Oxytetracycline injection and preparation method thereof

Abstract

The invention provides an oxytetracycline injection and a preparation method thereof, and belongs to the technical field of medicine preparation, the preparation method comprises the following steps: uniformly mixing 35-50 parts by weight of polyethylene glycol 400 and water, sequentially adding 0.4-0.6 part by weight of sodium formaldehyde sulfoxylate and 1.6-1.8 parts by weight of magnesium oxide, heating, adding 18-22 parts by weight of oxytetracycline, carrying out a complex reaction, adding monoethanolamine to adjust the pH value to 8.4-8.6, and uniformly stirring to obtain the oxytetracycline injection. And fixing the volume and filtering to obtain the oxytetracycline injection. The oxytetracycline injection prepared from the oxytetracycline can effectively improve the stability and the drug effect, prolong the half-life period of the drug and reduce the irritation.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and in particular relates to oxytetracycline injection and a preparation method thereof. Background Art

[0002] Oxytetracycline injection is an antibiotic drug, the main ingredient of which is oxytetracycline, which belongs to the tetracycline antibiotics. The mechanism of action is mainly through binding to the 30S subunit of the bacterial ribosome, preventing aminoacyl-tRNA from entering the A position, thereby inhibiting the synthesis of bacterial proteins and exerting an antibacterial effect. However, the prior art is to produce oxytetracycline by fermentation. In the post-process of producing oxytetracycline by fermentation, the product quality is often improved by optimizing the process conditions; however, due to the large amount of impurities such as proteins, metal ions and other organic by-products produced during the fermentation process, the overall product purity is poor, the impurity content is high, and the stability of the oxytetracycline injection produced later is poor. Summary of the invention

[0003] In view of the above problems, the present invention provides an oxytetracycline injection and a preparation method thereof.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is:

[0005] A oxytetracycline injection, the formula of which comprises, by weight: 18-22 parts of oxytetracycline, 35-50 parts of polyethylene glycol 400, 0.4-0.6 parts of sodium formaldehyde sulfoxylate and 1.6-1.8 parts of magnesium oxide.

[0006] Furthermore, the oxytetracycline is prepared by adding oxytetracycline raw material to a methanol aqueous solution, adding hydrochloric acid to dissolve it, adding modified activated carbon to remove impurities, filtering, adding ammonia water to the obtained filtrate and crystallizing, filtering, and drying.

[0007] Furthermore, the preparation process of the modified activated carbon is as follows:

[0008] Treating the activated carbon with hydrogen peroxide to obtain oxidized activated carbon;

[0009] Zinc sulfate is added to deionized water to dissolve, and the oxidized activated carbon is added, ultrasonic treatment is performed, and then potassium ferrocyanide is added, stirring is continued, filtering is performed, drying is performed, and calcination is performed to obtain the modified activated carbon.

[0010] Furthermore, in the process of preparing modified activated carbon, the weight ratio of the oxidized activated carbon to zinc sulfate and potassium ferrocyanide is 10:1.3-1.5:1.5-1.8.

[0011] Furthermore, in the process of preparing modified activated carbon, the calcination temperature is 300-320°C and the time is 3-4h.

[0012] Furthermore, in the process of preparing modified activated carbon, the ultrasonic treatment time is 2 to 3 hours, the stirring speed is 100 to 120 rpm, and the stirring time is 12 to 16 hours.

[0013] Further, the weight ratio of oxytetracycline raw material to modified activated carbon is 100:4-6;

[0014] The concentration of the methanol aqueous solution is 80 to 85 vol%.

[0015] Furthermore, the process of adding aqueous ammonia to the filtrate and performing crystallization is as follows: the filtrate is first cooled to 8-10°C for the first time, aqueous ammonia is slowly added to the filtrate until the pH is 4.3-4.6, then cooled to 0-4°C for the second time, maintained at 0-4°C for the first crystallization for 2-3 hours, filtered, aqueous ammonia is added to the filtrate for the second time until the pH is 6.8-7.2, and maintained at 0-4°C for the second crystallization for 3-4 hours.

