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WO2025049588A1 - Nouveaux traitements pour neuropathies optiques et glaucome - Google Patents

Nouveaux traitements pour neuropathies optiques et glaucome Download PDF

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WO2025049588A1
WO2025049588A1 PCT/US2024/044204 US2024044204W WO2025049588A1 WO 2025049588 A1 WO2025049588 A1 WO 2025049588A1 US 2024044204 W US2024044204 W US 2024044204W WO 2025049588 A1 WO2025049588 A1 WO 2025049588A1
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way
mitochondrial
optic
hrgcs
optic neuropathy
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Arupratan DAS
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Indiana University
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    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4172—Imidazole-alkanecarboxylic acids, e.g. histidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A61K9/0051—Ocular inserts, ocular implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present disclosure provides novel approaches and methods for treating various eye diseases or conditions including optic neuropathies.
  • the disclosed methods and treatments are directed to those eye diseases or conditions involving mitochondrial dysfunctions. More particularly, the methods and treatments provided can restore mitochondrial homeostasis.
  • Optic neuropathies are a group of neurodegenerative diseases that result in the dysfunction and death of retinal ganglion cells (RGCs), the projection neurons of the retina that deliver visual signals to the brain.
  • Glaucoma accounts for the majority of optic neuropathies, being a leading cause of blindness worldwide and a leading cause of blindness in individuals of African descent. See Leske, “Open-angle glaucoma-an epidemiologic overview,” (2007) Ophthalmic Epidemiol. 14(4): pp. 166-172; Quigley et al., “The number of people with glaucoma worldwide in 2010 and 2020,” (2006) Br J Ophthalmol. 90(3): pp. 262-267.
  • Glaucoma is largely axogenic which probably explains why significant vision loss occurs even with minor RGC body loss in non-human primate glaucoma model 4 .
  • This interval provides an obvious window of opportunity for neuroprotective therapeutic intervention, especially for patients who do not respond to hypotensive regimens or other treatments, including for secondary glaucoma patients.
  • Currently, our understanding on RGC intrinsic mechanisms that can safely be stimulated in combination with IOP management for novel neuroprotective therapies is limited.
  • IOP elevation first impairs the anterograde axonal transport and at later stages affects retrograde transport (brain to retina) with eventual cell body death 6, 10 , RGC sensitivity to metabolic disruption is due in part to the high ATP consumption need for unmyelinated intraretinal and laminar axons where transport is slow with a lack of saltatory conduction requiring densely packed mitochondria 11 ' 13 .
  • Depleted ATP supply first affects anterograde transport to onset distal axonopathy due to less efficient ATP hydrolysis by the anterograde motor kinesins than the retrograde motor dynein 6 .
  • hRGCs glaucomatous human stem cell differentiated RGC’s
  • OPTN mitophagy protein Optineurin
  • constitutive transport of mitochondria to distal axons can deplete supply to the proximal unmyelinated RGC axons that are particularly ATP-demanding.
  • daily supplementation with pyruvate or constitutive mito-fusion can both activate OXPHOS and in long run that can lead to mitochondrial damage and release of toxic cytochrome c and buildup of toxic oxidative byproducts above the basal level leading to neurodegeneration 19 ' 21 .
  • our long-term goal is to restore metabolic imbalance by improving mitochondrial health and regaining ATP homeostasis without constitutive changes.
  • Mitochondrial biogenesis relies on synthesis of 13 proteins from mitochondrial DNA (mtDNA) and -1000 proteins from the nuclear DNA.
  • Peroxisome proliferator-activated receptor gamma co-activator (PGCla) is a transcriptional co-activator that drives most of these nuclear genes, including mitochondrial transcription factor Tfam, which is required for mtDNA transcription.
  • Tfam mitochondrial transcription factor
  • PGCla is considered as the mitochondrial biogenesis master regulator. See Ventura-Clapier et al., “Transcriptional control of mitochondrial biogenesis: the central role of PGC-la,” (2008) Cardiovascular Research 79(2): pp. 208-217.
  • the inventors have discovered that under optic neuropathy RGCs need to produce more ATPs from a smaller number of functional mitochondria leading to mitochondrial damage, disrupted metabolic homeostasis, and neurodegeneration.
  • the inventors have discovered a novel pharmacological agent that restores mitochondrial homeostasis leading to the restoration of metabolic homeostasis with strong RGC protection effect in multiple glaucoma models. See FIG. 1.
  • one of these compounds provided robust RGC protection, axon regeneration and vision maintenance in mice long after ONC injury with no systemic toxicity.
  • the discovery that restoration of mitochondrial homeostasis could lead to RGC protection is innovative which led the inventors to identify a novel compound and its target signaling pathway without relying on a priori notions about which pathway and molecules are involved in RGC survival.
  • WAY-100635 maleate a serotonin receptor 5-HT1A antagonist that results in mitochondrial biogenesis activation, suppression of apoptosis in human stem cell derived glaucomatous RGCs (hRGCs).
  • hRGCs human stem cell derived glaucomatous RGCs
  • WAY provides robust neuroprotection, axon regeneration and vision maintenance in vivo.
  • the strong cell biology approaches for investigating RGC protection mechanisms using hRGCs and its application for developing in vivo neuroprotection is novel.
  • Inhibition of 5-HT1A receptor by WAY has been shown to alleviate learning and memory impairments in primates and is also indicative of beneficial effects for other neurodegenerative conditions.
  • the 5-HT1A antagonist, WAY-100635 ameliorates the cognitive impairment induced by fornix transection in the marmoset,” (1996) Psychopharmacology (Berl) 127(3): pp. 245-254; Harder et al.. “The 5-HT1A antagonist, WAY- 100635, alleviates cognitive impairments induced by dizocilpine (MK-801) in monkeys,” (2000) Neuropharmacology 39(4): pp. 547-552.
  • the inventors further disclose and have identified BX795 as an activator of mito- biogenesis and RTA-408 (omaveloxolone) as an activator of the oxidative stress response master regulator Nrf2 that led to robust reduction of cellular apoptosis in glaucomatous hRGCs.
  • RTA-408 omaveloxolone
  • Nrf2 oxidative stress response master regulator
  • I4AA, L-glutamic acid, ouabain, BX795 and RTA-408 also show suppression of cell death in hRGCs.
  • the cunent invention provides methods for treating various eye diseases or conditions including optic neuropathies.
  • the disclosed methods and treatments are directed to those eye diseases or conditions involving mitochondrial dysfunction.
  • the methods and treatments provided can restore mitochondrial homeostasis.
  • the eye diseases or conditions contemplated to be treated include glaucoma and optic neuropathies, where the optic neuropathies can include: ischemic optic neuropathy, anterior ischemic optic neuropathy, posterior ischemic optic neuropathy, radiation optic neuropathy, optic neuritis, compressive optic neuropathy, infiltrative optic neuropathy, traumatic optic neuropathy, mitochondrial optic neuropathy, nutritional optic neuropathy, toxic optic neuropathy, and hereditary or inherited optic neuropathy.
  • the methods and treatments disclosed herein include the administration of a therapeutically effective amount of a compound or pharmaceutical composition that includes one or more of WAY- 100635 maleate or other pharmaceutically acceptable salt.
  • FIG. 1 depicts that under optic neuropathy RGCs need to produce more ATPs from a smaller number of functional mitochondria to combat the stress leading to mitochondrial damage, metabolic disruption and neurodegeneration.
  • a novel pharmacological compound that transiently activates of mitochondrial biogenesis and/or other neuroprotective mechanisms that can restore mitochondrial health ultimately leading to the restoration of metabolic homeostasis for RGC protection therapy.
  • FIG. 2A. B shows that the small molecule screen identifies compound for increasing mitochondrial biogenesis.
  • FIG. 2A Small molecule screen on H7-hRGC WT (differentiated from H7-hESC reporter line) identified drugs showing >40% change (outside of blue box) to mitochondria mass by MTDR labeling, with WAY-100635 maleate (WAY) indicated by the arrow.
  • FIG. 2B Manual validation of WAY treatment shows increased MTDR labelled mitochondrial mass in H7- hRGC WT .
  • FIG. 3A - I depicts that WAY transiently activates cyclic AMP (cAMP) level and promotes hRGC survival.
  • FIG. 3A Illustration for mechanism of action by antagonist WAY or agonist DPAT through 5-HT1A GPCR.
  • FIG. 3B Representative max-projections and central z- plane of confocal IF images of H7-hRGC WT against cAMP (anti-cAMP antibody, R&D systems) and DAPI after indicated timepoints of 5 pM WAY treatments. Insets show perinuclear cAMP that increases with WAY treatment.
  • FIG. 3A Illustration for mechanism of action by antagonist WAY or agonist DPAT through 5-HT1A GPCR.
  • FIG. 3B Representative max-projections and central z- plane of confocal IF images of H7-hRGC WT against cAMP (anti-cAMP antibody, R&D systems) and DAPI after indicated timepoints of 5 pM WAY treatments. Insets show per
  • FIG. 3E Quantification of cAMP intensity per area from sum projections, normalized to control shows WAY pretreatment increases cAMP level but addition of DP AT suppresses it. DMSO treatment used for control as DP AT solubilizes in DMSO.
  • FIG. 4A-F shows that WAY promotes neuroprotection in mice against ONC injury.
  • FIG. 4A Schematic illustration of unilateral ONC in mouse.
  • FIG. 4B Representative tiled confocal image (10X/0.3NA) of mouse retinal flat mount with immunohistochemistry (IHC) against RGC specific RBPMS protein. Dotted circle and white boxes on the circles represent central and peripheral retinal regions for image acquisitions and quantifications.
  • FIG. 4C Representative high-resolution confocal images (40X/1.3NA) of RGCs from indicated retinas from peripheral white boxes (FIG. 4B).
  • FIG. 4D Quantification of RGC numbers/mm 2 from central and peripheral retina shows significant protection by WAY on 6 and 14 dpc.
  • FIG. 4E Regenerated or protected RGC axons are detected by confocal IHC images of optic nerve cryosections against growth associated protein 43 (GAP43) following published methods by Larry Benowitz lab 22, 23 . GAP43 antibody is shared by Larry Benowitz.
  • FIG. 4F Quantification of RGC axon numbers at 0.5 mm from crush site on serial cryosections.
  • FIG. 5A-F shows that WAY maintains visual acuity (VA) in mice after ONC injury.
  • FIG. 5A Schematic illustration of the WAY treatment, ONC and subsequent VA measurements.
  • FIG. 5B VA measured by scoring head movements at increasing spatial frequency but at fixed speed and contrast.
  • FIG. 5C, E Quantification of VA in response to both clockwise and counterclockwise rotation of the sinusoidal gratings. One-way ANOVA with Tukey’s correction.
  • FIG. 6D, F VA in response to the individual clockwise or counterclockwise movement of the gratings. Error bars are SEM. Two-way ANOVA with Tukey’s correction, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • n 4 mice/group
  • FIG. 6A-E shows that WAY maintains contrast sensitivity (CS) in mice after ONC injury.
  • CS measurements done on the same mice by OKR as illustrated in FIG. 5A.
  • FIG. 6A CS measured by scoring head movements at decreasing contrast but at fixed speed and spatial frequency.
  • FIG. 6B, D CS presented here as Michelson contrast (max-min/max+min); max, min represent maximum or minimum contrast that the mouse responded (Prusky et al. 2006).
  • FIG. 7C, E CS data in response to the individual clockwise or counterclockwise movement of the gratings. Error bars are SEM.
  • Two-way ANOVA with Tukey 's correction **p ⁇ 0.01. ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • n 4 mice/group.
  • FIG. 7A, B shows that WAY maintains visual depth perception after ONC.
