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Numéro de publicationWO2003078500 A1
Type de publicationDemande
Numéro de demandePCT/US2003/007697
Date de publication25 sept. 2003
Date de dépôt11 mars 2003
Date de priorité12 mars 2002
Numéro de publicationPCT/2003/7697, PCT/US/2003/007697, PCT/US/2003/07697, PCT/US/3/007697, PCT/US/3/07697, PCT/US2003/007697, PCT/US2003/07697, PCT/US2003007697, PCT/US200307697, PCT/US3/007697, PCT/US3/07697, PCT/US3007697, PCT/US307697, WO 03078500 A1, WO 03078500A1, WO 2003/078500 A1, WO 2003078500 A1, WO 2003078500A1, WO-A1-03078500, WO-A1-2003078500, WO03078500 A1, WO03078500A1, WO2003/078500A1, WO2003078500 A1, WO2003078500A1
InventeursChristopher G. Anderson
DéposantWatson Pharmaceuticals, Inc.
Exporter la citationBiBTeX, EndNote, RefMan
Liens externes:  Patentscope, Espacenet
Polyalkylene graft polymers
WO 2003078500 A1
Résumé
The present invention provides substantially pure, pharmaceutically acceptable polyaklylene graft polymers and processes for obtaining them. Liquid polyalkylene oxide, from about ten to fifteen equivalents of an acrylic monomer and a suitable catalyst are mixed in the absence of oxygen to form a reaction mixture. The substantially pure polymers are collected from the reaction mix. The polymers can be further purified. Further provided is a process to isolate or collect large molecular weight polymers from an aqueous/organic solvent system.
Revendications  (Le texte OCR peut contenir des erreurs.)
CLAIMSWe claim:
1. A process for preparing polyalkylene graft polymers, comprising the steps of:
a) adding to form a reaction mixture a fluid polyalkylene oxide, from about ten to fifteen equivalents of an acrylic monomer, a suitable catalyst, and in the absence of oxygen, to form said grafted polymers in said reaction mixture; and
b) collecting said grafted polymers from said reaction mixture.
2. The process of claim 1, further comprising purifying said collected graft polymers of step b).
3. The process of claim 2, wherein said purification step is crystallization at 0°C.
4. The process of claim 2, wherein said purification step comprises crystallizing said collected grafted polymer from an acetone and isopropyl alcohol solvent system.
5. The process of claim 4, wherein said suitable solvent is an acetone and isopropyl alcohol solvent system is in a ratio of about 1:1.
6. The process of claim 1, wherein said fluid polyalkylene oxide comprises a pendant functional group.
7. The process of claim 6, wherein said pendant functional group is propionic acid.
8. The process of claim 1, wherein said polyalkylene oxide is alkyl blocked.
9. The process of claim 6, wherein said pendant functional group further comprises a reactive group.
10. The process of claim 1, wherein said polyalkylene oxide is selected from the group consisting of polyethylene glycol, polypropylene glycol, polyethylene glycol monomethyl ether, polyethylene glycol dimethyl ether, and derivatives thereof.
11. The process of claim 1 , wherein said catalyst is a peroxide that forms free radicals.
12. The process of claim 1, wherein step a further comprises an effective amount of a dispersant.
13. The process of claim 12, wherein said dispersant is a hydrocarbon.
14. The process of claim 11, wherein said peroxide is selected from the group consisting of t-butyl peroxide, t-butyl peroxybenzoate, di-t-butyl peroxide, and t- butyl hydroperoxide.
15. The process of claim 1, wherein said collection of step b) is by ultrafiltration from an aqueous organic solvent.
16. A process for preparing polyalkylene graft polymers, comprising the steps of:
a) adding to fluid polyalkylene from about ten to fifteen equivalents of an acrylic monomer and a suitable catalyst and in the absence of oxygen to form a reaction mixture; and
b) collecting said polyalkylene graft polymers by ultrafiltration with an aqueous/organic solvent system; and
purifying said polymers produced by step b) by crystallization in an organic solvent solution.
17. The process of claim 16, wherein said organic solvent system of step c) is an acetone and isopropyl alcohol solvent system.
18. The process of claim 17, wherein said acetone and isopropyl alcohol solvent system is in a ratio of about 1:1.
19. Purified polyalkylene oxide graft polymers produced by the process of claim 18.
20. A process for separating large molecular weight polymers from an aqueous/organic solvent solution comprising ultrafiltration of said solution and collecting the separated polymers.
21. The process of claim 20, wherein said aqueous/organic solvent is an alcohol/water solvent system.
22. The process of claim 21, wherein said solvent system comprises at least 20% v/v alcohol.
23. A preparation of pharmaceutically acceptable substantially pure polyalkylene graft polymers.
24. A pharmaceutically acceptable, substantially colorless, polyalkylene graft polymer preparation of claim 23, wherein said preparation has less than about 1,000 ppm low molecular weight contaminants.
25. The preparation of claim 24, wherein said low molecular weight contaminants have a weight molecular weight of less than 14,000 daltons.
26. A pharmaceutical composition comprising the graft polymers of claim 23 and a pharmaceutically active agent or drug.
27. The composition of claim 26, further comprising a pharmaceutically acceptable carrier.
28. Use of the composition of claim 26 in the preparation of a medicament to treat, prevent or ameliorate the symptoms of a pathology.
Description  (Le texte OCR peut contenir des erreurs.)

POLYALKYLENE GRAFT POLYMERS

FIELD OF THE INVENTION

The present invention relates generally to the field of grafting polymers and copolymers, particularly polymers and copolymers of poly(oxyalkylene) compounds.

BACKGROUND

Polyalkylene oxides, polymers and copolymers thereof are multi-functional. They are used in a variety of industrial applications such as lubricants and cleaning agents. They are used in pharmaceutical applications including polymer-based drug delivery systems. For example, monomethoxypolyethyleneglycol ("mPEG") bound to various proteins, including asparaginase and interleukin-2, and attached via various spacer or coupling groups have been tested clinically. (See, Duncan, Anti-Cancer Drugs 3:175-210 (1992)). The presence of multiple functionalities on apolymer permits the attachment of larger quantities of therapeutic agents to the polymer, to improve efficacy of a polymer-drug system. See e.g., U.S. Patent No. 6,251, 866B1 to Prakash and Clemens.

Techniques for producing polymers and copolymers by grafting unsaturated monomers onto polymer backbones are generally known. However, the resultant graft polymers and copolymers are often contaminated with by-products from undesired side reactions, such as chain cleavage resulting in lower molecular weight polymeric aldehydes, alcohols and other oxidation products. For example, U.S. Patent No. 4,528,334 to Knopf et al. describes a method for preparing graft polymers by grafting 3 to 15 % by weight of acrylic acid onto poly(alkylene) compounds. The polymers obtained using this process are mixtures containing not only the desired polymer, but also degradation products derived from chain scission of the polymer and degradation products from the peroxide initiator. They also contain measurable amounts of the solvent and by-products derived from reaction of the peroxide initiator with solvent. Moreover, minor amounts of homopolymer derived from polymerization of the unsaturated monomer also appear to be present.

