Cefmenoxime-avibactam injection as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a cefmenoxime-avibactam injection as well as a preparation method and application thereof. A novel beta-lactamase antibiotic / beta-lactamase inhibitor cefmenoxime-avibactam compound antibiotic product for injection is developed, bacterial drug resistance can be remarkably improved, and the injection is stable in prescription process, good in reproducibility and high in dissolving speed and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a cefotaxime-avibactam injection, its preparation method, and its application. Background Technology

[0002] With the widespread use and abuse of antibiotics, bacterial resistance has become a major challenge in global public health, especially the prevalence of β-lactamase-producing resistant strains, which has led to a significant decline in the clinical efficacy of traditional β-lactam antibiotics. Cefotaxime hydrochloride is a third-generation cephalosporin antibiotic with a low incidence of adverse reactions, a broad antibacterial spectrum, and strong activity against both Gram-positive and Gram-negative bacteria. However, due to the widespread use and abuse of antibiotics, Acinetobacter baumannii, Proteus mirabilis, Escherichia coli, and Pseudomonas aeruginosa have all developed resistance to cefotaxime, and this resistance is increasing year by year. Therefore, in addition to developing new antibiotic products, seeking new combination antibiotic formulations is also a means to address bacterial resistance.

[0003] CN 115252561 A discloses a method for preparing ceftazidime avibactam sodium for injection, but studies have found that it is generally ineffective against drug-resistant Proteus mirabilis, Escherichia coli and Klebsiella pneumoniae enzyme-producing strains.

[0004] CN113413367A discloses an injectable avibactam-cefotaxime compound powder and its preparation method. The applicant's experimental verification showed that when no solubilizer is added to the formulation, the active ingredient, cefotaxime hydrochloride, has poor solubility in commonly used clinical infusion media, failing to meet clinically required solubility and severely impacting the practical application of the preparation. Secondly, this patent application employs a preparation process involving mixing raw materials, compressing, and then pulverizing. This process has significant shortcomings: on the one hand, it significantly increases the complexity of sterile preparation production and the difficulty of quality control; on the other hand, mechanical stress may be generated during tableting and pulverization, potentially adversely affecting the stability of the active ingredient. Summary of the Invention

[0005] The technical solution of the present invention is as follows: a cefotaxime-avibactam injection, comprising cefotaxime hydrochloride, avibactam sodium and a solubilizer.

[0006] In some embodiments, the co-solvent is selected from Tween-80, mannitol, sorbitol, lecithin, L-arginine, and anhydrous sodium carbonate, preferably anhydrous sodium carbonate and L-arginine.

[0007] In some embodiments, the ratio of the cosolvent anhydrous sodium carbonate to L-arginine is 1:1.

[0008] A cefotaxime-avibactam injection comprising 68% cefotaxime hydrochloride, 17% avibactam sodium, 7.5% L-arginine and 7.5% anhydrous sodium carbonate.

[0009] This invention also provides a method for preparing the cefotaxime-avibactam injection, comprising the following steps:

[0010] Weighing: Weigh cefotaxime hydrochloride, avibactam sodium, L-arginine and anhydrous sodium carbonate according to the prescription ratio.

[0011] Mixing: Add cefotaxime hydrochloride, avibactam sodium, L-arginine and anhydrous sodium carbonate into the mixing device at one time and mix directly; set the mixing speed to 20 r / min and the mixing time to 15 min.

[0012] The present invention relates to the use of cefotaxime-avibactam injection in the preparation of drugs for the prevention and / or treatment of fungal infections, wherein the bacteria are selected from Escherichia coli, Proteus mirabilis, Haemophilus influenzae, enzyme-producing strains of Klebsiella pneumoniae, or Acinetobacter baumannii.

[0013] The beneficial effects of this invention are as follows: This invention develops a novel cefotaxime-avibactam combination antibiotic product for injection, which can significantly improve bacterial resistance. The injection formulation of this invention has stable process, good reproducibility, and rapid dissolution, showing promising application prospects. Attached Figure Description

[0014] Figure 1 Detection of β-lactamase production in test strains Detailed Implementation

[0015] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0016] Avibactam sodium is a white to pale yellow powder. This compound is extremely soluble in water, slightly soluble in methanol, and practically insoluble in anhydrous ethanol or acetone. Avibactam sodium is hygroscopic; therefore, the influence of ambient humidity should be considered during research.

