Effects of Varying Intranasal Treatment Regimens in ST266-Mediated Retinal Ganglion Cell Neuroprotection

Introduction: Previous studies have shown that intranasally administered ST266, a novel biological secretome of amnion-derived multipotent progenitor cells containing multiple growth factors and anti-inflammatory cytokines, attenuated visual dysfunction and prevented retinal ganglion cell (RGC) loss in experimental optic neuritis. Long-term effects and dose escalation studies examined here have not been reported previously. Methods: Optic neuritis was induced in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE). EAE and control mice were treated once or twice daily with intranasal placebo/vehicle or ST266 beginning after onset of optic neuritis for either 15 days or continuously until sacrifice. Visual function was assessed by optokinetic responses (OKRs). RGC survival and optic nerve inflammation and demyelination were measured. Results: Both once and twice daily continuous intranasal ST266 treatment from disease onset to 56 days after EAE induction significantly increased OKR scores, decreased RGC loss, and reduced optic nerve inflammation and demyelination compared with placebo (saline, nonspecific protein solution, or cell culture media)-treated EAE mice. ST266 treatment given for just 15 days after disease onset, then discontinued, only delayed OKR decreases, and had limited effects on RGC survival and optic nerve inflammation 56 days after disease induction. Conclusions: ST266 is a potential neuroprotective therapy to prevent RGC damage, and intranasal delivery warrants further study as a novel mechanism to deliver protein therapies for optic neuropathies. Results suggest that once daily ST266 treatment is sufficient to sustain maximal benefits and demonstrate that neuroprotective effects promoted by ST266 are specific to the combination of factors present in this complex biologic therapy.

Basic and Translational Research: Bench to Bedside Section Editors: Jeffrey L. Bennett, MD, PhD Kenneth S. Shindler, MD, PhD Effects of Varying Intranasal Treatment Regimens in ST266-Mediated Retinal Ganglion Cell Neuroprotection Reas S. Khan, PhD, Kimberly Dine, BS, Howard Wessel, BS, MBA, Larry Brown, ScD, Kenneth S. Shindler, MD, PhD Introduction: Previous studies have shown that intranasally administered ST266, a novel biological secretome of amnion‐derived multipotent progenitor cells containing multiple growth factors and anti-inflammatory cytokines, attenuated visual dysfunction and prevented retinal ganglion cell (RGC) loss in experimental optic neuritis. Longterm effects and dose escalation studies examined here have not been reported previously. Methods: Optic neuritis was induced in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE). EAE and control mice were treated once or twice daily with intranasal placebo/vehicle or ST266 beginning after onset of optic neuritis for either 15 days or continuously until sacrifice. Visual function was assessed by optokinetic responses (OKRs). RGC survival and optic nerve inflammation and demyelination were measured. Results: Both once and twice daily continuous intranasal ST266 treatment from disease onset to 56 days after EAE induction significantly increased OKR scores, decreased RGC loss, and reduced optic nerve inflammation and demyelination compared with placebo (saline, nonspecific protein solution, or cell culture media)-treated EAE mice. ST266 treatment given for just 15 days after disease onset, then discontinued, only delayed OKR decreases, and had limited effects on RGC survival and optic nerve inflammation 56 days after disease induction. Scheie Eye Institute (RSK, KD, KSS), FM Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania; and Noveome Biotherapeutics, Inc. (HW, LB), Pittsburgh, Pennsylvania. Supported by NIH grant EY015014, Research to Prevent Blindness, and the FM Kirby Foundation. Portions of this work were subcontracted from grants to Noveome Biotherapeutics, Inc., through US Navy Contract # N62645-13-C-4014, Cell-Based Wound Therapeutics for Combat Casualties and The State of Pennsylvania, 2014 Bio-Technology Grant SAP#4100068500. H. Wessel and L. Brown are full-time employees of Noveome Biotherapeutics, Inc., which provided the ST266 product used in these studies. K. S. Shindler received research funding and has received consulting fees for discussions of clinical needs for optic neuritis from Noveome. Address correspondence