WO2012126231A1

WO2012126231A1 - Use of poly(4-styrene sulfonic acid-copoly-maleic acid) sodium salt and pharmaceutical composition thereof

Info

Publication number: WO2012126231A1
Application number: PCT/CN2011/078371
Authority: WO
Inventors: 吴稚伟; 邱敏; 孙亦彬; 蒋海燕
Original assignee: 苏州维赛生物医药有限公司; 南京大学
Priority date: 2011-03-18
Filing date: 2011-08-12
Publication date: 2012-09-27
Also published as: CN102160870B; CN102160870A

Abstract

Use of poly(4-styrene sulfonic acid-copoly-maleic acid) sodium salt in the preparation of pharmaceutical inhibiting infection of HIV-1 and pharmaceutical composition thereof. Said pharmaceutical composition comprises poly(4-styrene sulfonic acid-copoly-maleic acid) sodium salt as active ingredient and conventional pharmaceutical carrier.

Description

Use of poly(4-styrenesulfonic acid-co-maleic acid) sodium salt and pharmaceutical composition thereof

The present invention relates to the use of poly(4-styrenesulfonic acid-co-maleic acid) sodium salt, and to a pharmaceutical composition containing poly(4-styrenesulfonic acid-co-maleic acid) sodium salt as an active ingredient . Background technique

AIDS is caused by infection with human immunodeficiency virus (HIV). Invasion of target cells by HIV-1 is a specific biological process that requires the envelope protein gpl20 on the surface of the virus to bind to the cellular receptor CD4, induce a change in the conformation of _g pl20, and then bind to the cell surface co-receptor CCR5 or CXCR4. The conformational change of the fusion protein gp41, which was originally blocked by gpl20, exposes its hydrophobic N-terminal fusion peptide sequence and is inserted into the host cell membrane, thereby initiating fusion of the viral membrane with the cell membrane, eventually leading to the entry of the viral core structure into the cell, establishing an infection. . A number of inhibitors have been developed for this biological process, such as the adjuvant receptor inhibitors Maraviroc and PSC-RANTES, and the fusion process inhibitor T20 (which has been approved for marketing by the US FDA). Fuviridine) and C52L polypeptides are also promising candidates for microbicides.

The discovery of new compounds or natural products that inhibit HIV infection is important for the development of microbicides that effectively prevent HIV transmission. It is an object of the present invention to find innovative lead compounds and subsequently develop novel microbicides or drugs that effectively block HIV infection.

Poly (4-styrenesulfonic acid-co-maleic acid) sodium salt is a synthetic polymer compound, soluble in water, and the aqueous solution is colorless to Light yellow, good heat resistance. Since the compound carries a styrene sulfonic acid group and a maleic anhydride group, it has a high anion distribution density and is highly dispersible, and is a surface active material. Industrial synthesis of styrene sulfonic acid-maleic anhydride copolymer is very mature, usually divided into a first sulfonation polymerization method and a first polymerization sulfonation method: the former first sulfonated styrene monomer, and then in an inert solution Carry out horse The copolymerization of the anhydride is usually carried out at a temperature of 80 to 12 CTC; the latter is a copolymerization of styrene and maleic anhydride, followed by sulfonation of the copolymer with chlorosulfonic acid, concentrated sulfuric acid, sulfur trioxide or the like. Poly(4-styrenesulfonic acid-co-maleic acid) sodium salt has a wide range of applications in industry and agriculture, and can be used in detergents and dispersants, but there are few reports on its application in the field of medicine and health, especially It is used as an antiviral drug. Summary of the invention

A first object of the present invention is to provide a poly(4-styrenesulfonic acid-co-maleic acid) sodium salt for use in the preparation of a medicament for inhibiting HIV-1 infection;

A second object of the present invention is to provide a pharmaceutical composition for inhibiting HIV-1 comprising poly(4-styrenesulfonic acid-co-maleic acid) sodium salt as an active ingredient, which further comprises a conventional pharmaceutical carrier .

