A doxycycline hydrochloride and colistin sulfate injection solution and its preparation method

Abstract

This invention relates to the field of doxycycline hydrochloride and colistin sulfate preparations, and provides an injection solution of doxycycline hydrochloride and colistin sulfate and its preparation method. The injection solution comprises the following raw materials at final concentrations: doxycycline hydrochloride 0.02-0.05 g / mL, colistin sulfate 0.01-0.03 g / mL, antioxidant 0.003-0.005 g / mL, solubilizer 30-50 mL / 100 mL, complexing agent 0.02-0.05 g / mL, and the balance being water. The injection solution of this invention can cover a wider spectrum of pathogens, improve the efficiency and safety of clinical treatment, and ensure the safety of the injection solution when used at high doses, effectively solving the problems of poor stability and high toxicity of existing similar injection solutions. Therefore, this application provides an injection solution with good stability, low toxicity, significant efficacy, and broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of doxycycline hydrochloride and colistin sulfate preparations, and particularly to a doxycycline hydrochloride and colistin sulfate injection solution and its preparation method. Background Technology

[0002] In recent years, antibiotic resistance has become an increasingly serious problem, especially the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria, posing a severe challenge to clinical treatment. Currently, in veterinary clinical practice, particularly in the treatment of multidrug-resistant bacterial infections, existing single-antibiotic therapies often face problems such as insufficient efficacy and significant side effects.

[0003] Colistin sulfate, a key drug for treating multidrug-resistant Gram-negative bacteria, has achieved good clinical efficacy, but its nephrotoxicity and neurotoxicity limit its high-dose and widespread use, especially when high doses are required due to significant side effects. Meanwhile, doxycycline hydrochloride, while effective against Gram-positive bacteria and rickettsiae infections, shows relatively weaker antibacterial efficacy against Gram-negative bacteria, particularly drug-resistant ones. Furthermore, drug resistance is increasingly evident in the use of doxycycline hydrochloride, and its side effects are not negligible, especially in injectable form, where its poor stability and tendency to precipitate further affect its efficacy and safety.

[0004] Currently, in veterinary clinical practice, especially in the treatment of multidrug-resistant bacterial infections, existing single-antibiotic therapies often face problems such as insufficient efficacy and significant side effects. Combination antibiotic therapy, as an effective strategy, can broaden the antibacterial spectrum, delay the development of drug resistance, and to some extent reduce the side effects of single antibiotics through synergistic effects between drugs. However, although there have been studies on various antibiotic combination therapy regimens, research on the combined use of colistin sulfate and doxycycline hydrochloride remains relatively scarce. This invention, by combining colistin sulfate and doxycycline hydrochloride, can cover a wider range of pathogens and exert a synergistic effect in clinical treatment, significantly improving therapeutic efficacy and safety. Summary of the Invention

[0005] The purpose of this invention is to provide an injection solution of doxycycline hydrochloride and colistin sulfate, and its preparation method. By using colistin sulfate and doxycycline hydrochloride in combination, it is possible to more effectively combat drug-resistant strains and improve the success rate of clinical treatment. Furthermore, the injection solution provided by this invention not only has a broader antibacterial spectrum but also reduces the frequency of administration and adverse reactions through synergistic effects, significantly improving the efficiency and safety of clinical treatment, and has broad application prospects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an injection of doxycycline hydrochloride and colistin sulfate, comprising the following raw materials at the following final concentrations: doxycycline hydrochloride 0.02-0.05 g / mL, colistin sulfate 0.01-0.03 g / mL, antioxidant 0.003-0.005 g / mL, solubilizer 30-50 mL / 100 mL, complexing agent 0.02-0.05 g / mL, with the balance being water.

[0008] Preferably, the antioxidant is one or more of sodium metabisulfite, L-ascorbic acid, sodium thiosulfate, and sodium bisulfite.

[0009] Preferably, the co-solvent is one or more of polyethylene glycol 400, 1,2-propanediol, and polyethylene glycol 300.

[0010] Preferably, the complexing agent is one or more of magnesium chloride hexahydrate, disodium ethylenediaminetetraacetate, zinc oxide, calcium oxide, calcium lactate, magnesium sulfate, aluminum chloride, and aluminum oxide.

[0011] The present invention also provides a method for preparing the doxycycline hydrochloride and colistin sulfate injection, comprising the following steps:

[0012] (1) Mix water and antioxidant to obtain solution 1;

[0013] (2) Mix solution 1, solubilizer and complexing agent to obtain solution 2;

[0014] (3) Mix solution 2 with doxycycline hydrochloride to obtain solution 3;

[0015] (4) Mix solution 3 with colistin sulfate to obtain solution 4;

[0016] (5) After adjusting the pH of solution 4 to 4.0-5.0, filter it with a microporous membrane. The filtrate is doxycycline hydrochloride and colistin sulfate injection.

