Application of skull pill in preparation of drugs for treating lung cancer

Abstract

The application relates to the technical field of new drug use, and discloses application of Guanfupill in preparation of a drug for treating lung cancer, wherein 1793 kinds of compounds in Guanfupill are identified through component detection of Guanfupill, blood components are studied, and the blood components are combined with cisplatin to achieve tumor inhibition and synergistic effect. In conclusion, the technical scheme expands the drug for treating craniocerebral injury to the treatment of lung cancer, Guanfupill realizes the treatment of lung cancer through the synergistic effect of "multi-component-multi-target", provides a scientific basis for quality control, clinical application and further development of the action field of Guanfupill, and provides a reference paradigm for mechanism research on the treatment of lung cancer by traditional Chinese medicine.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of new uses of drugs, and particularly relates to application of Lufu Pills in preparation of a drug for treating lung cancer. BACKGROUND

[0002] According to the global cancer statistics report released by the International Agency for Research on Cancer (IARC) of the World Health Organization in 2022, the global incidence and mortality rate of lung cancer continues to rank first among all malignant tumors, with about 2.5 million new cases each year, accounting for 11.6% of the total number of new cases of global malignant tumors; 1.8 million deaths each year, accounting for 18.4% of the total number of global malignant tumor deaths, and the disease burden is extremely heavy.

[0003] With the innovation of medical technology, lung cancer treatment has moved from the traditional "one-size-fits-all" model to the era of precision and combined treatment, such as the combination of immune checkpoint inhibitors and chemotherapy, double-target inhibitor combination therapy and other schemes, which has significantly improved the prognosis of some patients. However, it is necessary to objectively recognize that the current various treatment methods still have limitations that cannot be ignored: surgical treatment as a radical means for early non-small cell lung cancer (stage I-II) depends on early diagnosis, and most patients are in locally advanced or advanced stage when diagnosed, losing the opportunity for surgery; chemotherapy can kill cancer cells through systemic administration, but its "enemy and me" mechanism is easy to cause serious side effects, such as nausea and vomiting (incidence rate over 70%), Ⅲ-Ⅳ degree myelosuppression (incidence rate about 30%) and neurotoxicity, resulting in nearly 20% of patients discontinuing treatment due to intolerance; radiotherapy technology (such as stereotactic radiotherapy) can accurately focus on tumors, but advanced equipment such as protons and heavy ions is scarce, and it is difficult for primary hospitals to carry out, and the incidence of normal tissue damage such as radiation pneumonitis and esophagitis still reaches 5%-15%; targeted therapy is only suitable for patients with clear driver mutations (such as EGFR and ALK mutations), but such patients only account for 30%-40% of non-small cell lung cancer, and drug resistance mutations are likely to occur after 1-2 years of treatment; although immunotherapy can activate the immune system to achieve long-term survival, only 20%-40% of patients can produce effective response, and PD-L1 negative or low expression has limited benefits, and although the incidence of immune-related adverse reactions (such as myocarditis and colitis) is low, the risk of death is high.

[0004] Lufu Pills (LFP) is a hospital preparation of Chongqing Municipal Hospital of Traditional Chinese Medicine, which is composed of eight traditional Chinese medicinal materials, i.e., cow-bezoar, borneol, Strychnos, Acorus, Yunnan rhizoma, Panax notoginseng, Astragalus and licorice, and has the effects of clearing heat and resolving toxins, resolving phlegm and removing mass, and refreshing brain and relieving pain, and is used for treating brain glioma. Clinical tests have proved that Lufu Pills can relieve neurological symptoms of patients, inhibit tumor progression and prolong survival of patients. However, as a complex traditional Chinese medicine preparation, Lufu Pills has problems such as unknown components and unclear action targets, which seriously limit the clinical application.

