WO2022028243A1 - 复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用 - Google Patents

复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用 Download PDF

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WO2022028243A1
WO2022028243A1 PCT/CN2021/107306 CN2021107306W WO2022028243A1 WO 2022028243 A1 WO2022028243 A1 WO 2022028243A1 CN 2021107306 W CN2021107306 W CN 2021107306W WO 2022028243 A1 WO2022028243 A1 WO 2022028243A1
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bacteria
antibiotics
amino acids
compound amino
amino acid
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李惠
彭博
彭宣宪
赖诗诗
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Sun Yat Sen University
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    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedicine. More specifically, it relates to the application of compound amino acids in the preparation of medicines for improving the sensitivity of bacteria to antibiotics.
  • Amino acids as small molecular compounds that widely exist in nature, are important substances that promote human growth, maintain normal body metabolism, and provide life activities. They are also the raw materials for synthesizing antibodies, hormones, enzymes and other tissues needed by the body, and the components that make up human proteins. basic unit. Existing studies have found that the combination of some amino acids with antibiotics can improve the sensitivity of bacteria to antibiotics.
  • Chinese patent application CN107308453A discloses a new application of cystine or cysteine, combining cystine or cysteine with Combination of antibiotics can improve the sensitivity of bacteria to antibiotics and improve the bactericidal effect of antibiotics, but the effect it achieves is limited, and it is aimed at sensitive bacteria such as Escherichia coli and Staphylococcus, and for bacteria that have developed drug resistance. , the effect of single amino acid combined with antibiotics needs to be improved.
  • the technical problem to be solved by the present invention is to overcome the limited effect of combining a single amino acid with antibiotics to improve the sensitivity of bacteria to antibiotics, especially the defects and deficiencies of unsatisfactory effects on bacteria that have developed drug resistance, and to explore a new improved bacteria Protocol for antibiotic susceptibility.
  • Another object of the present invention is to provide the application of compound amino acids in the preparation of medicines for improving the sensitivity of bacteria to antibiotics.
  • the present invention proposes the application of compound amino acids in the preparation of medicines for improving the susceptibility of bacteria to antibiotics.
  • the total amino acid content of the compound amino acid is 5-12%.
  • the total amino acid content of the compound amino acid is 5-8%; more preferably, the total amino acid content of the compound amino acid is 5%.
  • the compound amino acid contains glycine, serine, tryptophan, lysine and threonine.
  • the compound amino acid contains aspartic acid, glutamic acid, serine, histidine, glycine, threonine, alanine, arginine, tyrosine, cystine, valine, methyl alcohol thionine, tryptophan, phenylalanine, isoleucine, leucine, lysine acetate, and proline.
  • the molar concentration ratio of each amino acid to the total amino acids is: aspartic acid 2.65-2.8%, glutamic acid 4.09-4.23%, serine 2.37-4.64%, histidine 4.62-4.81% %, Glycine 11.25-11.6%, Threonine 5.06-5.22%, Alanine 19.57-20.11%, Arginine 6.88-7%, Tyrosine 0.16-0.27%, Cystine 0.09-0.21%, Valine Acid 6.67 ⁇ 6.8%, Methionine 4.04 ⁇ 4.17%, Tryptophan 0.98 ⁇ 1.04%, Phenylalanine 5.12 ⁇ 5.27%, Isoleucine 4.59 ⁇ 4.74%, Leucine 6.44 ⁇ 6.45%, Acetic Acid Lysine 6.59-6.64% and proline 6.23-6.32%.
  • the compound amino acid can also contain pharmaceutically acceptable auxiliary materials, and can be made into dosage forms such as oral liquid and injection.
  • antibiotics include beta-lactam antibiotics, quinolone antibiotics (balofloxacin) and aminoglycoside antibiotics (gentamicin).
  • ⁇ -lactam antibiotics include cephalosporin antibiotics (ceftazoline, ceftriaxone, ceftiofur), penicillin antibiotics (ampicillin) and carbapenem antibiotics (imipe, ropenem, Ertapenem).
  • bacteria include susceptible bacteria and resistant bacteria.
  • bacteria include Gram-positive bacteria and Gram-negative bacteria.
  • Gram-negative bacteria include Escherichia coli, Vibrio alginolyticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa; Gram-positive bacteria include Bacillus subtilis, Staphylococcus, Streptococcus, Neisseria pneumoniae, Bacillus anthracis , Tetanus bacillus.
  • the present invention also provides a medicine for improving the sensitivity of bacteria to antibiotics, which contains an effective amount of compound amino acids and antibiotics.
  • the mass ratio of the total amino acids in the antibiotic and the compound amino acid is 1:(12.5-1824).
  • a medicine for improving the sensitivity of bacteria to antibiotics comprises 5-12% compound amino acids, 0.01-4 mg/mL antibiotics and the balance of water.
  • the medicine for improving the sensitivity of bacteria to antibiotics may also include an appropriate amount of pharmaceutically acceptable excipients to prepare preparations such as oral liquid and injection.
  • the invention discloses for the first time the application of compound amino acids in the preparation of medicines for improving the sensitivity of bacteria to antibiotics.
  • the combination of compound amino acids and antibiotics can improve the sensitivity of bacteria to antibiotics, promote the entry of antibiotics into bacterial cells, and significantly increase intracellular antibiotics.
  • the content of antibiotics can improve the bactericidal effect of antibiotics, thereby reducing the survival rate of bacteria and treating diseases caused by bacterial infection.
  • the existing compound amino acid preparation products can be directly used, which are prepared from various amino acids, sugars, electrolytes, trace elements, vitamins and pH adjusters, etc., with high purity and low nitrogen content. , few adverse reactions and almost all can be used for protein synthesis and other advantages, is the basic nitrogen supply for parenteral nutrition.
  • compound amino acids are mainly used as supplementary nutritional preparations in clinical practice, which have passed clinical trials and are safe and reliable.
  • Figure 1 is a statistical diagram of the measurement results of the minimum inhibitory concentration (MIC) of the cefazolin Escherichia coli resistant bacteria obtained by artificial passage in Example 1.
  • FIG. 2 is a statistical graph of the results of the survival rate of the three compound amino acids in Example 1 in combination with cefazolin against Escherichia coli susceptible bacteria and Escherichia coli resistant bacteria.
  • FIG. 3 is a statistical diagram of the experimental results of improving the sensitivity of sensitive bacteria K12 and resistant bacteria K12-R CZO to cefazolin by compound amino acids in Example 1.
  • FIG. 4 is a statistical diagram of the effect of different concentration gradients of compound amino acids in Example 1 on improving the sensitivity of Escherichia coli to cefazolin.
  • Figure 5 is a statistical diagram of the effect of compound amino acids in Example 1 on improving the sensitivity of Escherichia coli cefazolin-resistant bacteria to cefazolin at different concentrations.
