Novel treatments for eye disorders

Abstract

The present invention relates to compounds and compositions comprising connexin modulators, and their use in dose regimens for treating non-healing or persistent defects and disorders of the ocular surface or cornea. In some embodiments, compounds or compositions for the modulation or inhibition of connexins, connexin gap junctions and / or connexin hemichannels are provided for treatment and administered as described herein.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Application No. 63 / 521,298 filed on 15 June 2023 and U.S. Provisional Application No. 63 / 450,597 filed on 7 March 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy, created on March 7, 2024, is named H7475-00103_SL.xml and has a size of 215,985 bytes.

[0003] field This invention relates to a connexin modulator and modulation, as well as defects or disorders of the ocular surface.

[0004] Embedding by reference All publications, patents, related applications, and other written or electronic materials mentioned, specified, or referenced herein (including all U.S. patents, published U.S. patent applications, non-U.S. patents, non-U.S. and PCT published applications, articles, and other documents cited or described herein), as well as all listed as references cited in any one or more patents issued herein, are incorporated herein by reference in their entirety. The incorporated information is part of this application, and all patents issued from this application or all patents claiming priority to this application are treated as part of the text and content of this application at the time of filing, and any patents issued from this application or any patents claiming priority to this application, as if all the text and other content were repeated in this application or patents, and any part of any material incorporated by reference may be incorporated herein by amendment as necessary. In the event of any conflicting use between this document and any document incorporated by reference, the use in the incorporated reference(s) should be deemed to supplement the use in this document. In the event of any irreconcilable discrepancies, including those involving definitions appearing in patents or patent applications, the usage in this document shall prevail. [Background technology]

[0005] Technical background The following contains information that may be useful in understanding the present invention. None of the information, publications, or documents specifically or implicitly referenced herein are considered to be prior art or of importance to the invention described herein and claimed herein.

[0006] Gap junctions are specialized intercellular connections found between most animal cell types. They are expressed in virtually all tissues of the body, with the exception of mature skeletal muscle and mobile cell types such as sperm and red blood cells, and provide regulated physical communication between cells by directly linking the interiors of adjacent cells, allowing various molecules, ions, and electrical impulses to pass through directly.

[0007] A gap junction channel consists of two connexin hemichannels (connexons) that connect across the intercellular space between adjacent cells. Each hemichannel of a gap junction is located on the adjacent cell membrane, and each hemichannel is formed by the covalent oligomerization of six individual connexin (Cx) proteins. See, for example, Yeager (1998) Structure of cardiac gap junction intercellular channels, J Struct Biol 121:231-245. A hemichannel can contain one or more different connexin proteins, but is usually in the form of a homohexamer.

[0008] The human connexin family of genes and proteins is described here as 21. They typically weigh 25–60 kDa and have an average length of 380 amino acids. All connexins share a common structure as four-transmembrane (TM) proteins containing several domains, namely a short intracellular N-terminus (NT), an intracellular loop (IL), and a C-terminus (CT) also localized in the cytoplasm, as well as two extracellular loops (EL1 and EL2) located outside the cell. The length of the cytoplasmic carboxyl terminus can vary considerably. See, for example, Unger et al. (1999) Electron cryo-crystallography of a recombinant cardiac gap junction channel, Novartis Found Symp 219:22-30 & discussion 31-43; Leith, E et al., The connexin 43 C-terminus: A tail of many tale. Biochimica et Biophysica Acta Vol.1860(1):48-64 (Jan 2018).

[0009] Connexin proteins are generally named according to their molecular weight; for example, Cx26 is a 26 kDa connexin protein, and Cx43 is 43 kDa. The main structural difference between connexin proteins is the length of the C-terminal cytoplasmic tail: connexin 26 has almost no tail (16 amino acids), while connexins 43 and 32 have long and intermediate tails (73 and 156 amino acids, respectively).

[0010] Genetic or acquired changes in the structure and function of connexin proteins are associated with a variety of diseases. See, for example, Delmar, M, Laird, DW et al., Connexins and Disease, Cold Spring Harb Perspect Biol 10:a029348 (2018); DW Laird and PD Lampe, Cellular mechanisms of connexin-based inherited diseases. Trends in Cell Biology Vol.32, Issue 1, pp. 58-69 (Jan 2022).

[0011] Studies have also linked connexins to various conditions and disorders. See, for example, Willebroads, J et al., Connexins and their channels in inflammation, Crit Rev Biochem Mol Biol. 51(6):413-439 (2016); Feng, J, Becker, DL, et al., Connexin 43 upregulation in burns promotes burn conversion through spread of apoptotic death signals, Burns 46(6):1389-1397 (Sept 2020); McDouall, A, Green, CR et al., Connexins, Pannexins and Gap Junction in Perinatal Brain Injury, Biomedicines 10:1445 (2022). Connexins have been proposed as therapeutic targets for several conditions, including spinal cord injury, perinatal brain injury, neurological disorders (e.g., Alzheimer's disease, Parkinson's disease), cardiac disorders (e.g., myocardial infarction), ocular disorders (e.g., age-related macular degeneration, diabetic macular edema), acute and chronic wounds (e.g., venous foot ulcers, diabetic foot ulcers), ischemia-reperfusion injury, inflammation, burns, and cancer. They were reviewed in Laird and Lampe, Therapeutic strategies targeting connexins, Nat Rev Drug Discov. 17(12):905-921 (Dec 2018); and Lampe and Laird, Recent advances in connexin gap junction biology, Faculty Reviews 27:11-14 (May 2022). See Becker DL, et al., Translating connexin biology into therapeutics. Semin. Cell Dev. Biol 50, 49-58 (2016).See also the articles in FEBS Letters Volume 588, Issue 8, Pages: i, 1185-1490 (April 17, 2014) in "Junctional Proteins," including Zhang J et al., "Connexin hemichannel induced vascular leak suggests a new paradigm for cancer therapy" (p. 1365-1371) and Martin PE et al., "Connexins: Sensors of epidermal integrity that are therapeutic targets" (p. 1304-1314). See also, for example, Van Campenhout R et al., "Mechanisms Underlying Connexin Hemichannel Activation in Disease." Int J Mol Sci. 22(7):3503 (Apr 2021) and U.S. publications 10,401,188 and 11,401,516 in "Channel Modulators."

[0012] Strategies for treating ocular wounds are outlined in Ziaei M, et al., Wound healing in the eye: Therapeutic prospects, Advanced Drug Delivery Reviews 126 (2018) 162-176. Ziaei et al. discuss, among other things, “traditional” treatment strategies including (1) steroids and (2) contact lenses, for example, as well as several “modern” treatment strategies including (3) growth factors (epidermal growth factor, insulin-like growth factor, nerve growth factor, and human growth hormone), (4) amniotic membrane-based products, (5) thymosine β4, (6) rebamipide, (7) stem cells and other blood-derived products, as well as (8) modulation of gap junction hemichannels. See U.S. Patent No. 8,034,789, "Antisense compounds targeted to connexins and methods of use thereof," issued on 11 October 2011, and Ormonde, S. et al., Regulation of connexin 43 gap junction protein triggers vascular recovery and healing in human ocular persistent epithelial defect wounds, J.Membr. Biol. 245 (2012) 381-388 (the potential for Cx43 modulation was considered in five cases of compassionate use, but this does not rule out the possibility of a "vehicle effect"). More recently, the use of topical insulin eye drops has been proposed to treat persistent epithelial defects (PEDs) of the eye that are resistant to conventional treatments in clinical practice. Diaz-Valle, D et al., Comparison of the efficacy of topical insulin with autologous serum eye drops in persistent epithelial defects of the cornea. Acta Ophthalmol 2022 Jun;100(4):e912-e919.Other possible treatments include the use of (10) punctal plugs, (11) cyanoacrylate adhesives, (13) debridement, and (14) fibronectin tarsal plate suturing (partial or complete eyelid closure). See also, for example, Katzman and Jeng, Management strategies for persistent epithelial defects of the cornea. Saudi Journal of Ophthalmology 28:168-172 (2014).

[0013] As described by Ziaei et al., many current and emerging treatments proposed for the healing of ocular surface wounds remain largely experimental or conceptual. For example, the use of gap junction regulators in the eye in humans is not defined in clinics, and there are no established doses, dose regimens, or methods for the modulation of connexins, connexin gap junctions, and / or connexin hemichannels to treat non-healing or persistent ocular or corneal surface defects or disorders. Such methods, doses, and dose regimens are described and claimed herein and provide long-needed means for treating subjects suffering from non-healing or persistent defects or disorders of the ocular surface, as well as other ocular diseases, disorders, and conditions described herein. In contrast to the treatment methods described and claimed herein, most current treatments for these eye diseases, disorders, and conditions have limited success because they fail to address their underlying causes, or multiple causes, which often include uncontrolled inflammation. [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] U.S. Patent No. 10,401,188 [Patent Document 2] U.S. Patent No. 11,401,516 [License 3] U.S. Patent No. 8,034,789 [Non-licensed literature]

[0015] [Non-licensed Document 1] Yeager(1998)Structure of cardiac gap junction intercellular channels, J Struct Biol 121:231-245 [Non-licensed Document 2] Ungerら(1999)Electron cryo-crystallography of a recombinant cardiac gap junction channel,Novartis Found Symp 219:22-30&discussion 31-43 [Non-licensed Document 3] Leith,Eら、The connexin 43 C-terminus:A tail of many tale.Biochimica et Biophysica Acta Vol.1860(1):48-64(Jan 2018 [Non-licensed Document 4] Delmar,M,Laird,DWらConnexins and Disease,Cold Spring Harb Perspect Biol 10:a029348(2018) [Non-licensed Document 5] DW Laird and PD Lampe,Cellular mechanisms of connexin-based inherited diseases.Trends in Cell Biology Vol.32,Issue 1,p58-69(Jan 2022) [Non-licensed Document 6] Willebrords, J et al., Connexins and their channels in Crit inflammation Rev Biochem Mol Biol.51(6):413-439(2016) [Non-Patent Document 7] Feng, J, Becker, DL, et al., Connexin 43 upregulation in burns promotes burn conversion through spread of apoptotic death signals, Burns 46(6):1389-1397(Sept 2020) [Non-Patent Document 8] McDouall, A, Green, CR et al. Connexins, Pannexins and Gap Junction in Perinatal Brain Injury. Biomedicines 10:1445(2022) [Non-Patent Document 9] Laird and Lampe, Therapeutic strategies targeting connexins, Nat Rev Drug Discov.17(12):905-921(Dec 2018) [Non-Patent Document 10] Lampe and Laird, Recent advances in connexin gap junction biology, Faculty Reviews 27:11-14 (May 2022) [Non-Patent Document 11] Becker DL, et al. Translating connexin biology into therapeutics.Semin.Cell Dev.Biol 50,49-58(2016) [Non-Patent Document 12] Zhang J et al., Connexin hemichannel induced vascular leak suggests a new paradigm for cancer therapy (p.1365-1371) [Non-Patent Document 13] Martin PE et al., Connexins: Sensors of epidermal integrity that are therapeutic targets (p. 1304-1314) [Non-Patent Document 14] The article is published in the "Junctional Proteins" issue of FEBS Letters Volume 588, Issue 8, Pages i, 1185-1490 (April 17, 2014). [Non-Patent Document 15] Van Campenhout R, et al., Mechanisms Underlying Connexin Hemichannel Activation in Disease.Int J Mol Sci.22(7):3503(Apr 2021) [Non-Patent Document 16] Ziaei M, et al., Wound healing in the eye: Therapeutic prospects, Advanced Drug Delivery Reviews 126 (2018) 162-176 [Non-Patent Document 17] Ormonde, S. et al., Regulation of connexin43 gap junction protein triggers vascular recovery and healing in human ocular persistent epithelial defect wounds, J.Membr.Biol.245(2012)381-388 [Non-Patent Document 18] Diaz-Valle, D et al., Comparison of the efficacy of topical insulin with autologous serum eye drops in persistent epithelial defects of the cornea. Acta Ophthalmol 2022 Jun;100(4):e912-e919 [Non-Patent Document 19] Katzman and Jeng,Management strategies for persistent epithelial defects of the cornea.Saudi Journal of Ophthalmology 28:168-172(2014) [Overview of the Initiative]

[0016] Brief Overview The inventions described and claimed herein include, but are not limited to, those described, explained, or referenced in this brief summary, and have many other attributes and embodiments. This is not intended to be exhaustive, and the inventions described and claimed herein are not limited to, or even limited by, the features or embodiments identified in this introduction, which are for illustrative purposes only and not limiting.

[0017] The object of the present invention is to provide methods, doses, dose regimens, compositions and kits for connexin modulation for the treatment of subjects for non-healing or persistent ocular surface or corneal defects or disorders. In some embodiments, compounds or compositions for the modulation or inhibition of connexins, connexin gap junctions and / or connexin hemichannels are provided for treatment and administered as described herein. In some embodiments, the non-healing ocular surface defect or disorder is a persistent epithelial defect (PED). In some embodiments, the non-healing ocular surface defect or disorder is a persistent corneal epithelial defect (PCED). In some embodiments, the subject is human.

[0018] In some embodiments, the connexin modulator modulates or inhibits connexin expression. In some embodiments, the connexin modulator inhibits or modulates the expression of connexin 43.

[0019] In some embodiments, the Connexin modulator is a Connexin gap junction modulator. In some embodiments, the Connexin gap junction modulator modulates a Connexin 43 gap junction.

[0020] In some embodiments, the connexin modulator is a connexin hemichannel modulator. In some embodiments, the connexin hemichannel modulator modulates the connexin 43 hemichannel. Modulation of the hemichannel may be carried out by any means. In some embodiments, for example, modulation may occur by inducing or promoting the closure of the hemichannel; by blocking, blocking, inhibiting or reducing the opening of the hemichannel; by suppressing the permeability of the hemichannel; by suppressing the release of ATP from the hemichannel; and / or by inducing, inducing or promoting the intracellularization of the hemichannel and / or gap junction. The hemichannel modulator comprises blockers and other compounds that interfere with the passage of molecules through the connexin hemichannel. The hemichannel modulator can block or reduce the release of molecules into the extracellular space via the hemichannel and / or block or reduce the entry of molecules into the intracellular space via the hemichannel. In some embodiments, the hemichannel modulator completely or partially blocks the opening of the hemichannel. In some embodiments, hemichannel modulators completely or partially block, delay, or inhibit the leakage or passage of molecules into or from the extracellular space via hemichannels. In some embodiments, hemichannel modulators are compounds that reduce the probability of hemichannels opening.

[0021] In some embodiments of the present invention, modulation of gap junctions and hemichannels is achieved by administering connexin expression modulators, connexin peptide mimes, and / or small molecule connexin modulators that modulate corneal epithelial connexin, corneal epithelial connexin gap junctions, or corneal epithelial connexin hemichannels, in dose regimens disclosed herein. In some embodiments of the present invention, modulation of gap junctions and hemichannels is achieved by administering connexin expression modulators, connexin peptide mimes, and / or small molecule connexin modulators that modulate corneal vascular connexin using dose regimens disclosed herein. In some embodiments of the present invention, modulation of gap junctions and hemichannels is achieved by administering connexin expression modulators, connexin peptide mimes, and / or small molecule connexin modulators that modulate (e.g., reduce, slow, inhibit, or eliminate) inflammation, in dose regimens disclosed herein.

[0022] In some embodiments, the connexin modulator, connexin gap junction modulator, and / or connexin hemichannel modulator used in the method of the present invention modulate one or more connexins in human corneal epithelium, namely connexin 26, connexin 30, connexin 30.3, connexin 31, connexin 31.1, connexin 32, connexin 43, connexin 45, connexin 50, and connexin 58.

[0023] In some embodiments, the connexin modulator comprises an antisense molecule. In some embodiments, the antisense molecule is a connexin 43 antisense oligonucleotide. In some embodiments, the connexin modulator may be a connexin 43 antisense polynucleotide comprising, essentially, or consisting of sequences according to SEQ ID NOs: 1-16 and / or modified versions thereof. In some embodiments, the antisense oligonucleotide comprises, essentially, or consists of 5'-GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC-3' (SEQ ID NO: 1; lufepirsen). In some embodiments, the antisense oligonucleotide may be chemically modified or may be an unmodified oligonucleotide, such as a modified or unmodified DNA oligonucleotide.

[0024] In some embodiments, the connexin modulator includes a connexin peptide mimetic. In some embodiments, the connexin peptide mimetic is a connexin 43 peptide mimetic. In some embodiments, the connexin peptide mimetic includes, essentially consists of, or consists of SRPTEKT (SEQ ID NO: 101). In some embodiments, the connexin peptide mimetic includes, essentially consists of, or consists of Gap19, XG19, Gap26, or Gap27. In some embodiments, the connexin peptide mimetic is a connexin 26 peptide mimetic (e.g., Gap26, i.e., VCYDKSFPISHVR (SEQ ID NO: 102)), a connexin 32 peptide mimetic (e.g., INCTLQPGCNSV (SEQ ID NO: 103) or 37,43 Gap27 is SRPTEKTIFII (SEQ ID NO: 104) or a connexin 45 peptide mimetic, or a connexin 50 peptide mimetic (e.g., TAT-Cx50L2, i.e., GGERAPLAADQGSVKKSSSSSKGTKK (SEQ ID NO: 105) or TAT-Cx50CT, i.e., SRARSDDLTV (SEQ ID NO: 106)).

[0025] In some embodiments, the connexin modulator includes a small molecule connexin, a connexin gap junction, or a connexin hemichannel modulator. In some embodiments, the small molecule connexin modulator inhibits or blocks the opening of the connexin hemichannel. In some embodiments, the small molecule connexin modulator mitigates ATP release from the connexin hemichannel. In some embodiments, the small molecule connexin modulator inhibits or blocks the opening of the Cx43 hemichannel, thereby mitigating ATP release from the Cx43 hemichannel. In some embodiments, the small molecule connexin modulator is a compound according to formula I (e.g., tonaversat). In some embodiments, the small molecule connexin modulator is a tonaversat prodrug compound according to formula II.

[0026] In some embodiments, the present invention relates to methods for modulating connexins, gap junction channels and / or hemichannels using therapeutically effective amounts of connexin modulators in the dose regimens described herein. In some embodiments, a therapeutically effective amount of a connexin antisense compound (e.g., lufepirsen) is administered. In some embodiments, a therapeutically effective amount of a connexin peptide mimetic compound (e.g., Peptide 5, Gap 19, XG 19, CXT 1-CXT 5, Antp / CXT 1-Antp / CXT 5, etc.) is administered. In some embodiments, a therapeutically effective amount of a small molecule connexin hemichannel blocker compound (e.g., tonaversat or tonaversat prodrug) is administered.

[0027] In some embodiments, the present invention relates to methods for the modulation of connexin, gap junction channels, and / or hemichannels using new specific doses of connexin modulators, gap junction modulators, and / or hemichannel modulators and newly discovered and clinically validated dose regimens for ocular surface restoration in subjects having non-healing or persistent ocular surface or corneal defects or disorders, or both. Non-healing ocular surface or corneal defects or disorders treated with the doses, compositions, dose regimens, and methods of the present invention include persistent epithelial defects (PEDs) and persistent corneal epithelial defects (PCEDs). These include PEDs and PCEDs from any cause, including chemical damage, thermal damage, disease, and inflammation.

[0028] In some embodiments, non-healing ocular surface defects or disorders are caused by chemical and / or thermal injury. In some embodiments, non-healing ocular surface defects or disorders are caused by physical trauma or injury. In certain embodiments, non-healing ocular surface defects or disorders are PED or PCED resulting from chemical and / or thermal injury. In some embodiments, the chemical and / or thermal injury resulting in PED or PCED is severe chemical and / or thermal injury. In some embodiments, severe ocular surface and / or corneal burns and injuries are those with more than 50% conjunctival involvement or more than 6 working hours of corneal margin involvement. These include ocular surface and corneal burns and injuries of 3 or higher on the Dua classification scale. In some embodiments, non-healing ocular surface or corneal defects or disorders are caused by inflammation or inflammatory disease. In some embodiments, non-healing ocular surface or corneal defects or disorders are caused by inflammation of the eye. In some embodiments, non-healing ocular surface or corneal defects or disorders are caused by inflammatory disease of the eye, ocular surface, or cornea.

[0029] All PED and PCED etiologies, regardless of the cause, can be treated using the methods and compositions of the present invention as described herein. Etiologies of PED and PCED include chemical, mechanical, and thermal causes, as well as inflammatory and inflammatory diseases, neurotrophic diseases and neurotrophic keratitis, and epithelial / corneal margin stem cell deficiency. In some embodiments, the methods, compositions, and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from chemical injury. In some embodiments, the methods, compositions, and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from thermal injury. In some embodiments, the methods and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from mechanical injury. In some embodiments, the methods and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from inflammatory or inflammatory diseases. In some embodiments, the methods, compositions, and / or dosages of the present invention are used to treat PED and / or PCED caused by or resulting from neurotrophic disorders (e.g., neurotrophic keratitis).

[0030] In some embodiments, the non-healing ocular surface defect or disorder is an ocular surface ulcer. In some embodiments, the non-healing ocular surface defect or disorder is a corneal ulcer. In some embodiments, the ocular surface ulcer or corneal ulcer is caused by bacteria, viruses, fungi, or parasites. In some embodiments, the ocular surface ulcer or corneal ulcer is due to Acanthamoeba keratitis, fungal keratitis, or herpes simplex keratitis. In other embodiments, the ocular surface ulcer or corneal ulcer is caused by trauma or a foreign body, such as an abrasion of the eye (e.g., a scratch or cut). In yet another embodiment, the ocular surface ulcer or corneal ulcer is caused by an allergic eye disease. In yet another embodiment, the ocular surface ulcer or corneal ulcer is caused by dry eye. In some embodiments, the non-healing ocular surface ulcer or corneal ulcer is caused by or resulting from inflammation or an inflammatory disease, disorder, or condition. In some embodiments, ocular surface ulcers or corneal ulcers are caused by other factors (e.g., injury or burns).

[0031] In some embodiments of the methods described herein and claimed, a connexin inhibitor or connexin modulator is administered to a subject with a non-healing surface defect or disorder on day 1, day 2, and about day 14. In some embodiments, the connexin inhibitor or connexin modulator is administered again to the subject on about day 28. In some embodiments, the connexin inhibitor or connexin modulator may be administered again to a subject with a non-healing surface defect or disorder on about day 35. In some embodiments, the connexin inhibitor or connexin modulator may also be administered to the subject on day 7 and / or day 21. In some embodiments, the non-healing surface defect or disorder treated is a PED. In some embodiments, the non-healing surface defect or disorder treated is a PCED. In some embodiments, the PED or PCED results from chemical and / or thermal damage or inflammation.

[0032] In some embodiments, a single dose of connexin modulator is administered in regimens for treating non-healing surface defects or disorders as described and claimed herein (e.g., single doses administered on day 1, day 2, and day 14, etc., including other dose regimens described herein). In some embodiments, the daily dose of connexin modulator for administration in the dose regimens of the present invention is administered in divided doses (e.g., half doses administered to the subject twice daily on days 1, 2, and day 14, etc.). In some embodiments, a single dose or divided dose of connexin modulator is administered on one day, more than one day, or all days in each of the dose regimens described herein.

[0033] In some embodiments of the present invention, a method for treating a subject for a non-healing ocular surface defect comprises administering a therapeutically effective amount of connexin modulator to the subject, the connexin modulator being administered at least once on day 1, at least once on day 2, and at least once on about day 14. In some embodiments, the connexin modulator is a connexin 43 modulator. In some embodiments, the connexin 43 modulator modulates the expression of connexin 43. In some embodiments, the connexin 43 expression modulator is an antisense molecule. In some embodiments, the connexin 43 antisense molecule is lufepirsen. In some embodiments, lufepirsen is administered in a composition comprising a nonionic polyoxyethylene-polyoxypropylene block copolymer carrier (e.g., Pluronic® F-127). In some embodiments, lufepirsen is administered in a composition comprising a cellulosic carrier, e.g., hydroxyethylcellulose, carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, or other pharmaceutically acceptable carriers.

[0034] In some embodiments, 3-4 doses of a connexin modulator, e.g., lufepirsen, are administered on days 1, 2, and 14 (and, if necessary, on day 28 or about 28). In other embodiments, 4-5 doses are administered, and in addition to days 1, 2, and 14 (or in addition to days 1, 2, 14, and, if necessary, about 28), one or more doses of a connexin modulator, e.g., lufepirsen, or another connexin antisense or modulator are also administered on day 35. In some embodiments of these dose regimens, doses of the connexin modulator, e.g., lufepirsen, may also be administered on about day 7 and / or day 21. In some embodiments, the connexin modulator dose, e.g., lufepirsen, is administered on days 1, 2, 14, and 28.

[0035] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 14, and 21.

[0036] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 7, 14, and 21.

[0037] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 7, 14, 21, and 28.

[0038] In some embodiments, the dose of the connexin modulator, e.g., lufepirsen, is administered on days 1, 2, 14, 28, and 35. In some embodiments, the dose of the connexin modulator, e.g., lufepirsen, is administered on days 1, 2, 7, 14, 28, and 35. In some embodiments, the dose of the connexin modulator, e.g., lufepirsen, is administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, the dose of the connexin modulator, e.g., lufepirsen, is administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, the dose is also administered on day 35 or later in one of these administration regimens. In some embodiments, the administration is for PED or PCED. In some embodiments, the connexin modulator comprises a connexin antisense molecule (e.g., lufepirsen). In some embodiments, the connexin modulator comprises a connexin peptide mimetic (e.g., Peptide 5, Gap 19, XG 19, aCT 1, etc.). In some embodiments, the connexin modulator comprises a small molecule connexin hemichannel blocker (e.g., Tonaversat). In some embodiments, the connexin modulator is a connexin 43 modulator. In some embodiments, the administration is for PED or PCED. The connexin modulator may be administered as a single dose or in divided doses on any administration day.

[0039] In some embodiments, to treat non-healing or persistent ocular or corneal surface defects in a subject, three doses of a connexin modulator (e.g., lufepirsen) are administered over 14 days, including, for example, on days 1 and 2. In some embodiments, four doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 28 days. In some embodiments, five doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 28 to approximately 35 days. In some embodiments, six to seven doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 35 days. For example, in one embodiment, the connexin modulator (e.g., lufepirsen) is administered four times: over days 1 and 2, and on approximately days 14 and 28. In another embodiment, the connexin modulator (e.g., lufepirsen) is administered five times: over days 1 and 2, on approximately day 14, on approximately day 28, and on approximately day 35. In another embodiment, the connexin modulator (e.g., lufepirsen) is administered five times: over days 1 and 2, on about day 7, on about day 14, and on about day 28. In yet another embodiment, the connexin modulator (e.g., lufepirsen) is administered six times: over days 1 and 2, on about day 7, on about day 14, on about day 21, and on about day 28. In yet another embodiment, the connexin modulator (e.g., lufepirsen) is administered seven times: over days 1 and 2, on about day 7, on about day 14, on about day 21, and on about day 28. References to “administration” in this specification, of course, refer to the administration of the connexin modulator in single or divided doses. In some embodiments of the present invention, modulation of connexins, gap junctions, and / or hemichannels is achieved by administering a connexin antisense oligonucleotide (e.g., lufepirsen) to the eye of a subject having a non-healing or persistent ocular surface or corneal defect or disorder (e.g., PED or PCED).

[0040] In some embodiments, the amount of connexin modulator administered to a subject having a non-healing ocular surface defect or disorder (e.g., PED) or a non-healing corneal surface defect or disorder (e.g., PCED) on any of the administration schedules described herein (e.g., days 1, 2, and 14; days 1, 2, and 14 and, if necessary, day 28; days 1, 2, 7, 14, and 21 and, if necessary, day 28; etc.) is a therapeutically effective dose.