[0005] Traditional Chinese medicine has a history of thousands of years of clinical application in China. From the theory of "supporting the healthy and eliminating the pathogenic" proposed in Huangdi Neijing to the syndrome differentiation and treatment system of Shanghan Zabing Lun, its efficacy has been fully verified in the conditioning of chronic diseases and the adjuvant treatment of difficult diseases. Although there is no modern medical term "cancer" in ancient Chinese medical books, the diagnosis and treatment experience of similar diseases such as "Xianwu" and "Ji Jufen" provides a theoretical basis for modern Chinese medicine anti-tumor research. In recent years, a large number of basic researches have confirmed that many Chinese medicines and their active ingredients (such as artemisinin derivatives, ginsenoside Rg3, curcumin, etc.) have significant anti-tumor potential and can play a role through inhibiting tumor cell proliferation, inducing tumor cell apoptosis, regulating tumor angiogenesis, etc.

[0006] It should be noted that cancer is not a local lesion of a single organ, but a complex systemic disease involving multiple factors, multiple pathways and multiple stages: its occurrence and development is not only related to the infiltration of immune suppressor cells (such as regulatory T cells and myeloid suppressor cells) in the tumor microenvironment, but also closely related to the decline of cell repair ability caused by aging, metabolic disorders (such as insulin resistance caused by obesity), nutritional depletion caused by cancer cachexia, cell cycle regulation affected by circadian rhythm disorders, interaction between nervous system and tumor (such as neurotransmitters promoting tumor invasion), metastasis risk caused by tumor-related thrombosis, and immune regulation abnormalities caused by imbalance of intestinal microbiome. This complexity determines that single-pathway targeted therapy is difficult to achieve complete cure, while the action characteristics of traditional Chinese medicine "multiple components-multiple targets-multiple pathways" are exactly matched with the systemic regulation needs of cancer, for example, a certain traditional Chinese medicine compound can regulate immune function, inhibit tumor angiogenesis and improve metabolic disorders. However, the complexity of traditional Chinese medicine components (a single traditional Chinese medicine contains dozens to hundreds of chemical components) and the diversity of action targets (the same component may act on multiple signaling pathways) also bring major challenges to the modernization research of traditional Chinese medicine. SUMMARY

[0007] The application intends to provide the application of Guanfu Pill in the preparation of a drug for treating lung cancer, aiming to expand the application field of Guanfu Pill and provide a new path for the adjuvant treatment of lung cancer.

[0008] To achieve the above-mentioned purpose, the application adopts the following technical scheme: the application of Guanfu Pill in the preparation of a drug for treating lung cancer.

[0009] Preferably, as an improvement, Guanfu Pill comprises the following raw materials in mass parts: ox gallstone 20-30 parts, borneol 1.0-1.5 parts, Strychnos 15-20 parts, Acorus 80-90 parts, Yunnan rhizoma 120-150 parts, Sanchi 120-150 parts, Astragalus 180-200 parts, and licorice 180-200 parts.

[0010] Preferably, as an improvement, the dosage form of Guanfu Pill is pill.

[0011] Preferably, as an improvement, the lung cancer cells are A594 cells or Lewis cells.

[0012] Preferably, as an improvement, the administration route is oral administration / gavage administration.

[0013] Preferably, as an improvement, the human dosage is 9-18 g / d; the administration dosage for mice is 2.34-9.36 g / kg·d.

[0014] Preferably, as an improvement, the skull pill is used for preparing a synergist for a drug containing cisplatin for treating lung cancer.

[0015] Preferably, as an improvement, the administration dosage of cisplatin is 2 mg / kg, and the administration frequency is once every other day; the administration dosage of the skull pill is 1.17 g / kg, and the administration frequency is once a day; the continuous administration lasts for 14 d.

[0016] The principle and advantages of the scheme are as follows: in actual application, the skull pill is a traditional clinically positioned drug for treating brain glioma, and the core efficacy is “activating blood circulation to resolve stasis, softening and resolving masses, and refreshing brain and relieving pain”, which is suitable for treating intracranial tumors such as brain glioma. The scheme creatively proposes, for the first time, to expand the use of the skull pill to lung cancer treatment in a cross-disease field, and to combine the skull pill with cisplatin, and it is found that the skull pill can not only be used in lung cancer treatment, but also can improve the anti-tumor effect of cisplatin. The new use of the skull pill in the scheme is not an empirical application, but a systematic scientific verification based on “component-target-pathway-efficacy”. First, the scheme identifies, through LC-MS / MS technology, that the skull pill contains 1793 kinds of compounds, and detects the blood components, which provides a “material accessibility” basis for the drug acting on the whole body tumor.

