Use of TTP488 in preparation of drug for inhibiting lymphocyte depletion

Abstract

Use of TTP488 in the preparation of a drug for inhibiting lymphocyte depletion. Disclosed is new use of TTP488 in inhibiting lymphocyte depletion, which provides a new research direction for improving the efficacy of tumor immune checkpoint inhibitors.

Description

Application of TTP488 in the preparation of drugs that inhibit lymphocyte depletion Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to the application of TTP488 in the preparation of drugs that inhibit lymphocyte depletion. Background Technology

[0002] The incidence and mortality rates of malignant tumors and severe infections are on the rise, becoming the second and third leading causes of death globally, respectively. One common characteristic of malignant tumors and severe infections is lymphocyte depletion, leading to persistent immunosuppression or tumor immune escape. Reducing lymphocyte depletion and enhancing lymphocyte vitality to promote the body's immune response helps improve overall survival. Because immunotherapy can enhance the body's immune capacity and has the potential for high specificity and low side effects, it shows great promise for future development.

[0003] Immune checkpoint inhibitors (ICIs) work by blocking the immunosuppressive ligand-receptor interactions involving CTLA-4 and PD-1. Immune checkpoint blockade (ICB), a representative of tumor immunotherapy, has revolutionized cancer treatment. While ICB treatment reduces major suppressor signals in T lymphocytes and enhances their potential T-cell-mediated anti-tumor capabilities, it is also accompanied by progressive exhaustion and death of tumor-reactive T cells, preventing them from exerting a sustained anti-tumor effect and thus impacting treatment efficacy.

[0004] For overall clinical treatment efficacy, it is necessary to improve the response rate and duration of the body's immune function to achieve better results. Therefore, specific small molecule inhibitors may not only inhibit key oncogenic signaling pathways but also maintain cell viability while enhancing lymphocyte response, thus playing a more durable role and serving as adjunctive therapy to existing immune checkpoint inhibitors. Current ICB therapy is only effective for a small number of cancer patients, and many patients cannot benefit from treatment long-term. Therefore, the scientific community urgently needs to find mechanisms to enhance immune cell viability and avoid immune cell depletion, thereby researching new target immune checkpoint inhibitors, combining immune checkpoint inhibitors with different targets, or combining immune checkpoint inhibitors with other treatment methods to enhance immune cell viability and reduce immune cell depletion.

[0005] Summary of the Invention

[0006] This application aims to at least solve one of the technical problems existing in the prior art. To this end, this application proposes the use of TTP488 in the preparation of a drug that inhibits lymphocyte depletion.

[0007] According to one aspect of this application, the use of TTP488 in the preparation of a drug for inhibiting lymphocyte depletion is proposed.

[0008] In some embodiments of this application, the TTP488 structure is shown in formula (I):

[0009] Its molecular weight is 532.116, and its chemical formula is C. 32 H 38 ClN3O2.

[0010] In some embodiments of this application, the lymphocytes include T lymphocytes and B lymphocytes.

[0011] In some embodiments of this application, the inhibition of lymphocyte depletion includes inhibiting lymphocyte death and / or inhibiting lymphopenia.

[0012] In some embodiments of this application, lymphocyte depletion includes lymphocyte depletion caused by severe infection.

[0013] In some embodiments of this application, TTP488 can be used alone or in combination with other drugs to enhance the efficacy of tumor immune checkpoint inhibitors.

[0014] In some embodiments of this application, the content of TTP488 in the drug is a therapeutically effective amount.

[0015] In some embodiments of this application, the content of TTP488 in the drug can be from 0.01% to 100%, for example, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%.

[0016] In some embodiments of this application, the concentration of TTP488 in the drug is from 2 mg / kg to 65 mg / kg.

[0017] In some embodiments of this application, the drug further includes pharmaceutically acceptable excipients.

[0018] In some embodiments of this application, the pharmaceutically acceptable excipients include at least one of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings.

[0019] In some embodiments of this application, the excipient includes water.

[0020] In some embodiments of this application, the filler includes at least one of starch and sucrose.

[0021] In some embodiments of this application, the adhesive includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

[0022] In some embodiments of this application, the wetting agent includes glycerin.

[0023] In some embodiments of this application, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.

[0024] In some embodiments of this application, the absorption enhancer includes a quaternary ammonium compound.

