HK1171366B - Acid salt forms of polymer-drug conjugates and alkoxylation methods - Google Patents

Description

Polymer-drug conjugates in acid salt form and alkoxylation process

Cross Reference to Related Applications

This application claims priority from each of the following applications in accordance with 35u.s.c. § 119 (e): U.S. provisional patent application serial No. 61/262,463, filed 11/18/2009, and U.S. provisional patent application serial No. 61/290,072, filed 12/24/2009, both of which are incorporated herein by reference in their entireties.

FIELD

The present disclosure relates generally to mixed acid salt compositions of water-soluble polymer drug conjugates, their pharmaceutical compositions, and methods of making, formulating, administering, and using such mixed acid salt compositions. The present disclosure also relates generally to alkoxylation processes for preparing alkoxylated polymeric materials from previously isolated alkoxylated oligomers, and to compositions comprising the alkoxylated materials, methods of using the alkoxylated polymeric materials, and the like.

Background

Over the years, various methods have been proposed for improving the stability and delivery of bioactive agents. Challenges associated with the formulation and delivery of pharmaceutical agents may include: poor water solubility, toxicity, low bioavailability, instability, and rapid in vivo degradation of the pharmaceutical agent, to name a few. Although many approaches have been devised to improve the delivery of pharmaceutical agents, none of the single approaches is free of its potential drawbacks. For example, drug delivery methods commonly employed aim to solve or at least ameliorate one or more of the following problems, including drug encapsulation, such as in a liposome, polymer matrix, or unimolecular micelle, covalent attachment to a water soluble polymer such as polyethylene glycol, use of gene targeting agents, salt structures, and the like.

Covalent attachment of water-soluble polymers can improve the water solubility of the active agent and alter its pharmacological properties. Certain exemplary polymer conjugates are described, inter alia, in U.S. patent No. 7,744,861. In another approach, active agents having acidic or basic functionality may be reacted with a suitable base or acid and sold in the form of a salt. More than half of all active molecules are sold as salts (Polymorphism in the Pharmaceutical Industry), Hilfiker, r., editors, Wiley-VCH publishing company, 2006). Challenges in using the salt form include finding the optimal salt and controlling the solid state behavior during processing. Biopharmaceutical salts can be amorphous, crystalline, and exist as hydrates, solvents, different polymorphs, and the like. Interestingly, the salt form of the polymer conjugate, let alone the mixed acid salt form, is rarely used in pharmaceutical formulations.

Another challenge associated with preparing active agent conjugates of water-soluble polymers is the ability to prepare relatively pure water-soluble polymers in a compatible and reproducible process. For example, for coupling to active agents (e.g., small molecules and proteins), poly (ethylene glycol) (PEG) derivatives activated with reactive functional groups are useful, thereby forming conjugates between PEG and the active agent. When an active agent is coupled to a polymer of poly (ethylene glycol) or "PEG," the coupled active agent is generally referred to as having been "pegylated.

The conjugated forms exhibit different, and often clinically beneficial, properties when compared to the safety and efficacy of the active agent in its unconjugated form. Commercial success of PEGylated active Agents, e.g.Pegylated interferon α -2a (Hoffmann-La Roche, Nutley, N.J.),pegylated interferon α -2b (Schering Corporation, Inc., Kennilworth, N.J.), andPEG-filgrastim (Amgen Inc., kilooak, ca) demonstrates a degree of potential for its pegylation to improve one or more properties of an active agent.

In preparing conjugates, polymeric reagents are typically used to allow for simpler synthetic methods for conjugate synthesis. By combining a composition comprising a polymeric reagent with a composition comprising an active agent, it is possible (under appropriate reaction conditions) to perform more convenient conjugate synthesis.

However, the preparation of polymeric reagents suitable for pharmaceutical product regulatory requirements is often challenging. Conventional polymerization processes result in less pure compositions and/or low yields. While such impurities and yields may not be an issue outside the pharmaceutical field, safety and cost represent important concerns in the context of pharmaceuticals for human use. Thus, conventional polymerization methods are not suitable for synthesizing polymeric reagents intended for the manufacture of pharmaceutical conjugates.

In the case of multi-armed polymers, there is a lack of available, desirable water-soluble polymers having well-controlled and well-defined properties without significant amounts of undesirable impurities. One can therefore easily obtain, for example, high molecular weight, multi-armed poly (ethylene glycol), but drug conjugates made from commercial polymers will have significant amounts (i.e., > 8%) of the polymer drug conjugate with either very low or very high molecular weight bioactive impurities. This range of active impurities in pharmaceutical compositions can render such compositions unacceptable, if not impossible, and thus render approval of such drugs challenging.

Summary of The Invention

In one or more embodiments of the invention, a composition is provided that includes mixed salts of a water-soluble polymer-active agent conjugate, wherein the active agent in the conjugate has at least one amine or other basic nitrogen-containing group, and further wherein the amine or other basic nitrogen-containing group is either protonated or unprotonated (i.e., as a free base), wherein any given protonated amine or other basic single group-containing group is an acid addition salt of either a strong mineral acid or a strong organic acid, such as, for example, trifluoroacetic acid (TFA).

Examples of strong inorganic acids include hydrohalic acids (e.g., hydrochloric acid, hydrofluoric acid, hydroiodic acid, and hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid, and nitrous acid.

In one or more embodiments of the invention, the protonated form comprises an addition salt of a hydrohalic acid.

In one or more embodiments of the invention, the protonated form comprises an addition salt of hydrochloric acid.

Examples of strong organic acids include organic acids having a pKa of less than about 2.00. Examples include trichloroacetic acid, dichloroacetic acid, as well as mixed haloacetic acids such as fluorodichloroacetic acid, fluorochloroacetic acid, difluorochloroacetic acid, and the like.

In one or more embodiments of the invention, the water soluble polymer is linear or multi-armed.

In one or more embodiments of the invention, the water soluble polymer is a poly (alkylene glycol) (e.g., poly (ethylene glycol)), or a copolymer or terpolymer thereof.

In one or more embodiments of the invention, the active agent is selected from the group consisting of small molecule drugs, peptides, and proteins.

In one or more embodiments of the invention, the active agent is camptothecin.

In one or more embodiments of the invention, the composition includes a mixed salt of a water-soluble polymer active agent conjugate corresponding to structure (I):

wherein n is an integer ranging from 20 to about 600 (no specific protonated amino nitrogen atoms and counter ions are shown), and for each amine group within each irinotecan, each amine group is either protonated or unprotonated, with any given protonated amine group being in the acid salt form of an inorganic or organic acid, such as trifluoroacetic acid.

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), the mole percentage of active agent amino groups (or other basic nitrogen atoms) in the composition that is protonated as the TFA salt is greater than each of the mole percentage of active agent amino groups in the composition that is protonated as the inorganic acid salt and the mole percentage of active agent amino groups in the composition in free base form.

In yet another alternative embodiment, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), the mole percentage of active agent amine groups (or other basic nitrogen atoms) in the composition that is protonated as the TFA salt is greater than the mole percentage of active agent amine groups in the composition in the free-base (i.e., unprotonated) form.

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), at least 20 mole percent of the active agent amine groups in the composition are protonated as TFA salts.

In one or more embodiments, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), at least 25 mole percent of the active agent amine groups in the composition are protonated as TFA salts.

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 20-45 mole percent of the active agent amino groups in the composition are protonated as the TFA salt.

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 24-38 mole percent of the active agent amino groups in the composition are protonated as TFA salts.

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 35-65 mole percent of the active agent amino groups in the composition are protonated as the TFA salt.

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 30-65 mole percent of the active agent amino groups in the composition are protonated as the inorganic acid salt (e.g., the HCl salt).

In yet one or more additional embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 32-60 mole percent of the active agent amino groups in the composition are protonated as the inorganic acid salt (e.g., the HCl salt).

In still one or more other embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 35-57 mole percent of the active agent amino groups in the composition are protonated as the inorganic acid salt (e.g., the HCl salt).

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 25-40 mole percent of the active agent amino groups in the composition are protonated as the inorganic acid salt (e.g., the HCl salt), and about 5-35 mole percent of the active agent amino groups in the composition are unprotonated (i.e., as the free base).

In one or more embodiments of the invention, with respect to the composition of the conjugate (e.g., the composition of a four-arm conjugate), about 32-60 mole percent of the active agent amino groups in the composition are protonated as the inorganic acid salt (e.g., the HCl salt), and about 5-35 mole percent of the active agent amino groups in the composition are unprotonated (i.e., as the free base).

In one or more embodiments of the invention, a trifluoroacetic acid/hydrochloric acid mixed salt of a conjugate is provided, the conjugate having the following structure:

where n is an integer ranging from about 20 to about 500 (including about 40 to about 500) (note that in the above structures, the particular basic nitrogen atom and corresponding anion in protonated form are not shown). In one or more embodiments of the invention, a portion of the amino groups in the conjugates encompassed within the above structures directly are non-protonated. Exemplary molar ratios of protonated and unprotonated forms are provided above and will further apply here to the above conjugates.

In one or more embodiments of the present invention, a method for providing a mixed salt of a water-soluble polymer active agent conjugate is provided, the method comprising the steps of: (i) deprotecting a protected form of an amine-active agent-containing inorganic acid salt by treatment with trifluoroacetic acid (TFA) or other organic acid deprotection reagent to form a deprotected active agent acid salt, (ii) coupling the deprotected active agent acid salt of step (i) with a water-soluble polymer reagent in the presence of a base (e.g., trimethylamine, triethylamine, and dimethylaminopyridine) to form a polymer active agent conjugate, and (iii) recovering the polymer active agent conjugate, wherein the recovered polymer active agent conjugate is characterized as having therein an active agent amino group alone in a form selected from the group consisting of: free base form (non-protonated), inorganic acid salt form, and TFA or other organic acid salt form. In one or more embodiments of the invention, the method further comprises determining the relative molar amounts of inorganic acid and TFA in the deprotected acid salt formed in step (i). In one or more embodiments of the invention, the inorganic acid salt in step (i) is a hydrohalic acid salt, e.g. a hydrochloride salt. In one or more embodiments of the invention, the amount of base in step (ii) ranges from 1.00 to 2.00 (moles TFA + moles acid). In one or more related embodiments, the amount of base in step (ii) ranges from 1.00 to 1.50 (TFA moles + moles of inorganic acid), wherein parentheses indicate the multiplication. In one or more related embodiments, the amount of base in step (ii) ranges from 1.00 to 1.20 (moles TFA + moles inorganic acid). In one embodiment, the number of equivalents of base is 1.05 (moles of TFA + moles of inorganic acid).

In one or more embodiments of the invention, the water-soluble polymer reagent is an activated polyethylene glycol ester (i.e., a polyethylene glycol reagent having at least one activated ester group). In one or more embodiments of the invention, the water-soluble polymer reagent is a polyethylene glycol reagent having three or more polymer arms.

In one or more embodiments of the invention, the active agent amine groups in the polymeric active agent conjugate are selected from the group consisting of secondary amine groups and tertiary amine groups. In one or more embodiments of the invention, the active agent amine group is a tertiary amino group. In yet another embodiment, the polymer active agent conjugate includes as its corresponding conjugate acid a pK in the range of about 10-11.5_aBasic nitrogen atom of (2).

In one or more embodiments of the invention, the active agent is selected from a small molecule, peptide, and protein. In one or more embodiments of the invention, the active agent is a camptothecin. Illustrative camptothecin molecules are selected from camptothecin, irinotecan, and 7-ethyl-10-hydroxy-camptothecin (SN-38). Exemplary sites for covalent attachment to water-soluble polymers include, among others, the 7-, 10-, and 20-ring positions of the camptothecin backbone.

In one or more embodiments of the present invention, there is provided a pharmaceutically acceptable composition comprising (i) a mixed salt according to any one or more embodiments described herein, and (ii) a lactate buffer, optionally 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 (e.g., vial), optionally containing a 100mg dose equivalent of irinotecan in unconjugated form.

In one or more embodiments of the invention, a method is provided of administering a conjugate-containing composition described herein (wherein the active agent is an anti-cancer agent) to an individual having one or more types of cancerous solid tumors, wherein the conjugate-containing composition is optionally dissolved in a 5% w/w glucose solution. In one or more embodiments of the invention, administration is achieved by intravenous infusion.

In one or more embodiments of the present invention, a process for preparing a mixed salt of a water-soluble polymer active agent conjugate is provided, the process comprising the steps of: (i) deprotecting t-Bocg glycine-irinotecan HCl by treatment with trifluoroacetic acid (TFA) to form a deprotected glycine-irinotecan HCl/TFA mixed salt, (ii) coupling the deprotected glycine-irinotecan HCl/TFA mixed salt with 4-arm-pentaerythrityl-polyethylene glycol-carboxymethyl succinimide in the presence of a base under effective conditions to form a conjugate, 4-arm-pentaerythrityl-polyethylene glycol-carboxymethyl glycine-irinotecan (also known as pentaerythrityl-4-arm- (PEG-1-methylene-2-oxo-vinylamide acetate linked irinotecan), and (iii) recovering the conjugate from step (ii), wherein the conjugate is a mixed salt comprising a combination of amine groups in the form of free base, HCl, and TFA salts. In one or more embodiments of the invention, the method further comprises purifying the conjugate (e.g., comprising recrystallizing the conjugate to form a recrystallized conjugate). In one or more embodiments of the present invention, a recrystallized product is provided which is a mixed acid salt comprising the active agent amino group, present as a combination of free base, HCl, and TFA salt forms.

In one or more embodiments of the invention, there is provided a method of treating a mammal suffering from cancer, the method comprising administering a therapeutically effective amount of a mixed salt of a water-soluble polymer-camptothecin conjugate, which conjugate comprises camptothecin having an amine or other basic nitrogen-containing group, both in free base and protonated form, wherein each protonated form is present as an acid addition salt of either a strong mineral acid and trifluoroacetic acid. The mixed acid salt administered to the mammal is effective to produce a reduction or inhibition of the growth of the solid tumor in the subject. In one or more embodiments of the invention, the cancerous solid tumor type is selected from the group consisting of: colorectal, ovarian, cervical, breast and non-small cell lung solid tumors.

In one or more embodiments of the invention, there is provided a mixed acid salt of an active agent conjugate as described herein, wherein the active is an anti-cancer agent for use in the manufacture of a medicament for the treatment of cancer.

In another aspect, a process is provided that includes the step of alkoxylating a pre-isolated oxyalkylatable oligomer in a suitable solvent to form an oxyalkylated polymer product, wherein the pre-isolated oxyalkylatable oligomer has a known and defined weight average molecular weight of greater than 300 daltons (e.g., greater than 500 daltons).

In one or more embodiments of the above aspects of the invention, there is provided a composition comprising an alkoxylated polymer product prepared by a process comprising the step of alkoxylating a pre-isolated oxyalkylatable oligomer to form an alkoxylated polymer product in a suitable solvent, wherein the pre-isolated oxyalkylatable oligomer has a known and defined weight average molecular weight of greater than 300 daltons (e.g., greater than 500 daltons).

In one or more embodiments of the invention, a composition is provided that includes an alkoxylated polymer product having a purity of greater than 92wt% and a total combined content of high molecular weight product and diol of less than 8wt% (e.g., less than 2 wt%), as determined by, for example, Gel Filtration Chromatography (GFC) analysis.

In one or more embodiments of the present invention, the alkoxylated polymer product has the following structure:

where each n is an integer from 20 to 1000 (e.g., from 50 to 1000).

In one or more embodiments of the present invention, there is provided a method comprising the steps of: (i) alkoxylating a pre-isolated oxyalkylatable oligomer in a suitable solvent to form an oxyalkylated polymeric material, wherein the pre-isolated oxyalkylatable oligomer has a known and defined weight average molecular weight of greater than 300 daltons (e.g., greater than 500 daltons), and (ii) optionally, further activating the oxyalkylated polymeric product to provide an activated oxyalkylated polymeric product that is useful, among other things, as a polymeric reagent for preparing polymeric drug conjugates.

In one or more embodiments of the present invention, a process is provided that includes the step of activating an alkoxylated polymer product obtained from and/or contained within a composition that includes an alkoxylated polymer product having a purity of greater than 90%, thereby forming an activated alkoxylated polymer product that is useful (among other things) as a polymeric reagent for preparing polymeric drug conjugates.

In one or more embodiments of the present invention, a process is provided comprising coupling an activated alkoxylated polymer product to an active agent, wherein the activated alkoxylated polymer product is prepared by a process comprising the step of activating alkoxylated polymer product obtained from and/or contained within a composition comprising alkoxylated polymer product having a purity of greater than 90% thereby forming an activated alkoxylated polymer product.

In one or more embodiments of the present invention, mixed salts of water-soluble polymer active agent conjugates are provided, the conjugate has been prepared by coupling (under coupling conditions) an amine-bearing active agent (e.g., deprotected glycine-irinotecan)) to a polymeric reagent (e.g., 4-arm pentaerythritolyl-poly (ethylene glycol) -carboxymethylsuccinimide) in the presence of a base to form a conjugate, wherein the conjugate is a mixed salt conjugate (e.g., the conjugate has nitrogen atoms, each of which will be protonated or unprotonated, wherein any given protonated amino group is an acid salt having one of two different anions), and further, wherein optionally, the polymeric reagent is prepared from an alkoxylation product prepared as described herein.

Further embodiments of the methods, compositions, and the like of the present invention will be apparent from the following description, drawings, examples, and claims. As can be appreciated from the foregoing and the following description, each and every feature described herein, and each and every combination of two or more such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. Furthermore, any feature or combination of features may be specifically excluded from any embodiment of the present invention. Further aspects and advantages of the invention are set out in the following description and claims, particularly when considered in conjunction with the accompanying examples and figures.

Brief description of the drawings

Figure 1 is a graph showing the results of a stress stability study on three different samples of 4-arm-PEG-Gly-Irino-20K, each with a different composition with respect to the relative amounts of trifluoroacetic acid and hydrochloride salts, along with the free base. The samples tested included >99% HCl salt (< 1% free base, triangles), 94% full salt (6% free base, squares), and 52% full salt (48% free base, circles). The samples were stored at 25 ℃ and 60% relative humidity and the graphs illustrate the degradation of the compounds over time, as detailed in example 3.

Figure 2 is a graph showing the increase in free irinotecan over time in samples of 4-arm-PEG-Gly-Irino-20K stored at 40 ℃ and 75% relative humidity, each with a different composition with respect to the relative amounts of trifluoroacetic acid and hydrochloride salts, along with the free base. As described in example 3, the test samples correspond to a product containing >99% HCl salt (< 1% free base, squares) and a product containing 86% full salt (14% free base, diamonds).

Figure 3 is a graph showing the increase over time of small PEG species (PEG degradation products) in samples of 4-arm-PEG-Gly-Irino-20K stored at 40 ℃ and 75% relative humidity, as detailed in example 3. The test samples correspond to products containing >99% HCl salt (< 1% free base, squares) and products containing 86% full salt (14% free base, diamonds).

Figure 4 is a superimposed compilation of chromatograms showing the release of irinotecan via hydrolysis from mono- (DS-1), di- (DS-2), tri- (DS-3) and tetra irinotecan-substituted (DS-4) 4-arm-PEG-Gly-Irino-20K, as described in detail in example 5.

FIG. 5 is a graph showing the results of hydrolysis of a different class of 4-arm-PEG-Gly-Irino-20K as described above in aqueous buffer in the presence of porcine carboxypeptidase B at pH8.4, in comparison to the hydrolysis kinetics modeling data as described in example 5. For the kinetic model, all kinds of hydrolysis were assumed to be class 1 kinetic. Disappearance of DS4 order 1 reaction Rate constant (0.36 hr)^-1) Is used to generate all curves.

FIG. 6 is a graph showing the hydrolysis of different species of 4-arm-PEG-Gly-Irino-20K as described above in human plasma, in comparison to hydrolysis kinetic modeling data. Details are provided in example 5. For the kinetic model, all kinds of hydrolysis were assumed to be class 1 kinetic. Order 1 reaction rate constant for disappearance of DS4 (0.26 hr)^-1) Is used to generate all curves.

Figure 7 is a chromatogram of gel filtration chromatography of a material prepared as described in example 8.

Figure 8 is a chromatogram of gel filtration chromatography of a material prepared as described in example 9.

Detailed Description

Various aspects of the present invention will now be described more fully hereinafter. These aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. In the event that the teachings of this specification are inconsistent with the art incorporated by reference, the teachings in this specification should take precedence.

It must be noted that, as used in this specification, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polymer" includes a single polymer along with two or more of the same or different polymers, reference to a "conjugate" refers to a single conjugate along with two or more of the same or different conjugates, reference to an "excipient" includes a single excipient along with the use of two or more of the same or different excipients, and the like.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

A "functional group" is a group that can be used under normal conditions of organic synthesis to form a covalent link between the entity to which it is attached and another entity, typically carrying another functional group. The functional group typically includes one or more multiple bonds and/or one or more heteroatoms. Preferred functional groups are described herein.

The term "reactive" means that a functional group reacts readily or at a substantial rate under normal conditions of organic synthesis. This is in contrast to those groups that do not react or require strong catalysts or impractical reaction conditions to react (i.e., "unreactive" or "inert" groups).

