RU2533232C2 - Using polycarboxylic fullerene deriative as microbiocidal antiviral agent - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely to using a polycarboxylic fullerene C₆₀ derivative as a microbiocidal antiviral agent for the herpes simplex virus (HSV) and cytomegalovirus (CMV) inhibition. The polycarboxylic fullerene C₆₀ derivative has the structural formula

.

EFFECT: invention provides the agent for the emergency prevention of HSV and CMV sexual transmission.

7 dwg, 2 tbl, 5 ex

Description

The invention relates to medicine, namely to the etiotropic chemotherapy of viral infections, and for the use of a compound based on fullerene C ₆₀ as a microbicidal agent for the inhibition of herpes virus infections (HBV) caused by herpes simplex virus (HSV) and cytomegalovirus (CMV), and can be used for emergency prevention of HSV and CMV infections, sexually transmitted infections.

The wide spread of infections caused by herpes simplex virus (HSV) and cytomegalovirus (CMV), sexually transmitted infections, makes it necessary to pay special attention to this area of human infectious pathology. However, the specific antiviral drugs used to treat herpes virus infections (GVI) have a number of significant drawbacks. One of them is high toxicity [Faulds D., Heel R.C., Ganciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections. Drugs 1990, V.39, P.597-638]. In addition, the use of known antiviral drugs is characterized by the development of drug resistance [Erice A. Resistance of human cytomegalovirus to antiviral drugs., Clin Microbiol Rev., 1999, V.12, P.286-297; Bhorade S.M., Lurain N.S., Jordan A., Leischner J., Villanueva J., Durazo R., Creech S., Vigneswaran W.T., Garrity E.R. Emergence of ganciclovir-resistant cytomegalovirus in lung transplant recipients, J Heart Lung Transplant. 2002, V. 21, P.1274-1282].

In this regard, a new approach is being developed - the creation of microbicides. Antiviral microbicides are a completely new category of drugs that inactivate or destroy microorganisms immediately before or during sexual contact and thus prevent the spread of sexually transmitted infections when applied topically: rectally or vaginally (emergency prevention). Medicines in the form of a gel, cream or vaginal (rectal) suppository to protect against human immunodeficiency virus (HIV) and other sexually transmitted infections can be a real alternative to condoms, and the efforts of scientists from different countries are focused on various aspects of this problem. A significant drawback of many of the proposed compounds is the lack of a well-established chemical structure, toxicity to human cells and low antiviral activity. This indicates the need to search for new compounds with microbicidal antiherpetic properties. Currently, the use of water-soluble derivatives of fullerenes is considered promising due to their high bioavailability, which can be used in medicine.

Closest to the invention are antiviral water-soluble amino acid or dipeptide derivatives of fullerene C ₆₀ [Miller GG, Kushch AA, Romanova BC Patent RU 2002101294/14, 2003]. As such fullerene derivatives, 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. These compounds, along with the C ₆₀ fullerene core, which provides hydrophilic-hydrophobic balance of the molecule, contain water-soluble radicals — amino and carboxyl groups, due to which the compound acquires the ability to dissolve in aqueous solutions. These in vitro compounds simultaneously inhibit the development of HIV and CMV infections in cell cultures. However, the presence of only one polar radical in the hydrophobic component of C _{60 is} insufficient to overcome the strong hydrophobic interaction between the molecules, which leads to a decrease in the effectiveness of the inhibitory properties of the compounds. In addition, these compounds have not been tested as microbicidal anti-HSV and anti-CMV agents used for emergency prophylaxis.

The problem to which the invention is directed, is to create a microbicidal agent based on a polysubstituted (polycarboxyl) C ₆₀ fullerene derivative and its use for emergency prevention, preventing the transmission of HSV and CMV infections during sexual contact.

In addition, the problem is solved by a therapeutic composition of antiviral action, in which the content of the polycarboxylic derivative of fullerene C ₆₀ 1-K is from 0.0001 to 99.9999 weight percent.

