Indocyanine green preparation, process for its preparation and use

Abstract

The application belongs to the technical field of pharmaceutical preparations, and particularly relates to an indocyanine green preparation and a preparation method and application thereof. The preparation is a liquid preparation or a freeze-dried preparation, raw materials for preparing the preparation include indocyanine green, phospholipid, cholesterol, a freeze-drying protective agent, water and a pH regulator, the pH regulator is selected from one or more of sodium citrate, potassium citrate, sodium tartrate, sodium acetate, sodium lactate and sodium succinate, and the pH of the preparation is adjusted to 6.0-8.5. By means of a specific organic acid salt buffer system and a specific preparation process (the freeze-drying protective agent is added before lipid film deposition), the application can specifically inhibit the growth of impurities with RRT 0.7 and RRT 0.3, respectively, and significantly improve the chemical stability of the preparation. Meanwhile, the preparation can realize accurate positioning of sentinel lymph nodes and second and third lymph nodes, and can be used for preparing a lymph tracer. The application effectively solves the technical problems of rapid impurity growth, poor stability and inaccurate multi-stage lymph positioning of the existing ICG preparation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to indocyanine green formulations, their preparation methods, and applications. Background Technology

[0002] Indocyanine green (ICG) is a near-infrared fluorescent dye with strong absorption and fluorescence emission around 800 nm. It has been widely used in liver function assessment, fundus angiography, lymphoillumination, surgical navigation, and photothermal therapy. However, while injectable ICG is indicated for lymphatic tracing, it cannot accurately locate sentinel lymph nodes and fine lymph nodes. Free ICG diffuses rapidly, leading to extensive staining of tissues near tumors and increased diffusion, making it impossible to accurately determine the location and fluorescence intensity of primary lymph nodes, let alone precisely locate secondary and tertiary lymph nodes.

[0003] Formulating ICG into nanoparticles to prolong in vivo circulation and enhance passive targeting capabilities has become a research hotspot in recent years. However, existing ICG liposomes generally face the following bottlenecks in industrialization and clinical translation:

[0004] First, the ICG molecule contains multiple easily degradable groups, and impurities grow rapidly, especially in aqueous solutions. The preparation process of nano-formulations is complex and requires a relatively long time, making it difficult to solve the technical challenge of significant impurity growth during the preparation of ICG nano-formulations. In high-performance liquid chromatography (HPLC) chromatograms for related substances detection, a significantly increased degradation impurity peak (hereinafter referred to as "RRT 0.7 impurity") often appears at approximately 0.7 relative retention time (RRT). This impurity is directly related to the degradation of ICG in aqueous solutions.

[0005] Second, existing ICG nanoformulations show significant impurity growth during stable storage. HPLC chromatograms often show a significantly increasing degradation impurity peak at approximately 0.3 RRT (hereinafter referred to as "RRT 0.3 impurity"), which is directly related to the degradation of ICG after lyophilization.

[0006] To address the aforementioned issues, the conventional approach in the art is to use universal pH adjustment systems such as sodium hydroxide (NaOH), hydrochloric acid (HCl), or phosphate buffer to adjust the pH of the formulation to the target range. The general understanding is that as long as the pH is controlled within a suitable range, ICG degradation can be inhibited. However, through extensive experimentation, the inventors unexpectedly discovered that simply controlling the pH value can stabilize free ICG, but not ICG nanoformulations—even when the NaOH, phosphate, and hydrochloric acid system is adjusted to the exact same pH as the pH adjustment buffer system of this invention, ICG degradation impurities continue to increase rapidly, and stability cannot be guaranteed. Therefore, the prior art has not recognized that the stability of ICG nanoformulations is not solely determined by pH value, and also lacks a systematic control strategy for the two key impurities, RRT 0.7 and RRT 0.3. Furthermore, another conventional strategy is to encapsulate ICG in a lipid carrier to isolate it from the aqueous phase.

[0007] Specifically, in the prior art, CN105854030A discloses nanoparticles that embed ICG in a lipid membrane. While this can improve the fluorescence intensity of ICG to some extent through the lipid environment, this patent completely fails to recognize the chemical stability problem caused by the significant increase of chemical impurities (especially RRT 0.7 and RRT 0.3 impurities) in ICG nanoparticles during liquid-phase preparation and freeze-drying solid-state storage. CN111135296A and CN115154602A employ albumin conjugates and prodrug cross-linked micelles as carrier strategies, respectively, but these also fail to address the problem of impurity surges caused by the lack of a specific buffering system in ICG liposomes. Meanwhile, at the clinical functional level, existing conventional injectable ICG and reported ICG liposomes (such as CN105854030A) mainly focus on improving the fluorescence intensity or resolution of tumor regions, but no systematic solution has been found for the precise, hierarchical localization of sentinel lymph nodes and their downstream multi-level lymph nodes (secondary and tertiary). Free ICG diffuses rapidly in the body and causes severe interference from the tissue background, making it unable to meet the precise requirements of multi-level lymphatic navigation in surgical procedures. Summary of the Invention