to Kenneth S. Shindler, MD, PhD, FM Kirby Center for Molecular Ophthalmology, Stellar-Chance Laboratories, 3rd Floor, 422 Curie Boulevard, Philadelphia, PA 19104; E-mail: [e-mail] Khan et al: J Neuro-Ophthalmol 2019; 39: 191-199 Conclusions: ST266 is a potential neuroprotective therapy to prevent RGC damage, and intranasal delivery warrants further study as a novel mechanism to deliver protein therapies for optic neuropathies. Results suggest that once daily ST266 treatment is sufficient to sustain maximal benefits and demonstrate that neuroprotective effects promoted by ST266 are specific to the combination of factors present in this complex biologic therapy. Journal of Neuro-Ophthalmology 2019;39:191-199 doi: 10.1097/WNO.0000000000000760 © 2019 by North American Neuro-Ophthalmology Society O ptic neuritis (ON) is an inflammatory demyelinating disease often associated with the central nervous system autoimmune disease multiple sclerosis (MS) (1). Neuronal damage and axonal damage also are key features of the pathophysiology of MS and ON, and result in long-term vision loss and neurologic disability in patients (2-4). Inflammation-mediated apoptotic retinal ganglion cell (RGC) loss occurs in eyes of patients with MS with or without a history of ON (5), and significant RGC loss also occurs in the experimental autoimmune encephalomyelitis (EAE) model of MS (6,7). Intravenous corticosteroids frequently are used to hasten visual recovery in acute ON but have no effect in preventing RGC loss and associated permanent visual deficits (8,9). No current therapy improves final visual outcomes in ON. Therefore, improved and safe potential neuroprotective therapies that prevent RGC loss from ON are needed. Studies of experimental ON in EAE animals have identified potential immunomodulatory and neuroprotective compounds effective in attenuating RGC loss during ON (10-12) and demonstrate the usefulness of this model. Our previous studies, for example, have demonstrated that pharmaceutical approaches that promote mitochondrial biogenesis and function using SIRT1 activating compounds or mitochondrial uncoupling drugs are beneficial in ON with improved cellular function, increased biomarkers, and reduced oxidative stress resulting in improved RGC preservation and visual outcomes 191 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research: Bench to Bedside (13-15). Recent advances in stem cell-based therapies offer a new, potentially multifactorial approach in the treatment of retinal and optic nerve diseases (16). Transplanted stem cells have shown significant neuroprotective effects in preclinical models of glaucoma (17), transient ischemia, and ocular hypertension models (18,19). Continuous intraocular secretion of several growth factors by stem cells, including brainderived neurotrophic factor (20), ciliary neurotrophic factor (21), and platelet-derived growth factor (22) provide immunomodulatory and neuroprotective effects in several retinal or optic neuropathies. Although stem cell therapies have shown promise in numerous studies, there are potential risks associated with transplantation, including immune rejection and unwanted cell growth. Further research has focused on stem cell secretion-based therapies that can be used in place of cells (23,24). Secretions from stem cells provide some key advantages, including the potential to be purified, stored, and dosed precisely compared with stem cells (25,26). ST266 (formerly Amnion-Derived Cellular Cytokine Solution) is the proprietary biologic secretome of amnion‐derived multipotent progenitor (AMP) cells. ST266 modulates inflammatory responses by decreasing vascular permeability, immune cell infiltration, and edema (27,28); accelerates wound healing (27,29,30); and attenuates the effects of a penetrating ballistic brain injury by inducing persistent motor improvement and ameliorating neuroinflammation (31,32). We have shown that intranasally delivered ST266 accumulates rapidly in rodent eyes and optic nerves 30 minutes after administration, not through systemic absorption but rather likely through direct central nervous system (CNS) entry (33). A single 6 mL intranasal drop of ST266 given once daily beginning after the onset of EAE ON attenuated visual dysfunction, prevented RGC loss, and reduced both inflammation and demyelination 6 weeks after induction of EAE, as compared to phosphate-buffered saline (PBS)-treated EAE mice (33). Thus, ST266 is a potential