Application of Pomalidomide in preparation of medicine for long-term immune reconstitution of HIV (Human Immunodeficiency Virus) patient

Abstract

The invention discloses an application of Pomalidomide in preparation of a medicine for long-term immune reconstitution of an HIV (human immunodeficiency virus) patient. According to the application disclosed by the invention, the research finds that pomalidomide can be used for remarkably inhibiting the replication of HIV-1 in MT-4 and Jurkat cell lines, primary CD4 + T cells and humanized mouse models for the first time, and the Pomalidomide can be used for increasing the quantity of CD4 + T and the ratio of CD4 to CD8, increasing the activation of T cells, reducing the depletion of the T cells and promoting immune reconstruction no matter in a cell model or a mouse model. The invention provides a theoretical basis for research and development of medicines for long-term immune reconstitution of HIV patients, opens up a new application of Pomalidomide, provides a brand new method for treating HIV infection and promoting long-term immune reconstitution of HIV patients, and has a wide application prospect in the technical field of treatment of HIV infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Pomalidomide in the preparation of long-term immune remodeling drugs for HIV patients. Background Technology

[0002] AIDS is a global, deadly infectious disease caused by the human immunodeficiency virus (HIV). The cause is that the virus specifically attacks and destroys the human immune system, leading to progressive immune failure and ultimately the body's inability to resist pathogen invasion and tumor development, eventually progressing to acquired immunodeficiency syndrome (AIDS). HIV primarily targets CD4+ cells in the human immune system. + T cells—as the core regulatory cells of the immune response—are responsible not only for activating B cells to produce antibodies and inducing the proliferation of cytotoxic T cells, but also for participating in the formation and maintenance of immune memory. After HIV invades cells by binding to CD4 molecules and co-receptors, it uses its own reverse transcriptase and integrase to reverse transcribe the viral genome from RNA into DNA and integrate it into the host cell genome. This directly leads to CD4... + T cell lysis and death, on the other hand, exacerbate immune function decline by inducing immune activation disorders, dendritic cell dysfunction, and regulatory T cell (Treg) abnormalities. The expert consensus on the clinical diagnosis and treatment of AIDS points out that CD4... + A T-cell count recovering to 500 / μL or close to that of healthy individuals can be considered a sign of good immune reconstitution after treatment. Therefore, developing methods to increase CD4 count in HIV-infected individuals is crucial. + The number of T cells and drugs that promote immune function reconstruction are crucial.

[0003] Although existing anti-HIV drugs can effectively suppress viral replication, reducing HIV levels in patients' plasma to below the clinical detection limit and significantly improving their quality of life, patients must take medication for life, and the virus rebounds rapidly once medication is stopped. Impaired immune reconstitution has become a bottleneck restricting treatment efficacy. Pomalidomide (Pom), with the molecular formula C... 13 H 11 N3O4, with a molecular weight of 273.25, belongs to the immunomodulatory imide class of drugs. It acts via a molecular gel and is a derivative of thalidomide, which inhibits tumor cell proliferation and induces apoptosis. It interacts with the E3 ubiquitin ligase cereblon (CRBN), inducing the degradation of essential transcription factors such as Ikaros. Currently, no studies have reported its use in anti-HIV treatment or in promoting immune reconstitution. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Pomalidomide in the preparation of long-term immune remodeling drugs for HIV patients, so as to solve the technical problems of rapid viral rebound and poor immune remodeling after HIV patients stop taking medication.

[0005] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: The first aspect of this invention provides the use of Pomalidomide or a derivative thereof in the preparation of a medicament for promoting long-term immune reconstitution in HIV patients.

[0006] Furthermore, the Pomalidomide or its derivatives also include pharmaceutically acceptable salts, hydrates, isomers, solvates, or crystalline forms.

