Preparations for use in the prevention and / or treatment of peripheral neuropathy and related diseases

Abstract

The present invention relates to the preparation and use of topically applied formulations of diaminophenothiazine compounds, such as 3,7-bis(dimethylamino)phenothiazine-5-ium chloride, in the prevention and / or treatment of peripheral neuropathy and related disorders, particularly ophthalmopathy. In a preferred embodiment, a topically applied formulation is prepared comprising 3,7-bis(dimethylamino)phenothiazine-5-ium chloride encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres, the microspheres being coated with a mucoadhesive agent, such as chitosan.

Description

INCORPORATION OF RELATED APPLICATIONS

[0001] This application claims priority to Singapore Patent Application No. 10201907677Q filed on August 20, 2019, the contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to preparations and methods of using the preparations in the prevention and / or treatment of peripheral neuropathy and related disorders, particularly ophthalmopathy. [Background technology]

[0003] The following discussion of the background of the invention is intended to facilitate an understanding of the present invention, but it should be understood that the discussion does not constitute an agreement or admission that any of the material referred to was published, known, or part of the general public in any jurisdiction as of the priority date of this application.

[0004] Our nervous system can be divided into two components: the central nervous system (CNS), which includes the brain and spinal cord, and the peripheral nervous system (PNS), which encompasses the dorsal root ganglia (DRGs) and nerve fibers outside the brain and spinal cord and neuroreceptors at their terminals. The PNS has two parts: an afferent part that transmits sensory information to the CNS, and an efferent part that transmits motor commands from the CNS to target effectors (cells, tissues, etc.). The PNS enables the CNS to communicate with the external environment.

[0005] Unlike the brain and spinal cord of the CNS, which are protected by a myelin sheath, vertebrae, and skull, the nerves of the PNS are often unmyelinated. Neurons are the cell bodies that make up the nervous system. Each neuron has a long projection known as an axon, which communicates with other neurons by transmitting electrochemical signals. These axon projections make up the PNS. The dorsal root ganglion is part of the PNS, and its nerve fibers carry information from sensory input to the brain for processing. Peripheral nerves provide important sensory units in the nerves of the hands, feet, and cornea. Peripheral neuropathy is damage to the PNS. Peripheral neuropathy often causes weakness, numbness, and pain. Peripheral neuropathy can result from trauma, infection, autoimmune disorders, metabolic disorders, genetic factors, aging, and exposure to toxins. One of the most common causes is diabetes, which results in hyperglycemia, or elevated glucose levels in the body. Hyperosmolarity is also a sequela of hyperglycemia, potentially leading to hyperosmolarity-induced peripheral neuropathy.

[0006] Diabetes mellitus (DM) is a group of metabolic disorders characterized by prolonged high blood glucose levels. As of 2017, an estimated 425 million people worldwide were living with diabetes (Diabetes Atlas 2017). DM can cause peripheral neuropathy, or complications associated with damage to the PNS. Diabetic peripheral neuropathy is one of the most common complications of DM, affecting all peripheral nerves, including sensory and motor neurons, and, less frequently, the autonomic nervous system. Currently, there is no reliable treatment for hyperglycemia (high glucose)-induced peripheral neuropathy other than successful blood glucose control. Managing blood glucose levels can be very challenging for some subjects, especially those who rely on insulin injections, due to the cost and pain of injections. With the exception of blood glucose control, the only treatment for diabetic neuropathy is reducing pain and other symptoms. Nerve fiber degeneration is common in diabetic subjects with type 2 diabetes, especially those with proliferative diabetic retinopathy (Gao et al. Int J Ophthalmol, Vol 8, No. 2, 2015, 358-364). DM subjects exhibit signs such as progressive loss of corneal nerve density and reduced corneal sensitivity (Han et al., Clinical Interventions in Aging, 2019, 14, 53-63). Corneal nerve fiber length measured by confocal microscopy has been shown to predict the development of diabetic peripheral neuropathy (Han et al., 2019). Hyperglycemia can also lead to a hyperosmolar ocular environment, resulting in hyperosmolality-induced corneal nerve damage. Better management and treatment of diabetic neuropathy, including prevention and / or treatment of peripheral nerve damage in the eye, particularly the corneal nerve and its branches, is needed.

[0007] Methylthioninium chloride (MTC) (also known as methylene blue (MB), methylthionine chloride, tetramethylthionine chloride, 3,7-bis(dimethylamino)phenothiazin-5-ium chloride, CI Basic Blue 9, tetramethylthionine chloride, 3,7-bis(dimethylamino)phenazathionium chloride, Swiss Blue, CI 52015, CI Solvent Blue 8, aniline violet, and Urolene Blue®) is a low molecular weight (319.86), water-soluble, tricyclic organic compound of the formula: [ka]

[0008] Various methods for synthesizing MTC are known and are summarized in WO 2006 / 032879. WO 2006 / 032879 also discloses numerous uses for methylene blue, including as a medical dye, a redox indicator, an antiseptic, for the treatment and prevention of kidney stones, and for the treatment of melanoma, malaria, and viral infections. Administered MTC has also been used as an oxidizing / reducing (redox) agent and as an antidote in cases of carbon monoxide, nitrite, and aniline poisoning. Administered MTC has also been proposed for the treatment of Alzheimer's disease, a disease affecting the central nervous system.

[0009] MTC is soluble in both water and alcohol. It is not recommended to dissolve MTC in saline, as it has been reported that methylene blue precipitates when diluted with 0.9% sodium chloride saline (due to the presence of chloride ions, which have been shown to reduce the solubility of methylene blue). It is reported to be incompatible with caustic alkalis, iodides, and dichromates, as well as with redox compounds. Injection of MTC into the spinal compartment of the cerebrospinal fluid has been reported to cause massive peripheral nerve damage, resulting in complete paraplegia (Shah et al., J. Neurol. Neurosurg., and Psychiatry, 1978, 41, 384-386).

[0010] The inadvertent use of higher concentrations of MTC in the eye rather than trypan blue during cataract surgery has resulted in irreversible blindness, corneal edema (Timsin et al., Arq. Bras. Oftalmol. Vol. 79, No. 2, 2016), bullous keratopathy resulting in corneal transplantation, and endophthalmitis progressing to corneal damage. Therefore, methylene blue is generally considered a toxic dye that should not be used in the eye. Furthermore, because MTC is a dye, its application to the skin or mucous membranes often results in unsightly blue stains or smears.

[0011] Thus, there is a need to develop formulations and methods of use that ameliorate at least one of the disadvantages outlined above. Summary of the Invention

[0012] The object of the present invention is to ameliorate some of the above-mentioned difficulties in the prevention and / or treatment of peripheral neuropathy, particularly ophthalmic diseases, preferably with preparations and methods of using the preparations.

[0013] One aspect of the present invention relates to a topically applied formulation for preventing and / or treating peripheral nerve injury and related disorders, comprising an effective amount of a diaminophenothiazine compound or tautomeric form of Formula I: [ka] wherein R1, R2, R3, R4, R5, R6, R1', R2', R3' and R4' are independently hydrogen, methyl or ethyl.

[0014] Another aspect of the present invention relates to the above-mentioned topically applied formulations for use in the prevention and / or treatment of peripheral nerve injuries and associated diseases.

[0015] Another aspect of the present invention relates to a method for preventing and / or treating peripheral nerve injury, comprising administering to a subject in need thereof a method for preventing and / or treating peripheral nerve injury and associated diseases. (a) an effective amount of a diaminophenothiazine compound of Formula I or a tautomeric form: [ka] provided that R1, R2, R3, R4, R5, R6, R1', R2', R3' and R4' are independently hydrogen, methyl or ethyl; (b) any of the above topically applied preparations; or (c) a topically applied formulation comprising an effective amount of a diaminophenothiazine compound of Formula I or a tautomeric form, and a carrier. The method comprises administering

[0016] Another aspect of the present invention relates to the use of a compound of formula I in the manufacture of a topically applied formulation for the prevention and / or treatment of peripheral nerve injury and diseases associated therewith.

