Nicotinamide for the treatment and prevention of ocular neurodegenerative diseases such as glaucoma

Abstract

The present invention relates to the use of pharmaceutical compositions comprising nicotinamide (NAM) and / or pyruvic acid as neuroprotective drugs or gene therapy in the treatment of axonal degeneration of neuronal tissue in neurodegenerative disorders, particularly eye-related neurodegenerative diseases, including glaucoma.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201680075950.8, whose application date is October 24, 2016 and invention name is "Niacinamide for the treatment and prevention of ocular neurodegenerative diseases such as glaucoma".

[0002] Statement Regarding Federally Funded Research

[0003] This invention was made with U.S. government support under grant number R01EY011721 awarded by the National Institutes of Health (NIH). The U.S. government has certain rights in this invention.

[0004] References to related applications

[0005] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 245,467, filed on October 23, 2015, and U.S. Provisional Application No. 62 / 366,211, filed on July 25, 2016, the entire contents of which are incorporated herein by reference in their entirety. Background Art

[0006] Axonal damage is an early event in neurodegenerative diseases. Neurodegenerative diseases are characterized by the dysfunction or loss of active nerve cells from the peripheral or central nervous system. In many cases, axonal degeneration has been shown to precede nerve loss, a process that is always more pronounced at the distal end of axonal processes rather than at the proximal end. The upstream molecular signals that trigger the neurodegenerative cascade in neurons remain unknown.

[0007] There are reports of using nicotinamide adenine dinucleotide (NAD) and its related compounds to reduce axonal degeneration. For example, US2006-0211744 A1 ('744 application) describes the use of agents including NAD, NADH and nicotinamide (NAM) to reduce chronic neurodegeneration. Using a cell culture model of transected dorsal root ganglion (DRG) neuron axons, the '744 application discloses that NAD provides protection against axonal degeneration for these neurons. The '744 application also discloses that NAM reduces neurodegeneration in mouse models for 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 diseases by administering an agent that increases NAD activity in damaged neurons. Using primary cell cultures of dorsal root ganglia (derived from spinal cord nerves) and axotomy (mechanical cutting) at a location close to the soma, the '326 patentee clearly concluded that niacin and NAM did not reduce axonal degeneration. In an ocular injection study, the '326 patentee again showed that NAM did not differ from control animals when injected intravitreally.

[0008] Glaucoma is one of the most common neurodegenerative diseases and represents the leading cause of irreversible blindness, affecting more than 70 million people worldwide (Quigley and Broman, Br J Ophthalmol 90, 262-267, 2006), especially in the elderly. Glaucoma is a complex multifactorial disease characterized by progressive dysfunction and loss of retinal ganglion cells (RGCs) that lead to vision loss. High intraocular pressure (IOP) and increased age represent susceptibility factors for glaucomatous neurodegeneration. Recent advances have hinted at various molecular changes that occur in glaucomatous tissues in the early stages of the disease (Howell et al., Journal of Clinical Investigation 121, 1429-1444, 2011; Nickells et al., Annu Rev Neurosci 35, 153-179, 2012). The early cellular and molecular mechanisms that trigger glaucomatous damage within RGCs are little known. It appears that RGCs are damaged at multiple sites in the early stages of glaucoma, including changes affecting their cell bodies, dendrites, and synapses in the retina, and axons in the optic nerve. It remains unclear how high IOP and aging contribute to neuronal vulnerability and the mechanisms that trigger glaucoma in humans.

[0009] Despite the availability of strategies to mitigate elevated IOP, there are no effective treatments or preventive measures for ocular neurodegeneration. Therefore, there is a continuing need to decipher the upstream molecular signals that trigger the initial neurodegenerative process and to identify novel molecular targets to provide therapeutic interventions that reduce retinal RGC damage and axonal degeneration, particularly for the treatment of glaucoma. Summary of the invention

[0010] The present invention is primarily based on our discovery that nicotinamide (NAM) or pyruvate or a combination thereof has the potential to protect neuronal cell bodies, axons and related cell types and is therefore beneficial in treating neurodegeneration (e.g., axonal degeneration) and ocular neurodegenerative diseases (e.g., glaucoma).

[0011] One object of the present invention is to provide a method for treating or preventing glaucoma, which method comprises the step of administering to an individual in need of treatment a pharmaceutical composition comprising a therapeutically effective amount of NAM and / or pyruvate.

[0012] It is an object of the present invention to provide a method of reducing intraocular pressure, for example in glaucoma, comprising the step of administering to a subject in need of treatment a pharmaceutical composition comprising a therapeutically effective amount of NAM and / or pyruvate.

[0013] One aspect of the present invention is to provide a method of improving visual function in a patient having or suffering from glaucoma, comprising the step of administering to an individual in need of treatment a pharmaceutical composition comprising a therapeutically effective amount of NAM and / or pyruvate.

[0014] Without being bound by a particular theory, the therapeutic treatments (drugs and / or gene therapy) of the present invention are believed to work by reducing intraocular pressure through two main mechanisms: 1) reducing aqueous humor production, and / or 2) increasing aqueous humor outflow. The present therapeutic methods can serve as an effective treatment by reducing high IOP, thereby protecting axons such as the optic nerve and preventing subsequent loss of visual function.

[0015] Therapeutic treatments of the invention may be used to treat or prevent neurodegeneration in glaucoma.

[0016] The glaucoma treatment of the present invention is effective in treating or preventing neurological dysfunction and neurodegeneration at the retinal (e.g., RGC) level. Glaucoma can be present at any intraocular pressure level (high IOP or normal IOP). The present therapy treatment provides an IOP-independent effect of neuroprotection of retinal cells such as retinal ganglion cells (RGCs).

[0017] In certain embodiments, the treatments of the invention prevent and / or slow the progression of glaucoma (eg, primary open angle glaucoma or POAG) by reducing intraocular pressure.Almost all current strategies for treating glaucoma are aimed at reducing or preventing increases in IOP.

[0018] In certain embodiments, the treatments of the invention prevent and / or slow the progression of glaucoma (eg, primary open angle glaucoma or POAG) by providing direct neuroprotection and reducing intraocular pressure.The present therapy provides an unexpected dual benefit in treating glaucoma.

[0019] In certain embodiments, the treatments of the invention prevent and / or slow the progression of glaucoma without reducing intraocular pressure.Thus, the therapies of the invention provide unexpected advantages in that the drug and / or gene therapy provides neuroprotection that is independent of IOP.

[0020] The treatment methods of the present invention provide treatment for glaucoma, such as primary open-angle glaucoma, by preserving the characteristic pattern of optic nerve changes and visual field loss. According to the AAO's preferred practice pattern, two of the three findings (elevated IOP, optic nerve damage, or visual field loss) must be present in the diagnosis of primary open-angle glaucoma.

[0021] The present invention provides a method for treating a neurodegenerative disorder (e.g., an ocular neurodegenerative disease, such as glaucoma) in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising increasing intracellular NADt levels or increasing intracellular NAD + , NADH, GSH, GSSG, PQQ or pyruvate levels.

[0022] In certain embodiments, neurodegenerative disorders (e.g., ocular neurodegenerative diseases, such as glaucoma) involve axonal degeneration, neuronal dysfunction, skin retraction, synaptic loss, dendritic atrophy, and / or normal neuronal aging. In certain embodiments, neurodegenerative disorders (e.g., ocular neurodegenerative diseases, such as glaucoma) involve axonal degeneration.

[0023] In certain embodiments, the neurodegenerative disorder (e.g., an ocular neurodegenerative disease such as glaucoma) involves Wallerian degeneration, Wallerian degeneration, or degeneration of the axon. For example, Wallerian degeneration is caused by neuronal damage. Neuronal damage may be caused by disease, trauma, chemotherapeutic drugs, or neuronal aging.

[0024] In certain embodiments, the neurodegenerative disorder is one or more of Alzheimer's disease, multiple sclerosis, diabetic neuropathy, traumatic brain injury, ischemia, peripheral neuropathy, or an ophthalmic disorder such as glaucoma or an age-related eye disease (e.g., age-related macular degeneration (AMD), Leber optic neuropathy, dominant optic atrophy, cataracts, diabetic eye disease / diabetic retinopathy, retinal degeneration, dry eyes, low vision).

[0025] In certain embodiments, the compounds of the invention comprise nicotinamide adenine dinucleotide (NAD + ) precursor (e.g., nicotinic acid (Na), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR) or a combination thereof).

[0026] In certain embodiments, the compounds of the invention include nicotinamide, pyruvic acid, or pyrroloquinoline quinone (PQQ). It is believed that the compounds of the invention replenish intracellular NADt or improve the mitochondrial electron transport chain.

[0027] In certain embodiments, compounds of the invention include: (a) NAM; (b) pyruvate; (c) PQQ; (d) NAM and pyruvate; (d) NAM and PQQ.

[0028] An object of the present invention is to provide a method of treating a neurodegenerative disorder (e.g., an ocular neurodegenerative disease, such as glaucoma) in an individual in need thereof, the method comprising administering to the individual a gene composition comprising a gene that increases Nmnat expression (e.g., Nmnat-1, Nmnat-2, or Nmnat-3).

[0029] In certain embodiments, the gene is NMNAT1.

[0030] In certain embodiments, the gene is Wld S .

[0031] In certain embodiments, the method comprises administering to an eye of the individual a gene encoding Nmnat (eg, Nmnat-1, Nmnat-2, or Nmnat-3) and / or Wld S polynucleotides.

[0032] In certain embodiments, the polynucleotide is administered topically to a subject.

[0033] In certain embodiments, the polynucleotide is administered to an individual on a viral vector (eg, an AAV vector, an adenoviral vector, a lentiviral vector, a retroviral vector, etc.).

[0034] In certain embodiments, the neurodegenerative disorder is glaucoma or an age-related eye disease; and the individual is a human.

[0035] The present invention provides a method for treating a neurodegenerative disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of one or more compounds that increase intracellular NADt levels, and further comprising administering a gene encoding and expressing Nampt, Nmnat (e.g., Nmnat-1) and / or Wld S polynucleotides.

[0036] An object of the present invention is to provide a method of treating glaucoma in a subject in need thereof, comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of nicotinamide (NAM), thereby treating glaucoma.

[0037] Another related but distinct object of the present invention is to provide a method of preventing glaucoma in an individual in need thereof, the method comprising the step of administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of nicotinamide (NAM), thereby preventing glaucoma.

[0038] In certain embodiments, the NAM is present in a therapeutically effective amount to reduce neurodegeneration in retinal ganglion cells. For example, in certain embodiments, the pharmaceutical composition contains about 0.5-10 grams of NAM, about 1-5 grams of NAM, or about 2.5 grams of NAM for daily consumption.

[0039] In certain embodiments, the NAM is present in a therapeutically effective amount to reduce intraocular pressure. For example, in certain embodiments, the pharmaceutical composition contains about 2-25 grams of NAM, about 10-20 grams of NAM, or about 10 grams of NAM for daily consumption.

[0040] In certain embodiments, the pharmaceutical composition further comprises pyruvic acid. Preferably, the NAM and pyruvic acid are present in a therapeutically effective amount to reduce neurodegeneration in retinal ganglion cells. For example, in certain embodiments, the pharmaceutical composition independently contains (1) about 0.5-10 grams of NAM, about 1-5 grams of NAM, or about 2.5 grams of NAM for daily consumption; and (2) about 0.5-10 grams of pyruvic acid, about 1-5 grams of pyruvic acid, or about 2.5 grams of pyruvic acid for daily consumption. Preferably, the NAM and pyruvic acid are present in a therapeutically effective amount to reduce intraocular pressure. For example, in certain embodiments, the pharmaceutical composition independently contains (1) about 2-25 grams of NAM, about 10-20 grams of NAM, or about 10 grams of NAM for daily consumption; and (2) about 2-25 grams of pyruvic acid, about 10-20 grams of pyruvic acid, or about 10 grams of pyruvic acid for daily consumption.

[0041] 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 riboside (NR). In certain embodiments, when PQQ is present, the pharmaceutical composition comprises about 10 mg-10 g, about 50 mg-1 g, or about 500 mg PQQ for daily consumption.

[0042] In certain embodiments, the methods of the invention (with or without pyruvate) further comprise the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1. In certain embodiments, the polynucleotide is in a viral vector, such as an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

[0043] 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.

[0044] In certain embodiments, the gene composition is administered intravitreally or intraocularly. Preferably, the gene composition is administered intravitreally.

[0045] In certain embodiments, the subject is a human subject.

[0046] In certain embodiments, the subject has an intraocular pressure of about 12-21 mmHg. In certain embodiments, the subject has an intraocular pressure of greater than 21 mmHg.

[0047] In certain embodiments, the individual has not yet developed neurodegenerative symptoms of glaucoma. In certain embodiments, the individual has already developed neurodegenerative symptoms of glaucoma.

[0048] In certain embodiments, the individual has developed a visual impairment

[0049] In certain embodiments, the method further includes applying another therapeutic agent to the individual. Exemplary other therapeutic agents include intraocular pressure reducers. In certain embodiments, the other therapeutic agent is a beta blocker, a non-selective adrenergic agonist, a selective alpha-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathomimetic agonist, carbachol or a combination thereof. In certain embodiments, the other therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinepherine, alaclodinidine, brimonidine, acetazolamide, methazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, echoiodide (echothiophate iodide), carbachol or a combination thereof.

[0050] The present invention also aims to provide a method for improving the visual function of an individual in need thereof, the method comprising the step of administering to the individual a pharmaceutical composition containing a therapeutically effective amount of nicotinamide (NAM), thereby improving the visual function.

[0051] In certain embodiments, the pharmaceutical composition further comprises pyruvate. In certain embodiments, the method further comprises the step of administering a genetic composition, wherein the genetic composition comprises a polynucleotide encoding NMNAT1.

[0052] In particular, the present invention relates to the following:

[0053] 1. A method of treating glaucoma in a subject in need thereof, the method comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of nicotinamide (NAM), thereby treating glaucoma.

[0054] 2. The method according to item 1, wherein the pharmaceutical composition further comprises pyruvate.

[0055] 3. The method according to item 2, wherein the pharmaceutical composition further comprises one or more compounds selected from nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD) and nicotinamide riboside (NR).

[0056] 4. The method of item 1, wherein the NAM is present in a therapeutically effective amount to reduce neurodegeneration in retinal ganglion cells.

[0057] 5. The method of item 1, wherein the NAM is present in a therapeutically effective amount to reduce intraocular pressure.

[0058] 6. The method of item 2, wherein the NAM and pyruvate are present in therapeutically effective amounts to reduce neurodegeneration in retinal ganglion cells.

[0059] 7. The method of item 2, wherein the NAM and pyruvate are present in therapeutically effective amounts to reduce intraocular pressure.

[0060] 8. The method of clause 1, wherein the individual is a human individual.

[0061] 9. The method according to item 1, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

[0062] 10. The method according to item 9, wherein the polynucleotide is in a viral vector.

[0063] 11. The method of item 10, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

[0064] 12. The method of item 10, wherein the viral vector is AAV.

[0065] 13. The method according to item 10, wherein the viral vector is AAV2.2.

[0066] 14. The method according to item 10, wherein the viral vector is a lentiviral vector.

[0067] 15. The method according to item 9, wherein the gene composition is administered intravitreally or intraocularly.

[0068] 16. The method of item 9, wherein the gene composition is administered intravitreally.

[0069] 17. The method according to item 2, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

[0070] 18. The method of any one of items 1-17, further comprising administering an additional therapeutic agent to the individual.

[0071] 19. The method of item 18, wherein the additional therapeutic agent is a beta blocker, a non-selective adrenergic agonist, a selective alpha-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathomimetic agonist, a carbachol, or a combination thereof.

[0072] 20. The method of item 18, wherein the additional therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinephrine, alaclonidine, brimonidine, acetazolamide, methazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, epididymidol, carbachol, or a combination thereof.

[0073] 21. A method of preventing glaucoma in an individual in need thereof, comprising the step of administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of nicotinamide (NAM), thereby preventing glaucoma.

[0074] 22. The method according to item 21, wherein the pharmaceutical composition further comprises pyruvate.

[0075] 23. The method according to item 22, wherein the pharmaceutical composition further comprises one or more compounds selected from nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD) and nicotinamide riboside (NR).

[0076] 24. The method of item 21, wherein the NAM is present in a therapeutically effective amount to reduce intraocular pressure.

[0077] 25. The method of item 22, wherein the NAM and pyruvate are present in therapeutically effective amounts to reduce intraocular pressure.

[0078] 26. The method of item 21, wherein the individual is a human individual.

[0079] 27. The method according to item 21, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

[0080] 28. The method of item 27, wherein the polynucleotide is in a viral vector.

[0081] 29. The method of item 28, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

[0082] 30. The method according to item 27, wherein the gene composition is administered intravitreally or intraocularly.

[0083] 31. The method of item 27, wherein the gene composition is administered intravitreally.

[0084] 32. The method according to item 22, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

[0085] 33. The method of any one of items 21-32, further comprising administering an additional therapeutic agent to the individual.

[0086] 34. The method of item 33, wherein the additional therapeutic agent is a beta blocker, a non-selective adrenergic agonist, a selective alpha-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathomimetic agonist, a carbachol, or a combination thereof.

[0087] 35. The method of item 33, wherein the additional therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinephrine, alaclonidine, brimonidine, acetazolamide, methazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, epididymidol, carbachol, or a combination thereof.

[0088] 36. A method of improving visual function in an individual in need thereof, comprising the step of administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of nicotinamide (NAM), thereby improving visual function.

[0089] 37. The method according to item 36, wherein the pharmaceutical composition further comprises pyruvate.

[0090] 38. The method according to any one of items 36-37, further comprising the step of administering a gene composition, wherein the gene composition comprises a polynucleotide encoding NMNAT1.

