CN104147596A - Method for prolonging treatment concentration by using non-covalent conjugated polymer of biological medicine and use - Google Patents
Method for prolonging treatment concentration by using non-covalent conjugated polymer of biological medicine and use Download PDFInfo
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Abstract
The invention relates to a non-covalent conjugate of a biological medicine, a preparation method and use of a pharmaceutical composition formed from the non-covalent conjugate. The biological medicine is conjugated to a high-molecular polymer at high concentration so as to form the non-covalent conjugate; the biological medicine is dissociated and released from the high-molecular polymer at low concentration, so that the pharmacological activity of the biological medicine takes effect. The method disclosed by the invention is characterized by being not only capable of stably reserving medicines and reducing the immunogenicity of medicines but also capable of relieving diffusion velocity and clearing velocity from blood, prolonging the time of keeping the effective treatment concentration of medicinal plasma and the half-life period, and basically eliminating medicine burst release. The invention further relates to use of the pharmaceutical composition containing the non-covalent conjugate of the biological medicine.
Description
Invention field
The present invention relates to a kind of polymer that carries affinity ligand and with method and the reagent of the non-covalent conjugate of thing medicine, belong to field of biological pharmacy.
Background of invention
Protein medicaments, has advantages of that target spot is clear and definite, evident in efficacy, be difficult for to form Drug resistance, side effect is little, but because of its Half-life in vivo shorter, therefore during clinical practice, for maintaining its effective blood drug concentration in vivo, need multiple dosing, patient compliance is poor.In order to address this problem, European patent EP 0539167 and US Patent No. 4904584 disclose the solution with the direct modified protein of PEG.PEG unit can be connected in a plurality of different amino sites of protein by covalency; Owing to there being a plurality of amino in albumen or peptide molecule, the covalent coupling thing of preparation is the mixture of multiple coupled product, the PEG of different molecular size and molecular structure is different on the impact of avtive spot, at specificity, by very large inhomogeneities of reverse side existence such as albumen space structure, product stability and its lytic activities after modifying.The mixture of conjugate position isomer [European patent EP 593868; Monkarsh etc., ACS.Symp.Ser., 680:207-216,1997], need loaded down with trivial details purification step to prepare the suitableeest conjugate.A kind of homogenization method that amino on IL28 and IL29 molecule N-end carries out PEGization is disclosed in patent PCT/US2004/025864, controlling reaction pH condition Selective Control reacts on the α of N-end amino such as reagent such as aldehyde (as methoxy poly (ethylene glycol) propionic aldehyde), and the ε that does not affect lysine residue is amino, prepare protein N-end modified product.Compared with the biological medicament of unmodified, PEG conjugate has higher solubility, stability, weak immunogenicity; Although but be connected to identification and the combination that PEG chain itself on pharmaceutical protein molecule amino acid residue does not participate in receptor, but the change sterically hindered and the protein molecular surface nature that PEGization causes of PEG chain has affected the combination with receptor, reduced the activity of albumen after modifying; With PEG chain length, increase and the raising of PEG degree of modification, biological activity reduction amplitude increases.As PEG-IL-15 only has 10% natural activity [Chinese patent application 201110095641.6];
(Peginterferon α-2b, Schering-Plough) only has natural activity approximately 10%28%, PEGASYS (IFN α-2a) 7% natural activity of only having an appointment.
In addition, chemical reaction, product inhomogeneity that protein PEGization is modified need purification not only to increase production stage, have caused product loss, have strengthened production cost.Therefore need the better method of development stability and prolong drug blood medicine valid density.
Summary of the invention
The present invention relates to a kind of high molecular polymer that carries affinity ligand, thing medicine can be with non-covalent form with it in conjunction with forming non-covalent conjugate; Preparation manipulation is simple, and loss of activity is little, is easy to preserve, more stable, with the comparison of natural goods medicine, has longer Half-life in vivo and effective blood drug concentration, medication number of times is few, therapeutic effect is long, medication is safer.
Specifically, the invention reside in a kind of high molecular polymer with affinity ligand is provided, thing medicine with it in conjunction with forming non-covalent conjugate, can add excipient, non-ionic surface active agent etc. by non-covalent form, filtration sterilization, subpackage, can deposit if needed in lyophilizing.Therefore this high molecular polymer is expected to exploitation becomes a kind of health promoting product or medicine or assists a ruler in governing a country medicine and for clinical.
Therefore, one object of the present invention is to provide a kind of non-covalent polymer of medicine-high molecular polymer.
It is characterized in that:
[1] on high molecular polymer molecule, contain covalently bound affinity ligand;
[2] high molecular polymer added in the pharmaceutical preparation stage, mixed, formed after degerming, filtration.
[3] described medicine include but not limited to recombinate biological medicament, extract biological medicament and synthesising biological medicine.
The non-covalent conjugate of the medicine-macromolecule of thing described in the present invention has following general formula:
BioPM ... Ligand mono-Polymer
In formula, the preferred molar ratio of described non-covalent conjugate is: BioPM: Ligand mono-Polymer=0.5-1.5: 0.5-2: 5
In formula, BioPM is described biological medicament finger protein/polypeptide drug: comprise erythropoietin (EPO), recombinant human granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), vaccine, interferon (INF), growth hormone (GH), insulin (Insulin), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-B), insulin like growth factor (IGF), vascular endothelial cell growth factor (VEGF), PDGF (PDGF), endothelial cell growth factor (ECGF) (ECGF), nerve growth factor (NGF), bone-derived growth factor (BDGF), bone morphogenetic protein(BMP) (BNIP), tissue polypeptide antigen (TPA), antibody (antibody), blood coagulation factor VIII (VIII) and IX genetic factor and the protein or the polypeptide that are used for the treatment of.
BioPM is that the medicine beyond described biological medicament finger protein/polypeptide class is: antisense nucleotide (anti-RNA), microRNA (RNAi), gene (DNA), antibody, vaccine or liposome.
Described biological medicament can be natural, recombiant protein or the gene mutation product equally with natural function, certainly, also comprise by product tissue culture, organic synthesis, that protein synthesis, cell culture (natural, reconstitution cell live mutant) method obtains.
Polymer is described high molecular polymer, refers to the polyamino acid of polyester, poly-anhydride or skeleton non-peptide bond, comprising: polylactic acid, polylactic acid one polyglycolic acid and combination thereof, also comprise Carmomer, hyaluronic acid, chondroitin sulfate, heparin, gelatin, collagen protein, Fibrinogen, fibrin, , glucosan, pi-allyl glucosan, agarose, cellulose, Polyethylene Glycol, the chimeric macromolecule of Polyethylene Glycol one polylactic acid, the chimeric macromolecule of Polyethylene Glycol one polyglycolic acid, the chimeric macromolecule of polyglycolic acid one Polyethylene Glycol one polyglycolic acid, the chimeric macromolecule of polylactic acid one Polyethylene Glycol one polylactic acid, the quick type gel of sugar and acid-sensitive type gel macromolecule, polybutylcyanoacrylate, poly phosphazene, poly phosphate, with the preferred PEG of the present invention, hyaluronic acid, heparin, chondroitin sulfate, glucosan, cellulose,
PEG peg molecule can be straight chain type or branching type, can be strand, two strands or multichain.Preferred straight chain type strand peg molecule.20 to 2000 of basic ethylene unit, is preferably the integer value between 500-1500, the integer between 700-1300 more preferably, and the molecular weight of Polyethylene Glycol, between 1kDa, 100kDa, is preferably between 200-50kDa, more preferably 30-40kDa.MPEG with the preferred 20-40KDa of the present invention;
Ligand is described affinity ligand, refer to have the molecular radical of combining target biological medicament, native ligand that can biological medicament, polypeptide ligand, drug molecule, organic molecule, and take biological medicament as synthetic the having in conjunction with described biological medicament ability molecular entity of shot design, comprise ethylenediamine, triethylene tetramine, 3-diethyl amino propylamine, 1, 2-diamino-cyclohexane, 5-amino-2, 2, 4-front three basic ring penta methylamine, heptyl amice, octylame, appoint amine, certain herbaceous plants with big flowers amine, Aminocyclopentane, cycloheptylamine, cyclooctylamine, ring is appointed amine, ring certain herbaceous plants with big flowers amine, histamine, color amine, tyramine, aniline, 3-amino-phenol, para-aminophenol, benzene methanamine, 3-aminopyridine, 2-amine methylpyrrole, 3, 5-diaminobenzoic acid, 2-aminobenzimidazole, 5-aminobenzimidazole, 4, 4 '-diaminodiphenyl ether, 3, 3 '-dimethylbenzidine,
Another object of the present invention is to provide a kind of high molecular polymer preparation method with affinity ligand.
