A fluorescence-magnetic resonance dual-modality contrast agent and its preparation method

Abstract

The present invention relates to the field of medical diagnostic imaging, and specifically discloses a fluorescence-magnetic resonance dual-modality contrast agent and a preparation method thereof. The dual-modality contrast agent is a lyophilized powder injection, which comprises the following compound of formula (I): #imgabs0# and a lyophilization excipient and an optional pH regulator. The present invention converts a less stable aqueous injection with the compound of formula (I) as the API component into a lyophilized powder injection, thereby avoiding the technical defect that the long-term stability of the aqueous injection with the compound of formula (I) as the API component is poor, resulting in a weakened or even ineffective fluorescence tracing effect, thereby ensuring the fluorescence-magnetic resonance dual-modality imaging efficacy of the API component; the lyophilized powder injection provided by the present invention can effectively solve the problem of a significant increase in the impurity RRT1.03 during the freeze-drying process to obtain the lyophilized powder and the problem of poor product stability after lyophilization, greatly improving the safety of clinical medication and further promoting the development of subsequent clinical application research of the compound of formula (I) pharmaceutical agent.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnostic imaging, and more particularly to a freeze-dried powder injection-type fluorescence-magnetic resonance dual-modality contrast agent and a preparation method thereof. Background Art

[0002] Cancer is currently one of the major threats to human health. Surgery remains the preferred treatment, with resection of the primary tumor being a common procedure. However, extensive clinical pathology has demonstrated that a significant proportion of early-stage cancers lack a clear focal area. Furthermore, tumors often grow invasively, blurring the boundary with normal tissue and making surgical resection extremely difficult. Incomplete tumor resection is a major contributor to the poor efficacy and high recurrence rates of current surgical treatments. Therefore, precise preoperative localization of tumors has become a pressing need in clinical treatment.

[0003] Magnetic resonance imaging (MRI) is an imaging technique that utilizes the magnetic resonance phenomenon generated by hydrogen nuclei (protons) in human tissue when stimulated by radiofrequency pulses in a magnetic field. This generates magnetic resonance signals, which are then processed by a computer to reconstruct an image of a specific layer of the human body. In clinical applications, MRI is widely used in hospitals and research institutions worldwide due to its advantages such as non-invasive diagnosis, strong soft tissue imaging capabilities, no ionizing radiation, and no detection angle restrictions. However, its low sensitivity and short circulation time of MRI contrast agents limit its effectiveness in imaging tumors. Furthermore, it cannot provide real-time imaging during surgery, which greatly restricts its practical clinical application. The applicant of the present invention successfully developed a new fluorescence-magnetic resonance dual-modal contrast agent compound in 2021 (patent publication number: WO2022242437A1), which has good magnetic resonance T1-weighted imaging angiography capability and in vivo fluorescence imaging capability, and has the advantages of high relaxation rate, excellent thermal stability, low toxic side effects, good correspondence between fluorescence and magnetic resonance signals, etc. It can be effectively used in magnetic resonance imaging and fluorescence imaging of living cells and living bodies. After 6 hours of administration, it can still significantly enhance the magnetic resonance signals of the liver and kidneys, significantly improve the resolution of organ boundaries, and the magnetic resonance signal intensity has a high correspondence with the fluorescence intensity, and has the preoperative diagnosis and intraoperative navigation effects required clinically.

[0004] Contrast agent compounds are generally prepared as aqueous injections or powder injections, which are injected intravenously through the gaps between tumor vascular endothelial cells to enhance tumor imaging. When the inventors of this application used the contrast agent API disclosed in patent WO2022242437A1 as the API component to prepare a contrast agent aqueous injection, they discovered that although the contrast agent API disclosed in patent WO2022242437A1 has good water solubility, its long-term stability after being formulated into an aqueous injection is unsatisfactory. In particular, the fluorescent groups in the API component are unstable in aqueous solution and easily aggregate, leading to fluorescence quenching, which weakens or even fails the fluorescent tracing effect during use. However, after the API component was directly dissolved in water for injection and then freeze-dried to prepare a lyophilized powder injection, HPLC analysis showed that the content of an unknown impurity at a relative retention time RRT of approximately 1.03 after lyophilization (referred to as impurity RRT1.03 in this application) was significantly higher than the impurity RRT1.03 content in the API component. Moreover, the content of impurity RRT1.03 tended to increase with prolonged storage time, increasing the clinical medication risk of the lyophilized powder injection. Summary of the Invention

