Pharmaceutical composition for synergistically inhibiting klebsiella pneumoniae and infectious pneumonia of klebsiella pneumoniae, preparation and application

Abstract

The invention relates to the technical field of pharmaceutical compositions, in particular to a pharmaceutical composition for synergistically inhibiting klebsiella pneumoniae and infectious pneumonia of the klebsiella pneumoniae, a preparation and application, and the composition is prepared from the following components in parts by weight: 50-200 parts of berberine, 10-100 parts of andrographolide, 10-100 parts of manuka honey and 10-50 parts of polymyxin B. In-vitro antibacterial study is carried out on the berberine, the andrographolide, the manuka honey and the polymyxin B, and it is found that the andrographolide, the berberine and the manuka honey can all improve the antibacterial effect of the polymyxin B and all generate a synergistic effect when being combined for use; besides, the combination of the four medicines provided by the invention is used for treating a mouse suffering from Klebsiella pneumonia infectious severe pneumonia, the effect of the combination of the four medicines is obviously superior to that of a single medicine, and the effect of treating the Klebsiella pneumonia infectious severe pneumonia can be obviously improved after the medicine composition provided by the invention is compounded.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical compositions, and particularly relates to a pharmaceutical composition for synergistically inhibiting Klebsiella pneumoniae and its infectious pneumonia, a preparation and application. BACKGROUND

[0002] Klebsiella pneumoniae (KP) as an opportunistic pathogen is a common pathogen causing hospital and community-acquired pneumonia, and carbapenem antibiotics are the most frequently selected drugs in the early stage of KP infection. With the global prevalence of KP, carbapenem antibiotic resistance has been widely observed in KP, and with the abuse of antibiotics, some multi-drug resistant Klebsiella pneumoniae has emerged, making the available antibiotics in the clinic less and less.

[0003] At present, in the treatment of carbapenem-resistant Klebsiella pneumoniae in China, there are mainly two approved antibiotics: tigecycline and polymyxin. In recent years, with the emergence and spread of pathogenic bacteria such as Escherichia coli carrying polymyxin resistance gene (MCR) plasmid, some Klebsiella pneumoniae has developed resistance to polymyxin, thereby gradually limiting the clinical application of polymyxin. Therefore, developing effective polymyxin compound preparations has gradually become an important direction for the clinical treatment of multi-drug resistant Klebsiella pneumoniae infection. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a pharmaceutical composition for synergistically inhibiting Klebsiella pneumoniae and its infectious pneumonia, a preparation and application.

[0005] Specifically, the present application provides, in a first aspect, a pharmaceutical composition for synergistically inhibiting Klebsiella pneumoniae, which comprises: polymyxin B and one or more combinations of the following drugs: berberine, andrographolide, and manuka honey.

[0006] The present application provides, in a second aspect, a pharmaceutical composition for synergistically inhibiting Klebsiella pneumoniae, which comprises: polymyxin B sulfate and one or more combinations of the following drugs: berberine, andrographolide, and manuka honey.

[0007] As a further illustration of the present application, the ratio of the berberine to the polymyxin B sulfate is (32-128) μg / mL: 1 μg / mL; or the ratio of the andrographolide to the polymyxin B sulfate is (256-1024) μg / mL: 1 μg / mL; or the ratio of the manuka honey to the polymyxin B sulfate is (3.125-12.5) %(w / v): 1 μg / mL.

[0008] The third aspect of the present application provides a pharmaceutical composition for treating Klebsiella pneumoniae infection pneumonia, which is composed of the following components: berberine, andrographolide, manuka honey and polymyxin B.

[0009] Berberine is also known as coptis, which is a quaternary ammonium alkaloid isolated from traditional Chinese medicine coptis, and is the main effective component of coptis antibacterial. Berberine has broad-spectrum antibacterial effect, and has inhibitory effect on many gram-positive and gram-negative bacteria. In recent years, many studies have found that berberine not only has the effects of clearing heat and detoxifying, anti-infection, but also has unexpected curative effect on many cardiovascular diseases and other diseases. Berberine has anticholinesterase effect on animals, and has peripheral antihypertensive and antipyretic effects. Studies have shown that berberine can combine with single / double stranded DNA of bacteria to form complex, and can inhibit the activity of topoisomerase, which may play an antibacterial role by inhibiting the function of DNA. Studies have shown that in addition to DNA binding, berberine can also interact with bacterial double-stranded RNA, especially at the AU base pairing site, which affects the normal function of RNA. It has been reported that berberine can inhibit the GTPase activity of Escherichia coli FtsZ, thereby inhibiting the formation of Z-ring. In summary, multiple studies have shown that the antibacterial mechanism of berberine is multifaceted, involving DNA, RNA and bacterial division proteins, etc. These mechanisms work together to effectively inhibit the growth and reproduction of many bacteria. Berberine is mainly used in the form of hydrochloride and sulfate in clinical application. Berberine hydrochloride (commonly known as hydrochloride coptis) has been widely used in the treatment of gastrointestinal inflammation, bacterial dysentery, etc., and has certain curative effect on pulmonary tuberculosis, scarlet fever, acute tonsillitis and respiratory tract infection.

