Lubricating composition comprising block copolymer and use thereof

Abstract

A lubricating block copolymer and a use thereof, and a composition comprising the lubricating block copolymer and a use thereof. Also provided is a method for imparting a suitable level of lubrication to biological tissue. The method comprises: bringing biological tissue into contact with said block copolymer and / or said composition so as to impart a suitable level of lubrication.

Description

Lubricating composition of block copolymer and use thereof Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a lubricating composition of a block copolymer and its use. Background Art

[0002] Lubricin is a glycosylated protein present in synovial fluid that plays a key role in joint boundary lubrication and the prevention of osteoarthritis. Lubricin reduces the coefficient of friction (COF) of articular cartilage in boundary mode by up to 70% (Gleghorn, JP et al., J. Orthop. Res. 2009, 27(6), 77). This effective lubricating ability stems from the structure of lubricin: the central mucin-like domain of lubricin is composed of an extensively glycosylated core protein that attracts and retains water near the molecule; the C-terminus of lubricin binds the protein to the cartilage surface (Zappone, B. et al., Langmuir 2008, 24(4), 1495). This structure is crucial for boundary mode lubrication of articular cartilage, because denaturation of any domain of lubricin will result in partial or complete loss of lubrication ability.

[0003] More than 50 million people in developed countries suffer from osteoarthritis (OA), and this number is expected to rise as median age and life expectancy increase. In the United States alone, the economic impact of osteoarthritis treatment exceeds $30 billion annually. This economic burden, along with other factors (i.e., quality of life, lost work time, etc.), has motivated the development of more effective treatments.

[0004] Current treatments for osteoarthritis (OA) include nonsteroidal anti-inflammatory drugs, intra-articular corticosteroid injections, and chondroitin sulfate or glucosamine supplements. However, these have little or no effect on disease progression. A recent treatment for OA is intra-articular injection of the natural synovial glycosaminoglycan, hyaluronic acid (HA) (e.g., Mabuchi et al., (1994) J. Biomed. Mat. Res. 28:865–70). HA is known to increase synovial fluid viscosity (e.g., viscosupplementation) to reduce the coefficient of friction in hydrodynamic mode lubrication (e.g., Tadmor et al., (2002) J. Biomed. Mat. Res. 61:514–23). Another major lubricating component of synovial fluid is lubricin, a high molecular weight glycoprotein that reduces the coefficient of friction in boundary mode lubrication.

[0005] In damaged cartilage, it is known that chondrocyte lubricin production is impaired and boundary-mode lubrication is reduced. Natural lubricants in nature, such as proteoglycan aggregates and mucins (e.g., lubricin), maintain the native surface as hydrophilic. Intra-articular injection of lubricin supplements and a truncated recombinant lubricin construct, LUB:1, slowed the progression of OA in rat models of the disease (e.g., Jay et al., (2010) Arthritis Rheum. 62:2382–91; Flannery et al., (2009) Arthritis Rheum. 60:840–7). However, large-scale recombinant production of lubricin and LUB:1 remains challenging to date due to the multiple amino acid repeats in the core structure of the proteins and their high degree of glycosylation (e.g., Jay (2004) Curr. Opin. Orthop. 15: 355–359; Jones et al. (2007) J. Orthop. Res. 25: 283-292). In the late stages of osteoarthritis, where direct bone-to-bone contact may occur, an effective bone lubricant is also needed separately. Therefore, an effective lubricant that can provide the same or similar boundary lubrication effect as lubricin or LUB:1 would be a major advance in this field.

[0006] Summary of the Invention

[0007] This application provides the design, synthesis, and use of a special block copolymer with a lubricin-mimicking structure that provides significant lubrication under boundary mode lubrication conditions. In certain embodiments, the block copolymer contains a lubricating block (e.g., Mn ~ 200 kDa) that mimics the mucin-like domain of lubricin and a smaller cartilage-binding block (e.g., Mn ~ 3 kDa) that mimics the hemoglobin-like domain. As disclosed below in this application, application of this type of polymer to biological tissue resulted in a significant reduction in the coefficient of friction (COF) compared to untreated controls.

[0008] In one aspect, the present application provides a block copolymer having the following structure:

[0009] wherein: R1 and R2 are each independently selected from hydrogen and methyl; X and X' are independently selected from -NR'-, -O-, and a bond, wherein R' is selected from hydrogen atoms and has at least 1 and at most 6 carbon atoms; Y is selected from polyalkylene glycols, sugars, and polyols; subscript a is an integer of at least 3, subscript b is at least twice a; and subscript c is an integer of at least 1; wherein, according to the laws of chemistry, the block copolymer is terminated at each end by an end group, and the total positive charge of the quaternary ammonium groups in the copolymer is offset by a total negative charge of equal magnitude provided by anions associated with the quaternary ammonium groups.

[0010] In certain embodiments, the anion is chloride, bromide, or iodide. In some embodiments, the anion is chloride.

[0011] In certain embodiments, wherein subscript b is at least twice as large as a. In certain embodiments, wherein subscript b is an integer greater than 40. In certain embodiments, wherein subscript b is an integer of at least 50. In certain embodiments, wherein subscript b is an integer of at least 100. In certain embodiments, wherein subscript b is an integer of 200-400 and subscript a is an integer of 20-96. In certain embodiments, wherein subscript b is 400 and subscript a is 24.

