WO2020228705A1 - 一种心脏治疗和保护的药物组合物 - Google Patents

一种心脏治疗和保护的药物组合物 Download PDF

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WO2020228705A1
WO2020228705A1 PCT/CN2020/089859 CN2020089859W WO2020228705A1 WO 2020228705 A1 WO2020228705 A1 WO 2020228705A1 CN 2020089859 W CN2020089859 W CN 2020089859W WO 2020228705 A1 WO2020228705 A1 WO 2020228705A1
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bmp10
complex
dimer
composition
leader peptide
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PCT/CN2020/089859
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French (fr)
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龚笑海
王若璋
金坚
曲秀霞
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龚笑海
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    • 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
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • 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
    • 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
    • A61K47/30—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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics

Definitions

  • the present invention relates to the field of biomedicine, and more specifically to a pharmaceutical composition for cardiac treatment and protection.
  • Anthracyclines represented by doxorubicin, epirubicin, daunomycin, etc. are common drugs for the treatment of hematological tumors and solid tumors.
  • the most common cardiotoxicity is cardiomyopathy associated with anthracycline antibiotics.
  • the use of anthracycline antibiotics can cause left ventricular systolic dysfunction and present a dose-dependent relationship.
  • repeated administration will cause permanent cell and intercellular substance damage, and even lead to heart failure.
  • dextroproimine can reduce the cardiotoxicity caused by doxorubicin .
  • dextropropimine may affect the chemotherapy effect of antitumor drugs and increase the risk of secondary tumors.
  • the antioxidant vitamin E is also believed to be able to resist the cardiotoxicity induced by doxorubicin; the peripheral vasodilator diazoxide can reduce the left ventricular diastolic pressure and eliminate the H 2 induced by doxorubicin O 2 , thereby protecting mitochondria from oxidative damage.
  • the purpose of the present invention is to provide a pharmaceutical composition for cardiac treatment and protection.
  • compositions for protecting the heart and/or treating heart damage containing one or more complexes selected from the following group as active ingredients:
  • the first complex is a complex of a BMP10 dimer and two BMP10 leader peptides, and each of the two monomers of the BMP10 dimer is connected with A BMP10 leader peptide is covalently linked;
  • the second complex is a complex of a BMP10 dimer and two BMP10 leader peptides, and a monomer of the BMP10 dimer is covalently linked to a BMP10 leader peptide , Another BMP10 leader peptide is non-covalently bound to the BMP10 dimer;
  • the third complex is a complex of a BMP10 dimer and two BMP10 leader peptides, and the two BMP10 leader peptides are both non-covalently bound to the BMP10 dimer.
  • the composition consists of or essentially consists of the first, second and/or third complex.
  • the BMP10 is human BMP10 (including wild type and mutant BMP10).
  • the mutant BMP10 lacks a Furin restriction site.
  • the Furin restriction site is mutated to reduce or eliminate Furin restriction.
  • the Furin restriction site RIRR 316 is mutated.
  • At least one of the RIRRs is mutated to Lys, His, or other natural amino acids.
  • the BMP10 after the restriction site mutation is no longer cleaved by Furin, and its activity is the same as the unmutated component, but the structure is more stable.
  • the amount of the first, second and/or third compound in the composition is at least 60% by weight of the total composition, preferably at least 70% by weight, more preferably at least 80% by weight. %, preferably at least 90%.
  • the composition contains a first complex and a third complex.
  • the composition contains the first complex and does not contain the second complex and the third complex.
  • connection site between the BMP10 dimer and the BMP10 leader peptide in the first complex is mutated.
  • the Furin restriction site contained in the first complex is mutated.
  • one, two, three, and four amino acids of the connection site are mutated.
  • the mutated amino acid position is R 313 and/or R 316 and/or R 315
  • the substituted amino acid after the mutation is Lys and/or His.
  • the mutated amino acid position is R 313 and/or R 316 and/or R 315
  • the substituted amino acid after the mutation is Asp and/or Glu.
  • the mutated amino acid position is R 313 and/or R 316 and/or R 315
  • the amino acid substituted after the mutation is Ala and/or Val and/or Leu and/or Ile and/or Pro and /Or Phe and/or Trp and/or Met.
  • the mutated amino acid position is R 313 and/or R 316 and/or R 315
  • the amino acid substituted after the mutation is Gly and/or Ser and/or Thr and/or Asn and/or Gln.
  • the mutated amino acid position is R 313
  • the substituted amino acid is Gly
  • the mutation is a knockout of the DNA encoding R 313 and/or R 316 and/or R 315 .
  • the mutation is the insertion of one or more amino acids between R 313 and/or R 316 and/or R 315 , so that the first complex cannot be recognized by Furin enzyme and cannot be cleaved by enzyme.
  • the first complex is a homodimer.
  • the first complex there is a non-covalent connection between the BMP10 leader peptide and the BMP10 dimer.
  • the BMP10 leader peptide surrounds the BMP10 dimer.
  • Z1 is the leader peptide of BMP10
  • Z2 is the BMP10 growth factor domain
  • the second compound has a structure of formula II:
  • Z1 is the leader peptide of BMP10
  • Z2 is the BMP10 growth factor domain
  • the third compound has a structure of formula III:
  • Z1 is the leader peptide of BMP10
  • Z2 is the BMP10 growth factor domain
  • the third complex there is no covalent connection between the BMP10 leader peptide and the BMP10 dimer.
  • composition further contains one or more minor ingredients selected from the following group:
  • the first essential component, the first essential component is a BMP10 leader peptide, and the BMP10 leader peptide is a free BMP10 leader peptide;
  • the second secondary component which is a complex formed by a BMP10 dimer and a BMP10 leader peptide, and a monomer of the BMP10 dimer is covalently linked to the BMP10 leader peptide ;
  • the third minor component, the third minor component is a complex formed by a BMP10 dimer and a BMP10 leader peptide, and the BMP10 leader peptide is bound to the BMP10 two in a non-covalent manner Aggregate
  • the second minor component has a structure of formula IV:
  • Z1 is the leader peptide of BMP10
  • Z2 is the BMP10 growth factor domain
  • the third minor component has a structure of formula V:
  • Z1 is the leader peptide of BMP10
  • Z2 is the BMP10 growth factor domain
  • the mole percentage P1 of the first compound is ⁇ 40%, wherein,
  • M1 is the number of moles of the first compound
  • M2 is the number of moles of the second compound
  • M3 is the number of moles of the third compound
  • Mf is the total number of moles of the minor components derived from proBMP10.
  • said P1 is ⁇ 50%, preferably ⁇ 60%, more preferably ⁇ 80%, and most preferably, ⁇ 90%, ⁇ 95% or ⁇ 98%.
  • the P1 is 50-99.99%, preferably 60-99.99%, more preferably 80-99.9%, most preferably 90-99.9%.
  • the mole percentage P2 of the second complex is ⁇ 40%, wherein,
  • M1 is the number of moles of the first compound
  • M2 is the number of moles of the second compound
  • M3 is the number of moles of the third compound
  • Mf is the total number of moles of the minor components derived from proBMP10.
  • said P2 ⁇ 30% preferably ⁇ 20%, more preferably ⁇ 10%.
  • the mole percentage of the third compound P3 ⁇ 40% in the composition, the mole percentage of the third compound P3 ⁇ 40%, wherein,
  • M1 is the number of moles of the first compound
  • M2 is the number of moles of the second compound
  • M3 is the number of moles of the third compound
  • Mf is the total number of moles of the minor components derived from proBMP10.
  • said P3 ⁇ 50% preferably ⁇ 60%, more preferably ⁇ 80%, most preferably, ⁇ 90%, ⁇ 95% or ⁇ 98%.
  • the mole percentage Ptotal of the first, second, and third complexes is greater than or equal to 70%, wherein,
  • M1 is the number of moles of the first compound
  • M2 is the number of moles of the second compound
  • M3 is the number of moles of the third compound
  • Mf is the total number of moles of the minor components derived from proBMP10.
  • the Ptotal is ⁇ 80%, preferably ⁇ 90%, more preferably ⁇ 95%, and most preferably, ⁇ 98%.
  • the first complex ie, proBMP10 homodimer
  • the first complex is a dimer composed of two proBMP10 monomers (preferably, the dimer is a homodimer).
  • the second complex (ie, BMP10&proBMP10 heterodimer) is a heterodimer of a proBMP10 and a growth factor domain, and the heterodimer is non-covalent with a BMP10 leader peptide Combine to form a complex.
  • the third complex (ie, BMP10 homodimer) is a homodimer of two BMP10 growth factor binding domains, and the homodimer is non-covalent with two BMP10 leader peptides. Combine to form a complex.
  • the "free BMP10 leader peptide" means that the BMP10 leader peptide does not belong to the proBMP10 dimer or the BMP10 complex, but exists independently.
  • the BMP10 dimer (ie, the fourth minor component) is connected by two BMP10 molecules through interchain disulfide bonds.
  • the BMP10 dimer contains three interchain disulfide bonds.
  • the BMP10 molecule is the growth factor domain of BMP10.
  • a pharmaceutical composition for protecting the heart and/or treating heart damage comprises the composition according to the first aspect of the present invention, and a pharmaceutically acceptable composition.
  • the dosage form of the pharmaceutical composition is selected from the following group:
  • Injections tablets, capsules, granules, powders, films, solutions, suspensions, emulsions, gels, suppositories, ointments, aerosols, sprays, patches.
  • the route of administration of the pharmaceutical composition is selected from the following group:
  • Injection administration oral administration, mucosal administration (nasal mucosa, sublingual, pulmonary administration, rectal mucosa, vaginal mucosa), transdermal administration.
  • the dosage form of the pharmaceutical composition includes a sustained-release agent.
  • a medicine kit in the third aspect of the present invention, includes:
  • the therapeutic drugs include antitumor drugs, sedative hypnotic drugs, antipsychotics and antidepressants, antishock drugs, antihypertensive drugs, vascular smooth muscle dilators, smooth muscle/striated muscle stimulants, central nervous system Stimulants, or combinations thereof.
  • the anti-tumor drugs include anthracycline antibiotics, fluoropyrimidines, taxanes, alkylated drugs, and tyrosine kinase inhibitors.
  • the anthracycline antibiotics include doxorubicin, epirubicin, daunorubicin, cyclophosphamide, and paclitaxel.
  • the sedative and hypnotic drugs include chloral hydrate, hymenone and hibernone.
  • the antipsychotics and antidepressants include phenothiazine antipsychotics (such as chlorpromazine, perphenazine, defluphenazine) and tricyclic antidepressants (such as Imipramine, clomipramine, doclopine).
  • phenothiazine antipsychotics such as chlorpromazine, perphenazine, defluphenazine
  • tricyclic antidepressants such as Imipramine, clomipramine, doclopine
  • the anti-shock drug includes epinephrine, norepinephrine, dopamine, and ephedrine hydrochloride.
  • the antihypertensive drug includes ridazine hydrochloride, guanethidine sulfate, reserpine, methyldopa, and guanidine sulfate.
  • the vasodilators for vascular smooth muscle include dihydralazine, elongatidine, dibazole, aminophylline, ephedrine, boliconyl, chlordiabetic tablets, and diabant.
  • the smooth muscle/striated muscle stimulant includes amine chloride, formylcholine, and galantamine.
  • the central stimulant includes lobeline hydrochloride, imipramine hydrochloride, atropine sulfate, belladonna tincture.
  • kit further includes instructions that describe the combined administration of the composition and the therapeutic drug, so as to protect the heart while treating diseases.
  • the fourth aspect of the present invention there is provided a use of the composition according to the first aspect of the present invention for preparing a medicine for protecting the heart and/or treating heart damage.
  • said protecting the heart includes protecting the systolic and diastolic functions of the heart.
  • said protecting the heart includes avoiding heart damage or heart failure.
  • the cardiac injury includes functional disease and organic injury of the heart.
  • the heart injury is selected from the following group:
  • a method for protecting the heart and/or treating heart damage comprising the steps:
  • composition according to the first aspect of the present invention or a medicine containing the composition as an active ingredient is administered to a subject in need.
  • the described is used for scientific research purposes.
  • the said method is used to protect the heart of an animal model during scientific research.
  • the subject includes humans and non-human mammals.
  • the object includes rodents, such as mice and rats.
  • Figure 1 shows the electrophoresis of fusion PCR point mutation nucleic acid.
  • Figure 1A is the electrophoresis of nucleic acid amplification of the fragments on both sides of the mutation site containing the point mutation site
  • Figure 1B is the electrophoresis of the long-segment fusion PCR electrophoresis
  • Figure 1C is the electrophoresis of the target fragment amplification
  • Figure 1D is the vector digestion electrophoresis Figure.
  • Figure 2 shows the plasmid map of pMH3-BMP10.
  • Figure 3 shows an immunoblot diagram of the construction of rhBMP10 expressing cell lines.
  • Figures 3A, 3B, and 3C are the results of the first, second, and third clone screening, respectively
  • Figure 3D is the expression after monoclonalization
  • Figure 3E is the Western blot map after monoclonalization.
  • Figure 4 shows a broken line diagram of the suspension acclimation of the CHO-BMP10 cell line.
  • Figure 5 shows the growth and product accumulation of the CHO-BMP10 cell line during the 3L shake flask fed culture process.
  • Figure 5A is a broken line graph of cell growth
  • Figure 5B is a Western blot graph of product accumulation.
  • Figure 6 shows the Q column purification results.
  • Figure 6A is the Q column purification operation map (A solution is 50mM NaPB, B solution is 1000mM NaCl),
  • Figure 6B is a Coomassie blue staining image of collected elution peaks, and
  • rhBMP10 lane is a 50mM NaPB elution peak containing 250mM NaCl
  • BMP10 (R&D) lane is the control substance purchased from R&D company.
