JPH07196925A - Reagent that forms peg hydrazone and peg oxime bonds and protein derivative thereof - Google Patents

Abstract

PURPOSE: To obtain a compound having a specified chemical structure which can modify a polypeptide through a bond of a water-soluble polymer without lowering the biological activity of the polypeptide.

CONSTITUTION: There is provided a compound having a chemical structure of the formula (wherein X is O or S; Q is NHNH₂ or C₆H₄-NHNH₂; Y is O, OCH₂, NH, NHNH, O-CO-CH₂CH₂, or NHCO-N-NHNH; and P is a water-soluble polymer). This compound is mixed with a polypeptide such as erythropoietin to obtain a water-soluble polymer-modified polypeptide.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

The present invention relates to water-soluble polymers, such as monomethoxy poly (ethylene glycol), which are modified to form hydrazone bonds with aldehyde groups on proteins and also modified with these water-soluble polymers. Regarding protein molecules.

The present invention relates to such water-soluble polymers modified to form oxime bonds, and protein molecules modified thereby.

Proteins and other similar organic molecules are
It can be chemically modified by covalent conjugation to water-soluble organic polymers such as polyethylene glycol (PEG). The generation of such a protein conjugate is
This is important because the attachment of water-soluble polymers imparts desirable properties to the polypeptide. These desirable properties include, for example, increased solubility in aqueous solutions, increased stability during storage, reduced immunogenicity, increased resistance to enzymatic degradation, compatibility with a wider variety of drug delivery systems. , And increased in vivo half-life. These properties, which result from derivatizing the polypeptide with PEG or other water-soluble polymers, are problematic when used as a therapeutic agent to inject the polypeptide into the body or in assays, usually immunoassays. It is especially important when used for the detection and / or quantification of compounds.

The attachment of reporter groups, ligands, etc. to proteins through the carbohydrate moiety of glycoproteins has been described [Weber, P. et al. And Hof, L .; (1975) Biochemical and Biophysical Research Communication (Biochem. Biophys. Res. Com.
m. ) 65 , 1298-1302; O'Shannessy, D.M. J. Dobersen, M .; J. And Quares (Q
arles), R.A. H. (1984) Immunology
Letters (Immunol. Lett.) 8 , 273-
277; O'Shannessy
y), D.I. J. And Quarles,
R. H. (1985) Jarbal of Applied
Biochemistry (J. Appl. Bioche
m. ) 7 , 347-355; Chua, M .;
-M. Fan, S.H. -T. , And Karush, F .; (1984) Bio Himika
Eto Biophysical Actor (Biochim.Bi
ophys. Acta), 800 , 291-300;
O'Shannessy, D.M.
J. And Wilchek, M .; (1
990) Analytical Biochemistry (Ana)
lyt. Biochem. ) 191 , 1-8; Koppel, G .; A. (1990) Bioconjugate Chemistry (Bioconjgate C)
hem. ) 1 , 13-23]. A number of groups, such as biotin, fluorescent probes, anti-cancer compounds, and solid supports were covalently bound in this way.

US Pat. No. 4,847,325 describes PE
It describes the possibility of synthesizing G-amine, PEG-hydrazide or PEG-hydrazine and coupling it to glycoproteins. However, what could be the synthesis of these PEG-derivatives, the ability of these PEG-derivatives to denature oxidized glycoproteins, and the resulting biological properties of these putative PEG-proteins? No experimental evidence of this has been given.

Publications by Kogan,
T. P. , Synthetic Communications (S
ynthetic Communications),
22 (16) , 2417-2424 (1992),
The synthesis of monomethoxy poly (ethylene glycol) -hydrazide is described.

The enzyme peroxidase has been modified with PEG through its carbohydrate group [Urrigoit (Urr
utigoity), M.A. And Soupp
e), J. (1989) Biocatalysis (Bioca
2 ), 145-149]. In this modification, PEG-diamine was reacted with oxidized peroxidase and the resulting imine was reduced with borohydride to form a stable bond between PEG and the carbohydrate group on the protein. Three molecules of PEG-20,000 were attached to this enzyme. A possible problem when using PEG-diamines in this method is the intermolecular crosslinking that occurs between the PEG diamine that functions as a crosslinker and the protein molecule. Another disadvantage of using PEG-diamine is that it consumes two available aldehyde groups for each PEG-diamine attached to the protein, thus reducing the potential number of sites for incorporation into PEG. .

In addition to the hydrazones that form mPEG derivatives, a series of oxime-forming mPEG derivatives are also synthesized. Oximes are formed by the reaction of hydroxylamine or oxylamine derivatives with aldehyde or ketone groups. Polystyrene substituted benzophenone oximes were used as supports for solid phase peptide synthesis [DeGrado, W. et al. F. And Kaiser, E .; T. (1980), Journal of Organic Chemistry (JO
rg. Chem. ) 45 , 1295-1300]. In this example, the growing peptide chain is coupled to the oxime group via an ester bond. The substituted oxime bond is very stable, and even the unsubstituted aldoxime is reacted in the presence of silica gel in the presence of 100 to cause the reaction.
It shows excellent stability against the Beckmann rearrangement, which requires 60 hours at 0 ° C. [March, J. et al.
(1985) Advanced Organic Chemistry (Advanced Organic Chemistry)
try), New York-John Willie &
Sands (John Wiley & Sons), p9
87-989]. The oxime bond was used to couple morpholinodoxorubicin to an antibody [Mueller, B. et al. M. , Lasidro (Wra
sidlo), W. A. And Reisfeld (Rei
sfeld), R.S. A. (1990) Bioconjugate Chemistry
m. ) 1 , 325-330]. In this example, the ketone group of morpholinodoxorubicin was reacted with aminooxyacetic acid. The newly coupled free acid group was activated, and morpholinodoxorubicin was attached to the free amino group of lysine on the monoclonal antibody.

A number of proteins have been modified by PEG. For an overview, see Inada, Y. et al. Yoshimoto, T .; Matsushima, A .; And Saito, Y. (19
86) Trends Biotechnology (Trends)
Biotechnol. ) 4: 68-73. In this field, a number of patents have been issued and applications have been published, as listed below: US Pat. No. 4,179,337; US Pat. No. 4,609,546.
U.S. Pat. No. 4,261,973; U.S. Pat. No. 4,
055,635; U.S. Pat. No. 3,960,830;
U.S. Pat. No. 4,415,665; U.S. Pat. No. 4,41
2,989; U.S. Pat. No. 4,002,531; U.S. Pat. No. 4,414,147; U.S. Pat.
948; U.S. Pat. No. 4,732,863; U.S. Pat. No. 4,745,180; European Patent (EP) No. 152,
847; European Patent (EP) No. 98,100, 198.
Published on January 11, 4th. The above patents and patent publications also describe the use of protein denaturants of other water-soluble polymers, including but not limited to: polypropylene glycol (PP
G), polyoxyethylated polyol (POP), heparin, heparin fragments, dextran, polysaccharides, polyamino acids such as proline, polyvinyl alcohol (PV
A) and other water-soluble organic polymers.

Recent patent specification (WO90 / 128)
74) describes the preparation of mPEG-EPO which contains genetically engineered cysteine residues. The cysteine-specific mPEG reagent is then covalently attached to the genetically engineered free sulfhydryl group. Only one mPEG molecule could be incorporated into EPO and no evidence of this incorporation was presented. Also, the biological or biophysical properties of the resulting mPEG-EPO were not described.

Erythropoietin is a glycoprotein that regulates the production of red blood cells. Erythropoietin exerts its biological properties by binding to receptors on erythrocyte precursors [Krants (Krant).
z), S.S. B. , Blood , 77: 419-.
434 (1991)]. Binding of erythropoietin to its receptor causes erythroid precursors to proliferate and differentiate into mature erythrocytes. Other growth factors, such as interleukin 3 or granulocyte-macrophage-colony stimulating factor, are also involved in erythropoiesis, along with cofactors such as iron, folic acid and vitamin B12. Erythropoietin is currently approved for use in anemia of chronic renal failure and HIV infection in both dialysis and predialysis patients and for use in combination with zidovudine treatment. Current researched uses of erythropoietin include anemia of cancer, pre-surgical blood self-donation, and peripheral surgical adjuvant treatment.

Erythropoietin consists of 165 amino acids containing two disulfide bridges. Erythropoietin has four carbohydrate chains emanating from the protein backbone. Three of the carbohydrate groups are N-linked and are attached to asparagine 24, 38 and 83. Also, there is one O-linked carbohydrate group fixed to serine 126. The carbohydrate chain is branched and consists of fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, and sialic acid. The carbohydrate composition of erythropoietin is described by Sasaki, H. et al. , Bosner,
B. Dell, A .; And Fukuda, M .; (1
987) Journal of Biological Chemistry (J. Biol. Chem.) 262 , 12059.
Inhomogeneous as determined by -12076.
Carbohydrate groups are about 40% by weight of protein. The carbohydrate groups on erythropoietin are believed to increase the solubility of erythropoietin and prolong its serum half-life.

There are several limitations regarding the ability of a polypeptide to be covalently conjugated to a water-soluble polymer and the extent to which the polypeptide can be modified. Different water-soluble polymeric reagents will vary with respect to the functional group that provides the coupling to the amino acid residue in the polypeptide in question. Specific functional groups couple the water-soluble polymer to specific amino acid residues.

MPEG reagents with different properties, such as succinimidyl carbonate-PEG, succinimidyl succinate-PEG, imidate-PEG, cyanuric chloride-PEG, carbonyldiimidazole-PE.
G, and PEG-phenyl carbonate derivative (4-
Modification of lysine residues using nitrophenol and 2,4,5-trichlorophenol) has been described. This application includes novel carbohydrate PEG modifiers that have different specificities for oxidized carbohydrate groups, similar to different lysine modified PEG derivatives.

Glycoproteins, ie, polypeptides covalently joined to one or more carbohydrate molecules,
The presence of carbohydrate molecules on the polypeptide provides an additional opportunity to provide different methods of derivatization of the water-soluble polymer of the polypeptide. The water-soluble polymeric reagent can be coupled directly to the glycoprotein's carbohydrate moiety, as opposed to the amino acid polypeptide backbone of the glycoprotein, ie, the various functional groups present on the polypeptide. Water-soluble reagents can be used as a polypeptide because of charge substitution, steric hindrance, different amino acid residues in the active site, and other problems that can disrupt the structure and function of the polypeptide component of water-soluble polymer-modified glycoproteins. It may be advantageous to couple to the carbohydrate portion of the glycoprotein, rather than to the backbone amino acids.

By providing a water-soluble polymeric reagent capable of coupling to the carbohydrate portion of a glycoprotein, the biological activity of the protein, which would be adversely affected through coupling at other amino acid residues, is substantially affected. It is possible to covalently attach a water-soluble polymer to a protein without adversely affecting it. The present invention relates to a water-soluble organic group that forms a hydrazone bond with an aldehyde group on a polypeptide or a group having similar chemical reactivity, such as a ketone, lactol, an activated carboxylic acid or an activated carboxylic acid derivative. Methods and compositions for modifying polypeptides with polymeric derivatives, ie, water-soluble polymeric reagents, are provided. Novel hydrazide, semicarbazide, aryl hydrazide, thiosemicarbazide, hydrazide carboxylate, carbonic dihydrazide, carbazide, and thiocarbazide derivatives of polyethylene glycol (PEG) and other water soluble polymers are provided. One or more water soluble polymer reagents can be coupled to individual polypeptides or similar organic molecules to form hydrazones that attach the polypeptides to the water soluble polymer.

Another aspect of the present invention is to provide proteins, especially glycoproteins, modified by covalent attachment of derivatives of water-soluble polymers with hydrazone linkages.

Also disclosed are methods and compositions for modifying a polypeptide with a derivative of a water-soluble organic polymer that forms an oxime bond with the aforementioned aldehyde or similarly reactive group. Provided are novel oxylamine derivatives of polyethylene glycol (PEG) and other water-soluble polymers, as listed below, wherein one or more water-soluble polymer reagents are added to individual polypeptides. Alternatively, it can be coupled to similar organic molecules to form oximes that attach the polypeptide to the water-soluble polymer.

Another aspect of the invention is to provide proteins, especially glycoproteins, modified by the covalent attachment of derivatives of oxylamine water-soluble polymers.

The water-soluble polymer reagent of the present invention is a homopolymer of polyethylene glycol, a homopolymer of polypropylene glycol, a copolymer of ethylene glycol and propylene glycol (wherein the homopolymer and copolymer are not substituted, or 1 Substituted at the ends with alkyl groups), polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, polyvinyl ethyl ethers, and α, β-poly [(2-
Hydroxyethyl) -DL-aspartamide] and other water-soluble organic polymer derivatives that form hydrazone and oxime bonds. The water-soluble polymer of polyethylene glycol is polyethylene glycol in which one of the terminal hydroxyl groups is modified with an R group,
That is, RO-PEG (where R is alkyl, aryl, alkylaryl, aroyl, alkanoyl, benzoyl, arylalkyl ether, cycloalkyl,
Can be cycloalkylaryl and the like). The water-soluble polymers listed are merely examples of those represented by P. Various derivatives of the specifically mentioned water-soluble polymers are also envisaged, provided that the derivatives are water-soluble. More preferably, the water-soluble polymer P is selected from the group consisting of polyethylene glycol and its derivatives, the monomethyl ether of polyethylene glycol (mPEG) being particularly preferred (to avoid cross-linking between proteins).

Polypeptides of interest for derivatization of water-soluble polymers with the water-soluble polymers of the present invention include hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies. Erythropoietin (EPO), especially recombinant erythropoietin,
Derivatization of water-soluble polymers of and their precursors, intermediates and mimetics is of particular importance.

Another aspect of the present invention is the partial oxidation and subsequent combination of (i) a semicarbazide derivative of monomethoxypoly (ethylene glycol) (mPEG) with 17-25 mP / molecule of erythropoietin.
Producing an mPEG-derivatized erythropoietin molecule containing an EG molecule (conjugated through a hydrazone bond),
(Ii) in combination with a carboxylate hydrazide derivative of mPEG to produce a derivatized EPO containing about 22-32 mPEG / EPO (conjugated through a hydrazone bond), and (iii) in combination with an oxylamine derivative of mPEG, About 3-36mPEG / EP
The purpose is to provide erythropoietin (all measured by retention time on gel filtration) that produces derivatized EPO containing O (conjugated through oxime bonds).

Another aspect of the invention is to provide a method of activating a polypeptide for covalent conjugation of a water-soluble polymer of the invention with a reagent.

Definitions The term "water-soluble polymer reagent" as used herein means a water-soluble polymer modified to contain a functional group that provides for the covalent attachment of the water-soluble polymer to the polypeptide. To do.

The term "polypeptide" as used herein means polypeptides of various sizes, including larger polypeptides (often called proteins), smaller peptides, and glycoproteins.