[0012] In certain embodiments, wherein Y comprises a polyalkylene glycol. In certain embodiments, wherein the polyalkylene glycol comprises polyethylene glycol. In certain embodiments, wherein the sugar comprises a monosaccharide. In certain embodiments, wherein the sugar contains at least two monosaccharide units. In certain embodiments, wherein the polyalkylene glycol, sugar or polyol is composed of at least 2 and at most 500 monomeric units. In certain embodiments, wherein the polyalkylene glycol, sugar or polyol is composed of at least 2 and at most 200 monomeric units. In certain embodiments, wherein the polyalkylene glycol, sugar or polyol is composed of at least 2 and at most 100 monomeric units. In certain embodiments, wherein the polyalkylene glycol, sugar or polyol is composed of at least 2 and at most 20 monomeric units. In certain embodiments, wherein at least one of the terminal groups is a sulfhydryl group.

[0013] In another aspect, the present application provides a composition comprising the block copolymer described herein.

[0014] In another aspect, the present application provides a use of the block copolymer and / or composition described herein for enhancing the lubricity of biological tissues or imparting an appropriate level of lubricity to biological tissues.

[0015] In another aspect, the present application provides a method for imparting an appropriate level of lubricity to biological tissue, the method comprising contacting the biological tissue with the block copolymer and / or the composition described herein to impart the appropriate lubricity.

[0016] In another aspect, the present application provides a block copolymer and / or composition described herein for use in treating dry eye.

[0017] On the other hand, the present application provides a block copolymer and / or composition described in , which is used for preparing a drug for treating dry eye.

[0018] In certain embodiments, the biological tissue is selected from the group consisting of joint, bone, eye tissue, nasal tissue, tendon, tendon bursa, and vaginal tissue.

[0019] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0021] Figure 1 shows a general representation of an exemplary diblock copolymer of the present application, with the tie block and lubricating block portions identified.

[0022] FIG2 shows a schematic diagram of the degradation of the block copolymer of the present application, wherein the degradation products thereof may include naturally occurring compounds.

[0023] FIG3A-C shows the PDI test results of the block copolymer of the present application, and the PDI value thereof is correlated to the type of counter ion.

[0024] FIG4 shows the lubrication ability of the exemplary block copolymers of the present application, which reduce the friction coefficient by at least 60% under different boundary conditions.

[0025] FIG5 shows the biocompatibility of the key intermediates of the block copolymer of the present application, and its cytotoxicity is correlated with the type of counter ions.

[0026] FIG6 shows the stability of the block copolymer of the present application. The methyl structure contained in the main chain can effectively inhibit the formation of degradation by-products during moist heat sterilization.

[0027] FIG7 shows the therapeutic effect of the exemplary block copolymer of the present application, which effectively reduced the corneal staining score in the mouse dry eye model and returned it to normal levels.

[0028] FIG8 shows the therapeutic effect of the exemplary block copolymer of the present application, which effectively reduces the expression levels of some inflammatory factors in a mouse dry eye model. DETAILED DESCRIPTION

[0029] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0030] Definition of terms

[0031] As used herein, the term "block copolymer," also known as a mosaic copolymer, generally refers to a specialized polymer formed by linking two or more polymer segments with distinct properties. For example, the block copolymers described herein may include a mucin-like domain and a C-terminal hemoglobin-like (PEX-like) domain. For example, the block copolymers described herein may include a binding domain and a lubricating domain.

[0032] In this application, the term "copolymer" refers to the presence of at least two polymer blocks. The copolymer can be, for example, a diblock copolymer, a triblock copolymer, a tetrablock copolymer, or a higher order block copolymer. The copolymer can, for example, contain polymer blocks containing positively or negatively charged side groups, as well as polymer blocks containing nonionic hydrophilic side groups, particularly hydrophilic groups containing ether and / or hydroxyl functional groups. In cases where the side groups are polymers, the copolymer can be further classified as a graft brush copolymer.

[0033] In this application, the term "polymer dispersity index" (PDI), also known as the polydispersity index or heterogeneity index, is generally used to describe the molecular weight distribution of polymers. It is the ratio of the weight-average molecular weight to the number-average molecular weight. A PDI value of 1 or greater indicates a broader molecular weight distribution, while a smaller PDI indicates a more uniform molecular weight distribution.

[0034] In this application, the term "pharmaceutically acceptable carrier" or equivalent terms refers to a pharmaceutically acceptable material, composition or carrier, which can be a liquid (diluent or excipient) or a solid filler. In this application, "pharmaceutically acceptable" refers to a compound, material, composition and / or dosage form that is suitable for contacting the tissues of humans and animals without excessive toxicity, irritation, allergic reaction or other problems or complications within the scope of reasonable medical judgment, in accordance with a reasonable benefit / risk ratio. In a pharmaceutical composition, the compound is usually dispersed in a physiologically acceptable vehicle, which is dissolved or emulsified by mixing (e.g., mixing with a solid vehicle in solid form) or in a liquid vehicle. The vehicle should be compatible with the other ingredients of the preparation and physiologically safe for the subject. Any vehicle known in the art is suitable for this application, depending on the mode of administration. Some examples of suitable vehicles include aqueous solutions, gelatin, fatty acids (e.g., stearic acid) and their salts, talc, vegetable fats or oils, gums and glycols, starch, dextran, etc.

[0035] The terms "sufficient amount," "therapeutically effective amount," and "effective amount" as used interchangeably herein refer to an amount of the copolymeric composition of the present invention that is sufficient to produce the following results: provide adequate lubrication to biological tissues, or prevent the development, recurrence, or onset of a disease or condition (e.g., osteoarthritis) or one or more of the above symptoms, enhance or improve the prophylactic effect of another therapy, reduce the severity and duration of a disease or condition, alleviate one or more symptoms of a disease or condition, prevent the development of a disease or condition, and / or enhance or improve the efficacy of an additional treatment.

[0036] Detailed Description of the Invention

[0037] The block copolymers and / or compositions comprising the block copolymers described herein have one or more of the following functions or advantages:

[0038] (1) When the counterion is Cl, it has improved cytotoxicity and biocompatibility;

[0039] (2) When the counter ion is Cl, the molecular weight is evenly distributed and has a smaller PDI (Polymer dispersity index).

