RU2270690C1 - Complex anti-hiv compound - Google Patents

Abstract

FIELD: biology, medicine.

SUBSTANCE: invention proposes the complex anti-HIV compound that comprises polyanionic matrix and chemical bound pseudoligand for HIV-1/2 gp120 that comprises a peptide fragment of HIV-1/2 CCR5 or CXCR4 co-receptor. Polyanionic matrix and bound peptide fragment of HIV-1/2 CCR5 or CXCR4 co-receptor are represented by the general formula:

. Invention provides target delivery of antiviral preparation to the focus (tissue) damaged with HIV-1/2. The compound inhibits the specific viral infection at its initial development stages and possesses the diminished toxicity also. Invention can be used in treatment and prophylaxis of HIV-infection and AIDS, and in research study in investigations of ligand-receptor interaction of surface viral proteins - cellular receptors type.

EFFECT: improved and valuable medicinal properties of complex compound.

3 cl, 2 tbl, 4 ex

Description

The invention relates to biology and medicine and can be used for the treatment and prevention of HIV infection and AIDS (a), as well as in research work to study ligand-receptor interactions (such as surface viral proteins - cellular receptors).

A complex anti-HIV compound is known, comprising a polymer matrix made in the form of nanoparticles, on the surface of which peptides, which are monoclonal anti-CD ₄ antibodies, are applied by passive adsorption (International application (WO) 94/24168, IPC C 07 To 17/08, publ. 10/27/1994) [1]. The specified complex compound can be used for the manufacture of a medicament for the prevention or treatment of pathological conditions associated with HIV infection.

However, this anti-HIV complex is a high molecular weight compound that is poorly utilized from the body, which can cause severe toxic effects when used.

Complex anti-HIV compounds are known, including a polyanionic matrix and a synthetic pharmacophore modifier Norbornen [2, 3, 4].

The closest technical solution (prototype) is another complex anti-HIV compound comprising a polyanionic matrix and chemically bonded pseudoligand for gp120 HIV-1/2. The pseudo-ligand for gp120 HIV-1/2 is a synthetic pharmacophore modifier, for example Adamantan [5, prototype].

The disadvantage of the above analog compounds, including the prototype, is the relatively narrow range of potential targets in the multi-stage process of interaction of HIV-1/2 - a sensitive cell. The interaction of the drug can occur in addition to the target agents with biologically important compounds and structures that are not involved in the development of HIV infection and AIDS. In addition, these compounds due to nonspecific binding of the pharmacophore to biologically active substances can lead to their inactivation and development due to this toxic manifestations. Thus, these chemical compounds cannot fully block the initial stages of cell infection with human immunodeficiency virus.

The technical result of the invention is the creation of less toxic complex chemical compounds that provide targeted delivery of an antiviral drug to the lesion (tissue) of HIV-1/2 and the suppression of specific viral infection at its initial stages of development.

This technical result is achieved in that in a complex anti-HIV compound comprising a polyanionic matrix and chemically bonded pseudo-ligand for gp120 HIV-1/2, according to the invention, the pseudo-ligand for gp120 HIV-1/2 contains a peptide fragment of the HIV-1/2 coreceptor CCR5 or CXCR4, and the polyanionic matrix and the peptide fragment of the HIV-1/2 CCR5 or CXCR4 coreceptor associated with it have the following general formula:

polyanion matrix

The peptide fragment of the HIV-1/2 CCR5 coreceptor (structure of the peptide fragment from the N-terminus (K-D2-E18) of the CCR5 chemokine receptor) has the following formula:

The peptide fragment of the HIV-1/2 CXCR4 coreceptor (the structure of the peptide fragment from the extracellular second loop (EXL2) region of the CxCR4 chemokine receptor (K-D187-V198) has the following formula:

Thus, to obtain the claimed complex product, targeted anti-HIV compounds are selected and synthesized based on peptide fragments of the HIV-1/2 CCR5 or CXCR4 coreceptors, which are chemically linked to the polyanion matrix into a complex preparation that provides a high level of protection for HIV-infected cells.

