Use of N-Fmoc-L-leucine as a drug for preventing cold exposure injury and its application in the preparation of a drug for preventing cold exposure injury

Abstract

The present invention provides an application of N-fluorenylmethoxycarbonyl-L-leucine in the preparation of a drug for preventing cold exposure injury and a drug for preventing cold exposure injury, and relates to the technical field of anti-freezing drugs. Through research, the present invention finds that N-fluorenylmethoxycarbonyl-L-leucine can be used to prepare a drug for preventing cold exposure injury, thereby providing an effective drug for personnel working in cold regions to potentially improve the body's cold resistance and anti-cold injury ability.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-freezing drugs, and in particular to the application of N-fluorenylmethoxycarbonyl-L-leucine in the preparation of drugs for preventing cold exposure injury and drugs for preventing cold exposure injury. Background Art

[0002] Cold exposure refers to the process in which the human body is exposed to a low-temperature environment for a long time, resulting in a series of physiological and biochemical reactions in the body. Such exposure can have various effects on the human body, including body temperature regulation, energy metabolism, and mental health.

[0003] China has a vast alpine region, covering large areas in the northeast, northwest, north China, etc. At the same time, China has a long border with North Korea, Russia, Mongolia, Pakistan, and India, which are important national defense strategic locations, and these locations are usually alpine regions. Among the operators performing various tasks such as national defense, transportation, and communication in alpine regions, frostbite caused by cold exposure occurs from time to time, posing a serious threat to the operation ability and life and health of the operators.

[0004] In view of this, the research and development of drugs for preventing cold exposure injury have become very necessary and urgent. Summary of the Invention

[0005] The purpose of the present invention is to provide a potential compound for preparing drugs to improve the body's cold resistance and anti-cold exposure injury, namely N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-L-leucine); and it has been proven by experiments that N-fluorenylmethoxycarbonyl-L-leucine can be used for the preparation of drugs for preventing cold exposure injury.

[0006] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0007] The present invention provides a compound that can be used as a drug for preventing cold exposure injury: N-fluorenylmethoxycarbonyl-L-leucine;

[0008] The structural formula of the N-fluorenylmethoxycarbonyl-L-leucine is:

[0009]

[0010] Further, the cold exposure injury is cold injury caused by a low-temperature environment.

[0011] Furthermore, the low temperature refers to 4 - 6 °C.

[0012] Further, the application refers to that N-fluorenylmethoxycarbonyl-L-leucine promotes adaptive thermogenesis of the body, alleviates the decrease in body temperature, and improves cold resistance in a cold exposure environment.

[0013] Further, the application is to administer a pharmaceutical dose of N-fluorenylmethoxycarbonyl-L-leucine preparation.

[0014] Furthermore, the administration method is to intraperitoneally inject the N-fluorenylmethoxycarbonyl-L-leucine preparation;

[0015] The administration dose of the intraperitoneal injection of the N-fluorenylmethoxycarbonyl-L-leucine preparation is Fmoc-L-leucine 0.4 mg / kg / d.

[0016] A drug for preventing cold exposure injury provided by the present invention, the drug comprises N-fluorenylmethoxycarbonyl-L-leucine and a pharmaceutically acceptable excipient.

[0017] Further, the pharmaceutically acceptable excipient includes one or more of a diluent, a binder, a wetting agent, a disintegrant, a lubricant, a solubilizer, a pH regulator, and an osmotic pressure regulator.

[0018] Further, the dosage form of the drug is an injection.

[0019] Furthermore, the administration route of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection, or subcutaneous injection, and preferably intraperitoneal injection.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] Through research, the present invention finds that N-fluorenylmethoxycarbonyl-L-leucine can be used to prepare a drug for preventing cold exposure injury, and thus provides a potential drug for improving the body's cold resistance and anti-cold injury ability for personnel working in cold regions. Description of the Drawings

[0022] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is a comparison chart of the core body temperature changes of mice in experimental group 1, positive control group, and negative control group provided in Example 1 of the present invention;

[0024] Figure 2 It is a representative infrared thermal imaging picture of mice in experimental group 1 and negative control group after 6 hours of cold exposure provided in Example 1 of the present invention;

[0025] Figure 3H&E staining images of brown adipose tissue (BAT), inguinal white adipose tissue (inguinal WAT), and epididymal white adipose tissue (eWAT) of experimental group 1 and negative control group provided in Example 2 of the present invention;

