CN1863549A - Conjugates of a polymer and a protein linked by an oxime linking group - Google Patents
Conjugates of a polymer and a protein linked by an oxime linking group Download PDFInfo
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- CN1863549A CN1863549A CNA2004800295229A CN200480029522A CN1863549A CN 1863549 A CN1863549 A CN 1863549A CN A2004800295229 A CNA2004800295229 A CN A2004800295229A CN 200480029522 A CN200480029522 A CN 200480029522A CN 1863549 A CN1863549 A CN 1863549A
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Abstract
The present invention relates to conjugates of hydroxyalkyl starch and a granulocyte colony stimulating factor protein (G-CSF) wherein these conjugates are formed by a covalent linkage between the hydroxyalkyl starch or a derivative of the hydroxyalkyl starch and the protein. The present invention also relates to the methods of producing these conjugates and the use of these conjugates.
Description
The present invention relates to by the polymer or derivatives thereof of amino oxygen base functionalization, or wherein the polymer of said functionalization by oxime linking group and the covalently bound conjugate of protein, be used to prepare the method for the polymer of this functionalization, be used to prepare the method for this conjugate, the polymer of the functionalization that can obtain with method of the present invention, the conjugate that can obtain with method of the present invention, be used for application that human or animal body is prevented or treats with the pharmaceutical composition that comprises at least a conjugate of the present invention and said conjugate and compositions.
With the polymer such as the polysaccharide of functionalization, for example starch with and derivant and glucosan with and derivant and Polyethylene Glycol with and derivant and human cytokines covalently boundly can prolong described protein in vivo cycle period, reduce its antigenicity and immunogenicity and improve it proteolysed repellence.These character have very big clinical benefit, and are especially true in relatively little proteinic situation, are associated with the reduction of renal clearance because be sure of the increase of Stoke ' s radius.
With biocompatible polymer protein being modified is known in the prior art.Up to now, great majority are undertaken by use Polyethylene Glycol or glucosan the modification that protein carries out always, and preferably use Polyethylene Glycol (PEG).In addition, it also is known with other polysaccharide albumen being modified in the prior art.
Wherein, WO 02/09766 discloses the biocompatible protein-polymer compound that engages preparation by the protein with biologic activity with the biocompatible polymer derivant.Its used biocompatible polymer is reactive very high branch polymer, and the conjugate of gained comprises long linking group between polymer derivant and protein.With regard to biocompatible polymer, to formula (P-OCH
2CO-NH-CHR-CO-)
nThe polymer of-L-Qk-A is described, and wherein P and Q are that polymer residue and k can be 1 or 0.For P and Q, mentioned Polyethylene Glycol, polypropylene glycol, polyethylene glycol oxide, polytrimethylene glycol, polylactic acid with and derivant, polyacrylic acid with and derivant, polyamino acid, polyvinyl alcohol, polyurethane, polyphosphazene, poly-(L-lysine), polyoxyalkylene, polyacrolein amide and water-soluble polymer such as glucosan or polysaccharide.Wherein, with regard to protein, α, β and IFN-, blood factor, cytokine such as interleukin, G-CSF, GM-CSF have been mentioned.In the embodiment of WO 02/09766, only disclose single-, two-and three-polyethyleneglycol derivative, these derivants only with interferon and epidermal growth factor and human growth hormone's coupling mutually.
WO 94/01483 discloses by the biocompatible polymer conjugate with the synthetic hydrophilic polymer covalent bond formation that inanimate object active polymer or polymer derivant and pharmacy is pure of the chemical bond of particular type.With regard to naturally occurring polymer with and derivant with regard to, polysaccharide such as hyaluronic acid are disclosed, Dan Baijutang such as chondroitin sulfate A, B and C, chitin, heparin, heparin sulfate, glucosan such as cyclodextrin, hydroxyethyl-cellulose, cellulose ether and starch, lipid such as triglyceride material and phospholipid substance.Wherein, with regard to synthetic polymer, described mean molecule quantity and be about 100 to about 100,000 polyethylene with and derivant.With regard to regard to protein that polymer or polymer derivant link to each other, the pair cell factor and somatomedin comprise that interferon, tumor necrosis factor, interleukin, colony stimulating factor, somatomedin such as osteogenic factor extract, epidermal growth factor, transforming growth factor, platelet-derived somatomedin, acid fibroblast growth factor or the like are described.In all processing and implementation examples of WO 94/01483, all use polyethyleneglycol derivative as polymer.
The protein compound that WO 96/11953 discloses the terminal chemical modification of N-with and preparation method thereof.Specifically the G-CSF component is described, this component is by the terminal coupling of the N-of water-soluble polymer and G-CSF is produced.In WO 96/11953, the terminal and link coupled interferon that agrees with of water-soluble polymer of N-is also disclosed.Though in WO 96/11953, enumerated many water-soluble polymers (copolymer of ethylene glycol and propylene glycol for example, carboxymethyl cellulose, glucosan, polyvinyl alcohol, polyvinylpyrrolidone, poly--1, the 3-dioxolanes, poly--1,3, the 6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid (homopolymer or random copolymer), poly-(N-vinyl pyrrolidone) Polyethylene Glycol, the polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer or polyoxy ethylization polyhydric alcohol), but the IFN compositions of in the processing and implementation example of WO 96/11953, only having described the G-CSF of PEGization or having agreed with.
WO 97/30148 relates to the polypeptide conjugate of the allergenicity with reduction, and it comprises the polymer support molecule with two or more peptide molecules link coupled with it.These conjugates are the part of personal care market composition therefor preferably.Said conjugate is by with the polymer support molecule activation, with two or more peptide molecules and this polymer support molecular reaction that is activated and block that active group remaining on this conjugate is prepared.With regard to the polymer support molecule, in WO97/30148, listed many carrier molecules, comprise for example natural or synthetic homopolymer such as polyhydric alcohol, polyamines, polycarboxylic acid and the heteropolymer that comprises at least two kinds of different linking groups etc.Wherein provide some examples, comprised star PEG, side chain PEG, polyvinyl alcohol, polycarboxylate, polyvinylpyrrolidone and poly--D, L-aminoacid.Wherein, hydrolyzate, starchy material such as the hetastarch of glucan material such as Sensor Chip CM 5, cellulose substances such as hydroxyethyl-cellulose or hydroxypropyl cellulose, chitosan or hydroxypropyl starch, glycogen, agarose, guar gum, inulin, Pullulan, xanthan gum, carrageenin, pectin, alginic acid or the like are also disclosed.With regard to polypeptide, some enzymes are only clearly disclosed.
Baldwin, J.E. wait the people, Tetrahedron, the 27th volume (1981), the 1723-1726 page or leaf has been described by glucosan and hetastarch being carried out chemical modification and has been obtained the polymer that some aldehyde replace, thereby these polymer can obtain the hemoglobin that combines with polymer of some solubilities with the hemoglobin reaction.Confirmed that these materials can be unsuitable for being used as blood substitute but the heart perfusion experiment clearly illustrates that this hemoglobin that combines with polymer in conjunction with oxygen.
WO 99/49897 has described some and has reacted the hemoglobin conjugate that forms by the amino with polysaccharide such as glucosan or hetastarch and hemoglobin.With regard to the functional group of this polysaccharide, use the aldehyde radical that produces by the open loop of oxidisability sugar.With regard to used preferred Reducing agent, the borine dimethylamine is disclosed.In addition, WO 99/49897 only only limits to hemoglobin.
WO 03/074087 relates to and a kind of protein and the deutero-modification of polysaccharides of starch is carried out link coupled method.Protein and polysaccharide---combination between the hydroxyalkyl starch be a kind of at said hydroxyalkyl starch molecule terminal aldehyde radical or by terminal aldehyde radical to it carry out forming between functional group that chemical modification produced and the described proteinic functional group covalently bound.With regard to proteinic reactive group, disclose amino, sulfydryl and carboxyl, but do not mentioned proteinic aldehyde radical.In addition, though in many probabilities and different chemical operation of enumerating the many different keys that provided the different link molecule that comprise different functional groups, suit in theory in the form, but its processing and implementation example has only been described two kinds of selections: first kind, use oxidized hetastarch and activate direct and protein coupling it with ethyl dimethylaminopropyl carbodiimide (EDC), perhaps use unoxidized hetastarch and directly with itself and protein coupling, thereby form a kind of Schiffs alkali, subsequently it is reduced into corresponding amine.
People such as Gaertner, Bioconjugate Chem.1996,7, being connected of the site specific that the Polyethylene Glycol (PEG) that 38-44 discloses functionalization and proteinic amino terminal carry out.With linking group-NHCO-CH
2-O-N=CH-couples together PEG and protein.
It is covalently bound to improve the used proteinic body-internal-circulation phase and to reduce the conjugate of the polymer of proteic antigenicity and immunogenic functionalization to the purpose of this invention is to provide some and protein.
The polymer of the functionalization by using formula I has been realized this purpose
" polymer "-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5(I)
Symbol wherein has following implication
" polymer " be the homopolymer of solubility straight or branched or random copolymer with and derivant, be selected from: aklylene glycol homopolymer, preferred ethylene glycol homopolymer (PEG), propylene glycol homopolymer; The aklylene glycol copolymer, preferred oxypropylene/oxyethylene copolymers; Polyvinyl alcohol; Polyvinylpyrrolidone; Gather-1, the 3-dioxolanes; Gather-1,3, the 6-trioxane; Ethylene/copolymer-maleic anhydride; Polyamino acid; And polysaccharide, be preferably selected from starch, cellulose, glucosan, arabic gum, xanthan gum, inulin, Ficus elastica (ghatti gum), pectin, guar gum, Tragacanth, agar, algin, karaya, carrageenin, scleroglycan, Furcellaran (fucellaran), arabinogalactan (arabinogalacton) and locust bean gum;
R
1、R
2、R
3、
R
4, R
5Being hydrogen, alkyl, aryl, more preferably is hydrogen;
M is 2 to 4, wherein at m group CR
1R
2In residue R
1And R
2Can be identical or different;
N is 0 to 20, preferably 0 to 10, and more preferably be 1 to 5, most preferably be 2;
O is 0 to 20, preferably 0 to 10, and more preferably be 0 or 2, wherein in the situation of n=0, o is not 0, in a preferred embodiment, o is 2 to 20, preferably 2 to 10, more preferably be 2, wherein at o group CR
3R
4In residue R
3And R
4Can be identical or different;
R is 0 or 1, preferably 1;
X is-(CR
8R
9)
pO-,-(CR
8R
9)
pS-,-(CR
8R
9)
pNR
6-,-(CR
8R
9)
pOC (O)-,-(CR
8R
9)
pC (O) O-,-(CR
8R
9)
pC (G) N (R
10) O-,-(CR
8R
9)
pN (R
11) O-,
Preferably
-(CR
8R
9)
pN (R
11) O-, more preferably
First-selected
With-(CR
8R
9)
pN (R
11) O-,
Wherein one or more groups-(CR
8R
9)-can be replaced by W, thus a kind of chemically rational group formed;
W is O, NR
12, C (G), preferably O, C (G);
G is S, O, NR
14, O preferably;
R
6、R
7、R
8、
R
9、R
10、
R
11, R
12, R
14Be hydrogen, alkyl, aryl, hydrogen preferably,
P is 0 to 20, preferably 0 to 10, more preferably be 0 to 5, most preferably be 0 to 4, polysaccharide with and the situation of derivant in more preferably be 0, and poly alkylene glycol with and the situation of derivant in more preferably 1 to 4, wherein at p group CR
8R
9In residue R
8And R
9Can be identical or different;
Group wherein
-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5Being positioned at intermediary group covalency with at least one end group of " polymer " or at least one links to each other.
About above-mentioned radicals X, preferably poly alkylene glycol with and the situation of derivant in, two group (CR
8R
9) can be replaced by two group W, preferably form group-N (R together with two group W
12) C (G)-mode replaced by two group W.In this case, X is more preferably
-N (R
12) C (G)-(CR
8R
9)
P-2-C (G) N (R
10) O-, or-N (R
12) C (G)-(CR
8R
9)
P-2-N (R
11) O-,
Wherein p is 2,3 or 4, preferably 3 or 4 and most preferably be 4, and R simultaneously
7, R
8, R
9, R
10, R
11, R
12Be hydrogen, alkyl, aryl, hydrogen preferably, in another preferred embodiment, X is-N (R
12) C (G)-(CR
8R
9)
P-3-G-C (G) N (R
10) O-, wherein p is 5, simultaneously R
7, R
8, R
9, R
10, R
11, R
12Be hydrogen, alkyl, aryl, preferably hydrogen.
About above-mentioned radicals X, preferably poly alkylene glycol with and the situation of derivant in, also be preferably as follows structure
-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5，
Wherein r=1 and X=-(CR
8R
9)
pO-,-N (R
12) C (G)-(CR
8R
9)
P-2-S-or-(CR
8R
9)
pS-is wherein at-N (R
12) C (G)-(CR
8R
9)
P-2In the situation of-S-, p is 2 to 20, preferably 2 to 10, and more preferably be 2 to 5, most preferably be 2 to 4.In these situations, also more preferably be n=0 and o=2 to 10, preferably 2 to 8 and more preferably be 2 to 6 as 2 or 4 or 6.
The polymer of functionalization of the present invention can be used as the precursor of preparation polymer-connexon-protein conjugate.
In the context of the present invention, term alkyl and aryl have following implication:
" alkyl " is straight chain, side chain or cyclic being substituted or unsubstituted C1 to C20, preferred C1 to C9, more preferably C1 to C4 alkyl.This alkyl can not be substituted or replaced by aryl, halogen, nitro, ether, alkoxyl, amino or carboxyl.This alkyl is preferably unsubstituted.In addition, the non-conterminous carbon atom of one or two of this alkyl hetero atom that can be selected from O, S and N replaces.Said hetero atom can randomly be replaced by hydrogen, above-mentioned alkyl or aryl according to its quantivalence.Preferred alkyl be methyl, ethyl, different-propyl group, just-propyl group, different-butyl, just-butyl, the second month in a season-butyl and the tert-butyl group.
" aryl " be the C6-aryl preferably.This aryl can not be substituted or replaced by above-mentioned straight chain, side chain or cyclic alkyl.This aryl can also comprise one or more hetero atoms in ring, said hetero atom preferably is selected from N, O and S.This aryl is phenyl or toluyl preferably." polymer "
" polymer " that suit is polysaccharide, is preferably selected from starch, cellulose, glucosan, arabic gum, xanthan gum, inulin, Ficus elastica, pectin, guar gum, Tragacanth, agar, algin, karaya, carrageenin, scleroglycan, Furcellaran, arabinogalactan and locust bean gum.In the context of the present invention, " polysaccharide " refer to above-mentioned polysaccharide with and derivant.The derivant of above-mentioned polysaccharide and preparation thereof and above-mentioned polysaccharide all are that those skilled in the art are well-known.Preferred polysaccharide is selected from starch, preferably hydroxyalkyl starch (HAS) and glucosan.
In the context of the present invention, term " hydroxyalkyl starch " (HAS) refers to the starch derivatives that has been replaced by at least one hydroxy alkyl.The preferred hydroxyalkyl starch of the present invention has the composition of formula (VI)
Wherein the reducing end under neutral of starch molecule illustrates with unoxidized form and terminal sugar unit illustrates with the form of hemiacetal, according to for example used solvent, this hemiacetal form can and the aldehyde form between have balance.Used in the context of the present invention abbreviation HAS ' refers to does not have the HAS of terminal sugar unit molecule on the reducing end under neutral of HAS molecule.
Used term hydroxyalkyl starch is not limited in for the sake of brevity and with wherein carbohydrate partly comprise hydroxy alkyl R, R ' and/or the R chemical compound of " chemical compound, but also refer to wherein the middle existence of remainder HAS ' at least one hydroxy alkyl R, R ' or R of carbohydrate part and/or starch molecule endways " endways of formula (VI) expression among the present invention.The hydroxyalkyl starch that comprises two or more different hydroxy alkyls also is suitable for.
