CN1593651A - Novel usage of human antalzyme in the process for preparing ophthalmopathy treating medicine - Google Patents
Novel usage of human antalzyme in the process for preparing ophthalmopathy treating medicine Download PDFInfo
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Abstract
The invention relates to the novel use of human lysozyme, in particular the use of human lysozyme in pharmaceutical industry, specifically the use of human lisozima in preparing ophthalmocace treating medicine, such as epidemic conjunctivitis, opthalmitis, ceratitis, corneal ulcer diseases caused by bacterium, Chlamydia, viruses and drug resistant medicines. The gene recombination lysozyme has rich source, the eye drops prepared thereby are easy to use.
Description
Technical field:
The present invention relates to the new purposes of human lysozyme, particularly the new purposes in pharmaceutical field.
Background technology:
At present very common by diseases such as antibacterial, virus, the various pink eye disease that cause, ophthalmia, keratitis, corneal ulcer, therapy generally is to adopt the antibiotics eye drop, but today of 21 century, the development of fastbacteria makes us startling, from the drug resistance development history of antibacterial as can be seen, after certain new antibiotic occurs, just there is a collection of Resistant strain to occur.Developing a kind of new antibiotic generally needs the time in about 10 years, and the generation of generation fastbacteria needs only the time in 2 years, and antibiotic development speed is unable to catch up with the development speed of fastbacteria far away.Use antibiotics many more, causing antibacterial is that to hide the mode that antibiotics makes a variation many more.Because antibiotic abuse, considerable drug tolerant bacteria occurs.In decades, the mankind have invented a large amount of antibacterials, are applied to clinical not following 200 kinds more than at present, and are still increasing with the speed that new antibacterials are come out more than 10 kinds every year on average.Though the development of new antibiotic constantly arranged and come into operation, in a short period of time, just have new endurance strain to produce.As if in the face of the antibacterial of " survival of the fittest is constantly evolved ", antibiotic is at one's wit's end.
Summary of the invention:
The purpose of this invention is to provide the application of a kind of human lysozyme in the medicine of preparation treatment ophthalmic.Effectively at by diseases such as antibacterial, virus, the various pink eye disease that cause, ophthalmia, keratitis, corneal ulcer, dosage form is reasonable, and is easy to use.
In fact the present invention relates to the application of human lysozyme in the medicine of the various oculopathy of preparation treatment.
Relate to the application of human lysozyme in the medicine of various pink eye disease that preparation treatment is caused by antibacterial, chlamydia, virus and fastbacteria, ophthalmia, keratitis, corneal ulcer disease.
Relate to the application of human lysozyme in the medicine of various pink eye disease that preparation treatment is caused by Salmonella and chlamydia, ophthalmia, keratitis, corneal ulcer disease.
Relate to the application of human lysozyme in the medicine of various pink eye disease that the herpes disease that preparation treatment is caused by I type herpesvirus causes, ophthalmia, keratitis, corneal ulcer disease.
Relate to the application of human lysozyme in the medicine that causes various pink eye disease, ophthalmia, keratitis, corneal ulcer disease that the preparation treatment is caused by Coxsackie virus.
Relate to the application of human lysozyme in the medicine that causes various pink eye disease, ophthalmia, keratitis, corneal ulcer disease that the preparation treatment is caused by line virus.
Relate to human lysozyme and read application in the coccigenic medicine that causes various pink eye disease, ophthalmia, keratitis, corneal ulcer disease by pathogenicity in preparation treatment.
Described medicine is an eye drop, contains active 300U～3,000,000 U/mL human lysozymes.
Described human lysozyme comprises that the recombinant human lysozyme of gene engineering expression, the aminoterminal of gene engineering expression human lysozyme have (glutamic acid-alanine)
2Or (glutamic acid-alanine)
3Human lysozyme, gene engineering expression or the chemosynthesis mutant recombinant human lysozyme modified.
Recombinant human lysozyme gene source people's peripheral blood.By the DNA recombinant technique, in the pUC19 plasmid vector, clone strain turns out to be the recombinant human lysozyme gene by the nucleotide DNA sequence analysis with gene clone again.It is a kind of effective antibacterial, and full name is: 1, and 4-β-N-lysozyme or title: mucopeptide N-acetyl group muramyl hydrolytic enzyme.The connection that it can cut off β-1,4 glycosidic bond between the N-acetylglucosamine and-acetylmuramic acid in the Peptidoglycan of bacteria cell wall destroys the Peptidoglycan support, and antibacterial cell spalling under the effect that internal penetration is pressed is opened, and causes the antibacterial cracking.The acellular wall construction of humans and animals cell does not also have Peptidoglycan, so lysozyme is to the human body cell free of toxic effects.Recombinant human lysozyme also has antiviral, antitumor, antiinflammatory and immunoregulation effect except that direct cracking antibacterial.
