Pharmaceutical composition containing linaclotide as well as preparation method and application of pharmaceutical composition

Abstract

The invention discloses a linaclotide-containing pharmaceutical composition as well as a preparation method and application thereof, and belongs to the technical field of medicine synthesis. The pharmaceutical composition containing linaclotide comprises linaclotide, polyethylene glycol, an electrolyte agent and a triazolyl derivative, in the synthesis of linaclotide, a side chain phenolic hydroxyl group of Fmoc-Tyr-OtBu is bonded with 2-chlorotrityl chloride resin, amino acids are sequentially coupled, a pair of disulfide bonds are oxidized on the resin, and the linaclotide is obtained through the processes of resin cracking and oxidation. In the preparation of the triazolyl derivative, triethylamine is used as an acid-binding agent, and 3-benzyl-4H-1, 2, 4-triazole and 4-phenylbutyryl chloride are subjected to a reaction to obtain the triazolyl derivative. The pharmaceutical composition containing linaclotide disclosed by the invention has excellent biocompatibility and an excellent effect of relieving constipation.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202510748934.1, filed on June 6, 2025, entitled "A pharmaceutical composition containing linaclotide and its preparation method and application". Technical Field

[0002] This invention relates to the field of pharmaceutical synthesis technology, specifically to a pharmaceutical composition containing linaclotide, its preparation method, and its application. Background Technology

[0003] Linaclotide is a guanylate cyclase C (GC-C) agonist with visceral analgesia and secretory-promoting effects. It can relieve abdominal pain and constipation symptoms caused by constipation-predominant irritable bowel syndrome, thereby improving the condition and enhancing the patient's quality of life. Its peptide chain is linked by cyclophosphamide (Cys) bonds. 1 -Cys 6 Cys 2 -Cys 10 Cys 5 -Cys 13 Currently, the synthesis of linaclotide mainly involves bonding the α-carboxyl group of tyrosine to resin. However, the process of using Wang resin to bond the α-carboxyl group of tyrosine is cumbersome, prone to generating racemic impurities when loading the first amino acid, and accompanied by piperazine-diketone cyclization (DKP) side reactions. While using 2-chlorotriphenylmethyl chloride resin (2-CTC resin) to react with the α-carboxyl group avoids racemization and DKP side reactions, the resin's poor stability leads to reduced yield. Utilizing the side-chain phenolic hydroxyl group to bond with the resin can make the resin more stable and avoid racemization and DKP side reaction impurities.

[0004] Linaclotide is a medication used to treat chronic constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation (CC). However, it can cause some common adverse reactions, including diarrhea, abdominal pain, intestinal gas, and headache. Therefore, there is a need for a pharmaceutical composition containing linaclotide that can relieve constipation symptoms while maintaining good biocompatibility. Summary of the Invention

[0005] The purpose of this invention is to provide a pharmaceutical composition containing linaclotide, its preparation method, and its application. This invention not only optimizes the synthesis process of linaclotide, providing a promising prospect for the preparation process of pharmaceutical compositions containing linaclotide, but also improves the biocompatibility and constipation-relieving effect of the pharmaceutical composition containing linaclotide.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A pharmaceutical composition containing linaclotide includes linaclotide, polyethylene glycol, and an electrolyte agent; the electrolyte agent includes 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, the disulfide bond is linked by Cys 1 -Cys 6 Cys 2 -Cys 10 Cys 5 -Cys 13 .

[0007] This invention discloses a pharmaceutical composition containing linaclotide, comprising linaclotide, polyethylene glycol, and an electrolyte agent; the electrolyte agent includes sodium bicarbonate, sodium chloride, and potassium chloride. In the synthesis of linaclotide, this invention uses Fmoc-Tyr-OtBu raw material coupled with 2-chlorotriphenylmethyl chloride resin, bonding the phenolic hydroxyl groups of the side chain to the resin. This differs from the conventional method of bonding α-carboxyl groups to the resin, significantly reducing steric hindrance during peptide chain synthesis, improving reaction efficiency, reducing the possibility of C-terminal amino acid deletion, increasing resin stability, and greatly improving the synthesis yield. Then, a pair of disulfide bonds is oxidized on the resin, reducing the mismatch rate during oxidation and improving product yield. The method for synthesizing linaclotide in this invention is simple and suitable for industrial production, providing a promising prospect for the preparation process of pharmaceutical compositions containing linaclotide.

