A pharmaceutical composition containing linaclotide, and its preparation method and application

Abstract

The present invention discloses a pharmaceutical composition containing linaclotide, a preparation method, and an application thereof, and belongs to the field of pharmaceutical synthesis technology. The pharmaceutical composition containing linaclotide of the present invention comprises linaclotide, polyethylene glycol, an electrolyte agent, and a triazole derivative. In the synthesis of linaclotide, the side chain phenolic hydroxyl group of Fmoc-Tyr-OtBu is first bonded to a 2-chlorotrityl chloride resin, followed by sequential coupling of amino acids, and then a pair of disulfide bonds are oxidized on the resin to obtain linaclotide through resin cleavage and oxidation. In the preparation of the triazole derivative, triethylamine is used as an acid-binding agent to react 3-benzyl-4H-1,2,4-triazole with 4-phenylbutyryl chloride to obtain the triazole derivative. The pharmaceutical composition containing linaclotide of the present invention has excellent biocompatibility and excellent constipation relief effect.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical synthesis, and in particular to a pharmaceutical composition containing linaclotide, a preparation method thereof, and an application thereof. Background Art

[0002] Linaclotide is a guanylate cyclase C (GC-C) agonist with visceral analgesic and secretory effects, which can relieve abdominal pain and constipation symptoms caused by constipation-type irritable bowel syndrome, thereby improving the condition and enhancing the quality of life of patients. 1 -Cys 6 , Cys 2 -Cys 10 , Cys 5 -Cys 13 . Currently, the synthesis of linaclotide mainly focuses on the bonding of the α-carboxyl group of tyrosine to the resin, but the process of using Wang Resin to bond to the α-carboxyl group of tyrosine is cumbersome, and racemic impurities are easily generated when the first amino acid is loaded, and it is accompanied by a diketopiperazine cyclization (DKP) side reaction. Although the use of 2-chlorotrityl chloride resin (2-CTC Resin) to react with the α-carboxyl group can avoid racemization and DKP side reactions, the resin has poor stability, resulting in a reduced yield. Using the side chain phenolic hydroxyl group to bond to the resin can make the resin more stable and avoid racemization and DKP side reaction impurities.

[0003] Linaclotide is a drug used to treat irritable bowel syndrome with chronic constipation (IBS-C) and chronic constipation (CC). However, it can cause common adverse reactions, including diarrhea, abdominal pain, intestinal gas, and headache. Therefore, there is a need for a pharmaceutical composition containing linaclotide that can alleviate constipation symptoms while exhibiting good biocompatibility. Summary of the Invention

[0004] The purpose of the present invention is to provide a pharmaceutical composition containing linaclotide, and a preparation method and application thereof, which not only optimizes the synthesis process of linaclotide and provides good prospects for the preparation process of the pharmaceutical composition containing linaclotide; but also improves the biocompatibility and constipation relief effect of the pharmaceutical composition containing linaclotide.

[0005] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0006] A pharmaceutical composition containing linaclotide, comprising linaclotide, polyethylene glycol and an electrolyte agent; the electrolyte agent comprises sodium bicarbonate, sodium chloride and potassium chloride; the amino acid sequence from the N-terminus to the C-terminus of the linear main chain of linaclotide is H-Cys 1 -Cys 2 -Glu 3-Tyr 4 -Cys 5 -Cys 6 -Asn 7 -Pro 8 -Ala 9 -Cys 10 -Thr 11 -Gly 12 -Cys 13 -Tyr 14 -OH, disulfide bond connection mode is Cys 1 -Cys 6 、Cys 2 -Cys 10 、Cys 5 -Cys 13 .

[0007] The present invention discloses a pharmaceutical composition containing linaclotide, comprising linaclotide, polyethylene glycol and an electrolyte agent; the electrolyte agent comprises sodium bicarbonate, sodium chloride and potassium chloride. In the synthesis of linaclotide, the present invention adopts Fmoc-Tyr-OtBu raw material to couple with 2-chlorotrityl chloride resin, and bonds the phenolic hydroxyl group of the side chain to the resin. Different from the general method of bonding the α-carboxyl group to the resin, the present invention greatly reduces the steric hindrance in the peptide chain synthesis process, improves the reaction efficiency, reduces the possibility of missing amino acids at the C-terminus, and has higher stability of the resin, and the synthesis yield can be greatly improved. Then, a pair of disulfide bonds are oxidized on the resin, which reduces the mismatch rate of the oxidation process and improves the yield of the product. The operation method for synthesizing linaclotide of the present invention is simple, suitable for industrial production, and provides a good prospect for the preparation process of the pharmaceutical composition containing linaclotide.

