Uses of lubricating block copolymers and biomimetic boundary lubricants

Abstract

Lubricating compositions and their use in methods for lubricating surfaces of biological tissue, particularly joints, cartilage and bone, are provided. [Solution] A method for lubricating biological tissue, such as joints, bones, ocular tissue, nasal tissue, tendons, aponeurosis, and intravaginal tissue, by contacting the biological tissue with an effective amount of at least lubricin or a block copolymer lubricating composition that performs better than lubricin. In particular embodiments, the method is used to treat osteoarthritis. In certain embodiments, the block copolymer has an ammonium-containing polymer block and a non-ionic hydrophilic polymer block, or the copolymer has a carboxylic acid-containing polymer block and a non-acid non-ionic hydrophilic polymer block.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 403,962, filed on 4 October 2016 under Section 119 of the U.S. Patent Act, which is incorporated herein by reference in its entirety.

[0002] Description of federally funded research This invention was made with government support under authorization number AR066667-01, granted by the National Institutes of Health. The government has certain rights in this invention.

[0003] This invention generally relates to pharmaceutically acceptable lubricating compositions and their use in methods of lubricating the surfaces of biological tissues, particularly joints, cartilage, and bone. More specifically, the invention relates to polymer compositions that mimic the action of labrisin, and more specifically, to methods of using such compositions to treat various conditions, such as osteoarthritis, in which lubrication is particularly beneficial in treating and alleviating the effects of a disease or condition. [Background technology]

[0004] Labrisin is a glycosylated protein found in synovial fluid that plays a central role in joint boundary lubrication and the prevention of osteoarthritis. Labrisin reduces the coefficient of friction (COF) of articular cartilage in boundary mode by as much as 70 percent (Non-Patent Literature 1). This powerful lubricating ability stems from the structure of labrisin (the central mucin-like domain of labrisin consists of a broadly glycosylated core protein that attracts and retains water near the molecule; the C-terminus of labrisin binds the protein to the cartilage surface) (Non-Patent Literature 2). This construction is essential for boundary mode lubrication of articular cartilage because denaturation of any domain of labrisin results in a partial or complete loss of lubricating ability.

[0005] Osteoarthritis (OA) affects more than 50 million people worldwide in developed countries, and this number is expected to rise as median age and life expectancy increase. The economic impact of osteoarthritis treatment exceeds $30 billion annually in the United States alone. Financial burdens, along with other factors (i.e., quality of life, lost working hours, etc.), are motivating the development of more effective treatments.

[0006] Current treatments for osteoarthritis include nonsteroidal anti-inflammatory drugs, intra-articular corticosteroid injections, and chondroitin sulfate or glucosamine supplements. However, all of these treatments have little to no effect on disease progression. A more recent approach to treating OA is intra-articular injection of hyaluronic acid (HA), a glycosaminoglycan from natural synovial fluid (e.g., Non-Patent Literature 3), where HA is known to reduce the coefficient of friction in the hydrodynamic mode of lubrication by increasing synovial fluid viscosity (e.g., intra-articular replacement therapy) (e.g., Non-Patent Literature 4). Another major lubricating component in synovial fluid is the high molecular weight glycoprotein labrisin, which reduces the coefficient of friction in the boundary mode of lubrication.

[0007] It is well known that in injured cartilage, chondrocyte production of labrisin is impaired, and boundary-mode lubrication is reduced. Natural lubricants such as proteoglycan aggregates and mucins (e.g., labrisin) maintain the hydrophilicity of the natural surface. Intra-articular injection of adjunctive labrisin and cleaved recombinant labrisin construct LUB:1 have been shown in rat models of the disease. It has been shown to delay the progress of OA (for example, Non-Patent Document 5; Non-Patent Document 6). However, to date, large-scale recombinant production of both lubricin and LUB:1 remains challenging due to multiple amino acid repeats in the protein core and high glycosylation (for example, Non-Patent Document 7; Non-Patent Document 8). Moreover, there is a separate need for an effective lubricant agent for bone in situations where direct bone-to-bone contact can occur, such as in advanced stages of osteoarthritis. As a result, an effective lubricant agent that can provide the same or similar boundary lubrication as lubricin or LUB:1 would be a significant advancement in this field.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0009] This disclosure is directed to the design, synthesis, and use of specialized block copolymers having a lubricin-mimicking structure and providing substantial lubricating ability under boundary mode lubrication conditions. In particular embodiments, the block copolymer comprises a lubricating block (e.g., M of about 200 kDa n ) that mimics the mucin-like domain of lubricin and a smaller chondro-binding block (e.g., M of about 3 kDa n ) that mimics the hemopexin-like domain. As disclosed later herein, applying this type of polymer to lubricin-deficient bovine articular cartilage or bone resulted in a significantly reduced coefficient of friction (COF) compared to untreated controls.

Means for Solving the Problems

[0010] In one aspect, the present invention is directed to a block copolymer having the following structure:

Chemical formula

[0011] In another embodiment, the present invention has the following structure: [ka] (In the formula: X is selected from -NR'-, -O-, and bonds, where R' is selected from hydrogen atoms and hydrocarbon groups having at least 1 and up to 6 carbon atoms; Y is selected from polyalkylene glycols, saccharides, and polyalcohols; R is a hydrogen atom, a hydrocarbon group (R) having 1 to 12 carbon atoms, or a cartilage-binding domain; the subscripts d and e are independently integers of at least 3; the subscript f is 0 or an integer of at least 1) The subject is a block copolymer having the following: In the formula, the hydrogen atom on the carboxylic acid group shown is optionally replaced by a positively charged metal ion or a positively charged organic group. According to the laws of chemistry, the block copolymer is terminated by terminal groups at the end opposite the thiol group.