[0005] The impurity level in a drug directly reflects the stability of the product quality. Lower impurity levels mean better product stability and higher safety, which is more conducive to patient medication safety. The purpose of the present invention is to provide a freeze-dried powder injection-type fluorescence-magnetic resonance dual-modality contrast agent with low impurity levels, good formulation stability, and safer medication.

[0006] The technical solution of the present invention is as follows: a fluorescence-magnetic resonance dual-modal contrast agent, wherein the dual-modal contrast agent is a lyophilized powder injection, which comprises a compound of the following formula (I):

[0007] and lyophilization excipients and optionally a pH adjuster.

[0008] The lyophilized powder injection is dissolved in water for injection to form a solution with a concentration of 10 to 40 mg / ml of the compound of formula (I) and a pH value of 5.0 to 7.0.

[0009] In the above technical solution, the freeze-dried powder injection contains 30 to 180 parts by weight of the freeze-dried excipient for every 40 parts by weight of the compound of formula (I).

[0010] In the above technical solution, the lyophilization excipient is a carbohydrate compound and / or a sugar alcohol compound; preferably, the carbohydrate compound is selected from one or more of dextran, anhydrous glucose, lactose, sucrose, trehalose and maltose, more preferably lactose, sucrose, trehalose, and even more preferably sucrose; the sugar alcohol compound is selected from one or more of sorbitol, mannitol and erythritol.

[0011] The present inventors studied the freeze-drying process and excipients including pH regulators and freeze-drying excipients when preparing a freeze-dried powder injection with a compound of formula (I) as the API component. After a large number of experimental explorations, they found that when specific carbohydrate compounds and / or sugar alcohol compounds were introduced as freeze-drying excipients, the growth of impurity RRT1.03 during the freeze-drying process to obtain the freeze-dried powder was significantly suppressed, effectively improving the drug quality stability and clinical drug safety of the freeze-dried powder injection with the compound of formula (I) as the API component. The function of these conventional freeze-drying excipients in this field is to give the freeze-dried powder injection a specific shape, but the technical effects they present in the present invention are completely unrelated to their conventional functions.

[0012] In the above technical solution, the pH adjuster is selected from NaOH, KOH, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or a combination thereof.

[0013] In the above technical solution, the solid content of the freeze-dried powder injection in the solution before freeze-drying is 2-12% (w / v), preferably 4-8% (w / v).

[0014] The term "solid content" refers to the percentage of the weight of a solid substance (e.g., the active compound of the present invention and all excipients used, weight / gram) added to a solvent (e.g., water for injection) and dissolved to obtain a solution (weight / volume percentage, e.g., g / 100ml).

[0015] The moisture content of the freeze-dried powder injection prepared by the above technical solution does not exceed 3%. The moisture content refers to the moisture content of the freeze-dried powder obtained through the freeze-drying process, and the moisture content of the freeze-dried powder injection during the storage period does not exceed 5%.

[0016] In the present invention, the symbol % can have a meaning that is easily understood by those skilled in the art, depending on the context in which it is used. For example, when referring to solid content, the symbol represents the percentage of weight / volume (w / v, such as g / 100ml); for another example, when referring to the "water content" in a lyophilized powder injection, for example, when the water content is below 3%, the symbol % represents the percentage of weight / weight (w / w, g / 100g). Generally speaking, when a solid is dispersed in a liquid, % represents the weight / volume percentage; when a solid is dispersed in a solid or a liquid is dispersed in a solid (such as the water content of a lyophilized powder injection), % represents the weight / weight percentage. In other cases, unless otherwise specified, the symbol % represents the weight / weight percentage.

