CN109820857A - The new opplication of compound DK45 - Google Patents

Abstract

The present invention relates to the new opplications of compound DK45 a kind of.Application of the compound DK45 in the drug that preparation promotes fat cell UCP1 expression.Promote the brown of fat cell that extra fat is consumed in the form of heat, it can promote glucose uptake simultaneously and promote the oxidation of fatty acid, the quantity and vigor of mitochondria are improved, the level of body glycolipid metabolism and energetic supersession is enhanced, to effectively treat fat and fatty liver.Therefore, compound DK45 has the function of promoting fat cell UCP1 expression, to effectively prevent and treat fat and fatty liver.

Description

The new opplication of compound DK45

Technical field

The present invention relates to field of pharmaceutical chemistry technology, more particularly to the new opplication of compound DK45 a kind of.

Background technique

Obesity refers to that apparent fat deposit is blocked up to a certain extent and overweight, is mostly the energy because of organism metabolism Caused by unbalance.Currently with the increasingly raising of economic development and people's living standard, life, the continuous of operating pressure add Greatly, the number of obese patient is global constantly soaring.Obesity be both an independent disease and diabetes B, angiosis, The risk factor of hypertension, apoplexy and kinds cancer is classified as one of the risk factor for leading to Disease Spectrum by the World Health Organization.

People recognize research and development while understanding obesity to health hazard, and safely and effectively slimming medicine is very urgent.No Good fortune is drug not only limited amount but also effect is also indefinite currently used for body weight control, is once used for there are many kinds of drug Drop weight treatment, but due to apparent side effect (such as hypertension, serious emotional handicap, apparent myocardium markers change and The increase of the death rate) and stopped treatment of having to.Therefore developing the fat drug of new treatment can not allow to delay.

Obese patient is mainly shown as that abdominal adipose tissue increases, this shows that fat cell is sent out in fat generating process Wave important function.Fat cell traditional concept thinks that adipose tissue in the mammalian body is divided into white adipose tissue (white Adipose tissue, WAT) and brown adipose tissue (BAT), WAT be divided into subcutaneous WAT and internal organ WAT again.Adipose tissue be by A large amount of mature fat cell and a small amount of PECTORAL LIMB SKELETON, macrophage and immunocyte and fibrous connective tissue, blood vessel, mind It is knitted etc. and to be constituted.Both WAT and BAT have significant difference in form, distribution, function, are embodied in the following: White adipocyte contains a big fat drips, occupies the cytoplasm volume of the overwhelming majority, brown fat cell then contains more A small fat drips；White adipocyte contains relatively small number of mitochondria, and brown fat cell then contains a large amount of mitochondria； WAT is mainly distributed on subcutaneous, epididymis, mesenterium and kidney week etc., and BAT is mainly distributed under neck, omoplate, between omoplate and actively Equal regions around arteries and veins；BAT is compared with WAT, and BAT has richer blood vessel, and by the domination of nervous system；WAT is mainly with trigalloyl The energy of the form excess of storage of glycerol, BAT mainly consume energy by way of heat production, adjust energy i (in vivo) metabolism, and It plays an important role in maintaining animal heat and energy balance；The WAT of the mankind since birth after just occur and gradually increase, and BAT occurs since foetal period is, and cradle peaks, and reduces with age；In white adipocyte not UCP1 is expressed, and high expression UCP1 in BAT, this is its major reason for playing biological effect and its is most important and unique Molecular labeling.So the major function of brown fat cell is exactly come heat production by UCP1 to consume energy without generating ATP (adenosine triphosphate): i.e. Intramitochondrial electron transport chain can be synthesized solution with ATP by UCP1 by brown fat cell Coupling, and then breathed by oxidative phosphorylation uncoupling, the energy that fatty acid beta oxidation generates is lost in the form of heat. Non- the trembling property heat production function of brown fat cell, can maintain thermostasis, body can also be consumed in a manner of thermal energy and taken the photograph The energy entered, to reduce fat generation.