[0012] In another embodiment, the present invention relates to a method for imparting a suitable level of lubrication to biological tissue by increasing the lubrication performance of the biological tissue by contacting the biological tissue with a sufficient amount of a lubricating composition. The lubricating composition may be, for example, any of the block copolymers described above. The biological tissue can be selected from, for example, joints, bones, eye tissues, nasal tissues, tendons, aponeuroses, and vaginal tissues. [Brief explanation of the drawing]

[0013] [Figure 1] This is a general representation of an exemplary diblock copolymer of the present invention in which the bonding block and lubrication block portions have been identified. [Figure 2] This graph plots the coefficient of friction (COF) results for phosphate-buffered saline (PBS) solution, the diblock copolymer shown in Figure 1, the binding block only, and the lubricating block only. [Figure 3] As part of competitive bonding studies, this graph plots the COF of solutions varying with the bonding block:diblock copolymer ratio. [Figure 4] This graph plots the COF of the diblock copolymer shown in Figure 1, and the random copolymer version of the diblock copolymer synthesized by random RAFT copolymerization of two block monomers, followed by quaternary ammonium conversion (i.e., the same monomer units, but incorporated into the copolymer in a random manner instead of blocks). [Figure 5A] This graph plots COF as a function of the concentration of the diblock copolymer shown in Figure 1. [Figure 5B] This graph plots COF as a function of incubation time, using a copolymer at a concentration of 1 mg / mL for different durations. The resulting graph can be considered a binding kinetics curve. [Figure 6] Figures 6A and 6B are graphs plotting the COF of cancellous bone and subchondral bone samples treated with diblock copolymer (3) solution (10 mg / mL for 2 hours or 1 mg / mL for 1 hour) or PBS solution, respectively. [Modes for carrying out the invention]

[0014] In a first embodiment, the present invention relates to block copolymers that mimic rubrisin by having mucin-like domains and C-terminal hemopexin-like (PEX-like) domains. The copolymer may contain, for example, polymer blocks containing positive or load-positive pendant groups, as well as polymer blocks containing pendant nonionic hydrophilic groups, particularly hydrophilic groups containing ether functional groups and / or hydroxyl functional groups. The copolymer may be further classified as a graft brush copolymer when the pendant groups are polymeric. The term “copolymer” as used herein refers to the presence of at least two polymer blocks. The copolymer may be, for example, a diblock copolymer, a triblock copolymer, a tetrablock copolymer, or a higher-order copolymer.

[0015] A first class of block copolymers contemplated herein has the following general structure: [Chemical formula] which is encompassed by:

[0016] The substituents R in formula (1) 1 , R 2 and R 3 are independently selected from hydrocarbon groups (R) having at least 1 and at most 12 carbon atoms. The substituents R 1 , R 2 and R 3 can be further specifically defined in some embodiments as having exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, or a particular range of those carbon atoms, for example, 1 - 10, 1 - 8, 1 - 6, 1 - 4, 1 - 3, 2 - 12, 2 - 10, 2 - 8, 2 - 6, 2 - 4, 3 - 12, 3 - 10, 3 - 8 or 3 - 6 carbon atoms. In some embodiments, R 1 , R 2 and R 3 are all the same, while in other embodiments, R 1 , R 2 and R 3 are not all the same (or at least two of R 1 , R 2 and R 3 are different). The hydrocarbon group R can be saturated or unsaturated, linear (straight-chain) or branched, and cyclic or acyclic.

[0017] In one set of embodiments, R 1 , R 2 and R 3At least one, two, or all of these are selected from hydrocarbon groups composed solely of carbon and hydrogen. These hydrocarbon groups may be, for example, alkyl groups, alkenyl groups, cycloalkyl groups, cycloalkenyl (aliphatic) groups, or aromatic groups. Some examples of linear alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl groups. Some examples of branched alkyl groups include isopropyl(2-propyl), isobutyl(2-methylpropa-1-yl), sec-butyl(2-butyl), t-butyl, 2-pentyl, 3-pentyl, 2-methylbuta-1-yl, isopentyl(3-methylbuta-1-yl), 1,2-dimethylpropa-1-yl, 1,1-dimethylpropa-1-yl, neopentyl(2,2-dimethylpropa-1-yl), 2-hexyl, 3-hexyl, 2-methylpenta-1-yl, 3-methylpenta-1-yl, and isohexyl(4-methylpenta-1-yl), where the "1-yl" suffix indicates the attachment point of the group. Some examples of linear olefinic groups include vinyl, propen-1-yl(allyl), 3-buten-1-yl (CH2=CH-CH2-CH2-) group, 2-buten-1-yl (CH2-CH=CH-CH2-) group, butadienyl, and 4-penten-1-yl group. Some examples of branched olefinic groups include propen-2-yl, 3-buten-2-yl (CH2=CH-CH.-CH3), 3-buten-3-yl (CH2=C.-CH2-CH3), 4-penten-2-yl, 4-penten-3-yl, 3-penten-2-yl, 3-penten-3-yl, and 2,4-penta Dien-3-yl is an example, where the dot in the illustrative formula above represents a radical (i.e., an attachment point of the group). Some examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be polycyclic (e.g., bicyclic) by having either a bond between two ring groups (e.g., dicyclohexyl) or a covalent (i.e., condensed) side (e.g., decalin and norbornane). Some examples of cycloalkenyl (aliphatic) groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, cyclooctenyl, cyclooctadienyl, and cyclooctatetraenyl. Some examples of aromatic groups include phenyl and benzyl. Unsaturated cyclic hydrocarbon groups may also be polycyclic groups (such as bicyclic or tricyclic polyaromatic groups) by having either a bond (e.g., biphenyl) or a covalent (i.e., condensed) side between two of the cyclic groups, as in naphthalene, anthracene, phenanthrene, phenalene, or indene.

