CN1767858A - Biologically active material conjugated with biocompatible polymer with 1:1 complex, preparation method thereof and pharmaceutical composition comprising the same - Google Patents

Biologically active material conjugated with biocompatible polymer with 1:1 complex, preparation method thereof and pharmaceutical composition comprising the same Download PDF

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CN1767858A
CN1767858A CNA2004800084834A CN200480008483A CN1767858A CN 1767858 A CN1767858 A CN 1767858A CN A2004800084834 A CNA2004800084834 A CN A2004800084834A CN 200480008483 A CN200480008483 A CN 200480008483A CN 1767858 A CN1767858 A CN 1767858A
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bioactive materials
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biocompatible polymer
csf
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朴明玉
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BiopolyMed Inc
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    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

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Abstract

The present invention relates to conjugates of biocompatible polymers and biologically active molecules wherein the activated biocompatible polymer is conjugated to a carboxyl group of biologically active material at a molar ratio of 1:1 and methods of preparation thereof and a pharmaceutical composition comprising the same.

Description

Carboxyl in bioactive materials and the biocompatible polymer combines in 1: 1 ratio, comprises preparation method and identical medicinal ingredient
Technical field
The invention relates to biocompatible polymer and combine, comprise preparation method and identical medicinal ingredient with the mol ratio of bioactive molecule by 1: 1.Especially the present invention combines by the mol ratio of the carboxyl in specific biocompatible polymer and the bioactive materials by 1: 1, comprises preparation method and identical medicinal ingredient.
Background technology
Protein and peptide are used as the restriction that medical product is subjected to several problems usually.Not equal to peptide or protein are very low at the organic intravital absorbance of living, its reason be the two in entering body in very short time just easily by protease hydrolysis or degraded, also can cause immunoreation during repetitive operation simultaneously.Therefore, most protein or peptide pharmaceutical products are injected one or many at least every day, and frequent injection can cause patient's pain and danger, and secular injection is expensive and inconvenient concerning the patient.
Need to attempt the more stable medicine of development to overcome the above problems, utilize the technology of biocompatible polymer improvement bioactive materials (as protein, polypeptide) existing development.Protein or medical active molecule combine with biocompatible polymer, can embody lot of advantages in the time of inside and outside being applied to organism.With after biocompatible polymer combines, it can demonstrate the surface property and the dissolubility of improvement to bioactive molecule by covalent bond, and this can improve its dissolubility in water and organic solvent.In addition, biocompatible polymer can make that also the protein or the polypeptide that are attached thereto are more stable in organism, increases proteinic biocompatible and reduces immunoreation, also can reduce intestinal, stomach, spleen, liver etc. to proteinic clearance rate.
Although bioactive materials (protein or peptide) and biocompatible polymer (as PEG) be combined with many benefits, there are some problems in institute's perception method in combination.
Most associated methods are that active PEG is bonded on the amino of amino acid residue (as lysine).When the active site with protein surface was connected on the PEG, because much proteinic one or more free lysine residues are frequently near active site, the biological activity of protein-polymer conjugate had lowered greatly.Simultaneously, react easily between proteinic lysine residue and the active PEG, obtain the PEG-protein conjugate, wherein two or more PEG molecules are connected on the protein molecule.For instance, when the PEG molecule that surpasses two was connected to cytokine (as interferon, CSF and interleukin) or polypeptide (as EGF, hGH, insulin) surface, the biological activity of conjugate reduced rapidly and causes losing efficacy.Simultaneously, this type of reaction can take place at random, thereby has caused multiple PEG-protein conjugate, and this makes that the purification of required conjugate is complicated and difficult.If too many polymer molecule is connected on target protein or the peptide, conjugate can lose whole or most biological activity.In addition, if used the apparent activity bridging agent or be connected to the amount of polymer deficiency of target protein molecule, then the therapeutic effect of conjugate reduces.
For overcoming above problem, attempted the employing genetic engineering polymer scale has been incorporated into proteinic specific part, replace with this biocompatible polymer is attached on the proteinic amino acid residue.Yet the method has changed proteinic original performance usually, and the genetic engineering molecule also haves much room for improvement as the safety of medical simultaneously.
Also once the specific part by chemical modifying bioactive materials conjugated polymer solves problem.United States Patent (USP) the 5951974th and having described for No. 5985263 is attached to the PEG molecule on the histidine residues of interferon, improves curative effect by prolong drug in organic intravital half-life.Yet this method has still been used active amino, and can produce the PEG-IFN isomer at random on some positions of histidine, and this need adopt ion exchange column to carry out extra purification step, and required 1: 1 conjugate is separated with PEG-IFN.In addition, than other amino amino, histidine combines the easy hydrolysis of imidazole radicals of PEG, makes histidine break away from from PEG one histidine conjugate easily.
United States Patent (USP) has been described combining of macromole and mutant for No. 5766897, and wherein cysteine residues connects active PEG molecule.Because the most protein molecule contains a disulfide bond freely, perhaps there is not remaining cysteine, so the aminoacid at nonactive position may change cysteine residues into and combines with polymer by sudden change.The method has been incorporated into the specific part of bioactive molecule with polymer scale, although have this advantage, than the conjugate of amino or carboxyl, this method product activity reduces greatly.
United States Patent (USP) has been described for No. 5985265 active site has been combined with the PEG molecule at the N of G-CSF and IFN end.Yet, this type of living polymer reactive relatively poor, and long time of reaction needed.In addition, the productive rate of this reaction is low and protein stability is poor.If the activity of proteins position is then amino in conjunction with causing biological activity to lower greatly or disappearing at the N end especially near the N end.
United States Patent (USP) has been described for No. 5824778 by PEG at amino or carboxyl in conjunction with G-CSF.Add the carboxyl in the excessive EDAC activation of protein, a large amount of PEG is attached on the pendant carboxylic group.The PEG-G-CSF that obtains is defined as uneven mixture, wherein contain a large amount of bonded PEG molecules, and the biological activity of conjugate reduces greatly.Therefore, if under special ratios, kept with its activity of bioactive molecule after polymer is connected, and obtain the specific part connector of homogeneity, then the clinical practice of this quasi-molecule significantly improves.
Summary of the invention
The present invention has prepared PEG-bioactive molecule conjugate in 1: 1 ratio, and wherein PEG is connected on the carboxyl of bioactive molecule.The carboxyl of bioactive molecule is lower than amino-reactive.According to observations, because this conjugate has longer half-life and higher stability than native protein (not combination), so its therapeutic effect will be higher than 20 times of native proteins.
The conjugate performance of 1: 1 ratio is better than those conjugates greater than 1: 1 ratio (in carboxyl or amino combination) simultaneously.
Therefore, the invention relates to combining of biocompatible polymer and bioactive molecule, wherein the carboxyl that is fit in polymer and the bioactive materials of active bio combines by 1: 1 mol ratio, comprises preparation method and identical medicinal ingredient.Conjugate of the present invention has kept the biological activity of bioactive molecule, and has strengthened stability, biological practicality and half-life.
Description of drawings
Fig. 1 is mPEG (5K)-H and the z-G-CSF conjugation by HPLC and SDS-PAGE.
Fig. 2 is the conjugation of (20K)-Hz-G-CSF by HPLC and SDS-PAGE.
Fig. 3 is that mPEG (the 5K)-Hz-IFN of 1: 1 ratio is on SDS-PAGE.
Fig. 4 is for making an addition to the amount of SDS-PAGE, the productive rate of mPEG (20K)-Hz-IFN conjugate corresponding to EDAC.
