New Treatment for Macular Degeneration

Abstract

Formulations and methods of treatment for the prevention and / or treatment of vision loss due to age-related macular degeneration are disclosed. The disclosed formulations include a botulinum neurotoxin (e.g., a botulinum neurotoxin or fragment thereof, in pure form or with one or more peptide fragments and / or neurotoxin-related proteins). In some embodiments, the disclosed formulations include one or more anti-VEGF agents. The disclosed formulations can be applied to the intraocular or extraocular regions of a patient. When applied to the extraocular region of a patient, the botulinum-based pharmaceutical formulations may be delivered to the intraocular region of a patient via intraaxonal transport, thereby allowing the active ingredient to penetrate the choroid, neural retina, and / or retinal pigment epithelium without direct injection into the eye, improving therapeutic safety by eliminating the risks of retinal detachment, retinal tears, retinal hemorrhage, and blindness associated with direct injection into the eye.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority from U.S. Provisional Patent Application No. 62 / 431,512, filed on December 8, 2016; U.S. Provisional Patent Application No. 62 / 449,914, filed on January 24, 2017; and U.S. Provisional Patent Application No. 62 / 533,961, filed on July 18, 2017, the contents of which are hereby incorporated by reference in their entirety.

Background Art

[0002] Age - related macular degeneration (AMD) is a leading cause of blindness in humans and generally occurs in people over 50 years old. This disease is genetic, and there is about a 50% chance that descendants will inherit a clinically significant disease from a parent who has become blind due to the disease. AMD accounts for up to 70% of irreversible blindness in the United States and is one of the most common problems encountered by ophthalmologists. Worldwide, the number of people predicted to have age - related macular degeneration in 2020 is 196 million, and it is predicted to increase to 288 million in 2040.

Summary of the Invention

[0003] New formulations and methods are disclosed herein for treating, and in some cases preventing, vision loss due to macular degeneration by administering botulinum toxin-based pharmaceuticals. Administration of the disclosed formulations can be intravitreal or extraocular, and in some embodiments can include subcutaneous, submuscular, intraneural, topical, intraosseous, and / or interfacial injection. As used herein, the term "intravitreal" means applying the formulation directly to the eye, and the term "extraocular" means applying the formulation to an area outside the eye (e.g., to the eyelid or orbit). When extraocular injection is employed, complications of intravitreal injection can be avoided. In some embodiments, repeated injections can be employed to maintain the biological effect up-to-date and practical. Improvement in vision can be subjectively reported after treatment by the disclosed methods, and in some cases, physical changes in the physical structure of the eye can be observed using SD-OCT, funduscopy, or other imaging techniques.

[0004] In some embodiments, the injection can penetrate the orbit and macula via a route that does not cause weakness of the extraocular muscles, thereby avoiding diplopia, apoptosis, and other neuromuscular effects that can cause complications. As described in detail below, the disclosed formulations and methods can be designed to target one or more of the choroid, neural retina, retinal pigment epithelium (RPE), peripheral nerves entering the eye, and / or other related tissues. The disclosed formulations and methods can be used, in some embodiments, to treat exudative macular degeneration (i.e., non-exudative with intraretinal fluid, blood, or subretinal fluid, or blood and which may lead to geographic atrophy).

[0005] Prior to administering the disclosed formulation to a patient, a clinical evaluation may be performed by a qualified practitioner to assess whether treatment by the disclosed method is appropriate. The clinical evaluation can be performed based on one or more of a family history, a fundus examination using photography, and SD OCT, along with a careful examination of the state of the retinal pigment epithelium for signs of impairment. Signs of impairment include, but are not limited to, the presence of pigment in funduscopy, pigment migration forward into the neurosensory retina (excess pigment within the retina), the presence and volume of drusen, focal intra retinal hyper reflection, subretinaloid deposits, subretinaloid hyperreflectivity, a dynamic decrease in drusen volume, second eye staging for severity, hypo reflectivity, choroidal neovascularization, hypopigmentation, discontinuity and disappearance of OCT reflectivity lines (e.g., IS-OS, outer nuclear layer, RPE layer), the thickness of the retina and choroid or related components, dynamic changes in any measurements, thickening of reflectivity lines, cyst formation, and the formation of any body fluids. In some cases, RPE activation can be a risk factor for the progression of macular degeneration and the risk of progression can be evaluated based on one or more of anatomical pathological findings, medical history, and the tempo of disease progression and the state of the second eye.

[0006] As will be described in detail below, the disclosed formulation and treatment method can slow RPE degeneration, preserve photoreceptors, treat or prevent high-risk leakage, treat and prevent angiogenesis, prevent cell apoptosis, treat and prevent RPE activation, treat and prevent RPE migration, treat and prevent RPE sheet distortion, prevent geographic atrophy, prevent retinal atrophy, prevent loss of rods and cones, convert the wet stage to the dry stage, maintain vision, and / or restore vision.

Brief Description of the Drawings

[0007]

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[0008] Macular degeneration generally destroys the central vision of the affected individual, making reading, driving, and the performance of independent productive life impossible. Rapid vision loss is generally associated with the exudative "or wet" form of the disease with leakage of fluid from newly formed pathological choroidal blood vessels through the biological barrier established by the retinal pigment epithelium. Leakage through the retinal pigment epithelium results in destruction of photoreceptors with changes to atrophic states associated with fibrotic scarring of the structure of the retinal pigment epithelium or associated photoreceptor destruction.

[0009] Current treatments for macular degeneration include intravitreal injection of protein - based antibodies (monoclonal antibodies) against vascular endothelial growth factor (VEGF and related targets), resulting in a reduction in leakage from regression of angiogenesis and new blood vessel growth with restoration of the important interface between the neurosensory photoreceptors and the retinal pigment epithelium. These current treatments require intravitreal injection due to the short half - life and molecular size of the existing drugs. Intravitreal injection performed in the field of ophthalmology is associated with many risks including damage to the intraocular contents (lens, retina, choroid and the potential for intraocular infections).

[0010] In contrast to previous treatment approaches for the treatment of macular degeneration, novel methods for treating, preventing, reducing, and / or reversing macular degeneration are disclosed. In the disclosed methods, botulinum toxin (in any known form, such as botulinum neurotoxin or a fragment thereof) or one or more of its peptide fragments or neurotoxin-associated proteins (accessory proteins) are injected into the intraocular region (i.e., the eyeball) and / or the extraocular region (i.e., outside the eyeball, such as the eyelid) of a patient. Applying the disclosed compounds to one or more extraocular regions of a patient can treat vision loss from either macular degeneration or its associated symptoms, or in some cases, can present. As described in detail herein, botulinum toxin and its fragments can undergo axonal transport. Thus, applying botulinum toxin and related compounds to the peribulbar or extraorbital regions of a patient can enable penetration into the intraocular region and into the choroid, neural retina, and / or retinal pigment epithelium without direct injection into the eye. In the disclosed remote administration format, botulinum toxin and related compounds can produce a barrier-enhancing effect and regression of the pathological process associated with macular degeneration without any of the potential complications associated with intraocular injection.

[0011] As used herein, the term "botulinum toxin" refers to any known form of botulinum toxin, including, but not necessarily limited to, pure botulinum neurotoxin, fragments thereof, and / or neurotoxin-associated proteins. For example, botulinum toxin can be produced by the bacterium Clostridium botulinum (e.g., by fermentation) or by recombinant techniques and includes engineered variants and fusion proteins. In some particular exemplary embodiments, botulinum toxin is produced using recombinant or synthetic chemistry techniques (e.g., recombinant peptides, fusion proteins, and / or hybrid neurotoxins prepared from subunits of different botulinum toxin serotypes). Botulinum toxin can be serotypes A - H, and in some embodiments, botulinum toxin is an isolated botulinum toxin molecule (e.g., C 6760 H 10447 N 1743 O 2010 S 32exists as the molecular formula of and botulinum neurotoxin type A with an atomic mass of 150 kDa. The formulation Xeomin® (incobotulinumtoxin A) is an example of a pure botulinum neurotoxin (without related accessory proteins). In embodiments containing isolated botulinum neurotoxin molecules, one or more exogenous stabilizers (e.g., albumin) may also be included in the formulation. In embodiments having a complex form of botulinum toxin (i.e., where hemagglutinin and related proteins are present), one or more exogenous stabilizers may also be present. In some specific exemplary embodiments, the botulinum toxin used in the disclosed formulations and methods includes one or more related proteins that do not contain pure neurotoxin. Some exemplary related proteins that do not contain pure neurotoxin include hemagglutinin derived from the fermentation process that produces the raw material for botulinum toxin-based pharmaceuticals (e.g., whole-strain fermentation of botulinum toxin type A), and non-hemagglutinin, non-neurotoxin derived from the fermentation of the same process, but are not limited thereto. Furthermore, hemagglutinin and fragments thereof having specific activity against cell adhesion proteins (e.g., cadherin or other related proteins) can be separated or genetically expressed in a carrier suitable for subsequent purification. The prototype of the fermentation process has been described (e.g., Borodic GE, Pearce LB, Johnson E, Schantz E: Clinical and Scientific Aspects of Therapeutic Botulinum Toxin Administrations, Ophthalmology Clinics of N America, September, Vol. 4, No. 3, 1991). In some embodiments, purification of the final product of fermentation can produce raw materials for related proteins. The protein can be expressed by a recombinant process from all or part of the identified gene corresponding to the related protein.

[0012] In some embodiments, the disclosed formulations may include botulinum toxin (including the pure neurotoxin form or neurotoxin-related proteins), hemagglutinin (in any known suitable form), and / or one or more anti-VEGF agents. In some embodiments, the botulinum toxin may be fused to the anti-VEGF agent present, and in other embodiments, the botulinum toxin may be separated from the anti-VEGF agent and be distinct (i.e., not fused).

[0013] Fusion proteins may be produced using genetic material corresponding to a protein or protein fragment. Here, the gene from one protein is ligated (via a suitable ligase) to one or more separate genes corresponding to another protein to create a protein hybrid while retaining the desired biological activity of each protein to produce a useful agent or drug. The fusion genetic material can often be amplified by PCR in the process, with the addition of a linker substance and the elimination of stop codons. In some embodiments, the targeting domain of the botulinum toxin may include selective neuronal uptake (near the carboxy terminus of the botulinum heavy chain, a fragment of the botulinum molecule, or an accessory molecule). It expresses a protein involved in forming or regulating the expression of a structural protein that links cells or regulates the cytoskeleton, or a protein involved in a protein that governs RPE function or rod cone function. Additionally, a protein having anti-VEGF activity can be fused to the botulinum toxin or a fragment thereof, or an accessory protein or fragment. Further, a monoclonal antibody targeting an inflammatory mediator such as complement or other inflammatory autacoids can be added to the fusion protein containing the botulinum fragment. In some embodiments, Rho and / or ROCK regulators can also be added to the fusion protein. In some embodiments, one or more fragments of the VEGF receptor, the entire receptor, a fragment of a nerve growth protein, a VEGF subtype or fragment that inhibits angiogenesis, and / or an immunoglobulin fraction that improves protein stability and reduces immunogenicity can also be added.

[0014] Unique aspects of the fusion protein relate to the fluorescent tag, which can be used in animal models (and clinically if possible) to study transport in order to further understand axonal trafficking targeting specific retinal and choroidal tissues from injection outside the eye and permeation of various structures such as peripheral nerves. In this disclosure, the fusion protein can be formed with a botulinum toxin-based carrier, which affects binding and transport via the peripheral nerves. In some embodiments, the fusion protein can include both a carrier portion of a botulinum subtype (a botulinum type fragment) and a fluorescent marker. Other additives having biological effects can be added to the fusion protein. Such compositions can be used to study the pharmacodynamic effects of botulinum toxin-based pharmaceuticals in vivo using standard photography used in ophthalmic practice (e.g., fluorescein angiography). In some such embodiments, the tag can also confirm that a drug appropriate for a lesion on the retina or choroid being treated has been delivered. Differential penetration of the target lesion by the therapeutic agent can also provide preclinical data necessary for important individualized dosing, general dosing, efficacy of the carrier protein, formulation, and qualification of the fusion protein for clinical use. The disclosed methods can also include direct visualization of retinal tissue in vivo or in vitro for penetration and localization to the retina and choroid.

[0015] In these and other embodiments, the disclosed formulations can also include a stabilizing excipient such as albumin. In embodiments where there is one or more accessory proteins (i.e., complex-forming proteins such as lectins), the concentration and / or activity of the accessory protein can be increased from naturally occurring levels. Given the present disclosure and the teachings provided herein, numerous configurations and variations will be apparent to those of ordinary skill in the art.

[0016] (Current methods for treating macular degeneration) Currently, effective treatments for age-related macular degeneration (AMD) are limited to the wet form treated with anti-vascular endothelial growth factor ("anti-VEGF") agents and related fusion proteins that involve both antibodies and receptors. The primary treatment for "wet AMD" is intravitreal injection with a VEGF inhibitor. Currently, ranibizumab (Lucentis®) has FDA approval, while bevacizumab (Avastin) is used off-label. Eylea® (aflibercept) was recently approved for macular degeneration and has a slightly improved duration of action. These drugs are each administered by intravitreal injection. The most recently FDA-approved agent, Eylea®, has achieved commercial sales of approximately $1 billion per quarter.

[0017] Macular degeneration occurs in stages and typically begins with visible changes in the retinal pigment epithelium by direct observation using a photograph taken through the pupil of a human eye and destruction of the cellular tissue of the retinal pigment epithelium by optical coherence tomography (OCT). Figures 1A - 1C provide explanatory diagrams of the various stages of macular degeneration. Figure 1A shows a normal macula. Figure 1B shows dry macular degeneration. Figure 1C shows wet macular degeneration.

[0018] Figure 1D is an image obtained using OCT and shows a disruption in the continuity of the retinal pigment epithelium such as occurs in early AMD (age-related macular degeneration). Disruption and destruction of retinal pigment epithelial cells can lead to structural barrier defects within the retinal pigment epithelium sheet and basement membrane (Bruch's membrane), and the growth of new blood vessels from the choroid layer of the posterior human eye. During AMD, retinal pigment epithelial cells are often seen to sequentially detach from adjacent cells while adapting to migrate onto the neural retina (as shown in Figure 1D). Discontinuity of the integrity of the retinal pigment epithelium is an important factor in the etiology of the disease. In the first stage of this disease, atrophy, migration, autolysis, and disintegration occur in the cells and associated pigments, resulting in an abnormal appearance of the macula with disruption of the normal pigment density and irregularization of the pigments characterized by irregular cell shapes around the fovea, and often a breakdown of the retinal epithelial barrier as the disease progresses. Changes in the retinal pigment epithelium (RPE) cause the formation of drusen (and drusenoid pseudodrusen), pigment aggregation, spot formation, vitelliform areas, and hypopigmentation. In some cases, these symptoms may appear before more destructive changes (e.g., geographic atrophy, choroidal neovascularization, and subretinal hemorrhage) occur.

[0019] As the disruption of cell-cell adhesion and cell-basement membrane adhesion progresses, the growth of new blood vessels from the choroidal capillary lamina through the pigment epithelial defect leads to more dramatic vascular and choroidal leakage, disruption of the neural retina and retinal pigment epithelium juxtaposition, and ultimately, a catastrophic breakdown of photoreceptors (rods and cones) accompanied by loss of vision characterized by a central scotoma and loss of a person's ability to read.

[0020] Figure 2 shows a dense disciform fibrous scar with geographic atrophy (GA) of end-stage macular degeneration. The eye shown in Figure 2 is legally blind. The disciform fibrous scar shown in Figure 2 is likely formed by the associated polarity of filamentous proteins from collagen and other cellular elements. The retinal pigment epithelium (RPE) shown in Figure 2 has undergone metaplasia (a process involving the conversion from epithelium to mesenchyme) into fibrous scarring as well as flattening and atrophy of the cells and degeneration. This is an irreversible (end-stage) form of macular degeneration and is difficult to treat.

[0021] Figure 3 is an image obtained using OCT technology and shows leakage of body fluid through the RPE under the neurosensory retina in the case of wet age-related macular degeneration. The type of leakage shown in Figure 3 is generally associated with rapid vision loss and requires immediate medical intervention. Wet age-related macular degeneration (as shown in Figure 3) can be treated using drugs such as Avastin®, Lucentis®, EYLEA®, and abicipar (Allergan). These current drugs contain different antibodies against various isoforms of vascular endothelial growth factor (VEGF), and said antibodies cause regression of developing angiogenesis and / or leakage, resulting in restoration or stabilization of vision with partial recovery of the structural disturbances in the retina accompanied by a decrease in subretinal fluid.

[0022] Treatment with these drugs (anti-VEGF agents) usually requires multiple injections and carries the risk of intraocular hemorrhage, infections (e.g., endophthalmitis threatening the eye), PVR (postoperative proliferative vitreoretinopathy), lens dislocation, cataract, glaucoma, and / or retinal holes or retinal detachment. These injections can also be painful. The more injections a patient is given, the higher the likelihood of complications associated with administration. Injections into the eye are more painful than the soft tissues surrounding the eye (e.g., eyelids, orbit, periorbital and / or extraocular muscles). Experts in the fields of monoclonal antibodies and genetically engineered proteins have attempted to extend the duration of anti-VEGF agent action using fusion proteins between anti-VEGF antibodies, fractions of VEGF receptors 1 and 2, and the Fc portion of immunoglobulins.

[0023] (Overview of the currently disclosed treatment approach) While not wishing to be bound by theory, using an agent with a very long duration of action, such as botulinum toxin, to enhance the duration and efficacy of anti-VEGF therapy may increase both safety and improvement in targeted mitigation of leakage, neovascularization, or structural instability of the continuity of the retinal pigment epithelium. Exophthalmic botulinum toxin can be used repeatedly with well-defined excellent safety results. Exophthalmic botulinum injections can, in some cases, rule out intraocular hemorrhage, infectious diseases (endophthalmitis), lens dislocation, cataract, and / or retinal holes and retinal detachment that can occur with existing treatment criteria.

