WO2020036658A2 - Drugs promoting retinal rod photoreceptor survival - Google Patents

Abstract

Described herein are methods and compositions for treating or preventing blindness in a subject. The method includes a step of administering to a subject having a disease resulting in photoreceptor death, retinal cell death, or a combination thereof one or more compounds of the present invention.

Description

DRUGS PROMOTING RETINAL ROD PHOTORECEPTOR SURVIVAL

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent application 62/663,496, filed April 27, 2018, which is hereby incorporated by reference for all purposes as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no. R01EY022810 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Retinitis pigmentosa (RP) is an inheritable retinal degenerative disease characterized by early onset night blindness, gradual loss of visual field, and eventual loss of central vision (Hamel, 2006)(Ferrari et al, 2011). Approximately 1 in 4000 are affected, with more than one million current patients worldwide (Hartong et al, 2006). The pathological features of the disease are selective progressive rod photoreceptor cell death. Rod photoreceptor cell death, and perhaps more specifically loss of rod-derived cone viability factor (RdCVF; (Leveillard et al, 2014), leads to subsequent loss of cone photoreceptors. Hundreds of mutations, in more than 70 genes, have been linked to RP (Dias et al, 2017). However, understanding of how the majority of these mutations affect the function of disease-linked genes (e.g. rhodopsin (rho) (Mendes et al, 2005)) or initiate aberrant photoreceptor cell loss, is incomplete.

Currently there are no effective therapies for ameliorating RP. As disease progression is relatively protracted, pharmacological interventions aimed at slowing rod photoreceptor cell death have been widely investigated. Neurotrophic factors, anti-apoptotic agents, nutritional supplements and anti-oxidants have been shown to provide neuroprotective effects in animal RP models (Dias et al, 2017). Unfortunately, these reagents have produced only limited benefits in patients to date. For example, ciliary neurotrophic factor (CNTF) had shown to be effective in protecting photoreceptors in chicken (Fuhrmann et al, 2003), mouse (Cayouette et al, 1998) and dog (Tao et al, 2002) models of retinal degeneration. However, CNTF failed to improve either visual acuity or field sensitivity in both short- and long-term clinical trials (Birch et al., 20l3)(Ho et al., 2015). Additionally, clinical trials of Vitamin A in combination with Vitamin E (Berson et al., 1993), docosahexaenoic acid (Berson et al, 2004a)(Berson et al, 2004b) or lutein (Berson et al, 2010) produced limited benefits and only in certain subpopulations. Moreover, unfortunately, the mild improvements observed were offset by adverse side effects associated with long-term use (Dias et al, 2017). As a means of discovering new therapeutic treatments, target-directed high- throughput screening (HTS) approaches have been immensely successful in identifying compounds that modulate disease-implicated molecules. However, many promising leads fail during late-stage testing in animal models or in clinical trials (Scannell et al, 20l2)(Munos, 2009)(Sams-Dodd, 2013). As such, there has been renewed interest in phenotypic drug discovery (PDD), a complementary chemical biology approach emphasizing evaluation of drug effects in cells or living disease models (Swinney, 20l3)(Lee et al., 20l2)(Bickle, 2010). For example, between 1999 and 2013 a number of first-in-class drugs were discovered using phenotypic screening (Swinney and Anthony, 20l l)(Eder et al., 2014), many being identified by conducting phenotypic assays directly in animal models (Swinney, 2014)

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for treatment or preventing retinitis pigmentosa of a subject. The method includes the step of administering to a subject with retinitis pigmentosa or prone to getting retinitis pigmentosa one or more compounds of Figs. 7 and 13 Treating or preventing the retinitis pigmentosa of the subject compared to a reference subject that has not been administered the one or more compounds of Figs. 7 and 13. Examples of compounds used in the present invention includes

CC(=0)CC(Cl=CC=CC=Cl)C2=C(C3=CC=CC=C30C2=0)0 (Warfarin);

CCl=CC(=0)N(C(=Cl)C2CCCCC2)0 (Ciclopirox olamine);

CC 1 CCC2C(C(OC3C24C 1 CCC(03)(004)C)0) (Dihydroartemisinin);

CC 1 CCC2C(C(=0)0C3C24C 1 CCC(03)(004)C)C (Artemisinin); C 1 =CC(=S)N(C=C 1 )[0- ].Cl=CC(=S)N(C=Cl)[0-].[Zn+2] pyrithione zinc, or a combination thereof. In some embodiments of the present invention a combination of compounds may be administered to a subject. An example of a combined therapy includes administering dhydroartemisinin, artemisinin, and ciclopirox olamine to a subject. The one or more compounds of Figs. 7 and 13 may administered to a subject by any suitable means such as eye drops delivering one or more compounds to the eye of a subject. A pharmaceutical composition of the one or more compounds of Figs. 7 and 13 may be formed prior to the administration to a subject.

Another embodiment of the present invention is a method of promoting photoreceptor and/or retinal cell survival. The method includes the steps of: administering one or more compounds of Figs. 7 and 13 to a photoreceptor, a retinal cell, or both having a disease or treated with an agent that causes cell death; and promoting the survival of the photoreceptor, retinal cell, or both compared to a reference photoreceptor, a reference retinal cell, or both that have not been administered one or more compounds of Figs 7 and 13. In some embodiments of the present invention the photoreceptor, the retinal cell, the reference photoreceptor, and the reference retinal cell used may have a disease, causing blindness such as RP as an example, prior to administering one or more compounds of Figs. 7 and 13. Other embodiments of the present invention use a photoreceptor, a retinal cell, or both that is treated with the agent that causes photoreceptor cell death, retinal cell death, or both after the administering of one or more compounds of Figs. 7 and 13. Suitable the agents used in the present invention tunicamycin, thapsigargin or a combination thereof, as examples.

Another embodiment of the present invention includes a method of treating or preventing blindness in a subject. The method includes the steps of: administering to a subject having a disease resulting in photoreceptor death, retinal cell death, or a combination thereof, one or more compounds of Figs. 7 and 13; and treating or preventing blindness of the subject compared to a reference subject that has not been administered the one or more compounds of Figs. 7 and 13.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features.

By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include retinitis pigmentosa (RP).

By "effective amount" is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject.

Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

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

A“reference” refers to a standard or control conditions such as a sample (human cells) or a subject that is a free, or substantially free, of an agent such as one or more of the compounds of Figs 7 and 13.

As used herein, the term "subject" is intended to refer to any individual or patient to which the method described herein is performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) , as well as other common animal model species (including fish, frogs, newts, and chickens) are included within the definition of subject.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

As used herein, the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for blindness such as RP, an eye disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, a marker (as defined herein), family history, and the like).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-1B. Primary Screen. A. Rod photoreceptor ablation at different Mtz concentrations. Five concentrations of Mtz from 10 mM to 0.625 mM were tested for the ability to induce rod photoreceptor ablation in rho:YFP-NTR larvae using a 48 hr treatment regimen from 5 to 7 dpf (days post-fertilization), ARQiv quantification of YFP loss, and a sample size of 24 per condition. The 2.5 mM Mtz treated group displayed progressive YFP loss, equivalent to higher concentrations by the second day of treatment, and was therefore chosen as the regimen for large-scale screening. B. Schematic of the primary screening process: 1) Large amounts of embryos were collected at 0 dpf. 2) Embryos were treated with PTU at 16 hpf (hours post-fertilization) to suppress melanization. 3) At 4 dpf, test compounds were dispensed into microtiter plates using robotic liquid handlers to titrate a 2-fold dilution series, from 4 mM to 125 nM, across a 96-well plate (16 wells per concentration). 4) At 5 dpf, larvae were dispensed into individual wells of compound containing 96-well plates. 5) After a 4-hour pre-exposure to test compounds, Mtz was added at a final concentration of 2.5 mM to ablate rod photoreceptor cells. 6) At 7 dpf, YFP signal of each larva was quantified by ARQiv (i.e. Tecan Ml 000 Pro microplate reader assay). 7) Data was collected and analyzed in near real-time to allow same day visual confirmation of compound effects. Data processing included a signal to background ratio plot, a SSMD score plot, a heat map of signal to background ratio within the plate, and SSMD score table. 8) Conditions producing an SSMD score >1 were visually inspected using stereo fluorescence microscopy. Conditions producing elevated YFP signals either from drug autofluorescence or deceased larvae were excluded.

