CN105311008B - The purposes of stearoyl amino acid or its pharmaceutical salts - Google Patents

Abstract

The invention discloses the use of a stearoyl amino acid or a pharmaceutically acceptable salt thereof in the preparation of weight-loss drugs. The stearoyl amino acid has a structural formula shown in the following formula (I), wherein R ₁ represents H or can be Aryl or C _1-4 straight chain or branched alkyl substituted by one or more substituents, the substituents are alcoholic or phenolic hydroxyl groups; R ₂ represents C _11-25 saturated or unsaturated Saturated aliphatic hydrocarbon group. The invention also discloses a pharmaceutical composition containing stearoyl amino acid or a pharmaceutically acceptable salt thereof. The stearyl amino acid or the medicinal salt thereof of the present invention has been confirmed by preclinical pharmacokinetic experiments, not only can significantly reduce the body weight of obese mice, but also has high safety, and is expected to become a clinical weight loss drug with broad application prospects.

Description

Use of stearoyl amino acid or its pharmaceutically acceptable salt

technical field

The invention relates to the technical field of medicine, in particular to a new application of a stearyl amino acid or a pharmaceutically acceptable salt thereof.

Background technique

Obesity is the primary cause of diseases such as diabetes, cardiovascular and cerebrovascular diseases, and non-alcoholic fatty liver. The global adult obesity rate shows a sharp increase year by year, reaching 22% in 2013. Although appetite suppressants such as sibutramine and rimonabant can effectively reduce weight, side effects such as depression eventually led to the withdrawal of these two weight loss drugs from the market. The FDA-approved weight-loss drug Orlistat also found rare cases of liver injury in Phase IV clinical monitoring. Due to the complex etiology of obesity, an ideal weight loss drug should have good safety and be able to promote fat decomposition and inhibit fat absorption and synthesis by regulating multiple related metabolic pathways in the body. At present, the research on obesity treatment drugs at home and abroad is mainly focused on the discovery of natural products that promote β-oxidation (the main pathway of fat catabolism), but such natural products often cannot obtain the desired curative effect in obese animal models. At the same time, their Safety evaluation results also limit the development of such drugs. Therefore, the discovery of weight loss drugs with novel mechanisms of action not only has a major social demand, but also has important theoretical significance.

The N-stearyl amino acid (such as N-stearyl tyrosine, NsTyr) or its pharmaceutically acceptable salt (such as N-stearyl tyrosine dipotassium salt, NST-2K) developed by our laboratory is an internal A novel analogue of anandamide (AEA), which exerts neuroprotective effects by interfering with the metabolism of the endocannabinoid system (ECS).

Previous studies on stearoyl amino acid and its salts focused on the neuroprotective effect of this compound, and there is no public report on stearoyl amino acid or its pharmaceutically acceptable salt used as a weight loss drug.

Contents of the invention

The present invention aims to solve the technical problem of lack of safe and effective weight-loss drugs at present, and provides a use of stearyl amino acid or a medicinal salt thereof in preparing weight-loss drugs.

In order to solve the problems of the technologies described above, the present invention is realized through the following technical solutions:

In one aspect of the present invention, a use of a stearoyl amino acid or a pharmaceutically acceptable salt thereof in the preparation of weight-loss drugs is provided, the stearoyl amino acid has a structural formula shown in the following formula (I):

Wherein, R ₁ represents H or an aryl group that may be substituted by one or more substituents or a C _1-4 straight chain or branched alkyl group, and the substituents are alcoholic or phenolic hydroxyl groups; R ₂ represents C _11-25 saturated or unsaturated aliphatic hydrocarbon group.

The above-mentioned C _1-4 alkyl group refers to a straight chain or branched chain alkyl group with 1 to 4 carbon atoms, for example: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl base, sec-butyl. An alkyl group having 1 to 2 carbon atoms is preferred.

The above-mentioned C _11-25 aliphatic hydrocarbon group refers to a saturated or unsaturated aliphatic hydrocarbon group with 11 to 25 carbon atoms, wherein the saturated aliphatic hydrocarbon group refers to a linear or branched alkyl or cycloalkyl group, such as dodecyl Alkyl, octadecyl, cyclododecyl, cyclooctadecyl, etc., unsaturated aliphatic hydrocarbon group refers to alkenyl, alkynyl or alkenyl, alkenyl such as 1-dodecenyl , 2-dodecenyl, alkynyl such as 1-octadecynyl, 2-octadecynyl, alkadienyl such as 1,3-octadecenyl, 7,9-octadecenyl, etc. A linear or branched alkyl group having 17 to 25 carbon atoms is preferred, and a linear alkyl group having 17 carbon atoms is particularly preferred.