At least one hydroxy alkyl that is comprised in HAS can comprise two or more hydroxyls.According to an embodiment preferred, at least one hydroxy alkyl that HAS comprised comprises a hydroxyl.
The statement of " hydroxyalkyl starch " also comprises wherein alkyl coverlet-or polysubstituted derivant.In this article, this alkyl is preferably by halogen, especially fluorine or replaced by aryl.In addition, the hydroxyl of this hydroxy alkyl can esterified or etherificate.
In addition, can also with straight or branched be substituted or unsubstituted alkylene replaces alkyl.
Hydroxyalkyl starch is the ether derivant of starch.Except that said ether derivant, can also use other starch derivatives in the context of the present invention.For example, can use and comprise esterified hydroxy derivative.These derivants can be list-or the derivants of dicarboxylic acids or its substituted derivant of for example unsubstituted 2-12 of having carbon atom.What be particularly useful is the derivant of derivant, especially acetic acid of the monocarboxylic acid of the unsubstituted 2-6 of a having carbon atom.In this article, preferred acetyl starch, butyl starch and propyl group starch.
In addition, the derivant that also preferably has the unsubstituted dicarboxylic acids of 2-6 carbon atom.
In the situation of the derivant of dicarboxylic acids, can also carry out esterification to second carboxyl of this dicarboxylic acids.In addition, in the context of the present invention, the derivant of dicarboxylic acids mono alkyl ester also is suitable for.
For the list that replaces-or dicarboxylic acids for, described substituent group preferably with the top identical substituent group of mentioning about the alkyl residue that replaces of substituent group.
The technology of starch being carried out esterification is known (referring to people such as for example Klemm D. in the prior art, Comprehensive Cellulose Chemistry, the 2nd volume, 1998, Whiley-VCH, Weinheim, New York, especially the 4.4th chapter, cellulosic esterification (Esterification of Cellulose) (ISBN 3-527-29489-9).
According to the preferred embodiment of the invention, use the hydroxyalkyl starch of above-mentioned formula (VI).In formula (VI), sugar ring that clearly is described and the residue that is represented as HAS ' are represented this preferred hydroxyalkyl starch molecule together.Other the sugared ring structure that is contained among the HAS ' can be identical or different with this sugar ring that clearly is described.
Residue R, R ' and R in the formula (VI) " there is not specific restriction.According to an embodiment preferred, R, R ' and R " is hydrogen or hydroxy alkyl, hydroxyaryl, hydroxyl aralkyl or the hydroxyl alkaryl that has 2 to 10 carbon atoms in alkyl residue separately independently.Preferred hydrogen and hydroxy alkyl with 2 to 10 carbon atoms.This hydroxy alkyl more preferably has 2 to 6 carbon atoms, more preferably has 2 to 4 carbon atoms and more preferably has 2 carbon atoms.Therefore, " hydroxyalkyl starch " preferably includes hetastarch, hydroxypropyl starch and hydroxyl butyl starch, wherein preferred especially hetastarch and hydroxypropyl starch, and hetastarch most preferably.
Said alkyl, aryl, aralkyl and/or alkaryl can be straight or branched and can suitably be replaced.
Therefore, R, R ' and R " preferably hydroxyl hexyl, hydroxyl amyl group, hydroxybutyl, hydroxypropyl such as 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxyl isopropyl, hydroxyethyl such as 2-hydroxyethyl, hydrogen and especially preferred 2-hydroxyethyl.
For all embodiments of the present invention who relates to starch, hetastarch (HES) most preferably.
Hetastarch (HES) is the derivant of naturally occurring amylopectin, and can be degraded by intravital α-Dian Fenmei.HES is the derivant of the replacement of carbohydrate polymer amylopectin, and described amylopectin is present in the corn starch with the concentration up to 95% weight.HES shows favourable biological property, and can be used as the blood volume substituting agent and be used for clinical hemodilution therapy (people such as Sommermeyer, 1987, Krankenhauspharmazie, 8 (8), 271-278; With people such as Weidler, 1991, Arzneim.-Forschung/Drug Res., 41,494-498).
Amylopectin is made up of glucose, wherein has α-1 in main chain, 4-glycosidic bond and can find α-1, the 6-glycosidic bond at component.The physical-chemical property of this molecule mainly is by the type decided of glycosidic bond.Because this nicked (nicked) α-1, the 4-glycosidic bond has produced the helicoidal structure that every circle has about six glucose monomers.Can be by replacing physical-chemical and the biochemical property that changes this polymer.Can introduce hydroxyethyl by alkaline hydroxyethylation.For hydroxyethylation,, can utilize the different reactivity of each hydroxyl in the unsubstituted glucose monomer by changing reaction condition.Because this fact, those of ordinary skill can influence substitute mode with limited degree.
The feature of HES is mainly molecular weight distribution and substitution value.Two kinds of possibilities when being described, substitution value are arranged:
1. can use for all glucose moieties, the shared ratio of substituted glucose monomer comes substitution value is described.
2. can come substitution value is described with the molar substitution form, wherein the ethoxy number to each glucose moiety be described.
In the context of the present invention, the substitution value that is represented as DS refers to molar substitution described above.
HES solution is that the form with heterogeneous dispersive composition exists, and wherein each molecule is differing from one another aspect the number of the degree of polymerization, branch part and pattern and the substitute mode.Therefore, HES is the mixture with chemical compound of different molecular weight.So specific HES solution is to be determined by the mean molecule quantity that obtains by statistical method.In this article, M
nBe calculated as the form of the arithmetic mean of instantaneous value that depends on molecular amounts.As the displaced selection of a kind of confession, M
w(or MW)---weight average molecular weight is represented the unit of the quality of a kind of HES of depending on.
In the context of the present invention, hetastarch preferably has 1 to 300kD mean molecule quantity (weight average).With regard to hydroxyethyl, hetastarch can also show 0.1 to 0.8 preferred molar substitution and 2 to 20 C
2: C
6Substitute proportion.
Used in the context of the present invention term " mean molecule quantity " refers to according to people such as Sommermeyer, and 1987, Krankenhauspharmazie, 8 (8), 271-278; With people such as Weidler, 1991, Arzniem.-Forschung/Drug Res., 41, the determined weight of 494-498.
Embodiment preferred according to the present invention, the mean molecule quantity of used hetastarch are 1 to 300kD, and more preferably 2 to 200kD, and more preferably 4 to 130kD, and more preferably 4 to 70kD.
The example of HES with mean molecule quantity of about 130kD is the Voluven that derives from Fresenius.Voluven is a kind of displaced artificial colloid of capacity used in the treatment indication of hypovolaemia that is used to for example treat and prevents.The mean molecule quantity that is characterized as of Voluven is 130,000+/-20, and 000D, molar substitution are 0.4, C2: C6 ratio is about 9: 1.
Therefore, the invention still further relates to a kind of aforesaid method and conjugate, wherein said hydroxyalkyl starch is that mean molecule quantity is 4 to 70kD hetastarch.
Preferred average molecular weight range is for for example 4 to 70kD or 10 to 70kD or 12 to 70kD or 18 to 70kD or 50 to 70kD or 4 to 50kD or 10 to 50kD or 12 to 50kD or 18 to 50kD or 4 to 18kD or 10 to 18kD or 12 to 18kD or 4 to 12kD or 10 to 12kD or 4 to 10kD.
The particularly preferred embodiment according to the present invention, the mean molecule quantity of used hetastarch is being higher than 4kD and is being lower than in the scope of 70kD, and 10kD according to appointment perhaps is 9 to 10kD or 10 to 11kD or 9 to 11kD; Perhaps about 12kD perhaps is 11 to 12kD or 12 to 13kD or 11 to 13kD; Or about 18kD, perhaps be 17 to 18kD or 18 to 19kD or 17 to 19kD; Or about 50kD, perhaps be 49 to 50kD or 50 to 51kD or 49 to 51kD.
With regard to related substitution value (DS), DS preferably is at least 0.1, and more preferably at least 0.2, more preferably at least 0.4 and more preferably at least 0.7.The preferable range of DS is 0.1 to 0.8, more preferably 0.2 to 0.8, more preferably 0.3 to 0.8 and more preferably 0.4 to 0.8, also more preferably 0.1 to 0.7, more preferably 0.2 to 0.7, more preferably 0.3 to 0.7 and more preferably 0.4 to 0.7.Particularly preferred DS value is for example 0.1,0.2,0.3,0.4,0.5,0.6,0.7 or 0.8, more preferably 0.2,0.3,0.4,0.5,0.6,0.7 or 0.8, more preferably 0.3,0.4,0.5,0.6,0.7 or 0.8, also more preferably 0.4,0.5,0.6,0.7 or 0.8, and for example preferred especially 0.4 and 0.7.
The molecular weight of hydroxyalkyl starch, preferred hetastarch with and the particularly preferred combination of substitution value DS be 10kD and 0.4 or 10kD and 0.7 or 12kD and 0.4 or 12kD and 0.7 or 18kD and 0.4 or 18kD and 0.7 or 50kD and 0.4 or 50kD and 0.7 for example.
In another preferred embodiment, the molecular weight of hydroxyalkyl starch, preferred hetastarch with and the combination of substitution value DS be 30kD and 0.7 or 30kD and 0.4.
The molecular weight of hydroxyalkyl starch, preferred hetastarch with and the another kind of preferred combination of substitution value DS be 10kD and 0.8,12kD and 0.8,18kD and 0.8,30kD and 0.8 and 50kD and 0.8.
With regard to C
2: C
6The ratio that replaces, said replacement is preferably 2 to 20, and more preferably 2 to 15 and more preferably 3 to 12.
According to another embodiment of the invention, can also use and have different mean molecule quantities and/or different substitution value and/or different C
2: C
6The mixture of the hetastarch of substitute proportion.Therefore, can use and have different mean molecule quantities and different substitution values and different C
2: C
6Substitute proportion or have different mean molecule quantities and different substitution value and identical or about identical C
2: C
6Substitute proportion or have different mean molecule quantities and identical or approximately identical substitution value and different C
2: C
6Substitute proportion or have identical or about identical mean molecule quantity and different substitution values and different C
2: C
6Substitute proportion or have different mean molecule quantities and identical or approximately identical substitution value and identical or about identical C
2: C
6Substitute proportion or have identical or approximately identical mean molecule quantity and different substitution value and identical or about identical C
2: C
6Substitute proportion or have identical or approximately identical mean molecule quantity and identical or approximately identical substitution value and different C
2: C
6Substitute proportion or have approximately identical mean molecule quantity and approximately identical substitution value and about identical C
2: C
6The hetastarch mixture of substitute proportion.
In the polymer and/or conjugate method of the different functionalization of the present invention, can use different hydroxyalkyl starchs, preferred different hetastarch and/or different hydroxyalkyl starch mixture preferably use different hetastarch mixture.
The glucosan that comprising the main D-glucose unit main chain that links to each other with other 1,3 branch point by α-D (1,6) key shown in the formula (VII) is mainly by antibacterial or synthesize and make.
The molecular weight of this glucosan is preferably 4kD to 300kD, more preferably 5kD to 100kD and most preferably be 6kD to 40kD.Its molecular weight is to measure with the GPC that uses the suitable molecular weight standard product that can obtain by commercial sources.
Other suitable polymer blend is to be selected from the aklylene glycol homopolymer, preferred ethylene glycol homopolymer (PEG) and propylene glycol homopolymer, aklylene glycol copolymer, preferred oxypropylene/oxyethylene copolymers, polyvinyl alcohol, polyvinylpyrrolidone, poly--1,3-dioxolanes, poly--1,3, the solubility straight or branched polymer of 6-trioxane, ethylene/copolymer-maleic anhydride and polyamino acid (homopolymer or random copolymer) with and derivant.In the context of the present invention, can use above-mentioned polymer with and derivant.Above-mentioned polymer and the preparation with and derivant all well known to a person skilled in the art.
Preferred polymer is the aklylene glycol homopolymer, preferred ethylene glycol homopolymer (PEG) and propylene glycol homopolymer.More preferably ethylene glycol homopolymer (PEG).
PEG is the PTMEG of formula (VIII)
HO-(CH
2CH
2O)
t-CH
2CH
2-OH (VIII)，
Wherein t is-(CH
2CH
2O)-number of group.It can be with various molecular weight and low dispersion (M
w/ M
n≤ 1.1, M wherein
wBe weight average molecular weight and M
nBe number-average molecular weight) form obtain by commercial sources.Though this polyether backbone chemically is being very inert, its primary hydroxyl can carry out derivatization.The molecular weight that is used to prepare the PEG of conjugate biology can change between 1000 to 20000Da, but in some cases, also can use the polymer with the molecular weight that is higher or lower than this scope.This molecular weight is to measure with the GPC that uses the suitable molecular weight standard product that can obtain by commercial sources.What preferably use is the monoalky lether of PEG, and wherein said alkyl is selected from C1 to C4 alkyl.More preferably use the monomethyl ether (mPEG) of PEG.But the end group of the unique derivatization that exists on other monoalky lether of mPEG or PEG has dropped to minimum level with crosslinked probability and has improved the homogeneity of prepared conjugate.
Except that the monoalky lether of PEG, preferred mPEG, can also use the PEG derivant of other functionalization in the present invention.The sulphonic acid ester, amino-PEG, diazanyl-PEG, sulfydryl-PEG, carboxyl-PEG that suitable example has halo derivatives, the PEG of PEG with and active ester, aldehyde-PEG, cyanuryl chloride-PEG and epoxide-PEG.At S.Zalipsky, Bioconj ugate Chem.1995,6, the preparation of said derivative and other derivant and preparation thereof are disclosed among the 150-165.In addition, some PEG derivants can obtain by commercial sources.
The functional group derivant of PEG generally prepares in the following way: (i) hydroxyl is directly changed into new target functional group, (ii) this polymer and difunctional molecule are reacted, thereby make functional group form and being connected of this polymer, and another still can further carry out chemical conversion.
More preferably use the monoalky lether of PEG in the present invention, preferred mPEG.
Radicals X in the polymer of the functionalization of formula I is a kind of linking group, and it is selected from-(CR
8R
9)
pO-,-(CR
8R
9)
pS-,-(CR
8R
9)
pNR
6-,-(CR
8R
9)
pOC (O)-,-(CR
8R
9)
pC (O) O-,-(CR
8R
9)
pC (G) N (R
10) O-,-(CR
8R
9)
pN (R
11) O-and
Wherein one or more-(CR
8R
9)-group can be replaced by W, thereby forms a kind of chemically rational group.
W is O, NR
12, C (G), preferred O, C (G), and G is S, O, NR
14, preferred O.
R in the polymer of the functionalization of formula I
6, R
7, R
8, R
9, R
10, R
11, R
12, R
14Be hydrogen, alkyl, aryl, preferably hydrogen independently.
P is 0 to 20, preferably 0 to 10, more preferably be 0 to 5, most preferably be 0 to 4, polysaccharide with and the situation of derivant in more preferably be 0, and poly alkylene glycol and
In the situation of its derivant more preferably 1 to 4, p group CR wherein
8R
9In residue R
8And R
9Can be identical or different.
X preferably
-(CR
8R
9)
pC (G) N (R
10) O-and-(CR
8R
9)
pN (R
11) O-,
More preferably be
-(CR
8R
9)
pN(R
11)O-，
Still more preferably be
When said polymer was the poly alkylene glycol or derivatives thereof, the preferred structure of linking group X was
V=1 to 10, preferred 1 to 4, more preferably 2 or 3.
Most preferably be
Wherein v is 1 to 10, and is preferred 1 to 4, more preferably 2 to 3.
