Source: https://patents.google.com/patent/US8273833
Timestamp: 2018-02-22 07:25:06
Document Index: 583351328

Matched Legal Cases: ['§119', 'Application No. 02', 'Application No. 2002352524', 'Application No. 2004', 'Application No. 4004336', 'Application No. 40044336', 'Application No. 2004', 'Application No. 2003', 'Application No. 2003', 'Application No. 2003', 'Application No. 2']

US8273833B2 - Branched Polymers - Google Patents
US8273833B2
US8273833B2 US12963170 US96317010A US8273833B2 US 8273833 B2 US8273833 B2 US 8273833B2 US 12963170 US12963170 US 12963170 US 96317010 A US96317010 A US 96317010A US 8273833 B2 US8273833 B2 US 8273833B2
US12963170
US20110077362A1 (en )
William Dudley Battle, III
The present invention is directed to branched reactive water-soluble polymers comprising at least two polymer arms, such as poly(ethylene glycol), attached to a central aliphatic hydrocarbon core molecule through ether linkages. The branched polymers bear at least one functional group for reacting with a biologically active agent to form a biologically active conjugate. The functional group of the branched polymer can be directly attached to the aliphatic hydrocarbon core or via an intervening linkage, such as a heteroatom, -alkylene-, —O-alkylene-O—, -alkylene-O-alkylene-, -aryl-O—, —O-aryl-, (—O-alkylene-)m, or (-alkylene-O—)m linkage, wherein m is 1-10.
This application is a continuation application of U.S. patent application Ser. No. 11/336,695, filed Jan. 20, 2006, now U.S. Pat. No. 7,872,072, which is a divisional application of U.S. patent application Ser. No. 10/290,082, filed Nov. 7, 2002, now U.S. Pat. No. 7,026,440, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 60/337,613, filed Nov. 7, 2001, all of which are incorporated herein by reference in their entireties.
This invention relates to branched, reactive water soluble polymers useful for conjugating to biologically active molecules and to methods for making an utilizing such polymers.
Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, is a highly advantageous method of increasing water solubility and bioavailability and extending the circulation time of many biologically active molecules, particularly hydrophobic molecules. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995). The total molecular weight of the polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.
Proteins and other molecules often have a limited number of reactive sites available for polymer attachment. Often, the sites most suitable for modification via polymer attachment play a significant role in receptor binding, and are necessary for retention of the biologically activity of the molecule. As a result, indiscriminate attachment of polymer chains to such reactive sites on a biologically active molecule often leads to a significant reduction or even total loss of biological activity of the polymer-modified molecule. To form conjugates having sufficient polymer molecular weight for imparting the desired advantages to a target molecule, prior art approaches have typically involved either (i) random attachment of numerous polymer arms to the molecule, thereby increasing the risk of a reduction or even total loss in bioactivity of the parent molecule, or (ii) attachment of one or two very long polymer chains. Unfortunately, the use of very high molecular weight linear polymer chains is problematic because of the difficulty and expense associated with their preparation, purification, and associated instability.
The present invention is based upon the development of branched, reactive water-soluble polymers useful for conjugation to biologically active molecules in a manner that tends to avoid a significant reduction in the biological activity of the molecule while still providing the benefits of water-soluble polymer conjugation. The branched polymers of the invention can be readily synthesized from a number of aliphatic core structures that do not require the presence of activating groups suitable for coupling to an activated linear polymer, such as succinimidyl carbonate end-capped poly(ethylene glycol) or the like, for building the branched water-soluble polymer. That is to say, the preparation of the polymers of the invention is not hampered by the need to utilize core structures having reactive functional groups necessary for coupling with the polymer arms, since the polymer portions of the molecule are generally built directly onto the core by polymerization of suitable monomer units.
The term “protected functional group” or “protecting group” or “protective group” refers to the presence of a moiety (i.e., the protecting group) that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. The protecting group will vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule, if any. Protecting groups known in the art can be found in Greene, T. W., et al., P ROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, New York, N.Y. (1999).
