Patent Application
20240009228
The invention belongs to the technical field of biomedicine, in particular, to a PEGylated rapamycin compound and a preparation method therefor and an application thereof.
In 2018, 8 of the TOP 10 drugs with the largest sales and sales growth in the world are monoclonal antibodies. The global biopharmaceutical industry is growing rapidly. Compared with small-molecule drugs, most biological drugs are immunogenic, and they will cause an immune response and produce anti-drug antibodies after entering the body, thereby neutralizing or changing the pharmacokinetics and biodistribution of biological drugs, and more seriously inducing life-threatening toxic side effects such as hypersensitivity reactions in the body. Therefore, inhibiting the production of antidrug antibodies during the treatment with the biological drugs is critical to improving the safety and efficacy of the biological drugs.
Immunosuppressants refer to drugs that inhibit the immune response of the body. They can inhibit the proliferation and function of cells related to the immune response (macrophages such as T cells and B cells), thereby reducing the antibody immune response. The immunosuppressants are mainly used for organ transplantation to resist rejection and for autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, dermatomycosis, membranous glomerulonephritis, inflammatory bowel disease, and autoimmune hemolytic anemia.
Rapamycin (RAPA), also called sirolimus, is a lipophilic triene nitrogen-containing macrolides antibiotic immunosuppressant produced by Streptomyces hygroscopicus, and can be used for adjuvant therapy of cancer, anti-graft rejection and other immune diseases. Rapamycin plays an immunosuppressive role by blocking the signal transduction involved in mammalian target of rapamycin (mTOR), preventing the differentiation of T cells and the maturation of dendritic cells.
Takashi K. Kishimoto first reported that immunosuppressant-rapamycin-loaded poly(lactic acid-co-glycolic acid, PLGA) nanoparticles are immunotolerant, which can effectively inhibit the production of anti-drug antibodies of biological drugs (Nature nanotechnology, 2016, 11(10): 890-899), and defined it as immunotolerant nanoparticles. The above literature reported that rapamycin PLGA nanoparticles prepared by embedding rapamycin on a PLGA carrier have the functions of inducing immune tolerance and inhibiting the production of anti-drug antibodies of biological drugs. PLGA is a synthetic high molecular weight compound, which itself has no pharmacological effect, and is a pharmaceutical excipient product.
A first objective of the invention is to provide a PEGylated rapamycin compound. The compound contains hydrophobic rapamycin moiety and hydrophilic PEG chain, and is a good carrier for the preparation of nanoparticles.
A second objective of the invention is to provide a preparation method for the above compound with mild reaction conditions and simple operations.
A third objective of the invention is to provide an application of the above compound. The compound is prepared into nano-drugs, freeze-dried preparations, pharmaceutical compositions and has a use in preparing drugs used to reduce the immune response.
A PEGylated rapamycin compound represented in formula (I), wherein n is 10-150.
The invention further discloses a preparation method for the above PEGylated rapamycin compound, which includes: dissolving mPEG-COOH in an organic solvent, adding catalysts EDC-HCl, DMAP and RAPA, and performing the reaction by stirring at 0-40° C. in the dark.
In an embodiment of the invention, a reaction temperature of the preparation method is preferably 20˜30° C.
In an embodiment of the invention, the organic solvent is one or both of dichloromethane and chloroform, preferably dichloromethane.
In an embodiment of the invention, a molar ratio of the mPEG-COOH to the RAPA is 5:1˜1:5, preferably 3:1-1:3.
In an embodiment of the invention, the preparation method further includes a separation and purification step after the reaction, wherein the separation and purification step is dialysis purification or silica gel column chromatography.
In an embodiment of the invention, the dialysis purification uses a dialysis bag and a dialysis solvent for separation and purification. A molecular weight cutoff of the dialysis bag is 500-5000, preferably 1500. The dialysis solvent is one or more of DMSO, halogenated hydrocarbon, tetrahydrofuran and ultrapure water, preferably one or both of DMSO and ultrapure water, more preferably DMSO and ultrapure water.
