CN104083381A - Medical application of ursodesoxycholic acid - Google Patents
Medical application of ursodesoxycholic acid Download PDFInfo
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- CN104083381A CN104083381A CN201410365971.6A CN201410365971A CN104083381A CN 104083381 A CN104083381 A CN 104083381A CN 201410365971 A CN201410365971 A CN 201410365971A CN 104083381 A CN104083381 A CN 104083381A
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Abstract
The invention relates to the field of medicines, and particularly relates to medical application of ursodesoxycholic acid and a medicinal composition of ursodesoxycholic acid. Obtained ursodesoxycholic acid can be used for preparing RF-6A cell proliferation inhibitors.
Description
Technical field
The present invention relates to Medicines and Health Product field, particularly relate to a kind of medical usage and pharmaceutical composition thereof of ursodesoxycholic acid.
Background technology
Retinal neovascularization disease refers to because the growth of intraretinal neovascularization, thereby and follow hemorrhage, ooze out, the ocular disease of pathologic change blinding such as hypertrophy.At present, retinal neovascularization disease has day by day become one of blinding oculopathy the most serious in world wide, comprise diabetic retinopathy (Diabetic retinopathy, DR), retinopathy of prematurity (Retinopathy of prematurity, ROP), age-related macular degeneration (age-related macular degeneration, AMD), retinal vascular occlusive disease, periphlebitis of retina etc.Studies confirm that, the activities such as the differentiation of vascular endothelial cell, propagation, migration, blood capillary formation are the key links of the whole pathological process of retinal neovascularization disease, so suppress vascular endothelial cell proliferation and neovascularization growth is the key of this class disease for the treatment of.
Diabetic retinopathy (Diabetic retinopathy, DR) is one of most important performance in diabetic microangiopathy, is a kind of retinopathy that specificity changes that has, and is one of severe complication of diabetes.Clinically according to whether occurring that retinal neovascularization is mark, the diabetic retinopathy that does not have retinal neovascularization to form is called to nonproliferative diabetic retinopathy (nonproliferative diabetic retinopathy, NPDR) (or claiming simple type or background type), and being called to proliferating diabetic retinopathy, the diabetic retinopathy that has retinal neovascularization to form becomes (proliferative diabetic retinopathy, PDR).
Retinopathy of prematurity (Retinopathy of prematurity, ROP) is due to the class disease due to neonatal retinal vessel abnormality proliferation, with premature labor, low birth weight, to accept the factors such as oxygen therapy relevant.Be common in birth latter 3～6 weeks, be divided into clinically active stage and fibrous membrane and form the phase.The treatment of retinopathy of prematurity mainly relies on the medical treatment such as surgical operation and neovascularization inhibitor.
Age-related macular degeneration (age-related macular degeneration, AMD) claim again senile degeneration of macula, for the Aging of macular area structure changes.To be retinal pigment epithelium engulf digestion power to acromere dish film to main manifestations declines, and result makes not by the dish film residual body retention of catapepsis in basilar part cell oleo stock, and discharges to extracellular, is deposited on Bruch film, forms glass-film wart.Glass-film wart causes this fracture of Bruch film then, and choriocapillary enters under RPE and under neuroepithelium of retina by the Bruch film breaking, and forms choroidal neovascularization.Due to the textural anomaly of new vessels wall, cause the seepage of blood vessel and hemorrhage, and then cause a series of Secondary cases pathological change.Age-related macular degeneration betides more than 45 years old mostly, and its prevalence increases with age growth, is the important diseases of current old people's blinding.Primary disease is divided into dryness and moist amphitypy clinically.The feature of wet age related macular degeneration is under pigment epithelium layer, to have active new vessels, thus cause a series ofly ooze out, hemorrhage, cicatrix changes.
Ursodesoxycholic acid (Ursodeoxycholic Acid, UDCA) is one of active component separating from Chinese medicine Fel Ursi, is cholest acid compounds, i.e. 3 α, 7 beta-dihydroxy-5 β cholanic acids.Study and show both at home and abroad, ursodesoxycholic acid has the effect of dissolving cholelithiasis, and has advantages of that toxicity is little, dosage is little.It is the active drug of having treated cholesterol type cholelithiasis since the seventies.In recent years find still to have blood fat reducing, blood sugar lowering, spasmolytic, convulsion, haemolysis and lipase facilitation.The clinical treatment for diseases such as cholelithiasis, acute, chronic hepatitis, cholecystitis, cholangitis.
