A lyophilized powder base for an immunosuppressant and its use

Abstract

In order to improve the stability of immunosuppressants and solve the problem of large differences between batches of whole blood matrix and ethical problems, the application provides a freeze-dried powder matrix of immunosuppressants and its application, belonging to the field of freeze-dried preparations. The matrix is divided into a component for stabilizing immunosuppressants and a component for reducing matrix effects, the former contains cyclodextrin and citric acid-sodium citrate buffer salt, and the latter contains hemoglobin and serum albumin. It has been verified through experiments that the matrix is suitable for different types and concentrations of immunosuppressants and has universality. In addition, the matrix has small differences between batches, the components are industrial products, the cost is low, and it has great application prospect and very high clinical value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of freeze-dried preparations, in particular to a freeze-dried powder matrix of an immunosuppressant and its use. BACKGROUND

[0002] The immune system maintains the normal immune response of the human body, but the human body's immunity may be too strong to cause autoimmune diseases. Immunosuppressants are important substances for inhibiting abnormal immune responses of the body, mainly used in rejection reactions in organ transplantation and the treatment of autoimmune diseases in clinical practice, and play a very important role in the prevention and treatment of organ transplantation rejection reactions. Different immunosuppressants have different degrees of toxic side effects, and the curative effects are different. Clinically, the combined use of drugs is usually used to reduce the dose of the drug and thus reduce the toxic side effects. The therapeutic window of immunosuppressants is narrow, and the individual differences in drug kinetics are large. The efficacy and toxic side effects of the drug are closely related to the dose. When the dosage is insufficient, an immune rejection reaction may occur, and when the dosage is too large, serious toxic side effects may occur. Therefore, the drug concentration in the patient's body needs to be monitored to ensure that the use of immunosuppressants is within a safe and effective treatment range. Immunosuppressants such as tacrolimus, cyclosporine, and rapamycin have shown significant efficacy in clinical practice, but also have certain adverse reactions. At present, high-performance liquid chromatography tandem mass spectrometry (LC-MS / MS) as the gold standard in the field of drug concentration monitoring can accurately monitor the concentration of immunosuppressants in the patient's body to obtain complete treatment information and facilitate the adjustment of individual drug administration plans. When monitoring the concentration of immunosuppressants by liquid chromatography tandem mass spectrometry, stable and reliable calibration quality control substances are needed to quantify clinical monitoring samples. At present, such calibration quality control substances are mainly whole blood matrix.

[0003] Patent CN110352062A discloses a stabilizer for immunomodulatory macrolides, which is prepared by mixing substantially cell-free human whole blood hemolysate and immunosuppressants to prepare a calibration solution. The stabilizer has strong stabilizing effect on most immunosuppressants.

[0004] Patent CN111856044A discloses a whole blood type freeze-dried powder immunosuppressant quality control substance, its preparation method and application. The calibration substance is mainly a freeze-dried powder prepared by mixing human whole blood, immunosuppressants and other additives. In the freeze-dried powder, 5.71 ng / ml concentration of rapamycin has a bias of 10.03% in the 7-day accelerated experiment at 37℃. A large number of literature reports that rapamycin has poor solubility and stability, and high-concentration rapamycin has poorer stability, which cannot meet the requirements of reagent kit shelf life in clinical detection.

[0005] Patent CN115236216A discloses a kit for detecting immunosuppressants in whole blood, and a preparation method and a detection method. The calibrant is mainly a freeze-dried powder prepared by mixing animal whole blood, hemoglobin, serum albumin and other additives. In this patent, high-concentration sheep red blood cells and additives are mixed to prepare a freeze-dried powder. Various immunosuppressants can be stored at 2-8℃ for 6 months. This scheme uses phosphate buffer to dilute high-concentration sheep red blood cells to 50% sheep red blood cells as the main material, supplemented with various additives to prepare a freeze-dried powder. The high-concentration red blood cells are extremely expensive and have high transportation requirements, and the shelf life is short, resulting in low economic benefits.

[0006] Patent CN117192135A discloses a freeze-dried powder of immunosuppressants in whole blood matrix and a preparation method thereof. The calibrant is mainly a freeze-dried powder prepared by mixing animal whole blood, immunosuppressants and additives. The repeated experiments of this patent found that the appearance of the experimental results is poor, and the detection results are not uniform, with a coefficient of variation > 10%. In the stability experiment, the bias of 5 ng / ml rapamycin after 12 months is 13.7%, and the stability problem of high-concentration rapamycin has not been solved.

