WO2018232750A1 - 重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物 - Google Patents

重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物 Download PDF

Info

Publication number
WO2018232750A1
WO2018232750A1 PCT/CN2017/089823 CN2017089823W WO2018232750A1 WO 2018232750 A1 WO2018232750 A1 WO 2018232750A1 CN 2017089823 W CN2017089823 W CN 2017089823W WO 2018232750 A1 WO2018232750 A1 WO 2018232750A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
bfgf
seq
fibroblast growth
growth factor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/089823
Other languages
English (en)
French (fr)
Inventor
方海洲
曲伟
戴佩旻
杨波
王新志
王振恒
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ZHUHAI ESSEX BIO-PHARMACEUTICAL Co Ltd
Original Assignee
ZHUHAI ESSEX BIO-PHARMACEUTICAL Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ZHUHAI ESSEX BIO-PHARMACEUTICAL Co Ltd filed Critical ZHUHAI ESSEX BIO-PHARMACEUTICAL Co Ltd
Priority to EP17914766.5A priority Critical patent/EP3643784A4/en
Priority to US16/625,308 priority patent/US11254723B2/en
Priority to PCT/CN2017/089823 priority patent/WO2018232750A1/zh
Priority to KR1020207002337A priority patent/KR102480402B1/ko
Priority to MYPI2019007650A priority patent/MY194351A/en
Priority to SG11201912799SA priority patent/SG11201912799SA/en
Priority to CN201780092441.0A priority patent/CN111315886A/zh
Priority to JP2020520691A priority patent/JP7044870B2/ja
Publication of WO2018232750A1 publication Critical patent/WO2018232750A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/04—Artificial tears; Irrigation solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof

Definitions

  • This invention relates to the field of DNA recombination and biopharmaceuticals. More specifically, the present invention relates to a mutant nucleic acid molecule encoding a recombinant human-basic fibroblast growth factor (rh-bFGF), a pharmaceutical composition comprising the rh-bFGF, and the pharmaceutical composition The use of the substance for the treatment of dry eye.
  • rh-bFGF human-basic fibroblast growth factor
  • Dry eye syndrome is a general term for various diseases caused by abnormal or kinetic abnormalities of tears caused by any cause, resulting in decreased tear film stability, accompanied by ocular discomfort and/or ocular surface tissue lesions, also known as keratoconjunctivitis .
  • tear film liquid tear production is reduced, mucus secretion is abnormal, and meibomian gland dysfunction and other causes, the absolute and relative lack of tear film components, abnormal distribution of tears on the ocular surface, tear fluid evaporation can cause dry eyes disease.
  • Common symptoms include dry eyes, easy fatigue, itchy eyes, foreign body sensation, burning sensation, thick secretions, fear of wind, photophobia, sensitivity to external stimuli; sometimes the eyes are too dry, the basic tears are insufficient, but stimulate the reflex Tears are secreted, causing frequent tearing; in severe cases, the eyes will be red, swollen, hyperemic, keratinized, and the corneal epithelium is broken and adhered to the filaments. This damage can cause keratoconjunctival lesions and affect vision.
  • preservatives are currently contained in commercially available products, and long-term use of preservative-containing drugs may cause damage to the ocular surface and may cause an increase in ocular surface diseases.
  • the invention provides a mutant nucleic acid molecule encoding recombinant human basic fibroblast growth factor (rh-bFGF).
  • the invention provides a recombinant human basic fibroblast growth factor (rh-bFGF) encoded by the mutant nucleic acid molecule.
  • rh-bFGF human basic fibroblast growth factor
  • the invention provides a pharmaceutical composition comprising the recombinant human basic fibroblast growth (rh-bFGF) and at least one pharmaceutically acceptable excipient.
  • rh-bFGF recombinant human basic fibroblast growth
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the recombinant human basic fibroblast growth (rh-bFGF) and at least one selected from the group consisting of glycine, histidine, arginine, Tween, heparin A stabilizer for sodium or human serum albumin (HSA).
  • rh-bFGF recombinant human basic fibroblast growth
  • HSA human serum albumin
  • the pharmaceutical composition is free of preservatives.
  • the invention provides a method of treating dry eye, the method comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition.
  • Figure 1 is a diagram showing the structure of the pET-30a(+) plasmid.
  • Figure 2 shows the results of Western Blot of rh-bFGF.
  • Figure 3 shows the purity of rh-bFGF detected by high performance liquid chromatography.
  • Figure 4 shows the molecular mass spectrum of the rh-bFGF intact protein.
  • Figure 5 shows the cDNA sequence, amino acid sequence of the native human bFGF and the mutated human bFGF amino acid sequence.
  • Figure 6 shows a four-parameter fit curve for rh-bFGF in vitro activity assay, where C is the EC50 value.
  • Figure 7 shows the effect of different amino acids, including histidine, glycine and arginine, and Tween on the purity of rh-bFGF under hot pressure conditions, 95% of which refers to the purity standard of rh-bFGF stock solution; A purity of less than 95% means that the purity is unacceptable.
  • Figure 8 shows the effect of HSA on the purity change of rh-bFGF under hot pressure conditions.
  • Figure 9 shows the effect of different concentrations of rh-bFGF eye drops on tear secretion in an alkali burned New Zealand rabbit dry eye model.
  • the symbol "*” or "**” indicates a significant difference (p ⁇ 0.05 or p ⁇ 0.01).
  • Figure 10 shows the effect of different concentrations of rh-bFGF eye drops on tear film rupture time in an alkali burned New Zealand rabbit dry eye model.
  • the symbol "*" or "**” indicates a significant difference (p ⁇ 0.05 or p ⁇ 0.01).
  • the inventors of the present application have for the first time developed a pharmaceutical composition comprising recombinant human basic fibroblast growth factor and one or more pharmaceutically acceptable excipients.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the recombinant human basic fibroblast growth factor (rh-bFGF) and at least one selected from the group consisting of glycine, histidine, arginine, Tween, A stabilizer for heparin sodium or human serum albumin (HSA).
  • rh-bFGF human basic fibroblast growth factor
  • HSA human serum albumin
  • the pharmaceutical composition is free of preservatives.
  • the invention provides a mutant nucleic acid molecule encoding recombinant human basic fibroblast growth factor (rh-bFGF).
  • the invention provides a mutant nucleic acid molecule encoding recombinant human basic fibroblast growth factor (rh-bFGF) comprising: selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. a sequence, or a sequence having at least 95%, 96%, 97%, 98 or 99% identity to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the invention provides a mutant nucleic acid molecule encoding recombinant human basic fibroblast growth factor (rh-bFGF) selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. the sequence of.
  • rh-bFGF recombinant human basic fibroblast growth factor
  • the invention provides a recombinant human basic fibroblast growth factor (rh-bFGF) encoded by the mutant nucleic acid molecule.
  • rh-bFGF human basic fibroblast growth factor
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the recombinant human basic fibroblast growth (rh-bFGF) and at least one pharmaceutically acceptable excipient.
  • the excipients include, but are not limited to, buffer systems, thickeners, stabilizers, neutralizing agents, humectants, and the like.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the recombinant human basic fibroblast growth (rh-bFGF) and at least one selected from the group consisting of glycine, histidine, arginine, Stabilizer for Tween, sodium heparin or human serum albumin (HSA).
  • rh-bFGF recombinant human basic fibroblast growth
  • HSA human serum albumin
  • the excipient comprises a buffer system.
  • the buffer system includes, but is not limited to, sodium dihydrogen phosphate-disodium hydrogen phosphate, citric acid-disodium hydrogen phosphate or boric acid-borax.
  • the buffer system is sodium dihydrogen phosphate-disodium hydrogen phosphate.
  • the buffer system is citrate-disodium hydrogen phosphate.
  • sodium dihydrogen phosphate and disodium hydrogen phosphate when sodium dihydrogen phosphate and disodium hydrogen phosphate are used as a buffer system, sodium dihydrogen phosphate and disodium hydrogen phosphate are respectively 0.25 mg/mL to 1.25 m, and 1.25 mg/mL to 3.75 mg/mL. The concentration is present.
  • sodium dihydrogen phosphate and disodium hydrogen phosphate are used as a buffer system
  • sodium dihydrogen phosphate and disodium hydrogen phosphate are present in concentrations of 0.2-2.5 mg/mL and 0.5-5.0 mg/mL, respectively.
  • sodium dihydrogen phosphate and disodium hydrogen phosphate are used as a buffer system
  • sodium dihydrogen phosphate and disodium hydrogen phosphate are present at a concentration of 0.425 mg/mL and 2.50 mg/mL, respectively.
  • the excipient comprises a thickening agent.
  • the thickening agent includes, but is not limited to, polyvinyl alcohol, sodium hyaluronate, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hypromellose, poloxamer or carbo Mu's thickener.
  • the thickening agent is polyvinyl alcohol.
  • the thickening agent is a carbomer.
  • the carbomer is, for example, a carbomer 940, a carbomer 934, a carbomer 974, a carbomer 980, etc., preferably a carbomer 980 series.
  • the thickening agent is typically present at a concentration of from 0.1 to 20.0 mg/mL.
  • the thickener is typically 5.0-15.0 mg/mL, such as 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, 10.0 mg/mL, 11.0. The concentrations of mg/mL, 12.0 mg/mL, 13.0 mg/mL, and 14.0 mg/mL were present.
  • the amount of polyvinyl alcohol in the composition is typically 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, 10.0. Mg/mL.
  • the amount of carbomer in the composition is typically 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL, 10.0. Mg/mL.
  • the excipient comprises a stabilizer.
  • stabilizers include, but are not limited to, heparin sodium, human serum albumin (HSA), glycine, histidine, arginine, Tween, or a combination of two or more thereof.
  • the stabilizer is sodium heparin and human serum albumin (HSA).
  • the stabilizer is human serum albumin (HSA).
  • the stabilizer is glycine.
  • the stabilizer is histidine.
  • the stabilizer is arginine.
  • the stabilizer is Tween, such as Tween-20.
  • the amount of sodium heparin in the composition is typically from 0.1 to 100 ⁇ g/mL, such as from 0.25 to 75.0 ⁇ g/mL, such as from 0.25 to 5.0 ⁇ g/mL, 25.0- 75.0 ⁇ g/mL, for example, 10 ⁇ g mL, 15 ⁇ g/mL, 20 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 35 ⁇ g/mL, 40 ⁇ g/mL, 45 ⁇ g/mL, 50 ⁇ g/mL, 55 ⁇ g/mL, 60 ⁇ g/mL, 70 ⁇ g/ mL, or 75 ⁇ g/mL; the amount of human serum albumin in the composition is usually 0.01-10.0 mg/mL, such as 0.03-10.0 mg/mL, 0.025-0.375 mg/mL, 0.1-0.375 mg/mL, for example 0.1 mg.
  • the amount of human serum albumin in the composition is typically from 0.01 to 10.0 mg/mL, such as from 0.03 to 10.0 mg/mL.
  • the amount of glycine, histidine, or arginine in the composition is usually from 2% to 5% (w/v), for example, 2%, 3%, 4% or 5%.
  • the excipient comprises a neutralizing agent.
  • the neutralizing agent includes, but is not limited to, triethanolamine, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate or borax. In one aspect, the neutralizing agent is triethanolamine.
  • the neutralizing agent is typically present at a concentration of from 1.25 to 12.5 mg/mL, such as 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, or 10 mg/mL.
  • the amount of triethanolamine in the composition is typically 5-12.5 mg/mL, such as 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL or 10 mg/mL.
  • the excipient comprises a humectant.
  • the humectant includes, but is not limited to, glycerin, propylene glycol or a mixture thereof.
  • the humectant is typically present at a concentration of from 0.1 to 50.0 mg/mL.
  • the amount of glycerin in the composition is typically from 12.5 to 50.0 mg/mL, such as from 12.5 to 25.0 mg/mL, from 25.0 to 50.0 mg/mL, such as 25.0 mg/mL.
  • the composition is buffered to a pH of between about 6.0 and 8.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6. a pH of 7.7, 7.8, 7.9 or 8.0 or any range defined between them.
  • the invention provides a method of treating dry eye, the method comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition.
  • the invention provides the pharmaceutical composition for treating dry eye.
  • the invention provides the use of the pharmaceutical composition for the treatment of dry eye.
  • the invention also provides the use of the composition in the manufacture of a medicament for the treatment of dry eye.