[0016] A method for preparing the above-mentioned oxytetracycline injection, the preparation method comprising: mixing polyethylene glycol 400 and water, adding sodium formaldehyde sulfoxylate and magnesium oxide in sequence, then heating, adding oxytetracycline, carrying out complexation reaction, adding monoethanolamine to adjust the pH to 8.4-8.6, fixing the volume, filtering, and obtaining the oxytetracycline injection.

[0017] Furthermore, when oxytetracycline is added, the temperature of the resulting system is 45-55°C; and the temperature of the complexation reaction is 60-65°C.

[0018] The beneficial effects of the oxytetracycline injection and the preparation method thereof of the present invention are:

[0019] The present invention prepares modified activated carbon by co-loading zinc sulfate and potassium ferrocyanide on activated carbon, and uses the modified activated carbon to treat oxytetracycline raw materials, so that metal ions, proteins and other impurities in oxytetracycline can be adsorbed by the activated carbon, and the potassium ferrocyanide loaded on the modified activated carbon forms a stable complex with heavy metal ions, and zinc sulfate combines with proteins, so that metal ions, proteins and other impurities in oxytetracycline can be effectively removed;

[0020] The modified activated carbon prepared by the present invention has a large number of pores, which is conducive to the occurrence of adsorption. In addition, since the activated carbon is modified by zinc sulfate and potassium ferrocyanide, the occurrence of complex reaction between the activated carbon and impurities can be further promoted during the adsorption process, thereby improving the impurity removal rate.

[0021] The entire oxytetracycline purification process of the present invention is simple and convenient, has a high recovery rate, and the oxytetracycline produced has high purity;

[0022] The present invention uses n-butanol containing hydrochloric acid as a solvent, which can not only effectively prepare oxytetracycline and separate oxytetracycline from impurities such as protein, but also prevent oxytetracycline from being hydrolyzed during purification and reaction, thereby effectively ensuring the recovery rate and purity of the prepared oxytetracycline;

[0023] The present invention can effectively remove impurities and ensure the recovery rate and purity of the prepared oxytetracycline by adopting a specific temperature control process;

[0024] The oxytetracycline prepared by the present invention is used to prepare oxytetracycline injection, which can effectively improve the stability and efficacy, prolong the half-life of the drug, and reduce irritation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the drug-time curve in rabbits in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0027] Example 1 A method for preparing oxytetracycline injection

[0028] The preparation method of oxytetracycline injection in this embodiment specifically comprises the following steps:

[0029] S1. Preparation of modified activated carbon

[0030] S11, taking activated carbon and immersing it in a hydrogen peroxide solution with a concentration of 15 vol%, ultrasonically treating it for 1.5 hours, filtering it, and drying it to obtain oxidized activated carbon.

[0031] S12. At room temperature, take 1.4 g of zinc sulfate and add it to 40 mL of deionized water to dissolve it, add 10 g of oxidized activated carbon, ultrasonically treat it for 2 hours, then start stirring, add 1.6 g of potassium ferrocyanide at a stirring speed of 100 rpm, continue stirring for 12 hours, filter, put it in a 105°C oven to dry, and then calcine it at 300°C in a muffle furnace for 4 hours to obtain modified activated carbon, marked as G1.

[0032] S2. Preparation of oxytetracycline

[0033] Take 100g of oxytetracycline raw material (95.83%) and add it to 500mL of 85vol% methanol aqueous solution, heat it to 45°C, add 20mL of 36% hydrochloric acid, and stir to dissolve while adding at 45°C for 30min. After the addition is completed, continue to stir at 45°C until it is almost completely dissolved, add 5g of modified activated carbon G1, maintain 45°C, stir to remove impurities for 1h, filter, and cool the filtrate to 10°C for the first time. Slowly add ammonia water dropwise until the pH value is 4.5, cool to 4°C for the second time, maintain 4°C for the first crystallization for 2 hours, filter, add ammonia water to the filtrate for the second time to pH 7.0, maintain 4°C for the second crystallization for 3 hours, filter, combine the solids obtained by the two filtrations, wash with a small amount of cold water, and dry at 75°C for 6 hours to obtain 91.62g of pure oxytetracycline (marked as M1), with a recovery rate of 91.62%, a purity of 99.34%, and an actual recovery rate of 94.98%.