[0017] In the above technical solution, the lyophilization excipient is sucrose, and the weight ratio of the compound of formula (I) to the lyophilization excipient is 1:(0.75-4), preferably 1:(1-4), and more preferably 1:4.

[0018] The inventors have discovered through extensive experiments that, when preparing a lyophilized powder injection with the compound of formula (I) as the API component, introducing sucrose as a lyophilization excipient and regulating the dosage ratio of the compound of formula (I) and sucrose can more effectively inhibit the growth of the impurity RRT1.03 during the freeze-drying process to obtain the lyophilized powder. The content of the impurity RRT1.03 in the prepared lyophilized powder injection does not exceed 1%, which is significantly lower than the impurity RRT1.03 content in the raw material of the compound of formula (I). This ensures the drug quality stability and clinical drug safety of the lyophilized powder injection with the compound of formula (I) as the API component, and the prepared lyophilized powder injection is more fluffy.

[0019] After a large number of experimental verifications, the inventors further found that when sucrose is used as a lyophilization excipient, the pH value of the drug solution before freeze-drying is adjusted to 5.5-6.7, which will further inhibit the growth of the impurity RRT1.03 during the freeze-drying process to obtain the lyophilized powder. The content of the impurity RRT1.03 in the prepared lyophilized powder injection can be reduced to as low as 0.32%, which is much lower than the impurity RRT1.03 content in the raw material of the compound of formula (I). Moreover, after storage for 6 months at 25°C±2°C and 60%±5%RH, the content of the impurity RRT1.03 is basically stable at a relatively low value, which greatly improves the clinical drug safety of the lyophilized powder injection with the compound of formula (I) as the API component.

[0020] The lyophilized powder injection prepared by the above technical scheme is dissolved in water for injection to form a solution with a concentration of 10 mg / ml of the compound of formula (I). The pH value is measured according to the method 0631 of the general rules of the fourth part of the 2020 edition of the Chinese Pharmacopoeia as 5.3-6.7.

[0021] In the above technical solution, the moisture content of the freeze-dried powder injection does not exceed 2%. The moisture content here also refers to the moisture content of the freeze-dried powder obtained through the freeze-drying process.

[0022] Another object of the present invention is to provide a method for preparing the above-mentioned fluorescence-magnetic resonance dual-modality contrast agent, comprising the following steps:

[0023] (a) Weigh the prescribed amount of excipients and place them in a preparation tank. Add an appropriate amount of water for injection and stir to dissolve.

[0024] (b) adding a prescribed amount of the compound of formula (I) to a preparation tank and stirring to dissolve it;

[0025] (c) Add water for injection to the preparation tank to the prescribed weight, stir evenly, measure the pH of the solution, and adjust the pH to 5-7 with a pH adjuster if necessary;

[0026] (d) Sterilize the liquid medicine, fill it into a vial according to the prescribed amount, freeze-dry it to remove moisture, and stopper it.

[0027] Preferably, the weight ratio of the compound of formula (I) to the excipient is 1:(0.75-4.5). Exemplarily, the lyophilized excipient is sucrose, and the weight ratio of the compound of formula (I) to the lyophilized excipient in the lyophilized powder injection is 1:(0.75-4), preferably 1:(1-4), and more preferably 1:4.

[0028] Preferably, the lyophilization excipient is a carbohydrate compound and / or a sugar alcohol compound; the carbohydrate compound is selected from one or more of dextran, anhydrous glucose, lactose, sucrose, trehalose and maltose, preferably lactose, sucrose, trehalose, and more preferably sucrose; the sugar alcohol compound is selected from one or more of sorbitol, mannitol and erythritol.

[0029] Preferably, the pH adjuster is selected from NaOH, KOH, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or a combination thereof; further preferably, the pH adjuster is a NaOH aqueous solution, and the concentration of the NaOH aqueous solution is 0.002 to 0.05 mol / L, preferably 0.005 to 0.02 mol / L.

