FORM OF SALT OF A DRUG-POLYMER CONJUGATE WITH MULTIPLE ARMS

专利摘要:
SALT FORM OF A MULTI-ARM DRUG-POLYMER CONJUGATE Among other aspects, provided herein is a hydrohalide salt of a water-soluble multi-arm polyethylene glycol-drug conjugate, in conjunction with related methods of making and using the same. The hydrohalide salt is stably formed, and appears to be more resistant to hydrolytic degradation than the corresponding free base form of the conjugate.
公开号:BR112012011947B1
申请号:R112012011947-8
申请日:2010-11-18
公开日:2021-05-18
发明作者:Anthony O. Chong；Seoju Lee；Bhalchandra V. Joshi；Brian Bray；Shaoyong Nie；Patrick Spence；Antoni Kozlowski；Samuel McManus；Sachin Tipnis；Greg Lavaty；David Swallow
申请人:Nektar Therapeutics；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] This application claims priority benefit under 35 USC §119(e), for each of US Provisional Patent Application Serial No. 61/262,463, filed November 18, 2009, and Application for US Provisional Patent Serial No. 61/290,072, filed December 24, 2009, both of which are incorporated herein by reference in their entirety. DOMAIN
[0002] This disclosure relates, in general, to salt forms of water-soluble polymer-drug conjugates, their pharmaceutical compositions, and methods of preparation, formulation, administration and use of these compositions of mixed acid salts. This disclosure also relates, in general, to alkoxylation methods for preparing alkoxylated polymeric materials from a previously isolated alkoxylated oligomer, as well as compositions comprising the alkoxylated polymeric material, methods of using the alkoxylated polymeric material, and the like. BACKGROUND
[0003] Over the years, numerous methods have been proposed to improve the stability and distribution of biologically active agents. Challenges associated with the formulation and delivery of pharmaceutical agents can include poor aqueous solubility of the pharmaceutical agent, toxicity, low bioavailability, instability, and rapid degradation in vivo, to name but a few. Although many approaches have been devised to improve the delivery of pharmaceutical agents, no single approach is without its drawbacks. For example, commonly employed drug delivery approaches aimed at solving or at least ameliorating one or more of these problems include drug encapsulation, such as in a liposome, polymer matrix, or unimolecular mycelium, covalently bonding to a soluble polymer in water, such as polyethylene glycol, use of selective gene approach agents, salt formation, and the like.
[0004] The covalent bonding of a water-soluble polymer can improve the water solubility of an active agent, as well as change its pharmacological properties. Certain exemplary polymer conjugates are described in U.S. Patent No. 7,744,861, among others. In another approach, an active agent with acidic or basic functionalities can react with a suitable base or acid and be marketed in salt form. More than half of all active molecules are marketed as salts (Polymorphism in the Pharmaceutical.1 Industry, Hilfiker, R., editor, Wiley-VCH, 2006). Challenges associated with salt forms include finding an optimal salt as well as controlling solid state behavior during processing. Biopharmaceutical salts can be amorphous, crystalline, and exist in the form of hydrates, solvents, various polymorphs, etc. It is interesting to note that rarely, if ever, are salt forms, let alone mixed acid salt forms, of polymer conjugates used in drug formulations.
[0005] Another challenge associated with the preparation of conjugates of active agents from water-soluble polymers is the ability to prepare relatively pure water-soluble polymers in a consistent and reproducible manner. For example, poly(ethylene glycol) (PEG) derivatives activated with reactive functional groups are useful for coupling to active agents (such as small molecules and proteins), thereby forming a conjugate between the PEG and the active agent. When an active agent is conjugated to a polymer of poly(ethylene glycol) or nPEG", the conjugated active agent is conventionally referred to as being "PEGylated".
[0006] Comparing with the safety and efficacy of the active agent in unconjugated form, the conjugated version exhibits different properties, and often clinically beneficial. The commercial success of PEGylated active agents such as PEGylated interferon alpha-2a PEGASYS'' (Hoffmann-La Roche, Nutley, NJ), PEGylated interferon alpha-2b PEG-INTRON® (Schering Corp., Kennilworth, NJ), and PEG- filgrastim NEULASTA' (Amgen Inc., Thousand Oaks, CA) demonstrates the extent to which PEGylation has the potential to improve one or more properties of an active agent.
[0007] When preparing a conjugate a polymeric reagent is typically employed to allow a relatively straightforward synthetic approach to conjugate synthesis. By combining a composition comprising a polymeric reagent with a composition comprising the active agent, it is possible - under the appropriate reaction conditions - to effect a relatively convenient conjugate synthesis.
[0008] However, the preparation of polymeric reagent adequate to regulatory requirements for drugs is often challenging. Conventional polymerization approaches yield relatively impure and/or low yield compositions. While these impurities and yields may not be a problem outside the pharmaceutical domain, safety and cost are important concerns in the context of medicines for human use. Thus, conventional polymerization approaches are not suitable for the synthesis of polymeric reagents for the manufacture of pharmaceutical conjugates.
[0009] Alternative methods of preparing polymeric reagents, in particular high molecular weight polymers, with relatively high yield and purity are needed in the field. In the case of multiarm polymers, there is a paucity of available and desirable water-soluble polymers that have well-controlled and well-defined properties, without significant amounts of unwanted impurities. Thus it is possible to easily obtain, for example, a poly(ethylene glycol) with multiple arms of high molecular weight, but drug conjugates made from commercial polymers can have significant amounts (i.e., > 8%) of polymer-conjugate. drug having biologically active impurities with very low or very high molecular weight. This extension of active impurities in a drug composition can be unacceptable and thus can make approval of these drugs challenging, if not impossible. ABSTRACT
[0010] In one or more embodiments of the invention, the present disclosure provides a hydrohalide salt form of
wherein n is an integer ranging from about 20 to about 600, or from about 20 to 500 (for example 40 to about 500) and, in terms of a composition comprising the above conjugate, greater than 95 per mole percent (and in some cases more than 96 mole percent, more than 97 mole percent, and even more than 98 mole percent) of basic nitrogens from the irinotecan portions of all conjugates contained in the composition are protonated in the form of hydrohalide salt (HX), wherein X is selected from fluoride, chloride, bromide, and iodide.
[0011] In one or more embodiments of the invention, the hydrohalide salt is a hydrochloride salt.
[0012] In one or more embodiments of the invention, n of a repeating monomer is an integer ranging from about 80 to about 150.
[0013] In one or more embodiments of the invention, n for any one occurrence of (OCH2CH2)n is about 113.
[0014] In one or more embodiments of the invention, the hydrohalide salt is a hydrochloride salt and the weight average molecular weight of the conjugate is about 23,000 Daltons.
[0015] In one or more embodiments of the invention, a method is provided for preparing a hydrohalide salt of a water-soluble polymer-active agent conjugate [such as the water-soluble polymer-active agent conjugate having the structure (I )], wherein the method comprises the following steps: (i) treating a glycine-irinotecan hydrohalide in protected form (II),
with a molar excess of hydrohalic acid, to thereby remove the protecting group to form glycine-irinotecan hydrohalide,
(ii) coupling the unprotected glycine hydrohalide-irinotecan from step (i) to the 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-succinimide,
in the presence of a base, to form 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide salt (also called pentaerythritolyl-(PEG-1-methylene-2-oxo-(vinylamino acetate linked to irinotecan) with 4 arms)),
(iii) recovering the 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide salt by precipitation. In connection with this method, the polymer reagent used to implement the method is not particularly limited, and the other polymer reagents containing an activated ester can replace the 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-succinimide.
[0016] In one or more embodiments of the invention, a recovered 4-armed pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide is contained in a composition in which more than 95 mole percent (and, in some cases, more than 96 mole percent, more than 97 mole percent, and even more than 98 mole percent) of basic nitrogens from the irinotecan moieties of all conjugates contained in the composition are protonated in hydrohalide salt (HX) form, where X is selected from fluoride, chloride, bromide, and iodide.
[0017] In one or more embodiments of the invention, the glycine-irinotecan hydrohalide in protected form is treated with a ten-fold or more molar excess of hydrohalic acid to thereby remove the protecting group, to form glycine-irinotecan hydrohalide .
[0018] In one or more embodiments of the invention, the glycine-irinotecan hydrohalide in protected form is treated with a molar excess of hydrohalic acid in a range of ten times to 25 times to thereby remove the protecting group, to form glycine-irinotecan hydrohalide.
[0019] In one or more embodiments of the invention, the hydrohalide of glycine-irinotecan in protected form is tert-butyloxycarbonyl(Boc)-glycine-irinotecan hydrochloride, wherein the amino group of glycine is protected with Boc.
[0020] In one or more embodiments of the invention, the glycine-irinotecan hydrohalide in step (i) is glycine-irinotecan hydrochloride in protected form, and the glycine-irinotecan hydrochloride in protected form is treated with hydrochloric acid, to remove the protective group.
[0021] In one or more embodiments of the invention, the glycine-irinotecan hydrochloride in protected form is treated with a solution of hydrochloric acid in dioxane.
[0022] In one or more embodiments of the invention, a method of preparing a hydrohalide salt of a water-soluble polymer-active agent conjugate also comprises isolating the glycine-irinotecan hydrohalide (e.g., by precipitation, by adding methyl tert-butyl ether, "MTBE") prior to the coupling step to a polymer reagent.
[0023] In one or more embodiments of the invention, the base used in the coupling step is an amine (for example, trimethylamine, triethylamine, and dimethylamino-pyridine).
[0024] In one or more embodiments of the invention, the coupling step is carried out in a chlorinated solvent.
[0025] In one or more embodiments of the invention, the step of recovering the hydrohalide salt of 4-armed pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan comprises adding tert-butyl methyl ether.
[0026] In one or more embodiments of the invention, the method of preparing a hydrohalide salt of a water-soluble polymer-active agent conjugate also comprises the step of (iv) analyzing (e.g., by means of ion chromatography ) the hydrohalide salt of pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan with 4 arms recovered in halide content and, in case the halide content is less than 95 mole percent, (v) dissolve the 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide salt recovered in ethyl acetate, and adding more hydrohalic acid to thereby form the 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide salt having one content halide greater than 95 mole percent.
[0027] In one or more embodiments of the invention, the added hydrohalic acid is in the form of a solution in ethanol.
[0028] In one or more embodiments of the invention, in a method in which the step of adding more hydrohalic acid is performed, the method also comprises recovering the 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide per means of precipitation (which can be done, for example, by means of cooling).
[0029] In one or more embodiments of the invention there is provided a hydrohalide salt of 4-armed pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan by carrying out the methods described herein.
[0030] In one or more embodiments of the invention a pharmaceutically acceptable composition is provided, wherein the composition comprises a hydrohalide salt (e.g. hydrochloride salt) of the compound corresponding to structure (I), pentaerythritol-polyethylene glycol-carboxymethyl- glycine-irinotecan with 4 arms, and a pharmaceutically acceptable excipient.
[0031] In one or more embodiments of the invention a composition is provided, wherein the composition comprises (i) a hydrochloride salt according to any one or more of the embodiments described herein, and (ii) lactate buffer, in lyophilized form, In one or more embodiments of the invention, the pharmaceutically acceptable composition is a sterile composition. In one or more embodiments of the invention, the pharmaceutically acceptable composition is optionally provided in a container (eg vial), optionally containing the equivalent of a 100 mg dose of irinotecan.
[0032] In one or more embodiments of the invention there is provided a method, wherein the method comprises administering a composition containing a conjugate described herein to a subject suffering from one or more types of solid cancerous tumors, wherein the composition containing a conjugate is optionally dissolved in a 5% w/w dextrose solution. In one or more embodiments of the invention, administration is via intravenous infusion.
[0033] In one or more embodiments of the invention there is provided a method of treating a mammal suffering from cancer, wherein the method comprises administering a therapeutically effective amount of a hydrohalide salt (such as a hydrochloride salt) of pentaerythritolyl-polyethylene 4-armed glycol-carboxymethyl-glycine-irinotecan. The hydrohalide salt is administered to the mammal in an amount effective to produce a slowing or inhibition of solid tumor growth in the subject. In one or more embodiments of the invention, the solid cancer tumor is selected from the group consisting of colorectal, ovarian, cervical, breast and non-small cell lung tumor.
[0034] In one or more embodiments of the invention there is provided a hydrohalide salt of pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan with 4 arms, wherein the salt is an anticancer agent for the manufacture of a drug intended for the treatment of cancer.
[0035] In one or more embodiments of the invention a composition is provided, wherein the composition comprises an alkoxylated polymeric product prepared by a method comprising the step of alkoxylation, in a suitable solvent, of a fit-to-be oligomer alkoxylated previously isolated to form an alkoxylated polymeric product, wherein the oligomer capable of being alkoxylated previously isolated has a known and defined weight average molecular weight greater than 300 Daltons (e.g., greater than 500 Daltons).
[0036] In one or more embodiments of the invention a composition is provided, wherein the composition comprises an alkoxylated polymeric product with a purity greater than 92% by weight, and the total combined content of high molecular weight products and diols is less than 8% by weight (e.g. less than 2% by weight), determined, for example, by means of gel filtration chromatography (GFC) analysis.
[0037] In one or more embodiments of the invention, the alkoxylated polymeric product has the following structure:
where each n is an integer from 2 0 to 10 00 (for example, from 50 to 1000).
[0038] In one or more embodiments of the invention a method is provided, wherein the method comprises the steps of (i) alkoxylating, in a suitable Solvent, an oligomer capable of being alkoxylated previously isolated to form an alkoxylated polymeric material, wherein the previously isolated alkoxylated oligomer has a known and defined weight average molecular weight of greater than 300 Dalton (e.g. greater than 500 Dalton), and (ii) optionally also activating the alkoxylated polymeric product to to provide an activated alkoxylated polymeric product that is useful (among other things) as a polymeric reagent to prepare polymer-drug conjugates.
[0039] In one or more embodiments of the invention a method is provided, wherein the method comprises the step of activating an alkoxylated polymeric product obtained from and/or contained in a composition comprising an alkoxylated polymeric product having a higher purity than 90%, to thereby form an activated alkoxylated polymeric product which is useful (among other things) as a polymeric reagent for preparing polymer-drug conjugates.
[0040] In one or more embodiments of the invention a method is provided, wherein the method comprises the step of conjugating an activated alkoxylated polymeric product to an active agent, wherein the activated alkoxylated polymeric product has been prepared by means of a method which comprises the step of activating an alkoxylated polymeric product obtained from and/or contained in a composition comprising an alkoxylated polymeric product having a purity greater than 90%, to thereby form an activated alkoxylated polymeric product.
[0041] In one or more embodiments of the invention a mixed salt of a water-soluble polymer-active agent conjugate is provided, wherein the conjugate was prepared by coupling (under conjugation conditions) of an active agent containing amine (eg an unprotected glycine-irinotecan) to a polymer reagent (eg a 4-armed pentaerythritolyl-poly(ethylene glycol)-carboxymethyl succinimide) in the presence of a base to form a conjugate, wherein the conjugate is in the form of a mixed salt conjugate (for example, the conjugate has nitrogen atoms, each of which will be protonated or non-protonated, where any given protonated amino group is an acid salt having one of two different anions), and also wherein, optionally, the polymer reactant is prepared from an alkoxylation product prepared as described herein.
[0042] Additional embodiments of the present method, compositions, and the like will be clear from the following description, figures, examples, and claims. As will be appreciated from the foregoing and following descriptions, each and every feature described herein, and each and every combination of two or more of these features, are included within the scope of this disclosure, provided that the features included in such combination are not mutually inconsistent. Additionally, any feature or combination of features may be specifically excluded from any embodiment of the present invention. Additional aspects and advantages of the present invention are set out in the following description and claims, in particular when considered in conjunction with the accompanying examples and figures. BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 is a graph illustrating the results of stability studies under accelerated stress on three different samples of "4-armed PEG-Gly-Irino-20K" (corresponding to 4-armed pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan), each having a different composition with regard to relative amounts of trifluoroacetic acid (TFA) and hydrochloride salts as well as the free base. Samples tested included >99% HCl salt (<1% free base, triangles), 94% total salt (6% free base, squares), and 52% total salt (48% free base, circles) . Samples were stored at 25°C and 60% relative humidity; the graph illustrates the degradation of the compound over time, as described in detail in Example 3.
[0044] FIG. 2 is a graph illustrating the increase in free irinotecan over time in 4-arm PEG-Gly-Irino-20K samples stored at 40 °C and 75% relative humidity, each having a different composition in regard to to relative amounts of trifluoroacetic acid and hydrochloride salts as well as the free base. The samples tested correspond to product containing >99% HCl salt (<1% free base, squares) and product containing 86% total salts (14% free base, diamonds), as described in Example 3.
[0045] FIG. 3 is a graph illustrating the increase over time in small PEG species (PEG degradation products) in 4-arm PEG-Gly-Irino-20K samples stored at 40°C and 75% relative humidity, such as described in detail in Example 3. The samples tested correspond to product containing >99% HCl salt (<1% free base, squares) and product containing 86% total salts (14% free base, diamonds).
[0046] FIG. 4 is a layered compilation of chromatograms showing release of irinotecan via hydrolysis from 4-arm PEG-Gly-Irino-20K with mono- (DS-1), di- (DS-2), tri- (DS) substitution -3) and tetra-(DS-4) irinotecan, as described in detail in Example 5.
[0047] FIG. 5 is a graph illustrating the hydrolysis results of various species of PEG-Gly-Irino-20K with. 4 arms, as described above, in aqueous buffer at pH 8.4 in the presence of porcine carboxypeptidase B compared to hydrolysis kinetics modeling data as described in Example 5. For the kinetics model, it was assumed that hydrolysis of all species was a kinetics of the 1st order. The 1st order reaction rate constant for DS4 disappearance (0.36 hours'1) was used to generate all curves.
[0048] FIG. 6 is a graph illustrating the hydrolysis of various species of 4-armed PEG-Gly-Irino-20K as described above in human plasma compared to hydrolysis kinetic modeling data. Details are provided in Example 5. For the kinetics model, it was assumed that the hydrolysis of all species was 1st order kinetics. The 1st order reaction rate constant for DS 4 disappearance (0.26 hours'1) was used to generate all curves.
[0049] FIG. 7 is a chromatogram after gel filtration chromatography of a material prepared as described in Example 7.
[0050] FIG. 8 is a chromatogram after gel filtration chromatography of a material prepared as described in Example 8. DETAILED DESCRIPTION
[0051] Various aspects of the invention will now be described more fully hereinafter. However, these aspects can be implemented in many different ways and should not be considered as limiting the embodiments presented here; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to practitioners.
[0052] All publications, patents and patent applications cited herein, either above or below, are hereby incorporated by reference in their entirety. In the event of an inconsistency between the teachings in this specification and the area incorporated by reference, the meaning of the teachings in this specification will prevail.
[0053] It should be noted that, as used in this specification, the singular forms "a", "an" and "the", "a" include plural referents, unless the context clearly dictates otherwise. Thus, for example, reference to a "polymer" includes a single polymer as well as two or more identical or different polymers, reference to a "conjugate" refers to a single conjugate as well as two or more identical or different conjugates, reference an "excipient" includes a single excipient as well as two or more identical or different excipients, and the like.
In describing and claiming the present invention, the following terminology will be used in accordance with the definitions described below.
[0055] A "functional group" is a group that can be used, under normal conditions of organic synthesis, to form a covalent bond between the entity to which it is attached and another entity, which typically contains another functional group. The functional group generally includes multiple bond(s) and/or heteroatom(s). Preferred functional groups are described here.
[0056] The term "reactive" refers to a functional group that reacts easily, or at a practical rate, under conventional conditions of organic synthesis. This contrasts with those groups that do not react, or else that require strong catalysts or impractical reaction conditions to react (ie, an "unreactive" or "inert" group).
[0057] A "protective group" is a moiety that prevents or blocks the reaction of a particular chemically reactive functional group on a molecule under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group to be protected, as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule. Functional groups which may be protected include, by way of example, carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups and the like. Representative protecting groups for carboxylic acids include esters (such as p-methoxybenzyl ester), amides and hydrazides; for amino groups, carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters; for thiol, thioethers and thioester groups; for carbonyl groups, acetals and ketals, and the like. These protective groups are well known to practitioners and are described, for example, in T.W. Greene and G.M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and in P.J. Kocienski, Protecting Groups, Third Edition, Thieme Chemistry, 2003, and references cited therein.
[0058] A functional group in "protected form" refers to a functional group containing a protecting group. As used herein, the term "functional group", or any synonym thereof, is intended to encompass its protected forms.
