Preparation method of high-content cefpodoxime proxetil nanoemulsion

Abstract

The invention relates to the technical field of pharmaceutical preparations, in particular to a preparation method of a high-content cefpodoxime proxetil nanoemulsion with remarkable in-vitro antibacterial effect difference of avian salmonella and pathogenic escherichia coli. The nanoemulsion is prepared from the following raw materials in percentage by weight: 0.1%-1.5% of cefpodoxime proxetil; the oil phase is 1%-12% of origanum oil; the surfactant is prepared from 18%-25% of polyoxyethylated castor oil 40; the cosurfactant is 0.4%-5% of 1, 2-propylene glycol; and the balance of water. The advantages of the nanoemulsion and the cefpodoxime proxetil are integrated, a preliminary prescription is screened out by adopting a traditional nanoemulsion preparation method, and an optimal nanoemulsion prescription is obtained by designing and testing by adopting an L9 (34) multi-index orthogonal test method. The cefpodoxime proxetil injection has the remarkable effects that the solubility of the cefpodoxime proxetil is remarkably improved, the minimum inhibitory concentration and the minimum bactericidal concentration of the cefpodoxime proxetil to avian salmonella and pathogenic escherichia coli are remarkably reduced by using the origanum oil as the oil phase, and a pharmaceutical foundation is laid for improving the bioavailability of the cefpodoxime proxetil.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparation, in particular to a high-content cefpodoxime proxetil nanoemulsion and a preparation method thereof. BACKGROUND

[0002] Cefpodoxime proxetil is a cephalosporin antibiotic, has a broad antibacterial spectrum, and has significant antibacterial effect in vivo and in vitro (including gram-negative bacteria and gram-positive bacteria). After oral administration, it can be hydrolyzed by intestinal wall esterase into cefpodoxime proxetil with antibacterial activity. Cefpodoxime proxetil binds to specific penicillin binding proteins 1A, 1B, 2 and 3, and interferes with bacterial cell wall synthesis.

[0003] Nanotechnology has brought revolutionary changes to the field of oral administration, especially for oral drugs with poor water solubility and weak enzyme stability. It has the advantages of improving drug bioavailability and physical stability. The absolute bioavailability of cefpodoxime proxetil is only 50%, and the reasons for the low bioavailability of cefpodoxime proxetil are: on the one hand, it is hydrolyzed by intestinal wall esterase into cefpodoxime proxetil which is difficult to absorb in the intestine, and on the other hand, the solubility in water is only 400 μg / mL, which is also the main reason.

[0004] At present, in the related research of cefpodoxime proxetil nanoemulsion at home and abroad, the self-nanoemulsion drug delivery system prepared in the published literature Design and evaluation of self-nanoemulsifying drug delivery systems (SNEDDS) force fpodoxime proxetil [J]. Int J Pharm, 2007. 329: 166 ~ 172 is applied to solve the problems of low bioavailability and pH-dependent solubility of cefpodoxime proxetil at high dose. The average particle size is 170 nm, the amount of surfactant in the prescription is less than 40%, and the cefpodoxime proxetil can be completely released within 20 minutes without the influence of the pH of the water phase. The published literature

Quandan and Gao Yuan. Research on self-emulsifying cefpodoxime proxetil preparation [J]. Strait Pharmacy, 19(3): 24 ~ 26

[0005] In order to overcome the above problems, the present application provides a high-content cefpodoxime proxetil nanoemulsion and a preparation method thereof.

[0006] In order to achieve the above technical purposes, the present application adopts the following technical solutions:

[0007] The first aspect of the present application provides a high-content cefpodoxime proxetil nanoemulsion, comprising, in mass percentage:

[0008] 0.1% to 0.5% cefpodoxime proxetil, 1% to 4% oregano oil, 18% to 25% polyoxyethylene castor oil-40, 0.4% to 5% 1,2-propanediol, and the balance water;

[0009] The second aspect of the present application provides a preparation method of the high-content cefpodoxime proxetil nanoemulsion of the first aspect, comprising:

[0010] mixing oregano oil and 1,2-propanediol to obtain a first mixture;

[0011] adding cefpodoxime proxetil to the first mixture to obtain a second mixture;

[0012] adding polyoxyethylene castor oil-40 and 1,2-propanediol to the second mixture to obtain a third mixture;

[0013] adding water to the third mixture to obtain the cefpodoxime proxetil nanoemulsion.