  • Figure 7 shows the results of Gel filtration column purification.
  • Fig. 7A is a purification operation chart of Gel Filtration column
  • Fig. 7B is a Coomassie blue staining chart of collected elution peaks (1 is ⁇ 12mL elution peak, 2 is ⁇ 19mL elution peak).
  • Figure 8 shows the Coomassie blue staining map, Western blot map and Native page map after Furin digestion.
  • Figure 8A is the post-translational processing structure diagram of BMP10
  • Figure 8B is the Coomassie brilliant blue staining diagram before and after purification of rhBMP10 Furin
  • Figure 8C is the anti-BMP10 and anti-BMP10 propeptide Western blot diagrams before and after the purification of rhBMP10
  • Figure 8D is the Native page map of purified rhBMP10
  • Figure 8E is the Native page anti-BMP10 Western blot map of purified rhBMP10
  • Figure 8F is the Native page anti-BMP10 propeptide Western blot map of purified rhBMP10.
  • Figure 9 shows the plasmid map of pGL6-BRE-Luciferase.
  • Figure 10 shows the luciferase report results.
  • Figure 10A shows the effect of different concentrations of serum in the culture medium on the signal (BMP10 purchased from R&D Systems)
  • Figure 10B shows the standard curve (rhBMP10 purchased from R&D Systems)
  • Figure 10C shows the activity test of the purified rhBMP10 protein.
  • Figure 11 shows the activity of rhBMP10 protein before and after Furin digestion.
  • Figure 12 shows the results of echocardiography.
  • Figure 12A is a representative M mode echocardiogram of each group of mice
  • Fig. 14 shows the structures of several complexes contained in the composition having cardioprotective and/or heart injury treatment functions of the present invention. Among them, 1 is the structure of the first complex of the present invention, 2 is the structure of the second complex, 3 is the structure of the third complex, and 4 is the free leader peptide.
  • Figure 15 shows the predicted natural structure of BMP10.
  • Figure 16A is a Western blot image of the anti-BMP10 leader peptide digested with purified rhBMP10 and rhBMP10 (313G) Furin
  • Figure 16B is a map of anti-BMP10 digested with purified rhBMP10 and rhBMP10 (313G) Furin.
  • Figure 17 shows the activity of purified rhBMP10 and rhBMP10 (313G) proteins after Furin digestion.
  • the inventors unexpectedly prepared a BMP10 complex derived from proBMP10, as well as compositions, kits, and applications containing the BMP10 complex for the first time.
  • the BMP10 complex of the present invention includes a first complex, a second complex and a third complex.
  • the above three complexes are composed of one BMP10 dimer and two BMP10 leader peptides that are covalently or non-covalently connected. .
  • the results show that the composition of the present invention has a positive effect on alleviating the cardiac function and organic damage caused by anti-tumor drugs.
  • the presence of the leader peptide in the complex of the present invention not only does not affect the original function of BMP10, but also prolongs the half-life of rhBMP10, so as to better exert its function.
  • Bone morphogenetic proteins are secreted extracellular signaling peptides belonging to the TGF- ⁇ superfamily, which bind to the type II transmembrane Ser/Thr kinase receptor complex in the form of dimers , Phosphorylation modifies the Smad family transcription factor, the phosphorylated Smad protein is transferred to the nucleus and regulates the transcription of specific target genes.
  • BMPs Bone morphogenetic proteins
  • BMPs can induce multiple key regulators of heart development such as Nkx2.5, Gata4, Tbx and Hand genes in the embryonic heart, and cooperate with multiple heart-specific transcription factors to regulate cardiomyocyte proliferation, induce cardiomyocyte differentiation, ventricular wall and The formation of heart valves.
  • BMP10 is a special member of the BMP family.
  • the expression of BMP10 is largely restricted to cardiomyocytes during development. It has been proved in articles that BMP10 can regulate the proliferation of cardiomyocytes and the formation of ventricular wall during heart development. From birth to adult mouse heart, the expression of BMP10 is restricted to the right atrium, and the secreted BMP10 protein molecule first enters the left ventricle through the blood circulation, and then is sent to the whole body. Therefore, there is a relatively high concentration of active BMP10 molecules in the left ventricle. It has certain nutritional and protective effects on ventricular cells. The functional research of BMP10 in the adult heart is not very sufficient.
  • BMP10 is highly expressed in cardiomyocytes of hypertensive myocardial hypertrophy rat models. Peripheral blood testing of some clinical acute myocardial infarction patients also found an up-regulation of BMP10 expression, suggesting that BMP10 plays a role in heart injury and repair.
  • the complete BMP10 molecule consists of 424 amino acid residues and is divided into three parts, namely 11-21, signal peptide, 222-316, leader peptide, 3317-424, growth factor domain.
  • the calculated molecular weight is 48kDa.
  • the synthetic form of BMP10 is a full-length precursor protein.
  • Anthracyclines represented by doxorubicin, epirubicin, daunomycin, etc. are common drugs for the treatment of hematological tumors and solid tumors.
  • the most common cardiotoxicity is cardiomyopathy associated with anthracycline antibiotics.
  • the use of anthracycline antibiotics can cause left ventricular systolic dysfunction and present a dose-dependent relationship.
  • repeated administration will cause permanent cell and intercellular substance damage, and even lead to heart failure.
  • Chronic cardiotoxicity caused by anthracycline antibiotics presents a dose-dependent relationship and is irreversible, and can be divided into early-onset chronic toxicity and late-onset chronic toxicity based on the time when symptoms appear.
  • 1Early-onset chronic toxicity appears after the start of treatment and can last one year after the end of treatment. 1.6%-2.1% of patients developed the disease during treatment or within one year after the end of treatment; 2The delayed chronic toxicity appeared one year after the end of treatment. In 1.6%-5% of patients, cardiac damage does not progress until 10-20 years after the end of chemotherapy.
  • the main feature of chronic cardiotoxicity caused by anthracycline antibiotics is ventricular systolic and diastolic dysfunction, which often leads to dilated cardiomyopathy and heart failure.
  • doxorubicin inducing cardiotoxicity 1 inhibiting the activity of DNA synthetase, or combining with the DNA of cells in the replication cycle to cause DNA fragmentation; 2 inhibiting the synthesis of DNA-RNA-protein Pathway; 3mediate the production of reactive oxygen species (ROS), leading to an increase in the level of cell oxidation, leading to lipid peroxidation or cell vacuolation; 4influencing the transcription process of important myocardial-specific genes .
  • ROS reactive oxygen species
  • the decrease in protein expression level and the degradation of myofilament eventually lead to damage to the sarcomere and cause sarcopenia of the heart.
  • anthracyclines and trastuzumab can exacerbate the damage of muscle fibers and DNA.
  • Anthracycline antibiotics can also disrupt the dynamic regulation of heart function, change the activity of adenylate cyclase, and affect calcium homeostasis.
  • Anthracycline antibiotics can cause the apoptosis or necrosis of cardiomyocytes.
  • the total number of cardiomyocytes will continue to decline, leading to ventricular remodeling, and the cardiomyocytes will eventually be transformed into fibrous tissue with inelasticity. replace.
  • the present invention provides a BMP10 complex.
  • the BMP10 complex includes a first complex, a second complex and a third complex.
  • the above three complexes are covalently or non-covalently linked by a BMP10 dimerization
  • the body is composed of two BMP10 leader peptides.
  • the BMP10 complex includes:
  • the first complex is a complex of a BMP10 dimer and two BMP10 leader peptides, and each of the two monomers of the BMP10 dimer is connected with A BMP10 leader peptide is covalently linked;
  • the second complex is a complex of a BMP10 dimer and two BMP10 leader peptides, and a monomer of the BMP10 dimer is covalently linked to a BMP10 leader peptide , Another BMP10 leader peptide is non-covalently bound to the BMP10 dimer;
  • the third complex is a complex of a BMP10 dimer and two BMP10 leader peptides, and the two BMP10 leader peptides are both non-covalently bound to the BMP10 dimer.
  • the BMP10 complex of the present invention further includes:
  • the second secondary component which is a complex formed by a BMP10 dimer and a BMP10 leader peptide, and a monomer of the BMP10 dimer is covalently linked to the BMP10 leader peptide ;
  • the third minor component, the third minor component is a complex formed by a BMP10 dimer and a BMP10 leader peptide, and the BMP10 leader peptide is bound to the BMP10 two in a non-covalent manner Aggregate
  • CHO-S engineered cells are used to express the biologically active rhBMP10, thereby preparing the BMP10 complex. Due to the lack of sufficient PC activity in CHO-S, rhBMP10 overexpressed by CHO-S cannot be completely cleaved intracellularly. The main secreted form of rhBMP10 is uncut and partially cleaved dimers. The mature dimer of rhBMP10 can only be detected in the last few days of culture.
  • leader peptide does not affect the activation of BMP10 in rat cardiac myoblasts (C2C12) and human pulmonary artery epithelial cells (HPAEC). Phosphorylation of the Smad1/5/8 signaling pathway; and other literature believes that the presence of the leader peptide will seriously affect the transcription process of BMP10 inducing the Id3 gene in C2C12 cells.
  • the invention also provides a pharmaceutical composition containing the BMP10 complex as an active ingredient, which is used to protect the heart while applying anti-tumor drugs.
  • the BMP10 complex can be directly used for disease treatment, and other therapeutic agents, such as anti-tumor drugs, can also be used at the same time.
  • the present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the BMP10 complex of the present invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powder, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions.
  • the pharmaceutical combination of the present invention can also be made into a powder for inhalation.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the BMP10 complex of the present invention can also be used together with other therapeutic agents.
  • composition of the present invention can be administered to desired subjects (such as humans and non-human mammals) in a conventional manner.
  • desired subjects such as humans and non-human mammals
  • Representative administration methods include (but are not limited to): oral, injection, aerosol inhalation, etc.
  • a safe and effective amount of the BMP10 complex is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases not more than about 8 mg/kg body weight, which is more Preferably, the dosage is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.
  • the BMP10 complex of the present invention has a positive effect on reducing cardiac function and organic damage caused by anti-tumor drugs
  • the leader peptide in the BMP10 complex of the present invention can extend the half-life of rhBMP10, so that it can better perform its functions.
  • the BMP10 complex of the present invention has a positive protective and reversal effect on the heart that has undergone functional disease and organic damage, that is, the heart that has a history of heart disease;
  • the BMP10 complex of the present invention has a positive preventive effect on possible heart diseases
  • the BMP10 complex of the present invention may be used as an adjuvant or adjuvant for cardiotoxic drugs, which can reduce the damage to the heart by the drug while the drug exerts its function.
  • the CHO-S cell line is the cell line retained in the laboratory, the C2C12 cell line was purchased from ATCC, and the mouse primary cardiomyocytes and special culture medium were purchased from Procell.
  • 70 healthy male C57BL/6J mice, 8 weeks old, weighing 22 ⁇ 2g, were purchased from Shanghai Slack Experimental Animal Center, and adaptively reared in the Experimental Animal Center of Jiangnan University School of Medicine, and the experiment started after one week.
  • the breeding environment is constant temperature and humidity (25 ⁇ 2°C, 50 ⁇ 10%), with daily light for 12 hours, and free food and water.
  • the main reagents used in the examples are all commercially available.
  • FL-5' and FL-3' are primers at both ends of the sequence
  • MID-5' and MID-3' are intermediate primers containing point mutation sites.
  • the wavy line is the restriction site
  • the underline is the mutation site.
  • FL-5' and MID-3' primers respectively, FL-3' and MID-5' primers amplify the first half of the cDNA sequence and the second half of the cDNA sequence covering the mutation site, and use 1.5% agarose for the PCR product Separate by gel electrophoresis, take pictures and recover, use the two recovered products as templates, FL-5' and FL-3' as primers, and then perform PCR. The products are separated by 2% agarose gel electrophoresis, photographed and recovered, using NanoDrop Measure the concentration in 2000 and put it in the refrigerator at -30°C for later use.
  • the PCR product was digested with EcoRI and NotI restriction enzyme sites for 2 hours, and the PCR product recovery kit was used for recovery.
  • the pMH3 vector was digested for 2 hours, separated by 1% agarose gel electrophoresis, photographed and recovered, and measured by NanoDrop2000. Concentration, then use T4 Ligase at a ratio of 4:1, connect at 22°C for 30 minutes, and immediately perform DH5 ⁇ conversion.
  • the DNA obtained by methods 2.1 and 2.2 of rhBMP10 were mutated to construct a variety of mutation forms: (1) the amino acid R at position 313 of the BMP10 protein was mutated to G; (2) the amino acid R at position 316 of the BMP10 protein was mutated to G; (3) Simultaneously mutate (1) and (2); (4) mutate the amino acid R at position 315 to G, and mutate the above (1)(2)(3) at the same time; (5) randomly mutate positions 313-316 .
  • the purpose is to obtain that Furin cannot recognize, but the activity of rhBMP10 after mutation is the same or better than before mutation.
  • the mutation method is the same as method 2.1 and 2.2.
  • the medium of CHO-S cells is D/F12 basal medium with 10% FBS, and the C2C12 cell medium is DMEM basal medium with 10% FBS and 1% double antibody.
  • CHO-S cells cultured in suspension take a sample to measure the cell density, and mix a certain volume of culture medium with a certain volume of fresh medium according to the experimental needs.
  • the suspension medium of CHO-S cells is a 1:1 configuration of M2 and M4, and glutamine with a final concentration of 50 mM is added.
  • the cells in good condition were digested and centrifuged after T75 resumed growth, inoculated into a 150mL shake flask at a density of 2.0 ⁇ 10 6 cells/mL, cultured on a cell shaker, and observed cell growth daily happening. Adjust the cell density to about 2.0 ⁇ 10 6 cells/mL until the cell density can double in 24 hours and the viability rate is ⁇ 95%.
