New treatment of ocular conditions

Abstract

The present invention relates to compounds and compositions comprising connexin modulators, and their use in dosage regimens for the treatment of non-healing or persistent ocular surface or corneal defects and disorders.

Description

Related applications

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

[0002] This application contains a sequence list electronically submitted in XML format and incorporated herein by reference in its entirety. The XML copy was created on March 7, 2024, named H7475-00103_SL.xml, and is 215,985 bytes in size. Technical Field

[0003] This invention relates to connexin modulators and the regulation of ocular surface defects or diseases. Incorporation

[0004] All publications, patents, related applications, and other written or electronic materials mentioned, identified, or cited herein, including every U.S. patent, U.S. patent application publication, non-U.S. patent, non-U.S. patent application, and PCT publication, articles, and other documents cited or referenced herein, and all material listed as cited in any patent granted herein, are incorporated herein by reference in their entirety. The incorporated information has the same effect as any part of this application and all patents for which it is granted or claims priority, and will be considered part of the text and content of this application at the time of filing, and of any patent for which it is granted or claims priority, as if all text and other content were repeated in this application or patent; and any portion of any material incorporated by reference may be incorporated herein by modification if necessary. In the event of any inconsistency in terminology between this document and any document incorporated by reference, the terminology of the incorporated reference shall be considered supplementary to the terminology of this document; in the event of irreconcilable inconsistencies (including definitions appearing in patents or patent applications), the terminology of this document shall prevail. Background Technology

[0005] The following contains information that may help in understanding the invention. This does not imply that any information, publications, or documents specifically or implicitly referenced herein are prior art to or important to the invention described and claimed herein.

[0006] Gap junctions are specialized intercellular connections that exist between most animal cell types. They are expressed in almost all tissues of the body, except for mature skeletal muscle and mobile cell types such as sperm and erythrocytes, and enable regulated physical communication between cells by directly connecting the interiors of adjacent cells, allowing a variety of molecules, ions, and electrical impulses to pass through directly.

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

[0008] The human connexin family of genes and proteins currently comprises 21 members. Their molecular weights typically range from 25 to 60 kDa, with an average length of 380 amino acids. All connexins share a common structure as four-transmembrane (TM) proteins, which contains multiple domains: a short intracellular N-terminus (NT), an intracellular loop (IL), and a C-terminus (CT) (also located in the cytoplasm), and two extracellular loops (EL1 and EL2) (located extracellularly). The length of the cytoplasmic C-terminus can vary significantly. See, for example, Unger et al. . (1999) Electron cryo-crystallography of a recombinantcardiac 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 tales. Biochimica et Biophysica Acta Vol. 1860(1):48-64 (Jan 2018).

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

[0010] Hereditary or acquired alterations in the structure and function of connexins are associated with a variety of diseases. See, for example, Delmar, M, Laird, DW, et al., Connexins and Disease. Cold Spring Harb Perspective Biol10:a029348 (2018); DW Laird and PD Lampe, Cellular mechanisms of connexin-basedinherited diseases. Trends in Cell Biology Vol. 32, Issue 1, p58-69 (Jan2022).

[0011] The study also found that connexins are associated with a variety of conditions and symptoms. See, for example, Willebrords, 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 inburns 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 Junctions in Perinatal Brain Injury. Biomedicines 10:1445 (2022). Connexins have been proposed as therapeutic targets for a variety of conditions, including spinal cord injury, perinatal brain injury, neurological disorders (e.g., Alzheimer's disease, Parkinson's disease), heart diseases (e.g., myocardial infarction), eye conditions (e.g., age-related macular degeneration, diabetic macular edema), acute and chronic wounds (e.g., venous leg ulcers, diabetic foot ulcers), ischemia-reperfusion injury, inflammation, burns, and cancer. See Laird and Lampe, Therapeutic strategies targeting connexins, for a review. Nat Rev Drug Discov . 17(12): 905-921 (Dec2018); 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. Biol50, 49-58 (2016). See also: FEBS Letters Articles in the journal *Connexins* (Vol. 588, No. 8, pp. 1185–1490) (April 17, 2014) include 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. Patent Nos. 10,401,188 and 11,401,516 are both authorized as “channel modulators”.

[0012] A review of treatment strategies for eye wounds is presented in Ziaei M et al., Wound healing in the eye: Therapeutic prospects. Advanced Drug Delivery Reviews 126 (2018) 162-176. Among other things, Ziaei et al. discussed “traditional” treatment strategies, including, for example, (1) steroids and (2) contact lenses, as well as a variety of “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) thymosin β4, (6) rebamipide, (7) stem cells and other blood-derived products, and (8) gap junction hemichannel regulation. See U.S. Patent No. 8,034,789, granted October 11, 2011, “Antisense Compounds Targeting Connexin Proteins and Methods of Using Them Therewith,” and 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 (The potential for Cx43 modulation was explored in 5 compassionate users, but the possibility of a “mediator effect” was not ruled out). Recently, studies have proposed the use of (9) topical insulin eye drops in clinical practice to treat persistent epithelial defects (PED) of the eye that are unresponsive to conventional treatment. See 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 suggested treatments include (10) punctal plugs, (11) cyanoacrylate glue, (13) debridement, and (14) fibronectin sutures (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 Ziaei et al. have stated, many existing and emerging therapies for ocular surface wound healing remain largely experimental or conceptual. For example, the application of gap junction modulators in the human eye has not been clinically established, and there are no established doses, dosing regimens, or methods for modulating connexins, connexin gap junctions, and / or connexin hemichannels to treat non-healing or persistent ocular or corneal surface defects or conditions.

[0014] Such methods, dosages, and dosing regimens are described and claimed herein, and provide a long-needed means for treating subjects with non-healing or persistent ocular surface defects or conditions, as well as other ocular diseases, conditions, and ailments described herein.

[0015] Unlike the treatments described and claimed in this article, most current treatments for these eye diseases, conditions, and ailments do not address their underlying causes (which often include uncontrolled inflammation), and therefore have limited success rates. Summary of the Invention

[0016] The invention described and claimed herein has many features and embodiments, including but not limited to those features and embodiments set forth, described or referenced in this description. It is not intended to cover all aspects, and the invention described and claimed herein is not limited to the features or embodiments mentioned in this introduction, which are included for illustrative purposes only and not for limiting purposes.

[0017] The object of this invention is to provide methods, dosages, dosing regimens, compositions, and kits for modulating connexins to treat non-healing or persistent ocular surface or corneal defects or conditions. In some embodiments, compounds or compositions are provided for modulating or inhibiting connexins, connexin gap junctions, and / or connexin hemichannels for treatment and administration as described herein. In some embodiments, the non-healing ocular surface defect or condition is a persistent epithelial defect (PED). In some embodiments, the non-healing ocular surface defect or condition is a persistent corneal epithelial defect (PCED). In some embodiments, the subject is a human.

[0018] In some embodiments, connexin regulators modulate or inhibit connexin expression. In some embodiments, connexin regulators inhibit or modulate the expression of connexin 43.

[0019] In some embodiments, the connexin regulator is a connexin gap junction regulator. In some embodiments, the connexin gap junction regulator modulates the gap junction of connexin 43.

[0020] In some embodiments, the connexin regulator is a connexin hemichannel regulator. In some embodiments, the connexin hemichannel regulator modulates the connexin 43 hemichannel. Hemichannel regulation can occur in any manner. For example, in some embodiments, regulation can occur by: inducing or promoting hemichannel closure; preventing, blocking, inhibiting, or reducing hemichannel opening; inhibiting hemichannel permeability; inhibiting ATP release from the hemichannel; and / or triggering, inducing, or promoting the internalization of the hemichannel and / or gap junctions. Hemichannel regulators include blockers and other compounds that interfere with the passage of molecules through connexin hemichannels. Hemichannel regulators can block or reduce the release of molecules through the hemichannel into the extracellular space, and / or block or reduce the entry of molecules through the hemichannel into the intracellular space. In some embodiments, the hemichannel regulator completely or partially blocks hemichannel opening. In some embodiments, the hemichannel regulator completely or partially blocks, slows down, or inhibits the leakage or transfer of molecules through the hemichannel into or from the extracellular space. In some embodiments, the hemichannel regulator is a compound that reduces the probability of hemichannel opening.

[0021] In some embodiments of the invention, the modulation of gap junctions and hemichannels is achieved by applying connexin expression regulators, connexin peptides, and / or small molecule connexin regulators, which modulate corneal epithelial connexins, corneal epithelial connexin gap junctions, or corneal epithelial connexin hemichannels according to the dosage regimens disclosed herein. In some embodiments of the invention, the modulation of gap junctions and hemichannels is achieved by applying connexin expression regulators, connexin peptides, and / or small molecule connexin regulators, which modulate corneal vascular connexins according to the dosage regimens disclosed herein. In some embodiments of the invention, the modulation of gap junctions and hemichannels, according to the dosage regimens disclosed herein, is achieved by applying connexin expression regulators, connexin peptides, and / or small molecule connexin regulators, which modulate (e.g., reduce, slow, inhibit, or eliminate) inflammation.

[0022] In some embodiments, the connexin modulators, connexin gap junction modulators, and / or connexin hemichannel modulators used in the method of the present invention modulate one or more connexins in the 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 regulator comprises an antisense molecule. In some embodiments, the antisense molecule is a connexin 43 antisense oligonucleotide. In some embodiments, the connexin regulator may be a connexin 43 antisense polynucleotide comprising, substantially comprising, or consisting of the sequence according to SEQ ID NO. 1-16 and / or its modified form. In some embodiments, the antisense oligonucleotide comprises, substantially comprises, 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 a chemically modified oligonucleotide or may be an unmodified oligonucleotide, such as a modified or unmodified DNA oligonucleotide.

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

[0025] In some embodiments, the connexin regulator comprises a small-molecule connexin, a connexin gap junction, or a connexin hemichannel regulator. In some embodiments, the small-molecule connexin regulator inhibits or blocks the opening of connexin hemichannels. In some embodiments, the small-molecule connexin regulator regulates the release of ATP from connexin hemichannels. In some embodiments, the small-molecule connexin regulator inhibits or blocks the opening of Cx43 hemichannels and regulates the release of ATP from Cx43 hemichannels. In some embodiments, the small-molecule connexin regulator is a compound according to Formula I (e.g., tonabersat). In some embodiments, the small-molecule connexin regulator is a tonabersat prodrug compound according to Formula II.

[0026] In some embodiments, the present invention relates to a method of modulating connexins, gap junction channels, and / or hemichannels using a therapeutically effective amount of a connexin modulator according to the dosage regimen described herein. In some embodiments, a therapeutically effective amount of a connexin antisense compound (e.g., lufifenesin) is administered. In some embodiments, a therapeutically effective amount of a connexin peptide compound (e.g., Peptide5, Gap19, XG19, CXT1 to CXT5, Antp / CXT1 to Antp / CXT5, etc.) is administered. In some embodiments, a therapeutically effective amount of a small molecule connexin hemichannel blocking compound (e.g., tonaboxa or tonaboxa prodrug) is administered.

[0027] In some embodiments, the present invention relates to a method for modulating connective proteins, gap junction channels, and / or hemichannels in subjects suffering from non-healing or persistent ocular surface or corneal defects or conditions (or both), the method using novel specific doses of connective protein modulators, gap junction modulators, and / or hemichannel modulators, and newly discovered and clinically proven dosing regimens to promote ocular surface recovery. Non-healing ocular surface or corneal defects or conditions treated using the doses, compositions, dosing regimens, and methods of the present invention include persistent epithelial defects (PED) and persistent corneal epithelial defects (PCED). These include PED and PCED caused by any of the following reasons: chemical damage, thermal damage, disease, and inflammation.

[0028] In some embodiments, the non-healing ocular surface defect or condition is caused by chemical and / or thermal injury. In some embodiments, the non-healing ocular surface defect or condition is caused by physical trauma or injury. In some embodiments, the non-healing ocular surface defect or condition is PED or PCED caused by chemical and / or thermal injury. In some embodiments, the chemical and / or thermal injury leading to PED or PCED is severe chemical and / or thermal injury. In some embodiments, severe ocular surface and / or corneal burns and injuries are cases where the conjunctival involvement exceeds 50% or the limbal involvement exceeds 6 o'clock positions. These include ocular surface and corneal burns and injuries with a Dua classification scale ≥3. In some embodiments, the non-healing ocular surface or corneal defect or condition is caused by inflammation or inflammatory disease. In some embodiments, the non-healing ocular surface or corneal defect or condition is caused by ocular inflammation. In some embodiments, the non-healing ocular surface or corneal defect or condition is caused by inflammatory disease of the eye, ocular surface, or cornea.

[0029] All causes of PED and PCED, regardless of their specific etiology, can be treated with the methods and compositions of the present invention described herein. The causes of PED and PCED include not only inflammation and inflammatory diseases, neurotrophic diseases, and neurotrophic keratitis, but also chemical, mechanical, thermal damage, and epithelial / limbic stem cell deficiency. In some embodiments, the methods, compositions, and / or dosages of the present invention are used to treat PED and / or PCED caused or resulting from chemical damage. In some embodiments, the methods, compositions, and / or dosages of the present invention are used to treat PED and / or PCED caused or resulting from thermal damage. In some embodiments, the methods and / or dosages of the present invention are used to treat PED and / or PCED caused or resulting from mechanical damage. In some embodiments, the methods and / or dosages of the present invention are used to treat PED and / or PCED caused or resulting from inflammation 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 or resulting from neurotrophic diseases (e.g., neurotrophic keratitis).

[0030] In some embodiments, the non-healing ocular surface defect or condition is an ocular surface ulcer. In some embodiments, the non-healing ocular surface defect or condition 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 caused by Acanthamoeba keratitis, fungal keratitis, or herpes simplex keratitis. In other embodiments, the ocular surface ulcer or corneal ulcer is caused by trauma (such as abrasions, scratches, or cuts) or an intraocular foreign body. In another embodiment, the ocular surface ulcer or corneal ulcer is caused by allergic eye disease. In another embodiment, the ocular surface ulcer or corneal ulcer is caused by dry eye syndrome. In some embodiments, the non-healing ocular surface ulcer or corneal ulcer is caused by or resulting from inflammation or an inflammatory disease, condition, or illness. In some embodiments, the ocular surface ulcer or corneal ulcer is caused by other causes (e.g., injury or burn).

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

[0032] In some embodiments, in the treatment regimens for non-healing surface defects or conditions described and claimed herein, a single dose of the connexin modulator is administered (e.g., a single dose administered on day 1, day 2, day 14, etc., as well as in other dosing regimens described herein). In some embodiments, in the dosing regimens of the present invention, for administration, the daily dose of the connexin modulator is administered in fractional form (e.g., half a dose, administered twice daily to the subject on day 1, day 2, day 14, etc.). In some embodiments, a single dose or fractional dose of the connexin modulator is administered over one or more or all of the days of each dosing regimen described herein.

[0033] In some embodiments of the invention, a method of treating a subject with non-healing ocular surface defects includes administering a therapeutically effective amount 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. 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 a loufi derivative. In some embodiments, the loufi derivative is administered in the form of a composition comprising a nonionic polyoxyethylene-polyoxypropylene block copolymer carrier (e.g., poloxamer F-127). In some embodiments, the loufi derivative is administered in the form of a composition comprising a cellulose carrier (e.g., hydroxyethyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, etc.) or other pharmaceutically acceptable carriers.

[0034] In some embodiments, three to four doses of a connexin modulator (e.g., rufi-derived) are administered on days 1, 2, and 14 (and optionally, on or about day 28). In other embodiments, four to five doses are administered, wherein a single or multiple dose of the connexin modulator (e.g., rufi-derived or other connexin antisense molecules or modulators) is administered on about day 35, in addition to days 1, 2, and 14 (or in addition to days 1, 2, 14, and about day 28). In some embodiments of these dosing regimens, multiple doses of the connexin modulator (e.g., rufi-derived) may also be administered on about day 7 and / or day 21. In some embodiments, the connexin modulator dose (e.g., rufi-derived) is administered on days 1, 2, 14, and 28.

[0035] In some implementations, the dose of the connective protein modulator (e.g., rufi derivative) is administered on days 1, 2, 14, and 21.

[0036] In some implementations, the dose of the connective protein modulator (e.g., rofi derivative) is administered on days 1, 2, 7, 14, and 21.

[0037] In some implementations, the dose of the connective protein modulator (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, and 28.

[0038] In some embodiments, the connexin modulator dose (e.g., rufi derivative) is administered on days 1, 2, 14, 28, and 35. In some embodiments, the connexin modulator dose (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 28, and 35. In some embodiments, the connexin modulator dose (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, the connexin modulator dose (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, in any of these dosing regimens, a dose may also be administered after day 35. In some embodiments, administration is to the PED or PCED. In some embodiments, the connexin modulator comprises a connexin antisense molecule (e.g., rufi derivative). In some embodiments, the connexin modulator comprises connexin peptides (e.g., Peptide5, Gap19, XG19, aCT1, etc.). In some embodiments, the connexin modulator comprises a small molecule connexin hemichannel blocker (e.g., tonaboxa). In some embodiments, the connexin modulator is a connexin 43 modulator. In some embodiments, administration is to the PED or PCED. On any administration day, the connexin modulator may be administered as a single dose or in multiple doses.

[0039] In some embodiments, a connexin modulator (e.g., rufi derivative) is administered three times over 14 days to treat a subject with non-healing or persistent ocular or corneal surface defects, including, for example, administration on days 1 and 2. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered four times over approximately 28 days. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered five times over approximately 28 to approximately 35 days. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered six to seven times over approximately 35 days. For example, in one embodiment, the connexin modulator (e.g., rufi derivative) is administered four times on days 1, 2, approximately 14, and approximately 28. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered five times on days 1, 2, approximately 14, approximately 28, and approximately 35. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered five times on days 1, 2, approximately 7, approximately 14, and approximately 28. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered six times on days 1, 2, approximately 7, approximately 14, approximately 21, and approximately 28. In yet another embodiment, the connexin modulator (e.g., rufi derivative) is administered seven times on days 1, 2, approximately 7, approximately 14, approximately 21, and approximately 28. Of course, "administer" as used herein refers to a single dose or multiple doses of the connexin modulator. In some embodiments of the invention, modulation of connexins, gap junctions, and / or hemichannels is achieved by administering a connexin antisense oligonucleotide (e.g., rufi derivative) to the eye of a subject suffering from non-healing or persistent ocular surface or corneal defects or conditions (e.g., PED or PCED).

[0040] In some implementations, in any of the dosage regimens described herein (e.g., days 1, 2, and 14; days 1, 2, 14, and optionally, day 28; days 1, 2, 7, 14, 21, and optionally, day 28; etc.), the amount of connective modulator administered to a subject with a non-healing ocular surface defect or condition (e.g., PED) or a non-healing corneal surface defect or condition (e.g., PCED) is a therapeutically effective amount.

[0041] In some embodiments, in any of the dosing regimens described herein, the amount of connexin modulator administered to a subject with a non-healing ocular surface defect or condition (e.g., PED) or a non-healing corneal surface defect or condition (e.g., PCED) is one or more of the doses described herein (e.g., mg / mL connexin modulator dose, μM connexin modulator concentration dose, mg connexin modulator dose, etc.). For example, the specific dose described herein may be administered to a subject with a non-healing ocular surface defect or condition (e.g., PED) or a non-healing corneal surface defect or condition (e.g., PCED) in the following dosage regimens: day 1, day 2, day 14 and optionally day 28 and / or day 35; day 1, day 2, day 14 and optionally day 7, day 21 and / or day 28; etc., or in any other dosing regimen described herein.

[0042] In some embodiments of the method of the present invention, the connexin modulator applied to the non-healing surface defect or lesion of the subject is 0.06% by weight of rufi derivative. In some embodiments, the connexin modulator applied to the subject suffering from a non-healing surface defect or lesion is 0.006% by weight of rufi derivative. In some embodiments, the applied connexin modulator is at least about 0.06% by weight of rufi derivative. In some embodiments, the connexin modulator applied to the subject suffering from a non-healing surface defect or lesion is at least about 0.006% by weight of rufi derivative. In some embodiments, the application is to a PED or PCED. In some embodiments, the application comprises or substantially comprises a pharmaceutically acceptable carrier and about 0.06% by weight of rufi derivative, about 0.006% by weight of rufi derivative, at least about 0.06% by weight of rufi derivative, or at least about 0.006% by weight of rufi derivative to heal the non-healing surface defect or lesion of the subject.

[0043] In some embodiments of the invention, a composition containing 0.6 mg / mL leucovorin is used to treat non-healing surface defects or conditions. In some embodiments of the invention, a composition containing 0.06 mg / mL leucovorin is used to treat non-healing surface defects or conditions. In some embodiments, the composition contains at least about 0.6 mg / mL leucovorin. In some embodiments, the composition contains at least about 0.06 mg / mL leucovorin. In some embodiments, administration is to a PED or PCED.

[0044] In some embodiments of the invention, according to the scheme described herein, a composition comprising or consisting essentially of 0.18 mg of rofi derivative is used to treat a subject's non-healing surface defect or condition. In some embodiments, according to the scheme described herein, each dose of rofi derivative administered to a subject's non-healing surface defect or condition contains at least about 0.18 mg of rofi derivative, or consists essentially of at least about 0.18 mg of rofi derivative. In some embodiments of the invention, according to the scheme described herein, a composition comprising or consisting essentially of 0.018 mg of rofi derivative is used to treat a non-healing surface defect or condition. In some embodiments, according to the scheme described herein, each dose of rofi derivative administered to a non-healing surface defect or condition contains at least about 0.018 mg of rofi derivative, or consists essentially of at least about 0.018 mg of rofi derivative. In some embodiments, administration is to a PED or PCED.

[0045] In some implementations, about 0.18 mg of rofidogen (or other connexin antisense molecules or connexin modulators), about 0.018 mg of rofidogen (or other connexin antisense molecules or connexin modulators), or about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or about 0.5 mg to about 1 mg of rofidogen (or another connexin antisense molecule or other connexin modulator) may be administered as a single dose or in two or more divided doses, such as twice daily for up to six doses administered on day 1, day 2, day 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 rufi derivative (or another connexin antisense molecule or other connexin modulator) is administered to the non-healing surface defect or condition as a single dose or in two or more fractionated doses, such as twice daily for up to 6 to 10 doses administered on days 1, 2, and 14, or using other dosing 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 rufi derivative (or another connexin antisense molecule or other connexin modulator) is administered as a single dose or in fractionated doses, such as twice daily for up to 14 doses administered on days 1, 2, and 14, and on one or more days, such as day 7, 21, 28, and / or day 35. In some embodiments, administration is to the subject's PED or PCED. In some embodiments, the connexin antisense molecule administered to a subject in the amounts described herein comprises or substantially comprises the antisense compound according to the sequence shown in SEQ ID NO:2-16. In some embodiments, the connexin antisense molecule administered to a subject in the amounts and on the dates described herein comprises or comprises another connexin 43 antisense compound.

[0046] In any embodiment of these dosages, dosing regimens, and methods, the luffi-derived dose may be administered to the subject's PED or PCED. In some embodiments of these dosages, dosing regimens, and methods, the administered connexin modulator dose comprises another connexin antisense molecule or modulator, such as another Cx43 antisense molecule or Cx43 modulator other than (or included in addition to) the luffi-derived dose, or a modulator of another ocular surface or corneal epithelial connexin other than Cx43. In some embodiments, the connexin antisense molecule administered to the subject in the amounts and on the dates described herein comprises or substantially comprises an antisense compound for modulating the expression of one or more connexins selected from 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 invention, the volume of the composition comprising or substantially consisting of a connexin modulator applied to the non-healing surface defect or lesion of the subject is about 300 μL. In other embodiments, the volume of the composition applied 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 lufifine derivative, or about 0.18 mg, 0.2 mg, or 0.5 mg to about 1 mg of lufifine derivative. In some embodiments, the composition comprises at least about 0.018 mg of lufifine derivative, at least about 0.18 mg of lufifine derivative, at least about 0.5 mg of lufifine derivative, or at least about 1.0 mg of lufifine derivative. In some embodiments, instead of or excluding ruby-derived compounds, 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, 0.2 mg, or 0.5 mg to about 1 mg of another connexin 43 antisense compound (or other connexin modulators, including, for example, other modulators of connexin 43 or other ocular surface or corneal epithelial connexins, peptide-like substances, small molecule connexin modulators). In some embodiments, instead of or excluding ruby-derived compounds, 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 molecule or other connexin modulator (e.g., other modulators of connexin 43 or other ocular surface or corneal epithelial connexins, peptide-like substances, and / or small molecule connexin modulators).