[0017] Preferably, the mixing temperature in step (1) is 50-70°C; the mixing time is 1-3 min; and the mixing speed is 1100-1300 r / min.

[0018] Preferably, the mixing time in step (2) is 4 to 6 minutes, and the mixing speed is 1100 to 1300 r / min.

[0019] Preferably, the mixing time in step (3) is 15 to 20 minutes, and the mixing speed is 1100 to 1300 r / min.

[0020] Preferably, the mixing method in step (4) is as follows: colistin sulfate is added to solution 3 in 5 to 7 portions; the mixing time is 25 to 35 minutes, and the mixing speed is 1100 to 1300 r / min.

[0021] Preferably, in step (5), a sodium hydroxide solution of 1-5 mol / L is used to adjust the pH of solution 4; the diameter of the microporous membrane is 0.22 μM; and the filtration is performed 2-3 times.

[0022] The beneficial effects of this invention are as follows:

[0023] The doxycycline hydrochloride and colistin sulfate injection prepared by this invention exhibits good stability, controllable quality, and superior therapeutic efficacy compared to single-agent doxycycline hydrochloride or colistin sulfate injections. Through synergistic effects between the drugs, it can cover a wider spectrum of pathogens, and also reduce the frequency of administration and adverse reactions, significantly improving the efficiency and safety of clinical treatment, thus showing broad application prospects.

[0024] The doxycycline hydrochloride and colistin sulfate injection of the present invention effectively improves the stability of the drugs through precise drug formulation and preparation methods. No visible crystals or precipitation are observed within two years, and the product quality remains stable. It can maintain efficacy for a longer period of time, solving the problems of poor stability and high toxicity of existing doxycycline hydrochloride and colistin sulfate injections in clinical applications. It provides a veterinary injection with better stability, lower toxicity and side effects, and significant efficacy. Detailed Implementation

[0025] This invention provides an injection of doxycycline hydrochloride and colistin sulfate, comprising the following raw materials at the following final concentrations: doxycycline hydrochloride 0.02-0.05 g / mL, colistin sulfate 0.01-0.03 g / mL, antioxidant 0.003-0.005 g / mL, solubilizer 30-50 mL / 100 mL, complexing agent 0.02-0.05 g / mL, with the balance being water.

[0026] In this invention, the concentration of doxycycline hydrochloride is preferably 0.02-0.05 g / mL, more preferably 0.03-0.04 g / mL.

[0027] In this invention, the concentration of the colistin sulfate is preferably 0.01 to 0.03 g / mL, and more preferably 0.02 g / mL.

[0028] In this invention, the concentration of the antioxidant is preferably 0.003 to 0.005 g / mL, and more preferably 0.004 g / mL.

[0029] In this invention, the concentration of the co-solvent is preferably 30-50 mL / 100 mL, and more preferably 40 mL / 100 mL.

[0030] In this invention, the concentration of the complexing agent is preferably 0.02 to 0.05 g / mL, and more preferably 0.03 to 0.04 g / mL.

[0031] In this invention, the antioxidant is preferably one or more of sodium metabisulfite, L-ascorbic acid, sodium thiosulfate, and sodium bisulfite.

[0032] In this invention, the co-solvent is preferably one or more of polyethylene glycol 400, 1,2-propanediol and polyethylene glycol 300.

[0033] In this invention, the complexing agent is preferably one or more of magnesium chloride hexahydrate, disodium ethylenediaminetetraacetate, zinc oxide, calcium oxide, calcium lactate, magnesium sulfate, aluminum chloride, and aluminum oxide.

[0034] The present invention also provides a method for preparing the doxycycline hydrochloride and colistin sulfate injection, comprising the following steps:

[0035] (1) Mix water and antioxidant to obtain solution 1;

[0036] (2) Mix solution 1, solubilizer and complexing agent to obtain solution 2;

[0037] (3) Mix solution 2 with doxycycline hydrochloride to obtain solution 3;

[0038] (4) Mix solution 3 with colistin sulfate to obtain solution 4;

[0039] (5) After adjusting the pH of solution 4 to 4.0-5.0, filter it with a microporous membrane. The filtrate is doxycycline hydrochloride and colistin sulfate injection.

[0040] In this invention, the mixing temperature in step (1) is preferably 50-70°C, more preferably 60°C; the mixing method is preferably stirring; the mixing time is preferably 1-3 min, more preferably 2 min; and the mixing speed is preferably 1100-1300 r / min, more preferably 1200 r / min.

[0041] In this invention, the mixing temperature in step (2) is preferably 50-70°C, more preferably 60°C; the mixing method is preferably stirring; the mixing time is preferably 4-6 min, more preferably 5 min; and the mixing speed is preferably 1100-1300 r / min, more preferably 1200 r / min.