[0040] (3) When the counter ion is Cl, the synthesis route is shorter and the synthesis is convenient.

[0041] (4) Having the ability to lubricate biological tissues;

[0042] (5) When the main chain contains methyl groups, it has stronger stability.

[0043] The block copolymers described herein have improved cytotoxicity and biocompatibility compared to other combinations. They share a similar structure to the naturally occurring compound choline, and no other similar structures are found in natural biological systems. Furthermore, as the number of hydrocarbon chains increases, the hydrophobicity of the amino groups in the block copolymers described herein increases, resulting in amphiphilic properties, a common feature in cell lysis reagents.

[0044] Typically, as the number of hydrocarbon chains increases, the hydrophobicity / amphiphilicity of the compound increases, which increases the synthetic challenge and leads to a wider range of molecular weight distribution. However, the block copolymers described in this application have a PDI close to 1 and show a uniform molecular weight distribution.

[0045] In one aspect, the present application provides a series of block copolymers having the structures shown below:

[0046] The variables X and X' in formula (1) are independently selected from -NR'-, -O- and a bond, wherein R' is selected from a hydrogen atom and a hydrocarbon group having at least 1 and at most 6 carbon atoms (selected from R groups). In certain 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 tert-butyl group, or a more specific selection thereof.

[0047] On the other hand, the present application provides a series of block copolymers having a structure as shown in formula (1), wherein: R1 and R2 are selected from hydrogen and methyl, and R1 and R2 are not methyl at the same time; X and X' are independently selected from -NR'-, -O- and a bond, wherein R' is selected from a hydrogen atom and has at least 1 and at most 6 carbon atoms; Y is selected from polyalkylene glycols, sugars and polyols; subscripts a and b are independently integers of at least 3; and subscript c is an integer of at least 1; according to the laws of chemistry, the block copolymer is terminated at each end by an end group, and the total positive charge of the quaternary ammonium group in the copolymer is offset by a total negative charge of equal magnitude provided by an anion associated with the quaternary ammonium group. In some embodiments, the anion is chloride, bromide or iodide. In some embodiments, the anion is chloride.

[0048] In certain embodiments, R1 and R2 are both hydrogen. In certain embodiments, R1 is hydrogen and R2 is methyl. In certain embodiments, R1 is methyl and R2 is hydrogen. In certain embodiments, R1 is methyl and R2 is methyl.

[0049] On the other hand, the present application provides a series of block copolymers having a structure as shown in formula (21):

[0050] wherein X and X' are independently selected from -NR'-, -O-, and a bond, wherein R' is selected from hydrogen and has at least 1 and at most 6 carbon atoms; Y is selected from polyalkylene glycols, sugars, and polyols; subscripts a and b are independently integers of at least 3; and subscript c is an integer of at least 1; wherein, according to the laws of chemistry, the block copolymer is terminated at each end by an end group, at least one of which is a thiol, and the total positive charge of the quaternary ammonium groups in the copolymer is offset by an equal amount of total negative charge provided by an anion associated with the quaternary ammonium groups. In some embodiments, the anion is chloride, bromide, or iodide. In some embodiments, the anion is chloride.

[0051] In certain embodiments, the variable Y in formula (1) is or includes a polyalkylene glycol group. The polyalkylene glycol group can be conveniently represented by the following structure: (-CR'2CR'2O-) n R', wherein for each instance of R' R' is independently selected from a hydrogen atom and a hydrocarbon group (e.g., a methyl or ethyl group), and n is at least 2, 3, 4, 5, or 6 and at most 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 a range bounded by any two of the foregoing values, wherein each of the foregoing values ​​corresponds to the number of alkylene oxide (-CR'2CR'2O-) monomer units. In particular embodiments, the polyalkylene glycol is a polyethylene glycol or polypropylene glycol group. Furthermore, the polyalkylene glycol group may or may not be incorporated 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.

[0052] In certain embodiments, the variable Y in formula (1) is or includes a glycosyl group. In this application, the term "sugar" includes monosaccharides (containing one monosaccharide unit) and saccharides containing at least or more than two monosaccharide units, such as disaccharides, trisaccharides, oligosaccharides (e.g., at least 4 and up to 20, 30, 40, 50 or 60 monosaccharide units) and polysaccharides (typically more than 60, 70 or 80 monosaccharide units, and up to, for example, 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 form can still be reasonably classified as a sugar 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), galacto-oligosaccharides (GOS) and manno-oligosaccharides (MOS). Some examples of polysaccharides include dextran, dextran sulfate, starch (for example, 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 certain embodiments, saccharide is selected to have total anionic charge, as in those saccharides with carboxylic acid, carboxylate, sulfate or sulfonate groups. Glycosyl may or may not be integrated into copolymer, as the copolymer containing oligosaccharide moiety and oligopeptides or polyacrylic acid moiety. In certain embodiments, the saccharide-containing copolymer contains a saccharide portion and an anionic polymer portion, such as polyacrylic acid (PAA), polyglutamic acid, or polyaspartic acid.