[0041] In some embodiments, the amount of connexin modulator administered to subjects having non-healing ocular surface defects or disorders (e.g., PED) or non-healing corneal surface defects or disorders (e.g., PCED) in any of the administration schedules described herein is one or more of the doses described herein (e.g., administration of 1 milligram / milliliter (mg / mL) of connexin modulator, administration of a micromolar (μM) concentration of connexin modulator, administration of milligrams (mg) of connexin modulator, etc.). Specific doses described herein may be administered to subjects having non-healing ocular surface defects or disorders (e.g., PED) or non-healing corneal surface defects or disorders (e.g., PCED) on, for example, days 1, 2, and 14, and optionally on days 28 and / or 35; on days 1, 2, and 14, and optionally on days 7, 21 and / or 28; etc., or in any other dose regimen described herein.

[0042] In some embodiments of the method of the present invention, the connexin modulator administered to a non-healing surface defect or disorder in a subject is 0.06 wt.% lufepirsen. In some embodiments, the connexin modulator administered to a subject having a non-healing surface defect or disorder is 0.006 wt.% lufepirsen. In some embodiments, the administered connexin modulator is at least about 0.06 wt.% lufepirsen. In some embodiments, the connexin modulator administered to a subject having a non-healing surface defect or disorder is at least about 0.006 wt.% lufepirsen. In some embodiments, the administration is for PED or PCED. In some embodiments, a pharmaceutically acceptable carrier and a composition comprising or essentially comprising about 0.06 wt.% lufepirsen, about 0.006 wt.% lufepirsen, at least about 0.06 wt.% lufepirsen, or at least about 0.006 wt.% lufepirsen are administered to heal non-healing surface defects or disorders in a subject.

[0043] In some embodiments of the present invention, non-healing surface defects or disorders are treated with a composition comprising 0.6 mg / mL of lufepirsen. In some embodiments of the present invention, non-healing surface defects or disorders are treated with a composition comprising 0.06 mg / mL of lufepirsen. In some embodiments, the composition comprises at least about 0.6 mg / mL of lufepirsen. In some embodiments, the composition comprises at least about 0.06 mg / mL of lufepirsen. In some embodiments, the administration is for PED or PCED.

[0044] In some embodiments of the present invention, non-healing surface defects or disorders in a subject are treated with a composition comprising or essentially comprising about 0.18 mg of lufepirsen in the regimen described herein. In some embodiments, each dose of lufepirsen applied to a non-healing surface defect or disorder within or on a subject comprises or essentially comprises at least about 0.18 mg of lufepirsen in the regimen described herein. In some embodiments of the present invention, non-healing surface defects or disorders are treated with a composition comprising or essentially comprising about 0.018 mg of lufepirsen in the regimen described herein. In some embodiments, each dose of lufepirsen applied to a non-healing surface defect or disorder comprises or essentially comprises at least about 0.018 mg of lufepirsen in the regimen described herein. In some embodiments, the administration is for PED or PCED.

[0045] In some embodiments, approximately 0.18 mg of lufepirsen (or other connexin antisense or connexin modulator), approximately 0.018 mg of lufepirsen (or other connexin antisense or connexin modulator), or approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or approximately 0.5 mg to approximately 1 mg of lufepirsen (or another connexin antisense or other connexin modulator) is administered as a single dose or in two or more divided doses (for example, twice daily with up to six doses administered on days 1, 2, and 14). In other embodiments, at least about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or 0.5 mg or at least about 1 mg of lufepirsen (or another connexin antisense, or other connexin modulator) is administered to non-healing surface defects or disorders in a single dose, or in two or more divided doses (e.g., twice daily doses with up to 6-10 doses administered on days 1, 2, and 14), or using other dose regimens described herein, including day 28 and / or 35. In other embodiments, at least about 0.5 mg or at least about 1 mg of lufepirsen (or another connexin antisense, or other connexin modulator) is administered in a single dose or in divided doses (e.g., twice daily doses with up to 14 doses administered on days 1, 2, and 14, and on one or more days, e.g., days 7, 21, 28, and / or 35). In some embodiments, the administration is for PED or PCED in the subject. In some embodiments, the connexin antisense administered to a subject in the amounts described herein comprises or is essentially derived from an antisense compound having a sequence according to SEQ ID NOs: 2-16. In some embodiments, the connexin antisense administered to a subject in the amounts described herein on the days described herein comprises or is essentially derived from another connexin 43 antisense compound.

[0046] In any of these doses and embodiments of the administration regimen and method, the lufepirsen dose may be administered to the subject's PED or PCED. In some embodiments of these doses and administration regimens and method, the administered connexin modulator dose includes another connexin antisense or modulator, e.g., another Cx43 antisense or Cx43 modulator other than or in addition to lufepirsen, or another ocular or corneal epithelial connexin modulator other than or in addition to Cx43. In some embodiments, the connexin antisense administered to the subject in the amount specified herein on the day specified herein includes or is essentially an antisense compound for modulating the expression of one or more connexins selected from the group consisting of connexin 26, connexin 30, connexin 30.3, connexin 31, connexin 31.1, connexin 32, connexin 43, connexin 45, connexin 50, and connexin 58.

[0047] In some embodiments of the present invention, the volume of a composition comprising or essentially comprising a connexin modulator administered to a non-healing surface defect or disorder in a subject is about 300 μL. In other embodiments, the volume of the administered composition is about 250 μL to about 1.0 mL. In some embodiments, the composition comprises about 0.01 mg, 0.018 mg, about 0.18 mg to about 0.2 mg of lufepirsen, or about 0.18 mg or 0.2 mg or 0.5 mg to about 1 mg of lufepirsen. In some embodiments, the composition comprises at least about 0.018 mg of lufepirsen, at least about 0.18 mg of lufepirsen, at least about 0.5 mg of lufepirsen, or at least about 1.0 mg of lufepirsen. In some embodiments, instead of or in addition to lufepirsen, the composition comprises about 0.01 mg, about 0.018 mg, about 0.18 mg to about 0.2 mg, or about 0.18 mg or 0.2 mg or 0.5 mg to about 1 mg of another connexin 43 antisense compound (or other connexin modulators, including other antisenses, peptide mimes, and / or small molecule connexin modulators that modulate connexin 43 or other ocular or corneal epithelial connexins). In some embodiments, instead of or in addition to lufepirsen, the composition comprises at least about 0.018 mg, at least about 0.18 mg, at least about 0.5 mg, or at least about 1.0 mg of another connexin antisense or other connexin modulators (e.g., other antisenses, peptide mimes, and / or small molecule connexin modulators that modulate connexin 43 or other ocular or corneal epithelial connexins).

[0048] In some embodiments, the composition for use in the method of the present invention comprises a reverse thermosetting gel. In some embodiments, the composition for use in the method of the present invention comprises Pluronic® gel, also known as poloxamer gel. In some embodiments, the composition for use in the method of the present invention comprises poloxamer F-127 (poloxamer 407 or Pluronic® F-127). In some embodiments, the composition for use in the method of the present invention comprises about 20-30 mg / mL of poloxamer F-127. In some embodiments, the composition for use in the method of the present invention comprises about 22-25 mg / mL of poloxamer F-127. In some embodiments, the composition for use in the method of the present invention comprises about 22.6 mg / mL of poloxamer F-127.

[0049] In some embodiments, the connexin modulator is lufepirsen in a composition comprising Pluronic® F-127, one or more buffers, and water for injection. The buffers incorporated into the lufepirsen composition may include dibasic sodium phosphate heptahydrate, (monobasic) potassium phosphate, or both. In some embodiments, the composition contains 0.06% or 0.006% lufepirsen, at least about 0.06% or at least about 0.006% lufepirsen. In some embodiments, the buffer composition contains 0.6 mg / mL or 0.06 mg / mL lufepirsen, at least about 0.6 mg / mL or at least about 0.06 mg / mL lufepirsen. In some embodiments, about 300 mL of the lufepirsen composition is administered to the non-healing surface injury on days 1, 2, and 14. If necessary or desired, in some embodiments, about 300 μL of lufepirsen is administered again to the subject with the non-healing surface injury on about day 28. If necessary or desired, in some embodiments, a dose of about 300 μL of lufepirsen is administered again to subjects with non-healing surface lesions on about 35 days. In other embodiments, if deemed necessary or desired, about 300 μL of the lufepirsen composition is also administered again to subjects with non-healing surface lesions on 7 days and / or about 21 days. In some embodiments, the non-healing surface lesion treated with the composition is PED or PCED. In some embodiments, the PED or PCED is caused by chemical and / or thermal injury or other trauma. In some embodiments, about 300 μL to 1.0 mL of the lufepirsen composition is administered to the non-healing surface lesion. In some embodiments, the connexin modulator administered in this composition contains another connexin 43 antisense compound. In some embodiments, the connexin modulator administered in this composition contains another antisense compound against an ocular or corneal epithelial connexin.In some embodiments, the connexin modulator administered in this composition includes another connexin 43 antisense compound, another antisense compound against an ocular or corneal epithelial connexin, and / or another connexin modulator (e.g., a peptide mimetic or small molecule connexin modulator).

[0050] In some embodiments of the present invention, modulation of gap junctions and hemichannels is achieved by administration of a small molecule hemichannel blocker that blocks or modulates the opening of corneal epithelial connexin hemichannels. In some embodiments, treatment of non-healing ocular surface defects by modulation of corneal epithelial hemichannels is achieved by topical, oral, or systemic administration of a small molecule hemichannel blocker. In some embodiments, a therapeutically effective amount of the small molecule is administered in the dose regimen described herein. In some embodiments, the dose of the small molecule is administered in the dose regimen described herein and is one or more of the dose described herein. In some embodiments, the small molecule hemichannel blocker is tonaversat.

[0051] In any embodiment of the present invention, administration of the connexin modulator may be topical. In some embodiments, a connexin antisense connexin modulator (e.g., lufepirsen) is administered topically. In some embodiments, a connexin peptide mimetic connexin modulator (e.g., XG19) is administered topically. In some embodiments, a small molecule connexin modulator (e.g., tonavelsat) is administered topically. In some embodiments, a combination of one or more compounds selected from the group consisting of a connexin antisense connexin modulator (e.g., lufepirsen), a connexin peptide mimetic connexin modulator (e.g., XG19), and a small molecule connexin modulator (e.g., tonavelsat) is administered topically. In some embodiments, one or more compounds selected from the group consisting of a connexin antisense connexin modulator (e.g., lufepirsen), a connexin peptide mimetic connexin modulator (e.g., XG19), and a small molecule connexin modulator (e.g., tonavelsat), along with one or more other therapeutic agents, are administered topically. In some embodiments, one or more other therapeutic agents are selected from the group consisting of antibiotics, antivirals, antiparasitic agents, antifungals, steroids, anti-inflammatory agents and immunosuppressants. In some embodiments, one or more other therapeutic agents are pannexin 1 (Panx1) inhibitors (e.g., probenecid, 10 The group consists of Panx1, inhibitors of its associated P2X receptor (or any of its isoforms), and inhibitors of the Panx1-P2X signalosome. In some embodiments, one or more compounds selected from the group consisting of a pannexin 1 (Panx1) inhibitor (e.g., probenecid), inhibitors of its associated P2X receptor (or any of its isoforms), and inhibitors of the Panx1-P2X signalosome are administered alone or in combination with a connexin modulator or another therapeutic agent (e.g., topically). Other useful pannexin inhibitors are described in U.S. Patent No. 10,465,188 ("Channel Modulators").

[0052] In some embodiments, the subject has amniotic membrane attached to the surface of the eye to be treated. In some embodiments, the amniotic membrane is a permanent surgical graft. In some embodiments, the amniotic membrane is a dehydrated sutureless graft. In some embodiments, the amniotic membrane is a cryopreserved sutureless graft.

[0053] In some embodiments, connexin modulators, gap junction modulators, and / or hemichannel modulators are administered to the subject subamniotically. In some embodiments, after applying the modulator to a non-healing surface defect or disorder, the amnion is attached to the subject's eye.

[0054] In some embodiments, the connexin modulator, gap junction modulator, and / or hemichannel modulator are administered to the subject under a contact lens. In some embodiments, the contact lens is applied to the subject's eye approximately simultaneously with the administration of the connexin modulator, gap junction modulator, and / or hemichannel modulator, either immediately after or some time after administration. In some embodiments, the contact lens is a silicone hydrogel contact lens. In some embodiments, the lens is loaded with the connexin modulator, gap junction modulator, and / or hemichannel modulator before the lens is applied to an intraocular or superocular non-healing surface defect or disorder of the subject. In some embodiments, the modulator is lufepirsen.

[0055] In some embodiments, the contact lens is a therapeutic contact bandage lens. Therapeutic contact bandage lenses are also called “bandage lenses” or “therapeutic bandage lenses.” In some embodiments, the therapeutic bandage lens is, for example, a soft contact lens, a gas-permeable (scleral) lens, or a 3D-printed bio-gel. Some suitable FDA-approved soft bandage lenses include Acuvue Oasys with Hydraclear Plus (Johnson & Johnson Vision), Air Optix Night & Day Aqua (Alcon), PureVision (Bausch+Lomb), and UCL 55% (United Contact Lens), and Kontur (Kontur Kontact Lens).

[0056] In some embodiments, the present invention relates to methods for modulating connexin, connexin gap junction channels and / or connexin hemichannels using doses and dose regimens of connexin modulators (e.g., including gap junction modulators and / or hemichannel modulators) for ocular surface restoration and / or corneal epithelial restoration. In some embodiments, modulation of gap junctions and hemichannels is achieved by administration of connexin 43 antisense oligonucleotides, connexin 43 peptide mimes and / or small molecule connexin 43 hemichannel blockers. In other embodiments, connexin 26, connexin 30, connexin 30.3, connexin 31, connexin 31.1, connexin 32, connexin 45, connexin 50 and / or connexin 58 modulators are used alone or in combination with or together with connexin 43 modulators.

[0057] In some embodiments, a gap junction modulator or hemichannel modulator (e.g., lufepirsen) is applied topically to the eye of a subject having non-healing or persistent ocular surface and / or corneal epithelial damage, and another topically administered gap junction modulator (e.g., Peptide 5, Ga19, XG19, or another "peptide mimetic") or a topically, orally, or systemically administered hemichannel modulator (e.g., Tonaversat, or another orally or systemically bioavailable gap junction and / or hemichannel modulator) is administered to the subject simultaneously with, before, or after the topically applied gap junction modulator or hemichannel modulator.

[0058] In some embodiments, the method involves co-administering a local connexin modulator and / or a gap junction modulator with a systemically available connexin-hemichannel modulator. The co-administration of the gap junction channel modulator can be performed simultaneously with, after, or before the administration of the connexin-hemichannel modulator. In some embodiments, compounds of formula I, e.g., tonaversat, compounds of formula II, and / or peptide mimes (e.g., Peptide 5, XG19, etc.) can be co-administered with a connexin expression modulator, e.g., lufepirsen or an anti-connexin peptide mime.

[0059] In some embodiments, the present invention provides the use of connexin modulators in the manufacture of pharmaceuticals for treating non-healing or persistent defects or impaired modulation of the ocular surface and / or cornea. In some embodiments, the pharmaceutical comprises or is essentially derived from a connexin antisense molecule (e.g., lufepirsen), including in amounts and / or concentrations as described herein. In some embodiments, the pharmaceutical comprises or is essentially derived from a connexin peptide mimetic, e.g., Peptide 5, Gap 19, XG 19, aCT1 peptide (e.g., CXT 1, CXT, CXT 3, CXT 4, CXT 5, or any other C-terminal connexin peptide mimetic with or without a cell-penetrating peptide attached to the C-terminus or N-terminus). In some embodiments, the pharmaceutical comprises or is essentially derived from a small molecule connexin hemichannel blocker, e.g., compounds according to formula I, including tonavelsat and caravelsat, and a prodrug, including a tonavelsat prodrug according to formula II.

[0060] In some embodiments, the connexin modulator and connexin 43 modulator include means for downregulating connexin transcription or translation of connexins, such as antisense molecules. Means for connexin modulation also include ZO-1 binding peptides, for example, in the case of connexin 43 (e.g., aCT1, αCT1, CXT1, CXT2, CXT3, CXT4, CXT5, etc.). Preferred gap junction channel modulators are connexin 43 expression modulators, connexin 43 gap junction modulators, and connexin 43 hemichannel modulators. Particularly preferred connexin 43 modulators include lufepirsen, XG19, and tonaversat.

[0061] In some embodiments, the present invention relates to pharmaceutical compositions and products including a kit with instructions for use, comprising a dose regimen for treating non-healing ocular and corneal surface defects and disorders using a therapeutically effective amount of a connexin modulator, and one or more connexin modulators, including a connexin expression modulator, a gap junction modulator, and / or a hemichannel modulator. In some embodiments, the kit includes or relates to instructions (e.g., online instructions) for administering and treating PEDs and / or PCEDs as described herein.

[0062] In some embodiments, a connexin modulator (e.g., including modulators of Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannels, e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators) is combined with a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutical composition. In some embodiments, suitable carriers and diluents include buffered aqueous solutions, isotonic saline solutions, e.g., phosphate-buffered saline, isotonic water, etc. In some embodiments, the carrier is Pluronic® or Poloxamer Gel. In some embodiments, the gel is Pluronic® F-127. In some embodiments, the pharmaceutical composition includes a buffer. In some embodiments, the buffer includes or is essentially dibasic sodium phosphate heptahydrate or monobasic sodium phosphate or both.

[0063] In some embodiments, the methods, doses, and dose regimens of the present invention described herein, as well as connexin modulators (e.g., including Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexin, gap junction, and / or hemichannel modulators, e.g., connexin expression modulator, connexin gap junction modulator, and connexin hemichannel modulator) are used to treat subjects for uveitis, including inflammation of the intermediate layer, uvea, and surrounding tissues of the eye. In some embodiments, connexin 43 modulator is used to treat one or more forms of uveitis. In some embodiments, other connexin modulators, including, for example, connexin 26 modulator, connexin 45 modulator, etc., are used alone or in combination. In some embodiments, anterior uveitis is treated. In some embodiments, intermediate uveitis is treated. In some embodiments, posterior uveitis is treated. In some embodiments, panuveitis is treated. In some embodiments, the uveitis treated is of any cause or is multifactorial. In each of these embodiments, one or more symptoms of uveitis are treated or mitigated, in whole or in part, using the methods, doses and dose regimens of the present invention, and connexin modulators as described herein.

[0064] In some embodiments, the methods of the present invention, any of the doses and dose regimens described herein, and any connexin modulator (e.g., including modulators of Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannels, e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators) are used to treat subjects for blepharitis.

[0065] In some embodiments, the methods, doses and dose regimens of the present invention described or referenced herein, as well as connexin modulators (e.g., including modulators of Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50 or Cx58 connexins, gap junctions and / or hemichannels, e.g., connexin expression modulators, connexin gap junction modulators and connexin hemichannel modulators) are used to treat subjects having any stage of dry eye disease (DED), also known as dry eye syndrome (DES), keratoconjunctivitis sicca (KCS), and keratitis sicca.

[0066] In other embodiments, the methods, doses and dose regimens of the present invention described or referenced herein, as well as the connexin modulator, are used, in whole or in part, to treat a subject for Sjögren's syndrome-related KCS, where one or more symptoms of Sjögren's syndrome-related KCS are treated or alleviated.

[0067] In other embodiments, the methods, doses and dose regimens of the present invention described or referenced herein, as well as the connexin modulator, are used to treat a subject for Fuchs dystrophy, the treatment resulting in the relief of all or part of one or more symptoms of Fuchs dystrophy.

[0068] In addition to the use of the present invention for the restoration of the ocular surface and / or corneal epithelium in some embodiments, the present invention relates to methods for modulating connexin, connexin gap junction channels and / or connexin hemichannels using dose and dose regimens of connexin modulators (e.g., including gap junction modulators and / or hemichannel modulators) for the restoration of the uvea, sclera, lacrimal glands and lacrimal ducts, meibomian glands, eyelids, etc. In some embodiments, modulation of gap junctions and hemichannels is achieved by administration of connexin 43 antisense oligonucleotides, connexin 43 peptide mimes and / or small molecule connexin 43 hemichannel blockers. In other embodiments, the connexin 26, connexin 30, connexin 30.3, connexin 31, connexin 31.1, connexin 32, connexin 45, connexin 50 and / or connexin 58 modulators are used alone or in combination with or together with the connexin 43 modulator and / or another therapeutic agent.

[0069] In some embodiments, doses of the connexin modulators described or referenced herein are administered together with or co-formulated with one or more effective amounts of compounds selected from the group consisting of antimicrobial agents, antibiotics, antiviral agents, antifungal agents, antiparasitic agents, mydriatic eye drops, steroidal anti-inflammatory agents, immunosuppressants, immunomodulatory agents, dry eye treatments (e.g., Tyrvaya, Rstasis, Cequa, Xiidra, Eysuvis), artificial tear compositions, and lubricants.

[0070] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antimicrobial agent for use in the methods and regimens described herein.

[0071] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antibacterial agent for use in the methods and regimens described herein.

[0072] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antiparasitic agent for use in the methods and regimens described herein.

[0073] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antifungal agent for use in the methods and regimens described herein.

[0074] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antiviral agent for use in the methods and regimens described herein. [Brief explanation of the drawing]

[0075] [Figure 1] Figure 1 shows dose regimens from connexin modulator clinical trials for the treatment of ocular surface defects and disorders, including non-healing persistent corneal epithelial defects. [Modes for carrying out the invention]

[0076] Detailed disclosure In some embodiments, the inventions described and claimed herein relate to connexins, connexin gap junctions and connexin hemichannel modulators, and their use in methods for treating non-healing ocular defects and disorders, including non-healing corneal defects and disorders, as well as doses and dose regimens therefor.

[0077] In some embodiments, the inventions described and claimed herein relate to connexins, connexin gap junctions and connexin hemichannel modulators, and their use in methods for treating other ocular defects, diseases, disorders and conditions, including uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy and any form of dry eye disease.

[0078] To maintain a smooth eye surface, the corneal epithelium must continuously regenerate itself to maintain its function as a barrier against the fluctuating external environment and various environmental injuries. After trauma, the cornea typically re-epithelializes rapidly, thereby minimizing the risk of infection, opacity, or perforation. Persistent epithelial defects or persistent corneal epithelial defects are called ocular or corneal epithelial lesions that do not heal after approximately two weeks of treatment with standard medical care. They can occur, for example, after exposure to toxic substances, chemicals, mechanical injury, thermal injury, and infections of the ocular surface, and are associated with a significant clinical morbidity in patients, resulting in discomfort or visual loss. The primary risk factor for most non-healing ocular surface defects or disorders is visual acuity loss.

[0079] In one embodiment, the present invention relates to pharmaceutical compositions, products, kits and methods for treating subjects having (or at risk of having) non-healing or persistent defects or disorders of the ocular surface or cornea, such as persistent epithelial defects of the eye or persistent corneal epithelial defects, by administering a therapeutically effective dose of at least one connexin modulator to the eye of the subject using a new clinically validated dosing regimen. The therapeutically effective dose may be used in conjunction with these new dosing regimens. New clinically validated doses may be used in conjunction with these new dosing regimens. These doses used in conjunction with these dosing regimens have been proven for very prolonged closure of non-healing defects or disorders of the ocular surface or cornea, including, for example, persistent epithelial defects of the eye and persistent corneal epithelial defects.

[0080] In one embodiment, a composition comprising a connexin modulator is applied topically to the target eye. In one embodiment, the connexin modulator is administered topically to the target eye, followed by the application of a bandage contact lens and / or amniotic membrane. In some embodiments, the connexin modulator is a connexin 43 modulator. In some embodiments, the connexin 43 modulator modulates the expression of connexin 43. In some embodiments, the connexin modulator (e.g., connexin 43 modulator) is placed on a bandage contact lens, which is then placed over the target eye.

[0081] In some embodiments of the method of the present invention, non-healing ocular surface defects or disorders are treated by pulse application of a connexin modulator in a dosing regimen as described herein (e.g., 3 to 7 times over 14 to about 35 days). In some embodiments, a connexin modulator is administered. In some embodiments, a connexin gap junction modulator is administered. In some embodiments, a connexin hemichannel blocker or modulator is administered. In some embodiments, the connexin modulator blocks or modulates connexin protein expression (e.g., connexin 43 protein expression). In some embodiments, the connexin gap junction modulator and / or a connexin hemichannel blocker or modulator blocks or modulates the gap junction and / or the hemichannel of connexin 43.

[0082] In some embodiments, the present invention relates to pharmaceutical compositions, products, and methods for treating non-healing ocular surface defects or disorders by administering a therapeutically effective amount of at least one connexin modulator to the target eye and / or by administering it orally or systemically, for example, in the case of a non-topically administered connexin modulator. In some embodiments, two or more connexin modulators are administered to the target eye and / or orally or systemically.

[0083] In some embodiments, the non-healing ocular surface defect or disorder is a persistent epithelial defect (PED). In some embodiments, the non-healing ocular surface defect or disorder is a persistent corneal epithelial defect (PCED). In the method of the present invention, administering at least one connexin modulator in the doses described herein or another therapeutically effective dose on days 1 and 2, subsequently on day 14, and optionally on day 28 or about 28 (and also optionally on day 35 or about 35), in a pulsed dosing format, is effective in healing persistent or non-healing ocular surface or corneal epithelial defects or disorders. See Example 2 below. The dose of the connexin modulator may also be administered on day 7 and / or day 21. In some embodiments, the connexin modulator is a connexin 43 modulator (e.g., a connexin expression modulator such as an antisense connexin expression modulator containing Cx43 antisense). Other embodiments include other connexin 43 gap junction modulators and connexin 43 hemichannel blockers or modulators (e.g., peptide mimes and small molecules).

[0084] In some embodiments, doses of the connexin modulator, e.g., lufepirsen, are administered on days 1, 2, and 14. In some embodiments, doses of the connexin modulator, e.g., lufepirsen, are administered on days 1, 2, 14, and 28 (e.g., if the surface defect does not heal after administration on days 1, 2, and 14). In some embodiments, doses of the connexin modulator, e.g., lufepirsen, are administered on days 1, 2, 7, 14, and 21 (e.g., if the surface defect does not heal after administration on days 1, 2, 14, and 28). In some embodiments, doses of the connexin modulator, e.g., lufepirsen, are administered on days 1, 2, 7, 14, 21, and 28. In some embodiments, doses of the connexin modulator, e.g., lufepirsen, are administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, doses are administered after day 35. In some embodiments, the therapeutically effective dose of the connexin modulator (e.g., lufepirsen) is administered on days 1, 2, and 14, and on one of days 7, 21, 28, and 35. In some embodiments, the administration using the described doses and dose regimens is for PED or PCED. In some embodiments, the connexin modulator comprises a connexin antisense molecule (e.g., lufepirsen). In some embodiments, the connexin modulator comprises a connexin peptide mimetic (e.g., Peptide 5, Gap 19, XG 19, Gap 26, Gap 27, aCT 1, etc.). In some embodiments, the connexin modulator comprises a small molecule connexin hemichannel blocker (e.g., tonaversat). In some embodiments, the connexin modulator is a connexin 43 modulator.

[0085] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 14, and 21.

[0086] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 7, 14, and 21.

[0087] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 7, 14, 21, and 28.