  • FIG. 7A Animals were tested in a visual cliff apparatus with approximately 2 feet tall cliff (deep) side with similar surface pattern under the glass for optical illusion.
  • FIG. 7B Latency time to step down from the platform. Each data point is average from 5 measurements.
  • One-way ANOVA with Dunnett’s, **, p ⁇ 0.005. n 3 mice/group.
  • FIG. 8A-D shows WAY prevents RGC and visual acuity loss in glaucoma mouse.
  • IOP Intraocular pressure
  • MB Polyscience, Inc.
  • PBS PBS
  • FIG. 8B Confocal IHC image of RBPMS positive RGCs
  • FIG. 8C RGC number counted 57 days after MB injection as in FIG. 4D.
  • FIG. 8D Visual acuity measured by OptoMotry (Cerebral Mechanics) where mouse exposed to 4 adjoining LCD monitors with moving sinusoidal gratings (12deg/sec clockwise or counterclockwise) with randomly varying frequency but 100% contrast. Spatial frequency in cycles per degree (c/deg) measured for different condition with mouse identity masked.
  • FIG. 8D 3 mice/group. Two-way ANOVA with Tukey’s, ****, p ⁇ 0.0001. ***, p ⁇ 0.001.
  • FIG. 9A-D shows robust 5-HT1A expression in hRGCs and in mouse RGCs.
  • FIG. 9A Confocal images show 5-HT1A membrane localization in H7-hRGCs and
  • FIG. 9C shows on ganglion cell layer of mouse retinal cryosection.
  • FIG. 9B Western blots detect 5-HT1 A protein expression in hRGCs
  • FIG. 9D detects 5-HT1A expression in RGCs isolated from mouse retina.
  • FIG. 10A-D depicts that WAY transiently activates mitochondrial biogenesis in hRGCs.
  • FIG. 10A Representative confocal images (63X/1.4NA) of H7-hRGC WT with Tom20, TdTomato, and DAPI after 5 pM WAY treatment at indicated timepoints.
  • FIG. 10C Representative max projections of confocal IF images of H7-hRGC WT against PGCl after indicated timepoints of 5 pM WAY treatments.
  • FIG. 1 1 A-F shows that WAY transiently activates mitochondrial biogenesis with neuroprotection effect to OPTN E50K .
  • H7-hRGCs with OPTN E50K mutation are treated with 5 pM WAY for indicated timepoints.
  • FIG. 11 A Images show Mitochondria by confocal IF against Tom20.
  • FIG. 11C Representative live cell confocal images of JC1 labelled mitochondria in H7-hRGC E50K .
  • FIG. 1 IF Shown are action potential firing spikes from a single MEA sensor monitored over 3 minutes for respective conditions.
  • FIG. 1 IB One-way ANOVA with Dunnett’s.
  • FIG. 11D Unpaired student’s t-test.
  • FIG. HE Two- way ANOVA with Tukey's. * p ⁇ 0.05, ** p ⁇ 0.01, **** p ⁇ 0.0001.
  • FIG. 12A, B shows WAY restores OXPHOS and glycolysis balance in OPTN E50K hRGCs.
  • hRGCs are treated with 5 pM WAY for 24 hr follow ed by seahorse analysis.
  • FIG. 12A Seahorse Mito Stress test measures mitochondrial ATP production from change in oxygen consumption rate (OCR) under sequential inhibition of ETC proteins.
  • FIG. 13A-C depicts CRISPR mediated gene KO in hRGCs using lentivirus (LV).
  • FIG. 13A Confocal image (63x/1.4NA) of WT H7-hRGCs transduced with the LV containing viral vector with EFla-GFP cassette or no GFP vector (Control LV).
  • FIG. 13C TBK1 KO in iPSC-hRGCs with OPTN E50K mutation by LV-U6-gRNA (TBK1) and the LV-EFla-Cas9. 8 days post transduction.
  • FIG. 14 shows that LC-MS chromatograms detect WAY in mice retina.
  • the blue filled peak is the analyte of interest.
  • WAY retention time is 5.5 minutes.
  • Representative chromatogram for treated retinal tissue is from 4 independent experiments.
  • FIG. 15 A, B shows that WAY does not show systemic toxicity in mice.
  • FIG. 16 shows that the small molecule screen identifies compound for activator of mitobiogenesis and mitophagy.
  • Error bars are SEM.
  • Arrows indicate compounds that show reduced apoptosis in H7-hRGCs in the secondary screen for potential RGC neuroprotection therapy.
  • FIG. 17A, B shows that L-Glutamic acid (L-Glut) promotes mitochondrial mass and lowers apoptosis in hRGCs.
  • L-Glut L-Glutamic acid
  • FIG. 17A Manual validation of L-Glut treatment at 5 pM for 24h shows increase in mitochondrial mass compared to the untreated control (CTR) in H7-hRGC x l that was identified from high throughput screen in FIG. 16.
  • CTR untreated control
  • Error bars are SEM. Unpaired student’s t-test.
  • FIG. 18 A, B shows that MAA promotes mitochondrial mass and lowers apoptosis in hRGCs.
  • FIG. 19 A, B shows that Ouabain promotes mitochondrial degradation and lowers apoptosis in hRGCs.
  • FIG. 19A Manual validation of ouabain treatment at 5 pM for 24h shows reduced mitochondrial mass in H7-hRGC WT compared to the DMSO control (CTR) that was identified from the high throughput screen in figure 15.
  • FIG. 20A-C shows that Nrf2 activation in OPTN E50K hRGCs is defective.
  • FIG. 20 A Western blot images of Nrf2 and p-Nrf2 in presence of 10 pM CCCP;
  • FIG. 21 A-D shows that the Nrf2 activator RTA-408 reduces ROS level in the WT and glaucoma causing OPTN E50K mutant hRGCs.
  • FIG. 20 A Max projection confocal z-stacks of hRGCs treated with lOOnM RTA-408 or DMSO for 24h. ROS level detected in the live cells by DCFDA which fluoresces green upon oxidation by ROS.
  • FIG. 22 shows that Nrf2 activation by RTA-408 reduces apoptosis in WT and glaucoma causing OPTN E50K hRGCs.
  • the inventors have discovered that under optic neuropathy RGCs need to produce more ATPs from a smaller number of functional mitochondria leading to mitochondrial damage, oxidative stress and neurodegeneration. Transient activation of mitochondrial biogenesis restores mitochondrial homeostasis, leading to RGC protection. See FIG. 1.
  • the present disclosure provides novel approaches and methods for treating various eye diseases or conditions including optic neuropathies.
  • the disclosed methods and treatments are directed to those eye diseases or conditions involving mitochondrial dysfunctions. More particularly, the methods and treatments provided can restore mitochondrial homeostasis.
  • items included in a list in the form of “at least one of A, B, and C” can mean (A); (B); (C): (A and B); (B and C); (A and C); or (A, B, and C).
  • items listed in the form of “at least one of A, B. or C” can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C).
  • the terms “treating” or “to treat” includes restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.
  • the term “patient” refers to a human.
  • optic neuropathy is damage to the optic nerve, which provides visual signals to the brain from the eye, from any cause. Damage and death of these nerve cells, or neurons, leads to characteristic features of optic neuropathy.
  • the main symptom is loss of vision, with colors appearing subtly washed out in the affected eye.
  • a pale disc is characteristic of long-standing optic neuropathy.
  • Optic neuropathy is often called optic atrophy, to describe the loss of some or most of the fibers of the optic nerve.
  • Optic neuropathies capable of being treating by the current invention include: [0058] Ischemic optic neuropathy in which there is insufficient blood flow (ischemia) to the optic nerve.
  • the anterior optic nerve is supplied by the short posterior ciliary artery and choroidal circulation, while the retrobulbar optic nen e is supplied intraorbitally by apial plexus, which arises from the ophthalmic artery, internal carotid artery, anterior cerebral artery, and anterior communicating arteries.
  • Ischemic optic neuropathies are classified based on the location of the damage and the cause of reduced blood flow, if known.
  • Anterior ischemic optic neuropathy includes diseases that affect the optic nerve head and cause swelling of the optic disc. These diseases often cause sudden rapid visual loss in one eye. Inflammatory diseases of the blood vessels, like giant-cell arteritis, polyarteritis nodosa, Churg-Strauss syndrome, granulomatosis with polyangiitis, and rheumatoid arthritis can cause arteritic AIONs (AAION).
  • AAION arteritic AIONs
  • NAION nonarteritic AIONs
  • NAION nonarteritic AIONs
  • NAION is often associated with diabetes mellitus, elevated intraocular pressure (acute glaucoma, eye surgery), high cholesterol, hypercoagulable states, a drop in blood pressure (bleeding, cardiac arrest, perioperative esp. cardiac and spine procedures), and sleep apnea.
  • amiodarone, interferonalpha, and erectile dysfunction drugs have been associated with this disease.
  • Posterior ischemic optic neuropathy is a syndrome of sudden visual loss with optic neuropathy without initial disc swelling with subsequent development of optic atrophy. This can occur in patients who are predisposed to AAION and NAION as described above as well as those who had cardiac and spine surgery or serious episodes of hypotension.
  • Radiation optic neuropathy is also thought to be due to ischemia of the optic nen e that occurs 3 months to 8 or more years after radiation therapy to the brain and orbit. It occurs most often around 1.5 years after treatment and results in irreversible and severe vision loss, which may also be associated with damage to the retina (radiation retinopathy). This is thought to be due to damage to dividing glial and vascular endothelial cells. RON can present with transient visual loss followed by acute painless visual loss in one or both eyes several weeks later. The risk of RON is significantly increased with radiation doses over 50 Gy.
  • Optic neuritis is inflammation of the optic nerve, which is associated with swelling and destruction of the myelin sheath covering the optic nerve. Young adults, usually females, are most commonly affected. Symptoms of optic neuritis in the affected eye include pain on eye movement, sudden loss of vision, and decrease in color vision (especially reds). Optic neuritis, when combined with the presence of multiple demyelinating white matter brain lesions on MRI, is suspicious for multiple sclerosis.
  • optic neuritis which are classified thusly: i) Single isolated optic neuritis; ii) relapsing isolated optic neuritis; iii) chronic relapsing inflammatory optic neuropathy; iv) neuromyelitis optica spectrum disorder; v) multiple sclerosis associated optic neuritis; and various unclassified optic neuritis.
  • Medical examination of the optic nerve with an ophthalmoscope may reveal a swollen optic nerve, but the nerve may also appear normal. Presence of an afferent pupillary defect decreased color vision, and visual field loss (often central) are suggestive of optic neuritis. Recovery of visual function is expected within 10 weeks. However, attacks may lead to permanent axonal loss and thinning of the retinal nerve fiber layer.
  • Compressive optic neuropathy includes tumors, infections, and inflammatory processes which can cause lesions within the orbit and, less commonly, the optic canal. These lesions may compress the optic nerve, resulting optic disc swelling and progressive visual loss. Implicated orbital disorders include optic gliomas, meningiomas, hemangiomas, lymphangiomas, dermoid cysts, carcinoma, lymphoma, multiple myeloma, inflammatory orbital pseudotumor, and thyroid ophthalmopathy. Patients often have bulging out of the eye (proptosis) with mild color deficits and almost normal vision with disc swelling.
  • Infiltrative optic neuropathy are disorders caused by infiltrative processes.
  • the optic nerve can be infiltrated by a variety of manners, including tumors, inflammation, and infections.
  • Tumors that can infiltrate the optic nerve can be primary 7 (optic gliomas, capillary 7 hemangiomas, and cavernous hemangiomas) or secondary 7 (metastatic carcinoma, nasopharyngeal carcinoma, lymphoma, and leukemia).
  • the most common inflammatory disorder that infiltrates the optic nerve is sarcoidosis.
  • Opportunistic fungi, viruses, and bacteria may also infiltrate the optic nerve.