[0042] In this invention, the mixing temperature in step (3) is preferably 50-70°C, more preferably 60°C; the mixing time is preferably 15-20 min, more preferably 18 min; and the mixing speed is preferably 1100-1300 r / min, more preferably 1200 r / min.

[0043] In this invention, the mixing temperature in step (4) is preferably 50-70°C, more preferably 60°C; the mixing method is preferably: colistin sulfate is added to solution 3 in 5-7 portions, more preferably in 6 portions; the mixing time is preferably 25-35 min, more preferably 30 min; the mixing speed is preferably 1100-1300 r / min, more preferably 1200 r / min.

[0044] In this invention, when adjusting the pH of solution 4 in step (5), a sodium hydroxide solution of 1 to 5 mol / L is preferably used, and a sodium hydroxide solution of 3 mol / L is even more preferred; the diameter of the microporous membrane is preferably 0.22 μM; the number of filtrations is preferably 2 to 3 times, and even more preferably 2 times.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1: Screening of raw materials and determination of reaction conditions in the formulation

[0047] 1. Solvent selection

[0048] Both doxycycline hydrochloride and colistin sulfate are water-soluble drugs; therefore, laboratory ultrapure water was chosen to dissolve both drugs.

[0049] Operating procedures: Place 100 mL of ultrapure water in a beaker, place the beaker on a magnetic stirrer, and stir at a constant speed. First, add 3.0 g of doxycycline hydrochloride. After the doxycycline hydrochloride is completely dissolved, add 1.5 g of colistin (45 million units) and dissolve it. The evaluation criteria are no precipitation, no turbidity, and a clear and transparent solution after 24 hours.

[0050] Results: After doxycycline hydrochloride was added and completely dissolved, colistin was added and stirred, but it did not dissolve, and the drug precipitated after stirring was stopped. It was speculated that the precipitation was caused by the order of drug addition. Therefore, the order of addition was changed: colistin was added first and completely dissolved, then doxycycline hydrochloride was added, but precipitation still occurred. Therefore, adding a solubilizer to dissolve both drugs was considered.

[0051] 2. Screening of cosolvents

[0052] One or two of 1,2-propanediol for injection, polyethylene glycol 300, and polyethylene glycol 400 were selected as cosolvents for the formulation, and a screening test for the cosolvents was conducted.

[0053] Procedure: In a 100 mL system, place the beaker on a magnetic stirrer, add 50 mL of ultrapure water, then add the co-solvent from Table 1 and stir until homogeneous. Next, add 3.0 g of doxycycline hydrochloride and dissolve completely, then add 1.5 g of colistin and dissolve completely. After standing at room temperature, observe for any precipitation or turbidity. The absence of precipitation, turbidity, and a clear, transparent solution after 24 hours is used as the evaluation criterion.

[0054] Table 1 Screening of cosolvents in formulations

[0055]

[0056]

[0057] Based on the results in Table 1, when using only one solubilizer to dissolve both drugs, some precipitation occurred after 24 hours. However, when polyethylene glycol 400 and 1,2-propanediol were used simultaneously, both drugs dissolved without precipitation after 24 hours. Therefore, polyethylene glycol 400 and 1,2-propanediol were selected as the solubilizers for this formulation.

[0058] 3. Screening of complexing agents

[0059] After dissolving doxycycline hydrochloride and colistin with a solubilizer, a precipitate formed in the solution after standing at room temperature for 7 days. This is likely due to the poor stability of doxycycline hydrochloride in aqueous solution. Therefore, it was considered to use a complexing agent to complex doxycycline hydrochloride to increase its stability in aqueous solution. One or two of the following pharmaceutically acceptable complexing agents—magnesium chloride hexahydrate, disodium oxotetraacetate, zinc oxide, calcium oxide, calcium lactate, magnesium sulfate, aluminum chloride, and aluminum oxide—were selected as complexing agents, and a screening test was conducted.

[0060] The specific operating steps are as follows: The formulation system is 100 mL. Place the beaker on a magnetic stirrer, first add 50 mL of ultrapure water, then add 20 mL of polyethylene glycol 400 and 20 mL of 1,2-propanediol and stir until homogeneous. Next, add the complexing agent as shown in Table 2 and dissolve completely. Add 3.0 g of doxycycline hydrochloride to complex with the complexing agent. After the doxycycline hydrochloride is completely dissolved, add 1.5 g of colistin and dissolve completely. After standing at room temperature, observe for any precipitation or turbidity. The evaluation index is the absence of precipitation, no colloid formation, and a clear, transparent solution after 7 days.

