WO2022042546A1 - Combined antibacterial pharmaceutical composition and use thereof - Google Patents

Abstract

A combined antibacterial pharmaceutical composition comprising berberine and an antibiotic. The antibiotic is one or two selected from sulbactam, tigecycline, amikacin, ciprofloxacin, meropenem and tetracycline. The drug combination of berberine and the antibiotic has a significant inhibitory effect on multi-drug resistant Acinetobacter baumannii. The antibacterial index is less than 1, which represents an additive or synergistic effect. The drug combination can effectively reduce the MIC value of the antibiotic, and can also resensitize the multi-drug resistant Acinetobacter baumannii to the antibiotic.

Description

A kind of combined antibacterial drug composition and application thereof

The present invention claims to enjoy the priority of the invention patent application submitted by the applicant to the State Intellectual Property Office of China on August 24, 2020, the application number is 202010868371.7, and the invention name is "a combined antibacterial drug composition and its application". The entirety of this prior application is incorporated herein by reference.

technical field

The invention relates to the field of pharmaceutical compositions, in particular to a combined antibacterial pharmaceutical composition comprising berberine and antibiotics and application thereof.

Background technique

Acinetobacter baumannii is a class of Gram-negative bacilli, non-motile, catalase-positive, oxidase-negative and strictly aerobic bacteria, which are considered to be one of the important pathogens causing nosocomial infections. Acinetobacter baumannii can cause a variety of acquired diseases, including pneumonia, skin and soft tissue infections, wound infections, urinary tract infections, etc. Among them, the infection with the highest mortality rate is pneumonia that requires ventilator-assisted treatment. Disease or patients who have undergone major surgical procedures are more common, accounting for about 40%-70%. Antibiotic resistance of Acinetobacter baumannii has increased significantly over the past decade, and a report from the Bacterial Resistance Surveillance System of the China Bacterial Resistance Surveillance Network shows that between 2005 and 2014, carbapenems The drug-resistant A. baumannii species increased from 31% to 66.7%. Considering the dilemma of increasing multidrug resistance in Multidrug-resistant Acinetobacter baumannii (MDRAb) and the relative difficulty of developing new antibiotics, combination therapy has emerged as an option to improve treatment outcomes and possibly prevent the emergence of new Potential options for drug resistance. At present, the clinical combination treatment plan mainly focuses on the combination of antibiotics and antibiotics. Based on the drug resistance spectrum of the infected bacteria, an appropriate combination of drugs can be selected.

Berberine, also known as berberine, is a pale yellow isoquinoline alkaloid that is widely present in the roots, rhizomes and bark of Berberis plants. Berberine hydrochloride, the most common form of berberine, is considered a drug with various medical potentials, including antibacterial, antiviral, antidiarrheal, antipyretic, and anti-inflammatory effects. In China, traditional Chinese medicines containing berberine, such as Coptis chinensis, are used as traditional antibacterial drugs for the treatment of gastroenteritis, abdominal pain and diarrhea. They have been used for more than 2000 years, and a large number of clinical research data have accumulated, indicating that berberine is a Safe clinical antibacterial agent.

With the increasing drug resistance of Acinetobacter baumannii, it has become resistant to traditional therapeutic antibiotics such as carbapenems, and it is difficult to use it alone. Some newer antibiotics, such as tigecycline or colistin, have relatively low resistance rates, but are expensive to treat and have certain side effects. At present, combination drugs are often used clinically to combat infections caused by multidrug-resistant Acinetobacter baumannii, but sometimes some strains have a relatively broad drug resistance spectrum, and the available antibiotic combinations are limited. Although there are related patent reports on the combination of meropenem and sulbactam or faropenem and tazobactam against multidrug-resistant Acinetobacter baumannii, the combination of the two has an unsatisfactory resensitization effect, and the antibiotic concentration is still in the bacterial resistance. within the scope of the medicine.

