PEGylated rapamycin compounds, methods for their preparation and uses

Abstract

The present invention belongs to the field of biopharmaceuticals and relates to a PEGylated rapamycin compound, its preparation method, and use. The compound is represented by formula (I), where n is 10-150. The preparation method involves dissolving mPEG-COOH in an organic solvent, adding EDC·HCl, DMAP, and RAPA as catalysts, and stirring the mixture at 0-40°C in the dark. The compound and its pharmaceutical formulations can be used to prepare medicines that reduce immune responses. The compound can effectively suppress the production of anti-drug antibodies in biopharmaceuticals, such as urate oxidase, and has promising clinical potential, particularly suitable for use in nanomedicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and relates to a PEGylated rapamycin compound, a preparation method thereof, and uses thereof.

Background Art

[0002] Among the top 10 best-selling drugs in the world in 2018 with growing sales, 8 are monoclonal antibodies. The global biopharmaceutical industry is growing rapidly. However, compared with small molecule drugs, most biopharmaceuticals are immunogenic, causing an immune response after entering the body, generating anti-drug antibodies, thereby neutralizing or changing the pharmacokinetics and biodistribution of biopharmaceuticals. In severe cases, it may induce life-threatening toxicities and side effects such as hypersensitivity reactions in the body. Therefore, suppressing the generation of anti-drug antibodies during the treatment with biopharmaceuticals is very important for enhancing the safety and effectiveness of biopharmaceuticals.

[0003] An immunosuppressant is a drug that has the effect of suppressing the immune response of the body. By suppressing the proliferation and function of cells related to the immune response (such as macrophages such as T cells and B cells), the antibody immune response can be reduced. Immunosuppressants are mainly used for the prevention of rejection reactions in organ transplantation and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, dermatomycosis, membranous glomerulonephritis, inflammatory bowel disease, and autoimmune hemolytic anemia.

[0004] Rapamycin (RAPA) is also called sirolimus and is a lipophilic triene nitrogen-containing macrolide antibiotic immunosuppressant produced by Streptomyces hygroscopicus. It can be applied to the adjuvant treatment of cancer, anti-graft rejection reactions, and other immune diseases. Rapamycin exerts an immunosuppressive effect by blocking the signal transduction involving the mammalian target of rapamycin (mTOR) in mammals, thereby preventing the differentiation of T cells and the maturation of dendritic cells.

[0005] Takashi K. Kishimoto first reported that poly(lactic acid-co-glycolic acid) (PLGA) nanoparticles loaded with the immunosuppressant rapamycin have immunotolerance and can effectively suppress the generation of anti-drug antibodies by biopharmaceuticals (Nature nanotechnology, 2016, 11(10): 890-899), and defined these as immunotolerant nanoparticles. The above-mentioned literature reported that rapamycin PLGA nanoparticles prepared by embedding rapamycin in a PLGA carrier have the function of inducing immunotolerance and suppressing the generation of anti-drug antibodies by biopharmaceuticals. PLGA is an artificially synthesized high-molecular-weight compound that itself has no pharmacological effect and is a pharmaceutical adjuvant.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The first object of the present invention is to provide a PEGylated rapamycin compound containing a hydrophobic rapamycin moiety and a hydrophilic PEG chain, which is a carrier suitable for the preparation of nanoparticles.

[0007] The second object of the present invention is to provide a method for preparing the above compound under mild reaction conditions and easy to operate.

[0008] The third object of the present invention is to provide the above compound, the nano-pharmaceuticals prepared using the above compound, freeze-dried preparations, pharmaceutical compositions, and their uses in the preparation of pharmaceuticals for reducing immune responses.

Means for Solving the Problems

[0009] A PEGylated rapamycin compound represented by formula (I), wherein n is 10 to 150. JPEG0007703023000001.jpg84170

[0010] The present invention also discloses a method for preparing the PEGylated rapamycin compound, which comprises dissolving mPEG-COOH in an organic solvent, adding EDC·HCl, DMAP, and RAPA as catalysts, and stirring and reacting at 0 to 40°C under light shielding.

[0011] In one embodiment of the present invention, the reaction temperature of the preparation method is preferably 20 to 30°C.

[0012] In one embodiment of the present invention, the organic solvent is one or two of dichloromethane or chloroform, preferably dichloromethane.

[0013] In one embodiment of the present invention, the molar ratio of mPEG-COOH to RAPA is 5:1 to 1:5, preferably 3:1 to 1:3.