The high molecular polymer that carries affinity ligand described in patent of the present invention can be by conventional chemical method preparation process: [1] polysaccharide polymer-affinity ligand synthesis step: accurately take polysaccharide some, according to calculating molal quantity divided by monosaccharide molal weight number (approximately 180).Then the NaOH that adds 0.1-1 times of monosaccharide molal quantity, the epoxychloropropane of 1 times of monosaccharide molal quantity or other bicyclic oxygen reagent, airtight after in 60 ℃ of stirring reaction 0.5-8 hour.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain epoxy activated polysaccharide polymer, measure epoxy radicals density; Get epoxy activated polysaccharide polymer some, calculate epoxy radicals molal quantity; Get the ligand molecular of 1-2 times of mole, be dissolved in buffer more than pH8, mix with activation polysaccharide polymer, airtight after in 60 ℃ of stirring reaction 10-24 hour.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain polysaccharide polymer-affinity ligand material;
[2] 1-3 chain mPEG-affinity ligand synthesis step: accurately take 1-3 chain PEG-active group (PEG molecular weight 2-50kd, active group is epoxy radicals, butanimide fat, succinimdyl carbonate, aldehyde radical, trifluoroethyl sulphonic acid ester, isocyanates, hydrazine, 2-mercaptothiazoline, benzotriazole, maleimide, two-2-pyridyl disulfide, vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan), adjacent two thiopyridines, iodoacetamide) some, calculated activity group molal quantity; Get the ligand molecular of 2-10 times of mole, be dissolved in reaction buffer, mix with 1-3 chain PEG-active group, airtight after under reaction temperature stirring reaction 10-24 hour.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain PEG-affinity ligand material;
Synthesizing of [3] three nitrogen piperazine skeleton affinity ligand-polysaccharide polymers
Get in the some 1-10%NaOH of being dissolved in of polysaccharide, add epoxychloropropane, mix, react 2 hours at airtight 60 ℃; Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, obtain epoxy activation-polysaccharide polymer,
Epoxy activated polysaccharide polymer is some to be dissolved in 1-5% ammonia, mixes, and reacts at airtight 60 ℃ and spends the night; Take out reactant, in fume hood, with 0.1NaHCO3 solution, in ultrafiltration mode, change buffer, to pH value and electrical conductivity and 0.1NaHCO3 solution phase ought, obtain amino-polysaccharide polymer, analyze amino density; Get the some 0.1NaHCO3 of the being dissolved in solution of amino-polysaccharide polymer, calculate amino molal quantity, the trichloride and triazine of getting 1-2 times of mole is dissolved in cold acetone, progressively add, and with saturated NaHCO3, pH is maintained between 7.0-7.5, reaction temperature maintains 0-4 ℃ and continues to stir 2h-4h, to white disappearance stopped reaction.With deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain three nitrogen piperazine activation-polysaccharide polymers; Take three nitrogen piperazine activation-polysaccharide polymers dissolving 0.1NaHCO3 solution, get the target amino-compound (R1) of 1-5 times of mole, be dissolved in a certain amount of distilled water, add and mix, airtightly react 24h with 50 ℃, in course of reaction, with saturated NaHCO3 and 1M acetic acid, pH value of solution is remained between 7-7.5; With 0.1NaHCO3 solution, in ultrafiltration mode, change buffer, work as to pH value and electrical conductivity and 0.1NaHCO3 solution phase; Take the target amino-compound (R2) of getting 1-5 times of mole, dissolve, add, 95 ℃ of reaction 24-60h, in course of reaction, remain between 7-7.5 pH value of solution with saturated NaHCO3 and 1M acetic acid; With deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, dried for standby.The synthetic double-basis of this method is rolled into a ball three nitrogen piperazine skeleton affinity ligand-polysaccharide polymers, and by different amino-compound numeral number called afters " Cx ", it is skeleton that three nitrogen piperazines are take in " C " representative, and " x " is amino number designation numeral number.
Described conventional chemical method, refers to that C.R.Lowe is at < < affinity chromatography introduction > > (Lip river (C.R.Lowe) work; Liu Yuxiu translates. Science Press, 1983 with " year May the 1st edition, 61-84 page) in polymer activation and the functional method mentioned; as the polymer of polysaccharide-based and hydroxyl; available cyanogen halides, triazine (all dichlorotriazine or three chlorotriazines), periodate oxidation, oxirane (Isosorbide-5-Nitrae-dihydroxy normal butane bisglycidyl ether); epoxy chloropropionate alcohol; epoxy bromopropyl alcohol, two aziridine, divinyl sulfone; benzoquinone compound is as quinone, and bromoacetyl bromide activates and functionalization; Part synthetic method can also list of references affinity ligand the structure activity relationship [J.Sep.Sci.2011,34,3145-3150] of the synthetic and adsorbed proteins of design.Thereby connect the affinity ligand needing on polymer, or the synthetic affinity ligand of original position.
A further object of the present invention, provides this pharmaceutical composition preparation method
The preparation process of non-covalent conjugate described in patent of the present invention: by the injection buffer of 2 times of concentration crude drug (BioPM) and 2 times of concentration polymer-parts (Ligand mono-Polymer) injection buffer, add excipient dissolving, non-ionic surface active agent, in sterile chamber after mix homogeneously, standing 2 hours, filtration sterilization, the preferred 4-10 ℃ of temperature, mol ratio is BioPM: Ligand mono-Polymer=1: 1.1～1: 5, BioPM crude drug is combined rate >=95% with polymer.Selection and the preparation method of preparation condition describe in detail in example.
Another object of the present invention has been to provide the adjuvant of preparing this pharmaceutical composition.
Described in patent of the present invention, excipient can be any polyhydric alcohol, produces the non-covalent conjugate preparations of stable biological medicament-high molecular polymer with together with other component of preparation.The example of polyhydric alcohol has mannitol, sorbitol, Sargassum sugar alcohol, maltose alcohol, xylitol and maltose alcohol.Preferred polyhydric alcohol excipient is mannitol.
According to another object of the present invention, the invention provides the non-ionic surface active agent of this pharmaceutical composition of preparation.
According to non-ionic surface active agent of the present invention.Ionic surfactant pack is drawn together polyol derivative, propylene-glycol diacetate, polyoxyethylene ester and ether, nonylplenyl ether, polyvinyl alcohol, poloxamer (PLURONICS F87), alkanolamide.Non-ionic surface active agent is poloxamer preferably, particularly preferably PLURONICS F87.
Can maintain the stable any buffer of this pharmaceutical composition and all can use, include but not limited to sodium-acetate buffer or PBS solution, preferably sodium-acetate buffer.In a preferred embodiment of the invention, PEG-ligand, the composition of liquid medicine of interferon-γ, surfactant, excipient and buffer, wherein said described buffer is sodium-acetate buffer, excipient is Sargassum sugar alcohol, and described surfactant is PLURONICS F87.
PBS solution preparation: accurately take sodium chloride 8g, potassium chloride 0.2g, sodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, be made into 1000mL solution with deionized water, subpackage, 121 ℃ of autoclaving 15min.