[0017] In summary, the scheme has the beneficial effects as follows:

[0018] 1. The scheme provides a “component-target-efficacy” paradigm reference for modernization research of traditional Chinese medicine by using multi-omics technology (LC-MS / MS) to determine the active components and body entry characteristics of traditional Chinese medicine compound.

[0019] 2. In the scheme, the skull pill is combined with cisplatin for treating lung cancer for the first time, and the results show that the combination of the skull pill and cisplatin can enhance the tumor inhibition effect of cisplatin. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 FIG. 1 is a Base Peak Chromatogram (BPC) of the skull pill in the positive ion mode.

[0021] Figure 2This is the Base Peak Chromatogram (BPC) of the present invention, Lufu Pill, in negative ion mode.

[0022] Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the determination of the content of key compounds in the Lufu Pill of the present invention.

[0023] Figure 4 This is a line graph showing the effect of the present invention's craniotomy pills on the body weight of lung cancer-bearing mice.

[0024] Figure 5 This is a line graph showing the effect of the present invention, Lufu Pill, on tumor growth in lung cancer-bearing mice.

[0025] Figure 6 This is a physical comparison image of the tumor size in lung cancer-bearing mice under different treatment groups according to the present invention.

[0026] Figure 7 The figure shows the tumor inhibition rate of the present invention's Lufu Pill on lung cancer-bearing mice.

[0027] Figure 8 This is a line graph showing the effect of the combination of the present invention, Lufuwan and cisplatin, on tumor growth in Lewis lung cancer mice.

[0028] Figure 9 This is a physical comparison image showing the tumor size of Lewis lung cancer mice in different treatment groups according to the present invention.

[0029] Figure 10 The figure shows the tumor inhibition rate of Lewis lung cancer mice when the clumping pills and cisplatin were used in combination.

[0030] Figure 11 This is a graph showing the change in body weight of mice during the acute toxicity experiment of the Lufu Pill of the present invention.

[0031] Figure 12 This is a HE-stained pathological section of the main organs of mice in the acute toxicity experiment of the Lufu Pill of the present invention. Detailed Implementation

[0032] The following detailed description provides further details on specific embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials and reagents used are all commercially available.

[0033] Overview of the plan:

[0034] Application of LFP (Lum-Fu Pill) in the preparation of drugs for treating lung cancer.

[0035] Chemical composition analysis of Lu Fu Wan in Experiment 1

[0036] 1. Composition and content identification of Guanfu Pill

[0037] By LC-MS / MS technology, 1793 compounds in Guanfu Pill were identified, and 43 chromatographic peaks were labeled in BPC (Figure 1) Figures 1-2 ), including cholic acid, strychnine, brucine, panax notoginseng saponins R1, sophorin, ginsenoside F1, chonglou saponin I, and other compounds, see Table 1 for specific compound information.

[0038] Table 1

[0039]

[0040] 2. Content identification of Guanfu Pill

[0041] According to the quality control components of each traditional Chinese medicine specified in the Chinese Pharmacopoeia, the contents of several representative compounds were detected by HPLC. The results showed that the content of strychnine was 0.038%, the content of brucine was 0.0208%, the content of cholic acid was 10.2061%, the content of ginsenoside Rg1 was 1.2562%, the content of panax notoginseng saponins R1 was 5.6846%, and the content of β-asarone was 0.1262%. The chromatogram is shown in Figure 2 Figure 3 .

[0042] 3. Blood component identification of Guanfu Pill

[0043] After the rats were given Guanfu Pill at a dose of 2.28 g / (kg / d) orally for 7 days, the rat serum was collected, and the compounds in the serum were identified by LC-MS / MS technology. A total of 76 blood compounds were identified, and the results are shown in Table 2.

