Methods of preventing or reducing cartilage loss and / or joint damage using nav 1.7 inhibitors

Abstract

The present application relates to a method for preventing or reducing cartilage loss joint damage and / or degeneration, and / or pain in a subject in need thereof, comprising administering to the subject an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

Description

Yale Ref. YV 8761WO || TP Ref.251609.000125 METHODS OF PREVENTING OR REDUCING CARTILAGE LOSS AND / OR JOINT DAMAGE USING Nav 1.7 INHIBITORS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No.63 / 548,458, filed on November 14, 2023 and U.S. Provisional Application No.63 / 680,333, filed on August 7, 2024, the disclosures of which are herein incorporated by reference in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 AR062207 and R01 AR078035 awarded by the National Institutes of Health and I50 RX002999 awarded by the US Department of Veterans Affairs. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The present application relates to a method of preventing or reducing cartilage loss, joint damage and / or degeneration, and / or pain in a subject in need thereof, comprising administering to the subject an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein. BACKGROUND

[0004] Osteoarthritis (OA) is a disabling, degenerative disorder distinguished by progressive joint failure3. Although it is currently unclear whether the primary cause of OA is cartilage damage, OA always involves cartilage breakdown and loss of the unique extracellular matrix that normally guarantees the compressive resilience essential for joint function1. OA chondrocytes undergo complex changes, including anabolic and catabolic alteration. Chondrocytes are central protagonists in this regulatory cascade as the target of external biomechanical and biochemical stimuli, as well as the source of proteases, cytokines and mediators that regulate the deterioration of articular cartilage4. Despite the high prevalence and morbidity of OA, effective disease- modifying treatments are not currently available, and the molecular mechanisms involved in OA remain poorly understood.Yale Ref. YV 8761WO || TP Ref.251609.000125 SUMMARY OF THE INVENTION

[0005] As specified in the Background section above, there is a great need in the art for identifying molecular mechanisms, agents, disease-modifying treatments, and / or compositions that can prevent or reduce cartilage loss, joint damage and / or degradation, and / or pain in various diseases (e.g., osteoarthritis). The present application addresses these and other needs.

[0006] In one aspect, provided herein is a method of preventing or reducing cartilage loss and / or joint damage in a mammal in need thereof, wherein the method comprises administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

[0007] In another aspect, provided herein is a method of regulating chondrocyte metabolism in a mammal in need thereof, wherein the method comprises administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein in the mammal’s chondrocytes.

[0008] In yet another aspect, provided herein is a method of preventing, delaying progression or treating osteoarthritis (OA) in a mammal in need thereof, wherein the method comprises administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

[0009] In some embodiments, the agent inhibits catabolism and / or enhances anabolism in the cartilage.

[0010] In some embodiments, the agent is a selective Nav 1.7 blocker.

[0011] In some embodiments, the agent is a non-selective Nav 1.7 blocker.

[0012] In some embodiments, the agent is selected from PF-04856264, ProTx II, PF-051980047, PF-05089771, PF-05153462, PF-05150122, PF-05186462, PF-05241328, PF-06456384, GNE- 3565, SSCI-1, GDC-0276, MK-2075, OLP-1002, iN1011-N17, ST-2262, CC8464, AM-8145, AM-0422, AM-6120, JzTx V, μ-theraphotoxin-Pn3a, GpTx-I, GpTx I-71, Huwentoxin-IV (HwTx-IV) or mutants thereof, m3-HwTx-IV, Df1a, GX-201, GX-044, GX-585, GX-674, MRL5, Cd1a, DSP-2230, GDC-0287 (RG7893), ST-2427, ST-2578, GDC-0310, NKTR-171, CNV-3000223, CNV-3000164, μ-SLPTX-Ssm6a, antibody that can bind to sensor S3-S4 loop, or derivatives thereof and any combinations thereof.

[0013] In some embodiments, the agent is PF-05089771 or derivatives thereof.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0014] In some embodiments, the agent is PF-04856264 or derivatives thereof.

[0015] In some embodiments, the agent is Pro Tx II or derivatives thereof.

[0016] In some embodiments, the agent is selected from carbamazepine (CBZ), oxcarbazepine (OXA), eslicarbazepine acetate (ESL), lacosamide (LCM), tetrodotoxin (TTX), XEN402 (funapide or TV-45070), vixotrigine (raxatrigine, CNV1014802 or BIIB074), lamotrigine, bupivacaine (BPV), vinpocetine (VPC), hardwickiic acid (HDA), lidocaine, phenytoin, ralfinamide (NW-1029, FCE-26742A, PNU-0154339E), saxitoxin (STX), phlotoxin-1, BIA 2- 093, BIA 2-024, CNV-1061436, or derivatives thereof and any combinations thereof.

[0017] In some embodiments, the agent is carbamazepine (CBZ) or derivatives thereof. In some embodiments, the agent is lacosamide (LCM) or derivatives thereof.

[0018] In some embodiments, the agent is oxcarbazepine (OXA) or derivatives thereof.

[0019] In some embodiments, the agent is selected from the group consisting of a small molecule, an antibody, a peptide, an antisense oligonucleotide, an RNAi molecule, a gene therapy molecule, an aptamer and any combinations thereof.

[0020] In some embodiments, the agent is a sulfonamide-based inhibitor.

[0021] In some embodiments, the agent is administered systemically.

[0022] In some embodiments, the agent is administered orally, intravenously, intraperitoneally, intradermally, intramuscularly, nasally, topically, or subcutaneously.

[0023] In some embodiments, the agent is administered locally.

[0024] In some embodiments, the agent is administered via an intra-articular delivery.

[0025] In some embodiments, the agent is administered via a skin patch or a transdermal delivery system.

[0026] In some embodiments, the agent is formulated as microspheres, hydrogels, nanoparticles, or liposomes.

[0027] In some embodiments, the agent is formulated together with a positively charged solvent or material.

[0028] In some embodiments, the agent is formulated for extended release, immediate release, controlled release, sustained release, delayed release, or pulse release of the agent.

[0029] In some embodiments, the agent is administered at a dose of 0.1-250 mg / kg body weight.

[0030] In some embodiments, the agent is administered at a dose of 1-50 mg / kg body weight.

[0031] In some embodiments, the agent is administered at a dose of 10-50 mg / kg body weight.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0032] In some embodiments, the agent is administered at a dose of 50-250 mg / kg body weight.

[0033] In some embodiments, the method further comprises assessing the cartilage loss and / or joint damage in the mammal before and / or after the treatment.

[0034] In some embodiments, the cartilage loss is assessed by assessing articular cartilage destruction by the OARSI grade and / or assessing osteophyte formation and / or assessing subchondral bone plate (SBP) thickness.

[0035] In some embodiments, the method further comprises assessing the distance of movement and / or mechanical allodynia in the mammal before and / or after the treatment.

[0036] In some embodiments, the method further comprises determining level of HSP70 and / or midkine in the blood of the mammal before and / or after the treatment.

[0037] In some embodiments, the method further comprises determining level of HSP70 and / or midkine in the chondrocyte secretome of the mammal before and / or after the treatment.

[0038] In some embodiments, the mammal is human.

[0039] In some embodiments, the mammal is a companion animal or a veterinary animal.

[0040] In another aspect provided herein is a method of assaying activity of Nav 1.7 ion channel in cells comprising low densities of sodium (Na) channels, said method comprising: (i) recording of Na currents produced by Nav 1.7 channel in a low-noise environment using a patch clamp; and (ii) enhancing the signal / noise ratio by averaging multiple runs for each voltage clamp trial.

[0041] In some embodiments of the method of assaying activity of Nav 1.7 ion channel, the cells comprising low densities of sodium (Na) channels are non-excitable cells.

[0042] In some embodiments, the method of assaying activity of Nav 1.7 ion channel further comprises using one or more pharmacological agents and subtraction to isolate the Na currents produced by Nav 1.7 channel.

[0043] In some embodiments of the method of assaying activity of Nav 1.7 ion channel, the one or more pharmacological agents are selected from small molecule Nav1.7 blockers and ProTx II. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figures 1A-1J show that TTX-S currents are present in OA chondrocytes and largely produced by Nav1.7 ProTx II-S channels. Fig.1A, Representative current traces in control buffer (left), in the presence of 1 µM TTX (middle), and resulting traces of the TTX-S current (right).Yale Ref. YV 8761WO || TP Ref.251609.000125 Fig.1B, Current voltage curves of peak current amplitudes in control solution (^) and in the presence of 1 µM TTX (^), and the I-V curve for the TTX-S current (light gray circle). Fig.1C, G / Gmax of the TTX-S current (mean ± s.e.m., n = 3) fitted with the Boltzmann equation. Fig. 1D, Single-exponential time constants of TTX-S current inactivation (mean ± s.e.m., n = 3). Fig. 1E, Time to peak of the TTX-S current (mean ± s.e.m., n=3). Fig.1F, Inhibition of fast- inactivating sodium current by 20 nM ProTx II. Averaged sodium current traces at 0 mV in control (black solid line) and in 20 nM ProTx II (black dotted line), and the resulting trace of their difference (ProTx II-S current, black solid thick line). Fig.1G, Inhibition of fast- inactivating sodium current by 1 µM TTX. Averaged traces of sodium currents evoked by 0 mV test voltage from -90 mV holding voltage in control solution (black solid line) and in the presence of 1 µM TTX (black dotted line), and the trace of their difference (TTX-S current, gray trace). Fig.1H, Overlays of TTX-S (gray) and ProTxII-S (black) current traces from (Figs.1F and 1G), normalized by peak current amplitudes. Fig.1I, (left) Inactivation time constants (mean ± s.e.m.) of TTX-S (n = 4) versus ProTx II-S (n = 5) currents. Fig.1I, (right) Time-to- peak of TTX-S (mean ± s.e.m., n = 4) versus ProTx II-S (mean ± s.e.m., n = 5) currents at 0 mV test voltage. Fig.1J, (left) Effect of 1 µM TTX on peak current amplitudes (mean ± s.e.m., n = 4) measured at 0 mV test voltage. Fig.1J, (right) Effect of 20 nM ProTx II on peak current amplitudes (mean ± s.e.m., n = 5) measured at 0 mV test voltage; averages of current amplitudes are shown for total, ProTx II-S and persistent sodium currents. n indicates cell number; P values by two-tailed Mann-Whitney test; n / s, not significant.

[0045] Figures 2A-2P show that ablation of chondrocyte Nav1.7 protects against OA and reduces pain. Fig.2A, Safranin O and Fast Green stained sections of knee joints of mice with the indicated genotype (n = 8). Scale bar, 50 µm. Histological analysis demonstrated that the deletion of Nav1.7 in both DRG neurons and chondrocytes significantly reduced cartilage loss, whereas DMM-induced cartilage degradation was prominent in littermate controls (indicated in black box). Figs.2B-2E, OARSI score (Fig.2B), osteophyte development (Fig.2C), subchondral bone plate (SBP) thickness (Fig.2D), and synovitis score (Fig.2E) in indicated mice 12 weeks after DMM (n = 8). Fig.2F, Traces of open field testing at 12 weeks after DMM. Figs.2G, and 2H, Two-minute travel distance (Fig.2G) and von Frey testing (Fig.2H) in DMM operated mice at the indicated time-points after surgery (n = 8). Fig.2I, Safranin O and Fast Green stained sections of knee joints (n = 8). Scale bar, 50 µm. Nav1.7 chondrocyte-deficientYale Ref. YV 8761WO || TP Ref.251609.000125 mice exhibited a marked reduction in DMM-induced cartilage loss compared to littermate controls (indicated in black box). Figs.2J-2M, OARSI score (Fig.2J), osteophyte development (Fig.2K), SBP thickness (Fig.2L), and synovitis score (Fig.2M) in indicated mice 12 weeks after DMM surgery (n = 8). Fig.2N, Traces of open field testing at 12 weeks after DMM surgery. Figs.2O, and 2P, Two-minute travel distance (Fig.2O) and von Frey testing (Fig.2P) in DMM-operated mice at the indicated time-points after surgery (n = 8). Figs.2B-2E, and 2J- 2M, Data are mean ± s.d., P values by two-tailed unpaired Student’s t-test (Figs.2G, 2H, 2O, and 2P). Data are mean ± 95% confidence interval (CI), P values by two-tailed multiple unpaired Student’s t-test with Welch’s correction.

[0046] Figures 3A-3H show that blockade of Nav1.7 regulates chondrocyte biology through enhancing HSP70 and midkine secretion. Fig.3A, Peptide-spectrum match (PSM)-based abundance of proteins identified from the 30-100kDa fraction of conditioned medium that are unique to (gray dots) or increased with (black dots) PF-04856264 and ProTx II treatment. Fig. 3B, PSM-based abundance of proteins (black dots), identified from the 10-30kDa fraction of conditioned medium that are unique to PF-04856264 and ProTx II treatment. Fig.3C, COL2 and ACAN mRNA levels in human C28I2 chondrocytes stimulated with conditioned medium collected from cells treated with ProTx II (Pro-CM) or PF-04856264 (PF-CM) in the absence or presence of control IgG or anti-HSP70 antibodies (n = 4 biological replicates). Fig.3D, MMP13 and ADAMTS5 mRNA levels in C28I2 chondrocytes stimulated with IL-1β and conditioned medium collected from cells treated with ProTx II (Pro-CM) or PF-04856264 (PF-CM) in the absence or presence of control IgG or anti-midkine antibodies (n = 4 biological replicates). Fig. 3E, Safranin O and Fast Green stained knee joint sections (n = 8). Ver, VER 155008. Scale bar, 50 µm. Treatment with the Nav1.7 blocker PF-04856264 (PF) significantly reduced cartilage loss in DMM mice. However, this protective effect was nearly abolished when combined with the HSP70 inhibitor VER 155008 (Ver) and the midkine inhibitor iMDK. Notably, both VER 155008 and iMDK alone also significantly diminished the protective effects of PF-04856264 against cartilage loss. Fig.3F, OARSI score from images represented in Fig.3E. Fig.3G and 3H, Two-minute travel distance and von Frey testing at the indicated time-points with indicated treatments after DMM surgery (n = 8). Data are mean ± 95% confidence interval, P values by two-way ANOVA with Bonferroni post-hoc test. *Vehicle versus PF, P < 0.05; **vehicle versus PF, P < 0.01;#PF versus PF + Ver + iMDK, P < 0.05;##PF versus PF + Ver + iMDK, P < 0.01.Yale Ref. YV 8761WO || TP Ref.251609.000125 (Figs.3C, 3D, and 3F), Data are mean ± s.d., P values by one way ANOVA with Bonferroni post hoc test.

[0047] Figures 4A-4M show Ca2+signaling in chondrocytes. Figs.4A, and 4B, F / F0 [PBS (^), Pro Tx II (▼), and PF (▲)] (Fig.4A) and area under the curve (AUC) (Fig.4B) of intracellular Ca2+in human OA chondrocytes following ATP stimulation, measured by plate reader. ATP present from black arrow. Figs.4C, and 4D, HSP70 (Fig.4C) and midkine (Fig. 4D) levels in conditioned medium of C28I2 cells pre-treated with BAPTA-AM, followed by ProTx II or PF-04856264 (PF). Fig.4E, F / F0 of intracellular Ca2+in KB-R7943 treated C28I2 chondrocytes following ATP stimulation, assayed by confocal fluorescence microscopy. [PBS (black solid line), KB-R7943 (gray solid line), and KB-R7943+PF (black dotted line)] Figs.4F, and 4G, HSP70 (Fig.4F) and midkine (Fig.4G) levels in conditioned medium of C28I2 cells treated with KB-R7943 in the presence or absence of PF-04856264. Fig.4H, Expression of NCX isoforms in C28I2 cells. Fig.4I, Knockdown efficiency of NCX1 in C28I2 cells. Figs.4J, and 4K, F / F0 (Fig.4J) and AUC of intracellular Ca2+(Fig.4K) following ATP stimulation, in C28I2 chondrocytes transfected with scramble or NCX1 siRNA measured by plate reader. Fig.4L, HSP70 and midkine levels in conditioned medium of chondrocytes transfected with scramble or NCX1 siRNA and treated with ProTx II or PF-04856264. Fig.4M, Model of mechanisms of chondrocyte- and DRG-expressed Nav1.7 in OA, and amelioration of OA and pain via Nav1.7 blockade. Figs.4B-4D, 4F, 4G, 4K, and 4L, Data are mean ± s.d., Figs.4B, 4F, and 4G, P values calculated by one way ANOVA with Bonferroni post-hoc test. Fig.4K, Two-tailed unpaired Student’s t-test. Figs.4C, 4D, and 4L, Two-way ANOVA with Bonferroni post-hoc test. Figs.4B, 4F, 4I, and 4K, n=3 biological replicates. Figs.4C, 4D, and 4L, n = 4 biological replicates.

[0048] Figures 5A-5H show that Nav1.7 is expressed in chondrocytes and upregulated in OA cartilage. Fig.5A, Relative expression of 9 distinct VGSCs in human C28I2 chondrocytes, assayed by PCR. Expression of GAPDH mRNA serves as internal control. Fig.5B, Relative expressions of VGSCs in human OA (n = 4) versus healthy (n = 3) cartilage, in RNA-sequencing data of human cartilage. Fig.5C, The expression levels of chondrocytes expressed VGSCs in human C28I2 cells treated with 10 ng / ml TNFα (gray circle) or IL-1β (dashed circle) for 24 hours, assayed by qRT-PCR (n = 4 biological replicates). Fig.5D, qPCR analysis of SCN9A in human cartilage from healthy individuals (n = 11) and from individuals with early stage (KLYale Ref. YV 8761WO || TP Ref.251609.000125 grade 1-2, n = 14) or late stage OA (KL grade 3-4, n = 22). Fig.5E, Expression of Nav1.7 in cartilage from healthy individuals and patients with OA with KL grade 1-2 or 3-4, assayed by Immunoblotting (n = 3 for each group). Fig.5F, Membrane location of Nav1.7 in human chondrocytes detected by Immunoblotting (n = 3 biological replicates). Fig.5G, Immunostaining of Nav1.7 in cartilage from healthy individuals and from individuals with late stage OA (n = 4 for each group). Scale bar = 100 µm. Cartilage remained intact in normal human samples, but was degraded in cartilage isolated from human OA patient (Fig.5G, upper panel). Immunohistochemical analysis further revealed elevated expression of Nav1.7 in OA cartilage (Fig.5G, lower panel). Fig.5H, Immunostaining of Nav1.7 in cartilage collected from mice subjected to sham or DMM surgery (n = 4 mice per group). Scale bar, 100µm. Following DMM OA surgery, cartilage loss progressively increased at 4, 8, and 12 weeks post-surgery (Fig.5H, upper panel). In parallel, Nav1.7 expression was upregulated at various stages after surgery compared to sham-operated control mice (Fig.5H, lower panel). Data are mean ± s.d., P values by two-tailed unpaired Student’s t-test in 5C and one way ANOVA with Bonferroni post-hoc test in Figs.5D and 5E. DMM, destabilization of medial meniscus; OA, osteoarthritis.

[0049] Figures 6A-6D show generation of conditional Nav1.7 knockout mouse. Fig.6A, Schematic of mouse breeding strategy to generate conditional knockout mice lacking Nav1.7 in chondrocyte (Nav1.7chondrocyte), DRG neurons (Nav1.7DRG) or both chondrocyte and DRG neurons (Nav1.7DRG;chondrocyte). Figs.6B-6D, Relative Nav1.7 mRNA level in chondrocyte and DRG neurons from Nav1.7chondrocyte(Fig.6B), Nav1.7DRG(Fig.6C), and Nav1.7DRG;chondrocyte(Fig.6D) mice (n = 3 biological replicates).10-week-old mice Agc1-CreERT2; Nav1.7flox / floxand Agc1-CreERT2;Nav1.8-Cre;Nav1.7flox / floxwere intraperitoneally injected with tamoxifen at a dose of 150 µg per gram of body weight, administered daily for 5 consecutive days. Basal levels of Nav1.7 in DRG neurons and chondrocytes from Nav1.7floxmice were set as 1, respectively. Data are mean ± s.d., P values by two-tailed unpaired Student’s t-test.

