CN101703503B - Application of organic dicarboxylic acid salts in preparing medicaments for preventing and treating cataract - Google Patents
Application of organic dicarboxylic acid salts in preparing medicaments for preventing and treating cataract Download PDFInfo
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- CN101703503B CN101703503B CN2009102126910A CN200910212691A CN101703503B CN 101703503 B CN101703503 B CN 101703503B CN 2009102126910 A CN2009102126910 A CN 2009102126910A CN 200910212691 A CN200910212691 A CN 200910212691A CN 101703503 B CN101703503 B CN 101703503B
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N NC(Cc1c[nH]cn1)C(O)=O Chemical compound NC(Cc1c[nH]cn1)C(O)=O HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- IGULOYSOGFNAFG-UHFFFAOYSA-N OC(C(C(O)=O)Oc1n[n](Cc2ccccc2)c2ccccc12)=O Chemical compound OC(C(C(O)=O)Oc1n[n](Cc2ccccc2)c2ccccc12)=O IGULOYSOGFNAFG-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention aims to provide application of organic dicarboxylic acid salts, namely new application in pharmacy, in particular application in preparing medicaments for preventing and treating cataract. New anti-cataract active components in No.1, No.2, No.3, No.4, No.6, NO.7, No.8 and No.9 compounds are discovered by screening rabbit and rat H2O2 induced cataract and rat xylose induced cataract models, and the medical application of the compounds in preparing the medicaments for resisting oxidative damage and sugar metabolism disorder induced cataract is discovered. The No.4 compound has strong and high effect of resisting phacoscotasmus, and has good medicinal prospect. The No.4 compound has obvious effects of improving rabbit lens water-soluble proteins (sol-pro) and total antioxidant capacity (T-AOC), and reducing malondialdehyde (MDA), so the effect of the No.4 compound of resisting H2O2 induced lens oxidative damage is related to the strong oxidation resistance of the No.4 compound.
Description
Technical field
The present invention relates to the purposes of one type of organic dicarboxylic acid salt, particularly relate to the application of one type of organic dicarboxylic acid salt in the cataractous medicine of preparation control.
Background technology
BENDAZACLYSINE: chemical name: L-lysine [1-benzyl-1H-Yin rattle away azoles-3-oxygen base] acetate L-lysine-mono [[[(1-phenylmethyl)-and 1H-indazol-3-yl] oxy] acetat] English name Bendazac Lysine (BDL).BDL is abroad through after zoopery widely and the clinical research, at first by Anglini pharmacy group, in Italy's listing (1983), two kinds of tablet and 0.5% the eye drops of 0.5g arranged as the cataractous active drug of treatment.This medicine is obtained production licence in Italy, Argentina, Spain, Portugal, Philippine, in Greece, Brazil and korean registered registration or carry out clinical research.
The raw material of this medicine and eye drop (trade name: Sha Puaisi) obtained New Drug Certificate and produce certification, [(97) are defended the accurate word X-219 of medicine number], [(97) are defended the accurate word X-220 of medicine number] in 1997.Zhejiang Sapuaisi Pharmacy Co., Ltd. (former Zhejiang Pinghu pharmaceutical factory) enjoys the power of producing without competition of crude drug and preparation, is domestic first national two kind new medicines [the accurate word of traditional Chinese medicines (1998) X445 (1) number] that are used for ophthalmology.Benzydalysine eye drop is country's two kind new medicines of Jiangsu Province Medicine Primary Institute and Zhejiang Sapuaisi Pharmacy Co., Ltd.'s research and development; Be " eight or five " national key scientific and technological project; The National Torch Plan project; Once won Zhejiang Province Drug Administration progress prize in scientific and collective technology scientific and technological achievement second prize, and regarded as the state key new product, (bullets: 98GO41D7000007) cure mainly the early-stage senile cataract by five ministries and commissions such as national science technology departments in 1998.Listed national medium-sized and small enterprises innovation funds project (plan numbering: 991118519) in 1999 in.Over 10 years, because its distinguished definite curative effect, increasing cataract patient is welcome this medicine, and the power of influence of these article on the thing of anti-cataract market risen with powerful advantage; Because the reaction of 50% user has the property a crossed burning sensation, part patient can not tolerate and abandoning cure, and this has just influenced the performance of its anti-cataract medicine advantage; So reduce the zest of Benzydalysine eye drop, increase compliance of patients, become the problem that this medicine presses for solution,
Not only in order to solve the irritating problem that reduces Benzydalysine eye drop; Also can further improve curative effect for the new product of expecting to improve the Benzydalysine eye drop of new generation after the chemical constitution; Can have more powerful competitiveness and wide market like this; Obtain obvious social and economic benefit, lay a solid foundation for further opening up the domestic and foreign markets.Its success must bring glad tidings for vast cataract patient.