Compared with the prior art, the present invention has the following advantages: poly(4-styrenesulfonic acid-co-maleic acid) sodium salt has a high negative charge and can be associated with HIV-1 cell membrane protein _g pl20 V3 The fragment (positive charge) interacts to block the interaction of the V3 fragment with the receptor CD4 to block the entry of HIV-1 virus into the cell or to inhibit the fusion of CH0-WT with MT-2 cells. In vitro antiviral experiments have demonstrated that poly(4-styrenesulfonate-co-maleic acid) sodium salt has strong anti-CD4-dependent HIV-1 infection and also performs well for some clinical isolates. The activity, which includes the B', B'C and CRF01-AE recombinant subtypes, indicates that the poly(4-styrenesulfonate-co-maleic acid) sodium salt has a broad spectrum of resistance to HIV-1. In addition, poly(4-styrenesulfonate-co-maleic acid) sodium salt also has good anti-infective ability against CD4-independent HIV-1 infection. In vitro toxicity tests on poly(4-styrenesulfonic acid-co-maleic acid) sodium salt revealed that the compound has very low toxicity. Finally, cell fusion experiments showed that poly(4-styrenesulfonate-co-maleic acid) sodium salt has a role in inhibiting the fusion process mediated by the HIV-1 viral envelope protein gpl20. Poly(4-styrenesulfonic acid-co-maleic acid) sodium salt is abundant in source and low in production cost, and has potential application value as a novel anti-HIV-1 drug and microbicide.

DRAWINGS Figure 1 shows poly(4-styrenesulfonic acid-co-maleic acid) sodium salt inhibits HIV-1 B subtype standard strain JR-FUR5 tropism) and HXB2U4 tropism) to CD4+ CCR5+ CXCR4+ Ghost (3 a graph of X4-Hi5 cell infection;

Figure 2 is a graph showing the inhibition of infection of CD4 ⁺ CCR5 + CXCR4 ⁺ Ghost (3) X4_Hi5 cells by a poly(4-styrenesulfonic acid-co-maleic acid) sodium salt;

Figure 3 is a poly(4-styrenesulfonic acid-co-maleic acid) sodium salt inhibiting the HIV-1 B subtype standard strain JR-FL (R5 tropism) and HXB2 (X4 tropism) on CD4- a graph of Caco_2 cell infection;

Figure 4 is a poly(4-styrenesulfonic acid-co-maleic acid) sodium salt inhibiting the HIV-1 B subtype standard strain JR-FL (R5 tropism) and HXB2 (X4 tropism) on CD4- a graph of HEC-A cell infection;

Figure 5 is a graph showing the toxicity of poly(4-styrenesulfonic acid-co-maleic acid) sodium salt on CD4-Caco-2 cells;

Figure 6 is a graph showing the toxicity of poly(4-styrenesulfonic acid-co-maleic acid) sodium salt on CD4- HEC-1-A cells;

Figure 7 is poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSM), dextran sulfate (Dextran sul f at e ), zidovudine (AZT), nevirapine (nev irapine) Inhibition of time-inhibition profiles of HIV-1 infected CD4+ CCR5+ CXCR4+ Ghost (3) Χ4-ΗΪ5 cells;

Figure 8 is a bar graph showing the inhibition of HIV-1 viral envelope protein gpl20 mediated cell fusion by poly(4-styrenesulfonate-co-maleic acid) sodium salt at various concentrations. detailed description

The invention is further illustrated below in conjunction with the drawings showing experimental results.

Experimental material

1.1 poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSM);

Its molecular formula is as follows:

Positive control samples: Dextran sulfate, Zidovudine (AZT), Nevirapine were purchased from SIGMA;

CCK-8, Dojindo Laboratories;

PEK linear, 25kD), purchased from Polyscience, formulated into a 5 mg/ml solution;

HIV-1 pseudotype virus

1.6.1 Pseudovirus backbone plasmid: PNL4-3, constructed with luciferase reporter gene

(lucif erase reporter gene ) , bow I from the United States Rockf eller university;

1.6.2 Pseudovirus envelope plasmids derived from different HIV-1 subtype gene sequences, cited from Tsinghua University AIDS Research Center;