  • Plating C2C12 cells in good growth condition take two 1.5mL EP tubes, add 25 ⁇ L Opti-MEM respectively, add 500 ⁇ g plasmid to one tube, add 1.5 ⁇ L Lipo3000 to the other tube, let stand at room temperature for 5min, mix well, Let it stand for 5 min, add it to the hole, and place it in a wet carbon dioxide incubator for static culture (37°C, 5% CO 2 ).
  • the CHO-BMP10 cell line was suspended and acclimated, and the well-growing seeds were inoculated into a 250 mL shake flask with a density of 1.0 ⁇ 10 6 cells/ml, a volume of 150 mL, 100 rpm, and culture at 37°C.
  • the density reaches 5.0-6.0 ⁇ 10 6 cells/ml
  • put the secondary seeds into a 3L shake flask and incubate at 100 rpm and 37°C.
  • the temperature is lowered to 34°C for culture.
  • Feed 4 feed medium according to the glucose concentration to maintain the glucose concentration in the system at about 3g/L.
  • the cell diameter is ⁇ 16 ⁇ m, pay close attention to the cell viability. When the viability drops suddenly (about 90%), stop the culture.
  • the purified product of Q column was concentrated with a 10kDa ultrafiltration tube.
  • the mobile phase used is 50mM NaPB+150mM NaCl, and the system flow rate is 0.5mL/min.
  • the Loop loop is loaded onto the column equilibrated with 50mM NaPB+150mM NaCl, and the elution peaks are collected. Pay attention to drop-to-drop connection during the connection of the chromatographic column to avoid air bubbles; monitor the column pressure during the system operation to prevent overpressure.
  • RhFurin was diluted to 4 ⁇ g/mL with PBS, and BMP10 was diluted to 100 ⁇ M. Mix the diluted rhFurin with rhBMP10 1:1, add 1% 100mM CaCl 2 , and incubate at 37°C for 1 hour.
  • Primary mouse cells are cultured in a special culture medium. After the cells grow to 70-80%, trypsinize the cells, inoculate the cells in two 24-well cell plates with 8000 cells per well, and place them in a cell incubator for regular culture 18-48h.
  • BMP10 Take out one of the 24-well cell plates, and add BMP10 with final concentrations of 0 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, and 10 ng/mL to the well plates, each with 4 replicate wells. After 24 hours, doxorubicin and paclitaxel were added for treatment, and the final concentration was 1 ⁇ g/mL, and the control group was taken out after 10 hours.
  • doxorubicin or paclitaxel Take out another 24-well cell plate, add doxorubicin or paclitaxel to the well plate for treatment, the final concentration of which is 1 ⁇ g/mL, the untreated group is used as the control group, 4h later, the final concentration is added to the well plate.
  • the concentration of BMP10 was 0 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, and 10 ng/mL. After 24 hours, the cell plate was taken out.
  • the Vevo 2100 small animal ultrasound system from Visual Sonics was used to perform echocardiography after the fifth week of the experiment.
  • Vaporizer nebulizer place the mouse in a closed transparent acrylic box to inhale isoflurane anesthetize, apply an appropriate amount of depilatory cream on the left chest of the mouse, and wipe off the hair with a wet cotton ball after a while. Then put the mouse in the supine position and put on the isoflurane breathing mask. Fix the limbs to collect ECG and respiratory signals. After the heart rate stabilizes, apply couplant on the left chest and connect the probe to collect data. Select the long-axis and short-axis slices respectively for acquisition.
  • the M mode ultrasound is intercepted from the B mode echocardiogram, and on this basis, the short axis shortening rate (fraction shortening, FS), ejection fraction (EF), left ventricular end diastolic diameter ( left ventricular internal diameter at diastole, LVID, d), left ventricular end systolic diameter (left ventricular internal diameter at systole, LVID, s), left ventricular end diastolic volume (left ventricular volume at diastole, LV Vol,d), left ventricle Parameters such as left ventricular volume (left ventricular volume at systole, LV Vol, s).
  • the short axis shortening rate fraction shortening, FS
  • EF ejection fraction
  • left ventricular end diastolic diameter left ventricular internal diameter at diastole, LVID, d
  • left ventricular end systolic diameter left ventricular internal diameter at systole, LVID
  • the experimental process is basically the same as 2.2.15.
  • the main reagents include R1 nuclear staining solution, R2 slurry staining solution, R3 color separation solution, R4 counterstaining solution and R5 washing solution.
  • the staining steps are as follows:
  • Routine slices are deparaffinized to water
  • R2 slurry dyeing solution is dyed for 40s, discarded, rinsed with washing solution for 30s;
  • Routine slices are deparaffinized to water
  • the linearized empty plasmid at ⁇ 8,000bp and ⁇ 1,300bp can be seen The original mount fragment.
  • the target fragment amplified by PCR was digested with EcoRI and NotI restriction enzymes at 37°C to obtain sticky ends, and the linearized empty load after digestion was ligated with T4 Ligase at 22°C for 30 minutes, and then DH5 ⁇ competent The cells were transformed, and positive clones were selected and inoculated in LB medium and sent to GeneWiz for sequencing to confirm that the pMH3 plasmid expressing rhBMP10 containing the target mutation was obtained.
  • Furin's specific catalytic cleavage recognition site is: Arg-XY-Arg carboxy terminal peptide bond, where X can be any amino acid and Y is Arg or Lys.
  • the mutation site of Furin in BMP10 is RIRR 316 , and the corresponding gene encodes CGAATCAGAAAGG, so this part of the sequence is mutated.
  • the mutated plasmid was sent to GeneWiz for sequencing to confirm that multiple pMH3 plasmids expressing rhBMP10 containing the target mutation were obtained.
  • the constructed multiple plasmids were transiently transfected into CHO cells with lipo3000, the cells were lysed, and the expression of BMP was detected by WB.
  • the 2-4 plasmids with uncut leader peptides were screened and reserved for further testing of the corresponding plasmid expression. BMP10 protein activity.
  • the pMH3 plasmid was electrotransfected into empty CHO-S cells, and G418 at a final concentration of 4.5 mg/mL was added for pressure screening. After about 10 days, a white translucent cell clone was visible to the naked eye at the bottom of the dish. The clones were picked to a 96-well plate and cultured until round clones were grown, then they were cultured with D/F basic medium for two days, and the expression level of rhBMP10 in the supernatant was detected by Dot blot.
  • the background is dark, which on the one hand indicates that the expression level of rhBMP10 is low, on the other hand, it indicates that the cells in a single well are cells of non-single source, and a certain percentage of cells cannot express rhBMP10.
  • the expression level of rhBMP10 showed an upward trend.
  • FIG. 1 shows the immunoblot of the construction of the CHO-BMP10 cell line.
  • the growth environment of the cells is worse than normal culture.
  • Cells in a well plate need to start dividing and growing from a single or a few cells, during which there is no communication between cells, crowded space, and serum-free (nutrient and growth factor deficiency) environment.
  • serum-free nutrient and growth factor deficiency
  • cells may turn off genes that do not affect survival through promoter methylation.
  • the cell line that can maintain a high level of stable expression of the target protein can be considered to have better resistance to harsh environments and high robustness, which is more conducive to long-term and stable culture.
  • the adherent method is mainly used for cell culture and screening. Compared with the suspension culture method, it is relatively difficult to achieve high-density culture in adherent culture. Need special perfusion equipment, consumables for high cultivation area, etc. Therefore, the large-scale production of rhBMP10 using the CHO-BMP10 cell line requires the use of suspension high-density culture. Before using the CHO-BMP10 cell line for the production and expression of rhBMP10, the CHO-BMP10 cell line needs to be subjected to suspension acclimation so that it can adapt to the suspended growth environment.
  • the suspension cultured cells are taken to inoculate the shake flask to prepare seeds.
  • the shake flask was cultured to a density of 4 ⁇ 10 6 cells/mL and seeds with a viability of 99.8% or more were inoculated into a 3L shake flask, and samples were taken daily for testing.
  • the cells are in a state of rapid growth at 0-3d. At this time, the cells are in the logarithmic growth phase, with vigorous cell proliferation, high viability and small cell diameter.
  • the glucose consumption is also fast, and the lactate level increases rapidly; start on the fourth day Feeding, while lowering the culture temperature to 34°C, to extend the plateau period and maintain a high-density growth state of cells.
  • the cell growth rate decreases and the survival rate also slowly decreases. Finally, the maximum density (9.1 ⁇ 10 6 cells/mL) was reached on the 8th day. Starting from the third day, the cell diameter gradually increased and reached 16.4 ⁇ m on the ninth day, indicating that the cells were senescent, and the viability rate dropped to ⁇ 94%, so the culture was ended on the tenth day.
  • Cultivation should be terminated promptly after cell senescence.
  • the main reason is that the production capacity of cells after aging decreases and the target protein is almost no longer expressed; 2to prevent cell autolysis and release protease to destroy the accumulated target protein; 3to prevent cell autolysates from polluting the culture supernatant and bring about subsequent purification unnecessary trouble.
  • the samples taken every day were added to 5 ⁇ SDS Loading Buffer and electrophoresed. The results are shown in Figure 5.
  • the rhBMP10 gradually accumulates with the increase of incubation time and presents the characteristics of multiple bands, among which the ⁇ 110kDa band is at 1 -2d is less, and then gradually increased; bands of ⁇ 68kDa and ⁇ 57kDa began to accumulate at 3d, while mature rhBMP10 dimers at ⁇ 25kDa began to appear at 7d, indicating that the CHO expression system lacks the relevant restriction digestion system. It is not enough to process the RIRR ⁇ 316 restriction site in rhBMP10.
  • the culture system was immediately centrifuged to remove insoluble cells and cell debris, and the cell-free culture supernatant was obtained. Filter the obtained culture to 0.22 ⁇ m to remove small cell debris.
  • the obtained liquid should be orange-yellow transparent and clear liquid.
  • the treated liquid is then subjected to ultrafiltration to remove salt in the medium and impurities less than 10kDa, and concentrate the system. The liquid after the end of ultrafiltration was concentrated from ⁇ 5L before the treatment to ⁇ 500mL, and the treated liquid appeared light yellow with a slightly sticky feeling.
  • the UV280 Use the concentrated culture supernatant to load the sample. After the sample is loaded, the UV280 rapidly rises to between 1400-2000mAU, and the electropositive protein is washed out because it cannot be combined with the filler. After loading the sample, it was rinsed with 50mM NaPB, and the UV280 dropped quickly and gradually returned to the baseline. Then directly use 50mM NaPB containing 250mM NaCl for elution. At this time, a peak of ⁇ 5800mAU appears on the UV280 spectrum, and the elution peak is collected for the next step of purification.
  • Coomassie Brilliant Blue staining results of the eluted peaks are shown in Figure 6, showing the characteristics of multiple bands, mainly including four bands-116kDa, -68kDa, -43kDa and -26kDa.
  • the loading volume should be controlled within 1% of the column volume, and the protein concentration of the sample should be controlled within 1 mg/mL.
  • the mobile phase used in gel filtration chromatography is 50 mM NaPB containing 150 mM NaCl.
  • the addition of NaCl is mainly to reduce the non-specific adsorption of proteins by the gel globules.
  • the purification results are shown in Figure 7. After loading the sample, an elution peak appeared at ⁇ 55mL, and the UV280 was about 500mAU; a small peak with UV280 of 110mAU appeared at ⁇ 95mL. Collect two elution peaks and prepare samples for electrophoresis. On the Coomassie brilliant blue stained gel, the electrophoresis result of the first main peak shows 4 bands, which are consistent with the result of Q column purification, indicating that rhBMP10 is secreted in various forms , And maintain a similar molecular weight. The specific results will be analyzed in 3.2.2. The electrophoresis result of the second peak is basically invisible, it should be a small molecular weight contaminated protein.
  • Fig. 17 The result is shown in Fig. 17. It was verified by Fig. 16 that the amino acid R at position 313 of rhBMP10 was mutated to G to produce rhBMP10 (313G), which was not cleaved by furin; the signal value of the uncut rhBMP10 (313G) was 67617 ⁇ 2620 RLU, still has biological activity, rhBMP10 can induce 85305 ⁇ 6419 RLU expression at the same concentration, BMP10 purchased from R&D induces 72923 RLU expression. It is proved that CHO-BMP10 is expressed and purified at 313 site mutation rhBMP10, its leader peptide The presence of rhBMP10 does not affect the activity or function of rhBMP10.
  • rhBMP10 expressed and purified by CHO-BMP10 on mouse cardiomyocytes
  • the mouse cells were treated with rhBMP10 before or after the cardiotoxic drug attack, and TUNEL was used to detect cardiomyocyte apoptosis.
  • the results showed that when mouse cardiomyocytes were treated with BMP10 and then treated with doxorubicin or paclitaxel, the apoptosis of cardiomyocytes was significantly reduced compared with cells that were not treated with BMP10.
  • BMP10 can not only protect mouse cardiomyocytes from the stimulation of cardiotoxic drugs, but also treat and reverse the damage of cardiotoxic drugs to cardiomyocytes.
  • TUNEL The main function of TUNEL is to detect the breakage of nuclear DNA during cell apoptosis.
  • dUTP is connected to the 3'-OH end of broken DNA in apoptotic cells under the action of deoxyribonucleotide terminal transferase; through the biotin label attached to dUTP, HRP-labeled streptavidin is used Signals can be reported; in the presence of diaminobenzidine (DAB), it produces a strong color response (dark brown), and specifically and accurately locates apoptotic cells. Normal or proliferating cells have almost no DNA breaks, so there is no 3'-OH formation, and negative results during the staining process.
  • DAB diaminobenzidine
  • the TUNEL test results are shown in Figure 13.
  • the proportion of cardiac apoptosis in the DOX group mice was significantly increased (p ⁇ 0.01), reaching 0.196%.
