Methods for treating osteoarthritis

Abstract

This disclosure provides compositions and methods for treating osteoarthritis, such as osteoarthritis, in subjects. The methods include, for example, blocking the opening of Cx43 hemichannels in chondrocytes by using a composition comprising an anti-connexin 43 antibody. In some embodiments of this disclosure, methods for treating osteoarthritis in subjects requiring treatment of osteoarthritis are provided herein, comprising administering at least one dose of anti-connexin 43 (Cx43) antibody to the subject.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 511,563, filed on 30 June 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporating sequence lists by reference The sequence listing named 172628-201003_PCT_SL.xml, created on June 19, 2024, with a size of 34,860 bytes, is incorporated herein by reference in its entirety. [Background technology]

[0003] background Osteoarthritis (OA) is a common type of arthritis affecting approximately 20% of adults in the United States. Osteoarthritis is characterized by the degeneration of the joint, including the articular cartilage and subchondral bone, leading to the loss of articular cartilage, narrowing of the joint cavity, increased joint friction, and potential structural remodeling. Current treatments include exercise, lifestyle changes, and pain medication. In severe cases, joint replacement surgery is usually performed. However, most existing treatments fail to address the underlying cause of osteoarthritis (i.e., cartilage deterioration).

[0004] Chondrocytes express connexin Cx43 hemichannels, which mediate the passage of small molecules (less than 1.2 kDa) between the inside and outside of the cell. Hemichannels are normally closed, but are activated and opened under certain conditions, such as mechanical stress and inflammatory states. Open Cx43 hemichannels in chondrocytes promote an inflammatory response, accompanied by the release of pro-inflammatory factors, such as prostaglandin E2 (PGE2) and ATP. Inhibition of Cx43 hemichannel opening in chondrocytes (e.g., by chemical reagents) may suppress inflammation and the development of osteoarthritis.

[0005] Therefore, there is still a need for effective treatment of osteoarthritis and other pathological conditions associated with increased hemichannel activity. [Overview of the project] [Means for solving the problem]

[0006] overview In some embodiments of this disclosure, a method for treating osteoarthritis in a subject requiring treatment for osteoarthritis is provided herein, comprising administering at least one dose of anti-connexin 43 (Cx43) antibody to the subject. In some embodiments, the anti-Cx43 antibody comprises the following heavy chain CDR sequence and light chain CDR sequence: HCDR1: Sequence ID 1; HCDR2: Sequence ID 2; HCDR3: Sequence ID 3; LCDR1: Sequence ID 4; LCDR2: Sequence ID 5; and LCDR3: Sequence ID 6.

[0007] In some embodiments, the method includes administering the first dose of anti-Cx43 antibody on day 1 and the second dose on day 8. In some embodiments, the method further includes administering the third dose of anti-Cx43 antibody on day 15. In some embodiments, the method further includes administering the fourth dose of anti-Cx43 antibody on day 22. In some embodiments, the method further includes administering subsequent doses weekly after the fourth dose.

[0008] In other aspects of this disclosure, a method for treating osteoarthritis in a subject requiring treatment for osteoarthritis is provided herein, comprising administering an effective amount of anti-connexin 43 (Cx43) antibody to the subject, wherein the anti-Cx43 antibody is administered according to the following dosing schedule: i) First dose on day 1; ii) Second dose on day 8; iii) The third dose on day 15; and iv) The fourth dose on day 21.

[0009] In some embodiments, the method further includes administering subsequent doses of anti-Cx43 antibody weekly after the fourth dose.

[0010] In some embodiments, the anti-Cx43 antibody comprises the following heavy chain CDR sequence and light chain sequence: HCDR1: Sequence ID 1; HCDR2: Sequence ID 2; HCDR3: Sequence ID 3; LCDR1: Sequence ID 4; LCDR2: Sequence ID 5; and LCDR3: Sequence ID 6.

[0011] In some embodiments of any of the methods of this disclosure, the first, second, third, fourth and / or subsequent doses are approximately 0.01 mg / kg to approximately 100 mg / kg. In some embodiments, the first, second, third, fourth and / or subsequent doses are approximately 15 mg / kg. In some embodiments, the first, second, third, fourth and / or subsequent doses are approximately 25 mg / kg. In some embodiments, the first, second, third, fourth and / or subsequent doses are approximately 50 mg / kg.

[0012] In some embodiments of any of the methods disclosed herein, the anti-Cx43 antibody is administered intravenously.

[0013] In some embodiments of any of the methods of the present disclosure, at least one indicator of the severity of osteoarthritis is assessed at least 1 day to 1 week after each dose is administered. In some embodiments, at least one indicator of the severity of osteoarthritis consists of (a) arthritis pain threshold, (b) gait score, or (c) weight-bearing difference. In some embodiments, at least one indicator of the severity of osteoarthritis is improved after administration of at least one dose of the anti-Cx43 antibody.

[0014] In some embodiments of any of the methods of the present disclosure, the anti-Cx43 antibody comprises the heavy chain variable sequence of SEQ ID NO: 7 and / or the light chain variable sequence of SEQ ID NO: 8. In some embodiments of any of the methods of the present disclosure, the anti-Cx43 antibody comprises any one of the heavy chain sequences of SEQ ID NOs: 9 and 11-18, and / or the light chain sequence of SEQ ID NO: 10. In some embodiments, the anti-Cx43 antibody comprises the heavy chain sequence of SEQ ID NO: 9 and / or the light chain sequence of SEQ ID NO: 10.

[0015] In some embodiments of any of the methods of the present disclosure, the anti-Cx43 antibody blocks the opening of Cx43 hemichannels in a subject.

[0016] In some embodiments of any of the methods of the present disclosure, the subject is human. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] [Figure 1] FIG. 1 shows the effect of treatment with an anti-Cx43 antibody on the walking score (foot pressure score) of a rat model of osteoarthritis. The rat model of osteoarthritis was established by partial medial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT). The anti-Cx43 antibody was administered intravenously once a week for a total of 4 times starting on days 15, 22, 29, and 36 after establishment of the osteoarthritis model (corresponding to days 1, 8, 15, and 22 of treatment). Three different dosages of the anti-Cx43 antibody were tested (15 mg / kg, 25 mg / kg, and 50 mg / kg). Duloxetine was used as a control substance and was administered orally daily at a dose of 5 mg / kg. The walking score (foot pressure score) was measured on days 16, 23, 30, and 37 after establishment of the osteoarthritis model. Each data point corresponds to the mean walking score (mean ± standard error of the mean [SEM], n = 8). When used in the drawings: control, control rats; model, rat model of osteoarthritis (vehicle-treated); and AM1, anti-Cx43 antibody ALMB-0166.

[0018] [Figure 2]Figure 2 shows the effect of treatment with anti-Cx43 antibody on the pain threshold of plantar pain in a rat model of osteoarthritis. The rat model of osteoarthritis was established by pMMx+ACLT. Anti-Cx43 antibody was administered intravenously once a week for a total of four times, starting on days 15, 22, 29, and 36 after establishment of the osteoarthritis model (corresponding to days 1, 8, 15, and 22 of treatment). Three different doses of anti-Cx43 antibody were tested (15 mg / kg, 25 mg / kg, and 50 mg / kg). Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. The pain threshold of plantar pain was measured on days 1, 15, 16, 23, 30, and 37 after establishment of the osteoarthritis model. Each data point corresponds to the mean gait score (±SEM, n=8). When used in drawings: control, control rat; model, osteoarthritis rat model (vehicle-treated); AM1, anti-Cx43 antibody ALMB-0166; and 50% MWT, positive reaction with a 50% probability under stimulation intensity.

[0019] [Figure 3-1]Figures 3A–3D show the effect of anti-Cx43 antibody treatment on the pain threshold of plantar pain in a rat model of osteoarthritis. The rat model of osteoarthritis was established by pMMx+ACLT. Anti-Cx43 antibody was administered intravenously once a week for a total of four times, starting on days 15, 22, 29, and 36 (corresponding to days 1, 8, 15, and 22 of treatment) after the establishment of the osteoarthritis model. Three different doses of anti-Cx43 antibody were tested (15 mg / kg, 25 mg / kg, and 50 mg / kg). Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. The figures show the pain threshold of plantar pain on days 16 (Figure 3A), 23 (Figure 3B), 30 (Figure 3C), and 37 (Figure 3D) after the establishment of the osteoarthritis model. Each data point corresponds to the mean gait score (±SEM, n=8). When used in drawings: control, control rat; model, osteoarthritis rat model (vehicle-treated); AM1, anti-Cx43 antibody ALMB-0166; 50% MWT, positive response with 50% probability under stimulation intensity; ##, p<0.01 compared to control rat; *, p<0.05 compared to osteoarthritis model rat; **, p<0.01 compared to osteoarthritis model rat. [Figure 3-2] Same as above.

[0020] [Figure 4]Figure 4 shows the effect of treatment with anti-Cx43 antibody on differences in hindlimb weight-bearing in a rat model of osteoarthritis. The rat model of osteoarthritis was established by pMMx+ACLT. Anti-Cx43 antibody was administered intravenously once a week for a total of four times, starting on days 15, 22, 29, and 36 after establishment of the osteoarthritis model (corresponding to days 1, 8, 15, and 22 of treatment). Three different doses of anti-Cx43 antibody were tested (15 mg / kg, 25 mg / kg, and 50 mg / kg). Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. Differences in hindlimb weight-bearing were measured on days 1, 15, 16, 23, 30, and 37 after establishment of the osteoarthritis model. Each data point corresponds to the mean gait score (±SEM, n=8). When used in drawings: control, control rat; model, osteoarthritis rat model (vehicle-treated); and AM1, anti-Cx43 antibody ALMB-0166.