[0008] Preferably, in the synthesis of linaclotide, a solid-phase synthesis technique is used. Fmoc-Tyr-OtBu is coupled with 2-chlorotriphenylmethyl chloride resin, followed by sequential coupling of amino acids with main chain protecting groups. Iodine solution is repeatedly added and stirred during the reaction. After treatment with lysis buffer and buffer solution, linaclotide is obtained.

[0009] More preferably, the amino acids coupled with the main chain protecting group are in the following order: 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 lysis buffer, TFA, EDT, TIS and deionized water are mixed evenly to obtain the lysis buffer.

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

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

[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 and 4-phenylbutyryl chloride to obtain the triazole derivative.

[0015] More preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole and 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 method for synthesizing linaclotide is as follows: S1. Weigh 2-CTC Resin and Fmoc-Tyr-OtBu, dissolve them in solvent, add DIEA and stir for 15-20 hours. After the reaction is complete, dry the reaction solution and wash it 2-5 times with detergent. Then add PIP solution and react for 15-45 minutes to obtain Tyr-resin-OtBu.

[0018] S2. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is complete, dry the reaction solution and then add PIP solution and react for 25-30 minutes to obtain Cys(Trt)-Tyr-resin-OtBu.

[0019] S3. Add Fmoc-Gly-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is complete, dry the reaction solution and then add PIP solution to react for 25-30 minutes to obtain Gly-Cys(Trt)-Tyr-resin-OtBu.

[0020] S4. Add Fmoc-Thr(tBu)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is complete, dry the reaction solution and then add PIP solution to react for 25-30 minutes to obtain Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0021] S5. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is complete, dry the reaction solution and then add PIP solution and react for 25-30 minutes to obtain Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0022] S6. Add Fmoc-Ala-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is complete, dry the reaction solution and then add PIP solution and react for 25-30 minutes to obtain Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0023] S7. Add Fmoc-Pro-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is complete, dry the reaction solution and then add PIP solution and react for 25-30 minutes to obtain Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0024] S8. Add Fmoc-Asn(Trt)-OH, DIC and Oxyma, add DMF and stir for 1-3 hours. After the reaction is complete, dry the reaction solution and 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.

[0025] S9. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir for 1-3 hours. After the reaction is complete, dry the reaction solution and 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.

[0026] S10, add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir to react for 1-3 hours. After the reaction is complete, dry the reaction solution and 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.

[0027] S11. Add Fmoc-Tyr(tBu)-OH, DIC and Oxyma, add DMF and stir for 1-3 hours. After the reaction is complete, dry the reaction solution and 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.

[0028] S12. Add Fmoc-Glu(OtBu)-OH, DIC and Oxyma, add DMF and stir for 1-3 hours. After the reaction is complete, dry the reaction solution and 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.

[0029] S13. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for 1-3 hours. After the reaction is complete, dry the reaction solution and 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.

[0030] S14. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir for 1-3 hours. After the reaction is complete, dry the reaction solution and 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.

[0031] S15. Add iodine solution and stir for 10-20 min. After the reaction is complete, remove the reaction solution from the vacuum. Add iodine solution again and stir for 10-20 min. After the reaction is complete, remove the reaction solution from the vacuum. Add iodine solution again and stir for 10-20 min. After the reaction is complete, remove the reaction solution from the vacuum. Wash the resin with DMF 2-5 times. Add lysis buffer and lyse for 2-6 h. Add buffer solution and stir for 10-40 h at 15-25℃. Separate the linaclotide by high performance liquid chromatography (HPLC).

[0032] More preferably, the degree of substitution of 2-CTC Resin in step S1 is 0.7-1.0 mmol / g.

[0033] More preferably, in step S1, the ratio of 2-CTC Resin to Fmoc-Tyr-OtBu is 1g:1-10mmol.

[0034] More preferably, in step S1, the solvent is DMF, and the ratio of Fmoc-Tyr-OtBu to the solvent is 1 mmol: 2-5 mL.

