WO2024213932A1 - Driving axon regeneration by novel enpp1 inhibitors - Google Patents

Abstract

A neurite growth stimulator is a small molecule ENPP1 inhibitor. The small molecule ENPP1 inhibitor allows treatment of a central nervous system (CNS) injury with promotion of axon regeneration. The ENPP1 inhibitor in a vehicle can be administered to a neurological injury to promote the regeneration of axons.

Description

DESCRIPTION

TITLE

DRIVING AXON REGENERATION BY NOVEL ENPP1 INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATION

The subject application claims the benefit of U.S. Provisional Application Serial No. 63/495,541, filed April 11, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Axons rarely undergo spontaneous regeneration after traumatic injury in the central nervous system (CNS), which leads to permanent sensory and motor dysfunction. Methods of promoting axon regeneration have focused on reducing extrinsic inhibitory substrates, such as CSPG, myelin-mediated inhibitory molecules, or depletion of glial scar, which have showed only modest effects. Alternative approaches have targeted intrinsic signaling pathways to promote robust axon regeneration. Examples of such intrinsic factors include PTEN, SOCS3, KLF, GSK3b and Lin28, However, these intrinsic targets do not have highly potent smallmolecule agonists or antagonists, which means manipulation requires gene therapy. The safety of gene therapy is still concerning for clinical practices. Additionally, some suppressors of axon regeneration also function as tumor suppressors. Deletion or inhibition of these genes may hyperactivate intrinsic pathways, causing damaged intracellular homeostasis, impaired neuronal functions or tumorigenesis.

Extrinsic as well as intrinsic factors contribute to the failure of axon regeneration in the adult mammalian CNS. The presence of inhibitory extrinsic factors was first demonstrated by transplantation studies where CNS axons were found to regenerate extensively into a peripheral nerve graft. After CNS injury, reactive astrocytes proliferate into the lesion site and secret chondroitin sulfate proteoglycans (CSPGs), which forms a formidable barrier to axon regeneration. Myelin associated inhibitors (MAIs) such as Nogo and MAG exposed during nerve injury also block axon regeneration in the CNS. Targeting these extrinsic inhibitory molecules and their receptors are commonly used to promote axon regeneration according to prior arts. However, counteracting these extrinsic factors such as digesting CSPG with the enzyme chABC, or antagonizing the signaling of MAIs can only result in limited axon regeneration. These findings highlight that modifying extrinsic factors is not sufficient, but that the neuronal intrinsic growth program must also be activated to drive axon regeneration.

Hence, the identification of clinically applicable targets for the treatment of CNS injuries remains an unmet need. The identification of therapeutic targets that are druggable and show significant axon regeneration effect are desirable.

BRIEF SUMMARY OF THE INVENTION

The identified therapeutic target for the treatment of CNS injuries is druggable and show significant axon regeneration effect through CRISPR/Cas9-mediated depletion. Enppl knockout mouse is viable, encouraging clinical use of the ectonucleotide pyrophosphatase/phosphodiesterase I (ENPP1) inhibitors for treatment. Advantageously, ENPP1 inhibition and stimulator of interferon genes (STING) activation do not have tumorigenic effects. This signaling pathway is the immunotherapy drug target for cancer. Systemic delivery of ENPP1 inhibitors and local delivery of STING agonist have been tested in clinical trials, indicating their safety for clinical translation.

Embodiments are directed to small molecule ENPP1 inhibitors and their use to target intrinsic pathways that promote axon regeneration. As desired, intravitreal injection of these ENPP1 inhibitors for retinal ganglion cells (RGCs) manipulation, can be restricted to the projecting neurons if there are fears of adverse side effects to other organs caused by systemic delivery. The small molecule ENPP1 inhibitors can be used to suppress ENPP1 activity, allowing the avoidance of gene therapy. ENPP1 inhibition and STING activation is achieved by ENPP1 inhibitor (compound) administration, such as, but not limited to, injection. The ENPP1 inhibitors, according to embodiments, are optimized derivatives and analogues of known ENPP1 inhibitors that demonstrate axon regeneration after CNS injury.