A "protecting group" is a moiety 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 being protected as well as the reaction conditions to be used and the presence of additional reactive or protecting groups in the molecule. By way of example, functional groups that may be protected include: carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups, and the like. Representative protecting groups for carboxylic acids include esters (e.g., a p-methoxybenzyl ester), amides, and hydrazides; for amino are carbamates (e.g. tert-butoxycarbonyl) and amides; ethers and esters for the hydroxyl group; for thiol groups, thioethers and thioesters; for carbonyl groups acetals and ketals; and the like. Such Protecting Groups are well known to those skilled in the art and are described, for example, in t.w.greene (gelinii) and g.m.wuts (wutz), Protecting Groups in organic synthesis, third edition, Wiley (willi), new york, 1999 and p.j.kocienski (corinski), Protecting Groups, third edition, Thieme Chemistry press, 2003 and references cited therein.

A functional group in "protected form" refers to a functional group that bears a protecting group. As used herein, the term "functional group" or any synonym thereof is intended to encompass its protected form.

As used herein, "PEG" or "poly (ethylene glycol)" is meant to encompass any water-soluble poly (ethylene oxide). Typically, the PEG used in the present invention will comprise one of two of the following structures: "(CH)₂CH₂O)_n- "or" (CH)₂CH₂O)_{n- 1}CH₂CH₂- "depending on whether one or more of the terminal oxygens has been replaced, for example, during a synthetic transformation. The variable (n) ranges from 3 to about 3000, and the terminal groups as well as the structure of the overall PEG can vary.

A water-soluble polymer may carry one or more "end-capping groups" (in which case the water-soluble polymer may be referred to as "end-capped". about the end-capping group, exemplary end-capping groups are generally carbon-containing or hydrogen-containing groups, typically comprising 1-20 carbon atoms and an oxygen atom covalently bonded to the group.

The end-capping group may also include a detectable label. When the polymer has an end-capping group comprising a detectable label, the amount or location of the polymer and/or moiety (e.g., active agent) to which the polymer is attached can be determined using a suitable detector. Such labels include, but are not limited to, fluorescent glossers, chemiluminescent agents, moieties used in enzyme labeling, colorimetric labels (e.g., dyes), metal ions, radioactive moieties, and the like.

In the context of the polymer of the present invention, "water-soluble" or "water-soluble polymer segment" refers to any segment or polymer that is at least 35% (by weight), preferably greater than 70% (by weight), and more preferably greater 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 the light from the same solution after the filtering.

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 a nucleophile on another molecule. This is in contrast to groups that require strong bases or highly impractical reaction conditions to react (i.e., a "non-reactive" or "inert" group).

"electrophile" refers to an ion or atom or neutral or ionic collection of atoms that has an electrophilic center, i.e., a center that seeks electrons or is capable of reacting with a nucleophile.

"nucleophile" refers to an ion or atom or a neutral or ionic collection of atoms that has a nucleophilic center, i.e., a center that seeks an electrophilic center or is capable of reacting with an electrophile.

The term "protected" or "protecting group" means that a moiety (i.e., such a protecting group) is present that prevents or prevents the reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Such protecting groups will vary depending on: the type of chemically reactive group that is protected, along with the reaction conditions employed, and the presence of additional reactive or protecting groups, if any, in the molecule. Protecting GROUPS known IN the art can be found IN Greene, T.W. et al, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (PROTECTIVE group IN ORGANIC SYNTHESIS), third edition, John Wiley & Son (John Willi-father publishing Co.), New York, NY (1999).

In the context of a water-soluble polymer (e.g., PEG), "molecular weight" refers to the weight average molecular weight of a polymer, typically determined by size exclusion chromatography in an organic solvent (like 1,2, 4-trichlorobenzene), light scattering techniques, or intrinsic viscosity measurements.

The term "spacer" or "spacer moiety" as used herein refers to an atom or a collection of atoms that may optionally be used to link multiple interconnecting moieties (e.g., an end of a series of monomers and an electrophile). The spacer moiety of the present invention may be hydrolytically stable or may comprise a physiologically hydrolyzable or enzymatically degradable linker.

A "hydrolyzable" bond is a relatively unstable bond that reacts with (i.e., is hydrolyzed by) water under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the overall type of linkage connecting the two central atoms, but also on the substituents attached to these central atoms. Illustrative hydrolytically labile linkages include carboxylates, phosphates, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, and oligonucleotides.

"enzymatically degradable linkage" means a linkage that is degraded by one or more enzymes.

A "hydrolytically stable" linkage or bond refers to a chemical bond that is substantially stable in water, that is, does not undergo any appreciable degree of hydrolysis under physiological conditions over an extended period of time. Examples of hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in an aliphatic chain), ethers, amides, carbamates, and the like. Generally, a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1% to 2% per day under physiological conditions. The hydrolysis rates of representative chemical bonds can be found in most standard chemical textbooks.

With respect to the geometry or overall structure of a polymer, "multiarmed" means that the polymer has 3 or more polymer-containing "arms" connected to a "core" molecule or structure. Thus, a multi-armed polymer may have 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms, or more, depending on its configuration and core structure. A particular type of multi-armed polymer is a highly branched polymer, known as a dendrimer or hyperbranched polymer, having an initiator core of at least 3 branches, an internal branching diversity of 2 or greater, a generation of 2 or greater, and at least 25 surface groups within a single dendrimer molecule. For purposes herein, a dendrimer is considered to have a structure that is different from the structure of a multiarm polymer. That is, a multiarm polymer as referred to herein expressly excludes dendrimers. In addition, a multiarm polymer as provided herein has a non-crosslinked core.

A "dendrimer" or "hyperbranched polymer" is a spherical, size monodisperse polymer in which all bonds are formed radially from a central focal point or core, with a regular branching pattern and with multiple repeating units (each contributing a branching point). Dendrimers are typically (although not necessarily) formed using a nanoscale multi-step structuring process. Each step produces a new "generation" of twice or more the complexity of the previous generation. Dendrimers exhibit certain dendritic state properties such as core encapsulation, making them unique from other types of polymers.

"branching point" refers to a bifurcation point comprising one or more atoms at which a polymer splits or branches from a linear structure into one or more additional polymer arms. A multi-armed polymer may have a branch point or branch points, as long as the branches are not regular repeats of forming a dendrimer.

"substantially" or "substantially" means almost entirely or completely, e.g., 95% or more of a given amount.

"alkyl" refers to a hydrocarbon chain having a length ranging from about 1 to 20 atoms. These hydrocarbon chains are preferably, but not necessarily, saturated and may be branched or straight. 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 or branched chain, as exemplified below: methyl, ethyl, n-butyl, isobutyl, and tert-butyl.

"cycloalkyl" refers to a saturated cyclic hydrocarbon chain, including bridged, fused, or spiro compounds, preferably consisting of 3 to about 12 carbon atoms, more preferably 3 to about 8.

"non-interfering substituents" are those groups that, when present in a molecule, are typically unreactive with other functional groups contained in the molecule.

The term "substituted," as in, for example, "substituted alkyl," refers to a moiety (e.g., an alkyl group) substituted with one or more non-interfering substituents such as, but not limited to: c₃-C₈Cycloalkyl groups such as cyclopropyl, cyclobutyl, and the like; halo, such as fluoro, chloro, bromo, and iodo; a cyano group; alkoxy, lower phenyl; substituted phenyl; and the like. For substitution on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).

"alkoxy" means an-O-R group, wherein R is alkyl or substituted alkyl, preferably C₁-C₂₀Alkyl (e.g., methoxy, ethoxy, propoxy, etc.), preferably C₁-C₇An alkyl group.

As used herein, "alkenyl" refers to a branched and unbranched hydrocarbon group of 1 to 15 atomic lengths containing at least one double bond, such as ethylene (vinyl), 2-propen-1-yl (allyl), isopropenyl, 3-buten-1-yl, and the like.

The term "alkynyl" as used herein refers to a branched and unbranched hydrocarbon group of from 2 to 15 atoms in length containing at least one triple bond, such as ethynyl, 1-propynyl, 3-butyn-1-yl, 1-octyn-1-yl, and the like.

The term "aryl" denotes an aromatic group having up to 14 carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl, phenanthryl, tetracenyl, and the like.

"substituted phenyl" and "substituted aryl" respectively denote a phenyl group substituted with one, two, three, four or five ((e.g., 1-2, 1-3, 1-4, or 1-5 substituents) and an aryl group, the substituents being selected from halogen (F, Cl, Br, I), hydroxy, cyano, nitro, alkyl (e.g., C)_1-6Alkyl), alkoxy (e.g. C)_1-6Alkoxy), benzyloxy, carboxyl, aryl, and the like.

The inorganic acid is an acid having no carbon atom. Examples include halogen acids, nitric acid, sulfuric acid, phosphoric acid, and the like.

"hydrohalic acid" refers to hydrogen halides such as hydrofluoric acid (HF), hydrochloric acid (HCl), hydrobromic acid (HBr), and hydroiodic acid (HI).

"organic acid" refers to any organic compound (i.e., having at least one carbon atom) having one or more carboxyl groups (-COOH). 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.

As used herein, "active agent" includes any agent, drug, compound, and the like that provides some pharmacological, often beneficial effect, which may be demonstrated in vivo or in vitro. As used herein, these terms further include any physiologically or pharmacologically active substance that produces a local or systemic effect in a patient. As used herein, particularly with respect to the synthetic methods described herein, "active agent" is meant to encompass its derivatized or linker-modified form, such that upon in vivo administration, the parent "biologically active" molecule is released.

"pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to an excipient that can be included in a composition that includes an active agent and that does not cause significant detrimental toxicological effects to the patient.

"pharmacologically effective amount," "physiologically effective amount," and "therapeutically effective amount" are used interchangeably herein to mean that the amount of active agent present in a pharmaceutical formulation is required to provide a desired level of active agent and/or conjugate in the bloodstream or in a target tissue or site in the body. The precise amount will depend on many factors, such as the particular active agent, the composition and physical characteristics of the pharmaceutical formulation, the intended patient population, and patient considerations, and can be readily determined by one of ordinary skill in the art based on the information provided herein and available in the relevant literature.

In the context of a polymer, "polyfunctional" refers to a polymer having 3 or more functional groups, wherein the functional groups may be the same or different, and are typically present at the polymer terminus. The multifunctional polymer will typically comprise from about 3-100 functional groups, or from 3-50 functional groups, or from 3-25 functional groups, or from 3-15 functional groups, or from 3 to 10 functional groups, i.e. comprise 3,4,5,6, 7, 8, 9 or 10 functional groups.

"difunctional" and "difunctional" are used interchangeably herein and refer to an entity, such as a polymer, having (typically at the end of the polymer) two functional groups contained therein. When these functional groups are the same, the entity is said to be homobifunctional or homobifunctional. When these functional groups are different, the entity is said to be heterobifunctional or heterobifunctional.

A basic or acidic reactant described herein includes neutral, charged, 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, domestic animals, sport animals, and pets. Such subjects typically suffer from or are predisposed to a condition that can be prevented or treated by administration of a water-soluble polymer active agent conjugate as described herein.

The term "about" (particularly with respect to a given amount) is intended to encompass deviations of plus or minus five percent.

"treatment" (treatment) of a particular disorder includes: (1) preventing a condition that causes the condition not to develop or to occur to a lesser extent or intensity in a subject that may be exposed to or susceptible to the condition but has not yet experienced or exhibited the condition, and (2) inhibiting the condition, i.e., halting the development or reversing the condition.

"optional" or "optionally" means that the subsequently described circumstance may, but need not, occur, such that the description includes instances where the circumstance occurs and instances where it does not.

A "small molecule" is an organic, inorganic, or organometallic compound typically having a molecular weight of less than about 1000, preferably less than about 800 daltons. Small molecules as mentioned herein encompass oligopeptides as well as other biomolecules having a molecular weight of less than about 1000.

A "peptide" is a molecule consisting of from about 13 to about 50 amino acids. Oligopeptides typically contain from about 2 to 12 amino acids.

Unless specifically stated to the contrary, as used herein, the terms "partial mixed salt" and "mixed salt" are used interchangeably, and in the context of polymer conjugates (and corresponding compositions comprising a plurality of such polymer conjugates) refer to conjugates and compositions comprising one or more basic amino (or other basic nitrogen-containing) groups, wherein (i) one of any given basic amino group in the conjugate or conjugate composition is either unprotonated or protonated, and (ii) with respect to any given protonated basic amino group, the protonated basic amino group will have one of two different counterions. (the term "partially mixed salt" refers to the feature where not all of the amino groups in the compound or composition are protonated-thus the composition is a "partial" salt, while "mixed" refers to the feature of multiple counterions). Mixed salts as provided herein encompass hydrates, solvates, amorphous phase forms, crystalline forms, polymorphs, isomers, and the like.

An amine (or other basic nitrogen) group in the "free base" form is one in which the amine group (i.e., primary, secondary, or tertiary) has a free electron pair. The amine is neutral, i.e., uncharged.

The amine groups present in "protonated form" are present as protonated amines, such that the amine groups are positively charged. As used herein, a protonated amine group can also be in the form of an acid addition salt formed from the reaction of an amine with an acid (e.g., an inorganic or organic acid).

"mole percent" of amino groups of an active agent refers to the percentage or fraction of amino groups in the active agent molecules contained in one form of polymer conjugate, or another, wherein the total mole percent of amino groups in the conjugate is 100%.

As used herein, "psi" refers to pounds per square inch.

To summarize: mixed salts, conjugates, alkoxylation processes, and products made from polymers using alkoxylation processes Compositions of conjugates prepared with prepared polymeric reagents (and mixed salt forms thereof)

Mixed salts:as indicated above, in one or more aspects of the invention, conjugates of a water-soluble polymer and an active agent are provided, wherein the conjugates are in the form of a mixed salt. Such an dollThe association exhibits a novel solid state form and is based, at least in part, on the discovery that: although an alkaline treatment is used in their formation, the conjugates precipitate as mixed salts. Furthermore, it has been found that these conjugates can be reliably and reproducibly produced as mixed salts-wherein within the conjugate (and within the active agent component of the conjugate) any given basic nitrogen atom is present in one of a variety of forms. Specifically, the conjugates provided herein have an active agent basic nitrogen atom, such as an amino group, each of which will be protonated or unprotonated, where any given protonated amino group is an acid salt having one of two different anions. Furthermore, it has been found that the conjugate in the form of a mixed salt has several unexpected and advantageous characteristics (i.e. greater stability to degradation of the polymer backbone, greater hydrolytic stability, etc.) when compared to the corresponding conjugate in the form of the free base or the mono-acid salt.

The alkoxylation process:as also indicated above, in one or more aspects of the invention, a process is provided that includes the step of alkoxylating a pre-isolated oxyalkylatable oligomer in a suitable solvent to form an oxyalkylated polymer product, wherein the pre-isolated oxyalkylatable oligomer has a known and defined weight average molecular weight of greater than 300 daltons (e.g., greater than 500 daltons). Among other advantages, the alkoxylation processes provided herein result in polymer products that exceed (e.g., in terms of consistency and purity) polymer products prepared by previously known processes. In one or more embodiments, the polymers formed by the alkoxylation process of the present invention may be advantageously used to prepare mixed acid salts as described herein.

Compositions of conjugates prepared from polymeric reagents prepared from polymer products using alkoxylation processes (and mixed salt forms thereof):as also noted above, in one or more embodiments of the present invention, there is provided a mixed salt of a water-soluble polymer active agent conjugate, wherein the mixed salt is formed by (in-situ) couplingUnder combined conditions) coupling an amine-bearing active agent (e.g., deprotected glycine-irinotecan)) to a polymeric reagent (e.g., 4-arm pentaerythrityl-poly (ethylene glycol) -carboxymethylsuccinimide) in the presence of a base to form a conjugate, wherein the conjugate is a mixed salt conjugate (e.g., the conjugate has nitrogen atoms, each of which will be protonated or unprotonated, wherein any given protonated amino group is an acid salt having one of two different anions), and further wherein, optionally, the polymeric reagent is prepared from an alkoxylation product prepared as described herein.

Conjugate-generally polymers

Water-soluble polymer the active agent conjugate (whether in the particular form employed, e.g., base form, salt form, mixed salt, etc.) comprises a water-soluble polymer. Typically, to form a conjugate, a water-soluble polymer (in the form of a polymeric reagent) is coupled (under coupling conditions) to an active agent at an electrophile or nucleophile contained within the active agent. For example, a water-soluble polymer (again, in the form of a polymeric reagent with, for example, an activated ester) can be coupled to an active agent having one or more basic amine groups (i.e., an amine having a pK value of from about 7.5 to about 11.5) (determined after coupling).

Representative polymers include poly (alkylene glycols), poly (alkenyl alcohols), poly (vinyl pyrrolidone), poly (hydroxyalkyl methacrylamides), poly (hydroxyalkyl methacrylates), poly (saccharides), poly (α -hydroxy acids), poly (acrylic acids), poly (vinyl alcohols), polyphosphazenes, polyoxazolines, poly (N-acryloylmorpholin), or copolymers or terpolymers thereof₂CH₂O)_n-wherein n ranges from about 3 to about 2700 or even more, or preferably from about 25 to about 1300. Typically, in partial mixingThe weight average molecular weight of the water-soluble polymer in the acid salt ranges from about 100 daltons to about 150,000 daltons. Illustrative overall molecular weights of the conjugates can range from about 800 to about 80,000 daltons, or from about 900 to about 70,000 daltons. Additional representative molecular weights range from about 1,000 to about 40,000 daltons, or from about 5,000 to about 30,000 daltons, or from about 7500 daltons to about 25,000 daltons, or even from about 20,000 to about 80,000 daltons for higher molecular weight embodiments of the mixed salts of the ready-to-use fraction.

The water-soluble polymer may be in any of a number of geometries or forms, including linear, branched, forked. In exemplary embodiments, the polymer is generally linear or multi-armed. Commercially available water-soluble polymers are referred to simply as water-soluble polymers. In addition, the water-soluble polymer in activated form, which may be conventionally obtained, is referred to as a polymeric reagent (which may be coupled to an active agent without further modification or activation). A description of water-soluble polymers and polymeric reagents can be found in Nektar's Advanced PEGylation catalog, 2005-2006, "Polyethylene glycols and Derivatives for Advanced PEGylation," otherwise commercially available from NOF corporation and Keykem technologies, Inc., USA (Jenkem Technology USA).

Exemplary branching polymers having two polymer arms in a branching mode are shown below, commonly referred to as PEG-2 or mPEG-2:

whereinRefers to the location of additional atoms forming any functional group suitable for reaction with an electrophile or nucleophile contained within the active agent. Exemplary functional groups include NHS estersAldehydes, and the like.

For the polymer structures described herein that contain the variable "n," such variable corresponds to an integer and represents the number of monomeric subunits within the repeating monomeric structure of the polymer.

Exemplary configurations for use in preparing these conjugates are multi-armed water-soluble polymeric reagents having, for example, 3,4,5,6, or 8 polymeric arms, each optimally bearing one functional group. The multi-arm polymeric reagents may have any number of cores (e.g., polyol cores) from which the polymer arms originate. Exemplary polyol cores include glycerol, glycerol dimer (3,3' -oxydipropane-1, 2-diol) trimethylolpropane, saccharides (e.g., sorbitol or pentaerythritol, pentaerythritol dimer), and glycerol oligomers (e.g., hexaglycerol or 3- (2-hydroxy-3- (2-hydroxyethoxy) propoxy) propane-1, 2 diol, and other glycerol condensation products.

In an illustrative embodiment, the water soluble polymer is a 4-arm polymer as shown above, wherein n may range from about 20 to about 500, or from about 40 to about 500.

In the multi-arm embodiments described herein, each polymer arm typically has a molecular weight corresponding to one of: 200. 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000, 10000, 12,000, 15000, 17,500, 18,000, 19,000, 20,000 daltons or more. The overall molecular weight for the configuration of the multiarm polymer described herein (that is, the molecular weight of the multiarm 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 more.

The water-soluble polymer, e.g., PEG, can be covalently attached to the active agent via an intermediate linker. The linker may contain any number of atoms. In general, the linker has an atomic length that satisfies 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 of atoms, only the atoms contributing to the total distance are considered. For example, having a structureCH₂-C(O)-NH-CH₂CH₂ O-CH₂ CH₂ O-C(O)-OThe linker of (a) is considered to have a chain length of 11 atoms, since it is believed that the substituent does not contribute significantly to the length of the linker. Illustrative linkers include bifunctional compounds such as amino acids (e.g., alanine, glycine, isoleucine, leucine, phenylalanine, methionine, serine, cysteine, sarcosine, valine, lysine, and the like). The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid. Suitable linkers also include oligopeptides.

The above multi-arm structure is drawn primarily to illustrate the polymer core with the PEG chains attached thereto, and although not explicitly drawn, the final structure may optionally include an additional ethylene group, -CH, depending on the nature of the active agent and the attachment chemistry used₂CH₂The ethylene group is attached to an oxygen atom at the end of each polymer arm, and/or optionally contains any number of intermediate linker atoms to facilitate attachment to the active agentIs covalently attached. In a specific embodiment, each of the PEG arms described above further includes a carboxymethyl group, -CH₂-C (O) O-, the carboxymethyl group being covalently attached to the terminal oxygen atom.

Novel alkoxylation process for improved polymer compositions

As indicated above, water-soluble polymers effective, for example, in preparing conjugates (along with mixed salt forms thereof) with active agents are commercially available. However, as further described herein, methods for preparing water-soluble polymers are provided that are distinct from the methods described above for preparing water-soluble polymers, and are particularly suitable for preparing conjugates with active agents (as well as their salt and mixed salt forms).