Summary of the invention
Technical problem: the object of the present invention is to provide the purposes of one type of organic dicarboxylic acid salt, i.e. new application in pharmacy is is especially preventing and treating the application in the cataractous medicine as preparation.
Technical scheme: organic dicarboxylic acid salt:
The eight kinds of compounds in the preparation of oxidative damage prevention and treatment of cataract and cataract induced glucose metabolism medicament.
Beneficial effect:, can find out that the present invention has the following advantages from pharmacological tests:
(1) the present invention is through rabbit and rat H
2O
2Cause cataract and rat xylose and cause the cataract model discrimination and found new anti-cataract active component in 1,2,3,4,6,7,8, No. 9 chemical compound, found the new purposes of this compounds in cataract medicine due to preparation anti-oxidative damage and the metabolism obstacles of blood glucose.
(2) the anti-lenticular opacity effect of No. 4 chemical compounds of the present invention, not only action intensity is big, and usefulness is also high.Indicating good prospect in medicine is being arranged.
(3) No. 4 chemical compounds of the present invention have significant rabbit crystalline lens water-solubility protein (sol-pro), anti-total oxidability (T-AOC) rising effect, and the decline effect of malonaldehyde (MDA).So anti-H of No. 4
2O
2Causing the effect of crystalline lens oxidative damage has stronger enhancing oxidation resistance relevant with it.
The specific embodiment
Embodiment 1
One type of organic dicarboxylic acid salt is eight kinds of monomeric compounds of deriving out according to BENDAZACLYSINE, its chemical structural formula and the numbering as follows:
BENDAZACLYSINE (BDL, No. 5) is provided by Zhejiang Sapuaisi Pharmacy Co., Ltd., and purity is greater than more than 98.5%; The 1-9 chemical compound is according to different tests, with different solution dissolving preparations; As: anxious malicious intravenous injection test; The chick chorioallantoic membrane irritation test, eye irritation test nictation is irritated the stomach test with 0.5% (WT) carboxymethyl cellulose liquid with 0.9%NaCl (WT) solution, anxious poison; Clean crystalline lens and use the pH7.2 phosphate buffer, hypersensitive test with commercially available Benzydalysine eye drop prescription and anti-crystalline lens oxidation test with the DMEM cell culture fluid etc.Under these conditions, be prone to be dissolved in water 1-5 number, be difficult for for 6-9 number water-solublely, more be difficult for water-soluble with No. 8 especially.Each compound quality concentration representes with WT that all volumetric concentration is represented with VT in following examples.
The method for preparing of above-mentioned each chemical compound is:
1:2-(1-benzyl-1H-indazole-3-oxygen base) malonate (C1)
In the 250mL three-necked bottle, add 12g (0.09mol) 3-hydroxyl-indazole, 3.94g (0.098mol) sodium hydroxide, 90mL water, 40 ℃ are stirred 10min down; Drip 0.076mol benzyl chlorine,, have solid to separate out at 70 ℃ of reaction 2hr; Filter, filter cake is used water washing, gets 1-benzyl-1H-indazole-3-alcohol (B1).
In the four-necked bottle of 100mL, add 40mL glycol dinitrate ether solvents, the potassium carbonate that adds 1-benzyl-1H-indazole-3-alcohol (10.5mmol) and 3.6g (26.1mmol) again mixes.Stir 10min under the gained mixed liquor room temperature, drip the bromo diethyl malonate (lark prestige company) of 3.0g (12.6mmol), reflux 4h then, reactant liquor becomes rufous by yellow, filters.To filtrate and concentrate column chromatography (developing solvent: petroleum ether (60-90 ℃): ethyl acetate=1: 1), obtain 2-(1-benzyl-1H-indazole-3-oxygen base) malonate (C1).
2:2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonate (C2)
With a benzyl chloride chlorine is raw material, and method obtains 2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonate (C2) with the preparation of C1.
3:2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonate (C3)
With a fluorine benzyl chlorine is raw material, and method obtains 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonate (C3) with the preparation of C1.
4:2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid (D1)
2-(1-benzyl-1H-indazole-3-oxygen base) malonate (C1) (2.7mmol) is joined in the 10mL aqueous solution that contains 0.3g potassium hydroxide (5.4mmol); Reflux 2h; Be adjusted to pH=2 with 1M hydrochloric acid then, the adularescent solid is separated out, and filters; With a small amount of washing, dry white solid 2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid (D1) that gets.
5:2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D2)
With 2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonate (C2) is raw material, and method obtains 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D2) with the preparation of D1.
6:2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D3)
With 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonate (C3) is raw material, and method obtains 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D3) with the preparation of D1.
The preparation (No. 3 chemical compounds) of 7:2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid disodium salt
2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid (D1) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol sodium hydroxide; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid disodium salt.
The preparation (No. 2 chemical compounds) of 8:2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid di-potassium
2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid (D1) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol potassium hydroxide; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid di-potassium.