[0010] Andrographolide is extracted from Andrographis paniculata of Acanthaceae, which can be extracted from the whole plant of Andrographis paniculata (including leaves, stems and fruits, etc.). Compared with stems and fruits, andrographolide is highest in Andrographis paniculata leaves. Andrographolide is the main and most representative diterpene lactone compound in Andrographis paniculata, and is also one of the core components of its pharmacological effects (such as anti-inflammatory, antiviral, antibacterial, liver protection and choleretic, immune enhancement, etc.). It has been reported that andrographolide can directly destroy the bacterial outer membrane, and has obvious inhibitory effect on gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae). It also has anti-inflammatory and immune regulation effects. It can reduce the release of TNF-α, IL-6 and other inflammatory factors by inhibiting the NF-κB pathway, and can enhance the phagocytic ability of macrophages by regulating TLR4 / MyD88 signal. In the process of clinical application, andrographolide tablets can be used for the adjuvant treatment of respiratory tract infection and sepsis, and viral pneumonia, etc.

[0011] Honey has been used to treat wounds and topical infections before the advent of antibiotics as a natural antibacterial agent, and in recent years, the bacteriostatic and antioxidant properties of honey have been widely recognized, and polyphenols, hydrogen peroxide, methylglyoxal, flavonoids and bee antimicrobial peptides in honey are considered to be the key components of its bacteriostatic activity. The nectar of Manuka honey is Manuka trees growing in the virgin forest of New Zealand, and compared with ordinary honey, Manuka honey is darker, yellow-brown or dark amber, which belongs to the medical grade honey that has been popularized in clinical use. A number of studies have shown that Manuka honey can inhibit different types of bacteria in vitro, in addition, Manuka honey also shows bacteriostatic activity against Mycoplasma penumoniae and Ureaplasma urealyticum with high levels of intrinsic and acquired drug resistance. In addition, it has been reported that Manuka honey can cause structural damage to bacterial cell membranes by reducing the expression of OprF gene of bacterial membrane protein, and the unique feature of Manuka honey is that it contains a large amount of methylglyoxal (methylglyoxal), which is much higher than other types of honey, and this substance can irreversibly modify bacterial proteins (such as DNA gyrase and phosphorylase), thereby achieving the purpose of sterilization, in addition, it can also decompose extracellular polysaccharide (PIA), thereby enhancing the penetration of antibiotics, so that Manuka honey has obvious antibacterial advantage over other ordinary honey.

[0012] Although there are research reports that berberine, andrographolide and Manuka honey have different degrees of antibacterial effect, but for the clinical treatment of Klebsiella pneumoniae, especially the refractory problem of severe pneumonia caused by it, there are few targeted studies on the antibacterial effect of Klebsiella pneumoniae and the treatment of infectious pneumonia after the combination of berberine, andrographolide and Manuka honey with antibiotics. The present application carries out in vitro antibacterial experiment research on berberine, andrographolide, Manuka honey and polymyxin B, and finds that andrographolide, berberine and Manuka honey can all enhance the bacteriostatic effect of polymyxin B, and all produce synergistic effect when combined. In addition, the four drugs provided by the present application are combined to treat Klebsiella pneumoniae infectious severe pneumonia mice, and it is found that the effect of the four drugs combined is obviously better than that of single drug, and the drug composition provided by the present application can significantly improve the effect of treating Klebsiella pneumoniae infectious severe pneumonia after compounding.

[0013] As a further description of the present application, the composition is composed of the following components by weight: berberine 50-200 parts, andrographolide 10-100 parts, Manuka honey 10-100 parts and polymyxin B 10-50 parts.

[0014] As a further description of the present application, the administration mode of the polymyxin B is intravenous injection, intraperitoneal injection or subcutaneous injection, and the administration mode of the berberine, the andrographolide and the Manuka honey is oral or intraperitoneal injection.

[0015] The fourth aspect of the present application provides a pharmaceutical preparation for treating Klebsiella pneumoniae infection pneumonia, which is prepared by the above-mentioned pharmaceutical composition and clinically acceptable adjuvant in a conventional preparation method.