[0026] Figure 4 UCP1 protein expression levels in BAT, iWAT, and eWAT tissues after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1 provided in Example 3 of the present invention;

[0027] Figure 5a Graph of thermogenic gene expression levels in BAT after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1 provided in Example 3 of the present invention;

[0028] Figure 5b Graph of thermogenic gene expression levels in iWAT after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1 provided in Example 3 of the present invention;

[0029] Figure 5c Graph of thermogenic gene expression levels in eWAT after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1 provided in Example 3 of the present invention;

[0030] Figure 6 UCP1 immunofluorescence analysis image of eWAT after intraperitoneal injection of Fmoc-L-leucine in experimental group 1 provided in Example 3 of the present invention;

[0031] Figure 7 Images of intracellular lipid droplets after administration of Fmoc-L-leucine (20 μM) for 48 h and mitochondrial imaging using mitochondrial probe Mito-Tracker in BODIPY staining assay provided in Example 4 of the present invention;

[0032] Figure 8 Graph of UCP1 expression levels after administration of Fmoc-L-leucine to primary inguinal adipocytes after differentiation and maturation provided in Example 4 of the present invention;

[0033] Figure 9 UCP1 immunofluorescence analysis image after administration of Fmoc-L-leucine to primary inguinal adipocytes after differentiation and maturation provided in Example 4 of the present invention;

[0034] Figure 10 Graph of thermogenic gene expression levels after administration of Fmoc-L-leucine to primary inguinal adipocytes after differentiation and maturation provided in Example 4 of the present invention. Detailed implementation manners

[0035] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] According to one aspect of the present invention, a compound that can be used as a drug for preventing cold exposure injury: N-Fmoc-L-leucine;

[0037] The structural formula of the N-Fmoc-L-leucine is as follows:

[0038]

[0039] Through research, the present invention finds that N-Fmoc-L-leucine can be used to prepare a potential drug for improving the body's cold resistance and preventing cold injury, thereby providing a basis for developing an effective drug for preventing cold injury for personnel working in cold regions.

[0040] It should be noted that N-Fmoc-L-leucine is a selective PPARγ regulator, with the molecular formula C 21 H 23 NO4, molecular weight 353.41, CAS number: 35661-60-0.

[0041] In a preferred embodiment of the present invention, the cold exposure injury includes cold injury caused by low temperature environment and frostbite caused by ultra-low temperature environment.

[0042] In the above preferred embodiment, the low temperature refers to 4 - 6°C.

[0043] In a preferred embodiment of the present invention, the application refers to that N-Fmoc-L-leucine promotes the body's adaptive thermogenesis, alleviates the body temperature drop, and improves the cold resistance in a cold exposure environment.

[0044] In a preferred embodiment of the present invention, the application is to administer a pharmaceutical dose of N-Fmoc-L-leucine preparation.

[0045] In the above preferred embodiment, the administration method is to intraperitoneally inject the N-Fmoc-L-leucine preparation;

[0046] The dosage of the intraperitoneal injection of the N-Fmoc-L-leucine preparation is Fmoc-L-leucine 0.4 mg / kg / d.

[0047] According to one aspect of the present invention, a drug for preventing cold exposure injury, the drug comprising N-fluorenylmethoxycarbonyl-L-leucine and a pharmaceutically acceptable excipient.

[0048] The drug for preventing cold exposure injury provided by the present invention, the drug comprising N-fluorenylmethoxycarbonyl-L-leucine and a pharmaceutically acceptable excipient. It has been verified that at the dosage of Fmoc-L-leucine 0.4 mg / kg / d by intraperitoneal injection of the N-fluorenylmethoxycarbonyl-L-leucine preparation, the above drug can promote adaptive thermogenesis of the body and relieve body temperature drop in a cold exposure environment.

[0049] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipient includes one or more of a diluent, a binder, a wetting agent, a disintegrant, a lubricant, a solubilizer, a pH regulator and an osmotic pressure regulator.

[0050] In a preferred embodiment of the present invention, the dosage form of the drug is an injection.

[0051] In the above preferred embodiment, the administration route of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, preferably intraperitoneal injection.

[0052] The technical solution of the present invention will be further described below in conjunction with examples.

[0053] Note: In the following examples of this application, Fmoc-L-leucine was purchased from Sigma-Aldrich, ≥97.0%, catalog number 47633.