In an embodiment of the invention, an elution mode of silica gel column chromatography is isocratic elution or gradient elution, preferably gradient elution.
In an embodiment of the invention, an eluent used in the gradient elution is two or more of dichloromethane, ethyl acetate and anhydrous methanol, preferably a mixed solvent of dichloromethane and anhydrous methanol. A ratio of dichloromethane to anhydrous methanol (v/v) is 100:1-10:1, preferably 50:1-20:1.
The invention further discloses a nano-drug, which includes the PEGylated rapamycin compound in an effective drug loading, preferably consisting of the PEGylated rapamycin compound in an effective drug loading and a free rapamycin.
In an embodiment of the invention, a particle size of the PEGylated rapamycin nanoparticle is 5-1000 nm, preferably 50-200 nm.
In an embodiment of the invention, a drug loading of the PEGylated rapamycin nanoparticle is 15%-100%, preferably 25%-85%.
The invention further discloses a preparation method for the nano-drug, which is prepared by a bottom-up approach. The preparation method for the PEGylated rapamycin nanoparticle includes emulsification/solvent evaporation, nanoprecipitation, thin film dispersion, self-assembly or SPG membrane emulsification, preferably emulsification/solvent evaporation.
In an embodiment of the invention, the emulsification/solvent evaporation includes: dissolving the PEGylated rapamycin compound or dissolving the PEGylated rapamycin compound and the rapamycin in an organic solvent to form an organic phase, which are then added to an aqueous phase containing polyvinyl alcohol to prepare an oil-in-water emulsion by high-speed stirring, sonication, vortexing and/or using a high-pressure homogenizer, and finally obtaining a PEGylated rapamycin nanoparticle solution by evaporating and removing the organic solvent.
In an embodiment of the invention, an organic solvent used in the emulsification/solvent evaporation is one or both of chloroform and CH2Cl2, preferably CH2Cl2.
In an embodiment of the invention, a concentration of the PEGylated rapamycin in the emulsification/solvent evaporation is 0.1-10 mg/mL in the organic phase, preferably 0.5-5 mg/mL.
In an embodiment of the invention, the emulsification/solvent evaporation further includes the free rapamycin, and a ratio of the free rapamycin to the PEGylated rapamycin is 0-20/1 (w/w), preferably 1/5-5/1 (w/w).
In an embodiment of the invention, a ratio of the organic phase to the aqueous phase in the emulsification/solvent evaporation is 1/1-1/100 (v/v), preferably 1/2-1/10 (v/v).
In an embodiment of the invention, a concentration of the polyvinyl alcohol in the emulsification/solvent evaporation is 0-5% (w/v) in the aqueous phase, preferably 0.5%-2% (w/v).
The invention further discloses a freeze-dried preparation, which is prepared by freeze-drying an aqueous solution of the PEGylated rapamycin nanoparticle and a freeze-drying protective agent.
In an embodiment of the invention, the freeze-drying protective agent is one or more of sucrose, lactose, mannitol, glucose, trehalose and maltose.
In an embodiment of the invention, a concentration of the freeze-drying protective agent in aqueous solution of the PEGylated rapamycin nanoparticle is 0.1%-20% (w/v), preferably 2%-8% (w/v).
In an embodiment of the invention, a pre-freezing temperature used in preparing the freeze-dried preparation is lower than −10° C., preferably −30° C.-−50° C. The pre-freezing methods are fast freezing and slow freezing, preferably fast freezing.
The invention further discloses a pharmaceutical composition, which includes a therapeutically effective dose of the PEGylated rapamycin compound and a pharmaceutically acceptable carrier.
In an embodiment of the invention, a biological drug is further included.
In an embodiment of the invention, the biological drug is a protein and polypeptide drug, one or more of urate oxidase, enzymes and coenzyme drugs, nucleic acid and degradation products and derivatives thereof, cell growth factors or cytokines, preferably urate oxidase.
