RU2196602C1 - Agent for inhibition of human immunodeficiency virus (hiv) and cytomegalovirus (cmv) infections and method of their inhibition - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: invention relates to etiotropic chemotherapy of viral infections, in part, agent used for inhibition of HIV and CMV infections and method of their inhibition. Method involves application of amino acid and dipeptide derivatives of fullerene of the general formula C₆₀-X where C₆₀ means fullerene nucleus; X means NH-CHR-COOH, NH-(CH₂)_n-COOH, NH-CHR-CO-NH-CHR-COOH where n = 2-6; R means by-side radical of natural amino acid or dipeptide as agent for inhibition of viral infections, in part, HIV and CMV infections. Method of application of water-soluble amino acid or dipeptide derivatives of fullerene of the general formula C_60'-X as agent for inhibition of viral infections involves its effect on infected cellular cultures in vitro with effective inhibitory dose. EFFECT: enhanced inhibitory activity of agent. 20 cl, 1 tbl

Description

 The invention relates to medicine, in particular to the etiotropic chemotherapy of viral infections, in particular to the use of amino acid and dipeptide derivatives of fullerene as a means of inhibiting the reproduction of human immunodeficiency virus (HIV) and human cytomegalovirus (CMV) in cell cultures in vitro and can be used in therapy diseases caused by these viruses.

 To date, there are no absolutely effective means and methods of inhibition using chemotherapy drugs, vaccines, and other drugs that can lead to a cure for HIV infection and AIDS. Difficult to treat and CMV infection. In AIDS, she accompanies the underlying disease as the most common opportunist. Most chemotherapeutic agents for the inhibition of HIV and CMV require the use of high doses over an extended period of time and are generally highly toxic. The most common for inhibition is a group of nucleoside analogues, such as AZT, DDI, and DDC, which blocks the reverse transcription process in the genome of HIV-infected cells. A disadvantage of the known group of nucleoside analogues and the method of inhibition is the need for initial phosphorylation of cellular enzymes for their activation. In addition, resistance to these drugs and their high toxicity are rapidly developing in patients with nucleoside therapy.

Recently, the viral protease (HIV), which is a dimer with a molecular weight of about 10 kD, has attracted the most attention as a target for inhibition in the treatment and prevention of HIV. The active center of the enzyme is formed by two amino acids Asp ₂₅ and Asp ₁₂₅ . HPIC specifically cleaves the precursors of polyproteins (Gag - pol), responsible for the synthesis of structural proteins and HIV enzymes. Therefore, an effective blockade of HIV-positive activity, which prevents the formation of an infectious virus, relieves the severity of infection in the body. However, since the HIV DNA provirus is integrated with the cellular genome, the possibility of producing an infectious virus as a result of the activation of the reverse transcription process using the critical enzyme for HIV replication, reverse transcriptase (RT), remains constant.

Currently, there is an idea about the need to use complex inhibition of HIV infection with the simultaneous use of both RT inhibitors [nucleoside analogues (NRTI) and non-nucleoside inhibitors (NNRTI)], and chemical protease inhibitors — saquinavir, indinavir, ritonavir and nelsinavir (SG Deeks , M. Smith, M. Holodniy et al., JAMA, 1977, v. 277, 12, p. 145-153; Ch. Carpenter, M. Fischi, S. Hammer et al., JAMA, 1997, v. 277 , N.24, p. 1962; P. Lorenzi, V. Masserei, B. Laubereau et al., "12 ^th World AIDS Conf. Geneva", 1998, abstr. 32453, p. 27).

 The disadvantage of complex inhibition is a significant decrease in efficiency due to complications arising from the excessive load of the patient’s body with high doses of three, four or more drugs, which are simultaneously used by the patient for a long time. The need to supplement the therapeutic course with drugs that suppress the activity of pathogens of concomitant infections, especially such as CMV, further complicates the treatment process. An additional obstacle for any type of therapy is the rather rapid development of chemical resistance in patients due to the high rate of mutagenic processes occurring in the structure of the viral genome and gene products.