The small molecule inhibitor allows identification of a neurite growth stimulator including an ENPP1 inhibitor such as 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-7V-(3,4- dimethoxyphenyl)acetamide (ENP001); and P-[2-[l-(6,7-dimethoxy-4-quinazolinyl)-4- piperidinyl]ethyl]-phosphonic acid (ENP002). The neurite growth stimulator can be formulated into a nerve treatment medication when included with a pharmaceutically active carrier for delivery of the nerve treatment medication. The pharmaceutically active carrier can be selected from: solvents; diluents; buffers, neutral buffered saline, phosphate buffered saline, Tris-HCl, acetate buffers, and phosphate buffers; oil-in-water emulsions; water-in-oil emulsions; aqueous compositions without cosolvents; aqueous compositions with organic co-solvents; solubilizers, Polysorbate 65, Polysorbate 80; colloids; dispersion media; fillers; chelating agents, EDTA, glutathione; amino acids; proteins; disintegrants; binders; lubricants; wetting agents; emulsifiers; sweeteners; colorants; flavorings; aromatizers; thickeners, carbomer, gelatin, or sodium alginate; coatings; preservatives, thimerosal, benzyl alcohol, and polyquaterium; antioxidants, ascorbic acid, and sodium metabisulfite; tonicity controlling agents; absorption delaying agents; adjuvants; bulking agents, lactose, and mannitol; and any combination thereof.

The nerve treatment medication can be employed for treatment of a neurological injury to regenerate axons by administrating the nerve treatment medication to an injured nerve and their cell bodies. The neurological injury can be a spinal cord injury, a traumatic brain injury, an optic neuropathy, a stroke, or glaucoma. Administering can be in the form of an aerosol or spray that is formulated in the form of a powder, particle, solution, suspension, or emulsion. Such formulations can include: saline; polyethylene glycol or glycols; DPPC; methylcellulose; powdered dispersing agents; fluorocarbons; propellants, dichlorodifluoromethane, propane, nitrogen, fluorocarbons; and any combination thereof. Another form of administration can be by intracranial, intravitreal, subcutaneous, or intramuscular injecting. In this mode the formulation may include a solution or suspension, which may 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 diphosphatidylcholine; squalene; or parenteral vegetable oil-in-water emulsion; dextrose; glycerol; phosphate buffered saline (PBS); triethanolamine; or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A shows sections of optic nerves from WT mice at 2 weeks post injury (WPI), where the vitreous body was injected with either PBS or lOmM of a commercial ENPP1 inhibitor (ENPP1 inhibitor C, Cayman chem 29809) immediately after optic nerve injury with a Scale bar of 200 pm.

Fig. IB is a bar graph giving the number of regenerating axons at indicated distances from the lesion site, where **p < 0.01, *p < 0.05, ANOVA followed by Tukey’s test, n = 5-7 mice.

Fig. 1C shows sections of optic nerves from WT mice at 2 WPI, where the vitreous body was injected with either vehicle (DMSO) or lOmM of ENP002 with a Scale bar of 200 pm. Fig. ID is a bar graph giving the number of regenerating axons at indicated distances from the lesion site, where **p < 0.01, *p < 0.05.

Fig. 2A shows sections of optic nerves from Rosa26-Cas9 mice at 2 WPI injected with either AAV-control-sgRNA or AAV-Enppl-sgRNA with a Scale bar of 200 pm.

Fig. 2B is a plot of the quantity of regenerating axons at indicated distances from the lesion site in Fig 2A, where **p < 0.01, ns, not significant, with ANOVA followed by Bonferroni’s test, n = 5 mice.

Fig. 3A shows a representative image of replated DRG neurons treated by the indicated concentration of ENP001 and ENP002 with a Scale bar: 400pm.

Fig. 3B shows a quantification of the longest neuron length in Fig 3 A.

