Zomib nano-micelle preparation and preparation method thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a zomib nano-micelle preparation and a preparation method thereof. The carfilzomib nano-micelle preparation comprises carfilzomib, a micelle carrier material and a stabilizer, wherein the mass ratio of the carfilzomib to the micelle carrier material to the stabilizer is 1: (15-30): (15-30); and the carrier material is PEG (Polyethylene Glycol)-DSPE (Distearoyl PEG-DSPE is used as a drug carrier to prepare the carfilzomib micelle preparation, the particle size is 8-20 nm, the encapsulation efficiency is high, and the carfilzomib micelle preparation has a very strong cell penetration effect and cannot be quickly cleared. Meanwhile, the preparation operation is simple, and freeze-drying operation is avoided. Compared with the existing carfilzomib preparation, the toxicity is reduced under the same dosage.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to a zolmib-type nano-micelle preparation and a preparation method thereof. Background Art

[0002] Carfilzomib (CFZ, structure as shown in Formula 1) is the second proteasome inhibitor (PI) after bortezomib. It was first launched in the United States in 2012 and in my country in 2021 (trade name: Kailos) is used for relapsed or refractory multiple myeloma (MM) that has been treated with at least carfilzomib and one immunomodulatory drug. The original research company is Onyx Pharmaceuticals Inc. Its mechanism of action is that the drug molecule irreversibly binds to the N-terminal threonine active site of the 20S proteasome (the proteolytic core particle in the 26S proteasome).

[0003]

[0004] Kailos is a lyophilized injection, which contains 60 mg of carfilzomib, 3000 mg of sulfobutyl-β-cyclodextrin, 57.7 mg of citric acid and an appropriate amount of sodium hydroxide. Due to the low solubility of carfilzomib (<2 μg / mL, pH 7.0), the dosage of cyclodextrin is as high as 50 times. It is generally recognized in the industry that cyclodextrin will increase the burden on the kidneys after entering the human blood circulation system, and can also interact with the cell membrane of the inner wall of blood vessels, causing cell damage, and then causing venous spasm and phlebitis (Non-clinical Study and Case Analysis of Drug-Induced Nephrotoxicity, Chinese Journal of Clinical Pharmacology, 2018, (19): 2362-2365). Although sulfobutyl-β-cyclodextrin has improved compared with cyclodextrin, its toxicity is still obvious. The Phase 3 clinical study PX-171-009 disclosed in the EU review report of carfilzomib showed that the incidence of venous embolism and thrombotic events in the CRd group (carfilzomib / lenalidomide / dexamethasone) was 15.3% (5.6% ≥ Grade 3), which was higher than the 9.0% (3.9% ≥ Grade 3) in the Rd group (lenalidomide / dexamethasone). Due to the safety risks of venous spasm and phlebitis, its instructions require that dexamethasone be given before each administration to prevent infusion reactions, which limits its clinical application.

[0005] In view of the above safety risks, it is necessary to develop an improved preparation for carfilzomib. CN115243674A discloses a cyclodextrin-free carfilzomib preparation, which avoids the use of cyclodextrin excipients that cause side effects by using solubilizers such as DMSO (dimethyl sulfoxide), DMA (N,N-dimethylacetamide), and PVP (povidone). DMSO and DMA are chemical reagents with high boiling points, and it is difficult to control them within the limits required by the pharmacopoeia when they are finally removed by freeze drying.

[0006] In addition, there are many related studies on the use of nanotechnology to prepare nanoformulations and the use of the EPR effect to passively target tumors for treatment. Table 1 below lists the relevant reports on carfilzomib nanoformulations (including liposomes and micelles) in recent years. The key quality indicator of encapsulation rate is generally very low, with the highest being only 72%, which is lower than the Chinese Pharmacopoeia's requirement that the encapsulation rate of nanoformulations must not be less than 80%, making it difficult to continue to develop into therapeutic drugs.