[0017] Another aspect of the present invention relates to a topically applied formulation for preventing and / or treating peripheral nerve injury and related disorders, comprising 150 μM or less of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride and a carrier medium that adjusts the viscosity of the formulation to a range of about 15 to 150 millipascals per second.

[0018] Another aspect of the present invention relates to a topical kit for use in a method for preventing and / or treating peripheral nerve injury and related disorders, more particularly ocular nerve injury associated with corneal nerve injury and related disorders, including neurotrophic keratopathy, comprising: (a) a topical composition comprising an effective amount of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride; and (b) a container for holding the composition.

[0019] Another aspect of the invention relates to a method of preparing a topically applied composition, comprising encapsulating 3,7-bis(dimethylamino)phenothiazin-5-ium chloride into microparticles.

[0020] Other aspects and features of the present invention will become apparent to those skilled in the art from the following description of specific embodiments of the invention, when viewed in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0021] In the exemplary drawings, embodiments of the invention are shown. [Figure 1] Figure 1 shows the test schedule. [Figure 2] Figure 2 shows cell viability by Hoechst 33342 nucleic acid staining in cells treated with methylene blue under glucose toxicity conditions. DRGs were treated with methylene blue for 1 hour prior to 150 mM glucose, and HTS analysis was performed 48 hours later. [Figure 3] Figure 3 shows limb counts per neuron by staining with beta III tubulin in cells treated with methylene blue under glucose toxicity conditions. DRGs were treated with methylene blue for 1 hour prior to 150 mM glucose, and HTS analysis was performed 48 hours later. [Figure 4] Figure 4 shows the total length per neuron stained with beta III tubulin in cells treated with methylene blue under glucose toxicity conditions. DRGs were treated with methylene blue for 1 hour prior to 150 mM glucose, and HTS analysis was performed 48 hours later. [Figure 5] Figure 5 shows cell viability by staining with WST8 in cells treated with methylene blue under glucose toxicity conditions. DRGs were treated with methylene blue for 1 hour prior to 150 mM glucose, and HTS analysis was performed 48 hours later. [Figure 6] FIG. 6 shows a sample of a micrograph (40x magnification) of the microspheres produced in Run 1. [Figure 7] FIG. 7 shows a sample of a micrograph (40x magnification) of the microspheres produced in run 2. [Figure 8] FIG. 8 shows a sample of a micrograph (40x magnification) of the microspheres produced in run 3. [Figure 9]FIG. 9 shows a graph of the 7-day release profile of encapsulated methylene blue for the 5 mg sample produced in Run 2. Detailed Description of the Invention

[0022] Specific embodiments of the present invention will now be described with reference to the accompanying drawings. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention. Additionally, unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For clarity and consistency, the same reference numbers will be used throughout the drawings whenever possible.

[0023] Various embodiments relate to topically applied formulations for preventing and / or treating peripheral nerve injury and related disorders, comprising a diaminophenothiazine compound or tautomeric form of Formula I: [ka] wherein R1, R2, R3, R4, R5, R6, R1', R2', R3' and R4' are independently hydrogen, methyl or ethyl.

[0024] In various embodiments, forms of the compounds of formula (1) include pharmaceutically acceptable salts, hydrates, and solvates thereof.

[0025] Various examples relate to topically applied formulations for protection against peripheral nerve injury comprising an effective amount of a diaminophenothiazine compound of Formula I or a tautomeric form, 5 μM or less of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride, and a physiological buffer: wherein in Formula I, R1, R2, R3, R4, R5, R6, R1', R2', R3', and R4' are independently hydrogen, methyl, or ethyl.

[0026] As used herein, diaminophenothiazine compounds are positively charged due to electron delocalization, resulting in partial positive charges located on both the nitrogen and sulfur atoms. Among various examples, Structure A shows the chemical structure of thionine, the simplest diaminophenothiazine, and the three tautomeric forms produced by electron delocalization. [ka]

[0027] In various embodiments, R1, R2, R3, R4, R5, and R6 are hydrogen, and R2, R5, R1', R2', R3', and R4' are independently hydrogen or ethyl.

[0028] In various embodiments, the diaminophenothiazine, sometimes referred to as a thiazine, is selected from the group consisting of Azure A, Azure B, Azure C, thionine, 3,7-bis(dimethylamino)phenothiazin-5-ium chloride (methylene blue), new methylene blue, and 1-9-dimethylmethylene blue.

[0029] In various embodiments, the diaminophenothiazine compound comprises 3,7-bis(dimethylamino)phenothiazin-5-ium chloride.

[0030] In various embodiments, the diaminophenothiazine compound comprises 150 μM or less of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride.

[0031] In various embodiments, 3,7-bis(dimethylamino)phenothiazin-5-ium chloride comprises the compound of formula II. [ka]

[0032] While methylene blue is strictly discouraged or even prohibited in the management of eye-related disorders, it has been devised that solutions buffered to a physiological pH range and containing methylene blue at sufficiently low concentrations are tolerable. Similarly, such solutions are also tolerable to peripheral nerves. Beneficially, low concentrations of methylene blue can be used in the management of eye-related and related disorders, particularly as a topically applied formulation, in various embodiments, for peripheral neuropathy caused by trauma, infection, autoimmune disorders, metabolic disorders, genetic factors, aging, exposure to toxins such as chemotherapy, hyperglycemia, and / or hyperosmolarity-induced peripheral neuropathy, or other causes known in the art that result in peripheral neuropathy.

[0033] As used herein, the term "topically applied formulation" refers to any formulation that is applied to a specific location on or on the body. It may refer to a solution, suspension, lotion, ointment, gel, powder, paste, dough, transdermal patch, or any other preparation that can be applied to a specific location on or on the body. In various embodiments, the specific location on or on the body refers to a body surface such as the skin or mucous membrane. In various embodiments, the specific location on or on the body directly refers to a nerve site, for example, a nerve site on the surface of the cornea. In various embodiments, the specific location on or on the body refers to the eye.

[0034] The terms "prevention" and "protection" from peripheral nerve injury are used herein to refer to preventing, inhibiting, or reducing damage or death of nerve cells, fibers, or outgrowths from the PNS. In various embodiments, this is achieved by reducing the undesirable sequelae resulting from glucose influx into neuronal mitochondria, which results in cellular damage. Neurons are known to have a greater amount of mitochondria than other cells. In various embodiments, this is achieved by reducing the non-enzymatic binding, i.e., glycation, of glucose to proteins or lipoproteins, which leads to peripheral nerve damage. In various embodiments, this is achieved by reducing toxic effects or toxins, such as reducing the undesirable neuronal effects of chemotherapy drugs. In various embodiments, this is achieved by increasing the antioxidant defense capacity of cells.

[0035] The term "treating" peripheral nerve injury is used herein to refer to preventing or reducing further damage or death of nerve cells, fibers, or outgrowths from the PNS and / or providing a suitable environment for the formation and growth of new nerve cells, fibers, or outgrowths from the PNS. In various embodiments, this is achieved by reducing the undesirable sequelae resulting from glucose influx into neuronal mitochondria, which results in cellular damage. Neurons are known to have a greater amount of mitochondria than other cells. In various embodiments, this is achieved by reducing non-enzymatic binding, i.e., the glycation of glucose to proteins or lipoproteins, which leads to peripheral nerve damage. In various embodiments, this is achieved by reducing toxic effects or toxins, such as reducing the undesirable effects of chemotherapy drugs on nerves. In various embodiments, this is achieved by increasing the antioxidant defense capacity of the cells and / or their nerve fibers.