The term "oxidative activatable group", as used herein, means a functional group, polyol, lactol, that reacts with the hydrazide or oxylamine moieties of the compounds of the present invention after exposure of the functional group to oxidizing conditions. , Amines, phenols, carboxylic acids or derivatives of carboxylic acids. The oxidatively activatable group present on the glycoprotein polypeptide can be present on the carbohydrate portion of the glycoprotein or on the amino acid residue portion of the glycoprotein. An example of an oxidatively activatable group is, but not limited to, a hydroxyl present on the carbohydrate portion of a glycoprotein. The hydroxyl group can be oxidized to a hydrazide-reactive aldehyde or an oxylamine-reactive aldehyde, depending on the derivative used.

The term "partial oxidation" as used herein means a process of oxidation that proceeds to such an extent that it does not completely destroy the biological activity of the polypeptide to be oxidized.

The term "activated for conjugation" as used herein in reference to a polypeptide means partial oxidation of the polypeptide, wherein the degree of oxidation is at least one oxidatively activatable group. Is sufficient to convert one of the water-soluble polymer reagents of the present invention to a functional group capable of chemically reacting with one hydrazide moiety or an oxylamine moiety (or a moiety of a similar functional group).

The term "biological activity" as used herein means the biologically relevant properties of a compound, including: enzymatic activity, binding to receptors (including antibodies). Ability to bind, bind ligand, elicit an immune response, therapeutic activity, etc.

The term "antibody" as used herein includes both polyclonal and monoclonal antibodies having the sequences of natural immunoglobulins, synthetic antibody derivatives and the like; antibodies may be labeled with various labels, fluorescent, radioactive,
It can be modified to conjugate to enzymes, biotin / avidin, etc. Synthetic antibodies are naturally-occurring immunoglobulin sequences selected for mutated and altered binding specificities, typically single-chain, of various immunizing agents produced by genetically modified bacteria. It includes globulin gene-derivatized polypeptides, antibodies modified to contain modified constant regions, etc .; an overview of synthetic antibody derivatives based on the principles of formation of such antibodies can be found in Winter and Mill. Stain
in), Nature , 349: 293.
-299 (1991).

The present invention is a novel water-soluble polymer, for example a derivative of PEG, ie hydrazine or oxylamine of polyethylene glycol, for use in modifying a polypeptide to bind to a water-soluble polymer. A polypeptide denaturing reagent is provided. The water soluble polymer reagents of the present invention can be used to covalently attach a variety of water soluble polymers to the polypeptide of interest. The hydrazine and oxylamine derivatives of the water-soluble polymers of the present invention, ie, the reagents of the water-soluble polymer, can be covalently attached to the protein through reaction with aldehyde groups or other suitable functional groups present on the protein of interest. . Aldehyde groups can be introduced by partially oxidizing the hydroxyl groups (or other oxidatively activatable groups) on the polypeptide. Examples of oxidatively activatable groups include hydroxyl groups present on the carbohydrate portion of glycoproteins. A suitable method of oxidation, ie partial oxidation, is to treat the polypeptide in question with an oxidizing agent, such as periodate or other oxidizing agents known to those skilled in the art, or a portion of the protein in question. It involves adding an enzyme capable of catalyzing the oxidation reaction above, for example, galactose oxidase. Another aspect of the present invention is a molecule modified by a reagent molecule, ie a hydrazine or oxylamine derivative of a water-soluble polymer of the present invention, so as to be covalently attached to one or more water-soluble polypeptides. To provide a peptide.

Preferred formulas for compounds useful for coupling water-soluble polymers to polypeptides are as follows:

[0033]

Embedded image Hydrazine derivative (I) P—O—CH ₂ —CO—NHNH ₂ hydrazine derivative; (II) P—O—CO—NHNH ₂ hydrazine carboxylate derivative; (III) P—NH—CO—NHNH ₂ semicarbazide derivative; (IV) P-NH- CS-NHNH 2 thiosemicarbazide derivative; (V) P-NHCO- NHNHCO-NHN
H ₂ carbonic dihydrazide derivative; (VI) P-NHNHCONHNH ₂ carbazide derivative; (VII) P-NHNHCSNHNH ₂ thiocarbazide derivatives; (VIII) P-NH- CO-C 6 H 4 -NHNH 2 aryl hydrazide derivative; (IX) P _{-O-CO-CH 2 CH 2} -CO-N
HNH ₂ hydrazide derivative; oxylamine derivative (XIX) P-O-CH ₂ CH ₂ -CO-ON
_{H 2; (XX) P-} O-CH 2 CH 2 -O-CO-ON
_{H 2; (XXI) P-} O-CH 2 CH 2 -NH-CO-O
_{NH 2; (XXII) P-} O-CH 2 CH 2 -O-CS-ON
H ₂ ; (XXIII) P-O-CH ₂ CH ₂ -ONH ₂ ; (XXIV) P-O-CH ₂ CH ₂ -NH-CO-C
_{H 2 ONH 2; (XXV)} P-O-CH 2 CH 2 -O-CO-CH
_{2 -ONH 2; (XXVI) P} -O-CH 2 CH 2 -CH (OH) -
CH ₂ -ONH _2; and (XXVII) P-O-CH 2 CH 2 -CO-CH 2 -
ONH ₂ .

P represents a water-soluble organic polymer in the above formula. The water-soluble organic polymer in question has a hydroxyl attached to the polymer backbone and can be selected from known water-soluble polymers, including, but not limited to, the following water-soluble polymers: (A) Dextran and dextran derivatives such as dextran sulfate, P-amino cross-linked dextrin, and carboxymethyl dextrin, (b) cellulose and cellulose derivatives such as methyl cellulose and carboxymethyl cellulose, (c) starch and dextrin, and derivatives of starch. And hydroiractes (hy
(d) polyalkylene glycol and its derivatives, such as polyethylene glycol, methoxy polyethylene glycol, homopolymers of polyethylene glycol, homopolymers of polypropylene glycol, copolymers of ethylene glycol and propylene glycol (wherein said homopolymers and copolymers). Is unsubstituted or substituted at one end with an alkyl group), (e) heparin and heparin fragments, (f) polyvinyl alcohol and polyvinyl ethyl ether, (g) polyvinyl pyrrolidone, (h) α, β-poly [(2-hydroxyethyl)-
DL-aspartamide], and (i) polyoxyethylated polyol. Preferably, the water-soluble polymer P is
It is selected from dextran and dextran derivatives, dextrins and dextrin derivatives, more preferably polyethylene glycol and its derivatives. Water-soluble polymers of polyethylene glycol include polyethylene glycol in which one of the terminal hydroxyls has been modified with an R group, namely RO-PEG, where R is alkyl, aryl, alkylaryl, aroyl, alkanoyl,
Can be benzoyl, arylalkyl ether, cycloalkyl, cycloalkylaryl, etc.)
Includes. The water-soluble polymers listed are only examples of water-soluble polymers represented by P. Various derivatives of the specifically mentioned water-soluble polymers are also envisaged, provided that the derivatives are water-soluble. More preferably, the water-soluble polymer P is selected from the group consisting of polyethylene glycol and its derivatives, the monomethyl ether of polyethylene glycol (mPEG) being particularly preferred (to avoid cross-linking between proteins). When the polypeptide modified with the water-soluble polymer reagent of the present invention is used as a drug, the polymer P should be non-toxic.

The compounds of formulas I-IX are generally of the formula:

[0036]

[Chemical formula 22] Wherein, X is O or S, Q is selected from the group consisting of -NHNH ₂ and -C ₆ H ₄ -NHNH _2, and Y is _{-O -, - OCH 2 -,} - NH -, - NHNH -,
_{-O-CO-CH 2 CH 2} - and -NHCO-N-NH
Selected from the group consisting of NH-, and P is a water-soluble polymer (as in compounds I-IX).

The compounds of formulas XIX-XXVII are generally represented by the formula:

[0038]

Embedded image In the formula, X is C═O, C═S, CH ₂ or CHOH, and Q is selected from the group consisting of —ONH ₂ — and —CH ₂ —ONH ₂ —. It is, and Y is _{-O-CH 2 CH 2 -,} -
_{O-CH 2 CH 2 -O -} , - O-CH 2 CH 2 -N -, - O
It is selected from the group consisting of -CH ₂ CH ₂ -S- and -O-CH ₂ CH ₂ CH-, and P is a water-soluble polymer (as in the compound XIX~XXVII).

Formulas I, II, III, IV, V, VI, V
In addition to the molecules of II, VIII and IX, the invention provides the compounds of the formulas I, II, III, IV, V, VI, VII, VIII
And polypeptides modified by reaction with molecules of IX. Formulas I, II, III, IV, V, VI,
Polypeptides modified with reagents of water-soluble polymers of VII, VIII and IX have formulas X, X, respectively.
I, XII, XIII, XIV, XV, XVI, XVI
Can be represented by I and XVIII:

[0040]

Embedded image hydrazide modified polypeptide (X) [P-O- CH 2 -CO-NHN = CH
-] _N- Z, (XI) [P-O-CO-NHN = CH-] _n-
Z, (XII) [P-NH-CO-NHN = CH-] _n
-Z, (XIII) [P-NH-CS-NHN = CH-] _n
-Z, (XIV) [P-NHCO-NH-NHNHCO-
NHN = CH-] _n- Z, (XV) [P-HNNHCON = CH-] _n- Z, (XVI) [P-HNNHCSN = CH-] _n- Z, (XVII) [P-NH-CO-C ₆ H ₄ -NHN = CH
-] _n -Z, and (XVIII) [P-O- CO-CH 2 CH 2 -CO-
NHN = CH-] _n- Z, _where P is a water-soluble polymer as described above, Z is a polypeptide as described above, and n is 1-x.
, Where x is the maximum number of oxidatively activatable groups present in polypeptide Z. The C in the hydrazone bond formed between the water-soluble polymer reagent and Z originally exists on Z and is not a water-soluble polymer reagent.

Formulas XIX, XX, XXI, XXII, XX
III, XXIV, XXV, XXVI and XXVII
In addition to the molecule of formula I, the invention also provides a compound of formula XIX, XX, XX
I, XXII, XXIII, XXIV, XXV, XXV
I and polypeptides modified by reaction with XXVII. Formulas XIX, XX, XXI, XXII,
XXIII, XXIV, XXV, XXVI and XXV
Polypeptides modified with the water-soluble polymeric reagent of II have formulas XXVIII, XXIX, XX, respectively.
X, XXXXI, XXXII, XXXIII, XXXI
Can be represented by V, XXXV and XXXVI.

[0042]

Embedded image oxylamine modified polypeptide (XXVIII) [P-O- CH 2 CH 2 -CO-ON
_{= CH-] n -Z; (XXIX} ) [P-O-CH 2 CH 2 -O-CO-
_{ON = CH-] n -Z; (} XXX) [P-O-CH 2 CH 2 -NH-CO
_{-ON = CH-] n -Z; (} XXXI) [P-O-CH 2 CH 2 -NH-CS
_{-ON = CH-] n -Z; (} XXXII) [P-O-CH 2 CH 2 -ON = CH
_{-] n -Z; (XXXIII)} [P-O-CH 2 CH 2 -NH-CO
_{-CH 2 -ON = CH-] n -Z} ; (XXXIV) [P-O-CH 2 CH 2 -O-CO-
_{CH 2 -ON = CH-] n -Z} ; (XXXV) [P-O-CH 2 CH 2 -CH (OH)
_{-CH 2 -ON = CH-] n -Z} ; and (XXXVI) [P-O- CH 2 CH 2 -CO-CH 2
-ON = CH-] _n- Z, _where P is a water-soluble polymer as previously described, Z is a polypeptide as previously described, and n is in the range 1
~ X, where X is the maximum number of oxidatively activatable groups present in the polypeptide. The carbon atom in the oxime bond formed between the water-soluble polymer reagent and Z originally exists on Z and is not a water-soluble polymer reagent.

The polypeptide is x per polypeptide molecule
Can be modified by water-soluble polymer coupling up to, but it may be desirable to modify a given polypeptide with less than x water-soluble polymer molecules. It is not desirable to derivatize a polypeptide with the maximum number of water-soluble polymers, ie x water-soluble polymer / polypeptide molecule. This is because, for a given polymer, increasing the number of water-soluble polymers per molecule of the polypeptide may reduce the biological activity as compared to the unmodified polypeptide. See, for example, Figures 2, 4, 5, 9, 10 and 11 for some results obtained with water soluble polymer modified EPO.

Formulas X, XI, XII, XIII, XIV,
A hydrazone-binding compound of XV, XVI, XVII and XVIII and a formula XXVIII, XXIX, XXX,
XXXI, XXXII, XXXIII, XXXIV, X
Different methods of measuring the number of water-soluble polymer molecules attached to glycoprotein molecules, such as in the oxime-binding compounds of XXV and XXXVI, may give different results. For the purposes of this application, when we say that a polypeptide is derivatized with a given number of water-soluble polymer molecules / protein molecules, the number of water-soluble polymers given is an experimental value determined by the retention time of gel filtration chromatography. It is the numerical value decided.

Formulas X, XI, XII, XIII, XIV,
Synthesis of the compounds of XV, XVI, XVII and XVIII can result in a mixture of reaction products that differ from each other with respect to the exact number of water-soluble polymers attached to the polypeptide through the hydrazone linkage and the site where these hydrazone linkages are present. is there. Similarly, formula XXVIII,
XXXIX, XXX, XXXI, XXXII, XXXII
Synthesis of compounds of I, XXXIV, XXXV and XXXVI can result in a mixture of reaction products that differ from each other with respect to the exact number of water-soluble polymers attached to the polypeptide through the oxime bond and the site where these oxime bonds are present. is there. It will be appreciated that the molecular weight of P can vary considerably, since the polymer P is composed of varying amounts of many identical units. Furthermore, when P is said to have a given molecular weight, that molecular weight is only an approximation and may reflect the average molecular weight of a population of different molecules P with respect to the number of subunits present in the molecule. Generally, P is about 200-200,000,
Preferably 700-30,000, more preferably 2,
It will have a molecular weight in the range of 000 to 12,000. Suitable molecular weights for P are those of formulas I, II, III,
Molecules of IV, V, VI, VII, VIII and IX and formulas XXVIII, XXIX, XXX, XXXI, X
When coupling a molecule of XXII, XXXIII, XXXIV, XXXV and XXXVI to a polypeptide, it will vary according to the particular polypeptide to be modified and the particular water-soluble polymer selected. Individual polypeptide molecules may be prepared by one or more different water-soluble polymers of formula I, II, III, IV, V, VI,
Compounds of VII, VIII and IX (hydrazone),
Or formulas XX, XX, XXI, XXII, XXII
It can be derivatized by reacting I, XXIV, XXV, XXVI and XXVII (oximes), or any combination of hydrazones and oximes with different embodiments.