This result is achieved by using the fullerene derivative 1-K as a microbicidal antiviral agent, in which phenoxybutyric acid residues are attached to solubilize the fullerene backbone. The specified compound was obtained in accordance with the methodology presented in example 1. This compound exhibits pronounced antiviral activity in vitro with both HSV and CMV infections in cell cultures. At the same time, the prevention of GVI is carried out according to the microbicidal method, which provides emergency prevention.

The molecular formula of compound 1-K is shown in figure 1.

Compound 1-K does not possess cytotoxicity up to 1000 μg / ml and exhibits pronounced antiviral activity in vitro in both HSV and CMV infections in cell cultures. Effective inhibitory doses (ID ₅₀ ) of 1-K are 18 μg / ml for HSV and 10 μg / ml for CMV. The chemotherapeutic index (CTI) exceeds 100 for both HSV and CMV infections. At the same time, the prevention of GVI is carried out according to the microbicidal method, which provides emergency prevention.

The present invention relates to the use of a derivative of fullerene C ₆₀ 1-K, which has high bioavailability and belongs to the group of non-toxic substances.

The essence of the proposed invention lies in the fact that the polysubstituted derivative of fullerene C ₆₀ 1-K has the ability to exert an inhibitory effect on both HSV and CMV in the microbicidal mode of use for emergency prevention.

Assessment of toxicity and study of the antiviral activity of the compound was carried out in sensitive in vitro cell cultures in stages. At the first stage, the cytotoxic properties of compound 1-K were determined. As a comparison, we used data on the cytotoxicity of reference drugs: acyclovir, which has anti-HSV activity, and ganciclovir, which has anti-CMV activity.

At the second stage, the antiviral properties of compound 1-K were tested against HSV and CMV infections in cell cultures in vitro. Herpesviridae family virus uses: herpes simplex virus type 1 (HSV-1), reference strain F, and human cytomegalovirus (CMV), reference strain AD169. The choice of herpes simplex virus type 1 is based on the results indicating that since the 90s, the cause of genital herpes in 70-90% of cases is HSV-1 [Nilsen A., Myrmel H. Changing trends in genital herpes simplex virus infection in Bergen, Norway. Acta Obstet. Gynecol. Scand. 2000; 79 (8), 693-696]. The antiviral activity of compound 1-K was studied in a microbicidal regimen, which consists in the fact that the test substance is introduced into the cell culture 1 hour before infection [Bernstein DI, Stanberry LR, Sacks S., Ayisi NK, Gong YH, Ireland J., Mumper RJ, Holan G., Matthews B., McCarthy T., Bourne N. Evaluations of unformulated and formulated dendrimer-based microbicide candidates in mouse and guinea pig models of genital herpes. Antimicrob Agents Chemother. 2003; 47 (12), 3784-3788]. To study, we took green monkey kidney cells (Vero), which are sensitive to HSV-1, and human diploid fibroblasts (PLL), which are sensitive to CMV. Cell cultures were obtained from the collection of tissue cultures of the FSBI “Research Institute of Virology named after DI. Ivanovsky "Ministry of Health and Social Development of Russia.

Brief Description of the Figures

For a clearer understanding of the claimed invention, which is reflected in the claims, as well as to demonstrate its features and advantages, the following is a detailed description with reference to the drawings.

Figure 1 shows the molecular formula of compound 1-K.

Figure 2 presents a graph of the dependence of the viability of Vero cells on the concentration of compound 1-K within 24 hours of exposure. The graph shows the determination of acute cytotoxic dose (OCD ₅₀ ) - 1-K concentration, at which 50% of the cells die after 24 hours of exposure. The OCD ₅₀ was 2300 μg / ml.

Figure 3 presents a graph of the dependence of the viability of Vero cells on the concentration of compound 1-K within 72 hours of exposure. The graph shows the determination of chronic cytotoxic dose (HCD ₅₀ ) - 1-K concentration, at which 50% of the cells die after 72 hours of exposure. ChCD ₅₀ was 2000 μg / ml.

Table 1 summarizes the data that characterize the cytotoxic effect of 1-K against Vero cells. For comparison, data on the cytotoxicity of the reference drug acyclovir used in clinical practice for the treatment of HSV infection are presented. As can be seen from the data presented, the cytotoxicity of compound 1-K is significantly lower than the toxicity of the known antiviral drug acyclovir.