[0036] The term "3,7-bis(dimethylamino)phenothiazin-5-ium chloride" is used herein to refer to methylthioninium chloride (MTC), a low molecular weight (319.86), water-soluble, tricyclic organic compound of the formula: (also known as methylene blue (MB), methylthionine chloride, tetramethylthionine chloride, CI Basic Blue 9, tetramethylthionine chloride, 3,7-bis(dimethylamino)phenazathionium chloride, Swiss blue, CI 52015, CI Solvent Blue 8, aniline violet, and Urolene Blue®). [ka]

[0037] In various embodiments, 3,7-bis(dimethylamino)phenothiazin-5-ium chloride may be prepared by any method known in the art.

[0038] In various embodiments, the topically applied formulation comprises between 0.0001 μM and 150 μM of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride. In various embodiments, the topically applied formulation comprises an MTC of 149.5 μM or less. In various embodiments, the topically applied formulation comprises an MTC of 59.5 μM or less. In various embodiments, the topically applied formulation comprises an MTC of 4.5 μM or less. In various embodiments, the topically applied formulation comprises between 0.0001 μM and 50 μM of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride. In various embodiments, the topically applied formulation comprises between 0.001 μM and 90 μM, between 0.005 μM and 0.5 μM, or between 0.0059 μM and 0.49 μM of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride. In various embodiments, the topically applied formulation comprises 0.05 μM 3,7-bis(dimethylamino)phenothiazin-5-ium chloride.

[0039] In various embodiments, the topically applied formulation further comprises a physiological buffering agent.

[0040] In various embodiments, the physiological buffer comprises an aqueous hydrogen ion solution consisting of a mixture of an acid and its conjugate base, which is capable of maintaining the physiological pH of the formulation at about pH 4 to about pH 8 when in contact with additives or components of the biological environment. In various embodiments, the physiological buffer is selected from the group consisting of bicarbonate, bicine, cacodate, HEPES, MES, MOPS, PIPES, phosphate, TEPS, TAPSO, TES, tricine, and Tris. In various embodiments, the physiological buffer comprises sodium bicarbonate and sodium chloride. Surprisingly, when sodium chloride is present in the physiological buffer along with sodium bicarbonate, diaminophenothiazine compounds such as MTC remain in solution, demonstrate peripheral nerve protection, and have a pH suitable for both direct peripheral nerve application and ophthalmic use, despite technical prohibitions against using MTC with sodium chloride. In various embodiments, the pH of the solution is 4 to 8. In various embodiments, the pH of the solution is 4 to 7.5. In various embodiments, the pH of the solution is 4. In various embodiments, the pH of the solution is 5. In various embodiments, the pH of the solution is 6. In various embodiments, the pH of the solution is 7. In various embodiments, the pH of the solution is 7.4. In various embodiments, the pH of the solution is 7.5. In various embodiments, the pH of the solution is 8.

[0041] In various embodiments, the physiological buffer preferably comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in the presence of sodium bicarbonate and sodium chloride. In various embodiments, the physiological buffer comprises a neurobasal medium.

[0042] In various embodiments, the topically applied formulation comprises a carrier. In various embodiments, the carrier is a carrier-vehicle that is liquid and / or water-soluble in nature. In various embodiments, the carrier or carrier-vehicle may comprise a polymer such as a high molecular weight hydrophilic polymer, polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer, cellulose derivatives (gellan gum, xanthan gum), dexamethasone, pilocarpine, 2-hydroxypropyl beta-cyclodextrin, poly(2-hydroxyethyl methacrylate), ethylene glycol dimethyl acrylate, acrylamide, N-vinylpyrrolidone, and ethyl acrylate, or a combination thereof. In various embodiments, the carrier or carrier-vehicle may comprise an excipient that enhances delivery of the drug to the eye, such as a chelating agent, a surfactant, or a cyclodextrin. In various embodiments, the carrier or carrier-vehicle may comprise a nanoparticle, a microparticle, a niosome, a discosome, a dendrimer, an in situ gel, a wafer, or an inclusion compound such as an embedding in a contact lens. In various embodiments, the carrier or carrier-vehicle may comprise a sugar molecule, such as sucrose, glucose, lactose, or D-glucose (dextrose). In various embodiments, endogenous glucose, which occurs naturally in the peripheral nerve environment, may form part of the carrier or carrier-vehicle in solution. This is particularly true in DM subjects with hyperglycemia, which results in high concentrations of glucose.

[0043] In various embodiments, the carrier or carrier medium comprises microparticles or microspheres.

[0044] In various embodiments, the effective amount of diaminophenothiazine compound comprises 3,7-bis(dimethylamino)phenothiazine-5-ium chloride preloaded into microparticles.

[0045] In various embodiments, the microparticles are coated with a mucoadhesive agent, such as chitosan. In various embodiments, the microparticles are suspended in a fluid medium. In various embodiments, the fluid medium is water-soluble in nature so that a diaminophenothiazine compound, such as 3,7-bis(dimethylamino)phenothiazin-5-ium chloride, can be dissolved in the fluid medium.

[0046] In various embodiments, the carrier or carrier medium adjusts the viscosity of the topically applied formulation to a range of about 15 to 150 millipascals per second.

[0047] In various embodiments, the carrier or carrier medium modulates the release profile of the diaminophenothiazine compound.

[0048] In various embodiments, the carrier or carrier medium adjusts the release profile of a diaminophenothiazine compound, such as 3,7-bis(dimethylamino)phenothiazine-5-ium chloride, over a predetermined time period. Any known carrier or carrier-vehicle capable of adjusting the rate of release of a diaminophenothiazine compound, such as MTC, into a peripheral nerve environment is suitable. In various embodiments, the carrier or carrier medium adjusts the release profile of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride to a predetermined rate that is less than or slower than the rate of release of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride itself. In various embodiments, suitable carriers or carrier-vehicles may comprise polymers such as high molecular weight hydrophilic polymers, polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer, cellulose derivatives (gellan gum, xanthan gum), dexamethasone, pilocarpine, 2-hydroxypropyl beta-cyclodextrin, poly(2-hydroxyethyl methacrylate), ethylene glycol dimethyl acrylate, acrylamide, N-vinylpyrrolidone, and ethyl acrylate, or combinations thereof. In various embodiments, suitable carriers or carrier-vehicles may comprise inclusion compounds such as nanoparticles, microparticles, niosomers, discosomes, dendrimers, in situ gels, wafers, or may be embedded in contact lenses. In various embodiments, the carrier or carrier-vehicle slows or reduces the release profile of a diaminophenothiazine compound, such as 3,7-bis(dimethylamino)phenothiazin-5-ium chloride, over a predetermined period of time.

[0049] In various embodiments, the carrier or carrier-vehicle adjusts the viscosity of the topically applied formulation to a range of about 15 to 150 millipascals / second. In various embodiments, the carrier or carrier-vehicle adjusts or increases the viscosity to achieve a range of about 15 to 150 millipascals / second. In various embodiments, the carrier or carrier-vehicle increases the viscosity to a range of about 15 to 100 millipascals / second. In various embodiments, the carrier or carrier-vehicle adjusts or increases the viscosity to achieve a range of about 15 to 50 millipascals / second. In various embodiments, the viscosities are at room temperature. Increasing the viscosity advantageously increases the contact time of a diaminophenothiazine compound, such as MTC, with the corneal surface, thereby increasing its bioavailability, particularly to the corneal nerves and their branches. In various embodiments, the carrier or carrier-vehicle, such as a polymer, sugar molecule, nanoparticle, microparticle, niometh, discosometh, or dendrimer, may adjust or increase the viscosity of the solution.