[0088] In some embodiments, to treat non-healing or persistent ocular or corneal surface defects in a subject, three doses of a connexin modulator (e.g., lufepirsen) are administered over 14 days, including, for example, on days 1 and 2. In some embodiments, four doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 28 days. In some embodiments, five doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 28 to approximately 35 days. In some embodiments, six to seven doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 35 days. For example, in one embodiment, the connexin modulator (e.g., lufepirsen) is administered four times: over days 1 and 2, and approximately on days 14 and 28. In another embodiment, the connexin modulator (e.g., lufepirsen) is administered five times: over days 1 and 2, approximately on day 14, approximately on day 28, and approximately on day 35. In another embodiment, the connexin modulator (e.g., lufepirsen) is administered five times: over days 1 and 2, on about day 7, on about day 14, and on about day 28. In yet another embodiment, the connexin modulator (e.g., lufepirsen) is administered six times: over days 1 and 2, on about day 7, on about day 14, on about day 21, and on about day 28. In yet another embodiment, the connexin modulator (e.g., lufepirsen) is administered seven times: over days 1 and 2, on about day 7, on about day 14, on about day 21, and on about day 28. References to “administration” in this specification, of course, refer to the administration of the connexin modulator in single or divided doses.

[0089] In some embodiments, the connexin modulator is a modulator of Cx26, Cx30, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, Cx57, or any other connexin in the eye or blood vessels.

[0090] In some embodiments, the connexin modulator is a modulator of Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx43, Cx45, Cx50, and Cx58, or any other connexin, in the corneal epithelium. Transcripts encoding these 10 connexin isoforms have been detected by reverse transcriptase polymerase chain reaction in both the central and peripheral corneal epithelium. Six (Cx26, Cx31, Cx32, Cx43, Cx45, and Cx58) were confirmed by laser scanning confocal microscopy. Cx26 was detected throughout the central corneal epithelium as well as in the intermediate and superficial layers of the corneal marginal epithelium. Cx43 and Cx45 were localized in basal and suprabasal epithelial cells. Cx58 was expressed in the superficial epithelium throughout the cornea, while Cx31 and Cx32 were expressed primarily in the central corneal epithelium. See Yuan, X, et al., Expression Pattern of Connexins in the Corneal and Limbal Epithelium of a Primate. Cornea 28(2):194-199 (Feb 2009); Zhai, J, et al., Connexin expression patterns in diseased human corneas. Exp.Ther.Med. 7, 791-798 (2014). In some embodiments, the connexin modulator is a Cx43 connexin modulator, e.g., a Cx43 expression modulator (e.g., anti-Cx43 antisense), a Cx43 gap junction modulator, or a Cx43 hemichannel modulator (e.g., an anti-connexin 43 peptide mime or small molecule). In some embodiments, the modulator is a modified or unmodified antisense polynucleotide or peptide mime, e.g., a modified or unmodified Cx43 antisense polynucleotide or C43 peptide mime, or other corneal epithelial, endothelial, or vascular connexin. In some embodiments, the modulator may include or exclude any of the aforementioned connexins.

[0091] In some embodiments, the amount of connexin modulator administered to a subject having a non-healing ocular surface defect or disorder (e.g., PED) or a non-healing corneal surface defect or disorder (e.g., PCED) in any of the administration schedules described herein is a therapeutically effective dose. In some embodiments, a therapeutically effective dose of connexin modulator is administered to the subject, for example, on (1) days 1, 2 and 14, or (2) days 1, 2 and 14, and optionally on day 28, or (3) days 1, 2, 7, 14, and about 21, and optionally on about 28 and 35, or (4) in addition to these administration regimens (1) to (3), on any of the other administration schedules listed herein. In some embodiments, a therapeutically effective dose of lufepirsen is administered.

[0092] In some embodiments, in any of the administration schedules described herein (for example, (1) days 1, 2, and 14, and optionally day 28 and / or 35; (2) days 1, 2, and 14, and optionally day 7, 21, and / or 28; (3) days 1, 2, and 14, and optionally day 7, 21, 28, and / or 35; (4) any more, more, or all of days 1, 2, and 14, and days 7, 21, and 28, and optionally day 35; or (5) any day described in other administration schedules listed herein), subjects having non-healing ocular surface defects or disorders (e.g., PED) or non-healing corneal surface defects or disorders (e.g., PCED) The amount of connexin modulator administered is one or more of the specific doses described herein, and includes, for example, a dose of milligrams / milliliter (mg / mL) of connexin modulator (e.g., a composition containing or essentially comprising about 0.6 mg / mL or at least about 0.6 mg / mL of connexin modulator), a dose of micromolar (μM) of connexin modulator (e.g., a composition containing or essentially comprising connexin modulator at a concentration of about or at least about 19 μM), a dose of milligrams (mg) of connexin modulator (e.g., a composition containing or essentially comprising about or at least about 0.18 mg of connexin modulator), and so on.

[0093] In some embodiments, the connexin modulator comprises an antisense molecule. See Examples 1 and 2 below. In some embodiments, the antisense molecule is a connexin 43 antisense oligonucleotide. In some embodiments, the connexin modulator may be a connexin 43 antisense polynucleotide comprising, essentially, or consisting of sequences by SEQ ID NOs: 1-3, SEQ ID NOs: 4-16 and / or modified versions thereof. In some embodiments, the antisense oligonucleotide comprises, essentially, or consists of 5'-GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC-3' (SEQ ID NO: 1; lufepirsen). In some embodiments, the antisense oligonucleotide may be chemically modified or unmodified oligonucleotide, e.g., modified or unmodified DNA oligonucleotide. In some embodiments, the oligonucleotide is modified by altering a phosphodiester bond, a ribose backbone, and / or one or more nucleic acid bases. Other modifications are described herein. Other modifications are known in the art and are described in various publications, for example, Shen, X. and Corey, DR., Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNA, Nuelic Acids Res. 2018 Feb 28;46(4):1584-1600. Manufacturing methods are also known in the art. For example, see Abramova T., Frontiers and Approaches to Chemical Synthesis of Oligodeoxyribonucleotides, Molecules 2013 Jan;18(1):1063-1075; and Hao M et al., Current and Emerging Methods for Synthesis of Single-Stranded DNA Genes (Basel) 2020 Jan 21;11(2):116.See Anwar, S. et al., "Enhancing the Effectiveness of Oligonucleotide Therapeutics Using Cell-Penetrating Peptide Conjugation, Chemical Modification, and Carrier-Based Delivery Strategy and Carrier-Based Delivery Strategies." Pharmaceutics 2023, 15, 1130.

[0094] In some methods of the present invention, the connexin modulator administered to a non-healing surface defect or disorder in a subject is 0.06% lufepirsen. In some embodiments, the connexin modulator administered to a subject having a non-healing surface defect or disorder is 0.006% lufepirsen. In some embodiments, the administered connexin modulator is at least about 0.06% lufepirsen. In some embodiments, the connexin modulator administered to a subject having a non-healing surface defect or disorder is at least about 0.006% lufepirsen. In some embodiments, the administration is for PED or PCED. In some embodiments, a pharmaceutically acceptable carrier and a composition comprising or essentially comprising about 0.06% lufepirsen, about 0.006% lufepirsen, at least about 0.06% lufepirsen, or at least about 0.006% lufepirsen is administered to heal a non-healing surface defect or disorder in a subject. The doses of lufepirsen described herein and below may be prepared as described in Example 1. In some embodiments, the connexin modulator administered in the amounts described is a connexin antisense molecule other than (or in addition to) lufepirsen. In some embodiments, the connexin modulator administered in the amounts described is a connexin peptide mimetic (e.g., XG19). In some embodiments, the connexin modulator administered in the amounts described is a connexin hemichannel blocker (e.g., Tonaversat).

[0095] In some embodiments, non-healing surface defects or disorders are treated with a composition comprising or essentially comprising 0.6 mg / mL of lufepirsen. In some embodiments of the present invention, non-healing surface defects or disorders are treated with a composition comprising or essentially comprising 0.06 mg / mL of lufepirsen. In some embodiments, the composition comprises or essentially comprises at least about 0.6 mg / mL of lufepirsen. In some embodiments, the composition comprises or essentially comprises at least about 0.06 mg / mL of lufepirsen. In some embodiments, the administration is for PED or PCED. In some embodiments, the connexin modulator administered in the described amounts is a connexin antisense molecule other than (or in addition to) lufepirsen. In some embodiments, the connexin modulator administered in the described amounts is a connexin peptide mimetic (e.g., XG19). In some embodiments, the connexin modulator in the described amounts is a connexin hemichannel blocker (e.g., Tonaversat).

[0096] In some embodiments, non-healing surface defects or disorders in a subject are treated with a composition comprising or essentially comprising about 0.18 mg of lufepirsen in the regimen described herein. In some embodiments, each dose of lufepirsen applied to a non-healing surface defect or disorder within or on a subject comprises or essentially comprises at least about 0.18 mg of lufepirsen in the regimen described herein. In some embodiments of the present invention, non-healing surface defects or disorders are treated with a composition comprising or essentially comprising about 0.018 mg of lufepirsen in the regimen described herein. In some embodiments, each dose of lufepirsen applied to a non-healing surface defect or disorder comprises or essentially comprises at least about 0.018 mg of lufepirsen in the regimen described herein. In some embodiments, the administration is for PED or PCED. In some embodiments, the connexin modulator administered in the amounts described herein is a connexin antisense molecule other than (or in addition to) lufepirsen. In some embodiments, the connexin modulator administered in the described amount is a connexin peptide mimetic (e.g., XG19). In some embodiments, the connexin modulator administered in the described amount is a connexin hemichannel blocker (e.g., Tonaversat).

[0097] In some embodiments, the therapeutically effective amount of modulators effective in the methods of the present invention, e.g., connexin modulators, e.g., connexin 43 modulators, connexin 43 gap junction modulators, and / or connexin 43 hemichannel modulators, comprises a composition having a concentration of approximately or at least approximately 2 micromolars (2 μM), 5 micromolars (5 μM), 10 micromolars (10 μM), 15 micromolars (15 μM), or 20 micromolars (20 μM), which includes a dose of a connexin modulator (e.g., lufepirsen) at approximately (or at least approximately) 1.9 micromolars (1.9 μM) or 1.9405 micromolars (1.9405 μM), and approximately (or at least approximately) 19-19.4 micromolars or 19.405 micromolars (19-19.4 μM or 19.405 μM), or any amount within two of these listed doses. Other effective doses effective in the methods of the present invention for the treatment of defects or disorders of the ocular surface (e.g., ocular PED and PCED) include a composition comprising a connexin modulator, e.g., a connexin 43 modulator, a connexin 43 gap junction modulator, and / or a connexin 43 hemichannel modulator (e.g., lufepirsen) at a concentration of about or at least about 30 micromolars (30 μM) or 40 micromolars (40 μM). In some embodiments, the therapeutically effective dose of connexin modulator administered as described is a connexin antisense molecule other than (or in addition to) lufepirsen. In some embodiments, the therapeutically effective dose of connexin modulator administered as described is a connexin peptide mimetic (e.g., XG19). In some embodiments, the therapeutically effective dose of connexin modulator described is a connexin hemichannel blocker (e.g., tonaversat).

[0098] In some embodiments, approximately 0.18 mg (or other connexin antisense), approximately 0.018 mg of lufepirsen (or other connexin antisense or connexin modulator), or approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or approximately 0.5 mg to approximately 1 mg of lufepirsen is administered to non-healing surface defects or disorders in a single dose or in two or more divided doses (e.g., twice daily doses with up to 6 doses administered on days 1, 2, and 14). In other embodiments, at least approximately 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or 0.5 mg, or at least approximately 1 mg of lufepirsen is administered to non-healing surface defects or disorders in a single dose or in two or more divided doses (e.g., twice daily doses with up to 6 to 10 doses administered on days 1, 2, and 14), or using other dose regimens described herein, including on day 28 and / or day 35. In other embodiments, at least about 0.5 mg or at least about 1 mg of lufepirsen is administered, for example, in a single dose or in divided doses (e.g., twice daily doses administered on days 1, 2 and 14, and on one or more days among days 7, 21, 28 and / or 35, with a maximum of 14 doses administered). In some embodiments, the administration is for PED or PCED in the subject. In some embodiments, other connexin modulators, including, for example, other connexin 43 modulators (and / or other connexin modulators), as well as other antisense compounds, peptide mimes, and / or small molecules, are administered at these doses on these days. In some embodiments, the connexin modulator administered at the amounts described is a connexin antisense molecule other than (or in addition to) lufepirsen. In some embodiments, the connexin modulator administered at the amounts described is a connexin peptide mime (e.g., XG19). In some embodiments, the described amount of connexin modulator is a connexin hemichannel blocker (e.g., tonaversat).

[0099] In any of these dosage and administration regimen and method embodiments, the lufepirsen dose may be administered to the target PED or PCED. In some embodiments of these dosage and administration regimen and method, the administered connexin modulator dose includes another connexin antisense or modulator, for example, another Cx43 antisense or Cx43 modulator other than or in addition to lufepirsen, or another ocular or corneal epithelial connexin modulator other than or in addition to Cx43.

[0100] In some embodiments of the present invention, the volume of a composition containing or essentially comprising a connexin modulator administered to a non-healing surface defect or disorder in a subject is about 300 μL. In other embodiments, the volume of the administered composition is about 250 μL to about 1.0 mL. In some embodiments, the composition contains about 0.01 mg, 0.018 mg, 0.18 mg to about 0.2 mg of lufepirsen, or about 0.18 mg, 0.20 mg, or 0.50 mg to about 1 mg of lufepirsen. In some embodiments, the composition contains at least about 0.018 mg of lufepirsen, at least about 0.18 mg of lufepirsen, at least about 0.5 mg of lufepirsen, or at least about 1.0 mg of lufepirsen. In some embodiments, other connexin modulators, including other connexin 43 modulators (and / or other connexin modulators) other than lufepirsen, as well as other antisense compounds, peptide mimes, and / or small molecules, are administered in these amounts.

[0101] In some embodiments, the modulator is a small molecule connexin antagonist. In some embodiments, the modulator is a small molecule connexin antagonist comprising a compound of formula I, including tonaversat. Tonaversat (benzoylaminobenzopyran) is a modulator of gap junction channel and hemichannel activity containing connexin 43. In particular, tonaversat can block or inhibit hemichannels containing connexin 43.

[0102] In some embodiments, the modulator is a connexin peptide mimetic. Connexin modulators containing Peptide 5 inhibit Cx43 hemichannel activity and / or ATP release during and after injury. Other connexin modulator peptide mimetics include Gap19 and XG19, as well as their analogues.

[0103] The present invention provides, in particular, (1) a method for treating a non-healing defect or disorder of the ocular surface or cornea (e.g., PED or PCED) in a subject by administering a therapeutically effective amount of a connexin modulator (e.g., a connexin expression modulator), a connexin gap junction modulator, and / or a connexin hemichannel modulator in a dose regimen described herein to close and heal said defect or disorder; (2) a method for treating a non-healing defect or disorder of the ocular surface or cornea (e.g., PED or PCED) in a subject by administering a dose of a connexin modulator described herein (e.g., in any dosing regimen described herein); and (3) a kit containing said pharmaceuticals, including or relating to instructions for the use of such modulators in the manufacture of pharmaceuticals (e.g., pharmaceutical compositions), including, for example, antisense modulators, peptide mimetic modulators, and small molecule modulators, and instructions for use in the methods of the present invention. See, for example, Example 2, which describes a clinically validated pulse dosing regimen for treating and healing a non-healing persistent ocular defect.

[0104] In some embodiments, the present invention provides, among other things, (1) a method for treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and all forms of dry eye disorders (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye) in a subject by administering a therapeutically effective dose of a connexin modulator (e.g., a connexin expression modulator, a connexin gap junction modulator, and / or a connexin hemichannel modulator) in a dose regimen described herein for treating the said disorder; (2) a method for treating uveitis in a subject by administering a dose of a connexin modulator described herein (e.g., in any dose regimen described herein); (3) Methods for treating blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy and all forms of dry eye disease (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-reduced dry eye); and (4) Pharmaceuticals (e.g., pharmaceutical compositions) for treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy and dry eye disease (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-reduced dry eye), as well as the use of such modulators, including antisense modulators, peptide mimetic modulators and small molecule modulators, in the manufacture of a kit containing such pharmaceuticals, including or relating to instructions for use in the methods of the present invention.

[0105] All non-healing or persistent ocular surface or corneal (e.g., corneal epithelium) defects or disorders of any etiology can be treated using the compositions and methods of the present invention, comprising doses and dose regimens. In some embodiments, the non-healing ocular surface defect or disorder is PED. In some embodiments, the non-healing ocular surface defect or disorder is PCED. In some embodiments, the non-healing ocular surface defect or disorder is a corneal ulcer. In some embodiments, the non-healing ocular surface or corneal defect or disorder is due to chemical and / or thermal injury. The non-healing ocular surface or corneal defect or disorder may occur after exposure to toxic agents, chemicals, mechanical injury, thermal injury, and ocular surface or corneal infection. In some embodiments, the non-healing ocular surface or corneal defect or disorder is due to inflammation or inflammatory disease. In some embodiments, the non-healing ocular surface or corneal defect or disorder is due to inflammation of the eye. In some embodiments, the non-healing ocular surface or corneal defect or disorder is due to inflammatory disease of the eye, ocular surface, or cornea. In some embodiments, non-healing ocular surface defects or disorders result from physical trauma or injury, including mechanical injury. In certain embodiments, non-healing ocular surface defects or disorders are PED or PCED resulting from chemical and / or thermal injury. In some embodiments, the chemical and / or thermal injury resulting in PCED is severe chemical and / or thermal injury. In some embodiments, ocular surface and / or corneal burns or injuries treated by the methods of the present invention have more than 50% conjunctival involvement or more than 6 working hours of corneal margin involvement. In some embodiments, ocular surface and corneal burns and injuries treated by the methods of the present invention are 3 or higher on the Dua classification scale. In some embodiments of the present invention, severe ocular and corneal burns and injuries are particularly responsive to treatment with a connexin 43 modulator (e.g., lufepirsen) and / or other connexin modulators using the methods described herein, and all etiologies of PED and PCED, regardless of cause, can be treated using the methods of the present invention as described herein.The etiologies of PED and PCED include not only inflammation and inflammatory diseases, but also chemical, mechanical, and thermal causes, neurotrophic keratitis, and corneal margin stem cell deficiency. In some embodiments, the methods and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from chemical injury. In some embodiments, the methods and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from thermal injury. In some embodiments, the methods and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from mechanical injury. In some embodiments, the methods and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from inflammation. In some embodiments, the methods and / or doses of the present invention are used to treat PED and / or PCED caused by or resulting from neurotrophic keratitis.

[0106] In some embodiments, the non-healing ocular surface defect or disorder is a corneal ulcer. In some embodiments, the corneal ulcer is caused by bacteria, viruses, fungi, or parasites. In some embodiments, the corneal ulcer is due to Acanthamoeba keratitis, fungal keratitis, or herpes simplex keratitis. In other embodiments, the non-healing ocular surface defect or disorder, e.g., a corneal ulcer, is caused by trauma or a foreign body, such as an abrasion of the eye (e.g., a scratch or cut). In yet another embodiment, the non-healing ocular surface defect or disorder, e.g., a corneal ulcer, is caused by allergies or allergic eye disease. In yet another embodiment, the non-healing ocular surface defect or disorder, e.g., a corneal ulcer, is caused by dry eye. In some embodiments, the non-healing ocular surface defect or disorder is a burn. In some embodiments, the non-healing ocular surface defect or disorder is a corneal burn or an eye or corneal burn ulcer.

[0107] In some embodiments, the methods, doses, and dose regimens of the present invention described herein, as well as connexin modulators (e.g., including Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexin, gap junction and / or hemichannel modulators, e.g., connexin expression modulator, connexin gap junction modulator, and connexin hemichannel modulator) are used for the treatment of uveitis, i.e., inflammation of the middle layer, uvea, and surrounding tissues of the eye. In some embodiments, connexin 43 modulator is used to treat one or more forms of uveitis. In some embodiments, other connexin modulators, including, for example, connexin 26 modulator, connexin 45 modulator, etc., are used alone or in combination.

[0108] In some embodiments, the uveitis treated as described herein is anterior uveitis (which, for example, affects the iris in the front of the eye).

[0109] In some embodiments, the uveitis treated as described herein is intermediate uveitis (which, for example, affects the ciliary body and vitreous humor).

[0110] In some embodiments, the uveitis treated as described herein is posterior uveitis (which, for example, affects the retina and choroid at the back of the eye).

[0111] In some embodiments, the uveitis treated as described herein is panuveitis (which affects, for example, all parts of the uvea from the anterior to the posterior part of the eye).

[0112] In some embodiments, the uveitis treated as described herein is caused, whole or in part, by one or more of the following: eye trauma, eye injury, eye surgery, infection (e.g., cytomegalovirus (CMV) retinitis, stoplasmosis, reactive arthritis, shingles, syphilis, toxoplasmosis, feline pruritus, herpes zoster, tuberculosis, etc.), inflammation, inflammatory diseases, and autoimmune disorders (e.g., AIDS, Behçet's disease, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, ulcerative colitis, Vogt-Koyanagi-Harada (VKH) disease, etc.). In some embodiments, the uveitis treated as described herein is caused, whole or in part, by autoimmune or inflammatory disorders affecting other parts of the body, such as sarcoidosis, systemic lupus erythematosus, or Crohn's disease. In some embodiments, the uveitis treated as described herein is caused, whole or in part, by ankylosing spondylitis. In some embodiments, uveitis treated as described herein occurs for unknown reasons.

[0113] In these embodiments of uveitis treatment, one or more symptoms of uveitis are alleviated, in whole or in part, using the methods, doses, and dose regimens of the present invention, as well as other agents for treating uveitis (e.g., antibiotics, antivirals, or antifungals; mydriatic eye drops; steroidal anti-inflammatory agents; immunosuppressants) and connexin modulators as described herein, which may be administered alone or in combination with (or co-formulated with) other agents. Symptoms of uveitis that are alleviated when treated as described herein include eye pain, conjunctival hyperemia, photosensitivity, blurred vision, dark / floating spots (floaters) in the visual field, and decreased visual acuity.

[0114] In some embodiments, the methods, doses, and dose regimens of the present invention described herein, as well as connexin modulators (e.g., including modulators of Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannels, e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators) are used for the treatment of blepharitis. In some embodiments, the subject is treated for anterior blepharitis. In some embodiments, the subject is treated for posterior blepharitis. In some embodiments, one or more connexin modulators described or referenced herein are administered to a subject together with, or formulated to be administered to or co-administered with, one more antimicrobial agent (e.g., erythromycin, bacitracin) and / or one or more immunomodulatory agents (e.g., cyclosporine) and / or blepharitis treatment agents for use in the methods and regimens described herein for treating blepharitis. In some embodiments, one or more connexin modulators described or referenced herein are administered together with, or co-formulated with, one more anti-inflammatory agent (e.g., steroid) for use in the methods and regimens described herein for treating blepharitis. In these embodiments of blepharitis treatment, one or more symptoms of blepharitis are alleviated, holistically or partially, using the methods and connexin modulators of the present invention as described herein, including doses and dose regimens. Symptoms of blepharitis that are relieved when treated as described herein include a feeling of something in the eye, burning or stinging of the eye, watery eye, itchy eye, sensitivity to light, redness and swelling of the eye or eyelid, dry eye, crusted eyelid or eyelashes upon waking, and blurred vision.

[0115] Dry eye is an ocular surface disease. In some embodiments, the methods, doses, and dose regimens of the present invention described or referenced herein, as well as connexin modulators (e.g., including modulators of connexin, gap junction, and / or hemichannel, e.g., connexin expression modulator, connexin gap junction modulator, and connexin hemichannel modulator) are used for the treatment of subjects having dry eye syndrome (DES), keratoconjunctivitis sicca (KCS), and any stage of dry eye disease (DED), also known as keratitis sicca. All stages of DED, including DED Stage 1, DED Stage 2, DED Stage 3, and DED Stage 4, can be treated using the methods, doses, dose regimens, compositions, and kits described herein. In some embodiments, the methods, doses, and dose regimens of the present invention, as well as the compositions of the present invention, may be used alone or in combination with artificial tears and / or systemic or topical anti-inflammatory agents for the treatment of DED. In some embodiments, the connexin modulator doses for use as described herein may be formulated together with or within an artificial tear composition, or together with an anti-inflammatory agent, for use in the treatment of DED. In some embodiments, the connexin modulator doses for use as described herein are formulated together with an anti-inflammatory compound or composition for topical administration in the treatment of DED, using a known effective dose of at least one inflammatory compound. In some embodiments, the connexin modulator doses for use as described herein are administered alone or in combination with one or more DED treatments (e.g., Tyrvaya, Rstasis, Cequa, Xiidra, Eysuvis).In some embodiments, a connexin modulator dose for use as described herein is formulated together with one or more DED treatments for co-administration (e.g., one or more connexin modulators together with one or more other dry eye treatments (e.g., Tyrvaya, Rstasis, Cequa, Xiidra, Eysuvis, etc.)). In some embodiments, a connexin modulator dose for use as described herein is formulated together with at least one corticotosteroid for co-administration. In these embodiments of dry eye treatments, one or more symptoms of dryness are alleviated, in whole or in part, using the methods and connexin modulators of the present invention as described herein, including doses and dose regimens. Symptoms of dry eye that are alleviated when treated as described herein include dryness of the eyes, a gritty, stinging or burning sensation, redness, watery or teary eyes, and mucus that causes the eyes to feel “stuck closed” after sleep. Many people with dry eyes also report a sensation in the eye or eye strain, blurred vision, or eye fatigue. Itching and photosensitivity are other symptoms of dry eyes.

[0116] In other embodiments, the methods, doses, and dose regimens of the present invention described or referenced herein, as well as the connexin modulator, are used, in whole or in part, to treat a subject for Sjögren's syndrome-related KCS, and one or more symptoms of Sjögren's syndrome-related KCS are treated or alleviated. Symptoms of Sjögren's syndrome-related KCS that are alleviated when treated as described herein include itching, burning, grittiness, pulling, or foreign body sensation and / or photophobia. Sharp, stinging pain, eye strain or fatigue, and blurred vision may also occur. Some patients notice excessive tearing after severe irritation.

[0117] In other embodiments, the methods, doses and dose regimens of the present invention described or referenced herein, as well as connexin modulators, are used to treat subjects for Fuchs dystrophy, and the treatment results in the relief of all or some of the symptoms of Fuchs dystrophy, one or more of the symptoms of Fuchs dystrophy. One of the first signs and symptoms of Fuchs dystrophy is blurred vision caused by fluid accumulation in the cornea. In the early stages of the disease, excess fluid accumulates overnight during sleep, causing blurred vision and / or discomfort that may last for several hours when awake in the morning. In later stages of Fuchs dystrophy, the duration of swelling, visual impairment and discomfort is longer, and may even last throughout the day. Other symptoms of Fuchs dystrophy include a rough or gritty sensation in the eye, sometimes accompanied by sharp eye pain, discomfort with bright light, vision that fluctuates throughout the day or from day to day, halos and / or glare from bright light, and blurred vision combined with poor color contrast.

[0118] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antimicrobial agent for use in the methods and regimens described herein.

[0119] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antibacterial agent for use in the methods and regimens described herein.

[0120] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antiparasitic agent for use in the methods and regimens described herein.

[0121] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antifungal agent for use in the methods and regimens described herein.

[0122] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more antiviral agent for use in the methods and regimens described herein.

[0123] In some embodiments, one or more connexin modulators described or referenced herein are formulated together with one more compound selected from the group consisting of antimicrobial agents, antibacterial agents, antiparasitic agents, antiviral agents, antifungal agents and lubricants.

