Application of 2-hydroxybutyric acid and derivatives thereof in preparation of drugs for preventing and / or treating metabolic diseases

Abstract

The invention discloses application of 2-hydroxybutyric acid and derivatives thereof in preparation of drugs for preventing and / or treating metabolic diseases, and belongs to the technical field of biological medicines. Experiments prove that the 2-hydroxybutyric acid and the derivatives thereof can effectively inhibit obesity induced by high fat diet and reduce body fat, and have good improvement effects on related glucose and lipid metabolism disorders, including reduction of fasting blood-glucose, improvement of glucose tolerance, reduction of liver lipid deposition and improvement of liver functions. More importantly, the 2-hydroxybutyric acid does not affect ingestion, the metabolism improvement effect of the 2-hydroxybutyric acid depends on activation of fat heat production, and energy consumption can be directly promoted, so that obvious gastrointestinal tract adverse reactions cannot be generated, and potential mental side effects of existing appetite inhibiting drugs do not exist.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of 2-hydroxybutyric acid and its derivatives in preparing drugs for preventing and / or treating metabolic diseases. Background Art

[0002] Obesity is a chronic non-communicable disease and a risk factor for other chronic diseases such as type 2 diabetes, atherosclerotic cardiovascular disease, non-alcoholic fatty liver disease, and tumors. Over the past fifty years, obesity and related metabolic diseases have become a global pandemic. According to statistics, the number of obese people in the world has reached 1 billion, accounting for about 1 / 8 of the total population. Therefore, obesity has become an important research topic and a key direction in the field of global health.

[0003] The pathological process of obesity is complex, mainly manifested as the excess energy caused by energy intake exceeding consumption is stored in fat in the form of triglycerides, leading to a series of metabolic disorders. Therefore, reducing energy intake and / or promoting energy consumption is the core way to inhibit obesity. Traditional obesity prevention and treatment strategies include lifestyle (diet, exercise) intervention, the use of weight loss drugs and necessary weight loss surgery, but each has its limitations and has failed to effectively curb the rapidly growing incidence. Diet and exercise intervention has limited effects and is difficult to adhere to, and weight is prone to rebound after stopping intervention; weight loss surgery has surgical risks and complications; existing weight loss drugs are mainly appetite suppressants, and there are many cardiovascular and psychiatric adverse reactions. For example, the GLP-1 receptor agonists that have been launched in recent years have significant weight loss effects, but require injections, are inconvenient for long-term use, have severe gastrointestinal reactions, and are intolerant to some users. Therefore, it is still necessary to develop new weight loss preparations that are safer, more effective, convenient to use, and suitable for population promotion.

[0004] Studies in recent years have shown that intestinal flora and its metabolites participate in host energy metabolism and have become new targets for the development of weight loss drugs. It has been reported that under cold stimulation, fat thermogenesis in mice is activated and energy metabolism is significantly increased, but this effect is inhibited in mice that have cleared intestinal flora, indicating the important role of intestinal flora in host energy metabolism, but it is not clear which intestinal microorganisms and corresponding metabolites play a key role. The applicant's research team's preliminary research found that cold stimulation can increase 2-hydroxybutyric acid (2-HB) in mouse plasma, and some studies have reported that some probiotics that can improve obesity metabolism can produce 2-hydroxybutyric acid, but there is no research report on whether 2-hydroxybutyric acid can directly treat / improve obesity. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to disclose for the first time a new medical use of 2-hydroxybutyric acid and its derivatives in preventing and / or treating obesity.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention discloses for the first time the use of 2-hydroxybutyric acid (2-HB) and its derivatives in preparing medicines for preventing and / or treating metabolic diseases.

[0008] Preferably, the 2-hydroxybutyric acid derivatives include pharmaceutically acceptable salts, esters, isomers and other derivatives thereof.

[0009] Preferably, the metabolic disease is obesity, hyperglycemia, diabetes, fatty liver disease.

[0010] Preferably, the drug is a drug for lowering fasting blood sugar.

[0011] More preferably, the drug is directed against blood sugar metabolism disorders and liver lipid metabolism disorders.