[0017] Cefotaxime hydrochloride is a white to pale yellow crystalline or crystalline powder. This compound is readily soluble in formamide, slightly soluble in methanol, very slightly soluble in water, insoluble in ethanol, and readily soluble in phosphate buffer at pH 6.8. Cefotaxime hydrochloride is hygroscopic; therefore, attention should be paid to ambient humidity and the mixing time should be minimized during research and production.

[0018] Formula and process research

[0019] 1.1 Types of Cosolvents

[0020] Cefotaxime hydrochloride is poorly soluble in aqueous solution, and its solubility can be significantly improved by adding a solubilizer. This invention compares the effects of various solubilizers, including Tween-80, mannitol, sorbitol, lecithin, L-arginine, and anhydrous sodium carbonate, on the dissolution time and solution stability of drug powder under the same conditions. Experimental results show that when both L-arginine and anhydrous sodium carbonate are added at 0.25 g, the sample with added anhydrous sodium carbonate yields a clear solution after shaking for 1 min, and the solution remains stable without precipitation for 30 min, demonstrating significantly stronger stability than when anhydrous sodium carbonate is used alone. However, the sample with 0.25 g of L-arginine requires shaking for 3 min, and the resulting solution remains turbid. When the amount of L-arginine is increased to 0.5 g, although a clear solution is obtained after shaking for 1 min, precipitation occurs after 30 min. The solubility effect of other solubilizers is not as good as the combination of L-arginine and anhydrous sodium carbonate. Comprehensive comparisons show that the combination of L-arginine and anhydrous sodium carbonate achieves better solubilization effect at lower dosages. Therefore, the combination of L-arginine and anhydrous sodium carbonate was chosen as the solubilizer for cefotaxime hydrochloride.

[0021] Investigation on the ratio of 1.2L-arginine to anhydrous sodium carbonate

[0022] With fixed mixing methods and times, L-arginine and anhydrous sodium carbonate were designed to have ratios of 2:1, 1:1, and 1:2. Cefotaxime hydrochloride and avibactam sodium were added according to the prescription ratio, mixed thoroughly, and the powder flowability and reconstitution time were examined. Water was added to prepare a solution containing approximately 0.1 g of cefotaxime and 0.025 g of avibactam per ml. The pH and clarity of the solution were determined according to General Chapters 0631 and 0901 of the 2020 edition of the Chinese Pharmacopoeia. The results showed that the powder with a 1:1 ratio of L-arginine to anhydrous sodium carbonate had the best flowability; when the ratio was 2:1, the powder reconstituted for a longer time, and precipitation occurred after 60 minutes; when the ratio was 1:2, the powder reconstituted quickly and was stable, but the solution pH was too high, posing a certain risk of injection; when the ratio was 1:1, the solution was clear and light in color, with a pH close to neutral, meeting the requirements.

[0023] Table 1. Screening results of L-arginine and anhydrous sodium carbonate ratio.

[0024]

[0025] 1.3 Powder Mixing Method

[0026] The active pharmaceutical ingredient (API) and excipients were mixed using both direct mixing and stepwise mixing methods. The advantages and disadvantages of the two mixing methods were evaluated by measuring the mixing uniformity and powder particle size distribution. Direct mixing involved adding cefotaxime hydrochloride, avibactam sodium, L-arginine, and anhydrous sodium carbonate to the mixing equipment all at once for direct mixing. Stepwise mixing involved first pre-mixing avibactam sodium with L-arginine and anhydrous sodium carbonate to form a premix, then adding the premix and cefotaxime hydrochloride to the mixing equipment for final mixing. To evaluate the mixing effect, six samples were randomly selected from the mixed samples, and the content of each component was determined by HPLC. The RSD was calculated to assess the mixing uniformity. A smaller RSD value indicated higher mixing uniformity and better mixing effect. The results are shown in Table 2: the RSD for avibactam content using the direct mixing method was 0.65%, and the RSD for cefotaxime content was 1.4%. The RSD of avibactam content using the stepwise mixing method was 1.1%, and the RSD of cefotaxime content was 1.6%, both less than 5.0%. Both direct mixing and stepwise mixing can achieve excellent mixing results; therefore, choosing the direct mixing method simplifies the process.

