Preparation composition of polypeptide conjugate fluorescence imaging agent as well as preparation method and application of preparation composition

Abstract

The invention relates to the technical field of freeze-dried powder injections, in particular to a polypeptide conjugate fluorescent developer preparation composition as well as a preparation method and application thereof. The polypeptide conjugate fluorescence imaging agent preparation composition is prepared from the following raw materials: a combination of a polypeptide conjugate fluorescence imaging agent raw material medicine, an excipient and a pH buffering agent, and at least one of a solubilizer and a chelating agent is included or not included. By improving the formula and the process of the freeze-dried powder injection, the problems that a polypeptide conjugate fluorescence imaging agent is insoluble and poor in stability are solved, and the formability, the resolubility and the stability are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of lyophilized powder injection technology, and in particular to a composition of a polypeptide conjugate fluorescent imaging agent formulation, its preparation method, and its application. Background Technology

[0002] Fluorescence imaging technology can be used in various scenarios such as intraoperative visualization, tumor labeling, and lymph node tracing in surgery, which can shorten operation time and improve surgical clearance rate. Currently, the types of fluorescence imaging agents used clinically are very limited, including only 5-aminolevulinic acid (Cysview, indicated for bladder cancer imaging), methylene blue, and indocyanine green (ICG), which lack targeting specificity, resulting in poor imaging specificity. These fluorescence imaging agents are all in single-molecule form, which is prone to irreversible excited-state molecular destruction under strong laser energy excitation, causing rapid decay and quenching of the fluorescence imaging agent, resulting in a photobleaching effect. In clinical applications, single-molecule fluorescence imaging agents have poor photostability, short imaging windows, and low signal-to-noise ratios, leading to high false positive and false negative rates, significantly affecting the precision of surgical procedures. Therefore, developing long-term stable targeted fluorescence imaging agents will help doctors identify occult tumors in real time, reduce the risk of positive surgical margins, and lower the probability of recurrence and secondary surgery for patients.

[0003] Chinese patent CN107648618A discloses a drug delivery system, its preparation method, and its applications. The drug delivery system includes a molecular recognition portion, an assembly portion, a response portion, a functional molecule portion, and an optional long-circulation portion. The peptide-conjugate fluorescent imaging agent prepared by the method of this invention can utilize the assembly-induced retention effect (AIR) within tumor cells to achieve efficient enrichment and retention of fluorescent molecules in tumor tissue, significantly improving the accuracy and stability of tumor imaging. This peptide-conjugate fluorescent imaging agent's highly specific visualization fluorescence imaging technology can change the traditional diagnostic and treatment model based on two-dimensional imaging, addressing clinical problems such as defining tumor boundaries, displaying small lesions, determining lymph node metastasis, and identifying residual lesions at the resection surface at the molecular and cellular levels. However, this peptide-conjugate fluorescent imaging agent is almost insoluble or insoluble in water, exhibiting high solubility at a specific pH, but its aqueous solution stability is poor. The intended clinical use of this drug is for in vivo tumor localization and intraoperative imaging; therefore, these characteristics limit its application.

[0004] Lyophilized powder injections are sterile powder injections produced using freeze-drying technology. They are mainly used for drugs that are unstable in aqueous solutions. The preparation process generally includes steps such as solution preparation, filtration, filling, pre-freezing, and vacuum drying, ultimately forming a well-defined, loosely structured solid formulation. For example, Chinese patent CN111388429A discloses a novel lyophilized powder injection of the broad-spectrum antiviral drug Triazavirin, composed of Triazavirin, β-cyclodextrin, low molecular weight dextran, disodium edetate, a pH adjuster, and an osmotic pressure adjuster. The lyophilized powder injection of Triazavirin prepared by this invention has advantages such as low moisture content, good appearance, and low levels of related impurities, making it suitable for long-term storage.

[0005] The formulation and preparation process of lyophilized powder injections must be based on the active pharmaceutical ingredient (API) to achieve activity protection or performance improvement. Currently, however, there is a lack of corresponding formulation processes for the drug delivery system disclosed in Chinese patent CN107648618A. Furthermore, formulation processes for other APIs are difficult to apply due to significant differences in their properties. Therefore, a formulation solution is needed to address the issues of insolubility and poor stability of the drug delivery system disclosed in Chinese patent CN107648618A. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a formulation composition of a polypeptide conjugate fluorescent imaging agent, its preparation method and application, and to solve the problems of insolubility and poor stability of the polypeptide conjugate fluorescent imaging agent by improving the formulation and process of the lyophilized powder injection, and to significantly improve its formability, resolubility and stability.

