Drug compositions for treating cancer

Abstract

This invention provides an application of the traditional Chinese medicine compound Huagai San in anti-tumor treatment and enhancing the efficacy of PD-1 inhibitors. The anti-tumor composition consists of HGS and a PD-1 inhibitor. HGS and PD-1 inhibitors have a synergistic effect in inhibiting tumor growth. Combined use can reduce the concentration of HGS alone and achieve better anti-tumor effects while reducing toxic side effects.

Description

Technical Field

[0001] This invention belongs to the field of cancer treatment technology, specifically relating to a pharmaceutical composition including a PD-1 inhibitor and the traditional Chinese medicine compound Huagai San. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, posing a serious threat to human life and health. In recent years, tumor immunotherapy, represented by programmed death protein-1 (PD-1) inhibitors, has made groundbreaking progress. PD-1 inhibitors block the binding of PD-1 to its ligand PD-L1, thereby relieving the immunosuppression of T cells by the tumor microenvironment and reactivating the body's own anti-tumor immune response. They have shown significant clinical benefits in the treatment of various malignant tumors, such as non-small cell lung cancer and liver cancer, and have become one of the standard treatment methods.

[0003] However, PD-1 inhibitors have significant limitations in clinical application. First, their efficacy as monotherapy is generally low, with an objective response rate of only about 20%, and a large number of patients exhibit primary resistance. Second, some patients with good initial responses develop acquired resistance during treatment, leading to disease relapse. Furthermore, in patients with advanced cancer, their immune systems are often suppressed or exhausted, further limiting the efficacy of PD-1 inhibitors. Therefore, improving the response rate of PD-1 inhibitors and overcoming resistance have become core challenges that urgently need to be addressed in the field of tumor immunotherapy.

[0004] To overcome these limitations, combination therapy is considered a key strategy. Currently, the mainstream approach involves combining PD-1 inhibitors with chemotherapy, targeted therapy, radiotherapy, or other immunotherapies. While these strategies have improved overall response rates to some extent, they have also introduced new challenges. The most prominent of these is the additive effect of toxicities. Chemotherapy drugs themselves have broad cytotoxicity; when combined with PD-1 inhibitors, not only do the toxic side effects of traditional chemotherapy (such as bone marrow suppression and gastrointestinal reactions) persist, but the incidence and severity of immune-related adverse events (irAEs) also increase significantly, such as immune-related pneumonia, colitis, hepatitis, and endocrine disorders. This severely impacts patients' treatment tolerance and quality of life, and may even lead to treatment interruption, limiting its widespread clinical application.

[0005] Therefore, there is an urgent clinical need to find a new combination therapy that can effectively enhance the efficacy of PD-1 inhibitors, while also having good safety and being able to reverse or delay drug resistance. Summary of the Invention

[0006] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides an application of the traditional Chinese medicine compound Huagai San in anti-tumor treatment and enhancing the efficacy of PD-1 inhibitors. To date, no published literature or patents have reported the use of Huagai San in enhancing the efficacy of PD-1 inhibitors. The purpose of this invention is to provide a novel tumor treatment strategy with synergistic efficacy and controllable toxicity.

[0007] According to one aspect of the present invention, a pharmaceutical composition is provided, comprising a PD-1 inhibitor and the traditional Chinese medicine compound Huagai San.

[0008] Preferably, the Huagai Powder is made from the following raw materials in parts by weight: 10-30 parts of stir-fried perilla seeds, 10-30 parts of red poria cocos, 10-30 parts of roasted mulberry bark, 10-30 parts of tangerine peel, 10-30 parts of stir-fried bitter almond, 10-30 parts of ephedra, and 5-15 parts of stir-fried licorice.

[0009] Preferably, the Huagai Powder is made from the following raw materials in parts by weight: 20 parts of stir-fried perilla seeds, 20 parts of red poria cocos, 20 parts of roasted mulberry bark, 20 parts of tangerine peel, 20 parts of stir-fried bitter almond, 20 parts of ephedra, and 10 parts of stir-fried licorice.

[0010] Preferably, the PD-1 inhibitor is selected from one of pembrolizumab, nivolumab, and toripalimab.