The specific embodiment
Following examples are used for describing in detail the present invention and effect thereof, but the present invention is not limited only to these embodiment, the scope that these embodiment do not limit the present invention in any way.Those skilled in the art can make various changes or modifications the present invention, but the similar change of doing or revise and to drop on equally the application's claims limited range within.
Embodiment 1 mPEG-TEQA is synthetic
Accurately weighing m PEG-epoxide group (mPEG-Epoxide, molecular weight 20KD) 10g is dissolved in 500ml 0.01M NaOH, measures triethylene tetramine 5ml and adds mixing, puts into 1L vial, and sealing covers tightly the imperial 60 ℃ of shaking tables reaction of rear defence and spends the night.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain PEG-TEQA;
Embodiment 2 diPEG-DaCH are synthetic
Accurately take double-stranded PEG-butanimide fat (diPEG-su, molecular weight 40KD) 10g is dissolved in 500ml 0.1M acetic acid-sodium acetate buffer, pH4.5, measure 1,2-diamino-cyclohexane 5ml adds mixing, adjust pH4.5, put into 1L vial, sealing covers tightly the imperial 50 ℃ of shaking tables reaction of rear defence and spends the night.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain diPEG-DaCH;
Embodiment 3 diPEG-APn are synthetic
Accurately take double-stranded PEG-butanimide carbonic ether (diPEG-su, molecular weight 60KD) 10g is dissolved in 500ml 0.1M acetic acid-sodium acetate buffer, pH4.5, measure Aminocyclopentane 5ml and add mixing, adjust pH4.5, put into 1L vial, sealing covers tightly the imperial 50 ℃ of shaking tables reaction of rear defence and spends the night.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain diPEG-APn;
Embodiment 4 diPEG-ABZMZ are synthetic
Accurately take double-stranded PEG-trifluoroethyl sulphonic acid ester (diPEG-TrS, molecular weight 50KD) 10g and be dissolved in 500ml 0.1M sodium bicarbonate buffer liquid, pH8.0, take 5-aminobenzimidazole 5g and add mixing, adjust pH4.5, put into 1L vial, sealing covers tightly the imperial 50 ℃ of shaking tables reaction of rear defence and spends the night.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain diPEG-ABZMZ;
Embodiment 5 diPEG-ABZMZ are synthetic
Accurately take double-stranded PEG-trifluoroethyl sulphonic acid ester (diPEG-TrS, molecular weight 10KD) 10g and be dissolved in 500ml 0.1M sodium bicarbonate buffer liquid, pH8.0, take 5-aminobenzimidazole 5g and add mixing, adjust pH4.5, put into 1L vial, sealing covers tightly the imperial 50 ℃ of shaking tables reaction of rear defence and spends the night.Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer washing, suitable with deionized water to pH value and electrical conductivity, obtain diPEG-ABZMZ;
Embodiment 6 HA-Lig's is synthetic
Take hyaluronic acid (Hyaluronan, hyaluronic acid) 15g and be dissolved in 4%NaOH, add epoxychloropropane 5ml, mix, react 2 hours at airtight 60 ℃; Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, be dried to obtain epoxy activation-hyaluronic acid 14g.Take epoxy activation-hyaluronic acid 9g and be dissolved in 5% ammonia, mix, react at airtight 60 ℃ and spend the night; Take out reactant, in fume hood,, with deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, be dried to obtain amino-hyaluronic acid 8g; Get amino-hyaluronic acid 5g and be dissolved in 0.1NaHCO3 solution, get 3g trichloride and triazine and be dissolved in cold acetone, progressively add, and use saturated NaHCO
3pH is maintained between 6.8-7.2, at 4 ℃ of stirring reaction 4h.Take out with deionized water and change buffer washing in ultrafiltration mode, suitable with deionized water to pH value and electrical conductivity, be dried to obtain three nitrogen piperazine activation-hyaluronic acid 5g; Taking three nitrogen piperazines activation--hyaluronic acid 4.5g dissolves with 0.1NaHCO3 solution 200ml, and the cycloheptylamine (R1) of getting 1g adds and mixes, and airtightly reacts 18h with 50 ℃, in course of reaction, uses saturated NaHCO
3and 1M acetic acid remains between 6.8-7.2 pH value of solution; Take out with deionized water and change buffer washing in ultrafiltration mode, suitable with deionized water to pH value and electrical conductivity, be dried to obtain cycloheptylamine base-chlorine three nitrogen piperazine-hyaluronic acid 4.7g; Take cycloheptylamine base-chlorine three nitrogen piperazine-hyaluronic acid 4g and dissolve with 0.1NaHCO3 solution 250ml, get 3,5-diaminobenzoic acid (R2) 1.2g and add dissolving to mix, 95 ℃ of reaction 24h, in course of reaction, use saturated NaHCO
3and 1M acetic acid remains between 7-8 pH value of solution; With deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, dried for standby, obtains cycloheptylamine base-3-amino-5-carboxyl phenylamino three nitrogen piperazine-hyaluronic acid 4.2g.
Embodiment 7 HP-Lig's is synthetic
Take heparin (heparin) 50g and be dissolved in 3%NaOH, add epoxychloropropane 10ml, mix, react 1.5 hours at airtight 65 ℃; Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, be dried to obtain epoxy activation-heparin 48g.Take epoxy activation-heparin 45g and be dissolved in 4% ammonia, mix, react at airtight 60 ℃ and spend the night; Take out reactant, in fume hood,, with deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, be dried to obtain amino-heparin 44g; Get amino-heparin 35g and be dissolved in 0.1NaHCO3 solution, get 10g trichloride and triazine and be dissolved in cold acetone, progressively add, and use saturated NaHCO
3pH is maintained between 6.5-7.5, at 2 ℃ of stirring reaction 6h.Take out with deionized water and change buffer washing in ultrafiltration mode, suitable with deionized water to pH value and electrical conductivity, be dried to obtain three nitrogen piperazine activation-heparin 35g; Take three nitrogen piperazine activation-heparin 30g 0.1NaHCO
3solution 200ml dissolves, and gets 1 of 10g, and 2-diamino-cyclohexane (R1) adds and mixes, and airtightly reacts 20h with 55 ℃, in course of reaction, uses saturated NaHCO
3and 1M acetic acid remains between 6.5-7.0 pH value of solution; Take out with deionized water and change buffer washing in ultrafiltration mode, suitable with deionized water to pH value and electrical conductivity, be dried to obtain the amino hexamethylene amino-chlorine of 2-three nitrogen piperazine-heparin 27g; Take the amino hexamethylene amino-chlorine three nitrogen piperazine-heparin 25g of 2-and dissolve with 0.1NaHCO3 solution 250ml, get 4,4 '-diaminodiphenyl ether (R2) 13g adds dissolving to mix, and 90 ℃ of reaction 48h, in course of reaction, use saturated NaHCO
3and 1M acetic acid remains between 7-8 pH value of solution; With deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, dried for standby, obtains amino hexamethylene amino-4-(the 4 '-amino phenylate) anilino--tri-nitrogen piperazine-heparin of 2-25g.
