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

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical synthesis, and particularly relates 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, which has visceral analgesic and secretagogue effects, and can relieve abdominal pain and constipation symptoms caused by constipation-predominant irritable bowel syndrome, so as to improve the condition and the quality of life of patients. The disulfide bond connection mode in its peptide chain is Cys 1 -Cys 6 ,Cys 2 -Cys 10 ,Cys 5 -Cys 13 。At present, the synthesis of linaclotide mainly focuses on the bonding of the α-carboxyl group of tyrosine with the resin. However, the process of bonding the Wang Resin with the α-carboxyl group of tyrosine is cumbersome, and racemic impurities are easily generated during the loading of the first amino acid, and a piperazinedione cyclization (DKP) side reaction will occur. Reacting 2-chlorotrityl chloride resin (2-CTC Resin) with the α-carboxyl group can avoid racemization and DKP side reactions, but the resin has poor stability, resulting in a reduced yield. Bonding the side-chain phenolic hydroxyl group with the resin can make the resin more stable and avoid racemization and DKP side reaction impurities.

[0003] Linaclotide is a drug 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, headache, etc. Therefore, a pharmaceutical composition containing linaclotide is needed, which has good biocompatibility while relieving constipation symptoms. Summary of the Invention

[0004] The purpose of the present invention is to provide a pharmaceutical composition containing linaclotide, a preparation method thereof, and an application thereof, which not only optimize the synthesis process of linaclotide and provide a good prospect for the preparation process of the pharmaceutical composition containing linaclotide, but also improve the biocompatibility and constipation-relieving effect of the pharmaceutical composition containing linaclotide.

[0005] The technical solution adopted by the present invention to achieve the above purpose is as follows: A pharmaceutical composition containing linaclotide, comprising 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 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, with the disulfide bond linkage being Cys 1 -Cys 6 、Cys 2 -Cys 10 、Cys 5 -Cys 13 。

[0006] The present invention discloses a pharmaceutical composition containing linaclotide, which comprises linaclotide, polyethylene glycol and an electrolyte agent; the electrolyte agent includes sodium bicarbonate, sodium chloride and potassium chloride. In the synthesis of linaclotide, the present invention uses Fmoc-Tyr-OtBu raw material to couple with 2-chlorotrityl chloride resin, bonding the phenolic hydroxyl group of the side chain to the resin, which is different from the general method of bonding the α-carboxyl group to the resin, greatly reducing the steric hindrance in the peptide chain synthesis process, improving the reaction efficiency, reducing the possibility of C-terminal missing amino acids, having higher resin stability, and being able to greatly improve the synthesis yield. Then, a pair of disulfide bonds are oxidized on the resin, reducing the mismatch rate in the oxidation process and improving the product yield. The operation method for synthesizing linaclotide in the present invention is simple, suitable for industrial production, and provides a good prospect for the preparation process of the pharmaceutical composition containing linaclotide.

[0007] Preferably, in the synthesis of linaclotide, solid-phase synthesis technology is adopted. After coupling Fmoc-Tyr-OtBu with 2-chlorotrityl chloride resin, amino acids with main chain protecting groups are sequentially coupled, and iodine solution is repeatedly added and stirred for reaction. After treatment with a cleavage solution and a buffer solution, linaclotide is obtained.

[0008] More preferably, the sequence 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.

[0009] More preferably, in the preparation of the iodine solution, iodine is dissolved in DMF to a final concentration of 0.1 - 1.0 g / mL.

[0010] More preferably, in the preparation of the lysis solution, TFA, EDT, TIS and deionized water are mixed evenly to obtain the lysis solution.

[0011] 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 ammonia water 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.

[0012] Preferably, the linaclotide-containing pharmaceutical composition comprises linaclotide, polyethylene glycol, an electrolyte agent and a triazolyl derivative. The linaclotide-containing pharmaceutical composition of the present invention has good biocompatibility. When the linaclotide-containing pharmaceutical composition acts on constipated mice, it can relieve mouse constipation by increasing the small intestine propulsion rate of the mice.

[0013] More preferably, 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 reacted to obtain the triazolyl derivative.

