WO2004067735A1

WO2004067735A1 - Human genic recombination lysozyme it’s a new use about preparation anti-bacteria resistant drug

Info

Publication number: WO2004067735A1
Application number: PCT/CN2004/000014
Authority: WO
Inventors: Mi An; Hua Zhang; Yuan Li; Xian Lu An
Original assignee: Changchun Qilong Technology Research Institution
Priority date: 2003-01-09
Filing date: 2004-01-05
Publication date: 2004-08-12
Also published as: CN1439427A; CN1227028C

Abstract

The present invention relates to a new use of human genic recombination lysozyme on the bacteria resistant drug, it to apply for as a drug to treat pneumonia, tracheitis, urethritis, cystitis and so on, because of bacteria-resistant. It can be made to an injection, tablet, capsule, drops, electuary and so on. The present invention explores a new medical treatment use about human genic recombination lysozyme, developing a new application area. It is safety, no side effect, and has a fine foreground in medicine. Also, the source of human genic recombination lysozyme is enriching, and the preparation is simple, expedience put to use.

Description

Technical field:

The invention relates to a new use of genetically recombined human lysozyme, in particular to a new use of genetically recombined human lysozyme in the field of pharmaceuticals against drug-resistant bacteria.

Background technique:

Antibiotics have created many medical miracles, making many diseases disappear, such as pneumonia, meningitis, puerperal fever, sepsis, tuberculosis and so on. Today in the 21st century, the development of drug-resistant bacteria is startling. For example, penicillin-resistant Streptococcus pneumoniae used to be very sensitive to drugs such as penicillin, erythromycin, and sulfa. Now it is almost "inaccessible". The resistance of Klebsiella pneumoniae to 16 high-grade antibiotics, such as Xilixin and Fudaxin, is as high as 51.85% to 100%. Methicillin-resistant Staphylococcus aureus (MRSA) has no cure except vancomycin. From the history of bacteria resistance development, it can be seen that after the emergence of a new antibiotic, a batch of drug-resistant strains appeared. It usually takes about 10 years to develop a new antibiotic, and the generation of drug-resistant bacteria takes only 2 years. The development of antibiotics is far behind the development of drug-resistant bacteria. There is an urgent need to develop a "super antibiotic" that is effective against different resistant strains for clinical treatment. '

Summary of the invention:

The object of the present invention is to provide a new use of genetically recombinant human lysozyme for resistance to drug-resistant bacteria, including resistance to Gram-positive bacteria and Gram-negative bacteria. The so-called drug-resistant bacteria is the resistance of the bacteria to the drug. After the bacteria has been in contact with the drug for many times, the sensitivity to the drug decreases or even disappears, resulting in the drug's sensitivity to bacteria being reduced or even ineffective. Its

1

Confirm this Medium resistant bacteria include Staphylococcus aureus MRS A, Staphylococcus epidermidis MRSE, Streptococcus pneumoniae, Streptococcus hemolyticus, Enterococcus, Escherichia coli, Klebsiella pneumoniae, Propionibacterium, Acinetobacter, Citrus E. coli, Serratia, Enterobacter cloacae, Pseudomonas aeruginosa, Candida albicans, etc. It is mainly resistant to the following drugs: clarithromycin, roxithromycin, amoxicillin, vancomycin, decomycin, broad-spectrum penicillin, methoxoline, cephalosporins, penicillin sodium, penicillin potassium , Sulfa.

Specific resistance standards are: (diameter of inhibition zone mm)

Golden yellow epidermal pneumonia, intestinal lysing, large lungs, citrus sand, shade, copper, white staphylococcus, streptococcus, and blood enteritis

MRSA MRSE Streptococcus sp

Carat ^ 1.2 07 12 si <: <: <:

15 14 10 12 17 17 18 12 10 1.2 12 Luo Hong 1.2 08 17;;: :: <; g: ;;: c

Amomycin 17 14 12 12 17 17 16 17 12 1.2 17 Amo 1.5 10 19 :: :::;::: 5; <: <: Xilin 19 16 15 15 22 22 21 19 15 1.5 19 Vanguard 3.2 27 32: sc; sc <:; <: <;:;; ¾ <: zero prime 34 37 30 32 35 35 33 32 30 3.2 32 Deco 30 25 <:: <: <;:: ξ :: c: ;;; < : ¾ <ς :: Mycin 3.2 33 32 28 28 27 33 34 32 28 30 32 Qingxiao 27 22 23 ¾::;; ¾ ::;; c:: ¾; Sodium 27 26 18 25 32 28 27 23 18 27 23 Penicillium 27 22 23 ¾:: si; <: <:;;:; <;: <;:;;: Elemental potassium 27 26 25 25 32 28 27 23 25 27 23 Broad spectrum 25 20 25 <: ; <:: G;: ξ: ξ <:: ξ s Penicillium 24 26 25 25 32 28 27 25 25 25 25

Cephalosporin 27 22 22 <:;: c <<:: <; <c: Class 27 24 25 23 24 26 26 22 25 27 22 Methoxy 28 23 23 :: ζ <; :: ζ <:: <:

Oxyline 22 30 05 26 32 27 27 23 05 28 23 category

Sulfa 9 10 9 :: ζ: 5: 9 <: ¾ <; <;

Class 10 12 15 11 18 15 16 18 18 13 In fact, the present invention relates to the application of genetically recombined human lysozyme as a medicine for treating or preventing pneumonia caused by drug-resistant Streptococcus pneumoniae and Klebsiella pneumoniae.

The invention relates to the application of gene recombinant human lysozyme as a medicine for treating or preventing bronchitis caused by drug-resistant Streptococcus pneumoniae and Klebsiella pneumoniae.

The invention relates to the application of genetically recombinant human lysozyme as a medicine for treating or preventing nephritis caused by resistant Escherichia coli.

The invention relates to the application of gene recombinant human lysozyme as a medicine for treating or preventing pharyngitis caused by resistant hemolytic streptococcus and Staphylococcus aureus MRSA.

The invention relates to the application of gene recombinant human lysozyme as a medicine for treating or preventing urethritis caused by drug-resistant Serratia and Escherichia coli.

Recombinant human lysozyme is involved in the preparation of medicines used to treat or prevent uterine inflammation caused by drug-resistant Serratia and Escherichia coli, Candida albicans.

The invention relates to the application of genetically recombined human lysozyme as a medicine for treating or preventing cystitis caused by resistant Escherichia coli.

The invention relates to the application of genetically recombined human lysozyme as a medicine for treating or preventing vaginitis caused by drug-resistant Serratia and Escherichia coli, Candida albicans.

The invention relates to the application of genetically recombinant human lysozyme as a medicine for treating or preventing flat adenitis caused by drug-resistant Staphylococcus aureus MRSA and Staphylococcus epidermidis MRSE.

The invention relates to the application of genetically recombinant human lysozyme as a medicine for treating or preventing stomatitis caused by drug-resistant Staphylococcus aureus MRSA and Staphylococcus epidermidis MRSE.

The invention relates to the application of gene recombinant human lysozyme as a medicine for treating or preventing otitis media caused by resistant hemolytic streptococcus. The invention relates to the application of gene recombinant human lysozyme as a medicine for treating or preventing acne caused by drug-resistant propionibacterium.

Recombinant human lysozyme involved as a preparation for treatment by resistant Staphylococcus aureus

Application of medicines caused by MRSA or preventing rhinitis

In order to better understand the essence of the present invention, the pharmacological tests and results of the recombinant human lysozyme will be used to explain its new application in the pharmaceutical field.

Genetically recombined human lysozyme is based on the preparation of 200 ml of culture medium, using 6 ml of phosphate, 3 g of magnesium sulfate, 4 g of potassium sulfate, 1 g of potassium hydroxide, 1.5 g of calcium sulfate, and adding distilled water to 200 ml. After autoclaving Inoculate glycerine tube seeds with a shaker speed of 250 revolutions per minute, a culture temperature of 20-35 ° C, and a 36-48 hour culture on a constant temperature bed. Seed tank culture was performed, and finally, production tank culture was performed. Extraction and purification of the fermentation broth was performed. The concentrated protein extract was lyophilized, and the protein was tested for protein activity. Dissolve qualified genetically recombinant human lysozyme lyophilized powder in water to prepare injections for use at 30,000 U / ml for later use; prepare sprays and drops for use at 30,000 U / ml for future use; prepare pastes (cream) for use at 30,000 U / g for future use. Prepared dry powder granules 30000U / g for later use.