[0025] In some embodiments of this application, the surfactant includes hexadecyl alcohol.

[0026] In some embodiments of this application, the adsorbent carrier includes at least one of kaolin and soap clay.

[0027] In some embodiments of this application, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0028] In some embodiments of this application, the dosage form of the drug is powder, ointment, drops, gel, lozenge, granule, suspension, syrup, patch, capsule, spray, tablet, pill, injection, gel, or oral liquid.

[0029] According to some embodiments of this application, at least the following beneficial effects are achieved: This application discloses a new use of TTP488 in inhibiting lymphocyte depletion, providing a new research direction for improving the efficacy of tumor immune checkpoint inhibitors. Attached Figure Description

[0030] The present application will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0031] Figure 1 shows the flow cytometry results in Example 1 of this application. In this figure, Vehicle represents the C57 spleen cell control group (containing an equal volume of DMSO equal to 0.25 μg / mL TTP488), IFNγ represents the C57 spleen cell IFNγ (50 ng / mL) stimulation group, TTP488 represents the C57 spleen cell control group treated with TTP488 (final concentration 0.25 μg / mL), and IFNγ+TTP488 represents the C57 spleen cell IFNγ (50 ng / mL) stimulation group treated with TTP488. PI + Represents dead cells, PI - CD3-FITC, CD4-AF700, and CD8-PE represent live cells; CD19-APC / Cy7 represents B lymphocytes.

[0032] Figure 2 is a statistical chart of flow cytometry detection results in Example 1 of this application, where Vehicle represents the control group (containing an equal volume of DMSO as 0.25 μg / mL TTP488), T 0.25 This represents a TTP488 concentration of 0.25 μg / mL, I 50 This represents an IFNγ concentration of 50 ng / mL, PI + Represents dead cells, PI - Represents living cells;

[0033] Figure 3 shows the flow cytometry results in Example 2 of this application, where DMSO represents the control group (containing the same volume of DMSO as 60 mg / kg TTP488), LPS concentration is 8 mg / kg, T4, T20, T40 and T60 represent TTP488 concentrations of 4 mg / kg, 20 mg / kg, 40 mg / kg and 60 mg / kg, respectively, CD3-FITC represents T lymphocytes, and CD19-APC / Cy7 represents B lymphocytes;

[0034] Figure 4 shows the flow cytometry results in Example 2 of this application, where DMSO represents the control group (containing the same volume of DMSO as 60 mg / kg TTP488), LPS concentration is 8 mg / kg, T4, T20, T40 and T60 represent TTP488 concentrations of 4 mg / kg, 20 mg / kg, 40 mg / kg and 60 mg / kg respectively, and CD4-AF700 and CD8-PE represent T lymphocytes;

[0035] Figure 5 shows CD3 in Embodiment 2 of this application. +The statistical graph of T lymphocyte detection results shows that DMSO represents the control group (containing the same volume of DMSO as 60 mg / kg TTP488), LPS concentration is 8 mg / kg, and T4, T20, T40 and T60 represent TTP488 concentrations of 4 mg / kg, 20 mg / kg, 40 mg / kg and 60 mg / kg, respectively. ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0036] Figure 6 shows CD4 in Embodiment 2 of this application. + Statistical graph of T lymphocyte detection results, where DMSO represents the control group (containing the same volume of DMSO as 60 mg / kg TTP488), LPS concentration is 8 mg / kg, T4, T20, T40 and T60 represent TTP488 concentrations of 4 mg / kg, 20 mg / kg, 40 mg / kg and 60 mg / kg respectively, **** is p < 0.0001;

[0037] Figure 7 shows CD8 in Embodiment 2 of this application. + The statistical graph of T lymphocyte detection results shows that DMSO represents the control group (containing the same volume of DMSO as 60 mg / kg TTP488), LPS concentration is 8 mg / kg, and T4, T20, T40 and T60 represent TTP488 concentrations of 4 mg / kg, 20 mg / kg, 40 mg / kg and 60 mg / kg, respectively. ** indicates p < 0.01, **** indicates p < 0.0001, and ns indicates no significant difference.