[0091] It should be understood that all embodiments described herein may be combined with any other embodiments, unless explicitly disclaimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Shown are the results of hierarchical clustering (HC) for defining molecularly determined stages of glaucoma in DBA / 2J (D2) mice at the initial stage of disease and with age-matched D2-Gpnmb + or young controls are morphologically indistinguishable. HC is based on RNA-sequencing in retinal ganglion cells (RGCs) isolated from D2 mice and controls. HC allows clustering of RGC samples into distinct groups, where controls and young samples are molecularly similar (Spearman rank correlation coefficient). Circles = samples from D2 RGCs, triangles = samples from D2-Gpnmb + Sample of RGCs. Insertion: D2-Gpnmb + The number of differentially expressed (DE) genes between the groups (q < 0.05).

[0093] Figure 2A Four D2 mouse groups (Group 1, Group 2, Group 3, and Group 4) and D2-Gpnmb are shown + Number of differentially expressed (DE) genes in the control group.

[0094] Figure 2B Heatmap correlations are shown for all samples (Spearman rank correlation coefficient, blue = highest correlation, red = lowest correlation). Figure 1 The dendrogram is shown in gray.

[0095] Figure 3A The total ratio of mitochondrial:nuclear reads was shown to increase with increasing distance from the HC in controls (9 month old mice). The results are consistent with the notion that mitochondrial dysfunction is an early driver of RGC damage in glaucomatous eyes.

[0096] Figure 3B Figure 3 shows the relationship between the expression of 9-month-old D2 mice (three 9-month-old D2 mice clusters - Group 2, Group 3 and Group 4) and age- and sex-matched wild-type controls (D2-Gpnmb +) RNA-seq data obtained from , several of the top significantly enriched pathways based on Ingenuity Pathway Analysis (IPA). The larger the -log p value, the greater the enrichment. Two of the top 3 enriched pathways are mitochondrial dysfunction and oxidative phosphorylation. Also see Table 3 for other significantly enriched pathways. Note that there are no differentially expressed pathways in D2 Group 1.

[0097] Figure 3C It is shown that as the HC distance of the D2 group from the control (all 9-month-old mice) increases, the transcript expression of nuclear-encoded mitochondrial proteins increases first. The results are again consistent with the view that mitochondrial dysfunction is an early driver of RGC damage in glaucoma. Points represent individual genes, gray or light points = not differentially expressed, red or dark points = differentially expressed at q<0.05. Genes were taken from mouse MitoCarta2.0 (Calvo et al., Nucleic Acids Res 44, D1251-1257, 2016).

[0098] Figure 3D RNA-sequencing identified increased mitochondrial fission gene transcripts in early stages of glaucoma (9-month-old mice). The results are consistent with the notion that mitochondrial dysfunction is an early driver of RGC damage in glaucoma.

[0099] Figure 3E Shown is the early mitochondrial unfolded protein response compared to controls. Data shown are for D2 Group 4 (9 month old mice). Results are consistent with the notion that mitochondrial dysfunction is an early driver of RGC damage in glaucomatous eyes.

[0100] Figure 3F The results of single gene expression plots showing metabolic and oxidative events in early glaucoma (9 month old mice) are shown. Points represent single genes, grey or light dots = not differentially expressed, red or dark dots = differentially expressed at q < 0.05. Again, oxidative phosphorylation genes were among the genes showing the highest levels of differential expression, and differential expression appeared to be gradually increased in the D2 group with increasing distance from the HC of the control (Group 4 > Group 3 and Group 2).

[0101] Figure 3G and 3H is a summary of the Western blotting protein validation from differentially expressed genes (n = 4 / age group) (respectively Fig. 3I and 3J ) is a bar graph of the data. Cytochrome c ( Figure 3G ) and eIF2a( Figure 3H) correlates with transcript abundance assessed by RNA-sequencing. The vertical axis in both bar graphs is relative to the density of β-actin. * = P < 0.05, ** = P < 0.01.

[0102] Figure 3K Oxidative phosphorylation genes are shown to be differentially expressed in all groups, with increasing expression in the D2 group as the distance from the HC of the control increases (Group 4 > Group 3 > Group 2 > Group 1). Red = highest expression, blue = lowest expression, IV = mitochondrial complex IV (listed in Table 1), G = D2-Gpnmb + , 1-4 = D2 groups 1-4.

[0103] Figure 4A and 4B D2 mice at 9 months of age show reduced cristae volume in RGC somatic and dendritic mitochondria. However, there was no significant difference in total mitochondrial size / volume. Scale bar = 350 nm. ** = P < 0.01, * = P < 0.001.

[0104] Figure 5A Display in NAM Lo NAD(t) levels were increased in D2 mice (n=22 / group) treated (550 mg / kg / day). *=P<0.05, ***=P<0.001.

[0105] Figure 5B The GSH / GSSG levels were shown to decrease with age (n=22 / group). *=P<0.05, ***=P<0.001.

[0106] Figure 5C Shown in D2-Gpnmb + NAD(t) levels in control mice (n=22 / group) also decreased with age. ***=P<0.001.

[0107] Figure 5D Showing age-related decrease in pyruvate levels restored by pyruvate treatment. D2 mice were treated with 500 mg / kg / d pyruvate in normal drinking water starting at 6 months of age.

[0108] Figure 5E The total NAD (NAD(t)) in the D2 retina (i.e., NAD + +NADH) decreased with age, and NAM treatment (550 mg / kg / day) or addition of Wld s Allele recovery. s The combination with NAM provides additional benefits.

[0109] Fig. 6A and6B It is shown that NAM treatment (550 mg / kg / day) alleviates HIF-1α upregulation in early glaucoma as assessed by immunostaining (n=6 / group). *=P<0.05, ***=P<0.001. The results suggest that NAM treatment can prevent the early destructive changes in D2 glaucoma.

[0110] Fig. 7A RNA-sequencing identifies changes in cellular metabolism, specifically, alterations in fatty acid metabolism genes in D2 glaucoma. Points represent individual genes, grey or light dots = not differentially expressed, red or dark dots = differentially expressed at q < 0.05.

[0111] Figure 7B Shows intraretinal extracellular lipid droplet formation in IOP-impaired aged D2 eyes stained with Oil Red O. Staining is present in D2 eyes with no optic nerve degeneration (NOE) or no severe (SEV) optic nerve degeneration. Extraocular fat was used as a positive control (n=6 / group). Scale bar=25 μm.

[0112] Fig. 8A and 8B Increased DNA damage levels assessed by γH2AX staining are shown (n=6 / group). Results are presented as a bar graph of maximal intensity (AU) of γH2AX staining. ***=P<0.001.

[0113] Fig.9A and 9B NAM was shown to block PARP activation in glaucoma. PARP is an NAD + PARP is a major consumer of NAD(t) and may contribute to the age-related decrease in NAD(t) in RGCs. PARP expression (pan-PARP immunostaining) was reduced in NAM-treated retinas (n=6 / group) following NAM administration (550 mg / kg / day). ***=P<0.001. Scale bar=15 μm.

[0114] Fig. 10A and 10C (IOP Profile), 10B and 10D (Clinical Manifestations of Iris Disease with Elevated IOP) show that the protective strategy did not alter clinical disease progression / manifestations in the treated eyes. Iris disease progressed at a similar rate and reached a severe state in all groups in the same time. NAM Lo =550mg / kg / day. NAM Hi 2000 mg / kg / day. Nmnatl1 = Gene therapy by expressing exogenous Nmnat1 (on a viral vector).

[0115] Fig.11ANAM is shown to protect against optic nerve degeneration. Green or lower portion of the bar = no glaucoma or early glaucoma (NOE) (stage with no nerve damage), yellow or middle portion of the bar = moderate damage (MOD), red or top portion of the bar = severe (SEV) damage. Fisher's exact test: ** = P < 0.01, *** = P < 0.001. NAM Lo =550mg / kg / day. NAM Hi = 2000 mg / kg / day. Early start = treatment started at 6 months of age (almost all eyes have pre-elevated IOP). Late start - treatment started at 9 months of age (after the onset of elevated IOP; at this time point most eyes have or already have elevated IOP).

[0116] Fig. 11B Nicotinamide protects against optic nerve degeneration in D2 glaucoma at 12 months of age. Graphs show the percentage of mice with no detectable glaucoma (NOE; bottom of bar), moderate glaucomatous damage (MOD; middle of bar), or severe glaucomatous damage (SEV; top of bar). From left, DBA / 2J control—D2 mice with standard drinking water; Nicotinamide (NAM Lo ) Early start - D2 mice were supplemented with 550 mg / kg / day NAM in standard drinking water from 6 months of age (before disease); nicotinamide (NAM Lo ) evening—D2 mice supplemented with 550 mg / kg / day NAM in standard drinking water from 9 months of age (during illness); nicotinamide (NAM Hi ) Early start - D2 mice supplemented with 2000 mg / kg / day NAM in standard drinking water from 6 months of age (before disease); Pyruvate - D2 mice supplemented with 500 mg / kg / d pyruvate in standard drinking water from 6 months of age (before IOP elevation); NAM Lo + Pyruvate - D2 mice supplemented with 550 mg / kg / day NAM + 500 mg / kg / day pyruvate in standard drinking water from 6 months of age (before disease); D2.Wld s — Carrying Wld in standard drinking water s Transgenic (altered NMNAT enzyme with enhanced enzyme activity) D2 mice; D2.Wld s + Niacinamide (NAM Lo )—Carryover of 550 mg / kg / day NAM in standard drinking water from 6 months of age (before IOP elevation) s Transgenic (altered NMNAT enzyme) D2 mice. It is important to note that in addition to treating glaucomatous neurodegeneration through intervention, NAM can also prevent glaucomatous neurodegeneration prophylactically.

[0117] Fig. 12ANAM and pyruvate are shown to prevent RGC soma loss (n=8 / group), retinal NFL and IPL thinning (n=8 / group), optic nerve degeneration (n>50 / group), and loss of anterograde axoplasmic transport (n=20 / group). The corresponding labels and color keys are located 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 at the ONH site.

[0118] Fig. 12B Shows restoration of axonal transport in NAM- and pyruvate-treated D2 mice. Loss of axonal transport is a prominent feature in glaucoma and can be used as an indicator of neuronal health. Axonal transport from the eye portion of retinal ganglion cells to the terminal (brain) ends of the cells 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 centers in the brain (LGN; lateral geniculate nucleus, Sup.Col.; superior colliculus). Second row: D2 mice have incomplete arrest of axonal transport within the optic nerve (white asterisk). No labeling is present in the visual centers of the brain. Third and fourth rows: NAM treatment (third row) or pyruvate treatment (fourth row) prevents loss of axonal transport.

[0119] Fig.13A NAM was shown to prevent RGC soma loss (n=8 / group, D2 and D2-Gpnmb + The density decrease between 2 and 100 μm is due to the stretching caused by pressure). *** = P < 0.001.

[0120] Fig. 13B Display NAM Lo (550 mg / kg / day) and pyruvate (500 mg / kg / day) prevented RGC soma loss (using a specific marker for RGCs; RBMPS).

[0121] Fig.14 Displays visual function tests based on the use of PERG amplitude (pattern electroretinogram), NAM (NAM Lo and NAM Hi ), pyruvic acid (500 mg / kg / day), Wld s Both NAM and its combination protected D2 mice from early visual function loss (n>20 / group). PERG is a very sensitive and early measure of glaucoma, so NAM or pyruvate treatment can even prevent very early stages of glaucoma. NAM Lo+ Combination of pyruvate and NAM Lo +Wld s All combinations showed additional restoration of PERG amplitude. Lo =550mg / kg / day. NAM Hi =2,000 mg / kg / day.

[0122] Fig.15 showed that even in old mice, NAM Lo (550 mg / kg / day) and pyruvate (500 mg / kg / day) also protected PERG. D2 from 6 and 12 months and 12 month NAM Lo Example traces of mice treated with NAM (550 mg / kg / day). Also shown are 12 month pyruvate treated mice and NAM Lo Processed Wld s Results in mice.

[0123] Fig.16A and 16B NAM was evaluated by SNAP-25 staining (n=6 / group) Lo Treatment (550 mg / kg / day) prevents synaptic loss in early glaucoma. ** = P < 0.01, *** = P < 0.001. Scale bar = 25 μm.

[0124] Fig.17A and 17B Display NAM Lo = 550 mg / kg / day) can prevent early mitochondrial dysfunction in dendritic mitochondria, such as Figure 4A and 4B These data correspond to early changes in PERG and previously reported synaptic loss in D2 retinas at 9 months. Thus, elevated IOP-induced mitochondrial dysfunction may contribute to early neurodegenerative changes. ** = P < 0.01, *** = P < 0.001. ns = not statistically significant. Scale bar = 350 nm.

[0125] Fig.18 NAM treatment (550 mg / kg / day) was shown to prevent lipid droplet formation in the inner retina (12 months; e.g. Figure 7B Scale bar = 25 μm.

[0126] Fig.19A and 19B The results were shown by γH2AX staining (n=6 / group), NAM treatment (NAM Lo =550 mg / kg / day) prevents DNA damage in early glaucoma. ***=P<0.001. Scale bar=25 μm.

[0127] Fig. 20A Represents individual gene expression plots showing restoration of metabolic and DNA damage pathways to normal in NAM-treated RGCs. Dots represent individual genes, grey or light dots = not differentially expressed, red or dark dots = differentially expressed with D2-Gpnmb + The differential expression was observed when q < 0.05 compared with the control.

[0128] Fig. 20B is a heat map of gene expression (all expressed genes) showing that NAM-treated RCGs are molecularly similar to controls.

[0129] Fig.21A NAM-treated RCGs are shown to be molecularly similar to young and age-matched non-glaucoma control RCGs. (Spearman rank correlation coefficient). Circles = samples from D2 RGCs, triangles = samples from D2-Gpnmb + Samples of RGCs, squares = from NAM-treated (NAM Lo =550 mg / kg / day) of RGC samples. Thus NAM treatment can prevent disease- and age-related molecular changes.

[0130] Fig.21B Summarizes the comparison with D2-Gpnmb + The number of differentially expressed genes in the D2 groups (group 1, group 2, group 3 and group 4) compared with the other groups.

[0131] Fig. 21C and 21F Display NAM treatment (NAM Lo =550 mg / kg / day) prevented the transcriptome imbalance and OXPHOS imbalance (mitochondrial: nuclear library size ratio) observed in D2 RGCs at 9 months. Fig.21F In the figure, red = highest expression, blue = lowest expression. IV = mitochondrial complex IV (listed in Table 1), G = D2-Gpnmb + , 1-4 = D2 groups 1-4, N = NAM.

[0132] Fig.21D : D2 mice (aged NAM) were treated with 550 mg / kg / day nicotinamide in normal drinking water starting at 6 months of age. RNA-seq of retinal ganglion cells from NAM-treated mice showed that NAM prevented age- and disease-related gene expression changes. NAM-treated retinal ganglion cells were most similar to young (young controls) rather than age-matched controls (aged controls). This suggests that NAM prevents age-dependent molecular changes.

[0133] Fig.21EHeatmap correlations are shown for all samples (Spearman rank correlation coefficient, blue = highest correlation, red = lowest correlation). Fig.21A The dendrogram is shown in grey.

[0134] Fig.22A and 22B NAM was shown to have a protective effect against neurodegeneration in response to treatments that mimicked glaucomatous injury. NAM was shown to have a protective effect against neurodegeneration in the ganglion cell layer (GCL) cells from death ( Fig.22A ) and nuclear shrinkage prior to apoptosis ( Fig. 22B ) showed an attractive dose-response effect in terms of damage to the retinal ganglion cells. In the axotomy culture model of retinal ganglion cell injury, the addition of nicotinamide (NAM) prevented cell shrinkage (a sign of apoptosis and dysfunction) in a dose-dependent manner (5 days after axotomy). The average nuclear diameter of DAPI-labeled nuclei from D2 mouse retinas exposed to different concentrations of NAM for 5 days was measured (including day 0 control (normal retina), untreated (5 days after axotomy), 100mM and 500mM NAM (5 days after axotomy). NAM-treated retinas were indistinguishable from undamaged baseline controls (day 0). Treatment of retinas with β-NAD & β-NMN (n=8 retinas / group) also had a significant protective effect. ***=P<0.001. Scale bar=20μm.

[0135] Figures 23A-23C Display NAM Lo =550 mg / kg / day) prevented PERG in TNFα-injected eyes 12 weeks after TNFα administration (n=20 / group) Fig.23A ) and somatic cell loss ( Fig. 23B and 23C ). * = P < 0.05. ns = not statistically significant. Scale bar = 20 μm.

[0136] Fig.24A and 24B Gene therapy was shown to strongly protect against glaucomatous neurodegeneration. At 5.5 months, D2 eyes were injected intravitreally with an AAV2.2 viral vector carrying a plasmid to overexpress murine Nmnat1 under the CMV promoter. Fig.24A Nmnat1 overexpression prevented RGC soma loss and loss of anterograde axoplasmic transport (n=10 / group). Fig. 12A As shown. Fig.24A The scale bar is 50 μm. Fig. 24B The thickness is 100 μm.

[0137] Fig.24C and 24Dshowed that Nmnat1 gene therapy also protected D2 eyes with elevated IOP from optic nerve degeneration (n>40 / group) ( Fig.24C ), somatic cell loss (n=8 / group) ( Fig.24D , upper figure) and PERG amplitude (n>20 / group) ( Fig.24D , below). Adding NAM (NAM Lo =550 mg / kg / day) provided additional protection against optic nerve degeneration (Nmnat1 vs. Nmnat1+NAM Lo =P<0.001, Fisher's exact test) ( Fig.24C ). **=P<0.01, ***=P<0.001.

[0138] Fig.25 Displays NAM and NMNAT1 / WLD S NAD cycle pathway.

[0139] Fig.26A and 26B Administration of PQQ was shown to prevent nuclear diameter shrinkage in excised retinas treated with PQQ ( Fig.26A ) and decreased cell density ( Fig.26B ).

[0140] Fig. 27 A-27H shows FACS sorted RGCs. Retinal samples were stained with an antibody cocktail (see Materials and Methods). Fig. 27 A shows gating to identify live cells ( Fig. 27 D) forward and side scatter ( Fig. 27 B and 27C). RGCs were identified as gated Th1.2 + (Cd11b - ,Cd11c - ,Cd31 - ,Cd34 - ,Cd45.2 - GFAP - ,DAPI - ) events. Only Cd11b, Cd45, and Thyl.2 are shown ( Fig. 27 E-27G). Fig. 27 In H, FACS-positive RGCs were plated and stained with SNAP-25 and β-tubulin to confirm RGC status. Scale bar = 25 μm. DETAILED DESCRIPTION

[0141] definition

[0142] The terms used in this application shall have the following meanings.

