Doxycycline hydrochloride and colistin sulfate injection and preparation method thereof

Abstract

The invention relates to the technical field of doxycycline hydrochloride and colistin sulfate preparations, and provides a doxycycline hydrochloride and colistin sulfate injection and a preparation method thereof.The injection is prepared from, by final concentration, 0.02 g / mL to 0.05 g / mL of doxycycline hydrochloride, 0.01 g / mL to 0.03 g / mL of colistin sulfate, 0.003 g / mL to 0.005 g / mL of antioxidant, 30 mL / 100 mL of cosolvent, 0.02 g / mL to 0.05 g / mL of complexing agent and the balance water. The injection provided by the invention can cover a wider pathogenic bacteria spectrum, can also improve the efficiency and safety of clinical treatment, ensures the safety of the injection in high-dose application, and effectively solves the problems of poor stability and high toxic and side effects of the existing similar injection. Therefore, the injection provided by the invention is good in stability, low in toxic and side effects, remarkable in effect and wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of doxycycline hydrochloride and colistin sulfate preparations, and particularly relates to an injection of doxycycline hydrochloride and colistin sulfate and a preparation method thereof. Background Art

[0002] In recent years, the problem of antibiotic resistance has become increasingly serious, especially the spread of multi-drug resistant (MDR) and extensively drug-resistant (XDR) bacteria, which poses a severe challenge to clinical treatment. At present, in veterinary clinics, especially when treating multi-drug resistant bacterial infections, existing single-antibiotic therapies often face problems such as insufficient efficacy and large side effects.

[0003] As a key drug for treating multi-drug resistant Gram-negative bacteria, although colistin sulfate has achieved good clinical efficacy, its nephrotoxicity and neurotoxicity limit its high-dose and wide application, especially when high doses are required, the toxic side effects are significant. At the same time, although doxycycline hydrochloride has good efficacy against infections such as Gram-positive bacteria and rickettsia, its antibacterial effect is relatively weak when treating Gram-negative bacteria, especially drug-resistant bacteria. In addition, the problem of drug resistance has gradually emerged in the use of doxycycline hydrochloride, and its side effects cannot be ignored. Especially in the form of an injection, due to its poor stability and easy formation of precipitation and other phenomena, it further affects the efficacy and safety of the drug.

[0004] At present, in veterinary clinics, especially when treating multi-drug resistant bacterial infections, existing single-antibiotic therapies often face problems such as insufficient efficacy and large side effects. As an effective strategy, combination antibiotic therapy can expand the antibacterial spectrum, delay the development of drug resistance, and to a certain extent reduce the side effects of single antibiotics through the synergistic effect between drugs. However, although there have been studies on various combination antibiotic therapy regimens, the research on the combined use of colistin sulfate and doxycycline hydrochloride is still relatively scarce. By combining colistin sulfate and doxycycline hydrochloride, the present invention can cover a wider spectrum of pathogenic bacteria and exert a synergistic effect in clinical treatment, significantly improving the treatment effect and safety. Summary of the Invention

[0005] The purpose of the present invention is to provide an injection of doxycycline hydrochloride and colistin sulfate and a preparation method thereof. By combining the use of colistin sulfate and doxycycline hydrochloride, it can more effectively deal with drug-resistant strains and improve the success rate of clinical treatment. In addition, the injection provided by the present invention not only has a wider antibacterial spectrum, but also can reduce the administration frequency through synergistic effects, reduce adverse reactions, and significantly improve the efficiency and safety of clinical treatment, and has broad application prospects.

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

[0007] The present invention provides an injection of doxycycline hydrochloride and colistin sulfate, comprising raw materials with 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.

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

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

[0010] Preferably, the complexing agent is one or several 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 preparation method of the injection of doxycycline hydrochloride and colistin sulfate, comprising the following steps:

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

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

[0014] (3) Mix Solution 2 and doxycycline hydrochloride to obtain Solution 3;

[0015] (4) Mix Solution 3 and 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, and the filtrate is the injection of doxycycline hydrochloride and colistin sulfate.