In this regard, a process is provided that includes the step of alkoxylating a pre-isolated oxyalkylatable oligomer in a suitable solvent to form an oxyalkylated polymer product, wherein the pre-isolated oxyalkylatable oligomer has a known and defined weight average molecular weight of greater than 300 daltons (e.g., greater than 500 daltons).

Alkoxylation step in a novel alkoxylation process

The alkoxylation step is carried out using alkoxylation conditions such that the sequential addition of the monomers is accomplished by repeated reactions of the oxirane compound. Where the oxyalkylatable oligomer initially has one or more hydroxyl functional groups, one or more of the hydroxyl groups in the oxyalkylatable oligomer are converted to a reactive alkoxide by reaction with a strong base. The oxirane compound then reacts with an oxyalkylatable functional group (e.g., one reactive alkoxide), thereby not only adding to the reactive alkoxide, but doing so in a manner that also terminates in another reactive alkoxide. Thereafter, the repeated reaction of the oxirane compound at the reactive alkoxide end of the above added and reacted oxirane compound effectively produces a polymer chain.

While each of the one or more oxyalkylatable functional groups is preferably a hydroxyl group, other groups (e.g., amine, thiol, and carboxylic acid hydroxyl groups) may also be used as acceptable oxyalkylatable functional groups. Furthermore, due to the acidity of the hydrogen of the α carbon atom in aldehydes, ketones, nitriles, and amides, the adducts of these groups at the α carbon atom can be used as acceptable oxyalkylatable functional groups.

The oxirane compound contains an oxirane group and has the following formula:

wherein (with respect to the structure):

R¹selected from the group consisting of: h and alkyl (preferably lower alkyl when alkyl);

R²selected from the group consisting of: h and alkyl (preferably lower alkyl when alkyl);

R³selected from the group consisting of: h and alkyl (preferably lower alkyl when alkyl); and is

R⁴Selected from the group consisting of: h and alkyl (preferably lower alkyl when alkyl).

With respect to the chemical formula of the above oxirane compound, R is particularly preferred¹、R²、R³And R⁴Are each H, and preferably R¹、R²、R³And R⁴Only one of which is an alkyl group (e.g., methyl or ethyl), and the remaining substituents are H. Exemplary oxirane compounds are ethylene oxide, propylene oxide, and 1, 2-butylene oxide. Is added to causeThe amount of oxirane compound for optimal alkoxylation conditions depends on a number of factors, including the amount of the initial oxyalkylatable oligomer, the desired size of the resulting alkoxylated polymeric material, and the number of oxyalkylatable functional groups on the oxyalkylatable oligomer. Thus, when a larger alkoxylated polymeric material is desired, relatively more oxirane compound is present under the alkoxylation conditions. Similarly, if (Oa) represents the amount of oxirane compound needed to achieve a given size of polymer "growth" on a single oxyalkylatable functional group, then 2x (Oa) would be required for an oxyalkylatable oligomer with two oxyalkylatable functional groups, 3x (Oa) would be required for an oxyalkylatable oligomer with three oxyalkylatable functional groups, 4x (Oa) would be required for an oxyalkylatable oligomer with four oxyalkylatable functional groups, and so on. In all cases, one of ordinary skill in the art can determine the appropriate amount of oxirane compound required for the alkoxylation conditions by taking into account the desired molecular weight of the alkoxylated polymeric material and the following routine experimentation.

The alkoxylation conditions include the presence of a strong base. The purpose of the strong base is to deprotonate every acidic hydrogen present in the oxyalkylatable oligomer (e.g., the hydrogen of a hydroxyl group) and form an alkoxide ionic species (or ionic species for a non-hydroxyl oxyalkylatable functional group). Preferred strong bases for use as part of the alkoxylation conditions are: alkali metals (e.g., metallic potassium, metallic sodium), and alkali metal mixtures (e.g., sodium-potassium alloys); hydroxides (e.g., NaOH and KOH); and toy oxides (e.g., adducts of the following oxirane compounds). Other strong bases can be identified and used by one of ordinary skill in the art. For example, a given base may be used as a strong base herein if the strong base can form an alkoxide ionic species (or an ionic species for a non-hydroxyl oxyalkylatable functional group) and also provide a cation that does not interfere with the alkoxide ionic species so as to hinder (or effectively hinder by unrealistically slowing) the reaction of the alkoxide ionic species with the ethylene oxide molecule. The strong base is generally present in a small and calculated amount, which may fall within one or more of the following ranges: from 0.001 to 10.0 weight percent, based on the weight of the total reaction mixture; and from 0.01 to about 6.0 weight percent, based on the weight of the total reaction mixture.

The alkoxylation conditions include a temperature suitable for alkoxylation to occur. Exemplary temperatures that may be suitable for alkoxylation to occur include those falling within one or more of the following ranges: from 10 ℃ to 260 ℃; from 20 ℃ to 240 ℃; from 30 ℃ to 220 ℃; from 40 ℃ to 200 ℃; from 50 ℃ to 200 ℃; from 80 ℃ to 140 ℃; and from 100 ℃ to 120 ℃.

Alkoxylation conditions include pressures suitable for alkoxylation to occur. Exemplary pressures that may be suitable for alkoxylation to occur include those falling within 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. Further, the alkoxylation pressure may be about atmospheric pressure at sea level (e.g., 14.696 psi +/-10%).

In some examples, the alkoxylation conditions include the addition of an oxirane compound in liquid form. In some examples, the alkoxylation conditions include adding the oxirane compound in vapor form.

Alkoxylation conditions may include the use of a suitable solvent. Most preferably, the system in which the alkoxylation conditions occur will not include any components (including any solvent) that can be deprotonated (or remain substantially protonated under the conditions of pH, temperature, etc. at 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, xylene, mesitylene, tetrachloroethylene, anisole, dimethylacetamide, and mixtures thereof. Less desirable solvents for use as partial alkoxylation conditions (but nonetheless contemplated) are acetonitrile, phenylacetonitrile and ethyl acetate; in some examples, the alkoxylation conditions will not include any of acetonitrile, phenylacetonitrile, and ethyl acetate as a solvent.

In one or more embodiments of the present invention, when the alkoxylation conditions are conducted in the liquid phase, the alkoxylation conditions are conducted such that both the oxyalkylatable oligomer and the desired alkoxylated polymeric material formed from alkoxylating the oxyalkylatable oligomer have not only similar solubilities (and preferably, substantially the same solubilities) in the suitable solvent used, but are also substantially soluble in the suitable solvent. For example, in one or more embodiments, the oxyalkylatable oligomer will be substantially soluble in the solvent used under the oxyalkylation conditions, and the resulting oxyalkylated polymeric material will also be substantially soluble in the oxyalkylation conditions.

In one or more embodiments, substantially the same solubility of the alkoxylated oligomer and alkoxylated polymeric material in a suitable solvent is opposite to the solubility of the precursor molecule in a suitable solvent (e.g., used in preparing a previously isolated alkoxylated oligomer), wherein the precursor molecule may have a lower (or even substantially lower) solubility in the suitable solvent than the alkoxylated oligomer and/or alkoxylated polymeric material. By way of example only, both the alkoxylated oligomer and the alkoxylated polymeric material will have a pentaerythritol core and will be substantially soluble in toluene, but pentaerythritol itself has limited solubility in toluene.

It is particularly preferred that the solvent used in the alkoxylation conditions is toluene. The amount of toluene used in the reaction is greater than 25wt% and less than 75wt% of the reaction mixture based on the weight of the reaction mixture after the completion of the addition of the oxirane compound. One of ordinary skill in the art can calculate the starting amount of the solvent by taking into account the desired molecular weight of the polymer, the number of sites at which alkoxylation will occur, the weight of the oxyalkylatable oligomer used, and the like.

Preferably, the amount of toluene is measured such that the amount is sufficient to provide the desired alkoxylation conditions for the alkoxylated polymeric material.

Furthermore, it is particularly preferred that these alkoxylation conditions are substantially free of water. It is therefore preferred that these alkoxylation conditions have a water content of less than 100ppm, more preferably 50ppm, still more preferably 20ppm, much more preferably less than 14ppm, and even still more preferably less than 8 ppm.

Alkoxylation conditions occur in a suitable reactor, typically a stainless steel reactor.

In one or more embodiments, oxyalkylatable oligomer and/or precursor molecules lacking an isocyanate group attached to a carbon bearing one alpha hydrogen are acceptable. In one or more embodiments, the oxyalkylatable oligomer and/or precursor molecule prepared above lacks isocyanate groups.

Oxyalkylatable oligomers in a novel oxyalkylation process

The oxyalkylatable oligomer used in the new oxyalkylation process must have at least one oxyalkylatable functional group. However, the oxyalkylatable oligomer may have one, two, three, four, five, six, seven, eight or more oxyalkylatable functional groups, preferably one oxyalkylatable oligomer has from one to six oxyalkylatable functional groups.

As described above, the oxyalkylatable functional groups within the oxyalkylatable oligomer may be independently selected from the group consisting of: hydroxyl, carboxylic acid, amine, thiol, aldehyde, ketone, and nitrile. In those instances where more than one oxyalkylatable functional group is present within the oxyalkylatable oligomer, typically each oxyalkylatable functional group is the same (e.g., each oxyalkylatable functional group within the oxyalkylatable oligomer is a hydroxyl group), although examples of different oxyalkylatable functional groups within the same oxyalkylatable oligomer are also contemplated. Where the oxyalkylatable functional group is a hydroxyl group, it is preferred that the hydroxyl group is a primary hydroxyl group.

The oxyalkylatable oligomer may take any of a number of possible geometries. For example, the oxyalkylatable oligomer may be linear. In one example of a linear oxyalkylatable oligomer, one end of the linear oxyalkylatable oligomer is a relatively inert functional group (e.g., a capping group) and the other end is an oxyalkylatable functional group (e.g., a hydroxyl group). An exemplary oxyalkylatable oligomer of this structure is methoxy-PEG-OH, or simply mPEG, where one terminus is the relatively inert methoxy group and the other terminus is the hydroxyl group. The structure of mPEG is given below.

CH₃O-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-OH

(wherein n is an integer from 13 to 100 only for the immediately preceding structures).

Another example of a linear geometry that can be employed by the oxyalkylatable oligomer is a linear organic polymer bearing oxyalkylatable functional groups (the same or different) at each end. Exemplary oxyalkylatable oligomers of this structure are alpha-, omega-dihydroxy poly (ethylene glycol), or

HO-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-OH

(wherein n is an integer from 13 to 100 only for the immediately preceding structures),

it can be represented by the simplified form HO-PEG-OH, wherein it is understood that the-PEG-symbol represents the following structural unit:

-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-

(wherein n is an integer from 13 to 100 only for the immediately preceding structures),

another geometry that the oxyalkylatable oligomer may have is a "multi-armed" or branched structure. With respect to this branched structure, one or more atoms in the oxyalkylatable oligomer serve as "branch point atoms" through which two, three, four or more (but typically two, three or four) different series of repeating monomers or "arms" are attached (either directly or through one or more atoms). At a minimum, "multi-arm" structures as used herein have three or more different arms, but may have as many as four, five, six, seven, eight, nine or more arms, with multi-arm structures of 4 to 8 arms being preferred (e.g., 4-arm, 5-arm, 6-arm, and 8-arm structures).

Exemplary multi-arm structures of oxyalkylatable oligomers are provided below:

wherein (for the immediately preceding structure only) the average value of n is from 1 to 50, such as from 10 to 50, or otherwise defined such that the molecular weight of the structure is from 300 daltons to 9,000 daltons (e.g., from about 500 daltons to 5,000 daltons);

wherein (for the immediately preceding structure only) the average value of n is from 2 to 50, such as from 10 to 50, or otherwise defined such that the molecular weight of the structure is from 300 daltons to 9,000 daltons (e.g., from about 500 daltons to 5,000 daltons);

wherein (for the immediately preceding structure only) the average value of n is from 2 to 35, such as from 8 to about 40, or otherwise defined such that the molecular weight of the structure is from 750 daltons to 9,500 daltons (e.g., from about 500 daltons to 5,000 daltons); and is

Where (for the immediately preceding structure only) n has an average value of from 2 to 35, such as from 5 to 35, or is otherwise defined such that the molecular weight of the structure is from 1,000 daltons to 13,000 daltons (e.g., from about 500 daltons to 5,000 daltons).

For each of the four immediately preceding configurations, it is preferred that the value of n be substantially the same in each instance. Thus, it is preferred that when considering all values of n for a given oxyalkylatable oligomer, all values of n for that oxyalkylatable oligomer are within three standard deviations, more preferably within two standard deviations, and still more preferably within one standard deviation.

In terms of molecular weight of the oxyalkylatable oligomer, the oxyalkylatable oligomer will have a known and defined weight average molecular weight. For use herein, the weight average molecular weight of an oxyalkylatable oligomer is only known and defined when the oxyalkylated oligomer is isolated from the synthetic environment in which it was generated. Exemplary weight average molecular weights of the oxyalkylatable oligomers will fall within one or more of the following ranges: greater than 300 daltons; greater than 500 daltons; from 300 daltons to 15,000 daltons; from 500 daltons to 5,000 daltons; from 300 daltons to 10,000 daltons; from 500 daltons to 4,000 daltons; from 300 daltons to 5,000 daltons; from 500 daltons to 3,000 daltons; from 300 daltons to 2,000 daltons; from 500 daltons to 2,000 daltons; from 300 daltons to 1,000 daltons; from 500 daltons to 1,000 daltons; from 1,000 daltons to 10,000 daltons; from 1,000 daltons to 5,000 daltons; from 1,000 daltons to 4,000 daltons; from 1,000 daltons to 3,000 daltons; from 1,000 daltons to 2,000 daltons; from 1,500 daltons to 15,000 daltons; from 1,500 daltons to 5,000 daltons; from 1,500 daltons to 10,000 daltons; from 1,500 daltons to 4,000 daltons; from 1,500 daltons to 3,000 daltons; from 1,500 daltons to 2,000 daltons; from 2,000 daltons to 5,000 daltons; from 2,000 daltons to 4,000 daltons; and from 2,000 daltons to 3,000 daltons.

For the purposes of the present invention, preference is given to isolating the oxyalkylatable oligomers beforehand. By pre-isolated is meant that the oxyalkylatable oligomer is present outside of the synthetic environment in which it is produced and isolated therefrom (most typically outside of the oxyalkylation conditions used to prepare the oxyalkylatable oligomer) and may optionally be stored for a longer period of time, or optionally stored for a shorter period of time without substantial change for subsequent use. Thus, for example, if it is enclosed in an inert environment, the oxyalkylatable oligomer is isolated beforehand. In this regard, the pre-isolated oxyalkylatable oligomer may be packaged in a container substantially devoid (e.g., less than 0.1 wt%) of oxirane compounds. Moreover, the previously isolated oxyalkylatable oligomer did not change its molecular weight by more than 10% over the course of 15 days. Thus, in one or more embodiments of the invention, the concept of "pre-isolated" as opposed to, for example, where uninterrupted and continuous alkoxylation reactions from precursor molecules are permitted, wherein the alkoxylation reactions result in structures corresponding to the oxyalkylatable oligomer, resulting in structures corresponding to the alkoxylated polymeric material, the concept of "pre-isolated" requires that the oxyalkylatable oligomer be present away from the conditions under which it is formed. However, according to the present invention, the pre-isolated oxyalkylatable oligomer, once added (as a separate step) to the oxyalkylation conditions, will undergo an oxyalkylation step.

Sources of oxyalkylatable oligomers in a novel oxyalkylation process

The oxyalkylatable oligomer may be obtained via synthetic means. In this regard, the oxyalkylatable oligomer is prepared by (a) oxyalkylating a precursor molecule having a molecular weight of less than 300 daltons (e.g., less than 500 daltons) to form a reaction mixture comprising the oxyalkylatable oligomer or prepolymer, and (b) separating the oxyalkylatable oligomer from the reaction mixture. The step of alkoxylating the precursor molecule largely meets the conditions and requirements of the alkoxylation step discussed above. The step of isolating the oxyalkylatable oligomer may be carried out using any art-known procedure, but may include allowing all of the oxirane compound to be consumed in the reaction, actively conducting a quenching step, and isolating the final reaction mixture by art-known methods (including, for example, distilling off all volatiles, removing solid reaction by-products by filtration or washing and applying chromatographic means).

In addition, the oxyalkylatable oligomers may be obtained from commercial sources. Exemplary commercial sources include NOF corporation (Tokyo, Japan) which provides SUNBRIGHTA poly (ethylene glycol) having a high degree of polymerization,GL glycerol, Tri (ethylene glycol) Ether, SUNBRIGHTPentaerythritol, tetra (ethylene glycol) ether,DG diglycerol, tetramer (ethylene glycol) ether, and SUNBRIGHTHexaglycerol, octapoly (ethylene glycol) etherAlkoxylated oligomers are also known. Preferred oxyalkylatable oligomers include those having SUNBRIGHTPentaerythritol, tetrapoly (ethylene glycol) ether (which has a weight average molecular weight of about 2,000 daltons) andDG-2000 diglycerol, tetrameric (ethylene glycol) ether (which has a weight average molecular weight of about 2,000 daltons).

The precursor molecule can be any small molecule having one or more oxyalkylatable functional groups (e.g., a molecular weight less than the weight average molecular weight of the oxyalkylatable oligomer).

Exemplary precursor molecules include polyols, which are small molecules (typically having a molecular weight of less than 300 daltons, e.g., less than 500 daltons) having a plurality of available hydroxyl groups. Depending on the number of polymer arms desired in the oxyalkylatable oligomer or prepolymer, the polyol used as a precursor molecule will typically comprise from 3 to about 25 hydroxyl groups, preferably from about 3 to about 22 hydroxyl groups, most preferably from about 4 to about 12 hydroxyl groups. Preferred polyols include glycerol oligomers or polymers such as, for example, hexaglycerol, pentaerythritol, and oligomers or polymers thereof (e.g., dipentaerythritol, tripentaerythritol, tetrapentaerythritol, and ethoxylated forms of pentaerythritol), and sugar-derived alcohols (e.g., sorbitol, arabitol, and mannitol). Moreover, many commercially available polyols, such as different isomers of inositol (i.e., 1,2,3,4,5, 6-hexahydroxycyclohexane), 2-bis (hydroxymethyl) -1-butanol, (2-amino-2- (hydroxymethyl) -1, 3-propanediol (TRIS), 2- [ bis (2-hydroxyethyl) amino ] -2- (hydroxymethyl) -1, 3-propanediol, { [ 2-hydroxy-1, 1-bis (hydroxymethyl) ethyl ] amino } acetic acid (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 } -1-butanesulfonic acid, and 2- [ bis (2-hydroxyethyl) amino ] -2- (hydroxymethyl) -1, 3-propanediol hydrochloride may be used as an acceptable precursor molecule. In those cases where the precursor molecule has ionizable groups or groups that would interfere with the alkoxylation step, those ionizable groups must be protected or modified prior to conducting the alkoxylation step.

Exemplary preferred precursor molecules include those selected from the group consisting of: glycerol, diglycerol, triglycerol, hexaglycerol, mannitol, sorbitol, pentaerythritol, dipentaerythritol, and tripentaerythritol.

In one or more embodiments of the present invention, it is preferred that neither the pre-isolated oxyalkylatable oligomer nor the oxyalkylated polymer product have the oxyalkylatable functional groups (e.g., hydroxyl groups) of the precursor molecule.

Alkoxylated polymeric materials produced by novel alkoxylation processes

The alkoxylated polymeric material prepared under the process described herein will have a basic architecture corresponding to the structure of the alkoxylated oligomer (i.e., linear alkoxylated oligomer results in a linear alkoxylated polymeric material, four-arm alkoxylated oligomer results in a four-arm alkoxylated polymeric material, etc.). As a result, the alkoxylated polymeric material will adopt any of a variety of possible geometries, including linear, branched, and multiarmed.

With respect to branched structures, a branched alkoxylated polymeric material will have three or more different arms, but may have as many as four, five, six, seven, eight, nine or more arms, with 4 to 8 arm branching structures being preferred (e.g., 4 arm branching structures, 5 arm branching structures, 6 arm branching structures, and 8 arm branching structures).

Exemplary branched structures of alkoxylated polymeric materials are provided below:

wherein (for the immediately preceding structure only) the average value of n satisfies one or more of the following ranges: from 10 to 1,000; from 10 to 500; from 10 to 250; from 50 to 1000; from 50 to 250; and from 50 to 120 (or alternatively define n such that the molecular weight of the structure is from 2,000 daltons to 180,000 daltons, e.g., from 2,000 daltons to 120,000 daltons);

where (for the immediately preceding structure only) the average value of n satisfies one or more of the following ranges: from 10 to 1,000; from 10 to 500; from 10 to 250; from 50 to 1,000; from 50 to 250; and from 50 to 120 (or alternatively define n such that the molecular weight of the structure is from 2,000 daltons to 180,000 daltons, e.g., from 2,000 daltons to 120,000 daltons);

wherein (for the immediately preceding structure only) the average value of n 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 otherwise defined such that the molecular weight of the structure is from 3,000 daltons to 200,000 daltons, e.g., from 12,000 daltons to 200,000 daltons); and is

Wherein (for the immediately preceding structure only) the average value of n satisfies one or more of the following ranges: from 10 to 600 and from 35 to 600 (or otherwise defined such that the molecular weight of the structure is from 4,000 daltons to 215,000 daltons, e.g., from 12,000 daltons to 215,000 daltons).