  • the DOX+rhBMP10 group had a similar degree of cardiac apoptosis in the Saline group, which were 0.0361% and 0.0136%, respectively.
  • the apoptotic ratio of cardiomyocytes was 0.0234%.

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Abstract

本发明提供了一种保护心脏和治疗心脏损伤的组合物。具体地，本发明提供了衍生自proBMP10的BMP10复合物，以及包含BMP10复合物的组合物、药盒、及其应用。其中，本发明的BMP10复合物包括第一复合物、第二复合物和第三复合物，上述三种复合物由共价或非共价连接的一个BMP10二聚体与两个BMP10前导肽构成。本发明的组合物可以减轻抗肿瘤药物所致心脏功能性和器质性损伤。

Description

一种心脏治疗和保护的药物组合物 技术领域
本发明涉及生物医药领域，更具体地涉及一种心脏治疗和保护的药物组合物。
背景技术
随着我国人口老龄化日益加剧，高血压、冠心病、糖尿病等老年慢性疾病的发病率逐年上升，心衰患者人数也随之迅速增长，成人患病率(35-74岁)约为0.9％，已成为65岁以上患者人群住院的首要原因，且患者的长期预后差，五年生存率小于50％，严重危害国民健康，也给公共医疗带来沉重的负担。对于临床心衰阶段病人，目前缺乏有效的治疗方案，临床常用的治疗性药物如ACEI/ARB、β-受体阻滞剂、醛固酮受体拮抗剂等的主要目标仍仅是预防心源性猝死和部分改善心功能，并不能有效控制和逆转心衰的发展进程。
以多柔比星、表柔比星、道诺霉素等为代表的蒽环类抗生素是临床上治疗血液系统肿瘤和实体瘤的常见药物。在癌症治疗中，最常见的心脏毒性便是与蒽环类抗生素相关的心肌病变。蒽环类抗生素的使用会导致左心室收缩功能紊乱，并呈现出剂量依赖关系。同时，重复多次的给药将会导致永久性的细胞和细胞间质损伤，甚至导致心力衰竭。
临床上需要寻找合适的药物，在不干扰蒽环类抗生素的抗肿瘤作用的前提下，拮抗其导致的心脏毒性。鉴于ROS和氧化应激在蒽环类诱导的心脏毒性中的重要作用，研究热点主要集中在寻找能够改善心肌细胞抗氧化防御能力的药物和天然化合物。包括抗氧化剂，铁螯合剂和降脂药物在内的多种药物已经在动物模型和人体中进行了测试，但一些药物的心脏保护作用仍然值得商榷。右丙亚胺是目前唯一批准用于预防蒽环类药物引起的心脏毒性的临床药物，尽管其作用机制尚不明确，大量临床证据表明，右丙亚胺可以减少多柔比星引起的心脏毒性。但右丙亚胺可能影响抗肿瘤药物的化疗效果和增加继发性肿瘤风险。在临床前研究中，抗氧化剂维生素E也被认为能够抵抗多柔比星诱导的心脏毒性；外周血管扩张剂二氮嗪能够减轻左心室舒张压，并消除由多柔比星诱导产生的H 2O 2，从而保护线粒体免受氧化损伤。
尽管关于心肌细胞的受损与死亡的分子机制已经研究的比较透彻，但目前仍未有有效的治疗方法来保护心肌细胞并阻止其死亡，本领域需要开发新的保护心肌细胞的药物和方法
发明内容
本发明的目的在于提供一种心脏治疗及保护的药物组合物。
在本发明的第一方面，提供了一种用于保护心脏和/或治疗心脏损伤的组合物，所述组合物含有选自下组的一种或多种复合物作为活性成分：
(a)第一复合物，所述的第一复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述BMP10二聚体的两个单体中每一个单体分别与一个BMP10前导肽共价连接；
(b)第二复合物，所述的第二复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述BMP10二聚体的一个单体与一个BMP10前导肽共价连接，另一个BMP10前导肽以非共价方式结合于所述BMP10二聚体；
(c)第三复合物，所述的第三复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述的两个BMP10前导肽均以非共价方式结合于所述BMP10二聚体。
在另一优选例中，所述组合物中由或基本上由第一、第二和/或第三复合物构成。
在另一优选例中，所述的BMP10是人BMP10(包括野生型和突变型BMP10)。
在另一优选例中，所述的突变型BMP10缺乏Furin酶切位点。
在另一优选例中，所述的BMP10中，Furin酶切位点被突变，从而降低或消除Furin酶切。
在另一优选例中，所述的proBMP10中，Furin酶切位点RIRR 316被突变。
在另一优选例中，所述的RIRR中至少一个被突变为Lys、His、或其他天然氨基酸。
在另一优选例中，所述的酶切位点突变后的BMP10不再被Furin切割，同时其活性同于未突变组份，但结构上更加稳定。
在另一优选例中，所述组合物中第一、第二和/或第三复合物的数量为所述组合物总量的至少60wt％，较佳地至少70wt％，更佳地至少80％，最佳地至少90％。
在另一优选例中，所述的组合物含有第一复合物和第三复合物。
在另一优选例中，所述的组合物含有第一复合物，并且不包含第二复合物和第三复合物。
在另一优选例中，所述的第一复合物中BMP10二聚体与BMP10前导肽的连接位点是被突变的。
在另一优选例中，所述的第一复合物中包含的Furin酶切位点是被突变的。
在另一优选例中，所述的连接位点有1个、2个、3个、4个氨基酸被突变。
在另一优选例中，突变的氨基酸位点为R 313和/或R 316和/或R 315，突变后替代的氨基酸为Lys和/或His。
在另一优选例中，突变的氨基酸位点为R 313和/或R 316和/或R 315，突变后替代的氨基酸为Asp和/或Glu。
在另一优选例中，突变的氨基酸位点为R 313和/或R 316和/或R 315，突变后替代的氨基酸为Ala和/或Val和/或Leu和/或Ile和/或Pro和/或Phe和/或Trp和/或Met。
在另一优选例中，突变的氨基酸位点为R 313和/或R 316和/或R 315，突变后替代的氨基酸为Gly和/或Ser和/或Thr和/或Asn和/或Gln。
在另一优选例中，突变的氨基酸位点为R 313，突变后替代的氨基酸为Gly。
在另一优选例中，突变是对R 313和/或R 316和/或R 315编码DNA的敲除。
在另一优选例中，突变是在R 313和/或R 316和/或R 315间插入一个或多个氨基酸，从而使得第一复合物无法被Furin酶识别，且无法被酶切。
在另一优选例中，所述的第一复合物为同二聚体。
在另一优选例中，所述的第一复合物中，所述的BMP10前导肽与BMP10二聚体之间存在非共价连接。
在另一优选例中。所述的非共价方式或非共价结合中，所述BMP10前导肽包围在BMP10二聚体周围。
在另一优选例中，所述的第一复合物具 有式I结构：
Figure PCTCN2020089859-appb-000001
式中，
Z1为BMP10前导肽；
Z2为BMP10生长因子结构域；
“-”为肽键；
Figure PCTCN2020089859-appb-000002
为两个Z2之间的链间二硫键。
在另一优选例中，所述的第二复合物具有式II结构：
Figure PCTCN2020089859-appb-000003
式中，
Z1为BMP10前导肽；
Z2为BMP10生长因子结构域；
“-”为肽键；
Figure PCTCN2020089859-appb-000004
为两个Z2之间的链间二硫键；
Figure PCTCN2020089859-appb-000005
为Z1和Z2之间的非共价键。
在另一优选例中，所述的第三复合物具有式III结构：
Figure PCTCN2020089859-appb-000006
式中，
Z1为BMP10前导肽；
Z2为BMP10生长因子结构域；
Figure PCTCN2020089859-appb-000007
为两个Z2之间的链间二硫键；
Figure PCTCN2020089859-appb-000008
为Z1和Z2之间的非共价键。
在另一优选例中，所述的第三复合物中，所述的BMP10前导肽与BMP10二聚体之间不存在共价连接。
在另一优选例中，所述的组合物还含有选自下组的一种或多种次要成分：
(f1)第一次要成分，所述第一次要成分为BMP10前导肽，并且所述的BMP10前导肽是游离的BMP10前导肽；
(f2)第二次要成分，所述第二次要成分为一个BMP10二聚体与一个BMP10前导肽形成的复合物，并且所述BMP10二聚体的一个单体与BMP10前导肽共价连接；
(f3)第三次要成分，所述第三次要成分为一个BMP10二聚体与一个BMP10前导肽形成的复合物，并且所述的BMP10前导肽以非共价方式结合于所述BMP10二聚体；
(f4)第四次要成分，所述第四次要成分为BMP10二聚体。
在另一优选例中，所述的第二次要成分具有式IV结构：
Figure PCTCN2020089859-appb-000009
式中，
Z1为BMP10前导肽；
Z2为BMP10生长因子结构域；
“-”为肽键；
Figure PCTCN2020089859-appb-000010
为两个Z2之间的链间二硫键。
在另一优选例中，所述的第三次要成分具有式V结构：
Figure PCTCN2020089859-appb-000011
式中，
Z1为BMP10前导肽；
Z2为BMP10生长因子结构域；
Figure PCTCN2020089859-appb-000012
为非共价键；
Figure PCTCN2020089859-appb-000013
为两个Z2之间的链间二硫键。
在另一优选例中，所述组合物中，第一复合物的摩尔百分比P1≥40％，其中，
P1＝M1/(M1+M2+M3+Mf)    (Q1)
式中，
M1为所述第一复合物的摩尔数量；
M2为所述第二复合物的摩尔数量；
M3为所述第三复合物的摩尔数量；
Mf为所述衍生自proBMP10的次要成分的摩尔数量总和。
在另一优选例中，所述的P1≥50％，较佳地≥60％，更佳地≥80％，最佳地，≥90％、≥95％或≥98％。
在另一优选例中，所述的P1为50-99.99％，较佳地60-99.99％，更佳地80-99.9％，最佳地90-99.9％。
在另一优选例中，所述组合物中，第二复合物的摩尔百分比P2≤40％，其中，
P2＝M2/(M1+M2+M3+Mf)     (Q1)
式中，
M1为所述第一复合物的摩尔数量；
M2为所述第二复合物的摩尔数量；
M3为所述第三复合物的摩尔数量；
Mf为所述衍生自proBMP10的次要成分的摩尔数量总和。
在另一优选例中，所述的P2≤30％，较佳地≤20％，更佳地≤10％。
在另一优选例中，所述组合物中，第三复合物的摩尔百分比P3≥40％，其中，
P3＝M3/(M1+M2+M3+Mf)    (Q1)
式中，
M1为所述第一复合物的摩尔数量；
M2为所述第二复合物的摩尔数量；
M3为所述第三复合物的摩尔数量；
Mf为所述衍生自proBMP10的次要成分的摩尔数量总和。
在另一优选例中，所述的P3≥50％，较佳地≥60％，更佳地≥80％，最佳地，≥90％、≥95％或≥98％。
在另一优选例中，所述组合物中，第一、第二、和第三复合物的摩尔百分比Ptotal≥70％，其中，
Ptotal＝(M1+M2+M3)/(M1+M2+M3+Mf)    (Q1)
式中，
M1为所述第一复合物的摩尔数量；
M2为所述第二复合物的摩尔数量；
M3为所述第三复合物的摩尔数量；
Mf为所述衍生自proBMP10的次要成分的摩尔数量总和。
在另一优选例中，所述的Ptotal≥80％，较佳地≥90％，更佳地≥95％，最佳地，≥98％。
在另一优选例中，所述第一复合物(即proBMP10同二聚体)为两条proBMP10单体构成的二聚体(较佳地，所述二聚体为同二聚体)。
在另一优选例中，所述的第二复合物(即BMP10&proBMP10异二聚体)为一条proBMP10与一条生长因子结构域的异二聚体，该异二聚体与一条BMP10前导肽非共价结合形成的复合物。
在另一优选例中，所述的第三复合物(即BMP10同二聚体)为两条BMP10生长因子结合域的同二聚体，该同二聚体与两条BMP10前导肽非共价结合形成的复合物。
在另一优选例中，所述的“游离的BMP10前导肽”指所述BMP10前导肽既不属于proBMP10二聚体，也不属于BMP10复合物，而是独立存在的。
在另一优选例中，所述的BMP10二聚体(即第四次要成分)是由两个BMP10分子通过链间二硫键相连。
在另一优选例中，所述的BMP10二聚体中包含3个链间二硫键。
在另一优选例中，所述的BMP10分子为BMP10的生长因子结构域。
在本发明的第二方面，提供了一种用于保护心脏和/或治疗心脏损伤的药物组合物，所述的药物组合物包含本发明第一方面所述的组合物，以及药学上可接受的载体。
在另一优选例中，所述的药物组合物的剂型选自下组：
注射剂、片剂、胶囊剂、颗粒剂、散剂、膜剂、溶液剂、混悬剂、乳剂、凝胶剂、栓剂、软膏剂、气雾剂、喷雾剂、贴剂。
在另一优选例中，所述的药物组合物的给药途径选自下组：
注射给药、口服给药、黏膜给药(鼻黏膜，舌下，肺部给药，直肠粘膜，阴道粘膜)，经皮给药。
在另一优选例中，所述的药物组合物的剂型包括缓释剂。
在本发明的第三方面，提供了一种药盒，所述的药盒包括：
(a)第一容器和置于第一容器中的本发明第一方面所述的组合物；和