The invention further discloses an application of the PEGylated rapamycin compound, the nano-drug, the freeze-dried preparation or the pharmaceutical composition in preparing a drug for reducing an immune response.
Compared with the conventional art, the present invention has the following beneficial effects:
In the invention, an amphiphilic PEGylated rapamycin is prepared by linking hydrophilic polyethylene glycol to hydrophobic rapamycin by esterification, and then the PEGylated rapamycin alone or the PEGylated rapamycin together with rapamycin are used to prepare the PEGylated rapamycin nanoparticle with good water solubility, so that the invention may be effectively used for inhibiting the production of anti-drug antibodies of biological drugs such as urate oxidase and the like, and has a good clinical application prospect.
The following further describes the present invention in combination with specific embodiments. It should be understood that these embodiments are only used for illustrating the present invention, but not for the limitation of the scope of the present invention. Improvements and adjustments made by those skilled in the art according to the invention in practical applications still belong to the protection scope of the invention.
The raw materials, reagents, instruments, etc. used in the invention may be purchased through commercial channels. The abbreviated terms involved are as follows: EDC·HCl: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DMAP: dimethylaminopyridine; DMSO: dimethyl sulfoxide; PVA: polyvinyl alcohol; DLS: dynamic light scattering.
mPEG-COOH (PEG for short, with an average molecular weight of about 2000, 0.2 g, 0.1 mmol) is dissolved in CH2Cl2 (8 mL), and then RAPA (0.1 g, 0.1 mmol), EDC·HCl (0.04 g, 0.2 mmol) and DMAP (0.024 g, 0.2 mmol) are added for oscillating/stirring to be fully dissolved; then the reaction is performed in the dark at room temperature for 36 hours, and a reaction solution is concentrated to obtain a concentrated solution, which is dissolved in DMSO (2 mL) to obtain a solution; the solution is placed in a dialysis bag (molecular weight cut-off: 1500 Da), dialyzed with DMSO and ultrapure water for 1 d and 2 d respectively while changing a dialysis medium every 4 h; finally, a dialysate is freeze-dried to obtain the PEGylated rapamycin of 0.074 g with a yield of 23.2%.
mPEG-COOH (PEG for short, with an average molecular weight of about 2000, 0.3007 g, 0.15 mmol) is dissolved in CH2Cl2 (50 mL), and then RAPA (0.2722 g, 0.30 mmol), EDC·HCl (0.0289 g, 0.15 mmol) and DMAP (0.0020 g, 0.15 mmol) are added for oscillating/stirring to be fully dissolved; then the reaction is performed in the dark at room temperature for 8 hours, and a reaction solution is concentrated to obtain a concentrated solution containing the PEGylated rapamycin with a crude yield of 12.1%.
mPEG-COOH (PEG for short, with an average molecular weight of about 2000, 1.196 g, 0.60 mmol) is dissolved in CH2Cl2 (50 mL), and then RAPA (0.2754 g, 0.30 mmol), EDC·HCl (0.1157 g, 0.60 mmol) and DMAP (0.0070 g, 0.06 mmol) are added for oscillating/stirring to be fully dissolved; then the reaction is performed in the dark at room temperature for 4 hours, and a reaction solution is concentrated to obtain a concentrated solution containing the PEGylated rapamycin with a crude yield of 39.8%.
mPEG-COOH (PEG for short, with an average molecular weight of about 2000, 1.8027 g, 0.90 mmol) is dissolved in CH2Cl2 (20 mL), and then RAPA (0.2758 g, 0.30 mmol), EDC·HCl (0.1727 g, 0.90 mmol) and DMAP (0.0114 g, 0.90 mmol) are added for oscillating/stirring to be fully dissolved; then the reaction is performed in the dark at room temperature for 24 hours, and a reaction solution is concentrated to obtain a concentrated solution containing the PEGylated rapamycin with a crude yield of 59.1%.
mPEG-GOOH (PEG for short, with an average molecular weight of about 2000, 1.8011 g, 0.90 mmol) is dissolved in CH2Cl2 (10 mL), and then RAPA (0.2754 g, 0.30 mmol), EDC·HCl (0.1720 g, 0.90 mmol) and DMAP (0.0105 g, 0.09 mmol) are added for oscillating/stirring to be fully dissolved; then the reaction is performed in the dark at room temperature for 18 hours, and a reaction solution is concentrated to obtain a concentrated solution containing the PEGylated rapamycin with a crude yield of 62.1%. The column chromatography is used to purify the concentrated solution containing the PEGylated rapamycin, with a flow shown in Embodiments 6 to 8 as below.