Embodiment 8 CS-Lig's is synthetic
Take chondroitin sulfate (Chondroitin sulfate) 80g and be dissolved in 3%NaOH600ml, add epoxychloropropane 60ml, mix, react 6 hours at airtight 55 ℃; Take out reactant, in fume hood, with deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, be dried to obtain epoxy activation-chondroitin sulfate 78g.Take epoxy activation-chondroitin sulfate 75g and be dissolved in 2% ammonia, mix, react 30 hours at airtight 60 ℃; Take out reactant, in fume hood,, with deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, be dried to obtain amino-chondroitin sulfate 74g; Get amino-chondroitin sulfate 70g and be dissolved in 0.2NaHCO
3solution, gets 20g trichloride and triazine and is dissolved in 200ml cold acetone, progressively adds, and uses saturated NaHCO
3pH is maintained between 6.5-7.0, at 7 ℃ of stirring reaction 4h.Take out with deionized water and change buffer washing in ultrafiltration mode, suitable with deionized water to pH value and electrical conductivity, be dried to obtain three nitrogen piperazine activation-chondroitin sulfate 74g; Take three nitrogen piperazine activation-chondroitin sulfate 60g 0.1NaHCO
3solution 300ml dissolves, and gets the 5-of 30g amino-2,2, and 4-front three basic ring penta methylamine (R1) adds and mixes, and airtightly reacts 30h with 48 ℃, in course of reaction, uses saturated NaHCO
3and 1M acetic acid remains between 6.5-7.0 pH value of solution; Take out with deionized water and change buffer washing in ultrafiltration mode, suitable with deionized water to pH value and electrical conductivity, be dried to obtain 5-amino-2,2,4-front three basic ring penta methylamine-chlorine three nitrogen piperazine-chondroitin sulfate 65g; Take 5-amino-2,2,4-front three basic ring, penta methylamino-chlorine, three nitrogen piperazine-chondroitin sulfate 55g dissolve with 0.1NaHCO3 solution 550ml, get 2-aminobenzimidazole (R2) 24g and add dissolving to mix, and 95 ℃ of reaction 36h, in course of reaction, use saturated NaHCO
3and 1M acetic acid remains between 7-8 pH value of solution; With deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, dried for standby, obtains 5-amino-2,2,4-front three basic ring, penta methylamino-benzimidazolyl-2 radicals-amino three nitrogen piperazine-chondroitin sulfate 57g.
Embodiment 9 TiPEG-Lig's is synthetic
Take 4 chain PEG amine (TiPEG, 60kD) 10g and be dissolved in 0.1NaHCO
3solution 200ml, gets 5g trichloride and triazine and is dissolved in 100ml cold acetone, progressively adds, and uses saturated NaHCO
3pH is maintained between 6.5-7.0, at 4 ℃ of stirring reaction 5h.Take out with deionized water and change buffer washing in ultrafiltration mode, suitable with deionized water to pH value and electrical conductivity, dry that three nitrogen piperazines activate-4 chain PEG amine (Amino-TiPEG) 9g; Take Amino-TiPEG8g 0.1NaHCO
3solution 100ml dissolves, and gets the 3-of 20g amino-1, and 5-benzene dicarboxylic acid (R1) adds and mixes, airtight prior to 50 ℃ of reaction 30h, in course of reaction, uses saturated NaHCO
3and 1M acetic acid remains between 6.5-7.0 pH value of solution; Be warming up to again 95 ℃, then react 24h, in course of reaction, use saturated NaHCO
3and 1M acetic acid remains between 7-8 pH value of solution; With deionized water, in ultrafiltration mode, change buffer, suitable with deionized water to pH value and electrical conductivity, dried for standby, obtains two (3,5-dicarboxyl toluidine) three nitrogen piperazine-4 chain PEG (TiPEG-Lig) 8g.
The HA-Lig preparation of embodiment 10 Recombulins
Insulin HA-Lig solution allocation: with the recombinant human insulin of 0.01mol/l pH7.4 phosphate buffer preparation 1U/ml, become recombinant human insulin-HA-Lig solution of recombinant human insulin-10mg/mlHA-Lig of 1U/ml with the HA-Lig solution mixing system of the 0.01mol/l pH7.4 phosphate buffer preparation recombinant human insulin of 2U/ml and the embodiment of 20mg/ml 6 preparations.
Mouse subcutaneous injection administration blood sugar lowering test: get 18 healthy male mices, be divided at random 3 groups.Fasting 12h before experiment, can freely drink water.Weigh, according to the subcutaneous solution of injecting respectively recombinant human insulin's solution of 1U/ml and recombinant human insulin-HA-Lig of 1U/ml of 0.5U/kg amount of insulin, in 0,15,30,60,90,120,180,240,300,360, during 480min, from tail venous blood sampling 20 μ l, add in 180 μ l 0.129mol/l sodium citrate anticoagulant solution, mix immediately the centrifugal 20min of 3000rpm, precision measures 20 μ l supernatants, by glucose oxidase method (GOD-PAP), measures blood glucose value.As a result, after insulin administration, 30min left and right blood glucose is down to low ebb, is 25% left and right of initial value; 3h after administration, blood glucose is returned to the more than 90% of initial value.After recombinant human insulin-HA-Lig administration, 280min left and right blood glucose is down to low ebb, is 65% left and right of initial value; 10h after administration, blood glucose is returned to the more than 90% of initial value.With area on trapezoidal method calculated curve (area above curve, AAC), the area of insulin of take is benchmark, calculates the biological activity percentage ratio of the relative natural insulin of recombinant human insulin-HA-Lig.Relative Recombulin group, the biological activity percentage ratio 87% of recombinant human insulin-HA-Lig.
Rat tail vein drug administration by injection blood sugar lowering test: get 12 healthy male SD rats, be divided at random 3 groups.Fasting 12h before experiment, can freely drink water.Weigh, tail vein is injected respectively (0.5U/kg) insulin and recombinant human insulin-HA-Lig solution, in 0,15,30,45,60,90,120,180,240,300, during 360,480min, from tail venous blood sampling 20 μ l, add in 180 μ l 0.129mol/l sodium citrate anticoagulant solution, mix immediately, the centrifugal 20min of 3000rpm, precision measures 20 μ l supernatants, by glucose oxidase method (GOD-PAP), measures blood glucose value.After Recombulin administration, 20min left and right blood glucose value is down to low ebb, is 50% left and right of initial value, and 90min left and right blood glucose is returned to initial level.After recombinant human insulin-HA-Lig administration, 100min left and right blood glucose is down to low ebb, is 55% left and right of initial value; 6h after administration, blood glucose is returned to 95% left and right of initial value.Relative Recombulin group, the biological activity percentage ratio 91% of recombinant human insulin-HA-Lig.
The diPEG-DaCH preparation of embodiment 11 Recombulins
Insulin DiPEG-DaCH solution allocation: with the recombinant human insulin of 0.01mol/l pH7.4 phosphate buffer preparation 1U/ml, become recombinant human insulin-DiPEG-DaCH solution of recombinant human insulin-10mg/mlDiPEG-DaCH of 1U/ml with the DiPEG-DaCH solution mixing system of the 0.01mol/l pH7.4 phosphate buffer preparation recombinant human insulin of 2U/ml and the embodiment of 20mg/ml 2 preparations.
Mouse subcutaneous injection administration blood sugar lowering test: get 18 healthy male mices, be divided at random 3 groups.Fasting 12h before experiment, can freely drink water.Weigh, according to the subcutaneous solution of injecting respectively recombinant human insulin's solution of 1U/ml and recombinant human insulin-DiPEG-DaCH of 1U/ml of 0.5U/kg amount of insulin, in 0,15,30,60,90,120,180,240,300,360, during 480min, from tail venous blood sampling 20 μ l, add in 180 μ l0.129mol/l sodium citrate anticoagulant solution, mix immediately the centrifugal 20min of 3000rpm, precision measures 20 μ l supernatants, by glucose oxidase method (GOD-PAP), measures blood glucose value.As a result, after insulin administration, 30min left and right blood glucose is down to low ebb, is 25% left and right of initial value; 3h after administration, blood glucose is returned to the more than 90% of initial value.After recombinant human insulin-DiPEG-DaCH administration, 260min left and right blood glucose is down to low ebb, is 65% left and right of initial value; 10h after administration, blood glucose is returned to the more than 90% of initial value.With area on trapezoidal method calculated curve (area above curve, AAC), the area of insulin of take is benchmark, calculates the biological activity percentage ratio of the relative natural insulin of recombinant human insulin-DiPEG-DaCH.Relative Recombulin group, the biological activity percentage ratio 82% of restructuring islets of langerhans-diPEG-DaCH.