Closest to the invention is a compound containing a water-soluble derivative of fullerene with the general formula C ₆₀ —X = HOC (O) (CH ₂ ) ₂ C (O) NH (CH ₂ ) ₂ (publ. WO 95/19949, C 07 C 49 / 223, 1995), and a method for its use, which consists in exposing a pharmaceutically effective amount of this compound to infected cells in vitro. A known compound is a derivative of methanofullerene. Due to the exact steric and chemical complementarity of the C ₆₀ molecule and the active region of the PVICH, a complex of the ligand – protein type is formed between them during the interaction. Since C ₆₀ and its derivatives are mainly hydrophobic compounds, a strong hydrophobic interaction takes place between C ₆₀ derivatives and the active region of HIV, sufficient to inhibit the activity of HIV. For different fullerene derivatives, the ligand – protein bond is 7–11 kcal / mol. Polar radicals provide water solubility to fullerene compounds due to solubilization. As substituents, any alkyl or aryl-alkyl substituents having nitrogen or oxygen in their composition and containing from 1 to 20 carbon atoms can be used. The effective therapeutic dose (ED ₅₀ ) for acutely infected HIV-1 primary human peripheral blood lymphocytes (LPCs) is 7.3 ± 5.9 μM, for chronically infected H9 cells - 10.8 ± 8.2 μM. The known inhibitor also exerts an effect on the recombinant RT HIV-1 p66 / 51, the inhibitory dose (ID ₅₀ ) of which is 4.6 ± 0.92 μM.

 A disadvantage of the known compound and method of its use is the lack of inhibitory activity - the ratio of Michaelis constant to the inhibition constant (Km = 15.9 μM, Ki = 5.3 μM) and, as a consequence, the use of high doses to achieve the effect of inhibition.

 The problem to which the invention is directed, consists in creating agents based on amino acid and dipeptide derivatives of fullerene and a method for using them to inhibit the reproduction of HIV (by the blocking mechanism of PVICH and RT enzymes) and simultaneously inhibit CMV infection (by the mechanism of suppressing the synthesis of late structural proteins virion).

The specified result is achieved by using the amino acid or dipeptide derivatives of fullerene of the general formula C ₆₀ -X, where C ₆₀ is the fullerene core, X = NH-CHR-COOH, NH- (CH ₂ ) _n COOH, NH-CHR-CO-NH-CHR- COOH;
n = 2 to 6;
R is a side radical of a natural amino acid or dipeptide,
as an agent for inhibiting viral infections, in particular HIV and CMV infections.

The indicated result is also achieved by the method of using amino acid or dipeptide derivatives of fullerene of the general formula C ₆₀ -X as a means for inhibiting viral infections, which consists in its effect on infected cell cultures in vitro with an effective inhibitory dose. The specified inhibition is carried out against viruses of the family Lentiviridae and family Herpesviridae simultaneously. As the virus of the Lentiviridae family, the human immunodeficiency virus type 1 (HIV-1) is used, in particular, the HIV-899 strain (Gelderloom, 1994) is used for acutely infected cells, and the HTHIV27 strain is used for chronically infected cells (G.G. Miller, 1994).

As a virus of the Herpesviridae family, human cytomegalovirus is used, in particular, the reference strain AD169 is used. In this case, the effect on HIV-1 is carried out by the mechanism of inhibition of protease and reverse transcriptase, the effect on human CMV is carried out by the mechanism of inhibition of late structural proteins. As the amino acid derivative of fullerene, C ₆₀ -6-aminocaproic acid or its sodium salt (C ₆₀ -ACA) or C ₆₀ -4-aminobutyric acid or its sodium salt (C ₆₀ -AMA) is used.

 The indicated result is also achieved by the fact that the inhibitory agents do not possess cytotoxicity up to 1500 μg / ml and have a selectivity index (SI) in the range of 267-2500.

The inhibitor (C ₆₀ -ACA) has a 50% inhibition of proliferation of primary lymphocytes and rapidly dividing cells at a concentration of 720 - 830 μg / ml. Its effective inhibitory doses (ED ₅₀ ) are 0.4–0.6 μM for acutely HIV-infected cells, 8.4–12.6 μM for chronically HIV-infected cells, and 2.16– for CMV-infected cells. 3.24 μM. The inhibitory dose for exerting a virucidal effect against HIV is 0.8-1.2 μM, and against CMV it is 8.4-12.6 μM. The inhibitory dose (ID ₅₀ ) on the activity of an isolated HIV protease is 2.3–2.9 μM, and that of the recombinant reverse transcriptase is 5.6–6.8 μM.