Traditional idea thinks, what WAT and BAT were entirely isolated in distribution, but in recent years studies have shown that two kinds of fat Tissue may be mixed distribution, and only classification dominant in different types of adipose tissue is different, i.e., in WAT In, in addition to prevailing white adipocyte, under the stimulation centainly adjusted, the fat that also will appear brown sample is thin Born of the same parents.This cell as BAT with heat production function found in WAT is become " cream-coloured fat cell " (beige by people Cell), this phenomenon is also considered as the brown of white adipocyte, i.e. enhancing brown fat cell marker gene uncoupling The expression of albumen 1 (UCP1).Classical brown fat cell and cream-coloured fat cell, although can express with calorigenic action UCP1, but the distribution of two kinds of brown fat cells is different, and classical brown fat cell is distributed mainly on rodent Intrascapular region, and cream-coloured fat cell is distributed mainly in the subcutaneus adipose tissue of inguinal region.It is brown for the mankind Color fat cell is primarily present in infants, but it has now been found that, also contain a great deal of on the neck of adult, clavicle Brown fat cell, and they are similar to the cream-coloured fat cell of rodent.Other than distributional difference, two kinds of brown fats The source of cell is also different.Classical brown fat cell and muscle cell is homologous.Promote white adipocyte brownization can be with It is effectively reduced fat and fatty liver generation.Therefore finding, which can promote the drug of white adipocyte brown to become, grinds Study carefully hot research discovery, such research is of great significance for the treatment of human obesity related metabolic diseases.

DK45 is a kind of activation estrogen receptor correlation found by Chinese Academy of Sciences's Guangzhou biological medicine with research institute Receptor (Estrogen-Related Receptor α, ERR α) activator, the entitled 8-ethyl-2-phenyl-4H- of English Pyrido [1,2-a] pyrimidin-4-one, molecular structural formula are as follows:

Have the function of promoting glucose absorption, but DK45 has promotion fat cell There is not been reported for the effect of UCP1 expression.

Summary of the invention

Based on this, it is necessary to aiming at the problem that treatment obesity and fatty liver, provide the new opplication of compound DK45 a kind of.

A kind of application of compound DK45 in the drug that preparation promotes fat cell UCP1 expression.

The fat cell includes white adipocyte and brown fat cell in one of the embodiments,.

The present invention also provides a kind of application of compound DK45 in the drug that preparation promotes white adipocyte brown.

The present invention also provides a kind of application of compound DK45 in the fat drug of preparation prevention and/or treatment.

The present invention also provides a kind of application of compound DK45 in the drug of preparation prevention and/or treatment fatty liver.

Fat drug is treated the present invention also provides a kind of, the active constituent of the drug includes compound DK45.

The present invention also provides a kind of drug for treating fatty liver, the active constituent of the drug includes compound DK45.

Since the major function of brown fat cell is come heat production by the expression of UCP1 to consume energy without generating Intramitochondrial electron transport chain can be synthesized uncoupling with ATP, and then pass through by UCP1 by ATP, i.e. brown fat cell The breathing of oxidative phosphorylation uncoupling, the energy that fatty acid beta oxidation generates is lost in the form of heat.Brown fat cell Non- trembling property heat production function, can maintain thermostasis, the energy of body intake can also be consumed in a manner of thermal energy, to drop Low fat generation.White adipocyte also will appear the fat cell of brown sample, it is considered to be white under the adjusting stimulation of DK45 The brown of color fat cell, and the expression of enhancing brown fat cell marker gene uncoupling proteins 1 (UCP1).Promote rouge The brownization of fat cell can effectively be consumed extra fat in the form of heat, at the same can promote glucose uptake and The oxidation for promoting fatty acid improves the quantity and vigor of mitochondria, enhances the level of body glycolipid metabolism and energetic supersession, thus Effectively treat fat and fatty liver.Therefore, compound DK45 has the function of promoting fat cell UCP1 expression, so as to Effectively prevent and treat fat and fatty liver.