Fig. 5 is for corresponding to the adding method of EDAC and make an addition to the amount of SDS-PAGE, the extent of reaction of mPEG (20K)-Hz-IFN conjugate.
Fig. 6 is for to purify by the SDS-PAGE of ion exchange column to mPEG (20K)-Hz-IFN.
Fig. 7 is the biological activity that compares mPEG (20K)-Hz-IFN conjugate, natural G-CSF and NeulastaTM (FDA passed through in 2002 for PEG-G-CSF, Amgen development) by the cell chemical examination.
Fig. 8 is that the plasma half-life of mPEG (20K)-Hz-IFN conjugate, natural G-CSF and NeulastaTM (FDA passed through in 2002 for PEG-G-CSF, Amgen development) compares.
Fig. 9 is the WBC of mPEG (20K)-Hz-IFN conjugate, natural G-CSF and NeulastaTM (FDA passed through in 2002 for PEG-G-CSF, Amgen development).
Figure 10 is the biological activity that compares mPEG (12K)-Hz-IFN conjugate, natural IFN and PEG-IFN (Schering-Plough development) by the cell chemical examination.
Figure 11 is the biological activity that compares mPEG (20K)-Hz-IFN conjugate, natural IFN by the cell chemical examination.
Figure 12 is for relatively Di-mPEG-Hz-IFN conjugate, two PEG molecules are connected to the biological activity that an IFN molecule, unidirectional mPEG-Hz-IFN, a PEG molecule are connected to an IFN molecule by cell chemical examination.
Figure 13 is that the plasma half-life of PEG (20K)-Hz-IFN conjugate, natural IFN and PEG-IFN (Schering-Plough development) compares.
Figure 14 is that the PEG-IGN connector compares in carboxyl and amino stability.
Figure 15 is the HPLC chromatogram that does not pass through the natural PTH of biological adaptation polymer modified.
Figure 16 is PTH and mPEG (20k)-Hz reactant mixture (unreacted PTH, mPEG (20K)-Hz-PTH, the HPLC chromatogram of mPEG (20K)-Hz) before purifying.(peak 1: the unreacted PTH that has PEG, peak 2:mPEG (20k)-Hz-PTH).
Figure 17 is that PTH combines after the HPLC chromatogram of the mPEG that purifies (20k0)-Hz-PTH with mPEG (20K)-Hz.
Figure 18 is SDS-PAGE, makes the product of PTH and mPEG (20k)-Hz (line 1:M sign with the blue dyeing in Kumasi, line 2:PTH, line 3:PTH, PTH-mPEG (the 20k)-Hz conjugate before purifying, line 4: the PTH-mPEG after the purification (20k)-Hz conjugate).
Figure 19 is the interior biological activity of the organism of PTH and PEG-PTH.
Figure 20 is the half-life of interior PTH of mouse body and PEG-PTH.
The specific embodiment
On the one hand, the invention relates to combining of biocompatible polymer and bioactive molecule, wherein the carboxyl that is fit in polymer and the bioactive materials of active bio combines by 1: 1 mol ratio.
On the other hand, the present invention relates to medicinal composition, comprise above biocompatible polymer-bioactive molecule conjugate with therapeutic effect, and the medicinal acceptable carrier of going up.
Furtherly, the present invention relates to the bonded preparation method of the mol ratio by 1: 1 of biocompatible polymer and bioactive molecule, wherein the suitable polymer of active bio is pressed with the carboxyl in the bioactive materials and is combined, comprise bioactive materials is attached to step on the biocompatible polymer, it progressively adds bonding agent under reaction condition, the mol ratio of bioactive materials and biocompatible polymer is 1: 1-1: 20, with the mol ratio of bonding agent be 1: 1-1: 50, pH value 2-5.
In said method, EDAC adds as bonding agent, and it is easy to hydrolysis in aqueous solution, progressively adds (optimum is 5 or 6 times) so should be no less than 5 times.
Said method is that biocompatible polymer combines with the mol ratio by 1: 1 of bioactive molecule.In other words, the present invention combines with the mol ratio by 1: 1 of bioactive materials by biocompatible polymer, carries out the specific part combination.Owing to stoped polymer scale to be incorporated on the active site, so this type of conjugate has kept the biological activity of bioactive materials.Simultaneously, the present invention has avoided the randomized response of many active residues, thereby various heterogeneous mixture can not occur by the combination of 1: 1 mol ratio.In addition, this type of conjugate also has other advantages: such as having improved the organism internal stability, improved biological activity, having prolonged the half-life by the biological adaptation polymer.Therefore, compare with additive method, homogeneity biocompatible polymer of the present invention-bioactive molecule conjugate saves time and saves cost.
WO92/16555 has described the reaction of ovalbumin in carboxyl or candy Ji Chu and PEG-hydrazides (comprising amino acid spacer region).Yet it has described the combination of PEG molecule, does not mention by 1: 1 the mol ratio bonded preparation method with biocompatible polymer and bioactive materials, the also biological activity of not mentioned conjugate.
In addition, United States Patent (USP) has been described for No. 5824779 PEG has been connected on the carboxyl of G-CSF, but according to said method the conjugate activity of preparation is very low, and reason is that several PEG molecules are connected in the agedoite acid or glutamic acid of G-CSF randomly.
There are some problems in the quantity of the connection polymer of determining for specific connection or according to the difference of amino acid whose Pka value in the reaction when controlling reaction condition.
When carrying out the amino reaction of lysine in pH value 7-8 scope, histidine is randomized response also.Simultaneously, when pH value is lower than 6-6.5, histidine than lysine easier with PEG reaction (United States Patent (USP) the 5951974th and No. 5985263).According to above method, smaller or equal to 3, under 1: 1 condition of mol ratio, PEG can be connected to carboxyl, especially the C of bioactive materials end at pH value.
Therefore, the present invention relates to combining of biocompatible polymer and bioactive molecule, and wherein active bio is fit to polymer and holds with C in the bioactive materials by 1: 1 mol ratio and combine.
On the other hand, the present invention relates to medicinal composition, comprise the medicinal acceptable conjugate quantity that,, wherein active bio is fit to polymer and combines with C end in the bioactive materials by 1: 1 mol ratio, and medicinal upward acceptable carrier.
Furtherly, the C end that the present invention relates at bioactive materials, with biocompatible polymer and bioactive materials the bonded preparation method of mol ratio by 1: 1, wherein the suitable polymer of active bio is pressed with the carboxyl in the bioactive materials and is combined, comprise bioactive materials is attached to step on the biocompatible polymer, it progressively adds bonding agent under reaction condition, the mol ratio of bioactive materials and biocompatible polymer is 1: 1-1: 20, with the mol ratio of bonding agent be 1: 1-1: 50, pH value 2-5.
Biocompatible polymer
Be used for bonded bond material one speech of bioactive molecule, its meaning is the biocompatible polymer that can be connected with bioactive molecule arbitrarily, as natural or synthetic polymer.
Biocompatible is meant and biological tissue or system's fit, nontoxic in vivo, not inflammation, do not cause immunoreation and do not have carcinogenesis.
Biocompatible polymer combines with bioactive materials, the polymer that the present invention adopts is soluble in all kinds of solvents, molecular weight is about 300-100000Da (weight preferably is 2000-40000Da), biocompatible polymer comprises followingly enumerates these, but is not limited thereto: Polyethylene Glycol (PEG), polypropylene glycol (PPG), polyoxyethylene (POE), poly-hydroquinone ethylene glycol, polylactic acid and derivant thereof, polypropylene acid and derivant thereof, poly-propylhomoserin, polyvinyl alcohol, polyurethane, many phosphides, polyethylene, polyalkylene oxides (PAO), polysaccharide, dextran, the polyethylene pyrrolin, polyacrylamide, copolymer and other non-immunogen polymer.