An advantage of the present invention is that it does not substantially diminish the biological activity of the polypeptide, or by similar chemical coupling methods and compounds known in the art, a similar number of identical water soluble polymers / polypeptides. It means that the polypeptide can be modified by the attachment of water-soluble polymers, with a reduced reduction in biological activity that is reduced by the attachment of molecules. Aspects of the biological activity of EPO include stimulating the formation of red blood cells. The biological activity of EPO is
Krantz, S.M. B. Blood (Bloo
d) , 77: 419-434 (1991).

Another advantage of the present invention is that of formulas I, II, II
Compounds of Formula I, IV, V, VI, VII, VIII and IX or Formulas XIX, XX, XXI, XXII, XXI
Polypeptides modified with compounds of II, XXIV, XXV, XXVI and XXVII are frequently used for modification of lysine (before the present invention) using active esters of mPEG to remove water soluble polymers. That is, by conjugating to a polypeptide, a greater degree of biological activity can be retained than when the same polypeptide is denatured to the same degree.
Thus, the present invention provides modified polypeptides that have the advantages associated with the covalent conjugation of water-soluble polymers while minimizing the loss of biological activity associated with denaturation. In the end, water-soluble polymers can be more highly derivatized, thus producing polypeptides having the advantages associated with otherwise higher degrees of derivatization, and these polypeptides being It has the same or a higher level of biological activity as the polypeptide derivatized with the water-soluble polymer to a lesser extent using the method.

Another advantage obtained when using hydrazone-forming derivatives of PEG (and other water-soluble polymers) instead of PEG-amines for coupling PEG to proteins is the hydrazide or oxylamine (or similar). Coupling of a compound) to an aldehyde yields a hydrazone or an oxime, respectively, but an amine-mediated coupling produces an imine, which is less stable than a hydrazone or an oxime, and is stable upon reduction. It is necessary to produce various derivatives. Thus, the use of amines instead of hydrazone-forming compounds requires an extra step.

Another advantage of the present invention is that higher levels of water-soluble polymers can be attached to glycoproteins when derivatives of other water-soluble polymers are used. The semicarbazide (formula III), thiosemicarbazide (formula IV) and carbonic acid dihydrazide (formula V) derivatives are of particular interest as they are more reactive than the comparable hydrazide derivatives of the present invention. Reactions involving the derivatization of EPO using the semicarbazides, thiosemicarbazides, and carboxylate hydrazides described herein can yield mPEs of up to about 31-34 for each molecule of EPO.
While resulting in the addition of G molecules, a similar reaction using the corresponding hydrazide derivative of EPO resulted in the addition of approximately 6-12 molecules of EPO for each molecule of EPO. Carbonic acid dihydrazide and hydrazide carboxylate derivatives and EPO
The reaction between and is up to 22 m for each molecule of EPO.
This resulted in the addition of PEG molecules. In order to allow the hydrazide derivative of mPEG to incorporate about 20 mPEG molecules for each molecule of EPO, very strong oxidizing conditions, eg
50 mM periodate, required 60 minutes incubation at room temperature. The subject mPEG semicarbazide and thiosemicarbazide derivatives produce EPO modified with PEG to a similar extent under more mild oxidizing conditions, such as 10 mM periodate, 5-15 minutes at 0 ° C. Could be used to Strong oxidizing conditions can adversely affect the structure and biological properties of many polypeptides, and thus PEG semicarbazide, carbonic dihydrazide, hydrazide carboxylate and thiosemicarbazide derivatives can cause the polypeptide to PEG (or other water soluble polymer). It may be a particularly suitable compound for modification with.

A new series of oxylamine derivatives of mPEG was synthesized and reacted with the oxidized carbohydrate groups of EPO. Some of the mPEG-oxylamine are
It showed high reactivity to oxidized carbohydrates. Also, lower numbers of mPEG can be introduced into EPO, and these are high in vivo as seen when using semicarbazide and carboxylate hydrazide (hydrazone forming) mPEG derivatives.
I was able to give it more activity. This lower number m
Incorporation of PEG is advantageous in that shorter and milder oxidizing conditions can be used. Also, smaller amounts of mPEG derivative can be used in the denaturing reaction.

Similarly, formula XXI, formula XXIV and formula X
When using the compound of XIII, a particularly high level of water-soluble polymer is attached to the glycoprotein compared to the formula XIX oxylamine derivative. Formula X described here
The reactions involving derivatization of EPO using XI and formula XXIV are 18 to 18 for each molecule of EPO, respectively.
The addition of mPEG / EPO and 31 mPEG molecules up to 19 resulted in the corresponding formulas XXII and X of EPO.
A similar reaction using IX oxylamine derivatives is described in EP
About 3-4 molecules of mPEG were added for each molecule of O. Perhaps more importantly, the biological activity of the resulting oxylamine-derivatized PEG-EPO is surprisingly high, even at more modest levels of attachment of the water-soluble polymer to EPO (description and figure below. 17). In this regard, the biological activity of the 12 mPEG isolated fractions of formula XXII is of particular importance (see Figures 16 and 17).

Long-acting mPEG- with high activity via coupling of mPEG to oxidised carbohydrate groups
The likelihood of generating EPO depends on the mPEG derivative chosen. Certain mPEG carbohydrate modified derivatives are not reactive enough to conjugate optimal amounts of mPEG onto EPO. Certain mPEG derivatives require large amounts of incorporation onto EPO due to the stability of the resulting bond. The optimal amount of mPEG incorporation is about 17-25 for semicarbazide derivatives, more preferably about 22.
And about 22-32 for the carboxylate hydrazide, more preferably about 31. mPEG-
The reactivity of oxylamine is about 3-36 mPEG / EPO
Is.

The water-soluble polymeric reagents of the present invention can be used to modify various polypeptides or similar molecules, where similar molecules are aldehyde groups or similar functional groups with similar chemical reactivity. Groups containing, for example, ketones, lactol, activated carboxylic acids or activated carboxylic acid derivatives, oxidation of hydroxyl groups, polypeptides of interest (carbohydrate moieties when the polypeptide is a glycoprotein, and in the primary sequence) Of other oxidative activatable groups present on amino acid residues present in, eg, serine, threonine, N-terminal of hydroxyl lysine), or hydrazine present on the polypeptide before and after oxidative treatment Or it can chemically react with the reactive groups of oxylamine. Polypeptides of interest include monoclonal antibodies, polyclonal antibodies, cytokines, growth factors, hormones, enzymes, protein or peptide ligands, and the like. Polypeptides of interest for modification with a molecule of a water-soluble polymeric reagent forming a hydrazone bond or oxime bond of the present invention may be derived from their natural source, genetically engineered cells such as E.
CHO transformed with expression vector for PO production
Can be isolated from cells, or in various
It can be produced by the in vitro synthesis method. Particularly preferred polypeptides for the purposes of the present invention are EPO and its precursors, intermediates and mimetics, whether human or recombinant.

Although most polypeptides can be modified using the water soluble polymeric reagents of the present invention,
It is particularly important to modify (1) the polypeptide for use as a drug, and (2) the polypeptide for use in assays. Polypeptides for use in the assay include specifically binding proteins, specifically binding proteins, and enzymes. Proteins that specifically bind include antibodies, hormone receptors, lectins and the like.

Various polypeptides may be coupled to different water-soluble polymers by the reagents of the water-soluble polymers of the invention and their method of use according to the invention.
It can be modified to different degrees of modification. Variable parameters, such as (1) number of water-soluble polymers coupled to individual polypeptide molecules, which is the activity of derivatized mPEG on EPO and the resulting mPEG-EPO.
The reactivity of about 3 to 36 molecules of mPEG / EPO, (2) the molecular weight of the water-soluble polymer, eg, 2,000 to 12,000 daltons,
(3) the structure of the water-soluble polymer, eg, monomethoxypoly (ethylene glycol), (4) the reaction conditions that carry out the reaction between the reagent of the water-soluble polymer and the polypeptide of interest, eg, temperature and duration, and (5) Oxidizing conditions that activate the polypeptide for denaturation for covalent conjugation, such as periodate at a concentration in the range of 10-40 μmol / mg protein, results in a water-soluble polymer-modified polypeptide. Can affect the biological properties of.

In a preferred embodiment of the invention, the activation of the polypeptide for covalent conjugation is carried out by denaturing the protein for denaturation with periodate (0.1-1,000 μmo).
(l / mg protein) and for a period ranging from 1 minute to 3 days,
More preferably 0.5-50 μmol periodate /
It is carried out by mixing with mg of protein for a range of 5 minutes to 180 minutes. In a preferred embodiment of the invention, the activation for conjugation is carried out by mixing the protein for denaturation with periodate at a temperature in the range of -10 to 50 ° C, preferably 0 to 30 ° C.

In a preferred embodiment of the present invention, when the protein for denaturation is EPO, EPO is represented by the formula II-
Derivatized with a compound of formula VII, preferably a compound of formula II-V, a compound of formula III, a semicarbazide, and a compound of formula I
The compound of I, a carboxylate hydrazide, is particularly preferred where the water soluble polymer P is monomethoxy polyethylene glycol (mPEG) and each molecule of EPO is 3-36, more preferably 17-25 molecules of monomethoxy polyethylene glycol. Derivatized (in the case of a semicarbazide), and more preferably with 22 to 32 molecules of monomethoxypolyethylene glycol (in the case of a carboxylate hydrazide), and the mPEG used has an average molecular weight of about 5000 daltons. Preferred reaction conditions for the preparation of mPEG5000 semicarbazide modified EPO are 5-60 minutes at 0-30 ° C. and 0.5-50 μmol periodate / mg EPO (using periodate as oxidizing agent). Is. EPO for modification by the water-soluble polymer reagents and methods of the invention is preferably obtained from genetically engineered cells, more preferably from CHO cells that have been genetically modified to produce EPO. EPO
By using the preferred water-soluble polymer reagents and methods for the modification of EPO, an unexpectedly extended biological half-life of EPO can be obtained, and increased hematocrit levels can be seen, eg , Figures 2, 4 and 5.

In another preferred embodiment, EPO is the formula X
X-XXVII compounds, more preferably formulas XXI-X
Derivatized with a compound of XIV, wherein the water-soluble polymer P is mPEG and each molecule of EPO has the formula XXI,
About 18 for XXIV and XXIII, respectively
Derivatize with -19 mPEG, 31 mPEG and 17 mPEG. These results show that the mPEG used is about 500
Obtained when it has an average molecular weight of 0 daltons. Formula XX
The compound of III had a fraction of 12mPEG, which fraction had comparable in vivo biological activity to 22mPEG semicarbazide and 31mPEG carboxylate hydrazide. Formulas XXI, XXIV and XXII
The reactivity for I (number of molecules of mPEG / EPO) is
It exceeded that for PEG hydrazide under the same reaction conditions. The preferred reaction conditions for the preparation of mPEG5000 oxime are more milder oxidation conditions than those of the corresponding hydrazides, such as shorter oxidation times or lower oxidizers, to produce higher in vivo biological activity. Can be a concentration of.

The present invention also provides a method of activating a polypeptide for conjugating a water-soluble polymer of the invention with a reagent, ie, covalent conjugation. These methods of activating the polypeptide for conjugation consist of partially oxidizing the piperonyl in question to oxidation. Partial oxidation involves the addition of oxidizing agents, such as periodate and other oxidizing agents known to those skilled in the art, or enzymes capable of catalyzing the oxidation reaction on the portion of the polypeptide in question, This can be achieved, for example, by adding galactose oxidase. A preferred method of partially oxidizing a polypeptide for activation for conjugation is 0.
Periodate at a concentration in the range of 1 to 1,000 μmol / mg protein for a period in the range of 1 minute to 3 days, more preferably at a concentration in the range of 0.5 to 50 μmol / mg protein. By adding the acid salt for a period ranging from 5 minutes to 180 minutes. The temperature at which the activation is carried out is preferably in the range of -10 to 50 ° C, more preferably 0 to 3
It is in the range of 0 ° C.

The salts of the macromolecules described herein, eg, polypeptides, water-soluble polymers and derivatives thereof, are present in (or isolated from) aqueous solutions of such molecules at various pH's. When) it will occur naturally. All salts of the indicated biologically active polypeptides and other macromolecules are considered to be within the scope of the present invention. Examples of such salts include alkali metal, alkaline earth metal and other metal salts of carboxylic acid residues, acid addition salts of amino residues (eg HC
l), and zwitterions formed by the reaction between carboxylic acid and amino residues in the same molecule. The compounds of the invention can be administered alone, but generally,
It will be administered in admixture with a pharmaceutical carrier or diluent selected with regard to the intended route of administration and standard pharmaceutical practice. The preparation can be injected parenterally, eg intraarterially or intravenously. This preparation
It can also be delivered by the oral, subcutaneous or intramuscular route. For parenteral administration, they can be used, for example, in the form of a sterile aqueous solution which contains other solutes, for example enough salts or glucose to render the solution isotonic. can do.

For the oral mode of administration, the target EPO
The composition can be used in the form of tablets, capsules, lozenges, powders, syrups, elixirs, aqueous solutions and suspensions. In the case of tablets, carriers that can be used include lactose, sodium citrate, and salts of phosphoric acid. Various disintegrants, such as starch,
And lubricants such as magnesium stearate are commonly used in tablets. For administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. Certain sweetening and / or flavoring agents can be added when the aqueous solution is required for oral use.

For administration to humans in the treatment of disease states responsive to treatment of EPO, the prescribing physician will ultimately determine the appropriate dose for a given human subject, and Is expected to vary according to body weight, age and individual response and the nature and severity of the patient's disease. The dosage of the pegylated form of the drug can generally be the amount used for the natural drug, although in some cases it is preferable to administer doses outside these limits, Or it may be necessary.

Also of the formulas I, II, III, IV, V, V
I, VII, VIII and IX, and formulas XIX, X
X, XXI, XXII, XXIII, XXIV, XX
It is important to provide the water-soluble polymeric reagents of V, XXVI and XXVII separately or in various combinations in the form of a kit to provide convenient and reproducible derivatization of the polypeptide in question. The kit in question is of formula I, II, III, IV, V, VI, VII, VIII
Or a reagent of a water-soluble polymer of IX, or formula XI
X, XX, XXI, XXII, XXIII, XXIV,
XXV, XXVI or XXVII water-soluble polymer reagents, buffers, oxidants, reaction indicator compounds,
A solution consisting of a protein concentration medium reagent, such as for the Bradford assay, can be included. The compounds included in the kit are designed to provide reproducibility and minimize errors.
It is preferably provided in a premedium portion and a premixed solution. The kit also preferably contains instructions. The instructions for use relate to various steps in practicing the subject method.

Synthesis of Derivatives of Water-Soluble Polymers The following synthesis of compounds of the invention is exemplary and not intended to limit the invention. Those skilled in organic chemistry will be able to devise various variations on the exemplified syntheses.

