KLRLEWNR nanoprobe kit and preparation method thereof

Abstract

The invention discloses a KLRLEWNR nano-probe medicine box and a preparation method thereof, the KLRLEWNR nano-probe medicine box comprises a plurality of single-dose KLRLEWNR nano-probe medicaments, and each 1g of the KLRLEWNR nano-probe medicaments comprise the following components: 50 to 150 [mu] g of KLRLEWNR, 2 to 5 mg of TPPTS, 3 to 5 mg of Tricine, 60 to 100 mg of excipient, and 0.01 to 1.00 mg of surfactant. The pH is adjusted to 6.2-6.8 through a pH buffer salt system; and the balance of water. The KLRLEWNR nanoprobe kit disclosed by the invention has good stability and marking rate, can meet the requirements of clinical application, and provides more effective support for disease diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoprobe medicine boxes, and in particular to a KLRLEWNR nanoprobe medicine box and a preparation method thereof. Background Art

[0002] As a new diagnostic and therapeutic tool, nanoprobes have great potential in the precise detection and targeted treatment of diseases. They can specifically identify diseased cells or tissues, achieve early diagnosis and precise treatment of diseases, thereby improving treatment effects and reducing side effects. However, there are many problems with the nanoprobe kits currently on the market, which limits their widespread clinical application.

[0003] In terms of stability, the existing nanoprobe kits are not stable enough and are easily affected by various factors during production, storage and application, resulting in unstable performance. For example, changes in temperature, humidity, light and other conditions may cause the drug components in the nanoprobe kit to degrade, denature or aggregate, thereby affecting its efficacy and safety.

[0004] In terms of labeling rate, the labeling rate of existing products is not high, which is difficult to meet the clinical needs for disease diagnosis and treatment. The low labeling rate means that the efficiency of the combination of nanoprobes and radionuclides is low, and the lesion site cannot be accurately located and detected, thus affecting the accuracy of diagnosis and the effect of treatment.

[0005] In terms of specificity, existing nanoprobe kits are not very specific and may bind to non-target tissues or cells non-specifically, leading to misdiagnosis or mistreatment. In addition, non-specific binding may also cause side effects, posing a potential threat to the patient's health.

[0006] The specific impacts of these deficiencies on clinical applications include: reduced diagnostic accuracy, which may lead to missed or misdiagnosis of the disease; unsatisfactory treatment effects, inability to effectively treat the lesion site, affecting the patient's recovery; increased side effects, causing unnecessary pain and risks to patients.

[0007] Therefore, developing a nanoprobe kit with good stability, high labeling rate and specificity has important clinical significance and application value.

[0008] KLRLEWNR (H10) has a structure of D-lysine-D-leucine-D-arginine-D-leucine-D-glutamic acid-D-tryptophan-D-asparagine-D-arginine. It is a new type of peptide nanoprobe that can specifically bind to the tumor marker human epidermal growth factor receptor 2 (HER2). The structural formula is as follows:

[0009]

[0010] HER2 stands for human epidermal growth factor receptor 2. It is a transmembrane protein receptor with tyrosine kinase activity that plays a role in the growth and differentiation of normal cells. However, when the HER2 gene is amplified or overexpressed, it can lead to uncontrolled cell growth and division, thereby promoting the occurrence and development of tumors. HER2 positivity is common in many cancers, especially breast cancer and gastric cancer. In breast cancer, about 15%-20% of patients are HER2-positive. HER2-positive breast cancer tends to be more invasive and metastatic, but it also provides a target for targeted treatment.

[0011] After research, it is found that there is no nanoprobe kit suitable for KLRLEWNR and can be directly used in the prior art. Summary of the invention

[0012] The purpose of the present invention is to provide a KLRLEWNR nanoprobe kit and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art and meet the needs of clinical application.

[0013] To achieve the above purpose, the technical solution provided by the present invention is:

[0014] A KLRLEWNR nanoprobe medicine kit includes a plurality of single-dose KLRLEWNR nanoprobe medicines. The prescription composition of the KLRLEWNR nanoprobe medicine is calculated per 1g, and the ingredients contained are as follows:

[0015] 50-150 μg KLRLEWNR, 2-5 mg TPPTS, 3-5 mg Tricine, 60-100 mg excipient, 0.01-1.00 mg surfactant; pH is adjusted to 6.2-6.8 by a pH buffer salt system; the balance is water.

