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CN1593652A - Usage of human lysozyme in preparation of dermics - Google Patents

Usage of human lysozyme in preparation of dermics Download PDF

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Publication number
CN1593652A
CN1593652A CN 200410020816 CN200410020816A CN1593652A CN 1593652 A CN1593652 A CN 1593652A CN 200410020816 CN200410020816 CN 200410020816 CN 200410020816 A CN200410020816 A CN 200410020816A CN 1593652 A CN1593652 A CN 1593652A
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human lysozyme
medicine
lysozyme
application
preparation
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张华�
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Abstract

The invention relates to the use of human lysozyme in pharmaceutical industry, in particular the use in preparing medicines for treating dermatosis, infection and ulceration caused by bacteria, fungus, viruses, allergic reaction, and occupational diseases, physical diseases, nerve dysfunctional diseases, and immunity diseases. The invention exploits the novel use of human lysozyme in medical treatment, the gene recombination lysozyme has rich source, the invention also features simple process for manufacturing, and ease in use.

Description

The new purposes of human lysozyme in the dermopathic medicine of preparation treatment
Technical field:
The present invention relates to the purposes of human lysozyme, the particularly human lysozyme new purposes in pharmaceutical field.
Background technology:
Dermatosis causes by multiple factor, and the internal and external factor of the generation of these diseases and development and body, host's factors such as reaction condition, heredity and individual quality are relevant.Dermatosis is slightly very easily out in the cold sometimes, can involve internal organs sometimes, as early diagnosis and early treatment can not cause serious consequence.A lot of dermatosiss are bacterial, and antibiosis have better curative effect.Antibiotic has been created many medical science miracles, makes the no track of numerous disease disappearance, as pneumonia, meningitis, lochiopyra, septicemia, tuberculosis etc.But today of 21 century, antibiotic external causes a large amount of fastbacteria to produce, and the development of fastbacteria makes us startling.As penicillin-fast streptococcus pneumoniae, the past is all very sensitive to medicines such as penicillin, erythromycin, sulfanilamide, now almost " armsproof ".Klebsiella pneumonia to 16 kinds of antibiotic drug resistance of top grade such as zinacef, fortums up to 51.85%-100%.Staphylococcus aureus (MRSA) pasts medical help except that vancomycin, and pseudomonas aeruginosa also pasts medical help clinically substantially.Be badly in need of at present that exploitation is a kind of all effectively not to produce chemical sproof novel " Zyvox " at different Resistant strains and be used for clinical treatment.
Summary of the invention:
The purpose of this invention is to provide the new purposes of a kind of human lysozyme in the dermopathic medicine of preparation treatment, effectively at the dermatosis that causes by antibacterial, fungus, virus, allergy, occupational, chemical, physical property, delayed ischemic neurological deficits, immunity, disease infects, festers.
In fact, the present invention relates to the application of human lysozyme in the dermopathic medicine of preparation treatment.
Relate to the application of human lysozyme in the medicine of the dermatosis that the preparation treatment is caused by herpes simplex virus and varicella zoster virus.
Relating to human lysozyme treats by the application in bacterial skin impetigo and the impetiginous medicine of newborn skin in preparation.
Relating to human lysozyme treats by the application in the bacterial ecthymatous medicine in preparation.
Relating to human lysozyme treats by the application in the medicine of bacterial pyoderma in preparation.
Relate to human lysozyme and cause application in contact dermatitis and the urticarial medicine by allergy in preparation treatment.
Relate to the application of human lysozyme in the medicine of pruritus that preparation treatment is caused by delayed ischemic neurological deficits and prurigo nodularis disease.
Relate to the application of human lysozyme in the medicine that the silver that the preparation treatment is caused by the immunity of organism disease is considered to be worth doing, the palm is wasted time impetigo and pemphigus.
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 perhaps claims: 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.Lysozyme removes directly cracking antibacterial, can also regulate the effect of neutrophilic granulocyte as immunomodulator, and protection inflammation part normal structure plays negative feedback (Leo IG, et al.J.Clin.Invist.1979 in regulating inflammatory reaction; 64:222).It may combine by the polysaccharide part with polymorphonuclear granulocyte film surface; suppressing polymorphonuclear granulocyte moves to inflammation part; and the inflammation part of can decaying is by the effect of the superoxides that polymorphonuclear granulocyte produced, the tissue injury that the protection body excessively takes place owing to immunne response when inflammatory reaction.
