Application of α-ketoglutaric acid in the preparation of drugs for alleviating gastrointestinal toxicity induced by chemotherapy

Abstract

The present invention discloses the application of α-ketoglutaric acid in the preparation of a drug for alleviating gastrointestinal toxicity caused by chemotherapy, and belongs to the field of medical technology. Experimental studies of the present invention show that α-ketoglutaric acid can improve the changes in small intestine length of mice induced by the chemotherapy drug irinotecan, significantly alleviate the intestinal damage of mice caused by irinotecan, significantly improve the intestinal barrier function of mice, and significantly alleviate the intestinal toxicity caused by irinotecan. Therefore, during tumor chemotherapy, α-ketoglutaric acid can be combined with intervention to alleviate the intestinal toxicity caused by clinical chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of alpha-ketoglutaric acid in the preparation of a drug for alleviating gastrointestinal toxicity caused by chemotherapy. Background Art

[0002] Irinotecan, a semisynthetic derivative of camptothecin, is commonly used to treat metastatic colorectal cancer that has recurred after standard chemotherapy. It is also widely used in chemotherapy for lung cancer, breast cancer, gastric cancer, ovarian cancer, and other cancers. Research results show that approximately 20% of patients receiving irinotecan-based combination chemotherapy regimens experience severe diarrhea. Gastrointestinal toxicities, represented by delayed diarrhea, have become the main factor limiting the clinical application of irinotecan. Therefore, the development of a drug that can improve irinotecan chemotherapy-related intestinal mucosal damage and reduce the risk of delayed diarrhea is of great clinical significance.

[0003] α-ketoglutarate is an important intermediate metabolite in the tricarboxylic acid cycle, where it can be converted into glutamate and glutamine. As a precursor to glutamine, α-ketoglutarate is the primary energy source for gastrointestinal cells, promoting their proliferation and playing a vital role in maintaining gastrointestinal health. α-ketoglutarate is also a crucial link between carbon and nitrogen metabolism, enhancing immune function, influencing calcium and phosphorus metabolism, increasing bone density and bone mineral content, and improving intestinal mucosal structure. Furthermore, α-ketoglutarate can increase skeletal muscle protein synthesis and deposition by activating the mammalian target of rapamycin (mTOR) signaling pathway, thereby improving growth performance. Recent studies have also revealed that α-ketoglutarate possesses antioxidant, anti-tumor, and immunomodulatory properties. Studies have shown that α-ketoglutarate can protect against apoptosis in H2O2-induced intestinal epithelial cell damage. α-ketoglutarate can also inhibit cyanide-induced neuronal apoptosis. Furthermore, an amino acid buffer containing α-ketoglutarate can protect against apoptosis in rats with cerebral ischemia-reperfusion injury. Therefore, α-ketoglutarate has a wide range of applications as a drug and is safe.

[0004] Although α-ketoglutarate is an important metabolic intermediate in the tricarboxylic acid cycle and is widely involved in regulating cell proliferation, differentiation, and fate, research on its regulation of the intestine has largely focused on animal husbandry, and there are currently no reports that it can alleviate the intestinal toxicity caused by chemotherapy drugs such as irinotecan. Summary of the Invention

[0005] Purpose of the Invention: This invention addresses the shortcomings of the prior art and provides a method for preparing a drug for alleviating chemotherapy-induced gastrointestinal toxicity. α-Ketoglutarate significantly improves the intestinal barrier function of mice and can alleviate the intestinal toxicity caused by irinotecan. Therefore, during tumor chemotherapy, α-ketoglutarate can be combined with intervention to alleviate the gastrointestinal toxicity caused by clinical irinotecan chemotherapy, providing a new option for the preparation of drugs to alleviate and inhibit chemotherapy-induced gastrointestinal toxicity.

[0006] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0007] The present invention provides the use of alpha-ketoglutaric acid in preparing a drug for alleviating gastrointestinal toxicity caused by chemotherapy.

[0008] Preferably, the chemotherapy drug is irinotecan.