Therefore, it is of great clinical significance to find a reasonable combination of antibacterial drugs.

SUMMARY OF THE INVENTION

In order to improve the above-mentioned technical problems, the present invention provides a combined antibacterial drug composition, which comprises a combination of berberine and an antibiotic;

Wherein, the antibiotic is selected from one or two of sulbactam, tigecycline, amikacin, ciprofloxacin, meropenem and tetracycline.

According to an embodiment of the present invention, the berberine may be in the form of its pharmaceutically acceptable salt, such as berberine hydrochloride or berberine sulfate.

According to an embodiment of the present invention, the weight ratio of the berberine to the antibiotic may be (1-4096): 1, such as (1-1024): 1, such as (2-512: 1), exemplarily 4: 1, 8:1, 16:1, 32:1, 64:1, 128:1, 256:1, 512:1, or 4096:1.

According to an embodiment of the present invention, the combined antibacterial pharmaceutical composition comprises one of the following combinations:

(1) the combination of berberine hydrochloride and sulbactam, the weight ratio of the two can be (2-512): 1, such as (4-256): 1, (8-128): 1, exemplarily 2:1, 4:1, 8:1, 16:1, 32:1, 64:1 or 128:1;

(2) the combination of berberine hydrochloride and tigecycline, the weight ratio of the two can be (1-4096): 1, for example (1-256): 1, (2-128): 1, (4- 64):1, exemplarily 8:1, 16:1, 32:1, 64:1 or 4096:1;

(3) the combination of berberine hydrochloride and ciprofloxacin, the weight ratio of the two can be (1-1024):1, such as (2-512:1), exemplarily 4:1, 8:1 , 16:1, 32:1, 64:1, 128:1, 256:1 or 512:1;

(4) The combination of berberine hydrochloride and meropenem, the weight ratio of the two can be (2-512): 1, such as (4-256): 1, (8-128): 1, exemplarily 8 :1, 16:1, 32:1, 64:1 or 128:1;

(5) the combination of berberine hydrochloride and amikacin, the weight ratio of the two can be (1-256):1, such as (2-128:1), exemplarily 4:1, 8:1 , 16:1, 32:1, 64:1, 128:1 or 256:1;

(6) the combination of berberine hydrochloride and tetracycline, the weight ratio of the two can be (1-256): 1, such as (2-128: 1), exemplarily 4: 1, 8: 1, 16: 1, 32:1, 64:1, 128:1 or 256:1.

Preferably, the combined antibacterial pharmaceutical composition comprises one of the combinations of (1)-(4) above.

According to an embodiment of the present invention, the weight percentage of the berberine in the composition may be 5.0%-90.0%, for example, 10.0%-85.0%, 15.0%-80.0%, 20%-75.0%, 25% %-70%; the weight percentage of the antibiotic in the composition can be 0.01%-50.0%, such as 0.05%-45.0%, 0.1%-40.0%, 0.2%-35%, 0.3%-30% .

According to an embodiment of the present invention, the combined antibacterial pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or solvent.

According to an embodiment of the present invention, the combined antibacterial pharmaceutical composition can be prepared into oral preparations or injections by conventional methods, such as tablets, granules, capsules, sustained-release preparations, injections, powders or infusions, and the like.

The pharmaceutically acceptable carriers or excipients include but are not limited to fillers, disintegrants, binders, wetting agents, lubricants, flavoring agents, pH adjusters, isotonicity adjusters, antioxidants, Metal ion chelating agents, etc.

Examples of fillers useful in the present invention include, but are not limited to, microcrystalline cellulose, lactose, mannitol, starch or dextrin, or a combination of two or more thereof.

Examples of disintegrants that can be used in the present invention include, but are not limited to, sodium carboxymethyl starch, croscarmellose sodium, sodium carboxymethyl starch, hydroxypropyl starch, crospovidone, low-substituted hydroxypropyl based cellulose or cornstarch, or a combination of two or more thereof.