Rat tail vein drug administration by injection blood sugar lowering test: get 12 healthy male SD rats, be divided at random 3 groups.Fasting 12h before experiment, can freely drink water.Weigh, tail vein is injected respectively (0.5U/kg) insulin and recombinant human insulin-DiPEG-DaCH solution, in 0,15,30,45,60,90,120,180,240,300, during 360,480min, from tail venous blood sampling 200 μ l, add in 180 μ l 0.129mol/l sodium citrate anticoagulant solution, mix immediately, the centrifugal 20min of 3000rpm, precision measures 200 μ l supernatants, by glucose oxidase method (GOD-PAP), measures blood glucose value.After Recombulin administration, 20min left and right blood glucose value is down to low ebb, is 50% left and right of initial value, and 90min left and right blood glucose is returned to initial level.After recombinant human insulin-DiPEG-DaCH administration, 130min left and right blood glucose is down to low ebb, is 60% left and right of initial value; 6h after administration, blood glucose is returned to 90% left and right of initial value.Relative Recombulin group, the biological activity percentage ratio 83% of recombinant human insulin-DiPEG-DaCH.
The diPEG-ABZMZ preparation of embodiment 12 interferon-alpha
The diPEG-ABZMZ solution allocation of interferon-alpha: prepare the interferon-alpha of 80 μ g/ml with 0.01mol/l pH7.4 phosphate buffer, become interferon-alpha-DiPEG-ABZMZ solution of interferon-alpha-15mg/mldiPEG-ABZMZ of 80 μ g/ml with the diPEG-ABZMZ solution mixing system of the 0.01mol/l pH7.4 phosphate buffer preparation interferon-alpha of 1.0mg/ml and the embodiment of 160 μ g/ml, 4 preparations.
Three dosage groups are established in test: interferon-alpha-DiPEG-ABZMZ low, in and a Senior Three dosage group, 10 μ g/kg (2 ♀/2 ♂), 40 μ g/kg (3 ♀/3 ♂) and 80 μ g/kg (2 ♀/2 ♂), in nape portion subcutaneous injection (s.c.), administration volume is 0.2mL/kg.After machin subcutaneous injection interferon-alpha-DiPEG-ABZMZ injection, respectively at different time, from the monkey forelimb saphena about 1.0mL that takes a blood sample, concrete blood sampling time point is: 0h (before medicine), 4,8,16,24,48,72,96,120 and 144h (after medicine); After adopting whole blood, mix anticoagulant immediately with 9ml 0.129mol/l sodium citrate anticoagulant solution dilution, the centrifugal 20min of 3000rpm, collects blood serum sample, preserves until measure for-70 ℃.
Adopt cytopathic effect method (CPE) to measure the antiviral activity of interferon-alpha-DiPEG-ABZMZ in machin body, i.e. interferon protection Wish cell is avoided the ability of VSV virus attack.Concrete operation method is:
(1) interferon standard substance is diluted to 1000IU/mL with mensuration culture fluid, again with measuring 4 times of serial dilutions of culture fluid (1: 1,1: 4,1: 16,1: 64,1: 256,1: 1024,1: 4096,1: 16384), its final concentration is respectively 1000,250,62.5,15.625,3.906,0.977,0.244 and 0.061IU/mL;
(2) get each time point blood serum sample of each dosage group and dilute by certain extension rate with measuring culture fluid, add in 96 porocyte plates;
(3) cell culture and inoculation are cultivated Wish cell to be monolayer in complete culture solution, adherent growth, and 3 times weekly, 1: 3 had digestive transfer culture is grown in complete culture solution.Get well-grown Wish cell, abandon after culture fluid, PBS washing 2 times, after digestion and collecting cell, adjusts cell suspending liquid to be adjusted to 3.0 * 10 with complete culture solution
5/ mL, is inoculated in 96 porocyte plates, every hole 100 μ L, and 37 ℃, 5%CO2 cultivates 5h;
(4) add standard substance or blood serum sample in cell plates, 100 μ L/ holes, 37 ℃, 5%CO2 cultivates 24h;
(5) counteracting toxic substances, abandons culture plate supernatant, dilutes VSV virus to 100TCID50,100 μ L/ holes, 37 ℃, 5%CO with counteracting toxic substances culture fluid
2overnight incubation is to till the cell death in positive control wells;
(6) dyeing and decolouring, abandon culture plate supernatant, adds dyeing liquor 50 μ L/ holes, and room temperature is placed 30min, rinses and remove dyeing liquor, and suck dry moisture, adds destaining solution 100 μ L/ holes, and room temperature is placed 3-5min;
(7) in microplate reader, measure the absorbance (OD) at 570nm place, reference wavelength is 630nm;
(8) result statistics: each sample replication is got its meansigma methods 3 times, and every cell microwell plate is prepared a standard curve is established cell contrast and VSV contrast simultaneously.In serum, interferon activity unit is defined as protecting half Wish cell to avoid the titre of VSV virus attack.Adopt four parametric regressions to calculate, clearance rate (CL), elimination half-life (t1/2 β); Peak time (Tmax) and peak concentration (Cmax) adopt measured value.
Result shows, after medicine, interior resisting virus activity raises gradually, arriving high antiviral active time T max is 16h, in time extend and reduce gradually thereafter, low, in and high dose at the high bioactivity (Cmax) of 16h, be respectively 35000,72000 and 150000IU/mL, drop to gradually 1500 of 144h, 4000 and 9000IU/mL, area under the drug-time curve AUC (0-144h) is respectively 1.2x10
6, 3.5x10
6and 7.3x10
6iU/mLh; Average clearance rate CL is 17mL/h/kg, on average eliminates the half-life (t1/2) and is about 43h, and mean residence time (MRT) is 76h.
The CS-Lig preparation of embodiment 13 recombination human interleukin-15
The CS-Lig solution allocation of recombination human interleukin-15: the I for preparing 10 μ g/ml with 0.01mol/l pH7.4 phosphate buffer
125labelling recombination human interleukin-15, become recombination human interleukin-15-CS-Lig solution of recombination human interleukin-15-5mg/mlCS-Lig of 10 μ g/ml with the CS-Lig solution mixing system that 0.01mol/l pH7.4 phosphate buffer is prepared 20 interleukin-15s of μ g/ml and the embodiment of 10mg/ml 4 preparations.
Pharmacokinetics comparative measurements: 12 of rats, male and female half and half, are divided into 2 groups, and single gives I respectively
125recombination human interleukin-15 of 10 μ g/kg of labelling and containing recombination human interleukin-15-CS-Lig solution of recombination human interleukin-15 of 10 μ g/kg, adopt the blood drug level after recombination human interleukin-15 radioimmunities (R1A) kit measurement subcutaneous injection recombination human interleukin-15 and recombination human interleukin-15-CS-Lig; At time point, be 0min, 30min, 1hr, 2h, 4h, when 8h, 12h, 16h, from tail venous blood sampling 200 μ l, add in 180 μ l 0.129mol/l sodium citrate anticoagulant solution, mix immediately, the centrifugal 20min of 3000rpm, precision measures 200 μ l supernatants, and-20 ℃ of preservations are used for the mensuration of blood drug level.Determination data shows that the medicine of recombination human interleukin-15 is for meeting first order kinetics two compartment models, average out to peak time T
peak(measured value) is 8.1 ± 1.7min, on average eliminates the half-life (t
1/2e) be 21.2 scholar 1.3min; Recombination human interleukin-15-CS-Lig group average out to peak time T
peak(measured value) is 12.3 ± 2.7h, on average eliminates the half-life (t
1/2e) be 35.3 ± 3.5h, than large nearly 100 times of the Increased Plasma Half-life of recombination human interleukin-15.