Summary of the invention
Object of the present invention aims to provide a kind of new medical usage and pharmaceutical composition thereof of ursodesoxycholic acid.
Specifically, a first aspect of the present invention has been to provide the application of ursodesoxycholic acid in preparation RF-6A inhibition of cell proliferation.
In a preference, described RF-6A cell proliferation is produced by VEGF induction.
A second aspect of the present invention has been to provide the application of ursodesoxycholic acid in the medicine of preparation control retinal neovascularization disease.
A third aspect of the present invention has been to provide the application of ursodesoxycholic acid in the medicine of preparation control diabetic retinopathy.
In a preference, described diabetic retinopathy is that proliferating diabetic retinopathy becomes.
A fourth aspect of the present invention has been to provide the application of ursodesoxycholic acid in the medicine of preparation control retinopathy of prematurity.
A fifth aspect of the present invention has been to provide ursodesoxycholic acid and has prevented and treated the application in the medicine of age-related macular degeneration in preparation.
In a preference, described age-related macular degeneration is wet age related macular degeneration.
A sixth aspect of the present invention has been to provide a kind of pharmaceutical composition that suppresses retinal neovascularization, and it comprises the ursodesoxycholic acid for the treatment of effective dose.
The details of various aspects of the present invention will be able to detailed description in chapters and sections subsequently.By below and the description of claim, feature of the present invention, object and advantage will be more obvious.
Brief description of the drawings
Fig. 1 CD31 detects the effect of UDCA to new vessels in STZ induction DR mice and normal mouse retina.Retina partial enlarged drawing (100 ×).
A. Normal group b. diabetic retinopathy model group c.UDCA1mg/kg d.UDCA10mg/kg, e. vascular counts cartogram (Data=Means ± SEM, n=6-8, with relatively * * * P<0.001 of Control group, compares with DR Model group
###p<0.001).
Fig. 2 CD31 detects the effect of UDCA to new vessels in OIR Mouse Retina.Retina partial enlarged drawing (100 ×).A. (Data=Means ± SEM, n=6-8, with relatively * * * P<0.001 of Control group, compare with OIR Model group Normal group b.OIR model group c.UDCA10mg/kg d.UDCA40mg/kg e. vascular counts cartogram
###p<0.001).Fig. 3 azovan blue (Evans Blue) detects the impact of UDCA on retinal tissue medium vessels seepage.Data=Means ± SEM, n=6-8, with relatively * * * P<0.001 of Control group, compares with OIR Model group
###p<0.001.
Detailed description of the invention
The part of coming out of the present invention is based on so unexpected discovery: ursodesoxycholic acid (UDCA) can significantly suppress the propagation of the retinal endothelial cell RF-6A of VEGF induction.Therefore the material that, above-claimed cpd can be expected to exploitation and becomes a kind of RF-6A of inhibition cell proliferation is RF-6A inhibition of cell proliferation.As known to persons skilled in the art, described " RF-6A inhibition of cell proliferation " can be various forms of materials, includes but not limited to: medicine, health product etc.And then, " the RF-6A inhibition of cell proliferation " prepared with ursodesoxycholic acid can be used for preventing and treating various retinal neovascularization diseases, these diseases include but not limited to: diabetic retinopathy (Diabetic retinopathy, DR), retinopathy of prematurity (Retinopathy of prematurity, ROP), age-related macular degeneration (age-related macular degeneration, AMD), retinal vascular occlusive disease and periphlebitis of retina etc.
And then a first aspect of the present invention has been to provide ursodesoxycholic acid or the application in preparation RF-6A inhibition of cell proliferation of its pharmaceutically acceptable salt, hydrate or prodrug.
Preferably, described RF-6A cell proliferation is produced by VEGF induction.
A second aspect of the present invention has been to provide ursodesoxycholic acid or the application in medicine or the health product of preparation control retinal neovascularization disease of its pharmaceutically acceptable salt, hydrate or prodrug.