[0008] Preferably, in the synthesis of linaclotide, solid-phase synthesis technology is used to couple Fmoc-Tyr-OtBu with 2-chlorotrityl chloride resin, and then amino acids with main chain protecting groups are coupled in sequence. Iodine solution is repeatedly added and stirred to react, and linaclotide is obtained after treatment with a cleavage solution and a buffer solution.

[0009] More preferably, the order of coupling amino acids with main chain protecting groups is Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Cys(Acm)-OH.

[0010] More preferably, in the preparation of the iodine solution, elemental iodine is dissolved in DMF to a final concentration of 0.1-1.0 g / mL.

[0011] More preferably, in the preparation of the lysate, TFA, EDT, TIS and deionized water are uniformly mixed to obtain the lysate.

[0012] More preferably, in the preparation of the buffer solution, a cysteine ​​hydrochloride solution and a DMSO solution are mixed, and then the pH is adjusted to 9-10 with aqueous ammonia to obtain a buffer solution; the concentration of the cysteine ​​hydrochloride solution is 0.1-1 mmol / L, the volume concentration of the DMSO solution is 20-40%, and the volume ratio of the cysteine ​​hydrochloride solution to the DMSO solution is 1:1-2.

[0013] Preferably, the pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, an electrolyte agent, and a triazole derivative. The pharmaceutical composition containing linaclotide of the present invention has good biocompatibility. When applied to constipated mice, the pharmaceutical composition containing linaclotide can alleviate constipation by increasing the small intestinal propulsion rate of the mice.

[0014] More preferably, in the preparation of the triazole derivative, triethylamine is used as an acid-binding agent to react 3-benzyl-4H-1,2,4-triazole with 4-phenylbutyryl chloride to obtain the triazole derivative.

[0015] More preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride is 1:1-2.

[0016] More preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1-6.

[0017] Preferably, the synthesis method of linaclotide is specifically as follows:

[0018] S1. Weigh 2-CTC Resin and Fmoc-Tyr-OtBu, add solvent to dissolve, then add DIEA and stir to react for 15-20 hours. After the reaction, drain the reaction solution, wash with detergent 2-5 times, then add PIP solution and react for 15-45 minutes to obtain Tyr-resin-OtBu.

[0019] S2. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Cys(Trt)-Tyr-resin-OtBu.

[0020] S3. Add Fmoc-Gly-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Gly-Cys(Trt)-Tyr-resin-OtBu.

[0021] S4. Add Fmoc-Thr(tBu)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0022] S5. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0023] S6. Add Fmoc-Ala-OH, DIC, and Oxyma, add DMF, and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0024] S7. Add Fmoc-Pro-OH, DIC, and Oxyma, add DMF, and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0025] S8. Add Fmoc-Asn(Trt)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0026] S9. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0027] S10. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, add DMF, and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0028] S11. Add Fmoc-Tyr(tBu)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0029] S12. Add Fmoc-Glu(OtBu)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0030] S13. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0031] S14. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25-30 minutes to obtain Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0032] S15. Add iodine solution and stir to react for 10-20 minutes. After the reaction is completed, drain the reaction solution, add iodine solution again and stir to react for 10-20 minutes. After the reaction is completed, drain the reaction solution, add iodine solution again and stir to react for 10-20 minutes. After the reaction is completed, drain the reaction solution, wash the resin with DMF 2-5 times, add lysis solution and lysis for 2-6 hours, add buffer, stir to react at 15-25°C for 10-40 hours, and separate by high performance liquid chromatography (HPLC) to obtain linaclotide.

[0033] More preferably, the degree of substitution of 2-CTC Resin in step S1 is 0.7-1.0 mmol / g.

[0034] More preferably, in step S1, the ratio of 2-CTC Resin to Fmoc-Tyr-OtBu is 1 g: 1-10 mmol.

[0035] More preferably, in step S1, the solvent is DMF, and the usage ratio of Fmoc-Tyr-OtBu to the solvent is 1 mmol:2-5 mL.