Contain active 300U~3,000,000 U/ml human lysozymes in the described medicine.Can make spray, drop, milk, Emulsion, cream, cream etc. according to general formulation method.
The present invention has excavated new medical application to gene recombinant human lysozyme, has opened up a new application.Evident in efficacy and safe without toxic side effect has good prospect in medicine.And the gene recombinant human lysozyme source is abundant, and preparation technology is simple, and is easy to use.
In order to understand essence of the present invention better, will its new purposes in pharmaceutical field be described with the pharmacological testing and the result of gene recombinant human lysozyme below.
To prepare 200 milliliters of culture medium is benchmark, uses H 3PO 4(phosphoric acid) 4-8 milliliter, MgSO 4(magnesium sulfate) 1-5 gram, K 2SO 4(potassium sulfate) 2-6 gram, KOH (potassium hydroxide) 1-3 gram, CaSO 42H 2O (calcium sulfate) 1-3.5 gram, adding distil water to 200 milliliter, inoculation glycerol pipe seed behind the autoclaving, the shaking table revolution is that per minute 250 changes, cultivation temperature is 20-35 ℃, cultivates 36-48 hour on the constant temperature bed.Carry out seed tank culture again, produce a jar cultivation then.The Pichia sp. bacteria culture fluid filtering and concentrating that will contain the human lysozyme of gene engineering expression, lyophilization is measured protein content, purity and lysozyme activity (purity 99.8% active 80,000 units/ml) preserve.With qualified gene recombinant human lysozyme lyophilized powder soluble in waterly make spray, drop 30000U/ml is standby; It is standby to make cream (frost) agent 30000U/g.
One, animal model test:
A, human lysozyme are to the analgesic activity (rat tail-flick method) of rat
Select 50 of 120-150 gram SD healthy rats for use, 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.TF-photo-thermal dolorimeter (light source is 12V, 50W) under the thermal exposure 10 second internal reaction rat, 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 ± 7.09 ± 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
The molten 30 10 5.7 12.3** 15.0** 13.8* 13.6* 11.7 of chicken
Bacterium enzyme ± 1.64 ± 4.6 ± 7.99 ± 7.99 ± 7.6 ± 6.9
The molten 120 10 5.8 14.2** 15.7** 14.5** 14.3* 13.5 of people
Bacterium enzyme ± 1.23 ± 6.54 ± 6.78 ± 7.11 ± 7.44 ± 7.69
The molten 60 10 5.6 12.6** 14.3** 14.0* 13.8* 12.5 of people
Bacterium enzyme ± 1.89 ± 4.05 ± 6.69 ± 6.9 ± 6.72 ± 6.65
The molten 30 10 5.7 10.0* 12.0* 10.7 10.1 11.2 of people
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.
B, 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, be divided into 5 groups at random, 10 every group.Animal freely drinks water.The test chamber temperature is controlled at about 22-28 ℃.Animal feed rat standard feed.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, 30 μ L and causes inflammation, (Oleum Tiglii, light brown oily liquid, pharmaceutical grade, lot number 000309 is produced with factory by the precious crude drug of Jishui County, Jiangxi China.Face with the preceding ethanol of using: water: it is standby that 25: 5: 70 mixed solvents of ether are mixed with 1% concentration.) 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 respectively after half an hour.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).The result shows 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.
C, gene recombinant human lysozyme (HLZ) inside and outside antibacterial action are estimated
1 vitro antibacterial activity
Medicine 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 be the 2001.4-2002.4 month 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:
1, Tris-HCl buffer: 0.1M Tris 100ml, 0.1M HCl 70ml, HighWater 800ml surveys pH value, transfers to PH7.2 with HCl solution, adds High Water to 1000ml.
2, a Tris-HCl agar culture medium: in the Tris-Cl buffer, add 116 ℃ of agaroses! Use the sterilization back.
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:
(1) 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.
(2) 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 (the mg/ml of 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 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
D, recombinant human lysozyme suppress Cavia porcellus bovine serum albumin anaphylaxis test model