Examples of binders useful in the present invention include, but are not limited to, hypromellose, povidone, methylcellulose, or sodium carboxymethylcellulose, or a combination of two or more thereof.

Examples of wetting agents useful in the present invention include, but are not limited to, water or aqueous ethanol.

Examples of lubricants useful in the present invention include, but are not limited to, magnesium stearate, talc, or micronized silica gel, or a combination of two or more thereof.

Examples of flavoring agents that can be used in the present invention include, but are not limited to, sucrose, stevioside, or aspartame, or a combination of two or more thereof.

Examples of pH adjusting agents useful in the present invention include, but are not limited to, sodium hydroxide, hydrochloric acid, phosphoric acid, phosphate, citric acid, citrate, citric acid, citrate, acetic acid, acetate, glycine or lysine. amino acid, or a combination of two or more thereof; according to an embodiment of the present invention, the pH adjusting agent adjusts the pH value between 5.5 and 8.5, preferably between 6.5 and 7.5.

Examples of isotonicity modifiers useful in the present invention include, but are not limited to, sodium chloride, dextrose, glycerol, sorbitol, maltose, mannitol, or propylene glycol, or a combination of two or more thereof.

Examples of antioxidants useful in the present invention include, but are not limited to, sodium sulfite, sodium bisulfite, or sodium metabisulfite, or a combination of two or more thereof.

Examples of metal chelating agents useful in the present invention include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or ethylenediaminetetraacetic acid sodium salt (EDTA-Na), especially ethylenediaminetetraacetic acid disodium salt.

A pharmaceutically acceptable solvent useful in the present invention is water for injection.

The present invention also provides the application of the combined antibacterial drug composition for preparing antibacterial drugs. Preferably, the antibacterial drug is used against infections caused by Acinetobacter baumannii, especially multidrug-resistant Acinetobacter baumannii.

beneficial effect

1. Berberine hydrochloride, a monomer component of traditional Chinese medicine selected by the present invention, is cheap and easy to obtain, and has accumulated more than 2,000 years of clinical experience, and basically has no toxic and side effects.

2. When berberine hydrochloride is used in combination with antibiotics, the working concentration is between 16-512 mg/L. For different strains, the working concentration is determined according to the MIC of berberine hydrochloride. For strain MDRAb-1 (MIC of berberine hydrochloride is 256 mg/L), the working concentration is between 16-128 mg/L, and the effect is significant when the concentration is 128 mg/L; for strain MDRAb-2 (MIC of berberine hydrochloride is 256 mg/L) For 1024mg/L), the working concentration is between 128-512mg/L, and the effect is significant when the concentration is 256 and 512mg/L.

3. The combination of berberine and antibiotics has a significant effect against multidrug-resistant Acinetobacter baumannii, and the bacteriostatic index is less than 1, showing superposition or synergy; the MIC value of antibiotics is reduced by at least 2 times; it can also make multidrug-resistant Acinetobacter baumannii is resensitized to antibiotics. Therefore, the combined antibacterial drug composition of the present invention has a significant inhibitory effect on drug-resistant bacteria, especially multidrug-resistant Acinetobacter baumannii.

detailed description

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the intended protection scope of the present invention.

Unless otherwise stated, the starting materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Example

Determination of antibiotics (sulbactam, tigecycline, amikacin, ciprofloxacin, meropenem, tetracycline) and berberine hydrochloride against multi-drug resistant Bowman immobility by 96-well plate microdilution method The minimum inhibitory concentration (MIC) value of Bacillus; on the basis of measuring the MIC value, the partial inhibitory index FIC of berberine hydrochloride and sulbactam, tigecycline, ciprofloxacin and meropenem was determined by using the checkerboard method. value, and then determine the synergistic bacteriostatic effect of the combination of the two and the effect of berberine hydrochloride in assisting antibiotics against multidrug-resistant Acinetobacter baumannii to restore sensitivity, that is, resensitization.