In the concentrated solution containing the PEGylated rapamycin, a ratio of each component (PEG-RAPA:RAPA: by-products of PEGylation of rapamycin) is 52:15:9 (w/w); the silica gel column chromatography is used, an eluent is dichloromethane/anhydrous methanol (50:1/30:1/20:1), and an elution time is 25 min/40 min/120 min; a yield of PEG-RAPA is 85.3%, a purity of PEG-RAPA is 95.9%, a removal rate of RAPA is 93.2%, and a removal rate of PEGylated by-products of rapamycin is 90.0%.
In the concentrated solution containing the PEGylated rapamycin, a ratio of each component (PEG-RAPA:RAPA: by-products of PEGylation of rapamycin) is 48:13:12 (w/w); the silica gel column chromatography is used, an eluent is dichloromethane/anhydrous methanol (50:1/30:1/10:1), and an elution time is 25 min/50 min/25 min; a yield of PEG-RAPA is 80.8%, a purity of PEG-RAPA is 95.1%, a removal rate of RAPA is 92.8%, and a removal rate of PEGylated by-products of rapamycin is 90.7%.
In the concentrated solution containing the PEGylated rapamycin, a ratio of each component (PEG-RAPA:RAPA: by-products of PEGylation of rapamycin) is 41:11:16 (w/w); the silica gel column chromatography is used, an eluent is dichloromethane/anhydrous methanol (50:1/40:1/30:1), and an elution time is 25 min/70 min/200 min; a yield of PEG-RAPA is 63.1%, a purity of PEG-RAPA is 98.5%, a removal rate of RAPA is 96.4%, and a removal rate of PEGylated by-products of rapamycin is 100.0%.
In the invention, a hydroxyl group on the 40th carbon atom on rapamycin molecule and mPEG-COOH with the structure shown in the following formula (II) is subjected to esterification to obtain the PEGylated rapamycin with the structure shown in the following formula (I), wherein the results of the structural characterization are shown in
In
Combined with the NMR spectra of mPEG-GOGH, rapamycin and PEGylated rapamycin, it is found in
The molecular structure of the PEGylated rapamycin is shown in
The structural formula of mPEG-COOH is:
20 mg PEG-RAPA and 2 mg RAPA are weighed, and added with 5 mL CH2Cl2 to be fully dissolved to obtain an organic phase; 50 mL of an aqueous solution containing 0.500 PVA is taken as an aqueous phase, and then the organic phase is dripped into the aqueous phase with a syringe while performing ultrasound by using probe ultrasound under ice bath conditions, so as to obtain a white emulsion after performing ultrasonic emulsification for 10 min. The organic solvent is removed by a rotary evaporator at 40° C., and centrifuged at 4000 r/min for 5 min to remove unencapsulated drugs and larger particles for taking a supernatant, so as to obtain the PEGylated rapamycin nanoparticle solution.
A particle size and a polydispersity index (PDI) of the PEGylated rapamycin nanoparticle solution are determined by DLS method.
100 μL PEGylated rapamycin nanoparticle solution is taken, added with acetonitrile until the volume reaches 1 mL, and then is subjected to ultrasound for 20 min to destroy the nanoparticle structure, so that the rapamycin is released free into the solution; then the solution is centrifuged at 12000 r/min for 10 min for taking a supernatant, which is filtered by 0.22 μm microporous membrane; finally, a concentration of RAPA is determined by HPLC analysis, and an encapsulation efficiency and a drug loading of the PEGylated rapamycin nanoparticle are determined.