[0053] In certain embodiments, the variable Y in formula (1) includes a polyol group. In this application, the term "polyol" generally refers to a non-sugar group having multiple (i.e., at least 2, 3, or 4) hydroxyl groups. A polyol can be, for example, a sugar alcohol or a polyhydric alcohol. Some examples of sugar alcohols include erythritol, xylitol, mannitol, glycerol, and sorbitol. In certain embodiments, a polyol is a polymer containing hydroxyl groups. A saccharide or polyol (Y) is typically connected to the block copolymer of formula (1) via one of its deprotonated hydroxyl groups, wherein X' can represent an oxygen atom of a Y group; or X' can represent a bond, with Y representing a saccharide or polyol bound to the C(O) group shown via its oxygen atom; or X' is -NR'-, with Y representing a saccharide or polyol bound to the -NR'- group via its carbon atom. In certain embodiments, the polymer containing hydroxyl groups can comprise, for example, 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 monomer units. Some examples of such polymers include polyvinyl alcohol, poly(hydroxyethyl methacrylate), and poly(hydroxypropyl methacrylate). The polyol group may or may not be incorporated into the copolymer, such as polyvinyl alcohol-polyacrylic acid (PVA-PAA) copolymer. In certain embodiments, the copolymer containing a polyol contains a polyol portion and an anionic portion, which may be an anionic polymer portion, such as polyacrylic acid (PAA), polyglutamic acid, or polyaspartic acid.

[0054] In certain embodiments, the subscripts a and b in formula (1) are independently an integer of at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 50. In certain embodiments, the subscripts a and b are independently no more than 30, 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 certain embodiments, the subscript a is selected to be smaller than the subscript b. For example, the subscript a can be in the range of 3-10, 3-20, 3-30, or 3-40, while the subscript b is 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 can be in the range of 30-500, 40-500, 50-500, or 60-500, while subscript b is in the range of 3-10, 3-20, 3-30, or 3-40.

[0055] In certain embodiments, the subscript a in formula (1) may be an integer of at least 3, and the subscript b may be at least 2 times a. In certain embodiments, the subscript b may be at least 5 times a. In certain embodiments, the subscript b may be at least 10 times a. In certain embodiments, the subscript b may be at least 20 times a. In certain embodiments, the subscript b may be at least 50 times a. In certain embodiments, the subscript b may be at least 100 times a. In certain embodiments, the subscript b may be at least 200 times a.

[0056] In certain embodiments, the subscript a in formula (1) may be an integer of at least 3, and the subscript b may be an integer greater than 40. In certain embodiments, the subscript b may be an integer of at least 50. In certain embodiments, the subscript b may be an integer of at least 100. In certain embodiments, the subscript b may be an integer of at least 200. In certain embodiments, the subscript b may be an integer of at least 300. In certain embodiments, the subscript b may be an integer of at least 400.

[0057] In certain embodiments, subscript a may be in the range of 3-10, 3-20, 3-30, or 3-40, and subscript b may be in the range of 40-500, 50-500, or 60-500. In certain embodiments, subscript a may be in the range of 3-30, and subscript b may be in the range of 40-500. In certain embodiments, subscript a may be in the range of 20-30, and subscript b may be in the range of 300-500. In certain embodiments, subscript a may be an integer from 10-100, and subscript b may be an integer from 100-500. In certain embodiments, subscript a may be an integer from 20-96, and subscript b may be an integer from 200-400. In certain embodiments, subscript a may be 24, and subscript b may be 400.

[0058] The subscript c in formula (1) is an integer of at least 1. In various embodiments, c is exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or c is within a range defined by any two of the aforementioned values. The value of c corresponds to the number of methylene groups to which c is assigned. That is, when the subscript c is 1, a CH2 linker is present between X and the quaternary ammonium group shown in formula (1); and when the subscript c is 2, a CH2CH2 linker is present between X and the quaternary ammonium group shown in formula (1).

[0059] Although not shown in formula (1), the structure shown in formula (1) necessarily (that is, by the laws of chemistry) includes end groups at each of the two ends of the copolymer. End groups are independently selected from, for example, hydrogen atoms, hydrocarbon radicals R or R' or groups containing heteroatoms (such as -OH, -OCH3), alkyl groups containing nitrile (such as provided by free radical initiators or chain transfer agents), sulfhydryl groups (-SH) or dithioester groups (provided by RAFT chain transfer agents). In some embodiments, at least one end group is a sulfhydryl group. End groups generally correspond to the groups initially present in the precursor reactants for synthesizing block copolymers, so the type of end groups generally depends on the chemical substances used to synthesize block copolymers. However, end groups can be suitably regulated by reacting the block copolymers initially produced to add specific end groups (for example, cartilage binding domains (for example, containing peptide groups)), which help to bind the block copolymer to the desired biological tissue. In some embodiments, the cartilage binding domain is attached to the block copolymer by -S- joints, such as RS- forms, where R can be a cartilage binding domain. In certain embodiments, the compound of formula (1) has terminal extremes R and R', which may be as shown in formula (2):

[0060] In certain embodiments, R may be as shown in formula (3):

[0061] In certain embodiments, R' can be as shown in formula (4):

[0062] Moreover, although also not shown in formula (1), the total positive charge of the quaternary ammonium groups in the copolymer shown in formula (1) is offset by an equivalent amount of total negative charge provided by anions associated with the quaternary ammonium groups. The anions can be selected from any biologically acceptable species, such as halides (e.g., chloride, bromide, or iodide), carbonates, bicarbonates, sulfates, bisulfates, bisulfites, carboxylates (e.g., acetates, propionates, butyrates, maleates, and citrates), and sulfonates (e.g., present between X shown in formula (1) and the quaternary ammonium group. In some embodiments, the anion is chloride, bromide, or iodide. In some embodiments, the anion is chloride.

[0063] The block copolymer represented by formula (1) described in the present application may include the compounds shown in the following table:

[0064] The block copolymers described herein can be synthesized by any suitable polymerization method. In a specific 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, in the presence of a polymerization initiator, the first polymer block is produced by reacting a first functionalized vinyl monomer with a RAFT transfer agent. Then, in the presence of a polymerization initiator, the first polymer block is reacted with a second functionalized vinyl monomer to attach the polymerized second functionalized vinyl monomer block to the first polymer block.