Synthesis of Hydrazone-Forming m-PEG Synthesis of mPEG-hydrazide There are several ways to synthesize mPEG-hydrazide. Two methods are shown.

MPEG5000 acid (20.8 g, 4 mm
ol) was dissolved in 30 ml dichloromethane and t-butylcarbazate (2.64 g, 8 mmol) in 15 ml dichloromethane was added, followed by 10 ml.
1.68 g (8 m) dissolved in dimethylformamide
(mol) dicyclohexylcarbodiimide was added. After allowing the reaction to proceed overnight at room temperature, the reaction mixture was filtered. The filtrate was concentrated and the resulting residue was taken up in dichloromethane. Add ether to add mPEG-
t-Butyl-carbazate was precipitated, which was filtered,
And dried. The product was placed in a dichloromethane / trifluoroacetic acid (1: 1) mixture. After 40 minutes, the solution was concentrated, redissolved in dichloromethane and ether was added. The product was collected by filtration. Yield 1
7.7 g. IR: (C = O) 1730, 1700. Analytical, calculated for N: 0.55. Found: N, 0.
44. An alternative method shown below converted the mPEG-alcohol to an ester. The mPEG-ester is then hydrolyzed with hydrazine to produce mPEG-hydrazide. mP
The synthesis of EG-esters is described by Royer,
G. P. And Anther Therma
aiah), G.I. M. (1979) Journal of
American Chemical Society (J. Am. C
hem. Soc. ), 101 , 3394-3395.

[0067]

[Chemical formula 26]

In a typical synthesis, mPEG-OH was dried in a high vacuum oven for about 5 hours at 85 ° C. After cooling, 5 g of mPEG-OH (molecular weight = 5000, 1 mm
ol) was dissolved in 5 ml of dry tetrahydrofuran. To 26.4 mg (1.1 mmol) sodium hydride was added 1 ml dry tetrahydrofuran. m
The PEG5000 solution was added dropwise to NaH. The mixture was stirred at room temperature for 1 hour under an argon atmosphere.
During this time the solution turned orange. Bromoacetyl acid benzyl ester (2.29 g, 10 mmol)
Was dissolved in 1 ml of dry tetrahydrofuran and this solution was added dropwise to the mPEG5000 mixture. The reaction mixture was stirred at room temperature under an atmosphere of argon overnight and then filtered. Add cold ether to the filtrate and add mP
The EG5000-benzyl ester was precipitated and the solid was collected and dried. Yield 4.5g. IR: (C
= O) 1752. This compound was purified by gel filtration on a LH-20 column and was added to methanol / methylene chloride (5:
Elute with 1).

The mPEG5000-benzyl ester was converted to hydrazide by treatment with hydrazine. In a typical experiment, 1.0 g mPEG5000-benzyl ester was dissolved in 3 ml methanol / methylene chloride (5: 1) under an argon atmosphere. Hydrazine (0.0
91 ml, 2.91 mmol) was added and the solution was stirred at room temperature for about 70 hours. Add this mixture to L
Placed on an H-20 column and eluted with methanol / methylene chloride (5: 1). The mPEG-hydrazide was separated from the hydrazide and precipitated with ether. The solid was collected by filtration. Yield 0.78g. IR (H ₂ N-C
= O): 1669. Analysis, calculated for N, 0.5
5. Found: N, 0.34.

In addition, mPEG2000-hydrazide, m
PEG6000-hydrazide, mPEG8500-hydrazide, and mPEG12000-hydrazide were synthesized by the procedure described above.

Of mPEG-hydrazinecarboxylate
Synthesis

[0072]

[Chemical 27]

The above mPEG5000-hydrazinecarboxylate was synthesized as follows. Methoxy polyoxyethylene imidazolyl carbonyl (from Sigma Chemical, 2.5 g,
0.49 mmol) was treated with hydrazine (0.077 ml, 2.45 mmol) in 10 ml methylene chloride. After 4 hours at room temperature, the reaction mixture was filtered and the filtrate treated with cold ether. Yield 2.22g. I
R (C = O): 1718. Analysis, calculated value for N,
0.55. Found: N, 0.595.

Synthesis of mPEG-semicarbazide

[0075]

[Chemical 28]

The above mPEG5000-semicarbazide was synthesized as follows. mPEG5000-amine was added to Rajasekharn Pirai
anPillai), V.I. N. And Mutter
ter), M.A. (1980) Journal of Organic Chemistry (J. Org. Chem.), 4
5 , 5364-5370. mPEG5000-amine (2 g, 0.4 mm
ol) was dissolved in 9 ml of dichloromethane and 0.28 ml of triethylamine was added. To this mixture was added phosgene (in toluene, 0.4
2 ml, 0.8 mmol) was added. The reaction was bubbled overnight and then bubbled with argon to remove excess phosgene. The solution was concentrated and the residue was dissolved in dichloromethane and 0.063 ml hydrazine (2m
mol) was added, followed by 2 ml of methanol. The reaction was bubbled for 4 hours, then cold ether was added,
Then the precipitate was filtered off and dried. Yield 1.51g. IR (C = O): 1683. Analysis, calculated for N, 0.83. Found: N, 0.56.

Also, using the procedure described above, mPEG2
000, mPEG8500, and mPEG12000
The following semicarbazide was synthesized.

Synthesis of mPEG-thiosemicarbazide 1.5 g mPEG500 in 5 ml dichloromethane.
To 0-amine, 0.1 ml of triethylamine (0.7
5 mmol) and 0.071 g (0.3 mmol) of di-2-pyridylthionocarbonate were added. The reaction was aerated overnight and then 0.047 ml (0.3 mmo
l) Hydrazine was added. After 4 hours, the mixture was filtered and the filtrate treated with cold ether. The product was collected by filtration. Yield 1.34g. IR (N = H stretch): 3332. Analysis, calculated value for N, 0.
83. Found: N, 0.255.

Synthesis of mPEG-carbonic acid dihydrazide In a reaction flask purged with argon, 2 ml dichloromethane, 0.084 ml triethylamine (0.6
mmol) and 0.03 g of triphosgene (0.1 m
t-butylcarbazate (0.04 g,
0.3 mmol) was added. After 5 minutes, 1.5 g of mPEG5000-amine (0.3 mmol) in 4 ml of dichloromethane was added. The reaction was bubbled overnight, then cold ether was added to precipitate the product. The product was isolated by filtration. Yield 1.44g. 2 ml of protected dihydrazide (0.61 g) at room temperature for 10 minutes
Of trifluoroacetic acid. Trifluoroacetic acid was removed and the resulting oil was dissolved in methylene chloride and concentrated. This process was repeated. This oil was dissolved in methylene chloride and the product was precipitated with cold ether. The product was isolated by filtration. Yield 0.41g. I
R (C = O): 1695. Analysis, calculated value for N,
1.37. Found: N, 0.41.

Synthesis of mPEG-aryl hydrazides

[0081]

[Chemical 29]

The above mPEG5000-arylhydrazide was synthesized as follows. By reacting 4-hydrazinobenzoic acid with di-t-butylpyrocarbonate in dioxane in the presence of a base at 0 ° C., B
The _{oc-NHNH-C 6 H 4} -COOH was prepared. Protected hydrazine arylate (0.378 g, 1.5 mmo
l) to mPEG-amine (1.5 g, 0.3 mmol)
And dichloromethane / dimethylformamide solution (4m
1, 1: 1). Also, dicyclohexylcarbodiimide (0.31 g, 1.5 mmol), 1-
Hydroxybenzotriazole (0.2g, 1.5mm
ol) and triethylamine (0.21 ml, 1.5
mmol) was added. The reaction was bubbled overnight and then the contents were filtered. The filtrate was treated with ether and the precipitate was collected. The precipitate is treated with trifluoroacetic acid and 30
After minutes, trifluoroacetic acid was removed. Ether was added to the oily residue to precipitate the mPEG-arylhydrazine product. The product was isolated by filtration. Yield 1.
19 g. IR (N-H): 3267; (C = O): 16
55; (C = O): 1606. Analytical, calculated for N, 0.82. Found: N, 0.50.

Synthesis of oxime-forming mPEG CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —CO—ONH
Synthesis of ₂

[0084]

[Chemical 30]

10 ml of mPEG-5000 succinimide ester (NHS) (2.0 g, 0.4 mmol)
Dissolved in dichloromethane. t-Butyl N-hydroxycarbamate (0.53 g, 4 mmol) was added, followed by 0.7 ml triethylamine (5 mmo.
l) was added. After allowing the reaction to proceed overnight, cold ether was added and the resulting precipitate was collected by filtration, washed and dried. The product (1.5 g) was placed in a dichloromethane / trifluoroacetic acid (1: 1) mixture. After 60 minutes, this solution was concentrated. LH
It was further purified by gel filtration on a -20 column and eluted with methanol / methylene chloride (5: 1). Yield 1.2
g. IR (C = O): 1741. Analysis, calculated for N, 0.28. Found: N, 0.13.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
Synthesis of O-CO-ONH ₂

[0087]

[Chemical 31]

MPEG5000-Oxycarbonylimidazole (2.0 g, 0.4 mmol) was dissolved in 10 ml of dichloromethane. t-Butyl N-hydroxycarbamate (0.53 g, 4 mmol) was added, followed by 0.7 ml triethylamine (5 mmol). After allowing the reaction to proceed overnight, cold ether was added and the resulting precipitate was collected by filtration, washed and dried. The product (1.5 g) was placed in a dichloromethane / trifluoroacetic acid (1: 2) mixture. After 60 minutes, this solution was concentrated. This compound was further purified by gel filtration on a LH-20 column, eluting with methanol / methylene chloride (5: 1). Yield 1.2g. IR (C
= O): 1741. Analysis, calculated value for N, 0.2
8. Found: N, 0.15.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
Synthesis of NH-CO-ONH ₂

[0090]

[Chemical 32]

MPEG5000-amine (2.0 g,
0.4 mmol) of carbonyl imidazole (0.23
g, 1.45 mmol) in 10 ml chloroform. This reaction is based on Ranucc
i), E. And Feruti, P .; (1
991) Macromoleculus (Macromolec)
ules) 24 , 3747-3752, followed by the procedure for activation of mPEG-alcohols with carbonylimidazole. The reaction mixture was stirred at room temperature for 2 hours, then 7 ml of water was added and the organic layer was extracted. The extraction with water was repeated 5 times. The organic layer was dried over sodium sulfate and the salt filtered. T-Butyl N-hydroxycarbamate (0.53 g,
4 mmol) was added, followed by 0.7 ml triethylamine (5 mmol). After stirring the reaction mixture overnight, cold ether was added and the resulting precipitate was collected by filtration, washed and dried. Product (1.
5 g) in dichloromethane / trifluoroacetic acid (1: 2)
Placed in the mixture. After 60 minutes, this solution was concentrated.
This compound was further purified by gel filtration on a LH-20 column, eluting with methanol / methylene chloride (5: 1). Yield 1.3g. IR (C = O): 1726. analysis,
Calculated for N, 0.55. Measured value: N, 0.4
3.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
Synthesis of NH-CS-ONH ₂

[0093]

[Chemical 33]

MPEG5000-amine (1.5 g,
0.3 mmol) was dissolved in 30 ml of dichloromethane. Triethylamine (0.1 ml, 0.75 mmo
1) was added, followed by di-2-pyridylthionocarbonate (0.028 g, 0.35 mmol).
The reaction mixture was stirred at room temperature for 2 hours, then 7 ml
Water was added and the organic layer was extracted. The extraction with water was repeated 5 times. The organic layer was dried over sodium sulfate and the salt filtered. T-Butyl N-hydroxycarbamate (0.53 g, 4 mmol) was added to the filtrate,
Then t-butyl N-hydroxycarbamate (0.5
3 g, 4 mmol) was added along with 0.5 ml triethylamine (3.75 mmol). After stirring the reaction mixture overnight, cold ether was added and the resulting precipitate was collected by filtration, washed and dried. The product (1.6 g) was placed in a dichloromethane / trifluoroacetic acid (1: 1) mixture. After 30 minutes, this solution was concentrated. This compound was further purified by gel filtration on an LH-20 column, methanol / methylene chloride (5:
Elute with 1). Yield 0.72g. IR (C = S): 1
684. Analysis, calculated for N, 0.55. Found: N, 0.30.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
Synthesis of ONH ₂

[0096]

[Chemical 34]

MPEG5000-Tresylate (2.0
g, 0.4 mmol) was dissolved in 10 ml of dichloromethane, to which t-butyl N-hydroxycarbamate (0.53 g, 4 mmol) and 0.7 ml of triethylamine (5 mmol) were added. 4 this mixture
Heat to 5 ° C. (reflux) and allow the reaction to proceed overnight.
Cold ether was added and the resulting precipitate was collected by filtration, washed and dried. The product (1.5 g) was placed in a dichloromethane / trifluoroacetic acid (3: 7) mixture. After 60 minutes, this solution was concentrated. This compound was further purified by gel filtration on a LH-20 column, eluting with methanol / methylene chloride (5: 1). Yield 1.6g. Analysis, calculated for N, 0.28. Found: N, 0.19.

Another synthesis is as follows. t-butyl N-
Hydroxy carbamate (0.53 g, 4 mmol) was placed in 1 ml of tetrahydrofuran and then NaH
(78 mg, 3.25 mmol) was added. After a few minutes,
This solution was added to mPEG5 in 5 ml of tetrahydrofuran.
000-Tresylate (1.0 g, 0.2 mmol) was added. The mixture was heated to 40 ° C. and the reaction was allowed to proceed overnight. Cold ether was added and the resulting precipitate was collected by filtration, washed and dried. The product (1.5 g) was placed in a dichloromethane / trifluoroacetic acid (3: 7) mixture. After 60 minutes, this solution was concentrated. This compound was further purified by gel filtration on an LH-20 column, methanol / methylene chloride (5:
Elute with 1). Yield 0.75g. Analysis, calculated for N, 0.28. Found: N, 0.10.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
Synthesis of NH-CO-CH ₂ -ONH ₂

[0100]

[Chemical 35]

MPEG5000-amine (2.0 g,
0.4 mmol) and Boc-aminooxyacetic acid (0.2 g, 1.05 mmol) were dissolved in 20 ml of dichloromethane. Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (1.1 g, 2 mmol) was added, followed by diisopropylethylamine (0.7 ml, 3.9 mmol).
Was added. The reaction mixture was stirred at room temperature for about 72 hours, then cold ether was added and the resulting precipitate was collected by filtration, washed and dried. Half of the collected precipitate was placed in a dichloromethane / trifluoroacetic acid (3: 7) mixture. After 60 minutes, this solution was concentrated. This compound was further purified by gel filtration on a LH-20 column, eluting with methanol / methylene chloride (5: 1). The resulting yellow precipitate was taken up in water and treated with decolorizing charcoal. After about 24 hours, the decolorizing charcoal was filtered and the clear filtrate was collected. The residue was dissolved in dichloromethane, dried over sodium sulphate, filtered and the filtrate treated with cold ether. A white product was obtained. Yield 0.5g. I
R (C = O): 1676. Analysis, calculated value for N,
0.55. Found: N, 0.50.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
Synthesis of O-CO-CH ₂ -ONH ₂

[0103]

[Chemical 36]

MPEG5000-alcohol (2.0
g, 0.4 mmol) and Boc-aminooxyacetic acid (0.2 g, 1.05 mmol) were dissolved in 20 ml of dichloromethane. Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (1.1 g, 2 mmol) was added, followed by diisopropylethylamine (0.7 ml, 3.09 mmo.
l) was added. The reaction mixture was stirred at room temperature for about 2 hours, then dimethylaminopyridine (0.244 g,
2 mmol) was added. After about 4 days cold ether was added and the resulting precipitate was collected by filtration, washed and dried. The precipitate was taken up in water and treated with decolorizing charcoal. After about 24 hours, the decolorizing charcoal was filtered and the clear filtrate was collected. The residue was dissolved in dichloromethane, dried over sodium sulphate, filtered and the filtrate treated with cold ether. Analysis, calculated for N, 0.28. Found: N, 0.14.

