Preparation method of indocyanine green for injection

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of indocyanine green for injection. The preparation method provided by the invention comprises the following steps: 1) pre-freezing; 2) primary drying; and 3) secondary drying. The preparation method disclosed by the invention does not need to use a freeze-drying excipient, and has the characteristics of simple process, convenience in operation and easiness in realizing large-scale production. The indocyanine green for injection prepared by the method has key quality parameters such as moisture, related substances, stability and the like which are obviously superior to those of commercially available original research products.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to a method for preparing indocyanine green for injection. Background Art

[0002] Indocyanine Green (ICG) for injection is a near-infrared fluorescent diagnostic reagent developed by Akorn Inc. in the United States. Due to its unique liver-selective metabolic properties and near-infrared optical activity, it has become a core diagnostic reagent for hepatobiliary function assessment, cardiovascular angiography, sentinel lymph node tracing for tumors, and minimally invasive surgical navigation. However, its clinical efficacy is highly dependent on the purity, stability, and lyophilization reconstitution properties of the formulation.

[0003] While indocyanine green (ICG) is readily soluble in water, it is unstable in aqueous solutions and contains oxidative impurities, which compromise the quality and stability of the resulting ICG for injection. Physical and chemical transformations of ICG are more likely to occur in the presence of light, high temperatures, and oxygen. Exposure to different types and intensities of light can also affect ICG degradation. Therefore, a method is needed to ensure the stability of ICG injection formulations while simultaneously controlling impurity levels within product requirements.

[0004] The current process for ICG injection includes adding stabilizers (such as lyoprotectants or chelating agents and other excipients) or optimizing the freeze-drying process. In order to ensure product stability, excipients such as ethylenediaminetetraacetic acid (EDTA), ascorbic acid, and histidine are often added to freeze-dried preparations to play multiple roles such as stabilizing drugs, inhibiting degradation, and assisting in corrosion protection; the freeze-drying process mostly uses multiple freeze-drying operations, or multi-stage cooling and tempering steps. For example, the preparation method disclosed in patent CN116997325A improves the storage stability of indocyanine green compositions by adding ethylenediaminetetraacetic acid (EDTA), histidine, and sodium chloride. The preparation method disclosed in patent CN119679972A can effectively maintain the thermosensitive structure of ICG, but this method requires steps such as primary freeze-drying, secondary freeze-drying, tertiary freeze-drying, and ultimate drying. Each freeze-drying step requires frequent annealing and sublimation operations, resulting in high energy consumption, low equipment utilization, and increased production costs. In addition, the increase in operating steps makes it difficult to ensure batch consistency of the preparation, making it difficult to meet the needs of industrial production.

[0005] Therefore, there is an urgent need to develop an efficient, stable and easy-to-scale preparation method for indocyanine green for injection, which can simplify the preparation method while taking into account the physical and chemical properties and long-term stability of the product. Summary of the Invention

[0006] The purpose of the present invention is to develop a method for preparing indocyanine green for injection that is efficient, stable and easy to scale up.

[0007] The present invention provides a method for preparing indocyanine green for injection, comprising the following steps:

[0008] 1) Prefreeze: Keep the drug solution at -35℃~-45℃ for 2~6 hours;

[0009] 2) Primary drying: Maintain vacuum at 0.15 mbar ± 0.05 mbar, raise the temperature to -8°C to 0°C, and maintain for 5 to 20 hours;

[0010] 3) Secondary drying: Maintain the vacuum degree at 0.15 mbar ± 0.05 mbar, raise the temperature to 30°C ~ 45°C, maintain for 2 to 6 hours, and then apply ultimate vacuum to the system.

[0011] In a preferred embodiment of the present invention, in step 1), the temperature is -38 to -42°C.

[0012] In one embodiment of the present invention, in step 1), the temperature is -40°C.

[0013] In a preferred embodiment of the present invention, in step 1), the concentration of the drug solution is 10 mg / mL.

[0014] In a preferred embodiment of the present invention, in step 1), the holding time is 2 to 4 hours.

[0015] In one embodiment of the present invention, in step 1), the holding time is 3 hours.

[0016] In a preferred embodiment of the present invention, in step 2), the heating rate is 5 to 15° C. / h; preferably 8 to 12° C. / h.

[0017] In one embodiment of the present invention, in step 2), the heating rate is 10° C. / h.

[0018] In a preferred embodiment of the present invention, in step 2), the temperature is raised to -8°C to -2°C.

[0019] In one embodiment of the present invention, in step 2), the temperature is raised to -5°C.

[0020] In one embodiment of the present invention, in step 2), the vacuum degree is 0.15 mbar.

