Regeneration of axons from novel ENPP1 inhibitors

Abstract

A neurite growth stimulant is a small molecule ENPP1 inhibitor. Small molecule ENPP1 inhibitors are capable of treating central nervous system (CNS) injury and promoting axonal regeneration. The ENPP1 inhibitor in the carrier can be administered at damaged nerves and their cell bodies to promote the regeneration of axons.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 495,541, filed April 11, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Following traumatic injury to the central nervous system (CNS), spontaneous axonal regeneration is rare, leading to permanent sensory and motor dysfunction. Methods to promote axonal regeneration focus on reducing external inhibitory components, such as CSPG, myelin-mediated inhibitory molecules, or glial scarring; however, these methods have shown limited effectiveness. Alternative approaches target internal signaling pathways to promote robust axonal regeneration. Examples of such endogenous factors include PTEN, SOCS3, KLF, GSK3b, and Lin28; however, there are no highly effective small-molecule agonists or antagonists for these endogenous targets, meaning manipulation via gene therapy is necessary. The safety of gene therapy remains a concern in clinical practice. Furthermore, some axonal regeneration targets are also closely related to tumorigenesis. The loss or inhibition of these genes can overactivate internal pathways, leading to impaired intracellular homeostasis, neuronal dysfunction, or tumorigenesis.

[0004] Exogenous and endogenous factors contribute to the failure of axonal regeneration in the central nervous system (CNS) of adult mammals. The presence of inhibitory exogenous factors was confirmed for the first time in a transplantation study, which found that CNS axons could proliferate extensively into peripheral nerve grafts. Following CNS injury, reactive astrocytes proliferate to the injury site and secrete chondroitin sulfate proteoglycans (CSPGs), forming a strong barrier that inhibits axonal regeneration. Myelin-associated inhibitors (MAIs) exposed during nerve injury, such as Nogo and MAG, also inhibit axonal regeneration in the CNS. According to current techniques, targeting these exogenous inhibitory molecules and their receptors is typically used to promote axonal regeneration. However, counteracting these exogenous factors, such as digesting CSPGs with the enzyme chABC or antagonizing MAI signaling, only elicits limited axonal regeneration. These findings highlight that altering exogenous factors is insufficient; it is also necessary to activate the neuronal's internal growth program to drive axonal regeneration.

[0005] Therefore, identifying clinically applicable therapeutic targets for CNS injury remains an unmet need. The aim is to identify druggable therapeutic targets with significant axonal regeneration effects. Summary of the Invention

[0006] The identified therapeutic target for treating CNS injury is druggable and has a significant effect on axonal regeneration by CRISPR / Cas9-mediated gene knockout. Enppl gene knockout mice are viable, which encourages clinical use of an exo-nucleotide pyrophosphatase / phosphodiesterase I (ENPP1) inhibitor for treatment. Advantageously, ENPP1 inhibition and stimulator of interferon genes (STING) activation do not have tumorigenic effects. This signaling pathway is an immunotherapeutic drug target for cancer. Systemic delivery of ENPP1 inhibitors and local delivery of STING agonists have been tested in clinical trials, indicating that they are safe for clinical translation.

[0007] Embodiments of the present application relate to small molecule ENPP1 inhibitors and their use for targeting the internal pathways that promote axonal regeneration. Depending on the need, if there is a concern that systemic delivery can have adverse side effects on other organs, intravitreal injection of these ENPP1 inhibitors can be limited to the scope of the target neurons for retinal ganglion cell (RGC) treatment. Small molecule ENPP1 inhibitors can be used to inhibit ENPP1 activity, thereby avoiding gene therapy. ENPP1 inhibition and STING activation are achieved by administering an ENPP1 inhibitor (compound), such as, but not limited to, injection. According to embodiments, the ENPP1 inhibitor is an optimized derivative and analog of known ENPP1 inhibitors that exhibits axonal regeneration after CNS injury.