The term “drug”, “biologically active molecule”, “biologically active moiety” or “biologically active agent”, when used herein means any substance which can affect any physical or biochemical properties of a biological organism, including but not limited to viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, oligonucleotides, polynucleotides, nucleic acids, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the invention include, but are not limited to, antibiotics, fungicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, and the like.
As described in greater detail below, for branched polymers of the invention comprising poly(alkylene glycol) polymer arms, such as PEG arms, the branched polymers are advantageously synthesized by polymerizing alkylene oxide monomer units, such as ethylene oxide units, directly onto an aliphatic hydrocarbon core molecule substituted with two or more mucleophilic groups (e.g., thiol, amino or hydroxyl groups). In this manner, expensive and time-consuming polymer purification steps associated with prior art methods are avoided.
Y-(X)p-R(—X′-POLY)q Formula I
The aliphatic hydrocarbon core, R, preferably comprises 3 to about 12 carbon atoms, more preferably 3 to about 7 carbon atoms, most preferably 3 to about 5 carbon atoms. Core structures of 3, 4, and 5 carbon atoms in length are particularly preferred. The aliphatic hydrocarbon core can be linear or branched and may include one or more heteroatoms in the hydrocarbon chain. In a preferred embodiment, the polymer arms, POLY, and the functional group, Y, are each attached to different carbon atoms of the core molecule. For example, in a three-carbon core embodiment, the POLY polymer arms are preferably attached at the 1- and 3-position and the Y functional group is preferably attached at the 2-position.
Although less preferred due to its multifunctional nature, the PEG polymer may alternatively comprise a forked PEG. Generally speaking, a polymer having a forked structure is characterized as having a polymer chain attached to two or more active agents via covalent linkages extending from a hydrolytically stable branch point in the polymer. An example of a forked PEG is represented by -PEG-YCHZ2, where Y is a linking group and each Z is an activated terminal group for covalent attachment to a biologically active agent. The Z group is linked to CH by a chain of atoms of defined length. International Application No. PCT/US99/05333, the contents of which are incorporated by reference herein, discloses various forked PEG structures capable of use in the present invention. The chain of atoms linking the Z functional groups to the branching carbon atom serve as a tethering group and may comprise, for example, an alkyl chain, ether linkage, ester linkage, amide linkage, or combinations thereof. In this embodiment of the invention, the resulting branched polymer is multifunctional, i.e., having reactive sites suitable for attachment to a biologically active molecule not only extending from the aliphatic core but also extending from the forked polymer arms(s). As in the above case, such forked polymers, if utilized to prepare the branched structures of the invention, are attached to the aliphatic core structures provided herein not by polymerization but typically by covalent attachment.
In addition to the above-described forms of PEG, the polymer arms (POLY) can also be prepared with one or more weak or degradable linkages in the polymer backbone, including any of the above described polymers. For example, PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight
Other hydrolytically degradable linkages, useful as a degradable linkage within a polymer backbone, include carbonate linkages; imine linkages resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi et al., Polymer Preprints, 38 (1):582-3 (1997), which is incorporated herein by reference); phosphate ester linkages formed, for example, by reacting an alcohol with a phosphate group; hydrazone linkages which are typically formed by reaction of a hydrazide and an aldehyde; acetal linkages that are typically formed by reaction between an aldehyde and an alcohol; ortho ester linkages that are, for example, formed by reaction between a formate and an alcohol; peptide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by, for example, a phosphoramidite group, e.g., at the end of a polymer, and a 5′ hydroxyl group of an oligonucleotide.
In one instance, the polymer arms having one or more hydrolyzable linkages contained therein are prepared in a two-step polymerization process which includes an intermediate step for inclusion of the desired hydrolyzable linkage. That is to say, polymerization of, e.g., ethylene oxide subunits, onto the central core is carried out to a certain desired chain length and the reactive polymer termini extending from the central core are then coupled to short polymer chains suitably functionalized at one end to react with the hydroxyl groups of the intermediate polymer arms extending from the core to introduce the hydrolyzable linkages(s). Further polymerization of ethylene oxide subunits onto the polymer arms, now containing one or more hydrolyzable linkages, is then carried out to prepare polymer arms of a desired chain length.