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

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

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

[0020] Preferably, the method of mixing 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 min, and the mixing speed is 1100 to 1300 r / min.

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

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

[0023] The doxycycline hydrochloride and colistin sulfate injection prepared by the present invention has good stability, controllable quality, and a better therapeutic effect than the single doxycycline hydrochloride or colistin sulfate injection. Through the synergistic effect between drugs, it can cover a wider spectrum of pathogenic bacteria, reduce the dosing frequency, reduce adverse reactions, significantly improve the efficiency and safety of clinical treatment, and has broad application prospects.

[0024] The doxycycline hydrochloride and colistin sulfate injection of the present invention effectively improves the stability of the drug through precise drug ratio and preparation method. There are no visible crystals and precipitates within two years, and the product quality is stable. It can maintain the drug effect for a long time, solve the problems of poor stability and high toxic and side effects of existing doxycycline hydrochloride and colistin sulfate injections in clinical applications, and provide a veterinary injection with better stability, low toxic and side effects and significant effects. Detailed implementation mode

[0025] The present invention provides a doxycycline hydrochloride and colistin sulfate injection, which comprises the following raw materials at the 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.

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

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

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

[0029] In the present invention, the concentration of the cosolvent is preferably 30 - 50 mL / 100 mL, more preferably 40 mL / 100 mL.

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

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

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

[0033] In the present invention, the complexing agent is preferably one or several 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 injection of doxycycline hydrochloride and colistin sulfate, comprising the following steps:

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

[0036] (2) Mix Solution 1, the cosolvent, and the complexing agent to obtain Solution 2;

[0037] (3) Mix Solution 2 and doxycycline hydrochloride to obtain Solution 3;

[0038] (4) Mix Solution 3 and colistin sulfate to obtain Solution 4;

[0039] (5) Adjust the pH of Solution 4 to 4.0 - 5.0, and then filter it through a microporous membrane. The filtrate is the injection of doxycycline hydrochloride and colistin sulfate.

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

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

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

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

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

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

[0046] Example 1 Screening of each raw material in the preparation and determination of reaction conditions

[0047] 1. Screening of solvents

[0048] Both doxycycline hydrochloride and colistin sulfate are water-soluble drugs. Therefore, laboratory ultrapure water is selected to dissolve the two drugs.

[0049] Operation steps: Take 100 mL of ultrapure water and place it in a beaker. Place the beaker on a magnetic stirrer and stir evenly. First, add 3.0 g of doxycycline hydrochloride. After the doxycycline hydrochloride is completely dissolved, then add 1.5 g of colistin (forty-five million units of colistin) to dissolve. Taking no precipitation, no turbidity, and clear and transparent solution after 24 h as the evaluation index.

[0050] Results: When adding colistin after the complete dissolution of doxycycline hydrochloride and stirring together, an insoluble phenomenon occurred, and the drug precipitated after stopping stirring. It is speculated that the possible reason for the drug precipitation is the addition order of the drugs. Therefore, the addition order of the drugs was changed: adding colistin completely dissolved first, and then adding doxycycline hydrochloride, and precipitation still occurred. Therefore, it was considered to increase a cosolvent to dissolve the two drugs.

[0051] 2. Screening of cosolvents

[0052] One or two of propylene glycol for injection, polyethylene glycol 300, and polyethylene glycol 400 were selected as co-solvents for the preparation, and a screening test for co-solvents was carried out.

[0053] Operating steps: In a 100 mL system, place the beaker on a magnetic stirrer, first add 50 mL of ultrapure water, then add the co-solvent in Table 1 and stir evenly. After that, add 3.0 g of doxycycline hydrochloride and dissolve it completely, then add 1.5 g of colistin and dissolve it completely. After standing at room temperature, observe whether there is precipitation, turbidity, etc. No precipitation, no turbidity, and a clear and transparent solution after 24 hours were used as the evaluation index.