Gene recombinant human lysozyme of the present invention source is abundant, and preparation technology is simple, and safe without toxic side effect has good prospect in medicine, makes eye drop, and is easy to use.Excavate new medical application, opened up a new application.
Essence for a better understanding of the present invention will illustrate its new purposes in pharmaceutical field with the pharmacological testing and the result of gene recombinant human lysozyme below.
Gene recombinant human lysozyme is as the criterion to prepare 200 milliliters of culture medium, with 6 milliliters of phosphoric acid, magnesium sulfate 3 grams, potassium sulfate 4 grams, potassium hydroxide 1 gram, calcium sulfate 1.5 grams, adding distil water to 200 milliliter, inoculation glycerol pipe seed behind the autoclaving, the shaking table revolution is that per minute 250 changes, and cultivation temperature is 20-35 ℃, cultivates 36-48 hour on the constant temperature bed.Carry out seed tank culture, produce a jar cultivation at last.The culture fluid that fermentation expression is finished extracts purification, and the protein concentrated solution that extracts purification is carried out lyophilizing, surveys albumen and surveys active the preservation.Pharmaceutical factory in GMP compatible, by the pharmacy rules, the gene recombinant human lysozyme of purity 95% is made 300U～3,000,000 U/mL, phosphate buffer 1 0～20mM (pH6.5～7.5) 80%, 5～25% propylene glycol, 5/10000ths Tween 80s, mix homogenizing at normal temperatures, make eye drop.
One, to the model test of animal:
A, gene recombinant human lysozyme (HLZ) inside and outside antibacterial action are estimated
(1) vitro antibacterial activity:
Be subjected to reagent product and reagent:
1, human lysozyme (Human Lysozyme HLZ): active unit: 30000 units/mL are provided by the strange imperial biotechnology research in Dalian.
2, the contrast lysozyme (contral Lysozyme, CLZ): white powder, active unit: 50000 units/mg, U.S. SIGMA company product, lot number: L6876.
3, clarithromycin: 948 μ that tire/mg, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's standard substance, lot number:
4, Roxithromycin: 878 μ that tire/mg, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's standard substance, lot number:
5, injection amoxicillin: Harbin Pharmaceutical Factory's product, lot number: 010504.
6, agarose (B10WEST AGAROSE):
7, Tris (Tris): Chengdu chemical test factory, lot number 010211
Test strain:
Bacterial strain uses therefor is 2001.4 ~ 2002.4 months in Sichuan, the Beijing area clinical separation pathogenic bacterium of collecting.After identifying again with the API method, this chamber is used for test.
Quality Control bacterial strain: staphylococcus aureus ATCC25923
Escherichia coli ATCC25922
Pseudomonas aeruginosa ATCC27853
Culture medium:
4, Tris-HCl buffer: 0.1MTris 100ml, 0.1MHCl 70ml, High Water 800ml surveys pH value, transfers to PH7.2 with HCl solution, adds High Water to 1000ml.
2, Tris-HCl agar culture medium: in the Tris-Cl buffer, add 116 ℃ of aseptic backs of agarose and use.
3, M-H culture medium: Nat'l Pharmaceutical ﹠ Biological Products Control Institute's product, the M-H broth bouillon: take by weighing 25g and add the 1000ml distilled water, heating for dissolving, packing, autoclaving, 116 ℃ 20 minutes.The M-H solid medium: take by weighing 36g, add the 1000ml distilled water, autoclaving, 116 ℃ 20 minutes, be used for the drug sensitive test of Grain-positive, negative aerobe.
4, blood meida, it is formulated promptly to add 5-10% defiber Sanguis Leporis seu oryctolagi in the M-H culture medium, is used for enterococcus, streptococcic drug sensitive test.