[0018] In another set of embodiments, R 1 , R 2 and R 3At least one of the is selected from hydrocarbon groups containing at least one heteroatom (i.e., a non-carbon and non-hydrogen atom), one or more heteroatoms selected from, for example, an oxygen atom, a nitrogen atom, a sulfur atom, and a halide atom, and a group containing one or more of these heteroatoms (i.e., a heteroatom-containing group). In some embodiments, the hydrocarbon group does not contain hydrogen atoms (e.g., all hydrogen atoms are replaced by heteroatoms, such as in -CF3), but in other embodiments, the hydrocarbon group contains at least one hydrogen atom. Some examples of oxygen-containing groups include hydroxyl groups (OH), alkoxy groups (OR), carbonyl-containing groups (e.g., carboxylic acid-functional groups, ketone-functional groups, aldehyde-functional groups, carboxylic acid ester-functional groups, amide-functional groups, and urea-functional groups), nitro groups (NO2), carbon-oxygen-carbon groups (ethers), sulfonyl groups, and sulfinyl groups (i.e., sulfoxides). Some special examples of alkoxy groups (-OR) include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, phenoxy, benzyloxy, 2-hydroxyethoxy, 2-methoxyethoxy, 2-ethoxyethoxy, vinyloxy, and allyloxy groups. In the case of ether groups, the ether group may be a polyalkylene oxide group (polyalkylene glycol) such as a polyethylene oxide group. Some examples of nitrogen-containing groups include primary amine groups, secondary amine groups, and tertiary amine groups (i.e., -NR'2 or NR'3). +Examples include nitrile (CN) groups, amide groups (i.e., -C(O)NR'2 or -NRC(O)R', where R' is independently selected from a hydrogen atom and the hydrocarbon groups described above), imine groups (e.g., -CR'=NR', where R' is independently H or a hydrocarbon group), urea groups (-NR'-C(O)-NR'2, where R' is independently H or a hydrocarbon group), and carbamate groups (-NR'-C(O)-OR', where R' is independently H or a hydrocarbon group). Some examples of sulfur-containing groups include mercapto groups (i.e., -SH), thioether groups (i.e., sulfides, e.g., -SR), disulfide groups (-RSSR), sulfoxide groups (-S(O)R), sulfone groups (-SO2R), sulfonate groups (-S(=O)2OR'', where R'' is H, a hydrocarbon group, or a cationic group), and sulfate groups (-OS(=O)2OR'', where R'' is H, a hydrocarbon group, or a cationic group). Some examples of halide atoms include fluorine, chlorine, bromine, and iodine. One or more of the heteroatoms listed above (e.g., oxygen, nitrogen, and / or sulfur) can be inserted between carbon atoms in any of the hydrocarbon groups listed above (e.g., as -O-, -NR'-, or -S-). Alternatively, or in addition, one or more heteroatom-containing groups may be inserted between carbon atoms on one or more hydrocarbon groups. It can be replaced by more than this number of hydrogen atoms. In some embodiments, one or more of the above groups are excluded.

[0019] In formula (1), the variable X and X' are selected independently of -NR'-, -O-, and bonds, where R' is selected from a hydrogen atom and a hydrocarbon group having at least one and up to six carbon atoms (selected from the R group). In some embodiments, R' is specifically selected from a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, and a t-butyl group, or a more specific selection therefrom.

[0020] In the first example, the variable Y in formula (1) is or contains a polyalkylene glycol group. The polyalkylene glycol group is conveniently represented by the following structure: (-CR'2CR'2O-) n R', where R' is independently selected from a hydrogen atom and a hydrocarbon group (e.g., methyl or ethyl) for each example of R', n is at least 2, 3, 4, 5 or 6 and at most, for example, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500, or n is within the range of being combined by any two of the aforementioned values, where each of the aforementioned values ​​corresponds to the number of alkylene oxide (-CR'2CR'2O-) monomer units. In special embodiments, the polyalkylene glycol is a polyethylene glycol group or a polypropylene glycol group. Furthermore, the polyalkylene glycol group may or may not be integrated into a copolymer, such as a copolymer containing polyethylene glycol and an anionic polymer moiety such as polyacrylic acid (PAA), polyglutamic acid or polyaspartic acid.

[0021] In the second example, the variable Y in formula (1) is or contains a saccharide group. The term “saccharide” as used herein includes monosaccharides (containing one monosaccharide unit) and saccharides containing at least two or more monosaccharide units, such as disaccharides, trisaccharides, oligosaccharides (e.g., at least four and up to 20, 30, 40, 50, or 60 monosaccharide units), and polysaccharides (generally more than 60, 70, or 80 monosaccharide units and up to, e.g., 100, 200, 300, 400, 500, or 1000 monosaccharide units). Saccharides can also be derivatized in such a manner (e.g., esterification, etherification, amination, or halogenation) that the derivatized version is still reasonably classified as a saccharide by those skilled in the art. Some examples of monosaccharides include glucose, galactose, fructose, mannose, sialic acid, glucosamine, N-acetylglucosamine, and galacturonic acid. Some examples of disaccharides include lactose, sucrose, maltose, trehalose, cellobiose, and mannobiose. Some examples of oligosaccharides include fructooligosaccharides (FOS), galactooligosaccharides (GOS), and mannanoligosaccharides (MOS). Some examples of polysaccharides include dextran, dextran sulfate, starch (e.g., amylose or amylopectin), cellulose, hemicellulose, polysialic acid, pectin, glycogen, mannan, galactomannan, xylan, pullulan, xanthan gum, carrageenan, guar gum, polygalacturonic acid, poly(N-acetylgalactosamine), heparin, hyaluronic acid, and chondroitin sulfate. In some embodiments, the saccharide is selected to have an overall anionic charge, such as a saccharide having a carboxylic acid group, a carboxylate group, a sulfate group, or a sulfonate group. The saccharide group is integrated into or not integrated into a copolymer, such as a copolymer containing an oligosaccharide moiety and an oligopeptide moiety or a polyacrylic acid moiety.In some embodiments, the saccharide-containing copolymer contains a saccharide moiety and an anionic polymer moiety, such as polyacrylic acid (PAA), polyglutamic acid, or polyaspartic acid.