[0014] In one embodiment of the present invention, it further includes a separation and purification step after the reaction, and the separation and purification step uses a dialysis purification method or a silica gel column chromatography method.

[0015] In one embodiment of the present invention, the dialysis purification method performs separation and purification using a dialysis bag and a dialysis solvent. Here, the fractionation molecular weight of the dialysis bag is 500 to 5000, preferably 1500. The dialysis solvent is one or more of DMSO, halogenated hydrocarbon, tetrahydrofuran, and ultrapure water, preferably one or two of DMSO and ultrapure water, more preferably DMSO and ultrapure water.

[0016] In one embodiment of the present invention, the elution method by the silica gel column chromatography method is isocratic elution or gradient elution, preferably gradient elution.

[0017] In one embodiment of the present invention, the eluent used for the gradient elution is two or more of dichloromethane, ethyl acetate, and anhydrous methanol, preferably a mixed solvent of dichloromethane and anhydrous methanol. Here, the ratio of dichloromethane to anhydrous methanol (v / v) is 100:1 to 10:1, preferably 50:1 to 20:1.

[0018] The present invention also discloses a nano-drug comprising an effective loading amount of the above-mentioned PEGylated rapamycin compound, preferably an effective loading amount of the above-mentioned PEGylated rapamycin compound and free rapamycin.

[0019] In one embodiment of the present invention, the particle size of the PEGylated rapamycin nanoparticles is 5 to 1000 nm, preferably 50 to 200 nm.

[0020] In one embodiment of the present invention, the loading amount of the PEGylated rapamycin nanoparticles is 15% to 100%, preferably 25% to 85%.

[0021] The present invention also discloses a method for preparing the above nano-drug by a bottom-up method. The method for preparing the PEGylated rapamycin nanoparticles includes an emulsification / solvent evaporation method, a nanoprecipitation method, a thin film dispersion method, a self-assembly method or an SPG membrane emulsification method, preferably an emulsification / solvent evaporation method.

[0022] In one embodiment of the present invention, in the emulsification / solvent evaporation method, the PEGylated rapamycin compound, or the PEGylated rapamycin compound and rapamycin are dissolved in an organic solvent to form an organic phase, and then added to an aqueous phase containing polyvinyl alcohol, and an oil-in-water emulsion is formed by high-speed stirring, ultrasonic waves, vortex shaking and / or a high-pressure homogenizer. Finally, the organic solvent is evaporated and removed to obtain a PEGylated rapamycin nanoparticle solution.

[0023] In one embodiment of the present invention, the organic solvent used in the emulsification / solvent evaporation method is one or two of chloroform and CH2Cl2, preferably CH2Cl2.

[0024] In one embodiment of the present invention, in the emulsification / solvent evaporation method, the concentration of the PEGylated rapamycin in the organic phase is 0.1 to 10 mg / mL, preferably 0.5 to 5 mg / mL.

[0025] In one embodiment of the present invention, free rapamycin is further included in the emulsification / solvent evaporation method, and the ratio of the free rapamycin / the PEGylated rapamycin is 0 to 20 / 1 (w / w), preferably 1 / 5 to 5 / 1 (w / w).

[0026] In one embodiment of the present invention, in the emulsification / solvent evaporation method, the ratio of the organic phase / the aqueous phase is 1 / 1 to 1 / 100 (v / v), preferably 1 / 2 to 1 / 10 (v / v).

[0027] In one embodiment of the present invention, in the emulsification / solvent evaporation method, the concentration of the polyvinyl alcohol in the aqueous phase is 0 to 5% (w / v), preferably 0.5% to 2% (w / v).

[0028] The present invention also discloses a lyophilized preparation obtained by lyophilizing an aqueous solution of the above PEGylated rapamycin nanoparticles and a lyoprotectant.

[0029] In one embodiment of the present invention, the lyoprotectant is one or more of sucrose, lactose, mannitol, glucose, trehalose, and maltose.

[0030] In one embodiment of the present invention, the concentration of the lyoprotectant in the aqueous solution of the PEGylated rapamycin nanoparticles is 0.1% to 20% (w / v), preferably 2% to 8% (w / v).

[0031] In one embodiment of the present invention, the pre-freezing temperature used for preparing the lyophilized preparation is < -10 °C, preferably -30 °C to -50 °C. The pre-freezing method is rapid freezing and slow freezing, preferably rapid freezing.