  • FIG. 6 is a statistical graph of the effect of compound amino acids in Example 1 on improving the sensitivity of Escherichia coli cefazolin-resistant bacteria to cefazolin at different times.
  • Figure 7 is a statistical diagram of the results of the investigation of the compound amino acids in Example 1 to increase the content of intracellular antibiotics in bacteria.
  • FIG. 8 is a statistical diagram of the results of the compound amino acids in Example 2 significantly improving the bactericidal efficiency of cephalosporin antibiotics.
  • FIG. 9 is a statistical diagram of the results of the compound amino acids in Example 2 significantly improving the bactericidal efficiency of penicillin antibiotics.
  • Figure 10 is a statistical diagram of the results of the compound amino acid in Example 2 significantly improving the bactericidal efficiency of quinolone antibiotics.
  • Figure 11 is a statistical diagram of the results of the compound amino acid in Example 2 significantly improving the bactericidal efficiency of aminoglycoside antibiotics.
  • FIG. 12 Example 3 after adding compound amino acid, the statistical chart of the survival rate of various bacteria to cefazolin; A is Vibrio alginolyticus, B is Vibrio parahaemolyticus, C is Pseudomonas aeruginosa, D is Bacillus subtilis .
  • Figure 13 is a statistical diagram of the results of the determination of the resistance of clinical Escherichia coli drug-resistant bacteria to cefazolin in Example 4.
  • Figure 14 is a statistical graph of the results of the determination of the survival rate of clinical Escherichia coli drug-resistant bacteria to cefazolin after adding compound amino acids in Example 4.
  • Figure 15 is a statistical chart of the determination results of intracellular cefazolin in clinical Escherichia coli drug-resistant bacteria after adding compound amino acids in Example 4.
  • Figure 16 is a statistical graph of the results of the determination of the survival rate of clinical Escherichia coli drug-resistant bacteria to meropenem after adding compound amino acids in Example 4.
  • Figure 17 is a statistical diagram of the results of the determination of the survival rate of clinical Escherichia coli drug-resistant bacteria to levofloxacin after adding compound amino acids in Example 4.
  • Figure 18 is a statistical graph of the results of the determination of the survival rate of cefazolin against clinical bacteria when a single amino acid of the compound amino acid component of Example 5 is added alone.
  • Figure 19 is a statistical diagram of the results of the determination of the survival rate of cefazolin on clinical bacteria when the five amino acids of the compound amino acid component of Example 5 are added in combination.
  • the present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • Example 1 Compound amino acid improves the sensitivity of cefazolin-resistant Escherichia coli bacteria to cefazolin
  • test subject
  • Preparation of bacterial samples Pick sensitive K12 or drug-resistant K12-R CZO monoclonal bacteria and inoculate them in 50 mL LB liquid medium, respectively, at 37 °C, 200 rpm for 17 h, and then centrifuge at 8000 rpm for 4 min to collect bacteria; use 0.85 The bacterial cells were washed twice with % physiological saline, suspended in 1 ⁇ M9 medium, adjusted to an OD 600 value of 0.6, and then dispensed into 5 mL test tubes for subsequent experimental research.
  • Compound amino acids with a total amino acid content of 5%, 8.5%, and 11.4% were configured as a solution with a final molar concentration of 2 mM total amino acids.
  • 2mg/mL) acted on bacteria for 6 hours, take 100 ⁇ L for doubling dilution, and take 10 ⁇ L of bacterial solution for plate counting; the data of colonies in the 20-200 range can be used for statistical analysis, and the bacterial survival rate is the bacterial CFU (colony in the treatment group). Forming units/ml) as a percentage of bacterial CFU in the control group, 3 biological replicates for each treatment; see Figure 2 for results.
  • the prepared bacterial samples were divided into 4 groups: control group, only 2mM 5% compound amino acid group, and only cefazolin antibiotic group (the concentration of K12 for sensitive bacteria was 80 ⁇ g/mL, and the concentration of K12-R CZO for resistant bacteria was 80 ⁇ g/mL). 2mg/mL) and cefazolin and 2mM amino acid compound group.
  • the bacteria were incubated at 37° C. and 200 rpm for 6 h, and then 100 ⁇ L was taken for doubling dilution, and 10 ⁇ L of bacterial liquid was taken from it for plate count, and the bacterial survival rate was calculated; the results are shown in FIG. 3 .
  • compound amino acids combined with cefazolin resulted in a 320-fold reduction in the survival rate of sensitive bacteria K12, and a 3,556-fold reduction in the survival rate of resistant bacteria K12-R CZO , indicating that compound amino acids can improve the resistance of bacteria, including artificially passaged resistant bacteria, to cefazolin. Sensitivity to morpholino.
  • the survival rate of Escherichia coli K12 was 89.6%, 54.9%, 0.705 after adding 0, 0.125, 0.5, 2.0, 8.0, 32, and 64 mM compound amino acids in the case of 80 ⁇ g/mL cefazolin. %, 0.0341%, 0.00301%, 0.00247%, 0.00104%; in the case of cefazolin Escherichia coli-resistant bacteria at 1,000 ⁇ g/mL cefazolin, the survival rates were 92.6%, 71.4%, 20.4%, 0.0211%, 0.0181%, 0.0167%, 0.0146%.
  • the sterilization times increased by 1.12 times, 1.68 times, 2.32 times, 674 times, 3,661 times, 4,909 times, and 4,527 times, respectively.
  • the concentration of cefazolin from 62.5 ⁇ g/mL to 2,000 ⁇ g/mL can make the bactericidal rate from 1.12x improved to 4,909x.
  • Bacterial death is related to the number of antibiotics that enter the bacterial cell, and a key reason for bacterial resistance is that the concentration of antibiotics entering the bacterial body is lower than the concentration that makes it dead. Therefore, the content of cefazolin in bacterial cells after adding compound amino acid was determined.
  • the prepared bacterial samples were added with 2mM compound amino acid and cefazolin (80 ⁇ g/mL for Escherichia coli K12 and 1,000 ⁇ g/mL for cefazolin Escherichia coli), incubated at 37°C, 200rpm for 6h; then The cells were collected by centrifugation, washed twice with normal saline, and the OD 600 of the bacteria was adjusted to 0.6; 30 mL of cells were collected, and after washing, an aqueous solution of acetonitrile with a volume ratio of 1:1 was added, and ultrasonically crushed (power 30%, crushed for 2 s, stopped for 3 s, totaling 4 min), centrifuged to take the supernatant, repeated centrifugation to take the supernatant, filtered through a 0.22 ⁇ m filter membrane, and detected by LC/MS. The results are shown in FIG. 7 .
