Retinal Neuroprotective Effects of Flibanserin, an FDA-Approved Dual Serotonin Receptor Agonist-Antagonist

Purpose To assess the neuroprotective effects of flibanserin (formerly BIMT-17), a dual 5-HT1A agonist and 5-HT2A antagonist, in a light-induced retinopathy model. Methods Albino BALB/c mice were injected intraperitoneally with either vehicle or increasing doses of flibanserin ranging from 0.75 to 15 mg/kg flibanserin. To assess 5-HT1A-mediated effects, BALB/c mice were injected with 10 mg/kg WAY 100635, a 5-HT1A antagonist, prior to 6 mg/kg flibanserin and 5-HT1A knockout mice were injected with 6 mg/kg flibanserin. Injections were administered once immediately prior to light exposure or over the course of five days. Light exposure lasted for one hour at an intensity of 10,000 lux. Retinal structure was assessed using spectral domain optical coherence tomography and retinal function was assessed using electroretinography. To investigate the mechanisms of flibanserin-mediated neuroprotection, gene expression, measured by RT-qPCR, was assessed following five days of daily 15 mg/kg flibanserin injections. Results A five-day treatment regimen of 3 to 15 mg/kg of flibanserin significantly preserved outer retinal structure and function in a dose-dependent manner. Additionally, a single-day treatment regimen of 6 to 15 mg/kg of flibanserin still provided significant protection. The action of flibanserin was hindered by the 5-HT1A antagonist, WAY 100635, and was not effective in 5-HT1A knockout mice. Creb, c-Jun, c-Fos, Bcl-2, Cast1, Nqo1, Sod1, and Cat were significantly increased in flibanserin-injected mice versus vehicle-injected mice. Conclusions Intraperitoneal delivery of flibanserin in a light-induced retinopathy mouse model provides retinal neuroprotection. Mechanistic data suggests that this effect is mediated through 5-HT1A receptors and that flibanserin augments the expression of genes capable of reducing mitochondrial dysfunction and oxidative stress. Since flibanserin is already FDA-approved for other indications, the potential to repurpose this drug for treating retinal degenerations merits further investigation.


Introduction
Inherited retinal dystrophies (IRDs), such as retinitis pigmentosa, are clinically and genetically diverse, and account for approximately 5% of the vision loss in the Western world. [1][2][3] There are no effective treatments for most of these disorders, but gene therapy shows promise for a small subset of IRDs. [4][5][6][7][8] However, with almost 180 genes and thousands of distinct mutations, developing gene replacement strategies for all IRD patients will be a long and difficult endeavor. [2] The development of gene-independent therapies is therefore essential in preserving retinal morphology and function until suitable gene therapies can be developed. Additionally, synergistic rescue may be achieved with neuroprotective agents in combination with gene or cell-based therapies.
Flibanserin, marketed as Addyi™ (Sprout Pharmaceuticals), was recently approved by the Food and Drug Administration (FDA) for the treatment of hypoactive sexual desire disorder (HSDD). [28][29][30][31][32][33] Seven phase III clinical trials demonstrated that a daily 100 mg dose of flibanserin was safe and resulted in an improvement of HSDD-related symptoms in pre-menopausal women. [30][31][32][33] In CHO cells, flibanserin has a high affinity for serotonergic 5-HT 1A and 5-HT 2A , as well as dopaminergic D 4 receptors, although D 4 receptor binding has yet to be verified in human brain tissue. [34][35][36][37][38][39] The determination of retinal protection and cell survival mechanisms conferred by neuroprotective agents, such as flibanserin, is aided by evaluation in models with widely-described cell death mechanisms. The relatively synchronous and rapid progression of apoptosis in lightinduced retinopathy has provided many insights into the mechanisms of cell death. [40] Therefore, to evaluate the neuroprotective potential of flibanserin and its mechanism of action, we tested it in the well-established light-induced retinopathy model. [20,[41][42][43][44][45]

Animals
All experiments and animal handling procedures were approved by and performed in compliance with the policies of the Institutional Animal Care and Use Committee at Oregon Health & Science University (IACUC Protocol Number IS03147) and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Albino BALB/cJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). 5-HT 1A -/mice (5-HT 1A knockout mice) were generously provided by Dr. René Hen (Columbia University, New York, NY). [46] 5-HT 1A -/mice were originally on a pure 129/Sv background, but backcrossed with albino BALB/c mice for nine generations (F9) before being used in experiments. Genotypes were confirmed using PCR. Mice utilized in these studies were males between two and three months old. Mice were housed in a 12-hour alternating light/dark cycle room. The light cycle (~15 lux) occurred from 9:00 PM to 9:00 AM, and dark cycle from 9:00 AM to 9:00 PM. Following data acquisition, all mice were euthanized via gradual CO 2 inhalation and cervical dislocation.