[0007] Table 1 List of representative literature

[0008] Serial number Encapsulation rate (%) literature 1 — International Journal of Pharmaceutics,2020,59,119885 2 72 Colloid and Polymer Science,2018,296,1827 3 — ACS Appl.Mater.Interfaces 2018,10,19355 4 55.9 Acta Biomaterialia 2018,80(15),288 5 ≤44.3 J Pharmacol Exp Ther.2015,355(2):168-73 6 30 Nanoscale, 2015, 7, 19722 7 — Journal of Biomaterials Science,Polymer Edition,2015,26,1178

[0009] (“—” indicates no mention in the literature)

[0010] The preparation of nanomicelles is very difficult, and factors such as carrier materials, stabilizers, and preparation processes will affect their preparation. In addition, drug leakage is also a problem that requires special attention. In order to solve the problem of solution stability, freeze-dried preparations are often used. However, freeze-dried preparations often contain a large amount of surfactants, and the operation is cumbersome. A large number of bubbles are easily generated during the re-dissolution process, and it takes a long time for the bubbles to disappear naturally, which is very unfavorable for clinical application. Summary of the invention

[0011] The content of the present invention is to overcome the deficiencies of the prior art, and the purpose of the present invention is to provide a novel micellar preparation containing carfilzomib and a preparation method thereof. The adverse reactions caused by sulfobutyl-β-cyclodextrin in ordinary preparations are avoided, thereby improving the safety of CFZ (carfilzomib); the preparation operation is simple, while improving its stability and facilitating clinical application, thereby realizing the improvement of ordinary lyophilized preparations of carfilzomib.

[0012] In order to solve the above problems, the technical solution provided by the present invention is as follows:

[0013] A zosomib nano-micelle preparation comprises carfilzomib, a micelle carrier material and a stabilizer; wherein the mass ratio of carfilzomib, the micelle carrier material and the stabilizer is 1:15-30:15-30; and the carrier material is PEG-DSPE.

[0014] Preferably, the micellar preparation containing carfilzomib is an injection, comprising the active ingredient carfilzomib, a micellar carrier, and a stabilizer, wherein the stabilizer is composed of two stabilizers, and the mass ratio of carfilzomib, the micellar carrier material and the stabilizer in each preparation unit is 1:15-30:15-25.

[0015] Preferably, the mass ratio of carfilzomib to micellar carrier material is 1:15-30, preferably 1:20.

[0016] Preferably, the mass ratio of carfilzomib to stabilizer is 1:15-25; preferably 1:20.

[0017] Preferably, the carrier material used is PEG-DSPE (methoxypolyethylene glycol-distearoylphosphatidylethanolamine, Formula 2).

[0018]

[0019] Preferably, the molecular weight of the polyethylene glycol in the PEG-DSPE is 1000-5000 Da; preferably, the molecular weight of the polyethylene glycol is 2000 Da.

[0020] Preferably, the stabilizer is composed of two stabilizers, wherein the first stabilizer is selected from one or a combination of glycine, glucose, and trehalose, preferably glycine, and the second stabilizer is selected from acetic acid, ascorbic acid, fumaric acid, citric acid, tartaric acid, and maleic acid, preferably citric acid.

[0021] Preferably, the first stabilizer accounts for 90.0-99.0% of the stabilizer; preferably, the first stabilizer accounts for 95.0-99.0% of the stabilizer; preferably, the mass ratio of the first stabilizer to the second stabilizer is 190:10-198:2.

[0022] Preferably, the method for preparing the nano-micelle preparation comprises the following steps:

[0023] (1) dissolving carfilzomib and PEG-DSPE in an organic solvent;

[0024] (2) removing the organic solvent in the mixed solution of step (1) by evaporation under reduced pressure to form a drug-containing lipid film;

[0025] (3) using water for injection to re-dissolve the drug-containing lipid film obtained in step (2), adding a stabilizer until the drug-containing solution is completely dissolved; or dissolving the stabilizer in water for injection and re-dissolving the drug-containing lipid film obtained in step (2);

[0026] (4) Aseptically filter the micelle solution obtained in step (3) to prepare a micelle injection.

