Pegylated sirolimus and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to a releasable pegylated sirolimus compound and application thereof. The invention provides a releasable PEGylated sirolimus compound. The compound can be self-assembled to form micelles, prepared into nanoparticles and targeted to immune cells, regulates the release behavior of drugs, reduces the system toxicity and prolongs the biological half-life period. And moreover, the sirolimus-hydrophilic PEG conjugate has a proper hydrophobic sirolimus end and a hydrophilic PEG chain end, can effectively release drugs in immune cells, and is a good carrier for preparing nanoparticles. The invention provides a releasable PEGylated sirolimus compound, a nano-drug and a pharmaceutical composition prepared from the releasable PEGylated sirolimus compound, and application of the releasable PEGylated sirolimus compound in preparation of drugs for reducing immune response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a PEGylated sirolimus compound, its preparation method and application. Background Technology

[0002] The global biopharmaceutical industry is experiencing rapid growth, but compared to small-molecule chemical drugs, most macromolecular drugs are immunogenic. Once in the body, they easily generate anti-drug antibodies, which can neutralize or alter the pharmacokinetics and biodistribution of the biopharmaceutical. More seriously, they may induce life-threatening toxic side effects such as hypersensitivity reactions. Therefore, inhibiting the formation of anti-drug antibodies during biopharmaceutical therapy is crucial for improving the safety and efficacy of biopharmaceuticals.

[0003] Immunosuppressants are drugs that inhibit the body's immune response. They suppress the proliferation and function of cells involved in the immune response, thereby reducing antibody-mediated immune responses. Immunosuppressants are mainly used to treat organ transplant rejection and autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, fungal skin diseases, nephritis, inflammatory bowel disease, and autoimmune hemolytic anemia.

[0004] Sirolimus is a lipophilic triene nitrogen-containing macrolide antibiotic immunosuppressant produced by Streptomyces hygroscopicus. It can be used as an adjunct therapy for cancer, graft rejection, and other immune diseases, and has an immunosuppressive effect.

[0005] Some researchers have combined sirolimus with nanocarriers to reduce the aforementioned excessive immune response. For example, patent application CN201480031937.3 discloses a combination of a therapeutic macromolecule not linked to a synthetic nanocarrier and an immunosuppressant linked to a synthetic nanocarrier for reducing undesirable humoral immune responses. However, the preparation process of sirolimus-nanocarrier conjugates is complex and costly. The nanocarriers have a wide particle size distribution, resulting in poor batch stability and unsatisfactory therapeutic effects, which is not conducive to industrial-scale production and practical application. Some researchers have prepared formulations or nano-formulations by PEGylating sirolimus, but this suffers from reduced drug activity and poor release from the target site. Summary of the Invention

[0006] The primary objective of this invention is to provide a PEGylated sirolimus compound that can self-assemble into micelles, form nanoparticles, target immune cells, regulate drug release behavior, reduce systemic toxicity, and increase biological half-life.

[0007] A second objective of this invention is to provide a PEGylated sirolimus compound having suitable hydrophobic sirolimus ends and hydrophilic PEG chain ends, which can effectively release drugs into immune cells and is a good carrier for preparing nanoparticles.

[0008] A third object of the present invention is to provide an application of the above-mentioned compound, its preparation as a nanomedicine, a pharmaceutical composition, and its use in the preparation of a medicament for reducing immune responses.

[0009] This invention discloses a polyethylene glycol-modified sirolimus, characterized in that the polyethylene glycol-modified sirolimus is composed of polyethylene glycol, a linker peptide, a linker, and sirolimus and its derivatives, wherein the polyethylene glycol and the linker peptide are linked at the N-terminal amino group, and the linker peptide is linked at the C-terminus via a linker and sirolimus and its derivatives, having the general structural formula: PEG—linker peptide—linker—sirolimus and its derivatives.

[0010] In this context, the N-terminus of a linker peptide refers to the amino terminus of the polypeptide, and the C-terminus of a linker peptide refers to the carboxyl terminus of the polypeptide.