The preparation (No. 1 chemical compound) of 9:2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid two lysinate
2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid (D1) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol lysine; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid two lysinate.
The preparation (No. 4 chemical compounds) of 10:2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid two histidine
2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid (D1) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol histidine; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-(1-benzyl-1H-indazole-3-oxygen base) malonic acid two histidine salt.
The preparation (No. 6 chemical compounds) of 11:2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonate disodium salt
2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D2) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol sodium hydroxide; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonic acid disodium salt.
The preparation (No. 7 chemical compounds) of 12:2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonic acid two lysinate
2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D2) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol lysine; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-[1-(3-chloro-benzyl)-1H-indazole-3-oxygen base] malonic acid two lysinate.
The preparation (No. 8 chemical compounds) of 13:2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonate disodium salt
2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D3) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol sodium hydroxide; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid disodium salt.
The preparation (No. 9 chemical compounds) of 14:2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid two lysinate
2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid (D3) 2.0mmol is joined in the 5mL water, stir 5min, drip the 5mL aqueous solution of 4.0mmol lysine; Stirring at room 4h, concentrating under reduced pressure dewaters, and obtains grease; Add the 20mL dehydrated alcohol then, separate out solid, filter; Use the 2mL absolute ethanol washing, vacuum drying, 2-[1-(3-fluoro-benzyl)-1H-indazole-3-oxygen base] malonic acid two lysinate.
The experimentation of embodiment 2 anti-crystalline lens oxidative damages
1. material
1.1. laboratory animal: rat, Cavia porcellus, rabbit, male and female dual-purpose.
1.2. main agents and medicine:
DMEM/high glucose (high sugar) culture fluid flies Si Er biochemistry Products Co., Ltd (Beijing) available from Sai Mo, vitamin C, 30% (VT) H
2O
2, FeCl
3, NaCl, Na
2HPO
4And NaH
2PO
4Deng being commercially available AR level reagent. xylose (D+xylose) is commercially available HGB level reagent.
1.3. key instrument equipment: superclean bench, CO
2Incubator, homogenizer, low-temperature and high-speed centrifuge, visible spectrophotometer, 12 well culture plates.
2. method
2.1. crystalline lens is cultivated: rat, rabbit are put to death; Take out eyeball with after containing the rinsing of 500u/mL penicillin normal saline, carefully cut eyeball open and remove ball wall and vitreous body, peel off Suspensory ligament from the way of escape; Take out crystalline lens; After containing 500u/mL penicillin, 0.5mg/mL streptomycin PBS liquid rinsing 5min, move ahead anatomical lens inspection of experiment, confirm crystalline lens do not have muddiness and other unusual after.Place 2mL DMEM/high glucose sterile medium sterilization 12 orifice plates of (containing penicillin 100u/mL, streptomycin 0.1mg/mL) are housed, add different 1-9 chemical compounds respectively at 37 ℃ of temperature, humidity 95%, 5%CO
2After cultivating 1h in advance in the incubator, in each hole, add again and contain H
2O
2And FeCl
3{ the H of every hole total concentration rabbit
2O
2Be 2% (VT), the H of rat
2O
2Be 0.5% (VT), FeCl
3Be 0.02% (WT) } or xylose every hole total concentration 20mmol/L (WT)) and sterile medium }, the 4mL culture fluid is contained in every hole altogether, uses H
2O
2The incubation time of cataract model was under equal conditions cultivated 24 hours above-mentioned.The incubation time that causes the cataract model with xylose was cultivated 72 hours under above-mentioned equal conditions, was divided into following several groups during two kinds of model tests, batch test, and every batch is provided with negative control group and model group.
1. negative control group: do not contain H
2O
2Or the DMEM culture fluid of xylose.
2. model group (oxidative damage group): contain H
2O
2And FeCl
3The DMEM culture fluid, or contain the DMEM culture fluid of xylose.
3. vitamin C group: except that each composition of model group, vitaminize C (ultimate density is 1mmol/L) in addition.
4. BENDAZACLYSINE (BDL, No. 5) is organized: except that each composition of model group, other adds BDL (ultimate density 0.5mmol/L).
5. be divided into each group of 1-9 sample: except that each composition of model group, each group adds 1-9 sample (ultimate density 0.5mmol/L) respectively in addition.
