Pharmaceutical composition for enhancing anti-melanoma effect of immune checkpoint inhibitor, preparation and application

Abstract

The invention relates to the technical field of traditional Chinese medicines, and provides a pharmaceutical composition for enhancing the anti-melanoma effect of an immune checkpoint inhibitor, a preparation and application. The pharmaceutical composition comprises a phenylethanoid glycoside extract and a total terpene extract in a mass ratio of (2-6): 1, the pharmaceutical preparation comprises the pharmaceutical composition and pharmaceutically acceptable auxiliary materials or pharmaceutically acceptable salts. The invention also provides an application of the pharmaceutical composition combined with an immune checkpoint inhibitor in preparation of drugs for treating melanoma. The immune checkpoint inhibitor comprises one or more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor and an LAG3 inhibitor. The pharmaceutical composition can improve the response rate of a PD-L1 inhibitor and enhance the anti-melanoma effect of an immune checkpoint inhibitor, and an efficient and low-toxicity combined scheme is provided for treating melanoma.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a pharmaceutical composition, preparation and application for enhancing the anti-melanoma effect of immune checkpoint inhibitors. Background Art

[0002] Melanoma is a malignant tumor that arises from melanocytes. It often develops in the skin, but can also be found in the eyes, leptomeninges, gastrointestinal tract, and oral, genital, and sinus mucosa. It is characterized by rapid spread, easy metastasis, poor prognosis, and a high mortality rate. When melanoma cells become invasive and begin to spread, treatment and prognosis are significantly impacted.

[0003] The occurrence of melanoma is related to many factors, including ultraviolet exposure, family history, genetic factors, and immune system suppression. Currently, the treatment of melanoma is based on surgery, combined with emerging therapies such as targeted and immune therapies. Drugs for the treatment of melanoma include interferon, interleukin-2, vemurafenib, dabrafenib, etc. These treatments have significantly improved the prognosis of some patients, but there are problems such as easy recurrence, easy development of drug resistance, large side effects, and insignificant efficacy in the late stage. In the field of tumor treatment, Chinese medicine monomer compounds, as single chemical components extracted and purified from traditional Chinese medicine, have shown broad application prospects due to their unique chemical structure and diverse biological activities. Although single Chinese medicine ingredients, such as betulinic acid, have certain anti-cancer activity, they have problems such as low bioavailability and narrow therapeutic window.

[0004] Tumor immune escape refers to the phenomenon in which tumor cells evade immune recognition and attack through various biological mechanisms, such as antigen loss and expression of immune checkpoint molecules, allowing them to survive, proliferate, and form tumors. PD-L1 is an immune checkpoint ligand widely expressed on the surface of tumor cells. By binding to the PD-1 receptor on T cells, it inhibits T cell proliferation, activation, and cytotoxicity, blocking the release of lytic enzymes and cytokines from T cells, reducing their ability to attack tumor cells. It also induces T cell exhaustion or apoptosis, allowing tumor cells to evade immune surveillance. PD-1 / PD-L1 inhibitors can block the binding of tumor cell PD-L1 and T cell PD-1, eliminating this immunosuppressive effect and relieving the suppression of T cell activity, allowing T cells to reactivate and restore their ability to recognize and kill tumor cells. Currently, PD-1 / PD-L1 inhibitors have become a cornerstone of cancer treatment. However, PD-L1 inhibitors have limited response rates (20%-30%) and are prone to drug resistance in melanoma, necessitating combination therapy for improved efficacy.

[0005] Traditional Chinese medicine ingredients reduce tumor cell resistance to immunotherapy by regulating the EMT process, metabolic reprogramming, and immune checkpoint molecules. They are often used in combination with surgery, radiotherapy, or immune checkpoint inhibitors to enhance tumor treatment efficacy and reduce side effects. Chinese patent publication number CN113577064A discloses that a combined pharmaceutical composition of rhein and berberine can significantly enhance the anti-tumor effect of immune checkpoint inhibitors and significantly prolong the survival of tumor-bearing mice. Chinese patent publication number CN118717782A discloses the use of a combination of ginsenoside CK and PI3K pathway inhibitors in the preparation of a drug for treating melanoma. However, research on using traditional Chinese medicine ingredients to improve the response rate and drug resistance of PD-L1 inhibitors and enhance the anti-tumor effect of immune checkpoint inhibitors is relatively lacking.

[0006] Therefore, the development of traditional Chinese medicine combination drugs that can improve the response rate of PD-L1 inhibitors, enhance the anti-melanoma effect of immune checkpoint inhibitors, and effectively treat melanoma remains a technical problem that needs to be solved urgently. Summary of the Invention

[0007] The purpose of the present invention is to provide a pharmaceutical composition, preparation and application for enhancing the anti-melanoma effect of immune checkpoint inhibitors. The pharmaceutical composition, preparation and application are combined with the immune checkpoint inhibitors to improve the response rate of the immune checkpoint inhibitors and enhance the anti-melanoma effect of the immune checkpoint inhibitors, thereby providing a high-efficiency and low-toxic combination regimen for the treatment of melanoma.

[0008] The embodiments of the present invention are achieved through the following technical solutions: The present invention provides a pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors, namely a composition of effective parts of Forsythia suspensa; The effective part composition of Forsythia suspensa comprises a phenylethanoid glycoside extract and a total terpene extract in a mass ratio of 2 to 6:1; The phenylethanoid glycoside extract includes forsythiaside A, and also includes at least one of forsythiaside E, forsythiaside B, forsythiaside H, forsythiaside I, and salidroside; The total terpene extract includes betulinic acid and at least one of ursolic acid and oleanolic acid.

