Pharmaceutical composition and preparation method thereof

Abstract

The present invention discloses a pharmaceutical composition and a preparation method thereof, comprising a therapeutically effective amount of an active ingredient, a solubilizer, an isotonic agent, an antibacterial agent, a pH regulator, and a solvent; wherein the active ingredient is a polypeptide compound that is a GLP-1R / GCGR dual-target agonist. The pharmaceutical composition and preparation method of the present invention can maintain long-term stability of the polypeptide compound that is a GLP-1R / GCGR dual-target agonist in a solution state, and meet the quality standards established for the product during its shelf life, thereby ensuring the safety, homogeneity, stability, and effectiveness of the drug.

Description

Technical Field

[0001] The present invention relates to a composition, in particular to a pharmaceutical composition and a preparation method thereof. Background Art

[0002] Non-alcoholic steatohepatitis (NASH) is a more severe subtype of non-alcoholic fatty liver disease (NAFLD). It is often considered an acquired metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility. Its main feature is excessive fat deposition in hepatocytes caused by non-excessive alcohol consumption and other clear liver damage factors. It is a clinical pathological syndrome. Because NASH can develop into liver fibrosis, cirrhosis, liver failure, and even hepatocellular carcinoma, it is a major cause of health problems. Literature reports that the prevalence of NASH has reached 1.9%-2.2%. The basic measures for treating NAFLD and NASH are mainly diet control and weight loss, and there is no specific treatment.

[0003] Viral hepatitis-associated liver fibrosis is caused by infection with hepatitis B virus (HBV) and hepatitis C virus (HCV). Hepatitis B is a global disease caused by HBV. More than 400 million people are chronically infected with HBV and are at risk for developing hepatocellular carcinoma (HCC) and cirrhosis. However, the incidence of HCC and cirrhosis in HBV infection varies greatly worldwide, and many factors may influence the incidence of these serious complications, including sex (HCC incidence is higher in men than in women), geographic region (HCC incidence is higher in East Asia, Southeast Asia, and sub-Saharan Africa than in other endemic regions), environmental factors (such as the presence of aflatoxins in food), age at infection, ethnicity, alcohol abuse, and smoking (McMahon BJ. The influence of hepatitis B virus genotype and subgenotype on the natural history of chronic hepatitis B. Hepatol Int. 2009; 3(2): 334-342.). During chronic HBV infection, the immune system's ineffective response to HBV leads to low-level liver cell destruction and prolonged regeneration, which in turn causes fibrosis, cirrhosis, fatty degeneration, and ultimately HCC (Chisari FV, Isogawa M, Wieland SF. Pathogenesis of hepatitis B virus infection. Pathol Biol (Paris). 2010; 58(4): 258-266.). The main treatment for cirrhosis caused by HBV and HCV is antiviral drug therapy. To date, nucleoside and nucleotide drugs are clinically recognized as effective drugs for treating chronic hepatitis B. These drugs target the reverse transcriptase region of HBV DNA polymerase and, after phosphorylation in vivo, act as triphosphate analogs of the natural substrate dNTP of reverse transcriptase, terminating viral DNA chain elongation and interfering with viral DNA synthesis, thereby exerting an antiviral effect and acting as competitive inhibitors of reverse transcriptase. Currently used nucleoside and nucleotide drugs can be divided into three types: L-nucleosides, D-cyclopentanes, and acyclic phosphates.Commonly used drugs include entecavir (ETV), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF), and telbivudine (LdT) (Delaney WE 4th, Locarnini S, Shaw T. Resistance of hepatitis B virus to antiviral drugs: current aspects and directions for future investigation. Antivir Chem Chemother. 2001; 12(1): 1-35.; Caliò R, Villani N, Balestra E, et al. Enhancement of natural killer activity and interferon induction by different acyclic nucleoside phosphonates. Antiviral Res. 1994; 23(1): 77-89.). Of all species, only humans, chimpanzees, and tree shrews are known to be infected with HBV.