Preferably, the stearoyl amino acid is N-stearyl tyrosine of the following formula (II):

Preferably, the stearoyl amino acid salt has the following structural formula (III) or formula (IV):

Wherein, M in formula (III) is a monovalent metal cation or NH ₄ ⁺ , and M in formula (IV) is a divalent metal cation.

Preferably, M in the formula (III) is selected from K ⁺ , Na ⁺ , or NH ₄ ⁺ .

Preferably, M in the formula (IV) is selected from Ba ²⁺ , Ca ²⁺ , or Mg ²⁺ .

In another aspect of the present invention, a pharmaceutical composition for weight loss is also provided, which comprises a safe and effective amount of stearyl amino acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The above-mentioned acceptable carrier is non-toxic, can assist administration and has no adverse effect on the therapeutic effect of stearoyl amino acid or its pharmaceutically acceptable salt. Such carriers can be any solid, liquid, semisolid or, in aerosol compositions, gaseous excipients commonly available to those skilled in the art. Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl stearyl, sodium chloride , Anhydrous skim milk, etc. Liquid and semisolid excipients can be selected from glycerin, propylene glycol, water, ethanol and various oils, including those derived from petroleum, animal, vegetable or synthetic oils, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc., preferably Liquid carriers, especially for injectable solutions, include water, saline, aqueous dextrose and glycol. In addition, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.

The stearoyl amino acid of the present invention or pharmaceutically acceptable salt thereof is administered in a therapeutically effective amount, and the mode of administration may be oral, systemic (for example, transdermal, nasal inhalation or suppository) or parenteral ( For example, intramuscularly, intravenously or subcutaneously). The preferred mode of administration is oral, which can be adjusted according to the extent of the disease.

The actual administration amount (i.e. active ingredient) of stearoyl amino acid or pharmaceutically acceptable salt thereof of the present invention depends on many factors, such as the severity of the disease to be treated, the age and relative health of the subject to be treated, the nature of the compound used, Potency, route and form of administration, and other factors.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional methods in the field of pharmacy. For example, the stearoyl amino acid salt (active ingredient) is mixed with one or more carriers, and then it is made into a desired dosage form, such as tablet, pill, capsule, semi-solid, powder, sustained-release dosage form, solution, Suspensions, formulations, aerosols, etc.

In another aspect of the present invention, a use of stearyl amino acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating fatty liver is also provided.

When the stearoyl amino acid or its pharmaceutically acceptable salt of the present invention is orally administered to KM mice in preclinical pharmacokinetic experiments, the results show that the stearoyl amino acid or its pharmaceutically acceptable salt can not only significantly reduce the body weight of obese mice , and with high safety, it is expected to become a clinical weight-loss drug, and its application prospect is very broad.

Description of drawings

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Fig. 1 is the diagram of the body weight changes of mice in each group during 11 weeks of dietary intervention in Example 1 of the present invention;

Fig. 2 is a histogram of the average food intake of mice in each group during 11 weeks of dietary intervention in Example 1 of the present invention;

Fig. 3 is the graph of the body weight change of each group of mice during the 4 weeks of administration in Example 2 of the present invention;

Fig. 4 is the histogram of the average food intake of mice in each group during the administration of Example 2 of the present invention for 4 weeks;

Fig. 5 is the mouse abdominal cavity fat photo figure of embodiment 3 of the present invention;

Fig. 6 is the H&E staining photomicrograph (scale bar=100 μm) figure of epididymis fat tissue section of embodiment 4 of the present invention;

Fig. 7 is a histogram of the size of epididymal tissue fat cells in Example 4 of the present invention;

Figure 8 is a micrograph (scale bar=50 μm) of liver tissue section H&E staining in Example 4 of the present invention;

Fig. 9 is the equilibrium solubility curve and the apparent lipid-water partition coefficient curve diagram of Example 5 of the present invention;

Fig. 10 is a graph showing blood drug concentration-time curves of NST-2K and NsTyr in rats in Example 5 of the present invention after oral gavage.

In Fig. 3-8, ND, standard feed blank control group; HFDⅠ, high-fat feed blank control group; HFDⅡ, low-dose NST-2K group (20mg/kg/day); HFDⅢ, medium-dose NST-2K group (60mg/kg/day); kg/day); HFDⅣ, high-dose NST-2K group (100mg/kg/day); HFDⅤ, positive control group (Orlistat100mg/kg/day).