[0050] Figures 7A-7L show that genetic ablation of Nav1.7 in both chondrocyte and DRG neuron ameliorates OA progression and alleviates pain in chemically induced OA model. Fig. 7A, Experimental scheme showing establishment of MIA model in mice harboring tamoxifen inducible Nav1.7 deletion in chondrocyte and DRG neuron. Fig.7B, Representative Safranin O and Fast Green stained knee joint sections of Nav1.7floxand Nav1.7DRG;chondrocytemale after saline or MIA injection at day 28 (n = 8 mice for each group). Scale bar = 50 µm. Safranin O stainingYale Ref. YV 8761WO || TP Ref.251609.000125 revealed that the deletion of Nav1.7 in both DRG neurons and chondrocytes significantly reduced cartilage loss, whereas MIA-induced cartilage degradation was prominent in littermate controls. Fig.7C, Quantification of OARSI score shown in Fig.7B. Fig.7D, Representative traces of open field testing at day 28 post MIA injection in Nav1.7floxand Nav1.7DRG;chondrocytemice. Figs.7E, and 7F, Quantitation of two minute travel distance (Fig.7E) and von Frey testing (Fig.7F) in Nav1.7floxand Nav1.7DRGmice at the indicated time-points post MIA injection (n = 8 mice per group). Figs.7G, 7H, and 7I, Immunohistochemical staining (Fig.7G) and corresponding quantification for COL2, Mmp13 (Fig.7H), Aggrecan neoepitope, and Comp fragment (Fig.7I) in knee joint sections of Nav1.7floxand Nav1.7DRG;chondrocytemice at day 28 post MIA injection (n = 6). Scale bar, 50 µm. Immunohistochemical staining of knee joints indicated that OA-associated loss of the anabolic marker type II collagen (COL2), and increase of MMP13, aggrecan neoepitope generated via cleavage by ADAMTS5, and COMP fragment observed in Nav1.7flox mice, were inhibited in Nav1.7DRG; chondrocyte mice in both surgically and chemically induced OA (Figs.7G, and 7J). Fig.7J, 7K, and 7L, (related to Figs. 2A-2H) Immunohistochemical staining (Fig.7J) and corresponding quantification of COL2, Mmp13 (Fig.7K), aggrecan neoepitope, and Comp fragment (Fig.7L) in knee joint sections of Nav1.7floxand Nav1.7DRG; chondrocytemice at 12 weeks after DMM surgery (n = 8). Scale bar, 50 µm. Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test (Fig.7C) and two-tailed unpaired Student’s t-test (Figs.7H, 7I, 7K, and 7L). Data are mean ± 95% confidence interval (CI), P values by two-tailed multiple unpaired Student’s t-test with Welch’s correction (Figs.7E, and 7F). * P < 0.05; ** P < 0.01.

[0051] Figures 8A-8N show that genetic ablation of Nav1.7 in DRG neuron contributes to reduce OA associated pain without affecting OA pathological progression in surgically induced OA model. Figs.8A, 8B and 8C, (related to Figs.2I-2P) Immunohistochemical staining of knee joints indicated deletion of Nav1.7 in chondrocytes increased the amount of the anabolic effector COL2 and simultaneously decreased the amount of DMM-induced catabolic effectors, including MMP13, aggrecan neoepitope and COMP fragment. (Fig.8A) and corresponding quantification of Col2, Mmp13, (Fig.8B) Aggrecan neoepitope, and Comp fragment (Fig.8C) in knee joint sections of Nav1.7floxand Nav1.7chondrocytemice at 12 weeks after DMM surgery (n = 8). Scale bar, 50 µm in Fig.8A. Fig.8D, Representative Safranin O and Fast Green stained sections of knee joints from DMM operated Nav1.7floxand Nav1.7DRGmice (n = 8 mice per group). ScaleYale Ref. YV 8761WO || TP Ref.251609.000125 bar, 50 µm. Safranin O staining showed that DMM surgery induced similar levels of cartilage loss in both control and Nav1.7DRG mice (indicated in black box). Figs.8E -8H, OARSI score (Fig.8E), osteophyte development (Fig.8F), SBP thickness (Fig.8G), and synovitis score (Fig. 8H) in Nav1.7floxand Nav1.7DRGmice 12 weeks after DMM surgery (n = 8 mice per group). Fig. 8I, Representative traces of open field testing at 12 weeks after DMM surgery in Nav1.7floxand Nav1.7DRGmice. Figs.8J, and 8K, Quantitation of two minute travel distance (Fig.8J) and von Frey testing (Fig.8K) in DMM operated Nav1.7floxand Nav1.7DRGmice at the indicated time- points after surgery (n = 8 mice per group). Figs.8L, 8M, and 8N, Immunohistochemical staining indicated no significant differences between Nav1.7floxand Nav1.7DRGDMM mice in the expression of cartilage markers such as COL2, MMP13, aggrecan neoepitope, and COMP fragment. (Fig.8L) and corresponding quantification for Col2, Mmp13(Fig.8M), Aggrecan neoepitope, and Comp fragment (Fig.8N) in knee joint sections of Nav1.7floxand Nav1.7DRGmice at 12 weeks after DMM surgery (n = 6). Scale bar, 50 µm in Fig.8L. Data are mean ± s.d., P values by two-tailed unpaired Student’s t-test in Figs.8B, 8E-8H, 8M, and 8N; data are mean ± 95% confidence interval (CI), P values by two-tailed multiple unpaired Student’s t-test with Welch’s correction in Figs.8J and 8K (** P < 0.01).

[0052] Figures 9A-9O show that pharmacological blockade of Nav1.7 through locally is therapeutic against OA and OA-related pain in surgically induced mouse models. Fig.9A, Schematic of the experimental design to determine the effects of intra-articular administrated PF- 04856264 (the selective Nav1.7 inhibitor) on OA progression and pain-related behaviors in the surgically induced DMM mouse model. Fig.9B, Representative Safranin O and Fast Green stained knee joint sections of DMM operated WT male mice treated with or without PF- 04856264 for 8 weeks (n = 8 mice for each group). Scale bar, 50 µm. Safranin O staining revealed that PF-04856264 treatment protected the structure of articular cartilage and maintained proteoglycan content in the cartilage compared with vehicle. Figs.9C-9E, OARSI score (Fig. 9C), osteophyte development (Fig.9D) and SBP thickness (Fig.9E) in DMM operated WT male mice treated with or without PF-04856264 for 8 weeks (n = 8 mice per group). Figs.9F, and 9G, Quantitation of two minute travel distance (Fig.9F) and von Frey testing (Fig.9G) in DMM- operated WT male mice treated with or without PF-04856264 at the indicated time-points after surgery (n = 8 mice per group). Figs.9H,and 9I, Immunohistochemical staining indicated that Col2 level was increased; by contrast, MMP13 and aggrecan neoepitope levels were significantlyYale Ref. YV 8761WO || TP Ref.251609.000125 reduced in the cartilage of mice with DMM OA that received intra-articular injections of PF- 04856264 compared with those treated with vehicle. (Fig.9H) and corresponding quantification (Fig.9I) of Col2, Mmp13, and Aggrecan neoepitope in knee joint sections of DMM operated WT male mice treated with or without PF-04856264 for 8 weeks (n = 6). Scale bar, 50 µm in Fig.9H. Fig.9J, Representative Safranin O and Fast Green stained knee joint sections of DMM operated WT female mice treated with or without PF-04856264 for 8 weeks (n = 8 mice for each group). Scale bar, 50 µm. Consistent with the observations in male mice, female mice treated with PF-04856264 exhibited less articular cartilage loss compared with untreated controls. Figs. 9K-9M, OARSI score (Fig.9K), osteophyte development (Fig.9L) and SBP thickness (Fig. 9M) in DMM operated WT female mice treated with or without PF-04856264 for 8 weeks (n = 8 mice per group). Figs.9N,and 9O, Quantitation of two minute travel distance (Fig.9N) and von Frey testing (Fig.9O) in DMM-operated WT female mice treated with or without PF-04856264 at the indicated time-points after surgery (n = 8 mice per group). Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test (Figs.9C, 9D, 9E, 9K, 9L, and 9M) and two-tailed unpaired Student’s t-test (Fig.9I). Data are mean ± 95% confidence interval (CI), P values by two-tailed multiple unpaired Student’s t-test with Welch’s correction in Figs.9F, 9G, 9N and 9O ( ** P < 0.01).

[0053] Figures 10A-10J show that oral delivery of PF-04756264 attenuates OA progression and alleviates pain in both chemically and surgically induced OA models. Fig.10A, A schematic representation highlights the experimental outline to determine the effects of PF-04856264 through oral delivery on OA progression and behavior changes in WT male and female mice with MIA model. Fig.10B, Representative Safranin O and Fast Green stained knee joint sections of WT male and female mice treated with or without PF-04856264 after saline or MIA injection at day 28 (n = 6 mice for each group). Scale bar, 50 µm. Safranin O staining showed that systemic delivery of PF-04856264 markedly decreased cartilage loss compared to vehicle treatment in both male and female mice in MIA induced OA model. Figs.10C,and 10D, OARSI score in male (Fig.10C) and female (Fig.10D) mice as shown in Fig.10B. Figs.10E,and 10F, Quantitation of two minute travel distance and von Frey testing in WT male (Fig.10E) and female (Fig.10F) mice treated with or without PF-04856264 at the indicated time-points post MIA injection (n = 6 mice per group). Figs.10G, and 10H, Immunohistochemical staining revealed that oral PF-04856264 delivery increased COL2 expression and decreased MMP13 andYale Ref. YV 8761WO || TP Ref.251609.000125 aggrecan neoepitope levels in cartilage compared with vehicle. (Fig.10G) and corresponding quantification (Fig.10H) for Col2, Mmp13, and Aggrecan neoepitope in knee joint sections of WT male mice treated with or without PF-04856264 at day 28 post MIA injection (n = 6). Scale bar, 50 µm in Fig.10G. Fig.10I, Representative Safranin O and Fast Green stained knee joint sections of DMM operated WT male mice orally treated without or with PF-04856264 for 8 weeks (n = 8 mice for each group). Scale bar, 50 µm. Safranin O staining demonstrated that oral administration of PF-04856264 significantly reduced cartilage loss in mice with DMM-induced OA compared to the vehicle-treated group. Fig.10J, OARSI score for Fig.10I. Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test (Figs.10C, 10D, and 10J) and two-tailed unpaired Student’s t-test (Fig.10H). Data are mean ± 95% confidence interval (CI), P values by two-tailed multiple unpaired Student’s t-test with Welch’s correction in Figs.10E and 10F (* P < 0.05; ** P < 0.01).

[0054] Figures 11A-11M show that carbamazepine (CBZ), a clinically used sodium chancel blocker, attenuates OA progression and pain in chemically and surgically induced OA models. Fig.11A, Schematic of timeline to analyze the effects of systemic oral delivery carbamazepine (CBZ) on OA progression and pain in MIA model. Fig.11B, Representative Safranin O and Fast Green stained knee joint sections of WT male mice treated with or without CBZ after saline or MIA injection at day 28 (n = 6 mice for each group). Scale bar, 50 µm. Fig.11C, OARSI score as shown in Fig.11B. Fig.11D, Representative traces of open field testing at day 28 post MIA injection in WT male mice with indicated treatment at day 28 post MIA injection. Figs.11E, and 11F, Quantitation of two minute travel distance (Fig.11E) and von Frey testing (Fig.11F) in WT male mice at the indicated time-points post MIA injection (n = 6 mice per group). Figs. 11G, and 11H, Immunohistochemical Safranin O staining demonstrated that systemic CBZ treatment attenuated cartilage loss (Fig.11B). It also conferred protection to chondrocytes by promoting pro-anabolic effects, as evidenced by the upregulation of COL2 expression, while reducing anti-catabolic activity by downregulating MMP13 and aggrecan neoepitope levels. (Fig.11G) and corresponding quantification (Fig.11H) for Col2, Mmp13, and Aggrecan neoepitope in knee joint sections of WT mice with indicated treatment at day 28 post MIA injection (n = 6). Scale bar, 50 µm in Fig.11G. Fig.11I, Schematic of timeline to analyze the effects of systemic oral delivery of various dosages of CBZ on OA progression and pain in surgically induced DMM model. Fig.11J, Representative Safranin O and Fast Green stainedYale Ref. YV 8761WO || TP Ref.251609.000125 knee joint sections of DMM operated WT male mice treated without or with 10 mg / kg body weight, 50 mg / kg body weight or 250mg / kg body weight CBZ for 8 weeks (n = 8 mice for each group). Scale bar, 50 µm. Safranin O staining demonstrated that in mice subjected to DMM surgery, treatment with a low dose of CBZ (10 mg / kg body weight) resulted in a significant reduction in cartilage loss 12 weeks post-surgery compared to the vehicle-treated group. Furthermore, medium (50 mg / kg) and high (250 mg / kg) doses of CBZ provided even greater protection against cartilage loss compared to the low dose. Fig.11K, OARSI score for Fig.11J. Figs.11L,and 11M, Quantitation of two minute travel distance (Fig.11L) and von Frey testing (Fig.11M) in DMM-operated WT male mice treated without or with various dosage of CBZ, as indicated, at specified time-points after surgery (n = 8 mice per group). Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test (Figs.11C, and 11K) and two-tailed unpaired Student’s t-test (Fig.11H). Data are mean ± 95% confidence interval (CI), P values by two-tailed multiple unpaired Student’s t-test with Welch’s correction in (Figs.11E, and 11F) and two way ANOVA with Bonferroni post-hoc test (Figs.11L and 11M). * P < 0.05; ** P < 0.01.

[0055] Figures 12A-12T show that pharmacological blockade or deletion of Nav1.7 enhances anabolic and inhibits catabolic metabolism in chondrocytes. Figs.12A-12D, mRNA levels of catabolism markers MMP13 (Fig.12A), ADAMTS5 (Fig.12B), COX2 (Fig.12C) and NOS2 (Fig.12D) in human C28I2 chondrocytes treated with or without 10 ng / ml IL-1β in the absence or presence of 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 hours, assayed by qRT-PCR (n = 4 biological replicates). Figs.12E-12H, mRNA levels of catabolism markers MMP13 (Fig.12E), ADAMTS5 (Fig.12F), COX2 (Fig.12G) and NOS2 (Fig.12H) in human C28I2 chondrocytes treated with or without 10 ng / ml TNFα in the absence or presence of 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 hours, assayed by qRT- PCR (n = 3 biological replicates). Figs.12I-12L, mRNA levels of catabolism markers MMP13 (Fig.12I), ADAMTS5 (Fig.12J), COX2 (Fig.12K) and NOS2 (Fig.12L) in human C28I2 chondrocytes treated with or without 500 ng / ml poly(I:C) in the absence or presence of 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 hours, assayed by qRT-PCR (n = 3 biological replicates). Figs.12M, and 12N, mRNA levels of anabolic markers COL2 (Fig. 12M) and ACAN (Fig.12N) in human C28I2 chondrocytes treated with 1 µM TTX, 25 nM ProTx II (Pro) or 1 µM PF-04856264 (PF) for 24 hours, assayed by qRT-PCR (n = 4 biological replicates). Figs.12O,and 12P, mRNA levels of Col2 (Fig.12O) and Acan (Fig.12P) inYale Ref. YV 8761WO || TP Ref.251609.000125 chondrocytes isolated from Nav1.7floxand Nav1.7chondrocytemice at P6 (n = 4). Figs.12Q-12T, mRNA levels of Mmp13 (Fig.12Q), Adamts5 (Fig.12R), Cox2 (Fig.12S) and Nos2 (Fig.12T) in chondrocytes isolated from Nav1.7floxand Nav1.7chondrocytemice at P6 which are treated with or without IL-1β for 24 hours. (n = 4) [PBS (black circle) and IL-1β (gray circle)]. Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test (Figs.12A-12N) and two- tailed unpaired Student’s t-test (Figs.12O-12T).

[0056] Figures 13A-13R shows that blocking Nav1.7 pharmacologically inhibit catabolism and enhance anabolism through regulating the chondrocyte secretion. Figs.13A-D, mRNA levels of MMP13 (Fig.13A), ADAMTS5 (Fig.13B), COX2 (Fig.13C) and NOS2 (Fig.13D) in primary human chondrocytes isolated from patients with last stage OA which are treated with or without 10 ng / ml IL-1β along with a serial doses of ProTx II (Pro) or PF-04856264 (PF) for 24 hours, assayed by qRT-PCR (n = 6). Figs.13E, and 13F, mRNA levels of COL2 (Fig.13E) and ACAN (Fig.13F) in primary human chondrocytes isolated from patients with late stage OA which are treated with a serial doses of ProTX II (Pro) or PF-04856264 (PF) for 24 hours, assayed by qRT-PCR (n = 6 donors). Figs.13G, and 13H, Proteins levels of MMP13 (Fig.13G) and PRG4 (Fig.13H) in the supernatants of full-thickness human OA cartilage explants, as determined by ELISA (n = 8 donors). Cartilage explant was cultured with 10 ng / ml IL-1β in the absence or presence of 25 nM ProTx II or 1 µM PF-04856264 for 5 days. Figs.13I-13L, mRNA levels of MMP13 (Fig.13I), ADAMTS5 (Fig.13J), COL2 (Fig.13K), and ACAN (Fig.13L) in the human OA cartilage explants cultured with 10 ng / ml IL-1β in the absence or presence of 25 nM ProTx II or 1 µM PF-04856264 for 5 days, assayed by qRT-PCR (n = 8 donors). Figs.13M, and 13N, mRNA levels of COL2 (Fig.13M) and ACAN (Fig.13N) in human C28I2 cells treated with conditioned medium collected from 25 nM Pro or 1 µM PF treated C28I2 cells (n = 4 biological replicates). Figs.13O-13R, mRNA levels of MMP13 (Fig.13O), ADAMTS5 (Fig. 13P), COX2 (Fig.13Q) and NOS2 (Fig.13R) in human C28I2 cells treated with conditioned medium collected from 25 nM Pro or 1 µM PF treated C28I2 cells as well as 10 ng / ml IL-1β for 24 hours (n = 4 biological replicates). Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test.

[0057] Figures 14A-14F shows the effects of conditioned medium on human chondrocyte anabolism and catabolism. Fig.14A, Schematic of the experimental strategy of conditioned medium production, fraction and characterization. Human C28I2 chondrocytes are expanded inYale Ref. YV 8761WO || TP Ref.251609.000125 DMEM supplemented with FBS until they are 90% confluent. The growth medium is then exchanged with medium supplemented with ITS and 25 nM ProTX II (Pro) or 1 µM PF- 04856264 (PF) for 2 days. The medium is then collected and separated into 4 fractions based on sized exclusion: <10 kDa, 10-30 kDa, 30-100 kDa, and >100 kDa using the centrifugal filter device. Fig.14B, mRNA levels of COL2 and ACAN in human C28I2 cells treated with 30-100 kDa fraction of the conditioned medium for 24 hours (n = 4 biological replicates). Fig.14C, mRNA levels of MMP13 and ADAMTS5 in human C28I2 cells treated with 30-100 kDa fraction of the conditioned medium in the absence or presence of 10 ng / ml IL-1β for 24 hours (n = 4 biological replicates). Fig.14D, mRNA levels of COL2 and ACAN in human C28I2 cells treated with 10-30 kDa fraction of the conditioned medium for 24 hours (n = 4 biological replicates). Figs.14E and 14F, mRNA levels of MMP13, ADAMTS5, (Fig.14E) COX2 and NOS2 (Fig. 14F) in human C28I2 cells treated with 10-30 kDa fraction of the conditioned medium in the absence or presence of 10 ng / ml IL-1β for 24 hours (n = 4 biological replicates). Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test (Figs.14B-14F)

[0058] Figures 15A-15P show that HSP70 enhances anabolism and midkine inhibits IL-1β induced catabolism in human chondrocytes. Figs.15A, and 15B, ELISA quantification of HSP70 in conditioned medium (Fig.15A) and cell lysate (Fig.15B) of human C28I2 cells treated with 25 nM ProTx II (Pro), 1 µM PF-04856264 (PF), or 10 µM CBZ for 48 hours. Figs.15C, and 15D, ELISA quantification of Midkine in conditioned medium (Fig.15C) and cell lysate (Fig. 15D) of human C28I2 cells treated with 25 nM Pro or 1 µM PF for 48 hours. Fig.15E, mRNA levels of COL2 and ACAN in human C28I2 cells treated with serial doses of HSP70 for 24 hours. Fig.15F, mRNA levels of MMP13 and ADAMTS5 in human C28I2 cells treated with serial doses of HSP70 and 10 ng / ml IL-1β for 24 hours. Fig.15G, mRNA levels of COL2 and ACAN in human C28I2 cells treated with serial doses of midkine for 24 hours. Fig.15H, mRNA levels of MMP13 and ADAMTS5 in human C28I2 cells treated with serial doses of midkine and 10 ng / ml IL-1β for 24 hours. n = 4 biological replicates; Data are mean ± s.d., P values two-tailed unpaired Student’s t-test. Figs.15I, and 15J, ELISA quantification of HSP70 (Fig.15I) and midkine (Fig.15J) in conditioned medium of primary chondrocytes isolated from Nav1.7floxand Nav1.7chondrocyemice at 12 weeks after DMM surgery (n = 4 biological replicates). Figs.15K, and 15L, Levels of HSP70 (Fig.15K) and midkine (Fig.15L) in mouse sera collected from sham surgery control and from DMM surgery WT mice at 12 weeks after surgery (n = 24),Yale Ref. YV 8761WO || TP Ref.251609.000125 assayed by ELISA. Figs.15M, and 15O, Levels of HSP70 (Fig.15M) and midkine (Fig.15O) in human sera collected from healthy individuals (n = 22) and from patients with OA (n = 165), assayed by ELISA. Figs.15N, and 15P, Correlation analysis (Pearson R and two-tailed P value) of HSP70 (Fig.15N) and midkine (Fig.15P) between matched sera and synovium fluids isolated from patients with OA (n = 35). Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test (Figs.15A-15H) and two-tailed unpaired Student’s t-test (Figs.15I- 15L, 15M, and 15O).