Figure 2. Hit Compound Confirmation Assay. 42 top-performing hit compounds underwent secondary validation tests involving newly sourced stocks and a minimum of 3 biological replicates. YFP signal of each larva in each treatment group was normalized to the signal window produced by the negative control (rod cell ablated fish treated with DMSO vehicle alone) and positive control (non-ablated fish) readings taken on the same day (See materials and methods) to calculate relative effect size. Experimental repeats were pooled and plotted to show relative effect sizes. Eleven confirmed hit compounds showed reproducibly elevated YFP signals, with effect sizes ranging from 10 to 38 percent, suggesting ameliorated rod photoreceptor cell loss. Student’s /-test with multiple comparisons correction was performed to calculate 95% confidence intervals and p values, shown in a table below the graph along with the sample size for each compound.

Figure 3. Intravital Imaging. Confocal z-stack projections images show YFP reporter signals in rho:YFP-NTR transgenic larval zebrafish eyes following Mtz-induced rod photoreceptor loss and treatment with hit compounds or vehicle control. At 7 dpf, non- ablated retinas displayed strong YFP expression in rod photoreceptors in the outer nuclear layer (ONL) with elevated signals in the ventral retina corresponding to a higher of rod cells in this region. Mtz-treated rho:YFP-NTR retinas showed reduced YFP levels when treated with 0.1% DMSO vehicle alone (negative control). Retinas treated with hit compounds exhibited enhanced YFP signals relative to the negative control, thus preservation of rod photoreceptor cells. High magnification images of hit compound treated retinas detail surviving rod cell morphologies, which ranged from healthy elongated rod cell structure to condensed cells, suggesting variable effects on preservation of rod photoreceptor function.

Figure 4. Cell Death Assay. TUNEL staining was used to quantify cell death. Non- ablated control retinas showed only occasional TUNEL-positive cells. In retinas treated with 2.5 mM Mtz, YFP signal was lost and an increased number of TUNEL-positive cells was observed in the ONL. Mtz-treated retinas exposed to hit compounds, e.g., dihydroartemisin (Dih), showed sustained YFP signals and a reduction in TUNEL-positive cells, suggesting suppression of cell death. The mean, SD and sample size of each group is listed. Student’s t- test was used to calculate the p value.

Figure 5. Additive Effects Assay. Combinatorial assays pairing 7 hit compounds at their maximal effective concentrations were used to test for additive effects. Ten pairs produced additive effect sizes (all green boxes) near or above the sum of their individual effects (italicized values in lower left). Moreover, eight pairs produced better than additive effects (dark green boxes).

Figure 6. Mouse Retinal Explant Assays. A subset of hit compounds was tested for photoreceptor survival promoting effects in mouse retinal degeneration 1 ( rdl ) retinal explant cultures. Retinal explants were isolated for postnatal day 10 rdl mice and cultured with DMSO or test compounds for 11 days. Explants were then harvested, fixed for nuclear staining using toluidine blue and the number of rows of photoreceptors in each retina counted (n= 3 to 4). ANOVA followed by Bonferroni/Dunn posthoc analysis was performed. Among 6 tested compounds, artemisinin (Art), dihydroartemisinin (Dih), and ciclopirox olamine (Cic) showed repeatable effects on photoreceptor preservation. (* indicated the concentration with p< 0.05).

Figure 7. Eleven hit compounds abbreviations. Figure 8. Mouse Primary Retinal Cell Culture Assays. Table summarizing protective effects of tested hit compounds on primary cultures of isolated mouse retinal cells treated with exogenous stressor compounds to induce cell death. (++: >2SD; +++: >3SD)

Figure 9. NTR Inhibition Assay. The effect of 11 hit compounds on NTR enzymatic activity was tested. NTR activity was estimated using CB1954 kinetics assay in the presence of test compounds at 300 mM. The NTR activity in each compound treated sample was compared to vehicle only controls. If the ratio was higher than 0.75, drugs were not considered to have an inhibitory effect. War, Cic, Cal and Sul were found to inhibit NTR activity at 300 mM. Using the same drug concentrations, NTR inhibitory effects of War, Cic, Cal was also tested using an Mtz kinetics assay. Subsequently, IC50 values were calculated. Relative NTR activity levels, standard deviation and IC50 in CB1954 kinetics assay, as well as NRT activity in Mtz kinetics assay are summarized in the table.

Figure 10. Rod Cell Fate Assay. Effects of 11 hit compounds on rod cell differentiation was tested. Larvae were treated with hit compounds at maximal

neuroprotective concentrations from 5 to 7 dpf. YFP signals were quantified at 7 dpf and compared to vehicle only controls. RA (retinoic acid) served as positive control compound. None of 11 hits showed improved YFP signals compared to the control. Instead, 3 drugs, Clo, Cic and Cor, displayed reproducible inhibitory effects (black asterisks, p-value <0.005). The positive control enhanced YFP signals (red asterisk). Student’s /-test with multiple comparisons correction was performed to calculate 95% confidence intervals and p values, shown in a table below the graph along with the sample size for each compound.

Figure 11. Rod Cell Regeneration Assay. Effects of 11 hit compounds on rod cell regeneration. Larvae were treated with lOmM Mtz from 5 to 6 dpf to induce rod cell loss.

Mtz was then rinsed out and larvae exposed to hit compound for 3 days (6 to 9 dpf). YFP signal was quantified at 9 dpf. All signals were compared to Mtz ablated controls treated with vehicle alone. Non-ablated larvae served as positive controls. None of the larvae treated with the hit compounds showed improved YFP signals compared to vehicle controls; two compounds (Clo and Cic) showed reduced YFP signals (black asterisks, p-value <0.005). Student’s /-test with multiple comparisons correction was performed to calculate 95% confidence intervals and p values, shown in a table below the graph along with the sample size for each compound.

Figure 12. Potential Hits Compounds. Compounds producing an SSMD >1 at any concentration were defined as potential hits and are listed here. The drug names,

concentrations producing SSMD >1, and SSMD scores are shown. Compounds are ordered by relative SSMD score. The corresponding effect subtype is also listed. YFP intensity in treated larvae was visually inspected to determine if there was a concentration-dependent trend (last column). The yellow highlighted drugs were selected for confirmation testing. The black dot denotes confirmed hit drugs.

Figure 13. Previously implicated retinal neuroprotectants tested via the primary screen process.

Figure 14. Possible signaling pathways for each hit compound. Searches were conducted in in PubChem (https://puhchem.ncbi.nlm.nih.gov/) to assess bioassay results for hit compounds, and previously implicated signaling pathways are summarized. Note that some drugs appear to modulate multiple pathways and many of the implicated signaling pathways overlap between hit compounds. For example, Tdpl inhibition was shared by 8 out of 11 hit compounds. Tdpl: Tyrosyl-DNA phosphodiesterase 1; Rorc: RAR related orphan receptor gamma; AR: androgen receptor signaling pathway; TR: thyroid receptor signaling pathway; VDR: vitamin D receptor; ER: estrogen receptor alpha signaling; AhR: aryl hydrocarbon receptor signaling; GR: glucocorticoid receptor; Dopa: dopamine related; HIF1 : Hypoxia-inducible factor 1 -alpha; SHH: Sonic hedgehog; COX: cyclooxygenase.

DETAILED DESCRIPTION OF THE INVENTION

Zebrafish are amendable to large-scale chemical screening due to their high fecundity rate, small size, and ease of visualizing phenotypes across gross morphological, tissue- specific, cell-specific, molecular, and behavioral levels of analysis (Mathias et al., 2012) (Zon and Peterson, 2005). Semi-automated low- to mid-throughput compound screens have been performed with zebrafish using high-content imaging (Selderslaghs et al, 2009)(Padilla et al, 20l2)(Ton et al, 2006) or behavioral assay platforms (Rihel et al., 20l0)(Kokel et al, 2010). The inventors established a complementary platform, automated reporter quantification in vivo (ARQiv; (Walker et al, 20l2)(White et al, 2016)) to enable high-throughput screening rates in whole-organism (Wang et al, 2015) and organoid (Vergara et al, 2017) disease models. In an inaugural ARQiv screen, was evaluated over 500,000 transgenic larval zebrafish to identify drugs that stimulated an increase in the number of insulin-producing pancreatic beta cells (Wang et al, 2015).

