Application of N-fluorenylmethoxycarbonyl-L-leucine in preparation of medicine for preventing cold exposure injury and medicine for preventing cold exposure injury

Abstract

The invention provides application of N-fluorenylmethoxycarbonyl-L-leucine in preparation of a medicine for preventing cold exposure injury and the medicine for preventing cold exposure injury, and relates to the technical field of medicines for preventing frostbite. Research finds that N-fluorenylmethoxycarbonyl-L-leucine can be used for preparing the medicine for preventing cold exposure injury, and then a potential effective medicine for improving the cold resistance and cold injury resistance of organisms is provided for cold region operation and combat personnel.

Description

Technical Field

[0001] The invention relates to the technical field of anti-freezing drugs, and in particular to 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. 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, which causes a series of physiological and biochemical reactions in the body. This exposure will have many effects on the human body, including temperature regulation, energy metabolism and mental health.

[0003] my country has a wide range of high-altitude cold regions, covering the Northeast, Northwest, North China and other vast areas. At the same time, my country has long borders with North Korea, Russia, Mongolia, Pakistan and India, which are important national defense strategic locations, and these locations are usually high-altitude cold regions. Frostbite caused by cold exposure sometimes occurs on a large scale in troops performing military missions in high-altitude cold regions, which can easily cause a large number of non-combat casualties and seriously weaken the combat capability of the troops.

[0004] In view of this, the research and development of drugs to prevent cold exposure injuries has become necessary and urgent. Summary of the invention

[0005] The purpose of the present invention is to provide a potential drug for preparing a drug for improving the body's ability to resist cold and resist cold exposure injury, namely N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-L-leucine); and experiments have proved that N-fluorenylmethoxycarbonyl-L-leucine can be used to prepare drugs for preventing cold exposure injury.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The present invention provides a compound that can be used as a drug for preventing cold exposure injury: N-fluorenylmethoxycarbonyl-L-leucine; The structural formula of the N-fluorenylmethoxycarbonyl-L-leucine is:

[0007] Furthermore, the cold exposure injury is cold injury caused in a low temperature environment.

[0008] Furthermore, the low temperature refers to 4~6°C.

[0009] Furthermore, 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.

[0010] Furthermore, the application is to administer a pharmaceutical dose of N-Fmoc-L-leucine preparation.

[0011] Furthermore, the administration method is intraperitoneal injection of the N-Fmoc-L-leucine preparation; The dosage of the intraperitoneal injection of N-Fmoc-L-leucine preparation is 0.4 mg / kg / d.

[0012] The invention provides a medicine for preventing cold exposure injury, which comprises N-fluorenylmethoxycarbonyl-L-leucine and pharmaceutically acceptable excipients.

[0013] Furthermore, the pharmaceutically acceptable excipients include 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.

[0014] Furthermore, the dosage form of the drug is an injection.

