CN103316018B - The application of latanoprost in the medicine for the preparation for the treatment of diabetes and/or hypertriglyceridemia and complication thereof - Google Patents
The application of latanoprost in the medicine for the preparation for the treatment of diabetes and/or hypertriglyceridemia and complication thereof Download PDFInfo
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Abstract
The invention discloses the purposes of latanoprost in the medicine for the preparation for the treatment of diabetes and/or hypertriglyceridemia and complication thereof, especially, described diabetes are the type 2 diabetes mellitus caused by insulin resistant.In described purposes, protein kinase (AMPK) agonist that latanoprost activates as nuclear receptor retinoic acid receptor X α (RXR α) antagonist and/or AMP.Latanoprost has antagonism to retinoic acid receptor X α (RXR α), can the transcriptional activity of specific antagonist PPAR γ: RXR α heterodimer, thus there is the activity suppressing the synthesis of adipose cell triglyceride and storage, it also shows the activity of the protein kinase (AMPK) that exciting AMP activates simultaneously, promotes the effect of the upper film of GLUT4 (GLUT4) and further glucose absorption.Latanoprost acid also shows the activity of obvious exciting AMPK in addition.
Description
Technical field
The present invention relates to pharmacotherapeutics field, be specifically related to latanoprost and acid derivative latanoprost acid or its pharmaceutical composition for the preparation for the treatment of diabetes and/or hypertriglyceridemia and complication thereof medicine in purposes, more specifically, the present invention relates to latanoprost and the acid of acid derivative latanoprost or its pharmaceutical composition thereof as nuclear receptor retinoic acid receptor X α (RXR α) antagonist, protein kinase (the AMP-activatedproteinkinase that AMP activates, AMPK) agonist and for the preparation for the treatment of diabetes and/or hypertriglyceridemia and complication thereof medicine in purposes.
Background technology
As the derivant of prostaglandin, the latanoprost with structure shown below is the propyl diester dinoprost that a kind of novel phenyl substitutes, and is selective prostaglandins F receptor (FP) agonist.It is the prodrug of isopropyl esterification, can be absorbed by cornea and be hydrolyzed into the activated latanoprost acid of tool, intraocular pressure (intraocularpressure, IOP) [DrugsFuture.17:691-704,1992 are reduced by the ah outflow increased through glucose sclera approach; JMedChem.36:243-248,1993].Latanoprost is mainly used in the treatment of open angle glaucoma and ocular hypertension clinically, at present by the Second line Drug as treatment open angle glaucoma.The major side effects of its performance is clinically: long-term prescription can cause iris color to change, and causes eyelid atrophy, and further microscopy finds the atrophy of eyelid adipose cell, and then causes eyelashes sagging.
Latanoprost
Retinoic acid receptor X (RXR) is the core member of nuclear receptor protein family, it self can not only form homodimer, heterodimer functionating can also be formed with other nuclear receptors, participate in many important physiological process such as Growth of Cells differentiation, Metabolism regulation, form generation and fetal development.The heterodimer that wherein RXR and PPAR γ is formed can participate in the adjustment of Adipocyte Differentiation and lipid metabolism, therefore this dimeric antagonist is except suppressing Adipocyte Differentiation, the content [JClinInvest108:1001-1013,2001] of triglyceride in white adipocyte, muscle cell and liver can also be reduced.Based on this, the selective antagonist of RXR α-PPAR γ has potential using value in the treatment of fat or hypertriglyceridemia.