[0007] In some embodiments, the term "pharmaceutical-grade salt" as used herein includes conventional salts formed from pharmaceutically acceptable inorganic or organic acids or inorganic or organic bases, as well as acid addition salts of quaternary ammonium compounds. More specific examples of suitable acid salts include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid, citric acid, pyric acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, tannic acid, etc. Other acids, such as oxalic acid, although not pharmaceutically acceptable on their own, can be used to prepare salts as intermediates to obtain the compounds of the present invention and their pharmaceutically-grade salts. More specific examples of suitable alkaline salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, and procaine salts. Hereafter, when referring to pharmaceutically acceptable salinomycin salts, it generally means that they are suitable for use in the pharmaceutical industry, harmless to the product or mammals, or have a reasonable or acceptable benefit / risk ratio.

[0008] In some embodiments, the hydrate refers to a compound that is combined with water. Those skilled in the art will understand that organic compounds can form complexes with solvents, react in that solvent, or precipitate or crystallize out of that solvent; these complexes are called solvates. When the solvent is water, the complex is called a hydrate.

[0009] In some embodiments, the isomers refer to compounds having the same molecular formula but different connectivity or spatial arrangement of the constituent atoms within the molecule. Isomers can include, for example, structural isomers and stereoisomers. Stereoisomers can be diastereomers or enantiomers. Enantiomers are isomers that cannot be superimposed on their mirror images, analogous to the relationship between the left and right hands, and are also known as optical isomers. When the chiral carbon atom has four different substituents, enantiomers are classified as R (Rectus; clockwise) or S (Sinister; counterclockwise). Diastereomers are stereoisomers that are not mirror images of each other; they can include cis-trans isomers caused by differences in atomic spatial arrangement.

[0010] In some embodiments, the solvate refers to a compound or its salt that is bound to a solvent and is typically formed by a solvent decomposition reaction. This physical association may include hydrogen bonding. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. In some cases, the solvate will be separable, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvates include solvates in solution and separable solvates.

[0011] In some embodiments, the crystalline form refers to the crystalline form of a compound with a specific crystal packing arrangement. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, photoelectric properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors can lead to the dominance of one crystalline form. Various polymorphs of the compound can be prepared by crystallization under different conditions.

[0012] In some embodiments, the derivative refers to a compound whose structure is modified or functionalized by chemical, physical, or biological methods, such as by introducing new functional groups, changing the molecular conformation, or combining with other molecules, thereby obtaining a compound with enhanced or expanded functions while maintaining the core structure and function of the parent compound. Exemplary derivatives include compounds obtained by introducing new functional groups into the parent compound, such as through esterification, acylation, or sulfonation.

[0013] Furthermore, the derivative is a functional compound obtained by chemically modifying the core structure of pomalidomide.

[0014] Furthermore, the derivatives include compounds as shown in formula (I) and formula (II).

[0015]

[0016] Equation (I)

[0017] Formula (II).

[0018] Furthermore, the concentration of the Pomalidomide or its derivative is 0.8 nM-100 μM.

[0019] Furthermore, the concentration of Pomalidomide or its derivatives is 0.01 μM-100 μM.

[0020] Furthermore, the concentration of Pomalidomide or its derivatives is 0.01 μM-10 μM.

[0021] In some embodiments, the medicament of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level can be determined based on the patient's disease type, severity, degree, drug activity, drug sensitivity, timing of administration, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors known in the medical field. The medicament of the present invention can be administered as a standalone therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple times. It is important to administer an amount that achieves the greatest effect with the least amount and without side effects, taking all the above factors into account; this can be readily determined by those skilled in the art.

[0022] Furthermore, the pomalidomide or its derivatives significantly increase CD4 levels after HIV infection. + The number of T cells and the CD4 / CD8 ratio can promote immune reconstitution in HIV patients.

[0023] Furthermore, the promotion of long-term immune reconstitution in HIV patients is defined as the ability of Pomalidomide or its derivatives to significantly increase CD4 levels post-HIV infection for at least 5 weeks after discontinuation. + T cell count and CD4 / CD8 ratio.