[0011] The polyethylene glycol-modified sirolimus disclosed in this invention, in some embodiments, refers to sirolimus and its derivatives as sirolimus, the structure of which is shown in Formula 1:

[0012]

[0013] Formula 1 (The structure contains three hydroxyl groups, located at carbon atoms at positions 10, 28, and 40, respectively)

[0014] In some embodiments, the sirolimus and its derivatives are sirolimus derivatives of Formula 1 that do not contain the hydroxyl groups at positions 28 and 10.

[0015] In some embodiments, the sirolimus and its derivatives are sirolimus derivatives of Formula 1 that do not contain a hydroxyl group at position 10.

[0016] In some embodiments, the sirolimus and its derivatives are sirolimus derivatives of Formula 1 that do not contain the hydroxyl group at position 28.

[0017] The linker of polyethylene glycolated sirolimus of the present invention is covalently bonded to the hydroxyl groups of sirolimus and its derivatives. In some embodiments, the linker has a structure in which the carbonyl group is bonded to the oxygen in the hydroxyl group of sirolimus by forming a carbonate bond.

[0018]

[0019] In some embodiments, the linker has a structure in which the oxygen atom is provided by the oxygen in the sirolimus hydroxyl group.

[0020]

[0021] The polyethylene glycol-modified sirolimus linker peptide of the present invention is characterized in that the linker peptide contains no more than 6 amino acids and their derivatives, and does not contain lysine; in some embodiments, the linker peptide is Val-Cit; in other embodiments, the linker peptide is Val-Ala; in still other embodiments, the linker peptide is Gly-Gly-Phe-Gly.

[0022] In this invention, the C-terminus of the linker peptide and the amino group of the linker are linked by an amide bond. In some embodiments, the linker peptide-linker has one of the following structures:

[0023]

[0024] Note: In the above structure, the N-terminus of the linker peptide is a free amino group. In the PEGylated sirolimus structure, the N-terminal free amino group of the linker peptide exists in the form of an amide bond formed with PEG.

[0025] In this invention, the polyethylene glycol of polystyromycin is linked to the N-terminal amino group of the linker peptide via an amide bond, wherein the polyethylene glycol is polyethylene glycol with an activating group selected from N-hydroxysuccinimide or acyl chloride;

[0026] In some embodiments, the polyethylene glycol is polyethylene glycol succinimide butyrate; in some embodiments, the polyethylene glycol is polyethylene glycol succinimide propionate; and in other embodiments, the polyethylene glycol is polyethylene glycol succinimide acetate.

[0027] The polyethylene glycol used in the preparation of this invention has a molecular weight not higher than 10 kDa, and in some embodiments it is 1 kDa, in some embodiments it is 2 kDa, in some embodiments it is 3 kDa, and in other embodiments it is 5 kDa.

[0028] The sirolimus and its derivatives in the polyethylene glycolated sirolimus of this invention are sirolimus (Formula 1) or sirolimus derivatives. In some embodiments, sirolimus and its derivatives contain a total of three hydroxyl groups located at carbon atoms at positions 10, 28, and 40 (Formula 1). In some embodiments, the linker in the polyethylene glycolated sirolimus is connected to one of the hydroxyl groups at carbon atoms at positions 10, 28, and 40, forming a polyethylene glycolated sirolimus containing one polyethylene glycol molecule. Romulus; in some embodiments, the two linkers in the PEGylated sirolimus are respectively linked to two of the hydroxyl groups on the carbon atoms at positions 10, 28, and 40, forming PEGylated sirolimus containing two polyethylene glycol molecules; in some embodiments, the three linkers in the PEGylated sirolimus are respectively linked to three hydroxyl groups on the carbon atoms at positions 10, 28, and 40, forming PEGylated sirolimus containing three polyethylene glycol molecules.

[0029] The present invention also discloses a nanomedicine formed by the self-assembly of any one or more of the above-mentioned polyethylene glycolated sirolimus components.

[0030] In some embodiments, the polyethylene glycolated sirolimus nanomedicine is formed from a single PEG-RAPA component with a linker connected to a hydroxyl group on the 40th carbon atom.

[0031] In some embodiments, the polyethylene glycolated sirolimus nanomedicine is formed from multiple PEG-RAPA components linked to one or more hydroxyl groups at carbon atoms 10, 28, and 40.

[0032] The present invention also discloses another nanomedicine comprising any of the above-mentioned PEGylated sirolimus compounds, together with free sirolimus.