The biocompatible polymer that the present invention adopts not only comprises linear polymer, also comprise following polymer: solubility, the nonantigenic polymer, it is connected on the active function group, can connect residue by aliphatic like this and carry out nucleophilic displacement of fluorine (No. the 5643575th, 5994455, United States Patent (USP)).Also have many support arms, single function, hydrolytic stability polymer in addition, it has two and connects segment (having the polymer support arm around the central carbon atom); An activable residue is to be connected on the bioactive materials, as protein; And side chain, it can be that hydrogen, methyl or other connect No. the 5932462nd, segment United States Patent (USP)).The biocompatible polymer that the present invention uses can also be the PEG that support arm is arranged, functional group wherein can combine with bioactive materials by the connection support arm that has residue (WO00/33881).
Wherein, PEG is a prevailing biocompatible polymer among the present invention.Usually, PEG is a kind of nontoxic, hydrophilic polymers, has repetitive HO-(CH 2CH 2O) n-H.Range protein with can be in blood plasma after PEG combines prolong half-life, improve stability, reduce immunogenicity.
The PEG molecular weight ranges that is connected on the bioactive materials (protein, peptide) is about 1000-100000Da, and the toxicity of PEG that surpasses 1000Da is very low.The PEG of 1000-6000Da scope is distributed in the whole body and removes in kidney.The support arm PEG of 40000Da is distributed in blood or the organ, comprises liver, and produce metabolic alterations in liver.
Because the PEG of various molecular weight ranges buys and obtains, so it is only biocompatible polymer.Each oxyethylene group unit is possess hydrophilic property all, can be in conjunction with 2-3 hydrone.PEG derives an end functional group from methoxy polyoxyethylene (being easy to synthesize), PEG is difficult for antigen-antibody reaction takes place, and its correlation technique has obtained large development.
Bioactive materials
Bioactive molecule be meant all can with the bonded nucleopilic reagent of biocompatible polymer, and in conjunction with after keep certain biological activity to I haven't seen you for ages.The biological activity of herein mentioning is not limited to physiology or the medicinal activity that goes up.For instance, some contain the nucleopilic reagent of enzyme, its conjugate can be in organic solvent catalytic reaction.Similarly, some comprise proteinic polymer conjugates, as canavaline or immunoglobulin, can be used as the usefulness of diagnosis in laboratory.Usually, bioactive molecule can separate from natural or synthetic material by reorganization or chemical method, comprises protein, peptide, polypeptide, enzyme, biological agent, gene, plasmid or organic residue.
Comprise protein, peptide, polypeptide but not only be confined to this: hemoglobin, serum albumin (as contain factor VII, VIII and IX blood factor), immunoglobulin, cytokine (as interleukin), α-, (β-gamma interferon, colony stimulating factor comprise G-CSF and GM-CSF, platelet from somatomedin (PDGF), phospholipase activator protein (PLAP), and thyroxin (PTH).Other gene biologicals or medical protein comprise: insulin, vegetable protein (as agglutinin and ricin), tumor necrosis factor (TNF) and relevant allele, growth factor is (as the factor TGF of organizational growth, TGF β and epidermal growth factor), hormones (stimulates hormone as folliculus, thyrotropin, vassopressin, pigment hormone, Alfasone-releasing hormone and derivant thereof), calcitonin, calcitonin-gene-related peptide (CGRP), artificial enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, prolactin antagonist, promoting sexual gland hormone, activator of plasminogen, peptide discharges somatomedin (GHRP), thymic humoral factor (THF), like that.Immunoglobulins comprises IgG, IgE, IgM, IgA, IgD and segment thereof.
When two or more biocompatible polymers were connected to low molecular weight polypeptide (as interferon, G-CSF), the biological activity of this conjugate was quite low.In addition, when the amino of relative high response connects two or more biocompatible polymers, separating and combining thing and being not easy in 1: 1 chemical compound.Yet, the invention provides the selectivity preparation of biocompatible polymer-IFN in 1: 1 chemical compound or biocompatible polymer-G-CSF, this type of conjugate biological activity height wherein, the half-life prolongs, the bioavailability excellence.
Bioactive materials of the present invention also comprises the polypeptide of any verified biologically active.This comprises aminoacid family, anti-sensitization oligomer, and antibody fragment, linear antigen (consulting United States Patent (USP) 4946778), binding molecule comprises antibody or pulsating fusion, polyclonal antibody, monoclonal antibody, catalytic antibody, nucleotide, oligonucleotide etc.
Bioactive materials also comprises enzyme.Enzyme comprises: carbohydrase, proteolytic enzyme, oxidoreductase, transferring enzyme, hydrolytic enzyme, lyases, isomerase and ligase; The specific enzymes of Xian Dinging does not comprise: asparaginase, arginase, arginine deaminase, ADA Adenosine deaminase, peroxidating dismutase, endotoxin enzyme, catalase, Chymotrypsin, lipase, uricase, adenosine deaminase, tryrosinase and Bilirubin oxidase etc.; Carbohydrase comprises: glucoseoxidase, glucosidase, Lac Bovis seu Bubali glycosidase, ceramide glucoside, glucoxylose enzyme etc.
Above-mentioned example is to be fit to and the bonded biological activity nucleophilic of biocompatible polymer reagent among the present invention.Even below do not mention, all suitable bioactive materials that have nucleophilic group all are contained in the present invention.The used bioactive materials of the present invention requires to have a carboxyl at least, makes it combine with polymer.
Conjugate biologically active of the present invention, and be purpose with the medical applications.The polymer conjugates that comprises bioactive materials, its effective medical dosage can be tested by mammal.
The preparation of biocompatible polymer-bioactive materials conjugate
For biocompatible polymer is combined with bioactive molecule, an end group of polymer need change into reactive functional group.This process is called activation, its products known as activated polymer.For instance, (the alkylene oxide PAO) with peptide or protein bound, can be converted into an one hydroxyl reactive functional group (as carbonization), obtains soluble activation PAO under the room temperature thus with polyethylene.This group comprises mono-substituted polyethylene (alkylene oxide) derivant, and as mPEG, perhaps other are suitable, contain Cl 4 end group alkyl replaces PAO.
Reactive functional group used herein refers to the activation biocompatible polymer, makes it the group or the residue that are connected with bioactive materials.
Reactive functional group of the present invention, from can with the functional group of carboxylic acid reaction and reactive carbonyl select.For instance, first-selected amine or hydrazine and hydrazides functional group (as acyl group hydrazides, carbonohydrazides, semicarbazides, sulfocarbazone etc.).
" carboxyl bonding agent " (hereinafter referred to as the bonding agent) herein mentioned, referring to can be with carboxyl in the bioactive materials and the bonded any reagent of biocompatible polymer (overactivation processing).
The used bonding agent of the present invention comprises following several, but be not limited thereto: EDAC[N-(3-dimethyl-aminopropyl)-N '-ethyl group carbodiimide hydrogen chlorine], DIC[1, the 3-DIC], DCC[dicyclohexyl carbodiimide] and EDC[1-ethyl group-3-(3-dimethylamino-propane base)-carbodiimide], optimum bonding agent is EDAC.