[0024] In some implementations, the drug may be administered to the subject via injection, topical administration, or oral administration. For example, the method may include administering the drug to the subject three times a day, once a day, or every two days. In some embodiments, injection may include subcutaneous injection, intramuscular injection, intravenous injection, etc. In some embodiments, injection may include injecting the drug directly into the lesion or an area near the lesion. In some embodiments, topical administration may include rectal administration, nasal administration, ear administration, intramedullary administration, intra-articular administration, intrapleural administration, etc., or any combination thereof. In some embodiments, the drug may be administered to the subject via a combination of different administration methods.

[0025] In some implementations, the patient / subject can be human or non-human and can include, for example, animal strains or species used as a "model system" for research purposes. Similarly, the patient / subject can include adults or adolescents (e.g., children). Furthermore, the patient / subject can refer to any living organism, preferably a mammal (e.g., human or non-human). Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (e.g., chimpanzees) and other apes and monkeys; livestock such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, etc.

[0026] The second aspect of the present invention provides for any of the following applications: 1) Use of Pomalidomide or its derivatives in the preparation of pharmaceutical compositions for promoting long-term immune reconstitution in HIV patients.

[0027] 2) The use of Pomalidomide or its derivatives in the preparation of pharmaceutical formulations for promoting long-term immune remodeling in HIV patients.

[0028] Furthermore, the pharmaceutical composition also includes other drugs that can be used to promote long-term immune reconstitution in HIV patients.

[0029] In this invention, the term "pharmaceutical composition" refers to a composition containing at least one bioactive compound. The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. The pharmaceutical compositions of this invention may contain any commonly used non-toxic, pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided the target tissue can be reached.

[0030] In some embodiments, the two drugs in the pharmaceutical composition can be administered simultaneously, separately, or sequentially. Simultaneous administration means that the two drugs are administered concurrently. If not administered simultaneously, they are administered sequentially within a time frame so that both can be therapeutically effective within the same time frame. Therefore, sequential administration allows for the administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours after administering one drug, provided that the circulating half-life of the first administered drug allows for a simultaneously therapeutically effective amount of both. The time delay between administrations of the components will vary depending on the exact nature of the components, their interactions, and their respective half-lives. This differs from simultaneous or sequential administration, which refers to a significant interval between the administration of one drug and another, meaning that when the second drug is administered, the first administered drug may no longer be present in the bloodstream at a therapeutically effective amount.

[0031] In some implementations, a pharmaceutical composition comprising at least one drug may conventionally be administered in unit doses. When used in a pharmaceutical composition, a unit dose refers to a physically discrete unit suitable as a unit dosage form for use in a subject, each unit containing a predetermined amount of the active substance, combined with a desired physiologically acceptable diluent (i.e., a carrier or delivery agent), calculated to produce the desired therapeutic effect.

[0032] Furthermore, the pharmaceutical preparation also includes pharmaceutically acceptable excipients.

[0033] In some embodiments, the pharmaceutically acceptable excipients of this invention include, but are not limited to: diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants. The diluents include, but are not limited to: lactose, sodium chloride, glucose, urea, starch, and water. The binders include, but are not limited to: starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginate, xanthan gum, hydroxypropylcellulose, and hydroxypropyl methylcellulose. The surfactants include, but are not limited to: polyethylene glycol sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol. The humectants include, but are not limited to: glycerin and starch. The adsorbents include, but are not limited to: starch, lactose, bentonite, silica gel, kaolin, and soap clay. The lubricants include, but are not limited to: zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc. The fillers include, but are not limited to: mannitol, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, sodium bicarbonate, etc. The disintegrants include, but are not limited to: crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0034] In some embodiments, the medicament described in this invention may also contain additives such as stabilizers, buffers, bactericides, isotonic agents, pH control agents, surfactants, and chelating agents.