[0143] As used herein, the term "glaucoma" refers to an eye disease that causes damage to the retina and optic nerve and visual dysfunction or vision loss. Glaucoma is more common in older people. Vision loss from glaucoma is permanent and irreversible.

[0144] As used herein, the term "individual / patient in need of treatment" is generally understood to be a person suffering from certain neurodegenerative diseases or disorders, particularly ocular neurodegeneration including glaucoma, or who simply feels in need of such treatment.

[0145] As used herein, the term "glaucoma" includes primary open-angle glaucoma, secondary open-angle glaucoma, normal-tension glaucoma, hypersecreting glaucoma, primary angle-closure glaucoma, secondary angle-closure glaucoma, high-altitude iris glaucoma, pigmentary glaucoma, combined glaucoma, developmental glaucoma, steroid-induced glaucoma, exfoliation glaucoma, amyloid glaucoma, neovascular glaucoma, malignant glaucoma, capsular glaucoma, high-altitude iris syndrome, and the like.

[0146] As used herein, the term "normal intraocular pressure" (normal "IOP") in humans refers to human individuals having an IOP value of 10 mmHg to 21 mmHg. However, some individuals may develop optic nerve damage despite having a normal IOP (called normal-tension glaucoma).

[0147] As used herein, the term "high intraocular pressure" (high IOP) in humans refers to a human individual with an IOP value greater than or equal to 21 nmHg (or 2.8 kPa). High IOP is known to be a risk factor for glaucoma. However, some individuals never develop optic nerve damage despite the high intraocular pressure that may occur for many years.

[0148] As used herein, the term "neuroprotective" or "neuroprotection" refers to the ability to protect neurons, their synapses, dendrites, soma or axons in the ocular nerve (e.g., optic nerve), central or peripheral nervous system from damage (including dysfunction) or death, or to delay the onset of neuronal damage or death, or to reduce the severity of the extent of neuronal damage / death in a neuronal population.

[0149] As used herein, the term "prevent" or "prevention" with respect to, for example, general neuronal damage or death or particular ocular neurodegenerative diseases (e.g., glaucoma) refers to the ability of a compound or agent of the invention to confer neuroprotection, preferably before such damage, death, or disease occurs. Thus, preventing glaucoma includes avoiding the development of glaucoma, reducing the risk or chance of eventually developing glaucoma, delaying the onset or progression of glaucoma, or reducing the severity of neuronal damage / neuronal death / loss in a population of neurons that eventually develop glaucoma.

[0150] As used herein, the term "treating" includes administering a compound or agent of the invention to a subject to alleviate, arrest or inhibit the development of a symptom or disorder associated with neurodegeneration, such as glaucoma.

[0151] As used herein, "improving vision or visual function" refers to the effect of the compounds or agents of the present invention in improving vision or visual function (e.g., visual field testing of RGCs or pattern electroretinogram (PERG) amplitude) in individuals to which these compounds or agents are administered, compared to control individuals to which these compounds or agents are not administered.

[0152] As used herein, the term "therapeutically effective amount" refers to an amount that produces the effect of its administration when administered to an individual. For example, a "therapeutically effective amount" when administered to an individual to inhibit neurodegeneration, reduce IOP elevation, and / or improve RGC function (e.g., prevent RGC function from deteriorating).

[0153] As used herein, the terms "subject" or "patient" are used interchangeably and refer to mammals, such as rodents, dogs, and humans. Therefore, the terms "subject" or "patient" as used herein refer to any mammalian patient or individual (e.g., human) to which the compounds of the invention can be administered.

[0154] As used herein, the term "drug" or "pharmaceutical composition" refers to a pharmaceutical preparation used to treat, cure or improve a disease or treat, improve or alleviate the symptoms of a disease.

[0155] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of achieving expression of the gene / nucleic acid molecule contained therein in cells compatible with these sequences. An expression vector generally includes at least a suitable promoter sequence and optionally a transcription termination signal.

[0156] The following abbreviations are used:

[0157] NAM - Niacinamide

[0158] NAMPT - Nicotinamide phosphoribosyltransferase

[0159] NMN or β-NMN-nicotinamide mononucleotide

[0160] NMNAT - Nicotinamide mononucleotide adenylyltransferase

[0161] NAD - Nicotinamide Adenine Dinucleotide

[0162] NaMN-Nicotinic acid mononucleotide

[0163] NR-Nicotinamide Riboside

[0164] Overview

[0165] The present invention is primarily based on the use of state-of-the-art molecular techniques (RNA-sequencing; RNA-seq) on retinal ganglion cells (RGCs), which led to the discovery that mitochondrial abnormalities are early drivers of neuronal dysfunction, occurring before detectable neurodegeneration. Using the DBA / 2J (D2) mouse model (chronic age-related hereditary glaucoma), the inventors further revealed a relationship between increasing age (a major risk factor for most glaucoma) and high IOP in driving neuronal degeneration in RGCs.

[0166] The inventors have developed a method for delivering therapeutic levels of nicotinamide adenine dinucleotide (NAD + ) to supplement NAD + The administration of the specific pharmaceutical compositions of the present invention (e.g., NAM and / or pyruvate) represents a novel approach to treating ocular neurodegeneration and glaucoma. The present therapeutic approach can be used to treat other neurodegenerative diseases, including diseases in which aging neurons are susceptible to disease-related damage.

[0167] In one aspect, the present invention provides therapeutic uses of targeted pharmaceutical compositions for treating or preventing age-dependent neurodegeneration, such as treating or preventing glaucoma.

[0168] In certain embodiments, the pharmaceutical composition is effective in treating or preventing glaucoma, preferably primary open-angle glaucoma, normal-tension glaucoma, and primary angle-closure glaucoma. In certain embodiments, the pharmaceutical formulation of the present invention is particularly effective in treating or preventing primary open-angle glaucoma.

[0169] In certain embodiments, the method involves administering a therapeutically effective amount of nicotinamide (NAM) to an individual in need of treatment / prevention of a neurodegenerative disorder (eg, glaucoma).

[0170] In certain embodiments, NAM is administered to an individual in combination with pyruvate. It is believed that NAM and / or pyruvate increase intracellular NADt levels (NAD + and total levels of NADH), and thereby treat or prevent a neurodegenerative disorder in an individual.

[0171] In one aspect, the invention provides for the therapeutic use of gene delivery to increase intracellular nicotinamide adenine dinucleotide to prevent neurodegeneration and thereby treat glaucoma.

[0172] In certain embodiments, the invention provides methods of gene therapy (e.g., driving expression of nicotinamide nucleotide adenylyltransferase 1 (Nmnat1). Expression of the Nmnat1 protein was found to be profoundly protective and synergistic with NAM (i.e., 84% of eyes were free of glaucomatous neurodegeneration).

[0173] One advantage of the present invention is the reduction of risk factors for developing glaucoma. Current studies clearly show that NAM alone or in combination with other drugs (such as pyruvate) can reduce the risk factor for glaucoma. In control D2 mice at 12 months of age, about 60% of the nerves had severe glaucoma. This represents a 0.6 risk factor for developing glaucoma. After low-dose NAM administration (550 mg / kg / day in mice), this risk factor was reduced to 0.36 (about a 2-fold reduction in risk factor), or when high-dose NAM (2,000 mg / kg / day in mice) was administered, it was reduced to 0.06 (a 10-fold reduction in risk factor).

[0174] An advantage of the present invention is the synergistic effect between NAM and gene therapy administration (e.g., NMNAT1 gene therapy). Gene therapy combined with NAM reduced the risk factor to 0.16 (about a 4-fold reduction in risk factor), compared to gene therapy alone which reduced the risk factor to 0.29 (about a 2-fold reduction in risk factor). Thus, NAM and gene therapy combined synergistically reduced the risk factor for glaucoma following high IOP.

[0175] The finding of a protective effect of NAM (alone or in combination with other agents such as pyruvate) is unexpected. This finding is in stark contrast to the report in the '326 patent, which states that NAM has no protective effect.

[0176] At least the following reasons provide a basis for the different findings. The first reason is that the '326 patentee used an experimental system that is not relevant to in vivo effects, especially in eye-related diseases such as glaucoma. In their experiments, the '326 patentee isolated dorsal root ganglion (ORG) neurons and induced neural injury by cutting their neuritis. This artificial culture-induced neural injury, in which the mechanical cutting occurs very close to the DRG neuron cell body, hardly resembles the in vivo neurodegeneration. It is recognized that the initial axonal damage in glaucoma occurs at a distance from the soma that is much greater than from the DRG neuritis. The second reason is that the '326 patentee's culture system did not use retinal ganglion cells (RGCs), which are highly relevant neurons in glaucomatous neurodegeneration. The third reason is that the '326 patentee used an artificial cell system instead of intact neural tissue (which is composed of various cell types that communicate and support each other).

[0177] Finally, the '326 patentee mentions an in vivo complete optic nerve transection model in which the nerve (including the sheath surrounding it) is mechanically cut. This abruptly damages the nerve and significantly alters the local environment in which the axons reside, which results in >90% retinal ganglion cell death within 14 days (see www.ncbi.nlm.nili.gov / pubmed / 21610673). Although the injury was in the correct location of the nerve, there were no cases of glaucoma. Overall, the approach taken by the '326 patentee is a very artificial method of rapidly destroying retinal ganglion cells that does not cause human glaucoma. Interestingly, in their optic nerve transection experiments, the '326 patentee intravitreally injected compounds (including NAM) to drive NAD production in the eye, and the '326 patentee clearly concluded that NAM had no protective effect against axonal degeneration in RGCs in this model.

[0178] All of this is particularly relevant. Our culture experiments are more relevant to glaucoma because we use intact retinal tissue that includes retinal ganglion cells in a normal relationship with other retinal cell types, and the retinal ganglion cell axons are cut in the same location where damage occurs in glaucoma. It is thought that the damage in glaucoma occurs in the true axons of the optic nerve head, where RGC axons leave the eye and become the optic nerve.

[0179] In contrast, the data provided in the present application clearly show the neuroprotective effect of NAM in glaucoma. The use of NAM alone or in combination with other agents (e.g., pyruvic acid) provides neuroprotection in eye-related diseases such as glaucoma. In the present application, the neuroprotective effect is demonstrated by at least one of the following parameters: (i) prevention of somatic cell loss (RBPMS staining); (ii) prevention of retinal thinning and nerve fiber layer loss (Nissl staining); (iii) prevention of optic nerve degeneration and axon loss (PPD staining); (iv) prevention of anterograde axoplasmic transport loss (Ct-B staining); (v) prevention of visual function loss (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).

[0180] The inventors further discovered that the use of NAMs, alone or in combination with other agents (e.g., pyruvate), influences intracellular events to provide neuroprotection, as demonstrated by: (i) reduction in HIF-1α activation (HIF-1α staining); (ii) reduction in synaptic loss (SNAP-25 staining); (iii) restoration of nuclear to mitochondrial transcript abundance (RNA-seq); and (iv) prevention of age-associated molecular / genetic changes (RNA-seq).

[0181] Another advantage of the present invention is the combined use of NAM and gene therapy to deliver NAD. It is believed that many clinical studies have shown that the gene therapy approach lasts at least 3-5 years. Gene therapy provides synergistic neuroprotection with NAM and / or pyruvate and lowers IOP in glaucoma.

[0182] With the invention generally described above, the following sections provide a more detailed description of other aspects of the invention.

[0183] DBA / 2J Mouse Model

[0184] The inventors chose to use the DBA / 2J (D2) mouse model because this mouse strain produces an inherited, age-related glaucoma that closely mimics human glaucoma. The D2 mouse is 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), the same location of key glaucomatous lesions in the optic nerve head, and the same topographic pattern of RGC death that occurs in human glaucoma. D2 mice suffer from iris disease and elevated intraocular pressure beginning at approximately 6-8 months of age. At 9 months of age, most eyes of D2 mice continue to have elevated intraocular pressure. D2 mice subsequently have progressive vision loss, optic nerve damage, and inner retinal dysfunction. At 12 months of age, when designed experiments are typically terminated, approximately 70% of D2 mouse eyes have severe disease based on histological examination of retinal and optic nerve glaucoma. Topical administration of compounds (e.g., memantine, timolol, or latanoprost) reduces the IOP of D2 mice and reduces the risk of neurodegeneration in human glaucoma. Control D2-Gpnmb + are age- and strain-matched mice that do not develop glaucoma.

[0185] Intraocular Pressure in Glaucoma

[0186] Intraocular pressure (IOP) can be measured using, for example, Goldmann applanation tonometry (Haag Streit, Bern, Switzerland). In humans, normal IOP is 12-21 mmHg. An IOP above 21 mmHg is considered high. Elevated IOP is a major risk factor for glaucoma. For patients with POAG (primary open-angle glaucoma, the most common glaucoma, accounting for more than 90% of cases), 25 mmHg is the median untreated baseline IOP.

[0187] The Baltimore Eye Study (www.ncbi.nlm.nih.gov / pub / 12049574) reported that high risk = IOP > 25.75 mmHg; intermediate risk = IOP 23.75-25.75 mmHg; low risk = IOP < 23.75 mmHg. Lowering IOP by 20% to levels below 24 mmHg would reduce the risk of progression from 9.5% to 4.4% at 5 years. The probability of blindness in one eye is 27% 10 years after diagnosis and 38.1% after 20 years. The probability of blindness in both eyes is 6% and 13.5%, respectively.

[0188] In human glaucoma patients, such as those with primary open-angle glaucoma (POAG), glaucoma is asynchronous and age-related (most common at >40 years of age). When the IOP is 21-25 mmHg, it takes an average of 14 years for untreated glaucoma to progress from the initial stage to the advanced stage of the disease. This rate of progression increases rapidly as the IOP increases (~3 years from early stage to advanced stage of the disease when the IOP is >30 mmHg).

[0189] In human patients, treatments that lower IOP (surgery or pharmacology) reduce the risk of neurodegeneration. Because patients are not diagnosed, the rate of disease progression and the percentage of blindness may be misrepresented. It is generally believed that high IOP does not always indicate glaucoma (about 30% blindness after 10 years, about 40% after 20 years). Lowering IOP cannot cure glaucoma, but it can indeed reduce risk factors by 58%. Despite conventional preventive measures to lower IOP, there is still a risk of vision loss or even blindness. The available neuroprotective strategies in glaucoma are limited. Glaucoma vision loss is irreversible. In fact, glaucoma is the leading case of irreversible blindness in the world.

[0190] Retinal ganglion cells (RGC)

[0191] Retinal ganglion cells (RGC) are the output neurons of the retina. They receive visual information from photoreceptors (i.e., rods and cones) through interneurons (i.e., bipolar cells and amacrine cells). This visual information starts with photons in the light, and the potential reaches the highest at the synapses of the retinal ganglion cells. RGC has long axons that leave the cell body and pass through the retina to reach the optic disc (i.e., the blind spot) that leaves the eye (optic nerve head). Outside the optic nerve 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 depends on the strain is ~ 50,000). The axons in the optic nerve eventually reach the terminal visual center in the brain, and then these signals are transmitted or processed by the information itself. The two important visual centers where the retinal ganglion cell axons terminate are the lateral geniculate nucleus (LGN) and the superior colliculus (sup.col. / SC).

[0192] Retinal ganglion cells are particularly affected (i.e., cell loss) in glaucoma. Damage to the RGCs may occur at the axons in the area of ​​the optic nerve head. It has been hypothesized that the optic nerve head is a "weak point" in the eye where mechanical retinal ganglion cell axonal injury may occur due to ocular pressure caused by abnormally high IOP. The axons are not the only point of injury in the retinal ganglion cells, as pressure throughout the eye must also affect the soma and dendrites. The exact mechanism of RGC injury is not completely understood.

[0193] In DBA / 2J (D2) mice, the inventors showed that at time points when IOP was high, there was no detectable axonal loss in the optic nerve (i.e., no ocular nerve degeneration). Surprisingly, the inventors found early mitochondrial and molecular changes, dendritic atrophy, and synaptic loss. This finding suggests that the effects of IOP are manifested in other compartments of the retinal ganglion cells, not just the axons in the optic nerve.

[0194] Neurodegeneration treatment

[0195] In certain embodiments, the agent comprises nicotinamide adenine dinucleotide (NAD + ) precursor (e.g., niacin, nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR) or a combination thereof), a Krebs cycle intermediate or a precursor thereof (e.g., pyruvate), or a combination thereof.

[0196] In certain embodiments, the agent comprises nicotinamide adenine dinucleotide (NAD + ) precursors, such as nicotinic acid, nicotinamide (NAM), nicotinamide mononucleotide, nicotinamide riboside, or a combination thereof. In certain embodiments, NAD + The precursor is nicotinamide or nicotinamide riboside.

[0197] Niacin (also known as nicotinic acid, nicotinic acid salt, vitamin B3, and vitamin PP) is a vitamin. Its corresponding amide is called nicotinamide or niacinamide. These vitamins are not directly interconvertible; however, both nicotinic acid salt and niacinamide are essential for the synthesis of the redox pair NAD + / NADH (nicotinamide adenine dinucleotide) and NADP + / NADPH (nicotinamide adenine dinucleotide phosphate) precursor. Nicotinate and nicotinamide metabolic pathways may be referred to herein as NAD + Synthetic pathway.

[0198] NAD is synthesized via two metabolic pathways + : salvage pathway and de novo pathway. In the salvage pathway, NAD + It can be synthesized from external sources of precursor compounds (e.g., nicotinic acid, nicotinamide, nicotinamide riboside, etc.). In the de novo pathway, NAD can be synthesized from quinolinic acid produced during amino acid metabolism. + (e.g. tryptophan, aspartic acid, etc.).

[0199] NAD of the present invention + Precursors include nicotinic acid, nicotinamide, nicotinamide riboside, and the like, and salts and analogs thereof. In certain embodiments, administration of the NAD + The precursor leads to an increase in intracellular NADt levels.

