CN103169719B - Application of anthracene nucleus antibiotic and its pharmaceutical salt for treating diabetic eye diseases - Google Patents

Abstract

The invention discloses an application of anthracene nucleus antibiotic and its pharmaceutical salt for treating diabetic eye diseases. The invention found that the anthracene nucleus antibiotic and its pharmaceutical salt have good effect on treatment of diabetic eye diseases, and especially the anthracene nucleus antibiotics can be prepared to prolonged action preparations such as a nano preparation, a microballoon preparation, liposome and hydrogel, the toxicity of anthracene nucleus antibiotic is reduced, and the anthracene nucleus antibiotic enables ophthalmic administration, administration dosage is simultaneously enlarged, the duration time of drug effect is long, the medicament curative effect is stronger than that of the common preparations of the anthracene nucleus antibiotic, when the ophthalmic administration is carried out, the anthracene nucleus antibiotic has good treatment effect for treating various eye diseases relative to diabetes, the medicine treatment is carried out when the diabetic eye diseases is found at initial stage, the deterioration of diseases can be avoided, the active treatment effect is provided, so that a surgery treatment method with large wound such as operation can be avoided.

Description

Anthracycline antibiotics and the purposes of officinal salt in treatment diabetic ophthalmopathy thereof

Technical field

The present invention relates to a kind of medicine for the treatment of diabetic ophthalmopathy, be specifically related to a series of traditional anti-tumor antibiotic anthracycline antibiotics and the novelty teabag of officinal salt in preparation treatment diabetic eye medicine thereof, especially, described anthracycline antibiotics and officinal salt preparation thereof become the durative action preparations such as nanometer formulation, microball preparation, liposome and hydrogel.

Background technology

Diabetes cause hypoinsulinism etc. and a series of metabolism disorder syndrome such as sugar, fat, protein, electrolyte of causing because the multiple paathogenic factor such as immunologic function disorder, inherited genetic factors, infected by microbes and toxin thereof, free radical toxin, Nervous and Mental Factors acts on body, the performance such as be main feature clinically with hyperglycemia, polyuria, polydipsia, polyphagia can appear in model case, become thin.Due to diabetes, patient Ke Yin microangiopathies causes multiple complications, and diabetic ophthalmopathy is then one of important chronic complicating diseases.The diabetic ophthalmopathy at initial stage shows eyestrain, visual deterioration.Patient very easily feels eyestrain, do not see thing, dark and dim eyesight when standing up, droopy eyelids, visual field narrows, and sees that thing is smudgy, and eyes are suddenly from presbyopia's phenomenon etc. that hypermetropia becomes myopia or do not have in the past, later stage nearly all oculopathy all may occur in it diabetics, as cataract, optic atrophy, degeneration of macula, glaucoma, optical fundus blood vessel tumor, dacryocystisis, retina shedding, retinal hemorrhage, vitreous opacity etc.

Ocular disease (i.e. diabetic ophthalmopathy) kind that diabetes cause is more, and each position of eyes is all likely subject to the impact of body disorder and falls ill.Main eye diabetic complication has diabetic retinopathy, diabetic choroidopathy, diabetic cataract, diabetic keratopathy, diabetic optic neuropathy, diabetic glaucoma, diabetic neuropathy, diabetic iridocyclitis, diabetic refraction change, and diabetes class eyeground haemorrhage etc., wherein diabetic retinopathy, diabetic choroidopathy and diabetic cataract more the patient of diabetic eye, these three kinds of pathological changes all can cause visual loss, be endanger in diabetic complication the most serious.

Derive because of diabetes the ocular disease and diabetic ophthalmopathy caused for treatment at present, take multiple therapy methods clinically at present.1, comprising insulin injection, hyperglycemia is reduced to the damage of vascular endothelial cell, pericyte etc.Thus control blood glucose; 2, blood fat is controlled, by statins antilipemic drugs protection blood retina barrier from destruction; 3, control blood pressure, reduced the generation of diabetic renal papillary necrosis by antihypertensive drugs; 4, the reactivity of neurogliocyte is reduced, by aldose reductase inhibitor, such as glycolylurea and flavonoid medicine; 5, increase retinal vascular permeability, utilize the biological preparation such as AGEs inhibitor, growth factor receptor inhibitors and kinases inhibitor to promote cardiovascular generation; 6, utilize oxygen free radical scavenger to regulate oxidative stress level, reduce the apoptosis of retina pericyte; 7, suppress the generation of inflammatory cytokine of diabetes-induced, reduce the retinal microglia release inflammatory mediator of activation thus delay the visual loss that diabetic ophthalmopathy causes, being worked by inflammation-inhibiting reaction medicine; 8, reduce the formation of thrombosis, play drug effect by antiplatelet drug; 9, improve Retinal hypoxia state, improve retinal microcirculation, by external physical therapy or some Chinese medicine preparation etc.These Therapeutic Method spininess are to diabetogenous retinopathy, and effect is not fine, the most important thing is that effect is not often had to other the diabetes associated ophthalmopathy such as diabetogenous choroidopathy and diabetic cataract, there is no the medicine of directly tackling these two kinds of pathological changes preferably at present.For diabetic cataract, there is no corresponding class of medications clinically at present, often take direct surgical operation, great pain is brought and often can only respite eye part disease to patient, and the patient of diabetic ophthalmopathy often suffers from multiple diabetes associated ophthalmopathy simultaneously, be not only one, mostly clinical treatment means applied at present is " only control one, do not control its 2345 ".