[0030] When NaOH aqueous solution is used to adjust the pH value of the pre-freeze-drying drug solution, if the pH value is out of range, it is preferably adjusted with hydrochloric acid solution.

[0031] Preferably, the fluorescence-magnetic resonance dual-modality contrast agent is a lyophilized powder injection.

[0032] Preferably, the solid content of the freeze-dried powder injection in the solution before freeze-drying is 2-12% (w / v), preferably 4-8% (w / v).

[0033] Preferably, the moisture content of the freeze-dried powder injection does not exceed 3%, and further preferably, the moisture content of the freeze-dried powder injection does not exceed 2%.

[0034] Preferably, the lyophilized powder injection is dissolved in water for injection to form a solution with a concentration of 10 mg / ml of the compound of formula (I), and its pH value is determined to be 5.3-6.7 according to the method 0631 of the General Rules of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0035] Preferably, in step (d), the freeze-drying to remove moisture includes pre-freezing the sterilized and filled medicinal solution below -40°C, maintaining the temperature for 0.5 to 4 hours when the medicinal solution temperature reaches below -40°C; heating it to above -20°C within 1 to 4 hours, maintaining it for 5 to 40 hours, and maintaining the vacuum degree <0.5 mbar; heating it to above 10°C within 1 to 6 hours, maintaining it for 1 to 20 hours, and then heating it to above 40°C within 0.5 to 5 hours, maintaining it for 2 to 10 hours, pressing the stopper, locking the aluminum cover to seal, and packaging.

[0036] Preferably, in step (d), the freeze-drying to remove moisture includes pre-freezing the sterilized and filled medicinal solution at -45°C, maintaining the temperature for 1 to 4 hours when the medicinal solution temperature reaches -45°C; heating it to -15°C within 1 to 4 hours, maintaining it for 10 to 40 hours, and maintaining the vacuum degree <0.5mbar; heating it to 20°C within 1 to 6 hours, maintaining it for 1 to 20 hours, and then heating it to above 40°C within 1 to 5 hours, maintaining it for 5 to 10 hours, pressing the stopper, locking the aluminum cover to seal, and packaging.

[0037] Beneficial effects of the present invention:

[0038] (1) The present invention converts the less stable aqueous injection containing the compound of formula (I) as the API component into a lyophilized powder injection, thereby avoiding the technical defect that the long-term stability of the aqueous injection containing the compound of formula (I) as the API component is poor, resulting in a weakened or even ineffective fluorescent tracing effect, thereby ensuring the fluorescence-magnetic resonance dual-modality imaging efficacy of the API component.

[0039] (2) The present invention selects a specific lyophilized excipient and regulates the dosage ratio of the lyophilized excipient and the API component and the pH value of the system, thereby not only avoiding the risk of an increase in the impurity RRT1.03 content caused by the freeze-drying process to obtain the lyophilized powder, but also giving the lyophilized powder injection a lower impurity RRT1.03 content. The prepared lyophilized powder has a full and uniform appearance, a low moisture content, and a high drug stability. When placed at 25°C±2°C / 60%±5%RH for 6 months or at 40°C / 75%RH for 30 days, the impurity RRT1.03 content in the lyophilized powder injection is basically maintained at the same level, with only occasional slight fluctuations.

[0040] (3) The lyophilized powder injection provided by the present invention can effectively solve the problem of significant increase of impurity RRT1.03 during the freeze-drying process to obtain lyophilized powder and the problem of poor stability of the product after lyophilization. In addition, the lyophilized powder injection not only has a short reconstitution time, but also has a very small amount of insoluble particles after reconstitution, which greatly improves the safety of clinical drug use and further promotes the development of subsequent clinical application research of the compound of formula (I). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1This is the HPLC spectrum of the impurity RRT1.03 content in the lyophilized powder injection prepared in Example 13.

[0042] Figure 2 This is the HPLC spectrum of the impurity RRT1.03 content of the lyophilized powder injection prepared in Example 13 after being stored for 12 months.