[0054] Table 1 Screening of co-solvents in the preparation

[0055]

[0056]

[0057] According to the results in Table 1, when using a single co-solvent to dissolve the two drugs, there was partial precipitation after standing for 24 hours. However, after using polyethylene glycol 400 and 1,2-propanediol simultaneously, both drugs were dissolved without precipitation after standing for 24 hours. Therefore, polyethylene glycol 400 and 1,2-propanediol were selected as the co-solvents for this preparation.

[0058] 3. Screening of complexing agents

[0059] After using co-solvents to dissolve doxycycline hydrochloride and colistin, precipitation occurred in the solution after standing at room temperature for 7 days. The reason may be the poor aqueous solution stability of doxycycline hydrochloride. Therefore, a complexing agent was considered to complex with doxycycline hydrochloride to increase its stability in aqueous solution. One or two of magnesium chloride hexahydrate, disodium ethylenediaminetetraacetate, zinc oxide, calcium oxide, calcium lactate, magnesium sulfate, aluminum chloride, and aluminum oxide that meet the pharmaceutical requirements were selected as the complexing agents for doxycycline hydrochloride, and a screening test for complexing agents was carried out.

[0060] The specific operating steps were as follows: The preparation system was 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 evenly. Then add the complexing agent in Table 2 and dissolve it completely. Add 3.0 g of doxycycline hydrochloride to complex with the complexing agent. After doxycycline hydrochloride was completely dissolved, add 1.5 g of colistin and dissolve it completely. After standing at room temperature, observe whether there is precipitation, turbidity, etc. No precipitation, no colloid formation, and a clear and transparent solution after 7 days were used as the evaluation index.

[0061] Table 2 Screening of the types of complexing agents in the preparation

[0062]

[0063]

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

[0065] 4. Temperature Screening of the Complexation Reaction of Doxycycline Hydrochloride

[0066] Since temperature may affect the complexing effect, we will experimentally screen the suitable temperature for doxycycline hydrochloride. Magnesium chloride hexahydrate meeting the requirements for injection is selected as the complexing agent for doxycycline hydrochloride, and different temperatures are investigated.

[0067] The specific operation steps are as follows: The preparation 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 evenly. Then add 3.0 g of magnesium chloride hexahydrate and dissolve it completely. Then add 3.0 g of doxycycline hydrochloride. The temperature is set as shown in Table 3. After complete dissolution, add 1.5 g of colistin and dissolve it completely. After standing at room temperature, observe phenomena such as whether there is precipitation and turbidity. Taking no precipitation, no colloid formation, clear and transparent solution, and stable preparation after 15 days as the evaluation index.

[0068] Table 3 Screening of the Complexing Temperature of the Preparation

[0069]

[0070] As shown in Table 3, the complexing stability of doxycycline hydrochloride and magnesium chloride hexahydrate is the best under the conditions of 50 °C and 60 °C. The solubility of doxycycline hydrochloride at 60 °C is the same as that at 50 °C. Considering that too high temperature will affect other drug components, 50 °C is adopted as the complexing temperature of doxycycline hydrochloride.

[0071] 5. Screening of the Complexing Time

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

[0073] The specific operation steps are as follows: The preparation system is 100 mL. Place the beaker on a magnetic stirrer, first add 50 mL of injection water, then add 20 mL of polyethylene glycol 400 and 20 mL of 1,2 - propanediol and stir evenly. Add 3.0 g of magnesium chloride hexahydrate. After dissolution, then add 3.0 g of doxycycline hydrochloride and dissolve completely. Set the complexation time as shown in Table 4. Finally, add 1.5 g of colistin. Throughout the process, under the condition of 50 °C, after complete dissolution, let it stand at room temperature and observe whether there is precipitation, turbidity and other phenomena. Take no precipitation, no colloid formation, and the solution being clear and transparent after 30 days as the evaluation index.