  • compositions of the invention can be administered by a variety of routes of administration, such as by ocular administration to a patient in need thereof, such as by conjunctival administration.
  • the pharmaceutical composition of the present invention can be administered in various dosage forms, for example, as a solution, a suspension, an emulsion, a microemulsion, a composite emulsion, a drop, a gel, a spray, an eye drop, an ophthalmic ointment, or an ophthalmoscope.
  • a solution for example, as a solution, a suspension, an emulsion, a microemulsion, a composite emulsion, a drop, a gel, a spray, an eye drop, an ophthalmic ointment, or an ophthalmoscope.
  • Lotions and injections for example, as a solution, a suspension, an emulsion, a microemulsion, a composite emulsion, a drop, a gel, a spray, an eye drop, an ophthalmic ointment, or an ophthalmoscope.
  • the pharmaceutical composition of the invention is an ophthalmic pharmaceutical composition.
  • the pharmaceutical composition of the invention is in the form of an eye drop or a gel.
  • an advantage of the pharmaceutical composition of the present invention is that the rh-bFGF and the stabilizer such as glycine, histidine, arginine, Tween, heparin sodium or human serum albumin (HSA) are unexpectedly It constitutes a specific combination of stability.
  • glycine, histidine, arginine, Tween, sodium heparin or human serum albumin (HSA) significantly improved the stability of rh-bFGF.
  • the humanized bFGF of the present invention is immunologically safer than non-human derived bFGF.
  • the cDNA sequence encoding the native human bFGF (SEQ ID NO: 4) was mutated without changing the amino acid sequence of the protein, and the three mutated cDNA sequences were designed and synthesized (SEQ ID NO: 1, SEQ ID, respectively).
  • the expression vector used was pET-30a (+) (see Figure 1), purchased from Merk KgsA (Cat. No. 69909-3), which carries the T7 promoter, T7 transcription initiation site, His Tag, Coding sequence, S Tag coding sequence, multiple cloning sites (MCS), T7 terminator, lactose coding sequence, kan resistance coding sequence and pBR322 replicon and f1 replicon.
  • the MCS comprises the cleavage sites Xho I, Not I, Eag I, HindIII, Sal I, Sac I, EcoR I, BamH I, EcoR V, Nco I, Kpn I, Bgl II, Nsp V, Nde I.
  • a Nde I restriction site was designed upstream of the start codon of SEQ ID NO: 1 and a HindIII cleavage site was designed near the terminator.
  • the 480bps sequence was completely synthesized by chemical method. The sequence was digested with Nde I and HindIII and inserted into the same double-digested expression vector pET-30a(+) to obtain a 5724bps recombinant plasmid.
  • DH5 ⁇ (TaKaRa, 9057) was transformed and cultured in LB medium containing 50 ⁇ g/mL kanamycin. Monoclones were selected and the correct transformants were screened by Nde I and HindIII double digestion. The correctness of the transformants was verified again by sequencing.
  • pET-28a (+), pET-23c (+) or pET-15b or the like can be used in place of the above pET-30a (+) vector.
  • the prokaryotic expression vector containing the protein sequence was transformed into E. coli BL21 (DE3), and soluble human bFGF was induced to be expressed by adjusting the cell culture temperature, induction temperature, pH range, glucose concentration and inducer concentration.
  • the cells are collected by hollow fiber washing and filtration; the fermenting cells are crushed by high-pressure homogenization and kept at a low temperature, and after adding an appropriate amount of nonionic surfactant, the supernatant is collected by low-temperature high-speed centrifugation, and the precipitate is discarded.
  • the recombinantly prepared human bFGF protein was analyzed by amino acid sequence, and it was confirmed that the amino acid sequence thereof was identical to the amino acid sequence of native human bFGF (see Fig. 5).
  • balb/c3T3 cells were tested for in vitro activity using balb/c3T3 cells. Cell proliferation was judged by MTT assay. The results showed that the proliferative effect of the obtained human bFGF on balb/c3T3 cells was consistent with the bFGF activity standard (NISCB) (see Fig. 6).
  • NISCB bFGF activity standard
  • the buffer system may be the above sodium dihydrogen phosphate and disodium hydrogen phosphate, or a boric acid-borax buffer system, a citric acid-disodium hydrogen phosphate buffer system or the like.
  • the pharmaceutical composition has a pH of from 6.5 to 7.5.
  • the thickener is polyvinyl alcohol, sodium hyaluronate, hypromellose, poloxamer, etc., preferably polyvinyl alcohol.
  • step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting with sterile water for injection, that is, obtaining;
  • the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
  • the carbomer is a series of carbomer 940, carbomer 934, carbomer 974, carbomer 980, etc., preferably carbomer 980 series;
  • the neutralizing reagent is sodium hydroxide, potassium hydroxide, potassium hydrogencarbonate, borax and triethanolamine, preferably triethanolamine.
  • step (3) Take appropriate amount of room temperature water for injection, add glycerin, human serum albumin, heparin sodium, recombinant human basic fibroblast growth factor, stir evenly, pass the 0.22 ⁇ m filter under sterile conditions, and step (2) The medium gel matrix is mixed, then quantified and stirred evenly.
  • Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
  • the buffer salt system may be the above sodium dihydrogen phosphate and disodium hydrogen phosphate, or a boric acid-borax buffer system, a citric acid-disodium hydrogen phosphate buffer system, etc., preferably the pH is 6.5-7.5. .
  • the thickener is polyvinyl alcohol, sodium hyaluronate, hypromellose, poloxamer, etc., preferably polyvinyl alcohol.
  • the humectant is glycerin, propylene glycol or a mixture thereof.
  • step (2) solution is filtered through a 0.22 ⁇ m microporous membrane and mixed with the solution obtained in the step (1), and made up to 1 mL with water for injection;
  • the volume of the container is in the range of 0.4 g/piece, that is, the finished product is obtained.
  • compositions were also prepared (see Table 2). Among them, the preparation of the eye drops was 100 ml, and the preparation of the external gel was 100 g.
  • step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
  • the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
  • step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
  • the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
  • step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
  • the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
  • step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
  • the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
  • step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
  • the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
  • step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
  • the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
  • step (3) Take appropriate amount of room temperature water for injection, add 2.50g glycerol, 30.0mg human albumin, 7.0mg heparin sodium, recombinant human basic fibroblast growth factor 450,000IU, stir evenly, and pass 0.22 ⁇ m filtration under sterile conditions.
  • the membrane is mixed with the gel matrix in step (2), quantified, and stirred uniformly.
  • Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
  • step (3) Take appropriate amount of room temperature water for injection, add 2.50g glycerin, 30.0mg human albumin, 7.0mg heparin sodium, recombinant human basic fibroblast growth factor 420000IU, stir evenly, and pass 0.22 ⁇ m filtration under sterile conditions.
  • the membrane is mixed with the gel matrix in step (2), quantified, and stirred uniformly.
  • Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
  • step (3) Take appropriate amount of room temperature water for injection, add 2.50g glycerol, 30.0mg human albumin, 7.0mg heparin sodium, recombinant human basic fibroblast growth factor 450,000IU, stir evenly, and pass 0.22 ⁇ m filtration under sterile conditions.
  • the membrane is mixed with the gel matrix in step (2), quantified, and stirred uniformly.
  • Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
  • Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
  • rh-bFGF stock solution itself is unstable in nature, polymerization precipitation tends to occur at room temperature. Therefore, different stabilizers are used to initially screen the stability of the stock solution. 5% and 2% mannitol, 5% and 2% glycine, and 2% dextran were used and allowed to stand at 25 ° C for 17 days. Protein concentrations were measured on days 0, 7, and 17, respectively, and the results are shown in Table 3.
  • the stability of the stock solution was further selected by using different amino acids (glycine, histidine, arginine) and Tween 20, and placed at 25 ° C for 18 h. The results are shown in Fig. 7.
  • New Zealand rabbit dry eye model was selected to observe the clinical indicators of the model (tea secretion, tear film rupture time), and to evaluate the clinical effect of dry eye treatment.
  • New Zealand rabbits were divided into negative control group (Negative control, untreated), model control group (Model, treated with alkali burn but no eye drops were added), sodium hyaluronate eye drops treatment group (HA group), and The group D (4.0 ⁇ g/mL), E (8.0 ⁇ g/mL), F (16.0 ⁇ g/mL), and G (32.0 ⁇ g/mL) which were used to prepare the rh-bFGF eye drop concentration of Example 3 were used.
  • the negative control group consisted of 72 rats in each group, and the remaining groups were 8 rats in each group at a dose of 300 ⁇ l/eye/day.
  • the secretion of tears from New Zealand rabbits was measured using the Schirmer I test. That is, the phenol red cotton thread was clamped with ophthalmology and placed in the outer crotch of the New Zealand rabbit. After 60 s, the wet length of the phenol red cotton thread was taken out. The phenol red cotton thread turns red after being wet, and the eye wetness is determined according to the wet length.
  • the experimental results are shown in Figure 9.
  • the wet length of the phenol red cotton thread of the model control group was significantly decreased as compared with the negative control eye.
  • the wet length of the phenol red cotton thread in the rh-bFGF eye drops D, E, F, and G groups was significantly longer, with statistically significant differences.
  • the tear burst time of the model versus group was significantly shorter than the negative control eyes on day 10 of administration compared to the negative control eyes.
  • the tear film rupture time of the rh-bFGF eye drops group D, E, F, and G groups was significantly prolonged on the 10th day after administration, with significant statistical difference.
  • the phenol red cotton tear secretion test results show that the eye drops of the present invention can improve the tear secretion of the model animals (see Figure 9); the tear film break time test shows that the tear film rupture time of the dry eye model is obvious Improve the effect (see Figure 10).
  • the increase of dose rh-bFGF eye drops have a significant improvement on the dry burn model of Alkali-burned New Zealand rabbits.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Inorganic Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种重组人碱性成纤维细胞生长因子（rh-bFGF）的突变核酸分子，和所述rh-bFGF的药物组合物及其用途。