The preparation method of conjugate of the present invention may further comprise the steps: contain the bioactive molecule and the activatory biocompatible polymer generation substitution reaction of nucleopilic reagent, and enough conjugates keep the intrinsic biological activity of a part of bioactive molecule at least.
Bioactive materials and excessive biocompatible polymer reaction make the bioactive materials-biocompatible polymer conjugate of 1: 1 ratio with this.For instance, in preparation protein-polymer, peptide-polymer, enzyme-polymer, antibody-polymer and drug-polymer process, bioactive materials and biocompatible polymer proportion control are 1: 1-1: 20, and ratio preferably is 1: 1-1: 10.Reagent in order to activation bioactive materials carboxyl can be done following selection, but is not limited thereto.For example: EDAC ([N-(3-dimethyl-aminopropyl)-N '-ethyl group carbodiimide hydrogen chlorine]); The water-soluble carbodiimide base is as 3-[2-morpholinyl-(4)-ethyl group]; The different azoles salt of 5-, as the p-toluene fulfonate, Wood is irrigated reagent K.
The mol ratio of bioactive materials and EDAC is controlled at 1 among the present invention: 1-1: 50, and ratio preferably is 1: 1-1: 30, optimum ratio is 1: 1-1: 20.Yet, progressively add (optimum is 5 or 6 times) 5 times and add when EDAC divides to be no less than, rather than 20 times of moles once adding fashionablely, the output of PEG-bioactive materials conjugate improves, and its reason is that hydrolysis takes place EDAC easily in aqueous solution.
The association reaction of bioactive materials and activated polymer relies on the pH value with aqueous solution (playing cushioning effect).Usually, the reaction of protein/polypeptide buffering pH value is 2-5, and scope preferably is 2.5-4.5.The present invention has obtained stable optimum reaction condition and the output of this type of material: the optimum temperature range of association reaction is 0-60 ℃, scope 4-30 preferably ℃.The temperature of solvent should not surpass the denaturation temperature of protein or polypeptide.Simultaneously, the suitable response time is 10min-5h, and the conjugate that makes can recover or purification by column chromatography or diafiltration (or the two combination).
Medicinal synthetic
The invention still further relates to medicinal synthesizing, comprise the effective dose of activation biocompatible polymer-bioactive materials conjugate as active component.
" medicinal acceptability " refers to can not cause irritated or similar reaction after the people takes.
Biocompatible polymer-bioactive materials conjugate can be independent as medicinal synthetic composition, and is perhaps compound with medicinal acceptable carrier, is used as disease prevention and treatment with this.
Medicinal acceptable carrier refers to medicinal acceptable molecule, composition or medium, as solution, diluent, excipient or solvent etc., utilizes it that bioactive materials is transmitted between organ or tissue.
Medicinal composition of the present invention can be carried out by following route: oral, local injection, injection, its prescription comprises the effective dose of biocompatible polymer-bioactive materials conjugate as active component.The present invention is used for oral prescription and comprises pill, tablet, coated tablet, granule, bag medicine wafer paper, elixir, hard and soft capsule, solution, syrup, Emulsion, suspension or spraying etc.; The injection prescription comprises: injection, microcapsule and other.
Can utilize the inorganic or organic additive of static property, determine medicinal formula according to known method.For instance, lactose, corn starch and derivant thereof, Talcum, stearic acid and stearate can be used for preparing pill, tablet and hard capsules; The additive of soft capsule and suppository can use oil, wax, semisolid or liquid polyhydric alcohol, natural or solid oil; The additive of solution and syrup can make water, sucrose, invertase, glucose and polyhydric alcohol; The additive of injection solution can make water, ethanol, glycerol, polyhydric alcohol, plant wet goods.Injection solution can be used as the combination of antiseptic, analgesic, solubilizing agent and stabilizing agent.The local combination that also can be used as anesthetis, diluent, lubricant and antiseptic with prescription.The additive of microcapsule or transplantation can use copolymer, carboxyl acetic acid or lactic acid.
The dosage of biocompatible polymer among the present invention-bioactive materials conjugate is with following factors vary, the absorbance of bioactive materials, dissolubility, patient age, sex, environment and disease degree etc.
The injection interval of biocompatible polymer among the present invention-bioactive materials conjugate from once a day, two days once to weekly, biweekly successively decrease.Therefore, the toxicity of medicine and influence detect the also corresponding reduction of frequency.
By following example the present invention is carried out deep elaboration and demonstration.The example of listing separately wherein has the variation that does not much deviate from spirit of the present invention and scope in order to explanation rather than limitation the present invention.
1. the carboxyl by bioactive materials prepares biocompatible polymer-bioactive materials conjugate
Example 1 preparation mPEG (12000)-Hz-G-CSF conjugate
1mgG-CSF solution (0.00005mmol, Dong-A Pharm.LEUCOSTIM), (Centricon-10, Amicon USA) handle, and make ultimate density reach 2mg/ml with the dialysis of the MES buffer solution (PH3.0) of 50mM.To this protein solution, and mPEG (12000)-Hz of adding 6.6mg (ISU Chemical, Korea, 0.0005mmol).Next 2mgEDAC is dissolved in the d-H of 20 μ l 2Preparation 2 μ l EDAC solution (0.001mmol, 20 times of molar excess) in 0.Be reflected under room temperature (20-25 ℃) stirring condition and carry out 1h.Behind the 1h, remove unreacted G-CSF and excessive reagent, obtain above mPEG (the 12000)-Hz-G-CSF conjugate of 0.3mg by removing post or ion exchange column.Change the EDAC consumption from 20-200 times of molar excess, mPEG (12000) consumption repeats experiment from 10-20 times of molar excess.When the EDAC consumption surpasses 50 times of molar excess, can be observed two or more mPEG (12000)-Hz and be connected on the G-CSF.
Example 2 preparation mPEG (5000)-Hz-G-CSF conjugates
1mgG-CSF solution (0.00005mmol), (Centricon-10, Amicon USA) handle, and make ultimate density reach 5mg/ml with the dialysis of the MES buffer solution (PH3.0) of 50mM.To this protein solution, and mPEG (5000)-Hz of adding 1.3mg (ISU Chemical, Korea, 0.00025mmol).Next 2mgEDAC is dissolved in the d-H of 20 μ l 2Preparation 2 μ l EDAC solution (0.001mmol, 20 times of molar excess) among the O.Be reflected under room temperature (20-25 ℃) stirring condition and carry out 1h.Behind the 1h, remove unreacted G-CSF and excessive reagent, obtain above mPEG (the 5000)-Hz-G-CSF conjugate of 0.3mg by removing post or ion exchange column.Fig. 1 is for passing through the made mPEGE of SDS-PAG (5000)-Hz-G-CSF conjugate product and HPLC profile (exclusion chromatography).
Example 3 preparation mPEG (20000)-Hz-G-CSF conjugates
1mgG-CSF solution (0.00005mmol), (Centricon-10, Amicon USA) handle, and make ultimate density reach 5mg/ml with the dialysis of the MES buffer solution (PH3.0) of 50mM.To this protein solution, and mPEG (20000)-Hz of adding 5mg (ISU Chemical, Korea, 0.00025mmol).Next 2mgEDAC is dissolved in preparation 2 μ l EDAC solution (0.001mmol, 20 times of molar excess) among the d-H20 of 20 μ l.Be reflected under room temperature (20-25 ℃) stirring condition and carry out 1h.Behind the 1h, remove unreacted G-CSF and excessive reagent, obtain above mPEG (the 20000)-Hz-G-CSF conjugate of 0.3mg by removing post or ion exchange column.Fig. 2 is for passing through the made mPEG of SDS-PAGE (20000)-Hz-G-CSF conjugate product figure (exclusion chromatography).