[0021] In a preferred embodiment of the present invention, in step 2), the maintenance time is 5 to 15 hours.

[0022] In a preferred embodiment of the present invention, in step 2), the maintenance time is 10 to 15 hours.

[0023] In a preferred embodiment of the present invention, in step 3), the heating rate is 25 to 35° C. / h; preferably 28 to 32° C. / h.

[0024] In one embodiment of the present invention, in step 3), the heating rate is 30° C. / h.

[0025] In a preferred embodiment of the present invention, in step 3), the temperature is raised to 32-40°C.

[0026] In one embodiment of the present invention, in step 3), the temperature is raised to 35°C.

[0027] In one embodiment of the present invention, in step 3), the vacuum degree is 0.15 mbar.

[0028] In a preferred embodiment of the present invention, in step 3), the maintenance time is 3 to 5 hours.

[0029] In one embodiment of the present invention, in step 3), the maintenance time is 4 hours.

[0030] In a preferred embodiment of the present invention, in step 3), the ultimate vacuum time is 1 to 3 hours.

[0031] In one embodiment of the present invention, in step 3), the ultimate vacuum time is 2 hours.

[0032] In the present invention, the indocyanine green solution is preferably prepared under nitrogen protection conditions.

[0033] In a preferred embodiment of the present invention, the drug solution needs to be sterilized and filtered through a 0.22 μm microporous filter membrane before the freeze-drying step.

[0034] In the present invention, the material of the microporous membrane includes but is not limited to nylon, polyvinylidene fluoride (PVDF) or polyethersulfone (PES), preferably PES.

[0035] In a further preferred embodiment of the present invention, the following steps are included:

[0036] (1) Under nitrogen protection, prepare a 10 mg / mL indocyanine green solution;

[0037] (2) The drug solution was sterilized and filtered through a 0.22 μm microporous filter membrane;

[0038] (3) Freeze-drying step:

[0039] 1) Prefreeze: Keep the drug solution at -35℃~-45℃ for 2~6 hours;

[0040] 2) Primary drying: Maintain a vacuum of 0.15 mbar ± 0.05 mbar, raise the temperature to -8°C to 0°C at a rate of 5-15°C / h, and maintain for 5-20 hours;

[0041] 3) Secondary drying: Maintain the vacuum degree at 0.15 mbar ± 0.05 mbar, increase the temperature to 30°C to 45°C at a heating rate of 25 to 35°C / h, maintain for 2 to 6 hours, and then apply ultimate vacuum. The ultimate vacuum time is 1 to 3 hours.

[0042] (4) Pressure plug.

[0043] In a further preferred embodiment of the present invention, the following steps are included:

[0044] (1) Under nitrogen protection, prepare a 10 mg / mL indocyanine green solution;

[0045] (2) The drug solution was sterilized and filtered through a 0.22 μm microporous filter membrane;

[0046] (3) Freeze-drying step:

[0047] 1) Prefreezing: Cool the mixture to -38-42°C at full speed and keep the mixture at -38-42°C for 2-4 hours;

[0048] 2) Primary drying: Maintain a vacuum of 0.15 mbar ± 0.05 mbar, raise the temperature to -8°C to -2°C at a rate of 8-12°C / h, and maintain for 5-15 hours;

[0049] 3) Secondary drying: Maintain the vacuum degree at 0.15 mbar ± 0.05 mbar, increase the temperature to 32-40°C at a heating rate of 28-32°C / h, maintain for 3-5 hours, and then apply ultimate vacuum. The ultimate vacuum time is 1-3 hours.

[0050] (4) Pressure plug.

[0051] The preparation method of the present invention eliminates the need for lyophilization excipients and is characterized by simplicity, ease of operation, and ease of scalable production. The indocyanine green for injection prepared using this method significantly outperforms the commercially available original product in key quality parameters such as moisture, related substances, and stability. Of particular note, the content of specific impurities A and B in the product is reduced by 79% and 85% respectively compared to the original product.

[0052] Impurity A:

[0053] Impurity B: DETAILED DESCRIPTION

[0054] Below in conjunction with specific embodiment, the present invention is further elaborated in detail, but those skilled in the art will understand that the embodiment described below is a part of embodiment of the present invention, rather than all embodiments, is only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise fall within the scope of protection of the present invention. The experimental method used in the following examples, unless otherwise specified, the experimental method of the specific conditions not specified in the examples, usually according to conventional conditions, the materials, reagents etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0055] Detection method for related substances: Refer to the analysis method for related substances under the indocyanine green variety in Part II of the 2020 edition of the Chinese Pharmacopoeia: Chromatographic column: octadecylsilane bonded silica gel as filler (Agilent Poroshell 120EC-C18, 4.6mm×100mm, 2.7μm); mobile phase: phosphate buffer (pH5.8)-acetonitrile-methanol (50:47:3); isocratic elution; detection wavelength: 263nm; injection volume 10μL.