[0036] More preferably, the molar ratio of Fmoc-Tyr-OtBu to DIEA in step S1 is 1:2-10.

[0037] More preferably, in step S1, the detergent is DMF, and the usage ratio of Fmoc-Tyr-OtBu to the detergent is 1 mmol:2-5 mL.

[0038] More preferably, in the preparation of the PIP solution in step S1, PIP is diluted with DMF to a final mass concentration of 10-30% to obtain a PIP solution.

[0039] More preferably, in step S1, the usage ratio of Fmoc-Tyr-OtBu and PIP solution is 1 mmol:2-10 mL.

[0040] More preferably, the molar ratio of Fmoc-Cys(Trt)-OH to DIC in step S2 is 1:1-2.

[0041] More preferably, the molar ratio of Fmoc-Gly-OH to DIC in step S3 is 1:1-2.

[0042] More preferably, the molar ratio of Fmoc-Thr(tBu)-OH to DIC in step S4 is 1:1-2.

[0043] More preferably, the molar ratio of Fmoc-Cys(Trt)-OH to DIC in step S5 is 1:1-2.

[0044] More preferably, the molar ratio of Fmoc-Ala-OH to DIC in step S6 is 1:1-2.

[0045] More preferably, the molar ratio of Fmoc-Pro-OH to DIC in step S7 is 1:1-2.

[0046] More preferably, the molar ratio of Fmoc-Asn(Trt)-OH to DIC in step S8 is 1:1-2.

[0047] More preferably, the molar ratio of Fmoc-Cys(Acm)-OH to DIC in step S9 is 1:1-2.

[0048] More preferably, the molar ratio of Fmoc-Cys(Trt)-OH to DIC in step S10 is 1:1-2.

[0049] More preferably, the molar ratio of Fmoc-Tyr(tBu)-OH to DIC in step S11 is 1:1-2.

[0050] More preferably, the molar ratio of Fmoc-Glu(OtBu)-OH to DIC in step S12 is 1:1-2.

[0051] More preferably, the molar ratio of Fmoc-Cys(Trt)-OH to DIC in step S13 is 1:1-2.

[0052] More preferably, the molar ratio of Fmoc-Cys(Acm)-OH to DIC in step S14 is 1:1-2.

[0053] More preferably, the molar ratio of DIC to Oxyma in steps S2 to S14 is 1:1-2.

[0054] More preferably, the usage ratio of DIC to DMF in steps S2 to S14 is 1 mmol:5-10 mL.

[0055] More preferably, in the preparation of the PIP solution in steps S2 to S14, PIP is diluted with DMF to a final mass concentration of 10-30% to obtain a PIP solution.

[0056] More preferably, the volume ratio of DMF to PIP solution in steps S2 to S14 is 1:1-2.

[0057] More preferably, in the preparation of the iodine solution in step S15, elemental iodine is dissolved in DMF to a final concentration of 0.1-1.0 g / mL to obtain an iodine solution.

[0058] More preferably, in the preparation of the lysis solution in step S15, TFA, EDT, TIS and deionized water are evenly mixed to obtain a lysis solution, the volume ratio of EDT to TFA is 1:15-20, the volume ratio of EDT to TIS is 1:0.2-1; and the volume ratio of TIS to deionized water is 1:1-2.

[0059] More preferably, in the preparation of the buffer solution in step S15, the cysteine ​​hydrochloride solution and the DMSO solution are mixed, and then the pH is adjusted to 9-10 with aqueous ammonia to obtain a buffer solution; the concentration of the cysteine ​​hydrochloride solution is 0.1-1 mmol / L, the volume concentration of the DMSO solution is 20-40%, and the volume ratio of the cysteine ​​hydrochloride solution to the DMSO solution is 1:1-2.

[0060] More preferably, in step S15, the volume ratio of the iodine solution to the lysis solution is 1:1-5.

[0061] More preferably, in step S15, the volume ratio of the iodine solution to the buffer solution is 1:10-20.

[0062] Preferably, a pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol and an electrolyte agent, wherein the electrolyte agent comprises sodium bicarbonate, sodium chloride and potassium chloride.

[0063] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500-1000.

[0064] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200-400.

[0065] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2-8.