The kinases (AMPK) that AMP activates is the induction apparatus of a competent cell self-energy state, can exercise its metabolism and energy adjustment effect in multiple tissue.In muscular tissue, can by promoting that on the cell of GLUT4 (GLUT4), film finally promotes the glucose absorption [AmJPhysiol273:E1107-1112 of muscular tissue after AMPK activation, 1997], the fatty acid oxidation of muscle cell can also be promoted by its stream substrates acetyl-CoA carboxylase (ACC) of phosphorylation, and then cause insulin sensitivity enhancing [JClinInvest108:1167-1174,2001], can also promote that the mitochondrion of muscle cell generates in addition, and improve the expression of GLUT4 gene; In hepatic tissue, AMPK can regulate and control carbohydrate metabolism in hepatic tissue and lipid metabolism, comprises lipogenesis, fat oxidation and cholesterol and generates [Nature414:799-786,2001; JClinInvest108:1167-1174,2001]; In fatty tissue, AMPK is except stimulating except the absorption of glucose by the upper film of promotion GLUT4, can also its stream substrates hormone sensitive lipase of phosphorylation, thus suppress it by the activation of PKA, the generation of final suppression free fatty with to the release [EurJBiochem179:249-254,1989] in blood; In islet tissue, the insulin releasing that the activation of AMPK can suppress glucose to stimulate, thus reduce the insulin level in blood.In sum, AMPK is the intracellular energy state sensor of a diverse in function, and can regulate the metabolic balance of body in nervus centralis and multiple peripheral tissues.Activate AMPK in muscle, liver and fatty tissue can stimulate metabolism, strengthen insulin sensitivity and regulate and control the effect that prevention and therapy type 2 diabetes mellitus is finally played in series of genes expression.
Latanoprost is the medicine being used to treat open angle glaucoma and ocular hypertension at present, and the present invention has found the potentiality that it is treated new indication further.
Summary of the invention
An object of the present invention is to provide the new medical usage of latanoprost and latanoprost acid, namely its for the preparation for the treatment of diabetes and/or hypertriglyceridemia and complication thereof medicine in purposes.Particularly, described diabetes are the type 2 diabetes mellitus caused by insulin resistant.
In described purposes, protein kinase (AMPK) agonist that latanoprost activates as nuclear receptor retinoic acid receptor X α (RXR α) antagonist and/or AMP.
Another object of the present invention is to provide latanoprost in preparation as the purposes in the medicine of nuclear receptor retinoic acid receptor X α (RXR α) antagonist.
Another object of the present invention is to provide latanoprost and the purposes of latanoprost acid in the medicine of preparation as protein kinase (AMPK) agonist of AMP activation.
Another object of the present invention is to provide the pharmaceutical composition comprising latanoprost or latanoprost acid and is preparing the purposes for the treatment of in the medicine of diabetes and/or hypertriglyceridemia and the complication thereof caused by insulin resistant, and described pharmaceutical composition comprises pharmaceutically acceptable adjuvant.
Present invention finds new action target spot and the drug effect of latanoprost, it has antagonism to nuclear receptor retinoic acid receptor X α (RXR α), has agonism to the protein kinase (AMPK) that AMP activates.Also there is the effect suppressing the synthesis of adipose cell triglyceride and store, strengthen glucose absorption simultaneously.This compound can be used for treating diabetes, hypertriglyceridemia and complication thereof.
Accompanying drawing explanation
Fig. 1 shows the associativity of latanoprost and RXR α-LBD albumen.
Fig. 2 shows the Antagonism of latanoprost to the transcriptional activation of PPAR γ: RXR α.
###.P < 0.05, compares with first DMSO group,
*.P < 0.05,
* *.P < 0.001, compared with second ROS group.One factor analysis of variance (One-wayANOVA).
Fig. 3 shows the impact of latanoprost on the content of triglyceride in adipose cell 3T3-L1 after differentiation.
* *.P < 0.001, compared with DMSO group.One factor analysis of variance.
Fig. 4 A shows latanoprost and the impact of latanoprost acid on the phosphorylation of AMPK.
Fig. 4 B is depicted as quantitative figure.
* *.P < 0.001, compared with DMSO group.One factor analysis of variance.
Fig. 5 shows the impact of latanoprost on film on GLUT4.
Fig. 6 shows the impact of latanoprost on adipose cell glucose absorption.Compare with first DMSO group,
*.P < 0.01,
* *.P < 0.001, one factor analysis of variance.