Detailed description of the invention

Fig. 1 is the expression for the UCP1 that DK45 promotes white adipocyte in vitro and the relational graph of dosage；

Fig. 2 is the expression for the UCP1 that DK45 promotes brown fat cell in vitro and the relational graph of dosage；

Fig. 3 is that DK45 promotes UCP1 that UCP1 is promoted to express in brown fat cell and white adipocyte in Mice Body Compare figure；

Fig. 4 is influence diagram of the DK45 to fat drop size in mouse fatty liver；

Fig. 5 is the influence diagram for the mouse glucose tolerance exception that DK45 induces high lipid food；

Fig. 6 is the influence diagram that DK45 resists the mouse islets element that high lipid food induces.

Specific embodiment

In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, to specific embodiment party of the invention Formula is described in detail.In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention.But this hair Bright to be implemented with being much different from other way described herein, those skilled in the art can be without prejudice in the present invention Similar improvement is done in the case where culvert, therefore the present invention is not limited by the specific embodiments disclosed below.

Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term " and or " used herein includes one or more phases Any and all combinations of the listed item of pass.

In one embodiment, application of the compound DK45 in the fat drug of preparation prevention and/or treatment.It is fat It is both an independent disease and diabetes B, angiosis, hypertension, the risk factor of apoplexy and kinds cancer, by generation Boundary's health organization, which is classified as, leads to one of risk factor of Disease Spectrum.

In one embodiment, application of the compound DK45 in the drug of preparation prevention and/or treatment fatty liver.Rouge The excessive lesion of fat accumulation in liver cell caused by fat liver (fatty liver) refers to due to various reasons is a kind of common Pathology of livers, rather than a kind of independent disease.

In one embodiment, the present invention provides a kind of compound DK45 and supports in preparation prevention and/or treatment insulin Application in anti-drug.Insulin resistance refers to that a variety of causes promotes insulin under glucose uptake and the efficiency utilized The hypersecretion insulin of drop, body compensatory generates hyperinsulinemia, to maintain the stabilization of blood glucose.Due to insulin resistance Metabolic syndrome is easily led to seek peace diabetes B, so it has also been found that, it is comprehensive that compound DK45 can also prevent and treat metabolism Simulator sickness and diabetes B.

In one embodiment, the present invention also provides a kind of compound DK45 in preparation prevention and/or treatment glucose Application in the drug of tolerance exception.Impaired glucose tolerance (IGT) is tolerance of the human body to glucose.Postprandial 2 is small When blood glucose be more than normal 7.8mmol/L, but impaired glucose tolerance be still not up to 11.1mmol/L diabetes diagnostic criterion it is (or empty Abdomen blood glucose rise, the not up to diagnostic criteria of diabetes, i.e., fasting blood-glucose is between 6.2~7.0) title impaired glucose tolerance (or it is empty Abdomen impaired).

Impaired glucose tolerance is a kind of transient state of normal person to diabetes, although this groups of people is not also sugar now Urine disease, but it is very high that diabetes B risk occurs in the future, it is possible to develop into diabetes B.In addition, glucose is resistance to It measures patients with abnormal and cardiovascular pathological changes occurs, as myocardial infarction, anginal risk also greatly improve.

Due to impaired glucose tolerance person's generation diabetes easier than normal person, so present invention discover that compound DK45 can also Prevent and treat diabetes.

It is below specific embodiment

Unless otherwise specified, reagent used in following embodiment derives from commercially available, and operating method is existing Conventional practices.

Embodiment 1DK45 is in the table that dose dependent promotes the UCP1 of white adipocyte and brown fat cell in vitro It reaches.

1. the separation of white adipose mesenchymal cell at groin

1) the adult UCP1-2A- luciferase mouse of about 10 week old is taken, the neck that breaks is put to death, and is soaked in 75% alcohol Bubble 5 minutes, is then transferred into station, takes out the white adipose at groin.

2) it is cleaned 3 times with PBS, is shredded adipose tissue with scissors, 1g adipose tissue is added 10ml clostridiopetidase A I solution and (uses D-Hanks solution is prepared, and 0.1g clostridiopetidase A I is added in 100ml), it places 37 DEG C and digests 40 minutes.