[0011] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0012] Preferably, the pharmaceutical composition is prepared as an oral or non-oral formulation. The oral formulation is selected from capsules, tablets, oral liquids, granules, pills, powders, or ointments; the non-oral formulation is an injection.

[0013] According to another aspect of the invention, the use of the traditional Chinese medicine compound Huagai San and a PD-1 inhibitor in the preparation of a medicament for treating cancer is provided.

[0014] According to another aspect of the present invention, the use of the traditional Chinese medicine compound Huagai San in the preparation of a medicament for enhancing the antitumor efficacy of PD-1 inhibitors is provided.

[0015] Preferably, the cancer is a cancer that is resistant to or poorly responsive to PD-1 inhibitors.

[0016] Preferably, the cancer is lung cancer.

[0017] The combination of an effective amount of PD-1 inhibitor and the traditional Chinese medicine compound Huagai San in this invention has a synergistic anti-tumor growth effect. The combined use achieves a better anti-tumor effect while reducing the required concentration of Huagai San. In this invention, Huagai San alone inhibits cancer cell growth in a dose-dependent manner.

[0018] Compared with existing technologies, this invention has the following beneficial effects: This invention reveals for the first time that the combined use of Huagai San and PD-1 inhibitors has a synergistic anti-tumor effect. This combination regimen provides a novel solution for overcoming primary and acquired resistance to PD-1 inhibitors. Huagai San is a natural Chinese herbal compound with good safety and low toxicity. Under the experimental conditions of this invention, no significant increase in toxicity was observed due to the combined use. Compared with existing combination regimens such as "PD-1 inhibitor + chemotherapy," this invention significantly improves efficacy while potentially significantly reducing immune-related adverse events and the toxicity of traditional cytotoxic drugs, thereby improving patients' quality of life and treatment tolerance. Attached Figure Description

[0019] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0020] Figure 1 This is a graph showing the change in tumor volume of mice in each treatment group as a function of drug administration time in Example 1 of the present invention.

[0021] Figure 2 This is a graph showing the tumor weight of mice in each treatment group after drug treatment in Example 1 of the present invention.

[0022] Figure 3 This is a graph showing the change in tumor volume of mice in each treatment group as a function of drug administration time in Example 2 of the present invention.

[0023] Figure 4 This is a graph showing the change in tumor volume of mice in each treatment group as a function of drug administration time in Example 3 of the present invention.

[0024] Figure 5 This is a graph showing the tumor weight of mice in each treatment group after drug treatment in Example 3 of the present invention.

[0025] Figure 6 This is a graph showing the results of Kyoto Genetics and Genome Encyclopedia (KEGG) enrichment analysis of ribonucleic acid (RNA) sequencing of tumor tissues from mice in the control group and the high-dose Huagai San group of Example 1 of this invention.

[0026] Figure 7 The image shows the results of quantitative photopolymerase chain reaction (qPCR) of tumor tissues from mice in each treatment group in Example 1 of this invention.

[0027] Figure label: " indicates "p<0.05"; " indicates "p<0.01"; " indicates "p<0.001". Detailed Implementation

[0028] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0029] In the context of this invention, Ctrl refers to the blank control.

[0030] In the context of this invention, HGS refers to Huagai Powder, which is made from the following raw materials in parts by weight: 10-30 parts of stir-fried perilla seeds, 10-30 parts of red poria cocos, 10-30 parts of processed mulberry bark, 10-30 parts of tangerine peel, 10-30 parts of stir-fried bitter almond, 10-30 parts of ephedra, and 5-15 parts of stir-fried licorice. Specifically, Huagai Powder is made from the following raw materials in parts by weight: 20 parts of stir-fried perilla seeds, 20 parts of red poria cocos, 20 parts of processed mulberry bark, 20 parts of tangerine peel, 20 parts of stir-fried bitter almond, 20 parts of ephedra, and 10 parts of stir-fried licorice. For example, 3.81g of stir-fried perilla seeds, 3.81g of red poria cocos, 3.81g of processed mulberry bark, 3.81g of tangerine peel, 3.81g of stir-fried bitter almond, 3.81g of ephedra, and 1.91g of stir-fried licorice. The product is prepared into freeze-dried powder by water extraction and rotary evaporation concentration, and dissolved in double-distilled water before use.

