Injection compound preparation as well as preparation method and application thereof

Abstract

The invention relates to the field of pharmacy, and discloses an injection compound preparation which at least contains semeglutide, thiamine pyrophosphate and normal saline, the preparation method comprises the following steps: selecting thiamine pyrophosphate powder according to the formula ratio, adding the thiamine pyrophosphate powder into normal saline, and uniformly mixing to obtain a mixed solution; and adding the semeglutide into the mixed solution to obtain the compound preparation, the invention also discloses application of the compound preparation in matrix weight loss and / or fatty liver treatment. According to the invention, the weight reducing effect of the semeglutide is improved, and the semeglutide has the function of treating the fatty liver to a certain extent, so that a new medicine variety is provided for treating the fatty liver.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and in particular to an injection compound preparation and a preparation method and application thereof. Background Art

[0002] Obesity can lead to a range of serious health problems, including cardiovascular, respiratory, and endocrine and reproductive system diseases, cancer, and intestinal flora imbalance. These problems not only affect an individual's quality of life but can also lead to serious health crises.

[0003] Nonalcoholic fatty liver disease (NAFLD), recently renamed metabolic-associated fatty liver disease (MAFLD), is one of the most common liver diseases worldwide. NAFLD is growing in tandem with the obesity epidemic, leading to the urgent need to manage fatty liver disease and overeating.

[0004] Semaglutide, a drug developed and produced by Danish pharmaceutical company Novo Nordisk, is used to treat adults with type 2 diabetes. Its significant weight-loss effect has led to its use in obese patients, but its effectiveness has been limited. To achieve even greater weight loss and fatty liver disease treatment, major laboratories have begun experimenting with combining weight-loss drugs, achieving a "1+1 > 2" effect.

[0005] However, the combination of drugs requires special attention to drug reactions and drug damage, especially since semaglutide is used by injection. Some drugs can cause double damage to the human body when used in combination, making it difficult to improve the efficacy of semaglutide. Summary of the Invention

[0006] The purpose of the present invention is to provide an injectable compound preparation and its preparation method and application, so as to solve the technical problems in the prior art that drug combination is difficult and drug efficacy is difficult to improve.

[0007] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0008] The present invention provides an injection compound preparation, which contains at least semaglutide, thiamine pyrophosphate and normal saline.

[0009] As a preferred embodiment of the present invention, the concentration of semaglutide is (8.2-12.3)*10 -2 mg / kg;

[0010] The dosage of the thiamine pyrophosphate is 150-200 mg / / kg.

[0011] As a preferred embodiment of the present invention, the compound preparation is an injection.

[0012] The present invention provides a method for preparing an injectable compound preparation, comprising the following steps:

[0013] According to the formula, thiamine pyrophosphate powder was selected and added to normal saline, and mixed evenly to obtain a mixed solution;

[0014] Semaglutide is added to the mixed solution to obtain the compound preparation.

[0015] As a preferred embodiment of the present invention, the mixing temperature is room temperature.

[0016] The present invention provides application of an injectable compound preparation for weight loss in mammals; the mammals are mice.

[0017] The present invention provides application of an injectable compound preparation in inhibiting or alleviating fatty liver.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a compound preparation that inherits the dual properties of semaglutide and thiamine pyrophosphate. After application to mice, it can reduce transaminase levels and reduce the degree of liver damage. For some thin patients who need to alleviate fatty liver disease, this compound preparation has excellent adaptability. By adjusting the ratio of thiamine pyrophosphate to semaglutide, it can achieve the goal of improving the therapeutic effect of fatty liver disease while ensuring that the patient's weight loss is within a reasonable range. It is suitable for selective preparation in the process of body weight loss or fatty liver relief.

[0020] The compound preparation provided by the present invention is simple to prepare and easy to use. Compared with simple drug combinations, the compound preparation causes less damage to the body during medication operation than when used alone.