The test results are shown in Table 1.
20 mg PEG-RAPA and 4 mg RAPA are weighed, and added with 20 mL CH2Cl2 to be fully dissolved to obtain an organic phase; 40 mL of an aqueous solution containing 1% PVA is taken as an aqueous phase, and then the organic phase is dripped into the aqueous phase with a syringe while performing ultrasound by using probe ultrasound under ice bath conditions, so as to obtain a white emulsion after performing ultrasonic emulsification for 20 min. The organic solvent is removed by a rotary evaporator at 40° C., and centrifuged at 4000 r/min for 5 min to remove unencapsulated drugs and larger particles for taking a supernatant, so as to obtain the PEGylated rapamycin nanoparticle solution.
The particle size and PDI of the PEGylated rapamycin nanoparticle solution are determined by DLS method.
100 μL PEGylated rapamycin nanoparticle solution is taken, added with acetonitrile until the volume reaches 1 mL, and then is subjected to ultrasound for 20 min to destroy the nanoparticle structure, so that the rapamycin is released free into the solution; then the solution is centrifuged at 12000 r/min for 10 min for taking a supernatant, which is filtered by 0.22 μm microporous membrane; finally, a concentration of RAPA is determined by HPLC analysis, and an encapsulation efficiency and a drug loading of the PEGylated rapamycin nanoparticle are determined.
The test results are shown in Table 2.
20 mg PEG-RAPA and 8 mg RAPA are weighed, and added with 40 mL CH2Cl2 to be fully dissolved to obtain an organic phase; 120 mL of an aqueous solution containing 2% PVA is taken as an aqueous phase, and then the organic phase is dripped into the aqueous phase with a syringe while performing ultrasound by using probe ultrasound under ice bath conditions, so as to obtain a white emulsion after performing ultrasonic emulsification for 30 min. The organic solvent is removed by a rotary evaporator at 40° C., and centrifuged at 4000 r/min for 5 min to remove unencapsulated drugs and larger particles for taking a supernatant, so as to obtain the PEGylated rapamycin nanoparticle solution.
The particle size and PDI of the PEGylated rapamycin nanoparticle solution are determined by DLS method.
100 μL PEGylated rapamycin nanoparticle solution is taken, added with acetonitrile until the volume reaches 1 mL, and then is subjected to ultrasound for 20 min to destroy the nanoparticle structure, so that the rapamycin is released free into the solution; then the solution is centrifuged at 12000 r/min for 10 min for taking a supernatant, which is filtered by 0.22 μm microporous membrane; finally, a concentration of RAPA is determined by HPLC analysis, and an encapsulation efficiency and a drug loading of the PEGylated rapamycin nanoparticle are determined.
The test results are shown in Table 3.
The PEGylated rapamycin nanoparticle solution prepared according to Embodiments 10 to 12 is centrifuged at 12000 r/min for 45 min for removing a supernatant and removing PVA, and then after the precipitate is resuspended with ultrapure water, a concentrated nanoparticle aqueous solution is obtained. 2 mL nanoparticle aqueous solution is taken into 10 mL penicillin vial respectively, and added with a freeze-drying protective agent (with a mass fraction of 5%) for freeze-drying.
(1) Preparation of Freeze-Dried Powder Injection of PEGylated Rapamycin Nanoparticle Using Different Freeze-Drying Protective Agents
The mass fraction of the freeze-drying protective agent is 5%. 0.1 g of freeze-drying protective agent is taken into 10 mL penicillin vial, and dissolved using 2 mL PEGylated rapamycin nanoparticle aqueous solution respectively for freeze-drying. The appearance, the reconstitution speed and the clarity of the prepared freeze-dried powder injection of nanoparticle are observed, and the particle size and PDI of the reconstituted nanoparticle are determined.
The evaluation on quality for the preparation of freeze-dried powder injection of PEGylated rapamycin nanoparticle using different freeze-drying protective agents is shown in Table 4.