Figure 4 presents a graph of the dependence of the viability of the cells of the PLEC from the concentration of compound 1-K within 24 hours of exposure. The graph shows the determination of acute cytotoxic dose (OCD ₅₀ ) - 1-K concentration, at which 50% of the cells die after 24 hours of exposure. The OCD ₅₀ was 1354 μg / ml.

Figure 5 presents a graph of the dependence of the viability of the cells of the PLEC from the concentration of compound 1-K within 72 hours of exposure. The graph shows the determination of chronic cytotoxic dose (HCD ₅₀ ) - 1-K concentration, at which 50% of the cells die after 72 hours of exposure. ChCD ₅₀ was 1269 mcg / ml.

Table 2 summarizes the data that characterize the cytotoxic effect of 1-K in relation to FLEC cells. For comparison, data on the cytotoxicity of the reference drug ganciclovir used in clinical practice for the treatment of CMV infection are presented. As can be seen from the data presented, the OCD ₅₀ and HCD ₅₀ for compound 1-K are lower than those for the well-known antiviral drug ganciclovir.

Figure 6 presents a graph of the suppression of plaque-forming ability of HSV on the concentration of 1-K. The graph shows the determination of a 50% inhibitory dose (ID ₅₀ ) - a 1-K concentration that causes inhibition of plaque formation by 50%. ID ₅₀ 1-K for HSV was 18 μg / ml.

Figure 7 presents a graph of the suppression of plaque-forming ability of CMV on the concentration of 1-K. The graph shows the determination of a 50% inhibitory dose (ID ₅₀ ) - a 1-K concentration that causes inhibition of plaque formation by 50%. ID ₅₀ 1-K for CMV was 10 μg / ml.

The invention is characterized by the following examples.

Example 1. Obtaining a derivative of fullerene 1-K

Chlorofullerene C ₆₀ Cl ₆ (0.933 g) was dissolved with stirring in dry 1,2-dichlorobenzene (250 ml) under argon. The solution was pumped out with gentle heating (30-40 ° C) to remove dissolved gases and traces of water. Phenoxybutyric acid ester (10 ml) distilled under vacuum was added to the solution, after which it was again evacuated until the evolution of gas bubbles ceased. Anhydrous tin tetrachloride (0.3 g) was added to the resulting solution, after which the system was rapidly heated to the temperature at which the reaction began (50-70 ° С). It was held at the indicated temperature for 30 minutes, after which it was cooled to room temperature.

The reaction mixture was poured into a separatory funnel containing 1 L of dilute hydrochloric acid and shaken to remove tin salts from the organic phase. The organic phase was washed 3-4 times with distilled water, after which it was separated and dried with calcined sodium sulfate or magnesium sulfate.

The selection of the target product 1A was carried out by column chromatography. For this, the dichlorobenzene solution containing the reaction products was diluted twice with petroleum ether and applied to a wide chromatographic column (diameter 30 mm, height of the silica gel layer 30 cm). Dichlorobenzene was removed by passing pure toluene, and then the products of the elimination of C ₆₀ Ar _2-4 were slowly separated by elution with a 0.4-0.5% solution of methanol in toluene. The target products were washed with 0.6-0.7% methanol in toluene, selecting individual fractions with a volume of 50-100 ml and controlling their purity by HPLC (reverse phase C 18, the solvent is a mixture of methanol with 30% toluene). Product fractions with a purity of more than 95% were collected together and evaporated on a rotary evaporator, and impure fractions were evaporated to a small volume and rechromatographed under the same conditions. After evaporation of the solutions, the residue was washed with methyl or ethyl alcohol and dried in air. 0.876 g of product 1a was obtained (yield 47%).

To carry out acid hydrolysis, compound 1a (0.876 g) was dissolved in toluene (200 ml / g) in an argon atmosphere or in air, after which an equal volume of acetic acid and concentrated 37% hydrochloric acid (40 ml) were added to the solution. The biphasic mixture was heated with vigorous stirring to 90-95 ° C for two to three days, adding 5-10 ml of hydrochloric acid every 3 hours.