Select 20 of healthy guinea pigs about body weight 250 gram for use, male, divide equally four groups at random, 5 every group, carry out the modeling of intravenous injection bovine serum albumin.Be divided into four groups more at random after 20 mices after the modeling are reconsolidated, set up three administration groups and a model group respectively.The concentration of medicinal liquid is 15000u/ml during the clinical treatment by aerosol of recombinant human lysozyme, and tentatively drafting dosage is 15000u/ time, every day 3~5 times, promptly 0.5mg/ time/day.(by body surface area calculate to Cavia porcellus dosage be 1.3 * 10 -3Mg/ is (250g weighing machine) only, because people's ventilation 9000ml/ divides 290 times that are equivalent to Cavia porcellus ventilation 31ml/ branch, human single dose 0.5mg/70kg is equivalent to mice dosage 1.3 * 10 -3280 times of mg/250g, more than the two is comparatively approaching).So set and mice carried out nebulae inhalation with clinical human concentration 0.5mg/ml, other considers that clinical human aerosol suction recombinant human lysozyme gas effciency is higher, and mice can only lean on self breathing suction pastille gas in atomization cylinder, so have only the respiratory time of prolongation just can guarantee to suck dose, be set at one hour.At 15000u/ml is liquor strength when preparing 30000u/ml, 60000u/ml two concentration as middle and high dosage group treatment on the low dosage concentration basis again.Gave previous hour of every Cavia porcellus intravenous injection bovine serum albumin at the 21 day, Cavia porcellus is placed the glass exsiccator after, atomize the recombinant human lysozyme liquid of variable concentrations to the Cavia porcellus prevention that atomizes with the PAR1.BOY compression sprayer.Give every Cavia porcellus intravenous injection bovine serum albumin 1mg then, between perusal Cavia porcellus metamorphosis situation and dead quantity are timely.
Behind the experimental result Cavia porcellus intravenous injection bovine serum albumin 10 second model group cough, tachypnea occur and scratch the lip phenomenon.The model group Cavia porcellus is all dead after 30 seconds.The still all survivals of Cavia porcellus in 30 hours of administration recombinant human lysozyme group.An administration recombinant human lysozyme group and a model group have obvious difference.See Table 4, see Table 5.
Table 4 aerosol was treated the 1st day
Number of animals
After the group medication after the medication in 10 seconds after the medication in 30 seconds 60 seconds
(only)
5400 of model control group
5555 of model high dose group
The dosage group is 5555 in the model
5555 of model low dose group
Table 5 aerosol treatment second day
Number of animals
After the group medication after the medication in 10 hours after the medication in 20 hours 30 hours
(only)
0000 of model control group
5555 of model high dose group
The dosage group is 5555 in the model
5555 of model low dose group
Comprehensive above experimental result shows, Cavia porcellus intravenous injection bovine serum albumin is carried out anaphylaxis test recombinant human lysozyme atomizing protection, and good protection preventive effect is arranged.Its protection preventive effect and using dosage do not have obvious dose-effect relationship.
E, recombinant human lysozyme anti-herpesvirus suppress Cavia porcellus keratitis test model
Select 20 of body weight 250 gram left and right sides healthy guinea pigs for use, male, 20 Cavia porcelluss are divided 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, provide I type herpesvirus 100~1000TCID with Sichuan Industrial Institute of Antibiotics's Experimental Animal Center 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 6, see Table 7.
Table 6
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 7
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 experimental result shows that 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.
Six, usage and consumption:
1, spray: directly nebulization is in the focus topical, and every day 1~6 time, each 1500u~30000u adult is identical with children's.
2, milk, Emulsion, drop: directly act on focus topical every day 1~6 time, each 1500u~30000u.
3, cream: directly act on focus topical 1500u~30000u/g external, every day 2~3 times, press the administration of lesions position size at every turn.
The specific embodiment:
Embodiment 1
The gene recombinant human lysozyme of purity 85%-99% 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, (in the pharmaceutical factory of GMP compatible, finishing by the pharmacy rules) makes spray.
Embodiment 2
With the gene recombinant human lysozyme of purity 85%-99% make, 300U~300U ten thousand/ml, phosphate buffer 10 (pH6.0) 80%, 5~20% propylene glycol, 6%2-pyrrolidone-5-carboxylic acid sodium, 0.9% water solublity azone, (, finishing) drop of 3/10000ths Tween 80s, milk, Emulsion by the pharmacy rules in the pharmaceutical factory of GMP compatible.
Embodiment 3
With the gene recombinant human lysozyme of 85%-99% make, 300U~300U ten thousand/ml/g, phosphate buffer 20mM (pH7.0) 85%, 5~20% propylene glycol, 3/10000ths Tween 80s, carbomer 1%, essence 0.3% (, finishing) cream by the pharmacy rules in the pharmaceutical factory of GMP compatible.