The ARQiv system was used to perform a large-scale whole-organism screen to identify potential new therapeutics for delaying photoreceptor loss in RP patients. Using an inducible zebrafish model of RP, over 300,000 larvae were screened to identify

“neuroprotective” compounds that promoted rod photoreceptor survival. A collection of approximately 3,000 largely human-approved compounds (the Johns Hopkins Drug Library; (Shim and Liu, 2014), was evaluated across six concentrations using quantitative HTS (qHTS; (Inglese et al, 2006b)) principles. The inventors identified 114 hits with and 42 of the top performing compounds were advanced through a series of confirmatory and orthogonal assays. Eleven compounds passed all secondary tests and moved forward as lead drug candidates. Subsequent tests in primary mouse retinal cell cultures and mouse rdl retinal explants {retinal degeneration 1, Pde6b mutant) validated three compounds as cross-species neuroprotectants, capable of promoting photoreceptor survival in both fish and mouse models of RP.

Material and Methods:

All animal studies described herein were performed in accordance with both the Association for Research in Vision and Ophthalmology (ARVO) statement on the“Use of Animals in Ophthalmic and Vision Research” and the National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW) policies regarding studies conducted in vertebrate species.

Fish maintenance and husbandry

All zebrafish procedures and studies described have been approved by the Johns Hopkins University Animal Care and Use Committee (ACUC). The transgenic line, Tg(rho:YFP- Eco.NfsB)gmc500 (hereafter, rho:YFP-NTR (Walker et al, 2012)) expresses the bacterial enzyme nitroreductase (NTR) and yellow fluorescent protein (YFP) selectively in rod photoreceptors. This line was propagated in a roy ^a9 ( roy ) background to facilitate YFP reporter signal quantification via our ARQiv plate reader assay. Non-transgenic roy fish were used to define reporter signal cutoff values for plate reader assays. All fish were maintained at 28.5°C with a 14: 10 light/dark cycle. Large numbers of eggs (6,000-12,000) were collected per screening day by grouped breeding of -300 adult rho:YFP-NTR fish using our customized mass breeding chambers (White et al, 2016).

Optimization of Mtz concentration to establish an inducible RP model

rho: YFP-NTR larvae were separated into 6 groups with 24 larvae in each group. Each group was treated with varying metronidazole (Mtz) concentrations (10, 5, 2.5, 1.25, 0.625, 0 mM) from 5-8 days post fertilizations (dpi). YPF signals were quantified every day by microplate reader (Infinite Ml 000 PRO from Tecan) to track changes in rod photoreceptor cell numbers relative to Mtz concentration. The 2.5mM treatment was the minimal concentration of Mtz that promoted maximal cell loss and was therefore selected for large-scale screening to minimize toxicity associated with long-term Mtz exposure.

Sample size estimation

rho: YFP-NTR larvae were treated with either 2.5mM Mtz/0. l% DMSO or 0.1% DMSO (control) from 5-7 dpf. Each group contained at least 48 larvae and three biological replicates were performed. YFP signals were measured using the TECAN microplate reader at 7 dpf. Power calculations were used to determine sample sizes across a range of error rates and effect sizes for both raw and log2 -transformed data, as per published methods (White et al, 2016). A sample size of 9 per condition was predicted to facilitate detection of an effect size of 50% of non-ablated controls with error rates of 0.05 for false-positives and false-negatives (type I and type II, respectively). However, to account for dispensing errors and in keeping with 96-well microtiter plate formats, a sample size of 16 was chosen for the primary screen.

Primary drug screening using ARQiv platform

For the primary screen, 2,934 compounds from John Hopkins Drug Library (JHDL) were screened across six concentrations (from 4mM to 125hM using a 2-fold dilution series). The JHDL consists of -2,200 human-approved drugs and an additional -800 in clinical trials (Chong et al, 2006). The ARQiv-based screening process has been detailed previously (White et al, 2016) and adapted here for large-scale quantification of YFP-expressing rod photoreceptors. rho:YFP-NTR embryos were collected and raised in zebrafish E3 embryo media (5 mM NaCl; .17 mM KC1; .33 mM CaCl; .33 mM MgS04). At 16 hours post fertilization (hpf), 0.2mM N-phenylthiourea (PTU) was added to E3 media (E3/PTU) to promote ocular transparency by inhibiting melanosome maturation in the retinal pigment epithelium. At 4 dpf, visual screens were performed to remove larvae with abnormal morphology or low retinal YFP levels. Stock drug and DMSO (negative control) solutions were automatically dispensed into and diluted across a 96-well plate containing E3/PTU using a robotic liquid handling system (Hudson Robotics), as previously described (White et al, 2016). At 5 dpf, a COPAS-XL (Complex Object Parametric Analyzer and Sorter, Union Biometrica) was used to were dispense a single larvae into individual wells containing either drug or DMSO, the final DMSO concentration was 0.1% across all conditions. After a 4 hour pre-exposure to test drugs or DMSO alone, larvae were treated with 2.5mM Mtz to induce rod photoreceptor death. Larvae were maintained under these conditions for two days until 7 dpf and then anesthetized by adding clove oil (final concentration 59 ppm). YFP signals were measured using TECAN Ml 000 PRO microplate reader (excitation 514 nm, bandwidth 5 nm; emission 538nm, bandwidth 10 nm; note these settings are optimized for the eYFP reporter present in the rho:YFP-NTR transgene, and different than previously published settings for tag YFP, (Wang et al, 2015) (White et al, 2016)). Larvae exposed to 2.5mM Mtz but treated only with 0.1% DMSO served as controls for maximal rod photoreceptor cell ablation.

Larvae treated with 0.1% DMSO but not exposed Mtz served as non-ablated controls to calculate maximal YFP signal levels. A schematic of the primary screening process is shown in Fig. 1B.

Secondary confirmatory test of potential hits

After the initial screen, 42 top-performing‘hit’ compounds were selected for confirmatory and orthogonal assays. For confirmation, hit drugs were obtained from new sources and tested across a wider range of concentrations, using a five-fold dilution series and a total of eight concentrations, with a sample size 30 fish per condition. Based on the toxicity profile of each drug, the starting concentration was either 100, 10 or 1 mM. Three biological replicates were conducted. The results were normalized and pooled to calculate effect sizes, confidence intervals, and p-values using Student’s t-test.

Confocal imaging

For in vivo imaging of YFP in the intact eye, non-ablated (positive control), 2.5mM Mtz- ablated (negative control) and Mtz+drug treated rho:YFP-NTR larvae were collected at 7 dpf (i.e., after a 48 hr exposure as per the primary screen). All compounds were tested at the maximal effective concentration. Three larvae from each group were embedded in 1% low melt agarose gel with either the left or right eye facing up. An Olympus Fluoview FV1000 confocal microscope with a 20x water immersion objective (0.95 NA) was used to take 30-40 images at 4 pm intervals. These image stacks were then processed to produce a maximal intensity projection of the entire retina. A region in the dorsal -nasal quadrant was imaged with a 60x water immersion objective (1.10 NA) to provide greater detail of rod

photoreceptor morphology, three images at 4.18 pm intervals. For cryosection imaging, antibody-stained slides were imaged under a 40x oil objective (1.30 NA) to obtain a Z-stack of three images spaced 3pm apart. All images from one eye were compiled into a single image using the maximum intensity projection method (Fiji software).

NTR inhibitory assay

The 42 top hit compounds were tested to eliminate false-positives that directly inhibited NfsB_Ec nitroreductase (NTR) activity using an established CB1954 reduction assay (Prosser et al, 2010). In the assay reaction, the following were combined: 250 pM CB1954, 1 pM NTR, 250 pM NADPH, 10 mM Tris (pH 7.0). In addition, the highest soluble concentration of the test drug (up to 300 pM maximum) or 0.1% DMSO (rate control) were added to the mixtures to determine any inhibitory effect on the ability of NTR to reduce CB1954.

Reactions were conducted in 100 pl volume in a 96-well plate format. CB1954 reduction kinetics were assayed at 420 nm unless the test compound absorbed at 420 nm as well. In those cases, NADPH depletion was monitored at 340 nm. The reaction rates of tested compound were compared to DMSO control. Compounds with NTR activity <75% of controls were deemed inhibitory and IC50 values (the concentration at which there is 50% NTR activity) determined.

Compounds were also evaluated for the ability to inhibit NTR-mediated reduction of Mtz. In the assay reaction, the following were combined: 500 mM Mtz, 17 pM NTR, 100 pM NADPH, 5 pM Glucose Dehydrogenase, 5 mM Glucose, 50 mM Sodium Phosphate Buffer (pH 7.0). The highest soluble concentration of the test drug (up to 300 pM maximum) or DMSO (rate control) were added to the mixtures to determine any inhibitory effect on the ability of NTR to reduce Mtz. Reactions were conducted in 100 pl volume and assayed at 340 nm using 1 mm quartz cuvettes (Rich et al, 2018).

