Nicotinamide for use in the treatment and prevention of ophthalmic neurodegenerative disorders (e.g., glaucoma)

Abstract

In retinal and axonal degeneration, particularly in the treatment of glaucoma, this provides a therapeutic intervention to reduce retinal ganglion cell damage. [Solution] The present invention provides the use of a pharmaceutical composition containing nicotinamide (NAM) and / or pyruvate as a neuroprotective drug or gene therapy in the treatment of axonal degeneration of neuronal tissue in neurodegenerative disorders, particularly ocular neurodegenerative diseases including glaucoma.

Description

[Technical Field]

[0001] Description of research funded by the federal government. This invention was developed with the support of the U.S. government under grant number R01 EY011721, granted by the National Institutes of Health (NIH). The U.S. government has certain rights to this invention.

[0002] References to related applications This application claims interest under 35 U.S.SC § 119(e) in relation to U.S. Provisional Application No. 62 / 245,467 filed on 23 October 2015 and U.S. Provisional Application No. 62 / 366,211 filed on 25 July 2016, and all of its contents are incorporated herein by reference in whole. [Background technology]

[0003] Axonal injury is an early event in neurodegenerative diseases. Neurodegenerative diseases are characterized by dysfunction or loss of surviving neurons in the peripheral or central nervous system. In many cases, axonal degeneration has been shown to precede neuronal loss, which is always a more pronounced process at the distal end of the axon than at the proximal end. The upstream molecular signals that trigger the neurodegenerative cascade in neurons remain unknown.

[0004] There are reports suggesting that nicotinamide adenine dinucleotide (NAD) and related compounds can reduce axonal degeneration. For example, U.S. Patent Application Publication 2006 / 0,211,744A1 ('744 application) describes the use of agents including NAD, NADH, and nicotinamide (NAM) to reduce chronic neurodegeneration. Using a cell culture model of severed dorsal root ganglion (DRG) neuron axons, the '744 application discloses that NAD provided a protective effect against axonal degeneration to these neurons. The '744 application further discloses that NAM reduces neurodegeneration in mouse models of experimental autoimmune encephalomyelitis (EAE), amyotrophic lateral sclerosis (ALS), and relapsing-remitting multiple sclerosis (RRMS). U.S. Patent No. 7,776,326 ('326 patent) discloses a method for treating axonal degradation in mammalian neurological disorders by administering agents that increase NAD activity in damaged neurons. The 326 patent holders, using primary cell cultures of dorsal root ganglia (derived from spinal nerves) and axonal transection (mechanical transection) near the nerve cell body, clearly concluded that nicotinic acid and NAM do not work to attenuate axonal degeneration. In ocular injection studies, the 326 patent holders again stated that NAM showed no difference compared to control animals when injected intravitreously.

[0005] Glaucoma is one of the most common neurodegenerative diseases and a leading cause of irreversible blindness, affecting more than 7 million people worldwide, particularly in the elderly (Quigley and Broman, Br J Ophthalmol 90, pp. 262-267, 2006). Glaucoma is a complex, multifactorial disease characterized by the progressive dysfunction and loss of retinal ganglion cells (RGCs) that lead to vision loss. High intraocular pressure (IOP) and aging are susceptibility factors for neurodegeneration in glaucoma. Recent advances have shown various molecular changes that occur in glaucoma tissue in the early stages of the disease (Howell et al., Journal of Clinical Investigation 121, pp. 1429-1444, 2011; Nickells et al., Annu Rev Neurosci 35, pp. 153-179, 2012). The initial cellular and molecular mechanisms that initiate glaucoma damage within RGCs are not well understood. RGC appears to affect multiple sites in the very early stages of glaucoma, including changes that impact cell bodies, dendrites, and synapses in the retina, as well as axons in the optic nerve. The mechanisms by which high IOP and aging lead to neuronal vulnerability and the onset of glaucoma in humans remain unclear. [Overview of the project] [Problems that the invention aims to solve]

[0006] While strategies exist to mitigate the rise in IOP, there are no effective treatments or preventive measures that target ophthalmic neurodegeneration. Therefore, it remains necessary to elucidate the upstream molecular signals that initiate the initial neurodegenerative process and identify novel molecular targets in order to provide therapeutic interventions that reduce RGC damage in retinal and axonal degeneration, particularly in the treatment of glaucoma. [Means for solving the problem]

[0007] This invention is primarily based on the discovery that nicotinamide (NAM) or pyruvate, or a combination thereof, has a potential effect of protecting nerve cell bodies, axons, and associated cell types, and is therefore beneficial in the treatment of neurodegeneration (e.g., axonal degeneration) and ophthalmic neurodegenerative diseases (e.g., glaucoma).

[0008] The object of the present invention is to provide a method for treating or preventing glaucoma, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition containing NAM and / or pyruvate to a subject requiring treatment.

[0009] The object of the present invention is to provide a method for reducing intraocular pressure in, for example, glaucoma, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition containing NAM and / or pyruvate to a subject requiring treatment.

[0010] One aspect of the present invention provides a method for improving the visual function of a patient who has or suffers from glaucoma, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition containing NAM and / or pyruvate to a subject requiring treatment.

[0011] This treatment (pharmaceuticals and / or gene therapy) is not bound by any specific theory, but is thought to work by reducing intraocular pressure through two main mechanisms: 1) reducing aqueous humor production, and / or 2) increasing aqueous humor outflow. This treatment serves as an effective measure by reducing high IOP, and therefore can preserve axons such as the optic nerve and prevent subsequent loss of visual function.

[0012] This treatment is useful for treating or preventing neurodegeneration in glaucoma. This glaucoma treatment is effective in treating or preventing neuronal dysfunction and neurodegeneration at the retinal level (e.g., RGCs). Glaucoma can be present at any level of intraocular pressure (high IOP or normal IOP). This treatment provides IOP-independent neuroprotective effects on retinal cells, such as retinal ganglion cells (RGCs).

[0013] In certain embodiments, this treatment prevents and / or delays the progression of glaucoma (e.g., primary open-angle glaucoma or POAG) by reducing intraocular pressure. Almost all current strategies for treating glaucoma are intended to reduce or prevent the rise in IOP.

[0014] In certain embodiments, this treatment prevents and / or delays the progression of glaucoma (e.g., primary open-angle glaucoma or POAG) by both providing direct neuroprotection and reducing intraocular pressure. This treatment offers an unexpected dual benefit in the treatment of glaucoma.

[0015] In certain embodiments, this treatment prevents and / or delays the progression of glaucoma without reducing intraocular pressure. Therefore, this treatment method offers unexpected benefits, in which case pharmaceuticals and / or gene therapies provide IOP-independent neuroprotective effects.

[0016] This treatment method provides a solution for glaucoma, including primary open-angle glaucoma, by protecting the characteristic patterns of optic nerve changes and visual field loss. According to the preferred practice pattern of AAO, two of three findings (elevated IOP, optic nerve damage, or visual field loss) must be present for the diagnosis of primary open-angle glaucoma.

[0017] The object of the present invention is to provide a method for treating neurodegenerative disorders (ophthalmic neurodegenerative diseases, such as glaucoma) in subjects requiring treatment, the method being to increase the intracellular level of NADt in the subject or NAD + The process includes administering a therapeutically effective amount of a pharmaceutical composition comprising one or more compounds that increase intracellular levels of NADH, GSH, GSSG, PQQ, or pyruvate.

[0018] In certain embodiments, neurodegenerative disorders (such as ophthalmic neurodegenerative diseases, e.g., glaucoma) are associated with axonal degeneration, neuronal dysfunction, cell body contraction, synaptic loss, dendritic atrophy, and / or normal neuronal aging. In certain embodiments, neurodegenerative disorders (such as ophthalmic neurodegenerative diseases, e.g., glaucoma) are associated with axonal degeneration.

[0019] In certain embodiments, neurodegenerative disorders (such as ophthalmic neurodegenerative diseases, e.g., glaucoma) are associated with Wallerian degeneration, Wallerian degeneration, or distal axonal degeneration. For example, Wallerian degeneration is caused by neuronal damage. Neuronal damage may be caused by disease, trauma, chemotherapy drugs, or neuronal aging.

[0020] In certain embodiments, the neurodegenerative disorder is one or more of the following eye disorders: Alzheimer's disease, multiple sclerosis, diabetic neuropathy, traumatic brain injury, ischemia, peripheral neuropathy, or glaucoma or senile eye disease (e.g., age-related macular degeneration [AMD], Leber's optic neuropathy, dominant optic atrophy, cataract, diabetic eye disease / diabetic retinopathy, retinal degeneration, dry eye, low vision).

[0021] In certain embodiments, the compound of the present invention is nicotinamide adenine dinucleotide (NAD + ) Contains precursors (e.g., nicotinic acid [Na], nicotinamide [NAM], nicotinamide mononucleotide [NMN], nicotinamide ribose glycoside [NR], or combinations thereof).

[0022] In certain embodiments, the compounds of the present invention include nicotinamide, pyruvate, or pyrroloquinoline quinone (PQQ). The compounds of the present invention are thought to replenish intracellular NADt or improve the mitochondrial electron transport chain.

[0023] In certain embodiments, the compounds of the present invention include: (a) NAM; (b) pyruvate; (c) PQQ; (d) NAM and pyruvate; and (d) NAM and PQQ. The object of the present invention is to provide a method for treating neurodegenerative disorders (ophthalmic neurodegenerative diseases, such as glaucoma) in subjects requiring treatment, the method comprising the step of administering a gene composition to the subject. The gene composition contains a gene that increases the expression of Nmnat (e.g., Nmnat-1, Nmnat-2, or Nmnat-3).

[0024] In certain embodiments, the gene is NMNAT1. In certain embodiments, the gene is Wld S That is the case. In certain embodiments, the method involves applying Nmnat (e.g., Nmnat-1, Nmnat-2, or Nmnat-3) and / or Wld to the target eye. S The process includes administering a polynucleotide encoding [the specified character].

[0025] In certain embodiments, polynucleotides are administered topically to the subject. In certain embodiments, polynucleotides are administered to the target via a viral vector (e.g., AAV vector, adenovirus vector, lentiviral vector, retroviral vector, etc.).

[0026] In certain embodiments, the neurodegenerative disorder is glaucoma or senile eye disease; the subject is human. The object of the present invention is to provide a method for treating neurodegenerative disorders in subjects requiring treatment, the method comprising the step of administering to a subject a therapeutically effective amount of one or more compounds that increase intracellular levels of NADt, the subject being Nampt, Nmnat (e.g., Nmnat-1), and / or Wld S The process further includes administering polynucleotides that encode and express [the specified character].

[0027] The object of the present invention is to provide a method for treating glaucoma in a subject requiring treatment, comprising the step of administering a pharmaceutical composition containing a therapeutically effective amount of nicotinamide (NAM) to the subject, thereby treating the glaucoma.

[0028] A further related, but characteristic, objective of the present invention is to provide a method for preventing glaucoma in a subject requiring prevention, comprising the step of administering a pharmaceutical composition containing a therapeutically effective amount of nicotinamide (NAM) to the subject, thereby preventing glaucoma.

[0029] In certain embodiments, the NAM is present in a therapeutically effective amount to reduce neurodegeneration of retinal ganglion cells. For example, in certain embodiments, the pharmaceutical composition contains approximately 0.5 to 10 g of NAM, approximately 1 to 5 g of NAM, or approximately 2.5 g of NAM per daily intake.

[0030] In certain embodiments, the NAM is present in a therapeutically effective amount for reducing intraocular pressure. For example, in certain embodiments, the pharmaceutical composition contains approximately 2 to 25 g of NAM, approximately 10 to 20 g of NAM, or approximately 10 g of NAM per daily intake.

[0031] In certain embodiments, the pharmaceutical composition further comprises pyruvate. Preferably, the NAM and pyruvate are present in therapeutically effective amounts to reduce neurodegeneration of retinal ganglion cells. For example, in certain embodiments, the pharmaceutical composition independently contains (1) about 0.5 to 10 g of NAM, about 1 to 5 g of NAM, or about 2.5 g of NAM per daily intake; and (2) about 0.5 to 10 g of pyruvate, about 1 to 5 g of pyruvate, or about 2.5 g of pyruvate per daily intake. Preferably, the NAM and pyruvate are present in therapeutically effective amounts to reduce intraocular pressure. For example, in certain embodiments, the pharmaceutical composition independently contains (1) about 2 to 25 g of NAM, about 10 to 20 g of NAM, or about 10 g of NAM per daily intake; and (2) about 2 to 25 g of pyruvate, about 10 to 20 g of pyruvate, or about 10 g of pyruvate per daily intake.

[0032] In certain embodiments, the pharmaceutical composition further comprises one or more compounds selected from the group consisting of nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD), and nicotinamide ribose (NR). In certain embodiments, if PQQ is present, the pharmaceutical composition contains approximately 10 mg to 10 g, approximately 50 mg to 1 g, or approximately 500 mg of PQQ per daily intake.

[0033] In certain embodiments, the method (with or without pyruvate) further comprises the step of administering a gene composition, the gene composition comprising a polynucleotide encoding NMNAT1. In certain embodiments, the polynucleotide is a viral vector, such as an adeno-associated virus (AAV) vector, an adenovirus vector, a lentiviral vector, or a retroviral vector.

[0034] In certain embodiments, the viral vector is AAV. In certain embodiments, the viral vector is AAV2.2. In certain embodiments, the viral vector is a lentiviral vector.

[0035] In certain embodiments, the gene composition is administered intravitreously or intraocularly. Preferably, the gene composition is administered intravitreously. In certain embodiments, the subject is a human subject.

[0036] In certain embodiments, the subject has an intraocular pressure of approximately 12 to 21 mmHg. In certain embodiments, the subject has an intraocular pressure greater than 21 mmHg. In certain embodiments, the subject does not develop neurodegenerative symptoms of glaucoma. In certain embodiments, the subject develops neurodegenerative symptoms of glaucoma.

[0037] In certain embodiments, the subject is suffering from visual impairment. In certain embodiments, the method further includes the step of administering an additional therapeutic agent to the subject. Typical additional therapeutic agents include agents that reduce intraocular pressure. In certain embodiments, the additional therapeutic agent is a β-receptor blocker, a non-selective adrenergic agonist, a selective α-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathomimetic agonist, a carbachol, or a combination thereof. In certain embodiments, the additional therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinephrine, apraclonidine, brimonidine, acetazolamide, metazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, ecothiophate iodide, carbachol, or a combination thereof.

[0038] Furthermore, an object of the present invention is to provide a method for improving visual function in a subject requiring improvement, comprising the step of administering a pharmaceutical composition containing a therapeutically effective amount of nicotinamide (NAM) to the subject, thereby improving visual function.

[0039] In certain embodiments, the pharmaceutical composition further comprises pyruvic acid. In certain embodiments, the method further comprises the step of administering a gene composition, the gene composition comprising a polynucleotide encoding NMNAT1.

[0040] Unless explicitly stated otherwise, all embodiments described herein should be understood to be combinable with any other embodiments. [Brief explanation of the drawing]