Peptide fragments of the HIV-1 CCR5 and CxCR4 cellular coreceptors play a key role in protein binding to the gp120 ligand.

Salient features of the invention are:

- design and synthesis of fragments of the original fragments of peptides of the co-receptors of HIV-1/2;

- use in the preparation of a polyanion matrix with a molecular weight of 20 KD in order to increase the charge and hydrophilicity of the complex compound;

- combining into a single macromolecular complex of targeted peptide fragments of HIV-1/2 coreceptors - CCR5 or CXCR4 and a polyanionic matrix.

This complex of the chemical and spatial structure of the macromolecular compound is unknown from available sources of information.

The drawing shows graphs of comparative anti-HIV efficacy (to protect cells from death) of the complex preparation As643 (matrix with attached peptide PZ), a mixture of the matrix with peptide P3 and one peptide P3 (simulator of the HIV co-receptor CxCR4).

Example 1. The synthesis of peptide fragments

The synthesis of peptide fragments imitating functionally significant sites of the CCR5 and CXCR4 coreceptors is carried out by the solid-phase method on a column of a chlorotrityl polymer using an alkaline labile 2- (4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc) group to temporarily protect the N-terminus [6]. Solid-phase synthesis is widely used to obtain biologically active peptides used in medicine, veterinary medicine, biotechnology and scientific research. The essence of solid-phase synthesis of peptides consists in the stepwise growth of the peptide chain through repeated cycles of chemical reactions starting from the C-terminal amino acid attached to an insoluble carrier. In this case, the target products of all reactions in the synthesis process remain bound to the carrier, and excess reagents and by-products are removed by filtration and washing of the carrier.

To conduct solid-phase synthesis of the peptide, the C-terminal amino acid of the target amino acid sequence protected by the α-amino group is covalently linked to the insoluble polymer carrier via the α-carboxyl group by formation of an amide or ether bond. Then, from the obtained Nα-protected aminoacyl polymer, the Nα-protective group is cleaved to obtain an aminoacyl polymer with a free α-amino group. Further, this polymer is acylated with the following amino acid protected at the α-amino group and an Nα-protected dipeptidyl polymer is obtained. Synthetic cycles consisting of the steps of cleaving the Nα-protecting group and acylating the free amino group of the peptidyl polymer with the next amino acid protected by the α-amino group are repeated until the complete amino acid sequence of the target peptide has been assembled.

Since large molar excesses of acylating reagents (2-10-fold with respect to free amino groups) are used in the process of solid-phase synthesis, all reactive groups in the side radicals of amino acids, in particular amino, carboxy, hydroxy, mercapto, guanidino groups must be blocked by protective groups. Protective groups for this purpose are selected in such a way as to ensure, on the one hand, complete and constant protection of amino acid side radicals under the conditions of acylation of the peptidyl polymer and cleavage of the temporary N-α protecting group and, on the other hand, be able to quantitatively and without damage to the structure the synthesized peptide to cleave these protective groups in one or two stages.

The synthesized peptides were purified by preparative HPLC (Altex, USA) on a 10 × 250 mm column with a SynChropak RPP 100, 10 μm phase (Eichrom Ind., USA) at a flow rate of 4 ml / min in a linear gradient of eluents from A to B in 2 h (Eluent A — 0.1% TFA in water; Eluent B — 0.1% TFA in 60% acetonitrile solution). At the output, peptides with 90-98% purity were obtained according to analytical HPLC. The amino acid lysine is introduced from the N-terminal fragment of the peptide, having an amino group sterically accessible for chemical modification with the aim of subsequent chemical binding of the peptides to the polycarboxylate matrix.