[0033] In some embodiments, the particle size of the nanomedicine described in this invention is 50–100 nm.

[0034] In some embodiments, the particle size of the nanomedicine described in this invention is 100–200 nm.

[0035] In some embodiments, the particle size of the nanomedicine described in this invention is 150–200 nm.

[0036] The present invention also discloses that the above-mentioned polyethylene glycolated sirolimus nanomedicine can be used to prepare drugs and compositions for reducing immune responses.

[0037] In some embodiments, the above-described polyethylene glycolated sirolimus nanomedicine is used in the preparation of a drug, which can be used in combination with a drug that has a strong immune response to reduce the immunogenicity of the drug.

[0038] In some embodiments, the drugs with strong immune responses include, but are not limited to, antibodies, peptides, proteases, nucleic acids and cell drugs and their conjugates, carrier drugs, etc.

[0039] In some embodiments, the PEGylated sirolimus nanoparticles of the present invention contain sirolimus compounds that are one of sirolimus linked to one PEG, two PEGs, or three PEGs, or a mixture of these structures. In rats, all of these compounds exhibit an inhibitory effect on phenylalanine lyase-induced immune responses, and the immunosuppressive effects are comparable at the same sirolimus dosage.

[0040] In some embodiments, the polyethylene glycol-modified sirolimus nanoparticles of the present invention exhibit a superior inhibitory effect on phenylalanine lyase-induced immune responses in rats compared to PEG-RAPA nanomedicines that do not contain linkers and linker peptides.

[0041] In some embodiments, the polyethylene glycol-modified sirolimus nanoparticles of the present invention exhibit a superior inhibitory effect on phenylalanine lyase-induced immune responses in rats compared to PEG-RAPA nanomedicines without linkers.

[0042] In some embodiments, the polyethylene glycol-modified sirolimus nanoparticles of the present invention exhibit a superior inhibitory effect on phenylalanine lyase-induced immune responses in rats compared to PEG-RAPA nanomedicines without linker peptides.

[0043] In some embodiments, the polyethylene glycol-modified sirolimus nanoparticles of the present invention exhibit superior inhibitory effects on phenylalanine lyase-induced immune responses in rats compared to PEG-RAPA nanomedicines containing other cleavable linkers and linkers, such as those from Immunomedics. A cleavable non-peptide linker.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] 1. The PEGylated sirolimus of the present invention has a well-defined structure and high purity, and can be used to directly prepare PEG-RAPA nanoparticles.

[0046] 2. The PEGylated sirolimus of the present invention has suitable hydrophobic sirolimus ends and hydrophilic PEG chain ends, and can be used as a carrier for preparing nanoparticles.

[0047] 3. The PEGylated sirolimus nanoparticles and PEGylated sirolimus / sirolimus combined nanoparticles disclosed in this invention can accumulate and release the drug in immune organs such as the spleen, effectively inhibiting the generation of anti-drug antibodies against biopharmaceuticals and inducing immune tolerance. Their effects are superior to non-release PEG-RAPA nanomedicines. Attached Figure Description

[0048] Figure 1 This demonstrates how PEG-RAPA nanoparticles, as described in Example 4, delay the production of antibodies against immunogenic drugs in rats. Detailed Implementation

[0049] Example 1: Preparation and purification of sirolimus (polyethylene glycol)

[0050] All raw materials, reagents, and instruments used in this invention can be purchased commercially. Specifically, 2K-PEG-SPA refers to polyethylene glycol succinimide propionate with a molecular weight of 2000 Da, and Val-Cit-PAB is valine-citrulline-PAB with the following structure: PEG-Val-Cit-PAB is the reaction product of polyethylene glycol and Val-Cit-PAB, PEG-RAPA is PEGylated sirolimus, and RAPA is sirolimus.

[0051] (1) Preparation of PEG-Val-Cit-PAB

[0052] Weigh 100 mg Val-Cit-PAB-OH and add it to 5 mL of 100 mmol / L Na2HPO4-NaH2PO4 buffer solution with a pH of 8.5–10.5. Dissolve the OH completely, add 600 mg 2K-PEG-SPA at a molar ratio of 1:1, add 60 mg triethylamine, and stir the mixture at room temperature for 2 h. After the reaction is complete, freeze-dry overnight to obtain approximately 600 mg of PEG-Val-Cit-PAB product with a recovery rate of approximately 85%.