At the end of the hydrolysis, the organic phase containing the polycarboxyl derivative 1b was separated and evaporated on a rotary evaporator. To remove traces of toluene, the dry residue was evaporated several times with glacial acetic acid. The bright red powder of product 1b was washed with diethyl ether and removed mechanically. Yield 1b was 96-99%. Potassium salt 1-K was obtained in quantitative yield by dissolving 1b in an aqueous solution of potassium carbonate (2.5 equivalents of K ₂ CO ₃ per equivalent of 1b).

Spectral characteristics:

1a. ¹ H NMR (600 MHz, CDCl ₃ , δ, ppm): 2.06 (m, 2H); 2.17 (m, 8H); 2.49 (t, 2H); 2.59 (t, 8H); 3.68 (s, 3H); 3.73 (s, 6H); 3.74 (s, 6H); 3.93 (t, 2H); 4.06 (m, 8H); 6.66 (d, 2H); 6.84 (d, 4H); 6.89 (d, 4H); 7.16 (d, 2H); 7.56 (d, 4H); 7.84 (d, 4H). ¹³ C NMR (150 MHz, CDCl ₃ , δ, ppm): 24.58 (CH ₂ ); 24.64 (CH ₂ ); 24.67 (CH ₂ ); 30.45 (CH ₂ ); 30.58 (CH ₂ ); 51.66 (OCH ₃ ); 51.72 (OCH ₃ ); 57.52 (sp ³ wireframe); 60.14 (sp ³ framework); 62.86 (sp ³ framework); 66.48 (OCH ₂ ); 66.72 (OCH ₂ ); 66.83 (OCH ₂ ); 113.66 (Ar); 114.56 (Ar); 114.67 (Ar); 114.80 (Ar); 127.74; 129.10; 129.25; 129.40; 129.76; 130.56; 131.03; 131.26; 136.45; 142.85; 143.42; 143.54; 143.75; 143.86; 144.09; 144.14; 144.24; 144.43; 144.49; 145.30; 145.42; 146.88; 147.25; 147.27; 147.40; 147.84; 148.14; 148.30; 148.45; 148.63; 148.68; 148.69; 148.76; 150.55; 151.34; 153.64; 156.86; 157.87; 158.38; 158.44; 173.65 (COOSN ₃ ); 173.67 (COOSN ₃ ); 173.71 (COOSN ₃ )

1b / 1-K ¹ H NMR (600 MHz, CS ₂ -acetone-D ₆ , δ, ppm): 2.00 (m, 2H), 2.09 (m, 8H), 2.45 ( t, 2H), 2.53 (m, 8H), 3.96 (t, 2H), 4.08 (m, 8H), 6.70 (d, 2H), 6.92 (d, 4H), 6.96 (d, 4H), 7.20 (d, 2H), 7.61 (d, 4H), 7.88 (d, 4H). ¹³ C NMR (150 MHz, CS ₂ -acetone-D ₆ , δ, ppm): 24.61 (CH ₂ ); 24.64 (CH ₂ ); 24.69 (CH ₂ ); 29.85 (CH ₂ ); 29.88 (CH ₂ ); 57.55 (sp ³ wireframe); 60.27 (sp ³ framework); 62.96 (sp ³ framework); 66.76 (CH ₂ ); 66.85 (CH ₂ ); 66.93 (CH ₂ ); 77.04 (C-Cl); 113.79 (Ar); 114.68 (Ar); 114.87 (Ar); 114.92 (Ar); 115.01 (Ar); 128.82; 129.20; 129.30; 129.66; 129.81; 130.35; 131.33; 136.03; 142.88; 143.59; 143.77; 143.83; 143.89; 144.17; 144.21; 144.28; 144.42; 144.52; 145.43; 145.57; 147.10; 147.23; 147.26; 147.39; 147.81; 148.09; 148.26; 148.42; 148.56; 148.64; 148.66; 148.73; 150.81; 151.53; 153.81; 157.03; 158.25; 158.79; 158.81; 173.55 (COOX); 173.68 (COOX); 173.85 (COOX).