"PEG" or "poly(ethylene glycol)" as used herein is intended to encompass any water-soluble poly(ethylene oxide). Typically, PEGs for use in the present invention will comprise one of the following two structures: " - (CH2CH2O)n-" or (CH2CH2O)I1.1CH2CH2-, " depending on whether the terminal oxygen(s) has(in) ) or not been displaced, for example, during a synthetic transformation Variable (n) ranges from 3 to about 3000, and the end groups and architecture of the overall PEG may vary.
[0060] A water-soluble polymer may contain one or more "end-capping group" (in which case it can be stated that the water-soluble polymer has "end-capping groups". capped")). With respect to end-protecting groups, exemplary end-protecting groups are, in general, groups containing carbon and hydrogen, typically comprised of 1-20 carbon atoms and an oxygen atom that is covalently bonded to the group. In this regard, the group is typically alkoxy (e.g., methoxy, ethoxy and benzyloxy) and, relative to the carbon-containing group, it may optionally be saturated or unsaturated, as well as aryl, heteroaryl, cyclo, heterocycle, and substituted forms of any of the above.
[0061] The terminal protection group may also comprise a detectable label. When the polymer has a terminal protecting group comprising a detectable label, the amount or location of the polymer and/or the fraction (eg, active agent) to which the polymer is attached can be determined using a suitable detector. Such labels include, without limitation, fluorescent agents, chemiluminescents, fractions used in enzymatic labeling, colorimetric agents (eg, dyes), metal ions, radioactive fractions, and the like.
[0062] "Water-soluble" in the context of a polymer of the invention, or a "water-soluble polymer segment" is any segment or polymer that is at least 35% (by weight), preferably more than 70 % (by weight) and more preferably more than 95% (by weight) soluble in water at room temperature. Typically, a water-soluble polymer or segment will transmit at least about 75%, more preferably at least about 95% of light, transmitted by the same solution after filtration.
[0063] The term "activated", when used in conjunction with a particular functional group, refers to a reactive functional group that readily reacts with an electrophile or nucleophile in another molecule. This contrasts with those groups that require strong bases or highly impractical reaction conditions to react (ie, an "unreactive" or "inert" group).
[0064] "Electrophil" refers to an ion or atom or a collection of neutral or ionic atoms having an electrophilic center, that is, a center that seeks electrons or is capable of reacting with a nucleophile.
[0065] "Nucleophile" refers to an ion or atom or a collection of neutral or ionic atoms having a nucleophilic center, that is, a center that seeks an electrophilic center or is capable of reacting with an electrophile.
[0066] The terms "protected" or "protecting group" or "protecting group" refer to the presence of a moiety (ie, the protecting group) that prevents or blocks the reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group to be protected, as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule, if any. Protective groups known in the area can be found in Greene, T.W., et al. , PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd edition, John Wiley & Sons, New York, NY (1999) ,
[0067] "Molecular mass", in the context of a water-soluble polymer such as PEG, refers to the weight average molecular weight of a polymer, typically determined by means of size exclusion chromatography, light scattering techniques, or determining intrinsic viscosity in an organic solvent such as 1,2,4-trichlorobenzene.
[0068] The terms "spacer" and "spacer fraction" are used here to refer to an atom or a collection of atoms optionally used to link interconnecting fractions, such as a terminus of a series of monomers and an electrophile. The spacer moieties of the invention may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable bond.
[0069] A "hydrolyzable" bond is a relatively unstable bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of bond connecting two core atoms, but also on the substituents attached to these core atoms. Illustrative hydrolytically unstable linkages include carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.
An "enzymatically degradable bond" means a bond that is subject to degradation by one or more enzymes.
[0071] A "hydrolytically stable" bond refers to a chemical bond that is substantially stable in water, that is to say, that does not undergo hydrolysis, under physiological conditions, to any appreciable extent for an extended period of time. Examples of hydrolytically stable bonds include but are not limited to the following: carbon-carbon bonds (for example, in aliphatic chains), ethers, amides, urethanes, and the like. In general, a hydrolytically stable bond is one that exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. Representative chemical bond hydrolysis rates can be found in most reference chemistry manuals.
[0072] "Multi-armed", with reference to the overall geometry or structure of a polymer, refers to a polymer that contains 3 or more "arms" containing polymer connected to a "core" molecule or structure. Thus, a multi-arm polymer can have 3 polymeric arms, 4 polymeric arms, 5 polymeric arms, 6 polymeric arms, 7 polymeric arms, 8 polymeric arms or more, depending on its configuration and core structure. A particular type of multiarm polymer is a highly branched polymer, called a dendritic polymer or hyperbranched polymer, having an initiator core with at least 3 branches, an inner branch multiplicity of 2 or more, a generation of 2 or more , and at least 25 surface groups on a single dendrimeric molecule. For the purposes presented herein, a dendrimer is considered to have a structure distinct from the structure of a multi-arm polymer. That is to say that a polymer with multiple arms, as explicitly stated here, excludes dendrimers. Additionally, a multiarm polymer, as provided herein, has an uncrosslinked core.
[0073] A "dendrimer" or "hyper-branched polymer" is a monodispersion-sized globular polymer in which all bonds emerge radially from a central focal point or core, with a regular branching pattern and with repeating units, wherein each contributes a branch point. Dendrimers are typically, though not necessarily, formed using a multi-step nanoscale fabrication process. Each step results in a new "generation" that is twice or more complex than the previous generation. Dendrimers exhibit certain properties of the dendritic state, such as core encapsulation, making them unique from other types of polymers.
[0074] "Branch point" refers to a bifurcation point comprising one or more atoms at which a polymer separates or branches from a linear structure into one or more additional polymeric arms. A polymer with multiple arms can have one branch point or multiple branch points, as long as the branches are not regular repeats resulting in a dendrimer.
[0075] "Substantially" or "essentially" means almost totally or completely, eg 95% or more of some given amount.
[0076] "Alkyl" refers to a hydrocarbon chain of length ranging from about 1 to 20 atoms. These hydrocarbon chains are preferably, but not necessarily, saturated and can consist of a branched or straight chain. Exemplary alkyl groups include methyl, ethyl, isopropyl, n-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, 3-methyl-3-pentyl, and the like.
"Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight-chain or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl and t-butyl.
"Cycloalkyl" refers to a saturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably consisting of 3 to about 12 carbon atoms, more preferably 3 to about 8.
[0079] "Non-interfering substituents" are those groups that, when present in a molecule, are typically non-reactive with other functional groups contained in the molecule.
[0080] The term "substituted" as, for example, in "substituted alkyl", refers to a moiety (for example, an alkyl group) substituted with one or more non-interfering substituents, such as but not limited to following: C3-.C8 cycloalkyl, for example, cyclopropyl, cyclobutyl, and the like; halo, for example, fluoro, chloro, bromo, and iodine; cyan; alkoxy, lower phenyl; substituted phenyl; and others. For substitutions on a phenyl ring, the substituents can be in any orientation (i.e., ortho, meta, or para).
"Alkoxy" refers to a group -O-R, where R is alkyl or substituted alkyl, preferably C1 -C20 alkyl (e.g., methoxy, ethoxy, propyloxy, etc.), preferably C1 - C7.
[0082] As used herein, "alkenyl" refers to branched and unbranched hydrocarbon groups 1 to 15 atoms in length, containing at least one double bond, such as ethenyl (vinyl), 2-propen-1-yl ( allyl), isopropenyl, 3-buten-1-yl, and the like.
[0083] The term "alkynyl", as used herein, refers to branched and unbranched hydrocarbon groups 2 to 15 atoms in length, containing at least one triple bond, such as ethynyl, 1-propynyl, 3-butyn- 1-ila, 1-octin-1-ila, and so on.
[0084] The term "aryl" means an aromatic group containing up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthracenyl, naphthacenyl, and the like.
[0085] "Substituted phenyl" and "substituted aryl" mean a phenyl group and an aryl group, respectively, substituted with one, two, three, four, or five (eg 1-2, 1-3, 1-4 , or 1-5 substituents) chosen from halo (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (for example C1 -ε alkyl), alkoxy (for example Cx -g alkoxy), benzyloxy, carboxy, aryl, and so on.
[0086] An inorganic acid is an acid devoid of carbon atoms. Examples include hydrohalic acids, nitric acid, sulfuric acid, phosphoric acid and the like.
"Hydrohalic acid" means a hydrogen halide, such as hydrofluoric acid (HF), hydrochloric acid (HC1), hydrobromic acid (HBr), and hydriodic acid (HI).
[0088] "Organic acid" means any organic compound (ie, containing at least one carbon atom) having one or more carboxy (-COOH) groups. Some specific examples include formic acid, lactic acid, benzoic acid, acetic acid, trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid, mixed chlorofluoroacetic acids, citric acid, oxalic acid, and the like.
[0089] "Active agent", as used herein, includes any agent, drug, compound, and the like that provides some pharmacological, often beneficial, effect that can be demonstrated in vivo or in vitro. As used herein, these terms also include any physiologically or pharmacologically active substance that produces a localized or systemic effect on a patient. As used herein, in particular with reference to synthetic approaches described herein, an "active agent" is intended to encompass its derivatized or modified versions with a linker such that, through in vivo administration, the parental "bioactive" molecule is released.
"Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to an excipient that can be included in a composition comprising an active agent and that does not cause significant toxicological adverse effects in the patient.
"Pharmacologically effective amount", "physiologically effective amount", and "therapeutically effective amount" are used interchangeably herein and mean the amount of an active agent present in a pharmaceutical preparation that is necessary to provide a desired level of agent active and/or conjugated in the bloodstream or at a tissue or target site in the body. The exact amount will depend on numerous factors, for example, the particular active agent, the components and physical characteristics of the pharmaceutical preparation, intended patient population, and patient considerations, and can be easily determined by the practitioner based on the information provided here and available in the relevant literature.
[0092] "Multifunctional", in the context of a polymer, means a polymer having 3 or more functional groups, where the functional groups may be the same or different, and are typically present at the polymer ends. Multifunctional polymers will typically contain between about 3-100 functional groups, or between 3-50 functional groups, or between 3-25 functional groups, or between 3-15 functional groups, or from 3 to 10 functional groups, i.e., contain 3 , 4, 5, 6, 7, 8, 9 or 10 functional groups.
[0093] "Difunctional" and "bifunctional" are used interchangeably herein and mean an entity, such as a polymer, with two functional groups contained therein, typically at the termini of the polymer. When the functional groups are the same, the entity is said to be homedifunctional or homobifunctional. When the functional groups are different, the entity is said to be heterodifunctional or heterobifunctional.
[0094] A basic or acidic reagent described herein includes a neutral, charged entity and any corresponding salt forms thereof.
The terms "subject", "individual" and "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. Mammals include but are not limited to murines, rodents, apes, humans, farm animals, sports animals and pets. Typically, such subjects suffer from or are prone to a condition that is preventable or treatable by administering a water-soluble polymer-active agent conjugate as described herein.
[0096] The term "about", in particular with reference to a given amount, is intended to encompass deviations of plus or minus five percent.
[0097] "Treatment" and "treating" a particular condition include: (1) preventing that condition, that is, causing the condition not to develop, or to occur with less intensity or to a lesser extent in a subject who may be exposed or predisposed to the condition but not yet feeling or exhibiting the condition, and (2) inhibiting the condition, that is, halting development or reversing the condition.
[0098] "Optional" or "optionally" means that the circumstance described below may but not necessarily occur, so the description includes cases where the circumstance occurs and cases where it does not.
[0099] A "small molecule" is an organic, inorganic, or organometallic compound that typically has a molecular weight less than about 1000, preferably less than about 800 Dalton. Small molecules, as referred to herein, encompass oligopeptides and other biomolecules with a molecular weight less than about 1000.
[0100] A "peptide" is a molecule composed of about 13 to 50 amino acids, or similar. An oligopeptide typically contains from about 2 to 12 amino acids.
[0101] Unless explicitly stated otherwise, the terms "partial mixed salt" and "mixed salt", as used herein, are used interchangeably, and in the case of a polymer conjugate (and corresponding compositions comprising a plurality of such polymer conjugates), refer to conjugates and compositions comprising one or more basic amino groups (or other basic nitrogen-containing groups), wherein (i) any of the given basic amino groups present in the conjugate or conjugate composition is not protonated or is protonated and (ii) relative to any given protonated basic amino group, the protonated basic amino group will have one of two different counterions. (The term "partial mixed salt" refers to the characteristic that not all amino groups present in the compound or composition are protonated - hence the composition is a "partial" salt, whereas "mixed" refers to the characteristic of multiple counterions). A mixed salt, as provided herein, encompasses hydrates, solvates, amorphous forms, crystalline forms, polymorphs, isomers, and the like.
[0102] An amine group (or other group with basic nitrogen) that is in "free base" form is such that the amine group, that is, a primary, secondary, or tertiary amine, has a pair of free electrons. The amine is neutral, that is, it has no charge.
[0103] An amine group that is in "protonated form" exists as a protonated amine, so the amino group is positively charged. As used herein, an amine group that is protonated can also be in the form of an acid addition salt resulting from the reaction of the amine with an acid, such as an inorganic acid or an organic acid.
[0104] The "molar percentage" of amino groups of an active agent refers to the fraction or percentage of amino groups in an active agent molecule contained in a polymer conjugate that are in one particular form or another, where the percentage total molar amino groups present in the conjugate is 100 percent.
[0105] As used herein, "psi" means pounds per square inch. Overview - Hydrohalide Salts, Alkoxylation Methods, and Conjugate Compositions (and Their Hydrohalide Salt Forms) Prepared from Polymer Reagents Prepared from Polymeric Products Prepared from Alkoxylation Methods
[0106] Hydrohalide Salts: As previously indicated, in one or more embodiments of the invention, a water-soluble polymer and active agent conjugate is provided, wherein the conjugate is in the form of a hydrohalide salt (e.g., a salt pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide with 4 arms). These conjugates represent new solid-state forms. A process for reproducibly preparing a composition containing an irinotecan conjugate, wherein - relative to all irinotecan conjugates present in the composition - more than 95 mole percent of all basic nitrogen atoms of irinotecan are protonated in a hydrohalide salt form (HX), has been discovered and is provided herein. It was further found that the hydrohalide salt demonstrates increased stability towards hydrolytic degradation, for example, as compared to the free base form of the conjugate. See, for example, Example 3.
[0107] By way of context, during the preparation of the 4-armed pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan, as described in detail in Example 1, it was found that, despite treatment with base, the product was generally formed in form of a salt of mixed acids having basic nitrogen atoms of irinotecan, e.g., amino groups, in protonated or non-protonated form, wherein any given protonated amino group was an acid salt having one of two different anions (eg, trifluoroacetate or chloride). In an attempt to further explore the resulting composition a method was devised to prepare substantially pure hydrohalide salt. As described generally above, the hydrohalide salt described herein possesses certain remarkable and advantageous properties. The structural characteristics, properties, method of preparation and use, and additional characteristics of the 4-armed pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide salt, among other characteristics, are described herein.
[0108] In summary, the characteristics of a hydrohalide salt, eg hydrochloride salt, of a water-soluble polymer-active agent conjugate typically include the following. Generally speaking, the compound is a multi-armed poly(ethylene glycol) polymer conjugate and irinotecan. Irinotecan, as is evident from its structure, has one or more basic amine groups (or other basic nitrogen atoms), that is, it has a pK from about 7.5 to about 11.5 after conjugation to the polymer core. multi-armed (i.e., the active agent has one or more amine or other basic nitrogen-containing groups after conjugation to the water-soluble polymer). The resulting conjugate is a hydrohalide salt, that is, in which the basic nitrogen atoms are protonated as a hydrohalide salt (HX, where X is selected from fluoride, chloride, bromide and iodide).
[0109] As used herein, a hydrohalide salt with greater than 95 mole percent basic nitrogen atoms of irinotecan protonated in the form of the hydrohalide salt refers to the "volume product" rather than necessarily referring to an individual molecular species contained in the product volume. Thus, individual molecular species contained in the salt, due to the number of polymeric arms of the conjugate structure, may contain a small number of amine groups which are in free base form as well as in protonated form, as described above. Furthermore, the core of the 4-armed PEG-carboxymethyl conjugate can generally have less than full substitution with covalently linked irinotecan, with this characteristic being described in more detail below.
[0110] Alkoxylation Methods: Also as previously indicated, in one or more embodiments of the invention, a method is provided, wherein the method comprises the step of alkoxylation, in a suitable solvent, of an oligomer capable of being alkoxylated previously isolated to form an alkoxylated polymeric product, wherein the previously isolated alkoxylated oligomer has a known and defined weight average molecular weight greater than 300 Daltons (e.g., greater than 500 Daltons). Among other advantages, the alkoxylation methods provided herein result in polymeric products that are superior (for example, in terms of consistency and purity) to polymeric products prepared by previously known methods.
[0111] Conjugate Compositions (and Their Hydrohalide Salt Forms) Prepared From Polymer Reagents Prepared From Polymeric Products Prepared From Alkoxylation Methods: Also as previously indicated, in one or more embodiments of the invention is A hydrohalide salt of a water-soluble polymer-active agent conjugate is provided, wherein the conjugate is prepared by coupling (under conjugation conditions) an amine-containing active agent (eg, an unprotected glycine-irinotecan) to a polymer reagent (eg, 4-armed pentaerythritolyl-poly(ethylene glycol)-carboxymethyl succinimide) in the presence of a base to form a conjugate, wherein the conjugate is a hydrohalide salt conjugate (eg, the conjugate has atoms of nitrogen, each of which will be protonated or non-protonated, wherein any given protonated amino group is a hydrohalide salt), and also wherein, optionally, the polymer reactant ro is prepared from an alkoxylation product prepared as described herein. Conjugates - The Polymer in General
[0112] Water-soluble polymer-active agent conjugates (regardless of the specific form taken, eg, a base form, salt form, mixed salt, and so on) include a water-soluble polymer. Typically, to form a conjugate, a water-soluble polymer - in the form of a polymer reagent - is coupled (under conjugation conditions) to an active agent in an electrophile or nucleophile contained in the active agent. For example, a water-soluble polymer (again, in the form of a polymer reagent containing, for example, an activated ester) can be coupled to an active agent having one or more basic amine groups (or other basic nitrogen atoms) , i.e., an amine with a pK of from about 7.5 to about 11.5 (determined after conjugation).
[0113] The water-soluble polymer component of the conjugate is typically a water-soluble, non-peptide polymer. Representative polymers include poly(alkylene glycol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharide), poly(α-hydroxy acid), poly(acrylic acid), poly( vinyl alcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), or their copolymers or terpolymers. A particular water-soluble polymer is polyethylene glycol or PEG comprising the repeating unit (CH2CH2O)n-, where n ranges from about 3 to about 2700 or even more, or preferably from about 25 to about 1300. Typically, the weight average molecular weight of the water soluble polymer present in the conjugate ranges from about 100 Daltons to about 150,000 Daltons. Illustrative overall molecular weights for the conjugate can range from about 800 to about 80,000 Daltons, or from about 900 to about 70,000 Daltons. Additional representative molecular weight ranges are from about 1,000 to about 40,000 Dalton, or from about 5,000 to about 30,000 Dalton, or from about 7,500 Dalton to about 25,000 Dalton, or even from about 20,000 to about 80,000 Daltons for higher molecular weight embodiments of the present salts.
[0114] The water-soluble polymer can be in any of a few geometries or shapes, including linear, branched, bifurcated. In exemplary embodiments, the polymer is often linear or has multiple arms. Water-soluble polymers can be obtained from the market simply in the form of the water-soluble polymer. Furthermore, water-soluble polymers can conveniently be obtained in an activated form as a polymer reagent (which optionally can be coupled to an active agent without further modification or activation). Descriptions of water-soluble polymers and polymer reagents can be found in the "Nektar Advanced PEGylation Catalog", 2005-2006, "Polyethylene Glycol and Derivatives for Advanced PEGylation", and are available for purchase from NOF Corporation and JenKem Technology USA, among others .
[0115] An exemplary branched polymer having two polymeric arms in a branched pattern is as follows, often called PEG-2 or mPEG-2:
on what
indicates the location of additional atoms to form any functional groups suitable for reaction with an electrophile or nucleophile contained in an active agent. Exemplary functional groups include NHS ester, aldehyde, and so on.
[0116] For polymer structures described here that contain the variable "n", this variable corresponds to an integer and represents the number of monomeric subunits within the repeating monomeric structure of the polymer.
[0117] An exemplary architecture for use in preparing the conjugates consists of multi-armed water-soluble polymer reagents having, for example, 3, 4, 5, 6 or 8 polymeric arms, each optimally containing a group functional. A multi-arm polymer reactant can have any of a few cores (eg, a polyol core) from which the polymeric arms emanate. Exemplary polyol cores include glycerol, glycerol dimer (3,3'-oxydipropane-1,2-diol), trimethylolpropane, sugars (such as sorbitol or pentaerythritol, pentaerythritol dimer), and glycerol oligomers such as hexaglycerol or 3-( 2-h.hydroxy-3-(2-hydroxyethoxy)propoxy)propane-1,2-diol, and other glycerol condensation products. As an example, the polymeric nuclei and arms that emanate from them may have the following formulas:


[0118] In an exemplified embodiment, the water-soluble polymer is a 4-arm polymer as shown above, wherein n can range from about 20 to about 500, or from about 40 to about 500.
[0119] In the multi-arm embodiments described herein, each polymeric arm typically has a molecular weight corresponding to one of the following: 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 , 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000, 10,000, 12000, 15000, 17500, 18000, 19000, 20,000 Dalton or greater. Overall molecular weights for the multi-arm polymer configurations described here (that is to say, the molecular weight of the multi-arm polymer as a whole) generally corresponds to one of the following: 800, 1000, 1200, 1600, 2000, 2400, 2800, 3200, 3600, 4000, 5000, 6000, 8000, 10 000, 12 000, 15 000, 16 000, 20 000, 24 000, 25 000, 28 000, 30,000, 32 000, 36 000, 40 000, 45 000, 48 000, 50 000, 60 000, 80 000 or 100 000 or greater.
[0120] The water-soluble polymer, for example, PEG, can be covalently linked to the active agent via an intervening linker. The linker can contain any number of atoms. Generally speaking, the linker length in atoms meets one or more of the following ranges: from about 1 atom to about 50 atoms; from about 1 atom to about 25 atoms; from about 3 atoms to about 12 atoms; from about 6 atoms to about 12 atoms; and from about 8 atoms to about 12 atoms. When considering the chain length in atoms, only the atoms that contribute to the global distance are considered. For example, a linker with the structure -CH2-C(O)-NH-CH2CH2O-CH2CH2O-C(O)-O- is considered to have a chain length of 11 atoms, as the substituents are not considered to contribute significantly for the length of the connector. Illustrative linkers include bifunctional compounds such as amino acids (for example, alanine, glycine, isoleucine, leucine, phenylalanine, methionine, serine, cysteine, sarcosine, valine, lysine, and the like). The amino acid can be a naturally occurring amino acid or an unnaturally occurring amino acid. Suitable linkers also include oligopeptides.
[0121] The multi-arm structures illustrated above are primarily drawn to illustrate the polymer core having PEG chains attached thereto, and although not explicitly drawn, depending on the nature of the active agent and binding chemistry employed, the final structure may optionally include an additional ethylene group, -CH 2 CH 2 -, attached to the oxygen atoms at the terminus of each polymeric arm, and/or may optionally contain any of a few intervening linker atoms to facilitate covalent attachment to an active agent. In a particular embodiment, each of the PEG arms illustrated above also comprises a carboxy methyl group, -CH 2 -C(O)-, covalently attached to the terminal oxygen atom. New Alkoxylation Method for Improved Polymer Compositions
[0122] As previously indicated, water-soluble polymers that have utility (for example) in the preparation of conjugates with active agents (as well as their salt forms) can be obtained on the market. However, as further described herein, methods of preparing water-soluble polymers are provided - wherein said methods are distinguished from previously described methods for preparing water-soluble polymers - which are particularly suitable for the preparation of conjugates with active agents (as well as their salt forms).
[01231 In this regard, a method is provided, wherein the method comprises the step of alkoxylation, in a suitable solvent, of a pre-isolated alkoxylated oligomer to form an alkoxylated polymeric product, wherein the alkoxylated oligomer is previously alkoxylated. isolate has a known and defined weight average molecular weight greater than 300 Dalton (eg greater than 500 Dalton). The Alkoxylation Step in the New Alkoxylation Method
[0124] The alkoxylation step is carried out using alkoxylation conditions, so that the sequential addition of monomers is effected through repeated reactions of an oxirane compound. When the alkoxylated oligomer initially has one or more hydroxyl functional groups, one or more of these hydroxyl groups present in the alkoxylated oligomer will be converted to a reactive alkoxide by reaction with a strong base. An oxirane compound then reacts with a functional group capable of being alkoxylated (eg, a reactive alkoxide), thereby not only adding to the reactive alkoxide but making it such that it also ends up in another reactive alkoxide. Then, repeated reactions of an oxirane compound at the reactive alkoxide terminus of the previously added and reacted oxirane compound effectively produces a polymer chain.
[0125] Although each of the one or more functional groups capable of being alkoxylated is preferably hydroxy, other groups, such as amines, thiols and the hydroxyl group of a carboxylic acid, can serve as an acceptable functional group capable of being alkoxylated. Furthermore, due to the acidity of the hydrogens of the alpha carbon atoms in aldehydes, ketones, nitriles and amides, the addition on the alpha carbon atoms of these groups can serve as a functional group capable of being acceptable alkoxylated.
[0126] The oxirane compound contains an oxirane group and has the following formula:
wherein (with respect to this structure):R1 is selected from the group consisting of H and alkyl (when alkyl, preferably lower alkyl); R2 is selected from the group consisting of H and alkyl (when alkyl, preferably is lower alkyl); R3 is selected from the group consisting of H and alkyl (when alkyl, preferably it is lower alkyl); and R4 is selected from the group consisting of H and alkyl (when it is alkyl it is preferably lower alkyl).
[0127] With regard to the formula of the oxirano compound above, it is particularly preferred that each of R1, R2, R3 and R4 is H, and it is preferred that only one of R1, R2, R3 and R4 is alkyl (for example, methyl and ethyl) and the remaining substituents are H. Exemplary oxirane compounds are ethylene oxide, propylene oxide and 1,2-butylene oxide. The amount of oxirane compound added to give optimal alkoxylation conditions depends on a number of factors, including the amount of starting alkoxylated oligomer, the desired size of the resulting alkoxylated polymeric material, and the number of alkoxylated functional groups present in the oligomer able to be alkoxylated. Thus, when a larger alkoxylated polymeric material is desired, relatively more oxirane compound will be present under the alkoxylation conditions. Similarly, if (Oa) represents the amount of oxirane compound required to achieve a given polymer "growth" size into a single alkoxylated functional group, then an alkoxylated oligomer containing two alkoxylated functional groups alkoxylated will require 2x(0a), an alkoxylated oligomer containing three alkoxylated functional groups will require 3x(0a), an alkoxylated oligomer containing four alkoxylated functional groups will require 4x(Oa) and so on. In all cases, the practitioner can determine an appropriate amount of oxirane compound required for alkoxylation conditions by taking into account the desired molecular weight of the alkoxylated polymeric material and following routine experimentation.
[0128] The alkoxylation conditions include the presence of a strong base. The purpose of the strong base is to deprotonate each acidic hydrogen (for example, the hydrogen of a hydroxyl group) present in the alkoxylated oligomer and form an ionic species of alkoxide (or an ionic species for functional groups capable of being alkoxylated other than hydroxyl ). Preferred strong bases for use as part of the alkoxylation conditions are: alkali metals such as metallic potassium, metallic sodium, and alkali metal mixtures such as sodium-potassium alloys; hydroxides such as NaOH and KOH; and alkoxides (for example, present after the addition of an oxirane compound). Other strong bases can be used and can be identified by the practitioner. For example, a certain base can be used here as a strong base if the strong base can form an ionic species of alkoxide (or an ionic species for functional groups capable of being alkoxylated other than hydroxyl) and also provides a cation that does not hamper the species. ionic alkoxide so as to stop the reaction (or effectively stop it by a mechanism so slow as to be impractical) of the ionic species of alkoxide with the oxirane molecule. The strong base is present in a generally small and calculated amount, and that amount may fall in one or more of the following ranges: from 0.001 to 10.0 percent by weight based on the weight of the total reaction mixture; and from 0.01 to about 6.0 percent by weight based on the weight of the total reaction mixture.
[0129] The alkoxylation conditions include a suitable temperature for the alkoxylation to occur. Exemplary temperatures that may be suitable for the alkoxylation to occur include those in one or more of the following ranges: from 10°C to 260°C; from 20°C to 240°C; from 30°C to 220°C; from 40°C to 200°C; from 50°C to 200°C; from 80°C to 140°C; and from 100°C to 120°C.
[0130] The alkoxylation conditions include a suitable pressure for the alkoxylation to occur. Exemplary pressures that may be suitable for alkoxylation to occur include those in one or more of the following ranges: from 10 psi to 1000 psi; from 15 psi to 500 psi; from 20 psi to 250 psi; from 25 psi to 100 psi. Additionally, the alkoxylation pressure can be about atmospheric pressure at sea level (for example, 14,696 pounds per square inch +/- 10%).
[0131] In some cases, alkoxylation conditions include the addition of the oxirane compound in liquid form. In some cases, alkoxylation conditions include addition of the oxirane compound in vapor form.
[0132] Alkoxylation conditions may include the use of a suitable solvent. Optimally, the system where the alkoxylation conditions occur will not include any component (including any solvent) that can be deprotonated (or remain substantially protonated under the conditions of pH, temperature, and so on under which the alkoxylation conditions will occur) . Suitable solvents for alkoxylation include organic solvents selected from the group consisting of tetrahydrofuran (THF), dimethylformamide (DMF), toluene, benzene, xylenes, mesitylene, tetrachloroethylene, anisole, dimethylacetamide, and mixtures of the foregoing. Less ideal (but still contemplated) solvents for use as part of the alkoxylation conditions are acetonitrile, phenylacetonitrile and ethyl acetate; in some cases, the alkoxylation conditions will include as solvent none of acetonitrile, phenylacetonitrile and ethyl acetate.
[0133] In one or more embodiments of the invention, when the alkoxylation conditions are conducted in the liquid phase, the alkoxylation conditions will be conducted in such a way that both the oligomer capable of being alkoxylated and the desired alkoxylated polymeric material, formed by means of alkoxylation of the oligomer capable of being alkoxylated, not only do they have similar solubilities (and preferably, Substantially the same solubility) in the suitable solvent used, but also are both substantially soluble in the suitable solvent. For example, in one or more embodiments, the alkoxylated oligomer will be substantially soluble in the solvent used under the alkoxylation conditions, and the resulting alkoxylated polymeric material will also be substantially soluble under the alkoxylation conditions.
[0134] In one or more embodiments, this substantially equal solubility of the alkoxylated oligomer and the alkoxylated polymeric material in a suitable solvent contrasts with the solubility of a precursor molecule (used, for example, in the preparation of the previously isolated alkoxylated oligomer) in the suitable solvent, wherein the precursor molecule may have a lower (and even substantially lower) solubility in the suitable solvent than the alkoxylated oligomer and/or the alkoxylated polymeric material. By way of example only, the alkoxylated oligomer and the alkoxylated polymeric material will both have a pentaerythritol core and will both be substantially soluble in toluene, but pentaerythritol itself has limited solubility in toluene.
[0135] It is particularly preferred that the solvent employed in the alkoxylation conditions is toluene. The amount of toluene used for the reaction is greater than 25% by weight and less than 75% by weight of the reaction mixture, based on the weight of the reaction mixture after the addition of the oxirane compound is complete. The skilled person can calculate the starting amount of solvent by taking into account the desired molecular weight of the polymer, the number of sites where alkoxylation will occur, the weight of the alkoxylated oligomer used, and so on.
[0136] It is preferred that the amount of toluene is measured so that the amount is sufficient for the alkoxylation conditions to provide the desired alkoxylated polymeric material.
[0137] Additionally, it is particularly preferred that the alkoxylation conditions have a substantially zero amount of water present. Thus, it is preferred that the alkoxylation conditions have a water content of less than 100 ppm, more preferably 50 ppm, even more preferably 20 ppm, much more preferably less than 14 ppm, and even more preferably less than than 8 ppm.
[0138] The alkoxylation conditions take place in a suitable reactor, typically a stainless steel reactor.
[0139] In one or more embodiments, it is acceptable that the alkoxylated oligomer and/or precursor molecule be devoid of an isocyanate group bonded to a carbon containing an alpha hydrogen. In one or more embodiments, the previously prepared alkoxylated oligomer and/or precursor molecule are devoid of an isocyanate group.
[0140] The alkoxylated oligomer used in the new alkoxylation method must have at least one functional group capable of being alkoxylated. However, the alkoxy-capable oligomer may have one, two, three, four, five, six, seven, eight or more alkoxy-capable functional groups, with preference to an alkoxy-capable oligomer having from one to six groups functional apt to be alkoxylated.
[0141] As previously stated, each alkoxylated functional group present in the alkoxylated oligomer can be independently selected from the group consisting of hydroxyl, carboxylic acid, amine, thiol, aldehyde, ketone, and nitrile. In those cases where there is more than one alkoxylated functional group in the alkoxylated oligomer, it is typical that each alkoxylated functional group is the same (for example, each alkoxylated functional group present in the alkoxylated oligomer to be alkoxylated is hydroxyl), although cases of different functional groups apt to be alkoxylated on the same oligomer apt to be alkoxylated are also contemplated. When the functional group capable of being alkoxylated is hydroxyl, it is preferred that the hydroxyl group is a primary hydroxyl.
[0142] The oligomer capable of being alkoxylated can have any of a few possible geometries. For example, the oligomer capable of being alkoxylated can be linear. In an example of a linear alkoxylated oligomer, one end of the linear alkoxylated oligomer is a relatively inert functional group (eg, a terminal protecting group) and the other end is a alkoxylated functional group ( for example, hydroxyl). An exemplary alkoxylated oligomer with this structure is methoxy-PEG-OH, or abbreviated to mPEG, in which one terminus is the relatively inert methoxy group while the other terminus is a hydroxyl group. The structure of mPEG is shown below.CH3O-CH2CH2O-(CH2CH2O) Q-CH2CH2-OH(wherein, for the immediately preceding structure only, n is an integer from 13 to 100).
[0143] Another example of a linear geometry that the oligomer capable of being alkoxylated can take is a linear organic polymer containing functional groups capable of being alkoxylated (the same or different) at each terminal. An exemplary alkoxylated oligomer with this structure is alpha-, omega-dihydroxylpoly(ethylene glycol), or HO - CH2 CH2O-(CH2CH2O)n-CH2CH2 - OH (wherein, for the immediately preceding structure only, n is a integer from 13 to 100), which may be represented in abbreviated form as HO-PEG-OH, it being understood that the symbol -PEG- represents the following structural unit: -CH2CH2O- (CH2CH2O) n-CH2CH2-(wherein, only for the immediately preceding structure, n is an integer from 13 to 100).
[0144] Another geometry that the alkoxylated oligomer can take is a "multi-armed" or branched structure. With respect to these branched structures, one or more atoms of the oligomer capable of being alkoxylated serve as a "branch point atom", through which two, three, four or more (but typically two, three or four) distinct sets of monomers of repeat or "arms" are connected (directly or through one or more atoms). At a minimum, a "multi-arm" structure, as used here, has three or more distinct arms, but can have as many as four, five, six, seven, eight, nine, or more atoms, with multi-arm structures containing 4 to 8 arms (such as a 4-arm frame, a 5-arm frame, a 6-arm frame, and an 8-arm frame).
[0145] Exemplary multi-arm structures for the alkoxylated oligomer are provided below:
wherein (for the immediately preceding structure only) the average value of n is from 1 to 50, for example from 10 to 50 (or defined otherwise such that the molecular weight of the structure is from 300 Dalton to 9 000 Dalton (for example, from about 500 Dalton to 5,000 Dalton));
wherein (for the immediately preceding structure only) the average value of n is from 2 to 50, for example from 10 to 50 (or defined otherwise such that the molecular weight of the structure is from 300 Dalton to 9,000 Dalton ( for example, from about 500 Dalton to 5,000 Dalton));
wherein (for the immediately preceding structure only) the average value of n is from 2 to 35, for example from 8 to about 40 (or defined otherwise such that the molecular weight of the structure is from 750 Dalton to 9 500 Dalton (for example from 500 Dalton to 5,000 Dalton)); and
wherein (for the immediately preceding structure only) the average value of n is 2 to 35, for example, from 5 to 35 (or defined otherwise such that the molecular weight of the structure is from 1,000 Dalton to 13,000 Dalton ( for example, from 500 Dalton to 5 000 Dalton)).
[0146] For each of the four immediately preceding structures, it is preferred that the value of n, in each case, is substantially equal. Thus, it is preferred that, when all values of n are considered for a given alkoxylated oligomer, all values of n for that alkoxylated oligomer are within three standard deviations, more preferably within two deviations standard, and even more preferably within one standard deviation.
[0147] In terms of the molecular weight of the alkoxylated oligomer, the alkoxylated oligomer will have a known and defined weight average molecular weight. For use herein, a weight average molecular weight can only be known and defined for an alkoxylated oligomer when the alkoxylated oligomer is isolated from the synthetic medium from which it was generated. Exemplary weight average molecular weights for the alkoxylated oligomer will fall in one or more of the following ranges: greater than 300 Daltons; greater than 5 00 Dalton,- from 30 0 Dalton to 15 000 Dalton; from 500 Dalton to 5,000 Dalton; from 300 Dalton to 10,000 Dalton; from 500 Dalton to 4000 Dalton; from 300 Dalton to 5,000 Dalton; from 500 Dalton to 3000 Dalton; from 300 Dalton to 2000 Dalton; from 500 Dalton to 2000 Dalton; from 300 Dalton to 1000 Dalton; from 500 Dalton to 1000 Dalton; from 1,000 Dalton to 10,000Dalton; from 1,000 Dalton to 5,000 Dalton; from 1,000Dalton to 4,000 Dalton; from 1000 Dalton to 3000 Dalton; from 1000 Dalton to 2000 Dalton; from 1500Daltons to 15,000 Daltons; from 1,500 Dalton to 5,000Dalton; from 1,500 Dalton to 10,000 Dalton; from 1500 Dalton to 4000 Dalton; from 1500 Dalton to 3000 Dalton; from 1500 Dalton to 2000 Dalton; from 2,000Daltons to 5,000 Daltons; from 2,000 Dalton to 4,000Dalton; and from 2000 Dalton to 3000 Dalton.
[0148] For purposes of the present invention, the oligomer capable of being alkoxylated is preferably previously isolated. By previously isolated is meant that the alkoxylated oligomer exists outside and separate from the synthetic medium from which it was generated (most typical is outside the alkoxylation conditions used to prepare the alkoxylated oligomer) and may optionally be stored for a relatively prolonged period of time, or optionally stored for a shorter period of time without substantial change for subsequent use. Thus, an oligomer capable of being alkoxylated was previously isolated if, for example, it is housed in an inert environment. In this regard, a previously isolated alkoxylated oligomer may be housed in a container substantially devoid (e.g., less than 0.1% by weight) of an oxirane compound. Furthermore, an oligomer capable of being alkoxylated previously isolated does not change its molecular weight by more than 10% over 15 days. Thus, in one or more embodiments of the invention, the concept of "previously isolated" contrasts (for example) with a situation in which an ongoing and uninterrupted alkoxylation reaction is allowed to proceed, from the precursor molecule, to a structure that corresponds to an oligomer capable of being alkoxylated, to a structure that corresponds to an alkoxylated polymeric material; the concept of "previously isolated" requires that the oligomer capable of being alkoxylated exists separately from the conditions under which it was formed. However, in accordance with the present invention, the previously isolated alkoxylated oligomer will be subjected to an alkoxylation step after being added, in a separate step, to the alkoxylation conditions. Alkoxylation
[0149] The oligomer capable of being alkoxylated can be obtained by synthetic means. In this regard, the alkoxylated oligomer is prepared by (a) alkoxylation of a precursor molecule with a molecular weight of less than 300 Dalton (e.g. less than 500 Dalton) to form a reaction mixture. comprising an alkoxylated oligomer or prepolymer, and (b) isolating the alkoxylated oligomer from the reaction mixture. The alkoxylation step of the precursor molecule largely follows the conditions and requirements of the previously discussed alkoxylation step. The step of isolating the alkoxylated oligomer can be carried out using any step known in the art, but may include allowing the consumption of all of the oxirane compound in the reaction, actively carrying out a quench step, separating the final reaction mixture by means of approaches known in the art (including, for example, removing by distillation all volatile materials, removing solid reaction by-products by filtration or washing, and applying chromatographic media).