A third aspect of the present invention has been to provide ursodesoxycholic acid or the application in medicine or the health product of preparation control diabetic retinopathy of its pharmaceutically acceptable salt, hydrate or prodrug.
Preferably, described diabetic retinopathy is that proliferating diabetic retinopathy becomes.
A fourth aspect of the present invention has been to provide ursodesoxycholic acid or the application in medicine or the health product of preparation control retinopathy of prematurity of its pharmaceutically acceptable salt, hydrate or prodrug.
A fifth aspect of the present invention has been to provide ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or prodrug and has prevented and treated the application in medicine or the health product of age-related macular degeneration in preparation.
Preferably, described age-related macular degeneration is wet age related macular degeneration.
A sixth aspect of the present invention has been to provide a kind of pharmaceutical composition that suppresses retinal neovascularization, and it comprises ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or the prodrug for the treatment of effective dose.
The present invention also provides a kind of health product that suppress retinal neovascularization, the ursodesoxycholic acid that it comprises effective dose or its pharmaceutically acceptable salt, hydrate or prodrug.
As known to persons skilled in the art, ursodesoxycholic acid of the present invention (UDCA) has following general structure:
Molecular formula: C
24h
40o
4molecular weight: 392.58
The present invention also comprises corresponding all pharmaceutically acceptable salt, hydrate or the prodrug of above-claimed cpd.These salt can by part positively charged in compound (for example, amido) with have contrary electrical electronegative (for example, trifluoracetic acid) form; Or for example, for example, formed by part electronegative in compound (, carboxyl) and positive charge (, sodium, potassium, calcium, magnesium).Compound can contain a nonaromatic pair of key, has one or more asymmetric centers.So these compounds can be used as racemic mixture, independent enantiomer, independent diastereomer, non-enantiomer mixture, cis or transisomer existence.All these isomers are all expected.Described " prodrug of ursodesoxycholic acid " is often referred to a kind of material, after using by suitable method, can in subject, carry out metabolism or chemical reaction and be transformed into ursodesoxycholic acid or its salt.
Ursodesoxycholic acid of the present invention can obtain from purchases such as Sigma companies by commercial sources, and its purity all meets medicinal standard.Or utilize marketable material, obtain by compou nd synthesis method traditional in prior art is synthetic.Those of ordinary skill in the art can synthesize compound of the present invention according to existing known technology.Synthetic compound can further be further purified by modes such as column chromatography, high performance liquid chromatography or crystallizations.
Synthetic chemistry transformation, protection functional group methodology (protect or go and protect) are helpful to synthetic application compound, and be technology commonly known in the art, as R.Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W.Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3
rded., John Wiley and Sons (1999); L.Fieser and M.Fieser, Fieser and Fieser ' s Reagents for Organic Synthesis, John Wiley and Sons (1994); And L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, has open in John Wiley and Sons (1995).
So that ursodesoxycholic acid of the present invention is made to medicine as example.Ursodesoxycholic acid of the present invention can use separately or use with the form of pharmaceutical composition.Pharmaceutical composition comprises ursodesoxycholic acid of the present invention and the pharmaceutically suitable carrier as active component.Preferably, the ursodesoxycholic acid of the present invention as active component that pharmaceutical composition of the present invention contains 0.1～99.9% percentage by weight." pharmaceutically suitable carrier " can not destroy the pharmaceutical active of ursodesoxycholic acid of the present invention, simultaneously its effective dose, and can bringing into play pharmaceutical carrier, to make the consumption of used time nontoxic to human body.
Described pharmaceutically suitable carrier includes but not limited to: soft phospholipid, aluminium stearate, aluminium oxide, ion exchange material, self-emulsifying drug delivery system, tween or other surfactants, serum albumin, buffer substance are if phosphate, glycine, sorbic acid, water, salt, electrolyte are as sulfate protamine, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, magnesium silicate, satisfied fatty acid partial glycerol ester admixture etc.
Other conventional excipient substances are as binding agent (as microcrystalline Cellulose), filler (as starch, glucose, Lactis Anhydrous and lactose beadlet), disintegrating agent (as cross-linked pvp, crosslinked carboxymethyl fecula sodium, cross-linking sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricant (as magnesium stearate) and absorption enhancer, absorption carrier, flavouring agent, sweeting agent, excipient, diluent, wetting agent etc.