[0027] Preferably, in step (1), the organic solvent is one or more of methanol, ethanol, acetone, tetrahydrofuran and dichloromethane.

[0028] Preferably, the organic solvent is ethanol. Preferably, the concentration of carfilzomib is 2-5 mg / ml.

[0029] The average particle size of the micelles was measured by dynamic light scattering (DLS) using a Malvern Zetasizer and was found to be approximately 11 nm. The negative staining method was used to prepare the sample, and the morphology of the drug-loaded micelles was observed using a transmission electron microscopy (TEM), which showed that the micelles were uniform in size.

[0030] The ultrafiltration centrifugation method was used and the HPLC detection was performed. The micellar encapsulation efficiency (EE%) was calculated according to formula (1):

[0031]

[0032] Preferably, the nano-micelle preparation is used in the preparation of a drug for treating multiple myeloma.

[0033] Compared with the prior art, the present invention has the following advantages: PEG-DSPE is used as a drug carrier to prepare a carfilzomib micelle preparation, the particle size is 8 to 20 nm, the encapsulation rate is high, and it has a strong cell penetration effect and will not be quickly cleared. At the same time, the preparation operation is simple and the freeze-drying operation is avoided. Compared with the existing carfilzomib preparation, the toxicity is reduced at the same dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the particle size distribution diagram of the carfilzomib micellar preparation of prescription 1-2;

[0035] Figure 2 This is the transmission electron microscopy image of the carfilzomib micelle preparation of formulation 1-2. DETAILED DESCRIPTION

[0036] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the following specific embodiments: It should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0037] Example 1: Preparation of micellar formulation containing carfilzomib

[0038] According to the prescription amount shown in Table 2, the micellar preparation was prepared according to the following process:

[0039] (1) Combine CFZ and PEG 2000 -DSPE was dissolved in ethanol, a volatile organic solvent, according to the mass ratio shown in Table 2;

[0040] (2) removing the solvent under reduced pressure at a temperature below 50°C to obtain a drug film;

[0041] (3) Dissolve the stabilizer in a corresponding amount of water for injection according to the mass ratio shown in Table 2;

[0042] (4) adding the aqueous solution in step (3) into the container in step (2), and stirring by magnetic stirring or ultrasonic oscillation until the drug-containing solution is completely dissolved into a clear and transparent solution; and filtering through a 0.22 μm filter membrane (nylon or mixed cellulose material) to obtain a carfilzomib micellar injection;

[0043] Table 2 Formulation design table

[0044]

[0045] Example 2: Quality test results of carfilzomib micellar preparation

[0046] 1. The micelle preparations prepared by each formulation in Table 2 were tested for encapsulation efficiency and average particle size, wherein the average particle size was tested by dynamic light scattering (DLS), and the detailed results are shown in Table 3. The micelle preparations prepared by the representative formulation 1-2# had an average particle size of about 11 nm, and it can be seen from the negative staining method of transmission electron microscopy that the particle size is uniform and spherical.

[0047] Table 3 Test results of micellar preparations

[0048]

[0049] From the above experimental results, it can be seen that when the ratio of material to drug reaches or exceeds 20 times, a better encapsulation efficiency (EE%>90%) can usually be obtained.

[0050] 2. Dilute the micellar preparations obtained from each prescription to 0.02 mg / ml with a phosphate buffer system (PBS) at pH 7.4, and visually observe the precipitation of the system at room temperature and 37°C. This method can be used to examine whether the system is stable. When the drug leaks from the micelle, it will precipitate due to its low solubility, and the system will appear turbid.

[0051] Minitab software was used to perform DOE orthogonal experimental design on the first stabilizer and the second stabilizer, wherein the first stabilizer was selected from one of glycine, glucose and trehalose, and the second stabilizer was selected from citric acid or fumaric acid.