[0016] To optimize the above technical solutions, the specific measures taken also include:

[0017] In the KLRLEWNR nanoprobe kit, the dosage of a single dose of the KLRLEWNR nanoprobe agent is 0.5 to 5 g.

[0018] Furthermore, the pH buffer salt system is a succinic acid solution system or a sodium hydroxide solution system.

[0019] Furthermore, the surfactant is polysorbate 80.

[0020] Furthermore, the excipient is trehalose.

[0021] The present invention also provides a method for preparing a KLRLEWNR nanoprobe kit, comprising the following steps:

[0022] Take a portion of the injection water for a single dose of KLRLEWNR nanoprobe agent, stir and introduce protective gas, add excipients, TPPTS and Tricine, stir and dissolve;

[0023] Stop the protective gas, add surfactant, and stir to dissolve;

[0024] Determine the pH value of the solution and adjust the pH value with a pH buffer salt system;

[0025] Add water for injection to make up to a single dose of KLRLEWNR nanoprobe agent;

[0026] Sterile filtration;

[0027] Filling;

[0028] Freeze drying.

[0029] Furthermore, the partial water for injection of a single dose of the KLRLEWNR nanoprobe medicament is 60% to 80% of the water for injection of a single dose of the KLRLEWNR nanoprobe medicament.

[0030] Furthermore, the protective gas is nitrogen.

[0031] Preferably, the pH value is adjusted by using a pH buffer salt system by using a 0.05-0.2 mol / L sodium hydroxide solution or a 0.05-0.2 mol / L succinic acid solution.

[0032] Furthermore, the freeze-drying step is as follows:

[0033] The product enters the freeze-drying chamber;

[0034] Freeze to below -40±2℃ and keep warm for 2 hours, turn on the vacuum pump to evacuate the front box to 30±5Pa, and heat and sublimate according to the following curve: set the heat transfer oil to heat up to -5℃ in 90 minutes and keep it for 8 hours; set the heat transfer oil to heat up to 25℃ in 1 hour and keep it for 5 hours;

[0035] Return nitrogen to the freeze-drying box before use; seal the lid after taking out of the box; and package.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention combines the problems existing in the current nanoprobe kit and the fact that there is no probe kit that can be directly applied to KLRLEWNR in the prior art, and develops a KLRLEWNR nanoprobe kit.

[0038] In view of the characteristics of KLRLEWNR, the present invention studies and designs the main components and dosages of the KLRLEWNR nanoprobe kit:

[0039] With KLRLEWNR as the core ligand, TPPTS and Tricine as the key ligands, and through the selection and coordination of excipients, surfactants and pH buffer salt systems, we finally obtained a KLRLEWNR nanoprobe preparation product with stable performance, high labeling rate, effective improvement of KLRLEWNR adsorption, good buffering capacity, good formability and good clarity.

[0040] The KLRLEWNR nanoprobe kit of the present invention has good stability and labeling rate, can meet the needs of clinical applications, fills the technical gap in the prior art that there is no nanoprobe kit suitable for KLRLEWNR and can be directly applied, and provides more effective support for disease diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : High performance liquid chromatogram of the KLRLEWNR nanoprobe agent prepared in Example 8.

[0042] Figure 2 : High performance liquid chromatogram of the KLRLEWNR nanoprobe agent prepared in Example 9.

[0043] Figure 3 : Example 9 The intermediate drug solution is heated and cooled at 1°C / min.

[0044] Figure 4 : The intermediate of Example 9 was heated and cooled at 10°C / min.

[0045] Figure 5 : Freeze-drying curve diagram of Example 9. DETAILED DESCRIPTION

[0046] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0047] The present invention provides a KLRLEWNR nanoprobe medicine kit, comprising a plurality of single-dose KLRLEWNR nanoprobe medicines.

[0048] In the KLRLEWNR nanoprobe kit of the present invention, the specific amount of a single dose of the KLRLEWNR nanoprobe agent can be set as required. Preferably, the single dose can be 50 to 150 μg.