Description

重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物 技术领域
本发明涉及DNA重组和生物制药领域。更具体地，本发明涉及编码重组人碱性成纤维细胞生长因子(recombinant human-basic fibroblast growth factor，rh-bFGF)的突变核酸分子、包含所述rh-bFGF的药物组合物以及所述药物组合物用于治疗干眼症的用途。
背景技术
干眼症是指任何原因造成的泪液质量异常或动力学异常，导致泪膜稳定性下降，并伴有眼部不适和/或眼表组织病变的多种疾病的总称，又称角结膜干燥症。
当泪膜液性泪液产生减少，粘液分泌异常，和睑板腺功能障碍等各种原因，引起泪膜各成分的绝对和相对缺乏，眼表泪液分布异常，泪液蒸发增加时，可以引起干眼症。
常见的症状包括眼睛干涩、容易疲倦、眼痒、有异物感、痛灼热感、分泌物黏稠、怕风、畏光、对外界刺激很敏感；有时眼睛太干，基本泪液不足，反而刺激反射性泪液分泌，而造成常常流泪；较严重者眼睛会红肿、充血、角质化、角膜上皮破皮而有丝状物黏附，这种损伤日久则可造成角结膜病变，并会影响视力。
如今干眼症患者日益增多，目前市售产品例如玻璃酸钠滴眼液、聚乙烯醇滴眼液、聚乙二醇滴眼液等能缓解干眼症状，但仅能起人工泪液的作用。天然泪液成分复杂，且天然泪膜三层结构的完整性是实施其有效功能的基础，人工泪液不能完全替代。
另外，目前市售产品中含有防腐剂，长期使用含防腐剂的药物会对眼表造成损害并且可能导致眼表疾病加重。
目前没有将bFGF用于治疗干眼症的产品，并且目前没有报道将人源的bFGF用于干眼症的治疗。
发明内容
在一个方面，本发明提供一种编码重组人碱性成纤维细胞生长因子(rh-bFGF)的突变核酸分子。
在一个方面，本发明提供一种由所述突变核酸分子编码的重组人碱性成纤维细胞生长因子(rh-bFGF)。
在一个方面，本发明提供一种药物组合物，其包含所述重组人碱性成纤维细胞生长(rh-bFGF)和至少一种药学上可接受的赋形剂。
在一个方面，本发明提供一种药物组合物，其包含所述重组人碱性成纤维细胞生长(rh-bFGF)和至少一种选自甘氨酸、组氨酸、精氨酸、吐温、肝素钠或人血白蛋白(HSA)的稳定剂。
在一个方面，所述药物组合物不含防腐剂。
在一个方面，本发明提供一种治疗干眼症的方法，所述方法包括向有需要的患者施用治疗有效量的所述药物组合物。
附图说明
图1为pET-30a(+)质粒结构图。
图2为rh-bFGF的Western Blot结果。
图3显示了通过高效液相色谱而检测的rh-bFGF纯度。
图4显示了rh-bFGF完整蛋白的分子量质谱图。
图5显示了天然人bFGF的cDNA序列、氨基酸序列以及经突变的人bFGF氨基酸序列。
图6显示了rh-bFGF体外活性检测的四参数拟合曲线，其中C为EC50值。
图7显示了在热压力条件下，不同氨基酸，包括组氨酸、甘氨酸和精氨酸以及吐温对rh-bFGF纯度变化的影响，其中95％LINE指rh-bFGF原液的纯度质量标准；原液纯度低于95％则表示纯度不合格。
图8显示了在热压力条件下，HSA对rh-bFGF纯度变化的影响。
图9显示了不同浓度的rh-bFGF滴眼液对碱烧伤新西兰兔干眼症模型泪液分泌的影响。符号“*”或“**”表示显著差异(p＜0.05或p＜0.01)。
图10显示了不同浓度的rh-bFGF滴眼液对碱烧伤新西兰兔干眼症模型泪膜破裂时间的影响。符号“*”或“**”表示显著差异(p＜0.05或p＜0.01)。
发明详述
在一个方面，本申请发明人首次开发了一种包含重组人碱性成纤维细胞生长因子和一种或多种药学上可接受的赋形剂的药物组合物。
在一个方面，本发明提供一种药物组合物，其包含所述重组人碱性成纤维细胞生长因子(rh-bFGF)和至少一种选自甘氨酸、组氨酸、精氨酸、吐温、肝素钠或人血白蛋白(HSA)的稳定剂。在这一方面，预料不到地发现，所述重组人碱性成纤维细胞生长因子与所述稳定剂构成了特定的稳定性组合。
在一个方面，所述药物组合物不含防腐剂。
在一个方面，本发明提供一种编码重组人碱性成纤维细胞生长因子(rh-bFGF)的突变核酸分子。在一个方面，本发明提供一种编码重组人碱性成纤维细胞生长因子(rh-bFGF)的突变核酸分子，其包含选自SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3的序列，或与SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3具有至少95％、96％、97％、98或99％同一性的序列。在一个方面，本发明提供一种编码重组人碱性成纤维细胞生长因子(rh-bFGF)的突变核酸分子，其为选自SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3的序列。
在一个方面，本发明提供一种由所述突变核酸分子编码的重组人碱性成纤维细胞生长因子(rh-bFGF)。
在一个方面，本发明提供一种药物组合物，其包含所述重组人碱性成纤维细胞生长(rh-bFGF)和至少一种药学上可接受的赋形剂。在一个方面，所述赋形剂包括，但不限于，缓冲体系、增稠剂、稳定剂、中和试剂、保湿剂等。
在一个方面，本发明提供一种药物组合物，其包含所述重组人碱性成纤维细胞生长(rh-bFGF)和至少一种选自甘氨酸、组氨酸、精氨酸、 吐温、肝素钠或人血白蛋白(HSA)的稳定剂。
在一个方面，所述赋形剂包括缓冲体系。所述缓冲体系包括，但不限于，磷酸二氢钠-磷酸氢二钠、枸橼酸-磷酸氢二钠或硼酸-硼砂。在一个方面，所述缓冲体系为磷酸二氢钠-磷酸氢二钠。在一个方面，所述缓冲体系为枸橼酸-磷酸氢二钠。
在一个方面，当磷酸二氢钠和磷酸氢二钠作为缓冲体系时，磷酸二氢钠、磷酸氢二钠分别以0.25mg/mL-1.25m妙mL、1.25mg/mL-3.75mg/mL的浓度存在。
在一个方面，当磷酸二氢钠和磷酸氢二钠作为缓冲体系时，磷酸二氢钠、磷酸氢二钠分别以0.2-2.5mg/mL、0.5-5.0mg/mL的浓度存在。
在一个方面，当磷酸二氢钠和磷酸氢二钠作为缓冲体系时，磷酸二氢钠、磷酸氢二钠分别以0.425mg/mL、2.50mg/mL的浓度存在。
在一个方面，所述赋形剂包括增稠剂。所述增稠剂包括，但不限于，聚乙烯醇，透明质酸钠、甲基纤维素、羟甲基纤维素、羟乙基纤维素、羟丙甲纤维素、泊洛沙姆或卡波姆的增稠剂。在一个方面，所述增稠剂为聚乙烯醇。在一个方面，所述增稠剂为卡波姆。在一个方面，所述卡波姆例如卡波姆940、卡波姆934、卡波姆974、卡波姆980等系列，优选卡波姆980系列。
在一个方面，所述增稠剂通常以0.1-20.0mg/mL的浓度存在。在一个方面，所述增稠剂通常以5.0-15.0mg/mL，例如5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL、10.0mg/mL、11.0mg/mL、12.0mg/mL、13.0mg/mL、14.0mg/mL的浓度存在。
在一个方面，当聚乙烯醇作为增稠剂时，组合物中聚乙烯醇的量通常为5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL、10.0mg/mL。
在一个方面，当卡波姆作为增稠剂时，组合物中卡波姆的量通常为5.0mg/mL、6.0mg/mL、7.0mg/mL、8.0mg/mL、9.0mg/mL、10.0mg/mL。
在一个方面，所述赋形剂包括稳定剂。所述稳定剂包括，但不限于，肝素钠、人血白蛋白(HSA)、甘氨酸、组氨酸、精氨酸，吐温(Tween)或其两种或更多种的组合。在一个方面，所述稳定剂为肝素钠和人血白蛋白(HSA)。在一个方面，所述稳定剂为人血白蛋白(HSA)。在一个方面，所述稳定剂为甘氨酸。在一个方面，所述稳定剂为组氨酸。在一个方面，所述稳定剂为精氨酸。在一个方面，所述稳定剂为吐温，例如吐温-20。
在一个方面，当肝素钠和人血白蛋白作为稳定剂时，组合物中肝素钠的量通常为0.1-100μg/mL，例如0.25-75.0μg/mL，例如0.25-5.0μg/mL、25.0-75.0μg/mL，例如10μgmL、15μg/mL、20μg/mL、25μg/mL、30μg/mL、35μg/mL、40μg/mL、45μg/mL、50μg/mL、55μg/mL、60μg/mL、70μg/mL、或75μg/mL；组合物中人血白蛋白的量通常为0.01-10.0mg/mL，例如0.03-10.0mg/mL、0.025-0.375mg/mL、0.1-0.375mg/mL，例如0.1mg/mL、0.15mg/mL、0.2mg/mL、0.25mg/mL或0.3mg/mL。
在一个方面，当人血白蛋白作为稳定剂时，组合物中人血白蛋白的量通常为0.01-10.0mg/mL，例如0.03-10.0mg/mL。
在一个方面，当甘氨酸、组氨酸、精氨酸作为稳定剂时，组合物中甘氨酸、组氨酸、精氨酸的量通常为2％-5％(w/v)，例如2％、3％、4％或5％。
在一个方面，所述赋形剂包括中和试剂。所述中和试剂包括，但不限于，三乙醇胺、氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、碳酸氢钾或硼砂。在一个方面，所述中和试剂为三乙醇胺。
在一个方面，所述中和试剂通常以1.25-12.5mg/mL，例如5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL或10mg/mL的浓度存在。在一个方面，当三乙醇胺作为中和试剂时，组合物中三乙醇胺的量通常为5-12.5mg/mL，例如5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL或10mg/mL。
在一个方面，所述赋形剂包括保湿剂。所述保湿剂包括，但不限于，甘油、丙二醇或其混合物。