Ursodesoxycholic acid of the present invention with and pharmaceutical composition can and can pass through intestinal or non-intestinal or topical routes by this area conventional method preparation.Oral formulations comprises capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.; Non-intestinal drug delivery agent comprises injection etc.; Local administration preparation comprises cream, patch, ointment, spray etc.Be preferably oral formulations.
Ursodesoxycholic acid of the present invention taking and the route of administration of pharmaceutical composition can be as oral, Sublingual, through muscle or subcutaneous, vein, urethra, vagina etc.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, the condition of conventionally advising according to normal condition or according to manufacturer.Unless otherwise indicated, otherwise all percent, ratio, ratio or umber by weight.
Unless otherwise defined, the same meaning that all specialties that use in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can combination in any.All features that patent specification discloses can with any composition forms use, each feature disclosing in description, can anyly provide the alternative characteristics of identical, impartial or similar object to replace.Therefore apart from special instruction, the feature disclosing is only the general example of equalization or similar features.
The activity of embodiment 1 ursodesoxycholic acid vitro inhibition retinal neovascularization
It is the main experimental methods of in-vitro evaluation angiogenesis that the propagation of vascular endothelial cell, migration, tube chamber form.We are on Rhesus Macacus retinal endothelial cell RF/6A cell, observe ursodesoxycholic acid important short Angiogenesis VEGF (Vascular endothelial growth factor, VEGF) has been promoted to the impact that vascular endothelial cell proliferation, migration and tube chamber form.In the RF/6A cell proliferation experiment of VEGF induction, ursodesoxycholic acid (10 μ M, 25 μ M) can significantly suppress the RF/6A cell proliferation of VEGF induction, and on the not significantly impact of the RF/6A cell of quiescent stage.Ursodesoxycholic acid (2.5 μ M, 5 μ M, 10 μ M, 25 μ M) can obviously suppress the RF/6A cell migration of VEGF induction.The tube chamber that ursodesoxycholic acid (2.5 μ M, 5 μ M, 10 μ M, 25 μ M) can obviously suppress the RF/6A cell of VEGF induction forms.As can be seen here, the retinal neovascularization that ursodesoxycholic acid can suppress to be induced by VEGF in vitro, and VEGF is the topmost angiogenesis promoting factor that participates in regulation and control retinal neovascularization.
1.1 experiment material
1.1.1 cell
RF-6A cell strain: be purchased from Shanghai Inst. of Life Science, CAS cell resource center.
1.1.2 medicine
Ursodesoxycholic acid is purchased from Sigma company of the U.S., and purity is more than 99%.
1.1.3 reagent
Gelatin and bromination-3 (4,5-dimethylthiazole-2) 2,5-xylyl tetrazole (MTT) is purchased from Sigma company of the U.S., and hyclone (FBS), Matrigel, RPMI1640 culture fluid, 0.25% trypsin are purchased from American I nvitrogen company.VEGF (VEGF) is purchased from PeproTech company of the U.S..In experiment, other reagent used is domestic analytical pure.
1.2. experimental technique:
1.2.1 medicine preparation
DMSO for ursodesoxycholic acid (DMSO) dissolves, and being made into mother liquid concentration is 0.1mol/L, and mother solution is all used primary sterilization filter (0.22 μ M) filtration sterilization.When dosing, prepare 5 required concentration by serum-free RPMI1640 culture medium, make DMSO total concentration in cultivation be no more than 0.1%.
1.2.2 ursodesoxycholic acid suppresses the RF/6A cell proliferation experiment of VEGF induction
The take the logarithm cell of trophophase, cell is made to cell suspension, spread in 96 orifice plates by certain concentration, in the time that cell attachment growth conditions is good, abandon former culture medium, change the RPMI1640 culture medium containing 1% hyclone, add medicine to be measured after 15 minutes, add VEGF (Vascular endothelial growth factor, VEGF) incubate altogether after 48 hours with cell, add 10 μ l MTT (final concentration 0.5mg/ml) to put incubator reaction 4 hours, add 50 μ l tri-agent (10%SDS-5% isobutanol-0.01M HCL) fully to dissolve more than 12 hours in incubator, finally measure light absorption value at OD570/630nm place.Cell survival rate (%)=administration group OD (570nm-630nm)/matched group OD (570nm-630nm) × 100%, matched group adds containing the cell culture fluid of equivalent DMSO and does not add any medicine.