new neuroprotective therapy for ON that may be delivered by a novel, noninvasive route of administration. However, the optimal dosage and frequency of administration of ST266 needed to achieve maximal therapeutic effect, and its ability to confer longer-term neuroprotective effects remain to be determined. Therefore, in the current studies, we examined the ability of different ST266 treatment regimens to prevent neuronal damage during EAE-induced experimental ON. In addition, we compared effects of ST266 with additional controls containing cell culture medium nutrients and high protein levels to confirm that ST266-mediated effects are due to its specific protein secretome composition. METHODS Animals Six-week-old female C57BL6/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed at an 192 approved animal facility at the University of Pennsylvania. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania and adhered to all applicable international, national, and institutional guidelines for the care and use of animals. Induction and Scoring of Experimental Autoimmune Encephalomyelitis EAE induction was performed as in our previous studies (7). Eight-week-old mice were anesthetized using isoflurane and were injected subcutaneously at 2 sites on the back with myelin oligodendrocyte glycoprotein (200 mg, MOG35-55 peptide; Genscript, Piscataway, NJ), emulsified in Complete Freund's Adjuvant (Difco, Detroit, MI) containing 2.5-mg/mL mycobacterium tuberculosis (Difco). An equal volume of PBS in complete Freund's adjuvant was injected into control mice. In addition, each animal received 200-ng pertussis toxin (List Biological, Campbell, CA) in 0.1-mL PBS by intraperitoneal (i.p.) injection at 0 and 48 hours after immunization. As in previous studies, severity of EAE spinal cord disease was monitored to confirm successful disease induction (data not shown) (13,14,34,35). ST266 Treatment ST266 (Noveome Biotherapeutics, Inc, Pittsburgh, PA) was aliquoted and stored at 220°C. A fresh aliquot was thawed and warmed to room temperature each day and administered to the mice as a single 6-mL drop placed in the nose (33,36). Mice were grouped and treated once or twice with one drop of ST266, PBS, STM100 (the cell culture medium used to culture AMP cells and collect ST266 protein secretions), or 0.5% human serum albumin (HSA) in PBS intranasally beginning after the onset of ON (Day 15 after immunization). Treatment was continued through Day 30, 42, or 56 as indicated in each experiment. Mice receiving ST266 for only Days 15-30 were treated with PBS during the interval when they were not receiving ST266 (Days 31-56), so that all control and treated mice received the same total number of days of intranasal fluid administration. In the 42-day treatment study, 5 mice were included in each treatment group (PBS-treated control nonEAE mice; PBS-treated EAE mice; STM100-treated EAE mice; and ST266-treated EAE mice). In the 56-day treatment study, the control non-EAE mouse group contained 4 mice, and each of the 6 experimental EAE groups listed in Table 1 contained 6 mice. No mice died or were otherwise removed from any of the studies; thus, all eyes of all mice in each experimental group were used in analyses of visual function and RGC survival. In the 56-day treatment study, the right optic nerve from 3 randomly selected mice in each treatment group was removed for protein isolation and Western blot analysis of inflammatory and apoptotic markers (data not shown due to undetectable levels). The left optic nerves of these 3 mice/groups, and both optic Khan et al: J Neuro-Ophthalmol 2019; 39: 191-199 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research: Bench to Bedside TABLE 1. ST266 treatment groups Group 1 2 3 4 5 6 7 Treatment Cohort ST266 Treatment Control (non-EAE) mice EAE mice EAE mice EAE mice EAE mice EAE mice EAE mice - - - Once/day, Days 15-30 Twice/day, Days 15-30 Once/day, Days 15-56 Twice/day, Days 15-56 Placebo Treatment PBS daily, PBS daily, 0.5% HSA PBS daily, PBS twice - - Days 15-56 Days 15-56 daily, Days 15-56 Days 31-56 daily, Days 31-56 EAE, experimental autoimmune encephalomyelitis; HSA, human serum albumin; PBS, phosphate-buffered saline. nerves from all other mice, were used for histopathologic analyses of optic nerve inflammation and demyelination. Measurement of Optokinetic Responses The methods for optokinetic responses (OKR) recording to assess visual function were similar to previous