U.S. Patent No. 3,546,321 to Nandenberg et al. describes a method for preparing graft copolymers of polyether polymers using monomers containing unsaturated ethylene groups. Polyether polymers having a hydrogen atom attached to a carbon atom alpha to an ether oxygen atom in the polymer backbone are first contacted with oxygen in the presence of a free radical initiator agent to form the hydroperoxide thereof. Graft polymers are then prepared from the polyether hydroperoxides by addition of one or more vinylidene monomers to a free radical on the polyether main chain created by decomposition of the hydroperoxy groups. However, this process is a two-stage process and more complex than desired for a number of applications. In addition, graft polymers and copolymers prepared in the presence of oxygen often contain colored impurities resulting in light yellow products. Thus, there remains a need in the art for improved methods for preparing graft polymers and copolymers and especially copolymers for pharmaceutical use that are simple and yield highly purified polymers and copolymers.

DISCLOSURE OF THE INVENTION

Substantially pure, pharmaceutically acceptable, graft polymers are provided by this invention. They are clear and colorless and contain less than 1,000 ppm impurities, e.g., they are substantially free of polyacrylic acid homopolymer, degradation products and other impurities, e.g., polyacrylic acid homopolymers, reactants and degradation products. Purity can be determined using conventional methods, e.g., chromatographic, spectrophotometric and by comprable and/or yet to be discovered methods, hi one aspect, the polymers of this invention are useful in the preparation of compositions for the targeted delivery of drugs. U.S. Patent No. 6,251, 866B1 to Prakash and Clemens describes pharmaceutical applications of the polymers.

The polymers optionally contain pendant groups that may or may not contain functional groups, like an acid group such as a propionic acid group. The number of pendant acid groups in the polymers also can vary. For example, the number can vary between: about one to twenty; about one to fifteen; about three to ten; or about two to eight. The number of pendant propionic acid groups in the polymer can also exceed eight.

In particular embodiments, the poly(oxyalkylene) graft polymer obtainable using the present invention include, but are not limited to polyethylene glycol monomethyl ether and polyethylene glycol dimethyl ether possessing pendant functionalities.

Processes for preparing and obtaining the pharmaceutically acceptable graft polymers are also provided. In one embodiment, the method requires adding to a liquid polyalkylene oxide from about ten to fifteen equivalents of an acrylic acid monomer and a suitable catalyst, in the absence of oxygen, to form grafted polymers in the reaction mixture. The process is optionally carried out in the presence of a sutiable dispersent. Grafted copolymers are then collected or isolated from the mix to yield substantially pure, pharmaceutically acceptable graft polymers. By way of example only, ultrafiltration can be used to isolate the substantially pure product.

Optionally, the collected polymers are further purified using a variety of techniques such as crystallization and/or flocculation.

Further provided is a method to separate or collect large molecular weight polymers from an aqueous/organic solvent or dispersent by ultrafiltration of the polymer/solvent mixture and collecting the separated polymer.

MODES FOR CARRYING OUT THE INVENTION

DEFINITIONS

To facilitate understanding of the present invention, a number of terms are defined below.

As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a composition containing "a pendant acid group" includes reference to one or more such pendant acid groups, which may be the same or different pendant acid groups.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. "Consisting of when used to define compositions and methods shall mean excluding more than trace elements of other ingredients and substantial method steps from the compositions and methods of the invention. Embodiments defined by each of these transition terms are within the scope of this invention.

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which may be varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are well known in the art.

As used herein, the terms "flocculation" or "crystallization" are used interchangeably and refer to a purification process that produces purified product from a suitable solvent system leaving the undesired products dissolved in the solvent system.

As used herein, the term "polymers" refers to high molecular weight compounds comprising linked monomer units, h certain aspects, the term may encompass homopolymers and copolymers.

As used herein, the terms "poly(oxyalkylene) graft polymer" and "polyalkylene oxide graft polymer" refer to the polymers obtainable by graft polymerizing unsaturated monomers onto the polymer backbone using the methods of the present invention.

As used herein, the terms "alkyl blocked" or "alkyl capped" polyalkylene oxide refers to the form of the polymer when one or more of the terminal hydroxyl groups are capped with an alkyl group. Thus, a mono-methyl blocked polyalkylene oxide refers to the form when one of the terminal hydroxyl groups is capped with a methyl group. As used herein, the term "mpPEG" refers to a polyethylene glycol monomethyl ether bearing pendant groups. The term "pPEG" refers to a polyethylene glycol bearing pendant groups. The term "dmpPEG" refers to a polyethylene glycol dimethyl ether bearing pendant groups.

As used herein, the term "mpPEG-8PA" refers to a mono-methyl blocked pendant polyethylene glycol having approximately eight pendant propionic acid moieties per mole of the polymer. As used herein, the term "pendant group" refers to groups attached onto a polymer backbone. In one aspect, the pendant group is a pendant acid group.

As used herein, the terms "pendant polyalkylene oxide," "pendant polyalkylene oxide glycol," or "polyalkylene oxide possessing pendant functionalities" refer to a graft polymer, wherein the polymer includes a plurality of pendant functional groups dispersed along the polymer chain. The pendant functional groups typically comprise reactive groups, or can be modified to comprise reactive groups to permit further modification and covalent attachment of other molecules to the polymer, e.g., targeting ligands and/or chemical agents.

As used herein, the terms "polyalkylene oxide derivative," "polyethylene glycol derivative" and "polypropylene glycol derivative" refer to derivatives that have been modified with a modifying group. For example, one or more of the terminal hydroxyl groups can be modified with a modifying group such as an alkyl group or a functional group. Glycols derivatized with one or more functional groups are termed pendant glycols, e.g., a pendant polyalkylene glycol.

An "acid group" intends those functionalities that possess an ionizable proton, such as a carboxylic acid functionality.

"Low molecular weight polymer or copolymer" intends those having a molecular weight less than 22,000, or alternatively greater than 20,000, or alternatively greater than 18,000, or alternatively, greater than 14,000 or alternatively greater than 12,000 or alternatively greater than 10,000. "High molecular weight polymer or copolymer" intends those having a molecular weight greater than 10,000, or alternatively greater than 12,000, or alternatively greater than 14,000, or alternatively, greater than 18,000 or alternatively greater than 20,000 or alternatively greater than 21,000.