The inhibitor (C ₆₀ -ABA) has a 50% inhibition of proliferation of primary lymphocytes and rapidly dividing cells at a concentration of 550-600 μg / ml. Its effective inhibitory dose for acutely HIV-infected cells is 8-12 μM, for CMV-infected cells - 0.48-0.72 μM. The inhibitory dose for exerting a virucidal effect against HIV is 15-22 μM, with respect to CMV it is 1.2-1.8 μM. The inhibitory dose (ID ₅₀ ) on the activity of an isolated HIV protease is (3.19-3.89) • 10 ^-8 M with an inhibition constant of 1.4 • 10 ^-8 M.

 The present invention relates to the use of the previously described compounds, fullerene derivatives of amino acids or dipeptides of the general formula I, which are known as adjuvants of the immune response stimulants (see RF patent 2129436), as agents for inhibiting HIV and CMV infections and a method for using them. Fullerene derivatives of amino acids and dipeptides of the general formula I represent a new class of compounds and belong to the group of non-toxic substances with high bioavailability.

Соединения получают одностадийным синтезом путем непосредственного присоединения остатков аминокислот или дипептидов к фуллереновому ядру. Для этого к раствору фуллерена в о-дихлорбензоле добавляют водный раствор натриевой или калиевой соли аминокислоты (в частности, аминокапроновой, аминомасляной и др.) и 18-краун-6. Реакционную массу перемешивают 6-8 часов при 60^oС. Затем растворители отгоняют, остаток фуллеренового производного обрабатывают насыщенным раствором KCl, после чего остаток фуллеренового производного промывают водой. Выход количественный. Полученное в виде твердого порошкообразного вещества N-(моногидро)-фуллерениламинокислотное производное растворимо в диметилсульфоксиде, диметилформамиде, пиридине и воде. Более детально способ получения описан в патентах РФ 2124022, 2129436. Использование в процессе синтеза натриевых солей аминокислот позволяет получить производное фуллерена с растворимостью в воде 20-30 мг/мл в отличие от средства-прототипа с растворимостью в воде около 1 мг/мл.

Compounds are prepared in a one-step synthesis by directly attaching residues of amino acids or dipeptides to the fullerene nucleus. For this, an aqueous solution of the sodium or potassium salt of an amino acid (in particular, aminocaproic, aminobutyric, etc.) and 18-crown-6 are added to a solution of fullerene in o-dichlorobenzene. The reaction mass is stirred for 6-8 hours at 60 ^o C. Then the solvents are distilled off, the residue of the fullerene derivative is treated with saturated KCl solution, after which the residue of the fullerene derivative is washed with water. The output is quantitative. The N- (monohydro)-fullerenylamino acid derivative obtained in the form of a solid powdery substance is soluble in dimethyl sulfoxide, dimethylformamide, pyridine and water. The production method is described in more detail in RF patents 2124022, 2129436. The use of amino acids in the synthesis of sodium salts allows to obtain a fullerene derivative with a solubility in water of 20-30 mg / ml, in contrast to the prototype agent with a solubility in water of about 1 mg / ml

The essence of the proposed invention lies in the fact that the known compounds of General formula I were discovered new properties, namely the ability to exert an inhibitory effect on HIV and CMV at the same time. Moreover, in contrast to monotherapeutic drugs, the compound can simultaneously inhibit the cytopathogenicity of HIV at two targets (HIV and RT), as well as block the synthesis of late structural CMV proteins (in particular, the gB protein), one of the main opportunists of HIV infection. The specified compound, along with the C ₆₀ fullerene core, providing the hydrophilic-hydrophobic balance of the molecule, contains water-soluble radicals — amino and carboxyl groups, due to which the compound has a solubility higher than methanofullerenes according to the prototype method.

To determine the antiviral activity, eight compounds, fullerene derivatives and amino acids, were synthesized:
C ₆₀ -6-aminocapronic acid, Na salt, (solubility 30 mg / ml);
C ₆₀ -4-aminooil acid, Na salt, (solubility 30 mg / ml);
C _60- DL-serine, Na salt, (solubility up to 20 mg / ml);
C _60- L-proline, Na salt, (solubility up to 2.0 mg / ml);
C _60- L-alanine, Na salt, (solubility up to 1.5 mg / ml);
C _60- L-lysine, Na salt, (solubility up to 1.5 mg / ml);
C _60- L-arginine, Na salt, (solubility up to 20 mg / ml);
C ₆₀ -glicine, Na salt, (solubility up to 20 mg / ml).