As mentioned above, although had medicine and the technology of a lot for the treatment of diabetes associated ophthalmopathies, but these current medicines and treatment technology are all only have effect for a single a kind of diabetes associated ophthalmopathy, than if any medicine only for retinopathy successful, such as, and for other oculopathy that diabetes cause, diabetic choroidopathy does not but have effect or is average in performance; Also some medicine may be effective for diabetic optic atrophy, but but have no effect for similar retinopathy.But, the occurrence cause of diabetic ophthalmopathy causes it and is only have single pathological changes, multiple pathological changes weave in often, patient not only shows retinopathy, also crystalline lens pathological changes is not only shown, also not only optic neuropathy or choroidopathy etc. is shown, but may to show simultaneously go up in these areas, if develop a kind of medicine can have therapeutic effect medicine to various or most of diabetes associated ocular disease, thus avoid the state of an illness to derive multiple associated ophthalmopathy or play the effect of thoroughly curing, so meaning is by far-reaching and great.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of anthracycline antibiotics and the novelty teabag of officinal salt in preparation treatment diabetic eye medicine thereof, especially described anthracycline antibiotics and officinal salt preparation thereof are become the durative action preparations such as nanometer formulation, microball preparation, liposome and hydrogel, better therapeutic effect can be obtained.

The concrete technical scheme that the present invention adopts is:

Anthracycline antibiotics of the present invention comprises the following compound of structure and stereoisomer thereof:

Wherein, R1 is H, OH, CH ₃, CH ₂oH or OCH ₃, R2 is COR1 or CR1R1, R3 is H, OH, CH ₃, CH ₂oH, OCH ₃, pyridine radicals, furyl, pyrrole radicals, thienyl or pyranose.

More preferably, described anthracycline antibiotics is doxorubicin, table doxorubicin, pyrans doxorubicin, mitoxantrone, daunorubicin, daunoblastin, idarubicin, aclarubicin, rubidomycin, aklavine or carminomycin.

Above-claimed cpd can by prior art disclosed method prepare, some compounds can be bought by commercial sources and obtain.

Anthracycline antibiotics and officinal salt thereof and pharmaceutically acceptable auxiliary material combination are prepared into various conventional preparation and are used for the treatment of diabetic eye disease by the present invention, and these preparations comprise: common flour injection, long-acting sustained-release injection.Preferably, described long-acting sustained-release injection can be nano particle preparations, microball preparation, aqueogel or Liposomal formulation.Preferably, described hydrogel is temperature-sensitive hydrogel preparation.Described anthracycline antibiotics is 0.5-2000 μ g/kg for the eye drops dosage of human body, preferred 1-1500 μ g/kg, more preferably 3-1000 μ g/kg.

When nanoparticle, microsphere, the aqueogel described in preparing, the pharmaceutically acceptable adjuvant adopted comprises macromolecular material, described macromolecular material is selected from: one or more in condensing model, polyoxyalkylene, polyamide, polyester, polyacrylic resin, polyethers or polysaccharide, or the copolymer between the different monomers being selected from described several macromolecular material.Preferably, described macromolecular material is selected from condensing model, polyvinyl alcohol, Polyethylene Glycol, condensing model-ethylene glycol copolymer, NIPA-acrylic copolymer, polybutylcyanoacrylate, polylactic acid, octadecane diacid acid anhydride-glycol copolymer, PLGA, polylactic-co-glycolic acid-ethylene glycol copolymer, PGA, Acetic acid, hydroxy-, bimol. cyclic ester lactide-ethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide triblock copolymer, polyether sulfone, gelatin, poly(hydrobutyl ester), poloxamer, collagen protein, Fibrinogen, albumin, cellulose, glucosan, alginate, dextran, hyaluronic acid, one or more in polypeptide or chitosan etc.

When the Liposomal formulation described in preparing, pharmaceutically acceptable adjuvant is adopted to comprise phospholipid that is natural or that synthesize, lipoid or its combination.

Said medicine preparation can be prepared by method disclosed in prior art, and the method recorded in the embodiment of the present invention also can be adopted to prepare.

At present, anthracycline antibiotics is usually used in treatment leukemia and antitumor, has less people to use it for treatment proliferative retinopathy.The present inventor has carried out the research of new indication to anthracycline antibiotics, and delightedly finds, anthracycline antibiotics has good effect to treatment diabetic ophthalmopathy, and can not produce anthracycline antibiotics very easily to the multiple side effect that eye produces.Research finds that anthracycline long-acting slow-release preparation not only has therapeutical effect to diabetic ophthalmopathy simultaneously, and have more targeting, it has long action time, toxic and side effects is little, can significantly improve the advantages such as bioavailability.At the model of action of this target slow-release administration, medicine can continuous action for a long time, the larger eye pair that significantly reducing anthracycline originally has does use, makes it to play new eye treatment effect, and loses eye toxic and side effects.

By anthracycline antibiotics with nanometer, liposome, after the form eye drops of the durative action preparation such as hydrogel or microsphere, not only there is effect for a single a kind of diabetes associated ophthalmopathy, for diabetic retinopathy, all successfuls such as diabetic choroidopathy and diabetic cataract, occurrence cause due to diabetic ophthalmopathy causes it and is only have single pathological changes, multiple pathological changes weave in often, the oculopathy of diabetics not only shows retinopathy, also crystalline lens pathological changes is not only shown, also not only optic neuropathy or choroidopathy etc. is shown, but may to show simultaneously go up in these areas, therefore this medicine is the medicine that all can produce therapeutic effect to various or most of diabetes associated ocular disease, thus avoid the state of an illness to derive multiple associated ophthalmopathy or play the effect of thoroughly curing.

Detailed description of the invention

Specific embodiment is described in further detail the present invention below, but the present invention not only limits to following examples.In following embodiment, method therefor is conventional method if no special instructions, test material used, if no special instructions, is the purchase of routine biochemistry reagent suppliers and obtains.