[0053] (2) Activation of sirolimus

[0054] The flask was heated and purged with nitrogen. 300 mg of sirolimus (Formula 1) and 50 mg of triphosgene (BTC) were dissolved in dry dichloromethane (DCM). The mixture was cooled to 0 °C, and 60 mg of triethylamine was added. The mixture was reacted at 0 °C for 6 hours to obtain the sirolimus activated reaction solution.

[0055] (3) Preparation and purification of polyethylene glycolated sirolimus (PEG-RAPA)

[0056] Dissolve PEG-Val-Cit-PAB from step 1 in 10 ml of dichloromethane, filter, add 300 mg of 4-dimethylaminopyridine (DMAP) to the above solution, add the reaction solution from step 2 (sirolimus activation reaction solution), and react at 0°C for 3–4 hours. After completion, concentrate the reaction solution to obtain a concentrated solution containing PEGylated sirolimus with a crude yield of 56%. The above concentrated solution is subjected to silica gel column chromatography with dichloromethane / anhydrous methanol (60:1–20:1) as the eluent. The PEG-RAPA yield is 62%, the PEG-RAPA purity is 98.5%, and the solution is concentrated to obtain a concentrated solution containing PEGylated sirolimus.

[0057] Example 2: Preparation of polyethylene glycol-modified sirolimus nanoparticle solution

[0058] Weigh 40 mg of PEG-RAPA and add 10 mL of CH2Cl2 to fully dissolve it to obtain the organic phase. Take 50 mL of an aqueous solution containing 0.5% PVA as the aqueous phase. Using a probe, sonicate the organic phase into the aqueous phase while sonicating in an ice-water bath for 10–20 min to obtain a white emulsion. Remove the organic solvent using a rotary evaporator. Centrifuge at 4000 r / min for 5–10 min and collect the supernatant to obtain the PEGylated sirolimus nanoparticle solution. Centrifuge the PEG-RAPA nanoparticle solution at 12000 r / min for 30–50 min, discard the supernatant, resuspend the precipitate in 20 mmol / L PB (pH 7.3) buffer, and then filter aseptically to obtain a concentrated nanoparticle solution.

[0059] The particle size and polydispersity index of PEGylated sirolimus nanoparticle solutions were determined using the DLS method.

[0060] Take a solution of PEGylated sirolimus nanoparticles, adjust the pH to 3-5, add cathepsin b and react for a period of time, then add acetonitrile, sonicate for 20 min to release sirolimus into the solution, centrifuge at 12000 r / min, take the supernatant, filter it through a 0.22 μm microporous membrane, and use HPLC to determine the RAPA concentration and the drug loading of PEGylated sirolimus nanoparticles.

[0061] Average particle size (nm) PDI RAPA drug loading 110.2 0.125 21.4％

[0062] Example 3: Preparation of a solution of polyethylene glycol-modified sirolimus and sirolimus combined nanoparticles

[0063] Weigh 40 mg of PEG-RAPA and 6 mg of RAPA, add 10 mL of CH2Cl2 to dissolve them completely to obtain the organic phase. Take 50 mL of an aqueous solution containing 0.5% PVA as the aqueous phase. Use a probe to sonicate under ice-water bath conditions, while sonicating, to add the organic phase to the aqueous phase. Sonicate for 10-20 min to obtain a white emulsion. Remove the organic solvent using a rotary evaporator. Centrifuge at 4000 r / min for 5-10 min, and collect the supernatant to obtain the PEGylated sirolimus and sirolimus combined nanoparticle solution. Take the PEG-RAPA and RAPA nanoparticle solution, centrifuge at 12000 r / min for 30-50 min, discard the supernatant, resuspend the precipitate in 20 mmol / L PB (pH 7.3) buffer, and then filter aseptically to obtain a concentrated nanoparticle solution.

[0064] The particle size and polydispersity index of PEGylated sirolimus nanoparticle solutions were determined using the DLS method.

[0065] Take a solution of PEGylated sirolimus nanoparticles, adjust the pH to 3-5, add acetonitrile after a period of time, sonicate for 20 min to release sirolimus into the solution, centrifuge at 12000 r / min, take the supernatant, filter it through a 0.22 μm microporous membrane, and use HPLC to determine the RAPA concentration and the drug loading of PEGylated sirolimus nanoparticles.