在一个方面，所述保湿剂通常以0.1-50.0mg/mL的浓度存在。在一个方面，当甘油作为保湿剂时，组合物中甘油的量通常为12.5-50.0mg/mL，例如12.5-25.0mg/mL、25.0-50.0mg/mL，例如25.0mg/mL。
在一个方面，将组合物缓冲至约6.0-8.0的pH，例如6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9或8.0的pH或其间的任何范围限定的pH。
在一个方面，本发明提供一种治疗干眼症的方法，所述方法包括向有需要的患者施用治疗有效量的所述药物组合物。
在一个方面，本发明提供用于治疗干眼症的所述药物组合物。
在一个方面，本发明提供所述药物组合物用于治疗干眼症的用途。
在一个方面，本发明还提供所述组合物在制备用于治疗干眼症的药物中的用途。
在一个方面，本发明的药物组合物可通过多种给药途径施用，例如向有需要的患者经眼给药，例如通过结膜给药。
本发明的药物组合物可以多种剂型给予，例如作为溶液剂、混悬剂、乳剂、微乳剂、复合型乳剂、滴剂、凝胶、喷雾剂、滴眼剂、眼用软膏剂、眼用洗剂和注射液。
在一个方面，本发明的药物组合物为眼用药物组合物。特别地，本发明的药物组合物为滴眼液或凝胶的形式。
本发明所述的药物组合物的优点在于，所述rh-bFGF与所述稳定剂例如甘氨酸、组氨酸、精氨酸、吐温、肝素钠或人血白蛋白(HSA)预料不到地构成了特定的稳定性组合。特别地，甘氨酸、组氨酸、精氨酸、吐温、肝素钠或人血白蛋白(HSA)明显改善rh-bFGF的稳定性。此外，与非人来源的bFGF相比，本发明人源化的bFGF在免疫学上具有更高安全性。
参考以下的非限制性实施例进一步描述本发明。
实施例
实施例1.核酸突变
在不改变蛋白氨基酸序列的前提下，将编码天然人bFGF的cDNA序列(SEQ ID NO：4)进行突变，设计并合成了3种突变的cDNA序列(分别为如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的突变的cDNA序列1、序列2和序列3)：
突变的cDNA序列1(SEQ ID NO：1)
Figure PCTCN2017089823-appb-000001
突变的cDNA序列2(SEQ ID NO：2)
Figure PCTCN2017089823-appb-000002
突变的cDNA序列3(SEQ ID NO：3)
Figure PCTCN2017089823-appb-000003
实施例2.原核表达以及蛋白质表征
2.1表达和纯化
所用的表达载体为pET-30a(+)(参见图1)，购自Merk KgsA公司(Cat.No.69909-3)，该载体带有T7启动子、T7转录起始位点、His Tag、编码序列、S Tag编码序列、多克隆位点(Multiple cloning sites，MCS)、T7终止子、乳糖编码序列、kan抗性编码序列和pBR322复制子和f1复制子。该MCS包含酶切位点Xho I、Not I、Eag I、HindIII、Sal I、Sac I、EcoR I、BamH I、EcoR V、Nco I、Kpn I、Bgl II、Nsp V、Nde I。
为了克隆方便，在序列1(SEQ ID NO：1)的起始密码子上游设计了Nde I酶切位点，在终止子附近设计了HindIII酶切位点。采用化学方法全基因合成该480bps序列，将此序列经Nde I和HindIII双酶切后插入到经相同双酶切的表达载体pET-30a(+)上，得到了5724bps的重组质粒，经热激转化DH5α(TaKaRa，9057)，在含有50μg/mL卡那霉素的LB培养基中培养。挑选单克隆，然后经Nde I和HindIII双酶切筛选正确的转化子。转化子的正确性通过测序再次验证。
在另一个实例中，可以使用pET-28a(+)，pET-23c(+)或pET-15b等代替上述pET-30a(+)载体。
将含有所述蛋白序列的原核表达载体转入大肠杆菌BL21(DE3)中，通过调节菌体培养温度、诱导温度、pH范围、葡萄糖浓度与诱导剂浓度，诱导表达可溶性人bFGF。通过中空纤维洗滤收集菌体；采用高压匀浆并保持低温而破碎发酵菌体，加入适量非离子表面活性剂后，低温高速离心收集上清液，弃去沉淀。
采用弱阳离子交换、肝素亲和层析等方法纯化，在纯化过程中加入适量保护剂，如巯基乙醇，DTT等。
样品经过上述步骤纯化后，纯度达到95％以上。经实验证实，每100g菌体可制备重组人bFGF蛋白约600mg，实现显著高效表达。
2.2纯度和分子量检测以及测序
15％SDS-PAGE电泳显示，所得的人bFGF蛋白为约18.5KD的 单一条带(参见图2)。高效液相色谱法C8反相柱检测结果显示，本发明所得人bFGF的纯度大于95％(参见图3)。
采用“超高分辨、超高准确、超高灵敏”Exactive Plus EMR进行精确分子量测定，4批rh-bFGF原液共检出6个组分(组分1、4、5比例基本＞10％，表1)，组分4为主要成分，分子量平均值为17121.02Da，批间RSD％为0.0007，与理论值的偏差≤41ppm，相对比例(以峰强度计算)为70.7％-79.0％。
表1：HPLC-Exactive Plus EMR质谱检测送检样本精确分子量
Figure PCTCN2017089823-appb-000004
另外，重组制备的人bFGF蛋白经氨基酸序列分析，结果证实其氨基酸序列与天然人bFGF的氨基酸序列一致(参见图5)。
2.3生物学活性测定
使用balb/c3T3细胞对样品进行体外活性检测。用MTT法判断细胞增殖情况。结果表明所得人bFGF对balb/c3T3细胞的促进增殖作用与bFGF活性标准品(NISCB)一致(参见图6)。
实施例3.药物组合物的制备
药物组合物1：
Figure PCTCN2017089823-appb-000005
Figure PCTCN2017089823-appb-000006
1、所述缓冲体系可以是上述磷酸二氢钠和磷酸氢二钠，也可以是硼酸-硼砂缓冲体系，枸橼酸-磷酸氢二钠缓冲体系等。优选药物组合物的pH值为6.5-7.5。
2、所述增稠剂为聚乙烯醇，透明质酸钠、羟丙甲纤维素、泊洛沙姆等，优选聚乙烯醇。
3、制备方法：
(1)将聚乙烯醇分散溶解于适量的注射用水中，于121℃高压灭菌30min，冷却至室温，备用；
(2)将重组人碱性成纤维细胞生长因子、人血白蛋白、肝素钠、氯化钠、磷酸二氢钠、磷酸氢二钠溶解于适量的注射用水中，用0.22μm滤膜无菌过滤；
(3)在无菌的条件下将步骤(1)和步骤(2)所得溶液混合均匀，用无菌注射用水定容，即得；
(4)无菌溶液使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4mL，即得成品。
药物组合物2：
Figure PCTCN2017089823-appb-000007
1、所述卡波姆为卡波姆940、卡波姆934、卡波姆974、卡波姆980等系列，优选卡波姆980系列；
2、所述中和试剂为氢氧化钠、氢氧化钾、碳酸氢钾、硼砂及三乙醇胺，优选三乙醇胺。
3、制备方法：
(1)将卡波姆分散于适量的注射用水中，搅拌均匀后过夜溶胀，备用；
(2)在卡波姆分散液中加入三乙醇胺，搅拌成透明均匀凝胶基质，于121℃高压灭菌30min，灭菌完成后放冷至室温，备用；
(3)取适量室温的注射用水加入甘油、人血白蛋白、肝素钠、重组人碱性成纤维细胞生长因子，搅拌均匀后，在无菌条件下通过0.22μm滤膜，并与步骤(2)中凝胶基质混合，然后定量，搅拌均匀。
(4)无菌凝胶使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4g，即得成品。
药物组合物3：
Figure PCTCN2017089823-appb-000008
1、所述缓冲盐体系可以是上述磷酸二氢钠和磷酸氢二钠，也可以是硼酸-硼砂缓冲体系，枸橼酸-磷酸氢二钠缓冲体系等，优选所述pH值为6.5-7.5。
2、所述增稠剂为聚乙烯醇，透明质酸钠、羟丙甲纤维素、泊洛沙姆等，优选聚乙烯醇。所述保湿剂为甘油、丙二醇或其混合物。
3、制备方法：
(1)将增稠剂、氯化钠、分散溶解于适量的注射用水中，于121℃高压灭菌30min；
(2)将重组人碱性成纤维细胞生长因子、人血白蛋白、保湿剂、磷酸二氢钠、磷酸氢二钠溶解于适量的注射用水中；
(3)将步骤(2)药液经过0.22μm微孔滤膜过滤后与步骤(1)所得药液混合，并用注射用水定容至1mL；
(4)灌装步骤(3)所得药液至不含抑菌剂的包装容器中，容器的容积范围为0.4g/支，即得到成品。
此外，还制备了以下药物组合物(参见表2)。其中，制备滴眼液均为100ml，制备外用凝胶均为100g。
Figure PCTCN2017089823-appb-000009
制备实施例1：
(1)将1.0g聚乙烯醇分散溶解于适量的注射用水中，高压灭菌，冷却至室温，备用；
(2)将重组人碱性成纤维细胞生长因子500000IU、人血白蛋白25mg、肝素钠2.5mg、氯化钠800mg、磷酸二氢钠42.5mg、磷酸氢二钠250mg溶解于适量的注射用水中，用0.22μm滤膜无菌过滤；
(3)在无菌的条件下将步骤(1)和步骤(2)所得溶液混合均匀，用无菌注射用水定容至100mL，即得；
(4)无菌溶液使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4mL，即得成品。
制备实施例2：
(1)将0.5g聚乙烯醇分散溶解于适量的注射用水中，高压灭菌，冷却至室温，备用；
(2)将重组人碱性成纤维细胞生长因子500000IU、人血白蛋白20mg、肝素钠2.0mg、氯化钠800mg、磷酸二氢钠42.5mg、磷酸氢二钠250mg溶解于适量的注射用水中，用0.22μm滤膜无菌过滤；
(3)在无菌的条件下将步骤(1)和步骤(2)所得溶液混合均匀，用无菌注射用水定容至100mL，即得；
(4)无菌溶液使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4mL，即得成品。