[0016] As a further illustration of the present application, the pharmaceutical preparation is any one of a powder, a pill, a granule, a capsule, a tablet, a dripping pill, a syrup, and an oral solution.

[0017] As a further illustration of the present application, the adjuvant includes one or more of starch, microcrystalline cellulose, sucrose, dextrin, lactose, glucose, stearic acid, sodium chloride, water, ethanol, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, sodium bicarbonate, citric acid, hydroxypropyl cellulose, agar, alginic acid, cellulose ether, polyglycolic acid, and polyamide.

[0018] The fifth aspect of the present application provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating Klebsiella pneumoniae infection pneumonia.

[0019] Compared with the prior art, the present application has the following beneficial technical effects: The present application carries out in-vitro antibacterial experiment research on berberine, andrographolide, manuka honey and polymyxin B, finds that andrographolide, berberine and manuka honey can all improve the bacteriostatic effect of polymyxin B, and all produce synergistic effect when used in combination. In addition, the four drugs provided by the present application are used in combination to treat Klebsiella pneumoniae infection pneumonia mice, and it is found that the effect after the four drugs are used in combination is obviously better than that of single drug use. The pharmaceutical composition provided by the present application can significantly improve the effect of treating Klebsiella pneumoniae infection pneumonia after being compounded. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The survival rate statistics of the mice in each group. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0022] I. Determining the in-vitro minimum inhibitory concentration MIC value of each component in the pharmaceutical composition on Klebsiella pneumoniae, and determining the synergistic antibacterial effect between polymyxin B and other components by using the checkerboard method.

[0023] 1. Experimental materials: Strain: Klebsiella pneumoniae, national standard strain, ATCC 13883.

[0024] Antibacterial samples: dehydroandrographolide (purity more than 99%), polymyxin B sulfate, berberine hydrochloride, manuka honey (high activity UMF 20+).

[0025] 2. Preparation of antibacterial dilutions: Dehydroandrographolide dilution: a small amount of dimethyl sulfoxide (DMSO) was used to dissolve dehydroandrographolide, and then the sterile MHB (Mueller-Hinton broth) was used to dilute to the working concentration of 16-1024 μg / mL by the double dilution method; Polymyxin B sulfate dilution: sterile MHB was used to dilute polymyxin B sulfate to the working concentration of 0.125-16 μg / mL by the double dilution method; Berberine hydrochloride dilution: a small amount of DMSO was used to dissolve berberine hydrochloride, and then sterile MHB was used to dilute to the working concentration of 8-512 μg / mL by the double dilution method; Manuka honey dilution: sterile MHB was used to dilute manuka honey to the working concentration of 3.125%-50% (w / v) by the double dilution method.

[0026] 3. Determination of minimum inhibitory concentration MIC The minimum inhibitory concentration MIC value of each component on Klebsiella pneumoniae was detected according to the standard method of the American Clinical and Laboratory Standards Association (CLSI): a single pure colony was picked and inoculated in MHB, and the bacteria were cultured in a 37°C shaking bed to the logarithmic growth phase, the bacterial liquid concentration was adjusted to 0.5 McFarland unit turbidity with a McFarland turbidimeter, and then diluted to 5×10 5 CFU / mL with MHB. 100 μL of a series of double dilutions of the above antibacterial dilutions (dehydroandrographolide dilution, polymyxin B sulfate dilution, berberine hydrochloride dilution, manuka honey dilution) was added to each well of the 96-well plate, and 100 μL of the diluted bacterial liquid was added to each well (100 μL per well). The negative control group only contained MHB; the positive control group only contained 100 μL of the bacterial liquid. After incubation in a 37°C incubator for 18 h, the results were observed, and the drug concentration contained in the clear well of the 96-well plate was the minimum inhibitory concentration MIC. The experimental results are shown in Table 1.

[0027]

[0028] 4. Determination of FICI value of polymyxin B combined with other drugs The FICI value of polymyxin B sulfate combined with other antibacterial drugs on Klebsiella pneumoniae was determined by using the chessboard method: polymyxin B sulfate as A drug, dehydroleucin, berberine hydrochloride and manuka honey as B drug, respectively, 8 concentrations (1 / 16 MIC to 8 MIC) of each MIC of the drugs were set around the MIC, 50 μL of MHB containing different concentrations of A and B drugs were added along the horizontal and vertical axes of the 96-well culture plate, respectively, then 100 μL of Klebsiella pneumoniae bacterial solution (final concentration about 5×10 5 CFU / mL) was added to each well, and the results were observed after incubation in a 37℃ incubator for 18h, and the interaction between the combined drugs was determined according to the combined inhibition index (FICI).