3) with 250 μm of membrane filtrations, cell culture fluid is added, is then transferred into centrifuge tube, 1000rpm is centrifuged 3 minutes.

4) the upper floating layer after being centrifuged is mature fat cell, is transferred in the centrifuge tube equipped with PBS solution, after mixing again Secondary centrifugation.The fat cell of the upper layer maturation separated again is used to extract RNA, albumen etc. after can collecting.

5) the bottom cell after being centrifuged for the first time is fat mesenchymal cell, outstanding with fresh culture solution after reject supernatant It rises, is taped against in culture plate, changes liquid within second day.

2. the differentiation of white adipose mesenchymal cell

1) DMEM (Hyclone) of the fat mesenchymal cell obtained in the high sugar of addition 10% fetal calf serum (Hyclone) Culture solution grow to it is full.

2) replacement culture solution be added to MDIR (0.5mM isobutyl group xanthine, 1 μM of dexamethasone, 87nM insulin and 0.5 μM of Rosiglitazone: IBMX+Dex+Insulin+rosiglitazone) induction broth (10% fetal calf serum of addition is high The culture solution of the DMEM of sugar), it cultivates 2 days.

3) after 2 days, culture solution is changed to only addition IR (87nM insulin and 0.5 μM of Rosiglitazone: Insulin+ Rosiglitazone induction broth (culture solution of the DMEM of the high sugar of 10% fetal calf serum of addition)), replacement one in every two days It is secondary, until cell is divided into fat cell completely.

4) culture medium is changed to normal culture medium (culture solution of the DMEM of the high sugar of 10% fetal calf serum of addition), simultaneously DK45 medicine irritation 24 hours for adding various concentration.Two days later, lytic cell measures the activity of cell fluorescence element enzyme, thus Determine the expression of UCP1 in cell.

3.DK45 promotes the expression of UCP1 in brown fat cell

Brown adipose tissue at 10 week old mouse omoplate fleshes is separated, and the white adipose tissue of surrounding is rejected, next Separation and differentiation method of the separation and differentiation method of brown fat cell with above-mentioned white adipocyte.

4. the luciferase vitality of cell detects

The fat cell of differentiation and maturation discards culture medium after the DK45 of respective concentration is handled 48 hours, is added 200 μ l's Luciferase lysis buffer lytic cell is placed on static 30min on ice, and at 4 DEG C, 12000rpm takes after being centrifuged 30 minutes Clearly.Luciferase assays are done respectively.Method particularly includes:

1) by taking 1 hole of 24 porocyte plates as an example, the culture solution of cell is removed, then cleans one with 500 microlitres of PBS Time, clean PBS is then removed as far as possible.

2) 100uL luciferase vitality is added and detects buffer, placement cracks half an hour on ice.Cell then is scraped off, is turned Into the EP pipe of 1.5ml.

3) 4 DEG C of low temperature, 12000rpm are centrifuged 20 minutes.

4) supernatant 20uL, DMEM in high glucose culture medium 20uL and 40 μ L Steady- are takenReagent (Steady- Luciferase Assay System, Promega) it mixes in the hole of real 96 orifice plate of bottom (PerkinElmer) of addition white.

5) it shakes 10 minutes.

6) it is measured on fluorescence illumination photometer；

7) protein concentration is measured with BCA method；

8) calculate vigor: uciferase activity is defined as the uciferase activity unit (Relative of every milligram of total protein Lueiferase Units, RLU).

As a result as shown in Figure 1, luciferase vitality measurement result shows that DK45 can promote white adipocyte in vitro Brown (i.e. enhancing white adipocyte in UCP1 expression)；And as the concentration of DK45 constantly increases, white adipose The expression of UCP1 also increases accordingly in cell, until reach highest at 20 μM, illustrate the expression of UCP1 to DK45 in dosage according to Lai Xing.