Claims (10)

1, the application of human lysozyme in the dermopathic medicine of preparation treatment.
2, the application of human lysozyme in the medicine of the dermatosis that the preparation treatment is caused by herpes simplex virus and varicella zoster virus.
3, human lysozyme is treated by the application in bacterial skin impetigo and the impetiginous medicine of newborn skin in preparation.
4, human lysozyme is treated by the application in the bacterial ecthymatous medicine in preparation.
5, human lysozyme is treated by the application in the medicine of bacterial pyoderma in preparation.
6, human lysozyme is caused application in contact dermatitis and the urticarial medicine in preparation treatment by allergy.
7, the application of human lysozyme in the medicine of pruritus that preparation treatment is caused by delayed ischemic neurological deficits and prurigo nodularis disease.
8, the application of human lysozyme in the medicine that the silver that the preparation treatment is caused by the immunity of organism disease is considered to be worth doing, the palm is wasted time impetigo and pemphigus.
9, according to the new purposes of the described human lysozyme of one of claim 1-8 in the dermopathic medicine of preparation treatment, it is characterized in that: medicine is spray, drop, milk, Emulsion or cream, cream.
10, according to the new purposes of the described human lysozyme of one of claim 1-8 in the dermopathic medicine 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 (glutamic acid-alanine) 2Or (glutamic acid-alanine) 3Human lysozyme, gene engineering expression or the chemosynthesis mutant recombinant human lysozyme modified.
CN 200410020816 2004-06-21 2004-06-21 Usage of human lysozyme in preparation of dermics Pending CN1593652A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079288A1 (en) * 2005-01-26 2006-08-03 Mi An Use of human lysozyme for preparing cosmetics against acne
CN100335127C (en) * 2005-05-20 2007-09-05 张华� Powder of human lysozyme, its production method and application
CN105770869A (en) * 2016-03-11 2016-07-20 浙江艾杰斯生物科技有限公司 Medicinal composition for treating psoriasis
CN105327342B (en) * 2015-11-20 2018-08-28 重庆苗秀生物科技有限公司 A kind of paste and preparation method thereof for treating skin eczema and insect dermatitis
WO2021012789A1 (en) * 2019-07-19 2021-01-28 广州新创忆药物临床研究有限公司 Pharmaceutical composition containing lysozyme and use thereof

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006079288A1 (en) * 2005-01-26 2006-08-03 Mi An Use of human lysozyme for preparing cosmetics against acne
CN100335127C (en) * 2005-05-20 2007-09-05 张华� Powder of human lysozyme, its production method and application
CN105327342B (en) * 2015-11-20 2018-08-28 重庆苗秀生物科技有限公司 A kind of paste and preparation method thereof for treating skin eczema and insect dermatitis
CN105770869A (en) * 2016-03-11 2016-07-20 浙江艾杰斯生物科技有限公司 Medicinal composition for treating psoriasis
CN112915198A (en) * 2016-03-11 2021-06-08 浙江艾杰斯生物科技有限公司 An ointment containing lysozyme for treating psoriasis
CN105770869B (en) * 2016-03-11 2021-06-08 浙江艾杰斯生物科技有限公司 Pharmaceutical composition for treating psoriasis
CN112915198B (en) * 2016-03-11 2022-05-24 浙江艾杰斯生物科技有限公司 An ointment containing lysozyme for treating psoriasis
WO2021012789A1 (en) * 2019-07-19 2021-01-28 广州新创忆药物临床研究有限公司 Pharmaceutical composition containing lysozyme and use thereof
CN112533628A (en) * 2019-07-19 2021-03-19 广州新创忆药物临床研究有限公司 Pharmaceutical composition containing lysozyme and application thereof

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