[0014] Even more preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride is 1:1 - 2.

[0015] Even more preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1 - 6.

[0016] Preferably, the synthesis method of linaclotide is specifically as follows. S1. Weigh 2-CTC Resin and Fmoc-Tyr-OtBu, add a solvent to dissolve, then add DIEA and stir for reaction for 15 - 20 h. After the reaction is completed, the reaction solution is dried by suction, washed 2 - 5 times with a detergent, and then a PIP solution is added and reacted for 15 - 45 min to obtain Tyr-resin-OtBu.

[0017] S2. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, the reaction solution is dried by suction, and then a PIP solution is added and reacted for 25 - 30 min to obtain Cys(Trt)-Tyr-resin-OtBu.

[0018] S3. Add Fmoc-Gly-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Gly-Cys(Trt)-Tyr-resin-OtBu.

[0019] S4. Add Fmoc-Thr(tBu)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0020] S5. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0021] S6. Add Fmoc-Ala-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0022] S7. Add Fmoc-Pro-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0023] S8. Add Fmoc-Asn(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0024] S9. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0025] S10. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0026] S11. Add Fmoc-Tyr(tBu)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0027] S12. Add Fmoc-Glu(OtBu)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min to obtain Glu(OtBu)-Tyr(tBu)-Cys(Trt)-Cys(Acm)-Asn(Trt)-Pro-Ala-Cys(Trt)-Thr(tBu)-Gly-Cys(Trt)-Tyr-resin-OtBu.

[0028] S13. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 - 30 min 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.

[0029] S14. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir for reaction for 1 - 3 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 - 30 min 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.

[0030] S15. Add iodine solution and stir for reaction for 10 - 20 min. After the reaction is completed, drain the reaction solution. Add iodine solution again and stir for reaction for 10 - 20 min. After the reaction is completed, drain the reaction solution. Add iodine solution again and stir for reaction for 10 - 20 min. After the reaction is completed, drain the reaction solution. Wash the resin with DMF for 2 - 5 times, add cleavage solution and cleave for 2 - 6 h, add buffer solution, stir and react at 15 - 25 °C for 10 - 40 h, and obtain linaclotide by separation with high performance liquid chromatography (HPLC).

[0031] More preferably, in step S1, the substitution degree of 2-CTC Resin is 0.7 - 1.0 mmol / g.

[0032] More preferably, in step S1, the dosage ratio of 2-CTC Resin to Fmoc-Tyr-OtBu is 1 g : 1 - 10 mmol.

[0033] More preferably, in step S1, the solvent is DMF, and the dosage ratio of Fmoc-Tyr-OtBu to the solvent is 1 mmol : 2 - 5 mL.

[0034] More preferably, in step S1, the molar ratio of Fmoc-Tyr-OtBu to DIEA is 1 : 2 - 10.

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

[0036] More preferably, in the preparation of the PIP solution in step S1, use DMF to dilute PIP to a final mass concentration of 10 - 30% to obtain the PIP solution.

[0037] More preferably, in step S1, the dosage ratio of Fmoc-Tyr-OtBu to the PIP solution is 1 mmol : 2 - 10 mL.

[0038] More preferably, in step S2, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1 : 1 - 2.

[0039] More preferably, in step S3, the molar ratio of Fmoc-Gly-OH to DIC is 1:1-2.

[0040] More preferably, in step S4, the molar ratio of Fmoc-Thr(tBu)-OH to DIC is 1:1-2.

[0041] More preferably, in step S5, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1-2.

[0042] More preferably, in step S6, the molar ratio of Fmoc-Ala-OH to DIC is 1:1-2.

[0043] More preferably, in step S7, the molar ratio of Fmoc-Pro-OH to DIC is 1:1-2.

[0044] More preferably, in step S8, the molar ratio of Fmoc-Asn(Trt)-OH to DIC is 1:1-2.

[0045] More preferably, in step S9, the molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1-2.

[0046] More preferably, in step S10, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1-2.

[0047] More preferably, in step S11, the molar ratio of Fmoc-Tyr(tBu)-OH to DIC is 1:1-2.