[0065] On the other hand, the application provides a composition comprising one or more block copolymers described herein. In certain embodiments, the composition can function as a lubricant. In certain embodiments, the composition can enhance lubricity or impart an appropriate level of lubricity to a biological tissue. In certain embodiments, the composition can function as a lubricant within or between biological tissues. The biological tissue is selected from the group consisting of joints, bones, eye tissue, nasal tissue, tendons, tendon bursae, and vaginal tissue.

[0066] In certain embodiments, the composition can be a pharmaceutical composition and can also include one or more pharmaceutically acceptable carriers. The pharmaceutical composition can also include one or more adjuvants, such as stabilizers, surfactants, salts, buffers, additives or combinations thereof, which are all well known in the pharmaceutical field. Stabilizers can be, for example, oligosaccharides (e.g., sucrose, trehalose, lactose or dextran), sugar alcohols (e.g., mannitol) or combinations thereof. Surfactants can be any suitable surfactants, including, for example, those containing polyalkylene oxide units (e.g., Tween 20, Tween 80, Pluronic F-68), which are generally included in an amount from about 0.001% (w / v) to about 10% (w / v). Salts or buffers can be any suitable salts or buffers, such as sodium chloride or sodium phosphate or potassium phosphate, respectively. Some examples of additives include, for example, glycerol, benzyl alcohol and 1,1,1-trichloro-2-methyl-2-propanol (e.g., chloroacetone or chlorobutanol). If necessary, the pH of the solution can be appropriately adjusted by including a pH regulator. Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, emulsions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desired. Pharmaceutical formulations can be in the form of sterile aqueous solutions containing one or more buffers, diluents and / or other suitable additives, such as, but not limited to, penetration enhancers and carrier compounds.

[0067] When biological tissue slides against the same tissue or other substances, an increase in lubricity generally corresponds to a decrease in friction (i.e., coefficient of friction, or COF). The coefficient of friction can be measured using a tribometer, which evaluates surface lubrication by linear oscillation of the sample at variable speeds (typically 0.1, 0.3, 1, 3, and 10 mm / s) and variable compressive normal stress (typically 250 to 300 kPa).

[0068] In another aspect, the present application provides use of the block copolymer and / or composition described herein for enhancing the lubricity of biological tissues or imparting an appropriate level of lubricity to biological tissues.

[0069] In another aspect, the present application provides a method for imparting a suitable level of lubricity to a biological tissue, the method comprising contacting the biological tissue with a block copolymer as described herein and / or a composition as described herein to impart suitable lubricity. In certain embodiments, the suitable lubricity may include an increase in the level of lubricity.

[0070] In certain embodiments, the increase in lubricity can include a decrease in friction. For example, the friction level of a biological tissue sliding against the same tissue or other substance is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, relative to before the block copolymers and / or compositions described herein are contacted with the biological tissue. The friction level can be measured by conventional means in the art. For example, the friction level can be reflected by measuring the coefficient of friction.

[0071] In certain embodiments, the suitable lubricity may include wear protection. In certain embodiments, the suitable lubricity may include wear protection. For example, the wear protection or wear protection may include slowing down or reducing the mass or volume loss due to wear between biological tissues. For example, the mass or volume loss of the biological tissue when sliding relative to the same tissue or other substance can be reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more over the same period of time relative to before the block copolymers and / or compositions described herein were contacted with the biological tissue. The effect of wear protection or wear protection can be tested by means commonly used in the art, for example, by friction and wear testing.

[0072] The biological tissue is selected from the group consisting of joints, bones, eye tissue, nasal tissue, tendons, tendon bursae and vaginal tissue.

[0073] In another aspect, the present application relates to a method for imparting lubricity to biological tissues (such as joints, cartilage, and bone) using the biomimetic block copolymers described herein. In the case of bone, the biomimetic copolymers can reduce the discomfort, pain, and additional damage sometimes caused by direct bone-to-bone contact in the late stages of osteoarthritis. According to this method, the biological tissue is contacted with a sufficient (i.e., effective or therapeutically effective) amount of any of the biomimetic block copolymers described herein to enhance lubricity or impart an appropriate level of lubricity to the biological tissue.

[0074] A therapeutically effective amount can be administered to the patient in one or more doses, the dose being sufficient to alleviate, relieve, stabilize, reverse or slow the progression of the disease or condition, or reduce the pathological consequences of the disease or condition, or reduce the symptoms of the disease or condition. The alleviation or reduction does not need to be permanent, but can be sustained for a period of time, ranging from at least one hour, at least one day, or at least one week or more. The effective amount is generally determined by the physician on a case-by-case basis, and this is within the skill of those skilled in the art. When determining the appropriate dosage to achieve an effective amount, several factors are generally considered. These factors include the patient's age, sex and weight, the condition being treated, the severity of the condition, as well as the route of administration, dosage form and regimen, and the desired results. In certain embodiments, the therapeutically effective amount is an amount that can effectively treat osteoarthritis, achieve pain relief, improve joint motion and flexibility, reduce friction in the joint, or improve other acceptable osteoarthritis measurement indicators over a period of time. The therapeutically effective amount can be adjusted by using concentration and injection volume. For example, the block copolymer described herein can be used at a concentration of 0.001 mg / mL to 100 mg / mL. For example, the use concentration of the block copolymer described herein can be 0.1mg / mL-100mg / mL. For example, the use concentration of the block copolymer described herein can be 1-5mg / mL. For example, the use concentration of the block copolymer described herein can be 1mg / mL-100mg / mL. For example, the block copolymer described herein can be administered with a dosage of about 0.1mL-about 10mL. For example, the block copolymer described herein can be administered with a dosage of about 0.1mL-about 3mL. In an exemplary embodiment, the scope of dosage level is to inject a volume of 0.1-10mL (for people) with a concentration of 0.1-100mg / mL, and more generally to inject a volume of 0.1-3mL with a concentration of about 1-5mg / mL.