Example 4 preparation mPEG (5000)-Hz-IFN conjugates
Four test tubes fill 200ugIFN solution (0.00001mmol, Korea Green Cross Corp., Green Alpha) respectively, respectively with the MES buffer solution (PH3.0) of 50mM dialysis (Centricon-10, Amicon USA) handles, and makes ultimate density reach 1mg/ml.To every test tube, add mPEG (5000)-Hz of 2.16mg.Next 2mgEDAC is dissolved in the d-H of 20 μ l 2Preparation 0.8 μ l EDAC solution (0.001mmol, 40 times of molar excess) among the O.Be reflected under room temperature (20-25 ℃) stirring condition and carry out 1h.Behind the 1h, remove unreacted IFN and excessive reagent by removing post or ion exchange column.Fig. 3 is for passing through the made mPEG of SDS-PAGE (5000)-Hz-IFN conjugate figure.
Example 5 preparation mPEG (12000)-Hz-IFN conjugates
1mgIFN solution (0.00005mmol), (Centricon-10, Amicon USA) handle, and make ultimate density reach 1mg/ml with the dialysis of the MES buffer solution (PH3.0) of 50mM.To this protein solution, add mPEG (12000)-Hz (0.0005mmol) of 6.6mg.Next 2mgEDAC is dissolved in the d-H of 20 μ l 2Preparation 2 μ l EDAC solution (0.001mmol, 20 times of molar excess) among the O.Be reflected under room temperature (20-25 ℃) stirring condition and carry out 1h.Behind the 1h, remove unreacted IFN and excessive reagent, obtain above mPEG (the 12000)-Hz-IFN conjugate of 0.3mg by removing post or ion exchange column.
Example 6 preparation mPEG (20000)-Hz-IFN conjugates
Four test tubes fill 200ugIFN solution (0.00001mmol) respectively, and dialysing with the MES buffer solution (PH3.0) of 50mM respectively, (Centricon-10, Amicon USA) handle, and make ultimate density reach 2mg/ml.To every test tube, and mPEG (20000)-Hz of adding 4.32mg (0.0002mmol, ISU Chemical, Korea).Next 2mgEDAC is dissolved in preparation 1 μ l (50 times of molar excess) or 4 μ l EDAC solution (200 times of molar excess) among the d-H20 of 20 μ l.In addition, add 50 times of molar excess and get sulfo-NHS with accelerated reaction and comparative result.Be reflected under room temperature (20-25 ℃) stirring condition and carry out 1h.The reaction condition of every group of sample is as shown in the table.Behind the 1h, remove unreacted IFN and excessive reagent by removing post or ion exchange column.The output of every group reaction by SDS-PAGE relatively.
The sample sequence number IFN mPEG-Hz Buffer solution Response time
#
1 2 mg/ml 20K(×20) 50mM MES pH4.4EDAC (×50) 1 hour
#
2 2 mg/ml 20K(×20) 50mM MES pH4.4EDAC (×50)EDAC(×30) 1 hour
#
3 2 mg/ml 20K(×20) 50mM MES pH4.4EDAC (×200) 1 hour
As a result of,, there is too many PEG to be connected on the carboxyl of IFN, thereby makes that the separation of 1: 1 molar ratio PEG-IFN conjugate is unsuccessful for the reaction of 200 times of molar excess EDAC, and the also bad estimation of the number of connection of PEG.For the reaction of 50 times of molar excess EDAC, 1: 1 molar ratio PEG-IFN conjugate causes the reason of this phenomenon to be that the EDAC consumption is excessive owing to it too disperses to be difficult for difference on SDS-PAGE.Reaction efficiency has been strengthened in the adding of sulfo-NHS, but with do not add sulfo-NHS (Fig. 4) in reaction control and indistinction.
In addition, following table has been listed the relation of reaction efficiency and EDAC interpolation number of times:
The sample sequence number IFN mPEG-Hz Buffer solution Response time
#
1 5 mg/ml 20K(×5) The every 10min of 50mM MES pH4.4 EDAC (* 10) adds 6 times. total molar excess: 60 times 1 hour
#
2 5 mg/ml 20K(×5) The every 10min of 50mM MES pH4.4 EDAC (* 10) adds 5 times. total molar excess: 50 times 1 hour
#
3 5 mg/ml 20K(×5) The every 10min of 50mM MES pH4.4 EDAC (* 5) adds 6 times. total molar excess: 30 times 1 hour
#
4 5 mg/ml 20K(×5) The every 5min of 50mM MES pH4.4 EDAC (* 3) adds 6 times. total molar excess: 18 times 1 hour
As a result of, when EDAC progressively joins in the reaction system, the output of 1: 1 molar ratio mPEG-IFN conjugate higher (Fig. 5).When the EDAC addition surpasses 50 times of molar excess, also have two or more PEG and be connected to IFN at random and go up (as shown in Figure 5) even progressively add EDAC.
Example 7 preparation mPEG (20000)-Hz-IFN conjugates
1mgIFN solution (0.00005mmol), (Centricon-10, Amicon USA) handle, and make ultimate density reach 5mg/ml with the dialysis of the MES buffer solution (PH2.5) of 50mM.To this protein solution, add mPEG (20000)-Hz (0.0005mmo, 10 times of molar excess) of 10.8mg.Next 2mgEDAC is dissolved in preparation 2 μ l EDAC solution (0.001mmol, 20 times of molar excess) among the d-H20 of 20 μ l.Be reflected under room temperature (20-25 ℃) stirring condition and carry out 1h.Behind the 1h, remove unreacted IFN and excessive reagent, obtain above mPEG (the 20000)-Hz-IFN conjugate of 0.3mg by removing post or ion exchange column.
The purification of 81: 1 molar ratio mPEG (20000)-Hz-IFN conjugates of example
MPEG (20000)-Hz-IFN conjugate (example 6) is diluted to 1mg/ml with 10mM sodium acetate buffer solution (PH4.4), then mPEG (20000)-Hz-IFN reactant mixture is placed the mobile fast post (5 * 50mm of SP-gel, total measurement (volume) 1ml) in, this equipment has utilized sodium acetate buffer solution (PH4.4) Balance Treatment mistake before using.After cleaning with the buffer solution of 3 times of column volumes, use the 10mM sodium acetate buffer solution (PH4.4) that contains 500mM NaCl, mPEG (the 20000)-Hz-IFN of 1: 1 molar ratio is separated from unreacted IFN.More than the mPEG of Ti Chuning (20000)-Hz-IFN is verified is conjugate, and one of them PEG has been connected on the carboxyl of IFN (Fig. 6).
The bioactive mensuration of example 9PEG-G-CSF conjugate, the CPE chemical examination follows these steps to carry out
Dish (RPMI-1640 medium, 10% FBS, 37 ℃, 5% CO in 60mm 2) go up and cultivate 2.5 * 10 6M-NFS-60 cell (5 * 10 5Cell/ml).G-CSF that each part is natural and mPEG (20000)-Hz-G-CSF (example 3) all is diluted to 1mg/ml, joins then that (each is 1 * 10 years old in 96 dishes 4And serial dilution cell).Plate was cultivated 2 days down at 37 ℃, and then each is all handled with 50XTT equipment (Roche, Germany), and handled 4h again in 37 ℃; Utilize the ELISA reader to read the O.D. value of plate at the 490nm place.