[0015] Furthermore, the administration route of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, preferably intraperitoneal injection.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention has found through research that N-Fmoc-L-leucine can be used to prepare a drug for preventing cold exposure injury, thereby providing a potential drug for improving the body's ability to resist cold and cold injury for personnel working and fighting in cold areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 A comparison chart of the core body temperature changes of mice in the test group 1, the positive control group and the negative control group provided in Example 1 of the present invention; Figure 2 Representative infrared thermal imaging images of mice in test group 1 and negative control group after 6 hours of cold exposure provided in Example 1 of the present invention; Figure 3 H&E staining images of brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and epididymal white adipose tissue (eWAT) sections of the test group 1 and the negative control group provided in Example 2 of the present invention; Figure 4 The expression level of UCP1 protein 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; Figure 5a This is a graph of the expression level of BAT thermogenic genes after cold exposure and intraperitoneal injection of Fmoc-L-leucine in Experimental Group 1 provided in Example 3 of the present invention; Figure 5b This is a graph of the expression level of thermogenic genes in iWAT after cold exposure and intraperitoneal injection of Fmoc-L-leucine in Experimental Group 1 provided in Example 3 of the present invention; Figure 5c This is a graph of the expression level of thermogenic genes in eWAT after cold exposure and intraperitoneal injection of Fmoc-L-leucine in Experimental Group 1 provided in Example 3 of the present invention; Figure 6 This is a UCP1 immunofluorescence analysis diagram of eWAT after intraperitoneal injection of Fmoc-L-leucine in Experimental Group 1 provided in Example 3 of the present invention; Figure 7 The pictures provided in Example 4 of the present invention are the measurement of intracellular lipid droplets by BODIPY staining 48 hours after administration of Fmoc-L-leucine (20 μM) and the imaging of mitochondria using the mitochondrial probe Mito-Tracker; Figure 8 This is a graph of UCP1 expression levels after Fmoc-L-leucine administration in primary inguinal adipocytes differentiated and matured as provided in Example 4 of the present invention; Fig. 9 This is a UCP1 immunofluorescence analysis diagram of primary inguinal adipocytes differentiated and matured after administration of Fmoc-L-leucine provided in Example 4 of the present invention; Fig.10 This is a graph of the thermogenic gene expression levels after Fmoc-L-leucine administration in primary inguinal adipocytes differentiated and matured as provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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 are within the scope of protection of the present invention.

[0020] According to one aspect of the present invention, a compound useful as a drug for preventing cold exposure injury: N-Fmoc-L-leucine; The structural formula of the N-fluorenylmethoxycarbonyl-L-leucine is:

[0021] The present invention has found through research that N-Fmoc-L-leucine can be used to prepare a potential anti-cold injury drug for improving the body's ability to resist cold, thereby providing a basis for developing an effective anti-cold injury drug for cold-region workers and combatants.

[0022] It should be noted that N-fluorenylmethoxycarbonyl-L-leucine (Fmoc-L-leucine) is a selective PPARγ modulator with a molecular formula of C 21 H 23 NO4, molecular weight 353.41, CAS number: 35661-60-0.

[0023] In a preferred embodiment of the present invention, the cold exposure injury includes cold injury caused in a low temperature environment and frostbite caused in an ultra-low temperature environment.

[0024] In the above preferred embodiment, the low temperature refers to 4-6°C.

[0025] In a preferred embodiment of the present invention, 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.

[0026] In a preferred embodiment of the present invention, the use is to administer a pharmaceutical dose of N-Fmoc-L-leucine preparation.

[0027] In the above preferred embodiment, 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 Fmoc-L-leucine 0.4 mg / kg / d.

[0028] According to one aspect of the present invention, a drug for preventing cold exposure injury comprises N-Fmoc-L-leucine and a pharmaceutically acceptable excipient.

[0029] The present invention provides a drug for preventing cold exposure injury, which includes N-fluorenylmethoxycarbonyl-L-leucine and a pharmaceutically acceptable excipient. It has been verified that at a dosage of 0.4 mg / kg / d of an intraperitoneal injection of an N-fluorenylmethoxycarbonyl-L-leucine preparation, the drug can promote adaptive heat production in the body and alleviate the decrease in body temperature in a cold exposure environment.

[0030] In a preferred embodiment of the present invention, the pharmaceutically acceptable excipients include 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.

[0031] In a preferred embodiment of the present invention, the dosage form of the drug is an injection.

[0032] 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.

[0033] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0034] Note: Fmoc-L-leucine in the following examples of this application was purchased from Sigma-Aldrich, ≥97.0%, catalog number 47633.