[0024] Reducing the number of injections at longer intervals would be an improvement over existing treatment approaches. Many of the complications of currently known treatments for macular degeneration are related to anti-VEGF intraocular injection procedures rather than to the side effects of the drugs. Botulinum toxin acts for a longer period than the known drugs currently used for this condition. Furthermore, a reduction in the injection frequency would provide a safer and more convenient treatment method for patients.

[0025] In some embodiments, botulinum toxin can be used with a VEGF antibody to further enhance the efficacy of an injectable. For example, in some cases, the treatment of macular degeneration can be achieved with one or more applications. Further, the disclosed botulinum toxin-based compounds can reduce or eliminate the need for frequent intraocular injections. Additionally, botulinum toxin can be used with other agents that promote actin polymerization, such as nerve growth factor. Botulinum toxin can, in some cases, affect and bind to the Rac1 system that acts on intracellular and extracellular actin with an enhancement of the barrier function along the cadherin protein, catenin polymer, and epithelial or endothelial surfaces. Botulinum toxin can also be transported by axoplasmic flow, a unique property that allows for transport into the eye without causing a paralytic neuromuscular effect on the extraocular muscles. Since direct diffusion of botulinum toxin-based compounds can cause paralysis of the extraocular muscles, the axoplasmic entry pathway provides a novel delivery method for intraocular diseases and can be used with any of the disclosed compounds. In embodiments where the axoplasmic delivery pathway is employed, the drug can be delivered via a nerve that enters the posterior part of the eye (posterior delivery) rather than the front of the eye (intravitreal delivery, topical drop delivery, or intracameral delivery).

[0026] In some embodiments, fragments of botulinum toxin can be fused to an anti-VEGF agent to provide for intraocular administration via axoplasmic flow, thereby avoiding the need for intraocular injection even for these agents that currently need to be used by more risky intraocular injection. Botulinum toxin can interact with mast cells and cause changes in the maintenance neurotransmitters, neuropeptides, trophic agents, and nerve growth factors that are important for maintaining a healthy retinal pigment epithelium. Other mechanisms of action are possible and are contemplated.

[0027] (Anatomical Structure of the RPE and Its Impact on Macular Degeneration) The RPE is a neural-derived structure in the eye, which forms a cell sheet with a cell structure that takes on a regular (equilateral hexagonal) configuration in RPE-RPE cell contacts. The apical surface has the form of microvilli, maximizing physical contact with photoreceptors (rods, cones), and the base of the RPE enables the physiological phagocytosis of the photoreceptor membrane while being tightly attached to its basement membrane (Bruch's membrane). This anatomical arrangement has been geometrically proven to maximize cell density and minimize cell surface connections. This evaluation is the same arrangement for a beehive, following the proposition (the Honey bee conjecture) by the Roman scholar Marus Terentius Varro more than 2,000 years ago (36 BC). The geometric proof followed Thomas Hales (University of Michigan) in 1999. This conjecture proposed that a regular hexagonal sheet maximizes the sheet area while minimizing the connection material. This anatomical structure enables economic efficiency by producing honey when bees build a beehive. This arrangement indicates that a functional barrier is important for RPE cells, and the biology maintaining this barrier effect is an extremely important target for the use of botulinum toxin in treating macular diseases.

[0028] Figures 4A-4C show the hexagonal structure of the RPE. In particular, Figure 4A shows healthy RPE, and Figure 4B shows the connected hexagonal structure. In the RPE, this structure allows for the production of actin, which is one of the major intracellular proteins that govern the adhesion of cell-cell adhesion, and the economy for the structural proteins that form the submembrane support for the hexagon. Furthermore, the microvilli on the RPE surface are also structurally supported by the protrusion and maintenance of intracellular actin and the RPE adhesion to the basement membrane. Actin also functions as a grout glue for the RPE sheet and attaches to other cell-cell proteins such as cadherin that support its functioning barrier effect. Disruptions in actin formation, the formation and arrangement of altered forms of actin and related proteins, and the regression of microvilli have been described as early changes in stage 1 age-related macular degeneration and related diseases. Figure 4C shows RPE affected by macular degeneration. As shown in Figure 4C, the actin and microvilli of the affected cells are deformed and no longer arranged as regular hexagons.

[0029] Without wishing to be bound by theory, botulinum toxin type A may act as a stimulator of actin on neural tissue. In other words, botulinum toxin may affect neurally derived RPE, potentially providing a unique opportunity to alter RPE cells in certain disease states, such as age-related macular degeneration. In some cases, cell-cell barrier function, increased microvilli surface area, or other related structural proteins may provide a way to maintain RPE structure and function. In some embodiments, botulinum toxin may slow the progression of various stages of macular degeneration and related retinal diseases. Genomic expression of actin may function to maintain RPE cells in a differentiated state, allowing adhesion and preventing separation from surrounding cells and their attachment to the basement membrane. Genomic effects may also drive RPE cells to continue to express other adhesion proteins (and functionally related proteins). Suppression of mRNA expression of proteins in RPE cells that govern motility, cell death, cell atrophy, or fibrocytic metaplasia may also be possible and may be used to treat various stages of macular degeneration. While this effect may be partially driven by other mechanisms, structural changes are important for the RPE (a neurodevelopmentally derived cell layer). The neural elements of the RPE may allow this beneficial interaction with botulinum neurotoxin, enabling and / or enhancing the therapeutic response.

[0030] Overview of Therapeutic Compounds and Related Methods In some embodiments of the present disclosure, therapeutic formulations are provided. For example, in some embodiments, the therapeutic formulation comprises botulinum toxin (e.g., types A - G botulinum toxins, specifically various subtypes of C2, C3 and / or A (e.g., A1 - A5)). The botulinum toxin included in the disclosed therapeutic formulation can be prepared by standardizing the bioactivity by dosing using methods that measure LD50, enzymatic cleavage of SNAP - 25, time - to - death assay, neuron - based assay, or other appropriate dosing to obtain bioactivity. Fragments of botulinum toxin or any fusion proteins added to the native structure to enhance efficacy can also be used in the disclosed formulations. In some embodiments, the disclosed formulations can also include penetration - enhancing peptides, or other molecules that can increase diffusion across membranes or duration of efficacy, such as polylysine polymers or albumin. Suitable adjuvants can include, but are not limited to, polycationic or polyionic peptides, hyaluronidase, and / or derivatives of local anesthetics (e.g., lidocaine, marcaine).

[0031] In some embodiments, the injection solution can be administered through the pars plana so as to avoid the retinal tissue, ciliary body or lens. In some such embodiments, the injected formulation may flow from the injection site into the vitreous. The formulation may then diffuse into the neural retina and subsequently into the retinal pigment epithelium. The toxin may then be taken up by the retinal pigment epithelium, neovascular membranes, or may diffuse through the Bruch's membrane, blood - retinal barrier, and / or defects within the choroid. Any appropriate level of activity can be utilized in such methods. The retinal pigment epithelium is highly active in vesicular cell uptake that interacts with the rods and cones of the neural retina and, in some cases, can readily take up the molecular botulinum toxin into its cytoplasm. Alternatively, the botulinum toxin may be administered in an upstream neural structure (e.g., the peripheral nervous system), which ultimately penetrates into the eye via axonal flow.

[0032] When the disclosed formulation is injected, one or more of the following results can be achieved. (1) Leakage from neovascularization with humoral outflow under the neurosensory retina or retinal pigment epithelium can be reduced. (2) Regression of new blood vessel growth can occur. (3) Retinal pigment epithelial degeneration can regress, leading to intracellular morphological changes including a decrease in retinal pigment epithelial activation. (4) The cell element polarity of the retinal pigment epithelium can be maintained with enhanced barrier function and metabolic activity, accompanied by an increase in density, length, and expression of microvilli. (5) Enhancement of tight junctions within the retinal pigment epithelium and enhancement of pigment epithelial attachment to its basement membrane can occur.

[0033] In some cases, the injection results can be measured using one or more of the following. (1) Visual acuity and / or a validated method of visual acuity measurement. (2) Contrast sensitivity. (3) Fundus photography. (4) Fluorescein fundus angiography (including OCT angiography). (5) OCT (e.g., examining any physical type of subretinal fluid, neovascularization under the retinal pigment epithelium, and neovascularization through it). (6) Changes in the RPE (drusen / drusenoid height and volume, density, distance from the basement membrane, migration, photoreceptor loss, loss of the IS-OS and outer nuclear layer, retinal and subretinal fluid accumulation, pseudodrusen density, pigment aggregation and tears, choroidal thickness, neurosensory retinal thickness, RPE atrophy, formation and leakage pattern of choroidal neovascularization, extent of geographic atrophy, hemorrhage, and the shape and regularity of retinal lines defined by OCT (e.g., ONL, IS-OS, RPE alignment)). (7) Amsler grid. (8) Autofluorescence from RPE lipofuscin. (9) Focal ERG (electroretinogram). (10) Changes in polarity, thickness, and shape in the retinal pigment epithelium using OCT. (11) Visual field. (12) A subjective device for evaluating patient satisfaction, validated against objective measurements. (13) Use of conventional clinical trial methods using controls and repeated injections. In some cases, continuous follow-up can be performed using patients, and if necessary, an assessment of the need for repeated injections can also be utilized. In these and other embodiments, fundus photography and OCT can be used to monitor the therapeutic effect.

[0034] (Effect of botulinum toxin on the intracellular cytoskeleton) Botulinum toxin may have important biological effects on endothelial cells and RPE, which play important roles in the etiology of degenerative and exudative human retinal diseases. The RPE has been studied using electron microscopy in the early and late stages of age-related macular degeneration. The study reveals that the condensation of the intracellular cytoskeleton near the basal membrane (base) of the cells leads to irregular cell shape, loss of polarity, disruption of cell-cell adhesion, accumulation of leaky proteins with membrane instability, and disruption of the apical-apical orientation of RPE cells with rod and cone cell structures, resulting in the destruction of the cell membrane. Distortion of the REP may lead to one or more of the following. (1) The RPE cannot maintain its supportive functional and metabolic interactions with the macular rods and cones and maintain the close barrier between the choroid and the neural retina that allows the release of reactive macromolecules from the neural retina to the choroid. Such exposure stimulates the release of angiogenic cracks and mediators from choroidal endothelial cells, nerves, and mast cells, causing subretinal fluid accumulation (characteristic of both "wet" and "dry" macular degeneration with RPE and neurosensory detachment). (2) Leakage from the tight junctions of the neovascular endothelium due to disruption of the cytoskeleton associated with the endothelial vascular system. (3) Exposure of the antigenic structures of the neural retina through the blood-retinal barrier, which elicits the reactivity of immune cells in the choroid with a limited response from the blood containing the cellular components circulating in the choroid. The immune response can include complement activation, which further damages the RPE structure. (4) Interruption of the nutrient delivery rate to the neural retina, which results in toxicity to the rods, cones, and RPE. (5) Loss of RPE microvilli, which is important for maintaining rod and cone function by removing photoreceptor degradation products. (6) Destruction of the rods and cones. (7) Formation of a geographic atrophy state of the RPE.

[0035] Figures 5A-5F are images of the RPE obtained using microscopy techniques. Specifically, Figures 5A-5F show membrane disruption, submembrane condensation, change in the hexagonal shape of the structure, self-degradation of the RPE, stress fiber formation from actin (shown in Figure 5A), disruption of the barrier function, and migration of the RPE from the barrier sheet. Note that the relationship between the structure and function of the neural retina is such that most exposures are one of isolation from blood components into the vitreous (a chamber containing hyaluronidate without transient perfusion). Defects in the retinal vascular system are quite consistent in creating retinal pathology. The blood-retinal barrier in the retinal and choroidal vessels is important for the health of the neural retina. The choroid is one of the most densely perfused tissues in the human body, and the RPE and photoreceptors are highly metabolically dependent on their close structural relationship with the choroid. The separation of the blood compartments by both the retinal and choroidal vessels is important for maintaining the health of the neural retina and the functional integrity of the photoreceptors. Furthermore, specific antigenic stimuli are exposed by barrier disruption and genetically cause individuals to react with immune responses at various levels. The various levels include, but are not limited to, complement activation, neuroreactivity, changes in regulatory autacoids, changes in cell functions unrelated to inflammation, accumulation of body fluids within the neural retina, barrier dysfunction of the choroidal and retinal vascular endothelium, and dysfunction of the RPE photoreceptor function.

[0036] Disruption of the cytoskeleton in endothelial and RPE cells is important for the etiology of macular degeneration with respect to leakage of blood, including fluid and membrane change mediators, and the function of the choroid of the eye. Generally, an increase in the generation of pathological arrangements of actin and microtubule proteins accumulates as the first step of macular degeneration associated with distortion of the RPE and endothelial membranes, which can be caused by the following: (1) Toxic leakage of subretinal fluid (wet macular degeneration). (2) Loss of cell-cell adhesion and cell-basement membrane adhesion, disruption of barrier function (drusen and drusenoid formation, RPE migration). (3) Loss of the polarized orientation of the RPE. This can be important for its role in supporting the structure and function of rods and cones (progressive dry degeneration), and can ultimately lead to loss and retraction of RPE microvilli. (4) Loss of the RPE's ability to remove photoreceptor degradation products (lipofuscin) at a rate sufficient to avoid photoreceptor toxicity (increased autofluorescence). (5) Pathological condensation of intracellular fibrous elements of the RPE, reflected by metaplasia of the RPE into white "fibrocystic" cell types that ultimately appear as "disciform scars" (see Figure 2) on fundus photographs, as well as the formation of geographic atrophy of the RPE and neurosensory retinal atrophy. (6) Disciform scars and geographic atrophy are commonly seen in patients blinded by macular degeneration and reflect the nature of the degenerative and damaging processes. Due to the degenerative and damaging processes, the macula is disrupted by changes in the accumulation orientation of pathological actin within cells and the accumulation of associated fibers, which fundamentally changes the RPE and causes RPE / photoreceptor death. The next result is the differentiation of the retinal pigment epithelium into fibroblast, migratory, and / or atrophic cells by differentiation of the type from epithelial to mesenchymal. The next events include substantial changes in mRNA expression by the RPE for reattached cell adhesion, basement membrane adhesion, pigment epithelial motility, and migration to the neurosensory retina. This process shows early changes in macular degeneration with a vascular response, and the growth of blood vessels into the RPE and choroid indicates a later stage (Figure 1, stages of macular degeneration). Following disruption of the barrier function, immune processes are ensured, which result in complement activation, mast cell activation, and neuropeptide release, further worsening the disrupted barrier and fluid accumulation. And / or (7) Genomic changes that result in altered RPE morphology, retinal layer tissue dysfunction, and loss of photoreceptors.

[0037] Botulinum toxin can enter cells through special receptors present on nerve cells, or by facilitation using an adjuvant protein in vivo or in pharmaceutical formulations. In some cases, due to the ability of this molecule to cause significant changes in cytoplasmic physiology or genomic responses and very low molecular concentrations, the vital concentration can be essentially low.

[0038] (Interaction between Botulinum and the Cytoskeleton) In some embodiments, the disclosed formulations and methods include the injection or topical application of botulinum toxin formulations for the treatment of macular degeneration and other related degenerative diseases. Botulinum toxin A can have a significant impact on cytoskeletal structures. The C3 version has been noted to interact with the Rho-actin polymerization system in experimental observations of cell biology. C2 and C3 toxins may not cause neuromuscular weakness, but these agents are cytotoxins that can cause cell death by mechanisms different from those of subtypes A, B, C1, D, E, F, and G. Further, as described in detail below, animal injection of type A botulinum into muscle cells can cause cell shrinkage related to morphometric diameters that are not proportional to the effects caused by denervation (neurogenic atrophy). This observation of a rapid rate (not previously reported) indicates that the A toxin has a basic direct effect on the cytoskeleton of muscle cells independent of the neuromuscular blockade related to blocked acetylcholine release at the neuromuscular junction. This action can interfere with the denaturation process that leads to cell death and significant dysfunction, and can interfere with the lysis and reorganization of cytoskeletal actin and related intracellular microtubules to the extent of preventing significant disease denaturation processes that maintain cell function. In some cases, it can inhibit caspases and cytoplasmic enzymes of apoptosis. Its action would be to maintain the polarity of living cell structures such as the RPE while maintaining the polarity of the cell structure and the related cells that functionally interact with the target cell population, and delaying or halting the accumulation of pathological cytoskeletal proteins.

[0039] In some embodiments, the health of endothelial cells, as well as the integrity of any cells undergoing a transformation process, can be maintained by an increase in the intracellular production of cytoskeletal proteins that disrupt important junctions and associated barriers, metabolite movement, neural retinal antigen exposure, or cell-cell relationships, or that impair a shape or important cellular component. Such changes can be caused by alterations in the expression of cell adhesion proteins, interactions with biological surface and internal receptors that govern cell metaplasia, apoptosis, epithelial-mesenchymal transition, relaxation of epithelial sheets and adhesion to the basement membrane, changes in the amounts of various isoforms of adhesion proteins (such as cadherin isoforms and related proteins), which can modify the barrier functions of epithelial and endothelial cells to inflammatory cytokines (such as VEGF) and related proteins. Important barrier functions in macular degeneration include endothelial-dominated leakage, epithelial cell-cell adhesion-dominated RPE barrier, choroidal neovascularization barrier function along neovascular endothelium. Also, botulinum toxin can suppress inflammatory autacoids such as mast cell function.