[0004] Liver fibrosis is the liver's wound healing response to liver damage caused by various causes. It manifests as the proliferation and deposition of connective tissue within the liver. It is a key pathological feature of chronic liver disease and a crucial step in the progression to cirrhosis. Any process that disrupts the liver's internal environment, including inflammation, toxic damage, altered liver blood flow, liver infection, inborn errors of metabolism, chemical and drug toxicity, hepatic circulatory disturbances and bile flow obstruction, and congenital anomalies, can lead to liver fibrosis. Cholestasis is a key factor in the development of liver fibrosis. Long-term chronic cholestasis, due to the effects of bile acids and bilirubin, leads to hepatocyte degeneration, necrosis, and liver fibrosis, ultimately leading to cirrhosis, a pathological condition known as biliary cirrhosis. Cholestatic cirrhosis of known cause is called secondary biliary cirrhosis (SBC); intrahepatic cholestatic cirrhosis of unknown cause is called primary biliary cirrhosis (PBC). The degree of liver fibrosis can play a good predictive role in cirrhosis (Angulo, P. et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology, 2015, 149, 389-397.e10.).

[0005] A large number of experiments have shown that the polypeptide compound of the GLP-1R / GCGR dual-target agonist has the effect of significantly inhibiting the physiological function activity of NASH.

[0006] Non-alcoholic fatty liver disease requires long-term continuous use within the course of treatment. If the polypeptide compound of the GLP-1R / GCGR dual-target agonist is prepared into a portable or self-usable preparation, such as an injection, and is prepared into a pharmaceutical preparation using a card pen or multi-dose packaging method of an injection pen, patients can inject the drug themselves at home, avoiding long-term and frequent trips to the hospital for treatment, thereby effectively improving patients' medication compliance.

[0007] Currently, after the preparation of the polypeptide compound of the GLP-1R / GCGR dual-target agonist, it will show coagulation and gradually precipitate from the solution over time in the solution state. In addition, the polypeptide compound injection of the GLP-1R / GCGR dual-target agonist gradually degrades during the shelf life of the drug, resulting in the problem of related substances exceeding the standard.

[0008] GLP-1R / GCGR dual-target agonist polypeptide compounds are generally unstable in solution and may be subject to degradation and aggregation. Therefore, there is an urgent need to provide a GLP-1R / GCGR dual-target agonist polypeptide compound that can maintain long-term stability in solution and meet the quality standards established for the product during its shelf life, thereby ensuring the safety, homogeneity, stability, and efficacy of the drug. Summary of the Invention

[0009] The object of the present invention is to provide a pharmaceutical composition and a preparation method thereof, wherein the pharmaceutical composition can enable a polypeptide compound of a GLP-1R / GCGR dual-target agonist to meet product quality standards within its shelf life in a solution state, thereby ensuring its safety, uniformity, stability and effectiveness.

[0010] To achieve the above-mentioned object, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient, a solubilizer, an isotonic agent, an antibacterial agent, a pH regulator, and a solvent; wherein the active ingredient is a polypeptide compound that is a GLP-1R / GCGR dual-target agonist, and the amino acid sequence of the parent peptide of the polypeptide compound is:

[0011] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Xaa16-Arg-Arg-Ala-Gln-Asp -Phe-Val-Gln-Trp-Leu-Xaa27-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-COR1(SEQ ID NO:1);

[0012] Wherein, R1=-NH2;

[0013] Xaa2=D-Ser or Aib;

[0014] Xaa16=Lys or Glu;

[0015] Xaa27=Met, Leu, Nle or Ile;

[0016] Wherein, when Xaa16 is Lys, the side chain of at least one Lys in the amino acid sequence is connected to a lipophilic substituent, and the connection method is that the lipophilic substituent forms an amide bond with the amino group of a bridging group through its carboxyl group, and the carboxyl group of the amino acid residue of the bridging group forms an amide bond with the amino group on the side chain of Lys at position 12 or 16 of the amino acid sequence of the parent peptide to connect to the parent peptide, and the bridging group is (PEG) m -Glu, (PEG) m -γGlu, (PEG) m -Asp or (PEG) m , wherein m is an integer of 1-10; the lipophilic substituent is selected from CH3(CH2) n CO- or HOOC(CH2) n CO-, wherein n is an integer from 10 to 24.