[0049] For the convenience of explanation and demonstration of the experiment, a single dose of 1 g of the KLRLEWNR nanoprobe agent is specifically described below, but the scope of the embodiments of the present invention is not limited thereto.

[0050] Example 1 Study on the type and dosage of surfactant

[0051] KLRLEWNR has high activity and certain surface activity, which means it can be adsorbed on the surface of objects it contacts. If adsorption occurs during production, storage or finished product application, the biological activity of the product will be reduced or even completely lost.

[0052] The invention adopts polysorbate 80 as a surfactant, which can improve the adsorption of KLRLEWNR and protect the protein from induced denaturation. Polysorbate 80 has been approved for use in injection.

[0053] The invention investigates the dosage of polysorbate 80 in KLRLEWNR preparation products and studies the influence on the adsorption of KLRLEWNR.

[0054] Specifically, the specific amount of polysorbate 80 is investigated according to Table 1.

[0055] Table 1 Investigation of the dosage of polysorbate 80

[0056]

[0057]

[0058] The effect of polysorbate 80 on the adsorption of KLRLEWNR is shown in Table 2.

[0059] Table 2 Effect of polysorbate 80 dosage on adsorption

[0060]

[0061]

[0062] According to the test results, the amount of polysorbate 80 used in the present invention has no obvious effect on the stability of the product, and the amount of polysorbate does not affect the product labeling rate; when the amount of polysorbate 80 is 0.005%, KLRLEWNR in the sample can prevent the product from being adsorbed. According to the principle of using as little excipient as possible, the amount of polysorbate 80 in this product can be 0.05 mg / tube.

[0063] Example 2 Study on buffer salt system

[0064] 99m When Tc-labeled coupled KLRLEWNR is used, a certain pH environment is required. According to the characteristics of KLRLEWNR, the present invention sets the following conditions for the selection of the pH buffer system:

[0065] It can maintain good buffering capacity after freeze-drying and reconstitution, and can remove volatile substances;

[0066] Buffer reagents do not participate 99m The reduction and complexation reaction of Tc can exclude the citrate buffer system;

[0067] The buffer reagents have no apparent toxicity.

[0068] In this example, various buffer solutions were used as the buffer salt system of the present invention, including citrate buffer, HEPES buffer and succinate system. The research process of the types and dosages of buffer salts is shown in Table 3.

[0069] Among them, after research, the pH range of citrate buffer is very suitable for radionuclides. 99m Tc-labeled buffer systems are also commonly used in clinical drugs. However, it is worth noting that citric acid itself can act as a bifunctional chelating agent to bind to the reduced valence state. 99m Tc is complexed and is widely used as a complexing agent for nuclides. 99m Tc is used in the labeling. 99m Tc-citrate ( 99m Tc-cirtric) is very simple to label and can be used as a renal imaging agent and an inflammatory lesion imaging agent. It will bring unexpected errors to this product, so citric acid is not suitable as a buffer for this kit.

[0070] Table 3 Investigation of buffer salt system

[0071]

[0072]

[0073] The test results show that the samples prepared with HEPES buffer solution showed opalescence after the freeze-dried product was reconstituted. When using the succinic acid buffer salt system, there was no significant difference in the related substances of the product on day 0 compared with the control group. The buffer salt system of this product prescription uses succinic acid and sodium hydroxide system.

[0074] Example 3 Study on the Type and Dosage of Excipients

[0075] The basic formula composition of freeze-dried powder injection (solution type) generally includes drugs and solvents. The solvent is usually selected according to the properties of the drug. The most common ones are water for injection or a composite solvent system of organic solvent and water. In addition, for small doses of drugs with low solid content, a certain amount of skeleton support agents such as mannitol need to be added.

[0076] The solid content refers to the content of all solids in the drug, including drugs, skeleton supports and additives. If the solid content in the drug solution is too low, the freeze-dried product is not easy to form, and the solid components will fly away with the escape of water vapor; if the solid content is too high, it will cause bottle cracking, bottom falling and other phenomena, and at the same time, energy consumption may increase. Because the moisture is from top to bottom during the freeze-drying process, the dried layer (upper layer) is too dense, which will make it difficult for the moisture in the lower layer to evaporate, resulting in prolonged freeze-drying time or even freeze-drying failure. Usually, a solid content in the range of 3% to 15% is more suitable for freeze-drying.