Test method:
Antibacterial activity in vitro (MIC) is measured: adopt the agar doubling dilution to measure and be subjected to the minimum inhibitory concentration (MIC) of reagent thing to test strain.To be subjected to reagent thing aseptic distillation water dissolution, suitably dilution.Get the 1ml medicinal liquid respectively and add the Tris-HCl agarose solid medium mixing that 9ml melts,, prepare serial pastille plate with doubling dilution.Every ware contained drug final concentration is respectively 4,2,1,0.5,0.25,0.125,0.06,0.03 ... 0.001mg/ml; (Denley A400 England) will be diluted to 10 to inoculate instrument with multiple spot
5The test organisms liquid of CFU/ml is inoculated in each pastille plate surface, place 37 ℃ to cultivate 8-10 hour, take out and draw the above-mentioned serial plate of M-H culture medium (50 ℃) the covering surface that 6ml melts respectively, place 37 ℃ to cultivate taking-up in 10 hours again, observed result is the minimum inhibitory concentration (MIC) of this bacterium with contained lowest concentration of drug in the no bacterial growth plate.
(2) vivo bacteria corrosion action evaluation:
Medicine: the human lysozyme source is the same.
The clarithromycin source is the same.
The Roxithromycin source is the same.
The source, amoxicillin is the same.
Antibacterial: golden yellow Portugal coccus 01193, the golden yellow coccus MRSA021923 of Portugal is clinical separation pathogenic bacterium.
Animal: Kunming mouse, body weight 18-22 gram is provided by animal housing of this institute.
Test method:
The endogenous protective test: a certain amount of lawn of picking is inoculated in the 2ml M-H fluid medium, cultivates after 6 hours for 37 ℃, takes out with sterilization dry yeast liquid and suitably dilutes (10
-1, 10
-2, 10
-3, 10
-4), get Kunming mouse again, random packet, every group of 5 Mus, respectively the different bacterium amounts of abdominal cavity infection tried bacterium liquid, measure the minimum that the causes mice 100% death bacterium amount (MLD) that causes death.By 1: 0.5 dose of spacing 5 dosage groups are set again, every group of 10 Mus, every mouse peritoneal infects the bacterium liquid 0.5ml of 1MLD bacterium amount, and intravenous injection at once is subjected to reagent 0.5ml after the infection, establishes and infects matched group (not administration), observes for 1 week, record dead mouse number.Press the Bliss method and calculate median effective dose ED
50And 95% fiducial limit.
The therapeutic evaluation of mouse skin burn infection model: Kunming mouse 26-30g, random packet, every group of 5 Mus, the skin burn infection model sprays staphylococcus aureus bacterium liquid 100ml respectively, and the bacterium amount is 10
8CFU/ml sprays 5 times (at interval 10 minutes spray once) continuously, and behind the last spray bacterium 6 hours, be applied to no medicine agar plate surface with aseptic cotton carrier picking skin burn infection model respectively, 37 ℃ of cultivations 18 hours, skin burn infection model bacterial infection number is＞10
3CFU/ml, and through Gram, light microscopic detect for the mice of staphylococcus aureus be infection model successfully.
Choose the skin burn infection model and infect successful mice random packet, every group of 10 Mus, be subjected to reagent thing 100 μ l/ time with the aerosol apparatus spraying respectively, 4 times/day, continuous 5 days, adopt the sub-smear method of pharynx examination every day, carry out count plate in agar plate surface, take statistics to learn and handle, make comparisons with infection matched group and clarithromycin group, Roxithromycin administration group.
Result of the test:
(1) human lysozyme sees Table 1 to the antibacterial activity in vitro of clinical isolates strain.
(2) clarithromycin, Roxithromycin, amoxicillin and human lysozyme the results are shown in Table 2 with the vitro antibacterial activity of 1: 1 drug combination.
(3) human lysozyme sees Table 3 to MIC50, the MIC90 of the clinical separation pathogenic bacterium of 379 strains.
Show according to above result of the test: human lysozyme is external to have certain antibacterial action, and the curative effect of in the body wound infection being festered is more definite.To the antibacterial activity of most bacterial strains less than clarithromycin, Roxithromycin and amoxicillin.Human lysozyme and clarithromycin, Roxithromycin coupling (1: 1) can make clarithromycin, Roxithromycin to more than antibacterial activity potentiation 2-16 times of its Resistant strain, and indivedual bacterial strain potentiation multiples are at 1000 times.Lysozyme is a kind of small protein, be present in tear, saliva, leukocyte and the serum of body, multiple gram positive bacteria and minority gram negative bacteria there is bactericidal action, the gene recombinant human lysozyme of being developed by the strange imperial biotechnology research in Dalian (Human Lysozyme) also has lysozyme same function mechanism, β-1 in the main cut-out Peptidoglycan between N-acetylglucosamine and the-acetylmuramic acid, the connection between 4 glycosidic bonds, destroy the Peptidoglycan support, cause the antibacterial cracking.