[0052] Table 4 PBS (pH 7.4) diluted to 0.02 mg / ml Visual observation results

[0053]

[0054] From the table above, it can be seen that when the first stabilizer (2-1#, 2-4#, 2-8#) is used alone, the micelles all precipitate, indicating that the second stabilizer is required. However, when the second stabilizer (2-9#) is used alone, even if it remains clear and transparent at room temperature, it precipitates again soon after incubation at 37°C, indicating that the micelles are still not stable enough in the presence of the second stabilizer alone. Only a reasonable combination of the two can achieve stability, such as 2-2#, 2-5#, and 2-7#.

[0055] Example 3: Use of PEG 5000 -DSPE as carrier material for the preparation of micelles

[0056] According to the prescription amount of prescription 1-2 shown in Table 2, PEG 5000 -DSPE micelles were prepared in the same way. The encapsulation efficiency and particle size were similar to those of PEG 2000 -DSPE similar.

[0057] Comparative Example 1 Preparation Stability

[0058] 1. The commercially available preparation (batch number: 1149962) and the micelle preparation were diluted to the same concentration (0.25 mg / ml) in a pH 7.4 aqueous solution, and placed at room temperature and 37°C, respectively, and the clarity of the solution was observed. The results are shown in Table 5:

[0059] Table 5 Comparison between homemade preparation and commercial preparation Kailos

[0060]

[0061] It can be seen that the stability of the prepared micelle preparation is better than that of the commercially available preparation. At the same time, it can be found from the instructions of Kairos that in clinical use, after the infusion is completed, the dropper needs to be rinsed with a large amount of normal saline, the purpose of which is to ensure that the precipitated drug can enter the body.

[0062] Example 4: Stability study of the preparation

[0063] According to the relevant requirements of the Appendix "9001 Guidelines for Stability Tests of APIs and Pharmaceutical Preparations" of the Chinese Pharmacopoeia, accelerated stability studies and long-term stability studies were conducted on homemade micelle preparations. The accelerated test conditions were: 25℃±2℃, RH60%±5%, and the long-term test conditions were: 5℃±3℃. At the same time, a long-term study was conducted on commercially available preparations. The results of related substances, visible foreign matter, and encapsulation efficiency are shown in Table 6:

[0064] Table 6 Stability test results

[0065]

[0066] (The day when the commercially available preparations are sampled and tested is taken as day 0; N / A means that the indicator is not applicable to common preparations)

[0067] From the above results, it can be seen that the representative micellar preparations have undergone 6-month accelerated test and long-term test stability studies. The finished products with different prescriptions have been subjected to accelerated test (25℃±2℃, RH60%±5%) for 6 months, and the content has shown a slight downward trend, but it is still within the qualified standard range, indicating that this product should be stored at low temperature: no significant changes were found in the long-term test content and related substance results.

[0068] Example 5: Cellular uptake experiment

[0069] Human multiple myeloma cells RPMI 8226 (lot number: 230602) were grown in a semi-adherent and semi-suspended state using IMDM complete medium.

[0070] Dilute it into three gradients: 2 × 10 4 / hole, 1×10 5 / hole, 5×10 5 / well cultured for 24 hours, and the cell concentration with a density of about 80% was selected for subsequent experiments. In a 12-well cell culture plate, 500 μL was inoculated in each well. The cells were incubated with the homemade product (2305054101, amplified according to prescription 1-2) and the commercial product (1149962) for 0.5, 1, 2, 4, and 6 hours, respectively. The cells were lysed with protein lysis buffer at each time point, and the drug concentration was measured. The results showed that when the cells acted with the drug for 2 hours, the cells took up the drug, of which the cell uptake of the control was 9.4%, and the uptake of the homemade product was 46.5%. The test results are as follows:

[0071] Table 7 Intracellular drug concentration determination results (Mean ± SEM, n = 3)

[0072]

[0073] Note: n: number of repetitions; “-” means the result is lower than the detection limit.