[0200] 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 is oxaloacetate, acetyl CoA, citric acid, CoA-SH, cis-aconitic acid, D-isocitrate, NAD + , oxalosuccinate, NADH, α-ketoglutarate, succinyl-CoA, GDP, ubiquinone, succinate, fumarate, L-malate, pyruvate, monosaccharides (such as glucose, galactose, fructose), disaccharides (such as sucrose, maltose, lactose), or a combination thereof.

[0201] Therefore, the present invention provides a pharmaceutical composition comprising nicotinic acid and / or nicotinamide ribonucleoside and / or nicotinamide and / or nicotinic acid metabolites. Nicotinic acid and / or nicotinamide ribonucleoside and / or nicotinamide and / or nicotinic acid metabolites can be used in free form. The term "free" as used herein with respect to a component means that the component is not incorporated into a larger molecular complex. In some embodiments, nicotinic acid can be contained in nicotinic acid. Nicotinic acid and / or nicotinamide ribonucleoside and / or nicotinamide and / or nicotinic acid metabolites can be in the form of a salt.

[0202] In some embodiments, any composition described herein may include salts, derivatives, metabolites, catabolites, anabolic metabolites, precursors and analogs thereof. For example, metabolites may include nicotinyl CoA, nicotinic acid, nicotinic acid ester mononucleotide, nicotinic acid adenine dinucleotide or nicotinamide adenine dinucleotide. In some embodiments, the composition comprises nicotinamide. In some embodiments, the composition may be substantially free of nicotinic acid metabolites.

[0203] In certain embodiments, the salt is selected from the group consisting of fluoride, chloride, bromide, iodide, formate, acetate, ascorbate, benzoate, carbonate, citrate, carbamate, formate, gluconate, lactate, methyl bromide, dimethyl sulfate, nitrate, phosphate, diphosphate, succinate, sulfate, and trifluoroacetate.

[0204] NAM / nicotinic acid analogs may also be used in the present invention. Suitable analogs of niacin include, for example, isonicotinamide and N-methylnicotinamide.

[0205] In certain embodiments, keto-, ethyl-, benzyl-, or other non-salt embodiments of NAMs may also be used in the present invention.

[0206] NAM / nicotinamide / NAD / NR / pyruvate has low toxicity and can be administered in relatively large amounts without toxic effects.

[0207] In mice, the compounds of the invention (e.g., NAM / nicotinamide / NAD / NR / pyruvate) can be administered at a daily dose of about 200-1,000 mg / kg / day to provide neuroprotection. Preferably, the daily dose is about 400-600 mg / kg / day. More preferably, the daily dose is about 550 mg / kg / day. At these doses, the compounds of the invention provide neuroprotection.

[0208] In mice, the compounds of the invention (e.g., NAM / nicotinamide / NAD / NR / pyruvate) can be administered at a dose of about 1,000-5,000 mg / kg / day to provide an IOP lowering effect. Preferably, the daily dose is about 1,500-3,000 mg / kg / day. More preferably, the daily dose is about 2,000 mg / kg / day. At these doses, the compounds of the invention provide an IOP lowering effect.

[0209] Regarding PQQ, it is tolerated in mice at least at 2,000 mg / kg / day. The neuroprotective effect provided by PQQ is about 20-2,000 mg / kg / day, about 100-1,000 mg / kg / day, or about 500 mg / kg / day in mice.

[0210] To convert a dose of a pharmaceutical composition expressed in mg / kg 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 provides approximate conversion factors based on the assumptions and constants in Freireich et al., Quantitative comparison of toxicity of anticancer agents in mouse, rat, dog, monkey and man, Cancer Chemother Rep. 50(4):219-244, 1966 (incorporated herein by reference).

[0211] Equivalent surface area dose conversion factor

[0212]

[0213] Therefore, a dose of 550 mg / kg in mice is equivalent to (assuming mg / m 2 Based on equivalence) 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 60 kg humans.

[0214] Some simplified conversion factors in the table above are based on the body mass to surface area ratio [km] of each species. If a more precise conversion is required, dose conversions can also be based on the km factors for each species listed below.

[0215] Representative surface area to weight ratio of each species [km]

[0216] Species Weight(kg) <![CDATA[Surface area (m 2 )]]> km factor Mouse 0.02 0.0066 3.0 Rat 0.15 0.025 5.9 monkey 3.0 0.24 12 dog* 8.0 0.40 20 Human (child) 20 0.8 25 Human (adult) 60 1.6 37

[0217] * Depending on the specific breed, the KM factor may be different. For example, in some dogs and other breeds that are susceptible to glaucoma, a KM factor of about 10 may be used.

[0218] Therefore, a dose of 550 mg / kg in mice is equivalent to 550 mg / kg x 3.0 / 37 = 44.6 mg / kg (or 2.68 g) in a 60 kg adult, and 550 mg / kg x 3.0 / 25 = 66 mg / kg (or 1.32 grams) in a 20 kg human child.

[0219] The table above can also be used to express the mg / kg dose for any given species as an equivalent mg / m2 dose by multiplying the dose by the appropriate km factor. For example, in adults, 100 mg / kg is equivalent to 100 mg / kg x 37 kg / sq.m. = 3700 mg / sq.m.

[0220] In humans (approximately 60 kg individuals), the compounds of the invention (e.g., NAM / nicotinamide / NAD / NR / pyruvate) can be administered at a daily dose of 0.5-10 grams to provide neuroprotection. Preferably, the daily dose is about 1-5 grams / day. Preferably, the daily dose is about 2-4 grams / day. More preferably, the daily dose is about 2.5 grams / day. At these doses, the compounds of the invention provide neuroprotection.

[0221] In humans (approximately 60 kg individuals), the compounds of the invention (e.g., NAM / nicotinamide / NAD / NR / pyruvic acid) can be administered at a daily dose of about 5-25 grams / day to provide an IOP lowering effect. Preferably, the daily dose is about 10-20 grams / day. Preferably, the daily dose is about 8-15 grams / day. More preferably, the daily dose is about 10 grams / day. At these doses, the compounds of the invention provide an IOP lowering effect.

[0222] In humans (~60 kg individuals), PQQ can be administered at a daily dose of about 2-160 mg / kg / day, or about 10-100 mg / kg / day, or about 50 mg / kg / day to provide neuroprotection. In certain embodiments, PQQ can be administered at a daily dose of about 10 mg-10 g per day, about 50 mg-1 g per day, or about 500 mg per day.

[0223] Ideally, a typical dosage can be once, twice or three times a day. The total daily dose can be given once, or given in two or three separate doses (e.g., each dose is 1 / 2 or 1 / 3 of the daily total). For multiple doses, each dose can be the same amount or a different amount. The pharmaceutical composition can be applied in the morning or evening. The pharmaceutical composition can be taken with or without meals.

[0224] Therapeutic agents for treating neurodegenerative diseases

[0225] In one aspect, the present invention provides a method for treating or preventing a neurodegenerative disorder in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a drug that increases intracellular NADt levels or increases intracellular NAD + , NADH, GSH, GSSG, pyruvate or PQQ levels.

[0226] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of NAM, nicotinamide mononucleotide (NMN or β-NMN), NAD, pyruvate, PQQ or glutathione.

[0227] The agent used in the pharmaceutical composition is a neuroprotective agent for treating or preventing such neurodegenerative disorders, such as glaucoma.

[0228] Many different types of injuries or neurodegenerative diseases or conditions can lead to neuronal damage or death, for example: metabolic stress caused by hypoxia, hypoglycemia, diabetes, loss of ion homeostasis, physical damage to neurons, exposure to toxic agents, and many diseases that affect the nervous system, including hereditary or non-hereditary neurodegenerative diseases. In certain embodiments, neurodegenerative conditions involve axonal degeneration. In certain embodiments, neurodegenerative conditions involve Wallerian degeneration or Wallerian degeneration. For example, Wallerian degeneration may be caused by neuronal damage, such as damage caused by disease, trauma, or chemotherapeutic agents.

[0229] It will be appreciated that this is only an illustrative list. The presence of a neuroprotectant will keep neurons alive when exposed to injury or disease conditions that would otherwise result in loss of functional integrity in unprotected neurons. Such drugs may also respond to injury by preventing neurons from being damaged by making them more resilient.

[0230] Pharmaceutical composition

[0231] Pharmaceutical formulations contain the pharmacologically active ingredient in a form that is not harmful to the subject to which it is administered, and additional ingredients designed to stabilize the active ingredient and affect its absorption into the circulation or target tissues.

[0232] The pharmaceutical composition according to the present invention can be formulated according to conventional techniques with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

[0233] Suitable pharmaceutically acceptable carriers include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents. Examples of solid carriers are lactose, carclazyte, sucrose, cyclodextrin, talcum, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid and the lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water.

[0234] The administration of the pharmaceutical composition according to the present invention can be by various routes, such as oral, parenteral (including subcutaneous, intramuscular, intradermal), intravitreal, intraocular (e.g., in the form of eye drops), etc. In a preferred embodiment, the pharmaceutical composition is used for oral administration (including administration of the pharmaceutical composition as part of a beverage). In a preferred embodiment, the pharmaceutical composition is used for intraocular administration.

[0235] Parenteral administration can be by subcutaneous, intramuscular, intraperitoneal or intravenous injection, by means of a syringe, an optional pen-shaped syringe. Alternatively, parenteral administration can be carried out by an infusion pump. As a further option, the preparation of the present invention can also be suitable for transdermal administration, for example by needleless injection or from a patch, optionally an iontophoresis patch or transdermal administration, for example buccal administration.

[0236] The pharmaceutical composition of the present invention can be administered in suitable dosage forms such as solutions, suspensions, emulsions, tablets, coated tablets, capsules, hard and soft gelatin capsules, drops, eye drops, eye ointments, eye rinses, injection solutions and the like.

[0237] The pharmaceutical compositions of the present invention can be further complexed or linked with drug carriers, drug delivery systems and advanced drug delivery systems through, for example, covalent, hydrophobic and electrostatic interactions to further enhance the stability of the composition, increase bioavailability, increase solubility, reduce side effects, achieve chronotherapy well known to those skilled in the art and increase patient compliance or any combination thereof.

[0238] 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, polyvinyl alcohol, acrylate and methacrylate polymers, polylactic and polyglycolic acid and block copolymers thereof, polyethylene glycol, carrier proteins such as albumin, gels such as thermogelling systems, such as block copolymer systems well known to those skilled in the art, micelles, liposomes, microspheres, nanoparticles, liquid crystals and dispersions thereof, L2 phases and dispersions thereof, phase behavior in lipid-water systems well known to those skilled in the art, polymer micelles, multiple emulsions, self-emulsification, self-microemulsification, cyclodextrins and derivatives thereof and dendrimers.

[0239] The pharmaceutical composition of the present invention can be used for compositions of controlled, sustained, extended, delayed and sustained-release drug delivery systems. More specifically, but not limited to, the pharmaceutical composition can be used for compositions of parenteral controlled-release and sustained-release systems well known to those skilled in the art (both systems result in many-fold reductions in the number of administrations). Even more preferably, it is a controlled-release and sustained-release system for subcutaneous administration. Without limiting the scope of the present invention, examples of useful controlled-release systems and compositions are hydrogels, oily gels, liquid crystals, polymer micelles, microspheres and nanoparticles.

[0240] Neurodegenerative diseases

[0241] It is expected that the pharmaceutical compositions of the present invention can be used to treat neurodegenerative diseases involving axonal degeneration, such as Wallerian degeneration. Examples of conditions where such degeneration may be important include diabetic neuropathy, motor neuron disease, multiple sclerosis, peripheral neuropathy, stroke and other ischemic conditions, traumatic brain injury, etc. This list is for illustrative purposes only and is not intended to be limiting or exhaustive.

[0242] In certain embodiments, the neurodegenerative disorder is one or more of diabetic neuropathy, traumatic brain injury, ischemia, peripheral neuropathy, or an ocular disorder such as glaucoma. In certain embodiments, the neurodegenerative disorder is glaucoma.

[0243] In one embodiment, the pharmaceutical composition is intended to be used as a neuroprotective drug for treating or preventing neurodegenerative disorders caused by neuronal damage. In a further embodiment, the modulator is intended to be used as a neuroprotective drug for treating neurodegenerative disorders involving axonal degeneration (e.g., Wallerian degeneration) caused by neuronal damage. As used herein, the term "damage" refers to damage to neurons, whether in the cell body or in axon or dendrite processes. This may be physical damage in the traditional sense, i.e., traumatic damage to the brain, spinal cord, or peripheral nerves caused by external forces applied to an individual. Other harmful external factors are, for example, environmental toxins, such as mercury and other heavy metals, arsenic, pesticides, and solvents. Alternatively, damage may be caused by damage to individual endogenous neurons, such as: oxygen and energy supply reduction in ischemic stroke and diabetic neuropathy, autoimmune attacks or oxidative stress in multiple sclerosis, and free radical production that is believed to be important in amyotrophic lateral sclerosis. Damage is also used to refer to any defect in the axonal transport mechanism.

[0244] In another embodiment, the pharmaceutical composition is intended for use as a neuroprotective drug, wherein the neurodegenerative disorder is caused by disease-induced neuronal damage.

[0245] In one embodiment, the neuronal damage is caused by aging.

[0246] In one embodiment, neuronal injury is caused by trauma. In one embodiment, the disease is neuronal injury induced by chemotherapeutic agents. Some drugs used for cancer chemotherapy, such as paclitaxel, velcade and vincristine, can cause peripheral neuropathy, which limits the maximum dose they can use. Recent studies have shown that neurons suffering from paclitaxel or vincristine toxicity experience Wallerian changes in their morphology and basic molecular events. In this case, suppressing Wallerian degeneration may be particularly effective because neurons are only temporarily exposed to neurotoxic agents. Therefore, paclitaxel or vincristine and the medicament that suppresses Wallerian degeneration can be administered simultaneously to allow the drug to be used in a significantly higher dose than currently possible, thereby further combating cancer.

[0247] Age is a common risk factor for most glaucoma, and current treatment methods can provide protection against age-related NAD decline and protect patients from developing glaucoma (i.e., preventing neurodegeneration). The present invention provides a combination therapy of administering NAM and / or pyruvate (to supplement NAD levels) and gene delivery of the NMNAT gene. In view of the amazing efficacy of NAM and / or pyruvate and gene therapy, combination therapy is an attractive method for treating and preventing glaucoma.

[0248] Gene therapy for neurodegeneration

[0249] Gene therapy is an effective method to overcome compliance issues and improve efficacy. The present invention provides a gene therapy method for treating neurodegeneration. Axonal degeneration is an area of ​​unmet therapeutic need.

[0250] Neurodegeneration causes symptoms in motor neuron disease, glaucoma, Alzheimer's disease, and multiple sclerosis. In diabetes, it causes neuropathic pain and distal sensory loss, which are the leading causes of limb amputation. It is also a dose-limiting side effect in cancer chemotherapy. Progressive axonal degeneration due to stretch injury is the primary pathology in traumatic brain injury, and failure to protect white matter limits treatment for stroke. Approximately half of the population will eventually suffer from one or more of these diseases, which can significantly reduce quality of life.

[0251] In certain embodiments, the present invention provides methods for treating neurodegeneration in the eye. The present invention provides methods for replacing copies of dysfunctional genes in the eye. The gene is introduced by viral gene delivery. In certain embodiments, the vector includes adenovirus, adeno-associated virus (AAV). In a preferred embodiment, AAV is AAV2.2. For the purposes of this application, it is intended to include other AAV serotypes, including AAV1, AAV2, AAV4, AAV5, AAV8, AAV9, etc. In other embodiments, the vector is a lentivirus, typically a pseudotyped HIV-based vector.

[0252] To target RGCs for gene delivery, a viral vector is introduced into the vitreous chamber. In a preferred embodiment, gene delivery is directly targeted to the proximal end of the inner retina. Intravitreal injections are typically performed during ophthalmic surgery and can be safely performed in an office / clinic.

[0253] The present invention offers viral gene delivery to the eye as an attractive prospect because it allows lifelong transfection of large numbers of cells, delivering targeted gene products. To the best of the inventors' knowledge, gene therapy in the eye represents a new approach to glaucoma treatment. This increase in NAD has applications in complex human diseases such as glaucoma. + Gene therapy represents a new treatment approach.

[0254] In one aspect, the invention provides methods for delivering genes to diseased eyes, thereby enhancing the expression of NAD. In certain embodiments, the gene therapy compositions of the invention (e.g., polynucleotides on viral vectors) are administered intravitreally or intraocularly. In a preferred embodiment, gene therapy is used for intravitreal administration. It should be understood that the preferred route will depend on the general condition and age of the individual to be treated, the nature of the condition to be treated, and the active ingredient selected.

[0255] In one aspect, the invention provides gene therapy for delivering a gene to the eye to increase Nmnat expression.

[0256] In certain embodiments, the gene that increases protein expression of Nmnat includes Nmnat-1, Nmnat-2 or Nmnat-3. In certain embodiments, the gene is Nmnat-1 (eg, human NMNAT1).

[0257] NMNAT

[0258] 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 nucleus. Alternative splicing of this gene results in multiple (at least three) transcript variants.

[0259] The NCBI reference sequence (RefSeq) for human NMNAT1 isoforms (1) includes: NM_022787.3 (nucleotides) and NP_073624.2 (protein), whose nucleotide sequences are known to be transcript variants (1) encoding longer NMNAT1 isoforms (1); and NM_001297778.1 (nucleotides) and NP_001284707.1 (protein), whose nucleotide sequences are known to be transcript variants (2) that differ in the 5'UTR region compared to variant 1. Variants 1 and 2 encode the same isoform (1) and can all be used in the methods of the present invention. All sequences are incorporated by reference.

[0260] The related nicotinamide nucleotide adenylyltransferase 1 (Nmnat2) is cytoplasmic and expressed primarily in the brain, unlike other human family members which are localized to the nucleus and expressed ubiquitously. Alternative splicing of this gene produces two transcript variants.

[0261] The NCBI reference sequence (RefSeq) of human NMNAT2 isoform (1) includes: NM_015039.3 (nucleotide) and NP_055854.1 (protein), the nucleotide sequence of which can be used in the methods of the present invention. All sequences are incorporated by reference.