[0074] Table 4 Screening of the reaction time between the complexing agent and doxycycline hydrochloride

[0075] Complexing time (min) After 30 days 5 A small amount of precipitation 10 A small amount of precipitation 15 Clear 20 Clear

[0076] As shown in Table 4, the complexing stability of doxycycline hydrochloride and magnesium chloride hexahydrate is the best under the conditions of 15 min and 20 min. The solubility of doxycycline hydrochloride at 20 min is the same as that at 15 min. Referring to the reference materials and combining with the physicochemical properties of this preparation, 15 min is used as the complexing time of doxycycline hydrochloride.

[0077] 6. Screening of antioxidants in the preparation

[0078] Although the addition of a complexing agent and doxycycline hydrochloride in the preparation increases its stability, since the aqueous solution of doxycycline hydrochloride is prone to oxidation reaction and the color changes. Therefore, it is necessary to add antioxidants to reduce the oxidation rate of doxycycline hydrochloride. Select one or two combinations that can dissolve in the solution, have no incompatibility with the main drug, and increase the antioxidant effect of this preparation from sodium thiosulfate, sodium sulfite, sodium bisulfite, sodium metabisulfite, and L - ascorbic acid.

[0079] The specific operation steps are as follows: The preparation 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 evenly. Add the antioxidants shown in Table 5. After that, add 2.64 g of magnesium chloride hexahydrate. After dissolution, add 3.0 g of doxycycline hydrochloride and complex for 15 min, and finally add 1.5 g of colistin. The whole process is at a temperature of 50 °C. After complete dissolution, let it stand at room temperature and observe whether there is precipitation, turbidity and other phenomena. Take no precipitation, no colloid formation, the solution being clear and transparent, and no obvious change in the solution color after 90 days as the evaluation index.

[0080] Table 5 Screening of antioxidants in the preparation

[0081]

[0082]

[0083] Since the aqueous solutions of sodium thiosulfate and sodium bisulfite are alkaline, which is not conducive to the stability of doxycycline hydrochloride, and sodium bisulfite has a sulfur dioxide odor, sodium metabisulfite and L-ascorbic acid are used as antioxidants for this preparation. Using only one of them as an antioxidant cannot achieve the goal of a long antioxidant time. Therefore, the two are used together, and the antioxidant effect obtained is remarkable.

[0084] 7. Screening of the concentration of sodium hydroxide solution for adjusting the pH of the preparation

[0085] Sodium hydroxide for injection is selected as the pH regulator for the preparation, and a concentration screening experiment is carried out.

[0086] Operating procedure: The preparation system is 100 mL, and the whole process temperature is 50 °C. 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 evenly. Add 0.2 g of sodium metabisulfite as an antioxidant 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 multiple times until completely dissolved. Use the concentrations in Table 6 to adjust the pH of the preparation to 4.0 (basic requirement for injection). After standing at room temperature, observe whether there is precipitation, turbidity, etc. No precipitation, no colloid formation, clear and transparent solution, and no obvious change in color after 24 h are used as evaluation indicators.

[0087] Table 6 Screening of the concentration of the pH regulator - sodium hydroxide for the preparation

[0088] Sodium hydroxide (mol / L) After 24 h 1.0 Dissolve 5.0 Dissolve 10.0 Precipitate

[0089] As shown in Table 6, when adjusting with 1 mol / L NaOH and 5 mol / L NaOH solutions, there will be no precipitation after standing for 24 h. When adjusting the pH of the compound injection with 10 mol / L NaOH, due to the relatively high alkalinity, local drug denaturation will occur during the addition process; while the concentration of the 1 mol / L NaOH solution is relatively low, and a large volume is used when adjusting the pH. Therefore, the best pH regulator is finally determined to be the 5 mol / L NaOH solution.