Rod photoreceptor development assay

To evaluate whether hit compounds promoted rod photoreceptor development, rho:YFP-NTR larvae were handled as described for the primary screen with the following exceptions. At 5 dpf, larvae were dispensed into 96-well plate and exposed solely to tested compounds; larvae were not treated with Mtz. YFP reporter signals were then quantified by microplate reader at 7 dpf. Each experiment was repeated at least twice, with tested groups containing

approximately 30 fish.

Rod photoreceptor regeneration assay

To determine whether compounds stimulated retinal regeneration, both non-transgenic and transgenic rho:YFP-NTR larvae were treated with PTU at 16 hpf. At 5 dpf, larvae were incubated with either lOmM Mtz/E3/TU or DMSO/E3/PTU for 24 hours. At 6 dpf, larvae were then placed in new DMSO/E3/PTU media containing test compounds (or DMSO alone) for three days. YFP signal intensity was measured by TECAN microplate reader at 9 dpf. Each experiment was repeated at least twice, with tested groups containing approximately 30 fish.

Cell death detection

Mtz-treated of DMSO only (control) rho:YFP-NTR larvae were collected and fixed in 4% paraformaldehyde (PFA) at 4^°C overnight. Larvae were washed five times with PBS the following day and then infiltrated with 30% sucrose overnight. Larvae were then embedded in OCT media (Fisher Scientific), cut into 10 pm sections using a Leica cryostat (CM 3050S), and placed on histological slides. TUNEL staining was performed according to the manufacture’s protocols (Roche, 12156792910). Briefly, sections were washed three times with lx PBS, followed by permeabilization with lx PBST (1 x PBS, 0.1% triton, 0.1% sodium citrate) on ice for two minutes. Slides were washed with lx PBS and incubated with manufacture’s“labeling solution" containing terminal deoxynucleotidyl transferase at 37^°C for 1 hour. Sections were then washed with lx PBS and mounted with VECTASHIELD antifade mounting medium with DAPI (Vector H-1200).

In vitro assay for the protection of mouse photoreceptors from exogenous stressors.

All mouse experimental procedures were approved by the Johns Hopkins ACUC. Primary mouse retinal cells were isolated and prepared for culture as previously described (Fuller et al, 2014). Briefly, murine retinas from the QRX-IRES-EGFP strain (Wang et al, 2004) were isolated at postnatal day four (P4). Retinal tissue was dissociated into a single cell suspension by incubating whole tissue in activated papain in Hibemate-E without Calcium (BrainBits) for 15 min at 37^°C . Cells were resuspended in culture media (Neurobasal, 2% B- 27, 0.5 mM L-Glutamine and lx final Penicillin/streptomycin; all Life Technologies) and seeded onto poly-D-lysine coated 384 well tissue culture plates. Tunicamycin and thapsigargin were used as stressor compounds (Oslowski and Urano, 20l l)(Zhang et al, 20l4)(Nakamura et al, 20l3)(Li et al, 2009)(Fan et al, 2017). Stressors as well as test compounds were added at the time of seeding. After 48 hours, cells are stained with Hoescht and Ethidium homodimers; nine field images were acquired via an automated imager (Cellomics Vti; ThermoFisher) using a 20x objective. Photoreceptor number and percent of retinal population per well were determined by quantifying the number of live Hoechst- stained, GFP-expressing cells using a custom algorithm (Neuronal Profiling software package; ThermoFisher).

Retinal explant assay using rdl mouse

The following procedures were approved by the Medical University of South Carolina (MUSC) IACUC. Mice were generated from retinal degeneration 1 ( Pde6brdl , hereafter rdl) breeding pairs (Dr. Deborah Farber; UCLA) and housed in the MUSC Animal Care Facility under a 12: 12 hour, lightdark cycle, with access to food and water ad libitum. All chemicals used for organ cultures were tissue culture grade and purchased from Invitrogen (Carlsbad, CA). Cultures of retina with attached retinal pigment epithelium (RPE) were grown according to published protocols (Rohrer and Ogilvie, 2003)(0gilvie et al, l999)(Pinz0n-Duarte et al, 2000) with modifications (Bandyopadhyay and Rohrer, 2010). Post-natal day 10 pups were deeply anesthetized by hypothermia and decapitated. Heads were rinsed in 70% ethanol; the eyeballs were dissected out and placed in ice-cold Hanks balanced salt solution plus glucose (6.5 g/L). Eyes were then incubated in 1 mL of high glucose Hanks balanced salt solution containing 0.5mg/mL proteinase K at 37°C for 7 min. The eyeballs were then placed in Neurobasal medium (Life Technologies) plus 10% fetal calf serum to stop enzymatic activity. After removing the anterior chamber, lens and vitreous, the retina with attached RPE attached was dissected free from the choroid and sclera. Relaxing cuts were made to flatten the tissue and transferred to the upper compartment of a Costar Transwell chamber using a drop of Neurobasal medium, RPE layer faced-down. A drop of fluid was used to flatten-out the retina. Neurobasal media with B-27 supplement (Life Technologies) was placed in the lower compartment. The cultures were kept in an incubator (5% C02, balanced air, 100% humidity, 37°C) and the media in the lower compartment changed every two days under dim red light. No antimitotics nor antibiotics were required.

For each of the cohorts, 3-4 individual retina/RPE sandwiches were placed in culture on PN10. Test compounds were added to the culture media and refreshed every 48 hrs for 11 days. At completion, cultures were fixed in 4% PFA, cut into 14 micron sections, and stained with .HE staining (Bandy opadhyay and Rohrer, 2010). For each culture, ten rows of photoreceptors along the length of each culture were counted; the ten values were averaged to give a value for each retina, while the average of all retinas provided the mean ± SEM of each culture condition.

Data analysis and statistics

Under the R environment, the ARQiv data analysis package was used to calculate sample size, quality control strictly standardized mean difference (SSMD) and hit selection SSMD scores as previously described (White et al, 2016). The following results of each drug were derived: 1) a plot of signal: background at all tested concentrations, 2) at table of SSMD hit selection scores, and 3) a signal intensity heat map of each drug plate. (96-well plate view).

To combine the data from different experiments, data normalization was conducted by multiplying a factor f, where f = (Si -Aneg)/( APOS -Aneg). Si is the signal of i^th reading, APOS is mean of positive controls, and A\_Cg is mean of negative controls. To perform two groups of data comparison, Student t tests were performed. Effect size, 95% confidence interval and p values were calculated.

Results

Inducible zebraflsh model of RP & Large-scale chemical screening platform, ARQiv A robotics-automated screening platform, ARQiv (Automated Reporter Quantification in vivo,· (Walker et al, 2012), was developed to enable large-scale whole-organism drug discovery (Wang et al, 2015) (White et al, 2016). Here, ARQiv was combined with an inducible zebrafish model of RP to identify neuroprotective compounds promoting rod photoreceptor survival. A transgenic line, Tg(rho:YFP-Eco.NfsB)gmc500 (hereafter, rho:YFP-NTR ), facilitating prodrug-inducible rod photoreceptor loss (Walker et al, 2012). In these fish, a 3.7kb rhodopsin (rho) promoter fragment (Hamaoka et al, 2002) drives expression of yellow fluorescent protein (YFP) and a bacterial nitroreductase (NTR, from the E. coli NfsB gene) exclusively in rod photoreceptor cells was created. When rho: YFP-NTR larvae are exposed to the prodrug metronidazole (Mtz), NTR reduces Mtz to a cellular toxin resulting in selective ablation of rod photoreceptors (Curado et al, 2007)(White and Mumm, 2013) mimicking the pathological onset of RP. We reasoned that combining rho: YFP-NTR fish with ARQiv, would facilitate a large-scale screen for compounds that sustain YFP reporter expression in the presence of Mtz; i.e., increase the survival of rod photoreceptor challenged by progressively toxic levels of DNA damage.