As used herein, the term "fluid" polyalkylene oxide refers to polyalkylene oxide whose physical state has been converted to a liquid or melted state. Conversion can be accomplished by application of a suitable energy source, examples of which include, but are not limited to heat, ultrasound, or radiation, e.g., microwave.

As used herein, the term "solid" refers to a state of matter that is rigid and having a defined shape and volume, unlike liquids or gases.

As used herein, the term "acrylic monomers" refers to monomers derived from acrylic acid, a polymerizable alkene having the structure H C=CH-COOH. Acrylic monomers include, but are not limited to acrylic acid and substituted acrylic acid monomers. As used herein, the term "substituted acrylic acid monomers" refers to acrylic acid that has been modified with a modifying group.

As used herein, the term "substantially purified" polymer refers to a polymer that is substantially free (e.g., about 70% to about 95% pure or about 80% to about 95% pure or at least 90% pure or at least 95% pure; or at least 97% pure) of impurities and undesired byproducts, such as reaction byproducts, solvent (dispersant), other volatile impurities, insoluble homopolymerized acrylic acid, degradation products, unreacted starting materials if any, and other impurities. Purity can be determined using any analytical method known in the art, that includes, but is not limited to, spectroscopic methods such as Nuclear Magnetic Resonance spectroscopy (NMR), Mass Spectroscopy, (FUR), and chromatographic methods such as Gel Permeation Chromatography (GPC) and Gas Chromatography (GC). In GPC, polymer molecules in a solvent are delayed in their passage through columns filled with porous particles depending on their ability to penetrate the pores. Larger molecules are unable to penetrate the membrane pores and pass through the column first, while smaller molecules penetrate into the pores and pass through the column at a later time. Thus, GPC separates molecules according to size. This separation technology is also referred to as size exclusion chromatography (SEC). Modern Size-Exclusion Liquid Chromato graphv. W.W. Yau, J. J. Kirkland, and D.D. Bly, John Wiley and Sons, New York, N.Y. (1979).

As used herein, the term "peroxide" refers to a compound having an oxygen- oxygen bond, and that produces free radicals to catalyze polymerization.

As used herein, the term "hydrocarbon" refers to a compound whose chemical structure is composed exclusively of carbon and hydrogen. Major classes of hydrocarbons are the alkanes, alkenes, alkynes, and the aromatic hydrocarbons. Alkanes are hydrocarbons that contain only single bonds. Alkenes are hydrocarbons that contain carbon-carbon double bonds. Alkynes are hydrocarbons with carbon- carbon triple bonds. Aromatic hydrocarbons are derivatives of benzene. Certain alkanes and aromatic hydrocarbons are useful dispersants for the reaction mixture used in this process.

As used herein, the term "reaction mixture" refers to the crude mixture obtainable by reacting an acrylic acid monomer with fluid polyalkylene oxide, prior to any purification.

As used herein, the term "absence of oxygen" refers to an absence of atmospheric oxygen, for example, a reaction condition that is maintained in an inert gas atmosphere such as nitrogen, helium or argon.

The present invention relates generally to the field of grafting water soluble, biocompatible copolymers, particularly copolymers of poly(oxyalkylene). Suitable polyalkylene oxides, catalysts, and acrylic acid monomers are discussed in detail in the Materials section, infra.

Carboxylated poly(oxyalkylenes) described in the prior art are mixtures containing not only polymer but also degradation products from reactants (see e.g., U.S. Patent No. 4,528,334 to Knopf et al.). In contrast, the graft polymers obtainable using the methods of the present invention are substantially pure (e.g., containing less than 1000 ppm contaminants) based upon GPC and GC results. Unlike prior art processes, the polymer is prepared in the absence of oxygen to prevent degradation products and improve color and subsequently collected. The addition of reagents is also carefully controlled which provides a product with reproducible physical and chemical properties.

Poly(oxyalkylene) graft polymers of the present invention are substantially free of polyacrylic acid homopolymer formed during the reaction. In one aspect, the removal of polyacrylic acid homopolymer is achieved using an acetone / isopropyl alcohol solvent system in the crystallization procedures. Polyacrylic acid homopolymer is soluble in acetone / isopropyl alcohol, while the poly(oxyalkylene) graft polymer is insoluble in the same solvent system at 0 °C.

The substantially pure and pharmaceutically acceptable copolymers are obtained by combining liquid polyalkylene oxide, a suitable catalyst and about ten to fifteen equivalents of an acrylic acid monomer in the absence of oxygen to form a reaction mixture. The grafted copolymers are then collected from the reaction mixture. Ultrafiltration is one means to collect the copolymer from the reaction mixture although any method that removes substantial amounts of lower molecular weight impurities, including chain scission products, unreacted reagents, degradation products, low molecular weight homopolymers, solvent or dispersant, and minor solvent reaction products is suitable for use in the process. Examples include, but are not limited to chromatographic techniques.

The molecular weight of the pendant polymer obtainable using the methods of the present invention is not limited to a particular number, but only by the relevant practical considerations such as crowding, handling, physical characteristics (e.g., viscosity, density, solubility, etc.).

In one aspect, the starting polymer is a blocked polyalkylene glycol, such as an alkyl blocked polyalkylene glycol. The number of carbons in the alkyl can vary, e.g. in ranges, from about one to twenty, three to eighteen, four to sixteen or twenty or less. For example, the starting polymer can be an alkyl blocked polyethylene glycol. hi one embodiment, the alkyl blocked polyethylene glycol starting material is selected from the group consisting of mono-methyl blocked polyethylene glycols and dimethyl blocked polyethylene glycols.

hi yet another aspect, the starting polymer is a linear polymer comprising terminal hydroxyl groups, with at least one of the tenninal hydroxyl groups capped with a nonreactive functional group such as an alkyl group. For example, the polymer can be mono-methyl blocked (i.e., having one terminal hydroxyl capped with a methyl group) or dimethyl blocked with a methyl capping group on each of the terminal hydroxyls.

In the case of a mono-alkyl blocked polyalkylene glycol, an acyl group such as acetyl or hemissucinyl can further block the remaining free hydroxyl groups (e.g., via an ester bond from reaction with a mono or dicarboxyhc acid or derivative), hi the case of hemisuccinyl or other diacyl compound, an additional reactive group (e.g., carboxyl groups) can be introduced for further derivatization. Alternatively, the non-alkylated polymer bearing pendant group containing two terminal hydroxyl moieties can be capped with two acyl or diacyl compounds such as acyl or hemisuccinyl to yield a bi- substituted bis-blocked polymer. When a hydroxyl group is capped with bis- hemisuccinyl or other bis-diacyl compound, two additional reactive groups (e.g., carboxyl groups) can be introduced at the ends of each polymer chain for further derivatization.