[0038] Figure 8 shows CD19 in Embodiment 2 of this application. + The statistical graph of B lymphocyte detection results shows that DMSO represents the control group (containing the same volume of DMSO as 60 mg / kg TTP488), LPS concentration is 8 mg / kg, T4, T20, T40 and T60 represent TTP488 concentrations of 4 mg / kg, 20 mg / kg, 40 mg / kg and 60 mg / kg, respectively. * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001, and ns indicates no significant difference. Detailed Implementation

[0039] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0040] Example 1: Application of TTP488 in inhibiting lymphocyte exhaustion

[0041] This embodiment demonstrates the application of TTP488 in inhibiting lymphocyte exhaustion through in vitro experiments. The specific steps are as follows:

[0042] (1) C57 mice (6-8 weeks old, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) were euthanized by cervical dislocation, and spleens were aseptically removed. The spleen cells were ground and filtered through a 40μm sterile filter to prepare a single spleen cell suspension.

[0043] (2) Single spleen cell suspension was centrifuged and the cells were settled at 4℃, 500×g, for 5 minutes.

[0044] (3) Discard the supernatant after centrifugation, add 1× erythrocyte lysis buffer (5 mL / spleen), and lyse the erythrocytes at room temperature for 8 minutes;

[0045] (4) Wash the cells with 5 mL of ice-cold 1×PBS, centrifuge at 4°C, 500×g, for 5 minutes, and repeat the washing 3 times.

[0046] (5) Discard the washed PBS and resuspend the washed cells in 2 mL of 1640 complete culture medium (fetal bovine serum containing 10% inactivated complement + 1% penicillin / streptomycin + 10 μmol / L β-mercaptoethanol);

[0047] (6) Filter the resuspended cells using a 40μm filter screen;

[0048] (7) Cell counting and cell viability assessment with 0.4% trypan blue. Cell viability must be ≥85% before proceeding to the next step.

[0049] (8) Adjust the cell count to 1×10 6 300 μL per well was seeded into a 48-well plate;

[0050] (9) Splenic cells were stimulated with IFNγ (final concentration of 50 ng / mL) and lymphocyte death was observed. The cells were divided into groups: C57 spleen cell control group (containing DMSO with an equal volume of 0.25 μg / mL TTP488), C57 spleen cell IFNγ (50 ng / mL) stimulation group, C57 spleen cell blank control group treated with TTP488 (final concentration of 0.25 μg / mL), and C57 spleen cell IFNγ (50 ng / mL) stimulation group treated with TTP488 (final concentration of 0.25 μg / mL) (the cells were pretreated with TTP488 (final concentration of 0.25 μg / mL) for 1 h and then IFNγ (final concentration of 50 ng / mL) was added directly to the pretreated cell well plate).

[0051] (10) After culturing in a 37℃, 5% CO2, saturated humidity incubator for 48h, the depletion of spleen T and B lymphocytes was detected by flow cytometry (cells were treated according to the instructions of the PI kit and the instructions of the CD3, CD4, CD8, and CD19 flow cytometry antibodies and then detected by flow cytometer).

[0052] The experimental results are shown in Figures 1 and 2. As can be seen from the figures, TTP488 can reduce lymphocyte death.

[0053] Example 2: Validation of TTP488's Inhibition of Lymphocyte Exhaustion in a Severe Infection Model

[0054] This embodiment demonstrates the application of TTP488 in inhibiting lymphocyte exhaustion in a severely infected mouse model through in vivo animal experiments. The C57 mice used in this experiment were 6-8 weeks old and purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. The specific steps are as follows:

[0055] Experimental groups: C57 saline control group (containing the same volume of DMSO as the experimental group 60 mg / kg TTP488) and experimental group. The experimental groups are as follows: C57-TTP488 (60 mg / kg) group, C57-LPS (8 mg / kg) group, C57-LPS (8 mg / kg) + TTP488 (4 mg / kg) group, C57-LPS (8 mg / kg) + TTP488 (20 mg / kg) group, C57-LPS (8 mg / kg) + TTP488 (40 mg / kg) group, C57-LPS (8 mg / kg) + TTP488 (60 mg / kg) group (TTP488 stock solution (dissolved in DMSO, storage concentration of 100 mg / mL) was dissolved in saline and sonicated until no precipitate was visible before proceeding to the next step of the experiment). There were 3 mice in each group (7-week-old mice were used in this example).