[0029] The MIC of each drug was recorded when the two drugs were used alone and in combination, and the FICI value was calculated according to the following formula.

[0030] FICI = (MIC A drug combination / MIC A drug alone) + (MIC B drug combination / MIC B drug alone) Judgment standard: FICI≤0.5, synergistic effect; 0.5<FICI≤1, additive effect; 1FICI≤2, no effect; FICI>2, antagonistic effect. The experimental results are shown in Table 2.

[0031] Table 2 FICI value of polymyxin B combined with other drugs

[0032] As can be seen from Table 2, in the in vitro antibacterial experiment, dehydroleucin, berberine hydrochloride and manuka honey can all enhance the antibacterial effect of polymyxin B, and the combination of the two drugs produces synergistic effect (FICI≤0.5). Among them, when polymyxin B is combined with dehydroleucin, the ratio is 1 μg / mL:512 μg / mL, the synergistic effect of the two is the best; when polymyxin B is combined with berberine hydrochloride, the ratio is 1 μg / mL:64 μg / mL, the synergistic effect of the two is the best; when polymyxin B is combined with manuka honey, the ratio is 1 μg / mL:6.25% (w / v), the synergistic effect of the two is the best.

[0033] II. Verify the therapeutic effect of the pharmaceutical composition provided by the application on Klebsiella pneumoniae infected mice with pneumonia through animal experiments.

[0034] 1. Experimental materials 1.1 Experimental animals 6-8 week old SPF level SD male mice were selected and purchased, with an average weight of (20g±2g), and after being purchased, they were adaptively fed for 1 week under standard laboratory conditions before subsequent experiments.

[0035] 1.2 Experimental agents: Strain: Klebsiella pneumoniae, national standard strain, ATCC 13883.

[0036] Antibacterial samples: dehydrated andrographolide (purity more than 99%), polymyxin B sulfate, berberine hydrochloride, manuka honey (high activity UMF 20+).

[0037] 2. Experimental method 2.1 Establishment of animal model, grouping and administration Strain culture: the standard strain of Klebsiella pneumoniae was successively subjected to resuscitation and activation, expansion culture (brain heart infusion broth (BHI) medium), spectrophotometry (OD 600 ) estimation of viable bacterial concentration and bacterial liquid concentration (from 10 10 → 10 11 CFU / mL), and finally obtained the target bacterial liquid with a concentration of 1×10 11 CFU / mL.

[0038] Animal grouping: the mice were randomly divided into blank group, model group, polymyxin B (DNJ) treatment group, polymyxin B + dehydrated andrographolide (DNJ+CXL) treatment group, polymyxin B + berberine hydrochloride (DNJ+HLS) treatment group, polymyxin B + manuka honey (DNJ+MLK) treatment group, polymyxin B + dehydrated andrographolide + berberine hydrochloride + manuka honey (DNJ+CXL+HLS+MLK) treatment group, 40 mice in each group, and the rats in the rest groups were subjected to the following modeling process except the blank group.

[0039] Modeling: after the mice were anesthetized, the bacterial liquid was sent into the trachea through tracheal intubation (one-time intratracheal instillation of 50 μL of Klebsiella pneumoniae bacterial liquid with a concentration of 1×10 11 CFU / mL), and then the mice were stood upright to allow the bacterial liquid to flow into the lungs naturally.

[0040] Drug administration: After 2h of injection of bacterial solution, the blank group, model group, DNJ group, DNJ+CXL group, DNJ+HLS group, DNJ+MLK group, DNJ+CXL+HLS+MLK group were given intraperitoneal injection of normal saline, normal saline, polymyxin B sulfate 30mg / kg, polymyxin B sulfate 30mg / kg+dehydrated andrographolide 50mg / kg, polymyxin B sulfate 30mg / kg+berberine hydrochloride 100mg / kg, polymyxin B sulfate 30mg / kg+manuka honey 50mg / kg, polymyxin B sulfate 30mg / kg+dehydrated andrographolide 50mg / kg+berberine hydrochloride 100mg / kg+manuka honey 50mg / kg, respectively, among which polymyxin B sulfate was given every 12 hours, and the rest of the drugs were given once a day, for 3 consecutive days.

[0041] 2.2 Observation index After 7 days of modeling, the survival rate of mice in each group was observed.

[0042] 3. Statistical analysis Statistical analysis was performed using SPSS 25.0 software, and chi-square test (χ2) was used for difference test of count data; the test method was P<0.05 or P<0.01, which was statistically significant.