[0035] Example 1 Body temperature maintenance and cold tolerance test of mice under cold exposure 1. Experimental animal grouping and drug administration method: Eight-week-old male C57BL / 6J mice were adapted for one week in an environment with a temperature of 23-26°C, a relative humidity of 50-60%, and a light-dark cycle of 12h / 12h. They were then randomly divided into five groups according to body weight, with eight mice in each group: Experimental group 1: daily intraperitoneal injection for 1 week, the dosage of Fmoc-L-leucine 0.4mg / kg / d; Experimental group 2: daily intraperitoneal injection for 1 week, the dosage of Fmoc-L-leucine 0.8mg / kg / d; Experimental group 3: daily intraperitoneal injection for 1 week, the dosage of Fmoc-L-leucine 0.2mg / kg / d; Positive control group: daily intraperitoneal injection for 1 week, β3-adrenaline receptor agonist CL316243 (purchased from Sigma-Aldrich, ≥98.0%, C5976), the dosage of 1mg / kg / d, β3 receptor mediated sympathetic neuron-induced adipocyte thermogenesis, CL316243 is an effective adipocyte lipolysis stimulator, which can increase the thermogenesis and metabolic rate of brown adipose tissue; Negative control group: daily intraperitoneal injection of 1 volume of control solvent as in the test group for 1 week.

[0036] (ii) After one week of feeding and dosing in step (1), the mice in each experimental group were transferred from the 23°C feeding environment to the 4°C cold exposure environment. Each mouse in the dosing group was placed in a single cage, and the cage was kept dry without mouse food and water. Other factors that interfere with body temperature were avoided 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).

[0037] The test results are shown in Table 1.

[0038] Table 1 Rectal temperature of mice in each group (℃) ):

[0039] *, P<0.05 compared with the cold-exposed negative control group; **, P<0.01 compared with the cold-exposed negative control group.

[0040] As can be seen from the table above, the rectal temperature of mice gradually decreased during the cold exposure process. Compared with the control group, the core body temperature of mice in the Fmoc-L-leucine-treated group in the first 6 hours of cold exposure decreased more slowly, which was close to that of the positive control group, and could better maintain the body temperature of mice and improve cold tolerance.

[0041] Figure 1 This is a comparison chart of the core body temperature changes of mice in the experimental group 1 provided in this example, and the positive control group and negative control group.

[0042] Compared with experimental group 1, experimental group 2 was also able to slow down the decline in rectal temperature, which was significantly better than the solvent control group (P < 0.01); The body temperature drop during cold exposure in test group 3 was close to that of the negative control group, and the drug effect could not be achieved. In other words, when the intraperitoneal injection of N-Fmoc-L-leucine preparation was administered at a dose lower than 0.4 mg / kg / d, it was difficult to alleviate the drop in body temperature during cold exposure.

[0043] Therefore, considering the efficacy and economy, a more appropriate dosage of 0.4 mg / kg / d is chosen.

[0044] (3) An infrared thermal imager (E75 Advanced Thermal Imaging Camera; FLIR) was used to take photos and measure the temperature of the scapula area of ​​the mice in the experimental group 1 and the negative control group after 6 hours of cold exposure in step (2). For specific temperature measurement results, see Figure 2 .

[0045] Figure 2 Representative infrared thermal imaging images of mice in test group 1 and negative control group after 6 hours of cold exposure.

[0046] Under cold exposure, activation of subscapular brown adipose tissue can participate in the body's non-shivering heat production. Figure 2 It can be seen that the temperature of the scapula area of ​​the mice in the experimental group 1 was significantly higher than that of the mice in the negative control group, which indicates that after the administration of Fmoc-L-leucine, the mice were able to maintain a higher temperature to resist the cold.

[0047] Example 2 Intraperitoneal injection of Fmoc-L-leucine can reduce lipid storage in adipose tissue of cold-exposed mice 1. Experimental animal grouping and drug administration method: After the completion of Example 1, the experimental group 1 and the negative control group were kept in a cold exposure environment (temperature 4-6°C, relative humidity 50-60%, light-dark cycle 12h / 12h) for one week under the condition of sufficient mouse food and water, and the mice were intraperitoneally injected with an equal volume of solvent to the control group, a corresponding dose of Fmoc-l-leucine to the drug-treated group, and CL316243 to the positive control group.