2.2. morphological observation: after cultivating 24h, gross examination of skeletal muscle lenticular opacity situation is taken pictures and is scored with white is decorated with different thicknesses at the end black+shape background at lining under 12 orifice plates, is divided into 4 ranks:
-be normal transparent crystal; Do not have muddy
+ be slight muddy (No. 1+word is slightly fuzzy, but visible, No. 2+word is high-visible); The I degree
++ be moderate muddy (but No. 2+word is slightly fuzzy visible, and No. 3+word is high-visible); The II degree
The whole lenticular opacity of +++be (No. 3+word can not be high-visible); The III degree
3. result:
3.1 lenticular opacity situation:
The anti-H of table one type of organic dicarboxylic acid salt of 2-1 (0.5mmol/L)
2O
2Cause the cataract screening and sum up (in vitro lagophthalmos)
This experimental evaluation standard: test in a large number according to model group, each group is produced the percentage rate of each degree of lenticular opacity and the percentage that model group produces each degree of lenticular opacity, be decided to be:
Slight take a turn for the better (√): ++ the percentage rate % of (being the IIIII degree) answers＜67%, and the percentage rate % of+(I degree) answers＞33%; Moderate improvement (√ √): ++ the percentage rate % of (IIIII degree) further reduces; And the percentage rate % of 0 (-) appears; (√ √ √) is clearly better: ++ (IIIII degree) and+the percentage rate % of (I degree) disappears the crystalline lens substantially transparent.
The above results explanation removes 7,8, and outside number screening, all the other chemical compounds judge that according to above-mentioned standard have inhibiting chemical compound to have to cataract: 4,5, No. 6,1,2,3, No. 9 effect is not obvious under the concentration of used dosage 0.5mmol/L.
The anti-H of table one type of organic dicarboxylic acid salt of 2-2 (0.5mmol/L)
2O
2Causing the cataract screening sums up
(the big rathole of in vitro)
Evaluation criterion: test in a large number according to model group, each group is produced the percentage rate of each degree of lenticular opacity and the percentage that model group produces each degree of lenticular opacity, be decided to be:
Slight take a turn for the better (√): ++ (being the IIIII degree) should＜72%+ (I degree) should＞20%; Moderate improvement (√ √): ++ the percentage rate % of (IIIII degree) further reduces, and the percentage rate % of 0 (-) occurs; (√ √ √) is clearly better ++ (IIIII degree) and+the percentage rate % of (I degree) disappears the crystalline lens substantially transparent.
The The above results explanation, except that No. 8 were screened, all the other chemical compounds judged have inhibiting chemical compound to have 3,4 to cataract according to above-mentioned standard, 5,6,7, No. 9 under the concentration of used dosage 0.5mmol/L.1, No. 2 effect is not obvious.
The anti-xylose of table one type of organic dicarboxylic acid salt of 2-3 (0.5mmol/L) causes the cataract screening and sums up (the big rathole of in vitro)
Evaluation criterion: test in a large number according to model group, each group is produced the percentage rate of each degree of lenticular opacity and the percentage that model group produces each degree of lenticular opacity, be decided to be:
Slight take a turn for the better (√): ++ (IIIII degree) answers＜64% ,+(I) answer＞30%; Moderate improvement (√ √): ++ the percentage rate % of (IIIII degree) further reduces, and the percentage rate % of 0 (-) occurs; (√ √ √) is clearly better: ++ the percentage rate % of (IIIII degree) and .+ (I degree) disappears, the crystalline lens substantially transparent.
The The above results explanation; Remove screen for 1, No. 2 outside, No. 7 effects are not obvious, all the other chemical compounds according to above-mentioned standard judgement, have inhibiting chemical compound to have to cataract under the concentration of used dosage 0.5mmol/L: 3,4,5,6,8, No. 9.
4. conclusion:
With the stripped crystalline lens of rat, rabbit through anti-H
2O
2And xylose causes three kinds of screening models of cataract and has proved:
(1). 4,5, No. 6 chemical compounds in one type of organic dicarboxylic acid salt (0.5mmol/L) 1-9 number, to rabbit and rat H
2O
2Causing cataract and rat xylose causes cataract antagonism is all arranged.
(2). No. 9 chemical compounds in one type of organic dicarboxylic acid salt (0.5mmol/L), only to rat H
2O
2Causing cataract and rat xylose causes cataract antagonism is arranged.
(3). No. 8 chemical compounds in one type of organic dicarboxylic acid salt (0.5mmol/L) only cause cataract to the rat xylose has antagonism.
(4). 1, No. 2 chemical compound in one type of organic dicarboxylic acid salt (0.5mmol/L) is to rabbit and rat H
2O
2Cause cataract and do not have obvious antagonism.
(5). in one type of organic dicarboxylic acid salt (0.5mmol/L), remove 1, No. 2 to rabbit and rat H
2O
2Causing cataract does not have outside the obvious antagonism, and the majority of compounds in 1-9 number is to rabbit and rat H
2O
2Causing cataract and rat xylose causes cataract antagonism is in various degree all arranged.
Embodiment 3 and commercially available Benzydalysine eye drop (No. 5) are to the eye irritation comparative test
1 materials and methods
11 given the test agent and reagent
In vitro tests: given the test agent 1-9 chemical compound is a reference substance comprising BENDAZACLYSINE, all is made into the concentration of 70mmol/L with 0.9%wtNaCl.Compare test with 0.9%wtNaCl solution, used chemical reagent is analytical pure.In vivo test 1-5 chemical compound: all use 0.9%wtNaCl to be mixed with achromatism and clarity, specification is: 5mL: the eye drop of 25mg.Do not do in vivo test 6-9 number.