[0009] The present invention also provides a pharmaceutical preparation for enhancing the anti-melanoma effect of immune checkpoint inhibitors, comprising the above-mentioned pharmaceutical composition and a pharmaceutically acceptable excipient or a pharmaceutically acceptable salt.

[0010] The present invention also provides a use of the above-mentioned pharmaceutical composition in combination with an immune checkpoint inhibitor in the preparation of a drug for treating melanoma.

[0011] Preferably, the immune checkpoint inhibitor includes one or more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor; preferably, the immune checkpoint inhibitor is a PD-L1 inhibitor; and the drug is a drug that improves the drug resistance of the PD-L1 inhibitor and increases the response rate of the PD-L1 inhibitor.

[0012] The technical solutions of the embodiments of the present invention have at least the following advantages and beneficial effects: The present invention significantly upregulates the expression of immune checkpoint inhibitor proteins, increases CD8+ T cell infiltration, and reshapes the tumor immune microenvironment through the effective part composition of Forsythia suspensa. That is, under the synergistic effect of the effective part composition of Forsythia suspensa and the immune checkpoint inhibitor, a closed-loop mechanism of "upregulating targets → enhancing blocking" can be formed, thereby enhancing the anti-melanoma effect of immune checkpoint inhibitors and improving their sensitivity and therapeutic response rate. In addition, the combination of the effective ingredient composition of Forsythia suspensa and immune checkpoint inhibitors can significantly downregulate the expression of TGF-β and IL-10 genes, upregulate the expression of IFN-γ and GZMB genes, improve the immune microenvironment, break immunosuppression, enhance immunotherapy sensitivity, reduce resistance to immune checkpoint inhibitors, alleviate melanoma growth, and prolong the growth period of mice with melanoma, showing better anti-melanoma effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 Schematic diagram of the effect of the active ingredient composition of Forsythia suspensa on the survival rate of melanoma cells (B16 / F10, A375, A2058); Figure 2 Schematic diagram of the effect of the active ingredient composition of Forsythia suspensa on the migration of melanoma cells (B16 / F10, A375, A2058); Figure 3 Schematic diagram of the effect of the active ingredient composition of Forsythia suspensa on apoptosis of melanoma cells (B16 / F10, A375, A2058); Figure 4 Schematic diagram showing the effect of the active ingredient composition of Forsythia suspensa on the tumor weight and volume of B16 / F10 melanoma mice; Figure 5Schematic diagram of the effect of the active ingredient composition of Forsythia suspensa on tumor tissues of B16 / F10 melanoma mice; Figure 6 This is a schematic diagram showing the effect of the Forsythia suspensa active ingredient composition of the present invention on the expression of PD-L1 in B16 / F10 melanoma mice; Figure 7 This is a schematic diagram showing the effect of the effective ingredient composition of Forsythia suspensa on the infiltration of CD8+ T cells in B16 / F10 melanoma-bearing mice; Figure 8 This is a schematic diagram showing the effect of the combination of the active ingredient composition of Forsythia suspensa of the present invention and a PD-L1 inhibitor on the tumor weight of B16 / F10 melanoma mice; Figure 9 This is a schematic diagram of the effects of the combination of the active ingredient composition of Forsythia suspensa of the present invention and a PD-L1 inhibitor on the expression of related genes in B16 / F10 melanoma mice. DETAILED DESCRIPTION

[0015] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0016] The following is a detailed description of a pharmaceutical composition, preparation, and application for enhancing the anti-melanoma effect of immune checkpoint inhibitors provided in an embodiment of the present invention.

[0017] The present invention provides a pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors, namely a composition of effective parts of Forsythia suspensa; The effective part composition of Forsythia suspensa comprises a phenylethanoid glycoside extract and a total terpene extract in a mass ratio of 2 to 6:1; The total content of phenylethanoid glycosides in the phenylethanoid glycoside extract is greater than 50%, and the total content of total terpenes in the total terpene extract is greater than 45%.

[0018] Preferably, the phenylethanoid glycoside extract includes forsythiaside A, and also includes at least one of forsythiaside E, forsythiaside B, forsythiaside H, forsythiaside I, and salidroside; the content of forsythiaside A in the phenylethanoid glycoside extract is not less than 30% of the phenylethanoid glycoside extract.

[0019] Preferably, the total terpene extract includes betulic acid and at least one of ursolic acid and oleanolic acid; the content of the betulic acid is not less than 35% of the total terpene extract.

[0020] Preferably, the mass ratio of the phenylethanoid glycoside extract to the total terpene extract is 3-5:1.

[0021] More preferably, the mass ratio of the phenylethanoid glycoside extract to the total terpene extract is 3:1.

[0022] Preferably, the preparation method of the effective fraction composition of Forsythia suspensa comprises the following steps: (1) After drying, crush the fruit of Forsythia suspensa and pass it through a 30-50 mesh sieve to obtain Forsythia suspensa powder; (2) Extracting the Forsythia suspensa powder with an alcohol solvent under reflux, and then purifying it with a polyamide resin, thereby obtaining the phenylethanoid glycoside extract; (3) After ultrasonic-assisted extraction of Forsythia suspensa powder, it was purified by macroporous resin to obtain the total terpene extract; (4) The phenylethanoid glycoside extract and the total terpene extract are combined and concentrated according to the mass ratio, and excipients are added to prepare a clinically applicable dosage form.

[0023] Preferably, in step (2), the preparation method of the phenylethanoid glycoside extract is as follows: adding Forsythia suspensa powder to 10-15 times the amount of 40-50% ethanol, extracting at least twice at 48-65°C for 1-1.5 hours each time, combining the extracts, filtering, concentrating, and re-filtering, and then purifying with polyamide resin. The purification conditions are: eluting with 12BV water and 8BV 30% ethanol at a flow rate of 1-3mL / min, respectively, collecting the 30% ethanol eluate, and concentrating to obtain the phenylethanoid glycoside extract.