With regard to above-mentioned radicals X, poly alkylene glycol with and the situation of derivant in, the linking group X that also preferably has following structure:
Wherein p is 2,3 or 4, preferred 3 or 4 and most preferably be 4, and R simultaneously
7, R
8, R
9, R
10, R
11, R
12Be hydrogen, alkyl, aryl, hydrogen preferably, in another preferred embodiment, X is-N (R
12) C (G)-(CR
8R
9)
P-3-G-C (G) N (R
10) O-, wherein p is 5, simultaneously R
7, R
8, R
9, R
10, R
11, R
12Be hydrogen, alkyl, aryl, preferably hydrogen.
More preferably Xia Mian structure:
V=1 to 10, preferred 1 to 4, more preferably 2 or 3.
Most preferably
Wherein v is 1 to 10, and is preferred 1 to 4, more preferably 2 to 3.
With regard to above-mentioned radicals X, poly alkylene glycol with and the situation of derivant in, preferred group also
-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5，
Wherein r=1 and X=-(CR
8R
9)
pO-or-(CR
8R
9)
pS-, wherein one or more groups-(CR
8R
9)-can be replaced by W, thus form a kind of at rational group chemically,
W is O, NR
12, C (G), preferably O, C (G) and G are S, O, NR
14, O preferably;
R
8, R
9And R
12Be hydrogen, alkyl, aryl, preferably hydrogen independently; And p is 2,3 or 4.
In these situations, the linking group X of especially preferred following structure
When polymer is a polysaccharide, when being preferably selected from above-mentioned glucosan or derivatives thereof or starch or derivatives thereof, X most preferably is
Or-N (H) O-.
Group-[(CR at formula I
1R
2)
mO]
n[CR
3R
4]
o-in, R
1, R
2, R
3And R
4Be hydrogen, above-mentioned alkyl or aryl independently, preferably methyl or hydrogen more preferably are hydrogen.M is 2 to 4, preferably 2, and m group CR wherein
1R
2In residue R
1And R
2Can be identical or different.-(CR
1R
2)
m-preferably-CH
2CH
2-or-CH (CH
3) CH
2-or-CH
2CH (CH
3)-,-(CR
1R
2)
m-more preferably be-CH
2CH
2-.
N is 0 to 20, preferably 0 to 10, and more preferably be 1 to 5, most preferably be 1 or 2 and more preferably be 1.
O is 0 to 20, preferably 0 to 10, and more preferably be 0 or 2, wherein in the situation of n=0, o is not 0, in a preferred embodiment, o is 2 to 20, preferably 2 to 10, more preferably be 2, o group CR wherein
3R
4In residue R
3And R
4Can be identical or different.
In the most preferred embodiment of the present invention ,-[(CR
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2OCH
2CH
2-.
In another embodiment preferred of the present invention ,-[(CR
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2CH (CH
3) CH
2CH
2-.
R among the formula I
5Be hydrogen, alkyl as defined above, aryl, preferably hydrogen.
Therefore, in the most preferred embodiment of the present invention, the group among the formula I-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5Be-CH
2CH
2OCH
2CH
2-ONH
2
Therefore, in another the most preferred embodiment of the present invention, the group among the formula I-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5Be-CH
2CH
2CH (CH
3) CH
2-ONH
2
Group-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5Being positioned at intermediary group covalency with at least one end group of described " polymer " or at least one links to each other.
Generally speaking, according to used polymer, this is covalently bound can to use any suitable group in this polymer.Most preferably by wrapping oxygen containing group in this polymer with group
-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5
Be connected with used polymer, said polymer is preferably selected from starch, glucosan and poly alkylene glycol or derivatives thereof, more preferably be hydroxyalkyl starch, the monomethyl ether (mPEG) of wherein preferred hetastarch (HES), glucosan and Polyethylene Glycol.The oxygen containing group of said bag can be a carbonyl, and preferably ketone group, hemiacetal group or aldehyde radical more preferably are hemiacetal group or aldehyde radical.In the situation of poly alkylene glycol, this wraps oxygen containing group can also be group OR " ; for example carboxylate or carbonic ester; it is to be produced by the reaction of poly alkylene glycol or derivatives thereof with alcohol; wherein preferred alcohol is selected from N-hydroxy-succinamide class such as N-hydroxy-succinamide or sulfo group-N-hydroxy-succinamide; the phenol such as the right-nitrophenol of the replacement that suit; neighbour, right-dinitrophenol,DNP, adjacent, adjacent '-dinitrophenol,DNP, trichlorophenol, 2,4,6,-T is as 2,4,6-trichlorophenol, 2,4,6,-T or 2,4, the 5-trichlorophenol, 2,4,6,-T, trifluoromethyl phenol is as 2,4,6-trifluoromethyl phenol or 2,4, the 5-trifluoromethyl phenol, pentachlorophenol, Pentafluorophenol, or hydroxyl azole compounds such as hydroxybenzotriazole.Especially preferred is the N-hydroxy-succinamide class, especially preferred N-hydroxy-succinamide and sulfo group-N-hydroxy-succinamide.But, can also realize and being connected of radicals X by other group except that the oxygen containing group of above-mentioned bag.Will be right hereinafter
-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5
Go through with being connected of this polymer.
The polymer of the functionalization of formula I is suitable for preparing and proteinic stable conjugate.
Therefore, in another preferred embodiment, the present invention relates to the conjugate of formula II,
Wherein symbol " polymer ", R
1, R
2, R
3, R
4, m, n, o, r, X and p have with aforementioned and decide the identical implication of implication, and
R
13Be hydrogen, alkyl, aryl, preferably hydrogen or methyl,
" protein " is by at least two aminoacid are reacted prepared aminoacid sequence, wherein in the conjugate of formula II, and group
To be positioned at intermediary group covalently bound with at least one end group of " polymer " and " protein " or at least one, preferably covalently bound with the oxidized-terminal amino acid of " protein " or oxidized carbohydrate side chain.
" protein "
Used term " protein " relates to and has at least 2 in the context of the present invention, preferably at least 5, more preferably at least 10, more preferably at least 15, more preferably at least 20, more preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45 and more preferably at least 50 amino acid whose any aminoacid sequences.
Described protein can be make with chemosynthesis operation or can derive from anyone or other mammal and can obtain by carrying out purification by natural origin.
According to the present invention, described protein can be somatomedin, cytokine, activator, inhibitor, enzyme, antibody, antigen, transport protein, Biosilica-adhesive protein, hormone, receptor, inhibitive factor or its functional deriv or segment.Used in the context of the present invention term " functional deriv or segment " refers to and can keep the required biological property of starting molecule or active derivant or segment wholly or in part, for example it can keep the required biological property of starting molecule or active at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80% and especially preferably at least 90%.The particularly preferred example of such segment is an antibody fragment for example.
Proteinic example has the people EPO (rhEPO) of erythropoietin (EPO) as reorganization, colony stimulating factor (CSF), the human G-CSF (rhG-CSF) of for example recombinating as G-CSF, alpha-interferon (IFN α), beta-interferon (IFN β) or gamma interferon (IFN γ), the people IFN α or the IFN β (rhIFN α or rhIFN β) that for example recombinate as IFN α and IFN β, interleukin, the human IL-2 or the IL-3 (rhIL-2 or rhIL-3) that for example recombinate as IL-2 or IL-3 of IL-1 to IL-18 for example, serum albumin such as prothrombin-XIII be the VIII factor for example, the VII factor, the IX factor, alpha antitrypsin (A1AT), activated PROTEIN C (APC), activator of plasminogen such as tissue plasminogen activator (tPA), as human tissue plasmin activator (hTPA), AT III is as the people AT III (rhATIII) of reorganization, Myoglobin, albumin such as bovine serum albumin (BSA), somatomedin is as epidermal growth factor (EGF), PDGF (PDGF), fibroblast growth factor (FGF), brain derived somatomedin (BDGF), nerve growth factor (NGF), B-cell growth factor (BCGF), brain derived neurotrophic growth factor (BDNF), ciliary neurotrophic factor (CNTF), transforming growth factor such as TGF α or TGF β, BMP (bone form formation property albumen), growth hormone such as human growth hormone, tumor necrosis factor such as TNF α or TNF β, somatostatin, growth hormone, somatomedin, hemoglobin, hormone or prohormone such as insulin, promoting sexual gland hormone, melanotropin (α-MSH), triptorelin, hypothalamic hormone such as vassopressin (ADH and oxytocin and releasing hormone and release inhibting hormone, parathyroid hormone, thyroxin such as thyroxine, thyrotropin, thyroliberin, prolactin antagonist, calcitonin, glucagon, glucagon-like peptide (GLP-1, GLP-2 or the like), exendines such as exendin-4, leptin, vassopressin, gastrin, secretin, integrin, glycoprotein hormones (LH for example, FSH or the like), short melanoside hormone, lipoprotein and apolipoprotein such as apo-B, apo-E, apo-La, immunoglobulin such as IgG, IgE, IgM, IgA, IgD with and segment, hirudin, the institutional approach inhibitor, vegetable protein such as phytohemagglutinin or Ricin, Venenum apis, snake venom, immunotoxin, E antigen, α-protease inhibitor, the artemisiifolia allergen, melanin (melanin), low polylysine albumen, the optional corresponding receptor of one of RGD albumen or these albumen; Or the functional deriv of any of these albumen or receptor or segment.
Preferred enzyme has for example carbohydrate specific enzyme, proteolytic enzyme, oxidase, oxidoreductase, transferring enzyme, hydrolytic enzyme, lyase, isomerase, kinases and ligase.Concrete limiting examples has asparaginase, arginase, the arginine deaminase, ADA Adenosine deaminase, glutaminase, glutaminase-asparaginase, phenylalanine, E.C. 4.1.99.1, tryrosinase, superoxide dismutase (SOD), endotoxin enzyme (endotoxinase), catalase, peroxidase, kallekrein, trypsin, Chymotrypsin, elastoser, thermolysin, lipase, uricase, adenosine deaminase, purine nucleotide phosphorylase, Bilirubin oxidase, glucoseoxidase, glucodase, the gluconate oxidase, tilactase, the glucocerebroside esterase, glycuronidase, hyaluronidase, tissue factor, streptokinase, urokinase, the MAP-kinases, the DNA enzyme, the RNA enzyme, lactoferrin with and functional deriv and segment.
Embodiment preferred according to the present invention, described protein is selected from EPO, G-CSF, the VII factor, the IX factor, IFN β, AT III, A1AT, the VIII factor, APC.
Compare with the protein before engaging, conjugate of the present invention can improve the used proteic body-internal-circulation phase and reduce described proteinic antigenicity and immunogenicity.
In another embodiment, the present invention relates to a kind of method for preparing the polymer of functionalization, it comprises the polymer with formula III
" polymer "-(CR
8R
9)
p-Y (III)
React with the chemical compound of formula IV,
Q-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5 (IV)，
Wherein symbol " polymer ", R
1, R
2, R
3, R
4, R
5, m, n, o the has implication identical with aforementioned implication, and
Thereby Y and Q be react each other the functional group that provides one of following linking group :-O-,-S-,-NR
6-,-OC (O)-,-C (O) O-,-C (G) N (R
10) O-,-N (R
11) O-,
Preferably
Wherein exist
Situation in, group-(CR
8R
9)-be
First-selected
Or-N (R
11) O-,
In another preferred embodiment, thus Y and Q be react each other provide linking group-O-or-functional group of S-;
Wherein in the polymer of formula III, one or more groups-(CR
8R
9)-can be replaced by W, thus a kind of chemically rational group formed;
W is O, NR
12, C (G), preferably O, C (G);
G is S, O, NR
14, O preferably;
R
6、R
7、R
8、
R
9、R
10、
R
11, R
12, R
14Be hydrogen, alkyl, aryl independently, hydrogen preferably,
P is 0 to 20, preferably 0 to 10, more preferably be 0 to 5, most preferably be 0 to 4, polysaccharide with and the situation of derivant in more preferably be 0, poly alkylene glycol with and the situation of derivant in more preferably 1 to 4, p group CR wherein
8R
9In residue R
8And R
9Can be identical or different;
Group wherein
-(CR
8R
9)
pY
With the end group of " polymer " or to be positioned at intermediary group covalently bound.
Wherein, Shi Yi group Y is following functional group:
The two keys of-C-C-or C-C-triple bond or aromatics C-C-key;
-sulfydryl or hydroxyl;
-alkyl sulfonic acid hydrazides, aryl sulfonic acid hydrazides;
-1,2-glycol (dioles);
-1,2-amino-sulfur alcohol;
-azide;
-1, the 2-amino alcohol;
-amino-NH
2Or comprise derivant such as aminoalkyl, aminoaryl, amino aralkyl or the alkaryl amino of the amino of construction unit-NH-;
-hydroxyl amino-O-NH
2Or comprise the derivant of the hydroxyl amino of construction unit-O-NH-, as hydroxy alkyl amino, hydroxyaryl amino, hydroxyl aralkyl amino or hydroxyl alkaryl amino;
-alkoxy amino, aryloxy group amino, aralkoxy amino or aryloxy alkyl amino, it all comprises construction unit-NH-O-;
-have carbonyl residue-Q '-C (=G)-M, wherein G is O or S, M for example is,
---OH or-SH;
--alkoxyl, aryloxy group, aralkoxy or aryloxy alkyl;
--alkylthio group, arylthio, aromatic alkylthio or alkane arylthio;
--alkyl carbonyl oxy, aryl-carbonyl oxygen, aralkyl carbonyl oxygen base or alkaryl carbonyl oxygen base;
--active ester as the ester of azanol such as N-hydroxy-succinamide with imine structure or azanol with construction unit O-N (wherein N is the part of heteroaryl compound) or, there are not time as aryloxy compound at G=O and Q ' with substituted aromatic yl residue such as pentafluorophenyl group, p-nitrophenyl or trichlorophenyl;
Wherein Q ' does not exist or NH or hetero atom such as S or O;
--NH-NH
2Or-NH-NH-;
--NO
2；
-itrile group (nitril);
-carbonyl such as aldehyde radical or ketone group perhaps can be converted to the group of aldehyde radical or ketone group, for example acetal or hemiacetal group or ketal or hemiketal base;
-carboxyl or ester group;
--N=C=O base or-the N=C=S base;
-ethylene halide base such as iodoethylene or bromo vinyl or triflate;
--C≡C-H；
--(C=NH
2Cl)-the O alkyl
-group-(C=O)-CH
2-Hal, wherein Hal is Cl, Br or I;
--CH＝CH-SO
2-；
-comprise-the disulphide group of S-S-structure;
-group
-group
Another kind of suitable group Y is a maleimide base group.
Preferred group Y is the functional group that comprises carbonyl, preferably comprises aldehyde radical, ketone group, carboxyl, ester group, halogen group, preferred Br or I or Otf (=O-SO
2CF
3), maybe can be converted to aldehyde radical or ketone group, for example the group of acetal or hemiacetal group or ketal or hemiketal base.
Also preferred group Y be residue-C with carbonyl (=G)-M, wherein G is O, M is an active ester as the hydroxylamine with imine structure such as the ester of N-hydroxy-succinamide.
Be polysaccharide at polymer, be preferably selected from starch with and derivant and glucosan with and the situation of derivant in, Y is aldehyde radical or acetal or hemiacetal group preferably.In this case, p preferably 0.
At polymer is in the situation of poly alkylene glycol such as Polyethylene Glycol, and Y is aldehyde radical or ester group preferably, preferably reactive ester group, as have the hydroxylamine of imine structure such as N-hydroxy-succinamide ester, hydroxyl (OH) or sulfydryl (SH).More preferably, be in the situation of hydroxyl or sulfydryl at Y, Q is a halogen group, preferably Br or I, or OTf base.At wherein X=-(CR of the present invention
8R
9)
pO-or-(CR
8R
9)
pIn the embodiment of S-, functional group-ONHR that the chemical compound of the formula (IV) that the used polymer with formula (III) reacts can have protected form
5, (III) with (IV) react after it can be gone protection.With regard to blocking group, that can mention has a phthalimide protecting group, and it can be removed by (III) and product (IV) and hydrazine are reacted, thereby obtains functional end-group-ONHR
5But, go the protection after can produce-ONHR
5Various suitable protection all can use.