[0017] The content of each component of the pharmaceutical composition of the present invention is calculated based on the volume of the pharmaceutical composition per milliliter.

[0018] The content of the active ingredient in the pharmaceutical composition of the present invention is 0.1-10 mg / mL, such as but not limited to 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, 3 mg / mL, 3.1 mg / mL, 3.2 mg / mL, 3.3 mg / mL, 3.4 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 3.7 mg / mL, 3.8 mg / mL, 3.9 mg / mL, 4 mg / mL, 4.1 mg / mL, 4.2 mg / mL, 4.3 mg / mL, 4.4 mg / mL, 4.5 mg / mL, 4.6 mg / mL, 4.7 mg / mL, 4.8 mg / mL, 4.9 mg / mL, 5 mg / mL, 5.1 mg / mL, 5.2 mg / mL, 5.3 mg / mL, 5.4 mg / mL, 5.5 mg / mL, 5.6 mg / mL, 5.7 mg / mL, 5.8 mg / mL, 5.9 mg / mL, 6 mg / mL, 6.1 mg / mL, 6.2 mg / mL, 6.3 mg / mL, 6.4 mg / mL, 6.5 mg / mL, 6.6 mg / mL, 6.7 mg / mL, 6.8 mg / mL, 6.9 mg / mL, 7 mg / mL, 7.1 mg / mL, 7.2 mg / mL, 7.3 mg / mL, 7.4 mg / mL, 7.5 mg / mL, 7.6 mg / mL, 7.7 mg / mL, 7.8 mg / mL, 7.9 mg / mL, 8 mg / mL, 8.1 mg / mL, 8.2 mg / mL, 8.3 mg / mL, 8.4 mg / mL, 8.5 mg / mL, 8.6 mg / mL, 8.7 mg / mL, 8.8 mg / mL, 8.9 mg / mL, 9 mg / mL, 9.1 mg / mL, 9.2 mg / mL, 9.3 mg / mL, 9.4 mg / mL, 9.5 mg / mL, 9.6 mg / mL, 9.7 mg / mL, 9.8 mg / mL, 9.9 mg / mL, 10 mg / mL.

[0019] The solubilizer of the pharmaceutical composition of the present invention is selected from at least one of Tween 80, Tween 60, Tween 40, Tween 20, polyoxyethylene castor oil, 15-hydroxystearate polyethylene glycol ester (kolliphor@HS15), and corn oil mono- and diglycerides; the content of the solubilizer is 1-300 mg / mL, for example, but not limited to, 1 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 70 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, and 300 mg / mL.

[0020] Kolliphor@HS15 is a mixture of mono- and diesters of 12-hydroxystearic acid polyethylene glycol formed by ethoxylation of 12-hydroxystearic acid and approximately 30% free polyethylene glycol. It can increase the solubility of the active ingredient. The chemical structure of its main component is:

[0021]

[0022] The antibacterial agent of the pharmaceutical composition of the present invention is selected from at least one of phenol, m-cresol, chlorobutanol, and chlorocresol; the content of the antibacterial agent is 0.1-10 mg / mL, for example, but not limited to, 0.1 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 1 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 2 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 3 mg / mL, 3.5 mg / mL, 3.6 mg / mL, 4 mg / mL, 4.5 mg / mL, 4.6 mg / mL, 5 mg / mL, 5.5 mg / mL, 5.6 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, and 10 mg / mL.