[0077] After research, it was found that the solid content of this product is relatively low, so the solution of this application is also designed to add excipients. Adding a certain amount of excipients is beneficial to the freeze-drying of the product.

[0078] Common antifreeze agents and water loss preventers in the final preparation of protein drugs include sugars, amino acids, polyols and polymers. This application selects sugars (trehalose), amino acids (glycine), and polyols (mannitol) for research, and selects one of them as the excipient of this product. The type and dosage of the excipients are shown in Table 4.

[0079] Table 4 Excipient types and dosages

[0080]

[0081] The results of the research on the types and dosages of excipients are analyzed as follows:

[0082] (1) Flocculent precipitates appeared in the solution after adding glycine, and glycine is not suitable as an excipient for this product.

[0083] (2) When mannitol was added, the drug solution showed aggregated white precipitate, and mannitol was not suitable as an excipient for this product.

[0084] (3) The solution of the drug with lactose added is slightly yellow, and the related substances have an increasing trend after preparation. Lactose is not used as an excipient for this product. (4) The solution of the drug with trehalose added is clear, and the related substances have no significant difference with the control group, so it can be used for subsequent experiments.

[0085] Therefore, the present application uses trehalose as an excipient, and the dosage of trehalose is shown in Table 5.

[0086] Table 5 Trehalose dosage investigation

[0087]

[0088]

[0089] The test results show that the dosage of trehalose has no obvious effect on the related substances of the product. When the dosage is above 60 mg, the product is formed into white loose blocks. The dosage can be selected as 60 mg to 100 mg per tube.

[0090] Example 4 Study on the dosage of core ligand KLRLEWNR

[0091] In the prescription of this product, KLRLEWNR is the key ligand of this product, and its dosage is related to the labeling rate. Therefore, this application studies the effect of the dosage of KLRLEWNR on the product, see Table 6.

[0092] Table 6 KLRLEWNR dosage investigation

[0093]

[0094]

[0095] The test results show that the dosage of KLRLEWNR in the prescription of this product can be selected as 50μg~150μg / vial, because when the dosage of KLRLEWNR is higher than 50μg / vial, it can meet the labeling rate of >90%, and when KLRLEWNR is above 50μg / vial, its labeling rate tends to be balanced.

[0096] Example 5 Study on the Dosage of the Core Ligand TPPTS

[0097] This kit needs to be used in conjunction with sodium pertechnetate; TPPTS can form a stable complex with the radioactive metal ion technetium. In radioactive diagnosis, it is usually necessary to combine the radionuclide with a suitable ligand to ensure the stability and targeting of the radionuclide in the body.

[0098] Strong complexing ability can prevent the premature dissociation of radionuclides in the body, reduce nonspecific uptake, and improve image quality and diagnostic reliability.

[0099] TPPTS has good biocompatibility and low toxicity to human tissues and organs. In radioactive diagnostic drugs, the biocompatibility of ligands is crucial because it directly affects the safety and tolerance of patients. Good biocompatibility can reduce the side effects of drugs and reduce patient discomfort and risks.

[0100] This application studies the dosage of the core ligand TPPTS, see Table 7.

[0101] Table 7 KLRLEWNR dosage investigation

[0102]

[0103] The test results show that when the dosage of KLRLEWNR and Tricine remains unchanged, when the dosage of TPPTS is between 0mg and 5mg, the radioactive labeling rate of the medicine box increases with the increase of TPPTS dosage, and when the dosage of TPPTS is ≥2mg, the labeling rate reaches more than 98%. In order to ensure that this product can be used normally during the shelf life, 2-5mg / vial can be selected.

[0104] Example 6 Study on the Dosage of the Core Ligand Tricine

[0105] This kit needs to be used in conjunction with sodium pertechnetate; Tricine has a high affinity for certain radionuclides and can form stable complexes with them. This affinity can improve the targeting of radionuclides, making them more likely to accumulate in specific tissues or organs, thereby improving the accuracy of diagnosis.