[0022] In the third example, the variable Y in formula (1) is or contains a polyalcohol group. The term “polyalcohol,” as used herein, refers to a non-saccharide group having a large number (i.e., at least two, three, or four) hydroxyl groups. The polyalcohol may be, for example, a sugar alcohol or a polyhydric alcohol. Some examples of sugar alcohols include erythritol, xylitol, mannitol, glycerin, and sorbitol. The saccharide or polyalcohol (as Y) is generally attached to the block copolymer of formula (1) by one of its hydroxyl groups in a deprotonated form, where X' can represent an oxygen atom from the Y group; or X' can represent a bond, and Y represents a saccharide or polyalcohol bonded to an indicated C(O) group by one of its oxygen atoms; or X' is -NR'-, and Y represents a saccharide or polyalcohol bonded to an -NR'- group by one of its carbon atoms. In other embodiments, the polyalcohol is a polymer containing a hydroxyl group. Hydroxy-containing polymers may contain, for example, at least 2, 3, 4, 5, or 6 monomer units, and up to, for example, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 monomer units. Some examples of such polymers include polyvinyl alcohol, poly(hydroxymethyl methacrylate), and poly(hydroxypropyl methacrylate). The polyalcohol group may or may not be integrated into the copolymer, such as polyvinyl alcohol-polyacrylic acid (PVA-PAA) copolymer. In some embodiments, the polyalcohol-containing copolymer contains a polyalcohol moiety and an anionic moiety, which may be an anionic polymer moiety, such as polyacrylic acid (PAA), polyglutamic acid, or polyaspartic acid.

[0023] In formula (1), the subscripts a and b are independently integers of at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 50. In some embodiments, the subscripts a and b are independently 30 or less, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, or 2000. In some embodiments, the subscript a is selected to be less than the subscript b. For example, subscript a may be in the range of 3-10, 3-20, 3-30, or 3-40, while subscript b may be in the range of 30-500, 40-500, 50-500, or 60-500. In other embodiments, subscript a is selected to be greater than subscript b. For example, subscript a may be in the range of 30-500, 40-500, 50-500, or 60-500, while subscript b may be in the range of 3-10, 3-20, 3-30, or 3-40.

[0024] The subscript c in formula (1) is at least an integer of 1. In different embodiments, c is exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or c is within the range combined by any two of the aforementioned values. The value of c corresponds to the number of methylene groups whose range is defined by c. That is, when the subscript c is 1, the CH2 linker exists between X and the quaternary ammonium group shown in formula (1); when the subscript c is 2, the CH2CH2 linker exists between X and the quaternary ammonium group shown in formula (1).

[0025] Although not shown in formula (1), the structure illustrated in formula (1) necessarily (i.e., by the laws of chemistry) contains a terminal group at each of the two ends of the copolymer. The terminal group may be, for example, a hydrogen atom, a hydrocarbon group R, or a heteroatom-containing group, such as -OH, -OCH3, a nitrile-containing alkyl (provided, for example, by a radical initiator or chain transfer agent), a thiol (-SH), or a dithioester group (provided by a RAFT chain transfer agent). ) are independently selected. In some embodiments, at least one of the terminal groups is a thiol group. The terminal groups often correspond to groups originally present in the precursor reactants used to synthesize the block copolymer, and therefore the type of terminal group is often dependent on the chemistry used to synthesize the block copolymer. Nevertheless, the terminal groups can be appropriately modified by reacting the initially produced block copolymer to add a specific terminal group, for example, a cartilage-binding domain (e.g., a peptide-containing group) that helps bind the block copolymer to a desired biological tissue. In some embodiments, the cartilage-binding domain is attached to the block copolymer via a -S-linker, as in morphology RS-, where R is the cartilage-binding domain. Furthermore, although not shown in formula (1), the total positive charge of the quaternary ammonium groups in the copolymer illustrated in formula (1) is balanced by a total negative charge of an equivalent magnitude provided by anions bonded to the ammonium groups. The anions can be selected from any species acceptable for administration to living organisms, such as halides (e.g., chlorides, bromides, or iodides), carbonates, bicarbonates, sulfates, bisulfates, bisulfites, carboxylates (e.g., acetates, propionates, butyrates, maleates, and citrates), and sulfonates (e.g., mesylates).

[0026] The second class of block copolymers considered herein includes the following comprehensive structure: [ka] It is encompassed by.

[0027] In formula (2), the variable X and Y in formula (2) are defined as provided above under formula (1). Variable R is a hydrogen atom, a hydrocarbon group (R) having 1 to 12 carbon atoms (or more specifically, at least 4, 5 or 6 and up to 7, 8, 9, 10, 11 or 12 carbon atoms), or a cartilage-binding domain. If R is a hydrocarbon group having 1 to 12 carbon atoms, the hydrocarbon group may more specifically be a linear or branched alkyl or alkenyl group. In some embodiments, the cartilage-binding domain is a peptide-containing group (or "peptide") which may be a monopeptide, dipeptide, tripeptide, or oligopeptide containing at least 4 and up to 5, 6, 7, 8, 9 or 10 peptide units. The cartilage-binding peptide may be, for example, TKKTLRT, SQNPVQP, WYRGRL, SYIRIADTN, or CQDSETRFY (SEQ ID NOs: 1-5, respectively), cholesterol or other sterol moieties, or any other moiety useful for binding the block copolymer to biological tissue. Conjugation chemistry for attaching cartilage-binding domains, hydrophobic alkyl chains, sterols, or other agents to the block copolymer is known to those skilled in the art. Although not shown, the structure in formula (2) necessarily includes a terminal group opposite the RS-terminal group. The other terminal group may be as described above under formula (1).