[0023] The pH regulator of the pharmaceutical composition of the present invention is selected from at least one of acetic acid, hydrochloric acid, L-glutamic acid, sodium hydroxide and L-arginine; the content of the pH regulator is 0.05-2.0 mg / mL, for example but not limited to 0.05 mg / mL, 0.1 mg / mL, 0.13 mg / mL, 0.15 mg / mL, 0.18 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.25 mg / mL, 0.28 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL .7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL,; the pH adjusting agent adjusts the pH value of the pharmaceutical composition to 3.0-8.5, for example, but not limited to, 3.0, 3.3, 3.4, 3.5, 3.7, 3.9, 4.0, 4.1, 4.2, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5.

[0024] The solvent of the pharmaceutical composition of the present invention is selected from at least one of water for injection and physiological saline; the content of the solvent is the remainder after preparing each mL of the pharmaceutical composition except for the active ingredient, solubilizer, isotonic agent, antibacterial agent and pH regulator.

[0025] The isotonic agent of the pharmaceutical composition of the present invention is selected from at least one of propylene glycol, glycerol, mannitol, and trehalose; the isotonic agent adjusts the osmotic pressure of the pharmaceutical composition solution to the normal osmotic pressure range of human plasma 280-310mOsm / kg, and the content of the isotonic agent is 0.1-50mg / mL, for example but not limited to 0.1mg / mL, 0.5mg / mL, 1mg / mL, 1.5mg / mL, 2mg / mL, 2.5mg / mL, 3mg / mL, 3.5mg / mL, 4mg / mL, 4.5mg / mL, 5mg / mL, 5.5mg / mL, 6mg / mL, 6.5mg / mL, 7mg / mL, 7.5mg / mL, 8mg / mL mL, 8.5mg / mL, 9mg / mL, 9.5mg / mL, 10mg / mL, 10.5mg / mL, 11mg / mL, 11.5mg / mL, 12mg / mL, 12.5mg / mL, 13mg / mL, 13.5mg / mL, 14mg / mL, 14.5mg / mL, 15mg / mL, 15.5mg / mL, 16mg / mL, 16.5mg / mL, 17mg / mL, 17.5mg / mL, 18mg / mL, 18.5mg / mL, 19mg / mL, 19.5mg / mL, 20mg / mL, 20.5mg / mL, 21mg / mL, 21.5mg / mL, 22m g / mL, 22.5mg / mL, 23mg / mL, 23.5mg / mL, 24mg / mL, 24.5mg / mL, 25mg / mL, 25.5mg / mL, 26mg / mL, 26.5mg / mL, 27mg / mL, 27.5mg / mL, 28mg / mL, 28.5mg / mL, 29mg / mL, 29.5mg / mL, 30mg / mL, 30.5mg / mL, 31mg / mL, 31.5mg / mL, 32mg / mL, 32.5mg / mL, 33mg / mL, 33.5mg / mL, 34mg / mL, 34.5mg / mL, 35mg / mL, 35.5mg / m L, 36mg / mL, 36.5mg / mL, 37mg / mL, 37.5mg / mL, 38mg / mL, 38.5mg / mL, 39mg / mL, 39.5mg / mL, 40mg / mL, 40.5mg / mL, 41mg / mL, 41.5mg / mL, 42mg / mL, 42.5m g / mL, 43mg / mL, 43.5mg / mL, 44mg / mL, 44.5mg / mL, 45mg / mL, 45.5mg / mL, 46mg / mL, 46.5mg / mL, 47mg / mL, 47.5mg / mL, 48mg / mL, 48.5mg / mL, 49mg / mL, 49.5mg / mL, 50mg / mL.