[0008] Small molecule inhibitors include ENPP1 inhibitors, such as 2-(3H-Imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (ENP001) and P-[2-[1-(6,7-dimethoxy-4-quinazolinyl)-4-piperidinyl]ethyl]- phosphonic acid (ENP002). The neurite growth stimulator can be formulated into a neurotherapeutic drug product when the neurite growth stimulator is included with a pharmaceutically active carrier for delivery of the neurotherapeutic drug product. The pharmaceutically active carrier can be selected from the group consisting of: a solvent; a diluent; a buffer, such as a neutral buffered saline, a phosphate buffered saline, Tris-HCl, acetate buffer, and phosphate buffer; an oil-in-water emulsion; a water-in-oil emulsion; an aqueous composition free of a co-solvent; an aqueous composition containing an organic co-solvent; a solubilizer, such as polysorbate 65, polysorbate 80; a colloid; a dispersion medium; a filler; a chelator, such as EDTA, glutathione; an amino acid; a protein; a disintegrant; a binder; a lubricant; a wetting agent; an emulsifying agent; a sweetener; a coloring agent; a flavoring agent; a fragrance; a thickening agent, carbomer, gelatin, or sodium alginate; a coating; a preservative, thiomersal, benzyl alcohol, and polyquaternium; an antioxidant, such as ascorbic acid and sodium metabisulfite; a tonicity control agent; an absorption delaying agent; an adjuvant; a bulking agent, such as lactose and mannitol; and any combination thereof.

[0009] The neurotherapeutic drug can be used to treat a nerve injury to regenerate an axon by administering the neurotherapeutic drug to the injured nerve and its cell body. The nerve injury can be a spinal cord injury, a traumatic brain injury, an optic neuropathy, a stroke, or glaucoma. The administration can be in the form of an aerosol or a spray, which can be formulated in the form of a powder, a granule, a solution, a suspension, or an emulsion. Such formulation can comprise: saline; polyethylene glycol or glycol; DPPC; methylcellulose; a powder dispersant; a fluorocarbon; a propellant, dichlorodifluoromethane, propane, nitrogen, a fluorocarbon; or any combination thereof. Other forms of administration can be intracranial, intravitreal, subcutaneous, or intramuscular injection. In this manner, the formulation can include a solution or a suspension, which can employ: mannitol; 1,3-butanediol; water; Ringer's solution; isotonic sodium chloride solution; synthetic mono- or diglycerides; fatty acids; oleic acid; 10% USP ethanol; 40% USP propylene glycol; polyethylene glycol 600; triethanolamine; dipalmitoyl diphosphatidyl choline; squalene; or parenteral vegetable oil emulsion; dextrose; glycerol; phosphate-buffered saline (PBS); triethanolamine; or any combination thereof. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1A Optic nerve sections of WT mice at 2 weeks post injury (2 WPI) are shown, where PBS or 10 mM of a commercial ENPP1 inhibitor (ENPP1 inhibitor C, Cayman chem 29809) was injected into the vitreous immediately after optic nerve injury, scale bar is 200 pm.

[0011] Figure 1B Bar graphs showing the number of regenerated axons at specified distances from the lesion site are shown, where **p < 0.01, *p < 0.05, followed by Tukey’s test after ANOVA, n = 5-7 mice.

[0012] Figure 1C Optic nerve sections of WT mice at 2 WPI are shown, where the vitreous was injected with vehicle (DMSO) or 10 mM of ENP002, scale bar is 200 pm.

[0013] Figure 1D Bar graphs showing the number of regenerated axons at specified distances from the lesion site are shown, where **p < 0.01, *p < 0.05.

[0014] Figure 2A Optic nerve sections of Rosa26-Cas9 mice at 2 WPI injected with AAV-control-sgRNA or AAV-Enppl-sgRNA are shown, scale bar is 200 pm.

[0015] Figure 2B For Figure 2A Graphs showing the number of regenerated axons specifying distance from the lesioned part of the mid-distance, where **p < 0.01, ns, not significant, Bonferroni test after ANOVA, n = 5 mice.

[0016] Figure 3A Representative images of replated DRG neurons treated with the indicated concentrations of ENP001 and ENP002 are shown, scale bar: 400 μm.

[0017] Figure 3B Representative images of replated DRG neurons treated with the indicated concentrations of ENP001 and ENP002 are shown, scale bar: 400 μm. Figure 3A Quantitative results of the longest neuron length in the mid-distance. DETAILED DESCRIPTION

[0018] Embodiments relate to inhibitors of ectonucleotide pyrophosphatase / phosphodiesterase I (ENPP1) that have superior biological activity on axonal growth, which has been demonstrated by primary neuronal cultures. These ENPP1 inhibitors promote axonal regeneration after injury. The therapeutic strategy is to promote axonal regeneration after CNS injury by modulating internal neural mechanisms.