[0021] [Figure 5-1] Figures 5A–5D show the effect of anti-Cx43 antibody treatment on weight-bearing differences in a rat model of osteoarthritis. The rat model of osteoarthritis was established by pMMx+ACLT. Anti-Cx43 antibody was administered intravenously once a week for a total of four times, starting on days 15, 22, 29, and 36 (corresponding to days 1, 8, 15, and 22 of treatment) after the establishment of the osteoarthritis model. Three different doses of anti-Cx43 antibody were tested (15 mg / kg, 25 mg / kg, and 50 mg / kg). Duloxetine was used as a control and administered orally daily at a dose of 5 mg / kg. The figures show the differences in hindlimb weight-bearing on days 16 (Figure 5A), 23 (Figure 5B), 30 (Figure 5C), and 37 (Figure 5D) after the establishment of the osteoarthritis model. Each data point corresponds to the mean gait score (±SEM, n=8). When used in drawings: control, control rat; model, osteoarthritis rat model (vehicle-treated); AM1, anti-Cx43 antibody ALMB-0166;##, p<0.01 compared to control rat;**, p<0.01 compared to osteoarthritis model rat. [Figure 5-2] Same as above. [Modes for carrying out the invention]

[0022] Detailed explanation This disclosure provides methods and compositions for treating osteoarthritis in a subject or patent requiring treatment for osteoarthritis, comprising administering at least one dose of an anti-Cx43 antibody to the subject or patent. In some embodiments, the anti-Cx43 antibody blocks or inhibits the opening of a Cx43 hemichannel. In some embodiments, the anti-Cx43 antibody comprises a specific CDR amino acid sequence. In some embodiments, the anti-Cx43 antibody is administered according to a dosing regimen.

[0023] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms.

[0024] It should be understood that the present invention is not limited to and is therefore subject to change the specific methodologies, protocols, and reagents described herein. The terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention as defined solely by the claims.

[0025] As used herein, the articles “a,” “an,” and “the” are used to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, “element” means one element or more elements.

[0026] The use of options (e.g., "or") should be understood to mean one of the options, both, or any combination thereof.

[0027] The terms "and / or" should be understood to mean either one or both of the options.

[0028] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by approximately 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the terms “about” or “approximately” refer to a range of a quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0029] As used herein, the terms “substantially” or “essentially” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. In one embodiment, the terms “essentially the same” or “substantially the same” refer to a range of quantities, levels, values, numbers, frequency, percentage, dimensions, size, volume, weight, or length that is approximately the same as a reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0030] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” are understood to mean including the steps or elements or groups of steps or elements described, but not excluding other steps or elements or groups of steps or elements. In certain embodiments, the terms “include,” “has,” “contains,” and “comprise” are used synonymously.

[0031] "Consisting of" means that what follows the phrase is included and limited to it. Therefore, the phrase "consisting of" indicates that the listed elements are necessary or essential, and that other elements cannot exist.

[0032] "Consisting essentially of" means including any elements listed after the phrase, but limited to other elements that do not interfere with or contribute to the activity or action of the listed elements as specified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but the other elements are not optional and may or may not be present depending on whether or not they affect the activity or action of the listed elements.

[0033] The term “to provide” is used in accordance with its usual meaning to supply or furnish for use. In some embodiments, a protein (e.g., an antibody) is provided directly by administering the protein, while in other embodiments, a protein (e.g., an antibody) is provided effectively by administering the nucleic acid encoding the protein. In certain embodiments, the present invention envisions compositions comprising various combinations of nucleic acids, antigens, peptides, and / or epitopes.

[0034] Throughout this specification, any reference to “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, the occurrence of the aforementioned phrases in various places throughout this specification does not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0035] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to molecules having amino acid residues covalently linked by peptide bonds. A polypeptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids in a polypeptide. As used herein, these terms refer to both short chains, also commonly called peptides, oligopeptides, and oligomers in the art, and long chains, also commonly called polypeptides or proteins in the art. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural polypeptides, recombinant polypeptides, synthetic polypeptides, or combinations thereof.

[0036] As used herein, the terms “percent sequence identity” or “sequence identity” refer to the degree of identity between any given query sequence and the target sequence. The percentage of identity of any query nucleic acid or amino acid sequence to another target nucleic acid or amino acid sequence can be determined using tools and techniques known in the art, such as NCBI BLAST.

[0037] The term "pharmaceutical preparation" refers to a preparation containing a therapeutic agent (e.g., an anti-Cx43 antibody). It is in a form that enables the biological activity of the antibody to be effective and does not contain additional components that are unacceptably toxic to the subject to whom the preparation is administered.

[0038] As used herein, the term "antigen" is a molecule that can be bound by an antibody or a T cell receptor. In certain embodiments, binding moieties other than antibodies are engineered to specifically bind to an antigen, such as aptamers, avimers, and the like.

[0039] As used herein, the term "specifically binds" is not intended to indicate that an antibody binds exclusively to its intended target. Rather, an antibody "specifically binds" when its affinity for its intended target is about 5-fold greater when compared to its affinity for non-target molecules. Appropriately, there is no significant cross-reaction or cross-linking with unwanted substances. The affinity of an antibody is, for example, at least about 5-fold, such as 10-fold, such as 25-fold, particularly 50-fold, particularly 100-fold or more greater for the target molecule than for non-target molecules. In some embodiments, specific binding between an antibody or other binding agent and an antigen is at least 10 -1 , -1 , -1 , -1 , -1 , -1 , 9 , 9 , 8 , 11 , 10 , 10 M -1 means the binding affinity of. An antibody is, for example, at least about 10 7 M -1 , for example, about 10 8 M -1 ~ about 10 9 M -1 , about 10 9 M -1 ~ about 10 10 M -1 , or about 10 10 M -1 ~ about 10 11 M -1Antibodies can bind with affinity. Antibodies can bind, for example, with an EC of 50 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or more preferably 10 pM or less. As is well known in the art, various immunoassay formats can be used to select antibodies that are specifically immunoreactive to an antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive to a protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.

[0040] Affinity-matured antibodies have one or more changes in their hypervariable regions, resulting in improved antibody affinity to an antigen compared to parent antibodies that do not have these changes. In one embodiment, affinity-matured antibodies have nanomolar or even picomolar affinity to a target antigen. Affinity-matured antibodies are produced by procedures known in the art. Marks et al., Biotechnology, 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDRs and / or framework residues has been described by Barbas et al., Proc Nat. Acad. Sci, USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol., 226:889-896 (1992).

[0041] "Arthritis" is used herein to refer to any disorder affecting the joints. Symptoms generally include one or more of the following: joint pain, stiffness, tenderness, redness, warmth, swelling, and reduced range of motion of the affected joint. In some types of arthritis, other organs are also affected. The onset of arthritis in a subject may be gradual or sudden (e.g., resulting from injury or infection). Exemplary types of arthritis include, but are not limited to, osteoarthritis, rheumatoid arthritis, gout, septic arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and Still's disease. Arthritis may be a secondary disorder resulting from another primary disease, such as Lyme disease, systemic lupus erythematosus, or Ehlers-Danlos syndrome.

[0042] As used herein, “osteoarthritis” refers to a type of arthritis characterized by wear and tear of the cartilage of a joint. Osteoarthritis results from the deterioration of articular cartilage, which is composed of specialized cells known as chondrocytes. Chondrocytes produce a collagenous extracellular matrix containing collagen and proteoglycans. Loss or destruction of cartilage increases friction between the bones of the joint, which can lead to the growth of bony projections (osteophytes) or the release of cartilage and bone fragments into the joint cavity. Symptoms of osteoarthritis include joint pain, stiffness, reduced range of motion, joint swelling, and weakness or numbness in the arms and legs. Osteoarthritis can occur in any joint, but most commonly in the hands and fingers, wrists, knees, hips, feet, neck, and back. The onset of osteoarthritis may be due to age (e.g., due to wear and tear of cartilage over many years) or may be triggered by joint injury or infection.

[0043] The term "affinity" refers to the strength of the binding reaction between the antibody's binding domain and its epitope. This is the sum of the attractive and repulsive forces acting between the binding domain and the epitope. As used herein, the term affinity refers to the dissociation constant KD.

[0044] The term “epitope” includes any determinant, preferably polypeptide determinant, capable of specific binding to immunoglobulins or T cell receptors. In certain embodiments, epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl molecules, and in certain embodiments, may have specific three-dimensional structural properties and / or specific charge properties. In one embodiment, an epitope is a region of an antigen to which an antibody binds. In certain embodiments, an antibody is said to bind specifically to an antigen if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Methods for epitope mapping are well known in the art, including X-ray cocrystallography, array-based oligopeptide scanning, site-directed mutagenesis, high-throughput mutagenesis mapping, and hydrogen-deuterium exchange. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids juxtaposed by the tertiary folding of proteins. Epitopes formed from adjacent amino acids are typically retained against exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly five or eight to ten, amino acids in their unique spatial conformation.

[0045] In this specification, the terms “reduce,” “inhibit,” and “block” as used synonymously refer to any statistically significant reduction in biological activity (e.g., hemichannel opening). For example, “reduce” may refer to a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of biological activity.

