Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
2000-07-21
2004-01-13
Naff, David M. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C435S180000, C435S181000, C435S188000, C530S815000, C530S816000
Reexamination Certificate
active
06677438
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for attaching or bonding a polyethylene glycol (PEG) compound to a macromolecule such as a protein, a carbohydrate or other polymeric material.
BACKGROUND OF THE INVENTION
An early detailed description of the synthesis of polyethylene glycol derivatives has been given by Harris in JMS. Rev. Macromol. Chem. Phys. C25, 325, 1985.
The modification of proteins and carbohydrates by the attachment of polyethylene glycol (PEG) is known and is described, for example, by Abuchowski et al in J. Biol. Chem. 252, 3578, 1977. The process is often referred to as PEGylation.
Various coupling reactions between amino groups of proteins and carbohydrate molecules and the monomethyl ether of PEG equipped with an electrophilic functional group have also been described, see Zalipsky, Advan. Drug Del. Rev. 16, 157, 1995). The composition of the resultant graft copolymeric system is dependent on the number of available attachment sites on the starting polypeptide (or carbohydrate), the reactivity of the PEG reagent, the excess of such a reagent and the reaction conditions.
Francis et al. discloses a method of bonding polyethylene glycol to proteins and other macromolecules under very mild conditions by activation with tresyl chloride, see Biotechnol. Appl. Biochem. 12, 119, 1990.
Various methods of PEGylation are also described in U.S. Pat. No. 4,179,337, U.S. Pat. No. 4,732,863, U.S. Pat. No. 4,917,888, WO-86/04145, WO-90/04606, WO-90/06952, WO-90/13540, WO-91/01758, EP-400472, EP400486 and EP-183503.
The therapeutic value of polyethylene glycol modified proteins has also been reviewed, see Nucci et al., Advan. Drug Del. Rev. 6, 133, 1991 and Inada et al., J. Bioactive Compat. Polymer 5, 343, 1990.
SUMMARY OF THE INVENTION
We have developed a novel method of attaching a polyethylene glycol compound to suitably functionalized macromolecular materials. By a polyethylene glycol compound we include polyethylene glycol (PEG) itself and derivatives thereof (PEG derivatives) in which one or both of the terminal hydroxyl groups in the polyethylene glycol molecule has been previously modified.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention there is provided a process for attaching a polyethylene glycol compound to a macromolecule to prepare a conjugate or adduct between the polyethylene glycol compound and the macromolecule which comprises the steps of:
(1) preparing an activated PEG or an activated PEG derivative by incorporating an acrylic ester, an acrylic thioester or an acrylamido group into the PEG or PEG derivative;
(2) reacting the activated PEG or PEG derivative with a macromolecular material comprising one or more sulphydryl groups, primary amino groups and/or secondary amino groups; and
(3) recovering the conjugate of the PEG or PEG derivative and the macromolecular material.
By a PEG derivative we mean a polyethylene glycol polymer in which one or both of the terminal hydroxyl groups found in polyethylene glycol itself has been modified. Examples of suitable modifications include replacing one or both hydroxyl group(s) with alternative functional groups, which may be protected or unprotected, with low molecular weight ligands, or with another macromolecule or polymer. Modification of the terminal hydroxyl groups in polyethylene glycol can be achieved by reacting the polyethylene glycol with compounds comprising complementary reactive. functional groups, i.e. functional groups which are able to undergo a reaction with the hydroxyl groups in polyethylene glycol.
Suitable PEG derivatives include compounds in which one of the terminal hydroxyl groups has been converted into a group having the formula RO— in which R is an alkyl, cycloalkyl, aryl, aralkyl or alkaryl group. Preferably R is alkyl to give a terminal alkoxy group. Preferred alkoxy groups are C
1-4
alkoxy, such as methoxy.