[0019] Guanosine monophosphate-adenosine monophosphate (cGAMP) is a second messenger involved in cGAMP synthase (cGAS) stimulator of interferon genes (STING) signaling, which promotes axonal regeneration in the CNS. The cGAS-STING pathway is recognized as a key mediator in innate immunity against pathogens. cGAS belongs to a class of DNA sensors known as pattern recognition receptors (PRRs). When cGAS detects cytosolic DNA, cGAS is activated and produces 2',3'-cGAMP as a second messenger, which in turn activates STING. Activated STING translocates from the endoplasmic reticulum to the Golgi apparatus, inducing autophagy. Activated STING also recruits TANK-binding kinase 1 (TBK1), leading to autophosphorylation of TBK1 and phosphorylation of interferon regulatory factor 3 (IRF3) and other transcription factors such as nuclear factor kappa-light chain enhancer of activated B cells, which in turn drives the transcription of various downstream target genes such as type I interferons. In addition to immune regulation, the cGAS-STING pathway has been shown to play a key role in neural functions such as neural development and nociception.

[0020] There are currently at least two clinical trials related to Enppl inhibitors. The first trial: see the article First-in-human experience using RBS2418, an oral ENPP1 inhibitor within an expanded access protocol in combination with pembrolizumab in a patient with metastatic adrenal cancer. Source: https: / / ascopubs.org / doi / abs / 10.1200 / JCO.2022.40.16_suppl.e14550. The first trial has been completed and the results show that RBS2418 caused complete enzyme inhibition throughout the trial.

[0021] The second trial: see the article A Study of SR-8541A (ENPP1 Inhibitor) in Advanced / Metastatic Solid Tumors. Source: https: / / clinicaltrials.gov / ct2 / show / NCT06063681. Although still ongoing, the efficacy of SR-8541A has been validated: Abstract: LB-118: SR8541A is a potent inhibitor of ENPP1 and exhibits dendritic cell-mediated antitumor activity.

[0022] Several clinical trials related to STING agonists have been completed or are ongoing. More information about these trials can be found in the articles “Trial watch: STING agonists in cancer therapy” (source: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC7466854 / ) and “STING: a master regulator in the cancer-immunity cycle” (source: https: / / molecular-cancer.biomedcentral.com / articles / 10.1186 / s12943-019-1087-y). The efficacy of STING agonists has been validated in both trials and animal studies.

[0023] ENPP1 is a type II transmembrane glycoprotein with nucleotide pyrophosphatase and phosphodiesterase activities. ENPP1 was identified as the major 2'3'-cGAMP hydrolyzing enzyme, while the anti-hydrolyzed form of 2'3'-cGAMP exhibits potent human STING (hSTING) agonist activity. Therefore, inhibition of ENPP1 can activate the cGAS-STING signaling axis to promote axonal regeneration. Two types of ENPP1 inhibitors have been developed. The first class is nucleotide-based ENPP1 inhibitors, which consist of substrate analogs, acting by competing with the natural substrate of ENPP1. However, this class of inhibitors has poor oral availability and high off-target potential, hindering their use in therapeutic applications. The other class is non-nucleotide-based ENPP1 inhibitors, which inhibit ENPP1 in both competitive and non-competitive modes of action, with a concentration range of kinase inhibition (Ki) from 0.00146 μΜ to 1400 μΜ. Although some of these inhibitors effectively inhibit ENPP1, most of the inhibitors are non-specific and affect multiple biological processes. For example, heparin is also a well-known anticoagulant. Suramin, which shows high potency against ENPP1, can also inhibit various receptors, such as epidermal growth factor receptor (EGFR) and follicle-stimulating hormone receptor (FSHR). Screening of a commercially available library of 1612 compounds found that 2-(3H-imidazo[4,5-b]pyridine-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide is an effective nucleotide pyrophosphatase / phosphodiesterase I NPP1 inhibitor (Ki value of 217 nM). A structure-activity relationship (SAR) study of the selected compound led to the development of a purine analog with high inhibitory potency (Ki value of 5.00 nM, evaluated against the artificial substrate p-Nph5'-TMP). However, the inhibitory potency was lower when tested against the natural substrate ATP (Ki value of 18 μΜ). Another cell-impermeable ENPP1 inhibitor showed high potency (Ki value of 33 nM) when evaluated against its natural substrate cGAMP.