[0054] Example 1 Mouse body temperature maintenance and cold tolerance test under cold exposure

[0055] (I). Grouping of experimental animals and administration method:

[0056] 8-week-old male C57BL / 6J mice were adaptively raised for one week at an environmental temperature of 23 - 26 °C, a relative humidity of 50 - 60%, and a light-dark cycle of 12 h / 12 h. Subsequently, they were randomly divided into 5 groups with 8 experimental mice in each group:

[0057] Experimental group 1: Intraperitoneal injection was administered daily for 1 week, and the administration dosage was Fmoc-L-leucine 0.4 mg / kg / d;

[0058] Experimental group 2: Intraperitoneal injection was administered daily for 1 week, and the administration dosage was Fmoc-L-leucine 0.8 mg / kg / d;

[0059] Experimental group 3: Intraperitoneal injection was administered daily for 1 week, and the administration dosage was Fmoc-L-leucine 0.2 mg / kg / d;

[0060] Positive control group: Administered intraperitoneally once a day for 1 week, with the β3-adrenergic receptor agonist CL316243 (purchased from Sigma-Aldrich, ≥98.0%, C5976) ， The dosage was 1 mg / kg / d. The β3 receptor mediates the thermogenesis of adipocytes induced by sympathetic neurons. CL316243 is an effective stimulant for adipocyte lipolysis, which can increase the thermogenesis and metabolic rate of brown adipose tissue;

[0061] Negative control group: Administered intraperitoneally with the same volume of the control solvent as in experimental group 1 once a day for 1 week.

[0062] (2) After one week of feeding and administration in the above step (1), transfer the mice in each experimental group from the breeding environment at 23°C to a cold exposure environment at 4°C. Each mouse in the administration groups was separately placed in a single cage, and the cage should be kept dry, without food and water, and other factors should be avoided to interfere with body temperature as much as possible. The rectal temperature of each group of mice was monitored and recorded every hour using a rectal probe connected to a digital thermometer (BAT-12 Microprobe-Thermometer; Physitemp).

[0063] See Table 1 for the test results.

[0064] Table 1 Rectal temperature of mice in each group (°C)

[0065]

[0066] *, P < 0.05 compared with the cold-exposed negative control group; **, P < 0.01 compared with the cold-exposed negative control group.

[0067] As can be seen from the above table, the rectal temperature of mice gradually decreased during cold exposure. Compared with the control group, in experimental group 1, the core body temperature of mice in the Fmoc-L-leucine treatment group decreased more slowly in the first 6 h before cold exposure, was close to that of the positive control group, could better maintain the body temperature of mice, and improve cold tolerance.

[0068] Figure 1 This is the comparison chart of the changes in the core body temperature of mice in experimental group 1, positive control group, and negative control group provided in this example.

[0069] Compared with experimental group 1, experimental group 2 could also slow down the decline in rectal temperature, which was significantly better than the solvent control group (P < 0.01);

[0070] The body temperature drop of experimental group 3 during cold exposure was close to that of the negative control group, and the drug efficacy could not be achieved. That is to say, when the administration dose of N-fluorenylmethoxycarbonyl-L-leucine preparation by intraperitoneal injection was less than 0.4 mg / kg / d, it was difficult to relieve the body temperature drop during cold exposure.

[0071] Therefore, considering the drug efficacy and economy, it is more appropriate to select the administration dose of 0.4 mg / kg / d.

[0072] (3) Use an infrared thermal imager (E75 Advanced Thermal Imaging Camera; FLIR) to take pictures and measure the temperature of the scapular region of the mice in experimental group 1 and the negative control group after 6 hours of cold exposure in the above step (2). For the specific temperature measurement results, see Figure 2 .

[0073] Figure 2 It is a representative infrared thermal image of the mice in experimental group 1 and the negative control group after 6 hours of cold exposure.

[0074] Under the stimulation of the cold exposure environment, the activation of brown adipose tissue under the scapula can participate in the non-shivering thermogenesis of the body. From the above Figure 2 it can be seen that the temperature of the scapular region of the mice in experimental group 1 was significantly higher than that of the mice in the negative control group, indicating that the mice could maintain a higher temperature to resist cold after administration of Fmoc-L-leucine.

[0075] Example 2 Intraperitoneal injection of Fmoc-L-leucine can reduce lipid storage in adipose tissue of cold-exposed mice

[0076] (I) Grouping of experimental animals and administration method:

[0077] After the completion of Example 1, experimental group 1 and the negative control group were continuously kept in a cold exposure environment (temperature 4 - 6 °C, relative humidity 50 - 60%, light-dark cycle 12 h / 12 h) with sufficient mouse food and water for one week. The control group was intraperitoneally injected with the same volume of solvent every day, the administration group was given the corresponding dose of Fmoc-l-leucine, and the positive control group was treated with CL316243.