[0034] Wherein, recovery rate (%) = weight of pure oxytetracycline / weight of raw oxytetracycline × 100%;

[0035] Actual recovery rate (%) = (weight of pure oxytetracycline × purity of pure oxytetracycline) / (weight of raw oxytetracycline × purity of raw oxytetracycline) × 100%.

[0036] S3. Preparation of oxytetracycline injection

[0037] Take 40g of polyethylene glycol 400 and 20g of deionized water and mix them evenly. Then add 0.5g of sodium formaldehyde sulfoxylate and 1.7g of magnesium oxide in turn. Stir and disperse at a speed of 1200rpm and raise the temperature to 50°C. Then add 20g of oxytetracycline and raise the temperature to 60°C for complexation reaction for 15min. Add monoethanolamine to adjust the pH to 8.5, add water to make up to 100mL, filter through a 0.22μm filter membrane, and aseptically fill to obtain oxytetracycline injection, which is marked as Y1.

[0038] Embodiment 2-5 A method for preparing oxytetracycline injection

[0039] Examples 2 to 5 are respectively a method for preparing oxytetracycline injection. According to the optimized method given in Example 1, the range of oxytetracycline and its injection that can be prepared is further explored to obtain the preparation methods in Examples 2 to 5, wherein the preparation methods of oxytetracycline injection in Examples 2 to 5 are basically the same as those in Example 1, except that some process parameters are different, as shown in Table 1 for details:

[0040] Table 1 List of process parameters in Examples 2 to 5

[0041]

[0042]

[0043] The contents and results of other parts of Examples 2 to 5 are basically the same as those of Example 1 and will not be repeated here.

[0044] Comparative Examples 1 to 5 Comparison of Oxytetracycline Preparation Process

[0045] Comparative Examples 1 to 5 are comparative tests of the preparation method of oxytetracycline injection in Example 1, and the steps are basically the same as those in Example 1, and the same batch of oxytetracycline (purity of 95.83%) in Example 1 is used, except that:

[0046] In step S2 of comparative example 1, 5g of unmodified activated carbon is used to replace 5g of modified activated carbon G1 to directly prepare oxytetracycline, and other process steps and raw material dosages remain unchanged, and 92.53g of oxytetracycline (labeled as DM1) is obtained, with a recovery rate of 92.53%, a purity of 96.47%, and an actual recovery rate of 93.15%. The obtained oxytetracycline DMT1 is used to prepare oxytetracycline injection according to the steps and raw material dosages of S3 in Example 1, labeled as DY1. By comparing comparative example 1 with Example 1, it can be seen that the purity of oxytetracycline is almost not improved by using unmodified activated carbon to prepare oxytetracycline, and the actual recovery rate is also reduced, which shows that the unmodified activated carbon is less effective in treating oxytetracycline, while the modified activated carbon can well adsorb impurities in oxytetracycline and does not adsorb oxytetracycline.

[0047] In step S12 of comparative example 2, only 0.5g zinc sulfate is added, and other process steps and raw material dosages remain unchanged to obtain modified activated carbon (marked as DG2), and modified activated carbon DG2 is used to prepare oxytetracycline according to the method in step S2 of embodiment 1, to obtain 91.03g oxytetracycline (marked as DM2), with a recovery rate of 91.03%, a purity of 97.34%, and an actual recovery rate of 92.46%. Oxytetracycline injection is prepared by using the obtained oxytetracycline DM2 according to the steps and raw material dosage of S3 in embodiment 1, marked as DY2. By comparing comparative example 2 with embodiment 1, it can be seen that reducing the amount of zinc sulfate will lead to a decrease in the actual recovery rate, but the degree of purity improvement is limited.