[0024] According to some embodiments, a set of three ENPP1 inhibitors is identified as potent neurite growth stimulators. In some embodiments, the ENPP1 inhibitors, in a vehicle such as but not limited to a solvent or solution, can have a concentration of 10 μΜ or higher at the time of delivery. Delivery can be by one or a series of administrations. For example, but not limited to, intracranial injection and intravitreal injection. The ENPP1 inhibitors are:

[0025]

[0026] 2-(3H-imidazo[4,5-b]pyridine-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (ENP001); and

[0027]

[0028] P-[2-[1-(6,7-dimethoxy-4-quinazolinyl)-4-piperidyl]ethyl] phosphonic acid (ENP002). In one embodiment, the subject composition is formulated as an oral consumable, such as, for example, a foodstuff, a capsule, a pill, or a drinkable liquid. An orally deliverable drug is any physiologically active substance that is delivered by initial absorption through the gastrointestinal tract or the oral mucosa. The topical composition can also be formulated as a solution that can be administered by, for example, injection, including intravenous injection, intraperitoneal injection, intramuscular injection, intrathecal injection, or subcutaneous injection.

[0029] In other embodiments, the subject composition is formulated for transdermal administration by a patch or for direct administration onto the skin for local or systemic effect. The composition can be administered sublingually, buccally, rectally, or vaginally. In addition, the composition can be sprayed into the nasal cavity for absorption through the nasal mucosa, atomized for inhalation through the mouth or nose, or administered into the eye or ear.

[0030] An oral consumable according to the present application is any formulation or composition suitable for consumption, nutrition, oral hygiene, or recreation, and is a preparation intended to be introduced into the oral cavity of a human or animal to remain in the oral cavity for a period of time before being swallowed (e.g., a ready-to-eat consumable or a pill) or removed from the oral cavity again (e.g., a chewing gum or an oral hygiene preparation or a medicinal mouthwash). While an orally deliverable drug can be formulated into an oral consumable, and an oral consumable can comprise an orally deliverable drug, the two terms are not to be used interchangeably herein.

[0031] An oral consumable includes all substances or preparations ingested by a human or animal in a processed, semi-processed, or unprocessed state. An oral consumable also includes substances added to an oral consumable during its production, handling, or processing and intended to be introduced into the oral cavity of a human or animal.

[0032] An oral consumable can also include substances intended to be swallowed by a human or animal in an unaltered, prepared, or processed state and then digested; thus, an oral consumable according to the present application also includes a casing, coating, or other packaging intended to be swallowed together with the preparation or expected to be swallowed.

[0033] In one embodiment, the oral consumable is a capsule, a pill, a syrup, an emulsion, or a liquid suspension containing the desired orally deliverable substance. In one embodiment, the oral consumable can include the orally deliverable substance in powder form, which can be mixed with water or other liquids to produce a drinkable oral consumable.