For each of the four structures just provided, it is preferred that the value of n be substantially the same in each instance. Thus, it is preferred that when considering all values of n for a given alkoxylated polymeric material, all values of n for the alkoxylated polymeric material, the oxyalkylatable oligomer or the prepolymer are within three standard deviations, more preferably within two standard deviations, and still more preferably within one standard deviation.

The alkoxylated polymeric material will have a known and defined number average molecular weight in terms of the molecular weight of the alkoxylated polymeric material. For use herein, the number average molecular weight will only be known and defined for materials isolated from the synthetic environment in which it is produced.

The overall molecular weight of the alkoxylated polymer product may be one that is suitable for the intended target. Acceptable molecular weights for any given target can be determined experimentally with error through routine experimentation. Exemplary molecular weights of the alkoxylated polymer product will have number average molecular weights falling within one or more of the following ranges: from 2,000 daltons to 215,000 daltons; from 5,000 daltons to 215,000 daltons; from 5,000 daltons to 150,000 daltons; from 5,000 daltons to 100,000 daltons; from 5,000 daltons to 80,000 daltons; from 6,000 daltons to 80,000 daltons; from 7,500 daltons to 80,000 daltons; from 9,000 daltons to 80,000 daltons; from 10,000 daltons to 80,000 daltons; from 12,000 daltons to 80,000 daltons; from 15,000 daltons to 80,000 daltons; from 20,000 daltons to 80,000 daltons; from 25,000 daltons to 80,000 daltons; from 30,000 daltons to 80,000 daltons; from 40,000 daltons to 80,000 daltons; from 6,000 daltons to 60,000 daltons; from 7,500 daltons to 60,000 daltons; from 9,000 daltons to 60,000 daltons; from 10,000 daltons to 60,000 daltons; from 12,000 daltons to 60,000 daltons; from 15,000 daltons to 60,000 daltons; from 20,000 daltons to 60,000 daltons; from 25,000 daltons to 60,000 daltons; from 30,000 daltons to 60,000; from 6,000 daltons to 40,000 daltons; from 9,000 daltons to 40,000 daltons; from 10,000 daltons to 40,000 daltons; from 15,000 daltons to 40,000 daltons; from 19,000 daltons to 40,000 daltons; from 15,000 daltons to 25,000 daltons; and from 18,000 daltons to 22,000 daltons.

For any given alkoxylated polymeric material, an optional step may be performed to further convert the alkoxylated polymeric material so that it bears the specified reactive groups to form a polymeric reagent. Thus, the alkoxylated polymeric material may be functionalized to include a reactive group (e.g., carboxylic acid, active ester, amine, thiol, maleimide, aldehyde, ketone, etc.) using techniques well known in the art.

In carrying out an optional step for further converting the alkoxylated product to bear a specific reactive group, such optional step is carried out in a suitable solvent. One of ordinary skill in the art can determine whether any particular solvent is suitable for any given reaction step. However, in general, the solvent is preferably a non-polar solvent or a polar solvent. Non-limiting examples of non-polar solvents include benzene, xylene, and toluene. Exemplary polar solvents include, but are not limited to, dioxane, Tetrahydrofuran (THF), t-butanol, DMSO (dimethyl sulfoxide), HMPA (hexamethylphosphoramide), DMF (dimethylformamide), DMA (dimethylacetamide), and NMP (N-methylpyrrolidone).

Other compositions of alkoxylated polymeric materials

Another aspect of the invention provided herein is a composition comprising an alkoxylated polymeric material, which encompasses not only any composition comprising an alkoxylated polymeric material, but also compositions wherein the alkoxylated polymeric material is further converted, for example, to a polymeric reagent, as well as compositions of conjugates formed by coupling such polymeric reagents with an active agent. Among other benefits, a benefit of the methods described herein is the ability to achieve compositions containing high purity alkoxylated polymeric materials. These compositions may be characterized as having: both a substantially low content of high molecular weight impurities (e.g., polymer-containing species having a molecular weight greater than the molecular weight of the desired alkoxylated polymeric material) and a low content of low molecular weight diol impurities (i.e., HO-PEG-OH), either impurity type (and preferably both impurity types) totaling less than 8wt%, and more preferably less than 2 wt%. Additionally or alternatively, the compositions may also be characterized by alkoxylated polymeric material having a purity of greater than 92wt%, greater than 93wt%, or greater than 94wt%, greater than 95wt%, preferably greater than 96wt%, and more preferably greater than 97wt% (along with compositions comprising polymeric reagents formed from the alkoxylated polymeric material, and compositions self-coupling conjugates formed from such polymeric reagents and active agents). The alkoxylated polymeric material may be characterized using Gel Permeation Chromatography (GPC) and Gel Filtration Chromatography (GFC). Those chromatographic methods allow the composition to be separated into its components according to molecular weight. Exemplary traces of GFC for the products described in examples 8 and 9 are provided as fig. 7 and 8.

Exemplary uses of alkoxylated polymeric materials and compositions formed therefrom

The alkoxylated polymeric materials provided herein are useful for coupling to, for example, an active agent, as well as those alkoxylated polymeric products (referred to herein as "polymeric reagents") that have been further modified to carry a particular reactive group. Preferred groups of bioactive 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 reaction with the polymeric reagents described herein are known to those of ordinary skill in the art. Accordingly, the present invention provides a method for making a conjugate comprising the step of contacting an active agent with a polymeric reagent described herein under coupling conditions.

Suitable coupling conditions are time, temperature, pH, reagent concentration, one or more functional groups of the reagent, available functional groups on the active agent, solvent, and the like sufficient to affect coupling between the polymeric reagent and an active agent. As is known in the art, the specific conditions depend on, among other things, the active agent, the type of coupling desired, the presence of other materials in the reaction mixture, and the like. Conditions sufficient to effect coupling in any particular case can be determined by one of ordinary skill in the art upon reading the disclosure herein, by reference to relevant literature, and/or by routine experimentation.

For example, when the polymeric reagent contains an active ester of N-hydroxysuccinimide (e.g., succinimide succinate, succinimide propionate, and succinimide butyrate) and the active agent contains an amine group, the coupling can be carried out at room temperature at a pH of from about 7.5 to about 9.5. In addition, when the polymeric reagent contains a vinylsulfone reactive group or a maleimide group and the pharmacologically active agent contains a thiol group, the conjugation can be carried out at room temperature at a pH of about 7 to about 8.5. Furthermore, when the reactive group associated with the polymeric reagent is an aldehyde or ketone and the pharmacologically active agent contains a primary amine, the coupling may be by reductive amination in which the primary amine of the pharmacologically active agent reacts with the aldehyde or ketone of the polymer. At a pH of from about 6 to about 9.5, reductive amination initially results in a conjugate in which the pharmaceutically active agent and the polymer are linked via an imine bond. Followed by a suitable reducing agent (e.g., NaCNBH)₃) The imine bond-containing conjugate is treated to reduce the imine to a secondary amine. For additional information concerning these and other coupling reactions, reference may be made to "Bioconjugate Techniques" by Hermanson (Hemanson), academic Press, 1996.

Exemplary coupling conditions include conducting the coupling 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 for 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 coupling can occur is typically, although not necessarily, in the range of from about 0 ℃ to about 40 ℃, and is generally at room temperature or lower. These coupling reactions are typically carried out using phosphate buffered solutions, sodium acetate, or similar systems.

With respect to reagent concentration, an excess of polymeric reagent is typically combined with the active agent. In some cases, however, it is preferred to have a stoichiometric amount of reactive groups on the polymeric reagent relative to the reactive groups of the reactive reagent. Thus, for example, one mole of polymeric reagent bearing four reactive groups is combined with four moles of active agent. Exemplary ratios of reactive groups of the polymeric reagent to the active agent include about 1:1 (reactive groups of the polymeric reagent: 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 coupling reaction is allowed to proceed until substantially no further coupling occurs, which can be determined generally by monitoring the progress of the reaction over time.

The progress of the reaction can be monitored by extracting aliquots from the reaction mixture at different time points and analyzing the reaction mixture by chromatographic methods, SDS-PAGE or MALDI-TOF mass spectrometry, NMR, IR, or any other suitable analytical method. Once a steady state is reached with respect to the amount of conjugate formed or the amount of unconjugated polymeric reagent remaining, the reaction is assumed to be complete. Typically, the time of the coupling reaction is anywhere from a few minutes to several hours (e.g., from 5 minutes to 24 hours or more). Preferably, but not necessarily, the mixture of products formed is purified to separate out excess active agent, strong base, condensing agent and reaction by-products and solvent. The resulting conjugates can then be further characterized using analytical methods (e.g., chromatographic methods, spectroscopic methods, MALDI, capillary electrophoresis, and/or gel electrophoresis). The polymer active agent conjugate can be purified to obtain/isolate different conjugated species.

With respect to the active agent, the alkoxylated polymeric material, and the polymeric reagent prepared from the alkoxylated polymeric material, can be combined under suitable coupling conditions to form the conjugate. In this regard, exemplary active agents may be active agents selected from the group consisting of: small molecule drugs, oligopeptides, peptides, and proteins. Active agents for use herein include, but are not limited to, the following: doxorubicin, gamma-aminobutyric acid (GABA), amiodarone, amitriptyline, azithromycin, benzphetamine, brompheniramine, carbinoxamine, calcitonin, chlorambucil, chloroprocaine, chloroquine, chlorpheniramine, cinnarizine, clarithromycin, clomiphene, cyclobenzaprine, cyclopentolate, cyclophosphamide, dacarbazine, daunomycin, demecycline, dibucaine, dicyclevine, diethylpropion, diltiazem, dimenhydrinate, diphenhydramine, piripramine, doxepin, doxycycline, doxylamine, dipyridamole, EDTA, erythromycin, flurazepam, methicone, hydroxychloroquine, imipramine, insulin, irinotecan, levomethadol, lidocaine, salpingine, mechlorethamine, melphalan, levopromethamine, levopromethazine, methotrexate, metoclopramide, minoxidine, naftifine, carpidine, mechlorethamine, and the like, Nizatidine, oxfenadrine, oxybutynin chloride, oxytetracycline, phenoxybenzamine, phentolamine, procainamide, procaine, promazine, promethazine, proparacaine, propoxycaine, dexpropoxyphene, ranitidine, tamoxifen, terbinafine, tetracaine, tetracycline, tramadol, trifluoroperazine, trimetprazine, trimethylbenzamide, trimipramine, tripelennamine, saprolactine, oleandomycin acetate, tyramine, uramustine, verapamil, and vasopressin.

Other exemplary active agents include those selected from the group consisting of: alvastigmine, amoxapine, astemizole, atropine, azithromycin, benazepril, benztropine, bicyclohexanol, bupivacaine, buprenorphine, buspirone, butorphanol, caffeine, camptothecin and molecules belonging to the camptothecin family, ceftriaxone, chlorpropham, ciprofloxacin, cladribine, clemastine, clindamycin, clofazimine, clozapine, cocaine, codeine, cyproheptadine, desipramine, hydridotetramine, difenidol, diphenoxylate, dipyridamole, docetaxel, doxepin, ergotamine, famciclovir, fentanyl, flavopiperite, fludarabine, fluvastatin, ganciclovir, granisetron, meperidine, flupiprolidine, homatropine, hydrocodone, hydromorphone, hydroxyzine, hyoscyamine, oxymatrimine, ketoconazole, ketorolac, troostine, levocabastine, mepiridine, doxepin, doxepinastine, doxycycline, clomazone, clomipramine, clozapine, Levorphanol, lincomycin, lomefloxacin, loperamide, losartan, loxapine, mazindol, chlorphenirazine, meperidine, mepivacaine, thiamphenizine, meglumine, urotropine, methimazole, levomepromazine, mesquite, metronidazole, minoxidil, mitomycin c, molindone, morphinan, nefazodone, nalbuphine, nalidixic acid, nalmefene, naloxone, naltrexone, naphazoline, nedocromil, nicotine, norfloxacin, ofloxacin, ondansetron, oxycodone, oxymorphone, paclitaxel, pentazocine, pentoxazine, pentoxifylline, phenazine, perphenazine, physostigmine, pilocarpine, pimozide, pramoxine, prazosin, prochlorperazine, procaine, quinidine, quinine, theobromine, vitamin b 78, carpidine, 2, rifolidone, rifabune, rifabutin, penoxepirubicin, pemphitin, pemphigenidine, pemphigomine, pramine, valorine, valfentanil, valorine, tacrine, terazosin, terconazole, terfenadine, thioridazine, thiothixene, ticlopidine, timolol, tolazamide, tolmetin, trazodone, thiethylperazine, trifluoroperazine, dipheny, isobutylazine, trimipramine, tubocurarine, vecuronium, vidarabine, vinblastine, vincristine and vinorelbine.

Still other exemplary active agents include those selected from the group consisting of: acetazolamide, Alvastigmine, Acyclovir, adenosine phosphate, allopurinol, alprazolam, oxychloride, amipridone, Acclonidine, azatadine, aztreonam, bisacodyl, bleomycin, brompheniramine, buspirone, butoconazole, molecules within the camptothecin and camptothecin families, carbinoxamine, cefamandole, ceftizoxime, cefmetazole, cefonicid, cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone, cefapine, chloroquine, chlorpheniramine, cimetidine, cladribine, clotrimazole, doxazosin, dipyridamole, doxazosin, econazole, enoxacin, estazolam, ethionamide, famciclovir, famotidine, fluconazole, fludarabine, folic acid, ganciclovir, bichloroquine, isoniazide, idoxuridine, idoxuriconazole, idonazole, idoxuriconazole, itraconazole, idoxuriconazole, amisol, amitraquinconazole, iodine-enriched, Ketoconazole, lamotrigine, lansoprazole, loratadine, losartan, mebendazole, mercaptopurine, methotrexate, metronidazole, miconazole, midazolam, minoxidil, nefazodone, nalidixic acid, nicotinic acid, nicotine, nizatidine, omeprazole, oxaprozin, oxiconazole, papaverine, pentostatin, phenazopyridine, pilocarpine, piroxicam, prazosin, primaquine, pyrazinamide, pyrimethamine, pyridoxine, quinidine, quinine, ribavirin, rifampin, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, terazosin, thiabendazole, thiamine, thioguanine, timolol, trazodone, triamterene, triazoline, trimethadione, trimethoprim, tripelenide, tripelennamide, tropinamide, tolnafide, and arabinose.

Still other exemplary active agents include those belonging to the camptothecin family of molecules. For example, the active agent may have the following general structure:

wherein R is₁、R₂、R₃、R₄And R₅Each independently selected from the group consisting of: hydrogen; halogen; an acyl group; alkyl (e.g., C1-C6 alkyl)) (ii) a A substituted alkyl group; alkoxy (e.g., C1-C6 alkoxy); a substituted alkoxy group; an alkenyl group; an alkynyl group; a cycloalkyl group; a hydroxyl group; a cyano group; a nitro group; an azide group; an amido group; hydrazine; an amino group; substituted amino groups (e.g., monoalkylamino and dialkylamino); a hydroxycarbonyl group; an alkoxycarbonyl group; an alkylcarbonyloxy group; an alkylcarbonylamino group; carbamoylalkoxy; an arylsulfonyloxy group; an alkylsulfonyloxy group; -C (R)₇)=N-(O)_i-R₈Wherein R is₇Is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, and R is₈Is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C (O) O-, wherein R₉Is halogen, amino, substituted amino, heterocycle, substituted heterocycle, or R₁₀-O-(CH₂)_m-, where m is an integer from 1 to 10 and R₁₀Is alkyl, phenyl, substituted phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or substituted heterocycle; or R₂And R₃Together or R₃And R₄Together form a substituted or unsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy group; r₆Is H OR OR 'wherein R' is alkyl, alkenyl, cycloalkyl, haloalkyl, OR hydroxyalkyl. Although not shown, in the just above structure, analogs having a hydroxyl group corresponding to a position other than position 20 (e.g., position 10 or 11, etc.) are included within the possible active agents.

One exemplary active agent is irinotecan.

Irinotecan

Another exemplary active agent is 7-ethyl-10-hydroxy-camptothecin (SN-38), the structure of which is shown below.

7-ethyl-10-hydroxy-camptothecin

Still other exemplary classes of active agents include those that belong to the taxane family of molecules. An exemplary active agent from this class of molecules is docetaxel, where the H:

the polymeric reagents described herein can be attached, either covalently or non-covalently, to a variety of entities including membranes, chemical separation and purification surfaces, solid supports, metal surfaces (e.g., gold, titanium, tantalum, niobium, aluminum, steel, and oxides thereof), silicon oxide, macromolecules (e.g., proteins, polypeptides, etc.), and small molecules. Additionally, these polymeric reagents can also be used in biochemical sensors, bioelectronic switches, and gates. These polymeric reagents can also be used as supports for peptide synthesis, for preparing polymer-coated surfaces and polymer grafts, for preparing polymer ligand conjugates for affinity partitioning, for preparing crosslinked or non-crosslinked hydrogels, and for preparing polymer cofactor adducts for bioreactors.

Optionally, the conjugate may be provided as a pharmaceutical composition for veterinary and for human medical use. Such a pharmaceutical composition is prepared by combining the conjugate with one or more pharmaceutically acceptable excipients, and optionally any other therapeutic ingredients.

Exemplary pharmaceutically acceptable excipients include, but are not limited to, those from the group consisting of: carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

A carbohydrate, e.g., sugar, derivatized sugar (e.g., sugar alcohol, aldonic acid), esterified sugar, and/or 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, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol (pyranosyl sorbitol), inositol, and the like.

The excipient may also include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and combinations thereof.

The composition may also include an antimicrobial agent for preventing or deterring 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, thimerosal (thimersol), and combinations thereof.

An antioxidant may also be present in the composition. Antioxidants are used to prevent oxidation and thus prevent deterioration of the conjugate or other components of the formulation. 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 thereof.

A surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as "tween 20" and "tween 80"; and pluronics such as F68 and F88 (both available from BASF (BASF corporation), olivary mountain, new jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposome form); fatty acids and fatty acid esters; steroids, such as cholesterol; and chelating agents such as EDTA, zinc and other such suitable cations.

The acid or base may be present in the composition as an excipient. Non-limiting examples of acids that may be used include those selected from the group consisting of: hydrochloric acid, acetic acid, phosphoric acid, citric acid, maleic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, but are not limited to, bases selected from the group consisting of: sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium fumarate, sodium sulfate, potassium fumarate, and combinations thereof.

The amount of conjugate in the composition (i.e., the conjugate formed between the active agent and the polymeric agent) will vary depending on a number of factors, but the composition is optimally a therapeutically effective dose when stored in a unit dose container, such as a vial. Alternatively, the pharmaceutical formulation may be contained in a syringe. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of conjugate in order to determine which amount produces a clinically desirable endpoint.

The amount of any individual excipient in the composition will vary depending on the activity of the excipient and the particular needs of the composition. Typically, the optimum amount of any individual excipient is determined by routine experimentation, i.e., by preparing compositions containing varying amounts of excipient (ranging from low to high), examining stability and other parameters, and then determining the range at which optimum performance is obtained without significant adverse effects.

However, generally 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 15% to about 95% by weight of excipient, with a concentration of less than 30% by weight being most preferred.

These aforementioned pharmaceutically acceptable excipients are described in "Remington: the science & Practice of Pharmacy, 19 th edition, Williams & Williams corporation, (1995), the "physicians' Desk Reference", 52 th edition, medical economics (medical economics), Montvale (monte waler), NJ (1998), and Kibbe, a.h., Handbook of pharmaceutical Excipients, 3 rd edition, American pharmaceutical association (American society of Pharmacy), washington, 2000.

These pharmaceutically acceptable compositions encompass all types of formulations and especially those suitable for injection, for example as a powder or lyophilizate which may be reconstituted with a liquid (lyoprotected). Examples of suitable diluents for reconstituting the solid composition 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 respect to liquid pharmaceutical compositions, solutions, and suspensions are contemplated.

The compositions of one or more embodiments of the invention are typically (although not necessarily) administered by injection and are thus generally liquid solutions or suspensions immediately prior to administration. The pharmaceutical preparation may also take other forms such as syrups, emulsions, ointments, tablets, powders, and the like. Other modes of administration are also included, such as pulmonary, rectal, transdermal, transmucosal, buccal, intrathecal, subcutaneous, intraarterial, and the like.

The invention also provides methods of administering a conjugate as provided herein to a patient having a condition responsive to treatment with the conjugate. The method comprises administering to the patient, generally by injection, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical composition). As noted above, these conjugates can be administered by intravenous injection, parenteral injection. Suitable formulation types for parenteral administration include: ready for injection solutions, dry powders for combination with a solvent prior to use, ready for injection suspensions, dry insoluble compositions for combination with a carrier prior to use, and emulsions and liquid concentrates, among others, for dilution prior to administration.

The method of administration can be used to treat any condition that can be treated or prevented by administration of the conjugate. Those of ordinary skill in the art understand which conditions a particular conjugate may effectively treat. Advantageously, the conjugate can be administered to the patient before, simultaneously with, or after the administration of the other active agent.

The actual dose to be administered may vary depending on the age, weight and general condition of the subject, as well as the severity of the condition to be treated, the judgment of the health professional, and the conjugate to be administered. Therapeutically effective amounts are known to those of ordinary skill in the art and/or described in the relevant documents and literature. In general, a therapeutically effective amount ranges from about 0.001mg to 100mg, preferably from 0.01 mg/day to 75 mg/day, and more preferably from 0.10 mg/day to 50 mg/day. A given dose may be administered periodically until, for example, the associated symptoms are reduced and/or eliminated altogether.