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

[0091] Since the stability of the aqueous solution of doxycycline hydrochloride is poor, different pH values will affect the stability of the preparation. Therefore, the pH value suitable for this preparation is selected.

[0092] The specific operation steps are as follows: The preparation system is 100 mL, and the temperature throughout the process is 50 °C. Place the beaker on a magnetic stirrer. First, add 50 mL of purified water, 20 mL of polyethylene glycol 400, and 20 mL of 1,2-propanediol. Then add 0.2 g of antioxidant sodium metabisulfite and 0.2 g of L-ascorbic acid. Next, add 2.64 g of magnesium chloride hexahydrate and dissolve completely. After that, add 3.0 g of doxycycline hydrochloride for a complexation reaction for 15 min. Finally, add 1.5 g of colistin in small portions and dissolve completely. Adjust the pH using 5.0 mol / L sodium hydroxide. After standing at room temperature, observe for phenomena such as precipitation and turbidity. The evaluation criteria are no precipitation, no colloid formation, clear and transparent solution, and no obvious color change after 24 h.

[0093] Table 7 Determination of the pH of the preparation

[0094] pH value After 24 h 4.0 Dissolve 4.5 Dissolve 5.0 Dissolve 5.5 Precipitate 6.0 Precipitate

[0095] Combined with the requirement that the pH value of this injection should be ≥ 4.0, doxycycline hydrochloride is stable under acidic conditions and not easily decomposed. It is comprehensively determined that the most stable pH value of the doxycycline hydrochloride injection prepared by this method is 4.0, and the production pH value is controlled between 4.0 and 5.0.

[0096] Example 2

[0097] The present invention prepares an injection of doxycycline hydrochloride and colistin sulfate. Each 1000 mL of the injection 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, and 26.4 g of magnesium chloride hexahydrate, with the balance being injection water.

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

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

[0100] Step 2: Under the condition of 50 °C, add polyethylene glycol 400 and 1,2-propanediol to the obtained Solution 1, and stir at a speed of 1200 r / min for 5 min until the mixture becomes viscous. After adding magnesium chloride hexahydrate and dissolving, obtain Solution 2.

[0101] Step 3: Under the condition of 50 °C, add doxycycline hydrochloride to Solution 2, and mix at a speed of 1200 r / min for 15 min to completely complex doxycycline hydrochloride and magnesium chloride hexahydrate until it is dissolved into a light yellow liquid to obtain Solution 3.

[0102] Step 4: At 50 °C, colistin sulfate was added to Solution 3 in small portions multiple times, with each addition being 2.5 g, for a total of 6 additions. Stirring was continued at a rotation speed of 1200 r / min for 30 min 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, it was filtered twice through a 0.22 μM microporous membrane;

[0104] Step 6: The filtrate obtained in Step 5 was filled into 5 mL vials, sealed with a disposable grade rubber stopper and an aluminum-plastic combination cap, and filled. The injection was prepared, which is the injection of doxycycline hydrochloride and colistin sulfate.

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

[0106] Test procedure: The obtained compound drug solution was placed under the conditions shown in the table, and the content change of the active ingredient was detected by high performance liquid chromatography at different time intervals.

[0107] High performance liquid chromatography detection conditions:

[0108] Instrument: 1260ⅡPrime LC;

[0109] Chromatographic column: Poroshell 120SB-C18, 4.6*100 mm, 2.7 μm;

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

[0111] Gradient elution:

[0112]

[0113]

[0114] Flow rate: 1 mL / min; Column temperature: 30 °C; Injection volume: 1 μL; Column pressure: about 255 bar; Detector: 215 nm (VWD); ELSD: 60 °C, 60 °C, 40 Hz.

[0115] Sample preparation:

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

[0117] Colistin sulfate solution: Accurately weigh 50 mg of colistin sulfate standard sample, dissolve it and make up the volume to 10 mL with 20 vt% 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 vt% acetonitrile aqueous solution to obtain a test standard solution with a final concentration of colistin sulfate of 0.5 mg / mL.