[0014] The third aspect of the present application provides the application of the cefpodoxime proxetil nanoemulsion of the first aspect and / or the cefpodoxime proxetil nanoemulsion prepared by the preparation method of the second aspect in an in-vitro bacteriostatic test.

[0015] The present application has the following beneficial effects:

[0016] (1) The cefpodoxime proxetil nanoemulsion can overcome the poor water solubility of cefpodoxime proxetil and prepare a high-content cefpodoxime proxetil nanoemulsion (0.1% to 0.5% cefpodoxime proxetil by mass fraction). The nanoemulsion is a colloidal dispersion system composed of an aqueous phase, an oil phase, a surfactant, and a co-surfactant, and the droplet size is usually 10 to 100 nm. Due to the extremely small droplet size of the nanoemulsion, the specific surface area is greatly increased, thereby increasing the contact area between the drug and the solvent and promoting the dissolution of the drug. In addition, the surface of the nanoemulsion particles is generally charged, and the electrostatic repulsion between these charges can prevent the coalescence of droplets, thereby maintaining the stability of the nanoemulsion. Therefore, the present application uses oregano oil as the oil phase, polyoxyethylene castor oil-40, and 1,2-propanediol to prepare a high-content cefpodoxime proxetil nanoemulsion by emulsification, which significantly improves the solubility of cefpodoxime proxetil and lays a foundation for improving the bioavailability of cefpodoxime proxetil.

[0017] ​(2) The surface activity and fat solubility of polyoxyethylene castor oil-40 and oregano oil increased the permeability of cefpodoxime proxetil to bacterial cell membranes, thereby significantly reducing the minimum inhibitory concentration and minimum bactericidal concentration of cefpodoxime proxetil for avian Salmonella Sendai and pathogenic Escherichia coli. The composition and physicochemical properties of the oily components in the nanoemulsion have a direct effect on its particle size. As the particle size of the nanoemulsion decreases, the antibacterial activity of the nanoemulsion increases. On the one hand, the reduction in particle size increases the contact probability between the nanoemulsion droplets and bacteria by increasing the specific surface area of ​​the droplets. On the other hand, the smaller particle size is more conducive to the uptake of the active ingredients in the nanoemulsion by cells and tissues.

[0018] (3) The present invention prepares cefpodoxime proxetil nanoemulsion by emulsification. The difficulty in preparing the nanoemulsion lies in the selection and ratio of the nanoemulsion oil phase, surfactant, and cosurfactant. In the present invention, oregano oil is selected as the oil phase, 1,2-propylene glycol is selected as the cosurfactant, and polyoxyethylene castor oil-40 is selected as the surfactant by the pseudo-ternary phase diagram method, thereby selecting a preliminary prescription.

[0019] (4) The difficulty of the present invention is to screen out the optimal formula that takes into account both stability and significant in vitro antibacterial effect on poultry Escherichia coli and Salmonella. In the experimental design, L9 (3 4 ) Orthogonal array design experiment was conducted. The inhibition zone diameter and nanoemulsion stability constant Ke were used as evaluation indicators. The mass fraction ratio of cefpodoxime proxetil to oregano oil (A), the pH value of the nanoemulsion aqueous phase (B), and the emulsification temperature (C) were selected as evaluation factors. The evaluation index data were analyzed by range analysis using PASW Statistics 18 software to screen out the optimal formulation of cefpodoxime proxetil nanoemulsion. For factor A, its influence on the diameter of the inhibition zone of Escherichia coli ranked first, so A1 was selected; its influence on Ke also ranked first, so A3 was selected. Therefore, A could be A1 or A3, but when A3 was selected, the stability of the nanoemulsion was improved by 82.86% compared with A1, while the diameter of the inhibition zone was only reduced by 50.01%. Therefore, A was selected as A3. B ranked second in the influence on the diameter of the inhibition zone and third in Ke. Therefore, B was selected as B2 based on the diameter of the inhibition zone, and C was selected as C2. The analysis method for screening the optimal formula for inhibiting Salmonella was the same as above. It was determined that A was A3, B was B2, and C was C2. Therefore, the optimal formula for inhibiting the two bacteria was A3B2C2. Based on this, cefpodoxime proxetil nanoemulsion was prepared. See Table 1 for details.