The branched polymers of the invention optionally include a linkage (i.e., X in Formula I) that joins a branching carbon of the aliphatic hydrocarbon central core molecule with the functional group, Y. The structure of the X linkage is typically determined by the structure of the aliphatic hydrocarbon core used to form the polymers of the invention and has an overall length of from 1 to about 40 atoms, preferably 1 to about 10 atoms, and most preferably 1 to about 5 atoms. Preferred linkages include heteroatoms such as —O— or —S—, -alkylene-, —O— alkylene-O—, -alkylene-O-alkylene-, -aryl-O— (e.g., -phenylene-O—), —O-aryl- (e.g., —O-phenylene), (—O-alkylene-)m, and (-alkylene-O—)m, wherein m is 1-10, preferably 1-5 (e.g., 1, 2, 3, 4, or 5). The alkylene groups of the X linkage are preferably C1-C6 alkylene, more preferably C1-C3 alkylene, including methylene and ethylene.
In one embodiment of the invention, the Y functional group is a protected functional group, such as a protected hydroxyl group of formula —O-Gp, wherein Gp is a protecting group. The Gp protecting group can be any of various hydroxyl protecting groups known in the art, such as benzyl or other alkylaryl groups (e.g., groups having the formula —CH2— Ar, wherein Ar is any aryl group), acetal, and dihydropyranyl. Other suitable protecting groups are described in Greene, T. W., et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley & Sons, New York, N.Y. (1999). As would be readily understood, the protecting group, Gp, can be readily displaced from the molecule to form a hydroxyl group, which can be further modified to form other functional groups using techniques known in the art.
In another embodiment of Formula Ia, each POLY is PEG end-capped with a methoxy as shown below:
In yet a further embodiment of Formula Ia, the X linkage is one of the specific linkages shown below:
The branched polymers of the invention are formed by attaching polymer arms to a heteroatom-substituted aliphatic hydrocarbon core molecule having at least three carbon atoms, such as propane, via heteroatom linkages (e.g., —NH—, —O—, or —S—). Although the polymer arms may be attached to the aliphatic hydrocarbon structure by simply reacting terminal functional groups on preformed purified polymers with reactive nucleophiles on the aliphatic hydrocarbon core without departing from the invention, for poly(alkylene glycol) polymers, it is preferable in many respects to directly polymerize alkylene oxide monomer units, such as ethylene oxide, propylene oxide or butylene oxide subunits, onto an aliphatic hydrocarbon core bearing at least two available hydroxyl groups (or other nucleophilic groups such as amino or thiol groups). As illustrated in the Examples, alkylene oxide units can be polymerized onto, for example, an alcohol molecule using a catalyzed reaction to form ether-linked polymer arms, preferably using base catalysis although other catalysts such as metal or acid catalysts could also be employed. By polymerizing the alkylene oxide directly onto a suitably functionalized aliphatic hydrocarbon core structure, the branched polymer can be formed without first forming and purifying high molecular weight polymers, which is technically challenging, expensive, and time-consuming.
Y′(X)p-R(—Nu)q Formula V
Y′ is a protected functional group, such as a protected hydroxyl group, wherein the presence of the protecting group prevents polymerization at the Y′ position on the aliphatic core, R:
D-L1-(X)p-R(—X-POLY)q Formula VI
L1 is a linkage resulting from the reaction of the functional group of the branched polymer (i.e., Y is Formula I) and a functional group on the biologically active molecule; and
D, L1 POLY, X and p are defined above.