[0028] In equation (2), the subscripts d and e are independently integers of at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 50. In some embodiments, the subscripts d and e are independently 30 or less, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, or 2000. In some embodiments, the subscript d is selected to be less than the subscript e. For example, the subscript d may be in the range of 3-10, 3-20, 3-30, or 3-40, while the subscript e may be in the range of 30-500, 40-500, 50-500, or 60-500. In other embodiments, the subscript d is selected to be greater than the subscript e. For example, the subscript d may be in the range of 30-500, 40-500, 50-500, or 60-500, while the subscript e may be in the range of 3-10, 3-20, 3-30, or 3-40.

[0029] The subscript f in equation (2) is at least an integer of 1. In different embodiments, f is exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or f is within the range that can be combined by any two of the aforementioned values. If the subscript f is 1, a CH2 linker exists. If the subscript f is 2, a CH2CH2 linker exists.

[0030] In formula (2), the hydrogen atoms on the carboxylic acid group shown can be replaced (i.e., are sometimes replaced) with positively charged metal ions or positively charged organic groups. Some examples of positively charged metal ions include lithium ions, sodium ions, and potassium ions. Some examples of positively charged organic groups include ammonium ions, such as dimethyl ions, trimethyl ions, or tetramethylammonium ions.

[0031] The block copolymers described above can be synthesized by any suitable polymerization method. In a particular embodiment, the block copolymer is synthesized by reversible addition-fragmentation-chain transfer (RAFT) polymerization, which is well known in the art. In the RAFT process, a first polymer block is produced by reacting a first functionalized vinyl monomer (e.g., 2-(dimethylamino)ethyl acrylate, i.e., DMAEA) with a RAFT transfer agent (e.g., 4-cyanopentanoic acid dithiobenzoate, i.e., CPADB) in the presence of a polymerization initiator (e.g., 4,4'-azobis(4-cyanopentanoic acid), i.e., ACPA). The first polymer block is then reacted with a second functionalized vinyl monomer (e.g., poly(ethylene glycol) methyl ether acrylate) in the presence of a polymerization initiator to add a polymerized block of the second functionalized vinyl monomer to the first polymer block.

[0032] The following scheme illustrates the RAFT process: [ka] This indicates.

[0033] In another embodiment, the present invention relates to pharmaceutical formulations comprising one or more block copolymers of the present invention and a pharmaceutically acceptable carrier (i.e., an excipient or diluent). Pharmaceutical formulations can be prepared for use in a variety of delivery forms, including intra-articular, intranasal, intravaginal, or ocular delivery. The phrase “pharmaceutically acceptable carrier” or equivalent terms, as used herein, refer to a pharmaceutically acceptable material, composition or vehicle, which may be a liquid (diluent or excipient) or a solid filler. The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, corresponding to a reasonable benefit / risk ratio. In pharmaceutical compositions, the compound is generally dispersed in a physiologically acceptable carrier by either being mixed (e.g., with a solid carrier in solid form) or dissolved or emulsified in a liquid carrier. The carrier should be compatible with the other components of the formulation and physiologically safe for the subject. Any carrier known in the art may be suitable herein, depending on the mode of administration. Some examples of suitable carriers include aqueous solutions, gelatin, and fatty acids (e.g., stearic acid). Examples include salts thereof, talc, vegetable fats or oils, gums and glycols, starch, and dextran.

[0034] The pharmaceutical composition may also contain one or more auxiliary agents, such as stabilizers, surfactants, salts, buffers, additives, or combinations thereof, all of which are well known in the pharmaceutical art. Stabilizers may be, for example, oligosaccharides (e.g., sucrose, trehalose, lactose, or dextran), sugar alcohols (e.g., mannitol), or combinations thereof. Surfactants may be any suitable surfactant, including, for example, those containing polyalkylene oxide units (e.g., Tween 20, Tween 80, Pluronic F-68), which are typically included in amounts from about 0.001% (w / v) to about 10% (w / v). Salts or buffers may be any suitable salt or buffer, for example, sodium chloride, or sodium phosphate or potassium phosphate, respectively. Some examples of additives include, for example, glycerin, benzyl alcohol, and 1,1,1-trichloro-2-methyl-2-propanol (e.g., chloreton or chlorobutanol). If necessary, the pH of the solution may be adjusted, appropriately, by the inclusion of a pH adjuster. Examples of pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, infusions, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, and thickeners may be necessary or desirable. The pharmaceutical formulation may be in the form of a sterile aqueous solution containing one or more buffers, diluents, and / or other suitable additives, for example, but not limited to, penetration enhancers and carrier compounds.

[0035] In another embodiment, the present invention relates to a method for imparting lubrication to biological tissues such as joints, cartilage, and bone by using biomimetic copolymers described above under formulas (1) and (2). In the case of bone, the biomimetic copolymers can reduce discomfort, pain, and additional injury caused by direct bone-to-bone contact, which sometimes occurs in advanced stages of osteoarthritis. According to this method, biological tissue is brought into contact with a sufficient (i.e., effective or therapeutically effective) amount of any of the copolymer compositions described above under formulas (1) and (2) to increase its lubrication or to impart an appropriate level of lubrication to the biological tissue. The increased level of lubrication generally corresponds to a lower level of friction (i.e., coefficient of friction, or coefficient of friction, COF) when the biological tissue slides against the same tissue or other material. The coefficient of friction can be measured using a friction meter that evaluates surface lubrication by linear vibration of the sample at variable speeds (generally 0.1 mm / s, 0.3 mm / s, 1 mm / s, 3 mm / s, and 10 mm / s) and normal stresses of variable compression (generally 250 kPa to 300 kPa).

[0036] As used herein, the terms “sufficient amount,” “therapeutic effective amount,” and “effective amount” are mutually interchangeable to refer to an amount of the copolymer composition of the present invention that is sufficient to provide sufficient lubrication to biological tissue, or to prevent the onset, recurrence, or development of a disease or condition (e.g., osteoarthritis) or one or more symptoms thereof, or to enhance or improve the preventive effect of another treatment, reduce the severity and duration of the disease or condition, alleviate one or more symptoms of the disease or condition, prevent the progression of the disease or condition, and / or enhance or improve the therapeutic effect of an additional treatment.