Five-Day Drug Time Course
Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. Mice were dosed with vehicle, 0.75, 1.5, 3.0, 6.0, 9.0 or 15 mg/kg flibanserin 48 hours, 24 hours and immediately prior to a one-hour bright light exposure, and 24 and 48 hours after light exposure. Naïve mice were not injected or exposed to bright light.

One-Day Drug Time Course
Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. A single dose of vehicle, 3, 6, 9 or 15 mg/kg of flibanserin was administered immediately before a one-hour bright light exposure in order to evaluate a shortened one-day treatment regimen. To assess 5-HT 1A -mediated effects, 10 mg/kg WAY 100635, a selective 5-HT 1A antagonist (K i = 2), [47] was administered to mice 30 minutes prior to 6 mg/kg flibanserin. Additionally, 5-HT 1A -/mice were also administered 6 mg/kg flibanserin immediately prior to bright light exposure. Because WAY 100635 is also a potent D 4 agonist (K i = 16), [47] 10 mg/kg WAY 100635 was injected alone prior to bright light exposure in order to assess whether a D 4 agonist, delivered at a concentration similar to that of 15 mg/kg flibanserin, could provide any neuroprotective effects against light-induced retinopathy. Naïve mice were not injected or exposed to bright light.

Light-Induced Retinopathy
Four compact fluorescent lamps (42 watts, 6500K) were installed in a custom-built light box producing 10,000 lux of uniform light, and capable of holding 16 mice. [20] Two hours into the 12-hour dark cycle, mice were placed into the light box and exposed to bright light for one hour. Temperature and humidity were regulated using a portable air conditioning unit. Mice were monitored throughout light exposure and were given access to water. Vehicleinjected control mice were used in each experiment to ensure the occurrence of light-induced retinopathy. [20] In Vivo OCT Imaging Retinas were imaged as previously reported. [20,48] Seven days following bright light exposure, mice were sedated using 1.5% isoflurane delivered via a nose cone, corneas were anesthetized with 0.5% proparacaine, and pupils were dilated using a combination of 1% tropicamide and 2.5% phenylephrine. Artificial tears were used to maintain corneal clarity. Mice were seated in a Bioptigen AIM-RAS holder and spectral domain optical coherence tomography (SD-OCT) images were obtained using an Envisu R2200-HR SD-OCT instrument (Bioptigen, Durham, NC). [20,48] Each eye was imaged using linear horizontal scans in the temporal and nasals quadrants and linear vertical scans in the superior and inferior quadrants.

Image Processing and Segmentation
SD-OCT scans were processed and segmented as previously reported. [20,48] Briefly, SD-OCT scans were converted into image files using ImageJ (version 1.48; National Institutes of Health, Bethesda, MD) and, using a custom designed SD-OCT segmentation program built in IGOR Pro (IGOR Pro 6.37; WaveMetrics Inc., Lake Oswego, OR), total retinal (TR) and receptor plus (REC+) thicknesses were measured. [20,48,49] The thickness from Bruch's membrane to the vitreous/retinal nerve fiber layer interface is defined as TR. REC+ is defined as the thickness from Bruch's membrane to the inner nuclear layer/outer plexiform layer interface. [20,48,49]

Electroretinograms (ERGs)
ERGs were performed as previously reported. [20,50,51] ERGs were recorded two to seven days after SD-OCT imaging. Dark-adapted mice were anesthetized with ketamine (100 mg/ kg)/xylazine (10 mg/kg), corneas were anesthetized, and pupils were dilated. Mice were placed on a heated platform (37°C) inside of a Ganzfeld dome coated with a highly reflective paint. Platinum signal electrodes were placed on the center of each cornea, and additional electrodes were placed in the forehead and tail as reference and ground, respectively. Finally, a lubricant, Goniovisc, Hypromellose 2.5% (Dynamic Diagnostics, Westland, MI), was applied to the corneas. ERG traces were averaged and analyzed using custom software. Light intensities ranged from -4.34 to 3.55 log cd•s/m 2 . [20,50,51] The number of trials at each light intensity was decreased as the intensity of the flashes increased in order to prevent light adaptation. The time between each flash was increased to allow for pigment regeneration. The minimum interstimulus interval was seven seconds, and the maximum was 70 seconds. Flashes were calibrated as previously described. [20,50,51]