[0008] This invention aims to overcome the technical challenge of accurately locating sentinel lymph nodes and micro-lymph nodes with injectable ICG. Free ICG diffuses rapidly, leading to extensive staining of tissues near the tumor and increased diffusion, making it impossible to accurately determine the location and fluorescence intensity of primary lymph nodes. The ICG formulation of this invention significantly reduces local diffusion and has lymphatic targeting properties, thus enabling precise localization of sentinel lymph nodes and secondary and tertiary lymph nodes in tumor models.

[0009] Meanwhile, this invention solves the problems of poor stability of ICG and the difficulty in balancing stability during preparation and storage. Unexpectedly, this invention reveals that the pH-adjusting buffer system and specific preparation process employed can solve the technical problem of significant impurity growth during the preparation and storage of ICG nanoparticles.

[0010] To achieve the above-mentioned objectives, this invention provides indocyanine green formulations, their preparation methods, and applications. The specific technical solutions are as follows:

[0011] In a first aspect, the present invention provides an indocyanine green formulation, which is a liquid formulation or a lyophilized formulation. The raw materials for preparing the formulation comprise the following components by weight: indocyanine green, 0.2-20 parts; phospholipids, 25-225 parts; cholesterol, 1-25 parts; a lyophilization protectant, 10-500 parts; and water, 1000-3000 parts. It also includes a pH adjuster for adjusting the pH of the formulation to 6.0-8.5. The pH adjuster is selected from one or more of sodium citrate, potassium citrate, sodium tartrate, sodium acetate, sodium lactate, and sodium succinate.

[0012] Preferably, the raw materials for preparing the formulation comprise the following components by weight: indocyanine green, 1-10 parts; phospholipids, 50-150 parts; cholesterol, 2-10 parts; lyophilization protectant, 50-200 parts; water, 1800-2200 parts; and the pH adjuster is used to adjust the pH of the formulation to 6.5-8.0.

[0013] More preferably, the phospholipid is selected from one or more of soybean phospholipids, egg yolk lecithin, hydrogenated soybean phospholipids, soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearate phosphatidylcholine, disqualyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, and phosphatidylethanolamine.

[0014] More preferably, the freeze-drying protectant is selected from one or more of sucrose, trehalose, mannitol, lactose, and glucose.

[0015] More preferably, the pH adjuster is selected from one or more of sodium citrate, potassium citrate, sodium tartrate, and sodium lactate.

[0016] More preferably, in the formulation, the content of RRT 0.3 impurity is ≤1.5%, and the content of RRT 0.7 impurity is ≤1.5%; wherein, the structural formula of the RRT 0.3 impurity is as shown in formula (I), and the structural formula of the RRT 0.7 impurity is as shown in formula (II):

[0017] Of the two impurities mentioned above, RRT0.3 is a major degradation impurity under light and high temperature conditions. An increase in this impurity will cause ICG to lose its fluorescence imaging ability, directly reducing the effectiveness of clinical diagnosis and increasing the risk of ICG sensitization. RRT0.7 is an oxidative degradation impurity of ICG; an increase in this impurity will reduce fluorescence imaging ability and increase toxicity.

[0018] The following literature has disclosed information on these two impurities: 1) CN114436938A An impurity in an indocyanine green drug, its preparation method and application; 2) CN118084770B A method for preparing impurities degraded by indocyanine green; 3) Mytych W, Bartusik-Aebisher D, Aebisher D. The medical basis for the photoluminescence of indocyanine green[J]. Molecules, 2025, 30(4): 888.DOI:10.3390 / molecules30040888. 4) Li DH, Smith B D. Deuterated indocyanine green (ICG) with extended storage shelf-life: chemical and clinical implications[J]. Chemistry– A European Journal, 2021. DOI:10.1002 / chem.202102816. 5) Kirchherr AK, Briel A, Mäder K. Stabilization of indocyanine greenby encapsulation within micellar systems[J]. Molecular Pharmaceutics, 2009, 6(2): 480-491. DOI:10.1021 / mp8001649.

[0019] In a second aspect, the present invention provides a method for preparing the indocyanine green formulation described in the first aspect, the method comprising the following steps: (1) Add the prescribed amounts of phospholipids, cholesterol, and indocyanine green to the solvent and mix; (2) Mix the mixture obtained in step (1) with the formulated amount of freeze-drying protectant, and heat to remove the solvent until a lipid film deposit is formed; (3) Mix the pH adjuster with water and then add it to the lipid film deposits obtained in step (2) for hydration reaction;

[0020] When the preparation is a lyophilized preparation, the preparation method further includes: step (4), lyophilizing the liquid obtained in step (3).

