CN104083763A - Application of histone deacetylase inhibitor in preparation of latent virus activator - Google Patents
Application of histone deacetylase inhibitor in preparation of latent virus activator Download PDFInfo
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Abstract
The invention discloses an application of a benzamide histone deacetylase inhibitor in preparation of virus reservoir reactivation drugs as well as a composition containing the benzamide histone deacetylase inhibitor. The composition has an obvious effect of activating a reservoir of multiple persistent infection viruses such as human immunodeficiency virus, hepatitis B virus and hepatitis C virus and can be combined with an antiviral agent to obtain a better treatment effect.
Description
Technical field
The present invention relates to a class for the reactivation medicine of storage vault in persistent infection virion, belong to microbiology field.
Background technology
Persistent virus infection refers to the long-term lasting Infection Status of body; because the virus of invasion can not be killed host cell; thereby form symbiosis balance between the two; the infected can band be malicious for a long time or throughout one's life; and the outside toxin expelling of frequent or changeable phase, but the normal appearance clinical symptom relevant with immunopathogenesis that lack.The storage vault of hiding that removing virus lays dormant infection cell forms is the key of persistent virus infection disease cured.The virus that causes mankind's persistent infection comprises HIV (human immunodeficiency virus) (Human Immunodeficiency Virus, HIV), hepatitis B virus (Hepatitis B Virus, HBV), hepatitis C virus (Hepatitis C Virus, HCV) and cytomegalovirus (Cytomegalovirus, CMV) etc.
HIV
Acquired immune deficiency syndrome (AIDS) (Acquired Immune Deficiency Syndrome, AIDS) is infected and is caused by HIV, has become the public health problem of serious threat people of the world health.Up to the present, the whole world has 3,400 ten thousand people and has infected HIV or suffered from AIDS the United Nations's data show, has caused so far more than 3,000 ten thousand people dead.Since 1996, Effective Anti retrovirus retrovirus therapy (Highly Active Anti-retroviral Therapy, HAART) combine and use three kinds or three kinds of above antiviral drugs, make AIDS become a kind of controlled chronic disease from a kind of fatal disease.Although HAART can effectively slow down disease process, can not fundamentally cure the infected, once just bounce-back immediately of drug withdrawal.The main cause that HIV can not be completely removed is that the storage vault of hiding that the latent infection cell due to HIV forms exists.HIV latent virus storage vault is mainly by the tranquillization CD4 of HIV latent infection
+the formations such as T lymphocyte, monocytes/macrophages, hematopoietic cell, its principal character: the one, after integrating, hide, the provirus producer being incorporated in host cell gene group is reticent, avoided the medicine of immune system and anti-reverse transcription virus to attack, once condition is suitable, virus can be recovered replication capacity again; The 2nd, though infected individuals carries latent infection cell quantity less, attenuation rate is slower, be impossible, so HIV latent virus storage vault is the huge obstacle that current clinical treatment can not thoroughly be removed HIV to such an extent as to only depend on HAART treatment that it is thoroughly removed.
Although reached common understanding, think that reactivation virus storage vault is that HIV cures the critical first step, still there is no in the world the marketed drug of storage vault reactivation at present.Antibiotic FR 901228 (HDACi), methylation inhibitor and Bryologs compound are three compounds that research at present has potentiality.
A kind of Buddhist ripple ester extracting in the medicinal plants Homolan thusnutans of medicine Prostratin Shi Cong Samoa.Prostratin can in conjunction with and activated protein kinase C, mediation stimulates transcribing of HIV, and then the virus of hiding in immunocyte is discharged.After this one group of Bryostatin analog Bryologs compound has been synthesized in design, and noval chemical compound is proved the effect that can equate or be better than former material and activates the HIV storage vault of hiding.
Epigenetics research discovery, the DNA cytosine methylation of infected cell contributes to maintain HIV hides, and lessens the curative effect.In vitro study data show, virus 5 ' long terminal repeat (LTR) of end has the CpG formation that methylates, and methylation inhibitor 5-aza-20-deoxycytidine can cause proviral reactivation.Proved that this class medicine is especially very effective with other drug use in conjunction.
HDACi class medicine also receives much concern, in clinical trial and different laboratory research stages.The sodium valproate of short-chain fat acids (VPA) is a kind of weak HDACi, can increase cell HIV In vitro culture, latent infection gene expression and virus replication.The Vorinostat (Vorinostat, SAHA) of hydroximic acid is that viral storage vault activates the comparatively forward HDACi of research propelling, in the clinical trial assessment stage, can induce the T cell line of the chronic HIV the infected's latent infection of part and static CD4
+t cellular replication HIV.