HIV-1 B subtype standard strain: JR-FL, HXB2;

HIV-1 clinical isolates: CNE6 (Β' subtype), CNE30 (Β' C subtype), CNE50 (B, C subtype) and Wokou CNE55 (CRF01-AE subtype), all isolated from HIV infection in China Including the major HIV-1 epidemic subtypes in China;

Luciferase as say system kit 贝勾自 Promega;

Cell

1.7.1 Human osteosarcoma cell line Ghost (3) Χ4-ΗΪ5, expressing CD4 receptor, CCR5 and CXCR4 co-receptors;

1.7.2 Human colon cancer epithelial cells Caco-2, does not express CD4 receptor;

1.7.3 Human cervical cancer epithelial cells HEC-1-A, does not express CD4 receptor;

1.7.4 Human T cell line Leukemia cell MT-2, expressing T cell surface receptor CD4, For detecting HIV-1 induced cell fusion;

1.7.5 Hamster ovary cells CH0-WT, expressing HIV-1 envelope protein gpl20, is used to detect HIV-1 induced cell fusion;

1.7.6 Human embryonic kidney cells 293T, used for packaging to produce HIV-1 pseudovirions; Cell culture medium: DMEM high glucose + 10% fetal bovine serum;

96-well cell culture plate: Corning;

0 24-well cell culture plate: Corning;

1 96-well fluorescence detection plate: Costar;

2 GloMax 96-well chemiluminescent microplate reader;

3 TECAN microplate reader;

4 Thermo C02 incubator. experimental method

Packaging of HIV-1 pseudovirions in human embryonic kidney cells 293T cells

The HIV-1 pseudoviral backbone plasmid pNL4-3 and different pseudovirus envelope plasmids were co-transfected into 293T cells using PEI transfection reagent. After 48 hours of culture, the supernatant was collected and stored in -75 °C. That is, a pseudovirus solution was obtained for subsequent experiments. Luciferase assay for the infectivity of pseudoviruses (TCID ₅ .)

To determine the infectious titer of the pseudovirus, firstly dilute the virus harvesting solution, add 100 ul to the 96-well plate, 4 replicate wells per gradient, and add lOOul Ghost (3) X4-Hi5 cell suspension, cell density 10 ⁵ After 48 hours of cell/mlo culture, the relative fluorescence value (RLU) of each 孑L was determined on a GloMax-96 Microplate Luminometer using a Promega GloMax Lysis Buffer and Luciferase assay system kit. The RLU was greater than 2.5 times the background value and was judged to be positive. The infectious titer of the pseudovirus (TCID ₅ ) was calculated using the Reed_Muench method. PSM antiviral activity test

The PSM was dissolved in double distilled water and sterilized by filtration. Dilute with DMEM complete medium At a certain concentration, perform a 3-fold gradient dilution in a 96-well plate at a maximum concentration of 60 μg/ml (3 μM and then add 50 μ 1 4000 TC ID ₅ ·/ml of virus dilution to each well to be tested. , mix, incubate for 30 min at 37 ° C. Finally add 10 ⁴ Ghost (3) X4_Hi5 cells (or 2 X 10 ⁴ Caco_2 and HEC-Bu A cells) to the well to be tested, 37 ° C, 5% ( 0 ₂ For 48 h, the IJ was tested for fluorescence intensity on a GloMax_96 Microplate Luminometer using a Promega GloMax Lysis Buffer and Luciferase assay system kit (the magnitude of the fluorescence intensity represents the extent of viral infection).

2.4 in vitro cytotoxicity experiment

2X 10 ⁴ Caco-2 and HEC-lA cells were inserted into 96-well plates per well, and cultured at 37 ° C, 5% CO _{2 for} 24 h. The PSM was serially diluted to a maximum concentration of 10 mg/ml and a dilution factor of 3 fold. The culture medium in the 96-well plate was discarded and replaced with DMEM complete medium containing a gradient concentration of PSM. Incubate at 37 °C, 5% CO ₂ to the detection time point (6h, 12h, 24h, 48h), and add 10 μl/well of CCK-8 reagent (Dojindo Laboratories) to continue to culture for 3 hours. Finally, the light absorption value was measured at 450 nm using a TECAN plate reader to calculate the cell survival rate.