Half of the collected precipitate was placed in a dichloromethane / trifluoroacetic acid (2: 1) mixture. After 30 minutes, this solution was concentrated. This compound was further purified by gel filtration on a LH-20 column, eluting with methanol / methylene chloride (5: 1). Yield 0.3g. IR (C
= O): 1734.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
CH (OH) Synthesis of -CH ₂ -ONH ₂

[0107]

[Chemical 37]

MPEG-5000-epoxide (1.0
g, 0.2 mmol) with 10 ml of 0.1 M NaOH
Dissolved in. t-Butyl N-hydroxycarbamate (0.53 g, 4 mmol) was added. The reaction was allowed to proceed overnight and the reaction mixture was extracted with dichloromethane. Sodium sulfate was added and filtered. Cold ether was added to a solution of dichloromethane and the resulting precipitate was collected by filtration, washed and dried. This compound is L
Further purification by gel filtration on an H-20 column, eluting with methanol / methylene chloride (5: 1). Protected m
The PEG-derivative (0.25 g) was placed in a dichloromethane / trifluoroacetic acid (1: 1) mixture. After 30 minutes, the solution was concentrated and taken up in dichloromethane. The compound was isolated from ether by precipitation. Yield 0.2g. IR (OH): 3447. Analysis, calculated for N, 0.28. Found: N, 0.21.

CH ₃ O— (CH ₂ CH ₂ ) _n —CH ₂ CH ₂ —
Synthesis of CO-CH ₂ -ONH ₂

[0110]

[Chemical 38]

MPEG-O-CH ₂ CH ₂ -CH (OH)
_{-CH 2 -ONH-Boc (0.3g,} 0.6mmo
l) in 3 ml of dry dimethylsulfoxide,
2 ml acetic anhydride was added. The reaction was allowed to proceed at room temperature for about 24 hours, then cold ether was added. The resulting precipitate was collected by filtration, washed and dried. The product was placed in a dichloromethane / trifluoroacetic acid (1: 1) mixture. After 30 minutes, this solution was concentrated. The compound was isolated from ether by precipitation. Yield 0.
18 g. IR (C = O): 1698. Analysis, calculated for N, 0.28. Found: N, 0.20.

Combination of mPEG-Ornithine Semicarbazide
The dissolved mPEG-000-amine (5.0 g, 1 mmol) was dissolved in 5 ml dry methylene chloride and 10 ml
Of dry dimethylformamide was added. Fmoc-O
rn (Boc) -OPfp (3.1 g, 5 mmol) was added. After 1 hour at room temperature, the solution was concentrated. Water was added to the residue, and the solution was filtered, centrifuged, and filtered to remove dispersed solids in the aqueous solution. The filtered aqueous solution was concentrated and the residue was taken up in methylene chloride and dried over sodium sulfate. The solution was filtered. The filtrate was treated with cold ether. The resulting precipitate was collected by filtration, washed and dried. Yield 3.3g. IR (C = O): 1713, 1680. 30% of this compound with 25% piperazine (in methylene chloride)
The Fmoc group was removed by treatment for minutes. Cold ether was added to this solution to precipitate the mPEG-derivative. The precipitate was collected, washed and dried. 1.
The free alpha amino group was acetylated by dissolving 4 g of mPEG-derivative in 3 ml of dichloromethane and adding 1 ml of acetic anhydride. After 1.7 hours at room temperature, the solution was concentrated. The resulting solid was treated with trifluoroacetic acid (5: 3) in methylene chloride. The solvent was removed and the resulting oil was taken up in methylene chloride and cold ether was added. The precipitate formed was collected, washed and dried. Yield 1.1g. IR
(C = O): 1685. The mPEG-derivative was dissolved in 3 ml dry methylene chloride and triethylamine (0.54 ml, 3.84 mmol) was added, followed by 1 ml phosgene (1.92 mmol) in toluene and an additional 2 ml dry methylene chloride. Was added. The reaction was allowed to proceed overnight at room temperature and then the solvent was removed.
The residue was dissolved in 3 ml dry methylene chloride and 0.2 ml hydrazone (5.76 mmol) was added. Dry methanol was added until the solution became clear (3.4 ml). After 4 hours at room temperature, the solution was clarified by centrifugation and concentrated. LH
It was further purified by gel filtration on a -20 column and eluted with methanol / methylene chloride (5: 1). Yield 0.7
g. IR (C = O): 1675.

Having described the invention, it is illustrated by the following examples. These examples do not limit the invention.

[0114]

EXAMPLES EPO denaturation (hydrazide method) In a typical experiment, EPO (0.5-1.0 mg)
(Ortho Biotech)
Was placed in a total of vlm 0.786 ml of 100 mM sodium acetate pH 5.6. A sufficient 10 mg / ml solution of sodium periodate was added to bring the final concentration of sodium periodate to 10 mM. Oxidation was allowed to proceed for 30 minutes at 0 ° C. in the dark, then 0.33 ml of 80 mM Na ₂ CO ₃ was added. After 5 minutes, the solution was concentrated and 100 in a microconcentrator.
It was washed 3 times with mM sodium acetate. After the last concentration
The oxidized EPO solution was made up to 1.0 ml with 100 mM sodium acetate. mPEG5000-hydrazide (5
0 mg) was added to the oxidized EPO. The mixture was stirred at room temperature overnight. Sephacryl S-200-HR with mPEG5000-EPO
Purified by gel filtration on a column (1 mm x 45 mm),
Elution with phosphate buffer containing 0.05% sodium azide. The amount of mPEG-modified EPO was determined by HPLC gel filtration Sol Bucks (Zorbax ^R) GF-250 or GF-450 column, it was used a phosphate buffer pH7 of 0.1 M. 6-12 molecules of mPEG
Was bound to each molecule of EPO.

The same procedure as the hydrazide method on the modification of EPO (semicarbazide method) was carried out, except that the reaction time of the oxidation was reduced to 5 minutes and a small amount compared to the amount of mPEG-hydrazide (50 mg). MPEG-
Semicarbazide (10 mg) was used. More (about 18) mPEG molecules bound to EPO despite shortening the oxidation time and adding a smaller amount of mPEG. If a longer oxidation time (15 minutes) is used and more mPEG-semicarbazide is added, about 30 mPEG molecules can be attached to EPO depending on the molecular weight of mPEG used. Thus mPEG-semicarbazide appears to be more reactive than mPEG-hydrazide, and it is possible under similar reaction conditions to attach a larger number of more mPEG molecules to EPO than is possible with mPEG-hydrazide.

A comparison of the effects of modifying EPO with mPEG on the carbohydrate groups or on the amino acid side chains is shown in FIG. The conditions for analytical HPLC gel filtration are the same as above. The chromatogram of native EPO is presented in Figure 1a. A peak with a retention time of 10.5 minutes is found. EPO on its carbohydrate group mPEG
When denatured at 5000 (FIG. 1b), a single large peak with a retention time of 9.4 minutes is seen for the crude reaction product. MPEG500 reacting with the side chain of lysine
When the succinimide ester of 0 is reacted with EPO, the reaction product heterogeneous methylene chloride is obtained (7.
5-10.5 min peak). A similar heterogeneous pattern was found for denaturation of mPEG using succinimide coupling to CSF-1, interleukin-2 and β-interferon (US Pat. No. 4,8,8).
47,325 and U.S. Pat. No. 4,917,888.
issue). Also, lower molecular weight impurities are present in the succinimide coupling (FIG. 1c). Coupling of active esters using succinimide derivatives of mPEG has been the preferred method of attaching mPEG to proteins [Nucci, M. et al. L. , Shor (Sh
orr), R.I. And Abuchow
ski), A. (1991) Advanced drug
Delivery Reviews (Adv. Drug Deli
Very Rev. ), 6 , 133-151]. Also,
EPO was treated with mPEG5 using the semicarbazide method described above.
About the result of biological experiment derivatized with 000 Fig. 4
See. Also, using the semicarbazide method described above, m
EPO modified with PEG12000 (see FIG. 3) and EPO modified with mPEG2000 (see FIG. 10) were obtained.

EPO was also modified with derivatives of thiosemicarbazide, hydrazide carboxylate and carbonic acid dihydrazide of mPEG. These derivatives of mPEG show similar performance to the semicarbazide derivatives of mPEG,
It was possible to obtain high levels of coupling of mPEG to EPO using these derivatives when compared to the hydrazide of mPEG. Other conditions of oxidation of EPO, such as increasing temperature, increasing concentration of sodium periodate, and increasing or decreasing reaction time, are used if these oxidizing conditions do not impair the biological activity of EPO. be able to. Large scale modification of EPO (semicarbazide or carboxyl
Sylate hydrazide method) EPO (12.0 mg) (Ortho Biotech (Ort
ho Biotech)) was placed in a total volume of 1.8 ml of 100 mM sodium acetate pH 5.5. Sodium periodate (0.215 ml)
Was added at a concentration of 40 mg / ml. Oxidation in the dark at 0 ° C
For 20 minutes, then 0.02 ml of ethylene glycol was added and the mixture was stirred at 0 ° C. for 10 minutes. The oxidized EPO Sephadex (Sephadex ^R) G-25 column (2.5 cm
X 9 cm) gel filtration and 100 mM
Eluted with sodium acetate buffer pH 4.3. The eluted oxidized EPO (10-11 ml) was pooled. mP
EG5000 semicarbazide (100 mg) was added to purified oxidized EPO. The mixture was stirred at room temperature overnight. mPEG5000-EPO was purified by gel filtration on a Sephacryl S-200-HR column, buffer pH 0.2M NaCl, 0.02M sodium citrate, 0.025% sodium azide pH 7. Elute at 0. The above denaturation was repeated using 200 mg of mPEG5000 semicarbazide. The reactivity was about 22 mPEG molecule / EPO molecule.

The above modification was repeated using 200 mg of the carboxylate hydrazide, using half the amount of the reaction components specified above. The reactivity was about 30 mPEG molecule / EPO molecule.

Modification of EPO (Oxime Method) A. _{mPEG-CH 2 CH 2 -NH-} CO-CH 2 -ON
The same procedure was performed for the large scale semicarbazide method on H ₂ . However, instead of the addition of mPEG5000 semicarbazide, mPEG5000-CH ₂ CH ₂ -
NH-CO-CH ₂ -ONH ₂ a (50 mg), 2.15
Mix with ml of oxidized EPO overnight at room temperature. mPE
Sephacryl G5000-EPO
l) purified by gel filtration on an S-200-HR column,
Elution with buffer pH 7 containing 0.2 M NaCl, 0.02 M sodium citrate, 0.025% sodium azide. Phenomenex Biosep (P
henomenex Biosep) -Sec-S40
H using a 00 column (30 cm x 0.017 cm)
About 31 molecules of mPEG as determined by PLC gel filtration
It was found that 5000 was bound to each molecule of EPO. A small fraction consisting of 25 molecules of mPEG-EPO was also isolated and used for biological studies.
See Figures 16, 18 and 19.

B. _{mPEG-O-CH 2 CH 2} -NH-C
Use _{O-ONH 2 mPEG-O-} CH 2 CH 2 -NH-CO-ONH 2, was repeated denaturation of EPO above. Approximately 18-19 molecules of mP determined using the method in Modification A above.
It was found that EG5000 was bound to each molecule of EPO. Biological data are shown in Figures 16, 18 and 19.

C. Use _{mPEG-O-CH 2 CH 2} -ONH 2 mPEG-O-CH 2 CH 2 -ONH 2, was repeated denaturation of EPO above. Approximately 17 molecules of mPEG5000 were determined to be EP using the method in Modification A above.
It was found to be bound to each molecule of O. 22 mPE
Two minor fractions of G, 12mPEG were also isolated (see Figures 16, 18 and 19).

D. _{mPEG-O-CH 2 CH 2} -CO-O
NH ₂ PEG-oxime derivative _{mPEG-O-CH 2 CH 2} -C
Use O-ONH _2, was repeated denaturation of EPO above. Determined using the method in Modification A above, about 3
It was found that the molecule mPEG5000 was bound to each molecule of EPO.

E. _{mPEG-O-CH 2 CH 2} -CH (O
H) -CH ₂ -ONH ₂ PEG- oxime derivative _{mPEG-O-CH 2 CH 2} -C
H (OOH) using the -CH ₂ -ONH _2, on the EPO
The denaturation of was repeated. Approximately 31 molecules of mPEG5000 were found to be attached to each molecule of EPO, as determined using the method in Modification A above.

F. _{mPEG-O-CH 2 CH 2} -NH-C
S-ONH ₂ PEG-oxime derivative _{mPEG-O-CH 2 CH 2} -N
Use H-CS-ONH _2, was repeated denaturation of EPO above. Determined using the method in Modification A above,
It was found that about 4 molecules of mPEG5000 were bound to each molecule of EPO.

Biological activity of mPEG-EPO The mPEG-EPO derivative was measured by measuring the increase in red blood cells generated after injection of the denatured protein.
Assayed for biological activity in vivo (Egrie, JC, Strickland (Str).
Ickland), T.W. W. , Lane,
J. Aoki, K .; , Cohen, A .;
M. Smalling, R .; , Trail, G.I. Lin, F .;
K. , Browne, J .; K. And Hines, D.H. K. (1986) Immunobiology. 172: 213-.
224). Briefly, mice (female CD-1, age 8) received 0.4 μg of protein 1 on 2 consecutive days.
It was injected intraperitoneally or subcutaneously once a day. Blood was drawn for hematocrit reading.