[0009] Preferably, the drug comprises α-ketoglutaric acid and its derivatives or pharmaceutically acceptable salts thereof.

[0010] Further preferably, the drug further comprises pharmaceutically acceptable excipients.

[0011] More preferably, the excipients include one or more of an emulsifier, a lubricant, a solvent, a diluent, a binder, a wetting agent, a preservative, an antioxidant, a solubilizer, and a disintegrant.

[0012] The emulsifier is selected from at least one of Tweens, Spans, glycerol fatty acid esters, gelatin, pectin, agar, sodium alginate and silicon dioxide.

[0013] The lubricant is selected from at least one of magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycols, and micro-powdered silica gel.

[0014] The solvent is selected from at least one of water, oil, ethanol, glycerol, dimethyl sulfoxide, fatty oil, propylene glycol, and polyethylene glycol.

[0015] The diluent is selected from at least one of starches, sugars, celluloses and inorganic salts.

[0016] The adhesive is selected from at least one of starch slurry, sodium carboxymethyl cellulose, povidone, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose.

[0017] The wetting agent is selected from at least one of water and ethanol.

[0018] The preservative is selected from at least one of benzoic acid and its salts, sorbic acid and its salts, and parabens.

[0019] The antioxidant is selected from at least one of ascorbic acid, sulfite, bisulfite, gallic acid and lipids thereof.

[0020] The solubilizing agent is selected from at least one of Tweens, polyoxyethylene fatty alcohol ethers, sulfates and sulfonates.

[0021] The disintegrant is selected from at least one of starch, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, and cross-linked polyvinyl pyrrolidone.

[0022] Preferably, the pharmaceutical dosage form is granules, tablets, capsules, oral liquids, pills, emulsions or suspensions.

[0023] Preferably, the pharmaceutical dosage form is an injection, an infusion or a spray.

[0024] Preferably, the drug is administered orally, by enema or by injection.

[0025] In the experiment of the present invention, mice were divided into a control group and an α-ketoglutaric acid administration group. The control group was given normal drinking water, and the α-ketoglutaric acid administration group was given drinking water containing α-ketoglutaric acid (1%). An irinotecan mouse diarrhea model was established. From day 1 to day 15, each group of mice was given normal drinking water and drinking water containing α-ketoglutaric acid, respectively; from day 2 to day 15, the mice were subjected to restraint stress (4 hours / day); on day 15, four mice were taken from each group, and the physiological and biochemical indicators of the mice were examined; from day 16 to day 23, each group of mice was gavage-administered irinotecan (drug concentration of 3 mg / ml, mouse dosage of 25 mg / kg), and the weight changes, diarrhea, and mortality of the mice in each group were observed; on day 23, the remaining mice were sacrificed, and the physiological and biochemical indicators and intestinal cell changes of the mice were examined.

[0026] Experimental studies in this paper demonstrate that α-ketoglutarate can improve irinotecan-induced intestinal barrier damage in mice and alleviate irinotecan-induced intestinal toxicity. Ileal pathology scores were reduced in mice treated with α-ketoglutarate; villus length and crypt height were significantly improved; and goblet cell and Paneth cell numbers increased. During tumor chemotherapy, α-ketoglutarate intervention can alleviate irinotecan-induced intestinal toxicity in clinical settings. Therefore, α-ketoglutarate has value in developing drugs that alleviate chemotherapy-induced gastrointestinal toxicity.