[0041] [Figure 1]This figure shows the results of hierarchical cluster analysis (HC) to define molecularly determined stages of glaucoma in DBA / 2J(D2) mice that are in the early stages of the disease and morphologically indistinguishable from D2-Gpnmb+ or younger controls of the same age. The HC was based on RNA sequencing of retinal ganglion cells (RGCs) isolated from D2 mice and controls. The HC allows for clustering of RGC samples into distinct groups containing molecularly similar control and younger samples (Spearman's ρ). Circles = D2 RGC-derived samples, triangles = D2-Gpnmb+ RGCS-derived samples. Inset: Number of differentially expressed (DE) genes between D2-Gpnmb+ and each group (q<0.05). [Figure 2A] This figure shows the number of differentially expressed (DE) genes among the four D2 mouse groups (Group 1, Group 2, Group 3, and Group 4) and the D2-Gpnmb+ control group. [Figure 2B] This figure shows the heatmap correlation of all samples (Spearman's ρ, blue = highest correlation, red = lowest correlation). The dendrogram from Figure 1 is shown in gray. [Figure 3A] This figure shows that the total mitochondrial:nuclear read ratio increases as the HC distance from the control increases (9-month-old mice). The results are consistent with the concept that mitochondrial dysfunction is an early driver of RGC damage in glaucoma. [Figure 3B] This figure shows several top pathways that were significantly enriched based on Ingenuity Pathway Analysis (IPA), using RNA-seq data obtained from RGCs of pre-disease D2 mice (9-month-old D2 mice, in three clusters: groups 2, 3, and 4) and wild-type controls (D2-Gpnmb+) of the same age and sex. A larger -logp value indicates greater enrichment. Two of the top three enriched pathways are mitochondrial dysfunction and oxidative phosphorylation. See Table 3 for additional significantly enriched pathways. Note that there are no differentially expressed pathways in D2 group 1. [Figure 3C]This figure shows that transcript expression increases, primarily for nuclear-encoded mitochondrial proteins, with increasing HC distance from the control to the D2 group (all 9-month-old mice). The results are again consistent with the concept that mitochondrial dysfunction is an early driver of RGC damage in glaucoma. Dots represent individual genes: gray or lighter dots = not differentially expressed, red or darker dots = differentially expressed with q < 0.05. Genes were obtained from mouse MitoCarta2.0 (Calvo et al., Nucleic Acids Res 44, pp. D1251-1257, 2016). [Figure 3D] This figure shows that RNA sequencing identifies mitochondrial fission gene transcripts that are increased in the early stages of glaucoma (9-month-old mice). The results are consistent with the concept that mitochondrial dysfunction is an early driver of RGC damage in glaucoma. [Figure 3E] This figure shows the early mitochondrial endoplasmic reticulum stress response compared to a control. The data shown are for group D2, subgroup 4 (9-month-old mice). The results are consistent with the concept that mitochondrial dysfunction is an early driver of RGC damage in glaucoma. [Figure 3F] This figure shows the results of individual gene expression plots illustrating metabolic and oxidative events in early glaucoma (9-month-old mice). The dots represent individual genes; gray or lighter dots = not differentially expressed, red or darker dots = differentially expressed (q < 0.05). Oxidative phosphorylation genes are among those showing the highest levels of differential expression, and differential expression appears to increase progressively in the D2 group as the HC distance from the control increases (group 4 > groups 3 and 2). [Figure 3G] This bar graph summarizes the Western blot protein validation data (Figure 3I) for differentially expressed genes (n=4 / age group). In cytochrome c, a general increase in protein was observed, correlated with the transcript abundance assessed by RNA sequencing. In the bar graph, the vertical axis represents the relative density to β-actin. *=P<0.05, **=P<0.01. [Figure 3H]This bar graph summarizes the Western blot protein validation data (Figure 3J) for differentially expressed genes (n=4 / age group). In eIF2α, there was an overall increase in protein that correlated with the transcript abundance evaluated by RNA sequencing. In the bar graph, the vertical axis represents the relative density to β-actin. *=P<0.05, **=P<0.01. [Figure 3I] Western blot protein validation data for genes expressed differentially (n=4 / month age group). [Figure 3J] Western blot protein validation data for genes expressed differentially (n=4 / month age group). [Figure 3K] This figure shows that oxidative phosphorylation genes are expressed differentially across all groups, with increasingly higher expression in the D2 group where the HC distance from the control increases (group 4 > group 3 > group 2 > group 1). Red = highest expression, blue = lowest expression, I~V = mitochondrial complexes I~V (as shown in Table 1), G = D2-Gpnmb+, 1~4 = D2 group 1~4. [Figure 4] Figures 4A and 4B show that 9-month-old D2 mice exhibited reduced cristal volume in RGC neuronal cell bodies and dendritic mitochondria. However, there was no significant difference in total mitochondrial size / volume. Scale bar = 350 nm. ** = P < 0.01, * = P < 0.001. [Figure 5A] This figure shows that NAD(t) levels increased in D2 mice (n=22 / group) treated with NAMLo (550 mg / kg / day). *=P<0.05, ***=P<0.001. [Figure 5B] This figure shows that GSH / GSSG levels decrease with age (n=22 / group). *=P<0.05, ***=P<0.001. [Figure 5C] This figure shows that NAD(t) levels also decreased with age in D2-Gpnmb+ control mice (n=22 / group). ***=P<0.001. [Figure 5D]This figure shows the age-related decrease in pyruvate levels that is restored by pyruvate treatment. D2 mice were treated with 500 mg / kg / day of pyruvate in normal drinking water from 6 months of age. [Figure 5E] This figure shows the age-related decline in total NAD (NAD[t]) (i.e., NAD++NADH) in the D2 retina, which is restored by NAM treatment (550 mg / kg / day) or the addition of the WldS allele. The combination of WldS and NAM yields further benefits. [Figure 6] Figures 6A and 6B show that NAM treatment (550 mg / kg / day) reduces the upregulation of HIF-1α in early glaucoma, as evaluated by immunohistochemistry (n=6 / group). *=P<0.05, ***=P<0.001. The results demonstrate that NAM treatment prevents early damage in D2 glaucoma. [Figure 7A] This figure shows that RNA sequencing identifies changes in cellular metabolism, particularly changes in fatty acid metabolism genes in D2 glaucoma. The dots represent individual genes: gray or lighter dots = not differentially expressed, red or darker dots = differentially expressed (q < 0.05). [Figure 7B] This figure shows extracellular lipid droplet formation in the inner retinal layer of aged D2 eyes invasive to IOP, as stained with Oil Red O. Staining was present in both D2 eyes with no optic nerve degeneration (NOE) and severe optic nerve degeneration (SEV). Extraocular fat was used as a positive control (n=6 / group). Scale bar = 25 μm. [Figure 8] Figures 8A and 8B show the increase in the level of DNA damage as evaluated by γH2AX staining (n=6 / group). The results are represented as a bar graph of the maximum γH2AX staining intensity (AU). ***=P<0.001. [Figure 9]Figures 9A and 9B show that NAM prevents PARP activation in glaucoma. PARP is a major consumer of NAD+ and may contribute to the age-related decline in NAD(t) in RGCs. After administration of NAM (550 mg / kg / day), PARP expression (total PARP immunostaining) decreased in NAM-treated retinas (n=6 / group). ***=P<0.001. Scale bar=15μm. [Figure 10A] Figure 10A (IOP profile) shows that protective strategies do not alter the progression / presentation of clinical disease in treated eyes. Iris disease progressed at a similar rate and reached a severe stage in all groups within the same timeframe. NAMLo = 550 mg / kg / day. NAMHi = 2000 mg / kg / day. Nmnat1 = gene therapy by expressing exogenous Nmnat1 (via a viral vector). [Figure 10B] Figure 10B (clinical findings of IOP-elevated iris disease) shows that protective strategies do not alter the progression / presentation of clinical disease in treated eyes. Iris disease progressed at a similar rate and reached a severe stage in all groups within the same timeframe. NAMLo = 550 mg / kg / day. NAMHi = 2000 mg / kg / day. Nmnat1 = gene therapy by expressing exogenous Nmnat1 (via a viral vector). [Figure 10C] Figure 10C (IOP profile) shows that protective strategies do not alter the progression / presentation of clinical disease in treated eyes. Iris disease progressed at a similar rate and reached a severe stage in all groups within the same timeframe. NAMLo = 550 mg / kg / day. NAMHi = 2000 mg / kg / day. Nmnat1 = gene therapy by expressing exogenous Nmnat1 (via a viral vector). [Figure 10D]Figure 10D (clinical findings of IOP-elevated iris disease) shows that protective strategies do not alter the progression / presentation of clinical disease in treated eyes. Iris disease progressed at a similar rate and reached a severe stage in all groups within the same timeframe. NAMLo = 550 mg / kg / day. NAMHi = 2000 mg / kg / day. Nmnat1 = gene therapy by expressing exogenous Nmnat1 (via a viral vector). [Figure 11A] This diagram shows that NAM protects against optic nerve degeneration. Green or bottom of the bar = no glaucoma or early glaucoma (NOE) (stage without nerve damage), yellow or middle of the bar = moderate damage (MOD), red or top of the bar = severe damage (SEV). Fisher's exact test: ** = P < 0.01, *** = P < 0.001. NAMLo = 550 mg / kg / day. NAMHi = 2000 mg / kg / day. Early initiation = treatment started at 6 months of age (before IOP elevation in almost all eyes). Late initiation - treatment started at 9 months of age (after onset of IOP elevation; at this point, the majority of eyes have or have had IOP elevation). [Figure 11B]This figure shows that nicotinamide provides protection against optic nerve degeneration in D2 glaucoma at 12 months of age. The graph shows the percentage of nerves with no detectable glaucoma (NOE; bottom of bar), moderate glaucoma damage (MOD; middle of bar), or severe glaucoma damage (SEV; top of bar). From left to right: DBA / 2J control - D2 mice fed standard drinking water; D2 mice fed standard drinking water supplemented with nicotinamide (NAMLo) 550 mg / kg / day from 6 months of age (pre-disease) starting early; D2 mice fed standard drinking water supplemented with nicotinamide (NAMLo) 550 mg / kg / day from 9 months of age (during disease) starting late; D2 mice fed standard drinking water supplemented with nicotinamide (NAMHi) 2000 mg / kg / day from 6 months of age (pre-disease) starting early; D2 mice fed standard drinking water supplemented with pyruvate 500 mg / kg / day from 6 months of age (before IOP increase); NAMLo + pyruvate - NAM from 6 months of age (pre-disease) starting early D2 mice fed standard drinking water supplemented with 550 mg / kg / day + pyruvate 500 mg / kg / day; D2 mice possessing the WldS transgene (modified NMNAT enzyme with enhanced enzyme activity) fed standard drinking water containing D2.WldS + nicotinamide (NAMLo) - D2 mice possessing the WldS transgene (modified NMNAT enzyme) fed standard drinking water containing NAM 550 mg / kg / day from 6 months of age (before the increase in IOP). In addition to intervening in the treatment of neurodegeneration in glaucoma, it should be noted that NAM also provides prophylactic protection against neurodegeneration in glaucoma. [Figure 12A] This figure shows that NAM and pyruvate protect against RGC cell body loss (n=8 / group), retinal NFL and IPL thinning (n=8 / group), optic nerve degeneration (n>50 / group), and loss of anterograde axonal protoplasmic transport (n=20 / group). Corresponding markers and color coding are below each column. Scale bars: RBPMS=20μm, Nissl=20μm, PPD=20μm, CT-β=100μm (retina), 200μm (LGN, Sup.Col.). ONH=optic nerve head, LGN=lateral geniculate nucleus, Sup.Col.=superior colliculus. White asterisks indicate loss of axonal transport in the ONH region. [Figure 12B] This figure shows the recovery of axonal transport in NAM and pyruvate-treated D2 mice. Loss of axonal transport is a prominent feature of glaucoma and can be used as a criterion for measuring neuronal health. Axonal transport from the ocular portion of retinal ganglion cells to the terminal end of the cells (brain) can be visualized using fluorescently labeled cholera toxin (Ct-B; green). Top row: D2.Gpnmb(wt) mice have normal, complete axonal transport from the retina to the visual center in the brain (LGN; lateral geniculate nucleus, Sup.col.; superior colliculus). Second row: D2 mice have incomplete axonal transport that terminates in the optic nerve (white asterisk). Labeling is absent in the visual center of the brain. Third and fourth rows: NAM treatment (third row) or pyruvate treatment (fourth row) prevents loss of axonal transport. [Figure 13A] This figure shows that NAM protects against RGC neuronal cell body loss (n=8 / group, the density decrease between D2 and D2-Gpnmb+ is due to pressure inducing stretching). ***=P<0.001. [Figure 13B] This figure shows that NAMLo (550 mg / kg / day) and pyruvate (500 mg / kg / day) prevent RGC neuronal cell body loss (using RGC-specific marker; RBMPS). [Figure 14] This figure shows that NAM (NAMLo and NAMHi), pyruvate (500 mg / kg / day), WldS, and combinations thereof all protect D2 mice from early visual function loss based on visual function tests (n>20 / group) using PERG (pattern electroretinography) amplitude. PERG is highly sensitive and is an early assessment criterion for glaucoma; therefore, NAM or pyruvate treatment prevents even the very early stages of glaucoma. Both the combination of NAMLo + pyruvate and the combination of NAMLo + WldS show further recovery of PERG amplitude. NAMLo = 550 mg / kg / day. NAMHi = 2000 mg / kg / day. [Figure 15]This figure shows that NAMLo (550 mg / kg / day) and pyruvate (500 mg / kg / day) protect PERG even in aged mice. Example figures from 6-month and 12-month D2 and 12-month NAMLo-treated mice (550 mg / kg / day) are shown. Results from 12-month pyruvate-treated mice and NAMLo-treated WldS mice are also shown. [Figure 16] Figures 16A and 16B show that NAMLo- treatment (550 mg / kg / day) prevents synaptic loss in early glaucoma, as evaluated by SNAP-25 staining (n=6 / group). **=P<0.01, ***=P<0.001. Scale bar = 25 μm. [Figure 17A] Figures 17A and 17B show that NAM (NAMLo = 550 mg / kg / day) prevents early mitochondrial dysfunction in dendritic mitochondria, as shown in Figures 4A and 4B. These data are consistent with early changes in PERG and previously reported synaptic loss in 9-month D2 retina. Therefore, mitochondrial dysfunction induced by elevated IOP may drive early neurodegenerative changes. ** = P < 0.01, *** = P < 0.001. ns = not statistically significant. Scale bar = 350 nm. [Figure 17B] Figures 17A and 17B show that NAM (NAMLo = 550 mg / kg / day) prevents early mitochondrial dysfunction in dendritic mitochondria, as shown in Figures 4A and 4B. These data are consistent with early changes in PERG and previously reported synaptic loss in 9-month D2 retina. Therefore, mitochondrial dysfunction induced by elevated IOP may drive early neurodegenerative changes. ** = P < 0.01, *** = P < 0.001. ns = not statistically significant. Scale bar = 350 nm. [Figure 18] This figure shows that NAM treatment (550 mg / kg / day) prevents lipid droplet formation in the inner retina (for 12 months; as shown in Figure 7B). Scale bar = 25 μm. [Figure 19]Figures 19A and 19B show that NAM treatment (NAMLo = 550 mg / kg / day) prevents DNA damage in early glaucoma, as evaluated by γH2AX staining (n=6 / group). ***=P<0.001. Scale bar = 25 μm. [Figure 20A] This figure shows individual gene expression plots illustrating metabolic and DNA damage pathways, indicating their return to normal in NAM-treated RGCS. The dots represent individual genes; gray or lighter dots indicate no differential expression compared to the D2-Gpnmb+ control, while red or darker dots indicate differential expression with q < 0.05. [Figure 20B] This is a heatmap of gene expression (all expressed genes) showing that NAM-treated RCGs are molecularly similar to the control group. [Figure 21A] This figure shows that NAM-treated RCG is molecularly similar to non-glaucoma control RCG in both young and same-age individuals (Spearman's ρ). Circles = D2 RGC-derived samples, triangles = D2-Gpnmb+RGC-derived samples, squares = NAM-treated (NAMLo=550 mg / kg / day) RGC-derived samples. Therefore, NAM treatment prevents disease and age-related molecular changes. [Figure 21B] This figure summarizes the number of genes that are differentially expressed in the D2 groups (groups 1, 2, 3, and 4) compared to the control D2-Gpnmb+ group. [Figure 21C] Figures 21C and 21F show that NAM treatment (NAMLo = 550 mg / kg / day) prevents transcriptome imbalance and OXPHOS imbalance (mitochondrial:nuclear library size ratio) observed in 9-month D2 RGCs. In Figure 21F, red = highest expression, blue = lowest expression. I-V = mitochondrial complexes I-V (as shown in Table 1), G = D2-Gpnmb+, 1-4 = D2 group 1-4, N = NAM. [Figure 21D]D2 mice were treated with 550 mg / kg / day of nicotinamide in normal drinking water from 6 months of age (aged NAM). RNA-seq of retinal ganglion cells from NAM-treated mice showed that NAM prevents age- and disease-related gene expression changes. NAM-treated retinal ganglion cells were most similar to younger controls (younger controls) than to controls of the same age (aged controls). This indicates that NAM prevents age-dependent molecular changes. [Figure 21E] This figure shows the heatmap correlation of all samples (Spearman's ρ, blue = highest correlation, red = lowest correlation). The dendrogram in Figure 21A is shown in gray. [Figure 21F] Figures 21C and 21F show that NAM treatment (NAMLo = 550 mg / kg / day) prevents transcriptome imbalance and OXPHOS imbalance (mitochondrial:nuclear library size ratio) observed in 9-month D2 RGCs. In Figure 21F, red = highest expression, blue = lowest expression. I-V = mitochondrial complexes I-V (as shown in Table 1), G = D2-Gpnmb+, 1-4 = D2 group 1-4, N = NAM. [Figure 22A] Figure 22A shows that NAM is protective against neurodegenerative treatments modeling the invasiveness of glaucoma. NAM showed an attractive dose-response effect protecting nerve cell layer cells (GCL cells) from death (Figure 22A) and preapoptotic nuclear contraction (Figure 22B). In axonal transection culture model of retinal ganglion cell injury, the addition of nicotinamide (NAM) resulted in protection against dose-dependent cell contraction (signs of cell apoptosis and dysfunction) (5 days after axonal transection). The mean nuclear diameter of DAPI-labeled nuclei in D2 mouse retinas exposed to different concentrations of NAM for 5 days was measured (including day 0 control [normal retina], untreated [5 days after axonal transection], and 100 mM and 500 mM NAM [5 days after axonal transection]). NAM-treated retinas were indistinguishable from intact, baseline controls (day 0). Significant protective effects were also observed when treating the retina with β-NAD and β-NMN (n=8 retinas / group). ***=P<0.001. Scale bar=20μm. [Figure 22B]Figure 22B shows that NAM is protective against neurodegenerative treatments modeling the invasiveness of glaucoma. NAM showed an attractive dose-response effect protecting nerve cell layer cells (GCL cells) from death (Figure 22A) and preapoptotic nuclear contraction (Figure 22B). In axonal transection culture model of retinal ganglion cell injury, the addition of nicotinamide (NAM) resulted in protection against dose-dependent cell contraction (signs of cell apoptosis and dysfunction) (5 days after axonal transection). The mean nuclear diameter of DAPI-labeled nuclei in D2 mouse retinas exposed to different concentrations of NAM for 5 days was measured (including day 0 control [normal retina], untreated [5 days after axonal transection], and 100 mM and 500 mM NAM [5 days after axonal transection]). NAM-treated retinas were indistinguishable from intact, baseline controls (day 0). Significant protective effects were also observed when treating the retina with β-NAD and β-NMN (n=8 retinas / group). ***=P<0.001. Scale bar=20μm. [Figure 23] Figures 23A-23C show that NAM (NAMLo = 550 mg / kg / day) prevents PERG (Figure 23A) and neuronal cell body loss (Figures 23B and 23C) in TNFα-injected eyes at 12 weeks after TNFα administration (n=20 / group). *=P<0.05. ns=not statistically significant. Scale bar=20μm. [Figure 24-1] Figures 24A and 24B illustrate that gene therapy provides strong protection against neurodegeneration in glaucoma. Eye D2 was intravitreously injected at 5.5 months with an AAV2.2 viral vector containing a plasmid for overexpression of mouse Nmnat1 under the CMV promoter. Figure 24A shows that Nmnat1 overexpression prevents RGC cell body loss and loss of anterograde axonal plasmotransport, as demonstrated in Figure 12A (n=10 / group). Scale bars are 50 μm in Figure 24A and 100 μm in Figure 24B. [Figure 24-2]Figures 24C and 24D show that Nmnat1 gene therapy also protects D2 eyes with elevated IOP against optic nerve degeneration (n>40 / group) (Figure 24C), neuronal cell body loss (n=8 / group) (Figure 24D, upper panel), and PERG amplitude (n>20 / group) (Figure 24D, lower panel). Addition of NAM to drinking water (NAMLo=550 mg / kg / day) provided further protection against optic nerve degeneration (Nmnat1 compared to Nmnat1+NAMLo = P<0.001, Fisher's exact test) (Figure 24C). **=P<0.01, ***=P<0.001. [Figure 25] This diagram shows the NAD regeneration paths using NAM and NMNAT1 / WLDS. [Figure 26] Figures 26A and 26B show that in axonally severed retinas treated with PQQ, PQQ administration prevents nuclear diameter reduction (Figure 26A) and decrease in cell density (Figure 26B). [Figure 27] Figures 27A–27H show FACS-sorted RGCs. Retinal samples were stained with an antibody cocktail (see Materials and Methods). Figure 27A shows gated forward and side scattering (Figures 27B and 27C) to identify live cells (Figure 27D). RGCs were identified as gated Thy1.2+ events (Cd11b-, Cd11c-, Cd31-, Cd34-, Cd45.2-, GFAP-, DAPI-). Only Cd11b, Cd45 and Thy1.2 plots are shown (Figures 27E–27G). In Figure 27H, FACS-positive RGCs were cultured on plates and stained with SNAP-25 and β-tubulin to confirm the RGC status. Scale bar = 25 μm. [Modes for carrying out the invention]

[0042] definition The terms used in this application shall have the following meanings: In this specification, the term "glaucoma" refers to an eye disease that causes damage to the retina and optic nerve, resulting in visual impairment or loss of vision. Glaucoma is common and widespread among older adults. Vision loss due to glaucoma is permanent and irreversible.