HBV
It is one of the most general problem jeopardizing human health that HBV infects.According to World Health Organization's statistics, in 6,000,000,000 populations of the whole world, approximately there are 2,000,000,000 people once to infect hepatitis B virus, approximately there are 400,000,000 Chronic HBV carriers, approximately there are l00 ten thousand people to die from HBV every year and infect relevant hepatic disease.China is the high Endemic Area that HBV infects, and approximately has 1.2 hundred million hepatitis B surface antigen carriers, wherein chronic hepatitis B patient approximately 2,000 ten thousand people.Two large class antiviral drugs nucleoside (acid) class medicines and the interferon of clinical treatment hepatitis B only can stop progression of disease at present, extend life cycle, but any class all can not be removed HBV completely, reach the object of radical cure hepatitis B.HBV has special reverse transcription replication form, and special replicative intermediate covalently closed circular DNA (covalently closed circular DNA, cccDNA).The viral minichromosomes of cccDNA and histone and nonhistones formation superhelix, stable existence is in the nucleus of liver, pancreas, kidney, bone marrow etc., become the intractable root of hepatitis B, after persistent viral infection, antiviral therapy, the aspect such as viral reactivation plays a crucial role.Promote the removing of cccDNA to become one of current antiviral important goal.
HCV
Hepatitis C is that the pandemic infection being caused by HCV is sick.Due to the factors such as variability, pantropic, immunologic tolerance and immunologic injury of HCV, cause HCV to infect height chronicity.At present, there are 1.7 hundred million people's HCV infection in the whole world, the existing the infected 4,000 ten thousand of China, and infection rate is 3.2%, is hepatitis C moderate country occurred frequently.
Adopt in recent years anti-HCV conjoint therapy, and can make most of patient obtain lasting virus sweep, but still had the interior lasting carrier state of hiding that forms of patients with chronic hepatitis C body.Remove the storage vault of hiding of virus, by the hope that brings disease thoroughly to cure for patient.
In sum, viruses molecule in scavenger precursor virus storage vault is the emphasis in treatment of viral infections, the viruses molecule how effectively to activate in viral storage vault is a difficult point of this treatment, although prior art has been found that the activator such as Vorinostat one class of hydroximic acid, but the problem due to its cytotoxicity and activation efficiency, in clinical practice, still exist problem demanding prompt solution, therefore prior art also needs activation efficiency higher, the latent virus activator that cytotoxicity is less.
Summary of the invention
The object of this invention is to provide a class for the reactivation medicine of storage vault in persistent infection virion.Based on above-mentioned purpose, first the present invention provides the application of benzamides Antibiotic FR 901228 in preparation reactivation persistent infection virus drugs.Described benzamides compound refers to the compound that contains the benzamides atomic groups such as phenyl acetamide, Benzoylamide in molecular structure.
In a preferred technical scheme; described benzamides Antibiotic FR 901228 antigen is chidamide (Chidamide), grace for his (Mocetinostat), AGK-2 (C23H13Cl2N3O2 of Nuo Te (Entinostat), Moses department; No. CAS: 304896-28-4), AR-42 (C18H20N2O3; No. CAS: 935881-37-1) or CI-994 (C15H15N3O2, No. CAS: 112522-64-2).
In a more preferred technical scheme, described benzamides Antibiotic FR 901228 is chidamide.
In technique scheme, described in cause that the virus of mankind's persistent infection can be Human Immunodeficiency Viruses, hepatitis B virus or hepatitis C virus.
The present invention also provides a kind of pharmaceutical composition, and described compositions comprises acceptable carrier on benzamides Antibiotic FR 901228 and pharmacopedics.
In a preferred technical scheme, described benzamides Antibiotic FR 901228 is chidamide, grace for Nuo Te, Moses department he, AGK-2, AR-42 or CI-994.
In a more preferred technical scheme, described benzamides Antibiotic FR 901228 is chidamide.
Preferably, the effective dose of described chidamide is 0.2-50 μ mol/L.
More preferably, the effective dose of described chidamide is 0.5-5.0 μ mol/L.
The present invention adopts HIV pseudovirus latent infection cell model, HIV virus lays dormant infection cell model to studies have shown that with HIV infected patient clinical samples: benzamides HDACi compares with the Antibiotic FR 901228 Vorinostat of hydroximic acid, and cytotoxicity is lower, activation is stronger.Described hydroximic acid Antibiotic FR 901228 refers to and contains hydroxamic acid group, the carboxylic acid derivates that the carboxyl bivalence oxygen in carboxylic acid molecules is replaced by oximido.Chidamide can not only effectively activate the HIV promoter of pseudovirus latent infection cell model A7 and the HIV promoter of pseudovirus latent infection cell model TZM-bl, HIV virus that can also reactivation latent infection cell line U1/HIV-1, and the effective reactivation HIV patient tranquillization CD4 of chidamide
+the HIV hiding in T cell.The HIV peripheral blood in patients CD4 of HAART (HAART)
+in tranquillization T cell, can't detect HIV RNA (copy number <50copies/mL); After 1 μ mol/L chidamide is processed, can reactivation CD4
+the HIV virus of hiding in tranquillization T cell, viral copy number reaches 1161copies/mL; And 0.5 μ mol/L Vorinostat processing fails effectively to activate latent virus activation.Benzamides HDACi comprises: chidamide, grace are for Nuo Te, Moses department he, AGK-2, AR-42 and CI-994.Its possible mechanism is to modify by epigenetics, makes the acetylation of histone of being combined with HIV virus LTR promoter region, forms the space conformation of partial relaxation, thereby make transcription complex and associated retroviral start the factor, is easy to enter, and startup is transcribed.
The virus that HIV, HBV, HCV and CMV etc. cause mankind's persistent infection can be hidden in infection cell after primary infection or treatment, forms in vivo the storage vault of hiding.Epigenetic modification medicine HDACi is by changing the acetylation state of albumen, and reactivation expressing viral, adjusts immune status, improves the therapeutic effect of antiviral drugs, finally reaches persistent virus infection disease cured.
Therefore, in aforementioned pharmaceutical compositions, can also contain anti-virus formulation, described anti-virus formulation can be one or more of nucleoside (acid) class reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitor, integrase inhibitor, fusion inhibitor, receptor antagonist and interferon.
HIV patient carries out conventional H AART treatment, peripheral blood HIV RNA copy number is very low or can't detect under the prerequisite of (copy number <50copies/ml), based on technique effect of the present invention, can proceed the following HIV latent virus of removing in viral storage vault of processing: selectively carry out on the one hand HIV vaccine active immunity (for example the preventative vaccine being formed by DNA vaccination and recombinant viral vector vaccine of the common development of Jilin University's vaccine research center and Changchun BCHT Pharmaceutical Co., Ltd.-
" compound anti AIDS virus vaccine "), the specific CD8 of human activin
+cytotoxic T lymphocyte; Give on the other hand chidamide, grace and process for Nuo Te, Moses department he, the benzamides HDACi such as AGK-2, AR-42 and CI-994, reactivation tranquillization CD4
+the HIV hiding in the cells such as T cell, mononuclear cell and macrophage, makes latent virus activation and expresses; Final CD8
+the cell of cytotoxic T lymphocyte specific killing HIV virus lays dormant, the HIV virus discharging is killed by antiviral treatment, finally reaches the object of healing or functional healing.
HIV infections medicine mainly can be divided into six large classes: 1. nucleoside (acid) class reverse transcriptase inhibitors, as zidovudine, lamivudine, stavudine, tenofovir, Abacavir, Combivir (lamivudine+zidovudine), three association dimension (Abacavir+zidovudine+lamivudine), emtricitabines, Abacavir+lamivudine, tenofovir+emtricitabine etc.; 2. non-nucleoside reverse transcriptase inhibitors, as efavirenz, nevirapine, etravirine etc.; 3. protease inhibitor, as gram force sesame (Lopinavir+ritonavir), atazanavir, Prezista etc.; 4. integrase inhibitor, replaces and draws Wei etc. as drawn; 5. fusion inhibitor, as En Fuwei peptide etc.; With 6. receptor antagonists, as MVC etc.The first-line treatment scheme of the free HAART of the current standard of China all comprises three kinds of antiviral therapy medicines, i.e. two kinds of ucleosides reverse transcriptase inhibitors and a kind of non-nucleoside reverse transcriptase inhibitors, for example tenofovir+lamivudine+efavirenz.
The treatment of HBV and HCV can activate and remove virus lays dormant storage vault by Antibiotic FR 901228 and clinical conventional medicament use in conjunction, reaches the object of persistent virus infection disease cured.
Treating hepatitis B virus infection drug main will divide two large classes, is respectively nucleoside (acid) medicine, as lamivudine, adefovir ester, Sebivo, Entecavir, tenofovir disoproxil etc.; And interferon, as common interferon-ALPHA, Polyethylene Glycol Intederon Alpha-2a, Polyethylene Glycol Interferon Alpha-2b etc.Can treat by the use in conjunction of nucleoside analog lamivudine and interferon two class medicines.
Infection with hepatitis C virus medicine can be Polyethylene Glycol interferon-ALPHA associating ribavirin use in conjunction, and non-structural protein 3/4A protease inhibitor (as telaprevir and boceprevir), non-structural 5B AG14361 and Non structural protein 5 A protein inhibitor etc.
Accompanying drawing explanation
Fig. 1. the impact of chidamide on HIV latent infection Growth of Cells;
Fig. 2. the HIV LTR promoter of chidamide reactivation A7 cell;
Fig. 3. the HIV LTR promoter of chidamide reactivation TZM-bl cell;
Fig. 4. the HIV virus of chidamide reactivation latent infection cell line U1/HIV-1;
Fig. 5. chidamide reactivation HIV patient tranquillization CD4
+hIV gene expression in T cell.
The specific embodiment
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, the scope of the claims in the present invention are not formed further and limited.
The impact of embodiment 1. chidamides on the growth of HIV virus lays dormant infection cell
The MTT cell proliferation experiment that adopts the present embodiment detects the impact of HDACi medicine chidamide on virus lays dormant infection cell A7 growth, the cytotoxicity of confirmation chidamide and for the concentration range of reactivation latent infection virus.
1.1 experimental technique
1.1.1 cell line:
HIV pseudovirus latent infection cell line J-Lat Tat-GFP Clone A7 (hereinafter to be referred as A7) is presented by the America NI H AIDS NIH AIDS of department research and Eric doctor Verdin of standard reagent project team.A7 cell is the people Jurkat T cell by viral vector stable integration LTR-Tat-IRES-GFP.LTR (long terminal repeats) plays the function of promoter and enhancer in virus transcription process; Tat gene coded protein can be combined with LTR, to increase viral gene transcription rate; The sign that GFP (Green Fluorescent Protein) activates as HIV promoter, can adopt flow cytometer to detect.A7 cell culture condition is the RPMI1640 culture medium that is added with 10%FBS.
1.1.2 experimental program:
The A7 cell of exponential phase is seeded in 96 well culture plates, and every hole 100 μ L are containing containing 5 * 10 in the RPMI1640 culture medium of 10%FBS
3individual cell.Then in every hole, add the chidamide of variable concentrations, concentration is respectively: 0.1 μ mol/L, 0.5 μ mol/L, 1.0 μ mol/L, 2.0 μ mol/L, 3.0 μ mol/L, 4.0 μ mol/L, 5.0 μ mol/L, 10.0 μ mol/L, using 0.5 μ mol/L Vorinostat as positive control, not add the negative contrast of medicine processed group.At 37 ℃ of 5%CO
2cultivate respectively after 2 days and 4 days, collecting cell detects the propagation situation of cell with MTT, and each treatment conditions is established 3 multiple holes.MTT detection method is: every hole adds the MTT10 μ L of 5mg/mL, and 37 ℃ are continued to hatch 4h, and then every hole adds 100 μ L10%SDS, hatches after 2h, at 570nm place, detects and record.
1.2 experimental result
According to MTT cell proliferation experiment result, make cell growth inhibition curve, chidamide has dosage and time dependence to A7 cell growth inhibition, the results are shown in Figure 1.Bibliographical information concentration is that the hydroximic acid Antibiotic FR 901228 Vorinostat activated viral effect of 0.5 μ mol/L is obvious, in an embodiment as positive control, the inhibitory rate of cell growth of drug treating 2 days and 4 days is respectively 17.3% ± 5.8% and 32.5% ± 13.0%, the cytotoxicity (chidamide process 2 days and 4 days inhibitory rate of cell growth be respectively 11.3% ± 2.9% and 11.2% ± 4.6%) that is much higher than same concentration chidamide, the cytotoxicity of a little higher than 3.0 μ mol/L chidamides (chidamide process 2 days and 4 days inhibitory rate of cell growth be respectively 16.0% ± 2.0% and 20.3% ± 4.4%).While advising accordingly chidamide reactivation latent virus, process the concentration≤3.0 μ mol/L of cell.
It is reference that this embodiment be take the Vorinostat latent virus reactivation concentration of bibliographical information, proves with the chidamide cytotoxicity of concentration lower.
Embodiment 2. chidamides activate the HIV promoter of pseudovirus latent infection cell model A7
The present embodiment adopts HIV pseudovirus latent infection cell model A7 to prove the reactivation effect of the HDACi such as chidamide to HIV viral promotors, explores HDACi for the preparation of the probability of the reactivation medicine of storage vault in persistent infection virion.Embodiment 1 is shown in the description of relevant A7 cell.
2.1 experimental technique
The HIV pseudovirus latent infection cell A7 of exponential phase is seeded in 12 well culture plates, and every hole adds 1 * 10 at 1mL containing in the RPMI1640 culture medium of 10%FBS
6cell.Then in every hole, add the chidamide of variable concentrations, concentration is respectively: 0.5 μ mol/L, 1.0 μ mol/L, 2.0 μ mol/L, using 0.5 μ mol/L Vorinostat as positive control, not add the negative contrast of medicine processed group.At 37 ℃ of 5%CO
2cultivate respectively collecting cell after 48h and 96h, with flow cytometer, detect GFP positive percentage.
2.2 experimental result
Flow cytometry demonstration, chidamide can obviously activate the expression of GFP in A7 cell, and has dosage and time dependence, the results are shown in Figure 2.Concentration is that the chidamide of 0.5 μ mol/L, 1.0 μ mol/L, 2.0 μ mol/L is processed after 48h, and in A7 cell, GFP positive rate is respectively 2.0%, 3.7% and 5.3%; The GFP positive rate of blank and positive control medicine Vorinostat is respectively 1.5% and 2.9%; Chidamide concentration 1.0 μ mol/L when above activation be better than Vorinostat.Concentration is that the chidamide of 0.5 μ mol/L, 1.0 μ mol/L, 2.0 μ mol/L is processed after 96h, and in A7 cell, GFP positive rate is respectively 2.6%, 5.1% and 10.5%; The GFP positive rate of blank and positive control medicine Vorinostat is substantially constant, is respectively 1.8% and 2.6%; Chidamide has concentration dependent to the reactivation ability of HIV latent virus, concentration 1.0 μ mol/L when above activation be better than Vorinostat.
This embodiment adopts pseudovirus latent infection cell model A7 proof chidamide can obviously activate HIV LTR promoter, reactivation latent virus storehouse, and also reactivation effect is better than positive control medicine Vorinostat.
Embodiment 3.HDACi activates the HIV promoter of pseudovirus latent infection cell model TZM-bl
The present embodiment adopts another HIV pseudovirus latent infection cell model TZM-bl further to prove the reactivation effect of the HDACi such as chidamide to HIV viral promotors.
3.1 experimental technique
3.1.1 cell line:
HIV pseudovirus latent infection cell model TZM-bl cell is presented by the America NI H AIDS NIH AIDS of department research and standard reagent project team, it is the Hela cell of stable integration HIV LTR promoter and luciferase gene (Luc), when HIV promoter is activated, in cell, just have luciferase and produce, can analyze by luciferase detection kit.The culture fluid of TZM-bl cell is the DMEM culture medium that is added with 10%FBS.
3.1.2 experimental program:
The HIV pseudovirus latent infection cell TZM-bl of exponential phase is seeded in 96 well culture plates, and every hole adds 1 * 10 at 100 μ L containing in the DMEM culture medium of 10%FBS
4cell.After cell attachment, every hole adds the chidamide of variable concentrations, and concentration is respectively: 0.5 μ mol/L, 1.0 μ mol/L, 2.0 μ mol/L, 3.0 μ mol/L, using 0.5 μ mol/L Vorinostat as positive control, not add the negative contrast of medicine processed group.Continuation is at 37 ℃ of 5%CO
2cultivate 48h with the cell pyrolysis liquid in luciferase detection system test kit by lysis, then 96 orifice plates and luciferase substrate are taken in bioluminescence instrument and are measured.
3.2 experimental results:
The demonstration of luciferase testing result, chidamide can obviously activate the expression of luciferase in TZM-bl cell, and has dose dependent, the results are shown in Figure 3.Compare with untreated fish group, after concentration is the chidamide effect TZM-bl cell of 0.5 μ mol/L, 1.0 μ mol/L, 2.0 μ mol/L, 3.0 μ mol/L, uciferase activity has increased respectively 1.24,1.44,1.65,1.82 times; Positive control medicine Fu Linuo processed group has increased 1.15 times.Presentation of results chidamide virus reactivation effect is better than Vorinostat, consistent with the result of pseudovirus latent infection cell model A7.
The HIV virus of embodiment 4.HDACi reactivation latent infection cell line U1/HIV-1
The present embodiment adopts virus lays dormant infection cell model TZM-bl to prove that can the HDACi such as chidamide reactivation be incorporated into the HIV provirus in host cell gene group.
4.1 experimental technique
4.1.1 cell line:
HIV latent infection cell line U1/HIV-1 is presented by the America NI H AIDS NIH AIDS of department research and standard reagent project team, it is the people U937 monoblast of dormant infection HIV-1-1, when HIV provirus is reactivated, to transcribe, translate virus protein, wherein p24 protein excretion is to cells and supernatant, can detect by the method for Enzyme-linked Immunosorbent Assay (ELISA), as the sign of latent virus reactivation.The culture fluid of U1/HIV-1 cell is the RPMI1640 culture medium that is added with 10%FBS.
4.1.2 experimental program:
The HIV latent infection cell line U1/HIV-1 of exponential phase is seeded in 12 well culture plates, and every hole adds 1 * 10 at 1mL containing in the RPMI1640 culture medium of 10%FBS
6cell.After cell attachment, every hole adds the chidamide of variable concentrations, and concentration is respectively: 1.0 μ mol/L, 2.0 μ mol/L, 5.0 μ mol/L, 10.0 μ mol/L, using 0.5 μ mol/L Vorinostat as positive control, not add the negative contrast of medicine processed group.Continuation is at 37 ℃ of 5%CO
2cultivate 48h, get cells and supernatant, with enzyme-linked immunosorbent assay kit, detect the content of viral p24.
4.2 experimental results:
Enzyme Linked Immunoadsorbent Assay demonstration, chidamide can obviously activate HIV provirus genomic expression in U1/HIV-1 cell, and has dose dependent, the results are shown in Figure 4.Concentration is that the chidamide of 1.0 μ mol/L, 2.0 μ mol/L, 5.0 μ mol/L, 10.0 μ mol/L is processed after 48h, and in cells and supernatant, the content of p24 virus protein reaches respectively 150.6pg/mL, 214.6pg/mL, 233.0pg/mL and 242.7pg/mL; The content of the p24 virus protein of blank and positive control medicine Vorinostat is respectively 99.1pg/mL and 122.9pg/mL.Presentation of results chidamide virus reactivation effect is better than Vorinostat.
This embodiment adopt HIV latent infection cell model proof chidamide obviously reactivation be incorporated into the HIV provirus in host cell gene group, and reactivation effect is better than positive control medicine Vorinostat.
The reactivation effect of embodiment 5.HDACi to HIV infected patient latent virus storehouse
Tranquillization CD4
+t lymphocyte is the main viral bunker of HIV latent infection, in order to verify the reactivation effect of the HDACi such as chidamide to latent virus in HIV patient's tranquillization T lymphocyte, tranquillization T lymphocyte in the first separated HIV peripheral blood mononuclear cells of the present embodiment, then adopt PCR and enzyme-linked immunosorbent assay to detect the expression of HIV gene and albumen after latent virus reactivation, correlated results is verified by virus load analytical system.
5.1 experimental techniques:
5.1.1 separated HIV patient's tranquillization CD4
+t lymphocyte
HIV patient's the set condition that enters is: accept conventional H AART treatment more than 1 year; In blood, can't detect virus load (HIV-1RNA copy number <50copies/mL) more than 1 year; Peripheral blood CD4
+t cell quantity is higher than 300/ μ L.The in the situation that of clinical informed consent, get the HIV peripheral blood in patients that carries out conventional H AART treatment, with lymphocyte separation medium density gradient centrifugation separating peripheral blood mononuclear cells.Then adopt the MACS magnetic field separation CD4 of German Mei Tian Ni company
+cD25
-cD69
-hLA-DR
-tranquillization T lymphocyte, basic operation is: at CD4
+in the negative sorting reagent of T cell, add CD25, CD69 and tri-kinds of antibody labeling magnetic beads of HLA-DR, be prepared into tranquillization CD4
+the negative sorting magnetic bead of T lymphocyte; Density－gradient centrifuga－tion method separation is obtained to PERIPHERAL BLOOD MONONUCLEAR CELL cell and tranquillization CD4 in MACS buffer
+the negative sorting magnetic bead of T lymphocyte mixes, by the negative sorting CD25 in MACS magnetic field
-cD69
-hLA
-dR
-tranquillization CD4
+t cell; And with CD4-FITC, CD25-PE, CD69-APC and HLA-DR-PerCP-Cy5 antibody, carry out the purification efficiency of labeled analysis cell.
5.1.2PCR detect the expression of HIV virus pol gene:
By the above-mentioned separated CD4 obtaining
+tranquillization T cell is inoculated into 12 porocyte culture plates, in the RPMI-1640 culture fluid of every hole 3mL containing 10U/mL IL-2 and 10% hyclone, adds 5 * 10
6cell.In culture hole, add respectively chidamide 1.0 μ mol/l and 2.0 μ mol/L to process, using 5.0 μ g/mL phytohaemagglutinin (PHA) or CD3 monoclonal antibody processing hole as positive control, with 0.5 μ mol/L Vorinostat medicine in contrast, not add the negative contrast in drug treating hole.Act on collecting cell culture supernatant after 6 days, extract nucleic acid.Design and synthesize the Auele Specific Primer in HIV-1 viral genome pol district, carry out conventional pcr amplification, can detection of drugs effect reactivation CD4
+the expression of latent virus in tranquillization T cell.
5.1.3 enzyme-linked immunosorbent assay detects the expression of HIV virus protein p24:
Above-mentioned cells and supernatant is respectively got to 1mL, comprise 1.0 μ mol/L and 2.0 μ mol/L chidamide processed group, 5.0 μ g/mL PHA processed group, CD3 monoclonal antibody processed group, 0.5 μ mol/L Vorinostat processed group and do not add medicine processed group.With enzyme-linked immunosorbent assay kit, detect the content of virus protein p24, PCR result is verified.
5.1.4 the variation of virus load after the T cell of virus load analytical system detection chidamide processing HIV latent infection:
Collect 1.0 μ mol/L chidamides and 0.5 μ mol/L Vorinostat processed group cell, isolating nucleic acid, adopts the NucliSENS EasyQ HIV-1 virus load analytical system (the linear kinetics detection range of viral RNA is 50-3000000copies/mL) of French Biomerieux SA to CD4
+after the effect of tranquillization T cell drug, HIV RNA copy number detects.Not add the negative contrast in drug treating hole.NucliSens EasyQ HIV-1 nucleic acid amplification has been used the special primer for the HIV-1RNA of street strain sequence and synthetic internal standard substance RNA sequence (EasyQ HIV-1Calibrator), and detection is to utilize the special molecular beacon of target to carry out.NucliSens EasyQ HIV-1 has applied two kinds of different molecular beacons, and one is specific for WT HIV-1 amplified matter, and another is specific for calibration amplified matter.The dynamic analysis of fluorescence signal has disclosed the transcription rate of WT and calibration RNA, just can release accordingly the quantity of HIV-1RNA in original sample.Like this, utilize in HIV-1 analysis software also n ary operation method quantitative virus load just of application data.
5.2 experimental results:
5.2.1MACS separation obtains HIV patient's tranquillization CD4
+t lymphocyte
The MACS magnetic field that adopts German Mei Tian Ni company obtains CD4 through negative sorting separation from carry out the HIV peripheral blood mononuclear cells of conventional H AART treatment
+cD25
-cD69
-hLA-DR
-tranquillization T lymphocyte, carries out the purification efficiency of labeled analysis cell with CD4-FITC, CD25-PE, CD69-APC and HLA-DR-PerCP-Cy5 antibody, result shows purification efficiency >95%.Because CD25 and CD69 content before and after sorting is all extremely low, Fig. 5 has listed the result of CD4-FITC and HLA-DR-PerCP-Cy5 labelling in wherein sorting experiment, and A is the HIV peripheral blood mononuclear cells before sorting, CD4
+hLA-DR
-cell accounts for 19.3%; B is the CD4 sub-electing
+tranquillization T lymphocyte, CD4
+hLA-DR
-cell accounts for 95.4%.
5.2.2 the effective reactivation HIV patient of chidamide tranquillization CD4
+the HIV hiding in T cell
The PCR testing result of HIV virus pol gene as shown in Figure 5 C, 1-6 in figure is respectively: 1: untreated fish group, 2:CD3 monoclonal antibody processed group, 3:5.0 μ g/mL PHA processed group, 4:0.5 μ mol/L Vorinostat processed group, 5:1.0 μ mol/L chidamide processed group and 6:2.0 μ mol/L chidamide processed group.1.0 μ mol/L and 2.0 μ mol/L chidamide processed group and PHA or CD3 monoclonal antibody processed group all amplify HIV virus pol gene specific band, and tranquillization CD4 is described
+the HIV hiding in T cell is reactivated; And do not add medicine processed group and negative for Vorinostat processed group result, illustrate that the latent infection virus in this group fails effectively to activate.
Adopt enzyme-linked immunosorbent assay to detect the expression of HIV virus protein p24,1.0 μ mol/L and 2.0 μ mol/L chidamide processed group and PHA or CD3 monoclonal antibody processed group all detect virus protein p24, are respectively: 1.3pg/mL, 1.3pg/mL, 1.1pg/mL and 4.8pg/mL; And do not add medicine processed group and p24 albumen do not detected for Vorinostat processed group.Result is consistent with PCR result.
HIV virus load detects and further confirms effectively reactivation HIV patient tranquillization CD4 of chidamide
+the HIV hiding in T cell.The HIV peripheral blood in patients CD4 of conventional H AART treatment
+in tranquillization T cell, can't detect HIV RNA (copy number <50copies/mL); After 1 μ mol/L chidamide is processed, can reactivation CD4
+the HIV virus of hiding in tranquillization T cell, viral copy number reaches 1161copies/mL; And 0.5 μ mol/L Vorinostat processing fails effectively to activate latent virus activation.
The present embodiment success is separated tranquillization T lymphocyte in HIV peripheral blood mononuclear cells, proves that the HDACi such as chidamide can effectively activate the HIV latent virus in tranquillization T lymphocyte; Even the not active case of control drug Vorinostat, the benzamides HDACi such as chidamide still have curative effect preferably.
Claims (10)
1. the application of benzamides Antibiotic FR 901228 in preparation reactivation persistent infection virus drugs.
2. application according to claim 1, is characterized in that, described benzamides Antibiotic FR 901228 is chidamide, grace for Nuo Te, Moses department he, AGK-2, AR-42 or CI-994.
3. application according to claim 2, is characterized in that, described benzamides Antibiotic FR 901228 is chidamide.
4. according to the arbitrary described application of claim 1-3, it is characterized in that, described in cause that the virus of mankind's persistent infection can be Human Immunodeficiency Viruses, hepatitis B virus or hepatitis C virus.
5. a pharmaceutical composition, described compositions comprises acceptable carrier on benzamides Antibiotic FR 901228 and pharmacopedics.
6. compositions according to claim 5, is characterized in that, described benzamides Antibiotic FR 901228 is chidamide, grace for Nuo Te, Moses department he, AGK-2, AR-42 or CI-994.
7. compositions according to claim 6, is characterized in that, described benzamides Antibiotic FR 901228 is chidamide.
8. compositions according to claim 7, is characterized in that, the effective dose of described chidamide is 0.2-50 μ mol/L.
9. compositions according to claim 8, is characterized in that, the effective dose of described chidamide is 0.5-5.0 μ mol/L.
10. according to the compositions described in arbitrary claim in claim 5-9, it is characterized in that, described compositions also contains anti-virus formulation, and described anti-virus formulation can be one or more of nucleoside (acid) class reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitor, integrase inhibitor, fusion inhibitor, receptor antagonist and interferon.
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| CN113289018A (en) * | 2020-02-21 | 2021-08-24 | 中国科学院上海药物研究所 | Application of old medicine such as auranofin and composition thereof in resisting single positive strand RNA virus |
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| IT202100005054A1 (en) * | 2021-03-04 | 2022-09-04 | Azienda Ospedaliero Univ Di Parma | PHARMACEUTICAL COMPOUND FOR USE IN THERAPEUTIC TREATMENT OF CHRONIC HBV INFECTION AND METHOD FOR IDENTIFICATION OF DEPLETED LYMPHOCYTES |
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