[0021] This invention proposes for the first time the weight loss mechanism of thiamine pyrophosphate. ENO1 aggravates hepatocyte lipid synthesis by activating cholesterol regulatory element binding protein. Thiamine pyrophosphate reduces ENO1 expression by accelerating ENO1 ubiquitination in the body. Therefore, after combining with semaglutide, thiamine pyrophosphate can be delivered specifically to the fatty liver site to further alleviate fatty liver in mice, providing a new type of drug for the treatment of fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0023] Figure 1 The present invention provides a schematic flow chart of a method for preparing an injectable compound preparation;

[0024] Figure 2 The present invention provides a comparison chart of the weight loss effect of mice in the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0025] Figure 3 The present invention provides a line graph of the degree of weight loss of mice in the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0026] Figure 4 The present invention provides a comparison chart of weight loss in mice after the end of medication in the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0027] Figure 5 The present invention provides a comparative diagram of the weight loss and fatty liver treatment effects of mice after the drug administration in the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0028] Figure 6 The present invention provides the fatty liver scores of mice in the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0029] Figure 7 The present invention provides a comparison chart of changes in serum triglycerides in mice in the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0030] Figure 8 The present invention provides a comparison chart of the changes in cholesterol in mice during the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0031] Figure 9 The present invention provides a comparison chart of the changes in blood glucose in mice during the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0032] Figure 10 The present invention provides a comparison chart of the degree of increase in serum transaminase in mice in the in vivo experiments of Examples 1-3 and Comparative Example 1;

[0033] Figure 11 The present invention provides a statistical graph of weight changes of mice in each group after intraperitoneal injection of thiamine pyrophosphate in comparative example 1 and comparative example 2;

[0034] Figure 12The present invention provides a statistical graph of the weight changes of mice in comparative example 1 and comparative example 2 in the last week;

[0035] Figure 13 The present invention provides gross liver specimens of mice in each group after TPP treatment in Comparative Example 1 and Comparative Example 2, HE staining and Oil Red staining images;

[0036] Figure 14 The present invention provides statistical graphs of mouse liver index in comparative example 1 and comparative example 2;

[0037] Figure 15 The present invention provides statistical graphs of NAS fatty liver scores in mice in comparative example 1 and comparative example 2;

[0038] Figure 16 The present invention provides a statistical graph of changes in total cholesterol levels in mouse serum in Comparative Example 1 and Comparative Example 2;

[0039] Figure 17 The present invention provides a statistical graph of changes in triglyceride content in mouse liver tissue in comparative example 1 and comparative example 2;

[0040] Figure 18 The present invention provides a statistical graph of the changes in serum ALT in mice in Comparative Example 1 and Comparative Example 2;

[0041] Figure 19 The present invention provides a statistical graph of changes in serum AST in mice in Comparative Example 1 and Comparative Example 2;

[0042] Figure 20 The present invention provides the oil red staining and Nile red staining images of HepG2 cells after FFA stimulation in vitro experiments in comparative examples 1 and 2;

[0043] Figure 21 The present invention provides a statistical graph showing changes in mRNA of related fatty acid synthesis genes and β-oxidation genes after FFA stimulation of HepG2 cells in Comparative Example 1 and Comparative Example 2;

[0044] Figure 22 The present invention provides a schematic diagram of the Lip-MS process in Comparative Example 1 and Comparative Example 2;

[0045] Figure 23 Provides pathway enrichment analysis diagrams of Lip-MS results in Comparative Example 1 and Comparative Example 2 of the present invention;

[0046] Figure 24 The present invention provides a schematic flow chart of non-targeted metabolomics in Comparative Example 1 and Comparative Example 2;

[0047] Figure 25The present invention provides a schematic diagram of the metabolite enrichment pathway in non-targeted metabolomics in Comparative Example 1 and Comparative Example 2;

[0048] Figure 26 The present invention provides SPR verification results of thiamine pyrophosphate binding to ENO1 in Comparative Example 1 and Comparative Example 2;

[0049] Figure 27 The present invention provides statistical graphs of ENO1 expression after CHX, MG132, and CQ stimulate Hep in comparative examples 1 and 2, respectively;

[0050] Figure 28 The present invention provides a schematic diagram of the changes in the YAP1 downstream signaling pathway after adding TPP in Comparative Examples 1 and 2. DETAILED DESCRIPTION

[0051] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0052] The present invention provides an injection compound preparation, which contains at least semaglutide, thiamine pyrophosphate and normal saline.

[0053] Thiamine pyrophosphate, also known as vitamin B1, acts as a catalyst in the decarboxylation of branched-chain amino acids and α-keto acids to produce energy. It also plays a protective role in the transmission of nerve impulses and in the maintenance of myelin. Deficiency of this water-soluble vitamin can affect the cardiovascular, nervous, and immune systems and is commonly seen in conditions such as wet beriberi, dry beriberi, and Wernicke-Korsakoff syndrome. Thiamine pyrophosphate is its active form.

[0054] Since the effect of thiamine pyrophosphate on obesity and fatty liver is unclear, there is currently no precedent for the combined use of semaglutide and vitamins. In the present invention, the use of thiamine pyrophosphate in the treatment of fatty liver is disclosed.