Table 1 human lysozyme, lysozyme of chicken, clarithromycin and Roxithromycin inside and outside antibacterial activity
MIC
Antibacterial HLZ CLZ CLA ROX
Mg/ml (mg/ml (ten thousand/μ ml) the mg/ml mg/ml of ten thousand μ/ml)
The gold MRSA02-22 of Portugal 0.25 (0.8) 0.008 (0.04)＞1＞1
The gold MRSA02-23 of Portugal 0.25 (0.8) 0.008 (0.04)＞1＞1
The gold MRSA02-26 of Portugal 0.25 (0.8) 0.004 (0.02)＞1＞1
The gold MRSA02-28 of Portugal 0.5 (1.5) 0.016 (0.08)＞1＞1
The gold 02-19-5 of Portugal 0.03 (0.1)＜0.002 (0.001)＞1＞1
The table MssE25 of Portugal 0.016 (0.05) 0.004 (0.02)＜0.001＜0.001
The table MRSE 02-29 of Portugal 0.063 (0.2) 0.5 (2.5) 0.03 0.5
The table MRSE 02-5 of Portugal＜0.001 (0.003)＜0.001 (0.003)＜0.001＜0.001
The table MRSE 02-6 of Portugal＜0.001 (0.005)＜0.001 (0.005)＜0.001＜0.001
The table MRSE 02-20-2 of Portugal＜0.001 (0.003)＜0.001 (0.005)＞11
The table MRSE 02-20-3 of Portugal＜0.001 (0.003) 0.004 (0.02)＞11
The table MRSE 02-20-4 of Portugal＜0.001 (0.003)＜0.001 (0.005)＞1＞1
The table MRSE 02-20-5 of Portugal 0.004 (0.012)＜0.001 (0.005)＞1＞1
The table MRSE 02-20-6 of Portugal 0.004 (0.012)＜0.001 (0.005)＞1＞1
The table MRSE 02-20-7 of Portugal＜0.001 (0.003)＜0.001 (0.005) 11
The table MRSE 02-20-8 of Portugal＜0.001 (0.003)＜0.001 (0.005)＜0.001＜0.001
The table MRSE 02-20-9 of Portugal＜0.001 (0.003)＜0.001 (0.005)＜0.001＜0.001
The table MRSE 02-20-1 of Portugal＜0.001 (0.003)＜0.001 (0.005)＜0.001＜0.001
The table MRSE 02-3 of Portugal 1 (3) 0.015 (0.08)＞1＞1
The table MRSE 02-4 of Portugal 0.004 (0.012) 0.008 (0.04) 0.5＞1
Continuous table 2
MIC
Antibacterial HLZ CLZ CLA ROX
Mg/ml (the mg/ml of ten thousand μ/ml) (the mg/ml mg/ml of ten thousand μ/ml)
The table MRSE 02-10 of Portugal 0.004 (0.012) 0.25 (1.25) 11
The table MRSE 02-12 of Portugal＜0.001 (0.003)＜0.001 (0.005) 0.008 0.125
The table MRSE 02-11 of Portugal＜0.001 (0.003)＜0.001 (0.005)＜0.001＜0.001
The table MRSE 02-15 of Portugal 0.008 (0.024) 0.002 (0.01) 1＞1
The table MRSE 02-17 of Portugal 0.004 (0.012)＜0.001 (0.005) 0.25＞1
The table MRSE 02-18 of Portugal＜0.001 (0.003)＜0.001 (0.005)＜0.001＜0.001
The table MRSE 02-20 of Portugal 0.008 (0.024)＜0.001 (0.005)＞1＞1
The table MRSE 02-21 of Portugal 0.016 (0.05) 0.25 (1.25)＜0.001＜0.001
The table MRSE 02-22 of Portugal 0.004 (0.012)＜0.001 (0.005)＜0.001＜0.001
Form staph 02-7-4 0.008 (0.02) 4 (20) 0.002 0.002
Form staph 02-7-5 0.008 (0.02) 0.25 (0.63)＜0.001＜0.001
The gold bacterium 02-7-6 of Portugal 0.008 (0.02) 0.063 (0.31) 0.008 0.25
The gold bacterium 02-7-9 of Portugal 0.063 (0.2) 0.125 (0.63) 0.008 0.016
The gold bacterium 02-7-7 of Portugal 0.125 (0.4) 0.016 (0.08) 0.032 0.25
The gold bacterium 01-2-22 of Portugal 0.032 (0.1) 0.5 (2.5) 11
The gold bacterium 01-2-30 of Portugal 0.063 (0.2) 4 (10)＞1＞1