[0124] definition It is understood that, for various reasons including the patient's ability to travel to a clinic, office, or site for drug administration, the patient's schedule, and drug availability, it may be necessary (and possible) to adjust the administration dates of connexin modulators according to the dosage regimens described herein. The term “about” when used in relation to administration dates for connexin modulators according to the dosage regimens described herein generally means plus or minus one day of that day. Thus, an “about day 28” administration means an administration on day 28, but also includes, for example, administrations on day 27 or day 29.

[0125] As used herein, the term “connexin modulator” (sometimes called a “connexin inhibitor” or “antagonist”) is a compound that blocks, inhibits and / or reduces the function or activity of connexin, connexin gap junction channels, or connexin hemichannels, either alone, together, or separately, including, for example, the blocking, inhibition and / or reduction of function and / or activity of connexin proteins, their transport and / or assembly, and / or the formation of connexin hemichannels and / or connexin gap junctions. Such functions and activities may include, for example, the docking of hemichannels on adjacent cells and opening to form gap junction channels. They may also include intercellular communication between cells and intercellular molecular flow through gap junction channels. Modulation of hemichannels is the modulation of one or more functions and / or activities of hemichannels, for example, the flow of molecules through hemichannels. Such functions and activities may include, for example, the flow of molecules from the extracellular space or environment to the cell via hemichannels, and / or the flow of molecules (e.g., adenosine triphosphate (ATP)) from the intracellular space or the cellular environment to the extracellular space or environment via hemichannels. Blocking, inhibiting, and / or reducing functions or activities may be direct or indirect (e.g., directly blocking the channel, inducing conformational changes, or altering the connexin phosphorylation state or open probability). Modulation of hemichannels also includes suppressing the permeability of hemichannels (e.g., reducing the flow of permeable substances through the hemichannels). In some embodiments, the permeable substance is adenosine triphosphate (ATP). Connexin modulators may be of any chemical nature. However, as an example, connexin modulators may be nucleic acids (including antisense molecules, RNAi molecules, morpholino, and other nucleic acids as described herein), peptides or peptide mimes, or small molecules or other chemical substances.In certain embodiments, a connexin modulator is a compound that targets one or more components of a gap junction, including a connexin or hemichannel, to inhibit or block its activity, expression, transport, and / or assembly. The terms “inhibit,” “block,” “antagonize,” or “modulate” should not be interpreted as meaning that the function, activity, expression, transport, and / or assembly of a connexin, connexin hemichannel (e.g., connexin 43 hemichannel), or gap junction is completely inhibited, blocked, completely antagonized, or modulated, although this may be preferable, and should be interpreted as including any reduction of the function, activity, expression, transport, and / or assembly of a connexin (including its transcription, translation, and / or expression), a connexin hemichannel (including its permeability to or opening to the extracellular environment, or release of ATP), or a gap junction (including opening to an adjacent cell, or creation from a hemichannel in an adjacent cell). The connexin modulators used herein include connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators.

[0126] In some embodiments, in addition to the Cx43 modulator, the connexin modulator is a modulator of any other connexin in the corneal epithelium, including Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx45, Cx50, and Cx58. In some embodiments, the connexin modulator may be a modulator of a connexin present in a blood vessel, e.g., the Cx37 modulator, Cx40 modulator, Cx43 modulator, and / or Cx45 modulator. Thus, as used herein, the term “connexin modulator” generally refers to a connexin modulator unless otherwise provided, but specifically refers to the connexin 43 modulator and other connexin modulators mentioned herein (including connexin 43 and other gap junction and hemichannel modulators), as well as modulators of other vascular, endothelial and epithelial (e.g., ocular and corneal epithelial) connexins, gap junctions, and hemichannels. In some embodiments, the connexin modulator is a connexin 43 modulator, e.g., a modulator of connexin 43 expression, a connexin 43 hemichannel modulator that inhibits or blocks hemichannel opening, or a connexin 43 peptide mimetic. In some embodiments, the gap junction modulator is a modulator of or includes other connexins found on the ocular surface, including the cornea and corneal epithelium, as well as their hemichannels and gap junctions. In other embodiments, the connexin modulator is a modulator of any other connexins in the eye or blood vessels, including Cx36 and Cx57, in addition to Cx37, Cx40, Cx43 and Cx45, as well as Cx26, Cx30, Cx31.1 and Cx50, which are also found in the corneal epithelium. In some embodiments, the modulating agent may include or exclude any of the above connexins, connexin gap junctions and / or connexin hemichannels described herein.In some embodiments, defects or disorders of the ocular surface, such as PED, PCED, and / or ocular ulcers (including, for example, inflammatory ocular surface ulcers, or ocular surface ulcers, disorders, or defects characterized at least partially by inflammation), are treated with a connexin modulator (e.g., a connexin expression modulator, a connexin gap junction modulator, and / or a connexin hemichannel modulator) to inhibit the activation of one or more inflammasomes. In some embodiments, the connexin modulator inhibits the activation of the inflammatory cascade by the inflammasome. In some embodiments, the activation and / or activity of the NLRP3 inflammasome is modulated (e.g., blocked or downmodulated) by the connexin modulator. In some embodiments, the activation of the inflammatory cascade by the NLRP3 inflammasome is modulated (e.g., blocked or downmodulated) by the connexin modulator. Inflammasome modulation assists in the closure of non-healing defects or disorders of the ocular surface or cornea, including those described herein, and in the treatment of other diseases, conditions, and disorders described herein. In some embodiments, defects or disorders of the ocular surface, such as PED, PCED, and / or ocular ulcers, are treated with inflammasome modulators (e.g., NLRP3 inflammasome modulators), which may not be connexin modulators (e.g., connexin 43 hemichannel modulators). Inflammasome modulators include, for example, those described by Leung and Lowery in *The patent landscape of inflammasome modulators*, *Nature Reviews Drug Discovery* 19, 158 (2020). See also Chauhan, D. et al., *Therapeutic modulation of inflammasome pathways*, *Immunol Rev. 297(1):123-138* (Sept 2020).The inflammasome modulator includes a selective G protein-coupled receptor 40 (GPR40) agonist (e.g., fasiglifam, which inhibits inflammasome activation by blocking the formation of apoptosis-associated speck-like proteins (ASCs) containing the caspase-mobilizing domain (CARD), an inflammasome component), and ethyl pyruvate, which significantly suppresses the activation of the NLRP3 inflammasome. Other inflammasome modulators unrelated to connexin and connexin hemichannels are known in the art. Modulation of the function of hemichannels and / or gap junction channels can be performed by any means. However, as merely an example, modulation may be carried out by one or more of the following: blocking, blocking, inhibiting, or reducing the formation of gap junctions by hemichannel docking; inducing or promoting hemichannel closure; blocking, blocking, inhibiting, or reducing hemichannel opening; blocking, blocking, inhibiting, or reducing hemichannel permeability; inducing or promoting the loss of coupling between hemichannels; or inducing, inducing, or promoting the intracellularization of hemichannels and / or gap junctions.Just as the use of words such as “blocking,” “inhibiting,” “preventing,” “reducing,” and “antagonizing” should not be interpreted as meaning complete blockage, inhibition, prevention, or antagonism, “induce” or “promote” should not be interpreted as meaning hemichannel docking; inducing or promoting complete closure of hemichannels; completely blocking, blocking, inhibiting, or reducing hemichannel opening; completely blocking, blocking, inhibiting, or reducing hemichannel permeability; completely inducing or promoting the loss of coupling between hemichannels; or the complete prevention, blockage, inhibition, or reduction of gap junction formation by inducing, inducing, or promoting the complete loss of coupling or complete internalization of connexin hemichannels and / or gap junctions (or groups of hemichannels and / or gap junctions), but should be interpreted as including partial induction or promotion of these. Connexins, hemichannels, and gap junctions can be present in any type of cell. Therefore, references to “connexins,” “hemichannels,” or “gap junctions” should be interpreted as including references to connexins, hemichannels, or gap junctions present in any cell type, unless otherwise specified in the context. In one embodiment, a hemichannel is an epithelial cell connexin, hemichannel, or gap junction (e.g., ocular or corneal epithelial cell connexin, hemichannel, or gap junction). In one embodiment, a connexin, hemichannel, or gap junction is a vascular connexin, hemichannel, or gap junction. In one embodiment, a connexin, hemichannel, or gap junction is a connexin, hemichannel, or gap junction found in vascular endothelial cells and / or vascular smooth muscle cells. A “hemichannel blocker” is a compound that prevents molecules from passing through connexin hemichannels. Hemichannel blockers can block or reduce hemichannel opening, block, reduce or inhibit hemichannel opening, block or reduce the release of molecules into the extracellular space via hemichannels, and / or block or reduce the entry of molecules into the intracellular space via hemichannels. Hemichannel blockers include compounds that completely or partially block hemichannel leakage (e.g., into or from the extracellular space) or the passage of molecules through hemichannels. Hemichannel blockers also include compounds that reduce the open probability of hemichannels. Open probability is a measure of the ratio of time a channel remains open to the time it is closed (seen in Goldberg GS et al., Selective permeability of gap junction channels, Biochimica et Biophysica Acta 1662 (2004) 96-101). Examples of hemichannel blockers include peptides, small molecules, antibodies, and antibody fragments. Hemichannel blockers also include hemichannel modulators.Hemichannel blockers can directly or directly interfere with the passage of molecules through connexin hemichannels, or with the permeability of hemichannels.

[0127] In some embodiments, the connexin modulator may be a modulator of a connexin hemichannel present in a blood vessel, e.g., a connexin 43 hemichannel modulator and / or a connexin 37 hemichannel modulator, a connexin 40 hemichannel modulator, a connexin 45 hemichannel modulator, or another vascular connexin hemichannel. In some embodiments, the connexin modulator may be a modulator of a connexin hemichannel present in the ocular epithelium, e.g., the corneal epithelium. In some embodiments, the connexin modulator may be a modulator of Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx43, Cx45, Cx50, and / or Cx58 hemichannels in the corneal epithelium, or any other connexin hemichannel. In other embodiments, the connexin modulator is a modulator of any other connexin hemichannel in the eye or blood vessels, including the hemichannels Cx37, Cx40, Cx43, and Cx45, which are also found in the corneal epithelium, as well as the hemichannels Cx36 and Cx57, in addition to the hemichannels Cx26, Cx30, Cx31.1, and Cx50 mentioned above.

[0128] A "bandage contact lens" is any contact lens that can be used to protect the surface of the eye. Bandage contact lenses are also called "therapeutic contact lenses" and "therapeutic bandage contact lenses." They can also help reduce pain and aid in healing. Bandage contact lenses allow for mechanical protection of the eye surface, such as the cornea, from eyelid movement, and also protect the eye surface, such as the corneal surface, from air exposure and further eye injury, and limit dehydration. Bandage lenses include hydrogel lenses and silicone hydrogel lenses, which are well known in the art. See Lim and Lim, Therapeutic Contact Lenses in the Treatment of Corneal and Ocular Surface Diseases - A Review Asia-Pacific Journal of Ophthalmology 9(6):524-532 (Nov-Dec 2020); Jacobs et al., CLEAR Medical use of contact lenses, Cont Lens Anterior Eye 2021 Apr;44(2):289-329. In some embodiments, the therapeutic bandage lens is, for example, a soft contact lens, a gas-permeable (scleral) lens, or a 3D-printed biogel. Suitable FDA-approved soft bandage lenses include Acuvue Oasys with Hydraclear Plus (Johnson & Johnson Vision), Air Optix Night & Day Aqua (Alcon), PureVision (Bausch+Lomb), and UCL 55% (United Contact Lens) and Kontur (Kontur Kontact Lens).

[0129] In this specification, "small molecules" are defined as compounds with a molecular weight of less than approximately 600 daltons and are generally organic compounds. Small molecule connexin modulators include compounds of formula I. Small molecules can be activators of prodrugs. Small molecule prodrugs include compounds of formula II, which are prodrugs of tonaversat, a small molecule hemichannel and connexin modulator.

[0130] As used herein, “treatment” (and its grammatical variations such as “to treat” or “to treat”) refers to a clinical intervention in an attempt to alter the course of the individual, tissue, or cell being treated, which may be done either for preventive purposes or during the course of a clinicopathological condition. The desired effects of treatment include, but are not limited to, ending a non-healing ocular defect or disorder (e.g., PED or PCED) and preventing the onset or recurrence of a non-healing ocular surface or corneal defect or disorder, alleviating signs or symptoms, reducing the direct or indirect pathological consequences of a non-healing ocular surface or corneal defect or disorder, slowing the rate of progression of a non-healing ocular surface or corneal defect or disorder, improving or mitigating a non-healing ocular defect or disorder, and achieving remission or improving prognosis. The term does not necessarily mean that the subject is treated until complete recovery, which is generally the case with the pulsed dose delivery method of the present invention for the treatment of a non-healing ocular surface or corneal defect or disorder. Therefore, “treatment” may also include maintaining or promoting a state of complete or partial remission of non-healing ocular defects or impairments. Very prolonged wound closure may be defined as 100% skin re-epithelialization without the need for drainage or bandaging at two consecutive evaluations of at least two weeks but no more than five months after closure following the last administration of a connexin modulator as described herein (e.g., on about 14, about 28, or about 35 days). In some embodiments, very prolonged wound closure refers to wound closure of one month (e.g., four weeks or 28 days). In some embodiments, “treatment” refers to treatments and clinical interventions as described above and herein, or treatment of uveitis, blepharitis, Sjögren’s syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and dry eye diseases (including evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye).

[0131] The term "block" means to block, improve, or control something, either entirely or partially.

[0132] As used herein, “effective dose” or “therapeutic effective dose” refers to an amount effective in the dosage and duration described in the dosage regimens herein to achieve a desired outcome in the treatment of non-healing ocular or corneal defects or disorders (e.g., PED, PCED, ocular ulcers, corneal ulcers, etc.). For example, but not limited to, “therapeutic effective dose” can refer to an amount of connexin modulator compounds or compositions, including but not limited to those disclosed herein, that can close a non-healing ocular defect or disorder when administered in the dosage regimens of the present invention. In other embodiments, the results of treatment with an “effective dose” are very long-lasting, and the non-healing ocular defect or disorder remains closed after treatment. The doses disclosed herein are therapeutic effective doses. However, the methods are not limited to those doses or dosages and include the use of other therapeutic effective doses. In some embodiments, “effective dose” or “therapeutic effective dose” refers to an amount effective in the dosage and duration described in the dosage regimens herein to achieve desired results in the treatment of uveitis, blepharitis, Sjögren’s syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and dry eye diseases (including evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye).

[0133] As used herein, “prophylactic effective dose” refers to an amount effective, for example, with an administration on about 28 days (and / or about 35 days if necessary) to achieve or ensure a desired outcome or desired prophylactic outcome, such as very long-lasting wound closure without recurrence of non-healing eye loss or impairment. In some embodiments, a prophylactic effective dose of connexin modulator is administered to a subject with a closed ocular surface disorder (e.g., closed PED or PCED). In some embodiments, an effective dose of connexin modulator is administered prophylactically to a subject with a closed ocular surface disorder (e.g., PED or PCED). In some embodiments, “preventive effective dose” means an amount effective in achieving a desired preventive outcome in the treatment of uveitis, blepharitis, Sjögren’s syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and dry eye diseases (including evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye), including during or after the treatment of uveitis, blepharitis, Sjögren’s syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and any form of dry eye disease, using the methods herein.

[0134] The terms “peptide,” “peptide mimetic,” and “mimetic” include synthetically or genetically produced chemical compounds that may have the same or substantially the same structural and functional characteristics as the protein region they mimic. For example, they may mimic the extracellular loops of opposing connexins involved in connexone-connexone docking and cell-cell channel formation, and / or the extracellular loops of hemichannel connexins, as well as the intracellular C-terminus, such as connexin 43. As used herein, “peptide mimetic” (also known as “peptide mimetic”), which includes peptides and peptide-like compounds, also includes such non-peptide-like compounds, such as peptide analogs. Peptide mimetics structurally similar to therapeutically useful peptides can be used to produce equivalent or enhanced therapeutic or prophylactic effects. For example, the initial extracellular loops of Cx37, Cx40, and Cx43 are mimicked by peptides under the Gap26 encoding. On the other hand, Gap27 and Peptide5 mimic the region of the second extracellular loop (both L2 and Gap19 also mimic the cytoplasmic loop but of Cx43). Gap27 targets Cx32, Cx40, and Cx43, while Peptide5 is used for Cx43 inhibition. Other peptide mimetic molecules include JM2 (VFFK-GVKDRVKGRSD; SEQ ID NO: 134), ΔSH3, CT9 (RPRDDLEI; SEQ ID NO: 135), and CT9-TAT, CT10 (SRPRDDLEI; SEQ ID NO: 136), and αCT (RQPKIWFPNRRKPWKK-RPRPDDLEI (SEQ ID NO: 137), where the inhibitor peptide contains the ZO-1 binding sequence of Cx43. Gap24 (containing C-terminal amino acids 374-382 (RPRPDDLEI; SEQ ID NO: 141), whose N-terminus is similarly bound to a 16-amino acid Antennapedia internalization vector), mimics the C-terminal tail of Cx43, Gap24 (GHGDPLHLEEVKC; SEQ ID NO: 138) reproduces the sequence of the cytoplasmic loop of Cx32, while TAT-Gap24 (YGRKKRRQRRRGHGDPLHLEEVKC; SEQ ID NO: 139) also mimics a portion of the intracellular loop of Cx43.Both L2 and Gap19 also mimic the cytoplasmic loop of Cx43. Peptides useful in the compositions and methods of the present invention are described and referenced herein and include connexin-mimicking peptides for the connexin extracellular loop and connexin intracellular region, as well as peptides that mimic the region of the connexin C-terminal tail (e.g., the C-terminus of connexin 43). Peptides and peptide mimes useful in the compositions and methods of the present invention include those shown in Tables II and III.

[0135] In some embodiments, the peptides and peptide mimes are modified. In some embodiments, the peptides and peptide mimes are unmodified. Peptide mimes are molecules that can mimic natural peptides and proteins. Generally, peptide mimes are structural or functional mimes (e.g., identical or similar) to paradigm polypeptides (i.e., polypeptides having biological or pharmacological function or activity), but peptide mimes may also be modified and may have one or more peptide bonds that are optionally replaced by bonds selected from the group consisting of, for example, -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO--. Peptide mimes may also be chemically modified by means of non-natural amino acid substitution, skeletal amide bond modification, rigid scaffolding, addition of hydrophobic residues, and other methods known in the art. Mimics may consist entirely of native amino acids, synthetic chemical compounds, or unnatural analogs of amino acids, or they may be chimeric molecules of partially native peptide amino acids and partially unnatural analogs of amino acids. Mimics may also contain any amount of conservative substitutions of native amino acids, as long as such substitutions do not substantially alter the mimic activity. In the case of connexins, these may mimic, for example, the extracellular loops of opposing connexins involved in connexone-connexone docking and cell-cell channel formation. For example, a mimic composition may be useful as a gap junction modifier if it can downmodulate the biological action or activity of hemichannels, such as by blocking the docking of hemichannels for forming gap junction-mediated intercellular communication, or by blocking the opening of hemichannels for exposing the cytoplasm of a cell to the extracellular environment. Peptide mimics include those described herein, as well as those known in the art, whether currently known or subsequently developed.Peptides and peptide-mimicking connexin modulators can also be modified to increase stability, improve bioavailability, and / or increase cell membrane permeability.

[0136] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained herein is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered. "Pharmaceutical composition" refers to a mixture of substances suitable for administration to a subject containing an active ingredient or pharmaceutical. For example, a pharmaceutical composition may include modified or unmodified antisense oligonucleotides and sterile aqueous solutions or poloxamers or Pluronic® carriers.

[0137] As used herein, “pharmaceutically acceptable carrier” refers to a component of a pharmaceutical preparation other than the active ingredient that can be safely administered to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, buffers, excipients, stabilizers, or preservatives. A pharmaceutically acceptable carrier includes poloxamer, e.g., Pluronic® F-127. A pharmaceutically acceptable carrier for ocular administration is an ophthalmologically acceptable carrier suitable for application to the eye (e.g., middle or upper eye).

[0138] Where used herein, terms such as “subject,” including “individual” and “patient,” may all be used interchangeably herein, but refer to any mammal, including humans, dogs, horses, cats, sheep, pigs, cattle, and other domesticated and farm animals, as well as zoo animals, wildlife park animals, sports animals, or pet animals. Preferred mammals herein are humans, including adults, children, and the elderly. Preferred sports animals are horses and dogs. Preferred pet animals are dogs and cats. Subjects may be aquatic park animals, such as dolphins, whales, seals, or walruses. In certain embodiments, subject, individual, or patient is human. Subject does not mean to include animals used in scientific experiments (e.g., mice and rats).

[0139] As used herein, “gap junction,” “connexin gap junction,” and “connexin gap junction channel” refer to two hemichannels that connect across the intercellular space between adjacent cells, allowing certain molecules to flow between those cells. They refer to intercellular channels or clusters that enable the direct diffusion of ions and small molecules between adjacent cells. Gap junctions have been studied for decades and are well known in the art.

[0140] As used herein, the term “hemichannel” refers to a part of a gap junction (two hemichannels or connexons that connect across the intercellular space between adjacent cells to form a gap junction) and is composed of several connexin proteins, typically hexamers of homomers or heteromers of connexin proteins that form a pore for a gap junction between the cytoplasm of two adjacent cells. A hemichannel is supplied by the cell on one side of the junction and by the cell on the other side, and the two hemichannels from the opposing cells usually come together to form a complete intercellular gap junction channel. However, in some cells, and in some circumstances, the hemichannel itself is active as a conduit between the cytoplasm and the extracellular space, allowing the movement of ions and small molecules (e.g., ATP). Like their gap junction counterparts, connexin hemichannels have been studied for many years and are well known in the art.

[0141] Hemichannels and gap junction channels can be present in any type of cell. References to “hemichannels” or “gap junction channels” should be interpreted as including references to hemichannels or gap junction channels present in any ocular cell type, as well as any ocular surface cell type, including corneal and / or corneal epithelial cells. In one embodiment of the present invention, hemichannels or gap junction channels are present in cells in the eye. In one embodiment of the present invention, hemichannels or gap junction channels are present in cells in the anterior part of the eye, i.e., the anterior segment. In some embodiments of the present invention, hemichannels or gap junction channels are present in cells in the cornea. In some embodiments of the present invention, hemichannels or gap junction channels are present in cells in the corneal epithelium. In some embodiments of the present invention, hemichannels or gap junction channels are present in cells in the microvascular system of the eye, including the corneal microvascular system. In some embodiments of the present invention, hemichannels or gap junction channels are present in cells in the uvea. In some embodiments of the present invention, hemichannels or gap junction channels are present in cells associated with dry eye disease, blepharitis (e.g., including cells of the eyelid margin or cells associated with the eyelid margin), Sjögren's syndrome-associated keratoconjunctivitis sicca (e.g., including cells of the conjunctiva or cells associated with the conjunctiva), and Fuchs dystrophy (e.g., including corneal endothelial cells).

[0142] "Non-healing ocular surface defects or disorders" means defects or disorders of the ocular surface that do not heal or close completely within approximately 10 days to 2 weeks of standard clinical treatment. Non-healing ocular surface defects or disorders include ocular ulcers, corneal ulcers, persistent epithelial defects (PEDs) of the eye, and persistent corneal epithelial defects (PCEDs). Ocular ulcers include corneal ulcers and include those described herein, including infectious ulcers, chemical ulcers, burn ulcers, traumatic or trauma-induced ulcers, inflammatory ulcers, and mixed-cause ulcers. "Non-healing" or "persistent" defects or disorders of the ocular surface or cornea (e.g., PEDs and PCEDs) result from failure of re-epithelialization and closure within 10 to 14 days, even with standard supportive care.

[0143] The phrase "essentially derived from" refers to materials that do not substantially affect the basic and novel features (or steps, in the case of methods) of a particular material and a pharmaceutical product. The basic and novel features of the present invention are described throughout this specification and include the ability of the connexin modulators, connexin modulator compositions and methods of the present invention to block or modulate connexins, connexin gap junctions and / or connexin hemichannels, for example, to attenuate, block or inhibit the production, function or activity of connexins, connexin gap junctions and / or connexin hemichannels, or inflammasome activity (e.g., hemichannel-mediated inflammasome activity, e.g., connexin hemichannel-mediated inflammasome activity or pannexin hemichannel-mediated inflammasome activity). The material variations of the basic and novel features of the present invention, including the pharmaceuticals and methods described herein, include undesirable or clinically undesirable, harmful, adverse or detrimental activity, delayed healing, reduced modulation and / or attenuation of connexin, connexin gap junctions and / or connexin hemichannels, or undesirable reduction in the activity or function of connexin, connexin gap junctions and / or connexin hemichannels. In one embodiment, the composition or pharmaceutical of the present invention includes, essentially consists of, or comprises connexin 43 hemichannel blockers, such as connexin 43 antisense molecules, connexin 43 peptide mimes or small molecule connexin 43 hemichannel blockers or their prodrugs.

[0144] Antisense modulator The gap junction and / or connexin polynucleotide or oligonucleotide may be selected from, for example, modified or unmodified connexin polynucleotide or oligonucleotide, such as modified or unmodified connexin 43 antisense polynucleotide or oligonucleotide. In some embodiments, the modified connexin antisense polynucleotide, or oligonucleotide or polynucleotide, comprises a mixture of modified and unmodified nucleotides. In some embodiments, the connexin 43 antisense compound used in the methods herein is an antisense oligonucleotide containing a naturally occurring nucleic acid base and an unmodified nucleoside bond.

[0145] In some embodiments, the connexin 43 antisense compound targets at least about eight nucleic acid bases of a nucleic acid molecule encoding connexin having a nucleic acid base sequence selected from SEQ ID NO: 17. Polynucleotides and oligonucleotides, such as the connexin 43 antisense compound, may have sequences of about 8 to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, or about 80 nucleotides of SEQ ID NO: 17, or a sequence complementary thereto, and / or the antisense polynucleotide or oligonucleotide may contain lengths in any range between any two of the enumerated lengths. The polynucleotides of the present invention include synthetic polynucleotides having a length of less than 80 nucleotides, for example, 12 to 18 to about 50 to 80 nucleotides, preferably about 30 nucleotides or less, for example, 12 to about 30 nucleotides, more preferably about 15 to about 30 nucleotides. In one example, the polynucleotide has 30 nucleotides. The methods of the present invention are characterized in some embodiments by the use of a connexin 43 antisense compound up to 40 nucleotides in length, for example, 15 to 40 nucleotides in length, which contains or is essentially derived from a nucleotide sequence selected from SEQ ID NOs: 1 to 17. The methods of the present invention are characterized in some embodiments by the use of a connexin 43 antisense compound up to 40 nucleotides in length, for example, 15 to 40 nucleotides in length, which contains a nucleotide sequence selected from SEQ ID NOs: 4 to 17.

[0146] Human Cx 43, α1 (SEQ ID NO: 17) LOCUS NM_000165 3088 bp mRNA linear PRI 26-OCT-2004 Definition: Homo sapiens gap junction protein, α1, 43 kDa (connexin 43) (GJA1), mRNA. [ka] [ka]

[0147] Modified oligonucleotides may include, for example, the following selected components: modified nucleoside bonds, such as phosphorothioate bonds, and modified sugar moieties, such as one or more of stereochemically distorted sugars, such as linked nucleic acids (LNAs) or cross-linked nucleic acids (BNAs).