[0150] Additionally, the oligomer capable of being alkoxylated can be obtained from commercial sources. Exemplary commercial sources include NOF Corporation (Tokyo Japan), which provides oligomers capable of being alkoxylated under the trademarks SUNBRIGHT DKH® poly(ethylene glycol), SUNBRIGHT® GL tri-poly(ethylene glycol) ether of glycerin, SUNBRIGHT PTE® ether of tetra-poly(ethylene glycol) pentaerythritol, SUNBRIGHT® DG tetra-poly(ethylene glycol) ether of di-glycerin, and SUNBRIGHT HGEO® octa-poly(ethylene glycol) ether of hexaglycerin. Preferred alkoxylated oligomers include those having the structures of SUNBRIGHT PTE®-2000 tetra-poly(ethylene glycol) ether of pentaerythritol (which has a weight average molecular weight of about 2000 Dalton) and SUNBRIGHT® DG-2000 tetra-poly(ethylene glycol) ether of diglycerin (which has a weight average molecular weight of about 2,000 Daltons).
[0151] Precursor molecules can consist of any small molecule (for example, a molecular weight less than the weight average molecular weight of the alkoxylated oligomer) having one or more alkoxylated functional groups.
[0152] Exemplary precursor molecules include polyols, which are small molecules (typically with a molecular weight less than 300 Dalton, eg, less than 500 Dalton) having a plurality of available hydroxyl groups. Depending on the desired number of polymeric arms in the alkoxylated oligomer or prepolymer, the polyol serving as the precursor molecule will typically comprise 3 to about 25 hydroxyl groups, preferably about 3 to about 22 hydroxyl groups, with lots of preferably about 4 to about 12 hydroxyl groups. Preferred polyols include glycerol oligomers or polymers, such as hexaglycerol, pentaerythritol and their oligomers or polymers (for example, dipentaerythritol, tripentaerythritol, tetrapentaerythritol, and ethoxylated forms of pentaerythritol), and alcohols derived from sugars, and mannitol, such as mannitol . In addition, many commercially available polyols such as various isomers of inositol (ie 1,2,3,4,5,6-hexahydroxycyclohexane), 2,2-bis(hydroxymethyl)-1-butanol,2-amino- 2-(hydroxymethyl)-1,3-propanediol (TRIS), 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol, acid {[2-hydroxy-1,1- bis(hydroxymethyl)ethyl]aminojaacetic (Tricine), 2-[(3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}propyl)amino]-2-(hydroxymethyl)-1,3- propanediol, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid (TES), 4-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}- acid 1-butanesulfonic acid, and 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol hydrochloride may serve as an acceptable precursor molecule. In those cases where the precursor molecule has an ionizable group or groups that will interfere with the alkoxylation step, those ionizable groups must be protected or modified before carrying out the alkoxylation step.
Exemplary preferred precursor molecules include those precursor molecules selected from the group consisting of glycerol, diglycerol, triglycerol, hexaglycerol, mannitol, sorbitol, pentaerythritol, dipentaerythritol, and tripentaerythritol.
[0154| In one or more embodiments of the invention, it is preferred that neither the previously isolated alkoxylated oligomer nor the alkoxylated polymeric product have a alkoxylated functional group (e.g., hydroxyl group) of the precursor molecule. Generated by the New Alkoxylation Method
[0155] The alkoxylated polymeric material prepared by the methods described herein will have a basic architecture corresponding to the structure of the alkoxylated oligomer (i.e., a linear alkoxylated oligomer will give rise to a linear alkoxylated polymeric material, a fit to be oligomer alkoxylated four-armed polymer material will give rise to a four-armed alkoxylated polymeric material, and so on.) Accordingly, the alkoxylated polymeric material will have any of a few possible geometries, including linear, branched, and multi-armed.
[0156] Regarding branched structures, a branched alkoxylated polymeric material will have three or more distinct arms, but may have as many as four, five, six, seven, eight, nine, or more arms, being preferred branched structures containing 4 to 8 arms (such as a 4-arm branched structure, a 5-arm branched structure, a 6-arm branched structure, and an 8-arm branched structure).
[0157] Exemplary branched structures for the alkoxylated polymeric material are provided below:
wherein (for the immediately preceding structure only) the mean value of n satisfies one or more of the following ranges: from 10 to 1000; from 10 to 500; from 10 to 250; from 50 to 1000; from 50 to 250; and from 50 to 120 (or defined otherwise such that the molecular weight of the structure is from 2,000 Dalton to 180,000 Dalton, for example, from 2,000 Dalton to 120,000 Dalton);
wherein (for the immediately preceding structure only) the mean value of n satisfies one or more of the following ranges: from 10 to 1000; from 10 to 500; from 10 to 250; from 50 to 1000; from 50 to 250; and from 50 to 120 (or defined otherwise such that the molecular weight of the structure is from 2,000 Dalton to 180,000 Dalton, for example, from 2,000 Dalton to 120,000 Dalton);
where (for the immediately preceding structure only) the mean value of n is / satisfies one or more of the following ranges: from 10 to 750; from 40 to 750; from 50 to 250; and from 50 to 120 (or defined otherwise such that the molecular weight of the structure is from 3,000 Dalton to 200,000 Dalton, for example, from 12,000 Dalton to
wherein (for the immediately preceding structure only) the average value of n is / satisfies one or more of the following ranges: from 10 to 600 and from 35 to 600 (or defined otherwise such that the molecular weight of the structure is from 4 000 Dalton to 215,000 Dalton, eg from 12 000 Dalton to 215,000 Dalton).
[0158] For each of the four structures immediately provided, it is preferred that the value of n, in each case, is substantially equal. Thus, it is preferred that, when all values of n are considered for a given alkoxylated polymeric material, all values of n for that alkoxylated polymeric material, oligomer or prepolymer capable of being alkoxylated, are within three standard deviations, more preferably within two standard deviations, and even more preferably within one standard deviation.
[0159] In terms of the molecular weight of the alkoxylated polymeric material, the alkoxylated polymeric material will have a known and defined number average molecular weight. For use here, a number average molecular weight can only be known and defined for a material that is isolated from the synthetic medium from which it was generated.
[0160] The total molecular weight of the alkoxylated polymeric product may be a suitable molecular weight for the intended purpose. An acceptable molecular weight for any particular purpose can be determined through trial and error via routine experimentation. Exemplary molecular weights for the alkoxylated polymeric product will have a number average molecular weight in one or more of the following ranges: from 2,000 Dalton to 215,000 Dalton; from 5,000 Dalton to 215,000 Dalton; from 5,000 Dalton to 150,000 Dalton; from 5,000 Dalton to 100,000 Dalton; from 5,000 Dalton to 80,000 Dalton; from 6,000 Dalton to 80,000 Dalton; from 7,500 Dalton to 80,000 Dalton; from 9,000 Dalton to 80,000 Dalton; from 10,000 Dalton to 80,000 Dalton; from 12,000 Dalton to 80,000 Dalton; from 15,000 Dalton to 80,000 Dalton; from 20,000 Dalton to 80,000 Dalton; from 25,000 Dalton to 80,000 Dalton; from 30,000 Dalton to 80,000 Dalton; from 40,000 Dalton to 80,000 Dalton; from 6 000 Dalton to 60 000 Dalton; from 7,500 Dalton to 60,000 Dalton; from 9,000 Dalton to 60,000 Dalton; from 10,000 Dalton to 60,000 Dalton; from 12,000 Dalton to 60,000 Dalton; from 15,000 Dalton to 60,000 Dalton; from 20,000 Dalton to 60,000 Dalton; from 25,000 Dalton to 60,000 Dalton; from 30,000 Dalton to 60,000; from 6,000 Dalton to 40,000 Dalton; from 9,000 Dalton to 40,000 Dalton; from 10,000 Dalton to 40,000 Dalton; from 15,000 Dalton to 40,000 Dalton; from 19,000 Dalton to 40,000 Dalton; from 15,000 Dalton to 25,000 Dalton; and from 18,000 Dalton to 22,000 Dalton.
[0161] For any given alkoxylated polymeric material, an optional step can be performed in order to further transform the alkoxylated polymeric material, so as to contain a specific reactive group to form a polymeric reagent. Thus, using techniques well known in the art, the alkoxylated polymeric material can be functionalized to include a reactive group (e.g., carboxylic acid, active ester, amine, thiol, maleimide, aldehyde, ketone, and so on).
[0162] When performing an optional step for the purpose of further transforming the alkoxylated polymeric product so as to contain a specific reactive group, this optional step is performed in a suitable solvent. The practitioner can determine whether any specific solvent is suitable for any given reaction step. However, often the solvent is preferably a non-polar solvent or a polar solvent. Non-limiting examples of non-polar solvents include benzene, xylenes and toluene. Exemplary polar solvents include but are not limited to dioxane, tetrahydrofuran (THF), t-butyl alcohol, DMSO (dimethyl sulfoxide), HMPA (hexamethyl phosphoramide), DMF (dimethylformamide), DMA (dimethylacetamide), and NMP (N-methylpyrrolidinone). Additional Alkoxylated Polymeric Material
[0163] Another aspect of the invention provided herein consists of compositions comprising the alkoxylated polymeric material, which include not only any compositions comprising the alkoxylated polymeric material, but also compositions in which the alkoxylated polymeric material is further processed, for example, into a reagent of polymer, as well as conjugate compositions formed by coupling these polymer reagents to an active agent. Among other things, a benefit of the method described here is the ability to achieve compositions containing high purity alkoxylated polymeric material. The compositions can be characterized by having: substantially low content of high molecular weight impurities (eg polymer-containing species with a molecular weight greater than the molecular weight of the desired alkoxylated polymeric material) and low content of low molecular weight diol impurities (i.e., HO-PEG-OH), wherein either type of impurity (and preferably both types of impurity) totals less than 8% by weight and more preferably less than 2% by weight. Additionally or alternatively, the compositions can also be characterized by having a purity of the alkoxylated polymeric material (as well as compositions comprising polymer reagents formed from the alkoxylated polymeric material, and compositions of conjugates formed from the conjugation of these polymer reagents and an active agent) greater than 92% by weight, more preferably greater than 97% by weight. Gel permeation chromatography (GPC) and gel filtration chromatography (GFC) can be used to characterize the alkoxylated polymeric material. These chromatographic methods allow the separation of the composition into its components according to molecular weight. Trace amounts of exemplary GFC products described in Example 7 and Example 8 are provided in the form of FIG. 7 and FIG. 8, respectively.Exemplary Uses of Alkoxylated Polymeric Materials and Compositions Formed From Them
[0164] The alkoxylated polymeric material provided herein, as well as those alkoxylated polymeric products which have been further modified to contain a specific reactive group (hereinafter referred to as "polymer reagent") are useful for conjugation, for example, to agents active. Preferred groups of biologically active agents suitable for reaction with the polymeric reagents described herein are electrophilic and nucleophilic groups. Exemplary groups include primary amines, carboxylic acids, alcohols, thiols, hydrazines and hydrazides. Such groups suitable for reacting with the polymeric reagents described herein are known to those skilled in the art. Thus, the invention provides a method of making a conjugate which comprises the step of contacting, under conjugating conditions, an active agent with a polymeric reagent described herein.
[0165] Suitable conjugation conditions are those conditions of time, temperature, pH, concentration of reagents, functional (functional) group(s) of the reagents, functional groups available in the active agent, solvent, and the like, sufficient to effect the conjugation between a polymeric reagent and an active agent. As is known in the art, the specific conditions depend, among other factors, on the active agent, the type of conjugation desired, the presence of other materials in the reaction mixture, and so on. Conditions sufficient to effect the conjugation in any particular case can be determined by the professional by reading the disclosure presented here, reference to the relevant literature, and/or by means of routine experimentation.
[0166] For example, when the polymeric reagent contains an active ester of N-hydroxysuccinimide (eg, succinimidyl succinate, succinimidyl propionate, and succinimidyl butanoate), and the active agent contains an amine group, conjugation can be performed at a pH of from about 7.5 to about 9.5 at room temperature. Additionally, when the polymeric reagent contains a vinyl sulfone reactive group or a maleimide group and the pharmacologically active agent contains a sulfhydryl group, the conjugation can be carried out at a pH of from about 7 to about 8.5 at room temperature. In addition, when the reactive group associated with the polymer reagent is an aldehyde or ketone and the pharmacologically active agent contains a primary amine, the conjugation can be carried out by means of reductive amination, in which the primary amine of the pharmacologically active agent reacts with the polymer aldehyde or ketone. Occurring at pH's from about 6 to about 9.5, reductive amination initially results in a conjugate in which the pharmacologically active agent and polymer are linked via an imine bond. Subsequent treatment of the conjugate containing an imine linkage with a suitable reducing agent such as NaCNBH3 reduces the imine to a secondary amine. For additional information concerning these and other conjugation reactions refer to Hermanson "Bioconjugate Techniques," Academic Press, 1996.
[0167] Exemplary conjugation conditions include carrying out the conjugation reaction at a pH of from about 4 to about 10, and, for example, at a pH of about 4.0, 4.5, 5.0, 5, 5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0. The reaction is allowed to proceed from about 5 minutes to about 72 hours, preferably from about 30 minutes to about 48 hours, and more preferably from about 4 hours to about 24 hours. The temperature at which conjugation can occur is typically, although not necessarily, in the range of from about 0°C to about 40°C, and often is room temperature or less. Conjugation reactions are often performed using a phosphate buffer solution, sodium acetate, or similar system.
[0168] With regard to the concentration of reactants, an excess of the polymer reactant is typically combined with the active agent. However, in some cases it is preferred to have stoichiometric amounts of reactive groups in the polymer reactant for the reactive groups in the active agent. Thus, for example, one mole of a polymer reactant containing four reactive groups is combined with four moles of active agent. Exemplary ratios between polymer reactant and active agent reactive groups include molar ratios of about 1:1 (polymer reactant reactive group:active agent), 1:0.1, 1:0.5, 1:1.5 , 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, OR 1:10. The conjugation reaction is allowed to proceed until substantially no more conjugation occurs, which can usually be determined by monitoring the progression of the reaction over time.
[0169] The progress of the reaction can be monitored by collecting aliquots of the reaction mixture at various times and analyzing the reaction mixture by means of chromatographic methods, SDS-PAGE or MALDI-TOF mass spectrometry, NMR, IR, or any other suitable analytical method . Once a plateau is reached with respect to the amount of conjugate formed or the amount of unconjugated polymer reagent remaining, the reaction is presumed to be complete. Typically, the conjugation reaction takes place for any period of time from minutes to several hours (for example, from 5 minutes to 24 hours or more). The resulting product mixture is preferably, but not necessarily, purified to separate excess active agent, strong base, condensing agents and reaction by-products and solvents. The resulting conjugates can then be further characterized using analytical methods, such as chromatographic methods, spectroscopic methods, MALDI, capillary electrophoresis, and/or gel electrophoresis. Polymer-active agent conjugates can be purified so that different species of conjugates are obtained/isolated.
[0170] With regard to an active agent, the alkoxylated polymeric material and a polymer reagent prepared from the alkoxylated polymeric material can be combined under suitable conjugation conditions to give rise to a conjugate. In this regard, exemplary active agents can consist of an active agent selected from the group consisting of a small molecule drug, an oligopeptide, a peptide, and a protein. The active agent for use herein may include but is not limited to the following: adriamycin, gamma-aminobutyric acid (GABA), amiodarone, amitriptyline, azithromycin, benzphetamine, bromopheniramine, cabinoxamine, calcitonin, chlorambucil, chloroprocaine, chloroquine, chlorpheniramine, chlorpromazine, cinnarizine , clarithromycin, clomiphene, cyclobenzaprine, cyclopentolate, cyclophosphamide, dacarbazine, daunomycin, demeclocycline, dibucaine, dicyclomine, diethylproprion, diltiazem, dimenhydrinate, diphenhydramine, disopyramide, doxepin, doxycycline, dibucaine, dicyclomine , imipramine, insulin, irinotecan, levomethadyl, lidocaine, loxarine, mechlorethamine, melphalan, methadone, methotimeperazine, methotrexate, metopelopramide, minocycline, naphthyfine, nicardipine, nizatidine, orphenadrine, oxybutine, promethinetrazine , proparacaine, propoxycaine, prop oxifene, ranitidine, tamoxifen, terbinafine, tetracaine, tetracycline, tramadol, triflupromazine, trimeprazine, trimethylbenzamide, trimipramine, tripelenamine, troleandomycin, tyramine, uracil mustard, verapamil, and vasopressin.
[0171] Other exemplary active agents include those selected from the group consisting of acrivastine, amoxapine, astemizole, atropine, azithromycin, benzapril, benztropine, biperiden, bupracaine, buprenorphine, buspirone, butorphanol, caffeine, camptothecin and molecules belonging to the camptothecin family, ceftriaxone, chlorpromazine, ciprofloxacin, cladarabine, clemastine, clindamycin, clofazamine, clozapine, cocaine, codeine, cyproeptadine, desipramine, dihydroergotamine, diphenidol, diphenoxylate, dipyridamole, flucetaxel, doxapram, fatantin, ergozamine ganciclovir, granisteron, guanethidine, haloperidol, homatropine, hydrocodone, hydromorphone, hydroxyzine, hyoscyamine, imipramine, itraconazole, ceterolac, ketoconazole, levocarbustine, levorphone, lincomycin, 1ornefloxacin, loperamide, mepinefloxacin, mepinefloxacin, mepinefloxacin, mepinal xalosartanin, mepinacidine, xalosartanin methdylazine, methenamine, methimazole, methotrimeperazine, methysergide, metronidazole, minoxidil, mitomycin c, molindone, morphine, nafzodone, nalbuphine, naldixic acid, nalmefene, naloxone, naltrexone, naphazoline, nedocromil, nicotine, norfloxacin, ofloxacin, ondansetron, oxycophenazine, pentazine physostigmine, pilocarpine, pimozide, pramoxin, prazosin, prochlorperazine, promazine, promethazine, quinidine, quinine, rauwolfia alkaloids., riboflavin, rifabutin, risperidone, rocuronium, scopalamine, sufentanil, thifentin, terazine, terazine, terazine , timolol, tolazamide, tolmetin, trazodone, triethylperazine, trifluopromazine, trihexylphenidyl, trimeprazine, trimipramine, tubecurarine, vecuronium, vidarabine, vinblastine, vincristine, and vinorelbine.
[0172] Still other exemplary active agents include those selected from the group consisting of acetazolamide, acrivastine, acyclovir, adenosine phosphate, allopurinal, alprazolam, amoxapine, amrinone, apraclonidine, azatadine, aztreonam, bisacodyl, bleomycin, bromopheniramine, buspirone, butoconazole and molecules of the camptothecin family, carbinoxarain, cefamandole, cefazol, cefixime, cefmetazol, cefonicide, cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone, cefapyrin, chloroquine, chlorpheniramine, cimetidine, cladarabine, cloxazoline, doxatrimazole , econazole, enoxacin, estazolam, ethionamide, famciclovir, famotidine, fluconazole, fludarabine, folic acid, ganciclovir, hydroxychloroquine, iodoquinol, isoniazid, itraconazole, ketoconazole, lamotrigine, lansoprazole, lorcetadine, losoprazole, mitramethopurtan midazolam, minoxidil, nafzodone, naldixic acid, niacin, n. icotine, nizatidine, omeprazole, oxaprozine, oxiconazole, papaverine, pentostatin, phenazopyridine, pilocarpine, piroxicam, prazosin, primaquine, pyrazinamide, pyrimethamine, pyroxidine, quinidine, quinine, ribaverine, rifampin, sulfadiassazole, sulfametoxazole, sulfamexazine, sulfametoxazole thiabendazole, thiamine, thioguanine, timolol, trazodone, triampterene, triazolam, trimethadione, trimethoprim, trimetrexate, tripelenamine, tropicamide, and vidarabine,
[0173] Still other exemplary active agents include those belonging to the camptothecin family of molecules. For example, the active agent can have the general structure:
wherein Rx, R2, R3, R< and R5 are each independently selected from the group consisting of the following; hydrogen; halo; acyl; alkyl (for example, C1-C6 alkyl); substituted alkyl; alkoxy (for example C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl; cycloalkyl; hydroxyl; cyan; nitro; azide; starch; hydrazine; amino; substituted amino (for example, monoalkylamino and dialkylamino); hydroxycarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino; carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; -C(R7)=N-(O)I-R8 where R7 is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, and R8 is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R$C(O)O- where R9 is halogen, amino, substituted amino, heterocycle, substituted heterocycle, or R10-O-(CH2) m- where m is an integer from 1-10 and R10 is alkyl , phenyl, substituted phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or substituted heterocycle; or R2 together with R3 or R3 together with R4 form substituted or unsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; Rg is H or OR', where R' is alkyl, alkenyl, cycloalkyl, haloalkyl, or hydroxyalkyl. Although not shown, analogs having a hydroxyl group corresponding to a position other than the 20 position (e.g., position 10, or 11, and so on) in the immediately preceding structure are encompassed under possible active agents.
[0174] An exemplary active agent is irinotecan.
Irinotecan
[0175] Another exemplary active agent is 7-ethyl-10-hydroxy-camptothecin (SN-38), and its structure is shown below.