  • Example 2 Compound amino acids improve the sensitivity of bacteria to antibiotics and have antibiotic specificity
  • cephalosporin antibiotics (10 ⁇ g/mL ceftriaxone, 200 ⁇ g/mL ceftiofur) acted on Escherichia coli K12 for 6 hours, and the bacterial survival rate effect; see Figure 8 for the results.
  • the survival rate of sensitive bacteria K12 decreased with the increase of compound amino acid concentration, which were 100%, 86.7%, 67.1%, 32.5%, 0.613%, 0.0516%, 0.00722% and 0.00493% in turn; ampicillin-resistant bacteria ( The survival rates of K12- RAMP ) were 100%, 87.2%, 56.1%, 28.9%, 0.109%, 0.0354%, 0.0101% and 0.00946%, respectively.
  • Adding compound amino acids reduced the survival rate of sensitive bacteria K12 by 1.29 times to 7,578 times, and decreased the survival rate of resistant bacteria K12-RAMP by 1.55 times to 9,314 times.
  • Example 3 The effect of compound amino acids in synergy with cefazolin on the survival rate of other species of bacteria
  • Example 4 Compound amino acid improves the sensitivity of clinical Escherichia coli resistant bacteria to cefazolin
  • Escherichia coli is one of the most common bacteria in the clinic.
  • the 35 strains of Escherichia coli obtained from the clinic are named Y1-12, Y15-24, Y27, Y28, respectively. Y30-40.
  • the minimum inhibitory concentrations (MICs) of these 35 clinically obtained Escherichia coli strains against cefazolin antibiotics were detected according to the NCCLS method. MIC was measured; the results of the measurement are shown in Figure 13 .
  • the MIC of 35 clinical strains of Escherichia coli was higher than that of Escherichia coli K12 (1.25 ⁇ g/mL) in 91.4% of the clinical strains.
  • the MIC of Y3, Y7, Y23 is 2.5 ⁇ g/mL
  • the MIC of Y8, Y15, Y16, Y19, Y22 is 5 ⁇ g/mL
  • the MIC of Y5 Y11, Y17 is 10 ⁇ g/mL, Y18, Y20, Y21, Y24
  • the MIC of Y40 is 20 ⁇ g/mL
  • the MIC of Y1, Y6, Y12, and Y12 are 40 ⁇ g/mL
  • the MIC of the remaining 37% of clinical Escherichia coli resistant bacteria is greater than 640 ⁇ g/mL, of which Y28, Y32, Y36 are as high as 5120 ⁇ g/mL.
  • cefazolin used was 8,000 ⁇ g/mL.
  • compound amino acids increased the sterilization ratio in order 23.1 times, 57.7 times, 4.2 times, 11.2 times, 1.7 times, 1.5 times, 4.4 times, 15.7 times, 16.4 times, 2.2 times, 2.1 times, 2.1 times and 68.3 times.
  • the antibiotic content in Y4, 16 and Y32 bacteria was determined by the method of 1.2.4, and the results are shown in Figure 15.
  • the antibiotic content of Y4 increased from 157.4ng/mL to 339.8ng/mL, and the intracellular antibiotic content increased by 182.4ng/mL; Y16 antibiotic content increased from 377.2ng/mL to 708.7ng/mL, The intracellular antibiotic content increased by 331.5ng/mL; the Y32 antibiotic content increased from 52.5ng/mL to 57.4ng/mL, and the intracellular antibiotic content increased by 4.9ng/mL.
  • the bactericidal multiples of compound amino acid synergistic antibiotics are 1.6 times, 8,085.7 times, 41.0 times, 175.5 times, 88.2 times, 30384.6 times, 485 times, 120.0 times and 25.9 times, respectively.
  • the bactericidal multiples of compound amino acid synergistic antibiotics are 1,226.7 times, 3.6 times, 2.3 times, 19.3 times, 8.3 times, 16.0 times, 326.7 times and 39.5 times respectively.
  • the bacterial survival rate is 85.04% when only cefazolin is added, and the survival rate when each amino acid in the 18 amino acids is added separately at the corresponding molar concentration is: alanine 404 ⁇ M (73.6%), valine Acid 136 ⁇ M (59.4%) M (78.8%), Leucine 95 ⁇ M (75.7%), Isoleucine 95 ⁇ M (69.6%), Proline 126 ⁇ M (85.4%), Phenylalanine 106 ⁇ M (98.8%), Tryptophan 20 ⁇ M (67.4%), Methionine 83 ⁇ M (91.5%), Glycine 233 ⁇ M (71.4%), Serine 47 ⁇ M (67.9%), Threonine, Tyrosine 5 ⁇ M (80.5%), Lysine Acetate 133 ⁇ M (68.6%), Arginine 140 ⁇ M (97.3%), Histidine 96 ⁇ M (97.6%), Aspartic acid 56 ⁇ M (94.5
  • the bacterial survival rate of the M9 control group added with antibiotics was 45.9%; the bacterial survival rate of the compound amino acid group after synergistic antibiotics was 0.001%, while the 5 amino acid combination group, after synergistic antibiotics, the bacterial survival rate was 0.15%, which was higher than the compound group.
  • the amino acid synergy fold is 148-fold lower.