[0055] However, in actual use, both thiamine pyrophosphate and semaglutide are injected into the body. If used together, they will cause double trauma to the patient. Therefore, the present invention combines thiamine pyrophosphate and semaglutide in a certain ratio to form a compound preparation, which not only achieves the effect of combined use but also reduces the trauma to the patient.

[0056] The compound preparation disclosed in the present invention can deliver drugs into the body through subcutaneous injection, and can achieve the effects of weight loss and treatment of fatty liver. By adjusting the drug ratio, its efficacy in inhibiting and alleviating fatty liver is greater than that of using semaglutide or thiamine pyrophosphate alone, and its weight loss effect can be customized according to specific weight loss needs.

[0057] The concentration of semaglutide can range from (8.2-12.3)*10 -2 The dosage of thiamine pyrophosphate can be selected within mg / kg, and the dosage range of thiamine pyrophosphate can be selected within 150-200 mg / / kg.

[0058] Furthermore, if Figure 1 As shown, the present invention provides a method for preparing an injectable compound preparation, comprising the following steps:

[0059] According to the formula, thiamine pyrophosphate powder was selected and added to normal saline, and mixed evenly to obtain a mixed solution;

[0060] Semaglutide is added to the mixed solution to obtain the compound preparation.

[0061] The compound preparation prepared by the present invention comprises dissolving thiamine pyrophosphate in physiological saline and compounding the mixed solution with semaglutide to obtain a finished product. When used, the product is subcutaneously injected into a living organism (such as a mouse) at a certain frequency and dosage to cause weight loss and treat fatty liver.

[0062] Example 1:

[0063] According to the formula, 20 mg of thiamine pyrophosphate (TPP) powder was weighed and added to 100 ml of normal saline, and mixed well to obtain a mixed solution;

[0064] 3nmol(12.3*10 -3 mg) semaglutide was added to the mixed solution, shaken and dissolved to obtain a compound preparation.

[0065] In Example 1, the concentration of semaglutide was 30 nmol / kg, and the concentration of thiamine pyrophosphate was 200 mg / kg.

[0066] Example 2:

[0067] The preparation steps are the same as those in Example 1, except that in Example 2, the concentration of semaglutide is 30 nmol / kg, and the concentration of thiamine pyrophosphate is 150 mg / kg.

[0068] Example 3:

[0069] The preparation steps are the same as those in Example 1, except that in Example 2, the concentration of semaglutide is 20 nmol / kg, and the concentration of thiamine pyrophosphate is 150 mg / kg.

[0070] Comparative Example 1:

[0071] The method is similar to that of Example 1, except that the drug solution contains only 30 nmol / kg semaglutide.

[0072] Comparative Example 2:

[0073] The same as the embodiment 1, except that the liquid medicine only contains 200 mg / kg of thiamine pyrophosphate.

[0074] Comparative Example 3:

[0075] The method is similar to that of Example 1, except that the medicinal solution only contains 150 mg / kg of thiamine pyrophosphate.

[0076] The functions of the embodiment are verified by a verification example as follows:

[0077] Verification Example 1: Functional verification of compound preparation in mice

[0078] (1) Compared with the use of semaglutide alone, the compound preparation has excellent fatty liver treatment / relief effect

[0079] In vivo experiments: Obese mice weighing 45 g were treated with the above drugs, injected subcutaneously, for 14 days (see the results). Figure 2 ),from Figure 3 、 Figure 4 as well as Figure 5 It can be seen that the weight loss effects of Examples 1 and 2 are better than those of semaglutide alone. Although the weight loss effect of Example 3 is lower than that of semaglutide, it is better than semaglutide in treating fatty liver.

[0080] Figure 3 This is the weight loss curve after taking the medicine. Figure 4 is the weight on the last day of medication; Figure 5 The changes of body weight and fatty liver of mice on the last day of different dosing regimens are as follows: Figure 6 It is the fatty liver score. The higher the score, the more severe the fatty liver.

[0081] Figure 7 、 Figure 8 、 Figure 9 These are the changes in serum triglycerides, cholesterol and blood sugar in mice after medication. It can be seen that the compound preparation has certain advantages.

[0082] Figure 10This is a statistical chart of changes in ALT and AST in mice, representing the severity of liver damage in mice, because fatty liver caused by obesity will have elevated transaminase.

[0083] According to the examples, after using the compound preparation disclosed in the present invention, the transaminase level of mice was reduced, and the compound preparation had a better effect.