[0037] The therapeutically effective dose of a block copolymer can be administered to a patient in one or more doses sufficient to alleviate, remit, stabilize, reverse or delay the progression of the disease or condition, or otherwise reduce the pathological outcome of the disease or condition, or reduce the symptoms of the disease or condition. Remission or reduction does not need to be permanent, but should range from at least one hour, at least one day, or at least one week, or longer. This can be for a certain period of time. The effective dose is generally determined by a physician on a case-by-case basis and is within the skill of those skilled in the art. Several factors are typically considered when determining an appropriate dosage to achieve an effective dose. These factors include the patient's age, sex and weight, the condition being treated, the severity of the condition, and the route of administration, dosage form, regimen, and desired outcome. In certain embodiments of the present invention, the therapeutic effective dose is an amount effective for treating osteoarthritis, achieving pain relief over a certain period of time, improving joint movement and flexibility, or reducing the degree of improvement of friction or other accepted osteoarthritis in the joint. In illustrative embodiments, the dose level ranges from about 0.1 to 10 mg / mL (for humans) in an injection volume of 0.1 to 10 mL, more typically from about 1 to 5 mg / mL in an injection volume of 0.1 to 3 mL.

[0038] The biological tissue to be lubricated can be brought into contact with either the block copolymer of formula (1) or (2) by any means well known in the medical technology field. The biological tissue can be brought into contact with the block copolymer, for example, by injecting, infusing, implanting, spraying or coating the block copolymer directly into or on the biological tissue, or indirectly into the biological tissue surrounding the tissue to be lubricated. Generally, contact with biological tissue means that the block copolymer is delivered to the tissue in any way that leads to surface coating with the copolymer or tissue immersion. In certain embodiments, the tissue is brought into contact by injection or infusion of the composition into the joint space, thereby leading to coating of the cartilage and / or meniscus found in the joint space. Furthermore, the volume used is at least in part dependent on the type of tissue to be contacted, whether the space is filled or the surface is coated, as can be determined by technicians in the medical technology field.

[0039] In a particular embodiment, the lubrication performance of a joint or bone is improved by injecting or infusing a block copolymer into or onto an arthritis or damaged joint or bone. Thus, the copolymer provides boundary lubrication. The treatment may specifically be aimed at treating or preventing osteoarthritis. Treatment of osteoarthritis or damaged joints, cartilage or bone preferably results in symptom reduction, improved mobility, reduced joint pain, and overall inhibition of disease progression, or, in the case of damaged joints, prevention. The method may also involve administering one or more of the block copolymers described above together with the simultaneous or sequential administration of another composition that, while outside the scope of formulas (1) and (2), functions to enhance or work in cooperation with the block copolymer. The enhancing (i.e., auxiliary) composition may be selected from, for example, hyaluronic acid, labrisin, synovial fluid, glycosaminoglycans, or other adjuvants. These other agents may also be administered, for example, by injection or infusion. In some embodiments, these other agents can work synergistically with one or more of the block copolymers described above to provide enhanced lubrication and wear protection.

[0040] In specific embodiments, the biological tissue is a joint, cartilage, or bone, more typically a damaged or arthritised joint, cartilage, or bone. In some embodiments, the joint is a weight-bearing joint, such as the hip, knee, or ankle joint. Many different joints, including the shoulder, elbow, wrist, hand, finger, and toe joints, can benefit from increased lubrication levels. Nevertheless, the biological tissue to be lubricated is not limited to joints, cartilage, and bone. Other biological tissues that can be lubricated by the use of the disclosed block copolymers include eye tissue, nasal tissue, and vaginal tissue. Thus, the use of the block copolymers described herein addresses a variety of conditions beyond those related to joints, cartilage, and bone. Some of these other conditions include, for example, dry eye syndrome, dry nose, postmenopausal vaginal dryness, carpal tunnel syndrome, and many others. Technicians in the medical technology field should be provided with appropriate delivery routes and methods for contacting specific biological tissues. The method can be determined. For example, for dry eyes, contact can be achieved by drip infusion of an intravenous solution; for a dry nose, contact can be achieved by nasal spray; for carpal tunnel syndrome, contact can be achieved by injection near or around the inflamed tendon and membrane; for postmenopausal dry vagina, pills, lozenges, or suppositories can be placed in or implanted in the vagina. Thus, by using this method, boundary mode lubrication can be achieved for any of the wide variety of biological tissues that can benefit from additional lubrication.

[0041] Examples are described below for illustrative purposes and to illustrate the best mode of the present invention. However, the scope of this invention is not limited in any way by the examples described herein. [Examples]

[0042] Synthesis and Characterization of Lubricating Diblock Copolymers In efforts to mimic the structure of labrisin, a large lubricating block (approximately 200 kDa M) is used to mimic the mucin-like domain of labrisin. n ) and small cartilaginous connective blocks (approximately 3-10 kDa M) that mimic the C-terminal domain. n Diblock copolymers containing ) were prepared here. Exemplary compositions exhibiting two mimetic domains are provided in Figure 1. The lubricating domain of the diblock copolymer shown in Figure 1 contained a polyacrylic acid skeleton grafted with polyethylene glycol (PEG) brushes. The aforementioned function aids in hydration and compressive resistance in the polymer. The binding domain of the diblock copolymer shown in Figure 1 contained a polyacrylic acid skeleton having pendant quaternary ammonium groups that nonspecifically interact with load electrocartilage components such as aggrecan. As further discussed below, application of this polymer to labricin-deficient bovine articular cartilage resulted in a significantly reduced coefficient of friction (COF).