A biologically active agent for use in coupling to a branched polymer of the invention may be any one or more of the following. Suitable agents may be selected from, for example, hypnotics and sedatives, psychic energizers, tranquilizers, respiratory drugs, anticonvulsants, muscle relaxants, antiparkinson agents (dopamine antagonists), analgesics, anti-inflammatories, antianxiety drugs (anxiolytics), appetite suppressants, antimigraine agents, muscle contractants, anti-infectives (antibiotics, antivirals, antifungals, vaccines) antiarthritics, antimalarials, antiemetics, anepileptics, bronchodilators, cytokines, growth factors, anti-cancer agents, antithrombotic agents, antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants, anti-asthma agents, hormonal agents including contraceptives, sympathomimetics, diuretics, lipid regulating agents, antiandrogenic agents, antiparasitics, anticoagulants, neoplastics, antineoplastics, hypoglycemics, nutritional agents and supplements, growth supplements, antienteritis agents, vaccines, antibodies, diagnostic agents, and contrasting agents.
Yield: 42 g. (93%). 1H nmr (400 MHz DMSO-d6), δ7.25-7.34 (m, 5H), 4.6 (s, 2H), 3.2-3.8 (m, 826H).
Yield: 4.2 g. 1H Nmr (400 MHz DMSO-d6), δ7.25-7.34 (m, 5H) 4.6 (s, 2H), 3.3-3.8 (m, 826H), 3.24 (s, 6H).
1H Nmr (300 MHz, DMSO-d6) δ4.76 (d, 1H), 3.3-3.8 (m, 826H), 3.24 (s, 6H).
Yield: 18.5 g 1H nmr (DMSO-d6): δ7.33 ppm (mult. —OCH2C6 H 5), δ 4.61 ppm (s. —OCH 2C6H5), δ 4.31 ppm (t, —OCH2CH 2OMs), 3.5 ppm (br. mult., PEG), δ 3.24 ppm (s, CH 3OPEG-).
Yield: 13.2 g; 1H nmr (DMSO-d6): δ 4.76 ppm (d. HO—CH—); δ3.5 ppm (br. mult., PEG), δ 3.24 ppm (s, CH 3OPEG-).
The solvent was evaporated to dryness under reduced pressure, the residue dried under vacuum for 2 hours, and finally redissolved in 60 ml of deionized water. The pH of the solution was adjusted to 2.0 with 10% H3PO4. After stirring at pH 2.0 for 15 min., the pH of the solution was adjusted to 12.0 with 1.0 N NaOH and stirred at pH 12.0 for 2 hours. The hydrolyzed solution was saturated with NaC1 and the pH adjusted to 3.0 with 10% H3PO4. The solution was extracted with dichloromethane (100 ml×2) and the combined extracts were dried over Na2SO4, filtered, evaporated and precipitated with Et2O (100 mL). The product was collected by vacuum filtration and dried in vacuum overnight.
1H nmr (DMSO-d6): δ3.5 ppm (br. mult., PEG), δ 3.24 ppm (s, CH 3OPEG-), δ 2.23 ppm (t, —OCH2CH2CH 2COOH), δ 1.70 ppm (mult. —OCH2CH 2CH2COOH).
Yield: 1.2 g; 1H nmr (DMSO-d6): δ 3.5 ppm (br. mult., PEG), δ 3.24 ppm (s, CH 3OPEG-), δ 2.80 ppm (s, —NHS), δ 2.70 ppm (t. —OCH2CH2CH 2COONHS), δ 1.81 ppm (mult. —OCH2CH 2CH2COONHS).
Example 3 Synthesis of (T-benzyloxyethoxy)ethyl-1,3-propanediol (BEEP)—An Illustrative Aliphatic Hydrocarbon Core Molecule Suitable for Use in Preparing a Branched Polymer
Yield: 22 g 1H nmr (DMSO-d6): δ 7.33 ppm (mult. —OCH2C6 H 5), δ 4.49 ppm (s. —OCH 2C6H5), δ 4.31 ppm (t, OCH2 CH2 OSO2—CH3), δ 3.69 ppm (t, OCH2 CH2OSO2—CH3), δ 3.59 ppm (mult., —OCH2CH2 O—), δ 3.24 ppm (s, OCH 2 CH2OSO2—CH3 ).