The in vivo biological activity (hematocrit level) of mPEG-EPO coupled via the hydrazide derivative of mPEG is shown in Figures 2, 4 and 5 and Tables I and II. Summarizing the findings, as shown in Figures 2 and 5, the optimal number of mPEG couplings is not clear and must be determined by synthetic and biological studies to obtain the best mPEG-EPO. . FIG. 4 compares the coupling of mPEG using hydrazide and semicarbazide linkers. mP
Higher and longer hematocrit levels for EG-EPO could be obtained using a semicarbazide linker. The results observed are, in part, the mP that can be obtained using a semicarbazide linker.
This is because of the higher level of EG incorporation. Table I
Is the number of mPEGs incorporated and the mPEGs used
The biological activity of mPEG-EPO as a function of the molecular weight of is shown.

Comparing the molecular weight of mPEG used and the amount of coupled mPEG, different hydrazide m
The biological activity of PEG-EPO is summarized in Table I.

The different mPEG hydrazide linkers used are compared in Table II. Optimal amount of mP
The biological activity of EPO modified with different mPEG5000-hydrazide derivatives incorporating EG is shown in Table II.
In summary. Not all carbohydrate mPEG derivatives exhibit the same biological activity when coupled to EPO due to the inability to couple well with optimal amounts of mPEG or other factors. Biological activity is the best value obtained for each linker.

[0129]

[Table 1]

[0130]

[Table 2]

EPO and mPEG50 in mice
The hematocrit levels of 00-EPO are shown in FIG.
12PEG-EPO was made from the coupling of mPEG5000-hydrazide, and this reflects the maximum incorporation achievable with this mPEG derivative under the experimental conditions described above. 18 PEG and 28 PEG EPO
Was made by coupling with mPEG5000-semicarbazide. The semicarbazide derivative of mPEG is
M that can be incorporated using this mPEG derivative
The large amount of PEG results in a much better biological activity than the hydrazide derivative of mPEG.
All three mPEG-EPOs show increased maximal activity and prolonged activity when compared to native EPO. Thus, PEG modification of protein carbohydrate groups can produce therapeutic proteins that are much more potent.

See FIGS. 8-15 for additional data on the effect of various mPEG hydrazide modified EPO used in the experiments on hematocrit levels. MPE used in the experiments shown in FIGS.
G-modified EPO was prepared using the appropriate water-soluble polymeric reagent essentially as described for the other mPEG-modified EPO molecules used in the experiments shown in Figures 2-5.

The in vivo biological activity (hematocrit level) of mPEG-EPO coupled via the oxime-forming derivative of mPEG is shown in FIGS. 16 and 17. In summary, mPEG-O-CH ₂ 16
_{CH 2 -NH-CO-ONH 2} ( "A") and mPEG
_{-O-CH 2 CH 2 -NH-} CO-CH 2 -ONH
Coupling of mPEG using a ₂ (“C”) linker is compared. The higher hematocrit level of mPEG-EPO is due to the "C" linker with 31 mPEG / EPO molecules (herein formula XXXIII.
(Corresponding to ## STR3 ##) can be obtained using the "A" linker with 18 mPEG molecules / EPO molecules (here corresponding to formula XXX). Also, 31mP
Notable is the higher hematocrit activity of the "C" linker with 25mPEG molecule / EPO molecule (herein formula XXXIII) compared to the same linker with EG molecule / EPO molecule.

In FIG. 17, 22, 17 and 12 m
PEG molecules / EPO molecule _{mPEG-O-CH 2 CH 2} -
The coupling of mPEG using ONH ₂ (formula XXIII) is compared. The highest hematocrit levels were obtained at the lowest degree of pegylation, and hematocrit decreased inversely with the degree of pegylation. However, m
In all mPEG linker to use _{PEG-O-CH 2 CH 2} -ONH 2, higher and period as compared to the native EPO increases.

Of particular note is the biological activity of the 12mPEG-EPO of formula XXIII. Hydrazide derivative 1
2mPEG-EPO does not produce the degree and duration of hematocrit increase as that of oxylamine-derivatized EPO.

[0136] Using the binding Kurinigen (Clinigen ^R) erythropoietin (EPO) EIA test kit of the antibody to the mPEG-EPO, mPEG-E
The antigenicity of PO was determined. Briefly, this assay consists of microtiter plates coated with a monoclonal antibody against EPO. EPO or mPEG
-EPO interacted with the coated plate. After washing the plate, a labeled polyclonal antibody against EPO was incubated on the plate. Read plate after development of substrate.

ELI for hydrazide-derivatized EPO
The results of SA assay are shown in FIG. mPEG-EPO
Is expressed as the approximate number attached for a given molecular weight mPEG. For example, 12PEG-5K is
This means that about 12 molecules of mPEG having a molecular weight of about 5000 are coupled to each molecule of EPO. As the data show, as the number of mPEG coupled to EPO increases, the antigenicity of the protein decreases. Similarly, as the number of mPEG coupled to EPO increases, the antigenicity of denatured EPO, ie, antibody binding, decreases. Reacting oxidized EPO with the hydrazide derivative of mPEG did not reach the high coupling levels seen with PEG derivatives of semicarbazide, thiosemicarbazide and carbonic dihydrazide. A decrease in the antigenicity of a protein correlates well with a decrease in the immunogenicity of the protein. Thus, mPEG-EPO coupled to the carbohydrate group of EPO may reduce the potential immunogenicity associated with proteins,
Derivatives of mPEG that can couple at high levels are most effective.

See FIG. 6 for additional data using mPEG hydrazide derivatives.

ELIS for oxime-derivatized EPO
The results of the A assay are shown in Figure 18. As the data show, as the number of mPEG coupled to EPO increases, the antigenicity of the protein decreases. Linkers (18P) that produced relatively low coupling levels
Reacting EG-A, 17PEG-B, 12PEG-B) with oxidized EPO, as seen with higher coupling level formulations (22PEG-B, 25PEG-C and 31PEG-C), It did not give a large reduction in immunogenicity. It should be noted that these differences in the ability of proteins to reduce immunogenicity appear to be determined largely on the basis of coupling level. (For example, comparison of 12PEG-B and 22PEG-B). Thus, mPEG coupled to the carbohydrate group of EPO through an oxime bond may reduce the potential immunogenicity associated with proteins, with derivatives of mPEG capable of high levels of coupling being most effective.

Denaturation of horseradish peroxidase:
Comparison of hydrazide / semicarbazide couplings Horseradish peroxidase (HRP) is a glycoprotein enzyme (oxide-reductase). HRP
MPEG5000-hydrazide or mPEG500
Denaturation with 0-semicarbazide revealed whether other glycoproteins other than EPO show denaturation differences between two different carbohydrate denaturing reagents. In a typical experiment, horseradish peroxidase (2 mg) was added to a total volume of 0.8 ml of 100 mM sodium acetate pH.
I put it in 5.6. Sufficient 10 mg / ml sodium periodate solution was added to bring the final concentration of sodium periodate to 10 mM. Oxidation at 0 ° C.
Proceed for minutes, then 0.33 ml of 80 mM Na ₂
The CO ₃ was added. After 5 minutes, the solution was concentrated and washed 3 times with 100 mM sodium acetate pH 4.2 in a microconcentrator. The oxidized horseradish peroxidase solution was then split in half, 30 mg mPEG5000-hydrazide was added to one half and 30 mg mPEG500 to the other half.
0-semicarbazide was added. The two oxidized horseradish peroxidase solutions were then stirred overnight at room temperature. Zorbax (Zorbax ^R) GF-
The degree of PEG denaturation was determined by HPLC gel filtration on a 250 column using 0.1 M phosphate buffer pH 7. Horseradish peroxidase modified with mPEG5000-hydrazide is approximately 7 PEG molecules / HR.
Had P; mPEG5000-semicarbazide modified horseradish peroxidase had approximately 19 PEG
Molecules / HRP. Thus, denaturing horseradish peroxidase with PEG attached to its carbohydrate group is even more effective when using the semicarbazide derivative of PEG over hydrazide under the same experimental conditions.

Half-life determination Half-life experiments were performed using male Sprague-Dawley rats weighing approximately 0.3 kg. Three rats were used for each compound. The details of the experiment are as follows. 1 μg for each rat
EPO or mPEG-EPO was injected IV (intravenous). For the hydrazide-derivatized mPEG-EPO, the mPEG-EPO used was mPEG5000-semicarbazide-18 and EPO using semicarbazide
Was prepared essentially as described in the section above. Blood was drawn from each rat at 2, 5, 15, 90 minutes and 3, 6, 24, 48, 54 hours. Blood was collected in heparinized tubes and plasma was isolated. Isolated plasma was tested for EPO biological activity in an EPO-dependent cell proliferation assay. The in vitro assay used the FDC-P1 / ER cell line. This murine cell line incorporates the EPO receptor and is dependent on EPO for growth.
This assay was performed as follows. Cells were grown for 24 hours in the absence of EPO (106 / ml), then EPO or mPEG-EPO at different concentrations were added to the cells. The cells were incubated for 42 hours and then tritiated thymidine was added to the cells. After 6 hours, cells were harvested and counted. Cell proliferation was determined by the increased absorption of thymidine. The results are shown in Fig. 7.

EPO-dependent cell proliferation assay was performed as previously described using oxime-derivatized mPEG-EPO. The results are shown in Fig. 19.

In vivo assay: Anemia mouse model
Dell In this assay, mice were placed in TNF for 5 consecutive days.
-Anemic by injection with alpha. To overcome this anemia, EPO or mPEG-EPO
SC at (0.03 μg / dose) over the same 5 days
Mice were injected (subcutaneously) or SC injected exactly 2 out of 5 days of receiving TNF-alpha.
The results are shown in Fig. 15.

Equivalent Embodiments All publications and patents mentioned in the above detailed description are herein incorporated by reference. The foregoing detailed description is considered to be sufficient for one skilled in the art to practice the present invention. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

The main features and aspects of the present invention are as follows.

1. formula:

[0147]

[Chemical Formula 39] Wherein, X is O or S, Q is selected from the group consisting of -NHNH ₂ and -C ₆ H ₄ -NHNH _2, and Y is _{-O -, - OCH 2 -,} - NH -, - NHNH -,
_{-O-CO-CH 2 CH 2} - and -NHCO-N-NH
A compound selected from the group consisting of NH-, and P is a water-soluble polymer.

2. The compound has the formula:

[0149]

Embedded image (I) Compound having P—O—CH ₂ —CO—NHNH ₂ , Formula:

[0150]

Embedded image (II) Compound having P—O—CO—NHNH ₂ , Formula:

[0151]

Embedded image (III) Compound having P—NH—CO—NHNH ₂ , Formula:

[0152]

Embedded image (IV) Compound having P—NH—CS—NHNH ₂ , Formula:

[0153]

(V) P-NHCO-N-NHN
A compound having HCO-NHNH ₂ , a formula:

[0154]

Embedded image (VI) Compound having P-NHNHCONHNH ₂ , Formula:

[0155]

Embedded image (VII) Compound having P-NHNHCSNHNH ₂ , Formula:

[0156]

Embedded image (VIII) A compound having P—NH—CO—C ₆ H ₄ —NHNH ₂ , and a formula:

[0157]

Embedded image (IX) P—O—CO—CH ₂ CH ₂
The compound according to item 1 above, which belongs to the group consisting of: a compound having —CO—NHNH ₂ .

3. The compound has the formula:

[0159]

(II) The compound according to the above-mentioned item _2, which has P—O—CO—NHNH ₂ .

4. The compound has the formula:

[0161]

(III) The compound according to the above-mentioned item _2, which has P—NH—CO—NHNH ₂ .

5. The compound has the formula:

[0163]

Embedded image (IV) The compound according to the above-mentioned item _2, which has P—NH—CS—NHNH ₂ .

6. The compound has the formula:

[0165]

(V) P-NHCO-N-NHN
A compound according to claim 2 having HCO-NHNH ₂ .

7. The polymer is a homopolymer of polyethylene glycol, a homopolymer of polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, wherein the homopolymer and copolymer are unsubstituted or substituted at one end with an alkyl group. ), Polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, α, β-poly [(2-hydroxyethyl)
-DL-aspartamide], RO-PEG (where R
Is alkyl, aryl, alkylaryl, aroyl,
Alkanoyl, benzoyl, arylalkyl ether, cycloalkyl, cycloalkylaryl), and a derivative of said polymer.

8. 8. The compound according to the above item 7, wherein the water-soluble polymer is polyethylene glycol or a derivative thereof.

9. 9. The compound according to the above item 8, wherein the water-soluble polymer is monomethoxy poly (ethylene glycol).

10. A step of mixing the polypeptide with a compound according to item 1 above for denaturation, wherein the water-soluble polymer-modified polypeptide is produced.

11. The polypeptide for denaturation is
The modified polypeptide according to claim 10 selected from the group consisting of hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies.

12. The polypeptide for denaturation is an antibody, and the method further comprises the step of combining the antibody with a compound capable of specifically binding to a binding site on the antibody before the mixing step. The modified polypeptide according to item 11.

13. 12. The modified polypeptide of claim 11, wherein the polypeptide for denaturation is an enzyme and the method further comprises the step of combining the enzyme with a substrate for the enzyme prior to the mixing step.

14. The modified polypeptide according to claim 11, wherein the polypeptide for denaturation is a glycoprotein.

15. 15. The modified polypeptide according to the above 14, wherein the glycoprotein is erythropoietin.

16. The modified polypeptide according to claim 9, wherein the method further comprises a step of adding an oxidizing agent before the mixing step.

17. A composition comprising the polypeptide according to the above item 10 and a pharmaceutically acceptable carrier.

18. A composition comprising the polypeptide according to the above item 15 and a pharmaceutically acceptable carrier.

19. formula:

[0179]

Embedded image (X) [P—O—CH ₂ —CO—NH
A compound having N = CH-] _n- Z, the formula:

[0180]

Embedded image (XI) [P—O—CO—NHN═C
H-] _n- Z, a compound having the formula:

[0181]

(XII) [P-NH-CO-NHN =
A compound having CH-] _n- Z, the formula:

[0182]

(XIII) [P-NH-CS-NHN =
A compound having CH-] _n- Z, the formula:

[0183]

Embedded image (XIV) [P-NHCO-NH-NH
A compound having NHCO-NHN = CH-] _n- Z, the formula:

[0184]

Embedded image (XV) [P-HNNHCON = CH-] _n- Z having the formula:

[0185]

(XVI) [P-HNNHCSN = CH-] _n- Z having the formula:

[0186]

Embedded image (XVII) [P-NH- CO-C 6 H 4 -N
HN = CH-] _n- Z, and the formula:

[0187]

Embedded image (XVIII) [P—O—CO—CH ₂ CH
A compound having _2- CO-NHN = CH-] _n- Z, wherein Z is a polypeptide, n is 1 to x, and x is the number of oxidatively activatable groups on Z, And P is a water-soluble polymer.