[0048] (ii) After one week of cold feeding in the above step (i), three adipose tissues were collected, namely scapular brown adipose tissue (BAT), inguinal white adipose tissue (inguinal WAT iWAT), and epididymal white adipose tissue (epididymal WAT), and immediately fixed in 4% paraformaldehyde, dehydrated, transparent, wax-impregnated, embedded, and paraffin-sectioned before hematoxylin-eosin staining.

[0049] Figure 3 H&E staining images of brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and epididymal white adipose tissue (eWAT) sections of test group 1 and negative control group provided in this example; Depend on Figure 3 It can be seen that in experimental group 1, the area of ​​BAT and iWAT adipocytes was significantly reduced, and lipid accumulation was significantly reduced, indicating that cold exposure can activate brown adipose tissue and induce browning of white adipose tissue. After administration, the diameter and area of ​​iWAT and eWAT adipocytes further decreased, 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 heat production in fat under cold exposure and slow down the drop in body temperature.

[0050] Example 3 Cold exposure and Fmoc-L-leucine administration promotes adipose tissue thermogenic activation (I) The expression of UCP1-related thermogenic marker genes in the adipose tissues of BAT, iWAT and eWAT after intraperitoneal injection of Fmoc-L-leucine was detected by WB, immunofluorescence and qPCR. The specific methods are as follows: Example 2: After one week of cold exposure, three fat tissues were taken from mice in the test group 1 and the negative control group. RNA was extracted using the Trizol method and reverse transcribed into cDNA. The levels of heat-related marker genes were determined by qPCR. The internal reference genes 36B4, 2 -ΔΔCt The data were analyzed by the method; the tissue samples of Example 2 were fixed and sliced, and then immunofluorescence staining was performed to examine the expression of UCP1; another tissue sample of Example 2 was taken, and an immunoblotting experiment was performed after protein extraction and BCA quantification.

[0051] Figure 4 The expression levels of UCP1 in BAT, iWAT and eWAT tissues after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1.

[0052] Depend on Figure 4 It can be seen that the expression of UCP1 in BAT and iWAT tissues increased significantly after cold exposure, and the expression of UCP1 in BAT, iWAT, and eWAT tissues further increased after cold exposure and Fmoc-l-leucine administration, suggesting that Fmoc-l-leucine activates brown adipose tissue and induces browning of white adipose tissue through UCP1-mediated proton uncoupling thermogenesis.

[0053] Figure 5a This is the expression level of BAT thermogenic genes after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1.

[0054] Figure 5b This is the expression level of thermogenic genes in iWAT after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1.

[0055] Figure 5c This is the expression level of thermogenic genes in eWAT after cold exposure and intraperitoneal injection of Fmoc-L-leucine in experimental group 1.

[0056] Depend on Figure 5a~Figure 5c It can be seen that the expression of UCP1-related thermogenic genes in three adipose tissues showed an increasing trend after cold exposure Fmoc-L-leucine administration as determined by qPCR, suggesting that administration can enhance the thermogenic capacity of adipose tissue.

[0057] Figure 6This is the UCP1 immunofluorescence analysis of eWAT after intraperitoneal injection of Fmoc-L-leucine in experimental group 1.

[0058] Depend on Figure 6 It can be seen that under cold exposure conditions, the UCP1 protein level was significantly increased after Fmoc-L-leucine administration (experimental group 1) compared with the negative control group, which was verified by wb and qPCR results, indicating that Fmoc-L-leucine activates adipose tissue thermogenesis and lipolysis through UCP1-mediated proton uncoupling, thereby improving the body's cold tolerance.