Preparation embodiment is as follows:

Embodiment 1

1. get 300mg PLGA (PLGA) to join in 1ml dichloromethane and be prepared into solution;

2. joined by 50mg aclarubicin in solution obtained 1., high speed homogenization device stirs;

3. with syringe, the solution in 2. is slowly injected in the Oleum Gossypii semen containing 0.05% egg yolk ovum Oletum Trogopterori, after 1000r/min stirs 20min, reduces rotating speed and continue to stir 4h to 200r/min;

4. to step 3. in add 20ml petroleum ether, the centrifugal 5min of 8000r/min after 30min;

5. microsphere is collected, petroleum ether and volatilizing.

Embodiment 2

1. get 200mg PLGA (PLGA) to join in 1ml tetrahydrofuran solution and be prepared into solution;

2. add in 0.2ml glycerol by 50mg mitoxantrone, join in 1. obtained solution after mixing, homogenizer obtains colostrum after stirring 1min;

3. joined in 2% polyvinyl alcohol (PVA) solution of 12ml, 1000r/min adds 120ml again 0.5%PVA solution after stirring 5min obtains emulsion;

4. after reducing rotating speed to 200r/min continuation stirring 4h, the centrifugal 5min of 8000r/min;

5. microsphere is collected, distilled water wash postlyophilization.

Embodiment 3

1. get 100mg condensing model-ethylene glycol copolymer (PSA-PEG) to join in 1ml dichloromethane solution and be prepared into solution;

2. 50mg doxorubicin is joined in solution obtained 1., ultrasonic mixing;

3. 2. obtained solution is joined in 15ml 1%PVA solution;

4. after reducing rotating speed to 200r/min continuation stirring 4h, the centrifugal 5min of 8000r/min;

5. collect microsphere, collect after distilled water wash.

Embodiment 4

1. get 160mg condensing model-ethylene glycol copolymer (PSA-PEG) to join in 2ml dimethyl sulfoxide and dichloromethane (8: 2) and be prepared into organic facies;

2. 40mg doxorubicin is joined 1., after mixing, be positioned in 1% polyvinyl alcohol ultrasonic;

3. will 2. in solution be placed in poly-vinyl alcohol solution and stir;

4. remove organic solvent 3., collected by centrifugation, obtains nanoparticle solution.

Embodiment 5

1. daunorubicin and polylactic-co-glycolic acid-ethylene glycol copolymer (PLGA-PEG) 800mg 3ml dichloromethane and 1ml DMF being acted in a water bath makes it dissolve and high-speed stirred;

2. joining in 1. containing 1% polyvinyl alcohol to 100ml, fully mix;

3. stirring at room temperature volatilization 2h, obtains nanoparticle solution;

4. at 4 DEG C, gained nanoparticle after centrifugal 20min, is collected;

5. will 4. in gains distilled water wash three times and be drug-carrying nanometer particle.

Embodiment 6

1. get 300mg condensing model (PSA) to join in 3ml dichloromethane and be prepared into organic facies;

2. 40mg idarubicin is joined in organic facies obtained 1.;

3. getting propylene glycol block polyether and Tween 80 (1: 1), to make containing emulsifying agent be the aqueous solution of 4%, and add macrodex wherein, regulates solution PH to be 8;

4. nanometer sedimentation is used obtained organic facies to be dropped in 3. obtained aqueous phase;

5. stir removing organic solvent, obtain nanoparticle solution, collected by centrifugation.

Embodiment 7

1. 0.01molL is used ^-1sodium hydroxide solution as solvent;

2. get 1. solvent 10ml and add 100 μ l hydroxypropyl emthylcelluloses, stir and make it even;

3. add 50 μ l carbomer 934s in the solution 2. obtained to step, stir and make its mix homogeneously;

4. add 40mg condensing model-ethylene glycol copolymer (PSA-PEG) in the solution 3. obtained to step, stir and make its mix homogeneously;

5. in above-mentioned solution, 1mg carminomycin is added;

6. take 1.8g poloxamer188 to join in 4. obtained solution, under magnetic agitation, make it be uniformly dispersed, 4 DEG C of conditions place more than 24 hours, make gel fully swelling, are uniformly dispersed and obtain clear and bright solution, both obtain carminomycin hydrogel.

Embodiment 8

1. 0.01molL is used ^-1sodium hydroxide solution as solvent;

2. get 1. solvent 10ml and add 80 μ l hydroxypropyl emthylcelluloses, stir and make it even;

3. add 50 μ l sodium alginates in the solution 2. obtained to step, stir and make its mix homogeneously;

4. add 40mg polylactic-co-glycolic acid-ethylene glycol copolymer (PLGA-PEG) in the solution 3. obtained to step, stir and make its mix homogeneously;

5. in above-mentioned solution, 1mg epirubicin is added;

6. take 1.8g poloxamer188 to join in 4. obtained solution, under magnetic agitation, make it be uniformly dispersed, 4 DEG C of conditions place more than 24 hours, make gel fully swelling, are uniformly dispersed and obtain clear and bright solution, both obtain epirubicin hydrogel.

Embodiment 9

1. take 0.9g phospholipid, 0.3g cholesterol in 50ml small beaker, add dehydrated alcohol 1-2ml, be placed in 65-70 DEG C of water-bath, be stirred to dissolve, rotate this small beaker and make the ethanol of phospholipid film forming on wall of cup, with rubber pipette bulb featheriness wind, ethanol is flung to;

2. pirarubicin solution 30ml is separately got in small beaker, with being placed in 65-70 DEG C of water-bath, insulation, stand-by;

3. get the pirarubicin solution 30ml of preheating, add in the small beaker containing phospholipid and cholesterol ester plasma membrane, 65-70 DEG C of stirred in water bath aquation 10min.Subsequently small beaker is placed on magnetic stirring apparatus, stirring at room temperature 30-60min, mixes and obtain Pirarubicin liposome.