[0050] In various embodiments, the carrier or carrier medium modulates the release profile of a diaminophenothiazine compound, such as 3,7-bis(dimethylamino)phenothiazine-5-ium chloride. In various embodiments, the carrier or carrier medium comprises microparticles with a sustained release profile. This has the advantage of increasing the viscosity of the formulation and also reduces the need for frequent eye drops by increasing the residence or residence time of the topically applied formulation on the precornea. Sustained-release formulations also have the advantage of reducing unsightly bluish stains on the skin, as the increased viscosity reduces dripping and / or tear-related release, eliminating staining and discoloration of the face. In various embodiments, the carrier or carrier medium microparticles with a sustained release profile comprise poly(lactide-co-glycolide) (PLGA) microspheres, although any microparticles known in the art to provide a slow / sustained release profile are suitable. In various embodiments, the PLGA microspheres have a polymer ratio of PLGA (50:50), PLGA (75:25), or PLGA (85:15). In various embodiments, the sustained release profile of a diaminophenothiazine compound, such as MTC, is tailored to 1 month, 3 months, or 6 months using different polymer ratios in the microspheres or using different sized microspheres.

[0051] In various embodiments, the topically applied formulation additionally comprises one or more compounds, including pharmaceutical agents.

[0052] Various embodiments relate to the use of the topically applied formulations described herein for the prevention and / or treatment of peripheral nerve injury.

[0053] In various embodiments, the topically applied formulations are suitable for use in animals, including humans, for the prevention and / or treatment of peripheral nerve injuries in a medical or veterinary setting.

[0054] In various embodiments, peripheral nerve damage results in or includes neurotrophic keratopathy, hi various embodiments, peripheral nerve damage includes peripheral neuropathy caused by trauma, infection, autoimmune disorders, metabolic disorders, genetic factors, aging, exposure to toxins such as chemotherapy, hyperglycemia-induced peripheral neuropathy, or any other cause known in the art to result in peripheral neuropathy.

[0055] In various embodiments, the topically applied formulation is for use on the eye, hi various embodiments, the topically applied formulation is in the form of eye drops or ointments.

[0056] Various embodiments relate to methods of preventing and / or treating peripheral nerve injury, including administering to a subject in need of preventing and / or treating peripheral nerve injury. (a) A diaminophenothiazine compound of formula I or a tautomeric form thereof: [ka] provided that R1, R2, R3, R4, R5, R6, R1', R2', R3' and R4' are independently hydrogen, methyl or ethyl; (b) a topically applied formulation according to any one of claims 1 to 13, or (c) A topically applied formulation comprising a diaminophenothiazine compound of formula I or a tautomeric form and a carrier-vehicle. The method comprises administering

[0057] Various embodiments relate to methods of preventing and / or treating peripheral nerve injury and related disorders, including administering to a subject in need of preventing and / or treating peripheral nerve injury and related disorders. (a) an effective amount of a diaminophenothiazine compound of Formula I or a tautomeric form: [ka] provided that R1, R2, R3, R4, R5, R6, R1', R2', R3' and R4' are independently hydrogen, methyl or ethyl; (b) a topically applied formulation according to any one of claims 1 to 13, or (c) a topically applied formulation comprising an effective amount of a diaminophenothiazine compound of formula I or a tautomeric form, and a carrier-vehicle; The method comprises administering

[0058] In various embodiments, the subject is an animal, such as a mammal, including humans, horses, cows, dogs, cats, and other animals in need of prevention and / or treatment of peripheral nerve damage.

[0059] Various embodiments relate to methods of preventing and / or treating peripheral nerve injury, including administering to a subject in need of preventing and / or treating peripheral nerve injury. (a) a topically applied formulation comprising 150 μM or less of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride and a physiological buffer; or (b) any of the above topically applied preparations; or (c) Topically applied formulations containing 150 μM or less of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride and a carrier-vehicle. The method comprises administering

[0060] In various embodiments, the topically applied formulation is an ophthalmic formulation.

[0061] As used herein, the term "subject" refers to a patient or individual who has been diagnosed with, is at risk for, suffers from, or is suspected of having peripheral neuropathy. In various embodiments, the patient or individual has been diagnosed with DM or prediabetes, is suspected of or at risk for DM, or is an individual with dry eye disease. In various embodiments, the patient or individual has been diagnosed with or is at risk for neurotrophic keratopathy. In various embodiments, the patient or individual suffers from peripheral neuropathy caused by trauma, infection, metabolic disorders, genetic factors, aging, exposure to toxins such as chemotherapy, hyperglycemia, and / or hyperosmolality-induced peripheral neuropathy, or other causes known in the art that result in peripheral neuropathy.

[0062] In various embodiments, the topically applied formulation is administered directly to a peripheral nerve site. In various embodiments, the topically applied formulation is administered to the eye.

[0063] In various embodiments, the method further comprises measuring corneal nerve parameters prior to administration of the topically applied formulation to determine whether the subject is in need of peripheral nerve injury prevention and / or treatment. In various embodiments, the measured parameters include nerve fiber length and nerve fiber density. In various embodiments, the parameters are measured by in vivo or in situ confocal microscopy of the cornea. Corneal nerve fiber status has been shown to correlate with DM and peripheral neuropathy in other parts of the subject's body (Han et al., 2019).

[0064] In various embodiments, the method further comprises measuring corneal nerve parameters before and / or after administration of the topically applied formulation. In various embodiments, the measured parameters include nerve fiber length and nerve fiber density. In various embodiments, the parameters are measured by in vivo or in situ confocal microscopy of the cornea, allowing for monitoring of the effectiveness of the prophylaxis and / or treatment.

[0065] Various examples relate to the use of a diaminophenothiazine compound of formula I or a tautomeric form in the manufacture of a topically applied formulation for the prevention and / or treatment of peripheral nerve injury.

[0066] In various embodiments, the compounds are prepared in topically applied formulations suitable for use in animals, including humans, for the prevention and / or treatment of peripheral nerve damage in a medical or veterinary setting.

[0067] In various embodiments, the peripheral nerve damage results in or includes neurotrophic keratopathy, hi various embodiments, the peripheral nerve damage includes peripheral neuropathy caused by trauma, infection, autoimmune disorders, metabolic disorders, genetic factors, aging, exposure to toxins such as chemotherapy, hyperglycemia-induced peripheral neuropathy, or any other cause known in the art that results in peripheral neuropathy.

[0068] Various examples relate to the use of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride in the manufacture of a topically applied formulation comprising 150 μM or less of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride and a physiological buffer or carrier or carrier-vehicle for the prevention and / or treatment of peripheral nerve injury.

[0069] In various examples, topically applied formulations for the prevention and / or treatment of peripheral nerve injuries are described.

[0070] In various embodiments, the use is ophthalmic. In various embodiments, the use is for the treatment of dry eye. In various embodiments, the use is for the prevention of corneal peripheral neuropathy.

[0071] Various embodiments relate to topically applied formulations for the prevention and / or treatment of peripheral nerve injury, comprising 150 μM or less of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride and a carrier or carrier-vehicle that adjusts or increases the viscosity of the formulation in the range of about 15 to 150 millipascals per second.