[0031] In the context of this invention, Cis refers to cisplatin.

[0032] In the context of this invention, PD-1 inhibitor refers to a PD-1 inhibitor.

[0033] PD-1 inhibitors are a class of innovative anti-tumor immunotherapies that restore and enhance the body's immune system's ability to recognize and kill tumor cells by blocking the binding activity of PD-1, thereby relieving immunosuppression of T lymphocytes. PD-1, as an important component of the immune checkpoint, is mainly expressed on the surface of activated T cells. Its physiological function is to maintain immune tolerance and prevent excessive autoimmune responses. However, tumor tissues can inhibit T cell function by activating PD-1 signaling, leading to immune escape and tumor progression. PD-1 inhibitors bind to the PD-1 receptor, blocking its negative regulatory signals, restoring T cell proliferation and cytotoxic function, and effectively activating a strong anti-tumor immune response. Clinically, representative PD-1 inhibitors include pembrolizumab, nivolumab, and toripalimab, which have been widely used in the treatment of various malignant tumors such as non-small cell lung cancer, melanoma, and renal cell carcinoma. Clinical trial data show that PD-1 inhibitors can significantly prolong progression-free survival and overall survival, demonstrating good efficacy and tolerability. Nevertheless, some patients develop primary and acquired resistance, limiting the sustainability of efficacy. Current research focuses on exploring various combination therapy strategies to enhance efficacy.

[0034] Lung cancer is a disease characterized by overall deficiency and local excess, a "deficiency in the root and excess in the branch" syndrome, representing a local manifestation of a systemic disease. It arises from a deficiency of the body's vital energy (Qi), an imbalance of Yin and Yang, and dysfunction of the internal organs. Pathogenic factors then invade and accumulate in the meridians and organs, impairing the lung's function of dispersing and descending Qi, obstructing the flow of Qi, blood, and body fluids, leading to the accumulation of phlegm, dampness, and blood stasis in the internal organs, resulting in tumor formation. The growth of the tumor and the long-term stagnation of Qi, blood, and body fluids further deplete the body's vital energy. Therefore, early-stage lung cancer is primarily characterized by excesses such as phlegm, toxins, and blood stasis. If lung cancer is left untreated or progresses, pathogenic factors can invade the brain, erode bones, and spread to other organs, ultimately leading to incurable disease. Therefore, "strengthening the body's resistance" and "eliminating pathogenic factors" are both opposing and complementary, forming a dialectical unity. In the HGS formula, licorice and red poria cocos have the functions of invigorating qi and strengthening the spleen, as well as harmonizing the effects of other herbs, and are classified as tonifying herbs. Ephedra, bitter almond, perilla seed, tangerine peel, and mulberry bark are mainly used to dispel cold from the lungs, lower qi and relieve asthma, and resolve phlegm and stop coughing, and are classified as expelling pathogens. The rational combination of tonifying and expelling herbs can not only relieve symptoms such as cough, asthma, excessive phlegm, and chest tightness, but also help improve lung function, reflecting the "tonifying and expelling" approach to cancer treatment.

[0035] This invention provides a pharmaceutical composition comprising a PD-1 inhibitor and the traditional Chinese medicine compound Huagai San. By evaluating the antitumor efficacy of HGS and HGS combined with a PD-1 inhibitor, this invention demonstrates that both HGS and HGS combined with a PD-1 inhibitor possess good tumor-inhibiting effects. Furthermore, it demonstrates that the antitumor effect of low-dose HGS combined with a PD-1 inhibitor is superior to that of a single substance or HGS combined with cis, providing important guidance for the development of highly effective tumor-inducing drugs.