2.5 drug inhibition phase experiment

This experiment was carried out using the Time-of-drug addition method. First, 10 ⁴ Ghost (3) X4-Hi5 cells were seeded in 96-well plates, and cultured at 37 ° C, 5% CO _{2 for} 12 h. Add 50 μl of 4000 TCID ₆₀ /ml virus dilution to the well to be tested. Then, PSM and positive control drugs were added at each time point and mixed. The PSM concentration was 60 μg/ml, AZT was 0.5 μg/ml, Nevirapine was 2 μg/ml, and dextran sulfate was 300 μg/ml. After 48 h of incubation, the fluorescence intensity was measured on a GloMax-96 Microplate Luminometer using Promega GloMax Lysis Buffer and Luciferase as say system kit (the magnitude of the fluorescence intensity represents the extent of viral infection). In this experiment, complete medium wells were added as control at each time point.

2.6 cell-cell fusion inhibition assay CH0-WT was cultured in GMEM-S medium containing 400 μM methionine sulf oximine. When the coverage reached 70%, sodium butyrate was added to a final concentration of 6 mM to stimulate _g pl60 trimer (envelope protein). Expression of gpl20 + fusion protein _g p41). After stimulation for 16 h, the cells were digested with 0.5 mM (EDTA + EGTA), counted, and inoculated with ⁵ × ^{10 5} CH0-WT per well in a 24-well plate. At the same time, the same number of MT-2 were inoculated into each well of a 24-well plate. Different concentrations of PSM (60, 20, 6.67, 2.22, 0.74, 0 ug/ml) were added, and cultured at 37 ° C, 5% CO _{2 for} 24 h, and the number of syncytia was observed and counted with an inverted microscope.

3. Experimental results

3.1 In Figure 1, the poly(4-styrenesulfonic acid-co-maleic acid) sodium salt was detected by a pseudovirus system carrying the luciferase gene to inhibit the HIV-1 B subtype standard strain JR-FL (R5 Sexuality HXB2 (X4 tropic) Infection of CD4+ CCR5+ CXCR4+ cells (Ghost (3) Χ4-ΗΪ5) was found to have a significant inhibitory effect and inhibit EC ₅ . They were 4.988 μg/ml and 5.260 g/ml, respectively. The poly(4-styrenesulfonic acid-co-maleic acid) sodium salt has substantially no inhibitory effect on the HIV-1 strain VSV-G having the envelope protein VSV.

3.2 In Figure 2, the poly(4-styrenesulfonic acid-co-maleic acid) sodium salt was detected by a pseudovirus system carrying the luciferase gene to inhibit HIV-1 clinical isolates against CD4 ⁺ CCR5 + CXCR4+ cells. Infection, HIV-1 clinical isolates include CNE6 (Β' subtype, R5 tropism), CNE30 (Β' C subtype, R5 tropism), CNE50 (Β' C subtype, R5 tropism) and CNE55 (CRF01-AE subtype, R5 tropism); The compound PSM was found to have a significant inhibitory effect on these clinical strains, inhibiting EC ₅ . 1.8757 μ g/ml,

3.1361 μ g/ml, 1.7050 μg/ml and 1.7064 μg/ml.

3.3 Detection of poly(4-styrenesulfonic acid-co-maleic acid) sodium sulphate-containing pseudovirus system Sodium salt inhibits HIV-1 B subtype standard strains JR-FL and HXB2 against CD4-cells ( Infection of human colon cancer epithelial cell Caco-2 and human cervical cancer glandular epithelial cell HEC-1-A) was found to have a strong inhibitory effect, as shown in Fig. 3 and Fig. 4: in Caco-2 The EC50 of the intracellular inhibition of JR-FL and HXB2 was 7.307 μg/ml and 542 μg/ml; inhibiting the EC50 of JR-FL and HXB2 on HEC-1-A was 2.050 μ g/ml and 3.951 μg/ml.