[0072] In various embodiments, the topically applied formulation comprises a carrier or carrier-vehicle. In various embodiments, the carrier or carrier-vehicle may comprise a polymer such as a high molecular weight hydrophilic polymer, polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer, cellulose derivatives (gellan gum, xanthan gum), dexamethasone, pilocarpine, 2-hydroxypropyl beta-cyclodextrin, poly(2-hydroxyethyl methacrylate), ethylene glycol dimethyl acrylate, acrylamide, N-vinylpyrrolidone, and ethyl acrylate, or a combination thereof. In various embodiments, the carrier or carrier-vehicle may comprise an excipient that enhances delivery or penetration of the drug into the eye, such as a chelating agent, surfactant, or cyclodextrin. In various embodiments, the carrier or carrier-vehicle may comprise an inclusion compound such as a nanoparticle, a microparticle, a niosome, a discosome, a dendrimer, an in situ gel, a wafer, or a contact lens. In various embodiments, the carrier or carrier-vehicle may comprise a sugar molecule, such as sucrose, glucose, lactose, or D-glucose (dextrose). In various embodiments, endogenous glucose, which occurs naturally in the peripheral nerve environment, may form part of the carrier or carrier-vehicle in solution. This is particularly true in DM subjects with hyperglycemia, which results in high concentrations of glucose.

[0073] In various embodiments, the carrier or carrier-vehicle modifies, e.g., reduces, the release profile of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride. Any known carrier or carrier-vehicle capable of modulating a slow release profile is suitable, such as a carrier or carrier-vehicle that can modulate, e.g., reduce, the rate of MTC release into the peripheral nerve environment. In various embodiments, the carrier or carrier-vehicle modulates, e.g., reduces, the release profile of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride to a predetermined rate that is less than or slower than the rate of release of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride alone. In various embodiments, suitable carriers or carrier-vehicles may comprise polymers such as high molecular weight hydrophilic polymers, polyvinyl alcohol, poloxamers, hyaluronic acid, carbomers, cellulose derivatives (gellan gum, xanthan gum), dexamethasone, pilocarpine, 2-hydroxypropyl betacyclodextrin, poly(2-hydroxyethyl methacrylate), ethylene glycol dimethyl acrylate, acrylamide, N-vinylpyrrolidone, and ethyl acrylate, or combinations thereof. In various embodiments, suitable carriers or carrier-vehicles may comprise nanoparticles, microparticles, niosomes, discosomes, dendrimers, in situ gels, inclusion compounds such as wafers, or may be embedded in contact lenses.

[0074] In various embodiments, the carrier or carrier-vehicle adjusts the viscosity, e.g., increases the viscosity to a range of about 15 to 150 millipascals / second. In various embodiments, the carrier or carrier-vehicle adjusts the viscosity, e.g., increases the viscosity to a range of about 15 to 100 millipascals / second. In various embodiments, the carrier or carrier-vehicle adjusts the viscosity, e.g., increases the viscosity to a range of about 15 to 50 millipascals / second. In various embodiments, the viscosities are at room temperature. Increasing the viscosity has the advantage of increasing the contact time of MTC with the corneal surface, thereby increasing its bioavailability. In various embodiments, the following carriers or carrier-vehicles adjust or increase the viscosity of the solution: polymers, sugar molecules, nanoparticles, microparticles, niosomes, discosomes, and dendrimers.

[0075] In various embodiments, the carrier or carrier-vehicle modifies, e.g., slows, the release profile of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride. In various embodiments, the carrier or carrier-vehicle comprises microparticles with a slow release profile. This has the advantage of increasing the viscosity of the formulation, which also reduces the need for frequent application of eye drops and improves patient acceptance. Slow-releasing formulations also have the advantage of reducing unsightly blue staining on the skin, as the increased viscosity reduces dripping and / or tearing release, eliminating staining and discoloration of the face. In various embodiments, the carrier or carrier-vehicle microparticles with a sustained / slow release profile comprise poly(lactide-co-glycolide) (PLGA) microspheres, although any microparticles known in the art to provide a slow / sustained release profile are suitable. In various embodiments, the PLGA microspheres have a polymer ratio of PLGA (50:50), PLGA (75:25), or PLGA (85:15). Such ratios allow for further tailoring, such as slowing down, the release profile of MTC. In various embodiments, the release profile of MTC is tailored to 1 month, 3 months, or 6 months by using different polymer ratios in the microspheres or by using different sized microspheres.

[0076] In various embodiments, the carrier or carrier-vehicle comprises a particulate as described herein.

[0077] In various embodiments, the carrier or carrier-vehicle comprises an aqueous fluid, such as a gel.

[0078] In various embodiments, the carrier or carrier-vehicle comprises a gel as described herein.

[0079] The term "gel" is used herein to refer to a crosslinked network comprising a three-dimensional semi-solid matrix that contains or can contain a high proportion of fluid.

[0080] Topically applied methylene blue has never been shown to be effective in PNS disorders in general. Topically applied formulations have been shown to manage peripheral neuropathy caused by trauma, infection, autoimmune disorders, metabolic disorders, genetic factors, aging, exposure to toxins such as chemotherapy, hyperglycemia-induced peripheral neuropathy, or other causes known in the art that result in peripheral neuropathy, which may be useful in the prevention and / or treatment of such disorders.

[0081] Various embodiments relate to a topically applied kit comprising: (a) a topically applied composition comprising an effective amount of a diaminophenothiazine, such as 3,7-bis(dimethylamino)phenothiazine-5-ium chloride; and (b) a container holding the composition for use in a method for preventing and / or treating peripheral nerve damage and associated disorders. In various embodiments, the peripheral nerve damage and associated disorders include ocular peripheral nerve damage associated with corneal nerve injury and associated disorders, including neurotrophic keratopathy.

[0082] In various embodiments, the container is configured to hold an effective amount of the topically applied composition to support treatment for at least 1 day, 7 days, 14 days, 21 days, or 30 days.

[0083] In various embodiments, the kit comprises an eye drop kit. In various embodiments, the container comprises a frangible seal.

[0084] Various examples relate to formulations of topically applied compositions that involve entrapment of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride in microparticles or other carrier-vehicles that may have a slow release profile.

[0085] In various embodiments, the microparticles or other carrier-vehicles that may have a slow release profile further comprise a fluid or aqueous medium.

[0086] In various embodiments, the carrier-vehicle can carry two or more compounds.

[0087] In various embodiments, the topically applied composition formulation comprises meeting at least some of the following conditions: (a) It does not contain any animal parts or products that are not halal under Islamic law or any animal parts or products that are not slaughtered in accordance with Islamic law; (b) It does not contain any najs under Islamic law; (c) It is safe for human use in the prescribed dosage, and is non-toxic, non-intoxicating, and harmless to health; (d) It is not compounded, processed, or manufactured using equipment contaminated with najs under Islamic law; (e) It does not contain any human parts or derivatives thereof that are not permitted under Islamic law; and (f) During compounding, processing, handling, packaging, storage, and distribution, the halal medicinal product is physically separated from other medicinal products that do not meet the requirements described in items (a), (b), (c), (d), or (e), or from other items that are not halal and najs under Islamic law.

[0088] Throughout the specification, unless indicated to the contrary, the terms "comprising," "consisting," and the like are intended to be inclusive, in other words, to mean "including but not limited to."

[0089] Throughout the specification, unless the context requires otherwise, the words "consist of," or "consisting of," "consisting of," and variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of other integers or groups of integers.

[0090] Throughout the specification, unless the context requires otherwise, the words "comprises," "including," "including," and variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of other integers or groups of integers.

[0091] The term "about" is used herein to typically mean ±5% of the stated value, more typically ±4% of the stated value, even more typically ±3% of the stated value, even more typically ±2% of the stated value, even more typically ±1% of the stated value, and even more typically ±0.5% of the stated value.

[0092] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this invention belongs. As used herein, the following definitions are provided to facilitate understanding of the invention.

[0093] Throughout this document, unless indicated to the contrary, the terms "comprising," "consisting," "having," and the like are intended to be inclusive, in other words, to mean "including but not limited to."

[0094] Furthermore, throughout the specification, unless the context requires otherwise, the words "comprises," "including," "including," and variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of other integers or groups of integers.