[0066] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5-10.

[0067] Preferably, a pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, an electrolyte agent and a triazole derivative, wherein the electrolyte agent comprises sodium bicarbonate, sodium chloride and potassium chloride.

[0068] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500-1000.

[0069] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200-400.

[0070] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2-8.

[0071] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5-10.

[0072] More preferably, the mass ratio of linaclotide to the triazole derivative is 1:200-400.

[0073] More preferably, the mass ratio of linaclotide to theaflavin-3-gallate is 1:100-200.

[0074] Preferably, a pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, an electrolyte, a triazole derivative, and theaflavin-3-gallate, wherein the electrolyte comprises sodium bicarbonate, sodium chloride, and potassium chloride. The present invention further utilizes theaflavin-3-gallate to form the pharmaceutical composition containing linaclotide, which not only further improves cell survival rate and optimizes the biocompatibility of the pharmaceutical composition containing linaclotide, but also helps further increase small intestinal propulsion rate and optimizes the effect of relieving constipation.

[0075] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500-1000.

[0076] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200-400.

[0077] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2-8.

[0078] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5-10.

[0079] More preferably, the mass ratio of linaclotide to the triazole derivative is 1:200-400.

[0080] More preferably, the mass ratio of linaclotide to theaflavin-3-gallate is 1:100-200.

[0081] More preferably, the preparation method of the triazole derivative is specifically as follows:

[0082] 3-Benzyl-4H-1,2,4-triazole was dissolved in tetrahydrofuran, 4-phenylbutyryl chloride was added dropwise at 0-5°C, and the mixture was stirred with triethylamine as an acid-binding agent for 18-30 hours. The solvent was removed by rotary evaporation, and the triazole derivative was purified by column chromatography.

[0083] More preferably, the usage ratio of 3-benzyl-4H-1,2,4-triazole to tetrahydrofuran is 1 mmol:10-40 mL.

[0084] More preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride is 1:1-2.

[0085] More preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1-6.

[0086] The present invention optimizes the synthesis of linaclotide, thereby achieving the following beneficial effects: The present invention utilizes Fmoc-Tyr-OtBu as a raw material coupled with 2-chlorotrityl chloride resin to bond the phenolic hydroxyl group of the side chain to the resin. This, unlike the conventional method of bonding the α-carboxyl group to the resin, significantly reduces steric hindrance during peptide chain synthesis, improves reaction efficiency, and reduces the likelihood of C-terminal amino acid deletions. The resin exhibits enhanced stability and significantly improves synthesis yield. Oxidation of a pair of disulfide bonds is then performed on the resin, reducing the mismatch rate during the oxidation process and improving the purity and yield of the product. The yield of linaclotide is 51.97-58.00%, and the purity is 98.66-98.72%. The present method for synthesizing linaclotide is simple and suitable for industrial production, offering promising prospects for the preparation of pharmaceutical compositions containing linaclotide.

[0087] The present invention utilizes linaclotide, polyethylene glycol, an electrolyte, a triazole derivative, and theaflavin-3-gallate to form a pharmaceutical composition containing linaclotide. The composition exhibits the following beneficial effects: The composition exhibits excellent biocompatibility, with cell survival rates of 87.7-98.8% after treatment; and exhibits excellent constipation-relieving effects, with small intestinal propulsion rates of 58.4-84.1% in constipated mice treated with the composition. Therefore, the present invention provides a pharmaceutical composition containing linaclotide with excellent biocompatibility and constipation-relieving effects, as well as its preparation method and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 The yield and purity of linaclotide. DETAILED DESCRIPTION

[0089] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0090] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0091] The Chinese and source information of the reagents used in the present invention are summarized in Table 1, including: material abbreviations, Chinese names and suppliers.