[0035] Furthermore, the dosage form of the pharmaceutical preparation includes oral dosage form, parenteral dosage form, or topical dosage form.

[0036] In some embodiments, the dosage forms described in this invention include, but are not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled-release formulations, aerosols, films, injections, intravenous infusions, transdermal absorption formulations, ointments, lotions, adhesive formulations, suppositories, pills, nasal preparations, pulmonary preparations, eye drops, etc.

[0037] A third aspect of the present invention provides the use of Pomalidomide or a derivative thereof in the preparation of a medicament for treating HIV infection.

[0038] Furthermore, the Pomalidomide or its derivatives achieve the effect of anti-HIV infection by inhibiting the replication of the HIV virus.

[0039] Furthermore, the inhibitory effect of Pomalidomide or its derivatives on HIV replication showed no significant rebound within at least 5 weeks after discontinuation of the drug.

[0040] Advantages and beneficial effects of the present invention: This invention is the first to discover that pomalidomide can significantly inhibit HIV in MT-4 and Jurkat cell lines, and primary CD4 cells. + Pomalidomide enhanced CD4 replication in both T cell and humanized mouse models, and in both cell and mouse models, it increased CD4 replication. + The increase in T cell count and CD4 / CD8 ratio enhances T cell activation, reduces T cell depletion, and promotes immune reconstitution. This invention provides a theoretical basis for the development of long-term immune reconstitution drugs for HIV patients, opens up new applications for Pomalidomide, and offers a novel method for treating HIV infection and promoting long-term immune reconstitution in HIV patients, showing broad application prospects in the field of HIV infection treatment. Attached Figure Description

[0041] Figure 1 This represents the in vitro inhibitory effect of pomalidomide (Pom) on HIV-1, where A represents primary CD4. + T cells, B is MT-4 cells, and C is Jurkat cells.

[0042] Figure 2 This is an in vitro cell proliferation assay using pomalidomide (Pom), where A represents primary CD4+. + T cells, B is MT-4 cells, and C is Jurkat cells.

[0043] Figure 3 This is an in vitro cytotoxicity assay for pomalidomide (Pom), where A represents primary CD4 cells. + T cells, B is MT-4 cells, and C is Jurkat cells.

[0044] Figure 4 The effect of pomalidomide (Pom) on the number of T cells in vitro, where A represents CD4+ in T cells. + The proportion of T cells, B being CD4. + The number of T cells, where C is the CD4 / CD8 ratio.

[0045] Figure 5The in vitro effects of pomalidomide (Pom) on T cell activation are shown in the figures, where A represents IL-2, B represents IL-6, C represents IL-1β, D represents T cell activation markers ITGA4 and HLA-C, and E represents T cell exhaustion markers LAG3 and TIGIT.

[0046] Figure 6 The effect of pomalidomide (Pom) on immune reconstitution in vivo, where A represents viral load and B represents CD4+. + The number of T cells, where C represents the CD4+ nucleotide count in T cells. + The proportion of T cells, D is CD25 + The number of T cells, E is CD8 + The number of T cells, F is the CD4 / CD8 ratio, G is the IL-2 expression level, H is the IL-6 expression level, and I is the IL-1β expression level. Detailed Implementation

[0047] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0048] Example 1: Experimental verification of pomalidomide's in vitro anti-HIV effect I. Experimental Methods 1. Experimental materials: MT-4 cells (NIH AIDS Reagent Program, ARP-120), Jurkat cells (ATCC, TIB-152), TZM-bl cells (ATCC, PTA-5659), HIV-NL4-3 (GenBank: MN685337.1), Pomalidomide (MCE, HY-10984).