[0059] Figures 16A-16H show that intracellular Ca2+signals are essential for the enhanced secretion of HSP70 and midkine following Nav1.7 blockade. Figs.16A,and 16B, Traces (F / F0) of intracellular Na+levels over time in human OA chondrocytes (Fig.16A) and C28I2 cells (Fig. 16B) following ATP stimulation, measured by confocal microscopy. ATP present from black arrows. Figs.16C, and 16D, Traces (F / F0) [PBS (black empty circle), ProTx II ( gray empty circle), and PF ( black solid circle)] (Fig.16C) and quantification of AUC (Fig.16D) of intracellular Ca2+levels in C28I2 chondrocytes following ATP stimulation, measured by plate reader (n = 3 biological replicates). Figs.16E, and 16F, Traces (F / F0) of intracellular Ca2+levels in human OA (Fig.16E) and C28I2 (Fig.16F) chondrocytes following ATP stimulation, assayed by confocal fluorescence microscopy. ATP present from black arrows. [PBS(black solid line), ProTx II(black dashed line), and PF (gray solid line)] Figs.16G, and 16H, ELISA quantification of HSP70 (Fig.16G) and midkine (Fig.16H) in conditioned medium of human C28I2 cells treated with or without Ionomycin in the absence or presence of 1 µM PF-04856264 (PF) for 48 hours. n = 3 biological replicates; Data are mean ± s.d., P values by one way ANOVA with Bonferroni post-hoc test in Figs.16D, 16G and 16H.

[0060] Figures 17A-17B show reduction in inflammatory cytokine induced catabolism by non-selective Nav channel inhibitors in C28I2 cells. DETAILED DESCRIPTION

[0061] Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here Nav1.7 was identified as an OA-associated VGSC and demonstrated that human OAYale Ref. YV 8761WO || TP Ref.251609.000125 chondrocytes express functional Nav1.7 channels, with a density of 0.1-0.15 channels / µm2and 350-525 channels / cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrated that Nav1.7 expressed in dorsal root ganglia (DRG) neuron is involved in pain, whereas Nav1.7 in chondrocytes regulated OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorated the progression of structural joint damage, and reduced OA pain behavior. Mechanistically, Nav1.7 blockers regulated intracellular Ca2+signaling and the chondrocyte secretome, which in turn affected chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte- expressed, OA-associated channel uncovers a dual target for the development of disease- modifying and non-opioid pain relief treatment for OA.

[0062] Alongside significant loss of articular cartilage, the dominant clinical symptom of OA is pain5. Specialized peripheral sensory neurons are abundant in joint tissues, including synovium and subchondral bone6, and contribute to OA pain. These neurons express unique repertoires of voltage-gated sodium channels (VGSCs)7. There are nine distinct VGSCs (Nav1.1-1.9), encoded by genes SCN1A-SCN11A8. Nav1.7, Nav1.8 and Nav1.9 are of particular interest as targets for pain treatment owing to preferential expression in peripheral sensory neurons within dorsal root ganglia (DRG), and their roles in action potential initiation and propagation within peripheral pain pathways7. A recent study reported that modulation of DRG- expressed Nav1.8 can attenuate OA pain9. The critical role of Nav1.7 in pain signaling10and genetic validation (severe pain with gain-of-function Nav1.7 mutations11,12, insensitivity to pain with loss-of-function Nav1.7 mutations13,14) have further supported Nav1.7 as a therapeutic target for pain. Notably, Nav1.7 gain-of-function mutations increase pain sensitivity in some patients with OA15. A role of Nav1.7 in inflammatory pain is supported by observations in global Nav1.7 knockout and DRG-specific knockout mice16,17. A role of DRG-expressed Nav1.7 in OA pain was supported by reduced OA pain following spinal administration of ProTx II, a Nav1.7-selective antagonist, in the monosodium iodoacetate (MIA) induced model of OA18.

[0063] A central canon of electrophysiology holds that voltage-gated sodium (Nav) channels produce explosive all-or-none action potentials, approximately 0.1 V in amplitude, that are essential for the operation of “excitable” cell-types such as neurons, muscle cells and cardiac myocytes40. Less well appreciated have been reports that Nav channels are also present within cell-types that do not produce action potentials and are traditionally considered non-excitable.Yale Ref. YV 8761WO || TP Ref.251609.000125 These include fibroblasts, astrocytes, macrophages and microglia19. Expression of Nav channels in these cells is dynamic, changing markedly depending on conditions. In at least some of these non-excitable cell-types, Nav channels are inserted within the cell membrane and are functional, producing sodium currents that are indistinguishable from those their counterparts in excitable cells43. However, the density of Nav channels within these cells is low, and the contribution of Nav channels to the behavior of non-excitable cells has remained enigmatic. Many of these observations have focused on cultured cells. Thus, there is a need to learn more about the roles of Nav channels in non-excitable cells, and to understand their roles in vivo. There is also a need to learn more about the role of Nav channels in the pathophysiology of non-excitable cells.

[0064] In the effort of the present inventors to identify novel, differentially expressed genes in OA, RNA sequencing (RNA-seq) analysis was performed on normal and arthritic cartilage, and Nav1.7 was identified as the only significantly upregulated OA-associated VGSC. Here it is demonstrated that distinct from DRG-expressed Nav1.7, which is only involved in OA pain signaling, chondrocyte-expressed Nav1.7 regulates chondrocyte biology and OA progression. It is also demonstrated herein that Nav1.7-blockade protects joints from deterioration, and Nav1.7- blockade mediates its chondroprotective effects, at least in part, by regulating intracellular Ca2+signaling and the chondrocyte secretome. Definitions

[0065] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present application. For purposes of the present application, the following terms are defined.

[0066] The terms “patient”, “individual”, “subject”, “mammal”, and “animal” are used interchangeably herein and refer to mammals, including, without limitation, humans, companion animals, veterinary animals (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.) and experimental animal models (e.g., mouse, rabbit, rat).

[0067] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at leastYale Ref. YV 8761WO || TP Ref.251609.000125 one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or sub- clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0068] The term “in need thereof” as used herein refers to a judgment made by a physician or other caregiver that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of the physician’s or caregiver’s expertise.

[0069] The terms “therapeutically effective amount” and “effective amount” are used interchangeably herein to refer to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount capable of having any detectable, positive effect on any symptom, aspect, or characteristic of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects, and it can be adjusted in connection with the dosing regimen and diagnostic analysis of the subject’s condition, and the like.

[0070] The term “pharmaceutically acceptable”, as used herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce undesirable reactions when administered to a mammal (e.g., a human or a veterinary or companion animal (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.)).

[0071] The term “carrier” or “a pharmaceutically acceptable carrier” as used herein, refers to any clinically useful solvents, diluents, adjuvants, excipients, recipients, vehicles and the like for use in preparing admixtures of a pharmaceutical composition.

[0072] As used herein, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that theYale Ref. YV 8761WO || TP Ref.251609.000125 term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0073] The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0074] The term “about X-Y” used herein has the same meaning as “about X to about Y.”

[0075] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0076] In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (Glover ed.1985); Oligonucleotide Synthesis (Gait ed.1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription And Translation (Hames and Higgins eds.1984); Animal Cell Culture (Freshney ed.1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al. eds., Current Protocols in Molecular Biology, John Wley and Sons, Inc.1994; among others.

[0077] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.Yale Ref. YV 8761WO || TP Ref.251609.000125 Nav 1.7 Blockers Or Nav 1.7 Inhibitors

[0078] In one aspect, provided herein are the agents that are capable of reducing or inhibiting a function or expression of the Nav 1.7 protein. In some embodiments, the agent described herein reduces or inhibits a function or expression of the Nav1.7 protein encoded by SCN9A gene.

[0079] In some embodiments, the agents that are capable of reducing or inhibiting a function or expression of Nav 1.7 include, but are not limited to, a small molecule, an antibody, a peptide, an anti-sense oligonucleotide, an RNAi molecule (e.g., an siRNA), a gene therapy, an aptamer or any combinations thereof.

[0080] In some embodiments, the agents that are capable of reducing or inhibiting the function or expression of Nav 1.7 have an antagonistic activity. In some embodiments, the agents that are capable of reducing or inhibiting the function or expression of Nav 1.7 are Nav inhibitors. The term “inhibitor” and “blocker” can be used interchangeably in the context of agents that are capable of reducing or inhibiting the function or expression of Nav 1.7 protein. Nav inhibitors can be selective or non-selective Nav inhibitors. Inhibitors described herein encompass blockers which are capable of blocking a function or expression of Nav 1.7 protein.

[0081] Selective Nav inhibitors are agents that specifically inhibit the Nav 1.7 protein and thereby reduce or inhibit a function or expression of Nav 1.7 protein. In some embodiments, the agents that specifically inhibit Nav 1.7 protein are sulfonamide-based inhibitors. In some embodiments, the selective Nav inhibitors are peptides, fragments, or variants thereof. In some embodiments, the selective Nav inhibitors are small molecules such as chemical compounds or derivatives thereof.

[0082] Non-limiting examples of selective Nav1.7 blockers or selective Nav 1.7 inhibitors include PF-04856264, ProTx II, PF-051980047, PF-05089771, PF-05153462, PF-05150122, PF- 05186462, PF-05241328, PF-06456384, GNE-3565, SSCI-1, GDC-0276, MK-2075, OLP-1002, iN1011-N17, ST-2262, CC8464, AM-8145, AM-0422, AM-6120, JzTx V, μ-theraphotoxin- Pn3a, GpTx-I, GpTx I-71, Huwentoxin-IV (HwTx-IV) or mutants thereof, m3-HwTx-IV, Df1a, GX-201, GX-044, GX-585, GX-674, MRL5, Cd1a, DSP-2230, GDC-0287 (RG7893), ST-2427, ST-2578, GDC-0310, NKTR-171, CNV-3000223, CNV-3000164, μ-SLPTX-Ssm6a, antibodies that can bind to sensor S3-S4 loop (Lee et al., Cell.2014 Jun 5;157(6):1393-1404), or derivatives thereof and any combinations thereof.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0083] Non-limiting examples of selective Nav1.7 blockers can also include the compounds that are described in International Patent Application Nos. WO 2007 / 109324; WO 2009 / 005460; WO 2009 / 000038;WO2009 / 145721; WO 2010 / 079443; WO 2012 / 004714; WO 2012 / 007868; WO 2012 / 007869; WO2012 / 004706; WO 2012 / 007877; WO 2012 / 004664; WO 2012 / 095781; WO2012 / 039657; WO 2012 / 035023; WO 2013 / 102826; WO 2013 / 134518; WO 2013 / 093688, and WO 2015 / 036734, the contents of which are incorporated herein by reference in their entirety.

[0084] In some embodiments, the agent is PF-05089771 or derivatives thereof. In some embodiments, the agent is PF-04856264 or derivatives thereof. In some embodiments, the agent is Pro Tx II or derivatives thereof.

[0085] Non-selective Nav inhibitors are agents that may inhibit or block other Nav channels in addition to the Nav 1.7 channel. In some embodiments, non-selective Nav inhibitors are pan-Nav blockers that ameliorate the progression of structural joint damage in animal models of osteoarthritis. In some embodiments, the non-selective Nav inhibitors are peptides, fragments, or variants thereof. In some embodiments, the non-selective Nav inhibitors are small molecules such as chemical compounds or derivatives thereof. Non-limiting examples of non-selective Nav1.7 blockers or non-selective Nav1.7 inhibitors include carbamazepine (CBZ), oxcarbazepine (OXA), eslicarbazepine acetate (ESL), lacosamide (LCM), tetrodotoxin (TTX), XEN402 (also known as funapide or TV-45070), vixotrigine (also known as raxatrigine, CNV1014802 or BIIB074), lamotrigine, bupivacaine (BPV), vinpocetine (VPC), hardwickiic acid (HDA), lidocaine, phenytoin, ralfinamide (formerly NW-1029, FCE-26742A, PNU- 0154339E), saxitoxin (STX), phlotoxin-1, BIA 2-093, BIA 2-024, CNV-1061436, or derivatives thereof and any combinations thereof. In some embodiments, the agent is carbamazepine (CBZ) or derivatives thereof. In some embodiments, the agent is lacosamide (LCM) or derivatives thereof. In some embodiments, the agent is oxcarbazepine (OXA) or derivatives thereof. In some embodiments, the agent is eslicarbazepine acetate (ESL) or derivatives thereof. In some embodiments, the agent is BIA 2-093 or derivatives thereof. In some embodiments, the agent is BIA 2-024 or derivatives thereof. In some embodiments, the agent is lamotrigine or derivatives thereof. In some embodiments, the agent is phenytoin or derivatives thereof.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0086] Non-limiting examples of Nav1.7 inhibitors are the inhibitors that are described in Eagles et al., Br J Pharmacol.2022;179:3592–3611; the contents of which are incorporated herein by reference in their entirety.

[0087] The Nav 1.7 inhibitors can be developed using rational design strategy to identify derivatives of natural bis-guanidinium toxins that specifically inhibit human Nav1.7 (hNaV1.7) over other off-target hNaV isoforms73.

[0088] In some embodiments, agents that are capable of reducing or inhibiting the function or expression of Nav 1.7 include a gene therapy. In some embodiments, the gene therapy is epigenetic. Non-limiting examples of gene editing tools that can be used are CRISPR and zinc finger nucleases. Efficacy of these gene tools can be determined by analyzing Nav 1.7 repression in cells. In some embodiments, the agent is a gene regulation system such as a CRISPR interference (CRISPRi) or CRISPRoff molecule. In some embodiments, the agent that is capable of reducing or inhibiting the function or expression of Nav 1.7 is a gene therapy such as a CRISPR interference (CRISPRi) or CRISPRoff molecule. In some embodiments, the agent that is capable of reducing or inhibiting the function or expression of Nav 1.7 is a chimeric antigen receptor (CAR) molecule. The CRISPR / Cas9 system can be used to cause a down regulation of SCN9A gene leading to the repression of Nav1.7 within chondrocytes.

[0089] Gene therapies may comprise recombinant vectors comprising a polynucleotide encoding a peptide or an antibody that can function as a Nav1.7 inhibitor. The terms “vector”, “cloning vector” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc. Vector can be a viral vector or non-viral vector. Non-limiting examples of viral vectors are a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, or a vaccinia virus vector. Non-limiting examples of non-viral vectors are a plasmid or a transposon (such as, for example, a Sleeping Beauty or a PiggyBac transposon).

[0090] The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become produced, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such asYale Ref. YV 8761WO || TP Ref.251609.000125 a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or transmembrane.

[0091] The agent may include an isolated host cell comprising a polynucleotide encoding a peptide, an antibody, or a CAR molecule that reduces or inhibits a function or expression of Nav 1.7 protein. The term “host cell” refers any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). In various embodiments, the host cell has been activated and / or expanded ex vivo. In some embodiment the polynucleotide can be an oligonucleotide, an siRNA, or an anti-sense oligonucleotide.

[0092] In some embodiments, the agent that is capable of reducing or inhibiting the function or expression of Nav 1.7 is an antibody or antigen-binding fragment(s) thereof. In some embodiments, an antibody or antigen-binding fragment(s) thereof may include any isotype of immunoglobulin (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA, IgE, IgM (e.g., IgM1, IgM2), IgD, and IgY). The antibody can be monomeric, polymeric, or chimeric forms. In some embodiments, an antibody or antigen-binding fragment(s) thereof may include polyclonal antibody, monoclonal antibody (mAb), or antibody-like polypeptide, such as a chimeric antibody or humanized antibody. In some embodiments, an antibody or antigen-binding fragment thereof may include Fab, F(ab’)2, Fc, Fabc, Fv molecule, scFv, disulfide-linked Fv (sdFv), intrabody, diabody, minibody, linear antibody, single domain antibody such as sdAb (either VL or VH), camelid nanobody (VHH domains), VNARs, or a multi-specific antibody formed from antibody fragments.

[0093] In some embodiments, the agent that is capable of reducing or inhibiting the function or expression of Nav 1.7 is a peptide, a functional fragment, or a derivative thereof. The term “polypeptide” or “peptide” as used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, peptide mimetics, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP- ribosylation, pegylation, biotinylation, etc.).

[0094] In some embodiments, the agent that is capable of reducing or inhibiting the function or expression of Nav 1.7 is an anti-sense oligonucleotide. Antisense oligonucleotides are RNA orYale Ref. YV 8761WO || TP Ref.251609.000125 single-stranded DNA molecules with nucleotide sequences complementary to a specified mRNA. Methods of producing and utilizing antisense RNA are well known to those of ordinary skill in the art (see, for example, Lichtenstein & Nellen (Editors), Antisense Technology: A Practical Approach, Oxford University Press, 1997; Agrawal & Crooke, Antisense Research and Application (Handbook of Experimental Pharmacology, Vol.131), Springer Verlag, 1998; Gibson, Antisense and Ribozyme Methodology: Laboratory Companion, Chapman & Hall, 1997; Mol & Van Der Krol, Antisense Nucleic Acids and Proteins, Marcel Dekker; Weiss, Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents, CRC Press, 1997; Stanley et al., (1993) Antisense Research and Applications, CRC Press; Stein & Krieg, (1998) Applied Antisense Oligonucleotide Technology).

[0095] Potency of the Nav 1.7 inhibitors can be assessed against species variants of Nav 1.7, including human, mouse, rat, monkey, dog, and cat. Non-limiting examples of analyzing potency include an IC50 evaluation, an EC50 evaluation, and / or an SPR analysis. Pharmaceutical Compositions

[0096] In one aspect, provided herein are compositions comprising the agent that reduces or inhibits a function or expression of Nav 1.7 protein and a pharmaceutically acceptable carrier and / or excipient, for preventing or reducing cartilage loss, joint damage, and / or joint degeneration in a subject (e.g., a mammal) in need thereof.

[0097] In another aspect, provided herein are compositions comprising the agent that reduces or inhibits a function or expression of Nav 1.7 protein and a pharmaceutically acceptable carrier and / or excipient, for regulating chondrocyte metabolism in a subject (e.g., a mammal) in need thereof.

[0098] In yet another aspect, provided herein are compositions comprising the agent that reduces or inhibits a function or expression of Nav 1.7 protein and a pharmaceutically acceptable carrier and / or excipient, for preventing, delaying progression or treating osteoarthritis (OA) in a subject (e.g., a mammal) in need thereof.

[0099] In yet another aspect, provided herein are compositions comprising the agent that reduces or inhibits a function or expression of Nav 1.7 protein and a pharmaceutically acceptable carrier and / or excipient, for preventing or reducing pain due to cartilage loss, joint damage, joint degradation, and / or osteoarthritis (OA) in a subject (e.g., a mammal) in need thereof.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0100] Agents that reduce or inhibit a function or expression of Nav 1.7 protein may include but are not limited to small molecules; antibodies or antigen binding fragments thereof; peptides, functional fragments, or derivatives thereof; anti-sense oligonucleotides; RNAi molecules such as siRNAs; gene therapies; aptamers or any combinations thereof.

[0101] In some embodiments, the pharmaceutical composition comprises a small molecule described herein, and a pharmaceutically accepted carrier and / or excipient. The small molecule can be a chemical compound that has an inhibitory or antagonistic activity against Nav 1.7 protein. The chemical compounds can have an inhibitory or antagonistic activity against other Nav channels in addition to Nav 1.7. Such compounds are pan Nav inhibitors that can also be used as agents according to the present disclosure.

[0102] In some embodiments, the pharmaceutical composition comprises a peptide, a functional fragment or derivative thereof; or an antibody or antigen-binding fragment thereof described herein, and a pharmaceutically accepted carrier and / or excipient.

[0103] In some embodiments, the pharmaceutical composition comprises a polynucleotide or a recombinant vector described herein, and a pharmaceutically accepted carrier and / or excipient.

[0104] Examples of pharmaceutical carriers include but are not limited to sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solutions, such as saline solutions and aqueous dextrose and glycerol solutions, are preferably employed as carriers, particularly for injectable solutions.

[0105] Compositions disclosed herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, dextrans, mannose, mannitol or sucrose; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0106] Compositions disclosed herein may comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants suchYale Ref. YV 8761WO || TP Ref.251609.000125 as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, acidic pH modifiers, basic pH modifiers, and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0107] The pharmaceutical compositions of the present disclosure may comprise the compounds described herein and a pharmaceutically acceptable carrier and / or excipient. Physiologically acceptable compounds can include, e.g., carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of glycopeptides, or excipients or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, e.g., phenol and ascorbic acid. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. Pharmaceutically acceptable carriers and formulations are known to the skilled artisan and are described in detail in the scientific and patent literature, see e.g., Gennaro et al., (1995) Remington's Pharmaceutical Sciences, Mack Publishing Company. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the route of administration of the composition, and on its particular physio-chemical characteristics.

[0108] In some embodiments, the compositions are formulated for parenteral administration, e.g., intravascular (intravenous), intra-articular, intraperitoneal, intraventricular, intrapleural or intramuscular administration. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile. In some embodiments, the composition is reconstituted from a lyophilized preparation prior to administration. In some embodiments, the compositions are formulated for inhalation or oral administration. In some embodiments, the compositions are formulated for subcutaneous, intradermal, transdermal, or skin-patch based administration.

[0109] In some embodiments, the compositions are formulated in lipid nanoparticle (LNP) form. Lipid nanoparticles are spherical and nanoscale delivery systems (typically, 80 – 200 nm in diameter) composed of several distinct types of lipids. Non-limiting examples ofYale Ref. YV 8761WO || TP Ref.251609.000125 components that can be used to formulate LNP based formulation are an ionizable lipid, a phospholipid, cholesterol, a lipid conjugated to polyethylene glycol (PEG-lipid), and any combinations thereof.