DETAILED DISCLOSURE OF THE INVENTION

Embodiments are directed to ectonucleotide pyrophosphatase/phosphodiesterase I (ENPP1) inhibitors having superior bioactivity towards axonal growth, demonstrated by primary neuronal culture. These ENPP1 inhibitors promote CNS axon regeneration after injury. The therapeutic strategy is promoting axon regeneration after CNS injury through modulating the intrinsic neuronal mechanism.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), a second messenger participating in the cGAMP synthase (cGAS) stimulator of interferon genes (STINGs) signaling, promotes CNS axon regeneration. The cGAS-STING pathway is identified as a key mediator of innate immunity to combat pathogens. cGAS belongs to a class of DNA sensors known as pattern recognition receptors (PRRs). When cGAS detects cytosolic DNA, it is activated and produces 2', 3 '-cGAMP as a second messenger, which activates STING. Activated STING translocated from the endoplasmic reticulum to the Golgi induces 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 (NFkB), in turn, driving the transcription of multiple downstream target genes, such as type I interferon. Apart from immune regulations, the cGAS-STING pathway has been shown to play critical roles in neuronal functions, such as neurodevelopment and nociception.

There are currently at least two clinical trials related to Enppl inhibitors. The first trial is: 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/JCQ.2022.40.16 suppl. e!4550). The Phase 1 trial has been completed, and the results showed that RBS2418 led to complete enzyme inhibition throughout the trial.

The second trial is: A Study of SR-8541A (ENPP1 Inhibitor) in Advanced/Metastatic Solid Tumors. Source: https://clinicaltrials.gov/ct2/show/NCT06063681). Although ongoing, the efficacy of SR-8541 A has been verified in: Abstract LB-118: SR8541 A is a potent inhibitor of ENPP1 and exhibits dendritic cell mediated antitumor activity.

Several clinical trials relate to STING agonists are complete or ongoing. More information on these trials can be found in: "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/sl2943-019-1087-y). The efficacy of STING agonists has been verified in both trials and animal studies.

ENPP1 is a type II transmembrane glycoprotein with nucleotide pyrophosphatase and phosphodiesterase activities. ENPP1 has been identified as the dominant 2'3'-cGAMP hydrolase, and that a hydrolysis resistant form of 2'3'-cGAMP showed strong human STING (hSTING) agonist activities. Therefore, inhibition of ENPP1 allows activating the cGAS- STING signaling axis to promote axon regeneration. Two types of ENPP1 inhibitors have been developed. The first type is nucleotide based ENPP1 inhibitors, which consist of substrate analogs that act by competition with natural substrates of ENPP1. However, poor oral availability and high off-target potentials preclude their use in therapeutic application. Another type is non-nucleotide based ENPP1 inhibitors, which inhibit ENPP1 in both competitive and non-competitive mode of action, with kinase inhibition (Ki) at concentrations ranging from 0.00146 to 1400 pM. While some of these inhibitors potently inhibit ENPP1, many are nonspecific and affect multiple biological processes. For example, heparin is also a well-known anticoagulant. Suramin, which shows high potency to ENPP1, also inhibits multiple receptors such as epidermal growth factor receptor (EGFR) and follicle-stimulating hormone receptor (FSHR). The screening of a commercial library consisting of 1612 compounds led to the discovery of a 2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-N-(3,4-dimethoxyphenyl)acetamide as a potent nucleotide pyrophosphatase/ phosphodiesterase I NPP1 inhibitor (Ki values of 217nM). The structure-activity relationships (SAR) study of this hit compound resulted in the development of a purine analogue with high inhibitory potency (Ki values of 5.00 nM evaluated against the artificial substrate p-Nph5’-TMP). However, the inhibitory potency was lower when tested against natural substrate ATP (Ki values of 18uM). Another cell-impermeable inhibitor of ENPP1 exhibits high potency (Ki values of 33nM) when evaluated against its natural substrate cGAMP.