[0049] 2. Experimental steps: (1) In vitro HIV replication inhibition experiment with Pomalidomide: CD4 isolated from peripheral blood of healthy individuals +T cells, MT-4 cells, and Jurkat cells were seeded in 24-well plates (3 × 10⁻⁶ cells / well). 5 Cells were cultured in 37°C, 5% CO2 incubators (cells / well). The following day, cells were infected with HIV-NL4-3 (MOI=0.1) and cultured for another 10 hours. After centrifugation at 1000 rpm for 5 minutes to discard the virus-containing culture medium, the cells were resuspended in culture medium containing different concentrations (0.01, 0.1, 1.0, 10.0, 100.0 μM) of pomalidomide (Pom) and then seeded into the corresponding wells. Cells were then cultured in 37°C, 5% CO2 incubators. After 48 hours, the cell culture supernatant was collected and centrifuged at 12000 rpm for 5 minutes to remove cell debris. 60 μL of the cell culture supernatant was seeded into 24-well plates, with three independent replicates for each concentration. 1 × 10⁶ cells were seeded into each well. 5 TZM-bl cells were cultured at 37°C in a 5% CO2 incubator. After 48 hours, the culture medium was discarded, and 500 μL of 0.25% trypsin was added to each well for digestion for 5 minutes. 1 mL of complete culture medium was then added to terminate the digestion reaction. The cells were centrifuged at 12000 rpm for 5 minutes to collect the TZM-bl cell pellet. The collected TZM-bl cell pellet was lysed with 60 μL of cell lysis buffer from a luciferase reporter assay kit at room temperature for 30 minutes, followed by centrifugation at 12000 rpm for 10 minutes. 4 μL of the supernatant was mixed with 20 μL of substrate reaction solution, and the chemiluminescence value was rapidly read using a GloMax 20 / 20 luminometer. The results were recorded, the virus inhibition rate was calculated, and the half-maximal inhibitory concentration (EC50) was calculated. 50 ).

[0050] (2) In vitro toxicity test of Pomalidomide: CD44 cells isolated from peripheral blood of healthy individuals + T cells, MT-4 cells, and Jurkat cells were seeded at 100 μL in 96-well plates (2 × 10⁶ cells per well). 4 Simultaneously, pomalidomide (Pom) stock solution was serially diluted with cell culture medium, and 100 μL was added to each well to achieve final concentrations of 0.001, 0.01, 0.1, 1.0, 10.0, and 100.0 μM, with three replicates for each concentration. A control well containing DMSO was also included. After incubation at 37°C and 5% CO2 for 24, 48, and 72 hours, 20 μL of CCK-8 assay reagent was added, and incubation continued for 1.5–2 hours. The absorbance of each well was measured at 450 nm using an ELISA reader. The results were recorded, and the cell count, 48-hour cell viability, and 48-hour half-maximal cytotoxicity concentration (CC50) were calculated. 50 ).

[0051] II. Experimental Results Experimental results showed that pomalidomide (Pom) was effective in inhibiting viral replication on CD4. + EC in T cells 50 The concentration of EC in MT-4 cells was 0.005 μM. 50 The concentration was 0.0008 μM in EC2 cells of Jurkat cells. 50 0.001 μM ( Figure 1 Furthermore, after adding Pom, CD4 + T cells, MT-4 cells, and Jurkat cells continued to proliferate normally without producing significant cytotoxicity. Figure 2 Pom in CD4 + CC in T cells 50 The concentration of CC in MT-4 cells was 25.862 μM. 50 The concentration of CC in Jurkat cells was 11.634 μM. 50 14.111 μM ( Figure 3 ).

[0052] Example 2: Experimental verification of pomalidomide's ability to promote immune reconstitution in vitro. I. Experimental Methods PBMC cells isolated from peripheral blood of healthy individuals were seeded into 6-well plates (2 × 10⁻⁶ cells / well). 4 Cells were cultured at 37°C in a 5% CO2 incubator for 48 hours and then infected with HIV-NL4-3 (MOI=0.1). Pomalidomide (Pom) was added to a final concentration of 2 μM. After culturing for another 72 hours, cells were collected by centrifugation at 1000 rpm for 5 minutes, resuspended in PBS, and stained with anti-human CD4 antibody (Biolegend, 344608) and anti-human CD8 antibody (Biolegend, 344704) at 4°C in the dark for 30 minutes. CD4 count was then detected by flow cytometry. + T cells, CD8 + The number of T cells was counted and the CD4 / CD8 ratio was calculated.