[0044] Among them, 6 peaks in the BPC map marked by traditional Chinese medicine are blood compounds, which are strychnine, p-hydroxyphenylpropionic acid, sophocarpine, ecdysone, glycyrrhizic acid, and 5,7,3,4-tetrahydroxy-6,8-diisopentenyl isoflavone; In addition, 21 compounds were only found in the serum of the drug administration group, suggesting that the compound may be an endogenous substance or metabolite of traditional Chinese medicine.

[0045] Table 2. Blood compound information table of Guanfu Pill

[0046]

[0047] Experimental Example Two: Study on the therapeutic effect of Guanfu Pill on A549 lung cancer mice

[0048] 1. Experimental materials

[0049] Animals: 50 male SPF-grade Balb / c nude mice, 4 - 6 weeks old, were purchased from Chengdu Medicilon Biotechnology Co., Ltd. The production license number is SCXK(Sichuan)2020 - 034. All animals were housed in the SPF animal facility of Huasen Pharmaceutical Co., Ltd. The facility use license number is SYXK(Chongqing)2023 - 0012, and the experimental animal ethics review approval number is 2025013. The feeding temperature was (22 ± 2)°C, the relative humidity was 40% - 70%, and the light-dark cycle was 12 h / 12 h. They had free access to food and water, and were subjected to the experiment after one week of adaptive feeding.

[0050] Cells: Human lung cancer cell line A549 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China), with the catalog number CL-001, and was cultured in F12k medium containing 10% FBS in an incubator at 37°C with 95% air, 5% CO2, and fully saturated humidity.

[0051] Skull Restoration Pills: Provided by the Preparation Center of Chongqing Traditional Chinese Medicine Hospital, with the drug approval number of Chongqing Z20051133; the clinical dosage is 18 g / d, and the equivalent dosage for mice is 2.34 g / kg·d. When used, it was prepared into a suspension of 0.23 g / mL with pure water.

[0052] 2. Experimental Methods

[0053] Human lung cancer cell line A549 was cultured, and the cell suspension was injected subcutaneously into the right axilla of nude mice at a density of 1×10 ,

[0056] , Figure 4 ,

[0055] , ,

[0054] , cells / mouse to establish a lung cancer xenograft mouse model. The tumor volume was considered to reach 3 50 mm 3 when the tumor became successfully established. After successful model establishment, the mice were grouped according to tumor size, with 10 mice in each group, namely the blank control group, the model group, the high-dose LFP group, the medium-dose LFP group, and the low-dose LFP group; the high, medium, and low-dose LFP groups were given intragastric administration at concentrations of 9.36 g / kg, 4.68 g / kg, and 2.34 g / kg respectively, twice a day; the model group was given intragastric administration of the same volume of normal saline. Administration was continued for 21 days. After the administration ended, the body weights of the mice in each treatment group and the tumor growth were detected and analyzed.

[0054] 3. Experimental Results<

[0055] 3.1 Effect of LFP on the body weight of mice

[0056] The results were as Figure 4 shown. The body weight of the mice in the model group was lower than that of the normal group; compared with the model group, the body weight of the mice in the low-dose LFP group decreased, but the difference was not statistically significant. The body weights of the mice in the high- and medium-dose LFP groups decreased significantly, and the difference was statistically significant starting from Day 14 (P < 0.05).

[0057] 3.2 Effect of LFP on Tumor Growth in Mice

[0058] The results are as Figures 5-7 shown. High and medium doses of LFP can significantly inhibit tumor growth, and the difference was statistically significant at Day 21 (P < 0.01); while there was no significant difference in tumor growth between the low-dose LFP group and the model group.

[0059] Experimental Example 3: Study on the Therapeutic Effect of Lufu Pills on Mice with Lewis Lung Cancer

[0060] 1. Experimental Method

[0061] Lewis cells in the logarithmic growth phase were suspended in sterile PBS solution, and a cell suspension of 1×10 7 cells per milliliter was inoculated into the right anterior axilla of 16 C57BL / 6 mice, 0.2 mL per mouse. After about 5 days, the average tumor volume was measured to be about 100 mm 3 , indicating successful modeling. The mice were divided into 4 groups, 4 mice in each group, namely the model group, cisplatin group, Lufu pill group, and cisplatin + Lufu pill group. The cisplatin group was intraperitoneally injected at 2 mg / kg every other day; the Lufu pill group was intragastrically administered at 1.17 g / kg once a day for 14 consecutive days. After the administration, all mice were sacrificed, and the spleen and tumor tissues were dissected and weighed.