Fig. 7 shows the impact of latanoprost on db/db mice fasting glucose.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further elaborated, but these embodiments should not be construed as restriction the present invention.
test example
Test example 1: latanoprost is to ligand binding region (RXR α ligandbindingdomain, RXR α-LBD) the binding activities determination experiment of RXR α
The present invention expresses in e. coli bl21 (DE3) and separation and purification obtains people source RXR α-LBD albumen, tests the binding activities of latanoprost to RXR α-LBD.Experiment shows, being combined with RXR α-LBD of latanoprost energy concentration dependent.
1, experimental principle
This experiment adopts surface plasma resonance (surfaceplasmonresonance, SPR) technology to detect the combination of latanoprost and RXR α-LBD.The principle of SPR technique is: generation be totally reflected when incident illumination incides the interface of two kinds of different transparent mediums (glass and air) with critical angle, if and dielectric surface plate one deck gold film after, incident illumination will cause the resonance of free electron in metal, the incident illumination at an angle incident resonance caused causes reflected light to disappear completely, and this angle is referred to as resonance angle.The change of the refractive index of the liquid phase that resonance angle can pass through with metal film surfaces and changing, and the molecular mass that the change of refractive index (representing with resonance units (responseunit, RU)) is combined with metal surface is directly proportional.Therefore, the mass change near sensor surface liquid level can be detected based on SPR technique, i.e. the change of refractive index.When the molecule in solution and target molecule in conjunction with time, quality increases, then Mass lost when dissociating.At present, SPR technique is widely used in the micromolecular compound screening and can be combined with target protein, and simulates micromolecule and macromolecular binding constant (K further
d).
2, experiment material and method
1) preparation of people source RXR α-LBD albumen: people source RXR α-LBD (221 ~ 458 amino acids) gene order is cloned on pET15b prokaryotic expression carrier (invitrogen company), be converted in e. coli bl21 (DE3) (invitrogen company) and express, RXR α-LBD the albumen obtained with His post (GE company) purification, dialyses, is concentrated to about 10mg/mL.
2) preparation of people source RXR α-LBD protein chip: the Biacore3000 instrument using GE company to produce completes this part work.All experiments are all carried out at 25 DEG C.RXR α-LBD is coupled to chip CM5 (GE company) on the surface by the method for standard amino coupled, concrete steps are as follows: first, preparation HBS-EP working buffer liquid (10mMHepes, 150mMNaCl, 3.4mMEDTA, 0.005% (v/v) surfactant P20 (surfactantP20), pH7.4), balancing machine is steady to baseline.During coupling, 0.2MN-ethyl-N '-dimethyl aminopropylcarbodiimide (N-ethyl-N '-dimethylaminopropylcarbodiimide) and 50mM N-Hydroxysuccinimide (N-hydroxysuccinimide) (EDC/NHS) 1: 1 (Sigma company) mix, with 5 μ L/min sample introduction, 7 minutes activation chip surfaces.RXR α-LBD is 25 μ g/mL with sodium acetate (Sigma company) solution dilution to the final concentration of 10mM (pH4.13), is fixed on chip surface with 5 μ L/min flow velocity sample introductions.Finally, the ethanolamine (ethanolamine) (Sigma company) of 1MpH8.5 was with 5 μ L/min flow velocity sample introduction 7 minutes, and carrying out closing of chip surface, is 8000RU to final coupling amount.After RXR α-LBD coupling completes, equilibrate overnight is steady to baseline, then carries out the dependent dynamics that latanoprost is combined with RXR α-LBD and studies.
3) latanoprost: available from Sigma.
4) compound binding activities determination experiment method: the binding activities measuring latanoprost and albumen RXR α-LBD by the method for SPR.Dilute latanoprost mother solution (20mM) to 20 μMs, 14 μMs, 9.8 μMs, 6.86 μMs, 4.8 μMs, 3.36 μMs, centrifugal rear auto injection with HBS-EP buffer respectively, detect the binding activities of variable concentrations latanoprost and RXR α-LBD.The interaction of latanoprost and RXR α-LBD carries out dynamic experiment with the dynamic analysis Wizard in Biacore3000 control software design respectively.During all micromolecule sample introductions, flow velocity is 30 μ L/min, sample introduction 1min, and dissociate 2min, then uses HBS-EP buffer solution elution 2min respectively, to complete a circulation.The data obtained carry out matching according to 1: 1Langmuir combination model in BIAevaluationsoftwareversion3.1 (Biacore) analysis software, obtain definite kinetic constant.
