Application of hypoxanthine nucleotide in preparing anti-infective medicament
阅读说明：本技术 次黄嘌呤核苷酸在制备抗感染药物中的应用 (Application of hypoxanthine nucleotide in preparing anti-infective medicament ) 是由 李惠 彭博 彭宣宪 赵贤亮 匡素芳 于 2020-12-30 设计创作，主要内容包括：本发明属于医药技术领域,具体涉及次黄嘌呤核苷酸在制备抗感染药物中的应用。本发明经过实验证明,次黄嘌呤核苷酸可以显著提高大肠埃希菌、肺炎克雷伯菌、金黄色葡萄球多重耐药菌、铜绿假单胞菌等细菌对阿莫西林、头孢哌酮、美罗培南、庆大霉素等抗生素的敏感性,可以与抗生素连用作为抗感染药物,在低浓度抗生素条件下杀灭细菌,达到较好的抗感染效果,同时减少细菌耐药性的产生。(The invention belongs to the technical field of medicines, and particularly relates to application of hypoxanthine nucleotide in preparation of anti-infective medicines. Experiments prove that the hypoxanthine nucleotide can obviously improve the sensitivity of bacteria such as Escherichia coli, Klebsiella pneumoniae, staphylococcus aureus multidrug resistant bacteria, pseudomonas aeruginosa and the like to antibiotics such as amoxicillin, cefoperazone, meropenem, gentamicin and the like, can be used as an anti-infective drug together with the antibiotics, kills the bacteria under the condition of low-concentration antibiotics, achieves a better anti-infective effect, and reduces the generation of drug resistance of the bacteria.)
1. Use of inosinic acid in preparing anti-infective medicine.
2. The use of claim 1, wherein the inosinic acid increases the sensitivity of the bacterium to antibiotics in anti-infective agents.
3. The use according to claim 2, wherein the bacteria are one or more of escherichia coli ATCC35218, klebsiella pneumoniae k. pneumoniae, staphylococcus aureus multidrug-resistant bacteria MRSA, pseudomonas aeruginosa p. aeruginosa, acinetobacter baumannii ATCC19606 a. baumannii, aeromonas hydrophila a ATCC19606, edwardsiella tarda EIB202, vibrio alginolyticus v. alginolyticus, vibrio parahaemolyticus, or streptococcus pyogenes s.
4. The use according to claim 2, wherein the antibiotic is one or more of amoxicillin, cefoperazone, meropenem, gentamicin, balofloxacin, ciprofloxacin, chlortetracycline, tetracycline, lincomycin.
5. An anti-infective composition comprising an inosine and an antibiotic.
6. The anti-infective composition of claim 5, wherein the antibiotic is one or more of amoxicillin, cefoperazone, meropenem, gentamicin, balofloxacin, ciprofloxacin, chlortetracycline, tetracycline, chloramphenicol, lincomycin.
7. The anti-infective composition of claim 5 or 6, wherein the weight ratio of the hypoxanthine nucleotide to the antibiotic is (300-5500): 1.
8. The anti-infective composition of claim 7, wherein the weight ratio of the inosinic acid to the antibiotic is (326-5223): 1.
9. An anti-infective agent comprising a composition as claimed in any one of claims 5 to 8.
10. The anti-infective drug of claim 9, wherein the anti-infective drug is in an oral dosage form or an injectable dosage form.
Technical Field
The invention belongs to the technical field of medicines. More particularly, it relates to the use of inosinic acid in the preparation of anti-infective drugs.
Background
As a main medicine for treating bacterial infectious diseases, antibiotics are the medicines with the widest application, the fastest development and the most varieties in the world. However, with the widespread use of antibiotics, resistance is becoming prominent. The World Health Organization (WHO) in 2007 world health reports clearly states that bacterial resistance is a major public health problem threatening human health.