[0053] The expression levels of IL-2, IL-6, and IL-1β in the cell supernatant were detected using an ELISA kit (Sangon Biotech). The mRNA levels of T cell activation markers ITGA4 and HLA-C, and T cell exhaustion markers LAG3 and TIGIT were detected using real-time quantitative PCR. The PCR primer sequences are shown in Table 1.

[0054] Table 1 Primer Sequences

[0055] II. Experimental Results The results showed that after the addition of pomalidomide (Pom), CD4 + The number of T cells increased by 1.2 times, and the CD4 / CD8 ratio increased by 2.2 times. Figure 4 Experimental results showed that pomalidomide (Pom) could increase CD4 counts in HIV-1-infected PBMCs. + T cells and CD4 / CD8 ratio.

[0056] After the addition of pomalidomide (Pom) to the infected group, IL-2 expression levels increased by 2.7-fold, while IL-6 levels decreased to half their original level and IL-1β expression levels decreased to three-fifths of their original level. The mRNA levels of T cell activation markers ITGA4 and HLA-C increased by 1.8-fold, while the mRNA levels of T cell exhaustion markers LAG3 and TIGIT decreased to three-tenths and one-half of their original levels, respectively. Figure 5 The results showed that pomalidomide (Pom) promoted T cell activation, reduced the inflammatory response, and decreased T cell exhaustion.

[0057] Example 3: Experimental verification of pomalidomide's in vivo anti-HIV infection and promotion of immune reconstitution. I. Experimental Methods This invention uses 4-6 week old female NPG-RF immunodeficient mice (Vitonda) as a model, and injects 100 μL of a solution containing 1×10⁻⁶ NPG-RF into the tail vein. 5 CD34 + Stem cell culture medium was used to construct humanized mice. Two weeks after infection, approximately 50 μL of blood was collected from the submandibular region via tail vein injection of 100 μL containing 20 ng of HIV-JRCSF (GenBank: M38429.1) viral stock solution. The blood sample was collected into a blood collection tube containing 0.5 M EDTA as an anticoagulant. Plasma was obtained by centrifugation at 1600 rpm for 8 minutes. 800 μL of Trizol solution was added to the mouse plasma, which was quickly dispersed by agitation. Then, 200 μL of chloroform was added, and the mixture was thoroughly mixed. The mixture was centrifuged at 12000 ×g for 20 minutes at 4°C. The supernatant was transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added. The mixture was precipitated at -20°C for 30 minutes. After centrifugation at 12000 ×g for 20 minutes at 4°C, the supernatant was discarded. 1 mL of 70% ethanol was added, and the mixture was centrifuged at 12000 ×g for 10 minutes at 4°C. Discard the supernatant, add 30 μL of DEPC water to dissolve the precipitate, obtain mouse plasma RNA, and perform reverse transcription using a reverse transcription kit to obtain cDNA.

[0058] According to the instructions of the Human Immunodeficiency Virus Type 1 Nucleic Acid Assay Kit (DaAn Gene, DA0331), a real-time quantitative PCR system was prepared, including standards, blank controls, positive controls, and the test sample—mouse plasma cDNA. Three replicates were prepared for each sample. The viral load of each mouse was obtained based on the PCR results. Mice were then divided into three groups based on their viral load: one group of six mice received antiretroviral therapy (ART) drugs (45 mg / kg CAB, 45 mg / kg RPV, 40 mg / kg 3TC, 40 mg / kg ABC); another group of six mice received Pom (30 mg / kg); and the third group served as a control group, receiving a solvent (solvent formulation: 5% DMSO, 40% PEG300, 5% Tween80, 50% (20% SED in saline W / V)). After one week of continuous daily administration, medication was discontinued when the viral load in the blood decreased below the detection line, and observation continued for another six weeks. Blood samples are collected weekly, and viral load is measured using a viral load assay kit. CD4 count is measured using flow cytometry. + T, CD8 + T, CD25 + The number of T cells was counted, the CD4 / CD8 ratio was calculated, and the expression levels of IL-2, IL-6, and IL-1β were detected using an ELISA kit.