Although acyl blocked polyalkylene glycols bearing pendant functionalities have similar advantages to alkyl blocked polyalkylene glycols bearing pendant functionalities, diacyl blocked polyalkylene glycols possessing pendant groups offer the advantage of introducing an additional reactive carboxyl group that can be further derivatized. This is a particular advantage when the pendant group contains a carboxyl moiety, since the possibility of differential reactivity between the hemisuccinyl carboxyl group and the pendant carboxyl group is created. Examples of diacyl blocked polyalkylene glycols bearing pendant groups include, but are not limited to graft polymers such as bis-hemisuccinyl polyethylene glycol and monomethyl-hemisuccinyl polyethylene glycol possessing pendant groups.

The addition of acrylic acid to liquid polyalkylene oxide can be performed with or without a dispersant. A suitable dispersent is one that is non-reactive and inert to the reactants. Hydrocarbon solvents, e.g., nonane, decane, dodecane and undecane are non- limiting examples. The polyalkylene oxide may or may not be partially or totally soluble in the dispersent.

If the polyalkylene oxide is not in liquid form, a suitable amount of an energy source is applied to the compound for an effective amount of time to convert it to a liquid. Heat, radiation (e.g., microwave) and ultrasound are non-limiting examples of suitable energy sources, h one embodiment, the compound is dispersed in a hydrocarbon solvent to form a dispersed mixture and heat is applied. Temperatures between about 100 °C and about 160 °C are sufficient for conversion to a fluid. Alternatively, temperatures between about 130 °C and about 150 °C will convert polyalkylene oxide.

Collection or isolation of the grafted copolymers from the reaction mixture can be by any means that separates large molecular weight copolymers from unreacted monomers and polyalkylene starting materials, copolymers formed from undesired side reactions as well as catalyst and dispersent if applicable. Ultrafiltration is one means to obtain a substantially pure grafted copolymer. As used herein, the term "ultrafiltration" refers to a pressure activated physical separation process, in which a porous membrane is used to separate lower molecular weight compounds from larger molecules. However, it is to be understood, although not always explicitly stated that other separation procedures that produce the same or similar result are intended to be included in the use of the term "ultrafiltration".

Ultrafiltration employs membrane separation techniques, which are based on molecular size. For example, a semi permeable membrane in an ultrafiltration system having an appropriate pore size will allow water and low molecular weight compounds to pass tlirough the pores as permeate, while the larger molecules and suspended solids flow across the membrane and become part of the retentate.

Ultrafiltration membranes are available with specific molecular weight cutoff values previously reported using water as solvent. For example an ultrafiltration membrane with a weight molecular weight cutoff value of 10 kiloDaltons will not allow a specified percentage of all molecules with a molecular weight greater than 10,000 Daltons to pass through the membrane, while allowing a specified percentage of all molecules with a molecular weight below 10,000 Daltons to pass through the membrane. Ultrafiltration membranes used in conjunction with aqueous organic solvent mixtures as in the present invention demonstrate higher molecular weight cutoff values than reported for water. Thus, use of membranes with lower molecular weight cutoff values with aqueous organic solvents is desirable.

More pure product can be obtained by further purification, e.g., by crystallization or flocculation. In one aspect, the flocculation procedure is in the presence of an acetone/isopropyl alcohol system.

Also provided is a pharmaceutically acceptable graft copolymer obtainable by a process of this invention. In one aspect, the graft copolymer is a grafted polyalkylene oxide copolymer. h a further aspect, the grafted copolymer contains pendant groups. In a further aspect the copolymer contains pendant functional groups. This invention also provides a pharmaceutical composition containing at least one or more of a copolymer selected from a grafted polyalkylene oxide copolymer, a pendant polyalkylene oxide copolymer and a functional pendent polyalkylene oxide copolymer and a pharmaceutically active agent or drug. A pharmaceutically acceptable composition comprising admixing a grafted copolymer obtainable by a process of this invention and a pharmaceutically active agent or drug is further provided herein. These compositions are suitable for the preparation of a medicament for the delivery of a pharmaceutically active agent.

A process for separating or collecting large molecular weight copolymers from an organic solvent or dispersant is further provided by ultrafiltration of the copolymer/solvent mixture and collecting the separated copolymer. In one aspect, the organic solvent comprises an aqueous/organic solvent system. Examples of organic solvent systems include, but are not limited to at least 20% alcohol/water such as methanol/water and ethanol/water. h another aspect, the % alcohol is increase to at least 25%o of the total volume. MATERIALS AND METHODS

Polyalkylene Oxides

Various water soluble, biocompatible polymers, e.g., polyalkylene oxides, can be used as the starting material. (See e.g., Duncan, "Drug-polymer conjugates: potential for improved chemotherapy" Anti-Cancer Drugs 3:175-210 (1992)). In one aspect, the water soluble polymer is a polyalkylene oxide. Within this group of substances are alpha-substituted polyalkylene oxide derivatives, such as methoxypolyethylene glycols or other suitable alkyl-substituted derivatives. Suitable alkyl-substituted derivates include, but are not limited to, Cι-C4 alkyl groups.

The polyalkylene oxide can be a monomethyl-substituted pendant PEG homopolymer. However, other poly(alkylene oxides) can also be used, including but not limited to, polyethylene glycol (PEG) homopolymers and derivatives thereof; polypropylene glycol homopolymers and derivatives thereof; alkyl-capped polyethylene oxides; bis-polyethylene oxides; copolymers of poly(alkylene oxides); and block copolymers of poly(alkylene oxides) and activated derivatives thereof. Other PEGs are branched, pendant and star PEGs, such as those commercially available from Shearwater Polymers, Inc. (Huntsville, AL). (Gnanou et al., Makromol. Chem. 189:2885 (1988); Rein et al., Acta Polymer 44: 225 (1993); Merrill, U.S.Patent 5,171,264; Poly(Ethylene Glycol) Chemistry in Biotechnical and Biomedical Application. J. Milton Harris, (1992).

The molecular weight of the polymer is not limited to a particular number, but only by the relevant practical considerations such as crowding, handling, physical characteristics (e.g., viscosity, density, solubility, etc.) and the composition for administration to a mammal. For example, in some aspects, the PEG-based polymers have average molecular weights of from about 200 to about 50,000. hi another embodiment, the PEG-based polymers have average molecular weights of from about 2,000 to about 20,000 are used. PEG is inexpensive, approved by the FDA for administration to humans, and is resistant to eliciting an antibody response. Polyethylene oxide) (PEO) is another water soluble polymer. Non-limiting examples are selected from the group consisting of polyethylene glycol, polypropylene glycol, polyethylene glycol monomethyl ether, polyethylene glycol dimethyl ether, copolymers of related compounds and derivatives thereof, such as any unsaturated system, e.g., an alkynyl, an alkynyl analog or a homolog thereof.