[0007] To achieve the above-mentioned objectives, the technical solution of this invention is as follows: On one hand, the present invention provides a formulation composition of a polypeptide conjugate fluorescent imaging agent, the raw materials of which include a combination of a polypeptide conjugate fluorescent imaging agent active pharmaceutical ingredient, an excipient, and a pH buffer; and includes, or excludes, at least one of a solubilizer and a chelating agent. The chemical structure of the polypeptide conjugate fluorescent imaging agent is: L-alanyl-L-valine-L-prolyl-L-isoleucyl-L-alanyl-L-glutamine-L-lysyl-L-aspartyl-L-glutamine-L-valine-L-aspartyl-L-lysyl-L-leucyl-L-valine-L-phenylalanyl-L-phenylalanyl-L-alanyl-L-glutamine-L-cysteyl-[1-(5-carboxypentyl)-2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindole-2-methylene)ethylene)-2-mercaptocyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-3H-indole-1-onium salt]-glycine; Preferably, the excipient is selected from at least one of carbohydrate excipients, polyol excipients, and polymer excipients; The pH buffer is selected from at least one of the following: phosphate buffer system, Tris buffer system, citrate buffer system, and acetate buffer system; The solubilizer is selected from at least one of Tween 20, Tween 80, polyoxyethylene 35 castor oil, 15-hydroxystearic acid polyethylene glycol HS15, 15-hydroxystearic acid polyethylene glycol ester, N,N-dimethylacetamide, glycerol, and propylene glycol. The chelating agent is selected from at least one of disodium edetate, DTPA, citric acid, gluconic acid, and EDDS.

[0008] Preferably, the content of each component in the raw material is as follows: Peptide conjugate fluorescent imaging agent raw material: 0.5-5 mg / mL; Excipients: 5-50 mg / mL; pH buffer: 0.001-0.2 mol / L ion concentration; Solubilizer: 0-10 mg / mL; Chelating agent: 0-1 mg / mL.

[0009] More preferably, the content of the polypeptide conjugate fluorescent imaging agent raw material in the raw material is 1-3 mg / mL.

[0010] More preferably, the content of excipients in the raw materials is 5-30 mg / mL, and more preferably 20-30 mg / mL.

[0011] More preferably, the pH buffer in the raw material has an ion concentration of 0.005-0.12 mol / L.

[0012] More preferably, the content of the solubilizer in the raw material is 0-5 mg / mL.

[0013] More preferably, the content of the chelating agent in the raw material is 0-1.0 mg / mL, and more preferably 0-0.5 mg / mL.

[0014] More preferably, the sugar excipient is selected from at least one of glucose, fructose, galactose, ribose, sucrose, maltose, lactose, trehalose, raffinose, inulin, honey, and cellobiose; The polyol excipients are selected from at least one of glycerol, mannitol, sorbitol, polyethylene glycol, inositol, thiols, propylene glycol, pentaerythritol, and 1,3-butanediol; The polymeric excipient is selected from at least one of polyvinylpyrrolidone, bovine serum albumin, dextran, polyethylene glycol, gelatin, β-cyclodextrin, cellulose, cellulose derivatives, and chitosan.

[0015] More preferably, the sugar excipient is selected from at least one of glucose, fructose, galactose, ribose, sucrose, maltose, lactose, trehalose, raffinose, and inulin; The polyol excipients are selected from at least one of glycerol, mannitol, sorbitol, polyethylene glycol, inositol, and thiols; The polymeric excipient is selected from at least one of polyvinylpyrrolidone, bovine serum albumin, dextran, polyethylene glycol, gelatin, β-cyclodextrin, and cellulose.

[0016] More preferably, the excipient is selected from at least one of mannitol, sucrose, and trehalose.

[0017] Most preferably, the excipient is mannitol.

[0018] Preferably, the pH buffer is a phosphate buffer system or an acetate buffer system.

[0019] More preferably, the pH buffer is a phosphate buffer system composed of disodium hydrogen phosphate and sodium dihydrogen phosphate or an acetate buffer system composed of acetic acid and sodium acetate.