[0007] The methods disclosed in these patents mainly use animal anticoagulant whole blood or human anticoagulant whole blood as the matrix supplemented with various additives to prepare a freeze-dried powder. There are generally the following problems: 1. Mixed whole blood calibrant has potential test substances, pathogens and ethical problems, high risk and high cost; 2. There is a large batch difference between different mixed whole blood; 3. The solubility of immunosuppressants is different, and the uniform and stable dissolution in mixed whole blood needs to be strictly controlled. 4. Some immunosuppressants, especially rapamycin, have poor solubility and stability, and the higher the concentration, the worse the stability. Therefore, there is an urgent need for an industrial product to replace mixed whole blood and other products with large batch differences, high health risks and even ethical risks, and to solve the stability problem of immunosuppressants. SUMMARY

[0008] In order to improve the stability of immunosuppressants and solve the problem of large batch difference of whole blood matrix and ethical problems, the present application provides a freeze-dried powder matrix of immunosuppressants and its application, which belongs to the field of freeze-dried preparations. The matrix is divided into a component for stabilizing immunosuppressants and a component for reducing matrix effect. The former contains cyclodextrin and citric acid-sodium citrate buffer salt, and the latter contains hemoglobin and serum albumin. Experimental verification shows that the matrix is suitable for different types and concentrations of immunosuppressants and has universality. In addition, the batch difference of the matrix is small, its components are industrial products, the cost is low, and it has great application prospect

[0009] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] In one aspect, the present application provides a lyophilized powder matrix of an immunosuppressant, the matrix comprising a stabilizer and a component to reduce matrix effect; the immunosuppressant includes, but is not limited to, any one or more of tacrolimus, cyclosporine, rapamycin, everolimus.

[0011] The tacrolimus, cyclosporine, rapamycin, everolimus mainly act on T cells, inhibit their activation and / or proliferation, and are the mainstay drugs for organ transplant anti-rejection treatment and are also used for certain autoimmune diseases. These immunosuppressants all have large cyclic core structures, and such large ring structures are crucial to their biological activity (binding to specific immunophilins) and solubility. At the same time, due to the presence of a large number of hydrophobic groups (such as alkyl chains, aromatic rings, methyl groups, etc.) on the large ring structure, they are highly lipophilic macromolecules; relatively, their solubility in water is extremely low, belonging to BCS class II (low solubility and high permeability) or class IV (low solubility and low permeability) drugs, which makes it necessary to use complex solubilization carriers during intravenous injection and oral administration, and the latter itself can cause adverse reactions. More importantly, the above-mentioned immunosuppressants have complex substructures and contain some unstable groups (such as lactone ring, alkene bond, etc.), which are generally sensitive to light, heat, and oxygen, especially rapamycin containing a triene structure, which is more likely to react with oxygen to generate peroxide under light (especially ultraviolet light), and then be degraded to produce a variety of inactive or reduced-activity impurities; at the same time, the double bond may undergo cis-trans isomerization, changing the molecular configuration and affecting the activity; in addition, rapamycin is also more sensitive to acid-base conditions. Therefore, it is necessary to contain a stabilizer in the matrix to stabilize the active ingredients in the immunosuppressant and solve the problems in production, storage, and application.

[0012] Based on the above-mentioned physicochemical commonalities, although the present application does not involve everolimus, but according to existing experience, the matrix can also be applied to everolimus. Similarly, analogs of tacrolimus, cyclosporine, and rapamycin mentioned in the present application can also be suitable for the matrix.

[0013] In some ways, compared with the existing whole blood matrix, the matrix provided by the present application can greatly improve the stability of rapamycin.

[0014] Further, the stabilizer is selected from the group consisting of citric acid, sodium citrate, cyclodextrin, glutathione, sodium pyrosulfite, and vitamin E.

[0015] Preferably, the stabilizer comprises citric acid, sodium citrate, and cyclodextrin.

[0016] Further, the concentration of citric acid is 40 mM ~ 200 mM, the concentration of sodium citrate is 40 mM ~ 200 mM, and the concentration of cyclodextrin is 1 ~ 12%.

[0017] Furthermore, the components that reduce matrix effects are selected from: hemoglobin, serum albumin, cholesterol, glucose, sodium chloride, lecithin, and glutamic acid.

[0018] Preferably, the component that reduces the matrix effect includes hemoglobin and serum albumin.