(b)第二容器和置于第二容器中的治疗药物，其中所述治疗药物具有心脏毒副作用或可引起药物性心肌病。
在另一优选例中，所述的治疗药物包括抗肿瘤药、镇静催眠药、抗精神病药和抗抑郁药、抗休克药、抗高血压药、血管平滑肌舒张药、平滑肌/横纹肌兴奋药、中枢兴奋药、或其组合。
在另一优选例中，所述的抗肿瘤药包括蒽环类抗生素、氟嘧啶、紫杉烷、烷基化药物、酪氨酸激酶抑制剂。
在另一优选例中，所述的蒽环类抗生素包括多柔比星、表柔比星、道诺霉素、环磷酰胺、紫杉醇。
在另一优选例中，所述的镇静催眠药包括水合氯醛、安眠酮、冬眠灵。
在另一优选例中，所述的抗精神病药和抗抑郁药包括吩噻嗪类抗精神病药(如氯丙嗪、奋乃静、癸氟奋乃静)和三环类抗抑郁药(如丙咪嗪、氯丙咪嗪、多氯平)。
在另一优选例中，所述的抗休克药包括肾上腺素、去甲肾上腺素、多巴胺、盐酸麻黄碱。
在另一优选例中，所述的抗高血压药包括盐酸井哒嗪、硫酸胍乙啶、利血平、甲基多巴、硫酸胍生。
在另一优选例中，所述的血管平滑肌舒张药包括双肼酞嗪、长压啶、地巴唑、氨茶碱、麻黄素、博利康尼、氯喘片、百喘朋。
在另一优选例中，所述的平滑肌/横纹肌兴奋药包括氯化胺、甲酰胆碱、加兰他敏。
在另一优选例中，所述的中枢兴奋药包括盐酸山梗菜碱、盐酸丙咪嗪、硫酸阿托品、颠茄酊。
在另一优选例中，所述的药盒还包含说明书，所述说明书中记载了联合给予所述的组合物和所述治疗药物的说明，从而在治疗疾病的同时保护心脏。
在本发明的第四方面，提供了一种本发明第一方面所述的组合物的用途，用于制备一种药物，所述的药物用于保护心脏和/或治疗心脏损伤。
在另一优选例中，所述的保护心脏包括保护心脏的收缩功能和舒张功能。
在另一优选例中，所述的保护心脏包括避免心脏损伤或心力衰竭。
在另一优选例中，所述的心脏损伤包括心脏的功能性病变和器质性损伤。
在另一优选例中，所述的心脏损伤选自下组：
心脏炎症、心衰竭，心率失常、限制性心肌病、扩张性心肌病、肥厚性心肌病、心脏纤维化、或其组合。
在本发明的第五方面，提供了一种保护心脏和/或治疗心脏损伤的方法，所述的方法包括步骤：
给需要的对象施用本发明第一方面所述的组合物，或含所述组合物作为活性成分的药物。
在另一优选例中，所述的用于科研用途。
在另一优选例中，所述的用于在科研过程中，保护动物模型的心脏。
在另一优选例中，所述的对象包括人和非人哺乳动物。
在另一优选例中，所述的对象包括啮齿动物，如小鼠、大鼠。
在本发明的第六方面，提供了一种制备本发明第一方面所述的组合物的方法，包括步骤：
(1)提供一CHO工程细胞，所述的CHO工程细胞表达外源的BMP10基因；和
(2)培养所述的CHO工程细胞，收集CHO工程细胞的培养上清，并从所述的 培养上清中分离纯化所述的组合物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了融合PCR点突变核酸电泳图。其中，图1A为包含点突变位点的突变位点两侧片段扩增核酸电泳图，图1B为长片段融合PCR电泳图，图1C为目的片段扩增电泳图，图1D为载体酶切电泳图。
图2显示了pMH3-BMP10质粒图谱。
图3显示了rhBMP10表达细胞株构建免疫印迹图。其中，图3A，3B，3C分别为第一、第二、第三次克隆筛选结果，图3D为单克隆化后的表达情况，图3E为单克隆化后的Western blot图。
图4显示了CHO-BMP10细胞株悬浮驯化折线图。
图5显示了CHO-BMP10细胞株在3L摇瓶流加培养过程中的生长和产物累积情况。图5A为细胞生长情况折线图，其中，图5B为产物累积情况Western blot图。
图6显示了Q柱纯化结果图。其中，图6A为Q柱纯化运行图谱(A液为50mM NaPB，B液为1000mM NaCl)，图6B为收集洗脱峰的考马斯亮蓝染色图，rhBMP10泳道为含有250mM NaCl的50mM NaPB洗脱峰，BMP10(R&D)泳道为从R&D公司购买的对照品。
图7显示了Gel filtration柱纯化结果。其中，图7A为Gel Filtration柱纯化运行图谱，图7B为收集洗脱峰的考马斯亮蓝染色图(1为～12mL洗脱峰，2为～19mL洗脱峰)。
图8显示了Furin酶切后的考马斯亮蓝染色图、Western blot图和Native page图。其中，图8A为BMP10的翻译后处理结构图，图8B为纯化rhBMP10 Furin酶切前后的考马斯亮蓝染色图，图8C为纯化rhBMP10 Furin酶切前后的anti-BMP10和anti-BMP10 propeptide Western blot图，图8D为纯化rhBMP10的Native page图，图8E为纯化rhBMP10的Native page anti-BMP10 Western blot图，图8F为纯化rhBMP10的Native page anti-BMP10 propeptide Western blot图。
图9显示了pGL6-BRE-Luciferase质粒图谱。
图10显示了荧光素酶报告结果。其中，图10A为培养基中不同浓度血清对信号的影响(BMP10购自R&D Systems)，图10B为标准曲线(rhBMP10购自R&D Systems)，图10C为纯化rhBMP10蛋白的活性测试。
图11显示了Furin酶切前后，rhBMP10蛋白的活性。
图12显示了超声心动图检查结果。其中，图12A为各组小鼠代表性M mode超声心动图，图12B为各组小鼠的FS和EF柱状图(n＝4)；其中“**” 代表p<0.01。
图13显示了TUNEL阳性率柱状图(n＝6)；其中**代表p<0.01。
图14显示了本发明具有心脏保护和/或心脏损伤治疗功能的组合物中包含的几种复合物的结构。其中，①为本发明的第一复合物的结构，②为第二复合物的结构，③为第三复合物的结构，④为游离的前导肽。
图15显示了推测的BMP10天然结构。
图16A为纯化rhBMP10和rhBMP10(313G)Furin酶切后的anti-BMP10前导肽蛋白印迹图，图16B为纯化rhBMP10和rhBMP10(313G)Furin酶切后的anti-BMP10图。
图17显示了Furin酶切后，纯化rhBMP10和rhBMP10(313G)蛋白的活性。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地制备了衍生自proBMP10的BMP10复合物，以及包含BMP10复合物的组合物、药盒、及其应用。其中，本发明的BMP10复合物包括第一复合物、第二复合物和第三复合物，上述三种复合物由共价或非共价连接的一个BMP10二聚体与两个BMP10前导肽构成。结果显示，本发明的组合物对于减轻抗肿瘤药物所致心脏功能性和器质性损伤的积极作用。并且，相对于成熟的BMP10二聚体，本发明的复合物中前导肽的存在，不仅不会影响BMP10的原有功能，还可以延长rhBMP10的半衰期，从而能够更好的发挥其功能。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
BMP10
骨形态发生蛋白(Bone morphogenetic proteins,BMPs)是一类属于TGF-β超家族的分泌型细胞外信号多肽，它以二聚体的形式结合到Ⅱ型跨膜Ser/Thr激酶受体复合物上，磷酸化修饰Smad家族转录因子，磷酸化的Smad蛋白转入细胞核并调控特异靶基因的转录。目前约有40余种成员，BMP家族成员在从昆虫到哺乳动物的各种物种的进化过程中介导各种各样的发育事件，在骨和软骨组织、眼、心脏、皮肤等多种器官和组织的早期发育、器官生长、重构以及损伤修复中起重要的调控作用。BMPs在胚胎心脏中可诱导Nkx2.5、Gata4、Tbx和Hand基因等多个心脏发育关键调节因子，并与多个心脏特异转录因子协同作用，调节心肌细胞的增殖、诱导心肌分化、心室壁和心脏瓣膜的形成。
BMP10是BMP家族一个比较特殊的成员。BMP10的表达在发育过程中很大程度的被限制在心肌细胞上，已有文章证明，在心脏发育过程中，BMP10可以调节心肌细胞的增殖，以及心室壁的形成。出生后至成年小鼠心脏中，BMP10的表达 被限制在右心房，分泌的BMP10蛋白分子通过血循环首先进入左心室，然后送往全身，因此在左心室中存在相对高浓度的活性BMP10分子，推测其对心室细胞具有一定的营养和保护作用。BMP10在成体心脏中的功能研究还不十分充分，有研究发现高血压性心肌肥厚大鼠模型的心肌细胞中BMP10高表达。对部分临床急性心梗病人的外周血检测也发现了BMP10表达的上调，提示BMP10在心脏损伤与修复中扮演一定角色。
完整的BMP10分子由424个氨基酸残基组成，分为三个部分，分别是①1-21位，信号肽，②22-316位，前导肽，③317-424位，生长因子结构域。计算所得的分子量为48kDa。BMP10生长因子结构域单体中存在7个半胱氨酸残基，可以形成三个分子内二硫键，同时通过和另一个BMP10分子形成链间二硫键，组成TGF-β超家族特征性的半胱氨酸结结构，形成具有活性的同源二聚体。在细胞内，BMP10的合成形式为全长的前体蛋白，①切除信号肽后，两个单体的BMP10分子将形成链间二硫键，形成同源二聚体，②前导肽的C端Arg-X-X-Arg序列在前蛋白转化酶(Propeptide Convertase，PC)的作用下发生切割，释放成熟的二聚体BMP10生长因子。
蒽环类抗生素
以多柔比星、表柔比星、道诺霉素等为代表的蒽环类抗生素是临床上治疗血液系统肿瘤和实体瘤的常见药物。在癌症治疗中，最常见的心脏毒性便是与蒽环类抗生素相关的心肌病变。蒽环类抗生素的使用会导致左心室收缩功能紊乱，并呈现出剂量依赖关系。同时，重复多次的给药将会导致永久性的细胞和细胞间质损伤，甚至导致心力衰竭。
由蒽环类抗生素引起的慢性心脏毒性则呈现剂量依赖关系，且不可逆转，并可以依据症状出现的时间分为早发性慢性毒性和迟发性慢性毒性。①早发性慢性毒性出现在治疗开始后，并可延续至治疗结束的一年后。1.6％-2.1％的病人在接受治疗过程中，或在治疗结束的一年内发病；②而迟发性慢性毒性则出现在治疗结束的一年以后。1.6％-5％的病人的心脏损伤甚至在在化疗结束后10-20年以后才会发生进展。蒽环类抗生素引起的慢性心脏毒性的主要特征是心室收缩和舒张功能失常，多导致扩张性心肌病和心力衰竭。
现在一般认为多柔比星引发心脏毒性的机制主要有①抑制DNA合成酶的活性，或是与在复制周期中的细胞的DNA结合，引起DNA的片段化；②抑制DNA-RNA-蛋白质的合成途径；③介导活性氧(reactive oxygen species,ROS)的产生，导致细胞氧化水平的上升，从而导致脂质过氧化或是细胞空泡化的发生；④影响重要的心肌特异性基因的转录过程。蛋白表达水平的下降以及肌丝的降解最终导致了肌节受损，引起心脏的肌减少症。同时，蒽环类和曲妥珠单抗的联合治疗会加剧肌纤维和DNA的破坏。蒽环类抗生素还能够破坏心脏功能的动态调节，改变腺苷酸环化酶活性，影响钙稳态。蒽环类抗生素能够导致心肌细胞的凋亡或是坏死，同时，由于心肌细胞有限的再生能力，心肌细胞的总数将会持续下降，导致心室重塑，心肌细胞最终被不具有弹性的纤维化组织取代。
BMP10复合物
本发明提供了一种BMP10复合物，所述的BMP10复合物包括第一复合物、第二复合物和第三复合物，上述三种复合物由共价或非共价连接的一个BMP10二聚体与两个BMP10前导肽构成，具体地，所述的BMP10复合物包括：
(a)第一复合物，所述的第一复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述BMP10二聚体的两个单体中每一个单体分别与一个BMP10前导肽共价连接；
(b)第二复合物，所述的第二复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述BMP10二聚体的一个单体与一个BMP10前导肽共价连接，另一个BMP10前导肽以非共价方式结合于所述BMP10二聚体；
(c)第三复合物，所述的第三复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述的两个BMP10前导肽均以非共价方式结合于所述BMP10二聚体。
在优选的实施方式中，本发明的BMP10复合物还包括：
(f2)第二次要成分，所述第二次要成分为一个BMP10二聚体与一个BMP10前导肽形成的复合物，并且所述BMP10二聚体的一个单体与BMP10前导肽共价连接；
(f3)第三次要成分，所述第三次要成分为一个BMP10二聚体与一个BMP10前导肽形成的复合物，并且所述的BMP10前导肽以非共价方式结合于所述BMP10二聚体；
(f4)第四次要成分，所述第四次要成分为BMP10二聚体。
在本发明的实施例中，利用CHO-S工程细胞表达了具有生物活性的rhBMP10，从而制备BMP10复合物。由于CHO-S体内缺乏足够的PC活性，CHO-S过表达的rhBMP10在胞内并不能够被完全切割。rhBMP10的主要分泌形式为未切割以及部分切割的二聚体，只有在培养的最后几天，才能够检测到rhBMP10成熟二聚体的存在。