[0020] More preferably, the pH buffer is a phosphate buffer system composed of disodium hydrogen phosphate and sodium dihydrogen phosphate.

[0021] Preferably, the pH of the raw material is adjusted to 2.5-9 by adding a pH buffer.

[0022] As an example, the pH may be 2.5-3, 3-5, 5-7, or 7-9. As some potentially more preferred examples, the pH may be 3-4, 4-5, 5-6, 6-7, 7-8, or 8-9.

[0023] More preferably, the pH of the pH buffer is 3-4 or 6-8.

[0024] As an example, the pH may be 2.5-3, 3-3.5, 3.5-4, 6-6.5, 6.5-7, 7-7.5, or 7.5-8. As some potentially more preferred examples, the pH may be 2.5, 3, 3.5, 4, 4.5, 6, 6.5, 7, 7.5, or 8.

[0025] More preferably, when the pH buffer is an acetate buffer system composed of acetic acid and sodium acetate, the pH of the raw material is adjusted to 3-4; when the pH buffer is a phosphate buffer system composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, the pH of the raw material is adjusted to 6-8.

[0026] Preferably, the structure of the polypeptide conjugate fluorescent imaging agent is as follows: .

[0027] In this invention, the structure and preparation process of the polypeptide conjugate fluorescent imaging agent can be found in the published patent CN107648618A.

[0028] Preferably, the polypeptide conjugate fluorescent imaging agent formulation composition further includes at least one of a pH adjuster, a solubilizer, and a chelating agent.

[0029] Preferably, the raw material further includes a pH adjuster, which is used to adjust the pH of the raw material to 2.5-9. The pH adjuster is added in trace amounts or not at all, to slightly correct to the target pH, and in most cases, it is not necessary to add it.

[0030] Preferably, the pH adjuster is at least one selected from hydrochloric acid, acetic acid, sulfuric acid, citric acid, sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate, and more preferably hydrochloric acid and sodium hydroxide. The concentration of the hydrochloric acid can be 0.1-20 mol / L, and the concentration of the sodium hydroxide can be 0.1-15 mol / L. As a specific example of the present invention, a 1 mol / L hydrochloric acid solution and a 1 mol / L sodium hydroxide solution are used.

[0031] Preferably, a solubilizer is added when the pH of the raw material is 3-6.

[0032] More preferably, when the pH of the pH buffer is 2.5-4, a solubilizer is added.

[0033] The solubilizer is selected from at least one of Tween 20, Tween 80, polyoxyethylene 35 castor oil, 15-hydroxystearic acid polyethylene glycol HS15, 15-hydroxystearic acid polyethylene glycol ester, N,N-dimethylacetamide, glycerol, and propylene glycol, and is more preferably at least one of Tween 20, Tween 80, polyoxyethylene 35 castor oil, and 15-hydroxystearic acid polyethylene glycol HS15.

[0034] Preferably, the chelating agent is selected from at least one of disodium edetate, DTPA (diethylenetriaminepentaacetic acid), citric acid, gluconic acid, and EDDS (ethylenediaminedisuccinic acid), and more preferably disodium edetate.

[0035] In another aspect, the present invention provides a method for preparing the above-mentioned polypeptide conjugate fluorescent imaging agent formulation composition, comprising the steps of: mixing the polypeptide conjugate fluorescent imaging agent and water for injection, sequentially adding the prescribed amounts of pH buffer, excipient, solubilizer, and chelating agent, and then lyophilizing to obtain the product.

[0036] Preferably, the water is water for injection.

[0037] Preferably, the freeze-drying process is as follows: Table 1

[0038] N / A means that an option, parameter, or data is irrelevant to the current situation and does not need to be filled in or marked.

[0039] More preferably, the freeze-drying process is as follows: Table 2

[0040] Preferably, the preparation method further includes the step of sterilization.

[0041] Preferably, the sterilization method includes, but is not limited to, filtration sterilization, ultraviolet sterilization, and high-temperature sterilization, with filtration sterilization being the most preferred.

[0042] More preferably, the filter membrane for filtration and sterilization is a PES filter membrane (polyethersulfone filter membrane).

[0043] More preferably, the filter membrane needs to undergo pre-saturation treatment.

[0044] The specific steps of the pre-saturation are as follows: After the medicine solution is prepared, connect the filter to filter the medicine solution into another container. At the beginning of the filtration stage, discard a small amount of medicine solution to pre-saturate the filter membrane.