Step Instructions:

1. Strain selection: Two multi-drug resistant Acinetobacter baumannii strains (MDRAb-1 and MDRAb-2) were selected in the experiment, which are resistant to traditional quinolones, cephalosporins, aminoglycosides, carbapenems, penicillin resistance to antibiotics such as tetracyclines and tetracyclines.

2. Preparation of antibacterial drugs:

All antibacterial drugs were prepared using sterile salt-free distilled water. The concentration of berberine hydrochloride, sulbactam and meropenem preparation mother solution is 2048mg/L, the concentration of tigecycline mother solution is 2560mg/L, the concentration of amikacin and tetracycline mother solution is 4096mg/L, and the concentration of ciprofloxacin mother solution is 64mg/L.

3. Determination of Minimum Inhibitory Concentration (MIC)

The MIC of antimicrobials was determined using the microbroth dilution method in accordance with the experimental guidelines of the American Society for Clinical Laboratory Standardization CLSI document M100, and all tests were performed in cation-adjusted Mueller Hinton broth (CAMHB). Add 100 μL of a series of two-fold dilutions of antimicrobial drug dilutions to each well of a 96-well plate, adjust the bacterial concentration to 0.5 McFarland turbidity (about 1.5×10 ⁸ CFU/mL), dilute 150 times, and then add to in each well (100 μL per well) such that the final bacterial concentration is approximately 5×10 ⁵ CFU/mL. Wells with bacterial suspension but no antibacterial agent added were used as positive growth controls and wells supplemented with CAMHB medium only were used as negative controls. Incubate the 96-well plate in an incubator at 35°C for 20-24 hours and observe the results. The test results are shown in Table 1.

The MIC is defined as the lowest concentration of antimicrobial agent that inhibits bacterial growth by more than 90%, while this reference is roughly equivalent to no visible bacterial growth. Three wells were replicated for each concentration and at least three independent assays were performed. Escherichia coli ATCC 25922 was used as the quality control strain.

Inhibition rate=(1-(OD _{600nm drug treatment group-} OD _{600nm negative control group} )/(OD _{600nm positive control group-} OD _{600nm negative control group} ))×100%

At present, there is no CLSI susceptibility point for tigecycline and sulbactam to A. baumannii. Therefore, this study used the CLSI criteria for the combination of ampicillin/sulbactam to determine the sulbactam sensitivity judgment point (≤4 mg/L, sensitive; 8 mg/L, intermediate; ≥16 mg/L, resistant); The criteria prescribed by the Food and Drug Administration (FDA) were used to determine the tigecycline sensitivity judgment points (≤2 mg/L, sensitive; 4-6 mg/L, intermediate; ≥8 mg/L, resistant).

Table 1 Minimum inhibitory concentration MIC of antibacterial agents

Note: R, resistant; M, intermediate; S, sensitive.

4. Determination of the partial inhibitory index FIC value of drug combination

A checkerboard design was used to determine the synergistic effect of antibiotics and berberine hydrochloride against multidrug-resistant Acinetobacter baumannii. The experimental procedure was as follows: A series of two-fold dilutions of the antibacterial agent were prepared based on previously determined MIC values. Then, 50 μL of a series of two-fold dilutions of antibiotics (sulbactam, tigecycline, ciprofloxacin, meropenem) were added horizontally and 50 μL of a series of two-fold dilutions were added vertically Berberine hydrochloride, and 100 μL of the bacterial suspension was added to a 96-well plate to a final concentration of approximately 5×10 ⁵ CFU/mL. Incubate the 96-well plate in an incubator at 35°C for 20-24 hours and observe the results. Interactions between combinations were determined according to the partial inhibition index (FIC).