As a result of, mPEG of the present invention (20000)-Hz-G-CSF has kept and mPEG (20000)-similar activity of G-CSF conjugate, wherein is connected on the amino of G-CSF (Fig. 7).
Example 10PEG-G-CSF conjugate half life determination
Utilize Sprague-Dawley Mus (5 the every group) anesthesia that he life/Rumpun of gram is big with 7 weeks, weigh the 220-240 gram, and PE is managed in the caval vein that inserts every Mus by operation.After treating that Mus recovers, utilize mPEG (20000)-Hz-G-CSF (example 3) injection of intravenous injection with 100ug/kg.As a comparison, the natural G-CSF of PBS and 100 μ g/kg is as placebo and controlling agent.
In the interval of injection back 0,0.5,1,2,4,6,12,24,48h, utilize intubate to extract the blood of 300 μ l.By centrifugal filtration (13000rpm, 10min, 4 ℃) separation of serum and in subzero 20 ℃ of storages to do further research.
Cell is after the G-CSF medium is cultivated 24 hours, and each in 96 dishes all adds 1.5 * 10 4Cell.Each blood serum sample of storing as stated above all dilutes 100 times, and the dilute sample of 50 μ l is joined in each dish.These are coiled at CO 2In 37 ℃ of cultivation 48h, each is all handled with 50XTT equipment (Roche, Germany) then, and handles 4h again in 37 ℃ under the atmosphere; Utilize the ELESA reader to read the O.D. value of plate at the 490nm place.
Fig. 8 is to (20000)-Hz-G-CSF (example 3) and natural G-CSF and Neulasta TMThe half-life of (PEG is connected to the N end of G-CSF) compares.MPEG of the present invention (20000)-Hz-G-CSF (example 3) has the longer half-life than natural G-CSF, with Neulasta TMHalf-life close.
Example 11PEG-G-CSF handles the leukocyte (WBC) of mice and measures
Buy the Sprague-Dawley Mus that big, heavy 220-240 of 7 weeks restrains from Charles River company (Atsugi, Japan), and mPEG (20000)-Hz-G-CSF (example 3) of 100ug/kg is expelled in the tail vein of mice.
The natural G-CSF of equal number and saline solution as controlling agent, are injected respectively.Extract blood sample in the interval of injection back 0,6,12,24,48,72,96h.WBC quantity utilizes automatic hematology analyzer shown in Figure 9 (Cysmex K-4500) to measure.The result shows that mPEG of the present invention (20000)-Hz-G-CSF is than natural G-CSF and Neulasta TMAll has higher WBC quantity.
The bioactive mensuration of example 12PEG-IFN conjugate
Utilize hemocytometer that the MDBK cell is calculated 7.5 * 10 5In cell/ml concentration, and be placed in 5% the FBS/MEM medium.It is 100IU (1mg/ml=2 * 10 that mPEG (12000)-Hz-IFN (example 5) is diluted to concentration 8IU).Add the FBS/MEM medium of 100 μ l 5% and the dilute sample of 100 μ l in each dish, and serial dilution.In each of 96 dishes, all add 100 μ l cell suspending liquids then, cultivate 20h in 37 ℃; The vesicular stomatitis virus (VSV, ATCC VR-158) that adds 100 times of 100 μ l dilutions in each dish is cultivated 20h in 37 ℃.Remove the culture medium solution that contains VSV in 96 dishes, in each dish, add 50 μ l, 0.05% crystal violet dye solution, utilize the ELISA reader to read the O.D. value of each plate at the 550nm place, determine the activity of IFN with this.The result shows that mPEG (12000)-Hz-IFN (example 5) has kept the activity of natural IFN 40-50%, has close activity with PEG-IFN (Schering-Plough, USA, PEG are connected on the amino of IFN, FDA by).(Figure 10)
Simultaneously, after measured, the activity of mPEG (20000)-Hz-IFN (example 6) is approximately 40% (Figure 11) of natural IFN.
In addition, utilize the CPE chemical examination, activity to Di-mPEG-Hz-IFN (two PEG are connected on the IFN) and unidirectional-PEG-IFN (1: 1 chemical combination, two PEG are connected on the IFN) compares, and the result is unidirectional-and PEG-IFN demonstrates high bioactivity (Figure 12).
Example 13PEG-IFN conjugate half life determination
The MDBK cell is calculated 7.5 * 10 5In cell/m concentration, and be placed in 5% the FBS/MEM medium.In each of 96 dishes, all add 100 μ l cell suspending liquids; By intravenous injection mPEG (20000)-Hz-IFN (example 6) is injected in the mice body, obtain serum thereafter and dilute 50 times.The serum that adds dilution in each dish is at CO 2Cultivate 20h in the incubator.
The vesicular stomatitis virus (VSV, ATCC VR-158) that adds 100 times of dilutions in each dish continues to cultivate 20h.Remove the viral based sols in each dish, add 50 μ l, 0.05% crystal violet dye solution, utilize the microplate reader to read the absorptance of 550nm, measure the half-life of IFN with this.Figure 13 has shown the half-life by the bonded mPEG of carboxyl (20000)-Hz-IFN (example 6), and compares with natural FIN and PEG-IFN.MPEG of the present invention (20000)-more natural FIN of Hz-IFN and longer half-life of PEG-IFN.
The stability of example 14PEG-IFN conjugate
Preparation and mPEG (20000)-Hz-IFN that purifies and the stability of PEG-IFN in the example 6 are wherein with the PEG (10K) of support arm 2-NHS (be connected according to the method in the document on the amino of IFN, utilize the removing post to separate unidirectional PEG-IFN then) measures by observing complete IFN dissociating on SDS-PAGE, is placed in the PBS solution before measuring and cultivates under 4 ℃ of conditions.The concentration of each sample is adjusted to 1mg/ml.The result shows, after about 2 weeks, 14% complete IFN is arranged approximately from by dissociating the bonded PEG-IFN of amino, yet the present invention is by the bonded mPEG of carboxyl (20000)-Hz-IFN, even also do not detect dissociate (Figure 14) of IFN after 6 months.
The preparation of example 15mPEG (5000)-Hz-PTH conjugate
With 1mg human body PTH (thyroxin, 0.00012mmol, 1-84aa, Dong-Kook Pharm. Korea) places the 50mM buffer solution MES (PH4.4) of 0.5ml with 3.0mg activation mPEG (5000)-Hz, react 10min under the stirring at room condition; With 2.5 μ l concentration is EDAC (0.00125mmol, the 10 times of molar excess) adding of 100ug/ μ l, and then reacts 1h under the stirring at room condition.Unreacted mPEG (5000)-Hz and PTH utilize Centricon-10, and (Amicon USA) removes, and makes mPEG (5000)-Hz-PTH of 0.4mg.
The preparation of example 16mPEG (12000)-Hz-PTH conjugate
With 1mg human body PTH (0.00012mmol) and 7.14mg activation mPEG (12000)-Hz (0.0006mmol, 5 times of molar excess, ISU Chemical Korea) places the 50mM buffer solution MES (PH4.4) of 0.5ml, reacts 10min under the stirring at room condition; With 2.5 μ l concentration is EDAC (0.00125mmol, the 10 times of molar excess) adding of 100ug/ μ l, and then reacts 1h under the stirring at room condition.Unreacted mPEG (12000)-Hz and PTH utilize Centricon-10, and (Amicon USA) removes, and makes mPEG (12000)-Hz-PTH of 0.3mg.