[0048] More preferably, in step S12, the molar ratio of Fmoc-Glu(OtBu)-OH to DIC is 1:1-2.

[0049] More preferably, in step S13, the molar ratio of Fmoc-Cys(Trt)-OH to DIC is 1:1-2.

[0050] More preferably, in step S14, the molar ratio of Fmoc-Cys(Acm)-OH to DIC is 1:1-2.

[0051] More preferably, in steps S2 to S14, the molar ratio of DIC to Oxyma is 1:1-2.

[0052] More preferably, in steps S2 to S14, the dosage ratio of DIC to DMF is 1 mmol:5-10 mL.

[0053] More preferably, for 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 the PIP solution.

[0054] More preferably, in steps S2 to S14, the volume ratio of the DMF and PIP solutions is 1:1 - 2.

[0055] More preferably, in the preparation of the iodine solution in step S15, iodine is dissolved in DMF to a final concentration of 0.1 - 1.0 g / mL to obtain the iodine solution.

[0056] More preferably, in the preparation of the lysis solution in step S15, TFA, EDT, TIS, and deionized water are mixed evenly to obtain the 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; the volume ratio of TIS to deionized water is 1:1 - 2.

[0057] More preferably, in the preparation of the buffer solution in step S15, a cysteine hydrochloride solution and a DMSO solution are mixed, and then the pH is adjusted to 9 - 10 with ammonia water 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.

[0058] More preferably, in step S15, the volume ratio of the iodine solution to the lysis solution is 1:1 - 5.

[0059] More preferably, in step S15, the volume ratio of the iodine solution to the buffer solution is 1:10 - 20.

[0060] Preferably, a linaclotide-containing pharmaceutical composition comprises linaclotide, polyethylene glycol, and an electrolyte agent, and the electrolyte agent comprises sodium bicarbonate, sodium chloride, and potassium chloride.

[0061] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500 - 1000.

[0062] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200 - 400.

[0063] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2 - 8.

[0064] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5 - 10.

[0065] Preferably, a linaclotide-containing pharmaceutical composition comprises linaclotide, polyethylene glycol, an electrolyte agent, and a triazolyl derivative, and the electrolyte agent comprises sodium bicarbonate, sodium chloride, and potassium chloride.

[0066] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500 - 1000.

[0067] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200 - 400.

[0068] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2 - 8.

[0069] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5 - 10.

[0070] More preferably, the mass ratio of linaclotide to the triazole-based derivative is 1:200 - 400.

[0071] More preferably, the mass ratio of linaclotide to theaflavin-3-gallate is 1:100 - 200.

[0072] Preferably, a linaclotide-containing pharmaceutical composition comprises linaclotide, polyethylene glycol, an electrolyte agent, a triazole-based derivative, and theaflavin-3-gallate. The electrolyte agent includes sodium bicarbonate, sodium chloride, and potassium chloride. The present invention further uses theaflavin-3-gallate to form a linaclotide-containing pharmaceutical composition, which can not only further improve the cell survival rate and optimize the biocompatibility of the linaclotide-containing pharmaceutical composition, but also help to further improve the small intestine propulsion rate and optimize the effect of relieving constipation.

[0073] More preferably, the mass ratio of linaclotide to potassium chloride is 1:500 - 1000.

[0074] More preferably, the mass ratio of potassium chloride to polyethylene glycol is 1:200 - 400.

[0075] More preferably, the mass ratio of potassium chloride to sodium bicarbonate is 1:2 - 8.

[0076] More preferably, the mass ratio of potassium chloride to sodium chloride is 1:5 - 10.

[0077] More preferably, the mass ratio of linaclotide to the triazole-based derivative is 1:200 - 400.

[0078] More preferably, the mass ratio of linaclotide to theaflavin-3-gallate is 1:100 - 200.

[0079] More preferably, the preparation method of the triazole-based derivative is specifically as follows: Dissolve 3-benzyl-4H-1,2,4-triazole in tetrahydrofuran, and dropwise add 4-phenylbutyryl chloride at 0 - 5 °C. Use triethylamine as an acid-binding agent and stir for reaction for 18 - 30 h. Rotate and evaporate to remove the solvent, and obtain the triazole-based derivative through column chromatography purification.