[0106] Tricine is a naturally occurring amino acid derivative with good biosafety. Its metabolic pathway in the human body is relatively clear and does not produce serious toxic side effects. High biosafety makes Tricine one of the ideal ligands in radioactive diagnostic drugs.

[0107] When the dosage of KLRLEWNR and TPPTS remained unchanged, the radioactive labeling rate of the kit increased with the increase of Tricine dosage, and when the dosage of Tricine was ≥4 mg, the labeling rate reached more than 99%.

[0108] Table 8 Investigation of the dosage of core ligand Tricine

[0109]

[0110]

[0111] Example 7 pH Study

[0112] During the preparation of injection, pH plays a decisive role in product stability. Its quality changes such as hydrolysis, oxidation, discoloration, decomposition and clarity are all related to pH. This application studies the suitable pH range of the product and examines the pH range required for the drug solution of this product.

[0113] Table 9 pH value investigation

[0114]

[0115]

[0116] The test results show that after the preparation of the drug solution, the clarity of pH 5.8 and pH 6.0 samples is slightly poor, the clarity of pH 6.2~pH 6.8 samples is good, there is no significant difference in related substances in the drug solution with different pH values ​​in the pH 6.2~pH 6.8 range, the labeling rate of pH 6.2~pH 6.8 samples is more than 99%, and there is no significant difference in related substances.

[0117] Example 8 Feeding Sequence Study

[0118] Table 10 Prescription composition of KLRLEWNR for injection

[0119]

[0120] Prescription Analysis:

[0121] (1) This product is a cold medicine box and needs to be mixed with sodium pertechnetate solution before use. KLRLEWNR, TPPTS and Tricine in this cold medicine box are core ligands, which are related to the product labeling rate.

[0122] (2) Nitrogen is a protective gas. Generally, nitrogen needs to be filled into the liquid preparation tank before all raw and auxiliary materials except water for injection are added.

[0123] (3) Initially, 60% to 80% of the total amount of injection water is generally used to dissolve the raw materials and excipients. This amount is related to the large-scale production preparation process (a portion of the water needs to be retained for rinsing weighing equipment, such as using a beaker to hold the excipients).

[0124] (4) Succinic acid and sodium hydroxide are pH regulators. If succinic acid and sodium hydroxide solids are added in the later stages of preparation, the local pH will be too low or too high, which is not conducive to product stability. Therefore, succinic acid and sodium hydroxide are added first to maintain the pH of the solution in a near-neutral range.

[0125] (5) Polysorbate 80 is a surfactant that can reduce the adsorption of the active ingredient KLRLEWNR on the surface of the device. It needs to be added before KLRLEWNR is added. Since nitrogen bubbling will cause polysorbate 80 to produce more bubbles, nitrogen bubbling can be suspended after adding polysorbate 80.

[0126] (6) Trehalose is an excipient and freeze-drying protective agent. In most cases, adding it at any time has no effect on product quality. It is usually added before the raw materials.

[0127] (7) TPPTS, Tricine, and KLRLEWNR are relatively important and are usually added later. However, KLRLEWNR has higher activity, two polypeptide chains, and more exposed amino and carboxyl groups. Therefore, it can be added last.

[0128] (8) This product is a sterile preparation. After preparation, it needs to be sterilized by filtration through a 0.22 μm pore size sterilization filter membrane. After sterilization, it is filled and half-stoppered, placed in a freeze dryer for freeze drying to remove moisture. After freeze drying, the stopper is pressed out of the box and the cap is rolled.

[0129] Example 9 Freeze-drying process study

[0130] The eutectic point of the prepared samples was determined:

[0131] The intermediate solution is heated and cooled at 1°C / min. Figure 3 As shown; the intermediate is heated and cooled at 10°C / min, as shown Figure 4 As shown. By counting the above two graphs, we get the following data:

[0132] Table 11DSC scanning results

[0133]

[0134] According to the DSC results, the freezing point of the drug solution is around -11 to -17°C; according to the DSC results of temperature rise, the eutectic point is around -2 to -3°C. From the spectrum analysis, the peak widths of the endothermic and exothermic peaks obtained by rapid temperature rise and fall are relatively large, indicating that there is a "relaxation" time for the drug solution to completely solidify during the rapid temperature drop, that is, the physical state change of the drug solution is sensitive to temperature.