RNA Extraction and RT-qPCR
Two hours into the 12-hour dark cycle, mice were injected intraperitoneally (10 mL/kg) under dim red light. Mice were dosed with vehicle or 15 mg/kg flibanserin 48 hours, 24 hours and immediately prior to a one-hour bright light exposure, and 24 and 48 hours after light exposure. Retinas from vehicle-injected mice and flibanserin-injected mice were harvested 48 hours and 72 hours after light exposure. Total RNA was extracted from retinas using an RNeasy Mini Kit (Qiagen, Hilden, Germany). One microgram of total RNA was converted to cDNA using a Bio-Rad iScript cDNA synthesis kit (Hercules, CA). Primers were designed with the Integrated DNA Technologies Realtime PCR Tool (S1 Table, Coralville, Iowa). PCR reaction mixtures consisted of 10 μL DyNAmo HS SYBR green (Thermo Scientific, Waltham, MA), 1 μL cDNA template, 1 μL of 200 μM gene-specific primer sets, and 8 μL distilled water. Using a Bio-Rad Chromo4 System (Hercules, CA), samples were initially denatured for 2 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and 58°C for 30 seconds. Gene expression was normalized to β-actin and relative gene expression in flibanserin-injected mice was compared to vehicle-injected mice using the ΔΔCt method.

Statistical Analysis
Average outer retinal thickness (REC+), total retinal thickness (TR), a-wave amplitudes and bwave amplitudes were calculated for each animal. Representative topographic distributions were an average of the REC+ thicknesses from the right eyes for all animals in each group, centered at the optic nerve. For scatterplot graphs, left and right eye data was combined for each animal and then averaged for each group. One-way analysis of variance (ANOVA) was used to compare REC+, TR and three different ERG measurements: b max,rod at -2.14 log cd•s/m 2 , and a max,rod+cone and b max,rod+cone at 3.55 log cd•s/m 2 using Prism (Prism 6.0; GraphPad Software Inc., La Jolla, CA). Multiple t-tests were performed to compare the expression of individual genes in flibanserin-and vehicle-injected mice at 48-and 72-hours after light exposure. For all analyses, a P value less than 0.05 was considered significant.

Flibanserin-Mediated Neuroprotection
Five-day time course. Bright light exposure resulted in thinning of the outer nuclear layer (ONL) and obscuration of the inner segment/outer segment junction (IS/OS) in retinas of vehicle-injected mice, and 0.75 and 1.5 mg/kg flibanserin-injected mice, as compared the normal morphology of naïve mice ( Fig 1A). Injections with 3, 6, 9, and 15 mg/kg of flibanserin not only preserved the ONL and IS/OS, but also revealed a dose-dependent preservation of ONL thickness (Fig 1A and 1B). Average REC+ thicknesses of 3, 6, 9, and 15 mg/kg flibanserin were significantly increased from vehicle-treated mice ( Table 1, Fig 1B), and administration of 15 mg/kg flibanserin provided maximum protection of retinal morphology, resulting in non-significant differences in REC+ thickness as compared to non-light damaged naïve mice.
ERGs demonstrated severe attenuation of retinal function in vehicle-injected mice versus naïve mice, as observed in the representative ERG waveforms and scotopic a-wave and b-wave amplitudes (Fig 2A and 2B, S1A Fig, Table 2). Intensity response curves and average a-and bwave amplitudes highlight the variable protection between treatment groups and a general dose-dependent effect (Fig 2B). 15 and 9 mg/kg flibanserin groups were not significantly different from the naïve group in b-wave responses (b max,rod , b max,rod+cone ), or a-wave responses (a max,rod+cone ) ( Fig 2B, S1A Fig, Table 2). The 1.5 and 0.75 mg/kg flibanserin groups failed to preserve retinal function entirely; ERG responses were not significantly increased from vehicleinjected mice. The 3 mg/kg and 6 mg/kg flibanserin-treated mice showed variable protection in ERG responses, but generally showed reduction of responses from naïve mice but improved from vehicle-treated mice.
One-day time course. In order to ascertain whether flibanserin has the ability to act quickly and effectively, a one-day time course was evaluated. A single dose of 3, 6, 9 or 15 mg/ kg flibanserin was injected immediately before light damage. Mice that received a 15, 9 or 6 mg/kg dose of flibanserin displayed complete morphological protection as assessed by OCT, and no statistical difference was found between their average REC+ thicknesses and those of naïve animals ( Fig 3A, Table 1). Interestingly, a single injection of 3 mg/kg flibanserin failed to protect retinal morphology, as REC+ thickness was not statistically different from the vehicleinjected mice.
In regard to retinal function, all responses (b max,rod , b max,rod+cone , and a max,rod+cone ) for the 6, 9 and 15 mg/kg flibanserin groups were statistically increased from the vehicle-treated group ( Fig 3B, S1B Fig, Table 2). A single 3 mg/kg flibanserin injection failed to protect retinal function, thus corresponding with OCT results.