[0148] Chemical modifications of antisense polynucleotides disclosed or referenced herein may enhance their resistance to nucleases and their ability to enter cells. For example, phosphorothioate oligonucleotides may be used. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates, and oligoribonucleotide phosphorothioates, as well as their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates. Alternatively, mixed-skeleton oligonucleotides ("MBOs") may be used. An MBO comprises a segment of phosphorothioate oligodeoxynucleotide and appropriately arranged segments of modified oligodeoxynucleotide or oligoribonucleotide. An MBO has a segment of phosphorothioate bonding and other segments of other modified oligonucleotides, such as methylphosphonate, which are nonionic and highly resistant to nucleases or 2'-O-alkyl oligoribonucleotides. Methods for preparing modified and mixed-skeleton oligonucleotides are known in the art.

[0149] In some embodiments, the antisense polynucleotides disclosed or referenced herein may include an oligonucleotide sugar moiety, which is a modified sugar moiety. In some embodiments, the modified sugar moiety may be a thiol-containing sugar moiety or a sugar moiety, which is a conformationally distorted sugar. In some embodiments, the conformationally distorted sugar may be a locked nucleotide (locked nucleic acid, or LNA). In some embodiments, the locked nucleotide can be selected from one of the following types: 2'-O-CH2-4' (oxy-LNA), 2'-CH2-CH2-4' (methylene-LNA), 2'-NH-CH2-4' (amino-LNA), 2'-N(CH3)-CH2-4' (methylamino-LNA), 2'-S-CH2-4' (thio-LNA), and 2'-Se-CH2-4' (seleno-LNA). In some embodiments, the conformationally distorted sugar may be a cross-linked nucleic acid (BNA). Some stereochemically distorted sugars can be locked nucleic acids, as shown in formulas III and IV of U.S. Patent No. 10,465,188.

[0150] The Specified Use of the Invention also features modified or unmodified epithelial, endothelial, corneal epithelial and / or vascular endothelial connexin antisense polynucleotides, comprising 8 to about 80 nucleotides in the connexin extracellular loop, intracellular domain, C-terminus, or other region, for use in the methods of the present invention.

[0151] In some embodiments of the present invention, connexin 43 or other antisense oligonucleotides or polynucleotides have at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% homology to a polynucleotide having a sequence selected from SEQ ID NOs: 1-17. The connexin modulators, which are oligonucleotides or polynucleotides, can have at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% homology to an 8 to 80 nucleotide portion of their respective sequences.

Table 1

[0152] Table I lists polynucleotide sequences of some embodiments of connexin 43 polynucleotide modulators useful in the methods of the present invention. When sequences such as SEQ ID NOs: 1-16 are recited, they and other Cx43 and other connexin antisense compounds represent both modified oligonucleotides or polynucleotides and unmodified oligonucleotides or polynucleotides. In some embodiments, the bonds between nucleotides, and the structure of the sugar portion of the nucleotides, can be modified. In some embodiments, the internucleoside bond between any two nucleotides can be a standard phosphodiester bond. In some embodiments, the internucleoside bond between any two nucleotides can be a phosphorothioate bond. For example, SEQ ID NO: 1 can be one of the following selected structures: G s T s A s A s TTGCGGCAAGAAGAATTGTTTC s T s G s T s C (SEQ ID NO: 356), wherein, " sThe symbol indicates a phosphorothioate bond between two nucleotides. As another non-limiting example, SEQ ID NO: 1 could be (G)(T)(A)(A)TTGCGGCAAGAAGAATTGTTTC(T)(G)(T)(C)(SEQ ID NO: 357), where the nucleotide in parentheses has a modified sugar moiety as described below. In some embodiments, the Cx43 antisense compound may be modified by substituting one or more thymine nucleotides in the sequence of SEQ ID NOs: 1-17 or another connexin (e.g., Cx26, C32, Cx45, etc.) with one or more uridine nucleotide residues.

[0153] Certain connexin modulators, including the connexin 43 modulator, provide downregulation of connexin expression (e.g., by downregulation of mRNA transcription or translation) or, in other ways, reduce or inhibit the activity of connexin proteins, connexin hemichannels, or gap junctions. In the case of downregulation, this has the effect of reducing direct intercellular communication via gap junctions or the exposure of the cell's cytoplasm to the extracellular space via hemichannels at the site where connexin expression is downregulated.

[0154] In certain embodiments, anticonnexin antisense compounds block, reduce, or alter the activity or function of hemichannels or gap junctions. As described herein, modulation of gap junction activity or function by anticonnexin antisense compounds can result in the closure of gap junctions, the closure of hemichannels, and / or the passage of molecules or ions through gap junctions and / or hemichannels.

[0155] Connexin modulators may also include, for example, one or more polynucleotides selected from the group consisting of morpholino oligonucleotides, RNAi molecules, siRNA molecules, PNA molecules, DNAzymes, and 5'-terminated U1 micronuclear RNA, as well as analogs thereof. These and other compounds may be used alone or in combination with one more connexin modulator.

[0156] Antisense polynucleotides and other anticonnexin polynucleotides, such as RNAi, siRNA, and ribozyme polynucleotides, as well as polynucleotides with modified and mixed backbones, can be synthesized. See, for example, Stein CA and Krieg AM (eds), Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Liss).

[0157] Antisense polynucleotides can inhibit the transcription and / or translation of connexin proteins (e.g., connexin 43). Antisense polynucleotides are generally antisense to connexin protein mRNA, e.g., connexin 43. Such polynucleotides can hybridize to connexin protein mRNA and thus inhibit connexin expression by interfering with one or more embodiments of connexin protein mRNA metabolism, including transcription, mRNA processing, mRNA transport from the nucleus, translation, or mRNA degradation. Antisense polynucleotides typically hybridize to connexin mRNA to form a double helix that can cause direct inhibition of mRNA translation and / or destabilization. Such double helixes may be susceptible to degradation by nucleases. Preferably, the polynucleotide is a specific inhibitor of transcription and / or translation from the connexin 43 gene or mRNA, and does not inhibit transcription and / or translation from other genes or mRNA.

[0158] Connexin modulator products can be attached to the connexin 43 gene or mRNA at (i) 5' relative to the coding sequence and / or (ii) relative to the coding sequence and / or (iii) 3' relative to the coding sequence. Antisense polynucleotides can hybridize to a portion of connexin protein mRNA, e.g., connexin 43 mRNA. Typically, antisense polynucleotides hybridize to the ribosome-binding region or coding region of connexin protein mRNA. Polynucleotides can be complementary to a region of connexin mRNA. For example, a polynucleotide can be the exact complement of a portion of connexin mRNA. However, absolute complementarity is not required, and polynucleotides with sufficient complementarity to form a double helix with melting temperatures above approximately 20°C, 30°C, or 40°C under physiological conditions are particularly suitable for use in the present invention. Therefore, polynucleotides are typically homologs of sequences complementary to mRNA. Polynucleotides can hybridize to connexin protein mRNA under medium to high stringency conditions such as 0.03 M sodium chloride and 0.03 M sodium citrate at approximately 50°C to 60°C.

[0159] Antisense polynucleotides may be part of a composition that may contain polynucleotides directed to more than one connexin protein. Preferably, the one connexin protein to which the polynucleotide is directed is connexin 43. Others include connexins found in the eye and / or corneal epithelium. Some embodiments of the present invention are described with reference to oligodeoxynucleotides. However, other suitable polynucleotides (such as RNA polynucleotides) may be used.

[0160] Peptide-mimicking modulator In some embodiments, the connexin modulators useful in the methods of the present invention include not only connexin oligonucleotides or polynucleotides such as connexin 43 antisense oligonucleotides or polynucleotides, but also connexin peptides or peptide mimetics, such as connexin 43 peptides or peptide mimetics sometimes called anticonnexin peptides or peptide mimetics, such as connexin 43 peptides or peptide mimetics, which are used in the methods of the present invention and may be administered according to one or more schedules described herein. They include, for example, peptides or peptide-mimicking connexin modulators that include or are essentially derived from sequences corresponding to portions of the connexin extracellular domain, connexin transmembrane region, and carboxy-terminal region of connexin. In some embodiments, the connexin, connexin gap junction, and connexin hemichannel modulators useful in the methods of the present invention include connexin 43 peptides or peptide mimetics (connexin 43 proteins) having sequences that conform to a portion of the amino acid sequence of SEQ ID NO: 100.

[0161] In some embodiments, a connexin modulator useful in the methods of the present invention for treating non-healing ocular defects or disorders (e.g., PED) in a subject, including non-healing corneal defects or disorders (e.g., PCED), is an anti-connexin 43 peptide or peptide mimetic. In some embodiments, anti-connexin 43 peptides or peptide mimetics that may be used in the methods of the present invention and administered according to one or more schedules described herein are peptides containing or essentially comprising a portion of the extracellular domain of connexin (e.g., connexin 43), and / or peptides containing or essentially comprising a portion of the carboxy-terminal portion of connexin (e.g., connexin 43), including those described and / or referenced herein.

[0162] The protein sequence of connexin 43 is shown below. Connexin 43 (SEQ ID NO: 100) [ka] [ka]

[0163] In some embodiments, the connexin 43 (Cx43) and other connexin peptide modulators useful for carrying out the methods of the present invention and administered according to one or more schedules described herein include peptides such as Peptide 5, i.e., VDCFLSRPTEKT (SEQ ID NO: 107), Gap 19, i.e., KQIEIKKFK (SEQ ID NO: 108), Gap 26, i.e., VCYDKSFPISHVR (SEQ ID NO: 102), Gap 27, a peptide called alpha-connexin carboxyl terminus 1 (αCT1), and other peptides, each targeting different binding sites with varying specificities and sizes. The connexin-mimicking peptide Gap27, which targets the SRPTEKTIFII sequence (SEQ ID NO: 104) (amino acids 204-214) on the second extracellular loop of Cx43 ("LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFII" disclosed as SEQ ID NO: 109), is a general-purpose inhibitor of connexin-mediated communication. In some embodiments, the connexin 43 modulator may include, or be, a peptide or peptide mimetic containing or essentially derived from, for example, SRPTEKTIF (SEQ ID NO: 110).

[0164] In addition to Peptide 5, Gap 19, Gap 26, and Gap 27, another peptide mimetic particularly useful in the compositions, dosages and administration methods and schedules, kits and products disclosed herein is a fusion peptide called XG19, i.e., lclrpvGGKQIEIKKFK, where lowercase letters represent the D-isomer [SEQ ID NO: 111]). The XG19 peptide mimetic and its connexin-modulating activity are described above in whole by reference in U.S. Patent No. 11,466,069.

[0165] In some embodiments, the present invention provides compositions for use in the present invention's methods for treating eye diseases or disorders, comprising a construct comprising (a) a targeted carrier peptide derived from the X protein of hepatitis B virus and (b) a peptide capable of interacting with the intracellular domain of connexin. In some embodiments, the targeted carrier peptide derived from the X protein of hepatitis B virus comprises an amino acid sequence selected from the group consisting of all targeted carrier peptides described in U.S. Patent No. 11,466,069. In some embodiments, the peptide capable of interacting with the intracellular domain interacts with one or more intracellular domains of connexin Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50 and Cx58 (including those described herein). In some embodiments, the peptide capable of interacting with the intracellular domain interacts with the intracellular domain of connexin 43. In some embodiments, peptides capable of interacting with the intracellular domain of connexin 43 include any of the connexin 43 intracellular interacting peptides described in U.S. Patent No. 11,466,069, as well as peptides described herein that interact with the intracellular domain of connexin 43.

[0166] In some embodiments, the connexin 43 modulators that can be used in the methods of the present invention and administered according to one or more of the schedules described herein may include, for example, peptides having sequences that include or are essentially derived from one or more of the following sequences: "Peptide 1"ADCFLSRPTEKT (SEQ ID NO: 112), "Peptide 2"VACFLSRPTEKT (SEQ ID NO: 113), "Peptide 11"VDCFLSRPTAKT (SEQ ID NO: 114), "Peptide 12"VDCFLSRPTEAT (SEQ ID NO: 115), "Peptide 5"VDCFLSRPTEKT (SEQ ID NO: 107), "Mod1"CFLSRPTEKT (SEQ ID NO: 116), and "Mod2"LSRPTEKT (SEQ ID NO: 117). In some embodiments, the carboxyl terminus of the anticonnexin peptide or peptide-mimicking modulator can be modified. In some embodiments, the carboxyl terminus modification may include an n-alkyl chain which can be further bonded to hydrogen or other parts as needed. In some embodiments, the connexin 43 peptide may include or exclude any of the peptides listed above or disclosed herein.

[0167] In some embodiments, the peptide or peptide mime is composed of or essentially consists of 7 to 40 amino acids of connexin, including, for example, SEQ ID NO: 101 (SRPTEKT) and SEQ ID NO: 107 (VDCFLSRPTEKT), and does not contain the connexin C-terminal peptide.

[0168] Anticonnexin agents include peptides having an amino acid sequence containing approximately 5 to 20 consecutive amino acids of a connexin protein such as connexin 43 (SEQ ID NO: 100), peptides having an amino acid sequence containing approximately 8 to 15 consecutive amino acids of connexin 43, or peptides having an amino acid sequence containing approximately 11 to 13 consecutive amino acids of connexin 43. Other anticonnexin agents include peptides having an amino acid sequence containing at least approximately 5, at least approximately 6, at least approximately 7, at least approximately 8, at least approximately 9, at least approximately 10, at least approximately 11, at least approximately 12, at least approximately 13, at least approximately 14, at least approximately 15, at least approximately 20, at least approximately 25, or at least approximately 30 consecutive amino acids of connexin 43. Other anticonnexin 43 modulators include peptides or peptide mimes containing or essentially derived from the extracellular domain of connexin 43, e.g., SRPTEKT (SEQ ID NO: 101) or VDCFLSRPTEKT (SEQ ID NO: 107).

[0169] In other anti-connexin compounds, the mimetic peptide is based on the extracellular domain of connexin 43, corresponding to the amino acids at positions 37-76 and 178-208 of the connexin 43 protein sequence. Therefore, specific anti-connexin peptides useful in the methods of the present invention have an amino acid sequence containing or essentially comprising about 7 to about 40 amino acids corresponding to the regions at positions 37-76 and 178-208 of the connexin 43 protein sequence. The peptide does not need to have the same amino acid sequence as the portion of the connexin 43 protein sequence, and conservative amino acid changes may be made so that the peptide retains binding or functional activity in assays described herein and otherwise known in the art. In other embodiments, the mimetic peptide is based on a peptide target region within the connexin protein other than the extracellular domain (e.g., the portion of the connexin 43 protein sequence not corresponding to positions 37-76 and 178-208).

[0170] For example, in addition to therapeutically effective modified or unmodified peptides or peptide mimetics containing or essentially comprising a portion of the extracellular or transmembrane domain or C-terminal domain of connexin 43, other embodiments include, for example, modified or unmodified peptides or peptide mimetics containing or essentially comprising a portion of the extracellular or transmembrane domain of one or more other connexins found in the eye and / or corneal epithelium, which may be used in the methods of the present invention and may be administered according to one or more schedules described herein. Other embodiments include, for example, modified or unmodified peptides or peptide mimetics containing a portion of the extracellular or transmembrane domain of one or more other connexins found in blood vessels including ocular and / or corneal blood vessels (e.g., endothelium).

[0171] In some embodiments, an anti-connexin peptide mime that is useful in the methods of the present invention and can be administered according to one or more of the schedules described herein is a connexin 45 peptide mime modulator containing a portion of the connexin 45 protein that antagonizes, inhibits, or blocks the connexin-connexin interaction. In some embodiments, the connexin 45 modulator may include a peptide or peptide mime containing, for example, SRPTEKT (SEQ ID NO: 101), which contains or is essentially derived from a portion of the E2 or C-terminal domain of connexin 45. The peptide or peptide mime may also include, for example, DCFISRPTEKT (SEQ ID NO: 118). Exemplary peptide sequences for connexin 45 peptides and peptide mime modulators useful in the methods of the present invention are also provided in Table 63 of U.S. Patent No. 10,465,188.

[0172] In some embodiments, the connexin modulator comprises a peptide having an amino acid sequence corresponding to a portion of the transmembrane region or the C-terminal region of connexin 45. In certain non-limiting embodiments, for example, the anticonnexin compound is a peptide having an amino acid sequence containing about 3 to about 30 consecutive amino acids of a known connexin 45 sequence, a peptide having an amino acid sequence containing about 5 to about 20 consecutive amino acids of a known connexin 45 sequence, a peptide having an amino acid sequence containing about 8 to about 15 consecutive amino acids of a known connexin 45 sequence, or a peptide having an amino acid sequence containing about 11, 12, or 13 consecutive amino acids of a known connexin 45 sequence. Other non-limiting embodiments include an anticonnexin compound which is a peptide having an amino acid sequence containing at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 consecutive amino acids of a known connexin 45 sequence. In certain anti-connexin compounds provided herein, the mimetic peptide is based on the extracellular domain of connexin 45, corresponding to the amino acids at positions 46-75 and 199-228 of the known connexin 45 sequence. Therefore, the specific peptides described herein have amino acid sequences corresponding to the regions at positions 46-75 and 199-228 of the known connexin 45 sequence. The peptide does not need to have an identical amino acid sequence to the portion of the known connexin 45 sequence. Conservative amino acid changes may be made, as otherwise known in the art, so that the peptide retains binding or functional activity in the assays described herein. In other embodiments, the mimetic peptide is based on a peptide target region within the connexin protein other than the extracellular domain (e.g., the portion of the known connexin 45 sequence not corresponding to positions 46-75 and 199-228). See International Publication 2006 / 134494, which discloses various connexin sequences.

[0173] Some peptide-mimicking connexin modulators useful in the method of the present invention include, for example, VDCFLSRPTEKT (SEQ ID NO: 107) and SRPTEKTIFII (SEQ ID NO: 104), which bind to the extracellular domain of Cx43.

[0174] In some embodiments, the connexin 26 peptide mimetic connexin modulator useful in the methods of the present invention and administered according to one or more schedules described herein is Gap26. In other embodiments, the anticonnexin peptide mimetic for use in the methods of the present invention is a connexin 32 peptide mimetic (e.g., INCTLQPGCNSV (SEQ ID NO: 103) or 32 Gap27, i.e., SRPTEKTIFII (SEQ ID NO: 104) or a connexin 50 peptide mimetic (e.g., TAT-Cx50L2, i.e., GGERAPLAADQGSVKKSSSSSKGTKK (SEQ ID NO: 105) or TAT-Cx50CT, i.e., SRARSDDLTV (SEQ ID NO: 106)).

[0175] In some embodiments, the peptides and peptide mimetics include peptides and peptide mimetics useful for inhibiting gap junction channels and hemichannels corresponding to the conserved QPG and SHVR (SEQ ID NO: 359) motifs of E1 (Gap26 peptide) and the SRPTEK motif (SEQ ID NO: 360) in E2 (Gap27 peptide), as well as specific sequences within the extracellular loops E1 and E2 involved in the cytoplasmic loop (Gap19 peptide). Useful peptide-mimicking connexin modulators are described in U.S. Patent No. 9,248,141 ("Methods of treatment by administering anti-connexin peptides and mimetics"). Other useful peptide connexin modulators, including XG19 and other constructs containing peptides that can interact with the intracellular domain of connexins (e.g., connexin 43) derived from the X protein of hepatitis B virus, are described in U.S. Patent No. 11,466,069 ("Methods of treatment and novel constructs").

[0176] Other connexin peptide modulators useful in the methods of the present invention are provided in Table 64 of U.S. Patent No. 10,465,188. Useful peptide modulators of connexin 43 (Cx43) and other connexins that can be administered in embodiments of the present invention are also referenced in Caufirez et al. and King, DR. et al., Mechanisms of Connexin Regulating Peptides Int. J. Mol. Sci. 22:10186 (Sept 2021) and in Figure 1 ("Schematic of the Cx43 protein in the plasma membrane with colored lines indicating the positions of described peptides targeting EL, IL and CT regions") and Table 1 ("Connexin Peptides").

[0177] In some embodiments, peptides may also be used as promoieties. See, for example, Vig, BS et al., Amino acids as promoieties in drug design and development. Advanced Drug Delivery Reviews 65(10):p 1370-1385 (2013); and Dhokchawle, B et al., Promoieties Used In Prodrug Design: A Review. Indian Journal of Pharmaceutical Education 48(2):35-40 (2013).

[0178] Gap junction modulators or anti-connexin hemichannel blocking peptides or peptide mimes may be unmodified or modified as desired (e.g., to increase stability, further stabilize peptide configuration, increase biological activity, increase cell permeability, etc.). See, for example, DeGruyter, JN et al., Residue-Specific Peptide Modification: A Chemist's Guide. Biochemistry 56, 30, 3863-3873 (2017); Boto, A, et al. Site-selective modification of peptide backbones. Org. Chem. Front. 8: 6720-6759 (2021) (review article). Therefore, for example, the peptide mimes used in the methods of the present invention may contain one or more modified amino acids, amino acid analogs, or may be modified in other ways to improve bioavailability or increase penetration across the cell membrane.

[0179] Other peptide sequences known to inhibit connexin-connexin binding that can modulate connexin activity include the cytoplasmic loop (amino acids 119-144) L2 peptide of connexin 43 and a subpart of the L2 peptide of connexin 43. In some embodiments, these peptides are, for example, the nine-amino acid sequence of Gap19, KQIEIKKFK (SEQ ID NO: 108); the natural Gap19 sequence, DGVNVEMHLKQIEIKKFKYGIEEHGK (SEQ ID NO: 119); and the His144→Glu of Gap19, as reported by Shibayama (Shibayama, J. et al., Biophys. J. 91, 405404063, 2006). L2 derivative, DGVNVEMHLKQIEIKKFKYGIEEQGK (SEQ ID NO: 120); TAT-Gap19 sequence, YGRKKRRQRRRKQIEIKKFK (SEQ ID NO: 121); SH3 binding domain, CSSPTAPLSPMSPPGYK (SEQ ID NO: 122) or its subpart PTAPLSPMSPP (SEQ ID NO: 123); C-terminal sequences of CT9 or CT10 peptides, with or without a TAT reader sequence to increase cell permeability, e.g., RPRDDEI (CT9; SEQ ID NO: 124), SRPRDLEI (CT10; SEQ ID NO: 125), YGRKKRRQRRRSRPRDDEI (TAT-CT9; SEQ ID NO: 126) or YGRKKRRQRRRRPRDDEI (TAT-CT10; SEQ ID NO: 127). Other peptide mimic sequences that may or may not be included in the compositions, methods, kits, or products disclosed herein are those reported by Dhein (Dhein, S., Naunyn-Schmiedeberg's Arch.Pharm., 350:174-184, 1994); AAP10 peptide, H2N-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH2 (SEQ ID NO: 128); and ZP123 peptide (rotigapeptide), Ac-D-Tyr-Pro-D-4Hyp-Gly-D-Ala-Gly-NH2 (SEQ ID NO: 129) (Dhein, S. et al. Cell Commun.Adhes. 10, 371-378, 2013). Rotigapeptide is composed of the D-form of the peptide for higher efficacy than the natural L-form of the peptide.

[0180] In some embodiments, the therapeutically effective modified or unmodified peptide or peptide mime is a portion of the E1 extracellular domain of a connexin, such as connexin 43 E1 (ESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVR; SEQ ID NO: 130) or connexin 45 E1 (GESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVR; SEQ ID NO: 131). In some embodiments, the therapeutically effective modified or unmodified peptide or peptide mime is a portion of the E2 extracellular domain of a connexin, such as connexin 43 E2 (LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKT; SEQ ID NO: 132) or connexin 45 E2 (LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKT; SEQ ID NO: 133).

[0181] In certain embodiments, the connexin 43 modulator peptide of the present invention may be linked to an internalized transporter at its amino or carboxyl terminus. The internalized transporter linked to the connexin 43 modulator peptide of the present invention may be any internalized sequence known or newly discovered in the art, or a conserved variant thereof. Non-limiting examples of internalized transporters and sequences include Antennapedia sequences, TAT, HIV-Tat, penetratin, Antp-3A (Antp variant), buphorin II, transportan, MAP (model amphiphilic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (Bis-guanidinium-spermidine-cholesterol), and BGTC (BisGuanidinium-Tren-cholesterol). The internalized transporters are useful for peptide mimes such as Gap19 and aCT peptides. Exemplary sequences of internalized peptides are known in the art. For example, see Table 65 of U.S. Patent No. 10,465,188; U.S. Patent No. 11,466,069.

[0182] In one embodiment, a modified peptide "C12-C12-Cxn43 MP" (SEQ ID NO: 358) capable of modulating connexin 43 can be prepared by conjugating the peptide sequence CFLSRPTEKT (SEQ ID NO: 116) or VDCFLSRPTEKT (SEQ ID NO: 107) to two dodecyl groups. See SEQ ID NO: 237 of U.S. Patent No. 10,465,188. The resulting structure "C12-C12-Cxn43MP" (SEQ ID NO: 358) is shown below. [ka] In the structure of C12-C12-Cxn43 MP (SEQ ID NO: 358), R1 and R2 can be hydrogen or alkyl groups. In some embodiments, R1=R2=n-dodecyl chain.

[0183] In some embodiments, the therapeutically effective modified or unmodified peptide or peptide mimetic includes a portion of the C-terminal domain of connexin, such as connexin 43 or connexin 45, preferably connexin 43. Some embodiments of anti-connexin 43 modulators useful in the methods of the present invention include the C-terminal region of connexin 43 or a modified version thereof. See, for example, O'Quinn, MP et al., A Peptide Mimetic of the Connexin 43 Carboxyl-Terminus Reduces Gap Junction Remodeling and Induced Arrhythmia Following Ventricular Injury. Circ Res. 108(6):704-715 (Mar 2011). C-terminal connexin peptide mimetic modulators containing αCT1 (alpha-connexin carboxy-terminal 1) peptide (also referred to as aCT1 or ACT1 peptide in publications) are described, for example, in Montgomery et al., Connexin 43-Based Therapeutics for Dermal Wound Healing Int. J. Mol. Sci. 2018, 19, 1778, and U.S. Patent No. 8,815,556 ("Compositions and methods for tissue engineering, tissue regeneration and wound healing"). See also International Publication No. 2006 / 069181. A preferred connexin carboxy-terminal polypeptide is the connexin 43 carboxy-terminal polypeptide. Such compounds are described in U.S. Patent Application Publication No. 20070042964 ("Compositions and methods for modulating connexin hemichannels"). If the connexin peptide or peptide-mimicking modulator contains a portion of the intracellular domain of connexin (e.g., aCT peptides, e.g., CT9, CT10, αCT1, etc.), the peptide may be conjugated to an intracellular transporter, including those described or referenced herein.In some embodiments, connexin peptide-mimicking modulators useful in the methods of the present invention can block occlusion zone (ZO-1) binding to connexin 43 and favorably modulate connexin gap junctions and hemichannels. See Figure 2 in Caufriez, A et al., Peptide-based targeting of connexins and pannexins for therapeutic purposes. Expert Opinion on Drug Discovery 15(10):1213-1222 (2020).

[0184] In some embodiments, the connexin modulator may be a gap junction closure compound and / or a hemichannel closure compound. In some embodiments, the gap junction closure compound and the hemichannel closure compound are a connexin 43 gap junction closure compound and a connexin 43 hemichannel closure compound (e.g., a Cx43 C-terminal peptide mime).