[0080] Even more preferably, the dosage ratio of 3-benzyl-4H-1,2,4-triazole to tetrahydrofuran is 1 mmol:10 - 40 mL.

[0081] Further preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride is 1:1-2.

[0082] Further preferably, the molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1-6.

[0083] Since the synthesis method of linaclotide of the present invention is optimized, the following beneficial effects are achieved: The present invention uses Fmoc-Tyr-OtBu raw material to couple with 2-chlorotrityl chloride resin, bonding the phenolic hydroxyl group on the side chain to the resin, which is different from the general method of bonding the α-carboxyl group to the resin, greatly reducing the steric hindrance in the peptide chain synthesis process, improving the reaction efficiency, reducing the possibility of C-terminal missing amino acids, having higher resin stability, and greatly improving the synthesis yield. Then, the oxidation of a pair of disulfide bonds is carried out on the resin, reducing the mismatch rate in the oxidation process, 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 operation method for synthesizing linaclotide of the present invention is simple and suitable for industrial production, providing a good prospect for the preparation process of the pharmaceutical composition containing linaclotide.

[0084] Since the pharmaceutical composition containing linaclotide of the present invention is composed of linaclotide, polyethylene glycol, electrolyte agent, triazolyl derivative and theaflavin-3-gallate, the following beneficial effects are achieved: The pharmaceutical composition containing linaclotide of the present invention has excellent biocompatibility, and the cell survival rate after being treated with the pharmaceutical composition containing linaclotide of the present invention is 87.7-98.8%; The pharmaceutical composition containing linaclotide of the present invention has excellent constipation-relieving effect, and the small intestine propulsion rate of constipated mice after being treated with the pharmaceutical composition containing linaclotide of the present invention is 58.4-84.1%. Therefore, the present invention provides a pharmaceutical composition containing linaclotide with excellent biocompatibility and good constipation-relieving effect, and its preparation method and application. Description of the Drawings

[0085] Figure 1 For the yield and purity of linaclotide. Detailed Embodiments

[0086] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0087] In the experimental methods of the following examples, unless otherwise specified, they are all conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0088] The Chinese names and source information of the reagents used in the present invention are summarized in Table 1, including: material abbreviation, Chinese name and supplier.

[0089] Table 1 English Abbreviation Comparison Table

[0090] Example 1: The synthesis method of linaclotide includes, S1. Weigh 2-CTC Resin and Fmoc-Tyr-OtBu, add a solvent to dissolve them, then add DIEA and stir for reaction for 15 h. After the reaction is completed, drain the reaction solution, wash it 3 times with a detergent, and then add a PIP solution and react for 25 min to obtain Tyr-resin-OtBu. The substitution degree of 2-CTC Resin is 0.8 mmol / g, and the dosage ratio of 2-CTC Resin to Fmoc-Tyr-OtBu is 1 g:4.8 mmol; the solvent is DMF, and the dosage 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 dosage ratio of Fmoc-Tyr-OtBu to the detergent is 1 mmol:3.3 mL; in the preparation of the PIP solution, dilute PIP with DMF to a final mass concentration of 20% to obtain the PIP solution; the dosage ratio of Fmoc-Tyr-OtBu to the PIP solution is 1 mmol:5 mL.

[0091] S2. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, and then add a PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol:6.6 mL; in the preparation of the PIP solution, dilute PIP with DMF to a final mass concentration of 20% to obtain the PIP solution; the volume ratio of DMF to the PIP solution is 1:1.5.

[0092] S3. Add Fmoc-Gly-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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.

[0093] S4. Add Fmoc-Thr(tBu)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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.

[0094] S5. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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.

[0095] S6. Add Fmoc-Ala-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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.

[0096] S7. Add Fmoc-Pro-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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.