[0021] Preferably, the solvent is selected from one or more of ethanol and chloroform.

[0022] Preferably, in step (2), the solvent is removed under vacuum and at a heating temperature of 40-50°C.

[0023] Preferably, in step (3), the hydrated liquid is further homogenized and filtered; preferably, the homogenization is performed 3-7 times; the filtration is performed using 0.45 µm and 0.22 µm filter membranes.

[0024] Preferably, in step (4), the freeze-drying process includes: pre-freezing at -40±5℃ for 160-170 min, drying at -5±5℃ for 1400-2100 min, and drying at 30±5℃ for 400-500 min.

[0025] Thirdly, the present invention provides the use of the indocyanine green formulation described in the first aspect in the preparation of a lymphatic tracer.

[0026] Furthermore, the lymphatic tracing is used for surgical navigation of tumors selected from the following: gastrointestinal tumors, hepatobiliary and pancreatic tumors, urinary tumors, gynecological tumors, thoracic tumors, and head, neck, ear, nose, and throat endoscopic or laparoscopic tumors.

[0027] Fourthly, the present invention provides a lymphatic tracer comprising the indocyanine green formulation described in the first aspect.

[0028] For example, the lymphatic tracer is generally a lyophilized powder, and the content of indocyanine green in the lyophilized powder is 0.2-20 mg / vial, with each vial containing 1g.

[0029] This invention is based on the following core findings:

[0030] First, regarding indications, the ICG formulation of this invention overcomes the technical challenge of accurately locating sentinel lymph nodes and fine lymph nodes with injectable ICG. The ICG formulation reduces local diffusion, has lymphatic targeting effects, and can achieve precise localization of sentinel lymph nodes and secondary and tertiary lymph nodes in tumor models.

[0031] Secondly, regarding the formulation, extensive experiments unexpectedly revealed that the stability of ICG formulations specifically depends on the pH-adjusting buffer system used in this invention, rather than general pH value control. Conventional pH-adjusting systems such as sodium hydroxide, phosphates, and carbonates, even when adjusted to the same pH as this buffer system, cannot achieve the same stabilizing effect. The pH-adjusting buffer system used in this invention has irreplaceable technical advantages. This buffer system is used to maintain the stability of the formulation in its liquid state during preparation and specifically inhibits the growth of RRT 0.7 degradation impurities.

[0032] Third, regarding the preparation process, the preparation process of this invention—adding the freeze-drying protectant before lipid film deposition—can specifically inhibit the significant increase of RRT 0.3 impurities in the freeze-dried product during stable storage. Attached Figure Description

[0033] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0034] Figure 1 Representative images of in vivo sentinel lymph node fluorescence imaging in mice in Example 5 of this invention; wherein, A is an image of the ICG conventional formulation group, B is an image of the ICG conventional formulation group, and C is an image of the ICG formulation group of this invention.

[0035] Figure 2 Representative fluorescence imaging images of secondary and tertiary lymph nodes in the tumor lymph node metastasis model of Example 5 of this invention; wherein, A is an image of the ICG conventional formulation group, B is an image of the ICG conventional formulation group, and C is an image of the ICG formulation group of this invention. Detailed Implementation

[0036] Those skilled in the art should understand that although the specific embodiments of this application only demonstrate and test certain types of phospholipids, lyophilization protectants and pH adjusters, the other optional components listed in claim 1 are based on the same or similar physicochemical properties and pharmaceutical mechanisms of action as the components in the embodiments. They are well-known in the art and can be directly substituted for each other. They can also achieve the technical effects of inhibiting impurity generation, improving chemical stability and achieving precise tracking of multi-level lymph nodes as expected by the present invention.

[0037] Specifically, regarding freeze-drying protectants, the sucrose, trehalose, mannitol, lactose, and glucose listed in the claims all contain abundant hydroxyl groups, which can form an amorphous glassy body during freeze-drying, effectively encapsulating and protecting the liposome bilayer membrane structure, preventing mechanical damage to vesicles by ice crystals, and also playing the same skeletal support role, thereby preventing the significant growth of RRT0.3 impurities during long-term stable storage.

[0038] Regarding the phospholipid components, the soybean phospholipids, egg yolk lecithin, hydrogenated soybean phospholipids, distearate phosphatidylcholine, dipalmitoyl phosphatidylcholine, etc. listed in the claims are all amphiphilic lipid membrane materials. They can all spontaneously assemble into closed phospholipid bilayer vesicles under the solvent-film dispersion and hydration process conditions of the present invention, thereby achieving effective encapsulation of indocyanine green and endowing the formulation with the characteristics of reducing tissue diffusion and precisely targeting lymph.