Discussion
This research demonstrates the novel finding that flibanserin, a dual serotonin receptor agonist and antagonist recently approved by the United States Food and Drug Administration for female HSDD, can completely protect the structural and functional integrity of albino BALB/c mouse retinas from light-induced retinopathy. Flibanserin's neuroprotective effects began with a 3 mg/kg dose following a five-day time course and a 6 mg/kg dose following a one-day time course. A 15 mg/kg dose of flibanserin, following either time course, fully prevented the photoreceptor degeneration caused by bright light exposure. Filbanserin demonstrated neuroprotection in a dose-dependent manner, consistent with the known pharmacokinetics of the drug. Borsini et al. showed that in the cortex, the 20% minimum receptor occupancy required for 5-HT 1A receptor activation was reached after a single 1 mg/kg dose, but that a 10 mg/kg dose was required to reach the same level of receptor occupancy in the hippocampus and dorsal raphe. [34] The dose needed to occupy receptors in the retina is currently unknown, but based on the finding that a single administration of 6 mg/kg or more was required to achieve neuroprotection, we hypothesize that the occupancy of 5-HT 1A receptors needed for neuroprotection in the retina is closer to that of the hippocampus and dorsal raphe.
In humans, three daily-administrations of flibanserin are required for the drug to reach steady state plasma concentrations. [33] The time to reach steady state concentrations of flibanserin is currently unknown in mice. However, due to flibanserin's differing half-life in humans (11 hours) versus rats (0.9 to 1.9 hours), the time to reach steady state concentrations in rodents likely takes longer than three days. [34] Since flibanserin was only injected three times prior to light damage, maximum plasma and retinal concentrations may not have been reached with lower doses. Therefore, we hypothesize that if the number of daily-injections prior to light damage was increased, the total concentration of flibanserin would also increase, subsequently enhancing the neuroprotective effects of even the low doses. receptor plus values demonstrates that pre-treatment with WAY 100635 prior to 6 mg/kg flibanserin (10 mg/kg WAY + 6 mg/kg Flibanserin, green) decreased average receptor plus thickness as compared to flibanserin-treatment without WAY 100635 (6 mg/kg Flibanserin, blue). A 6 mg/kg dose of flibanserin in 5-HT 1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, red) resulted in average receptor plus thickness that was not significantly different from vehicle-injected and naïve mice. The gray area indicates ± 2 SD of the naïve averaged data. (A ii, A iii) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that both pre-treatment of BALB/c mice with WAY 100635 prior to a flibanserin injection (10 mg/kg WAY + 6 mg/ kg Flibanserin, green) and a flibanserin injection in 5-HT 1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, red) significantly reduces the observed neuroprotective effects provided by a single 6 mg/kg dose of flibanserin (6 mg/kg Flibanserin, blue) in the (A ii) temporal quadrant and the (A iii) nasal quadrant. (B) Pre-treatment with WAY 100635 (10 mg/kg WAY + 6 mg/kg Flibanserin, green) mitigated the improvements to ERG a-and b-wave amplitudes provided by a single 6 mg/kg dose of flibanserin (blue). ERG a-and bwave amplitudes were not improved by a single dose of 6 mg/kg flibanserin when administered to 5-HT 1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, red). " †" indicates the flash intensity providing the b (max,rod) value at which statistical analyses was also performed and presented in S1C Fig. * indicates a significant difference from both the vehicle-treated group and the naïve group, P < 0.05. n.s. indicates non-significance with P > 0.05. Flibanserin has high affinity for the 5-HT 1A receptor (K i = 1 nM) and a lower affinity for the 5-HT 2A receptor (K i = 49nM), but lacks significant affinity for all other serotonergic and adrenergic receptors, allowing for the characterization of the neuroprotective targets, 5-HT 1A and 5-HT 2A , in combination. [15-20, 22-26, 34] Flibanserin does, however, have moderate affinity for the