[0097] S8. Add Fmoc-Asn(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, and then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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] S9. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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] S10. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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] S11. Add Fmoc-Tyr(tBu)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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] S12. Add Fmoc-Glu(OtBu)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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] S13. Add Fmoc-Cys(Trt)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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] S14. Add Fmoc-Cys(Acm)-OH, DIC and Oxyma, add DMF and stir for reaction for 2 h. After the reaction is completed, drain the reaction solution, then add PIP solution and react for 25 min 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 dosage ratio of DIC to DMF is 1 mmol: 6.6 mL; in the preparation of PIP solution, dilute PIP 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] S15. Add an iodine solution and stir for reaction for 15 min. After the reaction ends, drain the reaction solution. Add the iodine solution again and stir for reaction for 15 min. After the reaction ends, drain the reaction solution. Add the iodine solution again and stir for reaction for 15 min. After the reaction ends, drain the reaction solution. Wash the resin with DMF three times, add a lysis solution and lyse for 3 h, add a buffer solution, and stir for reaction at 20 °C for 20 h. Linaclotide is obtained by separation using high performance liquid chromatography (HPLC). In the preparation of the iodine solution, dissolve iodine in DMF to a final concentration of 0.5 g / mL. In the preparation of the lysis solution, mix TFA, EDT, TIS, and deionized water evenly to obtain the lysis 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. In the preparation of the buffer solution, mix a cysteine hydrochloride solution and a DMSO solution, and then adjust the pH to 10 with ammonia water to obtain the buffer solution; the concentration of the cysteine hydrochloride solution is 1 mmol / L, the volume concentration of the DMSO solution is 30%, and the volume ratio of the cysteine hydrochloride solution to the DMSO solution is 1:1. The volume ratio of the iodine solution to the lysis solution is 1:2.5, and the volume ratio of the iodine solution to the buffer solution is 1:15.

[0105] A pharmaceutical composition containing linaclotide, comprising 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; the mass ratio of potassium chloride to sodium chloride is 1:7.5.

[0106] Example 2: The synthesis method of linaclotide, compared with Example 1, except that in step S1, the stirring reaction time of adding DIEA is replaced with 18 h, and other conditions are the same as in Example 1.

[0107] A pharmaceutical composition containing linaclotide, compared with Example 1, except that linaclotide is replaced with the linaclotide prepared in this example, and other conditions are the same as in Example 1.

[0108] Example 3: The synthesis method of linaclotide, compared with Example 1, except that in step S1, the stirring reaction time of adding DIEA is replaced with 20 h, and other conditions are the same as in Example 1.

[0109] A pharmaceutical composition containing linaclotide, compared with Example 1, except that linaclotide is replaced with the linaclotide prepared in this example, and other conditions are the same as in Example 1.

[0110] Example 4: The synthesis method of linaclotide is the same as that in Example 1.

[0111] A preparation method of triazolyl derivatives, comprising, Dissolve 3-benzyl-4H-1,2,4-triazole in tetrahydrofuran, and dropwise add 4-phenylbutyryl chloride thereto at 0 °C. Using triethylamine as an acid-binding agent, stir and react for 24 h, rotary evaporate to remove the solvent, and purify by column chromatography to obtain triazolyl derivatives. The dosage ratio of 3-benzyl-4H-1,2,4-triazole to tetrahydrofuran is 1 mmol: 20 mL; the molar ratio of 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride is 1:1; the molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:3.

[0112] A pharmaceutical composition containing linaclotide, comprising linaclotide, polyethylene glycol, sodium bicarbonate, sodium chloride, potassium chloride and triazolyl derivatives. 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 triazolyl derivatives is 1:400.

[0113] Example 5: The synthesis method of linaclotide is the same as that in Example 1.

[0114] The preparation method of triazolyl derivatives is the same as that in Example 4.

[0115] A pharmaceutical composition containing linaclotide, compared with Example 4, except that the mass ratio of linaclotide to triazolyl derivatives is changed to 1:200, and other conditions are the same as those in Example 4.

[0116] Example 6: The synthesis method of linaclotide is the same as that in Example 1.

[0117] The preparation method of triazolyl derivatives is the same as that in Example 4.

[0118] A pharmaceutical composition containing linaclotide, comprising linaclotide, polyethylene glycol, sodium bicarbonate, sodium chloride, potassium chloride, triazolyl derivatives 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 triazolyl derivatives is 1:400; the mass ratio of linaclotide to theaflavin-3-gallate is 1:200.