[0118] Doxycycline hydrochloride solution: Prepared in the same way as the colistin sulfate solution, and the final concentration of doxycycline hydrochloride in the test standard solution is: 0.5 mg / mL.

[0119] Injection solution: Take 0.02 mL of the injection, dilute and make up the volume to 1 mL with 20 vt% acetonitrile aqueous solution (diluted 50 times).

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

[0121]

[0122] Conclusion: At day 0, the contents of colistin sulfate and doxycycline hydrochloride in the compound injection did not change at all. However, when placed at 40 °C and 60 °C for 30 days respectively, the contents both decreased, indicating that this injection should not be placed in an environment above 30 °C and should be stored in an environment below 30 °C to maintain the degradation of the drug content.

[0123] Experimental method for the inhibitory effect of combined drug FIC on different strains:

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

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

[0126] 3. Dilute the H row in a gradient upward, and the concentration after each dilution is half of that before dilution until the B row, and discard the remaining 100 μL.

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

[0128] 5. Dilute the first column in a gradient to the right, and the concentration after each dilution is half of that before dilution until the 11th column, 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 5 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]

[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 the 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 of the two drugs 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 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 shows that the combined application of colistin and doxycycline hydrochloride can effectively enhance the antibacterial effect against Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae. The experimental results show that this combination exhibits additive or synergistic effects in various strains. In addition, this combined regimen can achieve antibacterial activity at lower drug concentrations, which helps to reduce the dosage of single antibiotics, lower the risk of drug resistance, and improve the treatment effect simultaneously.

[0145] Example 3

[0146] The present invention prepares an injection of doxycycline hydrochloride and colistin for veterinary use. Each 1000 mL of the injection of doxycycline hydrochloride and colistin for veterinary use contains: 20 g of doxycycline hydrochloride, 10 g of colistin, 20 g of magnesium chloride hexahydrate, 1.5 g of sodium metabisulfite, 150 mL of polyethylene glycol 400, 150 mL of 1,2-propanediol, 1.5 g of L-ascorbic acid, and the balance is water for injection.

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

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

[0149] Step 2: At 60 °C, add polyethylene glycol 400 and 1,2-propanediol to the obtained Solution 1, stir at a rotation speed of 1100 r / min for 4 min until the mixture becomes viscous, add magnesium chloride hexahydrate and dissolve it to obtain Solution 2;

[0150] Step 3: At 60 °C, add doxycycline hydrochloride to Solution 2, mix at a rotation speed of 1100 r / min for 17 min to complete the complexation reaction of doxycycline hydrochloride and magnesium chloride hexahydrate until it is dissolved into a light yellow liquid to obtain Solution 3.

[0151] Step 4: At 60 °C, add colistin to Solution 3 in small amounts and multiple times, with each addition amount being 2 g, and add it 5 times in total. Continue to stir at a rotation speed of 1100 r / min for 25 min 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: Seal the filtrate obtained in Step 5 in 5 mL vials, and seal it with a disposable stopple and an aluminum-plastic combination cap, and seal it. The prepared injection is the injection of doxycycline hydrochloride and colistin.

[0154] Experimental Example 4

[0155] The present invention prepared an injection of doxycycline hydrochloride and colistin sulfate for veterinary use. Each 1000 mL of the injection of doxycycline hydrochloride and colistin sulfate for veterinary use contains: 50 g of doxycycline hydrochloride, 30 g of colistin sulfate, 50 g of magnesium chloride hexahydrate, 2.5 g of sodium metabisulfite, 250 mL of polyethylene glycol 400, 250 mL of 1,2 - propanediol, 2.5 g of L - ascorbic acid, and the balance is water for injection.