[0059] Example 4 1. Fmoc-L-leucine enhances the heat production capacity of fat cells: (1) Primary preadipocytes from iWAT (inguinal white adipose tissue) of 4-week-old C57BL / 6J mice were isolated and digested, and cultured in DMEM medium containing 10% FBS. When the cells grew to 80%-90%, it was recorded as Day 0. The induction differentiation medium (5 μg / mL insulin, 0.5 mM IBMX, 1 μM dexamethasone, 1 nM T3, 125 μM indomethacin, 1 μM rosiglitazone) was replaced. On Day 2 and Day 4, the medium was replaced with maintenance medium (5 μg / mL insulin, 1 nM T3).

[0060] After differentiation and maturation on Day 6, 20 μM Fmoc-L-leucine was administered, and BODIPY staining and Mitotracker staining were performed 48 hours later to observe the changes in lipid droplets and mitochondria after Fmoc-L-leucine administration. Figure 7 This example provides images of BODIPY staining to measure intracellular lipid droplets 48 hours after administration of Fmoc-L-leucine (20 μM) and imaging of mitochondria using the mitochondrial probe Mito-Tracker; Note: Figure 7 “Fmoc-L-leucine” in the table refers to the test group after 48 h of administration of Fmoc-L-leucine (20 μM); Figure 7 “Vehicle” in the table is the negative control.

[0061] Figure 7 It showed that the cells in the drug-treated group contained smaller lipid droplets than those in the control group, while the number of mitochondria increased. The most notable feature of thermogenic adipocytes was the presence of multi-chambered lipid droplets and high mitochondrial content, indicating that drug-treated groups could promote the browning of white adipocytes and improve the levels of heat production and energy metabolism.

[0062] (2) The protein expression level of UCP1 after Fmoc-L-leucine administration to the primary inguinal adipocytes differentiated and matured in step (1) was detected and fluorescence analysis was performed.

[0063] Figure 8 This is a graph showing the UCP1 protein expression level after Fmoc-L-leucine administration to primary inguinal adipocytes after differentiation and maturation. Figure 8 “Fmoc-L-leucine” in the table refers to the test group after 48 h of administration of Fmoc-L-leucine (20 μM); Figure 8 “Vehicle” in the table is the negative control.

[0064] Fig. 9 This is a UCP1 immunofluorescence analysis diagram of primary inguinal adipocytes after differentiation and maturation and administration of Fmoc-L-leucine. Fig. 9 “Fmoc-L-leucine” in the table refers to the test group after 48 h of administration of Fmoc-L-leucine (20 μM); Fig. 9 “Vehicle” in the table is the negative control.

[0065] (3) Detect the expression level of thermogenic genes after administration of Fmoc-L-leucine to the primary inguinal adipocytes differentiated and matured in step (1).

[0066] Fig.10 This is a graph showing the expression level of thermogenic genes after Fmoc-L-leucine administration in primary inguinal adipocytes after differentiation and maturation. Fig.10 “Fmoc-L-leucine” in the table refers to the test group after 48 h of administration of Fmoc-L-leucine (20 μM); Fig.10 “Vehicle” in the table is the negative control.

[0067] Through the above-mentioned staining, immunofluorescence, and qPCR detection of UCP1 and its related gene expression in this example, it can be seen that similar to the results of the in vivo experiment, Fmoc-L-leucine treatment of inguinal white adipocytes for 48 hours can upregulate the expression of the thermogenic marker protein Ucp1 and pathway-related genes, and the thermogenic capacity of adipocytes is enhanced.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to 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.

7. A drug for preventing cold exposure injury, characterized in that: The medicine comprises N-fluorenylmethoxycarbonyl-L-leucine and pharmaceutically acceptable auxiliary materials.

8. The drug for preventing cold exposure injury according to claim 7, characterized in that: The pharmaceutically acceptable excipients include one or more of diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH regulators and osmotic pressure regulators.

9. The drug for preventing cold exposure injury according to claim 7, characterized in that: The dosage form of the drug is injection.

10. The drug for preventing cold exposure injury according to claim 9, characterized in that: The administration route of the injection includes one of intravenous injection, intraperitoneal injection, intramuscular injection or subcutaneous injection, preferably intraperitoneal injection.

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