dopaminergic D 4 receptor (K i = 4-24 nm), where it acts as an antagonist at low doses, and a partial agonist at higher doses. [34] It has been reported that D 4 receptor agonists can inhibit oxidative stress-induced nerve cell death and protect against hypoxia/reoxygenation-induced cell death in cultured HT22 cells, which suggests that activation of the D 4 receptor could be neuroprotective. [52,53] Although D 4 receptors have been reported in rodent retina, the threshold at which flibanserin begins functioning as a dopaminergic agonist is not well described in the mouse. [34,54] Taken together, we considered the possibility that flibanserin-mediated neuroprotection could be due to D 4 receptor activation, but our results suggested otherwise. WAY 100635, a 5-HT 1A antagonist and a potent D 4 agonist, was tested in our light-induced retinopathy model, as it binds the D 4 receptor with similar affinity (K i = 16) as flibanserin. [34,47] WAY 100635 failed to protect BALB/c retinas, both structurally and functionally, from light-induced retinopathy when given at a molar equivalent dose of the most effective dose of flibanserin, 15 mg/kg (Fig 4A and 4B, S1C Fig). Because this degree of D 4 receptor activation did not protect the retina, it suggests that the observed neuroprotective effects of flibanserin are not likely mediated through D 4 receptors.
In HSDD, Flibanserin is thought to mediate its effect through modulation of serotonin, dopamine and norepinephrine levels in brain regions involved in activating dopaminergic reward and sexual cue integration. [33,35] Several studies suggest that Flibanserin's action in the brain occurs through 5-HT 1A serotonin receptors. [34,38,39] This is supported by the finding that WAY 100635, a selective 5-HT 1A antagonist, completely antagonized the effects of flibanserin on serotonin, dopamine and noradrenaline in the rat prefrontal cortex. [35] Our results similarly suggest that flibanserin's neuroprotective effects on the retina are mediated through the 5-HT 1A receptor. A single 10 mg/kg dose of WAY 100635 injected 30 minutes prior to a single 6 mg/kg injection of flibanserin severely reduced observed neuroprotective effects. In addition, 6 mg/kg of flibanserin failed to protect the retina from light-induced retinopathy in 5-HT 1A knockout mice. Whether flibanserin elicits any effect via the 5-HT 2A receptor remains to be elucidated.
The signaling cascade involved in 5-HT 1A -mediated neuroprotection has yet to be fully characterized, therefore we performed gene expression assays to elicit the mechanisms responsible for flibanserin-mediated neuroprotection. The cell death signaling implicated in lightinduced retinopathy involves the activation of nitric oxide synthase (NOS), increased intracellular calcium, disturbed mitochondrial function, and generation of oxidative stress, all of which can play a role in photoreceptor cell death in inherited retinal degeneration. [40,55,56] RT-qPCR results suggest that flibanserin provides neuroprotection by addressing the latter two components. In regard to mitochondrial dysfunction, the upregulation of Calpastatin (Cast1) and B-cell lymphoma 2 (Bcl-2) may lead to increased cell survival. Calpastatin is an endogenous inhibitor of calpains, which have been linked to caspase-dependent and caspase-independent cell death. [40,57,58] Arroba et al. demonstrated that maintained Cast1 expression, via treatment with insulin-like growth factor-I (IGF-I), can significantly reduce apoptosis. [59] This effect was shown in both 661W cone cells and C57BL/6 mouse retinal explants grown in the presence of excess Ca 2+ , as well as in naïve rd1 mouse retinal explants. [59] Bcl-2 has been described as an inhibitor of mitochondrial dysfunction during programmed cell death. [60] Additionally, transgenic expression of Bcl-2 under control of the rhodopsin promoter has been shown to provide retinal neuroprotection in a light-induced retinopathy model, rd1 mice, and S334ter-Rho mice. [61,62] Mitochondrial dysfunction often leads to oxidative stress, which occurs when reactive oxygen species capacity outweighs the antioxidant defense capacity. [63] Accordingly, antioxidants have been evaluated in several light-induced and inherited retinal dystrophy models, and