Application of loratadine in colistin antibiotic synergist
By designing loratadine-colistin composition and compound preparation, the problem of increased resistance to colistin is solved, the antibacterial activity of colistin is significantly enhanced, and the amount of use is effectively reduced, achieving a coordinated reversal of drug resistance.
Patent Information
- Application Number
- CN202411831961.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-03-07
AI Technical Summary
In recent years, due to the increase in colistin use and the abuse of other antibiotics, the effect of colistin treatment has decreased, and the problem of significant increase in colistin resistance has occurred.
A loratadine-colistin composition was designed, and the mass ratio of loratadine to colistin was (8-512): 1. It was used to prepare loratadine-colistin compound preparations. The content of loratadine was 16 μg/mL-64 μg/mL and the content of colistin was 0.125 μg/mL-2 μg/mL.
This compound preparation significantly enhanced the antibacterial activity of colistin against Klebsiella pneumoniae and Salmonella. The FICI was 0.0938-0.1563, both less than 0.5, indicating that the combination of the two showed significant synergistic effects, which can effectively reduce the amount of colistin used and synergistically reverse drug resistance.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medical application, and in particular relates to a new use of loratadine in a colistin antibiotic synergist. Background Art
[0002] Colistin is a class of polypeptide antibiotics produced by Bacillus polymyxa. They mainly exert their antibacterial effects by destroying the integrity of the outer membrane of Gram-negative bacteria and have strong antibacterial activity against most aerobic Gram-negative bacteria. Polymyxin drugs can be divided into five family members, A, B, C, D and E, according to their chemical structures. Currently, polymyxin B (PMB) and polymyxin E (also known as colistin, Polymycin E, Colistin, COL) are mainly used in clinical practice. Polymyxin drugs began to be used in clinical practice in the late 1950s, but due to their nephrotoxicity and neurotoxicity, as well as the continuous development of new antibacterial drugs, polymyxin drugs have gradually withdrawn from clinical practice. However, in recent years, due to the increasing incidence of multidrug-resistant and extensively drug-resistant Gram-negative bacilli infections, polymyxin drugs have been used again in the clinical frontline as important "last line of defense" drugs. In clinical applications, polymyxin B and E are mainly used to treat multidrug-resistant and extensively drug-resistant Gram-negative bacilli infections, especially when other antibiotics are ineffective.
[0003] Loratadine (LOR) is a commonly used anti-allergic drug and a second-generation antihistamine. It is mainly used to treat allergic rhinitis, acute or chronic urticaria, allergic conjunctivitis, hay fever and other allergic skin diseases. The chemical formula of loratadine is C 22 H 23 C l N2O2, with a molecular weight of 382.88 g / mol and a melting point of 134-136 °C, is a white or off-white crystalline powder, odorless and tasteless.
[0004] In recent years, with the increase in the use of colistin and the occurrence of abuse of other antibiotics, the therapeutic effect of colistin has been relatively reduced, and colistin resistance has emerged. According to the EUCAST European Union Susceptibility Test Standard, when the MIC value of colistin against Enterobacteriaceae is greater than 2 μg / mL, the bacteria show resistance. At present, colistin resistance is showing a significant upward trend. Therefore, it is of great clinical significance to reduce its resistance through synergists. Summary of the invention
[0005] In view of the above problems, the present invention provides a new use of loratadine in a colistin antibiotic synergist.
[0006] Based on the above new uses, the present invention designs a loratadine-colistin composition, wherein the mass ratio of loratadine to colistin is (8-512):1.
[0007] Based on the above composition, the present invention designs a loratadine-colistin compound preparation, wherein the content of loratadine is 16 μg / mL-64 μg / mL, and the content of colistin is 0.125 μg / mL-2 μg / mL.
[0008] Preferably, a colistin content of 2 μg / mL and a loratadine content of 16 μg / mL-64 μg / mL are used in combination.
[0009] Preferably, the colistin content is 0.125 μg / mL-1 μg / mL combined with the loratadine content is 16 μg / mL-64 μg / mL.
[0010] Another object of the present invention is to provide a new use of the above-mentioned loratadine-colistin composition and compound preparation in anti-Enterobacteriaceae drugs.
[0011] The present invention has the following beneficial effects: (1) When the compound preparation of the present invention is used, when the content of loratadine is 16 μg / mL-64 μg / mL, the content of colistin is 0.125 μg / mL-2 μg / mL, which can significantly enhance the antibacterial activity of colistin against Klebsiella pneumoniae and Salmonella.
[0012] (2) The combination of loratadine and colistin significantly enhanced the antibacterial activity of colistin against Enterobacteriaceae, with FICI ranging from 0.0938 to 0.1563 (all less than 0.5), indicating that the combination of the two showed a significant synergistic effect.