[0048] In step S12 of comparative example 3, only 0.5g of potassium ferrocyanide was added, and the other process steps and the amount of raw materials remained unchanged, and modified activated carbon (marked as DG3) was obtained, and modified activated carbon DG3 was used to prepare oxytetracycline according to the method in step S2 of embodiment 1, and 91.96g of oxytetracycline (marked as DM3) was obtained, with a recovery rate of 91.96%, a purity of 97.07%, and an actual recovery rate of 93.15%. The obtained oxytetracycline DM3 was used to prepare oxytetracycline injection according to the steps and raw material amounts of S3 in embodiment 1, marked as DY3. By comparing comparative example 3 with embodiment 1, it can be seen that reducing the amount of potassium ferrocyanide will cause the purity of oxytetracycline to decrease significantly.

[0049] In step S12 of comparative example 4, the calcination temperature is 250°C, and the other process steps and the amount of raw materials remain unchanged, and modified activated carbon (marked as DG4) is obtained, and modified activated carbon DG4 is used to prepare oxytetracycline according to the method in step S2 of example 1, and 91.71g oxytetracycline (marked as DM4) is obtained, with a recovery rate of 91.71%, a purity of 97.88%, and an actual recovery rate of 93.67%. The obtained oxytetracycline DM4 is used to prepare oxytetracycline injection according to the steps and raw material amounts of S3 in example 1, marked as DY4. By comparing comparative example 4 with example 1, it can be seen that changing the calcination temperature will cause the purity of oxytetracycline to decrease significantly.

[0050] The steps of step S2 in Comparative Example 5 are as follows:

[0051] Take 100g oxytetracycline raw material (95.83%) and add it to 500mL methanol aqueous solution with a concentration of 85vol%, heat it to 45°C, drip 20mL hydrochloric acid with a concentration of 36%, and stir and dissolve while dripping at 45°C, the dripping time is 30min, drip and finish, continue to maintain 45°C stirring until almost completely dissolved, add 5g modified activated carbon G1, maintain 45°C, stir to remove impurities for 1h, filter, and the resulting filtrate is directly cooled to 4°C, drip ammonia to pH 7.0, then maintain 4°C crystallization for 3h, filter, wash with a small amount of cold water, dry at 75°C for 6h, and obtain 88.64g oxytetracycline pure product (labeled as DM5), the recovery rate is 88.64%, the purity is 98.86%, and the actual recovery rate is 91.44%. By comparing Comparative Example 5 with Example 1, it can be seen that changing the dripping of ammonia and the crystallization mode can cause the oxytetracycline yield and purity to decrease significantly.

[0052] The obtained oxytetracycline DM5 was used to prepare oxytetracycline injection according to the steps and raw material dosages of S3 in Example 1, and was marked as DY5.

[0053] Experimental Example 1 Accelerated Experiment

[0054] The oxytetracycline injections Y1 and DY1 to DY5 prepared in Example 1 and Comparative Examples 1 to 5 were subjected to accelerated experiments, as follows:

[0055] Each oxytetracycline injection was placed at 60±2°C and RH75±5% for 28 days. Samples were taken on days 0, 7, 14 and 28, tested according to the stability test items, and compared with the data on day 0.

[0056] Observe its clarity and color, visible foreign matter or precipitation and other indicators. The specific test results are shown in the table below:

[0057] Table 2 Test results list

[0058]

[0059]

[0060] It can be seen from Table 2 that the oxytetracycline injection prepared in Example 1 can maintain good tolerance when stored under high temperature conditions, while the oxytetracycline injections prepared in Comparative Examples 1 to 5 are difficult to store under high temperature conditions. This may be related to the purity of the oxytetracycline raw material. It can be seen that the purity of oxytetracycline will affect the stability of the oxytetracycline injection prepared therefrom to a certain extent.