[0084] (2) Compared with the use of thiamine pyrophosphate alone, the compound preparation has excellent weight loss and fatty liver treatment / relief effects

[0085] In vivo experiments: Obese mice were treated with Comparative Example 2 and Comparative Example 3 by intraperitoneal injection (twice a week for two months). After medication, it was found that the weight of the mice in the treatment group was significantly reduced, and there was a dose-dependent effect ( Figure 11 、 Figure 12 ), after dissecting the liver specimens of mice, it was found that the fatty liver of mice in the treatment group was significantly alleviated ( Figure 13 ), obesity and fatty liver related biochemical metabolic indicators were significantly improved ( Figures 14-19 ), suggesting that thiamine pyrophosphate can alleviate fatty liver and achieve basal weight loss in animal experiments.

[0086] In vitro experiments: In cell experiments, free fatty acids (FFA) were used to simulate the external environment of fatty liver to stimulate HepG2 cells, causing HepG2 cells to produce lipid droplets and oxidative stress. After the cells were treated with different concentrations of TPP, it was seen that the production of lipid droplets and oxidative stress in HepG2 cells were alleviated to varying degrees ( Figure 20 、 Figure 21 ), demonstrating that TPP can alleviate lipid accumulation in hepatocytes in vitro.

[0087] Comparing the examples with the comparative examples, it can be seen that the compound preparation has excellent weight loss and fatty liver treatment / relief effects compared to the use of thiamine pyrophosphate alone. However, compared with the weight loss effect, this compound preparation mainly alleviates fatty liver by specifically inhibiting the formation of fatty liver.

[0088] Comparing the data of Verification Example 1 with that of Verification Example 2, this compound preparation has excellent adaptability for some thin patients who need to alleviate fatty liver disease. By adjusting the ratio of thiamine pyrophosphate and semaglutide, it can improve the therapeutic effect of fatty liver disease while keeping the patient's weight loss within a reasonable range.

[0089] Verification Example 3: TPP Regulation Mechanism

[0090] ENO1 can activate the expression of cholesterol regulatory element binding proteins (SREBPs), and the expression of SREBP1 can aggravate lipid synthesis in hepatocytes.

[0091] Through Limited proteolysis-small molecule mapping (LiP-Smap), the drug target of TPP is locked. Figure 22 、 23 ), and combined with metabolomics to further determine the target of TPP, ENO1 (Enolase1) ( Figure 24 、 25 ), and confirmed by SPR that ENO1 can indeed bind to TPP as the action site ( Figure 26 )

[0092] When HepG2 cells were treated with TPP, ENO1 protein expression decreased, however, ENO1 mRNA expression remained almost unchanged. We speculated that TPP may affect ENO1 expression at the protein level. We then used cycloheximide (CHX), proteasome inhibitor (MG132), and chloroquine (CQ) to confirm that TPP reduced ENO1 expression by accelerating ENO1 ubiquitination. Figure 27 ).

[0093] Through verification example 3, it can be seen that TPP can cause ENO1 to undergo ubiquitination degradation, that is, TPP can reduce the expression of SREBP1, thereby alleviating fatty liver ( Figure 28 ).

[0094] Through the injectable compound preparation of this embodiment and its preparation method and application, the weight loss effect of semaglutide can be improved, and it has the function of treating fatty liver to a certain extent, providing a new type of drug for the treatment of fatty liver.

[0095] The above embodiments are merely exemplary embodiments of the present application and are not intended to limit the scope of the present application. The scope of protection of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and scope of protection of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present application.

Claims

1. An injectable compound preparation, characterized in that: The compound preparation contains at least semaglutide, thiamine pyrophosphate and normal saline.

2. The injection compound preparation according to claim 1, characterized in that: The concentration of semaglutide is (8.2-12.3)*10 -2 mg / kg; The dosage of the thiamine pyrophosphate is 150-200 mg / / kg.

3. The injection compound preparation according to claim 1, characterized in that: The compound preparation is an injection.

4. A method for preparing the injectable compound preparation according to any one of claims 1 to 3, characterized in that: The steps include: According to the formula, thiamine pyrophosphate powder was selected and added to normal saline, and mixed evenly to obtain a mixed solution; Semaglutide is added to the mixed solution to obtain the compound preparation.

5. The method for preparing an injectable compound preparation according to claim 4, characterized in that: The mixing temperature was room temperature.

6. Use of the injectable compound preparation according to claims 1-5 for weight loss in mammals; The mammal is a mouse.

7. Use of the injectable compound preparation according to claims 1-5 in inhibiting or alleviating fatty liver.

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