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Abstract

本发明属于生物医药技术领域，具体涉及复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用。实验证明，将复方氨基酸与抗生素联用，可以提高细菌对抗生素的敏感性，并促进抗生素进入细菌细胞内，显著增加细胞内抗生素的含量，提高抗生素的杀菌效果，从而降低细菌生存率，治疗细菌侵染引起的疾病。

Description

复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用 技术领域
本发明属于生物医药技术领域。更具体地，涉及复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用。
背景技术
二十世纪初，抗生素的发现和应用使细菌感染疾病得到控制。然而，耐药菌、超级细菌的出现，抗生素治疗细菌性疾病出现疗效降低甚至无效的情况，人类又面临着新的挑战。目前细菌产生耐药性的速度逐渐加快，而新药研发的难度和时间都在逐渐增加，照此下去，在不久的将来很可能会出现无药可用的危险局面，严重威胁人类健康和生命安全。
氨基酸作为广泛存在于自然界中的小分子化合物，是促进人体生长、维持身体正常代谢、提供生命活动的重要物质，也是合成机体所需抗体、激素、酶类和其他组织的原料和构成人体蛋白质的基本单位。现有研究发现，一些氨基酸与抗生素联用，可以提高细菌对抗生素的敏感性，如中国专利申请CN107308453A公开了胱氨酸或半胱氨酸的新应用，将胱氨酸或半胱氨酸和抗生素联用可以提高细菌对抗生素的敏感性，提高抗生素的杀菌作用，但是其所达到的效果有限，并且针对的是大肠杆菌、葡萄球菌等细菌的敏感菌，而对于已经产生耐药性的细菌，单个氨基酸与抗生素联用所达到的效果还有待提高。
发明内容
本发明要解决的技术问题是克服单一氨基酸与抗生素联用提高细菌对抗生素敏感性的效果有限，特别是对已经产生耐药性的细菌效果不理想的缺陷和不足，探索一种新的提高细菌对抗生素敏感性的方案。
本发明的另一目的在于提供复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用。
本发明上述目的通过以下技术方案实现：
我们前期研究显示，丙氨酸、谷氨酰胺、L-天冬氨酸具有提高细菌对抗生素的敏感性的作用，然而并非所有氨基酸都有此作用，同时各种不同氨基酸作用于 机体后，发挥提高细菌对抗生素的敏感性的作用机理不尽相同，因此是否可以通过多种氨基酸的复配从而实现更大幅度地提升细菌对抗生素敏感性的能力，无法预料。我们经过大量的探索研究，组成了一种复方氨基酸组合，能够显著提升细菌对抗生素的敏感性，并促进抗生素进入细菌细胞内，显著增加细胞内抗生素的含量，提高抗生素的杀菌效果，从而降低细菌生存率，治疗细菌侵染引起的疾病，将复方氨基酸制剂应用于制备提高细菌对抗生素敏感性药物中，效果非常好。
因此，本发明提出复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用。
进一步地，所述复方氨基酸的总氨基酸含量为5～12％。优选地，所述复方氨基酸的总氨基酸含量为5～8％；更优选地，所述复方氨基酸的总氨基酸含量为5％。
更进一步地，所述复方氨基酸含有甘氨酸、丝氨酸、色氨酸、赖氨酸和苏氨酸。
优选地，所述复方氨基酸含有天门冬氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、苏氨酸、丙氨酸、精氨酸、酪氨酸、胱氨酸、缬氨酸、甲硫氨酸、色氨酸、苯丙氨酸、异亮氨酸、亮氨酸、醋酸赖氨酸和脯氨酸。
进一步地，所述复方氨基酸中，每种氨基酸占总氨基酸的摩尔浓度比为：天门冬氨酸2.65～2.8％、谷氨酸4.09～4.23％、丝氨酸2.37～4.64％、组氨酸4.62～4.81％、甘氨酸11.25～11.6％、苏氨酸5.06～5.22％、丙氨酸19.57～20.11％、精氨酸6.88～7％、酪氨酸0.16～0.27％、胱氨酸0.09～0.21％、缬氨酸6.67～6.8％、甲硫氨酸4.04～4.17％、色氨酸0.98～1.04％、苯丙氨酸5.12～5.27％、异亮氨酸4.59～4.74％、亮氨酸6.44～6.45％，醋酸赖氨酸6.59～6.64％和脯氨酸6.23～6.32％。
所述复方氨基酸还可以含有药学上可接受的辅料，制成口服液、注射剂等剂型。
更进一步地，所述抗生素包括β-内酰胺类抗生素、喹诺酮类抗生素(巴洛沙星)和氨基糖苷类抗生素(庆大霉素)。
其中，β-内酰胺类抗生素包括头孢菌素类抗生素(头孢唑啉、头孢曲松、头孢噻呋)、青霉素类抗生素(氨苄青霉素)和碳青霉烯类抗生素(亚胺培、南美罗培南、厄他培南)。
进一步地，所述细菌包括敏感菌和耐药菌。
更进一步地，所述细菌包括革兰氏阳性菌和革兰氏阴性菌。
其中，革兰氏阴性菌包括大肠杆菌、溶藻弧菌、副溶血弧菌、铜绿假单胞杆菌；革兰氏阳性菌包括菌枯草芽孢杆菌、葡萄球菌、链球菌、肺炎双球菌、炭疽杆菌、破伤风杆菌。
另外的，本发明还提供了一种提高细菌对抗生素敏感性的药物，含有有效量的复方氨基酸和抗生素。
进一步地，所述抗生素和复方氨基酸中总氨基酸的质量比为1:(12.5～1824)。
一种提高细菌对抗生素敏感性的药物，包括5～12％复方氨基酸、0.01～4mg/mL抗生素和余量的水。所述提高细菌对抗生素敏感性的药物还可以包括适量的药学上可接受的辅料，制成口服液、注射剂等制剂。
本发明具有以下有益效果：
本发明首次公开了复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用，将复方氨基酸与抗生素联用，可以提高细菌对抗生素的敏感性，并促进抗生素进入细菌细胞内，显著增加细胞内抗生素的含量，提高抗生素的杀菌效果，从而降低细菌生存率，治疗细菌侵染引起的疾病。
同时，基于本发明的方案，可直接利用现有的复方氨基酸制剂产品，由多种氨基酸、糖、电解质、微量元素、维生素及pH值调节剂等配制而成，具有纯度高、含氮量低、不良反应少和几乎全部可被利用于蛋白质合成等优点，是肠外营养的基本供氮物。目前复方氨基酸在临床上主要作为补充营养制剂，已通过临床试验，安全可靠。
附图说明