(2) Preparation of Freeze-Dried Powder Injection of PEGylated Rapamycin Nanoparticle Using Different Pre-Freezing Temperatures
The freeze-dried powder injection of PEGylated rapamycin nanoparticle is prepared under pre-freezing temperatures of −35° C. and −45° C. respectively. The appearance, the reconstitution speed and the clarity of the prepared freeze-dried powder injection of nanoparticle are observed, and the particle size and PDI of the reconstituted nanoparticle are determined.
The evaluation on quality for the preparation of freeze-dried powder injection of PEGylated rapamycin nanoparticle using different pre-freezing temperatures is shown in Table 5.
(3) Preparation of Freeze-Dried Powder Injection of PEGylated Rapamycin Nanoparticle Using Different Pre-Freezing Methods
The freeze-dried powder injection of PEGylated rapamycin nanoparticle is prepared by using fast freezing method and slow freezing method respectively. The appearance, the reconstitution speed and the clarity of the prepared freeze-dried powder injection of nanoparticle are observed, and the particle size and PDI of the reconstituted nanoparticle are determined.
The evaluation on quality for the preparation of freeze-dried powder injection of PEGylated rapamycin nanoparticle using different pre-freezing methods is shown in Table 6.
The materials used in the test are: the PEGylated rapamycin nanoparticle prepared in the invention (particle size of nanoparticle of 160.4 nm, and content of rapamycin of 663.8 μg/vial); the rapamycin PLGA nanoparticle (particle size of nanoparticle of 170.5 nm, and content of rapamycin of 294.8 g/vial); recombinant candida urate oxidase (content of 0.72 mg/mL*7 mL/vial), from Shenyang R&D Center of Shenyang 3sbio Inc.
45 mice are randomly divided into 3 groups according to body weight, 15 mice in each group. The groups and doses are shown in Table 7.
Group 1, group 2, and group 3 are administered twice a week for 4 consecutive weeks (according to the detection results on anti-urate-oxidase antibody, the administration period may be extended), and each group was administered intravenously to mice. Urate oxidase is mixed with the nanoparticle for administration in groups 2 and 3 before administration.
About 5 days after the last administration, 0.5 mL of mouse whole blood is collected in a non-anticoagulant tube, and the serum is separated and frozen for detection of anti-urate-oxidase antibodies.
The detection results on anti-urate-oxidase antibody are shown in Tables 8 and 9.
Mice in each group are administered by tail vein injection (the gray background indicates that when the tail vein could not be administered, the injection way is changed to intraperitoneal injection for 1-2 times per mouse) for 4 weeks (8 times), wherein Group 2 (PEGylated rapamycin polymer nanoparticle+urate oxidase) has the smallest titer of anti-urate-oxidase antibodies, and Group 3 (PLGA-loaded rapamycin nanoparticle+urate oxidase) is not significantly better than Control Group 1 (urate oxidase control group).
Mice in each group are administered by tail vein injection (the gray background indicates that when the tail vein could not be administered, the injection way is changed to intraperitoneal injection, and the injection should be administered no more than 5 times per mouse) for 6 weeks (12 times), wherein the results are basically the same as those under 4 weeks of administration, and Group 3 has 3 mice with increased titers of antibody.
The combined administration of urate oxidase protein and PEGylated rapamycin nanoparticle may effectively prevent the production of anti-urate-oxidase antibodies in mice, with effects obviously better than the combined administration of urate oxidase protein and rapamycin PLGA nanoparticle.
The preferred embodiments of the present invention disclosed above are only used to help the description of the present invention. The preferred embodiments do not describe all the details, and are not intended to limit the invention only to be the specific embodiments. It is obvious that various modifications and changes can be made to the content of the specification. The present invention selects and specifically describe the embodiments with the purpose of better explain the principle and practical use of the present invention, such that a person skilled in the art can well understand and utilize the present invention. The present invention is merely limited by the appended claims and the scope and equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110365285.9 | Apr 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/083907 | 3/30/2022 | WO |