[0110] In some embodiments, the compositions are formulated together with a positively charged solvent or material.

[0111] In some embodiments, the compositions are formulated as microspheres, hydrogels, nanoparticles, or liposomes. Microspheres can be a biodegradable nanofibrous temperature-responsive gelling microspheres. Poly(l-lactic acid)-b-poly(ethylene glycol)-b- poly(N-Isopropylacrylamide) (PLLA-PEG-PNIPAm) copolymer can be used to prepare nanofibrous gelling microspheres (NF-GMS). (See, Adv. Funct. Mater.2020, 30, 2000776; incorporated herein by reference in its entirety).

[0112] In some embodiments, the composition comprises an agent as described herein and an analgesic and / or anti-inflammatory agent with a pharmaceutically acceptable carrier and / or excipient. Non-limiting examples of analgesics and / or anti-inflammatory agents include acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and / or opioids. Non- limiting examples of NSAIDs include ibuprofen, naproxen, diclofenac, ketorolac, and any combinations thereof. Non-limiting examples of steroids include corticosteroids.

[0113] In some embodiments, the composition comprises an agent as described herein, a molecule that can reduce pain, and a pharmaceutically acceptable carrier and / or excipient. Non- limiting examples of the molecules that can reduce pain include corticosteroids, hyaluronic acid (HA), platelet-rich plasma (PRP), clodronate, saline, steroids such as intra-articular steroid injection (IASI), mesenchymal stem cells (MSCs), adipose-derived stromal cells (ADSCs), and any combinations thereof. In some embodiments, the composition comprising an agent as described herein is administered in combination with a corticosteroid injection, hyaluronic acid (HA), platelet-rich plasma (PRP), an intra-articular steroid injection (IASI), mesenchymal stem cells (MSCs), adipose-derived stromal cells (ADSCs), and any combinations thereof. (See, Testa, G. et al., J. Funct. Morphol. Kinesiol.2021, 6, 15; Ferrara et al., BMC Musculoskeletal Disorders 2021, 22(Suppl 2):997; Zhang et al., Arthritis Research & Therapy (2022) 24:260; incorporated herein by reference in its entirety)

[0114] Examples of corticosteroids include, but are not limited to, cortisone, prednisone, hydrocortisone, triamcinolone acetonide, triamcinolone hexacetonide, dexamethasone LA,Yale Ref. YV 8761WO || TP Ref.251609.000125 dexamethasone acetate, dexamethasone sodium, betamethasone, betamethasone sodium phosphate and acetate, hydrocortisone acetate, methylprednisolone acetate, and any combinations thereof.

[0115] Examples of hyaluronic acid include, but are not limited to, low (500–730 kDa), intermediate (800–2000 kDa), high (2000–6000 kDa) molecular weight hyaluronic acid, and any combinations thereof. Non-limiting examples of crosslinked formulations of HA that can be combined with the agent are 0.84% sodium hyaluronate, 1% sodium hyaluronate, 1.5% sodium hyaluronate, 0.8% hylan G-F20, 1% cross-linked hyaluronate, 2.2% cross-linked hyaluronan, 0.8% hexadecylamide derivative of hyaluronan, non-animal stabilized hyaluronic acid, and any combinations thereof. In some embodiments, the hyaluronic acid is exogenous hyaluronic acid or intra-articular hyaluronic acid (IAHA).

[0116] Examples of platelet-rich plasma (PRP) preparation include, but are not limited to, an autologous mixture of highly concentrated platelets and associated growth factors and other bioactive components produced by centrifugal separation of whole blood.

[0117] Examples of mesenchymal stem cells (MSCs) include, but are not limited to, adipose mesenchymal stem cells (ADMSCs), umbilical cord mesenchymal stem cells (UBMSCs), bone marrow mesenchymal stem cells (BMMSCs), and any combinations thereof.

[0118] In some embodiments, the compositions are controlled release or sustained release formulations. In some embodiments, the compositions are extended release formulations, immediate release formulations, delayed release formulations, or pulse release formulations.

[0119] In some embodiments, the compositions are controlled release formulations. Such controlled release formulations can be used for sustained release of the Nav 1.7 inhibitors in the subject’s system. In some embodiments, the controlled release formulations include at least some microparticles that can include an inert core; a layer of the agent as described herein disposed on the inert core; and a coating comprising at least one release rate controlling polymer disposed on the layer of the agent. Non-limiting examples of the release rate controlling polymers include polymethacrylates, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate phthalate, ethyl cellulose, polyvinyl acetate-phthalate, and any combinations thereof. In some embodiments, all of the microparticles include the inert core, the layer of an agent and the coating. In some embodiments, the controlled release formulations comprise a wax matrix. Non-limiting examples of wax matrix are carnauba wax, cetostearylYale Ref. YV 8761WO || TP Ref.251609.000125 alcohol, fatty acids, or any mixtures thereof. In some embodiments, the controlled release formulations comprise at least one matrix forming polymer. Non-limiting examples of matrix forming polymers are waxes, gums, polymethacrylates, polyethylene oxides, hydroxypropyl methylcelluloses, hydroxyethyl celluloses, hydroxypropyl celluloses, carbapols, and any combinations thereof. In some embodiments, the controlled release formulations comprise a water dispersing excipient. Non-limiting examples of water dispersing excipients are microcrystalline cellulose, silicified microcrystalline cellulose, starch, colloidal silicone dioxide, a super disintegrant, and any combinations thereof.

[0120] The controlled release formulations disclosed herein may comprise one or more of the following: a binder, a flow aid, lubricant, a solubility modifier, and a coloring agent.

[0121] In some embodiments, the compositions are prepared in such a manner that the activity of the agent is not disrupted during the process and the agent can reach its site of action without disruption. In some embodiments, the composition further comprises a carrier that promotes the delivery of the inhibitors of endocytosis to an area affected by the chronic pain. Exemplary carriers include liposomes, micelles, nanodisperse albumin and its modifications, polymer nanoparticles, dendrimers, inorganic nanoparticles of different compositions.

[0122] In some embodiments, the compositions of the agents are adjusted for pH. Buffer systems can be used to provide pH values in the desired range. Non-limiting examples of buffers include carboxylic acid buffers such as acetates, citrates, lactates and succinates. In some embodiments, the composition is formulated within a pH range of about 4.5 to about 9.0, including for example pH ranges of about any of 5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. In some embodiments, the pH of the composition is no less than about 6, including for example no less than about any of 6.5, 7, or 8 (such as about 8). In some embodiments, the pH of the composition is no more than 7.4. In some embodiments, the pH of the composition is about 7.4. In some embodiments, the pH of the composition is about 7.4 or less than about 7.4. In some embodiments, the composition of the agent is an isotonic composition. The composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol or salt. In some embodiments, the compositions of the agents are adjusted for about pH 7.4 for intra-articular delivery of the agent to the subject (e.g., a mammal) in need thereof. (See, Iannitti et al., Drugs R D.2011 Mar; 11(1): 13–27; incorporated herein by reference in its entirety).Yale Ref. YV 8761WO || TP Ref.251609.000125

[0123] In some embodiments, the compositions are prepared with an excipient(s) that are suitable for intra-articular, intravenous, subcutaneous, intradermal, intraperitoneal, intramuscular, transmucosal, intrastemal, infusion, transdermal, skin-patch, nasal, inhalation, oral, sublingually, buccally, intranasal, topical, intrarectal, lipid nanoparticle (LNP) based, viral and / or non-viral or cellular, delivery of the agent to the subject (e.g., a mammal) in need thereof. In certain embodiments, the compositions are prepared with an excipient(s) that are suitable for intra-articular delivery of the agent to the subject (e.g., a mammal) in need thereof.

[0124] Non-limiting examples of oral forms of the administration are pills, tablets, capsules, granules and powders. Non-limiting examples of infusion techniques are sterile injectable aqueous or non-aqueous solutions or suspensions. Non-limiting examples of nasal forms of the administration are inhalation spray, aerosol, mist, and nebulizer. Non-limiting examples of topical forms of the administration are a cream, an ointment, salve, a powder, and a gel. Non-limiting examples of transdermal forms of the administration include a patch. Non- limiting examples of rectal forms of the administration include suppositories.

[0125] In some embodiments, the compositions are administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps.

[0126] In some embodiments, the compositions are administered in a form suitable for a veterinary animal or a companion animal (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.). Therapeutic Applications

[0127] In one aspect, provided herein are methods of preventing or reducing cartilage loss, joint damage, and / or joint degradation in a subject in need thereof, the methods comprising administering to the subject an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

[0128] In certain embodiments, provided herein are methods of preventing or reducing cartilage loss, joint damage and / or joint degradation in a mammal in need thereof, the methods comprising administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0129] In another aspect, provided herein are methods of regulating chondrocyte metabolism in a subject in need thereof, the methods comprising administering to the subject an effective amount of an agent that reduces or inhibits function or expression of Nav 1.7 protein in the subject’s chondrocytes.

[0130] In certain embodiments, provided herein are methods of regulating chondrocyte metabolism in a mammal in need thereof, the methods comprising administering to the mammal an effective amount of an agent that reduces or inhibits function or expression of Nav 1.7 protein in the mammal’s chondrocytes.

[0131] The method of regulating chondrocyte metabolism includes a regulation of intracellular Ca2+signaling and the chondrocyte secretome. In some embodiments the methods of regulating chondrocyte metabolism include regulating chondrocyte catabolism and chondrocyte anabolism. In some embodiments, the agent regulates chondrocyte catabolism and chondrocyte anabolism. In some embodiments, the agent regulates chondrocyte catabolism and chondrocyte anabolism respectively by increasing secretion of HSP70 and midkine. An increase in secretion of HSP70 and midkine can be determined by analyzing intracellular Ca2+signaling. In some embodiments, the agent inhibits catabolism and / or enhances anabolism in the cartilage. In some embodiments, the agent inhibits catabolism in the cartilage. In some embodiments, the agent enhances anabolism in the cartilage.

[0132] In yet another aspect, provided herein are methods of preventing, delaying progression or treating osteoarthritis (OA) in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

[0133] In certain embodiments, provided herein are methods of preventing, delaying progression or treating osteoarthritis (OA) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

[0134] In some embodiments, the methods of preventing, delaying progression or treating osteoarthritis (OA) are disease-modifying and non-opioid pain relief treatments for OA. Osteoarthritis may include, but is not limited to knee osteoarthritis, hip osteoarthritis, shoulder osteoarthritis, wrist osteoarthritis (also known as wear and tear arthritis), and elbow osteoarthritis. In some embodiments, the osteoarthritis may be present in a veterinary animal or aYale Ref. YV 8761WO || TP Ref.251609.000125 companion animal (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.). In some embodiments, the osteoarthritis may include canine osteoarthritis. In some embodiments, the methods of preventing, delaying progression or treating osteoarthritis (OA) can be used to treat primary forms and / or secondary forms of osteoarthritis. Primary (or idiopathic) forms of osteoarthritis may occur due to lifestyle factors or aging; and secondary forms of osteoarthritis can be the consequence of several pathological conditions, e.g., developmental and / or metabolic disorders, infection, or joint injury.

[0135] In another aspect, provided herein are methods of preventing or reducing pain associated with cartilage loss, joint damage, joint degradation, and / or OA in a subject in need thereof, the methods comprising administering to the subject an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

[0136] In certain embodiments, provided herein are methods of preventing or reducing pain associated with cartilage loss, joint damage, joint degradation, and / or OA in a mammal in need thereof, the methods comprising administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

[0137] In some embodiments of the methods of the present disclosure, the methods comprise administering to the subject an effective amount of an agent capable of reducing or inhibiting the function or expression of a sodium voltage-gated channel alpha subunit 9 (SCN9A). In certain embodiments, the methods comprise administering to the mammal an effective amount of an agent capable of reducing or inhibiting the function or expression of a sodium voltage-gated channel alpha subunit 9 (SCN9A). In some embodiments of the methods of the present disclosure, the methods comprise modulating the function or expression of sodium voltage-gated channel alpha subunit 9 (SCN9A).

[0138] The term “administering” include dispensing, delivering, or applying an agent that reduces or inhibits a function or expression of Nav 1.7 protein to a subject (e.g., a mammal) in need thereof. Examples of administering which can be used in the practice of the present disclosure include via an intra-articular, intravenous, subcutaneous, intradermal, intraperitoneal, intramuscular, transdermal delivery system, skin-patch, inhalation, intranasal, oral delivery, lipid nanoparticle (LNP)-based delivery, cellular delivery, viral and / or non-viral delivery, or gene editing, or any combinations thereof.

[0139] In some embodiments, the agent is administered systematically or locally.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0140] In certain embodiments, the agent is administered via an intra-articular route. An intra-articular injection of the agent according to the methods illustrated herein may prevent or delay joint damage or degradation and may relieve pain. The intra-articular injection of the agent can be administered to those patients who are intolerant to pharmacological oral therapy, when drugs are no longer effective, or for the patients who want to delay or avoid surgical treatment.

[0141] In some embodiments, the methods comprising intra-articular administration of the agent have reduced pain associated with the administration or are painless.

[0142] In some embodiments, when the methods comprise administering to the subject an effective amount of an agent that reduces or inhibits function or expression of Nav 1.7 protein via the intra-articular route, the injection site is at or adjacent to a joint of the subject, e.g., a knee joint, a hip joint, a shoulder joint, a wrist joint, or an elbow joint. In some embodiments, the injection site is selected from lateral infrapatellar, medial infrapatellar, and suprapatellar. In some embodiments, the injection site is lateral infrapatellar. In some embodiments, the injection site is at or adjacent to the hip joint.

[0143] In some embodiments, when the method of preventing, delaying progression of, or treating hip osteoarthritis comprises administering to the subject an effective amount of an agent that reduces or inhibits function or expression of Nav 1.7 protein via the intra-articular route, the injection site is at or adjacent to the hip of the subject.

[0144] In some embodiments, when the method of preventing, delaying progression of, or treating knee osteoarthritis comprises administering to the subject an effective amount of an agent that reduces or inhibits function or expression of Nav 1.7 protein via the intra-articular route, the injection site is selected from lateral infrapatellar, medial infrapatellar and suprapatellar. In some embodiments, the injection site is lateral infrapatellar.

[0145] In some embodiments, the methods described herein can be combined with other surgical treatments. In some embodiments, the methods described herein can be combined with other nonsurgical treatments. In some embodiments, the methods of the present disclosure are combined with a physical therapy. In some embodiments, the method of preventing, delaying progression or treating osteoarthritis (OA) can be combined with stem cell-based therapy. In some embodiments of the methods of the present disclosure, the agent is administered in combination with compositions that can reduce pain. In some embodiments, the agent is administered in combination with opioid compositions.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0146] In some embodiments, the agent is administered in combination with analgesic and / or anti-inflammatory agents. In some embodiments, the analgesic and / or anti-inflammatory agents are non-opioid. In some embodiments, the agent is administered in combination with acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, and / or opioids. Examples of NSAIDs include, but are not limited to, ibuprofen, naproxen, diclofenac, ketorolac, and any combinations thereof. Examples of steroids include but are not limited to corticosteroids.

[0147] In some embodiments, the agent is administered in combination with corticosteroids, hyaluronic acid (HA), platelet-rich plasma (PRP), clodronate, saline, steroid such as intra-articular steroid injection (IASI), mesenchymal stem cells (MSCs), adipose-derived stromal cells (ADSCs), and any combinations thereof. In some embodiments, the agent is administered in combination with corticosteroid injection, hyaluronic acid (HA), platelet-rich plasma (PRP), intra-articular steroid injection (IASI), mesenchymal stem cells (MSCs), adipose- derived stromal cells (ADSCs), or any combinations thereof via an intra-articular route.

[0148] Examples of corticosteroids include, but are not limited to, cortisone, hydrocortisone, prednisone, triamcinolone acetonide, triamcinolone hexacetonide, dexamethasone LA, dexamethasone acetate, dexamethasone sodium, betamethasone, betamethasone sodium phosphate and acetate, hydrocortisone acetate, methylprednisolone acetate, and any combinations thereof. In some embodiments, the corticosteroid is administered at a dosage of 1-80 mg.

[0149] Examples of hyaluronic acid include, but are not limited to, low (500–730 kDa), intermediate (800–2000 kDa), high (2000–6000 kDa) molecular weight HA, and any combinations thereof. Non-limiting examples of crosslinked formulations of HA that can be combined with the agent are 0.84% sodium hyaluronate, 1% sodium hyaluronate, 1.5% sodium hyaluronate, 0.8% hylan G-F20, 1% cross-linked hyaluronate, 2.2% cross-linked hyaluronan, 0.8% hexadecylamide derivative of hyaluronan, non-animal stabilized hyaluronic acid, and any combinations thereof. In some embodiments, the hyaluronic acid is exogenous hyaluronic acid or intra-articular hyaluronic acid (IAHA). In some embodiments, hyaluronic acid is administered at a dosage of 1-80 mg.

[0150] Examples of platelet-rich plasma (PRP) preparation include, but are not limited to, an autologous mixture of highly concentrated platelets and associated growth factors and other bioactive components produced by centrifugal separation of whole blood.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0151] Examples of mesenchymal stem cells (MSCs) include, but are not limited to, adipose mesenchymal stem cells (ADMSCs), umbilical cord mesenchymal stem cells (UBMSCs), bone marrow mesenchymal stem cells (BMMSCs), and any combinations thereof.

[0152] In some embodiments, the methods of the present disclosure comprise administering an agent in combination with one or more additional therapeutic agents. The combination according to the present disclosure includes separate, continuous, or simultaneous administration of the agent that reduces or inhibits function or expression of Nav 1.7 protein with the one or more additional therapeutic agents. This combination can be provided in the form of a pharmaceutical composition.

[0153] Additional therapeutic agents can be administered simultaneously or staggered in time (i.e., at different times and at equal or different time intervals for any part of a kit). The ratio of the total amount of additional therapeutic agents administered in a combination can vary, e.g., to address the needs of a subpopulation of patients to be treated or the needs of a single patient, and different needs are the age of the patient, it can be due to gender, weight, etc. Non-limiting examples of additional therapeutic agents are acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, hyaluronic acid (HA), platelet-rich plasma (PRP), clodronate, saline, steroid such as intra-articular steroid injection (IASI), mesenchymal stem cells (MSCs), adipose-derived stromal cells (ADSCs), opioids, non-opioids, antibodies, and any combinations thereof. In some embodiments, the additional therapeutic agent is a TNFα inhibitor, an IL-1β inhibitor, or any combinations thereof. In some embodiments, the additional therapeutic agents are the drugs that are typically prescribed for the conditions that involve joint damage and / or pain according to generally accepted medical practice.

[0154] In some embodiments, the methods of the present disclosure reduce or prevent pain, inflammation, joint degeneration, joint damage, loss of articular cartilage, and / or immobility. In some embodiments, the methods of the present disclosure promote cartilage regeneration and repair of injured cartilage. In some embodiments, the methods of the present disclosure reduce or prevent loss of articular cartilage, and / or osteoarthritis associated pain.

[0155] In some embodiments, the agent that reduces or inhibits a function or expression of Nav 1.7 protein are administered to a subject (e.g., a mammal) over a defined time course. In some embodiments, the agents are administered to a subject in multiple doses. The multiple doses of the agent may include sequential administrations. The sequential administration mayYale Ref. YV 8761WO || TP Ref.251609.000125 include the administration of an agent to the subject (e.g., a mammal) at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). In some embodiments, the methods comprise sequentially administering to the subject (e.g., a mammal) a single initial dose of an agent, followed by one or more secondary doses of the agent, and optionally followed by one or more tertiary doses of the agent. The initial, secondary, and tertiary doses may all contain the same amount of the agent. The initial, secondary, and tertiary doses may differ from one another in terms of frequency of administration. In some embodiments, the amount of agent contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as loading doses followed by subsequent doses that are administered on a less frequent basis (e.g., maintenance doses). In some embodiments, the amount of an agent is gradually escalated over a defined time course to avoid or reduce the possibility of severe side effects.

[0156] In some embodiments, the agent can be administered at a frequency of every 8 hours, every 12 hours, every 16 hours, every 24 hours, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 8 days, every 8 days, every 10 days, once a week, twice a week, biweekly, once a month, twice a month, 3 times a month, 4 times a month, or 5 times a month.

[0157] The optimal therapeutically effective amount of an agent or composition of this invention may be determined experimentally, taking into consideration the exact mode of administration, the form in which the drug is administered, the indication toward which the administration is directed, the subject involved (e.g., body weight, health, age, sex, etc.), and the preference and experience of the physician or veterinarian in charge.

[0158] Following methodologies which are well-established in the art, effective doses and toxicity of the agents and compositions of the present invention, which performed well in in vitro tests, can be determined in studies using small animal models (e.g., mice, rats) in which they have been found to be therapeutically effective and in which these drugs can be administered by the same route proposed for the human trials and / or veterinary clinical trials.

[0159] For any pharmaceutical composition used in the methods of the invention, dose- response curves derived from animal systems can be used to determine testing doses forYale Ref. YV 8761WO || TP Ref.251609.000125 administration to humans or veterinary animals or companion animals (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.). In safety determinations for each composition, the dose and frequency of administration should meet or exceed those anticipated for use in any clinical trial.