图1为实施例1对人工传代获得的头孢唑啉大肠杆菌耐药菌最小抑菌浓度(MIC)的测定结果统计图。
图2为实施例1三种复方氨基酸协同头孢唑啉对大肠杆菌敏感菌及大肠杆菌耐药菌的生存率的结果统计图。
图3为实施例1复方氨基酸提高敏感菌K12和耐药菌K12-R CZO对头孢唑啉的敏感性考察实验结果统计图。
图4为实施例1复方氨基酸不同浓度梯度提高大肠杆菌对头孢唑啉敏感性影响结果统计图。
图5为实施例1复方氨基酸提高大肠杆菌头孢唑啉耐药菌对不同浓度头孢唑啉敏感性影响结果统计图。
图6为实施例1复方氨基酸提高大肠杆菌头孢唑啉耐药菌对头孢唑啉不同时间敏感性影响结果统计图。
图7为实施例1复方氨基酸增加细菌胞内抗生素的含量考察结果统计图。
图8为实施例2复方氨基酸显著提高头孢菌素类抗生素的杀菌效率结果统计图。
图9为实施例2复方氨基酸显著提高青霉素类抗生素的杀菌效率结果统计图。
图10为实施例2复方氨基酸显著提高喹诺酮类抗生素的杀菌效率结果统计图。
图11为实施例2复方氨基酸显著提高氨基糖苷类抗生素的杀菌效率结果统计图。
图12实施例3添加复方氨基酸后，多种细菌对头孢唑啉的生存率结果统计图；A为溶藻弧菌，B为副溶血弧菌，C为铜绿假单胞杆菌，D为枯草杆菌。
图13为实施例4临床大肠杆菌耐药菌对头孢唑啉耐药性测定结果统计图。
图14为实施例4添加复方氨基酸后，临床大肠杆菌耐药菌对头孢唑啉的生存率测定结果统计图。
图15为实施例4添加复方氨基酸后，临床大肠杆菌耐药菌胞内头孢唑啉的含量测定结果统计图。
图16为实施例4添加复方氨基酸后，临床大肠杆菌耐药菌对美罗培南的生存率测定结果统计图。
图17为实施例4添加复方氨基酸后，临床大肠杆菌耐药菌对左氧氟沙星的生存率测定结果统计图。
图18为实施例5复方氨基酸组分单一氨基酸单独添加时头孢唑啉对临床菌的生存率测定结果统计图。
图19为实施例5复方氨基酸组分5种氨基酸组合添加时头孢唑啉对临床菌的生存率测定结果统计图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技 术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1复方氨基酸提高头孢唑啉大肠埃希耐药菌对头孢唑啉的敏感性
1.1人工传代耐药菌-头孢唑啉大肠埃希耐药菌的获得
以大肠埃希敏感菌K12为起始菌株，在含1/2最小抑菌浓度(MIC)的头孢唑啉液体培养基中连续传代，每隔五代在平板上划线，获得单克隆并测定其对头孢唑啉的MIC；直至获得菌株的MIC为80μg/mL，即是起始菌株的64倍(具体参见图1)，将其命名为头孢唑啉大肠埃希耐药菌(K12-R CZO)。
1.2复方氨基酸提高大肠埃希敏感菌及头孢唑啉大肠埃希耐药菌对头孢唑啉敏感性
实验材料：三种总氨基酸含量分别为5％、8.5％、11.4％的复方氨基酸，三种复方氨基酸中每种氨基酸占总氨基酸的摩尔浓度比参见表1。
表1三种复方氨基酸中每种氨基酸占总氨基酸的摩尔浓度比
氨基酸 5％复方氨基酸 8.5％复方氨基酸 11.4％复方氨基酸
天门冬氨酸 2.80％ 2.69％ 2.65％
谷氨酸 4.23％ 4.09％ 4.14％
丝氨酸 2.37％ 4.64％ 4.58％
组氨酸 4.81％ 4.62％ 4.69％
甘氨酸 11.60％ 11.27％ 11.25％
苏氨酸 5.22％ 5.06％ 5.12％
丙氨酸 20.11％ 19.65％ 19.57％
精氨酸 7.00％ 6.92％ 6.88％
酪氨酸 0.27％ 0.16％ 0.18％
胱氨酸 0.21％ 0.12％ 0.09％
缬氨酸 6.80％ 6.74％ 6.67％
甲硫氨酸 4.17％ 4.04％ 4.09％
色氨酸 1.04％ 0.98％ 1.00％
苯丙氨酸 5.27％ 5.12％ 5.12％
异亮氨酸 4.74％ 4.59％ 4.65％
亮氨酸 6.45％ 6.45％ 6.44％
醋酸赖氨酸 6.64％ 6.61％ 6.59％
脯氨酸 6.27％ 6.23％ 6.32％
实验对象：
细菌样本的制备：挑取敏感菌K12或耐药菌K12-R CZO单克隆细菌，分别接种于50mL LB液体培养基中，37℃，200rpm培养17h后，8000rpm，4min离心收集菌体；用0.85％生理盐水洗涤菌体2次，悬浮于1×M9培养基中，调OD 600值为0.6，然后分装5mL于试管中，用于后续试验研究。
1.2.1复方氨基酸提高敏感菌K12和耐药菌K12-R CZO对头孢唑啉的敏感性
将总氨基酸含量分别为5％、8.5％、11.4％的复方氨基酸配置为最终总氨基酸摩尔浓度均为2mM的溶液，协同头孢唑啉(敏感菌使用浓度为80μg/mL，耐药菌使用浓度为2mg/mL)作用于细菌6小时后，取100μL进行倍比稀释，从中取10μL菌液进行平板计数；菌落在20-200区间的数据可用于统计分析，细菌存活率是处理组细菌CFU(菌落形成单位/毫升)与对照组细菌CFU的百分比，每种处理3个生物学重复；结果参见图2。
由图可见，5％、8.5％和11.4％的复方氨基酸协同头孢唑啉作用于敏感菌K12的生存率分别降低320倍、310倍和274倍；作用于耐药菌K12-R CZO的生存率分别降低3,556倍、4,299倍和4,273倍。说明三种配比的复方氨基酸均能有效恢复细菌对头孢唑啉敏感性，并且三者之间的协同杀菌效果无显著性差异。因此，后续实验采用最低浓度配比的5％复方氨基酸进行研究。
1.2.2 5％复方氨基酸提高敏感菌K12和耐药菌K12-R CZO对头孢唑啉的敏感性
将制备好的每种细菌样本分成4组：对照组，只加2mM 5％复方氨基酸组，只加头孢唑啉抗生素组(敏感菌K12浓度为80μg/mL，耐药菌K12-R CZO浓度为2mg/mL)和头孢唑啉和2mM复方氨基酸组。将细菌于37℃，200rpm孵育6h，然后取100μL进行倍比稀释，从中取10μL菌液进行平板计数，计算细菌存活率；结果参见图3。
由图可见，复方氨基酸协同头孢唑啉导致敏感菌K12的生存率降低320倍，耐药菌K12-R CZO的生存率降低3,556倍，说明复方氨基酸可提高细菌包括人工传代耐药菌对头孢唑啉的敏感性。
1.2.3复方氨基酸提高头孢唑啉耐药菌对头孢唑啉敏感性的重要条件研究
1.2.3.1复方氨基酸提高大肠埃希菌对头孢唑啉敏感性具有浓度依赖性