[0176] Yet another exemplary class of active agents includes those belonging to the taxane family of molecules. An exemplary active agent of this class of molecules is docetaxel, where the H at the 2* hydroxyl group is involved in forming the preferred multiarm polymer conjugate:

[0177] The polymer reagents described herein can be linked, covalently or non-covalently, to some entities, including films, chemical separation and purification surfaces, solid supports, metallic surfaces, such as gold, titanium, tantalum, niobium, aluminum , steel, and its oxides, silicon oxide, macromolecules (eg, proteins, polypeptides, and so on), and small molecules. Additionally, polymer reagents can also be used in biochemical sensors, bioelectronic switches, and digital ports. Polymer reagents can also be employed as carriers for peptide synthesis, for preparing polymer coated surfaces and polymer grafts, for preparing polymer-binder conjugates for affinity partitioning, for preparing cross-linked or non-cross-linked hydrogels, and for prepare polymer-cofactor adducts for bioreactors.
[0178] Optionally, the conjugate can be provided in the form of a pharmaceutical composition for human veterinary and clinical use. Such pharmaceutical composition is prepared by combining the conjugate with one or more pharmaceutically acceptable excipients, and optionally any other therapeutic ingredients.
[0179] Exemplary pharmaceutically acceptable excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
[0180] A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
[0181] The excipient may also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and combinations thereof.
[0182] The composition may also include an antimicrobial agent to prevent or deter microbial growth. Non-limiting examples of antimicrobial agents suitable for one or more embodiments of the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
[0183] An antioxidant may also be present in the composition. Antioxidants are used to prevent oxidation, thereby preventing deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite , and combinations of these.
[0184] A surfactant may be present as an excipient. Exemplary surfactants include: polysorbates such as "Tween 20" and "Tween 80," and Pluronic's such as F68 and F88 (both available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids such as cholesterol; and chelating agents such as EDTA, zinc and other such suitable cations.
[0185] Acids or bases may be present in the composition as an excipient. Non-limiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, sulfuric acid , fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, citrate sodium, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.
[0186] The amount of conjugate (i.e., the conjugate formed between the active agent and the polymeric reagent) in the composition will vary depending on a few factors, but optimally it will be a therapeutically effective dose when the composition is stored in a single-dose container (eg a vial). Additionally, the pharmaceutical preparation can be stored in a syringe. A therapeutically effective dose can be determined experimentally by repeatedly administering increasing amounts of the conjugate to determine what amount produces a clinically desired end result.
[0187] The amount of any individual excipient in the composition will vary depending on the excipient's activity and the particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, that is, preparing compositions containing varying amounts of excipient (ranging from low to high), examining stability and other parameters, and then determining the range in which optimal performance is achieved without significant adverse effects.
[0188] However, in general, the excipient will be present in the composition in an amount of from about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about about 15 to about 95% by weight of the excipient, with concentrations of less than 30% by weight being most preferred.
[0189] These prior pharmaceutical excipients, along with other excipients, are described in "Remington: The Science & Practice of Pharmacy", 19th edition, Williams & Williams (1995), the "Physician's Desk Reference", 52nd edition, Medical Economics , Montvale, NJ (1998), and Kibbe, AH, "Handbook of Pharmaceutical Excipients", 3rd Edition, American Pharmaceutical Association, Washington, DC, 2000.
[0190] Pharmaceutically acceptable compositions encompass all types of formulations and, in particular, those that are suitable for injection, for example, powders or lyophilisates that can be reconstituted as well as liquids. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, 5% dextrose in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With regard to liquid pharmaceutical compositions, solutions and suspensions are considered.
[0191] The compositions of one or more embodiments of the present invention are typically, though not necessarily, administered via injection and, therefore, are generally liquid solutions or suspensions immediately prior to administration. The pharmaceutical preparation may also take other forms such as syrups, creams, ointments, tablets, powders, and the like. Other modes of administration such as pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, subcutaneous, intraarterial, and so on are also included.
[0192] The invention also provides a method of administering a conjugate as provided herein to a patient suffering from a condition that responds to treatment with the conjugate. The method comprises administering to a patient, generally via injection, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical composition). As previously described, the conjugates can be administered parenterally by intravenous injection. Types of formulations suitable for parenteral administration include ready-to-inject solutions, dry powders for combination with a solvent before use, ready-to-inject suspensions, dry insoluble compositions for combination with a vehicle before use, and liquid emulsions and concentrates for dilution before use. administration, among others.
[0193] The method of administration can be used to treat any condition that can be remedied or prevented by administering the conjugate. Professionals appreciate what conditions a specific couple can effectively treat. Advantageously, the conjugate can be administered to the patient before, simultaneously with, or after administration of another active agent.
[0194] The actual dose to be administered will vary depending on the age, weight, and general condition of the subject, as well as the severity of the condition to be treated, the assessment of the health care provider, and the conjugate to be administered. Therapeutically effective amounts are known to practitioners and/or are described in relevant reference texts and literature. In general, a therapeutically effective amount will range from about 0.001 mg to 100 mg, preferably at doses from 0.01 mg/day to 75 mg/day, and more preferably at doses from 0.10 mg/day to 50 mg/day. A given dose may be administered periodically until, for example, symptoms are alleviated and/or completely eliminated.
[0195] Unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) may be administered in a variety of dosing schedules, depending on the clinician's judgment, the patient's needs, and so on. The specific dosing schedule will be known to practitioners or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration once a day, three times a week, twice a week, once a week, twice a month, once a month, and any combination thereof. Once the final clinical result has been achieved, the dosage of the composition is terminated,
[0196] An advantage of administering certain conjugates described here is that individual portions of the water-soluble polymer can be cleaved when a hydrolytically degradable bond is included between the residue of the active agent fraction and the water-soluble polymer. This result is advantageous when elimination from the body is a potential problem due to the size of the polymer. Optimally, cleavage of each portion of the water-soluble polymer is facilitated through the use of physiologically cleavable and/or enzymatically degradable bonds, such as amide, carbonate or ester containing bonds. In this way, conjugate elimination (via cleavage of individual portions of the water-soluble polymer) can be modulated by selecting the molecular size of the polymer and the type of functional group that provide the desired scavenging properties. The practitioner can determine the appropriate molecular size of the polymer as well as the cleavable functional group. For example, the practitioner, using routine experimentation, can determine an appropriate molecular size and cleavable functional group by first preparing a variety of polymer derivatives with different polymer weights and cleavable functional groups, and then obtaining the elimination profile ( for example, by periodically sampling blood or urine) by administering the polymer derivative to a patient and periodically sampling blood and/or urine. Once a series of clearance profiles has been obtained for each conjugate tested, a suitable conjugate can be identified.Hydrohalide Salts - Considerations Regarding the Active Agent, "D"
[0197] The hydrohalide salt compositions described herein comprise a water-soluble polymer-active agent conjugate, preferably a multi-armed polymer bioactive conjugate. Exemplary water-soluble polymers are described above. Turning now to the active agent, the active agent is a small molecule drug, an oligopeptide, a peptide, or a protein. The active agent, when conjugated to the water-soluble polymer, contains at least one basic nitrogen atom, such as an amine group (i.e., an amine or other group containing basic nitrogens that is not conjugated to the water-soluble polymer). In the hydrohalide salt, the basic nitrogen atoms are in protonated form in the form of the hydrohalide salt, that is to say, where at least 90 mole percent, or at least 91 mole percent, or at least 92 mole percent, or at least 93 mole percent, or at least 94 mole percent, or at least 95 mole percent, more preferably more than 95 mole percent of the drug's basic nitrogen atoms present in the conjugate are protonated in HX form.
[0198] Active agents containing at least one amino group or basic nitrogen atom suitable for providing a salt of mixed acids as described herein include but are not limited to the following: adriamycin, gamma-aminobutyric acid (GABA), amiodarone, amitriptyline, azithromycin , benzphetamine, bromopheniramine, cabinoxamine, calcitonin, chlorambucil, chloroprocaine, chloroquine, chlorpheniramine, chlorpromazine, cinnarizine, clarithromycin, clomiphene, cyclobenzaprine, cyclopentolate, cyclophosphamide, dacarbazine, daunomycin, demeclocydyl, chlorpromazine, dibucaine , doxepine, doxycycline, doxylamine, dipyridamole, EDTA, erythromycin, flurazepam, gentian violet, hydroxychloroquine, imipramine, insulin, irinotecan, levomethadyl, lidocaine, loxarine, mechlorethamine, melphalan, methadone, methotimeperazine, methotrexate, methotrexate , nizatidine, orphenadrine, oxybutine, oxytetracyclin a, phenoxybenzamine, phentolamine, procainamide, procaine, promazine, promethazine, proparacaine, propoxycaine, propoxyphene, ranitidine, tamoxifen, terbinafine, tetracaine, tetracycline, tramadol, triflupromazine, trimeprazine, trimethylbenzamide, trimipramine, tromarycin, tripelenamine verapamil, and vasopressin.
[0199] Additional active agents include those comprising one or more nitrogen-containing heterocycles such as acrivastin, amoxapine, astemizole, atropine, azithromycin, benzapril, benztropine, biperiden, bupracaine, buprenorphine, buspirone, butorphanol, caffeine, camptothecin and molecules belonging to the family of camptothecins, ceftriaxone, chlorpromazine, ciprofloxacin, cladarabine, clemastine, clindamycin, clofazamine, clozapine, cocaine, codeine, cyproheptadine, desipramine, dihydroergotamine, diphenidol, diphenoxycyclolate, dipyridamole, doxapram, flufenyl, doxapram, flufenyl, doxapram , ganciclovir, granisteron, guanethidine, haloperidol, homatropine, hydrocodone, hydromorphone, hydroxyzine, hyoscyamine, imipramine, itraconazole, ceterolac, ketoconazole, levocarbustine, levorphone, lincomycin, lomefloxacin, loperamide, meloperamide, loperamide, meloxalosar , methdylazine, methenamine, methimazole, methotrimeperazine a, methysergide, metronidazole, minoxidil, mitomycin c, molindone, morphine, nafzodone, nalbuphine, naldixic acid, nalmefene, naloxone, naltrexone, naphazoline, nedocromil, nicotine, norfloxacin, ofloxacin, ondansetron, pentazine, oxycodone, oxycodone physostigmine, pilocarpine, pimozide, pramoxine, prazosin, prochlorperazine, promazine, promethazine, quinidine, quinine, rauwolfia alkaloids, riboflavin, rifabutin, risperidone, rocuronium, scopalamine, sufentanil, thifendipine, terazine, terazine timolol, tolazamide, tolmetin, trazodone, triethylperazine, trifluopromazine, trihexylphenidyl, trimeprazine, trimipramine, tubecurarine, vecuronium, vidarabine, vinblastine, vincristine and vinorelbine.
[0200] Additional active agents include those comprising a nitrogen in the aromatic ring, such as acetazolamide, acrivastin, acyclovir, adenosine phosphate, allopurinal, alprazolam, amoxapine, amrinone, apraclonidine, azatadine, aztreonam, bisacodyl, bleomycin, bromopheniramine, butospirone, camptothecin and molecules of the camptothecin family, carbinoxamine, cefamandole, cefazol, cefixime, cefmetazol, cefonicide, cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone, cefapirin, chloroquine, chlorpheniramine, cimetidine, cladarabine, dicloxazoline, didotriazoline doxylamine, econazole, enoxacin, stazolam, ethionamide, famciclovir, famotidine, fluconazole, fludarabine, folic acid, ganciclovir, hydroxychloroquine, iodoquinol, isoniazid, itraconazole, ketoconazole, lamotrigine, lansoprazole, lorcondazole, metrotaniazole, mitrazole, losar , midazolam, minoxidil, nafzodone, naldixic acid, niacin, nicot ina, nizatidine, omeprazole, oxaprozine, oxiconazole, papaverine, pentostatin, phenazopyridine, pilocarpine, piroxicam, prazosin, primaquine, pyrazinamide, pyrimethamine, pyroxidine, quinidine, quinine, ribaverine, rifampin, sulfadiazine, sulfametoxazole, sulfametoxine thiabendazole, thiamine, thioguanine, timolol, trazodone, triampterene, triazolam, trimethadione, trimethoprim, trimetrexate, tripelenamine, tropicamide, and vidarabine.
[0201] A preferred active agent is an active agent belonging to the camptothecin family of molecules. For example, the active agent can have the general structure:
wherein R1-R5 are each independently selected from the group consisting of hydrogen; halo; acyl; alkyl (for example, C1-C6 alkyl); substituted alkyl; alkoxy (for example C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl; cycloalkyl; hydroxyl; cyan; nitro; azide; starch; hydrazine; amino,-substituted amino (for example, monoalkylamino and dialkylamino); hydroxycarbonyl; alkoxycarbonyl; alkylcarbonyloxy; alkylcarbonylamino; carbamoyloxy; arylsulfonyloxy; alkylsulfonyloxy; -C(R7)=N-(O)I-R8 where R7 is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, and Ra is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R9C(O)O- where R9 is halogen, amino, substituted amino, heterocycle, substituted heterocycle, or R10-O-(CH2) m- where m is an integer from 1-10 and R10 is alkyl, phenyl , substituted phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or substituted heterocycle; or R2 together with R3 OR R3 together with R4 form substituted or unsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy; R6 is H or OR', where R' is alkyl, alkenyl, cycloalkyl, haloalkyl, or hydroxyalkyl.
[0202] With reference to the above structure, although not shown, analogues having a hydroxyl group at a position other than the 20 position (eg 10 position, or 11 position, etc.) are similarly preferred.
[0203] In a particular embodiment, the active agent is irinotecan (structure shown immediately below),

[0204] In another embodiment, the active agent is irinotecan having a glycine linker at the 20-hydroxyl position (structure shown immediately below).

[0205] In yet another particular embodiment, the active agent is 7-ethyl-10-hydroxy-camptothecin (SN-38), a metabolite of irinotecan, and its structure is shown below.
In the above embodiment, covalent attachment of the active agent, SN-38, to the multi-armed polymer core similarly occurs at the 20-hydroxyl position, optionally via an intervening linker such as glycine, as shown below.
Hydrohalide Salts - Considerations Regarding Conjugates
[0206] Illustrative conjugates of a water-soluble polymer and an active agent may possess any of some structural characteristics, as described above. This is to say that the conjugate may have a linear structure, that is, having one or two molecules of the active agent covalently attached thereto, typically at the polymer terminus or termini, respectively. Alternatively, the conjugate may have a bifurcated, branched or multi-armed structure. Preferably, the conjugate is a multi-arm polymer conjugate.
[0207] An illustrative multi-arm polymer conjugate structure corresponds to the following:

[0208] The above structure is called here, in abbreviated way, ''PEG-Gly-Trino with 4 arms" (4-armed pentaerythritol-PEG-carboxymethylglycine irinotecan) ; a more complete name corresponds to "pentaerythritolyl-(PEG- 1-methylene-2-oxo-(vinylamino acetate linked to irinotecan)) with 4 arms". Basic amino and/or nitrogen groups in the active agent portion of the conjugate are shown above only in neutral form, with the understanding that the conjugate has the characteristics of a hydrohalide (HX) salt, as described in detail here. As can be seen from the above structure, the carboxymethyl-modified 4-armed pentaerythritolyl PEG reagent has an intervening glycine linker between the polymer portion and the active agent, irinotecan.
[0209] Typically, although not necessarily, the number of polymeric arms will correspond to the number of active agent molecules covalently bonded to the core of the water-soluble polymer. That is to say, in the case of a polymer reactant having a certain number of polymeric arms (eg corresponding to the variable "q"), each having a reactive functional group (eg carboxy, activated ester, such as ester of succinimidyl, benzotriazolyl carbonate, and so on) at its terminus, the optimized number of active agents (such as irinotecan) that can be covalently linked to that in the corresponding conjugate is most desirably "q" . That is to say, the optimized conjugate is considered to have a drug loading value of 1.00(q) (or 100%). In a preferred embodiment, the multi-arm polymer conjugate is characterized by a degree of drug loading of 0.90(q) (or 90%) or greater. Preferred drug loadings satisfy one or more of the following: 0.92(q) or greater; 0.93(q) or greater; 0.94(q) or greater; 0.95(q) or greater; 0.96(q) or greater; 0.97(q) or greater; 0.98(q) or greater; and 0.99(q) or greater. It is most preferable that the drug loading of a multi-arm polymer conjugate be one hundred percent. A composition comprising a hydrohalide salt of a multi-armed water-soluble polymer conjugate may comprise a mixture of molecular conjugates having an active agent attached to the polymer core, having two active agent molecules attached to the polymer core, having three active agents attached to the polymer core, and so on, up to and including a conjugate having "q" active agents attached to the polymer core. The resulting composition will have an overall drug loading value, averaged over the conjugate species contained in the composition. Ideally, the composition will comprise a majority (for example, more than 50%, but more preferably more than 60%, even more preferably more than 70%, even more preferably more than 80%, and most preferably more than 90 %) of polymer conjugates with full drug charge (ie, having "q" active agent molecules for "q" arms, a single active agent molecule for each arm).
[0210] As an illustration, in a case where the multi-armed polymer conjugate contains four polymeric arms, the idealized value of the number of drug molecules covalently linked by multi-armed polymer is four, and - for to describing the average in the context of a composition of such conjugates - there will be a value (i.e., percentage) of drug molecules loaded in the multiarm polymer ranging from about 90% to about 100% of the idealized value. That is to say, the average number of drug molecules covalently bonded to a given four-armed polymer (as part of a four-armed polymer composition) is typically 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, and 100% of the full charge value. This corresponds to an average number of D per multiarm polymer conjugate ranging from about 3.60 to 4.0.
[0211] In yet another embodiment, for a multi-armed polymer conjugate composition, for example, wherein the number of polymeric arms ranges from about 3 to about 8, the majority (e.g., more than 50 %, but more preferably more than 60%, even more preferably more than 70%, even more preferably more than 80% and most preferably more than 90%) species present in the composition are those which have an idealized number of drug molecules attached to the polymer core ("q") or those that have a combination of ("q") and ("q-1") drug molecules attached to the polymer core.
[0212] As described above, the hydrohalide salt (and compositions containing it) may comprise any one or more of the following structures, in addition to the fully charged drug structure (e.g., having an irinotecan molecule modified with glycine covalently bonded to each of the four polymeric arms):


For a given polymeric arm terminus shown above having a carboxylic acid (and therefore non-covalently linked to an active agent, e.g. irinotecan), other possible terminus extending from the PEG-CM arm ("CM" = -CH2C(0)-) with 4 arms include -OH, -OCH3,


[0213] For example, provided herein is a composition comprising a plurality of 4-armed pentaerythritol-tetrapolyethylene glycol-carboxymethyl conjugates, wherein at least 90% of the conjugates present in composition (i) have a structure encompassed by the formula:C- [CHz-O-(CH2CH20) n-CH2-C(0)-TERM] 4, wherein n, in each case, is an integer having a value from 20 to about 500, or from 40 to about 500, and TERM, in each case, is selected from the group that

and -NH-CH 2 -C(0)-O-Irino ("GLY-Irino"), wherein Irino is an irinotecan residue; and (ii) for each Term in at least 90% of the conjugates with four arms present in the composition, at least 90% of these are -NH-CH2-C(O)-O-Irino, and (iii) of the at least 90% of the -NH-CH2-C(O)-O-Irinotecan fractions present in the composition, at least 90 mole percent of the basic nitrogen atoms of irinotecan are protonated in hydrohalide form, as the hydrochloride salt. Preferably, of the at least 90% of -NH-CH 2 -C(O)-O-Irinotecan fractions present in the composition, at least 91 mole percent, or at least 92 mole percent, or at least 93 mole percent, or at least 94 mole percent or at least 95 mole percent or more than 95 mole percent of the basic nitrogen atoms of irinotecan are protonated in hydrohalide form, the hydrohalide content of which can be determined by means of ion chromatography.
[0214] The multi-armed polymer conjugate compositions provided herein are intended to encompass any and all stereoisomeric forms of the conjugates comprised in such compositions. In a particular embodiment of the conjugate, the C-20 stoichiometry of irinotecan, when in conjugated form, as in 4-armed PEG-Gly-Irino compositions, remains intact, i.e., C-20 retains its configuration ( S) when it is in its conjugated form. See, for example, Example 4.
[0215] A preferred multi-arm structure is a carboxymethyl-modified 4-arm pentaerythritolyl PEG having a glycine linker intervening between the polymer portion on each arm and the active agent (polymer portion and linker shown above), wherein the agent active is 7-ethyl-10-hydroxy-camptothecin. Again, included herein are embodiments in which the multiarm polymer is (i) fully charged, as well as (ii) three molecules of 7-ethyl-10-hydroxy-camptothecin covalently attached thereto, (iii) two 7-ethyl-10-hydroxy-camptothecin molecules covalently bonded thereto, and (iv) one 7-ethyl-10-hydroxy-camptothecin molecule covalently bonded to the core of the four-armed polymer. Typical drug loadings are as previously described.
[0216] Yet another representative multi-arm conjugate structure is a dimer of glycerol (3,3'-oxydipropane-1,2-diol) carboxymethyl-modified 4-armed PEG having 7-ethyl-10-hydroxy-camptothecin molecules (SN-38) covalently bonded to the polymer core. Included herein are embodiments in which the core of the multi-arm polymer is either fully loaded with drug (i.e., having four molecules of 7-ethyl-10-hydroxy-camptothecin covalently attached thereto), or has less charge than total (i.e., having one, two, or three molecules of 7-ethyl-10-hydroxy-camptothecin covalently attached thereto). Shown below is the conjugate having drug (i.e., 7-ethyl-10-hydroxy-camptothecin) covalently attached to each polymeric arm.