According to embodiments, a panel of three ENPP1 inhibitors are identified as effective towards neurite growth stimulators. In embodiments the delivery of the ENPP1 inhibitors in a vehicle, such as, but not limited to, a solvent or solution, can be included at a concentration of lOpM or more. Delivery can be conducted via one or a series of administrations. For example, but not limited to intracranial injection and intravitreal injection. The ENPP1 inhibitors are:

2-(3H-imidazo[4,5-b]pyridin-2-ylthio)-A-(3,4-dimethoxyphenyl)acetamide (ENP001); and

P-[2-[l-(6,7-dimethoxy-4-quinazolinyl)-4-piperidinyl]ethyl]-phosphonic acid (ENP002). In one embodiment, the subject compositions are formulated as an orally-consumable product, such as, for example a food item, capsule, pill, or drinkable liquid. An orally deliverable pharmaceutical is any physiologically active substance delivered via initial absorption in the gastrointestinal tract or into the mucus membranes of the mouth. The topic compositions can also be formulated as a solution that can be administered via, for example, injection, which includes intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously.

In other embodiments, the subject compositions are formulated to be administered via the skin through a patch or directly onto the skin for local or systemic effects. The compositions can be administered sublingually, buccally, rectally, or vaginally. Furthermore, the compositions can be sprayed into the nose for absorption through the nasal membrane, nebulized, inhaled via the mouth or nose, or administered in the eye or ear.

Orally consumable products according to the invention are any preparations or compositions suitable for consumption, for nutrition, for oral hygiene, or for pleasure, and are products intended to be introduced into the human or animal oral cavity, to remain there for a certain period of time, and then either be swallowed (e.g., food ready for consumption or pills) or to be removed from the oral cavity again (e.g., chewing gums or products of oral hygiene or medical mouth washes). While an orally-deliverable pharmaceutical can be formulated into an orally consumable product, and an orally consumable product can comprise an orally deliverable pharmaceutical, the two terms are not meant to be used interchangeably herein.

Orally consumable products include all substances or products intended to be ingested by humans or animals in a processed, semi-processed, or unprocessed state. This also includes substances that are added to orally consumable products (particularly food and pharmaceutical products) during their production, treatment, or processing and intended to be introduced into the human or animal oral cavity.

Orally consumable products can also include substances intended to be swallowed by humans or animals and then digested in an unmodified, prepared, or processed state; the orally consumable products according to the invention therefore also include casings, coatings, or other encapsulations that are intended to be swallowed together with the product or for which swallowing is to be anticipated.

In one embodiment, the orally consumable product is a capsule, pill, syrup, emulsion, or liquid suspension containing a desired orally deliverable substance. In one embodiment, the orally consumable product can comprise an orally deliverable substance in powder form, which can be mixed with water or another liquid to produce a drinkable orally-consumable product.

In some embodiments, the orally-consumable product according to the invention can comprise one or more formulations intended for nutrition or pleasure. These particularly include baking products (e.g., bread, dry biscuits, cake, and other pastries), sweets (e.g., chocolates, chocolate bar products, other bar products, fruit gum, coated tablets, hard caramels, toffees and caramels, and chewing gum), alcoholic or non-alcoholic beverages (e.g., cocoa, coffee, green tea, black tea, black or green tea beverages enriched with extracts of green or black tea, Rooibos tea, other herbal teas, fruit-containing lemonades, isotonic beverages, soft drinks, nectars, fruit and vegetable juices, and fruit or vegetable juice preparations), instant beverages (e.g., instant cocoa beverages, instant tea beverages, and instant coffee beverages), meat products (e.g., ham, fresh or raw sausage preparations, and seasoned or marinated fresh meat or salted meat products), eggs or egg products (e.g., dried whole egg, egg white, and egg yolk), cereal products (e.g., breakfast cereals, muesli bars, and pre-cooked instant rice products), dairy products (e.g., whole fat or fat reduced or fat-free milk beverages, rice pudding, yoghurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, and partly or wholly hydrolyzed products containing milk proteins), products from soy protein or other soy bean fractions (e.g., soy milk and products prepared thereof, beverages containing isolated or enzymatically treated soy protein, soy flour containing beverages, preparations containing soy lecithin, fermented products such as tofu or tempeh products prepared thereof and mixtures with fruit preparations and, optionally, flavoring substances), fruit preparations (e.g., jams, fruit ice cream, fruit sauces, and fruit fillings), vegetable preparations (e.g., ketchup, sauces, dried vegetables, deep-freeze vegetables, pre-cooked vegetables, and boiled vegetables), snack articles (e.g., baked or fried potato chips (crisps) or potato dough products and extrudates on the basis of maize or peanuts), products on the basis of fat and oil or emulsions thereof (e.g., mayonnaise, remoulade, and dressings), other readymade meals and soups (e.g., dry soups, instant soups, and pre-cooked soups), seasonings (e.g., sprinkle-on seasonings), sweetener compositions (e.g., tablets, sachets, and other preparations for sweetening or whitening beverages or other food). The present compositions may also serve as semi-finished products for the production of other compositions intended for nutrition or pleasure.