[0024] Preferably, in step (3), the preparation method of the total terpene extract is as follows: adding Forsythia suspensa powder to 20-30 times the amount of 70-90% ethanol, ultrasonically extracting at least twice at 40-60°C, each time for 1-1.5 hours, combining the extracts, filtering, concentrating, and re-filtering, and then purifying with a macroporous resin. Specifically, D101 macroporous resin can be selected. The purification conditions are: first eluting with deionized water, then eluting with 70% ethanol solution at a flow rate of 1-3 mL / min, collecting the 70% ethanol eluate, and concentrating to obtain the total terpene extract.

[0025] The present invention also provides a pharmaceutical preparation for enhancing the anti-melanoma effect of immune checkpoint inhibitors, comprising the above-mentioned pharmaceutical composition and a pharmaceutically acceptable excipient or a pharmaceutically acceptable salt.

[0026] Preferably, the dosage forms of the pharmaceutical preparation include oral preparations, injections and external preparations.

[0027] Preferably, the oral preparations include but are not limited to tablets, granules, pills, powders, suspensions, capsules and syrups; the injections include but are not limited to injection solutions, powder injections and injectable fluids; the external preparations include but are not limited to aerosols, creams, powders, gels and transdermal patches. Furthermore, the excipients include but are not limited to one or more of excipients, lubricants, preservatives, diluents, sweeteners, adhesives, colorants, solubilizers and surfactants; optionally include: starch, sodium carboxymethyl starch, talc, polyethylene glycol, micropowdered silica gel, starch slurry (paste), methyl cellulose, microcrystalline cellulose, magnesium stearate, povidone, powdered sugar, syrup, lactose, sucrose, mannitol, benzoic acid, sodium benzoate, glycerin, menthol, etc.

[0028] The present invention also provides a use of the above-mentioned pharmaceutical composition in combination with an immune checkpoint inhibitor in the preparation of a drug for treating melanoma.

[0029] The immune checkpoint inhibitor includes one or more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a TIM3 inhibitor, and a LAG3 inhibitor; preferably, the immune checkpoint inhibitor is a PD-L1 inhibitor.

[0030] Preferably, the drug is a drug that improves the drug resistance of PD-L1 inhibitors and increases the response rate of PD-L1 inhibitors.

[0031] The present invention significantly upregulates the expression of immune checkpoint inhibitor proteins, increases CD8+ T cell infiltration, and reshapes the tumor immune microenvironment through the effective part composition of Forsythia suspensa. That is, under the synergistic effect of the effective part composition of Forsythia suspensa and the immune checkpoint inhibitor, a closed-loop mechanism of "upregulating targets → enhancing blocking" can be formed, thereby enhancing the anti-melanoma effect of immune checkpoint inhibitors and improving their sensitivity and therapeutic response rate. In addition, the combination of the effective ingredient composition of Forsythia suspensa and immune checkpoint inhibitors can significantly downregulate the expression of TGF-β and IL-10 genes, upregulate the expression of IFN-γ and GZMB genes, improve the immune microenvironment, break immunosuppression, enhance immunotherapy sensitivity, reduce resistance to immune checkpoint inhibitors, alleviate melanoma growth, and prolong the growth period of mice with melanoma, showing better anti-melanoma effects.

[0032] Example 1 This embodiment provides a method for preparing a tablet that enhances the anti-melanoma effect of an immune checkpoint inhibitor, comprising the following steps: (1) After drying, crush the fruit of Forsythia suspensa and pass it through a 40-mesh sieve to obtain Forsythia suspensa powder; (2) Forsythia powder was added to 10 times the amount of 40% ethanol and extracted at 55°C for 3 times, each time for 1 hour. The extracts were combined, filtered, concentrated, and filtered again, and then purified with polyamide resin. The purification conditions were: 12BV water and 8BV 30% ethanol were used for elution at a flow rate of 2mL / min, respectively. The 30% ethanol eluate was collected and concentrated to obtain the phenylethanoid glycoside extract. (3) Forsythia powder was added to 25 times the amount of 80% ethanol and ultrasonically extracted three times at 50°C for 62 min each time. The extracts were combined, filtered, concentrated, and filtered again. Then, the extracts were purified using a D101 macroporous resin. The purification conditions were as follows: elution with deionized water and 70% ethanol solution at a flow rate of 2.0 mL / min, respectively. The 70% ethanol eluate was collected and concentrated to obtain the total terpene extract. (4) Take 240 g of phenylethanoid glycoside extract and 80 g of total terpene extract and mix them evenly. Add 64 g of microcrystalline cellulose as an adsorbent and stir evenly to prevent caking. Dry under reduced pressure at 60°C until the moisture content is less than 5%. Grind and pass through an 80-mesh sieve to obtain a mixed powder. Add 7 g of magnesium stearate as a lubricant and mix evenly. Use a tablet press to press into tablets to obtain tablets.

[0033] Example 2 The difference between this embodiment and embodiment 1 is that this embodiment provides a cream for enhancing the anti-melanoma effect of immune checkpoint inhibitors. In step (4), after the phenylethanoid glycoside extract and the total terpene extract are evenly mixed, 20 g of vaseline, 20 g of octadecyl alcohol, 20 g of lanolin, and 30 g of stearic acid are added, and the mixture is put into a reaction tank and heated to dissolve. The mixture is evenly stirred at a high speed until it is emulsified, and the mixture is filled to obtain a traditional Chinese medicine cream.

[0034] Example 3 The difference between this embodiment and embodiment 1 is that the mass ratio of the phenylethanoid glycoside extract to the total terpene extract is 4:1.