HTS-ready zebrafish assay for identifying neuroprotective compounds

To establish an HTS-ready assay with the rho: YFP-NTR line, we first determined optimal conditions for inducing loss of rod photoreceptors while maintaining larval health in a 96- well microtiter format. In unperturbed rho: YFP-NTR larvae, YFP expression was initially observed in a ventral patch of the retina at 3 dpf; this patch expanded to all regions by 4 dpf, with bright whole eye expression evident at 5 dpf. These results are consistent with developmental expression of rho (Raymond et al, 1995). Based on this expression pattern, and because retinal development is largely complete by day 5, rod photoreceptor ablation was initiated at 5 dpf. ARQiv could be used to quantify loss and regeneration kinetics of rod photoreceptors following a 24 hr exposure of lOmM Mtz from 5-6 dpf (Walker et al, 2012). However, confocal intravital imaging suggested maximal loss of YFP occurred two days after initiation of Mtz treatments (Walker et al, 2012). Consequently, a 48 hr Mtz exposure regimen would provide maximal YFP (rod photoreceptor) loss, thereby providing the broadest signal window to detect neuroprotective effects. Concluding the experiment at 7 dpf also avoids complications associated with exogenous feeding (see Walker et al. , 2012) as zebrafish can subsist on their yolk sac up to day 7 (Jardine and Litvak, 2003). However, lOmM Mtz treatments extending beyond 24 hrs are associated with general toxicity (Mathias et al, 2014). Alternatively, removing Mtz from microtiter wells after a 24 hr exposure would significantly complicate the screening process. Therefore, we first established a 48 hr Mtz treatment regimen which was sufficient for inducing maximal rod photoreceptor loss but showed no evidence of toxicity or deleterious morphological effects. Five concentrations were tested, from lOmM to 625 mM using a 2-fold dilution series, with treatments starting at 5 dpf. ARQiv was used to quantify changes in YFP reporter signal daily from 5-8 dpf. The data showed a concentration-dependent reduction in YFP reporter signal for all treatment regimens. Maximal YFP loss was observed at 7 dpf with 10, 5, and 2.5mM Mtz exposures (Figure 1 A). The 2.5mM Mtz treatment, in particular, displayed a linear decrease in YFP levels from 5 to 7 dpf, consistent with gradual rod photoreceptor loss (Figure 1A). Finally, no evidence of toxicity or deleterious effects on morphology were observed for 48 hr Mtz treatments of 2.5 mM or less. Accordingly, 2.5mM Mtz was selected as the treatment regimen for our large-scale primary screen as these results suggested ample opportunity for observing neuroprotective effects, due to progressive loss of YFP loss over two days, while eliminating toxicity issues associated with prolonged Mtz exposures at high concentrations.

To calculate sample size and evaluate assay quality, two 96-well plates of larvae were treated with 2.5mM Mtz (in 0.1% DMSO) or 0.1% DMSO (non-ablated control) at 5 dpf. ARQiv was then used to quantify YFP levels at 7 dpf. At a statistical power of 95% (type II-b error) and a significance level of .05 (type I-a error), it was determined that a sample size of nine larvae was sufficient to detect a 50% effect size between ablated and non-ablated controls. For ease of dispensing, microtiter formatting results, and to account for dispensing errors, the sample size was increased to 16 larvae per condition during the primary screen. Furthermore, the strictly standardized mean difference (SSMD) quality control (QC) score was calculated as 1.67, indicating the screen was of sufficient quality to justify a large-scale screening effort (Zhang, 2011).

To establish a positive control treatment, against which the performance of test compounds could be compared during the primary screen, 27 compounds and 1 compound ‘cocktail’ previously implicated in retinal neuroprotection (Fig. 13) for the ability to sustain YFP expression in Mtz-treated rho:YFP-NTR larvae were evaluated. Only XL880, a compound known to protect isolated mouse photoreceptors in cell culture following exposure to chemical stressors (Berlinicke et al, in preparation), was found to promote sustained YFP expression in a concentration-dependent manner. However, XL880 also affected general morphology (e.g., causing edema around the eye) thus making it unclear whether sustained YFP levels resulted from rescued rod photoreceptors or changes in detection of the reporter associated with edema. Nevertheless, as no other previously implicated neuroprotectant proved effective in our model, XL880 was included as a positive control for the detection of increased YFP signals in the primary screen.

Primary ARQiv-based screen

As per the inaugural large-scale ARQiv drug discovery effort (Wang et al, 2015), a compound library was screened comprised largely of human-approved drugs, the Johns Hopkins Drug Library (JHDL; (Shim and Liu, 2014). Consistent with a potential endpoint of repurposing existing drugs for a new indication, a high priority was placed on minimizing false discovery, in particular false-negatives rates. Accordingly, all compounds were tested at six concentrations (from 4mM to 625nM, across a two-fold dilution series), i.e., using quantitative HTS principals (qHTS; (Inglese et al, 2006a)). The screen largely followed published methodologies (Wang et al, 20l5)(White et al, 2016) with assay-specific details provided in Figure 1B (assay stages 1-8). In all, 2934 compounds were screened and

-350,000 zebrafish larvae evaluated. Real-time data analysis was performed as previously detailed (White et al, 2016) to generate: 1) YFP signal level plot, and 2) plot of SSMD scores across all tested concentrations, 3) a signal intensity heat map of each plate and 4) a SSMD score table (Fig. 1B, stage 7). Treatments resulting in a SSMD score > 1 were flagged for visual inspection to assess fluorescence and general morphology using a stereo fluorescence microscope. This step facilitated elimination of false-positive compounds producing autofluorescence in the YFP range due to either larval toxicity, larval‘staining’, or from the compound itself (Fig. 1B, stage 8). It also allowed visual confirmation of sustained YFP expression within the retina, indicative of a true hit compound. At the conclusion of the primary screen, 114 compounds (3.9% hit rate) were identified as potential hit compounds (Figure 11). Hits were classified according to the highest SSMD score achieved across all concentrations, and whether concentration-dependent effects were observed. Only a single drug was classified as having a‘strong effect’ (3>SSMD>2), four compounds produced ‘fairly strong’ (2>SSMD>l.645), 20 showed moderate (l.645>SSMD>l.28), and 89 were fairly moderate (l.28>SSMD>l) effects (Figure 11). Forty two drugs showed concentration- dependent responses, while 72 did not.

Validation Assay I: Confirmation

The results suggested that inflammation play key roles in regulating degenerative and regenerative processes in the retina (White et al, 2017)(Holly field et al, 2008)(Yoshida et al, 2013). Accordingly, in addition to prioritizing hit compounds based on SSMD score and evidence of concentration-dependent effects, several compounds known to inhibit inflammatory signaling (e.g., production of reactive oxygen species) were chosen. In addition, several compounds that did not produce concentration-dependent effects were selected to test whether this criterion was useful in predicting reproducibility. In all, 42 compounds were selected for confirmation assays, i.e., re-testing performance in the primary screen. All prioritized compounds were obtained from new sources to confirm authenticity; previously, we had noted differences in effective concentrations when we obtained compounds from new sources during validation tests (Wang et al., 2015). Accordingly, for secondary confirmation tests, compounds were assayed over a wider concentration range, a total of eight concentrations across a 5 -fold dilution series, to account for potential differences in reagent quality. Most compounds were evaluated from IOOmM to l.28nM. However, if toxicity was observed at higher concentrations, the dilution series was initiated at lOuM. Using this strategy, 11 out of 42 prioritized compounds (26.2%) were confirmed as hits (Figure 2). Visual analysis using fluorescence microscopy confirmed that preserved YFP signals were observed in the retina versus other parts of the body. Effect sizes for the 11 confirmed hit compounds ranged from 10-38% (Fig. 2). We next examined correlations between SSMD scores and concentration-dependent effects with confirmation rates. Among 19 selected drugs with high SSMD scores (SSMD>l.28), seven (36.8%) were confirmed; among 23 drugs with lower SSMD scores (l.28>SSMD>l), 4 (17.4%) were confirmed. Of 27 drugs with a concentration-dependent trend in the primary screen, 8 (29.6%) were confirmed. However, 3 of 15 drugs (20%) without a concentration-dependent trend in the primary screen were also confirmed. These results suggest that SSMD scores provide the highest predictive value; compounds with higher SSMD scores were two times more likely to be confirmed as hits. Concentration-dependent effects associated with a smaller predictive effect, about 1.5 times more likely to be confirmed.