[0036] It should be noted that the animal experiments conducted in Examples 1, 2, and 3 of this invention were independent experimental batches carried out at different times. Due to the inherent individual biological differences of experimental animals, as well as uncontrollable differences in cell passage, inoculation, and subtle environmental fluctuations in each experiment, the tumor growth load in the blank control group in each batch of experiments showed reasonable fluctuations within a certain range, which is a normal phenomenon in in vivo pharmacodynamic studies. Nevertheless, in all independent experimental batches, the core conclusions verified by this invention—that Huagai San alone has a dose-dependent tumor-suppressing effect, and that the combined use of Huagai San and PD-1 inhibitors can produce a synergistic effect—were verified. The specific processes and results of each example will be described below.

[0037] Example 1: Inhibitory effect of different concentrations of HGS on tumor growth

[0038] Establishment of a subcutaneous xenograft model of lung cancer: Mouse lung cancer cells LLC-1 were passaged in vitro, and approximately 5x cells were subcutaneously injected into the right forelimb of C57BL / 6J mice. One LLC-1 cell was observed, and the tumor grew to 50 mm in size after approximately 7 days. 3 The tumor volume is calculated as follows: (a× (a and b are the longest and shortest axes of the tumor, respectively), the lung cancer-bearing mouse model was successfully established. LLC-1 cell line was purchased from the ATCC Collection Center and cultured in DMEM medium containing 10% fetal bovine serum at 37°C with 5% and 2 / 2 (a and b are the longest and shortest axes of the tumor, respectively). Culture under the specified conditions. When the cells reach 80-90% confluence, passage them using 0.25% trypsin.

[0039] Administration: Twenty tumor-bearing mice were randomly divided into four groups of five each. One group served as a blank control, one group received a low-dose HGS (1.70 g / kg), one group received a high-dose HGS (5.09 g / kg), and one group received a Cis (2 mg / kg). The control and HGS groups received the medication via gavage once daily, while the Cis group received an intraperitoneal injection every three days for 15 days. After 15 days of administration, all mice were sacrificed, and the tumor weight was measured. The results are as follows: Figures 1-2 As shown.

[0040] from Figures 1-2 It can be seen that, compared with the blank control group, the low-dose HGS group, the high-dose HGS group, and the Cis group all inhibited tumor growth. On day 15 after tumor administration, the tumor volume in the blank control group was 2457±488 mm. 3 ( Figure 1 , Figure 2 All data are mean ± SD (n=5), and the tumor volume in the Cis group was 1246 ± 170 mm. 3 The tumor volume in the low-dose HGS group was 1458 ± 424 mm. 3 The tumor volume in the high-dose HGS group was 987±179 mm. 3 ( Figure 1 On day 15, the tumor weight was as follows: Figure 2 As shown, the tumor weight in the blank control group was 3.62±0.20 g, the tumor weight in the Cis group was 1.9±0.38 g, the tumor weight in the low-dose HGS group was 2.71±0.61 g, and the tumor weight in the high-dose HGS group was 1.28±0.44 g.

[0041] Example 2: Inhibitory effect of HGS combined with PD-1 inhibitor on tumor growth

[0042] Establishment of a subcutaneous xenograft model of lung cancer: Mouse lung cancer cells LLC-1 were passaged in vitro, and approximately 5x cells were subcutaneously injected into the right forelimb of C57BL / 6J mice. One LLC-1 cell was observed, and the tumor grew to 50 mm in size after approximately 7 days. 3 The tumor volume is calculated as follows: (a× (a and b are the longest and shortest axes of the tumor, respectively) / 2, establishing a lung cancer tumor-bearing mouse model. Male C57BL / 6J mice, 4-6 weeks old, were housed in SPF-grade animals at a controlled temperature of 20-26°C, relative humidity of 40-70%, and a 12-hour light-12-hour dark cycle. The environment was kept free of specific pathogens, with ventilation maintained at 15 times per hour or more. Drinking water and cages were sterilized by high-temperature sterilization, and feed was provided free of charge.