[0026] The polypeptide compound of the pharmaceutical composition of the present invention connects the lipophilic substituent to the side chain of Lys at position 12 or 16 via a bridging group to form one of the following structures:

[0027]

[0028]

[0029]

[0030] The amino acid sequence of the GLP-1R / GCGR dual-target agonist polypeptide compound of the pharmaceutical composition of the present invention is as follows:

[0031] Compound 1 (SEQ ID NO: 2):

[0032] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0033] H-(dS)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-RRAQDFVQWL-Nle-NTGGPSSGAPPPS-NH2

[0034] Compound 2 (SEQ ID NO: 3):

[0035] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0036] H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-RRAQDFVQWLLNTGGPSSGAPPPS-NH2

[0037] Compound 3 (SEQ ID NO: 4):

[0038] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0039] H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-RRAQDFVQWLLNTGGPSSGAPPPS-NH2

[0040] Compound 4 (SEQ ID NO: 5):

[0041] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0042] H-(d-S)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-RRAQDFVQWLMNTGGPSSGAPPPS-NH2

[0043] Compound 5 (SEQ ID NO: 6):

[0044] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2;

[0045] H-(dS)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-RRAQDFVQWLMNTGGPSSGAPPPS-NH2

[0046] Compound 6 (SEQ ID NO: 7):

[0047] His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2.

[0048] H-(dS)-QGTFTSDYSKYLD-K(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-RRAQDFVQWLINTGGPSSGAPPPS-NH2

[0049] The present invention also provides a method for preparing the pharmaceutical composition, comprising the following steps:

[0050] (1) Add the solubilizer to a solvent at 30-60°C and stir until dissolved;

[0051] (2) adding the isotonic agent and antibacterial agent to the solvent and stirring until dissolved;

[0052] (3) After mixing the solutions of step (1) and step (2), a solvent is added to 60-80% of the volume of the quantitatively prepared pharmaceutical composition and stirred, and then a therapeutically effective amount of the active ingredient is added, and the pH value of the pharmaceutical composition is adjusted to 3.0-8.5 with a pH regulator, and then the solvent is added to the fixed volume to obtain the quantitatively prepared pharmaceutical composition.

[0053] The present invention has at least the following beneficial effects:

[0054] (1) The pharmaceutical composition and preparation method of the present invention solve the problem of aggregation of GLP-1R / GCGR dual-target agonist polypeptide compounds in solution by introducing various components and adjusting the ratio of the components.

[0055] (2) The pharmaceutical composition and preparation method of the present invention reduce the degradation of the GLP-1R / GCGR dual-target agonist polypeptide compound in the solution state by introducing various components and adjusting the ratio of the components, thereby ensuring that the relevant substances of the GLP-1R / GCGR dual-target agonist polypeptide compound in the solution state meet the standard requirements within the validity period.

[0056] (3) The pharmaceutical composition and preparation method of the present invention ensure that the polypeptide compound of the GLP-1R / GCGR dual-target agonist is fully compatible with the excipients.

[0057] (4) The pharmaceutical composition and preparation method of the present invention have good physiological tolerance and stability of the components used, and can fully meet the process requirements of the sterile injection product of the polypeptide compound of the GLP-1R / GCGR dual-target agonist. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a picture of the pharmaceutical composition obtained in Example 1.

[0059] Figure 2 This is a picture of the pharmaceutical composition obtained in Example 4.

[0060] Figure 3 This is a picture of the pharmaceutical composition obtained in Example 7.

[0061] Figure 4 This is a picture of the pharmaceutical composition obtained in Example 10.

[0062] Figure 5 This is a picture of the pharmaceutical composition obtained in Example 15.

[0063] Figure 6 This is a picture of the pharmaceutical composition obtained in Comparative Example 1.

[0064] Figure 7 This is a picture of the pharmaceutical composition obtained in Comparative Example 2. DETAILED DESCRIPTION

[0065] The following is a detailed description of the embodiments of the present invention: This embodiment is implemented based on the technical solution of the present invention, and provides detailed implementation methods and processes. However, the protection scope of the present invention is not limited to the following embodiments. The experimental methods in the following embodiments where specific conditions are not specified are generally based on conventional conditions.