The gold bacterium 01-2-32 of Portugal 0.016 (0.05) 0.25 (1.25) 0.008 0.5
The gold bacterium 01-2-33 of Portugal 0.032 (0.1) 2 (10)＞1＞1
The gold bacterium 01-2-36 of Portugal 0.063 (0.2) 4 (20) 0.25 0.5
The gold bacterium 01-2-37 of Portugal 0.032 (0.1)＞4 (20)＞11
The gold bacterium 01-2-39 of Portugal 0.032 (0.1) 0.5 (2.5) 0.5 1
Continuous table 3
MIC
Antibacterial HLZ CLZ CLA ROX
Mg/ml (the mg/ml of ten thousand μ/ml) (the mg/ml mg/ml of ten thousand μ/ml)
Pseudomonas aeruginosa 01-2-41 0.032 (0.1) 0.5 (2.5)＞1＞1
Pseudomonas aeruginosa 01-2-42 0.016 (0.05) 0.5 (2.5) 0.063＞1
Pseudomonas aeruginosa 01-2-43＜0.001 (0.003)＞4 (20) 0.016 0.25
Pseudomonas aeruginosa 01-2-45 0.032 (0.1) 2 (10) 0.25 0.5
Pseudomonas aeruginosa 01-2-51 0.032 (0.1)＞4 (20)＞1＞1
Pseudomonas aeruginosa 01-2-52 0.032 (0.1)＞4 (20)＞1 0.25
Pseudomonas aeruginosa 01-2-53 0.008 (0.024) 4 (20) 0.03 0.25
Pseudomonas aeruginosa 01-2-54 0.032 (0.1) 1 (5)＞11
The gold bacterium 01-2-56 of Portugal 0.032 (0.1)＞4 (20) 0.016 0.25
The gold bacterium 01-2-57 of Portugal＜0.001 (0.003) 2 (10) 0.032 0.25
Escherichia coli 01-2-58＜0.001 (0.003) 4 (20)＞1 0.5
Escherichia coli 01-2-59 0.032 (0.1) 4 (20) 0.125 0.25
Serratieae 2-31-8 0.008 (0.02) 0.5 (0.25) 0.008 0.016
Serratieae 2-31-8 0.008 (0.02) 1 (5) 0.008 0.016
The gold bacterium 2-31-8 of Portugal 0.008 (0.02) 0.125 (0.63) 0.016 0.016
The gold bacterium 02-6-14 of Portugal 0.25 (0.8) 1 (5) 0.063＞1
The gold bacterium 02-6-18 of Portugal 0.5 (1.5)＞4 (20) 0.5 1
Annotate: HLZ refers to human lysozyme, and CLZ refers to contrast lysozyme (lysozyme of chicken), and CLA refers to clarithromycin, and ROX refers to Luo Hongsu
B, human lysozyme are to the analgesic activity (rat tail-flick method) of rat
120-150 gram SD healthy rat, male and female half and half, animal freely drinks water.The test chamber temperature is controlled at about 22-28 ℃, animal feed rat standard feed.Select for use at TF---the photo-thermal dolorimeter (light source is 12V, 50W) under the thermal exposure 10 second internal reaction 50 of rats, be divided into 5 groups at random, 10 every group, male and female half and half.If blank group, three dosage groups of human lysozyme (120,60,30IU/ only), lysozyme of chicken (positive control) 30IU/, all afterbody coating.Before the coating and pain reaction (whipping) time of surveying every rat behind the coating in 0.5-4 hour, do not interrupt irradiation during whipping, in order to avoid injured skin and foaming, and calculated with 30 seconds if the threshold of pain is elevated to 30 seconds of irradiation.Test repeats once.
Result of the test sees Table 17-21, and the result shows, smears human lysozyme 30,60, only in three hours, its threshold of pain obviously raises 120IU/, has analgesic activity.