在纯化过程中，考马斯亮蓝染色结果显示，Gel Filtration柱纯化后，从洗脱峰收集液中，可以电泳分离得到4个主要条带，其中分子量最小～25kDa，最大～110kDa。Gel Filtration的极限分辨率为分子量相差一倍的蛋白质。在rhBMP10的纯化过程中，分子量的差值为4.5倍，而其在Gel Filtration柱中的保留时间却保持一致，说明其发生了某种非共价作用力的结合，且这种结合力足够保证其在Gel Filtration中保持结构完整。在Western blot结果中，～110kDa、～68kDa和～25kDa的条带均具有anti-BMP10免疫原性，证明其均含有rhBMP10成熟肽结构域。间接说明了，前导肽在天然构象中，与成熟肽呈现非共价结合的复合体状态。在参考了TGF-β家族其他成员的天然构象后，发明人认为，BMP10的天然结构如图15所示，两个前导肽非共价地结合在成熟肽二聚体两侧，形成三元复合物。前导肽在BMP10中的作用仍然不明确，且存在争议，有文 献报道，在大鼠心肌成肌细胞(C2C12)和人肺动脉上皮细胞(HPAEC)中，前导肽的存在并不会影响BMP10激活并磷酸化Smad1/5/8信号通路；而另有文献则认为，前导肽的存在将会严重影响BMP10在C2C12细胞中诱导Id3基因的转录过程。
其后，为了获得均一的rhBMP10，进行了rhBMP10的酶切实验，使用Furin酶对表达并纯化的rhBMP10进行体外酶切，并检测了酶切前后的rhBMP10的生物活性。在萤火虫荧光素酶报告检测中发现，未被切割的rhBMP10具有与完全切割的rhBMP10相同水平的生物活性。这证明，至少在C2C12细胞上，rhBMP10是否切割并不显著影响其生物活性。
药物组合物和施用方法
本发明还提供了一种含有BMP10复合物作为活性成分的药物组合物，用于在应用抗肿瘤药物的同时保护心脏。
在本发明中，BMP10复合物可直接用于疾病治疗，还可同时使用其他治疗剂，如抗肿瘤药物药物等。
本发明还提供了一种药物组合物，它含有安全有效量的本发明BMP10复合物以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。本发明的药物组合也可以被制成粉剂用于雾化吸入。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明BMP10复合物还可与其他治疗剂一起使用。
对于本发明的药物组合物，可通过常规的方式施用于所需的对象(如人和非人哺乳动物)。代表性的施用方式包括(但并不限于)：口服、注射、雾化吸入等。
使用药物组合物时，是将安全有效量的BMP10复合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点包括：
(a)本发明的BMP10复合物对于减轻抗肿瘤药物所致心脏功能性和器质性损伤的积极作用；
(b)本发明的BMP10复合物中的前导肽可以延长rhBMP10的半衰期，从而能够更好的发挥其功能。
(c)本发明的BMP10复合物中的前导肽连接与否不影响rhBMP10活性的发挥；
(d)本发明的BMP10复合物对于已发生过功能性病变和器质性损伤的心脏， 即已有过心脏病史的心脏，具有积极的保护和逆转作用；
(e)本发明的BMP10复合物对于可能发生的心脏疾病具有积极的预防作用；
(f)本发明的BMP10复合物可能作为心脏毒性类药物的辅剂或辅料，在药物发挥功能的同时，减少药物对心脏的损伤。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用材料和方法
1实验材料
CHO-S细胞株为本实验室留存细胞株，C2C12细胞株购自ATCC，小鼠原代心肌细胞及专用培养液购自Procell。
实施例中使用了70只健康雄性C57BL/6J小鼠，8周龄，体质量22±2g，购于上海斯莱克实验动物中心，于江南大学医学院实验动物中心适应性饲养一周后开始实验，饲养环境恒温恒湿(25±2℃，50±10％)，每日光照12h，自由进食进水。
动物实验经由江南大学实验动物伦理委员会审核通过，审核编号为JN.No20180530c1851010[84]。
实施例中所用的主要试剂均为市售。
2实验方法
2.1融合PCR
由于rhBMP10的cDNA中1077位存在一个EcoRI识别位点(GAATTC)，使用融合PCR的方法进行点突变，突变后的序列为GAGTTC，不影响其编码的氨基酸序列和翻译效率。融合PCR所使用的引物和PCR程序如下
表1融合PCR引物
Figure PCTCN2020089859-appb-000014
其中FL-5'和FL-3'为序列两端引物，MID-5'和MID-3'为包含点突变位点的中间引物。波浪线为酶切位点，下划线为突变位点。
首先分别使用FL-5'和MID-3'引物，FL-3'和MID-5'引物扩增覆盖了突变位点的前半段cDNA序列和后半段cDNA序列，PCR产物用1.5％琼脂糖凝胶电泳分离，拍照并回收后，以两段回收产物作为模板，FL-5'和FL-3'为引物再进行PCR，产物用2％琼脂糖凝胶电泳分离，拍照并回收，用NanoDrop 2000测定浓度，置于-30℃冰箱备用。
2.2酶切连接
使用EcoRI和NotI限制性酶切位点对PCR产物酶切2h，使用PCR产物回收试剂盒进行回收，对pMH3载体酶切2h，1％琼脂糖凝胶电泳分离，拍照并回收，分别使用NanoDrop2000测定浓度，然后按4：1的比例使用T4 Ligase，22℃连接30min，并立即进行DH5α转化。
2.3Furin酶切位点突变
将rhBMP10经方法2.1和2.2得到的DNA进行突变，构建多种突变形式：(1)BMP10蛋白313位的氨基酸R突变为G；(2)BMP10蛋白316位的氨基酸R突变为G；(3)同时将(1)和(2)进行突变；(4)将315位的氨基酸R突变成G，同时突变以上(1)(2)(3)；(5)将313-316位进行随机突变。目的在于得到Furin无法识别，但突变后的rhBMP10活性与突变前无异或更佳。突变方法同方法2.1和2.2。
2.4细胞培养
(1)细胞复苏
将水浴锅打开，设定至37℃，从液氮罐中取出冻存管，迅速放入37℃水浴锅，摇动直至内容物完全融化。将冻存管转移至超净工作台中，取冻存液置15mL离心管中，并加入3倍体积的培养基稀释后离心(1000×g，5min)，弃上清，重悬后转移至T25/T75，置于湿式二氧化碳培养箱静置培养(37℃，5％CO 2)。CHO-S细胞的培养基为D/F12基础培养基，加入10％FBS，C2C12细胞培养基为DMEM基础培养基，加入10％FBS和1％双抗。
(2)细胞传代
对于贴壁培养的CHO-S细胞，弃上清，加入2-5mL胰酶浸润培养面，迅速倒出，加入1mL胰酶静置消化。待细胞变亮变圆后，加入5mL培养基终止消化，均匀吹散细胞，置于15mL离心管中离心，弃上清，重悬并按1：3-1：6比例传代。
对于悬浮培养的CHO-S细胞，取样测量细胞密度，按照实验需要取一定体积培养液与一定体积新鲜培养基混合即可。CHO-S细胞的悬浮培养基为M2与M4 1：1配置，并加入终浓度为50mM的谷氨酰胺。
(3)细胞计数
从摇瓶中取样或重悬细胞，取20μL细胞悬液与相同体积的台盼蓝染液混匀，加入一次性细胞计数板中，插入全自动细胞计数仪，静置片刻后读数。
(4)细胞冻存
取一定量悬浮培养的培养液，或消化贴壁细胞，离心，使用冻存液重悬，转移至冻存管中，放入平衡至室温的梯度降温冻存盒，置-80℃冰箱过夜后，转移 至液氮罐中。
2.5悬浮驯化
CHO-S细胞复苏后，在T75恢复生长后取状态良好的细胞消化、离心，以2.0×10 6cells/mL的密度接种入150mL摇瓶，置于细胞摇床培养，并每天观察细胞的生长情况。调整细胞密度在2.0×10 6cells/mL左右，直至细胞密度能够24h倍增且活率≥95％。
2.6细胞转染
取悬浮培养的状态良好的CHO-S细胞，计数，取约3.0×10 6cells/mL细胞，离心，用200μL PBS重悬，加入40μg质粒和10μg Salmon sperm混匀，将电转体系加入预冷的2mm电击杯中，置冰上5min，160V，15ms电击一次，置冰上一分钟，再电击一次，重复共三次。取两个100mm dish，分别加入10mL D/F完全培养基，将电击后的体系均匀分至两个dish中，置于湿式二氧化碳培养箱静置培养(37℃，5％CO 2)。第二天，换液，加入终浓度为4.5mg/mL的G418进行压力筛选，静置培养约10天。
取生长状态良好的C2C12细胞铺板(24wells)，取两个1.5mL EP管，分别加入25μL Opti-MEM，其中一管加入500μg质粒，另一管加入1.5μL Lipo3000，室温静置5min，混匀，静置5min，加入孔中，置于湿式二氧化碳培养箱静置培养(37℃，5％CO 2)。
2.7 CHO-BMP10流加培养
将CHO-BMP10细胞株悬浮驯化，将生长状态良好的种子接种于250mL摇瓶中，密度为1.0×10 6cells/ml，体积150mL，100rpm，37℃培养。至密度为5.0-6.0×10 6cells/ml时，将二级种子接入3L摇瓶中，，100rpm，37℃培养。根据生长情况将培养体积逐渐补充至1.5L，停止补加培养基后，待到细胞密度上升至9.0-10.0×10 6cells/ml时，降温至34℃培养。并根据葡萄糖浓度添加Feed 4补料培养基，维持体系中葡萄糖的浓度在3g/L左右。至细胞直径≥16μm时，密切关注细胞活率，当活率突然下降时(90％左右)，结束培养。
2.8 AKTA纯化
(1)培养上清处理
将5L培养上清4℃静置30min使大部分细胞沉淀，然后1000rpm离心10min，取上清，上清液继续8000rpm离心30min。用0.22μm微孔滤膜过滤上清，然后使用Millipore Pellicon超滤系统和10kDa膜包进行缓冲液置换。将体积超滤浓缩至500mL，然后加入4.5L 50mM NaPB，超滤浓缩至500mL，然后再加入4.5L 50mM NaPB，超滤浓缩至500mL。
(2)Q柱纯化
将3个5mL HiTrap Q HP柱串联，接入AKTA avant 25。所使用的流动相中，A液为50mM NaPB，B液为1M NaCl，系统流速为1mL/min。用A液冲平UV280，调零后上样，然后用A液淋洗，直至冲平UV280，用25％B液洗脱并收集洗脱峰。连接色谱柱过程中注意drop to drop connection，避免产生气泡；系统运行过程中注意监测柱压，防止超压。
(3)Gel Filtration纯化
用10kDa超滤管将Q柱纯化产物浓缩。所使用的流动相为50mM NaPB+150mM NaCl，系统流速为0.5mL/min。Loop环上样至用50mM NaPB+150mM NaCl平衡过的色谱柱上，并收集洗脱峰。连接色谱柱过程中注意drop to drop connection，避免产生气泡；系统运行过程中注意监测柱压，防止超压。
(4)Furin体外酶切
配置100×CaCl 2(100mM)溶液备用。用PBS将rhFurin稀释至4μg/mL，BMP10稀释至100μM。将稀释后的rhFurin与rhBMP10 1:1混匀，并加入1％的100mM CaCl 2，37℃孵育1h。
2.9考马斯亮蓝染色
(1)取CHO细胞的培养上清或纯化后的蛋白，适当稀释后，加入1/4体积的不含还原剂的5×Loading Buffer，混匀后100℃金属浴5min；
(2)取一块4-20％梯度预制胶板，加入缓冲液后每孔20μL上样，然后140V，70min电泳；
(3)修胶后，在150mm dish中加入适量考马斯亮蓝染液，室温摇床染色2h；
(4)加入脱色液，微波加热至微沸，4×20min脱色，拍照记录。
2.9 Western blot
(1)取CHO细胞的培养上清或纯化后的蛋白，适当稀释后，加入1/4体积的不含还原剂的5×Loading Buffer，混匀后100℃金属浴5min；
(2)取一块4-20％梯度预制胶板，加入缓冲液后每孔20μL上样，然后140V，70min电泳；
(3)修胶后，110V，70min转膜至NC膜；
(4)修膜，用TBST漂洗2次，加入5％脱脂奶粉(TBST)，室温摇床封闭1h；
(5)TBST漂洗，3×5min，加入用TBST稀释好的一抗，室温摇床孵育1.5h；
(6)TBST漂洗，3×5min，加入用TBST稀释好的二抗，室温摇床孵育1h；
(7)TBST漂洗，3×5min，ECL显色拍照。
2.10 Native Page
(1)在制样过程中，加入不含SDS和还原剂的Loading Buffer，直接上样电泳，同时电泳缓冲液也不加入SDS，然后常规考染脱色；
(2)在制样过程中，加入不含SDS和还原剂的Loading Buffer，直接上样电泳，同时电泳缓冲液也不加入SDS，然后正常转膜至NC膜，并按照Western blot的方法封闭和孵育抗体。
2.10 ELISA
(1)用PBS稀释Capture Antibody，取96孔板，每孔加入100μL，室温静置孵育过夜；
(2)用自动洗板机清洗3次，每孔加入300μL含3％牛血清白蛋白的PBS， 室温静置孵育过夜；
(3)用含3％牛血清白蛋白的PBS适当稀释样品，同时制作1000 pg/mL-15.6pg/mL标准溶液；
(4)用自动洗板机清洗3次，每孔加入100μL的样品或标准品，室温静置孵育2h；
(5)用自动洗板机清洗3次，每孔加入100μL Detection Antibody，室温静置孵育2h；
(6)用自动洗板机清洗3次，每孔加入100μL链霉亲和素-HRP，室温静置避光孵育20min；
(7)用自动洗板机清洗3次，每孔加入100μL底物溶液，室温静置避光孵育20min；
(8)每孔加入50μL终止液，温和摇匀，用酶标仪测定450nm吸光度，并用540nm矫正；