[0119] Example 7: The synthesis method of linaclotide is the same as that in Example 1.

[0120] The preparation method of the triazolyl derivative is the same as that in Example 4.

[0121] 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, and other conditions are the same as those in Example 6.

[0122] Comparative Example 1: The synthesis method of linaclotide is the same as that in Example 1.

[0123] 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; the mass ratio of linaclotide to theaflavin-3-gallate is 1:200.

[0124] Experimental Example: 1. Detection of the yield and purity of linaclotide The yields and purities of linaclotide prepared in Examples 1-3 were detected by HPLC respectively. 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 yields and purities of linaclotide are as Figure 1 shown.

[0125] It can be Figure 1 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.

[0126] 2. Biosafety experiment Mouse primary intestinal stem cells were used at 5×10 4The cells were inoculated at a density of 100 μg / mL in a 24-well plate and divided into 8 groups, namely a blank group, a control group and 6 experimental groups. 3L of deionized water was added to the pharmaceutical composition containing linaclotide in Example 1, Example 4-7 and Comparative Example 1, respectively, to obtain the dilutions of the pharmaceutical composition containing linaclotide in Example 1, Example 4-7 and Comparative Example 1. The blank group was not treated, the control group was treated with deionized water for 24 hours, and the 6 experimental groups were treated with the dilutions of the pharmaceutical composition containing linaclotide in Example 1, Example 4-7 and Comparative Example 1 for 24 hours. After the treatment, the cell survival rate of the experimental group was evaluated by thiazole blue colorimetry (MTT method), and the absorbance value of the solution at a wavelength of 570 nm was measured by an enzyme marker. Cell survival rate (%) = (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 survival rate is shown in Table 2.

[0127] Table 2 Cell survival rate (%)

[0128] As shown in Table 2, the cell survival rates of Examples 4-5 of the present invention are higher than those of Example 1 because the pharmaceutical compositions containing linaclotide in Examples 4-5 further include triazole derivatives; the cell survival rate of Example 4 is higher than that of Example 5 because the amount of triazole derivatives used in the pharmaceutical compositions containing linaclotide is different. This indicates that the pharmaceutical compositions containing linaclotide of the present invention can improve cell survival rates and have good cell compatibility. The cell survival rates of Examples 6-7 of the present invention are higher than those of Example 4 and Comparative Example 1 because in the pharmaceutical composition containing linaclotide, Examples 6-7 further use triazole derivatives and theaflavin-3-gallate to form the pharmaceutical composition containing linaclotide, Example 4 only uses triazole derivatives to form the pharmaceutical composition containing linaclotide, and Comparative Example 1 only uses theaflavin-3-gallate to form the pharmaceutical composition containing linaclotide; the cell survival rate of Example 6 is higher than that of Example 7 because the amount of theaflavin-3-gallate used in the pharmaceutical composition containing linaclotide is different. This shows that compared with the use of linaclotide and theaflavin-3-gallate alone, the synergistic use of linaclotide and theaflavin-3-gallate to form the pharmaceutical composition containing linaclotide can further improve the cell survival rate and improve the biocompatibility of the pharmaceutical composition containing linaclotide.

[0129] 3. Small Intestinal Propulsion Rate 3L of deionized water was added to the pharmaceutical compositions containing linaclotide of Example 1, Examples 4-7 and Comparative Example 1, respectively, to obtain the dilutions of the pharmaceutical compositions containing linaclotide of Example 1, Examples 4-7 and Comparative Example 1, respectively. The constipated mice were fasted for 12 hours, and a control group and 6 experimental groups were set up. The control group was fed with deionized water at a feeding dose of 10mL / kg; the 6 experimental groups were fed with the dilutions of the pharmaceutical compositions containing linaclotide of Example 1, Examples 4-7 and Comparative Example 1, respectively, at a feeding dose of 10mL / kg. After the feeding was completed, wait for 1d to allow the mice in the control group and the experimental group to defecate freely. After the treatment, mice in the control group and the six experimental groups were gavaged with 5% activated carbon powder and 5% gum arabic. After 25 minutes of gavage, 5% hydrated chloric acid was injected intraperitoneally for anesthesia. The mice were immersed in a 75% ethanol solution for 30 seconds. The intestinal tube from the pylorus to the ileocecal part of the mice was dissected, and the length of the ink-stained small intestine and the total length of the small intestine were measured. The small intestine propulsion rate (%) = ink propulsion length / total length of the small intestine × 100%. The measurement results are shown in Table 3.