Table 17-21 (A) human lysozyme is coated with the analgesic activity (tail-flick method) to rat outward
(result of the test for the first time)
The pain reaction time (second, X ± SD)
Group dosage number of animals
(IU/ only) (only) 0 0.5 1234 (h)
Blank-10 5.2 5.7 6.4 6.4 6.8 10.3
Contrast ± 1.69 ± 2.16 ± 2.46 ± 3.34 ± 3.91 ± 5.33
Chicken molten 30 10 5.5 11.4
*14.0
*14.7
*15.2
*12.7
*
Bacterium enzyme ± 1.72 ± 4.5 ± 6.93 ± 7.67 ± 7.45 ± 6.99
The people molten 120 10 5.3 13.6
*15.2
*13.4
*15.4
*14.9
Bacterium enzyme ± 1.77 ± 6.98 ± 7.42 ± 6.38 ± 6.62 ± 6.54
The people molten 60 10 5.3 9.8
*12.9
*12.7
*13.5
*12.4
Bacterium enzyme ± 1.89 ± 4.05 ± 6.69 ± 6.9 ± 6.72 ± 6.65
The people molten 30 10 5.2 8.6
*12.4
*10.9 11.7 10.1
Bacterium enzyme ± 1.93 ± 2.67 ± 7.49 ± 709 ± 6.89 ± 7.5
Annotate: learn by statistics and handle, compare * P＜0.05, * * P＜0.01 with the blank group.
Table 17-21 (B) human lysozyme is coated with the analgesic activity (tail-flick method) to rat outward
(result of the test for the second time)
The pain reaction time (second, X ± SD)
Group dosage number of animals
(IU/ only) (only) 0 0.5 1234 (h)
Blank-10 5.5 5.9 6.5. 6.6 6.9 9.8
Contrast ± 1.58 ± 1.66 ± 1.78 ± 4.95 ± 6.35 ± 6.73
Chicken molten 30 10 5.7 12.3
*15.0
*13.8
*13.6
*11.7
Bacterium enzyme ± 1.64 ± 4.6 ± 7.99 ± 7.99 ± 7.6 ± 6.9
The people molten 120 10 5.8 14.2
*15.7
*14.5
*14.3
*13.5
Bacterium enzyme ± 1.23 ± 6.54 ± 6.78 ± 7.11 ± 7.44 ± 7.69
The people molten 60 10 5.6 12.6
*14.3
*14.0
*13.8
*12.5
Bacterium enzyme ± 1.89 ± 4.05 ± 6.69 ± 6.9 ± 6.72 ± 6.65
The people molten 30 10 5.7 10.0
*12.0
*10.7 10.1 11.2
Bacterium enzyme ± 1.34 ± 4.47 ± 7.85 ± 4.27 ± 3.84 ± 4.5
Annotate: learn by statistics and handle, compare * P＜0.05, * * P＜0.01 with the blank group.
C, human lysozyme bring out the influence of mice auricle swelling to Oleum Tiglii
Select 50 of the healthy male mouse of kunming of body weight 27-30 gram for use, animal freely drinks water.The test chamber temperature is controlled at about 22-28 ℃, animal feed rat standard feed.Be divided into 5 groups at random, 10 every group.First group is the blank group; Second and third, four groups be human lysozyme, dosage is respectively 120,60,30IU/ only; The 5th group is lysozyme of chicken (positive control), and 30IU/ only.At first, each is organized the whole auris dextras of mice inboard and is coated with 1% Oleum Tiglii (lot number 000309 is produced with factory by the precious crude drug of Jishui County, Jiangxi China for Oleum Tiglii light brown oily liquid, pharmaceutical grade.Face with the preceding ethanol of using: water: 25: 5: 70 mixed solvents of ether are mixed with 1% concentration.) 30 μ L cause inflammation, use distilled water 20 μ l, human lysozyme (6000IU/ML) 20 μ l, human lysozyme (3000IU/ML) 20 μ l, human lysozyme (1500IU/ML) 20 μ l, lysozyme of chicken (1500IU/ML) 20 μ l to smear and respectively organize the mouse right ear inboard after half an hour respectively.Cause scorching back 4 hours mice is taken off cervical vertebra and cause death, two auricles about cutting along the auricle baseline sweep away two auricles with the 8mm card punch, accurately weigh, and the difference of left and right sides auricle weight is the swelling degree.Through the T check, the difference of comparative drug group and blank group, and obtain suppression ratio.Test repeats once.
Result of the test sees Table 17-22.Mice through outside only be coated with human lysozyme 120,60,30IU/, make mice bring out auricle swelling degree and obviously alleviate by Oleum Tiglii, through the T check, medicine group and the comparison of blank group have significant difference (P＜0.05).Illustrate that human lysozyme has antiinflammatory action.
Table 17-22 human lysozyme brings out the influence of mice auricle swelling to Oleum Tiglii
The 2nd test of the 1st test
Group dosage
(IU/ only) swelling degree suppression ratio swelling degree suppression ratio
(mg，X±SD) (％) (mg，X±SD) (％)
Blank-20.3 ± 3.40 20.1 ± 4.01
Human lysozyme 120 12.5 ± 5.56
*38.42 13.6 ± 3.78
*32.34
Human lysozyme 60 13.5 ± 4.67
*33.50 15.1 ± 2.42
*24.88
Human lysozyme 30 14.2 ± 3.77
*30.05 15.4 ± 4.22
*23.38
Lysozyme of chicken 30 14.6 ± 2.12
*28.08 15.1 ± 2.51
*24.88
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group.