[0185] Various useful peptide-mimicking peptides mimic the sequences of the extracellular regions of connexin. The first extracellular loops of Cx37, Cx40, and Cx43 are mimicked by peptides under the Gap26 encoding. Gap27 and Peptide5 mimic the regions of the second extracellular loop. Gap27 targets Cx32, Cx40, and Cx43, while Peptide5 is used for Cx43 inhibition. JM2, ΔSH3, CT9, CT10, and αCT mimic the C-terminal tail of Cx43, and Gap24 reproduces the sequence of the cytoplasmic loop of Cx32. Both L2 and Gap19 also mimic the cytoplasmic loop of Cx43.

[0186] The extracellular loops of connexin hemichannels are also good targets for peptide mimetic connexin inhibitors useful in the present invention due to their accessibility, in contrast to their full channel counterparts. Nevertheless, peptides containing the conserved motifs QPG and SHVR (SEQ ID NO: 359) of the first extracellular loop and the SRPTEK motif (SEQ ID NO: 360) of the second extracellular loop interfere with the formation of gap junctions. Thereby, peptide mimetics 43 Gap26, 37,40 Gap26, 32 Gap27, 40 Gap27, 43 Gap27 and 43 Peptide5 were developed (note that the superscripts in the naming of these peptide analogs refer to the Cx subtypes that can be targeted).

[0187] The conserved SHVR motif (SEQ ID NO: 359) of the first extracellular loop is incorporated into the sequences of useful Gap26 peptide mimetics. Two slightly different sequences are both classified as Gap26-coding, one targeting Cx37 and Cx40 and the other targeting only Cx43. Cells treated with either of these Gap26 peptides showed Cx hemichannel inhibition within minutes.

[0188] Three Gap27 peptides exist, each targeting a different Cx type, namely Cx32, Cx40, and Cx43, which are useful in the compositions and methods of the present invention. These peptides mimic the conserved SRPTEK motif (SEQ ID NO: 360) of the second extracellular loop, but have the same time-dependent effect on gap junction activity as Gap26. Similar to Gap27, Peptide5 contains the SRPTEK motif (SEQ ID NO: 360). However, the mimicked sequence of Peptide5 is shifted towards the N-terminal tail compared to that of Gap27. Peptide5 can inhibit the Cx43 hemichannel at concentrations of 5–10 μM, but incubation at higher concentrations (100 μM or higher) can also result in gap junction inhibition in some situations.

[0189] Other peptides useful in the compositions and methods of the present invention mimic the sequence of the intracellular region of connexin. The interaction between the cytoplasmic loop and the C-terminal tail mediates the gate mechanism of the Cx hemichannel and gap junction. In the absence of interaction between the C-terminal tail and the cytoplasmic loop, the gap junction remains open, but such interaction is crucial for Cx hemichannel opening. The CT10 peptide mimic replicates the last 10 amino acids of the C-terminal tail of Cx43. Inhibition of Cx43-mediated ATP release by a peptide mimic called TAT-L2 precisely identified its mimicked L2 region (amino acids 119-144) as an essential sequence of the cytoplasmic loop in its interaction with the Cx43 C-terminal tail. To date, two peptides mimicking the L2 region have been identified, namely 43 Gap19 and 32 Gap24 is available. 43 Gap19 inhibits the Cx43 hemichannel current by binding to the C-terminal tail, thereby inhibiting the cytoplasmic loop / C-terminal tail interaction. 43 Gap19 has the advantage of being a selective inhibitor because it does not affect gap junction or Panx1 channel activity. 32Gap24 is a peptide that mimics a 13 - amino - acid stretch of the L2 region of Cx32 and can also be used in the methods of the present invention. Cx32 is one of the 10 human corneal epithelial connexins that can be usefully targeted as described herein. In vitro studies have shown that Cx32 hemichannel - mediated ATP release is inhibited by Gap24 at a concentration of 17 μM without affecting gap junctions. 32 Gap24 - mediated inhibition was shown.

[0190] Peptides that target the intracellular region of Cx proteins need to access the intracellular environment. Cell - penetrating peptides for this purpose are described herein (e.g., TAT - peptides, oligoarginine tags, and Xentry peptides) and can be attached to Cx - derived peptide sequences to enhance their uptake into cells by endocytosis. 43 Gap19 can enter cells by itself due to the KKFK cell - translocation motif (SEQ ID NO: 361) of the L2 region. Nevertheless, for the inhibition of ATP release in glioma cells, 43 Gap19 itself (47 μM) and TAT - 43 Gap19 (7 μM) IC 50 (maximum half - inhibitory concentration) comparison showed that 43 the entry of Gap19 into cells can be improved by binding to the TAT tag. The five available peptides that mimic the C - terminal tail are all derived from Cx43.

[0191] αCT1 mimics the last 9 amino acids of Cx43 and is linked to the antennapedia sequence that promotes the intracellular internalization of peptide mimics. The interaction between Cx43 and the PDZ domain of ZO - 1, a region that has been suggested to be involved in the regulation of Cx trafficking and gap - junction assembly, is disrupted by αCT1. This interference has been reported to result in an increase in gap - junction plaque formation and a decrease in Cx hemichannel activity.

[0192] If the peptide or peptide-mimicking modulator contains a portion of the intracellular domain of connexin, the peptide may, in some embodiments, be conjugated to an intracellular transporter, and in some examples, block occlusion zone (ZO-1) binding to connexin 43.

[0193] In some embodiments of the present invention, the connexin modulator is a peptide or peptide mimeograph shown in Table II below (where E2 and T2 refer, for example, to the position of the peptide in the second extracellular domain or the second transmembrane domain). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0194] In some embodiments, the connexin 43 modulator may include a peptide or peptide mime, for example, SEQ ID NO: 101 (SRPTEKT). The peptide or peptide mime may also include, for example, SEQ ID NO: 168 (VDCFLSRPTEKT). The peptide may contain one or more modified amino acids, amino acid analogs, or may be otherwise modified to improve bioavailability or increase transmembrane permeability. For example, SEQ ID NO: 107 may be modified to obtain SEQ ID NOs: 177-191 and 311-313. In some embodiments, the peptide or peptide mime containing, for example, SEQ ID NO: 101 (SRPTEKT) or SEQ ID NO: 107 (VDCFLSRPTEKT) contains 7 to 40 amino acids or amino acid analogs and does not contain a C-terminal peptide. In some embodiments, the peptide may also be used as a promoety.

[0195] In some embodiments, the connexin 45 modulator may be a peptide or peptide mime containing a portion of the connexin 45 protein that antagonizes, inhibits, or blocks the connexin-connexin interaction. Exemplary peptide sequences for connexin 45 peptides and peptide mime modulators are provided in Table III. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0196] In some embodiments, the connexin 45 modulator can include a peptide or peptidomimetic that includes, for example, a portion of the E2 or C-terminal domain of connexin 45 that includes SEQ ID NO: 101 (SRPTEKT). The peptide or peptidomimetic can also include, for example, SEQ ID NO: 279 (DCFISRPTEKT). In some embodiments, the peptide can be only 3 amino acids in length and include SRL, PCH, LCP, CHP, IYY, SKF, QPC, VCY, APL, HVR, or more.

[0197] When a particular protein is referred to herein, derivatives, variants, and fragments are contemplated and included. Protein derivatives and variants are well understood by those skilled in the art and can include insertion, substitution, or deletion amino acid sequence variants known in the art.

[0198] Gap junction modulators and anti-connexin hemichannel blocking peptides or peptidomimetics are produced chemically, synthetically, or by other means.

[0199] Connexin hemichannel modulator Any modulators capable of inducing a desired inhibition of the passage (e.g., transport) of molecules through connexin gap junctions and / or connexin hemichannels in ocular or corneal blood vessels, and through connexin gap junctions and / or connexin hemichannels in ocular or corneal epithelium, may be used in embodiments of the present invention and may be administered according to one or more schedules described herein. Any connexin agents that modulate the passage of molecules through gap junctions or connexin hemichannels are also provided in specific embodiments (e.g., those containing ATP that modulate, block, or reduce the passage of molecules from the cytoplasm of a cell to the extracellular space or the cytoplasm of an adjacent cell). Such anti-connexin modulators may modulate the passage of molecules through gap junctions or connexin hemichannels with or without gap junction uncoupling (blocking the transport of molecules through gap junctions). Such compounds include, for example, binding proteins (e.g., scFv, antibodies, etc.), polypeptides (e.g., peptide mimes), and organic compounds (e.g., tonaversat and compounds of formula I and / or formula II) that can block the function or activity of gap junctions or hemichannels, either entirely or partially (e.g., by modulating the release of ATP from connexin hemichannels).

[0200] In some embodiments, the modulator used in the method of the present invention is a gap junction closure or blocking compound or a hemichannel closure or blocking compound (e.g., tonavelsat). In some embodiments, the modulator may be a small molecule which may also be referred herein to as anticonnexin, connexin, connexin gap junction, or connexin hemichannel modulator. In some embodiments, the method of the present invention is characterized by the use of a compound of formula I, e.g., tonavelsat and / or caravelsat, to directly and immediately block the Cx43 hemichannel and cause a concentration and time-dependent reduction of GJ coupling and / or hemichannel inhibition (e.g., blocking hemichannel opening and / or modulating or blocking ATP release from the connexin hemichannel). Caravelsat is N-[(3R,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-4-fluorobenzamide), also known as trans-(+)-6-acetyl-4-(S)-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-1-benzo[b]pyran-3R-ol, or hemihydrate. Tonavelsat is also known by the IUPAC name N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromen-4-yl]-3-chloro-4-fluorobenzamide or (3S-cis)-N-(6-acetyl-3,4-dihydro-3-hydroxy-2,2-(dimethyl-d6)-2H-1-benzopyran-4-yl)-3-chloro-4-fluorobenzamide.

[0201] In some embodiments, the anticonnexin or connexin or connexin gap junction or connexin hemichannel modulator is given by formula I: [ka]

[0202] (In the formula, Y is C-R1;

[0203] R1 is acetyl;

[0204] R2 is hydrogen, C 3-8 Cycloalkyl, with oxygen inserted as needed, or hydroxy, C 1-6 C substituted with alkoxy or substituted aminocarbonyl 1-6 Alkyl, C 1-6 Alkylcarbonyl, C 1-6 Alkoxycarbonyl, C 1-6 Alkylcarbonyloxy, C 1-6 Alkoxy, nitro, cyano, halo, trifluoromethyl, or CF3-S-; or group CF3-A- (wherein A is -CF2-, -CO-, -CH2-, CH(OH), SO2, SO, CH2-O or CONH); or group CF2H-A'- (wherein A' is oxygen, sulfur, SO, SO2, CF2 or CFH); trifluoromethoxy, C 1-6 Alkyl sulfinyl, perfluoro C 2-6 Alkyl sulfonyl, C 1-6 Alkyl sulfonyl, C 1-6 Alkoxysulfinyl, C 1-6 Alkoxysulfonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, phosphono, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulfinyl, heteroarylsulfinyl, arylsulfonyl, or heteroarylsulfonyl in which any aromatic moiety is substituted as needed, C 1-6 Alkylcarbonylamino, C 1-6 Alkoxycarbonylamino, C 1-6 Alkyl-thiocarbonyl, C 1-6 Alkoxy-thiocarbonyl, C 1-6 Alkyl-thiocarbonyloxy, 1-mercaptoC 2-7 Alkyl, formyl, or aminosulfinyl, aminosulfonyl, or aminocarbonyl; any amino portion consists of one or two C atoms. 1-6 Alkyl alkyl group, or C 1-6 Alkyl sulfinylamino, C 1-6Alkylsulfonylamino, C 1-6 Alkoxysulfinylamino or C 1-6 Substituted with alkoxysulfonylamino as needed, or C 1-6 Alkylcarbonyl, nitro, or cyano, or -C(C 1-6 Alkyl)NOH or -C(C 1-6 Ethyleneyl terminally substituted with alkyl)NNH2; or one or two C as needed. 1-6 Alkyl or C 2-7 It is an amino acid substituted with an alkanoyl group;

[0205] R3 and R4 are either hydrogen or C 1-4 It is alkyl, and the other is C 1-4 Alkyl, CF3, or CH2X a And here, X a Fluoro, chloro, bromo, iodine, C 1-4 Alkoxy, hydroxy, C 1-4 Alkylcarbonyloxy, -SC 1-4 Alkyl, nitro, one or two C 1-4 Amino, cyano, or C atoms are optionally substituted with alkyl groups. 1-4 It is an alkoxycarbonyl; or, R3 and R4 together are C 1-4 C substituted with alkyl as needed 2-5 It is a polymethylene compound;

[0206] R5 is C 1-6 Alkylcarbonyloxy, benzoyloxy, ONO2, benzyloxy, phenyloxy or C 1-6 It is an alkoxy, and R6 and R9 are hydrogen, or R5 is hydroxyl and R6 is hydrogen or C 1-2 It is an alkyl group, and R9 is hydrogen;

[0207] R7 is either heteroaryl or phenyl, and both of these can be independently chloro, fluoro, bromo, iodine, nitro, and C as needed. 1-4 Alkyl, cyano, azide, C1-4 a group selected from amino groups mono- or di-substituted with alkoxy, trifluoromethoxy and trifluoromethyl; or an atom substituted one or more times with such a group;

[0208] R8 is hydrogen, C 1-6 alkyl, OR 11 or NHCOR 10 (wherein R 11 is hydrogen, C 1-6 alkyl, formyl, C 1-6 alkanoyl, aroyl or aryl-C 1-6 alkyl, R 10 is hydrogen, C 1-6 alkyl, C 1-6 alkoxy, mono- or di-C 1-6 alkylamino, amino, amino-C 1-6 alkyl, hydroxy-C 1-6 alkyl, halo-C 1-6 alkyl, C 1-6 acyloxy-C 1-6 alkyl, C 1-6 alkoxycarbonyl-C 1-6 alkyl, aryl or heteroaryl); the R8-N-CO-R7 group is cis to the R5 group;

[0209] X is oxygen or NR 12 wherein R 12 is hydrogen or C 1-6 alkyl); and compounds thereof.

[0210] For any of the above Markush groups, in some embodiments, each group can include or exclude any of the species listed for that group.

[0211] In some embodiments, the small molecule connexin modulator can be tonabersat, carabersat, or SB-204269. SB-204269 is also known as (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol). Carabersat is also known as N-[(3R,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromen-4-yl]-4-fluorobenzamide. Tonabersat is also known as N-(6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromen-4-yl)-3-chloro-4-fluorobenzamide.

[0212] For any of the above Markush groups, the group can include or exclude any of the species listed for that group.

[0213] In some embodiments, the modulator can be a prodrug of any compound for use in the present invention. In one aspect, the connexin modulator prodrug of the present invention can be a compound of Formula II:

Chemical formula

[0214] (where

[0215] Q is O or an oxime,

[0216] R2 is H,

[0217] A is a direct bond, -C(O)O * -, -C(R3)(R4)O * -, -C(O)O-C(R3)(R4)O * -, or -C(R3)(R4)OC(O)O * (where * the atom marked is directly connected to R1,

[0218] R3 and R4 are independently H, fluoro, and C. 1-4 Alkyl and C 1-4 Selected from fluoroalkyl groups, or R3 and R4 together with the atoms to which they are bonded, form a cyclopropyl group.

[0219] R1 is selected from groups [1], [2], [2A], [3], [4], [5] and [6], in the formula, ** The marked atoms are directly connected to A. [ka]

[0220] During the ceremony, R5 and R6 are independently H and C 1-4 Alkyl, C 1-4 Selected from fluoroalkyl and benzyl;

[0221] R7 is independently H, C 1-4 Alkyl and C 1-4 Selected from fluoroalkyl;

[0222] The R8 is as follows: (i) H, C 1-4 Alkyl or C 1-4 Fluoroalkyl, (ii) side chains of natural or non-natural alpha-amino acids or peptides as described herein, and (iii) Biotin or substances chemically bonded to biotin Selected from;

[0223] R9 is H, -N(R 11 )(R 12 ), -N + (R 11 )(R 12 )(R 13 )X - , and -N(R 11 )C(O)R 14 Selected from;

[0224] In the formula, R 11 , R 12 and R 13 H, C 1-4 Alkyl and C 1-4 Selected independently from fluoroalkyls, R 14 H, C 1-4 Alkyl or C 1-4 It is a fluoroalkyl, R 15 C 1-4 Alkyl and C 1-4 Selected from fluoroalkyl groups, X - (This is a pharmaceutically acceptable anion.)

[0225] In some embodiments, R2 is BR 21 And in the formula, B is a direct bond, -C(O)O * -, -C(R 23 )(R 24 )O * , C(O)OC(R 23 )(R 24 ) * ,or -C(R 23 )(R 24 )OC(O)O * (In the formula, * The marked atom is R 21 (It is directly connected to) R 23 and R 24 These are independently H, fluoro, and C. 1-4 Alkyl and C 1-4 Selected from fluoroalkyl groups, R 21 is selected from groups

[21] ,

[22] , [22A],

[23] ,

[24] ,

[25] and

[26] , in the formula, ** The marked atoms are directly connected to B: [ka] In the formula, R5, R6, R7, R8, R9 and R 15 This is as defined herein.

[0226] For any of the Markush groups in Equation II above, the group may include or exclude any of the species listed for that group.

[0227] In some embodiments, the peptides described herein may be connexin modulators, calmodulin modulators, or pannexin modulators.

[0228] In some embodiments, Q is the expression = NOR 43 It is an oxime of, and in the formula, R 43 teeth, (i) H, C 1-4 Fluoroalkyl or optionally substituted C 1-4 Selected from alkyl, and (ii)-A 300 -R 300 (In the formula, A 300 This is a direct bond, -C(O)O * -, -C(R3)(R4)O * -, -C(O)OC(R3)(R4)O * -, or -C(R3)(R4)OC(O)O * - and in the formula, * The marked atoms are R 300 Directly connected, R3 and R4 are independently H, fluoro, and C. 1-4 Alkyl and C 1-4 Selected from fluoroalkyl groups, or R3 and R4 together with the atoms to which they are bonded, form a cyclopropyl group. R 300 is selected from groups [1], [2], [2A], [3], [4], [5] and [6], in the formula, ** The marked atom is A 300 Directly connected): [ka]

[0229] In the formula, R5 and R6 are, independently, H and C. 1-4 Alkyl, C 1-4 Selected from fluoroalkyl and benzyl; R7 is independently H, C 1-4 Alkyl and C 1-4 Selected from fluoroalkyl; The R8 is as follows: (iii) H, C 1-4 Alkyl or C 1-4 Fluoroalkyl, (iv) Side chains of natural alpha-amino acids, and (v) Biotin or a chemically bonded biotin Selected from; R9 is H, -N(R 11 )(R 12 ), -N + (R 11 )(R 12 )(R 13 )X - , and -N(R 11 )C(O)R 14 Selected from; In the formula, R 11 , R 12 and R 13 H, C 1-4 Alkyl and C 1-4 Selected independently from fluoroalkyls, R 14 H, C 1-4 Alkyl or C 1-4 It is a fluoroalkyl, R 15 C 1-4 Alkyl and C 1-4 Selected from fluoroalkyl groups, X - This is a pharmaceutically acceptable anion.

[0230] In one embodiment of formula II, R 43 C is substituted with a phosphate group as needed.1-4 Alkyl (P(O)OR 61 R 62 ). In one example of such an embodiment, OR 43 is -OCH2P(O)OR 61 Ure 62 And in the formula, R 61 and R 62 H or C 1-4 It is alkyl.

[0231] In another embodiment of Equation II, R 43 The structure is C(O)CH(R 100 ) an amino acid derivative having NH2, where the group R 100 These are side chains of natural or non-natural amino acids or peptides as described herein.

[0232] In some embodiments, the natural amino acid is selected from one of 22 standard amino acids. In some embodiments, the non-natural amino acid is selected from any amino acid other than one of the 22 standard amino acids. In some embodiments, the non-natural amino acid is (cis)-3-aminobicyclo[2.2.1]heptane-2-carboxylate hydrochloride, exo-cis-3-aminobicyclo[2.2.1]hepta-5-ene-2-carboxylate hydrochloride, cis-2-amino-2-methylcyclohexanecarboxylate hydrochloride, (R)-2-(Boc-amino)octanedioic acid, Boc-4-(Fmoc-amino)-L-phenylalanine, Boc-(2-indanyl)-Gly-OH, (R)-4-Boc-3-morpholine acetate, (S)-4-Boc-3-morpholine acetate, Boc-pentafluoro-D-phenylalanine , Boc-pentafluoro-L-phenylalanine, Boc-Phe(2-Br)-OH, Boc-Phe(4-Br)-OH, Boc-D-Phe(4-Br)-OH, Boc-D-Phe(3-Cl)-OH, Boc-Phe(4-NH2)-OH, Boc-Phe(3,5-F2)-OH, 2-(4-Boc-piperazino)-2-(2-fluorophenyl)acetic acid, 2-(4-Boc-piperazino)-2-(4-fluorophenyl)acetic acid, 2-(4-Boc-piperazino)-2-phenylacetic acid, 2-(4-Boc-piperazino)-2-(3-pyridyl)acetic acid, penicillamine, thiaidine. Kisscalic acid, canavanine. Selected from azetidine-2-carboxylic acid, carboxyglutamic acid, hydroxyproline, hypsin and pyroglutamic acid.

[0233] In one embodiment of formula II, OR 43 It is -OC(O)CH(CH(CH3)2)NH2.

[0234] In some embodiments, each of the Markush groups in Equation II above may include or exclude any of the species listed for that group.

[0235] In some embodiments, “promoety” refers to a species that acts as a protecting group, masking a functional group within the activator and thereby converting the activator into a prodrug. The activator may be either a modulator or an ophthalmic therapeutic as disclosed herein. Typically, the promoety binds to the drug via a bond(s) that are cleaved in vivo by enzymatic or non-enzymatic means, thereby converting the prodrug into its active form. In some embodiments, the promoety may be the activator. In some embodiments, the promoety may be bound to a connexin modulator, a connexin gap junction modulator, or a connexin hemichannel modulator. In some embodiments, the promoety may be bound to any of the polynucleotides, peptides or peptide mimes, small molecule antagonists, and / or other treatments disclosed herein. In some embodiments, the promoety may be bound to a compound of formula I. In some embodiments, the prodrug may be a compound of formula II.

[0236] In some embodiments, the promoety may be any peptide mime or peptide antagonist of the present disclosure. In some embodiments, the promoety is a single amino acid that is optionally protected on its functional group. In some embodiments, the promoety is a targeted species. In some embodiments, the promoety is a substrate of an influx or efflux transporter on the cell membrane, e.g., Gaudana, R. et al. The AAPS Journal, 12:3, 348-360 (2012). The promoety may be, for example, chemically bound biotin. The promoety may be, for example, chemically bound D-serine.

[0237] In some embodiments, the compounds of formula I or II, such as tonavelsat, caravelsat, or analogs, are nonionic and exist in the form of a free base, a free acid, or a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include hydrochloride salts, as well as salts derived from acids, including but not limited to hydrobromic acid, hydrochloric acid, phosphoric acid, acetic acid, fumaric acid, maleic acid, salicylic acid, citric acid, oxalic acid, lactic acid, malic acid, succinic acid, methanesulfonic acid, and p-toluenesulfonic acid, and salts of the acid itself. In one embodiment, the salt is a hydrochloride salt. In one embodiment, the salt is a succinate salt.

[0238] In other embodiments, compounds of formula I or II, such as tonavelsat, caravelsat or its analogs, one or more polymorphs, one or more isomers, and / or one or more solvates, may be used.

[0239] Other Connexin 43 modulators In addition to connexin antisense (e.g., lufepirsen), connexin peptide mimes (e.g., Peptide 5, XG19, etc.), and connexin hemichannel antagonists (e.g., tonaversat), connexin-binding proteins, including antibodies and antigen-binding antibody fragments, are also suitable connexin modulators for use in the methods of the present invention and are administered in therapeutically effective doses according to one or more administration schedules described herein. Binding proteins include, for example, monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, F(ab')2 and Fv fragments; single-chain antibodies; single-chain Fv; and single-chain binding molecules such as those containing, for example, binding domains, hinges, CH2 and CH3 domains, recombinant antibodies, and antibody fragments that can bind to antigenic determinants (i.e., that part of a molecule commonly called an epitope) that come into contact with a particular antibody or other binding molecule. These binding proteins, including antibodies, anti-binding antibody fragments, etc., may be chimeric or humanized, or otherwise modified to be less immunogenic in the target to which they are administered, or they may be synthesized and recombinant produced They may be produced by or in an expression library. Any binding molecules known or subsequently discovered in the art, e.g., those referenced herein and / or described in more detail in the art, are envisioned. For example, binding proteins include not only antibodies but also ligands, receptors, peptide mimes, or other binding fragments or molecules (e.g., produced by phage display) that bind to targets (e.g., connexin proteins or connexin hemichannel epitopes). Methods for synthesizing antibodies and binding fragments as well as peptides and polypeptides (including peptide mimes and peptide analogs) can also be carried out using appropriate methods.For example, see Lihu Yang et al., Proc.Natl.Acad.Sci.USA,1;95(18):10836-10841(Sept 1 1998); Harlow and Lane (1988) "Antibodies: A Laboratory Manuel," Cold Spring Harbor Publications, New York; Harlow and Lane (1999) "Using Antibodies," A Laboratory Manuel, Cold Spring Harbor Publications, New York.

[0240] Connexin modulators are antibodies that bind to connexin proteins and their binding fragments (e.g., scFv, human V). H or V L Domain, Humanized Camel V HH Domain, Ig NAR This also includes connexin peptides and polypeptides, including single-domain peptides, as well as peptide mimes and analogues of connexins that modulate the activity or function of hemichannels or gap junctions, and other gap junction blockers and gap junction protein phosphorylators. Connexin protein peptides and polypeptides can inhibit connexin function, for example, by binding to the connexin protein and inhibiting its function, or by mimicking the region of the connexin protein and inhibiting or disrupting its binding to other gap junction proteins. Strategies known in the art can be used to improve the naturally short half-lives of antibody fragments, including PEGylation, use of repeating peptide sequences, polysialylation, albumin or IgG binding or fusion, and other approaches.

[0241] The binding molecule generally possesses, but is not limited to, desired specificities including binding specificity and desired affinity. Affinity is, for example, about 10 4 M -1 The above is approximately 10 6 M -1 The above is approximately 10 7 M-1 The above is approximately 10 8 M -1 The above K a It is possible. 8 M -1 Super affinity, for example, about 10 9 M -1 , about 10 10 M -1 The above is approximately 10 11 M -1 and about 10 12 M -1 The affinity is appropriate. The affinity of a binding protein useful for treating a target according to the present invention can be easily determined using the prior art, for example, the technique described by Scatchard et al., 1949 Ann. NYAcad. Sci. 51:660.

[0242] Other compounds used to modulate, block, or close gap junctions (e.g., phosphorylated connexin 43 tyrosine and / or serine residues) are reported in U.S. Patent Nos. 7,153,822 and 7,250,397.

[0243] Connexin modulator combination and concomitant administration The methods, uses, and compositions of the present invention may involve the use of combinations of two or more connexin modulators, e.g., connexin antisense modulators (e.g., lufepirsen), connexin peptide mimes (e.g., Peptide 5, Gap 19, XG 19), and small molecules (e.g., compounds of formula I including compounds of formula II, e.g., tonaversat and / or its prodrugs). Two or more connexin modulators may be administered alone or together. In some embodiments, two or more separate pharmaceutical compositions, each containing one or more connexin modulators, are provided for administration. The pharmaceutical composition is also provided for co-administration in the form of a combination preparation, for example, as a mixture of two or more modulators, e.g., two or more gap junctions, connexins and / or hemichannel modulators, which may be modified or unmodified, e.g., one or more gap junctions, connexins and / or hemichannel modulator polynucleotides and one or more gap junctions, connexins and / or hemichannel modulator peptides or peptide mimetic products, and, optionally, one or more small molecule hemichannel blockers or inhibitors that can reduce the opening and function of hemichannels.