制备实施例3：
(1)将1.0g聚乙烯醇分散溶解于适量的注射用水中，高压灭菌，冷却至室温，备用；
(2)将重组人碱性成纤维细胞生长因子420000IU、人血白蛋白20mg、肝素钠2.0mg、氯化钠800mg、磷酸二氢钠42.5mg、磷酸氢二钠250mg溶解于适量的注射用水中，用0.22μm滤膜无菌过滤；
(3)在无菌的条件下将步骤(1)和步骤(2)所得溶液混合均匀，用无菌注射用水定容至100mL，即得；
(4)无菌溶液使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4mL，即得成品。
制备实施例4：
(1)将1.5g聚乙烯醇分散溶解于适量的注射用水中，高压灭菌，冷却至室温，备用；
(2)将重组人碱性成纤维细胞生长因子420000IU、人血白蛋白10mg、肝素钠1.0mg、氯化钠800mg、磷酸二氢钠42.5mg、磷酸氢二钠250mg溶解于适量的注射用水中，用0.22μm滤膜无菌过滤；
(3)在无菌的条件下将步骤(1)和步骤(2)所得溶液混合均匀，用无菌注射用水定容至100mL，即得；
(4)无菌溶液使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4mL，即得成品。
制备实施例5：
(1)将1.0g聚乙烯醇分散溶解于适量的注射用水中，高压灭菌，冷却至室温，备用；
(2)将重组人碱性成纤维细胞生长因子420000IU、人血白蛋白20mg、肝素钠2.0mg、氯化钠800mg、枸橼酸370.6mg、磷酸氢二钠5.9g溶解于适量的注射用水中，用0.22μm滤膜无菌过滤；
(3)在无菌的条件下将步骤(1)和步骤(2)所得溶液混合均匀，用无菌注射用水定容至100mL，即得；
(4)无菌溶液使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4mL，即得成品。
制备实施例6：
(1)将1.0g聚乙烯醇分散溶解于适量的注射用水中，高压灭菌， 冷却至室温，备用；
(2)将重组人碱性成纤维细胞生长因子500000IU、人血白蛋白25mg、肝素钠2.5mg、氯化钠800mg、枸橼酸370.6mg、磷酸氢二钠5.9g溶解于适量的注射用水中，用0.22μm滤膜无菌过滤；
(3)在无菌的条件下将步骤(1)和步骤(2)所得溶液混合均匀，用无菌注射用水定容至100mL，即得；
(4)无菌溶液使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4mL，即得成品。
制备实施例7：
(1)称取0.80g卡波姆940分散于适量的室温的注射用水中，搅拌60-120min，过夜溶胀，备用；
(2)在卡波姆940分散液中加入三乙醇胺0.60g，搅拌使成透明均匀凝胶基质，然后进行湿热灭菌(121℃，30min)，灭菌完成后放冷至室温，备用；
(3)取适量室温的注射用水加入2.50g甘油、30.0mg人血白蛋白、7.0mg肝素钠、重组人碱性成纤维细胞生长因子450000IU，搅拌均匀后，在无菌条件下通过0.22μm滤膜，并与步骤(2)中凝胶基质混合，并定量，搅拌均匀。
(4)无菌凝胶使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4g，即得成品。
制备实施例8：
(1)称取0.60g卡波姆940分散于适量的室温的注射用水中，搅拌60-120min，过夜溶胀，备用；
(2)在卡波姆940分散液中加入三乙醇胺0.50g，搅拌使成透明均匀凝胶基质，然后进行湿热灭菌(121℃，30min)，灭菌完成后放冷至室温，备用；
(3)取适量室温的注射用水加入2.50g甘油、30.0mg人血白蛋白、7.0mg肝素钠、重组人碱性成纤维细胞生长因子420000IU，搅拌均匀后，在无菌条件下通过0.22μm滤膜，并与步骤(2)中凝胶基质混合，并定量，搅拌均匀。
(4)无菌凝胶使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4g，即得成品。
制备实施例9：
(1)称取0.60g卡波姆974分散于适量的室温的注射用水中，搅拌60-120min，过夜溶胀，备用；
(2)在卡波姆974分散液中加入三乙醇胺0.50g，搅拌使成透明均匀凝胶基质，然后进行湿热灭菌(121℃，30min)，灭菌完成后放冷至室温，备用；
(3)取适量室温的注射用水加入2.50g甘油、30.0mg人血白蛋白、7.0mg肝素钠、重组人碱性成纤维细胞生长因子450000IU，搅拌均匀后，在无菌条件下通过0.22μm滤膜，并与步骤(2)中凝胶基质混合，并定量，搅拌均匀。
(4)无菌凝胶使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4g，即得成品。
制备实施例10：
(1)称取0.50g卡波姆974分散于适量的室温的注射用水中，搅拌60-120min，过夜溶胀，备用；
(2)在卡波姆974分散液中加入三乙醇胺0.50g，搅拌使成透明均匀凝胶基质，然后进行湿热灭菌(121℃，30min)，灭菌完成后放冷至室温，备用；
(3)取适量室温的注射用水加入2.50g甘油、20.0mg人血白蛋白、5.0mg肝素钠、重组人碱性成纤维细胞生长因子420000IU，搅拌均匀 后，在无菌条件下通过0.22μm滤膜，并与步骤(2)中凝胶基质混合，并定量，搅拌均匀。
(4)无菌凝胶使用吹、灌、封三合一灌装机进行一体化灌装，装量0.4g，即得成品。
实施例4.稳定性研究
1)氨基酸对rh-bFGF原液稳定性的影响研究
由于rh-bFGF原液本身性质不稳定，在室温下易发生聚合沉淀。因此选用不同的稳定剂对原液的稳定性进行初筛。选用5％与2％的甘露醇、5％与2％的甘氨酸以及2％葡聚糖，在25℃环境下放置17天。分别在第0、7与17天检测蛋白浓度，结果见表3。
表3：rh-bFGF原液稳定剂筛选试验的蛋白浓度变化率
样品 0天 7天 17天
无稳定剂 100.00％ 29.96％ 13.63％
5％甘露醇 100.00％ 49.80％ 17.31％
2％甘露醇 100.00％ 53.29％ 15.66％
5％甘氨酸 100.00％ 95.67％ 86.26％
2％甘氨酸 100.00％ 94.74％ 82.73％
2％葡聚糖 100.00％ 51.65％ 16.20％
结果表明5％甘氨酸可有效避免蛋白形成沉淀。蛋白在热破坏条件下放置17天仍可保持86.26％的蛋白。17天后，甘氨酸的效果显著优于甘露醇和葡聚糖，并且不加稳定剂的蛋白仅剩13.6％。
2)组氨酸对rh-bFGF原液稳定性的影响研究
进一步选用不同氨基酸(甘氨酸、组氨酸、精氨酸)和吐温20对原液的稳定性进行进一步的筛选，在25℃环境下放置18h，结果见图7。
结果表明与浓度为5％的甘氨酸相比，3％的组氨酸和0.03％的吐温20对于rh-bFGF原液的稳定性效果更好。蛋白在热破坏条件下放置18h天纯度仍可保持85％以上。组氨酸和吐温20对rh-bFGF的保护作用优于甘氨酸。
3)HSA对rh-bFGF原液稳定性的影响研究
由于HSA对蛋白含量测定有干扰，将含或不含HSA的原液在25℃ 环境下放置18h，使用高分辨率色谱检测经热破坏蛋白的纯度变化。结果显示在图8。
结果表明HSA能明显改善rh-bFGF的稳定性。以95％纯度变化率作为指标，不含HSA的样品，在压力条件下仅能维持4h，加入HSA后，可延长至15小时。证明对于bFGF，HSA为优秀的蛋白保护剂。
实施例5.动物干眼模型药效研究
本研究选用新西兰兔干眼模型，观察模型的临床指标(泪液分泌、泪膜破裂时间)，评价药物的干眼临床治疗效果。将新西兰兔分成阴性对照组(Negative control，未经处理)、模型对照组(Model，经碱烧伤处理但未加入任何滴眼液)、玻璃酸钠滴眼液处理组(HA group)，以及根据所用的制备实施例3的rh-bFGF滴眼液浓度而分成的组D(4.0μg/mL)、E(8.0μg/mL)、F(16.0μg/mL)、G(32.0μg/mL)。阴性对照组每组72只，其余组别每组8只，剂量300μl/眼/天。
(1)泪液分泌量(双眼)测定方法(酚红棉线湿润长度)：
采用Schirmer I实验(Schirmer I test)测量新西兰兔泪液的分泌。即，用眼科镊夹住酚红棉线，置于新西兰兔外眦部，60s后取出测量酚红棉线湿润的长度。酚红棉线湿润后变为红色，根据湿润长度判定眼睛湿润情况。实验结果显示在图9中。
如图9所示，与阴性对照眼相比，模型对照组的酚红棉线湿润长度均显著下降。与模型对照组相比，给药第10天，rh-bFGF滴眼液D、E、F、G组酚红棉线湿润长度均显著变长，具有显著统计学差异。
(2)泪膜破裂时间(双眼)测定方法：
使用可调移液器在新西兰兔眼下睑结膜囊内滴入2μl的0.5％荧光素钠溶液，以恒定力手动眨眼数次后，以恒定力撑开兔眼，用裂隙灯显微镜，钴蓝光观察角膜，当角膜绿色薄膜出现黑区时，表示泪膜破裂。连续测3次，取其平均值。泪膜破裂时间小于10秒，表明泪膜不稳定，是泪液中粘蛋白缺乏所致的KCS的突出标志，提示结膜的杯状细胞严重损害或丧失，容易造成干眼症。实验结果显示在图10中。
如图10所示，与阴性对照眼相比，给药第10天，模型对组泪膜破裂时间均显著短于阴性对照眼。与模型对照组相比，给药第10天，rh-bFGF滴眼液组D、E、F、G组泪膜破裂时间均显著延长，具有显著统计学差异。
综上，酚红棉线泪液分泌测试结果表明本发明的滴眼液能改善造模动物的泪液分泌量(见图9)；泪膜破裂时间测试显示对干眼模型的泪膜破裂时间有明显的改善作用(见图10)。rh-bFGF滴眼液随着剂量的升高，对碱烧伤新西兰兔干眼症模型有明显的改善作用。
虽然本发明某些特征已经在本文中阐释和描述，但本领域技术人员将想到许多修改、替代、变更和等同。因此，应理解的是，所附权利要求书意在涵盖落入本发明真实精神范围之内的所有此类修改和变更。