D, recombinant human lysozyme eye drop antiviral suppress Cavia porcellus keratitis test model
Select 20 of healthy guinea pigs about body weight 250 gram, malely divide equally four groups at random, 5 every group, carry out the eye drop modeling.At first on the cornea of Cavia porcellus, slightly mark three road vestiges, use I type herpesvirus 100～1000TCID then with fine needle
50Infect the cornea of Cavia porcellus, every day three times.Slight keratitis appearred in the guinea pig eye cornea in the 3rd day, and redness appearred in the guinea pig eye cornea in the 4th day, and keratitis increases the weight of.Be divided into four groups more at random after 20 Cavia porcelluss after the modeling are reconsolidated, set up three administration groups and a model group respectively.The recombinant human lysozyme eye drop is a liquor strength when preparing 30000u/ml (every 3000U), 60000u/ml (every 6000U) two concentration as middle and high dosage group treatment on the low dosage concentration basis again with 15000u/ml (every 1500U).Began to guinea pig eye cornea recombinant human lysozyme eye drop every day three times, one of each every eye, perusal Cavia porcellus metamorphosis situation and time at the 5th day.
Experimental result administration recombinant human lysozyme eye drop second day, the middle and high dosage group of administration recombinant human lysozyme eye drop therapeutic effect shows especially, and Cavia porcellus cornea redness alleviates.Administration recombinant human lysozyme eye drop the 3rd day, the therapeutic effect that takes a turn for the better also appears in administration recombinant human lysozyme eye drop low dose group.The improvement of all disappearing of administration recombinant human lysozyme eye drop the 6th day, basic, normal, high group of Cavia porcellus keratitis of administration model and redness.Model control group Cavia porcellus cornea redness, cornea inflammation increase the weight of and ulcer occurs.An administration recombinant human lysozyme group and a model group have obvious difference.See Table 4, see Table 5.
Table 4
Number of animals
Group medication medication in first day medication in second day the 3rd day
(only)
The red and swollen cornea of cornea
The red and swollen keratitis of the red and swollen keratitis cornea of 5 corneas of model group contrast
Scorching
The red and swollen cornea of cornea
5 rednesses of model high dose group alleviate redness and alleviate
Scorching
The red and swollen cornea of cornea
5 rednesses of dosage group alleviate redness and alleviate in the model
Scorching
The red and swollen cornea of cornea
The red and swollen keratitis redness of 5 corneas of model low dose group alleviates
Scorching
Table 5
Number of animals
Group medication medication in the 4th day medication in the 5th day the 6th day
(only)
Cornea redness, keratitis cornea redness, keratitis
The red and swollen keratitis of 5 corneas of model group contrast
The corneal ulcer corneal ulcer
The red and swollen keratitis of cornea
5 cornea inflammation of model high dose group alleviate recovers normal
Disappear
The red and swollen keratitis of cornea
5 cornea inflammation of dosage group alleviate recovery normally in the model
Disappear
The red and swollen keratitis of cornea
5 cornea inflammation of model low dose group alleviate recovers normal
Disappear
Comprehensive above interpretation, test recombinant human lysozyme eye drop is to anti-I type herpesvirus 100～1000TCID
50Infect the eye keratitis of Cavia porcellus, good therapeutic effect is arranged.Its therapeutic effect and using dosage have obvious dose-effect relationship.
Two, usage and consumption:
Each every eye drips every day 3～6 times.
The specific embodiment:
Embodiment 1
Gene recombinant human lysozyme is as the criterion to prepare 200 milliliters of culture medium, with 6 milliliters of phosphoric acid, magnesium sulfate 3 grams, potassium sulfate 4 grams, potassium hydroxide 1 gram, calcium sulfate 1.5 grams, adding distil water to 200 milliliter, inoculation glycerol pipe seed behind the autoclaving, the shaking table revolution is that per minute 250 changes, and cultivation temperature is 20-35 ℃, cultivates 36-48 hour on the constant temperature bed.Carry out seed tank culture, produce a jar cultivation at last.The culture fluid that fermentation expression is finished extracts purification, and the protein concentrated solution that extracts purification is carried out lyophilizing, surveys albumen and surveys active the preservation.Pharmaceutical factory in GMP compatible, by the pharmacy rules, the gene recombinant human lysozyme of purity 95% is made 30000U/mL, phosphate buffer 1 0～20mM (pH6.5～7.5) 80%, 5～25% propylene glycol, 5/10000ths Tween 80s, mix homogenizing at normal temperatures, make eye drop.