Example 2. Determination of the cytotoxic effect of the polycarboxylic derivative of fullerene 1-K on Vero cells.

Cytotoxicity was determined by three parameters: acute cytotoxic dose, chronic cytotoxic dose and maximum tolerated dose. Cells were incubated at a temperature of 37 ° C in an atmosphere of 4.7% CO ₂ in the presence of 1-K in increasing concentrations. Acute cytotoxicity was determined 24 hours after compound administration. Chronic cytotoxicity was determined after 72 hours, the maximum tolerated dose - 7 days after the introduction of the compound. Cell viability was determined by exclusion of the trypan blue vital dye. For this, the cells were stained with a 0.4% trypan blue solution and counted in the Goryaev chamber. Living cells remained unpainted, while those who died as a result of the toxic effect of the substance were stained in bright blue.

The acute cytotoxic dose (OCD ₅₀ ) 1-K corresponds to the concentration of the compound at which 50% of the cells died one day after the introduction of the substance. Chronic cytotoxic dose (HCD ₅₀ ) is the concentration of the compound at which 50% of the cells died three days after the substance was introduced. The maximum tolerated dose (MTD) is the concentration of the compound at which no cytopathogenic effect is detected within 7 days after the introduction of the substance. Based on the obtained data, graphs of the dependence of the number of viable cells on the concentration of the compound were constructed. The results of an experimental evaluation of the cytotoxic effect of compound 1-K are presented in FIGS. 2 and 3.

Table 1 shows the cytotoxic effect of compound 1-K on the viability of Vero cells. The values of OCD ₅₀ , HCD ₅₀ and MTD were 2300 μg / ml, 2000 μg / ml and 1000 μg / ml, respectively. As a comparison, data on the cytotoxicity of the reference drug acyclovir with anti-HSV activity are presented. As can be seen from the data presented, the cytotoxicity of compound 1-K is lower than the toxicity of the well-known antiviral drug acyclovir, which is used in clinical practice for the treatment of HSV infection.

Example 3. Determination of the cytotoxic effect of the polycarboxylic derivative of fullerene 1-K on the cells of VLEC.

Cytotoxicity was determined by three parameters: acute cytotoxic dose, chronic cytotoxic dose and maximum tolerated dose. Cells were incubated at a temperature of 37 ° C in an atmosphere of 4.7% CO ₂ in the presence of 1-K in increasing concentrations. Acute cytotoxicity was determined 24 hours after compound administration. Chronic cytotoxicity was determined after 72 hours, the maximum tolerated dose - 7 days after the introduction of the compound. Cell viability was determined by exclusion of the trypan blue vital dye. For this, the cells were stained with a 0.4% trypan blue solution and counted in the Goryaev chamber. Living cells remained unpainted, while those who died as a result of the toxic effect of the substance were stained in bright blue.

The acute cytotoxic dose (OCD ₅₀ ) 1-K corresponds to the concentration of the compound at which 50% of the cells died one day after the introduction of the substance. Chronic cytotoxic dose (ChCD ₅₀ ) is the concentration of the compound at which 50% of the cells died three days after the substance was introduced. The maximum tolerated dose (MTD) is the concentration of the compound at which no cytopathogenic effect is detected within 7 days after the introduction of the substance. Based on the obtained data, graphs of the dependence of the number of viable cells on the concentration of the compound were constructed. The results of an experimental assessment of the cytotoxic effect of compound 1-K are presented in FIGS. 4 and 5.

Table 2 shows data on the cytotoxic effect of compound 1-K on the viability of the VLC cells. The values of OCD ₅₀ , HCD ₅₀ and MTD were 1354 μg / ml, 1269 μg / ml and 10 μg / ml, respectively. As a comparison, data on the cytotoxicity of the reference drug ganciclovir with anti-CMV activity are presented. As can be seen from the data presented, the values of OCD ₅₀ and HCD _{50 of} compound 1-K are lower than those for the well-known antiviral drug ganciclovir, which is used in clinical practice for the treatment of CMV infection.

Testing results showed that OCD ₅₀ and HCD ₅₀ for the polycarboxyl derivative C ₆₀ 1-K exceed 1000 μg / ml. Thus, the data presented indicate a low cytotoxicity of compound 1-K.