1.2.3 migration experiment
Transwell cell is with after the coated 1h of 0.1% gelatin, and taking-up is dried, and the RF/6A cell serum-free medium having digested is washed 3 times, and dilution is 4 × 10
5/ ml, adds 100ul (4 × 10 in upper chamber
4cells/well) cell suspension.Lower chamber adds the serum-free medium of 600 μ l, and adds VEGF (10ng/ml), wears film 8 hours.Suck upper and lower chamber training liquid, with the fixing 30min of 4 DEG C of 4% paraformaldehyde room temperature, wipe not migrating cell of upper strata, interior chamber, 0.1% crystal violet room temperature dyeing 10min with cotton swab.Dye so that PBS rinsing is unnecessary.Under microscope, take pictures and count, finally using 10% acetic acid 100ul/ hole extracting 10min, with measuring OD value under microplate reader 600nm.
1.2.4 tube chamber forms experiment
Cold fluid matrix glue 30 μ l are spread in the 96 every holes of orifice plate, at 37 DEG C, solidify 45 minutes.Every hole adds 100 μ l cell suspension (1 × 10
4cells/well) add medicine or the contrast of variable concentrations simultaneously, hatch 4 hours.Poor as microscope document image with being inverted, calculate complete tube chamber and form number.
1.2.5 statistical procedures
Experimental data all uses meansigma methods ± standard error to represent, adopts SPSS11.5 statistical software to analyze, and carries out variance analysis in One-Way ANOVA mode, relatively adopts between two LSD method, and P<0.05 is for having statistical significant difference standard.
1.3 experimental result
1.3.1 the impact of ursodesoxycholic acid (UDCA) on VEGF (VEGF) induction RF/6A cell proliferation
From table 1, UDCA starts all can suppress the propagation of VEGF induction RF/6A cell significance in 5 μ M concentration, and is dose dependent.
The suppression ratio (n=10) of table 1 UDCA to VEGF induction RF/6A cell proliferation
With the comparison of solvent group, * * * P<0.001;
1.3.2. the impact of ursodesoxycholic acid (UDCA) on VEGF induction RF/6A cell migration
From table 2, UDCA starts all can suppress the migration of VEGF induction RF/6A cell explicitly in 5 μ M, and is dose dependent.
The impact (n=6) of table 2 UDCA on VEGF induction RF/6A cell migration
With the comparison of solvent group, * * P<0.01; * * P<0.001;
1.3.3. the impact that ursodesoxycholic acid (UDCA) forms VEGF induction RF/6A cell tube chamber
From table 3, UDCA starts all can suppress the tube chamber formation of VEGF induction RF/6A cell explicitly in 2 μ M, and is dose dependent.
The impact (n=6) that table 3 UDCA forms VEGF induction RF/6A cell tube chamber
With the comparison of solvent group, * * * P<0.001
Embodiment 2 ursodesoxycholic acid improve diabetic retinopathy drug activity
2.1 experiment material
2.1.1 animal:
SPF level C57 mice, male, body weight 18-24g, purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center.Animal quality certification numbering: SCXK (Shanghai) 2012-0002.Raise in the SPF of Shanghai Univ. of Traditional Chinese Medicine level Animal House, raise after one week and use.Raising condition is: 22 ± 1 DEG C of temperature, and humidity 55 ± 5%, 12 little time secretly circulated, and after feedstuff and water sterilization, freely absorbed.Experiment is used management rules to carry out in strict accordance with country and Shanghai Univ. of Traditional Chinese Medicine animal center animal.
2.1.2 the preparation of medicine:
Get appropriate ursodesoxycholic acid (Ursodeoxycholic Acid, UDCA) and be dissolved in DMSO, be mixed with the mother solution of 0.1M, with normal saline dilution pneumoretroperitoneum drug administration by injection, concentration is 1mg/kg and 10mg/kg.