studies (7). OKR experiments were performed using OptoMotry software and apparatus (Cerebral Mechanics Inc, Medicine Hat, AB, Canada). OKR function was determined by the highest spatial frequency at which mice track a 100% contrast grating projected at varying spatial frequencies, and data are reported as cycles/degree. length of each optic nerve section was examined with light microscopy by a blinded investigator and scored according to a 0-4 point scale: 0 = no infiltration; 1 = mild cellular infiltration of the optic nerve or optic nerve sheath; 2 = moderate infiltration; 3 = severe infiltration; and 4 = massive infiltration. Luxol fast blue staining was performed to evaluate demyelination in the optic nerve. The entire length of each optic nerve section was examined by light microscopy and scored on relative scale by a blinded investigator according to a 0-3 point scale: 0 = no demyelination; 1 = scattered foci of demyelination; 2 = prominent foci of demyelination; and 3 = large (confluent) areas of demyelination. Quantification of Retinal Ganglion Cell Survival RGCs were immunolabeled with Brn3a (RGC marker) antibody and counted as described previously (33). Briefly, mice were sacrificed at Day 42 or 56 as indicated, and retinas were isolated, prepared as flattened whole mounts, washed, and permeabilized in 0.5% Triton X 100 in PBS by freezing them for 15 minutes at 270°C. The specimens were incubated overnight at 4°C with goat anti-Brn3a antibody (Santa Cruz, CA) diluted 1:100 in blocking buffer (PBS, 2% bovine serum albumin, 2% Triton X 100). The retinas were then washed 3 times in PBS and incubated for 2 hours at room temperature with anti-goat secondary antibody diluted 1:500 in blocking buffer. Sections were washed in PBS and mounted vitreous side up on slides in antifading solution. Brn3a-labelled RGCs were photographed at 40· magnification in 12 standard fields: 1/6, 3/6, and 5/6 of the retinal radius from the center of the retina in each quadrant and counted by a masked investigator using image analysis software (Image-Pro Plus 5.0; Media Cybernetics, Silver Spring, MD). Evaluation of Optic Nerve Inflammation and Demyelination Inflammation and demyelination in the optic nerves were evaluated as described previously (10,33). Briefly, optic nerves were isolated, fixed in 4% paraformaldehyde for 15 min, embedded in paraffin, and then cut into 5-mm longitudinal sections. Hematoxylin and eosin staining was used to evaluate inflammation in the optic nerves. For histological analysis of inflammatory cell infiltration, the entire Khan et al: J Neuro-Ophthalmol 2019; 39: 191-199 Experimental Design and Statistics Mice were randomly numbered and assigned to treatment groups. All outcome measures including vision testing and tissue analysis were quantified in a masked fashion. Only female mice were used because they are more susceptible to the EAE disease, similar to human MS. Statistical comparisons were made using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Because ON occurs as an independent event that can affect one or both eyes, each eye was analyzed similar to previous studies (10,15,33). OKR responses measured across time were compared by analysis of variance (ANOVA) of repeated measures. RGC numbers and the level of optic nerve inflammation and demyelination were measured at a single time point, and differences were compared between mice in different treatment groups using 1-way ANOVA followed by Tukey's multiple comparison. Data are expressed as mean ± SEM. Differences were considered statistically significant at P , 0.05. RESULTS Ability of ST266 to Attenuate Experimental Optic Neuritis Was Mediated by Factors Secreted by Amnion-Derived Multipotent Progenitor Cells OKR responses significantly decreased in the eyes of PBSand STM100-treated EAE mice starting from Day 14 and progressed through Day 42, and ST266 treatment 193 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research: Bench to Bedside attenuated this decrease (Fig. 1A). After sacrifice at Day 42, RGC numbers were significantly decreased in the eyes of PBS- or STM100-treated EAE mice compared with control, non-EAE mice; and EAE mice that received daily ST266 showed a significant reduction in RGC loss (Fig. 1B). Continuous ST266 Treatment Promoted Longer-Term Preservation of Visual Function Table 1 indicates the specific treatment received by each experimental group. OKR responses progressively decreased in the eyes of