[0160] In some embodiments, the agent that reduces or inhibits a function or expression of Nav 1.7 protein are administered in a therapeutically effective amount. In some embodiments, the agent is administered to the subject at a dose of about 0.001-250 mg / kg of body weight. In some embodiments, the agent is administered to the subject at a dose of about 0.01-250 mg / kg of body weight. In some embodiments, the agent is administered to the subject at a dose of about 0.1-250 mg / kg of body weight. In some embodiments, the agent is administered to the subject at a dose of about 1-50 mg / kg of body weight. In some embodiments, the agent is administered to the subject at a dose of about 10-50 mg / kg of body weight. In some embodiments, the agent is administered to the subject at a dose of 50-250 mg / kg of body weight. In some embodiments, the agent is administered to the subject at a dose of about 50 mg / kg of body weight.

[0161] In some embodiments, the agent is administered to the subject at a dose of about 1 to 10, about 10 to 20, about 20 to 30, about 30 to 40, about 40 to 50, about 50 to 60, about 60 to 70, about 70 to 80, about 80 to 90, about 90 to 100, about 100 to 110, about 110 to 120, about 120 to 130, about 130 to 140, about 140 to 150, about 150 to 160, about 160 to 170, about 170 to 180, about 180 to 190, about 190 to 200, about 200 to 210, about 210 to 220, about 220 to 230, about 230 to 240, about 240 to 250 mg / kg.

[0162] In some embodiments, the methods of the present disclosure have reduced or no side effects (e.g., reduced or no systemic and / or autonomic side effects) while preventing, delaying progression, or treating osteoarthritis in a subject (e.g., a mammal) in need thereof. In some embodiments, the methods of the present disclosure have reduced or no side effects (e.g., reduced or no systemic and / or autonomic side effects) while regulating chondrocyte metabolism in a subject (e.g., a mammal) in need thereof. In some embodiments, the methods of the present disclosure have reduced or no side effects (e.g., reduced or no systemic and / or autonomic side effects) while preventing or reducing cartilage loss and / or joint damage in a subject (e.g., a mammal) in need thereof. In some embodiments, the methods that have reduced or no side effects (e.g., reduced or no systemic and / or autonomic side effects) comprise administering to the subject (e.g., a mammal) an effective amount of an agent that reduces or inhibits a function orYale Ref. YV 8761WO || TP Ref.251609.000125 expression of Nav 1.7 protein via an intra-articular route. Several studies have shown autonomic side effects such as hypotension after oral administration of Nav1.7 blockers (See Hackos et al, and Regan et al; incorporated herein by reference in its entirety). An intra-articular injection of the agent is expected to reach the chondrocytes and thereby can prevent side effects including autonomic side effects, such as hypotension, and / or systemic side effects. The substantial joint protection and reduction in pain were observed with carbamazepine, a non-selective Nav blocker, with a good safety profile (no hypotension at effective doses).

[0163] In some embodiments, the agent is administered at a dose not to exceed about 1 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 5 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 10 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 20 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 30 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 40 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 50 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 60 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 70 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 80 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 90 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 100 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 110 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 120 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 130 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 140 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 150 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 160 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 170 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 180 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 190 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 200Yale Ref. YV 8761WO || TP Ref.251609.000125 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 210 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 220 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 230 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 240 mg / kg body weight. In some embodiments, the agent is administered at a dose not to exceed about 250 mg / kg body weight.

[0164] In some embodiments, the agent is administered in a sustained release dosage form. Such sustained release dosage forms may provide a longer period of pharmacologic action after administration than the other dosage form. The sustained release dosage form of the Nav 1.7 inhibitor may allow to delay absorption of the inhibitor until it has reached certain site of action. Thus, sustained release dosage form can reduce incidences of adverse reactions. In some embodiments, the agent is administered in a sustained release dosage form via an intra-articular route to the subject (e.g., a mammal) in need thereof. The sustained intra-articular dosage form can provide better effects in preventing joint damage compared to systemic delivery.

[0165] Toxicity and therapeutic efficacy of agents can be analyzed by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50(the dose lethal to 50% of the subjects) or the ED50(the dose therapeutically effective in 50% of the subjects). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. While agents that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue, e.g., a joint, in order to minimize potential damage to unaffected cells and, thereby, reduce side effects.

[0166] The data obtained from in vitro assays and animal studies can be used in formulating a dose range for use in mammals (e.g., humans or veterinary or companion animals (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.)). For any agent used in the methods of the present disclosure, the therapeutically effective dose can be estimated initially from in-vitro assays. A dose can be formulated in animal models. Such information can be used to optimize efficacious doses for administration to mammals (e.g., humans or veterinary or companion animals (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.)).Yale Ref. YV 8761WO || TP Ref.251609.000125

[0167] The exact dose to be employed in the formulation will also depend on the seriousness of the disease or disorder, e.g., osteoarthritis, and should be decided according to the judgment of the practitioner and each subject’s circumstances. Normal dose ranges used for particular therapeutic agents employed for specific diseases can be found in the Physicians’ Desk Reference 54thEdition (2000).

[0168] In some embodiments, the methods of the present disclosure further comprise assessing the cartilage loss and / or joint damage in the subject (e.g., a mammal) before and / or after the treatment. In some embodiments, the cartilage loss is determined by assessing articular cartilage destruction by the OARSI grade, assessing osteophyte formation and / or assessing subchondral bone plate (SBP) thickness. In some embodiments, the methods of the present disclosure further comprise assessing the distance of movement and / or mechanical allodynia in the subject (e.g., a mammal) before and / or after the treatment. In some embodiments, the methods of the present disclosure further comprise determining level of HSP70 and / or midkine in the blood of the subject (e.g., a mammal) before and / or after the treatment. In some embodiments, the methods of the present disclosure further comprise determining level of HSP70 and / or midkine in the chondrocyte secretome of the subject (e.g., a mammal) before and / or after the treatment.

[0169] The inhibition of Nav1.7 protein after exposure to the agent capable of reducing or inhibiting a function or expression of Nav 1.7 protein may be determined in animal osteoarthritis models. Non-limiting examples of useful animal models for analyzing Nav 1.7 inhibition upon exposure to the agents described herein in osteoarthritis include surgical destabilization of the medial meniscus (DMM) model, monosodium iodoacetate (MIA) induced model, or any other animal models of osteoarthritis existing in the art.

[0170] In some embodiments, the subjects are the subjects with unilateral or bilateral symptomatic knee OA (as described by the American College of Rheumatology (ACR) criteria) visual analog score (VAS), or osteoarthritis according to Kellgren-Lawrence grade. Efficacy of the agent in preventing, delaying progression or treating osteoarthritis can be determined by analyzing VAS scores, WOMAC (Western Ontario and McMaster Universities Osteoarthritis) index of the total and safety indicators such as WOMAC Function, WOMAC Pain, WOMAC Stiffness, WOMAC Total, or incidence of adverse events (AE).Yale Ref. YV 8761WO || TP Ref.251609.000125

[0171] In some embodiments, the subject is a warm-blooded animal, preferably a mammal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal or a veterinary animal (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.). Methods Of Analyzing Activity Of Nav 1.7 Ion Channel In Cells

[0172] Although expression of VGSCs within excitable cells is well-known8, they have also been observed in cell-types that are not considered electrically excitable, including astrocytes, microglia, macrophages, and cancer cells19. Cartilage is avascular and aneural, but angiogenesis and sensory nerve growth into OA cartilage may contribute to OA pain20. In addition, the presence of tetrodotoxin (TTX) sensitive VGSCs in rabbit chondrocytes has been reported2. Nevertheless, although there is evidence that the aberrant activation of VGSCs contributes to OA pain21, the presence and function of VGSC(s) in chondrocytes and their roles in OA progression and pain remain essentially unknown.

[0173] In one aspect, provided herein is a method of assaying activity of Nav 1.7 ion channels in cells comprising low densities of sodium (Na) channels. In some embodiments, the method of assaying activity of Nav 1.7 ion channels in cells comprising low densities of sodium (Na) channels, said method comprising: (i) recording of Na currents produced by Nav 1.7 channels in a low-noise environment using a patch clamp; and (ii) enhancing a signal / noise ratio by averaging multiple runs for each voltage clamp trial.

[0174] In certain embodiments of the method of assaying activity of Nav 1.7 ion channels, the cells are non-excitable cells. The non-excitable cells can be the cells comprising low densities of sodium (Na) channels. In some embodiments, the non-excitable cells are chondrocytes, fibroblasts, astrocytes, macrophages, microglia, or cancer cells.

[0175] In some embodiments, the method of assaying activity of Nav 1.7 ion channels further comprises use of one or more pharmacological agents and subtraction to isolate the Na currents produced by Nav 1.7 channels.

[0176] In some embodiments, the pharmacological agents are the agents described herein that are capable of reducing or inhibiting a function or expression of Nav 1.7 protein. In some embodiments, the agents that are capable of reducing or inhibiting a function or expression ofYale Ref. YV 8761WO || TP Ref.251609.000125 Nav 1.7 include a small molecule, an antibody, a peptide, an anti-sense oligonucleotide, an RNAi molecule (e.g., an siRNA), a gene therapy, an aptamer or any combinations thereof.

[0177] In certain embodiments, the agents that are capable of reducing or inhibiting a function or expression of Nav 1.7 are selective Nav inhibitors or non-selective Nav inhibitors. Non-limiting examples of selective Nav1.7 blockers or selective Nav 1.7 inhibitors include PF- 04856264, ProTx II, PF-051980047, PF-05089771, PF-05153462, PF-05150122, PF-05186462, PF-05241328, PF-06456384, GNE-3565, SSCI-1, GDC-0276, MK-2075, OLP-1002, iN1011- N17, ST-2262, CC8464, AM-8145, AM-0422, AM-6120, JzTx V, μ-theraphotoxin-Pn3a, GpTx- I, GpTx I-71, Huwentoxin-IV (HwTx-IV) or mutants thereof, m3-HwTx-IV, Df1a, GX-201, GX- 044, GX-585, GX-674, MRL5, Cd1a, DSP-2230, GDC-0287 (RG7893), ST-2427, ST-2578, GDC-0310, NKTR-171, CNV-3000223, CNV-3000164, μ-SLPTX-Ssm6a, antibodies that can bind to sensor S3-S4 loop (Lee et al., Cell.2014 Jun 5;157(6):1393-1404), or derivatives thereof and any combinations thereof. In some embodiments, the agent is PF-04856264 or derivatives thereof. In some embodiments, the agent is Pro Tx II or derivatives thereof. In some embodiments, the agent is PF-05089771 or derivatives thereof. In some embodiments, the agents that are capable of reducing or inhibiting a function or expression of Nav 1.7 are sulfonamide- based inhibitors.

[0178] Non-limiting examples of non-selective Nav1.7 blockers or non-selective Nav1.7 inhibitors include carbamazepine (CBZ), oxcarbazepine (OXA), eslicarbazepine acetate (ESL), lacosamide (LCM), tetrodotoxin (TTX), XEN402 (also known as funapide or TV-45070), vixotrigine (also known as raxatrigine, CNV1014802 or BIIB074), lamotrigine, bupivacaine (BPV), vinpocetine (VPC), hardwickiic acid (HDA), lidocaine, phenytoin, ralfinamide (formerly NW-1029, FCE-26742A, PNU-0154339E), saxitoxin (STX), phlotoxin-1, BIA 2-093, BIA 2- 024, CNV-1061436, or derivatives thereof and any combinations thereof. In some embodiments, the agent is carbamazepine (CBZ) or derivatives thereof. In some embodiments, the agent is lacosamide (LCM) or derivatives thereof. In some embodiments, the agent is oxcarbazepine (OXA) or derivatives thereof. In some embodiments, the agent is eslicarbazepine acetate (ESL) or derivatives thereof. In some embodiments, the agent is BIA 2-093 or derivatives thereof. In some embodiments, the agent is BIA 2-024 or derivatives thereof. In some embodiments, the agent is lamotrigine or derivatives thereof. In some embodiments, the agent is phenytoin or derivatives thereof.Yale Ref. YV 8761WO || TP Ref.251609.000125 Kits

[0179] In one aspect, provided herein are kits comprising any of the various compositions described herein, including an agent that reduces or inhibits a function or expression of Nav1.7 protein.

[0180] In some embodiments, a kit may comprise an agent that reduces or inhibits a function or expression of Nav1.7 protein as described herein, a polynucleotide as described herein, or a vector as described herein, and (ii) optionally, packaging for the same and / or instructions for use.

[0181] In some embodiments, a kit can comprise: (a) a container that contains a pharmaceutical composition described herein, for example, a pharmaceutical composition in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstituting solution for the lyophilized formulation; and / or (c) optionally, instructions for (i) use of the solution or (ii) reconstitution and / or use of the lyophilized formulation.

[0182] In some embodiments, a kit further comprises, one or more of (i) a diluent, (ii) a buffer, (iii) a filter (iv) a syringe, and / or (v) a needle.

[0183] In some embodiments, kits may comprise a lyophilized formulation described herein in a suitable container and instructions for its reconstitution and / or use. Non-limiting examples of suitable containers include, e.g., syringes (such as dual chamber syringes), vials (such as dual chamber vials), bottles, and test tubes. In various embodiments, a container may be a multi-use container. The container can be formed from a variety of materials such as plastic or glass. The kit or container may contain instructions upon or accompanying the container which can denote directions for reconstitution of, e.g., a lyophilized formulation and / or use of the kit. In some embodiments, a label may denote that the lyophilized formulation is to be reconstituted to an appropriate concentration. The label may denote that the formulation is useful or intended for any route of administration disclosed herein.

[0184] The container containing the formulation may be a multi-use vial, which may allow for repeat administrations (e.g., from 2-6 administrations) of a reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (e.g., sodium bicarbonate solution).Yale Ref. YV 8761WO || TP Ref.251609.000125

[0185] Upon mixing of the diluent and a lyophilized formulation, a final concentration in the reconstituted formulation can be reached. The kit may further include other materials desirable from a commercial and / or user perspective, including, e.g., other filters, needles, syringes, buffers, diluents, and / or package inserts which may comprise, e.g., instructions for use.

[0186] Kits may contain a single container that contains the formulation of the pharmaceutical composition with or without other components (e.g., other compounds or pharmaceutical compositions of such other compounds) or may have a separate container for each component.

[0187] Kits may include a formulation of the disclosure packaged for use in combination with the co-administration of a second compound (such as adjuvants, diagnostic agent(s), or additional therapeutic agent(s)) or a pharmaceutical composition thereof. The components of the kit may pre-mixed and / or pre-complexed or each component of the kit may be in a separate distinct container prior to administration to a patient.

[0188] In some embodiments, the container of a therapeutic kit may be a vial, flask, test tube, bottle, syringe, or any other means of enclosing a solid or liquid. When there is more than one component, the kit may contain a second vial or other container, which may allow for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. In some embodiments, a kit may contain an apparatus (e.g., syringes, one or more needles, pipettes, eye droppers, etc.) which may permit administration of agents of the disclosure which are components of the kit. Materials and Methods Used in Illustration of the Present Invention Mice

[0189] C57BL / 6 and Agc1-CreERT2 mice were obtained from The Jackson Laboratory. Nav1.8-Cre;Nav1.7flox / flox mice were mated with transgenic mice expressing Agc1-CreERT2 to obtain inducible Nav1.7 knockout mice in chondrocytes and both chondrocytes and DRGs. For activation of CreERT2 in adult mice, 150 mg / kg body weight of tamoxifen (Sigma, T5648) in sunflower seed oil (Sigma, S5007) was injected intraperitoneally into 10-week-old mice once a day for 5 consecutive days. Littermate controls were used for all experiments. All animals were housed on a 12-hour light-dark cycle with ad libitum access to food and water in a specific pathogen-free environment. Animals were maintained on a C57BL / 6J background, and ageYale Ref. YV 8761WO || TP Ref.251609.000125 matched males typically at 12-weeks-of-age were used, unless otherwise specified in the figure legends. No statistical methods were used to predetermine sample size. All animal studies were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of New York University Grossman School of Medicine.

[0190] To establish the surgically induced DMM55model, after ketamine and xylazine anesthesia, the medial meniscotibial ligament in the right knee was sectioned with a blade to destabilize the medial meniscus. The chemically induced MIA OA model was established unilaterally via intra-articular injection of 0.1mg of MIA (Sigma, I2513) in 6 µl of 0.9% sterile saline with a 30-guage needle after anesthetization with ketamine and xylazine56. Mice with OA models were randomized to receive different treatment within a cage. PF-04856264 (Alomone labs, 1235397-05-3), at 30 µg / g body weight or 1.5 µg / g body weight was orally delivered or injected intra-articularly, respectively, daily over a 4-week period starting from the first day of MIA injection. PF-04856264 was injected intra-articularly at 1.5 µg / g body weight or orally delivered at 30 µg / g body weight every other day starting from 4 weeks after DMM surgery for a total of 8 weeks. CBZ (Sigma, C4024) was delivered through oral gavage at 250 mg / kg body weight daily over a 4-week period starting from the first day of MIA injection. Additionally, doses of 10, 50, or 250 mg / kg body weight CBZ were administered daily via oral gavage over an 8-week span, commencing 4 weeks after DMM surgery. To determine whether blocking HSP70 and / or midkine affected Nav1.7 blocker PF-04856264’s protective effects against OA in vivo, mice received oral administration of PF-04856264 at a dosage of 30 µg / g body weight every day, and they were simultaneously subjected to intra-articular injections of VER 155008 (Sigma, SML0271) at 0.55 µg / g body weight, iMDK (Tocris, 5126) at 0.9 µg / g, or a combined application of VER 155008 and iMDK daily starting from 4 weeks after DMM surgery for a total of 8 weeks. Behavior tests

[0191] OA-associated pain was measured using the von Frey assay and the open field travel analysis57three times before establishment of the OA model, and every week starting from 8 weeks after DMM surgery, or at day 2, 4, 8, 14, 20 and 28 post MIA injection. All the behavioral tests were conducted in a blinded manner and performed between the hours of 12:00 - 17:00. von Frey filaments (Stoelting) were applied with increasing force intensities on the plantar surface of the hind paw of the mouse which is placed in an elevated Plexiglas chamber with aYale Ref. YV 8761WO || TP Ref.251609.000125 metal grid floor that gave access to the plantar surface of the paws to determine the tactile pain threshold as based on a previous publication57. Rapid withdrawal of the hind paw was recorded as a positive response. Hind paws were subjected to 10 trials at a given intensity with a 30- second interval maintained between trials and the number of positive responses for each von Frey filament’s stimulus was recorded. Animals were considered to have reached tactile threshold when 5 out of 10 trials generated a positive response. For open field travel analysis, mice were placed individually in a square clear chamber (45 × 45 cm) and allowed to freely explore for 2 minute under normal lighting. Movement and trajectories of the mice were videoed and analyzed by a computerized system. Human subjects research

[0192] Human subjects research was performed according to the Institutional Review Boards at New York University Medical Center (institutional review board (IRB) Study Number i11-01488 and i9018)). Human OA cartilage samples were collected from patients receiving total knee joint replacement surgery for OA at New York University Langone Orthopaedic Hospital. Non-arthritic femoral condyle cartilage specimens were obtained from fresh osteochondral allografts discarded following donor plug collection during surgical osteochondral allograft implantation. Cartilage samples used are surgical discards, and no consent is required based on the approved IRB, as patient information was not collected except age, sex, and clinical diagnosis of the samples, such as Osteoarthritis. OA and non-arthritic cartilage specimens were stored in liquid nitrogen immediately after collection until protein or RNA extraction.