The unit dose of any given conjugate (again, preferably provided as part of a pharmaceutical formulation) may be determined on a regimen that varies depending upon the judgment of the clinician, the needs of the patient, and the like. Specific dosing regimens are known to those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing regimens include (without limitation): once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint is reached, administration of the composition is stopped.

One advantage afforded to certain conjugates described herein is that: when a hydrolytically degradable linkage is included between the residue of the active agent moiety and the water soluble polymer, the individual water soluble polymer moieties can be cleaved. Such a result is advantageous when clearance from the body is potentially a problem due to the size of the polymer. Optimally, cleavage of each water-soluble polymer moiety is facilitated by the use of physiologically cleavable and/or enzymatically degradable linkages (e.g., amide, carbonate, or ester-containing linkages). In this way, the clearance of the conjugate (by cleavage of the individual water-soluble polymer moieties) can be tailored by selecting the molecular size of the polymer and the type of functional group that will provide the desired clearance properties. One of ordinary skill in the art can determine the appropriate molecular size of the polymer and cleavable functional groups. For example, one of ordinary skill in the art can determine an appropriate molecular size and cleavable functional group by first preparing a plurality of polymer derivatives having different polymer weights and cleavable functional groups using routine experimentation, and then obtain the clearance profile (e.g., by periodic blood or urine sampling) by administering the polymer derivatives to a patient and periodically taking blood and/or urine samples. Once a series of clearance profiles for each tested conjugate is obtained, a suitable conjugate can be identified.

Mixed salts-considerations concerning active agents, "D"

As indicated above, the water-soluble polymer conjugates and compositions containing these conjugates can be provided as mixed salts. In the context of mixed salt conjugates and compositions, the active agent is a small molecule drug, oligopeptide, peptide, or protein, i.e., contains at least one basic nitrogen atom (e.g., an amine group (e.g., an amine or other basic nitrogen-containing group not coupled to a water-soluble polymer)) when coupled to a water-soluble polymer. In the mixed salts, the basic nitrogen atoms are each individually protonated or unprotonated, wherein the protonated nitrogen atoms are present as acid salts with two different anions.

Active agents containing at least one amine group or basic nitrogen atom suitable for providing a mixed acid salt as described herein include, but are not limited to, the following: doxorubicin, gamma-aminobutyric acid (GABA), amiodarone, amitriptyline, azithromycin, benzphetamine, brompheniramine, carbinoxamine, calcitonin, chlorambucil, chloroprocaine, chloroquine, chlorpheniramine, cinnarizine, clarithromycin, clomiphene, cyclobenzaprine, cyclopentolate, cyclophosphamide, dacarbazine, daunomycin, demecycline, dibucaine, dicyclavil, diethylpropion, diltiazem, dimenhydrinate, diphenhydramine, piripramine, doxepin, doxycycline, doxylamine, dipyridamole, EDTA, erythromycin, flurazepam, methicone, hydroxychloroquine, imipramine, insulin, irinotecan, levomethadol, lidocaine, oxapine, mechlorethamine, melphalan, levopromethazine, methotrexate, metoclopramide, minoxidine, naftifine, carpidine, meplate, cinnamyl, chlorpheniramine, bromamine, levopromethamine, brommetha, Nizatidine, oxfenadrine, oxybutynin chloride, oxytetracycline, phenoxybenzamine, phentolamine, procainamide, procaine, promethazine, proparacaine, propoxycaine, dexpropoxyphene, ranitidine, tamoxifen, terbinafine, tetracaine, tetracycline, tramadol, trifluoroperazine, trimetprazosin, trimethamamethyl, trimipramine, tripelennamine, oleandomycin acetate, tyramine, uramustine, verapamil, and vasopressin.

Additional active agents include those containing one or more nitrogen-containing heterocycles, such as atorvastatin, amoxapine, astemizole, atropine, azithromycin, benazepril, benztropine, bicyclohexanol, bupivacaine, buprenorphine, buspirone, butorphanol, caffeine, camptothecin, and molecules belonging to the camptothecin family, ceftriaxone, chlorpropham, ciprofloxacin, cladribine, clemastine, clindamycin, clofazimine, clozapine, cocaine, codeine, cyproheptadine, desipramine, hydridotetramine, difenidol, difenolate, dipyridamole, doxepin, ergotamine, famciclovir, fentanyl, flavoxate, fludarabine, fluphenazine, fluvastatin, ganciclovir, granisetron, guanethidine, flupiridol, homatropine, hydrocodone, hydromorphone, hydroxyzine, scopolamine, Oxymetamine, itraconazole, ketorolac, ketoconazole, levocabastine, levorphanol, lincomycin, lomefloxacin, loperamide, losartan, loxapine, mazindol, meclizine, pethidine, mepivacaine, thiamethoxam, urotropine, methimazole, levomepromazine, meccesteryl, metronidazole, minoxidil, mitomycin c, molindone, morphinan, nefazodone, nalbuphine, nalidixic, nalmefene, naloxone, naltrexone, naphazoline, nedoromil, nicotine, norfloxacin, ofloxacin, ondansetron, oxycodone, oxymorphone, pentazocine, hexanone, phenazine, physostigmine, pilocarpine, pimozide, pimozolomide, pramine, procaine, prochlorperazine, promethazine, quinine, 2, quinidine, vitamin B, and vitamin D, Rifabutin, risperidone, rocuronium, hyoscyamine, sufentanil, tacrine, terazosin, terconazole, terfenadine, thioridazine, thiothiflutolone, ticlopidine, timolol, tolazamide, tolmetin, trazodone, thiethylperazine, trifluoroperazine, trihexyphenidyl, isobutylazine, trimipramine, tubocurarine, vecuronium, vidarabine, vinblastine, vincristine and vinorelbine.

Additional active agents include those containing an aromatic ring nitrogen, such as acetazolamide, acrivastine, acyclovir, adenosine phosphate, allopurinol, alprazolam, oxypheniramine, amrinone, alcaladine, azatadine, aztreonam, bisacodyl, bleomycin, brompheniramine, buspirone, butoconazole, molecules within the camptothecin and camptothecin families, carbinoxamine, cefamandole, cefazolin, cefixime, cefmetazole, cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone, cefapirin, chloroquine, chlorpheniramine, cimetidine, cladribine, clotrimazole, cloxacillin, didanosine, dipyridamole, doxazosin, galantamine, econazole, enoxacin, estazolam, ethionamide, fametidine, famciclovir, flutolabine, doxoramide, doxoraabine, folic acid, Ganciclovir, hydroxychloroquine, diiodoquine, isoniazid, itraconazole, ketoconazole, lamotrigine, lansoprazole, loratadine, losartan, mebendazole, mercaptopurine, methotrexate, metronidazole, miconazole, midazolam, minoxidil, nefazodone, nalidixic acid, nicotinic acid, nicotine, nizatidine, omeprazole, oxaprozin, oxiconazole, papaverine, pentostatin, phenazopyridine, pilocarpine, piroxicam, prazosin, primaquine, pyrazinamide, pyrimethamine, pyridoxine, quinidine, quinine, ribavirin, rifampin, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, terazosin, thiabendazole, thiamine, thioguanine, timolol, trazodone, triamcinolone, trimethoprim, Tripelennamine, tropicamide, and vidarabine.

The preferred active agent is one of the molecules belonging to the camptothecin family. For example, the active agent may have the following general structure:

wherein R is₁-R₅Are each independently selected from the group consisting of: hydrogen; halogen; an acyl group; alkyl (e.g., C1-C6 alkyl); a substituted alkyl group; alkoxy (e.g., C1-C6 alkoxy); a substituted alkoxy group; an alkenyl group; an alkynyl group; a cycloalkyl group; a hydroxyl group; a cyano group; a nitro group; an azide group; an amido group; hydrazine; an amino group; substituted amino groups (e.g., monoalkylamino and dialkylamino); a hydroxycarbonyl group; an alkoxycarbonyl group; an alkylcarbonyloxy group; an alkylcarbonylamino group; ammoniaA formylalkoxy group; an arylsulfonyloxy group; an alkylsulfonyloxy group; -C (R)₇)=N-(O)_i-R₈Wherein R is₇Is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, and R is₈Is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and R₉C (O) O-, wherein R₉Is halogen, amino, substituted amino, heterocycle, substituted heterocycle, or R₁₀-O-(CH₂)_m-, where m is an integer from 1 to 10 and R₁₀Is alkyl, phenyl, substituted phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or substituted heterocycle; or R₂And R₃Together or R₃And R₄Together form a substituted or unsubstituted methylenedioxy, ethylenedioxy, or ethyleneoxy group; r₆Is H OR OR 'wherein R' is alkyl, alkenyl, cycloalkyl, haloalkyl, OR hydroxyalkyl.

With respect to the above structure, although not shown, analogs having a hydroxyl group at other than position 20 (e.g., position 10 or 11, etc.) are similarly preferred.

In a particular embodiment, the active agent is irinotecan (structure shown below).

In yet another embodiment, the active agent is 7-ethyl-10-hydroxy-camptothecin (SN-38), a metabolite of irinotecan, the structures of which are shown below.

Mixed salts-considerations relating to these conjugates

An illustrative mixed salt of a water-soluble polymer and an active agent can have any of the various structural features described above. That is, the conjugate may have a linear structure, i.e., having one or two active agent molecules covalently attached to a linear water-soluble polymer, typically at each end of the linear water-soluble polymer. Alternatively, the conjugate may have a forked, branched or multi-armed structure.

One exemplary multi-armed polymer conjugate corresponds to the following generalized structure: r (-Q-POLY)₁-X-D)_qWherein R is an organic group having from about 3 to about 150 carbon atoms, Q is a linker (preferably hydrolytically stable and may be-O-, -S-, -NH-C (O) -and-C (O) -NH-), POLY₁Is a water-soluble, non-peptidic polymer, X is a spacer comprising a hydrolyzable bond, D is an active agent moiety, and q ranges from 3 to 25 (e.g., 3 to 10, such as any of 3,4,5,6, 7, 8, 9, and 10).

Another exemplary multi-armed polymer conjugate corresponds to the following generalized structure: r (-Q-POLY)₁-CH₂C(O)-NH-CH₂-C(O)-O-D)_qWherein: r is an organic group having from 3 to 150 carbon atoms; q is a linker wherein Q is taken together to form R (-Q-)_qWhen R is the residue of a polyol or polythiol after removal of "q" hydroxyl or thiol protons, respectively, used to form a polymer for POLY₁The attachment point of (a); POLY₁Is a water soluble polymer selected from the group consisting of poly (alkylene glycols), poly (alkenyl alcohols), poly (vinyl pyrrolidones), poly (hydroxyalkyl-methacrylamides), poly (hydroxyalkyl methacrylates), poly (α -hydroxy acids), poly (acrylic acids), poly (vinyl alcohols), polyphosphazenes, polyoxazolines, poly (N-acryloylmorpholin), and copolymers or terpolymers thereof, D is camptothecin attached at its 10-, 11-, or 20-ring position, and q has a value from 3 to 50 (e.g., any of 3 to 10, e.g., 3,4,5,6, 7, 8, 9, and 10).

One illustrative multi-arm polymer conjugate corresponds to the structure:

the above structure is referred to herein in shorthand as "4-arm-PEG-Gly-Irino" (4-arm-pentaerythrityl-PEG-carboxymethylglycine irinotecan); a more complete name corresponds to "pentaerythrityl-4-arm- (PEG-1-methylene-2-oxo-vinylacetamide linked-irinotecan)". Basic amino and/or nitrogen groups in the active agent portion of the conjugate are shown above in only neutral form, with the understanding that the conjugate has the characteristics of a mixed salt of the portions as described in detail herein. As can be seen from the above structure, the carboxymethyl modified 4-arm pentaerythrityl PEG reagent has one glycine linker between the polymer moiety and the active agent irinotecan.

In certain examples, compositions comprising "4-arm-PEG-Gly-Irino" may be characterized as compositions comprising four-arm conjugates, wherein at least 90% of the four-arm conjugates in the composition:

(i) having a structure covered by the following chemical formula,

C-[CH₂-O-(CH₂CH₂O)_n-CH₂-C(O)-Term]₄，

wherein

n, in each instance, is an integer having a value from 5 to 150 (e.g., about 113), and

term, in each instance, is selected from the group consisting of: -OH, -OCH₃、-NH-CH₂-C(O)-OH、-NH-CH₂-C(O)-OCH₃、

and-NH-CH₂-c (O) -O-Irino ("GLY-Irino"), wherein Irino is a residue of irinotecan; and is

(ii) For each Term in at least 90% of the four-armed conjugates in the composition, at least 90% of it is-NH-CH₂-C(O)-O-Irino。

Typically, although not necessarily, the number of polymer arms will correspond to the number of active agent molecules covalently attached to the water-soluble polymer core. That is, where the polymeric reagents have a certain number of polymeric arms (e.g., corresponding to variable "q"), each having a reactive functional group (e.g., carboxyl, activated ester (e.g., succinimidyl ester, benzotriazolyl carbonate, etc.) at its terminus, the optimal number of active agents (e.g., irinotecan) that can be covalently attached thereto in the corresponding conjugate is most desirably "q". That is, 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 drug loading level of 0.90(q) (or 90%) or greater. Preferred drug loadings satisfy one or more of the following conditions: 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. Most preferably, the drug loading of the multi-arm polymer conjugate is one hundred percent. Compositions comprising multi-arm water-soluble polymer conjugates mixed acid salts can include mixtures of molecular conjugates having one 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 forth, up to and including conjugates having "q" active agents attached to the polymer core. The resulting composition will have an overall drug loading value that is the average over the conjugate species contained in the composition. Ideally, the composition will comprise predominantly (e.g., greater than 50%, but more preferably greater than 60%, still more preferably greater than 70%, still more preferably greater than 80%, and most preferably greater than 90%) drug-loaded polymer conjugates (i.e., "q" arms having "q" molecules of active agent, each arm having a single molecule of active agent).

As an illustration, in the example of the multi-armed polymer conjugate containing four polymer arms, the ideal value for the number of covalently attached drug molecules per multi-armed polymer is four, and with respect to the average number described in the context of the composition of such conjugate, there will be a value (i.e., percentage) of drug molecules loaded onto the multi-armed polymer that ranges from about 90% to about 100% of the ideal value. That is, the average number of drug molecules covalently attached to a given four-arm polymer (as part of the four-arm polymer composition) is typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% of the loading value. This corresponds to the average number of D per multi-armed polymer conjugate (ranging from about 3.60 to 4.0).

In yet another embodiment, for a multi-armed polymer conjugate composition, for example, where the number of polymer arms ranges from about 3 to about 8, (e.g., greater than 50%, but more preferably greater than 60%, still more preferably greater than 70%, still more preferably greater than 80%, and most preferably greater than 90%) of the species present in the composition are those having an idealized number ("q") of drug molecules attached to the polymer core or those having a combination of ("q") and ("q-1") drug molecules attached to the polymer core.

In certain examples, a multi-armed polymer conjugate (e.g., as described herein) is prepared, wherein the resulting conjugate exhibits a high degree of substitution or drug loading in the context of the ranges provided above. Illustrative conjugates thus prepared will generally have a drug loading value of at least 90%, and may typically have a drug loading value of greater than 91%, or greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and in some cases, at a 100% full loading value. In particular, multi-armed polymer conjugates prepared from multi-armed polymeric starting materials prepared, for example, according to the alkoxylation processes provided herein, can exhibit higher drug substitution values due, at least in part, to the purity of the polymeric starting material. As an example, on average, 4-arm PEG-CM-SCM (e.g., having a molecular weight greater than about 10 kilodaltons) prepared from the prepared 4-arm PEG-OH according to the alkoxylation methods provided herein may have a higher level of purity relative to the specific polymer species present in the 4-arm-PEG-CM-SCM reactant material as compared to results obtained with other commercially available 4-arm PEG-OH starting materials (e.g., having fewer low molecular weight polymer impurities). If undesirable polymeric materials present in the polymeric starting material are "carried over" into subsequent conversion steps, the level of purity of the multi-arm PEG starting material, especially those with higher molecular weights, can contribute to the purity of the final conjugate product. Particularly in synthetic methods that employ high-yield reaction steps (e.g., carboxymethylation) with coupling to an active agent (e.g., deprotected glycine-irinotecan), the use of polymeric starting materials with higher amounts of polymeric impurities can affect the purity and drug loading values (in some cases expressed as a few percent) of the resulting conjugate species. Furthermore, the presence of even a small percentage of low molecular weight polymer conjugate species in the final mixed salt conjugate can result in reduced bioavailability, as these small molecular weight conjugates will be cleared more rapidly. Polymer conjugates prepared from starting materials prepared using the alkoxylation methods described herein may therefore exhibit greater bioavailability compared to polymer conjugates prepared from commercially available multi-armed starting materials containing up to, for example, 20% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%) of low molecular weight or other polymeric impurities.

In light of the above, in addition to drug loaded structures (i.e., molecules of glycine-modified irinotecan having covalently attached to each of the four polymer arms), portions of the mixed salts (and compositions containing them) can include any one or more of the following structures:

for a given polymer arm end with one carboxylic acid shown above (and thus not covalently attached to a drug, e.g., irinotecan), from 4-arm-PEG-CM (-CH)₂Other possible ends of arm extension of C (O) -, include: -OH, -OCH₃、-NH-CH₂-C(O)-OH、NH-CH₂-C(O)-OCH₃、

The multi-armed polymer conjugate compositions provided herein are intended to encompass any and all stereoisomeric forms of the conjugates included in such compositions. In a particular embodiment of the conjugate, in the conjugated form (e.g. in the composition of 4-arm-PEG-Gly-Irino), the stereochemistry at C-20 of irinotecan remains intact, i.e. C-20 retains its (S) -configuration in its conjugated form. See, e.g., example 4.

Yet another preferred multi-arm structure is a carboxymethyl modified 4-arm pentaerythrityl PEG (polymer moieties and linkers shown above) with a glycine linker between the polymer moiety in each arm and the active agent, wherein the active agent is 7-ethyl-10-hydroxy-camptothecin. Again, embodiments are included herein wherein the multi-armed polymer is (i) fully loaded, along with (ii) three 7-ethyl-10-hydroxy-camptothecin molecules covalently attached thereto, (iii) two 7-ethyl-10-hydroxy-camptothecin molecules covalently attached thereto, and (iv) one 7-ethyl-10-hydroxy-camptothecin molecule covalently attached to a four-armed polymer core.

Yet another representative multi-arm conjugate structure is carboxymethyl modified 4-arm glycerol dimer (3,3' -oxydiprane-1, 2-diol) PEG with 7-ethyl-10-hydroxy-camptothecin (SN-38) covalently attached to the polymer core. Included herein are embodiments wherein the multi-armed polymer core is drug-loaded (i.e., has four 7-ethyl-10-hydroxy-camptothecin molecules covalently attached thereto), or less loaded (i.e., has one, two, or three 7-ethyl-10-hydroxy-camptothecin molecules covalently attached thereto). These conjugates with a drug (i.e. 7-ethyl-10-hydroxy-camptothecin) covalently attached to each polymer arm are shown below.

In yet another exemplary embodiment, the conjugate is a multi-arm structure comprising a carboxymethyl modified 4-arm glycerol dimer (3,3' -oxydiprane-1, 2-diol) PEG with irinotecan molecules covalently attached to a polymer core. Included herein are embodiments in which the multi-armed polymer core is drug-loaded (i.e., has four irinotecan molecules covalently attached thereto), or less loaded (i.e., has one, two, or three irinotecan molecules covalently attached thereto).

Parameters of mixed salt

The subject compositions can be, among other things, part of a mixed acid salt. That is, mixed salt conjugates are provided in the composition such that the basic nitrogen atoms of the conjugate (along with the bulk composition) may exist individually as either protonated or unprotonated forms, with these protonated nitrogen atoms (referred to as acid salts) having one of two different counter anions. One anion corresponds to the conjugate base of a strong inorganic acid (e.g., hydrohalic acid, sulfuric acid, nitric acid, phosphoric acid, nitrous acid, and the like) and the other anion corresponds to the conjugate base of a strong organic acid (e.g., trifluoroacetate). The subject mixed acid salt compositions are stable and can be prepared reproducibly.

A mixed acid salt as provided herein is characterized in terms of its bulk or macroscopic properties. That is, the basic nitrogen atom (i.e., amino group) in the conjugate exists individually in either neutral (unprotonated) or protonated form, with these protonated forms being associated with two different possible counterions. In characterizing the compositions of the invention based on bulk properties, different individual molecular species are contained within the bulk composition. Using the exemplary 4-arm polymer conjugate described in example 1, 4-arm-PEG-Gly-Irino-20K, the mixed acid salt product contains any of a variety of individual molecular species. A molecular species is one in which each polymer arm contains one molecule of irinotecan in neutral form, i.e., its amino groups are unprotonated. See structure I below. Another molecular species is one in which each polymer arm contains one molecule of irinotecan in its protonated form. See structure IV below. An additional molecular species is one in which three polymer arms contain the irinotecan molecule in its protonated form and one contains the irinotecan molecule in its neutral form (structure III). In yet another molecular species, two of the four polymeric arms contain irinotecan molecule in its neutral form (i.e., its amino groups are unprotonated), and two of the four polymeric arms contain irinotecan molecule in its protonated form (structure II). Within all of the above molecular species, it is possible for sub-species of the molecule to contain different combinations of counterions, except for the first "all neutral" form. The following schematic diagrams illustrate different possible combinations; the following table shows possible combinations of protonated acid salts corresponding to each structure.