Claims (17)

  1. 一种编码重组人碱性成纤维细胞生长因子的突变核酸分子，其包含选自SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3的序列，或与SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3具有至少95％、96％、97％、98或99％同一性的序列。
  2. 权利要求1的突变核酸分子，其序列如SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：3所示。
  3. 一种重组人碱性成纤维细胞生长因子，其由权利要求1或2的突变核酸分子编码。
  4. 一种药物组合物，其包含权利要求3的人碱性成纤维细胞生长因子和至少一种药学上可接受的赋形剂，所述赋形剂包括至少一种选自甘氨酸、组氨酸、精氨酸、吐温、肝素钠或人血白蛋白(HSA)的稳定剂。
  5. 权利要求4的药物组合物，其中所述稳定剂为甘氨酸。
  6. 权利要求4的药物组合物，其中所述稳定剂为组氨酸。
  7. 权利要求4-6的药物组合物，其中所述赋形剂还包括选自磷酸二氢钠-磷酸氢二钠、枸橼酸-磷酸氢二钠或硼酸-硼砂的缓冲体系。
  8. 权利要求4-7的药物组合物，其中所述赋形剂还包括选选自聚乙烯醇，透明质酸钠、甲基纤维素、羟甲基纤维素、羟乙基纤维素、羟丙甲纤维素、泊洛沙姆或卡波姆的增稠剂。
  9. 权利要求4-8中任一项的药物组合物，其中所述赋形剂还包括选自三乙醇胺、氢氧化钠、氢氧化钾、碳酸钠、碳酸氢钠、碳酸氢钾或硼砂的中和试剂。
  10. 权利要求4-9中任一项的药物组合物，其中所述赋形剂还包括选自甘油、丙二醇或其混合物的保湿剂。
  11. 权利要求4-10中任一项的药物组合物，将其缓冲至6.0-8.0的pH。
  12. 权利要求4-11中任一项的药物组合物，所述组合物为眼用组合物，优选为滴眼液或凝胶的形式。
  13. 权利要求4的药物组合物，其包含：
    Figure PCTCN2017089823-appb-100001
  14. 权利要求4的药物组合物，其包含：
    Figure PCTCN2017089823-appb-100002
  15. 权利要求4的药物组合物，其包含：
    Figure PCTCN2017089823-appb-100003
  16. 一种治疗干眼症的方法，所述方法包括向有需要的患者施用治疗有效量的权利要求4-15中任一项的药物组合物。
  17. 一种用于制备权利要求4-15中任一项的药物组合物的方法，其包括将所述人碱性成纤维细胞生长因子与至少一种药学上可接受的赋形剂混合。
PCT/CN2017/089823 2017-06-23 2017-06-23 重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物 Ceased WO2018232750A1 (zh)

Priority Applications (8)

Application Number Priority Date Filing Date Title
EP17914766.5A EP3643784A4 (en) 2017-06-23 2017-06-23 RECOMBINANT HUMAN BASIC FIBROBLASTIC GROWTH FACTOR (RH-BFGF) AND PHARMACEUTICAL COMPOSITION INCLUDING RH-BFGF
US16/625,308 US11254723B2 (en) 2017-06-23 2017-06-23 Recombinant human-basic fibroblast growth factor (rh-bFGF) and pharmaceutical composition comprising rh-bFGF
PCT/CN2017/089823 WO2018232750A1 (zh) 2017-06-23 2017-06-23 重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物
KR1020207002337A KR102480402B1 (ko) 2017-06-23 2017-06-23 재조합 인간-염기성 섬유아세포 성장 인자 (rh-bFGF) 및 rh-bFGF를 포함하는 약학적 조성물
MYPI2019007650A MY194351A (en) 2017-06-23 2017-06-23 Recombinant Human-Basic Fibroblast Growth Factor (Rh-Bfgf) and Pharmaceutical Composition Comprising Rh-Bfgf
SG11201912799SA SG11201912799SA (en) 2017-06-23 2017-06-23 Recombinant human-basic fibroblast growth factor (rh-bfgf) and pharmaceutical composition comprising rh-bfgf
CN201780092441.0A CN111315886A (zh) 2017-06-23 2017-06-23 重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物
JP2020520691A JP7044870B2 (ja) 2017-06-23 2017-06-23 組み換えヒト塩基性線維芽細胞増殖因子（ｒｈ－ｂＦＧＦ）およびｒｈ－ｂＦＧＦを含有する医薬組成物

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/089823 WO2018232750A1 (zh) 2017-06-23 2017-06-23 重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物

Publications (1)

Publication Number Publication Date
WO2018232750A1 true WO2018232750A1 (zh) 2018-12-27

Family

ID=64735425

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/089823 Ceased WO2018232750A1 (zh) 2017-06-23 2017-06-23 重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物

Country Status (8)

Country Link
US (1) US11254723B2 (zh)
EP (1) EP3643784A4 (zh)
JP (1) JP7044870B2 (zh)
KR (1) KR102480402B1 (zh)
CN (1) CN111315886A (zh)
MY (1) MY194351A (zh)
SG (1) SG11201912799SA (zh)
WO (1) WO2018232750A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116889545A (zh) * 2023-06-27 2023-10-17 上海腾瑞制药股份有限公司 一种重组人酸性成纤维细胞生长因子凝胶剂及其制备工艺