The polymer of formula III
" polymer "-(CR
8R
9)
pThe available method known to those skilled in the art of-Y (III) makes.
If use the monomethyl ether (mPEG) of poly alkylene glycol, preferred Polyethylene Glycol (PEG) or PEG, the proper method that then prepares the formula III polymer can be referring to S.Zalipsky, BioconjugateChem.1995,6,150-165.
If use polysaccharide, preferred starch or glucosan or derivatives thereof, then the polymer of formula HI is a polysaccharide, and group Y is aldehyde radical or hemiacetal group or the equilibrated form of the two preferably.
Preferably it is in the reducing end group in the balance of aldehyde and hemiacetal form to aldehyde radical Y in the polysaccharide.If polymer is a polysaccharide, then the p in the polymer of formula III most preferably is 0.The chemical compound of formula IV and polymer wherein are that connection between the polymer of formula III of polysaccharide or derivatives thereof is as shown in flow chart 2.
The chemical compound of formula IV and polymer wherein are that connection between the polymer of formula III of poly alkylene glycol or derivatives thereof is as shown in flow chart 1.
Be used for polysaccharide (" polymer "-(CR of formula III that the chemical compound with formula IV reacts in order to prepare
8R
9)
p-Y), and can be with the polysaccharide oxidation, thus a plurality of aldehyde radicals produced thereon.This oxidation can be finished with multiple oxidizing process, and preferred a kind of process is to react with periodate (sodium or potassium salt).This reaction can be in aqueous solution for example under 0 to 5 ℃, uses the sodium metaperiodate according to the selected Sq of required degree of oxidation to carry out at low temperature.
This reaction can be finished in about 10 minutes to 4 hours.Can remove undesired low-molecular-weight salt and polysaccharide component with ultrafiltration or dialysis, thereby the method for the molecular weight ranges of the oxidized polysaccharide that the chemical compound of a kind of control and formula IV reacts is provided.Can directly use this oxidized polysaccharide or suitably it is reclaimed, for example it be reclaimed, then it be dissolved again being used for and react with the chemical compound of formula IV by lyophilization.
In a word, preferably use the polysaccharide or derivatives thereof, and preferred polysaccharide is the above-mentioned polysaccharide of not handling by oxidation.The present inventor's discovery, needn't be handled polysaccharide during the chemical compound of especially above-mentioned preferred formula IV in advance when the chemical compound of use formula IV.
Suitable group Q also can be above-mentioned functional group, and wherein Y of functional group and Q select in the mode that can obtain one of following radicals :-O-,-S-,-NR
6-,-OC (O)-,-C (O) O-,-C (G) N (R
10) O-,-N (R
11) O-,
Q is H preferably
2N-O-.
Therefore, the group Y of formula III polymer preferably can with group-ONH
2The group of reaction.Therefore, preferred group Y is aldehyde radical, ketone group, carboxyl, carbonate group and activatory carboxyl, for example ester group, lactone group and amide groups.Other suitable group is halogenide and pseudohalide group or the like, for example Cl, Br, I and OTf.Y most preferably is aldehyde radical (being in the balance that is in the hemiacetal form in the situation of polysaccharide).In another preferred embodiment, Y is an active ester groups as the hydroxylamine with imine structure such as the ester of N-hydroxy-succinamide.
If Q is H
2N-O-, the then chemical compound of formula IV
Q-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5 (IV)
Most preferably be
According to another embodiment of the invention, if Q is H
2N-O-then also preferably has structure
The chemical compound of formula (IV).
As having two-O-NH
2The examples for compounds of base, the chemical compound that can mention also has
If Q is a halogen group, preferably Br or I or OTf group most preferably are Br or I, then the chemical compound of formula IV
Q-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5 (IV)
Most preferably be
Wherein o=2 to 10 more preferably is 2 to 8 and especially preferably 2 to 6 as 2,4 or 6, and wherein-O-NH
2Can exist as the form of being protected by the phthalimide blocking group with its protected form, thereby the chemical compound of the formula of making (IV) can be
Wherein o is that 2-6 and Q are people such as the preparation of chemical compound of aforesaid structural formula (IV) of Br such as Bauer, and J.Org.Chem.1963 is described in 28, the 1604 pages.The chemical compound that can prepare other formula (IV) with similar methods with structure as implied above.
Therefore, in another embodiment, the present invention relates to a kind of aforesaid method, the chemical compound of its Chinese style IV is
Therefore, still in another embodiment, the present invention relates to a kind of aforesaid method, the chemical compound of its Chinese style IV is
Therefore, still in another embodiment, the present invention relates to a kind of aforesaid method, the chemical compound of its Chinese style IV is
Wherein o=2 to 10 more preferably is 2 to 8 and especially preferably 2 to 6 as 2,4 or 6, and wherein-O-NH
2Can exist with its protected form.
The chemical compound of formula IV can be prepared with method known to those skilled in the art.
For example, people such as D.Boturyn, Tetrahedron 53 (1997) 5485-5492 disclose
Preparation.Can obtain the chemical compound of other formula IV with similar method.
For example, with regard to chemical compound
Synthetic, can be with reference to embodiment hereinafter.
Prepare in the preferred embodiment of method of polymer of functionalization in the present invention, with the polymer dissolution of formula III in organic solvent for example in dichloromethane, dimethyl formamide or the dimethyl acetylamide, perhaps be dissolved in aqueous systems for example the pH of sodium acetate buffer be 4.5 to 9, in preferred 5 to 8 the aqueous systems.The chemical compound that in the solution of this polymer, adds formula IV.This adds normally at 0 to 80 ℃, preferred 0 to 60 ℃, more preferably carries out under 20 to 40 ℃ the temperature.When this is reflected at when carrying out in the organic solvent, usually under said temperature, this mixture stirred or jolting 1 to 48 hour, preferred 2 to 24 hours, more preferably 2 to 16 hours, and when this is reflected at when carrying out in the aqueous systems, usually with its stirring or jolting 1 to 48 hour, preferred 2 to 24 hours, more preferably 2 to 16 hours.Make the product precipitation by adding product solvent or solvent mixture insoluble therein or that have a low dissolubility.Be used to make the sedimentary suitable solvent of product to depend on the character of product.In one embodiment, by adding alcohol, preferred 2-propanol or ethanol make said product precipitation, and usually with it at-60 to 20 ℃, be incubated under preferred-20 to 20 ℃.In another embodiment, by adding pure and mild low boiling-point and polarity organic solvent, for example the mixture of acetone makes said product precipitation.A kind of The suitable solvent mixture is ethanol and acetone, for example the mixture of ethanol and acetone 1: 1 (volume that shows said solvent equates) and usually with it at-60 to 20 ℃, be incubated under preferred-20 to 20 ℃.The product that collecting precipitation comes out, for example by (being generally 0 to 20 ℃ at low temperature, preferred 0 ℃) following centrifugal the collection, with its suspendible again, preferably carry out suspendible with solvent or solvent mixture, be generally-60 to 20 ℃ with being used to carry out sedimentary alcohol in one embodiment, preferred-20 to 20 ℃ temperature gets off to carry out suspendible, and it is incubated 0.5 to 20 hour under identical temperature, preferred 1 to 3 hour usually.Usually by centrifugal, with product be dissolved in the water, dialysis 12 to 72 hours in water, preferred 15 to 48 hours, more preferably 15 to 25 hours and its lyophilization come further the product of gained is carried out post processing.
In the flow chart below, provided by with the polymer of formula III with the chemical compound of formula IV, preferably
React the example (flow chart 1a and 2) of the method for the polymer for preparing functionalization.
In flow chart 1b, provided by with the polymer of formula III and the chemical compound of formula IV, preferred
React the example of the method for the polymer for preparing functionalization.
Flow chart 1a: poly alkylene glycol (PAG)-derivant
Flow chart 1b: poly alkylene glycol (PAG)-derivant
OR , OR " " and OR " derives from pure H-OR , H-OR " " and H-OR " wherein, said alcohol is preferably selected from N-hydroxy-succinamide class such as N-hydroxy-succinamide or sulfo group-N-hydroxy-succinamide; The suitable phenol that replaces such as right-nitrophenol, neighbour, right-dinitrophenol,DNP, neighbour, adjacent '-dinitrophenol,DNP, trichlorophenol, 2,4,6,-T be as 2,4,6-trichlorophenol, 2,4,6,-T or 2,4,5-trichlorophenol, 2,4,6,-T, trifluoromethyl phenol are as 2,4,6-trifluoromethyl phenol or 2,4,5-trifluoromethyl phenol, pentachlorophenol, Pentafluorophenol; Or hydroxyl azole compounds such as hydroxybenzotriazole.Especially preferably N-hydroxy-succinamide class, especially preferred N-hydroxy-succinamide and sulfo group-N-hydroxy-succinamide, v is 1 to 10, and is preferred 1 to 5, more preferably 2 or 3; T is Cl, Br, I or OTf, and PEG is the Polyethylene Glycol or derivatives thereof, preferred mPEG.PAG is the poly alkylene glycol or derivatives thereof, preferred PEG, more preferably mPEG.W is 1 to 10, and is preferred 1 to 5, more preferably is 4.
Flow chart 2: the reaction of the reducing end under neutral group of polysaccharide
R wherein
eBe 2H or O, R
aBe OH, R
b, R
c, R
d, R
fBe H, OH, O-alkyl, hydroxy alkyl, OC (O) R independently, wherein R is an alkyl, preferably O-acetyl group, OPO
3H
2, OSO
3H, ONO
2Or O-polysaccharide; For example for glucosan with and derivant for, R
fBe the O-polysaccharide, for starch with and derivant for, R
dIt is the O-polysaccharide.
Another embodiment of the invention relates to the polymer that can obtain with said method.Preferred embodiment to said method and used parent material is described in front.
The polymer of the functionalization that available said method obtains is the polymer of the functionalization of formula I preferably.
The polymer of functionalization of the present invention is suitable to the polymer of preparation functionalization and the parent material of proteinic conjugate.In said conjugate, with the linking group of formula (IV)
Q-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5(IV)，
Preferably use the linking group of following formula
Be selected from the homopolymer of straight or branched of solubility or random copolymer with and the polymer and the protein of derivant covalently bound, wherein the straight or branched homopolymer of said solubility or random copolymer with and derivant be selected from the aklylene glycol homopolymer, preferred ethylene glycol homopolymer (PEG), the propylene glycol homopolymer, the aklylene glycol copolymer, preferred oxypropylene/oxyethylene copolymers, polyvinyl alcohol, polyvinylpyrrolidone, poly--1, the 3-dioxolanes, poly--1,3, the 6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid and polysaccharide are preferably selected from starch, cellulose, glucosan, arabic gum, xanthan gum, inulin, Ficus elastica, pectin, guar gum, Tragacanth, agar, algin, karaya, carrageenin, scleroglycan, Furcellaran, arabinogalactan and locust bean gum.
In another preferred embodiment, said linking group has the structure of formula (IV)
In another embodiment, the present invention relates to a kind of method for preparing conjugate, it comprises that the protein with the functionalization of the polymer of functionalization of the present invention and formula V reacts,
" protein "-Z (V)
Wherein Z be comprise the group of carbonyl or be suitable for forming carbonyl group or another kind of can with the group of the polymer reaction of said functionalization, wherein at least one end group of Z and " protein " or at least one are positioned at intermediary group covalency and link to each other, and preferably link to each other with the oxidized-terminal amino acid of said " protein " or oxidized carbohydrate side chain covalency.The polymer of preferred functionalization and protein are as previously mentioned.
According to one embodiment of the invention, the described proteinic Z of functional group be can with had the end group-O-NHR that can react
5The group of polymer (polymer of the functionalization of preferred formula I) reaction of linking group functionalization, R wherein
5Be hydrogen, alkyl or aryl, preferably hydrogen.Therefore, preferred group Z is aldehyde radical, ketone group, carboxyl and activatory carboxyl, for example ester group and lactone group.The suitable group Z of other is halogenide or pseudohalide group or the like, for example Cl, Br, I or OTf.Z is aldehyde radical or ketone group preferably.Therefore, the present invention relates to a kind of aforesaid method and conjugate, the wherein said proteinic Z of functional group is aldehyde radical or ketone group.
Though the position to group Z does not have total restriction, be preferably placed at aldehyde radical or ketone group in the described protein, embodiment preferred according to the present invention, this aldehyde radical or ketone group are arranged in described proteinic carbohydrate side chain.Therefore, in the present embodiment, use by glycosylated protein.
In the context of the present invention, term " carbohydrate side chain " refers to covalently bound and be made up of the oligosaccharide of at least two " carbohydrate parts " with proteinic aminoacid.In the context of the present invention, term " carbohydrate part " refer to hydroxy aldehyde or hydroxy-ketone with and chemovar (see Rompp Chemielexikon, Thieme Verlag Stuttgart, Germany, the 9th edition, 1990, the 9 volumes, 2281-2285 page or leaf and the document of wherein being quoted).In addition, it also relates to the derivant of naturally occurring carbohydrate part as glucose, galactose, mannose, sialic acid or the like.
In a preferred embodiment, this aldehyde radical or ketone group are the galactose residues of carbohydrate side chain, preferably the terminal galactose residues of carbohydrate side chain.
Can carry out the oxidation of end carbon hydrate part with chemistry or Enzymology method.
The method that the carbohydrate of polypeptide is partly carried out chemical oxidation be well known in the prior art and comprise with periodate to its handle (people such as Chamow, 1992, J.Biol.Chem., 267,15916-15922).
In theory, chemical oxidation may be to being positioned on the terminal position or not locational endways any carbohydrate partly carries out oxidation.But, by selecting gentle reaction condition, but the terminal sialic acid of its preferential oxidation carbohydrate side chain, thus aldehyde radical or ketone group obtained.
Another embodiment preferred according to the present invention, the reaction condition of said gentleness relates to that (concentration of periodate is preferably 1 to 50mM with suitable periodate aqueous solution with described protein, more preferably 1 to 25mM and especially be preferably 1 to 10mM 1mM according to appointment) react and preferably react under 0 ℃ the reaction temperature according to appointment at 0 to 40 ℃, especially preferred 0 to 21 ℃, the preferred response time is 5 minutes to 5 hours, more preferably 10 minutes to 2 hours and especially be preferably 10 minutes to 1 hour 1 hour according to appointment.Periodate: proteinic preferred molar ratio is 1: 200 to 1: 1, and more preferably 1: 50 to 1: 5,1: 15 according to appointment.
If for example EPO is carried out chemical oxidation, then preferably with EPO at aqueous medium, in the preferred water with periodate solution with about 15: 1 periodate: the EPO mol ratio was reacted under about 0 ℃ reaction temperature preferred 1 hour, and working concentration is the periodate aqueous solution of about 1mM.
Therefore, the invention still further relates to a kind of aforesaid method and conjugate, wherein, before protein and polymer or polymer derivant are reacted, glycosylated protein and periodate solution are reacted, thereby obtain having the aldehyde radical that is arranged in oxidized carbohydrate side chain or the protein of ketone group, said reaction is preferably carried out in the oxidation reaction of gentleness.Opposite with the reaction temperature of exacting terms such as 10mM periodate solution and 20 to 25 ℃, used in this article term " gentle reaction condition " refers to the reaction temperature of 1mM periodate solution for example and 0 ℃.