  • the optic nerve may be elevated if the infiltration occurs in the proximal portion of the nerve. The appearance of the nerve on examination depends on the portion of the nerve that is affected.
  • Traumatic optic neuropathy includes disorders caused when the optic nerve is damaged when exposed to direct or indirect injury.
  • Direct optic nerve injuries are caused by trauma to the head or orbit that crosses normal tissue planes and disrupts the anatomy and function of the optic nerve; e.g., a bullet or forceps that physically injures the optic nerve.
  • Indirect injuries like blunt trauma to the forehead during a motor vehicle accident, transmit force to the optic nerve without transgressing tissue planes. This type of force causes the optic nerve to absorb excess energy at the time of impact.
  • the most common site of injury of the optic nerve is the intracanalicular portion of the nerve. Deceleration injuries from motor vehicle or bicycle accidents account for 17 to 63 percent of cases.
  • Mitochondrial optic neuropathies are described in detail herein. Mitochondria play a central role in maintaining the life cycle of retinal ganglion cells because of their high energy dependence. Mitochondria are made within the central somata of the retinal ganglion cell, transported down axons, and distributed where they are needed. Genetic mutations in mitochondrial DNA, vitamin depletion, alcohol and tobacco abuse, and use of certain drugs can cause derangements in efficient transport of mitochondria, which can cause a primary or secondary optic neuropathy.
  • Nutritional optic neuropathies may be present in a patient with obvious evidence of under-nutrition (weight loss and wasting). Months of depletion are usually necessary to deplete body stores of most nutrients. Undernourished patients often have many vitamin and nutrient deficiencies and have low serum protein levels. However, the optic neuropathy associated with pernicious anemia and vitamin B12 deficiency can even be seen in well-nourished individuals. Gastric bypass surgery' may also cause a vitamin B12 deficiency from poor absorption. Patients who have nutritional optic neuropathy may notice that colors are not as vivid or bright as before and that the color red is washed out. This normally occurs in both eyes at the same time and is not associated with any eye pain. They might initially notice a blur or fog, followed by a drop in vision. While vision loss may be rapid, progression to blindness is unusual. These patients tend to have blind spots in the center of their vision with preserved peripheral vision. In most cases, the pupils continue to respond normally to light.
  • Toxic optic neuropathies The most recognized cause of toxic optic neuropathy is methanol poisoning. This can be a life-threatening event that normally accidentally occurs when the person mistook or substituted, methanol for ethyl alcohol. The patient initially has nausea and vomiting, followed by respiratory distress, headache, and visual loss 18-48 hours after consumption. Without treatment, patients can go blind, and their pupils can dilate and stop reacting to light. Ethylene glycol, a component of automobile antifreeze, is a poison that is toxic to the whole body including the optic nen e. Consumption can be fatal, or recovery' can occur with permanent neurologic and ophthalmologic deficits.
  • Amiodarone is an anti arrhythmic medication commonly used for abnormal heart rhythms (atrial or ventricular tachyarrhythmias). Most patients on this medication get comeal epithelial deposits, but this medication has also been controversially associated with NAION. Patients on amiodarone with new visual symptoms should be evaluated by an ophthalmologist. Tobacco exposure, most commonly through pipe and cigar smoking, can cause optic neuropathy. Middle-aged or elderly men are often affected and present with painless, slowly progressive, color distortion and visual loss in both eyes. The mechanism is unclear, but this has been reported to be more common in individuals who are already suffering from malnutrition.
  • Hereditary or inherited optic neuropathies ty pically manifest as a symmetric bilateral central visual loss. Optic nerve damage in most inherited optic neuropathies is permanent and progressive.
  • Leber's hereditary optic neuropathy (LHON) is the most frequently occurring mitochondrial disease, and this inherited form of acute or subacute vision loss predominantly affects young males. LHON usually presents with rapid vision loss in one eye followed by involvement of the second eye (usually within months). Visual acuity often remains stable and poor (around or below 20/200) with a residual central visual field defect. Patients with the 14484/ND6 mutation are most likely to have visual recovery.
  • Dominant optic atrophy is an autosomal dominant disease caused by a defect in the nuclear gene OPAL
  • OPAL A slowly progressive optic neuropathy, dominant optic atrophy, usually presents in the first decade of life and is bilaterally symmetrical. Examination of these patients shows loss of visual acuity, temporal pallor of the optic discs, centrocecal scotomas with peripheral sparing, and subtle impairments in color vision.
  • Behr's syndrome is a rare autosomal recessive disorder characterized by early -onset optic atrophy, ataxia, and spasticity.
  • Berk-Tabatznik syndrome is a condition that shows symptoms of short stature, congenital optic atrophy and brachytelephalangy. This condition is extremely rare.
  • Small molecules discovered for novel use for optic neuropathy treatment [0071] Disclosed herein are several small molecules useful in various aspects of the current invention for RGC protection. Any embodiments disclosed herein can include the administration to a patient in need, a therapeutically effective amount of one or more of: WAY-100635 maleate or other pharmaceutically acceptable salt, L-Glutamic acid hydrochloride, Ouabain, 4- Imidazoleacetic acid hydrochloride (I4AA), BX-795 hydrochloride and RTA-408 (Omaveloxolone).
  • a method of treatment described herein comprises administering a pharmaceutical composition containing WAY- 100635 maleate for treating optic neurodegenerative diseases, such as glaucoma and inherited optic neuropathies.
  • optic neurodegenerative diseases such as glaucoma and inherited optic neuropathies.
  • the primary goal of this method is to achieve retinal ganglion cell (RGC) neuroprotection, axonal regeneration, and the maintenance of visual functions through various biological mechanisms.
  • RRC retinal ganglion cell
  • the pharmaceutical composition is administered intraperitoneally to a patient diagnosed with an optic neurodegenerative disease.
  • the administration may include daily doses, ranging from about 0. 1 mg/kg to about 45 mg/kg body weight, determined according to therapeutic requirements, patient's medical condition, and tolerance.
  • Alternative routes of administration including intravenous, subcutaneous, or oral, may also be considered based on formulation stability and patient compliance.
  • the methods disclosed herein comprise the administration of WAY-100635 maleate, which acts as a serotonin receptor 5-HT1A antagonist. Without being limited by theory, the inhibition of this receptor may lead to the transient activation of mitochondrial biogenesis in retinal ganglion cells. Consequently, there is a production of healthy mitochondria, which may be essential for sustaining cellular energy demands and overall cell survival.
  • WAY-100635 maleate may lead to the suppression of apoptosis in RGCs. Without being limited by theory. WAY- 100635 maleate may inhibit the progression of programmed cell death, preserving the cellular integrity of RGCs, which are crucial for transmitting visual information from the retina to the brain.
  • a pharmaceutical composition comprising WAY-100635 maleate may modulate neuroprotective signaling pathways that may enhance the survival and function of RGCs. This includes pathways potentially involving proteins such as PGC-lot, SIRT1, and CaMKII/p38 MAPK. which promote cellular survival and neuro-regeneration.
  • a pharmaceutical composition comprising WAY- 100635 maleate may assist in axonal regeneration following optic nerve injury.
  • a method of administering WAY-100635 maleate to a patient having optic nerve injun’ promotes the regeneration of damaged axons, facilitating the re-establishment of neural pathways necessary for vision restoration.
  • the method may include visual inspection, vision test, and MRI scans of patient’s brain to detect if visual signal is being properly processed in transmitted the brain.
  • a therapeutically effective dose of WAY-100635 maleate may range from approximately 0. 1 mg/kg to 45 mg/kg body weight. The determination of a therapeutically effective dose is based on clinical evaluations, pharmacokinetics, and patient-specific factors.
  • Administration Routes In some embodiments, the pharmaceutical composition is administered by intraperitoneal injection. Alternatively, or in addition to, the composition may be administered by other routes such as intravenous, subcutaneous, and oral administration are considered herein.
  • Subjects or patients who may benefit from this treatment include but are not limited to, encompass individuals diagnosed with optic neurodegenerative diseases, such as glaucoma, hereditary optic neuropathies, and other conditions characterized by the progressive degeneration of retinal ganglion cells (RGCs).
  • optic neurodegenerative diseases such as glaucoma, hereditary optic neuropathies, and other conditions characterized by the progressive degeneration of retinal ganglion cells (RGCs).
  • RRCs retinal ganglion cells
  • Specific patient populations include those: diagnosed with glaucoma, including primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), and angle-closure glaucoma, where increased intraocular pressure or other factors lead to RGC death and vision loss, exhibiting inherited optic neuropathies, such as Leber hereditary optic neuropathy (LHON) and dominant optic atrophy (DO A), which result in genetic mutations causing mitochondrial dysfunction and subsequent RGC deterioration, those suffering from optic nen e injuries, including traumatic optic neuropathy, such as where mechanical damage to the optic nerve results in axonal loss and impaired vision, those patients diagnosed with optic neuritis or anterior ischemic optic neuropathy (AION), where inflammation or ischemia leads to acute RGC damage and potential permanent vision loss, those patients experiencing toxic or nutritional optic neuropathies, where exposure to certain toxins or nutritional deficiencies cause mitochondrial damage and RGC apoptosis, or those patients presenting with secondary optic neuropathies due to systemic conditions like diabetes, multiple s
  • WAY- 100635 maleate and/or alternative compounds targeting mitochondrial dysfunction leading to enhanced mitochondrial biogenesis, reduction of apoptosis, and promotion of neuroprotective and regenerative pathways in retinal ganglion cells.
  • the resultant therapeutic outcomes include preservation of vision, improved visual acuity, and potentially the regeneration of damaged optic nerve fibers.
  • the pharmaceutical composition containing WAY-100635 maleate can be administered to individuals diagnosed with optic neurodegenerative diseases utilizing various administration routes.
  • Intraperitoneal injections involve delivering the pharmaceutical composition directly into the peritoneal cavity’, allowing for efficient absorption and systemic distribution. This route is commonly used in preclinical studies and provides consistent therapeutic dosing.
  • IV administration entails injecting the pharmaceutical composition directly into the bloodstream. This route ensures rapid onset of action and precise control over the administration dosage, making it suitable for acute therapeutic intervention.
  • Subcutaneous injections involve delivering the pharmaceutical composition into the subcutaneous tissue, which houses a network of small blood vessels for gradual absorption. This route is beneficial for sustained release formulations and enhanced patient compliance due to ease of administration.
  • Oral Administration involves ingestion of the pharmaceutical composition in the form of tablets, capsules, or liquid solutions. This route is non-invasive and highly convenient for patients, although it requires consideration of formulation stability and bioavailability. Various excipients and delivery systems may be employed to enhance solubility and absorption of WAY-100635 maleate.
  • Intravitreal Administration administer the pharmaceutical composition directly into the vitreous humor of the eye, potentially bypassing systemic circulation and delivering higher local concentrations to the retinal ganglion cells. This route is particularly effective for localized treatment of ocular conditions.
  • Topical ocular administration involves applying the pharmaceutical composition, such as eye drops or ophthalmic gels, directly to the ocular surface. This route may be used to target anterior segment conditions and offers non-invasive delivery, with considerations for periocular penetration.
  • pharmaceutical composition such as eye drops or ophthalmic gels
  • Each route of administration may be optimized based on therapeutic objectives, pharmacokinetics, and patient-specific factors. Additionally, formulation strategies, like encapsulation in nanoparticles or use of sustained release systems, may be employed to improve stability, efficacy, and patient compliance across different administration routes.
  • the administration of the therapeutic composition comprising WAY-100635 maleate may be executed in a manner optimized to achieve retinal ganglion cell (RGC) neuroprotection, axonal regeneration, and the maintenance of visual function.