12 animals and instrument
With 10 days Embryo Gallus domesticus age, kind Ji Chang provided by the southern suburbs, Nanjing.21 of white big ear rabbits are planted by New Zealand, body weight 2.5 ± 3.0kg, and male and female all have.Provide by Nanjing Medical University's Experimental Animal Center.OLYMPUS inverted microscope, Queue CO2 gas incubator.
2 test methods
2.1 in vivo test
The perusal cornea does not have muddiness, and conjunctiva does not have hyperemia, edema and secretions, and pupil is circular, and both sides etc. are big, and the good person's of light reflex rabbit is selected.
2.2 number of winks is measured
Selected healthy rabbits is divided into 5 groups every group 3, all drips in every animal right side eye conjunctival sac to be tried thing (0.1mL/ eye), drips 0.9%wtNaCl in the eye conjunctival sac of left side and compares.Compressing nasolacrimal duct during each administration, and make the passive closed 5～10s of eye, immediately write down number of winks in the 10min, calculate 3 animal images of left and right eyes average blink number of times respectively, estimate the zest (number of winks at most zest big) of medicine to eye.
2.2. in vitro tests: Embryo Gallus domesticus chorioallantois film test (HET-CAM test)
Hatching egg at the fertilization ovum gallinaceum in 10 day age is hatched in CO
2Incubator.37.0 ± 0.5 ℃ of temperature are put into water pond to keep humidity 40%～60% in the incubator, passage arranged.Regional with povidone iodine and alcohol disinfecting eggshell surface 2 * 3cm on the fertilization ovum gallinaceum in 10 day age; Draw at eggshell surface with emery wheel and to carve indenture, prick 1 duck eye in the egg plenum surface, drip a small amount of normal saline at the indenture place with prong; Gently take off recess egg skin with pin; Tear inner shell membrane gently, should sink by the CAM of place this moment, forms false air chamber (being different from the air chamber of egg self).Be sure not to damage CAM when being equipped with false air chamber.With the false air chamber of adhesive tape sealing label, form transparent windows, can supply to observe and feeding operations.Be ready to various measured objects after preparing false air chamber, tear adhesive tape gently, go into 1 * 1cm filter paper thus and make carrier.The concentration that on carrier, adds 100 μ l (70mmol/L) is tried thing, acts on after 20 seconds dashing with 5mL distillation water droplet.Observe the reactions change of CAM in the 5min; To observe with the anatomical lens observational method. the degree of impairment of different time sections CAM blood vessel after compound effects is index mark (table 1) haemolysis (hemolysis), hemorrhage (haemorrhage) and blood coagulation (coagulation) to occur.Through scoring and judgement stimulus intensity. after the end system for photographing is carried out in observed blood vessel variation.Each given the test agent detects with 3 ovum gallinaceums.
3. interpretation of result
3.1 in vivo test
Table 3-2 (drips behind the medicine number of winks in the 10min ± SD), n=3 to the influence of rabbit nictation test
| ? | Left eye (normal saline) | Right eye (chemical compound) | Estimate |
| 1 | 1.5±2.1 | 7.5±2.1 | * |
| 2 | 1.5±0.7 | 8±4.0 | * |
| 3 | 2.5±0.7 | 5.0±0 | √ |
| 4 | 0..5±0.7 | 2.0±2.8 | √ |
| 5 | 2±1.4 | 7.5±2.1 | * |
* dazzling number of times >=5 time/10min shows eye irritation is not reduced than No. 5
√ dazzling number of times≤5 times/10min shows has reduction to eye irritation than No. 5
3.2 in vitro tests
The scoring of table 3-31-9 compound H ET-CAM anatomical lens observational method
| Dosage | Negative | Hemorrhage | Haemolysis | Haemolysis, blood coagulation |
| 0.9％wt?NaCl | √ | ? | ? | ? |
| 1 | √ | ? | ? | ? |
| 2 | √ | ? | ? | ? |
| 3 | √ | ? | ? | ? |
| 4 | √ | ? | ? | ? |
| 5 | ? | 3-5 | ? | ? |
| 6 | √ | ? | ? | ? |
| 7 | √ | ? | ? | ? |
| 8 | √ | ? | ? | ? |
| 9 | ? | 7 | 5 | 5 |
4. conclusion
(1) disposable administration irritation test shows in the rabbit body: compare with BENDAZACLYSINE (No. 5), what zest reduced is 3, No. 4.
(2) external HET-CAM irritation test shows: compare with BENDAZACLYSINE (No. 5), what zest reduced is 1,2,3,4,6,7, No. 8.