[0217] In yet another illustrative embodiment, the conjugate is a multi-armed structure comprising a dimer of glycerol (3,3'-oxydipropane-1,2-diol) carboxymethyl-modified 4-armed PEG having attached irinotecan molecules covalently to the polymer core. Included herein are embodiments in which the multi-arm polymer core is either fully charged with drug (i.e., having four molecules of irinotecan covalently attached thereto), or is less than fully charged (i.e., having one, two, or three molecules of irinotecan covalently bonded thereto). Parameters of Hydrohalide Salts
[0218] The compositions in question are hydrohalide salts, typically hydrochloride salts. That is to say, conjugates such as those described above are provided in a composition such that at least 90% of the basic nitrogen atoms present in the conjugate (as well as in the volume of the composition) are present in a protonated form (ie, in the form of the hydrohalide salt). The hydrohalide salt compositions are stably and reproducibly prepared,
[0219] A hydrohalide salt as provided herein is characterized in terms of its bulk properties or macro properties. That is to say, basic nitrogen atoms (ie, amino groups) present in the conjugate exist in an almost totally protonated form. Although the present compositions are characterized based on bulk properties, different individual molecular species are typically contained within the bulk of the composition. Taking the exemplary 4-armed polymer conjugate described in Example 6, 4-armed PEG-Gly-Irino-20K hydrochloride, the salt product contains any of a few individual molecular species, although at least 90% of the total are protonated in the form of the hydrohalide salt. A molecular species is such that each polymeric arm contains an irinotecan molecule that is in neutral form, that is, its amino group is not protonated. See structure I below. Another molecular species is such that each polymeric arm contains an irinotecan molecule in protonated form. See structure IV below. An additional molecular species is such that three of the polymeric arms contain an irinotecan molecule which is in protonated form, and one polymeric arm contains an irinotecan molecule in neutral form (structure III). In another molecular species, two of the four polymeric arms contain an irinotecan molecule in neutral form (ie, its amino group is not protonated), and two of the four polymeric arms contain an irinotecan molecule that is in protonated form (structure II) .

[0220] As demonstrated in Example 1, certain polymer and prodrug conjugates can be obtained in the form of mixed acid salts of hydrochloric acid and trifluoroacetic acid. In Example 1, hydrochloric acid is introduced using an acid salt form of the active agent molecule to form the resulting polymer conjugate, while trifluoroacetic acid is introduced into the reaction mixture in a deprotection step. After covalently binding the active agent (or modified active agent, as illustrated in Example 1) to the water-soluble polymer reagent, and base treatment, even in cases where additional purification steps are performed, the resulting conjugate is obtained in form of a partial salt of mixed acids.
[0221] Conjugates of mixed acid salts generally contain defined proportions and ranges of each component (ie, free base, inorganic acid salt, organic acid salt). A positive correlation was observed between increased stability to hydrolysis and increased molar percentage of salt in the final conjugated product. Based on the slopes of the graphs it can be determined that as the free base content increases, the stability of the product decreases.
[0222] FIG. 2 further illustrates that stability (or resistance) against hydrolytic degradation is greater for conjugates having a higher degree of protonated amine groups (i.e., acid salt). For example, it was observed that conjugated product containing 14 mole percent or more free base was remarkably less stable by hydrolysis than the corresponding product rich in acid salt.
[0223] Additionally, as illustrated in FIG. 3, the hydrochloride salt-rich product appears to be somewhat more susceptible to poly(ethylene glycol) backbone cleavage under accelerated stress conditions than the mixed salt form containing a measurable amount of free base, despite the buffering of the final composition may be effective in improving this characteristic or trend.
[0224] These collective results indicate the unexpected advantages of a hydrohalide salt of a poly(ethylene glycol)-active agent conjugate (such as 4-armed PEG-Gly-Irino-20K) over the isolated free base. Hydrohalide Salts - Formation Methods
[0225] By formation and characterization of the mixed acid salt, a method was devised to synthesize a fully hydrohalide salt, that is, a salt having at least 90% of the basic nitrogen atoms of irinotecan protonated as a hydrohalide salt, as provided. in detail in Example 6. An acid salt of a water-soluble polymer conjugate can be prepared from commercially available starting materials considering the guidelines presented here, coupled with knowledge in the field of chemical synthesis.
[0226] Linear, branched, and multi-armed water-soluble polymer reagents are available from some commercial sources, as described above. Alternatively, PEG reagents, such as a multi-arm reactive PEG polymer, can be prepared synthetically as described herein. See, for example, Example 7 presented here.
[0227] The acid salt can be formed using known chemical coupling techniques for the covalent attachment of activated polymers, such as an activated PEG, to a biologically active agent (see, for example, " POLY(ETHYLENE GLYCOL) CHEMISTRY AND BIOLOGICAL APPLICATIONS ", American Chemical Society, Washington, DC (1997); and US Patent Publication Nos. 2009/0074704 and 2006/0239960). The selection of suitable functional groups, linkers and protective groups and the like to obtain a mixed acid salt according to the invention will depend, in part, on the functional groups present in the active agent and the polymer starting material and will be clear to the practitioner , based on the content of this disclosure. Considering certain characteristics of the acid salt, the method comprises the provision of an active agent containing amine (or other basic nitrogens) in the form of an inorganic acid addition salt, and a treatment step with inorganic acid. Reference to an "active agent" in the context of the synthetic method is intended to encompass an active agent optionally modified to have a linker covalently attached thereto for the purpose of facilitating binding to the water-soluble polymer.
[0228] In general, the method comprises the steps of (i) deprotecting an inorganic acid salt (hydrohalic) of an active agent containing amine (or other basic nitrogens) in protected form (e.g., glycine-irinotecan hydrohalide in form protected) by treatment with a molar excess of hydrohalic acid, thereby removing the protecting group to form a deprotected acid salt, such as glycine-irinotecan hydrohalide, (ii) coupling the deprotected inorganic acid salt from step (i) to a water-soluble polymer reagent, such as 4-armed pentaerythritol-polyethylene glycol-carboxymethyl-succinimide (or an equivalent chemically activated ester or the like), in the presence of a base to form a polymer-active agent conjugate, such as the hydrohalide salt of 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan (also called hydrohalide salt of pentaerythritolyl-(PEG-1-methylene-2-oxo-(vinylamino acetate linked to Irinotecan)) with 4 arms), and (iii) recovering the polymer-active agent conjugate, pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrohalide salt. with 4 arms, by means of precipitation.
[0229] The resulting polymer-active agent conjugate composition is characterized by having at least 90 mole percent of the conjugate active agent's basic amino groups, eg, irinotecan's basic amino groups, protonated as a hydrohalide salt. In general, the molar percentage of hydrohalide groups in the product is determined by means of ion chromatography.
[0230] Turning now to one of the preferred classes of active agents, camptothecins, since the hydroxyl group at position 20 of compounds of the camptothecin family is sterically hindered, it is difficult to carry out a one-step conjugation reaction with significant yields . As a result, a preferred method is to react the hydroxyl group at position 20 of the bioactive starting material, for example, irinotecan hydrochloride, with a small linker or spacer moiety containing a functional group suitable for reaction with a water-soluble polymer. This approach is applicable to many small molecules, in particular those that have a covalent binding site that is inaccessible to an incoming reactive polymer. Preferred linkers for reacting with a hydroxyl group to form an ester bond include t-BOC-glycine or other amino acids, such as alanine, glycine, isoleucine, leucine, phenylalanine, and valine having a protected amino group and an available carboxylic acid group (see Zalipsky et al., "Attachment of Drugs to Polyethylene Glycols", Eur. Polym.J., Volume 19, No. 12, pages 1177-1183 (1983)). Other spacer or linker moieties having an available carboxylic acid group, or another functional group reactive with a hydroxyl group, and having a protected amino group, can also be used in place of the amino acids described above.
[0231] Typical labile protecting groups, for example to protect the amino group of glycine, include t-BOC and FMOC (9-flourenylmethyloxycarbonyl). The t-BOC is stable at room temperature and is easily removed with dilute solutions of trifluoroacetic acid and dichloromethane. It can also be removed by treatment with an acid, such as an inorganic, hydrohalic acid. FMOC is a base-labile protecting group that is easily removed by concentrated solutions of amines (usually 20-55% piperidine in N-methylpyrrolidone).
[0232] In Example 6, directed to the preparation of 4-armed PEG20K-irinotecan hydrochloride, the carboxyl group of N-protected glycine reacts with the hydroxyl group at position 20 of irinotecan hydrochloride (or other suitable camptothecin such as 7-ethyl -10-hydroxy-camptothecin, or any other active agent) in the presence of a coupling agent (eg, dicyclohexylcarbodiimide (DCC)) and a base catalyst (eg, dimethylaminopyridine (DMAP) or other suitable base) to provide the agent active modified with the N-protected linker, eg t-Boc-glycine-irinotecan hydrochloride. Although hydrochloride is exemplified, other inorganic acid salts can be used. Preferably, each reaction step is conducted under an inert, dry atmosphere.
[0233] In a subsequent step, the amino protecting group, t-BOC (N-tert-butoxycarbonyl), is removed by treatment with hydrochloric acid (or other hydrohalic acid) under suitable reaction conditions. This differs from the mixed acid salt preparation, in which t-BOC is removed by treatment with trifluoroacetic acid as in Example 1. The resulting deprotected intermediate is active agent modified with linker, eg 20-glycine hydrochloride -irinotecan. Illustrative reaction conditions are described in Example 6 and can be further optimized by the practitioner through routine optimization. Usually a molar excess of acid is used to remove the protecting group. Preferably, protected glycine-irinotecan is treated with a ten-fold or more molar excess of hydrohalic acid to remove the protecting group. In some cases a 10-fold to 25-fold molar excess may be employed. The resulting deprotected drug salt is typically recovered from the reaction mixture, for example, by precipitation. As an example, the addition of methyl tert-butyl ether (MTBE) may be employed to precipitate the intermediate. The intermediate product is then isolated, for example by filtration, and dried.
[0234] The deprotected active agent (optionally modified with linker), eg 20-glycine-irinotecan HCl, is then coupled to a desired polymer reagent, eg, 4-armed pentaerythritol-PEG-succinimide (or any other corresponding of similarly activated ester, the nature of which was previously described) in the presence of a base (eg, DMAP, trimethyl amine, triethyl amine, etc.) to form the desired conjugate. The conjugation step can be conducted in the presence of excess base, for example, from about 1.1 to about 3.0 fold molar excess. Reaction yields from the coupling reaction are typically high, greater than about 90%.
[0235] The acid salt conjugate is recovered, for example, by precipitation with ether (eg, methyl tert-butyl ether, diethyl ether) or other suitable solvent. To ensure formation of the fully hydrohalide salt (ie at least 90 mole percent hydrohalide salt), the crude product is analyzed, for example, by ion chromatography, to determine the halide content. In case the halide content is less than desired, for example less than 90 mole percent, or less than 91, 92, 93, 94, or 95 mole percent, the acid salt of the conjugate is then dissolved in a suitable solvent, such as ethyl acetate or the like, and treated with additional hydrohalic acid. The product is then recovered as described above.
[0236] The acid salt product can be further purified by any suitable method. Purification and isolation methods include precipitation followed by filtration and drying, as well as chromatography. Suitable chromatographic methods include gel filtration chromatography, ion exchange chromatography, and Biotage Flash chromatography. Another method of purification is recrystallization. For example, the partial acid salt is dissolved in a suitable single solvent system or mixed solvents (eg isopropanol/methanol), and then allowed to crystallize. The recrystallization can be conducted multiple times, and the crystals can also be washed with a suitable solvent in which they are insoluble or only slightly soluble (for example, methyl tert-butyl ether or methyl tert-butyl ether/methanol). The purified product can optionally be additionally air-dried or vacuum-dried.
[0237] Preferably, the acid salt product is stored under conditions suitable to protect the product from exposure to any one or more of oxygen, moisture, and light. Any of a number of storage conditions or packaging protocols can be employed to adequately protect the acid salt product during storage. In one embodiment, the product is packaged under an inert atmosphere (eg, argon or nitrogen) by placing in one or more polyethylene bags, which are placed in a heat-sealable, aluminum-coated polyester bag.
[0238] Representative molar percentages of hydrochloric acid salt are provided in Example 6. As described herein, 4-armed pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan hydrochloride salt was prepared as the all-hydrochloride salt, ie, containing nearly 99 mole percent chloride. Hydrohalide Salts - Pharmaceutical Compositions Containing Hydrohalide Salt Conjugates
[0239] The acid salt may be in the form of a pharmaceutical formulation or composition for human veterinary or clinical use. An illustrative formulation will typically comprise the acid salt in combination with one or more pharmaceutically acceptable carriers and, optionally, any other therapeutic ingredients, stabilizers, or the like. The carrier(s) must be pharmaceutically acceptable(acceptable) in the sense of being compatible(compatible) with the other ingredients of the formulation and not being unduly harmful(harmful) to the recipient/ patient. The hydrohalic acid salt is optionally contained in bulk or unit dosage form in a container or receptacle that includes packaging that protects the product from exposure to moisture and oxygen.
[0240] The pharmaceutical composition may include polymeric excipients/additives or carriers, for example, polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficoll's (a polymeric sugar), hydroxyethyl starch (HES), dextrates (eg, cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin and sulfobutylether-p-cyclodextrin), polyethylene glycols, and pectin. Compositions may also include diluents, buffers, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), flavoring agents, taste masking agents, inorganic salts (eg, sodium chloride), antimicrobial agents (eg, chloride of benzalkonium), sweeteners, antistatic agents, surfactants (eg, polysorbates, such as "TWEEN 20" and "TWEEN 80", and Pluronics, such as F68 and F88, available from BASF), sorbitan esters, lipids (eg, phospholipids , such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters, steroids (eg cholesterol)), and chelating agents (eg EDTA, zinc and other suitable cations thereof). Other pharmaceutical excipients and/or additives suitable for use in the compositions according to the invention are listed in "Remington; The Science & Practice of Pharmacy", 19th edition, Williams & Williams (1995), and in "Physician's Desk Reference", 52nd edition, Medical Economics, Montvale, NJ (1998), and in "Handbook of Pharmaceutical Excipients", Third Edition, Editor AH Kibbe, Pharmaceutical Press, 2000 .
[0241] The acid salt can be formulated into a composition suitable for oral, rectal, topical, nasal, ophthalmic, or parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular injection) administration. The acid salt composition may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of associating the acid salt with a carrier which constitutes one or more accessory ingredients.
[0242] In a particular embodiment, the acid salt, eg hydrohalide of PEG-Gly-Irino-20K with 4 arms, is provided in lyophilized form in a sterilized single-use vial for use by means of injection. In one embodiment, the amount of conjugated product contained in the single-use vial is the equivalent of a 100 mg dose of irinotecan. More particularly, the lyophilized composition includes 4-armed PEG-Gly-Irino-20K hydrohalide salt combined with lactate buffer at pH 3.5. That is to say, the lyophilized composition is prepared by combining 4-armed PEG-Gly-Irino-20K hydrohalide, for example, in an amount equivalent to a dose of 100 mg irinotecan, with approximately 90 mg of acid lactic acid, and the pH of the solution is adjusted to 3.5 by adding acid or base. The resulting solution is then lyophilized under sterile conditions, and the resulting powder is stored at -20°C before use. Prior to intravenous infusion, the lyophilized composition is combined with a dextrose solution, for example a 5% (w/w) dextrose solution.
[0243] The amount of acid salt (ie, active agent) present in the formulation will vary depending on the specific active agent employed, its activity, the molecular weight of the conjugate, and other factors such as dosage form, target patient population , and other considerations, and in general will be easily determined by the professional. The amount of conjugate present in the formulation will be that amount necessary to deliver a therapeutically effective amount of the compound, for example, an alkaloid anticancer agent such as irinotecan or SN-38, to a patient in need of achieving at least one of the therapeutic effects associated with the compound. , for example, for the treatment of cancer. In practice, this will vary greatly depending on the particular conjugate, its activity, the severity of the condition being treated, the patient population, the stability of the formulation, and the like. Compositions will generally contain any amount from about 1% by weight to about 99% by weight of conjugate, typically from about 2% to about 95% by weight of conjugate, and more typically from about 5% to 85% by weight of conjugate, and will also depend on the relative amounts of excipients/additives contained in the composition. More specifically, the composition will typically contain at least about one of the following percentages of conjugate-. 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, or more by weight.
[0244] Compositions suitable for oral administration may be provided in the form of discrete units such as capsules, wafer capsules, tablets, lozenges, and the like, each containing a predetermined amount of the conjugate in the form of a powder or granules; or a suspension in an aqueous liquor or non-aqueous liquid, such as a syrup, an elixir, an emulsion, a drink preparation, and the like.
Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the prodrug conjugate, which may be formulated so as to be isotonic with the blood of the recipient.
[0246] Nasal spray formulations comprise purified aqueous solutions of the multi-arm polymer conjugate with preservative agents and isotonic agents. Preferably, these formulations are adjusted to a pH and isotonic state compatible with the nasal mucous membranes.
[0247] Formulations for rectal administration may be presented in the form of a suppository with a suitable carrier such as cocoa butter, or hydrogenated fats or hydrogenated fatty carboxylic acids.
[0248] Ophthalmic formulations are prepared by a method similar to nasal spray, with the exception that the pH and isotonic factors are preferably adjusted to match those of the eye.
[0249] Topical formulations comprise the multi-arm polymer conjugate dissolved or suspended in one or more media such as mineral oil, petroleum, polyhydroxy alcohols or other bases used for topical formulations. The addition of other accessory ingredients may be desirable, as noted above.
[0250] Pharmaceutical formulations are also provided that are suitable for administration in the form of an aerosol, for example, by inhalation. Such formulations comprise a solution or suspension of the desired multiarm polymer conjugate, or salt thereof. The desired formulation can be placed in a small, nebulized chamber. Nebulization can be achieved by means of compressed air or by means of ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the conjugates or salts thereof.Hydrohalide Salts - Methods of Using Hydrohalide Salt Conjugates
[0251] The acid salt described herein can be used to treat or prevent any condition that responds to the unmodified active agent in any animal, particularly in mammals, including humans. A representative acid salt, 4-armed pentaerythritol-PEG-glycine-irinotecan hydrochloride, comprising the anticancer agent irinotecan, is particularly useful in the treatment of various types of cancer.
[0252] The acid salt conjugate, in particular those in which the small molecule drug is an anticancer agent, such as a camptothecin compound as described herein (eg irinotecan or 7-ethyl-10-hydroxy-camptothecin) or other oncolytic, is useful in treating solid-type tumors such as breast cancer, ovarian cancer, colon cancer, gastric cancer, malignant melanoma, small cell lung cancer, non-small cell lung cancer, thyroid cancers, kidney cancer, bile duct cancer, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease, adrenocortical cancer, and the like. Additional cancers that can be treated with the acid salt include lymphomas, leukemias, rhabdomyosarcoma, neuroblastoma, and the like. As stated above, the conjugate in question is particularly effective in targeting and accumulating in solid tumors. The conjugate is also useful in treating HIV and other viruses.
[0253] It has also been shown that representative conjugates, such as 4-arm pentaerythritol-PEG-glycine-irinotecan, are particularly advantageous when used to treat patients with cancers found to be refractory to treatment with one or more anticancer agents.
The methods of treatment comprise administering, to a mammal in need thereof, a therapeutically effective amount of an acid salt composition or formulation as described herein.
[0255] Additional methods include the treatment of (i) metastatic breast cancer that is resistant to anthracycline and/or taxane based therapies, (ii) platinum resistant ovarian cancer, (iii) metastatic cervical cancer, and (iv) colorectal cancer in patients with a mutated K-Ras gene status by administering an acid salt composition as described herein.
[0256] In the treatment of metastatic breast cancer, an acid salt of a conjugate such as pentaerythritol-PEG-glycine-irinotecan with 4 arms, as provided herein, is administered to a patient with locally advanced metastatic breast cancer in an amount therapeutically effective, in which the patient had undergone no more than two previous (unsuccessful) treatments with anthracycline and/or taxane-based chemotherapeutics.
[0257] For the treatment of platinum-resistant ovarian cancer, a composition as provided herein is administered to a patient with locally advanced or metastatic ovarian cancer in a therapeutically effective amount, in which the patient had exhibited tumor progression during based therapy in platinum, with a progression-free interval of less than six months.
[0258] In yet another approach, a hydrohalic acid salt (eg, as in Example 6) is administered to a subject with locally advanced colorectal cancer, where the colorectal (colorectal) tumor(s) has ( have) a mutation in the K-Ras oncogene (mutant types of K-Ras) so that the tumor does not respond to EGFR inhibitors such as cetuximab. Subjects are those who have failed prior 5-FU-containing therapy, and who have also never received irinotecan.
[0259] A therapeutically effective dosage amount of any specific acid salt will vary from conjugate to conjugate, from patient to patient, and will depend on factors such as the patient's condition, the activity of the particular active agent employed, the type of cancer , and the route of distribution.
[0260] For active agents of the camptothecin type, such as irinotecan or 7-ethyl-10-hydroxy-camptothecin, dosages from about 0.5 to about 100 mg camptothecin/kg body weight are preferred, preferably from about 10.0 to about 60 mg/kg. When administered in conjunction with other pharmaceutically active agents, even a minor amount of the acid salt can be therapeutically effective. For administration of an acid salt of irinotecan as exemplified herein, the dosage amount of irinotecan will typically range from about 50 mg/m2 to about 350 mg/m2.
[0261] Methods of treatment also include administering a therapeutically effective amount of an acid salt composition or formulation as described herein (for example, where the active agent is a camptothecin-like molecule) in conjunction with a second anticancer agent . Preferably, such camptothecin-based conjugates in the form of an acid salt are administered in combination with 5-fluorouracil and folinic acid, as described in U.S. Patent No. 6,403,569.
[0262] The hydrohalic acid salt compositions can be administered once or several times a day, preferably once a day or less. The duration of treatment can be once a day for a period of two to three weeks and can continue for a period of months or even years. The daily dose may be administered by way of a single dose, in the form of an individual dosage unit, or several smaller dosage units, or by multiple administration of subdivided dosages at determined intervals.
[0263] It is to be understood that, although the invention has been described in conjunction with its specific preferred embodiments, the foregoing description as well as the following examples are intended to illustrate, not limit, the scope of the invention. Other aspects, advantages and modifications in the scope of the invention will be clear to professionals in the field to which the invention belongs. EXPERIMENTAL
[0264] The practice of the invention will employ, unless otherwise indicated, conventional techniques of organic synthesis and the like, which are within the scope of the field. These techniques are fully described in the literature. Reagents and materials are commercially available unless specifically stated otherwise. See, for example, MB Smith and J. March, March's Advanced Organic Chemistry: Reactions Mechanisms and Structure, 6th Edition (New York.-Wiley-Interscience, 2007), supra, and "Comprehensive Organic Functional Group Transformations II", Volumes 1 -7, Second Edition: "A Comprehensive Review of the Synthetic Literature 1995-2003" (Organic Chemistry Series), Editors Katritsky, AR, et al., ElsevierScience.
[0265] In the following examples, efforts have been made to ensure accuracy with respect to the numbers used (eg, amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, temperature is in degrees C and pressure is atmospheric pressure at or near sea level.
[0266] The following examples illustrate certain aspects and advantages of the present invention; however, it should in no way be considered that the present invention is limited to the particular embodiments described below.
[0267] ABBREVIATIONS
[0268] Arg argon
[0269] CM carboxymethyl or carboxymethylene (-CH2COOH)
[0270] DCC 1,3-dicyclohexylcarbodiimide
[0271] DCM dichloromethane
[0272] DMAP 4-(N,N-dimethylamino)pyridine
[0273] GLY glycine
[0274] hydrochloric acid HCl
[0275] RP-HPLC reverse phase high performance liquid chromatography
[0276] IPA isopropyl alcohol
[0277] IRT irinotecan
[0278] IPC ion pair chromatography
[0279] MeOH methanol
[0280] MTBE methyl tert-butyl ether
[0281] MW molecular weight
[0282] NMR nuclear magnetic resonance
[0283] PEG polyethylene glycol
[02841 AT room temperature
[0285] SCM succinimidylcarboxymethyl (-CH2- COO-N-succinimidyl)
[0286] TEA triethylamine
[0287] TFA trifluoroacetic acid
[0288] THF tetrahydrofuran
[0289] Materials and Methods
[0290] 4-ARM PEG2OK“OH based on pentaerythritol was obtained from NOF Corporation (Japan), 4 ARM PEG2OK-OH has the structure: C-(CH2O-(CH2CH2O) NH) 4, where each n is about 113 .
[0291] Additional vendors of pentaerythryl-based 4-arm PEG20K-OH (also called simply 4-arm PEG-OH) include Creative PEGWorks (Winston-Salem, NC), which also offers the succinimidyl-functionalized version, and JenKem Technology USA (Allen, Texas).
[0292] All LHRMN data were generated using an NMR spectrometer at 30 0 or 400 MHz manufactured by Bruker.EXAMPLE 1MIXED TRIFLUOROACETIC ACID SALT PREPARATION. VINYLAMINE ACETATE LINKED TO IRINOTECAN))-2OK WITH 4 ARMS "PEG-GLY-IRINO-20K WITH 4 ARMS"
[0293] Reaction Scheme