[0193] A total of 22 non-OA and 165 patients with knee symptomatic knee OA from the New York biomarker cohort58were included in this study according to the American college of rheumatology (ACR) criteria. The demographic data are summarized in Table 1. Informed consent was obtained from all participating subjects. The IRB of the New York University Grossman School of Medicine approved this study (No. i05-131).Yale Ref. YV 8761WO || TP Ref.251609.000125 Table 1. Demographic data of normal and Patients with OA

[0194] OA synovial fluid and serum samples were collected as part of an observational study to determine factors influencing knee OA pain improvement with hyaluronic acid visco- supplementation59. The synovial fluid samples were collected without joint lavage, and the volume ranged from 0.5 to 30 ml. The cell-free synovial fluids were prepared and frozen (−80 °C) within one hour of collection. Collection and storage of synovial fluids were approved (No.13-01257) by the IRB of the NYU Grossman School of Medicine. The demographic data are summarized in Table 2.Yale Ref. YV 8761WO || TP Ref.251609.000125 Table 2. Demographic data of Patients with OA with serum and synovial fluids

[0195] For the full-thickness cartilage explant assay, human tibia plateaus were obtained from 8 deidentified patients with OA undergoing total knee arthroplasty. For each individual patients with OA, 12 full-thickness cartilage explants were isolated from areas with various degrees of OA-related cartilage degeneration with a 3-mm biopsy punch and randomly distributed into three different groups and treated with 10 ng / ml IL-1β, 10 ng / ml IL-1β plus 25 nM ProTx II, or 10 ng / ml IL-1β plus 1 µM PF-04856264 in DMEM medium for 5 days. The supernatant was collected and spun at 200 g at 4 °C, followed by ELISA assay. Electrophysiology

[0196] Primary human chondrocytes from patients with OA were grown in 100 mm tissue culture dishes in growth medium DMEM (Gibco, 11995-065) supplemented with 10% FBS (Hyclone, SH30088.03) and 1x penicillin-streptomycin (ThermoFisher, 15070063). Chondrocytes were passaged every 5-7 days at 75-80% confluency no more than 3 times. Cells were plated at passaging into 12 mm round glass poly-D-lysin / laminin-coated coverslips (Corning, 354087) in 24-well plate format according to the following protocol: growth medium was removed, chondrocytes were rinsed once with 5 ml Ca2+and Mg2+-free DPBS (Gibco, 14190-144) and incubated for 3-5 mins with 1.5 ml 0.25% Trypsin / EDTA (Corning, 10222017), then cells were gently lifted off the dish and pipette-triturated in 8.5 ml of growth medium. Twenty-five microliters of homogenized chondrocytes suspension was diluted into 1 ml growth medium at each cover glass to reach optimal cell density and were maintained in growth medium for 3-6 days until electrophysiological recordings.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0197] Currents were recorded in whole-cell voltage clamp by Axopatch 200B amplifier (Molecular Devices). Recordings were low-pass filtered at 2 kHz and acquired at 100 kHz by Digidata 1440A DAC using Clampex 10.7 software (Molecular Devices). p / 4 leak subtraction protocol and sweep-averaging were used to subtract uncompensated leak and capacitance currents and to enhance signal / noise ratio. Pipettes were pulled from glass capillaries (PG52165- 4; WPI) and had resistance 2-3.5 MΩ when filled with intracellular solution (in mM): 140 CsF, 10 NaCl, 10 HEPES, 1 EGTA, 20 Dextrose, pH 7.3 with CsOH (328 mOsm / L with Sucrose). Extracellular solution contained (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES; 10 TEA-Cl, 10 Dextrose, pH 7.4 w / NaOH (327 mOsm / l). Solutions for sodium currents isolation were from10. The liquid junction potential was not compensated. Recordings were made at room temperature. Data were analyzed using pClamp 10.7 (Molecular Devices) and Origin 2022b (OriginLab) software. RNA extraction from human cartilage

[0198] For RNA isolation, about 1 g of cartilage was pulverized in liquid nitrogen and homogenized in Trizol at a concentration of 1 g tissue per 10 ml Trizol (Invitrogen, 15596026), followed by incubation at 4oC with rotating for 2 h. Samples were mixed with 0.2 volumes of chloroform, vortexed for 20 seconds, and centrifuged at 14000 rpm for 20 minutes at 4oC. The aqueous phase was collected and gently mixed with an equal volume of isopropanol, followed by centrifugation at 14000 rpm for 20 minutes at 4oC. The resulting pellet was suspended in 350 µl of RLT buffer and processed for cleanup using the RNeasy Mini Kit (Qiagen, 74104) following the manufacturer’s instructions. RNA assay by qRT-PCR

[0199] Total RNA extracted from chondrocytes or human cartilage was reverse- transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4387406). qRT-PCR was performed in triplicate with SYGR-Green (Applied Biosystems, A25780) using human or mouse primers to Acan, Col2, Mmp13, Adamts5, Cox2, Nos2 and Gapdh (Applied Biosystems Real-time PCR system). mRNA levels were normalized to Gapdh and reported as relative mRNA fold change. Histology

[0200] Human cartilage or mouse knee joints were fixed in 4% paraformaldehyde for 24 hours before decalcification in 10% w / v EDTA for 2 weeks before paraffin embedding. TheYale Ref. YV 8761WO || TP Ref.251609.000125 paraffin blocks were sectioned at a thickness of 5µm and serial sections were subjected to Safranin-O or haematoxylin and eosin (H&E) staining. Cartilage destruction was graded on Safranin-O stained sections by blinded observers using the OARSI histology scoring system (grade 0-6)60. Osteophyte development (grade 0-3)61was evaluated and the thickness of the subchondral bone plate62was measured on Safranin-O or H&E-stained sections. Synovitis (grade 0-9) was determined based on the synovial lining cell layer enlargement, resident cell density, and inflammatory infiltration on H&E-stained sections63.

[0201] For immunohistochemical staining, deparaffinized and hydrated sections were incubated with 0.1% trypsin for 30minutes at 37oC, followed by 0.25 U / ml chondroitinase ABC (Sigma-Aldrich, C3667) and 1 U / ml hyaluronidase (Sigma-Aldrich, H3560) for 60minutes at 37oC, respectively. After blocking, the sections were incubated with antibodies against Nav1.7 (1:50, Alomone Labs, ASC-008), COL2 (Invitrogen, cat. no. MA5-12789), COMP fragment64(1:200, affinity-purified monoclonal), aggrecan neoepitope (1:100, Millipore, AB8135), and MMP13 (1:200, Abcam, ab3208) overnight at 4oC. Detection was performed using the Vectastain Elite ABC kit (Vector Laboratories, PK6100), and the positive signal was visualized with 0.5 mg / ml 3,3-diaminobenzidine in 50 mM Tris-Cl substrate (Sigma-Aldrich, D12384) and then counterstained with 1% methyl green (Sigma-Aldrich, 67060). Images were acquired with a Zeiss microscope. Semi-quantification analysis of the density of immunohistochemical staining for Aggrecan neoepitope, COMP fragment and collagen X was performed by Image J, and the same signal threshold was used for each group of similar immunohistochemical images65. Cell culture

[0202] Primary articular chondrocytes were isolated from the femoral condyles and tibial plateaus of Nav1.7floxand Nav1.7chondrocytemice on postnatal day 666. Chondrocytes were maintained as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 50 U / ml penicillin, and 0.05 mg / ml streptomycin. Articular chondrocytes at culture day 2 were treated as indicated for each experiment.

[0203] Primary articular chondrocytes were isolated from the femoral condyles and tibial plateaus of Nav1.7floxand Nav1.7chondrocytemice at 12 weeks after DMM surgery. Chondrocytes were maintained as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 50 U / ml penicillin, and 0.05 mg / ml streptomycin. After 5 days, the conditioned medium was collected and spun at 200g at 4 °C, followed by ELISA assay.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0204] Human C28I2 chondrocytes were grown in DMEM medium supplemented with 10% FBS, 50 U / ml penicillin, and 0.05 mg / ml streptomycin. To knockdown Nav1.7, cells were transfected with commercially available siRNA (S534077, A134907) using Lipofectamine (Invitrogen, 13778100) as instructed by manufacture’s protocol. To fraction condition medium, C28I2 cells were stimulated with or without 25nM ProTx II or PF-04856264. In Brief, C28I2 cells were cultured with DMEM medium supplemented with 10% FBS, 50 U / ml penicillin and 0.05 mg / ml streptomycin. When cells reached 80% confluency, the medium was changed to DMEM supplemented with ITS Liquid Media Supplement (Sigma-Aldrich, I3146) containing 25nM ProTx II or PF-04856264 for 2 days. The conditioned medium was then collected. After centrifugation to get rid of the cell debris, the medium was then fractioned based on molecular weight to >100 kDa, 30-100 kDa, 10-30 kDa and <10 kDa using Amicon Ultra-2 Centrifugal Filter Units sequentially (Fisher Scientific, UFC210024, UFC203024 and UFC201024). PF- 04856264 and ProTx II have molecular weight of 437.492 and 3,826.65 Da, respectively. To exclude the effects of PF-04856264 and ProTx II in conditioned medium, ProTx II was depleted using Dialysis Tubing that allows the removal of molecules with molecular weights between 3.5- 5KD (Micro Float-A-Lyzer 3.5 - 5 kDa, F235053, Thomas Scientific), while PF-04856264 was simply removed through dialysis against the medium. Conditioned medium with molecular weight of 30-100 kDa and 10-30 kDa were then analyzed by mass spectrometry, performed by NYU Proteomics Laboratory. All MS / MS spectra were collected using the following instrument parameters: resolution of 15,000, automatic gain control (AGC) target of 1e5, maximum ion time of 120 ms, one microscan, 2 m / z isolation window, fixed first mass of 150 m / z, and normalized collision energy (NCE) of 27. MS / MS spectra were searched against a UniProt Human database using Sequest within Proteome Discoverer 1.4. Western blotting

[0205] Western blot analyses were conducted with protein lysates from primary human cartilage and C28I2 cells. To determine the membrane localization of Nav1.7 in chondrocytes, cytosolic and membrane fractions of human C28I2 cells were extracted with Men-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, 89842) and subjected to western blotting analysis. The following primary antibodies were used: Nav1.7 (1:500, Alomone Labs, ASC-008), TNFR2 (1:1,000, ProteinTech, 19272-1-AP), and GAPDH (1:5,000, ProteinTech, 60004-1-Ig).Yale Ref. YV 8761WO || TP Ref.251609.000125 ELISA

[0206] The levels of HSP70 and midkine in human sera and synovial fluid from healthy individuals and patients with OA, in conditioned medium and cell lysates of human C28I2 cells treated with 25 nM ProTx II or 1 µM PF-04856264 were measured by ELISA according to the manufacturer’s instructions, respectively (Abcam, ab133060, ab193761). Before ELISA analysis, human synovial fluids were digested with hyaluronidase at 1 unit per 100 µl synovial fluid for 1 hour at 37oC. The levels of PRG4 and MMP13 in supernatant of human OA cartilage explants were measured by ELISA according to the manufacturer’s instructions, respectively (R&D systems, DY9829-05; Abcam, ab100605). The levels of HSP70 and midkine in mouse sera and conditioned medium collected from primary chondrocytes were measured by ELISA according to the manufacture’s instruction (Abcam, ab133061, ab279416). Na+and Ca2+fluorescence imaging

[0207] Human OA chondrocytes or C28I2 cells were seeded on 8-well chamber (Thermo Fisher, 154461) and loaded with 5µM CoroNa Green (Invitrogen, C36676) or Fluo-8 (Abcam, ab112129) in Hanks' Balanced Salt Solution for 45 minutes at 37oC in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264 with or without 0.5 µM KB-R7943 (Tocris, 1244). CoroNa Green and Fluo-8 were excited at 488 nm and fluorescence images (525-530 nm) were acquired with 25x water-dipping objective on Zeiss 880 confocal microscope every 2 seconds during the experiment. Na+and Ca2+transients in chondrocytes were induced by 100nM ATP in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Fluorescence was expressed as the ratio of cytosolic fluorescence and initial intensity (F / F0). Measurement of intracellular Ca2+with plate reader

[0208] Intracellular Ca2+in human OA chondrocytes or C28I2 cells was measured using Fluo-8 Calcium Flux Assay Kit (Abcam, ab112129) according to the manufacturer’s instructions. In brief, cells seeded in black walled 96-well plate (Corning, 3904) were loaded with Fluo-8 in Hanks' Balanced Salt Solution for 30 minutes at 37oC and 30 minutes at room temperature in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Ca2+transient in chondrocytes were induced by 100 nM ATP in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Fluorescence was measured in a fluorescent plate reader with an excitation wavelength of 490 nm and emission wavelength of 525 nm, and expressed as the ratio of cytosolic fluorescence and initial intensity (F / F0).Yale Ref. YV 8761WO || TP Ref.251609.000125 Statistical analysis

[0209] All data are presented as mean ± s.d., unless otherwise specified in the figure or table legends. The numbers of mice used per genotype are indicated in figure legends. Comparisons between the two groups were analyzed using two-tailed unpaired Student’s t-test unless stated otherwise in the figure legends. ANOVA with post hoc Bonferroni test was used when comparing multiple groups as described in the figure legends. A value of P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 9. EXAMPLES

[0210] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. Example 1. Nav1.7 in chondrocytes, elevated in OA

[0211] The expression profile of VGSCs in human chondrocytes was assessed via reverse transcription polymerase chain reaction (RT-PCR) and found that, SCN2A, SCN3A, SCN4A, SCN8A, SCN9A, and SCN11A were expressed in chondrocytes (Fig.5A). In line with this data, analysis of the genes that were differentially regulated between OA cartilage (Kellgren- Lawrence (KL) Grade 3 or 4) and non-arthritic cartilage using the previous RNA-seq dataset (GSE168505)22with a particular interest in VGSCs, indicated that six VGSCs were expressed in chondrocytes (Fig.5B). However, encoding Nav1.7 mRNA (encoded by SCN9A), was the only VGSC transcript that was prominently upregulated (2.69 fold increased, P < 0.05) in OA cartilage compared with non-arthritic cartilage (Fig.5B). Among six VGSCs expressed in human chondrocytes, SCN9A expression was significantly induced by TNFα and IL-1β, pro- inflammatory cytokines associated with OA (Fig.5C)4. Quantitative PCR with reverse transcription (RT-qPCR) analysis of Nav1.7 expression using mRNAs obtained from independent cartilage specimens (11 non-arthritic, 14 KL grade 1-2 OA, and 22 KL grade 3-4 OA) confirmed that the mRNA expression of Nav1.7 mRNA expression was significantly increased in both KL grade 1-2 and KL grade 3-4 OA cartilage compared with non-arthritic cartilage (Fig.5D). Nav1.7 protein level was also increased in all grades of radiographic severity (KL 1-4) of human OA compared with non-arthritic controls (Fig.5E). Membrane localization of Nav1.7 in chondrocytes was confirmed following fractionation of human chondrocytes andYale Ref. YV 8761WO || TP Ref.251609.000125 western blotting by using cytosolic and membrane fractions (Fig.5F). Immunohistochemical staining demonstrated increased expression of Nav1.7 in both human and mouse OA cartilage (Fig.5G, 5H). Collectively, these data demonstrated that Nav1.7 was expressed in chondrocytes and associates with OA progression. Example 2. OA chondrocyte Nav1.7 electrophysiology

[0212] The presence of Nav1.7 currents was assessed in 77 human chondrocytes isolated from three patients with OA (Table 3). First, 1 µM TTX (Tetrodotoxin) which blocks all Nav channels except Nav1.5, Nav1.8 and Nav1.9 was used, to isolate TTX-sensitive (TTX-S) currents which were obtained by subtraction of TTX-resistant (TTX-R) currents from currents in control solution at the respective voltages, Fig.1A shows representative current traces in response to -50 mV, -30 mV, -10 mV and 10 mV test pulses in control solution (Fig.1A, left), in the presence of 1 µM TTX (Fig.1A, middle), and the resulting traces of TTX-S current (Fig.1A right). Sodium currents were elicited by test voltages (-60 mV to 50 mV in 10 mV increments) applied from -90 mV holding potential. Because the current amplitude was relatively low, the signal / noise ratio was enhanced by averaging eight runs for each current-voltage trial. Consistent with a substantial contribution of Nav1.7, TTX-S current began to activate at -40 mV threshold, and exhibited maximal peak amplitude at 0 mV and sodium current reversal potential (Er) = - 60.6 mV ± 2.3 mV (n = 3) (Fig.1B). G / Gmaxof TTX-S current was calculated from current- voltage data, averaged at the respective membrane voltages (n = 3), and fitted with the Boltzmann equation with voltage for half-maximal activation (V1 / 2) = -14.2 mV ± 0.8 mV, slope coefficient (k) = 6.1 mV ± 0.2 mV (n = 3) (Fig.1C). On kinetic analysis, the falling phases of TTX-S current traces were best fitted with a single exponential. Inactivation time constants (single exponential fits of the falling phase) were voltage-dependent, gradually decreasing with depolarizing membrane voltage from 3.3 ± 0.8 ms at -20 mV to 0.3 ± 0.1 ms (n = 3) at 50 mV membrane voltages (Fig.1D). Time to peak of TTX-S current was measured from the voltage step onset and gradually decreased from 1.8 ± 0.2 ms at -20 mV to 0.5 ± 0.1 ms (n = 3) at 50 mV membrane voltages (Fig.1E).Yale Ref. YV 8761WO || TP Ref.251609.000125 Table 3. Expression of fast-inactivation sodium currents in human OA chondrocytes

[0213] To further establish that the TTX-S currents included a Nav1.7 component, the time-course of the TTX-S and ProTx II (Protoxin-II) -sensitive (ProTx II-S) currents were compared. Inhibition by 1 µM TTX was observed in 4 / 4 cells studied. Inhibition by 20 nM ProTx II, which selectively blocked Nav1.7, was seen in 5 / 5 cells studied, confirmed the presence of Nav1.7.

[0214] To assess the effect of ProTx II, sodium currents were recorded before (Fig.1F, black solid trace), and after application of 20 nM ProTx II (Fig.1F, black dotted trace), each trace was an average of 15-30 consecutive sweeps with 2-3 s inter-sweep interval; ProTx II-S current (Fig.1F, black solid thick line) was obtained by point-by-point subtraction of current in the presence of ProTx II from the respective current recorded in control. Similarly, current sensitive to 1 µM TTX (Fig.1G, gray trace) was obtained. Supporting the notion that the TTX-S currents were largely produced by Nav1.7, overlays of TTX-S (Fig.1H, gray trace) and ProTx II-S (Fig.1H, black trace) current traces normalized by peak current amplitudes showed similar time-courses of TTX-S and ProTx II-S currents. Consistent with the observation of the similarities of TTX-S and ProTx II-S current time-courses, their kinetic parameters were not significantly different. Inactivation time constants, obtained from a single exponential fit of TTX-S and ProTx II-S currents elicited by 0 mV test voltage from -90 mV holding potential, were 1.0 ms ± 0.1 ms (n = 4) and 1.0 ms ± 0.2 ms (n = 5) (Fig.1I), respectively, and their difference was not statistically significant (P > 0.05). Time-to-peak values of TTX-S and ProTx II-S currents at 0 mV membrane voltage were 1.4 ms ± 0.3 ms (n = 4) and 1.5 ms ± 0.3 ms (n =Yale Ref. YV 8761WO || TP Ref.251609.000125 5) (Fig.1I), respectively, and were not statistically different (P > 0.05). These results added to the evidence that Nav1.7 contributes a substantial proportion of the Na+current in OA chondrocytes.1 µM TTX inhibited total sodium current (average amplitude 141 pA ± 11 pA; n = 4) by 61.8% ± 16.4% (n = 4); TTX-S current amplitude at 0 mV was 87 pA ± 13 pA (n = 4) (Fig. 1J); persistent (slow-inactivating, measured from control traces at 10 ms of test pulse onset) current amplitude was 41 pA ± 16 pA (n = 4).20 nM ProTx II reduced total sodium current (93 pA ± 14 pA) by 39.5% ± 13.5% (n = 5); ProTx II-S current amplitude at 0 mV was 37 pA ± 6 pA (n = 5); persistent (slow-inactivating, measured from traces in control at 10 ms of test pulse onset) current amplitude was 31 pA ± 9 pA (n = 5) (Fig.1J).

[0215] Together, these results established the presence of Nav1.7 in OA chondrocytes by demonstrating that Na+currents in these cells were sensitive to TTX, gating and kinetic properties similar to those known for Nav1.7, and inhibition by 20 nM ProTx II, a selective Nav1.7 blocker. The percentage of cells expressing Nav1.7 were seen from the patch clamp recordings, which were obtained from 77 human OA chondrocytes. Inward sodium currents with a fast-inactivating (millisecond time constant at 0 mV test voltage, Fig.1D) component and peak amplitude above 30 pA were evoked in 17% of cells. Average inward current amplitude at 0 mV test potential in these cells was 82 ± 19 pA (n = 13), and average current density was 2.4 ± 0.4 pA / pF (n = 13). A summary of the sodium current in human chondrocytes from three patients with OA is presented in Table 3. Example 3. Nav1.7 deletion in chondrocyte protected OA

[0216] To examine the role of Nav1.7 in chondrocytes in OA and determine the relative contribution of DRG- and chondrocyte-expressed Nav1.7 to OA progression and pain, mice were generated with Nav1.7 knockout in DRG neurons (hereafter referred to as Nav1.7DRG; Nav1.7 is encoded by Scn9a), chondrocytes (hereafter referred to as Nav1.7chondrocyte), and both DRG neurons and chondrocytes (hereafter referred to as Nav1.7DRG;chondrocyte), respectively, by crossing Nav1.7-floxed (Nav1.7flox) mice with Nav1.8-cre (Nav1.8 is encoded by Scn10a) mice and / or Agc1-CreERT2(Agc1 is also known as Acan) mice, in which Cre-mediated recombination was induced by tamoxifen (Fig.6A). Nav1.7 deletions in DRG and chondrocyte following tamoxifen administration in adult mice were confirmed (Figs.6B-6D).

[0217] The two most common methods to experimentally model OA in mice include surgical and chemical induction. As in humans, both surgically and chemically induced murineYale Ref. YV 8761WO || TP Ref.251609.000125 OA models exhibited articular cartilage erosion or loss and OA related pain23,24. Both surgically induced destabilization of the medial meniscus (DMM) (Fig.2) and chemically induced monoiodoacetate (MIA) models (Fig.7A) in Nav1.7floxand Nav1.7DRG;chondrocytemice were established. Histological analysis revealed that Nav1.7 deletion in both DRG neurons and chondrocytes substantially attenuated cartilage loss, and reduced the Osteoarthritis Research Society International (OARSI) score (Figs.2A, 2B, 7B, and 7C) in both DMM and MIA OA models. Notably, Nav1.7DRG;chondrocytemice exhibited markedly reduced osteophyte formation, thickening of subchondral bone plate, suggestive of sclerosis, and decreased synovitis score in the DMM model (Figs.2C-2E). The association of Nav1.7 with OA pain was evaluated by measuring open field movement activity and mechanical allodynia with von Frey testing. Nav1.7DRG;chondrocytemice exhibited greater overall distance of movement and significantly reduced mechanical allodynia throughout the three-month period after DMM surgery and the four weeks period post MIA injection relative to Nav1.7floxmice (Figs.2F-2H and 7D-7F). Immunohistochemical staining of knee joints indicated that OA associated loss of the anabolic marker type II collagen (Col2), and increase of MMP13, aggrecan neoepitope, generated via cleavage by ADAMTS5, and COMP fragment observed in Nav1.7floxmice, were inhibited in Nav1.7DRG;chondrocytemice in both surgically and chemically induced OA (Figs.7G, 7H, 7I, 7J, 7K and 7L). These findings indicated that chondrocyte- and DRG neuron- expressed Nav1.7 concurrently contributed to modulating OA progression and OA-associated pain.