[0048] In some embodiments, the composition used in the method of the present invention comprises a reverse thermosetting gel. In some embodiments, the composition used in the method of the present invention comprises Pronic gel, also known as poloxamer gel. In some embodiments, the composition used in the method of the present invention comprises poloxamer F-127 (poloxamer 407 or Pronic F-127). In some embodiments, the composition used in the method of the present invention comprises about 20-30 mg / mL of poloxamer F-127. In some embodiments, the composition used in the method of the present invention comprises about 22-25 mg / mL of poloxamer F-127. In some embodiments, the composition used 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 a luffi derivative in the form of a composition comprising Prönnick F-127, one or more buffers, and water for injection. The buffer contained in the luffi derivative composition may include disodium hydrogen phosphate heptahydrate or potassium dihydrogen phosphate (or both). In some embodiments, the composition comprises 0.06% or 0.006% luffi derivative, or at least about 0.06% or at least about 0.006% luffi derivative. In some embodiments, the buffer composition comprises 0.6 mg / mL or 0.06 mg / mL luffi derivative, or at least about 0.6 mg / mL or at least about 0.06 mg / mL luffi derivative. In some embodiments, about 300 mL of the luffi derivative composition is administered to the non-healing surface lesion on days 1, 2, and 14. In some embodiments, if necessary or required, about 300 μL of the luffi derivative dose is administered again to the subject with the non-healing surface lesion on about day 28. In some embodiments, if necessary or required, approximately 300 μL of the luffi-derived dose is administered again to the subject with the non-healing surface lesion on approximately day 35. In other embodiments, if deemed necessary or required, approximately 300 μL of the luffi-derived composition is also administered again to the subject with the non-healing surface lesion on day 7 and / or approximately day 21. In some embodiments, the non-healing surface lesion treated with the composition is PED or PCED. In some embodiments, PED or PCED is caused by chemical and / or thermal injury or other trauma. In some embodiments, approximately 300 μL to 1.0 mL of the luffi-derived composition is administered to the non-healing surface lesion. In some embodiments, the connexin modulator administered in the composition comprises another connexin 43 antisense compound. In some embodiments, the connexin modulator administered in the composition comprises an antisense compound targeting another ocular surface or corneal epithelial connexin. In some embodiments, the connexin modulator applied in the composition comprises another connexin 43 antisense compound, an antisense compound targeting another ocular surface or corneal epithelial connexin, and / or another connexin modulator (e.g., a peptide or small molecule connexin modulator).

[0050] In some embodiments of the invention, modulation of gap junctions and hemichannels is achieved by applying a small molecule hemichannel blocker that blocks or modulates the opening of corneal epithelial connexin hemichannels. In some embodiments, modulation of corneal epithelial hemichannels is achieved by topical, oral, or systemic application of the small molecule hemichannel blocker, thereby treating non-healing ocular surface defects. In some embodiments, a therapeutically effective amount of the small molecule is administered according to the dosage regimen described herein. In some embodiments, the dose of the small molecule administered according to the dosage regimen described herein is one or more of the doses described herein. In some embodiments, the small molecule hemichannel blocker is tonabothia.

[0051] In any embodiment of the invention, a connexin modulator may be applied topically. In some embodiments, a connexin antisense modulator (e.g., rofigen). In some embodiments, a connexin peptide-like connexin modulator (e.g., XG19). In some embodiments, a small molecule connexin modulator (e.g., tonaboxa). In some embodiments, a combination of one or more compounds selected from the group consisting of a connexin antisense modulator (e.g., rofigen), a connexin peptide-like connexin modulator (e.g., XG19), and a small molecule connexin modulator (e.g., tonaboxa). In some embodiments, one or more compounds selected from the group consisting of a connexin antisense modulator (e.g., rofigen), a connexin peptide-like connexin modulator (e.g., XG19), and a small molecule connexin modulator (e.g., tonaboxa), and one or more other therapeutic agents are applied topically. In some embodiments, one or more other therapeutic agents are selected from antibiotics, antiviral agents, antiparasitic agents, antifungal agents, steroids, anti-inflammatory drugs, and immunosuppressants. In some implementations, one or more other therapeutic agents are selected from pan-connector protein 1 (Panx1) inhibitors (e.g., probenecid, etc.). 10 Inhibitors of Panx1, its associated P2X receptor (and any subtype thereof), and the Panx1-P2X signaling neuron. In some embodiments, one or more compounds selected from pan-connector 1 (Panx1) inhibitors (e.g., probenecid), its associated P2X receptor (and any subtype thereof), and the Panx1-P2X signaling neuron are administered (e.g., topically), either alone or in combination with a pan-connector modulator or another therapeutic agent. Other available pan-connector inhibitors are described in U.S. Patent No. 10,465,188 (“Channel Modulators”).

[0052] In some embodiments, an amniotic membrane is 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, a connexin modulator, gap junction modulator, and / or hemichannel modulator is applied subamniotically to the subject. In some embodiments, after applying the modulator to a non-healing surface defect or condition, the amniotic membrane is attached to the subject's eye.

[0054] In some embodiments, a connexin modulator, a gap junction modulator, and / or a hemichannel modulator are applied to the subject under the contact lens. In some embodiments, the contact lens is worn on the subject's eye simultaneously with, immediately after, or at some later time following the application of the connexin modulator, gap junction modulator, and / or hemichannel modulator. In some embodiments, the contact lens is a silicone hydrogel contact lens. In some embodiments, the connexin modulator, gap junction modulator, and / or hemichannel modulator are loaded into the contact lens before it is worn on a non-healing surface defect or lesion within or on the surface of the subject's eye. In some embodiments, the modulator is a Luffi derivative.

[0055] In some implementations, the contact lens is a therapeutic bandage contact lens. Therapeutic bandage contact lenses are also known as “bandage lenses” and “therapeutic bandage lenses.” In some implementations, therapeutic bandage lenses are, for example, soft contact lenses, gas-permeable (scleral) lenses, or 3D-printed biogels. Some suitable FDA-approved soft bandage lenses include: Acuvue Oasyswith 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 connexins, connexin gap junction channels, and / or connexin hemichannels to achieve ocular surface restoration and / or corneal epithelial restoration by using dosages and dosing regimens of connexin modulators (including, for example, gap junction modulators and / or hemichannel modulators). In some embodiments, the modulation of gap junctions and hemichannels is achieved by administering connexin 43 antisense oligonucleotides, connexin 43 peptides, 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, either alone or in combination with or in combination with a connexin 43 modulator.

[0057] In some implementations, a gap junction modulator or hemichannel modulator (e.g., rofidin) is applied topically to the eye of a subject suffering from a nonhealing or persistent ocular surface and / or corneal epithelial condition, and at the same time as, before or after the application of the gap junction modulator or hemichannel modulator, another topically applied gap junction modulator (e.g., Peptide5, Gap19, XG19 or other peptide mimics) or a topically, orally or systemically administered hemichannel modulator (e.g., tonaboxa or other orally or systemically available gap junction and / or hemichannel modulators) is administered to the subject.

[0058] In some embodiments, the method includes co-administering a local connexin regulator and / or a gap connexin regulator, as well as a systemically available connexin hemichannel regulator. Co-administration of the gap connexin regulator may be performed simultaneously with, after, or before the administration of the connexin hemichannel regulator. In some embodiments, a Formula I compound (e.g., tonabosar), a Formula II compound, and / or a peptide (e.g., Peptide5, XG19, etc.) may be co-administered with a connexin expression regulator (e.g., rufi-derived) or an anti-connexin peptide.

[0059] In some embodiments, the present invention provides the use of connexin modulators in the preparation of medicaments for treating non-healing or persistent ocular surface and / or corneal defects or condition modulation. In some embodiments, the medicament comprises or is substantially composed of: connexin antisense molecules (e.g., luffi derivatives), including the amounts and / or concentrations described herein. In some embodiments, the medicament comprises or is substantially composed of: connexin peptides (e.g., Peptide5, Gap19, XG19, aCT1 peptide (e.g., CXT1, CXT2, CXT3, CXT4, CXT5, or other C-terminal connexin peptides that may be linked or not linked to cell-penetrating peptides at the C- or N-terminus) etc.). In some embodiments, the medicament comprises or is substantially composed of: small molecule connexin hemichannel blockers (e.g., compounds according to Formula I, including tonaboxa, karaboxa, and their prodrugs, including tonaboxa prodrugs according to Formula II).

[0060] In some embodiments, connexin regulators and connexin 43 regulators include, for example, substances for downregulating connexin transcription or translation, such as antisense molecules. Substances for regulating connexins also include, for example, ZO-1 binding peptides targeting connexin 43 (e.g., aCT1, αCT1, CXT1, CXT2, CXT3, CXT4, CXT5, etc.). Preferred gap junction channel regulators are connexin 43 expression regulators, connexin 43 gap junction regulators, and connexin 43 hemichannel regulators. Particularly preferred connexin 43 regulators include rufi derivatives, XG19, and tonaboza.

[0061] In some embodiments, the present invention relates to pharmaceutical compositions and articles comprising kits containing instructions for use, comprising therapeutically effective amounts of connexin modulators, and dosing regimens for treating non-healing ocular and corneal surface defects and conditions using one or more connexin modulators (including connexin expression modulators, gap junction modulators, and / or hemichannel modulators). In some embodiments, the kits include or are accompanied by instructions (e.g., online instructions) for administration and treatment of PED and / or PCED as described herein.

[0062] In some embodiments, connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators, including, for example, Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junction and / or hemichannel modulators) are mixed with a pharmaceutically acceptable carrier or diluent to produce 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 Pronickel or poloxamer gel. In some embodiments, the gel is Pronickel F-127. In some embodiments, the pharmaceutical composition comprises a buffer. In some embodiments, the buffer comprises or is substantially composed of disodium hydrogen phosphate heptahydrate or sodium dihydrogen phosphate (or both).

[0063] In some embodiments, the methods, dosages, and dosing regimens of the present invention described herein, as well as connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators, including, for example, Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannel modulators) can be used to treat uveitis in subjects, including inflammation of the inner lining of the eye (uvea) and surrounding tissues. In some embodiments, a connexin 43 modulator is used to treat one or more types of uveitis. In some embodiments, other connexin modulators are used, including, for example, connexin 26 modulators, connexin 45 modulators, etc., which may be 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 to be treated may be caused by any reason or be multifactorial. In each of these embodiments, the methods, dosages and dosing regimens of the present invention described herein, and connexin modulators may be used to partially / completely treat or alleviate one or more symptoms of uveitis.

[0064] In some embodiments, the methods, dosages and dosing regimens of the present invention described herein, and any connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators, including, for example, Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junction modulators, and / or hemichannel modulators) can be used to treat blepharitis in the subject.

[0065] In some embodiments, the methods, dosages, and dosing regimens of the invention described or cited herein, as well as connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators, including, for example, Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannel modulators) can be used to treat any stage of dry eye syndrome (DED), also known as dry eye syndrome (DES), keratoconjunctivitis sicca (KCS), and keratoconjunctivitis sicca.

[0066] In other embodiments, the methods, dosages and dosing regimens of the invention described or cited herein, as well as the connexin modulators, can be used to treat subjects with Sjögren's syndrome-associated KCS and to completely or partially treat or alleviate one or more symptoms of Sjögren's syndrome-associated KCS.

[0067] In other embodiments, the methods, dosages and dosing regimens of the invention described or cited herein, as well as the connexin modulators, can be used to treat subjects with Fuchs' dystrophy, and the treatment results in complete or partial relief of one or more symptoms of Fuchs' dystrophy.

[0068] In some embodiments, in addition to the use of this invention for ocular surface restoration and / or corneal epithelial restoration, this invention also relates to methods for modulating connexins, connexin gap junction channels, and / or connexin hemichannels using dosages and dosing regimens of connexin modulators (including, for example, gap junction modulators and / or hemichannel modulators) to achieve uveal restoration, scleral restoration, lacrimal gland and lacrimal duct restoration, meibomian gland restoration, eyelid restoration, etc. In some embodiments, the modulation of gap junctions and hemichannels is achieved by applying connexin 43 antisense oligonucleotides, connexin 43 peptides, 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, either alone or in combination with a connexin 43 modulator and / or another therapeutic agent.

[0069] In some embodiments, the connexin modulator described or cited herein is administered to the subject in dose, or is formulated and administered to the subject in combination with an effective amount of one or more compounds selected from the following: antimicrobial agents, antibiotics, antiviral agents, antifungal agents, antiparasitic agents, mydriatic eye drops, steroidal anti-inflammatory drugs, immunosuppressants, immunomodulators, dry eye treatments (e.g., Tyrvaya, Rstasis, Cequa, Xiidra, Eysuvis), artificial tear compositions, and lubricants.

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

[0071] In some embodiments, one or more ligand modulators described or referenced herein are formulated together with one or more antimicrobial agents for use in the methods and protocols described herein.

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

[0073] In some embodiments, one or more connective protein modulators described or referenced herein are formulated together with one or more antifungal agents for use in the methods and protocols described herein.

[0074] In some embodiments, one or more ligand regulators described or referenced herein are formulated together with one or more antiviral agents for use in the methods and protocols described herein. Attached Figure Description

[0075] Figure 1 The dosage regimen of a connexin modulator used in a clinical trial for the treatment of ocular surface defects and conditions including non-healing, persistent corneal epithelial defects was demonstrated. Detailed Implementation

[0076] In some respects, the invention described and claimed herein relates to modulators of connexins, connexin gap junctions and connexin hemichannels, and their use in methods of treating non-healing ocular surface defects and conditions, including non-healing corneal defects and conditions, as well as their dosages and dosage regimens.

[0077] In some embodiments, the invention described and claimed herein relates to modulators of connexins, connexin gap junctions, and connexin hemichannels, and their use in methods of treating other ocular surface defects, diseases, conditions, and illnesses, including uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye.

[0078] To maintain a smooth optical surface, the corneal epithelium must continuously renew itself to maintain its function as a barrier against external environmental factors and various forms of damage. Following trauma, the cornea typically re-epithels rapidly, minimizing the risk of infection, opacity, or perforation. Persistent epithelial defects or persistent corneal epithelial lesions generally refer to ocular surface or corneal epithelial lesions that fail to heal after approximately two weeks of standard treatment. These can occur, for example, after exposure to toxic agents, chemicals, mechanical injury, thermal injury, and ocular surface infections, and can lead to serious clinical complications, causing discomfort or vision loss. The primary risk factor for most non-healing ocular surface defects or conditions is vision loss.

[0079] In one aspect, the present invention relates to pharmaceutical compositions, articles, kits, and methods for treating subjects with (or at risk of having) non-healing or persistent ocular surface or corneal defects or conditions (e.g., persistent ocular surface epithelial defects or persistent corneal epithelial defects) by administering a therapeutically effective amount of at least one connexin modulator to the subject's eye using novel, clinically validated dosing regimens. Therapeuticly effective doses can be used in conjunction with these novel dosing regimens. These doses, used with these dosing regimens, demonstrate the ability to permanently close non-healing ocular surface or corneal defects or conditions, including, for example, persistent ocular surface epithelial defects and persistent corneal epithelial defects.

[0080] In one embodiment, a composition comprising a connexin modulator is applied topically to the subject's eye. In another embodiment, the connexin modulator is first applied topically to the subject's eye, followed by the application of a bandage contact lens and / or amnion. 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., a connexin 43 modulator) is loaded into a bandage contact lens before being placed in the subject's eye.

[0081] In some embodiments of the method of the present invention, non-healing ocular surface defects or conditions are treated by pulsed administration of a connexin modulator according to the dosage regimen described herein (e.g., 3 to 7 administrations over 14 to 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 expression (e.g., connexin 43 expression). In some embodiments, the connexin gap junction modulator and / or the connexin hemichannel blocker or modulator blocks or modulates the connexin 43 gap junction and / or the connexin 43 hemichannel.

[0082] In some embodiments, the present invention relates to pharmaceutical compositions, articles, and methods for treating non-healing ocular surface defects or conditions by administering a therapeutically effective amount of at least one connexin modulator to the eye of the subject, and / or, for example, by oral or systemic administration of a connexin modulator in the case of non-topical administration. In some embodiments, more than one connexin modulator is administered to the eye of the subject, and / or by oral or systemic administration.

[0083] In some embodiments, the non-healing ocular surface defect or condition is a persistent epithelial defect (PED). In some embodiments, the non-healing ocular surface defect or condition is a persistent corneal epithelial defect (PCED). In the method of the present invention, at least one connexin modulator is administered in pulsed doses at the dosage described herein or the therapeutically effective amount (administered on days 1 and 2, followed by day 14, and optionally on day 28 or about day 28 (and optionally on day 35 or about day 35), which can effectively cure persistent or non-healing ocular surface or corneal epithelial defects or conditions. See Example 2 below. 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, including Cx43 antisense molecules). Other embodiments include other connexin 43 gap junction modulators and connexin 43 hemichannel blockers or modulators (e.g., peptides and small molecules).

[0084] In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, and 14. In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, 14, and 28 (e.g., if the surface defect has not healed after administration on days 1, 2, and 14). In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, 7, 14, and 21 (e.g., if the surface defect has not healed after administration on days 1, 2, 14, and 28). In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, and 28. In some embodiments, the connexin modulator dose (e.g., rufi-derived) is administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, the dose is administered after day 35. In some embodiments, the therapeutically effective dose of the connexin modulator (e.g., rufi-derived) is administered on one or more days from days 1, 2, and 14, and days 7, 21, 28, and 35. In some embodiments, administration using the stated dose and dosing regimen is administered to a PED or PCED. In some embodiments, the connexin modulator comprises a connexin antisense molecule (e.g., rufi-derived). In some embodiments, the connexin modulator comprises connexin peptides (e.g., Peptide5, Gap19, XG19, Gap26, Gap27, aCT1, etc.). In some embodiments, the connexin modulator comprises a small molecule connexin hemichannel blocker (e.g., tonaboxa). In some implementations, the connexin regulator is a connexin 43 regulator.

[0085] In some implementations, the dose of the connective protein modulator (e.g., rufi derivative) is administered on days 1, 2, 14, and 21.

[0086] In some implementations, the dose of the connective protein modulator (e.g., rofi derivative) is administered on days 1, 2, 7, 14, and 21.

[0087] In some implementations, the dose of the connective protein modulator (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, and 28.

[0088] In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over 14 days in three doses to treat a subject's non-healing or persistent ocular or corneal surface defects, including, for example, administration on days 1 and 2. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over approximately 28 days. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over approximately 28 to approximately 35 days. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over approximately 35 days in six to seven doses. For example, in one embodiment, the connexin modulator (e.g., rufi derivative) is administered four times on days 1 and 2, approximately day 14, and approximately day 28. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered five times on days 1 and 2, approximately day 14, approximately day 28, and approximately day 35. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered five times on days 1 and 2, approximately day 7, approximately day 14, and approximately day 28. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered six times on days 1 and 2, approximately day 7, approximately day 14, approximately day 21, and approximately day 28. In yet another embodiment, the connexin modulator (e.g., rufi derivative) is administered seven times on days 1 and 2, approximately day 7, approximately day 14, approximately day 21, and approximately day 28. Of course, "administer" as used herein refers to a single dose or multiple doses of the connexin modulator.

[0089] In some implementations, the connexin regulator is a regulator 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 implementations, the connexin regulators are regulators 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 ten connexin isotypes were detected in both the central and peripheral corneal epithelium by reverse transcriptase-polymerase chain reaction. Six of these (Cx26, Cx31, Cx32, Cx43, Cx45, and Cx58) were confirmed by laser scanning confocal microscopy. Cx26 was detected throughout the central corneal epithelium and in the middle and superficial layers of the limbal epithelium. Cx43 and Cx45 were localized in the basal layer and basal epithelial cells. Cx58 was expressed throughout the superficial corneal epithelium, and Cx31 and Cx32 were primarily expressed in the central corneal epithelium. See Yuan, Exp. Ther. Med. 7,791–798 (2014). In some embodiments, the connexin regulator is a Cx43 connexin regulator, such as a Cx43 expression regulator (e.g., an anti-Cx43 antisense molecule), a Cx43 gap junction regulator, or a Cx43 hemichannel regulator (e.g., an anti-connexin 43 peptide or small molecule). In some embodiments, the regulator is a modified or unmodified antisense polynucleotide or peptide, such as a modified or unmodified Cx43 antisense polynucleotide or Cx43 peptide, or other corneal epithelial, endothelial, or vascular connexin. In some embodiments, the regulator may include or exclude any of the aforementioned connexins.

[0091] In some embodiments, the connexin modulatory dose administered to a subject with a non-healing ocular surface defect or condition (e.g., PED) or a non-healing corneal surface defect or condition (e.g., PCED) at any of the dosage regimens described herein is a therapeutically effective dose. In some embodiments, the therapeutically effective dose of the connexin modulator is administered to the subject, for example (1) on day 1, day 2, and day 14; or (2) on day 1, day 2, day 14, and optionally day 28; or (3) on day 1, day 2, day 7, day 14, and about day 21, and optionally about day 28 and day 35; or (4) on the number of days set forth in any other dosage regimen described herein, other than these dosage regimens (1)-(3). In some embodiments, a therapeutically effective dose of a rufi derivative is administered.

[0092] In some embodiments, the amount of connexin modulator administered to a subject with a non-healing ocular surface defect or condition (e.g., PED) or a non-healing corneal surface defect or condition (e.g., PCED) at any of the dosage regimens described herein is (e.g., (1) administered on day 1, day 2, day 14 and optionally day 28 and / or day 35; (2) administered on day 1, day 2, day 14 and optionally day 7, day 21 and / or day 28; (3) administered on day 1, day 2, day 14 and optionally day 28 and / or day 35). (3) Administer on day 7, day 21, day 28 and / or day 35; (4) Administer on day 1, day 2 and day 14 and on day 7, day 21 and day 28 or on day 35; or (5) the number of days as set forth in other dosing regimens described herein) is one or more of the specific doses described herein, including, for example, a dose of connective modulator in milligrams per milliliter (mg / mL) (e.g., a composition comprising or substantially comprising the following: about or at least about 0.6 mg / mL of connective modulator), a dose of connective modulator in micromolars (μM) (e.g., a composition comprising or substantially comprising the following: a connective modulator at a concentration of about or at least about 19 μM), a dose of connective modulator in milligrams (mg) (e.g., a composition comprising or substantially comprising the following: about or at least about 0.18 mg of connective modulator), etc.

[0093] In some embodiments, the connexin regulator 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 regulator may be a connexin 43 antisense polynucleotide comprising, substantially comprising, or consisting of the sequences shown in SEQ ID NO:1-3, SEQ ID NO:4-16, and / or their modified forms. In some embodiments, the antisense oligonucleotide comprises, substantially comprises, or consists of 5'-GTA ATT GCG GCA AGA AGA ATT GTTTCT GTC-3' (SEQ ID NO:1; Luffield derivative). In some embodiments, the antisense oligonucleotide may be a chemically modified oligonucleotide or may be an unmodified oligonucleotide, such as a modified or unmodified DNA oligonucleotide. In some embodiments, the oligonucleotide is modified by altering one or more of the phosphodiester bonds, ribose backbone, and / or nucleoside bases. Other modifications are described herein. Other modifications are known in the art and described in various publications, including, for example, Shen, X. and Corey, DR, Chemistry, mechanism and clinical status of antisenseoligonucleotides and duplex RNAs. Nuelic Acids Res 2018 Feb 28; 46(4): 1584–1600. The preparation method is also known in the art. See, for example, Abramova T, Frontiers and Approachesto Chemical Synthesis of Oligodeoxyribonucleotides, Molecules 2013 Jan; 18(1): 1063–1075; Hao M et al., Current and Emerging Methods for the Synthesis ofSingle-Stranded DNA Genes (Basel)2020 Jan 21;11(2):116. See also Anwar, S. et al. Enhancing the Effectiveness of Oligonucleotide Therapeutics Using Cell-Penetrating Peptide Conjugation, Chemical Modification, and Carrier-Based Delivery Strate and Carrier-Based Delivery Strategies. Pharmaceutics 2023, 15 , 1130.

[0094] In some methods of the present invention, the connexin modulator applied to a subject suffering from a non-healing surface defect or condition is 0.06% of rufi derivative. In some embodiments, the connexin modulator applied to a subject suffering from a non-healing surface defect or condition is 0.006% of rufi derivative. In some embodiments, the applied connexin modulator is at least about 0.06% of rufi derivative. In some embodiments, the connexin modulator applied to a subject suffering from a non-healing surface defect or condition is at least about 0.006% of rufi derivative. In some embodiments, the application is to a PED or PCED. In some embodiments, the application comprises or substantially comprises a pharmaceutically acceptable carrier and about 0.06% rufi derivative, about 0.006% rufi derivative, at least about 0.06% rufi derivative, or at least about 0.006% rufi derivative to heal the subject's non-healing surface defect or condition. The rufi derivative dosage described herein and hereinafter can be prepared as described in Example 1. In some embodiments, the connexin regulator applied in the stated amount is a connexin antisense molecule other than (or including in addition to) rufi derivatives. In some embodiments, the connexin regulator applied in the stated amount is a connexin peptide (e.g., XG19). In some embodiments, the connexin regulator applied in the stated amount is a connexin hemichannel blocker (e.g., tonabosar).