In addition, water-soluble dipeptide derivatives of fullerene of the following compositions were obtained:
C ₆₀ -glicil-L asparagine, Na salt, (solubility up to 1.5 mg / ml);
C ₆₀ -DL-alanil-DL-alanine, Na salt, (solubility up to 1.5 mg / ml);
C _60- L-alanil-L-alanine, Na salt, (solubility up to 1.5 mg / ml).

 Water-soluble derivatives of fullerene showed different antiviral activity against HIV-1 and CMV. The results for the determination of anti-HIV-1 and anti-CMV activity of drugs - amino acid and dipeptide derivatives of fullerene - are shown in the table.

Two compounds turned out to be the most effective: the sodium salt of C ₆₀ -6-aminocaproic acid and the sodium salt of C ₆₀ -4-aminobutyric acid.

 Evaluation of antiviral activity and the study of the pharmacological properties of compound I was carried out in sensitive in vitro cell cultures in stages.

 At the first stage, acute and chronic cytotoxicity and proliferative activity of uninfected cells treated with increasing concentrations of the tested substances are determined.

 At the second stage, substances are tested in acutely and chronically infected sensitive cell cultures and primary lymphocytes of human peripheral blood in vitro to determine the inhibitory dose.

 At the third stage, the mechanism of the inhibitory effect in the interaction of I with isolated viral enzymes (HIV and RT) is studied and the level of synthesis of virus-specific proteins for CMV using monoclonal antibodies (mAbs) to the late structural protein is determined (see table).

 First stage.

1. Determination of cytotoxicity of compounds With ₆₀ -ACA and C ₆₀ -AMA.

The toxic properties of the compounds are studied on cells sensitive to HIV and CMV, namely: human peripheral blood lymphocytes (MPCs), MT4 T-lymphoblastoid cells, diploid fibroblasts of the human lung embryo (PhEC). The viability of cells treated with increasing concentrations of C ₆₀ -ACA and C ₆₀ -ABA is determined by the exclusion of the trypan blue vital dye. After incubation at 37 ^o C in an atmosphere of 4.7% CO ₂ for 4-5 days, the cells are stained with trypan blue and counted in the Goryaev’s chamber. Living proliferating cells remain unpainted, while those killed as a result of the toxic effect of the substance are stained in bright blue.

 The test results show that the substances do not have cytotoxicity up to 160-1500 μg / ml according to different tests.

2. Determination of antiproliferative activity of C ₆₀ -ACA and C ₆₀ -ABA.

The antiproliferative effect of the compounds is studied on the same types of cells by monitoring cellular metabolism for 18-20 hours using a radioactive label. The test compounds at concentrations from 100 to 1000 μM were added to all cells placed in 96-well plates and incubated for 72 hours, after which ³ H-thymidine (H-TD) was introduced at a concentration of 2-5 μkCi / ml. The incorporation of ³ H-TD into the treated and untreated cells was counted using an LKB Ultrabetta-1210 liquid scintillation counter.

When counting the radioactive label, it was found that 50% inhibition of proliferation of primary lymphocytes and rapidly dividing cells occurs at a concentration of 720-830 μg / ml for C ₆₀ -ACA and 550 -600 μg / ml for C ₆₀ -ABA. The substance selectivity index is in the range of 267-2500 and differs significantly from the ganciclovir selectivity index (100-1000), a specific CMV inhibitor, and the AZT selectivity index (1000), a specific HIV inhibitor.

 Second phase.

1. Determination of the virus inhibitory activity of C ₆₀ -ACA and C ₆₀ -ABA in cell cultures in vitro.

The inhibitory activity of C ₆₀ -ACA and C ₆₀ -ABA was determined as a dose-dependent value expressed as the concentration of a substance necessary to inhibit virus reproduction by 50%.

In the process of analysis, the following methods were used to register the inhibitory activity of C ₆₀ -ACA and C ₆₀ -ABA:
- enzyme-linked immunosorbent assay (ELISA) to measure the level of production of p24 gag HIV antigen;
- the method of indirect immunofluorescence (NIF) for measuring the proportion of HIV antigen-containing cells after treatment with increasing concentrations of the active substance;
- method of immunocytochemical analysis of CMV viral proteins using monoclonal antibodies (mAbs) to late structural protein gB and early protein IE p72.