[0061] Table 2 Screening of Complexing Agents in Formulations

[0062]

[0063]

[0064] As shown in Table 2, among the screened complexing agents, magnesium chloride hexahydrate showed the best complexing effect on doxycycline hydrochloride. To screen out the best complexing effect of doxycycline hydrochloride, we will continue to investigate whether doxycycline hydrochloride has a better complexing effect on magnesium chloride hexahydrate at different temperatures.

[0065] 4. Temperature screening for the doxycycline hydrochloride complexation reaction

[0066] Because temperature may affect the complexation effect, we will conduct experiments to screen for the suitable temperature for doxycycline hydrochloride. Magnesium chloride hexahydrate, which meets the requirements for injection, was selected as the complexing agent for doxycycline hydrochloride, and its effectiveness at different temperatures was investigated.

[0067] The specific operating steps are as follows: The formulation system is 100 mL. Place the beaker on a magnetic stirrer, first add 50 mL of purified water, then add 20 mL of polyethylene glycol 400 and 20 mL of 1,2-propanediol and stir until homogeneous. Next, add 3.0 g of magnesium chloride hexahydrate and dissolve completely. Then add 3.0 g of doxycycline hydrochloride. The temperature setting is shown in Table 3. After complete dissolution, add 1.5 g of colistin and dissolve completely. After standing at room temperature, observe for any precipitation or turbidity. The evaluation indicators are no precipitation, no colloid formation, clear and transparent solution, and formulation stability after 15 days.

[0068] Table 3 Screening of complexation temperature of formulations

[0069]

[0070] As shown in Table 3, doxycycline hydrochloride and magnesium chloride hexahydrate exhibit the best complexation stability at 50℃ and 60℃. The solubility of doxycycline hydrochloride at 60℃ is the same as at 50℃. Considering that excessively high temperatures may affect other drug components, 50℃ was adopted as the complexation temperature of doxycycline hydrochloride.

[0071] 5. Screening of complexation time

[0072] Since the duration of complexation may affect the complexation effect, we will investigate and screen the appropriate time for the complexation of doxycycline hydrochloride with magnesium chloride hexahydrate.

[0073] The specific operating steps are as follows: The formulation system is 100 mL. Place the beaker on a magnetic stirrer, first add 50 mL of water for injection, then add 20 mL of polyethylene glycol 400 and 20 mL of 1,2-propanediol and stir until homogeneous. Dissolve 3.0 g of magnesium chloride hexahydrate, then add 3.0 g of doxycycline hydrochloride and dissolve completely. The complexation time is set as shown in Table 4. Finally, add 1.5 g of colistin. The entire process is carried out at 50℃ until complete dissolution. After standing at room temperature, observe for any precipitation or turbidity. The absence of precipitation, no colloid formation, and a clear, transparent solution after 30 days are used as evaluation indicators.

[0074] Table 4 Screening of reaction times between complexing agents and doxycycline hydrochloride

[0075] Complexation time (min) 30 days later 5 Small amount of sediment 10 Small amount of sediment 15 clarify 20 clarify

[0076] As shown in Table 4, doxycycline hydrochloride and magnesium chloride hexahydrate exhibited the best complexation stability at 15 min and 20 min. The solubility of doxycycline hydrochloride at 20 min was the same as at 15 min. Based on the literature and the physicochemical properties of this formulation, 15 min was selected as the complexation time for doxycycline hydrochloride.

[0077] 6. Screening of antioxidants in formulations

[0078] Although the addition of chelating agents and doxycycline hydrochloride to the formulation increases its stability, doxycycline hydrochloride aqueous solution is prone to oxidation, causing a color change. Therefore, it is necessary to add antioxidants to slow down the oxidation rate of doxycycline hydrochloride. Sodium thiosulfate, sodium sulfite, sodium bisulfite, sodium metabisulfite, and L-ascorbic acid are selected to screen for one or a combination of two that are soluble in solution, have no incompatibility with the active pharmaceutical ingredient, and enhance the antioxidant effect of the formulation.

[0079] The specific operating steps are as follows: The formulation system is 100 mL. Place the beaker on a magnetic stirrer, first add 50 mL of ultrapure water, then add 20 mL of polyethylene glycol 400 and 20 mL of 1,2-propanediol and stir until homogeneous. Add the antioxidants listed in Table 5, then add 2.64 g of magnesium chloride hexahydrate. After dissolving, add 3.0 g of doxycycline hydrochloride and complex for 15 minutes. Finally, add 1.5 g of colistin. The entire process is carried out at 50℃. After complete dissolution and standing at room temperature, observe for any precipitation or turbidity. The evaluation criteria are: no precipitation, no colloid formation, clear and transparent solution, and no significant color change after 90 days.