[0294] This example describes the synthesis of a mixed salt of TFA.HC1 acids of PEG-Gly-Irino-20K with 4 Arms.
[0295] All solvents used in the synthesis were anhydrous. Step 1. Conjugation of t-boc-glycine to Irinotecan*HCl salt (yield > 95%)
[0296] Irinotecan • HCl • trihydrate (1 mole or 677 g) and DMF (10 L) were charged to a distiller at 60°C. After dissolving the irinotecan-HC1•trihydrate in DMF, full vacuum was slowly applied to remove water from the irinotecan■HC1•trihydrate by azeotropic distillation at 60°C. After solids had formed from residual DMF, heptane (up to 60 L) was charged to the retort to remove residual DMF at 40 - 50°C. After removal of heptane, determined by observation, the azeotropic distillation was terminated and the solid (irinotecan-HC1) was allowed to cool to 17 ± 2°C. For the coupling reaction, t-boc-glycine (1.2 moles), 4-DMAP (0.1 moles) dissolved in DCM (1 L), and DCM (19 L) were loaded into the still. After the mixture was visually well dispersed, molten DCC (1.5 moles) was added and the reaction was allowed to proceed. The reaction was carried out under a blanket of argon or nitrogen, with sufficient mixing and vessel temperature at 17 ± 2°C.
[0297] After a reaction time of 2-4 hours, a sample was removed to measure the percentage of residual irinotecan peak area (IRT) by means of chromatography. It was determined that residual irinotecan was present in an amount not greater than 5%. DCU formed during the coupling reaction was removed by filtration, and washed with DCM. The resulting filtrates containing crude t-boc-glycine-irinotecan-HCl salt were combined and concentrated at less than 45°C under vacuum to remove DCM. When approximately 75% of its initial volume had been removed via distillation, IPA was then added to the concentrate to reach the initial volume, and the mixture was further distilled until the volume of condensates reached about 25% of its initial volume. The resulting clear solution was cooled to room temperature, followed by its addition to heptane with simultaneous mixing. The mixture was mixed for an additional period of 0.5 to 1 hour, during which time a precipitate formed. The precipitate was drained and filtered to a wet cake, then washed with heptane (up to 6 L). The wet cake was dried under vacuum to give t-boc-glycine-irinotecan powder for use in Step 2, Yield > 95%, Step 2. Deprotection of t-boc-glycine-irinotecan
[0298] The t-boc-glycine-irinotecan (1 mole) from Step 1 was dissolved in DCM with stirring to form a visually homogeneous solution. To this solution was added TFA (15.8 moles) over a period of 5 to 10 minutes, and the resulting solution was stirred for about 2 hours. Residual starting material was measured by RP-HPLC and was determined to be less than about 5%. Then acetonitrile was added to the reaction solution to form a visually homogeneous solution at RT. This solution was then added to MTBE (46.8 kg) with sufficient stirring at 35 °C to promote crystallization. Optionally to reduce the use of MTBE, DCM in the reaction solution was replaced with acetonitrile by distillation at 15 to 40°C. After solvent exchange, the product-containing solution was added to approximately 50% less volume of MTBE (23 kg) with sufficient stirring at the crystallization temperature (35°C). Mixing was continued for half to an hour. The resulting solid was filtered and the cake was washed with MTBE.
[0299] The wet cake was dried under vacuum, giving rise to the powdered glycine-irinotecan salt for use in Step 3. The trifluoroacetate and chloride content of the product was determined by ion chromatography with a conductivity detector. (Yield > 95%). Step 3. PEGylation of Glycine-irinotecan using 4-arm PEG-CM-SCM
[0300] The powdered glycine-irinotecan•TFA/HC1 salt from Step 2 was added to a reactor to which DCM had been added (approximately 23 L). The mixture was stirred for approximately 10 to 30 minutes to allow the glycine-irinotecan•TFA/HC1 salt to disperse in DCM. Triethyl amine (approximately 1.05 moles (HCl + TFA) moles in glycine-irinotecan salt TFA/HCl powder) was then added slowly, at a rate that kept the pot temperature at 24°C or less. The resulting mixture was stirred for 10 to 30 minutes to allow the free base of GLY-IRT (glycine-modified irinotecan) to dissolve.
[0301] Approximately 80% of the total amount (6.4 kg) of PEG-SCM-20kD with 4 arms was added to the reactor for a period of up to 30 minutes. After dissolution of the PEG reagent, the reaction progress was monitored by IPC. (In case the amount of unconjugated GLY-IRT was greater than 5% when the reaction appeared to plateau, the remaining 20% of 4-armed PEG SCM was then added to the reactor, and the progression of the reaction was monitored until a constant unreacted GLY-IRT value is observed.)
[0302] The crude product was precipitated by adding the reaction solution to MTBE (113.6 L) with stirring at room temperature for a period of 1 - 1.5 hours, followed by stirring. The resulting mixture was transferred to a filter drier with an agitator to remove the mother liquor. The precipitate (crude product) was partially dried under vacuum at approximately 10 to 25°C with minimal intermittent stirring.
[0303] The crude product was then placed in a reactor, to which IPA (72 L) and MeOH (8 L) were added, followed by stirring for a period of up to 30 minutes. Heat was applied to achieve visually complete dissolution (a clear solution) at the vessel temperature of 50°C, followed by stirring for 30 to 60 minutes. The solution was then cooled to 37°C, held at that temperature for several hours, followed by cooling to 20°C. The mixture was transferred to a stirred filter drier, and filtered to remove the mother liquor, giving a cake on a filter. The cake was washed with 70% MTBE in IPA and 30% MeOH and was partially dried under vacuum. This procedure was repeated two more times, with the exception that, prior to cooling, the clear IPA/MeOH solution containing PEG-Gly-IRT with 4 arms was filtered using a built-in filter (1 µm) at 50°C to remove any potential particulates in the last (3rd) crystallization.
[0304] Three representative samples were taken from the wet washed cake, and NHS levels were measured using NMR. The wet cake was dried under vacuum.
[0305] The product ("API") was packaged in double-sealed bags under an inert atmosphere, and was stored at -20°C without exposure to light. Product yield was approximately 95%.EXAMPLE 2 PRODUCT CHARACTERIZATION OF "PEG-GLY-IRINO-2 0K WITH 4 ARMS" FROM EXAMPLE 1 AS A MIXED SALT
[0306] The product of Example 1 was analyzed by ion chromatography (IC analysis). See Table 1 in
Based on the IC results provided in Table 1, it can be seen that the product formed in Example 1, PEG-Gly-Irino-20K with 4 arms, is a partial mixed salt of approximately 50 mole percent TFA salt, 30 mole percent HCl salt, and 20 mole percent free base, based on the conjugated irinotecan molecules in the product. Mixture of salts was observed even after repeated recrystallizations (1-3) of the product. In the various product lots analyzed above, it can be seen that about 35 - 65 mole percent of the irinotecan molecules present in the composition are protonated as the TFA salt, about 25-40 mole percent of the irinotecan molecules present in the composition are protonated in the form of the HCl salt, whereas the remaining 5-35 mole percent of the irinotecan is unprotonated (i.e., in the form of the free base).
[0307] The generalized structure of the product is shown below, where the irinotecan fractions are presented in free base form, and in association with HC1 and TFA - as an indication of the mixed salt nature of the product.
EXAMPLE 3 STABILITY STUDIES UNDER PEG-GLY-IRINO-20K STRESS WITH 4 ARMS
[0308] Accelerated stability studies were conducted in an attempt to evaluate the composition of the 4-arm PEG-Gly-Irino-20K product. Compositions containing varying amounts of protonated irinotecan as well as differing in the amount of TFA versus HCl salt were examined. Stress Stability Studies
[0309] The product formed in Example 1, PEG-Gly-Irino-20K with 4 arms, compound 5 (approximately 1-2 g) was weighed into PEG PE 'whirl top1' bags and placed in another 'whirl top' bag for simulate API marshaling conditions. In one study (results shown in FIG. 1), samples were placed in an environmental chamber at 25°C/60% Relative Humidity for 4 weeks. In another study, samples were placed in an environmental chamber at 40°C/75% Relative Humidity for a period of up to several months (results shown in FIG. 2 and FIG. 3). Samples were collected and analyzed on a periodic basis throughout the studies.
[0310] The results of the studies are presented in FIG. 1, FIG. 2 and FIG. 3. In FIG. 1, 4-arm PEG-Gly-Irino~20K peak area percentages for samples stored at 25°C and 60% relative humidity are plotted against time. Data shown are from samples consisting of >99% HCl salt (<1% free base, triangles), 94% total salt (6% free base, squares), and 52% total salt (48 % free base, circles). The slopes of the graphs indicate that as the free base content increases, the stability of the product decreases. Under the stress conditions employed (ie, 25°C for a period of up to 28 days), the fall in the peak area of PEG-Gly-Irino-20K with 4 arms correlated well with the increase in free irinotecan, indicating that the Decomposition mode is mainly via hydrolysis of the ester bond to release irinotecan. Based on the observed results, it appears that a greater amount of free base in the product leads to decreased stability to hydrolysis. Thus, a product containing a higher degree of protonated irinotecan appears to have greater stability against hydrolysis than a product containing less protonated irinotecan (on a mole percent basis).
[0311] FIG. 2 and FIG. 3 show another set of data obtained from a sample containing >99% HCl salt (<1% free base, squares) and a sample consisting of 86% total salts (14% free base, diamonds) that were stored at 40°C and 75% relative humidity. FIG. 2 shows the increase in free irinotecan over 3 months for both samples. These data are consistent with data from the previously described study (summarized in FIG. 1), which shows that a product with a higher content of free base is less stable with respect to hydrolysis. FIG. 3 shows the increase in smaller PEG species for the same samples over 3 months. The increase in smaller PEG species is indicative of decomposition of the PEG backbone, providing multiple PEG species. The data indicate that the corresponding HCl salt product is more prone to PEG backbone decomposition than the mixed salt sample containing 14% free base under accelerated stability conditions. Thus, not intending to be bound by theory, it appears that while the partial mixed salt can degrade mainly through hydrolytic drug release, the hydrochloride salt appears to degrade through a different mechanism, ie, main chain degradation of the polymer. However, the extent of main-chain degradation can be minimized, for example, by controlling storage conditions.
[0312] In summary, the two observed decomposition modes exhibit opposite trends regarding the salt/free base content. Although the hydrochloride salt demonstrated a higher degree of main-chain degradation under accelerated stability tests (possibly due to the acidity of the formulation), the hydrochloride salt has been shown to have greater hydrolytic stability than the free base or mixed salt of TFA.hydrochloride.EXAMPLE 4CHIRALITY STUDY
[0313] The chirality of carbon-20 doirinotecan in PEG-Gly-Irino-20K with 4 arms was determined.
[0314] As detailed in vendor documentation, the irinotecan hydrochloride starting material is optically active, with C-20 in its (S) configuration. The C-20 position of irinotecan contains a tertiary alcohol, which is not easily ionizable, hence it is not expected that this site will undergo racemization except under extreme (strongly acidic) conditions. To confirm the chirality at C-20 of the 4-arm PEG-Gly-Irino-2 0K, a chiral HPLC method was used to analyze the irinotecan released from the product via chemical hydrolysis.
[0315] Based on the resulting chromatograms, no (R)-enantiomer was detected for the 4-armed PEG-Gly-Irino-20K samples. After hydrolysis it was confirmed that the irinotecan released from the conjugate had the (S) configuration. EXAMPLE 5 HYDROLYSIS STUDY
[0316] All PEGylated irinotecan species are considered part of 4-armed PEG-Gly-Irino-20K (regardless of the particular form - free base, TFA.chloride mixed salt, or chloride salt); each species undergoes clean hydrolysis to produce irinotecan with >99% purity. In addition, the main species DS4 with full drug charge (irinotecan covalently bonded to each of the four polymeric arms) and the partially substituted species - species DS3 (irinotecan covalently bonded to three polymeric arms), DS2 (irinotecan bonded covalently to two of the polymeric arms) and DS1 (irinotecan covalently bonded to a single polymeric arm) - all hydrolyze at the same rate releasing the free drug, irinotecan.
[0317] Experiments were carried out to determine the fate of PEG species containing irinotecan in the mixed salt of TFA.PEG-Gly-Irino-20K chloride with 4 arms under conditions of transesterification (K2CO3 in CH3OH, 20°C) and aqueous hydrolysis (pH 10, 20°C). The transesterification reaction was >99% complete after 45 minutes. The aqueous hydrolysis reaction was >99% complete within 24 hours. For both types of reactions, control reactions using irinotecan were carried out under identical conditions, and some artifact peaks were observed. After adjustment for artifact peaks, in both cases, the produced irinotecans had chromatographic purities > 99%.
[0318] Based on these results, it was concluded that essentially all PEGylated species of the 4-armed PEG-Gly-Irino-20K, as the hydrochloride salt, release irinotecan. Overlays of HPLCs collected over time from the aqueous hydrolysis reaction show the conversion of DS4 to DS3 to DS2 to DS1 to irinotecan. All of these species hydrolyze releasing irinotecan, as illustrated in FIG. 4 which demonstrates the release of irinotecan via hydrolysis from PEG-Gly-Irino-20K species with 4 mono-, di-, tri- and tetra-substituted arms.
[0319] Additional experiments were conducted to measure the hydrolysis rates for the major component of 4-armed PEG-Gly-Irino-20K, DS4, and its less substituted intermediates, DS3, DS2 and DS1, in aqueous buffer (pH 8, 4) in the presence of porcine carboxypeptidase B and in human plasma. Hydrolysis in aqueous buffer (pH 8.4) in the presence of porcine carboxypeptidase B was an attempt to perform enzyme-based hydrolysis. The control experiment at pH 8.4 without the enzyme later showed that hydrolysis was pH driven and thus essentially chemical hydrolysis. Still, the data were valuable for comparison with data obtained from hydrolysis carried out in human plasma. These experiments showed that the hydrolysis rates of the various components are not significantly different and compare favorably with theoretical predictions. Additional experiments measured the hydrolysis rates for the major components (DS4, DS3, DS2 and DS1) of 4-arm PEG-Gly-Irino-20K in human plasma. These experiments also show that the various components are hydrolyzed at the same rate and compare favorably with theoretical predictions.
[0320] FIG. 5 and FIG. 6 present graphs showing theoretical hydrolysis rates versus experimental data for chemical hydrolysis (in the presence of enzyme) and plasma hydrolysis, respectively. In both cases, theoretical predictions are based on identical rates so that hydrolysis of each species produces the next lower homologue plus free irinotecan (ie, DS4>DS3>DS2>DS1). PEG-1-METHYLENE-2-Oxo-(VINYLAMINE ACETATE LINKED TO IRINOTECAN) )-20K WITH 4 ARMS "PEG-GLY-IRINO-2 0K WITH 4 ARMS"