[0034] In some embodiments, the oral consumable according to the present application can comprise one or more formulations intended to provide nutrition or recreation. These formulations include, in particular, baked goods (e.g., breads, dry biscuits, cakes and other pastries), confectionery (e.g., chocolates, chocolate bar formulations, other bar formulations, fruit-flavored gum, coated tablets, hard toffee, toffee and caramel, and chewing gum), alcoholic or non-alcoholic drinks (e.g., cocoa, coffee, green tea, black tea, black or green tea drinks enriched with green or black tea extract, rooibos tea, other herbal teas, fruit-containing lemonade, isotonic drinks, soft drinks, nectar / juice, fruit and vegetable juice, and fruit or vegetable juice formulations), instant drinks (e.g., instant cocoa drinks, instant tea drinks, and instant coffee drinks), meat products (e.g., ham, fresh or raw sausage formulations, and seasoned or cured fresh or salted meat products), egg or egg products (e.g., dehydrated whole egg, egg white, and egg yolk), cereal products (e.g., breakfast cereals, oat-nut energy bars, and pre-prepared instant rice products), dairy products (e.g., whole, low-fat or skimmed milk drinks, rice pudding, yogurt, quark, cream cheese, soft cheese, hard cheese, milk powder, whey, butter, buttermilk, and partially or totally hydrolyzed dairy protein-containing products), soy protein or other soy ingredient products (e.g., soy milk and products prepared therefrom, drinks containing isolated soy protein or enzyme-treated soy protein, drinks containing soy flour, formulations containing soy lecithin, fermented products such as tofu or soy fermented products prepared therefrom, and mixtures with fruit formulations and optional flavoring substances), fruit formulations (e.g., fruit jam, fruit ice cream, fruit sauce, and fruit filling), vegetable formulations (e.g., tomato sauce, sauce, dehydrated vegetables, frozen vegetables, pre-prepared vegetables, and boiled vegetables), snacks (e.g., baked or fried potato chips (crisps) or potato dough products, and corn- or peanut-based extrudates), products based on fats and oils or emulsions thereof (e.g., mayonnaise, egg yolk dressing, and salad dressing), other ready-to-eat meals and ready-to-eat soups (e.g., dry powder soups, instant soups, and pre-prepared soups), seasonings (e.g., sprinkling seasonings), sweetener compositions (e.g., tablets, sachets, and other formulations for sweetening or whitening drinks or other foods). The compositions of the present application can also be used as semi-finished products for the production of other compositions intended to provide nutrition or recreation.

[0035] The subject compositions can also comprise one or more pharmaceutically acceptable carriers and / or excipients, and can be formulated, for example, into formulations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, and aerosols.

[0036] The term "pharmaceutically acceptable" as used herein means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0037] The carriers and / or excipients according to the present application can include any and all solvents, diluents, buffers (e.g., such as neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), oil-in-water emulsions or water-in-oil emulsions, aqueous compositions with or without organic co-solvents suitable for, e.g., IV use, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavorings, aromatics, thickening agents (e.g., carbomer, gelatin or sodium alginate), coatings, preservatives (e.g., thimerosal, benzyl alcohol, polyquatemium salts), antioxidants (ascorbic acid, sodium metabisulfite), tonicity controls, absorption delaying agents, adjuvants, bulking agents (e.g., lactose, mannitol), and the like. The use of carriers and / or excipients in the field of pharmaceuticals and supplements is well known. Carriers or excipients can be considered for use in the subject compositions, except for any conventional medium or agent that is incompatible with the target health-promoting substance or with the composition.

[0038] In one embodiment, the compositions of the present application can be made into aerosol formulations, such that, for example, they can be nebulized or inhaled. Suitable pharmaceutical formulations for administration in aerosol or spray form are, for example, powders, granules, solutions, suspensions, or emulsions. Oral or nasal aerosol formulations or inhalation administration formulations can also be formulated with carriers including, for example, saline, polyethylene glycol or glycols, DPPC, methyl cellulose, or in admixture with powder dispersants or fluorocarbons. Aerosol formulations can be placed in pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Illustratively, delivery can be carried out with a disposable delivery device, a micro-atomizer, a breath-activated powder inhaler, a metered dose inhaler (MDI), or any other of the numerous nebulizer delivery devices available in the art. In addition, nebulizing tents or direct administration through endotracheal tubes can also be used.

[0039] In one embodiment, the compositions of the present application can be formulated as, for example, solutions or suspensions for administration by injection. Solutions or suspensions can comprise: a suitable non-toxic, parenterally acceptable diluent or solvent such as mannitol, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution; or a suitable dispersing or wetting agent and suspending agent such as sterile, non-irritating, non- toxic parenterally acceptable oil, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. One exemplary example of a vehicle for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600, and a balance of USP water for injection (WFI). Other exemplary vehicles for intravenous use include 10% USP ethanol and USP WFI; a 0.01% to 0.1% solution of triethanolamine in USP WFI; or a 0.01% to 0.2% solution of dipalmitoyl di-phosphatidyl choline in USP WFI; and 1% to 10% squalene or parenteral vegetable oil-in-water emulsion. Water or saline solutions and aqueous dextrose and glycerol solutions can be preferred as carriers, particularly for injectable solutions. Exemplary examples of vehicles for subcutaneous or intramuscular injection include phosphate buffered saline (PBS) solution, 5% dextrose in WFI solution and 0.01% to 0.1% triethanolamine in 5% dextrose solution or 0.9% sodium chloride in USP WFI solution, or a 1 :2 or 1 :4 mixture of 10% USP ethanol and 40% propylene glycol with a balance of acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or a 0.01% to 0.2% solution of dipalmitoyl di-phosphatidyl choline in USP WFI and 1% to 10% squalene or parenteral vegetable oil-in-water emulsion.