[0095] In some embodiments, a composition comprising or substantially consisting of the following is used to treat non-healing surface defects or conditions: 0.6 mg / mL rufi derivative. In some embodiments of the invention, a composition comprising or substantially consisting of the following is used to treat non-healing surface defects or conditions: 0.06 mg / mL rufi derivative. In some embodiments, the composition comprises or substantially consists of the following: at least about 0.6 mg / mL rufi derivative. In some embodiments, the composition comprises or substantially consists of the following: at least about 0.06 mg / mL rufi derivative. In some embodiments, administration is to a PED or PCED. In some embodiments, the connexin modulator administered in the stated amount is a connexin antisense molecule in addition to (or containing in addition to) rufi derivative. In some embodiments, the connexin modulator administered in the stated amount is a connexin peptide (e.g., XG19). In some embodiments, the connexin modulator administered in the stated amount is a connexin hemichannel blocker (e.g., tonaboxa).

[0096] In some embodiments, according to the scheme described herein, a non-healing surface defect or condition of a subject is treated with a composition comprising or substantially consisting of: about 0.18 mg of lufifine derivative. In some embodiments, according to the scheme described herein, each dose of lufifine derivative applied to a non-healing surface defect or condition of a subject, whether internal or external, comprises or substantially consists of: at least about 0.18 mg of lufifine derivative. In some embodiments of the invention, according to the scheme described herein, a non-healing surface defect or condition is treated with a composition comprising or substantially consists of: about 0.018 mg of lufifine derivative. In some embodiments, according to the scheme described herein, each dose of lufifine derivative applied to a non-healing surface defect or condition comprises or substantially consists of: at least about 0.018 mg of lufifine derivative. In some embodiments, administration is to a PED or PCED. In some embodiments, the connexin modulator applied in the stated amount is a connexin antisense molecule in addition to (or contained in addition to) lufifine derivative. In some embodiments, the connexin regulator applied in the stated amount is a connexin peptide (e.g., XG19). In some embodiments, the connexin regulator applied in the stated amount is a connexin hemichannel blocker (e.g., tonabosar).

[0097] In some embodiments, the modulator (e.g., a connexin modulator, such as a connexin 43 modulator, a connexin 43 gap junction modulator, and / or a connexin 43 hemichannel modulator) effective in the method of the invention is a composition comprising about or at least about 2 micromoles (2 μM), 5 micromoles (5 μM), 10 micromoles (10 μM), 15 micromoles (15 μM), or 20 micromoles (20 μM), including about (or at least about) 1.9 micromoles (1.9 μM) or 1.9405 micromoles (1.9405 μM) of a dose of a connexin modulator (e.g., Rufigen), or any amount of a connexin modulator (e.g., Rufigen) within or between these doses. Other effective doses for treating ocular surface defects or conditions (e.g., ocular surface PED and PCED) in the methods of the present invention include compositions comprising about or at least about 30 micromoles (30 μM) or 40 micromoles (40 μM) of a connexin modulator (e.g., a connexin 43 modulator, a connexin 43 gap junction modulator, and / or a connexin 43 hemichannel modulator (e.g., rubi-derived)). In some embodiments, the therapeutically effective amount of the connexin modulator administered as described is a connexin antisense molecule other than (or containing in addition to) rubi-derived. In some embodiments, the therapeutically effective amount of the connexin modulator administered as described is a connexin peptide (e.g., XG19). In some embodiments, the therapeutically effective amount of the connexin modulator administered as described is a connexin hemichannel blocker (e.g., tonaboxa).

[0098] In some embodiments, about 0.18 mg of rufi derivative (or other connexin antisense molecule), about 0.018 mg of rufi derivative (or other connexin antisense molecule or connexin modulator), or about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, or about 0.5 mg to about 1 mg of rufi derivative are administered to the non-healing surface defect or condition, either as a single dose or in two or more doses, such as twice daily for 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, 0.5 mg, or at least about 1 mg of rufi derivative are administered to the non-healing surface defect or condition, either as a single dose or in two or more doses, such as twice daily for up to six to ten doses administered on days 1, 2, and 14, or using other dosing regimens described herein that include days 28 and / or 35. In other embodiments, at least about 0.5 mg or at least about 1 mg of luffi derivatives are administered, either as a single dose or in multiple doses, such as twice daily for up to 14 doses administered on one or more days, for example, on days 1, 2, and 14, and on days 7, 21, 28, and / or 35. In some embodiments, administration is to the subject's PED or PCED. In some embodiments, other connexin modulators—including, for example, other connexin 43 modulators (and / or other connexin modulators), as well as other antisense compounds, peptides, and / or small molecules—are administered at these doses on these days. In some embodiments, the connexin modulator administered in the stated amount is a connexin antisense molecule other than (or containing in addition to) luffi derivatives. In some embodiments, the connexin modulator administered in the stated amount is a connexin peptide (e.g., XG19). In some embodiments, the connexin modulator administered in the stated amount is a connexin hemichannel blocker (e.g., tonaboxa).

[0099] In any embodiment of these dosages, dosing regimens, and methods, the luffi-derived dose may be administered to a subject suffering from PED or PCED. In some embodiments of these dosages, dosing regimens, and methods, the administered connexin modulator dose comprises another connexin antisense molecule or modulator, such as another Cx43 antisense molecule or Cx43 modulator other than (or contained in addition to) the luffi-derived dose, or another modulator of ocular surface or corneal epithelial connexin other than (or contained in addition to) Cx43.

[0100] In some embodiments of the invention, the volume of the composition comprising or substantially consisting of a connexin modulator is about 300 μL when applied to a subject suffering from a non-healing surface defect or condition. In other embodiments, the volume of the composition applied is about 250 μL to about 1.0 mL. In some embodiments, the composition comprises about 0.01 mg, 0.018 mg, 0.18 mg to about 0.2 mg of rufi derivative, or about 0.18 mg, 0.20 mg, or 0.50 mg to about 1 mg of rufi derivative. In some embodiments, the composition comprises at least about 0.018 mg of rufi derivative, at least about 0.18 mg of rufi derivative, at least about 0.5 mg of rufi derivative, or at least about 1.0 mg of rufi derivative. In some embodiments, other connexin modulators (including, for example, other connexin 43 modulators (and / or other connexin modulators) besides rufi derivative, as well as other antisense compounds, peptides, and / or small molecules) are applied 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 according to Formula I, including tonabolza. Tonabolza (a benzoylaminobenzopyran compound) is a modulator of the activity of gap junction channels and half-channels (including connexin 43). Specifically, tonabolza can block or inhibit half-channels containing connexin 43.

[0102] In some implementations, the regulator is a connexin peptide. Connexin regulators (including Peptide5) inhibit Cx43 half-channel activity and / or ATP release during and after injury. Other connexin regulator peptides include Gap19, XG19, and their analogues.

[0103] The present invention particularly provides: (1) a method for treating a non-healing ocular surface or corneal defect or condition (e.g., PED or PCED) in a subject, wherein the method closes and heals the defect or condition by administering a therapeutically effective amount of a connexin modulator (e.g., a connexin expression modulator), a connexin gap connexin modulator, and / or a connexin hemichannel modulator according to the dosage regimen described herein; (2) a method for treating a non-healing ocular surface or corneal defect or condition (e.g., PED or PCED) in a subject, wherein the method administers a connexin modulator at the dosage described herein (e.g., according to any dosage regimen described herein); and (3) the use of such modulators (including, for example, antisense modulators, peptide modulators, and small molecule modulators) in the preparation of a medicament (e.g., a pharmaceutical composition) and a kit containing said medicament, the kit including or accompanied by instructions for use for the method of the present invention. See, for example, Example 2, which describes a clinically proven pulse dosing regimen for treating and healing non-healing, persistent ocular surface defects.

[0104] In some embodiments, the present invention particularly provides: (1) a method for treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye), the method comprising treating the condition by administering a therapeutically effective amount of a connexin modulator (e.g., a connexin expression modulator, a connexin gap connexin modulator, and / or a connexin hemichannel modulator) according to the dosage regimen described herein; (2) a method for treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye (e.g., Methods for treating evaporative dry eye, aqueous-deficient dry eye, and dehumidified dry eye, by administering a connexin modulator at the dosage described herein (e.g., any dosage regimen described herein); and (3) the use of such modulators (including, for example, antisense modulators, peptide modulators, and small molecule modulators) in the preparation of a medicament (e.g., a pharmaceutical composition) and a kit containing said medicament, the kit including or accompanied by instructions for use for the methods of the present invention, the medicament being used to treat uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and dry eye (e.g., evaporative dry eye, aqueous-deficient dry eye, dehumidified dry eye).

[0105] All non-healing or persistent ocular surface or corneal (e.g., corneal epithelial) defects or conditions of any etiology can be treated with the compositions and methods of the present invention, including dosages and dosing regimens. In some embodiments, the non-healing ocular surface defect or condition is PED. In some embodiments, the non-healing ocular surface defect or condition is PCED. In some embodiments, the non-healing ocular surface defect or condition is a corneal ulcer. In some embodiments, the non-healing ocular surface or corneal defect or condition is caused by chemical and / or thermal injury. Non-healing ocular surface or corneal defects or conditions 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 condition is caused by inflammation or inflammatory disease. In some embodiments, the non-healing ocular surface or corneal defect or condition is caused by ocular inflammation. In some embodiments, the non-healing ocular surface or corneal defect or condition is caused by an inflammatory disease of the eye, ocular surface, or cornea. In some embodiments, the non-healing ocular surface defect or condition is caused by physical trauma or injury (including mechanical injury). In some embodiments, the non-healing ocular surface defect or condition is PED or PCED caused by chemical and / or thermal injury. In some embodiments, the chemical and / or thermal injury leading to PCED is severe chemical and / or thermal injury. In some embodiments, the ocular surface and / or corneal burns or injuries to be treated in the method of the present invention involve more than 50% of the conjunctiva or more than 6 o'clock positions of the limbus. In some embodiments, the ocular surface and corneal burns or injuries to be treated in the method of the present invention are ≥3 on the Dua classification scale. In some embodiments of the present invention, severe ocular and corneal burns and injuries respond particularly well to treatment with connexin 43 modulators (e.g., rofi derivatives) and / or other connexin modulators using the methods described herein. All causes of PED and PCED, regardless of the cause, can be treated with the methods of the present invention described herein. The causes of PED and PCED include not only inflammation and inflammatory diseases, but also chemical, mechanical, thermal causes, neurotrophic keratitis, and limbal stem cell deficiency. In some embodiments, the methods and / or dosages of the present invention are used to treat PED and / or PCED caused by or resulting from chemical damage. In some embodiments, the methods and / or dosages of the present invention are used to treat PED and / or PCED caused by or resulting from thermal damage. In some embodiments, the methods and / or dosages of the present invention are used to treat PED and / or PCED caused by or resulting from mechanical damage. In some embodiments, the methods and / or dosages 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 dosages 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 condition 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 caused by Acanthamoeba keratitis, fungal keratitis, or herpes simplex keratitis. In other embodiments, the non-healing ocular surface defect or condition (e.g., corneal ulcer) is caused by trauma, such as an abrasion (e.g., a scratch or cut) or an intraocular foreign body. In another embodiment, the non-healing ocular surface defect or condition (e.g., corneal ulcer) is caused by an allergy or allergic eye disease. In another embodiment, the non-healing ocular surface defect or condition (e.g., corneal ulcer) is caused by dry eye syndrome. In some embodiments, the non-healing ocular surface defect or condition is a burn. In some embodiments, the non-healing ocular surface defect or condition is a corneal burn or an ocular or corneal burn ulcer.

[0107] In some embodiments, the methods, dosages, and dosing regimens of the present invention described herein, as well as connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators, including, for example, Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannel modulators), can be used to treat uveitis, i.e., inflammation of the inner lining of the eye (uvea) and surrounding tissues. In some embodiments, a connexin 43 modulator is used to treat one or more types of uveitis. In some embodiments, other connexin modulators are used, including, for example, connexin 26 modulators, connexin 45 modulators, etc., which may be used alone or in combination.

[0108] In some implementations, the uveitis to be treated described herein is anterior uveitis (which affects, for example, the iris of the anterior segment of the eye).

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

[0110] In some implementations, the uveitis to be treated described herein is posterior uveitis (which affects, for example, the retina and choroid of the posterior part of the eye).

[0111] In some implementations, the uveitis to be treated described herein is panuveitis (which affects, for example, the entire uvea from the anterior to the posterior aspect).

[0112] In some embodiments, the uveitis to be treated described herein is caused entirely or partially by one or more of the following: ocular trauma, ocular injury, ocular surgery, infection (including, for example, cytomegalovirus (CMV) retinitis, histoplasmosis, reactive arthritis, herpes zoster, syphilis, toxoplasmosis, cat scratch disease, varicella-zoster virus infection, tuberculosis, etc.), inflammation, inflammatory diseases, and autoimmune diseases (including, for example, AIDS, Behcet's disease, lupus erythematosus, multiple sclerosis, psoriasis, rheumatoid arthritis, ulcerative colitis, Vogt-Koyanagi-Harada syndrome (VKH syndrome), etc.). In some embodiments, the uveitis to be treated described herein is caused entirely or partially by an autoimmune or inflammatory disease affecting other parts of the body, such as sarcoidosis, systemic lupus erythematosus, or Crohn's disease. In some embodiments, the uveitis to be treated described herein is caused entirely or partially by ankylosing spondylitis. In some implementations, the cause of the uveitis to be treated described herein is unknown.

[0113] In these uveitis treatment implementation schemes, the methods, dosages, and dosing regimens of the present invention described herein, along with a connexin modulator, completely or partially relieve one or more symptoms of uveitis, which may be administered alone or in combination with (or co-formulated with) other uveitis treatment agents (e.g., antibiotics, antiviral or antifungal agents; mydriatic eye drops; steroidal anti-inflammatory drugs; immunosuppressants). Uveitis symptoms that can be relieved after treatment as described herein include eye pain, redness of the eye, photophobia, blurred vision, the appearance of black / floating spots in the visual field (floaters), and decreased vision.

[0114] In some embodiments, the methods, dosages, and dosing regimens of the invention described herein, as well as connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators, including, for example, Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannel modulators) can be used to treat blepharitis. In some embodiments, anterior blepharitis is treated in the subject. In some embodiments, posterior blepharitis is treated in the subject. In some embodiments, one or more connexin modulators described or cited herein are administered to the subject, or formulated for administration or co-administration to the subject, in accordance with the methods and regimens for treating blepharitis described herein. In some embodiments, one or more connexin modulators described or cited herein are administered or co-formulated with one or more anti-inflammatory drugs (e.g., steroids) for use in the methods and regimens for treating blepharitis described herein. In these blepharitis treatment embodiments, the methods of the present invention described herein, along with the connexin modulators (including dosages and dosage regimens), completely or partially relieve one or more symptoms of blepharitis. Blepharitis symptoms that can be relieved after treatment as described herein include foreign body sensation, burning or stinging sensation in the eyes, tearing, itchy eyes, photophobia, redness and swelling of the eyes or eyelids, dry eye, crusting of the eyelids or eyelashes upon waking, and blurred vision.

[0115] Dry eye is an ocular surface disease. In some embodiments, the methods, dosages, and dosing regimens of the invention described or cited herein, as well as connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and connexin hemichannel modulators, including, for example, Cx26, Cx30, Cx30.3, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, or Cx58 connexins, gap junctions, and / or hemichannel modulators), can be used to treat any stage of dry eye (DED), also known as dry eye syndrome (DES), keratoconjunctivitis sicca (KCS), and keratoconjunctivitis sicca. All stages of DED can be treated with the methods, dosages, dosing regimens, compositions, and kits described herein, including DED stage 1, DED stage 2, DED stage 3, and DED stage 4. In some embodiments, the methods, dosages, dosage regimens, and compositions of the present invention can 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 dosage of the connexin modulator described herein can be formulated or loaded into an artificial tear composition, or formulated with an anti-inflammatory agent, and can be used to treat DED. In some embodiments, the dosage of the connexin modulator described herein can be formulated with an anti-inflammatory compound or composition (using a known effective dose of at least one anti-inflammatory agent) for topical application to treat DED. In some embodiments, the dosage of the connexin modulator described herein can be administered alone or in combination with one or more DED treatment agents (e.g., Tyrvaya, Rstasis, Cequa, Xiidra, Eysuvis). In some embodiments, the dosage of the connexin modulator described herein can be formulated and administered together with one or more DED treatment agents (e.g., one or more connexin modulators with one or more other dry eye treatment agents, such as Tyrvaya, Rstasis, Cequa, Xiidra, Eysuvis). In some embodiments, the dosage of the connexin modulator described herein may be formulated and co-administered with at least one corticosteroid. In these dry eye treatment embodiments, the methods and connexin modulators (including dosages and dosing regimens) of the present invention described herein provide complete or partial relief from one or more symptoms of dry eye. Dry eye symptoms that can be relieved by treatment as described herein include dryness, gritty feeling, stinging or burning sensation, redness, tearing, and a sticky feeling in the eyes upon waking. Many dry eye sufferers also experience a foreign body sensation or eye strain, blurred vision, or eye fatigue. Itchy eyes and photophobia are also other symptoms of dry eye.

[0116] In other embodiments, the methods, dosages and dosing regimens of the invention described or cited herein, as well as the connexin modulators, can be used to treat Sjögren's syndrome-related KCS in subjects, and to completely or partially treat or alleviate one or more symptoms of Sjögren's syndrome-related KCS. Symptoms of Sjögren's syndrome-related KCS that can be relieved after treatment as described herein include itchy, burning, gritty, pulling, or foreign body sensation in the eyes and / or photophobia. Severe stinging, eye strain or fatigue, and blurred vision may also occur. Some patients may experience excessive tearing after severe stimulation.

[0117] In other embodiments, the methods, dosages and dosing regimens of the invention described or cited herein, as well as the connexin modulators, can be used to treat Fuchs' dystrophy in subjects and to provide complete or partial relief from one or more symptoms of Fuchs' dystrophy. One of the early 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 upon waking, which may last for several hours. In later stages of Fuchs' dystrophy, swelling, impaired vision, and discomfort last longer, even throughout the entire day. Other symptoms of Fuchs' dystrophy include a rough or gritty feeling in the eyes (occasionally accompanied by severe eye pain), photophobia, fluctuating vision throughout the day or over several days, halos and / or glare in bright light, and blurred vision accompanied by decreased color contrast.

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

[0119] In some embodiments, one or more ligand modulators described or referenced herein are formulated together with one or more antimicrobial agents for use in the methods and protocols described herein.

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

[0121] In some embodiments, one or more connective protein modulators described or referenced herein are formulated together with one or more antifungal agents for use in the methods and protocols described herein.

[0122] In some embodiments, one or more ligand regulators described or referenced herein are formulated together with one or more antiviral agents for use in the methods and protocols described herein.

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

[0124] definition It should be understood that, for various reasons (including the patient's ability to travel to a clinic or treatment room or administration site, patient schedule, drug accessibility, etc.), the specific date of administration of the connexin modulator according to the dosage regimen described herein may need to be adjusted (and may be). The term "about," when used to describe the date of administration of the connexin modulator according to the dosage regimen described herein, generally refers to one day before or after that date. Thus, for example, administration "about day 28" means administration on day 28, but also includes administration on day 27 or day 29.

[0125] As used herein, the term "connein regulator" (sometimes also referred to as a connein "inhibitor" or "antagonist") refers to a compound that, alone, together, or separately, prevents, inhibits, and / or reduces the function or activity of conneins, connein gap junction channels, or connein hemichannels. This includes, for example, preventing, inhibiting, and / or reducing the function and / or activity and / or formation of connein hemichannels and / or connein gap junctions, including connein expression, their transport, and / or assembly. Such functions and activities may include, for example, docking of hemichannels on adjacent cells and opening to form gap junction channels. They may also include intercellular communication and the flow of molecules between cells through gap junction channels. Hemichannel regulation is the regulation of one or more functions and / or activities of a hemichannel, such as the flow of molecules through the hemichannel. Such functions and activities may include, for example, the entry of molecules from the extracellular space or environment into the cell through the hemichannel, and / or the entry of molecules (e.g., adenosine triphosphate (ATP)) from the intracellular space or environment of the cell into the extracellular space or environment through the hemichannel. Prevention, inhibition, and / or reduction of function or activity can be direct or indirect (e.g., but not limited to directly blocking the channel, inducing conformational changes, or altering the phosphorylation state or opening probability of the connexin). Hemichannel regulation also includes inhibiting the permeability of the hemichannel (e.g., reducing the flow of permeate through the hemichannel). In some embodiments, the permeate is adenosine triphosphate (ATP). Connexin regulators can have any chemical properties. However, for example, connexin regulators can be nucleic acids (including antisense molecules, RNAi molecules, morpholino oligonucleotides, and other nucleic acids described herein), peptides or peptide mimics, small molecules, or other chemical substances. In some embodiments, connexin regulators are compounds that target one or more components (including connexins, hemichannels) of gap junctions to inhibit or block their activity, expression, transport, and / or assembly. "Inhibition," "blocking," "antagonism," or "regulation" should not be construed as completely inhibiting, blocking, antagonizing, or regulating the function, activity, expression, transport, and / or assembly of connexins, connexin hemichannels (e.g., connexin 43 hemichannel), or gap junctions, although this may be preferred. Rather, it should be understood to include any reduction in the function, activity, expression, transport, and / or assembly of connexins (including their transcription, translation, and / or expression), connexin hemichannels (including their permeability or their opening or release of ATP into the extracellular environment), or gap junctions (including their opening to adjacent cells or formation by hemichannels in adjacent cells). As used herein, connexin regulators include connexin expression regulators, connexin gap junction regulators, and connexin hemichannel regulators.