[0046] II. Antibodies In this specification, the term “antibody” or “antibodies” is used in its broadest sense and specifically includes full-length antibodies, antibody peptides or immunoglobulins, monoclonal antibodies, chimeric antibodies, polyclonal antibodies, human antibodies, humanized antibodies, and antibodies derived from non-human species, including human antibodies derived from human germline immunoglobulin sequences transduced into non-human species such as mice, sheep, chickens, or goats, recombinant antigen-binding forms such as monobodies and diabodies, multispecific antibodies (e.g., bispecific antibodies), and individual antigen-binding fragments of antibodies or antibodies from which they are derived, including any of the aforementioned individual antigen-binding fragments, such as dAbs, Fv, scFv, Fab, F(ab)'2, Fab'.

[0047] Antibody-like conjugated peptide mimes are also intended to be prepared in the manner described herein. Liu et al. (2003) described “antibody-like conjugated peptide mimes” (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages such as a longer serum half-life and a less complicated synthesis method.

[0048] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the populations containing the individual antibodies are identical and / or bind to the same epitope. The modifier “monoclonal” indicates a characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0049] Antibody "antigen-binding fragments" preferably include at least the variable regions of the heavy and / or light chains of an anti-Cx43 antibody. For example, an antigen-binding fragment of an anti-Cx43 antibody may include the amino acid sequences of SEQ ID NOs. 7 and 8. Examples of such antigen-binding fragments include the Fab fragment, Fab' fragment, Fv fragment, scFv, and F(ab')2 fragment. Antibody antigen-binding fragments can be produced by enzymatic cleavage or recombinant techniques. For example, papain or pepsin cleavage may be used to produce the Fab or F(ab')2 fragment, respectively. Antibodies can also be produced in various truncated forms using antibody genes in which one or more stop codons are introduced upstream of the native stop site. For example, a recombinant construct encoding the heavy chain of the F(ab')2 fragment may be designed to include DNA sequences encoding the CH1 domain and hinge region of the heavy chain. In one embodiment, the antigen-binding fragment blocks or inhibits the opening of the Cx43 hemichannel and effects associated with the opening of the Cx43 hemichannel in a subject.

[0050] As used herein, “multispecific antibody” refers to an artificial antibody having two or more distinct parts, each part containing an antigen-binding region of the antibody against a different antigen or epitope. The parts of a multispecific antibody may be, for example, a full-length antibody or an antibody-binding fragment. A bispecific or bifunctional antibody is a multispecific antibody having two distinct parts (e.g., two different heavy and light chain pairs, or two different antibody-binding fragments) that bind to two different antigens or epitopes. Multispecific antibodies can be produced by various methods, including hybridoma fusion or ligation of antibody-binding fragments. For example, Songsivilai and Lachmann, Clin Exp Immunol, 79:315-21, 1990; Kostelny et al., J. Immunol., 148:1547-53 (1992). In some cases, a multispecific antibody (e.g., a bispecific antibody) comprises at least one portion of an anti-Cx43 antibody that blocks or inhibits the opening of Cx43 hemichannels and effects associated with Cx43 hemichannel opening in a subject.

[0051] A “therapeutic monoclonal antibody” is an antibody used for the treatment of human subjects. The therapeutic monoclonal antibodies disclosed herein include anti-Cx43 antibodies. Antibody “effector function” refers to the biological activity attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include C1q binding, complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downmodulation of cell surface receptors (e.g., B cell receptors; BCRs). To evaluate the ADCC activity of a molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337, may be performed.

[0052] The anti-Cx43 antibody of this disclosure may also be conjugated to at least one drug or moiety to form an antibody conjugate. The antibody may be linked to the drug by covalent or non-covalent bonds. The drug or moiety may include, but is not limited to, an effector or reporter molecule that can enhance the diagnostic or therapeutic potential of the antibody. The effector molecule may include a molecule having a desired activity (e.g., cytotoxic activity). Non-limited examples of effector molecules that may bind to the antibody include toxins, small molecule drugs, therapeutic enzymes, cytokines, antibiotics, radiolabeled nucleotides, etc. The reporter molecule may be a molecule that can be detected by assay and may include, but is not limited to, enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands (e.g., biotin).

[0053] Depending on the amino acid sequence of the heavy chain's constant domain, full-length antibodies can be assigned to different "classes." There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of antibodies are well known. The "light chains" of antibodies from any vertebrate species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0054] Parts of the present invention, such as polypeptides, peptides, antigens, or immunogens, may be covalently or noncovalently conjugated or linked to other parts, such as adjuvants, proteins, peptides, supports, fluorescent moieties, or labels. The terms “conjugate” or “immunoconjugate” are used broadly to define an effective association between one part and another drug and are not intended to refer only to any kind of effective association, and are not particularly limited to chemical “conjugations.”

[0055] As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody that is involved in antigen binding. The hypervariable region generally consists of amino acid residues derived from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain, and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or residues derived from the "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk This includes J.Mal.Biol.196:901-917(1987). A “framework region” or “FR” residue is a variable domain residue other than the hypervariable region residues as defined herein. The hypervariable region or its CDR can be transferred from one antibody chain to another antibody chain or another protein to confer antigen-binding specificity to the resulting (complex) antibody or binding protein.

[0056] As those skilled in the art will understand, the CDRs disclosed herein may also include variants. Generally, the amino acid identity between individual variant CDRs is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Thus, a “variant CDR” is one that has specific identity with the parent CDR of the present invention and shares biological functions including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and / or activity of the parent CDR.

[0057] Amino acid substitutions are typically single residues, and insertions are usually around 1 to 20 amino acid residues, although considerably larger insertions may be permitted. Deletions range from approximately 1 to 20 amino acid residues, but in some cases, deletions may be much larger.

[0058] Substitutions, deletions, insertions, or any combination thereof may be used to arrive at the final derivative or variant. Generally, these modifications are made to a small number of amino acids to minimize molecular changes, particularly to the immunogenicity and specificity of antigen-binding proteins. However, in certain circumstances, larger modifications may be acceptable.

[0059] As used herein, the terms “Fab” or “Fab region” mean polypeptides comprising the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this isolated region, or to this region in the context of a full-length antibody, antibody fragment, or Fab fusion protein, or any other antibody embodiment outlined herein.

[0060] As used herein, the terms “Fv,” “Fv fragment,” or “Fv region” refer to a polypeptide containing the VL and VH domains of a single antibody.

[0061] As used herein, the term "framework" refers to the region of the antibody variable domain excluding the region defined as the CDR. Each antibody variable domain framework may be further subdivided into adjacent regions separated by the CDR (FR1, FR2, FR3, and FR4).

[0062] The "humanization" of non-human (e.g., rodent) antibodies is a chimeric antibody containing the minimal sequence derived from the non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced with residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capabilities. In some cases, framework region (FR) residues of the human antibody are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in the recipient or donor antibody. These modifications are made to further improve the performance of the antibody. For further details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0063] "Isolated" antibodies are those identified, separated, and / or recovered from components of their natural environment. In certain embodiments, antibodies are purified to (1) more than 95% by weight of protein, or more than 99% by weight of protein, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies contain antibodies in insights within recombinant cells because at least one component of the antibody's natural environment is absent. However, isolated antibodies are typically prepared by at least one purification step.

[0064] Anti-Cx43 antibody Certain aspects of this disclosure provide compositions and methods for treating a target osteoarthritis. These methods and compositions include an anti-Cx43 antibody that specifically binds to a Cx43 hemichannel and blocks or inhibits its opening in chondrocytes.

[0065] The anti-Cx43 antibody may be any antibody known in the art that specifically binds to Cx43. In various embodiments, the anti-Cx43 antibody used in the method includes an HCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 1, an HCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 2, an HCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 3, and / or an LCDR1 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 4, an LCDR2 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 5, and an LCDR3 amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 6. In some specific embodiments, the anti-Cx43 antibody includes the same HCDR1 amino acid sequence as SEQ ID NO: 1, the same HCDR2 amino acid sequence as SEQ ID NO: 2, and the same HCDR3 amino acid sequence as SEQ ID NO: 3, and / or the same LCDR1 amino acid sequence as SEQ ID NO: 4, the same LCDR1 amino acid sequence as SEQ ID NO: 5, and the same LCDR3 amino acid sequence as SEQ ID NO: 6.

[0066] In various embodiments, the anti-Cx43 antibody used in this method comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 9 and 11-18, and / or a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 10. In some specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NOs: 9, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 10. In some other embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 11, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some other specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 12, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some further specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 13, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 10.In some further specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 14, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some further specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 15, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some further specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 16, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some further specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 17, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NO: 10. In some further specific embodiments, the anti-Cx43 antibody comprises a heavy chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 18, and a light chain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 10.

[0067] In some embodiments, the anti-Cx43 antibody includes the same heavy chain as SEQ ID NOs. 9 and any one of SEQ ID NOs. 11-18, and / or the same light chain as any one of SEQ ID NOs. 10. In preferred embodiments, the anti-Cx43 antibody includes the same heavy chain as SEQ ID NOs. 9 and the same light chain as any one of SEQ ID NOs. 10. In one embodiment, the anti-Cx43 antibody includes the same heavy chain as SEQ ID NOs. 11 and the same light chain as any one of SEQ ID NOs. 10. In another embodiment, the anti-Cx43 antibody includes the same heavy chain as SEQ ID NOs. 12 and the same light chain as any one of SEQ ID NOs. 10. In yet another embodiment, the anti-Cx43 antibody includes the same heavy chain as SEQ ID NOs. 13 and the same light chain as any one of SEQ ID NOs. 10. In yet another embodiment, the anti-Cx43 antibody includes the same heavy chain as SEQ ID NOs. 14 and the same light chain as any one of SEQ ID NOs. 10. In yet another embodiment, the anti-Cx43 antibody includes the same heavy chain as SEQ ID NOs. 15 and the same light chain as any one of SEQ ID NOs. 10. In yet another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 16 and the same light chain as any one of SEQ ID NO: 10. In yet another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 17 and the same light chain as any one of SEQ ID NO: 10. In yet another embodiment, the anti-Cx43 antibody comprises the same heavy chain as SEQ ID NO: 18 and the same light chain as any one of SEQ ID NO: 10.