[0062] 2. Experimental Results

[0063] The experimental results are as Figures 8-10 shown: Compared with the model group, there was no statistical significance in tumors in the cisplatin group and the Lufu pill group, while the tumors in the cisplatin + Lufu pill group were significantly smaller (P < 0.05), indicating that Lufu pills combined with cisplatin can enhance the antitumor effect of cisplatin.

[0064] Experimental Example 4: Acute Toxicity Study of Lufu Pills in Mice

[0065] 1. Experimental Materials

[0066] 1.1 Experimental Animals

[0067] 52 SPF-grade KM mice, 4 - 6 weeks old, half male and half female, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number: SCXK(Chuan)2023 - 0040. All animals were housed in the SPF animal room of Huasen Pharmaceutical Co., Ltd., facility use license number: SYXK(Yu)2023 - 0012, and the experimental animal ethics review approval number: 2025011. The feeding temperature was (22 ± 2)°C, the relative humidity was 40% - 70%, and the artificial lighting was 12 h / 12h light-dark alternation. They were fed freely with food and water, and the experiment was carried out after one week of adaptive feeding.

[0068] 1.2 Experimental drugs and reagents

[0069] Gu Fupill: provided by Chongqing Hospital of Traditional Chinese Medicine Preparation Center; when used, use pure water to configure into a suspension of 0.33 g / mL.

[0070] Reagents: mouse alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total protein (TP), total cholesterol (TC), triglyceride (TG), creatinine (CREA), urea (UREA) assay kit, purchased from Shenzhen Mindray Animal Medical Technology Co., Ltd., batch numbers are: 140124012, 140224006, 148324011, 148624005, 141624004, 141724019, 141124012, 141324021.

[0071] 1.3 Experimental instruments

[0072] BS-240VET automatic biochemical analyzer for veterinary use, Shenzhen Mindray Animal Medical Technology Co., Ltd.; CX22 optical microscope, Japan Olympus Corporation; RM2235 paraffin section machine, Germany Leica; BMJ-A embedding machine, PHY-III pathological tissue bleaching and drying instrument, Changzhou Zhongwei Electronic Instrument Co., Ltd.; NanoZoomer S60 digital pathology section scanning system, Japan Hamamatsu Photonics Corporation.

[0073] 2、Experimental method

[0074] 2.1 Pre-experiment

[0075] SPF KM mice 10, half male and half female, using the maximum gavage dose method, 24h within 4 times of gavage Gu Fupill 13.32g / kg (equivalent to 10.03 times of the clinical equivalent dose), every 2 times of gavage interval time more than 4h. After gavage, continuous observation for 14 days, no mice died, half lethal dose cannot be obtained, so the formal experiment uses the maximum gavage dose method for administration.

[0076] 2.2 Formal experiment

[0077] 42 SPF level KM mice, half male and half female, were divided into 3 groups: blank control group, high and low dose groups of skull pill, 14 in each group. Fasting for 12 h without water restriction, the high and low dose groups of skull pill were given skull pill 13.32 g / kg and 6.66 g / kg respectively by gavage within 24 h, and the blank control group was given pure water by gavage, with a volume of 10 mL / kg, divided into 4 times, with an interval of more than 4 h between each 2 times of gavage. After the gavage was completed, the animal state was observed for 30 min, then observed every 2 h, observed once a day after 24 h, and observed for 14 d, during which the general state of the animals such as mental state, behavior, excrement state, death, etc. were recorded, and the animal body weight was recorded every 2 days. At the end of the experiment, the eyeball was taken out to collect blood, and after the serum was separated, the biochemical indexes such as ALT, AST, TP, ALB, TG, TC, CREA, UREA, etc. were detected; the heart, liver, spleen, lung and kidney were observed by gross anatomy, and the abnormality and weight were recorded, and the organ coefficient (organ coefficient = organ weight / mouse weight x 100%) was calculated; the mouse heart, liver, spleen, lung and kidney tissues were fixed with 4% paraformaldehyde, dehydrated, fixed and sectioned by the conventional method, and HE staining was performed, and the histopathological changes were observed after sealing.