[0035] More preferably, the molar ratio of Fmoc-Tyr-OtBu to DIEA in step S1 is 1:2-10.

[0036] More preferably, in step S1, the detergent is DMF, and the ratio of Fmoc-Tyr-OtBu to detergent is 1 mmol: 2-5 mL.

[0037] More preferably, in the preparation of the PIP solution in step S1, the PIP is diluted with DMF to a final mass concentration of 10-30% to obtain the PIP solution.

[0038] More preferably, in step S1, the ratio of Fmoc-Tyr-OtBu to PIP solution is 1 mmol: 2-10 mL.

[0039] More preferably, in step S2, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1-2.

[0040] More preferably, the molar ratio of Fmoc-Gly-OH to DIC in step S3 is 1:1-2.

[0041] More preferably, in step S4, the molar ratio of Fmoc-Thr(tBu)-OH to DIC is 1:1-2.

[0042] More preferably, in step S5, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1-2.

[0043] More preferably, the molar ratio of Fmoc-Ala-OH to DIC in step S6 is 1:1-2.

[0044] More preferably, the molar ratio of Fmoc-Pro-OH to DIC in step S7 is 1:1-2.

[0045] More preferably, in step S8, the molar ratio of Fmoc-Asn(Trt)-OH to DIC is 1:1-2.

[0046] More preferably, in step S9, the molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1-2.

[0047] More preferably, in step S10, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1-2.

[0048] More preferably, in step S11, the molar ratio of Fmoc-Tyr(tBu)-OH to DIC is 1:1-2.

[0049] More preferably, in step S12, the molar ratio of Fmoc-Glu(OtBu)-OH to DIC is 1:1-2.

[0050] More preferably, in step S13, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1-2.

[0051] More preferably, in step S14, the molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1-2.

[0052] More preferably, the molar ratio of DIC and Oxyma in steps S2 to S14 is 1:1-2.

[0053] More preferably, the ratio of DIC to DMF used in steps S2 to S14 is 1 mmol: 5-10 mL.

[0054] More preferably, in steps S2 to S14, the PIP solution is prepared by diluting the PIP with DMF to a final mass concentration of 10-30% to obtain the PIP solution.

[0055] More preferably, the volume ratio of DMF and PIP solution in steps S2 to S14 is 1:1-2.

[0056] 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 the iodine solution.

[0057] More preferably, in the preparation of the lysis buffer in step S15, TFA, EDT, TIS and deionized water are mixed evenly to obtain the lysis buffer, 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.

[0058] More preferably, in the preparation of the buffer in step S15, the cysteine ​​hydrochloride solution and the DMSO solution are mixed, and the pH is adjusted to 9-10 with ammonia to obtain the 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.

[0059] More preferably, the volume ratio of iodine solution to lysis buffer in step S15 is 1:1-5.

[0060] More preferably, the volume ratio of iodine solution to buffer solution in step S15 is 1:10-20.

[0061] Preferably, a pharmaceutical composition containing linaclotide includes linaclotide, polyethylene glycol, and an electrolyte agent, wherein the electrolyte agent includes sodium bicarbonate, sodium chloride, and potassium chloride.

[0062] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500-1000.

[0063] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200-400.

[0064] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2-8.

[0065] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5-10.

[0066] Preferably, a pharmaceutical composition containing linaclotide includes linaclotide, polyethylene glycol, an electrolyte agent, and a triazole derivative, wherein the electrolyte agent includes sodium bicarbonate, sodium chloride, and potassium chloride.

[0067] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500-1000.

[0068] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200-400.

[0069] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2-8.

[0070] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5-10.

[0071] More preferably, the mass ratio of linaclotide to triazole derivative is 1:200-400.

[0072] More preferably, the mass ratio of linaclotide to theaflavin-3-gallate is 1:100-200.