3.4 In Figure 5 and Figure 6, the toxicity of poly(4-styrenesulfonate-co-maleic acid) sodium salt to related human cell lines was examined by CCK-8 method, and a high concentration of the compound PSM was found on human colon. Cancer epithelial cells (Caco-2) and human cervical cancer glandular epithelial cells (HEC-1-A) are less toxic.

3.5 In Figure 7, the poly(4-styrenesulfonate-co-maleic acid) sodium salt was determined to inhibit HIV-1 infection of CD4 ⁺ CCR5 + CXCR4+ cells using a pseudovirus system carrying the luciferase gene.

(Ghost (3) Χ4-ΗΪ5) The mode of action and principle, the results show that the poly(4-styrenesulfonate-co-maleic acid) sodium salt (PSM) and the dextran sulfate (Dextran sulfate) curve similar It can be speculated that the poly(4-styrenesulfonic acid-co-maleic acid) sodium salt acts in a similar manner to dextran sulfate, and achieves an antiviral action by inhibiting virus entry into cells.

3.6 Using CH0-WT-MT-2 co-culture fusion detection poly(4-styrenesulfonic acid-co-maleic acid) sodium salt inhibits gpl20 and CD4-mediated cell fusion, and in Figure 7, poly(4) was found. - Styrenesulfonic acid-co-maleic acid) Sodium salt can effectively inhibit the fusion of CH0-WT and MT-2 cells. Inhibition of HIV-1 viral envelope protein-mediated fusion from poly(4-styrenesulfonate-co-maleic acid) sodium salt, further illustrating poly(4-styrenesulfonic acid-co-maleic acid) sodium Salt prevents the HIV-1 virus from entering the cell to inhibit the virus.

Our study shows that poly(4-styrenesulfonate-co-maleic acid) sodium salt has a high negative charge and can interact with the V3 fragment (positive charge) of the membrane protein _g pl20 of HIV-1. The effect is to block the interaction of the V3 fragment with the receptor CD4 to block the entry of HIV-1 virus into the cell or to inhibit the fusion of CH0-WT with MT-2 cells. In vitro antiviral experiments have demonstrated that poly(4-styrenesulfonate-co-maleic acid) sodium salt has strong anti-CD4-dependent HIV-1 infection and some clinical isolates It also showed good activity, including B', B'C and CRF01-AE recombinant subtypes. This indicates that the poly(4-styrenesulfonic acid-co-maleic acid) sodium salt has a broad spectrum of resistance to HIV-1. In addition, poly(4-styrenesulfonate-co-maleic acid) sodium salt also has good anti-infective ability against CD4-independent HIV-1 infection. In vitro toxicity tests on poly(4-styrenesulfonic acid-co-maleic acid) sodium salt revealed that the compound has very low toxicity. Finally, cell fusion experiments showed that poly(4-styrenesulfonate-co-maleic acid) sodium salt has a role in inhibiting the fusion process mediated by the HIV-1 viral envelope protein gpl20. The poly(4-styrenesulfonic acid-co-maleic acid) sodium salt is rich in source and low in production cost, and has potential application value as a novel anti-HIV-1 drug and microbicide.

In the preparation of a pharmaceutical composition for inhibiting HIV-1 infection, a poly(4-styrenesulfonic acid-co-maleic acid) sodium salt is used as an active ingredient, and a conventional pharmaceutical carrier is added. The pharmaceutical composition can be formulated as an injection, a tablet, a capsule, an aerosol, a suppository, a film, a pill, a topical tincture, or a controlled release or sustained release dosage form or a nano preparation.

Claims

Claim

1. Use of poly(4-styrenesulfonic acid-co-maleic acid) sodium salt for the preparation of a medicament for inhibiting HIV-1 infection.

A pharmaceutical composition for inhibiting HIV-1 infection comprising a poly(4-styrenesulfonic acid-co-maleic acid) sodium salt as an active ingredient and comprising a conventional pharmaceutical carrier.