[0039] Regarding pH adjusters, the sodium citrate, potassium citrate, sodium tartrate, sodium acetate, sodium lactate, and sodium succinate listed in the claims all belong to organic acid salt buffer systems. Unlike inorganic acids and bases or phosphates, these organic acid salts can not only precisely control the pH of the formulation within the suitable range of 6.0 to 8.5, but their specific organic acid anions (such as carboxylic acid anions or hydroxy acid anions) can also generate specific interfacial compatibility and weak interactions with indocyanine green molecules or phospholipid membranes. This allows them to maintain the microenvironment during liquid preparation in a more gentle and stable manner, thus differentiating themselves from conventional inorganic salts and equally effectively inhibiting the abnormal growth of RRT0.7 impurities in indocyanine green nanoparticles during liquid-phase preparation and intermediate storage.

[0040] Furthermore, it should be noted in the description of this invention that, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0041] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0042] This embodiment presents four case studies and three comparative examples of experiments investigating the effect of formulation pH on related substances, as detailed below:

[0043] Example 1-1.

[0044] The formulation composition and dosage are shown in Table 1: Table 1. Components and content of the formulation Indocyanine Green 0.5 Soybean phospholipids 10.0 cholesterol 0.5 Sodium citrate Adjust the pH to 6.8 before filling. Mannitol 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200ml

[0045] Weigh out the prescribed amounts of soybean lecithin, cholesterol, and indocyanine green, mix them with the prescribed amount of anhydrous ethanol, and dissolve the raw materials by stirring. Then add the prescribed amount of mannitol, and while stirring, vacuum dry at 45°C to form a lipid film deposit, the lipid film state of which no longer changes. Add the obtained lipid film deposit to water for injection containing sodium citrate, and stir at 30°C until hydration is complete. Homogenize 5 times in a homogenizer. Filter the resulting homogenized solution through 0.45µm and 0.22µm filter membranes, fill, freeze-dry (pre-freeze at -40±1°C for 165 min, dry at -5±1°C for 2040 min, and dry at 30±1°C for 480 min, the same applies to the examples and comparative examples below), and cap.

[0046] Examples 1-2.

[0047] The formulation composition and dosage are shown in Table 2: Table 2. Components and content of the formulation Indocyanine Green 0.5 Egg yolk lecithin 15.0 cholesterol 0.75 Sodium tartrate Adjust the pH to 7.5 before filling. glucose 12.5 Chloroform (used in the process and eventually removed) Appropriate amount Water for Injection 200ml

[0048] Weigh out the prescribed amounts of egg yolk lecithin, cholesterol, and indocyanine green, mix them with the prescribed amount of chloroform, and dissolve the raw materials by stirring. Then add the prescribed amount of glucose, and while stirring, vacuum dry at 45°C to form a lipid film deposit, the lipid film state of which no longer changes. Add the obtained lipid film deposit to water for injection containing sodium tartrate, and stir at 40°C until hydration is complete. Homogenize four times in a homogenizer. Filter the resulting homogenized solution through 0.45µm and 0.22µm filter membranes, fill, freeze-dry, and cap.

[0049] Examples 1-3.

[0050] The formulation composition and dosage are shown in Table 3: Table 3. Components and content of the formulation Indocyanine Green 0.2 Soybean phospholipids 12.5 cholesterol 1.0 Sodium lactate Adjust the pH to 7.0 before filling. sucrose 10.0 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200ml

[0051] Weigh out the prescribed amounts of soybean lecithin, cholesterol, and indocyanine green, mix them with the prescribed amount of anhydrous ethanol, and dissolve the raw materials by stirring. Then add the prescribed amount of sucrose, and while stirring, vacuum dry at 50°C to form a lipid film deposit, the lipid film state of which no longer changes. Add the obtained lipid film deposit to water for injection containing sodium lactate, and stir at 25°C until hydration is complete. Homogenize 5 times in a homogenizer. Filter the resulting homogenized solution through 0.45µm and 0.22µm filter membranes, fill, freeze-dry, and cap.

[0052] Examples 1-4.

[0053] The formulation composition and dosage are shown in Table 4: Table 4. Components and content of the formulation Indocyanine Green 1.0 Hydrogenated soybean lecithin 7.5 cholesterol 0.5 Sodium citrate Adjust the pH to 7.8 before filling. sucrose 10.0 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200ml

[0054] Weigh out the prescribed amounts of hydrogenated soybean lecithin, cholesterol, and indocyanine green, mix with the prescribed amount of anhydrous ethanol, dissolve the raw materials by stirring, then add the prescribed amount of sucrose, and dry under vacuum at 50°C while stirring to form a lipid film deposit, the lipid film state no longer changing; add water for injection containing sodium citrate to the obtained lipid film deposit, stir at 25°C until hydration is complete; homogenize 5 times in a homogenizer; filter the obtained homogenized solution through 0.45µm and 0.22µm filter membranes, fill, freeze dry, and cap.