[0040] In the case of the RPE, tight junctions at the level of the RPE can be rendered incompetent by abnormal cytoskeletal protein accumulation. Thereby, barrier disruption along the tight junctions and subsequent antigen exposure of the neural retina to the choroid are caused. There is a potential for the release of various immunological and inflammatory proteins based on immunoreactivity, choroidal fluid, and subsequent photoreceptor death. Such processes both inferentially involve the release of histamine present in platelets and mast cells that are present in the choroid. Vasoactive intestinal peptide and CGRP may also play a role. Mast cells can interact with the autonomic nerves present in the choroid and yet another target of botulinum modulation or blockade action. The barrier function appears to be implicit, and the tissue composition of the retina and choroid and the disruption of this function can be seen as an upstream disorder occurring in macular degeneration. Note that retrograde movement (towards the central nervous system) and anterograde movement (away from the central nervous system) of botulinum toxin via peripheral nerves or veins occur during the use of the disclosed compositions and methods. Further, direct penetration of the disclosed formulations into the eye may encounter the natural barriers of the sclera and cornea. Prior to the filing of this application, botulinum toxin had not been proposed for intraocular diseases. This is at least in part due to the fact that the eye barrier was previously thought to prevent the entry of the neurotoxin into the eye.

[0041] (Interaction between RPE and photoreceptors) The phagocytic interaction of RPE on the rod and cone bodies is important for photoreceptor health. Damage to this interaction results in ultimate death with photoreceptor damage and vision loss. Driving this relationship at the subcellular level are the microtubules within the retinal pigment epithelium that enable phagocytic interactions at rapid cellular rates, and active actin and associated tubule polymerization enable the maintenance of photoreceptors. Defects in the maintenance and breakdown of the cytoskeletal assembly can lead to photoreceptor damage. Such defects can be reflected in disruptions of RPE cell polarity, as well as changes in the integrity of cell shape actin and tight junctions and cell relationships on the basement membrane (Bruch's membrane). Changes in early macular degeneration are associated with changes in RPE morphology and the accumulation of dense accumulations of subcellular fibers (drusen body accumulations) suggesting microfibril dysfunction. Autofluorescence is a sign of RPE dysfunction and indicates a decline in RPE associated with the accumulation of rhodopsin due to the poor metabolism and accumulation of lipofuscin, which is seen with the blue light filter of the fundus camera. Lipofuscin is an indication of dysfunction of functional RPE and often occurs in both wet macular degeneration and dry macular degeneration with geographic atrophy.

[0042] (Botulinum Toxin and Microtubule Degeneration and Microfibril Accumulation) Botulinum toxin has the ability to alter the accumulation formation of subcellular actin and microfibrils by suppressing the pathological accumulation polymerization of important cytoskeletal components to provide one or more of the following. 1. Maintain the barriers within the RPE essential for maintaining the integrity of the neural retina, and the rods and cones maintained by tight junctions. 2. Maintain polarization and the cytoskeleton to ensure continuous function. 3. Maintain endothelial integrity and suppress angiogenesis from the choroid. 4. Block or regulate mast cell activity and regulate the release of neuropeptides or other mediators within the choroid. It can damage or sustain photoreceptors. 5. Enlargement and strengthening of microvilli. 6. Barrier functions in RPE basement membrane attachment and RPE cell - to - cell adhesion. 7. Block exudative vascular leakage from the choroid.

[0043] (Botulinum interaction with the microvilli of the retinal pigment epithelium) The retinal pigment epithelium contains microvilli, which are an important structure for maintaining the physiological health of the rods and cones of the neural retina. The neural retina structure converts images and light into signals that can be transmitted to the brain via the optic nerve projection, enabling visual interpretation within the central nervous system. The effects of aging cause a microscopic anatomy of dysfunction between the rods and cones, ultimately reducing the extent, size, and integrity of the microvilli that lead to the initial stages of macular degeneration. The effect of botulinum toxin is accompanied by rejuvenation and reversal of the apical biological structure of the retinal pigment epithelium, which results in the cessation of degeneration and deterioration of the role of the choroid and retinal pigment epithelium on the neural retina, causing this deterioration, enhanced actin expression, and a shift in the associated protein polymerization.

[0044] The botulinum toxin species that cause this change act on the Rho kinase and ROCK intracellular systems, shift mRNA expression towards active protein expression, cause strong microvilli, and reverse or prevent the atrophic shift and apoptosis of RPE cells involved in macular degeneration. In genomic studies using ganglia with evaluation using strong cDNA fragments on gene chips, botulinum toxin elicited an mRNA response that regulates the production of proteins important for actin expression, intercellular adhesion molecules, and assimilatory proteins that govern the strengthened cell structure. Photoreceptor proteins have been shown to shift their expression after injecting botulinum toxin into cell cultures.

[0045] (Duration of action) The disclosed formulations and methods can provide a biological effect that extends the duration of action beyond existing therapies, with a potential effect lasting from 4 to 50 weeks and potentially longer with repeated injections. When intravitreal injection is used, the increased duration can enable fewer invasive procedures required to administer the drug. Botulinum toxins have various durations in clinical practice depending on the target tissue. The autonomic effects can last longer than the effects on heavily myelinated motor nerves. Most of the nerves within the choroid have minimal myelination and many represent autonomic nerves from ganglionic structures outside the orbit and are accessible to injections using the botulinum formulations described herein.

[0046] The duration of action of anti-VEGF agents (both FDA-approved and in development) is targeted to be longer because the need for intravitreal injections is associated with many complications. Fewer injections or a longer duration is more comfortable for the patient and reduces the risk of administration. The half-life of aflibercept EYLEA® in rabbits is approximately 7 days. In contrast, 0.5 mg of ranibizumab (Lucentis®) is approximately 2.88 days and 1.25 mg of bevacizumab (Avastin®) is 4.3 days.

[0047] Assuming that botulinum toxin-based pharmaceuticals inherently have a long duration of action, fewer injections may be required compared to known anti-VEGF agents. Due to the neuromuscular effect, the duration of action is generally 10 to 14 weeks. With some formulations, the period between treatments can reach 20 weeks. Additionally, for the autonomic effect, a maximum period of 24 weeks has been recorded. The botulinum technology provides a longer duration of action than expected with currently used anti-VEGF pharmaceuticals, so patient treatment convenience, as well as the potential for significant risk reduction and additive effects, are clear advantages. Further anti-VEGF agents are associated with vascular occlusive diseases (e.g., stroke and arterial occlusion). Despite complications with anti-VEGF drugs, the evaluation with botulinum toxin did not result in significant reported complications at conventional dosage levels (as defined by the FDA-approved dosage). The botulinum toxin-based pharmaceuticals described herein can act in the same manner as anti-VEGF agents and, in some embodiments, can increase the efficacy and duration of anti-VEGF agents (see Example 1).

[0048] (Dosage) The disclosed therapeutic formulations can, in some embodiments, include botulinum toxin or a fragment thereof. Any suitable form of botulinum toxin can be used in the disclosed formulations, for example, the disclosed formulations can include botulinum toxins A1 - A5, B, C1 - 3, D, E, F, G, and H. Also, LD50 units per cc of fluid can be used as a source of botulinum toxin, with or without a complexing protein.

[0049] The disclosed formulations can be prepared using an appropriate dosage of botulinum toxin. For example, in some embodiments, the disclosed formulations can be administered to a patient according to one or more of the following dosages: .01 to.5 LD50 units are administered via intravitreal, extraocular, periorbital, subconjunctival peribulbar injection, suprachoroidal injection, or topically administered. 0.5 to 5 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 5 to 10 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 10 to 20 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 20 to 40 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 40 to 80 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 80 to 160 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 160 to 320 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 320 to 640 LD50 units are administered via intraocular, extraocular, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 640 to 1280 LD50 units are administered via intraocular, extraocular, subconjunctival peribulbar injection, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 0.5 to 25,000 LD50 units are administered via intraocular, extraocular, subconjunctival peribulbar injection, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 0.01 to 3,000 LD50 units are administered via intraocular, extraocular, subconjunctival peribulbar injection, periorbital, subconjunctival peribulbar injection, supraocular injection, or administered topically. 1280 to 6,000 LD50 units are administered via injection into the eye, subconjunctival periorbital injection, injection around the eye, injection onto the eye, or administered topically.

[0050] In some exemplary methods, conventional dosages of botulinum toxin may be used. As used herein, the term "conventional dosage" refers to any FDA-approved dosage of botulinum toxin for indications of the head or neck. In selected embodiments, botulinum toxin at 300 LD50 units or less may be administered to a patient. For botulinum toxins with lower LD50 potencies, a table conditional on conversion evaluation and existing dosage conversions can be used. These exemplary dosages are given for the conventional form of botulinum toxin commercially available under the trademark BOTOX®.

[0051] (Topical and Subconjunctival Administration) As described herein, clinical efficacy can be derived from topical application of botulinum toxin-based pharmaceuticals. The dosage can range from 1 to 2,500 units using a botulinum toxin complex. To reduce unwanted toxicity, the botulinum toxin protein molecule can be modified to reduce or eliminate its neuromuscular effects. Thus, when it is applied to mucosal surfaces such as the ski, conjunctiva, or nasal mucosal surface, side effects such as oral paralysis and weakness do not occur. By eliminating the hemagglutinin protein derived from botulinum toxin represented as a botulinum toxin complex (e.g., BOTOX®), additional adjuvant proteins can be removed to limit toxicity from gastrointestinal absorption. Since the dosage is determined at LD50 in Swiss Webster mice, the unit is generally recognized, but it can be converted to alternative forms obtained from alternative assays or quantitative methods.

[0052] Importantly, delivery systems suitable for intraocular administration can be provided using penetration enhancers such as lidocaine, albumin, polylysine, or mechanical devices such as contact lenses, intraocular implants, subconjunctival implants, etc. Transconjunctival administration via the eyelid or bulbar conjunctiva can be used. Drying techniques on the ocular surface can also be used to enhance penetration. The use of goggles to enhance penetration on the ocular surface can be used to provide a high-pressure state such as that used in a positive pressure atmosphere or a hyperbaric oxygen chamber. Microperforation of the corneal and conjunctival epithelium followed by a botulinum toxin-based protein, with or without contact lenses, can increase corneal and ocular penetration. Depending on the specific stage of macular degeneration and clinically specific pathological findings or SD-OCT, various concentrations can be used to enable more effective uptake into the eye.

[0053] Anterior chamber (aqueous humor) injection is known to be safer than intravitreal injection and can provide an excellent method of increasing the intraocular botulinum concentration while reducing the potential for damage to the intraocular contents.

[0054] (Drugs in tests for dry non-exudative and wet exudative stages of macular degeneration) Tables 1 and 2 outline various agents under test or expected to be tested for the treatment of dry macular degeneration. [Table 1] JPEG0007716841000002.jpg107170 [Table 2]

[0055] It is noteworthy that there is no clear agent that consistently acts to suppress or halt dry age-related macular degeneration. Also worthy of note is the fact that the therapeutic agents being considered do not take into account botulinum toxin-based pharmaceuticals for the treatment of dry or wet degeneration. The mechanism of action is also described in Tables 1 and 2. Note that anti-VEFF, choroidal flow promoters, anti-amyloid antibodies, visual cycle regulators, antioxidants, apoptosis regulators, numerous anti-complement-directed antibodies, neuroprotective agents, nerve growth factors, phosphodiesterase inhibitors, stem cells, and anti-inflammatory agents have been tried. However, no review or study has considered or provided a rationale or reduction to practice for botulinum toxin-based pharmaceuticals. More recently, ranibizumab (Genetech, Inc.) has been reported to have failed at the 2017 American Academy of Ophthalmology in New Orleans. Recently, studies including the insertion of an intraocular implant complexed with corticosteroids for sustained release have been added to anti-VEGF agents (e.g., EYLEA®) for the treatment of wet age-related macular degeneration. Furthermore, newer agents such as angiopoietin are being tried in combination with anti-VEGF agents to increase the efficacy and duration of action of intravitreal pharmaceuticals.

[0056] At the 2017 Retina conference, a review of ocular delivery mechanisms that could not cite a transneural delivery mechanism for the treatment of choroidal or retinal diseases including AMD was conducted. The void gives credibility to this new component with respect to the formulations and treatment methods described herein (e.g., transneural delivery of Clostridium botulinum or its components to the choroid and choroidal ganglion, with favorable effects on the RPE and neural retina).

[0057] (Pharmacodynamic Delivery System for the Treatment of Human Macular Diseases (Axonal Flow from Extramocular Injection)) Described herein is not only a unique agent but also a unique delivery system for the treatment of human macular diseases. Botulinum toxin has the ability to diffuse from the injection site, which directly and volumetrically affects the local biological effects associated with dosing. The biological effects on structure are caused by genetic upregulation of proteins that govern cell adhesion such as actin and various cadherins, and have a direct effect on the differentiation of the polarity of the epithelial cytoplasm with respect to the structural proteins that govern membrane barrier function, cell adhesion to the basement membrane, and the function of the epithelial barrier, and can be further achieved by retrograde and anterograde axonal flow through the autonomic, sensory, and motor nerves that upregulate. In some embodiments, this unique feature is very useful in that intravitreal injection may not be necessary. Elimination of this step in the treatment of macular degeneration can reduce or eliminate the risks of vitreous hemorrhage, endophthalmitis, retinal detachment, traumatic cataract formation, glaucoma, retinal holes, and pain from direct injection into the eye. These complications can be devastating and can lead to blindness.

[0058] The pharmacological effects by injection of soft tissues around the eyes are not associated with more serious, potentially blindness-causing complications that can occur with direct injection into the eyes. These injection sites may also be less painful. The dosage of the disclosed formulation can vary from 1 to 3000 units, preferably from 1 to 300 units, and more preferably from 1 to 200 units (BOTOX®). Weaker formulations (Dysport, Xeomen, Myobloc or other formulations) can be used at higher dosages. The injection is generally performed over an area that includes the motor and sensory nerves entering the eye, particularly the trigeminal nerve, oculomotor nerve, and most specifically the autonomic nerves such as the pterygopalatine ganglion under the temporal muscle. Since the periorbital tissues in the anatomical regions of the forehead, eyelids and immediately surrounding them are directly drained into the orbit with collateral flow into the eye, transport via the venous system is also possible. The autonomic nerves also supply the human eye (pupillary fibers), and transport via the collateral autonomic nerves can act as a conduit for the delivery of biological intraocular effects from the nerves passing through the posterior pole of the eye above the macula (see orbital anatomy diagram). This conduit may provide a pathway for delivering botulinum or its fragments to the choroid and retinal pigment epithelium in a perhaps concentrated form at low concentrations. The sensory nerves can further direct this conduit to the target retinal pigment epithelium. Transcellular transport is possible by the penetration of botulinum material by the retinal pigment epithelium and neural retina structures that affect both the vascular endothelial growth factor, vascular permeability, and vascular responsiveness to the integrity of the retinal pigment epithelium barrier, as well as the leakage from immune cytokines released by the loss of integrity of the RPE barrier and the possibility of new blood vessel growth. Through this process, botulinum toxin enhances cell adhesion through possible regulatory effects on Rho kinase, ROCK, and other proteins important for maintaining the RPE actin cytoskeleton, intercellular adhesion molecules, cell-to-basement membrane adhesion molecules, and endothelial adhesion molecules that make the biological RPE barrier function more robust and inactive, and can improve physiological functions such as the processing, catabolism, and removal of rhodopsin protein. Furthermore, the action of botulinum toxin prevents vascular leakage and / or reduces and stabilizes vascular endothelial growth. Such effects can also include retinal vascular blood-retinal barriers that occur in macular edema from inflammation and diabetes.Furthermore, the autonomic nerves have been shown to integrate with the choroidal autonomic ganglion cells under the macula.

[0059] Prior to the filing of the present application, the intraocular action of botulinum toxin on the RPE retina was not known. Furthermore, injection of botulinum into the autonomic parasympathetic and sympathetic ganglia that project axons to the skin of the eyelid, face, forehead, facial bones, facial and jaw muscles, scalp paranasal mucosa, nasal mucosa, neck, mouth or palate, and eyes was not known to have any effect on the RPE / choroid. This information, alone, is novel and, when combined with the disclosed formulations and methods, can provide a dosing paradigm that is safer than intravitreal injection.

[0060] (Intraocular penetration by periorbital and peribulbar injection) Another unique aspect of the disclosed therapeutic formulations and methods is that the effect on the internal eye in the macular region can be achieved by peribulbar injection or periorbital injection. Not limited to the high-dose effect obtained by plantar injection (discussed for other embodiments), the opportunity to enter the eye by periorbital eyelid injection, or peribulbar injection and neck injection using axonal transport is a surgical improvement that avoids serious complications of other methods. Transscleral injection through the pars plana is readily possible and would be much easier for the patient. An extraocular injection targeting the upstream nerve away from the eye that ultimately enters the eye allows for a selective action on the intraocular contents without exposing muscle tissue to the action of the toxin. The toxin causes diplopia and a decrease in extraocular muscle strength with ptosis. Placing a syringe needle deep into the orbit with botulinum toxin-induced paralysis of the obtained extraocular muscles may not be desirable. Repeated intravitreal injections carry the risk of causing intraocular hemorrhage, endophthalmitis with globe destruction, retinal detachment or retinal breaks, lens dislocation, or increased intraocular pressure. The toxin can reach the target choroid and retina by unique mechanisms such as axonal flow, retrograde diffusion of veins, and / or direct diffusion from peribulbar injection and extraorbital injection. These indirect mechanisms for the treatment of AMD and related conditions provide a novel, selective entry into the eye via neural transport to avoid undesirable side effects due to muscle weakness.

[0061] In particular, the choroidal nerve fiber structure has been shown to be positive for a number of neuropeptides and related neurotransmitters. With age, a regression of the nerves within the choroid has been noted very close to the retinal pigment epithelium. Such denervation can provide trophic effects on the structure and function of the retinal pigment epithelium such that it causes RPE dysfunction, loss of cell-cell adhesion, and loss of the vital RPE barrier function, as well as other structural and functional degenerative changes. Certain neurotransmitters and neuropeptides present in the choroid can be extremely important for the health and function of the epithelium. Regression and depletion of such chemicals can lead to atrophy of the RPE, mesenchymal and migratory changes, loss of RPE-photoreceptor interaction, and ultimately photoreceptor damage with loss of retinal function and vision.