The preparation of example 17mPEG (20000)-Hz-PTH conjugate
(ISU Chemical Korea) places the 50mM buffer solution MES (PH4.4) of 0.5ml, reacts 10min under the stirring at room condition for 0.0006mmol, 5 times of molar excess with 1mg human body PTH (0.00012mmol) and 12mg activation mPEG (20000)-Hz; With 2.5 μ l concentration is EDAC (0.00125mmol, the 10 times of molar excess) adding of 100ug/ μ l, and then reacts 1h under the stirring at room condition.Unreacted mPEG (20000)-Hz and PTH utilize Centricon-30, and (Amicon USA) removes, and makes mPEG (20000)-Hz-PTH of 0.3mg.
The preparation of example 18mPEG (12000)-Hz-PTH conjugate
With 1mg human body PTH (0.00012mmol) and 14.4mg activation mPEG (12000)-Hz (0.0012mmol, 10 times of molar excess, ISU Chemical Korea) places the 50mM buffer solution MES (PH2.5) of 0.5ml, reacts 10min under the stirring at room condition; With 5 μ l concentration is EDAC (0.0025mmol, the 20 times of molar excess) adding of 100ug/ μ l, and then reacts 1h under the stirring at room condition.Unreacted mPEG (12000)-Hz and PTH utilize Centricon-10, and (Amicon USA) removes, and makes mPEG (12000)-Hz-PTH of 0.2mg.
The preparation of example 19mPEG (20000)-Hz-PTH conjugate
With 1mg human body PTH (0.00012mmol) and 24mg activation mPEG (20000)-Hz (0.0012mmol, 10 times of molar excess, ISU Chemical Korea) places the 50mM buffer solution MES (PH2.5) of 0.5ml, reacts 10min under the stirring at room condition; With 5 μ l concentration is EDAC (0.0025mmol, the 20 times of molar excess) adding of 100ug/ μ l, and then reacts 1h under the stirring at room condition.Unreacted mPEG (20000)-Hz and PTH utilize Centricon-30, and (Amicon USA) removes, and makes mPEG (20000)-Hz-PTH of 0.2mg.
Example 20mPEG-Hz-PTH conjugate is analyzed
PEG-PTH conjugate that obtains in the above-mentioned example and natural PTH adopt following HPLC condition to measure:
HPLC elution condition
Post: LiChroCART 125-4RP-8 (5um) (Merck, USA)
Solvent: A contains the deionized water of 0.1%TFA
B contains the acetonitrile of 0.1%TFA
Flow: 0.8ml/min
Detector: 220nmUV detector
Volume injected: 20 μ l
Sequence number Time (min) A(％) B(％) Flow (ml/min)
1 0.00 70.0 30.0 0.800
2 3.00 70.0 30.0 0.800
3 13.00 10.0 90.0 0.800
4 15.00 10.0 90.0 0.800
5 17.00 70.0 30.0 0.800
6 20.00 70.0 30.0 0.800
Utilization only detects PTH and other protein for the LiChroCART 125-4RP-8 (5um) of HPLC at 220nm, does not detect PEG.The RT of PTH measures by HPLC.PTH without polymer modified has a sharp-pointed peak at the 6.8min place, increase to 18min gradually and reduce (Figure 15) once more.
In the elution condition and range between last table 1,2, Zhi Bei mPEG-Hz-PTH carries out elution process at 6.8min and 7.3min place as stated above, removing unreacted PTH and PEG-PTH respectively, in the elution condition and range between last table 1,2.
In conjunction with after, have 3 kinds of products (unreacted PTH, mPEG (20000)-Hz-PTH, mPEG (20000)-Hz) before purifying, only detect unreacted PTH, mPEG (20000)-Hz-PTH at 220nm, do not detect unreacted mPEG (20000)-Hz (Figure 16).
MPEG (20000)-Hz-PTH after Figure 17 purifies for HPLC is upward final, Figure 18 are with the painted SDS-PAGE of Kumasi indigo plant after mPEG (20000)-Hz-PTH and the PTH reaction.
Example 21mPEG-PTH conjugate is in the active mensuration of biological external biological
According to the molecular weight of PEG, the use molecular weight is that the activation mPEG-Hz of 5000 (5K), 12000 (12K) and 20000 (20K) carries out biological activity determination.The external biological activity of coming more natural PTH, mPEG (5000)-Hz-PTHmPEG, (12000)-Hz-PTH and mPEG (20000)-Hz-PTH by the quantity of measuring c-AMP, wherein c-AMP is by using c-AMP kit (the Amersham Pharmacia of UMR-106 cell line, RPN225, USA) synthetic.Found that the increase along with the PEG molecular weight, the biological activity of mPEG-Hz-PTH reduces.Find 10 in addition -8The mPEG of molar concentration (5000)-Hz-PTH, mPEG (12000)-Hz-PTH and mPEG (20000)-Hz-PTH keep the activity (Figure 19) of 40%, 30%, 20% unreacted PTH respectively.
The half-life of example 22mPEG-Hz-PTH is measured
By intravenous injection, the PTH of 100ug/kg and mPEG-Hz-IFN (example 6) are injected respectively in each male mice (heavy 300-350g) body.In the interval of injection back 0,5,10,15,30,60 and 120min, utilize intubate to extract blood.By centrifugal filtration (13000rpm, 10min, 4 ℃) separation of serum, the half-life of mPEG-Hz-PTH directly measures by calculating the PTH concentration that keeps, wherein concentration is by passing through to use c-AMP kit (Amersham Pharmacia, RPN 225, and the concentration of USA) measuring c-AMP in the blood plasma obtains.
The result shows, does not detect unreacted PTH and mPEG (5000)-Hz-PTHmPEG behind the 15min, but detected mPEG (12000)-Hz-PTH and mPEG (20000)-Hz-PTH (Figure 20) behind 1h, the 2h respectively.
Discussed preferred unit of the present invention, under mental condition of the present invention, skilled operators also can adopt other equipment, and the true scope that also is included in statement herein is interior to improvement of the present invention and change.The concrete device in the scope of the invention has deeply been described, proved to above example.The example that provides separately is in order to illustrate rather than to limit the present invention, can much changing under mental condition of the present invention.
The present invention has provided the biocompatible polymer-bioactive materials conjugate of a kind of 1: 1 mol ratio, and wherein active bio is fit to polymer and combines with carboxyl in the bioactive materials, as protein or polypeptide; Also comprise preparation method simultaneously.This conjugate has strengthened biological practicality, has prolonged the half-life, and the body internal stability strengthens, and this can reduce frequency of utilization when making it as medical.

Claims (20)

1. the conjugate of a biocompatible polymer-bioactive materials, wherein the carboxyl that is fit in polymer and the bioactive materials of active bio combines by 1: 1 mol ratio.
2. conjugate according to claim 1, it is characterized by: biocompatible polymer can be done following selection: Polyethylene Glycol (PEG), polypropylene glycol (PPG), polyoxyethylene (POE), poly-hydroquinone ethylene glycol, polylactic acid and derivant, polypropylene acid and derivant thereof, poly-propylhomoserin, polyvinyl alcohol, polyurethane, many phosphides, polyethylene, polyalkylene oxides (PAO), polysaccharide, dextran, polyethylene pyrrolin, polyacrylamide and other copolymers.