为了解复方氨基酸浓度与杀菌效率之间是否存在梯度效应以及其最佳杀菌浓度，在固定头孢唑啉浓度(大肠埃希敏感菌K12为80μg/mL，头孢唑啉大肠埃希耐药菌为1,000μg/mL)的基础上加入不同浓度的复方氨基酸(0～64mM)作用6h；然后，进行活菌计数，并计算生存率，计算公式为：添加不同浓度复方氨基酸时活菌数/不添加复方氨基酸时活菌数×100％；结果参见图4。
由图可见，大肠埃希敏感菌K12在80μg/mL头孢唑啉情况下，添加0、0.125、0.5、2.0、8.0、32、64mM复方氨基酸后，其生存率依次为89.6％、54.9％、0.705％、0.0341％、0.00301％、0.00247％、0.00104％；头孢唑啉大肠埃希耐药菌在1,000μg/mL头孢唑啉情况下，生存率依次为92.6％、71.4％、20.4％、0.0211％、0.0181％、0.0167％、0.0146％。头孢唑啉敏感菌在复合氨基酸浓度为8mM时，与单独添加抗生素相比，协同杀菌倍数达29,807倍，继续增大氨基酸浓度，促进杀菌效果不再明显增强；头孢唑啉耐药菌在复合氨基酸浓度为2mM时，与单独添加抗生素相比，协同杀菌倍数达4,396倍，并且继续增大复方氨基酸浓度，促进杀菌效果不再明显增强。可见，为了提高耐药菌对抗生素的敏感性，2mM复方氨基酸可作为最佳协同杀菌浓度。
1.2.3.2复方氨基酸提高耐药菌对头孢唑啉敏感性具有抗生素浓度梯度效应
为探讨复方氨基酸提高耐药菌对抗生素的敏感性与抗生素浓度的关系，设置了8个头孢唑啉浓度梯度(0、62.5、125、250、500、1,000、2,000和4,000μg/mL)；然后，分别添加和不添加2mM复方氨基酸，6h后采用平板法进行活菌计数；比较在相同抗生素浓度情况下，未添加复方氨基酸(对照)和添加复方氨基酸后细菌的生存率；计算公式为：添加或不添加复方氨基酸时活菌数/6h后在M9中活菌数×100％；结果参见图5。
由图可见，在加入2mM复方氨基酸后，随着头孢唑啉浓度的升高，对耐药菌K12-R CZO杀菌效率大大提高。在不加复合氨基酸的62.5、125、250、500、1,000、2,000和4,000μg/mL头孢唑啉浓度下导致细菌的生存率依次为：87.5％、96.9％、83.3％、78.6％、83.3％、84.4％、69.8％；在添加复合氨基酸相应浓度头孢唑啉导致细菌的生存率依次为：78.1％、57.8％、35.9％、0.117％、0.0228％、0.0172％和0.0154％。添加复合氨基酸后增加杀菌倍数分别是1.12倍、1.68倍、2.32倍、674 倍、3,661倍、4,909倍、4,527倍，而头孢唑啉浓度从62.5μg/mL至2,000μg/mL可以使杀菌率从1.12倍提高至4,909倍。这些结果表明，头孢唑啉浓度在1000μg/mL以内，复方氨基酸促进杀菌能力随抗生素增加而显著增强；同时可知，1000μg/mL为头孢唑啉对耐药菌K12-R CZO的最佳杀菌浓度。
1.2.3.3复方氨基酸提高耐药菌对头孢唑啉敏感性的时间效应
为进一步了解复方氨基酸提高耐药菌对抗生素敏感性的作用是否具有时间效应，在添加2mM复方氨基酸和1000μg/mL头孢唑啉情况下，分别在1～48h内进行活菌计数，观察其杀菌效率与时间的关系，结果参见图6。
由图可见，头孢唑啉耐药菌K12-R CZO在添加头孢唑啉基础上再添加复方氨基酸后，活菌数随着时间的延长明显下降：从0、2、4、6、8、10、12、24、36和48h，细菌生存率依次为100％，18.2％，0.106％，0.0563％，0.0261％，0.0198％，0.00716％，0.00227％，0.00127％，0.000208％；复方氨基酸的协同杀菌倍数依次为1倍、4.97倍、765.3倍、1,297.3倍、2,483.3倍、3,201.6倍、6,951.2倍、6,713.8倍、10,632.0倍和50,000.0倍。以上结果说明，随着作用时间的不断延长，细菌生存率持续下降，4h复方氨基酸协同组的细菌生存率直线下降达两个数量级，并且在6h后到10h之前细菌生存率稳定在同一个数量级内，因此选择6h作为孵育时间，进行后续实验。
1.2.4复方氨基酸增加细菌胞内抗生素的含量
细菌死亡与进入细菌胞内的抗生素数量有关，细菌耐药的一个关键原因是由于进入细菌体内的抗生素浓度低于使其死亡的浓度。因此测定了添加复方氨基酸后细菌胞内的头孢唑啉含量。
将制备好的细菌样本加入2mM复方氨基酸和头孢唑啉(大肠埃希敏感菌K12为80μg/mL，头孢唑啉大肠埃希耐药菌为1,000μg/mL)，37℃，200rpm孵育6h；然后离心收集菌体，生理盐水洗涤两次，调细菌OD 600为0.6；收集30mL菌体，洗涤后加入体积比为1：1的乙腈水溶液，超声波破碎(功率30％，破碎2s，停3s，总计4min)，离心取上清，重复离心再取上清，用0.22μm滤膜过滤处理后，用LC/MS检测，结果参见图7。
复方氨基酸浓度为0、0.5、2、8mM与80μg/mL头孢唑啉协同处理敏感菌K12后，细菌生存率分别为：89.6％、0.705％、0.0341％和0.00301％(结果详见图4)；图7中对应的胞内抗生素含量分别为：48.2ng/mL、70.2ng/mL、101.6ng/mL和149.5ng/mL。
复合氨基酸浓度为0、0.5、2、8mM的复方氨基酸协同1000μg/mL头孢唑啉处理耐药菌K12-R CZO后，细菌生存率分别为92.6％、20.4％、0.0211％和0.0181％(结果详见图4)，图7中对应胞内抗生素含量分别为58.7ng/mL、141.8ng/mL、182.8ng/mL、189.3ng/mL。
上述结果表明，随着复合氨基酸浓度增加，胞内抗生素含量上升，细菌生存率下降。
实施例2复方氨基酸提高细菌对抗生素的敏感性具有抗生素特异性
2.1显著提高头孢菌素类抗生素的杀菌效率
进一步研究添加0.125、0.5、2.0、8.0、32、64mM复方氨基酸后，头孢类抗生素(10μg/mL头孢曲松、200μg/mL头孢噻呋)作用大肠埃希敏感菌K12 6h后，对细菌生存率的影响；结果参见图8。