A common method of combating bacterial resistance today is to kill bacteria by increasing their sensitivity to antibiotics such that an otherwise ineffective or ineffective antibiotic becomes effective or effective. For example, the chinese patent application CN104606219A discloses a small molecule metabolite inosine for improving antibiotic elimination of pathogenic bacteria, and the combination of inosine and antibiotic can significantly improve the bactericidal effect of antibiotic. However, in practical application, inosine is found to improve the sensitivity of some bacteria to some antibiotics, but the effect of inosine on other bacteria or antibiotics is not ideal.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects and the defects of few medicines for improving the antibiotic to eliminate pathogenic bacteria and limited effect on certain bacteria or antibiotics in the prior art, providing a new application of a small molecular substance, namely hypoxanthine nucleotide, in improving the sensitivity of bacteria to the antibiotics, and applying the hypoxanthine nucleotide in preparing anti-infective medicines.
The invention aims to provide application of hypoxanthine nucleotide in preparing anti-infective medicaments.
It is another object of the present invention to provide an anti-infective composition.
Another object of the present invention is to provide an anti-infective agent.
The above purpose of the invention is realized by the following technical scheme:
nucleotide is a compound composed of purine base or pyrimidine base, ribose or deoxyribose and phosphate, which is the basic unit of RNA and DNA and is the precursor of in vivo synthesized nucleic acid. The nucleotides are distributed in the nucleus and cytoplasm of organs, tissues and cells in organisms along with the nucleic acids, and participate in the inheritance, development and growth of organisms as the components of the nucleic acidsAnd waiting for basic life activities. Wherein, inosinic acid (IMP), which is the first nucleotide product in the biosynthesis process of purine nucleotide, is 6-hydroxypurine nucleotide, which is composed of aspartic acid, glycine, glutamine, CO2And one-carbon units (formyl and methine, carried by tetrahydrofolic acid), etc. are synthesized by 11 enzymatic reactions. Inosine (inosine) is converted from dephosphorylated inosine (inosine) to inosine (Hypoxanthine), which is produced by nucleotide cyclase.
Experiments prove that the hypoxanthine nucleotide can obviously improve the sensitivity of bacteria such as Escherichia coli E.coli ATCC35218, Klebsiella pneumoniae K.pneumoniae, staphylococcus aureus multidrug resistant bacteria MRSA, Pseudomonas aeruginosa P.aeruginosa to antibiotics such as amoxicillin, cefoperazone, meropenem, gentamicin and the like, can be used as an anti-infective drug together with the antibiotics, kills the bacteria under the condition of low-concentration antibiotics, achieves a better anti-infective effect, and reduces the generation of bacterial drug resistance. Moreover, experiments prove that the effect difference of the hypoxanthine nucleotide, the inosine and the hypoxanthine is obvious, and the hypoxanthine nucleotide can achieve better effect.
Therefore, the invention claims the use of inosinic acid in the preparation of anti-infective drugs.
Further, the inosinic acid can improve the sensitivity of bacteria to antibiotics in anti-infective drugs.
Preferably, the bacteria are one or more of escherichia coli ATCC35218, klebsiella pneumoniae k.pneumoniae, staphylococcus aureus multidrug-resistant bacteria MRSA, pseudomonas aeruginosa p.aeruginosa, acinetobacter baumannii ATCC19606 a.baumannii, aeromonas hydrophila a ATCC19606, edwardsiella tarda EIB202, vibrio-alginolyticus v.alginolyticus, vibrio parahaemolyticus v.parahaemolyticus, or streptococcus pyogenes s.pyogenenes. The bacteria are common pathogenic bacteria of human and cultured animals, wherein Staphylococcus aureus and Streptococcus pyogenes are gram-positive bacteria, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Aeromonas hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus and Vibrio alginolyticus are gram-negative bacteria.