[0095] As used in the specification and the appended claims, the singular forms "a" and "the" include plural references unless the context clearly dictates otherwise. [Example]

[0096] Example 1: Prevention of glucose-induced peripheral nerve damage Experiments based on in vitro cell assays demonstrated the ability of methylene blue to protect dorsal root ganglion neurons and their neural extensions from injury at high glucose concentrations after only 1 hour of pre-injury incubation.

[0097] The aim of this study was to explore the dose-dependent neuroprotective effects of methylene blue (MB) against high glucose-induced neurotoxicity using a cell-based, multiparameter protocol that reflects its mechanism of action. Assays were performed using freshly isolated rat dorsal root ganglion neurons (DRGs) cultured in 96-well plates that were pretreated with different concentrations of the compound (0.0005 μM, 0.005 μM, 0.05 μM, 0.5 μM, 5 μM, and 50 μM) prior to exposure to 150 mM glucose for 48 hours.

[0098] In vitro injury was induced in DRG cultures by 48 hours of glucose treatment (150 mM). Prior to glucose treatment, DRGs were incubated with six increasing concentrations of test compound (0.0005 μM, 0.005 μM, 0.05 μM, 0.5 μM, 5 μM, and 50 μM) for 1 hour, followed by 48 hours of co-culture with high glucose. After treatment, cells were fixed, stained with anti-beta III tubulin, and analyzed by BD Pathway 855 (Becton Dickinson). Using HCS technology, cellular parameters associated with neurite outgrowth—an indicator of pre-lethal cytotoxicity—were measured at the single-cell level, enabling high-throughput screening.

[0099] To compare the degree of neuroprotection of compounds at different concentrations, the level of neuroprotection at each concentration of compound was studied, and four different neuroprotection scores were established according to the change in level compared to control cells: no neuroprotection, low neuroprotection, medium neuroprotection, and high neuroprotection.

[0100] Primary cultures of dorsal root ganglion neurons were prepared from the dorsal root ganglia of neonatal SD rats on day 8. Ganglia were removed from the spine under a binocular microscope, and neurons were enzymatically dispersed twice with 1.25 mg / ml collagenase I (Worthington) for 1 hour at 37°C. They were then cultured at 30,000 cells per well in poly-L-lysine (Sigma-Aldrich Cat. P1524) containing neurobasal medium (Invitrogen Cat. 21103-049) supplemented with B27 (Invitrogen Cat. 17504-044). Cells were maintained in neurobasal medium supplemented with B27 in a humidified 5% CO atmosphere at 37°C for 8 days.

[0101] Methylene blue (Tocris) was dissolved in ethanol to a final concentration of 10 mM, and the test compound concentration was adjusted in neurobasal medium (Invitrogen Cat. 21103-049) supplemented with 20% B27 (Invitrogen Cat. 17504-044).

[0102] On day 8, cells were pretreated with six increasing concentrations of methylene blue (0.0005 μM, 0.005 μM, 0.05 μM, 0.5 μM, 5 μM, and 50 μM) in complete medium for 1 h, and the medium was replaced with neurobasal medium without B27 components (80%) or neurobasal medium supplemented with B27 components (20%) in the presence of 150 mM glucose, co-cultured with different concentrations of methylene blue for 48 h. See Figure 1 for the testing schedule.

[0103] Neurobasal medium is a basal medium formulated to meet the specific requirements of neuronal cells. It allows for the long-term maintenance of normal phenotype and growth of neuronal cells, maintaining a pure population of neuronal cells in a serum-free environment without the need for an astrocyte feeder layer. The use of this medium results in a methylene blue solution with a higher pH, ranging from 4 to 8, suitable for use with neuronal cells. The solution becomes more viscous.

[0104] [Table 1]

[0105] High-content screening assay: Methylene blue (MB) neuroprotection was measured by a high-content screening (HCS) assay, which includes the following endpoints: neurite outgrowth, cell number, and cell viability.

[0106] 1. Cell count: Hoechst 33342 nucleic acid dye was used to measure cell count. Cells were stained with 5 μg / ml Hoechst 33342 (Invitrogen # Cat. H1399), washed three times, and measured for fluorescence emission at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. This dye allows for highly sensitive cell count determination by fluorescence microscopy.

[0107] Glucose treatment prevented glucose-induced mortality in cocultures with methylene blue in a dose-dependent manner at all concentrations, except for the highest concentration (50 μM), which resulted in 54% neuronal mortality after exposure to a concentration of 150 mM. Furthermore, it was observed that addition of this concentration of the compound to glucose induced a further increase in mortality (Figure 2 and Table 1).

[0108] [Table 2]

[0109] Varying degrees of neuroprotection were observed at the remaining concentrations tested, thus methylene blue supplementation significantly reduced cell death and enhanced neuronal survival and neuroprotection.

[0110] 2. WST-8 assay: To detect viable cells, 10 μl of CCK-8 reagent (WST-8) was added to each well, and the plate was incubated at 37° C. After 1 hour, the absorbance at 450 nm was measured using a Synergy II microplate reader.

[0111] Although glucose treatment resulted in a 22% reduction in viability in DRGs as measured by WST8 after exposure, no compound prevented the glucose-induced reduction in viability (Figure 5). It was also observed that the highest concentration of compound (50 μM) added to glucose induced a reduction in viability.

[0112] 3. Beta III Tubulin Staining: Beta III tubulin staining was measured by immunohistochemistry (IHQ). Cells were washed with PBS (Sigma-Aldrich # Cat. D8537) and fixed with methanol for 10 minutes. After the fixation step, samples were washed three times with PBS and permeabilized with PBS + 0.3% Triton for 10 minutes. Then, samples were blocked with PBS + bovine serum albumin (BSA) for 30 minutes. Finally, anti-beta tubulin III antibody (Abcam # Cat. ab7751) was added at 1 / 1000 in PBS + 0.5% BSA for 60 minutes at room temperature. After three washing steps, a secondary antibody, anti-goat-anti-mouse Alexa 633 (Abcam # Cat. A21050), was added at 1 / 100 for 60 minutes to react with the primary antibody. Samples were then washed three times and measured by automated fluorescence microscopy. To investigate the role of neurite extensions, two geometric patterns were primarily used in this study: total neurite length (total length of the path from the neuron body) and limb number (number of terminal neurite segments per neuron).

[0113] At the end of the assay, cells were first stained with WST-8 for 1 hour to measure viability, then fixed with methanol for beta-III tubulin staining and imaged again using the BD Pathway 855. To obtain enough cells for analysis, nine fields per well were imaged. A 20x objective was used to collect images for distinct fluorescence channels. The dyes were excited, and fluorescence was monitored at their excitation and emission wavelengths with appropriate filter settings. Exposure times were adjusted during the setup to avoid overlapping emission between different probes. Collected images were analyzed using a module that allows simultaneous quantification of subcellular structures stained with different molecular probes to measure fluorescence intensities associated with defined nuclear and cytoplasmic compartments.

[0114] To investigate the role of neurite extension, two geometric patterns were primarily used in this study: total neurite length per neuron and limb count / neuron. Glucose treatment resulted in a 38% and 31% decrease in total neurite length and limb count, respectively, compared with untreated cells. On the other hand, methylene blue could reverse the neurite outgrowth lesions induced by 0.05 μM glucose (Figures 3 and 4, Tables 2 and 3). Thus, supplementation with methylene blue significantly promoted neurite outgrowth and enhanced neuronal survival.

[0115] [Table 3]

[0116] [Table 4]

[0117] It was also observed that the highest concentrations of compound added to glucose (5 and 50 μM) induced a reduction in neurite outgrowth.