[0262] The related nicotinamide nucleotide adenylyltransferase 3 (nmnat3) encodes a protein that is localized to mitochondria and may also function as a molecular chaperone for neuroprotection. Alternative splicing transcript variants (at least 6) encoding multiple isoforms (at least 5) have been observed for this gene.

[0263] The NCBI reference sequence (RefSeq) of human NMNAT3 isoform (3) includes: NM_001320510.1 (nucleotide) and NP_001307439.1 (protein), the nucleotide sequence of which is transcript variant 3 encoding the longest isoform (3) and can be used in the methods of the present invention. All sequences are incorporated by reference.

[0264] NMNAT2 is emerging as an important NAD-producing enzyme in axons and protects against axonal degeneration. Sustained stress negatively affects Nmnat2 expression in RGCs (q<0.05 in D2 glaucoma group 4, the last stage examined before glaucomatous degeneration). This decrease may be important in the transition to axonal degeneration in glaucomatous eyes. NMNAT2 expression is decreased in the brains of patients with Alzheimer's disease and has a highly variable repression in elderly postmortem human brains, which may contribute to variability in these diseases.

[0265] Many related nmnat sequences in different species of animals (eg, house mouse Mus musculus) are readily available from public databases such as NCBI RefSeq, GenBank, etc. All sequences are incorporated herein by reference.

[0266] It is thought that gene therapy using NAMPT will result in axonal cytotoxicity despite increasing NAD in cells + NAMPT converts NAM to NMN, which is then converted to NAD + Intracellular NAD + It is a key molecule associated with axonal degeneration. +When levels are low, axons degenerate rapidly. It has also been confirmed that NMN accumulates rapidly after axonal injury in sciatic nerve axons. In this axonal degeneration model, NMN levels increased within 12 hours, followed by nerve damage 36 hours after injury. Blocking the enzyme NAMPT that produces NMN using FK866 may inhibit this axonal degeneration, suggesting that NMN is toxic to neurons. Axonal degeneration appears to be NMN-dependent, and inhibiting the increase of NMN using FK866 prevents axonal degeneration. In a zebrafish model of axonal degeneration (two-photon laser cutting), FK866 effectively delayed axonal degeneration. In a cell culture model of neuronal degeneration (superior cervical ganglion; SCG), rapid removal of NMN (by overexpressing the bacterial enzyme NMN deamidase that converts NMN into NAMN) strongly protects against axonal degeneration.

[0267] Thus, the present invention represents the unexpected discovery that, unlike NAMPT, gene therapy using NMNAT is effective in protecting against ocular neurodegenerative diseases.

[0268] Therefore, we chose Nmnat1 instead of Nampt because Nampt overexpression drives NMN production, which is toxic to neurons if not properly cleared.

[0269] Wld S

[0270] Axonal degeneration is a common component of neurodegenerative diseases. Two models have attempted to explain this greater degree of distal axonal degeneration. The first is "retrograde degeneration," in which the degeneration propagates retrogradely from the nerve ending. The second is Wallerian degeneration, in which the degeneration spreads in either direction from the lesion site, depending on the type of lesion, ultimately leading to the loss of axons distal to the site of injury and leaving the proximal portion intact. Although strictly speaking Wallerian degeneration occurs only in response to physical injury to the axon, similar mechanisms operate in diseases in which no such injury has occurred. The latter is referred to as "Wallerian-like" degeneration. These two types of degeneration will be collectively referred to below as "Wallerian degeneration."

[0271] Recently discovered Wld S The study of mice has led to advances in the understanding of both processes. In these animals, Wallerian degeneration occurs about 10 times slower than in wild-type animals. Studies have shown that the mutation also delays a pathology thought to involve the "death of" axon terminals. Thus, Wld S Genes provide a mechanistic link between two models of axon degeneration.

[0272] Despite the identification and characterization of Wld S Although the study is ongoing, progress in understanding the molecular trigger of Wallerian degeneration has been limited. Knowledge of this trigger could have profound implications for understanding the early stages of "death-defying" neurodegenerative diseases.

[0273] With Wld S Mouse gene for delayed Wallerian degeneration. S The mutation is an autosomal dominant mutation occurring in mouse chromosome 4. The gene mutation is a naturally occurring 85-kb tandem triplet resulting in a mutated region containing two related genes: nicotinamide mononucleotide adenylyltransferase 1 (Nmnat-1) and ubiquitination factor e4b (Ube4b), as well as an 18-amino acid linker region. The resulting protein is localized in the nucleus and is undetectable in axons.

[0274] The mutation does not appear to cause harm to mice. The only known effect is that Wallerian degeneration is delayed by an average of three weeks after nerve injury. Recent studies suggest that the mutation protects axons through an unknown mechanism. While not wishing to be bound by any particular theory, it is possible that Wallerian degeneration may be caused by the mutation. S The mutation may result in overexpression and / or improved localization of the Nmnat (eg, Nmnat-1) protein and increased NAD synthesis.

[0275] In certain embodiments, the method comprises administering to the individual a gene encoding Nmnat (eg, Nmnat-1, -2, or -3) or Wld S polynucleotides.

[0276] In certain embodiments, the polynucleotide encoding Wld S Or its equivalent sequence.

[0277] In certain embodiments, the method comprises administering to the individual a NAM or precursor and a protein encoding Wld S polynucleotides.

[0278] In certain embodiments, the polynucleotide is administered topically to an individual. For example, to treat glaucoma, the agent can be delivered topically to the affected eye.

[0279] Carrier

[0280] In certain embodiments, the polynucleotide is administered to an individual on a viral vector. Exemplary suitable viral vectors include, but are not limited to, AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, etc. Preferably, the viral vector is an AAV vector or a lentiviral vector.

[0281] For example, polynucleotides can be introduced into individuals as exogenous genetic materials that can regulate the expression of one or more target genes of interest. This gene therapy can be used, for example, for methods for repairing damaged or diseased tissues such as neural tissue. In short, any suitable vector including adenovirus, slow virus or retroviral gene delivery vector (see below) can be used to deliver genetic information such as DNA and / or RNA to individuals. Technicians can replace or repair specific genes targeted in gene therapy. For example, normal genes can be inserted into non-specific positions in the genome of diseased cells to replace non-functional genes. In another example, abnormal gene sequences can be replaced by normal gene sequences through homologous recombination. Alternatively, selective back mutations can return genes to their normal functions. Another example is to change regulation (the degree of opening or closing genes of specific genes). In certain embodiments, target cells (such as neuronal stem cells) are treated in vitro by gene therapy methods and then transferred to mammals, preferably people who need treatment.

[0282] Any art-recognized method of genetic manipulation may be used, including transfection and infection by various types of nucleic acid constructs (eg, by viral vectors).

[0283] For example, chemical materials can be used to introduce heterologous nucleic acids (e.g., DNA) into an individual by physical treatment (e.g., electroporation, sonoporation, optical transfection, protoplast fusion, percussion infection, hydrodynamic delivery, nanoparticles, magnetic induction) or biological vectors (viruses). Chemical-based transfection can 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 (e.g., cationic liposomes, lipofection such as lipofection using Lipofectamine, etc.), or nanoparticles (with or without chemical or viral functionalization).

[0284] A nucleic acid construct comprises a nucleic acid molecule of interest and is generally capable of directing the expression of the nucleic acid molecule of interest in a cell into which it is introduced.

[0285] In certain embodiments, the nucleic acid construct is an expression vector in which a nucleic acid encoding a gene product (e.g., a polypeptide or antagonizing polypeptide expression (e.g., siRNA, miRNA, shRNA, antisense sequence, aptamer, ribozyme, antagomir, RNA sponge, etc.) is operably linked to a promoter capable of directing expression of the nucleic acid molecule in a target cell.

[0286] The DNA construct prepared for introduction into a specific cell generally includes a replication system recognized by the cell, an expected DNA segment encoding the desired polypeptide, and transcription and translation initiation and termination regulatory sequences operably connected to the polypeptide coding segment. When a DNA segment is in a functional relationship with another DNA segment, the DNA segment is "operably connected". For example, if a promoter or enhancer stimulates the transcription of a sequence, it is operably connected to a coding sequence. If the DNA of a signal sequence is expressed as a precursor protein that participates in polypeptide secretion, it is operably connected to the DNA encoding the polypeptide. Generally, the DNA sequence that is operably connected is continuous, and in the case of a signal sequence, it is continuous and in the reading stage. However, an enhancer does not need to be adjacent to the transcribed coding sequence that it controls. The connection is completed by connecting at a convenient restriction site or at an adapter or joint that replaces the insertion.

[0287] The selection of suitable promoter sequences generally depends on the host cell selected for expressing the DNA fragment. Examples of suitable promoter sequences include eukaryotic promoters known in the art (see, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, 2001). Transcriptional regulatory sequences generally include heterologous enhancers or promoters recognized by cells. Suitable promoters include CMV promoters. Expression vectors include replication systems, and insertion sites for transcriptional and translational regulatory sequences and polypeptide coding segments can be used. Sambrook and Russell (2001, supra) and Metzger et al. (1988) Nature, 334: 31-36 describe examples of feasible combinations of cell lines and expression vectors.

[0288] Some aspects of the present invention relate to the use of a nucleic acid construct or an expression vector comprising a nucleotide sequence as defined above, wherein the vector is a vector suitable for gene therapy. Vectors suitable for gene therapy are known in the art, for example, Anderson (Nature, 392: 25-30, 1998); Walther and Stein (Drugs, 60: 249-71, 2000); Kay et al. (Nat. Med., 7: 33-40, 2001); Russell (J. Gen. Virol., 81: 2573-604, 2000); Amado and Chen (Science 285: 674-6, 1999); Federico (Curr. Opin. Biotechnol., 10: 448-53, 1999); Vigna and Naldini (J. Gene Med., 2: 308-16, 2000); Marin et al. (Mol. Med. Today, 3: 396-403, 1997); Peng and Russell (Curr. Opin. Biotechnol., 10:454-7, 1999); Sommerfelt (J. Gen. Virol., 80:3049-64, 1999); Reiser (Gene Ther., 7:910-3, 2000) and those described in the references cited therein (all incorporated by reference). Examples include integrating vectors and non-integrating vectors, such as vectors based on retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), lentiviruses, poxviruses, alphaviruses, and herpes viruses.

[0289] Particularly suitable gene therapy vectors include adenovirus (Ad) and adeno-associated virus (AAV) vectors. These vectors infect a large number of dividing and non-dividing cell types. In addition, adenovirus vectors are capable of high levels of transgene expression. However, due to the additional properties of adenovirus and AAV vectors after cell entry, these viral vectors are most suitable for therapeutic applications that only require transient expression of transgenes (Russell, J. Gen. Virol., 81: 2573-2604, 2000; Goncalves, Virol J., 2 (1): 43, 2005), as described above. As described by Russell (2000, supra), preferred adenovirus vectors are modified to reduce host responses. The safety and efficacy of AAV gene transfer have been extensively studied in humans, with encouraging 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).

[0290] AAV2 is the best characterized serotype for gene transfer research in humans and experimental models. AAV2 presents a natural tropism for skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes. Other examples of non-integrating vectors based on adeno-associated viruses include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and pseudotyped AAV. The use of non-human serotypes, such as AAV8 and AAV9, may help overcome these immune responses in individuals, and clinical trials have just begun (ClinicalTrials dot gov Identifier: NCT00979238). In order to transfer genes into hepatocytes, adenovirus serotype 5 or AAV serotype 2, 7, or 8 have been shown to be effective vectors and are therefore preferred Ad or AAV serotypes (Gao, Molecular Therapy, 13: 77-87, 2006).

[0291] An exemplary retroviral vector for use in the present invention is a lentiviral expression construct. Lentiviral vectors have the unique ability to infect non-dividing cells (Amado and Chen, Science 285: 674-676, 1999). Methods for constructing and using lentiviral expression constructs are described in U.S. Pat. Nos. 6,165,782, 6,207,455, 6,218,181, 6,277,633, and 6,323,031, as well as Federico (Curr. Opin. Biotechnol. 10: 448-53, 1999) and Vigna et al. (J. Gene Med. 2: 308-16, 2000).

[0292] Typically, gene therapy vectors will be expression vectors as described above, meaning that they contain a nucleotide sequence encoding a gene product of the invention to be expressed (e.g., a polypeptide), whereby the nucleotide sequence is operably linked to an appropriate regulatory sequence as described above. Such a regulatory sequence will contain at least a promoter sequence. Suitable promoters for expressing the nucleotide sequence encoding the polypeptide from a gene therapy vector include, for example, the cytomegalovirus (CMV) intermediate early promoter, viral long terminal repeat promoters (LTRs), such as those from murine Moloney leukemia virus (MMLV) sarcoma virus or HTLV-1, simian virus 40 (SV40) early promoter, and herpes simplex virus thymidine kinase promoter. Other suitable promoters are described below.

[0293] Several inducible promoter systems have been described that can be induced by the application of small organic or inorganic compounds. Such inducible promoters include those controlled by heavy metals such as the metallothionein promoter (Brinster et al., Nature, 296:39-42, 1982; Mayo et al., Cell, 29:99-108, 1982), RU-486 (a progesterone antagonist) (Wang et al., Proc. Natl. Acad. Sci. USA, 91:8180-8184, 1994), steroids (Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607, 1993), tetracycline (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-5551, 1992; US Pat. No. 5,464,758; Furth et al. ... 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) and the tTAER system based on a multi-chimeric transactivator consisting of the tetR polypeptide as the activation domain of VP16 and the ligand binding domain of the estrogen receptor (Yee et al., 2002, US 6,432,705).

[0294] In addition to the above-mentioned polymerase II promoter, suitable promoters for nucleotide sequences encoding small RNAs for knocking out specific genes (see below) by RNA interference include polymerase III promoters. RNA polymerase III (pol III) is responsible for synthesizing a variety of small nuclear and cytoplasmic non-coding RNAs, including 5S, U6, adenovirus VA1, Vault, telomerase RNA and tRNA. The promoter structures of a large number of genes encoding these RNAs have been determined, and it has been found that RNA pol III promoters are divided into three types of structures (for review, 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). Particularly suitable for expressing siRNA is the type 3 RNA pol III promoter, in which transcription is driven by cis-acting elements found only in the 5' flanking region (i.e., upstream of the transcription start site). Upstream sequence elements include the traditional TATA box (Mattaj et al., Cell, 55: 435-442, 1988), the proximal sequence element and the distal sequence element (DSE; Gupta and Reddy, Nucleic Acids Res., 19: 2073-2075, 1991). Examples of genes controlled by type 3 pol III promoters are U6 small nuclear RNA (U6 snRNA), 7SK, Y, MRP, HI and telomerase RNA genes (see, e.g., Myslinski et al., Nucl. Acids Res., 21: 2502-09, 2001).

[0295] Gene therapy vectors can optionally include a second or one or more other nucleotide sequences encoding a second or another polypeptide. The second or other polypeptide can be a (selectable) marker polypeptide that allows identification, selection and / or screening of cells containing expression constructs. Suitable marker proteins for this purpose are, for example, fluorescent protein GFP and selective marker gene HSV thymidine kinase (for selecting on HAT culture medium), bacterial hygromycin B phosphotransferase (for selecting on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selecting on G418) and dihydrofolate reductase (DHFR) (for selecting on methotrexate), CD20 (low-affinity nerve growth factor gene). The source of these marker genes and their use methods are provided in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, 2001.

[0296] Alternatively, the second or additional nucleotide sequence can encode a polypeptide that provides a fail-safe mechanism that allows individuals from transgenic cells to be cured if deemed necessary. This nucleotide sequence, commonly referred to as a suicide gene, encodes a polypeptide that can convert a prodrug into a toxic substance that can kill transgenic cells expressing the polypeptide. Suitable examples of such suicide genes include, for example, the Escherichia coli cytosine deaminase gene or one of the thymidine kinase genes 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 an individual (see, for example, Clair et al., Antimicrob. Agents Chemother., 31: 844-849, 1987).

[0297] Administration routes for ocular gene therapy

[0298] In one aspect, the present invention provides a gene therapy method for treating ocular degenerative diseases. One skilled in the art will recognize approaches for gene therapy involving intravitreal administration or intraocular administration.

[0299] Intraocular administration delivers the gene composition to any place of the eyeball. In a preferred embodiment, the gene composition is injected intravitreally. Intravitreal administration is to deliver the gene composition directly to the vitreous compartment of the eye, i.e., directly adjacent to the retina, constituting the liquid at the back of the eye. In certain embodiments, the vitreous is removed during injection to leave room for the drug.

[0300] Intravitreal injections are usually performed in a doctor's office, but can also be done in a hospital operating room. Patients usually receive local anesthesia (usually eye drops or soaked sponges / swabs) and commonly use drugs such as proparacaine, lidocaine, etc.

[0301] In certain embodiments, the genetic composition is injected subretinal (ie, beneath the retina).

[0302] For injection, a 27-32 gauge needle can be used. In a preferred embodiment, the needle size is 30 gauge. Needle injection is becoming a common technique for injecting medications into the eye and does not pose any risks. The injection may be deep or shallow. The injection may be straight (i.e., 90 degrees to the surface of the eye), angled (45-60 degrees), or biplanar (angled, not straight).

[0303] Gene therapy in the eye

[0304] The present invention provides a method for increasing NAD in RGC by using a gene combination. + The eye is an ideal organ for gene therapy because of its small size (i.e., few cells to be transfected), easy access, partial / majority immune privilege (i.e., little / near chance of eliciting an immune response), and the fact that the contralateral eye serves as a contralateral control and backup.

[0305] Several eye-based gene therapy clinical trials are ongoing for blinding diseases. For example, gene therapy for Leber's congenital leucorrhea using an AAV2 vector is in Phase III clinical trials (NCT00481546, NCT00516477, NCT00643747, and NCT00999609); gene therapy for the MERTK gene using an AAV2 vector for retinitis pigmentosa is in Phase I (NCT014822195); gene therapy for the ABCA4 gene using an EIAV lentiviral vector for Stargardt's is currently in Phase II (NCTO 1367444); and gene therapy for the REP-1 gene using an AAV2 vector for chorioderemia is currently in Phase II (NCT02553135). Clinical studies in several clinics have shown that gene delivery to the eye appears to be safe and tolerated.