  • RRC retinal ganglion cell
  • the following provides exemplary therapeutic effective amounts; however, dosage ranges, frequency, and potential adjustments pursuant to individual patient responses and therapeutic requirements are considered by those having ordinary' skill in the art when w riting prescriptions and treatment plans.
  • the administration regimen for the pharmaceutical composition may comprise dosage ranges from about 0.1 mg/kg to about 45 mg/kg body weight. Specific dosage ranges within this spectrum include, but are not limited to about 0. 1 mg/kg to about 40 mg/kg, about 0.1 mg/kg to about 35 mg/kg, about 0.1 mg/kg to about 30 mg/kg, about 0.1 mg/kg to about 25 mg/kg, about 0. 1 mg/kg to about 20 mg/kg, about 0.1 mg/kg to about 15 mg/kg, about 0. 1 mg/kg to about 10 mg/kg, about 0. 1 mg/kg to about 5 mg/kg. about 0. 1 mg/kg to about 1 mg/kg, or about 0. 1 mg/kg to about 0.5 mg/kg.
  • Additional dosage ranges include about 0.5 mg/kg to about 40 mg/kg, about 0.5 mg/kg to about 35 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.5 mg/kg to about 25 mg/kg, about 0.5 mg/kg to about 20 mg/kg, about 0.5 mg/kg to about 15 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg. or about 0.5 mg/kg to about 1 mg/kg.
  • Additional dosage ranges include about 1 mg/kg to about 45 mg/kg, about 1 mg/kg to about 40 mg/kg, about 1 mg/kg to about 35 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 25 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, about 1 mg/kg to about 10 mg/kg, or about 1 mg/kg to about 5 mg/kg.
  • Additional dose ranges include about 5 mg/kg to about 45 mg/kg, about 5 mg/kg to about 40 mg/kg, about 5 mg/kg to about 35 mg/kg, about 5 mg/kg to about 30 mg/kg, about 5 mg/kg to about 25 mg/kg, about 5 mg/kg to about 20 mg/kg, about 5 mg/kg to about 15 mg/kg, or about 5 mg/kg to about 10 mg/kg.
  • Further dosage ranges include about 10 mg/kg to about 45 mg/kg, about 10 mg/kg to about 40 mg/kg, about 10 mg/kg to about 35 mg/kg, about 10 mg/kg to about 30 mg/kg, about 10 mg/kg to about 25 mg/kg, about 10 mg/kg to about 20 mg/kg, or about 10 mg/kg to about 15 mg/kg.
  • Further dosage ranges include about 15 mg/kg to about 45 mg/kg, about 15 mg/kg to about 40 mg/kg, about 15 mg/kg to about 35 mg/kg, about 15 mg/kg to about 30 mg/kg, about 15 mg/kg to about 25 mg/kg, about 15 mg/kg to about 20 mg/kg.
  • the dosage range administer in the methods disclosed herein may be modified to accommodate various factors, including but not limited to:
  • Patient-Specific Factors Variables such as age. weight, metabolic rate, and existing comorbidities may influence the dosage needed to reach therapeutic efficacy.
  • Adjustments to the dosage may be made based on the patient's response to the treatment and tolerance to the therapeutic agent. Continuous monitoring and evaluations by healthcare professionals may be employed to tailor the dosage to individual needs.
  • WAY-100635 maleate may follow a structured schedule to maximize therapeutic outcomes while minimizing potential adverse effects.
  • a method may comprise administered a pharmaceutical composition comprising WAY-100635 maleate as an initial dose, a maintenance dose, and/or long-term dosing.
  • Initial Dosing Phase Patients may begin treatment with a dose in the lower end of the effective range (0.1 mg/kg to 5 mg/kg per day). After initial administration, patients may be assessed for therapeutic response and tolerance. Adjustments to the dosing amount may be made based on observed efficacy and any adverse reactions.
  • Maintenance Dosing Depending on a patient response, the dosage may be gradually increased to maintenance levels within the specified range (0.5 mg/kg to 45 mg/kg per day). Maintenance dosing aims to stabilize mitochondrial biogenesis, reduce apoptosis, and promote neuroprotective pathways in RGCs.
  • Long-Term Treatment For sustained therapeutic outcomes, long-term administration of WAY- 100635 maleate may be administered, with periodic evaluations to ensure continued efficacy and safety. Long-term dosages may be maintained within the effective range, subject to adjustments based on clinical evaluations and patient-specific considerations.
  • Alternative Dosage Schedules Alternative routes, such as intravenous, subcutaneous, or oral administration, may adopt different schedules based on the pharmacokinetics associated with each route. Periodic breaks from administration may be considered in certain cases to assess the natural progression of the disease and the long-term efficacy of the treatment.
  • Pediatric and Geriatric Patients may require carefully tailored dosages due to differences in metabolism, drug absorption, and overall health status. Lower initial doses and gradual titration may be recommended in these populations to minimize potential adverse effects.
  • Concurrent Medications Patients receiving concurrent medications may be monitored for potential drug interactions that may influence the pharmacokinetics or dynamics of WAY- 100635 maleate. Dosage adjustments may be made based on comprehensive evaluations of all medications taken by the patient.
  • Dosages may be subject to change in response to the progression of optic neurodegenerative diseases, with more aggressive dosing schedules considered for rapidly advancing cases.
  • WAY-100635 maleate in combination with another compound, such as L-Glutamic acid hydrochloride, may provide a synergistic therapeutic effect for the treatment of optic neurodegenerative diseases. This combination approach may enhance the overall efficacy of the treatment by targeting multiple mechanistic pathways involved in neuroprotection and RGC survival.
  • the pharmaceutical composition comprising WAY- 100635 maleate may be administered with other compositions or may comprise additional compositions such as L- Glutamic acid hydrochloride, 4-Imidazoleacetic acid hydrochloride, BX-795 hydrochloride, RTA-408, or Ouabain.
  • the pharmaceutical composition comprises WAY- 100635 and L-Glutamic acid hydrochloride may be administered according to a regimen tailored to a patient's condition and therapeutic requirements.
  • the combination may be delivered via intraperitoneal injections, with dosages adjusted based on pharmacokinetic profiles and patient tolerance.
  • a pharmaceutical composition may comprise WAY- 100635 maleate in an amount of about 0.5 mg/kg to about 5 mg/kg body weight and L-Glutamic acid hydrochloride in an amount of about 10 mg/kg to about 50 mg/kg.
  • the pharmaceutical composition comprising WAY-100635 maleate in an amount of about 5 mg/kg to about 30 mg/kg body and L-Glutamic acid hydrochloride in an amount of about 50 mg/kg to about 100 mg/kg may be administered a patient in need thereof.
  • H7-hESCs Human embryonic stem cell line from WiCell, Madison. WI, was used as a reporter line.
  • the H7-hESCs were modified by CRISPR to introduce a multicistronic BRN3B- P2A-tdTomato-P2A-Thyl.2 construct into the endogenous BRN3B locus, specifically expressed in retinal ganglion cells (RGCs), serving as an isogenic control.
  • RRCs retinal ganglion cells
  • iPSCs patient- derived induced pluripotent stem cells
  • iPSC-E50K E50K mutation
  • iPSC-E50K corr iPSCs with the OPTN E ’ 0K mutation corrected to wild type
  • Routine clump passaging using Gentle Cell Dissociation Reagent was performed when the cells reached 70-80% confluency.
  • the media was aspirated, GD was added to the cells and incubated at 37°C in a 5% CO2 incubator for 5 minutes. After the aspiration of GD.
  • mT media was used to break up the colonies into small clumps through repeated pipetting and the clumps were then seeded onto new MG plates.
  • hRGC differentiation and immunopurification The stem cell colonies were split into individual cells using accutase for 10 minutes. The cells were treated with double the volume of mT containing 5 pM blebbistatin (blebb) to halt the accutase activity. Following this, the cells were centrifuged at 150 xg for 6 minutes and then resuspended in mT supplemented with blebb (5 pM). Cells were counted using a hemacytometer and subsequently, 100,000 cells were seeded in each well of a 24-well plate coated with matrigel (MG). The next day, the media was replaced with mT without blebb.
  • blebb blebbistatin
  • iNS media was added and differentiation with small molecules was carried out according to previous protocols. See Das et al. 2020.
  • the purified hRGCs were suspended in iNS media, counted using a hemocytometer and seeded onto MG-coated plates, coverslips, or MatTek dishes for subsequent experiments.
  • hRGCs Purified hRGCs were seeded at 30.000 cells per well of a 96-well MG-coated plate and maintained for 3 days. For measuring mitochondrial mass, hRGCs w ere labelled with mitochondria specific MTDR dye. To measure mitochondrial biogenesis, hRGCs were labeled with MTDR first and treated with WAY at different time points. After treatments, hRGCs were dissociated to singlecell suspension using accutase and analyzed using Attune NxT flow cytometer (Thermo Fisher). [00124] Western blot
  • the purified hRGCs were seeded at a density of 500,000 cells per well in 24-well plates coated with MG and were cultured for 3 days. Following this, the cells were subjected to WAY treatment at specified time points. As a control, distilled water (dFhO) was utilized since the WAY was dissolved in dFFO. Subsequently, the cells were lysed and collected in 100 pl of M-PER extraction buffer containing 5mM EDTA and protease inhibitors. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad) with bovine serum albumin (BSA) as a standard and quantified using a microplate reader.
  • DC Protein Assay Kit II Bio-Rad
  • BSA bovine serum albumin
  • the membranes were blocked in 5% skim milk in TBST (TBS buffer with 0.5% Tween20) for 2 h at room temperature and incubated overnight at 4°C in 1: 1000 dilution of primary antibodies for Tom20 (Santa Cruz), 5HT1A (Abeam), or GAPDH (CST). Membranes were then washed three times for 5 minutes each in TBST, followed by 2h of incubation in 5% milk in TBST containing anti-rabbit HRP-linked secondary antibody (CST) at 1 : 10,000 dilution. The membranes were washed three times with TBST and placed in Clarity Max Western ECL (Bio-Rad) substrate for 5 min.
  • the membranes were imaged in a BioRad ChemiDoc Gel Imager and the raw- integrated density 7 for each band was measured and normalized with respect to the corresponding GAPDH or actin loading control using Image J. In addition, the treatment conditions were further normalized to their respective controls within each experiment. The quantification of protein bands was performed based on the expected size of the proteins, as indicated in product datasheets and published literature.
  • hRGCs Purified hRGCs were cultured on MG coated coverslips (thickness 1.5). The cells were seeded at a density of 30,000-40,000 cells per coverslip and allowed to grow for 3 days. Later, the hRGCs w ere treated with the specified molecule (WAY) at specific time points. The culture media was removed after the treatment and the cells were w ashed with IX PBS. The cells were then fixed with 4% paraformaldehyde for 15 minutes at 37 °C. After fixation, the cells were permeabilized using 0.5% Triton-XlOO in PBS for 5 minutes and washed three times for 5 minutes with washing buffer (1% donkey serum and 0.05% Triton-XlOO in PBS).
  • the cells were blocked with a blocking buffer (5% donkey serum and 0.2% Triton-X 100 in PBS) for 1 hour.
  • Antibodies against various proteins including Tom20 (Santa Cruz), PGCla (Abeam), 5HT1A (Abeam), cAMP (Bio- Techne) were added to the coverslips at a dilution of 1:200 in blocking buffer.
  • the coverslips were then incubated overnight at 4°C and on the next day. washed three times for 5 minutes with the washing buffer and incubated with fluorophore-conjugated secondary antibodies specific to mouse or rabbit (dilution of 1 :500) in the dark at room temperature for 2 hours.