[0032] The present invention also discloses a pharmaceutical composition comprising a therapeutically effective amount of the above PEGylated rapamycin compound and a pharmaceutically acceptable carrier.

[0033] In one embodiment of the present invention, it further includes a biopharmaceutical.

[0034] In one embodiment of the present invention, the biopharmaceutical is one or more of protein and polypeptide pharmaceuticals, urate oxidase, enzyme and coenzyme pharmaceuticals, nucleic acids, their degradation products and derivatives pharmaceuticals, cell growth factors or cytokines, preferably urate oxidase.

[0035] The present invention also discloses the use of the above-mentioned PEGylated rapamycin compound, the above-mentioned nanopharmaceutical, the above-mentioned lyophilized preparation or the above-mentioned pharmaceutical composition in the preparation of a pharmaceutical for reducing the immune response.

Advantages of the Invention

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows. 1. The PEGylated rapamycin of the present invention has a clear structure, high purity, contains a hydrophobic rapamycin moiety and a hydrophilic PEG chain, and is a suitable carrier for the preparation of nanoparticles. 2. The PEGylated rapamycin nanoparticles of the present invention are composed of PEGylated rapamycin and rapamycin. PEGylated rapamycin also serves as a carrier. The rapamycin moiety in the PEGylated rapamycin molecule and the free rapamycin molecule constitute the hydrophobic core of the nanoparticles, and there is a hydrophilic PEG segment on the surface of the nanoparticles. Therefore, the dispersion stability in water is high. 3. The PEGylated rapamycin nanoparticles of the present invention have immune targeting properties, are concentrated in immune organs such as the spleen to release the pharmaceutical, and can effectively suppress the generation of anti-drug antibodies by biopharmaceuticals and cause immune tolerance. The PEGylated rapamycin nanoparticles of the present invention have a high rapamycin content, have a higher effect of suppressing the generation of anti-drug antibodies than rapamycin PLGA nanoparticles, avoid the toxicity and side effects caused by long-term high-dose administration of rapamycin, and do not contain pharmaceutical auxiliary materials such as PLGA. The present invention manufactures amphiphilic PEGylated rapamycin by esterifying hydrophilic polyethylene glycol to hydrophobic rapamycin through an esterification reaction. Then, PEGylated rapamycin alone or PEGylated rapamycin together with rapamycin is made into PEGylated rapamycin nanoparticles with good water solubility, which can effectively inhibit the generation of anti-drug antibodies such as urate oxidase in biopharmaceuticals and have promising clinical prospects.

Brief Description of the Drawings

[0037]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0038] Hereinafter, the present invention will be further described by specific examples. These examples are only for explaining the present invention and do not limit the patent scope of the present invention. All improvements and adjustments made by those skilled in the art based on the present invention when actually applying belong to the patent scope of the present invention.

[0039] All raw materials, reagents, and instruments used in the present invention are commercially available. Abbreviations and terms are as follows. EDC.HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP: dimethylaminopyridine; DMSO: dimethyl sulfoxide; PVA: polyvinyl alcohol; DLS: dynamic light scattering method.

[0040] I. Preparation and Purification of PEGylated Rapamycin

[0041] Example 1 mPEG-COOH (abbreviated as PEG, average molecular weight of about 2000, 0.2 g, 0.1 mmol) was dissolved in CH2Cl2 (8 mL), and RAPA (0.1 g, 0.1 mmol), EDC.HCl (0.04 g, 0.2 mmol), and DMAP (0.024 g, 0.2 mmol) were added. It was completely dissolved by shaking / stirring, reacted at room temperature in the dark for 36 hours, the reaction solution was concentrated to obtain a concentrated solution, the concentrated solution was dissolved in DMSO (2 mL), the obtained solution was put into a dialysis bag (molecular weight cut-off: 1500 Da), and dialysis was carried out sequentially with DMSO and ultrapure water for 1 day and 2 days, and the dialysis medium was exchanged once every 4 hours. The dialysis solution was lyophilized to obtain 0.074 g of PEGylated rapamycin, and the yield was 23.2%.

[0042] Example 2 mPEG-COOH (abbreviated as PEG, average molecular weight of about 2000, 0.3007 g, 0.15 mmol) was dissolved in CH2Cl2 (50 mL), and RAPA (0.2722 g, 0.30 mmol), EDC.HCl (0.0289 g, 0.15 mmol), and DMAP (0.0020 g, 0.15 mmol) were added. It was completely dissolved by shaking / stirring, stirred at room temperature in the dark for 8 hours to react, the reaction solution was concentrated to obtain a concentrated solution containing PEGylated rapamycin, and the crude yield was 12.1%.