Impact on lotus tumor Balb/c NK cells in mice activity: get 60 Balb/c mices, respectively at right side subcutaneous vaccination 1x10
6individual SP2/0 cell, after about 10d, form subcutaneous solid tumor, be divided into at random again 6 groups, every group 10, be respectively blank model group (the isopyknic normal saline of lumbar injection), recombination human interleukin-15 group (lumbar injection 0.1 μ g/ time), recombination human interleukin-15-CS-Lig group (lumbar injection 0.5 μ g/ time).Administering mode is: group 1 times/day, recombination human interleukin-15, and drug withdrawal in continuous 5 days is 2 days weekly, successive administration 5 weeks; Recombination human interleukin-15-CS-Lig group, is administered once weekly, successive administration 5 weeks.Within after administration the 21st, 28,35,40 days, carry out the detection of NK cell killing activity.The YAC-1 cell centrifugation of growth 24-48h washes twice, and 10% FCS-1640 (complete 1640) the adjustment concentration of take after counting is 2x10
5/ mL is as target cell; Get mouse blood Tris-NH
4cl dilution, after band erythrocytolysis, centrifugal-washing 3 times, counting, is adjusted to 8x10 with 10% FCS-1640
6/ mL, action effect cell; Get effector lymphocyte 100 μ l and add respectively each hole of cell plates, every part of laboratory sample six holes, in 3 holes wherein, respectively adding 100 μ l target cells is experimental port again, other 3 holes respectively add complete 1640 liquid 100 μ l effector lymphocyte control wells again.37 ℃, 5%CO
2incubator adds MTT after cultivating 2h, then cultivates 2h and add cosolvent, after overnight incubation, by microplate reader, measures absorbance A
570nmvalue.With formula [1-(experimental port A
570nm-effector lymphocyte control wells A
570nm)/ target cell control wells A
570nm] * 100% calculates NK cytoactive and represents with killing and wounding %.Data show model control group NK cytoactive average out to 21 ± 3.7%, and after group treatment of recombination human interleukin-15, NK cytoactive is increased to 40% ± 3.3, within the 5th week, starts fast-descending, and within the 6th week, while finishing, contrasting all close with model is 25 ± 3.3%; The rear NK cytoactive of recombination human interleukin-15-CS-Lig group group treatment improves and maintains stablizes 37% ± 3.1, and administration in the 45th day finishes latter the 10th day, and NK cell still maintains greater activity 33% ± 3.5.
The HP-Lig preparation of embodiment 14 recombination human interleukin-6
The HP-Lig solution allocation of recombination human interleukin-6: recombination human interleukin-6 of preparing 40 μ g/ml with 0.01mol/l pH7.4 phosphate buffer, become recombination human interleukin-6-HP-Lig solution of recombination human interleukin-6-5mg/mlCS-Lig of 40 μ g/ml with the HP-Lig solution mixing system that 0.01mol/l pH7.4 phosphate buffer is prepared 80 recombination human interleukin-6 of μ g g/ml and the embodiment of 10mg/ml 7 preparations.
Trichloroacetic acid (TCA) Precipitation Determination plasma drug level: get anticoagulation two parts of supernatant 10 μ l are respectively added in Eppendorf centrifuge tube, with 190 μ l distilled water dilutings, shake up, standing 10 minutes, add again 200 μ l 20%TCA, shake up standing 10 minutes, the centrifugal 30min of 5000rpm, abandons supernatant, use FT-2008 gamma activity calculating instrument to measure the radioactivity (cpm) of precipitation part, two-tube digital average.According to the specific radioactivity activity of each sample, radioactive decay, radioassay time and instrument counting efficiency, proofread and correct, be calculated to be in blood plasma
125the concentration of I-recombination human interleukin-6 (ng/ml).The curve tendency of several dosage is basically identical, meets linear process, recombination human interleukin-6-HP-Lig half-life.
Intravenous injection (iV) administration is measured: 20 of SD rats, be divided at random 4 groups, and every group 3 hero 2 is female, gives respectively I
125recombination human interleukin-6 of labelling group 20 μ g/kg and I
125recombination human interleukin-the 6-HP-Lig high (40 μ g/kg) of labelling, in (20 μ g/kg), low (3 μ g/kg) dosage group.By jugular vein intubate drug administration by injection, volume injected is 0.4ml/.Syringe needle is inserted in the tube chamber of the PE pipe of staying rats in vitro, with syringe, touch medicine is slowly injected to rat jugular vein, a small amount of normal saline that reinjects all pushes medicine in PE tube chamber in rat body.2,5,10,20,40min after injection, 1,2,4,6,8,24,48h cuts tail, and the blood of outflow splashes into rapidly in the Eppendorf centrifuge tube of heparinization, collects approximately 100 μ l, centrifugal 8000rpm then, 5min, draws supernatant, preserves 4 ℃.After sample collection, detect immediately.Determination data shows that medicine generation of recombination human interleukin-6 meets first order elimination model, on average eliminates the half-life (t
1/2e) be 5.2 scholar 1.7min; Average half-life (the t that eliminates of recombination human interleukin-6-HP-Lig group
1/2e) be 50.3 ± 2.3min, than large nearly 9 times of the Increased Plasma Half-life of interleukin-6.
Subcutaneous injection (sc) administration is measured: 20 of SD rats, be divided at random 4 groups, and every group 3 hero 2 is female, gives respectively I
125recombination human interleukin-6 of labelling group 20 μ g/kg and I
125recombination human interleukin-the 6-HP-Lig high (40 μ g/kg) of labelling, in (20 μ g/kg), low (3 μ g/kg) dosage by back subcutaneous injection administration, volume injected be 0.4ml/ only.2,5,10,20,40min after injection, 1,2,4,6,8,24,48,72h cuts tail, and the blood of outflow splashes into rapidly in the Eppendorf centrifuge tube of heparinization, collects approximately 100 μ l, centrifugal 8000rpm then, 5min, draws supernatant, preserves 4 ℃.After sample collection, detect immediately.Determination data shows that the medicine of recombination human interleukin-6-15 is for meeting first order kinetics two compartment models, average out to peak time T
peak(measured value) is 9.2 ± 1.5min, on average eliminates the half-life (t
1/2e) be 23.5 scholar 1.7min; Recombination human interleukin-6-HP-Lig group average out to peak time T
peak(measured value) is 15.7 ± 2.3h, on average eliminates the half-life (t
1/2e) be 37.3 ± 2.5h, than large nearly 95 times of the Increased Plasma Half-life of restructuring human interleukin-6.
Embodiment 15 recombinant human somatropin's diPEG-APn preparation
Recombinant human somatropin's (rhGH) diPEG-APn solution allocation: with the recombinant human somatropin of 0.01mol/l pH7.4 phosphate buffer preparation 0.5mg/ml, be mixed and made into the rhGH-diPEG-APn solution of the rhGH-10mg/mldiPEG-APn of 0.5mg/ml with the diPEG-APn of the 0.01mol/l pH7.4 phosphate buffer preparation recombinant human somatropin of 1.0mg/ml and the embodiment of 20mg/ml 3 preparations.
Measure rhGH and rhGH-diPEG-APn active: get serum-free, without hGH culture medium 100 μ l, add respectively in 96 each holes of hole flat-bottom microtiter plates; The rhGH and the rhGH-diPEG-APn solution example 100 μ l that get respectively 0.5mg/ml add in 96 hole flat-bottom microtiter plates the first rounds again, every kind of sample 3 holes, with the volley of rifle fire by sample blending in the first round, draw 100 μ l and add the second round, mix and draw again 100 μ l and add the 3rd round, be diluted to successively last round, mix and draw again 100 μ l and discard.Get NB2-11 cell 2x10
5the cell suspending liquid serum-free of/mL, without the culture medium washed twice of hGH, gets 50 μ l (containing 10 after Eddy diffusion
4individual cell) add respectively in 96 flat-bottom microtiter plates holes, hole, incubation adds cell proliferation reagent WST-1 in 48 hours again, then incubation 2.5 hours.In microplate reader, measure each hole optical density, as the function drawing of concentration, determine rhGH and the rhGH-diPEG-APn concentration value (EC50) of NB2-11 propagation 50%.Result shows that rhGH-diPEG-APn is with respect to the Bioactivity 65% of rhGH.