[0066] Conditions during stability observation: temperature 25±2°C, humidity 60±5%.

[0067] Purity testing instrument: HPLC: Ultimate 3000, chromatographic column: Agilent ZORBAX SB C18 (4.6 x 150 mm, 3.5 μm) or equivalent column, packed with octadecylsilane bonded silica. Mobile phase A: phosphate solution (100 mM potassium dihydrogen phosphate, pH adjusted to 2.6 with phosphoric acid) - acetonitrile (70:30), mobile phase B: phosphate solution (100 mM potassium dihydrogen phosphate, pH adjusted to 2.6 with phosphoric acid) - acetonitrile (50:50), flow rate: 0.6 mL / min; column temperature: 60°C; detection wavelength: 215 nm; injection volume: 20 μL; elution gradient: as shown in Table 1 below:

[0068] Table 1

[0069]

[0070]

[0071] Example 131 and Comparative Examples 1-8:

[0072] A solubilizing agent is added to a solvent and stirred until dissolved to obtain a solubilizing agent solution; an isotonic agent and a bacteriostatic agent are added to the solvent and stirred until dissolved to obtain a mixed solution; the solubilizing agent-containing solution and the mixed solution are mixed and then the solvent is added and stirred, and then a therapeutically effective amount of an active ingredient is added, and the pH value is adjusted with a pH regulator, and then the solvent is added to the volume to obtain a pharmaceutical composition. The components and addition amounts of the Examples and Comparative Examples are shown in Table 2.

[0073] Table 2

[0074]

[0075]

[0076]

[0077] Comparative Example 4:

[0078] Weigh 5.0g of mannitol and 4.0mg of citric acid into a beaker, then add 190μL of 0.1M sodium citrate. Finally, add 100mL of sterile water for injection and stir to dissolve. This is the excipient solution. Weigh 100mg of compound 3 sample into a beaker and add the excipient solution and stir to dissolve.

[0079] Comparative Example 5:

[0080] Weigh 5.0g of mannitol and 1.68mg of citric acid into a beaker, then add 80μL of 0.1M sodium citrate. Finally, add 100mL of sterile water for injection and stir to dissolve. This is the excipient solution. Weigh 100mg of compound 3 into a beaker and add the excipient solution and stir to dissolve.

[0081] Comparative Example 6:

[0082] Weigh 5.0g of mannitol, 135.10mg of citric acid, and 86.79mg of sodium citrate into a beaker. Add 650μL of 0.1M sodium citrate. Finally, add 100mL of sterile water for injection and stir to dissolve. This is the excipient solution. Weigh 100mg of compound 3 into a beaker and add the excipient solution and stir to dissolve.

[0083] Comparative Example 7:

[0084] Weigh 0.9g of sodium chloride into a beaker, add 100mL of sterile water for injection and stir to dissolve, thus obtaining the excipient solution. Weigh 100mg of compound 3 sample into a beaker, add the excipient solution and stir to dissolve.

[0085] Comparative Example 8:

[0086] Place 0.9g of sodium chloride, 11.21mg of sodium acetate, 6.87mg of acetic acid, and 1.4mL of propylene glycol in a beaker. Add 100mL of sterile water for injection and stir to dissolve. This will create an excipient solution. Weigh 100mg of compound 3 into a beaker and add the excipient solution, stirring to dissolve.

[0087] The pharmaceutical compositions obtained in Examples 1-31 and Comparative Examples 1-8 were sterile filtered using a PVDF filter membrane (purchased from Hangzhou Kebet Filter Equipment Co., Ltd.), and the filtrates were poured into cartridges. The stability of the resulting solutions was observed at 3 days, 5 days, 1 month, 2 months, 3 months, and 6 months. The results are shown in Table 3.