The bacteria are common pathogenic bacteria and drug-resistant strains thereof, and meanwhile, the Escherichia coli, the Pseudomonas aeruginosa and the Staphylococcus aureus are model bacteria for researching the drug resistance of the bacteria, so the bacteria are better representative bacteria of drug-resistant bacteria and non-drug-resistant bacteria. Although in the embodiments of the present invention, the exemplified bacteria include Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus multidrug-resistant bacteria, Pseudomonas aeruginosa, Acinetobacter baumannii, Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Streptococcus pyogenes. In particular, most of the verification tests of the present invention have been conducted on drug-resistant Escherichia coli. However, these bacteria should not be construed as limiting the scope of the present invention. This is because 1) Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus are model bacteria for studying the mechanism of drug resistance. 2) The bacteria belong to gram-negative bacteria and gram-positive bacteria respectively, wherein Staphylococcus aureus and Streptococcus pyogenes are gram-positive bacteria, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Aeromonas hydrophila, Edwardsiella tarda, Vibrio parahaemolyticus and Vibrio alginolyticus are gram-negative bacteria. And all human and cultured animal pathogenic bacteria can be classified according to the staining, so the bacteria have better representativeness. 3) The bacteria can have drug-resistant and non-drug-resistant states, i.e. drug-resistant and non-drug-resistant strains of the same bacteria, while the escherichia coli of the invention is in a relatively non-drug-resistant state, and the sensitivity to antibiotics is also improved after the addition of the inosinic acid. Therefore, many more species can be deduced from these species according to the above principles and are also suitable for the concept of the present invention.
Preferably, the antibiotic is one or more of amoxicillin, cefoperazone, meropenem, gentamicin, balofloxacin, ciprofloxacin, chlortetracycline, tetracycline, chloramphenicol, and lincomycin. Wherein, the ampicillin is penicillin antibiotic, and the cefoperazone is cephalosporin antibiotic (both are beta-lactam antibiotic); balofloxacin and ciprofloxacin are quinolone antibiotics; gentamicin is an aminoglycoside antibiotic; erythromycin is a macrolide antibiotic; chlortetracycline and tetracycline are tetracycline antibiotics; the chloramphenicol is amide alcohol antibiotic; lincomycin is lincosamide antibiotic. These include the major antibiotic types currently in clinical use.
In addition, the invention also provides an anti-infective composition, which contains the hypoxanthine nucleotide and the antibiotic.
Further, the antibiotic is one or more of amoxicillin, cefoperazone, meropenem, gentamicin, balofloxacin, ciprofloxacin, chlortetracycline, tetracycline, chloramphenicol and lincomycin. The above antibiotics should not be construed as limiting the scope of the present invention. This is because although the variety of antibiotics is hundreds, they can be classified according to their chemical structures and antibacterial mechanisms, and similar chemical structures have the same antibacterial mechanism, and thus do not need to be verified one by one. Currently, clinically common antibiotics are classified into: penicillin antibiotics, cephalosporin antibiotics, quinolone antibiotics, aminoglycoside antibiotics, amidoalcohol antibiotics, macrolide antibiotics, tetracycline antibiotics and lincosamide antibiotics. The ampicillin adopted by the invention is penicillin antibiotic, and the cefoperazone is cephalosporium antibiotic (both are beta-lactam antibiotics); balofloxacin and ciprofloxacin are quinolone antibiotics; gentamicin is an aminoglycoside antibiotic; erythromycin is a macrolide antibiotic; chlortetracycline and tetracycline are tetracycline antibiotics; the chloramphenicol is amide alcohol antibiotic; lincomycin is lincosamide antibiotic. Therefore, the antibiotic has good antibiotic representativeness. Those skilled in the art can easily deduce, based on the concept of the present invention, that other clinical antibiotics can be used in the method of the present invention.
Furthermore, the mass ratio of the hypoxanthine nucleotide to the antibiotic is (300-5500): 1.
Preferably, the mass ratio of the hypoxanthine nucleotide to the antibiotic is (326-5223): 1.
In addition, the invention also provides an anti-infective drug, which contains the composition.