20. formula:

[0189]

Embedded image (X) The compound according to the above item 19, which has [P—O—CO—NHN═CH—] _n —Z.

21. 21. The compound according to the above 20, wherein n is 22 to 32.

22. formula:

[0192]

Embedded image (XI) [P-NH-CO-NHN = C
20. The compound according to the above item 19, having H-] _n- Z.

23. 23. The compound according to the above item 22, wherein n is 17 to 25.

24. formula:

[0195]

(XII) [P-NH-CS-NHN = C
20. The compound according to the above item 19, having H-] _n- Z.

25. formula:

[0197]

(XIII) [P-NHCO-NH-NH
20. The compound according to the above item 19, having NHCO-NHN = CH-] _n- Z.

26. formula:

[0199]

(XIV) The compound according to the above item 19, which has [P-HNNHCON = CH-] _n- Z.

27. formula:

[0201]

(XV) The compound according to the above item 19, which has [P-HNNCSN = CH-] _n- Z.

28. P is a polyethylene glycol homopolymer, a polypropylene glycol homopolymer,
Copolymers of ethylene glycol and propylene glycol (wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, polyvinylethyl Ether, α, β-poly [(2-hydroxyethyl) -DL-
Aspartamide], RO-PEG (wherein R can be alkyl, aryl, alkylaryl, aroyl, alkanoyl, benzoyl, arylalkyl ether, cycloalkyl, cycloalkylaryl) and derivatives of said polymers. The compound according to the above item 19, which is selected from:

29. 20. The compound according to claim 19, wherein Z is selected from the group consisting of hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies.

30. The 29th above, wherein Z is a glycoprotein
The compound according to the item.

31. 31. The compound according to above item 30, wherein the glycoprotein is erythropoietin.

32. 23. The compound according to the above item 22, wherein Z is erythropoietin.

33. 33. The compound according to above item 32, wherein P is monomethoxypoly (ethylene glycol).

34. 34. The compound according to the above item 33, wherein the average molecular weight of monomethoxy poly (ethylene glycol) is in the range of 2000 to 12000.

35. The compound according to the above item 34, wherein n is 10 to 36.

36. The compound according to the above item 35, wherein n is 20 to 32.

37. 37. The compound according to the above item 36, wherein the average molecular weight of monomethoxy poly (ethylene glycol) is 5000.

38. Compound II, II, which comprises the step of mixing the polypeptide with an oxidizing agent for activation.
A method of activating a polypeptide for conjugation with a compound selected from the group consisting of I, IV and V.

39. 39. The method of claim 38, wherein the oxidizing agent is sodium periodate.

40. The periodate is 1 m of protein
The above third present in a concentration of 10-40 μmol per g
The method according to item 9.

41. 41. The method according to item 40 above, wherein the mixing step is performed at a temperature in the range of -10 to 50 ° C.

42. 42. The method according to item 41 above, wherein the mixing step is performed at a temperature in the range of 0 to 30C.

43. The method of claim 42, wherein said mixing step requires a period of 1 minute to 3 days.

44. 44. The method of paragraph 43, wherein the mixing step is performed for a period of 1 minute to 60 minutes.

45. A process for producing a water-soluble polymer-modified polypeptide, which comprises the step of mixing the polypeptide with the reagent compound of the water-soluble polymer according to the above item 1 for denaturation.

46. The polypeptide for denaturation is
45. The method of claim 44, wherein the method is selected from the group consisting of hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies.

47. 47. The method of claim 46, wherein the polypeptide for denaturation is a glycoprotein.

48. 48. The method of claim 47, wherein the glycoprotein is erythropoietin.

49. The forty-fourth aspect, further comprising the step of adding an oxidizing agent to the polypeptide for denaturation before the mixing step.
Method described in section.

50. The oxidizing agent is sodium periodate in a concentration range of 10 to 40 μmol, and P is 400
Having a molecular weight in the range of 0 to 12000, wherein the water-soluble polymer has the formula

[0225]

Embedded image (III) The method according to the above item 49, which is a compound of P—NH—CO—NHNH ₂ .

51. A kit for modifying a polypeptide with a water-soluble polymer, which comprises the water-soluble polymer according to the above-mentioned item 1.

52. 52. The kit of claim 51, further comprising an oxidizer.

53. 52. The kit of claim 51, further comprising a polypeptide for denaturation.

54. formula:

[0230]

Embedded image In the formula, X is C═O, C═S, CH ₂ or CHOH, and Q is selected from the group consisting of —ONH ₂ — and —CH ₂ —ONH ₂ —. It is, and Y is _{-O-CH 2 CH 2 -,} -
_{O-CH 2 CH 2 -O -} , - O-CH 2 CH 2 -N -, - O
It is selected from the group consisting of -CH ₂ CH ₂ -S- and -O-CH ₂ CH ₂ CH-, and P is a water-soluble polymer, a compound having a.

55. The compound has the formula:

[0232]

Embedded image (XIX) P—O—CH ₂ CH ₂ —CO—ONH ₂ compound, formula:

[0233]

Embedded image (XX) P—O—CH ₂ CH ₂ —O—
Compounds having a CO-ONH _2, wherein:

[0234]

Embedded image (XXI) P—O—CH ₂ CH ₂ —NH
Compounds having a -CO-ONH _2, wherein:

[0235]

Embedded image (XXII) P—O—CH ₂ CH ₂ —O—
A compound having CS-ONH ₂ , a formula:

[0236]

Embedded image (XXIII) compound having P—O—CH ₂ CH ₂ —ONH ₂ , formula:

[0237]

Embedded image (XXIV) P—O—CH ₂ CH ₂ —NH
Compounds having -O-CH ₂ ONH _2, wherein:

[0238]

Embedded image (XXV) P—O—CH ₂ CH ₂ —O—
Compounds having a CO-CH ₂ -ONH _2, wherein:

[0239]

[Of 77] (XXVI) P-O-CH 2 CH 2 -CH
(OH) compound having a -CH ₂ -ONH _2, and the formula:

[0240]

Embedded image (XXVII) P—O—CH ₂ CH ₂ —CO
The compound according to the above item 54, which belongs to the group consisting of compounds having —CH ₂ —ONH ₂ .

56. The compound has the formula:

[0242]

Embedded image (XXI) P—O—CH ₂ CH ₂ —NH
The compounds of the paragraph 55, further comprising a -CO-ONH _2.

57. The compound has the formula:

[0244]

(XXIII) The compound according to the above item 55, having P—O—CH ₂ CH ₂ —ONH ₂ .

58. The compound has the formula:

[0246]

Embedded image (XXIV) P—O—CH ₂ CH ₂ —NH
The compounds of the paragraph 55, further comprising a -CO-CH ₂ ONH _2.

59. The compound has the formula:

[0248]

(XIX) The compound according to the above item 55, having P—O—CH ₂ CH ₂ —CO—ONH ₂ .

60. The polymer is a homopolymer of polyethylene glycol, a homopolymer of polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, wherein the homopolymer and copolymer are unsubstituted or substituted at one end with an alkyl group. ), Polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ether, α, β-poly [(2-hydroxyethyl) -DL-aspartamide], RO-PEG (where R is alkyl, aryl, Alkylaryl, aroyl, alkanoyl, benzoyl, arylalkylether, cycloalkyl, cycloalkylaryl), and a derivative of said polymer according to claim 55. object.

61. 61. The compound according to the above 60, wherein the water-soluble polymer is polyethylene glycol or a derivative thereof.

62. 9. The compound according to the above item 8, wherein the water-soluble polymer is monomethoxy poly (ethylene glycol).

63. A step of mixing the polypeptide with a compound according to paragraph 61 above for denaturation, wherein the water-soluble polymer-modified polypeptide is prepared.

64. The polypeptide for denaturation is
64. A modified polypeptide according to claim 63 selected from the group consisting of hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies.

65. The polypeptide for denaturation is an antibody, and the method further comprises the step of combining the antibody with a compound capable of specifically binding to a binding site on the antibody before the mixing step. Item 64. The modified polypeptide according to Item 64.

66. 65. The modified polypeptide of claim 64, wherein the polypeptide for denaturation is an enzyme and the method further comprises the step of combining the enzyme with a substrate for the enzyme prior to the mixing step.

67. 65. A modified polypeptide according to claim 64, wherein said polypeptide for denaturation is a glycoprotein.

68. 68. The modified polypeptide according to above 67, wherein the glycoprotein is erythropoietin.

69. 63. The modified polypeptide of claim 62, wherein the method further comprises the step of adding an oxidizing agent prior to said mixing step.

70. A composition comprising the polypeptide according to the above 63 and a pharmaceutically acceptable carrier.

71. A composition comprising the polypeptide according to the above item 68 and a pharmaceutically acceptable carrier.

72. formula:

[0262]

Embedded image (XXVIII) [P—O—CH ₂ CH ₂ —
CO-ON = CH-] _n- Z having the formula:

[0263]

Embedded image (XXIX) [P—O—CH ₂ CH ₂ —O
-CO-ON = CH-] _n- Z having the formula:

[0264]

Embedded image (XXX) [P—O—CH ₂ CH ₂ —N
H-CO-ON = CH-] _n- Z having the formula:

[0265]

Embedded image (XXXI) [P—O—CH ₂ CH ₂ —N
H-CS-ON = CH-] _n- Z having the formula:

[0266]

Embedded image (XXXII) [P—O—CH ₂ CH ₂ —
ON = CH-] _n- Z having the formula:

[0267]

Embedded image (XXXIII) [P—O—CH ₂ CH ₂ —
Compounds having an _{NH-CO-CH 2 -ON =} CH-] n -Z, wherein:

[0268]

Embedded image (XXXIV) [P—O—CH ₂ CH ₂ —
_{O-CO-CH 2 -ON =} CH-] compounds with _n -Z, wherein:

[0269]

Embedded image (XXXV) [P—O—CH ₂ CH ₂ —
CH (OH) compound having a _{-CH 2 -ON = CH-] n -Z} , and wherein:

[0270]

Embedded image (XXXVI) [P—O—CH ₂ CH ₂ —C
O-CH ₂ - compound with ON = CH-] _n -Z, wherein, Z is a polypeptide, n is 1 to x, x
Is the number of oxidatively activatable groups on Z, and P is a water-soluble polymer.

73. formula:

[0272]

Embedded image (XXX) [P—O—CH ₂ CH ₂ —N
73. The compound according to clause 72, having H-CO-ON = CH-] _n- Z.

74. formula:

[0274]

Embedded image (XXXII) [P—O—CH ₂ CH ₂ —
73. The compound according to clause 72, having ON = CH-] _n- Z.

75. formula:

[0276]

Embedded image (XXXIII) [P—O—CH ₂ CH ₂ —
_{NH-CO-CH 2 -ON =} CH-] The compounds of the 72nd Claims having _n -Z.

76. formula:

[0278]

Embedded image (XXVIII) [P—O—CH ₂ CH ₂ —
73. The compound according to clause 72, having CO-ON = CH-] _n- Z.

77. P is a polyethylene glycol homopolymer, a polypropylene glycol homopolymer,
Copolymers of ethylene glycol and propylene glycol (wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, polyvinyls Ethyl ether, α, β-poly [(2-hydroxyethyl) -DL-
Aspartamide], RO-PEG (wherein R can be alkyl, aryl, alkylaryl, aroyl, alkanoyl, benzoyl, arylalkyl ether, cycloalkyl, cycloalkylaryl) and derivatives of said polymers. The compound according to paragraph 72, selected from:

78. 73. The compound according to claim 72, wherein Z is selected from the group consisting of hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies.

79. The 74th, wherein Z is a glycoprotein
The compound according to the item.

80. 80. The compound according to clause 79, wherein the glycoprotein is erythropoietin.

81. 77. The compound according to above item 76, wherein Z is erythropoietin.

82. 78. The compound according to the above 77, wherein P is monomethoxypoly (ethylene glycol).

83. 78. The compound according to the above item 78, wherein the average molecular weight of monomethoxy poly (ethylene glycol) is in the range of 2000 to 12000.

84. 83. The compound according to above 83, wherein n is 3-36.

85. 84. The compound according to the above item 84, wherein n is 8-31.

86. 88. The compound according to the above item 85, wherein the average molecular weight of monomethoxypoly (ethylene glycol) is 5000.

87. A step of mixing the polypeptide with an oxidizing agent for activation, the compound XIX, X
A method of activating a polypeptide for conjugation with a compound selected from the group consisting of XI, XXIII and XXIV.

88. 89. The method of claim 87, wherein the oxidant is sodium periodate.

89. The periodate is 1 m of protein
The eighth which is present at a concentration of 10-40 μmol per g
The method according to item 8.

90. 89. The method of paragraph 89, wherein the mixing step is performed at a temperature in the range of -10 to 50 ° C.

91. 91. The method of paragraph 90 above, wherein the mixing step is carried out at a temperature in the range of 0 to 30 ° C.

92. 92. The method of paragraph 91, wherein the mixing step requires a period of 1 minute to 3 days.

93. 93. The method according to clause 92 above, wherein the mixing step is performed for a period of 1 minute to 60 minutes.

94. A method for producing a water-soluble polymer-modified polypeptide, which comprises the step of mixing the polypeptide with the reagent compound of the water-soluble polymer according to the above item 54, for modification.

95. The polypeptide for denaturation is
95. The method of claim 94, selected from the group consisting of hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies.

96. 95. The method according to paragraph 94 above, wherein said polypeptide for denaturation is a glycoprotein.

97. 101. The method of claim 96, wherein the glycoprotein is erythropoietin.

98. 94. The method according to claim 94, further comprising the step of adding an oxidizing agent to the polypeptide for denaturation before the mixing step.
Method described in section.

99. The oxidizing agent is sodium periodate in a concentration range of 10 to 40 μmol, and P is 400
Having a molecular weight in the range of 0 to 12,000, wherein the water-soluble polymer has the formula

[0302]

Embedded image (XXI) P—O—CH ₂ CH ₂ —NH
98. The method according to above 98, which is a compound of —CO-ONH ₂ .

100. A kit for modifying a polypeptide with a water-soluble polymer, which comprises the water-soluble polymer according to the above item 54.

101. The 100th item, further including an oxidant.
Kit described in paragraph.

102. 101. The kit of paragraph 101, further comprising a polypeptide for denaturation.

[Brief description of drawings]

FIG. 1a shows an HPLC chromatogram of EPO. FIG. 1b shows an HPLC chromatogram of the hydrazine derivative of mPEG5000. Figure 1c shows mPEG
3 shows an HPLC chromatogram of EPO modified with 5000 succinimide ester.

FIG. 2: mPEG5 containing different amounts of conjugated mPEG (28, 18 and 12 mPEG / EPO molecules).
Figure 9 is a graph of hematocrit levels in mice treated with 000-EPO. 18 or 28 mPEG 5000
/ Molecule derivatized EPO is derivatized using the semicarbazide compounds of the invention. 12mPEG5000 /
Molecularly derivatized EPO is derivatized using the hydrazide compounds of the invention.