have established their worth as neuroprotective agents. [17,[64][65][66][67][68] Flibanserin's ability to upregulate antioxidant genes may play a significant role in cell survival. [40,69] We have shown that flibanserin increases expression of Superoxide dismutase 1 (Sod1), Catalase (Cat), and NAD(P) H quinone dehydrogenase 1 (Nqo1). Sod1 is an antioxidant responsible for the decomposition of free superoxide radicals to molecular oxygen and hydrogen peroxide, while Cat reduces the membrane-permeable hydrogen peroxide to water. [70,71] Nqo1, activated by the transcription factor Nrf2, is responsible for quinone detoxification. [72][73][74] Nqo1 operates by reducing ROS-generating quinones to hydroquinones, and has been reported to scavenge superoxides directly. [72,75,76] All three of these antioxidants have been able to provide protection against oxidative stress. For example, exogenous Sod1 and Cat provided protection against retinal ischemic injury following increased intraocular pressure in different in vivo models. [77,78] Adenovirus-mediated delivery of Cat was able to protect RPE cells from hydrogen peroxideinduced oxidative stress, as well as photoreceptors in an in vivo light damage model. [71] Additionally, Nqo1 has played a prominent role in neuroprotection against oxidative stress in neuronal HT22 cells. [79] Flibanserin treatment also led to significant increases in c-Jun and c-Fos. As a heterodimer, these proteins assemble into activator protein complex-1 (AP1), a transcription factor closely associated with apoptosis. [80] However, the role of the c-Fos/c-Jun heterodimer in apoptosis is complex. [80][81][82] While studies have shown that light-induced retinopathy can be prevented by the ablation of c-fos, [40,56] this phenomenon only occurs in mice with a methionine substitution at RPE65 codon 450 and not in BALB/c mice which have a leucine mutation at this position. [82] Knocking out c-fos also did not prevent cell death in rd1 mice. [56] Contrastingly, c-Fos and c-Jun have also been demonstrated to be anti-apoptotic. Gu et al. reported an absence of AP1 in dying photoreceptors, which argues against its role in photoreceptor apoptosis. AP1 activation was instead observed in Müller cells after light exposure, suggesting a pro-survival effect. [82] In summary, when light-damage occurs, there is an increase of intracellular calcium, disruption of mitochondrial function, and oxidative stress. [40] We hypothesize that flibanserin treatment leads to the activation of Creb, which can upregulate Cast1 and Bcl-2 and protect against mitochondrial dysfunction. Furthermore, Bcl-2 has the ability to modulate cellular calcium concentrations and increase antioxidant genes, such as Sod1, Cat and NqO1. [40,60,[83][84][85] Although we saw a flibanserin-mediated increases in c-Fos and c-Jun, the role they play in neuroprotection remains unclear.
Flibanserin was recently approved by the FDA and marketed as Addyi™ for the treatment of HSDD and its side effects are relatively benign in both men and women; the most frequent adverse events being dizziness, somnolence, nausea and fatigue. [29] The potential to repurpose flibanserin for retinal neuroprotection is exciting. However, much work remains to transition it into a viable treatment for inherited retinal dystrophies, such as evaluation of its neuroprotective effects in an animal model of inherited retinal degeneration. Other avenues of delivery, such as intraocular injection, could also be explored to reduce possible systemic side effects. With its documented safety profile and FDA-approved status, further investigation of flibanserin for the treatment of retinal degenerations is warranted. The ERG b (max, rod) response observed after a single dose of 6 mg/kg flibanserin was significantly reduced in both 5-HT 1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, red) and mice that received a pre-treatment of WAY 100635 (10 mg/kg WAY + 6 mg/kg Flibanserin, green). The averaged right and left eye data for each mouse is represented as a dot. Group averages are represented as mean ± standard error bar. Ã indicates a significant difference from both the vehicle-treated group and the naïve group, P < 0.05. n.s. indicates non-significance with P > 0.05. (TIF) S1