[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 connexins present in blood vessels, such as the Cx37 modulator, Cx40 modulator, Cx43 modulator, and / or Cx45 modulator. Therefore, as used herein, the term "connexin modulator" generally refers to a connexin modulator, and unless otherwise provided, specifically refers to the connexin 43 modulator and modulators of other connexins mentioned herein (including connexin 43 and other gap junction and hemichannel modulators), as well as other vascular, endothelial, and epithelial (e.g., ocular and corneal epithelium) connexins, gap junctions, and hemichannel modulators. In some embodiments, the connexin regulator is a connexin 43 regulator, such as a connexin 43 expression regulator, a connexin 43 hemichannel regulator that inhibits or blocks hemichannel opening, or a connexin 43 peptide. In some embodiments, the gap junction regulator is or includes regulators of other connexins present in the ocular surface (including the cornea and corneal epithelium) and their hemichannels and gap junctions. In other embodiments, the connexin regulator is a regulator of any other connexin 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 present in the corneal epithelium as mentioned above. In some embodiments, the regulator may include or exclude regulators of any connexins, connexin gap junctions, and / or connexin hemichannels described herein. In some embodiments, ocular surface defects or conditions, such as PED, PCED, and / or ocular surface ulcers (including, for example, inflammatory ocular surface ulcers or ocular surface ulcers, conditions, or defects characterized at least partially by inflammation), are treated with connexin modulators (e.g., connexin expression modulators, connexin gap junction modulators, and / or connexin hemichannel modulators) to inhibit the activation of one or more inflammasomes. In some embodiments, the connexin modulators inhibit the activation of inflammatory cascades via inflammasomes. In some embodiments, the connexin modulators modulate (e.g., block or downregulate) the activation and / or activity of the NLRP3 inflammasome. In some embodiments, the connexin modulators modulate (e.g., block or downregulate) the activation of NLRP3 inflammasome-mediated inflammatory cascades. Inflammasome modulation helps close non-healing ocular surface or corneal defects or conditions (including those described herein) and helps treat other diseases, conditions, and symptoms described herein.In some implementations, ocular surface defects or conditions, such as PED, PCED, and / or ocular surface ulcers, are treated with an inflammasome modulator (e.g., an NLRP3 inflammasome modulator), which may not be a connexin modulator (e.g., a connexin 43 hemichannel modulator). Inflammasome modulators include, for example, Leung and Lowery, *The patent landscape of inflammasome modulators*. Nature Reviews Drug Discovery Those described in 19, 158 (2020). See also Chauhan, D. et al., Therapeutic modulation of inflammasome pathways. Immunol Rev297(1): 123–138 (Sept 2020). Inflammasome regulators include selective G protein-coupled receptor 40 (GPR40) agonists (e.g., faglipizide, which inhibits inflammasome activation by blocking the formation of apoptosis-associated speckle-like protein (ASC) (an inflammasome component) containing a caspase recruitment domain (CARD)) and ethylpyruvate (which significantly inhibits NLRP3 inflammasome activation). Other inflammasome regulators unrelated to connexins and connexin hemichannels are known in the art. Regulation of hemichannel and / or gap junction channel function can occur in any manner. But by way of example only, regulation can occur by one or more of the following: preventing, blocking, inhibiting or reducing gap junction formation via hemichannel docking; inducing or promoting hemichannel closure; preventing, blocking, inhibiting or reducing hemichannel opening; preventing, blocking, inhibiting or reducing hemichannel permeability; inducing or promoting loss of coupling between hemichannels; triggering, inducing or promoting the internalization of hemichannels and / or gap junctions. Just as the use of terms such as “block,” “inhibit,” “prevent,” “reduce,” and “antagonize” should not be understood as meaning complete blockage, inhibition, prevention, or antagonism, “induction” or “promotion” should not be understood as meaning complete prevention, blockage, inhibition, or reduction of gap junction formation via hemichannel docking; induction or promotion of complete hemichannel closure; complete prevention, blockage, inhibition, or reduction of hemichannel opening; complete prevention, blockage, inhibition, or reduction of hemichannel permeability; complete induction or promotion of loss of coupling between hemichannels; or triggering, induction, or promotion of complete loss or internalization of coupling between connexin hemichannels and / or gap junctions (or a group of hemichannels and / or gap junctions), but should be understood to include partial induction or promotion of these processes. Connexins, hemichannels, and gap junctions can be present in any type of cell. Therefore, unless the context requires otherwise, references to “connexins,” “hemichannels,” or “gap junctions” should be understood to include references to connexins, hemichannels, or gap junctions present in any cell type. In one embodiment, the hemichannel is an epithelial cell connexin, hemichannel, or gap junction (e.g., an ocular or corneal epithelial cell connexin, hemichannel, or gap junction). In one embodiment, the connexin, hemichannel, or gap junction is a vascular connexin, hemichannel, or gap junction. In one embodiment, the connexin, hemichannel, or gap junction is a connexin, hemichannel, or gap junction present in vascular endothelial cells and / or vascular smooth muscle cells. A "half-channel blocker" is a compound that interferes with the flow of molecules through connexin hemichannels. Half-channel blockers can block or reduce half-channel opening, block, reduce, or inhibit half-channel opening, block or reduce the release of molecules through half-channels into the extracellular space, and / or block or reduce the entry of molecules through half-channels into the intracellular space. Half-channel blockers include compounds that completely or partially block half-channel leakage or the flow of molecules through half-channels (e.g., into or from the extracellular space).Half-channel blockers also include compounds that reduce the probability of half-channel opening. The probability of opening is a measure of the percentage of time a channel remains open compared to the percentage of time it remains closed (reviewed 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 include hemichannel modulators. Hemichannel blockers can directly or indirectly interfere with the flow of molecules through the hemichannel of a linker protein, or interfere with the permeability of the hemichannel.

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

[0128] "Bandage contact lenses" are any contact lenses that can be used to protect the ocular surface. Bandage contact lenses are also known as "therapeutic contact lenses" and "therapeutic bandage contact lenses." They also help relieve pain and promote healing. Bandage contact lenses provide mechanical protection to the ocular surface (e.g., the cornea) against damage caused by eyelid movement, and also protect the ocular surface (e.g., the corneal surface) from atmospheric exposure and further eye injury, and reduce dehydration. Bandage lenses include hydrogel lenses and silicone hydrogel lenses, and 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 Ophthalmology9(6): 524-532 (Nov-Dec 2020); Jacobs et al., CLEARMedical use of contact lenses, Cont Lens Anterior Eye 2021 Apr;44(2):289-329. In some implementations, therapeutic bandage lenses are, for example, soft contact lenses, gas-permeable (scleral) lenses, or 3D-printed biogels. 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] The term "small molecule" as defined herein includes compounds with a molecular weight below approximately 600 Daltons and is typically an organic compound. Small molecule connexin modulators include compounds of formula I. Small molecules can be active agents of prodrugs. Small molecule prodrugs include compounds of formula II, which are prodrugs of the small molecule hemichannel and connexin modulator tonabosar.

[0130] As used herein, “treatment” (and its grammatical variations such as “treat” or “treatment”) refers to a clinical intervention that attempts to alter the course of disease in the individual, tissue, or cells receiving the treatment, and may be used for prevention or during clinicopathological processes. Desired effects of treatment include, but are not limited to: closing non-healing ocular surface defects or conditions (e.g., PED or PCED) and preventing the occurrence or recurrence of non-healing ocular surface or corneal defects or conditions; relieving signals and symptoms; reducing any direct or indirect pathological consequences of non-healing ocular surface or corneal defects or conditions; slowing the progression of non-healing ocular surface or corneal defects or conditions; improving or alleviating non-healing ocular surface defects or conditions; and achieving disease remission or improved prognosis. This term does not necessarily mean that the subject needs to receive treatment until complete recovery, but this is generally the case with respect to the pulsed dose delivery method of the present invention for treating non-healing ocular surface or corneal defects or conditions. Therefore, “treatment” may also include maintaining or promoting complete or partial remission of non-healing ocular surface defects or conditions. Persistent wound closure can be defined as closure following the last application of the connexin modulator described herein (e.g., around day 14, around day 28, or around day 35), with 100% epithelial regeneration achieved in two consecutive assessments at least 2 weeks apart (but no more than 5 months) after closure, without the need for drainage or dressings. In some embodiments, persistent wound closure refers to wound closure lasting for 1 month (e.g., 4 weeks or 28 days). In some embodiments, “treatment” refers to the treatments and clinical interventions described above and herein, or to the treatment of uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and dry eye syndrome (including evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye).

[0131] The term "prevention" refers to complete or partial prevention, improvement, or control.

[0132] As used herein, "effective amount" or "therapeutic effective amount" means the effective amount that achieves the desired effect in treating non-healing ocular or corneal defects or conditions (e.g., PED, PCED, ocular surface ulcers, corneal ulcers, etc.) at the dosage and time period of the dosage regimens described herein. For example (and not limited to), "therapeutic effective amount" may refer to the amount of a conjugate protein modulator compound or composition (including, but not limited to, those disclosed herein) that, when administered according to the dosage regimens of the invention, closes a non-healing ocular surface defect or condition. In other embodiments, treatment with an "effective amount" results in a durable outcome, and the non-healing ocular defect or condition remains closed after treatment. The dosages disclosed herein are therapeutic effective amounts; however, the method is not limited to those dosages or amounts and includes the use of other therapeutically effective amounts. In some implementations, "effective dose" or "therapeutic effective dose" refers to the effective dose that achieves the desired effect in treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and dry eye syndrome (including evaporative dry eye, aqueous-deficient dry eye, and dehumidified dry eye) at the dose and time period described herein.

[0133] As used herein, "preventive effective amount" means, for example, an effective amount that achieves or ensures the desired effect or preventive effect (e.g., persistent wound closure and non-healing ocular surface defects or non-recurrence of the condition) when applied, for example, on about day 28 (and / or optionally about day 35). In some embodiments, a preventive effective amount of connexin modulator is applied to a subject with a closed ocular surface condition (e.g., closed PED or PCED). In some embodiments, an effective amount of connexin modulator is prophylactically applied to a subject with a closed ocular surface condition (e.g., PED or PCED). In some embodiments, "preventive effective amount" means, when treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and dry eye syndrome (including evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye) using the methods described herein, including during or after treatment of uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and any type of dry eye syndrome, an effective amount that achieves the desired preventive effect.

[0134] The terms “peptide,” “peptide mimic,” and “mimic” include synthetically produced or genetically engineered chemical compounds that may have the same or substantially the same structural and functional characteristics as the protein region they mimic. For example, these compounds may mimic the extracellular loops of pairing linker proteins involved in linker-linker docking and cell-cell channel formation, and / or the extracellular loops of hemichannel linker proteins, as well as the intracellular C-terminus of, for example, linker protein 43. As used herein, “peptide mimic” (also known as peptide mimic) includes peptides and peptide compounds, as well as such non-peptide compounds (such as peptide analogs). Peptides structurally similar to peptides with therapeutic effects can be used to produce equivalent or stronger therapeutic or preventative effects. For example, the first extracellular loop of Cx37, Cx40, and Cx43 is mimicked by the peptide designated Gap26. On the other hand, Gap27 and Peptide5 mimic the region of the second extracellular loop. Gap27 targets Cx32, Cx40, and Cx43, while Peptide5 inhibits Cx43. Other peptide mimics include JM2 (VFFK-GVKDRVKGRSD, SEQ ID NO:134), ΔSH3, CT9 (RPRDDLEI, SEQ ID NO:135), CT9-TAT, CT10 (SRPRDDLEI, SEQ ID NO:136), and αCT (RQPKIWFPNRRKPWKK-RPRPDDLEI, SEQ ID NO:137), among which the repressive peptides include Cx43 C-terminal amino acids 374-382 (RPRPDDLEI; SEQ ID NO:141), which contains a ZO-1 binding sequence that is then attached to a 16-amino acid antennal internalization vector at its N-terminus, mimicking the C-terminal tail of Cx43, and Gap24 (GHGDPLHLEEVKC, SEQ ID NO:138) which reproduces the sequence of the Cx32 cytoplasmic loop; while TAT-Gap24 (YGRKKRRQRRRGHGDPLHLEEVKC, SEQ ID NO:138) recreates the sequence of the Cx32 cytoplasmic loop; and TAT-Gap24 (YGRKKRRQRRRGHGDPLHLEEVKC, SEQ ID NO:137) NO:139 also mimics a portion of the intracellular loop of Cx43. L2 and Gap19 also mimic the intracellular loop of Cx43. Peptides useful in the compositions and methods of the present invention have been described and referenced herein, and include connexin mimics targeting the extracellular loop and intracellular regions of connexins, as well as peptides mimicking the C-terminal tail region of connexins (e.g., the C-terminus of connexin 43). Useful peptides and peptide mimics in the compositions and methods of the present invention include those listed in Tables II and III.

[0135] In some embodiments, the peptide and peptide mimicry are modified. In some embodiments, the peptide and peptide mimicry are unmodified. Peptides mimicry are molecules capable of mimicking natural peptides and proteins. Typically, peptide mimicry are structural or functional peptide analogs (e.g., identical or similar) of the prototype polypeptide (i.e., polypeptides with biological or pharmacological functions or activities), but peptide mimicry can also be modified, for example, by optionally replacing one or more peptide bonds with linkages selected from, for example, the following: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-. Peptides mimicry can also be chemically modified to include non-natural amino acid substitution, skeletal amide bond modification, rigid scaffolding, addition of hydrophobic residues, and other methods known in the art. Peptides mimicry can consist entirely of natural amino acids, synthetic chemical compounds, non-natural amino acid analogs, or can be chimeric molecules composed of a portion of natural peptide amino acids and a portion of non-natural amino acid analogs. Peptides mimicry can also contain any amount of conserved substitutions of natural amino acids, provided that such substitutions do not significantly alter the activity of the peptide mimicry. In the case of linker proteins, these peptide mimics can mimic the extracellular loop of paired linker proteins, for example, those involved in linker-linker docking and cell-cell channel formation. For instance, if a peptide mimic composition downregulates the biological function or activity of a half-channel, such as by preventing half-channel docking to form gap junction-mediated cell-cell communication, or by preventing half-channel opening to expose the cytoplasm to the extracellular environment, then the peptide mimic composition can be used as a gap junction regulator. Peptides mimicry include those described herein, as well as those known in the art (whether currently known or developed in the future). Peptides and peptide mimicry linker protein regulators may also be modified to improve stability, enhance bioavailability, and / or increase cell membrane permeability.

[0136] The term "pharmaceutical formulation" refers to a formulation in which the biological activity of the active ingredient contained therein is effective and free of any additional ingredients that would have unacceptable toxicity to the subject to which the formulation is to be administered. "Pharmaceutical composition" refers to a mixture of substances suitable for administration to a subject, containing an active ingredient or a pharmaceutical formulation. For example, a pharmaceutical composition may contain modified or unmodified antisense oligonucleotides and sterile aqueous solutions or poloxamer or pranic acid carriers.

[0137] As used herein, a “pharmaceuticalally acceptable carrier” refers to a component of a pharmaceutical preparation, other than the active ingredient, that can be safely administered to the target. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Pharmaceutically acceptable carriers include poloxamer (e.g., Pronek F-127). A pharmaceutically acceptable carrier for ocular application will be an ophthalmologically acceptable carrier suitable for application to the eye (e.g., intraocular or ocular surface).

[0138] As used herein, the terms “object”, etc. (including “individual” and “patient”, all of which are used interchangeably herein) refer to any mammal, including humans, livestock and farm animals, as well as animals in zoos, wildlife parks, sporting animals, or pets (such as dogs, horses, cats, sheep, pigs, cattle, etc.). Preferred mammals herein are humans, including adults, children, and the elderly. Preferred sporting animals are horses and dogs. Preferred pets are dogs and cats. Objects may be, for example, aquatic park animals (such as dolphins, whales, seals, or walruses). In some embodiments, the object, individual, or patient is a human. Objects are not intended to include animals used in scientific experiments (e.g., mice and rats).

[0139] As used herein, "gap junctions," "connexin gap junctions," and "connexin gap junction channels" refer to two hemichannels that bridge the intercellular gap between adjacent cells and allow specific molecules to flow between those cells. They are intercellular channels or clusters of intercellular channels that allow ions and small molecules to diffuse directly between adjacent cells. Gap junctions have been studied for decades and are well-known in the field.

[0140] As used herein, the term "half-channel" is part of a gap junction (two hemichannels or connexins that connect adjacent cells in the intercellular gap, forming a gap junction) and is composed of multiple connexins, typically homohexamers or heterohexamers of connexins, which form gap junction pores between the cytoplasm of two adjacent cells. A half-channel is provided by the cell on one side of the connection and the cell on the other side, where typically two hemichannels from opposing cells combine to form a complete intercellular gap junction channel. However, in some cells and under certain conditions, the half-channel itself can function as a channel between the cytoplasm and the extracellular space, allowing the transfer of ions and small molecules (e.g., ATP). Similar to their gap junction counterparts, connexin half-channels 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. The reference to "hemichannel" or "gap junction channel" should be understood to include hemichannels or gap junction channels present in any type of ocular cell and any type of ocular surface cell (including corneal and / or corneal epithelial cells). In one embodiment of the invention, hemichannels or gap junction channels are present in ocular cells. In one embodiment of the invention, hemichannels or gap junction channels are present in cells of the anterior segment (i.e., the anterior part of the eye). In some embodiments of the invention, hemichannels or gap junction channels are present in corneal cells. In some embodiments of the invention, hemichannels or gap junction channels are present in corneal epithelial cells. In some embodiments of the invention, hemichannels or gap junction channels are present in cells of ocular microvessels (including corneal microvessels). In some embodiments of the invention, hemichannels or gap junction channels are present in uveal cells. In some embodiments of the invention, a semi-channel or gap junction channel is present in cells associated with: dry eye, blepharitis (including, for example, eyelid cells or cells associated with the eyelid), Sjögren's syndrome-associated keratoconjunctivitis sicca (including, for example, conjunctival cells or cells associated with the conjunctiva), and Fuchs' dystrophy (including, for example, corneal endothelial cells).

[0142] "Non-healing ocular surface defects or conditions" refer to ocular surface defects or conditions that have not fully healed or closed after approximately 10 days to 2 weeks of standard clinical care. Non-healing ocular surface defects or conditions include ocular ulcers, corneal ulcers, persistent ocular epithelial defects (PED), and persistent corneal epithelial defects (PCED). Ocular ulcers include corneal ulcers, and also those described herein, including infected ulcers, chemical ulcers, burn ulcers, traumatic or trauma-induced ulcers, inflammatory ulcers, and ulcers of mixed etiology. "Non-healing" or "persistent" ocular surface or corneal defects or conditions (including, for example, PED and PCED) that fail to achieve epithelial regeneration and closure within 10–14 days, even with standard supportive care.

[0143] The phrase "basically composed of..." refers to the specified materials and materials that do not materially affect the essential and novel characteristics of the medicament (or, in the case of a method, the steps). The essential and novel characteristics of this invention are described throughout the specification and include the ability of the connexin modulators, connexin modulator compositions, and methods of this invention to: block or modulate connexins, connexin gap junctions, and / or connexin hemichannels, and to attenuate, block, or inhibit, for example, the generation, function, or activity of connexins, connexin gap junctions, and / or connexin hemichannels, or inflammasome activity (e.g., hemichannel-mediated inflammasome activity, such as connexin hemichannel-mediated or panconnexin hemichannel-mediated inflammasome activity). Substantial changes to the essential and novel features of this invention (including the drugs and methods described herein) include the following: unintended or clinically unfavorable, harmful, detrimental, or negative activity in the healing of the diseases, conditions, or illnesses described or mentioned herein; delayed healing; weakened regulation and / or inhibition of connexins, connexin gap junctions, and / or connexin hemichannels; or unintended weakening of the activity or function of connexins, connexin gap junctions, and / or connexin hemichannels. In one embodiment, the composition or drug of this invention comprises, is substantially composed of, or is composed of: a connexin 43 hemichannel blocker, such as a connexin 43 antisense molecule, a connexin 43 peptide, or a small molecule connexin 43 hemichannel blocker or a prodrug thereof.

[0144] Antisense modifier The gap linker and / or linker protein polynucleotide or oligonucleotide may be selected from, for example, modified or unmodified linker protein polynucleotides or oligonucleotides, such as modified or unmodified linker protein 43 antisense polynucleotides or oligonucleotides. In some embodiments, the modified linker protein antisense polynucleotide or oligonucleotide or polynucleotide comprises a mixture of modified and unmodified nucleotides. In some embodiments, the linker protein 43 antisense compound used in the methods herein is an antisense oligonucleotide comprising a naturally occurring nucleoside base and an unmodified nucleoside internucleotide bond.

[0145] In some implementations, the connexin 43 antisense compound targets at least about 8 nucleoside bases in a nucleic acid molecule encoding the connexin (having a nucleoside base sequence selected from SEQ ID NO:17). Polynucleotides and oligonucleotides (e.g., the antisense compound of connexin 43) may have 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, or their complementary sequences, as specified in SEQ ID NO:17; and / or the antisense polynucleotide or oligonucleotide may contain any length within any range between any two of the referenced lengths. The polynucleotides of the present invention include synthetic polynucleotides having a length of less than 80 nucleotides, for example from 12-18 to about 50-80 nucleotides, preferably about 30 nucleotides or shorter (e.g. from 12 to about 30 nucleotides), and more preferably from about 15 to about 30 nucleotides. In one example, the polynucleotide has 30 nucleotides. In some embodiments, the method of the present invention is characterized by using a linker protein 43 antisense compound (e.g., 15 to 40 nucleotides in length) with a length of up to 40 nucleotides, the compound comprising or substantially consisting of nucleotide sequences selected from SEQ ID NO:1-17. In some embodiments, the method of the present invention is characterized by using a linker protein 43 antisense compound (e.g., 15 to 40 nucleotides in length) with a length of up to 40 nucleotides, the compound comprising nucleotide sequences selected from SEQ ID NO:4-17.

[0146] Human Cx43, 1 (SEQ ID NO:17) LOCUS NM_000165 3088 bp mRNA linear PRI 26-OCT-2004 DEFINITION Homo sapiens gap connexin, α1, 43 kDa (connexin 43) (GJA1), mRNA.

[0147]

[0148]

[0149] The modified oligonucleotide may contain, for example, one or more of the following selected components: modified nucleoside internucleotides (e.g., phosphate thioester bonds) and modified sugar moieties (e.g., conformationally restricted sugars, such as locked nucleic acids (LNA) or bridged nucleic acids (BNA)).

[0150] Chemical modifications to the antisense polynucleotides disclosed or cited herein can enhance their resistance to nucleases and improve their ability to enter cells. For example, phosphate-thioester oligonucleotides can be used. Other deoxynucleotide analogs include methylphosphonates, aminophosphates, dithiophosphates, N3'P5'-aminophosphates, and oligonucleotide phosphate-thioesters and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates. Alternatively, mixed backbone oligonucleotides (“MBOs”) can be used. MBOs contain a phosphate-thioester oligodeoxynucleotide fragment and appropriately configured modified oligodeoxyribonucleotide or oligoribonucleotide fragments. MBOs have a phosphate-thioester bond fragment and other modified oligonucleotide fragments (such as nonionic and highly nuclease-resistant methylphosphonates, or 2'-O-alkyl oligoribonucleotides). Methods for preparing modified backbones and mixed backbone oligonucleotides are known in the art.

[0151] In some embodiments, the antisense polynucleotides disclosed or cited herein may include oligonucleotide sugar moieties as modified sugar moieties. In some embodiments, the modified sugar moieties may be thiol-containing sugar moieties or sugar moieties as conformationally restricted sugars. In some embodiments, the conformationally restricted sugar may be locked nucleotides (locked nucleic acids or LNAs). In some embodiments, the locked nucleotide may 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′ (sulfur-LNA), and 2′-Se—CH2-4′ (selenium-LNA). In some embodiments, the conformationally restricted sugar may be bridging nucleic acids (BNAs). Some conformationally restricted sugars that may be locked nucleic acids are shown in Formulas III and IV of U.S. Patent No. 10,465,188.

[0152] This document also provides modified or unmodified antisense polynucleotides of epithelial, endothelial, corneal epithelial, and / or vascular endothelial connexins that can be used in the methods of the present invention, comprising 8 to 80 nucleotides in the connexin extracellular loop, intracellular region, C-terminus, or other regions, for regulating 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. The polynucleotides include synthetic polynucleotides having a length of less than 80 nucleotides, for example from 12-18 to about 50-80 nucleotides, preferably about 30 nucleotides or shorter (e.g., from 12 to about 30 nucleotides), more preferably from about 15 to about 30 nucleotides. In one example, the polynucleotide has 30 nucleotides.

[0153] In some embodiments of the invention, linker protein 43 or other antisense oligonucleotides or polynucleotides have at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% homology with polynucleotides having sequences selected from SEQ ID NO: 1 to 17. Linker protein regulators, as oligonucleotides or polynucleotides, may have at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% homology with 8 to 80 nucleotide portions of their respective sequences.

[0154] Table I. List of some implementation schemes for linker protein 43 antisense oligonucleotides

[0155] Table I lists the polynucleotide sequences of several embodiments of linker protein 43 polynucleotide regulators that can be used in the methods of the present invention. When referring to sequences such as SEQ ID NO:1-16, they, along with other Cx43 and other linker protein antisense compounds, represent modified and unmodified oligonucleotides or polynucleotides. In some embodiments, the linking bonds between nucleotides and the sugar moiety structure of the nucleotides may be modified. In some embodiments, the internucleotide bond between any two nucleotides may be a standard phosphodiester bond. In some embodiments, the internucleotide bond between any two nucleotides may be a phosphate thioester bond. For example, SEQ ID NO:1 may 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), where " s" " indicates a phosphate thioester bond between two nucleotides. As another non-limiting example, SEQ ID NO:1 could be the structure (G)(T)(A)(A)TTGCGGCAAGAA GAATTGTTTC(T)(G)(T)(C) (SEQ ID NO:357), wherein the nucleotides within the brackets have a sugar moiety modified as described below. In some embodiments, the Cx43 antisense compound can be modified by replacing one or more thymidine nucleotides with one or more uridine nucleotide residues in the sequence of SEQ ID NO:1-17 or other linker proteins (e.g., Cx26, Cx32, Cx45, etc.).

[0156] Certain connexin regulators (including, for example, connexin 43 regulators) can downregulate connexin expression (e.g., by downregulating mRNA transcription or translation), or otherwise reduce or inhibit the activity of connexins, connexin hemichannels, or gap junctions. In the case of downregulation, this will have the effect of reducing direct cell-to-cell communication via gap junctions or reducing the exposure of the cytoplasm to the extracellular space via hemichannels at the site where connexin expression is downregulated.

[0157] In some implementations, anti-connector antisense molecules prevent, reduce, or alter the activity or function of hemichannels or gap junctions. As described herein, modulating the activity or function of gap junctions by anti-connector antisense compounds can lead to gap junction closure, hemichannel closure, and / or the flow of molecules or ions through gap junctions and / or hemichannels.