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

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

[0158] Step 2: Under the condition of 70 °C, add polyethylene glycol 400 and 1,2 - propanediol to the obtained Solution 1, stir at a speed of 1300 r / min for 6 min until the mixture becomes viscous, add magnesium chloride hexahydrate and dissolve it to obtain Solution 2;

[0159] Step 3: Under the condition of 70 °C, add doxycycline hydrochloride to Solution 2, mix at a speed of 1300 r / min for 17 min to complete the complexation reaction of doxycycline hydrochloride and magnesium chloride hexahydrate until it is dissolved into a light - yellow liquid to obtain Solution 3.

[0160] Step 4: Under the condition of 70 °C, add colistin sulfate to Solution 3 in small amounts and multiple times, a total of 7 times. The amount of colistin sulfate added in the first 6 times is 4.3 g, and the amount of colistin sulfate added in the 7th time is 4.2 g. Continue to stir at a speed of 1300 r / min for 35 min 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 3 times with a 0.22 μM microporous membrane;

[0162] Step 6: Seal the filtrate obtained in Step 5 in 5 - mL vials, seal it with a disposable grade rubber stopper and an aluminum - plastic combination cap, and perform potting. The prepared injection is the injection of doxycycline hydrochloride and colistin sulfate.

[0163] As can be seen from the above embodiments, the present invention provides a doxycycline hydrochloride and colistin sulfate injection and a preparation method thereof. The injection comprises the following raw materials in 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, cosolvent 30 - 50 mL / 100 mL, complexing agent 0.02 - 0.05 g / mL, and the balance is water. The injection of the present invention can cover a wider spectrum of pathogenic bacteria, improve the efficiency and safety of clinical treatment, ensure the safety of the injection when applied at high doses, and effectively solve the problems of poor stability and high toxic and side effects of existing similar injections. It can be seen that the present application provides an injection with good stability, low toxic and side effects, remarkable effects and broad application prospects.

[0164] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A doxycycline hydrochloride and colistin sulfate injection, characterized in that: The method comprises the following raw materials with final concentrations: 0.02-0.05 g / mL of doxycycline hydrochloride, 0.01-0.03 g / mL of colistin sulfate, 0.003-0.005 g / mL of antioxidant, 30-50 mL / 100 mL of cosolvent, 0.02-0.05 g / mL of complexing agent, and the balance is water.

2. The doxycycline hydrochloride and colistin sulfate injection according to claim 1, characterized in that: The antioxidant is one or more of sodium pyrosulfite, L-ascorbic acid, sodium thiosulfate and sodium bisulfite.

3. The doxycycline hydrochloride and colistin sulfate injection according to claim 2, characterized in that: The co-solvent is one or more of polyethylene glycol 400, 1,2-propylene glycol and polyethylene glycol 300.

4. The doxycycline hydrochloride and colistin sulfate injection according to claim 3, characterized in that: 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.

5. A method for preparing the doxycycline hydrochloride and colistin sulfate injection according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) mixing water and an antioxidant to obtain a solution 1; (2) mixing solution 1, a co-solvent and a complexing agent to obtain solution 2; (3) mixing solution 2 and doxycycline hydrochloride to obtain solution 3; (4) mixing solution 3 and colistin sulfate to obtain solution 4; (5) After adjusting the pH of solution 4 to 4.0-5.0, the solution is filtered through a microporous membrane, and the filtrate is the doxycycline hydrochloride and colistin sulfate injection.

6. The preparation method according to claim 5, characterized in that: 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.

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

8. The preparation method according to claim 7, characterized in that: The mixing time in step (3) is 15 to 20 minutes, and the mixing speed is 1100 to 1300 r / min.

9. The preparation method according to claim 8, characterized in that: The mixing method in step (4) is: adding colistin sulfate to solution 3 in 5 to 7 times; the mixing time is 25 to 35 minutes, and the mixing speed is 1100 to 1300 r / min.

10. The preparation method according to claim 9, characterized in that: In step (5), 1-5 mol / L sodium hydroxide solution 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.

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