[0306] Combination Therapy - Additional Therapeutic Agents

[0307] In certain embodiments, the present invention provides a method of treating neurodegeneration by combination therapy by administering to a person in need thereof a compound that increases the level of NAD in neural cells. + Intracellular levels of the compound (eg, RGC) and an additional therapeutic agent.

[0308] In certain embodiments, additional therapeutic agents of the present invention for treating glaucoma include, but are not limited to, agents that lower IOP. Exemplary additional therapeutic agents include, but are not limited to, beta blockers (e.g., timolol maleate, timolol hemihydrate, levobunolol hydrochloride, metipranolol, carteolol, betaxolol, etc.), non-selective adrenergic agonists (e.g., epinepherine, dipivefrin hydrochloride), selective alpha-2 adrenergic agonists (e.g., araclonidine hydrochloride, brimonidine tartrate, and brimonidine tartrate in sodium carbonate), or carbonic anhydrase inhibitors (CAI, e.g., acetazolamide (oral), acetazolamide (parenteral), methazolamide (oral), dorzolamide (topical), and brinzolamide (topical)).

[0309] In certain embodiments, additional therapeutic agents include prostaglandin analogs (e.g., latanoprost, travoprost, and bimatoprost (prostamide)), sympathomimetic stimulants (including direct cholinergic agonists such as pilocarpine HCL; and indirect cholinergics such as iodine, demercarium iodide, and physostigmine isoflurane), and carbachol (a mixed direct agonist / acetylcholine releaser).

[0310] In certain embodiments, the additional therapeutic agent comprises a fixed combination drug that provides the potential advantages of increased convenience, compliance, efficacy, and cost. The fixed combination may comprise a topical beta-blocker in combination with a prostaglandin analog, an alpha-adrenergic receptor agonist, or a topical carbonic anhydrase inhibitor. Exemplary fixed combinations include: (1) dorzolamide and timolol, such as 2% dorzolamide hydrochloride and 0.5% timolol maleate eye drops (e.g., Cosopt, now as a generic name); (2) brimonidine with timolol or brinzolamide, such as 0.2% brimonidine tartrate, 0.5% timolol maleate eye drops (e.g., Combigan) and 0.2% brimonidine tartrate and 1% brinzolamide (e.g., Simbrinza) or (3) latanoprost and timolol.

[0311] In certain embodiments, additional therapeutic agents include hyperosmotic agents such as oral glycerol, oral isosorbide, and intravenous mannitol, which can rapidly lower IOP by reducing vitreous volume. They do not cross the blood-ocular barrier and therefore exert colloid osmotic pressure that dehydrates the vitreous. Hyperosmotic agents are often used in acute situations to temporarily lower high IOP until more definitive treatment can be provided.

[0312] The invention is illustrated by the following non-limiting examples.

[0313] Example

[0314] Example 1 Identification of Glaucoma Susceptibility Genes / Pathways

[0315] (A) Selection of mouse models to mimic neurodegeneration in humans

[0316] We chose the DBA / 2J D2 (D2) mouse model to examine the pathogenesis of human neurodegeneration. The D2 mouse is a widely used glaucoma model that encompasses the hallmark features of human glaucoma. In the D2 mouse, two genes (Gpnmb R250X , Tyrp1 b ) causes a progressive iris disease. This iris disease has two major components: iris stromal atrophy (very similar to human idiopathic iris atrophy) and an iris pigment dispersion phenotype (very similar to human pigment dispersion syndrome). Both idiopathic iris atrophy and pigment dispersion syndrome predispose to elevated IOP and glaucoma in humans. Iris disease in D2 eyes results in age-related elevated intraocular pressure (IOP) in most eyes by 8-9 months of age. Optic nerve degeneration is essentially complete by 12 months of age (usually >70% of the nerve has severe damage).

[0317] In this study, D2 mice demonstrated that they develop ocular nerve degeneration following age-related increases in IOP. Specifically, we used D2 mice at three ages; namely, 4, 9, and 12 months of age.

[0318] (i) Demonstration that 4 month old D2 mice exhibit normal intraocular pressure (IOP) (i.e., 10-13 nmHg) and are free of glaucoma (i.e., these mice are at an age prior to the development of IOP and pre-glaucoma; no detectable neurodegeneration). At this age, D2 mice are indistinguishable from control mice.

[0319] (ii) Demonstrated that 9-month-old D2 mice exhibited high IOP (i.e., >21 nmHg) but no neurodegeneration. Despite the lack of traditional glaucoma at this stage of development (i.e., pre-neurodegeneration), 9-month-old D2 mice had molecular changes in their eyes that were defined herein as having "early glaucoma."

[0320] (iii) demonstrated that D2 mice at 12 months of age exhibited variably high IOP (ie, 14 to >21 nmHg) and neurodegeneration became evident (ie, glaucoma; >60% of eyes had severe neurodegeneration).

[0321] (iv) The control D2-Gpnmb was used in this study + mice, as these mice neither show high IOP nor neurodegeneration with age. R250X These mice were identical to D2 mice except for the mutant iris disease.

[0322] (B) Retinal ganglion cells (RGCs) were isolated from the retina of D2 mice.

[0323] We obtained 100 μg / mL of D2-Gpnmb from (i) 4-month-old DBA / 2J D2 mice, (ii) 9-month-old DBA / 2J D2 mice, and (iii) age-, sex-, and strain-matched D2-Gpnmb mice. + Retinas were collected from eyes obtained from wild-type controls.

[0324] Retinal samples were first stained with a cocktail of antibodies. Retinal ganglion cells (RGCs) were identified as Th1.2 + cells (and for Cd11b - 、Cd11c - 、Cd31 - 、Cd3 4- 、Cd45.2 - GFAP - , DAPF was negative). FACS-positive RCGs were plated and stained with SNAP-25 and β-tubulin (specific markers of RGCs) to confirm the RGC status.

[0325] Using fluorescence-activated cell sorting (FAC sorting), we then isolated RGCs from freshly harvested retinas in these three groups of mice. Fig. 27 A-27H.

[0326] (C) RNA sequencing

[0327] To identify susceptibility genes / pathways leading to glaucoma, we performed RNA sequencing (RNA-seq) of RNA from RGCs obtained from (B) to elucidate very early molecular changes within RGCs before neurodegeneration. Amplified dscDNA libraries (double-stranded copy DNA) were generated from RNA and read at a depth of 35 million reads per sample. Data analysis was performed with a false discovery rate (FDR, q) of q<0.05. Samples from all groups (B) were successfully amplified and sequenced.

[0328] We performed a randomized controlled trial of D2 and D2-Gpnmb from 4, 9, and 12 months old rats. + The neural retina of the eyes underwent additional metabolic analysis. Metabolic analysis was performed using targeted assays as recommended by the manufacturer. The following metabolites were analyzed: NAD+ / NADH (i.e., total NAD, NAD(t)), GSH / GSSG (i.e., total glutathione, glutathione(t)), and pyruvate.

[0329] (D) Hierarchical Clustering (HC)

[0330] In this study, we sequenced RNA from isolated RGCs to a depth of 35 million reads per sample. Unsupervised hierarchical clustering (HC) was used to define D2-Gpnmb in age-matched individuals at the initial stage of disease. + or molecularly determined glaucoma stages in samples that were morphologically indistinguishable from young controls.

[0331] HC identified 4 different groups of D2 samples at 9 months of age (i.e., Group 1, Group 2, Group 3, and Group 4). Group 1 clustered with all control samples and represented D2 RGCs with no detectable glaucoma at the molecular level. All samples in Groups 2 to 4 were at an early stage of disease, with increasing group numbers observed reflecting increasing distance from controls (greater disease progression at the transcriptome level) ( Figure 1 , 2A and 2B).

[0332] As the disease progresses, increases in transcript abundance are most pronounced for mitochondrial reads ( Figure 3A ). An imbalance in the relative proportions of mitochondrial molecules encoded by the nuclear and mitochondrial genomes has a negative impact on mitochondrial function. Differential expression of genes encoding mitochondrial proteins in the mitochondrial dysfunction and oxidative phosphorylation pathways and significant enrichment of differentially expressed genes in groups 2 to 4 of D2 (D2-Gpnmb matched for age and sex) + Compared with control RGCs) further pointed to mitochondrial abnormalities within RGCs ( Figures 3A-3J and 3K and Tables 1-3 below).

[0333] Total read abundance across the transcriptome (i.e., all transcribed genes) was divided into those reads derived from the nuclear transcriptome (i.e., encoded in the nucleus) or the mitochondrial transcriptome (i.e., encoded in the mitochondria). Read abundance increased in both transcriptomes in D2 RGCs but was most abundant in mitochondrial-derived reads, suggesting an imbalance that would favor mitochondrial dysfunction ( Figure 3A ).

[0334] Top enriched pathways (IPA) present in D2 groups 2, 3, and 4. There are no enriched pathways in D2 group 1, as there is only 1 differentially expressed (DE) gene. This D2 group 1 represents eyes without glaucomatous damage ( Figure 3B ).

[0335] We prepared a map of all mitochondrial proteins encoded by nuclear (but not mitochondrial) genes. DE genes are shown in red. Non-DE genes are shown in grey. Increased abundance of nuclear-derived transcripts encoding mitochondrial proteins further suggests an imbalance in mitochondrial turnover or function ( Figure 3C ).

[0336] Increased expression of mitochondrial fission genes (Dnml and Fisl) indicates pro-fission events in mitochondria in the early stages of glaucoma. Increased fission is associated with increased mitochondrial turnover and disease. Mutations that affect mitochondrial fusion / fission dynamics are often lethal or cause neurological disorders (including but not limited to dominant optic atrophy, Charcot-Marie-Tooth disease) ( Figure 3D ).

[0337] Increased expression of genes involved in the mitochondrial unfolded protein response, a stress response within cells that often precedes apoptosis (programmed cell death). Figure 3E ).

[0338] Individual gene maps in mitochondrial-related pathways were prepared. DE genes are shown in red and non-DE genes are shown in grey. Note the upregulation of mitochondrial genes across pathways, especially oxidative phosphorylation and reactive oxygen metabolism, which points to an energy crisis within the cell ( Figure 3F ).

[0339] Protein analysis (Western blot) of the mitochondrial pro-apoptotic molecule cytochrome c from the retina showed that cytochrome c was upregulated at both the gene and protein levels during early glaucoma (9 months) and was significantly upregulated by 12 months ( Figure 3G and 3I ).

[0340] Protein analysis also confirmed the upregulation of eIF2 in the retina ( Figure 3H and 3J ).

[0341] Table 1 from Figure 1 , 3A - List of mitochondrial complex genes of 3E, 3K, 4A and 4B

[0342]

[0343]

[0344]

[0345] Table 2 For Figure 1 , 3A -Genes included in pathway terms for 3E, 3K, 4A, 4B, 5A, 11-14, 20A, 20B

[0346]

[0347]

[0348]

[0349] (E) Top enriched pathways based on IPA analysis

[0350] Table 3 lists the 10 most enriched pathways based on IPA analysis. Figure 1 provided us with our knowledge on D2 mice and D2-Gpnmb + Results of preliminary analysis of top enriched pathways between controls (the disclosure of which is incorporated by reference).

[0351] Table 3 Ten most enriched pathways (IPA analysis)

[0352]

[0353]

[0354]

[0355]

[0356] Pathway analysis identified enrichment of eIF2 and mTOR signaling transcripts ( Figure 3B and 3F ), with eIF2 signaling being the most enriched pathway in cluster 2 (the first distinguishable stage from controls).

[0357] Significant upregulation of mitochondrial fission genes (Dnml and Fisl) Figure 3D ) and significant changes in the mitochondrial unfolded protein response (UPRmt) ( Figure 3E ), further indicating mitochondrial dysfunction.

[0358] (F) Morphology reveals mitochondrial abnormalities

[0359] In this study, we evaluated morphological alterations by performing electron microscopy (EM) in the retinas of these mice. EM currently provides us with the highest resolution available to study the morphology of intracellular organelles such as mitochondria. EM revealed dysfunctional mitochondria with reduced mitochondrial cristae volume in the dendrites of D2 RGCs, but not in the dendrites of control RGCs ( Figure 4A and 4B These mitochondrial EM findings were consistent with synaptic loss in D2 retinas at 9 months of age and an early decrease in pattern electroretinogram amplitude (PERG, a sensitive measure of RGC activity in human patients and animals) ( Fig.14 ) and increased levels of cytochrome c in the retina ( Figure 3G ) are consistent.

[0360] These data clearly indicate that mitochondrial perturbations are the earliest changes that occur in RGCs in vivo in hereditary age-related glaucoma. The present findings are consistent with reported in vitro studies in which cultured cells develop mitochondrial abnormalities when subjected to stress. However, in vitro studies have not been able to determine (although the present findings do) how early mitochondrial abnormalities occur in glaucoma in vivo.

[0361] In summary, the present RNA-seq study revealed transcriptional processing and mitochondrial dysfunction in RGCs of glaucoma-susceptible eyes with undetectable neurodegenerative phenotypes. Mitochondrial dysfunction was confirmed by targeted metabolic assays and EM, showing that early mitochondrial abnormalities in glaucomatous eyes are accompanied by an early energy crisis.

[0362] Example 2 Metabolic Analysis

[0363] To determine whether D2 mice (compared to control D2-Gpnmb + To investigate whether mitochondrial dysfunction / energetic crisis exists in glioma-bearing mice (compared with glioma-bearing mice), we performed metabolomic analysis of the neural retina before disease and at the earliest stages of disease (i.e., at 4 months of age (before glaucoma), at 9 months of age in the presence of high IOP without any neurodegeneration, and at 12 months of age when neurodegeneration is present / severe in most eyes).

[0364] We have 4, 9 and 12 months of D2 and D2-Gpnmb + Eyes underwent additional metabolic analysis of the neuroretina. Metabolic analysis was performed using targeted assays as recommended by the manufacturer. The following metabolites were analyzed: NAD+ / NADH (i.e., total NAD, NAD(t)), GSH / GSSG (i.e., total glutathione, glutathione(t)), and pyruvate. There was a significant age-related decline in all metabolites analyzed. These metabolic analysis changes occurred prior to any detectable neurodegenerative phenotype and also occurred in age-matched non-glaucoma controls D2-Gpnmb + In the retina ( Figure 5A -D).

[0365] As these are key molecules for cellular metabolism and protection from cellular stress, these age-dependent declines are expected to sensitize retinal neurons to disease-related stress and mitochondrial dysfunction.

[0366] Our data show that HIF-1α, a key metabolic regulator during perturbed redox status, is induced in the ganglion cell layer early in glaucoma (by RNA-seq and immunostaining: Fig. 6A and 6B This further suggests that RGCs undergo perturbed metabolism (and subsequent overproduction of reactive oxygen species) early in glaucoma.

[0367] To investigate the link between elevated IOP, mitochondrial dysfunction, metabolite depletion (particularly NAD), and cellular stress, we investigated the role of DNA damage and PARP (poly-ADP-ribose polymerase) in early glaucoma. PARP responds to DNA damage and is a major consumer of intracellular NAD.

[0368] RNA-seq datasets suggest that RGCs undergo a phase of mitochondrial stress and metabolite depletion, likely towards fatty acid metabolism (corresponding to the increase in lipid deposits in the retina during early glaucoma, Fig. 7A and 7B ).

[0369] One consequence of fatty acid β-oxidation is increased production of free radicals / reactive oxygen species (ROS). Therefore, we further assessed the retina for signs of ROS-induced DNA damage (by RNA-seq, Figure 3F ; and γ-H2AX immunostaining, a sensitive marker for dsDNA fragmentation). + The nuclei were present throughout the ganglion cell layer of D2 retinas, but not controls, suggesting that DNA damage is increased very early in glaucoma ( Fig. 8A and 8B ).

[0370] PARP activity was found to be induced in RGCs with age ( Fig.9A and 9B ), providing a link between DNA damage and increased metabolic stress within RGCs. Our findings are consistent with the hypothesis that PARP is induced by DNA damage and that they are the main NAD + Depletes enzymes and inhibits glycolysis through PAR inhibition of hexokinase.

[0371] The D2 mouse model clearly supports the paradigm that age is a major risk factor for glaucoma and various other neurodegenerative diseases. Our metabolic profiling data suggest that increasing age may increase neuronal susceptibility to injury through depletion of idiopathic neuronal metabolites.

[0372] In conclusion, the data presented herein support a novel model in which age-dependent declines in NAD and glutathione in the retina sensitize RGCs to glaucoma and possibly other age-related diseases. RGCs are particularly vulnerable to injury from injurious high IOP and glaucoma. Thus, age-dependent metabolite declines as well as PARP activation within RGCs work together to disrupt cellular metabolism and increase susceptibility to sustained IOP-induced stress, thereby leading to profound damage to RGCs.

[0373] In D2 mice, glaucomatous neurodegeneration is age-related, chronic, and heterogeneous. During the development of D2 glaucoma, RGCs undergo early loss of connections as well as metabolic and molecular changes, all of which precede overall cell loss, axonal loss, and optic nerve degeneration. These early changes are expected to reduce the reliability of cellular metabolism and increase the probability of cellular failure of RGCs with age under sustained IOP-induced stress.

[0374] In conclusion, based on RNA-sequencing and metabolic assays, we found that neurons in the retina undergo a metabolic crisis prior to degeneration. This suggests a key role for metabolic molecules that may alter or supplement mitochondrial function. Correcting these dysfunctional processes may be beneficial in glaucoma and related to more general age-related changes.

[0375] Data to date suggest that NAD decline is critical for age-related neuronal susceptibility to glaucoma.

[0376] Example 3 NAM protects against neuronal cell loss in axotomized cultures

[0377] In this line of research, we investigated whether increasing NAD levels in injured eyes could protect against neurodegeneration. Axotomy (severing axons) mimics the acute, severe injury seen in some forms of glaucoma and is an important model for examining these more severe injuries.

[0378] The study was based on the hypothesis that by reducing the likelihood of metabolic / energetic failure, it would make RGCs more flexible to external stresses.