2.2 experimental technique
2.2.1 zoopery:
(1) 0.1M citric acid solution and citric acid three sodium solution are mixed with the ratio of 14:11, adjust mixed liquor PH to 4.3-4.5, stand-by.Take appropriate STZ powder, lucifuge is dissolved, and makes it to become the solution of 5.5mg/ml, gives immediately the mouse peritoneal injection of fasting 12h, 0.1ml/10g, and the dosage that finally gives STZ is 55mg/kg, successive administration 5 days.
(2) last administration one Zhou Houyong cuts tail method and surveys mouse blood sugar concentration, and blood glucose value >=16.7mmol/L (250mg/dl) is considered as modeling success.Successful modeling mice is divided into 3 groups at random: DR model group (DR model group), UDCA1mg/kg group, UDCA10mg/kg group, 10 every group.In addition not 10 of i.p STZ normal C57 mices as normal group (Control group).Within every two weeks, detect body weight change, monthly detect change of blood sugar.
(3) modeling success started administration after 2 months, lumbar injection, 0.1ml/10g, UDCA1mg/kg group, UDCA10mg/kg group give respectively the UDCA solution of variable concentrations, whole dosage is respectively: 1mg/kg, and 10mg/kg, Model group gives the solvent contrast of same volume, administration was put to death after 2 months, got retina and observed retinal neovascularization situation.
2.2.2 retina paving sheet CD31 immunofluorescence detects ursodesoxycholic acid (Ursodeoxycholic Acid, UDCA) to diabetic retinopathy Mouse Retina angiogenesis Therapy situation
(1) retina separates: the eyeball of mice after putting to death is taken off, be fixed in 4% paraformaldehyde (in PBS), 4 DEG C are spent the night.Under anatomical lens, by detachment of retina, in PBS, omit rinsing 3-4 time.
(2) sealing: the retina after rinsing is put in the 1.5ml Epp pipe that Blocking Buffer (5%BSA, 0.5%Trixton X-100inPBS) is housed, and room temperature shaking table shakes slowly hatches 3h.
(3) primary antibodie is hatched: the retina having sealed is put in to (antibody 1:200 dilution, antibody diluent is 1%BSA, 0.5%Trixton X-100in PBS) in CD31 primary antibodie reactant liquor, and 4 DEG C of shaking tables shake slowly hatches 1-2 days.
(4) washing primary antibodie: at room temperature clean 6 times each 20min with Wash Buffer (0.5%Trixton X-100in PBS) on shaking table.
(5) two anti-hatching: retina is enclosed containing (1:200 dilution) in the two anti-reactant liquors of FITC conjugated anti-Rat IgG, and room temperature lucifuge is hatched 2h.
(6) washing two is anti-: with Wash Buffer (0.5%Trixton X-100in PBS) at room temperature on shaking table lucifuge clean 6 times, each 20min.
(7) mounting: under anatomical lens, retina is laid on microscope slide, cuts off into petal-shaped, drip several anti-fluorescence quenchers, with Gelatin moutin solution (2g/ml Gelatin) mounting, covered.
(8) take pictures: after retina immunofluorescence paving sheet is slightly dried, under fluorescence microscope, take pictures, observe the newborn situation of retina medium vessels.
(9) vessel density counting: choose CD31 immunofluorescence photograph (200 ×), each group is selected 3-5 to open photo, calculate optical center anyhow the number of blood vessel on axis of symmetry as vessel density.
2.2.3 data analysis
Data result represents with means ± SE.Experimental data adopts SPASS 16.0 software statistics, relatively adopts between two independent sample t inspection.When P<0.05, think and there is statistical significant difference.
2.3 experimental result
2.3.1 the impact of ursodesoxycholic acid (Ursodeoxycholic Acid, UDCA) on blood glucose in diabetic mice and body weight
As seen from Table 4, the each dosage group of UDCA all can not be improved alleviating of diabetic mice body weight, can not reduce the blood glucose of rising, illustrates that UDCA does not improve the drug activity of diabetes.
The impact of table 4 UDCA on blood glucose in diabetic mice and body weight
* *p<0.001 and normal phase ratio
2.3.2 the impact of ursodesoxycholic acid (Ursodeoxycholic Acid, UDCA) on diabetic mice retinal neovascularization
As seen from Figure 1, diabetic mice retina medium vessels is obviously newborn, and the each dosage group of UDCA can obviously reduce amphiblestroid angiogenesis.Prompting UDCA can suppress the release of angiogenic growth factor, suppresses amphiblestroid angiogenesis, thereby improves diabetic retinopathy.