[0043] The synthesis of the diblock copolymer began with the synthesis of a “pre-bonding” block by reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(dimethylamino)ethyl acrylate. n The subsequent RAFT polymerization added the lubricating block to the copolymer by using the “pre-binding” block as a macroinitiator. The tertiary amine in the “pre-binding” block was then converted to a quaternary ammonium group by treating the block copolymer with excess ethyl bromide, resulting in the final product (M of approximately 200 kDa). n A PDI of 1.8 was obtained. A general schematic diagram is shown below (R and R' represent the portion provided by the RAFT chain transfer agent): [ka] It will be provided.

[0044] Synthesis of poly(2-(dimethylamino)ethyl acrylate)(1) having 24 degrees of polymerization (DP) Anisole contains 14.0 mg (0.05 mmol) of 4,4'-azobis(4-cyanopentanoic acid) (ACPA) and 4-cyanopentanoic acid dithiobenzoate (CP) in 5 mL. To a solution containing 139.5 mg (0.5 mmol) of ADB, 4.30 g (30 mmol) of 2-(dimethylamino)ethyl acrylate (DMAEA) was added. The mixture was deoxygenated by five freeze-thaw cycles, and then heated to a maximum of 70°C for 48 hours. The reaction was then quenched by freezing the product in liquid nitrogen, and the residue was purified by inducing precipitation with the addition of hexane (repeated 5 times). The structure of the purified product is as described above: [ka] Having 1 Confirmed by 1H NMR.

[0045] Synthesis of block copolymer (2) by adding a PEG block to (1) To a solution containing 30.9 mg (0.009 mmol) of (1) and 0.5 mg (0.0018 mmol) of ACPA in 6 mL of anisole, PEG(9)-acrylate, methoxy-terminated (3.46 g, 7.2 mmol) was added. The mixture was deoxygenated by five freeze-thaw cycles, and then heated to a maximum of 65°C for 8 hours. The reaction was then quenched by freezing the product in liquid nitrogen, and the residue was purified by inducing precipitation with the addition of hexane (repeated 5 times). The structure of the purified product is as follows: [ka] Having 1 Confirmed by 1H NMR and GPC.

[0046] Synthesis of quaternary ammonium derivatives (3) of block copolymer (2) To a solution containing 865.9 mg of (2) in 3 mL of acetone, 0.3 mL of ethyl bromide was added dropwise at 0°C. The mixture was stirred at room temperature for 48 hours, and then quenched by evaporating the solvent with a nitrogen stream. The residue was dissolved in methylene chloride, and the product was first purified by inducing precipitation by adding hexane (repeated 5 times). The product (3) was then dissolved in 0.01 M PBS (phosphate-buffered saline) solution and further purified by dialysis in 0.01 M PBS for 24 hours and in deionized water for an additional 48 hours, after which it was lyophilized. The structure of the purified product (3) is as follows: [ka] Having 1 Confirmed by 1H NMR and GPC.

[0047] Evaluation of the lubricating ability of block copolymer (3) on cartilage To evaluate diblock copolymer (3) as a synthetic lubricant, the frictional behavior of the copolymer was determined using a custom-made cartilage-on-glass friction meter (Gleghorn, JP et al., J. Orthop. Res. 2009, 27(6), 77). Cartilage samples were obtained from the patellofemoral groove of the posterior knee joint of neonatal bovines, and labrisin was removed by incubation in 1.5 M NaCl. The cartilage surface was saturated by incubation of the samples in PBS and then in polymer solution for 120 minutes. After incubation, the samples were placed on a friction meter in a PBS bath under boundary mode conditions (30% compressive strain and a linear vibration velocity of 0.3 mm / s). To demonstrate the importance of diblock construction, individual blocks were also tested under the same conditions as bonding blocks only and lubricating blocks only. Figure 2 is a graph plotting the coefficient of friction (COF) results for PBS, diblock copolymer (3), bonding blocks only, and lubricating blocks only. In vitro boundary lubrication tests yielded results ranging from 0.391±0.020 to 0.088±0.039 (n=4~11). *This resulted in a decrease in COF to p<0.0001), which is comparable to the result in the Labricin treatment group (COF = 0.093 ± 0.011, dotted line in Figure 2) (see Gleghorn et al., above). Notably, a similar trend in reduced COF was not observed in treatments using either of the individual blocks, suggesting that both connective and lubricating blocks are required for boundary lubrication of articular cartilage.

[0048] The importance of bonding blocks for lubrication was further demonstrated through competitive bonding studies. 1 The COF of cartilage samples was characterized after exposure to solutions composed of combinations of binding domains and diblock copolymers in binding block:diblock copolymer molar ratios ranging from 0:1 to 1:1. Figure 3 is a graph plotting the COF of solutions with varying binding block:diblock copolymer (3) ratios. As shown by the data in Figure 3, the COF of the samples exhibited dose-response behavior. As expected, high concentrations of binding domains effectively inhibited lubrication by the diblock copolymer, suggesting that effective binding of polymers on the surface is essential for their success in effectively lubricating cartilage. As shown in Figure 3, the behavior follows an S-shaped dose-response (R 2 =0.87, IC 50 =13.45, n=4~6).

[0049] In another experiment, a random copolymer version of the diblock copolymer (i.e., the same monomer units but incorporated into the copolymer in a random manner instead of blocks) was synthesized by random RAFT copolymerization of two monomers, followed by quaternary ammonium conversion. Figure 4 is a graph plotting the COF (using the same method of frictional testing as described above) of the diblock copolymer (3) and the random copolymer version. In particular, as shown by the results in Figure 4, the random copolymer was significantly less capable of lubricating articular cartilage than the block copolymer. The failure of the random polymer to lubricate articular cartilage in the same test confirms the importance of the bonding block for providing significantly improved lubrication ability. In boundary lubrication modes, frictional properties are primarily governed by solid-solid interactions and are therefore highly dependent on the physical and chemical properties of the opposing surfaces. It is crucial that boundary mode lubricants support normal loading by forming a molecular layer that effectively coats the cartilage surface. Individual positively charged quaternary ammonium groups randomly distributed in the polymer backbone cannot effectively interact with the cartilage surface, which again demonstrates the importance of the diblock structure.