3, experimental result:
As shown in Figure 1, latanoprost can being combined with RXR α-LBD of concentration dependent for result, the K utilizing BIAevaluationsoftwareversion3.1 (Biacore) software to simulate latanoprost to be further combined with RXR α-LBD
dvalue is 3.2 μMs.
Test example 2: cellular level test compounds latanoprost is to the antagonism of RXR α: PPAR γ transcriptional activity
The luciferase reporter gene method for measuring that the present invention is regulated and controled by PPAR cis-acting elements (PPRE) on a cellular level tests the impact of latanoprost on the transactivation activity of RXR α: PPAR γ heterodimer.Result of study shows, latanoprost has the effect of concentration dependent ground antagonism RXR α: PPAR γ heterodimer transactivation activity.
1, experimental principle:
In Cytoplasm, PPAR γ and RXR α forms heterodimer and recruits the co-activation factor, then to enter on PPRE that core acts on target gene under the assistance of other associated transcription factors thus to start transcribing of this gene.PPRE is cloned in pGL3-promoter vector and builds recombinant vector pGL3-PPRE-Luc, in this recombinant vector, firefly luciferase gene transcribe the control being only subject to response element PPRE.Therefore, the activity of LUC Photinus pyralis LUC Photinus pyralis FL is just equivalent to the transactivation activity of PPAR γ: RXR α.By pCDNA3.1-PPAR γ, pCDNA3.1-RXR α, pGL3-PPRE-Luc and internal reference plasmid pRL-SV40 cotransfection are in cell, and by measuring LUC Photinus pyralis LUC Photinus pyralis FL and internal reference uciferase activity respectively, namely the ratio of the two characterize the transactivation activity of PPAR γ: RXR α.
2, experiment material and method
1) cell culture: (condition of culture is 37 DEG C to HEK-293 cell, 5%CO in DMEM (Gibco company) culture medium (10%FBS) inner cultivation
2).
2) luciferase reporter gene of transient transfection and the regulation and control of PPAR cis-acting elements measures: lipofection carries out with reference to Invitrogen company Lipo2000 reagent description.HEK-293 cell kind is on 24 orifice plates, when Growth of Cells to 50 ~ 70% density, utilize liposome (Invitrogen company) transient cotransfection pGL3-PPRE-Luc, pCDNA3.1-PPAR γ, pCDNA3.1-RXR α and internal reference plasmid pRL-SV40, culture medium was changed into DMEM (10%FBS) after 12 hours by transfection, add compound or DMSO (as negative control) hatches 18 hours simultaneously, suck culture medium, PBS washes once, the activity (Luciferase assay method carries out with reference to Promega company Luciferase test kit description) of LUC Photinus pyralis LUC Photinus pyralis FL and internal reference luciferase is measured with Luciferase test kit (Promega company) cell lysis.This experiment is positive compound with rosiglitazone (PPAR γ part, can the transcriptional activity of antagonism PPAR γ-RXR α heterodimer, available from Sigma).
Rosiglitazone (ROS) structural formula
3, experimental result
ROS is the part of PPAR γ, and the latter can be impelled after it is combined with PPAR γ to recruit the co-activation factor and form heterodimer with RXR α, this heterodimer acts on PPRE after entering core, thus promotes the expression in promoter with the gene of PPRE sequence.The luciferase reporter gene method for measuring that the present invention utilizes PPAR cis-acting elements to regulate and control determines the antagonistic activity of latanoprost to PPAR γ: RXR α heterodimer.Result as shown in Figure 2, compared with negative DMSO group, ROS (10 μMs) can the transcriptional activity of exciting PPAR γ: RXR α, latanoprost energy concentration dependent ground antagonism ROS, to the agonist activity of PPAR γ: RXR α trans-activation, shows as the antagonist of PPAR γ: RXR α heterodimer.