Furthermore, the anti-infective medicament can be added with auxiliary materials which are allowed to be added in pharmacy to prepare an oral preparation or an injection preparation.
The invention has the following beneficial effects:
the invention provides application of hypoxanthine nucleotide in preparing anti-infective drugs, and experiments prove that the hypoxanthine nucleotide can obviously improve the sensitivity of bacteria such as Escherichia coli, Klebsiella pneumoniae, staphylococcus aureus multidrug resistant bacteria and pseudomonas aeruginosa to antibiotics such as amoxicillin, cefoperazone, meropenem and gentamicin, can be used as anti-infective drugs together with the antibiotics, kills the bacteria under the condition of low-concentration antibiotics, achieves better anti-infective effect and reduces the generation of drug resistance of the bacteria.
Drawings
FIG. 1 is a data statistics chart of the results of inosine and hypoxanthine nucleotide increasing the sensitivity of Klebsiella pneumoniae to different antibiotics.
FIG. 2 is a data set of results of inosine and inosine nucleotide increasing the sensitivity of Pseudomonas aeruginosa and Acinetobacter baumannii to different antibiotics.
FIG. 3 is a data statistics chart showing the results of increasing the sensitivity of Escherichia coli to ampicillin by using xanthosine.
FIG. 4 is a data statistical chart showing the results of concentration gradients of inosinic acid increasing the sensitivity of Escherichia coli to ampicillin.
FIG. 5 is a statistical chart of the results of the increased ampicillin sensitivity of various bacteria with inosinic acid.
FIG. 6 is a data statistics plot of the results of inosinic acid increasing the sensitivity of Escherichia coli to various antibiotics.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 sample preparation
Inoculating single bacterial colony in 5mL LB culture medium, culturing at 37 deg.C (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus multiple drug-resistant bacteria, Pseudomonas aeruginosa, Acinetobacter baumannii, and Streptococcus pyogenes or at 30 deg.C (Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus) at 200rpm for 16 hr, transferring the bacterial colony to fresh LB culture medium at a ratio of 1:100, and culturing at 37 deg.C or 30 deg.C at 200rpm to OD600To 1.0, an appropriate amount of the bacterial solution was centrifuged at 8000rpm for 5min to collect the cells, the supernatant was removed and the cells were washed 3 times with an equal volume of 0.85% physiological saline and suspended in 1 XM 9 (containing 10mM sodium acetate, 2mM MgSO)4，0.1mM CaCl2) And adjusting the concentration of the bacterial liquid to 10 in an M9 culture medium6CFU/mL (diluted 1000-fold); subpackaging into 5mL test tubes for later use.
EXAMPLE 2 measurement of Effect of inosinic acid and inosine on improving the sensitivity of bacteria to antibiotics
Each bacterial sample prepared in example 1 was divided into 3 groups, 2 experimental groups were added with 10mM inosinic acid (IMP) and Inosine (Inosine), respectively, and the other control group was added with an equal volume of physiological saline; adding corresponding antibiotics into each test tube, and repeating for three times; incubating for 6 hours in a shaker at 37 ℃ and 200rpm, then taking 100 mu L of the mixed solution, respectively taking 10 mu L of the mixed solution to perform plate counting by adopting a serial dilution method, and calculating the CFU/mL (colony forming unit/mL) of bacteria; the data of bacterial colony number between 20 and 200 can be used for statistical analysis; the survival rate of bacteria (percent survival) was CFU of bacteria treated with antibiotic and inosine or xanthosine/the number of bacteria treated with antibiotic alone × 100%.