[0055] Comparative Example 1-1.

[0056] The formulation composition and dosage are shown in Table 5: Table 5. Components and content of the formulation Indocyanine Green 0.5 Soybean phospholipids 10.0 cholesterol 0.5 Sodium citrate Adjust the pH to 5.8 before filling. Mannitol 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200ml

[0057] Weigh out the prescribed amounts of soybean lecithin, cholesterol, and indocyanine green, mix them with the prescribed amount of anhydrous ethanol, and dissolve the raw materials by stirring. Then add the prescribed amount of mannitol, and while stirring, vacuum dry at 45°C to form a lipid film deposit, the lipid film state of which no longer changes. Add the obtained lipid film deposit to water for injection containing sodium citrate, and stir at 30°C until hydration is complete. Homogenize 5 times in a homogenizer. Filter the obtained homogenized solution through 0.45µm and 0.22µm filter membranes, fill, freeze dry, and cap.

[0058] Comparative Examples 1-2.

[0059] The formulation composition and dosage are shown in Table 6: Table 6. Components and Contents of the Formulation Indocyanine Green 0.5 Hydrogenated soybean lecithin 10.0 cholesterol 0.5 Sodium citrate Adjust the pH to 5.4 before filling. sucrose 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200ml

[0060] Weigh out the prescribed amounts of hydrogenated soybean lecithin, cholesterol, and indocyanine green, mix them with the prescribed amount of anhydrous ethanol, and dissolve the raw materials by stirring. Then add the prescribed amount of mannitol, and while stirring, vacuum dry at 45°C to form a lipid film deposit, the lipid film state of which no longer changes. Add the obtained lipid film deposit to water for injection containing sodium citrate, and stir at 30°C until hydration is complete. Homogenize 5 times in a homogenizer. Filter the obtained homogenized solution through 0.45µm and 0.22µm filter membranes, fill, freeze dry, and cap.

[0061] Comparative Examples 1-3.

[0062] The formulation composition and dosage are shown in Table 7: Table 7 Components and Contents of the Formulation Indocyanine Green 0.5 Soybean phospholipids 10.0 cholesterol 0.5 Sodium citrate Adjust the pH to 5.0 before filling. Mannitol 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200ml

[0063] Weigh out the prescribed amounts of soybean lecithin, cholesterol, and indocyanine green, mix them with the prescribed amount of anhydrous ethanol, and dissolve the raw materials by stirring. Then add the prescribed amount of mannitol, and while stirring, vacuum dry at 45°C to form a lipid film deposit, the lipid film state of which no longer changes. Add the obtained lipid film deposit to water for injection containing sodium citrate, and stir at 30°C until hydration is complete. Homogenize 5 times in a homogenizer. Filter the obtained homogenized solution through 0.45µm and 0.22µm filter membranes, fill, freeze dry, and cap.

[0064] For formulations using phospholipids as membrane materials, the mechanical flexibility of the phospholipid membrane directly determines the permeation performance in the filtration process. The process generally requires controlling the system temperature at around 40°C to bring the lipid membrane temperature into the phospholipid phase transition temperature range, reducing the rigidity of the phospholipid bilayer and improving the membrane's mobile phase and deformation properties. Simultaneously, scale-up production in the workshop needs to consider factors such as homogenization time, filtration time, and intermediate inspection time. Therefore, it is necessary to examine the stability of the samples in the liquid state before lyophilization to ensure the requirements of formulation preparation. Therefore, relevant substances were tested in the samples of Examples 1-1~1-4 and Comparative Examples 1-1~1-3 before lyophilization (0 h) and after being placed at 40°C for 24 h.

[0065] The methods for detecting the relevant substances are as follows (the same applies to all examples and comparative examples):

[0066] High-performance liquid chromatography (HPLC) was used for determination using a Shimadzu LC20A column. Octadecylsilane-bonded silica gel was used as the stationary phase (Ultimate® XB-C18, 4.6 mm × 250 mm, 5 μm or equivalent column). Mobile phase A consisted of phosphate buffer (4.17 g potassium dihydrogen phosphate and 0.435 g dipotassium hydrogen phosphate dissolved in water and diluted to 1000 ml) and mobile phase B consisted of phosphate buffer (4.17 g potassium dihydrogen phosphate and 0.435 g dipotassium hydrogen phosphate dissolved in water and diluted to 1000 ml) - organic phase [acetonitrile-methanol (35:15)] (30:70), with gradient elution according to Table 8. The detection wavelength was 263 nm; the sample chamber temperature was 4℃ or the test solution was freshly prepared; the injection volume was 20 μl; and the flow rate was 1.0 ml per minute. The detection results are shown in Table 9. Table 8 Gradient elution process 0 35 65 20 0 100 30 0 100 31 35 65 45 35 65 Table 9. Related substances of indocyanine green formulations prepared from organic acid salts at different pH values.