Detailed ways

The establishment of embodiment 1 obesity mouse model

1. Purpose: To establish an obese mouse model for testing the weight loss activity of stearoyl amino acid or its medicinal salt.

2. Methods: 60 C57BL/6 mice (3 weeks old, male, ~8.3 g) were adaptively fed with 12% kcal standard diet for one week, and randomly divided into 6 groups, 10 mice in each group. One group (ND) continued to be fed with standard feed, and the other five groups (HFDⅠ～Ⅴ) were fed with 45% kcal high-fat feed. Record the average body weight and food intake of each group every week until there is a significant difference in body weight between the two feed groups, while there is no significant difference in body weight between the five high-fat feed groups, and maintain this stable state for 3 weeks, which can be considered Modeling succeeded.

The mice were kept in the following environment during the whole experiment: temperature 23±3°C, humidity 50±5%, light and dark cycle 12h/12h, and the mice had free access to food and water throughout the experiment.

3. Results:

1) As shown in Figure 1, feed with different feeds until the 9th week (in Figure 1, the abscissa is 10, because the abscissa 0-1 stage is the adaptive feeding stage, and the six groups are all fed with standard feed at this stage , different feeds were used after the abscissa 1), the body weight of mice in the five high-fat diet groups (HFDⅠ～Ⅴ) was significantly higher than that in the standard diet group (ND), and the weight of mice in the five high-fat diet groups (HFDⅠ～Ⅴ) ), there was no significant difference in body weight of the mice. After the above-mentioned state of weight difference lasted for 3 weeks, the obesity model was successfully established (11 weeks of feeding with different feeds). At this time, the average body weight of the standard feed group (ND) was about 28.3 g, and the average body weight of the five high-fat feed groups (HFDⅠ-Ⅴ) was about It is 32.8g. In Fig. 1, all values are expressed in the form of mean ± standard error, n=10, and different letters indicate significant differences between the two (P<0.05).

2) As shown in Figure 2, during the 11-week period of dietary intervention (that is, during the feeding period of different feeds), the average daily food intake of the mice in the standard diet group (ND) was significantly higher than that of the high-fat diet groups (HFDⅠ-Ⅴ), but each There was no significant difference in the average daily food intake of the mice among the high-fat diet groups (HFDⅠ～Ⅴ). In Fig. 2, all values are expressed in the form of mean±standard error, n=10, *** indicates significant difference compared with ND (P<0.001).

4. Conclusion: The mouse obesity model was successfully established, and the appetite of the mice in the high-fat diet groups (HFDⅠ～Ⅴ) was similar in the modeling stage.

Embodiment 2 administration experiment

1. Purpose: To observe the effect of N-stearyl amino acid salt (this embodiment takes N-stearyl tyrosine dipotassium salt, NST-2K as an example) on the body weight of obese mice after administration, and compare with positive drug Orlistat Control Comparison.

2. Methods: ND and HFDⅠ were used as standard feed and high-fat feed blank control group respectively, and were given 0.5% sodium carboxymethylcellulose aqueous solution (5ml/kg/day); HFDⅡ～Ⅳ were used as the test group, and NST-2K ( 20, 60, 100 mg/kg/day); HFDⅤ was used as a positive control group, and Orlistat (100 mg/kg/day) was given. All the drugs were dissolved in 0.5% sodium carboxymethylcellulose aqueous solution and administered by intragastric administration. The administration lasted for 4 weeks, and the average body weight and food intake of each group were recorded every week until the end of the administration period.

3. Results:

1) As shown in Figure 3, during the administration period, the weight gain of the obese groups HFDⅡ, HFDⅢ and HFDⅤ was slower than that of the blank obese HFDⅠ, but at the end of the entire administration period, there was still no significant difference in body weight among the four groups of mice ; The body weight of HFDIⅣ in the obesity group showed a downward trend with the prolongation of the administration time, and at the end of the whole administration period, the body weight was significantly lower than that of HFDIⅣ. In Fig. 3, all values are expressed in the form of mean ± standard error, n=10, and different letters indicate significant differences between the two (P<0.05).

2) As shown in Figure 4, during the 4-week administration period, the average daily food intake of the mice in the standard diet group (ND) was significantly higher than that of the high-fat diet groups (HFDⅠ～Ⅴ), but between the high-fat diet groups ( There was no significant difference in the average daily food intake of HFDIⅠ～Ⅴ) mice. In Fig. 4, all values are expressed in the form of mean ± standard error, n=10, ^*** indicates significant difference compared with ND (P<0.001).

