A tetrastigma hemsleyanum extract and its use in the preparation of a medicament for preventing and / or reversing t cell exhaustion

Abstract

The application discloses a Radix Tetrastigae extract and application thereof in preparation of medicines for preventing and / or reversing T cell exhaustion, and belongs to the technical field of biological medicines. The Radix Tetrastigae extract is prepared by ultrasonic extraction and drying after mixing Radix Tetrastigae tubers with ethanol. Experiments prove that the Radix Tetrastigae extract provided by the application can inhibit and / or reverse the expression of T cell exhaustion markers, increase the expression of IL-2, TNF-alpha, IFN-gamma, GZMB and Perforin, promote the immune response of exhausted T cells, can obviously enhance the killing ability of T cells to tumor cells, and the application is a traditional Chinese medicine extract, compared with artificially synthesized medicines, has a comprehensive action mechanism of multiple pathways and multiple targets, has better treatment effect, lower side effects and better safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a Radix Tetrastigme extract and application thereof in preparation of a medicine for preventing and / or reversing T cell exhaustion. BACKGROUND

[0002] T cell exhaustion is an effector state of T cell functional decline caused by long-term exposure to high levels of antigens, which is particularly common in the microenvironment of solid tumors and is promoted by many factors, and is a key factor of immune dysfunction in cancer patients. As the main effector cells of anti-tumor immunity in the tumor microenvironment, CD8+ T cells gradually lose their function under persistent tumor antigen stimulation, and eventually exhibit T cell exhaustion. Functionally exhausted T cells mainly exhibit increased expression levels of T cell surface inhibitory receptors such as PD-1 (programmed death-1), Tim-3 (T cell immunoglobulin and mucin domain-3), CTLA-4 (cytotoxic T-lymphocyte-associated protein-4), LAG-3 (lymphocyte activation gene 3 protein) and TIGIT (T Cell immunoreceptor with Ig And ITIM domains), and reduced ability to produce and secrete effector cytokines such as IL-2, IFN-γ and TNF-α. Given the important role of T cells in anti-tumor immunity, targeting T cell exhaustion has become a very important direction of tumor immunotherapy. In particular, the emergence of anti-PD-1, PD-L1 and other immune checkpoint inhibitors (ICIs) has brought new hope for clinical tumor treatment.

[0003] At present, in the process of clinical immunotherapy of tumors, mainly PD-1, PD-L1 monoclonal antibodies are used to block the interaction between tumor cell PD-L1 and immune cell PD-1, thereby promoting the anti-tumor effect of immune cells. Although these monoclonal antibodies have shown great potential in the field of tumor treatment, only about 20%-40% of patients are sensitive to immunotherapy in clinical practice. As an important strategy for improving T cell function from an endogenous perspective, there is less research and development of small molecule inhibitors targeting T cell exhaustion. Given the potential toxicity of artificially synthesized drugs, natural drugs represented by traditional Chinese medicine have become a research hotspot for new drugs for tumor immunotherapy due to their comprehensive action mechanism of multiple pathways and multiple targets and fewer side effects, and have good research and development prospects.

[0004] Tetrastigma hemsleyanum Diels et Gilg, a rare medicinal plant of grape family and cliff climber genus in China, is used as underground tuber or whole herb, which has the effects of clearing heat and detoxifying, dispelling wind and reducing phlegm, activating blood and relieving pain, and is used for treating snake bite, tonsillitis, lymph node tuberculosis, sprains and injuries, and high fever convulsions in children.

[0005] CN113144051A discloses an application of a purified product of Tetrastigma hemsleyanum in preparation of a chemotherapy drug for inhibiting pancreatic cancer, and verifies that the purified product of Tetrastigma hemsleyanum can inhibit pancreatic cancer, thereby providing a new therapeutic target for preparation of a chemotherapy drug for inhibiting pancreatic cancer, a drug for inhibiting pancreatic cancer, and a therapeutic drug. CN106520901A discloses a method for studying Tetrastigma hemsleyanum extract in improving human costimulatory T cell proliferation, but there is no report on whether the Tetrastigma hemsleyanum extract mainly containing flavonoids can regulate T cell exhaustion and promote anti-tumor effect. SUMMARY

[0006] The present application aims to provide a Tetrastigma hemsleyanum extract and an application thereof in preparation of a drug for preventing and / or reversing T cell exhaustion.