[0080] Table 5 Antioxidant Screening of Formulations

[0081]

[0082]

[0083] Since sodium thiosulfate and sodium bisulfite solutions are alkaline, which is detrimental to the stability of doxycycline hydrochloride, and sodium bisulfite has a sulfur dioxide odor, sodium metabisulfite and L-ascorbic acid are used as antioxidants in this formulation. Using either one as an antioxidant alone is insufficient to achieve the goal of long-lasting antioxidant effect; therefore, using both together yields a significant antioxidant effect.

[0084] 7. Screening of sodium hydroxide solution concentration for pH adjustment in formulations

[0085] Sodium hydroxide for injection was selected as the pH adjuster for the formulation, and a concentration screening experiment was conducted.

[0086] Operating Procedures: The formulation system is 100 mL, and the temperature throughout the process is 50℃. Place the beaker on a magnetic stirrer, first add 50 mL of purified water, then add 20 mL of polyethylene glycol 400 and 20 mL of 1,2-propanediol and stir until homogeneous. Add 0.2 g of the antioxidant sodium metabisulfite and 0.2 g of L-ascorbic acid, then add 2.64 g of magnesium chloride hexahydrate. After complete dissolution, add 3.0 g of doxycycline hydrochloride and complex for 15 min. Finally, add 1.5 g of colistin in small amounts several times until completely dissolved. Adjust the pH of the formulation to 4.0 (basic requirement for injections) using the concentrations in Table 6. After standing at room temperature, observe for any precipitation or turbidity. The evaluation criteria are no precipitation, no colloid formation, clear and transparent solution, and no significant color change after 24 hours.

[0087] Table 6. Screening of pH adjuster-sodium hydroxide concentration in formulations.

[0088] Sodium hydroxide (mol / L) 24 hours later 1.0 Dissolve 5.0 Dissolve 10.0 Precipitation

[0089] As shown in Table 6, no precipitation occurred after 24 hours when adjusting the pH of the compound injection using 1 mol / L NaOH and 5 mol / L NaOH solutions. However, when adjusting the pH with 10 mol / L NaOH, the high alkalinity could cause local drug denaturation during addition; while the 1 mol / L NaOH solution had a lower concentration, requiring a larger volume for pH adjustment. Therefore, the optimal pH adjuster was determined to be 5 mol / L NaOH solution.

[0090] 8. Determination of the pH value of the formulation

[0091] Because doxycycline hydrochloride aqueous solution has poor stability, different pH values ​​can affect the stability of the formulation. Therefore, a suitable pH value should be selected for this formulation.

[0092] The specific operating steps are as follows: The formulation system is 100 mL, and the temperature throughout the process is 50℃. Place the beaker on a magnetic stirrer, first add 50 mL of purified water, then add 20 mL of polyethylene glycol 400 and 20 mL of 1,2-propanediol, then add 0.2 g of the antioxidant sodium metabisulfite, 0.2 g of L-ascorbic acid, and 2.64 g of magnesium chloride hexahydrate. Dissolve completely, then add 3.0 g of doxycycline hydrochloride and allow the complexation reaction to proceed for 15 min. Finally, add 1.5 g of colistin in small, repeated additions until completely dissolved. Adjust the pH using 5.0 mol / L sodium hydroxide. After standing at room temperature, observe for any precipitation or turbidity. The evaluation criteria are: no precipitation, no colloid formation, clear and transparent solution, and no significant color change after 24 hours.

[0093] Table 7. Determination of pH of Formulations

[0094] pH value 24 hours later 4.0 Dissolve 4.5 Dissolve 5.0 Dissolve 5.5 precipitation 6.0 precipitation

[0095] Considering that the specified pH value for this injection solution should be ≥4.0, and that doxycycline hydrochloride is stable and not easily decomposed under acidic conditions, the optimal pH value for the doxycycline hydrochloride injection solution prepared by this method is determined to be 4.0. The production pH value should be controlled between 4.0 and 5.0.

[0096] Example 2

[0097] This invention prepares doxycycline hydrochloride and colistin sulfate injection solutions. Each 1000 mL of doxycycline hydrochloride and colistin sulfate injection solution contains 30 g of doxycycline hydrochloride, 15 g of colistin sulfate, 2 g of L-ascorbic acid, 2 g of sodium metabisulfite, 240 mL of polyethylene glycol 400, 240 mL of 1,2-propanediol, 26.4 g of magnesium chloride hexahydrate, and the remainder is water for injection.

[0098] The preparation method of the above-mentioned doxycycline hydrochloride and colistin sulfate injection includes the following steps:

[0099] Step 1: Heat water for injection to 50°C, add L-ascorbic acid and sodium metabisulfite, and stir at 1200 r / min for 2 min until dissolved to obtain solution 1;

[0100] Step 2: At 50℃, polyethylene glycol 400 and 1,2-propanediol were added to the obtained solution 1. The mixture was stirred at 1200 r / min for 5 min until it became viscous. After dissolving the mixture in magnesium chloride hexahydrate, solution 2 was obtained.