[0012] Preferably, the drug is a drug for improving glucose tolerance.

[0013] Preferably, the drug is a drug for reducing liver lipid deposition.

[0014] Preferably, the drug is a drug for improving liver function.

[0015] Preferably, the drug is administered orally, by injection or by permeation.

[0016] Further preferably, when the administration route is oral, pharmaceutically acceptable excipients may be added, and the drug may be prepared into various forms such as tablets, oral liquids, granules, capsules, etc. according to conventional methods in the pharmaceutical field.

[0017] Further preferably, when the route of administration is injection, pharmaceutically acceptable excipients may be added, and the drug may be prepared into solution, suspension, emulsion, powder injection, lyophilized powder injection, large-volume injection, small-volume injection, and sterile powder for injection, etc., according to conventional methods in the pharmaceutical field.

[0018] Further preferably, when the route of administration is osmosis, an oral osmotic pump preparation (such as osmotic pump tablets) can be made. After oral administration, the drug is released at a constant rate in the gastrointestinal tract, maintaining a stable blood drug concentration, reducing the number of medications, and improving patient compliance.

[0019] Preferably, the structure of the 2-hydroxybutyric acid and its derivatives is shown in Formula I or Formula II below:

[0020]

[0021] In the formula, R1 is H, alkyl, alkoxy, cycloalkyl or aryl;

[0022] In formula I, R2 is H, alkyl, alkoxy, cycloalkyl or aryl;

[0023] In formula II, R2 is a metal ion, and the value of n is determined according to the valence state of the metal ion.

[0024] Preferably, 2-hydroxybutyric acid and its derivatives are L-form, D-form or a mixture of L-form and D-form.

[0025] Preferably, the 2-hydroxybutyric acid and its derivatives can be produced by chemical synthesis or bacterial fermentation.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discloses for the first time a new pharmaceutical use of 2-hydroxybutyric acid in metabolic diseases. Experiments have confirmed that 2-hydroxybutyric acid and its derivatives can effectively inhibit obesity induced by a high-fat diet, reduce body fat, and have good improvement effects on related sugar and lipid metabolism disorders, including reducing fasting blood sugar, improving glucose tolerance, reducing liver lipid deposition, and improving liver function. More importantly, 2-hydroxybutyric acid does not affect food intake. Its improvement effect on metabolism depends on the activation of fat thermogenesis, which can directly promote energy consumption, so it will not produce obvious gastrointestinal adverse reactions, and there are no potential psychiatric side effects of existing appetite suppressants.

[0028] Furthermore, 2-hydroxybutyric acid has no abnormal odor, good oral tolerance, high safety, no obvious side effects, good drugability and low cost. Therefore, oral drugs prepared with 2-hydroxybutyric acid and its derivatives as the main active ingredients for improving obesity and related metabolic diseases have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure shows the up-regulation effect of cold stimulation on 2-hydroxybutyrate in mouse plasma; (a) is the score diagram of principal component analysis (PCA) of mouse plasma metabolomics, and (b) is the concentration of 2-hydroxybutyrate (2-HB) in plasma.

[0030] Figure 2 Oral administration of 2-hydroxybutyrate inhibits high-fat diet-induced weight gain in mice.

[0031] Figure 3 Oral administration of 2-hydroxybutyrate reduces fat accumulation in mice induced by a high-fat diet; (a) is the body fat growth curve of mice, (b) is the body composition of mice, (c) is a representative adipose tissue image of mice, and (d) is a representative adipose tissue weight.

[0032] Figure 4Oral administration of 2-hydroxybutyrate improves blood glucose metabolism in obese mice; (a) is the fasting blood glucose level of mice, (b) is the oral glucose tolerance test, and (c) is the insulin tolerance test.

[0033] Figure 5 Oral administration of 2-hydroxybutyrate promotes the energy metabolism of mice; wherein, (a) is the energy consumption of mice per unit time, (b) is the cumulative energy consumption of mice during the day, night and the whole day, (c) is the oxygen consumption of mice per unit time, and (d) is the cumulative oxygen consumption of mice during the day, night and the whole day.