Indicating protonation

Representing unprotonated or neutral

As demonstrated in example 1 and in example 6, certain exemplary polymeric prodrug conjugates are obtained as mixed acid salts of both hydrochloric acid and trifluoroacetic acid. In example 1, hydrochloric acid is introduced by using an active agent molecule in the form of an acid salt to form the resulting polymer conjugate, while trifluoroacetic acid is introduced into the reaction mixture at the deprotection step (although any strong acid may be used). The following covalent attachment of the active agent (or modified active agent as illustrated in example 1) to the water-soluble polymer reagent, and treatment with a base, even in instances where an additional purification step is performed, the resulting conjugate is surprisingly and reproducibly obtained as part of a mixed acid salt having surprising and beneficial properties, which will be described in more detail below. Even after repeated purifications, it has been found that there is a persistent and reproducible association of exemplary strong mineral acids, hydrochloric acid, and trifluoroacetic acid in the resulting conjugates. See, e.g., example 2, table 1 and example 6, table 2.

The mixed acid salt conjugates described herein preferably contain each component (i.e., free base, inorganic acid salt, organic acid salt) in fairly well-defined ratios and ranges. The characteristics of the mixed acid salt product may of course vary depending on the changes in the synthesis conditions used. In looking at the compositions prepared according to the method described in example 1, the polymer conjugate mixed acid salt is consistently recovered with the largest relative molar amount of basic nitrogen atoms in protonated form (calculated relative to the basic nitrogen atoms in the active agent) compared to the free base (or unprotonated) nitrogen. Thus, if all of the basic nitrogens in the active agent portion of the conjugate are unprotonated, then the corresponding mole percent would be 100. In one embodiment, a partial mixed salt composition is characterized as having the maximum relative molar amount of TFA salt (as compared to the hydrochloride salt and the free base). In yet another specific embodiment, the partial mixed salt composition is typically characterized as comprising less relative molar amounts of hydrohalide salt (compared to TFA salt), and even less unprotonated (free base) nitrogen. In one embodiment, the portion of the mixed salt composition comprises about 30-75 mole percent of the TFA salt, about 15-45 mole percent of the hydrohalide salt, and 2-55 mole percent of the free base. These relative amounts can, of course, vary with variations in the processing conditions used to make the mixed acid salt. For example, in yet another embodiment, the mole percent of the trifluoroacetate salt ranges from about 45 to 70, the mole percent of hydrochloride salt ranges from about 20 to 38, and the mole percent of free base ranges from about 10 to 35. In general, for the earlier batches of prepared conjugates, the active agent basic nitrogen (e.g., amino) group in the conjugate is present at the highest mole percent as the trifluoroacetate salt, the second highest mole percent as the hydrochloride salt, and the third highest or lowest mole percent as the free base. In certain embodiments, the mole percentages of the hydrochloride salt and the free base in the conjugate are about the same. The relative molar amounts of trifluoroacetate, hydrochloride and free base in the conjugate were averaged over test batches and the product contained, on average, about 50 mole percent trifluoroacetate, about 30 mole percent hydrochloride and about 20 mole percent free base.

Turning now to example 6, it can be seen that mixed acid salt conjugates have been prepared in which the relative molar amounts of each of the TFA salt, the hydrochloride salt, and the unprotonated material exhibit a high level of identity in four different batches. Similar to the results in example 1, the polymer conjugate mixed acid salt was consistently recovered with the largest relative molar amount of basic nitrogen atoms in protonated form (calculated relative to the basic nitrogen atoms in the active agent) compared to the free base (or unprotonated) nitrogen. In the batches summarized in table 2, part of the mixed salt composition had the largest relative molar amount of HCl salt compared to TFA salt and free base. In yet another specific embodiment, the partial mixed salt composition may typically be characterized as including less relative molar amounts of TFA salt (as compared to HCl salt), and even less unprotonated (free base) nitrogen. In one embodiment, the portion of the mixed salt composition will comprise at least about 20 mole percent TFA, or at least about 25 mole percent TFA. Exemplary ranges for the TFA salt in the mixed salt composition can be from about 20-45 mole percent, or from about 24-38 mole percent, or even from about 35 to 65 mole percent. With respect to the hydrochloride salt, in certain embodiments, the composition can have from about 30 to 65 mole percent of the hydrochloride salt, or from about 32 to about 60 mole percent of the hydrochloride salt, or preferably, from about 35 to 57 mole percent of the hydrochloride salt.

In general, it has been found that the mixed acid salt conjugates described herein have greater stability than either the pure hydrochloride salt or the free base form of the conjugate. See, e.g., example 3 and fig. 1, illustrating the results of pressure stability tests on compositions containing varying amounts of salt and free base forms of an exemplary conjugate (4-arm-PEG-GLY-IRT). A positive correlation between increased stability to hydrolysis and increased mole percent of salt in the final conjugate product was observed. Based on the slope in the graph, it can be determined that as the free base content increases, the product stability decreases. A correlation was observed between the decrease in product and the increase in irinotecan over time, thereby leading to the determination that the mode of decomposition observed under the conditions used was hydrolysis of the ester bond.

Figure 2 further illustrates the greater stability (or tolerance) with respect to hydrolytic degradation for amine groups (i.e., acid salts) having a greater degree of protonation. For example, conjugate products containing 14 mole percent or more of the free base were observed to be significantly less stable to hydrolysis than the corresponding acid salt-rich products.

Additionally, as illustrated in fig. 3, the hydrochloride-rich product appears to be more prone to cleave the water-soluble polymer backbone than the mixed salt form, which contains measurable amounts of the free base. Indeed, the breakdown of mixed salt conjugates appears to be due primarily to the hydrolytic release of the drug rather than the cleavage of the polymer backbone. However, such skeletal decomposition appears to be only relevant under accelerated stress conditions.

Since the two observed modes of decomposition appear to show opposite trends with respect to degradation stability or tolerance versus salt/free base content, this may (but need not) indicate that the preferred range of salt constituents have greater overall stability than either the full salt or the extremes of the full free base. Furthermore, based on preliminary studies, the mixed salt appears to have somewhat greater stability than either the free base or the hydrochloride salt form, thus indicating its surprising superiority over any more traditional pure base or its single salt form.

In addition, mixed salt forms of the conjugates are prepared with high batch-to-batch consistency-that is, with a relatively consistent molar ratio of trifluoroacetate, halide (or other inorganic acid anion), and free base in the final conjugate product. As can be seen in table 1 of example 2, roughly 50 mole percent of the drug basic nitrogen groups were associated with trifluoroacetic acid. This mole percentage was observed fairly consistently from batch to batch. Similarly, roughly 30 mole percent of the conjugate drug amino (or other basic nitrogen) groups are fairly uniformly associated with hydrochloric acid, i.e., provided as a HCl salt. The free base form of the drug amino (or other basic nitrogen) group in the conjugate is then also stably and reproducibly prepared. Turning to the results provided by example 6, based on a slightly improved manufacturing process, it can be seen that despite the differences in the actual relative molar amounts of the protonated and unprotonated species, as well as in the protonated species, TFA versus hydrochloride, the mixed acid salt was reproducibly prepared.

These aggregated results demonstrate the unexpected advantage of a mixed salt of a portion of a water-soluble polymer-active agent conjugate (in one embodiment, 4-arm-PEG-Gly-Irino-20K) over the use of the free base alone or any salt lacking other ingredients. The mixed salt appears to have greater stability than either the free base or the hydrochloride salt, thus indicating that it is more pure than it is more common

The obvious advantages of either the alkali or pure salt forms.

Mixed salt conjugates-methods for forming

In view of the guidance presented herein, mixed acid salts of water-soluble polymer conjugates can be readily prepared from commercially available starting materials, in combination with what is known in the art. As described above, when in the conjugated form, the mixed salt polymer-active agent conjugate includes a water-soluble polymer covalently attached to one or more active agent molecules, each active agent molecule having one or more basic nitrogen atoms, such as amino groups. The amine groups in the resulting conjugates can be primary, secondary, or tertiary amino groups.

Linear, branched, and multiarm water-soluble polymeric reagents are available from a variety of commercial sources as described above. Alternatively, PEG reagents, such as multi-arm reactive PEG polymers, can be prepared synthetically as described herein.

The mixed acid salt of the moiety can be formed using known chemical coupling techniques for covalently attaching an activated polymer (e.g., an activated PEG) to a biologically active agent (see, e.g., for examplePOLY(ETHYLENE GLYCOL)CHEMISTRY AND BIOLOGICALAPPLICATIONS(poly (ethylene glycol) chemical and biological applications), American chemical society (American chemical society), Washington (1997; and U.S. patent publication Nos. 2009/0074704 and 2006/0239960). The selection of suitable functional groups, linkers, protecting groups, and the like to achieve a mixed acid salt according to the invention will depend in part on the functional groups on the active agent and the polymer starting material, and will be apparent to those skilled in the art based on this disclosure. In view of certain characteristics of the partial mixed salts, the method includes providing an amine (or other basic nitrogen) containing active agent in the form of an inorganic acid addition salt, and a trifluoroacetic acid treatment step. Alternatively, the conjugate product or intermediates in a synthetic pathway may be reacted with an inorganic acid to form an inorganic acid addition salt at a later stage of the process, thereby introducing a second counter ion (other than trifluoroacetate) into the reaction. Reference to "active agent" in the context of synthetic methods is meant to encompass active agents that are optionally modified to have a linker covalently attached thereto to facilitate attachment to the water-soluble polymer.

In general, the method comprises the steps of: (i) deprotecting an inorganic acid salt containing an amine (or other basic nitrogen) active agent in protected form by treatment with trifluoroacetic acid (TFA) to form a deprotected mixed acid salt, (ii) coupling the deprotected inorganic acid salt of step (i) with a water-soluble polymer reagent in the presence of a base to form a polymer-active agent conjugate, and (iii) recovering the polymer-active agent conjugate. The resulting polymer-active agent conjugate compositions are characterized as having one or more ammonia (or other basic nitrogen-containing) groups in a combination of free base, acid salt, and TFA salt forms. The product thus comprises both the inorganic acid salt and the trifluoroacetate salt, while a portion of the basic groups in the conjugate are in the unprotonated or free base form. Thus, the combined molar amount of inorganic acid salt and trifluoroacetate salt is less than the total number of basic amino or other nitrogen groups contained in the conjugate product.

Turning now to one of the preferred classes of active agents (camptothecins), significant yields are difficult to achieve with a single step conjugation reaction because the 20-hydroxy group of compounds within the camptothecin family is sterically hindered. As a result, a preferred method is to react the 20-hydroxyl group of a biologically active starting material (e.g., irinotecan hydrochloride) with a short linker or spacer moiety bearing a functional group suitable for reaction with a water-soluble polymer. Such a method is applicable to many small molecules, particularly those having a site that is difficult to covalently attach to an introduced reactive polymer. Preferred linkers for reaction with a hydroxyl group to form an ester linkage include t-BOC-glycine or other amino acids, such as alanine, glycine, isoleucine, leucine, and valine with a protected amino group and an available carboxylic acid group (see Zalipsky et al, "Attachment of drugs to Polyethylene glycol", Eur. Polymer. J. (J. Eur. Polymer), Vol. 19, No. 12, p. 1177-1183, (1983)). Other spacer or linker moieties may also be used in place of the above amino acids, these moieties having an available carboxylic acid group or other functional group reactive with a hydroxyl group and having a protected amino group.

Typical labile protecting groups include t-BOC and FMOC (9-fluorenylmethoxycarbonyl). t-BOC is stable at room temperature and is easily removed with dilute solutions of trifluoroacetic acid and dichloromethane. FMOC is a base-labile protecting group that is easily removed with concentrated solutions of amines (typically 20% -55% piperidine in N-methylpyrrolidone).

In an embodiment of the invention, the carboxyl group of N-protected glycine is reacted with the 20-hydroxyl group 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 (such as Dicyclohexylcarbodiimide (DCC)) and a base catalyst (such as Dimethylaminopyridine (DMAP) or other suitable base) to provide an N-protected linker modified active agent, such as t-Boc-glycine-irinotecan hydrochloride. While hydrochloride salts are illustrated, other inorganic acid salts may also be used. Preferably, each reaction step is carried out under an inert atmosphere.

In a subsequent step, the amino protecting group, t-BOC (N-tert-butoxycarbonyl), is removed by treatment with trifluoroacetic acid (TFA) under suitable reaction conditions. In this step, trifluoroacetic acid is typically introduced into the reaction mixture. The product is a linker modified active agent, such as 20-glycine-irinotecan TFA/HCl. Illustrative reaction conditions are described in example 1 and can be further optimized by one skilled in the art through routine optimization. Optionally, the molar amounts of mineral acid and trifluoroacetic acid in the uncoupled products are determined by suitable analytical methods (e.g., HPLC or ion chromatography) to allow for greater precision and product consistency in the coupling step.

The deprotected active agent (optionally modified by a linker) (e.g., 20-glycine-irinotecan TFA/HCl) is then coupled to a desired polymeric reagent, e.g., 4-arm pentaerythrityl-PEG-succinimide (or any other similar activated ester counterpart), in the presence of a coupling agent (e.g., Hydroxybenzotriazole (HOBT)) and a base (e.g., DMAP, trimethylamine, triethylamine, etc.). In one embodiment of the present method, the amount of base added in the coupling step is in the range of about 1.0 to 2.0 times, or from about 1.0 to 1.5 times, or from about 1.0 to 1.05 times the sum of the number of moles of TFA determined for the starting material (in this case 20-glycine-irinotecan TFA/HCl) and the number of moles of inorganic acid. By adjusting the amount of base to the acid salt content of 20-glycine-irinotecan TFA/HCl, a relatively consistent ratio of TFA, inorganic acid (e.g., HCl), and base is maintained during the coupling step to thereby form mixed acid salt conjugates with a consistently narrow range of TFA and inorganic acid contents. Preferably, the resulting partial mixed acid salt is reproducibly prepared such that the relative molar amounts of inorganic addition salt, trifluoroacetate salt and free base in the conjugate composition do not vary by more than about 25%, and even more preferably by more than about 15%, between batches. For the purpose of making such an assay, the above consistency measure is determined over at least five batches (e.g., from 5 to 7), with failed batches that are clearly outliers excluded from the calculation.

Although the coupling step is carried out in the presence of excess base, it has been unexpectedly found that the resulting conjugate is stably formed as a partial mixed acid salt, i.e., such that a significant amount of the basic amino groups or other nitrogen-containing groups in the conjugate are protonated and not in the free base form. The reaction yield of the coupling reaction is typically high, greater than about 90% (e.g., about 95% on average).

This portion of the mixed acid salt conjugate is recovered, for example, by precipitation with an ether (e.g., methyl tert-butyl ether, diethyl ether) or other suitable solvent. The product may 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. One preferred purification method is recrystallization. For example, a portion of the mixed acid salt is dissolved in a suitable single or mixed solvent system (e.g., isopropanol/methanol), and then allowed to crystallize. The recrystallization can be carried out several times and the crystals can also be washed with a suitable solvent in which they are insoluble or only slightly soluble, such as methyl tert-butyl ether or methyl tert-butyl ether/methanol. The purified product may optionally be further air or vacuum dried. Even upon repeated purification, the product is typically recovered as a mixed acid salt rather than as the free base. Even upon additional treatment with base, the conjugate remains in the form of a mixed acid salt of the moiety having the characteristics described herein.

The resulting conjugate is a partially mixed salt, i.e., one in which some basic nitrogen atoms are in neutral or free base form and others (e.g., amino groups) are protonated. The protonated amine groups are in the form of acid salts with different anions, one corresponding to the conjugate base of the inorganic acid and the other anion being trifluoroacetate (or the conjugate base of an organic acid as described above). As used herein, a partial mixed acid salt refers to the bulk product and not necessarily to the individual molecular species contained within the bulk product. Thus, depending on the particular conjugate structure, the individual molecular species contained within the mixed salt may contain amine groups in free base and protonated form as described above. Alternatively, the mixed salt may comprise a mixture of molecular species (e.g., having all amine groups in free base form, having all amine groups in protonated form, as a salt of an inorganic acid, as a salt of trifluoroacetic acid or other suitable organic acid, or as a mixture of both, as various combinations of the above, etc.), such that the characteristics of the bulk product are as described herein. If the conjugate is a polymer conjugate that includes only one active agent amine group, then the mixed salt must necessarily be such that the bulk product is a mixture of molecular species to achieve a mixed salt as generally described herein.

Preferably, the mixed 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 variety of storage conditions or packaging schemes can be used to suitably protect the acid salt product during storage. In one embodiment, the product is packaged under an inert atmosphere (e.g., argon or nitrogen) by placement in one or more polyethylene bags and placed in an aluminum-lined polyester heat sealable bag.

Representative mole percentages of TFA salts, hydrochlorides, and free bases determined across multiple batches of 4-arm-PEG-GLY-IRINO are summarized in table 1 (example 2) and table 2 (example 6). As can be seen, surprisingly, even after treatment with base and repeated purification, the conjugate product does not separate into a single non-protonated conjugate species, but rather as a mixed acid salt.

Mixed salt-mixed salt conjugates containing pharmaceutical compositions

Part of the mixed salts may be in the form of pharmaceutical formulations or compositions for veterinary use or for human medical use. Illustrative formulations will typically include a partial mixed acid salt conjugate in combination with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilizers, or the like. The carrier or carriers must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient/patient. The portion of the mixed acid salt conjugate is optionally contained in bulk or in unit dosage form in a container or vessel that includes packaging to protect the product from exposure to moisture and oxygen.

The pharmaceutical compositions may include polymeric excipients/additives or carriers such as polyvinylpyrrolidone, derivatized celluloses such as hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methylcellulose, ficols (a polymeric sugar), hydroxyethyl starch (HES), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-beta-cyclodextrin and sulfobutyl ether-beta-cyclodextrin), polyethylene glycol, and pectin. These compositions may further include diluents, buffers, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), flavoring agents, taste masking agents, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, surfactants (e.g., polysorbates such as "tween 20" and "tween 80", and pluronic such as F68 and F88 available from BASF, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty acid esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc and other suitable cations). other pharmaceutically acceptable excipients and/or additives suitable for use in The compositions according to The invention are listed in "Remington: The Science & Practice of pharmacy", 19 th edition, Williams & Williams company, (1995), the "Physician's Desk Reference", 52 th edition, Medical economies, Montvale (Monte Well), NJ (1998), and in the "Handbook of pharmaceutical excipients", 3 rd edition, KibbeA.H. eds, American pharmaceutical Association, 2000.

The mixed acid salt may be formulated into a composition suitable for oral, rectal, topical, nasal, ocular, or parenteral (including intraperitoneal, intravenous, subcutaneous, or intramuscular) administration. The mixed acid salt compositions may be conveniently 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 bringing the mixed acid salt into association with a carrier which constitutes one or more accessory ingredients.

In a specific embodiment, the mixed acid salt is provided in lyophilized form for use in a sterile disposable vial for administration by injection, e.g., 4-arm-PEG-Gly-Irino-20K. In one embodiment, the amount of conjugate product contained in the disposable vial is equivalent to a 100mg dose of irinotecan. More specifically, the lyophilized composition comprises 4-arm-PEG-Gly-Irino-20K conjugated to lactate buffer at pH 3.5. That is, a lyophilized composition is prepared by combining 4-arm-PEG-Gly-Irino-20K (e.g., irinotecan at a dose equivalent to 100 mg) with about 90mg of lactic acid, and adjusting the pH of the solution to 3.5 by adding either an acid or a base. The resulting solution was then lyophilized under sterile conditions and the resulting powder was stored at-20 ℃ prior to use. The lyophilized composition is combined with a glucose solution, such as a 5% (w/w) glucose solution, prior to intravenous infusion.

The amount of the mixed acid salt (i.e., active agent) in the formulation varies depending on the particular active agent used, 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 readily determined by those skilled in the art. The amount of conjugate in the formulation will be that amount necessary to deliver a therapeutically effective amount of the compound, e.g., an alkaloid anti-cancer agent (e.g., irinotecan or SN-38), to a patient in need thereof in order to achieve at least one of the therapeutic effects associated with the compound (e.g., for the treatment of cancer). In practice, this will vary widely depending on the particular conjugate, its activity, the severity of the condition to be treated, the patient population, the stability of the formulation, and the like. These compositions will generally comprise any number of conjugates from about 1% by weight to about 99% by weight, typically from about 2% to about 95% by weight of conjugates, and more typically from about 5% to 85% by weight of conjugates, and also depending on the relative amounts of excipients/additives included in the composition. More specifically, the composition typically includes a conjugate in at least about one of the following percentages: 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60% or more by weight.

Compositions suitable for oral administration may be provided in the form of: as discrete units, such as capsules, cachets, tablets, lozenges, and the like, each containing a predetermined amount of the conjugate as a powder or granules; or a suspension in an aqueous liquid or non-aqueous liquid such as a syrup, elixir, emulsion, drench, and the like.

Formulations suitable for parenteral administration suitably include a sterile aqueous preparation of the mixed acid salt conjugate, which may be formulated to be isotonic with the blood of the recipient.

Nasal spray formulations include purified aqueous solutions of the multi-arm polymer conjugates with preservatives and isotonic agents. Such formulations are preferably adjusted to a pH compatible with nasal mucosa and isotonic state.

Formulations for rectal administration may be presented as a suppository with a suitable carrier, for example cocoa butter or a hydrogenated fat or fatty carboxylic acid.

Formulations for ocular use are prepared by a method similar to nasal spray, except that the pH and isotonic factors are preferably adjusted to match the eye.