[0020] Table 1 Test plan and test results Note: The sum of the CFP and OR mass fractions in Experiments 1-9 was 3% of the nanoemulsion system. The drug control: 3% cefixime, with an inhibition zone diameter of 27.33 ± 2.52 mm for pathogenic Escherichia coli and 27.67 ± 1.16 mm for Salmonella.

[0021] (5) The nanoemulsion prepared by the present invention has good stability. The optimal nanoemulsion has a zeta potential of -9.96 ± 6.35 mv (pH 6.39), an average particle size of 18.94 nm, and a polydispersity index (PDI) of 0.258. It remains a clear, transparent, light yellow liquid after 12 months of storage at room temperature, with a shelf life of 21 months. Compared with the self-nanoemulsion drug delivery system prepared by previous researchers, it has significantly improved stability indicators such as particle size and polydispersity index. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 This is a comparison diagram of the pseudo-ternary phase diagram for surfactant screening among polyoxyethylene castor oil-40, polyoxyethylene hydrogenated castor oil-40, and a mixed surfactant of polyoxyethylene castor oil-40 / polyoxyethylene hydrogenated castor oil-40 with a mass ratio of 1:1.

[0024] Figure 2 is the particle size distribution diagram of the optimal formulation of cefpodoxime proxetil nanoemulsion prepared in the example;

[0025] Figure 3 This is a transmission electron microscopy image of the optimal formulation of cefpodoxime proxetil nanoemulsion prepared in the example;

[0026] Figure 4 is the UV absorption spectrum of cefpodoxime proxetil in the optimal formulation of cefpodoxime proxetil nanoemulsion prepared in the example; DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. It is indicated that all technical and scientific terms used in the present invention have the same meaning as those commonly used by those skilled in the art to which the present invention belongs. The same meaning is commonly understood.

[0028] A typical embodiment of the present invention provides a high-content cefpodoxime proxetil nanoemulsion, which comprises, by mass percentage:

[0029] Cefpodoxime proxetil 0.1%~0.5%; Oregano oil 1%~4%; Polyoxyethylene castor oil-40 18%~25%; 1,2-propylene glycol 0.4%~5%; The balance is water.

[0030] The preparation method of the cefpodoxime proxetil nanoemulsion comprises the following steps:

[0031] 1) Mix the formula amount of oregano oil and 1,2-propanediol with a glass rod;

[0032] 2) Add the formula amount of cefpodoxime proxetil to the above mixture, and continuously stir with a glass rod until the cefpodoxime proxetil is completely dissolved;

[0033] 3) Add the formula amount of polyoxyethylene castor oil-40 to the above mixture, and continuously stir with a glass rod until the mixture is uniformly mixed;

[0034] 4) Place the above mixture in a beaker, adjust the constant-temperature water bath to the optimum temperature between 25-35℃, and balance for one hour;

[0035] 5) Adjust the water to the required pH with strong acid, and add dropwise to the above mixture until the system is finally thin and has the same flowability as water after phase transition, and the preparation is completed.

[0036] The nanoemulsion prepared by the above method is subjected to quality evaluation, and the experimental method is as follows:

[0037] Centrifuge the nanoemulsion at 4,000 r / min for 30 min, and observe whether there are phenomena such as turbidity, stratification and drug precipitation;

[0038] Store the nanoemulsion at -20℃, 4℃, room temperature and 60℃ respectively, and take samples after 1, 3, 6 and 12 months to observe whether the above phenomena exist;

[0039] The nanoemulsion type identification experiment adopts dyeing method: take two portions of the same volume of nanoemulsion, and simultaneously add two drops of Sudan red III dye and methylene blue dye solution respectively, and observe the diffusion speed of the two dyes (red and blue) in the nanoemulsion and the change in appearance;

[0040] The light acceleration test of the nanoemulsion: place the nanoemulsion in a transparent bottle, seal it, and place it at room temperature, 40℃, 60℃ and (4000±500) Lx light respectively for 10 days, and then observe from the appearance.