[0073] Preferably, a pharmaceutical composition containing linaclotide includes linaclotide, polyethylene glycol, an electrolyte, a triazole derivative, and theaflavin-3-gallate, wherein the electrolyte includes sodium bicarbonate, sodium chloride, and potassium chloride. The present invention further utilizes theaflavin-3-gallate in the formulation of the linaclotide-containing pharmaceutical composition, which not only further improves cell viability and optimizes the biocompatibility of the linaclotide-containing pharmaceutical composition, but also helps to further improve small intestinal propulsion and optimize the effect of relieving constipation.

[0074] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500-1000.

[0075] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200-400.

[0076] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2-8.

[0077] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5-10.

[0078] More preferably, the mass ratio of linaclotide to triazole derivative is 1:200-400.

[0079] More preferably, the mass ratio of linaclotide to theaflavin-3-gallate is 1:100-200.

[0080] More preferably, the preparation method of the triazole group derivative is as follows: 3-Benzyl-4H-1,2,4-triazole was dissolved in tetrahydrofuran, and 4-phenylbutyryl chloride was added dropwise at 0-5℃. The reaction was stirred for 18-30 h with triethylamine as an acid-binding agent. The solvent was removed by rotary evaporation, and the triazole derivative was obtained by column chromatography.

[0081] More preferably, the ratio of 3-benzyl-4H-1,2,4-triazole to tetrahydrofuran is 1 mmol: 10-40 mL.

[0082] More preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole and 4-phenylbutyryl chloride is 1:1-2.

[0083] More preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1-6.

[0084] This invention optimizes the synthesis method of linaclotide, thus offering the following advantages: The invention couples Fmoc-Tyr-OtBu raw material with 2-chlorotriphenylmethyl chloride resin, bonding the phenolic hydroxyl groups of the side chain to the resin. This differs from the conventional method of bonding α-carboxyl groups to the resin, significantly reducing steric hindrance during peptide chain synthesis, improving reaction efficiency, reducing the possibility of C-terminal amino acid deletion, increasing resin stability, and greatly enhancing the synthesis yield. Furthermore, the oxidation of a disulfide bond on the resin reduces the mismatch rate during oxidation, improving product purity and yield. The yield of linaclotide is 51.97-58.00%, and the purity is 98.66-98.72%. The synthesis method of linaclotide in this invention is simple and suitable for industrial production, providing a promising prospect for the preparation process of linaclotide-containing pharmaceutical compositions.

[0085] This invention, by employing linaclotide, polyethylene glycol, an electrolyte agent, a triazole derivative, and theaflavin-3-gallate to form a linaclotide-containing pharmaceutical composition, possesses the following beneficial effects: The linaclotide-containing pharmaceutical composition of this invention exhibits excellent biocompatibility, with cell viability after treatment with this linaclotide-containing pharmaceutical composition reaching 87.7-98.8%; the linaclotide-containing pharmaceutical composition of this invention also exhibits excellent constipation-relieving effects, with small intestinal propulsion rate in constipated mice treated with this linaclotide-containing pharmaceutical composition reaching 58.4-84.1%. Therefore, this invention provides a biocompatible linaclotide-containing pharmaceutical composition with excellent constipation-relieving effects, its preparation method, and its applications. Attached Figure Description

[0086] Figure 1 To determine the yield and purity of linaclotide. Detailed Implementation

[0087] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0088] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0089] The Chinese names and source information of the reagents used in this invention are summarized in Table 1, including: material abbreviations, Chinese names and suppliers.

[0090] Table 1. English Abbreviation Comparison Table

[0091] Example 1: Methods for synthesizing linaclotide include, S1. Weigh 2-CTC Resin and Fmoc-Tyr-OtBu, dissolve them in solvent, add DIEA and stir for 15 hours. After the reaction is complete, dry the reaction solution, wash it 3 times with detergent, and then add PIP solution and react for 25 minutes to obtain Tyr-resin-OtBu. The degree of substitution of 2-CTC Resin was 0.8 mmol / g, and the molar ratio of 2-CTC Resin to Fmoc-Tyr-OtBu was 1 g: 4.8 mmol; the solvent was DMF, and the molar ratio of Fmoc-Tyr-OtBu to the solvent was 1 mmol: 3.3 mL; the molar ratio of Fmoc-Tyr-OtBu to DIEA was 1:6; the detergent was DMF, and the molar ratio of Fmoc-Tyr-OtBu to the detergent was 1 mmol: 3.3 mL; in the preparation of PIP solution, PIP was diluted with DMF to a final mass concentration of 20% to obtain PIP solution; the molar ratio of Fmoc-Tyr-OtBu to PIP solution was 1 mmol: 5 mL.