[0035] Example 4 The difference between this embodiment and embodiment 1 is that the mass ratio of the phenylethanoid glycoside extract to the total terpene extract is 5:1.

[0036] Experimental Example 1 - Effect of the Composition of Effective Fractions of Forsythia suspensa on Melanoma in Vitro 1.1 Effect of the combination of effective parts of Forsythia suspensa on the survival rate of melanoma cells (B16 / F10, A375, A2058) Experimental method: B16 / F10, A375, and A2058 cells in the logarithmic growth phase were collected and 1×10 4Cells were seeded at a density of 100 μL / well in a 96-well plate and placed in an incubator. After 24 hours, the cells were allowed to adhere and the old culture medium was discarded. 100 μL of DMEM high-glucose culture medium containing 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 mg / ml of the tablets prepared in Example 1 was added to B16 / F10 cells, 100 μL of DMEM high-glucose culture medium containing 0.1, 0.5, 1, 1.5, and 2 mg / ml of the tablets prepared in Example 1 was added to A375 cells, and 100 μL of DMEM high-glucose culture medium containing 0.1, 0.5, 1, 1.5, 2, 3, and 4 mg / ml of the tablets prepared in Example 1 was added to A2058 cells. 100 μL of DMEM high-glucose culture medium containing 0.01% DMSO was added to the control group. Three replicate wells were set up for each group. After incubation at 37°C for 48 hours, the original culture medium was discarded. Add 10 μL of DMEM high-glucose medium containing CCK8 reagent to each well and incubate at 37°C for 2 h. Measure and record the absorbance (OD) at 450 nm using a microplate reader to calculate cell viability.

[0037] Cell viability (%) = OD of experimental group / OD of control group × 100%.

[0038] The results are as follows Figure 1 As shown, Figure 1 A, B, and C represent the survival rates of B16 / F10, A375, and A2058 cells, respectively. Figure 1 It can be seen that the tablet prepared in Example 1 of the present invention has an inhibitory effect on the IC of B16 / F10, A375 and A2058 cells after 48 hours of treatment. 50 The values ​​were 2.091 mg / ml, 0.9538 mg / ml and 1.209 mg / ml, respectively. It can be seen that the tablet prepared in Example 1 of the present invention exhibited concentration-dependent inhibition of the proliferation of B16 / F10, A2058 and A375 cells, and its IC50 values ​​were all small, indicating that the effective part composition of Forsythia suspensa had a good inhibitory effect on B16 / F10, A375 and A2058 cells.

[0039] 1.2 Effects of the combination of effective fractions of Forsythia suspensa on the migration of melanoma cells (B16 / F10, A375, A2058) Experimental method: 0.25% trypsin-EDTA digestion solution was used to enzymatically dissociate the adherent B16 / F10, A2058 and A375 cells, and 5×10 4Cells were seeded at a density of 100 μg / well in a 6-well plate, with three replicates per group. After 24 hours, when the cells were fully attached, a straight line was drawn in the center of each well using a 200 μl pipette tip. After replacing the culture medium, the cells were observed and photographed under a microscope. B16 / F10, A2058, and A375 cells were treated with a 0 μM blank control group and different doses (25 μM low dose group and 50 μM high dose group) of the tablet solution prepared in Example 1 for 48 hours. The cells were observed and photographed under a microscope. The scratch test results are shown in the figure. Figure 2 As shown, Figure 2 The migration rates of B16 / F10, A375 and A2058 cells were expressed in The results of the B16 / F10 cell scratch test are as follows Figure 2 As shown in the figure, after the B16 / F10 cells were treated with the effective fraction composition solution of Forsythia suspensa for 48 hours, the cell migration rates of the blank control group, low-dose group, and high-dose group were 45.73%, 27.90%, and 16.29%, respectively. The migration rates of the high-dose group and the low-dose group were significantly lower than those of the blank control group (P<0.0001). The results of the A375 cell scratch test are shown in the figure. Figure 2 As shown in the figure, after 48 hours of treatment of A375 cells, the cell migration rates of the blank control group, low-dose group, and high-dose group were 47.85%, 27.42%, and 6.84%, respectively. The migration rates of the high-dose group and low-dose group were significantly lower than those of the blank control group (P<0.0001). The results of the A2058 cell scratch test are shown in the figure. Figure 2 As shown, after 48 hours of treatment with the effective fraction composition solution of Forsythia suspensa, the cell migration rates of A2058 cells were 38.20%, 28.50%, and 7.54% in the blank control, low-dose, and high-dose groups, respectively. The migration rates of the high-dose and low-dose groups were significantly lower than those of the blank control group (P<0.01). The results of the cell scratch assay showed that compared with the blank control group, the horizontal migration distance of melanoma cells treated with the effective fraction composition solution of Forsythia suspensa was significantly shortened, and the horizontal migration ability was reduced in a dose-dependent manner, indicating that the effective fraction composition solution of Forsythia suspensa can inhibit the horizontal migration of melanoma cells.

[0040] 1.3 Effects of the effective fraction composition of Forsythia suspensa on apoptosis of melanoma cells (B16 / F10, A375, A2058) Experimental method: Digest the cells with EDTA-free trypsin, transfer to a centrifuge tube, and collect the cells by centrifugation at 1000g for 5 minutes. Gently wash the cells twice with pre-cooled PBS solution, add 1*Binding buffer working solution to the cells, and culture them overnight to a cell concentration of 1*10 6 / mL. The tablet solution prepared in Example 1 was added to B16 / F10, A2058, and A375 cells in a blank control group, a low-dose group of 25 μM, and a high-dose group of 50 μM, respectively. After incubation in a 37°C, 5% CO2 incubator for 24 hours, 100 μL was aspirated and placed in a new tube. 5 μL of Annexin V-FITC and 5 μl of PI were added. After mixing, the cells were incubated in the dark at room temperature for 15 minutes, and then smears were made. FITC and PI were observed using the blue and green channels of a fluorescence microscope, respectively, and images were collected.