[0027] Table 2 Results of the investigation on the hybrid method

[0028]

[0029] 1.4 Determination of Formula and Process for Compound Preparations

[0030]

[0031] Weighing: Weigh cefotaxime hydrochloride, avibactam sodium, L-arginine and anhydrous sodium carbonate according to the prescription ratio.

[0032] Mixing: Cefotaxime hydrochloride, avibactam sodium, L-arginine, and anhydrous sodium carbonate were added to the mixing equipment at once and mixed directly; the mixing speed was set to 20 r / min and the mixing time to 15 min. The total cefotaxime intermediate content and the RSD of the mixing uniformity were determined.

[0033] Aseptic filling: The mixed powder is aseptically filled into sterilized vials, filled with a mixture of nitrogen, helium and carbon dioxide gas, and capped.

[0034] Test Example 1: Evaluation of Synergistic Antibacterial Effect

[0035] 1.1 Experimental Methods

[0036] 1.1.1 Preparation of the medicinal solution

[0037] Cefotaxime hydrochloride solution: Accurately weigh 5.0 mg of cefotaxime hydrochloride, dissolve it in ddH2O to a stock solution with a concentration of 1 mg / ml, filter it through a 0.22 μm aqueous phase filter membrane for sterilization, and dispense it. Prepare and use immediately.

[0038] Avibactam sodium solution: Accurately weigh 5.0 mg of avibactam sodium, dissolve it in ddH2O to a stock solution with a concentration of 1 mg / ml, filter it through a 0.22 μm aqueous phase filter membrane for sterilization, dispense it, and store it in a -60℃ refrigerator for later use.

[0039] Ceftazidime solution: Accurately weigh 5.0 mg of ceftazidime hydrochloride, dissolve it in ddH2O to a stock solution with a concentration of 1 mg / ml, filter it through a 0.22 μm aqueous phase filter membrane for sterilization, dispense it, and store it in a -60℃ refrigerator for later use.

[0040] 1.1.2 Preparation of working solution and culture medium

[0041] Cefotaxime working solution: Dissolve 1 mg of cefotaxime in 100 μl of high-quality DMSO, mix thoroughly, and prepare a 10 mg / ml stock solution. Aliquot and store protected from light. When using, add 10 μl of the stock solution to 190 μl of PBS buffer to prepare a 0.5 mg / ml cefotaxime working solution for detecting bacterial enzyme production.

[0042] Preparation of heme chloride stock solution: Weigh 10 mg of heme chloride powder, add 1 ml of PBS to prepare heme chloride stock solution (10 mg / ml), filter aseptically through a 0.22 μm filter membrane, aliquot and store in the dark. When using, add the heme chloride stock solution to BHIB medium at a volume ratio of 1:1000.

[0043] Luria–Bertani (LB) medium: Weigh 10g peptone, 5g yeast extract and 10g NaCl into a beaker, add ddH2O to dissolve, adjust the pH to 7.2-7.4, and then bring the volume to 1000ml. When preparing LB solid medium, add 1.5% agar strips for dispensing, autoclave and sterilize at 121℃ for 20min for later use.

[0044] BHIB medium: Weigh 3.85g of BHIB medium powder into 100ml of pure water and sterilize at 121℃ for 20min.

[0045] 1.1.3 Preparation of bacterial cultures

[0046] The frozen test strains were revived onto nutrient agar slants and incubated at 37°C for 18 hours. Colonies were then transferred to liquid test tubes using an inoculation loop and incubated at 37°C for 12–16 hours. The cultures were collected, and after centrifugation and discarding the supernatant, the precipitate was washed three times with sterile PBS. Finally, the bacteria were resuspended in sterile MH broth to a concentration of 2 × 10⁵ CFU / ml for later use.

[0047] 1.1.4 Screening of cefotaxime-resistant and intermediate-resistant strains

[0048] Screen strains resistant and intermediately resistant to cefmenoxime using a nitrocefin working solution. The specific method is as follows: Add the test bacterial solution dropwise to the nitrocefin working solution. If the color changes from yellow to pink (turning brownish after standing), it is determined as positive, indicating that the test strain produces β-lactamase and can hydrolyze β-lactam antibiotics. The color of high β-lactamase-producing strains changes rapidly to red, while that of medium-producing strains changes more slowly and the red color is lighter. The screened resistant and intermediately resistant strains are used for the checkerboard test later.