Perhaps, can carry out oxydasis to this carbohydrate side chain.The enzyme that is used for each carbohydrate side chain is carried out oxidation is known in the prior art, and for example in the situation of galactose, this enzyme is a beta-Galactose oxidase.If polypeptide is at the cell that sialic acid can be connected to carbohydrate chain mammalian cell or carried out genetic modification and thereby sialic acid can be connected in the cell on the carbohydrate chain and produce for example, if wish terminal galactose is partly carried out oxidation, then need to remove (partially or completely removing) terminal sialic acid.Being used to remove sialic chemistry or enzyme method is known (Chaplin and Kennedy (editor) in the prior art, 1996, carbohydrate analysis: practical approach (Carbohydrate Analysis:a practical approach), especially its 5th chapter Montreuill, glycoprotein (Glycoproteins), the 175-177 page or leaf; IRL Press practical approach series (Praeticalapproach series) (ISBN 0-947946-44-3)).
Another embodiment preferred according to the present invention, aldehyde radical or ketone group can be positioned on the proteinic N-terminal and can carry out suitable oxidation to it.Especially when the aminoacid that comprises hydroxyl such as threonine or serine are positioned at proteinic N-terminal on the 1-position, carry out the amino acid whose oxidation of said N-terminal and can produce said ketone group or aldehyde radical, preferably aldehyde radical.For example, threonine is preferably placed on the proteinic N-terminal, and described protein is for example eukaryotic cell such as mammal, especially the expression product that produces in people, insecticide or the yeast cells and by mammal or other eukaryote carbohydrate glycosylation.With regard to the method for suitable N-terminal aminoacid being carried out chemical oxidation, can use any conceivable method, preferably carry out oxidation, especially preferred gentle oxidizing condition with periodate.
Another embodiment preferred according to the present invention, with regard to the acid of N-terminal hydrogen base, the reaction condition of said gentleness relates to that (preferred periodate concentration is 1 to 50mM with described protein and suitable periodate aqueous solution, more preferably 1 to 25mM and especially be preferably 1 to 10mM 1mM according to appointment) at 0 to 40 ℃, especially preferred 0 to 21 ℃ is reacted under 0 ℃ the reaction temperature according to appointment, response time is preferably 5 minutes to 5 hours, more preferably 10 minutes to 2 hours and especially be preferably 10 minutes to 1 hour 1 hour according to appointment.Periodate: proteinic preferred molar ratio is 1: 200 to 1: 1 and more preferably 1: 50 to 1: 5 15: 1 according to appointment.
Therefore, the invention still further relates to a kind of aforesaid method and conjugate, wherein said aldehyde radical or ketone group are arranged in said proteinic carbohydrate side chain and/or are positioned on the said proteinic N-end group.
In the preferred embodiment of the method for the proteinic conjugate of the functionalization of preparation polymer of functionalization and formula V, proteinic aqueous solution to functionalization, be preferably placed at pH and be the aqueous solution that adds the polymer of functionalization in the solution in 5.0 to 5.5 the sodium-acetate buffer, be preferably placed at pH and be the solution in 5.0 to 5.5 the sodium-acetate buffer, add temperature and be generally 0 to 40 ℃, be preferably 0 to 25 ℃, more preferably 15 to 25 ℃.Then, will be incubated under the described in front temperature of this solution.This insulation was carried out 3 to 7 hours usually, preferred 8 to 48 hours, more preferably carried out 15 to 25 hours.It all is well known by persons skilled in the art carrying out the step of post processing and conjugate is carried out separation steps.Can further handle as post processing for example dialysis, centrifugal filtration or pressure filtration, ion exchange chromatography, reversed phase chromatography, HPLC, MPLC, gel filtration and/or lyophilization to this conjugate.
When pure by adding, when preferred 2-propanol or ethanol come the polymer to the functionalization of formula III to precipitate, the protein mol ratio of the functionalization of the polymer of the functionalization of formula III and formula V is generally 1-50: 1, be preferably 1-30: 1,1-20 more preferably: 1,1-15 more preferably: 1, and most preferably be 1-5: 1.
When coming polymer to the functionalization of formula III to precipitate by the solvent mixture that adds ethanol and acetone, the proteinic mol ratio of the functionalization of the polymer of the functionalization of formula III and formula V is generally 1-200: 1, be preferably 1-100: 1,1-50 more preferably: 1.
Can also begin to prepare conjugate of the present invention by the polymer of the functionalization of formula III with two-step method.
Therefore, in another embodiment, the present invention relates to a kind of method for preparing conjugate, it step that comprises has
A) with the polymer of formula III
" polymer "-(CR
8R
9)
p-Y (III)
React with the chemical compound of IV,
Q-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5 (IV)
Symbol wherein has following implication
" polymer " be the homopolymer of solubility straight or branched or random copolymer with and derivant, be selected from: the aklylene glycol homopolymer, preferred ethylene glycol homopolymer (PEG), the propylene glycol homopolymer, the aklylene glycol copolymer, preferred oxypropylene/oxyethylene copolymers, polyvinyl alcohol, polyvinylpyrrolidone, poly--1, the 3-dioxolanes, poly--1,3, the 6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid and polysaccharide are preferably selected from starch, cellulose, glucosan, arabic gum, xanthan gum, inulin, Ficus elastica, pectin, guar gum, Tragacanth, agar, algin, karaya, carrageenin, scleroglycan, Furcellaran, arabinogalactan and locust bean gum;
R
1、R
2、R
3、
R
4, R
5Be hydrogen, alkyl, aryl, preferably hydrogen;
M is 2 to 4, wherein at m group CR
1R
2In residue R
1And R
2Can be identical or different;
N is 0 to 20, preferably 0 to 10, and more preferably be 1 to 5, most preferably be 1 or 2, and more preferably be 1;
O is 0 to 20, preferably 0 to 10, and more preferably be 0 or 2, wherein in the situation of n=0, o is not 0, in a preferred embodiment, o is 2 to 20, preferably 2 to 10, more preferably be 2, wherein at o group CR
3R
4In residue R
3And R
4Can be identical or different;
Thereby Y and Q be suitable for reacting each other the functional group that provides one of following linking group :-O-,-S-,-NR
6-,-OC (O)-,-C (O) O-,-C (G) N (R
10) O-,-N (R
11) O-,
Preferably
-N(R
11)O-，-C(G)N(R
10)O-，
Wherein one or more groups-(CR
8R
9)-can be replaced by W, thus a kind of chemically rational group formed;
W is O, NR
12, C (G), preferably O, C (G);
G is S, O, NR
14, O preferably;
R
6、R
7、R
8、
R
9、R
10、
R
11, R
12, R
14Be hydrogen, alkyl, aryl, preferably hydrogen;
P is 0 to 20, preferably 0 to 10, more preferably be 0 to 5, most preferably be 0 to 4, polysaccharide with and the situation of derivant in more preferably be 0, and poly alkylene glycol with and the situation of derivant in more preferably 1 to 4, wherein at p group CR
8R
9In residue R
8And R
9Can be identical or different;
Group-(CR wherein
8R
9)
pAt least one end group of-Y and " polymer " and/or at least one are positioned at intermediary group covalency and link to each other, obtain thus a kind of functionalization polymer and
B) protein with the functionalization of the polymer of the functionalization that obtained in the step a) and formula V reacts,
" protein "-Z (V)
Wherein Z comprises the group of carbonyl or is suitable for forming the group of carbonyl or the other group that can react with the polymer of functionalization, wherein at least one end group of Z and " protein " and/or at least one are positioned at intermediary group covalency and link to each other, and preferably link to each other with the oxidized-terminal amino acid of described " protein " or oxidized carbohydrate side chain covalency.The protein of the functionalization of the polymer of preferred formula III, the chemical compound of formula IV and formula V as previously mentioned.
Step a) and b) reaction condition and the polymer of described preparation functionalization, to begin to prepare the described reaction condition of said conjugate (step b)) identical with being used for polymer by functionalization for the polymer of preferred formula I (step a)).The advantage of this two-step method is that the polymer that can omit the functionalization that obtained in step a) carries out separation steps.
Conjugate of the present invention itself or the pharmaceutical composition that comprises this conjugate can be used for human or animal body is treated.
In another embodiment, the present invention relates to be used for the conjugate of the present invention of method that human or animal body is treated or the conjugate that available method of the present invention obtains.
Pharmaceutical composition comprises conjugate of the present invention and acceptable diluents, auxiliary agent or the carrier for the treatment of effective dose.This pharmaceutical composition also randomly comprises other therapeutic component or galenical composition and auxiliary agent.Suitable auxiliary agent has for example diluent, buffer system, binding agent, surface active composition, thickening agent, lubricant and anti-distintegrant (comprising antioxidant).
The treatment effective dose is in order to improve, be enough to obtain in single in the scope of healing or prevent disease and the treatment carried out or the repetitive therapy amount of positive role.
Acceptable diluents is and conjugate of the present invention and all compatible diluent of human or animal body.
The form of pharmaceutical composition depends on required or suitable application process.The preferred application is parenteral application.Suitable parenteral application method is known in the prior art.Other possible application has intranasal application, intrachealic to use or topical application.Pharmaceutical composition can exist with the form of dosage unit and can be prepared with method well known in the prior art.
Pharmaceutical composition comprises the conjugate that the conjugate of the present invention for the treatment of effective dose or available method of the present invention obtain.
Described pharmaceutical composition also preferably comprises at least a acceptable diluents, auxiliary agent or carrier.Suitable diluent, auxiliary agent or carrier and other suitable composition all are well known by persons skilled in the art.
The present invention is further described with the following examples.
Embodiment
A) hydroxyl amino-PEG-derivant is synthetic
Aa) hydroxyl amino-PEG20's that carries out in aqueous solution is synthetic
With 200mg mPEG-butyraldehyde (mPEG-ButyrALD, MW20,000, Nektar, Huntsville, AL, USA) be dissolved in the 2mL 0.1M sodium-acetate buffer (pH5.2) and to wherein adding 1mmol O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine is (according to people such as D.Boturyn, Tetrahedron 53 (1997) 5485-92, the 5489-90 page or leaf is synthetic).It, is joined reactant mixture in the 45mL 2-propanol and with it to descend to be incubated 4 hours at-20 ℃ after 19 hours 22 ℃ of following joltings under-20 ℃.By under 0 ℃, carrying out the product that centrifugal collecting precipitation comes out, under-20 ℃, wash and it is descended to be incubated 1 hour at-20 ℃ with 15mL 2-propanol.After centrifugal, this product is dissolved in the 15mL water, water dialysis 21 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, PerbioSciences Deutschland GmbH, Bonn is D) and with its lyophilization.
Ab) hydroxyl amino-PEG20's that carries out in organic solvent is synthetic
With 200mg mPEG-butyraldehyde (mPEG-ButyrALD, MW20,000, Nektar, Huntsville, AL, USA) be dissolved in the 2mL dichloromethane and to wherein adding 1mmolO-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine is (according to people such as D.Boturyn, Tetrahedron 53 (1997) 5485-92, the 5489-90 page or leaf is synthetic).It, is joined reactant mixture in the 45mL 2-propanol and with it to descend to be incubated 4 hours at-20 ℃ after 19 hours 22 ℃ of following joltings under-20 ℃.By under 0 ℃, carrying out the centrifugal product that comes collecting precipitation to come out, with 15mL 2-propanol under-20 ℃, wash and with it-20 ℃ of insulations 1 hour down.After centrifugal, this product is dissolved in the 15mL water, water dialysis 21 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, PerbioSciences Deutschland GmbH, Bonn is D) and with its lyophilization.
B) hydroxyl amino-glucosan-derivant is synthetic
Synthesizing of the hydroxyl amino-glucosan 17 that in aqueous solution, carries out
The 500mg glucosan (is derived from Leuconostoc ssp., M
r～15000-20000D, Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) be dissolved in the 5mL 0.1M sodium-acetate buffer (pH5.2) and to wherein adding 1mmol O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine is (according to people such as D.Boturyn, Tetrahedron 53 (1997) 5485-92, the 5489-90 page or leaf is synthetic).It, is joined reactant mixture in the 45mL2-propanol and with it to descend to be incubated 4 hours at-20 ℃ after 19 hours 22 ℃ of following joltings under-20 ℃.By under 0 ℃, carrying out the centrifugal product that comes collecting precipitation to come out, with 15mL 2-propanol under-20 ℃, wash and with it-20 ℃ of insulations 1 hour down.After centrifugal, this product is dissolved in the 15mL water, water dialysis 21 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.
C) the EPO conjugate is synthetic
C1) by EPO being carried out gentle periodate processing N-n acetylneuraminic acid n residue is carried out periodate oxidation
To the EPO solution that remains on the 2.0mg/ml of 20mL altogether under 0 ℃ (EPO of the aminoacid sequence with people EPO of reorganization preparation and itself and product (Epoietin α: Erypo that can commercial acquisition, ORTHO BIOTECH, Jansen-Cilag or Epoietin β: NeoRecormon, Roche; Referring to EP 0 148 605, EP 0 205 564, and EP 0 411 678) characteristic similar or basic identical) in add the ice-cold 10mM metaperiodic acid sodium solution of 2.2mL, thereby produced the final concentration of 1mM sodium metaperiodate.This mixture is being incubated 1 hour on the ice bath in the dark under 0 ℃, is coming cessation reaction and it is incubated 5 minutes again by adding 40 μ l glycerol.
C2) be used for subsequently carrying out the buffer-exchanged of EPO of the periodate oxidation of derivatization with the hetastarch derivant of hydroxyl amino functionalization
Buffer-exchanged is to carry out with 20mL Vivaspin 20 concentrators (Vivaspin AG, Hannover, Germany) that use polyether sulfone (PES) film.By add 5mL 0.1M sodium-acetate buffer (pH5.5) come to these concentrator parts wash and with these concentrator parts under the 4000rpm under 6 ℃ at Megafuge 1.0R (Kendro Laboratory Equipment, Osterode, Germany) carry out centrifugal in.Subsequently, in these concentrator parts, add 20mL embodiment 1 periodate oxidation EPO solution and with its under 4000rpm centrifugal 25 minutes until obtaining a kind of 5-times concentrate.In this concentrate, add 15mL 0.1M sodium-acetate buffer (pH5.5) and carry out it centrifugal as mentioned above like that.Should be centrifugal circulation repeat 3 times, after these concentrator parts being washed 2 times (using 1mL) at every turn, take out last concentrate and it transferred in the aseptic plastic tube of 50mL with sodium-acetate buffer (pH5.5); The volume of this EPO is transferred to 26.7mL and by according to European Pharmacopoeia (erythropoietin concentrated solution with sodium-acetate buffer (pH5.5), the 4th edition, 2002, the 1123-1128 pages or leaves) absorbance (the specific absorption coefficient value is 7.43) under the description mensuration 280nm in is determined the protein concentration of the whole solution of EPO of this oxidation.The value that records the whole solution of EPO of this periodate oxidation is 1.378mg/ml (36.8mg EPO is equivalent to the ultimate yield of ≈ 90%).C3) the EPO-conjugate of mPEG aldehyde-reactive polymer and dextran aldehyde-reactive polymer is synthetic
In the oxidized solution (according to Embodiment C 1 carry out oxidation) of EPO in 0.1M sodium-acetate buffer (pH5.5) of 13.3 μ L, add the solution of 6.67 μ L aldehyde-reactive polymers in 0.1M sodium-acetate buffer (pH 5.5) and this solution is incubated 22 hours down at 22 ℃.Concentration below adopting:
For according to embodiment A a) and Ab) be 100mg/mL for aldehyde-reactive polymer of making
For according to Embodiment B) be 87.5mg/mL for dextran aldehyde-reactive polymer of making
In Embodiment C 1) to C3) in, protein spectra was understood successful joint to higher molecular weight migration table during the SDS page of Fig. 2 analyzed.The increase of spectral bandwidth is because the number of the molecular weight distribution of used glucosan and PEG-derivant and the HES derivant that links to each other with described protein causes.Opposite with hydroxyl amino-glucan derivative, corresponding PEG derivant also moves in the gel and also and is colored, and therefore makes that the video picture of protein spectra is complicated.