[0092] Table 1 English abbreviation comparison table

[0093]

[0094] Example 1:

[0095] A method for synthesizing linaclotide, comprising:

[0096] S1. Weigh 2-CTC Resin and Fmoc-Tyr-OtBu, add solvent to dissolve, then add DIEA and stir to react for 15 hours. After the reaction is completed, drain the reaction solution, wash it with detergent three times, and then add PIP solution to react for 25 minutes to obtain Tyr-resin-OtBu. The degree of substitution of 2-CTC Resin is 0.8 mmol / g, and the molar ratio of 2-CTC Resin and Fmoc-Tyr-OtBu is 1 g:4.8 mmol; the solvent is DMF, and the molar ratio of Fmoc-Tyr-OtBu to the solvent is 1 mmol:3.3 mL; the molar ratio of Fmoc-Tyr-OtBu to DIEA is 1:6; the detergent is DMF, and the molar ratio of Fmoc-Tyr-OtBu to the detergent is 1 mmol:3.3 mL; in the preparation of the PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain a PIP solution; the molar ratio of Fmoc-Tyr-OtBu to the PIP solution is 1 mmol:5 mL.

[0097] S2. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Cys(Trt)-Tr-resin-OtBu. The molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL. In the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution. The volume ratio of DMF to PIP solution is 1:1.5.

[0098] S3. Add Fmoc-Gly-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Gly-Cys(Trt)-Tr-resin-OtBu. The molar ratio of Fmoc-Gly-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL. In the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution. The volume ratio of DMF to PIP solution is 1:1.5.

[0099] S4. Add Fmoc-Thr(tBu)-OH, DIC, and Oxyma, then add DMF and stir to react for 2 hours. After the reaction, drain the reaction solution and then add PIP solution to react for 25 minutes to obtain Thr(tBu)-Gly-Cys(Trt)-Tr-resin-OtBu. The molar ratio of Fmoc-Thr(tBu)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0100] S5. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0101] S6. Add Fmoc-Ala-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Ala-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0102] S7. Add Fmoc-Pro-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Pro-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0103] S8. Add Fmoc-Asn(Trt)-OH, DIC, and Oxyma, add DMF, stir and react for 2 hours. After the reaction, drain the reaction solution, then add PIP solution and react for 25 minutes to obtain Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Asn(Trt)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0104] S9. Add Fmoc-Cys(Acm)-OH, DIC, and Oxyma, add DMF, and stir to react for 2 hours. After the reaction, drain the reaction solution, then add PIP solution and react for 25 minutes to obtain Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0105] S10. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL. In the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution. The volume ratio of DMF to PIP solution is 1:1.5.

[0106] S11. Add Fmoc-Tyr(tBu)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Tyr(tBu)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0107] S12. Add Fmoc-Glu(OtBu)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Glu(OtBu)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0108] S13. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction, drain the reaction solution and add PIP solution for 25 minutes to obtain Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the amount ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0109] S14. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir to react for 2 hours. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 minutes to obtain Cys(Acm)-Cys(Trt)-Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu. The molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1; the molar ratio of DIC to Oxyma is 1:1; the usage ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of the PIP solution, PIP is diluted with DMF to a final mass concentration of 20% to obtain a PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0110] S15. Add iodine solution and stir for 15 minutes. After the reaction, drain the reaction solution, add iodine solution again and stir for 15 minutes. After the reaction, drain the reaction solution, add iodine solution again and stir for 15 minutes. After the reaction, drain the reaction solution, wash the resin three times with DMF, add cleavage solution and cleave for 3 hours. Add buffer and stir at 20°C for 20 hours. Separate by high-performance liquid chromatography (HPLC) to obtain linaclotide. To prepare the iodine solution, dissolve iodine in DMF to a final concentration of 0.5 g / mL. To prepare the cleavage solution, mix TFA, EDT, TIS, and deionized water to obtain a cleavage solution. The volume ratio of EDT to TFA is 1:18, the volume ratio of EDT to TIS is 1:0.5, and the volume ratio of TIS to deionized water is 1:1. To prepare the buffer, cysteine ​​hydrochloride solution and DMSO solution were mixed and the pH was adjusted to 10 with aqueous ammonia to obtain the buffer. The concentration of the cysteine ​​hydrochloride solution was 1 mmol / L, the volume concentration of the DMSO solution was 30%, and the volume ratio of the cysteine ​​hydrochloride solution to the DMSO solution was 1:1. The volume ratio of the iodine solution to the lysate was 1:2.5, and the volume ratio of the iodine solution to the buffer was 1:15.

[0111] A pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, sodium bicarbonate, sodium chloride, and potassium chloride. The mass ratio of linaclotide to potassium chloride is 1:1000; the mass ratio of potassium chloride to polyethylene glycol is 1:280; the mass ratio of potassium chloride to sodium bicarbonate is 1:4; and the mass ratio of potassium chloride to sodium chloride is 1:7.5.