Topical formulations include the multi-arm polymer conjugate dissolved or suspended in one or more media (e.g., mineral oil, petroleum, polyhydric alcohols, or other bases used in topical formulations). It may be desirable to add other complexing agents as noted above.

Pharmaceutical formulations suitable for administration as aerosols, for example by inhalation, are also provided. The formulations comprise a solution or suspension of the desired multi-armed polymer conjugate or salt thereof. The desired formulation can be placed in a chamber and sprayed. Spraying may be accomplished by compressed air or by ultrasonic energy to form a plurality of droplets or solid particles comprising the conjugate or salt thereof.

Mixed salt-methods of using mixed salt conjugates

The mixed acid salts described herein can be used to treat or prevent any condition in any animal, particularly mammals (including humans), that is responsive to an unmodified active agent. Including a representative mixed acid salt of the anticancer agent (irinotecan), 4-arm-pentaerythrityl-PEG-glycine-irinotecan, is particularly useful in treating various types of cancers.

Some mixed acid salt conjugates, particularly where the small molecule drug is an anticancer agent (e.g., a camptothecin compound such as irinotecan or 7-ethyl-10-hydroxy-camptothecin, or other oncolytic drug as described herein), are 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 cancer, renal cancer, cholangiocarcinoma, brain cancer, cervical cancer, maxillary sinus cancer, bladder cancer, esophageal cancer, hodgkin's disease, adrenocortical cancer, and the like. Additional cancers treatable with the mixed acid salt include lymphoma, leukemia, rhabdomyosarcoma, neuroblastoma, and the like. As noted above, these mixed acid salt conjugates are particularly effective in targeting and accumulating within solid tumors. These mixed salt conjugates are also useful in the treatment of HIV (AIDS virus) and other viruses.

Representative conjugates (e.g., 4-arm-pentaerythrityl-PEG-glycine-irinotecan) have been shown to be particularly advantageous when used to treat patients with cancers that show difficulty in treatment with one or more anticancer agents.

The method of treatment comprises administering to a mammal in need thereof a therapeutically effective amount of a partial mixed acid salt composition or formulation as described herein.

Additional methods include treating (i) metastatic breast cancer resistant to anthracycline-based and/or taxane-based therapies, (ii) ovarian cancer resistant to platinum, (iii) metastatic cervical cancer, and (iv) colorectal cancer in a patient having a K-Ras mutated gene state by administering to the patient a partial mixed acid salt composition.

In the treatment of metastatic breast cancer, a mixed acid salt of a conjugate as provided herein (e.g., 4-arm-pentaerythrityl-PEG-glycine-irinotecan) is administered in a therapeutically effective amount to a patient having locally progressing metastatic breast cancer, wherein the patient has had no more than two prior (unsuccessful) treatments with anthracycline-and/or taxane-based chemotherapeutic agents.

For the treatment of platinum-resistant ovarian cancer, a composition as provided herein is administered in a therapeutically effective amount to a patient with locally advanced or metastatic ovarian cancer, wherein the patient has shown tumor progression during platinum-based treatment with a progression-free interval of less than six months.

In another method, a subject having locally advanced colorectal cancer is administered a mixed acid salt (e.g., as in example 1), wherein the one or more colorectal tumors have a K-Ras oncogene mutation (type of K-Ras mutant) such that the tumor is non-responsive to EGFR inhibitors such as cetuximab. The subjects were those who had failed a prior treatment with 5-FU and were also those who received irinotecan for the first time.

A therapeutically effective dose of any particular mixed acid salt will vary from conjugate to conjugate, patient to patient, and will depend on factors such as the condition of the patient, the activity of the particular active agent used, the type of cancer, and the route of delivery.

For camptothecin-type active agents (e.g., irinotecan or 7-ethyl-10-hydroxy-camptothecin), preferred doses are from about 0.5 to about 100mg camptothecin per kilogram body weight, preferably from about 10.0 to about 60 mg/kg. Even less of the mixed acid salt may be therapeutically effective when administered in combination with other pharmaceutically active agents. For administration of a mixed acid salt of irinotecan, the dose range of irinotecan will typically be from about 50mg/m²To about 350mg/m²。

The method of treatment also includes administering a therapeutically effective amount of a mixed acid salt composition or formulation as described herein (wherein the active agent is a camptothecin-type molecule) in combination with a second anticancer agent. Such camptothecin-based conjugates are preferably administered in combination with 5-fluorouracil and folinic acid as described in U.S. patent No. 6,403,569 (of course in the form of a mixed acid salt).

These mixed acid salt compositions may be administered once or several times a day, preferably once a day or less. The duration of treatment may be once daily for a period of from two to three weeks and may last for a period of months or even years. The daily dose can be administered in single dose form in the form of a single dosage unit or in the form of several smaller dosage units or by multiple administrations of subdivided doses at certain intervals.

It should be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description, together with the examples below, is intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention relates.

Experiment of

The practice of the present invention will employ, unless otherwise indicated, organic synthesis and similar conventional techniques, which are within the skill of the art. Such techniques are fully described in the literature. Unless specifically indicated to the contrary, reagents and materials are commercially available. See, e.g., M.B. Smith and J.March, March's Advanced organic Chemistry: Reactions Mechanisms and structures, 6 th edition, (New York: Wiley-Interscience, 2007), supra, and Comprehensive organic functional group Transformations II (Integrated organic functional group transformation II), volumes 1-7, second edition: a comprehensive review of the Synthetic Property 1995-: katritsky, a.r., et al, Elsevier Science.

In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should still be accounted for. Unless otherwise indicated, temperature is in degrees celsius (C) and pressure is atmospheric pressure at or near sea level.

The following examples illustrate certain aspects and advantages of the invention, however, the invention should not be construed as limited to the particular embodiments described below.

Abbreviations

Ar argon

CM Carboxymethyl or carboxymethylene (-CH)₂COOH）

DCC 1, 3-dicyclohexylcarbodiimide

DCM dichloromethane

DMAP 4- (N, N-dimethylamino) pyridine

GLY Glycine

HCl hydrochloric acid

RP-HPLC reversed-phase high performance liquid chromatography

IPA isopropyl alcohol

IRT irinotecan

IPC ion pair chromatography

MeOH methanol

MTBE methyl tert-butyl ether

MW molecular weight

NMR nuclear magnetic resonance

PEG polyethylene glycol

RT Room temperature

SCM succinimidyl carboxymethyl (-CH)₂-COO-N-succinimidyl)

TEA Triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

Materials and methods

Pentaerythrityl-yl 4-ARM-PEG from NOF corporation (Japan)_20K-OH。4-ARM-PEG_20K-OH has the following structure (where each n is about 113): c- (CH)₂O-(CH₂CH₂O)_NH)₄。

All of¹HNMR data were generated by a 300 or 400MHz NMR spectrometer manufactured by Bruker.

Example 1

Preparation 1: preparation of pentaerythrityl-4-arm- (PEG-1-methylene-2 oxo-vinylaminoacetate-linked irinotecan) -20K "4-arm PEG-GLY-irinotecan-20K" mixed acid salt

The reaction scheme is as follows:

all solvents used in the synthesis were anhydrous.

And (1).Coupling of t-boc-glycine to irinotecan HCl salt (>95% yield)

Irinotecan HCl trihydrate (1 mole or 677 g) and DMF (10L) were charged to a 60 ℃ still. Upon dissolution of irinotecan HCl trihydrate in DMF, full vacuum was slowly applied to remove water from irinotecan HCl trihydrate by azeotropic distillation at 60 ℃. Upon formation of a solid from residual DMF, heptane (up to 60L) was added to the still to remove residual DMF at 40-50 ℃. Upon removal of the heptane by visual inspection, the azeotropic distillation was stopped and the solid (irinotecan HCl) was allowed to cool to 17 ± 2 ℃. For the coupling reaction, t-boc-glycine (1.2 moles), 4-DMAP (0.1 moles), and DCM (19L) dissolved in DCM (1L) were added to the still. Once 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 thorough mixing and the pot temperature was 17 ℃ 2 ℃.

After a reaction time of 2-4 hours, samples were taken to measure the residual Irinotecan (IRT) peak area percentage by chromatography. Residual irinotecan was determined to be present in an amount no greater than 5%. DCU formed during the coupling reaction was removed by filtration and washed with DCM. The resulting filtrates containing the crude t-boc-glycine-irinotecan HCl salt were combined and concentrated under vacuum below 45 ℃ to remove DCM. Upon removal of about 75% of its initial volume by distillation, IPA was then added to the concentrate to reach the initial volume and the mixture was further distilled until the concentrated volume reached about 25% of its initial volume. The resulting clear solution was cooled to room temperature and then added to heptane with mixing. The mixture is mixed for an additional 0.5 to 1 hour, during which a precipitate is formed. The precipitate was drained and filtered to obtain a wet cake, and then washed with heptane (up to 6L). The wet cake was dried under vacuum to yield t-boc-glycine-irinotecan powder for step 2. The yield was > 95%.

And 2. step 2.Deprotection of t-boc-glycine-irinotecan

T-boc-glycine-irinotecan (1 mole) from step 1 was dissolved in DCM under stirring. TFA (15.8 moles) was added to the solution 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 determined to be less than about 5%. Acetonitrile was then added to the reaction solution to form a visually homogeneous solution at RT. This solution was then added to MTBE (46.8 kg) at 35 ℃ with thorough stirring to promote crystallization. Optionally, to reduce the use of MTBE, the DCM in the reaction solution was replaced by acetonitrile by distillation at 15 ℃ to 40 ℃. After solvent exchange, the product-containing solution was added to about 50% less by volume of well-stirred MTBE (23 kg) at crystallization temperature (35 ℃). Mixing is continued for half an hour to one hour. The resulting solid was filtered and the cake was washed with MTBE.

The wet cake was dried under vacuum to yield glycine-irinotecan salt powder for step 3. The trifluoroacetate and chloride contents of the product were determined by ion chromatography using a conductivity detector. (yield > 95%).

And 3. step 3.PEGylation of glycine-irinotecan using-arm-PEG-CM-SCM

Glycine-irinotecan TFA/HCl salt powder from step 2 was added to a reactor to which DCM (about 23L) was added. The mixture was stirred for about 10-30 minutes to allow glycine-irinotecan TFA/HCl to disperse in DCM. Triethylamine (glycine-irinotecan TFA/HCl salt powder moles at about 1.05 moles of (HCl + TFA)) was then slowly added at a rate to maintain the temperature of the kettle at 24 ℃ or less. The resulting mixture was stirred for 10 to 30 minutes to allow the GLY-IRT (glycine modified irinotecan) free base to dissolve.

About 80% of the total amount (6.4 kg) of 4-arm PEG-SCM was added to the reactor over the course of up to 30 minutes. After dissolving the PEG reagent, the reaction progress was monitored by IPC. (if the amount of unconjugated GLY-IRT is greater than 5% when the reaction appears to have reached a plateau, the remaining 20% of the 4-arm PEG SCM is added to the reactor and the reaction progress is monitored until a constant value of unreacted GLY-IRT is observed.

The crude product was precipitated by adding the reaction solution to MTBE (113.6L) stirred at room temperature over a period of from 1 to 1.5 hours, followed by stirring. The resultant mixture was transferred to a filter-drier having an agitator to remove the mother liquor. The precipitate (crude product) was partially vacuum dried at about 10 ℃ to 25 ℃ with minimal batch agitation.

The crude product was then placed in a reactor to which IPA (72L) and MeOH (8L) were added, followed by stirring for up to 30 minutes. Heat was applied to achieve complete visual dissolution at a pan temperature of 50 ℃ (clear solution) followed by stirring for 30 to 60 minutes. The solution was then cooled to 37 ℃, held there for several hours, and subsequently cooled to 20 ℃. The mixture was then transferred to an agitated filter dryer and filtered to remove the mother liquor, thereby forming a cake on the filter. The cake was washed with 70% MTBE and 30% MeOH in IPA and partially dried in vacuo. This step was repeated two additional times except that the clear IPA/MeOH solution containing 4-arm PEG-Gly-IRT was filtered using an in-line filter (1 um) filter at 50 ℃ to remove any potential particulates in the final (third) crystallization before cooling.

Three representative samples were taken from the washed wet cake and NHS levels were measured using NMR. The wet cake was dried under vacuum.

The product ("API") was packaged in double bags sealed under an inert atmosphere and stored at-20 ℃ in the dark. The product yield was about 95%.

Example 2

"4-arm-PEG-G as Mixed salt_LY-I_RINOCharacterization of the-20K "product

The product from example 1 was analyzed by ion chromatography (IC analysis). See Table 1 below for the results of IC analysis of different product batches of 4-arm-PEG-Gly-Irino-20K.

TABLE 1

Mole percent of irinotecan bound to PEG

Batch number	TFA salts	HCl salt	Free base
				010	59	36	5 (Low)
020	64 (high)	30	6
				030	27 (Low)	24	49 (high)

040	53	26	21
				050	54	26	20
060	57	28	15
				070	53	33	14
080	53	27	20
				090	44	19	36
100	33	41	26
				Average of last 7 batches	50	29	22

Based on the IC results provided in table 1, it can be seen that the product formed in example 1 (4-arm-PEG-Gly-Irino-20K) is a mixed salt based on the coupled irinotecan molecule in the product, about 50 mole percent TFA salt, 30 mole percent HCl salt, and 20 mole percent of a portion of the free base. Even after repeated (1-3) recrystallizations of the product, mixtures of salts were observed. In the different product batches analyzed above, it can be seen that about 35-65 mole percent of irinotecan molecules in the composition are protonated as the TFA salt, about 25-40 mole percent of irinotecan molecules in the composition are protonated as the HCl salt, while the remaining 5-35 mole percent of irinotecan is unprotonated (i.e., as the free base).

The general structure of the product is shown below, with the irinotecan moiety shown as the free base and associated with HCl and TFA-as one indication of mixed salts.

Example 3

4-arm-PEG-G_LY-I_RINOStress stability study at-20K

Stability studies were conducted in an attempt to evaluate the 4-arm-PEG-Gly-Irino-20K product composition. Compositions containing different amounts of protonated irinotecan, and differing in the amount of TFA versus HCl salt, were examined.

Study of stress stability

API packaging conditions were simulated by weighing (about 1-2 g) the product formed in example 1, 4-arm-PEG-Gly-Irino-20K, compound 5, into a PEG PE 'spin top' bag and into another 'spin top' bag. In one study (results are shown in fig. 1), the samples were placed in an environmental chamber at 25 ℃/60% RH for 4 weeks. In another study, the samples were placed in an environmental chamber at 40 ℃/75% RH for up to several months (results are shown in fig. 2 and 3). Samples were taken periodically during the study and analyzed.

Results

The results of the study are shown in fig. 1,2 and 3. In FIG. 1, the 4-arm-PEG-Gly-Irino-20K peak area percentage of samples stored at 25 ℃ and 60% relative humidity is plotted versus time. Data shown are for samples consisting of >99% HCl salt (< 1% free base, triangles), 94% full salt (6% free base, squares), and 52% full salt (48% free base, circles). The slope in the graph shows that the stability of the product decreases with increasing free base content. Under the stress conditions used (i.e. up to 28 days at 25 ℃), the decrease in the 4-arm-PEG-Gly-Irino-20K peak area is clearly correlated with an increase in free irinotecan, indicating that the mode of decomposition is primarily through hydrolysis of the ester bond to release irinotecan. Based on the observed results, it appears that a larger amount of free base in the product results in reduced stability to hydrolysis. Thus, irinotecan containing a greater degree of protonation appears to have greater stability (on a molar percentage basis) against hydrolysis than a product containing less protonated irinotecan.

Fig. 2 and 3 show another set of data obtained from samples stored at 40 ℃ and 75% relative humidity containing >99% HCl salt (less than 1% free base, squares) and samples consisting of 86% full salt (14% free base, diamonds). Figure 2 shows the increase in free irinotecan over 3 months for both samples. This data is consistent with the data from the previously described study (summarized in fig. 1), which shows that products with higher free base content are less stable to hydrolysis. Figure 3 shows a smaller increase in PEG species for the same samples over 3 months. The smaller increase in PEG species indicates that the PEG backbone breaks down to provide multiple PEG species. The data show that the product corresponding to the HCl salt is more prone to PEG backbone decomposition than the mixed salt sample containing 14% free base. Thus, while not intending to be bound by theory, it appears that while the mixed salt of the moiety degrades primarily by hydrolysis to release the drug, the hydrochloride salt appears to degrade by a different mechanism, i.e., to degrade the polymer backbone. Based on these preliminary results, part of the mixed salt product appeared to be superior to the hydrochloride salt.

In summary, the two observed decomposition modes show opposite tendencies with respect to salt/free base content. Surprisingly, these results indicate that there is a range of salt compositions where it can have overall stability enhanced beyond either of the traditional extremes of full salt and full free base. These results further indicate the unforeseen advantage of the mixed salt of the 4-arm-PEG-Gly-Irino-20K moiety over the free base alone or in the absence of either salt of the other. The mixed salt showed greater stability than either the free base or the hydrochloride salt, thus indicating its superiority over either the more common pure base or pure salt forms.

Example 4

Study of chirality

The chirality of carbon-20 of irinotecan in 4-arm-PEG-Gly-Irino-20K is determined.

As specified in the documents from the supplier, irinotecan hydrochloride starting material is optically active, with C-20 in its (S) -configuration. Irinotecan carries a tertiary alcohol at the C-20 position, which is not readily ionized, and therefore racemization at this site is not expected except under extreme (strong acid) conditions. To determine the chirality of C-20 in 4-arm-PEG-Gly-Irino-20K, irinotecan released from the product via chemical hydrolysis was analyzed using a chiral HPLC method.

Based on the resulting chromatogram, no (R) -enantiomer was detected for the 4-arm-PEG-Gly-Irino-20K sample. After hydrolysis, it was confirmed that irinotecan released from the conjugate was in the (S) -configuration.

Example 5

Hydrolysis study

All pegylated irinotecan is considered to be part of 4-arm-PEG-Gly-Irino-20K; each species clearly hydrolyses to yield irinotecan of greater than 99% purity. Furthermore, predominantly the drug-loaded DS4 species (drug covalently attached to each of the four polymer arms) and the partially substituted species-DS 3 (drug covalently attached to three polymer arms), DS2 (drug covalently attached to two polymer arms) and DS1 species (drug covalently attached to one single polymer arm) -all hydrolyze at the same rate to release free drug (irinotecan).

Experiments were performed to determine the degree of transesterification (in CH)₃K in OH₂CO₃) And the content of iritinic in the 4-arm-PEG-Gly-Irino-20K under the condition of aqueous hydrolysis (pH 10, 20℃)Results for healthy PEG species (fast). After 45 minutes, the transesterification reaction was complete>99 percent. Within 24 hours, the aqueous hydrolysis reaction was complete>99 percent. For both reaction types, a control reaction using irinotecan was carried out under the same conditions and some false peaks were observed. After adjustment of the peak of artifact, in both cases irinotecan is produced with>99% chromatographic purity.

Based on these results, it was concluded that substantially all pegylated species in 4-arm-PEG-Gly-Irino-20K released irinotecan. The superposition of multiple HPLC taken over time from the aqueous hydrolysis reaction shows the following conversions: DS4 to DS3 to DS2 to DS1 to irinotecan. All of these species hydrolyze to release irinotecan. Referring to fig. 4, it is demonstrated that irinotecan is released via hydrolysis from mono-, di-, tri-and tetra-substituted 4-arm-PEG-Gly-Irino-20K species.

Additional experiments were performed to measure the hydrolysis rate of the main component 4-arm-PEG-Gly-Irino-20K (DS 4), 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 in the presence of porcine carboxypeptidase B was one attempt to perform enzyme-based hydrolysis. A control experiment at pH8.4 but without enzyme later showed that hydrolysis was pH driven and thus was primarily a chemical hydrolysis. Nevertheless, this data is valuable for comparison with data obtained from hydrolysis performed in human plasma. These experiments show that the hydrolysis rates of the different components are not significantly different and better than predicted by theory. Additional experiments measured the hydrolysis rate of the major components of 4-arm-PEG-Gly-Irino-20K (DS 4, DS3, DS2, and DS 1) in human plasma. These experiments also show that the different components hydrolyze at the same rate and better than theoretically predicted.

Figures 5 and 6 present graphs showing theoretical hydrolysis rates of chemical hydrolysis (in the presence of enzymes) and plasma hydrolysis, respectively, versus experimental data. In both cases, theoretical predictions are based on the same rate of hydrolysis of each species to yield the next lower homolog plus free irinotecan (i.e., DS4> DS3> DS2> DS 1).

Example 6

Preparation 2: preparation of pentaerythrityl-4-arm- (PEG-1-methylene-2 oxo-vinylaminoacetate-linked irinotecan) -20K "4-arm PEG-GLY-irinotecan-20K" mixed acid salt

Step 1 Synthesis of Boc-glycine-irinotecan hydrochloride (Gly-IRT HCl)

Part 1: irinotecan hydrochloride trihydrate (irt. hcl.3h) ₂ O) drying

IRT HCl 3H₂O (45.05 g, 66.52 mmol) was added to one reactor. Anhydrous N, N Dimethylformamide (DMF) (666 mL, 14.7mL/g IRT HCl 3H₂O, DMF water content NMT 300 ppm) was added to the reactor. The reactor was heated to 60 ℃ (jacket temperature) with slow stirring. After Irinotecan (IRT) was completely dissolved (5-10 min), vacuum was slowly applied until 5-10 mbar was reached and DMF was distilled off. The vacuum was released when the volume of condensed Distillate (DMF) reached 85% -90% of the initial DMF charge. Heptane (1330 mL, 30.0mL/g IRT HCl 3H₂O, water content NMT50 ppm) was introduced into the reactor and the jacket temperature was reduced to 50 ℃ the heptane was vacuum distilled (100-150 mbar) until the volume of distillate was about 90% of the initial charge of heptane two more cycles of heptane distillation were performed (2 × 1330mL of heptane charge and distillation) the solvent phase sample was removed from the reactor and analyzed for DMF content using GC to ensure that the DMF content was less than 3% w/w (if residual DMF was present)>3.0% w/w, then a fourth azeotropic distillation cycle will be performed. The resultant slurry was used for the coupling reaction (part 2).