[0045] This invention discovers that pre-saturating the filter membrane can reduce the impact of filtration on the content.

[0046] In another aspect, the present invention provides the application of the above-mentioned polypeptide conjugate fluorescent imaging agent formulation composition in the preparation of tumor-targeting fluorescent imaging agents.

[0047] Preferably, the tumor is a solid tumor.

[0048] The term "solid tumor" refers to a localized, solid mass formed by abnormally proliferating cells within an organ or tissue. Unlike hematologic malignancies (such as leukemia), it has a clearly defined space-occupying structure and can be detected through imaging (such as CT and MRI) or palpation.

[0049] The solid tumors mentioned include bladder cancer, breast cancer, lung cancer, glioma, prostate cancer, liver cancer, ovarian cancer, stomach cancer, colorectal cancer, endometrial cancer, etc.

[0050] Finally, the present invention provides a tumor-targeted fluorescence imaging method, comprising administering the above-described polypeptide conjugate fluorescence imaging agent formulation composition to a subject via a route of administration.

[0051] Preferably, the injectable polypeptide conjugate fluorescent imaging agent formulation composition, after being diluted with a solvent, can be administered via local perfusion, local injection, or intravenous administration.

[0052] Preferably, the dosage of the lyophilized powder injection is 2-20 mg / person for local perfusion, 0.05-2 mg / kg for local injection, and 0.1-2 mg / kg for intravenous administration.

[0053] Here, "subject" refers to an individual suffering from a disease, symptom, or condition, including both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans; non-human primates (e.g., chimpanzees and other apes and monkeys); livestock such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-human mammals include, but are not limited to, birds and fish.

[0054] Preferably, the tumor is a solid tumor.

[0055] The solid tumors mentioned include bladder cancer, breast cancer, lung cancer, glioma, prostate cancer, liver cancer, ovarian cancer, stomach cancer, colorectal cancer, endometrial cancer, etc.

[0056] Compared with the prior art, the advantages of this invention are as follows: (1) The present invention provides an injectable polypeptide conjugate fluorescent imaging agent formulation composition, wherein the excipient can increase the stability of the active pharmaceutical ingredient and the formability of the surface of the lyophilized injection, and improve the shrinkage and collapse of the active pharmaceutical ingredient during lyophilization. At the same time, the pH adjuster solves the instability of polypeptide solubility and polymerization of the main component, making the polypeptide powder injection more stable.

[0057] (2) The present invention provides an injectable polypeptide conjugate fluorescent imaging agent formulation composition. Under specific excipient ratios and compositions, as shown in the examples, it is confirmed that the excipients provided by the present invention can significantly improve the shapeability and reconstitution properties of lyophilized injections. Furthermore, the examples demonstrate that, compared with the raw materials, the stability of the sample is significantly improved in various indicators, including the purity of the main component and the content of impurities. Through the examples, the present invention, through solution placement and stability tests under influencing factors (high temperature, high humidity, and light), shows that the injectable polypeptide lyophilized powder provided by the present invention has good stability after forced degradation stability tests, indicating that the addition of excipients contributes to the shapeability and stability of the lyophilized injections.

[0058] (3) The present invention provides an injectable polypeptide conjugate fluorescent imaging agent formulation composition. Under specific excipient and pH adjuster ratios and compositions, examples show that the excipient and pH adjuster provided by the present invention can work synergistically to significantly improve the solubility and stability of the polypeptide injection. This indicates that the injectable polypeptide lyophilized powder injection sample formulation under the ratios and compositions of the present invention is appropriate, has good stability, and can be predicted to be stable for 24 months of storage. Attached Figure Description

[0059] Figure 1 Appearance images of the lyophilized samples prepared for formulations 1-8, where numbers 1-8 correspond to formulations 1-8 respectively. Detailed Implementation

[0060] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention. Those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and these should also fall within the scope of protection claimed by the present invention. Unless otherwise specified, all percentages are by mass; unless otherwise specified, all operating temperatures are at room temperature; unless otherwise specified, all raw materials can be obtained through conventional purchasing channels or conventional methods.