Test example 3: cellular level test compounds latanoprost is on the impact of content of triglyceride in fat
The present invention is by detecting in the adipose cell 3T3-L1 after differentiation, and latanoprost process, on the impact of intracellular content of triglyceride, evaluates the impact of latanoprost on triglyceride in fat.Result shows, latanoprost can content of triglyceride in the suppression adipose cell of concentration dependent.
1, experimental principle
Adopt GPO-PAP method to detect content of triglyceride, its principle is: triglyceride is hydrolyzed into glycerol and fatty acid under the effect of esterase (LPL), and glycerol and ATP generate phosphoglycerol and ADP under glycerol kinase (GK) effect then; Phosphoglycerol and O
2di(2-ethylhexyl)phosphate hydroxypropanone-and H is generated under GPO (GPO) effect
2o
2; Final sum H
2o
2under the effect of peroxidase, red quinone compound and H is generated with 4-AA (4-AAP) and parachlorophenol
2o.The depth of quinones color generated is directly proportional to the content of triglyceride, respectively the absorbance of bioassay standard pipe and sample tube, calculates the content of triglyceride.
2, experiment material and method
1) cultivation of 3T3-L1 cell and differentiation-inducing: 3T3-L1 PECTORAL LIMB SKELETON DMEM (10%FBS) cultivates in 6 orifice plates, cultivation is continued 2 days after growing to 100% density, culture fluid changes the DMEM containing 10%FBS into and adds 167nM insulin (insulin), 0.5mM isobutylmethylxanthine (isobutylmethylxanthine, MIX) and 1 μM of dexamethasone (dexamethasone, Dex) (the whole available from Sigma of differentiation agents), induce 3 days, MIX and Dex is removed at the 4th day, with containing 10%FBS, the DMEM of 167nMinsulin continues induction 3 days, changed DMEM (10%FBS) into cultivate at the 7th day, within 8th day, compound latanoprost is hatched in differentiation, the content of triglyceride is measured after hatching 24 hours.
2) triglyceride detection kit: purchased from the safe clinical reagent company limited of Beijing Northization.
3) triglyceride detection method in cell: add the cell of compound treatment after 24 hours and add 1mLPBS (preheating) and wash twice, add 500 μ L trypsin preheatings) digestion (be placed in incubator and be about 2min), add 500 μ L culture medium (10%FBS) and stop; Be transferred to after cell is dispelled in 1.5mLEP pipe and (be placed on ice), centrifugal, 900g, 4 DEG C, 2min, abandons supernatant; Add 1mLPBS, dispelled by cell rear centrifugal, 900g, 5min, remove supernatant; Add 100 μ LRIPA cell lysis buffer solution, dispel, ice-bath ultrasonic (20-30s, interval 15s, 5-6 time); The centrifugal 15min of 12000rpm, draws intermediate layer clear liquid; Repeated centrifugation is (the centrifugal 15min of 12000rpm) once, sucts clearly for measuring the concentration of triglyceride and protein; When measuring triglyceride, the 96 every holes of orifice plate add 8 μ L samples, and add the buffer that 200 μ L test kits are joined, design temperature is 37 DEG C, reading after 5 minutes, and setting wavelength is that the value that Lm1500nM, Lm2660nM, Lm1-Lm2 obtain is test value; Measure total protein concentration by BCA method, after 595nM reading, calculate the protein concentration of each sample.
3, experimental result:
Research shows, the antagonist of RXR α-PPAR γ heterodimer, except suppressing Adipocyte Differentiation, can also reduce the content of triglyceride in white adipocyte, muscle cell and liver.Therefore, inventor have detected whether latanoprost can reduce triglyceride in adipose cell content as the antagonist of RXR α-PPAR γ heterodimer.As shown in Figure 3, latanoprost can reduce the content of triglyceride broken up in rear adipose cell 3T3-L1 by concentration dependent.