(9)根据标准曲线计算样品中rhBMP10的浓度。
2.11荧光素酶报告检测
(1)取100mm dish，并培养C2C12细胞至80％汇合度，10μg pGL6质粒，3μg pRL-SV40质粒，36μL Lipo3000转染，置于湿式二氧化碳培养箱静置培养过夜(37℃，5％CO 2)，并按照2.2.9筛选稳转细胞株，命名为C2C12-BRE；
(2)取C2C12-BRE细胞铺24孔板，每孔7×10 4cells，置于湿式二氧化碳培养箱静置培养过夜(37℃，5％CO 2)；
(3)每孔加入适当稀释的BMP10标准品或样品，置于湿式二氧化碳培养箱静置培养12h(37℃，5％CO 2)；
(4)每孔加入500μL PBS洗涤一次，弃上清，每孔加入100μL 1×PLB裂解液，均匀覆盖底面后室温摇床孵育15min，待细胞完全裂解后，将裂解物吸出，移至1.5mL EP管中，置于-80℃备用；
(5)取白底96孔板，每孔加入30μL裂解物，用排枪向每孔加入80μL LARII，立即用酶标仪测定发光值(1s)；
(6)取出孔板，进行数据分析处理。
2.12细胞TUNEL检测
小鼠原代细胞培养于专用培养液，待细胞细胞长至70-80％，胰酶消化处理细胞，将细胞以8000个每孔接种于两块24孔细胞板，放进细胞培养箱常规培养18-48h。
1.细胞损伤预防
取出其中一块24孔细胞板，向孔板中分别加入终浓度为0ng/mL、1ng/mL、2ng/mL、5ng/mL、10ng/mL的BMP10，每个浓度4个复孔。24h后，分别加入多柔比星和紫杉醇进行处理，其终浓度均为1μg/mL，不加要处理的作为对照组，10h后取出细胞孔板。
2.细胞损伤保护
取出另一块24孔细胞板，向孔板中加入多柔比星或紫杉醇进行处理，其终浓度均为1μg/mL，不加药处理的组作为对照组，4h后向孔板中分别加入终浓度为0ng/mL、1ng/mL、2ng/mL、5ng/mL、10ng/mL的BMP10，24h后取出细胞孔板。
取出处理后的两块细胞孔板，吸除培养基，PBS洗涤2-3次后，用4％的多聚甲醛固定30min，加入含0.1％Triton和0.5％BSA的PBS通透20min，每个切片滴加50μL TdT酶反应液，37℃避光反应1h；PBS中漂洗3×5min；每个切片滴加50μL荧光标记液，37℃避光反应1min；PBS中漂洗3×5min；荧光显微镜镜检拍照。
2.13动物分组以及多柔比星心肌损伤小鼠模型的建立
70只雄性健康C57小鼠随机分为4组，其中两组每组15只(Saline组，BMP10组)，其余两组每组20只(DOX组，DOX+rhBMP10组)。在实验开始前3天，Saline组和DOX每天注射PBS 200μL；rhBMP10组和DOX+rhBMP10组每天注射200μL rhBMP10(2μg)，直至第五周结束。Saline组和rhBMP10组每周注射一次PBS，共5次；DOX组和DOX+rhBMP10组每周注射一次DOX(5mg/kg)，共5次。第五周结束后，每组随机选取6只动物检测超声心动图，随后处死动物，分离血清检测心肌酶谱，心肌组织抽提RNA进行实时荧光定量PCR分析转录水平差异，抽提蛋白进行Western blot分析蛋白水平差异，组织切片Masson's染色观察纤维化情况，TUNEL检测凋亡水平。
2.14超声心动图
采用Visual Sonics公司的Vevo 2100小动物超声系统，在第五周实验结束后进行超声心动图检测。使用Vaporizer雾化罐，将小鼠置于密闭透明亚克力箱中使其吸入异氟烷麻醉，在小鼠左胸涂抹适量脱毛膏，稍等片刻后用湿棉球擦去毛发。然后仰卧位将小鼠固定物恒温手术台，套上异氟烷呼吸面罩。固定四肢以采集心电及呼吸信号，待心率稳定后在左胸涂抹耦合剂，接入探头采集数据。分别选取长轴和短轴切面进行采集。对于短轴切面，自B mode超声心动图中截取M mode超声，并以此为基础计算短轴缩短率(fraction shortening,FS)、射血分数(ejection fraction,EF)、左室舒张末期直径(left ventricular internal diameter at diastole,LVID,d)、左室收缩末期直径(left ventricular internal diameter at systole,LVID,s)、左室舒张末期容积(left ventricular volumn at diastole,LV Vol,d)、左室收缩末期容积(left ventricular volumn at systole,LV Vol,s)等参数。
2.15血清酶学检测
(1)血清的分离制备
小鼠眼球取血，并处死小鼠。全血室温静置30min后，3000rpm离心20min，分离上层血清，置-30℃冰箱备用；
(2)心肌酶谱
采用Advia 2400全自动生化分析仪检测；
(3)cTroponin和Myoglobin
使用上海鑫乐生物有限公司的ELISA试剂盒检测，实验过程与2.2.15基本一致。
2.16心肌组织切片制备
(1)固定
解剖并取出完整的小鼠心脏，用冰冷的生理盐水漂洗干净，置于4℃冰箱，用4％甲醛水溶液中固定一周，期间更换一次固定液；
(2)脱水
用流水冲洗固定后的小鼠心脏1h以去除固定剂，然后分别用70％乙醇、80％乙醇、95％乙醇I、95％乙醇II、无水乙醇I、无水乙醇II、二甲苯I和二甲苯II进行脱水，每步30min或更久；
(3)浸蜡包埋
分别在石蜡I、石蜡II和石蜡III中浸泡30min或更久，将心脏从包埋盒中取出，放入包埋模具中，灌入蜡液，覆盖上底座，稍等片刻后放入冷却台，使其冷却至凝固；
(4)切片：修块，切去多余蜡块，然后将蜡块固定在石蜡切片机上，调整位置后，步进至四腔室切面，然后以8μm厚度切片，裱于正电荷吸附载玻片上，滴超纯水展平，置于热台上过夜烘干，备用。
2.17 Masson's染色
采用南京建成生物工程有限公司的Masson's染色试剂盒，主要试剂包括R1核染液、R2浆染液、R3分色液、R4复染液和R5冲洗液，染色步骤如下：
(1)常规切片脱蜡至水；
(2)R1核染液染色60s，倒丢，冲洗液冲洗30s；
(3)R2浆染液染色40s，倒丢，冲洗液冲洗30s；
(4)R3黄色分色液分色8min，倒丢；
(5)直接用R4蓝色复染液染色5min，倒丢，用无水乙醇冲洗干净；
(6)中性树脂封固。
2.18组织切片TUNEL染色
(1)常规切片脱蜡至水；
(2)组化笔画圈，每个切片上滴加100μL Proteinase K工作液，37℃反应30min；
(3)PBS中漂洗3×5min；
(4)3％H2O2室温封闭10min；
(5)PBS中漂洗3×5min；
(6)每个切片滴加50μL TdT酶反应液，37℃避光反应1h；
(7)PBS中漂洗3×5min；
(8)每个切片滴加50μL Streptavidin-HRP反应液，37℃避光反应1min；
(9)PBS中漂洗3×5min；
(10)每个切片滴加50μL DAB工作液，室温镜下显色；
(11)PBS中漂洗3×5min；
(12)苏木素复染；
(13)常规脱水封固。
实施例1融合PCR及pMH3表达质粒构建
先使用带有突变位点的引物，将突变位点两边的片段扩增并电泳割胶回收，然后将回收产物和两端的引物一起进行PCR，15 cycles后制样电泳，凝胶在紫外下可以看到一条清晰的目的条带和一条模糊的非特异性条带；将位于～1,300bp的目的条带切胶回收后，再用FL-5'和FL-3'引物进行扩增，可在～1,300bp位置得到一条清晰的目的条带。使用EcoRI和NotI限制性内切酶在37℃酶切后，进行1％琼脂糖凝胶电泳分离，凝胶在紫外下可以看到位于～8,000bp的线性化空载质粒，以及位于～1,300bp的原装载片段。将PCR扩增所得目的片段用EcoRI和NotI限制性内切酶在37℃酶切以获得黏性末端，与酶切后的线性化空载在22℃使用T4 Ligase连接30min后，使用DH5α感受态细胞进行转化，并挑选阳性克隆接种在LB培养基中，送GeneWiz测序，确认获得包含目的突变的表达rhBMP10的pMH3质粒。
融合PCR点突变核酸电泳图如图1所示，pMH3-BMP10质粒图谱如图2所示。
实施例2 Furin突变的pMH3表达质粒的构建
考虑到Furin特异性催化切割识别位点为：Arg-X-Y-Arg羧基端肽键，其中X可为任意一种氨基酸，Y为Arg或Lys。BMP10中Furin的突变位点为RIRR 316，对应基因编码CGAATCAGAAGG，因而对这部分序列突变形式。
将突变后的质粒送GeneWiz进行测序，确认获得包含目的突变的表达rhBMP10的多个pMH3质粒。
将构建的多种质粒分别用lipo3000瞬时转染进CHO细胞后，裂解细胞，WB检测BMP的表达，筛选获得前导肽未被切割的表达2-4种质粒，留作进一步检测质粒所表达的对应BMP10蛋白的活性。
实施例3 CHO-BMP10稳定转染细胞株的构建
将pMH3质粒电转染至CHO-S空细胞，并加入终浓度为4.5mg/mL的G418压力筛选约10天后，可在dish底部观察到肉眼可见的白色半透明细胞克隆，在超净台中将克隆挑至96孔板并培养至圆形克隆长出后，换用D/F基础培养基培养两天，并用Dot blot检测上清中rhBMP10的表达水平。一次克隆检测中，背景较深，一方面说明rhBMP10的表达水平较低，另一方面说明单孔中的细胞为非单一来源细胞，有一定比例的细胞不能表达rhBMP10。二次克隆、三次克隆中，rhBMP10的表达水平呈上升趋势。选取三次克隆中表达最高的三个孔，将细胞消化、扩大培养后，用有限稀释的方法以每孔0.7个细胞的密度接种至96孔板，待克隆长出后再次进行Dot blot检测，选取表达量最高的孔，扩大至24孔板培养并再次检测，扩大培养表达量最高的孔并冻存，得到表达rhBMP10的CHO-S工程细胞株CHO-BMP10。构建CHO-BMP10细胞株的免疫印迹图如图3所示。
在稳定转染细胞株选育过程中，细胞的生长环境较正常培养时恶劣。细胞在 孔板中需要从单个或少数几个细胞开始分裂生长，期间存在无细胞间交流、空间拥挤、以及无血清(营养和生长因子缺乏状态)等环境。在恶劣的生长环境下，细胞有可能通过启动子甲基化等方式关闭不影响生存的基因。而在选育过程结束后仍然能够保持高水平稳定表达目的蛋白的细胞株，则可以认为其对恶劣环境的抗逆性比较好，鲁棒性高，更有利于长期和稳定培养。
实施例4 CHO-BMP10细胞株的悬浮驯化和摇瓶流加培养
在rhBMP10高表达CHO-S细胞株的构建过程中，主要使用贴壁的方法进行细胞培养和筛选，而与悬浮培养的方式相比，贴壁培养做到高密度培养相对比较困难。需要特殊的灌流设备，高培养面积的耗材等。因此，使用CHO-BMP10细胞株进行rhBMP10的大规模制备，需要使用悬浮高密度培养的方式进行。在使用CHO-BMP10细胞株进行rhBMP10的生产表达之前，需要对CHO-BMP10细胞株进行悬浮驯化，使其能够适应悬浮的生长环境。
结果如图4所示，在0-3d，细胞的密度呈下降趋势，3-10d细胞的生长速度逐渐加快，提示细胞已经逐步适应悬浮生长的新环境。～10d后，细胞基本可以实现24h倍增，并保持稳定的生长速率，提示悬浮驯化成功。台盼蓝染色后显微镜下观察，悬浮状态下的细胞基本呈现大小均一的球形，具备比较高的折光度，活率一般维持在99.0％以上。
悬浮驯化结束后，取悬浮培养的细胞接种摇瓶，制种子。并将摇瓶培养至密度为4×10 6cells/mL，活率在99.8％以上的种子接种至3L摇瓶，并每日取样检测。细胞在0-3d细胞呈现快速增长状态，此时细胞处于对数增长期，细胞增殖旺盛，活率高且细胞直径小，同时葡萄糖消耗也较快，乳酸水平迅速提高；在第四天开始进行补料，同时将培养温度下调至34℃，以延长平台期，维持细胞的高密度生长状态。由于养分的消耗和空间的挤占，细胞生长速度下降，活率也缓慢下降。最终在第8天达到最大密度(9.1×10 6cells/mL)。从第三天开始，细胞直径逐步增大，到第9天达到16.4μm，提示细胞衰老，同时活率下降至～94％，故选择在第十天结束培养。
细胞衰老后应及时结束培养。主要原因是①衰老后的细胞生产能力下降，几乎不再表达目的蛋白；②防止细胞自溶，释放蛋白酶破坏积累的目的蛋白；③防止细胞自溶物质污染培养上清，给后序纯化带来不必要的麻烦。
将每日所取样品加入5×SDS Loading Buffer制样后电泳，结果如图5所示，rhBMP10随着培养时间的增加逐步积累，同时呈现多条带的特征，其中～110kDa的条带在1-2d较少，随后逐渐增多；～68kDa和～57kDa的条带则在3d开始积累，而位于～25kDa的成熟rhBMP10二聚体则在7d开始出现，表明CHO表达体系缺乏相关的酶切体系，不足以处理rhBMP10中存在的RIRR ↓316酶切位点。
实施例5培养上清的处理和蛋白纯化
培养结束后，立即离心培养体系以除去不可溶的细胞和细胞碎片，取得不含细胞的培养上清。并对取得的培养进行0.22μm过滤，以除去细小的细胞碎片，得到的液体应为橙黄色透明澄清液体。随后将处理后的液体进行超滤，以除去培养基中的盐分，以及小于10kDa的杂质，并浓缩体系。超滤结束后的液体由处理前的～5L浓缩至～500mL，处理完成的液体呈现淡黄色，略有粘稠感。
使用浓缩后的培养上清上样，上样后UV280迅速上升至1400-2000mAU之间，电正性的蛋白由于不能与填料结合被冲出。上样结束后，使用50mM的NaPB进行淋洗，UV280迅速回落，并逐渐回归基线。然后直接使用含有250mM NaCl的50mM NaPB进行洗脱，此时UV280图谱上出现一个～5800mAU的尖峰，收集洗脱峰以用于下一步纯化。