PBS-treated EAE mice from Day 14 through Day 56 after induction (Fig. 2), with similar OKR decreases in EAE mice treated with 0.5% HSA (HSA is the most abundant protein in ST266 and is present at this concentration), which served as a control for potential nonspecific effects of high protein levels in solution. EAE mice that received either once or twice daily intranasal drops of ST266 starting from Day 15 and repeated continuously through Day 56 showed a significant preservation of OKR scores compared with PBS- or HSAtreated EAE mice (Fig. 2). By contrast, EAE mice that received once or twice daily ST266 drops only from Days 15-30 showed a delayed loss of OKR responses at Days 35-42 that was not maintained from Days 42-56, although twice daily treatment over this abbreviated period did show a trend toward improved OKR scores (Fig. 2). ST266 Treatment Reduced Retinal Ganglion Cell Loss in Experimental Autoimmune Encephalomyelitis Optic Neuritis EAE mice (treated as detailed in Table 1) were sacrificed on Day 56. RGC numbers were significantly decreased in the eyes of PBS- or HSA-treated EAE mice compared with control mice (Fig. 3). EAE mice that received either once or twice daily intranasal drops of ST266 starting from Day 15 and repeated continuously through Day 56 showed a significant preservation of RGCs compared with PBS- or HSA-treated EAE mice (Fig. 3). EAE mice that received twice daily ST266 drops only on Days 15-30 also showed a small but significant increase in RGC numbers compared with placebo-treated EAE mice, whereas once daily ST266 treatment limited to Days 15-30 failed to prevent RGC loss measured at Day 56 (Fig. 3). ST266 Treatment Reduced Inflammation in the Optic Nerve During Experimental Autoimmune Encephalomyelitis Optic Neuritis FIG. 1. ST266, not the media it is collected in, suppresses loss of vision and RGCs in experimental optic neuritis. A. OKR responses significantly (***P , 0.001) decreased in the eyes of PBS (N = 10)- or STM100-treated EAE mice (N = 10) compared with control (non-EAE) mice (N = 10) starting from Day 14 after immunization and progressing through Day 42. EAE mice that received intranasal ST266 once daily (N = 10) from Days 15-42 show significantly (@@P , 0.01 vs EAE treated with PBS, ^^^P , 0.001 vs EAE treated with STM100) higher OKR scores. B. RGCs were labeled and counted in retinas isolated 42 days after immunization. Data show a significant decrease in the number of RGCs in eyes of PBS (***P , 0.001, N = 10)- or STM100 (^^P , 0.01, N = 10)-treated EAE mice compared with control mice. EAE mice that received ST266 daily from Day 15-42 (N = 10) show significant (@P , 0.05 vs EAE treated with PBS, @P , 0.05 vs EAE treated with STM100) attenuation of RGC loss. EAE, experimental autoimmune encephalomyelitis; PBS, phosphate‐buffered saline; RGC, retinal ganglion cell; OKR, optokinetic responses. 194 Optic nerves from control mice showed normal histology, whereas optic nerves from PBS- and HSA-treated EAE mice showed significant inflammatory cell infiltration (Fig. 4). EAE mice that received either once or twice daily intranasal drops of ST266 starting from Day 15 and repeated continuously through Day 56 showed a significant reduction in optic nerve inflammation compared with PBS- or HSA-treated EAE mice, whereas EAE mice that received once or twice daily ST266 drops only from Days 15-30 showed only a nonsignificant trend toward decreased inflammation (Fig. 4). ST266 Treatment Reduced Demyelination in the Optic Nerve During Experimental Autoimmune Encephalomyelitis Optic Neuritis Optic nerves from control mice showed normal myelin staining, whereas optic nerves from PBS- and HSA-treated EAE mice showed significant areas of myelin loss (Fig. 5). EAE mice that received either once or twice daily intranasal drops of ST266 starting from Day 15 and repeated continuously through Day 56 showed a significant reduction in optic nerve demyelination compared with PBS- or HSAtreated EAE mice, whereas EAE mice that received once or twice daily ST266 drops only from Days 15-30 showed Khan et al: J Neuro-Ophthalmol 2019; 39: 191-199 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research: Bench to Bedside FIG. 2. Continuous ST266 treatment preserves OKR for 8 weeks in EAE optic neuritis. OKR responses significantly (***P , 0.001) decreased in the eyes of PBS (N = 12)- or HSA-treated EAE mice (N = 12) compared with control mice (N = 8), starting from Day 14 and progressing