[0068] In various embodiments, antibodies that bind to epitopes partially or completely located within the amino acid sequence of FLSRPTEKTI (SEQ ID NO: 19) are provided herein. In some embodiments, the epitope may comprise one or more amino acids selected from the group consisting of F1, S3, R4, P5, T6, E7, K8, T9, or I10 of SEQ ID NO: 19. In one embodiment, the epitope comprises F1, S3, R4, P5, T6, E7, K8, T9, and I10 of SEQ ID NO: 19. In some embodiments, the epitope may comprise all 10 amino acids of SEQ ID NO: 19. In certain embodiments, the epitope comprises all 10 amino acids of SEQ ID NO: 19.

[0069] The anti-Cx43 antibody is substantially pure and preferably substantially homogeneous (i.e., free from contaminating proteins, etc.). A “substantially pure” antibody means a composition containing at least about 90% by weight, at least about 95% by weight, or 97% by weight of the antibody, based on the total weight of the protein in the composition. A “substantially homogeneous” antibody means a composition containing a protein in which at least about 99% by weight of the protein is a specific antibody, such as an anti-Cx43 antibody, based on the total weight of the protein.

[0070] In some embodiments, the anti-Cx43 antibody is a humanized antibody. In some embodiments, the anti-Cx43 antibody is a monoclonal antibody. In some embodiments, the anti-Cx43 antibody is a humanized monoclonal antibody.

[0071] Pharmaceutical preparations containing anti-Cx43 antibodies One aspect of this disclosure provides a pharmaceutical formulation for treating a target osteoarthritis comprising an anti-Cx43 antibody. In some embodiments, the anti-Cx43 antibody blocks or inhibits the opening of Cx43 hemichannels in osteocytes. The anti-Cx43 antibody may be a complete antibody or its antigen-binding fragment.

[0072] As used herein, “pharmaceutically acceptable carrier” includes any solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic and absorption retardants, buffers, and other physiologically compatible excipients. Preferably, the carrier is suitable for parenteral, oral, or topical administration. Depending on the route of administration, the active compound, such as a small molecule or bioagent, may be coated onto the material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0073] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions, as well as conventional additives for the preparation of tablets, pills, capsules, and the like. The use of such media and agents for the formulation of pharmaceutically active substances is known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions provided herein is intended. Co-active compounds may also be incorporated into the compositions.

[0074] A pharmaceutically acceptable carrier may contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0075] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions provided herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, as well as injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, if necessary, by the use of coating materials such as lecithin, maintaining the required particle size in the case of dispersions, or by the use of surfactants such as polysorbates, sodium dodecyl sulfate, and nonionic surfactants. In many cases, it may be useful to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, or sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0076] These compositions may also contain functional additives such as preservatives, wetting agents, emulsifiers, and dispersants.

[0077] Therapeutic compositions are typically sterile, non-phylogenic, and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above, into a suitable solvent, and then by sterile means, such as microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method involves vacuum drying and lyophilization, thereby obtaining powders of the active ingredient and any additional desired components from its pre-sterile filtered solution. The activator can be mixed under sterile conditions with additional pharmaceutically acceptable carriers and any preservatives, buffers, or propellants as needed.

[0078] The prevention of microbial presence can be ensured by both the sterilization procedures described above and the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition. Furthermore, the inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin, can lead to longer absorption of the injectable pharmaceutical form.

[0079] The pharmaceutical compositions described herein may also have varying viscosities or osmotic pressures. Methods for measuring the viscosity of antibody preparations are known to those skilled in the art and may include, for example, a rheometer (e.g., an Anton Paar MCR301 Rheometer with any of 50 mm, 40 mm, or 20 mm plate accessories). Methods for measuring the volumetric osmolality of antibody preparations are known to those skilled in the art and may include, for example, an osmometer (e.g., a freezing point depression osmometer from Advanced Instrument Inc. 2020).

[0080] The pharmaceutical compositions described herein may also have various pH levels. The pH of the pharmaceutical compositions may be adjusted by any method known in the art, such as the addition of a buffer.

[0081] In some embodiments, the pharmaceutical composition comprises the anti-Cx43 antibody described herein, histidine / histidine hydrochloride buffer, polysorbate 80, and sucrose. In some embodiments, the pharmaceutical composition comprises about 40 mg / mL to about 60 mg / mL of the anti-Cx43 antibody described herein; about 10 mM to about 40 mM histidine / histidine hydrochloride buffer; about 0.005% w / v to about 0.05% w / v of polysorbate 80; and about 1% w / v to about 20% w / v of sucrose. In some embodiments, the pharmaceutical composition comprises about 50 mg / mL of the anti-Cx43 antibody described herein; about 20 mM histidine / aspartate buffer; about 0.02% w / v of polysorbate 80; and about 8% w / v of sucrose. In some embodiments, the pharmaceutical composition has a pH of about 5.4 to about 5.6. In some embodiments, the pharmaceutical composition has a pH of about 5.5.

[0082] For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions or unit dosage forms. As used herein, unit dosage forms refer to physically distinct units suitable as a unit dose for a patient being treated, each unit containing a predetermined amount of the active agent calculated to produce a desired therapeutic effect in combination with any necessary pharmaceutical carrier. The specifications of a unit dosage form are determined and directly depend on (a) the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) limitations inherent in the art for formulating such an active compound for the treatment of susceptibility in an individual.

[0083] The actual dosage levels of the active ingredients in the pharmaceutical compositions disclosed herein may vary to obtain an amount of the active ingredient that is not toxic to the patient and is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration. As used herein in the context of administration, “parenteral” typically means modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0084] As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral (i.e., via the gastrointestinal tract) and topical administration, typically by injection or infusion, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. Intravenous injections and infusions are frequently (but not limited to) used for antibody administration.

[0085] The formulations described herein are administered to subjects requiring treatment with anti-Cx43 antibodies, preferably humans, by known methods, for example, by intramuscular, intradermal, intraperitoneal, intracerebral, subcutaneous, intra-articular, bursal, intrathecal, oral, topical, or inhalation routes, for example, by intravenous administration as a bolus or by continuous infusion over a period of time.

[0086] In some embodiments, anti-Cx43 antibodies are administered to subjects by intravenous or subcutaneous (i.e., under the skin) administration. For such purposes, the formulation may be injected using a syringe. However, other devices are available for administering the formulation, such as injection devices (e.g., INJECT-EASE® and GENJECT® devices); pen-type injectors (e.g., GENPEN®); automated injection devices, needleless devices (e.g., MEDIJECTOR® and BIOJECTOR®); and subcutaneous patch delivery systems.

[0087] In certain embodiments, the disclosure relates to a kit for a single-dose dosing unit. Such a kit includes a container for an aqueous formulation of a therapeutic protein or antibody, which may include either a single-chamber or multi-chamber pre-filled syringe. An example of a pre-filled syringe is available from Vetter GmbH in Ravensburg, Germany.

[0088] III. Treatment methods for osteoarthritis Certain aspects of this disclosure provide a method for treating osteoarthritis in a subject. The method may include, for example, blocking the opening of Cx43 hemichannels in osteocytes in a subject where such blocking is required, by using an anti-Cx43 antibody. Cx43 modulation may be a standalone treatment for osteoarthritis or may be combined with other treatments. Osteoarthritis may affect any joint in a subject, such as the hands and fingers, wrists, knees, hips, feet, neck, and back.

[0089] Osteoarthritis is a common type of arthritis that causes degeneration of the joints, including the articular cartilage and subchondral bone. The pathophysiology of osteoarthritis is characterized by narrowing of the joint cavity, increased joint friction, and loss of articular cartilage, leading to potential structural remodeling. Current treatment options for osteoarthritis include exercise, lifestyle changes, pain medication, and, in severe cases, joint replacement. However, there is no specific pharmaceutical intervention for the treatment of osteoarthritis.

[0090] Various cells can communicate with each other and with the extracellular environment via hemichannels and gap junctions formed by the protein connexin. Connexin proteins are ubiquitously expressed throughout the body. Six connexin proteins constitute one hemichannel, and two hemichannels constitute one gap junction channel. Gap junctions are clusters of channels located in the plasma membrane between adjacent cells, and they mediate intercellular communication. Hemichannels are a distinct entity from gap junction channels. Hemichannels enable the exchange of molecules between intracellular compartments and the extracellular environment.

[0091] Chondrocytes express hemichannels known as connexin (Cx) 43 hemichannels. Under normal conditions, the Cx43 hemichannels in chondrocytes remain closed. Inflammatory states and mechanical stimuli can induce the opening of the Cx43 hemichannels, leading to the release of pro-inflammatory factors that contribute to inflammation associated with osteoarthritis. Therefore, inhibiting the opening of the Cx43 hemichannels in chondrocytes (e.g., using anti-Cx43 antibodies) may suppress the onset and progression of osteoarthritis in subjects.