Effect experimental is as follows:

Medicine is tested the therapeutic effect of diabetes rat eye different tissues

The structure of 1 diabetes animal model

Rat domestication nursing 1 week, fasting 12 hours.Streptozotocin is dissolved in the aseptic citric acid of 0.1mol/L, PH4.6, arrests in rafter acid sodium buffer, by the dosage injection abdominal cavity, rat lower-left of 50mg/kg body weight.After 1 week, Rat Fast 12 hours, blood is got in docking, and fasting glucose >=16.7mmol/L person elects diabetic groups as, feeding time 6 months.

2 groupings

Fasting glucose >=the 16.7mmol/L 120 of survival, after 3 months, is suffered from diabetic ophthalmopathy rat and is divided into model group (i.e. infected animal) at random by modeling; Treatment group: be respectively anthracycline antibiotics ordinary preparation medication group (after sodium carboxymethyl cellulose, mannitol hydrotropy doxorubicin, with suspended form injection), embodiment group (nanometer formulation of embodiment, microball preparation, aqueogel, Liposomal formulation).Except model group, treatment group equal intraocular injection 5 μ g medicine or the drug-carrying nanometer particle (microsphere, hydrogel or liposome) containing 5 μ g medicines.Normal group is the normal rat of blood sugar level.

3 check

Chemical colorimetry is adopted to measure the content of malonaldehyde (i.e. MDA), superoxide dismutase (i.e. SOD), glutathion peroxidase (i.e. GSH-Px) in retina and crystalline lens.

Adopt advanced glycation end products (i.e. AGE) level in fluorescence spectrometry retina and crystalline lens homogenate.

Staining is adopted to check retinal endothelial cell/pericyte's ratio (EC/PC) and retinal vessel density.

Adopt electron microscopic examination optic nerve form.

Check result sees the following form respectively:

The activity (n=10) of MDA, SOD, GSH-Px in table 1 treatment group and normal group retina

Compare with normal group ^#p < 0.05, ^##p < 0.01, compares * p < 0.05, * * p < 0.01 with model group

The activity (n=10) of MDA, SOD, GSH-Px in table 2 treatment group and normal group crystalline lens

Compare with normal group ^#p < 0.05, ^##p < 0.01, compares * p < 0.05, * * p < 0.01 with model group

As can be seen from the result of table 1 and table 2, suffer from rat retina and the reduction more obvious than normal group of crystalline lens oxidation resistance of diabetic ophthalmopathy (i.e. DM), MDA content significantly increases, SOD, GSH-Px reduced activity; After implementing anthracycline antibiotics ordinary preparation medication treatment, treatment group all can reduce MDA content, improve SOD, GSH-Px active, compare with model group and have significant difference, especially give the level of MDA, SOD, GSH-Px of the rat after the treatment of embodiment group close to normal group.Embodiment group comparatively anthracycline antibiotics ordinary preparation medication group has significant difference, better effects if.And the durative action preparation after macromolecular material parcel, water solublity strengthens, and is easy to implement ocular injection, and medicine goes directly focus, and toxicity is little, and the drug action time is long.

AGE level (n=10) in table 3 retina and crystalline lens homogenate supernatant

Compare with normal group ^#p < 0.05, ^##p < 0.01, compares * p < 0.05, * * p < 0.01 with model group

As can be seen from Table 3, DM rat retina and the rising more obvious than normal group of crystalline lens AGE level.Medicine group all can reduce rat retina and crystalline lens AGE level, compares have pole significant difference with model group, especially the AGE level closely normal group of embodiment group.Embodiment group comparatively anthracycline antibiotics ordinary preparation medication group has significant difference, better effects if.

Table 4 retinal endothelial cell/pericyte's ratio (EC/PC) and retinal vessel density (n=10)

Compare with normal group ^#p < 0.05, ^##p < 0.01, compares * p < 0.05, * * p < 0.01 with model group

As can be seen from the result of table 4, DM rat retina endotheliocyte/pericyte's ratio (EC/PC) and retinal vessel density compare with normal group, obviously raise.Medicine group all can reduce retinal endothelial cell/pericyte's ratio (EC/PC) and retinal vessel density, compares have significant difference with model group, especially the testing result of embodiment group closely normal group.Embodiment group comparatively anthracycline antibiotics ordinary preparation medication group has significant difference, better effects if.

Electron microscopic examination result is as follows:

Dynamic retinoscopy:

(1) normal group: vasoganglion complete as seen under low power lens, blood vessels caliber thickness is homogeneous.The dyeing of tremulous pulse trunk is comparatively dark, and caliber is comparatively thin, and vein blood vessel dyeing is more shallow, and caliber is comparatively thick, and arteriovenous is not with colleague, but after being sent by papilla of optic nerve radially alternately, capillary network is between arteriovenous.Under high power lens, Visible Core is comparatively large, and nuclear chromatin loosens, and the endotheliocyte and the nuclear chromatin that are generally positioned at blood capillary central part are finer and close, are positioned at the pericyte of blood capillary side; (2) model group: circuitous situation appears in visible blood capillary, form blood vessel and stumble, traveling is irregular.Blood capillary caliber is irregular, and tube chamber thickness differs, and tube wall edge is irregular, retina pericyte nuclear swelling, and dyeing shoals, and part nucleus is that shadow cell sample changes, and pericyte's quantity reduces; (3) anthracycline antibiotics ordinary preparation medication group: visible blood capillary circuitous situation alleviates, form the button knot of some, quantity is few compared with model group, the negligible amounts that blood vessel is stumbled, traveling is tending towards rule substantially, retinal microvascular clear in structure, vascularity fundamental rule, caliber is heterogeneity slightly, but expands without segmental or expand.Pericyte's form is without obvious change; (4) respectively to organize retinal capillary caliber thickness roughly homogeneous for embodiment, do not find the irregular situation in blood capillary tube wall edge, do not have button to tie and blood vessel is stumbled, slightly capillary tube circuitous situation; Anthracycline antibiotics ordinary preparation medication group capillary tube circuitous situation is heavier compared with embodiment group.Each treatment group all has amphiblestroid pathology damage caused by change diabetes in various degree, wherein, the effect of embodiment group is the most obvious, makes pathology damage reduce to minimum and have the normal trend of recovery, illustrates if life-time service can reach better therapeutic effect.