[0074] The results showed that the concentration of the homemade product in the cell was higher than that of the commercial product, proving that the homemade product can enter the cell more easily than the commercial product, indicating that the efficacy of the drug is improved.

[0075] Example 6: In vivo safety study

[0076] With reference to the Technical Guidelines for Single-dose Toxicity Studies of Drugs issued by the current National Medical Products Administration (NMPA), the homemade micelle preparation (2305054101) and the commercial preparation were subjected to acute toxicity experiments on SD rats. The experimental scheme was as follows: 24 SD rats were divided into 4 groups according to body weight, namely, the commercial product high (12 mg / kg), low dose group (8 mg / kg) and the homemade product high (12 mg / kg), low dose group (8 mg / kg), 6 rats in each group, half male and half female. All rats were given a single dose, the administration method was tail vein infusion, the infusion time was 10 minutes, and the rats were clinically observed within 7 days after administration, including survival status, appearance, physical signs, etc.

[0077] The results are shown in Table 8.

[0078] Table 8 Results of single intravenous toxicity test in SD rats

[0079]

[0080]

[0081] From the results of the acute toxicity test in rats, it can be seen that at low doses, the homemade micelle preparation is consistent with the commercially available preparation, and no toxic reactions are observed. At high doses, the toxicity of the homemade preparation is significantly better than that of the commercially available preparation, indicating that the micelle preparation has a safety advantage.

[0082] The protection scope of the present invention is not limited to the above-mentioned specific embodiments. The terms used in the examples are for describing specific embodiments, but not for limiting the protection scope of the present invention. The test methods without specific conditions in the examples are usually carried out under conventional conditions or methods commonly known to those skilled in the art.

Claims

1. A zolmib nano-micelle preparation, characterized in that: The nano micelle preparation comprises: carfilzomib, micelle carrier material and stabilizer; wherein the mass ratio of carfilzomib, micelle carrier material and stabilizer is 1:15-30:15-30; and the carrier material is PEG-DSPE.

2. The nano-micelle preparation according to claim 1, characterized in that The molecular weight of the polyethylene glycol in the PEG-DSPE is 1000-5000 Da.

3. The nano-micelle preparation according to claim 2, characterized in that The molecular weight of the polyethylene glycol in the PEG-DSPE is 2000Da.

4. The nano-micelle preparation according to claim 1, characterized in that The stabilizer comprises: a first stabilizer and a second stabilizer, wherein the first stabilizer is selected from one or more of glycine, trehalose or glucose, and the second stabilizer is selected from citric acid, acetic acid, ascorbic acid, tartaric acid, maleic acid or fumaric acid.

5. The nano-micelle preparation according to claim 4, characterized in that The mass ratio of the first stabilizer to the second stabilizer is 190:10 to 198:

2.

6. The nano-micelle preparation according to claim 4, characterized in that The first stabilizer is glycine, and the second stabilizer is citric acid.

7. A method for preparing a nano-micelle preparation according to any one of claims 1 to 6, comprising the following steps: (1) dissolving carfilzomib and PEG-DSPE in an organic solvent; (2) removing the organic solvent in the mixed solution of step (1) by evaporation under reduced pressure to form a drug-containing lipid film; (3) using water for injection to re-dissolve the drug-containing lipid film obtained in step (2), adding a stabilizer until the drug-containing solution is completely dissolved; or dissolving the stabilizer in water for injection and re-dissolving the drug-containing lipid film obtained in step (2); (4) Aseptically filter the micelle solution obtained in step (3) to prepare a micelle injection.

8. The preparation method according to claim 7, characterized in that: In the step (1), the organic solvent is one or more of methanol, ethanol, acetone, tetrahydrofuran and dichloromethane.

9. The preparation method according to claim 8, characterized in that: The organic solvent is ethanol.

10. Use of the nano-micelle preparation according to any one of claims 1 to 6 in preparing a drug for treating multiple myeloma.

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