[0135] The freezing temperature is set to about 20°C below the freezing point, which is about -40°C. The primary drying temperature should be lower than the eutectic point and can be set to -5°C (when estimating the time for the end of sublimation drying, it is generally roughly calculated as 1mm of thickness dried per hour. After the sublimation drying is completed, the partition temperature is usually maintained for a period of time (generally 1 to 2 hours) to completely remove the ice crystals in the product and the unevenness of the product drying, to ensure that the sublimation of the entire box of samples is completed). The product is intended to be stored in a refrigerator, and the secondary drying is set to 25°C (the amount of residual water in the product determines the temperature and time required for secondary drying. As far as preparations are concerned, it is generally believed that the moisture content of freeze-dried preparations is between 1% and 5%).

[0136] According to the above parameters, set the initial freeze-drying curve:

[0137] Freeze to below -40±2℃ and keep warm for 2 hours, turn on the vacuum pump to evacuate the front box to 30±5Pa, and heat and sublimate according to the following curve:

[0138] Set the heat transfer oil to rise to -5°C in 90 minutes and maintain for 8 hours;

[0139] Set the temperature of the heat transfer oil to 25°C in 1 hour and maintain it for 5 hours;

[0140] Return nitrogen to the freeze-drying box. Tighten the lid after unpacking. Pack.

[0141] Freeze drying curve Figure 5 As shown, after completion, the moisture content of the finished product was 1.2%.

[0142] According to the initial freeze drying, the product temperature reaches the set temperature in about 300 minutes of primary drying, which means that the product sublimation has been completed. Usually after the sublimation drying is completed, the partition temperature is usually maintained for a period of time (usually 2h to 3h) to completely remove the ice crystals in the product and the unevenness of the product drying, to ensure that the sublimation of the whole box of samples is completed. Therefore, the time for this stage is set to 300min+180min, that is, 8h.

[0143] After secondary drying for 5 hours, the moisture content is between 1% and 5%. The secondary drying of this product can be set to 25℃ for 5 hours.

[0144] The freeze-drying curve of this product can be set as follows:

[0145] Freeze to below -40±2℃ and keep warm for 2 hours, turn on the vacuum pump to evacuate the front box to 30±5Pa, and heat and sublimate according to the following curve:

[0146] Set the heat transfer oil to rise to -5°C in 90 minutes and maintain for 8 hours;

[0147] Set the temperature of the heat transfer oil to 25°C in 1 hour and maintain it for 5 hours;

[0148] Return nitrogen to the freeze-drying box. Tighten the lid after unpacking. Pack.

[0149] Example 10

[0150] A KLRLEWNR nanoprobe kit, the formula of which is as follows:

[0151] Table 12 Example 8 Kit Formula

[0152]

[0153] The preparation process of KLRLEWNR nanoprobe kit is as follows:

[0154] (1) Take 70% of the prescribed amount of water for injection, start stirring, and introduce nitrogen; add the prescribed amount to the batching tank;

[0155] Trehalose, TPPTS, Tricine, stir to dissolve, stirring time ≥ 5min;

[0156] (2) Stop nitrogen filling, add the prescribed amount of polysorbate 80 into the batching tank, stir to dissolve, and stir for ≥5 min;

[0157] (3) Determine the pH value of the solution and adjust the pH value with 0.1 mol / L sodium hydroxide solution or 0.1 mol / L succinic acid solution, with the target pH value being 6.2-6.8;

[0158] (4) Add the prescribed amount of KLRLEWNR to the batching tank and stir evenly for ≥5 min;

[0159] (5) Re-measure the pH value and adjust the pH value with 0.1 mol / L sodium hydroxide solution or 0.1 mol / L succinic acid solution, with the target pH value being 6.2-6.8;

[0160] (7) Add water to make up to 100% of the total volume, stir evenly, and obtain the intermediate solution for testing;

[0161] (8) Sterile filtration: The drug solution is filtered through two 0.22 μm filter membranes;

[0162] (9) Filling: Fill according to the labeled amount into 6 mL borosilicate glass injection vials, half-stopper, place on a plate, and send to a freeze dryer for freeze drying according to the freeze drying procedure;

[0163] (10) Freeze drying:

[0164] Products enter freeze drying chamber

[0165] Freeze to below -40±2℃ and keep warm for 2 hours, turn on the vacuum pump to evacuate the front box to 30±5Pa, and heat and sublimate according to the following curve:

[0166] Set the heat transfer oil to rise to -5°C in 90 minutes and maintain for 8 hours;

[0167] Set the temperature of the heat transfer oil to 25°C in 1 hour and maintain it for 5 hours;

[0168] Return nitrogen to the freeze-drying box; seal the lid after taking out of the box; and package.