The subject composition can further comprise one or more pharmaceutically acceptable carriers, and/or excipients, and can be formulated into preparations, for example, solid, semisolid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

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

Carriers and/or excipients according the subject invention can include any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of 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, aromatizers, thickeners (e.g. carbomer, gelatin, or sodium alginate), coatings, preservatives (e.g., Thimerosal, benzyl alcohol, polyquaterium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tonicity controlling agents, absorption delaying agents, adjuvants, bulking agents (e.g., lactose, mannitol) and the like. The use of carriers and/or excipients in the field of drugs and supplements is well known. Except for any conventional media or agent that is incompatible with the target health-promoting substance or with the composition, carrier or excipient use in the subject compositions may be contemplated.

In one embodiment, the compositions of the subject invention can be made into aerosol formulations so that, for example, it can be nebulized or inhaled. Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, powders, particles, solutions, suspensions, or emulsions. Formulations for oral or nasal aerosol or inhalation administration may also be formulated with carriers, including, for example, saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents or fluorocarbons. Aerosol formulations can be placed into pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Illustratively, delivery may be by use of a single-use delivery device, a mist nebulizer, a breath-activated powder inhaler, an aerosol metered-dose inhaler (MDI), or any other of the numerous nebulizer delivery devices available in the art. Additionally, mist tents or direct administration through endotracheal tubes may also be used.

In one embodiment, the compositions of the subject invention can be formulated for administration via injection, for example, as a solution or suspension. The solution or suspension can comprise suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3 -butanediol, water, Ringer's solution, or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, non-irritant, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. One illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI). Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01- 0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion. Water or saline solutions and aqueous dextrose and glycerol solutions may be preferably employed as carriers, particularly for injectable solutions. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.

In one embodiment, the compositions of the subject invention can be formulated for administration via topical application onto the skin, for example, as topical compositions, which include rinse, spray, or drop, lotion, gel, ointment, cream, foam, powder, solid, sponge, tape, vapor, paste, tincture, or using a transdermal patch. Suitable formulations of topical applications can comprise in addition to any of the pharmaceutically active carriers, for example, emollients such as carnauba wax, cetyl alcohol, cetyl ester wax, emulsifying wax, hydrous lanolin, lanolin, lanolin alcohols, microcrystalline wax, paraffin, petrolatum, polyethylene glycol, stearic acid, stearyl alcohol, white beeswax, or yellow beeswax. Additionally, the compositions may contain humectants such as glycerin, propylene glycol, polyethylene glycol, sorbitol solution, and 1,2,6 hexanetri ol or permeation enhancers such as ethanol, isopropyl alcohol, or oleic acid.

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. Furthermore, 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”. The transitional terms/phrases (and any grammatical variations thereof) “comprising”, “comprises”, “comprise”, “consisting essentially of’, “consists essentially of’, “consisting” and “consists” can be used interchangeably.