Part 2: coupling reaction: preparation of Boc-gly-IRT & HCl

Dichloromethane (1330 mL, 29.5mL DCM/g IRT HCl 3H₂O) Dry IRT to residual heptane with agitationHCl (1.0 eq) (approximate mass ratio of residual heptane to IRT HCl 3). The reaction ingredients were stirred for 15-30 minutes and the batch temperature was maintained at 17 ℃. Boc-glycine (14.0 g, 79.91mmol, 1.2 equiv.) and DMAP (0.81 g, 6.63mmol, 0.1 equiv.) were charged to the reactor as solids. A DCM solution of DCC (1.5 equivalents in 40mL of dichloromethane) was prepared and added to the reactor over a period of 15-30min and the resultant mixture was stirred at 17 ℃ (batch temperature) for 2-3 hr. The reaction was monitored by HPLC to ensure completion. A pre-made quenching solution was added to the reaction mixture to quench any remaining DCC. Briefly, the pre-quench solution was a premixed solution of TFA and IPA in dichloromethane by dissolving in DCM (15.3 mL, 0.34mL/g IRT HCl 3H)₂O) was mixed with TFA (1.53 mL, 0.034mL/g IRT HCl 3H)₂O) and IPA (3.05 mL, 0.068mL/g IRT HCl 3H₂O) and added to reactor V1 over a period of 5 to 10 minutes at a conversion of at least 97%. The ingredients were stirred for an additional 30-60min to allow for quenching. The reaction mixture containing DCU was filtered through a 1 micron filter into another reactor. The reaction filtrate was distilled to 1/3 of its volume under vacuum at 35 ℃. Isopropanol (IPA) (490.5 mL, 10.9mL/gIRT HCl 3H₂O) was added to the concentrated mixture and the mixture was stirred at 50 ℃ (jacket temperature) for 30-60 min. The resulting homogeneous solution was concentrated to about 85% of the initial IPA feed volume by vacuum distillation and the resultant concentrate was cooled to 20 ℃ (jacket temperature). The reaction mixture in IPA was transferred to heptane (1750 mL, 38.8mL heptane/g IRT HCl 3H) at 20 ℃ over 60-80min₂O) the resulting slurry containing the Boc-gly-IRT HCl precipitate was stirred for an additional 60-90 minutes and the product was collected by filtration with heptane (2 × 490mL, 20.0mL heptane/g IRT HCl 3H₂O) rinse the reaction flask and wash the product cake with rinse solution. The wet cake is dried at 20 ℃ to 25 ℃ under vacuum for at least 12 hr. Yield: 57.13g (110%, high due to residual solvent)

Step 2. Glycine-irinotecan hydrochlorideSynthesis (deprotection) of (Gly-IRT HCl-TFA) trifluoroacetate

To an appropriately sized reactor, dry Boc-gly-IRT. HCl (41.32 g, 52.5mmol, from step 1) was added under an inert atmosphere. Anhydrous DCM (347 mL, 8.4mL of DCM/g of Boc-gly-IRT. HCl) was added to the reactor and the ingredients were stirred at 17 ℃ until complete dissolution (about 15-30 min). TFA (61.98 mL, 691.5mmol, 1.5mL/g of Boc-gly-IRT. HCl) was added to the flask over 15-30min and mixing was continued for 3.0 h. The completion of the reaction was monitored by HPLC (limit: not less than 97%). The reaction was diluted with acetonitrile (347 mL, 8.4mL ACN/g Boc-gly-IRT. HCl). The jacket temperature was set to 15 ℃ and the reaction mixture was concentrated under vacuum until the final residual pot volume was about 85% of the initial acetonitrile charge (about 295-305 mL). The resulting acetonitrile solution was slowly added to a reactor containing methyl tert-butyl ether (MTBE, 1632mL, 39.5mL MTBE per g Boc-gly-IRT. HCl) over a period of 30-60 minutes. The precipitated product was gently mixed for 30 minutes, and the product was collected by filtration. The reactor was rinsed with MTBE (410 mL) and the gly-IRT HCl/TFA filter cake was washed with the rinse. The product was dried under vacuum at 17 ℃ for at least 12 hours. Yield: 42.1g (102%).

Step 3.4-arm PEG20K Synthesis of irinotecan hydrochloride-trifluoroacetate

Gly-IRT HCl-TFA (10.0 g) was added to the 250mL reactor and flushed with argon. The jacket temperature was set at 20 ℃. DCM (166 mL) and TEA (2.94 g) were added. The solution was mixed for 10 minutes. An initial charge of 4-arm PEG20K-SCM (47.6 g) was added and the reaction mixture was stirred for 30 minutes. Samples were taken and analyzed by HPLC. HPLC data showed 18% Gly-IRT remaining. A second charge of 4-arm PEG20K-SCM (10.7 g) was added to the reaction mixture and the solution was stirred for about 2 hours. Samples were taken for HPLC analysis. HPLC analysis data showed 1.5% Gly-IRT remaining. The reaction solution was then slowly added to MTBE (828 mL) to precipitate the product. The precipitate was stirred for 30 minutes and collected via filtration. The wet cake was washed with a mixture of 30% methanol/70% MTBE (830 mL). The product was then added to a reactor containing a mixture of 30% methanol/70% MTBE (642 mL) and the mixture was stirred at 20 ℃ for 20 minutes. The mixture was filtered and the wet cake was washed on the filter with a mixture of 30% methanol/70% MTBE (642 mL). The product was dried under vacuum at 20 ℃.

The dried product was added to a reactor containing ethyl acetate (642 mL). The mixture was heated to 35 ℃ to achieve complete dissolution. If it is necessary to remove the undissolved particles, the warm solution is filtered and then cooled to 10 ℃ with stirring. The precipitated 4-arm PEG 20K-glycine-irinotecan hydrochloride-trifluoroacetate product was filtered and the wet cake was washed on the filter with a mixture of 30% methanol/70% MTBE (642 mL). The product was then dried under vacuum at 20 ℃. Yield: 54g (about 85%).

Different batches prepared according to the above method were analyzed by ion chromatography on the salt composition.

TABLE 2

Mole percent of irinotecan bound to PEG

TABLE 3

Mean and standard deviation of the batches in Table 2

As can be seen from the results in table 2, batches prepared as described show consistent ratios of TFA salt, hydrochloride salt and free base. Based on reviewing batch information, it appears that higher chloride levels in the glycine-irinotecan TFA/HCl intermediate result in higher chloride levels in the final mixed salt conjugate product. By using a starting material with a fairly constant chloride content (e.g. irinotecan hydrochloride), glycine-irinotecan TFA/HCl salts with fairly constant chloride content can be prepared.

Based on a further review of batch information, it appears that the higher the number of equivalents of TEA utilized in step 3, the lower the TFA and to a lesser extent the content (chloride) in the final mixed salt conjugate product. Measurement of the chloride and TFA content of the intermediate (i.e. gly-irinotecan TFA/HCl) may be made easier by greater dissolution of the intermediate prior to analysis, e.g. ion chromatography may allow for more accurate determination of stoichiometry (e.g. in the amount of triethylamine added in the final reaction step).

Based on the above, the preferred range of TFA in the mixed acid salt is from about 20 to about 45 mole percent, preferably from about 22 to 40 mole percent, or from about 24 to 38 mole percent. With respect to the hydrochloride salt content, the preferred range of the mixed acid salt conjugate is from about 30 to 65 mole percent chloride, or from about 32 to 60 mole percent chloride, or from about 35 to 57 mole percent chloride.

Example 7

4-arm-PEG-G with different salt ratios_LY-I_RINOStress stability study of-20K Material

Short-term (4-week) stability studies were performed on 4-arm-PEG 20K-gly-irinotecan with varying salt concentrations, as summarized in table 4 below. The "pure" hydrochloride salt is shown in the left-most column, while the unprotonated, free base is shown in the right-most column, with a differential degradation in the middle. The study was performed over a range of temperatures (20 ℃ with no humidity control, 5 ℃ with no humidity control, 25 ℃ at 60% relative humidity, and 40 ℃ at 75% relative humidity) essentially as described in example 3.

TABLE 4

Sample information

For the HCl salt (batch a), the total product related species varied from 98.7% to 97% while the free irinotecan varied from 0.4% to 1.25% over the course of 4 weeks when evaluated over these temperature ranges. For the free base (batch D), the total product related species varied from 99.8% to 62.5% while the free irinotecan varied from 0.3% to 31.4% over the course of 4 weeks when evaluated over these temperature ranges.

When evaluated under cryogenic conditions, minimal degradation was observed for each material at-20 ℃ and-5 ℃ over the course of 4 weeks. Hydrolysis was observed in each test species using the free base material when evaluated at 25 ℃, thereby showing the most significant drug hydrolytic release. The same results were observed at 40 ℃, with the composition with the greatest amount of free base exhibiting a correspondingly faster rate of irinotecan hydrolysis. At high temperature, i.e. at 40 ℃, cleavage of the PEG backbone was detected.

Example 8

Preparation of pentaerythrityl-4-arm-PEG-20K on a 1.9KG Scale

Materials and methods. Very high grades of ethylene oxide with the lowest achievable water content should be used because the water content results in polymer diol impurities. Note that: ethylene oxide is a very reactive compound that reacts explosively with moisture and therefore leakage in the reaction and transfer equipment should be carefully avoided. Furthermore, care should be taken in handling, including having personnel work behind protective shields or in protective cabins.

Anhydrous toluene (4L) was refluxed in a two gallon jacketed stainless steel pressure reactor for two hours. Next, a portion of the solvent (3L) was distilled off at atmospheric pressure. The residual toluene was then vented and the reactor was dried overnight by passing steam through the reactor jacket and applying reduced pressure (3-5 mmHg). The reactor was then cooled to room temperature and filled with anhydrous Toluene (4L) and pentaerythrityl 4ARM-PEG-2K (SUNBRIGHT) was addedPentaerythritol, molecular weight about 2,000 daltons, NOF company, 200g, 0.100 mole) solvent was distilled off under reduced pressure, and then the reactor was cooled to 30 ℃ under a dry nitrogen atmosphere.one liter of molecular sieve dried toluene (water content about 5 ppm) and liquid sodium-potassium alloy (Na 22%, K78%; 1.2 g) were added to the reactor was heated to 110 ℃ and ethylene oxide (1,800 g) was added continuously over three hours, keeping the reaction temperature at 110-120 ℃ next, the reactor contents were heated at about 100 ℃ for two hours, and then cooled to about 70 ℃ after reduced pressure excess ethylene oxide and toluene were distilled off under reduced pressure.after distillation, the reactor contents were kept under reduced pressure, and a nitrogen sparge was performed to remove traces of ethylene oxide.phosphoric acid (1N) was added to neutralize the alkaline residue, and the product was dried under reduced pressure.the reactor, and after cooling the filtered 1.g the filtered 1.t. gave a filtered solid phase chromatography using a refractive index chromatography system providing a filtered flow chromatography of the product of pentaerythritol with a ph of 20-300 mm.8. the filtered product providing a filtered gfx-300 mm₃) The flow rate of (2) was 0.5 ml/min. The GFC chromatogram is shown in fig. 7.

GFC analysis showed that the 4-arm-PEG-20K product contained the following: high MW product 0.42%, 4 arm-PEG-20K 99.14%, HO-PEG (10K) -OH 0.44%.

Example 9

Analysis of commercially available 4-arm PEG-20K

NOF corporation is the current leader in providing commercial PEG. Thus, a fresh commercially available pentaerythritol-based 4-arm-PEG-20K (SUNBRIGHT) was obtainedMolecular weight of about 20,000 daltons, NOF corporation) and analyzed using Gel Filtration Chromatography (GFC) an Agilent1100HPLC system equipped with Shodex KW-803GFC column (300 × 8 mm) and differential refractometer detector was used mobile phase (0.1M NaNO)₃) The flow rate of (2) was 0.5 ml/min. The GFC chromatogram is shown in fig. 8.

GFC analysis showed that the commercial 4-arm-PEG-20K product contained: high MW product 3.93%, 4-arm-PEG-20K 88.56%, HO-PEG (10K) -OH 3.93%, HO-PEG (5K) -OH 3.58%.

Example 10

Preparation of oxyalkylatable oligomers: pentaerythrityl 4-arm-PEG-2K on a 15KG Scale

Twenty gallons of jacketed stainless steel pressure reactor were washed twice with 95kg of deionized water at 95 ℃. The wash water was removed and the reactor was dried overnight by passing steam through the reactor jacket and applying reduced pressure (3-5 mm Hg). 25kg of anhydrous toluene was charged into the reactor and a part of the solvent was distilled off under reduced pressure. The residual toluene was then vented and the reactor was maintained under reduced pressure. The reactor was then cooled to room temperature, filled with anhydrous toluene (15L), and pentaerythritol (1,020 g) was added. Part of the solvent (about 8L) was distilled off under reduced pressure, and then the reactor was cooled to 30 ℃ under a dry nitrogen atmosphere. Liquid sodium-potassium alloy (Na 22%, K78%; 2.2 g) was added to the reactor. Anhydrous ethylene oxide (14,080 g) was added continuously over three hours, maintaining the reaction temperature at 150 ℃ to 155 ℃. Next, the contents of the reactor were heated at about 150 ℃ for 30 minutes and then cooled to about 70 ℃. The excess ethylene oxide and toluene were distilled off under reduced pressure. After distillation, the reactor contents were kept under reduced pressure and a nitrogen sparge was performed to remove traces of ethylene oxide. Finally, the product is discharged from the reactor to giveGel Filtration Chromatography (GFC) was applied to characterize the product, pentaerythrityl 4-arm-PEG-2K. the analytical method provided chromatograms of compositions with separation of components according to molecular weight using an Agilent1100HPLC system equipped with a Shodex KW-803GFC column (300 × 8 mm) and a differential refractometer detector mobile phase (0.1 MNaNO)₃) The flow rate of (2) was 0.5 ml/min.

GFC analysis showed that the 4-arm-PEG-2K product was about 100% pure with low or high molecular weight impurities below detectable limits.

Example 11

Preparation of pentaerythritol-yl 4-arm-PEG-20K on a 20KG Scale

Twenty gallons of jacketed stainless steel pressure reactor were washed twice with 95kg of deionized water at 95 ℃. The water was drained and the reactor was dried overnight by passing steam through the reactor jacket and applying reduced pressure (3-5 mm Hg). 25kg of toluene was charged into the reactor and a part of the solvent was distilled off under reduced pressure. The residual toluene was then vented and the reactor was maintained under reduced pressure. The reactor was then cooled to room temperature, filled with anhydrous toluene (21L) and the previously isolated oxyalkylatable oligomer: pentaerythritol-based 4-arm-PEG-2K (2,064 g) from example 10 was added. Part of the solvent (16L) was distilled off under reduced pressure and then the reactor was cooled to 30 ℃ under a dry nitrogen atmosphere. Four liters of molecular sieve dried toluene (water content about 5 ppm) and liquid sodium-potassium alloy (Na 22%, K78%; 1.7 g) were added and the reactor was heated to 110 ℃. Next, ethylene oxide (19, 300 g) was continuously added over five hours, maintaining the reaction temperature at 145 ℃ and 150 ℃. Next, the contents of the reactor were heated at about 140 ℃ for 30 minutes and then cooled to about 100 ℃. Glacial acetic acid (100 g) was added to neutralize the catalyst. The excess ethylene oxide and toluene were distilled off under reduced pressure. After distillation, the reactor contents were kept under reduced pressure and a nitrogen sparge was performed to remove traces of ethylene oxide. The product was finally discharged from the reactor, giving 20,100Gel Filtration Chromatography (GFC) was applied to characterize the alkoxylated polymer product, pentaerythrityl 4-arm-PEG-20K. the analytical method provided chromatograms of compositions with component separation according to molecular weight using an Agilent1100HPLC mobile phase (0.1M NaNO) equipped with Shodex KW-803GFC column (300 × 8 mm) system and differential refractometer detector₃) The flow rate of (2) was 0.5 ml/min.

GFC analysis showed that the 4-arm-PEG-20K product contained the following: high MW product 0.75%, 4-arm-PEG-20K 97.92%, HO-PEG (10K) -OH 1.08%, HO-PEG (5K) -OH 0.48%.

The invention or inventions set forth herein have been described with respect to specific exemplary embodiments thereof. However, the above description is not intended to limit the invention to these exemplary embodiments, and those skilled in the art will recognize that many changes may be made within the spirit and scope of the invention, as described in the above description.

Claims (20)

1. A process for preparing a 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt having the structure:

wherein n is an integer from 40 to 500,

the method comprises the following steps:

(i) deprotecting the hydrochloride salt of glycine-irinotecan in protected form by treatment with trifluoroacetic acid to form a deprotected glycine-irinotecan hydrochloride/trifluoroacetic acid mixed salt, the trifluoroacetic acid being used in an amount corresponding to either a 13.2 molar excess or a 15.8 molar excess to the amount of glycine-irinotecan in protected form,

(ii) (ii) coupling the deprotected glycine-irinotecan hydrochloride/trifluoroacetic acid mixed salt of step (i) with 4-arm pentaerythritolyl-PEG-succinimide or other activated ester in the presence of a base to form a conjugate, and

(iii) recovering the conjugate, wherein the conjugate is a 4-arm-pentaerythritolyl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt having the one or more irinotecan amino groups present as a combination of free base, hydrochloride salt, and trifluoroacetate salt,

wherein 25 to 45 mole percent of the irinotecan amino groups are protonated as trifluoroacetate salts when the deprotection step (i) is conducted with 13.2 molar excess trifluoroacetic acid, and 35 to 65 mole percent of the irinotecan amino groups are protonated as trifluoroacetate salts when the deprotection step (i) is conducted with 15.8 molar excess trifluoroacetic acid.

2. The process of claim 1 further comprising determining the relative molar amounts of hydrochloric acid and trifluoroacetic acid in the deprotected glycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt formed in step (i).

3. The process of claim 2, wherein the amount of base in step (ii) is in the range of 1.00 to 2.00 times the total number of moles of hydrochloric acid and moles of trifluoroacetic acid determined for the deprotected glycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt.

4. The method of claim 1, wherein the glycine-irinotecan in protected form is t-boc-glycine irinotecan.

5. The process of claim 1, further comprising purifying the 4-arm-pentaerythritolyl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt recovered from step (iii).

6. The process of claim 5, wherein the purifying comprises recrystallizing the 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt to form a recrystallized product.

7. The process of claim 6, wherein the recrystallized product is also a 4-arm-pentaerythritolyl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt comprising irinotecan amino groups present as a combination of the free base, hydrochloride salt, and trifluoroacetate salt.

8. The process of claim 1 which is effective to reproducibly provide a 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt having relative molar amounts of the salt acid addition salt, the trifluoroacetate salt and the free base that vary consistently from batch to batch by no more than fifteen percent.

9. A 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt reproducibly prepared by the process of claim 1, having relative molar amounts of the salt acid addition salt, the trifluoroacetate salt and the free base that vary by no more than fifteen percent in batch-to-batch consistency.

10. Use of a 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt prepared by the process of claim 1 in the manufacture of a medicament for treating cancer in a mammalian subject in need thereof by administering to the mammalian subject a therapeutically effective amount of the mixed salt.

11. The use of claim 10, wherein said administering comprises parenterally administering said 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt.

12. Use of a 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt prepared by the process of claim 1 in the manufacture of a medicament for treating one or more cancerous solid tumors in a mammalian subject.

13. The use of claim 12, wherein the cancerous solid tumor is selected from the group consisting of: colorectal, ovarian, cervical, breast, and non-small cell lung tumors.

14. Use of a 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt prepared by the process of claim 1 in the manufacture of a medicament for treating a condition in a mammalian subject that is responsive to treatment with camptothecin.

15. A pharmaceutically acceptable composition in lyophilized form comprising (i) a 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt prepared by the process of claim 1, and (ii) a lactate buffer.

16. The composition of claim 15, which, when reconstituted, has a pH of 3.5.

17. A pharmaceutically acceptable, sterile composition in lyophilized form comprising (i) the 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt of claim 9 and (ii) a lactate buffer, the composition being contained in a single use vial.

18. The composition of claim 17, wherein the amount of the 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt contained in a vial is equivalent to a 100 milligram dose of irinotecan.

19. Use of a composition according to claim 17 or 18 in the manufacture of a medicament, wherein the composition is dissolved in a 5% w/w glucose solution and the medicament is administered by intravenous infusion for the treatment of one or more types of cancerous solid tumors.

20. A composition comprising a 4-arm-pentaerythrityl-polyethylene glycol-carboxymethylglycine-irinotecan trifluoroacetic acid/hydrochloric acid mixed salt obtainable by the process of any one of claims 1 to 4.