Embodiment 3 ursodesoxycholic acid improve the drug activity of oxygen induction oxygen-induced retinopathy
3.1 experiment material
3.1.1 animal
SPF level C57BL mouse, each 5 of male and female, 8 week age, purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center (quality certification number: SCXK (Shanghai) 2012-0002).Raise in Shanghai Univ. of Traditional Chinese Medicine's zoopery center, male and female random combine is 5 pairs breeds, 22 ± 1 DEG C of temperature, and humidity 55 ± 5%, freely absorbs after feedstuff and water sterilization, and 12 little time secretly circulated.Experiment is used management rules to carry out in strict accordance with country and Shanghai Univ. of Traditional Chinese Medicine animal center animal.
3.2 experimental technique
3.2.1 the preparation of medicine:
Get appropriate UDCA and be dissolved in DMSO, be mixed with the mother solution of 0.1M, with normal saline dilution pneumoretroperitoneum drug administration by injection, concentration is 20mg/kg.
3.2.2 OIR model is set up
After C57BL mouse birth the 7th day (P7), be divided at random normal group and anoxia group, 10 every group.Normal group mice is raised in normal oxygen environment; Anoxia group mice is raised in 75.5% partial pressure of oxygen oxygen cabin, oxygen environment is back in normal oxygen environment for 5 days afterwards on the 12nd day, continue raise 5 days to the 17th day (P17), during this time every day intraperitoneal injection, successive administration 5 days.
3.2.3 retina immunofluorescence paving sheet
Getting P17 days mices puts to death, win eyeball, in 4% paraformaldehyde (in PBS), 4 DEG C are fixedly spent the night, under anatomic microscope, cut off by iris scissors edge edge of cornea, remove crystalline lens and vitreous body, take out complete retina, PBS washes 2～3 times, Blocking Buffer (5%BSA, 0.5%Trixton X-100in PBS) seal 2 hours, hatch 2 days in 4 DEG C with CD31 antibody, Wash Buffer (0.5%Trixton X-100in PBS) washes 6 times, hatch 2 hours with two anti-lucifuges, Wash Buffer washes 6 times, cut retina and become petal-shaped, be laid on microscope slide, the fixing also mounting of gelatin (2g/ml), in fluorescence microscopy Microscopic observation and take pictures.
3.2.4 retina seepage experiment
Get P17 days mices, lumbar injection 2% azovan blue (Evans Blue) (in normal saline), 10ul/g body weight, circulation 2h; Cut thoracic cavity open, left ventricle lavation normal saline (37 DEG C); Win eyeball, under anatomic microscope, isolate retina; Retinal tissue, in 55 DEG C, claims dry weight after vacuum drying 5h; Add 120ul Formamide, 70 DEG C of water-bath 18h; Get supernatant, centrifugal: 10000g, 10min; Get supernatant 60ul, detect in microplate reader 620nm; Deduct Formamide control wells detected value, bring Evans Blue standard absorption curve into; Result is divided by retinal tissue dry weight, i.e. EvansBlue leakage values ng/g.
3.2.5 data analysis
Data result represents with means ± SE.Experimental data adopts SPASS 16.0 software statistics, relatively adopts between two independent sample t inspection.When P<0.05, think and there is statistical significant difference.
3.3 experimental result
3.3.1 the impact of UDCA on oxygen induction newborn mice retinal neovascularization
As seen from Figure 2, OIR model group Mouse Retina has a large amount of angiogenesiss, obviously reduces and give the blood vessel of hypertrophy after UDCA.Prompting UDCA can suppress OIR model group Mouse Retina angiogenesis, thereby improves retinopathy of prematurity.
3.3.2 the impact of UDCA on oxygen induction newborn mice retinal vessel seepage
As seen from Figure 3, OIR model group Mouse Retina has a large amount of azovan blue (Evans Blue) to leak out from blood vessel, obviously reduces and give the azovan blue (Evans Blue) of seepage after UDCA.Prompting UDCA can suppress the vascular permeability having raise in OIR model group mice, thereby improves retinopathy of prematurity.