[0092] S2. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react for 25 minutes to obtain 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0093] S3. Add Fmoc-Gly-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react for 25 minutes to obtain Gly-Cys(Trt)-Tyr-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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0094] S4. Add Fmoc-Thr(tBu)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react for 25 minutes to obtain Thr(tBu)-Gly-Cys(Trt)-Tyr-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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0095] S5. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0096] S6. Add Fmoc-Ala-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0097] S7. Add Fmoc-Pro-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0098] S8. Add Fmoc-Asn(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0099] S9. Add Fmoc-Cys(Acm)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0100] S10. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0101] S11. Add Fmoc-Tyr(tBu)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0102] S12. Add Fmoc-Glu(OtBu)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0103] S13. Add Fmoc-Cys(Trt)-OH, DIC, and Oxyma, then add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution, then add PIP solution and react 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 volume ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, DMF is used to dilute PIP to a final mass concentration of 20% to obtain PIP solution; the volume ratio of DMF to PIP solution is 1:1.5.

[0104] S14. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir for 2 hours. After the reaction is complete, dry the reaction solution and 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 volume 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] S15. Iodine solution was added and the mixture was stirred for 15 min. After the reaction, the reaction solution was dried under vacuum. Iodine solution was added again and the mixture was stirred for 15 min. After the reaction, the reaction solution was dried under vacuum. The resin was washed three times with DMF. Lysis buffer was added and the mixture was lysed for 3 h. Buffer solution was added and the mixture was stirred at 20 °C for 20 h. Linaclotide was obtained by high performance liquid chromatography (HPLC). In the preparation of the iodine solution, elemental iodine was dissolved in DMF to a final concentration of 0.5 g / mL. In the preparation of the lysis buffer, TFA, EDT, TIS, and deionized water were mixed evenly to obtain the lysis buffer. The volume ratio of EDT to TFA was 1:18, the volume ratio of EDT to TIS was 1:0.5, and the volume ratio of TIS to deionized water was 1:1. In the preparation of the buffer solution, cysteine ​​hydrochloride solution and DMSO solution were mixed, and the pH was adjusted to 10 with ammonia water. The concentration of cysteine ​​hydrochloride solution was 1 mmol / L, the volume concentration of DMSO solution was 30%, and the volume ratio of cysteine ​​hydrochloride solution to DMSO solution was 1:1. The volume ratio of iodine solution to lysis buffer was 1:2.5, and the volume ratio of iodine solution to buffer solution was 1:15.

[0106] A pharmaceutical composition containing linaclotide includes 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.

[0107] Example 2: The method for synthesizing linaclotide is the same as in Example 1, except that the stirring time for adding DIEA in step S1 is replaced with 18 hours.

[0108] A pharmaceutical composition containing linaclotide, which is the same as that in Example 1 except that the linaclotide is replaced with the linaclotide prepared in this example.

[0109] Example 3: The method for synthesizing linaclotide is the same as in Example 1, except that the stirring time for adding DIEA in step S1 is replaced with 20 hours.

[0110] A pharmaceutical composition containing linaclotide, which is the same as that in Example 1 except that the linaclotide is replaced with the linaclotide prepared in this example.

[0111] Example 4: The method for synthesizing linaclotide is the same as in Example 1.

[0112] Methods for preparing triazole derivatives include, 3-Benzyl-4H-1,2,4-triazole was dissolved in tetrahydrofuran, and 4-phenylbutyryl chloride was added dropwise at 0 °C. The mixture was stirred for 24 h with triethylamine as an acid-binding agent. The solvent was removed by rotary evaporation, and the triazole derivative was obtained by column chromatography. 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.

[0113] A pharmaceutical composition containing linaclotide includes 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.

[0114] Example 5: The method for synthesizing linaclotide is the same as in Example 1.

[0115] The preparation method of the triazole derivative is the same as in Example 4.

