CN-121991242-A - Freeze-dried preparation of PEGylated thrombopoietin and preparation method and application thereof

CN121991242ACN 121991242 ACN121991242 ACN 121991242ACN-121991242-A

Abstract

The application discloses a freeze-dried preparation of a PEGylated thrombopoietin peptide, a preparation method and application thereof, wherein the freeze-dried preparation comprises the PEGylated thrombopoietin peptide, histidine, glycine, mannitol and sucrose, the preparation method of the PEGylated thrombopoietin peptide comprises the step of coupling the PEGylated thrombopoietin peptide with the PEGylated thrombopoietin peptide to obtain the PEGylated thrombopoietin peptide, the thrombopoietin peptide is prepared by adopting a fusion protein, the amino acid sequence of the fusion protein is shown as Xm- (Y-Z) n, X is a insulin glargine mutant, Y is an enzyme cutting site, Z is the thrombopoietin peptide, m is 1 or 2, and n is 1 or 2. The fusion protein is efficiently expressed in a thallus in the form of inclusion bodies, and the expressed fusion protein is cut by adopting specific protease to release thrombopoietin, so that the integrity of the thrombopoietin can be ensured, and the cutting efficiency is high. The PEGylated thrombopoiesis-promoting peptide and the preparation thereof can effectively treat and prevent thrombocytopenia related to tumor drugs, primary immune thrombocytopenia, thrombocytopenia related to chronic liver diseases and acute radiation syndrome hematopoietic syndrome.

Inventors

FAN KAI
CHEN QING
ZENG XIN
QIN XIAOLAN
WU KUNCAN
BAI YANMIN
YANG HUI

Assignees

杭州派金生物医药科技有限公司

Dates

Publication Date: 20260508
Application Date: 20241101

Claims (12)