[0062] In the case of the ocular surface shown in the examples (filamentary keratitis), loss of barrier function with disruption of cell-cell adhesion and adhesion of epithelial cells to the basement membrane strands of the corneal epithelium forms filaments, exposes corneal sensory nerves, and leads to pain and pathological reactive changes (neovascularization). Filamentary keratitis is a problem common to corneal denervation and is a condition called neurotrophic keratitis. Neurotrophic keratitis results from trauma to sensory nerves, repeated infections with neurotrophic viruses (e.g., herpes simplex, varicella zoster), chronic infections, and dry eye with a lack of mucus and tears. As with macular degeneration, the epithelium often degenerates prior to loss of the corneal epithelium and neovascularization. In the cases described herein, local botulinum toxin resulted in repair and reduction of filaments over a time course consistent with known botulinum pharmacokinetics and repeated efficacy. Botulinum in this specification increases cell adhesion to surrounding cells and the basement membrane and stimulates nerve structures in a manner advantageous for both corneal epithelial function and elimination of filaments that disrupt the continuous corneal epithelial sheet. Botulinum stimulates the nerve / epithelial structure to produce actin and related adhesion molecules, thereby promoting epithelial barrier function and structure and at least partially restoring sensory nerve function over the pathological state.

[0063] In such cases of the cornea, the epithelial discontinuity progresses to new blood vessel growth. In the case of macular degeneration, the first stage of dry form macular degeneration progresses to new blood vessel growth that disrupts the interface between the RPE and photoreceptors and leads to blindness. Avoiding discontinuity by enhancing the nerve fiber effects from the choroid axons and ganglion cells is the mechanism described herein that is useful for the treatment of macular degeneration. The botulinum-enhanced choroidal nerve fiber effect on the RPE provides functional stability to the RPE, enabling enhancement of the barrier function, maintenance, and prevention of the degeneration of the retinal pigment epithelium over time. Defects in choroidal innervation result in the loss of important chemicals derived from peripheral nerves that are essential for the health of the RPE. The loss of certain neuropeptides such as vasoactive intestinal peptide (VIP) is known to be depleted in the case of macular degeneration. The infiltration of toxins through the nerves surrounding the RPE or the arteries entering the eye may also have the effect of blocking leakage from retinal arterioles, leading to the treatment of retinal vascular leakage.

[0064] Botulinum toxin by stimulation of peripheral sensory nerves to maintain and stimulate formative actin molecules, and related proteins, adhesion molecules, and important intracellular structures such as neurotransmitters and the RPE may be extremely important for maintaining the RPE and delaying the effects of macular degeneration. Botulinum toxin has a potent effect for stimulating actin-actin related protein formation in peripheral motor nerves, and such an effect can be carried over to the peripheral nerves that penetrate the human eye.

[0065] (Safety) The use of botulinum toxin around the eyes is also safe. Well-established dosing parameters for cosmetics, facial movement disorders (hemifacial spasm, blepharospasm, Meige syndrome, dystonia, bruxism, migraine, tension headache), fine wrinkles, forehead lines, glabellar lines, induced ptosis, and inflammatory facial conditions are designed to provide a very high safety record. Since this material is known to be very safe after repeated injections, there is a unique opportunity to provide an excellent opportunity to understand the risks and benefits of existing FDA-approved drugs (Eylea®, Lucentis®, and Avastin®) for patients with retinal and macular diseases. Most of the studies conducted over the past 30 years have undergone safety ophthalmic trials, and no serious irreversible eye complications have been confirmed. This opportunity is truly unique in clinical research and will serve as an impetus to proceed using various evaluation items such as visual acuity (such as those defined in the ETDRS - Early Treatment Diabetic Retinopathy Study) and other evaluation items described in this specification.

[0066] (Neurotransmission to the macula) In some embodiments disclosed herein, the peripheral nerves are utilized as conduits for delivering botulinum toxin-based pharmaceuticals to the eye, retina, and / or macula without using direct intraocular injection (which inherently increases the risk of complications). Like the vascular walls of the blood vessels of skeletal and cardiac muscle, the smooth muscle of the vascular wall of the choroid is innervated by both divisions of the autonomic nervous system, which forms a dense plexus of fibers (the "perivascular plexus") around the blood vessels. Axonal terminals are also found throughout the stroma and terminate in non-vascular smooth muscle, intrinsic choroidal neurons (ICNs), and possibly other cell types. There are also primary afferent sensory fibers that project to the trigeminal ganglion via the ophthalmic nerve. Some of these give rise to peptide-positive collaterals that terminate on and around blood vessels and intrinsic choroidal neurons.

[0067] Figure 6 shows the above-described human anatomical orbit. In Figure 6, the thin and dark arrows represent the arrangement of needles adjacent to the orbit. It should be noted that the proximity and presence of blood vessels and nerves in this region enable access to the posterior surface of the sclera of the botulinum toxin preparation with neurovascular penetration into the macula and choroidal pigment epithelium. The injection targets the autonomic and / or sensory nerves within the pterygopalatine fossa.

[0068] Figure 7 shows transcytosis with nerve penetration and ocular penetration after extraocular administration. Transport and transcytosis along axons in all directions achieve penetration into the eye (choroid, retinal pigment epithelium, and neural retina). Dendrite-axon penetration, cell transcytosis (retrograde penetration and transport) into new axons and dendrites can also be utilized in some embodiments.

[0069] The main parasympathetic input to the choroid is derived from the pterygopalatine ganglion located within the pterygopalatine fossa (Figure 6). These fibers are mainly cholinergic and are rich in vasodilatory factors, vasoactive intestinal polypeptide (VIP), and nitric oxide (NO). These nerves are the targets for the penetration and transport of botulinum toxin into the eye when an injection is given in the region of the pterygopalatine fossa (outside the eye and orbit). The sympathetic innervation of the choroid is derived from the superior cervical ganglion. These noradrenergic neurons terminate on blood vessels and mediate vasoconstriction. This anatomical arrangement enables neck injections to penetrate the eye by axonal flow.

[0070] The choroid has been shown to release peptides, such as substance P and calcitonin gene-related peptide, onto effector tissues using peptides in the central anterior reflex arc or axon reflex (a non-synaptic reaction that depolarizes sensory endings where local stimuli (chemical or mechanical) move to the nearest collateral (branch)). Evidence for this reflex has been found in the primary sensory projection pathways from the trigeminal ganglion of the uvea and choroid. The reflex may mediate changes in blood flow and various other functions. For example, in both mammals and birds, sensory fibers projecting from the choroid to the trigeminal ganglion via the ophthalmic branch of the trigeminal nerve induce vasodilation. These endings are positive for substance P and calcitonin gene-related peptide.

[0071] Botulinum toxin can be transported via any peripheral nerve pathway that allows collateral axonal flow and, in some cases, penetrate the choroid and retinal pigment epithelial structures via transcytosis to achieve a biological effect on target tissues in the macula (see Figure 7).

[0072] Intravenous delivery is not mutually exclusive with axoplasmic delivery to the eye. Diffusion into the cavernous sinus (a venous sinus with a carotid artery passing through its center) carries toxin molecules near the carotid siphon (sympathetic plexus) that contains sympathetic nerves across its entire surface. Binding of botulinum to the autonomic nerves surrounding the carotid surface of the cavernous sinus results in axonal flow along the ophthalmic artery into the orbit and ultimately to the posterior segment and macula, affecting the neuromuscular junction.

[0073] Veins flow from the periorbital region and nose and through the inferior orbital fissure near the orbital veins near the vortex veins that flow within the eye. Venous anastomoses allow for another conduit for delivery to the choroid and retina.

[0074] (Axonal flow (a unique conduit for entry into the choroid and retina)) In initial experiments using radiolabeled full-length BoNT / A, it was shown that upon intramuscular injection into the gastrocnemius muscle of cats, the toxin transfers to the anterior roots and adjacent spinal cord segments. Similarly, radiolabeled BoNT / A has been shown within the axoplasm of myelinated axons following peripheral injection into mice. Dose-dependent retrograde transport of BoNT / A in brainstem motor neurons has also been shown by electrophysiological and ultrastructural experiments in cats. Furthermore, segments of the botulinum toxin have also been noted to receive axonal flow (HcA segment). Both the full-length botulinum toxin and the binding segment form are capable of undergoing long-distance transport via axons. This phenomenon is utilized in one of the delivery mechanisms demonstrated in the present invention and the examples. In compartmental cultures of rat sympathetic neurons, BoNT / A moves retrogradely into the cell body when applied at high concentration to the distal compartment. However, retrograde transport of BoNT has mainly been inferred indirectly, i.e., by observing the appearance of radioactivity or substrates cleaved by BoNT away from the site of administration. Thus, the kinetics and intracellular pathways used for long-distance transport of BoNT remain unclear, although very recently, time-course tracking of SNAP 25 cleavage activity along neuronal axons and cell bodies has helped with initial observations. Transcellular transport has been demonstrated and is operable in various embodiments of the present invention.

[0075] In addition to axonal cytoplasmic transport and effects on choroidal and retinal structures, botulinum toxin A and its segments and related proteins are used either simultaneously or as part of a fusion protein complex comprising an anti-VEGF protein to enhance higher and more sustained biological effects, enhance barrier function, stop leakage, suppress angiogenesis and its pathological effects, and / or alter intracellular RPE structural protein expression. The combined molecular approaches provide an alternative method for delivering anti-VEGF drugs to the choroid without intravitreal injection and using a carrier protein that further targets cell mechanisms including retinal pigment epithelial integrity. Such formulations may include the use of botulinum toxin (e.g., subtype A) or fragments (e.g., HcA, binding domain), the fusion addition of an anti-VEGF agent (e.g., non-fusion addition of an anti-VEGF agent, Avastin® or another fusion protein having anti-VGF properties), and one or more of the stabilizing excipients known to facilitate stability and neuronal axonal uptake.

[0076] The anti-VEGF agent can be delivered by botulinum or its fragment that is involved by axonal flow and undergoes transcytosis with the anti-VEGF agent. The anti-VEGF agent causes reversal of leakage from new blood vessel growth, regression of new blood vessels, enhancement and promotion of strong inter-retinal pigment epithelial cell adhesion to the basement membrane and adjacent cells, and reversal of intracellular structural proteins that promote RPE degeneration. This formulation may also limit leakage from retinal vascular capillaries and post-capillary venules in reaction to the retinal vascular circulation.

[0077] The disclosed formulations can be used in conjunction with conventional para - plana injections, intravitreal injections, or other types of extra - ocular injections. Further, in some embodiments, one or more fusion proteins and axonal protoplasmic transport can be used to produce unique formulations. In some embodiments, the disclosed formulations can be used in conjunction with conventional para - plana (intravitreal) injections of anti - VEGF agents to produce an enhancement in efficacy with respect to the use of a single anti - VEGF alone (see examples). Further, one or more anti - VEGF agents can be included with the botulinum toxin formulations described herein and applied by the extra - ocular delivery methods (described herein) to enhance efficacy.

[0078] (Peri - orbital injection of botulinum toxin for AMD) As described above, extra - ocular injection of botulinum toxin can result in delivery of botulinum toxin to the macula via axonal flow. The anatomical placement that facilitates macular delivery involves placing the needle on the zygomatic arch, angled towards the pterygopalatine fossa and near the outer portion of the inferior orbital fissure. The inferior orbital fissure (unlike the superior orbital fissure) extends very anteriorly, allowing a 2 - cm needle to come very close to the fissure. The projection of the pterygopalatine ganglion protruding through this fissure supplies the eye with botulinum toxin and brings the botulinum toxin close to the autonomic synapses and the veins flowing towards the cavernous sinus. Penetration of botulinum toxin from this injection site into the eye can be facilitated by this anatomical placement. Toxin within the cavernous sinus can infiltrate the sympathetic autonomic nerves on the retina via the eye, retinal arteries, and ciliary arteries (the counter - current movement of botulinum toxin nerve - to - vein). Ganglia recorded in the human choroid are most likely to pick up innervation by the pterygopalatine ganglion. These ganglia are often seen close to the posterior pole of the eye. This unique injection location results in a very low - risk procedure as there are no major blood vessels or important structures. The sensory nerve (V2) and the autonomic nerve abut closely against the fissure, which can allow some penetration into the orbit and the eye. In these and other embodiments, other extra - ocular regions can also be used as injection points.

[0079] (Rho kinase) The unique aspects of the invention described herein are that botulinum toxin type A has Rho kinase regulatory effects and can affect the expression of actin and cadherin, which are important for preventing apoptotic changes in cell structure (programmed cell death cycle). Botulinum C3 has long been known to have very significant Rho kinase activity. Herein, the effect of immunotype A of botulinum toxin is to achieve a similar or identical effect at the local dosing level below that required to cause muscle weakness with accompanying dysfunctions such as diplopia and eyelid ptosis, which is an operable component of the invention. Rho kinase can effectively interact with the actin cytoskeleton to cause assimilatory gene expression and enhance the rapid turnover and expression of actin in such a way as to change and enhance cell, tissue, and organ function. The effects on other eye tissues related to retinal pigment epithelium interaction (filamentary keratitis) are considered with respect to Example 3.

[0080] The effect on the epithelial barrier is demonstrated in another eye condition known as filamentary keratitis, which can serve as a surface model for understanding the effect on the RPE. This is a condition often associated with dry eye syndrome and an inflammatory syndrome characterized by epithelial strains that separate from their attachment to the underlying basement membrane. The process in affected patients can be chronic and can be associated with vision loss, pain, photophobia, and involuntary eye closure. Botulinum toxin has been used in the past to close the eyelids to treat various forms of corneal ulcers in order to mimic tarsorrhaphy, a surgical procedure to close the space between the eyelids (palpebral fissure) to protect the ocular surface. In these descriptions, there is no mention of the specific effect of botulinum on the epithelium and epithelial cell structure, or on cell-cell adhesion and cell-basement membrane adhesion, either directly on adhesion molecules or on intracellular or extracellular proteins that cause an increase in the binding of actin-cadherin proteins or corneal epithelium.

[0081] This specification describes the unique effects on the corneal epithelium, where it increases the junctional integrity of corneal epithelial cells that improve the symptoms of filamentary keratitis with filamentary reduction and disappearance. This condition is being treated in accordance with the concepts described herein using topical botulinum drops. The botulinum drops gain access to the epithelial cells on the ocular surface through topical application to the defects caused by the disease. Botulinum toxin causes the expression of actin, enhances the expression and intracellular organization of actin, cadherin, and related proteins, which increases epithelial junction and integrity, resulting in reduced filament formation and improvement of the disease. A similar effect where the retinal pigment epithelium achieves increased integrity from cell-cell facilitation, cell-basement membrane facilitation, and enhanced specialization of intracellular-extracellular functions created by botulinum toxin-based agents is achieved in age-related macular degeneration. The topical (eyedrop) use of botulinum toxin can achieve beneficial effects on the epithelial structure.

[0082] In the case of filamentary keratitis, botulinum toxin is directly observed as follows. 1. Using a slit lamp microscope on the human eye, it strengthens the adhesion of the epithelial sheet to the ocular surface. 2. Reduction of the exposure of underlying nerves. 3. Reduction of corneal neovascularization in chronic diseases. 4. It covers the exposed nerve endings and reduces pain. 5. The enhanced and restored microvilli on the epithelial surface cause a significant enhancement of corneal integration with the tear film (required for oxygen transport). Such improvement of microvilli may be useful for administering botulinum toxin against dry eye syndrome and inflammatory conditions of the ocular surface, such as recurrent erosion. 6. Reduction of the recurrence rate

[0083] The same effects can be used to treat other forms of keratitis including the basement membrane, such as recurrent corneal erosion, basement membrane dystrophy (map-dot-fingerprint dystrophy), dystrophic corneal ulcer, herpes simplex keratitis, thyroid-related ocular surface disorders, corneal melting syndrome, chemical burns, ocular cicatricial pemphigoid, chronic dry eye syndrome, alcoholic keratitis, Stevens-Johnson syndrome, and exposure keratitis. Since many of the aforementioned conditions occur on or near the ocular surface, topical formulations can be devised.

[0084] Topical formulations containing higher concentrations of botulinum toxin can enter the eye and provide an administration method superior to intravitreal injection through the pars plana.

[0085] (Botulinum toxin preparation) Examples of botulinum toxin preparations include types A1-5, B, C1-C3, D, E, F, and / or G botulinum toxin. Fragments of botulinum toxin can be used to elicit special cellular effects including the isolated genomic expression of cellular components involved in enhancing the barrier effect, the action on the VEGF-related pathway, and the interaction with currently available anti-VEGF drugs, structural proteins, regulators of structural proteins, and inflammatory regulatory proteins. The disclosed formulations, in some embodiments, include stabilized proteins, polycationic proteins or osmotic substances (albumin or polycationic proteins), the use of lidocaine in the formulation or administration prior to injection, botulinum-derived protein derivatives with the SNAP-25 interaction moiety chemically removed, formulations containing enhanced hemagglutinin proteins typically found in botulinum complexes, enhancement by cadherin-binding proteins or drugs known to act on Rho kinase, and may include upstream and downstream metabolites and (ROCK). The regulation of ROCK by the botulinum toxin compositions described herein (e.g., type A toxin) contributes to the therapeutic effect against many of the conditions described herein.