 P24 antigen reduction was measured in acutely and chronically infected MT4 cells using a commercial ELISA test system Vironostika (Holland) in 96-well plates. The results were evaluated by the change in optical density (OD) at a wavelength of 450 nm on a multiscan (Labsystems Multiskan PLUS, Finland) (see graph).

a) Measurement of the virus inhibitory activity of C ₆₀ -ACA and C ₆₀ -AMK in acutely HIV-infected cells.

Increasing concentrations of a test substance capable of inhibiting the production of intracellular virus were incubated with acutely infected MT4 or LPC cells with a multiplicity of infection of approximately 100 TDC ₅₀ / ml HIV strain 899.

The effective dose for C ₆₀ -ACA was 0.49 ± 0.1 μM, for C ₆₀ -AMK-10 ± 2.0 μM.

The concentrations of C ₆₀ -ACA and C ₆₀ -ABA for the reduction of total HIV antigens in acutely infected cells, measured by the NIF method and calculated by the method of logarithmic interpolation, also amounted to 0.5 ± 0.1 μM and 10 ± 2 μM, respectively. Therefore, two different tests obtained the same concentration values for inhibiting HIV infectivity.

b) Measurement of the virus inhibitory activity of C ₆₀ -ACA and C ₆₀ -ABA in cells chronically infected with HIV.

 Chronically infected cells (strain HTHIV27) were used to calculate the inhibitory activity of the tested substances, which act on post-integration processes, such as gene expression and virus release from cells.

5 x 10 ⁵ cells per ml were placed in 1.5 ml Eppendorf conical tubes, centrifuged at 900-1000 rpm and twice washed with serum-free medium from virus released from cells. Supernatants were removed, increasing concentrations of C ₆₀ -ACA and C ₆₀ -ABA were added to the cells and incubated for 2 hours at 37 ^° C. After removal of the inhibitor solutions, the cells were placed in 24-well Nunc plates and 1 ml of RPMI 1640 medium was added with 10 % calf serum in each well. After 5 days of cultivation, 100 μl of culture fluid were taken from the control and treated wells and p24 antigen production was determined as described above. The analysis results showed that the effective dose for C ₆₀ -ACA is 10.5 ± 2.1 μM, which is about 20 times higher than for acutely infected cells.

c) Measurement of the virus inhibitory activity of C ₆₀ -ACA and C ₆₀ -ABA in CMV-infected cells.

Testing was performed on a phlebotomy strain (the strain was obtained from the state collection of cell cultures of the Institute of Cytology RAS, St. Petersburg) using the reference strain CMV - AD169. Cells infected with approximately 0.001 PFU / cell of CMV were placed in 25 ml Nunc vials in 10 ml of culture medium and incubated with increasing concentrations of C ₆₀ -ACA and C ₆₀ -ABA for 21 days. Immunocytochemical analysis of viral proteins in infected cells showed that the test substance effectively inhibit CMV cytopathic effect by inhibiting the synthesis of the late structural protein gB, but have no effect on the expression of p72 IE very early non-structural protein (for C ₆₀ -AKK) whereas C ₆₀ -AMK inhibits the synthesis of both late and early CMV proteins. The molecular mechanism of the action of this substance on CMV infection in vitro has not yet been studied.

Effective inhibitory dose calculated logarithmic interpolation accounted for C ₆₀ -AKK 2.7 ± 0.54 uM for C ₆₀ -AMK- 0.6 ± 0.12 uM.

2. Determination of the virucidal effect of C ₆₀ -ACA and C ₆₀ -ABA in relation to HIV and CMV.

Approximately 100 TCD ₅₀ / ml HIV and 0.001 PFU / cell CMV was incubated with increasing concentrations of C ₆₀ -ACA and C ₆₀ -ABA for 2 hours at 37 ^° C. Suspensions were centrifuged at 40,000 rpm for 30 minutes at 4 ^o C. Supernatants were removed, viral sediments resuspended in fresh nutrient medium and infected with them LPK and VLEC placed in 25 ml Nunc bottles in 10 ml of nutrient medium. After 5-6 days for HIV and after 21 days for CMV, the residual virus was analyzed by the NIF method and the immunocytochemical method, respectively.