[0019] In some preferred embodiments, the immunosuppressant lyophilized powder matrix comprises bovine hemoglobin, bovine serum albumin, glucose, sodium chloride, citrate-sodium citrate buffer (pH 5.0), preservative Proclin 300, and cyclodextrin.

[0020] More specifically, the immunosuppressant lyophilized powder matrix contains 10% bovine hemoglobin, 2% bovine serum albumin, 2% glucose, 0.5% sodium chloride, 100 mM citrate-sodium citrate buffer (pH 5.0), 200 mg / L preservative Proclin 300, and 5% cyclodextrin.

[0021] On the other hand, the present invention provides a lyophilized powder of an immunosuppressant, the lyophilized powder comprising the immunosuppressant and the above-described matrix.

[0022] Furthermore, the immunosuppressants include, but are not limited to, any one or more of tacrolimus, cyclosporine, rapamycin, and everolimus.

[0023] In some embodiments, the immunosuppressant is tacrolimus, cyclosporine, or rapamycin.

[0024] On the other hand, the present invention provides the use of a composition of citric acid, sodium citrate and cyclodextrin for stabilizing immunosuppressants and / or increasing the internal standard signal of immunosuppressants and / or decreasing the matrix signal and / or ensuring the consistency of the matrix signal, wherein the immunosuppressants include, but are not limited to, any one or more of tacrolimus, cyclosporine, rapamycin, and everolimus.

[0025] On the other hand, the present invention provides the use of the hemoglobin and serum albumin composition in at least one of the following aspects: enhancing the internal standard signal of immunosuppressants; reducing matrix effects; and ensuring matrix signal consistency.

[0026] Compared with existing technologies, this invention uses commercially available red blood cells and serum albumin as the main raw materials instead of a mixed whole blood matrix, and adds cyclodextrin to enhance the solubility and stability of immunosuppressants, then prepares them into lyophilized powders. This product exhibits good homogeneity, high stability and solubility, and is convenient to transport. It can be used as a calibrator for immunosuppressant kits and as a quality control material for immunosuppressant quality control. This invention solves the batch-to-batch variability, safety and hygiene risks, and ethical risks associated with using a mixed whole blood matrix as the main raw material for immunosuppressants. It also improves the stability of immunosuppressants, eliminates the need for refrigerated transport, and is convenient to use. This method results in minimal batch-to-batch variability, strong immunosuppressant stability, safety and reliability, and high clinical application value. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are shown in Table 1, and unless otherwise specified, they are commercially available reagents and materials.

[0029] Table 1. Materials and reagents involved in this invention

[0030]

[0031] Example 1: Experimental Method

[0032] 1.1 Preparation of lyophilized immunosuppressant powder products

[0033] The lyophilized immunosuppressant powder product comprises a lyophilized powder matrix and an immunosuppressant. The most preferred lyophilized powder matrix composition of this invention is as follows: 10% bovine hemoglobin, 2% bovine serum albumin, 2% glucose, 0.5% sodium chloride, 100 mM citrate-sodium citrate buffer (pH 5.0), 200 mg / L preservative Proclin 300, and 5% cyclodextrin.

[0034] Specifically, the preparation steps of the matrix are as follows: Weigh 10 g hemoglobin, 2 g serum albumin, 2 g glucose, 0.5 g sodium chloride, 0.80 g citric acid, and 1.5 g sodium citrate into a container (preferably a beaker), add 100 ml deionized water, and stir to dissolve; then add 20 mg proclin-300 and mix well; next, while stirring, add 5 g cyclodextrin, and stir to mix well to obtain the immunosuppressant matrix solution for later use.

[0035] The preparation steps of the lyophilized immunosuppressant powder product are as follows: Take 100 mL of the above immunosuppressant matrix solution, add 1 mL of immunosuppressant solution (tacrolimus 80 ng / ml, cyclosporine 800 ng / ml, rapamycin 80 ng / ml) prepared with acetonitrile, and stir to mix well; dispense 200 μl of the mixture into brown vials, pre-freeze at -40℃ for 2 hours, then raise the temperature to 0℃ within 1 hour and maintain for 30 hours; then raise the temperature to 30℃ at 1℃ / min and maintain for 2 hours, then cap and package.