[0049] 1.1.5 Study on the synergistic effect of different β-lactamase inhibitors on cefmenoxime

[0050] Use the checkerboard method to determine the combined antibacterial effect of avibactam with cefmenoxime or ceftazidime respectively. The specific steps are as follows:

[0051] (1) Preparation of bacterial solution: Dilute the cultured strain with MH broth medium to 2×105 CFU / ml for standby.

[0052] (2) Sampling: Pipette 100 μl of the bacterial solution into a 96-well plate, and then sequentially add different concentration gradients of cefmenoxime (50 μl / well, final concentrations of 0, 0.5, 1, 2, 4, 8, 16, 32 μg / ml, a total of 8 gradients) and β-lactamase inhibitor (50 μl / well, final concentrations of 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 μg / ml, a total of 11 gradients) to each well.

[0053] (3) Incubation: Place the 96-well plate in a 37 °C bacterial incubator for 16 h to observe the combined antibacterial effect of avibactam and ceftazidime or cefmenoxime.

[0054] (4) Calculate the FICI index: Calculate the fractional inhibitory concentration (FICI) index based on the measured minimum inhibitory concentration (MIC) values. FICI index = (MIC of drug A in combination / MIC of drug A alone) + (MIC of drug B in combination / MIC of drug B alone). Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 4, no interaction; FICI > 4, antagonistic effect. If the synergistic effect is good, the drug concentration can be further reduced.

[0055] 1.2 Experimental results

[0056] 1.2.1 Screening of cefmenoxime-resistant and intermediately resistant strains

[0057] The β-lactamase production of 50 clinically isolated Gram-negative bacilli from six species was detected using a cefotaxime-based chromogenic assay. Results showed that 16 strains tested positive. Rapid chromogenic reaction (strong positive) indicated high-producing strains, while delayed chromogenic reaction (weak positive) indicated medium-producing strains, suggesting potential resistance to penicillin-type drugs in these strains. Positive strains covered all tested species (see results below). Figure 1 ).

[0058] 1.2.2 Study on the synergistic effect of avibactam on cefotaxime

[0059] The combined use of cefotaxime and avibactam showed a synergistic effect on enzyme-producing strains of Escherichia coli, Proteus mirabilis, Haemophilus influenzae, and Klebsiella pneumoniae in high-producing β-lactamase strains; and a synergistic effect on enzyme-producing strains of Escherichia coli, Proteus mirabilis, Acinetobacter baumannii, and Klebsiella pneumoniae in medium-producing β-lactamase strains.

[0060] The combined use of ceftazidime and avibactam showed a synergistic effect only against Klebsiella pneumoniae that produces β-lactamase.

[0061] The above results indicate that the synergistic effect of cefotaxime + avibactam in combination with the strains selected in this invention for producing high and medium β-lactamases is significantly stronger than that of ceftazidime + avibactam.

[0062] Table 3 Summary of Bacterial Synergistic Index

[0063]

[0064] Note: Judgment criterion: 0

Claims

1. A cefmenoxime-avibactam injection, comprising cefmenoxime hydrochloride, avibactam sodium and a cosolvent.

2. The injection of claim 1, characterized in that The cosolvent is selected from Tween-80, mannitol, sorbitol, lecithin, L-arginine and anhydrous sodium carbonate, preferably anhydrous sodium carbonate and L-arginine.

3. The injection of claim 2, wherein, The ratio of the cosolvents anhydrous sodium carbonate and L-arginine is 1:

1.

4. The injection of claim 2, wherein, It comprises 68% cefmenoxime hydrochloride, 17% avibactam sodium, 7.5% L-arginine and 7.5% anhydrous sodium carbonate by weight percentage.

5. A preparation method of the cefmenoxime-avibactam injection of claim 1, comprising the following steps, weighing: weighing cefmenoxime hydrochloride, avibactam sodium, L-arginine and anhydrous sodium carbonate according to the prescription ratio; mixing: adding cefmenoxime hydrochloride, avibactam sodium, L-arginine and anhydrous sodium carbonate into a mixing device at one time, and directly mixing; setting the mixing speed at 20 r / min and the mixing time at 15 min.

6. Use of the cefmenoxime-avibactam injection of claim 1 in the preparation of a drug for preventing and / or treating bacterial infection diseases, wherein the bacteria are selected from Escherichia coli, Proteus mirabilis, Haemophilus influenzae and Klebsiella pneumoniae enzyme-producing strains or Acinetobacter baumannii.

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