(3) reduce in the chemical compound of eye irritation in the present embodiment; Though reduce in irritating chemical compound 1,2, No. 3 in the present embodiment on to eye or HET-CAM test; Its zest scoring all is lower than No. 5; But 1,2, No. 3 chemical compounds anti-cataract curative effect on embodiment 2 is not better than No. 5, thereby can think it is for No. 4 to reduce the screening targets that has value in the irritant compound most in this test.
No. 44, embodiment and commercially available Benzydalysine eye drop (No. 5) antagonism crystalline lens oxidative damage comparative test
1. material
1.1. laboratory animal: Cavia porcellus, rabbit, male and female dual-purpose.
1.2. main agents and medicine:
DMEM/high glucose (high sugar) culture fluid flies Si Er biochemistry Products Co., Ltd (Beijing) available from Sai Mo; No. 4 chemical compounds (purity 97.4%) are provided by this problem cooperative groups; Be that the drug research center Gou Shaohua of Southeast China University professor seminar provides; BENDAZACLYSINE (purity 98.5%BDL, No. 5) is provided by Zhejiang Sapuaisi Pharmacy Co., Ltd..Vitamin C, 30%H
2O
2, FeCl
3, NaCl, Na
2HPO
4And NaH
2PO
4Deng being commercially available AR level reagent. crystalline lens water-solubility protein (sol-pro), anti-total oxidability (T-AOC), malonaldehyde (MDA) test kit are all purchased and are built up bio-engineering research institute in Nanjing.
2. method
2.1. crystalline lens is cultivated:, add 2mL in each hole and contain H with embodiment 2
2O
2And FeCl
3(the H of every hole total concentration rabbit
2O
2Be 2%, the H of Cavia porcellus
2O
2Be 1.5%, FeCl
3Be 0.02%) test be divided into following several groups, batch test is established negative control group and model group for every batch.
A. negative control group: do not contain H
2O
2The DMEM culture fluid.
B. model group (oxidative damage group): contain H
2O
2And FeCl
3The DMEM culture fluid.
C. vitamin C group: except that each composition of model group, vitaminize C (ultimate density is 0.5mmol/L) in addition.
D. BENDAZACLYSINE (BDL, No. 5) is organized: establish variable concentrations, except that each composition of model group, other adds BDL (ultimate density 0.5-25mmol/L).
E.4 number sample sets: establish the variable concentrations group, except that each composition of model group, other adds No. 4 samples (ultimate density 0.635-25mmol/L)
2.2. morphological observation: with embodiment 2
2.3. rabbit crystalline lens water-solubility protein (sol-pro), anti-total oxidability (T-AOC), malonaldehyde (MDA) are measured: each group crystalline lens is taken out from culture fluid, and after the normal saline rinsing with 0.9%wt, the filter paper sassafras is dried weighs; Operation at low temperatures; The homogenate of 10%wt, the following 12000 rev/mins of centrifugal 10min of refrigerated centrifuge are processed in homogenate under the ice bath; Get supernatant ,-70 ℃ of preservations.During mensuration, undertaken by the test kit rule of operation.
3. result:
3.1 lenticular opacity situation:
Show 4-14 number and No. 5 antagonism H
2O
2Cause the comparative test of crystalline lens oxidative damage
(in vitro lagophthalmos)
Evaluation criterion: test in a large number according to model group, each group is produced the percentage rate of each degree of lenticular opacity and the percentage that model group produces each degree of lenticular opacity, be decided to be:
Slight take a turn for the better (√): ++ (IIIII degree) answers＜70% ,+(I) answer＞30%; Moderate improvement (√ √): ++ (IIIII) the percentage rate % of degree further reduces, and the percentage rate % of 0 (-) occurs; (√ √ √) is clearly better: ++ (IIIII) the percentage rate % of degree and .+ (I) degree disappears the crystalline lens substantially transparent.
Above data proves:
1.4 No. 5 its curative effects all have with concentration and increase and the trend strengthened.
Though strengthen than model group 2.5 number reach the 0.5-2.5mmol/L curative effect, but still it is muddy crystalline lens (IIIII) degree to occur in concentration;
Reach its curative effect of 5--20mmol/L in concentration and strengthen, crystalline lens (IIIII) degree muddiness occurring has and progressively reduces trend;
Reach 25mmol/L (IIIII) degree in concentration and do not occur, but still it is muddy (I) degree to occur.
3.4 number reaching the 0.625mmol/L curative effect in concentration strengthens than model group, but still it is muddy crystalline lens (IIIII) degree to occur;
Reach its curative effect of 1.25--10mmol/L in concentration and strengthen, crystalline lens (IIIII) degree muddiness occurring has and progressively reduces trend;
When concentration reaches 12.5-25mmol/L, I II, the III degree does not all occur.
4.4 number anti-lenticular opacity effect, compare with No. 5, not only action intensity is big, and usefulness is also high.