Model test of a pair of mice:

A. Clarithromycin inhibition zone for drug-resistant pneumococcus 12. Preparation of mouse pneumonia model caused by drug-resistant pneumococcal upper respiratory tract infection:

Ten healthy Kunming mice were selected, male and female, weighing 18-22 grams. Pick a certain amount of drug-resistant pneumococcus to infect the upper respiratory tract of the mice by spraying, causing 10 mice with pneumonia, and intravenous injection after infection The test drug was 30,000U / ml / 20g mice, observed for one week, and administered three times a day to record the effective rate of the mice. The experimental results show that the gene recombinant human lysozyme pairs Pneumonia in mice caused by drug-resistant pneumococcal upper respiratory tract infection has a significant effect. The results are as follows:

B. Antibacterial staphylococcus amoxicillin zone of inhibition 15. Preparation of model of bronchitis and bronchitis in mice caused by drug-resistant Staphylococcus aureus upper respiratory tract infection-20 healthy Kunming mice, half male and half male, weighing 18-22 grams, were divided into two groups of 10 mice each, Select a certain amount of drug-resistant Staphylococcus aureus to infect 10 mice with tracheitis and 10 bronchitis by spraying the upper respiratory tract. Immediately after the infection, the test drug 30000U / ml / 20g was weighed, and observed for one week. Dosing three times a day, the effectiveness of the mice was recorded. The experimental results show that the recombinant human lysozyme has a significant effect on tracheitis and bronchitis in mice caused by drug-resistant Staphylococcus aureus upper respiratory tract infection. The results are as follows:

C. Preparation of pyelonephritis in mice caused by urethral infection of drug-resistant E.coli cephalosporins 25

Ten healthy Kunming mice were selected, half male and half male, weighing 18-22 grams. A certain amount of drug-resistant E. coli was selected to inject 10 mouse kidney nephritis by injection, and intravenous injection was performed immediately after infection. The drug weighs 30000U / ml / 20g mice. Observe for one week and administer it three times a day to record the effective rate of the mice. The experimental results show that the recombinant human lysozyme has a significant effect on pyelonephritis in mice caused by drug-resistant E. coli urethral infection. The results are shown in the table below-Diseased test animals (only) 10 7 3 0 100

D. Antibiotic-resistant streptococcus erythromycin inhibitory zone 12. Cephalosporin inhibitory zone 2.5 The preparation of acute and chronic suppurative laryngitis model in mice caused by drug-resistant streptococcus pyogenes upper respiratory tract infection:

Ten healthy Kunming mice, half male and half male, weighing 18-22 grams, were selected. A certain amount of drug-resistant Streptococcus pyogenes was used to infect the mice with spray and 10 cases of acute and chronic pharyngitis caused by upper respiratory infection. Immediately after the infection, the test drug 30000U / ml / 20g was injected into the mice, and the mice were observed three times a day for one week to record the effective rate of the mice. The experimental results show that the recombinant human lysozyme has obvious curative effect on acute and chronic suppurative laryngitis in mice caused by drug-resistant upper respiratory tract infection of Streptococcus pyogenes. The results are as follows:

E. Drug-resistant Escherichia coli penicillin sodium bacteriostatic zone 27, Preparation of mouse urethritis model caused by drug-resistant Escherichia coli urethral infection-10 healthy Kunming mice, half male and half male, weight 18 -22 grams, pick a certain amount of drug-resistant Escherichia coli to inject 10 mice with urethritis caused by injection, and immediately inject intravenously the test drug 30,000U / ml / 20g mouse weight after infection, observe for one week, three times a day Dosing was recorded and the mouse's effectiveness was recorded. The experimental results show that the recombinant human lysozyme has a significant effect on urethritis in mice caused by drug-resistant E. coli urethral infection. The results are as follows:

F. Drug-resistant Serratia penicillin potassium inhibitory zone 27 and Escherichia coli sulfonamide inhibitors Circle 0.5, Candida albicans decomycin bacteriostasis circle 3.0 Preparation of drug-resistant Serratia and Escherichia coli, Candida albicans intrauterine infection model in mice:

Ten healthy Kunming mice were selected, half male and half male, weighing 18-22 grams. Pick a certain amount of drug-resistant E. coli and inject 10 mice with uterine cavity infection by injection, causing veins immediately after infection. The test drug was injected with 30,000 Uml / 20g mouse weight, observed for one week, and administered three times a day to record the effective rate of the mice. The experimental results show that the recombinant human lysozyme has obvious curative effect on hysteritis in mice caused by drug-resistant E.coli uterine infection. The results are as follows:

G. Broad-spectrum penicillin inhibition zone of drug-resistant Escherichia coli 27. Preparation of mouse cystitis model caused by drug-resistant Escherichia coli urethral infection:

Ten healthy Kunming mice were selected, male and female, weighing 18-22 grams, and a certain amount of drug-resistant streptococci was used to infect the mouse bladder with injections to cause cystitis in mice. The injections were tested immediately after infection. The drug weighs 30000U / ml / 20g mice. Observe for one week and administer it three times a day to record the effective rate of the mice. The experimental results show that the recombinant human lysozyme has a significant effect on cystitis in mice caused by drug-resistant Streptococcus urinary tract infection. The results are as follows:

H. Broad-spectrum penicillin inhibition zone of drug-resistant Escherichia coli 27. Preparation of mouse vaginitis model caused by drug-resistant Escherichia coli infection:

Select 10 healthy Kunming mice, half male and half male, weighing 18-22 grams. Pick After the infection, the test drug 30000U / ml / 20g was injected intravenously, and the mice were observed three times a day for one week to observe the effective rate of the mice. The experimental results show that the recombinant human lysozyme has a significant effect on vaginitis in mice caused by drug-resistant Streptococcus infection. The results are as follows:

I. Preparation of drug-resistant Staphylococcus aureus MRSA methicillin-like inhibitory zone 2. 7, Preparation of a mouse model of striatum caused by drug-resistant Staphylococcus aureus MRSA upper respiratory tract infection =

Ten healthy Kunming mice, half male and half male, weighing 18-22 grams, were selected. A certain amount of Streptococcus pyogenes was sprayed to infect 10 mice with upper tract striatum caused by spray. The test drug was injected intravenously at a weight of 30,000u / ml / 20g, and the mice were observed three times a day for one week, and the effective rate of the mice was recorded. The experimental results show that the recombinant human lysozyme has obvious curative effect on striatum caused by Streptococcus pyogenes infection. The results are as follows:

J. Broad-spectrum penicillin inhibition zone of drug-resistant hemolytic streptococcus 27. Preparation of otitis media model in mice caused by drug-resistant hemolytic streptococcus upper respiratory tract infection:

Ten healthy Kunming mice, half male and half male, weighing 18-22 grams, were selected. A certain amount of Streptococcus pyogenes was used to infect 10 mice with otitis media in the upper respiratory tract by spraying, and the test drug 1500 was titrated after infection. The mice weighed 1 / 20g, observed for a week, and administered six times a day to record the effective rate of the mice. Experimental results show that recombinant human lysozyme is resistant to purulent streptococci Otitis caused by bacterial infection has obvious curative effect. The results are as follows:

The spray is directly sprayed on the lesion and administered locally, 1-6 times a day, each time 1500u, the same for adults and children,

The drops are 1-6 times a day, each time 1500u-3000u.

The ointment (cream) is 1500u-10000u / g for external use 2-3 times a day, each time according to the size of the lesion.

From the above results, it can be concluded that the advantages of the present invention are:

(1) The present invention discovers new medical uses of genetically recombinant human lysozyme, and opens up a new field of application.

(2) The recombinant human lysozyme of the present invention is safe and has no toxic or side effects, and has good medicinal properties.

(3) The present invention has rich sources of genetically recombinant human lysozyme, simple preparation process, and can be made into injection forms, tablets, capsules, sprays, drops, pastes, etc., and is convenient to use. Two usages and dosages:

Intravenous injection: 1 to 3 times a day for adults, 1 million to 3 million U each time, and 1 to 3 times a day for children, 500,000 U to 2 million U each time. Spray: Direct spraying is applied locally to the lesion, 1 to 6 times a day, 1500u to 30,000u each time. Adults are the same as children.