Many aspects involved in the present invention have been done as above and have been set forth.But, it should be understood that before not departing from spirit of the present invention and put, those skilled in the art can be equal to and change and modify it, and described change and modification fall into the coverage of the application's claims equally.
Claims (10)
1. ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or the prodrug application in preparation RF-6A inhibition of cell proliferation.
2. application as claimed in claim 1, is characterized in that, described RF-6A cell proliferation is produced by VEGF induction.
3. ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or the prodrug application in medicine or the health product of preparation control retinal neovascularization disease.
4. ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or the prodrug application in medicine or the health product of preparation control diabetic retinopathy.
5. application as claimed in claim 4, is characterized in that, described diabetic retinopathy is that proliferating diabetic retinopathy becomes.
6. ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or the prodrug application in medicine or the health product of preparation control retinopathy of prematurity.
7. ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or prodrug are prevented and treated the application in medicine or the health product of age-related macular degeneration in preparation.
8. application as claimed in claim 7, is characterized in that, described age-related macular degeneration is wet age related macular degeneration.
9. the pharmaceutical composition that can suppress retinal neovascularization, is characterized in that, it comprises ursodesoxycholic acid or its pharmaceutically acceptable salt, hydrate or the prodrug for the treatment of effective dose.
10. the health product that can suppress retinal neovascularization, is characterized in that, the ursodesoxycholic acid that it comprises effective dose or its pharmaceutically acceptable salt, hydrate or prodrug.
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| CN201410365971.6A CN104083381A (en) | 2014-07-29 | 2014-07-29 | Medical application of ursodesoxycholic acid |
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| CN201410365971.6A CN104083381A (en) | 2014-07-29 | 2014-07-29 | Medical application of ursodesoxycholic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110177556A (en) * | 2017-02-09 | 2019-08-27 | 柳氏生物制药公司 | The composition for being used to prevent or treat vision disorder containing ursodesoxycholic acid |
| CN113164499A (en) * | 2018-12-18 | 2021-07-23 | 参天制药株式会社 | Therapeutic or prophylactic agent for presbyopia containing ursodeoxycholic acid |
| US11331326B2 (en) | 2016-09-30 | 2022-05-17 | Amicogen Pharma Inc. | Composition for prevention or treatment of inflammatory skin diseases or severe pruritus comprising the aqueous solubilized ursodeoxycholic acid |
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| WO2006086452A1 (en) * | 2005-02-10 | 2006-08-17 | Regents Of The University Of Minnesota | Methods for treating visual disorders |
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| WO2006086452A1 (en) * | 2005-02-10 | 2006-08-17 | Regents Of The University Of Minnesota | Methods for treating visual disorders |
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| SE JOON WOO等: "Ursodeoxycholic Acid and Tauroursodeoxycholic Acid Suppress Choroidal Neovascularization in a Laser-Treated Rat Model", 《JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11331326B2 (en) | 2016-09-30 | 2022-05-17 | Amicogen Pharma Inc. | Composition for prevention or treatment of inflammatory skin diseases or severe pruritus comprising the aqueous solubilized ursodeoxycholic acid |
| CN110177556A (en) * | 2017-02-09 | 2019-08-27 | 柳氏生物制药公司 | The composition for being used to prevent or treat vision disorder containing ursodesoxycholic acid |
| RU2723988C1 (en) * | 2017-02-09 | 2020-06-18 | Ю'С Биофарм Инк. | Composition for preventing or treating vision disorders, containing ursodeoxycholic acid |
| CN113164499A (en) * | 2018-12-18 | 2021-07-23 | 参天制药株式会社 | Therapeutic or prophylactic agent for presbyopia containing ursodeoxycholic acid |
| EP3900728A4 (en) * | 2018-12-18 | 2022-08-17 | Santen Pharmaceutical Co., Ltd. | Ursodeoxycholic acid-containing agent for treating or preventing presbyopia |
| US11419880B2 (en) | 2018-12-18 | 2022-08-23 | Santen Pharmaceutical Co., Ltd. | Ursodeoxycholic acid-containing agent for treating or preventing presbyopia |
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