FIG. 3: EPO, hydrazide mPEG5000-EPO (12PEG / EPO), which binds to a monoclonal antibody specific for EPO in an ELISA assay.
Hydrazide mPEG12000-EPO (6PEG / E
PO), thiosemicarbazide mPEG5000-EPO
(25PEG / EPO), semicarbazide mPEG12
000-EPO (14PEG / EPO), and mPE
It is a graph which shows the capability of G12000-EPO (29PEG / EPO).

FIG. 4: mPEG-hydrazide (HY) or mPEG
-Graph showing a comparison of the biological activity of EPO when modified with semicarbazide (SC). Two different molecular weights of mPEG (8500 and 5000) were used in the comparison. Mouse albumin was used as a control.

FIG. 5: mPEG8500-EP containing different amounts of conjugated mPEG (34, 20 and 12 mPEG).
9 is a plot showing hematocrit levels in mice treated with O. EPO derivatized with 34 or 20 mPEG8500 / molecule is derivatized using the semicarbazide compounds of the invention. EPO derivatized with 12mPEG8500 / molecule is derivatized with a hydrazide compound of the invention. Mouse albumin is used as a control.

FIG. 6 Clinigen ^R EPO E
FIG. 6 is a graph showing the results of an ELISA assay for mPEG-modified EPO using the IA test kit. In the legend, SC5-24 means a semicarbazide mPEG5000 modified EPO with 24 molecules of mPEG per molecule of EPO and SC5-18 means a semicarbazide mPEG5000 modified EPO with 18 molecules of mPEG semicarbazide per molecule of EPO.

FIG. 7 is a graph showing the circulating half-life of EPO in plasma. In the legend, SC5-18-iv means semicarbazide mPEG5000 modified EPO with 18 molecules of mPEG per molecule of EPO injected intravenously, EP
O-iv means intravenous injection.

FIG. 8 is a graph showing changes in hematocrit levels in response to EPO injection. In the legend, the term SC5-18 means a semicarbazide mPEG5000 modified EPO having 18 molecules of mPEG per EPO molecule, and the term SC5-22 means a semicarbazide mPEG5000 modified EPO having 22 molecules of mPEG5000 per EPO molecule. , The term HY5-8
Means hydrazide mPEG5000 modified EPO with 8 molecules of mPEG5000 per molecule of EPO.

FIG. 9 is a graph showing changes in hematocrit levels in response to EPO injection. In the legend, the term SC5-24 means a semicarbazide mPEG5000 modified EPO with 24 molecules of mPEG per molecule of EPO and the term TS5-25 means a thiosemicarbazide mPEG5000 modified EPO with 25 molecules of mPEG5000 per molecule of EPO. The term DH5
-22 is mPEG50 of 22 molecules per molecule of EPO
00 means dihydrazide mPEG5000 modified EPO having M.O. A. Means a control for mouse albumin.

FIG. 10 is a graph showing changes in hematocrit levels in response to EPO injection. In the legend, the term TS5-17 refers to thiosemicarbazide mPEG with 17 molecules of mPEG5000 per molecule of EPO.
Means 5000 modified EPO, term SC8.5-12 means thiosemicarbazide mPEG8500 modified EPO with 12 molecules of mPEG8500 per molecule of EPO, term SC2-15 has 15 molecules of mPEG2000 per molecule of EPO Semicarbazide mPEG2
000 modified EPO, and M.P. A. Means a control for mouse albumin.

FIG. 11 is a graph showing changes in hematocrit levels in response to injections of EPO. The legend is as follows: The term SC5-18 means mPEG5000 semicarbazide modified EPO with 18 molecules of mPEG5000 per molecule of EPO and the term SC12-14.
Is 14 molecules of mPEG12,000 per molecule of EPO
A thiosemicarbazide mPEG 12,000 modified EPO having 0, the term SC5-28 means a semicarbazide mPEG 5000 modified EPO having 28 molecules of mPEG5000 per molecule of EPO, and the term HY12-.
6 is 6 molecules of mPEG12,000 per EPO molecule
Mean hydrazide mPEG 12,000 modified EPO having 0. A. Means a control for mouse albumin.

FIG. 12 is a graph showing changes in hematocrit levels in response to EPO injection. In the legend, the term SC8.5-34 is 34 per molecule of EPO.
Semicarbazide mPEG with molecular mPEG8500
8500 means modified EPO and the term SC12-29 is E
A semicarbazide mPEG 12,000 modified EPO having 29 molecules of mPEG 12,000 per molecule of PO; A. Means a control for mouse albumin.

FIG. 13 is a graph showing hematocrit levels in mice injected subcutaneously or intravenously with EPO or EPO derivatives. The legend is as follows: EPO-S
C means EPO injected subcutaneously, EPO-iv means EPO injected intravenously, SC5-28-SC means mPEG50 of 28 molecules per molecule of EPO injected subcutaneously.
Semicarbazide mPEG5000 modified EPO with 00
SC5-28-iv means intravenously injected EPO
Of semicarbazide mPEG5000 modified EPO having 28 molecules of mPEG5000 per molecule of
12-6-SC is 6 per molecule of subcutaneously injected EPO
Hydrazide dimPE with molecular mPEG 12,000
G12,000 means EPO modified, HY12-6-i
v is 6 molecules of mP per molecule of EPO injected intravenously
Hydrazide mPEG 12,000 with EG 12,000
0-modified EPO, and M.O. A. -Sc means a control of subcutaneously injected mouse albumin; A. -Iv means a control for albumin in mice injected intravenously.

FIG. 14 is a graph showing hematocrit levels in mice injected with multiple / single doses of 0.1 μg EPO. The legend is as follows: EPO × 3sc means EPO injected subcutaneously 3 times / week, EPO × iv
Means EPO injected intravenously 3 times / week, SC5-
22 × 1sc means semicarbazide mPEG5000 modified EPO having 22 molecules of mPEG5000 per molecule of EPO injected subcutaneously once / week, SC5-22
X1 iv means semicarbazide mPEG5000-modified EPO having 22 molecules of mPEG5000 per molecule of EPO injected intravenously once / week, A. X3 means a control of albumin in mice injected intravenously 3 times / week.

FIG. 15 is a graph showing hematocrit levels in tumor necrosis factor α (TNFα) -induced anemia mice injected with EPO or EPO derivatives. The legend is as follows: TNF (5) was TN injected over 5 days.
F, T + EPO (5) means TNF and EPO co-injected over 5 days, T + EPO
(2) means TNF injected over 5 days and EPO injected on days 1 and 4, T + SC5-1
8 (5) refers to semicarbazide mPEG5000 modified EPO with 18 molecules of mPEG5000 per molecule of EPO injected on days 1 and 4 concurrently with injected TNF over 5 days, MA refers to a control of mouse albumin. means.

FIG. 16 is a graph showing changes in hematocrit levels in response to EPO injection. In the legend, 18PEG-A is _{mPEG-O-CH 2 CH 2} -NH-CO with mPEG of 18 molecules per molecule of EPO
-ONH ₂ (Formula XXI of the present invention) means EPO modified, 31PEG-C is mP with 31PEG / EPO
_{EG-O-CH 2 CH 2} -NH-CO-CH 2 -ONH
₂ (Formula XXIV of the present invention) means EPO modified,
25PEG-C is _{mPEG-O-CH 2 -CH 2} -NH-CO-CH 2 with mPEG / EPO molecules 25 molecules -
Means EPO modified with ONH ₂ (formula XXIV of the invention).

FIG. 17 is a graph showing changes in hematocrit levels in response to EPO injection. In the legend, the oxime-derivatized 22PEG-B has a 22mPEG molecules / EPO molecule _{mPEG-O-CH 2 CH 2} -ONH
₂ (Formula XXIII of the invention) means EPO modified, 17PEG-B means EPO modified with the same oxime linker with 17mPEG molecule / EPO molecule, and 12PEG-B 12mPEG molecule / EPO.
Mean EPO modified with the same oxime linker with molecules.

FIG. 18. EPO, mPE binding to a monoclonal antibody specific for EPO in an ELISA assay.
_{G-O-CH 2 CH 2} -NH-CO-ONH
₂ (“A”), mPEG5000 EPO (18PEG
/ EPO), mPEG-O- CH 2 CH 2 -ONH
₂ (“B”) mPEG5000 EPO (22PEG /
EPO), mPEG-O-CH 2 CH 2 -ONH
₂ (“B”) mPEG5000 EPO (17PEG /
EPO), mPEG-O-CH 2 CH 2 -ONH
₂ (“B”) mPEG5000 EPO (12 PEG /
EPO), mPEG-O-CH 2 CH 2 -NH-CO-C
H ₂ -ONH ₂ ( "C") mPEG5000 EPO (3
1PEG / EPO), and mPEG-O-CH ₂ CH _{2
-NH-CO-CH 2 -ONH 2} ( "C") MPEG50
Figure 9 is a graph showing the performance of 00 EPO (25 PEG / EPO).

FIG. 19 is a graph showing EPO-dependent cell proliferation using mPEG-EPO. In the legend, 18 PEG
-A is mPEG-O-CH ₂ with 18 PEG / EPO
_{CH 2 -NH-CO-ONH 2} E modified with (Formula XXI)
22PEG-B means 22mPEG molecule / E
_{MPEG-O-CH 2 CH 2} -ONH with PO molecules
₂ (formula XXIII) means EPO modified with 17P
EG-B has the formula XXIII in 17 mPEG / EPO.
Means 12PEG-B means formula XXIII in 12mPEG / EPO, 31PEG-C means 31PE.
MPEG-O-CH ₂ CH ₂ in G / EPO -NH-
CO-CH ₂ -ONH ₂ EPO modified with (Formula XXIV)
, And 25PEG-C is 25mPEG / EP
Means formula XXIV in O.

FIG. 20 is a graph showing EPO-dependent cell proliferation using mPEG-EPO. In the legend, 31mPE
G is an oxime-derivatized mPEG-O-CO-NHNH ₂ having 31 mPEG / EPO molecules (formula I of the present invention
It means EPO modified in I).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // A61K 38/00 38/22

Claims (14)

[Claims] 1. The formula: Wherein, X is O or S, Q is selected from the group consisting of -NHNH ₂ and -C ₆ H ₄ -NHNH _2, and Y is _{-O -, - OCH 2 -,} - NH -, - NHNH -,
_{-O-CO-CH 2 CH 2} - and -NHCO-N-NH
A compound selected from the group consisting of NH-, and P is a water-soluble polymer. 2. A water-soluble polymer-modified polypeptide produced by a process comprising the step of: mixing the polypeptide with the compound of claim 1 for modification. 3. A composition comprising the polypeptide of claim 2 and a pharmaceutically acceptable carrier. 4. The formula: (X) [P—O—CH ₂ —CO—NHN
= CH-] _n- Z, Formula: (XI) [P-O-CO-NHN = CH
-] _N- Z, a compound of the formula: (XII) [P-NH-CO-NHN = C
H-] _n- Z, a compound of formula: (XIII) [P-NH-CS-NHN = C
H-] _n- Z, a compound of the formula: (XIV) [P-NHCO-NH-NHN
A compound having HCO-NHN = CH-] _n- Z, a compound having the formula: embedded image (XV) [P-HNNHCON = CH-] _n- Z, a compound having the formula: embedded image (XVI) [P- compounds having HNNHCSN = CH-] _n -Z, wherein: embedded image (XVII) [P-NH- CO-C 6 H 4 -NH
N = CH-] compounds with _n -Z, and formula: 10] (XVIII) [P-O- CO-CH 2 CH
A compound having _2- CO-NHN = CH-] _n- Z, wherein Z is a polypeptide, n is 1 to x, and x is the number of oxidatively activatable groups on Z, And P is a water-soluble polymer. 5. A step, mixing the polypeptide with an oxidizing agent for activation,
A method of activating a polypeptide for conjugation with a compound of claim 1, which comprises: 6. A method for producing a water-soluble polymer-modified polypeptide, which comprises the step of mixing the polypeptide with the reagent compound of the water-soluble polymer according to claim 1 for modification. 7. A kit for modifying a polypeptide with a water-soluble polymer, which comprises the water-soluble polymer according to claim 1. 8. formula: 11] in P-Y-X-Q formula, X is C = O, C = S, CH 2 or CHOH, Q is -ONH ₂ - and -CH ₂ -ONH ₂ - is selected from the group consisting of, and Y is -O-CH ₂ CH ₂ -, -
_{O-CH 2 CH 2 -O -} , - O-CH 2 CH 2 -N -, - O
It is selected from the group consisting of -CH ₂ CH ₂ -S- and -O-CH ₂ CH ₂ CH-, and P is a water-soluble polymer, a compound having a. 9. A water-soluble polymer-modified polypeptide produced by a process comprising the step of: mixing the polypeptide with the compound of claim 8 for modification. 10. A composition comprising the polypeptide of claim 9 and a pharmaceutically acceptable carrier. 11. A compound of the formula: (XXVIII) [P—O—CH ₂ CH ₂ —
Compounds having a _{CO-ON = CH-] n -Z} , formula: 13] (XXIX) [P-O- CH 2 CH 2 -O
Compounds having a _{-CO-ON = CH-] n -Z} , formula: 14] (XXX) [P-O- CH 2 CH 2 -N
Compounds having an H-CO-ON = CH-] n -Z, formula: 15] (XXXI) [P-O- CH 2 CH 2 -N
Compounds having an H-CS-ON = CH-] n -Z, formula: 16] (XXXII) [P-O- CH 2 CH 2 -
Compounds having ON = CH-] _n -Z, formula: 17] (XXXIII) [P-O- CH 2 CH 2 -
_{NH-CO-CH 2 -ON =} CH-] compounds with _n -Z, formula: 18] (XXXIV) [P-O- CH 2 CH 2 -O-C
O-CH ₂ -ON = CH-] compounds with _n -Z, formula: 19] (XXXV) [P-O- CH 2 CH 2 -CH (O
H) a compound having a _{-CH 2 -ON = CH-] n -Z} , and formula: 20] (XXXVI) [P-O- CH 2 CH 2 -C
O-CH ₂ - compound with ON = CH-] _n -Z, wherein, Z is a polypeptide, n is 1 to x, x is a number from oxidation activatable groups on Z , And P is a water-soluble polymer. 12. A step, mixing the polypeptide with an oxidizing agent for activation,
9. A method of activating a polypeptide for conjugation with a compound of claim 8 which comprises. 13. A method for producing a water-soluble polymer modified polypeptide, which comprises the step of mixing the polypeptide with the reagent compound of the water-soluble polymer according to claim 8 for modification. 14. A water-soluble polymer according to claim 8,
A kit for modifying a polypeptide with a water-soluble polymer.