Show 4-24 number and No. 5 antagonism H
2O
2Cause the comparative test of crystalline lens oxidative damage
(in vitro guinea pig eye)
Evaluation criterion: test in a large number according to model group, each group is produced the percentage rate of each degree of lenticular opacity and the percentage that model group produces each degree of lenticular opacity, be decided to be:
Slight take a turn for the better (√): ++ (IIIII degree) should＜80%.+ (I degree) should＞20%;
Moderate improvement (√ √): ++ the percentage rate % of (IIIII degree) further reduces, and the percentage rate % of 0 (-) occurs;
(√ √ √) is clearly better: ++ the percentage rate % of (IIIII degree) and .+ (I degree) disappears, the crystalline lens substantially transparent.
Above data proves:
1.4 No. 5 its curative effects all increase with concentration and the trend strengthened.
Though strengthen than model group 2.5 number reach its curative effect of 1.5-5mmol/L, but still it is muddy crystalline lens (IIIII) degree to occur in concentration;
3.4 number reaching its curative effect of 0.625-2.5mmol/L in concentration obviously strengthens, but still it is muddy crystalline lens (IIIII) degree to occur.Reaching 5mmol/L (I) crystalline lens (IIIII) degree in concentration does not occur.When concentration reached 10mmol/L, it is muddy that (I) degree only appears in crystalline lens, has 25% not become turbid.
4.4 number anti-lenticular opacity effect, compare with No. 5, not only action intensity is big, and usefulness is also high.
3.2 rabbit crystalline lens water-solubility protein (sol-pro), anti-total oxidability (T-AOC), malonaldehyde (MDA) are measured:
Show antagonism H 4-34 number
2O
2The biochemical indicator that causes the crystalline lens oxidative damage is measured the result
| Group mmol/L | Soluble protein mg/mL (n=8) | MDA (nmol/mg albumen) (n=6) | T-AOC u/mg albumen (n=6) |
| Contrast | 22.36±2.16 | 0.07±0.03 | 0.66±0.54 |
| Model | 7.40±0.73## | 0.46±0.27## | 0.04±0.05## |
| VC-5 | 8.59±1.13 | ? | ? |
| 5-5 | 9.34±1.65 | 0.22±0.10 | 0.17±0.05** |
| 4-5 | 12.99±1.95** | 0.04±0.02** | 0.21±0.12** |
| 4-10 | 16.78±5.96** | 0.03±0.02** | 0.64±0.30** |
| 4-15 | 19.04±1.03** | 0.08±0.04** | 0.34±0.16** |
* compared with model group, P＜0.01
##compared with matched group, P＜0.01
4. sum up
1.4 number anti-H
2O
2Cause the effect of crystalline lens oxidative damage, compare with No. 5, not only action intensity is big, and usefulness is also high.
2.4 number anti-H
2O
2Cause the effect of crystalline lens oxidative damage and be better than No. 5, have stronger enhancing oxidation resistance relevant with it.
No. 54 hypersensitive tests of embodiment
1, test material:
1.1 supply the reagent thing: No. 4 chemical compounds, the drug research center Gou Shaohua of Southeast China University professor seminar provides, by commercially available Benzydalysine eye drop prescription, concentration 1%.
1.2 animal: Cavia porcellus, 16 of sums, body weight 350-400g, male and female half and half are provided credit number by Nanjing Medical University's Experimental Animal Center: SCXK (Soviet Union) 2008-0004.
2. pilot project:
2.1 initiatively irritated:
Method: get 6 of Cavia porcelluss, respectively at the 1st, 3,5 days intraperitoneal injections of experiment, once a day, each 0.5mL/ only carries out sensitization; Behind the 14th day of last administration,, attack with 2mL/ intravenous injection.Observed 3 hours.Table 5-1 is seen in dosage and grouping, and observation index and judgment criteria are seen table 5-2 table 5-3.
Show 5-14 number to Cavia porcellus hypersensitive test dosage and grouping
Table 5-2 symptoms of allergic
| 1 is restless | 5 sneezes | 9 defecation | 13 purpuras | 17 spasm |
| 2 perpendicular hairs | 6 coughs | 10 shed tears | 14 instability of gait | 18 rotations |
| 3 tremble | 7 rapid breathing | 11 dyspnea | 15 jump | 19 Cheyne-Stokes respiration |
| 4 scratch nose | 8 urinate | 12 wheezing sounds | 16 pant | 20 death |
Table 5-3 whole body sensitization evaluation criterion
| 0 | - | Anaphylaxis is negative |
| The 1-4 symptom | + | Anaphylaxis is weak positive |
| The 5-10 symptom | ++ | Anaphylaxis is positive |
| The 11-19 symptom | +++ | The anaphylaxis strong positive |
| 20 | ++++ | The extremely strong positive of anaphylaxis |
Result: do not meet quick reaction symptom.