[0041] The results of cell fluorescence experiments are as follows Figure 3 As shown, the FITC signal shows green fluorescence, representing phosphatidylserine (PS) externalization, indicating early apoptotic cells, and the PI signal shows red fluorescence, representing cell membrane damage, indicating late apoptotic or necrotic cells. Figure 3 A to C in the figure represent the apoptosis effects of B16 / F10, A375 and A2058, respectively. Figure 3 Results A to C in Figure 3 showed that after 24 hours of treatment of B16 / F10, A2058 and A375 cells with the effective part composition of Forsythia suspensa, the number of early apoptotic cells in the group treated with the effective part composition of Forsythia suspensa significantly increased, which was significantly different from that in the control group, indicating that the effective part composition of Forsythia suspensa induced early apoptosis of B16 / F10, A2058 and A375 cells, and the induction of apoptosis of B16 / F10, A2058 and A375 cells was concentration-dependent, indicating that Forsythia suspensa extract can trigger early apoptosis of melanoma cells, thereby exerting an anti-tumor mechanism.

[0042] Experimental Example 2 Effect of the Composition of Effective Fractions of Forsythia suspensa on Melanoma in Vivo 2.1 Model Construction and Grouping: Twenty SPF-grade C57BL / 6 female mice, 6 weeks old, weighing 18-20 g were used. Five mice were housed in individual cages with free access to water and food. The cages, bedding, drinking water, and food were sterilized. Melanoma cell suspension (B16 / F10) was prepared by suspending B16 / F10 cells in the logarithmic growth phase in serum-free DMEM and adjusting the concentration to 5 × 10 6Cells / mL were drawn from the cell suspension using a 1mL syringe and inoculated into each group. 0.1mL was inoculated into the subcutaneous tissue of the right axilla (forelimb) of each mouse to establish a mouse melanoma model. Mice were randomly divided into a blank control group, a cisplatin group, a low-dose group of the effective fraction composition of Forsythia suspensa, and a high-dose group, with 5 mice in each group. Three days after inoculation of the cell suspension, drug administration began, with daily dosing for 15 consecutive days. The blank control group received an equal volume of normal saline by gavage; the positive control group received an intraperitoneal injection of 2 mg / kg cisplatin; and the low-dose and high-dose groups received different doses of the tablets prepared in Example 1 (1g / kg and 2g / kg, using normal saline as the vehicle). Starting on the seventh day after cell inoculation, the long diameter (a) and short diameter (b) of the mouse tumors were measured every two days, and tumor volume was calculated (V = 1 / 2 × a × b²). For irregularly shaped tumors, a was measured as the longest diameter, and b was measured as the midpoint of the short diameter perpendicular to the longest diameter. After 18 days, the mice were sacrificed by cervical dislocation, and the tumors were removed and weighed. Inhibition rate = [(blank control group volume - experimental group volume) / control group volume] × 100%.

[0043] The results of the effect of the active ingredient combination of Forsythia suspensa on the tumor weight and volume of B16 / F10 melanoma mice are shown in Figure 4 , Figure 4 A shows the tumor mass results of B16 / F10 melanoma mice; Figure 4 B is the result of the inhibition rate of the active ingredient composition of Forsythia suspensa on mouse melanoma; Figure 4 C is the tumor volume result of B16 / F10 melanoma mice; Figure 4 As shown in A and C, after 15 days of administration of the low-dose group, high-dose group and positive control group of the effective fraction composition of Forsythia suspensa, the tumor weight and volume of mice were significantly different from those of the blank control group (P<0.01), indicating that the growth of melanoma was significantly inhibited. Figure 4 The tumor inhibition rates of the low-dose group, high-dose group and positive control group of the effective part composition of Forsythia shown in B were 30.93%, 47.38% and 52.88%, respectively. The tumor inhibition rate of the positive control group was significantly different from that of the low-dose group (P<0.05), and there was no significant difference in inhibition rate with that of the high-dose group (P>0.05), indicating that the treatment with the effective part composition of Forsythia can significantly delay the growth of melanoma, and the high-dose group has a significant inhibitory effect on melanoma growth.

[0044] 2.2 HE staining was used to evaluate the effect of the effective fraction composition of Forsythia suspensa on melanoma tissue in vivo.

[0045] Experimental method: After the tumor tissues of the blank control group, positive control group, high-dose group and low-dose group were separated, they were immediately placed in 4% paraformaldehyde fixative for 24 hours, then embedded in paraffin, sliced, preheated to 85℃, fixed on the slice rack of tumor tissue paraffin, and placed in the oven at 85℃ for 30 minutes; the baked slices were immediately placed in xylene twice, each time for 15 minutes, and then dewaxed in alcohol gradient, in order of 5 minutes in anhydrous ethanol, 5 minutes in 95% ethanol, 5 minutes in 80% ethanol, and 5 minutes in 70% ethanol. After rinsing with PBS, they were stained with hematoxylin for 1 minute, then rinsed with running water for 1 minute, and finally stained with eosin for 30 seconds. The excess dye was washed away with running water; the stained slices were passed through 70% ethanol for 10 seconds, 80% ethanol for 10 seconds, 95% ethanol for 30 seconds, anhydrous ethanol for 1 minute, xylene-I for 1 minute, and xylene-II for 1 minute. Dehydrate and make transparent for 1 minute; place the transparent sections in a dark box and let them air dry naturally. Then, use a yellow pipette tip to dip a small amount of neutral gum on the stained tissue, gently touch the cover slip to the slide, and then slowly cover it. The next day, take pictures under a microscope and record the results.