[0092] Cx43, also known as gap junction alpha-1 protein (GJA1), is a 43.0 kDa protein composed of 382 amino acids (NCBI reference sequence: NP 000156.1). GJA1 contains a long C-terminal tail, an N-terminal domain, and multiple transmembrane domains. The protein passes through the phospholipid bilayer four times, leaving its C-terminus and N-terminus exposed to the cytoplasm. The C-terminal tail consists of 50 amino acids and contains post-translational modification sites, as well as binding sites for transcription factors, cytoskeletal elements, and other proteins. As a result, the C-terminal tail is central to functions such as pH gating and channel assembly regulation. In particular, the DNA region of the GJA1 gene (NCBI gene ID: 2697) encoding this tail is highly conserved, indicating that it is resistant to mutations or becomes lethal if mutated. The N-terminal domain, on the other hand, is involved in channel gating and oligomerization and can therefore control the switching between the open and closed states of channels. The transmembrane domain forms a gap junction channel, and the extracellular loop facilitates proper channel docking. Furthermore, the two extracellular loops interact with the two hexamers to form a disulfide bond that completes the gap junction channel.

[0093] "Treatment" refers to a therapeutic treatment. Those requiring treatment include individuals who already have a disease. Therefore, subjects treated herein, such as humans, may have been diagnosed with a disease, such as osteoarthritis. A disease, such as osteoarthritis, is "inhibited" or "treated" if at least one symptom of the condition (determined by responsive / unresponsiveness, or by indicators known in the art and described herein) is alleviated, terminated, slowed, minimized, or prevented. The terms "patient" and "subject" are used synonymously herein.

[0094] The terms "subject" or "patient" refer to human or non-human animals, such as primates, mammals, and vertebrates. In certain embodiments, the subject is human.

[0095] Treatment may be appropriately administered to subjects, particularly humans, who have, are suffering from, are susceptible to, or are at risk of developing osteoarthritis. The determination of these “at-risk” subjects may be made by diagnostic tests or by any objective or subjective determination based on the opinion of the subject or healthcare provider. Identification of subjects requiring such treatment may be at the discretion of the subject or healthcare professional and may be subjective (e.g., opinion) or objective (e.g., measurable by tests or diagnostic methods).

[0096] Anti-Cx43 antibodies for use in any of the methods disclosed herein may be stored as lyophilized solids, aqueous formulations, or any other form known in the art. In the case of anti-Cx43 antibodies stored as lyophilized solids, the antibody is reconstituted in a solution such as water (e.g., for injection) before administration. When prepared for injection from either the lyophilized form or an aqueous formulation, for example, the final concentration of the reconstituted anti-Cx43 antibody (e.g., in saline, Ringer, or a 5% dextrose infusion system) after dilution may be about 0.1 mg / ml to about 80 mg / ml for administration. The final concentrations may be about 0.1 mg / ml to about 80 mg / ml, about 0.5 mg / ml to about 70 mg / ml, about 1 mg / ml to about 60 mg / ml, about 5 mg / ml to about 50 mg / ml, about 10 mg / ml to about 40 mg / ml, about 15 mg / ml to about 30 mg / ml, or about 20 mg / ml to about 25 mg / ml. In some embodiments, the final dosage form may have concentrations of approximately 0.1 mg / ml, approximately 0.5 mg / ml, approximately 1 mg / ml, approximately 2 mg / ml, approximately 3 mg / ml, approximately 4 mg / ml, approximately 5 mg / ml, approximately 10 mg / ml, approximately 15 mg / ml, approximately 20 mg / ml, approximately 25 mg / ml, approximately 30 mg / ml, approximately 35 mg / ml, approximately 40 mg / ml, approximately 45 mg / ml, approximately 50 mg / ml, approximately 55 mg / ml, approximately 60 mg / ml, approximately 65 mg / ml, approximately 70 mg / ml, approximately 75 mg / ml, approximately 80 mg / ml, or greater than 80 mg / ml.

[0097] As used herein, the term “effective dose” refers to the amount of a drug, such as an anti-Cx43 antibody, that, when administered to a patient or subject, is sufficient to treat, prognose, or diagnose osteoarthritis. The dosing regimen may be adjusted to provide an optimal therapeutic response. The effective dose is also the amount at which any toxic or adverse effects (side effects) of the drug are minimized and / or the beneficial effects outweigh them. The therapeutically effective dose varies depending on the patient and disease state being treated, the patient’s weight and age, the severity of the disease state, the method of administration, the course of the condition, the patient’s medical history and response to the anti-Cx43 antibody, etc., which can be readily determined by those skilled in the art. For example, an effective dose or one dose of anti-Cx43 antibody ranges from about 0.01 mg / kg to about 1000 mg / kg. In some embodiments, the effective amount or dose of anti-Cx43 antibody is approximately 0.01 mg / kg to approximately 900 mg / kg, approximately 0.1 mg / kg to approximately 800 mg / kg, approximately 0.5 mg / kg to approximately 700 mg / kg, approximately 1 mg / kg to approximately 600 mg / kg, approximately 1.5 mg / kg to approximately 500 mg / kg, approximately 2 mg / kg to approximately 400 mg / kg, approximately 5 mg / kg to approximately 300 mg / kg, approximately 10 mg / kg to approximately 200 mg / kg, approximately 15 mg / kg to approximately 100 mg / kg, approximately 20 mg / kg to approximately 50 mg / kg, approximately 25 mg / kg to approximately 45 mg / kg, or approximately 30 mg / kg to approximately 40 mg / kg. In some embodiments, the effective dose or amount of anti-Cx43 antibody is approximately 0.01 mg / kg, approximately 0.1 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 5 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 20 mg / kg, approximately 25 mg / kg, approximately 30 mg / kg, approximately 35 mg / kg, approximately 40 mg / kg, approximately 45 mg / kg, approximately 50 mg / kg, and approximately 55 mg / kg. Approximately 60 mg / kg, approximately 65 mg / kg, approximately 70 mg / kg, approximately 80 mg / kg, approximately 90 mg / kg, approximately 100 mg / kg, approximately 200 mg / kg, approximately 300 mg / kg, approximately 400 mg / kg, approximately 500 mg / kg, approximately 600 mg / kg, approximately 700 mg / kg, approximately 800 mg / kg, approximately 900 mg / kg, approximately 1000 mg / kg, or more than 1000 mg / kg.

[0098] In specific embodiments, the effective dose or one dose of anti-Cx43 antibody is approximately 0.01 mg / kg to approximately 100 mg / kg. In some embodiments, the effective dose or one dose of anti-Cx43 antibody is 15 mg / kg. In some embodiments, the effective dose or one dose of anti-Cx43 antibody is 25 mg / kg. In some embodiments, the effective dose or one dose of anti-Cx43 antibody is 50 mg / kg.

[0099] In some embodiments, an effective amount or dose of anti-Cx43 antibody may range from about 1 mg to about 800 mg. In some embodiments, the effective amount or dose of anti-Cx43 antibody is about 2 mg to 7000 mg, about 5 mg to 6000 mg, about 10 mg to 5000 mg, about 15 mg to 4000 mg, about 20 mg to 3000 mg, about 25 mg to 2000 mg, about 30 mg to 1000 mg, about 40 mg to 900 mg, about 50 mg to 800 mg, about 60 mg to 700 mg, about 70 mg to 600 mg, about 80 mg to 500 mg, about 90 mg to 400 mg, about 100 mg to 300 mg, or about 150 mg to 250 mg. In some embodiments, the effective amount or dose of anti-Cx43 antibody is less than 1 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 5 50 mg, approximately 600 mg, approximately 650 mg, approximately 700 mg, approximately 750 mg, approximately 800 mg, approximately 850 mg, approximately 900 mg, approximately 950 mg, approximately 1000 mg, approximately 1250 mg, approximately 1500 mg, approximately 1750 mg, approximately 2000 mg, approximately 2250 mg, approximately 2500 mg, approximately 2750 mg, approximately 3000 mg, approximately 4000 mg, approximately 5000 mg, approximately 6000 mg, approximately 7000 mg, approximately 8000 mg, or more than 8000 mg.

[0100] In the methods described herein, an effective dose or at least one dose of anti-Cx43 antibody is administered approximately once daily, approximately every two days, approximately every three days, approximately every four days, approximately every five days, approximately every six days, approximately every week, approximately every eight days, approximately every nine days, approximately every ten days, approximately every eleven days, approximately every twelve days, approximately every thirteen days, and approximately every two weeks. Approximately once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 3 weeks, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 4 weeks, once every 29 days, once every 30 days, once every 31 days, The drug is administered approximately every 32 days, every 33 days, every 34 days, every 5 weeks, every 36 days, every 37 days, every 38 days, every 39 days, every 40 days, or every 41 days, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks or every 6 months, or every 24 weeks or every 6 months.

[0101] In some specific embodiments, an effective dose or one dose of anti-Cx43 antibody is administered approximately once a week. In one particular embodiment, one dose of anti-Cx43 antibody is administered once every week. In another specific embodiment, one dose of anti-Cx43 antibody is administered once a week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or longer.

[0102] According to certain embodiments of the present disclosure, multiple doses of anti-Cx43 antibody (or a pharmaceutical composition comprising a combination of anti-Cx43 antibody and any additional therapeutic activators as described herein) may be administered to a subject over a defined time course. A method according to this aspect of the present disclosure includes sequentially administering multiple doses of the anti-Cx43 antibody of the present disclosure to a subject.