[0118] For all parameters studied, a variation of at least 20% in fluorescence intensity or in the corresponding morphological parameter relative to untreated cultures was considered. To compare the degree of neuroprotection provided by methylene blue, four different scores were assigned to neuroprotection according to the level of variation compared to control cells: 0 (no neuroprotection or less than 20% variation), 1 (20-40% variation), 2 (40-60% variation), 3 (60-100% variation), and 4 (100-200% variation). Furthermore, some concentrations exhibited some damage, possibly due to their toxicity or their toxicity in combination with glucose. Similar scores were assigned to damage according to the level of variation compared to glucose-treated cells: -1 (20-40% variation), -2 (40-60% variation), and -3 (40-60% variation). The individual scores were summed to obtain a total level of neuroprotection for each compound, defined as the degree of neuroprotection. From this calculation, a scale of neuroprotection was established: high (>6), moderate (4-5), low (1-3), and no neuroprotection (0).

[0119] To compare the degree of neuroprotection of compounds tested at different concentrations, we created different scores according to the level of change (the criteria for forming groups are described in the Materials and Methods section) and assigned a value to each parameter. The sum of all values ​​for each parameter and tested concentration was calculated, and four separate groups were created according to their level of neuroprotection. From this analysis, we defined the degree of neuroprotection for each compound investigated (Table 4).

[0120] [Table 5]

[0121] Briefly, methylene blue was rated as moderately neuroprotective at 0.05 μM, poorly neuroprotective at 0.0005 μM, 0.005 μM, and 0.5 μM, non-neuroprotective at 5 μM, and toxic at 50 μM.

[0122] The neuroprotective effects of methylene blue are shown in Table 5 below:

[0123] [Table 6]

[0124] Depending on all parameters investigated, MB showed some neuroprotection against glucose-induced DRG toxicity at concentrations ranging from 0.0005 μM to 0.5 μM, and moderate neuroprotective effect against glucose-induced DRG toxicity at 0.05 μM.

[0125] In this study, glucose toxicity resulted in a decrease in neurite outgrowth and cell number and viability. The preventive effect of MB against glucose toxicity was associated with an increase in cell viability and regeneration of neurite outgrowth. Considering all parameters investigated, MB supplementation provided a moderate level of neuroprotection at 0.05 μM.

[0126] Example 2: Testing the (in vivo) tolerance of topically applied methylene blue formulations in the human eye To determine whether topical methylene blue formulations, up to and including 0.005% (160 μM), are suitable for human ocular use, a range of ocular formulations were formulated and tested. The methylene blue compound (Bleu de Methylene 1%, STEROP) was dissolved in sterile lubricating eye gel (Systane Gel Drops) in 10 ml volumes, up to a final maximum concentration of 160 μM. The concentrations of the topical formulations were adjusted to 0.05 μM, 0.5 μM, 5 μM, 50 μM, 60 μM, 150 μM, and 160 μM. The lubricating eye gel (Systane Gel Drops) was used to increase the viscosity of the eye drops. One drop of each topical formulation was instilled into a human eye once daily. Eyes were examined immediately, 6 hours, and 24 hours after instillation using standard ophthalmoscopic scanning. No adverse effects were noted at any time point.

[0127] It has been observed that in all topical formulations containing methylene blue at concentrations below 150 μM, the topical formulations are fully tolerated without causing ocular damage or discomfort. At concentrations above 150 μM, there is a slight stinging sensation, causing mild discomfort and self-limiting effects. The viscosity of the gel-like topical formulation allows the topical formulation to remain in the eye and not run off the eyelid and streak the face. This "drip-free" effect is highly desirable.

[0128] Example 3: Preparation of a topically applied formulation Dispensing A Methylene blue (Tocris) is dissolved in sterile water to a final concentration of 10 mM. Test compound concentrations are adjusted to 0.05 μM, 0.5 μM, 5 μM, 50 μM, or 100 μM in 10 mM HEPES buffer containing sodium bicarbonate and sodium chloride.

[0129] Dispensing B First, a fluid gel is prepared by dissolving low-acrylic gelator gum (Kelcogel CG LA, Azelis, UK) in deionized water. A 1% (w / v) solution is obtained by adding the correct proportion of gelator powder to room-temperature deionized water. The sol is heated to 70 °C while stirring on a hot plate equipped with a magnetic stirrer until the polymer is completely dissolved. Once dissolved, the gelator sol is added to the cup of a rotary flowmeter equipped with a cup-and-vane geometry (cup: 35 mm diameter, vane: 28 mm diameter). The entire assembly is then cooled to 40 °C. Methylene blue (Tocris) is dissolved in sterile water to a final concentration of 10 mM. The test compound is adjusted to 0.05 μM, 0.5 μM, 5 μM, 50 μM, or 100 μM in phosphate-buffered saline (PBS) (4.76 mg / ml) and sodium chloride (0.2 M). Following this, the mixture is cooled at a rate of 1°C / min under shear (450 / s) to a final temperature of 20°C. Samples are then removed and stored at 4°C until further use. For fluid gels without methylene blue compounds, the proportions are adjusted so that the final eye drops have a composition of 0.9% (w / v) gelling agent and 10 mM NaCl.

[0130] Dispensing C PLGA encapsulation requires a compound with high solubility in DI water. Therefore, the solubility of MTC was tested. 20 ml of solvent was placed in a glass vial on a magnetic stirrer. 88.0% pure methylene blue (Merck) was added to the vial until it reached or exceeded the saturation point. Stirring was continued for as long as necessary for the slow-dissolving 88.0% pure methylene blue (Merck). The amount of dissolved 88.0% pure methylene blue (Merck) was measured to obtain the solubility of 88.0% pure methylene blue (Merck) in a particular solvent.

[0131] Solubility results for 88.0% pure methylene blue (Merck) in: DI water: 41.432 mg / ml Dichloromethane (DCM): < 0.1 mg / ml Acetonitrile (ACN): 0.5675 mg / ml

[0132] Solubility studies of methylene blue surprisingly suggested that it was initially incompatible with the PLGA carrier, despite the ultimate success.

[0133] Encapsulation Microsphere production was performed on three separate occasions in three separate runs as follows. The case of methylene blue is unique; the dye is distinct in color, and visual inspection can be performed to confirm that the methylene blue was successfully loaded into the PLGA microspheres. Three production runs were performed to optimize the loading efficiency of methylene blue into the microspheres.

[0134] Oil-in-water emulsions were generated using glass capillary microfluidics. An axisymmetric coaxial glass capillary flow-focusing device was assembled using square and circular capillaries. The surface of the circular capillary was hydrophilized by treatment with oxygen plasma (100 watts) for 120 seconds. An aqueous continuous phase was created by mixing PVA (1% w / v) with water. The dispersed phase (O) was perfused with 88.0% pure methylene blue (Merck). One of the following concentrations of 88.0% pure methylene blue (Merck) was used: (i) 0.5 μM, (ii) 50 μM, and (iii) 150 μM, respectively. The PLGA concentration (50 mg / mL) in dichloromethane (DCM) was kept constant in all three cases. The W and O phases were injected from the two ends of the square capillary tube through the outer coaxial region using a syringe pump (Harvard PHD22 / 2000 series) at flow rates of 150 and 50 μL / min, respectively. The fluids were hydrodynamically focused through the nozzle of the round capillary tube, resulting in the formation of emulsion droplets. A 6 cm ID glass bore was used for sample collection. To prevent droplet coalescence, approximately 300 μL of O / W emulsion was dispensed directly into the glass bore containing a preformed film (3 mL) of the continuous phase. The solvent was allowed to evaporate at room temperature (24 °C). Uniform microspheres were collected to encapsulate methylene blue and were confirmed using microscopic images.

[0135] Characterization of microspheres To maximize the performance of PLGA microspheres, the optimal microsphere size is in the range of 10-25 μm. 100 microspheres were measured using the software ImageJ and characterized by reporting the mean value and standard deviation.