These poly(oxyalkylene) compounds can be made by processes known in the art by reacting an alkylene oxide or mixtures of alkylene oxides with a compound having from one to four active hydrogen atoms, such as water, monohydroxylic alcohols such as ethanol and propanol, dihydroxylic alcohols such as ethylene glycol and monoethyl ether of glycerine, trihydroxylic alcohols such as glycerine and trimethylolpropane, tetrahydroxylic alcohols such as pentaerythritol, hexahydroxylic alcohols such as sorbitol. The poly(oxyalkylene) products of such reactions can have linear or branched oxyalkylene or oxyethylene-higher oxalkylene chains. Such chains can terminate with hydroxyl groups. Some or all of these hydroxyl groups can be etherified by reaction with a dialkyl sulfate such as diethyl sulfate.

Pendant Polymers

Poly(oxyalkylene) graft polymers of the present invention can comprise a pendant functional group. The functional group typically comprises a reactive group, or can be modified to comprise a reactive group to permit further modification and covalent attachment of other molecules to the polymer. For example, the pendant group can be an acid group, such as a propionic acid group.

The number of pendant acid groups in the polymer can vary. For example, the number of pendant acid groups in the polymer can be from about two to eight. In other embodiments, the number of pendant acid groups in the polymer exceeds eight. A direct correlation between the number of terminal hydroxyl groups and grafting efficiency has also been established. Polyethylene glycol) is more efficiently grafted than poly(ethylene glycol) monomethyl ether, which is in turn more efficiently grafted than poly(ethylene glycol) dimethyl ether. hi one aspect, the polymer is a blocked pendant polyalkylene glycol, such as an alkyl blocked pendant polyalkylene glycol. The polymer is preferably an alkyl blocked pendant polyethylene glycol. For example, the alkyl blocked pendant polyethylene glycol can be a mono-methyl blocked pendant polyethylene glycol, or a dimethyl blocked pendant polyethylene glycol.

In yet another aspect, the pendant polymer is a linear polymer comprising terminal hydroxyl groups. At least one of the terminal hydroxyl groups is preferably capped with a nonreactive functional group such as an alkyl group. For example, the polymer can be mono-methyl blocked (i.e., having one terminal hydroxyl capped with a methyl group) or dimethyl blocked with a methyl capping group on each of the terminal hydroxyls.

hi the case of a mono-alkyl blocked polyalkylene glycol, the remaining free hydroxyl groups can be further blocked by an acyl group such as acetyl or hemisuccinyl (e.g., via an ester bond from reaction with a mono or dicarboxyhc acid or derivative). In the case of hemisuccinyl or other diacyl compound, additional reactive groups

(carboxyl groups) can be introduced for further derivatization. Alternatively, the non- alkylated pendant polymer containing two terminal hydroxyl groups can be capped with two acyl or diacyl compounds such as acyl or hemisuccinyl to yield a bi-substituted bis- blocked polymer. When hydroxyl groups are capped with bis-hemisuccinyl or other bis-diacyl compound, two additional reactive groups (e.g., carboxyl groups) can be introduced at the ends of each polymer chain for further derivatization.

Blocked pendant polyalkylene glycols can be made using synthetic methods available in the art. Pendant PEGs can also be obtained commercially, such as from Innophase Corporation (Westbrook, CT). Alkyl-blocked pendant polyalkylene glycols are generally prepared by alkoxylation of monoalkylalkylene glycols using alkylene oxide and pendant groups attached by methods available in the art. The monomethyl PEGs are also commercially available. Dialkyl blocked pendant polyalkylene glycols are generally prepared from monoalkyl PEGs by reaction with dialkyl sulfate and a strong base, or via the tosylate ester by reaction with alkoxide and subsequent attachment of pendant groups by methods available in the art (see, for example, Advanced Organic Chemistry, J. March, Wiley: New York, Fourth Edition (1992) pp. 386-387). Acyl and diacyl blocked pendant PEGs can be prepared, for example by reaction of activated carboxyl derivatives such as acyl or cyclic anhydrides with the pendant polyalkylene glycols or monoalkyl blocked pendant polyalkylene glycols (See Advanced Organic Chemistry, J. March, Wiley: New York, Fourth Edition (1992) pp. 392-396).

The use of alkyl blocked pendant polyalkylene glycol, such as an alkyl blocked pendant polyethylene glycol is advantageous. The multiple pendant groups on the polymer permit the attachment of plural chemical agents to the conjugate, to improve efficacy of the conjugate. For example, the polymer can include from two to nine molecules of the chemical agent. Preferably, the polymer includes from three to six molecules of the chemical agent, h some embodiments, the polymer can include more than nine molecules of the chemical agent.

Although acyl blocked pendant polyalkylene glycols have similar advantages to alkyl blocked pendant polyalkylene glycols, diacyl blocked pendant polyalkylene glycol offers the advantage of introducing additional reactive carboxyl groups that can be further derivatized. This is a particular advantage when the pendant groups contain carboxyl moieties, since the possibility of differential reactivity between the hemisuccinyl carboxyl groups and the pendant carboxyl groups is created. Examples of diacyl blocked pendant polyalkylene glycols include, but are not limited to, bis- hemisuccinyl pendant polyethylene glycol and monomethyl-hemisuccinyl pendant polyethylene glycol.

Functional Pendent Grafted Polymers

The graft polymers obtainable using the methods of the present invention are non-toxic and colorless, making them suitable in biotechnology and pharmaceutical applications. The polymers are non-immunogenic and optionally can contain multiple functionalities to which other substances can be chemically attached to form conjugates. Varying the amounts of reagents used for synthesis control the presence of multiple functionalities.

The number of multiple functionalities or pendant groups on a single polymer can vary. In one aspect, the polymer can include 2, 3, 4, 5, 6, 7, 8, or 9 or more molecules of the chemical agent, hi another aspect, the polymer can include at least 3, at least 4, at least 5 or at least 6 molecules of the chemical agent.

A relatively large number of chemical agents can be attached to the polymer by chemical attachment onto the functional groups present on the polymer. The chemical agent can be any substance that has a selected effect either in vivo or in vitro. Examples of biological effects include a cytotoxic effect or an effect on gene regulation. A "transforming nucleic acid" (RNA or DNA) can be replicated and/or expressed by a cell. Other nucleic acids can interact with regulatory sequences or regulatory factors by the cell to influence gene expression by the cell in a selected manner. Detectable labels identify cells that have interacted with particular substrates by detection of the label. Drugs or pharmacologically active compounds can be used to ameliorate pathogenic effects or other types of disorders. Particularly useful chemical agents include polypeptides. Some chemical agents are active fragments of biologically active proteins, or are specific antigenic fragments (e.g., epitopes) of antigenic proteins. Thus, chemical agents include cytotoxins, gene regulators, transforming nucleic acids, labels, detectable labels, antigens, drugs, and the like. For example, drugs can be attached to a functional group in the polymer to form a drug conjugate for the treatment of various diseases. (See e.g., U.S. Patent No. 6,251,866 to Prakash et al).