[0050] Next, key lubrication parameters of some diblock copolymers were evaluated and compared to those of natural lubricin. Briefly, drug studies were performed using cartilage samples treated with solutions of (3) ranging in concentration from 0.01 to 10 mg / mL. Figure 5A is a graph plotting COF as a function of concentration of diblock copolymer (3). As shown in Figure 5A, the COF of the samples showed that high concentrations of copolymer (3) were comparable to that of lubricin (EC2). 50 Effective lubrication of cartilage (EC) at a level greater than 0.030 mg / mL under similar conditions. 50 Dose-response behavior (R = 0.404 mg / ml) resulting in this behavior 2The result was 0.89). Figure 5B is a graph plotting COF as a function of incubation time using 1 mg / mL diblock copolymer (3) for different durations. The resulting graph can be considered a binding kinetics curve. The concentration was selected using the inflection point of the S-shaped dosing curve. A one-phase decay, followed by a plateau model (R 2 When fitted to =0.95, the binding kinetics curve (Figure 5B) revealed a binding time constant (τ) of 7.19 mins, which is comparable to that of natural labrisin (approximately 9 mins). See, for example, Gleghorn et al., above.

[0051] The above results demonstrate the success of the design of a diblock copolymer whose construction mimics the lubricating protein rubrisin. As evaluated by a custom friction meter, the block copolymer (3) successfully reduced the coefficient of friction of articular cartilage in the boundary mode (0.088 ± 0.039) to a level comparable to that of natural rubrisin (0.093 ± 0.011). In addition, the EC of this polymer 50 Both the binding time constant (0.404 mg / mL) and the binding time constant (7.19 min) are comparable to the corresponding parameters of labrisin (>0.03 mg / mL, approximately 9 min). Like labrisin, the outstanding tribological properties of this diblock copolymer can be explained by its molecular structure. In particular, the effective binding of this polymer to articular cartilage has been shown to be essential for effective lubrication. Unexpectedly, the diblock copolymer (3) has been shown to possess at least the lubricating capacity of labrisin, which is significant. It shows clinical potential.

[0052] Evaluation of the lubrication ability of block copolymer (3) on bone. In this study, the same block copolymer (3) was tested for its lubrication ability on bone. Bone samples were obtained from the femoral condyloid process of the posterior knee joint of neonatal bovines. Bone plugs were extracted from the cartilage layer down to the growth plate using a 6 mm diameter drill, and then scraped down to a height of 2 mm. By removing the cartilage layer, the subcostal surface was exposed, and cancellous bone plugs were obtained by cutting the medial portion of the drilled bone plug. Bone samples were incubated in PBS solution and then in a polymer solution containing (3) (10 mg / mL for 2 hours or 1 mg / mL for 1 hour). Tribological properties were measured using a custom-made bone-on-glass friction meter (Gleghorn et al., see above) under a normal load of 450 g and under linear vibration velocities of 0.3 mm / s, 1 mm / s, and 3 mm / s. The coefficient of friction was calculated as the mean shear force while sliding, divided by the normal force. Mathematical significance between treatments was determined using one-way ANOVA and Student's t-test with adjusted sliding speed.

[0053] It is well known that the coefficient of friction (COF) of both cancellous bone and subchondral bone is significantly increased compared to cartilage under the same conditions. Figures 6A and 6B are graphs plotting the COF of cancellous bone and subchondral bone samples treated with diblock copolymer (3) solution (10 mg / mL for 2 hours or 1 mg / mL for 1 hour) or PBS solution, respectively. The COF of cartilage in PBS or synovial fluid in boundary mode is also shown. As the data in Figures 6A and 6B show, bone plug samples incubated in polymer solution exhibited significantly lower COF than the PBS control (ΔCOF of approximately -0.2 for cancellous bone, p<0.05; ΔCOF of approximately -0.15 for subchondral bone, p<0.05). As can also be confirmed by the data in Figures 6A and 6B, lubrication is further improved using higher concentrations of polymer solutions (e.g., greater than 1 mg / mL, or at least 2 mg / mL, 5 mg / mL, or 10 mg / mL) and / or longer incubation times (e.g., greater than 1 hour, or at least 1.5 or 2 hours). The results demonstrate that the block copolymers described herein effectively lubricate cartilage or bone to a level comparable to, or even superior to, the COF of cartilage in PBS in boundary mode. Without being constrained by theory, the diblock copolymers of the present invention are thought to strongly interact with the negatively charged mineral components of cartilage or bone and resist vertical compression thanks to their bottle-brush-like construction.

[0054] While preferred embodiments of the present invention and those currently considered are shown and described, those skilled in the art can make various variations and modifications that remain within the scope of the invention as defined by the appended claims.

Claims

1. It is a block copolymer with the following structure: 【Chemistry 1】 (In the formula: X is selected from -NR'-, -O-, and bonds, where R' is selected from a hydrogen atom and a hydrocarbon group having at least one and up to six carbon atoms; Y is a polyalkylene glycol; R is a hydrogen atom, a hydrocarbon group (R) having 1 to 12 carbon atoms, or a cartilage-binding domain, the cartilage-binding domain being selected from cartilage-binding peptides and cholesterol or other sterol moieties shown in Sequence IDs 1 to 5; The subscripts d and e are independently integers of at least 3; (The subscript f is an integer of 0 or at least 1.) It has, The hydrogen atoms on the carboxylic acid group shown may be replaced by positively charged metal ions or positively charged organic groups; According to the laws of chemistry, the block copolymer is terminated by terminal groups at the end opposite the thiol group. A composition for lubricating biological tissue, comprising the aforementioned block copolymer.

2. The composition according to claim 1, wherein R is a cartilage-binding domain.

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