[0061] Example 1 The preparation steps of the injectable polypeptide conjugate fluorescent imaging agent formulation composition (lyophilized powder for injection) are as follows: S1 Solution Preparation: Weigh the prescribed amount of active pharmaceutical ingredient and add water for injection (temperature controlled at 20-30℃). Then, while stirring, add the prescribed amount of pH buffer, excipient, solubilizer (if any), and chelating agent (if any). Adjust the pH of the solution with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide, and add water for injection to bring the volume to the prescribed amount.

[0062] S2 filtration: The above solution is filtered through a 0.22μm microporous membrane for sterilization. The filtrate is then dispensed into vials and partially sealed with rubber stoppers.

[0063] S3 Freeze-drying: Set the parameters of the vacuum freeze dryer according to the table below and perform freeze-drying.

[0064] Table 3

[0065] S4 Preservation: After freeze-drying, stopper the sample, remove it from the box, crimp the cap, and affix a label.

[0066] Based on the above preparation method and the formulations described in Tables 4 and 5, the relevant excipients of the injectable polypeptide conjugate fluorescent imaging agent formulation composition were investigated.

[0067] Table 4

[0068] Table 5

[0069] Detection method: The sample solutions prepared according to the prescriptions in Tables 4 and 5 were left at room temperature (25°C) for 0 h, 4 h, and 20 h. The filtrate obtained in step S2 was collected, and its properties, clarity, pH, and related substances were observed. Subsequently, each group of samples was lyophilized, and the appearance of the product was observed.

[0070] The properties and clarity are determined by visual observation. The standard for "clear" and "clarity" is: the solution should be clear. If it appears turbid, it should not be more concentrated than the No. 1 turbidity standard solution.

[0071] The relevant substances and their content detection methods are as follows: Use a high-performance liquid chromatograph equipped with a UV detector (or PDA / DAD detector) or equivalent. The chromatographic conditions are as follows: Table 6

[0072] Test results: The results of the excipient investigation are shown in Tables 7 and 8.

[0073] In an acetic acid / sodium acetate buffer system at pH 3.0, compared with the group without lyophilization excipients, all three excipients—mannitol, sucrose, and trehalose—improved the solution stability of the active ingredient and reduced the impurity content. Based on the appearance of the lyophilized samples ( Figure 1 It can be seen that the sample without excipients has poor formability and the lyophilized powder shrinks severely; the sample with added mannitol has good formability and the lyophilized powder is in block form without shrinkage or collapse; the sample with added sucrose and trehalose has good formability, the lyophilized powder shrinks slightly but does not collapse significantly.

[0074] Table 7

[0075] In a pH 7.0 disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system, compared with the group without lyophilization excipients, all three excipients—mannitol, sucrose, and trehalose—could improve the solution stability of the active ingredient and reduce the impurity content. However, based on the appearance of the lyophilized samples ( Figure 1 It can be seen that the sample without excipients has poor formability and the lyophilized powder shrinks severely; the sample with added mannitol has good formability and the lyophilized powder is in block form without shrinkage or collapse; the sample with added sucrose and trehalose has good formability, the lyophilized powder shrinks slightly but does not collapse significantly.

[0076] Table 8

[0077] Example 2 This embodiment provides the effect of adding metal ion chelating agents on the stability of formulation solutions of different peptide conjugate fluorescent imaging agents. Since the introduction of metal ions during the production of lyophilized powder injections may accelerate the degradation of peptide drugs, the effect of adding the chelating agent disodium edetate to different formulations on solution stability was investigated.

[0078] According to the preparation method of the injectable polypeptide conjugate fluorescent imaging agent formulation composition described in Example 1, 0.05% of disodium edetate by total mass was added to formulation 2 (acetate buffer system) and formulation 6 (phosphate buffer system), respectively. The filtrate obtained in step S2 was taken to investigate the change of impurities after being placed at room temperature (25°C) for 24 h. The investigation method was the same as in Example 1.

[0079] Table 9

[0080] The test results are shown in Table 10. Compared with the changes in impurities in formulations 2 and 6 in the examples, the addition of disodium edetate in the phosphate buffer system had no significant effect on the stability of the sample. However, the addition of disodium edetate in the acetate buffer system significantly reduced the generation of impurities, and the formulation had better stability.

[0081] Table 10

[0082] Example 3 This embodiment demonstrates the effect of different raw material ratios on the stability of lyophilized powder injection samples.