[0158] Connector regulators may also contain one or more polynucleotides selected from, for example, morpholino oligonucleotides, RNAi molecules, siRNA molecules, PNA molecules, deoxyribonucleases (DNAzymes), and 5'-mutated U1 small nuclear RNAs, as well as analogs thereof. These and other compounds may be used alone or in combination with one or more connector regulators.

[0159] It can synthesize antisense polynucleotides and other anti-linking protein polynucleotides (such as RNAi, siRNA, and ribozyme polynucleotides), as well as polynucleotides with modified and mixed backbones. See, for example, Stein CA and Krieg A.M. (eds.), Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Liss).

[0160] Antisense polynucleotides can inhibit the transcription and / or translation of connexins (e.g., connexin 43). Antisense polynucleotides are typically antisense connexin mRNA (e.g., connexin 43). Such polynucleotides are capable of hybridizing with connexin mRNA and thus can inhibit connexin expression by interfering with one or more embodiments of connexin mRNA metabolism, including transcription, mRNA processing, mRNA transport from the nucleus, translation, or mRNA degradation. Antisense polynucleotides typically hybridize with connexin mRNA to form a duplex that directly inhibits translation and / or causes mRNA instability. This duplex may be readily degraded by nucleases. Preferably, the polynucleotide is a specific inhibitor of the transcription and / or translation of the connexin 43 gene or mRNA and does not inhibit the transcription and / or translation of other genes or mRNAs.

[0161] The connexin regulator product can bind to the connexin 43 gene or mRNA: (i) binding to the 5' end of the coding sequence and / or (ii) binding to the coding sequence and / or (iii) binding to the 3' end of the coding sequence. An antisense polynucleotide can hybridize with a portion of the connexin mRNA, such as connexin 43 mRNA. Typically, the antisense polynucleotide hybridizes with the ribosome-binding region or coding region of the connexin mRNA. The polynucleotide can be complementary to a region of the connexin mRNA. For example, the polynucleotide can be completely complementary to a portion of the connexin mRNA. However, complete complementarity is not required, and polynucleotides with sufficient complementarity to form a double helix (with a melting temperature greater than about 20°C, 30°C, or 40°C under physiological conditions) are particularly suitable for use in this invention. Therefore, the polynucleotide is generally a homolog of the sequence complementary to the mRNA. The polynucleotide can be a polynucleotide that hybridizes with the connexin mRNA under medium to high stringency conditions (such as about 50°C to about 60°C, 0.03M sodium chloride, and 0.03M sodium citrate).

[0162] Antisense polynucleotides may be included as part of the composition, and may contain polynucleotides targeting more than one connexin. Preferably, the connexin targeted by the polynucleotide is connexin 43. Others include connexins present in the ocular and / or corneal epithelium. Some embodiments of the invention are described using oligodeoxynucleotides as examples. However, other suitable polynucleotides (such as RNA polynucleotides) may also be used.

[0163] Peptide modulators In some embodiments, the connector modulators (including connectors, connector gap junctions, and connector hemichannel modulators) that can be used in the methods of the present invention include not only connector oligonucleotides or polynucleotides (such as connector 43 antisense oligonucleotides or polynucleotides), but also connector peptides or peptide mimics (such as connector 43 peptides or peptide mimics, sometimes also referred to as anti-connector peptides or peptide mimics, e.g., anti-connector hemichannel blocking peptides or peptide mimics), which can be used in the methods of the present invention and can be administered according to one or more of the regimens described herein. They include, for example, peptides or peptide mimics comprising, or substantially comprising, a sequence corresponding to portions of the connector extracellular domain, the connector transmembrane region, and the connector C-terminal region. In some embodiments, the connectors, connector gap junctions, and connector hemichannel modulators that can be used in the methods of the present invention include connector 43 peptides or peptide mimics (a sequence having a portion of the amino acid sequence according to SEQ ID NO: 100 (connector 43)).

[0164] In some embodiments, the connexin modulator used in the methods of the present invention for treating non-healing ocular defects or conditions (e.g., PED) (including non-healing corneal defects or conditions, such as PCED) is an anti-connexin 43 peptide or peptide mimicry. In some embodiments, the anti-connexin 43 peptide or peptide mimicry used in the methods of the present invention and administered according to one or more of the regimens described herein is a peptide comprising or substantially composed of a portion of the extracellular domain of a connexin (e.g., connexin 43), and / or a peptide comprising or substantially composed of a portion of the carboxyl-terminal portion of a connexin (e.g., connexin 43), including those described and / or referenced herein.

[0165] The protein sequence of linker protein 43 is shown below.

[0166] Connector protein 43 (SEQ ID NO:100)

[0167]

[0168] In some embodiments, connexin 43 (Cx43) and other connexin peptide modulators that can be used to implement the methods of the present invention and administered according to one or more of the regimens described herein include peptides such as Peptide5 (i.e., VDCFLSRPTEKT, SEQ ID NO:107), Gap19 (i.e., KQIEIKKFK, SEQ ID NO:108), Gap26 (i.e., VCYDKSFPISHVR, SEQ ID NO:102), Gap27, and a peptide called α-connexin C-terminal 1 (αCT1), each peptide targeting different binding sites with varying specificity and size. The connexin peptide Gap27, targeting 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 universal inhibitor of connexin-mediated communication. In some embodiments, the linker protein 43 regulator may comprise or consist substantially of a peptide or peptide-like substance, such as SRPTEKTIF (SEQ ID NO: 110).

[0169] In addition to Peptide5, Gap19, Gap26, and Gap27, another peptide mimic that is particularly useful in the compositions, dosages, administration methods and regimens, kits, and articles disclosed herein is a fusion peptide named XG19, namely lclrpvGGKQIEIKKFK, where the lowercase letters represent the D-isomer [SEQ ID NO:111]. As described above, the regulatory effects of the XG19 peptide and its connexins are described in U.S. Patent No. 11,466,069, which is incorporated herein by reference in its entirety.

[0170] In some embodiments, the present invention provides a composition for treating an eye disease or condition, the composition comprising a construct comprising (a) a targeting carrier peptide derived from the X protein of hepatitis B virus, and (b) a peptide capable of interacting with an intracellular domain of a connexin. In some embodiments, the targeting carrier peptide derived from the X protein of hepatitis B virus comprises an amino acid sequence selected from all the targeting carrier peptides described in U.S. Patent No. 11,466,069. In some embodiments, the peptide capable of interacting with an intracellular domain may interact with one or more intracellular domains of connexins 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 an intracellular domain may interact with an intracellular domain of connexin 43. In some embodiments, peptides capable of interacting with the intracellular domain of connectin 43 include any connectin 43 intracellular interacting peptides described in U.S. Patent No. 11,466,069, as well as those peptides described herein that interact with the intracellular domain of connectin 43.

[0171] In some embodiments, the linker protein 43 modulator that can be used in the methods of the present invention and administered according to one or more of the schemes described herein may comprise a peptide having the following sequence, which comprises or is substantially composed of the following: for example, one or more of the following sequences: “Peptide1” (ADCFLSRPTEKT, SEQ ID NO: 112), “Peptide2” (VACFLSRPTEKT, SEQ ID NO: 113), “Peptide11” (VDCFLSRPTAKT, SEQ ID NO: 114), “Peptide12” (VDCFLSRPTEAT, SEQ ID NO: 115), “Peptide5” (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 linker protein peptide or peptide modulator may be modified. In some embodiments, the carboxyl terminus modification may comprise an n-alkyl chain, which may optionally be further linked with hydrogen or other portions. In some embodiments, the linker protein 43 peptide may include or exclude any peptides listed above or disclosed herein.

[0172] In some embodiments, the peptide or peptide-like substance comprises or is substantially composed of 7 to 40 amino acids of a connexin, including, for example, SEQ ID NO:101 (SRPTEKT) and SEQ ID NO:107 (VDCFLSRPTEKT), and does not contain a C-terminal peptide of the connexin.

[0173] Anti-connector agents include peptides comprising about 5 to 20 consecutive amino acids of a connector protein (such as connector 43, SEQ ID NO: 100), about 8 to 15 consecutive amino acids of connector 43, or about 11 to 13 consecutive amino acids of connector 43. Other anti-connector agents include peptides comprising at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, or at least about 30 consecutive amino acids of connector 43. Other anti-connector 43 modulators comprise the extracellular domain of connector 43, for example, peptides or peptide-like substances comprising or substantially consisting of the following: SRPTEKT (SEQ ID NO: 101) or VDCFLSRPTEKT (SEQ ID NO: 107).

[0174] In other anti-connector protein compounds, the peptide-mimicking is based on the extracellular domain of connector 43, corresponding to amino acids 37–76 and 178–208 of the connector 43 protein sequence. Therefore, certain anti-connector protein peptides usable in the methods of the present invention have an amino acid sequence comprising or substantially consisting of about 7 to about 40 amino acids corresponding to the region at positions 37–76 and 178–208 of the connector 43 protein sequence. The amino acid sequence of the peptide need not be identical to those portions of the connector 43 protein sequence, and conserved amino acid substitutions are permissible such that these peptides retain binding or functional activity in assays described herein and otherwise known in the art. In other embodiments, the peptide-mimicking is based on peptide-targeting regions of the connector protein other than the extracellular domain (e.g., portions of the connector 43 protein sequence not corresponding to positions 37–76 and 178–208).

[0175] In addition to therapeutically effective modified or unmodified peptides or peptide mimics comprising, for example, a portion of the extracellular domain, transmembrane domain, or C-terminal domain of connexin 43, other embodiments include modified or unmodified peptides or peptide mimics comprising, for example, a portion of the extracellular domain or transmembrane domain of one or more other connexins present in the ocular and / or corneal epithelium, which can be used in the methods of the present invention and administered according to one or more of the regimens described herein. Other embodiments include modified or unmodified peptides or peptide mimics comprising, for example, a portion of the extracellular domain or transmembrane domain of one or more other connexins present in blood vessels (e.g., endothelium, including ocular and / or corneal vessels).

[0176] In some embodiments, the anti-negotiation peptide that can be used in the methods of the present invention and administered according to one or more of the methods described herein is a connegotiation 45 peptide modulator, which comprises a portion of connegotiation 45 protein that antagonizes, inhibits, or blocks connegotiation-negotiation interactions. In some embodiments, the connegotiation 45 modulator may comprise, for example, a peptide or peptide consisting of, or substantially consisting of, a portion of the E2 domain or C-terminal domain of connegotiation 45, such as a peptide or peptide comprising SRPTEKT (SEQ ID NO: 101). The peptide or peptide may also comprise, for example, DCFISRPTEKT (SEQ ID NO: 118). Exemplary peptide sequences of connegotiation 45 peptides and peptide modulators that can be used in the methods of the present invention are also provided in Table 63 of U.S. Patent No. 10,465,188.

[0177] In some embodiments, the connexin regulator comprises a peptide containing an amino acid sequence corresponding to a portion of the transmembrane region of connexin 45 or the C-terminal region of connexin 45. For example, in certain non-limiting embodiments, the anti-connexin compound is a peptide whose amino acid sequence comprises about 3 to about 30 consecutive amino acids of a known connexin 45 sequence, a peptide whose amino acid sequence comprises about 5 to about 20 consecutive amino acids of a known connexin 45 sequence, a peptide whose amino acid sequence comprises about 8 to about 15 consecutive amino acids of a known connexin 45 sequence, or a peptide whose amino acid sequence comprises about 11, 12, or 13 consecutive amino acids of a known connexin 45 sequence. Other non-limiting embodiments include: the anti-connexin compound is a peptide whose amino acid sequence comprises 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 some of the anti-connector protein compounds described herein, the peptide mimicry is based on the extracellular domain of connector 45, which corresponds to amino acids 46–75 and 199–228 of the known connector 45 sequence. Therefore, the amino acid sequence of certain peptides described herein corresponds to the region of 46–75 and 199–228 of the known connector 45 sequence. The amino acid sequence of the peptide need not be identical to those portions of the known connector 45 sequence, and conserved amino acid substitutions may be made such that these peptides retain binding or functional activity in assays described herein and otherwise known in the art. In other embodiments, the peptide mimicry is based on peptide-targeting regions in the connector protein other than the extracellular domain (e.g., portions of the known connector 45 sequence that do not correspond to positions 46–75 and 199–228). See WO2006 / 134494, which discloses various connector protein sequences.

[0178] Some peptide-linking protein modulators that can be used in the methods 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.

[0179] In some embodiments, the connexin 26 peptide-modifying agent that can be used in the methods of the present invention and administered according to one or more of the regimens described herein is Gap26. In other embodiments, the anti-connexin peptide that can be used in the methods of the present invention is a connexin 32 peptide-modifying agent (e.g., INCTLQPGCNSV (SEQ ID NO: 103); or 32Gap27, namely SRPTEKTIFII (SEQ ID NO:104) or connexin 50 peptide (e.g., TAT-Cx50L2, namely GGERAPLAADQGSVKKSSSSSKGTKK (SEQ ID NO:105) or TAT-Cx50CT, namely SRARSDDLTV (SEQ ID NO:106)).

[0180] In some embodiments, the peptides and peptide mimics include peptides and peptide mimics that can be used to inhibit gap junction channels and hemichannels, corresponding to specific sequences within extracellular loops E1 and E2 involving the conserved QPG and SHVR (SEQ ID NO: 359) motifs of E1 (Gap26 peptide) and the SRPTEK motif (SEQ ID NO: 360) of E2 (Gap27 peptide), as well as an intracellular loop (Gap19 peptide). Useful peptide mimic connexin modulators are described in U.S. Patent No. 9,248,141 (“Therapeutic Treatment by Administration of Anti-Binding Protein Peptides and Peptides Mimics”). Other useful peptide connexin modulators are described in U.S. Patent No. 11,466,069 (“Therapeutic Treatment and Novel Constructs”), including XG19 and other constructs containing a targeting carrier peptide derived from the X protein of hepatitis B virus, and peptides capable of interacting with intracellular domains of connexins (e.g., connexin 43).

[0181] Other peptide-linking protein modulators that can be used in the methods of the present invention are provided in Table 64 of U.S. Patent No. 10,465,188. Also see Caufirez et al. and King, DR et al., Mechanisms of Connexin Regulating Peptides, for details on useful peptide modulators of connexin 43 (Cx43) and other connexins that can be administered in embodiments of the present invention. Int. J. Mol. Sci. 22:10186 (Sept 2021) and Figure 1 ("Schematic diagram of Cx43 protein in plasma membrane, with colored lines marking the locations of the peptides targeting the EL, IL and CT regions") and Table 1 ("Binding protein peptides").

[0182] In some implementations, these peptides may also be used as prodrug components. See, for example, Vig, BS et al., Amino acids as promoies in drug design and development. Advanced Drug Delivery Reviews65(10): p 1370-1385 (2013); Dhokchawle, B et al., PromoietiesUsed In Prodrug Design: A Review. Indian Journal of Pharmaceutical Education 48(2):35-40 (2013).

[0183] Gap join regulators or anti-joint protein hemichannel blocking peptides / peptides can be unmodified or modified as needed (e.g., to improve stability, further stabilize peptide conformation, enhance biological activity, improve 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 peptides used in the methods of the present invention may contain one or more modified amino acids, amino acid analogs, or may be otherwise modified to improve bioavailability or improve transcellular membrane permeability.

[0184] Other known peptide sequences that can inhibit connexin binding (and thus modulate connexin activity) are the intracellular loop (amino acids 119–144) L2 peptide of connexin 43 and fragments of the L2 peptide of connexin 43. In some embodiments, these peptides may include or exclude sequences such as: the 9-amino acid sequence of Gap19 KQIEIKKFK (SEQ ID NO: 108); the native Gap19 sequence DGVNVEMHLKQIEIKKFKYGIEEHGK (SEQ ID NO: 119); and the His144→Glu L2 derivative of Gap19 reported by Shibayama (Shibayama, J et al.). Biophys. J91, 405404063, 2006), DGVNVEMHLKQIEIKKFKYGIEEQGK (SEQ ID NO:120); TAT-Gap19 sequence, YGRKKRRQRRRKQIEIKKFK (SEQ ID NO:121); SH3 binding domain, CSSPTAPLSPMSPPGYK (SEQ ID NO:122) or fragment PTAPLSPMSPP (SEQ ID NO:123); C-terminal sequence of CT9 or CT10 peptide (with or without TAT guide sequence for enhancing cell permeability), such as RPRDDIEI (CT9, SEQ ID NO:124), SRPRDDLEI (CT10, SEQ ID NO:125), YGRKKRRQRRRSRPRDDEI (TAT-CT9, SEQ ID NO:126) or YGRKKRRQRRRRPRDDEI (TAT-CT10; SEQ ID NO:127). Other peptide-like sequences that may be included or excluded in the compositions, methods, kits, or articles 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). Rotigatide is composed of D-type peptides, which are more potent than natural L-type peptides.

[0185] In some embodiments, the therapeutically effective modified or unmodified peptide / peptide mimic comprises a portion of the E1 extracellular domain of a connexin, such as connexin 43 E1 (ESAWGDEQSAFRCNTQQPGCEN VCYDKSFPISHVR; SEQ ID NO: 130) or connexin 45 E1 (GESIYYDEQSKFVCNTEQPGCE NVCYDAFAPLSHVR; SEQ ID NO: 131). In some embodiments, the therapeutically effective modified or unmodified peptide / peptide mimic comprises a portion of the E2 extracellular domain of a connexin, such as connexin 43 E2 (LLIQWYIYGFS LSAVYTCKRDPCPHQVDCFLSRPTEKT; SEQ ID NO: 132) or connexin 45 E2 (LIGQYFLY GFQVHPFYVCSRLPCHPKIDCFISRPTEKT; SEQ ID NO: 133).

[0186] In some embodiments, the linker protein 43 regulatory peptide of the present invention may be linked to an internalization transporter at its amino or carboxyl terminus. The internalization transporter linked to the linker protein 43 regulatory peptide of the present invention may be any internalization sequence known or newly discovered in the art, or a conserved variant thereof. Non-limiting examples of internalization transporters and sequences include antennapedia sequences, TAT, HIV-Tat, transmembrane peptides, Antp-3A (Antp mutant), Buforin II, transport peptides, MAP (model amphiphilic peptide), K-FGF, Ku70, prions, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidino-spermine-cholesterol), and BGTC (bis-guanidino-triethylenetetramine-cholesterol). The internalization transporter may be used for peptide mimics such as Gap19 and aCT peptides. The sequences of exemplary internalization peptides are known in the art. See, for example, Table 65 of U.S. Patent No. 10,465,188; and U.S. Patent No. 11,466,069.

[0187] In one embodiment, the peptide sequence CFLSRPTEKT (SEQ ID NO: 116) or VDCFLSRPTEKT (SEQ ID NO: 107) can be conjugated with two dodecyl groups to form a modified peptide “C12-C12-Cxn43 MP” (SEQ ID NO: 358) that modulates connector protein 43. 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.

[0188]

[0189] In the structure C12-C12-Cxn43 MP (SEQ ID NO:358), R1 and R2 can be hydrogen or alkyl. In some respects, R1=R2=a dodecyl chain.

[0190] In some embodiments, the therapeutically effective modified or unmodified peptide or peptide mimic comprises a portion of the C-terminal domain of a connexin (such as connexin 43 or connexin 45, preferably connexin 43). Embodiments of some anti-connexin 43 modulators that can be used in the methods of the present invention comprise the C-terminal region of connexin 43 or a modified form thereof. See, for example, O'Quinn, MP et al., A Peptide Mimetic of the Connexin43 Carboxyl-TerminusReduces Gap Junction Remodeling and Induced Arrhythmia Following Ventricular Injury. Circ Res 108(6): 704-715 (Mar 2011). C-terminal linker peptide modulators, including αCT1 (α-linker C-terminal 1) peptide (also referred to as aCT1 or ACT1 peptide in publications), as described, for example, in Montgomery et al., Connexin 43-Based Therapeutics for Dermal Wound Healing Int. J. Mol. Sci. 2018, 19 See also U.S. Patent No. 1778 and U.S. Patent No. 8,815,556 (“Compositions and Methods for Tissue Engineering, Tissue Regeneration, and Wound Healing”). See also WO2006 / 069181. A preferred connexin C-terminal peptide is the connexin 43 C-terminal peptide. Such compounds are described in U.S. Patent Publication No. 20070042964 (“Compositions and Methods for Modulating Connexin Hemichannels”). If the connexin peptide or peptide modulator contains a portion of an intracellular domain of the connexin (e.g., an aCT peptide, such as CT9, CT10, αCT1, etc.), the peptide can bind to internalized transporters (including those mentioned or cited herein). In some embodiments, the connexin peptide modulator used in the methods of the present invention can block the binding of tight connexin (ZO-1) to connexin 43 and advantageously modulate connexin gap junctions and hemichannels. See Caufriez, A et al., Peptide-based targeting of connexins and pannexins for therapeutic purposes. Expert Opinion on Drug DiscoveryFigure 2 in 15(10):1213-1222(2020).

[0191] In some embodiments, the connexin regulator may be a gap-closing compound and / or a hemichannel-closing compound. In some embodiments, the gap-closing compound and the hemichannel-closing compound are connexin 43 gap-closing compounds and connexin 43 hemichannel-closing compounds (e.g., Cx43 C-terminal pseudopeptide).

[0192] Various useful peptide mimics simulate the sequences of extracellular regions of connective proteins. The peptide under the designation Gap26 mimics the first extracellular loop of Cx37, Cx40, and Cx43. Gap27 and Peptide5 mimic the region of the second extracellular loop. Gap27 targets Cx32, Cx40, and Cx43, while Peptide5 inhibits Cx43. JM2, ΔSH3, CT9, CT10, and αCT mimic the C-terminal tail of Cx43, and Gap24 reproduces a segment of the intracellular loop of Cx32. L2 and Gap19 also mimic the intracellular loop of Cx43.

[0193] The extracellular loops of connector hemichannels are also good targets for the peptide-mimicking connector inhibitors useful in this invention because they are more accessible than their intact 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 gap junction formation. This leads to peptide-mimicking... 43 Gap26 37,40 Gap26 32 Gap27 40 Gap27 43 Gap27 and 43 The development of Peptide5 (note that the superscript in the names of these peptide analogs refers to the Cx subtypes they can target).

[0194] The conserved SHVR motif (SEQ ID NO:359) of the first extracellular loop was incorporated into the sequence of the useful Gap26 peptide. Two slightly different sequences were both classified under the designation Gap26, one targeting Cx37 and Cx40, and the other targeting only Cx43. Cells treated with one of these Gap26 peptides showed Cx half-channel inhibition within minutes.

[0195] There are three Gap27 peptides, each targeting different Cx types, namely Cx32, Cx40, and Cx43, which can be used 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-joint activity as Gap26. Similar to Gap27, Peptide5 contains an SRPTEK motif (SEQ ID NO:360). However, compared to Gap27, the mimicked sequence of Peptide5 is directed towards... N End-to-end directional shift. Peptide 5 can inhibit Cx43 half-channels at concentrations of 5–10 μM, but incubation at higher concentrations (100 μM or higher) can also lead to inhibition of gap junctions in some cases.

[0196] Other peptides that can be used in the compositions and methods of the present invention mimic sequences of intracellular regions of connective proteins. The interaction between the intracellular loop and the C-terminal tail mediates the gating mechanism of the Cx hemichannel and gap junction. When there is no interaction between the C-terminal tail and the intracellular loop, the gap junction is open, and this interaction is crucial for the opening of the Cx hemichannel. The CT10 peptide mimic recreates the last 10 amino acids of the C-terminal tail of Cx43. Inhibition of Cx43-mediated ATP release by a peptide mimic named TAT-L2 indicates that its mimicked L2 region (amino acids 119 to 144) is a key sequence in the intracellular loop that interacts with the Cx43 C-terminal tail. To date, two peptides mimicking the L2 region have been obtained, namely… 43 Gap19 and 32 Gap24. 43 Gap19 inhibits intracellular loop / C-tail interactions by binding to the C-tail and suppressing the Cx43 half-channel current. 43 The advantage of Gap19 is that it is a selective inhibitor because it does not affect the activity of gap junctions or pan-connective protein 1 channels. 32 Gap24 is a 13-amino acid long peptide that mimics the L2 region of Cx32 and can also be used in the method of this invention. Cx32 is one of the ten human corneal epithelial connexins that can be effectively targeted as described herein. In vitro studies have shown that... 32 Gap24 at a concentration of 17 μM can inhibit Cx32 half-channel-mediated ATP release without affecting gap junctions.