Example 2. Obtaining a polymer matrix

Below is a diagram of a three-stage synthesis of a polycarboxylate matrix from maleic anhydride (a more detailed description of the synthesis is in [5]), as well as a scheme for covalent attachment of a peptide fragment:

Example 3. Obtaining a comprehensive anti-HIV compound

Take the polycarboxylate matrix of the following formula

abbreviated formula

To modify the matrix, its anhydride form is used, which is reactive with respect to synthetic peptides. The total degree of matrix modification was limited to no more than 20% to maintain the required number of unmodified carboxyl groups of the matrix, which are responsible for providing a negative charge and its specific adjuvant properties. These carboxyl groups were obtained after preliminary steps of modification with peptide pharmacophores (the scheme is shown in Example 2) by exhaustive hydrolysis of the remaining unmodified anhydride matrix cycles. The hydrolysis products were purified from residues of organic solvents and unreacted reagents by ultrafiltration of aqueous solutions using a 3KD membrane permeability. The final products in the form of a sodium hemisphere were isolated by freeze drying.

Example 4. The study of anti-HIV activity of the claimed complex compounds

Preparations. For research, we used preparations synthesized by organic and polymer synthesis at the Research Center for Biomodulators and Medicinal Compounds of the NIF Health. In preparation for biological tests, the preparations were thoroughly cleaned of possible low molecular weight impurities by the method of selective ultrafiltration: four cycles of tenfold washing-concentration on ultrafiltration membranes from Sigma (USA) with a permeability of up to 1 or 3 KD (NMLW; 1,000 and 3,000, respectively). Purified polymer-associated substances were isolated by freeze drying in the form of white highly porous powders soluble in water, dimethyl sulfoxide and alcohol.

Virus. To evaluate the effectiveness of anti-HIV compounds, we used the macrophagotropic strain HIV-1 _EVK (STB 4005), the macrophage-T-lymphocytotropic syncytium forming the HIV-1 _Z strain, the CXCR4-tropic variant of HIV-1 (X4 virus) —LAV strain .04; CCR5-tropic variant of HIV-1 (R5 virus) - strain SF162, provided by the National Institute of Health (USA).

Cell lines. Highly sensitive to HIV-1/2 infection, transplantable culture of human lymphoblastoid cells MT-4. MT-4 cells were cultured in complete growth medium RPMI-1640, containing 0.06% L-glutamine, 10% serum of cow embryos, lincomycin (60 units / ml) and gentamicin (100 μg / ml) at a temperature of 37 ° C, 5% CO ₂ for 4 days. The inoculum cell concentration was 0.5 × 10 ⁶ cells / ml. On the 4th day of cultivation, the number of cells was taken into account, diluted with growth medium to a seed concentration and cultured again.

PHA-stimulated human peripheral blood mononuclear cells (PBMCs). MCPC was isolated from the venous blood of a healthy donor according to the method described by Fotino et al. [2]. The primary lymphocytes were cultured in RPMI-1640 medium with the addition of 10% serum of cow embryos, 4 μg / ml PHA, 5 units / ml IL-2, 60 units / ml lincomycin and 100 μg / ml gentamicin in 50 mm ³ plastic mattresses at 37 ° C with 5% CO ₂ in the atmosphere. After 4 days of incubation of the culture under these conditions, the nutrient medium was replaced with a new one, similar in composition, but no longer containing PHA. For this, the cells were besieged by centrifugation at 200 g and diluted to inoculum concentration with fresh medium.

The toxicity analysis of the studied compounds was carried out on transplantable lymphoblastoid cell line of human MT-4. The toxicity of the drugs was judged by the viability of the cells in the presence of the tested compounds in a wide range of concentrations, proliferation index and morphology of culture growth. The experimental design is similar to that given below to evaluate the anti-HIV activity of the compounds, but without infection of the cells with the virus.