The macromolecular material may contain just sulphydryl, primary amino or secondary amino groups or it may contain a mixture of such groups. Suitable macromolecular materials for conjugation to a polyethylene glycol compound include therapeutic proteins such as interleukins, albumins, growth hormones, aspariginase, superoxide dismutase, monoclonal antibodies, as well as carbohydrates, such as starch and dextran. Many of these macromolecules are of biological origin. The macromolecular material may also be a polymer of biological origin which has been previously modified by a reagent to introduce a sulphydryl group or a primary or secondary amine group. A preferred macromolecular material is a protein or a poly(amino) sugar.
The preparation of a PEG or PEG derivative carrying an acrylic thioester group can be carried out using conventional techniques in which a PEG or PEG derivative containing a sulphydryl group is reacted with acryloyl chloride. The reaction is typically conducted in the presence of a base such as a tertiary amine, e.g. triethylamine, or an aqueous solution of sodium hydroxide at a temperature in the range of from 0 to 5° C. to avoid radical polymerisation between the acrylic double bonds. Suitable solvents for the reaction include aliphatic and aromatic hydrocarbons, chloroform and other halogenated hydrocarbon solvents. (See J. March, Advanced Organic Chemistry, John Wiley & Sons, New York, 3rd Edition (1985), page 362.)
The preparation of a PEG or PEG derivative carrying an acrylic ester group can be carried out using conventional techniques in which a PEG or PEG derivative containing a hydroxyl group is reacted with acryloyl chloride. The reaction is typically conducted in the presence of a base such as a tertiary amine, e.g. triethylamine, or an aqueous solution of sodium hydroxide at a temperature in the range of from 0 to 5° C. to avoid radical polymerisation between the acrylic double bonds. Suitable solvents for the reaction include aliphatic and aromatic hydrocarbons, chloroform and other halogenated hydrocarbon solvents. (See J. March, Advanced Organic Chemistry, John Wiley & Sons, New York, 3rd Edition (1985), page 346.)
The preparation of a PEG or PEG derivative carrying an acrylamido group can be carried out using conventional techniques in which a PEG or PEG derivative containing a primary or secondary amino group is reacted with acryloyl chloride. The reaction is typically conducted in the presence of a base such as a tertiary amine, e.g. triethylamine, or an aqueous solution of sodium hydroxide at a temperature in the range of from 0 to 5° C. to avoid radical polymerisation between the acrylic double bonds. Suitable solvents for the reaction include aliphatic and aromatic hydrocarbons, chloroform and other halogenated hydrocarbon solvents. (See J. March, Advanced Organic Chemistry, John Wiley & Sons, New York, 3rd Edition (1985), page 370.)
An example of such preparations is reported by Bignotti et al. (Macromol. Rapid Communic. 15, 659, 1994). Acrylating agents other than acryloyl chloride may also be used, such as 1-acryloylbenzotriazole and N-acryloyloxysuccinimide. However, acryloyl chloride is the preferred acrylating reagent.
The activated PEG or PEG derivative can be characterized by Gel Permeation Chromatography (GPC), FT-IR and UV spectroscopy. The degree of activation can be determined by end-group titration which involves adding an excess of 2-mercaptoethanol and titrating excess thiol with a calibrated KI/I
2
solution, or by UV spectroscopy, after calibration with a standard acrylamide or acrylic ester such as N-acryloylmorpholine or tetraethyleneglycol diacrylate, conducted at 233 nm.
Activated PEGs or activated PEG derivatives can be stored for several months at T≦4° C. in the presence of a desiccant. A radical inhibitor such as 4-methoxyphenol may also be added to prevent radical polymerization.
The reaction of the activated PEG or activated PEG derivative with the macromolecular material is preferably performed at pH≧8, usually in the range 8<pH<9, in aqueous media. Below pH 7.5 the reaction rate tends to be slow. Alcohols or alcohol/water mixtures can also be used.
A
Bignotti Fabio
Davis Stanley Stewart
Ferruti Paolo
Garnett Martin Charles
Naff David M.
Oppenheimer Wolff & Donnelly LLP
University of Nottingham
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