C4) hydroxyethyl starch and EPO conjugate is synthetic
C4.1) the HES derivant of hydroxyl amino functionalization is synthetic
O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine is according to people such as Boturyn, and Tetrahedron 53 (1997) the described methods of 5485-5492 page or leaf are in two steps by can be synthetic by the material that commercial sources obtains.
C4.1) (a) hydroxyl amino-HES 10/0.4 synthetic
With 2g HES 10/0.4 (MW=10000 D, DS=0.4, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D) be dissolved in the 17mL 0.1M sodium-acetate buffer (pH5.2) and to wherein adding 20mmol O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine.It, is joined reactant mixture in the mixture of 1: 1 ice-cold acetone of 100mL and ethanol (v/v) after 19 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is dissolved in the 50mL water again 21 hours (SnakeSkin Dialysis tubings of water dialysis, 3.51kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.
C4.1) (b) hydroxyl amino-HES 10/0.7 synthetic
With 2g HES10/0.7 (MW=10000 D, DS=0.7, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D) be dissolved in the 18mL 0.1M sodium-acetate buffer (pH5.2) and to wherein adding 20mmol O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine.It, is joined reactant mixture in the mixture of 1: 1 ice-cold acetone of 100mL and ethanol (v/v) after 19 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is dissolved in the 50mL water again 21 hours (SnakeSkin Dialysis tubings of water dialysis, 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.
C4.1) (c) hydroxyl amino-HES 50/0.4 synthetic
With 2g HES50/0.4 (MW=50000 D, DS=0.4, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D) be dissolved in the 20mL 0.1M sodium-acetate buffer (pH5.2) and to wherein adding 4mmol O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine.It, is joined reactant mixture in the mixture of 1: 1 ice-cold acetone of 100mL and ethanol (v/v) after 19 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is dissolved in the 50mL water again 21 hours (SnakeSkin Dialysis tubings of water dialysis, 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.
C4.1) (d) hydroxyl amino-HES 50/0.7 synthetic
With 2g HES50/0.7 (MW=50000D, DS=0.7, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D) be dissolved in the 20mL 0.1M sodium-acetate buffer (pH 5.2) and to wherein adding 4mmol O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine.It, is joined reactant mixture in the mixture of 1: 1 ice-cold acetone of 70mL and ethanol (v/v) after 17.5 hours 22 ℃ of following joltings.By the centrifugal product that is precipitated out of collecting under 0 ℃, with ice-cold 1: 1 the acetone of 30mL and the mixture of ethanol (v/v) it is washed, it is dissolved in the 50mL water again, 19.5 hours (SnakeSkin Dialysis tubings of water dialysis, 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.
C4.2) the HES-EPO conjugate is synthetic
In Embodiment C 4.2) (a) to C4.2) (d) in, protein spectra was understood successful joint to higher molecular weight migration table during the SDS page of Fig. 1 analyzed.The increase of spectral bandwidth is because the molecular weight distribution of used HES derivant and the number of the HES derivant that links to each other with described protein cause.
C4.2) (a) with implementing C4.1) (a) hydroxyl amino-HES 10/0.4 carry out synthetic
EPO the solution in 0.1M sodium-acetate buffer (pH5.5) oxidized to 3.63mL (makes according to Embodiment C 2; 1.378mg/ml) in add 83mg according to Embodiment C 4.1) the hydroxyl amino HES10/0.4 that (a) makes and with this solution 22 ℃ of following joltings 16.5 hours.
C4.2) (b) with Embodiment C 4.1) (b) hydroxyl amino-HES 10/0.7 carry out synthetic
Make to the oxidized solution (according to Embodiment C 2) of EPO in 0.1M sodium-acetate buffer (pH5.5) of 3.63mL; 1.378mg/ml) in add 83mg according to Embodiment C 4.1) the hydroxyl amino HES10/0.7 that (b) makes and with this solution 22 ℃ of following joltings 16.5 hours.
C4.2) (c) use Embodiment C 4.1) (c) hydroxyl amino-HES 50/0.4 carry out synthetic
Make to the oxidized solution (according to Embodiment C 2) of EPO in 0.1M sodium-acetate buffer (pH5.5) of 3.63mL; 1.378mg/ml) in add 416mg according to Embodiment C 4.1) the hydroxyl amino HES 50/0.4 that (c) makes and with this solution 22 ℃ of following joltings 16.5 hours.
C4.2) (d) use Embodiment C 4.1) (d) hydroxyl amino-HES 50/0.7 carry out synthetic
Make to the oxidized solution (according to Embodiment C 2) of EPO in 0.1M sodium-acetate buffer (pH5.5) of 3.63mL; 1.378mg/ml) in add 416mg according to Embodiment C 4.1) the hydroxyl amino HES 50/0.7 that (d) makes and with this solution 22 ℃ of following joltings 16.5 hours.
D) EPO's that modifies of the purification of the EPO that modifies of HES-and the HES-derivant of reacting and HES-separates
Carrying out Embodiment C 4.2) (a) to C4.2) after (d) the HES-coupling operation, at room temperature with being furnished with pump P-903, having blender M-925, the Monitor pH/C-900 of 0.6mL chamber, KTA Explorer 10 systems of pump P-950 (sample pump) and 3.21 editions Software Unicorn and come all samples are carried out purification.280,260 and 220nm under detect with the Monitor UV-900 with 10mm flow cell.
With the insulation mixture dilute with the buffer A (20mM transfers to pH with NaOH 8.0 N-morpholino propane sulfonic acid) of 10 times of volumes and with its with sample on 0.8mL/ minute the flow velocity to the pillar that comprises 4mL Q-Sepharose Fast Flow (Amersham Pharmacia Biotech); Used the buffer A of 7 column volumes (CV) that this post has been carried out balance before.Then, pillar washed with 1.0mL/ minute flow velocity with the buffer A of 6CV and (0.5M is arranged in the NaCl of 20mM sodium phosphate, pH6.5) carries out eluting with 0.6mL/ minute flow velocity with the buffer B of 2.5CV.Then, (1.5M is arranged in the NaCl of 20mM sodium phosphate, pH6.5) with 0.6mL/ minute flow velocity this post is washed, and the buffer A by making 7CV again it carries out balance by this post with 1.0mL/ minute flow velocity with the buffer C of 2.5CV.
Be incubated the sample that is obtained with (activity) hydroxyl amino HES derivant and under 220nm, all produced tangible absorption.Sample with hydroxyl amino HES 10/0.4 and 10/0.7 insulation does not have under 280nm and can detectedly absorb, and with the sample absorption that produced 800mAU * mL and 950mAU * mL respectively of hydroxyl amino HES 50/0.7 and 50/0.4 insulation.Reclaimed nearly all bonded protein in eluent 1, volume is 6.5-8.0mL, and the peak area that eluent 2 comprises is less than 2% of the peak area of total eluting peak of surveying under 280nm.The protein recovery of all EPO samples is (about 85%) quite.
Use 5mL Vivaspin concentrator (10, the 000MW cutoff value) and under 4000rpm, under 6 ℃, carry out centrifugally coming EPO that HES-is modified and the EPO that obtains by suitable contrast insulation to carry out buffer-exchanged like that by foregoing.With these samples (1-3mg epo protein) be concentrated into 0.5-0.7mL and with the saline (PBS) of phosphate-buffered (pH7.1) it is diluted to 5mL and by centrifugal with its concentrated 10 times.Each sample is carried out concentrating for three times-the dilution circulation.At last, these samples are taken out and the concentrator parts are washed with 2 * 0.5mL PBS.These samples are freezing in liquid nitrogen with the protein concentration of about 1.2mg/ml.
E) cross-linking compounds is synthetic
E1) O-[2-(2-amino oxygen base-ethyoxyl)-ethyl]-hydroxylamine (
1) synthetic
As mentioned embodiment A a) as described in, chemical compound (
1) be according to people such as D.Boturyn, Tetrahedron 53 (1997) 5485-92, the description of 5489-90 page or leaf is carried out synthetic.E2) 1,6-two-amino oxygen base-3-methylpentane (
4) synthetic
With 20g (82.0mmol) 1,5-two bromo-4-methylpentane (Acros Organics BVBA, Geel, B) at room temperature be added drop-wise in the 36.72g (204.9mmol)-N-hydroxyl-5-norborene-2,3-diformamide (Lancaster Synthesis GmbH, Frankfurt/Main, D) and 36.7g potassium carbonate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) at 140mL N, dinethylformamide (DMF) (the synthetic level of peptide, Biosolve, Valkenswaard, NL) in the suspension in, and with reactant mixture stirring 90 hours.Remove tiny precipitation by centrifugal, collect clarifying DMF supernatant and its vacuum concentration.With the thorough vacuum drying of crude product, be dissolved in 140mL ethanol (DAB quality, Sonnenberg, Braunschweig, D) in and with its under nitrogen with 16.2mL (333.3mmol) hydrazine hydrate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen D) refluxed 2 hours together.Under vacuum, remove and desolvate and remaining hydrazine hydrate, with crude product be suspended in 150mL t-butyl methyl ether (MTBE) (Acros Organics BVBA, Geel, B) in and it was at room temperature stirred 1 hour.Precipitation is leached, wash and it is discarded with MTBE.With collected filtrate vacuum concentration and with column chromatography to this product carry out purification (dichloromethane solution of 3% methanol, the two is all from Acros Organics BVBA, Geel, B), obtain 38% colorless oil form (
4).δ
H(300；CDCl
3)0.89(3H，d，J 6.5)，1.37-1.44(2H，m)，1.55-1.66(3H，m)，3.64-3.71(4H，m)，5.30(4H，s)；δ
C(75.5MHz；CDCl
3)19.7，27.2，35.4，74.3。
E3) 2-(2-bromine oxethyl)-3a, 7a-dihydro-iso-indoles-1, the 3-diketone (
5) synthetic
Chemical compound (5) is according to Bauer, L. and Suresh, K.S., J.Org.Chem.1963,28,1604 pages description by 16.3g (100mmol) N-hydroxyphthalimide (Acros OrganicsBVBA, Geel, B), 17.0mL (196.9mmol) 1,2-Bromofume (Lancaster SynthesisGmbH, Frankfurt/Main, D) and 28mL (197.6mmol) triethylamine (Acros OrganicsBVBA, Geel, B) in 120mL DMF (the synthetic level of peptide, Biosolve, Valkenswaard, synthetic in NL).By removing by filter formed post precipitation, make the crude product precipitation with 800mL water, collect and it is carried out the purification (petroleum ether solution of 25% t-butyl methyl ether by filtration with column chromatography, the two is all from Acros Organics BVBA, Geel, B), obtain 7.58g (29%) rice white solid, shaped (
5).δ H (400MHz; CDCl
3) 3.63 (J 6.9 for 3H, t), 4.46 (J 6.9 for 3H, t), 7.75 (2H, dd, J 3.1 and 5.5), 7.83 (2H, dd, J 3.1 and 5.5).
E4) 2-(6-bromo-hexyloxy)-3a, 7a-dihydro-iso-indoles-1, the 3-diketone (
6) synthetic
Chemical compound (6) is according to Bauer, L. and Suresh, K.S., J.Org.Chem.1963,28,1604 pages description is by 16.3g (100mmol) N-hydroxyphthalimide (Acros OrganicsBVBA, Geel, B), 30.8mL (200mmol) 1,6-dibromo-hexane (Aldrich, Sigma-AldrichChemie GmbH, Taufkirchen, D) and 28mL (197.6mmol) triethylamine (AcrosOrganics BVBA, Geel is B) at 120mL DMF (the synthetic level of peptide, Biosolve, Valkenswaard, synthetic in NL).With column chromatography carry out purification (petroleum ether solution of 10% t-butyl methyl ether, the two is all from Acros Organics BVBA, Geel, B), obtain 12.28g (38%) rice white solid, shaped (
6).δH(400MHz；CDCl
3)1.48-1.60(4H，m)，1.77-1.84(2H，m)，1.87-1.93(2H，m)，3.42(2H，t，J 6.8)，4.20(2H，t，J 6.5)，7.71-7.76(2H，m)，7.81-7.85(2H，m)；δC(100.6MHz；CDCl
3)24.9，27.9，28.0，32.7，33.7，78.4，123.5，129.1，134.5，163.7。
F) preparation of the reactive polymer-derivant of hydroxylamine cross-linking agent
F1) by cross-linking agent (
4) and hydroxyl amino-glucosan 17.5 of carrying out of Dextran 15-20kD (
A) synthetic.
With 204mg Dextran 15-20kD (MW=15000-20000D, Fluka, Sigma-Aldrich Chemie, Taufkirchen, D) be dissolved in the 2mL 0.1M sodium-acetate buffer (pH5.2) and to wherein add 0.57mmol (
4).It, is joined reactant mixture in the ice-cold 2-propanol of 40mL and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, be suspended in it in 15mL 2-propanol and by filtering it collected once more.Crude product is dissolved in the 15mL water, with its water dialysis 43.5 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio SciencesDeutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 82%.
F2) by cross-linking agent (
4) and hydroxyl amino-HES10/0.4 of carrying out of HES 10/0.4 (
B) synthetic
With 403mg HES10/0.4 (MW=10000D, DS=0.4, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D) be dissolved in the 4mL 0.1M sodium-acetate buffer (pH5.2) and to wherein add 4mmol (
4).It, is joined reactant mixture in the ice-cold 2-propanol of 40mL and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, be suspended in it in 15mL 2-propanol and by filtering it collected once more.Crude product is dissolved in the 15mL water, and water dialysis 46 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 52%.
F3) by cross-linking agent (
1) and hydroxyl amino-PEG10 of carrying out of mPEG-NHS ester 10kD (
C) synthetic.
With 607mg α-methoxyl group-PEG-ω-succinimido ester (MW=10000D, RappPolymere, T ü bingen, D) be dissolved in the 6mL dichloromethane (Acros Organics BVBA, Geel, B) in and to wherein adding 3.2mmol
1It, is joined reactant mixture in the ice-cold 2-propanol of 40mL and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, be suspended in it in 15mL 2-propanol and by filtering it collected once more.Crude product is dissolved in the 15mL water, and water dialysis 43.5 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 82%.
With this product of 200mg be dissolved in the 2mL dichloromethane (Acros Organics BVBA, Geel, B) in and to wherein add the 0.2mmol morpholine (Acros Organics BVBA, Geel, B).With it 22 ℃ of following joltings after 17 hours, with reactant mixture join the 40mL t-butyl methyl ether (AcrosOrganics BVBA, Geel, B) in and with it-20 ℃ of insulations 1 hour down.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, be suspended in it in 15mL t-butyl methyl ether and by filtering it collected once more.Crude product is dissolved in the 10mL water, and water dialysis 45 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 79%.
F4) by cross-linking agent (
4) and hydroxyl amino-PEG10 of carrying out of mPEG-NHS ester 10kD (
D) synthetic.
With 206mg α-methoxyl group-PEG-ω-succinimido ester (MW=10000D, RappPolymere, T ü bingen, D) be dissolved in the 6mL dichloromethane (Acros Organics BVBA, Geel, B) in and to wherein add 1mmol (
4).It, is joined reactant mixture in the ice-cold 2-propanol of 40mL and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, be suspended in it in 15mL 2-propanol and by filtering it collected once more.Crude product is dissolved in the 15mL water, and water dialysis 43.5 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 86%.