[0197] Peptides targeting intracellular regions of Cx proteins require entry into the intracellular environment. Cell-penetrating peptides for this purpose (e.g., TAT peptides, oligoarginine tags, and Xentry peptides) are described herein and can be anchored to Cx-derived peptide sequences to enhance cellular uptake via endocytosis. 43Gap19 itself can enter cells because of the KKFK cell transport motif in the L2 region (SEQ ID NO:361). Nevertheless, for 43 Gap19 itself (47 μM) and TAT- 43 A comparison of the IC50 (half-maximal inhibitory concentration) of Gap19 (7 μM) in inhibiting ATP release in glioma cells showed that linkage with the TAT tag improved [the inhibition rate]. 43 Gap19 enters cells. All five available C-terminal-mimicking peptides are derived from Cx43.

[0198] αCT1 mimics the last nine amino acids of Cx43 and is linked to an antennal foot protein sequence that promotes peptide internalization. αCT1 disrupts the interaction between Cx43 and the PDZ domain of ZO-1 (a region thought to be involved in the regulation of Cx transport and gap junction assembly). This disruption has been reported to lead to increased gap junction plaque formation and decreased Cx half-channel activity.

[0199] If the peptide or peptide modulator contains part of the intracellular domain of a linker protein, in some embodiments the peptide may bind to an internalization transporter and, in some cases, may block the binding of tight junction protein (ZO-1) to Cx43.

[0200] In some embodiments of the invention, the linker protein regulator is a peptide or peptide-like substance as shown in Table II below (E2 and T2 refer to, for example, the position of the peptide in a second extracellular domain or a second transmembrane domain).

[0201] Table II

[0202]

[0203]

[0204]

[0205] In some embodiments, the connexin 43 regulator may comprise, for example, a peptide or peptide mimic comprising SEQ ID NO:101 (SRPTEKT). The peptide or peptide mimic may also comprise, 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 transmembrane permeability. For example, SEQ ID NO:107 may be modified to obtain SEQ ID NO:177-191 and 311-313. In some embodiments, the peptide or peptide mimic comprising, for example, SEQ ID NO:101 (SRPTEKT) or SEQ ID NO:107 (VDCFLSRPTEKT) comprises from 7 to 40 amino acids or amino acid analogs and does not contain a C-terminal peptide. In some embodiments, these peptides may also be used as a prodrug moiety.

[0206] In some embodiments, connexin 45 regulators may be peptides or peptide mimics comprising a portion of the connexin 45 protein, which antagonize, inhibit, or block connexin-connexin interactions. Exemplary peptide sequences of connexin 45 peptides and peptide mimic regulators are provided in Table III.

[0207] Table III. Sequences of the connector protein 45 regulatory peptide or peptide-like peptide

[0208]

[0209] In some embodiments, the connexin 45 regulator may comprise, for example, a peptide or peptide-like substance comprising a portion of the E2 domain or C-terminal domain of connexin 45, such as the peptide or peptide-like substance comprising SEQ ID NO:101 (SRPTEKT). The peptide or peptide-like substance may also comprise, for example, SEQ ID NO:279 (DCFISRPTEKT). In some embodiments, the peptide may be only 3 amino acids long, including SRL, PCH, LCP, CHP, IYY, SKF, QPC, VCY, APL, HVR, or longer.

[0210] When a particular protein is referred to herein, its derivatives, variants, and fragments are considered and covered. Protein derivatives and variants are well known to those skilled in the art and may include inserted, substituted, or deleted amino acid sequence variants known in the art.

[0211] Gap junction modulators and anti-connector protein hemichannel blocking peptides or peptides are manufactured by chemical, synthetic or other means.

[0212] Connecting protein hemichannel regulators Any modulator capable of producing a desired inhibition of the entry (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 is permitted for use in embodiments of the invention and for administration in one or more of the methods described herein. In certain embodiments, any connexin agent capable of modulating the entry of molecules through gap junctions or connexin hemichannels (e.g., those that modulate, block, or reduce the entry of molecules from the cytoplasm into the extracellular space or adjacent cell cytoplasm, including ATP) is also provided. Such anti-connexin modulators can modulate the entry of molecules through gap junctions or connexin hemichannels with or without gap junction uncoupling (blocking molecule transport through gap junctions). Such compounds include, for example, binding proteins (e.g., single-chain antibodies (scFv), antibodies, etc.), peptides (e.g., peptide mimics), and organic compounds (e.g., tonabothia and compounds of formula I and / or II) that can, for example, wholly or partially block the function or activity of gap junctions or hemichannels (e.g., by modulating the release of ATP from connexin hemichannels).

[0213] In some embodiments, the modulators used in the methods of the present invention are gap junction closure or blocking compounds or hemichannel closure or blocking compounds (e.g., tonabosar). In some embodiments, the modulators may be small molecules, and may also be referred to herein as anti-connector proteins, or connector protein modulators, or connector protein gap junction modulators, or connector protein hemichannel modulators. In some embodiments, the methods of the present invention are characterized by the use of a compound of formula I (e.g., tonabosar and / or carabersat) to directly and immediately block the Cx43 hemichannel, resulting in a concentration- and time-dependent reduction in GJ coupling and / or hemichannel inhibition (e.g., blocking hemichannel opening and / or regulating or blocking the release of ATP from the connector protein hemichannel). Kalabosa is N-[(3R,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromene-4-yl]-4-fluorobenzamide, and is also known as trans-(+)-6-acetyl-4-(S)-(4-fluorobenzoamide)-3,4-dihydro-2,2-dimethyl-2H-1-benzo[b]pyran-3R-ol hemihydrate. Tonabosar is also known by the IUPAC name N-[(3S,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydrochromene-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.

[0214] In some embodiments, the anti-connector protein, connector protein modulator, connector protein gap junction modulator, or connector protein hemichannel modulator is a compound according to Formula I: (I), Where Y is C—R1; R1 is an acetyl group; R2 is hydrogen, C 3-8 cycloalkyl, C 1-6 Alkyl groups (optionally interrupted by oxygen or by hydroxyl groups, C 1-6 alkoxy or substituted amino carbonyl), C 1-6 alkyl carbonyl, C 1-6 alkoxycarbonyl, C 1-6 Alkyl carbonyl group, C 1-6 Alkyl, nitro, cyano, halogen, trifluoromethyl, or CF3-S-; or the group CF3-A-, wherein A is —CF2—, —CO—, —CH2—, CH(OH), SO2, SO, CH2—O, or CONH; or the group CF2H-A′-, wherein A′ is oxygen, sulfur, SO, SO2, CF2, or CFH; trifluoromethoxy, C 1-6 Alkyl sulfinyl, perfluorinated C 2-6 alkylsulfonyl, C 1-6 alkylsulfonyl, C 1-6 Alkoxysulfinyl, C 1-6 Alkoxysulfonyl, aryl, heteroaryl, arylcarbonyl, heteroarylcarbonyl, phosphono, arylcarbonyloxy, heteroarylcarbonyloxy, arylsulfinyl, heteroarylsulfinyl, arylsulfonyl or heteroarylsulfonyl (where any aromatic moiety is optionally substituted), C 1-6 Alkyl carbonyl amino, C 1-6 alkoxycarbonylamino, C 1-6 alkyl-thiocarbonyl, C 1-6 alkoxy-thiocarbonyl, C 1-6 Alkyl-thiocarbonyloxy, 1-mercapto-C 2-7 alkyl, formyl, or aminosulfinyl, aminosulfonyl, or aminocarbonyl (wherein any amino moiety is optionally surrounded by one or two Cs) 1-6 Alkyl substitution), or C 1-6 alkylsulfinylamino, C 1-6 alkylsulfonylamino, C 1-6 alkoxysulfinylamino or C 1-6 alkoxysulfonylamino, or terminally C 1-6 Alkyl carbonyl, nitro or cyano-substituted vinylene, or -C(C 1-6 alkyl)NOH or -C(C 1-6 Alkyl)NNH2; or optionally with one or two C 1-6 Alkyl or C2-7 Alkyl-substituted amino groups; One of R3 and R4 is hydrogen or C. 1-4 Alkyl group, and another one is C. 1-4 Alkyl, CF3 or CH2X a , where X a It contains fluorine, chlorine, bromine, iodine, and C. 1-4 Alkoxy, hydroxy, C 1-4 Alkyl carbonyl group, -S-C 1-4 Alkyl, nitro, optionally marked with one or two Cs 1-4 Alkyl-substituted amino, cyano or C 1-4 alkoxycarbonyl; or R3 and R4 together are optionally C 1-4 Alkyl-substituted C 2-5 Polymethylene; R5 is C 1-6 Alkyl carbonyloxy, benzoyloxy, ONO2, benzyloxy, phenoxy, or C 1-6 Alkyl group, and R6 and R9 are hydrogen, or R5 is hydroxyl group, and R6 is hydrogen or C. 1-2 Alkyl group, and R9 is hydrogen; R7 is a heteroaryl or phenyl group, both of which are optionally substituted once or multiple times by a group or atom independently selected from the following groups or atoms: chlorine, fluorine, bromine, iodine, nitro, optionally substituted by C. 1-4 Alkyl substitution once or twice for amino, cyano, azide, C 1-4 Alkyloxy, trifluoromethoxy, and trifluoromethyl; R8 is hydrogen, C 1-6 Alkyl, OR 11 or NHCOR 10 , where R 11 It is hydrogen, C 1-6 Alkyl, formyl, C 1-6 Alkyl, aryl or aryl-C 1-6 Alkyl, and R 10 It 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, Halogenated -C 1-6 Alkyl, C 1-6 Acyloxy-C 1-6 Alkyl, C 1-6 Alkoxycarbonyl-C 1-6 Alkyl, aryl, or heteroaryl; R8—N—CO—R 7 The group and the R5 group are cis; And X is oxygen or NR. 12 , where R12 Is it hydrogen or C? 1-6 alkyl.

[0215] For any of the above Markush groups, in some implementations, each group may include or exclude any of the kinds listed in that group.

[0216] In some implementations, the small molecule linker protein regulator may be tonaboza, carabosa, 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). Carabosa is also known as... N -[(3R,4S)-6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromene-4-yl]-4-fluorobenzamide. Tonabosar is also known as... N -(6-acetyl-3-hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromene-4-yl)-3-chloro-4-fluorobenzamide.

[0217] For any of the above Markush groups, the group may include or exclude any of the kinds listed in the group.

[0218] In some embodiments, the modulator can be a prodrug of any compound used in this invention. In one aspect, the connexin modulator prodrug of this invention can be a compound of formula II: (II) in, Q is either O or oxime; R2 is H; A is a direct bond, -C(O)O*-, -C(R3)(R4)O*-, -C(O)OC(R3)(R4)O*- or -C(R3)(R4)OC(O)O*-, where the atom marked with * is directly connected to R1; R3 and R4 are independently selected from H, fluorine, and C. 1-4 Alkyl and C 1-4 Fluoroalkyl groups, or R3 and R4 together with the atoms they are attached to, form cyclopropyl groups; R1 is selected from groups [1], [2], [2A], [3], [4], [5], and [6], where the atom marked with ** is directly connected to A: , in, R5 and R6 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Fluorinated alkyl and benzyl groups; R7 is independently selected from H and C. 1-4Alkyl and C 1-4 Fluorinated alkyl groups; R8 is selected from: (i) H, C 1-4 Alkyl or C 1-4 Fluorinated alkyl groups; (ii) Side chains of natural or non-natural α-amino acids, or peptides as described herein; (iii) Biotin or chemically linked to biotin; R9 is selected from H, –N(R) 11 (R) 12 ), –N + (R 11 (R) 12 (R) 13 )X - and –N(R 11 )C(O)R 14 ; Where R 11 R 12 and R 13 Independently selected from H and C 1-4 Alkyl and C 1-4 Fluoroalkyl, R 14 Selected from H, C 1-4 Alkyl or C 1-4 Fluoroalkyl, R 15 Selected from C 1-4 Alkyl and C 1-4 Fluoroalkyl groups, and X - It is a pharmaceutically acceptable anion.

[0219] In some implementation schemes, R2 is BR 21 ,in, 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*, where the atom marked with * is directly related to R 21 connect, R 23 and R 24 Independently selected from H, fluorine, and C 1-4 Alkyl and C 1-4 Fluoroalkyl, R 21Selected from groups

[21] ,

[22] , [22A],

[23] ,

[24] ,

[25] , and

[26] , wherein the atom marked with ** is directly connected to B: , Among them, R5, R6, R7, R8, R9 and R 15 As defined in this article.

[0220] For any Markush group of Formula II above, the group may include or exclude any of the kinds listed in the group.

[0221] In some implementations, the peptides described herein may be connexin regulators, calmodulin regulators, or panconnexin regulators.

[0222] In some implementation schemes, Q is the formula = NOR 43 Oxime, of which R 43 yes (i) Selected from H, C 1-4 Fluoroalkyl or optionally substituted C 1-4 Alkyl, and (ii)-A 300 -R 300 ,in A 300 It is a direct bond, -C(O)O*-, -C(R3)(R4)O*-, -C(O)OC(R3)(R4)O*-, or -C(R3)(R4)OC(O)O*-, where the atom marked with * is directly bonded to R. 300 connect, R3 and R4 are independently selected from H, fluorine, and C. 1-4 Alkyl and C 1-4 Fluoroalkyl groups, or R3 and R4 together with the atoms they are attached to, form cyclopropyl groups. R 300 Selected from groups [1], [2], [2A], [3], [4], [5] and [6], wherein the atom marked with ** is directly related to A. 300 connect: , R5 and R6 are each independently selected from H and C. 1-4 Alkyl, C 1-4 Fluorinated alkyl and benzyl groups; R7 is independently selected from H and C. 1-4 Alkyl and C 1-4 Fluorinated alkyl groups; R8 is selected from: (iii) H, C 1-4 Alkyl or C 1-4 Fluoroalkyl, (iv) The side chains of natural α-amino acids, and (v) Biotin or chemically linked to biotin; R9 is selected from H, -N(R) 11 (R) 12 ), –N + (R 1l (R) 12 (R) 13 )X - and -N(R) 11 )C(O)R 14 ; Where R 11 R 12 and R 13 Independently selected from H and C 1-4 Alkyl and C 1-4 Fluoroalkyl, R 14 Selected from H, C 1-4 Alkyl and C 1-4 Fluoroalkyl, R 15 Selected from C 1-4 Alkyl and C 1-4 Fluoroalkyl groups, and X - It is a pharmaceutically acceptable anion.

[0223] In one embodiment of Formula II, R 43 It is optionally covered with phosphate groups (P(O)OR) 61 R 62 ) replaced by C 1-4 Alkyl group. In one example of this embodiment, OR 43 Yes-OCH2P(O)OR 61 OR 62 , where R 61 and R 62 Independently, it is H or C 1-4 alkyl.

[0224] In another embodiment of Formula II, R 43 It has the structure C(O)CH(R) 100 ) NH2 amino acid derivatives, wherein the group R 100 These are the side chains of natural or non-natural amino acids or peptides described in this article.

[0225] In some embodiments, the natural amino acid is selected from one of the 22 standard amino acids. In some embodiments, the non-natural amino acid is selected from any amino acid other than the 22 standard amino acids. In some embodiments, the non-natural amino acid is selected from: (cis)-3-aminobicyclo[2.2.1]heptane-2-carboxylic acid hydrochloride, exo-cis-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid hydrochloride, cis-2-amino-2-methylcyclohexane carboxylic acid hydrochloride, (R)-2-(Boc-amino)octanoic acid, Boc-4-(Fmoc-amino)-L-phenylalanine, Boc-(2-indanyl)-Gly-OH, (R)-4-Boc-3-morpholinoacetic acid, (S)-4-Boc-3-morpholinoacetic acid, 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-piperazinyl)-2-(2-fluorophenyl)acetic acid, 2-(4-Boc-piperazinyl)-2-(4-fluorophenyl)acetic acid, 2-(4-Boc-piperazinyl)-2-phenylacetic acid, 2-(4-Boc-piperazinyl)-2-(3-pyridyl)acetic acid, penicillamine, thiolysine, quisqualine, canavalialine, azacyclobutane-2-carboxylic acid, carboxyglutamic acid, hydroxyproline, arginine, and pyroglutamic acid.

[0226] In one implementation of Formula II, OR 43 It is -OC(O)CH(CH(CH3)2)NH2.

[0227] For any Markush group of Formula II above, in some implementations, each group may include or exclude any of the kinds listed in that group.

[0228] In some embodiments, a "prodrug moiety" refers to a substance acting as a protective group that masks functional groups in the active agent, thereby converting the active agent into a prodrug. The active agent can be any modulator or ocular therapeutic agent disclosed herein. Typically, the prodrug moiety attaches to the drug via an enzymatically or non-enzymatically cleavable bond, thereby converting the prodrug into its active form. In some embodiments, the prodrug moiety can also be an active agent. In some embodiments, the prodrug moiety can bind to a connexin modulator, connexin gap junction modulator, or connexin hemichannel modulator. In some embodiments, the prodrug moiety can bind to any polynucleotide, peptide or peptide mimic, small molecule antagonist, and / or other therapeutic agent disclosed herein. In some embodiments, the prodrug moiety can bind to a compound of Formula I. In some embodiments, the prodrug can be a compound of Formula II.

[0229] In some embodiments, the prodrug moiety may be any peptide malignant or peptide antagonist of this disclosure. In some embodiments, the prodrug moiety is a single amino acid whose functional group is optionally protected. In some embodiments, the prodrug moiety is a targeting substance. In some embodiments, the prodrug moiety is a substrate for influx- or efflux transporters on cell membranes, such as those used by Gaudana, R. et al. The AAPS Journal Those described in, 12:3, 348-360 (2012). The prodrug moiety can be, for example, chemically linked biotin. The prodrug moiety can also be, for example, chemically linked D-serine.

[0230] In some embodiments, the compound of formula I or II (e.g., tonaboza, karabosa, or analogues thereof) is nonionic, in the form of a free base, a free acid, or a pharmaceutically acceptable salt. For example, pharmaceutically acceptable salts include hydrochloride salts, 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 compound itself. In one embodiment, the salt is a hydrochloride salt. In one embodiment, the salt is a succinate salt.

[0231] In other embodiments, one or more polymorphs, one or more isomers and / or one or more solvates of a compound of formula I or II (e.g., tonaboza, karabosa or the like) may be used.

[0232] Other linker protein 43 regulators In addition to connexin antisense molecules (e.g., rofi-derived), connexin peptides (e.g., Peptide5, XG19, etc.), and connexin hemichannel antagonists (e.g., tonaboxa), connexin-binding proteins (including antibodies, antigen-binding antibody fragments, etc.) are also connexin modulators suitable for the methods of the present invention and can be administered in therapeutically effective amounts according to one or more dosage regimens described herein. Binding proteins include, for example, monoclonal antibodies, polyclonal antibodies, antibody fragments (including, for example, Fab, F(ab')2, and Fv fragments; single-chain antibodies; single-chain Fv; and single-chain binding molecules, such as those comprising, for example, binding domains, hinge regions, CH2 and CH3 domains, recombinant antibodies, and antibody fragments capable of binding antigenic determinants (i.e., that portion of the molecule, commonly referred to as epitopes) that are in contact with a specific antibody or other binding molecule. These binding proteins (including antibodies, anti-binding antibody fragments, etc.) may be chimeric or humanized or otherwise reduced in their effect on the target body. The immunogenicity of the antibody can be synthesized, recombinantly generated, or generated in an expression library. Any binding molecules known in the art or to be discovered in the future, such as those cited herein and / or described in detail in the art, are envisioned. For example, binding proteins include not only antibodies, but also ligands, receptors, peptide-like molecules, or other binding fragments or molecules (e.g., generated via phage display) that can bind to targets (e.g., linkers or linker hemichannel epitopes). Methods for synthesizing antibodies and binding fragments, as well as peptides and polypeptides (including peptide-like molecules and peptide analogs), can also be performed using suitable methods. See, for example, 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.

[0233] Connector protein regulators also include antibodies and their binding fragments (e.g., scFv, human V). H or V L Structural domain, humanized camel V HH domain, Ig NARThese antibodies, including single-domain conjugates and their binding fragments, bind to conjugate proteins, as well as conjugate peptides and polypeptides, including peptide mimics and peptide analogs of conjugate proteins that regulate the activity or function of half-channels or gap junctions, and other gap junction blockers and gap junction protein phosphorylators. For example, conjugate peptides and polypeptides can bind to conjugate proteins to inhibit their function, or inhibit conjugate function by mimicking specific regions of conjugate proteins to inhibit or disrupt their binding to other gap junction proteins. The naturally short half-life of antibody fragments can be improved using strategies known in the art, including PEGylation, use of repeating peptide sequences, polysialylation, albumin or IgG binding or fusion, and other methods.

[0234] The bound molecules will typically possess the desired specificity (including but not limited to binding specificity) and the desired affinity. For example, the affinity can be greater than or equal to about 10. 4 M -1 , greater than or equal to approximately 10 6 M -1 , greater than or equal to approximately 10 7 M -1 , greater than or equal to approximately 10 8 M -1 K a Even greater than about 10 8 M -1 Affinity also applies, such as equal to or greater than about 10. 9 M -1 Approximately 10 10 M -1 Approximately 10 11 M -1 Peace Treaty 10 12 M -1 The affinity of the binding protein for therapeutic subjects in this invention can be easily determined using conventional techniques, such as those described in Scatchard et al., 1949. Ann. NY Acad. Sci. Those described in 51:660.

[0235] Other compounds used to modulate, block, or close gap junctions (e.g., compounds that phosphorylate tyrosine and / or serine residues of junctionin 43) have been reported in U.S. Patent Nos. 7,153,822 and 7,250,397.

[0236] Combination and combined administration of connective protein regulators The methods, uses, and compositions of the present invention may comprise a combination of two or more connexin modulators, such as connexin antisense modulators (e.g., rofi derivatives), connexin peptides (e.g., Peptide5, Gap19, XG19), and small molecules (e.g., compounds of formula I, such as tonaboxa and its prodrugs, including compounds of formula II). The two or more connexin modulators may be administered alone or together. In some embodiments, two or more separate pharmaceutical compositions are provided for administration, each composition comprising one or more connexin modulators. Co-administered pharmaceutical compositions in combined formulations are also provided, such as mixtures of two or more modulators, such as modified or unmodified gap junction modulators, connexin modulators, and / or hemichannel modulators, such as one or more gap junction, connexin, and / or hemichannel modulator polynucleotides, and one or more gap junction, connexin, and / or hemichannel modulator peptides or peptides, and optionally one or more small molecule hemichannel blockers or inhibitors that reduce hemichannel opening and function.

[0237] Treatment of the ocular condition described herein with one or more pharmaceutical compositions of the present invention (e.g., anti-connector antisense molecules and gap junction modulators, such as connector hemichannel blockers (e.g., peptides or peptide mimics), or a first anti-connector reagent and a second anti-connector reagent) may include simultaneous, separate, sequential, or continuous administration of these compositions.

[0238] The term "combination formulation" includes "kit of parts" or "product," meaning that the combination partners defined above can be administered independently or by using different fixed combinations (i.e., fixed amounts of combination partners (a) and (b)), i.e., administered simultaneously, separately, or sequentially, whether in drug form (e.g., topical or oral) or dressing / matrix form (e.g., a bandage contact lens impregnated with a conjugate modulator), or both, all in accordance with the methods, dosages, and daily dosing regimens of this invention. The multiple parts of the kit can then be administered, for example, simultaneously or staggered in time sequence (i.e., administered at different time points, and the intervals between the parts of the multipart kit can be the same or different).

[0239] In one embodiment, a combination formulation is administered, wherein two or more individual connexin modulator compositions are administered to a subject according to the methods, dosages, and dosing regimens of the invention, wherein the first composition comprises a therapeutically effective amount of a modulator (such as a gap junction modulator, a connexin modulator, e.g., an anti-connexin 43 polynucleotide, a peptide or peptide mimic, or a hemichannel closure compound), and the second composition comprises a therapeutically effective amount of a second modulator (such as a gap junction modulator, a hemichannel modulator, and / or a connexin modulator, e.g., an anti-connexin 43 polynucleotide, a peptide or peptide mimic, a hemichannel closure compound, and / or an ocular therapeutic agent). In another embodiment, a third composition is administered, comprising one or more anti-connexin polynucleotides, peptides or peptide mimics, hemichannel closure compounds, and / or an ocular therapeutic agent.

[0240] Modifiers (including connexin modifiers, connexin hemichannel modifiers, and connexin gap junction modifiers) can be administered, applied, or formulated according to the methods of the present invention described herein.

