WO2003078500A1 - Polymeres greffes a base de polyalkylene - Google Patents

Polymeres greffes a base de polyalkylene Download PDF

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Publication number
WO2003078500A1
WO2003078500A1 PCT/US2003/007697 US0307697W WO03078500A1 WO 2003078500 A1 WO2003078500 A1 WO 2003078500A1 US 0307697 W US0307697 W US 0307697W WO 03078500 A1 WO03078500 A1 WO 03078500A1
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polymers
polyalkylene
polymer
molecular weight
pendant
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PCT/US2003/007697
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English (en)
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Christopher G. Anderson
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Watson Pharmaceuticals, Inc.
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Priority to AU2003220228A priority Critical patent/AU2003220228A1/en
Publication of WO2003078500A1 publication Critical patent/WO2003078500A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F283/00—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G
    • C08F283/06—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polyethers, polyoxymethylenes or polyacetals
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/10—Definition of the polymer structure
    • C08G2261/12—Copolymers
    • C08G2261/128—Copolymers graft
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2205/00—Polymer mixtures characterised by other features
    • C08L2205/05—Polymer mixtures characterised by other features containing polymer components which can react with one another

Definitions

  • the present invention relates generally to the field of grafting polymers and copolymers, particularly polymers and copolymers of poly(oxyalkylene) compounds.
  • 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.
  • 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.
  • this process is a two-stage process and more complex than desired for a number of applications.
  • graft polymers and copolymers prepared in the presence of oxygen often contain colored impurities resulting in light yellow products.
  • improved methods for preparing graft polymers and copolymers and especially copolymers for pharmaceutical use that are simple and yield highly purified polymers and copolymers.
  • 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.
  • 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.
  • 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.
  • ultrafiltration can be used to isolate the substantially pure product.
  • the collected polymers are further purified using a variety of techniques such as crystallization and/or flocculation.
  • 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.
  • 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.
  • locculation 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.
  • polymers refers to high molecular weight compounds comprising linked monomer units, h certain aspects, the term may encompass homopolymers and copolymers.
  • 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.
  • 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.
  • a mono-methyl blocked polyalkylene oxide refers to the form when one of the terminal hydroxyl groups is capped with a methyl group.
  • mpPEG refers to a polyethylene glycol monomethyl ether bearing pendant groups.
  • pPEG refers to a polyethylene glycol bearing pendant groups.
  • dmpPEG refers to a polyethylene glycol dimethyl ether bearing pendant groups.
  • mpPEG-8PA refers to a mono-methyl blocked pendant polyethylene glycol having approximately eight pendant propionic acid moieties per mole of the polymer.
  • pendant group refers to groups attached onto a polymer backbone. In one aspect, the pendant group is a pendant acid group.
  • pendant polyalkylene oxide 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.
  • polyalkylene oxide derivative 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.
  • 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.
  • solid refers to a state of matter that is rigid and having a defined shape and volume, unlike liquids or gases.
  • Acrylic monomers include, but are not limited to acrylic acid and substituted acrylic acid monomers.
  • substituted acrylic acid monomers refers to acrylic acid that has been modified with a modifying group.
  • 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).
  • GPC Gel Permeation Chromatography
  • GC Gas Chromatography
  • SEC Size exclusion chromatography
  • peroxide refers to a compound having an oxygen- oxygen bond, and that produces free radicals to catalyze polymerization.
  • 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.
  • reaction mixture refers to the crude mixture obtainable by reacting an acrylic acid monomer with fluid polyalkylene oxide, prior to any purification.
  • 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.).
  • 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.
  • 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.
  • 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.).
  • 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.
  • the starting polymer can be an alkyl blocked polyethylene glycol.
  • the alkyl blocked polyethylene glycol starting material is selected from the group consisting of mono-methyl blocked polyethylene glycols and dimethyl blocked polyethylene glycols.
  • 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.
  • 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.
  • 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.
  • 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.
  • two additional reactive groups e.g., carboxyl groups
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Ultrafiltration employs membrane separation techniques, which are based on molecular size.
  • 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.
  • the flocculation procedure is in the presence of an acetone/isopropyl alcohol system.
  • a pharmaceutically acceptable graft copolymer obtainable by a process of this invention.
  • the graft copolymer is a grafted polyalkylene oxide copolymer.
  • the grafted copolymer contains pendant groups.
  • 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.