[0043] Drug administration: Twenty tumor-bearing mice were randomly divided into four groups of five each. One group served as a blank control; one group received a low-dose HGS (1.70 g / kg); one group received a PD-1 inhibitor (10 mg / kg); and one group received a combination therapy (1.70 g / kg HGS + 10 mg / kg PD-1 inhibitor). The control group, HGS treatment group, and combination therapy group received the drug via gavage once daily for 15 days. The PD-1 inhibitor group and combination therapy group received an intraperitoneal injection of the PD-1 inhibitor every three days. After 15 days of administration, all mice were sacrificed, and the tumor weight was measured. The results are as follows: Figures 6-7 As shown. The PD-1 inhibitor used is toripalimab.

[0044] from Figure 3 It can be seen that, compared with the blank control group, the low-dose HGS group, the PD-1 inhibitor group, and the combination therapy group all inhibited tumor growth. On day 15 after tumor administration, the tumor volume in the blank control group was 2535±321 mm. 3 ( Figure 3 All data are mean ± SD (n=5). The tumor volume in the PD-1 inhibitor group was 1850 ± 471 mm. 3 The tumor volume in the low-dose HGS group was 1625±221 mm. 3 The tumor volume in the combination therapy group was 903±231 mm. 3 ( Figure 3 This shows that the combination therapy group used only 1 / 3 of the HGS dose, yet achieved a superior effect compared to high-dose HGS monotherapy. Based on volume, the tumor inhibition rates of each group were calculated: the average volume of the control group was 2535 mm³, the inhibition rate of the PD-1 inhibitor group was (2535-1850) / 2535 = 685 / 2535 ≈ 27.0%, the inhibition rate of the low-dose HGS group was (2535-1625) / 2535 = 910 / 2535 ≈ 35.9%, and the inhibition rate of the combination therapy group was (2535-903) / 2535 = 1632 / 2535 ≈ 64.4%. The synergy index (SI) was calculated as 64.4% / (27.0%+35.9%) ≈ 1.02. This finding provides a new strategy and experimental basis for developing highly effective and low-toxicity tumor immunotherapy combination therapies.

[0045] Example 3: Inhibitory effects of HGS combined with PD-1 inhibitor and HGS combined with Cis on tumor growth

[0046] Establishment of a subcutaneous xenograft model of lung cancer: Mouse lung cancer cells LLC-1 were passaged in vitro, and approximately 5x cells were subcutaneously injected into the right forelimb of C57BL / 6J mice. One LLC-1 cell was observed, and the tumor grew to 50 mm in size after approximately 7 days. 3 The tumor volume is calculated as follows: (a× (a and b are the longest and shortest axes of the tumor, respectively) / 2, establishing a lung cancer tumor-bearing mouse model. Male C57BL / 6J mice, 4-6 weeks old, were housed in SPF-grade animals at a controlled temperature of 20-26°C, relative humidity of 40-70%, and a 12-hour light-12-hour dark cycle. The environment was kept free of specific pathogens, with ventilation maintained at 15 times per hour or more. Drinking water and cages were sterilized by high-temperature sterilization, and feed was provided free of charge.

[0047] Drug administration: Fifteen tumor-bearing mice were randomly divided into three groups of five each. One group served as a blank control; one group received a combination of low-dose HGS (1.70 g / kg) and a PD-1 inhibitor (10 mg / kg); and one group received a combination of low-dose HGS (1.70 g / kg) and Cis (2 mg / kg). The control group, the HGS and PD-1 inhibitor combination group, and the HGS and Cis combination group received the drug via gavage once daily for 15 days. The HGS and PD-1 inhibitor combination group received an intraperitoneal injection of the PD-1 inhibitor every three days. The HGS and Cis combination group received an intraperitoneal injection of Cis every three days. After 15 days of administration, all mice were sacrificed, and the tumor weight was measured. Toripalimab was used as the PD-1 inhibitor.

[0048] from Figure 4 It can be seen that, compared with the blank control group, both the HGS combined with PD-1 inhibitor group and the HGS combined with Cis group showed inhibitory effects on tumor growth. On day 15 after tumor administration, the tumor volume in the blank control group was 2412±349 mm. 3 ( Figure 4 All data are mean ± SD (n=5). The tumor volume in the HGS and Cis combined group was 1561 ± 85 mm. 3 The tumor volume in the HGS combined with PD-1 inhibitor group was 837±260 mm. 3 ( Figure 4 On day 15, the tumor weight was as follows: Figure 5 As shown, the tumor weight in the blank control group was 4.07±0.76 g, the tumor weight in the HGS and Cis combination group was 1.95±0.30 g, and the tumor weight in the HGS and PD-1 inhibitor combination group was 1.37±0.26 g.