[0034] Figure 6 Oral administration of 2-hydroxybutyrate improves liver lipid metabolism in obese mice; wherein, (a) is the weight of mouse liver, (b) is the triglyceride content in liver, and (c) is hematoxylin-eosin staining of liver paraffin sections.

[0035] Figure 7 The effects of long-term intervention with 2-hydroxybutyrate on the main functional organs of mice were used to evaluate possible side effects; (a) is food intake, (b) is representative muscle weight, (c) is spleen weight, (d) is heart weight, (e) is serum alanine aminotransferase level, and (f) is serum aspartate aminotransferase level.

[0036] Figure 8 2-Hydroxybutyrate directly promotes energy metabolism in adipocytes; (a) is a seahorse cell mitochondrial stress test, and (b) is the expression level of Ucp1, a key gene for thermogenesis, in mature adipocytes after 2-Hydroxybutyrate intervention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. 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 creative work should fall within the scope of protection of the present invention.

[0038] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0040] The structural formula of 2-hydroxybutyric acid and its derivatives is shown in the following formula I or formula II:

[0041]

[0042] R1 in Formula I or Formula II may be H, alkyl, alkoxy, cycloalkyl or aryl;

[0043] R2 in Formula I can be H, alkyl, alkoxy, cycloalkyl or aryl;

[0044] R2 in Formula II can be a metal ion (such as Na + , K + , Ca 2+ wait);

[0045] The value of n in formula II is determined according to the valence state of the metal ion.

[0046] The 2-hydroxybutyric acid used in the following examples of the present invention was purchased from MedChemExpress (MCE) with a product number of HY-113381.

[0047] Example 1 Effect of cold stimulation on plasma 2-HB levels in mice

[0048] 1. Experimental methods:

[0049] Twenty 10-week-old C57BL / 6J male mice (purchased from Saiye Bio) were kept in an SPF-level environment with an environmental humidity of 50%-60%, a temperature of 20±2℃, a circadian rhythm of 12h / 12h, and free access to water and food. After one week of adaptive feeding, they were randomly divided into three groups: control group (22℃; n=7), 4℃ cold stimulation 6-hour group (4℃-6h; n=6), and 4℃ cold stimulation 48-hour group (4℃, 48h; n=7), and were given corresponding temperature stimulation. At the end of the experiment, mouse heart blood was collected and placed in an EDTA-coated centrifuge tube, centrifuged at 4℃ and 4000rpm for 15 minutes, and plasma was separated. The plasma metabolomics was detected by LC-MS technology (metabolic omics detection was completed by Matt Spectrum Biotechnology (Shanghai) Co., Ltd.).

[0050] 2. Analysis of experimental results:

[0051] Cold stimulation significantly altered the plasma metabolomics of mice, such as Figure 1 As shown in (a); compared with the control group (22°C), 4°C cold stimulation for 6 hours significantly increased the level of 2-HB in plasma. Although the change was not as obvious as that of 6 hours, the 48-hour cold stimulation still showed an upward trend compared with 22°C. Figure 1 As shown in (b).

[0052] Example 2 Effect of 2-HB intervention on obesity in mice

[0053] 1. Experimental Principle of the Embodiment

[0054] This experiment is based on the fact that long-term high-fat diet induces weight and fat gain, energy metabolism imbalance, and abnormal glucose and lipid metabolism in mice. The changes in weight and fat, energy metabolism, glucose metabolism, and liver lipid metabolism were detected in the diet-induced obese mouse model to evaluate the preventive effect of oral supplementation of 2-hydroxybutyrate on obesity in mice.

[0055] 2. Experimental Materials and Methods

[0056] (1) Preparation of 2-hydroxybutyric acid (2-HB) solution.

[0057] Add 2-HB to PBS, dissolve and mix thoroughly to make a 10 mg / mL solution for immediate use.

[0058] (2) Constructing animal disease models and implementing interventions

[0059] 1) Feed source:

[0060] The food used to induce the disease animal model was a 60% high-fat feed for mice, produced by Ruidi Biological Company, and the feed ratio was Ruidi Biological RD21120802.