Drops: Locally administered directly to the lesion 1 to 6 times a day, 15001 each time! ~ 30000u. Ointment (cream): Direct application to the lesion for local administration 15001! ~ 30000u / g for external use, daily

2-3 times, each time according to the size of the lesion site.

Tablets and capsules: Adults 1 to 3 times a day, each time 1 million 11 to 3 million u. Children 1 to 3 times a day, 500,000 to 11 to 1.5 million each.

detailed description:

The following further describes the present invention in combination with the embodiments:

Example 1:

After autoclaving the culture medium, inoculate the human lysozyme strain, the rotation speed of the shaker is 250 revolutions per minute, the culture temperature is 20 ° C, and the culture is performed on a constant temperature bed for 36 hours. Seed tank culture is performed, and finally, production tank culture is performed. Extraction and purification of the culture solution after fermentation expression is completed, and the extracted and purified protein concentrate is lyophilized, and the protein activity is measured and stored. Take human lysozyme lyophilized powder 1%, phosphate buffer 5mM (pH5.0) 90%; made into injection, mainly used for resistance to diseases caused by Gram-positive bacteria and Gram-negative bacteria . Such as bronchitis, pneumonia and other diseases. '

Example 2:

After autoclaving the culture medium, inoculate the human lysozyme strain, incubate it on a constant temperature bed for 40 hours, and then purify the culture solution to prepare human lysozyme containing lyophilized powder 0.1%, phosphate buffer 20mM (pH7.0) 80%, 25% glycerol, 5 / 10,000 Tween 80 spray.

Example 3: After autoclaving the culture medium, inoculating a human lysozyme strain and incubating it on a constant temperature bed for 36 hours, and then purifying the culture solution to prepare a human lysozyme containing lyophilized powder 0.005%, phosphate buffer solution 10 (pH 6.0) 80%, 20% glycerol, 6% 2 monopyrrolidone-5 sodium carboxylate, 0.9% water-soluble azolone, 3 / 10,000 Tween 80 drops.

Example 4:

After autoclaving the culture medium, inoculating a human lysozyme strain and incubating it on a constant temperature bed for 48 hours, and then purifying the culture solution to prepare a lyophilized powder containing human lysozyme 0.1%, and a phosphate buffer solution 20mM (pH7.0) 8%, 20% glycerol, 1% Kappa resin, 0.3% ointment. Example 5:

After autoclaving the culture medium, inoculating a human lysozyme strain, incubating it on a constant temperature bed for 40 hours, and then purifying the culture solution into capsules and tablets. It contains human lysozyme lyophilized powder 10%, medicinal starch 50, medicinal dextrin 25%, phosphate buffer 10mM (pH6.0), and sucrose 5 ° /. .

u

Claims

Rights request

1. Application of genetically recombined human lysozyme in the preparation of medicines for treating pneumonia or preventing pneumonia caused by drug-resistant Streptococcus pneumoniae and Klebsiella pneumoniae.

2. Application of genetically recombined human lysozyme in the preparation of medicines for treating or preventing bronchitis and pharyngitis caused by drug-resistant hemolytic streptococcus and staphylococcus aureus MRSA.

3. Application of genetically recombined human lysozyme in the preparation of medicines caused by resistant Escherichia coli or prevention of nephritis.

4. Application of genetically recombinant human lysozyme in the preparation of medicines for treating or preventing cystitis and urethritis caused by drug-resistant Serratia and Escherichia coli.

5. Application of genetically recombined human lysozyme in the preparation of medicines for treating or preventing vaginitis and hysteritis caused by drug-resistant Serratia and Escherichia coli, Candida albicans.

6. Application of genetically recombined human lysozyme in the preparation of medicines for the treatment or prevention of flat adenitis and pharyngitis caused by drug-resistant Staphylococcus aureus MRSA and Staphylococcus epidermidis MRSE.

7. Application of genetically recombined human lysozyme in the preparation of a medicine for treating or preventing otitis media caused by resistant hemolytic streptococcus.

8. Application of genetically recombinant human lysozyme in the preparation of medicines for treating or preventing acne caused by resistant Propionibacterium.

9.Application of genetically recombinant human lysozyme in the preparation of medicines for the treatment or prevention of rhinitis caused by drug-resistant Staphylococcus aureus MRSA