In another aspect, the polymer may comprise more than one chemical agent which may be the same or different. For example, in one aspect, the polymer may comprise a number of doxorubicin molecules and a number of cyclosporin molecules.

Monomers

The choice of monomers for copolymerization can include any ethylenically unsaturated monomers, characterized by the presence therein of at least one polymerizable ethylenic group, hi one embodiment, the monomers for copolymerization comprise acrylic acid, hi an alternative embodiment, the monomers for copolymerization comprise substituted acrylic acid monomers. Examples of substituted acrylic acid monomers include, but are not limited to, methylacrylate, methyl methacrylate, cyclohexyl methacrylate, benzyl methacrylate, isopropyl methacrylate, octyl methacrylate, methacrylonitrile, methyl alpha-chloracrylate, ethyl alphaethoxyacrylate, methyl alpha-acetaminoacrylate, butyl acrylate, 2-ethylhexyl acrylate, phenyl acrylate, phenyl methacrylate, alpha-chloroacrylonitrile, and the like.

U.S. Patent No. 3,968,089 to Cuscurida et al. describes other ethylenically unsaturated monomers. These compounds include, but are not limited to, hydrocarbon monomers such as butadiene, isoprene, 1,4 -pentadiene, 1,6-hexadiene, 1,7-octadiene, styrene, α-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, isopropylstyrene, butylstyrene, phenylstyrene, cyclohexylstyrene, benzylstyrene, and the like; substituted styrenes such as chlorostyrene, 2,5-dichlorostyrene, bromostyrene, fluorostyrene, trifluoromethylstyrene, iodostyrene, cyanostyrene, nitrostyrene, N,N- dimethylaminostyrene, p-vinyl diphenyl sulfide, p-vinylphenyl phenyl oxide, and the like; the vinyl esters and vinyl ethers, such as vinyl acetate, vinyl chloracetate, vinyl butyrate, isopropenyl acetate, vinyl formate, vinyl acrylate, vinyl methacrylate, vinyl methoxy acetate, vinyl benzoate, vinyl naphthalene, vinyl bromide, vinyl fluoride, vinylidene bromide, 1 -chloro- 1-fluoroethylene, vinylidene fluoride, vinyl methyl ether, vinyl ethyl ether, vinyl propyl ethers, vinyl butyl ethers, vinyl 2-ethylhexyl ether, vinyl phenyl ether, vinyl 2-methoxyethyl ether, methoxybutadiene, vinyl 2-butoxyethyl ether, 3,4-dihydro-l, 2-pyran, 2-butoxy-2'-vinyloxy diethyl ether, vinyl 2-ethylmercaptoethyl ether, vinyl methyl ketone, vinyl ethyl ketone, vinyl phenyl ketone, and the like; dimethyl fumarate, dimethyl maleate, monomethyl itaconate, dimethylaminoethyl methacrylate, glycidyl acrylate, dichlorobutadiene, vinyl pyridine, and the like.

Catalysts

The choice of graft copolymerization catalyst can include any known free radical initiator. In one embodiment, the copolymerization catalyst comprises any peroxide that forms free radicals to initiate the free radical polymerization process. Suitable peroxides include, but are not limited to, t-butyl peroxybenzoate, di-t-butyl peroxide, and t-butyl hydroperoxide. For example, the peroxide catalyst is t-butyl peroxybenzoate.

Other examples of peroxides that can be used to practice the methods of the present invention include, but are not limited, to aliphatic peroxides, organic peroxides, aromatic peroxides, hydrogen peroxide, hydroperoxides or hydrogen peroxide derivatives wherein one hydrogen is substituted for an alkyl group or another organic atom group, and organic peroxides. U.S. Patent Nos. 5,070,167 and 5,011,981 to Tsuboniwa et al. provide examples of hydroperoxides including, but not limited to, t- butyl hydroperoxide, cumene hydroperoxide, diisopropylbenzene hydroperoxide, p- mentane hydroperoxide, peracetic acid, 2,5-dimethyl-2,5-dihydroperoxyhexane, 2,5- dimethyl-2,5-dihydroperoxyhexane-3 and the like. (See U.S. Patent Nos. 4,588,798 and 4,605,717 to Heitner; and U.S. Patent No. 5,612,426 to Nakano et al. for examples of other peroxide initiator systems).

The following examples are intended to illustrate, not limit the invention described herein, hi the experimental disclosure which follows, the following abbreviations apply: mol (moles); mmol (millimoles); g (grams); mg (milligrams); mm (millimeter); 1 or L (liters); ml (milliliters); °C (degrees Centigrade); rpm (revolutions per minute); Mp (polymer molecular weight); b.p. (boiling point); o.d. (outer diameter); IPA (isopropyl alcohol); MeOH (methanol); H2O (water); KD (kilodalton); PA (propionic acid); COOH (carboxylic acid); NaOH (sodium hydroxide); LiBr (lithium bromide); AcOH (acetic acid); PEG (polyethylene glycol); DMF (dimethylformamide); HPLC (high pressure liquid chromatography); FTIR (Fourier Transform Infrared spectroscopy); GPC (gel permeation chromatography) and SEC (Size Exclusion Chromatography) . EXAMPLE I

Preparation of 20KD mpPEG-8PA

One aspect of the invention can be illustrated using Shearwater Polymers mPEG-20,000 as a starting material (Lot# 611559, Mp = 21,569). Nonane (Aldrich) was chosen as a dispersant (b.p. = 151 °C) to lower viscosity and tert-butyl peroxybenzoate (Aldrich) as the peroxide catalyst.

The reaction vessel consisted of a 500 ml indented cylindrical resin kettle equipped with a head that possessed three 24/40 joints in a line and a 10/30 joint offset to one side. Into each of the other 24/40 joints was placed an offset tubing adapter for V inch tubing through which a piece of !4 inch Teflon tubing was introduced. Latex eyedropper bulbs were placed over the exposed outside ends of the XΛ inch Teflon tubing. The rubber bulbs were pierced with the aid of a 16 gauge syringe needle through which 1/16 inch o.d. Teflon tubing was inserted to the point that it exited the ! inch tubing by approximately 2 mm. The opposite ends of the 1/16 inch tubing were each attached through 18-gauge stainless steel needles to two 10 ml syringes incorporated into two digital syringe infusion pumps. A 16-gauge needle was inserted into each of the rubber bulbs. A 250 °C glass thermometer was placed via a Teflon adapter into the final joint. One syringe was charged with 8 ml of acrylic acid, and the other syringe charged with 3 ml solution of 100 mg tert-butyl peroxybenzoate per ml in nonane. The resin kettle was immersed in an oil bath which was controlled by a solid state Barnant temperature controller equipped with a Type T probe capable of maintaining a temperature of 150 °C +/- 0.5 °C. The probe was immersed in the oil bath.