over time until sacrifice at Day 56 after immunization. EAE mice that received either once or twice daily doses of intranasal ST266 from Days 15-30 (N = 12) showed a delay in OKR decreases but no significant preservation of OKR by Day 56. EAE mice that received either once (@P , 0.05) or twice (@@P , 0.01) daily doses of intranasal ST266 continuously from Day 15-56 (N = 12) showed a significant preservation of OKR compared with both PBS- and HSA-treated EAE mice. EAE, experimental autoimmune encephalomyelitis; HSA, human serum albumin; PBS, phosphate‐buffered saline; RGC, retinal ganglion cell; OKR, optokinetic responses. only a nonsignificant trend toward decreased demyelination (Fig. 5). DISCUSSION The current studies demonstrated that intranasal delivery of the novel biologic therapy ST266 promoted beneficial effects on multiple features of experimental ON, including attenuating vision loss, promoting RGC survival, and reducing the degree of optic nerve inflammation and demyelination. Results are consistent with previous studies of ON (33) and neuroprotective effects mediated by ST266 in a model of traumatic optic neuropathy (36). Importantly, the current results suggest that once daily ST266 dosing is sufficient to promote maximal neuroprotective effects, as twice daily treatment led to equivalent outcomes. That is, continuous treatment with either once or twice daily ST266 Khan et al: J Neuro-Ophthalmol 2019; 39: 191-199 for 6 weeks beginning after onset of ON (Days 15-56) significantly improved OKR responses and reduced RGC loss compared with placebo-treated EAE mice, whereas there was no significant difference in OKR responses or RGC numbers between once daily ST266-treated and twice daily ST266-treated mice. In a pevious study, ST266 was able to promote continued attenuation of ON 6 weeks after EAE induction even when treatment was given for only 15 days after disease onset (Days 15-30 after immunization) and then was discontinued (33). Here, using the same mouse model, results suggest that the protective effects of ST266 are diminished by 8 weeks after EAE induction when treatment is stopped at Day 30, but benefits are maintained with continuous treatment through Day 56. The MOGinduced EAE model in C57BL6/J mice used is known to induce an acute onset of ON with peak inflammation by Day 15 after immunization but also exhibits continued lowlevel inflammation for several weeks (7). This ongoing inflammation may explain why continuous ST266 treatment is needed to maintain effects in this model. In the human disease, ON is typically felt to be a self-limited inflammatory condition, and thus, it is likely that ST266 therapy may be able to promote improved long-term outcomes if it is administered only during the period of active inflammation. Interestingly, anti-MOG antibodies increasingly have been recognized in a subset of human patients with ON, and some studies have suggested these patients may have distinct phenotypes from typical MS-related ON (37). Although the MOG-induced EAE disease in C57BL/ 6J mice recapitulates many clinical features of human MS and ON, it is unlikely that this model specifically relates to human anti-MOG antibody-positive ON because this EAE model is known to be mediated by MOG-reactive T cells, as opposed to B cells/antibodies (38). Although specific mechanisms mediating transport of proteins from the nose to the eye and optic nerve are not known, previous studies showed that radiolabeled ST266 proteins administered intranasally accumulated at high levels in the optic nerve and vitreous within 30 minutes of treatment (33). ST266 contains numerous factors, including cytokines and growth factors, secreted by AMP cells, such as angiogenin, platelet-derived growth factor BB, vascular endothelial growth factor, transforming growth factor-beta 2, amphiregulin, decorin, secreted protein, acidic and rich in cysteine, and hyaluronic acid (39). ST266 also contains anti-inflammatory factors, including macrophage inhibitory cytokine-1, macrophage migration inhibitory factor, the protease dipeptidyl peptidase-IV, and the lipid resolvin D1, as well as anti-apoptotic factors such as soluble tumor necrosis factor receptor 1, soluble tumor necrosis factor-related apoptosis-inducing ligand receptor3, Axl, and tissue inhibitors of metalloproteinases (39). Individual proteins in ST266 are found at concentrations in the pg/mL to ng/mL range, with the total concentration 195 