With this product of 100mg be dissolved in the 2mL dichloromethane (Acros Organics BVBA, Geel, B) in and to wherein add the 0.2mmol morpholine (Acros Organics BVBA, Geel, B).With it 22 ℃ of following joltings after 17 hours, with reactant mixture join the 40mL t-butyl methyl ether (AcrosOrganics BVBA, Geel, B) in and with it-20 ℃ of insulations 1 hour down.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL t-butyl methyl ether and by centrifugal once more it is collected.Crude product is dissolved in the 10mL water, and water dialysis 45 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 61%.
F5) by cross-linking agent (
4) and the hydroxyl amino HES10/0.4 that carries out of oxidized HES10/0.4 (
E) synthetic
HES10/0.4 (the MW=10000D that 400mg is oxidized, DS=0.4, SupramolParenteral Colloids GmbH, Rosbach-Rodheim, D) be dissolved in the anhydrous DMSO (Fluka of 4mL 80 ℃ of following heating in vacuum 17 hours and with it, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) in.In this solution, add 4mmol (
4).It, is joined reactant mixture in the ice-cold 2-propanol of 35mL and with it to descend to be incubated 1 hour at-20 ℃ after 5 days 65 ℃ of following insulations.Tiny product by coming collecting precipitation to come out in centrifugal 9 hours down at 4 ℃ is dissolved in it in 10mL water again, 47 hours (SnakeSkin Dialysis tubings of water dialysis, 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.F6) by cross-linking agent (
1) and hydroxyl amino-PEG10 of carrying out of mPEG-aldehyde 10kD (
F) synthetic.
With 604mg α-methoxyl group-PEG-ω-aldehyde (MW=10000D, Rapp Polymere, T ü bingen, D) be dissolved in the 6mL 4mL 0.1M sodium-acetate buffer (pH5.2) and to wherein add 2.2mmol (
1).It 22 ℃ of following joltings after 17 hours, is concentrated reactant mixture under vacuum, with its be dissolved in again the 6mL dichloromethane (Acros Organics BVBA, Geel, B) and join in the ice-cold 2-propanol of 40mL, with it-20 ℃ of insulations 1 hour down.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL 2-propanol and by centrifugal once more it is collected.Crude product is dissolved in the 15mL water, and water dialysis 42 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 87%.
F7) by cross-linking agent (
4) and hydroxyl amino-PEG10 of carrying out of mPEG-aldehyde 10kD (
G) synthetic.
With 200mg α-methoxyl group-PEG-ω-aldehyde (MW=10000D, Rapp Polymere, T ü bingen, D) be dissolved in the 6mL 4mL 0.1M sodium-acetate buffer (pH5.2) and to wherein add 1mmol (
4).It, is joined reactant mixture in the ice-cold 2-propanol of 40mL and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL 2-propanol and by centrifugal once more it is collected.Crude product is dissolved in the 15mL water, and water dialysis 43.5 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 60%.
F8) by cross-linking agent (
6) and hydroxyl amino-PEG10 of carrying out of mPEG-mercaptan 10kD (
H) synthetic.
With 300mg α-methoxyl group-PEG-ω-mercaptan (Tbingen D) is dissolved in 3mL N for MW=10000D, Rapp Polymere, dinethylformamide (DMF) (the synthetic level of peptide, Biosolve, Valkenswaard, NL) in and remove deoxidation with nitrogen current.With this solution under nitrogen, join 0.4mmol (
6) and the 0.44mmol cesium carbonate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) in.With its 22 ℃ down stir 2.5 hours after, with reactant mixture join the 40mL t-butyl methyl ether (Acros Organics BVBA, Geel, B) in and with it-20 ℃ of insulations 1 hour down.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL t-butyl methyl ether, by centrifugal it is collected and with its vacuum drying once more.Do not measure the yield of the product of being separated.
With this crude product of 150mg be dissolved in the 1.5mL dichloromethane (Acros Organics BVBA, Geel, B) in and to wherein add the 0.15mmol hydrazine hydrate (Fluka, Sigma-Aldrich ChemieGmbH, Taufkirchen, D).It, is joined reactant mixture in the 40mL t-butyl methyl ether and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL t-butyl methyl ether and by centrifugal once more it is collected.Crude product is dissolved in the 10mL water, and water dialysis 41 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 63%.
F9) by cross-linking agent (
5) and hydroxyl amino-PEG10 of carrying out of mPEG-mercaptan 10kD (
I) synthetic.
With 300mg α-methoxyl group-PEG-ω-mercaptan (T ü bingen D) is dissolved in 3mL N for MW=10000D, Rapp Polymere, dinethylformamide (DMF) (the synthetic level of peptide, Biosolve, Valkenswaard, NL) in and remove deoxidation with nitrogen current.With this solution under nitrogen, join 0.4mmol (
5) and the 0.44mmol cesium carbonate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D) in.With its 22 ℃ down stir 2.5 hours after, with reactant mixture join the 40mL t-butyl methyl ether (Acros Organics BVBA, Geel, B) in and with it-20 ℃ of insulations 1 hour down.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL t-butyl methyl ether, by centrifugal it is collected and with its vacuum drying once more.Do not measure the yield of the product of being separated.
With this crude product of 150mg be dissolved in the 1.5mL dichloromethane (Acros Organics BVBA, Geel, B) in and to wherein add the 0.15mmol hydrazine hydrate (Fluka, Sigma-Aldrich ChemieGmbH, Taufkirchen, D).It, is joined reactant mixture in the 40mL t-butyl methyl ether and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL t-butyl methyl ether and by centrifugal once more it is collected.Crude product is dissolved in the 10mL water, and water dialysis 41 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 70%.
F10) by cross-linking agent (
1) and the hydroxyl amino HES18/0.5 that carries out of oxidized HES18/0.5 (
J) synthetic
The HES18/0.5 that 200mg is oxidized (MW=18000D, DS=0.5) 80 ℃ of following heating in vacuum 17 hours and with its be dissolved in the anhydrous DMSO of 2mL (Fluka, Sigma-AldrichChemie GmbH, Taufkirchen, D) in.In this solution, add 2mmol (
1).It, is joined reactant mixture in the ice-cold 2-propanol of 20mL and with it to descend to be incubated 1 hour at-20 ℃ after 5 days 65 ℃ of following insulations.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, the 2-propanol ice-cold with 42mL washs it, it is dissolved in the 10mL water again, 27 hours (SnakeSkin Dialysis tubings of water dialysis, 3.5kD cutoff value, Perbio Sciences DeutschlandGmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 72%.
F11) by cross-linking agent (
1) and the hydroxyl amino HES10/0.4 that carries out of HES10/0.4 (
K) synthetic
With 0.8g HES10/0.4 (MW=10000D, DS=0.4, Supramol ParenteralColloids GmbH, Rosbach-Rodheim, D) be dissolved in the 8mL 0.1M sodium-acetate buffer (pH5.5) and to wherein add 8mmol (
1).It, is joined reactant mixture in the 40mL 2-propanol after 19 hours 22 ℃ of following joltings under-20 ℃.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is dissolved in the 50mL water again 45 hours (SnakeSkin Dialysis tubings of water dialysis, 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 73%.
F12) by cross-linking agent (
6) and hydroxyl amino-PEG10 of carrying out of mPEG-alcohol 5kD (
L) synthetic
With 1g α-methoxyl group-PEG-alcohol (MW=5000D, Fluka, Sigma-Aldrich ChemieGmbH, Taufkirchen is D) with 257mg powdered potassium hydroxide (Riedel-de Ha n, Sigma-Aldrich Chemie GmbH, Taufkirchen D) is suspended in 10mL anhydrous dimethyl sulphoxide (DMSO) (Fluka, Sigma-Aldrich Chemie GmbH under nitrogen, Taufkirchen, D) in and reactant mixture is heated to 70 ℃.To wherein add the 978mg solid form (
6) also this mixture was stirred 2 hours down at 70 ℃.With the solution of gained join the 200mL t-butyl methyl ether (Acros Organics BVBA, Geel, B) in.With the gumminess product that is precipitated out 100mL t-butyl methyl ether extracting twice, it is dissolved in the 40mL water 47 hours (SnakeSldn Dialysis tubings of water dialysis, 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn is D) and with its lyophilization.The yield of the product of being separated is 98%.
With this crude product of 482mg be dissolved in the 5mL dichloromethane (Acros Organics BVBA, Geel, B) in and to wherein add the 1mmol hydrazine hydrate (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D).It, is joined reactant mixture in the 40mL t-butyl methyl ether and with it to descend to be incubated 1 hour at-20 ℃ after 17 hours 22 ℃ of following joltings.By under 4 ℃, carrying out the centrifugal product that is precipitated out of collecting, it is suspended in the 15mL t-butyl methyl ether and by centrifugal once more it is collected.Crude product is dissolved in the 10mL water and water dialysis 41 hours (the SnakeSkin Dialysis tubing, the 3.5kD cutoff value, Perbio Sciences Deutschland GmbH, Bonn, D).With its centrifugal 17 hours, thereby remove formed tiny precipitation between dialysis period.With clarifying supernatant lyophilization, the yield of the product of separating is 67%.
G) reactive polymer-derivant and oxidized hEPO's engages
G1) the oxidized solution of hEPO in 0.1M sodium-acetate buffer (pH5.5) is to use C1) and C2) described in method obtain.The final concentration of the hEPO that this is oxidized is transferred to 2mg/mL.
G2) with embodiment F 1) to F12) the EPO conjugate that carries out of the polymer that makes synthetic
In the oxidized solution (as embodiment G1 obtain) of hEPO in 0.1M sodium-acetate buffer (pH5.5) of 20 μ L, add the solution of 20 μ L aldehyde-reactive polymers in 0.1M sodium-acetate buffer (pH5.5) and this solution is incubated 22 hours down at 22 ℃.The concentration of used aldehyde-reactive polymer is shown in following table 1:
Table 1
| Clauses and subclauses | Polymer derivant | As described in the following embodiment, be synthesized | Equivalent | Concentration [mg/ml] |
| 1 | ( A) | F1) | 5 | 5.83 |
| 2 | ( B) | F2) | 5 | 3.33 |
| 3 | ( C) | F3) | 5 | 3.33 |
| 4 | ( D) | F4) | 5 | 3.33 |
| 5 | ( E) | F5) | 50 | 33.3 |
| 6 | ( F) | F6) | 50 | 33.3 |
| 7 | ( G) | F7) | 50 | 33.3 |
| 8 | ( H) | F8) | 5 | 3.33 |
| 9 | ( I) | F9) | 5 | 3.33 |
| 10 | ( J) | F10) | 50 | 60.0 |
| 11 | ( K) | F11) | 5 | 3.33 |
| 12 | ( L) | F12) | 50 | 33.3 |
| 13 | No polymer |
Protein spectra was understood successful joint to higher molecular weight migration table during the SDS page of Fig. 3-6 analyzed.The increase of spectral bandwidth is owing to used HES-, glucosan-cause with the molecular weight distribution of PEG-derivant and the number of the polymer derivant that links to each other with described protein.Opposite with hydroxyl amino-glucosan or hydroxyl amino-HES derivant, corresponding PEG derivant is moved in gel and also is colored, and therefore makes that the video picture of protein spectra is complicated.
Brief Description Of Drawings
Fig. 1
Fig. 1 represents according to Embodiment C 4.2) the SDS page of the HES-EPO conjugate that makes analyzes.For gel electrophoresis, use XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Turnhout, B).According to the explanation of manufacturer under reducing condition, use 10%Bis-Tris gel and MOPS SDS electrophoretic buffer (the two is all from Invitrogen GmbH, Karlsruhe, D).
Swimming lane A: protein labeling SeeBlue Plus2 (Invitrogen GmbH, Karlsruhe, D) molecular weight marker from the top to the bottom: 188kD, 98kD, 62kD, 49kD, 38kD, 28kD, 17kD, 14kD, 6kD, 3kD
Swimming lane B: Embodiment C 4.2) crude reaction product (a)
Swimming lane C: Embodiment C 4.2) crude reaction product (b)
Swimming lane E: Embodiment C 4.2) crude reaction product (d)
Swimming lane F: Embodiment C 4.2) crude reaction product (c)
Swimming lane G: oxidized EPO Embodiment C 2).
Fig. 2
Fig. 2 represents according to Embodiment C 3) the SDS page of the HES-EPO conjugate that makes analyzes.For gel electrophoresis, use XCell Sure Lock Mini Cell (Invitrogen GmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Turnhout, B).According to the explanation of manufacturer under reducing condition, use 10% Bis-Tris gel and MOPS SDS electrophoretic buffer (the two is all from Invitrogen GmbH, Karlsruhe, D).
Swimming lane A: protein labeling Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D) molecular weight marker from the top to the bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD.
Swimming lane B: oxidized hEPO with as embodiment Aa) as described in the polymer derivant that the makes crude product after engaging.
Swimming lane C: oxidized hEPO with as embodiment Ab) as described in the polymer derivant that the makes crude product after engaging.
Swimming lane D: oxidized hEPO with as Embodiment B) as described in the polymer derivant that the makes crude product after engaging.
Swimming lane E: reaction pair shines: the hEPO that does not have polymer derivant.
Swimming lane F: as embodiment Aa) polymer derivant that makes as described in.
Swimming lane G: as embodiment Ab) polymer derivant that makes as described in.
Fig. 3-6
Fig. 3-6 represents that separately the SDS page of the HES-EPO conjugate that makes according to embodiment G analyzes.For gel electrophoresis, use XCell Sure Lock Mini Cell (InvitrogenGmbH, Karlsruhe, D) and Consort E143 power supply (CONSORTnv, Turnhout, B).According to the explanation of manufacturer under reducing condition, use 10%Bis-Tris gel and MOPS SDS electrophoretic buffer (the two is all from Invitrogen GmbH, Karlsruhe, D).
Fig. 3
Swimming lane A: protein labeling Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D) molecular weight marker from the top to the bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD.
Swimming lane B: oxidized hEPO and polymer derivant
ACrude product behind the joint.
Swimming lane C: polymer derivant
A
Swimming lane D: oxidized hEPO and polymer derivant
BCrude product behind the joint.
Swimming lane E: polymer derivant
B
Swimming lane F: oxidized hEPO and polymer derivant
CCrude product behind the joint.
Swimming lane G: polymer derivant
C
Swimming lane H: oxidized hEPO and polymer derivant
DCrude product behind the joint.
Swimming lane I: polymer derivant
D
Swimming lane K: reaction pair shines: the hEPO that does not have polymer derivant.
Fig. 4
Swimming lane A: protein labeling Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D) molecular weight marker from the top to the bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD.
Swimming lane B: oxidized hEPO and polymer derivant
HCrude product behind the joint.
Swimming lane C: polymer derivant
H
Swimming lane D: oxidized hEPO and polymer derivant
ICrude product behind the joint.
Swimming lane E: polymer derivant
I
Swimming lane F: oxidized hEPO and polymer derivant
ECrude product behind the joint.
Swimming lane G: polymer derivant
E
Swimming lane K: reaction pair shines: the hEPO that does not have polymer derivant.
Fig. 5
Swimming lane A: protein labeling Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D) molecular weight marker from the top to the bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD.
Swimming lane B: oxidized hEPO and polymer derivant
FCrude product behind the joint.
Swimming lane C: polymer derivant
F
Swimming lane D: reaction pair shines: the hEPO that does not have polymer derivant.
Swimming lane E: polymer protein labeling Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D) molecular weight marker from the top to the bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD.
Swimming lane F: oxidized hEPO and polymer derivant
GCrude product behind the joint.
Swimming lane G: polymer derivant
G
Swimming lane K: reaction pair shines: the hEPO that does not have polymer derivant.
Fig. 6
Swimming lane A: protein labeling Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D) molecular weight marker from the top to the bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD.
Swimming lane B: oxidized hEPO and polymer derivant
MCrude product behind the joint.
Swimming lane C: polymer derivant
M
Swimming lane D: oxidized hEPO and polymer derivant
JCrude product behind the joint.