由图可见，0.125、0.5、2.0、8.0、32、64mM复方氨基酸协同头孢曲松后，细菌生存率依次为73.1％、54.3％、13.5％、0.115％、0.00827％、0.00345％和0.00173％，K12生存率降低倍数达1.35倍～42352倍；协同头孢噻呋后，细菌生存率依次为61.9％、77.7％、20.7％、0.00907％、0.00589％、0.00213％和0.000711％，敏感菌K12生存率降低倍数达0.797倍～87143倍。这些结果说明复方氨基酸可以提高细菌对头孢类抗生素的敏感性。
2.2显著提高青霉素类抗生素的杀菌效率
以大肠埃希菌敏感菌K12和氨苄青霉素人工传代耐药菌(K12-R AMP)为研究对象，前者氨苄青霉素浓度为40μg/mL，后者氨苄青霉素浓度为2000μg/mL。考察0.125、0.5、2.0、8.0、32、64mM复方氨基酸分别协同上述浓度抗生素对敏感菌和相应耐药菌生存率的影响，结果参见图9。
由图可见，敏感菌K12的生存率随复合氨基酸浓度增加而下降，依次为100％、86.7％、67.1％、32.5％、0.613％、0.0516％、0.00722％和0.00493％；氨苄青霉素耐药菌(K12-R AMP)的生存率依次为100％、87.2％、56.1％、28.9％、0.109％、0.0354％、0.0101％和0.00946％。添加复方氨基酸降低敏感菌K12的生存率倍数达1.29倍～7,578倍；降低耐药菌K12-R AMP的生存率达1.55倍～9,314倍。
2.3提高对喹诺酮类抗生素的杀菌效率
以大肠埃希敏感菌K12和巴洛沙星人工传代耐药菌(K12-R BLFX)为研究对 象，前者巴洛沙星浓度为0.2μg/mL，后者巴洛沙星浓度为25μg/mL。考察不同浓度复方氨基酸分别协同上述浓度抗生素对敏感菌和相应耐药菌生存率的影响，结果参见图10。
由图可见，0.125、0.5、2.0、8.0、32、64mM复方氨基酸协同巴洛沙星导致敏感菌K12的生存率依次为100％、80.9％、97.1％、117.9％、113.3％、71.7％、65.9％和64.7％；导致巴洛沙星耐药菌(K12-R BLFX)的生存率依次为100％、90.2％、85.4％、85.4％、80.8％、59.9％、10.4％和13.4％。结果表明仅在复方氨基酸浓度大于8mM时，对巴洛沙星杀菌有一定的促进效果。
2.4提高对氨基糖苷类抗生素的耐药性
以大肠埃希敏感菌K12和庆大霉素人工传代耐药菌(K12-R Gen)为研究对象，前者庆大霉素1μg/mL，后者庆大霉素8μg/mL。考察不同浓度复方氨基酸分别协同上述浓度抗生素对敏感菌和相应耐药菌生存率的影响，结果参见图11。
由图可见，0.125、0.5、2.0、8.0、32、64mM复方氨基酸协同氨基糖苷类抗生素庆大霉素，导致敏感菌K12的生存率依次为100％、33.9％、15.9％、48.6％、71.2％、140.1％和151.4％；导致庆大霉素耐药菌(K12-R Gen)的生存率依次为100％、8.20％、11.7％、54.6％、207.7％、269.2％、和302.3％。结果表明在庆大霉素作用下，高浓度的复方氨基酸对敏感菌和耐药菌有明显的保护作用。
实施例3复方氨基酸协同头孢唑啉对其他种类细菌生存率的影响
选择其他革兰氏阴性菌如溶藻弧菌、副溶血弧菌和铜绿假单胞杆菌，以及革兰氏阳性菌枯草芽孢杆菌为研究对象，考察复方氨基酸协同头孢唑啉对它们的杀菌效果；其中，复合氨基酸浓度为2mM，头孢唑啉为80μg/mL，孵育6h，计算细菌的相对生存率；结果参见图12。
由图可见，2mM复方氨基酸添加后，头孢唑啉导致溶藻弧菌的生存率从62.1％降低为13.9％，协同倍数达4.5倍(图12A)；导致副溶血弧菌的生存率从79.3％降低为1.54％，协同倍数达51.6倍(图12B)；导致铜绿假单胞杆菌的生存率从85.9％降低为5.4％，协同倍数达15.8倍(图12C)；导致枯草杆菌的生存率从58.0％降低为5.1％，协同倍数达11.3倍(图12D)。这些结果说明复方氨基酸协同头孢唑啉的杀菌作用对其他种类细菌亦有效果。
实施例4复方氨基酸提高临床大肠埃希耐药菌对头孢唑啉的敏感性
4.1临床大肠埃希耐药菌对头孢唑啉的耐药性分析
大肠埃希菌(Escherichia coli，E.coli)是临床中最常见的一种细菌，将从 临床上获得的35株大肠埃希菌，分别命名为Y1-12，Y15-24，Y27，Y28，Y30-40。根据NCCLS方法检测这35株临床获得的大肠埃希菌对头孢唑啉抗生素的最小抑菌浓度(MIC)，同时用大肠埃希敏感菌K12以及头孢唑啉耐药菌K12-R CZO作为对照，测定MIC；测定结果见图13。
由图13可见，35株临床大肠埃希菌的MIC与大肠埃希敏感菌K12相比，91.4％临床菌的MIC都比K12(1.25μg/mL)高。其中，Y3、Y7、Y23的MIC为2.5μg/mL，Y8、Y15、Y16、Y19、Y22的MIC为5μg/mL，Y5、Y11、Y17的MIC为10μg/mL，Y18、Y20、Y21、Y24、Y40的MIC为20μg/mL，Y1、Y6、Y12、的MIC为40μg/mL，其余37％的临床大肠埃希耐药菌的MIC均大于640μg/mL，其中Y28、Y32、Y36高达5120μg/mL。结果表明临床大肠埃希菌中，82.8％的菌株对头孢唑啉的MIC高敏感菌4倍以上，表明对头孢唑啉耐药。
4.2复方氨基酸提高临床菌对头孢唑啉的敏感性
参考1.2类似的方法制备临床耐药菌样本，分装5mL于试管中，在添加2mM复方氨基酸基础上，添加和不添加头孢唑啉孵育6h，然后进行活菌计数，并计算生存率；计算公式为：添加抗生素时活菌数/不添加抗生素时活菌数×100％；结果参见图14。
由图可见，使用头孢唑啉浓度为200μg/mL，对于MIC较低的Y1、Y3、Y4、Y5、Y6、Y7、Y8、Y9、Y10、Y11、Y12、Y13、Y15、Y16、Y17、Y18、Y19、Y20、Y21、Y23、Y24、Y40菌株，复方氨基酸提高杀菌倍数依次为138.6倍、1,881.7倍、16,521.7倍、91.3倍、1,326.9倍、13,700倍、1,182.8倍、5,128.2倍、3,043.5倍、723倍、2,547.1倍、284.7倍、48.1倍、166.7倍、108.3倍、337.4倍、4.6倍、1,867.5倍、3,176.5倍、16.3倍、3,331.0倍和5,444.4倍。