[0036] 1.2 Homogeneity Verification of Immunosuppressant Lyophilized Powder Products

[0037] Ten vials of the same batch of lyophilized immunosuppressant powder (preparation method as described above) were randomly selected; after reconstitution of the lyophilized powder with 200 μL of water, the vials were pretreated according to the instructions of the Tacrolimus / Cyclosporine A / Rapamycin Detection Kit (Liquid Chromatography-Tandem Mass Spectrometry) developed and manufactured by Disais Diagnostics. Each vial was tested once, and the CV value was calculated.

[0038] 1.3 Accelerated stability verification of lyophilized immunosuppressant powder products

[0039] Each of the lyophilized immunosuppressant powder products (containing matrix and immunosuppressants, including tacrolimus 80 ng / ml, cyclosporine 800 ng / ml, and rapamycin 80 ng / ml) was divided into three aliquots and stored at -80℃, 55℃, and 37℃, respectively. Samples stored at 55℃ were tested on day 3, and samples stored at 37℃ were tested on day 14. Samples stored at -80℃ served as controls. After reconstitution, the samples were analyzed according to the instructions of the Tacrolimus / Cyclosporine A / Rapamycin Assay Kit (Liquid Chromatography-Tandem Mass Spectrometry) developed and manufactured by Disais Diagnostics. Results are presented as recoveries, i.e., the percentage of the accelerated product's result relative to the product stored at -80℃.

[0040] 1.4 Reconstitution stability of lyophilized immunosuppressant powder products

[0041] Divide the lyophilized immunosuppressant powder product described in step “1.1” into two portions. Add 200 μL of water to one portion to reconstitute it, and then store it at 2-8℃. Measure it after 30 days. Store the other portion at -80℃, and take it out to reconstitute it after 30 days for measurement. Record the bias value.

[0042] 1.5 Matrix consistency verification of immunosuppressant lyophilized powder products

[0043] Take the lyophilized immunosuppressant powder products described in step "1.1" (tacrolimus 80 ng / ml, cyclosporine 800 ng / ml, rapamycin 80 ng / ml), national standard materials (national standards for cyclosporine A, tacrolimus, sirolimus, and everolimus in frozen human whole blood), and human whole blood samples. Perform the measurements according to the specifications of the tacrolimus / cyclosporine A / rapamycin detection kit (liquid chromatography-tandem mass spectrometry) developed and produced by Disais Diagnostics, and record the signal intensity of the isotope internal standard. The ratio of the internal standard signal of the lyophilized immunosuppressant powder to that of the whole blood sample = (internal standard signal of the lyophilized immunosuppressant powder / internal standard signal of the human whole blood sample) × 100%

[0044] Example 2: Optimization of stabilizing components in the lyophilized immunosuppressant powder matrix

[0045] The aforementioned lyophilized immunosuppressant powder matrix contains two functional components: a stabilizer to stabilize the immunosuppressant and a component to address matrix consistency issues. Immunosuppressants are macrolides or cyclic peptides with poor water solubility, almost insoluble in water. They are also prone to oxidation and ring-opening degradation in aqueous solutions, especially high concentrations of rapamycin. This is due to: increased density of active molecules per unit volume, enhanced light absorption efficiency, and an exponentially increasing photodegradation rate; degradation products (such as epoxides and peroxides) that may trigger chain reactions, further catalyzing the decomposition of surrounding molecules; the triene structure being easily attacked by oxygen, with higher absolute amounts of dissolved oxygen in high-concentration formulations; free radicals generated by localized oxidation forming autocatalytic cycles, accelerating overall degradation; and increased molecular collision probability at high concentrations, leading to a higher rate of lactone ring hydrolysis, especially in acidic / alkaline environments.

[0046] Based on this, the present invention uses a 14-day accelerated experiment at 37°C (specific steps as described in Example 1) to verify the effects of different stabilizers (citric acid-sodium citrate buffer, cyclodextrin, glutathione, sodium metabisulfite, and vitamin E), and then optimizes the types of stabilizers in the freeze-dried powder matrix. The specific results are shown in Table 2. Currently, products on the market mainly use anticoagulated bovine blood as a matrix component. This embodiment compares the matrix provided by the present invention with products on the market to highlight the effect of the matrix provided by the present invention.

[0047] Table 2 Optimization of stabilizers

[0048]

[0049] Note: Bold text indicates stabilizers.