[0112] Example 2:

[0113] The synthesis method of linaclotide is the same as that of Example 1, except that the stirring reaction time of adding DIEA in step S1 is replaced with 18 hours, and other conditions are the same as those of Example 1.

[0114] A pharmaceutical composition containing linaclotide, compared with Example 1, except that linaclotide is replaced by linaclotide prepared in this example, other conditions are the same as Example 1.

[0115] Example 3:

[0116] The synthesis method of linaclotide is the same as that of Example 1, except that the stirring reaction time of adding DIEA in step S1 is replaced with 20 hours, and other conditions are the same as those of Example 1.

[0117] A pharmaceutical composition containing linaclotide, compared with Example 1, except that linaclotide is replaced by linaclotide prepared in this example, other conditions are the same as Example 1.

[0118] Example 4:

[0119] The synthesis method of linaclotide is the same as that in Example 1.

[0120] The preparation method of triazole derivatives comprises:

[0121] 3-Benzyl-4H-1,2,4-triazole was dissolved in tetrahydrofuran, and 4-phenylbutyryl chloride was added dropwise at 0°C. The reaction was stirred for 24 hours with triethylamine as an acid binder. The solvent was removed by rotary evaporation, and the product was purified by column chromatography to obtain a triazole derivative. The molar ratio of 3-benzyl-4H-1,2,4-triazole to tetrahydrofuran was 1 mmol:20 mL; the molar ratio of 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride was 1:1; and the molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine was 1:3.

[0122] A pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, sodium bicarbonate, sodium chloride, potassium chloride, and a triazole derivative. The mass ratio of linaclotide to potassium chloride is 1:1000; the mass ratio of potassium chloride to polyethylene glycol is 1:280; the mass ratio of potassium chloride to sodium bicarbonate is 1:4; the mass ratio of potassium chloride to sodium chloride is 1:7.5; and the mass ratio of linaclotide to the triazole derivative is 1:400.

[0123] Example 5:

[0124] The synthesis method of linaclotide is the same as that in Example 1.

[0125] The preparation method of triazole derivatives is the same as that in Example 4.

[0126] A pharmaceutical composition containing linaclotide, compared with Example 4, except that the mass ratio of linaclotide to the triazole derivative is changed to 1:200, other conditions are the same as Example 4.

[0127] Example 6:

[0128] The synthesis method of linaclotide is the same as that in Example 1.

[0129] The preparation method of triazole derivatives is the same as that in Example 4.

[0130] A pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, sodium bicarbonate, sodium chloride, potassium chloride, a triazole derivative, and theaflavin-3-gallate. The mass ratio of linaclotide to potassium chloride is 1:1000; the mass ratio of potassium chloride to polyethylene glycol is 1:280; the mass ratio of potassium chloride to sodium bicarbonate is 1:4; the mass ratio of potassium chloride to sodium chloride is 1:7.5; the mass ratio of linaclotide to the triazole derivative is 1:400; and the mass ratio of linaclotide to theaflavin-3-gallate is 1:200.

[0131] Example 7:

[0132] The synthesis method of linaclotide is the same as that in Example 1.

[0133] The preparation method of triazole derivatives is the same as that in Example 4.

[0134] A pharmaceutical composition containing linaclotide, compared with Example 6, except that the mass ratio of linaclotide to theaflavin-3-gallate is changed to 1:100, other conditions are the same as Example 6.

[0135] Comparative Example 1:

[0136] The synthesis method of linaclotide is the same as that in Example 1.

[0137] A pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, sodium bicarbonate, sodium chloride, potassium chloride, and theaflavin-3-gallate. The mass ratio of linaclotide to potassium chloride is 1:1000; the mass ratio of potassium chloride to polyethylene glycol is 1:280; the mass ratio of potassium chloride to sodium bicarbonate is 1:4; the mass ratio of potassium chloride to sodium chloride is 1:7.5; and the mass ratio of linaclotide to theaflavin-3-gallate is 1:200.