[0046] The results of the effect of the active ingredient combination of Forsythia suspensa on the tumor tissue of B16 / F10 melanoma mice are shown in Figure 5 ;Depend on Figure 5 It can be seen that the tumor cells in the blank group mice grew well, the tumor cells were of varying sizes, the cell nuclei were clear, the cells were arranged neatly and tightly, and were distributed in a nested manner; compared with the blank group, the number of tumor cells in the positive control group, low-dose group, and high-dose group mice decreased, the tumor cells ruptured and necrotic, and vacuoles appeared, among which the tumor cells in the high-dose group showed large areas of necrosis.

[0047] 2.3 Effect of the active fraction composition of Forsythia suspensa on PD-L1 expression in B16 / F10 melanoma-bearing mice Experimental method: Approximately 30 mg of tumor tissue was weighed from the blank control group, high-dose group, and low-dose treatment group. After washing with pre-chilled PBS, 300 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added to extract total protein, and protein quantification was performed using the standard BCA method. Electrophoresis was performed using a 10% SDS-PAGE gel, and then electrophoresis was transferred to a PVDF membrane. After blocking with 5% skim milk powder for two hours, the corresponding primary antibody (PD-L1 was diluted at 1:2000, GAPDH was diluted at 1:5000) was added and incubated at 4°C overnight. After washing three times with TBST, the secondary antibody was added and incubated at room temperature for 1.5 hours. After washing three times, ECL luminescent solution was added, and the bands were collected using a chemiluminescence imaging system. The relative expression of PD-L1 was calculated based on the internal reference protein (GAPDH) (grayscale value of the target band / grayscale value of the internal reference).

[0048] PD-L1 protein expression results are as follows Figure 6 As shown by Figure 6 The results showed that compared with the blank control group, the PD-L1 protein expression levels in the low-dose and high-dose groups of the Forsythia suspensa effective fraction composition were significantly increased (P < 0.01) in a concentration-dependent manner, indicating that the Forsythia suspensa effective fraction composition can significantly upregulate PD-L1 protein abundance. In individuals with high PD-L1 expression, PD-L1 inhibitors can relieve immunosuppression and reactivate T cell killing function. PD-L1 protein detection results indicate that the combination of the Forsythia suspensa effective fraction composition and PD-L1 inhibitors can significantly improve T cell killing efficiency and enhance their anti-melanoma effect by blocking more PD-L1 proteins. Combining with PD-L1 inhibitors can form a closed-loop mechanism of "upregulating the target → enhancing blockade," thereby improving the response rate to PD-L1 inhibitor therapy.

[0049] 2.4 Effect of the effective fraction composition of Forsythia suspensa on CD8+ T cell infiltration in B16 / F10 melanoma-bearing mice by flow cytometry Experimental Methods: Mice were dislocated from the neck and soaked in 75% alcohol for 2 minutes. Tumor tissue was then removed from the blank control, high-dose, and low-dose groups. After washing in pre-chilled PBS, the tumor tissue was placed in a dish and minced into 1-3 mm particles using ophthalmic scissors. The tissue was then added to 4 mL of DMEM medium containing DNase I, Collagenase IV, Hyaluronidase, and Dispase II (biosharp). The tube was transferred to a 15 mL centrifuge tube and digested at 37°C for 30 minutes. Digestion was terminated by adding 3 volumes of DMEM medium containing 10% FBS. The tube was filtered through a 70 μm cell strainer, and incompletely digested tissue was triturated using a 20 mL syringe rubber plunger. The tube was centrifuged at 350 g for 5 minutes, and the supernatant was discarded. The tube was then lysed with 4 mL of red blood cell lysis buffer at room temperature for 5 minutes. Lysis was terminated by adding 6 volumes of 2% FBS in PBS. The tube was filtered through a 40 μm cell strainer and centrifuged at 350 g for 5 minutes. The cells were resuspended for cell counting and viability testing to a cell concentration of 1 × 10 cells. 7 Cells were collected at a concentration of 100 μL / mL. Transfer 100 μL of cell suspension to a new centrifuge tube and add 1 μL of CD16 / 32 antibody. Incubate on ice for 10 minutes. Add 1 mL of flow cytometry staining buffer to terminate labeling and centrifuge for 5 minutes. Next, resuspend the cells in 100 μL of flow cytometry buffer and label CD8+ T cells with antibodies to CD45, CD3e, and CD8a. Incubate on ice in the dark for 40 minutes. Terminate labeling by adding 1 mL of flow cytometry buffer. Wash twice with pre-chilled PBS, resuspend in 300–500 μL of PBS, and analyze on an analyzer.

[0050] The results are as follows Figure 7 As shown by Figure 7The results showed that compared with the blank control group, the UR quadrant content in the low-dose and high-dose groups treated with the effective fraction composition of Forsythia suspensa significantly increased, indicating an increase in CD8+ T cell infiltration. Increased CD8+ T cell infiltration can reshape the tumor immune microenvironment, improve the anti-melanoma effect of PD-L1 inhibitors, and enhance the sensitivity and response rate of PD-L1 inhibitors. Flow cytometry results indicate that the effective fraction composition of Forsythia suspensa can improve the tumor immune microenvironment by promoting the enrichment of CD8+ T cells, thereby enhancing the sensitivity and response rate of PD-L1 inhibitors.