Validation Assay II: Confocal intravital microscopy

To verify neuroprotective effects of confirmed hit compounds on rod photoreceptor cell preservation, we used intravital confocal microscopy. Rod photoreceptor cells in the non- ablated retina (positive control) and surviving rod photoreceptors in Mtz-ablated retina (negative control) displayed a typical elongated morphology (Fig. 3). As predicted, Mtz- ablated retinas exhibited reduced YFP signal in both the dorsal and ventral domains. In 11 drug treated retinas, varying degrees of preservation of YFP-expressing rod photoreceptor cells was observed (Fig. 3). Rescued cells often displayed typical rod photoreceptor morphologies, i.e. resembled healthy rod photoreceptors (Fig. 3; e.g., cortexolone,“Cor” treatment). However, some remaining cells appeared rounded, suggesting that the process of degeneration was not fully inhibited (Fig. 3; e.g., miconazole,“Mic” treatment).

Validation Assay III: NTR inhibition

As rod photoreceptor loss is dependent on a prodrug converting enzyme (NTR) in our RP model, it is possible that some hit compounds act by suppressing NTR activity directly rather than protect cells from DNA damage-induced cell death. To address this issue, NTR prodrug conversion kinetics assays were performed in the presence of each hit compound in vitro using the prodrug CB1954 (Prosser et al, 2010). To ensure any potential for NTR inhibition was accounted for, all compounds were tested at 300 mM (~l 00-fold greater than effective concentrations, on average). A compound was deemed inhibitory if the percentage of NTR- mediated CB1954 reduction was less than 75% of the activity of NTR alone. Seven of eleven confirmed hit compounds showed no NTR inhibitory effects (Supplementary Figure 1). Four compounds (warfarin, ciclopirox olamine, calcimycin and sulindac) showed evidence of NTR inhibition at 300 mM. These drugs showed similar results in NTR/Mtz reduction assays at 300 mM. Subsequent ICso assays determined inhibitory activities ranging from 150 mM (for ciclopirox olamine) to 350 mM (for sulindac). However, effective concentrations in fish were substantially lower (ranging from 0.4 to 20 mM, see Fig. 2), diminishing the possibility of direct inhibitory effects on NTR in neuroprotective assays. Therefore, we analyzed all hit compounds in additional NTR-independent RP models to further confirm neuroprotective effects (see Mouse model validation assays, below).

Validation Assay IV: Rod photoreceptor development

To investigate possible effects in promoting rod photoreceptor development, rho:YFP-NTR larvae were exposed to hit drugs alone (i.e. no Mtz induced cell ablation) from 5 to 7 dpf and YFP levels evaluated by ARQiv. Retinoic acid (RA, 1.25 mM) was used as a positive control. RA treated fish displayed significantly increased YFP signals (Fig. 10). In contrast, none of the eleven hit compound treated retinas exhibited increased YFP expression compared to untreated controls, suggesting that the compounds do not promote rod photoreceptor cell fate (Fig. 10). Interestingly, three hit compounds produced reproducibly lower YFP signals than controls (Clo, Cic, and Cor; p-value <0.05), suggesting negative effects on rod photoreceptor development.

Validation Assay V: Regeneration

It is well known that the zebrafish retina regenerates after injury and cell loss (Wan and Goldman, 2016). To determine whether hit compounds act by stimulating retinal

regeneration, we used a previously described ARQiv assay designed to detect changes in regeneration kinetics (White et al, 2017). Briefly, rho:YFP-NTR larvae were first treated with 10 mM Mtz at 5 dpf for 24 hrs to induce rapid rod-photoreceptor specific cell death (Walker et al, 2012). At 6 dpf, Mtz was washed out and larvae were treated with hit compounds at concentrations corresponding to maximal neuroprotective effects. YFP expression levels were then quantified by ARQiv at 9 dpf. The results showed that none of the compounds increased YFP levels in comparison to Mtz alone controls (Fig. 11). In fact, two compounds actually inhibited regeneration (Clo and Cic). These results indicate that the identified hit compounds do not increase YFP expression in larvae by inducing retinal regeneration.

Validation Assay VI: Programmed cell death

To determine whether hit compounds inhibit rod-photoreceptor cell death, rho:YFP-NTR larvae were pre-treated with hit compounds 4 hrs prior to 2.5 mM Mtz, as per the primary screen, and collected at 6 dpf, one day after initiation of Mtz treatment. Larvae were then sectioned and analyzed by TUNEL staining (Fig 4). There were significantly more TUNEL+ cells in Mtz ablated retina (7.3 ±4.0) versus DMSO controls (0.6 ±0.7; /?=0.00l9). TUNEL± cells were restricted to the ONL, consistent with diminished YFP signal observed in rho:YFP-NTR treated with Mtz (Fig. 4). The addition of hit drugs, such as

dihydroartemisinin, significantly decreased the number of dying cells (1.7 ±2.0, p=0.0055), correlating well with preserved YFP signals seen in previous experiments (Figs. 2 and 3). Combinatorial assay

PubChem (https://puhchem.ncbi.nim.mh.gov ') searches indicated that many of these compounds affected multiple signaling pathways involved in cell death protection

(Supplementary Fig. 3). An assay was designed to test whether hit compounds acted via complementary mechanisms and may produce additive effects. Seven hits were paired using optimal effective concentrations, a total of 21 paired conditions, and tested for additive effects using the confirmation assay protocol. Ten of 17 viable pairs exhibited additive effects (Fig. 5, all green boxes). Moreover, eight pairs produced better than additive effects (Fig. 5, dark green boxes). These results suggest that multiple signaling pathways might be involved in photoreceptor degeneration and that combinatorial therapeutic strategies may provide improved outcomes for RP patients.

Mouse Model Validation I: retinal cell cultures treated with stressor compounds

The overall goal of these studies is to identify potential new therapeutics for RP patients. We reasoned that compounds showing neuroprotective effects in both fish and mammalian models would increase the likelihood of translating to human RP patients. Therefore, we next sought to determine hit compound efficacy in mouse cell culture models of retinal degeneration.

Retinal cells were isolated from P4 QRX-IRES-EGFP mice and grown in cell culture as previously described (Wang et al, 2004). To induce retinal cell death, tunicamycin or thapsigargin were applied. These“stressor” compounds are known to induce endoplasmic reticulum (ER) stress (Oslowski and Urano, 20l l)(Zhang et al., 2014) and the unfolded protein response (UPR), respectively; two pathways implicated in the etiology of RP. Hit compounds were added to the culture media to test for the capacity to protect murine retinal cells from stress-induced cell death. Five concentrations (4, 2, 1, 0.5, 0.25 mM) of each drug were tested in primary retinal cell cultures, and seven concentrations (3-fold dilute down from 30 to 0.04 pM) of each drug were evaluated in photoreceptor cultures. After two days in culture, the number of remaining“retinal cells” (all cells) and photoreceptors (GFP± cells) was assessed using the live/dead kit. Compounds were considered effective if the mean number of surviving cells was two standard deviations greater than the control (non-treated) mean. Four hit compounds (warfarin, ciclopirox olamine, pyrithione zinc and dihydroartemisinin) showed protective effects in either whole retinal cell or photoreceptor alone cultures under ER stress (Fig. 8). In addition, both ciclopirox olamine and pyrithione zinc protected photoreceptors from ER stress, with pyrithione zinc also promoting photoreceptor survival after induction of UPR (Fig. 8). Many compounds showing efficacy in in vivo fish models of RP failed to show efficacy in isolated retinal cell cultures.

Mouse model validation II: using rdl mouse retinal explant

The final validation studies examined the effects of hit drugs in mouse retinal explants isolated from retinal degeneration 1 {rdl) mice. This mutant mouse line exhibits early onset retinal degeneration caused by a mutation in Pde6b gene (Chang et al., 2002); the

orthologous human gen & PDE6B also leads to RP in humans (McLaughlin et al, l993)(Gal et al, l994)(Bayes et al, l995)(Hmani-Aifa et al, 2009). In these mice, photoreceptor degeneration begins around P10 and by P21, only a few rows of photoreceptor cells remain in the ONL (LaVail and Sidman, 1974). For these experiments, retinal explants from P10 rdl mice were isolated and cultured ex vivo as previously described (Bandy opadhyay and Rohrer, 2010). In the absence of exogenous factors, photoreceptor degeneration proceeds rapidly under these conditions. Six hit drugs were selected to test for neuroprotective effects in this model system. All were evaluated at three concentrations across a 5-fold dilution series centered on the most effective concentration determined in fish RP models. After 11 days in culture, explants were fixed and retinal cells stained with 0.1% toluidine blue. Ten counts of rows of photoreceptors in the center of each explant were performed; the average count is the mean of each retina; and the average of all retinas provided the mean ± SEM per culture condition. Neuroprotective effects were defined as a concentration-dependent increase in the number of photoreceptor rows remaining in the ONL relative to untreated controls that achieved a p-v alue of <0.05 (Fig. 6). An average of 1.2+0.19 rows of photoreceptor cell bodies remained in the ONL of non-treated control explants cultured for 11 days. Four of six drugs tested, warfarin, ciclopirox olamine, dihydroartemisinin and artemisinin increased the number of surviving photoreceptor layers, suggesting cross-species conservation of neuroprotective effects (Figure 5; note that high concentrations of ciclopirox olamine, 15 mM, lead to disruption of retinal histology due to proliferation in the INL and ONL).