  • coverslips were washed three times (5 minutes each) with the washing buffer, the second wash included the addition of 1.43 pM DAPI (a fluorescent nuclear stain). Finally, the coverslips were mounted onto slides using DAKO mounting medium. Confocal immunofluorescence microscopy using Zeiss LSM700 with 63x/1.4 oil objective visualized the above proteins and nucleus. Analysis was carried out using ImageJ.
  • hRGCs were seeded on MG-coated glass bottom (1.5 thickness) MatTek dishes at 40,000 cells per dish and maintained for 3 days. Cells were treated with WAY for the designated time durations. Cells w ere washed with iNS media and then incubated with 250 pl of JC1 media (1 : 100 in iNS) for 30 min in the incubator. The JC1 media was then removed, cells were washed again and 2ml of new iNS was added to the dish. The dish was then transferred to the live cell chamber (5% CO2, 37°C, Tokai Hit) and confocal z-stacks were acquired using Zeiss LSM700 with 63x/1.4 oil objective.
  • hRGCs were seeded in MG coated 96 well Seahorse plate at 250,000 cells per well and maintained for 2 days. 24h before measurements, media was exchanged with 100 pl iNS with 5 pM WAY or equivalent vehicle control H2O. The day before the experiment the sensor cartridge was hydrated by submerging it in 200 pl of sterile water and kept in a non-CCty 37 °C incubator overnight. The next day, the sterile water was replaced with pre-warmed XF calibrant buffer (Agilent) and the sensors were submerged again and incubated in the same conditions for 45-60 minutes.
  • pre-warmed XF calibrant buffer Algilent
  • Seahorse media was made by adding stock solutions to XF DMEM to have final concentrations of 21.25mM glucose, 0.36mM sodium pyruvate and 1.25 mM L-glutamine (Agilent), with pH adjusted to 7.38-7.42.
  • solutions of 20 pM Oligomycin (Oligo), 20 pM FCCP, 2.5 pg/ml Rotenone plus 5 pM Antimycin A (Rot/AA) and/or 175 mM 2-deoxy -d-glucose (2-DG) were prepared in Seahorse media.
  • the iNS media was carefully replaced with Seahorse media, by removing 60 pl of iNS from each well and adding 140 pl of Seahorse media. Then, 140 pl of the mixed media was pipetted out and an additional 140 pl of Seahorse media was added to each well to reach a final volume of 180 pl. Any empty wells were filled with 180 pl of Seahorse media.
  • the plate was placed in Incucyte S3 (Sartorius) and one image of each well was taken for cell area normalization using brightfield and red fluorescence (tdTomato) channels.
  • the hRGC Seahorse plate was transferred to a non-CCh, 37°C incubator for at least 45 minutes.
  • the appropriate reagents were added to their respective ports in the cartridge, resulting in final concentrations in the wells as follows: for the ATP rate assay.
  • the hRGC Seahorse plate was placed in the machine and the selected assay (ATP rate assay, glycolytic rate assay, or Mito stress test) was run. Cell area from Incucyte images was measured using Image J and extrapolated for the total cell area in each well for normalization. Finally, the assay results were exported to the appropriate Excel macro using Seahorse Wave Desktop software (Agilent) for analysis.
  • Assay ATP rate assay, glycolytic rate assay, or Mito stress test
  • hRGCs Human stem cell differentiated RGCs
  • MOI multiplicity of infection
  • Quantitative PCR Approximately 50,000 hRGCs were seeded into 96 well plate and cultured for 3 days. LV was added at the indicated MOIs for Cas9, along with polybrene (2 pg/ml). The dish was centrifuged at 800G for 60min and then placed into the incubator (37°C, 5%CO2). Cell pellets were collected on day 4 after transduction and qPCR was performed for Cas9.
  • hRGCs were plated at a density of 25,000 cells per well in a 96-well clear bottom blackwalled plate and cultured for 3 days. Following this, the cells were treated with WAY at specified time points. The caspase activity was measured using the ApoTox-Glo Triplex assay kit (Promega). 100 pl of Caspase-Gio® 3/7 reagent was added to each well and incubated for 30 minutes at room temperature. Luminescence was then measured to quantify caspase activity (referred to as Caspase measurement). The measured values were normalized to the control condition.
  • ONC ONC
  • WAY body weight
  • MB microbead induced high IOP glaucoma
  • mice were placed under anesthesia using 2% isoflurane and 0.5% proparacaine hydrochloride ophthalmic drops were administered to the eyes.
  • Ophthalmic ointment was applied to both eyes to prevent dryness of the corneas throughout the procedure.
  • a small incision was made in the upper conjunctiva, allowing for gentle outward retraction of the eyeball using fine forceps.
  • Another pair of forceps w as used to expose the optic nerve by opening the connective tissues covering it.
  • the microscope w as adjusted to focus on the optic nerve and a specific pair of forceps was employed to crush the optic nerve for a duration of 5 seconds, approximately 1 mm behind the eyeball. Following the ONC, the eyeball was carefully placed back into the eye socket and the mouse's head was released.
  • Erythromycin ophthalmic ointment USP 0.5% (Baush + Lomb, Laval, Canada) was given to prevent infection and lubricate the eye during recovery. After the procedure, the animals were closely monitored until they regained full consciousness.
  • mice pupils are dilated with a drop of phenylephrine hydrochloride ophthalmic solution (10%) (Akom, Lake Forest, Illinois) followed by local anesthesia with a drop of proparacaine (0.5%) (Bausch and Lomb, Bridgewater, NJ) under whole body anesthesia with isoflurane inhalation (1.5 - 2%) (Akom, Lake Forest, Illinois).
  • phenylephrine hydrochloride ophthalmic solution (10%)
  • proparacaine 0.5%)
  • isoflurane inhalation 1.5 - 2%)
  • Injection is done at a rate of 5 pL/min controlled by the experimenter.
  • erythromycin ophthalmic ointment 0.5%) (Bausch and Lomb, Bridgewater, NJ) is applied on eye and the animal is allowed to recover for 24-48 hours prior to resumption of IOP measurements.
  • Retinal tissues are collected and immersed in ice-cold PBS before being fixed with a 4% paraformaldehyde solution. Then tissues were incubated overnight in guinea pig RBMS antibody at a dilution of 1:200. To detect the primary antibody, an Alexa Fluor 647-labeled goat anti-guinea pig secondary antibody was utilized. Nuclei were stained with DAPI during the final wash step. Eye cups were cut in a flow er shape and flat mounted over the slide using 0.45 pm black MCE membrane. Coverslips were then placed over the slides, securing them with Aqua- Poly/Mount mounting medium.
  • the visualization of the samples was performed using confocal immunofluorescence microscopy, using a Zeiss LSM700 microscope with a 40X/1.3NA oil objective. A total of eight 40X z-stacked images were captured for each retinal flat mount, with images taken from four quadrants located in both the central and peripheral regions of the retina. Manual quantification of RBPMS -positive retinal ganglion cells (RGCs) was carried out by analyzing the maximum projection of each image using imageJ software.
  • the eye cup and optic nerve were fixed in 4% paraformaldehyde in 0.1M phosphate buffer (PB. NaHPCL and Na2HPO4 dissolved in MilliQ H2O. pH7.4) for 6h at RT. After fixation, the tissues were transferred to a 5% sucrose solution in phosphate buffer for 30 minutes. Subsequently, they underwent a series of sucrose w ashes, with increasing concentrations of sucrose (10%, 15% and 20%) in 0.1M PBS. To prepare the tissues for cryosection, they were incubated overnight at 4°C in a solution consisting of 20% sucrose and optimum cutting temperature compound (OCT compound) in a 2: 1 ratio.
  • OCT compound optimum cutting temperature compound
  • tissue sections were aligned in Tissue-Tek cryo molds and frozen in isopentane cooled by liquid N2. Frozen tissue sections were then obtained using a Leica CM1850 cryostat with a thickness of 12pm for the retina and 14pm for the optic nerve. The sections were collected on microscopy slides. After drying, the slides were stored at - 80°C until further use. When needed, the slides were taken out from the -80°C storage and warmed using a 37°C incubator. The sections were encircled using an ImmEdge pen (Vector Laboratories).
  • Sections were rehydrated with PBS (in mM: 137 NaCl, 2.7 KC1, 8 Na2HPO4, 2 KH2PO4) containing 0.1% Triton X-100 (PBST) and blocked in a 5% normal goat serum diluted in PBST solution for 30min at RT. After the blocking, the sections were washed three times with PBST for 5 minutes each and incubated with the primary antibody (diluted 1:200) at 4°C for 14- 16 hours. Subsequently, the sections were washed again with PBST (3x5 minutes). Antibodies against 5HT1A was used.
  • PBS in mM: 137 NaCl, 2.7 KC1, 8 Na2HPO4, 2 KH2PO4
  • the sections were incubated with fluorophore- conjugated anti-mouse or anti-rabbit secondary antibodies (1:500 dilution) and DAPI dye (1: 1000 dilution) in PBST for 1 hour at RT. After this incubation, the sections underwent an additional three washes with PBST for 5 minutes each. To mount the sections, a drop of mounting medium or DAKO was added to a coverslip and the coverslip was carefully placed over the sections. To secure the coverslip, nail polish was used to seal the edges. The sections were allowed to dry at RT for 30 minutes or overnight before they were ready for imaging.
  • Sections were first washed with Tris-buffered saline (TBS. pH 7.4) for 5 minutes. Then, they were pre-incubated in 100% methanol for 10 minutes. Subsequently, the sections were blocked using a solution of 10% donkey serum in TBS. The primary antibody against growth - associated protein 43 (GAP43) was applied to the sections (1: 1,000; from sheep). See Benowitz et al., ‘‘Anatomical distribution of the growth-associated protein GAP-43/B-50 in the adult rat brain,” (1988) J Neurosci 8(1): pp. 339-352; Xie et al..
  • the sections were rinsed first in TBS with Tween 20 (TBS2T, containing 300 mM NaCl and 0.1% Tween 20, pH 7.4) for 1 hour at 4°C, followed by a rinse in solution A for 1 hour at room temperature. Lastly, the sections were washed again in TBS2T for 1 hour at room temperature. After washing, sections were incubated with an AlexaFluor-488-conjugated donkey secondary antibody to sheep IgG (1 :500) for 2hr at room temperature, washed in TBS2T (2X5 min, room temperature) and TBS (5min, room temperature). To mount the sections, one drop of DAKO was added to each section and a cover slip was placed over the section.
  • TBS2T Tween 20
  • a cover slip was placed over the section.
  • the coverslip was sealed with nail polish and the sections were allowed to dry’ overnight at room temperature before imaging.
  • the number of GAP43-positive axons crossing at 0.5 mm from the crush site is counted following published methods by Larry’ Benowitz lab 22, 23 .
  • the width of each section w as measured and four sections per nerve were analyzed.
  • the cross-sectional width of the optic nerve was measured at the point at which the counts were taken and was used to calculate the number of axons per millimeter of nerve width. The number of axons per millimeter can then be averaged over the four 14pm sections.
  • the total number of axons present at a distance ‘ ’ (Laa) having a radius ‘r’ can be measured by summing over all sections having thickness t (14
  • im) (Zaa 7rr x [ - — - ⁇ /t).
  • mice were pretreated with WAY (5mg/kg) for 5 days daily. After that, unilateral ONC injury' was performed, they were treated with WAY daily for the next 13 days post-ONC and visual cliff tests were done on Day 13.
  • Mice were tested in a visual cliff apparatus (square arena approximately 20cm tall with 62cm sides and a transparent bottom/floor). Half of this arena was placed on a table approximately’ 2 feet tall and the other half was suspended in air to simulate a cliff. A vinyl sheet with a black and w hite checkerboard pattern w as also placed under the apparatus and allowed to drape over the table to enhance the illusion of a steep drop-off.