[0043] Figure 3 This is the HPLC spectrum of the impurity RRT1.03 content in the lyophilized powder injection prepared in Example 18. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below by specific embodiments. It should be understood by those skilled in the art that the embodiments are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] In the following examples, except for the components of the compound of formula (I) used, which were prepared by the applicant according to the synthesis method described in WO2022242437A1, the remaining raw materials and reagents are commercially available and can be purchased through commercial channels. Specific techniques or conditions not specified in the examples were carried out in accordance with conventional techniques in the art or in accordance with the product instructions.

[0046] Example 1

[0047] Prescription composition (1000 tubes): 40g of compound of formula (I), 40g of mannitol;

[0048] Preparation method: a) dissolve mannitol in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 5.50 with a 0.01 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 1 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 1 ml / vial, half-press the butyl rubber stopper, and send it into a freeze dryer for freeze drying: the filled solution is pre-frozen at -50°C, and when the solution temperature reaches -50°C, it is maintained for 2 hours; within 2.5 hours, it is heated to -10°C and maintained for 35 hours, with a vacuum degree of <0.5 mbar; then, within 4 hours, it is heated to 20°C and maintained for 3 hours, and then within 2 hours, it is heated to 40°C and maintained for 5 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0049] Example 2

[0050] Prescription composition (1000 tubes): 40g of compound of formula (I), 80g of mannitol;

[0051] Preparation method: a) dissolve mannitol in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 5.50 with a 0.01 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 1 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 1 ml / vial, half-press the butyl rubber stopper, and send it to a freeze dryer for freeze drying: the filled solution is pre-frozen at -50°C, and when the solution temperature reaches -50°C, it is maintained for 2 hours; the temperature is raised to -10°C within 2.5 hours and maintained for 35 hours, with a vacuum degree of <0.5 mbar; the temperature is then raised to 20°C within 4 hours and maintained for 3 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 6 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0052] Example 3

[0053] Prescription composition (1000 tubes): 40g of compound of formula (I), 80g of trehalose;

[0054] Preparation method: a) dissolve trehalose in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 5.50 with a 0.01 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 1 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 1 ml / vial, half-press the butyl rubber stopper, and send it to a freeze dryer for freeze drying: the filled solution is pre-frozen at -40°C, and when the solution temperature reaches -40°C, it is maintained for 3 hours; the temperature is raised to -15°C within 2 hours and maintained for 40 hours, and the vacuum degree is less than 0.5 mbar; then the temperature is raised to 20°C within 4 hours and maintained for 3 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 6 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0055] Example 4

[0056] Prescription composition (1000 tubes): 40g of compound of formula (I), 40g of lactose;

[0057] Preparation method: a) dissolve lactose in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 5.50 with a 0.005 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it into a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 3 hours; the temperature is raised to -15°C within 2 hours and maintained for 30 hours, and the vacuum degree is less than 0.3 mbar; then the temperature is raised to 20°C within 4 hours and maintained for 4 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 8 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0058] Example 5

[0059] Prescription composition (1000 tubes): 40g of compound of formula (I), 40g of sucrose;

[0060] Preparation method: a) dissolve sucrose in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 5.50 with a 0.005 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it to a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 2.5 hours; then the temperature is raised to -15°C within 2 hours and maintained for 30 hours, with a vacuum degree of <0.5 mbar; then the temperature is raised to 20°C within 4.5 hours and maintained for 4 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 8 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0061] Example 6

[0062] Prescription composition (1000 tubes): 40g of compound of formula (I), 80g of sucrose;

[0063] The preparation method is the same as Example 5.

[0064] Example 7

[0065] Prescription composition (1000 tubes): 40g of compound of formula (I), 120g of sucrose;

[0066] The preparation method is the same as Example 5.

[0067] Example 8

[0068] Prescription composition (1000 tubes): 40g of compound of formula (I), 160g of sucrose;

[0069] The preparation method is the same as Example 5.

[0070] Example 9

[0071] Prescription composition (1000 tubes): 40g of compound of formula (I), 180g of sucrose;

[0072] The preparation method is the same as Example 5.