Crystalline lens checks:

1 normal group: frontal cortex region is lens fibers marshalling under low power lens, diameter is consistent, and fiber becomes flaggy sample to arrange.Under mirror in projection ridge on fiber long axes dash forward sample arrangement, pleurapophysis forms complementary surface between adjacent two fibers.Fiber cross-sectional is long hexagon, and fiber surface has digitation, and the substrate in it is homogenizing non-structure shape; 2 model group: lens fibers arrangement disorder under low power lens of same area, diameter thickness is not etc., the flaggy sample arrangement of fiber is very not obvious, the flaggy of some positions fiber has destruction, fibres visible swelling under high power lens, in prominent low flat, the form of pleurapophysis and size are irregular, fiber cross-sectional has not been the hexagon of rule, and is hard to tell interfibrous boundary, and the substrate in it has been that cotton-shaped network sample changes; 3 anthracycline antibiotics ordinary preparation medication groups: have no fiber swelling under mirror, in prominent low flat, arrange still neat under the lens fibers low power lens in frontal cortex region, diameter is consistent, fiber becomes flaggy sample to arrange, and the form of pleurapophysis and size be rule all, has less fiber cross-sectional display hexagon; 4 embodiment groups: visible frontal cortex region crystalline lens marshalling, diameter is consistent, fiber becomes flaggy sample to arrange, in fiber cross-sectional, existing part recovers the hexagon of rule, and distinguish interfibrous boundary, the cotton-shaped network sample of the less appearance of substrate in it changes, and the sample arrangement of dashing forward of middle projection ridge on fiber long axes is visible.In treatment group embodiment group comparatively anthracycline antibiotics ordinary preparation medication group have fiber cross-sectional to recover better.By the observation to crystalline lens pathological section, find that the anthracycline antibiotics of different preparation is all improved the effect of crystalline lens pathological change, but action effect has difference, embodiment group pathological change not only can be made to reduce to minimum and also pathologic condition close with normal group.Illustrate that medicine can enter agents area and play a role, and embodiment group plays its targeting acts on efficiently.

Optic nerve checks:

1 normal group: under mirror, visible neurogliocyte film is complete, in aixs cylinder, neurofilament arrangement is comparatively neat, nerve fiber by interior, in, outer three layers of sheath wrap up, nerve fiber is very thin wavy, the glial cell core of visible arrangement bunchiness between fibre bundle, mitochondrion is complete without significant change, optic nerve myelinated fiber is evenly densely distributed, and Medullary sheath is complete, and myelin thickness is more even, single fiber is full, 2 model group: under mirror, optic nerve fiber level is not obvious, glial cell core obviously reduces, the astrocyte of glial scar and arrangement disorder in visible Mus optic nerve, and optic nerve is out of shape, axonal atrophy, partial nerve occurs that myelin flaggy is separated, serious appearance demyelination change, myelin to myelin inner-outer stripper lose points from, in neural axis, microfilament dissolves more obvious, the neural axis mitochondrial swelling of myelinoclasis, ridge gap is expanded, and on ridge, granule reduces, 3 anthracycline antibiotics ordinary preparation medication groups: nerve fiber level is slightly clear, the glial cell core of component arrangement bunchiness can be seen, the myelin of optic nerve fiber still has part myelin abnormal, but quantity is few compared with model group, myelin flaggy Density inhomogeneity, some flaggy local still have loose, but do not see serious myelin to depigmentation phenomenon inside and outside myelin, neurogliocyte core between nerve fibre bundle has no showed increased, neurogliocyte compressing causes myelin to have no thinning, have no more obvious nerve fibre bundle interval broadening, anthracycline antibiotics ordinary preparation group comparatively diabetic model group obviously alleviates, 4 embodiment groups: embodiment group compared with the depigmentation phenomenon of anthracycline antibiotics ordinary preparation medication group show lighter, in visible optic nerve, optic nerve fiber and neurogliocyte arrange major part and are tending towards normal.In aixs cylinder, neurofilament arrangement is comparatively neat, and mitochondrion is complete without significant change, and pathologic condition is very close to normal group.The pathological examination of optic nerve shows, embodiment group is obviously better than other drug treatment group to the therapeutic outcome of care of patients with diabetic ocular lesion tissue, this illustrates that macromolecular material durative action preparation or Liposomal formulation can strengthen the drug effect of medicine, and bioavailability can be made higher, and drug effect is more of a specified duration.

Medicine is to the pharmacodynamic observation of STZ diabetic cataract

1 laboratory animal

Healthy adult male Wistar rat 110, body weight 160-180g, ordinary circumstance is good, sees that all animal crystalline lenses are completely transparent, without any muddiness, and without other ophthalmic under slit lamp.

2 experimental techniques

2.1 modeling method

Normal adult male Wistar rat, lens of both eyes is completely transparent, without any muddiness.The all rats by intraperitoneal injection STZ (50mg/kg) of diabetic groups after overnight fasting, normal rats gives isopyknic citrate buffer solution.After 4 days, glucose oxidase method surveys rat tail vein blood glucose, and fasting glucose >=16.7mmol/L person is for experiment.Every 10 days all animal mydriasis once, observe the rank of sobering animal len's opacity under slit lamp, detect blood glucose and the body weight change of animal simultaneously, after 70 days, all animals of sacrificed by decapitation, rear capsule method takes out crystalline lens, avoid laceration of lens capsule, after weighing-80 DEG C frozen.