[0169] Use a 1mL sterile syringe to draw 1mL of sodium pertechnetate diluent and add it to the medicine box. Mix well until the sample is dissolved, and heat it upright in a boiling water bath for 20 minutes. After taking it out, cool it naturally to room temperature to obtain the product. Using a high-performance liquid chromatography-radioactivity detector combined system, the detection labeling rate is 99.19%.

[0170] The stability of this product was tested, and the test data are as follows:

[0171] Table 13 Stability test

[0172]

[0173]

[0174] Embodiment 11

[0175] A KLRLEWNR nanoprobe kit, during the preparation process, strictly controls the operating conditions and parameters of each step to ensure the quality and stability of the product.

[0176] Table 14 Example 9 Kit Formula

[0177]

[0178] The preparation process of KLRLEWNR nanoprobe kit is as follows:

[0179] (1) Take 70% of the prescribed amount of water for injection, start stirring, and introduce nitrogen; add the prescribed amount to the batching tank;

[0180] Trehalose, TPPTS, Tricine, stir to dissolve, stirring time ≥ 5min;

[0181] (2) Stop nitrogen filling, add the prescribed amount of polysorbate 80 into the batching tank, stir to dissolve, and stir for ≥5 min;

[0182] (3) Determine the pH value of the solution and adjust the pH value with 0.1 mol / L sodium hydroxide solution or 0.1 mol / L succinic acid solution, with the target pH value being 6.2-6.8;

[0183] (4) Add the prescribed amount of KLRLEWNR to the batching tank and stir evenly for ≥5 min;

[0184] (5) Re-measure the pH value and adjust the pH value with 0.1 mol / L sodium hydroxide solution or 0.1 mol / L succinic acid solution. The target pH value is 6.4-6.8.

[0185] (7) Add water to make up to 100% of the total volume, stir evenly, and obtain the intermediate solution for testing;

[0186] (8) Sterile filtration: The drug solution is filtered through two 0.22 μm filter membranes;

[0187] (9) Filling: Fill according to the labeled amount into 6 mL borosilicate glass injection vials, half-stopper, place on a plate, and send to a freeze dryer for freeze drying according to the freeze drying procedure;

[0188] (10) Freeze drying:

[0189] Products enter freeze drying chamber

[0190] Freeze to below -40±2℃ and keep warm for 2 hours, turn on the vacuum pump to evacuate the front box to 30±5Pa, and heat and sublimate according to the following curve:

[0191] Set the heat transfer oil to rise to -5°C in 90 minutes and maintain for 8 hours;

[0192] Set the temperature of the heat transfer oil to 25°C in 1 hour and maintain it for 5 hours;

[0193] Return nitrogen to the freeze-drying box before use; seal the lid after taking out of the box; and package.

[0194] Use a 1mL sterile syringe to draw 1mL of sodium pertechnetate diluent and add it to the medicine box. Mix well until the sample is dissolved, and heat it upright in a boiling water bath for 20 minutes. Take it out and cool it naturally to room temperature to obtain the product. Using a high-performance liquid chromatography-radioactivity detector combined system, the detection labeling rate is 99.02%.

[0195] This product was tested for stability, and the test data are as follows:

[0196] Table 15 Stability test

[0197]

[0198]

[0199] In summary, the KLRLEWNR nanoprobe kit designed and prepared by the present invention has the following characteristics:

[0200] (1) Good stability: By designing the formula of the nanoprobe kit, including selecting appropriate surfactants, buffer salts, excipients, etc., the kit has good stability and can maintain stable performance during production, storage and application.