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2019013210A (es) 2017-05-05 2020-07-20 Trefoil Therapeutics Inc Factores de crecimiento recombinantes modificados de fibroblastos y usos terapéuticos de los mismos.
CN116710568A (zh) * 2020-06-26 2023-09-05 特里福伊尔治疗公司 重组修饰成纤维细胞生长因子及其治疗用途
CN111647089A (zh) * 2020-06-30 2020-09-11 安徽九川生物科技有限公司 一种重组类人弹性蛋白及其组合物
TWI865835B (zh) * 2020-11-16 2024-12-11 雅祥生技醫藥股份有限公司 穩定的酸性纖維母細胞生長因子組合物
CN113274486A (zh) * 2021-04-15 2021-08-20 上海腾瑞制药股份有限公司 一种稳定的酸性成纤维细胞生长因子制剂及其制备方法和应用
CN116392441B (zh) * 2023-04-21 2023-11-28 上海腾瑞制药股份有限公司 一种酸性成纤维细胞生长因子滴眼液制剂
CN117447580B (zh) * 2023-12-18 2024-04-30 朗肽生物制药股份有限公司 一种碱性成纤维细胞生长因子改构蛋白在护肤产品中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1448510A (zh) * 2002-04-04 2003-10-15 北京三元基因工程有限公司 一种重组人碱性成纤维细胞生长因子的高效表达
CN104606666A (zh) * 2015-02-12 2015-05-13 珠海亿胜生物制药有限公司 重组牛碱性成纤维细胞生长因子滴眼液
CN104984326A (zh) * 2015-07-14 2015-10-21 珠海亿胜生物制药有限公司 不含抑菌剂的重组人碱性成纤维细胞生长因子凝胶剂及其制备方法

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01291797A (ja) * 1987-06-26 1989-11-24 Progen Biotechnik Gmbh 成長因子をコードする合成遺伝子
KR100484653B1 (ko) 2004-05-06 2005-04-20 주식회사 대웅 원핵세포에서 활성형의 가용성 단백질을 제조하는 방법 및 이를 위한 폴리시스트론 벡터
CN1634567A (zh) 2004-09-28 2005-07-06 广州暨南大学医药生物技术研究开发中心 一种含有碱性成纤维生长因子凝胶剂的制备工艺及配方
CN1733294A (zh) * 2005-08-10 2006-02-15 南海朗肽制药有限公司 一种重组人碱性成纤维细胞生长因子凝胶剂及其制备方法
US20070185014A1 (en) 2006-02-09 2007-08-09 The Schepens Eye Research Institute, Inc. Methods and compositions for modulating conjunctival goblet cells
JP5553832B2 (ja) 2009-07-24 2014-07-16 泰彦 田畑 角膜内皮細胞増殖促進剤
CN101658491B (zh) 2009-09-24 2011-04-13 哈尔滨医科大学 一种治疗角膜碱烧伤的羊膜滴眼液
CN101947309B (zh) * 2010-04-12 2012-09-19 南海朗肽制药有限公司 人碱性成纤维细胞生长因子滴眼液及其制备方法
US20110300097A1 (en) 2010-06-04 2011-12-08 Al-Qahtani Ahmed H Method And Composition For The Treatment Of Moderate To Severe Keratoconjunctivitis Sicca
CN102389392B (zh) 2011-11-02 2012-10-24 珠海亿胜生物制药有限公司 重组牛碱性成纤维细胞生长因子眼用凝胶
CN103667329A (zh) * 2012-09-17 2014-03-26 北京双鹭药业股份有限公司 一种高效制备重组人碱性成纤维细胞生长因子的方法
GB2510407A (en) 2013-02-04 2014-08-06 Kalvista Pharmaceuticals Ltd Aqueous suspensions of kallikrein inhibitors for parenteral administration
US20160120693A1 (en) 2013-05-08 2016-05-05 Optometric Technology Group Eye Care Devices and Methods
CN106038350A (zh) 2016-05-31 2016-10-26 南海朗肽制药有限公司 一种复合生长因子护肤凝胶及其制备方法
CN107320716B (zh) 2017-06-27 2019-03-05 珠海亿胜生物制药有限公司 碱性成纤维细胞生长因子囊泡及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1448510A (zh) * 2002-04-04 2003-10-15 北京三元基因工程有限公司 一种重组人碱性成纤维细胞生长因子的高效表达
CN104606666A (zh) * 2015-02-12 2015-05-13 珠海亿胜生物制药有限公司 重组牛碱性成纤维细胞生长因子滴眼液
CN104984326A (zh) * 2015-07-14 2015-10-21 珠海亿胜生物制药有限公司 不含抑菌剂的重组人碱性成纤维细胞生长因子凝胶剂及其制备方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116889545A (zh) * 2023-06-27 2023-10-17 上海腾瑞制药股份有限公司 一种重组人酸性成纤维细胞生长因子凝胶剂及其制备工艺
CN116889545B (zh) * 2023-06-27 2023-12-29 上海腾瑞制药股份有限公司 一种重组人酸性成纤维细胞生长因子凝胶剂及其制备工艺
WO2025001036A1 (zh) * 2023-06-27 2025-01-02 上海腾瑞制药股份有限公司 一种重组人酸性成纤维细胞生长因子凝胶剂及其制备工艺
EP4652987A4 (en) * 2023-06-27 2026-04-08 Shanghai Tenry Pharmaceutical Co Ltd RECOMMENDED HUMAN ACID FIBROBLAST GROWTH FACTOR GEL AND ITS PREPARATION PROCESS

Also Published As

Publication number Publication date
KR20200021521A (ko) 2020-02-28
CN111315886A (zh) 2020-06-19
JP7044870B2 (ja) 2022-03-30
KR102480402B1 (ko) 2022-12-22
MY194351A (en) 2022-11-29
SG11201912799SA (en) 2020-01-30
EP3643784A4 (en) 2021-05-05
EP3643784A1 (en) 2020-04-29
US11254723B2 (en) 2022-02-22
JP2020533015A (ja) 2020-11-19
US20200157164A1 (en) 2020-05-21

Similar Documents

Publication Publication Date Title
WO2018232750A1 (zh) 重组人碱性成纤维细胞生长因子(rh-bFGF)以及包含rh-bFGF的药物组合物
US20220305083A1 (en) Stable peptide compositions
CN110945123B (zh) 生产可溶性重组人碱性成纤维细胞生长因子(rh-bFGF)的方法
JP7670704B2 (ja) 絹由来タンパク質の安定した配合物
WO2016169454A1 (zh) 一种神经生长因子组合物及注射粉剂
CN102336812B (zh) 一种具有抑制血管生成活性的多肽
CN101537172A (zh) 一种含有重组人角质细胞生长因子-2滴眼液及其制备方法
CN117205299A (zh) 一种重组载脂蛋白j在制备治疗眼科疾病药物中的应用
AU2018428269B2 (en) Novel artificial tears containing recombinant human lysozyme and recombinant human EGF
HK40031774A (zh) 重组人碱性成纤维细胞生长因子(rh-bfgf)以及包含rh-bfgf的药物组合物
CN104926933B (zh) Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用
RU2860337C2 (ru) Офтальмологическая фармацевтическая композиция и ее применение
US20250000936A1 (en) Treatment of geographic atrophy
WO2025236782A1 (zh) 一种用于治疗眼部病症的组合物
HK40027218A (zh) 生产可溶性重组人碱性成纤维细胞生长因子(rh-bfgf)的方法
JP2023517459A (ja) 眼科用医薬組成物及びその使用
CN119930748A (zh) 一种提高药物生物利用度的多肽及其应用
JP2025520875A (ja) 中等度／重度のドライアイ関連眼症状の処置のための安定なペプチド組成物およびその使用方法
HK40027218B (zh) 生产可溶性重组人碱性成纤维细胞生长因子(rh-bfgf)的方法
WO2021120937A1 (zh) 转甲状腺素蛋白进入眼内以及在制备滴剂中的应用
HK40052983B (zh) 含有高浓度vegf受体融合蛋白的制剂

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17914766

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020520691

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 20207002337

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2017914766

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2017914766

Country of ref document: EP

Effective date: 20200123

WWG Wipo information: grant in national office

Ref document number: 11201912799S

Country of ref document: SG

WWP Wipo information: published in national office

Ref document number: 11201912799S

Country of ref document: SG