[0067] The results above show that as the pH of the injection solution increases, the stability of impurities in RRT0.7 of the indocyanine green preparation increases after being placed at 40℃ for 24 hours before lyophilization. The stability of the indocyanine green preparation meets the requirements within the pH range of 6.5-8.0.

[0068] Conclusion: Based on product characteristics, pH range, pharmacodynamic experimental results, and clinical applications, the final pH control range is set at 6.5-8.0; this meets the requirements of the 2025 edition of the Chinese Pharmacopoeia and satisfies the clinical application needs of this product.

[0069] Example 2

[0070] This embodiment presents three comparative experiments investigating the effect of the type of pH adjuster in the formulation on related substances, as detailed below:

[0071] Comparative Example 2-1.

[0072] The formulation composition and dosage are shown in Table 10: Table 10 Components and Contents of the Formulation Indocyanine Green 0.5 Soybean phospholipids 10.0 cholesterol 0.5 Sodium hydroxide Adjust the pH to 6.8. Mannitol 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200 ml The preparation method is the same as in Example 1-1.

[0073] Comparative Example 2-2.

[0074] The formulation composition and dosage are shown in Table 11: Table 11 Components and Contents of the Formulation Indocyanine Green 0.5 Soybean phospholipids 10.0 cholesterol 0.5 disodium hydrogen phosphate Adjust the pH to 6.8. Mannitol 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200 ml The preparation method is the same as in Example 1-1.

[0075] Comparative Examples 2-3.

[0076] The formulation composition and dosage are shown in Table 12: Table 12 Components and Contents of the Formulation Indocyanine Green 0.5 Soybean phospholipids 10.0 cholesterol 0.5 Sodium carbonate Adjust the pH to 6.8. Mannitol 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200 ml The preparation method is the same as in Example 1-1.

[0077] The relevant substances of the samples before freeze-drying (0 h) and after being placed at 40°C for 24 h in Examples 1-1 to 1-4 and Comparative Examples 2-1 to 2-3 were tested respectively, and the results are shown in Table 13: Table 13. Related substances in ICG formulations prepared with different pH adjusters

[0078] The results above show that even when the pH value is controlled within the range of 6.5-8.0, the growth rate of RRT 0.7 impurities in samples prepared with organic salts such as sodium citrate and sodium lactate as pH adjusters after being placed at 40℃ for 24 h is significantly different from the growth trend of related substances with inorganic salts such as sodium hydroxide as pH adjusters.

[0079] Conclusion: Organic salts, such as sodium citrate and sodium lactate, can effectively control the stability of samples before freeze-drying during the preparation process, ensuring that the necessary liquid storage requirements are met even with extended time for each process in the workshop and intermediate inspection. In contrast, inorganic salts, such as sodium hydroxide, disodium hydrogen phosphate, and sodium carbonate, cannot effectively control the stability of ICG formulations in the liquid state during the preparation process.

[0080] Example 3

[0081] This embodiment investigates the pH regulation performance of a conventional lyophilized ICG formulation using sodium hydroxide as a pH adjuster and an ICG formulation obtained by the method of this invention, as detailed below:

[0082] Comparative Example 3-1.

[0083] The formulation composition and dosage are shown in Table 14: Table 14 Components and Contents of the Formulation Indocyanine Green 0.5 Sodium hydroxide Adjust the pH to 6.8. Water for Injection 200 ml

[0084] Weigh the prescribed amount of indocyanine green, add water for injection and stir to dissolve, adjust the pH with sodium hydroxide solution; filter through 0.45µm and 0.22µm filter membranes, fill, freeze dry, and cap to obtain the final product.

[0085] The relevant substances prepared in Comparative Examples 2-1 and 3-1 were tested respectively, and the results are shown in Table 15: Table 15 Related substances of conventional lyophilized ICG formulations and ICG formulations prepared according to the method of this invention (with different pH adjusters)

[0086] The results above show that, even when sodium hydroxide is used as a pH adjuster to the same pH value, the growth trends of related substances (RRT 0.7) in the ICG formulation of Comparative Example 3-1 and Comparative Example 2-1 differed significantly after being placed at 40°C for 24 hours. In the ICG formulation of Comparative Example 3-1, sodium hydroxide effectively controlled the stability of the ICG sample before lyophilization; however, in the ICG formulation of Comparative Example 2-1, sodium hydroxide could not effectively control the stability of the ICG nano-formulation sample before lyophilization.

[0087] Conclusion: The ICG formulation prepared using the method of this invention, combined with conventional pH adjusters, differs from ordinary ICG formulations. The pH control methods used in ordinary ICG formulations cannot meet the requirements for preparing the ICG formulation of this invention. This further illustrates that the use of organic salts, represented by sodium citrate, as pH adjusters, and the control of the pH value within the range of 6.5-8.0, as discovered in this invention, are specific conditions to ensure the stability of the ICG formulation samples before lyophilization, rather than a general method for controlling the stability of ICG formulation samples before lyophilization.