The study of the anti-HIV activity of the preparations was carried out according to the following scheme: a solution of the drug in concentrations from 0.1 to 500 μg / ml (three wells per concentration) was introduced into cell culture (MT-4, MCPC), the virus was introduced and incubated in for 4 days. The multiplicity of infection was 0.2-1 infectious units per cell. Cell cultures were incubated for 4 days at a temperature of 37 ° C in a humid atmosphere of 5% CO ₂ . HIV-infected cells without the addition of antiviral drugs and uninfected cells served as controls.

Anti-HIV activity was evaluated after incubation to reduce the viability of the culture and to suppress the production of virus-specific p24 HIV-1 protein. The proportion of viable cells was taken into account by trypan blue exclusion (Sigma, USA) or by the MTT dye reduction method (ICN, USA) with the accumulation of formazan in living cells. The amount of virus-specific p24 protein in the culture medium was evaluated using enzyme-linked immunosorbent assay (Vecto HIV-1 p24-antigen test system, sensitivity 20 × 10 ^-12 g / ml, manufactured by Vector-Best CJSC, Russia). Based on the experimental data, dose-dependent curves were constructed and drug concentrations were calculated that protect infected cells from death by 50% (EC ₅₀ ) and 90% (EC ₉₀ ); and also determined the concentration of the drug, 50% (IC ₅₀ ) and 90% (IC ₉₀ ) reducing the accumulation of virus-specific protein p24.

To search for the EU ₅₀ , EU ₉₀ , IC ₅₀ , IC _90, dose-response curves were constructed and the corresponding concentrations and errors were calculated from their linear sections using the reverse regression formulas [N. Draper, G. Smith, "Applied Regression Analysis", Moscow: Finance and Statistics, 1986]. For each concentration value, three different measurements were carried out simultaneously, the normality of the distribution was checked by the R / s criterion, and the variance was equal by the Kochren criterion. Comparison of the effectiveness of the drugs was carried out by comparing the effects at a fixed concentration (10 μg / ml) by the method of univariate analysis of variance. In this case, the reproducibility error estimated from three independent measurements was taken into account.

Experimental data on the anti-HIV activity of monocomponents of complex membrane-active drugs on MT4 cells with HIV-1 _Z strain showed that both peptide fragments of HIV-1/2 P1, P3 coreceptors and the polyanion matrix did not have a significant protective effect on infected HIV-1 cells. According to the data obtained, a mixture of P1 peptides with a matrix (As470 compound) in any P / M ratio (1: 1, 1: 5, 1:10) does not give a visible change in the antiviral activity of the latter. However, with the combination of P3 peptides with a polycarboxylate matrix in a P / M ratio of 1:10, the efficiency of suppressing the reproduction of the virus by As470 significantly increases. The drawing shows that the As643 preparation, in which the P3 peptide is covalently linked to the matrix, shows the highest anti-HIV activity among the compared compounds (polyanionic matrix and a mixture of the matrix with the peptide) in the entire concentration range studied.

Tables 1 and 2 present the results of a study of the antiviral activity of the effectiveness of complex compounds with peptide fragments and a polyanionic matrix.

Table 1
Comparative anti-HIV efficacy of complex membrane-active drugs and basic compounds (MT-4 cells, HIV-1 _z strain). A drug Structure EC ₅₀ μg / ml EC ₉₀ , mcg / ml IC ₅₀ μg / ml IC ₉₀ , mcg / ml As470 M 41.6-74.7 > 70 23.9-68.2 ≥100.0 As337 M-ad 2.9-9.7 2: 8.1 1.5-2.3 5.9-9.5 As639 M-P1P2 2.0-45.0 2: 68.4 ^ 1,0 ≥30.0 As641 M-P5 (Plns) 0.8-9.0 2: 21.5 6.0-9.7 ≥100.0 As643 M-RZ 0.4-0.6 7.1-39.2 0.6-1.0 3.6-11.7 As645 M-P4 6.3-13.7 2: 6.5 0.3-8.9 ≥5.7 Note: M is a polymer matrix; M-Ad - a complex drug matrix-adamantane (prototype); M-P1 (or P2 or P3 or P4 or P5) is the inventive complex preparation with different peptide fragments. table 2
Comparative anti-HIV efficacy of complex membranotropic drugs and basic compounds (MT-4 cells, HIV-levk strain) A drug The composition of the drug EC ₅₀ μg / ml EC ₉₀ , mcg / ml IC ₅₀ μg / ml IC ₉₀ , mcg / ml As470 M 14.0-28.0 ≥70 15.0-70.0 > 100 As337 M-ad 1.3-2.9 3,5 1.6-2.3 5.0-8.5 As639 M-P1P2 4.4-5.6 26.0-47.0 10.0-12.0 ≥30-≥100 As641 M-P5 (Plns) 0.1-1.2 0.26-10.5 2.0-3.7 7.8-14.0 As643 M-P3 1.3-1.8 6.1-7.8 0.9-1.0 12.0-20.0 As645 M-P4 2.0-3.4 12.0-18.0 1.2-1.5 9.3-20.0