[0073] Examples 10 to 16

[0074] The prescription composition (1000 tubes) is: 40g of the compound of formula (I), 160g of sucrose;

[0075] The preparation method is the same as that of Example 8, except that the pH value of the drug solution in Example 10 is adjusted to 5.00 before freeze-drying, the pH value of the drug solution in Example 11 is adjusted to 5.3, the pH value of the drug solution in Example 12 is adjusted to 6.00, the pH value of the drug solution in Example 13 is adjusted to 6.50, the pH value of the drug solution in Example 14 is adjusted to 7.00, the pH value of the drug solution in Example 15 is adjusted to 6.70, and the pH value of the drug solution in Example 16 is adjusted to 7.20.

[0076] Example 17

[0077] Prescription composition (1000 tubes): 40g of compound of formula (I), 120g of sucrose, 40g of mannitol;

[0078] Preparation method: a) dissolve sucrose and mannitol in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 6.50 with a 0.05 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it to a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 2.5 hours; then the temperature is raised to -15°C within 2 hours and maintained for 30 hours, with a vacuum degree of <0.5 mbar; then the temperature is raised to 20°C within 4 hours and maintained for 4 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 8 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0079] Example 18

[0080] Prescription composition (1000 vials): 40g of the compound of formula (I), NaOH solution and water for injection;

[0081] Preparation method: a) dissolve the compound of formula (I) in an appropriate amount of water for injection, stir to dissolve, and adjust the pH value of the solution to 6.50 with a 0.05 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it into a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 2.5 hours; the temperature is raised to -15°C within 2 hours and maintained for 30 hours, and the vacuum degree is less than 0.5 mbar; then the temperature is raised to 20°C within 4.5 hours and maintained for 4 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 8 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0082] Example 19

[0083] Prescription composition (1000 tubes): 40g of compound of formula (I), 160g of sucrose;

[0084] Preparation method: a) dissolve sucrose in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 6.50 with a 0.05 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it to a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 2.5 hours; then the temperature is raised to -15°C within 2 hours and maintained for 30 hours, with a vacuum degree of <0.5 mbar; then the temperature is raised to 20°C within 4.5 hours and maintained for 3 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 4 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0085] Example 20

[0086] Prescription composition (1000 tubes): 40g of compound of formula (I), 30g of sucrose;

[0087] Preparation method: a) dissolve sucrose in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), stir to dissolve, and adjust the pH value of the solution to 6.50 with a 0.05 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it into a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 2.5 hours; then the temperature is raised to -15°C within 2 hours and maintained for 30 hours, with a vacuum degree of <0.5 mbar; then the temperature is raised to 20°C within 4.5 hours and maintained for 4 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 8 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0088] Example 21

[0089] Prescription composition (1000 tubes): 40g of compound of formula (I), 25g of sucrose;

[0090] Preparation method: a) dissolve sucrose in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 6.50 with a 0.05 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it to a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 2.5 hours; then the temperature is raised to -15°C within 2 hours and maintained for 30 hours, with a vacuum degree of <0.5 mbar; then the temperature is raised to 20°C within 4.5 hours and maintained for 4 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 8 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0091] Example 22

[0092] Prescription composition (1000 tubes): 40g of compound of formula (I), 20g of sucrose;

[0093] Preparation method: a) dissolve sucrose in an appropriate amount of water for injection, stir to dissolve, then add the compound of formula (I), continue stirring to dissolve, and adjust the pH value of the solution to 6.50 with a 0.05 mol / L NaOH aqueous solution, and then make up the balance with water for injection to a total volume of 4 L of the solution; after filtering and sterilizing the solution, fill it into a vial at a dose of 4 ml / vial, half-press the butyl rubber stopper, and send it to a freeze dryer for freeze drying: the filled solution is pre-frozen at -45°C, and when the solution temperature reaches -45°C, it is maintained for 2.5 hours; then the temperature is raised to -15°C within 2 hours and maintained for 30 hours, with a vacuum degree of <0.5 mbar; then the temperature is raised to 20°C within 4.5 hours and maintained for 4 hours, and then the temperature is raised to 40°C within 2 hours and maintained for 8 hours, the stopper is pressed, the aluminum cap is locked, and the package is packaged.