[0041] The morphology, particle size and physicochemical parameters of the nanoemulsion are observed: take the nanoemulsion and drop it on a copper mesh covered with a support film, stand still for 10 min, then absorb it with filter paper, and then add 2% phosphotungstic acid (pH 7.4) solution to the copper mesh and negatively stain for 3 min, and then dry naturally. Observe and take photos with a transmission electron microscope. Use a Malvern laser particle size analyzer to detect the particle size. Take the nanoemulsion and measure the Zeta potential.

[0042] Long-term test, the nanoemulsion was placed at 25±2℃ and 60%±5% relative humidity for 12 months. At 0, 3, 6, 9, and 12 months, the appearance was observed, the content of cefpodoxime proxetil was monitored, the content-time regression equation was established, and the effective period of the drug was calculated.

[0043] The nanoemulsion stored at each temperature was clear and transparent and had no stratification phenomenon at 1, 3, 6, and 12 months. The centrifugal test and the light test showed that the nanoemulsion was stable and had no stratification, flocculation, transformation, demulsification, rancidity, and other phenomena. The staining results showed that the nanoemulsion was O / W type. Transmission electron microscopy observation showed that the nanoemulsion droplets were spherical and uniformly distributed. Particle size analysis results showed that the average particle size of the nanoemulsion was 18.94 nm, and the polydispersity coefficient (PDI) was 0.258. At an ambient temperature of 25℃, the Zeta potential was -9.96±6.35 mv (pH value was 6.39). The long-term test results showed that the nanoemulsion at each time point was a clear and transparent light yellow liquid. The cefpodoxime proxetil content-time regression equation was y=-0.428x+100.04 (R2=0.989). According to the Pharmacology (Cui Fude. 2008. Pharmacology. Sixth Edition. Beijing: People's Medical Publishing House), the quality control index was set as a 10% reduction in drug content. The time effective period of cefpodoxime proxetil was 23.45 months, and therefore the effective period of the nanoemulsion was 23 months.

[0044] A double-wavelength ultraviolet spectrophotometry method was established for determining the content of cefpodoxime proxetil nanoemulsion.

[0045] Preparation of the test solution: 50 mg of CFP raw material was accurately weighed, 1 mL of acetonitrile was added, and then 0.1 mol / L dilute hydrochloric acid was added to dilute to 100 mL to obtain a 500 μg / mL CFP stock solution;

[0046] Method specificity test: the cefpodoxime proxetil raw material test solution was diluted with 0.1 mol / L dilute hydrochloric acid to contain 20 μg / mL of cefpodoxime proxetil, the nanoemulsion test solution was diluted with distilled water to 20 μg / mL (calculated as CFP), and the dilution multiple of the cefpodoxime proxetil blank excipient was the same as that of the nanoemulsion. Ultraviolet spectrophotometry was performed at a wavelength range of 200 nm to 400 nm, and the ultraviolet scanning spectra were compared and analyzed.

[0047] Preparation of the standard curve: the cefpodoxime proxetil stock solution was diluted to gradient concentrations with 0.1 mol / L dilute hydrochloric acid, the absorbance was determined at 263 nm and 289.8 nm wavelengths by UV2450 ultraviolet-visible spectrophotometer, the absorbance difference (△A) at the two wavelengths was taken as the ordinate, and the concentration (C) was taken as the abscissa to draw the standard curve and calculate the regression equation.

[0048] Recovery test: dilute test No. 1-9 with distilled water to contain cefepime concentration of 20 μg / mL, 30 μg / mL, 40 μg / mL, and measure the absorbance, each sample is repeated 3 times, according to the standard curve equation, the average recovery and relative standard deviation (RSD) is calculated.