Nonane (75 ml) was added to the reaction vessel containing 150 g of mPEG- 20,000 (6.95 mmole). The system was purged with argon, and the oil bath temperature taken to

150 °C. When the entire solid had melted, stirring was initiated at a rate of 225 rpm. The internal temperature of the suspension varied between 137 °C to 138 °C at equilibrium. The syringe pump containing the 100 mg/ml of tert-butyl peroxybenzoate in nonane was programmed to deliver 2.0 ml of solution (200 mg, 1.03 mmole) over a period of 1.5 hours. The second syringe was programmed to deliver 6.7 ml acrylic acid over the same period.

Each of the pumps was operated briefly after filling the syringes, but before introducing the tubes into the apparatus with the reagents to remove slack from the drive mechanism. At least two drops of each of the reagents were collected, and the pumps turned off before introducing the tubes into the apparatus. The power to the pumps was turned back on to reset the mechanism so that the exact programmed amounts would be delivered when the pumps were activated. The pump containing the tert-butyl peroxybenzoate was activated first. As soon as the first drop of solution was released, the acrylic acid pump was activated. As the solutions were added, the reaction mixture became less viscous over the 1.5 hour period. After the addition was complete, stirring was continued for one hour while maintaining the temperature of the oil bath at 150 °C.

The oil bath was lowered, excess oil blotted from the resin kettle using paper towels, and the apparatus dismantled. The mixture was carefully poured into a 2 L round bottom flask through a glass powder funnel. The volatiles were then removed on a rotary evaporator using a boiling water bath, initially at aspirator pressure and finally with the aid of a vacuum pump at 1 mm pressure. When the product on the lip of the resin kettle had solidified, it was chipped off with a spatula and added to the 2 L round bottom flask. The same was done with the solidified material in the powder funnel. Water (100 ml) was added to the residue left in the resin kettle. The apparatus was reassembled, and the oil bath temperature adjusted to 100 °C. The apparatus was re- immersed in the oil bath, and the mixture stirred from time to time until the product had dissolved. The aqueous solution was transferred to the 2 L round bottom flask through the powder funnel, and the resin kettle rinsed several times with a total of 100 ml of water, which was also added to the round bottom. Approximately 100 ml of water were removed in vacuo, resulting in a thick solution, which was diluted with 900 ml of HPLC grade methanol, heated and agitated until a homogeneous solution was obtained. The hot solution was filtered under vacuum through sharkskin filter paper into a 2 L suction flask, and the funnel washed with 300 ml of 90/10 v/v MeOH/H2O. Finally, the filtrate was diluted to a volume of 1800 ml with 90/10 MeOH/H2O. The solution can be stored in the refrigerator overnight.

Initial attempts to purify the 20KD mpPEG-8PA using ultrafiltration technology involved the use of standard membranes in a tangential flow system and aqueous solutions of the crude mixture. When a crude product solution of high enough concentration in water was prepared that allowed reasonable working volumes (5 to 10% solids content), the viscosity of the solution was so high that the flux rate, i.e., rate of flow across the membrane, was so slow as to be prohibitive. In addition pressures on both the retentate and permeate sides were at maximum values, thus decreasing the useable life of the membrane. Removing the water in vacuo required higher temperatures, was slow and resulted initially in considerable foaming making, isolation of the product difficult.

The use of lower viscosity water-miscible organic solvents such as methanol, ethanol, acetone, etc. as substitutes for water were considered as solvents to potentially solve the problem of viscosity and increase the solids content of the solution. No literature could be found that indicated ultrafiltration membranes could be used with organic solvents. Inspection of solvent compatibility tables from several manufacturers of ultrafiltration membranes revealed, according to manufacturer's specifications, that none of the membranes were compatible with pure organic solvents, hi fact all of the solvent compatibility tables indicated that the membranes were compatible only with water-miscible organic solvents when mixed with large volumes of water. For example some of the membranes were compatible with ten percent ethanol or methanol in water or two to five percent acetone in water, h addition none of the manufacturers had any data on how the pore size of the membrane was affected by the use of organic solvent/water mixtures. Initial experiments using ten percent methanol in water resulted in the same problems associated with the use of pure water. Some of the membranes evaluated also contained materials that were soluble to some degree in the dilute aqueous solution resulting in an additional leaching problem.

The most inert membranes of those that were evaluated were Pall Filtron silicone encapsulated OMEGA polyethersulfone and silicone encapsulated REGEN regenerated cellulose membrane cassettes. Both systems appeared compatible with 25 % methanol in water and no leachable materials could be found in the solvent system after prolonged use.

The cassettes were then evaluated using 90/10 v/v methanol/water. Much to our surprise, after a relatively short break in period, no leachable substances were found in the solvent. In addition, the viscosity's of solutions of crude 20KD mpPEG-8PA containing five to ten percent solids were reasonable, the flux rates were high and system pressures were workable. The durability's of the membrane cassettes were established by re-equilibrating with water after prolonged use in our system, measuring the NWP's (normalized water permeabilities) of the membranes and demonstrating that the values had changed little. Repeated use over months revealed a slow but steady small increase in NWP, but less for the OMEGA membrane than the REGEN membrane. Both membranes were found to be compatible with 100 % methanol at lower pressures for short periods of time, which could be used for cleaning purposes. It was empirically determined that by using a membrane with a molecular weight cutoff (MWCO) of 3 kDa with our product, which demonstrates a weight average molecular weight (Mw) of approximately 20 kDa, that lower molecular weight material with an Mw range of approximately 10,000 to 14,000 was removed in the permeate.

The 1800 ml of solution at room temperature was diafiltered through a PALL FILTRON® ultrafiltration system (Pall Corporation) until 2.0 L of permeate had been collected. The PALL FILTRON® ultrafiltration system consisted of a Pall Filtron No. FS012K10 Centrainate PE (cassette holder) with a 2 pressure gauge fitting package and a Pall Filtron No. SPP990629 Centramate 3K cassette, REGEN (regenerated cellulose), T-screen channel format, 1 ft2 membrane area with silicone encapsulant. An FMI metering pump (Q2CKC pump head with a QD motor) (Fluid Metering, hie.) was used to deliver solvent.

With the retentate valve wide open, the flow rate was adjusted to give a retentate backpressure of 10 psi. The system was equilibrated for 15 minutes with the permeate valve closed. The permeate valve was opened and the retentate valve closed until a permeate backpressure of 20 psi was obtained. The purification was conducted under these conditions, with minor adjustments of the retentate valve from time to time, to maintain the backpressure at 20 psi until 2 L of permeate were collected.