[0244] Treatment of the ocular conditions described herein by the present invention with one or more pharmaceutical compositions, such as anticonnexin antisense and connexin hemichannel blockers, gap junction modulators such as peptides or peptide mimetic compounds, or a first anticonnexin agent and a second anticonnexin agent, may include simultaneous, separate, sequential, or continuous administration thereof.

[0245] The term “combination formulation” includes “kit of components” or “product” in the sense that the combination partners defined above can be administered independently or by using different fixed combinations, in distinct amounts of combination partners (a) and (b), i.e., simultaneously, separately, or sequentially, whether in pharmaceutical form (e.g., topical or oral), dressing / matrix form (e.g., connexin modulator-impregnated bandage contact lens), or both, according to the methods, doses, and daily administration schedules of the present invention. The components of the kit can then be administered, for example, simultaneously or staggered in time, i.e., at different time points, at equal or different time intervals for any component of the kit.

[0246] In one embodiment, two or more separate connexin modulator compositions are administered to a subject according to the method, dosage, and administration schedule of the invention, wherein a combination formulation is administered, in which the first composition comprises a therapeutically effective amount of a modulator, e.g., a gap junction, a connexin modulator, e.g., an anticonnexin 43 polynucleotide, a peptide or peptide mimetic, or a hemichannel occlusion compound, and the second composition comprises a therapeutically effective amount of a second modulator, e.g., a gap junction, a hemichannel, and / or a connexin modulator, e.g., an anticonnexin 43 polynucleotide, a peptide or peptide mimetic, a hemichannel occlusion compound, and / or an ophthalmic treatment agent. In another embodiment, a third composition comprising one or more anticonnexin polynucleotides, peptides or peptide mimetics, hemichannel occlusion compounds, and / or an ophthalmic treatment agent is administered.

[0247] Modulators comprising connexin, connexin hemichannel, and connexin gap junction modulator can be dosed, administered, or formulated in the manner of the present invention as described herein.

[0248] Dosage and dose regimen Examples of effective doses that may be used for the treatment of non-healing ocular surface defects or disorders (e.g., ocular PED), non-healing corneal defects or disorders (e.g., PCED), and other eye diseases, disorders and conditions (e.g., uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and all forms of dry eye diseases (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye) or similar or related diseases, disorders and conditions are described herein and claimed. In some embodiments, therapeutically effective doses of modulators effective in the methods of the present invention, such as connexin modulators, such as connexin 43 modulators, are used. The connexin 43 modulator, connexin 43 gap junction modulator and / or connexin 43 hemichannel modulator comprises a composition having about or at least about 0.1 mg, 0.2 mg or 0.3 mg of connexin modulator (e.g., lufepirsen), including doses of about (or at least about) 0.18 mg and about (or at least about) 0.018 mg of connexin modulator (e.g., lufepirsen), or any amount within or between any two of these enumerated doses. Another effective dose effective in the method of the present invention for the treatment of ocular surface defects or disorders (e.g., ocular PED and PCED) comprises about or at least about 1.0 mg of connexin modulator, e.g., connexin 43 modulator, connexin 43 gap junction modulator and / or connexin 43 hemichannel modulator (e.g., lufepirsen).

[0249] In some embodiments, the therapeutically effective dose of a modulator, e.g., a connexin modulator, e.g., a connexin 43 modulator, a connexin 43 gap junction modulator, and / or a connexin 43 hemichannel modulator in the method of the present invention comprises a composition in a dose of approximately or at least approximately 2 micromolar (2 μM), 5 micromolar (5 μM), 10 micromolar (10 μM), 15 micromolar (15 μM), or 20 micromolar (20 μM) (including approximately (or at least approximately) 1.9 micromolar (1.9 μM) or 1.9405 micromolar (1.9405 μM) of a connexin modulator (e.g., lufepirsen), and approximately (or at least approximately) 19-19.4 micromolar (19-19.4 μM) or 19.405 micromolar (19-19.4 μM or 19.405 μM)), or any two or any amount in between these enumerated doses. Other effective doses effective in the method of the present invention for the treatment of defects or disorders of the ocular surface (e.g., ocular PED and PCED) include compositions comprising a connexin modulator, e.g., a connexin 43 modulator, a connexin 43 gap junction modulator, and / or a connexin 43 hemichannel modulator (e.g., lufepirsen) at a concentration of about or at least about 30 micromolars (30 μM) or 40 micromolars (40 μM).

[0250] In some methods of the present invention, the connexin modulator administered to a non-healing surface defect or disorder in a subject is 0.06% lufepirsen. In some embodiments, the connexin modulator administered to a subject having a non-healing surface defect or disorder is 0.006% lufepirsen. In some embodiments, the administered connexin modulator is at least about 0.06% lufepirsen. In some embodiments, the connexin modulator administered to a subject having a non-healing surface defect or disorder is at least about 0.006% lufepirsen. In some embodiments, the administration is for PED or PCED. In some embodiments, a pharmaceutically acceptable carrier and a composition comprising or essentially comprising about 0.06% lufepirsen, about 0.006% lufepirsen, at least about 0.06% lufepirsen, or at least about 0.006% lufepirsen is administered to heal a non-healing surface defect or disorder in a subject. The doses of lufepirsen described herein and below may be prepared as described in Example 1.

[0251] In some embodiments, non-healing surface defects or disorders are treated with a composition comprising or essentially comprising 0.6 mg / mL of lufepirsen. In some embodiments of the present invention, non-healing surface defects or disorders are treated with a composition comprising or essentially comprising 0.06 mg / mL of lufepirsen. In some embodiments, the composition comprises or essentially comprises at least about 0.6 mg / mL of lufepirsen. In some embodiments, the composition comprises or essentially comprises at least about 0.06 mg / mL of lufepirsen. In some embodiments, the administration is for PED or PCED.

[0252] In some embodiments, non-healing surface defects or disorders in a subject are treated with a composition comprising or essentially comprising about 0.018 to about 0.18 mg of lufepirsen in the regimens described herein. In some embodiments, each dose of lufepirsen applied to a non-healing surface defect or disorder in or on a subject comprises or essentially comprises at least about 0.18 mg of lufepirsen or at least about 0.18 mg of lufepirsen in the regimens described herein. In some embodiments of the present invention, non-healing surface defects or disorders are treated with a composition comprising or essentially comprising about or at least about 0.01 mg, about or at least about 0.018 mg, about or at least about 0.18 mg to about or at least about 0.2 mg of lufepirsen, or about or at least about 0.18 mg, 0.20 mg, or 0.50 mg to about or at least about 1 mg of lufepirsen in the regimens described herein. In some embodiments, each dose of lufepirsen applied to a non-healing surface defect or disorder comprises or essentially consists of at least about 0.15 mg, at least about 0.2 mg, at least about 0.25 mg, at least about 0.3 mg, at least about 0.35 mg, at least about 0.4 mg, at least about 0.45 mg, or at least about 0.5 mg of lufepirsen in the regimens described herein. In some embodiments, the administration is for PED or PCED.

[0253] These doses are also useful in treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and all forms of dry eye disease (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye), or similar or related diseases, disorders, and conditions.

[0254] In some embodiments, the above doses are administered topically to the eye of a subject having a non-healing or persistent defect or disorder of the eye or corneal surface. In some embodiments, the doses referred to herein are administered intraocularly, including by injection (e.g., intravitreal, anterior chamber, etc.). Connexin modulators administered by routes other than topical administration (e.g., orally or parenterally) are adjusted as necessary to approximate these topical doses.

[0255] In some embodiments, a certain dose of lufepirsen (or other connexin antisense or connexin modulator) is administered to non-healing surface defects or disorders in a single dose or in two or more divided doses (e.g., up to 6 doses administered twice daily on days 1, 2, and 14, or up to 8 doses administered twice daily on days 1, 2, 14, and 28). In other embodiments, about or at least about 0.1–0.5 mg, or at least about 1 mg of lufepirsen is administered in a single dose or divided dose (e.g., up to 14 doses administered twice daily on days 1, 2, and 14, and on one or more days, e.g., 7, 21, 28, and / or 35). In some embodiments, the administration is for PED or PCED in the subject.

[0256] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 14, and 21.

[0257] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 7, 14, and 21.

[0258] In some embodiments, the dose of the connexin modulator, for example, lufepirsen, is administered on days 1, 2, 7, 14, 21, and 28.

[0259] In some embodiments, to treat non-healing or persistent ocular or corneal surface defects in a subject, three doses of a connexin modulator (e.g., lufepirsen) are administered over 14 days, including, for example, on days 1 and 2. In some embodiments, four doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 28 days. In some embodiments, five doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 28 to approximately 35 days. In some embodiments, six to seven doses of a connexin modulator (e.g., lufepirsen) are administered over approximately 35 days. For example, in one embodiment, the connexin modulator (e.g., lufepirsen) is administered four times: over days 1 and 2, and on approximately days 14 and 28. In another embodiment, the connexin modulator (e.g., lufepirsen) is administered five times: over days 1 and 2, on approximately day 14, on approximately day 28, and on approximately day 35. In another embodiment, the connexin modulator (e.g., lufepirsen) is administered five times: over days 1 and 2, on about day 7, on about day 14, and on about day 28. In yet another embodiment, the connexin modulator (e.g., lufepirsen) is administered six times: over days 1 and 2, on about day 7, on about day 14, on about day 21, and on about day 28. In yet another embodiment, the connexin modulator (e.g., lufepirsen) is administered seven times: over days 1 and 2, on about day 7, on about day 14, on about day 21, and on about day 28. References to “administration” in this specification, of course, refer to the administration of the connexin modulator in single or divided doses.

[0260] Whenever the dose of a connexin modulator is not specified in the dose regimen or method of the present invention for treating non-healing ocular or corneal surface defects or disorders (e.g., a dose of lufepirsen or another connexin expression modulator, connexin peptide mimetic and / or hemichannel blocker), the administration of a therapeutically effective dose of the connexin modulator(s) is intended.

[0261] In some embodiments, one or more connexin modulators are administered to subjects having ocular diseases, disorders, and conditions selected from the group consisting of uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and all forms of dry eye disease (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye) using one or more dose regimens described herein.

[0262] Whenever a dose of a connexin modulator is specified for day 7, 14, 21, 28, or 35 in a dose regimen or method of the present invention for the treatment of non-healing ocular or corneal surface defects or disorders, or other diseases, disorders, or conditions (e.g., a certain dose of lufepirsen or another connexin expression modulator, connexin peptide mimetic, and / or hemichannel blocker), it is intended that a therapeutically effective dose of the connexin modulator(s) be administered on approximately that day.

[0263] Administration of pharmaceutical compositions comprising or essentially consisting of connexin modulators (e.g., lufepirsen) and connexin modulators(s), is provided in specific dose regimens. In one embodiment, a composition comprising one or more gap junctions, hemichannels and / or connexin modulator polynucleotides (and / or other connexin modulators(s), e.g., peptide mimes or small molecule connexin modulators) is administered on days 1 and 2 of treatment, and subsequently on day 14. In another embodiment, the composition is administered on days 1 and 2 of treatment, and subsequently on days 14 and 28. In yet another embodiment, the composition is administered on days 1 and 2, and subsequently on days 14, 28 and 35. In other embodiments of these administration regimens, the composition is also administered on day 7 and / or 21, if necessary.

[0264] In some embodiments, the disease, disorder, or condition treated is a non-healing ocular surface defect or disorder, specifically a persistent epithelial defect (PED). In some embodiments, the non-healing ocular surface defect or disorder is a persistent corneal epithelial defect (PCED). In some embodiments, the non-healing ocular surface defect or disorder is a corneal ulcer. In some embodiments, the disease, disorder, or condition treated is selected from the group consisting of uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and all forms of dry eye disease (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye). In the method of the present invention, administering a therapeutically effective amount of at least one connexin modulator in pulsed dose form on days 1 and 2, subsequently on day 14, and optionally on day 28 or about day 28 (and optionally on day 35 or about day 35) is effective in healing a non-healing ocular surface defect or disorder, or other disease, disorder, or condition. See Example 2 below. The dose of the connexin modulator may also be administered on day 7 and / or day 21. In some embodiments, the connexin modulator is a connexin 43 modulator (e.g., a connexin expression modulator such as an antisense connexin expression modulator containing Cx43 antisense). Other embodiments include other connexin 43 gap junction modulators and connexin 43 hemichannel blockers or modulators (e.g., peptide mimes and small molecules).

[0265] In some embodiments, doses of the connexin modulator (e.g., lufepirsen or another modulator) are administered on days 1, 2, and 14. In some embodiments, doses of the connexin modulator (e.g., lufepirsen) are administered on days 1, 2, 14, and 28. In some embodiments, doses of the connexin modulator (e.g., lufepirsen) are administered on days 1, 2, 14, 28, and 35. In some embodiments, doses of the connexin modulator (e.g., lufepirsen) are administered on days 1, 2, 7, 14, and 21. In some embodiments, doses of the connexin modulator (e.g., lufepirsen) are administered on days 1, 2, 7, 14, 21, and 28. In some embodiments, doses of the connexin modulator, e.g., lufepirsen, are administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, doses are administered after day 35. In some embodiments, the administration using the described doses and dose regimens is for PED or PCED. In some embodiments, the connexin modulator comprises a connexin antisense molecule (e.g., lufepirsen). In some embodiments, the connexin modulator comprises a connexin peptide mimetic (e.g., Peptide 5, Gap 19, XG 19, Gap 26, Gap 27, aCT 1, etc.). In some embodiments, the connexin modulator comprises a small molecule connexin hemichannel blocker (e.g., tonaversat). In some embodiments, the connexin modulator is a connexin 43 modulator.

[0266] All descriptions relating to administration apply to the modulators of the present invention, including connexin modulators, connexin gap junction modulators, and connexin hemichannel modulators, unless otherwise explicitly stated. All descriptions relating to administration also apply to all indications described or referenced herein for treatment, including ocular surface defects and corneal surface defects, unless otherwise specifically stated. All descriptions relating to administration apply to the treatment of uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and all forms of dry eye disorders (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye), unless otherwise specifically stated.

[0267] formulation The pharmaceutical compositions of the present invention include various desired or appropriate delivery forms and formulations, including formulations for topical administration, as well as forms and formulations for drug administration suitable for systemic administration (e.g., oral and enteral), parenteral administration (e.g., injection, infusion, transplantation), intraocular administration, etc.

[0268] Such delivery forms and formulations include those for the treatment of subjects as disclosed herein. Pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient for ocular administration may be an ophthalmologically acceptable excipient. In some embodiments, the formulation may provide sustained delivery of a connexin modulator and / or ophthalmic treatment agent to a selected segment or compartment of the eye. In some embodiments, the formulation provides high ocular pharmacobioavailability (including by topical or oral administration), is safe and non-toxic, and / or has little to no systemic side effects or complications at the site of administration. Exemplary polynucleotide formulations for use in the methods of the present invention have ease of topical delivery and ease of administration.

[0269] Such delivery forms and formulations include those for the treatment of subjects as disclosed herein. Pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient for ocular administration may be an ophthalmologically acceptable excipient. In some embodiments, the formulation may provide sustained delivery of a connexin modulator and / or ophthalmic treatment agent to a selected segment or compartment of the eye. In some embodiments, the formulation provides high ocular pharmacobioavailability (including by topical or oral administration, for example), is safe and non-toxic, and / or has little to no systemic side effects or complications at the site of administration. Exemplary polynucleotide formulations for use in the methods of the present invention have ease of topical delivery, ease of administration, and a “no side effects” profile.

[0270] In some embodiments, the pharmaceutical formulations of the present invention may include any modulator described herein, such as gap junctions, hemichannels, and / or connexin modulators, such as modified or unmodified connexin 43 antisense oligonucleotides or polynucleotides, or modified or unmodified connexin 43 peptides or peptide mimes. In some embodiments, the connexin 43 antisense oligonucleotides included in the formulation may be unmodified connexin 43 antisense oligodeoxynucleotides or modified connexin 43 antisense oligodeoxynucleotides. In some embodiments, the pharmaceutical composition may include or exclude any of the above.

[0271] Modulators, including connexins, connexin hemichannels, and connexin gap junction modulators, may be present in substantially isolated forms in the formulation. It will be understood that the product may be mixed with a carrier or diluent that does not interfere with the intended purpose of the product and is still considered substantially isolated. The product of the present invention may also be in a substantially purified form, in which case it generally contains about 80%, 85%, or 90%, for example, at least about 88%, at least about 90%, 95%, or 98%, or at least about 99%, of polynucleotides, e.g. (or other connexin modulators such as connexin 43 modulators) or the dry mass of the formulation. As described in Example 1, the lufepirsen composition used in the clinical trial for treating non-healing eye defects described in Example 2 contained 94.2% pure lufepirsen (106% assay) and had a final product purity of 94.9% (97% assay).

[0272] Pharmaceutical formulations for use in the administration, dosage regimens, and methods of the present invention may comprise one or more pharmaceutically acceptable excipients suitable for delivering a modulator (e.g., lufepirsen) including a connexin, a connexin hemichannel, and a connexin gap junction modulator to the eye.

[0273] Modulators comprising gap junctions, hemichannels, and / or connexin modulators of the present invention can be formulated into microparticles (microspheres, Mps) or nanoparticles (nanospheres, Nps) formulations, or both. In some embodiments of the present invention, nanoparticles or microparticles are used. Such particles comprise poly(lactic acid-co-glycolic acid) ("PLGA") loaded with gap junctions, connexins, and / or hemichannel modulators, e.g., connexin 43 modulator. The modulator may be loaded in the particle volume, on the outer surface of the particle, or both. The particle formulation may be administered, for example, topically to the eye or subconjunctivally. In some embodiments, the particle formulation of any of the gap junctions, hemichannels, and / or connexin modulators (e.g., connexin 43 modulator) of the present disclosure may also comprise liposomes.

[0274] Modulators, such as connexins, connexin hemichannels, and connexin gap junction modulators, may be administered alone or in combination with one or more additional components, and may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients, diluents, and / or carriers. pharmaceutically acceptable diluents, carriers, and / or excipients include substances useful for the preparation of pharmaceutical compositions, are generally safe, non-toxic, and not biologically or otherwise undesirable. pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for veterinary use and human pharmaceutical use. Examples of diluents, carriers, and / or excipients include solutions, solvents, dispersions, retarders, polymers, and lipids, emulsions, etc. Further examples include liquid carriers particularly suitable for injection, such as water, saline solutions, and dextrose solutions, and vehicles such as liposomes, which are also particularly suitable for drug administration.

[0275] Suitable carriers and diluents include buffered aqueous solutions, saline solutions, dextrose, glycerol, isotonic saline solutions, e.g., phosphate-buffered saline, isotonic water, and combinations thereof. In some embodiments, the carrier may include propylene glycol, dimethyl isosorbide, and water, and more specifically, phosphate-buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetric alcohols. In some embodiments, pharmaceutically acceptable carriers or diluents may be or contain thermosetting poloxamers (which may be liquid or gel depending on temperature), carboxycellulose (e.g., carboxymethylcellulose), collagen (e.g., type I collagen), collagenous materials including tropocollagen, hyaluronane or derivatized hyaluronic acid, and / or oils (e.g., emu oil). Suitable carriers may be large, slowly metabolized polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. The pharmaceutical compositions of the present invention do not contain a connexin modulator in sterile water as the sole vehicle. In some embodiments, the formulation comprises a connexin modulator, for example, a connexin 43 modulator, and the 43 antisense oligonucleotide contained in the formulation may, in some embodiments, be unmodified or modified connexin 43 antisense oligodeoxynucleotide.

[0276] The composition may take the form of any standard known dosage form, including tablets, pills, capsules, semi-solids, powders, sustained-release formulations, liquids, suspensions, elixirs, aerosols, injectable solutions, gels, creams, transdermal delivery devices (e.g., transdermal patches), implants (e.g., ophthalmic implants), or any other suitable composition. Those skilled in the art to whom the present invention relates will readily understand, without any excessive experimentation, the most appropriate dosage form, taking into account the state being treated and the properties of the activator used (e.g., antisense, peptide mimetic, small molecule, etc.).

[0277] Preferably, the modulator of the present invention, such as a connexin modulator, is used in combination with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. The connexin modulator can be formulated as a composition for any desired route of administration, including topical, oral, systemic, transdermal, transnasal, sublingual, buccal, etc., and including injectable formulations (e.g., intracavitary injection, subcutaneous injection, intramuscular injection, intravenous injection, etc.).

[0278] Pharmaceutically acceptable salts may exist, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate; and salts of organic acids such as citrate, acetate, propionate, malonate, and benzoate.

[0279] In one embodiment, the inverse thermosetting gel may be liquid at low temperatures, for example, 2–8°C, and undergo a reversible liquid-to-gel transition at temperatures above about 15°C. Therefore, in some embodiments, the carrier may be liquid at temperatures below about 15°C, but may form a gel at temperatures above about 15°C, such as room temperature or body temperature. In some examples, the gel is a nonionic polyoxyethylene-polyoxypropylene copolymer gel. In some embodiments, the gel is a Pluronic® gel. The Pluronic® gel may be, for example, poloxamer 407, sometimes referred to as Pluronic® F-127 (BASF). In some embodiments, the formulation of the present invention may contain about 15–30% (w / v) of gel. In some embodiments, the formulation of the present invention may contain about 20–25% (w / v) of gel. In some embodiments, the formulation of the present invention may contain about 22.6% (w / v) of poloxamer 407 gel. In some embodiments, the composition or formulation contains 226.0 mg / mL of poloxamer (e.g., poloxamer 407). See Example 1.

[0280] Other suitable formulations include Pluronic® gel formulations, hydroxymethylcellulose formulations, hydroxyethylcellulose formulations, carboxymethylcellulose (CMC) formulations, and hydroxypropylmethylcellulose (HPMC) formulations. The compositions can be formulated for delivery in any desired form, including topical, ophthalmic, parenteral, intramuscular, subcutaneous, or transdermal administration. Other useful formulations include sustained-release or delayed-release formulations.

[0281] Furthermore, if desired, substances such as wetting agents or emulsifiers, stabilizers or pH buffers, or preservatives may also be present. In some embodiments, the pharmaceutical composition of the present invention includes suitable ophthalmologically acceptable buffers such as acetic acid buffers, citrate buffers, phosphate buffers, borate buffers, and mixtures thereof. In some embodiments, buffers useful in the present invention include boric acid, sodium borate, sodium phosphate (including monobasic, dibasic, and tribasic phosphates, e.g., monobasic sodium phosphate monohydrate and dibasic sodium phosphate heptahydrate), and mixtures thereof. In some embodiments, the preservative may be stabilized chlorine dioxide, a cationic polymer, or a quaternary ammonium compound. In some embodiments, the pharmaceutical composition may include wetting agents, nutrients, viscosity enhancers, antioxidants, etc., such as disodium ethylenediaminetetraacetate, alkali metal hexametaphosphate, citric acid, sodium citrate, sodium metabisulfite, sodium thiosulfate, N-acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, polyvinyl alcohol, polyoxomer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, and mixtures thereof, as well as mixtures thereof. In some embodiments, the pharmaceutical formulations of the present invention do not contain preservatives. In some embodiments, the connexin modulator composition or formulation includes dibasic sodium phosphate heptahydrate or monobasic potassium phosphate, or both. See Example 1.

[0282] When modulators, such as gap junctions, connexins, and / or hemichannel modulators, are nucleic acids such as polynucleotides, the uptake of nucleic acids by mammalian cells can be enhanced by known transfection techniques, including the use of transfection agents. Such techniques may be used in conjunction with certain anticonnexin agents containing polynucleotides. The administered formulation may contain such transfection agents. Examples of useful transfection agents include cationic agents (e.g., calcium phosphate and DEAE-dextran), lipofectants (e.g., lipofectam® and transfectam®), and surfactants.

[0283] Connexins, gap junctions, and / or hemichannel modulators, such as the connexin 43 modulator, may also be formulated to provide controlled release to the eye. In some embodiments of the present invention, the formulation may be an immediate-release or extended or sustained-release dosage form for release, for example, within a few hours, within a day, or within, for example, one to two days.

[0284] The composition is, for example, Gennaro AR: Remington: The Science and Practice of Pharmacy, 20 th It can be formulated according to standard techniques known in the art, including those that can be found in standard references such as Lippincott, Williams & Wilkins, 2000.

[0285] Any container suitable for storing and / or administering the pharmaceutical composition may be used for the combined product of the present invention. Suitable containers will be understood by those skilled in the art. Examples of such containers include vials and syringes. The containers may be properly sterilized and airtight sealed.

[0286] In some embodiments, a connexin modulator (e.g., connexin 43 modulator or connexin 45 modulator, preferably connexin 43 modulator, e.g., lufepirsen) is administered to the target eye, providing a therapeutically effective amount of connexin modulator to the eye or a specific compartment or portion of the eye by desired and appropriate administration, including but not limited to topical administration, depending on the properties of the connexin modulator.

[0287] The therapeutically effective dose includes, but is not limited to, the doses described herein. The doses described and other therapeutically effective doses are administered in one or more therapeutically effective dose regimens as described herein.

[0288] Administration The administration of modulators, such as connexin modulators, gap junction channel modulators, and / or hemichannel modulator compounds and compositions, may be by one of the following routes: oral, topical, systemic (including intravenous, intra-arterial, intraperitoneal, transdermal, intranasal, or suppository), or parenteral (including intramuscular, subcutaneous, or intravenous or intra-arterial injection). In some embodiments, connexin modulators, gap junction channel modulators, and / or hemichannel modulator compounds and compositions are administered topically. In some embodiments, connexin modulators, gap junction channel modulators, and / or hemichannel modulator compounds and compositions are administered systemically. In some embodiments, connexin modulators, gap junction channel modulators, and / or hemichannel modulator compounds and compositions are administered orally. In some embodiments, compounds and compositions of connexin modulators, gap junction channel modulators, and / or hemichannel modulators are administered by intrachorally injectable formulations and routes of administration, which may be preferred routes of administration (or when topical administration is less effective) for, for example, the treatment of uveitis and Fuchs dystrophy.

[0289] In some embodiments of the methods of the present invention, the connexin modulator, connexin gap junction modulator, and / or connexin-hemichannel modulator may be administered topically by topical administration to the eye of interest. In some embodiments, the connexin modulator is administered topically, by corneal and / or subconjunctival administration, or by local injection. Topical formulations of gap junctions, hemichannels, and / or connexin modulators may include ointments, gels, which may be, for example, thermosetting gels, drops, sprays, liquids, and powders, or sustained-release or non-sustained-release formulations. Bioavailable systemically administered connexin modulators, connexin gap junction modulators, and / or connexin hemichannel modulators may be administered, with or without simultaneous local administration to the eye, for example, by oral, intravenous, enteral (e.g., oral, rectal, sublingual, buccal), parenteral (e.g., intravenous, intramuscular, subcutaneous, transdermal), intranasal, nasal inhalation, and oral inhalation. In some embodiments, non-local administration, including intraperitoneal, oral, or parenteral administration, may be used, provided that the therapeutically effective dose comes into contact with the eye or a portion thereof (e.g., the cornea) to be healed. In some embodiments, in addition to the administration of connexin modulators by local administration, orally available connexin modulators (e.g., tonaversat) may also be administered intraperitoneally, orally, or parenterally, provided that the therapeutically effective dose comes into contact with the eye.