[0049] Injection repeatability test: dilute the nanoemulsion with distilled water to contain cefepime concentration of 20 μg / mL for 6 times, calculate the average mass concentration and average recovery and RSD.

[0050] Precision test: prepare nanoemulsion containing cefepime of 2.5 μg / mL, 7.5 μg / mL, 12.5 μg / mL, respectively, at 1, 5, 10, 15, 20, 24 h for intra-day precision test. Select a fixed time every day, and continuously measure for 5 d for inter-day precision test.

[0051] Method specificity investigation results: by Figure 4 It can be seen that the maximum absorption peaks of cefepime and blank emulsion are 263 nm and 234.8 nm, respectively, and the blank emulsion has certain absorption at the maximum absorption peak of cefepime. The blank emulsion is determined to have an equal absorption point wavelength of 289.8 nm by UV2450 selected point detection function. Select 263 nm as the CFP detection wavelength and 289.8 nm as the reference wavelength. The linear range of cefepime content determination is good.

[0052] Table 2 Linear equation of cefepime content determination Drug Linear equation Correlation coefficient (r 2 )]]> Linear range (μg / mL) Cefpodoxime proxetil A = 0.010 41C + 0.002 73 0.999 22 1 ~100

[0053] Recovery test results: meet the requirements of 70%~110%, which proves that the dual-wavelength ultraviolet spectrophotometry for establishing the content analysis method of cefepime nanoemulsion has high accuracy, and is suitable for the quality control of the drug, see Table 3.

[0054] Table 3 Recovery test results (n=3)

[0055] Injection repeatability test results: the RSD of cefepime determination of each test number is less than 0.6%, and the injection repeatability is good, see Table 4.

[0056] Table 4 Injection repeatability test results (n=6)

[0057] Precision test results: the intra-day precision RSD of cefepime determination in prepared nanoemulsion is less than 1%, and the inter-day precision RSD is less than 5%, and the intra-day and inter-day precision is good, see Table 5.

[0058] Table 5 Precision test results

[0059]

[0060] In vitro bacteriostatic test:

[0061] Culture medium preparation: general broth medium, 20 g of proteose peptone, 7 g of beef extract, 5 g of NaCl, 1 g of K2HPO4, 1000 ml of distilled water, heated and boiled to dissolve, pH adjusted to 7.5-7.6 with 1 mol / L NaOH, 121℃ high pressure sterilization for 20 min; general slope 1000 ml of general broth medium, 12 g of agar, heated and boiled to dissolve, poured into test tubes, sealed with cotton plugs, 121℃ high pressure sterilization for 20 min, placed on the slope after solidification, stored at 4℃ for standby; general flat plate 1000 ml of general broth medium, 12 g of agar, heated and boiled to dissolve, 121℃ high pressure sterilization for 20 min, poured into flat plates, stored at 4℃ for standby.

[0062] Preparation of drug solution: using the steps

[0031] to

[0036] to prepare cefpodoxime proxetil nanoemulsion, diluting with distilled water to contain 512 μg / mL of cefpodoxime proxetil for standby; dissolving and diluting cefpodoxime proxetil, oregano oil and drug control cefixime to 512 μg / mL with 60% anhydrous ethanol for standby.

[0063] Preparation of bacterial solution: inoculate the avian Salmonella and pathogenic Escherichia coli strains into general broth, cultivate at 37℃ on a shaking table for 24 h, and examine under a microscope. Determine the bacterial concentration with McFarland turbidity tube, and dilute with general broth to 1×10 5 CFU / mL for standby.

[0064] Using the double dilution method: add 200 μL of culture medium to the first hole of the 96-well plate as negative control, add 100 μL of culture medium to the second to twelfth holes in turn, then add 100 μL of drug solution to the second hole, mix well by repeatedly blowing, then transfer 100 μL to the third hole for dilution by a factor of two to the eleventh hole, discard 100 μL of drug solution from the eleventh hole, and add 100 μL of bacterial solution to the twelfth hole as positive control.