[0094] Examples 23 to 28

[0095] The prescription composition (1000 tubes) is: 40g of the compound of formula (I), 160g of sucrose;

[0096] The preparation method is the same as that of Example 13, except that the concentration of the pH adjuster used is different. In Example 23, a 0.01 mol / L NaOH aqueous solution is used to adjust the pH value of the solution to 6.50. In Example 24, a 0.02 mol / L NaOH aqueous solution is used to adjust the pH value of the solution to 6.50. In Example 25, a 0.025 mol / L NaOH solution is used to adjust the pH value of the solution to 6.50. In Example 26, a 0.03 mol / L NaOH solution is used to adjust the pH value of the solution to 6.50. In Example 27, a 0.1 mol / L NaOH solution is used to adjust the pH value of the solution to 6.50. In Example 28, a 0.15 mol / L NaOH solution is used to adjust the pH value of the solution to 6.50.

[0097] Detection and analysis methods:

[0098] (1) Quality testing of freeze-dried powder injection

[0099] For each batch of freeze-dried powder injection obtained in Examples 1 to 22, their quality / property indicators such as moisture content, API content, pH value and impurity RRT1.03 content were tested according to the following methods.

[0100] 1) Determination of API content: One vial of each test sample was taken and analyzed by HPLC using the following method: flow rate 0.9 ml / min, injection volume 10 μl, detection wavelength 230 nm, chromatographic column Phenomenex Gemini NX-C18 4.6 × 150 mm × 3 μm, 35 mM ammonium acetate as mobile phase A, methanol:acetonitrile (3:7) as mobile phase B, and mobile phase gradient as follows:

[0101]

[0102] The test results of the corresponding products in each embodiment are shown in Table 1.

[0103] 2) Determination of the content of impurity RRT1.03: Take one vial of each test sample and perform HPLC detection using the following method: flow rate 0.8 mL / min, injection volume 10 μL, detection wavelength 254 nm, chromatographic column Xtimate C18 Welch 250 × 4.6 mm × 3 μm, eluent A is 120 mM ammonium acetate + 5 mM citric acid solution (pH 6.0), eluent B is acetonitrile, diluent is methanol, and the mobile phase gradient is as follows:

[0104]

[0105] Using the above method, the RRT1.03 impurity content in the homemade compound of formula (I) used in the examples was determined to be 1.80%. The test results of the corresponding products in each example are detailed in Table 1.

[0106] 3) pH value: Take one sample each, prepare a solution of the compound of formula (I) with water at a concentration of 40 mg / ml for Examples 1 to 3, and prepare a solution of the compound of formula (I) with water at a concentration of 10 mg / ml for Examples 4 to 22. The pH value was determined according to the method 0631 of the General Rules of Part IV of the Chinese Pharmacopoeia 2020. The test results are detailed in Table 1.

[0107] 4) Moisture content: Take an appropriate amount of the sample and accurately weigh it. Then dry it at 105°C to a constant weight. Calculate the moisture content (%) of the sample based on the weight loss. See Table 1 for detailed test results.

[0108] 5) Insoluble particles: Take one vial of each test sample and test according to the insoluble particle examination method (second method, microscopic counting method) in the 2020 edition of the Chinese Pharmacopoeia, Part III, General Chapter 0903.

[0109] (2) Accelerated experiment

[0110] The lyophilized powder injections prepared in Examples 1-22 were placed in an accelerated test chamber. Samples were taken at regular intervals and the RRT1.03 impurity content was determined by HPLC. The accelerated test parameters were: temperature 40±2°C, humidity 75%±5%, and durations of 0, 5, 10, and 30 days. The experimental results are shown in Table 1.