洗脱峰的考马斯亮蓝染色结果如图6所示，呈现多条带的特征，主要包括～116kDa、～68kDa、～43kDa和～26kDa四个条带。
使用截留分子量为10kDa的50mL Amicorn超滤离心管浓缩Q柱洗脱蛋白，并脱盐，然后使用2mL Loop环进行上样。凝胶过滤层析主要通过分子量的差异对样品进行分离，不同分子量的物质在凝胶中的运动路径不同，分子量较大的蛋白由于不能进入凝胶内部，因此运动路程最短，最快被洗脱；而分子量较小的蛋白则可以进入凝胶球并在其内部网状结构内运动，因此路程较远，最慢被洗脱。上样时为了保证良好的分辨率，上样体积控制在柱体积的1％以内，样品的蛋白浓度控制在1mg/mL以内。凝胶过滤层析所使用的流动相为含有150mM NaCl的50mM NaPB，其中NaCl的加入主要是减少凝胶球对蛋白的非特异性吸附。
纯化结果如图7所示，上样后，在～55mL处出现洗脱峰，UV280约为500mAU；在～95mL处出现一个UV280为110mAU的小峰。收集两处洗脱峰，并制样电泳，在考马斯亮蓝染色凝胶上，第一个主峰的电泳结果呈现4个条带，与Q柱纯化的结果保持一致，说明rhBMP10以多种形式分泌，并保持类似的分子量。具体的结果将在3.2.2中进行分析。第二个峰的电泳结果基本不可见，应为小分子量的杂蛋白。
实施例6 BMP10的结构验证及Furin酶切
有文献报道，BMP10的体内酶切过程主要由Furin完成。由于rhBMP10在CHO-BMP10细胞中过表达，CHO自有的酶系可能不足以提供足够的处理能力，因此得到的蛋白并不是均一的蛋白，而是部分酶切的混合体系。为了获得均一的，酶切完全的蛋白，尝试将编码Furin的cDNA转染至CHO-BMP10细胞中，但Furin的过表达将会影响CHO细胞的活性，且很难获得稳定表达Furin的细胞株。因此使用了体外酶切的方法处理rhBMP10。同时，由于Furin的活性依赖于Ca 2+的存在，因此，根据相关文献结果，在PBS中加入1mM的Ca 2+作为反应的缓冲液。
结果如图8所示。酶切1h后，在考马斯亮蓝染色图中可以看到，位于～110 kDa的全长二聚体和位于～68kDa的部分酶切的二聚体条带在lane 2中不可见，证明Furin的加入可以识别未剪切的RIRR ↓316位点并进行切割，并释放成熟的二聚体(～26kDa)。这一结果在Western blot(anti-BMP10)中得到进一步验证，在lane 1中，～116kDa处和～68kDa均出现条带，而酶切后，在lane 2中，上述两处的条带均消失，证明剪切得到产物是成熟的rhBMP10二聚体。
而在Native page中，考马斯亮蓝染色结果可以观察到两条清晰的主带，在Western blot中，anti-BMP10抗体的杂交结果显示，接近顶端的条带具有BMP10免疫原性，而anti-BMP10 propeptide杂交结果则证明，两个条带均具有BMP10propeptide免疫原性。以上结果可以说明，在天然状态下，成熟的BMP10二聚体与前导肽非共价地结合在一起，形成复合物。这种结合在常规的SDS-PAGE制样电泳中会被破坏。
结果图16所示，酶切1h后，在蛋白印迹图中可以看出，在图16A的anti-BMP10前导肽和图16B的anti-BMP10中，rhBMP(313G)都仅有位于～110kDa的全长二聚体可见。证明突变后的rhBMP(313G)已经不能够被Furin识别到R ↓316位点并进行切割，也不能释放部分剪切二聚体和成熟的二聚体(～26kDa)。
实施例7荧光素酶报告活性
Ten Dijke Peter等人从C2C12细胞Id1基因的启动子中分离并合成了可以被BMP家族成员特异性激活的一段核酸序列，这种能够被BMP家族成员特异性激活的核酸序列被称为BRE(BMP responsive element,BRE)。将BRE序列整合到Minimal TA promoter前，在BMP10的存在下，便可以特异性的启动其下游编码蛋白的转录。构建了包含BRE元件的pGL6萤火虫荧光素酶报告质粒(图9)，将其稳定转染至C2C12细胞中，并利用这种方法来检测BMP10的生物活性。
在发明人构建的C2C12报告细胞株中，血清会对报告系统造成比较大的影响。培养基中10％的FBS可以导致约80,000 R.L.U.的非特异性信号。因此将检测过程的培养基血清含量降低至0.1％，以保证较高的信噪比。同时，在浓度依赖性检测中，该检测方法在BMP10浓度不高于40ng/mL时拥有较好的线性。最终，在检测过程中使用10ng/mL的rhBMP10浓度来测定rhBMP10的生物活性。
结果图10所示，未转染质粒的空白对照、加入CHO-S空细胞培养上清、加入TGF-β以及加入PBS的C2C12细胞几乎不表达萤火虫荧光素酶，说明该检测系统的特异性较好，噪音值较低。从R&D购买的BMP10 growth factor domain二聚体在10ng/mL的浓度时的信号值为84306±4938 R.L.U.(Relative luciferase units)，而同浓度下，CHO-BMP10表达并纯化的rhBMP10可以诱导表达99700±6065 R.L.U.。证明CHO-BMP10表达并纯化的rhBMP10具有很好的生物活性。
结果如图11所示，经图8的furin酶切验证后的rhBMP10进行活性验证表明纯化后的rhBMP10即使不经酶切，其活性与酶切后的活性无统计学差异，表明前导肽的存在不影响rhBMP10活性或功能的发挥。
结果图17所示，经图16验证rhBMP10第313位氨基酸R突变为G生成 rhBMP10(313G)后，未被furin酶切；在10ng/mL的浓度时未酶切的rhBMP10(313G)信号值为67617±2620 R.L.U.，仍具有生物活性，同浓度下rhBMP10可以诱导表达85305±6419 R.L.U.，从R&D购买的BMP10诱导表达72923 R.L.U..证明CHO-BMP10表达并纯化的313位点突变的rhBMP10，其前导肽的存在不影响rhBMP10活性或功能的发挥。
实施例8细胞TUNEL检测
为了检测CHO-BMP10表达并纯化的rhBMP10对小鼠心肌细胞的保护和治疗作用，将小鼠细胞在心脏毒性的药物攻击前或后分别用rhBMP10进行处理，并用TUNEL检测心肌细胞凋亡情况。结果显示，先将小鼠心肌细胞用BMP10作用，再用阿霉素或紫杉醇进行处理，与未事先用BMP10作用的细胞相比，心肌细胞的凋亡明显减少，并随BMP10浓度的增加，凋亡减少；与此同时，先用阿霉素和紫杉醇刺激而未用BMP10处理的心肌细胞对照组，心肌细胞凋亡比例超50％，而经BMP10处理后心肌细胞的凋亡情况明显逆转，并随着BMP10浓度的增加，逆转程度增加。
表明：BMP10的存在不仅可以保护小鼠心肌细胞免受心脏毒性药物的刺激，也可以治疗并逆转心脏毒性药物对心肌细胞带来的损伤。
实施例9超声心动图
为了评价多柔比星对小鼠心脏收缩和舒张功能的损伤，使用经胸廓超声心动图来检测小鼠的心功能。事实上，通过Pressure–volume loop(PV-loop)对左室的血压和容量进行在线监测是评价心脏功能的金标准，但是由于PV-loop需要进行血管插管手术，为侵入性监测手段，同时实验所需的时间较长(1～2只每天)和耗材价格较为高昂，故采用了超声的方法进行检测。通过使用超高频率的超声探头，并配合M mode超声，可以无损地对小鼠的左室功能进行定量分析。
结果如图12和表1所示，与对照组相比，DOX模型组的小鼠心脏功能出现了明显下降，其短轴缩短率(40.371±3.184)和射血分数(73.054±3.829)均出现了显著下降，其中提示多柔比星模型组小鼠心肌的收缩功能受损，心功能下降。在rhBMP10组和DOX+rhBMP10联合给药组小鼠的超声结果中，小鼠的心脏功能与对照组相比没有明显差异。
上述结果说明，注射rhBMP10可以显著减轻由多柔比星导致的小鼠心功能下降问题。
表1超声心动图结果列表
(n＝4)其中“*”和“**”分别代表与Saline组相比，p<0.05和p<0.01；
Figure PCTCN2020089859-appb-000015
和
Figure PCTCN2020089859-appb-000016
分别代表与DOX组相比，p<0.05和p<0.01
Figure PCTCN2020089859-appb-000017
Figure PCTCN2020089859-appb-000018
实施例10心肌切片TUNEL
TUNEL主要作用是检测细胞在凋亡过程中细胞核DNA的断裂情况。dUTP在脱氧核糖核苷酸末端转移酶的作用下，连接到凋亡细胞中断裂的DNA的3'-OH末端；通过连接在dUTP上的生物素标记，运用HRP标记的链霉亲和素便可以报告信号；在二氨基联苯胺(DAB)的存在下，产生很强的颜色反应(深棕色)，特异准确地定位正在凋亡的细胞。而正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3'-OH形成，染色过程中呈现阴性结果。
TUNEL检测结果如图13所示，DOX组小鼠的心脏凋亡比例显著提高(p<0.01)，达到0.196％。而DOX+rhBMP10组的小鼠心脏凋亡程度Saline组与接近，分别为0.0361％和0.0136％。在rhBMP10对照组中，心肌细胞的凋亡比例为0.0234％。
上述结果说明，注射rhBMP10可以减轻显著由多柔比星导致的小鼠心肌细胞凋亡的问题。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种用于保护心脏和/或治疗心脏损伤的组合物，其特征在于，所述组合物含有选自下组的一种或多种复合物作为活性成分：
    (a)第一复合物，所述的第一复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述BMP10二聚体的两个单体中每一个单体分别与一个BMP10前导肽共价连接；
    (b)第二复合物，所述的第二复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述BMP10二聚体的一个单体与一个BMP10前导肽共价连接，另一个BMP10前导肽以非共价方式结合于所述BMP10二聚体；
    (c)第三复合物，所述的第三复合物为一个BMP10二聚体与两个BMP10前导肽的复合物，并且所述的两个BMP10前导肽均以非共价方式结合于所述BMP10二聚体。
  2. 如权利要求1所述的组合物，其特征在于,所述组合物中由或基本上由第一、第二和/或第三复合物构成。
  3. 如权利要求1所述的组合物，其特征在于,所述的第一复合物中包含的Furin酶切位点是被突变的，从而降低或消除Furin酶切。
  4. 如权利要求1所述的组合物，其特征在于,所述的第一复合物中，所述的BMP10前导肽与BMP10二聚体之间存在非共价连接。
  5. 如权利要求1所述的组合物，其特征在于,所述的组合物还含有选自下组的一种或多种次要成分：
    (f1)第一次要成分，所述第一次要成分为BMP10前导肽，并且所述的BMP10前导肽是游离的BMP10前导肽；
    (f2)第二次要成分，所述第二次要成分为一个BMP10二聚体与一个BMP10前导肽形成的复合物，并且所述BMP10二聚体的一个单体与BMP10前导肽共价连接；
    (f3)第三次要成分，所述第三次要成分为一个BMP10二聚体与一个BMP10前导肽形成的复合物，并且所述的BMP10前导肽以非共价方式结合于所述BMP10二聚体；
    (f4)第四次要成分，所述第四次要成分为BMP10二聚体。
  6. 如权利要求1所述的组合物，其特征在于,所述组合物中，第一复合物的摩尔百分比P1≥40％，其中，
    P1＝M1/(M1+M2+M3+Mf)    (Q1)
    式中，
    M1为所述第一复合物的摩尔数量；
    M2为所述第二复合物的摩尔数量；
    M3为所述第三复合物的摩尔数量；
    Mf为所述衍生自proBMP10的次要成分的摩尔数量总和。
  7. 如权利要求1所述的组合物，其特征在于,所述组合物中，第一、第二、和第三复合物的摩尔百分比Ptotal≥70％，其中，
    Ptotal＝(M1+M2+M3)/(M1+M2+M3+Mf)    (Q1)
    式中，
    M1为所述第一复合物的摩尔数量；
    M2为所述第二复合物的摩尔数量；
    M3为所述第三复合物的摩尔数量；
    Mf为所述衍生自proBMP10的次要成分的摩尔数量总和。
  8. 一种用于保护心脏和/或治疗心脏损伤的药物组合物，其特征在于，所述的药物组合物包含权利要求1所述的组合物，以及药学上可接受的载体。
  9. 一种药盒，其特征在于，所述的药盒包括：
    (a)第一容器和置于第一容器中的权利要求1所述的组合物；和
    (b)第二容器和置于第二容器中的治疗药物，其中所述治疗药物具有心脏毒副作用或可引起药物性心肌病。
  10. 一种权利要求1所述的组合物的用途，用于制备一种药物，所述的药物用于保护心脏和/或治疗心脏损伤。
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