[0043] In this specification, the term “subject / patient requiring treatment” is generally understood to mean a person suffering from or simply feeling the need for treatment for a specific neurodegenerative disease or condition, particularly ophthalmic neurodegeneration including glaucoma.

[0044] In this specification, the term "glaucoma" includes primary open-angle glaucoma, secondary open-angle glaucoma, normal-tension glaucoma, hypersecretion glaucoma, primary closed-angle glaucoma, secondary closed-angle glaucoma, plateau iris glaucoma, pigmentary glaucoma, mixed glaucoma, developmental glaucoma, steroid glaucoma, exfoliation glaucoma, amyloid glaucoma, neovascular glaucoma, malignant glaucoma, capsule glaucoma, plateau iris syndrome, etc.

[0045] In this specification, the term "normal intraocular pressure" ("normal IOP") in humans refers to human subjects with an IOP value of 10 mmHg to 21 mmHg. However, some patients may develop optic nerve damage despite having a normal IOP (known as normal-tension glaucoma).

[0046] In this specification, the term "high intraocular pressure" (high IOP) in humans refers to a human subject with an IOP value exceeding 21 mmHg (or 2.8 kPa). High IOP is a known risk factor for glaucoma. However, some patients may have high IOP for many years without developing any optic nerve damage.

[0047] In this specification, the terms “neuroprotective” or “neuroprotection” refer to the ability to protect neurons, their synapses, dendrites, cell bodies, or axons in the ophthalmic nerve (e.g., the optic nerve), central or peripheral nervous system from damage (including functional failure) or death, or to delay the onset of neuronal damage or death, or to reduce the severity / extent of neuronal damage / death within a population of neurons.

[0048] In this specification, the terms “prevent” or “prevention” mean, for example, the ability of the compounds or agents of the present invention to provide neuroprotection before such damage, death, or disease occurs, with respect to neuronal damage or death in general or particularly ophthalmic neurodegenerative diseases (e.g., glaucoma). Accordingly, prevention of glaucoma includes avoiding the onset of glaucoma, reducing the risk or likelihood of eventually developing glaucoma, delaying the onset or progression of glaucoma, or reducing the extent of neuronal damage / neuronal death / loss in a population of neurons that would eventually develop glaucoma.

[0049] In this specification, the terms “to treat” or “treatment” include administering the compound or agent of the present invention to a subject to reduce, suppress, or inhibit the onset of symptoms or conditions associated with neurodegeneration, such as glaucoma.

[0050] In this specification, “improves visual acuity or visual function” means the effect of the compound or agent of the present invention on improving visual acuity or visual function (such as visual field testing or RGC pattern electroretinogram [PERG] amplitude) in a subject administered with such compound or agent compared to a control subject that has not been administered with such compound or agent.

[0051] In this specification, the term “therapeutic dose” means the amount administered to a subject that produces the effect it is intended to produce. For example, when administered to a subject to inhibit neurodegeneration, the “therapeutic dose” is the amount that reduces IOP elevation and / or improves RGC function (e.g., prevents deterioration of RGC function).

[0052] In this specification, the terms “subject” or “patient” are used interchangeably and refer to mammals such as rodents, dogs, and humans. Accordingly, in this specification, the terms “subject” or “patient” mean any mammalian patient or subject (e.g., human) to whom the compounds of the present invention can be administered.

[0053] In this specification, the terms "pharmaceutical" or "pharmaceutical composition" refer to pharmaceutical preparations used to treat, cure, or improve a disease, or to treat, improve, or alleviate the symptoms of a disease.

[0054] In this specification, the term “expression vector” refers to a nucleic acid molecule capable of performing the expression of a gene / nucleic acid molecule contained in a cell that is compatible with such a sequence. Expression vectors generally include at least a suitable promoter sequence and, optionally, a transcription termination signal.

[0055] The following abbreviations are used: NAM-Nicotinamide NAMPT-nicotinamide phosphoribosyltransferase NMN or βNMN-nicotinamide mononucleotide NMNAT-nicotinamide mononucleotide adenylyltransferase NAD - Nicotinamide adenine dinucleotide NaMN-nicotinic acid mononucleotide NR-Nicotinamide Ribose Glycoside overview This invention is primarily based on the use of state-of-the-art molecular technology (RNA sequencing; RNA-seq) on retinal ganglion cells (RGCs), which led to the discovery that mitochondrial abnormalities occurring before detectable neurodegeneration are the initial drivers of neuronal dysfunction. Using the DBA / 2J(D2) mouse model (chronic senile, hereditary glaucoma), the inventors further elucidated the relationship between aging, a key risk factor for most glaucomas, and high IOP in driving neuronal degeneration in RGCs.

[0056] The inventors have found that therapeutic levels of nicotinamide adenine dinucleotide (NAD) can be delivered to the retina. + ) delivers NAD to lower levels + A method for replenishing the specifics of the present invention was developed. The administration of pharmaceutical compositions (e.g., NAM and / or pyruvate) represents a novel approach in the treatment of ophthalmic neurodegeneration and glaucoma. This therapeutic method may be useful in treating other neurodegenerative conditions, including the reduction of areas where aging neurons are susceptible to disease-related trauma.

[0057] In one embodiment, the present invention provides therapeutic use of targeted pharmaceutical compositions for treating or preventing age-dependent neurodegeneration, for example, for treating or preventing glaucoma. In certain embodiments, the pharmaceutical composition is effective in treating or preventing glaucoma, preferably primary open-angle glaucoma, normal-tension glaucoma, and primary closed-angle glaucoma. In certain embodiments, the pharmaceutical formulation of the present invention is particularly effective in treating or preventing primary open-angle glaucoma.

[0058] In certain embodiments, the method includes the step of administering a therapeutically effective dose of nicotinamide (NAM) to a subject requiring treatment / prevention of a neurodegenerative disorder (e.g., glaucoma). In certain embodiments, the subject is administered NAM in combination with pyruvate. NAM and / or pyruvate are NADt(NAD + It is thought to increase intracellular levels of (and total levels of NADH), and therefore treat or prevent neurodegenerative disorders in the subjects.

[0059] In one embodiment, the present invention provides a therapeutic use of gene delivery to increase intracellular nicotinamide adenine dinucleotide to prevent neurodegeneration and thereby treat glaucoma.

[0060] In certain embodiments, the present invention provides a method for gene therapy (e.g., driven expression of nicotinamide nucleotide adenylyltransferase 1 [Nmnat1]). Expression of the Nmnat1 protein has been found to be highly protective and to act synergistically with NAM (i.e., 84% of eyes are free from glaucoma neurodegeneration).

[0061] The advantage of this invention is to reduce the risk factors for developing glaucoma. This study clearly demonstrates that NAM alone or in combination with other drugs (such as pyruvate) reduces the risk factors for glaucoma. In 12-month-old control D2 mice, approximately 60% of the nerves had severe glaucoma. This corresponds to a risk factor of 0.6 for developing glaucoma. After administration of low-dose NAM (550 mg / kg / day to mice), this risk factor decreased to 0.36 (a reduction of approximately half the risk factor), or to 0.06 (a reduction of one-tenth the risk factor) when high-dose NAM (2000 mg / kg / day to mice) was administered.

[0062] The advantage of the present invention lies in the synergistic effect of administering NAM and gene therapy (e.g., NMNAT1 gene therapy). Compared to gene therapy alone, which reduces the risk factor to 0.29 (a reduction of approximately half in the risk factor), gene therapy combined with NAM reduces the risk factor to 0.16 (a reduction of approximately one-quarter in the risk factor). Therefore, the combination of NAM and gene therapy synergistically reduces the risk factors for developing glaucoma after an increase in IOP.

[0063] This discovery of a protective effect for NAM (alone or in combination with other drugs such as pyruvate) is unexpected. This finding is in stark contrast to that reported in the '326 patent, which states that NAM has no protective effect.

[0064] The basis for differing findings can be found for at least the following reasons. The first reason is that the '326 patent holder used experimental systems that have little relevance to in vivo effects, particularly to eye-related diseases such as glaucoma. In those experiments, the '326 patent holder isolated spinal dorsal root ganglion (DRG) neurons and induced nerve damage by severing their neuritis. The nerve damage induced by this artificial culture, in which the mechanical severance occurred very close to the DRG neuron cell body, bears little resemblance to neurodegeneration in vivo. Early axonal damage in glaucoma is recognized to occur at a greater distance from the nerve cell body than DRG neuritis. The second reason is that the '326 patent holder's culture system did not use retinal ganglion cells (RGCs), which are neurons highly associated with neurodegeneration in glaucoma. The third reason is that the '326 patent holder used an artificial cell system instead of complete nerve tissue (which is composed of various cell types that communicate and support each other).

[0065] Finally, the '326 patent holder refers to a complete in vivo optic nerve cross-section model in which the nerve (including its surrounding sheath) is mechanically severed. This causes rapid damage to the nerve and substantially alters the local environment in which the axon is located, and this alteration causes the death of over 90% of retinal ganglion cells within 14 days (see www.ncbi.nlm.nih.gov / pubmed / 21610673). Despite the invasiveness being in the correct location on the nerve, there have been no cases of glaucoma. Overall, the method employed by the '326 patent holder is a highly artificial and rapid way to destroy retinal ganglion cells that does not reproduce human glaucoma. Interestingly, in its optic nerve cross-section experiment, the '326 patent holder injected compounds (including NAM) intravitreously to drive NAD production in the eye, and the '326 patent holder explicitly concluded that NAM was not protective against axonal degeneration of RGCs in this model.

[0066] All of this is particularly relevant. Our culture experiments are more relevant to glaucoma because they used complete retinal tissue containing retinal ganglion cells that have a normal relationship with other retinal cell types, and the retinal ganglion cell axons were severed at the same locations where damage occurs in glaucoma. Damage in glaucoma is thought to occur in the true axons of RGC axons in the optic head, where they emerge from the eye and become the optic nerve.

[0067] In contrast, the data presented in this application clearly demonstrate the neuroprotective effects of NAM in glaucoma. The use of NAM alone or in combination with other drugs (e.g., pyruvate) yields neuroprotective effects in ophthalmic diseases such as glaucoma. In this application, the neuroprotective effect is demonstrated by at least one of the following parameters: (i) prevention of neuronal cell body loss (RBPMS staining); (ii) prevention of retinal thinning and nerve fiber layer loss (Nissl staining); (iii) prevention of optic nerve degeneration and axonal loss (PPD staining); (iv) prevention of loss of anterograde axonal protoplasmic transport (Ct-B staining); (v) prevention of loss of visual function (PERG); (vi) prevention of abnormal mitochondrial cristae (EM); (vii) reduction of lipid droplets (Oil Red O staining); and (viii) reduction of PARP activation (PARP staining).

[0068] The inventors have further discovered that the use of NAM, either alone or in combination with other agents (e.g., pyruvate), affects intracellular events and provides neuroprotective effects, as evidenced by: (i) reduced HIF-1α activation (HIF-1α staining); (ii) reduced synaptic loss (SNAP-25 staining); (iii) restoration of nuclear transcript abundance relative to mitochondria (RNA-seq); and (iv) prevention of senile molecular / genetic changes (RNA-seq).

[0069] Another advantage of the present invention is the combined use of NAM and gene therapy in NAD delivery. Gene therapy approaches are considered to persist for at least 3 to 5 years, as proven by what numerous clinical studies have shown. Gene therapy provides a synergistic neuroprotective effect with NAM and / or pyruvate in reducing glaucoma and IOP.

[0070] Having generally described the invention above, the following sections provide a more detailed description for further aspects of the present invention. DBA / 2J mouse model The inventors chose to use this mouse strain because the DBA / 2J (D2) mouse model develops hereditary senile glaucoma that well mimics human glaucoma. D2 mice are one of the most studied glaucoma models with many established similarities to human glaucoma, including the induction of the same disease-mediating molecules (e.g., complement component molecules) that occur in human glaucoma, the same site of key glaucomatous attack at the optic nerve head, and the same topographical pattern of RGC death. D2 mice have iris disease and elevated intraocular pressure starting at about 6 to 8 months of age. By 9 months of age, elevated intraocular pressure was persistent in the eyes of most D2 mice. D2 mice subsequently develop progressive vision loss, optic nerve damage and inner retinal dysfunction. At 12 months of age, when designed experiments are completed as normal, approximately 70% of D2 mouse eyes have severe disease based on histological examination of the retina and optic nerve. Topical administration of compounds (e.g., memantine, timolol or latanoprost) reduces IOP and decreases the risk of developing neurodegeneration in D2 mice, just as it does in human glaucoma. Control D2-Gpnmb + are mice of the same age and strain that do not develop glaucoma.

[0071] Intraocular pressure in glaucoma Intraocular pressure (IOP) can be determined using, for example, the Goldmann applanation tonometry (Haag Streit, Bern, Switzerland). In humans, normal IOP is 12–21 mmHg. IOP above 21 mmHg is considered high. Elevated IOP is a major risk factor in glaucoma. In POAG (Primary Open-Angle Glaucoma, the most common type of glaucoma, accounting for over 90% of cases), 25 mmHg is the median baseline IOP without treatment.

[0072] The Baltimore Eye Study (www.ncbi.nlm.nih.gov / pub / 12049574) reported high risk = IOP > 25.75 mmHg; moderate risk = IOP 23.75–25.75 mmHg; and low risk = IOP < 23.75 mmHg. Reducing IOP by 20% to a level of 24 mmHg or less reduces the risk of progression from 9.5% to 4.4% at 5 years. The likelihood of blindness in one eye is 27% at 10 years after diagnosis and 38.1% at 20 years. The likelihood of blindness in both eyes is 6% and 13.5%, respectively.

[0073] In human glaucoma patients, such as those with primary open-angle glaucoma (POAG), glaucoma is asynchronous and age-related (usually occurring after age 40). With an IOP of 21–25 mmHg, untreated glaucoma takes an average of 14 years to progress from early to late stage. As the IOP increases, this rate of progression increases rapidly (approximately 3 years for progression from early to late stage with an IOP > 30 mmHg).

[0074] In human patients, procedures that reduce IOP (surgical or pharmacological) decrease the risk of developing neurodegeneration. The rate of disease progression and the percentage of patients likely to go blind are likely to be misrepresented due to the undiagnosed nature of the disease. High IOP is generally not considered to indicate glaucoma (approximately 30% blindness after 10 years, and approximately 40% blindness after 20 years). Reducing IOP does not treat glaucoma, but it reduces the risk factor by up to 58%. Despite conventional IOP reduction preventive measures, the risk of vision loss and even blindness still exists. Neuroprotective strategies available for glaucoma are limited. Vision loss in glaucoma is irreversible. In fact, glaucoma is the leading cause of irreversible blindness worldwide.

[0075] Retinal ganglion cells (RGCs) Retinal ganglion cells (RGCs) are output neurons of the retina. They receive visual information from photoreceptors (i.e., rod and cone photoreceptor cells) via interneurons (i.e., bipolar and amacrine cells). This visual information begins as photons in light and becomes an electrical potential at the retinal ganglion cell synapses. RGCs have long axons that leave behind cell bodies and cross the retina to the optic disc (i.e., the blind spot) where they exit the eye (optic head). Beyond the optic head (myelin transition zone), the retinal ganglion cell axons become myelinated and form the optic nerve (i.e., the optic nerve is a bundle of retinal ganglion cell axons, which in mice is approximately 50,000 depending on the strain). The axons in the optic nerve eventually reach the terminal visual centers in the brain, which then relay these signals or process this information itself. The two important visual centers where retinal ganglion cell axons terminate are the lateral geniculate nucleus (LGN) and the superior colliculus (sup.col. / SC).

[0076] Retinal ganglion cells are particularly affected in glaucoma (i.e., cell loss). Damage to the RGC is most likely to occur in the axons of the optic nerve head. Due to the stress induced in the eye by abnormally high IOP, the optic nerve head is presumed to be a "weak point" of the eye where mechanical invasion of retinal ganglion cell axons can occur. Since the pressure throughout the eye should similarly affect the cell bodies and dendrites, the axons are not the only point of invasion in retinal ganglion cells. The exact pathogenesis of RGC damage is not at all clear.

[0077] In DBA / 2J(D2) mice, the inventors demonstrated that at high IOP levels, there was no detectable axonal loss in the optic nerve (i.e., no ophthalmic nerve degeneration). Surprisingly, the inventors found the presence of early mitochondrial and molecular changes, dendritic atrophy, and synaptic loss. This finding suggests that the effects of IOP manifest not only in axons in the optic nerve but also in other compartments of retinal ganglion cells.

[0078] Neurodegenerative treatment In certain embodiments, the drug is nicotinamide adenine dinucleotide (NAD + This includes precursors (e.g., nicotinic acid, nicotinamide [NAM], nicotinamide mononucleotide [NMN], nicotinamide ribose glycoside [NR], or combinations thereof), Krebs cycle intermediates or their precursors (e.g., pyruvate), or combinations thereof.

[0079] In certain embodiments, the drug is nicotinamide adenine dinucleotide (NAD + ) Precursors, for example, nicotinic acid, nicotinamide (NAM), nicotinamide mononucleotide, nicotinamide ribose glycoside, or combinations thereof. In certain embodiments, NAD + The precursor is nicotinamide or nicotinamide-ribose glycoside.

[0080] Nicotinic acid (also known as niacin, nicotinate, vitamin B3, and vitamin PP) is a vitamin. Its corresponding amide is called nicotinamide or niacinamide. These vitamins are not directly interconvertible; however, both nicotinate and nicotinamide are redox counterparts of NAD. + / NADH (nicotinamide adenine dinucleotide) and NADP + It is a precursor in the synthesis of NADPH (nicotinamide adenine dinucleotide phosphate). The nicotinate and nicotinamide metabolic pathway is, here, NAD + This can be called a synthesis pathway.

[0081] NAD + It is synthesized through two metabolic pathways: the salvage pathway and the nascent pathway. In the salvage pathway, NAD + It can be synthesized from an external source of precursor compounds (e.g., nicotinic acid, nicotinamide, nicotinamide-ribose glycosides, etc.). NAD + It can be synthesized from quinolinic acids (e.g., tryptophan, aspartic acid, etc.) produced during amino acid metabolism in the nascent pathway.