[0083] According to the preparation method of the injectable polypeptide conjugate fluorescent imaging agent formulation composition described in Example 1, the injectable polypeptide conjugate fluorescent imaging agent formulation composition was prepared according to formulations 11-14 in Table 11 and formulations 15-18 in Table 12, respectively.

[0084] Table 11

[0085] Table 12

[0086] Detection method: The sample solutions prepared according to the prescription in Table 11 were placed at room temperature (25°C) for 0 h, 4 h, and 24 h. The filtrate obtained in step S2 was collected, and its properties, clarity, pH, and related substances were observed. Subsequently, each group of samples was lyophilized, and the appearance of the product was observed.

[0087] The investigation method is the same as in Example 1.

[0088] The test results for acetate buffer system formulations 11-14 are shown in Table 13. Under the condition that the lyophilized solutions of formulations 11-14 contain the same types of solubilizers, buffers, excipients, and chelating agents, the different proportions of the four excipients and the active pharmaceutical ingredient of the polypeptide conjugate fluorescent imaging agent had little effect on the impurity levels of the solution, and there was no significant difference in solution stability. The lyophilized samples exhibited good shape retention.

[0089] Table 13

[0090] The test results for phosphate buffer system formulation 15-18 are shown in Table 14. Under the condition that the lyophilized solution of formulation 15-18 contains the same types of solubilizers, buffers, and excipients, the different proportions of the three excipients and the active pharmaceutical ingredient of the peptide conjugate fluorescent imaging agent have little effect on the level of impurities in the solution, and there is no significant difference in solution stability. The lyophilized samples exhibit good shape retention.

[0091] Table 14

[0092] Comparative Example 1 This embodiment provides a comparison of the shapeability and stability of injectable peptide conjugate fluorescent imaging agent formulations with different excipients in an acetate buffer system formulation.

[0093] According to the preparation method of the injectable polypeptide conjugate fluorescent imaging agent formulation composition described in Example 1, the injectable polypeptide conjugate fluorescent imaging agent formulation compositions were prepared according to the comparative formulations 1-3 in Table 15.

[0094] Compared to Formula 2, the difference between Formula 1 and Formula 2 is that the excipient is replaced with β-cyclodextrin; all other aspects are the same.

[0095] Compared to Formula 2: The difference is that the excipient is replaced with cellulose, otherwise they are the same.

[0096] Compared to prescription 2, the difference in prescription 3 is that the excipient is replaced with maltose, otherwise they are the same.

[0097] Table 15

[0098] Result detection: Each group of sample solutions was placed at room temperature (25℃) for 0 h, 4 h, and 24 h. The filtrate obtained in step S2 was collected, and its properties, clarity, pH, and related substances were observed. Subsequently, each group of samples was freeze-dried to observe the product appearance.

[0099] The investigation method is the same as in Example 1.

[0100] The test results of the acetate buffer system compared with formulations 1-3 are shown in Table 16. Compared with the mannitol excipient group in formulation 2, under the same buffer system, the stability of the three excipients β-cyclodextrin, cellulose and maltose on the lyophilized powder injection samples differed. The impurity levels in the three comparative formulations increased with time, and the lyophilized powder had poor appearance and shape retention, with severe shrinkage and collapse.

[0101] Table 16

[0102] Comparative Example 2 This embodiment provides a comparison of the shapeability and stability of injectable peptide conjugate fluorescent imaging agent formulations with different excipients in a phosphate buffer system formulation.

[0103] According to the preparation method of the injectable polypeptide conjugate fluorescent imaging agent formulation composition described in Example 1, the injectable polypeptide conjugate fluorescent imaging agent formulation compositions were prepared according to the comparative formulations 4-6 in Table 17.

[0104] Compared to prescription 6, the difference in prescription 4 is that the excipient is replaced with β-cyclodextrin; otherwise, they are the same.

[0105] Compared to prescription 6, the difference in prescription 5 is that the excipient is replaced with cellulose; otherwise, they are the same.

[0106] Compared to prescription 6: The difference is that the excipient is replaced with maltose, otherwise they are the same.

[0107] Table 17

[0108] Result detection: Each group of sample solutions was placed at room temperature (25℃) for 0 h, 4 h, and 24 h. The filtrate obtained in step S2 was collected, and its properties, clarity, pH, and related substances were observed. Subsequently, each group of samples was freeze-dried to observe the product appearance.

[0109] The investigation method is the same as in Example 1.