[0056] (1) The mice were weighed and the control group mice were injected intraperitoneally with 100 μL of physiological saline (containing the same volume of DMSO as 60 mg / kg TTP488). The mice in each treatment group were injected intraperitoneally with 100 μL of TTP488 at doses of 4 mg / kg, 20 mg / kg, 40 mg / kg, and 60 mg / kg, respectively.

[0057] (2) One hour after intraperitoneal injection of TTP488, mice were given a non-lethal dose of lipopolysaccharide LPS (8 mg / kg) intraperitoneally according to their grouping.

[0058] (3) 72 hours after modeling, mouse spleens were harvested and ground to prepare single spleen cells (the sampling method was the same as in Example 1). Lymphocyte death was analyzed using the following method:

[0059] 1) The mice were euthanized by cervical dislocation, and the spleen was aseptically removed. The spleen was then ground and filtered through a 40μm sterile filter to prepare a single spleen cell suspension.

[0060] 2) Centrifuge the single spleen cell suspension to settle the cells. The centrifugation conditions are 4℃, 500×g, and 5 minutes.

[0061] 3) Discard the supernatant after centrifugation, add 1× erythrocyte lysis buffer (5 mL / spleen), and incubate at room temperature for 8 minutes to lyse the erythrocytes;

[0062] 4) Wash the cells with 5 mL of 1×PBS on ice, centrifuge at 4°C, 500×g, for 5 minutes, and repeat the washing 3 times.

[0063] 5) Discard the washed PBS and resuspend the washed cells in 2 mL of 1640 complete culture medium (fetal bovine serum containing 10% inactivated complement + 1% penicillin / streptomycin + 10 μmol / L β-mercaptoethanol);

[0064] 6) Filter the resuspended cells through a 40μm filter;

[0065] 7) Cell counting and assessment of cell viability using 0.4% trypan blue; cell viability must be ≥85% before proceeding to the next step.

[0066] 8) Adjust the cell count to 1×10⁻⁶. 6 300 μL per well was seeded into a 48-well plate;

[0067] 9) Stimulate spleen cells with IFNγ (50 ng / mL) to observe lymphocyte death in spleen cells;

[0068] 10) After culturing at 37℃, 5% CO2, and saturated humidity for 48 h, the depletion of spleen T and B lymphocytes was detected by flow cytometry (cells were treated according to the instructions of the PI kit and the instructions of the CD3, CD4, CD8, and CD19 flow cytometry antibodies, and then detected by flow cytometer).

[0069] The experimental results are shown in Figures 3 to 8. Animal experiments showed that TTP488 can reduce lymphocyte death.

[0070] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the protection of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

1. Use of TTP488 in the preparation of a drug for inhibiting lymphocyte exhaustion.

2. Use according to claim 1, characterized in that, The lymphocytes include T lymphocytes and B lymphocytes.

3. Use according to claim 1, characterized in that, The inhibiting lymphocyte exhaustion includes inhibiting lymphocyte death and / or inhibiting lymphocyte reduction.

4. Use according to claim 1, characterized in that, The lymphocyte exhaustion includes lymphocyte exhaustion caused by severe infection.

5. The use according to claim 1, characterized in that, TTP488 can be used alone or in combination with other drugs in the drug.

6. Use according to claim 1, characterized in that, The content of TTP488 in the drug is a content reaching a therapeutically effective amount. Preferably, the content of TTP488 in the drug can be 0.01% to 100%.

7. Use according to claim 1, characterized in that, The drug further includes a pharmaceutically acceptable excipient.

8. Use according to claim 7, characterized in that, The pharmaceutically acceptable excipient includes at least one of a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.

9. Use according to claim 8, characterized in that, The excipient includes water. And / or, the filler includes at least one of starch and sucrose. And / or, the binder includes at least one of a cellulose derivative, an alginate, a gelatin, and a polyvinylpyrrolidone. And / or, the humectant includes glycerol. And / or, the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate. And / or, the absorption promoter includes a quaternary ammonium compound. And / or, the surfactant includes cetyl alcohol. And / or, the adsorption carrier includes at least one of kaolin and soap clay. And / or, the lubricant includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

10. The use according to claim 1, characterized in that, The dosage form of the drug is powder, ointment, drop, gum, tablet, granule, suspension, syrup, patch, capsule, spray, tablet, pill, injection, gel, or oral liquid.

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