[0075] In certain embodiments, the therapeutically effective amount is effective to reduce the level of friction in biological tissue. In certain embodiments, the therapeutically effective amount is effective to provide wear protection in biological tissue. In certain embodiments, the therapeutically effective amount is effective to provide lubricity in biological tissue to be lubricated.

[0076] Biological tissue to be lubricated can be contacted with any block copolymer described herein by any method known in the medical field. Block copolymers can be delivered directly to or on biological tissues, or indirectly to biological tissues around the tissue to be lubricated, such as by injection, infusion, implantation, spraying or coating, so that the biological tissue is contacted with block copolymers. Generally, contacting biological tissue refers to delivering block copolymers to tissues using any method of coating surfaces with copolymers or soaking tissues. In certain embodiments, the composition is contacted with tissues by injection or infusion into the joint space, thereby resulting in coating of cartilage and / or meniscus present in the joint space. Moreover, the volume used depends at least in part on the type of tissue contacted, whether to fill the gap, or to coat the surface, which can be determined by those skilled in the art of medicine.

[0077] In certain embodiments, the block copolymer is injected or infused into or on an arthritis or injured joint or bone to improve the lubricity of the joint or bone. In this way, the copolymer provides boundary lubrication. This treatment can be specifically used to treat or prevent osteoarthritis. The treatment of osteoarthritis or injured joints, cartilage or bone preferably results in the following results: alleviating symptoms, improving mobility, reducing joint pain and overall suppressing disease progression or preventing in the case of joint damage. The method may also include giving one or more of the above-mentioned block copolymers and simultaneously or sequentially giving another composition, which serves to enhance or work together with the block copolymer, and is outside the scope of the block copolymer described herein. Enhancement (i.e., auxiliary) compositions may be selected from, for example, hyaluronic acid, lubricant, synovial fluid, glycosaminoglycans or other adjuvants. These other agents may also be administered by, for example, injection or infusion. In certain embodiments, these other agents may synergize with one or more of the above-mentioned block copolymers to provide enhanced lubrication and wear protection.

[0078] In a specific embodiment, the biological tissue is a joint, cartilage or bone, more typically an injured or arthritic joint, cartilage or bone. In certain embodiments, the joint is a weight-bearing joint, such as a hip joint, knee joint or ankle joint. Many different joints can benefit from increased lubricity levels, including shoulder, elbow, wrist, hand, finger and toe joints. However, the biological tissues to be lubricated are not limited to joints, cartilage and bone. Other biological tissues, including eye tissue, nasal tissue and vaginal tissue, can be lubricated using the disclosed block copolymers. Thus, by using the block copolymers described herein, various conditions other than those associated with joints, cartilage and bone can be treated. Some of these other conditions include, for example, dry eyes, dry nose, postmenopausal vaginal dryness, carpal tunnel syndrome, etc. Those skilled in the art of medicine can determine the appropriate delivery route and method for contacting with a specific biological tissue. For example, for dry eyes, contact can be achieved by instilling drops; for dry nose, contact can be achieved through a nasal spray; for carpal tunnel syndrome, contact can be achieved by injection near or around inflamed tendons and bursae; and for postmenopausal vaginal dryness, a pill, lozenge, or suppository can be placed or implanted in the vagina. Thus, this method can be used to achieve boundary mode lubrication in any variety of biological tissues that can benefit from additional lubrication.

[0079] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0080] Example

[0081] Example 1 Synthesis of block copolymer

[0082] In order to mimic the structure of lubricin, the present application prepared a diblock copolymer comprising a larger lubricating block (Mn~200kDa) that mimics the mucin-like domain of lubricin and a smaller cartilage-binding block (Mn~3-10kDa) that mimics the C-terminal domain. An exemplary composition showing two mimicking domains is provided in Figure 1. The lubricating domain of the diblock copolymer shown in Figure 1 comprises a polyacrylic acid backbone grafted with a polyethylene glycol (PEG) brush. The aforementioned features contribute to the hydration and compression resistance of the polymer. The binding domain of the diblock copolymer shown in Figure 1 comprises a polyacrylic acid backbone having quaternary ammonium side groups that non-specifically interact with negatively charged cartilage components (such as aggrecan).

[0083] The general synthetic route of the block copolymers described in this application is as follows:

[0084] wherein R1 is hydrogen or methyl, R2 is methyl or ethyl, and X is Cl, Br, or I. n is 24, and m is 400. R is as shown in formula (3): R' is shown in formula (4):

[0085] In some embodiments, the block copolymers described herein can be synthesized via a shorter synthetic route. When the counter ion is chloride ion, the synthesis requires only two steps. The synthetic route is as follows:

[0086] Specifically, six compounds were synthesized in Example 1, and their structures are shown in the figure below. The differences between them are mainly reflected in whether there is a -CH3 substitution on the main chain and whether the counter ions of the quaternary ammonium salt are different halogen ions, namely chloride, bromide, and iodide.

[0087] 1.1 Synthesis of intermediates

[0088] To form a block copolymer, it is first necessary to synthesize a key intermediate, the corresponding structure of which is shown below:

[0089] The above key intermediates can be synthesized in two steps according to the general synthesis route, taking AI intermediate as an example:

[0090] Reaction i: Synthesis of poly(2-(dimethylamino)ethyl acrylate) (1)

[0091] 2-(Dimethylamino)ethyl acrylate (DMAEA) (4.30 g, 30 mmol) was added to a 5 mL anisole solution containing 14.0 mg (0.05 mmol) of 4,4'-azobis(4-cyanovaleric acid) (ACPA) and 139.5 mg (0.5 mmol) of 4-cyanopentanoic acid dithiobenzoate (CPADB). After deoxygenation by nitrogen for 30 min, it was heated to 70 ° C for 48 hours. The reaction was then frozen and quenched at -80 ° C, and the residue was purified by inducing precipitation by adding n-hexane (repeated 5 times). The structure of the purified product was confirmed by 1H NMR.