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research: Bench to Bedside FIG. 3. ST266 treatment reduces RGC loss in EAE optic neuritis. A. RGC counts 56 days after immunization were significantly (***P , 0.001) decreased in the eyes of PBS (N = 12)- or HSA-treated EAE mice (N = 12) compared with control mice (N = 8). EAE mice that received once daily doses of intranasal ST266 from Day 15-30 (N = 12) showed no significant preservation of RGCs, whereas twice daily ST266 given only from Day 15-30 (N = 12) did promote a significant increase (@P , 0.05) in RGC survival. EAE mice that received either once (N = 12) or twice daily (N = 12) intranasal doses of ST266 continuously from Day 15- 56 showed a significant (@@@P , 0.001) preservation of RGC numbers by Day 56 compared with PBS- or HSA-treated EAE mice. B. Representative images show RGCs (green) in one retina from each group (original magnification ·40). EAE, experimental autoimmune encephalomyelitis; HSA, human serum albumin; PBS, phosphate‐buffered saline; RGC, retinal ganglion cell. of secretome proteins approximating 100 mg/mL (27). The consistent reproducibility of specific constituents of ST266 previously have been demonstrated, including similar levels of PDGF, VEGF, angiogenin, TGF-b2, TIMP-1, and TIMP-2 across multiple lots of ST266 (27), and reproducible function in MMP-9 inhibition and Schwann cell pro196 liferation assays (data not shown). ST266 also has shown no serious adverse effects in 8 human clinical trials using topical, oral, intravenous, and ocular applications, including topical skin application to reduce UV light-induced damage (40) and ophthalmic drops for allergic conjunctivitis (Unpublished data: NCT02978183, Evaluation of the Effectiveness of ST266 Ophthalmic Drops Compared to Placebo to Treat Allergic Conjunctivitis, available at: https:// clinicaltrials.gov/ct2/show?term=Noveome&rank=6). Like other cell culture media, the STM100 growth medium that is used to grow the AMP cells contains HSA, amino acids, vitamins, salts, and buffers. Therefore, to rule out the possibility that observed neuroprotective effects are due to some components in the STM100 medium other than the cell secretions in ST266, effects of intranasal delivery of STM100 alone were compared with ST266 in EAE ON. HSA, a major component of STM100 and ST266, which is not expected to affect neuronal survival, was used in a separate experiment to rule out the possibility that high levels of nonspecific protein may induce any beneficial effects. Results revealed that neither HSA nor STM100 exerted any effects on inflammation, demyelination, and RGC loss-mediated visual loss in EAE ON mice, as predicted. These controls further support the idea that it is a combination of the multiple growth factors and antiinflammatory cytokines present in ST266 that promote its anti-inflammatory and neuroprotective effects. Although it is likely that multiple factors are needed to exert combined effects, it will be useful to identify and assess the most prominent components of ST266 as potential mediators of RGC neuroprotection in future studies. The potential role of intranasal delivery of proteins to the cribriform plate as a novel route to target the eye and optic nerve is supported by the current studies, although specific mechanisms mediating delivery to these tissues remain to be determined. The rapid protein accumulation in rodent eyes within 30 minutes after intranasal administration reported previously (33) suggests proteins spread by local diffusion after gaining direct access to the CNS. Published studies have demonstrated that intranasal delivery of proteins to the CNS primarily occurs through absorption by the olfactory nerves adjacent to the cribriform plate, and the trigeminal nerve (41-43), with deposition of radiolabeled interferon found at lower levels throughout the brain and in the optic nerve 60 minutes after intranasal administration (42). The mechanism of absorption of intranasally applied macromolecules seems to bypass the blood-brain barrier. Instead, the macromolecules rapidly enter the primate CNS along olfactory- and trigeminal-associated extracellular pathways. In similar intranasal delivery studies in cynomolgus monkeys using Evans Blue Dye bound to ST266 proteins, the dye deposited along the nasal turbinates to the cribriform plate, the olfactory bulb and olfactory nerve, and in the orbits surrounding the eyes (unpublished data). It seems that the general