[0241] Dosage and Dosing Regimen This document describes and claims examples of effective doses for treating non-healing ocular defects or conditions (e.g., ocular PED), non-healing corneal defects or conditions (e.g., PCED), and other ocular diseases, conditions, and illnesses (e.g., uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye syndrome (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye) or similar or related diseases, conditions, and illnesses. In some embodiments, a therapeutically effective amount of the modulator (e.g., a connexin modulator, such as a connexin 43 modulator, a connexin 43 gap junction modulator, and / or a connexin 43 hemichannel modulator) in the method of the invention comprises a composition having about or at least about 0.1 mg, 0.2 mg, or 0.3 mg of a connexin modulator (e.g., rofigen), including about (or at least about) 0.18 mg and about (or at least about) 0.018 mg. A dose of 1 mg of connexin modulator (e.g., rufi derivative), or any amount within or between any two of these doses. Other effective doses effective in the methods of the present invention for treating ocular surface defects or conditions (e.g., ocular PED and PCED) include about or at least about 1.0 mg of connexin modulator, such as connexin 43 modulator, connexin 43 gap junction modulator, and / or connexin 43 hemichannel modulator (e.g., rufi derivative).

[0242] In some embodiments, the therapeutically effective amount of the modulator (e.g., a connexin modulator, such as 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 of about or at least about 2 micromoles (2 μM), 5 micromoles (5 μM), 10 micromoles (10 μM), 15 micromoles (15 μM), or 20 micromoles (20 μM) (including about (or at least about) 1.9 micromoles (1.9 μM) or 1.9405 micromoles (1.9405 μM) and about (or at least about) 19 to 19.4 micromoles or 19.405 micromoles (19 to 19.4 μM or 19.405 μM)) of a connexin modulator (e.g., Rufi derivative), or any amount within or between any two of these doses. Other effective doses for treating ocular surface defects or conditions (e.g., ocular PED and PCED) in the methods of the present invention include compositions comprising about or at least about 30 micromoles (30 μM) or 40 micromoles (40 μM) of a connexin modulator (e.g., a connexin 43 modulator, a connexin 43 gap junction modulator, and / or a connexin 43 hemichannel modulator (e.g., a rofi derivative)).

[0243] In some methods of the present invention, the connexin modulator applied to a subject suffering from a non-healing surface defect or condition is 0.06% rubexin derivative. In some embodiments, the connexin modulator applied to a subject suffering from a non-healing surface defect or condition is 0.006% rubexin derivative. In some embodiments, the applied connexin modulator is at least about 0.06% rubexin derivative. In some embodiments, the connexin modulator applied to a subject suffering from a non-healing surface defect or condition is at least about 0.006% rubexin derivative. In some embodiments, the application is to a PED or PCED. In some embodiments, the subject is given a composition comprising or substantially comprising: a pharmaceutically acceptable carrier and about 0.06% rubexin derivative, about 0.006% rubexin derivative, at least about 0.06% rubexin derivative, or at least about 0.006% rubexin derivative to heal the non-healing surface defect or condition. The rubexin derivative dosage described herein and below can be prepared as described in Example 1.

[0244] In some embodiments, non-healing surface defects or conditions are treated with a composition comprising or substantially composed of: 0.6 mg / mL rufi derivative. In some embodiments of the invention, non-healing surface defects or conditions are treated with a composition comprising or substantially composed of: 0.06 mg / mL rufi derivative. In some embodiments, the composition comprises or substantially composed of: at least about 0.6 mg / mL rufi derivative. In some embodiments, the composition comprises or substantially composed of: at least about 0.06 mg / mL rufi derivative. In some embodiments, administration is to a PED or PCED.

[0245] In some embodiments, according to the scheme described herein, a non-healing surface defect or condition of a subject is treated with a composition comprising or substantially consisting of: about 0.018 to about 0.18 mg of lufifine derivative. In some embodiments, according to the scheme described herein, each dose of lufifine derivative applied to a non-healing surface defect or condition of a subject, whether internal or external, comprises or substantially consists of: at least about 0.18 mg of lufifine derivative or at least about 0.18 mg of lufifine derivative. In some embodiments of the invention, according to the scheme described herein, a non-healing surface defect or condition is treated with a composition comprising or substantially consisting of: 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 lufifine derivative, or from about or at least about 0.18 mg, or 0.20 mg, or 0.50 mg to about or at least about 1 mg of lufifine derivative. In some embodiments, each dose of rufi derivative applied to a non-healing surface defect or lesion, as described herein, comprises or consists essentially 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 rufi derivative. In some embodiments, administration is to a PED or PCED.

[0246] These doses may also be used to treat uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye) or similar or related diseases, conditions, and illnesses.

[0247] In some embodiments, the doses described above are applied topically to the eye of a subject suffering from non-healing or persistent ocular or corneal surface defects or conditions. In some embodiments, the doses described herein are administered intraocularly, including by injection (e.g., intravitreal injection, intra-anterior chamber injection, etc.). Connectin modulators administered via non-topical routes (e.g., oral or parenteral) will need to be adjusted as needed to approximate these topical doses.

[0248] In some embodiments, the dose of rufi derivative (or other connexin antisense molecules or connexin modulators) applied to non-healing surface defects or conditions may be a single dose or two or more fractionated doses, such as twice daily for up to six doses administered on days 1, 2, and 14; or twice daily for up to eight doses administered on days 1, 2, 14, and 28 (for non-healing surface defects or conditions). In other embodiments, about or at least about 0.1 to 0.5 mg or at least about 1 mg of rufi derivative may be administered as a single dose or fractionated dose, such as twice daily for up to 14 doses administered on days 1, 2, and 14, and on days 7, 21, 28, and / or 35. In some embodiments, the administration is to the subject's PED or PCED.

[0249] In some implementations, the dose of the connective protein modulator (e.g., rufi derivative) is administered on days 1, 2, 14, and 21.

[0250] In some implementations, the dose of the connective protein modulator (e.g., rofi derivative) is administered on days 1, 2, 7, 14, and 21.

[0251] In some implementations, the dose of the connective protein modulator (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, and 28.

[0252] In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over 14 days in three doses to treat a subject's non-healing or persistent ocular or corneal surface defects, including, for example, administration on days 1 and 2. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over approximately 28 days. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over approximately 28 to approximately 35 days. In some embodiments, a connexin modulator (e.g., rufi derivative) is administered over approximately 35 days in six to seven doses. For example, in one embodiment, the connexin modulator (e.g., rufi derivative) is administered four times on days 1 and 2, approximately day 14, and approximately day 28. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered five times on days 1 and 2, approximately day 14, approximately day 28, and approximately day 35. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered five times on days 1 and 2, approximately day 7, approximately day 14, and approximately day 28. In another embodiment, the connexin modulator (e.g., rufi derivative) is administered six times on days 1 and 2, approximately day 7, approximately day 14, approximately day 21, and approximately day 28. In yet another embodiment, the connexin modulator (e.g., rufi derivative) is administered seven times on days 1 and 2, approximately day 7, approximately day 14, approximately day 21, and approximately day 28. Of course, "administer" as used herein refers to a single dose or multiple doses of the connexin modulator.

[0253] In the dosage regimens or methods of the present invention for treating non-healing ocular or corneal surface defects or conditions, if the dosage of the connexin modulator is not specified (e.g., the dosage of rofigen or other connexin expression modulators, connexin peptides and / or hemichannel blockers), the aim is to administer a therapeutically effective amount of the connexin modulator.

[0254] In some implementations, one or more connexin modulators are administered to subjects suffering from the following ocular diseases, conditions, and ailments, according to one or more dosage regimens described herein: uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidified dry eye).

[0255] In the dosage regimens or methods of the present invention for treating non-healing ocular or corneal surface defects or conditions or other diseases, symptoms or conditions, if the dose of the connexin modulator (e.g., the dose of rofi-derived or other connexin expression modulators, connexin pseudopeptides and / or hemichannel blockers) is specified as day 7, day 14, day 21, day 28 or day 35, then the therapeutically effective amount of the connexin modulator is intended to be administered before or after that day.

[0256] In certain dosing regimens, administration of a connexin modulator (e.g., rufi derivative, etc.) and a pharmaceutical composition comprising or substantially consisting of a connexin modulator is provided. In one embodiment, a composition comprising one or more gap junction, hemichannel, and / or connexin modulator polynucleotides (and / or other connexin modulators, e.g., peptide-like or small molecule connexin modulators) is administered on days 1 and 2 of treatment, followed by administration on day 14. In another embodiment, the composition is administered on days 1 and 2 of treatment, followed by administration on days 14 and 28. In yet another embodiment, the composition is administered on days 1 and 2, followed by administration on days 14, 28, and 35. In other embodiments of these dosing regimens, the composition is optionally also administered on days 7 and / or 21.

[0257] In some embodiments, the disease, condition, or ailment to be treated is a non-healing ocular surface defect or ailment, namely persistent epithelial defect (PED). In some embodiments, the non-healing ocular surface defect or ailment is persistent corneal epithelial defect (PCED). In some embodiments, the non-healing ocular surface defect or ailment is a corneal ulcer. In some embodiments, the disease, condition, or ailment to be treated is selected from uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye syndrome (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye). In the method of the present invention, a therapeutically effective amount of at least one connexin modulator is administered in pulsed doses on days 1 and 2, then on day 14, and optionally on day 28 or about day 28 (and also optionally on day 35 or about day 35), which can effectively heal non-healing ocular surface defects or ailments or other diseases, conditions, or ailments. See Example 2 below. The connexin regulator dosage may also be administered on day 7 and / or day 21. In some embodiments, the connexin regulator is a connexin 43 regulator (e.g., a connexin expression regulator, such as an antisense connexin expression regulator, including Cx43 antisense molecules). Other embodiments include other connexin 43 gap junction regulators and connexin 43 hemichannel blockers or regulators (e.g., peptides and small molecules).

[0258] In some embodiments, the connexin modulator (e.g., rufi derivative or other modulators) is administered on days 1, 2, and 14. In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, 14, and 28. In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, 14, 28, and 35. In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, 7, 14, and 21. In some embodiments, the connexin modulator (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, and 28. In some embodiments, the connexin modulator dose (e.g., rufi derivative) is administered on days 1, 2, 7, 14, 21, 28, and 35. In some embodiments, the dose is administered after day 35. In some embodiments, administration according to the stated dose and dosing regimen is to the PED or PCED. In some embodiments, the connexin modulator comprises a connexin antisense molecule (e.g., rufi derivative). In some embodiments, the connexin modulator comprises connexin peptides (e.g., Peptide5, Gap19, XG19, Gap26, Gap27, αCT1, etc.). In some embodiments, the connexin modulator comprises a small molecule connexin hemichannel blocker (e.g., tonaboxa). In some embodiments, the connexin modulator is a connexin 43 modulator.

[0259] Unless otherwise expressly stated, all descriptions of dosage apply to the modulators of this invention, including connexin modulators, connexin gap junction modulators, and connexin hemichannel modulators. Unless otherwise expressly stated, all descriptions of dosage also apply to all therapeutic indications described or cited herein, including ocular surface defects and corneal surface defects. Unless otherwise expressly stated, all descriptions of dosage apply to the treatment of uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye).

[0260] preparation The pharmaceutical compositions of the present invention include various desired or suitable delivery forms and formulations, including topical formulations, as well as drug administration forms and formulations suitable for systemic administration (e.g., oral and enteral), parenteral administration (e.g., injection, infusion, implantation, etc.), intraocular administration, etc.

[0261] Such delivery forms and formulations include those used to treat the objects disclosed herein. The pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients for ocular application may be ophthalmologically acceptable excipients. In some embodiments, the formulation provides sustained delivery of connexin modulators and / or ocular therapeutic agents to specific segments or compartments of the eye. In some embodiments, the formulation provides high ocular drug bioavailability (including, for example, by topical or oral administration) and is safe and non-toxic, and / or has virtually no systemic side effects or complications at the site of application. Exemplary polynucleotide formulations that can be used in the methods of the present invention are characterized by convenient local delivery and ease of administration.

[0262] Such delivery forms and formulations include those used to treat the objects disclosed herein. The pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients for ocular administration may be ophthalmologically acceptable excipients. In some embodiments, the formulations provide sustained delivery of connexin modulators and / or ocular therapeutic agents to specific segments or compartments of the eye. In some embodiments, the formulations provide high ocular drug bioavailability (including, for example, by topical or oral administration) and are safe and non-toxic, and / or have virtually no systemic side effects or complications at the site of application. Exemplary polynucleotide formulations that can be used in the methods of the present invention are characterized by convenient local delivery, ease of administration, and “no side effects”.

[0263] In some embodiments, the pharmaceutical formulations of the present invention may comprise any of the modifiers described herein, such as gap join modifiers, hemichannel modifiers, and / or joinin modifiers, such as modified or unmodified joinin 43 antisense oligonucleotides or polynucleotides, or modified or unmodified joinin 43 peptides or peptide mimics. In some embodiments, the joinin 43 antisense oligonucleotide included in the formulation may be unmodified joinin 43 antisense oligodeoxynucleotide or modified joinin 43 antisense oligodeoxynucleotide. In some embodiments, the pharmaceutical composition may include or exclude any of the above-described substances.

[0264] Modifiers (including connexin modifiers, connexin hemichannel modifiers, and connexin gap junction modifiers) may be present in the formulation in a substantially segregated form. It should be understood that the product may be mixed with a carrier or diluent that does not interfere with its intended use and is still considered to be in a substantially segregated form. The product of the present invention may also be in a substantially purified form, in which case, for example, it typically constitutes about 80%, 85%, or 90% of the mass of the polynucleotide (or other connexin modifier, such as connexin 43 modifier) ​​or dry matter of the formulation, for example, at least about 88%, at least about 90%, 95%, or 98%, or at least about 99%. The luffi-derived composition used in the clinical trial for the treatment of non-healing ocular defects described in Example 2, as described in Example 1, comprised 94.2% pure luffi-derived (content determined to be 106%) and had a final pharmaceutical purity of 94.9% (content determined to be 97%).

[0265] Pharmaceutical formulations used in the dosages, dosing regimens, and methods of the present invention may comprise one or more pharmaceutically acceptable excipients suitable for delivering modulators (e.g., luffi derivatives) (including connexin modulators, connexin hemichannel modulators, and connexin gap junction modulators) to the eye.

[0266] The modulators of the present invention (including gap junction modulators, hemichannel modulators, and / or connexin modulators) can also be formulated as microparticles (microspheres, Mp) or nanoparticles (nanospheres, Np), or both. In some embodiments of the invention, nanoparticles or microparticles are used. Such particles comprise poly(lactic-co-glycolic acid) (“PLGA”) loaded with gap junction modulators, connexin modulators, and / or hemichannel modulators (e.g., connexin 43 modulators). The modulators may be loaded inside the particle body, on the outer surface of the particle, or both. The particle formulation can be applied topically, for example, to the eye or subconjunctival region. In some embodiments, particle formulations of any gap junction modulator, hemichannel modulator, and / or connexin modulator (e.g., connexin 43 modulators) of the present disclosure may also comprise liposomes.

[0267] Modifiers (e.g., connexin modifiers, connexin hemichannel modifiers, and connexin gap junction modifiers) can be administered alone or in combination with one or more other ingredients, and can 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 that can be used to prepare pharmaceutical compositions and are generally safe, non-toxic, and have no adverse biological or other effects. Pharmaceutically acceptable diluents, carriers, and / or excipients include those suitable for veterinary use as well as for human pharmaceutical use. For example, diluents, carriers, and / or excipients include solutions, solvents, dispersion media, delay agents, polymeric and lipid reagents, emulsions, etc. As further examples, suitable liquid carriers (especially solutions for injection) include water, saline solutions, glucose solutions, etc., and mediators (such as liposomes) are also particularly suitable for reagent administration.

[0268] Suitable carriers and diluents include buffered aqueous solutions, physiological saline, glucose, glycerol, isotonic saline solutions such as 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 symmetrical alcohols. In some embodiments, pharmaceutically acceptable carriers or diluents may be or contain thermosetting poloxamer (which may be liquid or gel depending on temperature), carboxycellulose (e.g., carboxymethyl cellulose), collagen (e.g., type I collagen), collagen-like materials containing procollagen, hyaluronic acid or derived hyaluronic acid, and / or oils (e.g., emu oil). Suitable carriers may be large, slowly metabolizing macromolecules 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 protein-linking modulators mediated solely by sterile water. In some embodiments, the formulation will contain a connexin regulator (e.g., a connexin 43 regulator). For example, in some embodiments, the connexin 43 antisense oligonucleotide contained in the formulation may be an unmodified or modified connexin 43 antisense oligodeoxynucleotide.

[0269] The composition may be in any standard known dosage form, including tablets, pills, capsules, semi-solid preparations, powders, sustained-release preparations, solutions, suspensions, elixirs, aerosols, injectable liquids, gels, creams, transdermal delivery devices (e.g., transdermal patches), implants (such as ocular implants), or any other suitable composition. Those skilled in the art to which this invention pertains can readily determine the most suitable dosage form based on the nature of the condition to be treated and the properties of the active reagents used (e.g., antisense molecules, peptides, small molecules, etc.) without excessive experimentation.

[0270] Preferably, the modulator of the present invention (e.g., a connexin modulator) is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. The connexin modulator can be formulated into compositions suitable for any desired route of administration, including topical, oral, systemic, transdermal, nasal, sublingual, buccal, etc., as well as injectable formulations (e.g., intra-anterior chamber injection, subcutaneous injection, intramuscular injection, intravenous injection, etc.).

[0271] Pharmaceutically acceptable salts may also be present, such as inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.); and organic acid salts (e.g., citrate, acetate, propionate, malonate, benzoate, etc.).

[0272] In one aspect, the antithermosetting gel can be liquid at low temperatures (e.g., 2-8°C) and undergoes a reversible liquid-to-gel transition at temperatures above about 15°C. Therefore, in some embodiments, the carrier can be liquid at temperatures below about 15°C but can form a gel at temperatures above about 15°C (e.g., room temperature or body temperature). In some cases, the gel is a nonionic polyoxyethylene-polyoxypropylene copolymer gel. In some embodiments, the gel is a Pranic gel. The Pranic gel can be, for example, poloxamer 407, sometimes also referred to as Pranic F-127 (BASF). In some embodiments, the formulation of the present invention may contain about 15% to about 30% (w / v) of gel. In some embodiments, the formulation of the present invention may contain about 20% to about 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.

[0273] Other suitable formulations include those based on Pranic gel, hydroxymethylcellulose, hydroxyethylcellulose, carboxymethylcellulose (CMC), and hydroxypropyl methylcellulose (HPMC). The compositions can be formulated into any desired delivery method, including topical, intravenous, parenteral, intramuscular, subcutaneous, or transdermal administration. Other useful formulations include slow-release or delayed-release formulations.

[0274] In addition, substances such as wetting agents or emulsifiers, stabilizers or pH buffers, or preservatives may be present, if desired. In some embodiments, the pharmaceutical compositions of the present invention will comprise suitable ophthalmologically acceptable buffers, such as acetate 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 ternary phosphates, such as sodium dihydrogen phosphate monohydrate and disodium hydrogen 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 further comprise humectants, nutrients, thickeners, antioxidants, etc., such as disodium ethylenediaminetetraacetate, alkali metal salts of hexametaphosphate, citric acid, sodium citrate, sodium metabisulfite, sodium thiosulfate, N-acetylcysteine, butylated hydroxyanisole, butylated hydroxytoluene, polyvinyl alcohol, poloxamer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, and mixtures thereof. In some embodiments, the pharmaceutical formulation of the present invention is preservative-free. In some embodiments, the connexin regulator composition or formulation comprises disodium hydrogen phosphate heptahydrate or potassium dihydrogen phosphate, or both. See Example 1.

[0275] When the modulator (e.g., gap junction modulators, connexin modulators, and / or hemichannel modulators) is a nucleic acid (such as a polynucleotide), the uptake of nucleic acid by mammalian cells can be enhanced by known transfection techniques (including the use of transfection agents). Such techniques can be used with certain anti-connexin reagents (including polynucleotides). The applied formulation may contain such transfection agents. Useful examples of transfection agents include cationic reagents (e.g., calcium phosphate and DEAE-glucan) and lipid transfection agents (e.g., lipofectam™ and transfectam™), as well as surfactants.

[0276] Connector protein modulators, gap junction modulators, and / or hemichannel modulators (e.g., connector protein 43 modulators) may also be formulated to provide controlled release to the eye. In some embodiments of the invention, the formulation may be an immediate-release, extended-release, or sustained-release dosage form, for example, released over several hours, one day, or, for example, one to two days.

[0277] The composition can be formulated according to standard techniques known in the art, for example, see Gennaro AR: Remington: The Science and Practice of Pharmacy, 20 th Those are those in the standard references such as those in ed., Lippincott, Williams & Wilkins, 2000, etc.

[0278] Any container suitable for storing and / or administering the pharmaceutical composition may be used in the combination products of this invention. Those skilled in the art will recognize suitable containers. For example, such containers include vials and syringes. Containers should be properly sterilized and sealed.

[0279] In some implementations, the application of a connexin modulator (e.g., a connexin 43 modulator or a connexin 45 modulator, preferably a connexin 43 modulator, such as a rofi derivative) to the target eye can deliver a therapeutically effective amount of the connexin modulator to the eye or a specific cavity or site of the eye by selecting the desired and applicable method of application (including, but not limited to, topical application) according to the properties of the connexin modulator.

[0280] Therapeutic effective doses include, but are not limited to, the doses described herein. These doses and other therapeutically effective doses must be administered according to one or more therapeutically effective dose regimens described herein.

[0281] application The administration of modulatory compounds and compositions (e.g., connexin modulators, gap junction channel modulators, and / or hemichannel modulators) can be carried out via one of the following routes: oral administration, local administration, systemic administration (including intravenous administration, arterial administration, intraperitoneal administration, transdermal administration, nasal administration, or suppository administration), parenteral administration (including intramuscular injection, subcutaneous injection, or intravenous or arterial injection), etc. In some embodiments, connexin modulators, gap junction channel modulators, and / or hemichannel modulator compounds and compositions are administered locally. 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, connexin modulators, gap junction channel modulators, and / or hemichannel modulator compounds and compositions are administered via anterior chamber injection formulation and route of administration, as this may be a preferred route of administration (or used when local administration is less effective) for the treatment of, for example, uveitis and Fuchs' dystrophy.

[0282] In some embodiments of the method of the present invention, connexin modulators, connexin gap junction modulators, and / or connexin hemichannel modulators can be locally administered by topical application to the target eye. In some embodiments, connexin modulators are administered by topical application, corneal application, and / or subconjunctival application, or by local injection. Topical formulations of gap junction modulators, hemichannel modulators, and / or connexin modulators may comprise ointments, gels (e.g., which may be thermosetting gels), drops, sprays, liquids, and powders, or sustained-release or non-sustained-release dosage forms. Systemically applicable and bioavailable connexin modulators, connexin gap junction modulators, and / or connexin hemichannel modulators can be administered with or without accompanying topical application to the eye, for example, by oral administration, intravenous administration, enteral administration via the gastrointestinal system (e.g., oral, rectal, sublingual, buccal administration), parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration), nasal administration, nasal inhalation administration, and oral inhalation administration. In some embodiments, non-topical application (including intraperitoneal, oral, or parenteral administration) may be used, provided that a therapeutically effective dose of the drug comes into contact with the eye or a portion thereof (e.g., the cornea) to be healed. In some embodiments, in addition to topical application, orally available connexin modulators (e.g., tonaboxa) may be applied via intraperitoneal, oral, or parenteral administration, provided that a therapeutically effective dose of the drug comes into contact with the eye.

[0283] In some embodiments, application of a connexin modulator (e.g., a connexin expression modulator, a peptide-like or small molecule gap junction modulator, and / or a hemichannel modulator (e.g., a connexin 43 modulator, or a modulator of any other connexin in the eye, cornea, corneal epithelium, or blood vessels)) to the subject eye provides a therapeutically effective amount of the connexin modulator to the eye. In some embodiments, a bandage contact lens is applied to the eye after application of the connexin modulator, whether by topical or other routes of application. In some embodiments, the connexin modulator is placed within a bandage contact lens, which is then applied to the eye of a subject with a non-healing ocular surface defect or condition (e.g., PED or PCED) or other diseases, conditions, or ailments described or cited herein. In some embodiments, the connexin modulator is applied subamniotically. In some embodiments, amniotic membrane is applied to the eye after application of the connexin modulator. The modulator may be a connexin 43 modulator. In some embodiments, the connexin modulator is embedded in a matrix containing 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) can slowly or continuously release the connexin modulator contact lens. As used herein, "matrix" includes, for example, matrices (such as polymer matrices, biodegradable or non-biodegradable matrices) and other carriers that can be used to prepare implants or application structures to deliver connexin modulators for the treatment of non-healing ocular surface and / or corneal surface conditions or neuropathological conditions. Compositions and methods for preparing drug-containing contact lenses have been developed and are known in the art. See, for example, Wang Z et al., Novel Contact Lenses Embedded with Drug-Loaded Zwitterionic Nanogels for Extended Ophthalmic Drug Delivery. Nanomaterials 11:2328 (2021).