[0201] (2) High labeling rate: The appropriate dosage of each component is determined, such as the dosage of the core ligand KLRLEWNR, TPPTS, Tricine, and the dosage of the surfactant polysorbate 80, so that the labeling rate of the drug kit is high and can meet the needs of clinical applications. (3) Avoid adsorption problems: Selecting polysorbate 80 as a surfactant can improve the adsorption of KLRLEWNR, protect the protein from induced denaturation, and avoid the reduction or loss of biological activity of the product due to adsorption during production, storage or application. (4) Buffering capacity: After research, succinic acid and sodium hydroxide system were selected as the buffer salt system, which can maintain good buffering capacity after freeze-drying and reconstitution, and will not participate in 99m The reduction and complexation reaction of Tc can avoid unexpected errors in the drug. (5) Formability: Through the study of excipients such as sugars (trehalose), amino acids (glycine), and polyhydroxy compounds (mannitol), trehalose was determined to be a suitable excipient, which is conducive to the freeze-drying of the product. When the dosage is above 60 mg, the product can be formed into a white loose mass.

[0202] (6) Appropriate pH value: Within the pH range of the present invention, the clarity of the prepared solution is good, the labeling rate is above 99%, the product stability is good, and the product quality can be guaranteed.

[0203] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A KLRLEWNR nanoprobe kit, characterized in that: It includes several single doses of KLRLEWNR nanoprobe medicines, and the prescription composition of the KLRLEWNR nanoprobe medicine is calculated per 1g, and the ingredients contained are as follows: 50-150 μg KLRLEWNR, 2-5 mg TPPTS, 3-5 mg Tricine, 60-100 mg excipient, 0.01-1.00 mg surfactant; pH is adjusted to 6.2-6.8 by a pH buffer salt system; the balance is water.

2. The KLRLEWNR nanoprobe kit according to claim 1, characterized in that: In the KLRLEWNR nanoprobe kit, the dosage of a single dose of the KLRLEWNR nanoprobe agent is 0.5 to 5 g.

3. The KLRLEWNR nanoprobe kit according to claim 1, characterized in that: The pH buffer salt system is a succinic acid solution system or a sodium hydroxide solution system.

4. The KLRLEWNR nanoprobe kit according to claim 1, characterized in that: The surfactant is polysorbate 80.

5. The KLRLEWNR nanoprobe kit according to claim 1, characterized in that: The excipient is trehalose.

6. The method for preparing the KLRLEWNR nanoprobe kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: Take a portion of the injection water for a single dose of KLRLEWNR nanoprobe agent, stir and introduce protective gas, add excipients, TPPTS and Tricine, stir and dissolve; Stop the protective gas, add surfactant, and stir to dissolve; Determine the pH value of the solution and adjust the pH value with a pH buffer salt system; Add water for injection to make up to a single dose of KLRLEWNR nanoprobe agent; Sterile filtration; Filling; Freeze drying.

7. The method for preparing the KLRLEWNR nanoprobe kit according to claim 6, characterized in that: The partial water for injection of a single dose of the KLRLEWNR nanoprobe medicament is 60% to 80% of the water for injection of a single dose of the KLRLEWNR nanoprobe medicament; and the protective gas is nitrogen.

8. The method for preparing the KLRLEWNR nanoprobe kit according to claim 6, characterized in that: The pH value is adjusted by using a pH buffer salt system, by using a 0.05-0.2 mol / L sodium hydroxide solution or a 0.05-0.2 mol / L succinic acid solution.

9. The method for preparing the KLRLEWNR nanoprobe kit according to claim 6, characterized in that: The specific steps of freeze drying are: the product enters the freeze drying box; freezes to below -40±2°C and keeps warm for 2 hours, turns on the vacuum pump to evacuate the front box to 30±5Pa, and sublimates according to the set curve; returns nitrogen to the freeze drying front box; seals the lid after leaving the box; and packages.

10. The method for preparing the KLRLEWNR nanoprobe kit according to claim 9, characterized in that: The temperature rise sublimation curve is: the heat transfer oil is set to heat up to -5°C in 90 minutes and maintained for 8 hours; the heat transfer oil is set to heat up to 25°C in 1 hour and maintained for 5 hours.

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