[0056] Example 1 Study on the stability of drug solutions at different concentrations

[0057] Indocyanine green was dissolved in water to obtain 5 mg / mL, 6.25 mg / mL and 10 mg / mL concentration solutions, respectively, and the growth trend of related substances at different concentrations was investigated.

[0058] Table 1 Results of stability study of different concentrations of drug solution

[0059]

[0060] Example 2

[0061] Dissolve 25 mg of indocyanine green in 5 mL of water to obtain a 5 mg / mL concentration of the drug solution. After filtering through a 0.22 μm microporous filter membrane, fill into 5 mL to 10 mL borosilicate glass injection vials. After half-stoppering, place in a LYO-0.5 freeze dryer and perform freeze drying according to the following freeze drying process parameters:

[0062] Pre-freezing: Turn on the front box plate refrigeration and reduce the inlet temperature of the heat transfer oil to -40°C at full speed (60°C / h). Maintain the inlet temperature of the heat transfer oil for 3 hours after reaching the temperature to completely freeze the product.

[0063] Vacuuming: After pre-freezing is completed, turn on the vacuum pump and evacuate the vacuum to reduce the vacuum degree of the front box to 0.100 mbar.

[0064] Primary drying: Control the vacuum degree of the front box to 0.100 mbar. Raise the temperature from -40°C to -5°C at a heating rate of 10°C / h and maintain for 14 hours.

[0065] Secondary drying: Raise the inlet temperature of the thermal oil from -5°C to 40°C in 90 minutes, maintain it for 4 hours, then stop vacuum control and apply ultimate vacuum to the system for 2 hours.

[0066] Pressing: Lift the freeze dryer plate layer to press the plug, and then press again, take the box out, and press the cap.

[0067] Example 3

[0068] Dissolve 25 mg of indocyanine green in 2.5 mL of water to obtain a 10 mg / mL concentration solution. The remaining steps are the same as those in Example 2.

[0069] Example 4

[0070] Pre-freezing: Turn on the front box plate refrigeration and reduce the heat transfer oil inlet temperature to -40℃ at full speed. Maintain the temperature for 3 hours after the heat transfer oil inlet temperature reaches the temperature; then increase the temperature to -10℃ at a rate of 1℃ / min and maintain it for 2 hours; continue to freeze it to -40℃ at a cooling rate of 1℃ / min and maintain it for 2 hours to completely freeze the product.

[0071] The remaining steps are the same as in Example 2.

[0072] Example 5

[0073] Dissolve 25 mg of indocyanine green in 2.5 mL of water to obtain a 10 mg / mL concentration solution.

[0074] Primary drying: Control the vacuum degree of the front box to 0.100 mbar. Raise the temperature from -40°C to 5°C at a heating rate of 10°C / h and maintain for 4.5 hours.

[0075] The remaining steps are the same as in Example 2.

[0076] Results: The properties of the prepared samples were poor. The samples were obviously shrunk, the surface was uneven, there were high and low levels, some cakes were broken, some cakes were crushed, and there was flying powder.

[0077] Table 2 Summary of results of Examples 2 to 4

[0078]

[0079] Example 6

[0080] Dissolve 500g of indocyanine green in 20kg of water for injection. After nitrogen displacement for 3 minutes, add more water for injection to 50kg and stir to mix the solution. The solution is filtered through 0.22μm and transported to the filling room. The filling volume is adjusted according to the content of the intermediate product. Fill, half-stopper, and enter the freeze drying chamber.

[0081] Freeze-drying was performed according to the following freeze-drying process parameters:

[0082] Pre-freezing: Turn on the front box plate refrigeration and reduce the inlet temperature of the heat transfer oil to -40℃ at full speed. Maintain the inlet temperature of the heat transfer oil for 3 hours after reaching the temperature to completely freeze the product.

[0083] Cooling and vacuuming the condenser: After pre-freezing is completed, confirm that the condenser temperature has dropped below -45°C, turn on the vacuum pump, and vacuumize the front box to reduce the vacuum degree to 0.15mbar.

[0084] Primary drying: Add sterile nitrogen and adjust the vacuum control range of the front box to 0.15 mbar ± 0.05 mbar. Raise the temperature from -40°C to -5°C at a rate of 10°C / h and maintain for 12 hours.

[0085] Secondary drying: Raise the inlet temperature of the thermal oil from -5°C to 35°C in 80 minutes, maintain for 4 hours, then stop vacuum control and apply ultimate vacuum to the system for 2 hours.