[0013] (3) The synergist of the present invention can effectively reduce the amount of colistin used. The amount of colistin used alone is 4-32 times the amount of loratadine and colistin used in combination.
[0014] (4) The combined use of loratadine and colistin can play a synergistic role in reversing drug resistance, with a FICI of 0.0938-0.1563. The combined use showed a significant bactericidal effect after 2-4 hours. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 The time-killing curve of the combination of loratadine and colistin against Klebsiella pneumoniae JX21CTR26 in Example 3 of the present invention; Figure 2 This is the time-killing curve of the combination of loratadine and colistin against Salmonella MLG in Example 3 of the present invention. DETAILED DESCRIPTION
[0016] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.
[0017] The term "synergistic effect" in this patent means that the effect of two drugs combined is greater than the algebraic sum of the effects of the single drugs.
[0018] The term "additive effect" in this patent means that the effect of two drugs taken together is equal to the algebraic sum of their separate effects.
[0019] The term "antagonism" in this patent means that the effect of two drugs taken together is less than the sum of their separate effects. Example 1: MIC test
[0020] (1) Drug testing Loratadine (LOR): Shanghai Aladdin Biochemical Technology Co., Ltd., purity: 98%.
[0021] Colistin (COL): Dalian Meilun Biotechnology Co., Ltd., purity: 88.7%.
[0022] Loratadine was prepared with dimethyl sulfoxide to a concentration of 5120 μg / mL. Colistin was prepared with deionized water to a concentration of 5120 μg / mL. The tested drugs were stored at -20 °C. Before the test, the drug storage solution was taken out and thawed at room temperature, mixed thoroughly, and the pharmacodynamic tests were performed separately. Previous tests have confirmed that dimethyl sulfoxide has no antibacterial effect on the test strains.
[0023] (2) Strains Clinically isolated Enterobacteriaceae strains: Klebsiella pneumoniae JX21CTR26 and Salmonella MLG were provided by the Pharmacology Laboratory of Henan Agricultural University. Klebsiella pneumoniae was activated by streaking on MacConkey medium, and Salmonella was activated by streaking on SS agar medium, cultured at 37°C for 16-18 h, and stored at 4°C for later use.
[0024] (3) Culture medium LB broth medium: produced by Beijing Aoboxing Biotechnology Co., Ltd., prepare 1000 ml according to the instructions of this product, sterilize at 121℃ for 30 minutes, and store at 4℃ for later use. MacConkey medium: produced by Beijing Aoboxing Biotechnology Co., Ltd., prepare 1000 ml according to the instructions of this product, heat and boil to fully dissolve and sterilize, cool to 60℃, pour into sterilized plates, and store at 4℃ for later use.
[0025] SS agar medium: produced by Beijing Aoboxing Biotechnology Co., Ltd., prepare 1000 ml according to the instructions of this product, heat and boil to fully dissolve and sterilize, cool to 60℃, pour into sterilized plates and store at 4℃ for later use.
[0026] MHB broth medium: produced by Beijing Aoboxing Biotechnology Co., Ltd., prepared 1000 ml according to the instructions of this product, sterilized at 121℃ for 30 minutes, and then stored at 4℃ for later use.
[0027] (4) Instruments High-pressure steam autoclave (Shanghai Shen'an Medical Instrument Factory); AB204-N electronic analytical balance (Mettler-Toli Instrument Shanghai Co., Ltd.); HZQ-R air bath oscillator (Beijing Liuyi Instrument Factory); DH-600 electric constant temperature incubator (Beijing Kewei Yongxing Instrument Co., Ltd.); DHG-9140A vacuum drying oven (Shanghai Yiheng Experimental Instrument Co., Ltd.); SW-CJ-JC clean bench (Suzhou Antai Air Technology Co., Ltd.).
[0028] (5) Preparation of bacterial culture Pick a single colony of Klebsiella pneumoniae from MacConkey medium stored at 4°C, pick a single colony of Salmonella from SS agar medium stored at 4°C, inoculate into 5 mL LB broth medium, and culture at 37°C with shaking at 180 rpm to activate the bacteria in the late exponential growth phase. Take the bacterial solution into MHB broth medium and dilute the original bacterial solution to 10 5 CFU / mL. Add corresponding concentrations of loratadine to the bacterial suspension for later use. The final concentrations of loratadine in the bacterial suspension are: 16 μg / mL, 32 μg / mL, and 64 μg / mL.