[0049] Mechanism analysis

[0050] Enrichment analysis was performed on the Kyoto Genetic Encyclopedia (KEGG) sequences of tumor tissues from mice in the control group and the high-dose Huagai San group of Example 1 of the present invention, and quantitative photopolymerase chain reaction (qPCR) was performed on the tumor tissues of mice in each treatment group of Example 1. Figure 6 KEGG enrichment analysis showed that, compared with the blank control group, the high-dose HGS group significantly enriched the following pathways, including: antigen processing and presentation; Th1 and Th2 cell differentiation; cell adhesion molecules; Th17 cell differentiation; PD-L1 expression and the PD-1 cancer checkpoint pathway; T cell receptor signaling pathway; cytokine-cytokine receptor interaction; phagosomes; natural killer cell-mediated cytotoxicity; and chemokine signaling pathways. PD-L1 expression and the PD-1 cancer checkpoint pathway were particularly prominent, and this was further validated. Figure 7 The qPCR results can verify Figure 6 KEGG enrichment analysis was performed. Compared with the blank control group, the high-dose HGS group significantly upregulated the mRNA expression levels of PD-L1 and PD-1, further demonstrating that HGS treatment resulted in a strong immunosuppressive signal. At this point, blocking this checkpoint with a PD-1 inhibitor and reactivating the T-cell immune response is a reasonable and effective treatment strategy.

[0051] KEGG enrichment analysis and qPCR results together indicate that HGS treatment activates the tumor immune microenvironment through multiple pathways, leading to increased T cell infiltration and activation (e.g., (Cell proliferation and activation). As a negative feedback regulatory mechanism of the body, activated T cells and tumor cells compensatorily upregulate the expression of PD-L1 and PD-1 in an attempt to suppress excessive immune responses, which ironically leads to a stronger state of immunosuppression. At this time, the combined use of PD-1 inhibitors can precisely block this compensatory activation of immune checkpoints, thereby maximizing the release of the anti-tumor activity of T cells activated by HGS. Therefore, HGS pretreatment creates a more favorable immune environment for PD-1 inhibitors, and the sequential combination strategy produces unexpected synergistic therapeutic effects.

[0052] Examples 1, 2, and 3 above represent multiple experimental batches conducted independently at different times. Those skilled in the art should understand that in in vivo experiments based on live animals, due to inherent individual biological differences in experimental animals, complex factors such as tumor cell inoculation status, and microenvironment, fluctuations in the baseline tumor growth of the blank control group in different batches of experiments are normal. However, in all independent experiments, the core findings of this invention were consistently confirmed: Huagai San monotherapy effectively inhibits tumor growth in a dose-dependent manner (as shown in Example 1); the combination of Huagai San and a PD-1 inhibitor significantly outperformed either monotherapy or the combination of HGS and Cis in antitumor therapy, demonstrating a clear synergistic effect (as shown in Examples 2 and 3).

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of the traditional Chinese medicine compound Huagai San in combination with a PD-1 inhibitor in the preparation of a drug for the treatment of non-small cell lung cancer, wherein the traditional Chinese medicine compound Huagai San is made from the following raw materials in parts by weight: 20 parts of stir-fried perilla seed, 20 parts of red poria cocos, 20 parts of roasted mulberry bark, 20 parts of tangerine peel, 20 parts of stir-fried bitter almond, 20 parts of ephedra, and 10 parts of stir-fried licorice; the dosage of the traditional Chinese medicine compound Huagai San is 1.70 g / kg, and the dosage of the PD-1 inhibitor is 10 mg / kg; the PD-1 inhibitor is toripalimab.

2. The use according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that, The traditional Chinese medicine compound Huagai San is prepared into an oral preparation, which is selected from capsules, tablets, oral liquids, granules, pills, powders or ointments; the PD-1 inhibitor is an injection.

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