proximity of the olfactory nerves within the Khan et al: J Neuro-Ophthalmol 2019; 39: 191-199 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research: Bench to Bedside FIG. 4. ST266 treatment reduces optic nerve inflammation during EAE optic neuritis. A. Optic nerves from PBS (N = 9)- and HSA-treated EAE mice (N = 9) showed significant (*P , 0.05) levels of inflammation compared with controls (N = 5). EAE mice that received either once (N = 9) or twice (N = 9) daily doses of intranasal ST266 from Days 15-30 showed a trend toward, but no significant, attenuation of inflammation. EAE mice that received either once (N = 9) or twice (N = 9) daily intranasal doses of ST266 continuously from Day 15-56 showed a significant reduction of inflammation scores compared with PBS- or HSA-treated EAE mice (@P , 0.05). B. Representative images of H&E stained optic nerves from each group show the relative cell density (original magnification ·20). EAE, experimental autoimmune encephalomyelitis; H&E, hematoxylin and eosin; HSA, human serum albumin; PBS, phosphate‐buffered saline. brain to the optic nerves allows for extracellular transport by diffusion, but additional studies are needed to better understand these mechanisms. Development of potential neuroprotective therapies for optic neuropathies depends not only on identifying drugs capable of preventing RGC damage, but also on the ability to deliver such drugs to the RGC cell bodies in the retina or axons in the optic nerve in a timely manner. A major issue with targeting therapeutic agents to these tissues is the presence of the blood-brain and bloodretinal barriers, which impede large molecular therapeutics from gaining access to the injured retina and optic nerve. Intravitreal injections are now a common delivery method for retinal therapy, currently used to treat millions of patients with degenerative retinal diseases includKhan et al: J Neuro-Ophthalmol 2019; 39: 191-199 ing macular degeneration and diabetic retinopathy (44). However, repeat injections are required, and each injection increases the potential for complications including endophthalmitis, retinal detachment, ocular hypertension, inflammation, or systemic toxicity (45-48). For optic neuropathies possibly requiring neuroprotective therapies on a daily basis, intravitreal delivery is not a viable method. The intranasal delivery method allows for large molecular therapeutics to gain access to the eye and CNS in a rapid noninvasive manner in rodents, making it a potential drug delivery system for targeting neuroprotective compounds to the retina and optic nerve that warrants further investigation in humans. Overall, results confirm significant neuroprotection mediated by intranasal delivery of ST266 in experimental ON and 197 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Basic and Translational Research: Bench to Bedside ACKNOWLEDGMENTS The authors thank Noveome Biotherapeutics, Inc., for providing ST266 for all studies reported in this manuscript. REFERENCES FIG. 5. ST266 treatment reduces demyelination in the optic nerve during EAE optic neuritis. A. Optic nerves from PBS (N = 9)- and HSA-treated EAE (N = 9) mice showed significant (*P , 0.05) levels of demyelination compared with controls (N = 5). EAE mice that received either once (N = 9) or twice (N = 9) daily doses of intranasal ST266 from Days 15-30 showed a trend toward, but no significant, attenuation of demyelination. EAE mice that received either once (N = 9) or twice (N = 9) daily intranasal doses of ST266 continuously from Day 15-56 showed a significant reduction of demyelination scores compared with PBS- or HSA-treated EAE mice (@P , 0.05). B. Representative images of LFB stained optic nerves images from each group show the relative level of myelin staining (original magnification ·20). EAE, experimental autoimmune encephalomyelitis; HSA, human serum albumin; LFB, luxol fast blue; PBS, phosphate‐buffered saline. demonstrate that effects are mediated specifically by ST266 as opposed to growth factors present in STM100 cell culture media or any nonspecific effects of any protein such as HSA. Results further demonstrate that once daily treatment is sufficient to sustain maximal benefits of this novel therapy. STATEMENT OF AUTHORSHIP Category 1: a. conception and design: R. S. Khan, H. Wessel, L. Brown, and K. S. Shindler; b. acquisition of data: R. S. Khan, K. Dine, and K. S. 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