Step 1. Synthesis of Boc-Glycine-irinotecan Hydrochloride (Gly-IRT HC1) Part 1: Drying of Irinotecan Hydrochloride Trihydrate (IRT.HC1.3H2O)
[0321] IRT-HCl-3H2O (45.05 g, 66.52 mmol) was charged to a reactor. Anhydrous N,N-Dimethylformamide (DMF) (666 mL, 14.7 mL/g IRT•HC1•3H2O, DMF water content not greater than 300 ppm) was charged into the reactor. With slow stirring, the reactor was heated to 60°C (jacket temperature). After the irinotecan (IRT) was completely dissolved (5-10 minutes), vacuum was slowly applied until reaching 5-10 mbar and the DMF was removed by distillation. When the volume of condensed distillate (DMF) reached 85 - 90% of the initial DMF charge, the vacuum was released. Heptane (1330 mL, 30.0 mL/g IRT■ HCl ■ 3H2O, water content not greater than 50 ppm) was introduced into the reactor and the jacket temperature was decreased to 50°C. The heptane was distilled under vacuum (100-150 mbar) until the distillate volume was about 90% of the initial heptane charge. Two more heptane distillation cycles (2 X 1330 mL of heptane charges and distillations) were performed. A sample of the solvent phase was removed from the reactor and analyzed for DMF content using gas chromatography to ensure that the DMF content of the sample was no greater than 3% w/w. (In case the residual DMF is >3.0% w/w, a fourth cycle of azeotropic distillation is performed.) The resulting slurry was used for the coupling reaction. Part 2: Coupling reaction
[0322] Dichloromethane (1330 mL, 29.5 mL DCM/g IRT • HCl ■ 3H2O) was charged to the reactor where dry IRT-HC1 slurry (1.0 equivalent) in residual heptanes (the approximate mass ratio between heptanes residuals and IRT-HCL was 3) was being shaken. The reaction contents were stirred for 15 - 30 minutes, and the batch temperature was maintained at 17°C. Boc-glycine (14.0 g, 79.91 tnmol, 1.2 equivalent) and DMAP (0.81 g, 6.63 mmol, 0.1 equivalent) were loaded into the reactor as solids. A DCM solution of DCC (1.5 equivalents in 40 mL of dichloromethane) was prepared and added over 15-30 minutes, and the resulting reaction mixture was stirred at 17°C (batch temperature) for 2-3 hours . The reaction was monitored by HPLC for completeness. A pre-prepared quick-cooling solution was loaded into the reaction mixture to quickly cool any remaining DCC. In summary, the pre-prepared quick-cool solution is a pre-mixed solution of TFA and IPA in dichloromethane, prepared by mixing TFA (1.53 mL, 0.034 mL/g IRT-HC1•3H2O) and IPA (3 .05 mL, 0.068 mL/g IRT•HC1•3H2O) in DCM (15.3 mL, 0.34 mL/g IRT-HC1•3H2O), and was added to the VI reactor over 5 - 10 minutes when the conversion was at least 97%. the contents were stirred for an additional 30-60 minutes to allow for rapid cooling. The reaction mixture containing DCU was filtered through a 1 micrometer filter into another reactor. The reaction filtrate was distilled to 1/3 volume under vacuum at 35°C. Isopropyl alcohol (IPA) (490.5 mL, 10.9 mL/g IRT ■ HCl • 3H 2 O) was added to the concentrated mixture and the mixture was stirred for 30 - 60 minutes at 50°C (jacket temperature). The resulting homogeneous solution was concentrated, via vacuum distillation, to approximately 85% of the volume of the initial IPA charge and the resulting concentrate was cooled to 20°C (jacket temperature). The reaction mixture in IPA was transferred over 60-80 minutes to heptane (1750 mL, 38.8 mL heptane/g IRT • HCl • 3H2O) at 20°C. The resulting slurry containing precipitate of Boc-gly-IRT HCl was stirred for an additional 60-90 minutes and the product was collected by filtration. The reaction flask was rinsed with heptane (2 X 490 mL, 20.0 mL Heptane/g IRT•HC1•3H2O) and the product cake was washed with the rinse. The wet cake was dried at 20°C to 25°C under vacuum for a minimum period of 12 hours. Yield: 57.13 g (110%, high due to residual solvents). Step 2. Synthesis of Glycine-irinotecan Hydrochloride (Gly-IRT HC1)
[0323] A 100 ml round bottom flask was charged with BOC-Gly-IRT (2.34 g, 0.003 moles) and IPA (12 ml), to which was added HCl (12 ml, 4 M, in dioxane, 0.045 moles) over a period of 10 minutes. The reaction mixture was stirred at RT for 6 hours (and was monitored by HPLC for the completeness of the reaction), followed by addition of dry acetonitrile (12 ml). The resulting reaction mixture was slowly added (5 minutes) to a stirred solution of MTBE (140 ml). The solid thus obtained was filtered and dried under vacuum to give the HCl salt of Gly-IRT as a yellow powder. Yield: 2.17 g. Step 3. Synthesis of 4-Arm Irinotecan PEG20K-Glycine Hydrochloride
[0324] Gly-IRT HC1 (5.04 g, 14.61 wt% HC1) was loaded into a 100 mL reactor and was flushed with argon. The jacket temperature was set at 20°C, DCM (100 ml) and TEA (4 ml) were added. The solution was stirred for 10 minutes.
[0325] An initial charge of 4-armed PEG20K-SCM (26.5 g) was added and the reaction mixture was stirred for 30 minutes. A sample was collected and analyzed via HPLC. HPLC data revealed 6.1% Gly-IRT.HC1 remaining. A second batch of 4-armed PEG20K-SCM (1.68 g) was added to the reaction mixture and the solution was stirred for approximately 2 hours. A sample was taken for HPLC analysis. Data from HPLC analysis revealed 1.2% Gly-IRT HCl remaining.
[0326] The reaction solution was then slowly added to MTBE (800 mL) to precipitate the product. The precipitate was stirred for 30 minutes and was collected via filtration. The wet cake was washed with MTBE (200 mL) twice. The product was dried under vacuum. The 4-armed PEG20K hydrochloride-irinotecan crude intermediate was analyzed by ion chromatography for chloride content.
[0327] Table 2 summarizes the salt content analysis of the resulting 4-arm PEG20K-irinotecan hydrochloride intermediate (IC chromatography).
Salt adjustment and isolation with ethyl acetate:
[0328] The crude 4-armed PEG20K-irinotecan hydrochloride intermediate (29.1 g, 83.3 mol% Cl) was dissolved in 600 ml of ethyl acetate at 35°C. The solution was stirred for 15 minutes after visible dissolution of solids. A 0.1N solution of HCl in ethanol (8.5 ml) was charged into the solution and stirred for 30 minutes. The flask was immersed in an ice bath with strong agitation. Visible solids precipitated out of solution after 10 minutes. The mixture was stirred for a total of 60 minutes in the bath. The precipitate was collected by filtration on a glass frit by applying a slight vacuum. The wet cake was washed with a 30% MeOH/70% MTBE solution (400 mL). The product was placed under vacuum to dry. Yield: 28.3 g
[0329] The chloride content of the final product (determined by ion chromatography) was as follows: IRT content, % by weight Chloride, % molar 9.8% 98.8%
[0330] It was determined, by means of ion chromatography, that another batch prepared by the above process has % chloride of 103.8% mol (that is, it was totally in the form of the hydrochloride salt). When stored and evaluated over a period of 4 weeks at 40°C, total product-related species ranged from 98.7% to 97.0%, whereas free irinotecan ranged from 0.4% to 1, 25%, indicating the stability of the hydrochloride salt (i.e. resistance) to hydrolytic degradation. Under these same conditions, cleavage of the polyethylene glycol backbone was detected after 4 weeks, but was not measurable.EXAMPLE 7PREPARATION OF PENTAERITITOL-BASED 4-ARM PEG-20K. TO THE SCALE OF 1.9 KG
[0331] Materials and Methods. A very high quality of ethylene oxide having the lowest attainable water content must be used, as the water content leads to impurities of polymeric diols. CAUTION; Ethylene oxide is a very reactive compound that can explosively react with moisture; thus, leakages in the reaction and transfer apparatus must be carefully avoided. Care must also be taken in operations to include personnel working behind protective shields or in bunkers.
[0332] Anhydrous toluene (4 L) refluxed for two hours in a two-gallon jacketed stainless steel pressure reactor. Then, a part of the solvent (3 L) was removed by distillation under atmospheric pressure. The residual toluene was then discharged and the reactor was dried overnight by passing steam through the jacket of the reactor and applying reduced pressure of 3 - 5 mm Hg. Then, the reactor was cooled to room temperature, filled with anhydrous toluene (4 L) and 4-ARM PEG-2K based on pentaerythritol (SUNBRIGHT PTE®-2000 pentaerythritol, molecular weight about 2000 Dalton) was added , NOF Corporation; 200 g, 0.100 moles). The solvent was distilled off under reduced pressure, and then the reactor was cooled to 30 °C under a dry nitrogen atmosphere. One liter of dry toluene with molecular sieves (water content ~ 5 ppm) and liquid sodium-potassium alloy (Na 22%, K 78%; 1.2 g) was added to the reactor. The reactor was heated to 110°C and ethylene oxide (1800 g) was continuously added for three hours, keeping the reaction temperature at 110 - 120°C. Then the reactor contents were heated for two hours at -100 °C, and then the temperature was lowered to ~70 °C. Excess ethylene oxide and toluene were removed by distillation under reduced pressure. After distillation, the contents of the reactor remained under reduced pressure and sparged with nitrogen to remove traces of ethylene oxide. Phosphoric acid (1N) was added to neutralize the basic residue and the product was dried under reduced pressure. Finally, the product was drained from the reactor and filtered, yielding, after cooling, 1900 g of white solid. Gel Filtration Chromatography (GFC) was applied to characterize the alkoxylated polymeric product, PEG-20K with 4 ARMS based on pentaerythritol. This analytical method provided a chromatogram of the composition, with separation of components according to molecular weight. An Agilent 1100 HPLC system equipped with a Shodex KW-803 GFC column (300 x 8 mm) and differential refractometer detector was used. The mobile phase flow (0.1 M NaNO3 ) was 0.5 ml/min. The GFC chromatogram is shown in FIG. 7.
[0333] GFC analysis showed that the PEG-20K product with 4 ARMS contained the following: High MW product 0.42%, PEG-20K with 4 ARMS 99.14%, HO-PEG(10K)-OH 0 .44%.EXAMPLE 9 PEG-20K ANALYSIS WITH 4 ARMS AVAILABLE ON THE MARKET
[0334] NOF Corporation is currently a leader in the provision of commercial PEGs. Thus, a commercially available 4-ARM PEG-20K based on pentaerythritol (SUNBRIGHT PTE®-20,000, molecular weight about 20,000 Dalton, NOF Corporation) was obtained and analyzed using Gel Filtration Chromatography (GFC). An Agilent 1100 HPLC system equipped with a GFC Shodex KW-803 column (300 x 8 mm) and differential refractometer detector was used. The mobile phase flow (0.1 M NaNO3) was 0.5 ml/minute. The GFC chromatogram is shown in FIG. 8.
[0335] GFC analysis showed that this commercial 4 ARM PEG-20K product contained: High MW products 3.93%, 4 ARM PEG-20K 88.56%, HO-PEG(lOK)-OH 3 .93%, HO-PEG(5K)-OH 3.58%.EXAMPLE 10PREPARATION OF THE ALCOXYLABLE OLIGOMER; PEG-2K WITH 4 ARMS BASED ON PENTAERYTHRITOL AT 15 KG SCALE
[0336] A twenty gallon jacketed stainless steel pressure reactor was washed twice with 95 kg of deionized water at 95°C. The wash water was removed and the reactor was dried overnight by passing steam through the jacket of the reactor and applying reduced pressure (3-5 mm Hg). The reactor was filled with 25 kg of anhydrous toluene and a part of the solvent was distilled off under reduced pressure. The residual toluene was then discharged and the reactor was kept under reduced pressure. Then, the reactor was cooled to room temperature, filled with anhydrous toluene (15 L) and pentaerythritol (1020 g) was added. Part of the solvent (-8 L) was distilled off under reduced pressure, and then the reactor was cooled to 30°C under a dry nitrogen atmosphere. Liquid sodium-potassium alloy (Na 22%, K 78%; 2.2 g) was added to the reactor. Anhydrous ethylene oxide (14,080 g) was continuously added for three hours, keeping the reaction temperature at 150-155 °C. Then the reactor contents were heated for 30 minutes to ~150°C, and then the temperature was lowered to ~70°C. Excess ethylene oxide and toluene were removed by distillation under reduced pressure. After distillation, the contents of the reactor remained under reduced pressure and sparged with nitrogen to remove traces of ethylene oxide. Finally, the product was drained from the reactor, giving rise to 14,200 g of viscous liquid. Gel Filtration Chromatography (GFC) was applied to characterize the product, 4 ARM PEG-2K based on pentaerythritol. This analytical method provided a chromatogram of the composition, with separation of components according to molecular weight. An Agilent 1100 HPLC system equipped with a GFC Shodex KW-803 column (300 x 8 mm) and differential refractometer detector was used. The mobile phase flow (0.1 M NaNO 3 ) was 0.5 ml/min.
[0337] GFC analysis showed that the 4-arm PEG-2K product was ~100% pure, with low or high molecular weight impurities below detectable limits. Â 20 KG SCALE
[0338] A twenty gallon jacketed stainless steel pressure reactor was washed twice with 95 kg of deionized water at 95°C. The water was discharged and the reactor was dried overnight by passing steam through the reactor jacket and applying reduced pressure of 3 - 5 mm Hg. The reactor was filled with 25 kg of toluene and a part of the solvent was distilled off under reduced pressure. The residual toluene was then discharged and the reactor was kept under reduced pressure. The reactor was then cooled to room temperature, filled with anhydrous toluene (21 L) and previously isolated alkoxylated oligomer added: 4-ARM PEG-2K based on pentaerythritol of Example 10 (2064 g). Part of the solvent (16 L) was removed by distillation under reduced pressure, and then the reactor was cooled to 30°C under a dry nitrogen atmosphere. Four liters of dry toluene with molecular sieves (water content ~5 ppm) and liquid sodium-potassium alloy (Na 22%, K 78%; 1.7 g) were added, and the reactor was heated to 110°C. Then, ethylene oxide (19300 g) was continuously added for five hours, keeping the reaction temperature at 145 - 150 °C. Then the reactor contents were heated for 30 minutes at ~140 °C, and then the temperature was lowered to ~100 °C. Glacial acidic acid (100 g) was added to neutralize the catalyst. Excess ethylene oxide and toluene were removed by distillation under reduced pressure. After distillation, the contents of the reactor remained under reduced pressure and sparged with nitrogen to remove traces of ethylene oxide. Finally, the product was drained from the reactor, yielding 20 100 g of white solid. Gel Filtration Chromatography (GFC) was applied to characterize the alkoxylated polymeric product, PEG-20K with 4 ARMS based on pentaerythritol. This analytical method provided a chromatogram of the composition, with separation of components according to molecular weight. An Agilent 1100 HPLC system equipped with a GFC Shodex KW-803 column (300 x 8 mm) and differential refractometer detector was used. The mobile phase flow (0.1 M NaNO3) was 0.5 ml/minute.
[0339] GFC analysis showed that the PEG-20K product with 4 ARMS contained the following: High MW product 0.75%, PEG-20K with 4 ARMS 97.92%, HO-PEG(10K)-OH 1.08%, HO-PEG(SK) -OH 0.48%.
[0340] The invention(s) presented herein have been described with respect to particular exemplified embodiments. However, the foregoing description is not intended to limit the invention to the exemplified embodiments, and the skilled person will recognize that variations may be made within the spirit and scope of the invention as described in the foregoing specification.

权利要求:
Claims (10)
[0001]
1. Hydrohalide salt form of a polymer-active agent conjugate corresponding to structure (I):
[0002]
2. Form of hydrohalide salt, according to claim 1, characterized in that the hydrohalide salt is a hydrochloride salt.
[0003]
3. Method of preparing a composition comprising a hydrohalide salt of a polymer-active agent conjugate corresponding to structure (I),
[0004]
4. Method according to claim 3, characterized in that it also comprises (iv) analyzing the hydrohalide salt of pentaerythritolyl-polyethylene glycol-carboxymethyl-glycine-irinotecan with 4 arms recovered for halide content, and, in the case of the halide content is less than 95 mole percent, (v) dissolve the recovered 4-armed hydrohalide salt of pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan in ethyl acetate, and add additional hydrohalic acid.
[0005]
5. Composition characterized in that it comprises a hydrohalide salt of pentaerythritol-polyethylene glycol-carboxymethyl-glycine-irinotecan with 4 arms prepared according to the method defined in any one of claims 3 and 4.
[0006]
6. Pharmaceutically acceptable composition characterized in that it comprises the hydrohalide salt as defined in any one of claims 1, 2, or 5 and a pharmaceutically acceptable excipient.
[0007]
7. Use of a pharmaceutically acceptable composition as defined in claim 6, characterized in that it is for the manufacture of a drug for the treatment of cancer.
[0008]
8. Composition according to claim 5, characterized in that the polymer reagent containing an active ester can be obtained by a method comprising: alkoxylation, in a suitable solvent, of an oligomer capable of being alkoxylated previously isolated to forming an alkoxylated polymeric material, wherein the pre-isolated alkoxylated oligomer has a known and defined weight average molecular weight greater than 300 Daltons; modification of the alkoxylated polymeric material, in one or more steps, so as to contain an active ester, thereby forming a polymer reagent containing an active ester.
[0009]
9. Composition according to claim 8, characterized in that the polymer reagent containing an active ester has the following structure:
[0010]
10. Composition comprising hydrochloride salts of four-armed polymer conjugates, characterized in that at least 90% of the four-armed conjugates present in the composition: (i) have a structure encompassed by the formula, C-[CH2-O-(CH2CH2O) )n-CH2-C(O)-Therm]4, where n, in each case, is an integer with a value from 5 to 150, and eTerm, in each case, is selected from the group consisting of -OH, -OCH3, and -NH-CH2-C(O)-O-Irino ("GLY-Irino"), wherein Irino is an irinotecan residue; and (ii) for each Term in at least 90% of the conjugates with four arms present in the composition, at least 90% of these are -NH-CH2-C(O)-O-Irino, and additionally, for each amino group in each Irino in at least 90% of the four-armed conjugates present in the composition, each amino group is protonated or non-protonated, wherein any given protonated amino group is a hydrochloride salt.

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法律状态:
2018-09-25| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001 |

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2020-05-05| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-05-12| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-10-13| B15K| Others concerning applications: alteration of classification|Free format text: AS CLASSIFICACOES ANTERIORES ERAM: A61K 47/48 , A61P 35/00 Ipc: A61K 47/60 (2017.01), C08G 65/329 (2006.01), C08G |

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2021-01-12| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-03-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-18| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 18/11/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

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2021-05-25| B16C| Correction of notification of the grant|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 18/11/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US26246309P| true| 2009-11-18|2009-11-18|

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US61/262,463|2009-11-18|

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US29007209P| true| 2009-12-24|2009-12-24|

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US61/290,072|2009-12-24|

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PCT/US2010/057292|WO2011063158A1|2009-11-18|2010-11-18|Salt form of a multi-arm polymer-drug conjugate|

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