Test example 4: cellular level test compounds latanoprost and the agonism of latanoprost acid to AMPK activity in fat
AMPK is the sufficient condition of exciting AMPK activity in the phosphorylation of the 172nd threonine, therefore promotes the phosphorylation of 172 threonine, namely represents the excitement activity of AMPK.The present invention tests the impact of latanoprost on AMPK phosphorylation in adipose cell after differentiation by the experimental technique of Westernblot, result shows that latanoprost can the phosphorylation level of increase AMPK of concentration dependent, namely illustrates that latanoprost can the activity of AMPK in exciting cell.And further test also proves that its carboxylic acid derivative latanoprost acid (structure See Figure) also has the activity of AMPK in exciting cell.
Latanoprost acid (latanoprostacid)
1, experimental principle
Westernblot tests: the expression that there is AMPK in adipose cell.Differentiation 3T3-L1 cell serum is hungry also uses compound treatment after 8 hours simultaneously, ice-cold PBS washes 3 times, add SDS sample buffer (60 μ g/ hole) cell lysis, by the cell harvesting of cracking, with the antibody test AMPK phosphorylation level of AMPK172 position phosphorylation, glyceraldehyde 3-phosphate dehydro-genase (GAPDH) and AMPK the albumen in contrast increase of detection compound on the phosphorylation of AMPK are not affect it to express.
2, experiment material and method
1) cultivation of 3T3-L1 cell and differentiation-inducing: with test example 3.
2) Westernblot tests major parameter: primary antibodie: p-AMPK α Thr172 (1: 1000, Cellsignaling company), AMPK α (1: 1000, Cellsignaling company) GAPDH (1: 10,000, Kang Cheng is biological); Two resist: rabbit resists (anti-Rabbit) (1: 5,000, Jackson company), mouse-anti (anti-mouse) (1: 10,000, AbCam company limited).
3, experimental result:
As shown in Figure 4, compared with contrast DMSO processed group, latanoprost can the phosphorylation of enhancing AMPK of concentration dependent, but does not affect its total protein for result.Latanoprost acid also can strengthen the phosphorylation of AMPK, does not affect AMPK total protein.
Test example 5: cellular level test compounds latanoprost is to the facilitation of the upper film of GLUT4 in adipose cell
GLUT4 be transported on cell membrane be AMPK activate after downstream effect, therefore can promote the experimental result of AMPK activity based on latanoprost, inventor have detected the effect of this compound to film on GLUT4.The present invention passes through the impact of attached cell immunofluorescence experiment technical testing latanoprost on film on GLUT4 in adipose cell after differentiation, and result shows that the latanoprost of 20 μMs can increase the upper film of GLUT4.
1, experimental principle
Immunologic fundamental reaction is antigen-antibody reaction.Because antigen-antibody reaction has the specificity of height, so when antigen-antibody reacts, as long as know one of them factor, just another factor can be found.Immunofluorescence technique is exactly not affect the fluorochrome mark of antigen-antibody activity on antibody (or antigen), after its corresponding antigen (or antibody) combines, presents the reaction of a kind of specificity fluorescent under fluorescence microscope.Based on this technology, inventor utilizes the GLUT4 albumen in the antibody characterization adipose cell of GLUT4 on Cytoplasm and cell membrane, evaluates the impact of latanoprost on the upper film of GLUT4 albumen.
2, experiment material and method
1) cultivation of 3T3-L1 cell and differentiation-inducing: with test example 3.
2) immunofluorescence test:
A, preparation: coverslip 1MHCl (commercially available concentrated hydrochloric acid is about 12M) is in 50 DEG C of soaked overnight, after the abundant rinsing of distilled water, put into dehydrated alcohol and soak 4-5h, being clipped in afterwards on filter paper makes it dry, finally coverslip is placed in clean culture dish, irradiates in super-clean bench medium ultraviolet and spend the night; Solution preparation: TBS-T; Concentrated hydrochloric acid+Tween-20 (0.2%); 3%BSA; 4% paraformaldehyde; 0.2%TritonX-100 (preparing with PBS); PBS.