[0103] As used herein, “sequential administration” means that each dose of anti-Cx43 antibody is administered to a subject at different times, for example, on different days separated by a predetermined interval (e.g., several hours, several days, several weeks, or several months). This disclosure includes a method comprising sequentially administering to a patient a single initial dose of anti-Cx43 antibody, followed by a second dose, followed by a third dose, followed by a fourth dose, followed by one or more subsequent doses of anti-Cx43 antibody as needed. Anti-Cx43 antibody may be administered in doses ranging from 0.01 mg / kg to approximately 1000 mg / kg.

[0104] The terms “first dose,” “second dose,” “third dose,” and “fourth dose” refer to the chronological order of administration of the anti-Cx43 antibody in this disclosure. Thus, the “first dose” is the dose administered at the start of the treatment regimen (also called the “baseline dose”); the “second dose” is the dose administered after the first dose; the “third dose” is the dose administered after the second dose; and the “fourth dose” is the dose administered after the third dose. The first, second, third, and fourth doses may all contain the same amount of anti-Cx43 antibody, but in general they may differ from one another in terms of the frequency of administration. However, in certain embodiments, the amounts of anti-Cx43 antibody contained in the first, second, third, and fourth doses and / or doses after the fourth dose may differ from one another during the course of treatment (e.g., adjusted up or down as needed).

[0105] In some embodiments of the methods disclosed herein, the method includes administering a first dose and a second dose of anti-Cx43 antibody. In some embodiments, the first dose of anti-Cx43 antibody is administered on day 1 of treatment. In some embodiments, the second dose of anti-Cx43 antibody is administered on day 8 of treatment. In some embodiments, the method disclosed herein includes administering a third dose of anti-Cx43 antibody. In some embodiments, the third dose of anti-Cx43 antibody is administered on day 15 of treatment. In some embodiments, the method further includes administering a fourth dose of anti-Cx43 antibody. In some embodiments, the fourth dose of anti-Cx43 antibody is administered on day 22 of treatment. In some embodiments, the method further includes administering subsequent doses of anti-Cx43 antibody after the fourth dose. In some embodiments, subsequent doses of anti-Cx43 antibody are administered weekly after the fourth dose.

[0106] In some embodiments of the method of the present disclosure, the method comprises administering an effective amount of anti-Cx43 antibody according to the following dose regimen: a first dose on day 1 of treatment, a second dose on day 8 of treatment, a third dose on day 15 of treatment, and a fourth dose on day 22 of treatment. In some embodiments, the method further comprises administering subsequent doses of anti-Cx43 antibody weekly after the fourth dose.

[0107] An effective dose or at least one dose of anti-Cx43 antibody may be administered to the patient at any time after diagnosis, or at any point in time after diagnosis. Anti-Cx43 antibody may be administered as a sole treatment or in combination with other drugs or treatments useful for the treatment of osteoarthritis.

[0108] In the methods described herein, an effective amount or one dose of anti-Cx43 antibody may be administered to a patient over a period of less than 5 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 150 minutes, about 180 minutes, or more than 180 minutes. In some specific embodiments, an effective amount or dose of anti-Cx43 antibody may be administered to a patient over a period of about 30 minutes. In a particular embodiment, an effective amount or dose of anti-Cx43 antibody may be administered intravenously to a patient over a period of 30 minutes.

[0109] The methods described herein involve administering an anti-Cx43 antibody to a patient. If the anti-Cx43 antibody is in a solid, for example, dry formulation, the administration process may include a step of converting the formulation into a liquid. In one embodiment, the dry formulation may be reconstituted with a liquid, for example, as described above, for use by injection, for example intravenous, intradermal, intramuscular, intraperitoneal, or subcutaneous injection. In another embodiment, the solid or dry formulation may be administered topically, for example, as a patch, cream, aerosol, or suppository.

[0110] In the methods described herein, the anti-Cx43 antibody may be administered to the subject alone or in combination with another treatment. The anti-Cx43 antibody may be administered before, with, or after the administration of additional treatment. In one embodiment, the dose of the co-administered treatment may be reduced over time or tapered off completely during the treatment period with the anti-Cx43 antibody. In some embodiments, the additional treatment may include one or more of the following: physiotherapy, adjuvant agents, steroids, surgery (e.g., joint replacement), cell-based therapy, or small molecule drugs.

[0111] In one embodiment, the improved efficacy of the combination according to this disclosure may be demonstrated by achieving a therapeutic synergy. The terms “therapeutic synergy” or “synergistic effect” are used when a combination of two products at a given dose is more effective than the best of each of the two products at the same dose. For example, therapeutic synergy may be assessed by comparing the combination to the best single agent using estimates obtained from a two-way analysis of variance with repeated measures (e.g., time factor) for a severity parameter of osteoarthritis.

[0112] Patients or subjects are examined for osteoarthritis-related parameters using techniques and methods known in the art before, during, or after treatment with anti-Cx43 antibodies. Non-limiting examples of common medical techniques and methods used to examine and diagnose osteoarthritis include physical examination by a physician, physical fitness tests, blood tests, X-rays, magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, the Western University of Ontario and McMaster University Arthritis and Osteoarthritis (WOMAC) index, the Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire, quality of life (QOL) assessments (e.g., using the SF-12 scale or the Quality of Life Assessment-6D or "AQoL-6D" method), the Patient's General Change Impression (PGIC) questionnaire, maximum voluntary isometric strength measurement (e.g., using a dynamometer), dynamic weight-bearing (DMB) tests, observational gait scores (OGS), and numerical rating scales for assessing pain levels. The selection of techniques and methods, as well as the frequency of examinations, may be determined and / or adjusted by those skilled in the art based on the specific condition of the patient or subject.

[0113] In some embodiments, subjects are examined for improvement in osteoarthritis, assessed by at least one index of osteoarthritis severity, before, during, or after treatment with anti-Cx43 antibody. In some embodiments, at least one index of osteoarthritis severity is assessed at least 1 day to 1 week after an effective dose or 1 dose of anti-Cx43 antibody is administered to the subject. In some embodiments, at least one index of osteoarthritis severity is assessed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week after an effective dose or 1 dose of anti-Cx43 antibody is administered to the subject. In some embodiments, at least one index of osteoarthritis severity includes differences in arthritis pain threshold, gait score, and / or weight-bearing.

[0114] In some embodiments, treatment with anti-Cx43 antibodies for osteoarthritis improves disease symptoms, such as differences in arthritis pain threshold, gait score, and / or weight-bearing, compared to a control population that may include, but is not limited to, osteoarthritis patients who receive or fail the same series of treatments as the subjects but do not receive anti-Cx43 antibodies for osteoarthritis.

[0115] In some embodiments, subjects treated with an anti-Cx43 antibody for osteoarthritis have a response period of at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 1 month, at least 2 months, at least 3 months or longer.

[0116] In some embodiments, subjects treated with anti-Cx43 antibodies for osteoarthritis have response times of less than 3 months, less than 2 months, less than 1 month, less than 25 days, less than 20 days, less than 15 days, less than 10 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day.

[0117] As used herein, “pain threshold” refers to the lowest intensity at which a given stimulus is perceived as painful and is relatively constant across individuals in response to a given stimulus. Injury or disease (e.g., osteoarthritis) may result in a decrease in the pain threshold of an object at a particular location (e.g., a limb). The pain threshold may be a mechanical pain threshold or a pressure pain threshold and may be measured locally (e.g., at the affected joint) or remotely (e.g., on an adjacent muscle) by any suitable method known in the art.

[0118] In some embodiments, treatment of a subject having osteoarthritis with at least one dose of anti-Cx43 antibody improves the arthritis pain threshold in the subject. In some embodiments, treatment of a subject having osteoarthritis with at least one dose of anti-Cx43 antibody increases the arthritis pain threshold by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more.

[0119] As used herein, “gait” refers to the manner in which a limb moves during locomotion. The gait of a subject may be influenced by the presence of a disease or impairment affecting locomotion (e.g., osteoarthritis). “Gait score” refers to any measure or scoring system known in the art for evaluating and analyzing the gait of a subject. Depending on the gait scoring system used, an improvement in the subject's gait may be reflected as an increase or decrease in the subject's gait score.

[0120] In some embodiments, treatment of subjects with osteoarthritis with an anti-Cx43 antibody improves the subjects' walking score. In some embodiments, treatment of subjects with osteoarthritis with at least one dose of an anti-Cx43 antibody improves the subjects' walking score by 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.2, at least 1.5, at least 1.8, at least 2, at least 2.2, at least 2.5, at least 3, at least 3.5, or more. In some embodiments, treatment of a subject with osteoarthritis with at least one dose of anti-Cx43 antibody improves the subject's walking score by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more.

[0121] As used herein, “weight-bearing” refers to the amount of weight or force that a particular limb can bear. Weight-bearing can be determined by any method known in the art. A decrease in weight-bearing may be observed in cases of injury or disease (e.g., osteoarthritis) and / or may be recommended by a physician to reduce the risk of injury or pain to the subject. As used herein, “difference in weight-bearing” refers to the difference in weight-bearing of a limb during an injury or disease (e.g., osteoarthritis) compared to the weight-bearing of the corresponding limb without injury or disease. A decrease in the difference in weight-bearing indicates that the weight-bearing of the limb is close to its total body weight.

[0122] In some embodiments, treatment of subjects with osteoarthritis with an anti-Cx43 antibody improves the weight-bearing difference of the subjects. In some embodiments, treatment of subjects with osteoarthritis with at least one dose of an anti-Cx43 antibody reduces the weight-bearing difference of the subjects by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%, or more.