[0379] To identify candidate drugs that could antagonize the mitochondrial dysfunction / energy crisis observed in D2 mice, a large number of drugs with effects at the mitochondria were tested in axotomy cultures. This screen identified nicotinamide (NAM) as having the most robust protective effect against nuclear shrinkage, a hallmark feature of nuclear remodeling prior to apoptosis. Fig.22A and 22B .

[0380] The above in vitro study results are consistent with the in vivo results in Example 3.

[0381] Example 4 NAM alleviates glaucoma in D2 mice

[0382] We used a large population of D2 mice for in vivo experiments.

[0383] In this experiment, the optic nerve was divided into three levels of damage: NO (no glaucoma), MOD (moderate damage) and SEV (severe damage).

[0384] Experimental D2 mice were divided into the following groups and given:

[0385] W = water (standard mouse water)

[0386] NAM or NAM Lo = 550 mg / kg / d NAM in drinking water

[0387] Early = early start = pre-glaucoma = 6 months of age (i.e. preventive)

[0388] Late = Late onset = During glaucoma = 9 months of age (when mice already have high IOP, ie invasive, and more relative to human glaucoma)

[0389] NAD levels were increased by administering nicotinamide (NAM; NAD precursor) to D2 mice (see Figure 5A , 11-14, 20A, and 20B). The mice were then evaluated for optic nerve damage, soma loss, visual function, and axonal transport at 12 months.

[0390] Administration of NAM (550 mg / kg / d, NAM Lo ) prevented the decline in NAD levels up to 12 months, the standard endpoint for assessing neurodegeneration in this glaucoma model ( Figure 5A and 5E ).

[0391] (A) NAM protected mice from all detectable glaucoma symptoms

[0392] Supporting the neuronal vulnerability hypothesis, NAM did not alter IOP ( Figures 10A-10D ), but it strongly protects against glaucoma, i.e., NAM is a neuroprotectant.

[0393] Importantly, NAM was protective both preventively (starting early, at 6 months; before IOP increased in the vast majority of eyes in the group) and as an interventional protective agent (starting late, at 9 months; when IOP continued to be elevated in the majority of eyes). This suggests that NAM can both prevent neuronal damage from occurring and limit neuronal damage to neurons that are already damaged ( Fig.11A and 11B ). This is important for human diseases where treatment is not initiated until the disease becomes symptomatic and detectable.

[0394] These data support that NAMs could be used to preventively treat glaucoma in humans, for example, in individuals at risk for glaucoma due to family history, ocular trauma, known genetic mutations, or those with elevated IOP but no glaucomatous damage.

[0395] (B) NAM can effectively prevent retinal ganglion cell dysfunction and degeneration

[0396] NAM significantly reduced the incidence of optic nerve degeneration ( Fig.11A and 12A ), preventing RGC soma loss and retinal thinning (detectable by common methods in the human clinic - e.g., OCT, funduscopy) ( Fig. 12A and 13A ), restoration of anterograde axonal transport (assessed by Ct-β tracing; and an important early marker of axonal dysfunction and degeneration) ( Fig. 12A ), and by pattern electroretinography (PERG) ( Fig.14 and 15 ) Visual function assessed by PERG. PERG has been shown to be a very sensitive and early indicator of visual dysfunction in glaucomatous eyes in humans and animal models (Saleh et al., Invest Ophthalmol Vis Sci 48, 4564-4572, 2007); therefore, NAM can prevent the early symptoms of glaucoma.

[0397] (C) NAM prevents early synaptic loss and lipid droplet formation in the retina

[0398] Administration of NAM also prevented the early synaptic loss (SNAP-25 immunostaining) that occurs in this model ( Fig.16A and 16B ), and was sufficient to inhibit the formation of dysfunctional mitochondria with abnormal cristae, as assessed by EM ( Fig.17A and 17B ).

[0399] Also prevented the formation of lipid droplets in the aged D2 retina ( Fig.18 ).

[0400] (D) NAM reduces PARP activation and prevents molecular symptoms of glaucoma

[0401] NAM also reduced PARP activation, limiting the level of DNA damage and transcriptional induction of HIF-1α ( Fig.9A , 9B , 19A, and 19B), reflecting less perturbed cellular metabolism. NAMs may prevent these destructive mechanisms by correcting upstream effectors—namely, metabolite consumption and redox buffering within mitochondria.

[0402] NAM prevented even the earliest molecular symptoms of glaucoma in most treated eyes, as assessed by RNA-seq. NAM-treated samples were molecularly similar to controls and clustered within age-matched and young control samples ( Fig. 20A , 20B and 21A-21F).

[0403] (E) NAM prevents age-related gene expression in RGCs

[0404] NAM even prevented most of the age-related gene expression changes in RGCs (n = number of DE genes; 4-month D2-Gpnmb + For 9 months D2-Gpnmb + =4699, 9 months D2-Gpnmb + For NAM = 4437, 4 months D2-Gpnmb + =83 for NAM). This remarkable degree of molecular protection highlights the unexpected efficacy of NAM in reducing the incidence of metabolic disorders and glaucoma in eyes with high IOP. Since age is a major risk factor in the pathogenesis of glaucoma, inhibition of age-related damaging changes has the potential to prevent neurodegeneration in many human glaucoma and other slowly degenerative neurodegenerative diseases.

[0405] In the majority of eyes treated, administration of NAMs completely prevented glaucoma, including based on very sensitive measures of early disease such as PERG. In addition, many ocular and neurodegenerative diseases occur in the elderly because of age-dependent molecular opportunities that increase susceptibility to damage. NAM treatment prevented age-related molecular changes assessed by gene expression, a very sensitive measure of these changes.

[0406] Furthermore, axonal degeneration and somatic atrophy may represent common components of some neurodegenerative diseases. At least in glaucoma treatment, NAM prevents these changes according to the above data. Furthermore, the above data show that NAM prevents axonal degeneration and somatic atrophy in glaucoma.

[0407] Example 5 Adding dietary NAM further reduces the degree of IOP elevation in D2 mice

[0408] NAM is considered to be safely tolerated even at high doses. Some studies have shown that hepatotoxicity occurs in humans at daily doses >4 g NAM. However, these were due to impurities in older preparations. In a recent survey of 6000 patients using high doses of niacinamide or niacin, only 3 cases of jaundice were reported. In one of these cases (6 g niacin / day), the problem resolved after stopping a different but concurrently administered drug (but not niacin), and in the other case, the problem resolved even with continued niacin treatment. With regard to higher doses of niacinamide, it has been suggested that abnormal liver enzyme tests do not indicate hepatocellular damage but rather represent changes in liver enzyme expression that are rapidly reversible when the drug is discontinued. Rare cases of hepatotoxicity may reflect individual genetic susceptibility or other individual factors. Although the long-term effects of high doses require further evaluation, experience suggests that the risk-benefit ratio of long-term niacinamide therapy will be very favorable.

[0409] To further reduce the incidence of glaucoma and protect more eyes from IOP-induced damage, we used an increased dose of NAM (representing 4 times the original lower dose, 2000 mg / kg / d NAM Hi ) was given to D2 mice.

[0410] Notably, NAM (at this dose) was found to be extremely protective in 93% of treated eyes without glaucomatous optic nerve damage ( Fig.11A ). This represents a 10-fold reduction in the risk factor for developing glaucoma. The degree of protection afforded by administration of this single molecule is unprecedented and completely unexpected.

[0411] Mice treated with 550 mg / kg / d NAM showed clear neuroprotection (as IOP was not altered), and increasing doses of 2000 mg / kg / d reduced the extent of IOP elevation ( Fig. 10A and 10B ). This suggests that NAM may protect other cell types, RGCs, from age-related pathogenic processes.

[0412] Current data suggest that NAM may protect against both IOP elevation and neuronal vulnerability, thus having dual benefits and great clinical potential in the treatment of human glaucoma. Even at the lower dose of 550 mg / kg / d, where IOP is unchanged, IOP-lowering treatments (such as surgery or eye drops) may be combined with NAM supplementation to provide greater protection against neurodegeneration.

[0413] Example 6 NAM is effective in two models of RGC death in glaucomatous injury

[0414] Glaucoma is a complex disease involving multiple insults. It has a wide range of etiologies, in which RGC compartments (e.g., soma, axon, dendrite) may be affected differently. Mechanical axonal injury and local inflammation are two important factors in RGC degeneration during glaucoma.

[0415] To evaluate the effectiveness of NAM treatment in different situations, we tested NAM efficacy in two models of RGC death. The first model of glaucomatous injury involved a tissue culture model using axotomy, and the second model of glaucomatous injury involved the use of intravitreal injections of soluble murine TNFα, which drives local inflammation and is implicated in glaucoma.

[0416] NAM strongly protects cultured retinas from RGC somatic degeneration ( Fig.22A and 22B NAM also prevented the amplitude loss and cell loss of PERG in TNFα-injected eyes ( Figures 23A-23C ).

[0417] Given these protections against severe acute injury and the commonalities between glaucoma and other neurodegenerative diseases, NAMs may have broad implications for the treatment of glaucoma and other age-related neurodegenerative diseases.

[0418] Example 7 Nmnat1 gene therapy alleviates glaucoma in D2 mice

[0419] In this study, we demonstrated that NAD + Production( Fig.25 Overexpression of Nmnat1, a key enzyme in NAD + The cellular mechanisms of production.

[0420] We injected AAV2.2 vector containing Nmnat1 gene and GFP reporter under CMV promoter expressed as a single transcript into the eyes of D2 mice at 5.5 months. Mice were anesthetized and injected with 1.5 μL viral vector (3x10 8 U / mL) was injected intravitreally (i.e., behind the limbus, into the vitreous chamber at an angle of 45-60 degrees, avoiding the lens and central retina). Both eyes were injected. Moisturizing eye drops were applied topically immediately after injection, and the mice were allowed to wake up under a heating lamp. The eyes were clinically examined at multiple time points after injection to confirm that there was no damage to the eyes. Visual function was assessed (by PERG) before and after injection to confirm that there were no adverse effects of the initial injection and viral transfection.

[0421] Nmnat1 expression was detectable at least in the first week after AAV2.2 injection (assessed by GFP expression) and was robust in RGCs (expressed in >83% of RGCs) by 2 weeks after injection and remained robust until the final time point (12 months). The vast majority of RGCs were transduced and expressed the virally delivered gene product, as evidenced by GFP fluorescence in RGC cell bodies in the retina and their endpoints in the brain, including the lateral geniculate nucleus (LGN) and superior colliculus (sup.col.).

[0422] Overexpression of Nmnat1 is sufficient to prevent axon and soma loss ( Figures 24A-24C and Fig.24D , upper panel) and preserves axoplasmic transport and electrical activity in RGCs (PERG) ( Fig. 24B and Fig.24D - lower panel). Glaucomatous neurological damage was absent in >70% of treated eyes. This robust protection encourages the use of similar gene therapy strategies to prevent glaucoma in humans.

[0423] Example 8 Combination therapy using Nmnat1 gene therapy and NAM

[0424] In this study, we examined the relationship between Nmnat1 and NAM (NAM Lo Briefly, mice received gene therapy as above, and after a 1-week viral shedding period, mice were returned to our regular colony and administered NAM 550 mg / kg / d in normal drinking water.

[0425] This combination provided significant additional protection, with 84% of eyes having no detectable glaucomatous damage at 12 months of age. This represents an approximately 4-fold reduction in the risk of developing IOP-induced glaucomatous neurodegeneration compared with untreated D2 controls.

[0426] Increasing the dose of NAM combined with gene therapy was more protective.

[0427] Embodiment 9 s Alleviating glaucoma in D2 mice

[0428] Wallerian slow allele (Wld s ) has been shown to partially protect against D2 glaucoma. S The protein is a modified NMNAT1 protein (an enzyme that converts NMN to NAD). S The cells showed increased enzyme activity, allowing NMN to be converted to NAD more quickly or more efficiently.

[0429] In this experiment, we confirmed that in the presence of Wld s NAM and Wld in mutant D2 mice s Combination vs. single Wld s Better prevention of glaucoma. Apply NAM and carry Wld s The mutant mice showed significant protection from glaucomatous damage (-95% glaucoma-free).

[0430] In addition to using the slow allele with Wallerian degeneration (Wld s The experiment was basically the same as in Example 4 except that D2 mice were grown in the presence of 1% β-cells. The results are also shown in Fig. 11B Obviously, whether using Wld alone s Still, NAM alone can prevent D2 glaucoma, but Wld s The combination with NAM significantly outperformed Wld alone s .

[0431] Specifically, NAM alone provided about 70% protection against glaucoma. s The protection rate against glaucoma is -95%. NAM and NAM+Wld sTherefore, although NAM and Wld s Individually they partially protected, but the combination showed a significant synergistic effect (ie from -70% protection to -95%).

[0432] Although not wishing to be bound by any particular theory, it is believed that by using Wld s The mutation increases Nmnat expression allowing more NAM to be converted to NADt, and the combination synergistically protects against neurodegeneration in glaucoma.

[0433] Consistent with this theory, NADt and other TCA / Krebs cycle components (e.g., pyruvate, Figure 5D ) show age- and disease-related declines in cellular levels. Therefore, cellular levels of NADt (NAD + / NADH).

[0434] We found that in NAM-treated mice as well as in Wld s NADt levels in mice + / NADH) is restored. When Wld s When mice were further treated with NAM, in NAM+Wld s The restoration of NADt levels in treated mice was synergistic (see Figure 5E ).

[0435] The results of this example are consistent with those in Example 8.

[0436] NADt (NAD + NAMs, NAM derivatives and / or NMNAT enzymes may be protective in a wide range of aging and disease phenotypes.

[0437] Example 10 Pyruvate alleviates / prevents glaucoma in D2 mice

[0438] This example demonstrates that nicotinamide (vitamin B3) and / or in combination with pyruvate (a simple metabolite of glucose) strongly protects DBA / 2J mice from vision loss, loss of axonal transport, retinal and optic nerve damage. Gene expression experiments demonstrate that mice treated with nicotinamide are more molecularly matched to young control mice than to age-matched non-glaucomatous mice. The data suggest that nicotinamide may work in part through an age-dependent mechanism, delaying the age-dependent increase in susceptibility to glaucoma. The degree of protection provided by NAM is completely unexpected and surprising.

[0439] Pyruvate is an important simple α-keto acid that participates in the metabolism that produces it from glucose. Pyruvate is converted into acetyl-CoA, which is the main input of the Kreb cycle (citric acid cycle). Under normoxic conditions (normal conditions) pyruvate increases NADH levels.

[0440] With aging, pyruvate levels in the retina decrease, making retinal neurons sensitive to glaucomatous damage under high intraocular pressure ( Figure 5D Mice given pyruvate in their normal drinking water had increased pyruvate levels in the retina ( Figure 5D Mice given pyruvate in their normal drinking water were resistant to optic nerve axonal degeneration, cell loss in glaucoma, and visual dysfunction (assessed by PERG) ( Fig. 11B , 12B , 13B and 14).

[0441] The above findings showed increased efficacy in mice administered NAM and pyruvate, suggesting a synergistic effect between NAM- and pyruvate-treatment to prevent neuronal degeneration.

[0442] Example 11 PQQ prevents nuclear diameter shrinkage and reduces cell density

[0443] Additional neuroprotective agents are also tested to determine their role in neurodegenerative diseases.

[0444] 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 biogenesis, and its neuroprotective function is believed to depend on its function as an antioxidant. Figure 26 shows that PQQ is protective in retinal tissue culture.

[0445] Example 12: Glutathione levels in the retina decrease with age

[0446] N-acetylcysteine ​​(N-acetyl-L-cysteine ​​or NAC) is a precursor of L-cysteine, a precursor of glutathione, a potent biological antioxidant and a major redox buffer in neurons. Figure 5B Data from the retina showed that glutathione levels decrease with age.

[0447] The synthesis of GSH from GSSG requires NADPH (generated from NAD). Therefore, NAD and GSH levels are intrinsically linked.

[0448] Materials and methods

[0449] 1. Mouse strains, breeding, and animal husbandry

[0450] As previously reported, mice were housed in a 14-hour light / 10-hour dark cycle with food and water available ad libitum (Howell et al., Journal of Clinical Investigation 121, 1429-1444, 2011). All breeding and experimental procedures were performed in accordance with the Association for Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic Research. The Institutional Biosafety Committee and the Animal Care and Use Committee of the Jackson Laboratory approved this study.

[0451] C57BL / 6J(B6), DBA / 2J(D2) and DBA / 2J-Gpnmb were used Rl50X (D2-Gpnmb + ) strains and are described in detail elsewhere (Anderson et al., Nature Genetics 30, 81-85, 2002). D2-Gpnmb + The mice do not develop high IOP and therefore do not develop glaucomatous neurodegeneration (Libby et al., Vis Neurosci 22, 637-648, 2005). They are genetically matched controls of DBA / 2J mice.

[0452] For the elderly glaucoma experiment, NAM was administered in food and / or water at 6 months (preventive, before IOP elevation in almost all eyes) or 9 months (when most eyes had high IOP and molecular changes, but no detectable neurodegeneration) (Howell et al., Journal of Clinical Investigation 121, 1429-1444, 2011). These molecular changes precede PERG deficiency and loss of anterograde axoplasmic transport. Low doses of NAM (NAM Lo , 550 mg / kg / d, PanReac AppliChem) dissolved in normal acidic drinking water (350 mL) and replaced weekly. Hi , 2000mg / kg / d), NAM was provided in both water (550mg / kg / d) and food (1450mg / kg / d) and changed weekly. NAM food was prepared in a previously disclosed custom Western diet for palatability (LabDiet) (Graham et al., Sci Rep 6, 21568, 2016). Control mice received the same diet without the addition of NAM. This diet had no effect on the eye health of mice.