[0027] Beneficial effects:

[0028] The α-ketoglutaric acid described in the present invention can be used as an active ingredient in drugs that alleviate chemotherapy-induced gastrointestinal toxicity. This opens up new uses for α-ketoglutaric acid and provides a new option for preparing drugs that alleviate and inhibit chemotherapy-induced gastrointestinal toxicity. α-ketoglutaric acid has good stability and solubility, and after meeting the body's normal requirements, it does not cause additional residue, thus causing minimal harm to the body. When used in the prevention and treatment of chemotherapy-induced gastrointestinal toxicity, the drugs containing α-ketoglutaric acid have many advantages, including safe dosage, rapid and reliable effects, low cost, no resistance to drug development by pathogenic microorganisms, and no concerns about drug residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure shows the technical route of mouse modeling, the changes in mouse weight and ileum length. Figure 1 A is the technical roadmap for the irinotecan-induced mouse diarrhea model and drug intervention; Figure 1 B is the result of the change of small intestine length in model mice; Figure 1 C is the curve of the percentage change in body weight of model mice.

[0030] Figure 2 The HE staining comparison of ileum pathological sections of mice in each group. Figure 2 A is the HE staining result of mouse ileum pathological section before irinotecan administration; Figure 2 B is the result of ileum pathology scoring; Figure 2 C is the HE staining results of the ileum pathological sections of mice in each group on the 7th day after irinotecan administration; Figure 2 D is the result diagram of ileal villus length. Figure 2 E is the result diagram of ileal crypt height.

[0031] Figure 3 The AB staining comparison of ileum pathological sections of mice in each group. Figure 3 A is a comparison of AB staining of mouse ileum pathological sections before irinotecan administration; Figure 3 B is the average number of positive cells in the ileal crypts; Figure 3 C is a comparison of AB staining of ileum pathological sections of mice in each group on day 7 after irinotecan administration; Figure 3 D is the average number of positive cells in the ileal crypts.

[0032] Figure 4 This is a comparison of immunofluorescence staining of ileum sections of mice in each group. Figure 4 A is a comparison of Chga staining; Figure 4 B is a comparison of Olfm4 staining; Figure 4 C is a comparison diagram of Lyz staining. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0034] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0036] Example 1

[0037] 1. Effect of α-ketoglutarate on body weight and small intestine length in mice

[0038] (1) Experimental materials

[0039] Animals: Healthy specific pathogen-free (SPF) female Balb / c mice, 6 weeks old, weighing 16-18 g, were purchased from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd. and used in the experiments after adaptive training.

[0040] Reagents:

[0041] Irinotecan hydrochloride (CPT-11·HCl·3H2O) raw material was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: C1822032. Irinotecan hydrochloride injection was prepared according to the literature: 12 mL of water for injection and 34 μL of lactic acid were thoroughly mixed at 85°C. Irinotecan hydrochloride powder (3 mg / mL, CPT-11·HCl·3H2O) was added, followed by 24 mL of water for injection, and the mixture was thoroughly stirred. 112.5 mg of D-sorbitol powder was added and stirred until completely dissolved. 4 mL of water for injection was then added to obtain a clear yellow solution. The solution was filtered through a 0.22 μm filter membrane in the dark and stored at 4°C until ready for use.

[0042] Among them, lactic acid (C3H6O3) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: #G2017197, sorbitol (C6H 14 O6) was purchased from Shanghai Sanen Chemical Technology Co., Ltd., batch number: DC120008.

[0043] α-Ketoglutaric acid (C5H6O5) raw material was purchased from Sigma, batch number 75890-25G. Its solution was prepared as follows: the α-ketoglutaric acid raw material was dissolved in Wahaha drinking water. After dissolution, sodium hydroxide (NaOH, purchased from Xilong Science Co., Ltd., batch number 1810211) was used to adjust the pH to neutral, and the α-ketoglutaric acid concentration was 1%.

[0044] Instruments: laser confocal microscope (Olympus FV3000), upright fluorescence microscope (BX53), electronic balance, pH meter.

[0045] (2) Experimental methods

[0046] Animal Experiments: Mice were adaptively housed for one week (24±2°C, 12 / 12h dark / light cycle) to allow them to fully acclimate. Mice were randomly divided according to body weight into a control group and an α-ketoglutarate-treated group (n=14 mice per group). Starting on the first day of the experiment, the control and α-ketoglutarate-treated groups were given normal drinking water or drinking water supplemented with 1% α-ketoglutarate, respectively, for 15 consecutive days.