As shown in FIGS. 1-2, it can be seen that, for Klebsiella pneumoniae R10, inosine has an increased sensitivity to gentamicin by about 2.35 times, while IMP has an increased sensitivity to gentamicin by 4.95 times; inosine increased its sensitivity to cefoperazone by about 31.83 times, while IMP increased by 0.88 times. For klebsiella pneumoniae R3, inosine can improve the sensitivity to gentamicin by about 3.61 times, while IMP can improve by 1.71 times; inosine increased its sensitivity to cefoperazone by about 2.45 times, while IMP increased by 1.64 times. For klebsiella pneumoniae R12, inosine can improve the sensitivity to gentamicin by about 8.33 times, while IMP can improve by 5.47 times; inosine increased its sensitivity to cefoperazone by about 2.45-fold, while IMP increased by 1.75-fold (figure 1).
For pseudomonas aeruginosa A2, inosine can improve the sensitivity to gentamicin by about 2.48 times, while IMP can improve by 15.09 times; inosine increased its sensitivity to cefoperazone by about 3.91 times, while IMP increased by 5 times. For acinetobacter baumannii 9, inosine can improve the sensitivity to gentamicin by about 1.28 times, and IMP can improve by 2.14 times; inosine increased its sensitivity to amoxicillin by about 2.95-fold, while IMP increased by 1.64-fold (fig. 2).
From the above results, there is a significant difference between inosine and inosinic acid to increase the sensitivity of bacteria to antibiotics, as shown in: (1) for the same antibiotic, inosine and inosinic acid have different effects, some bacteria have inosine effect superior to inosinic acid, and some bacteria have inosinic acid effect superior to inosine; (2) inosine and inosinic acid also have different effects on different antibiotics for the same bacterium.
Example 3 inosinic acid increases the sensitivity of Escherichia coli to ampicillin
Dividing prepared samples of Escherichia coli K12 into 3 groups, adding 7.5mM inosinic acid (IMP) and Hypoxanthine (Hypoxanthine) into 2 experimental groups, respectively, adding equal volume of normal saline into a control group, adding 100 micrograms/milliliter of ampicillin into each test tube, and repeating three biology procedures; after incubation for 6 hours at 37 ℃ in a shaker at 200rpm, 100. mu.L of the resulting suspension was serially diluted and 10. mu.L of the suspension was individually subjected to plate counting to calculate bacterial CFU/mL (colony forming units/mL). Data from 20-200 bacterial colonies can be used for statistical analysis. The survival rate of bacteria (percent survival) is CFU/initial bacteria count x 100% of bacteria after treatment with hypoxanthine compound and/or antibiotic.
As shown in FIG. 3, the survival rate of the bacteria only added with ampicillin was 75.98%, the survival rate of the bacteria decreased to 28.65% after the addition of hypoxanthine on the basis of the addition of ampicillin, and the sensitivity increased by 2.65 times; the survival rate of the bacteria is reduced to 0.41 percent and the sensitivity is improved by 182 times after the addition of the hypoxanthine nucleotide on the basis of the addition of the ampicillin. The results show that inosinic acid can greatly improve the sensitivity of Escherichia coli to ampicillin.
Example 4 inosinic acid increase of the sensitivity of Escherichia coli to ampicillin concentration-dependent on IMP
In order to research whether a gradient effect exists between the concentration of the hypoxanthine compound and the sterilization efficiency and the optimal sterilization concentration, the Escherichia coli K12 strain is taken as an object, the hypoxanthine compound (0.938 mM-15 mM) with different concentrations is added for 6 hours on the basis of adding 100 micrograms/ml of ampicillin, then viable bacteria counting is carried out, the survival rate is calculated, and the formula is that the viable bacteria number/the initial bacteria number is multiplied by 100% when the hypoxanthine compound with different concentrations is added.
Referring to fig. 4, it can be seen that the survival rate of the bacteria in the control group (i.e. without adding inosinic acid) was 69.97%, while the survival rate of the bacteria decreased from 5.97% to 0.27% and the sterilization efficiency increased from 11.7 times to 263 times as the concentration of the added inosinic acid increased. These results show that inosinic acid has concentration dependency for increasing the sensitivity of Escherichia coli to ampicillin, while inosine does not have concentration dependency, i.e., has no concentration dependency, and thus shows that the effect of inosine is insignificant.