  • the organic solvent comprises an aqueous/organic solvent system.
  • organic solvent systems include, but are not limited to at least 20% alcohol/water such as methanol/water and ethanol/water.
  • the % alcohol is increase to at least 25%o of the total volume.
  • the water soluble polymer is a polyalkylene oxide.
  • 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, C ⁇ -C 4 alkyl groups.
  • the polyalkylene oxide can be a monomethyl-substituted pendant PEG homopolymer.
  • 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.
  • PEGs are branched, pendant and star PEGs, such as those commercially available from Shearwater Polymers, Inc. (Huntsville, AL). (Gnanou et al., Makromol. Chem.
  • 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.
  • the PEG-based polymers have average molecular weights of from about 200 to about 50,000.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the alkyl blocked pendant polyethylene glycol can be a mono-methyl blocked pendant polyethylene glycol, or a dimethyl blocked pendant polyethylene glycol.
  • 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.
  • 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.
  • 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).
  • an acyl group such as acetyl or hemisuccinyl (e.g., via an ester bond from reaction with a mono or dicarboxyhc acid or derivative).
  • additional reactive groups such as acetyl or hemisuccinyl (e.g., via an ester bond from reaction with a mono or dicarboxyhc acid or derivative).
  • 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.
  • two additional reactive groups e.g., carboxyl groups
  • 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.
  • 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).
  • alkyl blocked pendant polyalkylene glycol such as an alkyl blocked pendant polyethylene glycol
  • the multiple pendant groups on the polymer permit the attachment of plural chemical agents to the conjugate, to improve efficacy of the conjugate.
  • the polymer can include from two to nine molecules of the chemical agent.
  • 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.
  • 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.
  • diacyl blocked pendant polyalkylene glycols include, but are not limited to, bis- hemisuccinyl pendant polyethylene glycol and monomethyl-hemisuccinyl pendant polyethylene glycol.
  • 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.
  • 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.
  • chemical agents are active fragments of biologically active proteins, or are specific antigenic fragments (e.g., epitopes) of antigenic proteins.
  • chemical agents include cytotoxins, gene regulators, transforming nucleic acids, labels, detectable labels, antigens, drugs, and the like.
  • 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).
  • the polymer may comprise more than one chemical agent which may be the same or different.
  • the polymer may comprise a number of doxorubicin molecules and a number of cyclosporin molecules.
  • 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.
  • 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, ⁇ -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, cyanost
  • the choice of graft copolymerization catalyst can include any known free radical initiator.
  • 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.
  • the peroxide catalyst is t-butyl peroxybenzoate.
  • 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.
  • 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.
  • 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.
  • 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 X ⁇ 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.
  • 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
  • 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.
  • 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/H 2 O. Finally, the filtrate was diluted to a volume of 1800 ml with 90/10 MeOH/H 2 O.
  • the solution can be stored in the refrigerator overnight.
  • 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 ft 2 membrane area with silicone encapsulant.
  • An FMI metering pump Q2CKC pump head with a QD motor
  • Fluid Metering was used to deliver solvent.
  • 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.
  • the GPC chromatogram of the product eluting with DMF/LiBr/AcOH was symmetrical indicating an M p 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 M p 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, 5 ⁇ m (Viscotek p/n 17392), exclusion limit PS 4M, using a mobile phase consisting of 0.1 ⁇ 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.
  • Table 1 summarizes the reactants and products of Examples II through XII, above.

Abstract

La présente invention concerne des polymères greffés à base de polyalkylène, sensiblement purs, de qualité pharmaceutique, ainsi que leurs procédés de fabrication. Ces procédés consistent à mélanger un oxyde de polyalkylène liquide avec dix à quinze parties équivalentes environ d'un monomère acrylique et un catalyseur adapté, en l'absence d'oxygène, pour former un mélange réactionnel. Le procédé consiste ensuite à récupérer les polymères sensiblement pures à partir du mélange réactionnel. Les polymères obtenus peuvent être davantage purifiés. L'invention concerne également un procédé servant à isoler ou à récupérer des polymères de masse moléculaire élevée à partir d'un système eau-solvant organique.
PCT/US2003/007697 2002-03-12 2003-03-11 Polymeres greffes a base de polyalkylene WO2003078500A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5626863A (en) * 1992-02-28 1997-05-06 Board Of Regents, The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US5858746A (en) * 1992-04-20 1999-01-12 Board Of Regents, The University Of Texas System Gels for encapsulation of biological materials

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5626863A (en) * 1992-02-28 1997-05-06 Board Of Regents, The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
US5858746A (en) * 1992-04-20 1999-01-12 Board Of Regents, The University Of Texas System Gels for encapsulation of biological materials

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