[0061] Experimental Example 2 Local irritation test

[0062] Oxytetracycline injections Y1 and DY1 to DY5 prepared in Example 1 and Comparative Examples 1 to 5 were used to conduct irritation experiments using rabbits, as follows:

[0063] 21 adult rabbits were randomly divided into 7 groups, 3 in each group, namely a blank control group, an experimental group, and a comparison group 1 to 5. The rabbit hair was cut off from the left quadriceps femoris of the rabbit, and after disinfection with 75% ethanol and 2% iodine tincture, 1 mL of sterile saline was injected into the left posterior quadriceps femoris of the adult rabbits in the blank control group, 0.5 mL of oxytetracycline injection Y1 prepared in Example 1 was injected into the left posterior quadriceps femoris of the adult rabbits in the experimental group, and 1 mL of oxytetracycline injection DY1 to DY5 prepared in Comparative Examples 1 to Comparative Examples 5 were correspondingly injected into the left posterior quadriceps femoris of the adult rabbits in the comparison group 1 to 5, wherein the comparison group 1 was injected with oxytetracycline injection DY1 prepared in Comparative Example 1, and the comparison group 2 was injected with oxytetracycline injection DY2 prepared in Comparative Example 2, and so on.

[0064] After injection, the mature rabbits were fed with the same diet and drinking water for 15 days. One rabbit from each group was killed at 2, 7, and 15 days after administration, and the quadriceps muscle was removed after dissection and cut longitudinally to observe the changes in the muscle tissue at the injection site.

[0065] The evaluation was conducted according to the assessment criteria for muscle tissue irritation reaction: Grade 0 irritation reaction: no obvious reaction at the administration site; Grade 1 irritation reaction: mild congestion at the administration site, with a diameter of less than 0.5 cm; Grade 2 irritation reaction: moderate congestion at the administration site, with a diameter of less than 1.0 cm; Grade 3 irritation reaction: moderate congestion, redness and swelling at the administration site, and muscle degeneration; Grade 4 irritation reaction: brown degeneration of the muscle, with necrosis less than 0.5 cm; Grade 5 irritation reaction: severe muscle degeneration, with large areas of necrosis.

[0066] Table 3 List of local irritation test results

[0067]

[0068] It can be seen from Table 3 that the purity of oxytetracycline will affect the irritation of the oxytetracycline injection prepared therefrom to a certain extent, and the oxytetracycline injection prepared by the present invention has less irritation.

[0069] Experimental Example 3 Pharmacokinetic Experiment

[0070] The oxytetracycline injection Y1 prepared in Example 1 was used to conduct a pharmacokinetic experiment using rabbits, as follows:

[0071] Four healthy rabbits, half male and half female, weighing about 2.5 kg, were randomly selected and fasted for 12 hours. A single dose of oxytetracycline injection Y1 was injected intramuscularly.

[0072] At 0 min (before administration), 15 min, 30 min, 60 min, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h after administration, 1.5 mL of blood was collected from the ear vein, added to a test tube containing sodium heparin, allowed to stand for 2 h, centrifuged at 3000 r / min for 10 min, and the upper serum was collected and stored at -20°C.

[0073] Take 400 μL of the upper layer, add 100 μL of 20% perchloric acid solution, shake for 10 seconds, centrifuge at 3000 rpm for 10 minutes, take the supernatant, filter with a 0.45 μm oily filter membrane, and take the filtrate.

[0074] Take 20 μL of the filtrate for high performance liquid chromatography analysis, where the chromatographic conditions are:

[0075] Chromatographic column: Welchrom-C18, 5μm, 4.6×250mm; mobile phase: N,N-dimethylformamide-sodium citrate-EDTA buffer, ultrasonic degassing; flow rate: 1mL / min; injection volume: 20μL; column temperature: 30℃; isocratic elution, retention to 14min; detection wavelength: 268nm.