[0040] In one embodiment, the compositions of the present application can be formulated for administration by topical application to the skin, for example, as a topical composition including a rinse, spray, or drop, lotion, gel, ointment, cream, foam, powder, solid, scrub, tape, vapor, paste, tincture, or using a transdermal patch. Suitable topical application formulations can include, in addition to any pharmaceutically active carrier, for example, an emollient such as carnauba wax, cetyl alcohol, cetyl esters wax, emulsifying wax, hydrated lanolin, lanolin, lanolin alcohols, microcrystalline wax, paraffin wax, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax, or yellow beeswax. In addition, the composition can include a humectant such as glycerin, propylene glycol, polyethylene glycol, sorbitol solution, and 1,2,6 hexanetriol; or a penetration enhancer such as ethanol, isopropyl alcohol, or oleic acid.

[0041] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms "including," "includes," "having," "has," "with," or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0042] The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured, i.e., the limitations of the measurement system. In cases where a certain amount comprises a composition containing an amount of an ingredient, that amount of the ingredient is present within + / - 10% of the stated amount (X ± 10%). In other cases, the term "about" is used to provide flexibility to a given value to account for variations in the value that would be expected by a person of ordinary skill in the art, such as those that would result from a change in measurement technique or the inherent variability of the value. It is apparent that such a variation is a range of values that is higher or lower than the given value by 10% (X ± 10%). For example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%.

[0043] In this disclosure, ranges are presented in abbreviated form to avoid having to set out and describe each and every value and all values within the range. Where appropriate, any suitable value within the range can be selected as the upper value, lower value, or terminal value of the range. For example, the range 0.1 to 1.0 is intended to include the terminal values 0.1 and 1.0, as well as the intermediate values 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, and 0.7 to 1.0, etc. It is contemplated that values having at least two significant digits within a range, for example, the range 5 to 10 is intended to include all values between 5.0 and 10.0, as well as between 5.00 and 10.00, including the terminal values. Combinations and subcombinations of ranges, as well as specific embodiments therein, are expressly included within the scope of the disclosure when ranges are used herein.

[0044] As used herein, "treatment," "treating," "palliating," and "ameliorating" (and grammatical variations of these terms) are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. Therapeutic benefit is achieved with a resolution or improvement of one or more physiological symptoms associated with the underlying disease or its complications, and as such, the patient experiences an improvement in health despite the fact that the patient can still be afflicted with the disease or its complications.

[0045] As used herein, the term "subject" refers to an animal in need of or desiring the benefits provided by a therapeutic composition. The animal can be a primate or a rodent. The animal can be, for example, a human, a pig, a horse, a goat, a cat, a mouse, a rat, a dog, an ape, a fish, a chimpanzee, an orangutan, a guinea pig, a hamster, a cow, a sheep, a bird, a chicken, and any other vertebrate or invertebrate. The benefits can include, but are not limited to, treating a health condition, a disease, or a disorder; preventing a health condition, a disease, or a disorder; immunizing health; enhancing the function of an organ, a tissue (e.g., solid tissue), or a system of the body. The subject can be of any age or developmental stage, including an infant, a toddler, an adolescent, a young adult, an adult, or an elderly adult. The terms "subject" and "patient" can be used interchangeably.

[0046] "Decreasing" refers to a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0047] "Increasing" refers to a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0048] The following are examples illustrating methods of practicing the present application. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight percent, and all solvent mixtures are by volume.

[0049] Materials and Methods

[0050] ENPP1 depletion promotes axonal regeneration.

[0051] Adult mice received intravitreal injections of an ENPP1 inhibitor and optic nerve crush. As shown in Figure 1A and Figure 1B at 2 weeks post-injury, ENPP1 inhibitor C stimulated significant axonal regeneration in a dose-dependent manner. The structure of ENPP1 inhibitor C is:

[0052] .

[0053] In addition, as shown in Figure 1C and Figure 1DAs shown, intravitreal injection of another ENPP1 inhibitor, ENP002, significantly promoted axon regeneration 2 weeks after optic nerve crush.