2.2 passive hypersensitive test:
Method: select 10 Cavia porcelluss, be divided into intact skin group and damaged skin group, 5 every group.With back part of animal left side QUMAO 3 * 4cm
2, wherein injured skin group Cavia porcellus makes " # " font cut with blade.For guaranteeing that medicine well contacts with local skin,, make to reach 6 hours the time of contact of medicine with the dressing parcel and with non-stimulated immobilization with adhesive tape.The sensitization stage: get No. 4, by dosage 0.5mL/ only, be coated in depilation district, back part of animal left side, the 1st day, 7 days and each administration in 14 days 1 time, totally 3 times.Excitation phase: after 14 days, only be applied to depilation district, right side, back by 0.5mL/ in the last administration, observe variation in 24,48,72 hours with No. 4.At percutaneous drug delivery simultaneously, its eye mucosa is pressed percutaneous drug delivery program eye drip administration (0.05mL), merge the allergy effect of observing the eye mucosa, the result judges: the anaphylaxis standards of grading are seen table 5-4.
Table 5-4 skin allergy standards of grading
| Dermoreaction | Score value |
| Erythema forms the slight erythema moderate erythema of no erythema severe erythema edematous erythema | 0 1 2 3 4 |
| Edema forms no edema Mild edema intermediate edema severe edema | 0 1 2 3 |
| Total mark (full marks) | 7 |
The result: in the sensitization stage, experimental guinea pig is not seen the constitutional IR; In excitation phase, also not show speckle, edema anaphylaxis.The result sees table 5-5,5-6
Show 5-54 number to Cavia porcellus anaphylaxis result of the test
Show 5-64 number to guinea pig eye mucosa result of the test
3. hypersensitive test brief summary:
No. 4 Cavia porcellus is carried out anaphylaxis and test or allergic response with cavy.
No. 64 acute toxicity tests of embodiment
1, test material:
1.1 supply the reagent thing:
No. 4 chemical compounds: irritate stomach: 1%CMC preparation, 1.33g-5mL, intravenous injection: normal saline preparation, 0.535g-7.5mL;
BENDAZACLYSINE (BDL): irritate stomach: 1%CMC preparation, 0.89g-5mL, intravenous injection: normal saline preparation, 0.367g-7.5mL;
No. 4 for the second time: irritate stomach: 1%CMC preparation, 1.25g-6mL, 0.4mL/20g;
BENDAZACLYSINE (BDL): irritate stomach: 1%CMC preparation, 0.852g-6mL, 0.4mL/20g;
1.2 animal: the ICR mice, 110 of sums, body weight 18-22g, male and female half and half are provided credit number by Nanjing Medical University's Experimental Animal Center: SCXK (Soviet Union) 2008-0004.
2. experimental technique and result:
Adopt BDL and No. 4 molar dose toxicity relative methods such as chemical compound, with two kinds of approach of intravenous injection and filling stomach, all observed for 2 weeks respectively, estimate the chemical compound acute toxicity No. 4.Route of administration, dosage and dead result see table 5-7, table 5-8,5-9.
Table 5-7 intravenous injection approach dosage and dead result
| Medicine | Dosage g/kg | Concentration g/mL | Volume mL/10g | Death toll (n/n) |
| BDL (No. 5) | 0.73 | 0.0489 | 0.15 | 10/10 |
| BDL (No. 5) | 0.47 | 0.049 | 0.096 | 10/10 |
| No. 4 | 1.069 | 0.0713 | 0.15 | 1/10 |
| No. 4 | 1.348 | 0.0713 | 0.188 | 4/10 |
Show 5-84 number and irritate stomach approach dosage and dead result
Table 5-9BDL (No. 5) irritates stomach approach dosage and dead result
Irritate stomach LD No. 5
50Be 2.091g/kg, be limited to 2.271g/kg on 95% fiducial limit,
Be limited to 1.925g/kg under 95% fiducial limit
Sum up:
No. 4 and BDL (No. 5) near etc. intravenous injection (No. 4 0.73g/kg, No. 5 1.069g/kg) under the molar dose condition, No. 4 toxicity all is lower than BDL.No. 4 oral maximum tolerated doses are 4.16g/kg, No. 5 filling stomach LD
50Be 2.091g/kg, be limited to 2.271g/kg on 95% fiducial limit, be limited to 1.925g/kg under 95% fiducial limit.It is thus clear that under two kinds of approach of intravenous injection and filling stomach, No. 4 toxicity all is lower than BDL (No. 5).
Claims (1)
1. organic dicarboxylic acid salt:
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| CN1985801A (en) * | 2006-12-14 | 2007-06-27 | 宁夏康亚药业有限公司 | Benzydalysine eye drop and its preparing method |
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| CN1985801A (en) * | 2006-12-14 | 2007-06-27 | 宁夏康亚药业有限公司 | Benzydalysine eye drop and its preparing method |
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