Swimming lane E: polymer derivant
J
Swimming lane F: oxidized hEPO and polymer derivant
KCrude product behind the joint.
Swimming lane G: polymer derivant
K
Swimming lane H: reaction pair shines: the hEPO that does not have polymer derivant.
Swimming lane I: protein labeling Roti-Mark STANDARD (Carl Roth GmbH+Co.KG, Karlsruhe, D) molecular weight marker from the top to the bottom: 200KD, 119KD, 66KD, 43KD, 29KD, 20KD, 14.3KD.
Swimming lane J: oxidized hEPO and polymer derivant
LCrude product behind the joint.
Swimming lane K: polymer derivant
L
Swimming lane L: reaction pair shines: the hEPO that does not have polymer derivant.
Claims (35)
1. the polymer of the functionalization of formula I,
" polymer "-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5(I)
Symbol wherein has following implication
" polymer " is for being selected from aklylene glycol homopolymer, aklylene glycol copolymer, polyvinyl alcohol, polyvinylpyrrolidone, poly--1,3-dioxolanes, poly--1,3, the homopolymer of the solubility straight or branched of 6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid and polysaccharide or random copolymer with and derivant;
R
1、R
2、R
3、
R
4, R
5Be hydrogen, alkyl, aryl;
M is 2 to 4, wherein at m group CR
1R
2In residue R
1And R
2Can be identical or different;
N is 0 to 20;
O is 0 to 20, and wherein in the situation of n=0, o is not 0, wherein at o group CR
3R
4In residue R
3And R
4Can be identical or different;
R is 0 or 1;
X is-(CR
8R
9)
pO-,-(CR
8R
9)
pS-,-(CR
8R
9)
pNR
6-,-(CR
8R
9)
pOC (O)-,-(CR
8R
9)
pC (O) O-,-(CR
8R
9)
pC (G) N (R
10) O-,-(CR
8R
9)
pN (R
11) O-,
Wherein one or more groups-(CR
8R
9)-can be replaced by W, thus a kind of chemically rational group formed;
W is O, NR
12, C (G);
G is S, O, NR
14
R
6、R
7、R
8、
R
9、R
10、
R
11, R
12, R
14Be hydrogen, alkyl, aryl,
P is 0 to 20, wherein at p group CR
8R
9In residue R
8And R
9Can be identical or different;
Group wherein
-(X)
r-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5Being positioned at intermediary group covalency with at least one end group of " polymer " or at least one links to each other.
2. the polymer of functionalization as claimed in claim 1, wherein X is
-(CR
8R
9)
pN (R
11) O-or-(CR
8R
9)
pC (G) N (R
10) O-.
3. the polymer of functionalization as claimed in claim 1 or 2, wherein said polymer is hydroxyalkyl starch, glucosan or ethylene glycol homopolymer.
4. as the polymer of any described functionalization in the claim 1 to 3, group-[(CR wherein
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2OCH
2CH
2-.
5. the conjugate of formula II
Symbol wherein has following implication
" polymer " is for being selected from aklylene glycol homopolymer, aklylene glycol copolymer, polyvinyl alcohol, polyvinylpyrrolidone, poly--1,3-dioxolanes, poly--1,3, the homopolymer of the solubility straight or branched of 6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid and polysaccharide or random copolymer with and derivant;
R
1、R
2、R
3、
R
4Be hydrogen, alkyl, aryl;
M is 2 to 4, wherein at m group CR
1R
2In residue R
1And R
2Can be identical or different;
N is 0 to 20;
O is 0 to 20, and wherein in the situation of n=0, o is not 0, wherein at o group CR
3R
4In residue R
3And R
4Can be identical or different;
R is 0 or 1;
X is-(CR
8R
9)
pO-,-(CR
8R
9)
pS-,-(CR
8R
9)
pNR
6-,-(CR
8R
9)
pOC (O)-,-(CR
8R
9)
pC (O) O-,-(CR
8R
9)
pC (G) N (R
10) O-,-(CR
8R
9)
pN (R
11) O-,
Wherein one or more groups-(CR
8R
9)-can be replaced by W, thus a kind of chemically rational group formed;
W is O, NR
12, C (G);
G is S, O, NR
14
R
6、R
7、R
8、
R
9、R
10、
R
11, R
12, R
14Be hydrogen, alkyl, aryl, preferred hydrogen,
P is 0 to 20, wherein at p group CR
8R
9In residue R
8And R
9Can be identical or different;
R
13Be hydrogen, alkyl, aryl;
" protein " is the aminoacid sequence that makes by at least 2 amino acid whose reactions group wherein
Being positioned at intermediary group covalency with at least one end group of " polymer " and " protein " or at least one links to each other.
6. conjugate as claimed in claim 5, wherein X is
-(CR
8R
9)
pN (R
11) O-or-(CR
8R
9)
pC (G) N (R
10) O-.
7. as claim 5 or 6 described conjugates, wherein said polymer is hydroxyalkyl starch, glucosan or ethylene glycol homopolymer.
8. as any described conjugate in the claim 5 to 7, wherein said " protein " is selected from EPO, G-CSF, the VII factor, the IX factor, IFN β, AT III, A1AT, the VIII factor and APC.
9. as any described conjugate, wherein group-[(CR in the claim 5 to 8
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2OCH
2CH
2-.
10. method for preparing the polymer of functionalization, it comprises the polymer with formula III
" polymer "-(CR
8R
9)
pThe step that the chemical compound of-Y (III) and formula IV reacts,
Q-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5(IV), symbol wherein has following implication
" polymer " is for being selected from aklylene glycol homopolymer, aklylene glycol copolymer, polyvinyl alcohol, polyvinylpyrrolidone, poly--1,3-dioxolanes, poly--1,3, the homopolymer of the solubility straight or branched of 6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid and polysaccharide or random copolymer with and derivant;
R
1、R
2、R
3、
R
4, R
5Be hydrogen, alkyl, aryl;
M is 2 to 4, wherein m group CR
1R
2In residue R
1And R
2Can be identical or different;
N is 0 to 20;
O is 0 to 20, and wherein in the situation of n=0, o is not 0, wherein at o group CR
3R
4In residue R
3And R
4Can be identical or different;
Thereby Y and Q be suitable for reacting each other the functional group that provides one of following linking group :-O-,-S-,-NR
6-,-OC (O)-,-C (O) O-,-C (G) N (R
10) O-,-N (R
11) O-,
Wherein one or more groups-(CR
8R
9)-can be replaced by W, thus a kind of chemically rational group formed;
W is O, NR
12, C (G);
G is S, O, NR
14
R
6、R
7、R
8、
R
9、R
10、
R
11, R
12, R
14Be hydrogen, alkyl, aryl;
P is 0 to 20, wherein at p group CR
8R
9In residue R
8And R
9Can be identical or different; Group-(CR wherein
8R
9)
pThe end group of-Y and " polymer " or be positioned at intermediary group covalency and link to each other.
11. method as claimed in claim 10, thus wherein Y and Q are suitable for reacting each other the functional group that provides following linking group:
-N (R
11) O-or-C (G) N (R
10) O-.
12. as claim 10 or 11 described methods, wherein said polymer is hydroxyalkyl starch, glucosan or ethylene glycol homopolymer.
13. as any described method, wherein group-[(CR in the claim 10 to 12
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2OCH
2CH
2-.
14. can use the polymer of the functionalization that any described method obtains in the claim 10 to 13.
15. prepare the method for conjugate, it comprises the step that the protein as the functionalization of the polymer of any described functionalization in claim 1 to 4 or 14 and formula V is reacted,
" protein "-Z (V) wherein Z be comprise the group of carbonyl or be suitable for forming carbonyl group or can with the other group of the polymer reaction of functionalization, wherein at least one end group of Z and " protein " or at least one are positioned at intermediary group covalency and link to each other.
16. method as claimed in claim 15 is wherein used the polymer as any described functionalization in the claim 2 to 4.
17. as claim 15 or 16 described methods, wherein " protein " is selected from EPO, G-CSF, the VII factor, the IX factor, IFN β, AT III, A1AT, the VIII factor and APC.
18. prepare the method for conjugate, it step that comprises has
A) with the polymer of formula III
" polymer "-(CR
8R
9)
p-Y (III) reacts with the chemical compound of formula IV,
Q-[(CR
1R
2)
mO]
n[CR
3R
4]
o-ONHR
5(IV) symbol wherein has following implication
" polymer " is for being selected from aklylene glycol homopolymer, aklylene glycol copolymer, polyvinyl alcohol, polyvinylpyrrolidone, poly--1,3-dioxolanes, poly--1,3, the homopolymer of the solubility straight or branched of 6-trioxane, ethylene/copolymer-maleic anhydride, polyamino acid and polysaccharide or random copolymer with and derivant;
R
1、R
2、R
3、
R
4, R
5Be hydrogen, alkyl, aryl;
M is 2 to 4, wherein m group CR
1R
2In residue R
1And R
2Can be identical or different;
N is 0 to 20;
O is 0 to 20, and wherein in the situation of n=0, o is not 0, wherein at o group CR
3R
4In residue R
3And R
4Can be identical or different;
Thereby Y and Q be suitable for reacting each other the functional group that provides one of following linking group :-O-,-S-,-NR
6-,-OC (O)-,-C (O) O-,-C (G) N (R
10) O-,-N (R
11) O-,
Wherein one or more groups-(CR
8R
9)-can be replaced by W, thus a kind of chemically rational group formed;
W is O, NR
12, C (G);
G is S, O, NR
14
R
6、R
7、R
8、
R
9、R
10、
R
11, R
12, R
14Be hydrogen, alkyl, aryl;
P is 0 to 20, wherein at p group CR
8R
9In residue R
8And R
9Can be identical or different; Group-(CR wherein
8R
9)
pAt least one end group of-Y and " polymer " and/or at least one are positioned at intermediary group covalency and link to each other, obtain thus a kind of functionalization polymer and
B) protein with the functionalization of the polymer of the functionalization that obtains in the step a) and formula V reacts,
" protein "-Z (V) wherein Z be comprise the group of carbonyl or be suitable for forming carbonyl group or can with the other group of the polymer reaction of functionalization, wherein at least one end group of Z and " protein " and/or at least one are positioned at intermediary group covalency and link to each other.
19. can use any conjugate that described method obtains in the claim 15 to 18.
20. be used for human or animal body Therapeutic Method as any described conjugate in claim 5 to 9 or 19.
21. comprise the pharmaceutical composition as any described conjugate in claim 5 to 9 or 19 for the treatment of effective dose.
22. pharmaceutical composition as claimed in claim 21, it also comprises at least a acceptable diluents, auxiliary agent or carrier.
23. as the polymer of any described functionalization in the claim 1 to 3, wherein o is 2 to 20.
24. as the polymer of any described functionalization in the claim 1 to 3, wherein group-[(CR
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2(CH
3) CH
2CH
2-.
25. as any described conjugate in the claim 5 to 8, wherein o is 2 to 20.
26. as any described conjugate, wherein group-[(CR in the claim 5 to 8
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2(CH
3) CH
2CH
2-.
27. as any described method in the claim 10 to 12, wherein o is 2 to 20.
28. as any described method, wherein group-[(CR in the claim 10 to 12
1R
2)
mO]
n[CR
3R
4]
o-be-CH
2CH
2(CH
3) CH
2CH
2-.
29. can use polymer as the functionalization of claim 27 or 28 described methods acquisitions.
30., wherein will react as the protein of the functionalization of the polymer of any described functionalization in the claim 23,24 or 29 and formula V as claim 15 or 17 described methods.
31. method as claimed in claim 18, wherein o is 2 to 20.
32. the conjugate that can obtain with method as claimed in claim 30.
33. be used for human or animal body Therapeutic Method as any described conjugate in the claim 25,26 or 32.
34. comprise the pharmaceutical composition as any described conjugate in the claim 25,26 or 32 for the treatment of effective dose.
35. pharmaceutical composition as claimed in claim 34, it also comprises at least a acceptable diluents, auxiliary agent or carrier.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EPPCT/EP03/08858 | 2003-08-08 | ||
| EPPCT/EP03/08829 | 2003-08-08 | ||
| PCT/EP2003/008829 WO2004024776A1 (en) | 2002-09-11 | 2003-08-08 | Method of producing hydroxyalkyl starch derivatives |
| EPPCT/EP03/08859 | 2003-08-08 | ||
| US60/552,157 | 2004-03-11 | ||
| EP04005872.9 | 2004-03-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1863549A true CN1863549A (en) | 2006-11-15 |
Family
ID=36994619
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2004800295229A Pending CN1863549A (en) | 2003-08-08 | 2004-08-06 | Conjugates of a polymer and a protein linked by an oxime linking group |
| CN2004800226500A Expired - Fee Related CN1832762B (en) | 2003-08-08 | 2004-08-06 | Conjugates of hydroxyalkyl starch and g-csf |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2004800226500A Expired - Fee Related CN1832762B (en) | 2003-08-08 | 2004-08-06 | Conjugates of hydroxyalkyl starch and g-csf |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN1863549A (en) |
| TW (1) | TWI356065B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102573920A (en) * | 2009-07-27 | 2012-07-11 | 利普森技术有限公司 | Glycopolysialylation of non-blood coagulation proteins |
| CN103269723A (en) * | 2010-12-22 | 2013-08-28 | 巴克斯特国际公司 | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
| CN103370082A (en) * | 2010-07-30 | 2013-10-23 | 巴克斯特国际公司 | Nucleophilic catalysts for oxime linkage |
| CN104530182A (en) * | 2009-07-27 | 2015-04-22 | 利普森技术有限公司 | Glycopolysialylation of non-blood coagulation proteins |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2070950A1 (en) * | 2007-12-14 | 2009-06-17 | Fresenius Kabi Deutschland GmbH | Hydroxyalkyl starch derivatives and process for their preparation |
| WO2014080730A1 (en) * | 2012-11-22 | 2014-05-30 | 株式会社糖鎖工学研究所 | Glycosylated linker, compound containing glycosylated linker moiety and physiologically active substance moiety or salt thereof, and methods for producing said compound or salt thereof |
| CN104017069A (en) * | 2014-06-23 | 2014-09-03 | 山东齐都药业有限公司 | Hydroxyethyl starch derivative modified bovine serum albumin conjugate containing aldehyde groups and preparation method thereof |
| US20180092993A1 (en) * | 2016-09-01 | 2018-04-05 | Life Technologies Corporation | Compositions and methods for enhanced fluorescence |
-
2004
- 2004-08-06 CN CNA2004800295229A patent/CN1863549A/en active Pending
- 2004-08-06 TW TW093123642A patent/TWI356065B/en not_active IP Right Cessation
- 2004-08-06 CN CN2004800226500A patent/CN1832762B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102573920A (en) * | 2009-07-27 | 2012-07-11 | 利普森技术有限公司 | Glycopolysialylation of non-blood coagulation proteins |
| CN104530182A (en) * | 2009-07-27 | 2015-04-22 | 利普森技术有限公司 | Glycopolysialylation of non-blood coagulation proteins |
| CN103370082A (en) * | 2010-07-30 | 2013-10-23 | 巴克斯特国际公司 | Nucleophilic catalysts for oxime linkage |
| CN103269723A (en) * | 2010-12-22 | 2013-08-28 | 巴克斯特国际公司 | Materials and methods for conjugating a water soluble fatty acid derivative to a protein |
| CN103269723B (en) * | 2010-12-22 | 2017-04-05 | 百深有限责任公司 | For couple water soluble derivative of fatty acid and the material and method of protein |
Also Published As
| Publication number | Publication date |
|---|---|
| TWI356065B (en) | 2012-01-11 |
| CN1832762B (en) | 2012-09-05 |
| TW200519127A (en) | 2005-06-16 |
| CN1832762A (en) | 2006-09-13 |
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