[0054] CRISPR technology was used to knock out Enppl in RGCs and assess its effect on axon regeneration. sgRNAs expressed in adeno-associated virus (AAV) targeting Enppl (sgEnppl) with a mCherry tag were injected into the eyes of mice constitutively expressing the Cas9 enzyme. As Figure 2A and Figure 2B As shown, AAV-sgEnppl but not AAV expressing sgRNAs targeting LacZ (sgCtrl) promoted axon regeneration after injection into Cas9 mice.

[0055] ENPP1 inhibitors that promote neurite growth in the DRG replating assay were identified.

[0056] The adult mouse dorsal root ganglion (DRG) replating assay was used to test the neurite growth promoting activity of two ENPP1 inhibitors, ENP001 and ENP002. The assay included the purine analog ENP001 and the ENPP1 inhibitor ENP002. Primary cultured DRG neurons were treated with the vehicle / ENPP1 inhibitor at a concentration range of 1 μΜ to 10 μΜ. Neurons were replated at one day after treatment and continued to be cultured for 20 hours under continued exposure to the inhibitors to assess the elongation rate of neurites. Both ENP001 and ENP002 at a concentration of 10 μΜ significantly promoted neurite growth compared to the control group treated with vehicle only (p < 0.05, n = 3, one-way ANOVA with Tukey’s multiple comparison test). Figure 3A and Figure 3B ).

[0057] It is to be understood that the embodiments and examples described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Moreover, any element or limitations of any application or embodiment disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) of any other application or embodiment disclosed herein, and all such combinations are to be included within the scope of the application and are to be considered part of the application.

[0058] All patents, patent applications, provisional applications, and publications referred to or cited herein, including all patents, patent applications, provisional applications, and publications of the inventor(s), are incorporated by reference in their entirety, to the extent permitted by law.

[0059] Exemplary Embodiments

[0060] The application can be better understood by reference to some illustrative examples, including but not limited to the following:

[0061] Embodiment 1. A neurite growth stimulant comprising an ENPP1 inhibitor selected from the group consisting of:

[0062] 2-(3H-imidazo[4,5-b]pyridine-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (ENP001); and P-[2-[1-(6,7-dimethoxy-4-quinazolinyl)-4-piperidinyl]ethyl]-phosphonic acid (ENP002).

[0063] Embodiment 2. A neurotherapeutic pharmaceutical product comprising:

[0064] a neurite growth stimulant according to Embodiment 1; and

[0065] a pharmaceutically active carrier.

[0066] Embodiment 3. The neurotherapeutic pharmaceutical product according to Embodiment 2, wherein the pharmaceutically active carrier is selected from the group consisting of: a solvent; a diluent; a buffer, a neutral buffered saline such as phosphate buffered saline, Tris-HCl, acetate buffer, and phosphate buffer; an oil-in-water emulsion; a water-in-oil emulsion; an aqueous composition free of a cosolvent; an aqueous composition containing an organic cosolvent; a solubilizing agent such as polysorbate 65, polysorbate 80; a colloid; a dispersion medium; a filler; a chelating agent, EDTA, glutathione; an amino acid; a protein; a disintegrant; a binder; a lubricant; a wetting agent; an emulsifying agent; a sweetening agent; a coloring agent; a flavoring agent; a perfuming agent; a thickening agent, carbomer, gelatin, or sodium alginate; a coating; a preservative such as thimerosal, benzyl alcohol, and polyquaternium; an antioxidant such as ascorbic acid and sodium pyrosulfite; a tonicity control agent; an absorption delaying agent; an adjuvant; a bulking agent such as lactose and mannitol; and any combination thereof.

[0067] Embodiment 4. A method of treating a nerve injury to regenerate axons comprising:

[0068] obtaining a neurotherapeutic pharmaceutical product according to Embodiment 2; and

[0069] administering the neurotherapeutic pharmaceutical product to the injured nerve and its cell body.

[0070] Embodiment 5. The method according to Embodiment 4, wherein the nerve injury is a spinal cord injury, a traumatic brain injury, an optic neuropathy, a stroke, or glaucoma.

[0071] Embodiment 6. The method according to Embodiment 4, wherein the mode of administration is an aerosol or a spray.