The retentate solution was transferred to a 5 L round bottom flask, and the system flushed with 3 x 150 ml of methanol, which was combined with the retentate. The combined solution was concentrated in vacuo at aspirator pressure until no more solvent distilled, and finally concentrated at 1 mm with the aid of a vacuum pump. The thick syrup was heated with 850 ml of acetone plus 850 ml of isopropyl alcohol, with intermittent agitation until a homogeneous solution was obtained. Subsequently, the warm solution was placed in a refrigerator at 0 °C. After sitting overnight, the product was filtered, washed with 3 x 100 ml of 50/50 acetone/IPA at 0 °C, and finally with 3 x 200 ml of ether at room temperature. When solvent ceased passing through the filter, the damp solid was transferred back to the 5 L round bottom flask and the flocculation repeated as above. The product was air-dried and vacuum dried overnight to yield 103 to 106 g (69-71%) of 20KD mpPEG-8PA.

Titration of 150 to 170 mg samples weighed to the nearest 0.1 mg in duplicate with 0.010 N NaOH showed the presence of 8.4 +/- 0.2 moles COOH/mole polymer (18.0 +/- 0.6 mg COOH/g polymer) in the purified product. An FTIR spectrum was consistent with structure. The UN spectrum at a concentration of 1 mg/ml in 0. IN ΝaOH above 300 nm was flat, indicating the absence of color in the product. Water content by Karl Fischer analysis proved to be 0.300% by weight.

The GPC chromatogram of the product eluting with DMF/LiBr/AcOH was symmetrical indicating an Mp of 22,200 ± 584 and a polydispersity of 1.366 +/- 0.036 when calibrated against linear standards. The GPC chromatogram of the lower molecular weight molecules, degradation products, dispersant, etc. isolated from permeate from the ultrafiltration (lower molecular weight compounds) was unsymmetrical showing tailing on the low molecular weight side of the peak and demonstrating an Mp of 10,368 ± 700 and a polydispersity of 1.46. GPC utilized a Water's instrument equipped with a GMHHR-M mixed bed column, 7.8 x 300 mm, 5μm (Viscotek p/n 17392), exclusion limit PS 4M, using a mobile phase consisting of 0.1Ν LiBr in DMF containing 0.1% acetic acid at a flow rate of 0.50 ml/min at 50 °C using RI for detection. Samples were injected at a concentration of 4 mg/ml. A calibration curve was constructed using linear polyethylene oxide standards.

Analysis by gas chromatography indicated the presence of only trace amounts of volatile compounds such as IP A, acetone, nonane, and tert-butanol (<1000 ppm). Gas Chromatographic Conditions: Column, 30 m x 0.32 mm I.D., Restek RTx-624, 1.80 μm df, Oven Temperature Program- Initial Temperature 40 °C, Initial Time 6.00 minutes, Temperature Ramp 15 °C/minute, Final Temperature 240 °C, Final Time 5.00 minutes, Detector Temperature 300 °C, Injection Temperature 180 °C, Injection Volume 1 μL, Injection Type-Splitless, Flow Rates: Column Helium Flow- Approximately 1 - 3 mL/min, Hydrogen Flow (FID)-Instrument specifications, Air Flow (FID)-l strument specifications.

EXAMPLE II

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 13.5 equivalents acrylic acid in 75 ml of tert-butylbenzene.

Molecular weight of product was 23,490 and 37.3% yield was obtained. See Table 1.

EXAMPLE III

Using the procedure of Example I, mp PEG-8A having a molecular weight of 23,160 was added to 14.5 equivalents acrylic acid in the absence of solvent. Molecular weight of product was 31,880 and 65 % yield was obtained. See Table 1.

EXAMPLE IV

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 13.5 equivalents acrylic acid in the absence of solvent. Molecular weight of product was 20,370 and 76 % yield was obtained. See Table 1. EXAMPLE V

Using the procedure of Example I, mp PEG-8A of unknown molecular weight was added to 14.3 equivalents acrylic acid in the absence of solvent. Molecular weight of product was 26,660 and 76 % yield was obtained. See Table 1.

EXAMPLE VI

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 13.5 equivalents acrylic acid in 75 ml of tert-butylbenzene. 61 % yield was obtained. See Table 1.

EXAMPLE VII

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 13.5 equivalents acrylic acid in 40 ml of tert-butylbenzene. 60 % yield was obtained. See Table 1.

EXAMPLE VIII

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 13.5 equivalents acrylic acid in 75 ml nonane. 71 % yield was obtained. See Table 1.

EXAMPLE IX

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 14.0 equivalents acrylic acid in 75 ml of nonane. 70 % yield was obtained. See Table 1. EXAMPLE X

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 14.0 equivalents acrylic acid in 75 ml of nonane. 71 % yield was obtained. See Table 1.

EXAMPLE XI

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 14.0 equivalents acrylic acid in 75 ml of nonane. 72 % yield was obtained. See Table 1.

EXAMPLE XII

Using the procedure of Example I, mp PEG-8A having a molecular weight of 21,570 was added to 14.0 equivalents acrylic acid in 75 ml of nonane. 70 % yield was obtained. See Table 1.

Table 1 summarizes the reactants and products of Examples II through XII, above.

TABLE 1

150 gram scale cDiafiltration conducted using 90/10 MeOH/water; 6 liters permeate collected during diafiltration d250 gram scale eDiacetate prepared in situ by reaction with acetic anhydride after formation of pPEG-8PA Software problem prevented calculation of value gDiafϊltration conducted using 50/50 MeOH/water; 1 liter permeate collected hDiafϊltration conducted using 90/10 MeOH/water; 2 liters permeate collected 'Calculated using Mp JNot yet determined kProduct was very turbid 'Based upon molecular weight of starting material

"Product melt was very light yellow, but final product was white "Crude product in 90/10 MeOH H2O treated with activated charcoal to remove turbidity °Tert-butylperoxybenzoate reduced by 40% and added as solution in 2.0 ml nonane pRe-flocculation of this material gave 98.5% yield of material with 8.5 moles COOH/mole polymer.

The above example is presented to enable those skilled in the art to more clearly understand and practice the present invention. It is to be understood that specific embodiments used to describe the invention are illustrative of the methods of the present invention, and are not intended to limit the scope of the invention. Other aspects of the invention will be apparent to those skilled in the art to which the invention pertains. The disclosures of all publications and patents referred to herein, are incorporated herein by reference in their entirety.

Citations de brevets
Brevet cité Date de dépôt Date de publication Déposant Titre
US5626863 *27 janv. 19956 mai 1997Board Of Regents, The University Of Texas SystemPhotopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US5858746 *25 janv. 199512 janv. 1999Board Of Regents, The University Of Texas SystemGels for encapsulation of biological materials
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