[0076] The pharmacokinetic parameters of oxytetracycline injection Y1 prepared by the present invention in rabbits determined by the above method are shown in Figure 1 and Table 4:

[0077] Table 4 Summary of pharmacokinetic results in rabbits

[0078] Chamber parameters unit Mean <![CDATA[t 1 / 2α ]]> h 9.37 <![CDATA[t 1 / 2β ]]> h 100.46 <![CDATA[C max ]]> ng / mL 3837.21 <![CDATA[T max ]]> h 1.98 <![CDATA[AUC (0-t) ]]> ng / (mL*h) 87354.27 <![CDATA[AUC (0-∞) ]]> ng / (mL*h) 92463.31 <![CDATA[T 1 / 2Ka ]]> h 0.36

[0079] Depend on Figure 1 As can be seen from Table 4, the oxytetracycline injection prepared by the present invention has a longer half-life in rabbits, indicating that the formulation of the present invention can prolong the half-life of the drug.

[0080] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A oxytetracycline injection, characterized in that: The formula for preparing the oxytetracycline injection includes, by weight: 18 to 22 parts of oxytetracycline, 35 to 50 parts of polyethylene glycol 400, 0.4 to 0.6 parts of sodium formaldehyde sulfoxylate and 1.6 to 1.8 parts of magnesium oxide.

2. The oxytetracycline injection according to claim 1, characterized in that: The oxytetracycline is prepared by adding oxytetracycline raw material to a methanol aqueous solution, dropping hydrochloric acid to dissolve it, adding modified activated carbon to remove impurities, filtering, dropping ammonia water to the obtained filtrate and performing crystallization, filtering, and drying.

3. The oxytetracycline injection according to claim 2, characterized in that: The preparation process of the modified activated carbon is as follows: Treating the activated carbon with hydrogen peroxide to obtain oxidized activated carbon; Zinc sulfate is added to deionized water to dissolve, and the oxidized activated carbon is added, ultrasonic treatment is performed, and then potassium ferrocyanide is added, stirring is continued, filtering is performed, drying is performed, and calcination is performed to obtain the modified activated carbon.

4. The oxytetracycline injection according to claim 3, characterized in that: In the process of preparing modified activated carbon, the weight ratio of the oxidized activated carbon to zinc sulfate and potassium ferrocyanide is 10:1.3-1.5:1.5-1.

8.

5. The oxytetracycline injection according to claim 3 or 4, characterized in that: In the process of preparing modified activated carbon, the calcination temperature is 300-320°C and the time is 3-4 hours.

6. The oxytetracycline injection according to claim 3 or 4, characterized in that: In the process of preparing modified activated carbon, the ultrasonic treatment time is 2 to 3 hours, the stirring speed is 100 to 120 rpm, and the stirring time is 12 to 16 hours.

7. The oxytetracycline injection according to any one of claims 2 to 4, characterized in that: The weight ratio of oxytetracycline raw material to modified activated carbon is 100:4-6; The concentration of the methanol aqueous solution is 80 to 85 vol%.

8. The oxytetracycline injection according to any one of claims 2 to 4, characterized in that: The process of adding aqueous ammonia to the filtrate and performing crystallization is as follows: the filtrate is first cooled to 8-10°C for the first time, aqueous ammonia is slowly added to the filtrate until the pH is 4.3-4.6, then cooled to 0-4°C for the second time, maintained at 0-4°C for the first crystallization for 2-3 hours, filtered, aqueous ammonia is added to the filtrate for the second time until the pH is 6.8-7.2, and maintained at 0-4°C for the second crystallization for 3-4 hours.

9. A method for preparing the oxytetracycline injection according to any one of claims 1 to 8, characterized in that: The preparation method comprises the following steps: mixing polyethylene glycol 400 and water, adding sodium formaldehyde sulfoxylate and magnesium oxide in sequence, heating the mixture, adding oxytetracycline to carry out a complexation reaction, adding monoethanolamine to adjust the pH to 8.4 to 8.6, fixing the volume, filtering, and obtaining the oxytetracycline injection.

10. The preparation method according to claim 9, characterized in that: When oxytetracycline is added, the temperature of the system is 45-55°C; the temperature of the complexation reaction is 60-65°C.

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