[0050] As shown in Table 2, under the same immunosuppressant and stabilizer, the matrix containing anticoagulated bovine whole blood has a better stabilizing effect on immunosuppressants than the matrix containing bovine hemoglobin + bovine serum albumin. This may be because anticoagulated bovine blood itself has the function of stabilizing immunosuppressants, which also highlights the difficulty of stabilizing immunosuppressants in non-whole blood matrices.

[0051] In terms of stabilizer types, the selected stabilizers and their combinations can generally stabilize tacrolimus and cyclosporine. However, when the target of protection is rapamycin, compared with the control, the stabilizing effects of glutathione, sodium metabisulfite, vitamin E, and their combinations are not significantly different in whole blood background or non-whole blood background (bovine hemoglobin + bovine serum albumin). The addition of citrate-sodium citrate buffer and / or cyclodextrin can further improve the stability of tacrolimus and cyclosporine, and also improve the stability of rapamycin by 12% to 20%. This is because rapamycin has poor water solubility, and the stability of rapamycin dissolved in aqueous solution is also poor. The structural cavity of cyclodextrin molecules can encapsulate rapamycin molecules, thereby increasing the solubility and stability of rapamycin. Citrate buffer can further adjust the pH of the cyclodextrin solution and provide a certain ionic strength to maintain the stability of rapamycin aqueous solution. Furthermore, the combination of citrate-sodium citrate buffer and cyclodextrin has the best stabilizing effect, followed by cyclodextrin. Subsequent experiments using low concentrations (1.5 ng / ml) and ultra-high concentrations (100 ng / ml) of rapamycin yielded the same conclusion. Therefore, the combination of citrate-sodium citrate buffer and cyclodextrin is preferred as a stabilizer for immunosuppressants.

[0052] Next, after confirming the optimal stabilizer components, based on a formulation of 10% bovine hemoglobin + 2% bovine serum albumin + 2% glucose + 0.5% sodium chloride + citrate-sodium citrate buffer + cyclodextrin + 0.02% proclin 300, the concentrations of citrate-sodium citrate buffer and cyclodextrin were optimized to further improve the stability of the immunosuppressant in the matrix. The specific results are shown in Tables 3 and 4.

[0053] Table 3 Optimization of Citric Acid-Sodium Citrate Buffer Concentration

[0054]

[0055] Note: The matrix formulation used in this table is 10% bovine hemoglobin + 2% bovine serum albumin + 2% glucose + 0.5% sodium chloride + citrate-sodium citrate buffer + 5% cyclodextrin + 0.02% proclin 300

[0056] Table 4 Optimization of cyclodextrin concentration

[0057]

[0058] Note: The matrix formulation used in this table is 10% bovine hemoglobin + 2% bovine serum albumin + 2% glucose + 0.5% sodium chloride + 100 mM citrate-sodium citrate buffer + cyclodextrin + 0.02% proclin 300

[0059] Table 3 shows the stability (recovery rate) results of different concentrations of citrate-sodium citrate buffer salt at 37℃ for 14 days. The results show that when the concentration of the buffer salt is greater than or equal to 100 mM, the stability of rapamycin is greater than 95%. This is because the higher the ionic strength of the buffer salt, the stronger its ability to stabilize the pH of the buffer solution system. When the concentration of the citrate buffer salt solution reaches 100 mM, the acid-base system of the solution tends to be stable and is not easily affected by external factors. Therefore, the concentration of citrate-sodium citrate buffer salt is preferably not less than 100 mM, and more preferably 100~200 mM.

[0060] Table 4 shows the results of accelerated stability (recovery rate) at 37℃ for 14 days at different cyclodextrin concentrations. As shown in Table 5, when the cyclodextrin concentration is greater than or equal to 5%, the recovery rate of each immunosuppressant is greater than 95%. This is because cyclodextrin, as a co-solvent for immunosuppression, has a molecular cavity that can encapsulate immunosuppressant molecules. The higher the concentration of cyclodextrin, the stronger its ability to encapsulate immunosuppressant molecules, and the higher the encapsulation degree. Therefore, the preferred concentration of cyclodextrin is not less than 5%, and more preferably, 5-12%.

[0061] Example 3: Optimization of components that reduce matrix effect in the lyophilized immunosuppressant powder matrix

[0062] Since the detection involves immunosuppressants in whole human blood, and the matrix in the calibrators and quality control samples is not whole blood, the matrix may exhibit matrix effects (i.e., the signal intensity produced by the non-whole blood matrix differs from that produced by whole human blood). Matrix effects may affect the sensitivity and accuracy of quantification. Therefore, some components (bovine hemoglobin, bovine serum albumin, cholesterol, glucose, and sodium chloride) need to be introduced into the matrix to simulate whole blood and reduce matrix effects. Based on this, this embodiment detected the intensity of isotopic internal standard signals in different components to screen for the component that best reduces matrix effects. The specific experimental steps are the same as described in Example 1, and the specific results are shown in Table 5.