[0007] To achieve the above-mentioned object, the technical solution adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a Tetrastigma hemsleyanum extract prepared by mixing Tetrastigma hemsleyanum tubers with ethanol, ultrasonic extraction, and drying.

[0009] Preferably, the volume fraction of the ethanol is 75-85%, and more preferably 80%.

[0010] Preferably, the mass-volume ratio of the Tetrastigma hemsleyanum tubers to ethanol is 1:15-25, and more preferably 1:20.

[0011] Preferably, the ultrasonic extraction is performed at an ultrasonic power of 550-650 W for 20-40 min, and more preferably at an ultrasonic power of 600 W for 30 min.

[0012] Preferably, before the ultrasonic extraction, the Tetrastigma hemsleyanum tubers are dried at low temperature and crushed, and the low temperature is not more than 60℃.

[0013] In some embodiments, the extraction method of the Tetrastigma hemsleyanum extract is as follows:

[0014] (1) drying the Tetrastigma hemsleyanum tubers at low temperature, crushing, and obtaining medicinal powder;

[0015] (2) according to the ratio of 1:15-25 (W / V), 75-85v / v% ethanol is added to the powder and mixed, 550-650W ultrasonic extraction is carried out for 20-40min, the filter residue is filtered, the extraction is repeated for 3 times, the filtrate is combined, concentrated to constant weight at low temperature and reduced pressure, and freeze-dried to obtain the extract of Smilax china.

[0016] The low temperature in the application is not more than 60 DEG C, preferably 60 DEG C.

[0017] In the second aspect, the application provides the use of the aforementioned extract of Smilax china in the preparation of a drug for preventing and / or reversing T cell exhaustion.

[0018] The extract of Smilax china in the application can inhibit and / or reverse the expression of T cell exhaustion markers, preferably the T cell exhaustion markers are at least one of PD-1, TIGIT, LAG-3 and CTLA-4.

[0019] Preferably, the extract of Smilax china can increase the expression of at least one of IL-2, TNF-alpha, IFN-gamma, GZMB and Perforin.

[0020] The T cell exhaustion in the application is related to cancer. Preferably, the use is the use of the aforementioned extract of Smilax china in the preparation of a drug for tumor immunotherapy.

[0021] In the third aspect, the application provides a drug comprising the aforementioned extract of Smilax china and a pharmaceutically acceptable excipient.

[0022] The selection of the excipient in the application is determined according to the conventional dosage form in the art.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] (1) The experiment proves that the extract of Smilax china provided in the application can inhibit and / or reverse the expression of T cell exhaustion markers, increase the expression of IL-2, TNF-alpha, IFN-gamma, GZMB and Perforin, promote the immune response of exhausted T cells, and can significantly enhance the killing ability of T cells to tumor cells.

[0025] (2) The application is a traditional Chinese medicine extract, which has a more comprehensive action mechanism of multiple pathways and multiple targets compared with artificially synthesized drugs, has better treatment effect and lower side effects, and has good safety. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The flow cytometry detection and statistical results of the influence of the extract of Smilax china in Example 2 of the application on the exhausted T cell markers of peripheral blood PBMC of breast cancer.

[0027] Figure 2 Flow cytometry detection and statistical results of the effect of the extract of Tripterygium wilfordii Hook.f. in Example 2 of the present application on the effect cytokine secretion of breast cancer peripheral blood PBMC exhausted T cells.

[0028] Figure 3 Effect of the extract of Tripterygium wilfordii Hook.f. in Example 2 of the present application on the clonal formation of breast cancer peripheral blood PBMC exhausted T cells.

[0029] Figure 4 Effect of the extract of Tripterygium wilfordii Hook.f. in Example 3 of the present application on the expression level of exhausted marker of mouse CD8+ exhausted T cells.

[0030] Figure 5 Effect of the extract of Tripterygium wilfordii Hook.f. in Example 3 of the present application on the expression level of effector factor and toxic molecule in mouse CD8+ exhausted T cells.

[0031] Figure 6 Effect of the extract of Tripterygium wilfordii Hook.f. in Example 3 of the present application on the killing ability of mouse 4T1 tumor cells by mouse CD8+ exhausted T cells. DETAILED DESCRIPTION

[0032] The present application will be described in greater detail by way of specific embodiments, so that the technical solutions of the present application are more easily understood and mastered, but the present application is not limited thereto, and the described embodiments are only some of the embodiments of the present application, rather than all the embodiments.