[0043] Example 3 mPEG-COOH (abbreviated as PEG, average molecular weight of about 2000, 1.196 g, 0.60 mmol) was dissolved in CH2Cl2 (50 mL), and RAPA (0.2754 g, 0.30 mmol), EDC.HCl (0.1157 g, 0.60 mmol) and DMAP (0.0070 g, 0.06 mmol) were added. It was completely dissolved by shaking / stirring, stirred at room temperature in the dark for 4 hours for reaction, the reaction solution was concentrated to obtain a concentrated solution containing PEGylated rapamycin, and the crude yield was 39.8%.

[0044] Example 4 mPEG-COOH (abbreviated as PEG, average molecular weight of about 2000, 1.8027 g, 0.90 mmol) was dissolved in CH2Cl2 (20 mL), and RAPA (0.2758 g, 0.30 mmol), EDC.HCl (0.1727 g, 0.90 mmol) and DMAP (0.0114 g, 0.90 mmol) were added. It was completely dissolved by shaking / stirring, stirred at room temperature in the dark for 24 hours for reaction, the reaction solution was concentrated to obtain a concentrated solution containing PEGylated rapamycin, and the crude yield was 59.1%.

[0045] Example 5 mPEG-COOH (abbreviated as PEG, average molecular weight of about 2000, 1.8011 g, 0.90 mmol) was dissolved in CH2Cl2 (10 mL), and RAPA (0.2754 g, 0.30 mmol), EDC.HCl (0.1720 g, 0.90 mmol) and DMAP (0.0105 g, 0.09 mmol) were added. It was completely dissolved by shaking / stirring, stirred at room temperature in the dark for 18 hours for reaction, the reaction solution was concentrated to obtain a concentrated solution containing PEGylated rapamycin, and the crude yield was 62.1%. As shown in Examples 6 to 8 below, the concentrated solution containing PEGylated rapamycin was purified by column chromatography.

[0046] Example 6 In the concentrated solution containing PEGylated rapamycin, the ratio of each component is PEG-RAPA:RAPA:by-products of rapamycin PEGylation = 52:15:9 (w / w). Silica gel column chromatography was adopted, the eluent was dichloromethane / anhydrous methanol (50:1 / 30:1 / 20:1), the elution time was 25 min / 40 min / 120 min, the yield of PEG-RAPA was 85.3%, the purity of PEG-RAPA was 95.9%, the removal rate of RAPA was 93.2%, and the removal rate of by-products of rapamycin PEGylation was 90.0%.

[0047] Example 7 In the concentrated solution containing PEGylated rapamycin, the ratio of each component is PEG-RAPA:RAPA:by-products of rapamycin PEGylation = 48:13:12 (w / w). Silica gel column chromatography was adopted, the eluent was dichloromethane / anhydrous methanol (50:1 / 30:1 / 10:1), the elution time was 25 min / 50 min / 25 min, the yield of PEG-RAPA was 80.8%, the purity of PEG-RAPA was 95.1%, the removal rate of RAPA was 92.8%, and the removal rate of by-products of rapamycin PEGylation was 90.7%.

[0048] Example 8 In the concentrated solution of PEGylated rapamycin, the ratio of each component is PEG-RAPA:RAPA:by-products of rapamycin PEGylation = 41:11:16 (w / w). Silica gel column chromatography was adopted, the eluent was dichloromethane / anhydrous methanol (50:1 / 40:1 / 30:1), the elution time was 25 min / 70 min / 200 min, the yield of PEG-RAPA was 63.1%, the purity of PEG-RAPA was 98.5%, the removal rate of RAPA was 96.4%, and the removal rate of by-products of rapamycin PEGylation was 100.0%.