[0088] Table 3

[0089]

[0090]

[0091]

[0092]

[0093] *If the properties do not meet the requirements, the relevant substances will not be tested, that is, the purity results in Table 3 will be displayed as NA. If the properties meet the requirements, the sample purity will be tested starting from the first month.

[0094] From Table 3 and Figure 1-7 The results show that the pharmaceutical composition and preparation method of the present invention solve the problem of coagulation of the polypeptide compound of the GLP-1R / GCGR dual-target agonist in the solution state by introducing various components and adjusting the ratio of the components, reduce the degradation of the polypeptide compound of the GLP-1R / GCGR dual-target agonist in the solution state, ensure that the relevant substances of the polypeptide compound of the GLP-1R / GCGR dual-target agonist in the solution state meet the standard requirements within the validity period, ensure that the polypeptide compound of the GLP-1R / GCGR dual-target agonist is fully compatible with the excipients, and each component used has good physiological tolerance and stability, which can fully meet the process requirements of the sterile injection product of the polypeptide compound of the GLP-1R / GCGR dual-target agonist.

[0095] Of course, the present invention may have many other embodiments and variations thereof. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and variations based on the present invention, but these corresponding changes and variations should all fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The invention is composed of a therapeutically effective amount of an active ingredient, a solubilizer, an isotonic agent, an antibacterial agent, a pH regulator, and a solvent; wherein the active ingredient is a polypeptide compound that is a GLP-1R / GCGR dual-target agonist, and the amino acid sequence of the polypeptide compound that is a GLP-1R / GCGR dual-target agonist is selected from at least one of the following: Compound 1 (SEQ ID NO: 2): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Nle-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2; Compound 2 (SEQ ID NO: 3): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2; Compound 3 (SEQ ID NO: 4): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Leu-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2; Compound 4 (SEQ ID NO: 5): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 16 CO2H)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2; Compound 5 (SEQ ID NO: 6): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2; Compound 6 (SEQ ID NO: 7): His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Lys(PEG2-PEG2-γGlu-CO(CH2) 14 CH3)-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Ile-Asn-Thr-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2; The solubilizing agent is selected from at least one of Tween 80, Tween 60, Tween 40, Tween 20, polyoxyethylene castor oil, 15-hydroxystearate polyethylene glycol, and corn oil mono- and diglycerides; the content of the solubilizing agent is 1-300 mg / mL; The antibacterial agent is selected from at least one of phenol, m-cresol, chlorobutanol, and chlorocresol; the content of the antibacterial agent is 0.1-10 mg / mL; The pH adjuster is selected from at least one of acetic acid, hydrochloric acid, L-glutamic acid, sodium hydroxide and L-arginine; the content of the pH adjuster is 0.05-2.0 mg / mL; the pH adjuster adjusts the pH value of the pharmaceutical composition to 3.0-8.5; The isotonic agent is selected from at least one of propylene glycol, glycerol, mannitol, and trehalose; the content of the isotonic agent is 0.1-50 mg / mL; The content of the active ingredient is 0.1-10 mg / mL.

2. The pharmaceutical composition according to claim 1, characterized in that The solvent is selected from at least one of water for injection and physiological saline; the content of the solvent is the remainder after the active ingredient, solubilizer, isotonic agent, antibacterial agent and pH regulator are prepared per mL of the pharmaceutical composition.

3. A method for preparing the pharmaceutical composition according to any one of claims 1 to 2, comprising the following steps: (1) Add the solubilizer to a solvent at 30-60°C and stir until dissolved; (2) adding the isotonic agent and antibacterial agent to the solvent and stirring until dissolved; (3) After mixing the solutions of step (1) and step (2), a solvent is added to 60-80% of the volume of the quantitatively prepared pharmaceutical composition and stirred, and then a therapeutically effective amount of the active ingredient is added, and the pH value of the pharmaceutical composition is adjusted to 3.0-8.5 with a pH regulator, and then the solvent is added to the fixed volume to obtain the quantitatively prepared pharmaceutical composition.

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