[0082] NAD of the present invention + The precursors include nicotinic acid, nicotinamide, nicotinamide-ribose glycosides, and their salts and analogs. In certain embodiments, the NAD of the present invention + Administration of the precursor causes an increase in intracellular levels of NADt.

[0083] In certain embodiments, the agent comprises a Krebs cycle intermediate or precursor thereof, or a combination thereof. For example, the Krebs cycle intermediate or precursor may be oxaloacetate, acetyl-CoA, citrate, CoA-SH, cis-aconitic acid, D-isocitric acid, or NAD. +These include oxalosuccinate, NADH, α-ketoglutaric acid, succinyl-CoA, GDP, ubiquinone, succinic acid, fumarate esters, L-malic acid, pyruvate, monosaccharides (glucose, galactose, fructose, etc.), disaccharides (sucrose, maltose, lactose, etc.), or combinations thereof.

[0084] Accordingly, the present invention provides pharmaceutical compositions comprising nicotinic acid and / or nicotinamide-ribose glycosides and / or nicotinamide and / or nicotinic acid metabolites. Nicotinic acid and / or nicotinamide-ribose glycosides and / or nicotinamide and / or nicotinic acid metabolites can be used in free form. In this specification, the term "free" with respect to an element indicates that the element is not incorporated into a larger molecular complex. In some embodiments, nicotinic acid may be contained in niacin. Nicotinic acid and / or nicotinamide-ribose glycosides and / or nicotinamide and / or nicotinic acid metabolites may be in salt form.

[0085] In some embodiments, any of the compositions described herein may be salts, derivatives, metabolites, catabolic products, anabolic products, precursors, and analogs thereof. For example, metabolites may include nicotinyl-CoA, nicotinuric acid, nicotinate mononucleotide, nicotinate adenine dinucleotide, or nicotinamide adenine dinucleotide. In some embodiments, the composition contains nicotinamide. In some embodiments, the composition may be substantially free of nicotinic acid metabolites.

[0086] In certain embodiments, the salt is selected from the group consisting of fluoride, chloride, bromide, iodide, formic acid, acetic acid, ascorbic acid, benzoate, carbonic acid, citric acid, carbamic acid, formic acid, gluconic acid, lactic acid, methyl bromide, methyl sulfate, nitric acid, phosphoric acid, diphosphate, succinic acid, sulfuric acid, and trifluoroacetate salts.

[0087] NAM / nicotinic acid analogs may also be used in the invention of the subject matter. Suitable analogs of nicotinic acid include, for example, isonicotinamide and N-methylnicotinamide. In certain embodiments, keto, ethyl, benzyl, or other non-salt embodiments of NAM may also be used in the present invention.

[0088] NAM / nicotinamide / NAD / NR / pyruvic acid has low toxicity and can be administered in relatively large doses without toxic effects. In mice, this compound (e.g., NAM / nicotinamide / NAD / NR / pyruvic acid) can be administered at a daily dose of approximately 200-1000 mg / kg / day to produce neuroprotective effects. Preferably, the daily dose is approximately 400-600 mg / kg / day. More preferably, the daily dose is approximately 550 mg / kg / day. At these doses, the compound provides neuroprotective effects.

[0089] In mice, administering this compound (e.g., NAM / nicotinamide / NAD / NR / pyruvic acid) at a dose of approximately 1000-5000 mg / kg / day can produce an IOP-reducing effect. Preferably, the daily dose is approximately 1500-3000 mg / kg / day. More preferably, the daily dose is approximately 2000 mg / kg / day. At these doses, this compound produces an IOP-reducing effect.

[0090] Regarding PQQ, it is tolerated in mice at a dose of at least 2000 mg / kg / day. The neuroprotective effects obtained by PQQ in mice range from approximately 20 to 2000 mg / kg / day, approximately 100 to 1000 mg / kg / day, or approximately 500 mg / kg / day.

[0091] To convert the dosage of a pharmaceutical composition expressed in mg / kg units in one species (e.g., mouse) to an equivalent surface area dose expressed in mg / kg in another species (e.g., human), the following table is used by Freireich et al., Quantitative comparison of toxicity of anticancer agents in Based on the assumptions and constants in *mouse, rat, dog, monkey and man*, Cancer Chemother Rep. 50(4): pp. 219-244, 1966 (incorporated herein by reference), approximate coefficients for the conversion are provided.

[0092] [Table 1]

[0093] Therefore, a dose of 550 mg / kg in mice is equivalent to 550 mg / kg × 1 / 4 = 137.5 mg / kg in monkeys, and 550 mg / kg × 1 / 12 = 45.8 mg / kg (or 2.85 g) in a 60 kg human (mg / m 2 (We assume equivalence based on this.)

[0094] The somewhat simplified conversion factors in the table above are based on the weight-to-surface-area ratio [km] for each species. For more accurate conversions, dosage conversions can also be based on the km coefficients for each species listed below.

[0095] [Table 2]

[0096] Therefore, a dose of 550 mg / kg in mice is equivalent to 550 mg / kg × 3.0 / 37 = 44.6 mg / kg (or 2.68 g) in a 60 kg human adult, and 550 mg / kg × 3.0 / 25 = 66 mg / kg (or 1.32 g) in a 20 kg human child.

[0097] Using the table above, the mg / kg dose can also be expressed as an equivalent mg / m² dose in any given species by multiplying the dose by an appropriate km factor. For example, in a human adult, 100 mg / kg is equivalent to 100 mg / kg × 37 kg / m² = 3700 mg / m².

[0098] In humans (patients weighing approximately 60 kg), neuroprotective effects can be obtained by administering this compound (e.g., NAM / nicotinamide / NAD / NR / pyruvic acid) at a daily dose of 0.5 to 10 g. Preferably, the daily dose is about 1 to 5 g / day. Preferably, the daily dose is about 2 to 4 g / day. More preferably, the daily dose is about 2.5 g / day. At these doses, this compound provides neuroprotective effects.

[0099] In humans (patients weighing approximately 60 kg), administering this compound (e.g., NAM / nicotinamide / NAD / NR / pyruvic acid) at a daily dose of approximately 5 to 25 g / day can achieve an IOP reduction effect. Preferably, the daily dose is approximately 10 to 20 g / day. Preferably, the daily dose is approximately 8 to 15 g / day. More preferably, the daily dose is approximately 10 g / day. At these doses, this compound provides an IOP reduction effect.

[0100] In humans (patients weighing approximately 60 kg), PQQ can produce neuroprotective effects when administered at daily doses of approximately 2-160 mg / kg / day, approximately 10-100 mg / kg / day, or approximately 50 mg / kg / day. In certain embodiments, PQQ can be administered at daily doses of approximately 10 mg-10 g, approximately 50 mg-1 g, or approximately 500 mg.

[0101] Ideally, a typical dose may be one, two, or three times a day. The total daily dose may be administered as a single dose or in two or three divided doses (for example, each dose being half or one-third of the total daily dose). In the case of multiple doses, each dose may be the same or different. The pharmaceutical composition may be administered in the morning or evening. The pharmaceutical composition may be taken with or without food. Therapeutic drugs for treating neurodegenerative disorders In one embodiment, the present invention provides a method for treating or preventing neurodegenerative disorders in subjects requiring treatment or prevention, wherein the method involves increasing the intracellular level of NADt in the subject or NAD + The process includes administering a therapeutically effective dose of a drug that increases intracellular levels of NADH, GSH, GSSG, pyruvate, or PQQ.

[0102] In another embodiment, the present invention provides a pharmaceutical composition comprising therapeutically effective amounts of NAM, nicotinamide mononucleotide (NMN or β-NMN), NAD, pyruvate, PQQ, or glutathione.

[0103] The drugs used in pharmaceutical compositions are neuroprotective agents used to treat or prevent neurodegenerative disorders such as glaucoma. Numerous different types of invasive or neurodegenerative diseases or conditions affecting the nervous system, such as hypoxia, hypoglycemia, diabetes, metabolic stress due to loss of ion homeostasis, physical injury to neurons, exposure to toxins, and hereditary or non-hereditary neurodegenerative disorders, can result in neuronal damage or death. In certain embodiments, neurodegenerative disorders include axonal degeneration. In certain embodiments, neurodegenerative disorders include Wallerian degeneration or Wallerian degeneration. For example, Wallerian degeneration may result from neuronal injury such as disease, trauma, or injury caused by chemotherapy drugs.

[0104] Needless to say, this is merely an illustrative list. The presence of neuroprotective agents allows neurons to survive exposure to invasive or disease-related conditions that could lead to loss of functional integrity in unprotected neurons. Such agents can also prevent neurons from being damaged in response to invasive conditions by making them stronger.

[0105] Pharmaceutical composition Pharmaceutical formulations include a pharmacologically active ingredient in a form that is not harmful to the target organism, and additional components designed to stabilize the active ingredient and affect its absorption into the bloodstream or target tissue.

[0106] The pharmaceutical compositions according to the present invention may be formulated with pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients in accordance with conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th edition, edited by Gennaro, Mack Publishing Co., Easton, PA, 1995.

[0107] Suitable pharmaceutically acceptable carriers include inert solid diluents or fillers, sterile aqueous solutions, and various organic solvents. Examples of solid carriers include lactose, clay, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and lower alkyl ethers of cellulose. Examples of liquid carriers include syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, and water.

[0108] The pharmaceutical composition according to the present invention can be administered via various routes, such as orally, parenterally (including subcutaneous, intramuscular, and intradermal), intravitreally, or intraocularly (e.g., in the form of eye drops). In one preferred embodiment, the pharmaceutical composition is for oral administration (including administration of the pharmaceutical composition as part of a beverage). In one preferred embodiment, the pharmaceutical composition is for intraocular administration.

[0109] Parenteral administration can be performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe, or optionally a pen-type syringe. Alternatively, parenteral administration can be performed using an infusion pump. Furthermore, as a further option, the formulation of the present invention can also be adapted for transdermal administration, for example by needleless injection or patch, optionally from an iontophoresis patch, or transmucosal administration, for example, oral administration.

[0110] The pharmaceutical composition of the present invention can be administered in an appropriate dosage form, such as a liquid, suspension, emulsion, tablet, coated tablet, capsule, hard capsule and soft capsule, intravenous drip, eye drop, eye ointment, ophthalmic rinse, injectable solution, etc.

[0111] The pharmaceutical compositions of the present invention can be further compounded or added to drug carriers, drug delivery systems, and advanced drug delivery systems, for example, by covalent, hydrophobic, and electrostatic interactions, to further enhance the stability of the composition, increase bioavailability, increase solubility, reduce adverse effects, achieve time-based therapy known to the art, improve patient adherence to medication, or any combination thereof.

[0112] Examples of carriers, drug delivery systems, and advanced drug delivery systems include, but are not limited to, polymers such as cellulose and derivatives, polysaccharides such as dextran and derivatives, starch and derivatives, poly(vinyl alcohol), acrylate and methacrylate polymers, polylactic acid and polyglycolic acid and their block copolymers, polyethylene glycol, carrier proteins such as albumin, gels such as thermal gelling systems such as block copolymer systems well known to the art, micelles, liposomes, microspheres, nanoparticles, liquid crystals and their dispersions, L2 phases and their dispersions well known to the art in lipid-water systems for their phase behavior, polymerized micelles, multilayer emulsions, self-emulsifying and self-micro-emulsifying cyclodextrins and their derivatives, and dendrimers.

[0113] The pharmaceutical compositions of the present invention may be useful for compositions of controlled, sustained, extended, delayed, and sustained-release drug delivery systems. More specifically, but not limited to, the pharmaceutical compositions may be useful for parenteral controlled-release and sustained-release system compositions (both systems reducing the number of doses to a fraction of the original number), as is well known to the art. Even more preferably, controlled-release and sustained-release systems are administered subcutaneously. Examples of useful controlled-release systems and compositions, without limiting the scope of the present invention, include hydrogels, oily gels, liquid crystals, polymerized micelles, microspheres, and nanoparticles.

[0114] Neurodegenerative diseases The pharmaceutical compositions of the present invention are expected to be useful in treating neurodegenerative disorders, including axonal degeneration such as Wallerian degeneration. Examples of disorders in which these degenerations may be significant include diabetic neuropathy, motor neuron disease, multiple sclerosis, peripheral neuropathy, stroke and other ischemic disorders, traumatic brain injury, etc. This list is for illustrative purposes only and is not restrictive or comprehensive.

[0115] In certain embodiments, the neurodegenerative disorder is one or more of the following: diabetic neuropathy, traumatic brain injury, ischemia, peripheral neuropathy, or ocular disorders such as glaucoma. In certain embodiments, the neurodegenerative disorder is glaucoma.

[0116] In one embodiment, the pharmaceutical composition is intended for use as a neuroprotective drug in the treatment or prevention of neurodegenerative disorders resulting from neuronal injury. In a further embodiment, the modulator is intended for use as a neuroprotective drug in the treatment of neurodegenerative disorders related to axonal degeneration (e.g., Wallerian degeneration) resulting from neuronal injury. Herein, the term “injury” means damage inflicted on a neuron, whether to the cell body, axon, or dendrite. This may be physical injury in the conventional sense, i.e., traumatic injury to the brain, spinal cord, or peripheral nerves caused by external force applied to the subject. Other external causes of injury include, for example, mercury and other heavy metals, arsenic, pesticides, and environmental toxins such as solvents. Alternatively, injury may result from intrusion into neurons originating from within the subject, e.g., reduced oxygen and energy supply as in ischemic stroke and diabetic neuropathy, autoimmune attacks as in multiple sclerosis, or oxidative stress and free radical generation, which are considered important in amyotrophic lateral sclerosis. Furthermore, injury is used herein to refer to any defect in the mechanism of axonal transport.

[0117] In another embodiment, the pharmaceutical composition in question is intended for use as a neuroprotective drug, and the neurodegenerative disorder is caused by neuronal damage resulting from the disease. In one embodiment, neuronal damage is caused by aging.

[0118] In one embodiment, neuronal damage is caused by trauma. In another embodiment, the damage is neuronal damage induced by chemotherapy drugs. Certain drugs used in cancer chemotherapy, such as Taxol, Velcade, and Vincristine, are causes of peripheral neuropathy that limits the maximum dose that can be used. Recent studies suggest that neurons suffering from Taxol or Vincristine toxicity undergo Waller-like changes in their morphology and underlying molecular events. Since neurons are only transiently exposed to neurotoxic drugs, inhibiting Waller degeneration may be particularly effective in this condition. Therefore, the co-administration of a Waller degeneration inhibitor with Taxol or Vincristine may allow the use of the drug at substantially higher doses than currently considered possible, thus enabling a greater fight against cancer.

[0119] Age is a common risk factor for most cases of glaucoma, and this treatment method provides prevention against age-related decline in NAD levels, protecting patients from the onset of glaucoma (i.e., preventing neurodegeneration). This invention provides a combination therapy of administration of NAM and / or pyruvate (to replenish NAD levels) and gene delivery of the NMNAT1 gene. Given the remarkable efficacy of NAM and / or pyruvate and gene therapy, the combination therapy is an attractive approach for treating and preventing glaucoma.

[0120] Gene therapy to treat neurodegeneration Gene therapy is an attractive method for overcoming medication adherence problems and improving efficacy. This invention provides a gene therapy method for treating neurodegeneration. Axonal degeneration is an area where treatment needs are not being met. Neurodegeneration causes symptoms in motor neuron diseases, glaucoma, Alzheimer's disease, and multiple sclerosis. In diabetes, it causes neuropathic pain and distal loss of sensation, and it is a leading cause of limb amputation. It is also a dose-limiting side effect in cancer chemotherapy. Progressive axonal degeneration due to stretch injury is a major pathology in traumatic brain injury, and the inability to protect the white matter limits the treatment of stroke. Approximately half of the human population will eventually suffer one or more of these disorders, which significantly reduces their quality of life.

[0121] In certain embodiments, the present invention provides a method for treating neurodegeneration in the eye. The present invention provides a method for replacing a dysfunctional gene copy in the eye. The gene is introduced using viral gene delivery. In certain embodiments, the vector is an adenovirus, an adeno-associated virus (AAV). In a preferred embodiment, the AAV is AAV2.2. For the purposes of this application, other AAV serotypes are to be included, including AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, etc. In other embodiments, the vector is a lentivirus, a vector generally based on pseudoHIV.

[0122] To deliver the gene to the targeted RGC, the viral vector is introduced into the vitreous cavity. In a preferred embodiment, gene delivery is directly targeted to the proximal inner retinal layer. Intravitreal injection is performed as is standard practice in ophthalmic surgery and can be safely administered in a clinic / medical setting.

[0123] Since viral gene delivery can deliver targeted gene products to a large number of cells to be transfected over a lifetime, the present invention offers the attractive prospect of viral gene delivery to the eye. To the best of the inventor's knowledge, gene therapy in the eye represents a novel approach to treating glaucoma. NAD applicable to human complex diseases such as glaucoma. + Such gene therapy to increase [the gene] would represent a novel treatment method.

[0124] In one embodiment, the present invention provides a method for delivering a gene to an affected eye to thereby enhance NAD expression. In certain embodiments, the gene therapy composition according to the present invention (e.g., polynucleotides on a viral vector) is administered intravitreously or intraocularly. In a preferred embodiment, the gene therapy is administered intravitreously. It goes without saying that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the disease to be treated, and the selected active ingredient.

[0125] In one embodiment, the present invention provides a gene therapy that delivers a gene to the eye to increase the expression of Nmnat. In a particular embodiment, the gene that increases the protein expression of Nmnat may be Nmnat-1, Nmnat-2, or Nmnat-3.

[0126] In certain embodiments, the gene is Nmnat-1 (e.g., human NMNAT1). NMNAT Nicotinamide nucleotide adenylyltransferase 1 (Nmnat1 [mouse], NMNAT1 [human]) encodes an enzyme that catalyzes a key step in the biosynthesis of nicotinamide adenine dinucleotide (NAD). The encoded enzyme is one of several nicotinamide nucleotide adenylyltransferases and is specifically localized to the cell nucleus. Alternative splicing of this gene results in multiple (at least three) transcript variants.

[0127] The NCBI reference sequences (RefSeq) for human NMNAT1 isoform (1) include: NM_022787.3 (nucleotide) and NP_073624.2 (protein), whose nucleotide sequences are known as transcription variant (1) encoding the longer NMNAT1 isoform (1); and NM_001297778.1 (nucleotide) and NP_001284707.1 (protein), whose nucleotide sequences are known as transcript variant (2) that differ in the 5'UTR region compared to variant 1. Variants 1 and 2 encode the same isoform (1) and can both be used in the method of the present invention. All sequences are incorporated by reference.