[0116] A pharmaceutical composition containing linaclotide, compared with Example 4, except that the mass ratio of linaclotide to triazole derivative is changed to 1:200, and all other conditions are the same as in Example 4.

[0117] Example 6: The method for synthesizing linaclotide is the same as in Example 1.

[0118] The preparation method of the triazole derivative is the same as in Example 4.

[0119] A pharmaceutical composition containing linaclotide includes linaclotide, polyethylene glycol, sodium bicarbonate, sodium chloride, potassium chloride, a triazole derivative, and theaflavin-3-gallic acid ester. 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-gallic acid ester is 1:200.

[0120] Example 7: The method for synthesizing linaclotide is the same as in Example 1.

[0121] The preparation method of the triazole derivative is the same as in Example 4.

[0122] A pharmaceutical composition containing linaclotide, compared with Example 6, except that the mass ratio of linaclotide and theaflavin-3-gallate is changed to 1:100, and all other conditions are the same as in Example 6.

[0123] Comparative Example 1: The method for synthesizing linaclotide is the same as in Example 1.

[0124] A pharmaceutical composition containing linaclotide includes 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.

[0125] Experimental example: 1. Linaclotide yield and purity detection 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 for 20 min; flow rate: 3 mL / min; detection wavelength: 220 nm; column temperature: room temperature. The yield and purity of linaclotide were as follows: Figure 1 As shown.

[0126] Depend on Figure 1It can be seen that the yield of linaclotide is 51.97-58.00%, and the purity is 98.66-98.72%. This indicates that the present invention can synthesize linaclotide with high yield and high purity.

[0127] 2. Biosafety Experiment Primary intestinal stem cells from mice were used at a concentration of 5 × 10⁻⁶. 4 Cells were seeded at a density in 24-well plates and divided into 8 groups: a blank group, a control group, and 6 experimental groups. 3 L of deionized water was added to the linaclotide-containing drug compositions of Examples 1, 4-7, and Comparative Example 1, respectively, to obtain the corresponding dilutions. The blank group received no treatment, the control group was treated with deionized water for 24 h, and the 6 experimental groups were treated sequentially with the dilutions of the linaclotide-containing drug compositions of Examples 1, 4-7, and Comparative Example 1 for 24 h. After treatment, cell viability in the experimental groups was evaluated using the MTT assay, and the absorbance of the solution at 570 nm was measured using a microplate reader. Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. Cell viability is shown in Table 2.

[0128] Table 2 Cell viability (%)

[0129] As shown in Table 2, the cell viability of Examples 4-5 of the present invention is higher than that of Example 1. This is because the linaclotide-containing pharmaceutical compositions of Examples 4-5 also include a triazole derivative. The cell viability of Example 4 is higher than that of Example 5 because the amount of the triazole derivative used in the linaclotide-containing pharmaceutical compositions is different. This indicates that the linaclotide-containing pharmaceutical compositions of the present invention can improve cell viability and have good cell compatibility. The cell viability rates of Examples 6-7 of this invention are higher than those of Examples 4 and Comparative Example 1 because, in the linaclotide-containing pharmaceutical compositions, Examples 6-7 further utilize a triazole derivative and theaflavin-3-gallate, while Example 4 only utilizes a triazole derivative, and Comparative Example 1 only utilizes theaflavin-3-gallate. The cell viability rate of Example 6 is higher than that of Example 7 because the amount of theaflavin-3-gallate used in the linaclotide-containing pharmaceutical compositions differs. This indicates that, compared to using linaclotide and theaflavin-3-gallate alone, the synergistic use of linaclotide and theaflavin-3-gallate in the linaclotide-containing pharmaceutical composition can further improve cell viability and enhance the biocompatibility of the linaclotide-containing pharmaceutical composition.