[0092] These reaction conditions are suitable for the generation of precursors for A-Cl and A-Br intermediates. The synthesis of the MA-Cl / Br / I intermediate precursor requires replacing the reaction substrate with the corresponding monomer, 2-(dimethylamino)ethyl methacrylate (DMAEMA).

[0093] Reaction ii: Synthesis of quaternary ammonium salt derivatives of poly(2-(dimethylamino)ethyl acrylate) (2)

[0094] At 0 °C, 4 mL of iodomethane was added dropwise to a 15 mL acetone solution containing 1.6 g of (1). The mixture was stirred at room temperature for 48 hours and then quenched by evaporating the solvent using a nitrogen stream. The residue was dissolved in methanol and the product was purified by inducing precipitation by adding acetone (repeated 3 times). The structure of the purified product (2) was confirmed by 1H NMR and GPC.

[0095] These reaction conditions are applicable to the formation of A-I and MA-I intermediates. When synthesizing other precursors, the reaction substrate needs to be replaced with the corresponding haloalkane.

[0096] 1.2 Synthesis of the final product

[0097] After obtaining the intermediate (2), methoxy-capped PEG(9)-acrylate (7.2 mmol) or methoxy-capped PEG(9)-methacrylate (7.2 mmol) was added to a methanol solution containing 0.009 mmol of the key intermediate (2) and 0.5 mg (0.0018 mmol) of the initiator. It was deoxygenated with nitrogen and then heated to 50 - 65 °C for reaction for 1 - 8 hours. Then, the reaction was quenched by freezing at -80 °C and the residue was purified by inducing precipitation by adding n-hexane (repeated 5 times). The structure of the purified product was confirmed by 1H NMR and GPC.

[0098] Example 2 Characterization of block copolymers

[0099] 2.1 Characterization of block copolymer properties

[0100] The product obtained in Example 1 was characterized by GPC. The test conditions for gel permeation chromatography were as follows: mobile phase: 0.1 M triethylamine and 1% glacial acetic acid solution (HPLC grade); flow rate: 0.5 mL / min; temperature: 30 °C; gel chromatography column: TSKgel G5000PWXL-CP, 7.8 mm ID x 30 cm; this method measures the absolute molecular weight of the sample by refractive index and small-angle light scattering.

[0101] After purification of the final product and characterization by GPC, it was found that among compounds of the same type, the PDI size was affected by the type of counterion, showing Cl < Br ≈ I. This trend was manifested in both types of compounds with an unsubstituted main chain and a main chain with methyl substitution. The results are shown in Figures 3A - C. The data summary of Figures 3A - 3C is shown in Table 1 as follows:

[0102] Table 1

[0103] This example shows that the molecular weight distribution of the block copolymers described in this application is more uniform.

[0104] 2.2 Evaluation of the lubricating ability of block copolymers on different biological tissues.

[0105] This example illustrates that the block copolymer described in this application exhibits a successful lubricating effect on different material interfaces.

[0106] In order to evaluate the diblock copolymer as a synthetic lubricant, the tribological behavior of the copolymer was evaluated using an Anton Paar M302e rheometer and its T-PID / 44 tribology module. The tribology module uses cylindrical PDMS with a diameter of 6 mm and a smooth glass surface as the interface material. Under the boundary conditions of 25°C and a normal force of 5 N, the coefficient of friction (COF) was measured in PBS solution and a PBS solution containing the block copolymer (10 mg / ml) at different sliding speeds (1-100 mm / s). The results of the in vitro lubrication test showed that the block copolymer exhibited good lubrication at different speeds, reducing the COF by more than 60%, from the range of 0.756-1.155 to 0.316-0.398 (Figure 4).

[0107] Example 3 Intermediate cytotoxicity experiment

[0108] This example demonstrates that the block copolymers described herein have improved cytotoxicity and biocompatibility.

[0109] This example investigates the in vitro toxicity of intermediates containing different counterions to human articular chondrocytes (HC-a). Considering that these intermediates ultimately become important components of the final product, these results can, to some extent, reflect the toxicity trends of the final products with different structures.

[0110] The experimental operation process is as follows:

[0111] Logarithmic-phase HC-a cells were seeded into 96-well plates (4000 cells / well) and cultured at 37°C and 5% CO2 for 12 hours. Different concentrations of the intermediate (1 mg / ml, 5 mg / ml, and 20 mg / ml) were added and treated for 24 hours. Cell viability was then measured. After removing the cell supernatant, 100 μl of fresh DMEM medium containing 10% CCK8 was added to each well. After incubation at 37°C and 5% CO2 for 2 hours, the absorbance of each well was measured at 450 nm. The results are shown in Figure 5.

[0112] As shown in Figure 5, the A-Cl intermediate exhibited superior in vitro cellular safety compared to other halide-containing intermediates. In the 1 mg / ml control group, the cell viability of the A-Cl intermediate sample group far exceeded that of the A-Br / I intermediate sample group and was closer to the negative control group. When the concentration was increased to 5 mg / ml, although the cell viability of the A-Cl intermediate sample group decreased somewhat, it still statistically exceeded that of the other sample groups. The cell viability advantage of the A-Cl intermediate over the other samples did not disappear until the sample concentration was increased to 20 mg / ml.

[0113] Example 4 Stability Test

[0114] This example investigates the stability of different compounds under moist heat sterilization conditions.