[0128] Unlike other human family members whose expression is localized and eccentrically distributed in the nucleus, the related nicotinamide nucleotide adenylyltransferase 2 (nmnat2) is cytoplasmic and primarily expressed in the brain. Alternative splicing of this gene results in two transcript variants.

[0129] The NCBI reference sequences (RefSeq) for the human NMNAT2 isoform (1) are: NM_015039.3 (nucleotide) and NP_055854.1 (protein), the nucleotide sequences of which can be used in the method of the present invention. All sequences are incorporated by reference.

[0130] The related nicotinamide nucleotide adenylyltransferase 3 (nmnat3) encodes a protein localized to mitochondria and may play a neuroprotective role as a molecular chaperone. Alternatively, spliced ​​transcript variants (at least six) encoding multiple isoforms (at least five) of this gene have been observed.

[0131] The NCBI reference sequences (RefSeq) for human NMNAT3, isoform (3) are: NM_001320510.1 (nucleotide) and NP_001307439.1 (protein). The nucleotide sequence is transcription variant 3, which encodes the longest isoform (3), and can be used in the method of the present invention. All sequences are incorporated by reference.

[0132] NMNAT2 appears as a crucial NAD-producing enzyme in axons, protecting against axonal degeneration. Sustained stress in the RGC negatively impacts NMNAT2 expression (D2 glaucoma group 4, q<0.05 at the final stage detected before glaucoma degeneration). This decline may be important for the transition to axonal degeneration in glaucoma. NMNAT2 expression is reduced in the brains of Alzheimer's disease patients and shows a large variability in the postmortem human brains of older individuals, and this variability may contribute to diverse vulnerabilities to these pathological conditions.

[0133] Numerous relevant nmnat sequences from different animal species (such as the house mouse) are readily available from publicly accessible databases such as NCBI RefSeq and GenBank. All sequences are incorporated herein by reference.

[0134] Gene therapy using NAMPT involves NAD in cells. + It is thought that this increases levels but leads to axonal cytotoxicity. NAMPT functions to convert NAM to NMN, and then NMN is converted to NAD + Converted to intracellular NAD. + NAD is a key molecule associated with axonal degeneration. +At low levels, axons degenerate rapidly. It has also been established that NMN rapidly accumulates after axonal injury in sciatic nerve axons. In this model of axonal degeneration, NMN levels rise within 12 hours, followed by nerve damage 36 hours after injury. Blocking the NMN-producing enzyme NAMPT using FK866 potentially inhibits this axonal degeneration, suggesting that NMN is toxic to neurons. Axonal degeneration is NMN-dependent, and inhibiting the increase of NMN using FK866 appears to protect against axonal degeneration. In a zebrafish model of axonal degeneration (two-photon laser axon cutting), FK866 strongly delayed axonal degeneration. In a cell culture model of neurite degeneration (superior cervical ganglion; SCG), rapid removal of NMN (by overexpression of the bacterial enzyme NMN deamidase, which converts NMN to NAMN) strongly protects against axonal degeneration.

[0135] Therefore, the present invention presents the unexpected finding that gene therapy using NMNAT, unlike that using NAMPT, is effective in protecting against ophthalmic neurodegeneration. Overexpression of Nampt drives the production of NMN, and since NMN is toxic to neurons if not properly removed, Nmnat1 was therefore chosen over Nampt.

[0136] Wld S Axonal degeneration is a common component of neurodegenerative diseases. There are two models that attempt to explain this greater extent of distal axonal degeneration. The first is “distal,” where the degeneration spreads retrogradely from the nerve terminal. The second is Wallerian degeneration, in which the degeneration spreads in any direction from the site of the lesion, depending on the type of lesion, ultimately resulting in the loss of axons distally to the site of the lesion, with the proximal portion remaining completely intact. Strictly speaking, Wallerian degeneration occurs only in response to physical damage to the axon, and a similar mechanism is at work in diseases where such damage does not occur. The latter is called “Wallerian” degeneration. Both types of degeneration will be referred to collectively as “Wallerian degeneration” below.

[0137] Recently discovered WldS Mice have advanced our understanding of these two processes. In these animals, Wallerian degeneration occurs approximately 10 times slower than in wild-type animals. Studies have shown that this mutation also delays lesions thought to be related to the "distality" of axon terminals. Therefore, Wld S Genes provide a mechanical link between the two models of axonal degeneration.

[0138] Wld S Despite gene identification and characterization, progress toward understanding the molecular triggers of Wallerian degeneration remains limited. Understanding these triggers could have a significant impact on our understanding of the early stages of "distal" neurodegenerative diseases.

[0139] Wld S Mice carrying the gene showed delayed Wallerian degeneration. Wld S The mutation is an autosomal dominant mutation occurring on mouse chromosome 4. The gene mutation is a naturally occurring 85kb series triplication, resulting in a variant region containing two related genes: nicotinamide mononucleotide adenylyltransferase 1 (Nmnat-1) and ubiquitination factor e4b (Ube4b), as well as a linker region encoding 18 amino acids. The resulting protein is localized in the nucleus and undetectable in the axon.

[0140] The mutation does not appear to cause harm to mice. The only known effect is that Wallerian degeneration is delayed by an average of up to three weeks after nerve injury. Recent studies suggest that the mutation protects axons through a mechanism that is not fully understood. Not bound by any particular theory, but Wld S Mutations are likely to result in overexpression and / or improved localization of Nmnat proteins (e.g., Nmnat-1), leading to increased NAD synthesis.

[0141] In certain embodiments, the method involves applying Nmnat (e.g., Nmnat-1-2 or -3) or Wld to the target. SThe process includes administering a polynucleotide encoding [the specified character]. In certain embodiments, polynucleotides are Wld S Or it codes for the equivalent human sequence.

[0142] In a particular embodiment, the method involves targeting NAM or a precursor, and Wld S The process includes administering a polynucleotide encoding [the specified character]. In certain embodiments, polynucleotides are administered topically to a subject. For example, to treat glaucoma, the drug may be delivered topically to the affected eye.

[0143] vector In certain embodiments, polynucleotides are administered to the target via a viral vector. Typical suitable viral vectors include, but are not limited to, AAV vectors, adenovirus vectors, lentiviral vectors, retroviral vectors, etc. Preferably, the viral vector is an AAV vector or a lentiviral vector.

[0144] For example, polynucleotides can be introduced into a target as exogenous genetic material capable of modulating the expression of one or more target genes. Such types of gene therapy can be used, for example, in methods used to repair damaged or diseased tissue, such as neuronal tissue. In summary, genetic information such as DNA and / or RNA can be delivered to a target using any suitable vector, including adenoviruses, lentiviruses, or retroviral gene delivery media (see below). Those skilled in the art can replace or repair specific genes targeted in gene therapy. For example, a non-functional gene can be replaced by inserting a normal gene into a nonspecific location in the genome of a diseased cell. In another example, an abnormal gene sequence can be replaced with a normal gene sequence by homologous recombination. Alternatively, selective reverse mutation can restore a gene to its normal function. A further example is modifying the regulation of a particular gene (to the extent to which the gene is turned on or off). In certain embodiments, target cells (such as neuronal stem cells) are treated ex vivo by a gene therapy technique and subsequently transferred to a mammal, preferably a human requiring treatment.

[0145] Any method of genetic engineering recognized by those skilled in the art can be used, including transfection and infection with various nucleic acid constructs (e.g., by viral vectors).

[0146] For example, heterologous nucleic acids (e.g., DNA) can be introduced into a target using chemical materials or biological vectors (viruses) by physical procedures (e.g., electroporation, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, nanoparticles, magnetfection). Chemical transfection may be based on calcium phosphate, cyclodextrin, polymers (e.g., cationic polymers such as DEAE dextran or polyethyleneimine), highly branched organic compounds such as dendrimers, liposomes (lipofections such as those using cationic liposomes, Lipofectamine, etc.), or nanoparticles (with or without chemical or viral functionalization).

[0147] Nucleic acid constructs generally contain the target nucleic acid molecule and can instruct the expression of the target nucleic acid molecule within the cell into which they are introduced. In a particular embodiment, the nucleic acid construct is an expression vector in which a nucleic acid molecule encoding a gene product such as a polypeptide or a nucleic acid that antagonizes the expression of a polypeptide (e.g., siRNA, miRNA, shRNA, antisense sequence, aptamer, ribozyme, antagonist miRNA, RNA sponge, etc.) is operationally linked to a promoter capable of directing the expression of the nucleic acid molecule in target cells.

[0148] DNA constructs prepared for introduction into specific cells generally include a replication system recognized by the cell, an intended DNA segment encoding a desired polypeptide, and transcription and translation initiation and termination regulatory sequences operatively ligated to the polypeptide-encoding segment. A DNA segment is "operatively ligated" if it is placed in a functional relationship with another DNA segment. For example, a promoter or enhancer is operatively ligated to a coding sequence if it stimulates the transcription of the sequence. DNA for a signal sequence is operatively ligated to the polypeptide-encoding DNA if it is expressed as a preprotein involved in polypeptide secretion. Generally, operatively ligated DNA sequences are contiguous, and in the case of a signal sequence, both are contiguous and in the read-frame phase. Enhancers, however, do not need to be contiguous with the coding sequence that controls transcription. Ligation is completed by ligation at a convenient restriction site or, instead, an adapter or linker inserted.

[0149] The selection of a suitable promoter sequence is generally determined by the host cell selected for the expression of the DNA segment. Examples of suitable promoter sequences include eukaryotic promoters well known to those skilled in the art (see, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Part 3, 2001). Transcriptional regulatory sequences generally include heterologous enhancers or promoters recognized by the cell. A suitable promoter is the CMV promoter. Expression vectors containing transcriptional and translational regulatory sequences together with the replication system and the polypeptide coding segment insertion site can be used. Examples of viable combinations of cell lines and expression vectors are described in Sambrook and Russell (2001, above) and Metzger et al. (1988), Nature, 334: pp. 31-36.

[0150] Some aspects of the present invention relate to the use of nucleic acid constructs or expression vectors comprising the nucleotide sequences defined above, wherein the vectors are suitable for gene therapy. Suitable vectors for gene therapy are described by Anderson (Nature, 392: pp. 25-30, 1998); Walther and Stein (Drugs, 60: pp. 249-71, 2000); Kay et al. (Nat. Med., 7: pp. 33-40, 2001); Russell (J. Gen. Virol., 81: pp. 2573-604, 2000); This is publicly known to those skilled in the art, as described in Amado and Chen (Science 285: pp. 674-676, 1999); Federico (Curr. Opin. Biotechnol., 10: pp. 448-453, 1999); Vigna and Naldini (J. Gene Med., 2: pp. 308-316, 2000); Marin et al. (Mol. Med. Today, 3: pp. 396-403, 1997); Peng and Russell (Curr. Opin. Biotechnol., 10: pp. 454-457, 1999); Sommerfelt (J. Gen. Virol., 80: pp. 3049-3064, 1999); Reiser (Gene Ther., 7: pp. 910-93, 2000); and the references cited therein (all incorporated by reference). Examples include embedded and non-embedded vectors based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), lentiviruses, poxviruses, alphaviruses, and herpesviruses.

[0151] Particularly suitable gene therapy vectors include adenovirus (Ad) and adeno-associated virus (AAV) vectors. These vectors infect the vast majority of dividing and non-dividing cell types. In addition, adenovirus vectors allow for high levels of transgene expression. However, as described in Russell (J. Gen. Virol., 81:2573-2604, 2000; Goncalves, Virol J., 2(1):43, 2005), due to the episomal nature of adenovirus and AAV vectors after cell entry, these viral vectors are best suited for therapeutic applications requiring only transient transgene expression. As reviewed by Russell (2000, above), preferred adenovirus vectors are modified to reduce the host response. The safety and efficacy of AAV gene transfer have been thoroughly studied in humans, along with promising results in the liver, muscle, CNS, and retina (Manno et al., Nat. Medicine, 2006; Stroes et al., ATVB, 2008; Kaplitt, Feigin, Lancet, 2009; Maguire, Simonelli et al., NEJM, 2008; Bainbridge et al., NEJM, 2008).

[0152] AAV2 is the most characterized serotype for gene transfer studies in both human and experimental models. AAV2 exhibits a natural tropism toward skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes. Other examples of adeno-associated virus-based non-embedded vectors include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and pseudotype AAV. The use of non-human serotypes such as AAV8 and AAV9 may be useful in overcoming these immunological responses in subjects, and clinical trials have just begun (ClinicalTrials dot gov Identifier:NCT00979238). For gene transfer into hepatocytes, adenovirus serotype 5 or AAV serotypes 2, 7, or 8 have been shown to be effective vectors and therefore preferred Ad or AAV serotypes (Gao, Molecular Therapy, 13:77-87, 2006).

[0153] A typical retroviral vector for application in the present invention is a lentivirus-based expression construct. Lentiviral vectors have an inherent ability to infect non-dividing cells (Amado and Chen, Science 285: pp. 674-676, 1999). Methods for constructing and using lentivirus-based expression constructs are described in U.S. Patents 6,165,782, 6,207,455, 6,218,181, 6,277,633 and 6,323,031, as well as by Federico (Curr. Opin. Biotechnol. 10: pp. 448-53, 1999) and Vigna et al. (J. Gene Med. 2: pp. 308-16, 2000).

[0154] In general, a gene therapy vector is an expression vector that contains a nucleotide sequence encoding the gene product of the present invention (e.g., a polypeptide) to be expressed, and in the sense that the nucleotide sequence is operatively linked to a suitable regulatory sequence as described above, such a regulatory sequence will include at least a promoter sequence. Suitable promoters for expressing a polypeptide-encoding nucleotide sequence from a gene therapy vector include, for example, the initial promoter in cytomegalovirus (CMV), viral terminal repeat sequence promoters (LTRs) such as those derived from mouse Moloney's leukemia virus (MMLV), Roussarcoma virus, or HTLV-1, the initial promoter of Simian virus 40 (SV40), and the herpes simplex virus thymidine kinase promoter. Further suitable promoters are described below.

[0155] Several inducible promoter systems that can be induced by the administration of small organic or inorganic compounds have been described. Such induceable promoters include heavy metals such as metallothionine promoter (Brinster et al., Nature, 296: pp. 39-42, 1982; Mayo et al., Cell, 29: pp. 99-108, 1982), RU-486 (progesterone antagonist) (Wang et al., Proc. Natl. Acad. Sci. USA, 91: pp. 8180-8184, 1994), steroids (Mader and White, Proc. Natl. Acad. Sci. USA, 90: pp. 5603-5607, 1993), and tetracyclines (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89: pp. 5547-5551, 1992; U.S. Patent No. 5,464,758; Furth et al., Proc. Natl. (Acad. Sci. USA, 91:9302-9306, 1994; Howe et al., J. Biol. Chem., 270:14168-14174, 1995; Resnitzky et al., Mol. Cell. Biol., 14:1669-1679, 1994; Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526, 1995) In addition, there are systems regulated by the tTAER system (Yee et al., 2002, U.S. Patent No. 6,432,705) based on a multi-chimeric transactivator composed of the tetR polypeptide and the ligand-binding domain of the estrogen receptor as the activation domain of VP16.

[0156] In addition to the polymerase II promoter mentioned above, the polymerase III promoter is also suitable for nucleotide sequences encoding small RNAs used to knock down specific genes by RNA interference (see below). RNA polymerase III (pol III) is involved in the synthesis of a wide variety of nuclear and cytoplasmic non-coding small RNAs, including 5S, U6, adenovirus VA1, Vault, telomerase RNA, and tRNA. The promoter structures of numerous genes encoding these RNAs have been determined, and it has been found that RNA pol III promoters can be classified into three structural types (see Geiduschek and Tocchini-Valentini, Annu. Rev. Biochem., 57:873-914, 1988; Willis, Eur. J. Biochem., 212:1-11, 1993; Hernandez, J. Biol. Chem., 276:26733-36, 2001). The RNA pol III promoter type 3 is particularly suitable for siRNA expression, as transcription is driven by a cis-acting element found only in the 5'-adjacent region, i.e., upstream of the transcription start site. Upstream sequence elements include the conventional TATA box (Mattaj et al., Cell, 55:435-442, 1988), proximal sequence elements, and distal sequence elements (DSE; Gupta and Reddy, Nucleic Acids Res., 19:2073-2075, 1991). Examples of genes under the control of the type 3 pol III promoter include U6 nuclear small RNA (U6 snRNA), 7SK, Y, MRP, HI, and telomerase RNA genes (see, for example, Myslinski et al., Nucleic Acids Res., 21:2502-09, 2001).

[0157] Gene therapy vectors may optionally contain a second or one or more additional nucleotide sequences encoding a second or further polypeptide. The second or further polypeptide may be a (selectable) marker polypeptide that enables the identification, selection, and / or screening of cells containing the expression construct. Suitable marker proteins for this purpose include, for example, the fluorescent protein GFP, as well as selectable marker genes such as HSV thymidine kinase (for selection in HAT medium), bacterial hygromycin B phosphotransferase (for selection for hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection for G418), and dihydrofolate reductase (DHFR) (for selection for methotrexate), CD20, and the low affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring. Provided by Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, 2001.

[0158] Alternatively, if deemed necessary, a second or further nucleotide sequence may encode a polypeptide that provides a double safety mechanism enabling the subject to be cured of transgenic cells. Such nucleotide sequences, often referred to as suicide genes, encode a polypeptide capable of converting a prodrug into a toxic substance that can kill transgenic cells expressing the polypeptide. Suitable examples of such suicide genes include, for example, the E. coli cytosine deaminase gene or one of the thymidine kinase genes derived from herpes simplex virus, cytomegalovirus, and varicella-zoster virus, in which case ganciclovir can be used as a prodrug to kill IL-10 transgenic cells in the subject (see, e.g., Clair et al., Antimicrob. Agents Chemother., 31: pp. 844-849, 1987).

[0159] Routes of administration for gene therapy in the eye In one embodiment, the present invention provides a method of gene therapy for treating ocular degenerative diseases. Those skilled in the art will recognize pathways of gene therapy in the eye, including intravitreous or intraocular administration.

[0160] Intraocular administration delivers the gene composition to any part of the eyeball. In a preferred embodiment, the gene composition is injected intravitreously. Intravitreous administration delivers the gene composition directly to the vitreous compartment of the eye, i.e., the fluid that makes up the posterior part of the eye, immediately adjacent to the retina. In a particular embodiment, the vitreous humor is removed during injection to create space for the drug.

[0161] Intravitreal injections are generally performed in clinics, but can also be done in hospital operating rooms. Patients typically receive local anesthesia (usually eye drops or a sponge / cotton swab) with common medications such as propalacaine, lidocaine, or others.