[0078] 3. Experimental results

[0079] 3.1 Effect of LFP on the general state of mice

[0080] During the experiment, no death was observed in mice of each group, and the mice in the control group were in good mental state, with normal behavior and soft and lustrous fur, and the feces were normal granular; the mice in the high and low dose groups of LFP showed reduced spontaneous activity and slight piloerection after administration, which gradually relieved at 3-4 h after administration, and recovered to normal the next day, and the feces of mice in both groups were normal granular during the observation period.

[0081] 3.2 Effect of LFP on the body weight of mice

[0082] In male mice, there was no significant difference in body weight between the high and low dose groups of LFP and the control group during the observation period; the body weight of female mice was significantly lower than that of male mice, and there was no significant difference in body weight between the high and low dose groups of LFP and the control group during the observation period (see Figure 11 ). The results showed that LFP had no significant effect on the body weight of mice.

[0083] 3.3 Effect of LFP on liver and kidney function of mice

[0084] Compared with the control group, there were no significant differences in serum AST, TC, and CREA levels in the LFP group mice. In female mice, the ALT and TP levels were significantly increased in the low-dose Lufuwan group (P < 0.05). In male mice, the TP, ALB, TC, and UREA levels were significantly increased after LFP administration, with the UREA level in the low-dose Lufuwan group showing a highly significant difference (P < 0.01). Detailed results are shown in Table 3.

[0085] Table 3 Biochemical indicators of mice in each group (x ± s, n=8)

[0086]

[0087] 3.4 Effects of LFP on organ coefficient in mice

[0088] After the observation period, the mice were dissected, and no obvious abnormalities were observed in any of the organs by visual inspection. The liver, heart, spleen, lungs, and kidneys were removed in sequence, weighed, and the organ coefficients were calculated. The results are shown in Table 4. Compared with the control group, there were no significant differences in the organ coefficients of liver, heart, spleen, lungs, and kidneys between male and female mice in the LFP group (P>0.05).

[0089] Table 4. Organ coefficients of mice in each group (x ± s, n=8)

[0090]

[0091] 3.5 Effects of LFP on the pathological morphology of various organs in mice

[0092] The results are as follows Figure 12 As shown, compared with the control group, no pathological changes were observed in the liver, heart, spleen, lungs, and kidneys of both male and female mice in the LFP group. The liver lobule structure was intact, the hepatic cord structure was clear, and the hepatocytes and cardiomyocytes showed normal morphology and uniform staining, with no obvious inflammation. The spleen tissue showed tightly packed cells with a clear boundary between the red and white pulp. The bronchial epithelial cells and alveoli of the lungs showed normal morphology, with no obvious inflammation. The kidney tissue showed abundant cortical glomeruli and normal tubular morphology, with no obvious inflammation. This indicates that at this dosage, LFP (Lulufuwan) did not cause acute damage to the major organs of the mice.

[0093] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. The use of skullcap pill in the preparation of a drug for treating lung cancer, characterized in that: The pill is composed of the following raw materials in mass fraction: ox gallstone 20-30 parts, borneol 1.0-1.5 parts, Strychnos 15-20 parts, Acorus 80-90 parts, Paris 120-150 parts, Panax 120-150 parts, Astragalus 180-200 parts, and licorice 180-200 parts. ​ 2. The use of skullcap pill in the preparation of a synergist for treating lung cancer, characterized in that: The pill is composed of the following raw materials in mass fraction: ox gallstone 20-30 parts, borneol 1.0-1.5 parts, Strychnos 15-20 parts, Acorus 80-90 parts, Paris 120-150 parts, Panax 120-150 parts, Astragalus 180-200 parts, and licorice 180-200 parts.

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