The virucidal effect of C ₆₀ -ACA in relation to HIV has a concentration of 1.0 ± 0.2 μM, in relation to CMV - 10.5 ± 2.1 μM; With ₆₀ -ABA in relation to HIV it has a concentration of 18.5 ± 3.5 μM, in relation to CMV - 1.5 ± 0.3 μM.

 The third stage.

1. Determination of the virus inhibitory effect of C ₆₀ -ACA and C ₆₀ -ABA on the activity of an isolated HIV protease (HIV).

The inhibitory effect of the tested substances against an isolated protease was determined to study the mechanism of inhibition. In this case, the inhibitor concentration was used, which halves the enzymatic reaction rate — ID ₅₀ . As an isolated HPPI, a prudic extract of HPIC obtained by genetic engineering in Esherichia coli was used. The study of the enzymatic reaction was carried out at (0.1-0.3) • 10 ^-3 M substrate and 2.7 • 10 ^-8 M HIV-1 protease without inhibitor and in its presence.

где S=0,15•10^-3 M - концентрация субстрата;
Km=205•10^-6 М - константа Михаэлиса- Ментена.To characterize the type of inhibition, the inhibition constant (Ki) was determined, which was 1.52 • 10 ^-6 M for C ₆₀ -ACA at an inhibitor concentration of 3.125 • 10 ^-6 M, which indicates a competitive type of inhibition and a high affinity of the inhibitor-viral enzyme complex . The inhibitory effect of HPIC was determined on the basis of spectrophotometric measurement of absorbance by optical density (OD) at a wavelength of 599 nm upon hydrolysis of the Lys-Ala-Arg-Val-Nle-Nph-Glu-Ala-Nle-NH ₂ peptide. Since this inhibition is of a competitive type, the formula was used to calculate ID ₅₀

where S = 0.15 • 10 ^-3 M is the concentration of the substrate;
Km = 205 • 10 ^-6 M - Michaelis-Menten constant.

The inhibitory dose for C ₆₀ -ACA was (2.6 ± 0.3) • 10 ^-6 M.

The determination of the inhibitory effect of C ₆₀ -ABA on PVIC was evaluated spectrophotometrically by measuring the absorption by hydrolysis of the Lys-Ala-Arg-Val-Leu-Nph-Glu-Ala-Met peptide. The Michaelis-Menten constant for C ₆₀ -AMK was 98 • 10 ^-6 M, the inhibition constant was 1.4 • 10 ^-8 M. The inhibitory dose calculated by the above formula was (3.54 ± 0.35) • 10 ^-8 M.

Compounds C ₆₀ -ACA and C ₆₀ -ABA at the same concentration of 3.125 μM did not show anti-pepsin activity at a concentration of 0.032 mM, thus confirming the specificity of the effect of the chosen inhibitor on HIV protease.

2. Determination of the virus-inhibitory effect of C ₆₀ -ACA on the activity of recombinant HIV reverse transcriptase (HIV HIV).

The inhibitory effect of compound C ₆₀ -ACA on the activity of recombinant HIV RT (p65 / 51) was determined using oligo (dT) _12-18 poli (rA) _n primers. The substance C ₆₀ -ACA showed efficacy at ID ₅₀ equal to (6.2 ± 0.6) • 10 ^-6 M. This value is in the intermediate interval between ID ₅₀ values for acutely and chronically infected cells and 62 times higher than selective positive control of AZT (0.1 μM).

 The substance showed no selective activity and cytotoxic effect against cellular DNA alpha polymerase.

 3. Determining the level of synthesis of virus-specific proteins for CMV using monoclonal antibodies to the late structural protein gB.

Inhibition of the late structural protein gB under the action of C ₆₀ -ACA is carried out similarly as described for a specific CMV inhibitor - ganciclovir (KK Biron et al., 1985). As regards C ₆₀ -ABA, the mechanism of inhibition of early and late CMV proteins has not yet been studied.

 Indicators are effective pharmaceutical doses, presented as average values from three experiments performed. The correlation coefficient was more than 0.96. The deviation from the average was less than 20%.