[0162] In certain embodiments, the gene composition is injected subretin (i.e., beneath the retina). For injections, needle sizes of 27-32 gauge can be used. In a preferred embodiment, the needle size is 30 gauge. Injection is a popular technique for injecting medication into the eye and is completely safe. The injection can be deep or superficial. The injection can be perpendicular (i.e., at 90° to the surface of the eye), oblique (45-60°), or double-pane (oblique, perpendicular to the outward direction).

[0163] Gene therapy in the eye This invention uses a gene composition to enable NAD in RGCs. + The gene therapy provides a boost to [specific gene]. The eye is an ideal organ for gene therapy because it is small in size (i.e., has few cells to transfect), easily accessible, partially / almost immune-privileged (i.e., unlikely / almost never to trigger an immune response), and the other eye can serve as a contralateral control and reserve.

[0164] Several eye-based gene therapy clinical trials have been conducted for disorders leading to blindness. For example, gene therapy of the RPE65 gene using an AAV2 vector to treat Leber congenital amaurosis is in a Phase 3 clinical trial (NCT00481546, NCT00516477, NCT00643747, and NCT00999609); gene therapy of the MERTK gene using an AAV2 vector to treat retinitis pigmentosa is in Phase 1 (NCT014822195); gene therapy of the ABCA4 gene using an EIAV lentiviral vector to treat Stargardt disease is currently in Phase 2 (NCT01367444); and gene therapy of the REP-1 gene using an AAV2 vector to treat chorioderemia is currently in Phase 2 (NCT02553135). Clinical studies in several clinics demonstrate that gene delivery to the eye appears to be safely tolerated.

[0165] Combination therapy - additional therapeutic drugs In certain embodiments, the present invention provides NAD in nerve cells (e.g., RGCs) to humans who require it. + This invention provides a method for the combined therapeutic treatment of neurodegeneration by administering compounds that enhance intracellular levels and additional therapeutic agents.

[0166] In certain embodiments, further therapeutic agents of the present invention for treating glaucoma include, but are not limited to, agents that reduce IOP. Further typical therapeutic agents include, but are not limited to, β-receptor blockers (such as timolol maleate, timolol hemihydrate, levobunolol HCl, metipranolol, carteolol, betaxolol, etc.), non-selective adrenergic agonists (such as epinephrine, dipivefrin HCl), selective α-2 adrenergic agonists (such as apraclonidine HCl, brimonidine tartrate, and brimonidine tartrate in Prite), or carbonic anhydrase inhibitors (CAIs such as acetazolamide [oral], acetazolamide [parenteral], metazolamide [oral], dorzolamide [topical], and brinzolamide [topical]).

[0167] In certain embodiments, further therapeutic agents include prostaglandin analogs (such as latanoprost, travoprost, and bimatoprost [prostamide]), parasympathetic agonists (including direct cholinergic agonists such as pilocarpine HCl; as well as indirect cholinergic agents such as ecothiophate iodide, demercarium iodide, and physostigmine isofluorophate), and carbachol (a mixed direct agonist / acetylcholine-releasing agent).

[0168] In certain embodiments, further therapeutic agents include combination drugs that offer potential advantages in terms of convenience, adherence, efficacy, and cost. Combinations may include topical β-receptor blockers in combination with prostaglandin analogs, α-adrenergic agonists, or topical carbonic anhydrase inhibitors. Typical combinations include: (1) dorzolamide and timolol (e.g., Cosopt, now available as a generic), such as 2% dorzolamide hydrochloride and 0.5% timolol maleate eye drops; (2) brimonidine containing timolol or brinzolamide, such as 0.2% brimonidine tartrate and 0.5% timolol maleate eye drops (e.g., Comvigan) and 0.2% brimonidine tartrate and 1% brinzolamide (e.g., Simbrinza); or (3) latanoprost and timolol.

[0169] In certain embodiments, further therapeutic agents include hyperosmolar agents such as oral glycerin, oral isosorbide, and intravenous mannitol, which can rapidly reduce IOP by decreasing vitreous volume. They do not cross the blood-ocular barrier and therefore do not exert colloidal osmotic pressure that dehydrates the vitreous humor. Hyperosmolar agents are commonly used in acute situations to temporarily reduce high IOP until a more definitive treatment can be provided.

[0170] The present invention is illustrated by the following non-limiting examples. [Examples]

[0171] [Example 1] Identification of glaucoma susceptibility genes / pathways (A) Selection of mouse models that mimic human neurodegeneration We selected the DBA / 2J D2 (D2) mouse model to investigate the pathogenesis of neurodegeneration in humans. The D2 mouse is a widely used model of glaucoma and outlines the prominent features of human glaucoma. In D2 mice, two genes (Gpnmb) R250X , Tyrp1 b Mutant alleles in ) cause progressive iris disease. This iris disease has two main causes. The main features include interstitial iris atrophy (very similar to essential iris atrophy in humans) and iris depigmentation phenotype (very similar to depigmentation syndrome in humans). Essential iris atrophy and depigmentation syndrome induce elevated IOP and glaucoma in humans. Iris disease in D2 eyes results in senile elevated intraocular pressure (IOP) in the majority of eyes by 8-9 months of age. Optic nerve degeneration is almost complete by 12 months of age (generally, more than 70% of nerves have severe damage).

[0172] This study demonstrated that D2 mice undergo ophthalmic neurodegeneration after age-related increases in IOP. Specifically, D2 mice at three different ages—4, 9, and 12 months—were used. (i) We verified that 4-month-old D2 mice had normal intraocular pressure (IOP) (i.e., 10-13 mmHg) and did not exhibit glaucoma (i.e., these mice were at an age before the onset of IOP and pre-glaucoma; there was no detectable neurodegeneration). At this age, D2 mice were indistinguishable from control mice.

[0173] (ii) We verified that 9-month-old D2 mice exhibited high IOP (i.e., greater than 21 mmHg) but did not show neurodegeneration. Conventional glaucoma is not present at this developmental stage (i.e., preneurodegeneration), but the eyes of 9-month-old D2 mice undergo molecular changes that are defined herein as having "early glaucoma".

[0174] (iii) Verify that 12-month-old D2 mice exhibit various high IOPs (i.e., 14 to >21 mmHg), revealing neurodegeneration (i.e., glaucoma; over 60% of eyes have severe neurodegeneration).

[0175] (iv) Control D2-Gpnmb + These mice were used in this study because they do not exhibit age-related high IOP or neurodegeneration. These mice were selected for Gpnmb, which causes iris disease. R250X Aside from the correction of mutations, it is identical to the D2 mouse.

[0176] (B) Isolation of retinal ganglion cells (RGCs) from the retina of D2 mice. (i) 4-month-old DBA / 2J D2 mice, (ii) 9-month-old DBA / 2J D2 mice, and (iii) D2-Gpnmb mice of the same age, sex, and strain. + Retinas were collected from the eyes of wild-type controls.

[0177] The retinal sample was first stained with an antibody cocktail. Retinal ganglion cells (RGCs) were stained with Thy1.2 + Cells (and Cd11b - , Cd11c - , Cd31 - , Cd34 - , Cd45.2 - GFAP - , DAPI - It was identified as negative for FACS. FACS-positive RCG was cultured on a plate and stained with SNAP-25 and β-tubulin (a specific marker for RGC) to confirm the status of RGC.

[0178] RGCs were subsequently isolated from freshly collected retinas of these three groups of mice using fluorescence-activated cell sorting (FAC sorting). See Figures 27A–27H.

[0179] (C) RNA sequencing To identify susceptibility genes / pathways leading to glaucoma, RNA sequencing (RNA-seq) was performed on the RNA of RGCs obtained above in (B) to describe very early molecular changes within RGCs preceding neurodegeneration. Amplified dscDNA libraries (double-stranded copy DNA) were generated from RNA and read at a read depth of 35,000,000 per sample. Data analysis was performed at (FDR, q) with a false discovery rate q < 0.05. All samples (B) from all groups were successfully amplified and sequenced.

[0180] 4, 9, and 12-month-old D2 and D2-Gpnmb +Further metabolic profiling was performed on ocular neuroretina. Metabolic profiling was carried out using targeted assays according to the manufacturer's recommendations. The following metabolite was profiled: NAD + / NADH (i.e., total NAD, NAD[t]), GSH / GSSG (i.e., total glutathione, glutathione[t]), and pyruvate.

[0181] (D) Hierarchical cluster analysis (HC) In this study, isolated RGC-derived RNA was sequenced with a read depth of 35,000,000 per sample. Unsupervised hierarchical clustering (HC) was used to sequence D2-Gpnm in the early stages of the disease and of the same age. + Alternatively, molecularly determined stages of glaucoma were defined between samples that were morphologically indistinguishable from young controls.

[0182] HC identified four distinct groups (i.e., Group 1, Group 2, Group 3, and Group 4) in the 9-month-old D2 samples. Group 1 clustered with all control samples and represented D2 RGC without molecularly detectable glaucoma. All samples in Groups 2–4 were in the early disease stage, and it was observed that the increase in group number reflected an increase in distance from the control (greater disease progression at the transcriptome level) (Figures 1, 2A, and 2B).

[0183] As the disease progressed, the most significant increase in transcript abundance relative to mitochondrial reads was observed (Figure 3A). The imbalance in the relative proportions of mitochondrial molecules encoded in the nuclear and mitochondrial genomes negatively impacts mitochondrial function. In groups D2-4, differential expression of genes encoding mitochondrial proteins in mitochondrial dysfunction and oxidative phosphorylation pathways, as well as significant enrichment of differentially expressed genes (D2-Gpnmb, same age and sex). + (Compared to the control RGCS) further indicates mitochondrial abnormalities within the RGC (Figures 3A-3J and 3K, and Tables 1-3 below).

[0184] The total read abundance of the entire transcriptome (i.e., all transcribed genes) was split into reads obtained from the nuclear transcriptome (i.e., those encoded in the nucleus) or the mitochondrial transcriptome (i.e., those encoded in mitochondria). D2 Read abundance in RGCs increased in both transcriptomes, but was most abundant in mitochondrial-derived reads. This suggests an imbalance that may promote mitochondrial dysfunction (Figure 3A).

[0185] The enriched higher pathway (IPA) appears in D2 groups 2, 3, and 4. Since there is only one differentially expressed (DE) gene, there is no enriched pathway in D2 group 1. This D2 group 1 represents eyes that were not affected by glaucoma (Figure 3B).

[0186] A plot was created showing all mitochondrial proteins encoded by nuclear genes (but not by mitochondria). DE genes are shown in red. Non-DE genes are shown in gray. Increased abundance of nuclear-derived transcripts encoding mitochondrial proteins further indicates an imbalance in mitochondrial turnover or function (Figure 3C).

[0187] Increased expression of mitochondrial fission genes (Dnm1 and Fis1) indicates premitotic events in mitochondria in the early stages of glaucoma. Increased fission is associated with increased mitochondrial turnover and disease. Mutations affecting mitochondrial fusion / fission dynamics are generally lethal or cause neurological pathologies (including, but not limited to, optic atrophy dominant and Charcot-Marie-Tooth disease) (Figure 3D).

[0188] The expression of genes related to the mitochondrial endoplasmic reticulum stress response increased. This is an intracellular stress response that generally often precedes apoptosis (programmed cell death) (Figure 3E).

[0189] Individual plots were generated for genes in pathways associated with mitochondria. Differentially expressed genes are shown in red, and non-differentially expressed genes are shown in gray. It should be noted that the upregulation of mitochondrial genes across the entire pathway, particularly in oxidative phosphorylation and reactive oxygen species metabolism, indicates an intracellular energy crisis (Figure 3F).

[0190] Protein analysis (Western blot) of the retina-derived mitochondrial pro-apoptotic molecule cytochrome C shows that cytochrome C is upregulated at both the gene and protein levels during early-stage glaucoma (9 months), and is significantly upregulated by 12 months (Figures 3G and 3I).

[0191] Protein analysis also confirms that there is upregulation of eIF2 in the retina (Figures 3H and 3J).

[0192]

Table 3-1

[0193]

Table 3-2

[0194]

Table 3-3

[0195]

Table 4-1

[0196]

Table 4-2

[0197]

Table 4-3

[0198] (E) Enriched higher pathways based on IPA analysis Table 3 lists the 10 most enriched pathways based on IPA analysis. Figure 1 of USSN62 / 366,211, filed July 25, 2016, shows D2 mice and D2-Gpnmb + We provide the results of a preliminary analysis of the enriched higher pathways among controls (the disclosure is incorporated by reference).

[0199] [Table 5-1]

[0200] [Table 5-2]

[0201] Pathway analysis identified enrichment of eIF2 and mTOR signaling transcripts (Figures 3B and 3F), with eIF2 signaling being the most enriched pathway in group 2 (the first step distinguishable from the control).

[0202] Significant upregulation of mitochondrial fission genes (Dnm1 and Fis1) (Figure 3D) and significant changes in the mitochondrial endoplasmic reticulum stress response (UPRmt) (Figure 3E) further indicated mitochondrial dysfunction.

[0203] (F) Mitochondrial abnormalities are revealed by morphology. In this study, morphological alternations were evaluated by performing electron microscopy (EM) on the retinas of these mice. EM currently provides the highest resolution available for studying the morphology of intracellular organelles (such as mitochondria). EM revealed dysfunctional mitochondria with reduced mitochondrial cristae volume in the dendrites of D2 RGCs, which were not present in control RGCs (Figures 4A and 4B). These mitochondrial EM findings are consistent with synaptic loss, an initial decrease in pattern electroretinogram amplitude (PERG, a highly sensitive indicator of RGC activity in both human patients and animals) (Figure 14), and an increase in retinal cytochrome c levels (Figure 3G) in 9-month-old D2 retinas.

[0204] These data show that mitochondrial disorder is associated with hereditary senile glaucoma. This clearly demonstrates that it is one of the first changes to occur within the vivo mitochondrial regulatory system (RGC). This finding is consistent with in vitro studies that have reported that cultured cells undergo mitochondrial abnormalities when subjected to pressure. However, in vitro studies cannot prove how early in vivo mitochondrial abnormalities occur in glaucoma (whereas this finding does).

[0205] In summary, this RNA-seq study revealed transcriptional processing and mitochondrial dysfunction in RGCs of glaucoma-prone eyes with undetectable neurodegenerative phenotypes. Mitochondrial dysfunction was confirmed by targeted metabolic assays and EM, indicating early abnormal mitochondria in glaucoma, along with an early energy crisis.

[0206] [Example 2] Metabolic profiling Mitochondrial dysfunction / energy crisis occurs pre- and very early in the disease (i.e., 4 months [pre-glaucoma], 9 months if high IOP is present without any neurodegeneration, and 12 months if neurodegeneration is present / severe in the majority of eyes), (control D2-Gpnmb) +To determine whether (compared with mice) it exists in D2 mice, metabolic profiling of the neural retina was performed.

[0207] 4-, 9-, and 12-month-old D2 and D2-Gpnmb + Further metabolic profiling was performed on neural retinas derived from eyeballs. Metabolic profiling was performed using a targeted assay according to the manufacturer's recommendations. The following metabolites were profiled: NAD + / NADH (i.e., total NAD, NAD[t]), GSH / GSSG (i.e., total glutathione, glutathione[t]), and pyruvate. A significant age-related decrease was observed in all profiled metabolites. These changes in metabolic profiles occurred even before any detectable neurodegenerative phenotype, and were also observed in age-matched, non-glaucomatous control D2-Gpnmb + retinas (Figures 5A to 5D).

[0208] Since these are key molecules in cellular metabolism and protection against cellular stress, this age-dependent decline is thought to sensitize retinal neurons to disease-related stress and mitochondrial dysfunction.

[0209] The data show that HIF-1α, a key metabolic regulator during disrupted redox status, was induced in the ganglion cell layer in early-stage glaucoma (by RNA-seq and immunostaining: Figures 6A and 6B). This further suggests that RGCs undergo metabolic disruption (and subsequent excessive production of reactive oxygen species) in early glaucoma.

[0210] To study the link between elevated IOP, mitochondrial dysfunction, metabolite depletion (particularly NAD) and cellular stress, the role of DNA damage and PARP (poly ADP-ribose polymerase) in early glaucoma was investigated. PARP responds to DNA damage and is a major consumer of intracellular NAD.

[0211] RNA-seq datasets suggest that RGCs potentially transition to fatty acid metabolism after a period of mitochondrial stress and metabolite removal (consistent with increased lipid deposition in the retina during early glaucoma, Figures 7A and 7B).

[0212] One consequence of fatty acid β-oxidation is increased free radical / reactive oxygen species (ROS) production. Therefore, the retina was further evaluated for signs of ROS-induced DNA damage (RNA-seq, Figure 3F; and γ-H2AX immunostaining, a highly sensitive marker of dsDNA cleavage). γ-H2AX + The nuclei were found to be present throughout the ganglion cell layer of the D2 retina but absent in the control, indicating that DNA damage increases very early in glaucoma (Figures 8A and 8B).

[0213] PARP activity was found to be induced in RGCs with age (Figures 9A and 9B), providing a link between DNA damage and increased metabolic stress in RGCs. This finding suggests that PARP is induced by DNA damage and is a major NAD activity. + It is a consuming enzyme, and this is consistent with the hypothesis that it inhibits glycolysis by inhibiting hexokinase PAR.

[0214] The D2 mouse model clearly supports the paradigm that age is a major risk factor for glaucoma and various other neurodegenerative diseases. Metabolic profiling data suggest how aging may increase neuronal vulnerability to damage by depleting key neuronal metabolites.

[0215] Overall, the data presented herein support a novel model in which age-dependent declines in NAD and glutathione in the retina make RGCs sensitive to glaucoma and possibly other age-related diseases. RGCs are particularly susceptible to harmful high IOP and glaucoma. Thus, age-dependent metabolite declines and PARP activation within RGCs lead to disruptions in cellular metabolism and increased susceptibility to sustained IOP-induced stress, resulting in critical damage to RGCs.

[0216] In D2 mice, glaucoma neurodegeneration is senile, chronic, and asynchronous. During the development of D2 glaucoma, regenerative cells (RGCs) undergo early loss of connectivity, as well as metabolic and molecular changes, all of which precede the loss of whole cell bodies, axons, and optic nerve degeneration. These early changes appear to reduce the certainty of cellular metabolism and increase the probability of age-related cellular failure when RGCs are under sustained IOP-induced stress.

[0217] In summary, based on RNA sequencing and metabolic assays, we discovered that neurons in the retina undergo a metabolic crisis before degeneration. This suggests that metabolic molecules capable of altering or supplementing mitochondrial function play a key role. Correcting these dysfunctional processes may have benefits beyond glaucoma and are likely related to more general age-related changes.