B. Synthesis of C6H5—CH2O—CH2CH2OCH2CH2—CH(COOCH5)2
Yield: 6 g. 1H nmr (DMSO-d6): δ 7.33 ppm (mult. —OCH2C6 H5 ), δ 4.48 ppm (s. —OCH 2C6H5), δ 4.10 ppm (mult. OCH2 CH3), δ 3.51 ppm (mult., —OCH2CH 2O—CH2 CH2—, —CH(CO2—C2H5)2), δ 2.01 ppm (mult. —OCH2 CH2 —CH(CO2—C2H5)2), δ1.16 ppm (t, —OCH2 CH3 ).
C6H5—CH2O—CH2CH2OCH2CH2—CH(COOC2H5)2 (5 g) from Step B was dissolved in 200 ml of toluene and 29.5 ml of LiA1H4 (1 M in THF) was added at 0-5° C. After stirring overnight at room temperature, 1 ml of water was added followed by 1.0 ml of 15% NaOH and 3.0 ml of water. The insoluble material was filtered and the filtrate was evaporated to dryness. The product was purified by flash chromatography on a silica gel column eluted with ethyl acetate. Combined fractions were evaporated to dryness. The final product was dried under vacuum overnight.
Yield: 1.5 g 1H nmr (CDCl3): δ 7.29 ppm (mult. —OCH2C6 H 5), δ 4.55 ppm (s. —OCH2 C6H5), δ 3.61 ppm (mult., C6H5CH2—OCH2CH2 CH2 CH2—, —CH(CH2 OH)2, δ 1.81 ppm (mult. —OCH2CH2—CH(CH2OH)2), δ1.65 ppm (mult. —OCH2 CH2 —CH(CH2OH)2).
Yield: ˜0.9 g 1H nmr (DMSO-d6): δ 7.31 ppm (mull. —OCH2C6 H 5), δ 4.48 ppm (s. —OCH 2C6H5), δ 4.43 ppm (s. br. —CH(CH2OH)2).), δ 3.67 ppm (t., —OCH2 CH 2O—CH(CH2OH)2), δ 3.54 ppm (t., —OCH2 CH2O—CH(CH2OH)2), δ3.41 ppm (mult. —OCH2CH2O—CH(CH 2OH)2), δ3.29 ppm (mult. —OCH2CH2O—CH(CH2OH)2).
Example 5 PEGylation of Lysozyme with Branched PEG Polymer
Samples from the reactions of pH 5.5 and 6.5 showed the presence of lysozyme, mono-, and di-pegylated lysozyme by MALDI-TOF. Samples from the reaction conducted at pH 7.5 indicated the presence of di-pegylated lysozyme only by MALDI-TOF. After 24 h all reaction product mixtures contained lysozyme, mono-, and di-pegylated lysozyme, and, tri-pegylated lysozyme.
Y—(X)p-R(—X-POLY)q
Y is a functional group reactive with an electrophilic or nucleophilic group;
X′ is —O—;
each POLY is a water soluble and non-peptidic polyethylene glycol (PEG) polymer that terminates with a hydroxyl or methoxy group,
2. The reactive polymer of claim 1, wherein each water soluble and non-peptidic polymer is a PEG that terminates with a hydroxyl group.
3. The reactive polymer of claim 1, wherein each water soluble and non-peptidic polymer is a PEG that terminates with a methoxy group.
4. The reactive polymer of claim 1, wherein Y selected from the group consisting of hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrate, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, hydrazide, thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.
5. The reactive polymer of claim 1, wherein p is 1 and X is selected from the group consisting of a heteroatom, -alkylene-, —O-alkylene-O—, -alkylene-O-alkylene-, -aryl-O—, —O-aryl-, (—O-alkylene-)m, and (-alkylene-O—)m, wherein m is 1-10.
6. The reactive polymer of claim 1, wherein p is 0 and Y is hydroxyl.
7. The reactive polymer of claim 1, wherein the branched polymer has a molecular weight of about 20,000 Da.
8. The reactive polymer of claim 1, wherein the branched polymer has a molecular weight of about 40,000 Da.
9. The reactive polymer of claim 1, wherein the branched polymer has a molecular weight of about 60,000 Da.
10. The reactive polymer of claim 1, having the structure:
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