The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured, z.e., the limitations of the measurement system. In the context of compositions containing amounts of ingredients where the term “about” is used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X ± 10%). In other contexts, the term “about” is used provides a variation (error range) of 0-10% around a given value (X ± 10%). As is apparent, this variation represents a range that is up to 10% above or below a given value, for example, X ± 1%, X ± 2%, X ± 3%, X ± 4%, X ± 5%, X ± 6%, X ± 7%, X ± 8%, X ± 9%, or X ± 10%. In the present disclosure, ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7- 1.0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. When ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range) and specific embodiments therein are explicitly included.

“Treatment”, “treating”, “palliating” and “ameliorating” (and grammatical variants of these terms), as used herein, are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease or symptom thereof such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the disease or symptom thereof.

As used herein, the term “subject” refers to an animal, needing or desiring delivery of the benefits provided by a therapeutic composition. The animal may be a primate or rodent. The animal may be, for example, humans, pigs, horses, goats, cats, mice, rats, dogs, apes, fish, chimpanzees, orangutans, guinea pigs, hamsters, cows, sheep, birds, chickens, as well as any other vertebrate or invertebrate. These benefits can include, but are not limited to, the treatment of a health condition, disease, or disorder; prevention of a health condition, disease, or disorder; immune health; enhancement of the function of an organ, tissue (e.g., solid tissue), or system in the body. The subject can be of any age or stage of development, including infant, toddler, adolescent, teenager, adult, or senior. The terms “subject” and “patient” can be used interchangeably.

By “reduces” is meant a negative alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

By “increases” is meant as a positive alteration of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%. Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

MATERIALS AND METHODS

ENPP1 depletion promotes axon regeneration.

Adult mice received ENPP1 inhibitor intravitreous injection and optic nerve crush. At 2 weeks after injury, ENPP1 inhibitor C stimulated significant axon regeneration, in a dosedependent manner, as indicated in Fig. 1 A and Fig. IB. The structure of ENPP1 Inhibitor C is:

In addition, intravitreous injection of ENP002, another inhibitor of ENPP1, significantly promoted axon regeneration 2 weeks after optic nerve crush, as shown in Fig. 1C and Fig. ID.

The CRISPR technique was used to knock out Enppl in RGCs and assessed the effect on axon regeneration. Adeno-associated virus (AAV)-expressing sgRNAs targeting Enppl (sgEnppl) with mCherry tag was injected into the eyes of mice constitutively expressing the Cas9 enzyme. AAV-sgEnppl but not AAV-expressing sgRNA targeting LacZ (sgCtrl) promoted axon regeneration after injection into Cas9 mice, as indicated in Fig. 2A and Fig. 2B).

ENPP1 inhibitors identified to promote neurite growth in DRG replating assay.

Adult mice dorsal root ganglion (DRG) replating assay were used to test two ENPP1 inhibitors ENP001 and ENP002 for neurite growth promoting activity. The purine analogue ENP001 and the ENPP1 inhibitor ENP002 were included in the assay. Primary cultured DRG neurons were treated with vehicle/ENPPl inhibitors at concentration ranging from 1 to lOpM. One day after treatment, neurons were replated and further cultured for 20 hours under continue exposure of inhibitors to assess neurite elongation. Treatment with both ENP001 and ENP002 at a concentration of lOpM resulted in a significant promotion of neurite growth compared to the control group treated with vehicle only. (Fig. 3 A and Fig. 3B). It should be understood that the examples and embodiments 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 the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

EXEMPLARY EMBODIMENTS

The invention may be better understood by reference to certain illustrative examples, including but not limited to the following:

Embodiment 1. A neurite growth stimulator, comprising an ENPP1 inhibitor selected from:

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

Embodiment 2. A nerve treatment medication, comprising; the neurite growth stimulator according to Embodiment 1; and a pharmaceutically active carrier.