Note: M is a polymer matrix; M-Ad - a complex drug matrix-adamantane (prototype); M-P1 (or P2 or P3 or P4 or P5) is the inventive complex preparation with different peptide fragments.

An analysis of the anti-HIV activity of complex preparations carried out on MT4 human lymphoid cells and the HIV-1 _Z strain showed their clear advantage over the use of monocomponent formulations: a peptide mimic or a polyanionic matrix. The presence of a synergistic anti-HIV effect with the simultaneous use of a matrix and peptides imitating HIV-1 coreceptors and an increase in the anti-HIV effectiveness of complex preparations with the inclusion of covalently linked peptides for the studied HIV-1 strains allows us to draw the following conclusion: components representing pseudoligands for the protein HIV-1 gp120, provide competitive inhibition of the interaction of gp120 with cellular coreceptors during viral adsorption-fusion and their inclusion in the composition, ensures the effectiveness of antiviral drugs.

The advantages of the proposed method compared to the known one are that the complex compounds provide targeted delivery of the antiviral drug to the lesion site of HIV-1/2 and suppression of specific viral infection by including peptide fragments in the structure and, in comparison with prototypes, affect a wider range of targets in the early stages of the virus-cell interaction.

Information sources

1. International application (WO) 94/24168, IPC C 07 K 17/08, publ. 10/27/1994.

2. Kolocouris N., Foscolos G. B., Kolocouris A. et al. - Synthesis and antiviral activity evaluation of some new aminoadamantane derivatives. J Med Chem 1994; 37: 2896-2902.

3. Serbin A.V., Stotskaya L.L., Krenzel B.A. et al. Copolymers based on furan and maleic anhydride and the prospects for their use in medicine. Sat Polymers for medical use. M .: INKS 1988; 127-152.

4. Timofeev D.I., Perminova N.G., Serbin A.V. and others. Membranotropic compounds and drugs that affect the early stages of HIV infection. Antibiotics and chemotherapy 2003; 48: 2: 29-41.

5. US patent No. 5880154, A. IPC C 08 F 22/06, publ. 03/09/1999.

6. RF patent No. 2079491, IPC C 07 C 317/48, publ. 05/20/1997.

Claims (3)

1. A complex anti-HIV compound comprising a polyanionic matrix and chemically linked pseudo-ligand for gp120 HIV-1/2, characterized in that the pseudo-ligand for gpl20 HIV-1/2 contains a peptide fragment of the HIV-1/2 co-receptor CCR5 or CXCR4, and the polyanionic matrix and its associated peptide fragment of the HIV-1/2 CCR5 or CXCR4 coreceptor has the following general formula: polyanion matrix

2. The complex anti-HIV compound according to claim 1, characterized in that the peptide fragment of the HIV-1/2 CCR5 coreceptor has the following formula:

3. The complex anti-HIV compound according to claim 1, characterized in that the peptide fragment of the HIV-1/2 CXCR4 coreceptor has the following formula: