CN112107571A

CN112107571A - Application of ingenol compounds and derivatives thereof in anti-HIV treatment

Info

Publication number: CN112107571A
Application number: CN201910539237.XA
Authority: CN
Inventors: 朱豫琪; 张龙飞; 杨鹤; 李玄; 朱焕章
Original assignee: Shanghai Xin Hao Biological Technology Co
Current assignee: Shanghai Xin Hao Biological Technology Co
Priority date: 2019-06-20
Filing date: 2019-06-20
Publication date: 2020-12-22

Abstract

The invention provides application of ingenol compounds and derivatives thereof in anti-HIV treatment. Specifically, the present invention provides the use of ingenol compounds, or pharmaceutically acceptable salts thereof, for (a) interfering with HIV latency; (b) inhibiting infection by HIV; and/or (c) reduces the expression of HIV-infected surface receptors of CD4 cells. The compound of the invention can be used together with AIDS therapeutic agents (such as anti-retroviral drugs, immunomodulators, therapeutic antibodies or the combination thereof) to accelerate the elimination of latent virus reservoirs, which is helpful for improving the cure rate of AIDS.

Description

Application of ingenol compounds and derivatives thereof in anti-HIV treatment

Technical Field

The invention relates to the field of medicines, in particular to application of ingenol compounds and derivatives thereof in anti-HIV treatment.

Background

Acquired Immune Deficiency Syndrome (AIDS) is an infectious disease caused by HIV infection that seriously compromises people's life and health. According to WHO statistics, the number of AIDS patients is more than 4000 ten thousand, 500 ten thousand patients are newly added every year, and 300 ten thousand deaths are caused every year. At present, the clinical treatment method of AIDS is mainly high-efficiency antiretroviral therapy (HAART), and by combining several types of anti-AIDS drugs with different action mechanisms, the combined antiretroviral therapy blocks the generation of HIV from various stages of virus entry, virus reverse transcription, virus replication, virus release and the like, thereby obviously reducing plasma viruses of patients, improving the health of HIV patients, prolonging the life of the patients, greatly reducing the transmission risk of HIV and leading the AIDS to become a chronic controllable disease. People hope to completely eliminate HIV in vivo by HAART, thereby achieving the aim of completely curing AIDS. However, subsequent practice has shown that, although HAART can maximally inhibit viral replication in patients and reduce plasma viral load (virus load) to levels that cannot be detected by conventional detection methods, the virus persists in infected subjects and, once drug therapy is stopped, the viral load returns to pre-therapy levels. On the other hand, serious side effects and drug resistance caused by long-term administration are also problems that cannot be ignored. Research shows that the drug resistance of HIV is obviously improved by 30 years, so that complete elimination of AIDS is still necessary. One important reason for the difficulty of complete elimination of HIV in vivo is that HIV-1 can be latent in resting memory CD4+ T cells, which are generated by transformation of a small fraction of HIV-infected activated CD4+ T cells, whose integrated provirus lacks transcriptional activity and, therefore, is not attacked by the immune system and antiretroviral drugs. Although infected individuals carry a small number of latently infected cells, the rate of depletion is so slow that complete elimination of HIV by HAART therapy alone is not possible during the life of the individual, therefore, latently HIV-infected resting CD4+ T cells constitute a major part of the viral reservoir (reservoir) in the body and are a great obstacle to complete elimination of HIV by current clinical therapy. The molecular mechanism of HIV-1 latent infection cell formation is thought to be related to chromatin state at the integration site, the presence of inhibitory nucleosome nuc-1, epigenetic modifications represented by acetylation, host transcription factors such as NF-. kappa.B and viral transcriptional activator Tat. Based on the mechanism, a therapeutic strategy for clearing the latent virus reservoir is proposed, namely, the latent virus is activated again by inducing the proviral expression of HIV latent infected cells through drugs, and the activated latent infected cells are killed under the action of the human immune system in combination with the high-efficiency antiretroviral therapy, so that the clearing of the virus reservoir is accelerated. Although several treatment schemes are available clinically, the result is still not satisfactory, whether the activator is ineffective or effective but has great toxic and side effects, and only one new anti-AIDS drug with independent intellectual property rights is on the market at present in China. Therefore, the research and development of a novel intervention drug which has independent intellectual property rights, safety, effectiveness and low price and can clear the HIV-1 virus repository is of great significance.

The traditional Chinese medicine is a huge treasure in Chinese medical treasury, has a long history for thousands of years, and is widely used for treating various diseases and maintaining the health of human bodies. The Euphorbiaceae plant is widely distributed in the world, has remarkable pharmacological action, and has effects of clearing away heat and toxic materials, and resisting tumor. Euphorbiaceae plants recorded in pharmacopoeia include euphorbia kansui, Euphorbia lathyris, Euphorbia fischeriana, Euphorbia pekinensis, Euphorbia hirta and Euphorbia hirta, wherein euphorbia kansui is widely cultivated in Shanxi and other areas of China. Kansui mainly contains diterpene alcohols, triterpenes and steroids, and researches show that kansui has various pharmacological effects of resisting tumor, oxidation and fertility, resisting virus, inducing diuresis by purgation, treating pancreatitis and the like, has the characteristics of small dosage and high activity in clinic, has higher research value and is more and more concerned by modern medicine and pharmacology.

Therefore, there is an urgent need in the art to develop a drug which can significantly interfere with the latency of HIV, effectively activate latent HIV, and can be used as an entry inhibitor, can inhibit the infection of cells by HIV, and has strong anti-HIV activity.

Disclosure of Invention

The invention aims to provide a medicament which can obviously intervene in the latency of HIV, effectively activate the latent HIV, can be used as an entry inhibitor, can inhibit the infection of HIV on cells and has strong anti-HIV activity.

The present invention provides in a first aspect the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a composition or formulation for (a) intervening in HIV latency; (b) inhibiting HIV infection; and/or (c) reduces the expression of CD4 cell surface receptors:

in the formula (I), the compound is shown in the specification,

R₁selected from H, substituted or unsubstituted COCH₃Substituted or unsubstituted CO- (CH ═ CH)₂-(CH₂)₄-CH₃Substituted or unsubstituted COCH (CH)₃)CH(CH₃)₂Or substituted or unsubstituted OCC₆H₅；

R₂Selected from H, substituted or unsubstituted COCH₃Substituted or unsubstituted COCH (CH)₃)CH(CH₃)₂Or substituted or unsubstituted OCC₆H₅；

R₃Selected from H, substituted or unsubstituted COCH₃Substituted or unsubstituted CO (C H ═ CH)₂-(CH₂)₄-CH₃Substituted or unsubstituted COCH (CH)₃)CH(CH₃)₂Substituted or unsubstituted OCC₆H₅Substituted or unsubstituted C₆H₁₂O₅Or substituted or unsubstituted SO₃H；

R₄Selected from the group consisting of: CH (CH)₂OR₅、-C(O)R₆Substituted or unsubstituted C₁-C₆Alkyl, or combinations thereof; wherein R is₅Selected from H, C₁-C₆Alkyl, CO- (CH ═ CH)₂-(CH₂)₄-CH₃、COCH₃、OCH₂CH₃、CO(CH₂)₁₀CH₃Or CO (CH)₂)₁₄CH₃；R₆Selected from the group consisting of: H. substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₁-C₆Alkoxy, -OH, substituted or unsubstituted C₁-C₆An ester group, or a combination thereof;

or, R₁And R₂、R₂And R₃、R₃And R₄Each or both of which independently form a 5-7 membered substituted or unsubstituted heterocyclic ring with the adjacent carbon atom, said heterocyclic ring containing 1-3 heteroatoms selected from N, O, or S;

wherein "substituted" means that H in the group is substituted with one or more substituents selected from the group consisting of: halogen, C₁-C₆Alkyl radical, C₁-C₆Haloalkyl, C₂-C₆Alkenyl radical, C₂-C₆An alkynyl group;

with the proviso that R₁Is CO- (CH ═ CH)₂-(CH₂)₄-CH₃When R is₂、R₃Not H at the same time.

In another preferred embodiment, the "substituted" means that H in the group is substituted with one or more substituents selected from the group consisting of: halogen, C₁-C₄Alkyl radical, C₁-C₄Haloalkyl, C₂-C₄Alkenyl radical, C₂-C₄Alkynyl group.

In another preferred embodiment, R is₄Selected from the group consisting of: CH (CH)₂OR₅、-C(O)R₆Substituted or unsubstituted C₁-C₄Alkyl, or combinations thereof; wherein R is₅Selected from H, C₁-C₄Alkyl, CO- (CH ═ CH)₂-(CH₂)₄-CH₃、COCH₃、OCH₂CH₃、CO(CH₂)₁₀CH₃Or CO (CH)₂)₁₄CH₃；R₆Selected from the group consisting of: H. substituted or unsubstituted C₁-C₄Alkyl, substituted or unsubstituted C₂-C₄Alkenyl, substituted or unsubstituted C₂-C₄Alkynyl, substituted or unsubstituted C₁-C₄Alkoxy, -OH, substituted or unsubstituted C₁-C₄An ester group, or a combination thereof.

In another preferred embodiment, R₁And R₂、R₂And R₃、R₃And R₄Each independently form a 5-7 membered substituted or unsubstituted heterocyclic ring containing 1-2 heteroatoms selected from N, O, or S.

In another preferred embodiment, R₁And R₂、R₂And R₃、R₃And R₄Each independently form a 5-7 membered substituted or unsubstituted heterocyclic ring containing 1-2O atoms.

In another preferred embodiment, R₁Is CO- (CH ═ CH)₂-(CH₂)₄-CH₃When R is₄Selected from the group consisting of: CH (CH)₂OR₅、-C(O)R₆Substituted or unsubstituted C₁-C₆Alkyl, or combinations thereof; wherein R is₅Is selected from C₁-C₆Alkyl, CO- (CH ═ CH)₂-(CH₂)₄-CH₃、COCH₃、OCH₂CH₃、CO(CH₂)₁₀CH₃Or CO (CH)₂)₁₄CH₃；R₆Selected from the group consisting of: H. substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₂-C₆Alkenyl, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₁-C₆Alkoxy, -OH, substituted or unsubstituted C₁-C₆An ester group, or a combination thereof; wherein "substituted" means that H in the group is substituted with one or more substituents selected from the group consisting of: halogen, C₁-C₆Alkyl radical, C₁-C₆Haloalkyl, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl group.

In another preferred embodiment, R₁Is CO- (CH ═ CH)₂-(CH₂)₄-CH₃When R is in the above-mentioned range₄Selected from the group consisting of: CH (CH)₂OR₅、-C(O)R₆Substituted or unsubstituted C₁-C₄Alkyl, or combinations thereof; wherein R is₅Is selected from C₁-C₄Alkyl, CO- (CH ═ CH)₂-(CH₂)₄-CH₃、COCH₃、OCH₂CH₃、CO(CH₂)₁₀CH₃Or CO (CH)₂)₁₄CH₃；R₆Selected from the group consisting of: H. substituted or unsubstituted C₁-C₄Alkyl, substituted or unsubstituted C₂-C₄Alkenyl, substituted or unsubstituted C₂-C₄Alkynyl, substituted or unsubstituted C₁-C₄Alkoxy, -OH, substituted or unsubstituted C₁-C₄An ester group, or a combination thereof; wherein "substituted" means that H in the group is substituted with one or more substituents selected from the group consisting of: halogen, C₁-C₄Alkyl radical, C₁-C₄Haloalkyl, C₂-C₄Alkenyl radical, C₂-C₄Alkynyl group.

In another preferred embodiment, the halogen is selected from the group consisting of: F. cl, Br, or I.

In another preferred embodiment, the compound is an optical isomer or racemate.

In another preferred embodiment, the compound is selected from the group consisting of:

in another preferred embodiment, the composition or formulation is also useful for (d) inhibiting HIV binding entry; and/or (e) treating AIDS; and/or (f) in combination with an AIDS therapeutic agent (e.g., an antiretroviral agent) to thereby treat AIDS.

In another preferred embodiment, the intervention of HIV latency comprises: inducing HIV proviral expression, and/or activation of latent HIV in a latently HIV infected cell; and/or

The treatment of AIDS comprises anti-HIV latent treatment.

In another preferred embodiment, the antiretroviral drug is selected from the group consisting of: a reverse transcriptase inhibitor, a protease inhibitor, a co-receptor antagonist, a retroviral integrase inhibitor, a viral adsorption inhibitor, a specific viral transcription inhibitor, a cyclin dependent kinase inhibitor, an anti-HIV antibody, or a combination thereof.

In another preferred embodiment, said inhibiting HIV comprises: inhibiting HIV-mediated cell fusion, inhibiting HIV invasion of cells, inhibiting HIV replication, and inhibiting HIV infection activity.

In another preferred embodiment, the composition or preparation further comprises other medicines for intervening HIV latency.

In another preferred embodiment, said other drug for intervening in HIV latency is selected from the group consisting of: histone acetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors, bromase inhibitors, protein kinase c (pkc) agonists, cytokines and chemokines, positive transcriptional elongation factor b (P-TEFb) activators, or combinations thereof.

In another preferred embodiment, the composition or formulation further comprises an additional aids therapeutic agent.

In another preferred embodiment, the additional aids therapeutic is selected from the group consisting of: an antiretroviral drug, an immunomodulator, a therapeutic antibody, or a combination thereof.

In another preferred embodiment, the immunomodulator is selected from the group consisting of: a TLR (Toll-like receptor) agonist, an anti-PD-1 or anti-PD-L1 monoclonal antibody, a chemokine receptor agonist, an mTOR (mammalian target of rapamycin) inhibitor, a JAK kinase 1and 2(Janus kinases 1and 2) inhibitor, or a combination thereof.

In another preferred embodiment, the chemokine receptor agonist is selected from the group consisting of: IL-7; IL-15 upgrade (ALT-803), IFN-alpha, or a combination thereof.

In another preferred example, the JAK kinase 1and 2 inhibitor comprises Ruxolitinib.

In another preferred embodiment, the therapeutic antibody is selected from the group consisting of: a therapeutic antibody Targeting CD4, a therapeutic antibody Targeting the V3 loop, a therapeutic antibody Targeting the V1/V2 region, a therapeutic antibody Targeting the membrane proximal region (MPER), bNabs, DARTs (parent-Affinity Re-Targeting molecules), CARs (Chimeric antigen receptors), or a combination thereof.

In another preferred embodiment, the CD 4-targeting therapeutic antibody is selected from the group consisting of: VRC01, 3BNC117, VRC07-523, N6, or combinations thereof.

In another preferred embodiment, the therapeutic antibody targeting the V3 loop is selected from the group consisting of: 10-1074, PGT121, or a combination thereof.

In another preferred embodiment, the therapeutic antibody targeting the V1/V2 region is selected from the group consisting of: PGDM1400, CAP256, or a combination thereof.

In another preferred embodiment, the therapeutic antibody targeting the membrane proximal region (MPER) comprises 10E 8.

In another preferred embodiment, the compound of formula I, or a pharmaceutically acceptable salt thereof, is chemically synthesized.

In another preferred embodiment, the compound of formula I, or a pharmaceutically acceptable salt thereof, is extracted from plants.

In another preferred embodiment, the extraction comprises extracting by a method selected from the group consisting of: solvent extraction, and chromatography.

In another preferred embodiment, the plant comprises a plant of the family euphorbiaceae.

In another preferred embodiment, the euphorbiaceae plant comprises Euphorbia kansui (Euphorbia kansui t.n.liou ex s.b.ho).

In another preferred embodiment, the composition comprises a pharmaceutical composition, a food composition or a nutraceutical composition.

In another preferred embodiment, the medicament further comprises an additional component selected from the group consisting of: an antiretroviral active ingredient or an active ingredient that enhances immunity.

In another preferred embodiment, the pharmaceutical composition comprises (I) a compound of formula I or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier or adjuvant.

In another preferred embodiment, the component (i) is 0.001-99.9 wt%, preferably 0.1-99 wt%, more preferably 1-90 wt% of the total weight of the pharmaceutical composition.

In another preferred embodiment, the composition or medicament comprises: oral and non-oral formulations.

In another preferred embodiment, the formulation comprises: powder, granule, capsule, injection, tincture, oral liquid, tablet or buccal tablet.

In another preferred embodiment, the pharmaceutically acceptable carrier or adjuvant comprises a solubilizing carrier.

In another preferred embodiment, the solubilizing carrier is selected from the group consisting of: ethanol, propylene glycol, glycerol, sorbitol, lecithin-n-butanol-acetylated monoglyceride fatty acid ester-water, lecithin-polyethylene glycol, distearoylphosphatidylethanolamine (PEG-DSPE) -soybean oil-water, polyethylene glycol-phospholipid-cholesterol-vitamin E, emulsifier in combination with co-emulsifiers (emulsifier: polyethylene glycol hydroxystearate Solutol HS15, Poloxamer 188, polyoxyethylene castor oil and lecithin; co-emulsifiers: medium chain triglycerides MCT, soybean oil and oleic acid), PEG-polyester based block copolymers (including polyglycine, polylactic acid PLA, polycaprolactone PCL and polylactic-polyglycolic acid PLGA), or combinations thereof.

In another preferred embodiment, the pharmaceutically acceptable carrier or excipient comprises a solubilizing excipient.

In another preferred embodiment, the solubilizing adjuvant is selected from the group consisting of: beta-cyclodextrin, polyethylene glycol, povidone, sorbitol, benzoic acid, citric acid, tween 80, starch, lactose, sucrose, mannose, hydroxymethyl cellulose, acacia, corn starch, gelatin, dextrin, or a combination thereof.

In another preferred embodiment, the composition is an oral formulation.

In another preferred embodiment, the composition (e.g., pharmaceutical composition) is administered to the mammal by: oral, intravenous, or topical injection.

In another preferred embodiment, the mammal includes a mammal having Acquired Immune Deficiency Syndrome (AIDS).

In another preferred embodiment, the mammal comprises a human or non-human mammal.

In another preferred embodiment, the non-human mammal comprises a rodent, such as a mouse, a rat, or a primate, such as a monkey.

In a second aspect, the present invention provides a pharmaceutical composition comprising:

(a1) a first active ingredient for use in intervening HIV latency and/or inhibiting HIV infection, said first active ingredient being a compound of formula I or a pharmaceutically acceptable salt thereof; and

(a2) a second active ingredient for inhibiting HIV replication, said second active ingredient being an aids therapeutic; and

(b) a pharmaceutically acceptable carrier;

wherein the definition of the compound of formula I is as described in the first aspect of the invention.

In another preferred embodiment, the pharmaceutical composition further comprises other drugs for intervening in HIV latency (or HIV latency activation).

In another preferred embodiment, said other agent interfering with HIV latency (or HIV latency activation) is selected from the group consisting of: histone acetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors, bromase inhibitors, protein kinase c (pkc) agonists, cytokines and chemokines, positive transcriptional elongation factor b (P-TEFb) activators, or combinations thereof.

In another preferred embodiment, the aids therapeutic is selected from the group consisting of: an antiretroviral drug, an immunomodulator, a therapeutic antibody, or a combination thereof.

In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is between 0.1% and 99%, preferably between 20% and 80%.

In another preferred embodiment, the pharmaceutical dosage form is an oral administration or a non-oral administration dosage form.

In another preferred embodiment, the oral administration form is tablet, powder, granule or capsule, or emulsion or syrup.

In another preferred embodiment, the non-oral administration dosage form is injection or injection.

In another preferred embodiment, the concentration of the compound of formula I or the pharmaceutically acceptable salt thereof is 0.001 ng/ml to 1000000ng/ml, preferably 0.01 ng/ml to 1000ng/ml, and more preferably 0.1 ng/ml to 100 ng/ml.

In a third aspect the present invention provides a method for non-therapeutically inducing expression of an HIV provirus in a latently HIV infected cell in vitro, comprising the steps of: culturing a cell latently infected with the HIV virus in the presence of a compound of formula I as defined in the first aspect of the invention, such that the latently located HIV provirus is expressed, thereby activating the latently located HIV virus.

In another preferred example, the method further comprises: killing or inhibiting activated HIV.

In a fourth aspect, the present invention provides an in vitro non-therapeutic method of activating latent HIV and/or inhibiting HIV infection, comprising the steps of: administering to a subject in need thereof a compound of formula I as defined in the first aspect of the invention, thereby activating latent HIV and/or inhibiting HIV infection.

In a fifth aspect, the present invention provides a method for preparing a pharmaceutical composition for preventing and/or treating aids, comprising the steps of: a compound of formula I as defined in the first aspect of the invention, or a pharmaceutically acceptable salt thereof, is admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

In another preferred embodiment, the method further comprises the step of mixing with an aids therapeutic agent.

In another preferred embodiment, the method further comprises the step of mixing with other drugs interfering with HIV latency (or HIV latency activation).

In a sixth aspect, the invention provides a method of activating latent HIV; and/or inhibiting HIV infection, characterized in that a safe and effective amount of a compound of formula I as defined in the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention is administered to a subject in need thereof.

In another preferred embodiment, the subject in need thereof is a mammal, such as a human.

In another preferred embodiment, the dosage of the administration is 0.1-2000 mg per day for an adult, preferably 1-300 mg per day.

According to a seventh aspect of the present invention there is provided a kit comprising:

a formulation comprising a compound of formula I as defined in the first aspect of the invention or a pharmaceutically acceptable salt thereof;

a preparation containing AIDS therapeutic agent; and

and (6) instructions.

In another preferred embodiment, the kit further comprises a formulation containing other drugs that interfere with HIV latency (or HIV latency activation).

In another preferred embodiment, the dosage form of the formulation containing a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof or the formulation containing other therapeutic agents or the formulation containing drugs that interfere with HIV latency (or HIV latency activation) comprises capsules, tablets, suppositories, or intravenous injections, respectively.

In another preferred embodiment, the concentration of the compound of formula I as defined in claim 1 or an acceptable salt thereof in the formulation containing the compound of formula I as defined in claim 1 or an acceptable salt thereof is from 0.001 μ g to 1000000 μ g/ml, preferably from 0.01 μ g to 1000 μ g/ml.

In another preferred embodiment, the antiretroviral drug comprises a reverse transcriptase inhibitor, a protease inhibitor, a fusion inhibitor, an integrase inhibitor, or a combination thereof.

In another preferred embodiment, the description describes the following method of use:

(I) administering to a subject in need thereof a formulation comprising a compound of formula I as defined in claim 1 or an acceptable salt thereof;

(II) administering to said subject a formulation comprising a therapeutic agent for aids 5-120 hours, preferably 24-96 hours, more preferably 72-96 hours, after step (I); and optionally (III) repeating steps (I) - (II).

In another preferred embodiment, said step (I) further comprises administering to a subject in need thereof an additional formulation comprising a drug that interferes with HIV latency (or HIV latency activation).

In an eighth aspect, the present invention provides an in vitro non-therapeutic method of inhibiting and/or killing latent HIV comprising the steps of:

(i) culturing cells latently infected with HIV in the presence of a compound of formula I as defined in the first aspect of the invention or a pharmaceutically acceptable salt thereof, thereby obtaining activated HIV-containing cells and inhibiting and/or killing latently HIV;

(ii) optionally, an aids therapeutic is added to the activated HIV-containing cells of (I) to inhibit and/or kill latent HIV.

In another preferred embodiment, the incubation time in step (i) is 5-120 hours, preferably 24-96 hours, more preferably 48-72 hours.

In a ninth aspect, the present invention provides a method of inhibiting HIV binding entry comprising the steps of: administering to a subject in need thereof a compound of formula I as defined in the first aspect of the invention, thereby inhibiting HIV binding entry.

In another preferred embodiment, the subject in need thereof comprises a human or non-human mammal.

In another preferred embodiment, the subject is a mammal, such as a human.

In another preferred embodiment, the non-human mammal includes a rodent (e.g., mouse, rat), primate (e.g., monkey).

In a tenth aspect, the present invention provides a method of treating latent HIV infection, comprising the steps of:

(I) administering to a subject in need thereof a formulation comprising a compound of formula I as defined in the first aspect of the invention or a pharmaceutically acceptable salt thereof;

(II) optionally, 5-120 hours, preferably 24-96 hours, more preferably 48-96 hours, after step (I), administering to said subject a formulation comprising a therapeutic agent for aids; and optionally (c) a second set of instructions,

(III) repeating steps (I) - (II);

thereby treating latent HIV infection.

In an eleventh aspect, the present invention provides a method for the prevention and/or treatment of aids by administering to a subject in need thereof a safe and effective amount of a compound of formula I as defined in the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the induced activation of HIV latency by EK-5A under fluorescent microscopy;

wherein, FIG. 1 specifically shows the green fluorescence expression in the cells observed by a fluorescence microscope after C11 cells are untreated or treated by 5ng/ml EK-5A for 48 h; the group without drug (FIG. 1-1 is a white light photograph, FIG. 1-2 is a fluorescence photograph of the same visual field), the group treated with EK-5A (FIG. 1-3 is a white light photograph, FIG. 1-4 is a fluorescence photograph of the same visual field); the fluorescent photograph is decolorized so that the fluorescent cells are white for easy recognition on the printed text.

FIG. 2 shows the efficiency of flow cytometry to detect the HIV latency-induced activation by EK-5A;

in which, FIG. 2 specifically shows the proportion of fluorescent cells obtained by flow cytometry analysis after C11 cells are untreated or treated with 5ng/ml EK-5A for 48 h.

FIG. 3 shows a comparison of the activation efficiency of EK-5A and the control PEP005 on latent HIV in C11 cells;

among them, flow cytometry examined the activation effect of EK-5A and PEP005 at different concentrations on latent HIV in C11 cell line, and plotted the concentration-dependent curve. All data results are the average of data obtained from 3 independent experiments.

FIG. 4 shows a comparison of the activation efficiency of EK-5A and the control PEP005 on latent HIV in J-Lat-A10.6 cells;

wherein, the flow cytometry is used for detecting the activation effect of EK-5A and PEP005 with different concentrations on latent HIV in a J-Lat-A10.6 cell line and drawing a concentration-dependent curve. All data results are the average of data obtained from 3 independent experiments.

FIG. 5 shows the effect of EK-5A on CD4\ CCR5 and CXCR4 receptor expression on the cell surface of CD 3;

wherein, the flow cytometry is used for detecting the expression of CD4, CCR5 and CXCR4 receptors on the cell surface of human CD3 by EK-5A with different concentrations. All data results are the average of data obtained from 3 independent experiments.

FIG. 6 shows the HIV inhibitory effect of EK-5A;

wherein, the inhibition ability of EK-5A against R5 type virus strain Bal is detected by using M7 cells through P24-based ELISA, and CD34 is a positive drug. All data results are the average of data obtained from 3 independent experiments.

Detailed Description

The present inventors have conducted extensive and intensive studies to screen a large number of compounds, and have surprisingly found for the first time that the ingenol compounds and derivatives thereof (compounds of formula I) of the present invention have (a) effects on HIV latency; (b) inhibiting HIV infection; and/or (c) the effect of reducing the expression of CD3 cell surface receptor, and in addition, the ingenol compound and the derivative thereof also have the effects of (d) preventing and/or treating AIDS; (e) inhibiting binding entry of HIV; and/or (f) the use in combination with an antiretroviral drug for the treatment of AIDS. On this basis, the present inventors have completed the present invention.

Specifically, the inventor screens ingenol compounds and derivatives thereof (compounds in formula I) by an original activity screening method, and the ingenol compounds and the derivatives can be used as anti-AIDS therapeutic drugs. The ingenol compound and the derivative thereof (the compound shown in the formula I) have the functions of interfering HIV latency and inhibiting HIV infection, or can eliminate activated latency infected cells, so that the elimination of a latency virus storage is accelerated, and a new way is provided for the complete cure of AIDS.

Radical definition

The term "substituted or unsubstituted" as used herein means that the group may be unsubstituted or that H in the group is substituted with one or more (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3, most preferably 1 to 2) substituents.

As used herein, the term "substituted" or "substituted" means that the group has one or more (preferably 1 to 6, more preferably 1 to 3) substituents selected from the group consisting of: halogen, C₁-C₆Alkyl radical, C₁-C₆Haloalkyl, C₂-C₆Alkenyl radical, C₂-C₆Alkynyl group.

As used herein, the term "C₁-C₆Alkyl "means a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

As used herein, the term "C₁-C₄Alkyl "means a straight or branched chain alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

As used herein, the term "C₂-C₆Alkenyl "means having 2 to 6 carbon atomsLinear or branched alkenyl groups of the subgroups, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "C₂-C₄Alkenyl "means a straight or branched chain alkenyl group having 2 to 4 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "C₂-C₆Alkynyl "means a straight or branched chain alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, or the like.

As used herein, the term "C₂-C₄Alkynyl "means a straight or branched chain alkynyl group having 2 to 4 carbon atoms, such as ethynyl, propynyl, or the like.

As used herein, the term "C₁-C₆Haloalkyl "refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, e.g., halomethyl, haloethyl, halopropyl, haloisopropyl, or the like, with hydrogen substituted with 1 or more than 1 halogen.

As used herein, the term "C₁-C₄Haloalkyl "refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms with hydrogen substituted with 1 or more than 1 halogen, for example, halomethyl, haloethyl, halopropyl, haloisopropyl, or the like.

As used herein, the term "C₁-C₆Alkoxy "means having (C)₁-C₆Alkyl) -O-structural radicals, e.g. CH₃-O-、C₂H₅-O-、C₃H₈-O-, or the like.

As used herein, the term "C₁-C₄Alkoxy "means having (C)₁-C₄Alkyl) -O-structural radicals, e.g. CH₃-O-、C₂H₅-O-、C₃H₈-O-, or the like.

As used herein, the term "C₁-C₆The "ester group" means having (C)₁-C₆Radicals of alkyl) -COO-structure, e.g. CH₃COO-、C₂H₅COO-、C₃H₈COO-、(CH₃)₂CHCOO-、nC₄H₉COO-、tC₄H₉COO-, or the like.

As used herein, the term "C₁-C₄The "ester group" means having (C)₁-C₄Radicals of alkyl) -COO-structure, e.g. CH₃COO-、C₂H₅COO-、C₃H₈COO-、(CH₃)₂CHCOO-、nC₄H₉COO-、tC₄H₉COO-, or the like.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine, preferably fluorine and chlorine.

The term "halogenated" as used herein refers to a group substituted with the same or different one or more of the above-mentioned halogen atoms, which may be partially halogenated or fully halogenated, for example, trifluoromethyl, pentafluoroethyl, heptafluoroisopropyl, or the like.

The compounds of the present invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and individual diastereomers. Asymmetric centers that may be present depend on the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the invention. The present invention includes all isomeric forms of the compounds.

Active ingredient

As used herein, the terms "active ingredient of the invention", "ingenol compound of the invention and derivatives thereof", "anti-HIV-latently active ingredient of the invention" and "compound of formula I of the invention" are used interchangeably and refer to an active ingredient extracted from the plant euphorbia kansui which has the effect of inducing proviral expression of HIV-latently infected cells and/or inhibiting HIV infection. In the invention, the anti-HIV active ingredient can be extracted from euphorbia plant and can be obtained by chemical synthesis.

In the present invention, the active ingredient of the present invention has the general formula shown in formula I:

in the formula (I), the compound is shown in the specification,

R₄Selected from the group consisting of: CH (CH)₂OR₅、-C(O)R₆Substituted or unsubstituted C₁-C₆Alkyl, or combinations thereof; wherein R is₅Selected from H, C₁-C₆Alkyl, CO- (CH ═ CH)₂-(CH₂)₄-CH₃、COCH₃、OCH₂CH₃、CO(CH₂)₁₀CH₃Or CO (CH)₂)₁₄CH₃；R₆Selected from the group consisting of: H. substituted or unsubstituted C₁-C₆Alkyl, substituted or unsubstituted C₂-C₆Alkene(s)Radical, substituted or unsubstituted C₂-C₆Alkynyl, substituted or unsubstituted C₁-C₆Alkoxy, -OH, substituted or unsubstituted C₁-C₆An ester group, or a combination thereof;

in another preferred embodiment, the compound of formula I, or a pharmaceutically acceptable salt thereof, is chemically synthesized or extracted.

Inhibition of HIV infection

The research of the invention discovers that HIV infected cells have a plurality of links, including links of virus and immune cell receptor (CD4, CCR5 and CXCR4) combination entry, reverse transcription, integration, transcription, replication, assembly and secretion and the like, and any link is blocked, namely the virus replication can be inhibited, so that the HIV infection is inhibited.

In the present invention, the compounds of the present invention reduce the expression of immune cell receptors (CD4, CCR5, CXCR4) and, thus, block HIV binding entry, thereby inhibiting HIV infection, and thus, the compounds of the present invention are HIV entry inhibitors. The compound can be combined with a plurality of inhibitors such as reverse transcriptase inhibitor, protease inhibitor, integrase inhibitor and the like, so that a stronger HIV infection inhibiting effect is obtained.

Activity screening assay

The invention adopts HIV latent cell model to carry out activity screening test, the result shows that the ingenol compound shown in formula I and the derivative thereof have HIV latent effect, and corresponding excipients are used for preparing an applicable dosage form according to the conventional method to carry out HIV latent effect test, and the result proves that the ingenol compound shown in formula I can enable HIV latent in infected cells to express, has the effect of HIV latent intervention, and can clear activated latent infected cells by being used together with antiretroviral drugs so as to accelerate the clearing of latent virus storage.

The invention relates to an HIV latent cell model, which comprises the following components: an established HIV latent screening model C11 clone is obtained by sorting and activating HIV slow virus infected T cells for 2 times (Chinese patent: 200810038851. X); another clone of human T cell 10.6 clone (J-Lat-A10.6) carrying the green fluorescent protein gene was obtained from the American national institute of health and wellness, AIDS reference reagent, Proc, and was established by doctor Eric Verdin, university of California. Human T cell Jurkat cells transfected with the HIV-R7/E-/GFP retroviral vector carrying the green fluorescent protein gene were sorted (Jordan A, Bisgrove D, Verdin E. HIV replication assay proteins a late infection after acid infection of T cells in vitro. EMBO J22: 1868-1877, 2003).

HIV infection inhibition assay

The present invention uses M7 cell model for HIV inhibition test, M7 cell is provided by the American national health research institute AIDS reference reagent program, established by Stephanie M.Brandt, California university, is CEM lymphocyte line stably integrating HIV-1LTR and GFP reporter genes, and can stably express CD4 and CXCR4, CCR5 receptor (Brandt SM, Mariani R, Holland AU, Hotpe J, Landau NR. Association of chemical-mediated block to HIV entry with receptor interaction. J Biol Chem 2002; 277: 17291- & gt 17299.) results show that the ingenol compound and its derivatives shown in formula I have anti-HIV infection effect, and suitable dosage forms are made by using corresponding excipients according to conventional methods to perform anti-HIV infection effect test, and the results prove that the ingenol compound shown in formula I has anti-HIV infection effect.

Compositions and methods of administration

As used herein, the term "composition" includes (a) compositions for the treatment and/or prevention of aids, (b) compositions for the activation of latent HIV. In addition, the composition includes a pharmaceutical composition, a food composition or a nutraceutical composition.

The anti-HIV latent active ingredient has the function of interfering HIV latency. Thus, when the anti-HIV latent active ingredient of the present invention is therapeutically administered or administered, the expression of latent HIV is promoted, thereby activating the latent HIV. Generally, the active ingredients of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The anti-HIV latent active ingredient of the invention has the effect of inhibiting HIV infection activity. Therefore, when the anti-HIV active ingredient of the present invention is therapeutically administered or administered, HIV infection can be inhibited, and an antiviral effect can be achieved. Generally, the active ingredients of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: oral, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

The invention also provides a pharmaceutical composition comprising a safe and effective amount of the active ingredient of the invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1ng to 10 mg per kg of body weight per day, preferably formula I or a derivative thereof may be used: the daily dosage of the adult is 0.1-20000 ng, preferably 1-3000 ng/day.

Can be used as medicine for preventing and treating AIDS, and can be made into oral and non-oral preparations. The oral administration can be made into tablet, powder, granule, capsule, etc., and the excipient can be one or more of starch, lactose, sucrose, mannose, hydroxymethyl cellulose, etc. The disintegrating agent can be one or more of potato starch, hydroxymethyl cellulose, etc. The binder can be one or more of acacia, corn starch, gelatin, dextrin, etc. The oral preparation can be made into emulsion, syrup, etc. besides the above dosage forms.

The non-oral preparation can be made into injection, or made into injection with water for injection, normal saline, and glucose solution, or added with ethanol, propanol, and ethylene glycol at a certain ratio.

Furthermore, the active ingredients of the invention are particularly suitable for use in combination with other anti-HIV agents. The active component of the invention can be used alone or in combination with other antiretroviral drugs/HIV latent infection activating drugs to eliminate the latent infected cells, thereby being used for treating AIDS.

The invention further aims to provide a preparation method of a medicine for treating AIDS, which adopts the ingenol compound shown in the formula I and the derivative thereof as raw materials to prepare oral and non-oral preparations by using corresponding excipients according to a conventional method, wherein the ingenol compound shown in the formula I and the formula IA and the derivative thereof can be used in the following dosage: the medicine is taken by adults for 1-5 times a day, wherein the daily dosage is 0.1-20000 ng, preferably 1-3000 ng/day; the dosage and frequency of children are decreased on adult basis.

Medicine box

The present invention also provides a kit (or a medicament for treating AIDS according to the first aspect of the present invention), comprising:

-formulations containing ingenol compounds of formula I and derivatives thereof;

-formulations containing aids therapeutic agents; and

-description.

In a preferred embodiment, the kit further comprises a formulation comprising an additional HIV latent activator drug.

The preparation containing the ingenol compound shown in the formula I and the derivative thereof can be a unit dosage form containing the ingenol compound and the derivative thereof, the preparation containing the AIDS therapeutic agent can be a unit dosage form containing the AIDS therapeutic agent, and the preparation containing other HIV latent activation medicines can be a unit dosage form containing HIV latent activation medicines.

The kit contains at least one unit dosage form containing ingenol compound and its derivatives, and unit dosage form containing AIDS therapeutic agent and HIV latent activating medicine; preferably 4-10 each.

As used herein, the term "unit dosage form" refers to a composition that is formulated for convenient administration into a dosage form required for a single administration, including, but not limited to, various solid dosage forms (e.g., tablets), liquid dosage forms, capsules, sustained release formulations.

The description of the kit of the invention describes the following methods of use:

(I) administering to a subject in need thereof a formulation comprising an ingenol compound and derivatives thereof;

(II) and/or 5-50 hours, preferably 10-48 hours, more preferably 15-24 hours after step (I), administering to said subject a formulation comprising a therapeutic agent for aids;

(III) repeating steps (I) - (II).

In addition, the preparation of the aids therapeutic agent that can be used in the kit of the present invention may be one or more, preferably, the aids therapeutic agent may be a plurality of, more preferably, a pharmaceutical combination of the aids therapeutic agent cocktail therapy well known to those skilled in the art.

Methods for activating latent HIV and/or inhibiting HIV infection

The invention also provides methods of activating latent HIV and/or inhibiting HIV, both in vitro non-therapeutically and in vivo therapeutically.

Wherein the method for non-therapeutically activating latent HIV (or inducing HIV proviral expression in HIV latently infected cells) in vitro comprises the steps of:

culturing said HIV latently infected cells in the presence of ingenol compounds and derivatives thereof (or ingenol compounds and derivatives thereof and other HIV latently activating drugs) to reactivate latently HIV.

When, after this step, the ingenol compound or derivative thereof itself has an effect of inhibiting the infection by reactivating latent HIV, and therefore, in vitro and in vivo research systems, the ingenol compound or derivative thereof is used in combination with aids therapeutic agents (such as antiretroviral drugs, immunomodulators, therapeutic antibodies, etc.), the method of inhibiting or killing HIV after activation of the present invention is obtained.

Wherein the method for non-therapeutic in vitro inhibition of HIV comprises the steps of:

the inhibitory ability against HIV strains was tested in the presence of ingenol compounds and derivatives thereof.

A method of treatment suitable for use in vivo, comprising the steps of:

(I) administering to a subject in need thereof an ingenol compound or a derivative thereof;

(II) optionally, administering to said subject a formulation comprising an aids therapeutic 5-120 hours, preferably 24-96 hours, more preferably 72-96 hours, after step (I); and optionally (III) repeating steps (I) - (II).

In a preferred embodiment, step (I) further comprises administering a formulation of another HIV latently activating drug.

The main advantages of the invention include:

1. through a large amount of screening, the invention discovers the derivatives of the ingenol compound capable of obviously intervening HIV for the first time.

2. The invention provides a new application of the ingenol compound for the first time, and the ingenol compound can obviously express latent HIV in infected cells at low concentration; the ingenol compound can inhibit HIV infection at low concentration.

3. The invention discovers for the first time that the ingenol compound and the derivative thereof have the function of inhibiting HIV infection at low concentration by reducing the expression of CD4 cell surface HIV infection receptors CD4, CCR5 and CXCR4, thereby inhibiting the entry of HIV and preparing the anti-HIV medicament.

4. The invention discovers for the first time that ingenol compounds are treated by C₆H₁₂O₅To obtain SO₃H has the effect of increasing water solubility after modification, or is combined with a solubilizing carrier andor auxiliary materials to increase water solubility.

5. The compound of the invention can be further used for preparing drugs for treating AIDS, and provides a new way for completely curing AIDS.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

EXAMPLE 1 preparation of ingenol Compound EK-5A of the present invention

Extracting root and rhizome of kansui root (E.kansui) 5Kg with 8 times of 95% ethanol under reflux twice, each for two hours, and recovering the obtained extract until there is no alcohol smell to obtain extract. The mixture was diluted to 1000mL with water, extracted three times with an equal volume of methylene chloride, and the methylene chloride solvent was recovered to obtain 101.2g of a methylene chloride extract.

The silica gel column chromatography conditions are as follows: the volume ratio of the adopted mobile phase is 10-2: 1 of petroleum ether-ethyl acetate. The conditions of the reverse phase chromatographic purification are as follows: octadecyl bonded silica gel is adopted as a filler, a mobile phase is a methanol-water solution with the volume ratio of 90 percent, isocratic elution is carried out, and a compound EK-5A (80mg) is obtained in 14 minutes and is a faint yellow sticky solid, and the ultraviolet absorption is 268 nm.

The separation and purification comprises the following steps: concentrating the extractive solution, performing silica gel column chromatography, detecting with thin layer chromatography to obtain desired fraction, and purifying with reverse phase chromatography to obtain compound EK-5A, 80 mg.

1H NMR(400MHz,CDCl3)7.71(ddd,J＝23.0,15.6,4.8Hz,1H)；6.16(p,J＝11.1Hz,2H)；6.08(t,J＝7.1Hz,1H)；6.02–5.88(m,2H)；5.62(d,J＝10.7Hz,1H)；5.32(s,1H)；4.77(d,J＝12.5Hz,1H)；4.50(d,J＝12.5Hz,1H)；4.11(dd,J＝11.7,4.2Hz,1H)；3.90(s,1H；3.52(d,J＝17.6Hz,2H)；2.60–2.44(m,1H)；2.37–2.18(m,4H)；2.08(s,3H)；1.97(s,1H)；1.86–1.78(m,4H)；1.51–1.39(m,3H)；1.39–1.24(m,6H)；1.08(d,J＝9.3Hz,7H)；1.00(t,J＝7.8Hz,4H)；0.97(dd,J＝8.1,3.6Hz,1H)；0.94–0.87(m,4H)；0.79–0.66(m,1H).

EXAMPLE 2 preparation of ingenol Compound EK-1A of the present invention

The silica gel column chromatography conditions are as follows: the volume ratio of the adopted mobile phase is 10-2: 1 of petroleum ether-ethyl acetate. The conditions of the reverse phase chromatographic purification are as follows: octadecyl bonded silica gel is adopted as a filler, a mobile phase is a methanol-water solution with the volume ratio of 86% to be eluted isocratically, and a compound EK-1A (30mg) is obtained in 15 minutes and is a faint yellow sticky solid with ultraviolet absorption of 268 nm.

The separation and purification comprises the following steps: concentrating the extractive solution, performing silica gel column chromatography, detecting with thin layer chromatography to obtain desired fraction, and purifying with reverse phase chromatography to obtain compound EK-1A, 30 mg.

1H NMR(400MHz,CDCl3)：7.74–7.57(m,1H)；6.24–6.13(m,1H)；6.10(d,J＝8.8Hz,1H)；6.03–5.83(m,3H)；4.84(d,J＝12.8Hz,1H)；4.62(d,J＝12.8Hz,1H)；4.45(d,J＝10.5Hz,1H)；4.30–4.00(m,3H)；3.84(s,1H)；3.78(s,1H)；3.71(s,1H)；2.49–2.00(m,8H)；1.86(t,J＝9.8Hz,4H)；1.83–1.70(m,2H)；1.51–1.39(m,2H)；1.39–1.25(m,5H)；1.17–1.04(m,6H)；0.98(dd,J＝12.5,7.8Hz,4H)；0.91(t,J＝6.9Hz,3H)；0.72(dd,J＝15.0,8.5Hz,2H).

EXAMPLE 3 the ingenol compounds and derivatives thereof of the present invention efficiently activate the expression of HIV-1 in latent cells

J-Lat-A10.6 or C11 cells were plated at 2X 10E4 cells per well in 96-well plates, and 100. mu.l of 1640 medium (Gibco) containing 10% FBS (Gibco) was added per well. After the cells are treated by the compounds of examples 1and 2 for 48 hours, the green fluorescence expression condition of the cells is observed under a fluorescence microscope, the cells are collected for flow cytometry detection, and the proportion of the fluorescent cells is analyzed.

The results are shown in FIGS. 1and 2. The results show that HIV latently infected cells without inducer treatment had only about 1-2% background activation of fluorescence positive cells (FIGS. 1-1 and 1-2). After being treated by 5ng/ml EK-5A (namely 5A), the proportion of cells expressing green fluorescence in a cell model is obviously increased; among them, J-Lat-A10.6 cells treated with the compound of example 1 showed a green fluorescence positive cell rate of up to 90% (FIGS. 1-3, 1-4, and 2), and C11 cells also showed a green fluorescence positive cell rate of nearly 80%.

The results show that the activation rate of HIV increases 32-45 fold after the treatment with the compounds of examples 1and 2, compared with the control group.

Example 4 comparison of the efficiency of ingenol compounds and derivatives thereof with PKC activator PEP005 in the activation of latent HIV-1

J-Lat-A10.6 or C11 cells were plated at 2X 10E4 cells per well in 96-well plates, and 100. mu.l of 1640 medium (Gibco) containing 10% FBS (Gibco) was added per well. After the cells are respectively treated by the compounds with different concentrations (such as the compounds in examples 1and 2) and PEP005 for 48 hours, the cells are collected for flow cytometry detection, and the proportion of fluorescent cells is analyzed.

The results are shown in FIGS. 3 and 4.

The results show that the better activation concentrations of ingenol compounds and derivatives thereof range from 1 to 100ng/ml and have a dose-response relationship in both cell models. Ingenol compounds 5A activate EC of latent HIV-1 on C11 cells₅₀0.23ng/ml and PEP005 0.31 ng/ml; whereas on J-Lat-A10.6 cells ingenol compounds and their derivatives (e.g. 1A,5A) activate the lowest EC of latent HIV-1₅₀0.12ng/ml and PEP005 0.82 ng/ml. EC of ingenol compounds and derivatives thereof on two latent cell lines₅₀Are all lower than PEP 005. The ingenol compound and its derivatives have extremely high activity for activating latent HIV-1, and can induce high level activation of HIV-1 at extremely low concentration.

Example 5 the ingenol compounds and derivatives thereof according to the present invention significantly reduced the expression of CD4, CCR5, CXCR4 receptors on the cell surface of CD4

After Human primary PBMC is treated by EK-5A and PEP005 with different concentrations for 48 hours, cells are collected, PBS is washed twice, 100 mu l of PBS is used for resuspending the cells, Human PE conjugated anti-CD4, Human FITC conjugated anti-CCR5 and Human FITC conjugated anti-CXCR4 antibodies are respectively added, the cells are incubated for 30 minutes on ice in a dark place, PBS is washed twice, 200 mu l of PBS is used for resuspending the cells, and the expression conditions of CD4, CCR5 and CXCR4 on the cell surfaces are detected by a flow cytometer.

The results are shown in FIG. 5.

The results show that EK-5A can reduce the expression of CD4, CCR5 and CXCR4 molecules at its effective activating concentration, indicating that EK-5A has the effect of reducing HIV infection.

Example 6 ingenol compounds and derivatives thereof of the present invention are effective in reducing HIV infection

Adding 50 mu LBal virus (diluted by serum-free 1640) and 50 mu L EK-5A (diluted by serum-free 1640) diluted by a multiple ratio (4 times) into each well of a 96-well plate, incubating at 37 ℃ for 30 minutes, adding 100 mu L cells (105 cells/ml, diluted by the 1640 culture solution added with 10% serum), using a well added with only M7 cells with the same final concentration as a negative control, adding Bal virus and M7 cells with the same final concentration, and using a well added with no drug as a positive control; 150 μ L of supernatant was aspirated every well the next day, 150 μ L of fresh 1640 medium containing 10% serum was added, 50 μ L of supernatant was collected the fifth day, 50 μ L of PBS containing 5% triton was added to the supernatant, mixing and left overnight at 4 ℃, the amount of P24 in the supernatant was measured by ELISA to quantify HIV-1 infection with M7, and P24 was measured by coating a half-area 96-well plate (Corning) with HIV IgG (5 μ g/ml, diluted with PBS) in the evening before the day, left at 4 ℃ for 12 hours, washing the plate and blocking with 2% PBS milk, and left at 37 ℃ for 2 hours. Adding 10 mu L of the mixture of the collected supernatant and PBS added with triton after washing the plate, then adding 40 mu L of LPBS, standing for 1 hour at 37 ℃, adding 50 mu L of primary antibody (1.5 mu g/ml, 183 antibody extracted by a laboratory per se) after washing the plate, standing for one hour at 37 ℃, adding 50 mu L of secondary antibody (diluted 3000 times, Dako Denmark rabbit anti-mouse monoclonal antibody) after washing the plate, standing for one hour at 37 ℃, adding developing solution after washing the plate, stopping reaction after 3-5 minutes by adding sulfuric acid, and detecting OD450 of each hole by using a microplate reader (TECAN infinite M200 pro) to obtain data; the inhibition rate and effective inhibitory concentration of each concentration sample were calculated according to the method described above.

As shown in fig. 6.

The results show that the compound (such as EK-5A) has remarkable activity of inhibiting HIV Bal virus.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a compound of formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a composition or formulation for (a) intervening in HIV latency; (b) inhibiting HIV infection; and/or (c) reduces the expression of CD4 cell surface receptors:

in the formula (I), the compound is shown in the specification,

2. The use according to claim 1, wherein R is₄Selected from the group consisting of: CH (CH)₂OR₅、-C(O)R₆Substituted or unsubstituted C₁-C₄Alkyl, or combinations thereof; wherein R is₅Selected from H, C₁-C₄Alkyl, CO- (CH ═ CH)₂-(CH₂)₄-CH₃、COCH₃、OCH₂CH₃、CO(CH₂)₁₀CH₃Or CO (CH)₂)₁₄CH₃；R₆Selected from the group consisting of: H. substituted or unsubstituted C₁-C₄Alkyl, substituted or unsubstituted C₂-C₄Alkenyl, substituted or unsubstituted C₂-C₄Alkynyl, substituted or unsubstituted C₁-C₄Alkoxy, -OH, substituted or unsubstituted C₁-C₄An ester group, or a combination thereof.

3. The use according to claim 1, wherein the compound is selected from the group consisting of:

4. the use of claim 1, wherein the composition or formulation further comprises an additional aids therapeutic agent.

5. A pharmaceutical composition, comprising:

(b) a pharmaceutically acceptable carrier;

6. A method for non-therapeutically inducing HIV proviral expression in HIV latently infected cells in vitro comprising the steps of: culturing a cell latently infected with the HIV virus in the presence of a compound of formula I as defined in claim 1, such that the latently HIV provirus is expressed, thereby activating the latently HIV virus.

7. An in vitro non-therapeutic method of activating latent HIV and/or inhibiting HIV infection comprising the steps of: administering to a subject in need thereof a compound of formula I as defined in claim 1, thereby activating latent HIV and/or inhibiting HIV infection.

8. A method for preparing a pharmaceutical composition for preventing and/or treating aids, comprising the steps of: mixing a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier, thereby forming a pharmaceutical composition.

9. A kit, comprising:

a formulation comprising a compound of formula I as defined in claim 1 or a pharmaceutically acceptable salt thereof;

a preparation containing AIDS therapeutic agent; and

and (6) instructions.

10. An in vitro non-therapeutic method of inhibiting and/or killing latent HIV comprising the steps of:

(i) culturing cells latently infected with HIV in the presence of a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof, thereby obtaining activated HIV-containing cells and inhibiting and/or killing latently HIV;