[0088] Example 4

[0089] This embodiment examines the effect of another preparation process on related substances in the formulation, as detailed below:

[0090] Comparative Example 4-1.

[0091] The formulation composition and dosage are shown in Table 16: Table 16 Components and Contents of the Formulation Indocyanine Green 0.5 Soybean phospholipids 10.0 cholesterol 0.5 Sodium citrate Adjust the pH to 6.8. Mannitol 12.5 Anhydrous ethanol (used in the process and ultimately removed) Appropriate amount Water for Injection 200 ml

[0092] Weigh out the prescribed amounts of soybean lecithin, cholesterol, and indocyanine green, mix them with the prescribed amount of anhydrous ethanol, and dissolve the raw materials by stirring. Under stirring, vacuum dry at 45°C to form a lipid film deposit until the lipid film state no longer changes. Add the obtained lipid film deposit to water for injection containing sodium citrate, stir at 30°C until hydration is complete, then add mannitol and stir to dissolve. Homogenize 5 times in a homogenizer. Filter through 0.45 µm and 0.22 µm filter membranes, fill, freeze dry, and cap to obtain the final product.

[0093] The relevant substances of Examples 1-1 and Comparative Example 4-1 were tested after preparation for 0 days and after being placed under influencing factors (high temperature, high humidity and light) for 10 days. The results are shown in Table 17: Table 17 Results of experimental investigation on influencing factors of ICG formulations prepared by different processes

[0094] The above results show that even with the same formulation composition and proportions, the stability of ICG formulations obtained by different preparation methods varies significantly. The preparation method of this invention—adding the lyophilization protectant before lipid film deposition—is a necessary condition to ensure the stability of the RRT 0.3 impurity in the ICG formulation during storage, while conventional preparation methods (adding the lyophilization protectant after hydration) cannot guarantee the stability of the ICG formulation.

[0095] Conclusion: The preparation method of the present invention can guarantee the stability of the RRT 0.3 impurity in the ICG formulation during storage, while conventional preparation methods cannot guarantee the stability of the ICG formulation.

[0096] Example 5

[0097] This embodiment examines the efficacy evaluation of ICG formulations in a mouse tumor lymph node metastasis model using lymph node tracing, as detailed below:

[0098] The lyophilized formulations of Examples 1-1 and 3-1 were used to prepare drug solutions for efficacy experiments. The drug concentrations in the solutions are shown in Table 18. A Balb / c mouse tumor lymph node metastasis model was established using 4T1 cells. The mice were fed with tumor-bearing cells for 12 days until the average tumor volume reached 50 mm. 3 Subsequent experiments were then conducted. Twenty-four Balb / c mice were selected and numbered according to their initial weight. After 7 days of acclimatization, they were randomly divided into three groups of eight mice each, based on the uniformity of tumor volume in the left hind paw: a standard ICG formulation group (0.1 mg / mouse), a standard ICG formulation group (0.05 mg / mouse), and the ICG formulation of this invention group (0.05 mg / mouse). Grouping and administration parameters are detailed in Table 18. Table 18 Experimental Groups and Dosage

[0099] Before the experiment, the skin of the left hind paw and popliteal fossa of mice was prepared. A needle was inserted subcutaneously near the tip of the left toe to the center of the foot. Forty-five minutes after drug administration, urethane anesthetic solution was injected intraperitoneally at a dose of 7.5 mL / kg of mouse body weight. One hour after drug administration, fluorescence signals from the popliteal lymph nodes were collected using an in vivo fluorescence imaging system, and the fluorescence intensity was semi-quantitatively analyzed. Visible fluorescence signals in the lymph nodes were considered positive for tracing. The number of positive lymph nodes in each group was counted, and the detection rate was calculated. The diffusion area ratio (total fluorescent positive area / area of ​​the core fluorescent dense area) was used as an indicator to evaluate the degree of fluorescence diffusion around the lymph nodes; a higher ratio indicates a larger diffusion range of the dye tissue, and vice versa.

[0100] Imaging of sentinel lymph nodes in animals was performed 1 hour after drug administration. Results are shown below. Figure 1 Eight animals each were included in the standard ICG regimen groups (0.1 mg / animal and 0.05 mg / animal). After injection, the fluorescent signal diffused from the tumor site to the entire hind limb. In both dosage groups, only one sentinel lymph node could be clearly distinguished, with a detection rate of 12.5%. In the ICG-Lip group (0.05 mg / animal), the sentinel lymph node was clearly observed and identified, with a detection rate of 100%. After a 1-hour observation period, the degree of fluorescence diffusion around the sentinel lymph node was observed. The results showed that, compared with ICG, the ICG-Lip fluorescent signal was concentrated in the sentinel lymph node and diffused less to the surrounding area.