[0115] The experimental procedure was as follows: First, six compounds with different counterions and backbone structures were dissolved in 1X PBS at a concentration of 30 mg / ml. Once fully dissolved, the samples were transferred to vials and sterilized with moist heat at 121°C for 15 minutes. After sterilization, the sample solutions were lyophilized and sent for NMR analysis. The proportion of hydrolysis products produced during the moist heat sterilization process was calculated by integrating the corresponding characteristic peaks.

[0116] The results are shown in Figure 6. When the backbone structure has methyl substitutions on the main chain, the hydrolysis product is significantly reduced compared to the structure without methyl substitutions on the main chain. This conclusion is also reflected in the three types of counterions Cl / Br / I. Therefore, the presence of methyl groups in the backbone structure can improve the stability of the block compound.

[0117] Example 5 Dry Eye Animal Model

[0118] Based on the good cytotoxicity results of A-Cl, this example further verified its potential therapeutic effect in a dry eye mouse model.

[0119] The experimental operation process is as follows:

[0120] This experiment used C57BL / 6J mice, and the animals had been acclimatized to the environment in the animal room for at least 5 days before the start of the experiment. After the start of the experiment (D1-D26), the modeled mice were placed in a low humidity environment (≤40% RH) and scopolamine hydrobromide (0.75mg / ml) was injected subcutaneously in the back of the neck twice a day, 0.3ml each time, with an injection interval of 6 hours. Starting from 5 days after modeling (D6-D26), the eye drops were administered twice a day for 21 days. The corneal fluorescence staining score is shown in Figure 7. It can be seen that A-Cl statistically significantly reduced the corneal fluorescence staining score from the 14th day, and there was no statistical difference with the healthy control group. The effect lasted at least until the 21st day. No obvious side effects or irritation were observed during the entire administration period, and the weight of the mice was not affected.

[0121] After the experiment, the eyeballs of the animals were removed, and the cornea and conjunctiva of each eye were collected and stored at -80°C. Protein content in the cornea and conjunctiva was then measured using the BCA assay, and the levels of various immune factors were measured using a multiplex protein detection kit (t-multiplex-m18h) suitable for immunoassays, as shown in Figure 8. The results showed that at least four immune factors were significantly reduced in the test group relative to the model control group, further validating the therapeutic efficacy of P002 in the dry eye model.

[0122] While there has been shown and described what are presently considered to be the preferred embodiments of the invention, various changes and modifications may be made by those skilled in the art which remain within the scope of the invention as defined by the appended claims.

Claims

1. A block copolymer having the following structure: in: R1 and R2 are each independently selected from hydrogen and methyl; X and X' are independently selected from -NR'-, -O- and a bond, wherein R' is selected from hydrogen atoms and having at least 1 and at most 6 carbon atoms; Y is selected from polyalkylene glycols, sugars and polyols; subscript a is an integer of at least 3, and subscript b is at least twice a; and Subscript c is an integer at least 1; Therein, according to the laws of chemistry, the block copolymer is terminated at each end by an end group, and the total positive charge of the quaternary ammonium groups in the copolymer is offset by the total negative charge of equal magnitude provided by the anions associated with the quaternary ammonium groups. 2 . The block copolymer according to claim 1 , wherein subscript b is an integer greater than 40. 3 .

3. The block copolymer according to any one of claims 1-2, wherein subscript b is an integer of at least 50. 4 . The block copolymer according to claim 1 , wherein subscript b is an integer of at least 100.

5. The block copolymer according to any one of claims 1 to 4, wherein subscript b is an integer of 200-400, and subscript a is an integer of 20-96. The block copolymer according to any one of claims 1 to 5, wherein subscript b is 400 and subscript a is 24.

7. The block copolymer according to any one of claims 1 to 6, wherein Y comprises a polyalkylene glycol.

8. The block copolymer of claim 7, wherein the polyalkylene glycol comprises polyethylene glycol.

9. The block copolymer of any one of claims 1-8, wherein the sugar comprises a monosaccharide.

10. The block copolymer according to any one of claims 1 to 9, wherein the sugar contains at least two monosaccharide units.

11. The block copolymer according to any one of claims 1 to 10, wherein the polyalkylene glycol, sugar or polyol consists of at least 2 and at most 500 monomer units.

12. The block copolymer according to any one of claims 1 to 11, wherein the polyalkylene glycol, sugar or polyol consists of at least 2 and at most 200 monomer units.

13. The block copolymer according to any one of claims 1 to 12, wherein the polyalkylene glycol, sugar or polyol consists of at least 2 and at most 100 monomer units.

14. The block copolymer according to any one of claims 1 to 13, wherein the polyalkylene glycol, sugar or polyol consists of at least 2 and at most 20 monomer units.

15. The block copolymer according to any one of claims 1 to 14, wherein at least one of the terminal groups is a thiol group.

16. A composition comprising the block copolymer of any one of claims 1 to 15.

17. The composition of claim 16, comprising one or more pharmaceutically acceptable carriers.

18. Use of the block copolymer according to any one of claims 1 to 15 and / or the composition according to any one of claims 16 to 17 for enhancing the lubricity of biological tissues or imparting a suitable level of lubricity to biological tissues.

19. Use of the block copolymer according to any one of claims 1 to 15 and / or the composition according to any one of claims 16 to 17 for treating dry eye.

20. Use of the block copolymer according to any one of claims 1 to 15 and / or the composition according to any one of claims 16 to 17 for preparing a drug for treating dry eye.

21. A method for imparting a suitable level of lubricity to a biological tissue, the method comprising contacting the biological tissue with the block copolymer according to any one of claims 1 to 15 and / or the composition according to claim 16 to impart a suitable level of lubricity.

22. The use according to claim 17 or the method according to claim 18, wherein the biological tissue is selected from the group consisting of joints, bones, eye tissue, nasal tissue, tendons, tendon bursae and vaginal tissue.

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