4 Conclusion:

1) It can be seen from Figure 3 that low and medium doses of NST-2K (20, 60mg/kg/day) and the positive drug Orlistat (100mg/kg/day) can inhibit the weight gain of obese mice, and high doses of NST-2K (100mg/kg/day) kg/day) can reduce the body weight of obese mice. At the same dose (100mg/kg/day), the weight loss activity of NST-2K is significantly better than that of Orlistat.

2) It can be seen from Figure 4 that during the administration period, the appetites of the mice in the high-fat diet groups (HFD Ⅰ-Ⅴ) were similar, indicating that neither NST-2K nor Orlistat exerted weight loss effects by suppressing appetite.

Example 3 Collection and Observation of Experimental Animal Fat and Liver Tissue

1. Purpose: To observe the fat content in the abdominal cavity of mice in each group, and to separate the liver and adipose tissue for subsequent H&E staining observation.

2. Method: After the administration, the mice were fasted overnight, and the abdominal cavity was opened to observe the abdominal fat content of the mice in each group, and the epididymal fat, perirenal fat and liver were separated and weighed.

3. Results:

1) As shown in Figure 5, the body size and abdominal fat pad of the mice in the standard diet group (ND) were significantly smaller than those in the high-fat diet groups (HFDⅠ～Ⅴ). The fat pad showed a shrinking trend compared with the blank obesity group (HFDⅠ), and the effect of HFDⅣ (high-dose NST-2K group) was the most obvious, which was better than that of HFDⅤ (Orlistat group).

2) As shown in Table 1, there was no significant difference in the percentage mass of liver among the high-fat diet groups (HFDⅠ～Ⅴ), but it was significantly lower than that in the standard diet group (ND); The weight of fat was significantly lower than that of the blank obesity group (HFD Ⅰ); compared with HFD Ⅰ, there was no significant difference in the fat percentage of epididymis and perirenal in HFD Ⅱ (low dose NST-2K group).

Table 1 The mass percentage of mouse liver and adipose tissue ^[a]

[a] All values are expressed as mean ± standard error, n=10. ^** , ^*** indicate significant difference compared with ND respectively (P<0.01, P<0.001). ^# 、 ^## 、 ^### P<0.001 means significant difference compared with HFDI (P<0.05, P<0.01, P<0.001). ND, standard feed blank control group; HFDⅠ, high-fat feed blank control group; HFDⅡ, low-dose NST-2K group (20mg/kg/day); HFDⅢ, medium-dose NST-2K group (60mg/kg/day); HFDⅣ , high-dose NST-2K group (100mg/kg/day); HFDⅤ, positive control group (Orlistat100mg/kg/day).

4. Conclusion: NST-2K can reduce the abdominal fat mass of obese mice, but has no significant effect on the liver mass. At the same dose (100mg/kg), NST-2K has a better effect on reducing abdominal fat mass than Orlistat.

Example 4 Observation of Fat and Liver Tissue Morphology by H&E Staining

1. Objective: To microscopically observe the morphology of fat and liver tissue.

2. Methods: Epididymis fat and liver were fixed in 10% neutral formalin solution for 48 hours, embedded in paraffin, cut into 5 μm sections, stained with hematoxylin-eosin (H&E staining), observed the size of fat cells with light microscope, liver Cell structure and fat accumulation.

3. Results:

1) As shown in Figure 6 and Figure 7, HFDI fat cells were significantly larger than ND, while HFD III, HFD IV and HFD V fat cells in the treatment group were significantly reduced compared with HFD I; low-dose NST-2K (HFD II) treatment could not significantly reduce fat cells. In Figure 7, all values are expressed in the form of mean ± standard error, n=10, ^*** indicates that there is a significant difference compared with ND (P<0.001), ^### indicates that there is a significant difference compared with HFDI (P <0.001).

2) As shown in Figure 8, HFDI liver tissue has severe fat accumulation, and after NST-2K or Orlistat administration, the hepatic fat accumulation tends to alleviate as the dosage increases.

4. Conclusion: NST-2K can reduce the size of adipocytes in obese mice, and alleviate the fatty changes of liver in obese mice.

The test experiment of embodiment 5N-stearyl amino acid salt physicochemical constant and pharmacokinetic parameter

1. Equilibrium solubility and apparent lipid-water partition coefficient

Determination of NsTyr (N-stearyl tyrosine), NST-2K (N-stearyl tyrosine dipotassium salt), NST-2Na (N-stearyl tyrosine dipotassium salt) at 37°C by shake flask method Sodium salt) Equilibrium solubility in water. The solubility of NsTyr is lower than 100μg/100ml water, the solubility of NST-2K and NST-2Na are 513.704μg/100ml water and 926.551μg/100ml water respectively; the apparent lipid-water partition coefficient logp of NST-2K and NST-2Na are 1.512, 1.477. The equilibrium solubility test data of NST-2K and NST-2Na at different pHs are shown in Table 2, and the apparent lipid-water partition coefficients are shown in Table 3. The equilibrium solubility curve and the apparent lipid-water partition coefficient curve are shown in Figure 9.