Embodiment 2
According to embodiment 1 described preparation method, pharmaceutical factory in GMP compatible, by the pharmacy rules gene recombinant human lysozyme of purity 95% is made 1,000,000 U/mL, phosphate buffer 1 0～20mM (pH6.5～7.5) 80%, 5～25% propylene glycol, 5/10000ths Tween 80s, mix homogenizing at normal temperatures, make eye drop.
Embodiment 3
According to embodiment 1 described preparation method, pharmaceutical factory in GMP compatible, by the pharmacy rules gene recombinant human lysozyme of purity 95% is made 2,000,000 U/mL, phosphate buffer 1 0～20mM (pH6.5～7.5) 80%, 5～25% propylene glycol, 5/10000ths Tween 80s, mix homogenizing at normal temperatures, make eye drop.
Claims (9)
1, the application of human lysozyme in the medicine of the various oculopathy of preparation treatment.
2, the application of human lysozyme in the medicine of various pink eye disease that preparation treatment is caused by antibacterial, chlamydia, virus and fastbacteria, ophthalmia, keratitis, corneal ulcer disease.
3, the application of human lysozyme in the medicine of various pink eye disease that preparation treatment is caused by Salmonella and chlamydia, ophthalmia, keratitis, corneal ulcer disease.
4, the application of human lysozyme in the medicine of various pink eye disease that the herpes disease that preparation treatment is caused by I type herpesvirus causes, ophthalmia, keratitis, corneal ulcer disease.
5, the application of human lysozyme in the medicine that causes various pink eye disease, ophthalmia, keratitis, corneal ulcer disease that the preparation treatment is caused by Coxsackie virus.
6, the application of human lysozyme in the medicine that causes various pink eye disease, ophthalmia, keratitis, corneal ulcer disease that the preparation treatment is caused by line virus.
7, human lysozyme is read application in the coccigenic medicine that causes various pink eye disease, ophthalmia, keratitis, corneal ulcer disease in preparation treatment by pathogenicity.
8, according to the application of the described human lysozyme of one of claim 1-7 in the medicine of the various oculopathy of preparation treatment, it is characterized in that: medicine is an eye drop, contains active 300U～3,000,000 U/mL human lysozymes.
9, according to the application of the described human lysozyme of one of claim 1-7 in the medicine of the various oculopathy of preparation treatment, it is characterized in that: human lysozyme is that the recombinant human lysozyme of gene engineering expression, the aminoterminal of gene engineering expression human lysozyme have human lysozyme, gene engineering expression or the chemosynthesis mutant recombinant human lysozyme of (glutamic acid-alanine) 2 or (glutamic acid-alanine) 3 modifications.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2134355A4 (en) * | 2007-03-02 | 2012-01-11 | Saint Simeon Lda | Novel ophthalmic compositions containing human recombinant lysozyme and use thereof for treating eye conditions and as contact lens solutions |
| CN103182074A (en) * | 2011-12-30 | 2013-07-03 | 沈阳兴齐眼药股份有限公司 | Ophthalmic preparation containing lysozyme |
| CN105999239A (en) * | 2016-05-11 | 2016-10-12 | 山东司邦得制药有限公司 | Lysozyme hydrochloride eye drops and preparation method and application thereof |
-
2004
- 2004-06-21 CN CNB2004100208152A patent/CN100536916C/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2134355A4 (en) * | 2007-03-02 | 2012-01-11 | Saint Simeon Lda | Novel ophthalmic compositions containing human recombinant lysozyme and use thereof for treating eye conditions and as contact lens solutions |
| CN103182074A (en) * | 2011-12-30 | 2013-07-03 | 沈阳兴齐眼药股份有限公司 | Ophthalmic preparation containing lysozyme |
| CN103182074B (en) * | 2011-12-30 | 2016-03-30 | 沈阳兴齐眼药股份有限公司 | A kind of ophthalmic preparation containing lysozyme |
| CN105999239A (en) * | 2016-05-11 | 2016-10-12 | 山东司邦得制药有限公司 | Lysozyme hydrochloride eye drops and preparation method and application thereof |
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| CN100536916C (en) | 2009-09-09 |
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