[0065] Record the drug solution concentration corresponding to the last dilution hole with no bacterial growth as the MIC of the drug. Inoculate the culture solution of all the holes with complete clarity and no bacterial growth into general nutrient agar flat plates, cultivate at 37℃ for 24 h, and the last flat plate with no more than 5 colonies corresponds to the MBC of the drug solution in the 96-well plate. Repeat each drug solution and each bacterial strain for 3 times. Use PASW Statistics 18 software for single factor analysis of variance.

[0066] In vitro bacteriostatic test results:

[0067] * and # The number indicates that cefpodoxime proxetil nanoemulsion is significantly different (P<0.05) compared with cefpodoxime proxetil, oregano oil and cefixime

[0068] The above merely describes preferred embodiments of the present application but is not intended to limit the present application, and various modifications and changes can be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A high-content cefpodoxime proxetil nanoemulsion, characterized in that: The raw materials of the cefpodoxime proxetil nanoemulsion include the following components in percentage by weight: 0.1% to 0.5% of cefpodoxime proxetil; 1% to 4% of oregano oil; 8% to 25% of polyoxyethylene castor oil-40; 0.4% to 5% of 1,2-propylene glycol; and the balance is water.

2. The high-content cefpodoxime proxetil nanoemulsion for poultry according to claim 1, characterized in that: The method for preparing a cefpodoxime proxetil nanoemulsion comprises the following steps: S2, based on the screening results, thoroughly mixing a formulated amount of oregano oil and 1,2-propylene glycol, adding a formulated amount of cefpodoxime proxetil, and continuously stirring. After the cefpodoxime proxetil is completely dissolved, adding a formulated amount of an optimal surfactant, mixing evenly, and then adding a formulated amount of distilled water dropwise until the system undergoes a phase change and becomes thin and has the fluidity of water, thereby screening a preliminary formulation of the cefpodoxime proxetil nanoemulsion. S3, selecting the mass fraction ratio of cefpodoxime proxetil to oregano oil (A), the pH value of the aqueous phase (B), and the emulsification temperature (C) as formulation screening factors, preparing nine samples, numbered 1-9, and using the diameter of the inhibition zone of poultry Salmonella and the stability constant Ke of each nanoemulsion as evaluation indicators for the nine nanoemulsions. The bacterial solution concentration is counted using the viable plate count method, and the evaluation indicator data are subjected to range analysis using PASW Statistics 18 software to screen the final formulation.

3. The high-content cefpodoxime proxetil nanoemulsion according to claim 1, characterized in that: The surfactant includes polyoxyethylene castor oil-40.

4. The high-content cefpodoxime proxetil nanoemulsion according to claim 1, characterized in that: The cosurfactant includes 1,2-propylene glycol.

5. The high-content cefpodoxime proxetil nanoemulsion according to claim 1, characterized in that: The oil phase includes oregano oil.

6. The high-content cefpodoxime proxetil nanoemulsion according to claim 1, characterized in that: The balance of water is selected from distilled Water, purified water or deionized water.

7. The high-content cefpodoxime proxetil nanoemulsion according to claim 1, characterized in that: The minimum inhibitory concentrations against poultry-derived Salmonella and pathogenic Escherichia coli were 6.67 μg / mL and 5.33 μg / mL, respectively, which were significantly different from those of cefpodoxime proxetil, oregano oil, and cefixime (P < 0.05).

8. The high-content cefpodoxime proxetil nanoemulsion according to claim 1, characterized in that: The minimum bactericidal concentration for poultry-derived Salmonella and pathogenic Escherichia coli was 42.67 μg / mL, which was significantly different from that of cefpodoxime proxetil, oregano oil, and cefixime (P < 0.05).

9. The high-content cefpodoxime proxetil nanoemulsion for poultry according to claim 1, characterized in that: Transmission electron microscopy observation showed that the nanoemulsion droplets were spherical and evenly distributed, with a Zeta potential of -9.96±6.35 mv, an average particle size of 18.94 nm, and a polydispersity index (PDI) of 0.258.

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