[0453] 2. FAC sorting of RCS

[0454] Before cell collection, all surfaces and volumes were cleaned with 70% ethanol and RNaseZap (ThermoFisherScientific) solution and then with dHO.Mice were euthanized, eyes were removed and immediately placed in ice-cold HBSS.Retinas were cut from eyes (4 or 9 months old, without axonal degeneration confirmed by PPD staining, data not shown) in HBSS on ice, and directly placed in 100 μL conventional HBSS (Gibco), dispase (5U / mL) (Stemcell Technologies), DNase I (2000U / mL) (Worthington Biochemical) and SUPERase (1U / μL) (ThermoFisherScientific) solutions.By PPD staining and analysis, all retinas were from eyes without glaucoma axonal degeneration, (see optic nerve assessment below).Retinas were incubated at 37°C for 20 minutes and vibrated at 350RPM in Eppendorf Thermomixer R, then ground gently using 200 microliters of pipettes. Samples were blocked in 2% BSA, SUPERase (1U / μL) in HBSS and stained with conjugated antibodies against Cd11b, Cd11c, Cd34, Cd45.2, GFAP, Thy1.2, and DAPI. This mix allowed other retinal cell types to be precisely removed during FACS.

[0455] FACS was performed on a FACS Aria (BD Biosciences).Thy1.2 (negative for all other markers) RGCs were sorted into 300 μl buffer RLT + 1% β-MEM, vortexed and frozen at -80°C until further processing.

[0456] 3. RNA Sequencing

[0457] The FAC-sorted RGC samples were thawed on ice and homogenized in RLT buffer (total volume 300 μL) by syringe. According to the manufacturer's protocol (Qiagen), total RNA was isolated using the RNeasy micro kit, including an optional DNA enzyme treatment step, and the quality was assessed using an Agilent 2100 bioanalyzer. Concentration was determined using Ribogreen Assay (Invitrogen). Amplified dscDNA libraries were generated using Nugen Ovation RNA-seq System V. SPIA dscDNA was sheared to a length of 300 bp using a Diogenode disruptor. Quality control was performed using an Agilent 2100 bioanalyzer and DNA 1000 chip analysis.

[0458] The resulting library sizes were analyzed by qPCR using the Library Quantitation Kit / Illumina GA / ABIPrism (Kapa Biosystems). Six samples per lane were barcoded, pooled, and sequenced on a HiSeq 2500 sequencer (Illumina) at a depth of 30–35 million reads per sample.

[0459] 4. Differential Gene Expression and Pathway Analysis

[0460] Samples were subjected to quality control analysis by custom quality control python scripts. Readings of 70% of bases with base quality scores ≥ 30 were retained for further analysis. Reading comparisons were performed using TopHat v 2.0.7 (Kim et al., Genome Biol 14, R36, 2013), and expression estimates were performed using HTSeq (Anders et al., Bioinformatics 31, 166-169, 2015) and default parameters (build-mm10) for DBA / 2J mouse genomes that provide annotations. Mapping statistics were calculated using Bamtools v 1.0.2 (Barnett et al., Bioinformatics 27, 1691-1692, 2011).

[0461] Differential gene expression analysis between groups was performed using edgeR v 3.10.5 (Robinson et al., Bioinformatics 26, 139-140, 2010), followed by batch correction using RUVSeq, by removing genes with less than five reads in more than two samples to remove outlier samples and lowly expressed genes. Normalization was performed using the truncated mean of the M value (TMM).

[0462] Unsupervised HC was performed in R (1-cor, Spearman rank correlation coefficient). A total of 72 samples were successfully amplified and sequenced in all groups, and 9 samples were removed as outliers after the HC pipeline. Adjustment for multiple testing was performed using the false discovery rate (FDR). Genes were considered significantly differentially expressed at FDR < 0.05.

[0463] To investigate age-related changes, D2-Gpmnb at 4 and 9 months of age were compared. + Samples and NAM LoSamples. For pathway analysis, QIAGEN's Ingenuity Pathway Analysis (IPA, Qiagen) was used for network generation across significantly differentially expressed genes. The list of significantly differentially expressed genes was uploaded to IPA and mapped to Mus musulus Entrez gene symbols using the IPA knowledgebase. These objects will be overlaid onto canonical pathways developed based on the information contained in the IPA knowledgebase. IPA's built-in network scoring algorithm will be used to grade the generated network.

[0464] 5. Metabolic phenotype

[0465] For GSH / GSSG and NAD + For quantification of GSH / GSSG, Cayman Chemical; NAD / NADH, retinas were dissociated (as above) and quantified according to the manufacturer's instructions (GSH / GSSG, Cayman Chemical; NAD + Compounds were measured using the standard curve generated using the kit standards. The final metabolite concentration of each sample was normalized to the total protein concentration measured by the Bradford assay.

[0466] 6. Whole Retinal Explant Culture

[0467] The mice were euthanized, eyes were removed and immediately placed in ice-cold HBSS. The retina was excised from the eye in HBSS on ice, and the ganglion cell layer was placed flat on a cell culture insert (MiUipore) and immersed in tissue culture medium containing Neurobasal-A, 1% penicillin-streptomycin (10000U / mL), 1% glutamine (100x), 1% N-2 (100x) and 1% B-27 (50x) (all ThermoFisher Scientific).

[0468] For drug-treated retinas, tissue culture medium was prepared as above and supplemented with one of the following compounds (all Sigma unless otherwise stated): β-NAD, NAM (PanReac AppliChem), β-MEM. Retinas were incubated for 5 days at 37°C and 4% CO2 in 6-well plates, then fixed in 4% PFA and stained with DAPI. (For untreated "day 0" controls, retinas were excised and placed directly into 4% PFA.) Retinas were imaged on a Zeiss AxioObserver.

[0469] 7. Soluble Mouse TNFα Injection

[0470] To induce delayed retinal ganglion cell degeneration, soluble mouse TNFα (1 ng / μL) was injected intravitreally as previously described (Nakazawa et al., J Neurosci 26, 12633-12641, 2006). 10-week-old B6 mice were pretreated with NAM for 2 weeks before TNFα injection and PERG was performed at 8, 10 and 12 weeks of age. Mice were sacrificed at 12 weeks and RGC counts were performed.

[0471] 8. Clinical phenotype

[0472] The elevated IOP in D2 mice is a consequence of pigment-dispersed iris disease. In all experiments, the progression of iris disease and intraocular pressure in mutant or drug-treated mice was compared with control D2 mice as previously described (John et al., Invest Ophthalmol Vis Sci 39, 951-962, 1998). In each experiment, iris disease and intraocular pressure were assessed.

[0473] Iris disease was assessed at two-month intervals starting at 6 months of age until completion of the experiment.

[0474] IOP was measured at 45-day intervals starting at 8.5-9 months of age until the completion of the experiment.

[0475] 9. Pattern electroretinogram (PERG)

[0476] PERG was recorded subcutaneously from the snout as previously reported. Briefly, graphic stimuli (0.05 cycles / degree, 100% contrast gratings) generated on an LED panel were presented to each eye separately, with slightly different frequencies around 1 Hz. Waveforms were retrieved using the asynchronous averaging method (Chou et al., Invest Ophthalmol Vis Sci 55, 2469-2475, 2014).

[0477] Mice were anesthetized using ketamine / xylazine (Savinova et al., BMC Genet 2, 12, 2001), and body temperature was maintained at 37°C in a feedback-controlled heating phase monitored by a rectal thermometer.

[0478] 10. Optic nerve assessment and determination of glaucomatous damage

[0479] The processing of the optic nerve and staining with p-phenylenediamine (PPD) is known (Smith et al., Systematic evaluation of the mouse eye. Anatomy, pathology and biomethods. CRC Press, Boca Raton, 2002). PPD stains the myelin sheaths of all axons but faintly stains the axoplasm of only damaged axons. It is well established to provide a very sensitive method for measuring optic nerve damage (Smith et al., Systematic evaluation of the mouse eye. Anatomy, pathology and biomethods. CRC Press, Boca Raton, 2002).

[0480] Briefly, the intracranial portion of the optic nerve was fixed in 4% PFA at room temperature for 48 hours, processed and embedded in plastic. Optic nerve segments within a 1 mm area from the posterior surface of the sclera were excised (1 μm thick sections) and stained with PPD. Typically 30-50 sections were taken from each nerve.

[0481] When determining the extent of damage, multiple parts of each nerve are considered. The optic nerve is analyzed and determined to have one of the following 3 levels of damage:

[0482] (1) No injury or early injury (NOE) - less than 5% axonal damage and no gliosis. This degree of damage is seen in age- and sex-matched non-glaucomatous mice and is not due to glaucoma. Although none of these eyes show glaucomatous neurological damage, this level of damage is called no glaucoma or early glaucoma because some of these eyes have early molecular changes before neurodegeneration. These molecular changes can be detected by gene expression studies (Howell et al., Journal of Clinical Investigation 121, 1429-1444, 2011). When discussing metabolic, mitochondrial, and gene expression changes here, eyes with these early molecular changes but no degeneration are considered to have early glaucoma.

[0483] (2) Moderate damage (MOD) - average 30% axonal loss and early gliosis.

[0484] (3) Severe (SEV)->50% axonal loss and lesions with prominent gliosis.

[0485] 11. Anterograde axonal transport

[0486] Mice were anesthetized with ketamine / xylazine and injected intravitreally with 2 μL of AF488 or AF594 cholera toxin subunit B (1 mg / mL in PBS) (ThermoFisher Scientific). After 72 hours, mice were anesthetized and euthanized by 4% PFA cardiac perfusion. Brains and eyes were postfixed in 4% PFA for another 24 hours, cryoprotected in 30% sucrose in PBS overnight (ON), frozen embedded in OCT and sectioned at 20 μm. AF488 was visualized using Zeiss AxioObserver or Zeiss Axiolmager.

[0487] 12. Histology

[0488] For immunofluorescence staining, mice were euthanized by cervical dislocation, their eyes were enucleated and placed in 4% PFA ON. Retinas were excised and mounted on slides, permeabilized with 0.1% Triton-X for 15 min, blocked with 2% BSA in PBS and stained in primary antibodies at room temperature (see antibody list below).

[0489] After primary antibody incubation, retinas were washed five times in PBS and stained with secondary antibodies for 4 h at room temperature. Slides were then washed five more times with PBS, stained with DAPI for 15 min, mounted on a fluorometer, covered with a coverslip and sealed with nail polish.

[0490] For retinal sections, eyes were cryoprotected in 30% sucrose ON, frozen in OCT and cold-sectioned at 18 μm. Slides were warmed to room temperature and the above procedure was followed. Retinas were imaged on a Zeiss AxioObserver to obtain low-resolution counts. Retinal sections were imaged on a Leica SP8 to obtain higher-resolution images.

[0491] For Nissl staining, cryosections were warmed to room temperature, placed in 1:1 alcohol:chloroform ON, and rehydrated through a series of alcohol gradients. Slides were washed once in distilled water and stained in 0.1% cresyl violet in distilled water for 15 min, then differentiated in 95% alcohol, dehydrated in 100% alcohol, and cleared in xylene. Slides were prepared as above. Nissl-stained retinal sections were imaged using a Nikon Eclipse E200.

[0492] For Oil Red O staining, slides were warmed to room temperature, quickly washed in 60% isopropanol, incubated in Oil Red O and hematoxylin for 15 min at room temperature, washed again in 60% isopropanol, mounted and coverslipped. Oil Red O-stained retinal sections were imaged using a Nikon Eclipse E200.

[0493] 13. Western Blot

[0494] Mice were euthanized by cervical dislocation, eyes were removed and immediately placed in ice-cold HBSS. The retina was excised from the eyes in HBSS, directly placed in RIPA buffer and homogenized. Protein extracts were separated by SDS-PAGE in precast gels (Bio-Rad) and transferred to PVDF membranes using iBlot 2 (ThermoFisher Scientific). The membranes were blocked in 5% milk or 5% BSA in 0.1% PBS-Tween and incubated with antibodies in blocking solution. Antibody staining was detected using ECL. Protein concentration was assessed by Bradford assay and normalized to housekeeping proteins using densitometry.

[0495] 14. Electron Microscope

[0496] Mice were euthanized, eyes were removed, and ONs were fixed in Smith-Rudt (0.8% PFA and 1.2% glutaraldehyde in 0.1 M phosphate buffer). The cornea and lens were excised, and the remaining posterior eye cup (containing the retina and ONH) was post-fixed in 2% aqueous osmium tetroxide and dehydrated in ethanol. The samples were then immersed and flat-mounted in Embed812 / Araldite resin (Electron Microscopy Sciences) to allow surface sectioning. The blocks were polymerized at 60°C for 48 hours, and 90nm microtome sections (Leica UC6, Leica) were cut onto 300 mesh copper grids. The grids were stained with 1% aqueous uranyl acetate / Reynolds lead citrate and observed on a JEOL JEM 1230TEM. Images were collected on an AMT 2K camera.

[0497] 15. Gene therapy

[0498] For virally delivered gene therapy, mice were anesthetized (as above) and transfected with 1.5 μL (3.1×10 10 gc / mL) AAV2.2 murine Nmnat1 was injected intravitreally. Mice were injected at 5.5 months of age in a BSL2 laboratory and moved to our conventional mouse colony 2 weeks later.

[0499] For Nmnatl mice that underwent additional NAM treatment, mice were given 550 mg / kg / d of NAM in normal drinking water at 6 months of age. Mice were euthanized at 12 months of age.

[0500] 16. Antibodies

[0501] The antibodies used in the above examples are listed below. IF: Immunofluorescence. WB: Western Blot

[0502] Antibody use concentration Manufacturer Table of contents# CYCS WB 1：1000 Abcam ab90529 EIF2 WB 1：1000 Cell Signaling 9722S H2AX IF 1：500 Millipore 05-636 HIF1A IF 1：500 Novus Biologicals NB 100-105 PARP IF 1：500 BD Pharmingen 556362 RBPMS IF 1：500 Novus Biologicals NBP2-20112 SNAP-25 IF 1：250 Abcam ab24737

[0503] 17. Statistical Analysis

[0504] Sample size (number of eyes, n) is indicated in each figure legend. Graphs and statistical analyses were performed in R. Student's t-test was used for pairwise analysis in quantitative graphs, and error bars represent standard error of the mean unless otherwise stated.

[0505] All cited references are incorporated by reference.While specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that numerous equivalents, modifications, substitutions and variations may be made thereto without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Use of a combination of a therapeutically effective amount of nicotinamide (NAM) and pyruvic acid in the preparation of a pharmaceutical composition for reducing axonal degeneration of retinal ganglion cells in glaucoma, wherein the therapeutically effective amount of nicotinamide (NAM) and pyruvic acid in the pharmaceutical composition is sufficient to be administered at a dose of 1-5 g / day to effectively reduce axonal degeneration of retinal ganglion cells in glaucoma.

2. The use according to claim 1, wherein the pharmaceutical composition further comprises a therapeutically effective amount of one or more compounds selected from nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD) and nicotinamide riboside (NR).

3. The use according to claim 1, wherein the step of reducing the axonal degeneration of retinal ganglion cells in glaucoma further comprises the step of administering a gene composition, wherein the gene composition comprises a gene encoding NMNAT1 polynucleotides.

4. The use according to claim 3, wherein the polynucleotide is in a viral vector.

5. The use according to claim 4, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector or a retroviral vector.

6. The use according to claim 5, wherein the viral vector is an AAV vector.

7. The use according to claim 5, wherein the viral vector is AAV2.

2.

8. The use according to claim 5, wherein the viral vector is a lentiviral vector.

9. The use according to claim 3, wherein the gene composition is administered intravitreally or intraocularly.

10. The use according to claim 3, wherein the gene composition is administered intravitreally.

11. The method according to claim 1, wherein the step of reducing the axonal degeneration of retinal ganglion cells in glaucoma further comprises the step of administering a gene composition, wherein the gene composition comprises a gene encoding NMNAT1 polynucleotides.

12. The use according to any one of claims 1-11, wherein said reducing axonal degeneration of retinal ganglion cells in glaucoma further comprises administering an additional therapeutic agent.

13. The use according to claim 12, wherein the additional therapeutic agent is a beta blocker, a non-selective adrenergic agonist, a selective alpha-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathomimetic agonist, a carbachol, or a combination thereof.

14. The use of claim 12, wherein the additional therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinephrine, alaclonidine, brimonidine, acetazolamide, methazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, epididymidol, carbachol, or a combination thereof.

15. Use of a combination of a therapeutically effective amount of nicotinamide (NAM) and pyruvic acid in the preparation of a pharmaceutical composition for preventing axonal degeneration of retinal ganglion cells in glaucoma, wherein the therapeutically effective amount of nicotinamide (NAM) and pyruvic acid in the pharmaceutical composition is sufficient to be administered at a dose of 1-5 g / day to effectively reduce axonal degeneration of retinal ganglion cells in glaucoma.

16. The use according to claim 15, wherein the pharmaceutical composition further comprises a therapeutically effective amount of one or more compounds selected from nicotinamide mononucleotide (NMN), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD) and nicotinamide riboside (NR).

17. The use according to claim 15, wherein the step of reducing the axonal degeneration of retinal ganglion cells in glaucoma further comprises the step of administering a gene composition, wherein the gene composition comprises a gene encoding NMNAT1 polynucleotides.

18. The use according to claim 17, wherein the polynucleotide is in a viral vector.

19. The use according to claim 18, wherein the viral vector is an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector or a retroviral vector.

20. The use according to claim 17, wherein the gene composition is administered intravitreally or intraocularly.

21. The use according to claim 17, wherein the gene composition is administered intravitreally.

22. The use according to claim 15, wherein the step of reducing axonal degeneration of retinal ganglion cells in glaucoma further comprises the step of administering a gene composition, wherein the gene composition comprises a gene encoding NMNAT1 polynucleotides.

23. The use of any one of claims 15-22, wherein said reducing axonal degeneration of retinal ganglion cells in glaucoma further comprises administering an additional therapeutic agent.

24. The use according to claim 23, wherein the additional therapeutic agent is a beta blocker, a non-selective adrenergic agonist, a selective alpha-2 adrenergic agonist, a carbonic anhydrase inhibitor, a prostaglandin analog, a parasympathomimetic agonist, a carbachol, or a combination thereof.

25. The use of claim 24, wherein the additional therapeutic agent is timolol, levobunolol, metipranolol, carteolol, betaxolol, epinephrine, alaclonidine, brimonidine, acetazolamide, methazolamide, dorzolamide, brinzolamide, latanoprost, travoprost, bimatoprost, pilocarpine, epididymidol, carbachol, or a combination thereof.

Images