[0101] Step 3: At 50℃, add doxycycline hydrochloride to solution 2 and mix at 1200 r / min for 15 min to allow the complexation reaction between doxycycline hydrochloride and magnesium chloride hexahydrate to be complete until it dissolves into a pale yellow liquid, thus obtaining solution 3.

[0102] Step 4: At 50℃, add colistin sulfate in small amounts several times to solution 3, each time adding 2.5g, for a total of 6 times. Continue stirring at 1200r / min for 30min until completely dissolved to obtain solution 4.

[0103] Step 5: After adjusting the pH of solution 4 to 4.0 with 5 mol / L sodium hydroxide solution, filter it twice with a 0.22 μM microporous membrane;

[0104] Step 6: Fill the filtrate obtained in Step 5 into 5mL vials, seal them with a washable, graded rubber stopper and an aluminum-plastic combination cap, and then fill the vials. The resulting injection solution is doxycycline hydrochloride and colistin sulfate injection.

[0105] The test results of the doxycycline hydrochloride and colistin sulfate injections prepared in this experimental example are as follows:

[0106] Experimental procedure: The obtained compound drug solution was placed under the conditions shown in the table, and the changes in the content of its active ingredients were detected by high performance liquid chromatography at different time periods.

[0107] High performance liquid chromatography (HPLC) detection conditions:

[0108] Instrument: 1260ⅡPrime LC;

[0109] Column: Poroshell 120SB-C18, 4.6*100mm, 2.7um;

[0110] Mobile phase A: 0.05% dilute TFA solution; Mobile phase B: acetonitrile; Mobile phase C: 50% acetonitrile-water solution; Mobile phase D: 90% methanol-water solution.

[0111] Gradient elution:

[0112]

[0113]

[0114] Flow rate: 1 mL / min; column temperature: 30℃; injection volume: 1 μL; column pressure: approximately 255 bar; detector: 215 nm (VWD); ELSD: 60℃, 60℃, 40 Hz.

[0115] Sample preparation:

[0116] Solvent: 20 wt% acetonitrile aqueous solution;

[0117] Colistin sulfate solution: Accurately weigh 50 mg of colistin sulfate standard, dissolve and dilute to 10 mL of 20 wt% acetonitrile aqueous solution to obtain a 5 mg / mL colistin sulfate acetonitrile aqueous solution; then dilute the colistin sulfate acetonitrile aqueous solution 10 times with 20 wt% acetonitrile aqueous solution to obtain a test standard solution with a final colistin sulfate concentration of 0.5 mg / mL;

[0118] Polytetracycline hydrochloride solution: Prepared in the same way as colistin sulfate solution, the final concentration of polytetracycline hydrochloride in the test standard solution is 0.5 mg / mL;

[0119] Injection solution: Take 0.02 mL of injection solution and dilute it to 1 mL with 20 wt% acetonitrile aqueous solution (dilute 50 times).

[0120] Table 8. Content changes of compound injection solution at different temperatures.

[0121]

[0122] Conclusion: At day 0, the contents of colistin sulfate and doxycycline hydrochloride in the compound injection did not change. However, after 30 days at 40℃ and 60℃ respectively, the contents decreased. This indicates that this injection should not be stored in environments above 30℃, but should be stored in environments below 30℃ to prevent drug degradation.

[0123] Inhibition experiments of FIC combined with other drugs on different bacterial strains:

[0124] 1. Add 100 μL of MH medium (MHB, purchased from Beijing Luqiao Technology Co., Ltd.) to each well of a 96-well plate;

[0125] 2. Add 100 μL of colistin sulfate (drug A) to each well from H1 to H12. The final concentration of colistin sulfate in each well from H1 to H12 is 8 μg / mL.

[0126] 3. Perform a gradient dilution from H to B, with each dilution resulting in half the original concentration, until the final concentration is reached. Discard the remaining 100 μL.

[0127] 4. Add 100 μL of doxycycline hydrochloride (drug B) to each well in column A1 to H1. The final concentration of doxycycline hydrochloride in each well in column A1 to H1 is 16 μg / mL.

[0128] 5. Perform a serial dilution from column 1 to the right, with each dilution resulting in half the original concentration. Dilute to column 11 and discard the remaining 100 μL.

[0129] 6. Add the test bacterial solution into each well of the 96-well plate. The amount of the test bacterial solution added is 100 μL / well, and the final content of bacteria in each well is 5×10

[0141] ,

[0142] ,

[0139] , ,

[0140] ,

[0144] ,

[0138] , , , , , , , ,

[0143] CFU.