[0061] 2) Establishment of obese mouse model:

[0062] Fourteen 10-week-old C57BL / 6J male mice (purchased from Saiye Bio) were kept in an SPF-grade environment with an ambient humidity of 50%-60%, a temperature of 20±2°C, a circadian rhythm of 12h / 12h, and free access to water and food. A 60% high-fat diet (HFD) was fed for 8 weeks to induce an obesity disease model with obesity, hyperlipidemia, hyperglycemia, insulin resistance, etc.

[0063] 3) 2-HB intervention

[0064] Before high-fat diet induction, mice were randomly divided into a control group (n=6) and a 2-HB intervention group (2-HB group, n=8) according to body weight, body fat, and blood glucose. They were given PBS and 100 mg / kg body weight 2-HB by gavage once a day for 8 weeks.

[0065] 4) Monitoring indicators

[0066] During the intervention period, the body weight, drinking water and food intake of mice were monitored weekly. The body fat content of mice was detected weekly using the awake animal body composition analyzer Echo MRI E26 340M. In the fourth week of intervention, the awake animal energy monitoring system was used to analyze the energy consumption and oxygen consumption of mice. In the fifth week of intervention, mice were fasted overnight and an oral glucose tolerance test was performed. In the sixth week of intervention, mice were fasted for 5 hours and an insulin tolerance test was performed. In the eighth week of intervention, mice were killed and the main tissues and organs were separated and weighed, and the triglyceride content of the liver was detected. Paraffin sections of liver tissue were HE stained to evaluate liver lipid metabolism. The levels of alanine aminotransferase and aspartate aminotransferase in plasma were detected.

[0067] 3. Analysis of experimental results.

[0068] After 8 weeks of high-fat diet feeding, mice treated with 2-hydroxybutyrate (2-HB) were significantly protected against high-fat diet-induced weight gain ( Figure 2 ); At the same time, body fat analysis showed that 2-hydroxybutyrate significantly slowed down the increase in body fat caused by high fat, such as Figure 3 As shown in (a), the main reason for 2-HB to reduce weight is the reduction of fat content, without affecting muscle content, such as Figure 3 As shown in (b), the figure includes three indicators: body weight (BW), body fat (fat) and lean meat (lean); the size and weight of adipose tissue of mice after 2-HB intervention were significantly lower than those of the control group, as shown in Figure 3 As shown in (c), (d) and (e).

[0069] In addition, 2-HB significantly reduced fasting blood glucose in obese mice. Figure 4 As shown in (a), improving glucose tolerance, such as Figure 4In the insulin tolerance test, 2-HB also reduced fasting blood sugar, but had no significant effect on insulin sensitivity, as shown in (b). Figure 4 As shown in (c). The analysis of mouse energy metabolism showed that 2-HB significantly promoted the energy and oxygen consumption of mice and improved energy metabolism. The results are shown in Figure 5 .

[0070] In addition, the liver weight of mice in the 2-HB intervention group was reduced compared with that in the control group. Figure 6 As shown in (a); the detection of triglyceride content in the liver showed that 2-HB significantly reduced the lipid content in the liver, such as Figure 6 As shown in (b); HE staining of liver tissue showed that 2-HB improved obesity-related liver steatosis. Figure 6 More importantly, 2-HB has no special smell, and mice have no adverse reactions during oral administration, and the acceptance is good. Moreover, 2-HB intervention does not affect the food intake of mice ( Figure 7 (a)), muscle content ( Figure 7 (b)) nor did it cause spleen enlargement ( Figure 7 (c)) and cardiac hypertrophy ( Figure 7 (d)), and reduced serum alanine aminotransferase levels ( Figure 7 (e)) and aspartate aminotransferase content ( Figure 7 These results indicate that 2-HB does not cause liver damage but has a liver-protective effect.