[0130] 3. Small intestinal propulsion rate 3 L of deionized water was added to the linaclotide-containing pharmaceutical compositions of Examples 1, 4-7, and Comparative Example 1, respectively, to obtain the corresponding diluted linaclotide-containing pharmaceutical compositions of Examples 1, 4-7, and Comparative Example 1. Constipated mice were fasted for 12 hours, and six experimental groups were established. The control group was fed deionized water at a dose of 10 mL / kg; the six experimental groups were fed the diluted linaclotide-containing pharmaceutical compositions of Examples 1, 4-7, and Comparative Example 1, respectively, at a dose of 10 mL / kg. After feeding, 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 administered 5% activated charcoal powder and 5% gum arabic via gavage, respectively. Twenty-five minutes after gavage, the mice were anesthetized by intraperitoneal injection of 5% chloric acid hydrate. The mice were then immersed in a 75% ethanol solution for 30 seconds. The intestinal tract from the pylorus to the ileocecal junction was dissected, and the length of the stained portion and the total length of the small intestine were measured. The small intestinal propulsion rate (%) was calculated as: (Length of ink propulsion / Total length of small intestine) × 100%. The results are shown in Table 3.

[0131] Table 3 Small intestinal propulsion rate (%)

[0132] As shown in Table 3, the 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 containing linaclotide, while the control group was treated with deionized water. The intestinal propulsion rates of Examples 4-5 were higher than those of Example 1 because the linaclotide-containing pharmaceutical compositions of Examples 4-5 also included a triazole derivative. The intestinal propulsion rate of Example 4 was higher than that of Example 5 because the amount of the triazole derivative used in the linaclotide-containing pharmaceutical compositions was different. This indicates that the linaclotide-containing pharmaceutical composition of the present invention can improve the intestinal propulsion rate. The small intestinal propulsion rate of Examples 6-7 of this invention is higher than that of Examples 4 and Comparative Example 1 because, in the linaclotide-containing pharmaceutical compositions, Examples 6-7 further use a triazole derivative and theaflavin-3-gallate to form the linaclotide-containing pharmaceutical composition, while Example 4 only uses a triazole derivative, and Comparative Example 1 only uses theaflavin-3-gallate. The small intestinal propulsion rate of Example 6 is higher than that of Example 7 because the amount of theaflavin-3-gallate used in the linaclotide-containing pharmaceutical composition is different. This indicates that, compared to using linaclotide and theaflavin-3-gallate alone, the synergistic use of linaclotide and theaflavin-3-gallate to form the linaclotide pharmaceutical composition can further improve the small intestinal propulsion rate.

[0133] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0134] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition containing linaclotide, comprising linaclotide, polyethylene glycol, an electrolyte, a triazole derivative, and theaflavin-3-gallate; wherein the electrolyte comprises sodium bicarbonate, sodium chloride, and potassium chloride.

2. The pharmaceutical composition according to claim 1, characterized in that, In the synthesis of linaclotide, solid-phase synthesis technology is used. Fmoc-Tyr-OtBu is coupled with 2-chlorotriphenylmethyl chloride resin, and then amino acids with main chain protecting groups are coupled sequentially. Iodine solution is added repeatedly and the reaction is stirred. After treatment with lysis buffer and buffer, linaclotide is obtained.

3. The pharmaceutical composition according to claim 2, characterized in that, The amino acids coupled with the main chain protecting group are in the following order: 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. The pharmaceutical composition 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 according to claim 2, characterized in that, In the preparation of the buffer solution, cysteine ​​hydrochloride solution and DMSO solution are mixed, and then the pH is adjusted to 9-10 with ammonia water to obtain the buffer solution; The concentration of cysteine ​​hydrochloride solution is 0.1-1 mmol / L, the volume concentration of DMSO solution is 20-40%, and the volume ratio of cysteine ​​hydrochloride solution to DMSO solution is 1:1-2.

6. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of linaclotide to theaflavin-3-gallate is 1:100-200.

7. The pharmaceutical composition according to claim 1, characterized in that, In the preparation of the triazole derivative, triethylamine is used as an acid-binding agent to react 3-benzyl-4H-1,2,4-triazole and 4-phenylbutyryl chloride to obtain the triazole derivative.

8. The pharmaceutical composition according to claim 7, characterized in that, The molar ratio of 3-benzyl-4H-1,2,4-triazole and 4-phenylbutyryl chloride is 1:1-2; and / or The molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1-6.

9. Use of the pharmaceutical composition according to any one of claims 1-8 in the preparation of a medicament for relieving constipation.

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