[0029] (6) Preparation of drug sensitivity plates Use the bacterial suspension in step 5 to perform drug sensitivity test. Take a sterile 96-well reaction plate, add 126 μL of freshly prepared bacterial suspension containing loratadine and 14 μL of 5120 μg / mL colistin to well 1 in the first row; add 70 μL of freshly prepared bacterial suspension containing loratadine to wells 2-12; add 70 μL of freshly prepared bacterial suspension without loratadine to well 1 in the second row, and add 70 μL of MHB broth to well 2 as a blank control. Perform serial dilutions of wells 1-12 in the first row, so that the final colistin concentrations of wells 1-12 in the first row are 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL, respectively; well 1 in the second row is MHB bacterial suspension without drugs, as a positive control; well 8 is MHB broth without drugs, as a negative control. The drug sensitivity plate was incubated at 37°C for 16-18 h.
[0030] (7) Determination of MIC value After the 96-well plate containing bacteria was incubated at 37 °C for 16-18 h, the MIC (minimum inhibitory concentration) results were read. The drug MIC (minimum inhibitory concentration) value is the drug concentration of the clear and transparent well before the turbid well in the 96-well plate. The above experiments were performed in parallel 3 times, and the MIC values were accepted only when they could be accurately repeated; when the MIC values differed by more than one concentration, a new test was required until the requirements were met. The results are shown in Table 1.
[0031] Table 1 MIC of loratadine combined with colistin against the tested strains
[0032] The results showed that the MIC values of colistin against strains JX21CTR26 and MLG were 16 and 4 μg / mL, respectively, and the MIC values of loratadine against strains JX21CTR26 and MLG were both 512 μg / mL. When 1 / 32 MIC (16 μg / mL), 1 / 16 MIC (32 μg / mL), and 1 / 8 MIC (64 μg / mL) of loratadine were added, the MIC of colistin against JX21CTR26 was 2 μg / mL; the MICs against MLG were 1, 0.125, and 0.125 μg / mL, respectively.
[0033] Compared with the MIC value of colistin alone, the MIC of colistin against strain JX21CTR26 decreased by 8 times when 16 μg / mL, 32 μg / mL, and 64 μg / mL of loratadine were added, respectively; the MIC of colistin against strain MLG decreased by 4-32 times.
[0034] The results showed that loratadine at a concentration of 16-64 μg / mL could significantly enhance the antibacterial activity of colistin against Klebsiella pneumoniae JX21CTR26 and Salmonella MLG.
[0035] Example 2: Testing of FICI (1) Drug testing: Same as Example 1.
[0036] (2) Strains Same as Example 1.
[0037] (3) Culture medium Same as Example 1.
[0038] (4) Instruments Same as Example 1.
[0039] (5) Preparation of bacterial culture Same as Example 1.
[0040] (6) Preparation of drug sensitivity plates Based on the microbroth dilution method, we obtained the MIC values of LOR and COL alone, and selected 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, 1 / 640, 1 / 1280 times the MIC of LOR and 1 / 160, 1 / 320, 1 / 640, 1 / 1280 times the MIC of COL for 8×8 combination, and added the prepared bacterial suspension to each well of the 96-well plate, and used LOR and COL single drug MIC as well as negative and positive controls. The 96-well plate was placed in a constant temperature incubator at 37 ℃ for 16-18 hours, and the results were observed and recorded. The experiment was repeated three times.
[0041] The fractional inhibitory concentration index (FICI) was used as the criterion for the chessboard susceptibility test. The FICI of two compounds A and B was defined by the following formula: FICI=FIC A +FIC B =MIC AB / MIC A +MIC BA / MIC B MIC A is the MIC of compound A alone, MIC AB is the MIC of A when used in combination, MIC B is the MIC of compound B alone, MIC BA is the MIC of B when used in combination. FICI < 0.5 indicates synergistic effect; 0.5 ≤ FICI ≤ 1 indicates additive effect; 1 < FICI ≤ 2 indicates irrelevant effect; FICI > 2 indicates antagonistic effect. The results are shown in Table 2.
[0042] Table 2 Combined antibacterial effect of loratadine and colistin
[0043] The results showed that loratadine alone had a weak antibacterial effect on the tested strains, and when used in combination with colistin, it showed a synergistic effect on colistin, with FICI of 0.0938-0.1563, all <0.5, that is, the combination of the two showed a synergistic effect.
[0044] Example 3: Time Kill Curve Determination (1) Drug testing: Same as Example 1.
[0045] (2) Strains Same as Example 1.
[0046] (3) Culture medium Same as Example 1.
[0047] (4) Instruments Same as Example 1.
[0048] (5) Preparation of bacterial culture Same as Example 1.
[0049] (6) Time-killing curve Klebsiella pneumoniae JX21CTR26 was selected for the time-kill curve test. A single colony was inoculated into fresh LB broth, placed in a shaker at 37°C, 180 r / min, and cultured overnight; the bacterial solution was then transferred to 20 mL of fresh LB broth at a ratio of 1:100 and divided into 5 test groups: Group A: Control; Group B: 1 / 2 MIC COL; Group C: 1 / 16 MIC LOR; Group D: 1 / 2 MIC COL+1 / 32 MIC LOR; Group E: 1 / 2 MIC COL+1 / 16 MIC LOR.