[0130] For all the combined antibacterial experiments, except for the different strains used, other operation steps are the same.

[0131] Table 9 Synergistic effect of colistin sulfate and doxycycline hydrochloride in combination against Escherichia coli

[0132] <0机器翻译错误的地方,我会纠正为正确的翻译

[0133] Note: MIC is the minimum inhibitory concentration;

[0134] The principle of FIC determination is based on the minimum inhibitory concentration MIC, that is, the lowest concentration at which a drug can inhibit the growth of microorganisms. By testing the MIC values of the drug used alone and the drug combination, the FIC index is calculated, and then the nature of the drug interaction is analyzed.

[0135] The calculation formula of the FIC index is:

[0136] FIC = (MIC of drug A in combination) / (MIC of drug A alone) + (MIC of drug B in combination) / (MIC of drug B alone)

[0137] When FICI ≤ 0.5, the mode of action of the two drugs is synergistic, indicating that the combined action of the two drugs is significantly greater than the sum of their individual actions;

[0138] When 0.5 < FICI ≤ 1, the mode of action of the two drugs is additive, indicating that the activity when the two drugs act in combination is equal to the sum of the two individual antibacterial activities;

[0139] When 1 < FICI ≤ 2, the mode of action of the two drugs is irrelevant, indicating that the activity of the two drugs acting in combination is equal to their individual activities;

[0140] When FICI > 2, the mode of action of the two drugs is antagonistic, indicating that the combined action of the two drugs is significantly lower than their individual antibacterial activities.

[0141] Table 10 Synergistic effect of colistin sulfate and doxycycline hydrochloride in combination against Klebsiella pneumoniae

[0142]

[0143]

[0144] This study demonstrates that the combined use of colistin sulfate and doxycycline hydrochloride effectively enhances the inhibitory effect against Gram-negative bacteria such as *Escherichia coli* and *Klebsiella pneumoniae*. Experimental results show that this combination exhibits additive or synergistic effects in a variety of bacterial strains. Furthermore, this combination regimen achieves antibacterial activity at lower drug concentrations, helping to reduce the dosage of single antibiotics, lower the risk of drug resistance, and improve therapeutic efficacy.

[0145] Example 3

[0146] This invention provides a veterinary doxycycline hydrochloride and colistin sulfate injection solution. Each 1000 mL of the veterinary doxycycline hydrochloride and colistin sulfate injection solution contains: 20 g doxycycline hydrochloride, 10 g colistin sulfate, 20 g magnesium chloride hexahydrate, 1.5 g sodium metabisulfite, 150 mL polyethylene glycol 400, 150 mL 1,2-propanediol, 1.5 g L-ascorbic acid, and the remainder is water for injection.

[0147] The preparation method of the above-mentioned doxycycline hydrochloride and colistin sulfate injection includes the following steps:

[0148] Step 1: Heat water for injection to 60°C, add L-ascorbic acid and sodium metabisulfite, and stir at 1100 r / min for 1 min until dissolved to obtain solution 1;

[0149] Step 2: At 60℃, polyethylene glycol 400 and 1,2-propanediol were added to the obtained solution 1. The mixture was stirred at 1100 r / min for 4 min until it became viscous. After dissolving the mixture in magnesium chloride hexahydrate, solution 2 was obtained.

[0150] Step 3: At 60℃, add doxycycline hydrochloride to solution 2 and mix at 1100 r / min for 17 min to allow the complexation reaction between doxycycline hydrochloride and magnesium chloride hexahydrate to be complete until it dissolves into a pale yellow liquid, thus obtaining solution 3.

[0151] Step 4: At 60℃, add colistin sulfate in small amounts multiple times to solution 3, with each addition being 2g, for a total of 5 additions. Continue stirring at 1100r / min for 25min until completely dissolved to obtain solution 4.

[0152] Step 5: After adjusting the pH of solution 4 to 4.5 with 3 mol / L sodium hydroxide solution, filter it twice with a 0.22 μM microporous membrane;

[0153] Step 6: Fill the filtrate obtained in Step 5 into 5mL vials, seal them with a washable, graded rubber stopper and an aluminum-plastic combination cap, and then fill the vials. The resulting injection solution is doxycycline hydrochloride and colistin sulfate injection.

[0154] Experiment Example 4

[0155] This invention prepares a veterinary doxycycline hydrochloride and colistin sulfate injection solution. Each 1000 mL of the veterinary doxycycline hydrochloride and colistin sulfate injection solution contains: 50 g doxycycline hydrochloride, 30 g colistin sulfate, 50 g magnesium chloride hexahydrate, 2.5 g sodium metabisulfite, 250 mL polyethylene glycol 400, 250 mL 1,2-propanediol, 2.5 g L-ascorbic acid, and the remainder is water for injection.