[0218] To distinguish the contributions of Nav1.7 expressed by chondrocyte- and DRG neurons towards progression of OA pathology and OA-associated pain, the DMM OA model in Nav1.7floxand Nav1.7chondrocyteor Nav1.7DRGmice was established. Similar to the observations in Nav1.7DRG;chondrocyteDMM mice, Nav1.7chondrocytemice also displayed substantial reductions in DMM-induced cartilage loss with reduced osteophyte formation, subchondral bone plate thickening, and synovitis score compared with Nav1.7floxmice (Fig.2I-2M). Deletion of Nav1.7 in chondrocytes reduced the loss of open field movement activity and mechanical allodynia (von Frey test) in the DMM model (Fig.2N-2P). In addition, deletion of Nav1.7 in chondrocytes increased the amount of anabolic effector Col2, and simultaneously decreased the levels of DMM-induced catabolic effectors including MMP13, aggrecan neoepitope, and COMP fragment (Figs.8A, 8B, and 8C). By contrast, deletion of Nav1.7 in DRG neurons reduced DMM-induced pain without affecting structural abnormalities such as cartilage destruction, osteophyteYale Ref. YV 8761WO || TP Ref.251609.000125 formation, subchondral bone plate thickening, and synovitis score (Figs.8D-8K). Immunohistochemical staining revealed that there was no difference between Nav1.7floxand Nav1.7DRGDMM mice in terms of the amounts of COL2, MMP13, aggrecan neoepitope, and COMP fragment in cartilage (Figs.8L, 8M, and 8N), supported the notion that Nav1.7 deletion in DRG neurons was not involved in the regulation of cartilage loss. In sum, Nav1.7 expressed in chondrocytes modulated OA progression and resultant OA-associated pain behavior, whereas Nav1.7 expressed by DRG neurons contributed to OA-associated pain without affecting OA progression. Example 4. Nav1.7 inhibition protected against OA

[0219] It was then sought to determine whether pharmacological Nav1.7 blockade with PF-04856264, a selective Nav1.7 blocker25, could inhibit cartilage destruction and alleviate pain. To this end, first DMM OA model in 12-week old C57BL / 6 wild-type (WT) male mice was established, followed by intra-articular injection of vehicle or PF-04856264 at 1.5 µg / g body weight every other day beginning 4 weeks post-operatively for a total of 8 weeks (Fig.9A). Safranin O staining revealed that PF-04856264 remarkably protected the structure of articular cartilage and maintained proteoglycan content in the cartilage compared with vehicle (Fig.9B). PF-04856264 treatment was associated with significantly lower OARSI scores, osteophyte development and subchondral bone plate thickness relative to vehicle treatment (Figs.9C-9E). In addition, DMM-induced OA pain behavior was reduced and COL2 level was increased in Nav1.7 blocker-treated mice (Figs.9F, and 9G). Accordingly, Col2 level was increased; by contrast, MMP13 and aggrecan neoepitope levels were significantly reduced in the cartilage of mice with DMM OA that received intra-articular injections of PF-04856264when compared with those treated with vehicle (Figs.9H, and 9I). To explore potential sex differences, the DMM OA model was replicated in female WT mice using the same treatment approach. Consistent with the observations in male mice, female mice treated with PF-04856264 exhibited less articular cartilage loss, lower OARSI score, and significant reduction in pain behavior as compared with untreated controls (Figs.9J-9O).

[0220] To further demonstrate the effectiveness of Nav1.7 blockade in preventing OA, the MIA model was established in WT male and female mice (Fig.10A). Since the route of drug delivery largely determines achieved drug concentrations at the target site and therapeutic efficacy, and because systemic delivery was well accepted by patients, the therapeutic effects ofYale Ref. YV 8761WO || TP Ref.251609.000125 systemic administration of PF-04856264 through oral gavage at 30 µg / g body weight in the MIA model were determined (Fig.10A). Similar to the results from local delivery, systemic delivery of PF-04856264 also markedly slowed OA progression as demonstrated by less cartilage loss, and improved OARSI score compared to vehicle treatment (Figs.10B-10D). Furthermore, systemic PF-04856264 substantially increased the distance of movement in the open field test and von Trey hind-paw withdrawal threshold (Figs.10E, and 10F). No apparent sex differences were observed, as PF-04856264 elicited cartilage loss protection and pain relief in both male and female mice (Figs.10B-10F). Immunohistochemical (IHC) staining revealed that oral PF- 04856264 increased COL2 expression and decreased MMP13 and aggrecan neoepitope levels in cartilage compared with vehicle (Figs.10G, and 10H). Oral delivery of PF-04856264 also reduced cartilage loss in mice with DMM-induced OA (Figs.10I, and 10J). Example 5. CBZ reduced cartilage loss and pain

[0221] Carbamazepine (CBZ) is a clinically used Na channel inhibitor that is known to act on Nav1.726and has been approved by the US Food and Drug (FDA)for indications in epilepsy, bipolar disorder and neuropathic pain. To determine whether this sodium channel inhibitor had therapeutic effects on OA, CBZ was systemically delivered into MIA mice by oral gavage (250mg / kg body weight, daily), which was equivalent to dosing used in treating epilepsy in humans taking species differences into account27, and was suggested to alleviate disease severity in a metaphyseal chondrodysplasia Schmid type mouse model28(Fig.11A). Systemic CBZ treatment closely recapitulated the therapeutic effects of systemic PF-04856264 treatment on OA in terms of attenuation of cartilage loss, alleviation of OA pain, and protection against changes in pro-anabolic and anti-catabolic effects in chondrocytes by upregulating COL2 expression and downregulating the expression of matrix-degrading enzymes (Figs.11B-11H). The therapeutic effects of varying doses of CBZ administered orally in DMM model were also evaluated (Fig.11I). In mice that underwent DMM surgery, a low dose of CBZ (10 mg / kg body weight) led to a significant reduction in cartilage loss 12 weeks after surgery. However, there was no significant difference in pain behavior compared to untreated DMM mice (Figs.11J- 11M). Medium (50 mg / kg body weight) and high (250 mg / kg body weight) doses of CBZ provided stronger protection against cartilage loss and significantly reduced OA-associated pain behavior compared with low dose CBZ (Figs.11J-11M). These findings suggested that non-Yale Ref. YV 8761WO || TP Ref.251609.000125 specific VGSC blockers such as CBZ may hold promise as novel disease-modifying treatments for OA, and did not simply act in an analgesic manner. Example 6. Nav1.7 blockade regulated chondrocyte biology

[0222] Given that Nav1.7 blockade led to pro-anabolic and anti-catabolic effects on chondrocytes in OA models, it was next sought to determine whether Nav1.7 blockade would affect chondrocyte biology in vitro. Expression of genes encoding catabolic molecules induced by IL-1β, including MMP13, ADAMTS5, COX2 (also known as PTGS2), and NOS2, were inhibited by 1 µM TTX (Figs.12A-12D). To further demonstrate the importance of Nav1.7 in chondrocyte biology, a pharmacological approach was used using Nav1.7 selective blockers and a loss-of-function Nav1.7 mutant approach. Two reported Nav1.7- selective inhibitors, ProTx II (a 30-residue peptide with IC50 = 0.3 nM)29and PF-04856264 (a small molecule Nav1.7 inhibitor with IC50 = 28 nM)25, were used to inhibit Nav1.7 pharmacologically. Similar to TTX, blockade of Nav1.7 with 25 nM ProTx II or 1 µM PF-04856264 (Figs.12A-12D) demonstrated the importance of Nav1.7 in inhibiting IL-1β-induced catabolism. To further assess whether Nav1.7 blockade effectively inhibited chondrocyte catabolism in an inflammatory OA environment in general, the effects of these inhibitors on chondrocyte catabolism in the presence of the additional inflammatory stimuli TNFα30and polyinosinic–polycytidilic acid (poly(I:C))31were evaluated. Simultaneous treatment of Nav1.7 blockers also significantly inhibited TNFα- and poly(I:C)- induced chondrocyte catabolism (Figs.12E-12L). The VGSC blockade with TTX, ProTx II or PF-04856264 also significantly induced the expressions of genes encoding anabolic molecules, such as collagen II (COL2) and aggrecan (ACAN)( Figs.12M, and 12N). Chondrocytes were also isolated from Nav1.7floxand Nav1.7chondrocytemice, and found that Nav1.7 deletion blocked IL-1β induced catabolism. Notably, Nav1.7 deletion did not change anabolism under physiological conditions (Figs.12O-12T).

[0223] To establish the relevance of the present findings in a human context, the effects of Nav1.7 on primary human chondrocytes isolated from six patients with KL 3-4 knee OA were characterized. It was confirmed that both ProTx II and PF-04856264 decreased IL-1β induced expressions of MMP13, ADAMTS5, COX2, and NOS2 (Figs.13A-13D), and increased the expressions of COL2 and ACAN (Figs.13E, and 13F). To further determine the effects of Nav1.7 blockers on human OA cartilage, an ex vivo cartilage explant assay was used using full- thickness OA cartilage from tibia plateaus of patients with OA undergoing total kneeYale Ref. YV 8761WO || TP Ref.251609.000125 arthroplasty. In line with the results obtained from in vitro monolayer culture, supernatants of explants treated with Nav1.7 blockers contained significantly lower levels of the cartilage catabolic marker MMP13, and higher levels of cartilage matrix component lubricin (also known as proteoglycan 4 (PRG4))32compared with those of vehicle-treated controls (Figs.13G, and 13H). In addition, Nav1.7 blockers decreased the expression of catabolic markers MMP13 and ADAMTS5, and increased the expression of anabolic markers COL2 and ACAN in human OA cartilage explants cultured under inflammatory conditions (Figs.13I-13L).

[0224] VGSCs are transmembrane proteins that open when the membrane potential in their vicinity become depolarized. There is increasing evidence that dynamic membrane potential influenced a wide range of biological functions in both excitable and non-excitable cells, and regulated secretion is one such function that has been widely studied33,34. In light of these observations, it was hypothesized that alternations in secretion / cross-membrane transport of proteins might contribute to the regulation of chondrocyte biology induced by Nav1.7 inhibition. It was examined whether incubation with conditioned medium (CM), collected from chondrocytes treated with Nav1.7 selective inhibitors, induced the same regulatory effects upon chondrocyte biology observed with Nav1.7 inhibition. It was found that conditioned medium collected from PF-04856264- or ProTx II-treated chondrocytes significantly promoted the expressions of anabolic markers and abolished IL-1β induced expressions of catabolic molecules, similar to direct treatment of chondrocytes with these Nav1.7 inhibitors (Figs.13M-13R). These findings suggested that secreted molecules in conditioned medium were responsible for the effects of Nav1.7 blockade on chondrocyte biology.

[0225] It was next attempted to isolate and characterize the molecules in conditioned medium that mediate Nav1.7 regulation of chondrocyte anabolism and catabolism. To this end, the components of conditioned medium were separated into 4 fractions based on sized exclusion: <10 kDa, 10-30 kDa, 30-100 kDa, and over100 kDa, and tested their effects on chondrocyte biology (Fig.14A). It was found that the 30-100 kDa fraction was capable of promoting chondrocyte anabolism without affecting catabolism, whereas the 10-30 kDa fraction only inhibited cytokine-induced catabolism (Figs.14B-14F). These findings suggested that one or more secreted protein(s) in the 30-100 kDa and the 10-30 kDa molecular weight range in conditioned medium from cells treated with selective Nav1.7 blockers were responsible for enhancing anabolism and inhibiting catabolism, respectively.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0226] These two fractions of conditioned medium were then analyzed by using tandem mass spectrometry(MS / MS). The MS / MS spectra were searched against the UniProt database, using Sequest within Proteome Discoverer. Candidate targets were defined on the basis of the following properties: (i) having a molecular weight between 10-30 kDa or between 30 and 100 kDa; (ii) exhibiting unique or more than two fold increased expression after treatment with both PF-04856264 and ProTx II relative to vehicle treatment; and (iii) being secreted proteins. Of the six hits that met these criteria in the 10-30 kDa and 30-100 kDa fractions of both PF-04856264- and ProTx II-treated conditioned medium, respectively (Figs.3A, and 3B), the focus was on HSP70 (encoded by HSPA1A and HSPA1B) and midkine (encoded by MDK) because these two proteins were previously reported to be implicated in chondrocyte biology, and over-expression of HSP7035and treatment with recombinant midkine36have been reported to protect against OA. After treatment with selective Nav1.7 blockers and the pan-Nav blocker CBZ, which is known to act on Nav1.726, both HSP70 and midkine levels were significantly up-regulated in the medium, even though the amount of HSP70 in the cell lysate remained unchanged. Midkine was also upregulated in the cell lysate (Figs.15A-15D), suggested that Nav1.7 blockers increased medium HSP70 and midkine through modulating the chondrocyte secretion. To further validate the biological regulations of HSP70 and midkine, chondrocytes were treated with recombinant proteins. HSP70 dose-dependently enhanced chondrocyte anabolism without affecting catabolism (Figs.15E, and 15F), whereas midkine dose-dependently inhibited IL-1β induced chondrocyte catabolism without having an effect on anabolism (Figs.15G, and 15H). Notably, specific blockade of HSP70 and midkine with their specific antibodies in conditioned medium, respectively, abolished the effects of conditioned medium on chondrocyte biology (Figs.3C, and 3D).

[0227] Given the finding that Nav1.7 blockers regulated chondrocyte biology by increasing secretion of HSP70 and midkine in vitro led to ask whether blocking HSP70 and / or midkine affected Nav1.7 inhibition mediated protective effects against OA in vivo. Administration of the Nav1.7 blocker PF-04856264 resulted in a reduction in cartilage loss and a decrease of OA-associated pain behavior in DMM mice (Figs.3E-3H). However, this protective effect was nearly abolished by combined application of the HSP70 inhibitor VER 155008 and the midkine inhibitor iMDK (Figs.3E-3H). Of note, HSP70 inhibitor VER 155008 or midkine inhibitor iMDK alone also significantly reduced the protective effects of PF-04856264 againstYale Ref. YV 8761WO || TP Ref.251609.000125 OA without significantly affecting OA-associated pain (Figs.3E-3H). Collectively, these findings underscored the importance of HSP70 and midkine in maintaining the protective effects of Nav1.7 inhibition against OA in vivo.

[0228] Consistent with the observations that Nav1.7 blockers upregulated medium HSP70 and midkine in human chondrocytes, it was found that genetic deletion of Nav1.7 increased the secretion of HSP70 and midkine in mouse chondrocytes isolated from DMM mice (Figs.15I, and 15J). In addition, serum levels of HSP70 and midkine were markedly increased in DMM surgery mice compared with sham surgery mice (Figs.15K, and 15L). To verify the clinical relevance of these findings, the levels of HSP70 and midkine were measured in serum and synovial fluid from healthy individuals and patients with symptomatic knee OA (Tables 1, 2). It was found that serum HSP70 and midkine levels were significantly higher in patients with OA than in healthy controls (Figs.15M, and 15O). For a subset of patients with symptomatic knee OA for whom both serum and synovial fluid samples were available, serum levels of HSP70 and midkine positively correlated with synovial fluid levels, respectively (Figs.15N, and 15P). Collectively, these results revealed that Nav1.7 blockade regulated chondrocyte anabolism and catabolism, at least in part, by regulating the secretions of HSP70 and midkine, respectively. Example 7. Nav1.7 blockade altered chondrocyte Ca2+

[0229] Previous studies have shown that VGSCs contributed to the regulation of intracellular Ca2+signaling in non-excitable glial cells37. Ca2+serves as a crucial second messenger, with a pivotal role in dynamic regulation of diverse cellular processes, including protein secretion38. To investigate the role of Nav1.7 blockade-mediated intracellular Ca2+signaling in regulation of HSP70 and midkine secretion in chondrocytes, initially, the effect of Nav1.7 inhibition on the ATP-triggered increases in intracellular Na+in human OA chondrocytes and C28I2 chondrocytes was assessed. Inhibition of Nav1.7 with PF-04856264 attenuated the increase in intracellular Na+(Figs.16A, and 16B). Inhibition of Nav1.7 with ProTx II or PF- 04856264 was followed by an increase in intracellular Ca2+levels in both human OA chondrocytes and C28I2 cells. Specifically, Nav1.7 blockade decreased the initial intracellular Ca2+surge triggered by ATP stimulation within approximately 100 seconds. Subsequently, Nav1.7 blockade resulted in higher sustained Ca2+levels than in control cells. These observations were confirmed using confocal microscopy and plate reader measurements (Figs. 4A, 4B, and 16C-16F). To validate the importance of intracellular Ca2+levels in the modulationYale Ref. YV 8761WO || TP Ref.251609.000125 of HSP70 and midkine secretion in chondrocytes, experiments on C28I2 cells were conducted using the Ca2+ionophore ionomycin, and the cell-permeant Ca2+chelator BAPTA-AM. The results demonstrated that higher Ca2+signals contributed to the regulation of HSP70 and midkine secretion via Nav1.7 blockade. Notably, there was a loss of the enhanced secretion of HSP70 and midkine by Nav1.7 blockade in chondrocytes when BAPTA-AM was present (Figs.4C, 4D, 16G, and 16H). The involvement of Na+ / Ca2+exchange in the modulation of Ca2+signals by VGSC blockade in astrocytes and microglial cells was previously documented37,39. To assess the role of NCX family proteins (also known as solute carrier family 8 proteins) in regulating intracellular calcium levels after Nav1.7 blockade in chondrocytes, the pharmacological inhibitor KB-R7943 was used to inhibit NCX prior to ATP stimulation. These experiments showed that KB-R7943 significantly decreased the ATP-induced surge of intracellular Ca2+within the first 100s. Additionally, when NCX was blocked with KB-R7943, PF-04856264 had minimal effect on Ca2+levels in chondrocytes. These results indicated that NCX contributed to the regulation of intracellular Ca2+signals by Nav1.7 blockade (Fig.4E). Consistent with these results, NCX inhibition abolished the enhanced secretion of HSP70 and midkine caused by Nav1.7 blockade (Figs.4F, 4G). RT-PCR was also used to examine the expression of NCX1 (also known as SLC8A1), NCX2 (SLC8A2), and NCX3 (SLC8A3) in C28I2 cells. This experiment demonstrated clear expression of NCX1 mRNA in the chondrocytes, whereas NCX2 and NCX3 were undetectable (Fig.4H). It was then showed that PF-04856264-mediated regulation of Ca2+signals and secretion of HSP70 and midkine were essentially lost in chondrocytes following short interfering RNA (siRNA) knockdown of NCX1 (Figs.4I-4L). Collectively, these findings indicated that intracellular Ca2+signals were essential for the enhanced secretion of HSP70 and midkine following Nav1.7 blockade. Moreover, these findings underscore the critical role of NCX1 in regulating calcium signaling and the associated protein secretion by Nav1.7 blockade in chondrocytes. Discussion

[0230] Although classically considered as the substrate for action potential initiation and propagation40, low densities of VGSCs have been reported in multiple cell types that have traditionally been considered as non-excitable, including macrophages, microglia, and astrocytes, where they may contribute to regulation of effector functions such as phagocytosis, motility, cytokine release and response to injury41-43. Chondrocytes express a variety of ion channel typesYale Ref. YV 8761WO || TP Ref.251609.00012544,45that participate in diverse physiological processes, including setting resting potential, mechanoresponsiveness, volume regulation, calcium signaling, bone development, intracellular pH regulation, cellular biosynthesis, and proliferation46-51. Notably, the expression of several ion channel types is known to be altered in OA chondrocytes44,45,52, whereas cartilage-specific knockout of mechanosensory ion channel decreases age-related OA53.

[0231] Sugimoto et al. reported a TTX-S current in rabbit articular chondrocytes; however, its molecular identity was unknown2. Here it was demonstrated that human OA chondrocytes expressed TTX-S sodium current that was mainly produced by functional Nav1.7. A sodium current density of 2.4 pA / pF (39.7 pS / pF) in human chondrocytes was observed. Assuming single-channel conductance of 6.4 pS54and open probability of 0.4-0.6 at 0 mV10,54, this suggested a channel density of 0.1-0.15 channels / µm2and 350-525 channels / cell. Notably, TTX-S currents in human OA chondrocytes comprised 62% of the total sodium current on average, whereas ProTx II-S current contributed 40%. This result highlighted the existence of Nav1.7 sodium channels in primary human OA chondrocytes, and pointed to the possibility of a fractional presence of non-Nav1.7 TTX-S sodium channels in this cell type.