[0043] Experimental results

[0044] Note: # compared with the model group P<0.05, ## compared with the model group P<0.01; * compared with the DNJ group P<0.05, ** compared with the DNJ group P<0.01; Δ compared with the DNJ+CXL+HLS+MLK group P<0.05, ΔΔ compared with the DNJ+CXL+HLS+MLK group P<0.01, n=40.

[0045] From the above table 3 and Figure 1As can be seen, compared with the model group, the survival rates of the mice in the DNJ group, the DNJ+CXL group, the DNJ+HLS group, the DNJ+MLK group and the DNJ+CXL+HLS+MLK group are all significantly improved, and the comparison between groups has a statistical difference (P<0.05), which indicates that the treatment of antibiotic polymyxin B alone or the combined treatment of antibiotic and other antibacterial drugs can significantly improve the survival rate of the mice; compared with the DNJ group, the survival rates of the mice in the DNJ+CXL group, the DNJ+HLS group and the DNJ+CXL+HLS+MLK group are all significantly improved, and the comparison between groups has a statistical difference (P<0.05, only the comparison between the DNJ+MLK group and the DNJ group has P>0.05), which indicates that the curative effect of the combined treatment of antibiotic and other antibacterial drugs is better than that of the treatment of antibiotic polymyxin B alone; compared with the DNJ+CXL+HLS+MLK group, the survival rates of the mice in the DNJ+CXL group, the DNJ+HLS group and the DNJ+MLK group are all significantly reduced, and the comparison between groups has a statistical difference (P<0.05), which indicates that the four drugs provided in the application have obvious synergistic effect, and when one single drug is removed, the final curative effect is not ideal, thereby reflecting the unique compounding advantage of the drug composition provided in the application.

[0046] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for synergistically inhibiting Klebsiella pneumoniae, comprising, The composition comprises: polymyxin B and one or more of the following drugs in combination: berberine, andrographolide, manuka honey.

2. A pharmaceutical composition for synergistically inhibiting Klebsiella pneumoniae, comprising, The composition comprises: polymyxin B sulfate and one or more of the following drugs in combination: berberine, andrographolide, manuka honey.

3. The pharmaceutical composition for synergistic inhibition of Klebsiella pneumoniae as described in claim 2, characterized in that, The ratio of the berberine to the polymyxin B sulfate is (32-128) μg / mL:1 μg / mL; Or the ratio of the andrographolide to the polymyxin B sulfate is (256-1024) μg / mL:1 μg / mL; Or the ratio of the manuka honey to the polymyxin B sulfate is (3.125~12.5) % (w / v):1 μg / mL.

4. A pharmaceutical composition for treating pneumonia caused by Klebsiella pneumoniae infection, characterized by, The composition comprises: berberine, andrographolide, manuka honey and polymyxin B.

5. The pharmaceutical composition for use in the treatment of Klebsiella pneumoniae infection pneumonia according to claim 4, wherein The composition comprises: berberine 50-200 parts, andrographolide 10-100 parts, manuka honey 10-100 parts and polymyxin B 10-50 parts by weight.

6. The pharmaceutical composition for use in the treatment of Klebsiella pneumoniae infection pneumonia according to claim 4, wherein The polymyxin B is administered by intravenous injection, intraperitoneal injection or subcutaneous injection; the berberine, the andrographolide and the manuka honey are administered orally or by intraperitoneal injection.

7. A pharmaceutical preparation for treating pneumonia caused by Klebsiella pneumoniae infection, characterized by, The pharmaceutical preparation is prepared from the pharmaceutical composition of any one of claims 4-6 and clinically acceptable adjuvants by conventional preparation methods.

8. The pharmaceutical preparation for use in the treatment of Klebsiella pneumoniae infection pneumonia according to claim 7, wherein The pharmaceutical preparation is any one of a powder, a pill, a granule, a capsule, a tablet, a dripping pill, a syrup, an oral liquid.

9. The pharmaceutical preparation for use in the treatment of Klebsiella pneumoniae infection pneumonia according to claim 7, wherein The adjuvants include one or more of starch, microcrystalline cellulose, sucrose, dextrin, lactose, glucose, stearic acid, sodium chloride, water, ethanol, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone, sodium bicarbonate, citric acid, hydroxypropyl cellulose, agar, alginic acid, cellulose ether, polyglycolic acid, polyamide.

10. Use of the pharmaceutical composition of any one of claims 4-6 in the preparation of a medicament for treating Klebsiella pneumoniae infection.

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