[0290] In some embodiments, a therapeutically effective amount of connexin modulator(s) is provided to the eye by administering a connexin modulator, such as a connexin expression modulator, a peptide-mimicking or small molecule gap junction modulator, and / or a hemichannel modulator (e.g., a connexin 43 modulator or modulator or any other connexin in the eye, cornea, corneal epithelium, or blood vessels). In some embodiments, a bandage contact lens is applied to the eye after administration of the connexin modulator(s), whether locally or by another route of administration. In some embodiments, the connexin modulator is placed in a bandage contact lens applied to the eye of a subject having a non-healing eye defect or disorder (e.g., PED or PCED) or other disease, disorder, or condition described or referenced herein. In some embodiments, the connexin modulator is administered under the amniotic membrane. In some embodiments, the amniotic membrane is applied to the eye after administration of the connexin modulator. The modulator may be a connexin 43 modulator. In some embodiments, the connexin modulator is embedded in a matrix comprising a bandage contact lens or other implantable device. In some embodiments, the connexin modulator matrix (e.g., the connexin modulator matrix of a bandage contact lens) provides sustained or prolonged release of the connexin modulator contact lens. As used herein, “matrix” includes, for example, a polymer matrix, a biodegradable or non-biodegradable matrix, and other carriers useful for fabricating implantable or applicable structures for delivering connexin modulators for non-healing ocular and / or corneal surface disorders or neuropathic treatments. Compositions and methods for preparing drug-containing contact lenses have been developed and are known in the art.For example, see Wang Z et al., Novel Contact Lenses Embedded with Drug-Loaded Zwitterionic Nanogels for Extended Ophthalmic Drug Delivery. Nanomaterials 11:2328 (2021).

[0291] In some embodiments of the present invention, subjects having a non-healing surface defect or disorder, such as PCED, have an amniotic graft. In some embodiments, a gap junction modulator or hemichannel modulator, such as lufepirsen, is administered under the amniotic graft. In some embodiments, the amniotic graft is applied to the non-healing surface disorder, such as PCED, after the application of the gap junction modulator or hemichannel modulator, such as lufepirsen.

[0292] Manufactured products / kits In another embodiment of the present invention, a product or “kit” is provided which comprises materials useful for treating non-healing defects and disorders of the ocular surface or cornea (e.g., PED or PCED). In another embodiment of the present invention, a product or “kit” is provided which comprises materials useful for treating uveitis, blepharitis, Sjögren’s syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy and / or dry eye diseases (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-decreased dry eye).

[0293] The kit includes a container containing a composition comprising one or more modulators, such as connexin antisense, connexin peptide mimetic and / or small molecule hemichannel blockers. The kit may further include a label or accompanying leaflet on or associated with the container, which includes instructions for administration as described herein. The term “accompanying leaflet” refers to instructions typically included in the market packaging of a therapeutic product, which include information regarding indications, usage, dosage, administration, contraindications and / or warnings relating to the use of the modulator, which may be provided, for example, physically or via an online link. Suitable containers include, for example, bottles, vials, etc. Containers may be formed from a variety of materials, such as glass or plastic. In some embodiments, the label or accompanying leaflet indicates that the composition is used to treat non-healing defects and disorders of the ocular or corneal surface (e.g., PED or PCED). In some embodiments, the label or accompanying information indicates that the composition is used to treat uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs dystrophy, and all forms of dry eye disease (e.g., evaporative dry eye, tear-deficient dry eye, and wettability-reduced dry eye).

[0294] The kit may contain one or more pharmaceutical compositions in separate vessels or partitioned containers, along with packaging and instructions for use. The kit may also contain a pharmaceutically acceptable carrier. In some embodiments, the kit may also contain components for administering the pharmaceutical compositions, such as syringes, needles, or microneedles. In some embodiments, the kit may include a bandage contact lens for application to a target after administration of the connexin modulator or for use in the application of the connexon modulator. The connexin modulators may be administered on separate days and may include packaging and / or instructions for staggered administration according to the method of the present invention.

[0295] manufacturing The polynucleotides of the present invention can be prepared using solid-phase chemistry for synthesizing oligonucleotides, chemistry known in the art for synthesizing and preparing peptides and peptide mimetic compounds, and chemistry known in the art for synthesizing organic compounds (e.g., tonaversat, and other compounds of formulas I and II). In one embodiment, the formulation of the present invention comprises a salt of the polynucleotide of the present invention, for example, a sodium salt of the polynucleotide of the present invention. The kit may also comprise a pharmaceutically acceptable carrier. In one embodiment, the formulation may comprise, for example, a sodium salt of a polynucleotide having any one of SEQ ID NOs: 1 to 16 or a portion of SEQ ID NO: 17. In some embodiments, the polynucleotide having any one of SEQ ID NOs: 1 to 16 may be a modified oligodeoxynucleotide having any one of SEQ ID NOs: 1 to 16 or a portion of SEQ ID NO: 17.

[0296] In some embodiments, the formulations of the present invention are substantially pure. Substantially pure means that the formulation contains about 10%, 5%, or less than 1%, preferably less than 0.1%, of any nucleotide or non-nucleotide impurities. In some embodiments, the total impurities, including metabolites of the connexin 43 regulator, are 15% or less. In some embodiments, the total impurities, including metabolites of the connexin 43 regulator, are 12% or less. In some embodiments, the total impurities, including metabolites of the connexin 43 regulator, are 11% or less. In other embodiments, the total impurities, including metabolites of the connexin 43 regulator, are 10% or less. See, for example, Example 1, which describes the preparation of a lufepirsen composition having a final formulation purity of 94.9%.

[0297] A sterile composition comprising the connexin 43 modifier of the present invention, prepared by aseptic treatment using the dissolution of the anti-connexin modifier in a formulation vehicle. In one embodiment, the formulation may also be sterilized by filtration. The excipients used in the manufacture of the formulation of the present invention are widely used in pharmaceuticals and are published in pharmacopoeia standards. [Examples]

[0298] The following studies were conducted to evaluate whether connexin modulation can be used in patients to treat non-healing ocular defects and disorders, including persistent ocular epithelial defects and persistent corneal epithelial defects.

[0299] The study described in the examples was conducted over several years in multiple countries in a randomized, double-blind, placebo-controlled human clinical trial, with the aim of discovering and identifying previously unknown but clinically useful therapeutic doses and dosing regimens.

[0300] The successful results of this research and the efforts made are described.

[0301] Example 1 Preparation of LUFEPIRSEN® composition for topical application in subjects with persistent corneal epithelial defects. As shown in the table below, an ocular topical administration composition containing the connexin 43 modulator lufepirsen (5'-GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC-3' [SEQ ID NO: 1]) was prepared by dissolving an anticonnexin modulator in a formulation vehicle and adding a buffer. [Table 4]

[0302] A composition containing 0.6 mg / ml or 0.06% lufepirsen connexin 43 modulator was prepared to a final volume of 300 μL for administration by dissolving 0.18 mg of lufepirsen or 19.40491591203 micromolar concentration (μM) [005 API (Lot C06A14001)] in a buffer in a poloxamer pharmaceutical vehicle.

[0303] A composition containing 0.06 mg / ml or 0.006% lufepirsen connexin 43 modulator was prepared to a final volume of 300 μL for administration by dissolving 0.018 mg of lufepirsen or 1.940491591203 micromolar concentration (μM) [005 API (Lot C06A14001)] in a poloxamer pharmaceutical vehicle containing a buffer.

[0304] The lufepirsen purity was 94.2% in a 106% assay, and the formulation purity was 94.9% in a 100% assay. The lufepirsen composition was used in clinical trials as described in Example 2.

[0305] Example 2 A phase 2, randomized, prospective, double-blind, vehicle-controlled study to evaluate the efficacy and safety of topically applied LUFEPIRSEN® (LUFEPIRSEN) in subjects with persistent epithelial defects (PED) resulting from chemical and / or thermal damage to the eye. This example describes the use of an exemplary connexin modulator (lufepirsen) in a randomized, prospective, vehicle-controlled, double-blind, human clinical trial to treat non-healing ocular surface defects. Lufepirsen is a connexin 43 antisense oligonucleotide by SEQ ID NO: 1. The vehicle control drug used in the trial was Pluronic® F-127.

[0306] Study Participants: A total of 35 subjects were enrolled in this clinical trial. All eligible subjects presented with non-infectious persistent epithelial defect (PED) of the cornea as a result of chemical and / or thermal eye injury, which was resistant to current standard care for at least 14 days. Of the 35 subjects, 24 received topical ocular doses of LUFEPIRSEN, and 11 received vehicle therapy. Twelve subjects either did not re-epithelialize after 28 days or remained unre-epithelialized, and these received either one (day 1 of salvage) or two (day 8 of salvage) doses of 0.06% LUFEPIRSEN in an open-label manner. The results are shown below.

[0307] Study Objective: The objective of this clinical protocol was to evaluate the efficacy and safety of two topical ocular dose concentrations of LUFEPIRSEN in a novel protocol for the treatment of non-healing corneal PED resulting from severe chemical and / or thermal eye injury.

[0308] Study endpoints: The primary, secondary, and other endpoints evaluated in this human clinical trial are listed below: Primary endpoints: ● Corneal epithelial recovery, defined as cornea that has re-epithelialized by day 28 of the procedure, after the initial re-epithelialization was first recorded, and remains re-epithelialized for at least 28 days, as assessed by the principal investigator. Secondary endpoint: ● The time to epithelialization is defined as the time from randomization to the first re-epithelialization. ● Improvement in visual acuity from baseline during the study period. ● The number of LUFEPIRSEN treatment doses required for corneal epithelial recovery. Exploratory endpoints: ● Change from baseline in the PED area, defined as the maximum diameter multiplied by the maximum diameter perpendicular to it, within the scope of the epithelial defect. ● Corneal re-epithelialization is defined as a cornea that has re-epithelialized by 28 days after treatment. ● Changes in ocular symptoms from baseline. ● Primary, secondary, and exploratory endpoints were also evaluated for the open-label sections of the study (where applicable). ● Descriptive comparison of intracorneal re-epithelialization in the other eye of subjects with bilateral lesions. Safety: ● Treatment rate for emergency adverse events.

[0309] Treatment Administration: The study included a 28-day treatment period to evaluate both safety and re-epithelialization status (durability), followed by a post-treatment follow-up period of at least 28 days, both of which were double-blind (i.e., double-blind). Once determined eligible, subjects were randomized to receive either LUFEPIRSEN 0.06%, LUFEPIRSEN 0.006%, or a vehicle in a 1:1:1 ratio. At the following time points during the treatment period, the study product was applied under a bandage contact lens (BCL) and to the fornix of the subject's eye: ● Day 1 ● Day 2 ● Day 14 (If the cornea has not re-epithelialized)

[0310] Salvage administration: However, regardless of the investigational product assignment at randomization, if a subject's cornea did not fully re-epithelialize by day 28 of the treatment period, the subject was enrolled in the open-label period of the study and received up to two salvage doses of LUFEPIRSEN 0.06% applied on day 1 of salvage in the open-label section of the study, and, if re-epithelialization had not yet been achieved, on day 8 of salvage, applied to the lower BCL and both fornixes of the eye. Twelve subjects receiving salvage treatment were followed up to 14 days after the first salvage dose to determine if re-epithelialization had occurred (day 41), and then returned to more than 28 days to confirm that re-epithelialization was maintained (endurance).

[0311] For each subject, after any corneal re-epithelialization was first recorded, a 28-day masked treatment period was completed following up to three masked applications of the test product, or the subject entered the open-label section of the study as a result of unhealed PED on day 28.

[0312] Subjects who achieved re-epithelialization immediately began a 28-day post-healing follow-up period (no further investigational drugs were administered). The purpose of the post-epithelialization follow-up period was to evaluate the durability of the epithelium.

[0313] In subjects who achieved re-epithelialization but did not sustain it during the 28-day follow-up period after treatment, they were eligible to receive a salvage dose of LUFEPIRSEN 0.06% and enter the open-label section of the study (salvage day 1). If re-epithelialization was not achieved by salvage day 8 in the open-label section of the study, a second dose of LUFEPIRSEN 0.06% was administered. If re-epithelialization occurred within 14 days of the initial salvage dose, a final assessment of epithelial durability was performed at 28 days.

[0314] Participants were also terminated from the study if epithelialization was not achieved within 14 days of receiving a salvage dose of LUFEPIRSEN 0.06% in the open-label section of the study. In the NEX-PED-005 study, 12 subjects received salvage (high-dose open-label LUFEPIRSEN). Eleven subjects received one dose on day 28, and one subject received two doses (on days 28 and 35).

[0315] The maximum duration of study participation for each subject was 98 days (+2 days). This allowed for anticipated re-epithelialization on day 28 of the masked treatment period, enabling the initiation of the post-healing follow-up period. If durability did not persist after 28 days, the subject was eligible for a 0.06% LUFEPIRSEN salvage dose(s), and could participate in the open-label section of the study for an additional 41 days (14 days + 28 days (+2 days)). Table 1 shows the evaluation schedule for the visit schedule and the detailed procedures to be performed during each research visit.

[0316] Investigational product administration for randomized subjects: Each investigational product dose application contained a volume that filled the BCL (100 μL) and covered the upper and lower fornix surfaces (100 μL was injected per fornix). The eye was then covered with a double eye pad and taped closed for approximately 8 hours.

[0317] Standard local ocular care procedures were not permitted two hours prior to or eight hours after administration of the investigational drug. Outside of this period, subjects continued to receive the standard care regimen prescribed by the principal investigator.

[0318] Three treatment arms were evaluated in the study. Randomization was in a 1:1:1 ratio: ● Group A - LUFEPIRSEN 0.06% ● Group B - LUFEPIRSEN 0.006% ● Group C-Vehicle

[0319] The investigational drug was administered once on days 1 and 2. If re-epithelialization did not occur, a potential additional administration of the investigational drug was scheduled for day 14.

[0320] If re-epithelialization of the eyes in the study did not occur at the end of the 28-day masked treatment period, or if the durability of the initial re-epithelialization was not maintained, the subjects received up to two salvage doses of LUFEPIRSEN 0.06% in the open-label section of the study.

[0321] Study Description: Potentially eligible subjects presenting clinically non-infectious PED as a result of severe chemical and / or thermal injury resistant to standard care. Clinical signs of ocular injury include deepithelialization, conjunctivitis (conjunctivitis), limbal ischemia, and corneal edema.

[0322] Prior to consideration of an entry, the PED must have been present for at least 14 days, with the day of injury counted as the first day, and in the opinion of the principal investigator, the defect must not have shown clinically meaningful improvement in healing despite conventional standard care for at least 14 days. Conventional standard care includes antibiotics, steroid drops, ciliary muscle paralyzers, ascorbic acid, amniotic membrane placement, and debridement of necrotic epithelium.

[0323] The principal investigator or a qualified designated person obtained informed consent from participants before any procedures specific to the clinical trial protocol were performed. Eligibility assessments and baseline assessments were performed prior to day 1 of the treatment period, and final eligibility checks (such as confirmation of a negative urine pregnancy test in women of childbearing potential) and pre-treatment measurements of PED dimensions were performed before randomization of participants to the treatment. Eligibility assessments and day 1 visits may be combined at the discretion of the principal investigator if the participant's recorded medical history confirms that the epithelial defect has been present for more than 14 days and is unresponsive to the SOC. Visits may be combined assuming that the medical history is confirmed and the participant is experiencing logistical challenges, such as needing to travel a long distance to the site or due to transportation difficulties and restrictions as a result of the COVID-19 pandemic, and all necessary assessments prior to randomization were provided, recorded, and participant eligibility was confirmed.

[0324] The first administration of the investigational drug was performed on day 1 of the treatment period after randomization, and the second administration was administered on day 2. For each single administration, a BCL filled with 100 μL of the investigational product was immediately applied to the subject's eye, followed by 100 μL applied to the upper and lower fornix, respectively. The eye was then covered with a double eye pad and taped closed for approximately 8 hours. Importantly, subjects continued their prescribed SOC throughout the treatment period, except for 2 hours before and 8 hours after IP application.

[0325] Next, each subject visited the clinic twice a week for evaluation until corneal re-epithelialization occurred.

[0326] If re-epithelialization of the defect did not occur by day 14, another single dose of the masked investigational drug was administered.

[0327] If corneal re-epithelialization did not occur by day 28 of the masked treatment period, subjects received up to two salvage doses of LUFEPIRSEN 0.06% in the open-label section of the study. If re-epithelialization did not occur within 14 days of the first salvage dose of LUFEPIRSEN 0.06%, subjects exited the open-label section of the study.

[0328] If re-epithelialization occurred, regardless of the time frame within the masked treatment period, subjects entered a post-healing follow-up period and continued to wear BCL for two weeks to allow new epithelium to anchor to the basement membrane and to avoid premature traumatic detachment of the cell layer. To confirm epithelial durability, subjects were re-evaluated 28 days after the initial re-epithelialization. Subjects then completed all hospital visits and concluded the study.

[0329] If epithelial breakdown occurred within a 28-day post-treatment follow-up period after initial epithelialization, subjects received up to two salvage doses of LUFEPIRSEN 0.06%, administered at 7-day intervals, in an open-label section of the study.

[0330] If re-epithelialization of the cornea occurred within 14 days of the initial open-label salvage dose application, these subjects were also followed for 28 days to assess the persistence of re-epithelialization, but were analyzed outside of the masked study population. (See reference) Figure 1 shows that from the time of each study visit after randomization until the end of the treatment period, participants underwent evaluations detailed in the evaluation schedule. These evaluations included slit-lamp examination to assess the condition of the anterior segment of the eye (including the eyelids, sclera, conjunctiva, cornea, anterior chamber, and lens), photography, measurement of epithelial defects, visual acuity, intraocular pressure, and ocular symptom questionnaires. The occurrence of treatment-related adverse events was also evaluated at each study visit and up to 30 days after the final administration of the investigational drug.

[0331] Standard and open-label sections of care during the study's treatment period: From the start of day 1 to the end of the treatment period, subjects received the standard care regimen prescribed by the principal investigator at the study site, except for 2 hours before and 8 hours after administration of the investigational drug. Details of administered drugs, doses, and administration regimens were recorded in source documents and case report forms.

[0332] Bandage contact lenses (BCLs) for IP administration were provided by the organizer, worn during the treatment period, and replaced only when a new BCL was used for IP administration on the day of administration, or when indicated, for example, in case of discomfort or BCL dislodgement.

[0333] Systemic standard care was not defined by the protocol, but rather by the routine practice of the principal investigator or the research site. However, systemic administration and regimens for the treatment of PED remained unchanged throughout the study until re-epithelialization or end of the study.

[0334] The use of local anesthetic droplets was permitted only for ophthalmic evaluations performed at each research visit.

[0335] Selection Criteria: Participants were eligible to participate in the study only if all of the following criteria were met. 1. Men and women of any age. 2. Clinically non-infectious persistent corneal epithelial defect (PED) resulting from severe chemical and / or thermal eye injury to one or both eyes. 3. A record that the PED has not responded to current standard care for at least 14 days, with day 1 being the day of injury. 4. Epithelial defect measuring at least 2 mm along its maximum diameter on day 1 of the treatment period. 5. To provide written informed consent and the ability to adhere to the hospital visit and administration schedule.

[0336] Analysis was performed when blinding was lifted and 35 randomized subjects (LUFEPIRSEN 0.06% (n=12), LUFEPIRSEN 0.006% (n=12)) completed the study. Using the full dataset, 66.7% (8 out of 12) of the LUFEPIRSEN 0.06% subjects and 66.7% (8 out of 12) of the LUFEPIRSEN 0.006% subjects achieved corneal epithelial recovery. In contrast, only 27.3% (3 out of 11) of the vehicle subjects achieved recovery. Each of these three subjects who recovered received only two doses of the vehicle on days 1 and 2, while the three subjects who did not recover also received two doses, and the remaining non-recovering vehicle cohort subjects received three doses of the vehicle.

[0337] Therefore, subjects with non-healing ocular surface defects treated with the connexin modulator LUFEPIRSEN (n=24) had nearly 2.5 times (2.44 times) greater likelihood of healing at the time of connexin modulator application and showed a 39.4% improvement in corneal epithelial recovery compared to the VEH group (n=11). These findings were clinically significant and approached statistical significance despite the small sample size (Fisher's exact test: p=0.065). In general, the treatment was well tolerated, and TEAEs across all three treatment groups were similarly characterized as being mild to moderate in severity and not thought to be related to / unrelated to the study drug. SAEs that did not appear to be related to the study drug were reported.

[0338] These data from randomized, prospective, vehicle-controlled, double-blind, human clinical trials for treating non-healing ocular surface defects support the use of connexin modulators (e.g., LUFEPIRSEN) in dose regimens described herein using the doses described herein, or in dose regimens described herein using different therapeutically effective doses, for the sustained healing of non-healing or persistent ocular surface defects, including persistent corneal epithelial defects. The data establish both safety and clinical proof of concept for the doses and dose regimens of the proprietary connexin modulators described herein and claimed herein, and confirm their usefulness in the treatment management of patients with non-healing ocular surface or corneal defects or disorders. *****

[0339] All patents, publications, scientific papers, websites, and other documents and materials referenced or mentioned herein represent the level of skill of those skilled in the art to which the present invention relates, and each such referenced document and material is incorporated by reference either individually or in whole herein to the same extent as if it were included herein. The applicant reserves the right to physically incorporate into this specification any and all materials and information from such patents, publications, scientific papers, websites, electronically available information, and other referenced materials or documents. Any reference herein to any application, patent and publication is not, and should not be construed as, an acknowledgment or suggestion in any form that they constitute valid prior art or form part of common general knowledge in any country of the world.

[0340] The specific methods and compositions described herein are representative of preferred embodiments and are illustrative; they are not intended to limit the scope of the invention. Other purposes, aspects, and embodiments will be conceivable to those skilled in the art in consideration of this specification and will fall within the spirit of the invention as defined by the claims. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described exemplary herein may be adequately carried out without any one or more elements or limitations, which are not specifically disclosed herein as essential. Accordingly, for example, in each example herein, any of the terms “comprising,” “consisting essentially of,” and “consisting of” in embodiments or examples of the invention may be replaced with any of the other two terms herein. Furthermore, terms such as “comprising,” “including,” and “containing” should be read broadly and without limitation. The methods and processes described herein as exemplary may be appropriately carried out in a different order of steps, and are not necessarily limited to the order of steps set forth herein or in the claims. Furthermore, where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Under no circumstances shall the patent be construed as being limited to any specific example or embodiment or method specifically disclosed herein. Under no circumstances shall the patent be construed as being limited by any statement made by any examiner or other officer or employee of the Patent and Trademark Office, except where such statement is specifically and expressly adopted in the applicant’s written response without qualification or reservation. Furthermore, titles or headings, etc., are provided to enhance the reader’s understanding of this document and should not be read as limiting the scope of the invention.Any examples of aspects, embodiments, or components of the present invention referenced herein should be considered non-limiting.

[0341] The terms and expressions used are for illustrative purposes only, not limitation, and in using such terms and expressions there is no intention to exclude equivalents of the exhibited and described features or any part thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, although the invention is specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art, and it will be understood that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0342] The present invention is broadly and generally described herein. Each of the narrower groups of species and subgenera included in the general disclosure also forms part of the present invention. This includes the general description of the invention with conditions or negative limitations of excluding any subject from a genus, whether or not the excised material is specifically described herein.

[0343] Other embodiments are within the scope of the following claims. Furthermore, whereever a feature or embodiment of the present invention is described in relation to the Markush group, a person skilled in the art will recognize that the present invention is also described in relation to any individual member or subgroup of a member of the Markush group.

Claims

1. A method for treating a subject for a non-healing ocular surface defect, comprising administering a therapeutically effective dose of a connexin modulator to the subject, wherein the connexin modulator is administered at least once on day 1, at least once on day 2, and at least once on about day 14.

2. The method according to claim 1, wherein the non-healing ocular surface defect is a persistent corneal epithelial defect or a persistent epithelial defect of the eye.

3. The method according to claim 1, wherein the non-healing ocular surface defect is caused by chemical damage, thermal damage, or inflammation.

4. The method according to claim 1, wherein the subject has an amniotic membrane graft.

5. The method according to claim 1, wherein the connexin modulator includes a connexin 43 modulator.

6. The method according to claim 1, wherein the connexin modulator comprises a connexin 43 antisense compound.

7. The method according to claim 6, wherein the antisense compound comprises lufeprirsen (SEQ ID NO: 1).

8. The method according to claim 6, wherein the antisense compound targets at least about eight nucleic acid bases of a nucleic acid molecule encoding a connexin having a nucleic acid base sequence according to Sequence ID No. 17, and has a nucleic acid base length of 15 to 35.

9. The method according to claim 8, wherein the antisense compound is a modified antisense oligonucleotide, and the modified antisense oligonucleotide comprises at least one modification selected from the group consisting of a modified internucleoside bond including a phosphorothioate bond, a modified sugar moiety, and a modified nucleic acid base.

10. The method according to claim 1, wherein the antisense compound connexin modulator is a modulator of corneal epithelial connexin.

11. The method according to claim 7, wherein the antisense compound is selected from the group consisting of 0.06% lufeprisen and 0.006% lufeprisen.

12. The method according to claim 7, wherein the antisense compound comprises at least about 0.006% lufeprisen or at least about 0.06% lufeprisen.

13. The method according to claim 1, wherein the connexin modulator is administered by local administration, oral administration, or by injection.

14. The method according to claim 1, further comprising the step of placing a bandage contact lens on the non-healing ocular surface defect.

15. The method according to claim 6, wherein the antisense compound is formulated together with Pluronic® gel.

16. The method according to claim 1, wherein the connexin modulator is a connexin 43 peptide mimetic.

17. The method according to claim 16, wherein the connexin peptide mimetic is selected from the group consisting of Peptide 5, Gap 19, Gap 20, Gap 22, XG 19, and CT peptide.

18. The method according to claim 1, wherein the connexin modulator is a connexin 43 connexin hemichannel modulator.

19. The method according to claim 18, wherein the connexin hemichannel modulator is a compound according to formula I or formula II.

20. The method according to claim 26, wherein the Connexin hemichannel modulator includes Tonaversat.

21. A method for treating a subject with persistent corneal epithelial defect, comprising administering to the subject a composition containing about 0.06% or about 0.006% lufepirsen on day 1, day 2, and about day 14.

22. The method according to claim 21, further comprising administering to the subject a composition containing about 0.06% or about 0.006% lufeprisen on about 7 days and / or about 21 days.

23. The method according to claim 21, further comprising administering to the subject a composition containing about 0.06% or about 0.006% lufeprisen on about 28 days and / or about 35 days.

24. The method according to claim 22, further comprising administering to the subject a composition containing about 0.06% or about 0.006% lufeprisen on about 28 days and / or about 35 days.

25. A pharmaceutical composition comprising approximately 0.6 or 0.06 mg / mL of lufeprisen, approximately 226.9 mg / mL of poloxamer 407, approximately 0.99 mg / mL of dibasic sodium phosphate heptahydrate, and approximately 0.25 mg / mL of monobasic potassium phosphate.

26. The pharmaceutical composition according to claim 39, having a final volume of approximately 300 μL.

27. A method for treating a subject with a non-healing ocular surface defect, comprising administering the pharmaceutical composition according to claim 26 to the subject's eye at least once on day 1, at least once on day 2, and at least once on about 14 days.

28. The method according to claim 27, further comprising administering the pharmaceutical composition to the eye of the subject on approximately the 7th day and approximately the 21st day.

29. The method according to claim 28, further comprising administering the pharmaceutical composition to the eye of the subject on approximately the 28th day.

30. The method according to claim 28, further comprising administering the pharmaceutical composition to the eye of the subject on approximately the 35th day.

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