1. A fusion protein, characterized in that the fusion protein has an amino acid sequence shown in a structure of a formula (I), Xm-(Y-Z)n(I); Wherein X is an insulin glargine mutant, and the amino acid sequence of the insulin glargine mutant is shown in any one of SEQ ID NO1 and 9-11; Y is an enzyme cutting site; z is thrombopoietin peptide; m is 1 or 2; n is 1 or 2; the fusion protein is adapted to be cleaved by a cleaving enzyme to form the free thrombopoietic peptide, which is not cleaved by the cleaving enzyme.
2. The fusion protein of claim 1, wherein the fusion protein has at least one of the following characteristics: i) The cleaving enzyme is selected from at least one of recombinant lysine endopeptidase, enterokinase, recombinant lysine endopeptidase and recombinant carboxypeptidase B; ii) the thrombopoietin peptide has the amino acid sequence from N-terminus to C-terminus: Cys-(Gly)p-Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala-(Gly)q-Ile-Glu-Gly-Pro-Thr-Le u-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala; wherein p and q are each independently any integer between 5 and 10; iii) m is 1; iv) n is 1.
3. The fusion protein of claim 1, wherein the thrombopoietin peptide has an amino acid sequence as shown in SEQ ID NO. 2; and/or the cleaving enzyme is selected from recombinant lysine endopeptidase; and/or the amino acid sequence of the enzyme cutting site is DDDDK or K; And/or, the structure of the formula (I) is X-Y-Z, or X- (Y-Z) 2 ; Preferably, the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO 3-5.
4. Use of the fusion protein according to any one of claims 1 to 3 for the preparation of thrombopoietin or pegylated thrombopoietin.
5. A method of preparing a thrombopoietic peptide comprising: Obtaining inclusion bodies of thalli, wherein the inclusion bodies comprise the fusion protein of any one of claims 1-3; performing enzyme digestion treatment on the fusion protein in the inclusion body to obtain the thrombopoiesis-promoting peptide; Optionally, the method has at least one of the following features: i) The enzyme digestion treatment is carried out by adopting a cutting enzyme; ii) prior to the cleavage treatment, subjecting the fusion protein to a denaturation treatment in advance; iii) Reducing the enzyme digestion treatment product; Preferably, the amino acid sequence of the cleavage site in the fusion protein is K, and the cleavage enzyme is selected from recombinant lysine endopeptidase; preferably, in the enzyme digestion treatment system, the final concentration of the fusion protein is 5 mg/ml-10 mg/ml; preferably, the mass ratio of the fusion protein to the cutting enzyme is 1 (1000-5000); preferably, the temperature of the enzyme digestion treatment is 25-35 ℃ and the time is 8-24 hours; preferably, the denaturation treatment is carried out under denaturing agent and detergent conditions; Preferably, the denaturant comprises 2-8 mol/L urea and 20-80 mM Tris, and the pH value of the denaturant is 8.5-9.5; preferably, the reduction treatment is carried out in a reducing agent; preferably, the reducing agent is selected from at least one of TCEP, DTT, β -mercaptoethanol, more preferably TCEP; preferably, the molar ratio of the thrombopoietin peptide to the reducing agent is (1-3): 1 Optionally, the method further comprises subjecting the reduction treatment product to a purification treatment; Preferably, the purification treatment is performed using chromatography.
6. A process for preparing a pegylated thrombopoietic peptide, characterized by comprising the following steps: coupling the thrombopoietin with polyethylene glycol to obtain the polyethylene glycol thrombopoietin; The thrombopoietin is prepared by the fusion protein of any one of claims 1-3 or according to the method of claim 5.
7. The method of claim 6, wherein the method comprises at least one of the following features: a) The molar ratio of the polyethylene glycol to the thrombopoietin is 1 (0.4-0.6); b) The coupling treatment is carried out in a phosphate buffer solution, and the pH value of the phosphate buffer solution is 5.5-7.5; c) Prior to the coupling treatment, the thrombopoietic peptide is pre-mixed with TCEP; d) Further comprising subjecting the coupled treatment product to a purification treatment; e) The molecular weight of the polyethylene glycol is 5-50 kDa; Preferably, the molar ratio of the polyethylene glycol to the thrombopoietic peptide is 1 (0.4-0.5); preferably, the pH value of the phosphate buffer solution is 5.5-6.5; Preferably, the molar ratio of the thrombopoietin to TCEP is 1 (0.5-2.0); preferably, the molar ratio of the thrombopoietin to TCEP is 1 (1.0-1.5); preferably, the purification treatment is performed using chromatography; preferably, the molecular weight of the polyethylene glycol is 15-25 kDa.
8. An injection liquid, in particular to an injection liquid, characterized by comprising the following steps: pegylated thrombopoietin, buffer salts, and protectants.
9. The injection according to claim 8, wherein the injection has at least one of the following characteristics: a) The pegylated thrombopoietin is prepared by the fusion protein of any one of claims 1-3 or according to the method of claim 6 or 7; b) The final concentration of the PEGylated thrombopoietin is 0.2 mg/ml-1.0 mg/ml based on the total mass of the injection; c) The pH value of the injection is 2.5-4.5; d) The buffer salt is selected from at least one of histidine, glycine, citric acid-sodium citrate or acetic acid-sodium acetate; e) The final concentration of the buffer salt in the injection is 10-100 mmol/L; f) The protective agent is at least one selected from sucrose, trehalose and mannitol; g) The mass percentage of the protective agent is 1% -10% based on the total mass of the injection; Preferably, the buffer salt comprises histidine, glycine; More preferably, the buffer salt comprises 20mmol/L glycine and 20mmol/L histidine; preferably, the mass percentage of the protective agent is 6% -8% based on the total mass of the injection; Preferably, the protective agent is selected from sucrose and mannitol; Preferably, the mass ratio of the sucrose to the mannitol is 1 (1-2); preferably, the injection further comprises a pH adjuster; Preferably, the injection comprises: 0.2-1.0 mg/ml of PEGylated thrombopoietin, 2% (W/w) of sucrose, 4% (W/w) mannitol, 20Mmol/L of histidine was added to the mixture, 20Mmol/L glycine, and the concentration of the glycine, Wherein the pH value of the injection is 2.5-4.5.
10. A lyophilized preparation obtained by subjecting the injection according to claim 8 or 9 to lyophilization treatment.
11. Use of a pegylated thrombopoietin, an injection according to claim 8 or 9, or a lyophilized formulation according to claim 10 for: treating and/or preventing diseases, and/or Preparing a medicament for the treatment and/or prophylaxis of a disease; Wherein the diseases include treatment and prevention of tumor drug-related thrombocytopenia, primary immune thrombocytopenia, chronic liver disease-related thrombocytopenia, acute radiation syndrome hematopoietic syndrome.
12. Use according to claim 11, wherein the pegylated thrombopoietin is prepared using a fusion protein according to any one of claims 1-3 or according to the method of claim 6 or 7; Optionally, the pegylated thrombopoietin is provided in a lyophilized formulation; optionally, the effective dose of the pegylated thrombopoietin is 6-100 mug; Optionally, the pegylated thrombopoietin is administered in a manner that treats primary immune thrombocytopenia, treats and prevents acute radiation syndrome hematopoietic syndrome, either once in a chemotherapy cycle, or once a week prior to surgery in a patient with chronic liver disease.