[0111] (3) Long-term stability test

[0112] The lyophilized powder injections prepared in Examples 1-22 were placed in a long-term sample storage chamber. Samples were taken at regular intervals and assayed for the RRT1.03 impurity content by HPLC. The temperature of the sample storage chamber was 25±2°C, and the humidity was RH 60%±10%. The observation periods were 0, 1, 2, 3, 6, and 12 months. The experimental results are shown in Table 1.

[0113] Table 1

[0114]

[0115]

[0116] It is obvious from the experimental data in Table 1 that the impurity RRT1.03 content of the lyophilized powder injection with the addition of lyophilized excipients is significantly lower than the impurity RRT1.03 content in the lyophilized powder injection without lyophilized excipients, whether it is the impurity RRT1.03 content after the lyophilization process or the impurity RRT1.03 content after the accelerated test or the long-term stability test (Example 18); the impurity RRT1.03 content in the lyophilized powder injection prepared by using sucrose as the lyophilization excipient is significantly lower than the impurity RRT1.03 content in the lyophilized powder injection using other excipients as lyophilization excipients, and it is found that as the period of the accelerated test and the long-term stability test is extended, the impurity RRT1.03 content in the lyophilized powder injection has only slight fluctuations, and finally remains in a stable state at a low content.

[0117] The foaming conditions of the lyophilized powder injections prepared in Examples 1 to 22 after reconstitution with water for injection and the test results of insoluble particles in the reconstituted solution are shown in Tables 2 and 3.

[0118] Table 2

[0119]

[0120] Table 3

[0121]

[0122] The present inventors also unexpectedly discovered that when sucrose is selected as the excipient, the concentration of the pH regulator - NaOH aqueous solution has a significant effect on the resolubility of the prepared lyophilized powder injection, as shown in Table 4.

[0123] Table 4

[0124]

Claims

1. A fluorescence-magnetic resonance dual-modality contrast agent, characterized in that: The dual-modality contrast agent is a lyophilized powder injection, which comprises the following formula (I) compound: and lyophilization excipients and optionally a pH adjuster; The lyophilization excipient is sucrose; The weight ratio of the compound of formula (I) to the lyophilized excipient contained in the lyophilized powder injection is 1:(1-4); The lyophilized powder injection is dissolved in water for injection to form a solution with a concentration of 10 to 40 mg / ml of the compound of formula (I) and a pH value of 5.0 to 7.

0.

2. The fluorescence-magnetic resonance dual-modality contrast agent according to claim 1, characterized in that: The pH adjuster is selected from NaOH, KOH, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or a combination thereof.

3. The fluorescence-magnetic resonance dual-modality contrast agent according to claim 1, characterized in that The lyophilized excipient is sucrose, and the weight ratio of the compound of formula (I) to the lyophilized excipient in the lyophilized powder injection is 1:

4.

4. The fluorescence-magnetic resonance dual-modality contrast agent according to claim 1, characterized in that The lyophilized powder injection is dissolved in water for injection to form a solution with a concentration of 10 mg / ml of the compound of formula (I), and its pH value is measured according to the method 0631 of the general rules of the fourth volume of the Chinese Pharmacopoeia 2020 edition, which is 5.3-6.

7.

5. The fluorescence-magnetic resonance dual-modality contrast agent according to claim 1 or 4, characterized in that: The pH regulator is a NaOH aqueous solution, and the concentration of the NaOH aqueous solution is 0.005-0.02 mol / L.

6. A method for preparing the fluorescence-magnetic resonance dual-modality contrast agent according to any one of claims 1 to 5, characterized in that: The steps include: (a) Weigh the prescribed amount of excipients and place them in a preparation tank. Add an appropriate amount of water for injection and stir to dissolve. (b) adding a prescribed amount of the compound of formula (I) to a preparation tank and stirring to dissolve it; (c) Add water for injection to the preparation tank to the prescribed weight, stir evenly, measure the pH of the solution, and adjust the pH to 5-7 with a pH adjuster if necessary; (d) Sterilize the liquid medicine, fill it into a vial according to the prescribed amount, freeze-dry it to remove moisture, and stopper it.

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