[0033] The endpoints of the ranges and any numerical values included in the disclosure are not limited to the precise values stated. These ranges and values should be construed and interpreted as being inclusive of values adjacent to the recited ranges and / or values, unless the context clearly indicates otherwise. For numerical ranges having an upper and lower limit, the endpoints are included in the range. For ranges having an upper limit only, the upper limit is included in the range. For ranges having a lower limit only, the lower limit is included in the range. For ranges having no upper or lower limit, the range is inclusive of all values subsumed under the range.

[0034] The "medicament" described in the present application includes the aforementioned extract and pharmaceutically acceptable excipients. In specific embodiments, the extract described in the present application is provided in the medicament in an effective amount (e.g., a therapeutically effective amount).

[0035] The "pharmaceutically acceptable excipients" described in the present invention include inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coatings, sweetening, flavoring, and perfuming agents can also be present in the pharmaceutical compositions.

[0036] The "medicaments" described in the present invention can be prepared by any method known in the art of pharmacy. Generally, these methods of preparation include bringing the aforementioned extract (i.e., the first active ingredient) into association with a carrier or excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0037] The medicaments of the present invention can be prepared according to known methods, such as those set forth in the General Rules for Preparations of the Japanese Pharmacopoeia, 16th Edition, the United States Pharmacopoeia, and the European Pharmacopoeia, 9th Edition. Depending on the dosage form.

[0038] The first active ingredient, pharmaceutically acceptable excipients in the "medicaments" described in the present invention will vary depending on the identity, size, and / or condition of the subject being treated and further depending on the route of administration of the composition. The medicaments can comprise between 0.1% and 100% (w / w) of the first active ingredient.

[0039] The "treatment" described in the present invention means, unless otherwise indicated, reversing, alleviating, lessening, inhibiting progression of, or preventing the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition, as the case can be. The term "treatment" as used in the invention refers to the act of treating, as "treatment" is defined immediately above.

[0040] The "effective amount" described in the present invention means an amount sufficient to elicit the desired biological response. The effective amount of the active ingredients of the present invention can vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disorder being treated, the mode of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. The effective amount is the amount of the first active ingredient described in the present invention in a single dose. In certain embodiments, the effective amount is the combined amount of the active ingredients described in the present invention in multiple doses.

[0041] A "therapeutically effective amount" as used herein is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or to delay or minimize one or more symptoms associated with the disorder. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of a disorder, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount that is sufficient to treat any of the diseases or disorders set forth.

[0042] The term "subject" or "patient" as used herein is art-recognized and used interchangeably herein, and refers to a mammal, including a dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and most preferably, a human. The term does not pertain to a specific age or gender. Thus, adult and neonatal subjects, whether male or female, are encompassed.

[0043] All other embodiments that would be obvious to one of ordinary skill in the art based on the embodiments disclosed herein are intended to be within the scope of the present application. The experimental methods described in the following examples are routine methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

[0044] Example 1 Preparation of extract TFT from Radix Tetrastigme

[0045] Example 1-1

[0046] Dried Radix Tetrastigme tubers were purchased from Zhejiang Tongjun Tang Herbal Pieces Co., Ltd. (Place of production: Zhejiang). The tubers were dried in an oven at 60°C, and then ground after drying to constant weight. The tuber powder was accurately weighed, and 80 v / v% ethanol was added at a solid-liquid ratio of 1:20 (W / V) and mixed well. After 24 h of soaking, the mixture was extracted by heating and refluxing for 3 times, 60 min each time. The filtrates were combined and concentrated to constant weight at 60°C under reduced pressure. The extract TFT1 was obtained by freeze-drying.

[0047] Example 1-2

[0048] Dried Radix Tetrastigme tubers were purchased from Zhejiang Tongjun Tang Herbal Pieces Co., Ltd. (Place of production: Zhejiang). The tubers were dried in an oven at 60°C, and then ground after drying to constant weight. The tuber powder was accurately weighed, and 80 v / v% ethanol was added at a solid-liquid ratio of 1:20 (W / V) and mixed well. After 24 h of soaking, the mixture was extracted by heating and refluxing for 3 times, 60 min each time. The filtrates were combined and concentrated to constant weight at 60°C under reduced pressure. The extract TFT1 was obtained by freeze-drying.