Description

Freeze-dried preparation of PEGylated thrombopoietin and preparation method and application thereof Technical Field The application relates to the field of biological pharmacy, in particular to a freeze-dried preparation of PEGylated thrombopoiesis-promoting peptide, a preparation method and application thereof. Background Thrombocytopenia is a clinically commonly encountered disease commonly found in the treatment and prevention of oncological drug-related thrombocytopenia (CANCER TREATMENT-induced thrombocytopenia, CTIT), primary immune thrombocytopenia (Primary immune thrombocytopenia, ITP), chronic liver disease-related thrombocytopenia syndrome, myelodysplastic syndrome (Myelodysplastic syndrome, MDS), acute radiation syndrome (acute radiation syndrome, ARS), and the like. Thrombocytopenia associated with oncology drugs is one of the common adverse reactions of antitumor drugs, with a incidence of up to 21.8% in patients treated with antitumor drugs. The median time to first drop in platelet count is 1-2 weeks after chemotherapy, and part of drug-induced CIT can also occur 2 weeks after chemotherapy. Most CIT patients also have other thrombocytopenia associated therewith, with a single thrombocytopenia incidence of about 17.7% in thrombocytopenia solid tumor patients. Chemotherapeutic agents that readily result in CTIT include gemcitabine, topotecan, temozolomide, and the like. The combination chemotherapy regimen is more prone to CTIT than single-dose chemotherapy. In the multi-drug combination regimen, the risk of developing CTIT is higher for GP (gemcitabine, cisplatin/carboplatin), EP (etoposide, cisplatin), CODOX-M/IVAC, GEMOX, ICE, MAID, etc. Thrombocytopenia usually begins around 5d after chemotherapy, reaches a minimum on days 7-14, and then gradually rises, and the platelet count returns to baseline on days 28-35. Acute radiation syndrome hematopoietic syndrome (acute radiation syndrome, ARS) is an acute disease caused by irradiation of the whole body with high doses of penetrating radiation in a short period of time, which results in bone marrow suppression in patients, and thus, a sufficient number of new white blood cells, red blood cells and platelets cannot be produced, and is frequently caused by a radiological accident or explosion of nuclear weapons. Primary immune thrombocytopenia (primary immune thrombocytopenia, ITP) is an acquired autoimmune hemorrhagic disease characterized primarily by isolated peripheral thrombocytopenia count without clear causes. The clinical manifestations of the disease are greatly changed, and asymptomatic thrombocytopenia, skin mucous membrane bleeding, severe visceral bleeding and fatal intracranial bleeding can all occur. The risk of fatal hemorrhage in elderly patients is significantly higher than in young patients. Some patients have symptoms of hypodynamia and anxiety. The Chinese guidelines for diagnosis and treatment of primary immune thrombocytopenia of adults (2020 edition) recommend two-line treatment means, contain platelet generation promoting medicines, the effective rate of the medicines is more than 60% in 1-2 Zhou Qixiao, and the curative effect cannot be maintained after stopping medicines, so that individuation maintenance treatment is needed. For patients with 1 antiplatelet agent being ineffective or intolerant, replacement of other antiplatelet agents or sequential therapy may benefit the patient. Chronic liver disease-related thrombocytopenia is internationally defined as platelet counts below 150 x 10 9/L and domestic is generally defined as platelet counts below 100 x 10 9/L. Liver disease-related thrombocytopenia is a multifactorial involved complex pathophysiological process involving a variety of mechanisms including reduced platelet production, increased destruction, abnormal distribution, and increased consumption. Major interventions for thrombocytopenia include platelet infusion, spleen arterial embolism and spleen resection, and thrombopoietin receptor agonists (TPO-RA). Platelet infusion is a therapeutic measure for the fastest rise of platelet count, but only improves platelet count briefly, and the improvement of platelet count is unpredictable and uncontrollable, and after repeated infusion of platelets, a part of thrombocytopenic patients may generate ineffective platelet infusion caused by alloimmunization, and platelet infusion may also bring about infusion risks such as infection, fever, non-hemolytic reaction and the like. Furthermore, given that its short shelf life, lack of clinical supply, etc. also limit its clinical application, it is suggested to be used only for patients at risk of life-threatening bleeding and who need to receive emergency surgery. The TPO-RA drugs currently marketed in China for promoting thrombopoiesis mainly comprise recombinant human thrombopoietin (recombinant human thrombopoietin, rhTPO), small-molecule TPO-RA drugs (Ai Qubo Pa, heteropap, avrotrapa and Lutrepa) and peptidomimetic TPO-