Half-life in vivo is measured: 10 of Wistar rats (200-250g), be divided at random 2 groups, every group 3 hero 2 is female, every rat is distinguished intravenous injection 0.25mg hGH or 0.25mg rhGH-diPEG-APn, gathers blood 100 μ l and adds in the Eppendorf centrifuge tube of heparinization, then centrifugal 8000rpm in 0.5,3,24,48,72 and 96 hour Sublingual, 5min, draw supernatant, preserve-80 ℃, with hGH ELISA test kit (DSL), measure each sample rhGH concentration.The curve calculation that time is drawn, rhGH half-life 22min, rhGH-diPEG-APn half-life 11h, is 30 times of rhGH.
The TiPEG-Lig preparation of embodiment 16 thymic peptide-5s
The TiPEG-Lig solution allocation of thymic peptide-5 (TP5): prepare the thymic peptide-5 (TP5) of 100 μ g/ml with 0.01mol/l pH7.4 phosphate buffer, be mixed and made into the TP5-TiPEG-Lig solution of the TP5-10mg/ml TiPEG-Lig of 100 μ g/ml with the TiPEG-Lig of the 0.01mol/l pH7.4 phosphate buffer preparation thymic peptide-5 (TP5) of 1.0mg/ml and the embodiment of 200 μ g/ml, 9 preparations.
BALB/c mouse is divided into 3 groups at random: TP5-TiPEG-Lig sample sets, TP5 standard substance group and blank group.Intraperitoneal injection of mice, every 0.6ml (60 μ g), simultaneously every lumbar injection 0.5ml sheep red blood cell (SRBC).Blank group, every lumbar injection 0.6ml sheep red blood cell and 0.6ml PBS.After 4d, kill Mus and get spleen to survey hemolysis plaque active, LTA, and immune mouse spleen cell secretion IL-2 activity according to document [Jin Baiquan. basic medical immunological experiment instructs. the 1st edition. Beijing: world book publishing house, 1990] operation.As a result, every 1,000,000 splenocytes of experiment with computing group respectively and matched group include the meansigma methods of plaque cellulation number.T check through between group, TP5 group and the comparison of TP5-TiPEG-Lig sample sets, equal significance (the t=5.1 of difference, 5.3 and 7.3, P < 0.01), blank group, TP5 group and TP5-TiPEG-Lig sample sets hemolysis plaque activity are respectively 30 ± 8,78 ± 15,352 ± 37PFC/10
6splenocytes, compares with TP5, and TP5-TiPEG-Lig activity exceeds nearly 4 times; LTA measurement result shows, TP5 group and TP5-TiPEG-Lig sample sets have improved the lymhocyte transformation rate of mice.Through between group, check, TP5 group and TP5-TiPEG-Lig sample sets and the equal significance of blank group comparing difference (t=5.5,4.2, P < 0.01), blank group, TP5 group and TP5-TiPEG-Lig group mouse lymphocyte conversion ratio are respectively 1.05 ± 0.3,1.7 ± 0.8,7.5 ± 0.7, TP5-TiPEG-Lig LTA is 4.4 times of TP5; The mouse boosting cell of respectively organizing of getting in lymphocyte transformation test adds 24 well culture plates with debita spissitudo, cultivate 8h, get 5 times of dilutions of its supernatant as testing sample, detect its secretion IL-2 active, data show, blank group, the activity of TP5 group and TP5-TiPEG-Lig sample sets secretion IL-2 is respectively 0.25 ± 0.07, and 1.6 ± 0.8,3.7 ± 1.2IL-2IU/ml, compare with TP5, TP5-TiPEG-Lig secretion IL-2 activity exceeds nearly 1.3 times;
E-rosettes is measured: ABLB/c mice (body weight 22 ± 2), male and female half and half, randomized grouping, 1 group of immunologic hypofunction mice, 1 group of TP5 control mice, 1 group of TP5-TiPEG-Lig experimental mice, every group of 8 mices.
The preparation of immunologic hypofunction mice: give mouse subcutaneous injection hydrocortisone, every daily dose is 20mg/kg, continuous use 6 days.Medication: the 7th day, negative control group mice gave 0 every day.1%CMC-Na solution gavage; Positive controls lumbar injection every day TP5, every daily dose is 3.0mg/kg, successive administration 7 days; Experimental group injection 21mg/kg TP5-TiPEG-Lig solution once; 4 rabbit erythrocyte suspension preparations: under aseptic condition, in ear edge tremulous pulse, extract Sanguis Leporis seu oryctolagi 1ml, with heparin sodium aqua anticoagulant, add D-Hanks liquid 5ml washing, centrifugal (5000rpm, 5 minutes), abandon supernatant, with method washed twice again, finally erythrocyte is suspended from D-Hanks liquid, finally obtain concentration and be 1% rabbit erythrocyte suspension.Mouse lymphocyte suspension preparation: get the about 3ml of blood by plucking eyeball of mouse, heparin sodium aqua, as anticoagulant, adds 3ml D-Hanks liquid.In glass centrifuge tube, add lymphocyte separation medium, Mouse Blood is slowly added on separating medium along centrifugal tube wall, use horizontal centrifuge centrifugal 20 minutes with 2000rpm.Careful separating medium and the middle milky mononuclearcell of the erythrocyte layer drawn, moves in another centrifuge tube, with 5ml D-Hanks liquid washing 3 times, centrifugal 10 minutes of each 1500rpm.Lymphocyte is suspended to D-Hanks liquid again, first, with blood cell counting plate counting, with D-Hanks liquid, adjusts lymphocyte concentration to be adjusted to 2x10
8individual/ml.E-rosettes experiment: get rabbit erythrocyte suspension and each 0.1ml of mouse lymphocyte suspension, mix homogeneously.Put into 37 ℃ and hatch 10 minutes, 500rpm, after centrifugal 6 minutes, puts into 4 ℃ of Refrigerator store 2-4 hour.From refrigerator takes out, first suck part supernatant, add 0.1m 0.8% glutaraldehyde solution, fix after 20 minutes for 4 ℃, pressure-vaccum mixes sedimentation cell gently.Then obtained cell suspension smear, natural drying, 95% ethanol is fixed, after washing with the dyeing of haematoxylin-eosin stain, microscopy counting.Three above rabbit erythrocytes of T cell absorption for E garland formation lymphocyte, by counting 200 lymphocytes, calculate E-rosette forming rate.
E-rosette forming rate (%)=E-Flos Rosae Rugosae number of rings/counting total lymphocyte count * 100%
Data show immunologic hypofunction group 6.5 ± 1.3, and TP5 group 12 ± 3, TP5-TiPEG-Lig group 30 ± 5, compares with TP5 group, and TP5-TiPEG-Lig group has improved 1.5 times;
The TiPEG-Lig preparation of embodiment 17 L-Aspartic acid enzymes
Erwinia (Erwinia) L-asparaginase (Laps) solution allocation: the Laps for preparing 100 μ g/ml with 0.01mol/l pH7.4 phosphate buffer; With the TiPEG-Lig of the 0.01mol/l pH7.4 phosphate buffer preparation Laps of 1.0mg/ml and the embodiment of 200 μ g/ml, 9 preparations, be mixed and made into the Laps-TiPEG-Lig solution of the TP5-10mg/ml TiPEG-Lig of 100 μ g/ml.
External enzymatic determination of activity: the hydrolyzable aminosilane enzymatic activity of measuring the ammonia quantitative determination L-ASP that altheine enzyme catalysis L mono-agedoite discharges with Nesslerization.20mML-agedoite that 50 μ L enzymatic solution and 50mM boric acid are received in buffer (pH8.6) is mixed is incorporated in incubation 10min at 37 ℃.The Nessler reagent cessation reaction that adds 200 μ L.Then measure absorbance A
450nm, from ammonium sulfate concentrations standard curve, calculate described activity.Result Laps specific activity 650U/mg albumen, Laps-TiPEG-Lig specific activity 623U/mg albumen, is 95.8% of L-asparaginase (Laps) specific activity; Can think and substantially there is no loss of activity.