[0049] II. Structural Characterization of PEGylated Rapamycin

[0050] Example 9 In the present invention, mPEG-COOH having a structure represented by the following formula (II) is esterified with the hydroxy group at the 40th carbon atom on the rapamycin molecule to obtain PEGylated rapamycin having a structure represented by the following formula (I), and the structural characterization results are shown in FIGS. 1 and 2. In FIG. 1, 1 Calibration was performed with the hydrogen atom (a) on the methylene of the -CH2-COOH structure in the corresponding mPEG-COOH molecule in the 1H NMR spectrum (the peak area was calibrated to 2.00), and the peak area corresponding to the hydrogen atom (b) in the repeating structural unit of the mPEG-COOH molecule was 183.86. As a result of calculation, the degree of polymerization of mPEG-COOH was 46, and the molecular weight of mPEG-COOH was 2114. In FIG. 2, from the nuclear magnetic resonance spectra of mPEG-COOH, rapamycin, and PEGylated rapamycin, no significant change was observed in the 10-C δ (97.47) of rapamycin, no significant change was observed in the 28-C δ (75.70) of rapamycin, and a significant change was observed in the 40-C δ (79.31) of rapamycin. This indicated that PEGylated rapamycin is an esterification product of the OH linked to 40-C in the rapamycin molecule and mPEG-COOH. The molecular structure of PEGylated rapamycin is shown in FIG. 3. JPEG0007703023000002.jpg86170Structural formula of mPEG-COOH: TIFF0007703023000003.tif42170

[0051] III. Preparation of PEGylated Rapamycin Nanoparticle Solution Example 10 Weighed 20 mg of PEG-RAPA and 2 mg of RAPA, added 5 mL of CH2Cl2 and dissolved them sufficiently to obtain an organic phase. Collected 50 mL of an aqueous solution containing 0.5% PVA as the aqueous phase. Using a probe ultrasonic wave, under ice bath conditions, while performing ultrasonic treatment, the organic phase was dropped into the aqueous phase with a syringe, and ultrasonic emulsification was carried out for 10 min to obtain a white emulsion. Removed the organic solvent with a rotary evaporator at 40 °C, centrifuged at 4000 r / min for 5 min to remove unencapsulated pharmaceuticals and particles with large particle diameters, and obtained a PEGylated rapamycin nanoparticle solution as the supernatant. The particle diameter and polydispersity index (PDI) of the PEGylated rapamycin nanoparticle solution were measured by the DLS method. Added acetonitrile to 100 μL of the PEGylated rapamycin nanoparticle solution to make it 1 mL, carried out ultrasonic treatment for 20 min to destroy the nanoparticle structure, released rapamycin and discharged it into the solution, centrifuged at 12000 r / min for 10 min, filtered the supernatant through a 0.22 μm microporous filter membrane, then measured the RAPA concentration by HPLC analysis, and measured the encapsulation rate and loading amount of the PEGylated rapamycin nanoparticles. The detection results are shown in Table 1. TIFF0007703023000004.tif23170

[0052] Example 11 Weighed 20 mg of PEG-RAPA and 4 mg of RAPA, added 20 mL of CH2Cl2 and dissolved them sufficiently to obtain an organic phase. Collected 40 mL of an aqueous solution containing 1% PVA as the aqueous phase. Adopted a probe ultrasonic wave, under ice bath conditions, while performing ultrasonic treatment, the organic phase was dropped into the aqueous phase with a syringe, and ultrasonic emulsification was carried out for 20 min to obtain a white emulsion. Removed the organic solvent with a rotary evaporator at 40 °C, centrifuged at 4000 r / min for 5 min to remove unencapsulated pharmaceuticals and particles with large particle diameters, and obtained a PEGylated rapamycin nanoparticle solution as the supernatant. The particle diameter and PDI of the PEGylated rapamycin nanoparticle solution were measured by the DLS method. 100 μL of the PEGylated rapamycin nanoparticle solution was added with acetonitrile to make 1 mL, sonicated for over 20 min to disrupt the nanoparticle structure, releasing rapamycin into the solution, centrifuged at 12,000 r / min for 10 min, and the supernatant was filtered through a 0.22-μm microporous filter membrane. Then, the RAPA concentration was measured by HPLC analysis to determine the encapsulation efficiency and loading amount of the PEGylated rapamycin nanoparticles. The detection results are shown in Table 2. TIFF0007703023000005.tif28170