[0047] Starting on the second day of the experiment, each group of mice was subjected to chronic restraint stress (4 hours per day) for 14 consecutive days. The chronic restraint stress test involves confining mice in a restraint tube so that the length of the tube is less than their body length, ensuring that only the head can move freely while the limbs are restricted. Restraint is performed for a fixed period of 4 hours per day.

[0048] On the fifteenth day of the experiment, four mice in each group were killed, and ileal samples were collected (the terminal 1-2 cm of the ileum was placed in a 4% paraformaldehyde solution (paraformaldehyde was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., batch number 20190322. 1L of 4% paraformaldehyde solution was prepared by weighing 40g of paraformaldehyde powder and adding it to 1L of PBS solution. The solution was heated in a 55-60°C water bath until dissolved and stored at room temperature in the dark) for later use; another 2cm of the terminal ileum was cut open longitudinally on filter paper and placed in a -80°C refrigerator for later use; at the same time, chronic restraint stress was stopped for the remaining mice, and they were all given normal drinking water and irinotecan hydrochloride injection at a dose of 25 mg / kg for 7 consecutive days. On the 23rd day, the mice were bled from their orbits and then sacrificed by cervical dislocation, and tissue samples were collected for subsequent operations. The body weight of the mice was weighed and recorded every day after administration of irinotecan. The technical route for the irinotecan-induced diarrhea model in mice and drug intervention can be found in the literature. Figure 1 A. Chronic restraint stress for 14 days followed by irinotecan administration for 7 days can better induce intestinal epithelial damage and ileitis in mice, creating a combined model.

[0049] (3) Data processing

[0050] All data were expressed as mean ± SEM and processed using GraphPad Prism software. Two-sided unpaired t-test was used to analyze the statistical significance. P < 0.05 was considered a significant difference.

[0051] (4) Experimental results

[0052] Figure 1 Figure 1 shows the mouse modeling technology route, mouse weight changes, and ileum length changes. As can be seen from the figure, α-ketoglutarate did not improve the weight loss caused by irinotecan in mice, but it did improve small intestine length, alleviating the shortening of small intestine length caused by irinotecan in mice.

[0053] 2. Effect of α-ketoglutarate on irinotecan-induced histopathological changes in the ileum of mice

[0054] (1) Experimental materials: Same as those in “Effects of α-ketoglutaric acid on body weight and small intestine length in mice”.

[0055] (2) Experimental methods

[0056] The animal experimental method is the same as that of “Experiment on the Effect of α-ketoglutaric acid on the Body Weight and Small Intestine Length of Mice”.

[0057] Histopathological Examination: Mouse ileum tissue samples fixed in 4% paraformaldehyde were sent to the Department of Pathology at Jiangsu Provincial Hospital of Traditional Chinese Medicine for histopathological examination according to standard procedures. Following standard laboratory procedures, tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE) and alcian blue (AB). Histopathological changes were observed and photographed under an upright fluorescence microscope (BX53).

[0058] (3) Data processing

[0059] All data were expressed as mean ± SEM and processed using GraphPad Prism software. Two-sided unpaired t-test was used to analyze the statistical significance. P < 0.05 was considered a significant difference.

[0060] (4) Experimental results

[0061] Figure 2 The HE staining comparison of ileum pathological sections of mice in each group. Figure 2 A is the HE staining result of mouse ileum pathological section before irinotecan administration; Figure 2 B is the result of ileum pathology scoring; Figure 2 C is the HE staining results of the ileum pathological sections of mice in each group on the 7th day after irinotecan administration; Figure 2 D is the result diagram of ileal villus length. Figure 2 E is the result diagram of ileal crypt height.

[0062] Figure 3 The AB staining comparison of ileum pathological sections of mice in each group. Figure 3 A is a comparison of AB staining of mouse ileum pathological sections before irinotecan administration; Figure 3 B is the average number of positive cells in the ileal crypts; Figure 3 C is a comparison of AB staining of ileum pathological sections of mice in each group on day 7 after irinotecan administration; Figure 3 D is the average number of positive cells in the ileal crypts.