[0059] II. Experimental Results The results are as follows Figure 6 As shown, after drug withdrawal, the viral load in the ART treatment group gradually rebounded and returned to a level comparable to the control group by week 5. In contrast, the viral load in the Pom group fluctuated around the minimum detection line without a significant rebound. Meanwhile, the CD4 count in the Pom group... + T, CD8 + T, CD25 + The number of T cells remained high in both groups, and the CD4 / CD8 ratio was also higher than that in the ART group and the control group. The IL-2 expression level in the Pom group remained high after treatment, while the IL-2 expression levels in the ART group and the control group gradually decreased after drug withdrawal and were lower than those in the Pom group, which was also related to their CD4 expression levels. + The proportion of T cells gradually decreased after drug withdrawal, consistent with the trend. The expression levels of IL-6 and IL-1β in the Pom group were lower than those in the ART group and the control group. These results indicate that Pom can inhibit viral rebound in mice and increase CD4+ expression. + T, CD8 + T, CD25 + The number and function of T cells reduced the inflammatory response and promoted immune function reconstruction.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of Pomalidomide or its derivatives in the preparation of drugs for promoting long-term immune reconstitution in HIV patients.

2. The application according to claim 1, characterized in that, The Pomalidomide or its derivatives also include pharmaceutically acceptable salts, hydrates, isomers, solvates, or crystalline forms.

3. The application according to claim 1, characterized in that, The derivative is a functional compound obtained by chemically modifying the core structure of pomalidomide; Preferably, the derivatives include compounds as shown in formula (I) and formula (II); Equation (I) Formula (II).

4. The application according to claim 1, characterized in that, The concentration of Pomalidomide or its derivatives is 0.8 nM-100 μM; Preferably, the concentration of Pomalidomide or its derivative is 0.01 μM-100 μM; Preferably, the concentration of Pomalidomide or its derivative is 0.01 μM-10 μM.

5. The application according to claim 1, characterized in that, The pomalidomide or its derivatives significantly increase CD4 levels after HIV infection. + The number of T cells and the CD4 / CD8 ratio can promote immune reconstitution in HIV patients; Preferably, the promotion of long-term immune reconstitution in HIV patients is defined as the ability of Pomalidomide or its derivatives to significantly increase post-HIV infection CD4 levels for at least 5 weeks after discontinuation. + T cell count and CD4 / CD8 ratio.

6. Any of the following applications: 1) The use of Pomalidomide or its derivatives in the preparation of pharmaceutical compositions for promoting long-term immune reconstitution in HIV patients; 2) The use of Pomalidomide or its derivatives in the preparation of pharmaceutical formulations for promoting long-term immune remodeling in HIV patients.

7. The application according to claim 6, characterized in that, The pharmaceutical composition also includes other drugs that can be used to promote long-term immune reconstitution in HIV patients.

8. The application according to claim 6, characterized in that, The pharmaceutical preparation also includes pharmaceutically acceptable excipients; Preferably, the dosage form of the pharmaceutical preparation includes an oral dosage form, a parenteral dosage form, or a topical dosage form.

9. Use of Pomalidomide or its derivatives in the preparation of drugs for treating HIV infection.

10. The application according to claim 9, characterized in that, The Pomalidomide or its derivatives achieve their anti-HIV infection effect by inhibiting the replication of the HIV virus. Preferably, the inhibitory effect of the pomalidomide or its derivatives on HIV replication does not show significant rebound within at least 5 weeks after discontinuation of the drug.

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