[0053] Example 12 20 mg of PEG-RAPA and 8 mg of RAPA were weighed, 40 mL of CH2Cl2 was added and dissolved thoroughly to obtain an organic phase. 120 mL of an aqueous solution containing 2% PVA was taken as the aqueous phase. Using a probe sonicator under ice bath conditions, the organic phase was dropped into the aqueous phase with a syringe while sonicating, and sonicated for emulsification for 30 min to obtain a white emulsion. The organic solvent was removed by a rotary evaporator at 40 °C, and centrifuged at 4000 r / min for 5 min to remove unencapsulated pharmaceuticals and large particle-sized particles, obtaining a PEGylated rapamycin nanoparticle solution as the supernatant. The particle size and PDI of the PEGylated rapamycin nanoparticle solution were measured by the DLS method. 100 μL of the PEGylated rapamycin nanoparticle solution was added with acetonitrile to make 1 mL, sonicated for over 20 min to disrupt the nanoparticle structure, releasing rapamycin into the solution, centrifuged at 12,000 r / min for 10 min, and the supernatant was filtered through a 0.22-μm microporous filter membrane. Then, the RAPA concentration was measured by HPLC analysis to determine the encapsulation efficiency and loading amount of the PEGylated rapamycin nanoparticles. The detection results are shown in Table 3. TIFF0007703023000006.tif30170 IV. Preparation of PEGylated rapamycin nanoparticle freeze-dried powder injection

[0054] Example 13 The PEGylated rapamycin nanoparticle solution prepared by the methods of Examples 10 to 12 above was centrifuged at 12,000 r / min for 45 min. The supernatant was discarded, PVA was evaporated and removed, and the precipitate was resuspended in ultrapure water to obtain a concentrated aqueous nanoparticle solution. 2 mL of the aqueous nanoparticle solution was placed in a 10 mL vial, a lyoprotectant (5% by mass) was added, and lyophilization was carried out. (1) Preparation of PEGylated rapamycin nanoparticle lyophilized powder injection using various lyoprotectants The lyoprotectant was set at 5% by mass. 0.1 g of the lyoprotectant was placed in a 10 mL vial, dissolved in 2 mL of the aqueous PEGylated rapamycin nanoparticle solution, and then lyophilized. The appearance, redissolution rate, and clarity of the prepared nanoparticle lyophilized powder injection were observed, and the particle size and PDI of the nanoparticles after redissolution were measured. Table 4 shows the evaluation of the quality of PEGylated rapamycin nanoparticle lyophilized powder injections prepared using various lyoprotectants. TIFF0007703023000007.tif109170Note: 1. Appearance: + Shrinks, collapses severely, and does not completely fall off. ++ Partially shrinks, collapses are observed, and adheres slightly to the wall. +++ Does not shrink or collapse and can fall out completely. 2. Redissolution rate: + Ultrasonic treatment for 1 min is required until complete redissolution. ++ Ultrasonic treatment for 30 s is required until complete redissolution. +++ Immediately redissolves. 3. Clarity: + Poor opalescence, obvious turbidity. ++ There is opalescence but there is a little turbidity. +++ Opalescence is clear and there is no turbidity. (2) Preparation of PEGylated rapamycin nanoparticle lyophilized powder injection at various precryogenic temperatures PEGylated rapamycin nanoparticle lyophilized powder injections were prepared at precryogenic temperatures of -35°C and -45°C, respectively. The appearance, redissolution rate, and clarity of the prepared nanoparticle lyophilized powder injections were observed, and the particle size and PDI of the nanoparticles after redissolution were measured. Table 5 shows the evaluation of the quality of PEGylated rapamycin nanoparticle lyophilized powder injections prepared at various precryogenic temperatures. TIFF0007703023000008.tif53170Note: 1. Appearance: + Atrophied, severely collapsed, and cannot be completely detached. ++ Partially atrophied, with collapse observed, and slightly adherent to the wall. +++ Can fall off completely without atrophy or collapse. 2. Redissolution rate: + Ultrasonic treatment for 1 min is required until complete redissolution. ++ Ultrasonic treatment for 30 s is required until complete redissolution. +++ Redissolves immediately. 3. Clarity: + Poor opalescence, with obvious turbidity. ++ There is opalescence but slightly turbid. +++ Opalescence is distinct and there is no turbidity (3) Preparation of lyophilized powder injection of PEGylated rapamycin nanoparticles by various precryogenic methods Lyophilized powder injections of PEGylated rapamycin nanoparticles were prepared by rapid freezing method and slow freezing method respectively. The appearance, redissolution rate, and clarity of the prepared nanoparticle lyophilized powder injections were observed, and the particle size and PDI of the nanoparticles after redissolution were measured Table 6 shows the evaluation of the quality of lyophilized powder injections of PEGylated rapamycin nanoparticles prepared by various precryogenic methods TIFF0007703023000009.tif49170Note: 1. Appearance: + Atrophied, severely collapsed, and cannot be completely detached. ++ Partially atrophied, with collapse observed, and slightly adherent to the wall. +++ Can fall off completely without atrophy or collapse. 2. Redissolution rate: + Ultrasonic treatment for 1 min is required until complete redissolution. ++ Ultrasonic treatment for 30 s is required until complete redissolution. +++ Redissolves immediately. 3. Clarity: + Poor opalescence, with obvious turbidity. ++ There is opalescence but slightly turbid. +++ Opalescence is distinct and there is no turbidity