[0218] Data to date have shown that a decline in NAD is central to senile neuronal vulnerability to glaucoma. [Example 3] NAM protects against neuronal cell loss in axonal transection culture. In this series of studies, we investigated whether increasing NAD levels could protect affected eyes from neurodegenerative changes. Axonal transection (i.e., severing axons) mimics the acute, severe invasiveness seen in some glaucomas and is an important model for testing these more severe forms of invasiveness.

[0219] This study is based on the hypothesis that reducing the probability of metabolic / energy deficiency should provide stronger RGC (Regional Regulatory Capacity) against external stress. Large doses of drugs acting in mitochondria were tested in axon-stretched cultures to identify candidate drugs that potentially antagonistize mitochondrial dysfunction / energy crisis observed in D2 mice. Screening identified nicotinamide (NAM) as providing the most potent protection against nuclear contraction, a prominent feature of nuclear remodeling preapoptosis. Figures 22A and 22B.

[0220] The above in vitro findings are consistent with the in vivo results in Example 3. [Example 4] NAM reduces glaucoma in D2 mice. We conducted in vivo experiments using a large cohort of D2 mice.

[0221] In this experiment, the optic nerve was classified into three levels of damage: NO (no glaucoma), MOD (moderate damage), and SEV (severe damage). The experimental D2 mice were divided into the following groups and given to the mice: W = Water (standard mouse water) NAM or NAM Lo = 550 mg / kg / day of NAM in drinking water Early stage = Early initiation = Pre-glaucoma = 6 months of age (i.e., preventative) Late stage = late stage onset = glaucoma in progress = 9 months of age (when mice already have high IOP, i.e., interventional, more related to human glaucoma) NAD levels were increased in D2 mice by administering nicotinamide (NAM; a precursor of NAD) (see Figures 5A, 11–14, 20A, and 20B). Mice were then evaluated at 12 months for optic nerve damage, cell body loss, visual function, and axonal transport.

[0222] NAM administration in drinking water (550 mg / kg / day, NAM Lo This prevented a decline in NAD levels up to 12 months (the standard final stage for evaluating neurodegeneration in this glaucoma model) (Figures 5A and 5E).

[0223] (A) NAM protects mice from all detectable signs of glaucoma. Supporting the neuronal vulnerability hypothesis, NAM did not alter IOP (Figures 10A-10D) but provided strong protection against glaucoma, i.e., NAM is a neuroprotective agent. Importantly, NAM was protective both prophylactically (started at 6 months, early initiation; before IOP elevation in the majority of eyes in the colony) and as an intervention (started at 9 months, late initiation; when the majority of eyes had persistent IOP elevation). This means that NAM can prevent both neuronal injury from occurring and limit neuronal injury to already affected neurons (Figures 11A and 11B). This is important in human diseases where treatment is only initiated when the disease becomes symptomatic and therefore detectable.

[0224] These data support the possibility of using NAM prophylactically to treat human glaucoma, for example, in subjects at risk of glaucoma due to family history, eye trauma, known genetic mutations, or those who appear to have a higher IOP but lack glaucoma damage.

[0225] (B) NAM strongly prevents retinal ganglion cell dysfunction and degeneration. NAM significantly reduced the incidence of optic nerve degeneration (Figures 11A and 12A), prevented RGC cell body loss and retinal thinning (detectable in human clinics by conventional methods, e.g., OCT and fundus examination) (Figures 12A and 13A), and restored anterograde axonal transport (assessed by Ct-β tracking; an important early marker of axonal dysfunction and degeneration) (Figure 12A) and visual function (PERG) (Figures 14 and 15) as assessed by pattern electroretinography. PERG is highly sensitive and has been shown to be an early assessment criterion for visual impairment in glaucoma in both human and animal models (Saleh et al., Invest Ophthalmol Vis Sci 48, pp. 4564-4572, 2007); therefore, NAM prevents the earliest signs of glaucoma.

[0226] (C)NAM protects against early synaptic loss and lipid droplet formation in the retina. NAM administration also protected against the initial synaptic loss occurring in this model (SNAP-25 immunostaining) (Figures 16A and 16B) and was sufficient to inhibit the formation of dysfunctional mitochondria with abnormal cristae as assessed by EM (Figures 17A and 17B).

[0227] Lipid droplet formation was also prevented in aged D2 retinas (Figure 18). (D)NAM reduces PARP activation and prevents molecular signs of glaucoma. NAM also reduces PARP activation, limited levels of DNA damage, and HIF-1α transcription induction, reflecting less disrupted cellular metabolism (Figures 9A, 9B, 19A, and 19B). NAM likely prevents these damage mechanisms by modifying upstream effectors, namely by removing metabolites from mitochondria and providing redox buffering.

[0228] NAM prevented even the earliest molecular signs of glaucoma in the majority of treated eyes, as assessed by RNA-seq. NAM-treated samples were molecularly similar to controls and clustered within both same-age and younger control samples (Figures 20A, 20B, and 21A–21F).

[0229] (E)NAM prevents senile gene expression in RGCs. NAM also prevented the majority of age-related gene expression changes in RGCs (n=DE gene count; 4-month D2-Gpnmb + vs 9 months D2-Gpnmb + =4699, 9 months D2-Gpnmb + Relative to NAM = 4437, 4-month D2-Gpnmb +(vs NAM = 83). This remarkable degree of molecular protection highlights the unexpected efficacy of NAM in reducing metabolic disruption and the probability of glaucoma in high-IOP eyes. Age is a major risk factor in the pathogenesis of glaucoma, and inhibiting damage to senile changes has the potential to prevent neurodegeneration in many cases of human glaucoma and other gradually degenerating neurodegenerative diseases.

[0230] In the majority of treated eyes, NAM administration completely prevents glaucoma, including results based on highly sensitive assessment criteria for early disease such as PERG. In addition, many ocular and neurodegenerative diseases occur in older adults due to age-dependent molecular potentials that increase susceptibility to damage. NAM treatment prevents senile molecular changes assessed by gene expression (highly sensitive assessment criteria for these changes).

[0231] Furthermore, axonal degeneration and cell body shrinkage may represent common elements in several neurodegenerative diseases. NAM prevents these changes, at least in glaucoma treatment, based on the data above. Moreover, the data above indicates that NAM prevents axonal degeneration and cell body shrinkage in glaucoma.

[0232] [Example 5] Increasing food-derived NAM further reduces the degree of IOP elevation in D2 mice. NAM is considered to be safely tolerable even at high doses. Several studies have suggested an incidence of hepatotoxicity in humans with NAM doses exceeding 4 g per day. However, these were attributed to impurities in stale preparations. In a recent study of 6,000 patients with high doses of nicotinamide or niacin, only three cases of jaundice were reported. In one of these cases (6 g / day niacin), the jaundice resolved after discontinuing a different but concurrently administered drug (not niacin), while in another, the jaundice resolved with continued niacin treatment. For high doses of nicotinamide, abnormal liver enzyme tests did not show liver cell damage, but rather altered liver enzyme expression, suggesting that this rapidly returned to normal upon discontinuation of the drug. Rare cases of hepatotoxicity may reflect individual genetic susceptibility or other individual factors. While the long-term effects of very high doses require further evaluation, experience suggests that the risk-benefit ratio of long-term nicotinamide treatment is very favorable.

[0233] In an attempt to further reduce the probability of glaucoma and protect more eyes from IOP-induced invasiveness, the dose of NAM administered to D2 mice was increased (2000 mg / kg / day, which is four times the original lower dose). Hi ) was used.

[0234] Surprisingly, NAM (at this dose) proved highly protective in 93% of treated eyes without glaucoma-induced optic nerve damage (Figure 11A). This represents a reduction of approximately one-tenth of the risk factors for developing glaucoma. The degree of protection provided by administering this single molecule is unprecedented and quite surprising.

[0235] In mice, NAM at 550 mg / kg / day demonstrated a clear neuroprotective effect (assuming no alteration of IOP), and an increased dose of 2000 mg / kg / day reduced the degree of IOP elevation (Figures 10A and 10B). This indicates that NAM protects against senile pathogenesis in further cell types of RGC.

[0236] These data demonstrate that NAM protects against both increased IOP and neuronal fragility, thus offering a dual benefit and significant clinical potential in the treatment of human glaucoma. Even at a lower dose of 550 mg / kg / day where IOP is not modified, combining NAM supplementation with IOP-reducing measures (such as surgery or eye drops) may provide greater protection against neurodegeneration.

[0237] [Example 6] NAM is effective in two models of RGC death due to glaucoma invasiveness. Glaucoma is a complex disease involving multiple invasive factors. It has a wide range of pathogenesis factors that can affect different RGC compartments (e.g., nerve cell bodies, axons, and dendrites). Mechanical axonal injury and local inflammation represent two important factors in RGC degeneration during glaucoma.

[0238] To evaluate the efficacy of NAM treatment in different contexts, the effectiveness of NAM was tested in two models of RGC death. The first glaucoma invasion model involved the use of a tissue culture model of axonal transection, and the second glaucoma invasion model involved the use of intravitreal injection of soluble mouse TNFα, which drives local inflammation and is associated with glaucoma.

[0239] NAM strongly protected cultured retinas from degeneration of RGC neuronal cell bodies (Figures 22A and 22B). NAM also protected against loss of PERG amplitude and cell loss in TNFα-injected eyes (Figures 23A-23C).

[0240] Given the protection against severe acute trauma and the commonalities between glaucoma and other neurodegenerative diseases, NAM may have broad implications for the treatment of glaucoma and other senile neurodegenerative diseases.

[0241] [Example 7] Nmnat1 gene therapy reduces glaucoma in D2 mice. In this study, Nmnat1, NAD + Overexpression of the key enzyme in production (Figure 25) leads to NAD + This demonstrates support for the cellular mechanism of production.

[0242] 5.5-month-old D2 mice were given a single injection of an AAV2.2 vector containing the Nmnat1 gene and GFP reporter, expressed as a single transcript under the CMV promoter. The mice were anesthetized and the viral vector (3 × 10⁻¹⁴⁻¹ 8 1.5 μL of (U / mL) was injected intravitreously (i.e., posterior to the limbus at a 45-60° angle into the vitreous cavity, avoiding the lens and central retina). Both eyes were injected. Immediately after injection, hydrated eye drops were administered topically, and the mice were kept awake under a sun lamp. The eyes were clinically examined at multiple time points after injection to confirm that there was no eye damage. Visual function was assessed (by PERG) before and after injection to confirm that there were no adverse effects from the initial injection and viral transfection.

[0243] Nmnat1 expression (assessed by GFP expression) was detectable at least 1 week after AAV2.2 injection, was potent in RGCs 2 weeks after injection (expressed in over 83% of RGCs), and remained potent until the final stage (12 months). The majority of RGCs were transduced and expressed the virus-delivered gene product as evidenced by GFP fluorescence in the RGC cell neuronal bodies in the retina and at their terminal points in the brain, including the lateral geniculate nucleus (LGN) and superior colliculus (sup.col.).

[0244] Overexpression of Nmnat1 was sufficient to prevent axonal and neuronal cell body loss (Figures 24A-24C and 24D, upper panel) and preserve axonal plasmotransport and electrical activity (PERG) in RGCs (Figures 24B and 24D, lower panel). Glaucoma nerve damage was absent in over 70% of the treated eyes. This strong protection encourages the use of similar gene therapy strategies to prevent human glaucoma.

[0245] [Example 8] Combination therapy using Nmnat1 gene therapy and NAM In this study, by combining the experiments detailed in Example 4 and Example 7, Nmnat1 and NAM(NAM LoThe effects of the combination of therapies were investigated. Briefly, mice received gene therapy as described above, and after a one-week viral shedding period, the mice were returned to normal colonies and administered 550 mg / kg / day of NAM in normal drinking water.

[0246] This combination provided significant additional protection, with 84% of eyes showing no detectable glaucoma damage at 12 months of age. This represents a reduction of approximately one-quarter in the risk of developing IOP-induced glaucoma neurodegeneration compared to untreated D2 controls.

[0247] Increasing the dose of NAM in combination with gene therapy is even more protective. [Example 9] Wld S This reduces glaucoma in D2 mice. Allele with slow Waller degeneration (Wld S ) showed partial protection in D2 glaucoma. WLD S The protein is a modified NMNAT1 protein (an enzyme that converts NMN to NAD). The mutant protein is WLD. S Cells containing this substance show increased enzyme activity, converting NMN to NAD more quickly or efficiently.

[0248] In this experiment, Wld S In D2 mice with mutations, NAM and Wld S The combination is Wld S It was demonstrated that it provides better protection against glaucoma than when administered alone. When NAM was administered, Wld S Mice carrying the mutation show significant protection from glaucoma damage (approximately 95% are glaucoma-free).

[0249] The experiment was basically carried out in the same manner as in Example 4, but the allele with slower Waller degeneration (Wld S D2 mice with the following characteristics were used. The results are also shown in Figure 11B. Wld S It is clear that either NAM alone or NAM alone provides protection against D2 glaucoma, but Wld S The combination of and NAM is WldS It is significantly superior to single-agent use.

[0250] In particular, NAM alone is approximately 70% protective against glaucoma. In contrast, the combination of NAM and Wld S is approximately 95% protective against glaucoma. Both NAM and NAM+Wld S also protect all parts of cells and the optic nerve (data not shown). Therefore, although NAM and Wld S each exert partial protective effects, the combination exhibited a significant synergistic effect (that is, from approximately 70% protection to approximately 95% protection).

[0251] Without being bound by any particular theory, Wld S the increase in Nmnat expression caused by the mutation is believed to allow conversion of more NAM into NADt, and the combination provides synergistic protection against neurodegeneration in glaucoma.

[0252] Consistent with this theory, in DBA / 2J mice, age- and disease-related reductions are present at the cellular level of NADt and other TCA / Krebs cycle components (such as pyruvate, Figure 5D). Therefore, cellular levels of NADt (NAD + / NADH) were determined in treated animals.

[0253] Levels of NADt (NAD + / NADH) were found to be restored in NAM-treated mice and Wld S mice. When Wld S mice are further treated with NAM, the restoration of NADt levels is synergistic in NAM+Wld S treated mice (see Figure 5E).

[0254] The results of this example are consistent with those of Example 8. NADt (NAD + ​N / NADH levels are thought to change in aging and neurodegenerative diseases. The data obtained herein demonstrate that administration / supplementation of NAM, NAM derivatives and / or NMNAT enzymes may be protective across a wide range of aging and disease phenotypes.

[0255] [Example 10] Pyruvate reduces / prevents glaucoma in D2 mice. This example demonstrates that a combination of nicotinamide (vitamin B3) and / or pyruvate (a simple metabolite of glucose) provides potent protection to DBA / 2J mice from vision loss, loss of axonal transport, and retinal and optic nerve damage. Gene expression experiments demonstrate that mice treated with nicotinamide are more molecularly contiguous to younger control mice than non-glaucoma mice of the same age. The data suggest that nicotinamide works to some extent through an age-dependent mechanism and may delay the age-dependent increase in susceptibility to glaucoma. The scale of protection provided by NAM is entirely unexpected and astonishing.

[0256] Pyruvate is a simple α-keto acid that is important for metabolism, as it is produced from glucose. Pyruvate is converted to acetyl coenzyme A, the main input to the Krebs cycle (citric acid cycle). Under normal oxygen conditions, pyruvate increases NADH levels.

[0257] Pyruvate levels in the retina decrease with age and make retinal neurons more sensitive to glaucoma damage caused by high intraocular pressure (Figure 5D). Mice administered pyruvate in normal drinking water showed increased pyruvate levels in the retina (Figure 5D). Mice administered pyruvate in normal drinking water were resistant to optic nerve axonal degeneration, cell loss, and visual dysfunction in glaucoma (assessed by PERG) (Figures 11B, 12B, 13B, and 14).

[0258] The above findings demonstrate increased efficacy in mice administered both NAM and pyruvate, illustrating the synergistic effect of NAM and pyruvate treatment for preventing neuronal degeneration. [Example 11] PQQ prevents nuclear diameter shrinkage and decrease in cell density. Further neuroprotective agents were tested, and their roles in neurodegenerative diseases were also determined.

[0259] In one experiment, a similar experiment was set up to determine the neuroprotective function of pyrroloquinoline quinone (PQQ). PQQ is an important redox cofactor, similar to nicotinamide. PQQ promotes mitochondrial biosynthesis, and its neuroprotective function is thought to be determined by its function as an antioxidant. Figure 26 shows that PQQ is protective in retinal tissue culture.

[0260] [Example 12] Glutathione levels in the retina decrease with age. N-acetylcysteine ​​(N-acetyl-L-cysteine ​​or NAC) is a precursor to L-cysteine ​​and glutathione, a strong biological antioxidant, and a major redox buffer in neurons. The data in Figure 5B shows that glutathione levels in the retina decrease with age.

[0261] NADPH (produced from NAD) is required for GSH synthesis from GSSG. Therefore, NAD and GSH levels are essentially linked. Materials and methods 1. Mouse strains, breeding, and management Mice were reared with continuous feeding of feed and water in a 14-hour light / 10-hour dark cycle, as previously reported (Howell et al., Journal of Clinical Investigation 121, pp. 1429-1444, 2011). All breeding and experimental procedures were carried out in accordance with the Association for Research for Vision and Ophthalmology Statement for Use of Animals in Ophthalmic Research. This study was approved by the Institutional Safety Committee and Animal Experimentation Committee of Jackson Laboratory.

[0262] C57BL / 6J(B6), DBA / 2J(D2) and DBA / 2J-Gpnmb R1 50X (D2-Gpnmb + ) Using the same system, which was described in detail elsewhere (Anderson et al.) , Nature Genetics 30, pp. 81-85, 2002). D2-Gpnmb + The mice did not develop high IOP and therefore did not develop glaucoma neurodegeneration (Libby et al., Vis Neurosci 22, pp. 637-648, 2005). They are genetically matched controls to DBA / 2J mice.

[0263] In experiments involving aged glaucoma, mice were given NAM in diet and / or water, starting at 6 months (prophylactically, before IOP elevation in almost all eyes) or 9 months (when the majority of eyes had high IOP and molecular changes, but no detectable neurodegeneration) (Howell et al., Journal of Clinical Investigation 121, pp. 1429-1444, 2011). These molecular changes preceded PERG deficiency and loss of anterograde axonal protoplasmic transport. Low-dose NAM (NAM) Lo 550 mg / kg / day of PanReac AppliChem was dissolved in 350 mL of regular acidic drinking water and changed once a week. High-dose NAM (NAM HiFor a dosage of 2000 mg / kg / day, NAM was available in both water (550 mg / kg / day) and feed (1450 mg / kg / day), and was changed once a week. The NAM feed was prepared using a Western-style diet specially ordered for palatability, as previously published (LabDiet) (Graham et al., Sci Rep 6, pp. 21568, 2016). Control mice received the same diet without NAM supplementation. This diet had no effect on the eye health of the mice.