  • Optokinetic response test [00157] After ONC injury (unilateral left crush) on 5-day WAY (5mg/kg) pretreated mice, they were treated with WAY daily for additional 21 days. Optokinetic response (OKR) measurements were done on Day 6 and 21 post ONC. In this test, the mouse is surrounded by 4 computer screens with moving (12deg/sec clockwise or counterclockwise) sinusoidal gratings of either increasing frequency or decreasing contrast separately. A mouse with healthy vision is expected to respond (move head) towards the moving stimulus with higher spatial frequency (high visual acuity (VA)) or decreasing contrast (high contrast sensitivity (CS)) respectively. VA was measured by scoring head movements at increasing spatial frequency but at fixed speed and contrast.
  • VA visual acuity
  • CS contrast sensitivity
  • CS was measured by scoring head movements at decreasing contrast but at a fixed speed and spatial frequency.
  • CS is presented here as Michelson contrast (max-min/max+min); max, min represents the maximum or minimum contrast that the mouse responded. See Pruskey et al. 2004; Prusky et al., “Enhancement of vision by monocular deprivation in adult mice,” (2006) J Neurosci 26(45): pp. 11554-11561. Quantification of VA and CS in response to both clockwise and counterclockwise along with the combined rotation of the sinusoidal gratings was recorded.
  • kidney and liver tissues from the naive, ONC, ONC+WAY mice were collected and fixed in 10% formaldehyde and processed for paraffin sectioning. Paraffin-embedded sections were stained with hematoxylin and eosin to study the histological changes under a light microscope.
  • IP intraperitoneal
  • mice mice were sacrificed and blood and eye tissue were collected at 4hr and 24hr after injection.
  • 75uL of blood was collected into heparinized microcapillary tubes and spun at 5000rpm for 5 minutes. After that, plasma was transferred to a tube and frozen at -80°C.
  • the retina was collected from dissected eye tissue, quick frozen in liquid nitrogen and stored at -80°C. Mass spectrometry was performed from collected serum and retinal tissue.
  • each treatment and corresponding time points are performed on cells grown on individual wells, hence each group is independent from others.
  • Seven to ten independent biological repeats on hRGCs can be performed for western blots, mitochondrial mass measurements by flow cytometry, cellular apoptosis measurements, qPCR, and seahorse analysis.
  • Thirty to forty hRGC neurons for each condition can be analyzed for high-resolution confocal images and transmission electron microscopy (TEM) acquired over 4-6 biological repeats for each condition.
  • TEM transmission electron microscopy
  • Statistical tests can be done by student’s t-test (for two samples) or ANOVA (three or more conditions) with Tukey's post-hoc tests to adjust for multiple testing among all groups or Dunnett’s tests for comparisons of multiple groups against a control.
  • Nonparametric tests can be used if the data cannot be transformed to meet the t-test/ ANOVA assumptions.
  • High content screening identifies a neuroprotective compound.
  • RGC differentiation is judged by tdTomato expression and RGC purification is done by CD90 (Thy 1.2) conjugated microbeads and magnetic activated cell sorting (MACS) with 95-99% purity.
  • CD90 Thi 1.2 conjugated microbeads
  • MCS magnetic activated cell sorting
  • hRGCs human stem cell differentiated RGCs
  • LOP AC library Sigma
  • MTDR MitoTracker Deep Red
  • MTDR entry into mitochondria depends on the mitochondrial membrane potential which may get affected by the drug treatments leading to erroneous mitochondrial mass measurements.
  • the inventors labeled mitochondria in hRGCs with the MTDR dye first and then treated cells with the drug library' in presence of the dye followed by flow cytometrybased measurements of the mitochondrial mass. Inhibition of mitophagy could also lead to increased mitochondrial mass, however mitophagy inhibition is toxic to hRGCs and causes mitochondrial membrane potential damage which can inhibit MTDR dye binding to mitochondria. See Das et al., “Programmed switch in the mitochondrial degradation pathways during human retinal ganglion cell differentiation from stem cells is critical for RGC survival,” (2020) Redox Biol 34: 101465; Georgakopoulos et al.. “The pharmacological regulation of cellular mitophagy.” (2017) Nat Chem Biol 13: pp. 136-146. Thus, compounds that can promote more MTDR binding to mitochondria are due to the biogenesis of healthy' mitochondria or due to improving membrane potential (health) of existing mitochondrial population.
  • FIG. 2A, B shows that the small molecule screen identifies compound for increasing mitochondrial biogenesis.
  • Drugs showing >40% change outside of blue box.
  • the competitive reaction to a GPCR target is a standard method for showing target specificity which we have shown here for WAY and DP AT in hRGCs and was reported for other systems as well 45 .
  • WAY binding to 5-HT1 A receptor is well characterized in-vivo 32 as well as in-vitro 46 .
  • D4 dopamine receptor D4
  • DP AT DP AT as agonist to the 5-HT7 49, 50 . Based on cAMP modulation by WAY and DP AT, this is unlikely that the above targets are engaged in hRGCs.
  • FIG. 3A - I depict that WAY transiently activates cyclic AMP (cAMP) level and promotes hRGC survival.
  • FIG. 3A Illustration for mechanism of action by antagonist WAY or agonist DPAT through 5-HT1A GPCR.
  • FIG. 3B Representative max-projections and central z- plane of confocal IF images of H7-hRGC WT against cAMP (anti-cAMP antibody, R&D systems) and DAPI after indicated timepoints of 5 pM WAY treatments. Insets show perinuclear cAMP that increases with WAY treatment.
  • FIG. 3A Illustration for mechanism of action by antagonist WAY or agonist DPAT through 5-HT1A GPCR.
  • FIG. 3B Representative max-projections and central z- plane of confocal IF images of H7-hRGC WT against cAMP (anti-cAMP antibody, R&D systems) and DAPI after indicated timepoints of 5 pM WAY treatments. Insets show perin
  • FIG. 3E Quantification of cAMP intensity per area from sum projections, normalized to control shows WAY pretreatment increases cAMP level but addition of DPAT suppresses it.
  • DMSO treatment used for control as DPAT solubilizes in DMSO.
  • ONC not as a glaucoma model per se but a robust RGC degeneration model that can be used to test the above neuroprotection strategy.
  • ONC axonal degeneration precedes cell body loss
  • WAY mediated RGC protection in ONC. which is an acute axonal injury model, is likely to be efficacious in glaucoma as well as for other mitochondrial optic neuropathies such as LHON and DOA. See Howell et al., “Intrinsic axonal degeneration pathways are critical for glaucomatous damage,” (2013) Exp Neurol. 246: pp. 54-61.
  • WAY is neuroprotective in vivo
  • FIG. 4B - D shows that WAY promotes neuroprotection in mice against ONC injury.
  • FIG. 4A Schematic illustration of the daily WAY (5mg/kg) IP injection in C57BL/6J mouse, unilateral ONC done on DO.
  • FIG. 4B Representative tiled confocal image (10X/0.3NA) of mouse retinal flat mount with immunohistochemistry (IHC) against RGC specific RBPMS protein. Dotted circle and white boxes on the circles represent central and peripheral retinal regions for image acquisitions and quantifications.
  • FIG. 4C Representative high-resolution confocal images (40X/1.3NA) of RGCs from indicated retinas from peripheral and central white boxes (FIG. 4B).
  • mice were treated with WAY at a low dose of 5mg/kg body weight 5 days pre and post ONC by IP injection and retinas were collected on day 6 post ONC for IHC.
  • RGC numbers were counted both at the peripheral and central retina as marked by white boxes on retinal flat mount IHC image (FIG. 4B).
  • the inventors noted drastic RGC loss in the ONC retina.
  • WAY provides robust RGC protection against ONC injury at early (6 dpc) and later time points (14 dpc) establishing its therapeutic potential (FIG. 4C, D).
  • WAY provides neuroprotection to both the ONC, and contralateral eyes is of high clinical relevance.
  • [00191] WAY promotes ganglion cell axon regeneration in vivo.
  • WAY also promotes axon regeneration after ONC.
  • RGC axon regeneration was measured in the optic nerve by IHC against GAP43 protein on the transverse cryosections followed by confocal imaging as developed by others. See Cheng et al., “Transcription factor network analysis identifies REST/NRSF as an intrinsic regulator of CNS regeneration in mice.” (2022) Nature Communications 13(1): p. 4418.
  • GAP43 is a critical player for axonal growth and plasticity discovered by Dr. Benowitz and his colleagues who also developed a robust antibody against GAP43 which has been widely used and shared with the inventors.
  • FIG. 4E, F show that WAY promotes RGC axon regeneration.
  • C57BL/6J mice were treated with 5mg/kg WAY daily 5 days prior to ONC until 25 days post crush.
  • FIG. 5A-F shows that WAY maintains visual acuity (VA) in mice after ONC injury.
  • FIG. 5A Schematic illustration of the WAY treatment, ONC and subsequent VA measurements.
  • FIG. 5B VA measured by scoring head movements at increasing spatial frequency but at fixed speed and contrast.
  • FIG. 5C, E Quantification of VA in response to both clockwise and counterclockwise rotation of the sinusoidal gratings. One-way ANOVA with Tukey’s correction.
  • FIG. 5D, F VA in response to the individual clockwise or counterclockwise movement of the gratings. Error bars are SEM. Two-way ANOVA with Tukey’s correction, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • n 4 mice/group
  • FIG. 6A-E shows that WAY maintains contrast sensitivity (CS) in mice after ONC injury.
  • CS measurements done on the same mice by OKR as illustrated in FIG. 5A.
  • FIG. 6A CS measured by scoring head movements at decreasing contrast but at fixed speed and spatial frequency.
  • FIG. 6B. D CS presented here as Michelson contrast (max-min/max+min); max, min represent maximum or minimum contrast that the mouse responded to (Prusky et al. 2006).
  • VA visual acuity
  • CS contrast sensitivity
  • WAY protects vision after acute ONC injury and performed a visual cliff test 13 days post ONC while mice were under daily WAY treatment (5mg/kg) from day -5 to +13 where day 0 is the day of ONC.
  • This experiment was done in a masked manner by the IUSM Behavioral Phenotyping Core as mice were provided with masked identity.
  • a circular metal platform approximately 2 cm tall and 10 cm wide was placed in the center of the apparatus so that half of it was on the ‘cliff side and the other half on the ‘safe' side as shown in FIG. 7 A.
  • FIG. 7A, B shows that WAY maintains visual depth perception after ONC.
  • FIG. 7A Animals were tested in a visual cliff apparatus with approximately 2 feet tall cliff (deep) side with similar surface pattern under the glass for optical illusion.
  • FIG. 7B Latency time to step down from the platform. Each data point is average from 5 measurements.
  • One-way ANOVA with Dunnetf s, **, p ⁇ 0.005. n 3 mice/group.
  • WAY provides glaucoma neuroprotection
  • FIG. 8 A-C shows WAY prevents RGC and visual acuity loss in glaucoma mouse.
  • IOP Intraocular pressure
  • MB Polyscience, Inc.
  • PBS PBS
  • FIG. 8B Confocal IHC image of RBPMS positive RGCs
  • FIG. 8C RGC number counted 57 days after MB injection as in FIG. 4D.
  • FIG. 8D Visual acuity measured by OptoMotry (Cerebral Mechanics) where mouse exposed to 4 adjoining LCD monitors with moving sinusoidal gratings (12deg/sec clockwise or counterclockwise) with randomly varying frequency but 100% contrast. Spatial frequency in cycles per degree (c/deg) measured for different condition with mouse identity masked.
  • FIG. 8D 3 mice/group. Two-way ANOVA with Tukey’s, ****, p ⁇ 0.0001, ***, p ⁇ 0.001.
  • 5-HT1 A expresses in mouse and human RGCs
  • FIG. 9A-D shows robust 5-HT1 A expression in hRGCs and in mouse RGCs.