[0086] ROCK1 is a protein serine / threonine kinase also known as rho-associated coiled-coil containing protein kinase 1. Other common names are ROKβ and P160ROCK. ROCK1 is a major downstream effector of the small molecular weight GTPase RhoA and a regulator of the actomyosin cytoskeleton that promotes the generation of contractile force. ROCK1 plays a role in cancer, particularly in cell motility, metastasis, cell adhesion, and angiogenesis. ROCK1 has diverse functions in the body. It is an important regulator of actin-myosin contraction, stability, and cell polarity. These contribute to many processes such as the regulation of morphology, gene transcription, proliferation, differentiation, apoptosis, and oncogenic transformation. Other functions include smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion, and motility. The regulation and / or inhibition of ROCK1 affects the reduction of stress fiber formation in RPE cells. Stress fibers formed by actin condensation in the RPE cytoplasm often occur in age-related macular degeneration and cause disruption of the RPE barrier function, and neovascularization, impaired RPE fluid pump activity, immune exposure of the neural retina, influx of neuropeptides, cytokines, and complement. Stress fibers in RPE cells are depicted in FIGS. 14A-14D, as well as FIGS. 4C, 5A, and 5C. Furthermore, the botulinum toxin formulations described herein can be considered to promote the regeneration of retinal nerve cells by altering Rho activity. In some cases, these formulations can include a botulinum complex typically known as BOTOX® (botulinum toxin type A complex).

[0087] The formulation is preferably administered by injection, but may also be delivered as an eye drop. Eye drop delivery can vary between 10 and 10,000 units, but less than 3,000 units is preferred. Modifications to the dosing for different formulations can be derived from the literature.

[0088] Preferably, the safety of the dosage form is well established for existing formulations, but type A (or subtype) or type B is used because other subtypes and non-neuromuscular subtypes or chemically modified types of botulinum A are expected to be useful.

[0089] Botulinum toxin formulations containing only hemagglutinin proteins without neurotoxins can also be used to isolate and enhance the effect of hemagglutinin on adhesion proteins such as cadherin isoforms and related intracellular proteins in order to allow for greater biological effects not limited by the attenuation and paralytic effects of the neurotoxin. Additionally, in unique embodiments, formulations containing neurotoxins with cleavage portions that remove SNAP-25 and the neuromuscular weakening effect but preserve the effect on actin and cell adhesion functions can be used therapeutically. Such formulations have been cited and studied in the past, but their use in medical applications such as macular degeneration or their use on the membrane barrier function beneficial for the treatment of the diseases described herein has not been suggested.

[0090] Formulations consisting of toxin derivatives with cleaved SNAP25 activity can also be used as carrier molecules for anti-VEGF agents and in combination with accessory proteins.

[0091] Botulinum toxin formulations containing an enhanced amount of botulinum-related hemagglutinin protein along with the neurotoxin can be used to isolate and enhance the effect on cadherin and related adhesion proteins and related intracellular proteins in order to allow for greater biological effects not limited by the attenuation and paralytic effects of the neurotoxin.

[0092] (Epithelial to mesenchymal transition and the effect of botulinum toxin) Generally, most forms of macular degeneration are associated with metaplasia of the retinal pigment epithelium. This process has been described as the conversion of RPE cells into fibrocytic cells that can migrate into the neural retina or vitreous of the eye. Importantly, this process involves the transformed RPE cells with reduced cell - cell adhesion detaching from their continuous sheet, enabling membrane disruption (see Figures 1B and 5D - 5F) and possible antigen recognition by inflammatory cells within the choroid, initiating leakage and growth of new blood vessels. Further growth of new blood vessels from the choroid most often leaks, causing cytokine accumulation and toxic release into the neural retina.

[0093] The botulinum toxin formulations described herein have the effect of essentially slowing or even reversing this process by causing the expression and / or regulation of actin, maintaining cell differentiation and cell structure to maintain barrier function, halting the epithelial - mesenchymal transition of the retinal pigment epithelium, and effectively preventing both major forms (wet and dry) of macular degeneration.

[0094] Due to the impairment of the conversion of retinal pigment epithelium to mesenchymal type by the botulinum toxin formulation, it is possible to treat or prevent proliferative vitreoretinopathy following various forms of retinal detachment, which is a leading and blind complication of corrective retinal detachment surgery.

[0095] (Treatment goals in the clinical setting) The disclosed formulations and methods can, in some cases, improve and / or maintain the vision of patients suffering from macular degeneration. Additionally, botulinum toxin can be used to reduce anatomical changes in populations at risk of macular degeneration.

[0096] Functional measurements can include various forms of visual acuity testing, contrast sensitivity testing, visual fields, measurement of anatomical results using coherence retinal tomography or fluorescein angiography, color vision, OCT, light - dark adaptation measurement, or any other visual function measurement.

[0097] The disclosed formulations can be used in one or more of the following. 1. Prevention in high-risk groups determined by genetic testing or strong family history. 2. Stop the progression of dry degeneration to the neovascular stage with concomitant leakage. 3. Promote dryness in the wet stage and reduce choroidal leakage from regression of choroidal neovascularization.

[0098] Approaches for treating dry age-related macular degeneration include maintaining the barrier between the neurosensory retina and the choroid (the source of neovascularization). Such applications include performing injections with barrier-enhancing agents at the level of the retinal pigment epithelium. Since such injections need to be extraocular to achieve a risk-benefit ratio suitable for repeated dosing in patients with age-related macular degeneration having leakage at the first stage or earlier stage, botulinum toxin by extraocular injection would be ideal. The safety profile is well known to be favorable for periorbital and facial injections at the dosing levels described herein, so botulinum toxin by extraocular injection would be ideal. Such repetition can provide prevention for early age-related macular degeneration cases that progress to the second stage (wet changes) associated with a rapid decline in visual acuity and reading ability.

[0099] (Botulinum toxin hemagglutinin in a complex without muscle-weakening neurotoxin, and role in macular applications (VEGF effect)) ) During the first few decades of the use of botulinum drugs in humans, the toxin has been administered as a complex of neurotoxin associated with non-covalently bound proteins. The type A molecule consists of a neurotoxin, a hemagglutinin protein, and a non-hemagglutinin, non-neurotoxin protein. Most publications to date have shown that the latter two proteins do not play a role in the clinical use of injectable botulinum toxin for various medical conditions and cosmetic applications.

[0100] Non-hemagglutinin can stabilize formation up to the shelf life. Hemagglutinin is important for trans-epithelial penetration and the toxicity against orally ingested botulinum toxin, and has been shown to affect the toxicity of orally ingested substances. Hemagglutinin makes the complex more toxic by promoting gastric absorption. In summary, these proteins, when used partially or in combination, can enhance the effect on the human retina and bring benefits to macular degeneration.

[0101] In contrast, the application of botulinum toxin to the macula and other epithelia is affected by the adjuvant protein in the formulation. In fact, such proteins can be used as independent pharmaceuticals to enhance the botulinum efficacy against epithelial structures including ocular application, and can have substantial directed biological effects even when used in the absence of the neurotoxin.

[0102] The examples shown herein used BOTOX® which is a complex with hemagglutinin adjuvant protein. Hemagglutinin derived from botulinum toxin directly acts on cleaving cadherin E, an important protein that maintains tight junctions between gastric epithelial cells, increasing the uptake of botulinum neurotoxin and thus increasing its toxicity. Even more and still more notably compared to the application to the retina and eye, this effect (contrary to the publications) can affect various cadherin types that cause significant interactions with important receptors involved in endothelial cell proliferation (neovascularization). Example 1 showed evidence of improvement in the leakage and regression of subepithelial neovascular membranes related to the improvement of the prognosis of macular degeneration progression. Furthermore, the effect of BOTOX® prevents the differentiation of RPE cells into mesenchymal fibroblasts accompanied by the death of photoreceptors in the neural retina.

[0103] Cadherin cell adhesion proteins are involved in many retinal diseases, including juvenile macular dystrophy, butterfly dystrophy, Usher syndrome, and autosomal recessive rod-cone dystrophy. Many typed polymorphisms in the cadherin genes are associated with these macular and retinal conditions, which result in the appearance and degeneration of the RPE. The unexpected result is that cadherin activity via the botulinum complex or lectin, which is known to act on cadherin lysis, can actually cause the re-expression of cadherin cell adhesion proteins that enhance barrier activity.

[0104] Cadherin VE is known to be an important protein contained within vascular endothelium that is important for vascular integrity and the growth of new blood vessels. Cadherin VE not only mediates adhesion between endothelial cells but is also required for the survival and maintenance of endothelial cells. Vascular endothelial growth factor (VEGF) requires the form of cadherin to bind to its receptor tyrosine kinase in order to maintain and activate endothelial proliferation. To this extent, lectins with or without complexes having neurotoxins can act as anti-VEGF that can enhance the effects of Avastin®, EYLEA®, or other forms of anti-VEGF drugs. Using botulinum toxin with lectins can act on VEGF function to inhibit vascular activity and growth. This effect also enhances the application of retinal pigment epithelial barrier function, adhesion to the basement membrane, cell polarity, microvilli protrusion, desmosome integrity, and the function of the retinal pigment epithelium produced by botulinum toxin-based formulations.

[0105] These serendipitous observations and applications are that a certain amount of hemagglutinin has been present in Botox-Occulinum® for years and has been tested in numerous clinical trials with a very high safety factor indicating very high safety. This complex has been demonstrated to dissociate rapidly from the neurotoxin component when injected into a subject, and the free complex-forming protein has been shown to be well tolerated and not to cause complications or substantial adverse events. Further isolation of the hemagglutinin protein via ion exchange or other forms of protein separation enables the development of a potentially more targeted pharmaceutical formulated as a specific anti-VEGF for the treatment of macular degeneration. Without being limited to the mechanism, the case reports presented herein have demonstrated a basis for formulating theory and practice from observations, unexpected pharmacodynamics (ocular penetration), and important pharmacologic effects consistent with the novel uses described herein.

[0106] The hemagglutinin derived from botulinum toxin can be recombinantly produced and purified by removing the neurotoxin from the formulation. For botulinum toxin type A and its various subtypes, the final product generated for potency can be tested using local and mouse LD50 assays to confirm that no residual neurotoxin remains in the formulation. The formulation can be administered in a periorbital or intravitreal form around the eye at a dosage that does not affect hemagglutination but can inhibit angiogenesis and retinal pigment epithelial cell leakage and vascular proliferation under the retinal pigment epithelium in the dosage form.

[0107] (Botulinum toxin complex-forming protein) All naturally occurring serotypes of botulinum toxin (types A–G) have non-covalently associated complexing proteins and form toxin complexes. The complexing proteins are encoded by two gene clusters that are located in close proximity to each other on the botulinum chromosome. The first cluster encodes the botulinum toxin itself and the non-toxic non-hemagglutinin (NTNHA) protein, and the second cluster encodes three hemagglutinin (HA) proteins (HA1, HA2, and HA3), where HA3 is cleaved post-translationally into two smaller components (HA3a and 3b) in serotype A. In botulinum toxin serotypes A–D and G, these components form two different toxin complexes, i.e., a medium-sized toxin complex that contains the botulinum toxin and NTNHA (300 kDa) and a large toxin complex that also contains three HA molecules (500–600 kDa). In contrast, serotypes E and F produce only the medium-sized toxin complex. Serotype A also forms a third complex with a higher molecular weight (900 kDa). The detailed molecular structure of the botulinum toxin D-type large toxin complex has been visualized and contains a 14-subunit complex of the neurotoxin, NTNHA, three HA3 molecules (70 kDa molecules, also known as HA-70), three HA2 (also known as HA-17), and six HA1 (HA-33). The denaturing capillary electrophoresis method can determine the subunits that form the very large or higher molecular weight toxin complexes of botulinum toxin type A, which contains a single copy of the 150 kDa neurotoxin and NTNHA subunits, as well as 5–6 HA-17, 4–5 HA-23, 3–4 HA-48, and 8–9 HA-34 subunits and has a total mass of 880–1000 kDa.

[0108] Any component of the botulinum toxin hemagglutinin is a candidate for evaluation of biological activity as an anti-VEGF agent, an intercellular, cell-to-basement membrane or cytoskeleton stabilizer, or an agent useful for the applications to eye diseases described herein. The formulation may contain a neurotoxin having a complex protein, any one or more complex proteins, or components of the complex protein.

[0109] It is expected that increasing the amount of hemagglutinin in existing formulations would be useful for the treatment of spastic conditions (post-stroke and cerebral palsy), blepharospasm, hemifacial spasm, torticollis, prostatic hypertrophy, plantar fasciitis, bruxism, arthritic conditions, myofascial pain, migraine, tension headache, major depressive disorder (MDD), distress, and wound healing. The inventor has observed inflammation as a sensitizer to exacerbate many of the aforementioned conditions. This can be addressed by a higher amount or an increased amount of botulinum toxin-derived hemagglutinin relative to existing formulations in order to achieve a more potent effect.

[0110] (Dosage of HA and dosing at a higher concentration than previously used and expected) Since botulinum has been used for decades as Botox®, the amount of HA derived from botulinum toxin type A complex (see Schantz therapy with botulinum toxin) is expected to vary between generally used levels in the range of 5 U to 8000 U (1 U = LD50 of white mouse). The amount of hemagglutinin in the range of 5 to 4000 U is most preferred.

[0111] (Use of a formulation injectable for a topical formulation of isolated botulinum toxin-derived hemagglutinin) Since the lethal component (neurotoxin) of the complex is absent and systemic weakening is not limited by dosage, higher dosages of botulinum toxin-related hemagglutinin (dosages related to botulinum complexes exceeding 800 U) are possible. This concept essentially enables a unique dosage form without paralytic toxins.

[0112] The topical formulation of botulinum toxin-derived hemagglutinin is an effective composition at the dosage described herein for limiting neovascularization from various infectious diseases (herpes virus, syphilis, ocular cicatricial pemphigoid, traumatic injury, exposure keratitis, corneal transplant rejection, alkali burns, socket inflammation) and scarring of the human cornea, or other infectious degenerations or dystrophies of the human cornea. Aerosol botulinum-derived hemagglutinin can prevent vascular leakage and treat scarring of the lung, upper respiratory system, esophagus, pharynx, intestine, nasal mucosa, rectal area. Intraperitoneal injection can be used to prevent scarring of the peritoneum as well as the surfaces of the large and small intestines. Intravenous injection can be used to reduce new blood vessel growth in malignant tumors that promote angiogenesis, such as metastatic tumors to the liver, spleen, lung, brain, and other organs. Use in allergies is also anticipated, similar to autoimmune diseases such as Graves' disease and autoimmune thyroid diseases. Use in various forms of uveitis to prevent exudation and leakage is anticipated by peribulbar, intravitreal, or intravenous injection. Treatment of leaking blood vessels associated with diabetic retinopathy and blinding diabetic angiogenesis can be targeted by the "anti-VEGF" component action of botulinum toxin hemagglutinin activity. In some embodiments, chronic asthma with vascular leakage and scarring can also be targeted. Eczema and inflammatory skin diseases can be targeted. Various forms of rhinitis can be targeted for treatment. Use in IgE-mediated edema can also be targeted. Other inflammatory conditions can be anticipated for anti-VEGF effects.

[0113] The novel use of isolated hemagglutinin for macular degeneration avoids problems associated with paralysis induced by the neurotoxic component of the molecule and allows for a higher dosage of hemagglutinin than when the hemagglutinin is used in complex with a muscle-paralyzing neurotoxin.

[0114] (Expansion of the invention to other forms of diseases involving the retinal pigment epithelium) Other forms of retinal diseases can be targeted by botulinum toxin delivered by extraorbital and / or periorbital methods and include the following. 1. Retinitis pigmentosa (RP), degenerative, X-linked and dominant types 2. Best disease 3. Stargardt disease 4. Pattern retinal and macular dystrophy 5. Chloroquine retinopathy 6. Lattice dystrophy (with or without retinal breaks) 7. Retinal pigmentary streaks 8. Birdshot retinochoroidopathy 9. Central serous chorioretinopathy 10. Ocular histoplasmosis syndrome 11. Irvine-Gass syndrome 12. White dot syndromes 13. Trauma to the retinal pigment epithelium 14. Ocular toxoplasmosis syndrome 15. Ocular symptoms associated with pseudoexfoliation syndrome 16. PVR (postoperative proliferative vitreoretinopathy) 17. RPE damage associated with choroiditis 18. Macular holes (partial and complete) 19. Early and late retinal detachment (rhegmatogenous - hole-related and nonrhegmatogenous, non-hole-related both) 20. Diabetic macular edema 21. Diabetic retinopathy (at any stage) (both enhanced retinal vascular barrier effect by botulinum toxin and enhanced RPE in barrier and fluid leakage functions)

[0115] In each of the above diseases, destruction of the retinal pigment epithelium can occur, causing damage to photoreceptors due to leakage of choroidal fluid containing cytokines, white blood cells, antibodies, and various immunoreactive agents that destroy photoreceptors, leading to vision loss and blindness.

[0116] Agents that increase the epithelial barrier induce an improvement in the integrity of the pigment epithelial barrier that enhances the protection of photoreceptors and visual function even in situations not associated with age-related macular degeneration. Further, botulinum toxin can affect neuropeptides and other agents of neuroinflammation, which, when transported to the choroid, act to suppress barrier damage and subsequent vision loss associated with macular degeneration as well as other forms of degenerative and inflammatory diseases. Further, even if the effects do not address genetic causes or other processes, enhancement of the RPE photoreceptor system can be neuroprotective to photoreceptors by a mechanism of enhancing RPE function that supports phagocytosis and transport of the apical rod-cone structure. <(

[0117] In the case of retinal degeneration such as retinitis pigmentosa, the defect may mainly involve photoreceptors, and retinal pigment epithelial changes follow excessive degenerated rod and cone material, which undergoes phagocytosis, toxicity accumulates in retinal pigment epithelial cells, and subsequent RPE degeneration and dysfunction follow. Agents that can enhance RPE resistance to toxic protein accumulation will delay visual deterioration based on RPE loss. Other mechanisms at the photoreceptor level can play a role. Stabilization of the barrier membrane by endothelial cells can also be effective in preventing or transitioning the progression of photoreceptor damage and protection. The cause of macular edema in RP is probably related to inflammatory autacoids and antibodies entering the neurosensory retina and inducing rupture of the blood-retinal barrier in the retinal circulation. The edema suggests that RPE leakage from the neovascularized choroid may be important in the progression of RP. Further intrinsic functions of the RPE, such as preservation of microvilli, improvement in the efficiency of phagocytosis based on actin stimulation in the submembrane region, and increased turnover of accumulated dysfunctional rhodopsin protein in photoreceptors, play a role in reducing vision loss over time in various forms of retinitis pigmentosa.