[0071] Example 3 Effect of 2-HB on adipocyte energy metabolism

[0072] 1. Experimental methods:

[0073] (1) Seahorse cell energy metabolism: Immortalized mouse brown adipocytes (DE-2-3) were seeded in 10 cm culture plates using the culture medium: DMEM + 10% fetal bovine serum + 1% penicillin and streptomycin. When the cell density reached 80%, the cells were subcultured into Seahorse XF 96-well cell culture plates and subjected to a cell mitochondrial stress test (XF Cell Mito Stress Test) using the Seahorse XFe96 Energy Metabolism Analyzer (Agilent).

[0074] (2) Thermogenesis of mature adipocytes: Immortalized mouse brown adipocytes (DE-2-3) were inoculated in 10 cm culture plates, and the growth medium was: DMEM + 10% fetal bovine serum + 1% penicillin and streptomycin. When the cell density reached 80%, the cells were subcultured and inoculated into 12-well cell culture plates. Fresh medium was replaced every 2 days until the cell density reached 80%, and the differentiation medium (growth medium + 20nM insulin + 1nM triiodothyronine) was replaced. After 2 days, the induction medium (differentiation medium + 0.5mM 3-isobutyl-1-methylxanthine + 0.5uM dexamethasone + 0.125mM indomethacin) was replaced. After 48 hours, the culture medium was replaced with differentiation medium, and fresh medium was replaced every 2 days until the cells were fully differentiated (after 6 days of differentiation). Mature adipocytes were treated with PBS, 25uM 2-HB, 50uM 2-HB, 100uM 2-HB, 250uM 2-HB, and 2500uM 2-HB for 24 hours, respectively. The cells were harvested, RNA was extracted by Trizol method, and the expression of Ucp1, a key gene for thermogenesis, was detected by q-PCR.

[0075] 2. Analysis of experimental results:

[0076] 2-HB can directly promote the energy metabolism of fat cells, and this effect is gradient-dependent, such as Figure 8 As shown in (a); at the same time, 2-HB can upregulate the level of Ucp1, a key gene for thermogenesis in mature adipocytes, indicating that 2-HB may promote energy consumption by promoting thermogenesis in adipocytes, such as Figure 8 As shown in (b).

[0077] In summary, 2-hydroxybutyrate (2-HB) can be upregulated in response to energy metabolism needs when exposed to cold stimulation; unlike existing weight loss drugs, or other short-chain fatty acids (such as butyrate) and organic acids (such as 3-hydroxybutyrate) that achieve weight loss by suppressing appetite and limiting energy intake, 2-HB has good oral tolerance and can directly promote fat thermogenesis without affecting food intake, accelerate energy consumption, significantly improve obesity and related sugar and lipid metabolism disorders, have no potential mental side effects, and have a good protective effect on the liver, good safety, and excellent application prospects and drug development potential.

[0078] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Use of 2-hydroxybutyric acid and its derivatives in the preparation of drugs for preventing and / or treating metabolic diseases.

2. The use according to claim 1, characterized in that The metabolic diseases are obesity, hyperglycemia, diabetes, and fatty liver disease.

3. The use according to claim 1, characterized in that The medicine is a medicine for lowering fasting blood sugar.

4. The use according to claim 1, characterized in that The medicine is a medicine for improving glucose tolerance.

5. The use according to claim 1, characterized in that The medicine is a medicine for reducing liver lipid deposition.

6. The use according to claim 1, characterized in that The medicine is a medicine for improving liver function.

7. The use according to claim 1, characterized in that The drug is administered orally, by injection or by penetration.

8. The use according to claim 7, characterized in that The drug is prepared into tablets, granules, oral liquid or capsules according to pharmaceutically acceptable dosage forms.

9. The use according to any one of claims 1 to 8, characterized in that The structure of the 2-hydroxybutyric acid and its derivatives is shown in Formula I or Formula II below: In the formula, R1 is H, alkyl, alkoxy, cycloalkyl or aryl; In formula I, R2 is H, alkyl, alkoxy, cycloalkyl or aryl; In formula II, R2 is a metal ion, and the value of n is determined according to the valence state of the metal ion.

10. The use according to claim 9, characterized in that 2-Hydroxybutyric acid and its derivatives are L-type, D-type or a mixture of the two.

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