使用的头孢唑啉浓度为8,000μg/mL，对于MIC较高的Y2、Y27、Y28、Y30、Y31、Y32、Y33、Y34、Y35、Y36、Y37、Y38、Y39菌株，复方氨基酸提高杀菌倍数依次为23.1倍、57.7倍、4.2倍、11.2倍、1.7倍、1.5倍、4.4倍、15.7倍、16.4倍、2.2倍、2.1倍、2.1倍和68.3倍。
上述结果表明复方氨基酸能有效恢复临床耐药大肠杆菌对头孢唑啉的敏感性，其倍数与细菌本身耐药性程度有关。
4.3复方氨基酸增加临床耐药菌胞内抗生素的含量
采用1.2.4方法测定Y4、16和Y32菌内的抗生素含量，结果参见图15。
由图可见，复方氨基酸添加后，Y4抗生素含量从157.4ng/mL增加至339.8ng/mL，胞内抗生素含量增加了182.4ng/mL；Y16抗生素含量从377.2ng/mL增加至708.7ng/mL，胞内抗生素含量增加了331.5ng/mL；Y32抗生素含量从52.5ng/mL增加至57.4ng/mL，胞内抗生素含量增加了4.9ng/mL。
4.4复方氨基酸提高临床菌对其他抗生素的敏感性
4.4.1提高临床菌对美罗培南的敏感性
随机选取Y2、Y6、Y16、Y17、Y21、Y23、Y27、Y31、Y35等9株临床菌株为研究对象，使用2mM复方氨基酸及40μg/mL美罗培南进行处理，结果参见图16。
由图可见，复方氨基酸协同抗生素杀菌倍数分别是1.6倍、8,085.7倍、41.0倍、175.5倍、88.2倍、30384.6倍、485倍、120.0倍和25.9倍。
4.4.2提高临床菌对喹诺酮类左氧氟沙星的敏感性
选取Y6、Y16、Y17、Y21、Y23、Y27、Y31、Y35等8株临床菌株为研究对象，使用2mM复方氨基酸及62.5μg/mL左氧氟沙星处理，结果参见图17。
由图可见，复方氨基酸协同抗生素杀菌倍数分别是1,226.7倍、3.6倍、2.3倍、19.3倍、8.3倍、16.0倍、326.7倍和39.5倍。
实施例5复合氨基酸组分对大肠埃希头孢唑啉耐药菌生存率的影响
5.1单一氨基酸组分提高大肠埃希头孢唑啉耐药菌对头孢唑林敏感性研究
用2mM复方氨基酸中每一种氨基酸相应的浓度单独添加，通过细菌生存率，研究每种氨基酸协同头孢唑啉提高细菌对抗生素的敏感性，结果参见图18。
由图可见，仅仅添加头孢唑啉时细菌生存率为85.04％，而18种氨基酸中的每种氨基酸以相应摩尔浓度单独添加时的生存率分别是：丙氨酸404μM(73.6％)、缬氨酸136μ105μM(59.4％)M(78.8％)、亮氨酸95μM(75.7％)、异亮氨酸95μM(69.6％)、脯氨酸126μM(85.4％)、苯丙氨酸106μM(98.8％)、色氨酸20μM(67.4％)、甲硫氨酸83μM(91.5％)、甘氨酸233μM(71.4％)、丝氨酸47μM(67.9％)、苏氨酸、酪氨酸5μM(80.5％)、醋酸赖氨酸133μM(68.6％)、精氨酸140μM(97.3％)、组氨酸96μM(97.6％)、天冬氨酸56μM(94.5％)、谷氨酸85μM(94.5％)、胱氨酸4μM(94.1％)。与仅仅添加头孢唑林抗生素进行比较，单独添加18种氨基酸中的任何一种氨基酸，与仅仅添加头孢唑林对照组无明显差异。这些结果说明，单独使用的氨基酸不能提高细菌对 抗生素的敏感性。
5.2氨基酸组合提高大肠埃希头孢唑啉耐药菌对头孢唑林敏感性研究
选取上述18种氨基酸效果较好的5种氨基酸组合：甘氨酸233μM(71.4％)、丝氨酸47μM(67.9％)、色氨酸20μM(67.4％)、赖氨酸133μM(69.6％)和苏氨酸105μM(59.4％)，探讨它们的组合使用对头孢唑啉杀菌效果的影响。这5种氨基酸的总浓度为约0.5mM，该浓度协同1mg/mL头孢唑啉处理头孢唑啉耐药菌6h，计算细菌的生存率；结果参见图19。
由图可见，M9对照添加抗生素组细菌生存率为45.9％；复方氨基酸组协同抗生素后细菌生存率为0.001％，而5种氨基酸组合组，协同抗生素作用后，细菌生存率为0.15％，比复方氨基酸协同倍数低148倍。这些结果表明，即使是效果较好的5种氨基酸组合，其协同抗生素作用效果显著低于18种氨基酸的效果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 复方氨基酸在制备提高细菌对抗生素敏感性药物中的应用。
  2. 根据权利要求1所述应用，其特征在于，所述复方氨基酸的总氨基酸含量为5～12％。
  3. 根据权利要求1所述应用，其特征在于，所述复方氨基酸含有甘氨酸、丝氨酸、色氨酸、赖氨酸和苏氨酸。
  4. 根据权利要求2所述应用，其特征在于，所述复方氨基酸含有天门冬氨酸、谷氨酸、丝氨酸、组氨酸、甘氨酸、苏氨酸、丙氨酸、精氨酸、酪氨酸、胱氨酸、缬氨酸、甲硫氨酸、色氨酸、苯丙氨酸、异亮氨酸、亮氨酸、醋酸赖氨酸和脯氨酸。
  5. 根据权利要求4所述应用，其特征在于，所述复方氨基酸中，每种氨基酸占总氨基酸的摩尔浓度比为：天门冬氨酸2.65～2.8％、谷氨酸4.09～4.23％、丝氨酸2.37～4.64％、组氨酸4.62～4.81％、甘氨酸11.25～11.6％、苏氨酸5.06～5.22％、丙氨酸19.57～20.11％、精氨酸6.88～7％、酪氨酸0.16～0.27％、胱氨酸0.09～0.21％、缬氨酸6.67～6.8％、甲硫氨酸4.04～4.17％、色氨酸0.98～1.04％、苯丙氨酸5.12～5.27％、异亮氨酸4.59～4.74％、亮氨酸6.44～6.45％，醋酸赖氨酸6.59～6.64％和脯氨酸6.23～6.32％。
  6. 根据权利要求1～5任一所述应用，其特征在于，所述抗生素包括β-内酰胺类抗生素、喹诺酮类抗生素和氨基糖苷类抗生素。
  7. 根据权利要求1～5任一所述应用，其特征在于，所述细菌包括敏感菌和耐药菌。
  8. 根据权利要求7所述应用，其特征在于，所述细菌包括革兰氏阳性菌和革兰氏阴性菌。
  9. 一种提高细菌对抗生素敏感性的药物，其特征在于，含有有效量的复方氨基酸和抗生素。
  10. 根据权利要求9所述药物，其特征在于，所述抗生素和复方氨基酸中总氨基酸的质量比为1:(12.5～1824)。
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