[0051] Experimental Example 3 Study on the synergistic effect of the effective fraction composition of Forsythia suspensa combined with PD-L1 inhibitors on the anti-proliferative effect of melanoma 3.1 Model construction and grouping Twenty SPF-grade C57BL / 6 female mice, 6 weeks old, weighing 18-20 g were used. Five mice were housed in individual cages with free access to water and food. The cages, bedding, drinking water, and food were sterilized. Melanoma cell suspensions were prepared by suspending B16 / F10 cells in the logarithmic growth phase in serum-free DMEM at a concentration of 5 × 10 6 The cell suspension was drawn with a 1 mL syringe and inoculated into each group. 0.1 mL was inoculated into the subcutaneous tissue of the right axilla (forelimb) of each mouse to establish a mouse melanoma model. Mice were randomly divided into a blank control group, a PD-L1 inhibitor group, a Forsythia suspensa effective fraction composition group, and a Forsythia suspensa effective fraction composition + PD-L1 inhibitor group (referred to as the composition + inhibitor group), with 5 mice in each group. Three days after inoculation of the cell suspension, drug administration began, with administration occurring once daily for 15 consecutive days. The blank control group received an equal volume of normal saline by intraperitoneal administration; the PD-L1 inhibitor group received an intraperitoneal injection of 2 mg / kg of the PD-L1 inhibitor; and the Forsythia suspensa effective fraction composition group received an intraperitoneal injection of 2 g / kg of the Forsythia suspensa effective fraction composition prepared in Example 3.

[0052] 3.2 Effects of the Forsythia suspensa active ingredient composition of the present invention combined with a PD-L1 inhibitor on tumor weight and survival of B16 / F10 melanoma mice Test Method: 18 days after administration, mice were sacrificed by cervical dislocation, and tumors were removed and weighed. Inhibition rate = [(blank control group weight - experimental group weight) / control group weight] × 100%.

[0053] The results of the effect of the combination of the active ingredient composition of Forsythia suspensa and PD-L1 inhibitor on the tumor weight of B16 / F10 melanoma mice are shown in Figure 8 ,in, Figure 8 A is the effect of the combination of the active ingredient composition of Forsythia suspensa of the present invention and a PD-L1 inhibitor on the tumor mass of B16 / F10 melanoma mice; Figure 8B is the melanoma inhibition rate of mice treated with the combination of the active ingredient composition of Forsythia suspensa of the present invention and a PD-L1 inhibitor; Figure 8 C is the effect of the combination of the active ingredient composition of Forsythia suspensa of the present invention and a PD-L1 inhibitor on the survival of B16 / F10 melanoma mice; Figure 8 As shown in the results of A, 15 days after administration of the effective part composition of Forsythia suspensa, the PD-L1 inhibitor group, and the effective part composition of Forsythia suspensa + PD-L1 inhibitor group, the tumor weight of the mice was significantly different from that of the blank control group (P<0.05), and the growth of melanoma was significantly inhibited. There was a significant difference between the PD-L1 inhibitor group and the effective part composition of Forsythia suspensa + PD-L1 inhibitor group (P<0.05). Figure 8 The tumor inhibition rates of the Forsythia suspensa effective part composition, PD-L1 inhibitor group and Forsythia suspensa effective part composition + PD-L1 inhibitor group shown in B were 41.68%, 57.74% and 80.32%, respectively. There were significant differences between the PD-L1 inhibitor group and the Forsythia suspensa effective part composition + PD-L1 inhibitor group (P<0.01). Figure 8 As shown in Figure C, the survival of mice treated with the effective fraction composition of Forsythia suspensa, the PD-L1 inhibitor, and the combination of the effective fraction composition of Forsythia suspensa and the PD-L1 inhibitor was prolonged, with mice in the combination of the effective fraction composition of Forsythia suspensa and the PD-L1 inhibitor having even longer survival. Tumor weight and survival test results showed that compared with the groups treated with the effective fraction composition of Forsythia suspensa and the PD-L1 inhibitor alone, the combination of the effective fraction composition of Forsythia suspensa and the PD-L1 inhibitor significantly prolonged the survival of mice, demonstrating a superior anti-melanoma effect, indicating that the effective fraction composition of Forsythia suspensa can enhance the anti-melanoma effect of the PD-L1 inhibitor.

[0054] 3.3 Effects of the combination of active ingredients from Forsythia suspensa and PD-L1 inhibitors on the expression of related genes in B16 / F10 melanoma-bearing mice Experimental Methods: Total RNA was extracted from melanoma tissue supernatant after lysis with lysis buffer using the Takara Mini BEST kit, and RNA concentration was measured using a micro-nucleic acid analyzer. Reverse transcription was performed using the Prime Script TMRT kit with gDNA Eraser to synthesize cDNA. The mixture (including cDNA, primers, TB Green Premix Ex Taq II, and ROX plus) was reacted in the Step One Plus Real-Time PCR System. Primer sequences are shown in Table 1.

[0055] Gene expression levels were measured using the Step One Plus Real-Time PCR System, using GAPDH as an internal control. -△△Ct Method for analysis.

[0056] Table 1 Primer sequence information

[0057] TGF-β is a key immunosuppressive factor in the tumor microenvironment, promoting the differentiation of regulatory T cells (Tregs), inhibiting effector T cell activity, and promoting immune escape. Downregulating TGF-β may disrupt the immunosuppressive state and enhance immune cell infiltration and cytotoxicity. IL-10 is an immunosuppressive cytokine whose excessive secretion promotes T cell exhaustion. Its downregulation may reduce the immunosuppressive phenotype of tumor-associated macrophages and restore anti-tumor immune responses. IFN-γ is a core cytokine that activates anti-tumor immune responses, promoting MHC-I expression on tumor cells and inhibiting regulatory T cell (Treg) activity. Its upregulation suggests that the combination regimen may activate the effector function of CD8+ T cells and disrupt tumor immune escape. GZMB is a key effector molecule released by cytotoxic T cells and NK cells that can directly induce tumor cell apoptosis. Increased GZMB expression after combined therapy indicates a significant enhancement of T cell cytotoxicity.