[0050] After grouping, the cells were placed at 37 °C and shaken at 180 r / min. 100 μL of the bacterial suspension was taken at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h, respectively. 100 μL of the bacterial suspension was diluted 10-fold in a series with PBS buffer, and 100 μL of the dilution was spread on the LB agar plate. The plates were cultured at 37 °C for 16 to 18 h. Finally, the colonies on the agar plate were counted, and the time-killing curve was plotted with time as the horizontal axis and log10 CFU / mL as the vertical axis. The experiment was repeated three times.
[0051] Results: At 24 h, compared with single drugs, a decrease of ≥2log10 in the colony count of the combination group was defined as a synergistic effect, a decrease of <1log10 in the colony count was defined as an irrelevant effect, and a decrease of 1log10-2log10 in the colony count was defined as an additive effect. Figure 1-2 shown.
[0052] The experimental results showed that the bactericidal curve of single drug 1 / 16 MIC LOR against the strain was almost consistent with that of the blank control (Control), indicating that the antibacterial effect of LOR was weak; single drug 1 / 2 MIC COL initially showed a slight antibacterial effect on the strain, but then the growth trend was consistent with the blank control and 1 / 16 MIC LOR; for Klebsiella pneumoniae JX21CTR26, the bactericidal curve of the combination of 1 / 32 MICLOR and 1 / 2 MIC COL showed obvious bactericidal effect after 2 hours, and then gradually increased. At 24 hours, the colony count of the combination group was reduced by ≥2log10 compared with the single drug group; the bactericidal curve of the combination of 1 / 16 MIC LOR and 1 / 2 MIC COL showed obvious bactericidal effect after 4 hours, and then gradually increased. At 24 hours, the colony count of the combination group was reduced by ≥2log10 compared with the single drug group, both showing synergistic effects. For Salmonella MLG, the bactericidal curve of 1 / 32 MIC LOR combined with 1 / 2 MIC COL showed obvious bactericidal effect after 2 hours, and then gradually increased. At 24 hours, the colony count of the combined drug group was reduced by ≥2log10 compared with the single drug group; the bactericidal curve of 1 / 16 MIC LOR combined with 1 / 2 MIC COL killed all bacteria after 4 hours, showing obvious bactericidal effect. The time-bactericidal curve results were basically consistent with the combined drug sensitivity results, that is, the antibacterial effect of LOR alone was weak, and the combination of 1 / 32 MIC LOR or 1 / 16 MIC LOR and COL could significantly reduce the MIC value of COL against drug-resistant bacteria, showing a good synergistic reversal of drug resistance.
[0053] In summary, loratadine at a concentration of 16-64 μg / mL can significantly enhance the antibacterial activity of colistin against Klebsiella pneumoniae JX21CTR26 and Salmonella MLG. The combination of loratadine and colistin showed significant synergistic effects, with FICI of 0.0938-0.1563, all <0.5, that is, the combination of the two showed significant synergistic effects. The results of the time-kill curve showed that the combination showed significant bactericidal effects after 2 h or 4 h. The colony count of the combination group at 24 h was reduced by ≥2log10 compared with the single-drug group, showing a synergistic effect, which was basically consistent with the combined drug sensitivity results, that is, the antibacterial effect of LOR alone was weak, and the combination of 1 / 32 MIC LOR or 1 / 16 MIC LOR with COL could significantly reduce the MIC value of COL against resistant bacteria, showing a good synergistic reversal of resistance.
[0054] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. Application of loratadine in colistin antibiotic synergist.
2. A loratadine-colistin composition, characterized in that: The mass ratio of loratadine to colistin is (8-512):
1.
3. A loratadine-colistin compound preparation, characterized in that: The content of loratadine was 16 μg / mL-64 μg / mL, and the content of colistin was 0.125 μg / mL-2 μg / mL.
4. The loratadine-colistin content preparation according to claim 3, characterized in that: The content of colistin is 2 μg / mL, and the content of loratadine is 16 μg / mL-64 μg / mL.
5. The loratadine-colistin content preparation according to claim 3, characterized in that: The content of colistin is 0.125 μg / mL-1 μg / mL, and the content of loratadine is 16 μg / mL-64 μg / mL.
6. Use of the loratadine-colistin composition according to claim 2 in the preparation of an anti-Enterobacteriaceae drug.
7. Use of the loratadine-colistin compound preparation according to claim 3 in the preparation of an anti-Enterobacteriaceae drug.