B, process of the test: by sterile cover slips folder in 24 orifice plates, inoculating cell, attach overnight; Agent-feeding treatment is serum starvation 8h (insulin process 10min, latanoprost process 8h) simultaneously; After drug treating terminates, exhaust culture fluid, every hole adds 4% paraformaldehyde to covering coverslip completely, fixing 15min; PBS washs 3 times, puts shaking table 5min at every turn; Every hole adds 0.2%TritonX-100 to covering coverslip completely, permeabilized cells 15min; PBS washs 3 times, puts shaking table 5min at every turn; Every hole adds the BSA of 3% to covering coverslip completely, closes 15min; Parafilm film drips primary antibodie (with the BSA preparation of 3%, often open coverslip and be about 5-10 μ L, as GLUT41: 50, ABCAM), carries out labelling for different sample; The each port lid slide of careful gripping, cell placed face down, on correspondence position primary antibodie solution, hybridizes 1h in wet box; Careful clamping cover slide, cell faces up and places back in each hole of original porous plate, and TBS-T washs 3 times, puts shaking table 5min at every turn; Parafilm film drips fluorescence two anti-(with the BSA preparation of 3%, often open coverslip and be about 5-10 μ L, as Mouse488, dilute at 1: 200), carries out labelling for different sample; The each port lid slide of careful gripping, cell placed face down, on the anti-solution of correspondence position two, hybridizes 1h in wet box; Careful clamping cover slide, cell faces up and places back in each hole of original porous plate, and TBS-T washs 3 times, puts shaking table 5min at every turn; Microscope slide drips mountant 1-1.5 μ L, after blotting coverslip surplus liquid with absorbent paper, cell faces down and is placed on microscope slide, fluorescence microscope.
3, experimental result:
As shown in Figure 5, compared with contrast DMSO processed group, insulin can the upper film of obvious stimulation GLUT4, and latanoprost also shows the effect of film on obvious stimulation GLUT4 simultaneously for result.
Test example 6: cellular level test compounds latanoprost is to the facilitation of the absorption of glucose in fat
The present invention evaluates the effect of latanoprost to adipose cell glucose absorption by the experiment of 3T3-L1 adipose cell glucose absorption, and result display latanoprost can the ability of increase adipose cell glucose absorption of concentration dependent.
1, experimental principle
3T3-L1 adipose cell glucose absorption experiment be add in the culture fluid breaking up 3T3-L1 adipose cell completely 2-[
3h]-deoxyglucose, by the ability of isotope calculating instrument count detection Cell uptake glucose.Based on this, inventor adopts this technology for detection latanoprost on the impact of adipose cell glucose absorption.
2, experiment material and method
1) cultivation of 3T3-L1 cell and differentiation-inducing: with test example 3.
2) hungry agent-feeding treatment 8h (DMEM+0.5%BSA) of the 3T3-L1 adipose cell broken up; At 7.5h, DMEM containing 0.5%BSA in culture medium pours out, and rinse 2-3 time with the PBS of temperature, then Krebs (the 0.25mL/ hole containing 0.5%BSA is added, 24 orifice plates), now need the medicine adding same concentration, while desaccharide, add insulin stimulating (concentration is 16.7nM); Add HOT, 5min before experiment terminates, successively every hole add 23 μ LHOT (HOT formula:
3h-2DOG3.2 μ L+2DOG16 μ L+DDW600 μ L); Reaction terminating: wash cell 3 times with ice PBS, adds 0.1%Triton150 μ L/ hole, lysis at room temperature 1h or with rifle piping and druming, 2min/ hole; Collecting cell, gets 100 μ L cell pyrolysis liquids and adds 400 μ L scintillation solution countings.
3, experimental result:
As shown in Figure 6, compared with contrast DMSO processed group, latanoprost can the glucose absorption of promotion adipose cell of concentration dependent for result.
Test example 7: compound latanoprost is on the impact of db/db mice fasting glucose
The present invention, by measuring lumbar injection to the fasting blood glucose level of type 2 diabetes mellitus model mice after compound latanoprost (db/db mice), studies compound latanoprost to the treatment of type 2 diabetes mellitus or improvement result.Result shows that compound latanoprost has good hypoglycemic activity.