[0118] Genetic defects are associated with retinitis pigmentosa, which is a genetic disease associated with night blindness leading to progressive often-endless vision loss and degeneration of rods and cones in the neurosensory retina.

[0119] (Proliferative vitreoretinopathy (PVR) and epithelial-mesenchymal transition (EMT)) PVR is one of the most devastating complications that occur after retinal detachment surgery. The reaction of the RPE here is to undergo EMT, where the cells proliferate into the vitreous body and transform into fibroblasts, resulting in a traction membrane that causes recurrent retinal detachment. Recurrent retinal detachment is inadequately treated by existing means. The application of botulinum toxin by extraocular or intraocular administration stabilizes the retinal pigment epithelium from fibrotic metaplastic transformation and results in the alleviation of the fibrotic transformation surrounding retinal detachment surgery. Application as a prophylactic agent before, during, and after surgery has proven to be a useful means to reduce the incidence and progression of this complication.

[0120] Pseudoexfoliation syndrome is yet another condition associated with abnormalities in cell adhesion. Here, the migration of pigment epithelial cells from the iris causes glaucoma, often due to cell accumulation in the trabecular meshwork. The use of botulinum toxin by intraocular or extraocular injection can strengthen the adhesion between pigment cells and reduce pigment dispersion, enabling a novel approach to treating this disease. Additionally, this condition may be associated with a higher rate of cataract surgery complications from lens and zonular dislocation. This agent can be used to stimulate a tighter connection between the pigment epithelium and the zonules.

[0121] In some embodiments, a formulation containing botulinum toxin can be injected or topically applied to a patient for the treatment of surface epithelial ulcers and the stabilization of biological tissue barriers. Botulinum toxin has been conventionally used for the treatment of spastic muscle contractions, relaxation of muscles that affect muscle tone, blockade of the autonomic functions that cause secretion, reduction of the sensation of pain such as headaches from various causes, and smoothing of skin wrinkles generated in muscles. Application to non-muscular parts and areas of the skin can cause epithelial tightening by the mechanisms described herein.

[0122] Another novel application of botulinum toxin, when used locally or by injection, results in the rapid healing of epithelial ulcers or stabilizes biological tissue barriers disrupted by various disease processes other than macular degeneration. Its effects center on new biological observations. In these observations, actin and related intracellular elements of the cell structure are stimulated by botulinum toxin, causing upregulation of cell construction proteins after injection, enhancing the cytoskeleton, maintaining the intracellular structure, and enhancing cell-cell adhesion, actin production, and conservation of the cell structure by enhancing the microtubule cross-links between cells that support and strengthen the biological barrier.

[0123] The targeted ulcers occur in the colon, skin along the extremities and lower legs, and include pressure ulcers, decubitus ulcers, oral and tongue ulcers, esophageal ulcers, gastric ulcers, poorly healing surgical and skin wounds, burn-induced wounds, vasculitis-induced ulcers, infections by bacteria and fungi, periprosthetic surgically induced, rectal ulceration, radiation-induced ulceration, oral and gingival ulcers, gingival recession, conjunctival ulcers, post-infection ulcers. For these injections, injectable local delivery methods are effective.

[0124] Important epithelial / endothelial barriers include not only the retinal pigment epithelial barrier, but also the integrity of the corneal epithelium, the uroepithelial barrier in the urethra and bladder, the blood-brain barrier, the endothelial barrier in blood vessels and the cornea, the repair of endothelial microvilli by the GI tract, and the strengthening of the dental gingival barrier important for the development of dental caries and periodontal disease. The biological barrier is strengthened by enhancing the cytoskeleton of actin and related proteins, which causes enhancement of the integrity of the barrier necessary for maintaining the health of the target organs and related tissues.

[0125] Botulinum toxin has been conventionally used to treat spastic muscles and to temporarily denervate glands (exocrine and sebaceous glands, salivary glands, prostate, lacrimal glands, mucus secretions from nasal mucosa, acidic secretions in gastric acid). The muscle target was to induce neuromuscular blockade that causes neurogenic muscle atrophy by blocking acetylcholine release by a blocker of vascular release of acetylcholine. The target includes binding of the heavy chain of botulinum toxin to the presynaptic membrane via the C-terminus of the heavy chain to a membrane receptor accompanied by penetration of the light chain into the cytoplasm that causes cleavage of SNAP-25 (a mechanofusion protein essential for exocytosis). The blockade at the neuromuscular junction occurs in a dosage-dependent area around the injection so as to target the area where the action is involved and prevent unwanted spread that causes complications. Besides these uses, botulinum toxin is used herein to enhance the integrity of the epithelial surface so as to enhance the integrity between cells on the surface, enhance the barrier function of the surface, and fulfill the function of maintaining the surface from degenerative changes occurring in the process of aging or disease.

[0126] The cellular effect enhances the application of botulinum toxin to novel indications that are difficult and often impossible to treat effectively and definitively. The present invention is derived from the previously described "side effects" associated with neuromuscular injection blockade. The injection of botulinum toxin results in side effects that block exocytosis of acetylcholine from presynaptic vesicles, flaccid muscle paralysis, and subsequent atrophy of muscle cells, including nerve sprouting around the neuromuscular blockade with growth of sprouts away from the neuromuscular junction. Previous observers interpreted this cellular response as merely secondary to neuromuscular blockade, but this explanation ignores the following findings. That is, this effect is a direct effect of botulinum toxin that enhances actin and related cell construction proteins, and actin and related cell construction proteins are directly stimulated by the toxin and related proteins to increase protein synthesis and expression of actin and related cell construction proteins that cause sprouting. This finding is applicable to the invention and clinical applications described herein and defines the intracellular process by which the toxin brings benefits to the target tissue.

[0127] Actin and related adhesion and related protein cell structures are important for the function, lifespan, and barrier integrity of many cells and tissues. Programmed cell death can occur by the spontaneous destruction of cytoskeletal proteins, such proteins being important for cell polarity, specificity, and cell adhesion. The upregulation of actin and related proteins in damaged cells and tissues is caused by inflammation, degeneration, infection, metabolic abnormalities, trauma, or burns and helps cells and tissues resist death and destruction. This essential effect of botulinum toxin is important for the implementation of the use of botulinum toxin to assist in the healing process, enhance wound healing, enhance epithelial healing, and the healing rate. Cytoskeletal enhancers can be very useful for protecting cells from destruction based on various causes.

[0128] (Botulinum toxin types that achieve cytoskeletal changes similar to C2 and C3 (topical application and injection)) Botulinum toxin exists as types A(1 - 5), B, C, C2, C3, D, E, F, G. C2 and C3 type toxins cause cytotoxic effects leading to cell death by affecting the integrity that causes actin lysis, increased tissue and cell permeability, and cytotoxic effects. Other neurotoxins cause organism death by flaccid paralysis, asphyxiation from respiratory paralysis. The essential component of the present invention includes using various forms of non - C2 type, C3 type toxins (e.g., various forms of type A toxin) to achieve intracellular and intercellular enhancement effects on actin and related protein production as a cell - protective effect of botulinum use by application at lower dosages. In fact, botulinum toxins in various forms and dosages (concentrations) can have opposing effects on cytoskeletal proteins depending on tissue type and cell cycle. This finding and derived applications are essential for understanding the implementation of the present invention. Type A botulinum toxin can be protective and not toxic at the predetermined dosages and application methods described herein by enhancing and preserving the cytoskeleton. This concept runs counter to intuition regarding the known effects of specific isoforms of botulinum toxin such as type A.

[0129] (Epithelial surface) The epithelial surface tends to have requirements for the integrity of cells and tissues that are important for health and resilience to various forms of disease and trauma.

[0130] The skin and mucosa are obvious epithelial surfaces of the human body. The skin functions to maintain the body's water volume and prevent life-threatening dehydration due to changes in humidity, temperature, and convection. The skin contains squamous epithelial cells that are important for the function of the biological barrier. These cells arise from embryonic cells. The embryonic cells are in close contact with each other on the basement membrane and in a plane perpendicular to the epithelial surface. Actin and related microtubular structures are strongly expressed in the cytoplasm of these cells and respond to various injuries such as burns, viral diseases, trauma, autoimmune diseases, degenerative conditions, and genetic defects. Intracellular elements important for the contribution of the skin as a functional barrier include numerous adhesions including transcellular tubulization, desmosomes, and hemidesmosomes, as well as the actin and microtubular organization of skin cells that allows for the integrity of the cell membrane. Diseases and genetic experimental models involving abnormalities of actin and related proteins cause barrier disruption resulting in structural changes, dehydration, protein loss and damage, and deterioration of the structural appearance of the skin.

[0131] Described herein are approaches to modify the actin and related microtubular elements of skin cells as follows. 1. The integrity of the skin barrier is maintained by local or injectable botulinum toxin such that evaporation, protein leakage, release of protease enzymes, immunoglobulins, and white blood cells can be harmful to the epithelial barrier. 2. Enhance the integrity and proliferation of epithelial cells so as to more effectively heal ulceration and other forms of skin discontinuity. 3. Function as a preventive therapy to prevent the formation of pressure ulcers of the skin, exposure ulcers of the eyeball due to facial nerve paralysis or exophthalmos, esophageal ulcers of the esophageal mucosa due to reflux, bladder ulcers due to irritants such as radiotherapy and chemotherapy, peptic ulcers in patients with active or past duodenal ulcers, gingival extrusion due to breakdown of the gingival epithelium from bacteria or genetic predisposition, etc.

[0132] The mucosal surface is also exposed to functional disorders associated with ulcer formation and loss of barrier integrity. Such loss of integrity can lead to the leakage of enzymes, immunoglobulins, and related cellular elements, such as polymorphonuclear leukocytes, which can further damage the barrier function and other cellular functions of the mucosal surface. Botulinum toxin can function to enhance the integrity of the mucosal epithelial barrier by causing microtubule changes in mucosal cell structures and enabling improvement of the barrier function of epithelial cells when applied by injection or topically.

[0133] Described herein is an approach that modifies the actin and related microtubule elements of mucosal and cell adhesion protein (cadherin) cells as follows: (1) The integrity of the skin barrier is maintained by locally or injectable botulinum toxin such that evaporation, protein leakage, release of protease enzymes, immunoglobulins, and leukocytes can be harmful to the epithelial barrier. And / or, (2) enhance the integrity and proliferation of epithelial cells such that ulcer formation and other forms of skin discontinuity can be more effectively healed.

[0134] Examples of applicable mucosal surfaces include, but are not limited to, the conjunctiva, vagina, rectum, alveoli, glomeruli and renal tubules, intestine, stomach, esophagus, nasal mucosa, oral mucosa, dental-gingival mucosa (periodontal disease), bronchiole and tracheal mucosa, bladder mucosa, urethral mucosa, ureteral mucosa, and / or gallbladder mucosa and bile duct mucosa.

[0135] Conventionally, botulinum toxin has been used to remove dynamic lines and wrinkles based on its neuromuscular weakening effect. This approach has been employed for decades and is the source of a billion-dollar revenue market. This approach has also been the target of the US FDA approval pathway for these indications using forced glabellar lines as an endpoint. Intramuscular injection is described as the target of injection for producing favorable aesthetic results.

[0136] The disclosed formulations and methods of treatment may, in some cases, enhance intercellular adhesion at epithelial surfaces, providing more insight and utility for aesthetic applications. The application of botulinum toxin by injection into non-muscular regions at multiple puncture sites on a surface remote from muscle tissue is beneficial to the texture of the skin and may be effective in removing non-dynamic wrinkles (wrinkles not caused by muscle tension or muscle contraction at rest). The disclosed formulations may be delivered along multiple puncture sites that are much lower than the sites necessary to produce a muscle weakening effect.

[0137] (Botulinum toxin action on Rho protein family) Certain immunotypes of botulinum toxin are known to cause cytotoxicity, acting as cytotoxins that cause intoxication by non-neuromuscular mechanisms. These are types C2 and C3 botulinum, which are characterized by both chemical and cellular effects. These toxins are known to enhance actin depolymerization by actin and to enhance the disruption of tight junctions associated with vascular leakage, hemodynamic instability, and death. This toxin acts as an ADP-ribosylating toxin that interferes with actin formation and integrity. Recently, botulinum toxin type A has been shown to interfere with the migration and function of fibroblasts and to reduce skin scarring. Observations have been made by observers that other botulinum toxins affect actin cytoskeletal elements in such a way as to impair actin formation and actin-related cellular functions (e.g., cell motility), as well as the functional integrity of tissues. These biological effects are negative when the toxin is administered at high doses to cause impairment of cellular function.

[0138] Cell motility requires rapid actin polymerization and depolymerization to achieve this function from members of the Rho protein family (Cdc2, Rac, Rho). These proteins are also involved in maintaining cell polarity, motility, which is important for many tissue barrier functions.

[0139] Conversely, the invention described herein has a positive effect on barrier cells, thereby improving and strengthening cell-to-cell contact and cell-to-basement membrane contact for non-motile epithelial cells. Non-motile epithelial cells form a biological barrier and enhance (without suppressing) cell migration when there are defects, or enhance a biological barrier important in the disease process when there are defects in epithelial adhesion and transformation. The improvement in barrier function is due to the cell-constructing effect of the toxin. Botulinum toxin type A is associated with the reorganization of actin fibers in nerve-derived cell cultures and shows an effect opposite to that of the related C2, C3, and D toxins. Instead of disrupting cell-to-cell contact, type A toxin can reorganize the cytoskeleton in actin and related proteins in a configuration that promotes epithelial and endothelial proliferation to strengthen cell-to-cell contact, enhance the integrity of the biological barrier, improve the function of the epithelial barrier, and seal defects in endothelial and epithelial cell barriers. Its mechanism may involve interacting with Rho-family-like enzymes to regulate the relative rate of reorganization of actin and related proteins, whereby tight junctions are improved, and the biological interaction of actin with its adhesion protein cadherin and special intermediate-sized cell filaments may be related to improving cell function and barrier function.

[0140] While the above may seem contrary to the disclosed reports, this is simply because the neuromuscular effect is controlled by the dosage. These cytological effects are also affected by the dosages conventionally used to treat the medical conditions described herein. Such dosages can regulate the actin cytoskeleton to improve barrier and cell adhesiveness and enhance functions within the epithelial barrier to reduce the disease process based on a subliminal effect on the cytoskeleton involving improved adhesion from actin-cadherin interaction.

[0141] (Complex vs. pure neurotoxin) Current efforts in pharmaceutical design have sought to remove accessory proteins from botulinum toxin preparations. These proteins include hemagglutinins and non-hemagglutinin non-neurotoxin proteins. Recently, the botulinum-related protein hemagglutinin has been associated with the interaction and weakening of cadherin proteins in tissue types. Cadherin interactions may be important for maintaining the integrity of nerve synapses. This disruption further breaks down the actin cellular elements of the presynaptic neuron, enhancing the uptake of botulinum toxin in the presynaptic structure, causing a more effective penetration of botulinum toxin uptake, and is crucial for enhancing the potency and effectiveness of injectable or topically applied botulinum preparations. The interaction with cadherin proteins may induce a genomic response that causes an enhancement of cadherin and related proteins for cell and tissue repair.

[0142] Clinicians have observed that the effectiveness of some formulations of botulinum toxin is not equivalent to that of the botulinum complex (BOTOX® vs XEOMEN). Any membrane interacting substance that can enhance the permeability of botulinum toxin into neurons may be useful for enhancing potency. Recently, there have been two studies on laryngeal spasm and adult-onset spasmodic torticollis reporting an increase in efficacy based on an adjuvant polylysine (poly-cation) designed to increase penetration to the axonal tips of motor neurons. Alternative methods are described herein for increasing the concentration of hemagglutinin to enhance the effect of the preparation from muscle to axonal nerve tip cell adhesion proteins to increase neuronal penetration to the axonal tip and improve efficacy.

[0143] (Preventive therapy for stage 1 macular degeneration) The therapies disclosed herein include, in some embodiments, benign placement alternatives to intravitreal injection, which represents the current placement method when using anti-VEGF pharmaceuticals such as Eylea®, Lucentis®, and Avastin®. In some embodiments, the disclosed methods provide an opportunity for a novel treatment approach that provides prophylactic treatment to high-risk patients, patients diagnosed with early-stage AMD, and / or patients with high-risk characteristics that progress to geographic atrophy or late-stage (exudative) AMD.

[0144] In current practice, age-related macular degeneration (AMD) is often diagnosed at an early stage before the disease progresses to a late-stage degeneration with rapid vision loss that includes new blood vessel growth and leakage within and under the retina due to new blood vessel growth under or on the retinal pigment epithelium. In some embodiments, a method of preventing any stage of AMD is provided that includes identifying high-risk patients with AMD based on genomic testing for high-risk polymorphic genetic constructs that have been implicated in AMD. In these and other embodiments, the method continues to provide extraocular injections to the orbital, extraorbital, periorbital regions (sinus or temporal), and / or the lateral orbital region outside the pterygopalatine fossa to enable a botulinum effect on the posterior eye, macula, or intraocular structures. This method can continue to monitor patients using the methods described herein for AMD assessment and ultimately reduce the incidence of AMD in the targeted eye or eyes.

[0145] There are risk factors for the development and progression of AMD that may be used in connection with the disclosed methods. For example, potential risk factors may include the number and volume of drusen and drusenoid lesions, the extent and location of geographic atrophy in the target or contralateral eye, the number and location of hyperreflective foci in the neurosensory retina (which location is overlying the drusen-drusenoid), loss of continuity and disruption of the IS-OS line and outer nuclear layer, hyperpigmentation or hypopigmentation, hyperreflectivity and deposits within drusen, dynamic changes in the number and size of drusen, hyperreflective foci, IS-OS line ONL, and the presence and number of pseudodrusen, but are not limited thereto. Further, genetic testing may be employed in connection with the disclosed methods to evaluate polymorphisms associated with severe macular degeneration and complement factors and other genes associated with serious diseases.