2.2 group technology

After modeling, the fasting glucose blood glucose fasting glucose >=16.7mmol/L person of survival is divided into 12 groups at random, and namely matched group (i.e. normal rat), model group (namely suffering from diabetes rat), treatment group comprise: anthracycline antibiotics medication group (the common epirubicin preparation of intraocular injection), embodiment group.Each treatment group equal intraocular injection 1 μ l medicine or the drug-carrying nanometer particle (microsphere, hydrogel, liposome) containing 1 μ l medicine except matched group, model group gives isopyknic PBS solution.

3 test indexs and method

3.1 medicines are on the impact of diabetic rat cataract len's opacity

Within after rat modeling the 10th, 30,70 day, respectively organize the crystalline lens situation of rat by slit lamp observation, and according to cataract grade scale, rat cataract lenticular opacity degree is marked.

Sugar cataract grade scale

0 grade: crystalline lens is completely transparent, without any muddiness.

1 grade (vesicle initial stage): tiny vesicle or vesicle group appear in crystalline lens periphery.

1.3 ambituss are dispersed in vesicle, for joining together.Ambitus vesicle joins together, but width shared by vesicle prolongs 1/4 radius.Shared by the vesicle of ambitus, width prolongs 1/3 radius.

2 grades (vesicle phase): tiny vesicle, medium vesicle gradually by ambitus forward pole cortex expand, formed endless belt, endless belt is the broadening 1/3-2/3 to lens diameter gradually, with or expand without " Y ".

Width shared by 2.3 vesicles is for prolonging 1/2 radius.

Width shared by 2.6 vesicles is hair 2/3 radius.

Shared by 2.9 vesicles, width has exceeded 2/3 of diameter, is almost covered with whole anterior lens capsule.

3 grades (cortex phase): cortex presents the flocculent turbidity stove of irregular striated or JIANZHU shape polar radiation forward, and forms diffusivity Lycoperdon polymorphum Vitt gradually to linen flocculent turbidity, and nucleus lentis is still transparent.

There is slight flocculent turbidity or the muddy stove of JIANZHU shape in 3.3 cortex, especially central part.

There is moderate flocculent turbidity stove in 3.6 cortex.

3.9 cortex flocculent turbidities are severe.

4 grades (core muddy phase): crystalline lens is except cortex flocculent turbidity, and cuclear density also increases gradually, color by Lycoperdon polymorphum Vitt to greyish white, white development, with or without blister and " Y " expansion.

4.3 core little cloudy, gray, now can have a small amount of blister to exist.

4.6 core moderate muddinesses are in canescence, and this obviously expands with " Y " often.

4.9 core severe muddinesses, in white, are often expanded with " Y ".

5 grades (period of maturation): cortex of lens starts to be developed to density white muddiness by flocculent turbidity further, and final and core combines together in white sphere.

5.3 cores are in white, and cortex muddiness is in canescence, and the two demarcation line is clear.

5.6 cores are in white, and the further muddiness of cortex is creamy white, and the two boundary is unclear.

5.9 cores and cortex muddiness are in white, and the two combines together completely, without any demarcation line.

3.2. medicine is to diabetic cataract rat lens weight and crystalline lens/body weight ratios affect

Rat feeding is after 70 days, and all animals of sacrificed by decapitation, rear capsule method takes out crystalline lens, avoids laceration of lens capsule, claims to go crystalline lens weight, and obtains the ratio of crystalline lens weight and rat body weight.

3.3 rat lens water-solubility protein (WSP) assays

3.3.1. water-solubility protein preparation of samples

Get crystalline lens homogenate supernatant deionized water dilute 500 times after for subsequent use.

3.3.2 protein standard

Get 4mg/ml BSA protein standard, respectively with deionized water and the dilution of 10 times, SM carbamide, and then by coordinative solvent doubling dilution 5 concentration: 200,100,50,25,12.5 μ g/ml.

3.3.3. coomassie brilliant blue surveys protein concentration

20 μ l testing samples or protein standard add 180 μ l Coomassie brilliant blue dye liquors, and room temperature is determined at spectrophotometric value (the i.e. OD under 595nm after leaving standstill 5min ₅₉₅nm).

3.3.4. calculate

Drawing standard curve, calculates WSP content.

4 statistical procedures data x ± s represent, compare t inspection between employing group between two groups.

5 results

5.1. medicine is on the impact of diabetic rat cataract len's opacity

Normal rats crystalline lens is completely transparent all the time, and without any muddiness, this experimental diabetes model group animal len's opacity is apparently higher than normal group (p < 0.01).Compared with model group, embodiment group has obvious retarding action (p < 0.01) to len's opacity progress, and intraocular injection common anthracycline preparation and each phase turbidity of intraocular injection long-acting anthracycline antibiotics preparation group more also have different (p > 0.05) with model group.

5.2. medicine is to diabetic cataract rat lens weight and crystalline lens/body weight ratios affect

Table 5 rat lens weight and crystalline lens/body weight ratio (n=10)

Compare with matched group ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compares * p < 0.05, * * p < 0.01 with model group

Normal rat is crystalline body weight about 39.01 ± 1.19mg, crystalline lens is 0.89 ± 0.04 with the ratio average of body weight.Model group crystalline lens (49.85 ± 1.05mg) obviously overweights normal group (P < 0.001), crystalline lens/body weight ratio, up to 1.60 ± 0.06, is obviously greater than normal group animal ratio (p < 0.001).Compared with model group, each treatment group all has impact in various degree to crystalline lens weight and crystalline lens/body weight ratio.