[0138] Experimental example:

[0139] 1. Linaclotide yield and purity test

[0140] The yield and purity of linaclotide prepared in Examples 1-3 were determined by HPLC. The chromatographic conditions were as follows: mobile phase A: water (containing 0.1% TFA); mobile phase B: acetonitrile (containing 0.1% TFA); mobile phase gradient: mobile phase B 15-25%, gradient elution 20 min, flow rate 3 mL / min, detection wavelength 220 nm; column temperature: room temperature. Figure 1 shown.

[0141] Depend on Figure 1 It can be seen that the yield of linaclotide is 51.97-58.00%, and the purity is 98.66-98.72%. This shows that the present invention can synthesize linaclotide with high yield and high purity.

[0142] 2. Biosafety Experiment

[0143] Mouse primary intestinal stem cells were cultured at 5×10 4Cells were seeded at a density of 100 μg / mL in 24-well plates and divided into eight groups: a blank group, a control group, and six experimental groups. 3 L of deionized water was added to the linaclotide-containing pharmaceutical compositions of Example 1, Examples 4-7, and Comparative Example 1, respectively, to obtain dilutions of the linaclotide-containing pharmaceutical compositions of Example 1, Examples 4-7, and Comparative Example 1, respectively. The blank group received no treatment. The control group was treated with deionized water for 24 hours. The six experimental groups were treated with the dilutions of the linaclotide-containing pharmaceutical compositions of Example 1, Examples 4-7, and Comparative Example 1, respectively, for 24 hours. After treatment, cell viability in the experimental groups was assessed using the MTT colorimetric assay (MTT) by measuring the absorbance of the solution at 570 nm using a microplate reader. Cell viability (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%. The cell viability is shown in Table 2.

[0144] Table 2 Cell survival rate (%)

[0145]

[0146] As shown in Table 2, the cell survival rates of Examples 4-5 of the present invention were higher than those of Example 1 because the linaclotide-containing pharmaceutical compositions of Examples 4-5 also included a triazole derivative. The cell survival rate of Example 4 was higher than that of Example 5 because the amount of the triazole derivative used in the linaclotide-containing pharmaceutical composition was different. This demonstrates that the linaclotide-containing pharmaceutical composition of the present invention can improve cell survival rates and has good cell compatibility. The higher cell survival rates in Examples 6-7 of the present invention compared to those in Example 4 and Comparative Example 1 are due to the fact that Examples 6-7 further utilize triazole derivatives and theaflavin-3-gallate in the linaclotide-containing pharmaceutical compositions, while Example 4 utilizes only triazole derivatives, and Comparative Example 1 utilizes only theaflavin-3-gallate. The higher cell survival rate in Example 6 compared to Example 7 is due to the different amounts of theaflavin-3-gallate used in the linaclotide-containing pharmaceutical compositions. This suggests that the synergistic use of linaclotide and theaflavin-3-gallate in the linaclotide-containing pharmaceutical composition can further improve cell survival and enhance the biocompatibility of the linaclotide-containing pharmaceutical composition, compared to the use of linaclotide and theaflavin-3-gallate alone.

[0147] 3. Small intestinal propulsion rate

[0148] 3 L of deionized water was added to each of the linaclotide-containing pharmaceutical compositions of Example 1, Examples 4-7, and Comparative Example 1, respectively, to obtain dilutions of the linaclotide-containing pharmaceutical compositions of Example 1, Examples 4-7, and Comparative Example 1, respectively. Constipated mice were fasted for 12 hours and assigned to a control group and six experimental groups. The control group was fed deionized water at a dose of 10 mL / kg; the six experimental groups were fed 10 mL / kg dilutions of the linaclotide-containing pharmaceutical compositions of Example 1, Examples 4-7, and Comparative Example 1, respectively. After feeding, the mice in the control and experimental groups were allowed to defecate freely for 1 day. After treatment, mice in the control group and six experimental groups were gavage-treated with 5% activated carbon powder and 5% gum arabic. Twenty-five minutes after gavage, they were anesthetized with 5% chloric acid hydrate intraperitoneally. The mice were immersed in 75% ethanol solution for 30 seconds. The intestinal tract from the pylorus to the ileocecal region was dissected, and the length of the ink-stained small intestine and the total length of the small intestine were measured. The small intestinal propulsion rate (%) = ink-stained propulsion length / total small intestine length × 100%. The results are shown in Table 3.