[0146] (Macular edema) Numerous causes of macular edema are known. For example, macular edema is often associated with diabetes, where damaged blood vessels in the retina begin to leak fluid containing small amounts of blood into the retina. This is the most common cause of vision loss associated with diabetes. Fat deposits may also leak inside the retina. This leakage swells the macula. In this situation, the biological barrier is restricted by retinal vascular endothelium and supported by pericytes in the retinal circulation.

[0147] Ophthalmic surgeries, including cataract surgery, can increase the risk of developing macular edema because blood vessels are stimulated to leak fluid. Macular edema that develops after cataract surgery is called cystoid macular edema (CME). Some other causes of macular edema include type 1 and type 2 diabetes, age-related macular degeneration (AMD), uveitis, retinal vein occlusion (branch and central retinal vein occlusion - Example 8), occlusion of retinal venules by radiation, macular telangiectasia, side effects of certain drug therapies, and specific genetic disorders such as retinal detachment or retinitis pigmentosa, dyschromatosis. The disclosed formulations are methods that can be used to treat, prevent, or cure macular edema caused by one or more of these conditions.

[0148] By the mechanisms described herein, the barriers (endothelial cells and pericytes) that occur around retinal blood vessels can be increased to result in less leakage, less macular edema, and / or maintenance of vision. For these indications, injections can be performed in a manner similar to that described for macular degeneration, via the pars plana (intraocular injection) or via soft tissue injection around the eye. Higher dosages of topical application used to achieve greater penetration can also be used. Such higher dosages are in the range of 1 to 5,000 units.

[0149] (renal function (barrier function) and nephrotic syndrome) Nephrotic-range proteinuria is a loss of more than 3 grams of protein per day into the urine, or the presence of 2 g of protein per gram of urinary creatinine in a single urine or single urine collection. Nephrotic syndrome is a combination of nephrotic-range proteinuria with low serum albumin levels and edema. Nephrotic syndrome has many causes, including primary kidney diseases such as minimal change nephrosis, focal segmental glomerulosclerosis, and membranous nephropathy. Nephrotic syndrome can also result from systemic diseases that affect organs other than the kidney, such as diabetes, amyloidosis, and systemic lupus erythematosus. Nephrotic syndrome can affect both males and females, adults and children of all races. It can occur in a typical form or in association with nephrotic syndrome. The latter includes glomerular inflammation with hematuria and renal dysfunction.

[0150] Nephrotic syndrome can be a primary kidney-specific disease or a secondary manifestation of a systemic disease. In many cases, glomerular damage is an important feature. Kidney diseases that affect the tubules or interstitium, such as interstitial nephritis, do not cause nephrotic syndrome.

[0151] The main causes of nephrotic syndrome include, in approximate order of frequency, minimal change nephrosis, focal segmental glomerulosclerosis, membranous nephropathy, and hereditary nephropathy. Secondary causes include, in approximate order of frequency, diabetes, lupus erythematosus, viral infections (e.g., hepatitis B, hepatitis C, human immunodeficiency virus [HIV]), amyloidosis and paraproteinemia, preeclampsia, and alloantibodies from enzyme replacement therapy.

[0152] Proteinuria in the nephrotic range may occur in other kidney diseases such as IgA nephropathy. In its common glomerular diseases, one-third of patients may have proteinuria in the nephrotic range. Nephrotic syndrome occurs in people with sickle cell disease and may progress to renal failure. Membranous nephropathy may complicate bone marrow transplantation in relation to graft-versus-host disease. From a therapeutic perspective, nephrotic syndrome can be classified as steroid-sensitive, steroid-resistant, steroid-dependent, or frequently relapsing.

[0153] In healthy individuals, less than 0.1% of plasma albumin can pass through the glomerular filtration barrier. There is controversy regarding the sieving of albumin that has crossed the glomerular permeability barrier. Based on studies in experimental animals, the following has been proposed. The ongoing passage of albumin into the urine occurs in amounts of several grams per day, and there is a substantial tubular uptake of albumin equivalent, such that the urine contains less than 80 mg of albumin per day.

[0154] However, studies in humans with tubular transport disorders suggest that the glomerular urinary space albumin concentration is approximately 3.5 mg / L. At this concentration and with a normal daily glomerular filtration rate (GFR) of 150 liters, a maximum of 525 mg of albumin per day in the final urine is expected. Since most of the filtered albumin is reabsorbed by the tubules, in a healthy state, urinary albumin is less than 50 mg / day. Amounts exceeding 500 mg / day usually indicate a glomerular disease.

[0155] The glomerular capillaries are lined by fenestrated endothelium located on the glomerular basement membrane, which is in turn covered by the glomerular epithelium, or podocytes, which are cells with extensions called foot processes that wrap around the capillaries. Between the feet are filtration slits. These three structures (fenestrated endothelium, glomerular basement membrane, and glomerular epithelium) are the glomerular filtration barrier. A schematic diagram of the glomerular barrier is provided in Figure 8.

[0156] Figure 8 shows a schematic diagram of the glomerular barrier. In Figure 8, the abbreviation "GBM" refers to the glomerular basement membrane and "ESL" refers to the endothelial cell surface layer (often called the glycocalyx). The ultrafiltrate is formed by filtration of plasma fluid across the glomerular barrier (arrow). In humans, the glomerular filtration rate (GFR) is 125 mL / min. The plasma flow rate (Qp) is nearly 700 mL / min and the filtration fraction is 20%. The concentration of albumin in serum is generally 40 g / L, but the estimated concentration of albumin in the ultrafiltrate is 4 mg / L, or 0.1% of its concentration in plasma.

[0157] Filtration of plasma water and solutes is extracellular and occurs through endothelial fenestrae and filtration slits. The importance of podocytes and filtration slits is demonstrated by genetic diseases. Finnish-type congenital nephrotic syndrome results from mutations in the gene for nephrin, a protein of the filtration slit, and leads to nephrotic syndrome in infancy. Similarly, podocin, a protein of podocytes, can be abnormal in many children with steroid-resistant focal segmental glomerulosclerosis.

[0158] Structural changes in the glomerulus that can cause proteinuria are damage to the endothelial surface, glomerular basement membrane, or podocytes. One or more of these mechanisms can be seen in any one type of nephrotic syndrome. Albuminuria alone may occur, or with greater damage, leakage of all plasma proteins (i.e., proteinuria) can occur.

[0159] Proteinuria that is more than 85% albumin is selective proteinuria. Albumin has a net negative charge, and it has been proposed that loss of the negative charge of the glomerular membrane may be important in causing albuminuria. Nonselective proteinuria, which is glomerular leakage of all plasma proteins, involves a generalized defect in permeability rather than a change in the net charge of the glomerulus. This configuration does not allow a clear separation of the causes of proteinuria, except for minimal change nephrosis where proteinuria is selective.

[0160] The renal tubules are also governed by a barrier function in cell-cell adhesion and adhesion to the basement membrane. Targeting the kidney or the nerves entering the kidney may be useful in treating kidney diseases where the barrier function is essential. Since botulinum toxin can stimulate proteins essential for cell-cell adhesion and adhesion to the basement membrane, enhancement of adhesion complexes in the glomerular barrier and renal tubules may be useful in the treatment of kidney diseases. Since the kidney is retroperitoneal, from the middle to near the lumbar region of the back, this organ can be accessed by injection through the back muscles and the nerves with axoplasmic transport through the innervated organs. The needle can access the kidney from a back injection and diffusion through a dosage nomogram. In some cases, the purpose of treatment may be to delay the progression of the need for dialysis due to diabetes, or to treat or prophylactically treat glomerular diseases in high-risk patients (e.g., advanced diabetic patients, patients with systemic lupus erythematosus, patients with systemic amyloidosis, paraproteinemia, patients with systemic amyloidosis, or patients with primary nephrotic syndrome).

[0161] (Tooth loss due to periodontal disease) Teeth are attached to the surrounding supporting alveolar bone by periodontal ligament (PDL) fibers. The PDL fibers enter from the bone and naturally into the cementum that exists over the entire root surface of the tooth. They are also attached to the gingival (gum) tissue that covers the alveolar bone by an attachment device. Since this attachment is superficially present relative to the top or height of the alveolar bone, it is called the supracrestal attachment device. This device is subject to deterioration in periodontal disease.

[0162] The supracrestal attachment apparatus consists of two layers, namely the coronal junctional epithelium and the more apical gingival connective tissue fibers. The two layers together form the thickness of the gingival tissue, and this dimension is called the biologic width. Plaque-induced periodontal diseases are generally classified as either destructive or non-destructive. Clinical attachment loss is a sign of destructive (physiologically irreversible) periodontal disease. The quality of the epithelial layer limits the extent and progression of periodontal disease.

[0163] The barrier function of the epithelial layer serves to prevent and delay periodontal disease. Repeated use of botulinum toxin by topical application or local injection can result in a tighter seal by increasing cell-cell adhesion that protects the quality of bone loss and the integrity of the PDL. In gingivitis, inflammation localized to the supracrestal region of the periodontal tissue results in ulceration of the junctional epithelium. This is technically a loss of clinical attachment, but the term loss of clinical attachment is used almost exclusively to refer to the loss of connective tissue attachment. The use of repeated botulinum injections enables the prevention and treatment of ulceration, epithelial erosion, subsequent loss of PDL integrity, connective tissue attachment, and bone loss.

[0164] (Example 1 - Exudative (wet) age-related macular degeneration unresponsive to conventional treatment) A 71-year-old male was diagnosed with progressive macular degeneration due to substantial subretinal and subfoveal fluid, which was unresponsive to repeated intravitreal injections of Avastin® and Eylea (10 injections) (Figs. 9A - 9E). The patient was treated with 100 units of periocular botulinum toxin type A (BOTOX®) in the forehead, orbicularis, and deep temporal fossa, which resulted in a substantially enhanced response to anti-VEGF agents with substantial resolution of subfoveal fluid with subsequent injections (see Figs. 9A - 9E).

[0165] The patient recognized that his vision was enhanced after the combination of anti-VEGF and botulinum preparations compared to his previous failed anti-VEGF therapy. The interpretation was that the botulinum toxin given prior to the next anti-VEGF enhanced the response and (by the preceding botulinum toxin injection) converted this patient's wet age-related macular degeneration to a dry state.

[0166] Anatomical improvements in this patient included retinal flattening, reduction of subretinal and intraretinal fluid, reduction of choroidal neovascular membranes, and RPE hypertrophy, as recorded by optical coherence tomography (Figure 9F). These anatomical findings are typical of a positive response to exudative (WET) age-related macular degeneration.

[0167] (Example 2 - Non-exudative macular degeneration (dry macular degeneration)) The results of this example are shown in Figures 10A and 10B. An elderly female with exudative macular degeneration well documented in each eye and having approximately 20 / 40 vision in each of the left and right eyes receives a botulinum injection. The botulinum injection includes a total of approximately 100 units into the head, periorbital region, and the region within the pterygopalatine fossa targeting autonomic and sensory ganglion structures within this region. The patient notices a slow improvement in contrast sensitivity and clarity that persists for approximately three months. She desires another injection of botulinum toxin type A (BOTOX-A®, Allergan) to maintain her vision. Ophthalmic examinations were unable to establish any other reason for the subjective improvement in vision in the pre- and post-injection examinations.

[0168] Optical coherence tomography shows flattening and regression of drusen bodies and improvement in the surface regularity of the retinal pigment epithelium (Figure 10). The findings occurred concurrently with the subjective improvement in vision.

[0169] Without wishing to be bound by theory, injection into the periorbital nerve structures allows axoplasmic transport into the eye, thereby improving the functionality of the retinal pigment epithelium and perhaps the structure to enable improvement in vision.

[0170] (Example 3 - Filamentary keratitis (improvement in corneal epithelial integrity and adhesion based on direct surface examination of the epithelial sheet)) A 71-year-old male received treatment for blepharospasm for 10 years. He showed improvement after receiving injections of 40 - 80 units. At the same time, he was diagnosed with filamentary keratitis. After administration of botulinum in the form of eye drops (20 units) and by injection to the eyelids, the filaments disappeared or showed significant improvement with regard to reduced photosensitivity, reduced pain, improved vision, and increased epithelial regularity (shown by computerized reflected corneal topography). The increased epithelial adhesion improved his corneal surface, resulting in improved vision, reduced surface distortion, and reduced pain accompanied by the resolution of associated detached filaments.

[0171] (Example 4) An 82-year-old female with long-term stage 1 macular degeneration had been followed for about 4 years due to stage 1 macular degeneration. She noted a slight decrease in vision in her left eye over a one-year period. OCT (Zeiss) showed accumulation of intraretinal fluid on the degenerated retinal pigment epithelium as a change from the previous scan (see Figures 11A - 11D). The previous scan showed dry degeneration with irregularities in the sheet configuration of the RPE, evidenced by discontinuity and splitting of the RPE due to migration of the neurosensory retina RPE (local hyperreflective lesions migrating to the neurosensory retina) (Figure 11A).

[0172] After advice on side effects to the patient, botulinum toxin injections were performed using 70 units sub-temporally in several sites and the periorbital region (multiple dosing injections). The region of the pterygopalatine fossa was also targeted for the diffusion effect. Figure 11B shows leakage around the fovea with conversion to the wet type of macular degeneration.

[0173] A treatment plan using Avastin® or Eylea® was carried out within 2 weeks. Submuscular injection into the pterygopalatine fossa was performed using 70 IU of type A botulinum toxin. When the OCT scan was repeated 10 days later, no breakdown of the body fluid was shown (Figure 11C). After 14 days, the body fluid was completely broken down (see Figure 11D).

[0174] This case demonstrated an effect tempo that took approximately 14 days, consistent with the delay expected with axonal flow. This case showed conversion of stage 2 macular degeneration (wet type) to stage 1. Intravitreal injections of Eylea® or Avastin® were not necessary and were discontinued. The patient received continuous monitoring.

[0175] (Example 5) An 87-year-old woman with hemifacial spasm. She developed dry macular degeneration 4 years ago. Approximately 2 years after she converted to wet degeneration with leakage into the subretinal space and neurosensory retina, several injections of Avastin® resulted in drying of the neurosensory retina with improvement in vision. She remained stable for approximately 2 years and it was found on routine OCT examination that there was reaccumulation of fluid in the perifoveal area. Botulinum toxin injections were performed for her hemifacial spasm at a daily dosage for this condition. Additionally, 20 - 30 units of deep injection were directed towards the pterygopalatine fossa towards the ganglion. Her pre-injection photograph is shown in Figure 12A.

[0176] A2 weeks later, improvement in perifoveal fluid was noted (Figure 12B). Visual acuity in the left eye improved from 20 / 40 to 20 / 25. Note that the accumulation of fluid on both sides of the fovea was significantly reduced 2 weeks later. Additionally, the structural regularity was enhanced (the surface smoothness of the RPE was increased and the white and black, as well as the outer limiting membrane and the IS-OS interface, were made clearer). At 10 weeks, the end of the duration of the botulinum toxin was known and fluid accumulation began to recur. When the injection was repeated after fluid reaccumulation at 10 weeks, a second cycle response was obtained with complete dissipation of the intraretinal edema. The dosing injection was increased to 100 units.

[0177] (Example 6 - Macular Edema) A 90-year-old male with a 35-year history of type 2 diabetes developed macular edema 5 years after cataract surgery. Microaneurysms / leakage were recorded in the macula by examination and angiography. Macular edema was recorded by OCT. 40 U of botulinum toxin type A was injected into the area of the pterygopalatine fossa outside the eye and orbit. Three weeks later, the macular edema completely dissipated. The experimental results of this example are provided in FIGS. 13A and 13B. In particular, FIG. 13A shows the macular edema before injection. FIG. 13B shows a reduction in visible macular edema symptoms (by spatial computer registration) 3 weeks after the lateral temporal injection into the pterygopalatine fossa. Repeated injections are planned.

[0178] (Example 7 - Retrospective study) After the initial observation (Example 1), a retrospective study of several hundred general ophthalmology patients who had received treatment for blepharospasm and spasmodic torticollis (treated with botulinum toxin), which are diseases of the elderly, was conducted for progressive age-related macular degeneration. While repeating the botulinum injection, there were no patients with progression of macular degeneration. It is rare for patients over 60 years old not to have this common problem, suggesting a causal relationship with the combined use of botulinum treatment. The dosage range for these patients was typically 10 - 600 units.

[0179] (Example 8 - Central vein occlusion) An 84-year-old female with central vein occlusion OD who could not receive anti-VEGF therapy for 5 months due to other medical reasons presented with extreme macular edema and hand movement vision. 50 units of botulinum toxin were injected into the involved side. Two weeks later, the macular edema decreased by 60 - 70% on SD OCT, and there was some visual improvement (CF 3 ft) in the involved eye. A repeated dosage of 100 units was given to the patient.

[0180] (Selected exemplary embodiments) In some embodiments, methods for preventing and delaying the onset of macular degeneration are provided. In some such embodiments, the method comprises administering to a human or mammalian patient suffering from macular degeneration or at risk of blindness, a formulation comprising botulinum neurotoxin, a fragment thereof, and / or a neurotoxin-related protein. In these and other embodiments, the botulinum neurotoxin, a fragment thereof, and / or a neurotoxin-related protein is selected from the group consisting of botulinum toxins A1 - A5, B, C1 - 3, D, E, F, G, and H.

[0181] In another exemplary embodiment, a method for enhancing the activity of an anti-VEGF injection is provided. In some such embodiments, the method comprises administering to a patient suffering from the exudative form of macular degeneration, a formulation comprising botulinum neurotoxin, a fragment thereof, and / or a neurotoxin-related protein. The formulation is administered to the patient via an intravitreal injection or a subconjunctival injection, and an anti-VEGF agent is administered to the patient. In selected embodiments, the formulation comprises a fusion protein containing a botulinum neurotoxin or a fragment thereof and an anti-VEGF agent. In these and other embodiments, the formulation is administered to the patient separately from the anti-VEGF agent. In certain cases, the anti-VEGF agent is selected from the group consisting of ranibizumab, bevacizumab, and aflibercept.