5.3 medicines are on the impact of diabetes rat crystalline lens WSP content

Normal rat crystalline lens WSP content is 220.41 ± 11.49, and in diabetes rat crystalline lens, WSP obviously declines (157.77 ± 24.37, p < 0.001).The decline for the treatment of group to crystalline lens WSP all has some improvement, the effect of embodiment group is the most remarkable, about 25% is strengthened compared with the WSP content of model group, in embodiment group crystalline lens, WSP content has notable difference (p < 0.01) compared with model group, specifically in table 6.

Table 6 diabetes rat crystalline lens WSP content (n=10)

Group	WSP content
		Normal group	220.41±11.49

Model group	157.77±24.37 ###
		Anthracycline antibiotics ordinary preparation medication group	174.33±13.66*
Embodiment 1	209.47±10.51**
		Embodiment 2	211.52±9.54*
Embodiment 3	213.31±8.99**
		Embodiment 4	209.36±9.97**
Embodiment 5	212.36±11.37**
		Embodiment 6	216.16±13.00**
Embodiment 7	214.36±10.05**
		Embodiment 8	212.63±9.45**
Embodiment 9	215.45±8.57**

Compare with matched group ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compares * p < 0.05, * * p < 0.01 with model group

Result shows, treatment group all can suppress the content of WSP to decline, but medicine group action effect, and be not as obvious as embodiment group, this illustrates that the action effect of embodiment group is ideal when using the medicine of same dose.Medicine is to diabetic retinopathy rebirth blood vessel function

1 laboratory animal

Select Mus to be age the healthy Wistar rat 110 of 6 weeks, body weight is 200 ± 20g.Rearing conditions remains on room temperature 18-25 DEG C, air circulation, relative humidity 55%-70%, and 12 h light maintain.

2 experimental techniques

2.1 modeling method

Normal adult male Wistar rat, lens of both eyes is completely transparent, without any muddiness.The all rats by intraperitoneal injection STZ (50mg/kg) of diabetic groups after overnight fasting, normal rats gives isopyknic citrate buffer solution.After 4 days, glucose oxidase method surveys rat tail vein blood glucose, and fasting glucose >=16.7mmol/L person is for experiment.Every 10 days all animal mydriasis once, observe the rank of sobering animal len's opacity under slit lamp, detect blood glucose and the body weight change of animal simultaneously, after 70 days, all animals of sacrificed by decapitation, rear capsule method takes out crystalline lens, avoid laceration of lens capsule, after weighing-80 DEG C frozen.

2.2 group technology

After modeling, the fasting glucose blood glucose fasting glucose >=16.7mmol/L person of survival is divided into 12 groups at random, and namely matched group (i.e. normal rat), model group (namely suffering from diabetes rat), treatment group comprise: anthracycline antibiotics ordinary preparation medication group (the common carminomycin preparation of intraocular injection), embodiment group.Each treatment group equal intraocular injection 1 μ l medicine or the drug-carrying nanometer particle (microsphere, hydrogel, liposome) containing 1 μ l medicine except matched group, model group gives isopyknic PBS solution.

The preparation of 2.3 paraffin sections

One and a half months after each group of rat experiment administration, puts to death with overdose of sodium pentobarbital anesthesia lumbar injection, extracts eyeball immediately, eyeball is fixed on 48h in 10% neutral formalin, through the dehydration of ethanol routine, the transparent rear waxdip embedding of dimethylbenzene, 4 μm of sections are used for immunohistochemical staining.

2.4 Immunohistochemical detection

Detect with SABC method immunohistochemical staining test kit, concrete operation step: 1. dewaxing and aquation; 2. 3%H ₂o ₂1 part adds 10 parts of distilled water mixing, and drip in section, room temperature leaves standstill 10min; 3. antigen retrieval; 4. the bovine serum albumin confining liquid of 5% is dripped; 5. drip primary antibodie RabbitAnti-VEGF, bFGF, 4 DEG C are spent the night; 6. biotinylated goat antimouse IgG is dripped; 7. reagent strepto-avidin+Radix Cochleariae officinalis enzyme labelling biotin is dripped; 8. Radix Cochleariae officinalis chromogenic enzyme substrate; 9. haematoxylin redyeing, dehydration, transparent, mounting, microscopy.

2.5 statistical method

Data all represent with (x ± s), compare and adopt t inspection between group, adopt SPSS statistical software to add up.

3 results

3.1 ImmunohistochemistryResults Results

Matched group: VEGF (i.e. VEGF), all no positive expression of fibroblast growth factor (i.e. bFGF), retina cell marshalling, cellular morphology is normal; All visible VEGF, bFGF strong positive of model group retina holostrome is expressed, retina cell arrangement disorder, and ganglion cell's degeneration increases the weight of, structural fuzzy; The all visible weak positive expression of VEGF, bFGF of medicine group holostrome, retina cell arrangement disorder is not obvious, and ganglion cell's degeneration is better than model group; Visible VEGF, bFGF positive expression of embodiment group retina holostrome is the lightest, and dyeing is weaker than other groups.

3.2 computer aided video systems detect the average optical density value of each group of VEGF, bFGF positive products

VEGF, bFGF positive expression district average optical respectively organized by table 7

Compare with matched group ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compares * p < 0.05, * * p < 0.01 with model group

The result of upper table demonstrates treatment group all curative effect to a certain degree, but the curative effect of embodiment group is best, reaches the object realizing best therapeutic effect by low dose.

Medicine is to the inhibitory action of the blood vessel hyperplasia that artificial diabetes choroidopathy causes

1 laboratory animal

Select Mus to be age the healthy Wistar rat 110 of 6 weeks, body weight is 200 ± 20g.Rearing conditions remains on room temperature 18-25 DEG C, air circulation, relative humidity 55% ~ 70%, and 12 h light maintain.