[0086] Pressing: Lift the freeze dryer plate layer to press the plug, and then press again, take the box out, and press the cap.

[0087] Table 3 Comparison of related substances in Example 6 and commercially available preparations (from Akorn Inc, USA)

[0088]

[0089] The two batches of samples from Example 6 were subjected to stability tests with commercially available preparations. The long-term stability test conditions were 25°C ± 2°C, 60% RH ± 5% RH; the intermediate test conditions were 30°C ± 2°C, 65% RH ± 5% RH.

[0090] Table 4 Long-term stability test results

[0091]

[0092]

[0093] Table 5 Intermediate test stability test results

[0094]

Claims

1. A method for preparing indocyanine green for injection, comprising the following steps: 1) Prefreeze: Keep the drug solution at -35℃~-45℃ for 2~6 hours; 2) Primary drying: Maintain vacuum at 0.15 mbar ± 0.05 mbar, raise the temperature to -8°C to 0°C, and maintain for 5 to 20 hours; 3) Secondary drying: Maintain the vacuum degree at 0.15 mbar ± 0.05 mbar, raise the temperature to 30°C ~ 45°C, maintain for 2 to 6 hours, and then apply ultimate vacuum to the system.

2. The preparation method according to claim 1, characterized in that In step 1), the temperature is -38 to -42°C; preferably, the temperature is -40°C; Or / and, in step 1), the holding time is 2 to 4 hours; preferably, the holding time is 3 hours.

3. The preparation method according to claim 1, characterized in that In step 1), the concentration of the drug solution is 10 mg / mL.

4. The preparation method according to claim 1, characterized in that In step 2), the heating rate is 5 to 15°C / h; preferably, the heating rate is 8 to 12°C / h; more preferably, the heating rate is 10°C / h; Or / and, in step 2), the temperature is raised to -8°C to -2°C; preferably, the temperature is raised to -5°C.

5. The preparation method according to claim 1, characterized in that In step 2), the vacuum degree is 0.15 mbar; Or / and, in step 2), the maintenance time is 5 to 15 hours; preferably, the maintenance time is 10 to 15 hours.

6. The preparation method according to claim 1, characterized in that In step 3), the heating rate is 25 to 35°C / h; preferably, the heating rate is 28 to 32°C / h; more preferably, the heating rate is 30°C / h; Or / and, in step 3), heating to 32-40°C; preferably, heating to 35°C; Or / and, in step 3), the vacuum degree is 0.15 mbar; Or / and, in step 3), the maintenance time is 3 to 5 hours; preferably, the maintenance time is 4 hours.

7. The preparation method according to claim 1, characterized in that In step 3), the ultimate vacuum time is 1 to 3 hours; preferably, the ultimate vacuum time is 2 hours.

8. The preparation method according to claim 1, characterized in that Under nitrogen protection, prepare indocyanine green solution.

9. The preparation method according to claim 1, characterized in that Before the freeze-drying step, the drug solution must be sterilized and filtered through a 0.22μm microporous filter membrane.

10. The preparation method according to claim 1, characterized in that The steps include: (1) Under nitrogen protection, prepare a 10 mg / mL indocyanine green solution; (2) The drug solution was sterilized and filtered through a 0.22 μm microporous filter membrane; (3) Freeze-drying step: 1) Prefreeze: Keep the drug solution at -35℃~-45℃ for 2~6 hours; 2) Primary drying: Maintain a vacuum of 0.15 mbar ± 0.05 mbar, raise the temperature to -8°C to 0°C at a rate of 5-15°C / h, and maintain for 5-20 hours; 3) Secondary drying: Maintain a vacuum degree of 0.15 mbar ± 0.05 mbar, raise the temperature to 30°C to 45°C at a heating rate of 25-35°C / h, maintain for 2-6 hours, then apply ultimate vacuum for 1-3 hours; (4) tamponade; Preferably, the method comprises the following steps: (1) Under nitrogen protection, prepare a 10 mg / mL indocyanine green solution; (2) The drug solution was sterilized and filtered through a 0.22 μm microporous filter membrane; (3) Freeze-drying step: 1) Prefreezing: Cool the mixture to -38-42°C at full speed and keep the mixture at -38-42°C for 2-4 hours; 2) Primary drying: Maintain a vacuum of 0.15 mbar ± 0.05 mbar, raise the temperature to -8°C to -2°C at a rate of 8-12°C / h, and maintain for 5-15 hours; 3) Secondary drying: Maintain a vacuum degree of 0.15 mbar ± 0.05 mbar, raise the temperature to 32-40°C at a heating rate of 28-32°C / h, maintain for 3-5 hours, then apply ultimate vacuum for 1-3 hours; (4) Pressure plug.

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