[0055] V. Test on the effect of reducing anti-uricase antibody in mouse body by the combined use of rapamycin nanoparticles and uricase Example 14 The PEGylated rapamycin nanoparticles prepared in the present invention (nanoparticle particle size: 160.4 nm, rapamycin content: 663.8 μg per unit), rapamycin PLGA nanoparticles (nanoparticle particle size: 170.5 nm, rapamycin content: 294.8 μg per unit), and recombinant Candida uricase (content: 0.72 mg / mL * 7 mL per unit) are manufactured by the Shenyang Research and Development Center of Shenyang Sansheng Pharmaceutical Co., Ltd. Forty-five mice were divided into three groups of 15 each according to body weight. The grouping and dosage are shown in Table 7. TIFF0007703023000010.tif100170In groups 1, 2, and 3, the administration was carried out twice a week for 4 consecutive weeks (the administration period may be extended according to the detection results of anti-uricase antibodies), and each group was administered by intravenous injection to mice. In groups 2 and 3, uricase and nanoparticles were mixed before administration. Approximately 5 days after the last administration, 0.5 mL of mouse whole blood was collected into a non-anticoagulant tube. After serum separation, it was frozen and stored to detect anti-uricase antibodies. The detection results of anti-uricase antibodies are shown in Tables 8 and 9. JPEG0007703023000011.jpg133170Mice in each group were administered by tail vein injection (the gray background indicates that when tail vein injection cannot be performed, it was changed to intraperitoneal injection, 1 - 2 times per mouse, for 4 weeks (8 times)). In group 2 (PEGylated rapamycin polymer nanoparticles + uricase), the anti-uricase antibody titer was the lowest. In group 3 (PLGA-loaded rapamycin nanoparticles + uricase), no significant superiority was observed compared with control group 1 (uricase control group). JPEG0007703023000012.jpg133170Mice in each group were administered by tail vein injection (the gray background indicates that when tail vein injection cannot be performed, it was changed to intraperitoneal injection, 5 times or less per mouse, for 6 weeks (12 times)). As a result, it was substantially the same as when administered for 4 weeks. Moreover, in group 3, the antibody titers of 3 mice increased. By using uricase protein and PEGylated rapamycin nanoparticles in combination, the generation of anti-uricase antibodies in the mouse body can be effectively blocked, and moreover, an effect significantly superior to that obtained when uricase protein and rapamycin PLGA nanoparticles are used in combination can be obtained.

[0056] The preferred embodiments of the present invention disclosed above are only for explaining the present invention. Details are not specifically described in the preferred embodiments, and the present invention is not limited to the specific embodiments. Of course, various modifications and changes are possible based on the content of this specification. The purpose of explaining these embodiments in detail in this specification is to effectively explain the principle and actual application of the present invention so that those skilled in the art can well understand and utilize the present invention. The present invention is limited only by the scope of the claims and the entire scope and equivalents thereof.

Claims

1. A method for preparing a PEGylated rapamycin compound, wherein the PEGylated rapamycin compound is represented by formula (I), n is 10 to 150, dissolve mPEG-COOH in an organic solvent, add EDC·HCl, DMAP, and RAPA as catalysts, and stir and react at 0 to 40 °C under light shielding to prepare a PEGylated rapamycin compound. A method for preparing a PEGylated rapamycin compound, characterized in that.

2. The method for preparing a PEGylated rapamycin compound according to claim 1, wherein the organic solvent is one or two of dichloromethane or chloroform.

3. The method for preparing a PEGylated rapamycin compound according to claim 1, wherein the molar ratio of mPEG-COOH to RAPA is 5:1 to 1:

5.

4. The method for preparing a PEGylated rapamycin compound according to claim 1, further comprising a separation and purification step after the reaction, wherein the separation and purification step uses a dialysis purification method or a silica gel column chromatography method.

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