2.1 modeling method

Normal adult male Wistar rat, lens of both eyes is completely transparent, without any muddiness.The all rats by intraperitoneal injection STZ (50mg/kg) of diabetic groups after overnight fasting, normal rats gives isopyknic citrate buffer solution.After 4 days, glucose oxidase method surveys rat tail vein blood glucose, and fasting glucose >=16.7mmol/L person is for experiment.Every 10 days all animal mydriasis once, observe the rank of sobering animal len's opacity under slit lamp, detect blood glucose and the body weight change of animal simultaneously, after 70 days, all animals of sacrificed by decapitation, rear capsule method takes out crystalline lens, avoid laceration of lens capsule, after weighing-80 DEG C frozen.

2.2 group technology

After modeling, the fasting glucose blood glucose fasting glucose >=16.7mmol/L person of survival is divided into 12 groups at random, and namely matched group (i.e. normal rat), model group (namely suffering from diabetes rat), treatment group comprise: common anthracycline antibiotics ordinary preparation medication group (the common powder injection formulation of intraocular injection doxorubicin), embodiment group.Each treatment group equal intraocular injection 1 μ l medicine or the drug-carrying nanometer particle (microsphere, hydrogel, liposome) containing 1 μ l medicine except matched group, model group gives isopyknic PBS solution.

The preparation of 2.3 paraffin sections

One and a half months after each group of rat experiment administration, often group gets the execution of 5 rat overdose of sodium pentobarbital anesthesia lumbar injections, extracts eyeball immediately, eyeball is fixed on 48h in 10% neutral formalin, through the dehydration of ethanol routine, the transparent rear waxdip embedding of dimethylbenzene, 4 μm of sections are used for HE dyeing.

The observation of 2.4 blood vessel hyperplasia areas

The size in observation experiment diabetic choroidopathy median nexus film blood vessel hyperplasia region.Often organize and separately get 5 rat overdose of sodium pentobarbital intraperitoneal administrations execution, extract experimental eye immediately, respectively the choroidal blood vessel hyperplasia area of accurate measurement.

3 results

3.1 histological examination

Light microscopic drag group neovascularization resulting, free companion's inflammatory cell infiltration.Medicine group is compared with model group, and new vessels is more rare; Embodiment group is compared with common drug group, and choroidal neovascularization resulting obviously reduces and has no film disengaging and inflammatory cell infiltration.The durative action preparation targeting of microsphere, nanometer, hydrogel and liposome is stronger, directly arrives affected area and works, and more obviously and lasting, and medicine group works to affected area and do not have embodiment group obvious in effect, and onset is slow.

3.2 blood vessel hyperplasia area result see the following form

Table 8 treatment group and model group choroidal neovascularization area (unit: mm ²n=5)

* p < 0.05, * * p < 0.01 is compared with model group

The area of blood vessel hyperplasia directly reflects the situation that pathological changes occurs.Model group choroid generation blood vessel hyperplasia area is larger.Each treatment group respectively different limit reduce blood vessel hyperplasia area.Show the experiment of rat ocular vascular proliferation area, each treatment group all can reduce choroidal blood vessel hyperplasia area, and embodiment group obviously can reduce the choroidal blood vessel hyperplasia area of pathological changes.

Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give exhaustive to all embodiments.Every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.

Claims (10)

1. anthracycline antibiotics and the purposes of officinal salt in preparation treatment diabetic eye medicine thereof, is characterized in that described treatment diabetic ophthalmopathy is for treat retinopathy, crystalline lens pathological changes, optic neuropathy and choroidopathy simultaneously.

2. purposes according to claim 1, described anthracycline antibiotics is selected from: doxorubicin, table doxorubicin, pyrans doxorubicin, mitoxantrone, daunorubicin, daunoblastin, idarubicin, aclarubicin, rubidomycin, aklavine or carminomycin in one or more.

3. the purposes according to any one of claim 1-2, is characterized in that described medicine is prepared by described anthracycline antibiotics and officinal salt thereof and pharmaceutically acceptable adjuvant.

4. purposes according to claim 3, it is characterized in that described medicine is common flour injection or long-acting sustained-release injection, wherein said long-acting sustained-release injection is the preparation of nano particle preparations, microball preparation, aqueogel or liposomal form.

5. purposes according to claim 4, the adjuvant adopted when it is characterized in that prepared by described nano particle preparations, microball preparation, aqueogel comprises macromolecular material, described macromolecular material is selected from: one or more in condensing model, polyoxyalkylene, polyamide, polyester, polyacrylic resin, polyethers, polyphosphazene, starch, collagen, polypeptide or polysaccharide, or the copolymer between the different monomers being selected from described macromolecular material; The adjuvant adopted time prepared by described Liposomal formulation comprises phospholipid that is natural or that synthesize, lipoid or its combination.

6. purposes according to claim 5, described macromolecular material is selected from: condensing model, polyvinyl alcohol, Polyethylene Glycol, condensing model-ethylene glycol copolymer, NIPA-acrylic copolymer, polybutylcyanoacrylate, polylactic acid, octadecane diacid acid anhydride-ethylene glycol block copolymer, PLGA, polylactic-co-glycolic acid-ethylene glycol copolymer, PGA, Acetic acid, hydroxy-, bimol. cyclic ester lactide-ethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide triblock copolymer, polyether sulfone, gelatin, poly(hydrobutyl ester), poloxamer, collagen, Fibrinogen, albumin, cellulose, glucosan, alginate, dextran, one or more in hyaluronic acid or chitosan.

7. purposes as claimed in one of claims 1-6, is characterized in that anthracycline antibiotics and officinal salt thereof adopt the mode of eye drops.

8. purposes according to claim 7, is characterized in that anthracycline antibiotics is 0.5-2000 μ g/kg for the eye drops dosage of human body.

9. purposes according to claim 8, is characterized in that anthracycline antibiotics is 1-1500 μ g/kg for the eye drops dosage of human body.

10. purposes according to claim 8, is characterized in that anthracycline antibiotics is 3-1000 μ g/kg for the eye drops dosage of human body.