[0110] The test results of the phosphate buffer system compared with formulations 4-6 are shown in Table 18. Compared with the mannitol excipient group in formulation 6, under the same buffer system, the stability of the three excipients β-cyclodextrin, cellulose and maltose for lyophilized powder injection samples differed. The impurity levels in the three comparative formulations increased with time, and the lyophilized powder had poor appearance and shape retention, with severe shrinkage and collapse.

[0111] Table 18

[0112] Comparative Example 3 This embodiment provides a comparison of the stability of injectable peptide conjugate fluorescent imaging agent formulations using phosphate buffer systems and acetate buffer systems with different pH values.

[0113] According to the preparation method of the injectable polypeptide conjugate fluorescent imaging agent formulation composition described in Example 1, the injectable polypeptide conjugate fluorescent imaging agent formulation compositions were prepared according to comparative formulations 7 and 8 in Table 19, respectively.

[0114] Table 19

[0115] Result detection: Each group of sample solutions was placed at room temperature (25℃) for 0 h, 4 h, and 24 h. The filtrate obtained in step S2 was collected, and its properties, clarity, pH, and related substances were observed. Subsequently, each group of samples was freeze-dried to observe the product appearance.

[0116] The investigation method is the same as in Example 1.

[0117] The test results for comparative formulations 7 and 8 are shown in Table 20. The results indicate that, compared to formulation 6, under the same buffer system, comparative formulation 7 exhibits poor stability in a solution at pH 10.0, with a significant increase in impurity content over time. Compared to formulation 2, under the same buffer system, comparative formulation 8 exhibits poor stability in a solution at pH 2.0, with a significant increase in impurity content over time. pH has a relatively small impact on the shape retention of the lyophilized samples; both comparative formulations 7 and 8 showed good shape retention with no significant collapse or shrinkage.

[0118] Table 20

[0119] Example 4 This embodiment provides a stability study of injectable lyophilized polypeptide powder under room temperature, high temperature and light conditions.

[0120] According to the preparation method of the injectable polypeptide conjugate fluorescent imaging agent formulation composition described in Example 1, lyophilized powders were prepared according to formulations 2 and 6. The lyophilized powders prepared according to formulations 2 and 6 were placed under room temperature, high temperature (40 degrees Celsius), and light irradiation conditions for 0 days, 5 days, 10 days, and 30 days to determine their properties, related substances, and contents. The test methods are described in Example 1.

[0121] Result detection: The lyophilized powders prepared by formulations 2 and 6 were tested at room temperature, high temperature, and light exposure for 0, 5, 10, and 30 days, respectively. The results are shown in Table 21. The lyophilized powder formulations of peptide-coupled cyanine dye fluorescent imaging agents with added excipients and buffer salts can be stably stored at room temperature. High temperature and light exposure have a certain impact on the stability of the formulations, with the content decreasing and the total impurities increasing over time.

[0122] Table 21

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide conjugate fluorescent imaging agent formulation composition, characterized in that, Its raw materials include a combination of peptide conjugate fluorescent imaging agent active pharmaceutical ingredient, excipient and pH buffer; and at least one of solubilizer and chelating agent, with or without inclusion; The chemical structure of the polypeptide conjugate fluorescent imaging agent is: L-alanyl-L-valine-L-prolyl-L-isoleucyl-L-alanyl-L-glutamine-L-lysyl-L-aspartyl-L-glutamine-L-valine-L-aspartyl-L-lysyl-L-leucyl-L-valine-L-phenylalanyl-L-phenylalanyl-L-alanyl-L-glutamine-L-cysteyl-[1-(5-carboxypentyl)-2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindole-2-methylene)ethylene)-2-mercaptocyclohexyl-1-en-1-yl)vinyl)-3,3-dimethyl-3H-indole-1-onium salt]-glycine.

2. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 1, characterized in that, The excipient is selected from at least one of carbohydrate excipients, polyol excipients, and polymer excipients; The pH buffer is selected from at least one of the following: phosphate buffer system, Tris salt buffer system, citrate buffer system, and acetate buffer system; The solubilizer is selected from at least one of Tween 20, Tween 80, polyoxyethylene 35 castor oil, 15-hydroxystearic acid polyethylene glycol HS15, 15-hydroxystearic acid polyethylene glycol ester, N,N-dimethylacetamide, glycerol, and propylene glycol. The chelating agent is selected from at least one of disodium edetate, DTPA, citric acid, gluconic acid, and EDDS.

3. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 1, characterized in that, The content of each component in the raw material is as follows: Peptide conjugate fluorescent imaging agent raw material: 0.5-5 mg / mL; Excipients: 5-50 mg / mL; pH buffer: 0.001-0.2 mol / L ion concentration; Solubilizer: 0-10 mg / mL; Chelating agent: 0-1 mg / mL.

4. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 2, characterized in that, It also includes a pH adjuster used to adjust the pH to 2.5-9; The pH adjuster is selected from at least one of hydrochloric acid, acetic acid, sulfuric acid, citric acid, sodium hydroxide, potassium hydroxide, sodium carbonate, and sodium bicarbonate.

5. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 2, characterized in that, The content of the polypeptide conjugate fluorescent imaging agent raw material in the raw materials is 1-3 mg / mL.

6. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 2, characterized in that, The sugar excipient is selected from at least one of glucose, fructose, galactose, ribose, sucrose, maltose, lactose, trehalose, raffinose, inulin, honey, and cellobiose; The polyol excipients are selected from at least one of glycerol, mannitol, sorbitol, polyethylene glycol, inositol, thiols, propylene glycol, pentaerythritol, and 1,3-butanediol; The polymeric excipient is selected from at least one of polyvinylpyrrolidone, bovine serum albumin, dextran, polyethylene glycol, gelatin, β-cyclodextrin, cellulose, cellulose derivatives, and chitosan.

7. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 6, characterized in that, The excipient is selected from at least one of mannitol, sucrose, and trehalose, and its content in the raw material is 5-30 mg / mL.

8. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 7, characterized in that, The excipient is mannitol, and its content in the raw material is 20-30 mg / mL.

9. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 2, characterized in that, The pH buffer is a phosphate buffer system composed of disodium hydrogen phosphate and sodium dihydrogen phosphate or an acetate buffer system composed of acetic acid and sodium acetate, and the ion concentration in the raw material is 0.005-0.12 mol / L.

10. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 4, characterized in that, The pH adjuster is selected from at least one of hydrochloric acid and sodium hydroxide.

11. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 9, characterized in that, When the pH buffer is a phosphate buffer system composed of disodium hydrogen phosphate and sodium dihydrogen phosphate, the pH of the raw material is adjusted to 6-8.

12. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 9, characterized in that, When the pH buffer is an acetate buffer system composed of acetic acid and sodium acetate, the pH of the raw materials is adjusted to 3-4.

13. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 2, characterized in that, The solubilizer is selected from at least one of Tween 20, Tween 80, polyoxyethylene 35 castor oil, and 15-hydroxystearic acid polyethylene glycol HS15, and its content in the raw material is 0-5 mg / mL.

14. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 13, characterized in that, The solubilizer is selected from Tween 20 and Tween 80, and its content in the raw material is 0-5 mg / mL.

15. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 2, characterized in that, The chelating agent is selected from at least one of disodium edetate, DTPA, and EDDS, and its content in the raw material is 0-1.0 mg / mL.

16. The polypeptide conjugate fluorescent imaging agent formulation composition according to claim 15, characterized in that, The chelating agent is disodium edetate, and its content in the raw material is 0-0.5 mg / mL.

17. A method for preparing the polypeptide conjugate fluorescent imaging agent formulation composition according to any one of claims 1-16, characterized in that, The steps include: mixing the peptide conjugate fluorescent imaging agent and water for injection, then sequentially adding the prescribed amounts of pH buffer, excipient, solubilizer, and chelating agent, followed by lyophilization to obtain the product.

18. The preparation method according to claim 17, characterized in that, The preparation method further includes the step of sterilization. When the sterilization method is filtration sterilization, the filter membrane needs to be pre-saturated.

19. The use of the polypeptide conjugate fluorescent imaging agent formulation composition according to any one of claims 1-16 in the preparation of tumor-targeting fluorescent imaging agents.

20. The application according to claim 19, characterized in that, The tumor is a solid tumor.

21. The application according to claim 20, characterized in that, The solid tumor is selected from at least one of bladder cancer, breast cancer, lung cancer, glioma, prostate cancer, liver cancer, ovarian cancer, stomach cancer, colorectal cancer, and endometrial cancer.

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