Thus, exposure to a pharmaceutically effective amount of water-soluble amino acid or dipeptide fullerene derivatives of the general formula C ₆₀ -X allows simultaneous inhibition of HIV and CMV infections in cell cultures in vitro. The loss of infectiousness is accomplished by binding this substance to the active site of the HIV molecule and / or inhibiting HIV RT, as well as blocking the synthesis of late structural CMV protein - gB. Substances C ₆₀ -X can be used for the treatment and prevention of AIDS, as well as CMV infections, since they have effective antiviral properties, are non-toxic, water soluble, and do not inhibit cell proliferation at doses higher than therapeutic.

Claims (20)

1. The use of water-soluble amino acid or dipeptide derivatives of fullerene of the General formula
From the _60s ,
where C _{60 is a} fullerene core,
X = NH-CHR-COOH, NH- (CH ₂ ) _n COOH, NH-CHR-CO-NH-CHR-COOH;
n is 2-6;
R is a side radical of an amino acid or dipeptide,
as a means for inhibiting viral infections, in particular HIV and CMV infections.  2. A method of inhibiting viral infections against viruses of the family Lentiviridae and family Herpesviridae, which consists in exposing the infected cell cultures to in vitro agents according to claim 1, with an effective pharmaceutical dose.  3. The method according to p. 2, characterized in that as the virus of the family Lentiviridae use the human immunodeficiency virus type 1 (HIV-1).  4. The method according to PP. 2 and 3, characterized in that the HIV 899 strain is used as HIV-1 for acutely infected cells.  5. The method according to PP. 2 and 3, characterized in that the strain HTHIV27 is used as HIV-1 for chronically infected cells.  6. The method according to p. 2, characterized in that as a virus of the family Herpesviridae use human cytomegalovirus (CMV).  7. The method according to p. 6, characterized in that the reference strain AD 169 is used as human CMV.  8. The method according to PP. 2-5, characterized in that the effect on infected cell cultures is carried out by the mechanism of protease inhibition (HIV) and HIV reverse transcriptase (RT) with an effective inhibitory dose.  9. The method according to PP. 2, 6 and 7, characterized in that the effect on infected cell cultures is carried out by the mechanism of inhibition of the late structural protein of human CMV gB. 10. The method according to PP. 2-9, characterized in that as the amino acid derivative of fullerene using the sodium salt of C ₆₀ -6-aminocaproic acid. 11. The method according to PP. 2-9, characterized in that the sodium salt of C ₆₀ -4-aminobutyric acid is used as the amino acid derivative of fullerene.  12. The method according to PP. 10 and 11, characterized in that the agent for inhibition does not have cytotoxicity up to 160-1500 μm, has a selectivity index (SI) in the range of 267-2500.  13. The method according to p. 10, characterized in that the agent for inhibition causes 50% inhibition of proliferation of primary lymphocytes and rapidly dividing cells at a concentration of 720-830 μm. 14. The method according to p. 10, characterized in that the effective pharmaceutical dose (EB ₅₀ ) is for acute HIV-infected cells (0.5 ± 0.1) μM, for chronically HIV-infected - (10.5 ± 2, 1) μM, for CMV-infected - (2.7 ± 0.54) μM.  15. The method according to p. 10, characterized in that the concentration of the agent for exerting a virucidal effect against HIV is (1.0 ± 0.2) μM, with respect to CMV- (10.5 ± 2.1) μM. 16. The method according to p. 10, characterized in that the inhibitory dose (ID ₅₀ ) on the activity of an isolated HIV protease is 2.6 • 10 ^-6 M and on the activity of a recombinant reverse transcriptase is 6.2 • 10 ^-6 M.  17. The method according to p. 11, characterized in that the means for inhibiting causes 50% inhibition of proliferation of primary lymphocytes and rapidly dividing cells at a concentration of 160-1500 μm. 18. The method according to p. 11, characterized in that the effective pharmaceutical dose (ED ₅₀ ) is for acute HIV-infected cells (10 ± 2.0) μM, for CMV-infected - (0.6 ± 0.12) μM .  19. The method according to p. 11, characterized in that the concentration of the inhibitor for exerting a virucidal effect against HIV is (18.6 ± 3.72) μM, with respect to CMV- (1.5 ± 0.3) μM. 20. The method according to p. 11, characterized in that the inhibitory dose (ID ₅₀ ) on the activity of an isolated HIV protease is 3.54 • 10 ^-8 M.

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