[0063] HE pathological scoring criteria: The histological criteria of Neurath et al. were used for scoring, and the lesions were scored from mild to severe as 1 point (mild; no destruction of villus structure, low-level inflammatory infiltration), 2 points (mild; deepening of intestinal crypts, thickening of intestinal wall, moderate level of inflammatory infiltration, no ulcer), 3 points (moderate; vascular hyperplasia, thickening of intestinal wall, high level of inflammatory infiltration, no ulcer) and 4 points (severe: vascular hyperplasia, deepening and deformation of intestinal crypts, thickening of intestinal wall and invasion of muscular layer, obvious inflammatory infiltration, accompanied by ulcer). No obvious lesions were scored as 0 points.

[0064] The length of ileal villi and crypt height were measured using ImageJ software.

[0065] from Figure 2 A and Figure 2 As can be seen in B: the ileal villi structure of the mice in the α-ketoglutarate group was more complete than that in the control group, the degree of inflammation was lower, and the pathological score was significantly lower than that in the control group, indicating that α-ketoglutarate can improve the inflammation caused by restraint. Figure 2 C and Figure 2 As can be seen from D, the ileal villus structure of mice in the α-ketoglutarate group was more complete than that in the control group, the degree of inflammation was lower, and the villus length and crypt height were significantly higher than those in the control group, indicating that α-ketoglutarate has an improving effect on intestinal damage caused by irinotecan in mice.

[0066] The average number of AB-positive spots (representing goblet cells) in each crypt was counted manually and analyzed graphically. Figure 3 B and Figure 3 As can be seen in D: the number of AB-positive spots in the crypts of the ileum of mice in the α-ketoglutarate group was significantly higher than that in the control group. The mucin secreted by goblet cells is an important component of the intestinal barrier. The reduction of goblet cells indicates that the intestinal barrier is damaged. Figure 3 The results showed that α-ketoglutarate can significantly alleviate the intestinal barrier function damage caused by irinotecan in mice.

[0067] Example 2 Effect of α-ketoglutarate on the number of mouse ileal stem cells and Paneth cells

[0068] (1) Experimental materials: Same as those in “Effects of α-ketoglutaric acid on body weight and small intestine length in mice”.

[0069] (2) Experimental methods

[0070] The animal experimental method is the same as that of “Experiment on the Effect of α-ketoglutaric acid on the Body Weight and Small Intestine Length of Mice”.

[0071] Immunofluorescence staining:

[0072] The mouse ileum (1-2 cm) was cut open longitudinally on filter paper, fixed, embedded in OCT glue, and prepared into frozen sections. PBS (1 L PBS solution was prepared: weighed potassium chloride (KCl, purchased from Nanjing Chemical Reagent Co., Ltd., batch number: 171116678F) powder 0.2 g, potassium dihydrogen phosphate (KH2PO4, purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., batch number: 20190322) powder 0.2 g, sodium chloride (NaCl, purchased from Xilong Scientific Co., Ltd., batch number: 2204022) powder 8 g, disodium hydrogen phosphate dodecahydrate (Na2HPO4· The slides were washed with PBS containing 10% serum derived from the secondary antibody (donkey serum was used in the present invention and purchased from Beijing Solebow Technology Co., Ltd.; product number SL050) and 0.3% Triton-X (Shanghai Biyuntian Biotechnology Co., Ltd.; product number ST795) at room temperature for 1 h. The blocking solution was removed and the slides were incubated with primary antibodies (Chga, purchased from Abcam, product number ab283625; Olfm4, purchased from Cell Signaling, product number 39141S; Lyz, purchased from Abcam, product number ab108508) at 4°C overnight. Slides were removed from the refrigerator and equilibrated at room temperature for 1 hour. Slides were then washed five times for 5 minutes with PBST (PBS containing 0.1% Tween-20, purchased from Nanjing Chemical Reagent Co., Ltd., Lot No. 211228916F). Secondary antibody (AlexaFluor 555, purchased from Abcam, Catalog No. ab150062, used in dark) was diluted in blocking buffer and blocked for 1 hour. Slides were then washed five times for 5 minutes with PBST, dried, and mounted with DAPI (Abcam; Catalog No. ab104139). After 5 minutes, coverslips were applied, dried, and images were captured under a laser confocal microscope (Olympus FV3000).

