Flibanserin’s neuroprotective effects are 5-HT_1A receptor-mediated.

<p>(<b>A i</b>) A spider graph representing average right-eye receptor plus values demonstrates that pre-treatment with WAY 100635 prior to 6 mg/kg flibanserin (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) decreased average receptor plus thickness as compared to flibanserin-treatment without WAY 100635 (6 mg/kg Flibanserin, <i>blue</i>). A 6 mg/kg dose of flibanserin in 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>) 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. (<b>A ii, A iii</b>) 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, <i>green</i>) and a flibanserin injection in 5-HT_1A knockout mice (5-HT1A KO + 6 mg/kg Flibanserin, <i>red</i>) significantly reduces the observed neuroprotective effects provided by a single 6 mg/kg dose of flibanserin (6 mg/kg Flibanserin, <i>blue</i>) in the <b>(A ii)</b> temporal quadrant and the <b>(A iii)</b> nasal quadrant. (<b>B</b>) Pre-treatment with WAY 100635 (10 mg/kg WAY + 6 mg/kg Flibanserin, <i>green</i>) mitigated the improvements to ERG a- and b-wave amplitudes provided by a single 6 mg/kg dose of flibanserin (<i>blue</i>). ERG a- and b-wave 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, <i>red</i>). “†” indicates the flash intensity providing the b_(max,rod) value at which statistical analyses was also performed and presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159776#pone.0159776.s001" target="_blank">S1C Fig</a>. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05.</p

A 5-day time course of flibanserin protects retinal morphology from bright light exposure in a dose-dependent manner.

<p>(<b>A</b>) Representative linear SD-OCT scans of nasal retina show increasing outer nuclear layer thickness with flibanserin treatment, in a dose-dependent manner. A naïve non-light damaged mouse shows an easily distinguishable OPL, ONL, OLM, IS/OS, and RPE. GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, OPL-outer plexiform layer, ONL-outer nuclear layer, OLM-outer limiting membrane, IS/OS-inner segment/outer segment junction, RPE-retinal pigment epithelium. (<b>B i</b>) A spider graph of average right-eye receptor plus values demonstrates that flibanserin-mediated protection increases in a dose-dependent manner starting at 3 mg/kg, with a 15 mg/kg dose showing comparable receptor plus thicknesses to naïve (non-light damaged) mice. The gray area indicates ± 2 SD of the naïve averaged data. (<b>B ii, iii</b>) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that 15 mg/kg flibanserin achieves complete morphological protection in the <b>(B ii)</b> temporal quadrant and the <b>(B iii)</b> nasal quadrant. Group averages are represented as mean ± standard error bar. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05.</p

A one-day time course of flibanserin can preserve retinal morphology and function.

<p><b>(A i)</b> A spider graph representing average right-eye receptor plus values demonstrates that a single dose of 6 mg/kg flibanserin or greater can significantly preserve retinal morphology to thicknesses comparable to naïve mice. The gray area indicates ± 2 SD of the naïve averaged data. (<b>A ii, iii</b>) Receptor plus thicknesses, with each dot representing average right and left eye thickness from one mouse, demonstrate that 6 mg/kg flibanserin provides morphological protection in the <b>(A ii)</b> temporal quadrant and the <b>(A iii)</b> nasal quadrant. Group averages are represented as mean ± standard error bar. <b>(B)</b> Mice treated with doses of 6 mg/kg and greater of flibanserin showed significantly higher ERG b-wave and a-wave responses at the highest flash intensity (3.55 log cd•s/m²) as compared to vehicle-treated mice. “†” indicates the greatest flash intensity that elicits a rod-only response (b_(max,rod)). Group differences at the “†” flash intensity were statistically evaluated and are represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159776#pone.0159776.s001" target="_blank">S1B Fig</a>. Individual ERGs were averaged with ERGs for mice within group and are represented as mean ± standard error. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05.</p

Summary of OCT findings.

<p>Summary of OCT findings.</p

A 5-day time course of flibanserin preserves retinal function in a dose-dependent manner, as measured by ERG.