[0049] Example 2 Effect of Radix Tetrastigme extract on T cell exhaustion in peripheral blood of breast cancer patients

[0050] (1) Isolation and culture of peripheral blood mononuclear cells: the peripheral blood white membrane layer of the first diagnosis breast cancer patients in Jiaxing City Second Hospital was separated, Ficoll separation liquid was purchased from Beijing Solaybao Technology Co., Ltd., then red blood cells were removed with red blood cell lysis solution and washed with PBS. The eluted PBMC was resuspended with RPMI 1640 medium containing 10% FBS and 100 U / mL rhIL-2, the density was adjusted to 1x10 6 6 / mL, then inoculated in 96-well plates, placed in a 37°C, 5% carbon dioxide incubator.

[0051] (2) Intervention of Tetrastigma hemsleyanum extract: PBMC of breast cancer patients were intervened with 0, 25 μg / mL TFT1 and 25 μg / mL TFT2, after 72 h of culture, the culture supernatant and cells were collected respectively.

[0052] (3) Flow cytometry was used to detect the expression of PD-1, LAG-3, TIGIT and other T cell exhaustion markers in CD3 + CD8 + T cells in PBMC after 72 h of culture and intervention.

[0053] The results are shown in Figure 1 , compared with the Control group, the content of PD-1, LAG-3 and TIGIT in CD8 + T cells in the TFT2 group had significant difference (P<0.05), while the content of PD-1, LAG-3 and TIGIT in CD8 + T cells in the TFT1 group had no significant difference (P>0.05), which indicated that Tetrastigma hemsleyanum extract TFT2 had good inhibition and reversal effect on the exhaustion of CD8 + T cells, while TFT1 had no obvious improvement effect on the exhaustion of CD8 + T cells.

[0054] (4) ELISA method was used to detect the concentration of IL-2, TNF-α and IFN-γ in cell culture supernatant.

[0055] The results are shown in Figure 2 , compared with the Control group, the concentration of TNF-α and IFN-γ in the supernatant of CD8 + T cells in the TFT2 group had significant difference (P<0.05), the concentration of IL-2 had no significant difference (P>0.05), while the concentration of TNF-α and IFN-γ in the supernatant of CD8 +The concentrations of IL-2, TNF-alpha and IFN-gamma in the T cell supernatant had no significant difference (P>0.05), and the results showed that the TFT2 extract of Radix Tetrastigme could promote the secretion of TNF-alpha and IFN-gamma in CD8+T cells, while TFT1 had no effect on promoting the secretion of TNF-alpha and IFN-gamma in CD8+T cells.

[0056] (5) Microscopically observe the cell clone formation and take photos.

[0057] The results are shown in Table 1. Figure 3 As shown in Table 1, compared with the Control group, the number of T cells in the TFT1 group and the TFT2 group was significantly increased (P<0.05), and the effect of the TFT2 group was better than that of the TFT1 group, which indicated that the TFT1 and TFT2 extracts of Radix Tetrastigme could significantly promote the proliferation and clone formation of T cells, and the effect of the TFT2 extract of Radix Tetrastigme was better than that of the TFT1 extract of Radix Tetrastigme.

[0058] Example 3 Effect of Radix Tetrastigme extract on CD8 + T cells of mice

[0059] (1) Lymphocyte culture: CTLL-2 mouse CD8 + T lymphocytes were cultured in RPMI 1640+1% Glutamax+100 U / mL mouse recombinant IL-2+10% FBS+1% P / S medium, and were passaged at a ratio of 1:2 to 1:3, and cells of passages 2-6 were used for subsequent experiments.

[0060] (2) Induction of lymphocyte immunosuppression: CTLL-2 cells in the logarithmic growth phase were activated with mouse anti-CD3 / CD28 antibodies, and 24 h later, 3x10 5 cells were inoculated in each well of a 12-well plate, and an immunosuppressive environment was simulated according to the method in the literature to culture and model. Specifically, 20 μM CGS-21680 (an adenosine selective receptor agonist) was used for induction culture, and the cells were induced and cultured for 72 h to simulate functionally exhausted T cells.

[0061] (3) Effect of Radix Tetrastigme extract on immunosuppressive cells: CTLL-2 cells in the logarithmic growth phase were inoculated in a 12-well plate, and were intervened with Radix Tetrastigme extract (25 μg / mL) for 2 h, and then 20 μM CGS-21680 was added to simulate an immunosuppressive environment.