In summary, we demonstrate that out of 2,934 compounds tested, dihydroartemisinin, ciclopirox olamine, and artemisinin exhibited neuroprotective effects in both zebrafish and mouse RP models of retinal degeneration. All drugs were tested in at least two animal models of RP. Lead candidates promoted the survival of rod photoreceptor cells in both fish and mouse models of RP. The former is a model in which rod photoreceptor cell death is inducible upon exposing transgenic fish to the prodrag substrate, metronidazole. The latter involved isolated photoreceptor cell cultures (collaboration with Dr. Donald Zack, JHU) and retinal explant cultures from retinal degeneration 1 (rdl) mouse models of RP (collaboration with Dr. Barbel Rohrer, MU SC). The list of 4 current lead drug candidates includes:

1) Warfarin - SMILES: CC(=0)CC(C1=CC=CC=C1)C2=C(C3=CC=CC=C30C2=0)0

2) Ciclopirox - SMILES: CC1=CC(=0)N(C(=C1)C2CCCCC2)0

3) Dihydroartemisinin - SMILES: CC1CCC2C(C(0C3C24C1CCC(03)(004)C)0)C

4) Artemisinin - SMILES: CC1CCC2C(C(=0)0C3C24C1CCC(03)(004)C)C

Discussion:

In this study, a large-scale in vivo drug screen involving the ARQiv platform and a zebrafish model of RP was performed to identify neuroprotectants capable of protecting rod photoreceptors from DNA damage-induced cell death. A total of 2,984 compounds from the JHDL was screened in more than 350,000 transgenic zebrafish larvae using qHTS principals to identify 119 potential neuroprotective compounds. A series of follow-on confirmatory and orthogonal assays identified 42 prioritized hits and validated 11 compounds as lead drug candidates. This validation rate (26%) is substantially lower than our previous large-scale screen of the JHDL (62%) focused on identifying compounds that increased pancreatic beta cell mass (Wang et al, 2015). The reasons for this are unknown, however, SSMD scores were somewhat compressed here compared to the prior screen; high SSMD scores correlate well with successful validation. Limiting both screens to the highest 25 SSMD scoring compounds results in more comparable validation rates (62% in (Wang et al, 2015); 37% here). Subsequent cross-species assays utilizing in vitro and ex vivo mouse model systems confirmed three of the eleven lead drug candidates as potential neuroprotectants.

Interestingly, validation rates in the rdl retinal explant model were higher (50%, 3 of 6 tested) than isolated retinal cell cultures (17%, 4 of 24 evaluated for survival of all retinal cells and 25%, 2 of 8, evaluated specifically for photoreceptor survival). These results suggest that neuroprotective mechanisms of action underlying the effects of the lead drug candidates may require an intact retinal model system; i.e., act via indirect cross-talk between retinal cell types rather than directly on damaged photoreceptor cells. Alternatively, the zebrafish inducible RP model and rdl retinal explants may share a common mechanism, or mechanisms, of cell death that differs from the stressor-induced mechanisms used for in vitro assays. The type of cell death incurred by the NTR/prodrug cell ablation system is not well characterized. Initial reports suggested this system caused a p53-dependent apoptotic cell death(Iyengar et al, 20l5)(Pisharath et al, 2007). However, use of methods such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) may not be appropriate as the cytotoxins produced upon prodrug reduction by NTR cause DNA cross-linking and subsequent fragmentation (Grasl-Kraupp et al. 1995). Thus, TUNEL labeling may represent a false-positive marker for apoptosis in this case. Others have associated activation of caspase 3 with NTR/prodrug-induced cell death (Chen et al, 2011).

Combinatorial assays demonstrated additive effects of -50% tested pairs, indicating that complementary signaling pathways regulate cell death during retinal degeneration.

Furthermore, the neuroprotective effect of three lead candidates (dihydroartemisinin, artemisinin and ciclopirox olamine) were verified in mouse retinal degeneration models, underscoring the evolutionary conservation of the affected signaling mechanisms in terms of regulating retinal cell death.

Understanding how photoreceptors die during RP has been informed by recent discoveries regarding cell death signaling. Numerous mutations have been identified which could lead to rod photoreceptor cell death followed by the secondary cone death through different signaling pathways or combined signaling (Sancho-Pelluz et al, 2008)(Arango- Gonzalez et al, 2014). In addition, At least three major cell death pathways have been linked to photoreceptor cell death observed in inherited RP— necroptosis, apoptosis and parthanotos (Sancho-Pelluz et al, 2008) (Sato et al, 2013) (Yang et al, 2017) (Arango-Gonzalez et al, 2014). Necroptosis was thought to mainly contribute to secondary cone cell death (Murakami et al, 2012) (Murakami et al, 2015) while apoptosis was thought to be the mechanism for primary rod photoreceptor cell death observed during RP (Chang et al, 1993)(Portera- Cailliau et al, l994)(Doonan et al, 2003)(Zeiss et al, 2004). However, attempts to block rod photoreceptor apoptosis in rod/cone photoreceptor degeneration mouse models were not successful (Hamann et al, 2009)(Yoshizawa et al, 2002).

Many of the early experiments which identified apoptosis as the cell death pathway in photoreceptors relied on TUNEL staining. Later reports determined that TUNEL staining did not always distinguish apoptosis from other types of cell death, such as necrosis (Grasl- Kraupp et al, 1995). Later studies examining multiple apoptosis-related markers showed that apoptotic cell death only occurred in a minority of RP models; the majority occurred through non-apoptosis pathway (Sancho-Pelluz et al, 2008)(Arango-Gonzalez et al, 2014).

Furthermore, a common pathway appears to be active in RP mouse models deficient for pde6a pde6b, rho,prph2, cpfll, cngbl, cngb3, rpe65 (Jiao et al, 20l6),(Paquet-Durand et al, 2007). In these models, retinal cell death is characterized by the excessive activation of histone deacetylase (HD AC), poly-ADP-ribose-polymerase (PARP), calpain and the abnormal accumulation of cyclic guanosine monophosphate (cGMP) and poly-ADP-ribose (PAR). The application of PARP inhibitors significantly protect the photoreceptor cells from cell death in rdl model (Paquet-Durand et al, 2007)(Sahaboglu et al, 2016). Moreover, photoreceptor cell death in the degenerating retina of PARP 1 knockouts were significantly reduced (Sahaboglu et al, 2010). In our study, Pubchem searches identified the majority of our hits as Tyrosyl-DNA phosphodiesterase 1 (Tdpl) inhibitors; Tdpl is a DNA repair enzyme that helps repair topoisomerase I-induced damage. Tdpl acts in conjunction with PARP1 to repair DNA damage in mice, so therefore, Tdpl inhibitors may inhibit PARP as well (Murai et al, 2014). Thus, it is thought the neuroprotective compounds identified inhibit PARP mediated cell death during RP.

It is possible that multiple cell death mechanisms are active during RP. For example, in S334ter Rho transgenic rats, changes in signaling activity was observed for markers of apoptosis (BAX, crytochrome c, caspase-9, cleaved caspase-3) and non-apoptosis pathways (cGMP, HDAC, PARP and PAR)(Arango-Gonzalez et al, 2014). Additionally, PARP activation has been observed in four different Pde6a mouse mutants which also exhibit strong TUNEL staining (Jiao et al, 2016). Interestingly, when all four models were treated with PJ34, a known PARP inhibitor that suppresses photoreceptor degeneration in Pde6a^rdl mice, while all showed significant decreases in cell death, the level of photoreceptor survival varied in each mutant model (Jiao et al, 2016). Specifically, a greater effect was seen in mutants which serve as slow death models versus rapidly degenerating models, which also demonstrated strong TUNEL staining. The combination of strong TUNEL staining and PARP activation may indicate that multiple cell death pathways are active during rapid RP degeneration models. As such, combinatorial treatment approach may provide the best therapeutic outcome. The results indicate an additive effect for seven compound pairs. One caveat of this approach is that while combining compounds may increase treatment efficacy, drug concentration may need to be decreased to avoid the systemic side effects.