[0149] Table 3 Small intestinal propulsion rate (%)

[0150]

[0151] As shown in Table 3, the small intestinal propulsion rate of Example 1 of the present invention was higher than that of the control group because Example 1 was treated with a diluent of the pharmaceutical composition containing linaclotide, while the control group was treated with deionized water. The small intestinal propulsion rates of Examples 4-5 were higher than those of Example 1 because the pharmaceutical compositions containing linaclotide in Examples 4-5 also included a triazole derivative. The small intestinal propulsion rate of Example 4 was higher than that of Example 5 because the amount of the triazole derivative used in the pharmaceutical composition containing linaclotide was different. This demonstrates that the pharmaceutical composition containing linaclotide of the present invention can improve the small intestinal propulsion rate. The higher small intestinal propulsion rates in Examples 6-7 of the present invention than in Example 4 and Comparative Example 1 are due to the fact that, in the pharmaceutical compositions containing linaclotide, Examples 6-7 further incorporate triazole derivatives and theaflavin-3-gallate, while Example 4 incorporates only triazole derivatives, and Comparative Example 1 incorporates only theaflavin-3-gallate. The higher small intestinal propulsion rate in Example 6 than in Example 7 is due to the different amounts of theaflavin-3-gallate used in the pharmaceutical compositions containing linaclotide. This suggests that the synergistic use of linaclotide and theaflavin-3-gallate in pharmaceutical compositions containing linaclotide can further improve the small intestinal propulsion rate compared to the use of linaclotide and theaflavin-3-gallate alone.

[0152] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0153] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A pharmaceutical composition containing linaclotide, comprising linaclotide, polyethylene glycol, an electrolyte agent, and a triazole derivative; the electrolyte agent comprises sodium bicarbonate, sodium chloride, and potassium chloride; the amino acid sequence from the N-terminus to the C-terminus of the linear backbone of linaclotide is H-Cys 1 -Cys 2 -Glu 3 -Tyr 4 -Cys 5 -Cys 6 -Asn 7 -Pro 8 -Ala 9 -Cys 10 -Thr 11 -Gly 12 -Cys 13 -Tyr 14 -OH, disulfide bond connection mode is Cys 1 -Cys 6 、Cys 2 -Cys 10 、Cys 5 -Cys 13 The triazole derivative is prepared by the following preparation steps: dissolving 3-benzyl-4H-1,2,4-triazole in tetrahydrofuran, adding 4-phenylbutyryl chloride dropwise at 0-5°C, stirring and reacting for 18-30 hours with triethylamine as an acid binder, removing the solvent by rotary evaporation, and purifying by column chromatography to obtain the triazole derivative; the amount ratio of the 3-benzyl-4H-1,2,4-triazole to tetrahydrofuran is 1 mmol:10-40 mL; the molar ratio of the 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride is 1:1-2; the molar ratio of the 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1-6.

2. A pharmaceutical composition containing linaclotide according to claim 1, characterized in that: In the synthesis of linaclotide, solid-phase synthesis technology is used to couple Fmoc-Tyr-OtBu with 2-chlorotrityl chloride resin, and then amino acids with main chain protecting groups are coupled in sequence. Iodine solution is repeatedly added and stirred to react. Linaclotide is obtained after treatment with a cleavage solution and a buffer solution.

3. A pharmaceutical composition containing linaclotide according to claim 2, characterized in that: The order of the coupled amino acids with main chain protecting groups is Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-Cys(Acm)-OH.

4. A pharmaceutical composition containing linaclotide according to claim 2, characterized in that: In the preparation of the iodine solution, elemental iodine is dissolved in DMF to a final concentration of 0.1-1.0 g / mL.

5. The pharmaceutical composition containing linaclotide according to claim 2, characterized in that: In the preparation of the lysate, TFA, EDT, TIS and deionized water are uniformly mixed to obtain the lysate.

6. The pharmaceutical composition containing linaclotide according to claim 2, characterized in that: In the preparation of the buffer solution, a cysteine ​​hydrochloride solution and a DMSO solution are mixed, and then the pH is adjusted to 9-10 with aqueous ammonia to obtain a buffer solution; The concentration of the cysteine ​​hydrochloride solution is 0.1-1 mmol / L, the volume concentration of the DMSO solution is 20-40%, and the volume ratio of the cysteine ​​hydrochloride solution to the DMSO solution is 1:1-2.

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