[0073] (3) Data processing

[0074] All data were expressed as mean ± SEM and processed using GraphPad Prism software. Two-sided unpaired t-test was used to analyze the statistical significance. P < 0.05 was considered a significant difference.

[0075] (4) Experimental results

[0076] Figure 4 This is a comparison of immunofluorescence staining of ileum sections of mice in each group. Figure 4 A is a comparison of Chga staining; Figure 4 B is a comparison of Olfm4 staining; Figure 4 C is a comparison of Lyz staining. The average number of positive staining spots per intestinal crypt was manually counted and plotted for analysis. As shown, the number of Olfm4 and Lyz-positive cells in the ileum of mice treated with α-ketoglutarate significantly increased compared to the control group, indicating that α-ketoglutarate significantly increases the number of intestinal stem cells and Paneth cells in mice.

[0077] Intestinal stem cells promote the development, renewal, and regeneration of intestinal tissue and are the primary source of self-renewal, replenishment, and repair of stress-induced damage. The intestinal epithelium has a rapid cell renewal capacity, approximately every 3 to 5 days. The production of new intestinal epithelial cells is achieved through the continuous proliferation and differentiation of intestinal stem cells.

[0078] Paneth cells are rare cells in the small intestine that provide host defense against microbial invasion. Their function is similar to that of neutrophils. When bacteria or bacterial antigens invade the body, Paneth cells secrete antimicrobial molecules, such as defensins, into the villi lining the small intestinal lumen to help maintain the gastrointestinal barrier. Increased numbers of intestinal stem cells and Paneth cells suggest that α-ketoglutarate can effectively alleviate irinotecan-induced small intestinal barrier damage in mice.

[0079] In summary, α-ketoglutarate can improve irinotecan-induced changes in small intestinal length in mice, significantly alleviate irinotecan-induced intestinal damage in mice, significantly improve intestinal barrier function in mice, and significantly alleviate irinotecan-induced intestinal toxicity. Therefore, during cancer chemotherapy, α-ketoglutarate can be combined with intervention to alleviate clinical chemotherapy-induced intestinal toxicity.

[0080] α-ketoglutaric acid has excellent stability and solubility. After meeting the body's normal needs, it leaves no excess residue and poses minimal harm to the body. The α-ketoglutaric acid concentration in the present invention is 1%, and the drug is widely used in animal husbandry, ensuring safety. Furthermore, α-ketoglutaric acid can alleviate irinotecan-induced enterotoxicity 23 days after administration and one week after irinotecan administration. Therefore, the α-ketoglutaric acid-containing drug of the present invention offers numerous advantages for preventing and treating chemotherapy-induced gastrointestinal toxicity, including safe dosage, rapid and reliable effects, low cost, resistance to pathogenic microorganisms, and no drug residue.

[0081] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. Application of α-ketoglutaric acid in the preparation of drugs for alleviating intestinal toxicity induced by irinotecan chemotherapy.

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

3. The use according to claim 2, characterized in that The auxiliary materials include one or more of emulsifiers, lubricants, solvents, diluents, adhesives, wetting agents, preservatives, antioxidants, solubilizers, and disintegrants.

4. The use according to claim 1, characterized in that The pharmaceutical dosage form is granules, tablets, capsules, oral liquids, pills, emulsions or suspensions.

5. The use according to claim 1, characterized in that The pharmaceutical dosage form is an injection or a spray.

6. The use according to claim 1, characterized in that The administration route of the drug is oral administration, enema administration or injection administration.

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