[0101] Animals were dissected 1 hour after injection to observe the detection of lymph nodes at all levels. Secondary and tertiary lymph nodes were detectable in all groups. Figure 2However, visual observation revealed high background fluorescence after administration of ICG (0.1–0.05 mg / animal); in the ICG formulation group of this invention (0.05 mg / animal), the background of the tissue surrounding the secondary and tertiary lymph nodes was clean, with less interference from fluorescence signals and clear localization. Both visual observation and statistical results of lymph nodes at each level showed that, compared to ICG, the ICG-Lip fluorescence signal was concentrated in the sentinel lymph nodes and diffused less into the surrounding tissues.

[0102] Furthermore, based on the pharmacodynamic data from Example 5, it can be deduced that when ICG is used as a free small molecule drug (i.e., the conventional ICG formulation group), it diffuses rapidly, resulting in extensive staining and high diffusion in the tissue near the tumor, making it impossible to accurately distinguish the location of the sentinel lymph nodes. In contrast, the ICG formulation prepared in this invention can significantly reduce local diffusion, concentrating the fluorescence signal highly on the sentinel lymph nodes and accurately locating secondary and tertiary lymph nodes. This physical behavior of "reduced diffusion and precise targeting" is precisely the core characteristic that distinguishes liposome / nanoformations from free small molecule drugs. Therefore, based on the pharmacodynamic results, the formulation of this invention should possess a nanocarrier structure.

[0103] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. An indocyanine green formulation, wherein the formulation is a liquid formulation or a lyophilized formulation, characterized in that, The raw materials for preparing the formulation comprise the following components by weight: indocyanine green, 0.2-20 parts; phospholipids, 25-225 parts; cholesterol, 1-25 parts; lyophilization protectant, 10-500 parts; water, 1000-3000 parts; and also include a pH adjuster for adjusting the pH of the formulation to 6.0-8.5; the pH adjuster is selected from one or more of sodium citrate, potassium citrate, sodium tartrate, sodium acetate, sodium lactate, and sodium succinate.

2. The indocyanine green formulation according to claim 1, characterized in that, The raw materials for preparing the formulation comprise the following components by weight: indocyanine green, 1-10 parts; phospholipids, 50-150 parts; cholesterol, 2-10 parts; lyophilization protectant, 50-200 parts; water, 1800-2200 parts; the pH adjuster is used to adjust the pH of the formulation to 6.5-8.

0.

3. The indocyanine green formulation according to claim 1 or 2, characterized in that, The phospholipids are selected from one or more of soybean phospholipids, egg yolk lecithin, hydrogenated soybean phospholipids, soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearate phosphatidylcholine, disqualyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, and phosphatidylethanolamine.

4. The indocyanine green formulation according to claim 1 or 2, characterized in that, The freeze-drying protectant is selected from one or more of sucrose, trehalose, mannitol, lactose, and glucose.

5. The indocyanine green formulation according to claim 1 or 2, characterized in that, The pH adjuster is selected from one or more of sodium citrate, potassium citrate, sodium tartrate, and sodium lactate.

6. The indocyanine green formulation according to any one of claims 1-5, characterized in that, In the formulation, the content of RRT 0.3 impurity is ≤1.5%, and the content of RRT 0.7 impurity is ≤1.5%; wherein, the structural formula of the RRT 0.3 impurity is shown in formula (I), and the structural formula of the RRT 0.7 impurity is shown in formula (II):

7. The method for preparing the indocyanine green formulation according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Add the prescribed amounts of phospholipids, cholesterol, and indocyanine green to the solvent and mix; (2) Mix the mixture obtained in step (1) with the formulated amount of freeze-drying protectant, and heat to remove the solvent until a lipid film deposit is formed; (3) Mix the pH adjuster with water and then add it to the lipid film deposits obtained in step (2) for hydration reaction; When the preparation is a lyophilized preparation, the preparation method further includes: step (4), lyophilizing the liquid obtained in step (3).

8. The preparation method according to claim 7, characterized in that, In step (2), the solvent is removed under vacuum and heating temperature of 40-50°C; and / or, in step (3), the hydrated liquid is further homogenized and filtered; preferably, the homogenization is performed 3-7 times; the filtration is performed using 0.45 µm and 0.22 µm filter membranes.

9. Use of the indocyanine green formulation according to any one of claims 1-6 or the indocyanine green formulation obtained by the preparation method according to claim 7 or 8 in the preparation of a lymphatic tracer.

10. A lymphatic tracer, characterized in that, The indocyanine green formulation included in any one of claims 1-6 or indocyanine green formulation obtained by the preparation method described in claim 7 or 8.

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