The equilibrium solubility of table 2NST-2K, NST-2Na in the buffer solution of different pH

pH value NST-2K(mg/L) NST-2Na(mg/L) 2.0 49.290 66.938 3.0 58.504 66.334 4.0 188.155 195.371 5.0 220.693 232.102 6.0 243.482 274.629 7.0 251.430 318.016 7.4 270.414 374.484 8.0 285.935 463.902 9.0 369.807 512.644

Table 3 Apparent lipid-water partition coefficients of NST-2K and NST-2Na in different pH buffers

pH value NST-2K Ko/w LogP NST-2Na Ko/w LogP 2.0 76.020 1.881 49.913 1.698 3.0 30.571 1.485 28.980 1.462 4.0 16.020 1.205 15.224 1.183 5.0 21.934 1.341 18.710 1.272 6.0 28.935 1.461 19.963 1.300

7.0 37.855 1.578 31.436 1.497 7.4 16.674 1.222 29.286 1.467 8.0 13.797 1.140 28.704 1.458 9.0 13.140 1.119 21.872 1.340

From the data of equilibrium solubility and apparent lipid-water partition coefficient in Table 2 and Table 3 above, as well as Figure 9, it can be seen that the low pH in the stomach causes a large amount of NST-2K and NST-2Na to be reduced to the original drug NsTyr, which passes through the biomembrane Absorption exerts the drug effect; after entering the intestinal segment, its equilibrium solubility increases sharply, and the apparent lipid-water partition coefficient drops sharply. The drug mainly exists in the form of ions, and it is difficult to absorb into the blood through the biomembrane. Compared with the original drug NsTyr, NST-2K, NST-2Na solubility has been improved.

2. Absorption Kinetic Data

Ten SD rats of 150-180 g were randomly divided into 2 groups, 5 in each group, both male and female. After intragastric administration of 200 mg/kg of NsTyr and NST-2K normal saline solution, the average blood drug concentration-time curves are shown in Figure 10, respectively. See Table 4 for the metabolic kinetic parameters of SD rats after oral administration of 200 mg/kg of NsTyr and NST-2K.

Table 4 The metabolic kinetic parameters after oral administration of 200mg/kg NsTyr and NST-2K in rats

parameter NsTyr NST-2K AUClast(h*μg/ml) 0.13 3.49 AUCinf(h*μg/ml) 0.14 3.69 MRTlast(h) 1.38 2.19 T1/2(h) 1.49 2.90 Tmax(h) 0.75 0.60 Cmax(μg/ml) 0.11 2.40

By comparing the metabolic kinetic parameters of the two, the area under the curve (AUC) of NST-2K increased by 30 times compared with NsTyr, the relative bioavailability was significantly improved, and the half-life was significantly prolonged (nearly twice); the peak concentration Cmax was greatly improved (about 20 times) and peak time Tmax sharply decreased, suggesting that NST-2K has a high absorption rate in the body, which can appropriately reduce the dosage, help reduce the occurrence of toxic and side effects, and increase the treatment window and safety factor. In preparation of formulations, limited by solubility, NsTyr can be made into suspension; salts with high solubility, NST-2K and NST-2Na, can be prepared into homogeneous solutions.

The above-mentioned embodiments only express the implementation manner of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (5)

1. Use of N-stearyl tyrosine or a pharmaceutically acceptable salt thereof shown in formula (II) in the preparation of weight-loss drugs 2. The use according to claim 1, characterized in that the pharmaceutically acceptable salt of said N-stearyl tyrosine has the structural formula of the following formula (III) or formula (IV): Wherein, M in formula (III) is a monovalent metal cation or NH ₄ ⁺ , and M in formula (IV) is a divalent metal cation. 3. The use according to claim 2, characterized in that, M in the formula (III) is selected from K ⁺ , Na ⁺ or NH ₄ ⁺ . 4. The use according to claim 2, characterized in that M in the formula (IV) is selected from Ba ²⁺ , Ca ²⁺ or Mg ²⁺ . 5. Use of N-stearyl tyrosine represented by formula (II) or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating fatty liver