[0130] Table 3 Small intestinal propulsion rate (%)

[0131] As shown in Table 3, the small intestinal propulsion rate of Example 1 of the present invention is higher than that of the control group because Example 1 is treated with a diluent of the pharmaceutical composition containing linaclotide, while the control group is treated with deionized water; the small intestinal propulsion rate of Examples 4-5 is higher than that of Example 1 because the pharmaceutical compositions containing linaclotide in Examples 4-5 also include triazole derivatives; the small intestinal propulsion rate of Example 4 is higher than that of Example 5 because the amount of triazole derivatives used in the pharmaceutical composition containing linaclotide is different. This shows that the pharmaceutical composition containing linaclotide of the present invention can improve the small intestinal propulsion rate. The small intestinal propulsion rate of Examples 6-7 of the present invention is higher than that of Example 4 and Comparative Example 1 because in the pharmaceutical composition containing linaclotide, Examples 6-7 further use triazole derivatives and theaflavin-3-gallate to form the pharmaceutical composition containing linaclotide, while Example 4 only uses triazole derivatives to form the pharmaceutical composition containing linaclotide, and Comparative Example 1 only uses theaflavin-3-gallate to form the pharmaceutical composition containing linaclotide; 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 pharmaceutical composition containing linaclotide is different. This shows that compared with the use of linaclotide and theaflavin-3-gallate alone, the synergistic use of linaclotide and theaflavin-3-gallate to form the pharmaceutical composition containing linaclotide can further improve the small intestinal propulsion rate.

[0132] The routine operations in the operation steps of the present invention are well-known to those skilled in the art and will not be elaborated here.

[0133] The above-described embodiments have detailed 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 used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the principle scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A drug composition containing linaclotide, comprising linaclotide, polyethylene glycol, and an electrolyte agent; the electrolyte agent includes sodium bicarbonate, sodium chloride, and potassium chloride; the amino acid sequence of the linear main chain of linaclotide from the N-terminus to the C-terminus 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, and the disulfide bond connection mode is Cys 1 -Cys 6 、Cys 2 -Cys 10 、Cys 5 -Cys 13 。 2. The pharmaceutical composition containing linaclotide according to claim 1, characterized in that, In the synthesis of linaclotide, solid-phase synthesis technology is adopted. After coupling Fmoc-Tyr-OtBu with 2-chlorotrityl chloride resin, amino acids with main-chain protecting groups are successively coupled. The iodine solution is repeatedly added and stirred for reaction, and linaclotide is obtained after treatment with a cleavage solution and a buffer solution.

3. The pharmaceutical composition containing linaclotide according to claim 2, characterized in that, The sequence of coupling amino acids with main-chain protecting groups is successively 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 containing linaclotide according to claim 2, wherein In the preparation of the iodine solution, 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, wherein In the preparation of the cleavage solution, TFA, EDT, TIS, and deionized water are mixed evenly to obtain the cleavage solution.

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 ammonia water 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.

7. The pharmaceutical composition containing linaclotide according to claim 1, characterized in that, The pharmaceutical composition containing linaclotide includes linaclotide, polyethylene glycol, an electrolyte agent, and a triazolyl derivative.

8. The pharmaceutical composition containing linaclotide according to claim 7, wherein 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 reacted to obtain the triazolyl derivative.

9. The pharmaceutical composition containing linaclotide according to claim 8, wherein The molar ratio of 3-benzyl-4H-1,2,4-triazole to 4-phenylbutyryl chloride is 1:1 - 2.

10. The pharmaceutical composition containing linaclotide according to claim 8, wherein The molar ratio of 3-benzyl-4H-1,2,4-triazole to triethylamine is 1:1 - 6.

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