Embodiment 3. The nerve treatment medication according to Embodiment 2, wherein the pharmaceutically active carrier is selected from: solvents; diluents; buffers, neutral buffered saline, phosphate buffered saline, Tris-HCl, acetate buffers, and phosphate buffers; oil-in-water emulsions; water-in-oil emulsions; aqueous compositions without cosolvents; aqueous compositions with organic co-solvents; solubilizers, Polysorbate 65, Polysorbate 80; colloids; dispersion media; fillers; chelating agents, EDTA, glutathione; amino acids; proteins; disintegrants; binders; lubricants; wetting agents; emulsifiers; sweeteners; colorants; flavorings; aromatizers; thickeners, carbomer, gelatin, or sodium alginate; coatings; preservatives, thimerosal, benzyl alcohol, and polyquaterium; antioxidants, ascorbic acid, and sodium metabisulfite; tonicity controlling agents; absorption delaying agents; adjuvants; bulking agents, lactose, and mannitol; and any combination thereof. Embodiment 4. A method of treatment of a neurological injury to regenerate axons, comprising: obtaining a nerve treatment medication according to Embodiment 2; and administering the nerve treatment medication to an injured nerve and its cell bodies.

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

Embodiment 6. The method according to Embodiment 4, wherein administering is in the form of an aerosol or spray.

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

Embodiment 8. The method according to Embodiment 7, wherein the formulation comprises: saline; polyethylene glycol or glycols; DPPC; methylcellulose; powdered dispersing agents; fluorocarbons; propellants, dichlorodifluoromethane, propane, nitrogen, fluorocarbons; or any combination thereof.

Embodiment 9. The method according to Embodiment 4, wherein the administering is intracranial, intravitreal, subcutaneous, or intramuscular injecting.

Embodiment 10. The method according to Embodiment 9, wherein a formulation for injecting the nerve treatment medication comprises a solution or suspension.

Embodiment 11. The method according to 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 diphosphatidylcholine; squalene; or parenteral vegetable oil-in-water emulsion; dextrose; glycerol; phosphate buffered saline (PBS); triethanolamine; or any combination thereof. REFERENCES

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Claims

CLAIMS We claim:

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

2. A nerve treatment medication, comprising; the neurite growth stimulator according to claim 1; and a pharmaceutically active carrier.

3. The nerve treatment medication according to claim 2, wherein the pharmaceutically active carrier is selected from: solvents; diluents; buffers, neutral buffered saline, phosphate buffered saline, Tris-HCl, acetate buffers, and phosphate buffers; oil-in-water emulsions; water-in-oil emulsions; aqueous compositions without cosolvents; aqueous compositions with organic co-solvents; solubilizers, Polysorbate 65, Polysorbate 80; colloids; dispersion media; fillers; chelating agents, EDTA, glutathione; amino acids; proteins; disintegrants; binders; lubricants; wetting agents; emulsifiers; sweeteners; colorants; flavorings; aromatizers; thickeners, carbomer, gelatin, or sodium alginate; coatings; preservatives, thimerosal, benzyl alcohol, and polyquaterium; antioxidants, ascorbic acid, and sodium metabisulfite; tonicity controlling agents; absorption delaying agents; adjuvants; bulking agents, lactose, and mannitol; and any combination thereof.

4. A method of treatment of a neurological injury to regenerate axons, comprising: obtaining a nerve treatment medication according to claim 2; and administering the nerve treatment medication to an injured nerve and its cell bodies.

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

6. The method according to claim 4, wherein administering is in the form of an aerosol or spray.

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

8. The method according to claim 7, wherein the formulation comprises: saline; polyethylene glycol or glycols; DPPC; methylcellulose; powdered dispersing agents; fluorocarbons; propellants, dichlorodifluoromethane, propane, nitrogen, fluorocarbons; or any combination thereof.

9. The method according to claim 4, wherein the administering is intracranial, intravitreal, subcutaneous, or intramuscular injecting.

10. The method according to claim 9, wherein a formulation for injecting the nerve treatment medication comprises a solution or suspension.

11. The method according to claim 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 diphosphatidylcholine; squalene; or parenteral vegetable oil-in-water emulsion; dextrose; glycerol; phosphate buffered saline (PBS); triethanolamine; or any combination thereof.