Enzymatic determination of activity in body: enzyme is L-ASP and azanol by L mono-aspartoyl-β-different hydroxyl hydroxamic acid (AHA) substrate hydrolysis, with oxine condensation, be oxidized to indooxine again, measure absorbance A 710nm quantitative, calculate enzyme activity (Analytical Biochem.309 (2002): 117-126).
Pharmacokinetic property: mice (B6D2F1) (female tool immunocompetence), every group of 8 animals, according to 5,25 or the Laps single intravenous injection administration of 50U/kg Laps-TiPEG-Lig and 250U/kg, 90min, 2h, 4h, 6h, 8h, 24h, 48h, 96h, 144h, 192h, 240h and 288h after 1h and administration before administration, from eye frame, collect plasma sample respectively, measure blood plasma activity of residual enzyme and L mono-agedoite level; By methanol extraction, remove sample plasma protein, the derivative wherein free amino acid of phenyl isothiocyanate for supernatant, then walk amount with RP-HPLC.Calculate half-life time=-ln (2* time)/ln (described time point blood plasma enzyme activity/initial blood plasma enzyme activity); Area under curve (AUC) represents the time integral of blood plasma activity of residual enzyme, represents gross effect.It is 75U/L that result L-asparaginase (250U/kg) maximum plasma concentration appears at 6h, AUC1200, and enzyme activity maximum length in time 18h can maintain in blood plasma L mono-agedoite level lower than 5 μ M concentration in 48h; It is 230U/L that Laps-TiPEG-Lig (50U/kg) maximum plasma concentration appears at 7h, AUC35374, and enzyme activity maximum length in time 288h can maintain in blood plasma L mono-agedoite level lower than 5 μ M concentration in 144h.
Claims (17)
1. a non-covalent polymer for medicine-high molecular polymer, is characterized in that:
(1) on high molecular polymer molecule, contain covalently bound affinity ligand;
(2) high molecular polymer and medicine mix rear formation.
2. the non-covalent polymer of medicine-high molecular polymer according to claim 1, is characterized in that, described medicine include but not limited to recombinate biological medicament, extract biological medicament and synthesising biological medicine.
3. restructuring biological medicament according to claim 2 includes but not limited to erythropoietin (EPO), Filgrastim (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), vaccine, interferon (INF), interleukin (IL), growth hormone (GH), insulin (Insulin), epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-B), insulin like growth factor (IGF), vascular endothelial cell growth factor (VEGF), PDGF (PDGF), endothelial cell growth factor (ECGF) (ECGF), nerve growth factor (NGF), bone-derived growth factor (BDGF), bone morphogenetic protein(BMP) (BNIP), tissue polypeptide antigen (TPA), antibody (antibody), blood coagulation factor VIII (VIII) and IX, genetic factor, streptokinase, with the protein being used for the treatment of or polypeptide, vaccine is containing recombinant hepatitis B vaccine.
4. synthesising biological medicine according to claim 2 includes but not limited to antisense nucleotide (anti-RNA), microRNA (RNAi), gene (DNA), bacitracin, pentagastrin, propylamine Rayleigh, oxytocin, calcitonin, octreotide, thymosin, somatostatin, glutathion, atrial natriuretic peptide, Bestatin, presses down pepsin BPTI, Desmopressin, ubenimex, ampeptide elemente.
5. extraction biological medicament according to claim 2 refers to that blood products includes but not limited to albumin, immunoglobulin, thrombin, Fibrinogen, thrombin, antitrypsin Alpha-1.
6. extraction biological medicament according to claim 2 refers to that animal viscera extraction product include but not limited to kallidinogenase, Lumbrukinase, Chymosin, pancreatin, trypsin, Chymotrypsin, hyaluronidase, Hyaluronidase, Elastase, aprotinin, asparaginase I, asparaginase I, cytochrome C, urokinase, ulinastatin, kininogenase, menotrophin, HMg, chorionic gonadotrophin, bromelain, bromelain, superoxide dismutase (SOD), Serrapeptase, lysozyme, Defibrase, Effect of Agkistrodon acutus Enzyme, transfer factor.
7. the non-covalent polymer of medicine-high molecular polymer according to claim 1, is characterized in that, described medicine includes but not limited to that people source, animal sources comprise pig, cattle, sheep, horse, chicken, duck, Columba livia, fish.
8. the non-covalent polymer of medicine-high molecular polymer according to claim 1, it is characterized in that described high molecular polymer includes but not limited to the polyamino acid of polyester, poly-anhydride or skeleton non-peptide bond, comprising: polylactic acid, polylactic acid one polyglycolic acid and combination thereof, also comprise Carmomer, hyaluronic acid, chondroitin sulfate, heparin, gelatin, collagen protein, Fibrinogen, fibrin, , glucosan, pi-allyl glucosan, agarose, cellulose, Polyethylene Glycol, the chimeric macromolecule of Polyethylene Glycol one polylactic acid, the chimeric macromolecule of Polyethylene Glycol one polyglycolic acid, the chimeric macromolecule of polyglycolic acid one Polyethylene Glycol one polyglycolic acid, the chimeric macromolecule of polylactic acid one Polyethylene Glycol one polylactic acid, the quick type gel of sugar and acid-sensitive type gel macromolecule, polybutylcyanoacrylate, poly phosphazene, poly phosphate.
9. the non-covalent polymer of medicine-high molecular polymer according to claim 1, it is characterized in that described affinity ligand includes but not limited to by chemical group ethylenediamine, triethylene tetramine, 3-diethyl amino propylamine, 1, 2-diamino-cyclohexane, 5-amino-2, 2, 4-front three basic ring penta methylamine, heptyl amice, octylame, appoint amine, certain herbaceous plants with big flowers amine, Aminocyclopentane, cycloheptylamine, cyclooctylamine, ring is appointed amine, ring certain herbaceous plants with big flowers amine, histamine, color amine, tyramine, aniline, 3-amino-phenol, para-aminophenol, benzene methanamine, 3-aminopyridine, 2-amine methylpyrrole, 3, 5-diaminobenzoic acid, 2-aminobenzimidazole, 5-aminobenzimidazole, 4, 4 '-diaminodiphenyl ether, 3, 3 '-dimethylbenzidine and combination thereof.
10. affinity ligand according to claim 9, is characterized in that with conventional chemical method direct covalently bound single aglucon molecule on polymer molecule.
11. affinity ligands according to claim 9, is characterized in that on polymer, by multi-active base, rolling into a ball skeleton combines the fixedly synthetic labyrinth part of multiple compounds step by step.
The synthetic method of 12. affinity ligand combinations according to claim 11, is characterized in that multi-active base group skeleton includes but not limited to that trichloride and triazine, Solid-phase Polypeptide synthesize.
13. pharmaceutical compositions, in the non-covalent polymeric acceptor that comprises medicine-high molecular polymer according to claim 1, after injection, the treatment effect level in blood plasma maintains more than at least 2 hours.
14. pharmaceutical compositions according to claim 12, is characterized in that adding and produce the non-covalent conjugate preparations of thing medicine-high molecular polymer described in steady right 1 together with other component of excipient and preparation.
15. excipient according to claim 13, is characterized in that including but not limited to that polyhydric alcohol contains mannitol, sorbitol, Sargassum sugar alcohol, maltose alcohol, xylitol and maltose alcohol.Preferred polyhydric alcohol excipient is mannitol.
16. pharmaceutical compositions according to claim 12, is characterized in that adding and produce the non-covalent conjugate preparations of thing medicine-high molecular polymer described in steady right 1 together with other component of surfactant and preparation.
17. surfactants according to claim 15, is characterized in that including but not limited to polyol derivative, propylene-glycol diacetate, polyoxyethylene ester and ether, nonylplenyl ether, polyvinyl alcohol, poloxamer (PLURONICS F87), alkanolamide.
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| CN110343245A (en) * | 2019-07-16 | 2019-10-18 | 天津大学 | Glycopeptide of epsilon-polylysine-graft-hydrophobic amino acid-graft-trehalose and preparation method thereof |
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