1, experimental principle:
Db/db mice (leptin receptor deficiency) belongs to spontaneous type 2 diabetes mellitus animal model, is by C57BL/KsJ inbred strain autosomal recessive inheritance, AR derivation.The animal model of the present invention using the mice of this strain as the anti-type 2 diabetes mellitus of assessing compound.
2, experiment material and method
1) animal origin: genotype Spontaneous Diabetic db/db mice purchased from American Jackson company.
2) animal condition of culture: SPF level Animal House is raised; Temperature: 22-24 DEG C; Humidity: 45-80%; Illumination: 150-300Lx, within 12 hours, day alternates with night.It is raised, administration, blood sugar detection and the guidance (with reference to Shanghai City management of laboratory animal regulations) of putting to death all in strict accordance with zoopery and welfare
3) animal grouping and administration: db/db Mouse feeder in SPF level Animal House, after adaptability raises and train one week.Mice is divided into blank group, positive controls and tested material group by the average according to measuring fasting fasting glucose result after 6 hours, often organizes 9.Each group of mice 10:00-11:00 in morning every day respectively lumbar injection gives solvent (5%Tween80, Veh group), 200mg/kg positive compound (metformin Met, Pos group), 15mg/kg compound latanoprost (latanoprost H group) and 5mg/kg compound latanoprost (latanoprost L group).
4) observation index:
Long term to mouse blood sugar: monitor weekly fasting glucose once during administration, fasting glucose be mice fasting can't help 6h after water (from the morning 9:30-10:30 to 3:30-4:30 in afternoon) after blood glucose value, and add up each group of average blood sugar.
Date processing and statistical analysis: data represent with means standard deviation (mean ± sem), adopt one factor analysis of variance method to carry out statistical analysis to data.
3, experimental result:
Compound latanoprost is to the impact of db/db mice fasting glucose (fastingbloodglucose, FBG)
During blank group mouse experiment, fasting glucose maintains relatively high level always.The fasting glucose of positive controls is in below group of solvents always, and this phenomenon is continued until that experiment terminates.Latanoprost H and L group also showed from first week the phenomenon be in below group of solvents, except second week fasting glucose is than except group of solvents height, this phenomenon is continued until that experiment terminates, and especially at latter 3 weeks, administration group shows good hypoglycemic activity (accompanying drawing 7).
Generally speaking, through random screening, the present invention finds that latanoprost is retinoic acid receptor X α (retinoidXreceptor α, RXR α) antagonist, can the transcriptional activity of specific antagonist PPAR γ: RXR α heterodimer, thus there is the activity suppressing the synthesis of adipose cell triglyceride and storage, it also shows the activity of the protein kinase (AMPK) that can be activated by exciting AMP thus the effect of the upper film of promotion GLUT4 (GLUT4) and further glucose absorption simultaneously.Therefore, latanoprost can be used for the medicine preparing treatment diabetes, hypertriglyceridemia and complication thereof.Meanwhile, in type 2 diabetes mellitus mouse model, latanoprost also shows good hypoglycemic activity, demonstrates the medicine that it may be used for preparing treatment type 2 diabetes mellitus further.
Claims (6)
1. latanoprost and the purposes of latanoprost acid in the medicine for the preparation for the treatment of diabetes and complication thereof.
2. purposes according to claim 1, wherein, described diabetes are the type 2 diabetes mellitus caused by insulin resistant.
3. purposes according to claim 1 and 2, wherein, the protein kinase agonist that latanoprost activates as AMP.
4. comprise the purposes of pharmaceutical composition in the medicine preparing treatment diabetes and complication thereof for the treatment of effective dose latanoprost or latanoprost acid, described pharmaceutical composition comprises pharmaceutically acceptable adjuvant.
5. purposes according to claim 4, wherein, described diabetes are the type 2 diabetes mellitus caused by insulin resistant.
6. the purposes according to claim 4 or 5, wherein, the protein kinase agonist that latanoprost activates as AMP.
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