[0062] (4) Detection of T cell exhaustion markers: After 72 h of intervention with Radix Tetrastigme extract, the cells in each group were collected, total RNA was extracted from the cells by TRIzol method, and the expression of T cell exhaustion markers PD-1, TIGIT and CTLA-4 was detected by RT-qPCR method.

[0063] (5) Results as shown in Figure 4 Figure 6, compared with the Control group, the expression levels of T cell exhaustion markers PD-1, TIGIT and CTLA-4 in the CGS-21680 group were significantly increased (P<0.05), indicating that the modeling of functional exhausted T cells was successful.

[0064] After 72h of intervention of Sanjieqing extract, compared with the CGS-21680 group, the expression levels of PD-1, TIGIT and CTLA-4 in the T cells in the CGS-21680+TFT2 group were significantly reduced (P<0.05), and the expression levels of PD-1 and CTLA-4 in the T cells in the CGS-21680+TFT1 group were significantly reduced (P<0.05). The results showed that Sanjieqing extract TFT2 could significantly reduce the expression levels of exhaustion-related markers PD-1, TIGIT and CTLA-4 in immunosuppressive T cells, and had a better effect on reversing T cell exhaustion than Sanjieqing extract TFT1.

[0065] (6) Detection of the expression levels of T cell functional effector factors: the mRNA expression levels of effector cytokines and toxic molecules such as IL-2, TNF-α, IFN-γ, GZMB and Perforin were detected by RT-qPCR.

[0066] Results as shown in Figure 5 Figure 7, compared with the Control group, the levels of IL-2 and GZMB in the T cells in the CGS-21680 group were significantly reduced (P<0.05), and after intervention of Sanjieqing extract, the levels of IL-2 and GZMB had significant differences compared with the CGS-21680 group (P<0.05); compared with the Control group, there was no significant difference in the levels of TNF-α, IFN-γ and Perforin in the T cells in the CGS-21680 group (P>0.05), and after intervention of Sanjieqing extract, the levels of TNF-α, IFN-γ and Perforin in the T cells in the TFT1 and TFT2 groups were significantly increased (P<0.05), and the effect of the TFT2 group was obviously better than that of the TFT1 group.

[0067] The results showed that Sanjieqing extract could significantly increase the expression levels of effector factors and toxic molecules in immunosuppressive T cells, promote the immune response of exhausted T cells, and the effect of TFT2 was obviously better than that of TFT1.

[0068] (7) Mouse 4T1 breast cancer cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and CTLL-2 cells were cultured in the same way as described above, and cells in logarithmic growth phase were used in subsequent experiments. CTLL-2 cells (effector) were intervened with trifolious knotweed extract TFT2 (25 μg / mL) and 20 μM CGS-21680 in advance, and cells were collected after 72 h of culture. CFSE-labeled 4T1 cells (target) were inoculated in a 24-well plate, and after overnight culture, CTLL-2 cells were added at an optimized effector: target (E:T) ratio of 2:1, and a tumor cell killing experiment was performed, after which PI staining was performed and fluorescence microscopy was performed.

[0069] Results are shown in Table 2, and the results show that the trifolious knotweed extract TFT2 of the present application can significantly enhance the killing ability of T cells on mouse 4T1 tumor cells. Figure 6

[0070] It should be finally pointed out that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application, and simple modifications or equivalent replacements of the technical solutions of the present application by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.​

Claims

1. An extract of Tetrastigma hemsleyanum for use in the manufacture of a medicament for the treatment of breast cancer associated with CD8 + T cell exhaustion, characterised in that, The extraction method of the extract of Smilax sieboldi is as follows: (1) drying the tuber of Smilax sieboldi at low temperature, crushing to obtain medicinal powder; (2) adding 75-85% (v / v) ethanol to the medicinal powder according to the solid-liquid ratio of 1:15-25, mixing uniformly, and extracting for 20-40 min under ultrasonic wave with the power of 550-650 W, filtering the residue, repeating the extraction for 3 times, combining the filtrates, concentrating to constant weight under low temperature and reduced pressure, and freeze-drying to obtain the extract of Smilax sieboldi; The low temperature in steps (1) and (2) is not more than 60 °C.

2. Use according to claim 1, characterized in that, The volume fraction of the ethanol in step (2) is 80%, and the solid-liquid ratio of the tuber of Smilax sieboldi to ethanol is 1:

20.

3. Use according to claim 1, characterized in that, The ultrasonic extraction in step (2) is performed at the power of 600 W for 30 min.

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