Determination of the antiviral properties of 1-K in a microbicidal regimen for use in cell systems in vitro.

To establish the antiviral effect of 1-K, the concentration of the substance, causing the suppression of viral activity by 50%, was determined. Viral activity was detected by immunocytochemical staining using monoclonal antibodies interacting with viral proteins, followed by counting of antigen-containing cells (plaques).

Example 4. Determination of the antiviral properties of the derivative of fullerene 1-K against HSV-1 in a microbicidal regimen.

Compound 1-K was applied to the Vero cell monolayer at various concentrations and incubated for 1 hour at 37 ° C. Then the cells were infected with HSV-1, reference strain F, with a multiplicity of infection of 10 ^-3 PFU / cell. The control was infected cells not treated with compound 1-K. 3 days after infection, the number of virus-specific plaques in the control and experimental samples was counted. Antiviral activity of the compound was evaluated by suppressing the ability of HSV-1 to plaque and was expressed as a percentage of the control. Figure 6 presents the dependence of the suppression of plaque-forming ability of HSV-1 on concentrations of 1-K. The concentration of the compound causing 50% inhibition of plaque formation relative to the control was taken as the inhibitory dose (ID ₅₀ ). ID ₅₀ 1-K for HSV-1 was 18 μg / ml.

Example 5. Measurement of the antiviral properties of the 1-K fullerene derivative with respect to CMV in a microbicidal regimen.

Compound 1-K in various concentrations was applied to the cell monolayer of fibroblasts and incubated for 1 hour at 37 ° C. Then the cells were infected with CMV, reference strain AD 169, with a multiplicity of infection of 10 ^-4 PFU / cell. The control was infected cells not treated with compound 1-K. 5 days after infection, the number of virus-specific plaques in the control and experimental samples was counted. The antiviral activity of the compound was evaluated by suppressing the ability of CMV to plaque formation and expressed as a percentage of the control. Figure 7 presents the dependence of the suppression of plaque-forming ability of CMV on concentrations of 1-K.

The concentration of the compound causing 50% inhibition of plaque formation relative to the control was taken as the inhibitory dose (ID ₅₀ ). ID ₅₀ 1-K for CMV was 10 μg / ml.

Chemotherapeutic evaluation of compound 1-K

To assess the prospects of using compound 1-K as a possible chemotherapeutic agent, a chemotherapeutic index (CTI) was calculated, which characterizes the specific antiviral activity of the drugs.

HTI = HCD ₅₀ / ID ₅₀ ,

where HCD ₅₀ is the concentration of the compound, causing a 50% decrease in cell viability 72 hours after the introduction of the substance.

ID ₅₀ - the concentration of the compound, inhibiting the plaque-forming ability of the virus by 50%.

It was established that the CTI of compound 1-K is 111 for HSV infection and 127 for CMV infection. Exceeding the value of CTI equal to 100 indicates a high antiviral activity of the patented compound 1-K.

Thus, compound 1-K, for which it is claimed to be used as a microbicidal antiviral agent, has low cytotoxicity in in vitro cell cultures. The chronic cytotoxic dose of this compound is 2000 μg / ml and 1269 μg / ml in relation to Vero cells and VLCF, respectively. Compound 1-K exhibits pronounced anti-HSV and anti-CMV activity when used in an in vitro microbicidal regimen. The inhibitory dose (ID ₅₀ ) is 18 μg / ml for HSV-1 and 10 μg / ml for CMV. 1-K is characterized by a high chemotherapeutic index (CTI) of more than 100 for both HSV and CMV. An important advantage of this compound is its ability to simultaneously inhibit HSV and CMV infections, unlike most drugs with a monotherapeutic effect.

The data presented confirm the promise of using compound 1-K as a chemotherapeutic agent or biologically active substance in the form of antiviral drugs for preventing infection during sexual contact for emergency prevention of HSV and CMV infections.

Claims (1)

The use of a polycarboxyl derivative of fullerene C ₆₀ having the structural formula

as a microbicidal antiviral agent for inhibiting herpes simplex and cytomegalovirus viruses.

Images