[0078] (II) After one week of feeding in the cold environment in the above step (I), three adipose tissues were sampled, namely brown adipose tissue (BAT) under the scapula, inguinal white adipose tissue (inguinal WAT iWAT), and epididymal white adipose tissue, and immediately fixed in 4% paraformaldehyde. After dehydration, clearing, wax infiltration, embedding, and paraffin sectioning, hematoxylin-eosin staining was performed.

[0079] Figure 3H&E staining images of brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and epididymal white adipose tissue (eWAT) sections of experimental group 1 and negative control group provided in this example;

[0080] It can be seen from Figure 3 that in experimental group 1, the adipocyte area of BAT and iWAT was significantly reduced, and lipid accumulation was significantly reduced, indicating that cold exposure can activate brown adipose tissue and induce the browning of white adipose tissue. After administration, the adipocyte diameter and area of iWAT and eWAT were further reduced, suggesting that intraperitoneal injection of Fmoc-L-leucine can further reduce lipid storage in adipose tissue of cold-exposed mice. This shows that Fmoc-L-leucine is an active compound that can stimulate adaptive thermogenesis in adipose tissue and slow down body temperature decline under cold exposure.

[0081] Example 3 Cold exposure and Fmoc-L-leucine administration promote thermogenic activation of adipose tissue

[0082] (1). Detect the expression of UCP1-related thermogenic marker genes in BAT, iWAT, and eWAT adipose tissues after intraperitoneal injection of Fmoc-L-leucine by wb, immunofluorescence, and qPCR. The specific methods are as follows:

[0083] For the mice in experimental group 1 and negative control group after one week of cold exposure experiment in Example 2, three adipose tissues were taken, RNA was extracted by the Trizol method, reverse transcribed into cDNA, and then the thermogenic-related marker gene levels were measured by qPCR. The internal reference gene 36B4 was selected, and 2 -ΔΔCt methods were used to analyze the data; after the tissue samples in Example 2 were fixed and sectioned, immunofluorescence staining was performed to investigate the expression of UCP1; another tissue sample in Example 2 was taken, and after protein extraction and BCA quantification, immunoblotting experiments were carried out.

[0084] Figure 4 shows the UCP1 expression levels in BAT, iWAT, and eWAT tissues after intraperitoneal injection of Fmoc-L-leucine in experimental group 1 under cold exposure.

[0085] It can be seen from Figure 4 that after cold exposure, the expression levels of UCP1 in BAT and iWAT tissues increased significantly. After cold exposure and Fmoc-l-leucine administration, the expression of UCP1 in BAT, iWAT, and eWAT tissues further increased, suggesting that Fmoc-l-leucine activates brown adipose tissue and induces the browning of white adipose tissue through UCP1-mediated proton uncoupling thermogenesis.

[0086] Figure 5a It is the graph of the expression levels of BAT thermogenic genes after intraperitoneal injection of Fmoc-L-leucine in experimental group 1 under cold exposure.

[0087] Figure 5b It is the graph of the expression levels of iWAT thermogenic genes after intraperitoneal injection of Fmoc-L-leucine in experimental group 1 under cold exposure.

[0088] Figure 5c It is the graph of the expression levels of eWAT thermogenic genes after intraperitoneal injection of Fmoc-L-leucine in experimental group 1 under cold exposure.

[0089] From Figures 5a to 5c it can be seen that after administration of Fmoc-L-leucine under cold exposure, the expression of UCP1-related thermogenic genes in the three adipose tissues measured by qPCR shows an increasing trend, suggesting that the drug administration can enhance the thermogenic capacity of adipose tissue.

[0090] Figure 6 It is the immunofluorescence analysis graph of UCP1 in eWAT after intraperitoneal injection of Fmoc-L-leucine in experimental group 1.

[0091] From Figure 6 it can be seen that under cold exposure conditions, the UCP1 protein level in the Fmoc-L-leucine-administered group (experimental group 1) is significantly higher than that in the negative control group, which is verified with the wb and qPCR results, indicating that Fmoc-L-leucine activates adipose tissue thermogenesis and lipolysis through UCP1-mediated proton uncoupling, improving the cold tolerance of the body.