[0063] Table 5 Optimization of components to reduce matrix effect

[0064]

[0065] From the perspective of immunosuppressant types, the signal intensity of tacrolimus and cyclosporine is significantly affected by the matrix components. Among the components, hemoglobin and serum albumin have the greatest impact on the matrix signal, followed by glucose and citrate-sodium citrate buffer. Sodium chloride has a less significant effect, but it can enhance ionic strength, improve precipitation during pretreatment, make the extract cleaner, and reduce column operating pressure. However, the addition of cholesterol, used to simulate lipids in whole blood, actually decreased the signal of the internal standard in the matrix. This may be because cholesterol competes with immunosuppressants for cyclodextrin molecule cavities, affecting system equilibrium. In summary, the optimal combination is bovine hemoglobin + bovine serum albumin + glucose + sodium chloride + citrate-sodium citrate buffer + cyclodextrin + proclin 300.

[0066] Next, after identifying the component that best reduces the matrix effect, based on the above-mentioned preferred formulation, this embodiment optimizes the concentrations of bovine hemoglobin and bovine serum albumin to further ensure the consistency of the matrix signal. The specific results are shown in Tables 6 and 7.

[0067] Table 6 Optimization of bovine hemoglobin concentration

[0068]

[0069] Note: The matrix formulation in this table is bovine hemoglobin + 2% bovine serum albumin + 2% glucose + 0.5% sodium chloride + 100 mM citrate-sodium citrate buffer + 5% cyclodextrin + 0.02% proclin 300

[0070] Table 7 Optimization of bovine serum albumin concentration

[0071]

[0072] Note: The matrix formulation in this table is 10% bovine hemoglobin + bovine serum albumin + 2% glucose + 0.5% sodium chloride + 100 mM citrate-sodium citrate buffer + 5% cyclodextrin + 0.02% proclin 300

[0073] Table 6 shows the internal standard signal ratios of lyophilized immunosuppressant powder containing different concentrations of hemoglobin to human whole blood samples. The table shows that as the hemoglobin concentration increases, the deviation of the internal standard signal in the lyophilized powder relative to human whole blood is relatively small. This may be because the immunosuppressant partially binds to the protein, affecting the extraction efficiency of the immunosuppressant. When the hemoglobin concentration increases to 10%, the matrix effect is lowest. At this point, further increasing the hemoglobin concentration does not significantly reduce the matrix effect, possibly because the degree of binding between the immunosuppressant and the protein reaches its maximum.

[0074] Table 7 shows the internal standard signal ratios of lyophilized immunosuppressant powder containing different concentrations of serum albumin to human whole blood samples. The table shows that when serum albumin is selected at 2%, the difference in internal standard signal between the non-whole blood matrix and the human whole blood matrix is ​​small. In summary, the optimal concentrations of bovine hemoglobin and bovine serum albumin are 10% or higher and 2% or higher, respectively.

[0075] Example 4: Verification of the homogeneity of lyophilized immunosuppressant powder products

[0076] Based on the optimization results of Examples 2-3, this example verifies the uniformity of the immunosuppressant lyophilized powder product containing the optimized matrix (formulation as described in Example 1) and immunosuppressant. The specific steps are described in Example 1, and the results are shown in Table 8.

[0077] Table 8. CV values ​​of lyophilized immunosuppressant powder products

[0078]

[0079] The experimental results showed that the CV values ​​of different lyophilized immunosuppressant powder products were all no greater than 5%, proving that the three batches of products had good uniformity and could be used for mass production.

[0080] Example 5: Accelerated stability verification of immunosuppressant lyophilized powder products

[0081] This embodiment verifies the stability of an immunosuppressant lyophilized powder product containing an optimized matrix (formulation as described in Example 1) and an immunosuppressant. The specific steps are described in Example 1, and the results are shown in Tables 9 and 10. The data in the tables represent the recovery rate, which is the percentage of the immunosuppressant concentration after acceleration relative to the immunosuppressant concentration stored at -80°C.