EXAMPLE 5 inosinic acid increases the sensitivity of various bacteria to ampicillin
In order to investigate whether inosinic acid is effective against various bacteria, a study was made to improve the sensitivity of various bacteria to ampicillin by inosinic acid. Bacterial species include escherichia coli (e.coli ATCC35218), klebsiella pneumoniae (k.pneumoconiae), staphylococcus aureus multidrug resistant bacteria (MRSA), pseudomonas aeruginosa (p.aeruginosa), acinetobacter baumannii ATCC19606(a.baumannii), aeromonas hydrophila (a.hydrophila ATCC19606), edwardsiella tarda (EIB202), vibrio (vibrio alginolyticus, vibrio parahaemolyticus) and streptococcus pyogenes (s.pyogens), specific bacteria and antibiotic ampicillin dosages used are shown in table 1.
TABLE 1 bacteria and ampicillin dosages used therein
Each bacterial sample was prepared according to example 1, separately and distributed in 5mL tubes, and each bacterium was treated with a different ampicillin dose as shown in Table 1, with or without the addition of 15mM inosinic acid for 6 hours, and then viable cell count was performed and the survival rate was calculated as CFU/number of starting bacteria of inosinic acid and/or antibiotic-treated bacteria X100%.
Referring to FIG. 5, it can be seen that the ampicillin sensitivity of these bacteria was generally improved by the addition of inosinic acid as follows: escherichia coli ATCC35218 is increased by 39.77 times, Klebsiella pneumoniae is increased by 4.82 times, Staphylococcus aureus Multidrug Resistance (MRSA) is increased by 3.19 times, Pseudomonas aeruginosa is increased by 169.94, Acinetobacter baumannii is increased by 10.76 times, Aeromonas hydrophila is increased by 95 times, Edwardsiella tarda is increased by 26.88 times, Vibrio alginolyticus is increased by 21.33 times, Vibrio parahaemolyticus is increased by 4.47 times, and Streptococcus pyogenes is increased by 6.46 times.
Example 6 inosinic acid increases the sensitivity of Escherichia coli to other antibiotics
To investigate whether or not Escherichia coli is effective against antibiotics other than ampicillin after adding inosinic acid, samples of Escherichia coli K12 were prepared as in example 1, and 15mM of inosinic acid and antibiotics (antibiotics: Amoxicillin, cefoperazone, meropenem, gentamicin, balofloxacin, ciprofloxacin, aureomycin, tetracycline, chloramphenicol, and lincomycin, respectively) were added, each at the dosage shown in Table 2. Counting the number of live bacteria after 6 hours of action, and calculating the survival rate.
TABLE 2 antibiotics and dosages thereof
Antibiotic
Chinese character
Dosage used (μ g/mL)
Amoxicillin
Amoxicillin
100
Cefoperazone
Cefoperazone
100
Meropenem
Meropenem
10
Gentamicin
Gentamicin
10
Baloflocacin
Balofloxacin hydrate
10
Ciprofloxacin
Ciprofloxacin
2
Chlortetracycline
Aureomycin
200
Tetracycline
Tetracycline derivatives
20
Chloramphenicol
Chloromycetin
200
Lincomycin
Lincomycin
10
Referring to FIG. 6, it can be seen that inosinic acid can improve the sensitivity of the starting bacteria to various antibiotics as follows: the amoxicillin is improved by 89.84 times, the cefoperazone is improved by 156.47 times, the meropenem is improved by 24.37 times, the gentamicin is improved by 58.17 times, the balofloxacin is improved by 17.4 times, the ciprofloxacin is improved by 6.75 times, the aureomycin is improved by 2.98 times, the tetracycline is improved by 3.57 times, the chloramphenicol is improved by 1.37 times, and the lincomycin is improved by 1.66 times.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.