The formula for calculating the FIC value is as follows:

FIC=(Antibiotic combined with MIC)/(Antibiotic alone MIC)+(Berberine hydrochloride combined with MIC)/(Berberine hydrochloride alone MIC)

FIC values were defined as follows: synergistic effect, FIC≤0.5; additive effect, 0.5<FIC≤1; irrelevant effect, 1<FIC≤2; antagonism, FIC>2.

Table 2 The range of FIC values for the combination of berberine hydrochloride and antibiotics

Note: The concentrations of berberine hydrochloride and antibiotics are 16, 32, 64, 128mg/L (MDRAb-1) and 64, 128, 256, 512mg/L (MDRAb-2)

Table 3 Some Bacteriostatic Index FIC Values of Berberine Hydrochloride Combined with Antibiotics

Note: (1) R, resistant; M, intermediate; S, sensitive.

(2) Mark a as complex sensitive combination and related data.

Table 4 FIC values of berberine hydrochloride combined with antibiotics on different Acinetobacter baumannii strains

According to Table 4, it can be seen that berberine hydrochloride reduces the MIC value of antibiotics by up to 64 times, while the range of reduction times in the comparative literature is 2-16 times. The combination of the present invention can restore the sensitivity of some bacteria to the corresponding antibiotics, that is, resensitization (combination in bold font in Table 4).

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (9)

1. A combined antibacterial pharmaceutical composition comprising berberine and an antibiotic;

   Wherein, the antibiotic is selected from one or two of sulbactam, tigecycline, amikacin, ciprofloxacin, meropenem and tetracycline.
2. The composition of claim 1, wherein the berberine can be in the form of its salt, such as berberine hydrochloride or berberine sulfate.
3. The composition according to claim 1 or 2, wherein the weight ratio of the berberine to the antibiotic can be (1-4096):1, such as (1-1024):1, (2-512) ):1;
4. The composition according to any one of claims 1-3, characterized in that it comprises one of the following combinations:

   According to an embodiment of the present invention, the combined antibacterial pharmaceutical composition comprises one of the following combinations:

   (1) the combination of berberine hydrochloride and sulbactam, the weight ratio of the two can be (2-512): 1, for example (4-256): 1, (8-128): 1;

   (2) the combination of berberine hydrochloride and tigecycline, the weight ratio of the two can be (1-4096): 1, for example (1-256): 1, (2-128): 1, (4- 64):1;

   (3) the combination of berberine hydrochloride and ciprofloxacin, the weight ratio of the two can be (1-1024): 1, such as (2-512: 1);

   (4) the combination of berberine hydrochloride and meropenem, the weight ratio of the two can be (2-512): 1, such as (4-256): 1, (8-128): 1;

   (5) the combination of berberine hydrochloride and amikacin, the weight ratio of the two can be (1-256): 1, such as (2-128: 1);

   (6) the combination of berberine hydrochloride and tetracycline, the weight ratio of the two can be (1-256): 1, such as (2-128: 1);

   Preferably, the combined antibacterial pharmaceutical composition comprises one of the combinations of (1)-(4).
5. The composition according to any one of claims 1-4, wherein the weight percentage of the berberine in the composition can be 5.0%-90.0%, such as 10.0%-85.0%, 15.0% %-80.0%, 20%-75.0%, 25%-70%; the weight percentage of the antibiotic in the composition can be 0.01%-50.0%, for example, 0.05%-45.0%, 0.1%-40.0% , 0.2%-35%, 0.3%-30%.
6. The composition according to any one of claims 1-5, wherein the combined antibacterial drug composition further comprises a pharmaceutically acceptable carrier, excipient or solvent.
7. The composition according to any one of claims 1-6, wherein the combined antibacterial drug composition can be made into oral preparations or injections by conventional methods.
8. Use of the composition of any one of claims 1-6 for preparing an antibacterial drug.
9. The application according to claim 8, characterized in that, the antibacterial drug is used to fight against infection caused by Acinetobacter baumannii, especially multidrug-resistant Acinetobacter baumannii.