  • FIG. 9A Confocal images show 5-HT1A membrane localization in H7-hRGCs and
  • FIG. 9C shows on ganglion cell layer of mouse retinal cryosection.
  • FIG. 9B Western blots detect 5-HT1 A protein expression in hRGCs
  • FIG. 9D detects 5-HT1A expression in RGCs isolated from mouse retina.
  • the inventors next undertook a series of studies to demonstrate the mechanisms of 5- HT1 A inhibition mediated mitochondrial biogenesis activation in hRGCs.
  • EXAMPLE 7 restores mitochondrial health and provides protection to glaucoma causing OPTN E50K hRGCs.
  • PGCla is considered to be the master regulator of mitochondrial biogenesis, which, upon translocation to the nucleus, drives the expression of nuclear-encoded mitochondrial electron transport chain (ETC) proteins and mitochondrial transcription factor Tfam 51 .
  • ETC mitochondrial electron transport chain
  • Tfam 51 mitochondrial transcription factor
  • JC1 dye fluoresces green (monomers) when bound to damaged (depolarized) and red (J aggregates) when bound to healthy (polarized) mitochondria 55 .
  • WAY treatment significantly increased JC1 labelled red-to-green mitochondria ratio (FIG.
  • FIG. 11A - F shows that WAY transiently activates mitochondrial biogenesis with neuroprotection effect to OPTN E50K .
  • H7-hRGCs with OPTN E50K mutation are treated with 5 pM WAY for indicated timepoints.
  • FIG. 11 A Images show Mitochondria by confocal IF against Tom20.
  • FIG. 11C Representative live cell confocal images of JC1 labelled mitochondria in H7-hRGC E50K .
  • FIG. 1 IF Shown are action potential firing spikes from a single MEA sensor monitored over 3 minutes for respective conditions.
  • FIG. 1 IB One-way ANOVA with Dunnett’s.
  • FIG. 1 ID Unpaired student’s t-test.
  • FIG. HE Two- way ANOVA with Tukey’s. * p ⁇ 0.05, ** p ⁇ 0.01, **** p ⁇ 0.0001.
  • WAY maintained high mitoATP production in OPTN E50K hRGCs which is measured from mitochondrial oxygen consumption rate (OCR)
  • OCR mitochondrial oxygen consumption rate
  • WAY restored mitoOCR/glycoPER ratio close to the Wt hRGCs as detected by Seahorse glycolytic rate assay (FIG. 12B). This indicates a metabolic reprograming by WAY that regains energetic and biosynthetic balance between OXPHOS and glycolysis to combat glaucomatous OPTN E50K stress.
  • FIG. 12A, B shows WAY restores OXPHOS and glycolysis balance in OPTN E50K hRGCs.
  • hRGCs are treated with 5 pM WAY for 24 hr followed by seahorse analysis.
  • FIG. 12A Seahorse Mito Stress test measures mitochondrial ATP production from change in oxygen consumption rate (OCR) under sequential inhibition of ETC proteins.
  • WAY treatment transiently activates mitochondrial biogenesis with neuroprotection effects in the OPTN E50K hRGCs. They have also shown WAY treatment to hRGCs transiently activates cAMP which can activate mitobiogenesis master regulator PGCla as well as provide neuroprotection through mitochondria independent mechanisms. If WAY mediated RGC protection is through PGCla then suppressing PGCl activity would suppress the mitochondrial biogenesis and neuroprotection. Based on this rationale, they can first knock out (KO) PGCla in the OPTN E50K hRGCs (as done in FIG. 13A-C but for TBK1) and then measure mitochondrial mass, mitochondrial membrane potential, cellular apoptosis, excitotoxicity at different WAY (5 pM) exposure time points (th to 24h).
  • WAY treatment transiently activates mitochondrial biogenesis with neuroprotection effects in the OPTN E50K hRGCs. They have also shown WAY treatment to hRGCs transiently activates
  • FIG. 13A-C depicts CRISPR mediated gene KO in hRGCs using lentivirus (LV).
  • FIG. 13A Confocal image (63x/1.4NA) of WT H7-hRGCs transduced with the LV containing viral vector with EFla-GFP cassette or no GFP vector (Control LV).
  • FIG. 13C TBK1 KO in iPSC-hRGCs with OPTN E50K mutation by LV-U6-gRNA (TBK1) and the LV-EFla-Cas9. 8 days post transduction.
  • WAY can promote RGC protective mechanisms in the absence of PGC1 a, they can study if this is through transiently activating cAMP-responsive element binding protein (CREB) transcription factor 57 .
  • cAMP can directly activate CREB whose transient activation is known to be neuroprotective 57 .
  • WAY is a robust antagonist of serotonin receptor 5-HT1 A and their binding efficiency has been established both biochemically and in vivo. See for example. Fletcher et al., “Electrophysiological, biochemical, neurohormonal and behavioural studies with WAY-100635, a potent, selective and silent 5-HT1A receptor antagonist/’ (1996) Behav Brain Res 73(1-2): pp. 337-353; Kolan et al., “Growth-inhibition of cell lines derived from B cell lymphomas through antagonism of serotonin receptor signaling,” (2019) Sci Rep 9(1): p.
  • WAY is well tolerated in humans, a radiolabeled form of WAY has been used in multiple clinical trials to map the serotonergic system by positron emission tomography (PET) for patients undergoing serotonin receptor targeted treatments (NCT00603018, NCT02810717).
  • PET positron emission tomography
  • EXAMPLE 8 [00229] WAY crosses blood-retinal barrier
  • WAY Since the inventors observed robust RGC protection against ONC injury by WAY via intraperitoneal injection, they then sought to determine if the effect is direct or indirect. To have a direct effect, WAY needs to cross the blood-retinal barrier and present in the retina. WAY can cross the blood-brain barrier following IP injection in mice. See Markowitz et al., “Drug-induced glomerular disease: direct cellular injury,” (2015) Clin J Am Soc Nephrol 10(7): pp. 1291-1299. To test if WAY crosses the blood-retinal barrier the inventors tested IP injection at 30mg/kg body weight dose (100 pl PBS) to C57BL/6J mice followed by plasma and retina collection at 4hr and 24hr for detection by mass spectrometry. A significant amount of WAY was measured in the retina and plasma within 4h after injection (FIG. 14).
  • FIG. 14 shows that LC-MS chromatograms detect WAY in mice retina.
  • the blue filled peak is the analyte of interest.
  • WAY retention time is 5.5 minutes.
  • Representative chromatogram for treated retinal tissue is from 4 independent experiments.
  • WAY concentration in retina was maximum after 4h (2006.41+/- 263.75 ng/gm) and reduced (17.05+/-3.03 ng/gm) 24h after the I.P injection at 30mg/kg dose.
  • the inventors used once a daily IP injection, however, since they observed robust RGC protection against ONC at a much lower dose, 5mg/kg body weight was chosen. This suggests WAY could be efficacious even at a much lower dose and/or capable of maintaining neuroprotective effect with intermittent treatments which can be further tested.
  • FIG. 15 A, B shows that WAY does not show systemic toxicity in mice.
  • the inventors intend to identify 5-HT1 A s cognate G protein and its target AC involved in WAY response as to resolve their direct effect on cAMP activation and RGC protection, and if in the absence of corresponding G protein and AC, WAY loses its beneficial effects.
  • the Gi/o family’ includes Gai 1. Gai2. Gai3, and Gao proteins which have been reported to couple with 5-HT1A receptor 36 . The activation of any of these G proteins could be suppressed by the antagonist (WAY) binding to 5-HT1 A GPCR. Loss of specific inhibitory G protein involved in WAY signaling can disrupt the transient cAMP activation. Inventors can test this prediction by knocking out (KO) individual Gail, Gai2, Gai3, and GaO protein in hRGCs then measure cAMP level to compare between WT and KO in the absence and presence of WAY. Inventors can perform CRISPR/Cas9 based knockout in hRGCs by LV-gRNA as shown for TBK1 knockout for feasibility’ (FIG. 13).
  • Identify ACs involved There are nine transmembrane ACs (tmACs) and one soluble AC (sAC) in mammals 59 , that may be involved in WAY -mediated cAMP modulation. This is important to identify the specific AC involved as it has been reported that sAC activation can promote RGC survival and axon growth 39 . Active sAC can also directly translocate to mitochondria and maintain organelle health independent of cytosolic mechanisms 60 ’ 61 .
  • hRGCs pan-tmAC inhibitor dDADO 62 and two sAC specific inhibitor KH7 63 and 2HE 63 in 1 - 10 mM range and measure if this blocks WAY mediated transient activation of cAMP level as measured in FIG. 3B-E.
  • AC inhibitors are commercially available from Sigma-Aldrich and are well characterized for their inhibition effect in cultured cells with above concentration range 64 .
  • Inventors’ data shows WAY transiently activates mito-biogenesis and cAMP level with improved mitochondrial health and reduced hRGC apoptosis. They intend to resolve three aspects of WAY-mediated effects in hRGCs: (a) mechanisms for WAY-mediated transient mito- biogenesis activation, (b) They can test whether WAY maintains mitochondrial health independent of its effect on mito-biogenesis. (c) They can test if WAY exerts its neuroprotective effects in the absence of mitochondrial
  • Biogenesis master regulator PGClcc activation by cAMP could occur via few mechanisms.
  • cAMP can bind and activate CaMKII (pThr286) 65, 66 through autophosphorylation.
  • Active CaMKII either can phosphorylate and activate P38 MAPK (pThr!80) 52 ’ 67, 68 or Sirtl (pSer47) 69 .
  • active P38 MAPK can phosphorylate PGC1 a for activation, or active SIRT1 can deacetylate PGCla to block proteasomal degradation leading to activation 70 ' 72 .
  • Inventors can measure the activation of above proteins in hRGCs treated with WAY over a time course (Ih, 6h, 18h, 24h) by western bloting against the native and phosphorylated (active) forms. Based on activation of above molecular players, they can suppress the involved protein such as by CRISPR knockout as in FIG. 13 and test if that blocks WAY effect on biogenesis.
  • WAY activates sAC for translocating it into the mitochondria for a local cAMP dependent mitochondrial membrane potential (health) maintenance 60, 61 independent of PGCla dependent mechanisms.
  • inventors can knockout PGCla in hRGCs by transducing LV-U6-gRNA (PGCla) (ThermoFisher) and LV-Cas9. Then can measure mitochondrial membrane potential in hRGCs under WAY treatment by live cell mitochondrial membrane potential dye JC1 (demonstrated in FIG. 11C, D and the reference 54 from our lab).
  • Inventors can validate the findings by another live cell mito-membrane potential dye TMRM (ThermoFisher) which enters mitochondria if membrane potential is high or healthy. They can analyze mitochondrial ultrastructure in PGCla knockout hRGCs to study if WAY rescues cristae area, morphology’ and mitochondrial shape (elongation, sphericity) as done by them previously 54 which directly reflects organelle health and OXPHOS competency 74 . The inventors have extensive experience in analyzing mitochondrial ultrastructure 54 . Restoration of mitochondrial health by WAY independent of PGCla would indicate a biogenesis independent mechanism such as byreduced oxidative stress or by direct effect of sAC 60, 61 .
  • TMRM live cell mito-membrane potential dye
  • inventors can test if sAC translocates to mitochondria in hRGCs by confocal IF (anti-sAC antibody. Millipore Sigma) under WAY treatment and whether the protective effect is abolished by' sAC inhibitor KH7 63
  • the inventors expect to identify the specific AC and Gi/o involved in WAY mediated RGC protection. Based on neuroprotection effect of sAC 39 , they expect that WAY can activate sAC and suppression of sAC activity can suppress WAY effect on cAMP activation, mitobiogenesis and cellular apoptosis.
