.6-2% solution of sodium nucleospermate in water or another pharmaceutically acceptable solvent for the body person.  16. The method according to any one of paragraphs.12-15, characterized in that the preparation containing sodium nulcospermatum is administered rectally 2-3 times a week, two single doses per day, for example, in the morning and in the evening, or with a break between administration at 5-12 hours  17. The method of treatment according to any one of paragraphs.12-16, characterized in that at least one drug, for example thymazide, is additionally introduced into the human body.  18. The method of treatment according to any one of paragraphs 12-17, characterized in that the sodium nucleospermate is introduced into the human body in the form of an active ingredient associated with a molecular carrier through a biological membrane and an element of a sustained release dosage form, in the form, for example, of free or liposomes polymerized, in particular liposomes with encapsulated sodium nucleospermate, surrounded by a phospholipid layer (s) and with an outer shell of polyethylene glycol and / or as an element of the conventional liposome obtained in astnosti from glitserilfosfolipidov substitution with glycerol, for example ribose, and which is the basis carboxylate skeleton; and / or as an element associated with a peptide or peptide-like (peptide derivative or monoclonal antibody repeating the peptide properties) delivery system according to the principle of receptor-mediated transcytosis.

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