[0123] As used herein, “response” or “responsiveness” to a treatment refers to a subject having an improvement in at least one parameter of disease progression. A subject may have a partial or complete response to a treatment. Response to a treatment may be determined based on methods known in the art. Those skilled in the art may determine an appropriate method based on the type of disease being evaluated. Non-limiting examples of methods include arthritis pain thresholds, gait scores, and weight-bearing differences, as well as other methods described herein. [Examples]

[0124] Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to function well in carrying out the present invention and, therefore, may constitute a preferred mode for its implementation. However, those skilled in the art should understand that, in light of this disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention, and similar or comparable results can still be obtained.

[0125] Example 1. Effect of treatment with anti-Cx43 antibody on a rat model of osteoarthritis. This example describes the evaluation of the effect of anti-Cx43 treatment in a rat model of osteoarthritis.

[0126] Research objectives The purpose of this study was to investigate the effect of the anti-Cx43 antibody ALMB-0166 on the symptoms of osteoarthritis in a rat model of osteoarthritis caused by partial meniscectomy and anterior cruciate ligament resection (pMMx+ACLT) when administered intravenously once a week for four weeks.

[0127] Regulatory Compliance The experiment followed standard operating procedures (SOPs).

[0128] material and method Test materials The anti-Cx43 antibody used in the study was ALMB-0166 (AlaMab Therapeutics Inc.). Duloxetine hydrochloride (MedChemExpress) was used as a positive control. Other materials used in the study included isoflurane (RWD Life Science, Inc.), phosphate-buffered saline (Thermo), and normal saline (Anhui Shuanghe Pharmaceutical Co., Ltd.).

[0129] For administration to animals, 0.5 mg / mL of duloxetine hydrochloride was prepared by dissolving the drug in physiological saline, and two ALMB-0166 solutions, one at 3 mg / mL and the other at 5 mg / mL, were prepared by diluting the antibody stock solution with PBS.

[0130] device The equipment used in this study included a non-rebreathing anesthesia machine (Midmark Corporation, Model VWR), a Von Frey filament (IITC, Life Science, USA), and a Linton incapacitance tester (UK).

[0131] Animals and breeding The study used male Sprague Dolly (SD) rats (specified pathogen-free grade; Shanghai Slac Laboratory Animal Co. Ltd). Each rat's weight was at the start of treatment, with an average weight ranging from 200 to 220 grams. Upon arrival at the laboratory, the animals were housed in cages of five rats each and acclimatized for 5 to 7 days before the start of treatment.

[0132] The animals were kept in an environment set to maintain a temperature of 23±2°C, humidity of 40-70%, and a 12-hour light / 12-hour dark cycle. The 12-hour dark cycle was temporarily interrupted to accommodate the research procedure. SPF Rat Growth Breeding Feed (Beijing Keao Xieli Feed Co., Ltd) was freely available throughout the study's lifespan. Reverse osmosis water was freely available. The animals had free access to both food and water throughout the entire study.

[0133] The animals used in the study were selected based on their overall health and adaptation to the cage, with a total of 48 rats chosen to participate in the study.

[0134] research design Animal groups and drugs For the study, animal groups were formed as shown in Table 1 below. Each procedure was initiated one week after partial medial meniscectomy in combination with anterior cruciate ligament amputation (pMMx+ACLT) in rats. [Table 1]

[0135] Models and Methods After acclimatizing the animals, they were assessed for normal pain threshold (day 1 of the study) using the Von Frey or lift-and-drop method, and the bipedal balance degree of the surgical knee joint (right hind limb) was assessed using a bipedal balance tester. The rats were then anesthetized with isoflurane, and the knee joints were dehaired and disinfected. Osteoarthritis was induced in the animals by partial medial meniscectomy combined with anterior cruciate ligament amputation (pMMx+ACLT). Briefly, the skin and joint cavity near the medial side of the right knee were cut, the knee joint was placed in maximum flexion, the joint cavity was exposed, and the medial meniscus was separated. The medial 1 / 3 meniscus and anterior cruciate ligament were then cut. For the control group (no osteoarthritis induction), only the skin was cut to open the joint cavity without removing any ligaments or menisci. The establishment date of the osteoarthritis model was day 8 of the study.

[0136] One week after model establishment (day 15 of the study), prior to treatment, the basic pain threshold for plantar pain was assessed on the surgical side for each animal. Eight animals were then evenly divided into osteoarthritis model rats according to their basic Von Frey response score (2-5) into three groups: the osteoarthritis model group (vehicle treatment), the positive control drug (duloxetine) 5 mg / kg group, and the ALMB-0166 15 mg / kg, 25 mg / kg, and 50 mg / kg dose groups. After grouping, drug treatment was initiated on day 15 of the study. For each of the ALMB-0166 treatment groups, the treatment was administered intravenously once a week at a total of four doses (corresponding to days 15, 22, 29, and 36 of the study, and days 1, 8, 15, and 22 of the treatment). For the positive control group, duloxetine was administered orally daily until the end of the experimental cycle.

[0137] Body weight was measured for each animal in the study on days 15, 22, 29, and 36. On days 16, 23, 30, and 37, gait, changes in plantar pain threshold induced by the knee joint lesion on the surgical side, and weight-bearing on both lower limbs were measured for each animal and scored according to the Coderre scoring method, the lift-and-drop method, and the bipedal balance pain test, respectively.

[0138] Coderre scoring method The Coderre gait test was used to measure the gait of each animal. Gait was measured according to the following scoring system: Standard (Grade 0: normal gait, 0 points); Level 1 (sight claudication, slight flexion below the knee, 2 points); Level 2 (lameness, affected limb can touch the ground, 4 points); Level 3 (severe lameness, affected limb is off the ground and the animal walks on three legs, 6 points).

[0139] Measurement of mechanical pain threshold The lift-and-drop method was used to measure the plantar pain threshold. Following the lift-and-drop method, each rat was placed in a transparent enclosure with a sieve-like metal plate at the bottom, and the animals were allowed to adapt for approximately 15 minutes until autonomous exploring and grooming activities ceased. Then, using a Von Frey probe, the center of the hind plantar of each rat was stimulated vertically to slightly S-shape the plantar for 5 seconds, and the paw retraction response was observed. A positive response was observed if the paw was retracted abruptly or flinched upon removal. The interval between each measurement was approximately 10 seconds. If no response was observed after probe stimulation, it was considered a negative response.

[0140] For mechanical pain scoring, rats were stimulated with a series of Von Frey probes of 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, and 15 g. The test began with a 2 g probe, and if a positive response occurred, it was recorded as "X," and the test continued with a 1.4 g probe. If no positive response was observed with the 2 g probe, it was recorded as "O," and the test continued with a 4 g probe. Accordingly, the test continued using probes continuously without avoidance. Each response was recorded, and if "OX" or "XO" appeared for the first time, it was tested four times to obtain a sequence such as "OXOXOX." If the rat did not show a positive response after using the 15 g probe, the 50% withdrawal threshold (50% MWT) was recorded as 15 g. If a positive response was also induced by the 0.4 g probe, the 50% MWT was recorded as 0.4 g. If 15g and 0.4g probes are not used, calculate according to the following formula: 50% MWT(g) = (10[Xf+kδ]) / 10000, where "50% MWT" is defined as a positive response with a 50% probability at that stimulus intensity, "Xf" is the value (in log units) of the last used Von Frey probe, "K" is a value that can be obtained by searching the table according to the measurement sequence, and "δ" is the mean value of the difference between a series of stimuli (in log units defined as 0.224).

[0141] Bipedal balance measurement A bipedal balance pain tester was used to assess the weight-bearing capacity of each animal's lower limbs. Each animal was placed on the plexiglass cover of the bipedal balance pain device. After the animal completed its exploratory behavior, the rat's forelimbs were placed on the device's incline and its hind limbs were placed on the left and right balance plates, respectively. Once the animal became inactive, the average weight-bearing capacity of the left and right hind limbs was measured within 5 seconds of inactivity. Each animal was measured three times with a 10-second interval between each repeated measurement.

[0142] statistical analysis Differences in gait scoring, plantar pain threshold, and weight-bearing were expressed as mean and standard error (mean ± SD). Data between the model group and the treatment group at multiple time points were analyzed using repeated measures ANOVA with SPSS Statistics 21 software, and Tamhane's method for subsequent analysis. P<0.05 was considered statistically significant, and p<0.01 was considered very significant. Final data were plotted using GraphPad Prism 6 software.