[0156] The preparation method of the above-mentioned doxycycline hydrochloride and colistin sulfate injection includes the following steps:

[0157] Step 1: Heat water for injection to 70°C, add L-ascorbic acid and sodium metabisulfite, and stir at 1300 r / min for 3 min until dissolved to obtain solution 1;

[0158] Step 2: At 70℃, polyethylene glycol 400 and 1,2-propanediol were added to the obtained solution 1. The mixture was stirred at 1300 r / min for 6 min until it became viscous. After dissolving the mixture in magnesium chloride hexahydrate, solution 2 was obtained.

[0159] Step 3: At 70℃, add doxycycline hydrochloride to solution 2 and mix at 1300 r / min for 17 min to allow the complexation reaction between doxycycline hydrochloride and magnesium chloride hexahydrate to be complete until it dissolves into a pale yellow liquid, thus obtaining solution 3.

[0160] Step 4: At 70℃, add colistin sulfate to solution 3 in small amounts several times, for a total of 7 times. The amount of colistin sulfate added in the first 6 times is 4.3g, and the amount of colistin sulfate added in the 7th time is 4.2g. Continue stirring at 1300r / min for 35min until completely dissolved to obtain solution 4.

[0161] Step 5: After adjusting the pH of solution 4 to 5.0 with 1 mol / L sodium hydroxide solution, filter it three times through a 0.22 μM microporous membrane;

[0162] Step 6: Fill the filtrate obtained in Step 5 into 5mL vials, seal them with a washable, graded rubber stopper and an aluminum-plastic combination cap, and then fill the vials. The resulting injection solution is doxycycline hydrochloride and colistin sulfate injection.

[0163] As can be seen from the above embodiments, the present invention provides an injection solution of doxycycline hydrochloride and colistin sulfate, and a method for preparing the same. The injection solution comprises the following raw materials at the following final concentrations: doxycycline hydrochloride 0.02-0.05 g / mL, colistin sulfate 0.01-0.03 g / mL, antioxidant 0.003-0.005 g / mL, solubilizer 30-50 mL / 100 mL, complexing agent 0.02-0.05 g / mL, and the balance being water. The injection solution of the present invention can cover a wider spectrum of pathogens, improve the efficiency and safety of clinical treatment, and ensure the safety of the injection solution when used at high doses, effectively solving the problems of poor stability and high toxicity of existing similar injection solutions. Therefore, this application provides an injection solution with good stability, low toxicity, significant efficacy, and broad application prospects.

[0164] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A doxycycline hydrochloride and colistin sulfate injection solution, characterized in that, The raw materials include the following final concentrations: doxycycline hydrochloride 0.02~0.05 g / mL, colistin sulfate 0.01~0.03 g / mL, antioxidant 0.003~0.005 g / mL, solubilizer 30~50 mL / 100 mL, complexing agent 0.02~0.05 g / mL, and the balance is water; The antioxidants are sodium metabisulfite and L-ascorbic acid; The co-solvent is polyethylene glycol 400 and 1,2-propanediol; The complexing agent is magnesium chloride hexahydrate.

2. A method for preparing the doxycycline hydrochloride and colistin sulfate injection solution according to claim 1, characterized in that, Includes the following steps: (1) Mix water and antioxidant to obtain solution 1; (2) Mix solution 1, cosolvent and complexing agent to obtain solution 2; (3) Mix solution 2 with doxycycline hydrochloride to obtain solution 3; (4) Mix solution 3 with colistin sulfate to obtain solution 4; (5) After adjusting the pH of solution 4 to 4.0~5.0, filter it with a microporous membrane. The filtrate is doxycycline hydrochloride and colistin sulfate injection.

3. The preparation method according to claim 2, characterized in that, The mixing temperature in step (1) is 50~70℃; the mixing time is 1~3min; and the mixing speed is 1100~1300r / min.

4. The preparation method according to claim 3, characterized in that, The mixing time in step (2) is 4 to 6 minutes, and the mixing speed is 1100 to 1300 r / min.

5. The preparation method according to claim 4, characterized in that, The mixing time in step (3) is 15~20 min, and the mixing speed is 1100~1300 r / min.

6. The preparation method according to claim 5, characterized in that, The mixing method in step (4) is as follows: add colistin sulfate to solution 3 in 5 to 7 portions; the mixing time is 25 to 35 minutes and the mixing speed is 1100 to 1300 r / min.

7. The preparation method according to claim 6, characterized in that, In step (5), a sodium hydroxide solution of 1-5 mol / L is used to adjust the pH of solution 4; the diameter of the microporous membrane is 0.22 μM; and the filtration is performed 2-3 times.

Images