[0136] result The sizes of the microspheres produced in each production run are as follows: Run 1: 25.45±1.13μm Run 2: 20.46±1.12μm Run 3: 16.69±0.74μm

[0137] Photomicrographs of each run are shown in Figures 6, 7 and 8. Runs 2 and 3 meet the acceptance criteria.

[0138] Example 4: Release profile of Formulation C 5 mg of dry microspheres prepared in Run 2 above were used in a 7-day release study. The following time points were measured to assess the release profile: 0, 3, 6, 9, 12, 24, 48, 72, 96, 120, 144 and 168 hours.

[0139] A small sample of the dry microspheres was also measured to assess the methylene blue loading.

[0140] The concentration of each sample was determined using HPLC. result The results for the 7-day release of methylene blue from the microspheres produced in Run 2 are as follows:

[0141] [Table 7]

[0142] A target size range was established for successful microsphere fabrication by encapsulating methylene blue within PLGA microspheres. The results were confirmed in a 7-day release study showing uniform release of methylene blue. The microspheres were coated with a mucoadhesive coating.

[0143] In production runs, encapsulation resulted in substantial loading of methylene blue into the PLGA microspheres, which was confirmed using HPLC, and the release profiles are shown in Table 6 and Figure 9.

[0144] Example 5: In vivo prevention and / or treatment of ocular peripheral nerve injury Methylene blue compound (Tocris) was dissolved in sterile water to a final concentration of 10 mM. The test compound was adjusted to 0.05 μM, 0.5 μM, 5 μM, 50 μM, or 100 μM in 10 mM HEPES buffer containing sodium bicarbonate and sodium chloride (Preparation A). The untreated group contained only 10 mM HEPES buffer containing sodium bicarbonate and sodium chloride. A second group of the same test solution was prepared with 5 g / L gelling gum to adjust and increase the viscosity (Preparation B). A third group of the same test solution was prepared with slow-release microparticles to adjust and increase the viscosity (Preparation C).

[0145] The solution was instilled into the eyes of diabetic mice once daily for two weeks. Intravital confocal microscopy was performed using a modified Heidelberg Retina Tomography Rostock Corneal Module. Before scanning, mice were anesthetized with a single injection of 50 mg / kg ketamine and 5 mg / kg xylazine, followed by a single drop of topical 0.5% eye drops (Bausch & Lomb, Tampa, FL). Lubrication of the contralateral eye was maintained using Refresh Plus (Allergan, Irvine, CA). To improve epithelial visualization, a silicone washer was placed on the outer edge of the TomoCap (Heidelberg, Germany), creating a thin space between the tip of the TomoCap and the epithelial surface. The washer dimensions were as follows: outer diameter 1.2 cm, inner diameter 3 mm, and thickness 600 mm (Specialty Silicone Products, Barston Spa, NY). Image acquisition was performed as previously described. The cornea was scanned at a lens speed of 30 mm per second, increasing the speed by 1 mm per step. A minimum of three scans were taken for each cornea.

[0146] Repeated treatments are contemplated to increase the efficacy of topical methylene blue eye drops in a dose-dependent manner, and increasing the viscosity of the solution to 5 μM, 50 μM, and 100 μM is also contemplated to increase the efficacy of methylene blue eye drops.

[0147] Those skilled in the art should further appreciate that variations and combinations, rather than substitutions or permutations, of the above-described features may be combined to form still further embodiments that fall within the intended scope of the present invention.

[0148] As will be appreciated by those skilled in the art, each embodiment may be used in combination with other embodiments or with several embodiments.

Claims

1. 1. A topically applied formulation for preventing and / or treating peripheral nerve damage at nerve sites on the surface of the cornea, comprising an effective amount of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride and a carrier-vehicle that increases the viscosity of the topically applied formulation, the carrier-vehicle comprising: A topically applied formulation comprising a polymer selected from the group consisting of polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer, gellan gum, xanthan gum, 2-hydroxypropyl betacyclodextrin, poly(2-hydroxyethyl methacrylate), ethylene glycol dimethyl acrylate, acrylamide, N-vinylpyrrolidone, and ethyl acrylate, or a combination thereof.

2. 2. The topical formulation of claim 1, wherein the topical formulation comprises between 0.0001 μM and 60 μM of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride, or wherein the topical formulation comprises 0.0005 μM, 0.005 μM, 0.05 μM, 0.5 μM, 5 μM, 50 μM or 100 μM of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride.

3. 10. The topical formulation of claim 1, wherein the topical formulation comprises 150 μM or less of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride.

4. The method further comprises the step of: a) sodium bicarbonate and sodium chloride; b) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; or c) A neurobasal medium, comprising glycine, L-alanine, L-arginine hydrochloride, L-asparagine-H 2 O, L-cysteine, L-histidine hydrochloride-H 2 O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, choline chloride, calcium D-pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, lipoflavin, thiamine hydrochloride, vitamin B12, i-inositol, calcium chloride (CaCl 2 ) (anhydrous), iron nitrate (Fe(NO 3 ) 3 .9H 2 O), magnesium chloride (anhydrous), potassium chloride (KCl), sodium bicarbonate (NaHCO 3 ), sodium chloride (NaCl), sodium dihydrogen phosphate (NaH 2 PO 4 -H 2 O), zinc sulfate (ZnSO 4 .7H 2 O), D-glucose (dextrose), HEPES, phenol red or sodium pyruvate.

5. 10. The topical formulation of claim 1, wherein the carrier-vehicle comprises a gel.

6. 10. The topical formulation of claim 1, wherein the carrier-vehicle adjusts the viscosity of the topical formulation to be in the range of 15 to 150 millipascals per second.

7. 2. The topically applied formulation of claim 1, wherein the peripheral nerve damage at the nerve site on the surface of the cornea consists of neurotrophic keratopathy.

8. 2. The topically applied formulation of claim 1, wherein the application is to the eye.

9. 9. The topically applied formulation of claim 8, wherein the topically applied formulation is an eye drop or an ointment.

10. 10. Use of 3,7-bis(dimethylamino)phenothiazin-5-ium chloride and a carrier-vehicle in the manufacture of a topically applied formulation according to claim 1 for preventing and / or treating peripheral nerve damage on nerve sites on the surface of the cornea, wherein the carrier-vehicle increases the viscosity of the topically applied formulation.

11. The use according to claim 10, wherein the peripheral nerve damage at the nerve site on the surface of the cornea consists of neurotrophic keratopathy.

12. 12. The use according to claim 10 or claim 11, which is for use on the eyes.

13. The use according to claim 12, wherein the topically applied formulation is an eye drop or an ointment.

14. A topically applied formulation for preventing and / or treating peripheral nerve damage at nerve sites on the surface of the cornea, comprising 150 μM or less of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride and a carrier-vehicle that adjusts the viscosity of the formulation to the range of 15 to 150 millipascals per second.

15. 15. The topically applied formulation of claim 14, wherein the carrier-vehicle modulates the release profile of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride.

16. A topical formulation according to claim 14 or claim 15, wherein the carrier-vehicle consists of a gel.

17. A kit for topical application for use in preventing and / or treating peripheral nerve damage at nerve sites on the surface of the cornea, the kit comprising:

1. A kit comprising: (a) a topically applied composition comprising an effective amount of 3,7-bis(dimethylamino)phenothiazine-5-ium chloride and a carrier-vehicle that increases the viscosity of the topically applied formulation; and (b) a container holding said composition for preventing and / or treating peripheral nerve injury at a nerve site on the surface of the cornea.

18. 20. The kit of claim 17, wherein the container is configured to hold an amount of the topically applied composition to support treatment for at least 1 day, 7 days, 14 days, 21 days, or 30 days.

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