The large-scale whole-organism drug screening using a zebrafish RP model and ARQiv platform successfully identified three novel rod photoreceptor neuroprotective compounds that were effective in mouse retinal cell death models. These drugs appear to be reducing PARP mediated cell death in retinal photoreceptors. Combinatorial assays suggested that therapies targeting complementary neuroprotective signaling pathways provide an improved strategy for RP treatment. Embodiments of the disclosure concern methods and/or compositions for treating and/or preventing Retinitis pigmentosa (RP). In certain embodiments, individuals with Retinitis pigmentosa (RP) are administered with one or more compounds of Figs. 7 and 13.

An individual known to have RP, suspected of having RP, or at risk for having RP may be provided an effective amount of one or more compounds of Figs. 7 and 13. Those at risk for RP may be those individuals having one or more genetic factors, may be of advancing age, and/or may have a family history, for example.

In particular embodiments of the disclosure, an individual is given an agent for RP therapy in addition to the one or more compounds of Figs. 7 and 13. When combination therapy is employed with one or more compounds of Figs. 7 and 13, the additional therapy may be given prior to, at the same time as, and/or subsequent to the one or more compounds of Figs. 7 and 13.

Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise an effective amount of one or more the one or more compounds of Figs. 7 and 13, dissolved or dispersed in a

pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that comprises at least one or more compounds of Figs. 7 and 13 or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21 ^st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards. As used herein,

"pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated. The one or more compounds of Figs. 7 and 13 may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present compositions can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

The one or more compounds of Figs. 7 and 13 may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as

isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.

Further in accordance with the present disclosure, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.

In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of a

pharmaceutical lipid vehicle compositions that include the one or more compounds of Figs. 7 and 13, and an aqueous solvent. As used herein, the term“lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester- linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the one or more compounds of Figs. 7 and 13 may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes. The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.

Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

In other non-limiting examples, a dose may also comprise from about 1

microgram/kg/body weight, about 5 microgram/kg/body weight, about 10

microgram/kg/body weight, about 50 microgram/kg/body weight, about 100

microgram/kg/body weight, about 200 microgram/kg/body weight, about 350

microgram/kg/body weight, about 500 microgram/kg/body weight, about 1

milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500

milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

Alimentary Compositions and Formulations

In one embodiment of the present disclosure, the one or more compounds of Figs. 7 and 13 are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al, 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, com starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

For oral administration the compositions of the present disclosure may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically - effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively, the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

Additional formulations that are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%. Parenteral Compositions and Formulations

In further embodiments, inducer of expression of PGC-l□ may be administered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety). Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" l5th Edition, pages 1035-1038 and 1570- 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.

Miscellaneous Pharmaceutical Compositions and Formulations In other preferred embodiments of the invention, the one or more compounds of Figs.

7 and 13 may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin.

Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins

(Takenaga et al, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a

polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject’s age, weight and the severity and response of the symptoms.

Kits of the Disclosure

Any of the compositions described herein may be comprised in a kit. In a non- limiting example, the one or more compounds of Figs. 7 and 13 may be comprised in a kit. The kits may comprise a suitably aliquoted the one or more compounds of Figs. 7 and 13 and, in some cases, one or more additional agents. The component(s) of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed.

However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the one or more compounds of Figs. 7 and 13 and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The one or more compounds of Figs. 7 and 13 composition(s) may be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

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All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms“a” and“an” and“the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms“comprising,”“having,”“including,” and “containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims:

1. A method of using one or more compounds of Figs. 7 and 13 for the treatment or prevention of retinitis pigmentosa of a subject comprising the steps of:

administering to a subject with retinitis pigmentosa or prone to getting retinitis pigmentosa one or more compounds of Figs. 7 and 13; and

treating or preventing the retinitis pigmentosa of the subject.

2. The method of claim 1 wherein the one or more compounds are selected from the group consisting of CC(=0)CC(Cl=CC=CC=Cl)C2=C(C3=CC=CC=C30C2=0)0

(Warfarin); CCl=CC(=0)N(C(=Cl)C2CCCCC2)0 (Ciclopirox olamine);

CC 1 CCC2C(C(OC3C24C 1 CCC(03)(004)C)0) (Dihydroartemisinin);

CC 1 CCC2C(C(=0)0C3C24C 1 CCC(03)(004)C)C (Artemisinin); C 1 =CC(=S)N(C=C 1 )[0- ].Cl=CC(=S)N(C=Cl)[0-].[Zn+2] pyrithione zinc, or a combination thereof.

3. The method of claim 2 wherein the one or more compounds comprises

dhydroartemisinin, artemisinin, and ciclopirox olamine.

4. The method of claim 1 wherein the one or more compounds of Figs. 7 and 13 are administered to the eye of the subject.

5. The method of claim 1 wherein a pharmaceutical composition of the one or more compounds of Figs. 7 and 13 are administered to the subject.

6. A method of using one or more compounds of Figs.7 and 13 to promote photoreceptor and/or retinal cell survival:

administering one or more compounds of Figs. 7 and 13 to a photoreceptor, a retinal cell, or both having a disease or treated with an agent that causes death; and

promoting the survival of the photoreceptor, retinal cell, or both compared to a reference photoreceptor, a reference retinal cell, or both that have not been administered one or more compounds of Figs 7 and 13.

7. The method of claim 6 wherein the photoreceptor, the retinal cell, the reference photoreceptor, and the reference retinal cell has a disease prior to administering one or more compounds of Figs. 7 and 13.

8. The method of claim 6 wherein the photoreceptor, the retinal cell, or both is treated with the agent that causes photoreceptor cell death, retinal cell death, or both after the administering of one or more compounds of Figs. 7 and 13.

9. The method of claim 8 wherein the agent is tunicamycin, thapsigargin or a combination thereof.

10. The method of claim 6 wherein the one or more compounds are selected from the group consisting of CC(=0)CC(Cl=CC=CC=Cl)C2=C(C3=CC=CC=C30C2=0)0 (Warfarin); CCl=CC(=0)N(C(=Cl)C2CCCCC2)0 (Ciclopirox olamine);

CC 1 CCC2C(C(OC3C24C 1 CCC(03)(004)C)0) (Dihydroartemisinin);

CC 1 CCC2C(C(=0)0C3C24C 1 CCC(03)(004)C)C (Artemisinin); C 1 =CC(=S)N(C=C 1 )[0- ].Cl=CC(=S)N(C=Cl)[0-].[Zn+2] pyrithione zinc, or a combination thereof.

11. The method of claim 10 wherein the one or more compounds comprises dhydroartemisinin, artemisinin, and ciclopirox olamine.

12. A method of using one or more compounds of Figs. 7 and 13 to treat or prevent blindness in a subject comprising the steps of:

administering to a subject having a disease resulting in photoreceptor death, retinal cell death, or a combination thereof, one or more compounds of Figs. 7 and 13; and treating or preventing blindness of the subject compared to a reference subject that has not been administered the one or more compounds of Figs. 7 and 13.

13. The method of claim 12 wherein the one or more compounds are selected from the group consisting of CC(=0)CC(Cl=CC=CC=Cl)C2=C(C3=CC=CC=C30C2=0)0

(Warfarin); CCl=CC(=0)N(C(=Cl)C2CCCCC2)0 (Ciclopirox olamine);

CC 1 CCC2C(C(OC3C24C 1 CCC(03)(004)C)0) (Dihydroartemisinin);

CC 1 CCC2C(C(=0)0C3C24C 1 CCC(03)(004)C)C (Artemisinin); C 1 =CC(=S)N(C=C 1 )[0- ].Cl=CC(=S)N(C=Cl)[0-].[Zn+2] pyrithione zinc, or a combination thereof.

14. The method of claim 13 wherein the one or more compounds comprises dhydroartemisinin, artemisinin, and ciclopirox olamine.

15. The method of claim 12 wherein the one or more compounds of Figs. 7 and 13 are administered to the eye of the subject.

16. The method of claim 12 wherein a pharmaceutical composition of the one or more compounds of Figs. 7 and 13 are administered to the subject.