[0284] In some embodiments of the invention, a subject with a non-healing surface defect or condition (e.g., PCED) receives an amniotic graft. In some embodiments, a gap junction modifier or hemichannel modifier (e.g., Rufi derivative) is applied beneath the amniotic graft. In some embodiments, the amniotic graft is applied to the non-healing surface defect (e.g., PCED) after the application of the gap junction modifier or hemichannel modifier (e.g., Rufi derivative).

[0285] Products / Reagent Kits In another embodiment of the invention, an article of manufacture or "kit" is provided comprising materials for treating non-healing ocular surface or corneal defects and conditions (e.g., PED or PCED). In another embodiment of the invention, an article of manufacture or "kit" is provided comprising materials for treating uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and / or dry eye syndrome (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidifying dry eye).

[0286] The kit includes a container containing a composition comprising one or more modulators (e.g., connexin modulators, such as connexin antisense molecules, connexin peptides, and / or small molecule hemichannel blockers). The kit may further include a label or instruction manual, affixed to or associated with the container, containing the dosing instructions described herein. The term "instruction manual" refers to the instruction document typically included in the commercial packaging of a therapeutic product, containing information about the indications, usage, dosage, administration, contraindications, and / or warnings regarding the modulator, which may be provided, for example, in physical form or via an online link. Suitable containers include, for example, bottles, vials, etc. Containers may be made of a variety of materials, such as glass or plastic. In some embodiments, the label or instruction manual indicates that the composition is intended for the treatment of non-healing ocular surface or corneal surface defects and conditions (e.g., PED or PCED). In some implementations, the label or package insert indicates that the composition is used to treat uveitis, blepharitis, Sjögren's syndrome-associated keratoconjunctivitis sicca, Fuchs' dystrophy, and all types of dry eye (e.g., evaporative dry eye, aqueous-deficient dry eye, and dehumidified dry eye).

[0287] The kit may contain one or more pharmaceutical compositions in individual or separate 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 composition, such as a syringe, needle, microneedle, etc. In some embodiments, the kit includes a bandage contact lens for application to a subject after administration of a connexin modulator, or for administering the connexin modulator. The connexin modulator needs to be administered on different dates, and the kit may include packaging and / or instructions for use to guide the chronologically staggered administration according to the method of the present invention.

[0288] preparation The polynucleotides of the present invention can be prepared using methods such as solid-phase chemistry (for synthesizing oligonucleotides), chemical methods known in the art (for synthesizing and preparing peptides and peptide-like compounds), and chemical methods known in the art (for synthesizing organic compounds (e.g., tonabosar and other compounds of formula I and II)). In one aspect, the formulations of the present invention will comprise a salt of the polynucleotides of the present invention, such as a sodium salt of the polynucleotides of the present invention. The kit may also comprise a pharmaceutically acceptable carrier. In one embodiment, the formulation may comprise a sodium salt of a polynucleotide having, for example, any one of SEQ ID NO:1-16 or a portion of SEQ ID NO:17. In some embodiments, the polynucleotide having any one of SEQ ID NO:1-16 may be a modified oligodeoxynucleotide having any one of SEQ ID NO:1-16 or a portion of SEQ ID NO:17.

[0289] In some embodiments, the formulations of the present invention are substantially pure. "Substantially pure" means that the formulation contains less than about 10%, 5%, or 1%, and preferably less than about 0.1%, of any nucleotide or non-nucleotide impurities. In some embodiments, the total impurities of the metabolites including the connexin 43 regulator do not exceed 15%. In some embodiments, the total impurities of the metabolites including the connexin 43 regulator do not exceed 12%. In some embodiments, the total impurities of the metabolites including the connexin 43 regulator do not exceed 11%. In other embodiments, the total impurities of the metabolites including the connexin 43 regulator do not exceed 10%. See, for example, Example 1, which describes the preparation of a Luffi-derived composition with a final pharmaceutical purity of 94.9%.

[0290] The sterile composition of the present invention comprising a connexin 43 modulator is prepared by aseptic process by dissolving the anti-connexin modulator in a formulation medium. In one embodiment, the formulation may also be sterilized by filtration. The excipients used to prepare the formulations of the present invention are widely used in pharmaceutical products and conform to pharmacopoeia standards. Example

[0291] The following work was conducted to evaluate whether connexin modulation could be used to treat patients with non-healing ocular defects and conditions, including persistent ocular epithelial defects and persistent corneal epithelial defects.

[0292] The work described in the examples has been carried out for several years in multiple countries, working to discover and identify previously unknown but clinically useful therapeutic doses and dosing regimens in randomized, double-blind, placebo-controlled human clinical trials.

[0293] It describes the successful results achieved by this work and related efforts.

[0294] Example 1 LUFEPIRSEN is used for local application to subjects with persistent corneal epithelial defects. ® Preparation of (Rufie-derived) compositions Compositions containing the anti-connector protein 43 modulator Luffi derivative (5'-GTA ATT GCG GCA AGAAGA ATT GTT TCT GTC-3' [SEQ ID NO:1]) for topical ocular application are prepared by dissolving the anti-connector protein modulator in a formulation medium and adding a buffer, as shown in the table below.

[0295]

[0296] A composition containing 0.6 mg / mL or 0.06% of lufifide connexin 43 modulator for administration was prepared by dissolving 0.18 mg lufifide or 19.40491591203 μM [005 API (batch number C06A14001)] in a buffered poloxamer pharmaceutical formulation medium.

[0297] A composition containing 0.06 mg / mL or 0.006% of lufifide connexin 43 modulator for administration was prepared by dissolving 0.018 mg lufifide or 1.940491591203 μM [005 API (batch number C06A14001)] in a buffered poloxamer pharmaceutical formulation medium.

[0298] The purity of the rofi derivative was 94.2% (106% by assay), and the purity of the pharmaceutical product was 94.9% (100% by assay). The rofi derivative composition was used in the clinical trial described in Example 2.

[0299] Example 2 Phase 2, randomized, prospective, double-blind, mediator-controlled study Assessment of LUFEPIRSEN ® (Lufie derivative) Efficacy and safety of topical application to subjects with persistent epithelial defects (PED) caused by ocular chemical and / or thermal damage. This embodiment describes the use of an exemplary connexin modulator (Lufi derivative) to treat non-healing ocular surface defects in a randomized, prospective, mediator-controlled, double-blind human clinical trial. Lufi derivative is a connexin 43 antisense oligonucleotide according to SEQ ID NO:1. The mediator control in the trial was Prunic F-127.

[0300] Research subjectsThis clinical trial enrolled 35 participants. All eligible participants had non-infectious, persistent epithelial defects (PED) of the cornea due to ocular chemical and / or thermal injury and had not responded to current standard of care for at least 14 days. Of the 35 participants, 24 received topical ocular application of rufi derivative, and 11 received a mediator. Twelve participants failed to achieve reepithelialization or maintain reepithelialization for 28 days; these participants received one (day 1 of salvage therapy) or two (day 8 of salvage therapy) applications of 0.06% rufi derivative on an open-label basis. Results are described below.

[0301] Research Objective The purpose of this clinical protocol is to evaluate the efficacy and safety of two ocularly administered concentrations of leucovorin in a novel regimen for the treatment of nonhealing corneal PED caused by severe ocular chemical and / or thermal injuries.

[0302] Study endpoints The primary endpoint, secondary endpoints, and other endpoints assessed in this human clinical trial are described below: Primary endpoint : • Corneal epithelial repair is defined as corneal re-epithelialization achieved on day 28 of treatment, and the re-epithelialization is maintained for at least 28 days after the first recorded re-epithelialization, as assessed by the investigator.

[0303] Secondary endpoint : • Epithelialization time is defined as the time from the time of random grouping to the first re-epithelialization.

[0304] • The degree of improvement in visual acuity from baseline during the study period.

[0305] • The number of doses and sessions of rofi-derived treatment required for corneal epithelial repair.

[0306] Exploratory endpoint : • The change in PED area from the baseline within the epithelial defect is defined as the product of the maximum diameter and the maximum diameter perpendicular to that diameter.

[0307] • Corneal re-epithelialization is defined as the re-epithelialization that occurs on day 28 of corneal treatment.

[0308] • Changes in eye symptoms compared to baseline.

[0309] • The open-label portion of the study also assesses (if applicable) the primary endpoint, secondary endpoints, and exploratory endpoints.

[0310] • A descriptive comparison of intra-orbital corneal reepithelialization in bilaterally injured subjects with that in the contralateral eye.

[0311] Security : • Incidence of adverse events during treatment.

[0312] Therapeutic administration The study included a 28-day treatment period, followed by at least a 28-day follow-up period to assess safety and epithelialization status (persistence). Both phases were double-blind (i.e., mutually unknown). Eligible participants were randomly assigned in a 1:1:1 ratio to receive either 0.06% of the study product, 0.006% of the study product, or the mediator. During the treatment period, the study product was applied infrabanded to the bandage-contact lens (BCL) and to the fornix of the study eye at the following time points: • Day 1 • Day 2 • Day 14 (if corneal re-epithelialization has not been achieved) rescue drug administration However, if the cornea had not fully re-epithelialized by day 28 of the treatment period, regardless of the randomized study product allocation, the subject entered the open-label phase of the study and received up to two salvage doses of 0.06% rubefaline (applied below the BCL and in the fornix of both eyes). The 0.06% rubefaline was applied on day 1 of the salvage treatment, and if re-epithelialization was still not achieved, it was applied on day 8 of the open-label phase of the salvage treatment. Twelve subjects who received salvage treatment were followed up from the first salvage dose to day 14 to confirm re-epithelialization (day 41), and then returned for follow-up at >28 days to confirm maintenance of re-epithelialization (persistence).

[0313] For each subject, the 28-day double-blind treatment period ended when either of the following conditions was met: corneal re-epithelialization was first recorded after a maximum of three double-blind applications of the study product, or the subject entered the open-label phase of the study due to failure of PED healing on day 28.

[0314] Those subjects who achieved re-epithelialization immediately entered a 28-day post-healing follow-up period (without further application of the study product). The purpose of the post-epithelialization follow-up period was to assess the persistence of the epithelium.

[0315] For subjects who achieved corneal 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 0.06% leufi derivative and entered the open-label phase of the study (day 1 of salvage treatment). If corneal re-epithelialization was not achieved by day 8 of salvage treatment in the open-label phase of the study, a second dose of 0.06% leufi derivative was administered. If re-epithelialization occurred within 14 days of the initial salvage treatment dose, epithelial persistence would be finally assessed after 28 days.

[0316] If a subject failed to achieve epithelialization within 14 days of receiving a salvage dose of 0.06% rufi derivative during the open-label phase of the study, they were withdrawn from the study. In the NEX-PED-005 study, a total of 12 subjects received salvage therapy (high-dose open-label rufi derivative). Eleven subjects received one dose on day 28, and one subject received two doses (on days 28 and 35).

[0317] The longest study participation time for participants was 98 days (+2 days). This duration was based on the assumption that re-epithelialization was achieved on day 28 of the double-blind treatment period, and the follow-up period would begin immediately after healing. If persistence was not maintained after 28 days, participants were eligible for a 0.06% lufifine-derived salvage dose, and thus could participate for an additional 41 days (14 days + 28 days (+2 days)) during the open-label phase of the study. Table 1 shows the assessment timeline, detailing the schedule and procedures for each study visit.

[0318] Application of research products of randomized objects Each dose of the study product was administered, including a volume (100 µL) for filling the BCL and a volume (100 µL per fornix) for covering the inner surfaces of the upper and lower fornixes. The eye was then covered with a double-layered eye pad and secured with adhesive tape for approximately 8 hours.

[0319] No topical ocular treatments should be used within 2 hours before and 8 hours after application of the study product. Outside of this period, subjects should continue to receive the standard treatment regimen prescribed by the investigator.

[0320] This study evaluated three treatment groups. The randomization ratio was 1:1:1. • Group A – 0.06% Rufoi Derivative • Group B – 0.006% Rufoi Derivative • Group C – Medium.

[0321] The study product is applied once on day 1 and day 2. If no re-epithelialization occurs by day 14, an additional application of the study product is planned.

[0322] If no reepithelialization of the study eye occurs at the end of the 28-day double-blind treatment period, or if the persistence of initial reepithelialization is not maintained, the subject will receive up to two salvage doses of 0.06% rufi derivative during the open-label phase of the study.

[0323] Research Description Potentially eligible candidates are those with clinically non-infectious PED resulting from severe chemical and / or thermal injury that has not responded to standard treatment. Clinical signs of ocular injury include epithelial shedding, conjunctivitis (congestion), limbal ischemia, and corneal edema.

[0324] Before considering object inclusion in the group, PED must already exist. At least The treatment period was 14 days (the day of injury was counted as day one), and researchers determined that the defect must not show clinically significant improvement in healing despite the subject receiving at least 14 days of conventional standard treatment. Conventional standard treatment included antibiotics, steroid eye drops, mydriatics, ascorbic acid, amniotic membrane transplantation, and debridement of necrotic epithelium.

[0325] Before proceeding with any protocol-specific procedures, informed consent must be obtained from the participants by the principal investigator or a designated person with the appropriate qualifications. Enrollment eligibility assessment and baseline assessment are conducted before Day 1 of treatment. Final eligibility checks are required before randomization to treatment, such as a urine pregnancy test (confirmed negative) for women of childbearing age and pre-treatment measurement of PED size. If a participant's medical history confirms that the epithelial defect has been present for more than 14 days and is unresponsive to SOC, the investigator may decide to combine the eligibility assessment visit with the Day 1 visit. Visits may also be combined if the medical history is confirmed and the participant faces practical difficulties (e.g., the participant needs to travel long distances to the base, or transportation inconveniences and restrictions due to the COVID-19 pandemic), provided that all assessments and records required before randomization have been completed and eligible participants have been confirmed.

[0326] The first application of the study product was performed on day 1 of the treatment period after randomization, and the second application was performed on day 2. For each single application: BCL filled with 100 μL of study product was immediately applied to the study eye, followed by 100 μL of study product on each side of the superior and inferior fornix. The eye was then covered with a double-layer eye pad and secured with tape for approximately 8 hours. Importantly, during the treatment period, subjects continued to receive their prescribed SOC except for 2 hours before and 8 hours after IP application.

[0327] Then, each subject underwent assessment visits twice a week until corneal reepithelialization occurred.

[0328] If the defect has not re-epithelialized by day 14, an additional single application of the double-blind study product will be administered.

[0329] If corneal re-epithelialization does not occur by day 28 of the double-blind treatment period, subjects will receive up to two salvage doses of 0.06% rufi derivative during the open-label phase of the study. If re-epithelialization does not occur within 14 days after the first salvage dose, the subject will be withdrawn from the open-label phase of the study.

[0330] Once re-epithelialization occurs (regardless of the time point within the double-blind treatment period), the subject enters the post-treatment follow-up period and continues to wear the BCL for 2 weeks to anchor the newly formed epithelium to the basement membrane and prevent premature detachment of the cell layer due to trauma. The subject is re-evaluated 28 days after the initial re-epithelialization to confirm epithelial persistence. The subject then withdraws from the study after completing all visits.

[0331] If the initial epithelialization is followed by epithelial shedding during the 28-day follow-up period after treatment, the subject will receive up to two salvage doses of 0.06% rufi derivative during the open-label phase of the study, 7 days apart.

[0332] If corneal re-epithelialization occurs within 14 days of the first open-label salvage dose, these subjects should also be followed up for 28 days to assess the persistence of epithelialization; however, this is not included in the analysis of double-blind study populations. See also Figure 1 At each study visit from randomization to the end of the treatment period, participants will be assessed, as detailed in the assessment schedule. Assessments include: slit-lamp examination to assess the condition of the anterior segment (including eyelids, sclera, conjunctiva, cornea, anterior chamber, and lens), ocular photography, epithelial defect measurement, visual acuity testing, intraocular pressure measurement, and an ocular symptom questionnaire. At each study visit, any adverse events occurring during treatment will also be assessed, and participants will be followed up for up to 30 days after the last administration of the study product.

[0333] Standard treatment during the treatment and open-label phases of the study: From day 1 to the end of the treatment period, except for 2 hours before and 8 hours after administration of the study product, subjects may continue with the standard treatment regimen prescribed by the investigator at the study site. Details of the administered medication, dosage, and dosage regimen must be recorded in the original documents and case report form.

[0334] The bandage contact lens (BCL) used for IP application is provided by the sponsor and must be worn during the treatment period; the BCL should only be replaced when a new BCL is used for IP application on the day of administration, or if necessary (e.g., in case of discomfort or BCL dislodgement).

[0335] Systemic standard treatment is not defined in the protocol but is determined by the investigator or the research site's routine procedures. However, the systemic medications and regimens used to treat PED must remain unchanged during the study until the subject re-epithels or withdraws from the study.

[0336] Local anesthetic eye drops are permitted only during ophthalmological evaluations at each study visit.

[0337] Inclusion criteria: Subjects are eligible for inclusion in the study only if they meet all of the following criteria: 1. Men or women of any age.

[0338] 2. Clinically non-infectious, persistent corneal epithelial defects (PED) caused by severe ocular chemical and / or thermal damage to one or both eyes.

[0339] 3. Records of PED not responding to current standard treatment for at least 14 days (day 1 is counted as the day of injury).

[0340] 4. On day 1 of treatment, the epithelial defect is measured to be at least 2 mm along the maximum diameter.

[0341] 5. Provide written informed consent and have the ability to follow the visit and medication schedule.

[0342] After the 35 randomly assigned participants (0.06% of the Ruffi derivative group (n=12) and 0.006% of the Ruffi derivative group (n=12)) completed the study, the data were unblinded and analyzed. Using the complete dataset, 66.7% (8 / 12) of the Ruffi derivative 0.06% and 0.006% of the Ruffi derivative group achieved corneal epithelial repair. In contrast, only 27.3% (3 / 11) of the mediator group achieved repair, and each of the three repaired participants received two doses of mediator on days 1 and 2, while the three participants who did not repair also received two doses of mediator, and the remaining participants in the mediator group who did not repair received three doses of mediator.

[0343] Therefore, the subjects with non-healing ocular surface defects who received the connexin modulator Lufi derivative (n=24) showed approximately a 2.5-fold (2.44-fold) greater probability of healing compared to the VEH group (n=11) after connexin modulator application, and demonstrated a 39.4% relative improvement in corneal epithelial repair. These findings were clinically significant and, despite the small sample size, were close to statistical significance (Fisher's exact test: p=0.065). Overall, treatment was well tolerated, with similar TEAEs across all three treatment groups, characterized by mild to moderate severity, and unlikely to be associated with or unrelated to the study drug. One case of SAE was reported but determined to be unrelated to the study drug.

[0344] Data from this randomized, prospective, mediator-controlled, double-blind human clinical trial for the treatment of non-healing ocular surface defects demonstrate that the use of connexin modulators (e.g., rufi derivatives) at the doses described herein, or at different, therapeutically effective doses described herein, can provide sustained healing of non-healing or persistent ocular surface defects, including persistent corneal epithelial defects. These data both confirm the safety of the unique connexin modulator doses and dosing regimens described herein and claimed, provide a clinical proof of concept, and validate their use in the treatment management of patients with non-healing ocular surface or corneal defects or conditions.

[0345] ***** All patents, publications, scientific articles, websites, and other documents cited or referenced herein reflect the technical level of those skilled in the art to which this invention pertains, and each such cited document is incorporated herein by reference to the same extent as if it were individually and completely cited or listed herein. The applicant reserves the right to substantially incorporate all material and information from any such patents, publications, scientific articles, websites, electronically available information, and other cited materials or documents into this specification. References to any application, patent, or publication in this specification should not and must not be construed as an admission or suggestion of any kind that such documents constitute valid prior art or are part of general knowledge in any country of the world.

[0346] The specific methods and compositions described herein represent preferred embodiments and are exemplary, not intended to limit the scope of the invention. Other objects, aspects, and embodiments will arise in those skilled in the art upon reading this specification, all of which are included within the spirit of the invention as defined by the claims. It will be 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 herein by way of example may be practiced in the absence of any one or more elements or limitations not expressly disclosed herein as necessary. Therefore, for example, in each case herein, in embodiments or examples of the invention, any terms “comprising,” “substantially consisting of,” and “consisting of” in the specification may be replaced with any of the other two terms. Furthermore, the terms “comprising,” “including,” “containing,” etc., should be understood to be broad and not restrictive. The methods and processes described herein by way of example may be practiced in different sequences of steps and are not necessarily limited to the sequence of steps shown herein or in the claims. It should also be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Under no circumstances should this patent be construed as limited to the specific embodiments, implementations, or methods disclosed herein. Under no circumstances should this patent be construed as limited by any statement made by an examiner or other official of the Patent and Trademark Office (unless the applicant expressly and unreservedly acknowledges such statement in their response). Furthermore, headings, subheadings, etc., are provided to enhance the reader's understanding and should not be construed as limiting the scope of the invention. Any examples of aspects, embodiments, or components of the invention mentioned herein should be considered non-limiting.

[0347] The terms and expressions used are descriptive and not restrictive, and their use does not preclude any equivalents of the shown and described features or portions thereof. However, it should be understood that various modifications are possible within the scope of the invention. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, modifications and variations can be made by those skilled in the art using the concepts disclosed herein, and such modifications and variations are considered to be included within the scope defined by the appended claims.

[0348] This document provides a broad and general description of the invention. Each narrower group of species and subgenera falling within the scope of this general disclosure also constitutes part of the invention. This includes general descriptions of the invention where anything is excluded by provisos or negative limitation of genus, regardless of whether the excluded content is specifically enumerated herein.

[0349] Other embodiments are included in the following claims. Furthermore, if the features or embodiments of the invention are described in the form of a Markush group, those skilled in the art will recognize that the invention is also described by any single member or subgroup of that Markush group.

Claims

1. A method for treating a subject having a non-healing ocular surface defect, the method comprising administering to the subject a therapeutically effective amount of a connexin modulator, 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 of claim 1, wherein the non-healing ocular surface defect is a persistent corneal epithelial defect or a persistent ocular epithelial defect.

3. The method of claim 1, wherein the non-healing ocular surface defect is caused by a chemical injury, a thermal injury, or inflammation.

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

5. The method of claim 1, wherein the connexin modulator comprises a connexin 43 modulator.

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

7. The method of claim 6, wherein the antisense compound comprises a LuF derivative (SEQ ID NO: 1).

8. The method of claim 6, wherein the antisense compound targets at least about 8 nucleobases of a nucleic acid molecule encoding a connexin having a nucleobase sequence according to SEQ ID NO: 17, and the antisense compound is 15 to 35 nucleobases in length.

9. The method of claim 8, wherein the antisense compound is a modified antisense oligonucleotide, and the modified antisense oligonucleotide comprises at least one modification selected from a modified internucleoside linkage containing a phosphorothioate linkage, a modified sugar moiety, and a modified nucleobase.

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

11. The method of claim 7, wherein the antisense compound is selected from 0.06% LuF derivative and 0.006% LuF derivative.

12. The method of claim 7, wherein the antisense compound comprises at least about 0.006% LuF derivative or at least about 0.06% LuF derivative.

13. The method of claim 1, wherein the connexin modulator is administered by topical administration or oral administration or by injection.

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

15. The method of claim 6, wherein the antisense compound is formulated with a pluronic gel.

16. The method of claim 1, wherein the connexin modulator is a connexin 43 peptidomimetic.

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

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

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

20. The method of claim 26, wherein the connexin hemichannel modulator comprises tonabersat.

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

22. The method of claim 21, further comprising administering to the subject a composition comprising about 0.06% or about 0.006% Rufinamide on about day 7 and / or about day 21.

23. The method of claim 21, further comprising administering to the subject a composition comprising about 0.06% or about 0.006% Rufinamide on about day 28 and / or about day 35.

24. The method of claim 22, further comprising administering to the subject a composition comprising about 0.06% or about 0.006% Rufinamide on about day 28 and / or about day 35.

25. A pharmaceutical composition comprising about 0.6 or 0.06 mg / mL Rufinamide, about 226.9 mg / mL Poloxamer 407, about 0.99 mg / mL Disodium Phosphate Dibasic Heptahydrate, and about 0.25 mg / mL Potassium Phosphate Monobasic.

26. The pharmaceutical composition of claim 39, having a final volume of about 300 μL.

27. A method for treating non-healing ocular surface defects in a subject, the method comprising administering to the eye of the subject a pharmaceutical composition of claim 26 at least once on day 1, at least once on day 2, and at least once on about day 14.

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

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

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

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