[0058] The results of the effect of the combination of the active ingredient composition of Forsythia suspensa and PD-L1 inhibitor on the expression of related genes in B16 / F10 melanoma mice are shown in Figure 9 ,in, Figure 9 A is the relative expression level of TGF-β; Figure 9 B is the relative expression level of IFN-γ; Figure 9 C is the relative expression level of GZMB; Figure 9 D is the relative expression level of IL-10; Figure 9 As shown in A, the Forsythia suspensa active ingredient composition, PD-L1 inhibitor and combination group (i.e., composition + inhibitor group) all significantly downregulated the expression of TGF-β gene (P<0.001), and the difference between the PD-L1 inhibitor and the combination group was significant (P<0.05), indicating that the Forsythia suspensa active ingredient composition can promote the anti-melanoma effect of PD-L1 inhibitor. TGF-β is a key immunosuppressive factor in the tumor microenvironment that can promote the differentiation of regulatory T cells (Treg), inhibit the activity of effector T cells, and promote immune escape. Down-regulating TGF-β levels indicates that the drug-treated group may break the immunosuppressive state and enhance the infiltration and killing function of immune cells. Figure 9As shown in Figure B, the combination of Forsythia suspensa active ingredient and PD-L1 inhibitor group upregulated the expression of IFN-γ gene, but there was no significant difference compared with the blank control group (P>0.05). The combination group significantly upregulated the expression of IFN-γ gene (P<0.001), and was significantly different from the PD-L1 inhibitor group (P<0.001). IFN-γ is a core cytokine that activates anti-tumor immune response, which can promote the expression of MHC-I in tumor cells and inhibit the activity of regulatory T cells (Treg). Its upregulation indicates that the combination regimen may activate the effector function of CD8+ T cells and break the tumor immune escape. Figure 9 As shown in Figure C, the combination of active ingredients from Forsythia suspensa, the PD-L1 inhibitor, and the combination group all significantly upregulated GZMB gene expression (P < 0.05). The difference between the PD-L1 inhibitor and combination groups was significant (P < 0.0001). GZMB is a key effector molecule released by cytotoxic T cells and NK cells that can directly induce tumor cell apoptosis. Increased GZMB expression after combined use indicates a significant enhancement of T cell cytotoxic activity. Figure 9 As shown in Figure D, the combination of Forsythia suspensa active ingredient, PD-L1 inhibitor and combination group significantly downregulated the expression of IL-10 gene (P<0.0001), and the difference between the PD-L1 inhibitor and combination groups was significant (P<0.05). IL-10 is an immunosuppressive cytokine, and excessive secretion can promote T cell exhaustion. Its downregulation may reduce the immunosuppressive phenotype of tumor-associated macrophages and restore anti-tumor immune response.

[0059] This suggests that PD-L1 inhibitors relieve T cell exhaustion by blocking PD-1 / PD-L1 signaling, but some patients have limited response due to insufficient effector T cell function. The simultaneous downregulation of TGF-β and IL-10 following combined administration of Forsythia suspensa components suggests that the combination of Forsythia suspensa active ingredient combination with PD-L1 inhibitors can improve the immune microenvironment, break down immunosuppression, and enable PD-L1 inhibitors to more effectively activate T cell responses. The synergistic upregulation of IFN-γ and GZMB may indicate that Forsythia suspensa components may enhance the efficiency of T cell reactivation by PD-L1 inhibitors by modulating γc family cytokine signaling (e.g., IL-2 sensitivity), thereby reshaping the immune microenvironment, reducing the risk of immune escape, enhancing immunotherapy sensitivity, and reducing PD-L1 inhibitor resistance.

[0060] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors, characterized in that: The pharmaceutical composition is a composition of effective parts of Forsythia suspensa, and comprises, by weight, a phenylethanoid glycoside extract and a total terpene extract in a mass ratio of 2 to 6:

1.

2. The pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors according to claim 1, characterized in that: The mass ratio of the phenylethanoid glycoside extract to the total terpene extract is 3 to 5:

1.

3. The pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors according to claim 1, characterized in that: The phenylethanoid glycoside extract includes forsythiaside A, and also includes at least one of forsythiaside E, forsythiaside B, forsythiaside H, forsythiaside I, and salidroside.

4. The pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors according to claim 3, characterized in that: The content of the forsythiaside A is not less than 30% of the phenylethanoid glycoside extract.

5. The pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors according to claim 1, characterized in that: The total terpene extract includes betulinic acid and at least one of ursolic acid and oleanolic acid.

6. The pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors according to claim 5, characterized in that: The content of betulinic acid is not less than 35% of the total terpene extract.

7. The pharmaceutical composition for enhancing the anti-melanoma effect of immune checkpoint inhibitors according to claim 1, characterized in that: The total content of phenylethanoid glycosides in the phenylethanoid glycoside extract is greater than 50%, and the total content of total terpenes in the total terpene extract is greater than 45%.

8. A pharmaceutical preparation for enhancing the anti-melanoma effect of immune checkpoint inhibitors, characterized in that: The pharmaceutical composition comprises the pharmaceutical composition according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient or a pharmaceutically acceptable salt.

9. Use of the pharmaceutical composition according to any one of claims 1 to 7 in combination with an immune checkpoint inhibitor in the preparation of a medicament for treating melanoma.

10. Use of the pharmaceutical composition according to claim 9 in combination with an immune checkpoint inhibitor in the preparation of a drug for treating melanoma, characterized in that: The immune checkpoint inhibitors include one or more of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, and LAG3 inhibitors.

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