[0092] Example 4

[0093] (1). Fmoc-L-leucine enhances the thermogenic capacity of adipocytes:

[0094] (1). Isolate and digest the primary preadipocytes of iWAT (inguinal white adipose tissue) from 4-week-old C57BL / 6J mice, and culture them in DMEM medium containing 10% FBS. When the cells grow to 80%-90%, it is recorded as Day0, and the induction differentiation medium (5 μg / mL insulin, 0.5 mM IBMX, 1 μM dexamethasone, 1 nM T3, 125 μM indomethacin, 1 μM rosiglitazone) is replaced. On Day2 and Day4, the maintenance medium (5 μg / mL insulin, 1 nM T3) is replaced.

[0095] After differentiation and maturation on Day6, 20 μM of Fmoc-L-leucine is administered, and after 48 h, BODIPY staining and Mitotracker staining are performed to observe the changes in lipid droplets and mitochondria after Fmoc-L-leucine administration

[0096] Figure 7 The picture shows the intracellular lipid droplets after 48 h of administration of Fmoc-L-leucine (20 μM) provided in this example and the imaging of mitochondria using the mitochondrial probe Mito-Tracker;

[0097] Note: Figure 7 In “Fmoc-L-leucine”, it refers to the experimental group after 48 h of administration of Fmoc-L-leucine (20 μM); Figure 7 In “Vehicle”, it refers to the negative control.

[0098] Figure 7 It shows that the cells in the administration group contain smaller lipid droplets than those in the control group, while the number of mitochondria increases. The most significant characteristics of thermogenic adipocytes are the presence of multi-chamber lipid droplets and high mitochondrial content, indicating that the administration can promote the browning of white adipocytes and improve the levels of thermogenesis and energy metabolism.

[0099] (3) Detect the protein expression level of UCP1 after administration of Fmoc-L-leucine to the differentiated and mature primary inguinal adipocytes in step (1), and perform fluorescence analysis.

[0100] Figure 8 It is the graph of the protein expression level of UCP1 after administration of Fmoc-L-leucine to the differentiated and mature primary inguinal adipocytes. Among them: Figure 8 In “Fmoc-L-leucine”, it refers to the experimental group after 48 h of administration of Fmoc-L-leucine (20 μM); Figure 8 In “Vehicle”, it refers to the negative control.

[0101] Figure 9 It is the immunofluorescence analysis graph of UCP1 after administration of Fmoc-L-leucine to the differentiated and mature primary inguinal adipocytes. Among them: Figure 9 In “Fmoc-L-leucine”, it refers to the experimental group after 48 h of administration of Fmoc-L-leucine (20 μM); Figure 9 In “Vehicle”, it refers to the negative control.

[0102] (4) Detect the expression level of thermogenic genes after administration of Fmoc-L-leucine to the differentiated and mature primary inguinal adipocytes in step (1).

[0103] Figure 10 It is the graph of the expression level of thermogenic genes after administration of Fmoc-L-leucine to the differentiated and mature primary inguinal adipocytes. Among them: Figure 10In "Fmoc-L-leucine", it is the experimental group after administering Fmoc-L-leucine (20 μM) for 48 h; Figure 10 In "Vehicle", it is the negative control.

[0104] From the above detection of UCP1 and the expression of its related genes by staining, immunofluorescence, and qPCR in this example, it can be seen that similar to the results of in vivo experiments, after treating inguinal white adipocytes with Fmoc-L-leucine for 48 h, the expression of the thermogenic marker protein Ucp1 and the genes related to the pathway can be up-regulated, and the thermogenic ability of adipocytes is enhanced.

[0105] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of N-Fmoc-L-leucine in the preparation of drugs for preventing cold exposure injury.

2. The use according to claim 1, characterized in that: The cold exposure injury refers to the cold injury caused in a low temperature environment.

3. The use according to claim 2, characterized in that: The low temperature is 4-6°C.

4. The use according to claim 1, characterized in that: The application refers to N-Fmoc-L-leucine promoting the body's adaptive heat production, alleviating the drop in body temperature, and improving the ability to resist cold in a cold exposure environment.

5. The use according to claim 1, characterized in that: The application is to administer a pharmaceutical dose of N-Fmoc-L-leucine preparation.

6. The use according to claim 5, characterized in that: The administration method is intraperitoneal injection of N-Fmoc-L-leucine preparation; The dosage of the intraperitoneal injection of N-Fmoc-L-leucine preparation is 0.4 mg / kg / d.

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