As a result as shown in Fig. 2, luciferase vitality measurement result shows that DK45 can enhance brown fat cell in vitro The expression of middle UCP1；And as the concentration of DK45 constantly increases, the expression of UCP1 is also increased accordingly in brown fat cell, says The expression of bright UCP1 is in dose dependent to DK45.

The mouse of 2 10 week old of embodiment stomach-filling DK45 (20mg/kg weight), control group while high lipid food is fed The PBS of intragastric administration on mice corresponding dosage after mouse 8 weeks, dissects mouse, white adipose tissue at groin in detection Mice Body (iWAT) and at omoplate flesh the UCP1 of brown adipose tissue (iBAT) expresses the variation with fatty liver.

The luciferase signal of 1 adipose tissue detects

The adipose tissue that mouse separates is weighed into 50mg, 200 μ L HEPES are then added, are smashed with electric homogenizer, so After be transferred in EP pipe；Remaining step is identical as the luciferase vitality measuring method of the cell of embodiment 1.

The detection of 2 hepatic pathology sections

Mouse will be dissected after mouse overnight fasting, separates liver organization, and be fixed in formalin solution, is used Conventional method carries out paraffin section and HE dyeing, the size variation of fat drop in optical microphotograph microscopic observation liver.

As a result as shown in figure 3, luciferase vitality measurement result shows the DK45 stomach-filling compared with blank control group (CON) The expression of brown adipose tissue (iBAT) and the UCP1 in white adipose tissue (iWAT) significantly improves at the omoplate flesh of mouse, That is: DK45 can promote expression of the UCP1 in vivo in white adipocyte and brown fat cell.

As a result as shown in figure 4, HE coloration result show to be distributed in blank control group (CON) mouse liver it is a large amount of larger The fat drop of volume, and the volume and content of fat drop are obviously reduced in DK45 stomach-filling group mouse liver, this shows that DK45 can Effectively treat fatty liver.

The above result shows that DK45 can effectively treat fat and fatty liver.

The mouse of 3 10 week old of embodiment stomach-filling DK45 (10mg/kg weight), control group while high lipid food is fed The PBS of intragastric administration on mice corresponding dosage after 8 weeks, carries out glucose-tolerant and insulin sensitivity experiment.

1 glucose tolerance test (IGTT)

With the glucose solution of normal saline 20%, mouse then is injected according to 2g glucose/kg weight dosage, Mouse of the intraperitoneal injection through fasting in 12 hours (at night 9 points~next day morning 9 points).With blood glucose meter monitoring injection before (0 minute) with And the blood sugar concentration (15 minutes, 30 minutes, 60 minutes, 120 minutes) of mouse in different time points after injection, time error control Within 5 seconds.

2 insulin resistants test (ITT)

Insulin dose is 0.5 unit/kg weight, but fasting time is 6 hours at 3 points (morning in 9 points~afternoon), remaining Operating method is consistent with above-mentioned glucose tolerance test.

As a result as it can be seen in figures 5 and 6, IGTT and ITT the experimental results showed that, compared with blank control group, DK45 stomach-filling mouse It is significantly reduced in the blood glucose level of various time points, the impaired glucose tolerance of DK45 stomach-filling mouse has with insulin-resistant states It is obviously improved, shows that DK45 can be very good treatment impaired glucose tolerance and insulin resistance.

Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, all should be considered as described in this specification.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

Claims (7)

1. application of the compound DK45 in the drug that preparation promotes fat cell UCP1 expression, the molecular formula of the DK45 are, Structural formula is following formula (1):

2. application according to claim 1, which is characterized in that the fat cell includes white adipocyte and brown rouge Fat cell.

3. application of the compound DK45 in the drug that preparation promotes white adipocyte brown.

4. application of the compound DK45 in the fat drug of preparation prevention and/or treatment.

5. application of the compound DK45 in the drug of preparation prevention and/or treatment fatty liver.

6. a kind of treat fat drug, which is characterized in that the active constituent of the drug includes compound DK45.

7. a kind of drug for treating fatty liver, which is characterized in that the active constituent of the drug includes compound DK45.