  • CRISPR KO of a gene in hRGCs causes cell death
  • inventors can take gene knockdown approach by lentiviral shRNA delivery.
  • the inventors can identify the lowest dose required for longer vision, RGC and optic nen e protection. Next, they can test how long the drug efficacy remains by only pretreating mice 5 days before ONC with above identified low dose and then performing vision tests, retinal tissue collection for above experiments on day +7 and +14. This can help a determination as how frequently treatment is needed for optic nerve and vision protection.
  • FIG. 26 shows that the small molecule screen identifies compound for activator of mitobiogenesis and mitophagy'.
  • Error bars are SEM.
  • Red arrows indicate compounds that show reduced apoptosis in H7- hRGCs in the secondary screen.
  • L-Glut glutamate neurotransmitter
  • FIG. 17 A, B shows that L-Glutamic acid (L-Glut) promotes mitochondrial mass and lowers apoptosis in hRGCs.
  • L-Glut L-Glutamic acid
  • FIG. 17A Manual validation of L-Glut treatment at 5 pM for 24h shows increase in mitochondrial mass compared to the untreated control (CTR) in H7-hRGC l that was identified from high throughput screen in FIG. 16.
  • CTR untreated control
  • Error bars are SEM. Unpaired student's t-test.
  • MAA increased mitochondrial mass in hRGCs from the screening (FIG. 16) and the result was reproduced when tested manually (FIG. 18 A).
  • FIG. 18 A, B shows that MAA promotes mitochondrial mass and lowers apoptosis in hRGCs.
  • FIG. 18A Manual validation of MAA treatment at 5 pM for 24h shows increase in mitochondrial mass compared to the untreated control (CTR) in H7-hRGC WT that was identified from high throughput screen in FIG. 16. MAA is water soluble hence we used untreated cells as control.
  • MAA also activates mitobiogenesis in hRGCs with potential neuroprotection effect. Indeed, it was observed that robust reduction of cellular apoptosis occurred for the WT and E5 OK-corrected hRGCs when treated with MAA (FIG. 18B). MAA has been shown to antagonize the GAB Ac receptors in white perch retinal neurons. See Tunni cliff et al., “Pharmacology and function of imidazole 4-acetic acid in brain,” (1998) Gen Pharmacol 31(4): pp. 503-509. To date, there has been no study indicating that MAA could serve as RGC protective compound. Thus, the current discovery 7 that MAA reduces apoptosis in hRGCs is novel, hence MAA and/or its derivatives that target GAB Ac receptor provide novel therapeutic agent(s) for optic neuropathy therapy development.
  • FIG. 19A, B shows that ouabain promotes mitochondrial degradation and lowers apoptosis in hRGCs.
  • FIG. 19A Manual validation of ouabain treatment at 5 pM for 24h shows reduced mitochondrial mass in H7-hRGC WT compared to the DMSO vehicle control (CTR) that was identified from the high throughput screen in figure 16.
  • Ouabain is a steroid hormone which is a classic inhibitor of Na + /K + -ATPase membrane channel and upon binding ouabain activates cell growth, proliferation and cell survival pathw ays. See Xie et al., “Na(+)/K(+)-ATPase as a signal transducer.” (2002) Eur J Biochem 269(10): pp. 2434-2439. It has been shown that in primary rat RGC culture ouabain treatment increased cell survival by suppressing inflammatory pathways when isolated from naive as well as from retinas following optic nerve axotomy.
  • ouabain is neuroprotective for human RGCs and if in vivo under optic nerve damage conditions.
  • the inventor’s data showing ouabain reduces apoptosis in human hRGCs is novel. Further studies can include identifying the optimum dose, treatment duration and the mechanism of RGC protection in mice ONC and in high IOP glaucoma conditions. Thus, based on these data a carefully tested dose of ouabain and/or its derivatives could provide strong RGC protection for optic neuropathy therapy development which is an unmet area.
  • RTA-408 (Omaveloxolone) as a therapeutic drug for optic neuropathy
  • Oxidative Damaged mitochondria are the primary source for cellular ROS (mtROS). See Murphy, “How mitochondria produce reactive oxygen species,” (2009) Biochem J 417(1): pp. 1-13. Increased ROS level have been widely found in the primary open-angle glaucoma (POAG) patients’ retina with high eye pressure. Mitochondrial biogenesis and antioxidant response pathways involve a common player, Nrf2. Mitochondrial biogenesis requires mtDNA transcription and activated PGCla stimulates Nrfl and Nrf2 which drive the expression of mitochondrial transcription factor Tfam. See Piantadosi et al.. “Redox regulation of mitochondrial biogenesis.” (2012) Free Radic Biol Med 53(11): pp. 2043-2053.
  • Nrf2 is also the master regulator for ROS removal pathways. See Holstrom et al. 2016. In basal conditions, Nrf2 is rapidly degraded by the Keapl-Cullin3 (Cul3)/Rbx E3 ubiquitin ligase system and proteasome pathway. Under elevated ROS. cysteine sensors of Keapl react with the ROS electrophiles leading to Nrf2 release and stabilization. See Dinkova-Kostova et al., “Keapl, the cysteine-based mammalian intracellular sensor for electrophiles and oxidants,” (2017) Arch Biochem Biophys 617: pp. 84-93.
  • Nrf2 activators are under FDA clinical trials for mitochondrial dysfunction related neurodegenerative conditions such as Parkinson’s, Alzheimer’s and Huntington’s disease. See Robledinos-Anton et al., “Activators and Inhibitors of NRF2: A Review of Their Potential for Clinical Development,” (2019) Oxid Med Cell Longev 2019: p. 9372182. Over-expression ofNrf2 have been shown to be RGC protective in the mouse optic nerve crush model. See Yang et al., “Antioxidant Treatment Limits Neuroinflammation in Experimental Glaucoma,” (2016) Invest Ophthalmol Vis Sci 57(4): pp. 2344-2354.
  • RTA- 402 Pharmacological Nrf2 activators bardoxolone (RTA- 402) and sulforaphane have been shown to be RGC protective against anterior ischemic optic neuropathy while CDDO-Im and dimethyl fumarate (DMF) have been shown to protect RGCs against ONC in mouse.
  • Nrf2 Activators Bardoxolone Methyl and Omaveloxolone, on Retinal Ganglion Cell Survival during Ischemic Optic Neuropathy,” (2021) Antioxidants (Basel) 10(9): epub 20210915; Pan et al., “Sulforaphane Protects Rodent Retinas against Ischemia-Reperfusion Injury through the Activation of the Nrf2/HO-l Antioxidant Pathway,” (2014) PLOS ONE 9(12): el 14186.; Himori et al., “Critical role of Nrf2 in oxidative stress-induced retinal ganglion cell death/’ (2013) J Neurochem 127(5): pp.
  • Damaged mitochondria are the primary source for cellular reactive oxygen species (ROS) which at excess levels cause cellular apoptosis.
  • ROS reactive oxygen species
  • the transcription factor, nuclear factor erythroid-2 related factor 2 (Nrf2) protein is the master regulator of mitochondrial ROS (mtROS) clearance through activation of Antioxidant Response Element (ARE) gene. See Holmstrom et al., “The multifaceted role of Nrf2 in mitochondrial function,” (2016) Curr Opin Toxicol 1: pp. 80-91.
  • Nrf2 oxidative stress response master regulator
  • FIG. 20A-C shows that Nrf2 activation in OPTN E50K hRGCs is defective.
  • FIG. 20A Western blot images ofNrf2 and p-Nrf2 in presence of 10 pM CCCP.
  • the inventors have shown glaucomatous hRGCs with OPTN E50K mutation suffer oxidative stress by the live cell ROS sensor DCFDA (FIG. 21 A, B). Thus, they seek to determine if specific Nrf2 activators can reduce oxidative stress and promote pro-survival pathway in the OPTN E50K hRGCs and tested several Nrf2 activators such as sulphorafane, DMF and RTA-408. The inventors have discovered that RTA-408, showed robust reduction of ROS level as revealed by reduced DCFDA green signal in both the Wt and in OPTN E50K hRGCs (FIG. 21C, D).
  • FIG. 21A-D shows that the Nrf2 activator RTA-408 reduces ROS level in hRGCs.
  • FIG. 21A Max projection of confocal z-stacks, of hRGCs treated with 100 nM RTA-408 or vehicle control DMSO for 24h. ROS level detected in the live cells by DCFDA which fluoresces green upon oxidation by ROS.
  • RTA-408 shows robust reduction of cellular apoptosis in both the WT and glaucomatous OPTN E ’ 0K hRGCs (FIG. 22), establishing its novel therapeutic application for glaucoma and or mitochondrial dysfunction related optic neuropathy.
  • RTA-408 is an FDA approved drug used for Friedreich ataxia treatment (Omav el oxoIone).
  • FIG. 22 shows that Nrf2 activation by RTA-408 reduces apoptosis in hRGCs.
  • Metabolic vulnerability disposes retinal ganglion cell axons to dysfunction in a model of glaucomatous degeneration. JNeurosci 30, 5644-5652 (2010). Calkins, D.J. Critical pathogenic events underlying progression of neurodegeneration in glaucoma. ProgRetin Eye Res 31. 702-719 (2012). Calkins, D.J. Adaptive responses to neurodegenerative stress in glaucoma. Prog Retin Eye Res 84, 100953 (2021). Harun-Or-Rashid, M. et al. Structural and Functional Rescue of Chronic Metabolically Stressed Optic Nerves through Respiration. The Journal of Neuroscience 38, 5122-5139 (2016). Crish, S.D.
  • the Mammalian-Specific Protein Annex 1 Regulates Mitochondrial Transport during Axon Regeneration. Neuron 92, 1294-1307 (2016). Zheng, Y.R.. Zhang. X.N. & Chen, Z. Mitochondrial transport serves as a mitochondrial quality control strategy in axons: Implications for central nervous system disorders. CNS Neurosci Ther 25, 876-886 (2019). Harder, J.M. et al. Disturbed glucose and pyruvate metabolism in glaucoma with neuroprotection by pyruvate or rapamycin. Proc Natl Acad Sci U SA 117, 33619-33627 (2020). Tribble, J.R. et al.
  • Nicotinamide provides neuroprotection in glaucoma by protecting against mitochondrial and metabolic dysfunction.
  • Chemel, B.R., Roth, B.L., Armbruster, B.. Watts, V.J. & Nichols. D.E. WAY-100635 is a potent dopamine D4 receptor agonist.
  • 8-OH-DPAT as a 5-HT7 agonist phase shifts of the circadian biological clock through increases in cAMP production.

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Abstract

La présente divulgation concerne de nouvelles approches et méthodes de traitement de diverses maladies ou pathologies oculaires, y compris des neuropathies optiques. En particulier, les méthodes et les traitements divulgués concernent celles de ces maladies ou pathologies oculaires qui impliquent un dysfonctionnement mitochondrial. Plus particulièrement, les méthodes et les traitements divulgués peuvent restaurer l'homéostasie mitochondriale.
PCT/US2024/044204 2023-08-29 2024-08-28 Nouveaux traitements pour neuropathies optiques et glaucome Pending WO2025049588A1 (fr)

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US20040147581A1 (en) * 2002-11-18 2004-07-29 Pharmacia Corporation Method of using a Cox-2 inhibitor and a 5-HT1A receptor modulator as a combination therapy
US20120077818A1 (en) * 2009-05-13 2012-03-29 Sunovion Pharmaceuticals Inc. Compositions comprising transnorsertraline and serotonin receptor 1a agonists/antagonists and uses thereof
US20200390780A1 (en) * 2011-02-18 2020-12-17 The Scripps Research Institute Directed differentiation of oligodendrocyte precursor cells to a myelinating cell fate
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