3. conjugate according to claim 1, it is characterized by: bioactive materials can be done following selection: hemoglobin, serum albumin (as contain factor VII, VIII and IX blood factor), immunoglobulin, cytokine, α-, (β-gamma interferon, colony stimulating factor comprise G-CSF and GM-CSF, platelet from somatomedin, phospholipase activator protein (PLAP), and thyroxin (PTH).Other gene biologicals or medical protein comprise: insulin, vegetable protein (as agglutinin and ricin), tumor necrosis factor (TNF) and relevant allele, growth factor is (as the factor TGFec of organizational growth, TGF β and epidermal growth factor), hormones (stimulates hormone as folliculus, thyrotropin, vassopressin, pigment hormone, Alfasone-releasing hormone and derivant thereof), calcitonin, calcitonin-gene-related peptide, artificial enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, prolactin antagonist, promoting sexual gland hormone, activator of plasminogen, peptide discharges somatomedin, thymic humoral factor.
4. conjugate according to claim 1, it is characterized by: bioactive materials can be selected interferon, G-CSF or thyroxin.
5. medicinal synthetic conjugate (according to statement 1) and the medicinal acceptable carrier that comprises medicinal acceptable number.
6. biocompatible polymer and bioactive materials by 1: 1 the bonded preparation method of mol ratio, wherein active bio is fit to polymer and combines with carboxyl in the bioactive materials, comprise bioactive materials is attached to step on the biocompatible polymer, it progressively adds bonding agent under reaction condition, the mol ratio of bioactive materials and biocompatible polymer is 1: 1-1: 20, with the mol ratio of bonding agent be 1: 1-1: 50, pH value 2-5.
7. method as claimed in claim 6 is characterized by: biocompatible polymer need through can with the functional group activation of carboxylic acid and reactive carbonyl reaction, can with carboxylic acid and reactive carbonyl reaction.
8. method as claimed in claim 6 is characterized by: biocompatible polymer can be done following selection: Polyethylene Glycol (PEG), polypropylene glycol (PPG), polyoxyethylene (POE), poly-hydroquinone ethylene glycol, polylactic acid and derivant, polypropylene acid and derivant thereof, poly-propylhomoserin, polyvinyl alcohol, polyurethane, many phosphides, polyethylene, polyalkylene oxides (PAO), polysaccharide, dextran, polyethylene pyrrolin, polyacrylamide and other copolymers.
9. method as claimed in claim 6, it is characterized by: bioactive materials can be done following selection: hemoglobin, serum albumin (as contain factor VII, VIII and IX blood factor), immunoglobulin, cytokine (as interleukin), α-, (β-gamma interferon, colony stimulating factor comprise G-CSF and GM-CSF, platelet from somatomedin, phospholipase activator protein (PLAP), and thyroxin (PTH).Other gene biologicals or medical protein comprise: insulin, vegetable protein (as agglutinin and ricin), tumor necrosis factor (TNF) and relevant allele, growth factor is (as the factor TGFec of organizational growth, TGF β and epidermal growth factor), hormones (stimulates hormone as folliculus, thyrotropin, vassopressin, pigment hormone, Alfasone-releasing hormone and derivant thereof), calcitonin, calcitonin-gene-related peptide (CGRP), artificial enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, prolactin antagonist, promoting sexual gland hormone, activator of plasminogen, peptide discharges somatomedin, thymic humoral factor.
10. according to the biocompatible polymer-bioactive materials conjugate of claim 6 preparation, its two mol ratio is 1: 1.
11. biocompatible polymer-bioactive materials conjugate, wherein biocompatible polymer was connected the C end of bioactive materials in 1: 1 ratio.
12. conjugate as claimed in claim 11, it is characterized by: biocompatible polymer can be done following selection: Polyethylene Glycol (PEG), polypropylene glycol (PPG), polyoxyethylene (POE), poly-hydroquinone ethylene glycol, polylactic acid and derivant, polypropylene acid and derivant thereof, poly-propylhomoserin, polyvinyl alcohol, polyurethane, many phosphides, polyethylene, polyalkylene oxides (PAO), polysaccharide, dextran, polyethylene pyrrolin, polyacrylamide, and other copolymers.
13. conjugate as claimed in claim 11, it is characterized by: bioactive materials can be done following selection: hemoglobin, serum albumin (as contain factor VII, VIII and IX blood factor), immunoglobulin, cytokine (as interleukin), α-, (β-gamma interferon, colony stimulating factor comprise G-CSF and GM-CSF, platelet from somatomedin, phospholipase activator protein (PLAP), and thyroxin (PTH).Other gene biologicals or medical protein comprise: insulin, vegetable protein (as agglutinin and ricin), tumor necrosis factor (TNF) and relevant allele, growth factor is (as the factor TGFec of organizational growth, TGF β and epidermal growth factor), hormones (stimulates hormone as folliculus, thyrotropin, vassopressin, pigment hormone, Alfasone-releasing hormone and derivant thereof), calcitonin, calcitonin-gene-related peptide (CGRP), artificial enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, prolactin antagonist, promoting sexual gland hormone, activator of plasminogen, peptide discharges somatomedin, thymic humoral factor.
14. conjugate as claimed in claim 13 is characterized by: bioactive materials can be selected interferon, G-CSF or thyroxin.
15. medicinal synthetic conjugate (according to 11) and the medicinal acceptable carrier that comprises medicinal acceptable number.
16. the mol ratio by 1: 1 of biocompatible polymer and bioactive materials is held bonded preparation method at the C of bioactive materials, wherein active bio is fit to polymer and combines with carboxyl in the bioactive materials, comprise bioactive materials is attached to step on the biocompatible polymer, it progressively adds bonding agent under reaction condition, the mol ratio of bioactive materials and biocompatible polymer is 1: 1-1: 20, with the mol ratio of bonding agent be 1: 1-1: 50, pH value 2-5.
17. method as claimed in claim 16, biocompatible polymer need through can with the functional group activation of carboxylic acid and reactive carbonyl reaction, can with carboxylic acid and reactive carbonyl reaction.
18. according to the conjugate of claim 16, biocompatible polymer can be done following selection: Polyethylene Glycol (PEG), polypropylene glycol (PPG), polyoxyethylene (POE), poly-hydroquinone ethylene glycol, polylactic acid and derivant, polypropylene acid and derivant thereof, poly-propylhomoserin, polyvinyl alcohol, polyurethane, many phosphides, polyethylene, polyalkylene oxides (PAO), polysaccharide, dextran, polyethylene pyrrolin, polyacrylamide and other copolymers.
19. conjugate according to claim 16, bioactive materials can be done following selection: hemoglobin, serum albumin (as contain factor VII, VIII and IX blood factor), immunoglobulin, cytokine (as interleukin), α-, (β-gamma interferon, colony stimulating factor comprise G-CSF and GM-CSF, platelet from somatomedin, phospholipase activator protein (PLAP), and thyroxin (PTH).Other gene biologicals or medical protein comprise: insulin, vegetable protein (as agglutinin and ricin), tumor necrosis factor (TNF) and relevant allele, growth factor is (as the factor TGFec of organizational growth, TGF β and epidermal growth factor), hormones (stimulates hormone as folliculus, thyrotropin, vassopressin, pigment hormone, Alfasone-releasing hormone and derivant thereof), calcitonin, calcitonin-gene-related peptide (CGRP), artificial enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, prolactin antagonist, promoting sexual gland hormone, activator of plasminogen, peptide discharges somatomedin, thymic humoral factor.
20. according to the biocompatible polymer-bioactive materials conjugate of claim 16 preparation, wherein to be connected to the mol ratio of bioactive materials C end be 1: 1 to biocompatible polymer.
CNA2004800084834A 2003-03-28 2004-03-27 Biologically active material conjugated with biocompatible polymer with 1:1 complex, preparation method thereof and pharmaceutical composition comprising the same Pending CN1767858A (en)

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