[0072] Embodiment 7. The method of embodiment 7, wherein the formulation of the aerosol or spray is in the form of a powder, granule, solution, suspension, or emulsion.

[0073] Embodiment 8. The method of embodiment 7, wherein the formulation comprises: saline; polyethylene glycol or glycol; DPPC; methylcellulose; a powder dispersant; a fluorocarbon; a propellant, dichlorodifluoromethane, propane, nitrogen, fluorocarbon; or any combination thereof.

[0074] Embodiment 9. The method of embodiment 4, wherein the administration is an intracranial, intravitreal, subcutaneous, or intramuscular injection.

[0075] Embodiment 10. The method of embodiment 9, wherein the formulation of the injection of the neurotherapeutic drug product comprises a solution or a suspension.

[0076] Embodiment 11. The method of embodiment 10, wherein the solution or suspension comprises: mannitol; 1,3-butanediol; water; Ringer’s solution; isotonic sodium chloride solution; synthetic mono- or diglycerides; fatty acids; oleic acid; 10% USP ethanol; 40% USP propylene glycol; polyethylene glycol 600; triethanolamine; dipalmitoyl diphosphatidyl choline; squalene; or parenteral vegetable oil emulsion; dextrose; glycerin; phosphate buffered saline (PBS); triethanolamine; or any combination thereof.

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Claims

1. A neurite growth stimulant comprising an ENPP1 inhibitor selected from: 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (ENP001); and P-[2-[1-(6,7-dimethoxy-4-quinazolinyl)-4-piperidinyl]ethyl]phosphonic acid (ENP002).

2. A neurotherapeutic drug, comprising: The neurite growth stimulant according to claim 1; and Pharmaceutically active carrier.

3. The neurotherapeutic drug according to claim 2, wherein the pharmaceutically active carrier is selected from: solvents; diluents; Buffers, such as neutral buffered saline, phosphate buffered saline, Tris-HCl, acetate buffer, and phosphate buffer; oil-in-water emulsions; water-in-oil emulsions; aqueous compositions without cosolvents; aqueous compositions containing organic cosolvents; solubilizers, such as polysorbate 65 and polysorbate 80; colloids; dispersion media; fillers; chelating agents, such as EDTA and glutathione; amino acids; proteins; disintegrants; binders; lubricants; wetting agents; emulsifiers; sweeteners; colorants; flavoring agents; aroma agents; Thickeners, such as carbomer, gelatin, or sodium alginate; coatings; preservatives, such as thimerosal, benzyl alcohol, and polyquaternium salts; antioxidants, such as ascorbic acid and sodium metabisulfite; tension control agents; absorption delay agents; adjuvants; compatibilizers, such as lactose and mannitol; and any combination thereof.

4. A method for treating nerve injury to regenerate axons, comprising: Obtain the neurotherapeutic medicine according to claim 2; as well as The neurotherapeutic drug is applied to the damaged nerves and their cell bodies.

5. The method according to claim 4, wherein the nerve injury is spinal cord injury, traumatic brain injury, optic neuropathy, stroke, or glaucoma.

6. The method of claim 4, wherein the form of application is an aerosol or spray.

7. The method according to claim 7, wherein the aerosol or spray formulation is in the form of a powder, granules, solution, suspension or emulsion.

8. The method of claim 7, wherein the formulation comprises: brine; polyethylene glycol or ethylene glycol; DPPC; methylcellulose; powder dispersant; fluorocarbon; propellant, dichlorodifluoromethane, propane, nitrogen, fluorocarbon; or any combination thereof.

9. The method of claim 4, wherein the administration is an intracranial, intravitreal, subcutaneous, or intramuscular injection.

10. The method of claim 9, wherein the formulation of the injected neurotherapeutic agent comprises a solution or a suspension.

11. The method of claim 10, wherein the solution or suspension comprises: mannitol; 1,3-butanediol; water; Ringer's solution; isotonic sodium chloride solution; synthetic monoglycerides or diglycerides; fatty acids; oleic acid; 10% USP ethanol; 40% USP propylene glycol; polyethylene glycol 600; triethanolamine; dipalmitoyldiphosphatidylcholine; squalene; or parenteral water-in-oil emulsion; glucose; glycerol; phosphate-buffered saline (PBS); triethanolamine; or any combination thereof.

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