[0182] In other embodiments, methods for reducing progressive vision loss due to retinitis pigmentosa are provided. The method may, in some cases, comprise administering to a patient suffering from retinitis pigmentosa, a formulation comprising botulinum toxin or a fragment thereof. Here, the formulation is administered to the patient by intravitreal injection or subconjunctival injection.

[0183] In other embodiments, methods are disclosed for reducing vision loss due to diabetic macular edema, central vein occlusion or branch vein occlusion, degenerative retinal diseases, retinitis pigmentosa (RP) retinal diseases, or uveitis caused by diabetes. In some cases, this method may include administering a formulation containing botulinum toxin or a fragment thereof to a patient suffering from macular edema due to diabetes, branch vein occlusion, or uveitis. Here, the formulation is administered to the patient via intravitreal injection or subconjunctival injection.

[0184] In selected embodiments, a method for preventing age-related macular degeneration in a patient is described. The method may include administering to the patient a formulation containing botulinum toxin or a fragment thereof. Here, the formulation is administered to the patient via intravitreal injection or subconjunctival injection. In these and other embodiments, the patient may be at risk of macular degeneration as determined by medical history or genetic evaluation.

[0185] Methods for treating a patient's periodontal disease and tooth loss are also described herein. The disclosed method includes administering to the patient a formulation containing botulinum toxin or a fragment thereof. Here, the formulation is injected or topically applied to the gingiva, peripheral nerves, oral mucosa, or the skin of the facial or perioral region.

[0186] In another exemplary embodiment, a method for treating a patient's chronic nephrotic syndrome is described. The disclosed method includes administering to the patient a formulation containing botulinum toxin or a fragment thereof. Here, the formulation is injected or topically applied to the kidney or the surrounding area containing one or more nerves entering the kidney. Given the present disclosure, many other exemplary embodiments will be apparent to those skilled in the art.

[0187] (Definitions and Abbreviations) Unless otherwise defined herein, the following terms have the stated definitions.

[0188] AMD - Age-related macular degeneration

[0189] VEGF - Vascular endothelial growth factor. VEGF binds to two members of the receptor tyrosine kinase family (VEGF receptors (VEGFR)-1 and VEGFR-2). VEGFR-2 is considered the main VEGF receptor and mediates the proliferative action of VEGF on vascular endothelial cells. VEGF bound to VEGFR-2 induces receptor dimerization and subsequent autophosphorylation by the intracellular kinase domain, leading to mitogenic and proliferative signals. VEGF-C and VEGF-D bind to VEGFR-3, which is another member of the receptor tyrosine kinase of this family.

[0190] Botulinum toxin - Any immunotype, fragment, or subtype of botulinum derived from Clostridium botulinum species by gene expression in a fermentation or recombinant system.

[0191] HA - Hemagglutinin produced by Clostridium botulinum in fermentation or other natural processes, or recombinant, or derived from any other expression system (accessory protein).

[0192] RPE - Retinal pigment epithelium in mammals. HA is also a botulinum accessory protein.

[0193] OCT - Spectral domain, or any other version or improvement of optical coherence tomography.

[0194] NHNT - Non-neurotoxin, a non-hemagglutinin protein produced by fermentation or recombinant production of botulinum. NHNT is also an accessory protein.

[0195] Anti-VEGF - Any known VEGF monoclonal or fusion protein, or a VEGF agent that suppresses angiogenesis and / or leakage. As used herein, the anti-VEGF term refers to an agent that recognizes multiple isoforms of VEGF. The agent may include a fragment of the VEGF receptor or the entire receptor structure.

[0196] Complement protein - Any complement factor involved in the complement activation cascade.

[0197] Administration of botulinum toxin (and, where applicable, other compounds) in any required form or with microneedles.

[0198] Symptoms treated by periorbital administration of botulinum toxin for blepharospasm (usually in a dosage range of 10 to 300 units).

[0199] Neuropeptides - Any known neuropeptides including, but not limited to, substance P, CGRP, and VIP.

[0200] ELM - External limiting membrane of the retina

[0201] IS / OS - Line defining the inner and outer segments of photoreceptors.

[0202] Stress fiber - Condensation of contractile actin and associated proteins that distort the cell membrane and disrupt the barrier effect of a given tissue or epithelial layer.

[0203] CRVO - Central retinal vein occlusion

[0204] BRVO - Branch retinal vein occlusion

[0205] nAMD - Stage 2 and 3 AMD with angiogenesis (active angiogenic stage with leakage). Biological barrier - Any biological barrier that depends on cell - to - cell adhesion and cell - to - basement membrane adhesion to maintain tissue function.

[0206] mRNA - Messenger RNA

[0207] Conventional dosage - Any FDA - approved dosage of botulinum toxin for indications of the head or neck.

[0208] Formulation - As used herein, the term "formulation" means a composition of one or more biological agents, with or without excipients.

[0209] Addition of one or more proteins industrially produced for the purpose of preserving the biological activity of each protein in order to enhance the usefulness of the fusion protein - composition. Generally, a fusion protein represents the fusion of linked genes or gene fragments and is expressed in a suitable cell line often using PCR to enhance the amount of gene present in the expression system.

[0210] Macromolecule - A large molecule having a relatively large molecular weight, such as nucleic acid, protein, carbohydrate, or lipid.

[0211] Activity - The term "activity" refers to the specific activity or biological activity of a given compound measured using methods recognized in the conventional industry. The activity of a compound is used to quantify purity or concentration and is calculated as a unit per mass.

[0212] Rho - Rho family GTPase.

[0213] Ras - A related C3 botulinum toxin substrate that maintains epithelial differentiation, cytoskeletal reorganization, and cell proliferation.

[0214] Ras 2 - A protein involved in cytoskeletal reorganization.

[0215] Ras 3 - A protein involved in intracellular signaling pathways.

[0216] ROCK1 - A protein kinase regulator of the actin cytoskeleton that promotes the generation of important contractile forces in angiogenesis and cell motility. A major downstream effector of RhoA.

[0217] IU - 20 - 30 gm Botulinum LD50 for Swiss Webster mice, "mouse unit".

[0218] HcA - A fragment of the heavy chain of type A botulinum that functions as a binding domain to nerve cells.

[0219] SNAP-25 - Synaptosomal-associated protein 25 is a component of the trans-SNARE complex, which is proposed to cause the specificity of membrane formation and to directly effect fusion by forming a tight complex that brings synaptic vesicles and the plasma membrane together. Substrate for L-chain botulinum activity.

[0220] Regular hexagon - A six-sided closed figure with equal sides.

[0221] Ophthalmologist - A physician trained to treat medical and surgical diseases of the eye. Duties include injections into the eyeball and the periorbital region.

[0222] EMT - Epithelial-mesenchymal transition.

[0223] GA - Geographic atrophy (end-stage form of dry AMD).

[0224] RPE atrophy - Contraction, flattening, and loss of important physiological functions of the retinal pigment epithelium.

[0225] CNV - Choroidal neovascularization (neovascular proteins that leak fluid and blood). Occurs under the RPE, subretinally, and within the neurosensory retina.

[0226] Leakage - Abnormal, pathological movement of fluid through a biological barrier into intraocular structures. The term "leakage" is used herein to refer to fluid accumulation in the neurosensory retina, subretinal space, or choroid (under the RPE). Such leakage is generally associated with visual distortion and photoreceptor occlusion.

Claims

1. A method of using botulinum neurotoxin in the manufacture of a medicament for treating and / or delaying the progression of wet age-related macular degeneration in a patient, wherein the botulinum neurotoxin is injected into the periorbital or extraorbital region of the patient, without being injected into the patient's eye or subconjunctival region, the botulinum neurotoxin is injected into the patient at a site other than the patient's eye or subconjunctival region, and the botulinum neurotoxin is injected near the extraorbital region of the patient, and an anti-VEGF agent selected from the group consisting of bevacizumab and aflibercept, ranibizumab and abicipar is administered to the patient in combination with the botulinum neurotoxin, the botulinum neurotoxin that undergoes transcytosis together with the anti-VEGF agent reaches the patient's eye through a nerve that affects the blood flow, which increases the efficacy or duration of action of the anti-VEGF agent, administration of the botulinum neurotoxin reduces wet and exudative age-related macular degeneration, characterized in that it is a method of using botulinum neurotoxin.

2. The patient has the presence of geographic atrophy near the fovea, numerous and large drusen, the presence of soft drusen or drusenoids, cyst formation, hyperreflective lesions in the neurosensory retina, pigment in the macula, disruption of the continuity of the inner segment-outer segment (IS-OS) line, genetic polymorphisms associated with severe age-related macular degeneration, polymorphic genes including complement, geographic atrophy in the contralateral eye, and has one or more of the risk factors of a genetic evaluation indicating an increased risk factor, characterized in that it is the method of use according to Claim 1.

3. Wet age-related macular degeneration is diagnosed by OCT, OCT-A, and / or fluorescein fundus angiography, characterized in that it is the method of use according to Claim 1 or 2.

4. The botulinum neurotoxin is injected into the extraorbital region and targets the pterygopalatine fossa, characterized in that it is the method of use according to any one of Claims 1 to 3.

5. The injected botulinum neurotoxin avoids diplopia and ptosis, characterized in that it is the method of use according to any one of Claims 1 to 4.

6. Any FDA-approved dosage of botulinum toxin for head or neck indications ranges from 5 to 2000 units, characterized in that it is the method of use according to any one of Claims 1 to 5.

7. The botulinum neurotoxin is administered such that the method of use further comprises one or more repeated injections of the botulinum neurotoxin into the periorbital or extraorbital region of the patient. The method of use according to any one of claims 1 to 6, characterized in that.

8. Since changes in the cytoskeletal assembly within the retinal pigment epithelium occur, the barrier within the retinal pigment epithelium is maintained by attachment to the basement membrane, rejuvenation of the apical portion of the retinal pigment epithelium occurs, thereby causing cessation of degeneration within the choroid. The method of use according to claim 1, characterized in that.

9. The botulinum neurotoxin is injected into the outer portion of the inferior orbital fissure of the patient. The method of use according to any one of claims 1 to 8, characterized in that.

10. The botulinum neurotoxin is transported into the intraocular region of the patient by one or more of axonal flow within the autonomic and sensory nerves entering the eye, retrograde and anterograde movement of the toxin along nerve fibers, and neurotransmission to the macula choroid and retinal pigment epithelium. The method of use according to any one of claims 1 to 9, characterized in that.

11. A method of using botulinum neurotoxin in the manufacture of a medicament for the treatment of age-related macular degeneration in a human or mammalian patient suffering from or at risk of age-related macular degeneration, The botulinum neurotoxin is injected into the patient in a region other than the intraocular region or the subconjunctival region, and the botulinum neurotoxin is injected into or near the extraorbital region of the patient. After administration of the botulinum neurotoxin, an anti-VEGF agent selected from the group consisting of bevacizumab, aflibercept, ranibizumab, and abicipar is administered to the patient in combination. The botulinum neurotoxin, which undergoes transcytosis together with the anti-VEGF agent, reaches the intraocular region of the patient through the bloodstream, increasing the efficacy or duration of action of the anti-VEGF agent. The administration of the botulinum neurotoxin reduces wet and exudative age-related macular degeneration. A method of use, characterized in that.

12. The botulinum neurotoxin reduces complications due to intraocular injection, including intraocular hemorrhage, endophthalmitis, retinal detachment, retinal hole, lens dislocation, cataract development, and increased intraocular pressure. The method of use according to claim 11, characterized in that.

13. The botulinum neurotoxin is administered as a dosage compatible with conventional dosing. The method of use according to claim 11 or 12, characterized in that.

14. The method of use according to any one of claims 11 to 13, wherein the administered botulinum neurotoxin improves and / or maintains the patient's vision.

15. The method of use according to any one of claims 11 to 14, wherein the botulinum neurotoxin is selected from the group consisting of botulinum toxins A1 to A5, B, C1 to 3, D, E, F, G, and H.

16. The method of use according to any one of claims 11 to 15, wherein the botulinum neurotoxin is administered together with a polycationic protein or macromolecule.

17. The method of use according to any one of claims 11 to 16, wherein the botulinum neurotoxin is administered together with one or more accessory proteins.

18. The method of use according to any one of claims 11 to 17, wherein the botulinum neurotoxin is administered as a pure neurotoxin.

19. The method of use according to any one of claims 11 to 18, wherein the botulinum neurotoxin is injected into the patient using a needle placed above the patient's zygomatic arch, and the needle is directed towards the patient's pterygopalatine fossa and near the outer part of the inferior orbital fissure.

20. The method of use according to any one of claims 11 to 19, wherein the botulinum neurotoxin is injected into the patient's submuscular region.

21. The method of use according to any one of claims 11 to 20, wherein the method of use avoids diplopia.

22. The method of use according to any one of claims 11 to 21, wherein the progression from dry age-related macular degeneration to wet degeneration is reduced by enhancing the integrity of the retinal pigment epithelial barrier.

23. The method of use according to any one of claims 11 to 22, wherein the administered botulinum neurotoxin does not cause neuromuscular paralysis of the extraocular muscles.

24. A method of using a botulinum neurotoxin in the manufacture of a medicament for the treatment of age-related macular degeneration in a human or mammalian patient suffering from or at risk of age-related macular degeneration, wherein an anti-VEGF agent selected from the group consisting of bevacizumab and aflibercept is administered to the patient prior to the botulinum neurotoxin. The botulinum neurotoxin is type A botulinum neurotoxin, and the botulinum neurotoxin is combined with the anti-VEGF agent and injected into the periorbital or extraorbital region of a patient, The botulinum neurotoxin is administered to a patient to avoid diplopia, Reaching the intraocular region of the patient through the nerves around blood vessels that affect the blood flow of the administered botulinum neurotoxin undergoing transcellular transport together with the anti-VEGF agent increases the efficacy or duration of action of the anti-VEGF agent. A method of use characterized by this.

25. The periorbital and extraorbital regions include one or more of the forehead, eyelids, face, facial bones, jaw muscles, neck, nasal mucosa, scalp, paranasal sinus mucosa, mouth, palate, and pterygopalatine fossa. The method of use according to claim 24, characterized by this.

26. The botulinum neurotoxin is transported to the intraocular region by axonal flow in the autonomic and sensory nerves entering the eye. The method of use according to claim 24 or 25, characterized by this.

27. The botulinum neurotoxin is injected into the outer part of the inferior orbital fissure of the patient. The method of use according to any one of claims 24 to 26, characterized by this.

28. The administered botulinum neurotoxin reduces the injection frequency of the administered anti-VEGF agent. The method of use according to any one of claims 24 to 27, characterized by this.

29. A method of using botulinum neurotoxin in the manufacture of a medicament for treating and / or delaying the progression of an intraocular disease in a patient, The botulinum neurotoxin is injected into the periorbital or extraorbital region of the patient, but not into the intraocular or subconjunctival region of the patient, and an anti-VEGF agent is administered to the patient, The intraocular diseases include one or more of central serous chorioretinopathy, central retinal vein occlusion, diabetic retinopathy, diabetic macular edema, neovascular diabetic retinopathy, retinal vascular leakage, retinitis pigmentosa, retinal pigment epithelial damage associated with choroiditis, cystoid macular edema associated with intraocular surgery, uveitis, macular telangiectasia, proliferative vitreoretinopathy, intraocular pseudoexfoliation syndrome, and retinal detachment. Reaching the intraocular region of the patient through the nerves around blood vessels that affect the blood flow of the botulinum neurotoxin undergoing transcellular transport together with the anti-VEGF agent increases the efficacy or duration of action of the anti-VEGF agent. A method of use characterized by this.

30. The administered botulinum neurotoxin is The method of use according to claim 29, having an intraocular effect including one or more of enhancing photoreceptor neuroprotection by enhancing retinal pigment epithelial phagocytosis, removing apical rod and cone material, and enhancing resistance to the accumulation of retinal pigment epithelial toxic proteins from toxic photoreceptor-related proteins.

31. The method of use according to claim 30, wherein the photoreceptor comprises a related protein including dysfunctional rhodopsin or an accumulation of proteins derived from incomplete retinal pigment epithelial phagocytosis.

32. An injectable formulation for treating and / or delaying the progression of dry age-related macular degeneration in a patient, wherein the formulation comprises a fusion protein of an anti-VEGF agent and botulinum toxin, a botulinum neurotoxin that undergoes transcytosis together with the anti-VEGF agent, and a stabilizing excipient, and is an injectable formulation characterized by the anti-VEGF agent being selected from the group consisting of ranibizumab, abicipar, bevacizumab, or aflibercept.

33. The injectable formulation according to claim 32, wherein the botulinum toxin is selected from the group consisting of botulinum toxins A1 - A5, B, C1 - 3, D, E, F, G, and H.

34. An injectable formulation for treating and / or delaying the progression of dry age-related macular degeneration in a patient, wherein the formulation comprises a non-covalent additive of an anti-VEGF agent, a botulinum toxin that undergoes transcytosis together with the anti-VEGF agent, and a stabilizer.

35. The injectable formulation according to claim 32, wherein the botulinum toxin is selected from the group consisting of botulinum toxins A1 - A5, B, C1 - 3, D, E, F, G, and H.

36. The injectable formulation according to claim 34, wherein the anti-VEGF agent is ranibizumab, abicipar, bevacizumab, or aflibercept.

37. The injectable formulation according to any one of claims 32 - 36, wherein the progression of dry age-related macular degeneration includes the conversion from dry age-related macular degeneration to wet age-related macular degeneration.

38. The injectable formulation according to any one of claims 32 - 37, wherein the botulinum neurotoxin is in the form of a complex with an erythroagglutinin protein or a fragment thereof.

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