[0143] result Common clinical symptoms in rats after administration None of the animals exhibited any visible abnormalities during the experimental period. Effect of treatment with anti-Cx43 antibody on body weight in pMMx+ACLT-induced osteoarthritis rats

[0144] The body weight of the animals in each test group increased over time with the administration of each treatment (Table 2). No significant differences were observed between the groups. [Table 2]

[0145] Effect of anti-Cx43 antibody treatment on gait in pMMx+ACLT-induced osteoarthritis rats Osteoarthritis model rats exhibited lameness on day 16 of the study period (one week after surgery), with a mean gait score of 3.8 between the test groups (Table 3 and Figure 1). The gait score improved over the course of treatment, and by day 37 of the study, the gait score of the osteoarthritis model rats was comparable to that of the control rats (Table 3 and Figure 1). A significant difference in gait score was observed in the osteoarthritis model rat test group compared to control (non-osteoarthritis) rats (multivariate study, p<0.05, α=0.05; Table 3). A decrease in gait score over time was observed in the ALMB-0166 and duloxetine treatment groups, but no significant difference was observed compared to the vehicle-treated control group (multivariate study, p>0.05, α=0.05; Table 3). [Table 3]

[0146] Effect of treatment with anti-Cx43 antibody on mechanical pain in pMMx+ACLT-induced osteoarthritis rats One week after osteoarthritis induction, baseline levels of plantar pain threshold (50% MWT) were observed in osteoarthritis model rats at approximately 3.5 g. An increase in plantar pain threshold (50% MWT) was observed over time after the start of each treatment, compared to day 15 of the study (day 1 of treatment) (Tables 4-5 and Figure 2). Improvement in plantar pain threshold correlated with the number of ALMB-0166 doses administered (Table 5 and Figure 2). [Table 4] [Table 5]

[0147] Compared to model rats (without osteoarthritis), a significant difference in plantar pain threshold was observed in all osteoarthritis model test groups (multivariate study, p<0.01, α=0.05; Table 4). Furthermore, when comparing plantar pain threshold over time, a significant increase was observed in the ALMB-0166 25 mg / kg and 50 mg / kg treatment groups, as well as in the duloxetine 5 mg / kg test group, compared to the osteoarthritis model (vehicle-treated) test group (multivariate study, p<0.05, α=0.05; Table 4).

[0148] Analysis of plantar pain thresholds at each time point revealed a significant increase in plantar pain thresholds at days 23, 30, and 37 of the study in the ALMB-0166 25 mg / kg and 50 mg / kg groups, as well as the duloxetine group (p<0.05, α=0.05; Table 4). An increase in plantar pain threshold was also observed in the ALMB-0166 15 mg / kg group over the course of treatment, but the difference did not reach a significant threshold compared to osteoarthritis (vehicle-treated) animals (p=0.52, α=0.05; Table 4). Furthermore, the increase in plantar pain threshold resulting from ALMB-0166 treatment showed a dose-dependent effect (Figures 3A-3D).

[0149] Effect of anti-Cx43 antibody on differences in hindlimb weight-bearing in pMMx+ACLT-induced osteoarthritis rats The difference in hindlimb weight-bearing decreased over time in all osteoarthritis treatment groups (Table 6 and Figure 4). Compared to the osteoarthritis model (vehicle treatment) treatment group, the reduction in hindlimb weight-bearing difference was significant in all three ALMB-0166 treatment groups and the duloxetine 5 mg / kg treatment group (multivariate study, p<0.05, α=0.05; Table 6). Analyzing hindlimb weight-bearing at each time point, a significant reduction in weight-bearing difference was observed in all ALMB-0166 treatment groups and the duloxetine treatment group on days 16, 23, 30, and 37 of the study (multivariate study, p<0.01, α=0.05; Table 6). Furthermore, the reduction in hindlimb weight difference attributable to ALMB-0166 treatment showed a dose-dependent effect (Figures 5A-5D). [Table 6]

[0150] conclusion Treatment of rat models of osteoarthritis (induced by pMMX + ACLT) with the anti-CX43 antibody ALMB-0166 at doses of 15 mg / kg, 25 mg / kg, and 50 mg / kg resulted in improvements in gait scores over time. Furthermore, treatment of osteoarthritis model rats with the anti-CX43 antibody ALMB-0166 at doses of 15 mg / kg, 25 mg / kg, and 50 mg / kg also resulted in significant improvements in mechanical pain threshold and differences in hindlimb weight-bearing. The analgesic effect observed in osteoarthritis rats treated with the anti-CX43 antibody ALMB-0166 was dose-dependent, with analgesia obtained from treatment with 25 mg / kg and 50 mg / kg of ALMB-0166 being equivalent to or better than that obtained from treatment with 5 mg / kg of duloxetine. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

Claims

1. A method for treating osteoarthritis in a subject requiring treatment for osteoarthritis, comprising administering at least one dose of anti-connexin 43 (Cx43) antibody to the subject, wherein the anti-Cx43 antibody comprises the following heavy chain CDR sequence and light chain CDR sequence: HCDR1: Sequence ID 1; HCDR2: Sequence ID No. 2; HCDR3: Sequence ID 3; LCDR1: Sequence ID 4; LCDR2: Sequence ID 5; and LCDR3: Sequence ID 6.

2. The method according to claim 1, comprising administering the first dose of the anti-Cx43 antibody on day 1 and the second dose on day 8.

3. The method according to claim 2, further comprising administering the third dose of the anti-Cx43 antibody on the 15th day.

4. The method according to claim 3, further comprising administering the fourth dose of the anti-Cx43 antibody on day 22.

5. The method according to claim 4, further comprising administering subsequent doses weekly after the fourth dose.

6. The method according to any one of claims 1 to 5, wherein the first, second, third, fourth and / or subsequent doses are approximately 0.01 mg / kg to approximately 100 mg / kg.

7. The method according to any one of claims 1 to 6, wherein the first, second, third, fourth and / or subsequent doses are approximately 15 mg / kg.

8. The method according to any one of claims 1 to 6, wherein the first, second, third, fourth and / or subsequent doses are approximately 25 mg / kg.

9. The method according to any one of claims 1 to 6, wherein the first, second, third, fourth and / or subsequent doses are approximately 50 mg / kg.

10. The method according to any one of claims 1 to 9, wherein the anti-Cx43 antibody is administered intravenously.

11. The method according to any one of claims 1 to 10, wherein at least one indicator of the severity of osteoarthritis is evaluated at least 1 day to 1 week after each dose is administered.

12. The aforementioned at least one indicator of the severity of osteoarthritis is (a) The pain threshold of the arthritis, (b) Walking score, or (c) Differences in weight load The method according to claim 11, including the method described in claim 11.

13. The method according to any one of claims 1 to 12, wherein at least one indicator of the severity of osteoarthritis is improved after administration of at least one dose of the anti-Cx43 antibody.

14. The method according to any one of claims 1 to 13, wherein the subject is a human.

15. The method according to any one of claims 1 to 14, wherein the anti-Cx43 antibody comprises the heavy chain variable sequence of SEQ ID NO: 7 and / or the light chain variable sequence of SEQ ID NO:

8.

16. The method according to any one of claims 1 to 15, wherein the anti-Cx43 antibody comprises one of the heavy chain sequences of SEQ ID NOs. 9 and 11 to 18, and / or the light chain variable sequence of SEQ ID NO.

10.

17. The method according to any one of claims 1 to 16, wherein the anti-Cx43 antibody comprises the heavy chain sequence of SEQ ID NO: 9 and / or the light chain variable sequence of SEQ ID NO:

10.

18. A method for treating osteoarthritis in a subject requiring treatment for osteoarthritis, comprising administering an effective amount of anti-connexin 43 (Cx43) antibody to the subject, wherein the anti-Cx43 antibody is administered according to the following dosage schedule: i) First dose on day 1; ii) Second dose on day 8; iii) Third dose on day 15; and IV) Fourth dose on day 21.

19. The method according to claim 18, further comprising administering subsequent doses of the anti-Cx43 antibody weekly after the fourth dose.

20. The method according to claim 18 or claim 19, wherein the first, second, third, fourth and / or subsequent doses are approximately 0.01 mg / kg to approximately 100 mg / kg.

21. The method according to any one of claims 18 to 20, wherein the first, second, third, fourth and / or subsequent doses of the anti-Cx43 antibody are approximately 15 mg / kg.

22. The method according to any one of claims 10 to 20, wherein the first, second, third, fourth and subsequent doses of the anti-Cx43 antibody are approximately 25 mg / kg.

23. The method according to any one of claims 18 to 20, wherein the first, second, third, fourth and subsequent doses of the anti-Cx43 antibody are approximately 50 mg / kg.

24. The method according to any one of claims 18 to 23, wherein the anti-Cx43 antibody is administered intravenously.

25. The method according to any one of claims 18 to 24, wherein at least one indicator of the severity of osteoarthritis is evaluated at least 1 day to 1 week after each dose is administered.

26. The aforementioned at least one indicator of the severity of osteoarthritis is (a) The pain threshold of the arthritis, (b) Walking score, or (c) Differences in weight load The method according to claim 25, including the method described in claim 25.

27. The method according to any one of claims 18 to 26, wherein at least one indicator of the severity of osteoarthritis is improved after administration of at least one dose of the anti-Cx43 antibody.

28. The method according to any one of claims 18 to 27, wherein the subject is a human.

29. The method according to any one of claims 18 to 28, wherein the anti-Cx43 antibody comprises the following heavy chain CDR sequence and light chain sequence: HCDR1: Sequence ID 1; HCDR2: Sequence ID No. 2; HCDR3: Sequence ID 3; and LCDR1: Sequence ID 4; LCDR2: Sequence ID 5; and LCDR3: Sequence ID 6.

30. The method according to any one of claims 18 to 29, wherein the anti-Cx43 antibody comprises the heavy chain variable sequence of SEQ ID NO: 7 and / or the light chain variable sequence of SEQ ID NO:

8.

31. The method according to any one of claims 18 to 30, wherein the anti-Cx43 antibody comprises one of the heavy chain sequences of SEQ ID NOs. 9 and 11 to 18, and / or the light chain variable sequence of SEQ ID NO.

10.

32. The method according to any one of claims 18 to 31, wherein the anti-Cx43 antibody comprises the heavy chain sequence of SEQ ID NO: 9 and / or the light chain variable sequence of SEQ ID NO:

10.

33. The method according to any one of claims 1 to 32, wherein the anti-Cx43 antibody blocks the opening of the Cx43 hemichannel in the subject.

Images