[0264] 2. RGC FAC sorting Prior to cell collection, all surfaces and volumes were cleaned with 70% ethanol and RNaseZap (ThermoFisher Scientific) solution, followed by dH2O. Mice were euthanized, their eyes were removed, and immediately placed in ice-cold HBSS. The retinas were dissected from the eyes into the ice-cold HBSS (4 or 9 months of age, confirmed to be free of axonal degeneration by PPD staining; data not shown) and placed directly into 100 μL of specially ordered HBSS (Gibco), dispase (5 U / mL) (Stemcell Technologies), DNase I (2000 U / mL) (Worthington Biochemical), and SUPERase (1 U / μL) (ThermoFisher Scientific) solution. All retinas were derived from eyes free of glaucoma and axonal degeneration by PPD staining and analysis (see optic nerve assessment below). The retina was incubated at 37°C for 20 minutes, shaken at 350 RPM in an Eppendorf Thermomixer R, and then gently ground using a 200 μL pipette. The samples were blocked with 2% BSA, SUPERase (1 U / μL) in HBSS and stained with conjugated antibodies against Cd11b, Cd11c, Cd34, Cd45.2, GFAP, Thy1.2, and DAPI. This cocktail allowed for precise removal of other retinal cell types during FACS.

[0265] FACS was performed using FACSAria (BD Biosciences). Thy1.2 +RGCs (and negative for all other markers) were sorted in 300 μL of buffer RLT + 1% β-ME, vortexed, and frozen at -80°C until further processing.

[0266] 3. RNA sequencing FAC-sorted RGC samples were thawed on ice and homogenized in RLT buffer using a syringe (total volume 300 μL). Total RNA was isolated using the RNeasy Microkit (Qiagen) according to the manufacturer's procedure, including the optional DNase treatment step, and its quality was assessed using an Agilent 2100 Bioanalyzer. Concentrations were determined using Ribogreen Assay (Invitrogen). Amplified dscDNA libraries were prepared using the Nugen Ovation RNA-seq System V. SPIA dscDNA was analyzed using Diogenode The samples were sheared to a length of 300 bp using a Disruptor. Quality control was performed using an Agilent 2100 Bioanalyzer and a DNA 1000 chip assay.

[0267] The size of the produced libraries was analyzed using qPCR with the Library Quantitation Kit / Illumina GA / ABI Prism (Kapa Biosystems). The libraries were barcoded, pooled, and then analyzed using HiSeq. Using a 2500 sequencer (Illumina), six samples were sequenced per lane, resulting in a read depth of 30,000,000 to 35,000,000 per sample.

[0268] 4. Differential gene expression and pathway analysis The samples were subjected to quality control analysis using a custom-ordered quality control Python script. Reads with 70% of the bases having a base quality score of ≥30 were retained for further analysis. Read alignment was performed using TopHat v2.0.7 (Kim et al., Genome). Expression evaluation was performed using Biol 14, R36, 2013, and HTSeq (Anders et al., Bioinformatics 31, pp. 166-169, 2015) with the annotations and initial settings given to the DBA / 2J mouse genome (build-mm10). Mapping statistics were calculated using Bamtools v 1.0.2 (Barnett et al., Bioinformatics 27, pp. 1691-1692, 2011).

[0269] After batch correction using RUVSeq to remove underexpressed genes by removing outlier samples and genes with fewer than 5 reads in samples with more than 2 reads, differential gene expression analysis between groups was performed using edgeR v3.10.5 (Robinson et al., Bioinformatics 26, pp. 139-140, 2010). Standardization was performed using the trimmed mean M value (TMM).

[0270] Unsupervised HC was performed in R (1cor, Spearman's ρ). A total of 72 samples across all groups were successfully amplified, sequenced, and after the HC pipeline, 9 samples were removed as outliers. Adjustments for multiple trials were made using the false discovery rate (FDR). Genes were considered to have significantly differential expression if FDR < 0.05.

[0271] To study age-related changes, a comparison was made between 4-month and 9-month D2-Gpmnb+ samples and NAM. LoThe analysis was performed across samples. For pathway analysis, QIAGEN's Ingenuity Pathway Analysis (IPA, Qiagen) was used to generate a network of all significantly differentially expressed genes. A list of significantly differentially expressed genes was uploaded to IPA and mapped to mouse Entrez Gene Symbols using the IPA knowledge base. These targets were then superimposed onto standard pathways developed from the information contained in the IPA knowledge base. The generated networks were ranked using IPA's built-in network scoring algorithm.

[0272] 5. Metabolic phenotypic testing GSH / GSSG and NAD + For NADH quantification, the retina was isolated (as described above), and the compound was measured according to the manufacturer's instructions (GSH / GSSG, Cayman Chemical; NAD + (NADH, Biovision). Results were calculated using standard curves generated by using the kit standards. Final metabolite concentrations for each sample were standardized against the total protein concentration measured by the Bradford assay.

[0273] 6. Whole Retinal Explant Culture Mice were euthanized, their eyes were removed, and immediately placed in ice-cold HBSS. The retina was dissected from the eye into the ice-cold HBSS, placed flat on a cell culture insert (Millipore) with the ganglion cell layer facing upward, and immersed in tissue culture medium containing Neurobasal-A, 1% penicillin streptomycin (10000 U / mL), 1% glutamine (100×), 1% N-2 (100×), and 1% B-27 (50×) (all ThermoFisher Scientific).

[0274] For drug-treated retinas, the tissue culture medium was prepared as described above and supplemented with one of the following compounds: β-NAD, NAM (PanReac AppliChem), or β-NMN (Sigma unless otherwise specified). The retinas were incubated in a 6-well plate at 37°C and 4% CO2 for 5 days, then fixed in 4% PFA and stained with DAPI. (For untreated "day 0" controls, the retina was dissected and placed directly in 4% PFA.) The retinas were imaged using a Zeiss AxioObserver.

[0275] 7. Soluble mouse TNFα injection To induce delayed retinal ganglion cell degeneration, soluble mouse TNFα (1 ng / μL) was injected intravitrically as described above (Nakazawa et al., J Neurosci 26, pp. 12633-12641, 2006). Ten-week-old B6 mice were pre-treated with NAM for two weeks prior to TNFα injection, and PERG was performed at 8, 10, and 12 weeks of age. Mice were sacrificed at 12 weeks, and RGC counting was performed.

[0276] 8. Clinical Phenotyping Elevated IOP in D2 mice follows pigment-dispersive iris disease. In all experiments, the progression of iris disease and intraocular pressure in mutant or drug-treated mice was compared to control D2 mice as described above (John et al., Invest Ophthalmol Vis Sci 39, pp. 951-962, 1998). Iris disease and intraocular pressure were evaluated in each experiment.

[0277] Iris disease was evaluated at 2-month intervals, starting at 6 months of age and continuing until the end of the experiment. Intraocular pressure was measured at 45-day intervals, starting at 8.5-9 months of age and continuing until the end of the experiment. 9. Pattern electroretinography (PERG) As previously reported, PERG was recorded subcutaneously through the nasal or oral region. Briefly, a pattern stimulus generated by an LED panel (0.05 grid periods / degree, 100% contrast) was shown separately to each eye at slightly different frequencies of approximately 1 Hz. The waveforms were read using an asynchronous averaging method (Chou et al., Invest Ophthalmol Vis Sci 55, pp. 2469-2475, 2014).

[0278] Mice were anesthetized using ketamine / xylazine (Savinova et al., BMC Genet 2, p. 12, 2001), and their body temperature was maintained at 37°C on a feedback-controlled heating platform monitored by a rectal thermometer.

[0279] 10. Assessment and determination of optic nerve damage in glaucoma. The processing of optic nerves and staining with paraphenylenediamine (PPD) have been published (Smith et al., *Systematic evaluation of the mouse eye*, *Anatomy, pathology and biomethods*, CRC Press, Boca Raton, 2002). PPD stains the myelin sheath of all axons, but darkens the protoplasm of damaged axons. It has been established to provide a highly sensitive evaluation criterion for optic nerve damage (Smith et al., *Systematic evaluation of the mouse eye*, *Anatomy, pathology and biomethods*, CRC Press, Boca Raton, 2002).

[0280] In short, the intracranial portion of the optic nerve was fixed in 4% PFA for 48 hours using RT, processed, and embedded in plastic. Sections of the optic nerve from within 1 mm of the posterior surface of the sclera were prepared (1 μm thick sections) and stained with PPD. Generally, 30 to 50 sections were taken from each nerve.

[0281] When determining the level of damage, multiple sections of each nerve were considered. The optic nerve was analyzed and determined to have one of three levels of damage: (1) No damage or early damage (NOE) - Less than 5% of axons are damaged and there is no gliosis. This level of damage is seen in non-glaucoma mice of the same age and sex and is not due to glaucoma. None of these eyes exhibit glaucoma nerve damage, but some of these eyes have early molecular changes that precede neurodegeneration, so this level of damage is called no-glaucoma or early glaucoma. These molecular changes can be detected by gene expression studies (Howell et al., Journal of Clinical Investigation 121, pp. 1429-1444, 2011). In this specification, when discussing metabolic, mitochondrial, and gene expression changes, eyes with these early molecular changes but no degeneration are considered to have early glaucoma.

[0282] (2) Moderate injury (MOD) - average 30% axonal loss and early gliosis. (3) Severe (SEV) → Injury with 50% axonal loss and significant gliosis. 11. Anterior axonal transport Mice were anesthetized with ketamine / xylazine and intravitreously injected with 2 μL of AF488 or AF594 cholera toxin subunit B (1 mg / mL in PBS) (ThermoFisher Scientific). After 72 hours, the mice were anesthetized and euthanized by 4% PFA cardiac perfusion. The brains and eyes were fixed in 4% PFA for a further 24 hours, frozen overnight (ON) in 30% sucrose in PBS, cryoembedded using OCT, and sectioned to 20 μm. AF488 was visualized using Zeiss AxioObserver or Zeiss AxioImager.

[0283] 12. Tissue examination For immunofluorescence staining, mice were euthanized by cervical dislocation, their eyes were removed, and placed in 4% PFA for ON staining. The retina was dissected, placed flat on a slide, permeabilized with 0.1% Triton-X for 15 minutes, blocked with 2% BSA in PBS, and stained with RT in primary antibody for ON staining (see below for antibody list).

[0284] After primary antibody incubation, the retina was washed five times with PBS and stained with secondary antibody in RT mode for 4 hours. The slide was then washed five more times with PBS, stained with DAPI for 15 minutes, mounted in a fluoromount, covered with a coverslip, and sealed with nail polish.

[0285] For retinal sections, the eye was kept freezable in 30% sucrose, frozen using OCT, and prepared as 18 μm frozen sections. The slides were warmed to room temperature and the procedure described above was followed. The retina was imaged with a Zeiss AxioObserver at low resolution count. The retinal sections were imaged with a Leica SP8 at high resolution.

[0286] For Nissl staining, frozen sections were warmed to room temperature, placed in a 1:1 alcohol:chloroform solution, and rehydrated by a continuous alcohol gradient. The slides were washed once with distilled water, separated with 95% alcohol before staining with 0.1% cresyl violet in distilled water for 15 minutes, dehydrated with 100% alcohol, and cleared with xylene. The slides were prepared as described above. Nissl-stained retinal sections were imaged using a Nikon Eclipse E200.

[0287] For oil red O staining, slides were warmed to room temperature, quickly washed with 60% isopropanol, incubated in oil red O and hematoxylin at RT for 15 minutes, washed again with 60% isopropanol, mounted, and covered with a coverslip. Retinal sections stained with oil red O were imaged using a Nikon Eclipse E200.

[0288] 13. Western blotting Mice were euthanized by cervical dislocation, their eyes were removed, and they were immediately placed in ice-cold HBSS. The retinas were dissected from the eyes into the HBSS, directly added to RIPA buffer, and homogenized. Protein extracts were separated by SDS-PAGE on a molded gel (Bio-Rad) and transferred to a PVDF membrane using iBlot 2 (ThermoFisher Scientific). The membranes were blocked in 5% milk or 5% BSA in 0.1% PBS-Tween, and antibody incubation was performed in the blocking solution. Antibody staining was detected using ECL. Protein concentrations were evaluated by the Bradford assay and normalized to housekeeping proteins using densitometry.

[0289] 14. Electron Microscope Observation Mice were euthanized, their eyes were removed, and then ON-fixed in Smith-Rudt (0.8% PFA and 1.2% glutaraldehyde in 0.1M phosphate buffer). The cornea and lens were dissected, and the remaining posterior eye-cup (containing the retina and ONH) was post-fixed with 2% aqueous osmium tetroxide and dehydrated with ethyl alcohol. The samples were then infiltrated and placed flat on Embed 812 / Araldite resin (Electron Microscopy Sciences) to form frontal sections. The blocks were polymerized at 60°C for 48 hours, and 90 nm microtome sections were cut onto a 300-mesh copper grid (Leica UC6, Leica). The grids were stained with 1% aqueous uranyl acetate / Reynold lead citrate and viewed with a JEOL JEM 1230 TEM. Images were captured using an AMT 2K camera.

[0290] 15. Gene therapy In the case of gene therapy delivered by a virus, mice are anesthetized (as described above), and 1.5 μL (3.1 × 10⁻¹) of AAV2.2 mouse Nmnat1 under the CMV promoter is administered along with the GFP reporter. 10The drug was injected intravitreously at a concentration of gc / mL (referred to as Nmnat1 in the main text; Vector Biolabs#ADV-265880). Mice were injected at 5.5 months of age in a BSL2 laboratory and moved to a normal mouse colony two weeks later.

[0291] In Nmnat1 mice undergoing further NAM treatment, the mice were given 550 mg / kg / day of NAM in normal drinking water starting at 6 months of age. The mice were euthanized at 12 months of age. 16. Antibodies The antibodies used in the above examples are listed below. IF: Immunofluorescence. Wb: Western blot.

[0292] [Table 6]

[0293] 17.Statistical analysis The sample size (number of eyes, n) is shown in the legend for each figure. Graphing and statistical analysis were performed using R. Student's t-test was used for pairwise analysis in quantitative plots, and error bars refer to the standard error of the mean unless otherwise specified.

[0294] All referenced documents are incorporated by reference. While specific embodiments of the present invention have been described above, it will be acknowledged to those skilled in the art that many equivalents, modified forms, substitutions, and variations thereof can be obtained without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for treating glaucoma in a subject requiring treatment, comprising the step of administering a pharmaceutical composition containing a therapeutically effective amount of nicotinamide (NAM) to the subject, thereby treating the glaucoma.

2. The method according to claim 1, wherein the pharmaceutical composition further comprises pyruvic acid.

3. The method according to claim 2, wherein the pharmaceutical composition further comprises one or more compounds selected from the group consisting of nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD), and nicotinamide ribose (NR).

4. The method according to claim 1, wherein the NAM is present in a therapeutically effective amount for reducing neurodegeneration of retinal ganglion cells.

5. The method according to claim 1, wherein the NAM is present in a therapeutically effective amount for reducing intraocular pressure.

6. The method according to claim 2, wherein the NAM and pyruvate are present in therapeutically effective amounts for reducing neurodegeneration of retinal ganglion cells.

7. The method according to claim 2, wherein the NAM and pyruvate are present in therapeutically effective amounts for reducing intraocular pressure.

8. The method according to claim 1, wherein the subject is a human subject.

9. The method according to claim 1, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

10. The method according to claim 9, wherein the polynucleotide is located within the viral vector.

11. The method according to claim 10, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, a lentiviral vector, or a retrovirus vector.

12. The method according to claim 10, wherein the viral vector is AAV.

13. The method according to claim 10, wherein the viral vector is AAV2.

2.

14. The method according to claim 10, wherein the viral vector is a lentiviral vector.

15. The method according to claim 9, wherein the gene composition is administered intravitreously or intraocularly.

16. The method according to claim 9, wherein the gene composition is administered into the vitreous body.

17. The method according to claim 2, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

18. The method according to any one of claims 1 to 17, further comprising the step of administering an additional therapeutic agent to the subject.

19. The method according to claim 18, wherein the additional therapeutic agent is a β-receptor blocker, a non-selective adrenergic agonist, a selective α-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathetic agonist, a carbachol, or a combination thereof.

20. The method according to claim 18, wherein the additional therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinephrine, apraclonidine, brimonidine, acetazolamide, metazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, ecothiophate iodide, carbachol, or a combination thereof.

21. A method for preventing glaucoma in a subject requiring prevention, comprising the step of administering a pharmaceutical composition containing a therapeutically effective amount of nicotinamide (NAM) to the subject, thereby preventing glaucoma.

22. The method according to claim 21, wherein the pharmaceutical composition further comprises pyruvic acid.

23. The method according to claim 22, wherein the pharmaceutical composition further comprises one or more compounds selected from the group consisting of nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD), and nicotinamide ribose (NR).

24. The method according to claim 21, wherein the NAM is present in a therapeutically effective amount for reducing intraocular pressure.

25. The method according to claim 22, wherein the NAM and pyruvate are present in therapeutically effective amounts for reducing intraocular pressure.

26. The method according to claim 21, wherein the subject is a human subject.

27. The method according to claim 21, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

28. The method according to claim 27, wherein the polynucleotide is located within the viral vector.

29. The method according to claim 28, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenovirus vector, a lentiviral vector, or a retrovirus vector.

30. The method according to claim 27, wherein the gene composition is administered intravitreously or intraocularly.

31. The method according to claim 27, wherein the gene composition is administered intravitreously.

32. The method according to claim 22, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

33. The method according to any one of claims 21 to 32, further comprising administering an additional therapeutic agent to the subject.

34. The method according to claim 33, wherein the additional therapeutic agent is a β-receptor blocker, a non-selective adrenergic agonist, a selective α-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathetic agonist, carbachol, or a combination thereof.

35. The method according to claim 33, wherein the additional therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinephrine, apraclonidine, brimonidine, acetazolamide, metazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, ecothiophate iodide, carbachol, or a combination thereof.

36. A method for improving visual function in a subject requiring improvement, comprising the step of administering a pharmaceutical composition containing a therapeutically effective amount of nicotinamide (NAM) to the subject, thereby improving visual function.

37. The method according to claim 36, wherein the pharmaceutical composition further comprises pyruvic acid.

38. The method according to any one of claims 36 or 37, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

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