[0082] Table 9. Results of accelerated stability verification of lyophilized immunosuppressant powder products at 55℃ for 3 days.

[0083]

[0084] Table 10 Results of accelerated stability verification of lyophilized immunosuppressant powder products at 37℃ for 14 days

[0085]

[0086] Experimental results show that the bias of different lyophilized immunosuppressant powders after accelerated testing at 55°C for 3 days and 37°C for 14 days is less than 5%, proving that the products have extremely high stability. Based on experience, it is estimated that this product can be stored at 2-8°C for at least one year or longer. Furthermore, this invention also demonstrates that the matrix provided can stabilize different types and concentrations of immunosuppressants, exhibiting a certain degree of universality.

[0087] Example 6: Stability Verification of Reconstitution of Lyophilized Immunosuppressant Powder Products

[0088] This embodiment verifies the reconstitution stability of an immunosuppressant lyophilized powder product containing an optimized matrix (formulation as described in Example 1) and an immunosuppressant. The specific steps are described in Example 1, and the results are shown in Table 11. The data in the table are recovery rates, which are the percentage values ​​of the immunosuppressant concentration in the lyophilized powder product 30 days after reconstitution relative to the immunosuppressant concentration in the lyophilized powder product immediately after reconstitution.

[0089] Table 11. Results of stability verification of lyophilized immunosuppressant powder products after 30 days of reconstitution.

[0090]

[0091] Experimental results show that different types and batches of lyophilized immunomodulatory powder products can be stored at 2-8℃ for at least 30 days or longer after being reconstituted with water, indicating that the product has high reconstitution stability.

[0092] Example 7: Matrix consistency verification of immunosuppressant lyophilized powder products

[0093] This embodiment verifies the matrix consistency of an immunosuppressant lyophilized powder product containing an optimized matrix (formulation as described in Example 1) and an immunosuppressant. The specific steps are described in Example 1, and the results are shown in Table 12. The data in the table are the recovery rates, namely tacrolimus 119%, cyclosporine 90%, and rapamycin 89%.

[0094] Table 12 Results of matrix consistency evaluation for lyophilized immunosuppressant powder products

[0095]

[0096] Table 12 shows that, compared with the national standard reference material, the internal standard signals of the immunosuppressant lyophilized powder products mixed with human whole blood samples all showed certain differences. This is mainly because the red blood cells in the human whole blood samples were not fragmented, and the three drugs (tacrolimus, cyclosporine, and cyclosporine) were distributed evenly in the whole blood samples, resulting in a matrix effect. This proposed method, by adjusting the ratio of hemoglobin, serum albumin, and salts, controls the matrix effect within a smaller range, thus enhancing the reliability of the detection method.

[0097] In summary, the lyophilized powder matrix provided by this invention can not only stabilize different types and concentrations of immunosuppressants, but also minimize matrix effects, and has great application potential.

[0098] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention, and no designation in the claims should be construed as limiting the scope of the claims.

Claims

1. An immunosuppressant lyophilized powder matrix, characterized in that, The matrix consists of a stabilizer and a component that reduces matrix effect; the immunosuppressant includes, but is not limited to, any one or more of tacrolimus, cyclosporine, and rapamycin; the stabilizer is citric acid, sodium citrate, and cyclodextrin; the component that reduces matrix effect is bovine hemoglobin and bovine serum albumin; the concentration of citric acid is 40 mM to 200 mM, the concentration of sodium citrate is 40 mM to 200 mM, and the concentration of cyclodextrin is 5% to 12%; the concentration of bovine hemoglobin is 10% to 12%, and the concentration of bovine serum albumin is 2% to 4%.

2. The lyophilized immunosuppressant powder matrix as described in claim 1, characterized in that, The matrix also includes glucose, sodium chloride, and preservatives.

3. A lyophilized powder of an immunosuppressant, characterized in that, The lyophilized powder comprises an immunosuppressant and a matrix as described in any one of claims 1 to 2, wherein the immunosuppressant includes, but is not limited to, one or more of tacrolimus, cyclosporine, and rapamycin.

4. The use of the lyophilized immunosuppressant powder matrix according to any one of claims 1 to 2 for preparing reagents that stabilize immunosuppressants and / or enhance the internal standard signal of immunosuppressants and / or reduce the matrix signal and / or ensure the consistency of the matrix signal, characterized in that, The immunosuppressants include, but are not limited to, any one or more of tacrolimus, cyclosporine, and rapamycin.

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