[0232] Nav1.7 is known to regulate cytokine secretion in dendritic cells42. Here, it was demonstrated that Nav1.7 blockers affected chondrocyte anabolic and catabolic processes through regulation of the chondrocyte secretome. The combination of fractioning conditioned medium and subsequent proteomics analysis led to identification of HSP70 and midkine as key molecules in conditioned medium from cells treated with Nav 1.7 inhibitors that participate in the control of chondrocyte metabolism. Increased release of HSP70 and midkine by blockade of Nav1.7 in Nav1.7-expressing chondrocytes can have both autocrine and paracrine effects and enabled both Nav1.7 expressing and non-expressing chondrocytes to integrate signaling from the local environment to orchestrate the anabolic and catabolic processes that contribute to OA. It has been demonstrated by the present inventors that increased intracellular Ca2+signals were essential for the enhanced secretion of HSP70 and midkine induced by Nav1.7 blockade in chondrocytes. This effect was nullified by pharmacological inhibition and genetic ablation of NCX1, highlighting the crucial role of NCX1 in regulating intracellular Ca2+signaling and subsequent secretion of HSP70 and midkine in response to the Nav1.7 blockade. Of note, Nav1.7 blockade elicited a distinct sequence of intracellular calcium level changes in chondrocytes. It initially reduced Ca2+levels stimulated by ATP, which was counteracted by NCX1 inhibition.Yale Ref. YV 8761WO || TP Ref.251609.000125 Subsequently, a sustained increase of Ca2+levels was potentially orchestrated by a series of protein-protein and protein / lipid interactions. These results suggested that Nav1.7 blockers hold promise as therapeutic agents to both protect against cartilage loss and attenuate pain in OA. It was demonstrated in multiple animal models that CBZ, a sodium channel blocker currently in clinical use, prevented cartilage loss in animal models of OA, an effect beyond purely blocking pain perception. These results highlighted the potential clinical application of a currently available, FDA-approved sodium channel blocker that might be repurposed for the treatment of OA.

[0233] In conclusion, Nav1.7 was identified as an OA associated ion channel with dual roles in pain and cartilage homeostasis, with DRG neuron-expressed Nav1.7 being involved in pain and chondrocyte-expressed Nav1.7 governing chondrocyte biology, cartilage loss and resultant pain in OA through a powerful effect on the chondrocyte secretome (Fig.4M). Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated gene uncovered a target for the development of therapies that may provide both disease-modifying and non- addictive pain relief treatment for OA. Example 8. Nav1.7: Low Density but Large Role in Chondrocytes and Osteoarthritis

[0234] Osteoarthritis (OA) represents human need, especially as human lifespan is being extended1. The Nav1.7 channel was previously known to be present at high levels within peripheral sensory (dorsal root ganglion, DRG neurons) and is known to play a central role in pain-signaling in peripheral neurons. However, its expression within chondrocytes – cells essential for the integrity of cartilage – had not been suspected. This study began with the observation of Nav1.7 channel expression within human chondrocytes, and the finding of profound upregulation of Nav1.7 mRNA and protein within chondrocytes from human arthritic OA. Building on these findings, patch-clamp recording was used to determine whether Na currents with the attributes of currents produced by Nav1.7 channels, were present in these cells. What turned out to be a very low density of channels (0.1-0.15 channels / µm2within the cell membrane and 350-525 channels / cell) was challenging but, recording in a low-noise environment and enhancing the signal / noise ratio by averaging multiple runs for each voltage clamp trial, allowed to demonstrate currents with properties precisely matching those of Nav1.7; it was then obtained definitive confirmation of the molecular identity of the channels by demonstrating blockade of the currents by ProTx II-S, a specific blocker of Nav1.7.Yale Ref. YV 8761WO || TP Ref.251609.000125

[0235] To explore the role of Nav1.7 in OA, multiple lines of mice were constructed, with Nav1.7 knocked out in chondrocytes, in DRG neurons, or both. Two mouse models of OA were studied, surgically- and chemically-induced22. It was found that Nav1.7 knockout within chondrocytes protected joints from cartilage loss, reduced osteophyte formation and subchondral bone plate thickening, and improved synovitis score, along with an improvement in open field movement. Similar protection was provided by intra-articular injection of a Nav1.7-specific inhibitor, PF-04856264. Carbamazepine (CBZ), a clinically employed non-selective Na channel inhibitor known to act on Nav1.7 that is widely used clinically in epilepsy, bipolar disorder and neuropathic pain was then studied. Protective effects, similar to those seen with Nav1.7 knockout or PF-04856264, suggested that this agent, or other non-selective Nav blockers, can hold promise as novel disease-modifying treatments for OA.

[0236] As the mechanism for the effect of Nav1.7 deletion and blockade was explored, it was found that Nav1.7 knockout and blockers regulated chondrocyte anabolism and catabolism by increasing secretion of HSP70 and midkine, respectively. This in vitro observation led to do in vivo experiments where it was observed that Nav1.7 blockade regulated chondrocyte anabolism and catabolism, at least in part, by regulating the secretion of HSP70 and midkine. Measuring intracellular ion concentrations, it was able to link Nav1.7 activity to these changes in HSP70 and midkine, via a cascade in which intracellular Ca2+signals reflected the activity of the sodium-calcium exchanger NCX1 to regulate secretion of HSP70 and midkine following Nav1.7 blockade.

[0237] This study has implications for ion channel biology, for chondrocyte biology, and for the development of new therapeutics for OA, a major unmet medical need. It was seen in this study that Nav channels can play a role in vivo, in regulating the behavior of chondrocytes which are usually considered to be non-excitable. This approach may provide a template for studying the “below the surface” roles of Nav channels in other non-excitable cell-types that do not generate action potentials. The cell-type that was the focus of this study – the chondrocyte – is a major participant in the pathogenesis of OA. The new observations raised the possibility that existing non-specific Nav blockers, or Nav1.7-specific blockers, may offer a new avenue for disease modification in OA, protecting joints from damage and thereby preventing a very common form of disability.Yale Ref. YV 8761WO || TP Ref.251609.000125 Example 9. Nav Channel Inhibitors Reduce Catabolism

[0238] C28I2 cells (human chondrocyte cell lines) were treated with or without 10 ng / ml IL-1β along with 10 nM lacosamide (LCM), oxcarbazepine (OXC) or carbamazepine (CBZ) for 24 hours. All three non-selective Nav channel inhibitors significantly reduced inflammatory cytokine induced catabolism. Among them, LCM was the most potent inhibitor, effectively reduced inflammatory cytokine-induced catabolism at the same concentration (Figs.17A-17B).

[0239] OA is a multifactorial disease, defined by a combination of radiographic cartilage loss and chronic pain. Identification of potential genes that concurrently control OA disease progression and pain is a long-sought goal and remains a big challenge67. The present invention illustrated herein pinpoints Nav1.7 as a molecule with such dual roles in OA. Data from multiple genetically-engineered mice revealed the relative contribution of chondrocyte-, and DRG neuron-expressed Nav1.7 to cartilage protection and pain relief in OA. Genetic ablation of Nav1.7 in chondrocytes concurrently attenuated cartilage loss and alleviated OA pain, while deletion of Nav1.7 in DRG neurons only reduced OA pain without disease-modifying effects in OA cartilage; and deletion of Nav1.7 in both DRG neurons and chondrocytes conferred prominent protection against OA progression and OA pain. The fact that Nav1.7 deletion in DRG neurons only alleviates OA pain further emphasized the importance of chondrocyte- expressed Nav1.7 in regulating cartilage homeostasis during the pathogenesis of OA.

[0240] OA pain is believed to arise from many sources68. Although there is an established link between cartilage loss and OA pain69, the contribution of cartilage loss to OA pain is controversial given the weak correlation between radiographic evidence of OA and the main clinical presentation of OA pain70. The genetic studies illustrated herein indicate that OA pain can be attenuated by specific deletion of Nav1.7 in chondrocytes by a mechanism that involves protection against cartilage loss, suggesting that the association of cartilage loss with pain could be pathogenically important.

[0241] Serum HSP70 and midkine levels were elevated in Patients with OA compared to healthy controls. This paradoxical observation aligned with evidence that OA chondrocytes also exhibit increased anabolic activity, such as increased levels of anabolic growth factors71,72.

[0242] The results obtained in the experiments illustrated herein suggest that Nav1.7 blockers hold promise as therapeutic agents that can both protect against cartilage loss in OA and attenuate OA pain. It was demonstrated in multiple animal models that CBZ, a sodium channelYale Ref. YV 8761WO || TP Ref.251609.000125 blocker currently in clinical use, prevented cartilage loss in animal models of OA, an effect beyond purely blocking pain perception. These results highlighted the potential clinical application of a currently available, FDA-approved sodium channel blocker which might be repurposed for the treatment of OA.

[0243] Genetic deletion and pharmacological blockade of Nav1.7 provided proof-of- principle that targeting of Nav1.7 can protect against cartilage destruction, ameliorate OA progression, and attenuate OA-associated pain. These findings provided the first evidence demonstrating that chondrocytes express functional Nav1.7 channels, and demonstrated the importance of Nav1.7 in the regulation of chondrocytes and OA. The experimental observations described herein also suggest a new avenue for the development of Nav1.7 blockers as novel disease-modifying drugs for treating OA pathologically and symptomatically, thereby expanding their clinical utility beyond that of relief from neuronal hyperexcitability. Example 10. Treatment of Veterinary or Companion Animals

[0244] A veterinary or companion animal (such as for example and not limitation, e.g., cats, dogs, cows, horses, sheep, pigs, avian species, amphibians, reptiles, etc.), is administered a Nav 1.7 inhibitor or a pharmaceutical composition comprising a Nav 1.7 inhibitor as described herein. The administration can be an intra-articular injection or an injection near a joint affected by osteoarthritis. The pharmaceutical composition can be provided in a therapeutically effective amount as described herein. For example and not limitation, a canine having osteoarthritis of a joint, e.g., of a stifle (knee) joint, hip joint, elbow joint, wrist joint, and / or hock (ankle) joint, is administered an injection, optionally an intra-articular injection, of a Nav 1.7 inhibitor or a pharmaceutical composition comprising a Nav 1.7 inhibitor as described herein, optionally in a therapeutically effective amount as described herein. For example and not limitation, an equine or bovine having osteoarthritis of a joint, e.g., of a fetlock joint, carpal (knee) joint, stifle joint, hip joint, elbow joint, sacroiliac joint, and / or hock (tarsal) joint, is administered an injection, optionally an intra-articular injection, of a Nav 1.7 inhibitor or a pharmaceutical composition comprising a Nav 1.7 inhibitor as described herein, optionally in a therapeutically effective amount as described herein.Yale Ref. YV 8761WO || TP Ref.251609.000125 References 1 Katz, J. N., Arant, K. R. & Loeser, R. F. Diagnosis and Treatment of Hip and Knee Osteoarthritis: A Review. JAMA 325, 568-578, doi:10.1001 / jama.2020.22171 (2021). 2 Sugimoto, T., Yoshino, M., Nagao, M., Ishii, S. & Yabu, H. Voltage-gated ionic channels in cultured rabbit articular chondrocytes. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 115, 223-232, doi:10.1016 / s0742-8413(96)00091-6 (1996). 3 Loeser, R. F. et al. 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The therapeutic effect of rhMK on osteoarthritis in mice, induced by destabilization of the medial meniscus. Biol Pharm Bull 37, 1803-1810, doi:10.1248 / bpb.b14- 00470 (2014). 37 Persson, A. K. et al. Contribution of sodium channels to lamellipodial protrusion and Rac1 and ERK1 / 2 activation in ATP-stimulated microglia. Glia 62, 2080-2095, doi:10.1002 / glia.22728 (2014). 38 Anantharam, A. & Kreutzberger, A. J. B. Unraveling the mechanisms of calcium- dependent secretion. J Gen Physiol 151, 417-434, doi:10.1085 / jgp.201812298 (2019). 39 Pappalardo, L. W., Samad, O. A., Black, J. A. & Waxman, S. G. Voltage-gated sodium channel Nav 1.5 contributes to astrogliosis in an in vitro model of glial injury via reverse Na+ / Ca2+exchange. Glia 62, 1162-1175, doi:10.1002 / glia.22671 (2014). 40 Hodgkin, A. L. & Huxley, A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117, 500-544, doi:10.1113 / jphysiol.1952.sp004764 (1952). 41 Craner, M. J. et al. Sodium channels contribute to microglia / macrophage activation and function in EAE and MS. Glia 49, 220-229, doi:10.1002 / glia.20112 (2005). 42 Kis-Toth, K. et al. Voltage-gated sodium channel Nav1.7 maintains the membrane potential and regulates the activation and chemokine-induced migration of a monocyte-derived dendritic cell subset. J Immunol 187, 1273-1280, doi:10.4049 / jimmunol.1003345 (2011). 43 Sontheimer, H., Black, J. A. & Waxman, S. G. Voltage-gated Na+ channels in glia: properties and possible functions. Trends Neurosci 19, 325-331, doi:10.1016 / 0166- 2236(96)10039-4 (1996). 44 Lewis, R., May, H., Mobasheri, A. & Barrett-Jolley, R. Chondrocyte channel transcriptomics: do microarray data fit with expression and functional data? Channels (Austin) 7, 459-467, doi:10.4161 / chan.26071 (2013).Yale Ref. YV 8761WO || TP Ref.251609.000125 45 Mobasheri, A. et al. The chondrocyte channelome: A narrative review. Joint Bone Spine 86, 29-35, doi:10.1016 / j.jbspin.2018.01.012 (2019). 46 Wilson, J. R., Duncan, N. A., Giles, W. R. & Clark, R. B. A voltage-dependent K+ current contributes to membrane potential of acutely isolated canine articular chondrocytes. J Physiol 557, 93-104, doi:10.1113 / jphysiol.2003.058883 (2004). 47 Maleckar, M. M., Clark, R. B., Votta, B. & Giles, W. R. The Resting Potential and K(+) Currents in Primary Human Articular Chondrocytes. Front Physiol 9, 974, doi:10.3389 / fphys.2018.00974 (2018). 48 Phan, M. N. et al. Functional characterization of TRPV4 as an osmotically sensitive ion channel in porcine articular chondrocytes. Arthritis Rheum 60, 3028-3037, doi:10.1002 / art.24799 (2009). 49 Qian, N. et al. TRPM7 channels mediate spontaneous Ca(2+) fluctuations in growth plate chondrocytes that promote bone development. Sci Signal 12, doi:10.1126 / scisignal.aaw4847 (2019). 50 Savadipour, A., Nims, R. J., Katz, D. 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Arthritis Res 3, 107-113, doi:10.1186 / ar148 (2001). 73 Walker JR, et al. Marked difference in saxitoxin and tetrodotoxin affinity for the human nociceptive voltage-gated sodium channel (NaV1.7) Proc. Natl. Acad. Sci. USA. 2012;109:18102–18107. * * *

[0245] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0246] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

Yale Ref. YV 8761WO || TP Ref.251609.000125 Claims 1. A method of preventing or reducing cartilage loss and / or joint damage in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

2. A method of regulating chondrocyte metabolism in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein in the mammal’s chondrocytes.

3. A method of preventing, delaying progression or treating osteoarthritis (OA) in a mammal in need thereof, the method comprising administering to the mammal an effective amount of an agent that reduces or inhibits a function or expression of Nav 1.7 protein.

4. The method of any one of claims 1-3, wherein said agent inhibits catabolism and / or enhances anabolism in the cartilage.

5. The method of any one of claims 1-4, wherein said agent is a selective Nav 1.7 blocker.

6. The method of any one of claims 1-4, wherein said agent is a non-selective Nav 1.7 blocker.

7. The method of claim 5, wherein said agent is selected from PF-04856264, ProTx II, PF- 051980047, PF-05089771, PF-05153462, PF-05150122, PF-05186462, PF-05241328, PF-06456384, GNE-3565, SSCI-1, GDC-0276, MK-2075, OLP-1002, iN1011-N17, ST- 2262, CC8464, AM-8145, AM-0422, AM-6120, JzTx V, μ-theraphotoxin-Pn3a, GpTx-I, GpTx I-71, Huwentoxin-IV (HwTx-IV) or mutants thereof, m3-HwTx-IV, Df1a, GX- 201, GX-044, GX-585, GX-674, MRL5, Cd1a, DSP-2230, GDC-0287 (RG7893), ST- 2427, ST-2578, GDC-0310, NKTR-171, CNV-3000223, CNV-3000164, μ-SLPTX-Yale Ref. YV 8761WO || TP Ref.251609.000125 Ssm6a, antibody that can bind to sensor S3-S4 loop, or derivatives thereof and any combinations thereof.

8. The method of any one of claims 1-5 or 7, wherein said agent is PF-05089771 or derivatives thereof.

9. The method of any one of claims 1-5 or 7, wherein said agent is PF-04856264 or derivatives thereof.

10. The method of any one of claims 1-5 or 7, wherein said agent is Pro Tx II or derivatives thereof.

11. The method of any one of claims 1-4 or 6, wherein said agent is selected from carbamazepine (CBZ), oxcarbazepine (OXA), eslicarbazepine acetate (ESL), lacosamide (LCM), tetrodotoxin (TTX), XEN402 (funapide or TV-45070), vixotrigine (raxatrigine, CNV1014802 or BIIB074), lamotrigine, bupivacaine (BPV), vinpocetine (VPC), hardwickiic acid (HDA), lidocaine, phenytoin, ralfinamide (NW-1029, FCE-26742A, PNU-0154339E), saxitoxin (STX), phlotoxin-1, BIA 2-093, BIA 2-024, CNV-1061436, or derivatives thereof and any combinations thereof.

12. The method of any one of claims 1-4,6, or 11, wherein said agent is carbamazepine (CBZ) or derivatives thereof.

13. The method of any one of claims 1-4,6, or 11, wherein said agent is lacosamide (LCM) or derivatives thereof.

14. The method of any one of claims 1-4, 6, or 11, wherein said agent is oxcarbazepine (OXA) or derivatives thereof.

15. The method of any one of claims 1-14, wherein said agent is selected from the group consisting of a small molecule, an antibody, a peptide, an antisense oligonucleotide, an RNAi molecule, a gene therapy molecule, an aptamer and any combinations thereof.Yale Ref. YV 8761WO || TP Ref.251609.000125 16. The method of any one of claims 1-4, wherein said agent is a sulfonamide-based inhibitor.

17. The method of any one of claims 1-16, wherein said agent is administered systemically.

18. The method of any one of claims 1-16, wherein said agent is administered orally, intravenously, intraperitoneally, intradermally, intramuscularly, nasally, topically, or subcutaneously.

19. The method of any one of claims 1-16, wherein said agent is administered locally.

20. The method of any one of claims 1-16, wherein said agent is administered via an intra- articular delivery.

21. The method of any one of claims 1-16, wherein said agent is administered via a skin patch or a transdermal delivery system.

22. The method of any one of claims 1-21, wherein said agent is formulated as microspheres, hydrogels, nanoparticles, or liposomes.

23. The method of any one of claims 1-22, wherein said agent is formulated together with a positively charged solvent or material.

24. The method of any one of claims 1-23, wherein the said agent is formulated for extended release, immediate release, controlled release, sustained release, delayed release, or pulse release of the agent.

25. The method of any one of claims 1-24, wherein said agent is administered at a dose of 0.1-250 mg / kg body weight.

26. The method of claim 25, wherein said agent is administered at a dose of 1-50 mg / kg body weight.Yale Ref. YV 8761WO || TP Ref.251609.000125 27. The method of claim 26, wherein said agent is administered at a dose of 10-50 mg / kg body weight.

28. The method of claim 25, wherein said agent is administered at a dose of 50-250 mg / kg body weight.

29. The method of any one of claims 1-28, further comprising assessing the cartilage loss and / or joint damage in the mammal before and / or after the treatment.

30. The method of claim 29, wherein the cartilage loss is assessed by assessing articular cartilage destruction by the OARSI grade and / or assessing osteophyte formation and / or assessing subchondral bone plate (SBP) thickness.

31. The method of any one of claims 1-30, further comprising assessing the distance of movement and / or mechanical allodynia in the mammal before and / or after the treatment.

32. The method of any one of claims 1-31, further comprising determining level of HSP70 and / or midkine in the blood of the mammal before and / or after the treatment.

33. The method of any one of claims 1-32, further comprising determining level of HSP70 and / or midkine in the chondrocyte secretome of the mammal before and / or after the treatment.

34. The method of any one of claims 1-33, wherein the mammal is human.

35. The method of any one of claims 1-33, wherein the mammal is a companion animal or a veterinary animal.

36. A method of assaying activity of Nav 1.7 ion channel in cells comprising low densities of sodium (Na) channels, said method comprising:Yale Ref. YV 8761WO || TP Ref.251609.000125 (i) recording of Na currents produced by Nav 1.7 channel in a low-noise environment using a patch clamp; and (ii) enhancing the signal / noise ratio by averaging multiple runs for each voltage clamp trial.

37. The method of claim 36, wherein the cells comprising low densities of sodium (Na) channels are non-excitable cells.

38. The method of claim 36 or claim 37, wherein the method further comprises using one or more pharmacological agents and subtraction to isolate the Na currents produced by Nav 1.7 channel.

39. The method of claim 38, wherein the one or more pharmacological agents are selected from small molecule Nav1.7 blockers and ProTx II.

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