[0066] Average particle size (nm) PDI RAPA drug loading 130.1 0.136 40.5％

[0067] Example 4: Delayed antibody production of immunogenic drugs in rats by polyethylene glycol-modified sirolimus nanoparticles.

[0068] The ability of PEG-RAPA nanoparticles to induce immune tolerance was evaluated. The evaluation samples included the degradable PEG-RAPA nanoparticle solution prepared in this invention (Example 2), the PEG-RAPA and RAPA combined nanoparticle solution (Example 3), and the non-degradable PEG-RAPA nanoparticle solution (without the degradable linker peptide and linker of this invention, the hydroxyl groups of polyethylene glycol and sirolimus are linked by ester bonds, the nanoparticle preparation method is as in Example 2, and the nanoparticle size is 100.4 nm); recombinant phenylalanine lyase solution (content: 10 mg / mL * 1 mL / bottle), Chongqing Paijin Biotechnology Co., Ltd.

[0069] Forty rats were randomly divided into four groups of 10 each, based on their body weight. Groups 1-4 received subcutaneous injections of recombinant phenylalanine lyase solution once a week for three consecutive weeks. Group 4 served as the control group. Groups 1-3 received intravenous injections of degradable PEG-RAPA nanoparticle solution (Example 2), a combination of PEG-RAPA and RAPA nanoparticle solution (Example 3), and a non-degradable PEG-RAPA nanoparticle solution, respectively, once a week at a dose of 2.5 mg / kg (calculated based on sirolimus). The interval between administration of these solutions and the administration of the recombinant phenylalanine lyase solution was no more than 30 minutes. Serum was collected seven days after each administration, and ADA was detected using an ELISA method.

[0070] The results showed that the combined use of degradable PEG-RAPA nanoparticle solution (Example 2), PEG-RAPA and RAPA combined nanoparticle solution (Example 3), and recombinant phenylalanine lyase effectively inhibited the production of anti-phenylalanine lyase antibodies in mice, and the effect was significantly better than that of non-degradable PEG-RAPA nanoparticle solution (see Example 3). Figure 1 ).

Claims

1. A sirolimus-releasing polyethylene glycol-modified compound, characterized in that, The PEGylated sirolimus is composed of polyethylene glycol, a linker peptide, a linker, and a sirolimus analogue, wherein the polyethylene glycol and the linker peptide are linked at the N-terminal amino group, and the linker peptide is linked at the C-terminus via a linker and a sirolimus analogue, conforming to the general formula: polyethylene glycol—linker peptide—linker—sirolimus analogue.

2. According to claim 1, the sirolimus analogues include sirolimus and its derivatives, wherein the structure of sirolimus is as follows:

3. According to claims 1 and 2, the linker of the polyethylene glycol-modified sirolimus is covalently bonded to the hydroxyl groups of the sirolimus and its derivatives, and the linker includes, but is not limited to, the following structures:

4. The linker peptide of PEGylated sirolimus according to claim 1, characterized in that, The linker peptide contains no more than 6 amino acids and their derivatives, and does not contain lysine, including but not limited to Val-Cit, Val-Ala, and Gly-Gly-Phe-Gly.

5. According to claim 1, the amino groups of the linker peptide C-terminus and the linker of the polyethylene glycolated sirolimus are linked by an amide bond.

6. According to claim 1, the polyethylene glycol of the polyethylene glycolated sirolimus is linked to the N-terminal amino group of the linker peptide via an amide bond, wherein the polyethylene glycol is a polyethylene glycol with an activating group selected from N-hydroxysuccinimide or acyl chloride.

7. According to claims 1 and 6, the molecular weight of the polyethylene glycol is not higher than 10 kDa.

8. According to claims 1 to 7, any of the polyethylene glycolated sirolimus can be used to prepare nanomedicines.

9. According to claim 8, the average particle size distribution of the nanomedicine is a diameter of 100 nm to 200 nm.

10. Any of the polyethylene glycolated sirolimus compounds according to claims 1 to 7, and the nanomedicine according to any one of claims 8 to 9, can be used to prepare drugs and compositions for the purpose of reducing immune responses.

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