<p>(<b>A</b>) Representative ERG traces at the 3.55 log cd•s/m² light intensity demonstrating dose-dependent improvements in ERG responses as compared to vehicle-injected mice. (<b>B</b>) Mice treated with doses of 3 mg/kg or greater of flibanserin showed significantly higher ERG b-wave and a-wave responses at the highest ERG flash intensity (3.55 log cds/m²) as compared to vehicle-treated mice. Individual ERGs were averaged with ERGs for mice within its respective group and are represented as mean ± standard error. * indicates a significant difference from both the vehicle-treated group and the naïve group, <i>P</i> < 0.05. n.s. indicates non-significance with <i>P</i> > 0.05. The responses at the ERG b_(max,rod) light intensity, indicated by the “†”, were statistically evaluated and are represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159776#pone.0159776.s001" target="_blank">S1A Fig</a>.</p

Flibanserin increases anti-apoptotic and antioxidant gene expression.

<p><b>(A)</b> RT-qPCR shows that 48 hours following light-exposure, expression of <i>Creb</i>, <i>Bcl-2</i>, <i>Cast1</i>, <i>Sod1</i>, and <i>Cat</i> are significantly increased in flibanserin-injected mice versus vehicle-injected mice. <b>(B)</b> After 72 hours, expression of <i>Creb</i>, <i>c-Jun</i>, <i>c-Fos</i>, <i>Bcl-2</i>, <i>Cast1</i>, <i>Nqo1</i>, and <i>Sod1</i> are significantly increased in flibanserin-injected mice versus vehicle-injected mice. Analysis was performed using the ΔΔCt method with β-actin as the internal control. Significance was determined using a multiple t-test analysis (* indicates <i>P</i> < 0.05). cAMP Response Binding-element Protein (<i>Creb</i>), Cyclin D1 (<i>Cd1</i>), B-cell lymphoma 2 (<i>Bcl-2</i>), Calpastatin (<i>Cast1</i>), Nitric Oxide Synthase (<i>Nos1</i>), NAD(P)H quinone dehydrogenase 1 (<i>Nqo1</i>), Superoxide Dismutase 1 (<i>Sod1</i>), Catalase (<i>Cat</i>), Metallothionein 1 (<i>Mt1</i>).</p

Summary of ERG parameters.

<p>Summary of ERG parameters.</p

Goldmann Visual Fields and Respective SD-OCT scans of the Central Retina in a Treatment Responder and a Non-Responder.

<p>(A) Normal subject. The horizontal midline scan shows segmentation lines separating Vitreous/Retinal Nerve Fiber Layer (RNFL), RNFL/Retinal Ganglion Cell Layer (RGC), Inner Plexiform Layer (IPL)/Inner Nuclear Layer (INL), INL/Outer Plexiform Layer (OPL), Inner segment (IS)/Outer segment (OS), OS/ Retinal Pigment Epithelium (RPE), Bruch’s Membrane (BM)/Choroid. The OS layer lies between the IS/OS line and the OS/RPE line (see Hood et al. 2009 Ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143846#pone.0143846.ref019" target="_blank">19</a>). (B) SD-OCT foveal scan at screening (length of OS layer in the central 20° was 23.1 μm), (C) Goldmann Visual Field (GVF) at screening visit (retinal area of primary isopter = 26 mm²) and at month 1.5 (D) (retinal area of primary isopter = 81 mm²) in a RP patient who showed a treatment response (subject 110 OS, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143846#pone.0143846.t001" target="_blank">Table 1</a>). (E) SD-OCT foveal scan at screening (length of OS layer in the central 20° was 6.2 μm), (F) Goldmann Visual Field (GVF) at screening visit (retinal area of primary isopter = 55 mm²) and at month 1 (G) (retinal area of primary isopter was 44 mm²) of a non-responder (subject 402 OS, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143846#pone.0143846.t001" target="_blank">Table 1</a>).</p

Functional Retinal Area Responders for Primary Isopter (top) and Visual Acuity Responders (bottom).

<p>^a For at least 2 consecutive visits starting within 2 months from the start of QLT091001 treatment.</p><p>Note: The responder categories include one patient who was not able to read any letters on the ETDRS chart at baseline but who was on-chart post-treatment (both eyes of patient 401 with RPE65 deficiency); inclusion criteria required either best corrected visual acuity (BCVA) of at least 3 letter score <u>or</u> evidence from OCT/FAF of a viable photoreceptor layer.</p><p>Functional Retinal Area Responders for Primary Isopter (top) and Visual Acuity Responders (bottom).</p

Percent of Treatment Responders for Functional Retinal Area (A, top) and Visual Acuity (B, bottom).

<p>Response in functional retina area was defined as an increase in visual field area from baseline of ≥20% increase in the visual field area in the primary isopter in one or both eyes at two or more visits within 2 months of treatment. Visual acuity response was defined as an increase from baseline in visual acuity of ≥5 ETDRS letter score at two or more visits within 2 months of treatment.</p