A low molecular weight dextran sulphate, ILB®, for the treatment of amyotrophic lateral sclerosis (ALS): An open-label, single-arm, single-centre, phase II trial.

BACKGROUND: Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig´s disease, is a rare neurological condition and is the most common motor neurone disease. It is a fatal disease with specific loss of motor neurons in the spinal cord, brain stem, and motor cortex leading to progressive paralysis and usually death within five years of diagnosis. There remains no cure for ALS, and management is focused on a combination of neuroprotective medication, respiratory support, and management by multidisciplinary clinics. PATIENTS AND METHODS: This prospective, single-arm, open-label phase II clinical trial of sustained weekly administration of 2 mg/kg ILB® (a low-molecular weight dextran sulphate) was conducted in a single UK hospital. Eligible patients were at least 18 years and had a definite diagnosis of ALS according to El Escorial Criteria. The co-primary outcomes were safety, tolerability, and quantity of ILB® administered. EudraCT number. 2018-000668-28. FINDINGS: Between 18-Apr-2019 and 27-Mar-2020, 11 patients were recruited and treated for up to 38 weeks. There were no treatment terminations or withdrawals. One serious adverse event was reported, which was not related to ILB® and resolved without sequalae. 270 mild/moderate adverse events were reported with no intolerable events occurring during the trial. The total number of ILB® treatments administered per patient ranged from 4 to 38, with a cumulative dose ranging from 745 to 6668 mg. As a result of the COVID-19 pandemic and the high-risk status of study participants, recruitment and treatment was suspended early in Mar-2020. At the long-term follow-up, three patients had died after the trial was halted, between 53 and 62 weeks after their final ILB® injection. INTERPRETATION: Long-term weekly ILB® injections of 2 mg/kg was well tolerated and had an acceptable safety profile in patients with ALS. TRIAL REGISTRATION: EudraCT: 2018-000668-28. clinicaltrials.gov: NCT03705390. This trial adheres to the principles of GCP in the design, conduct, recording and reporting of clinical trials as listed in part 2, "Conditions and Principles which apply to all Clinical Trials" under the header "Principles based on Articles 2 to 5 of the EU GCP Directive" in the Medicines for Human Use Clinical Trials Regulations (as amended in SI 2006/1928). For clarity, the study did not conform to all aspects of the International Conference on Harmonisation (ICH) E6 R2 Guidelines for GCP (also known as 'ICH GCP'). Of note, we did not use an external database, perform 100% source data verification, and only primary outcome data were analysed in parallel by a second, independent statistician.

Optical coherence tomography angiography analysis methods: a systematic review and meta-analysis.

Optical coherence tomography angiography (OCTA) is widely used for non-invasive retinal vascular imaging, but the OCTA methods used to assess retinal perfusion vary. We evaluated the different methods used to assess retinal perfusion between OCTA studies. MEDLINE and Embase were searched from 2014 to August 2021. We included prospective studies including ≥ 50 participants using OCTA to assess retinal perfusion in either global retinal or systemic disorders. Risk of bias was assessed using the National Institute of Health quality assessment tool for observational cohort and cross-sectional studies. Heterogeneity of data was assessed by Q statistics, Chi-square test, and I2 index. Of the 5974 studies identified, 191 studies were included in this evaluation. The selected studies employed seven OCTA devices, six macula volume dimensions, four macula subregions, nine perfusion analyses, and five vessel layer definitions, totalling 197 distinct methods of assessing macula perfusion and over 7000 possible combinations. Meta-analysis was performed on 88 studies reporting vessel density and foveal avascular zone area, showing lower retinal perfusion in patients with diabetes mellitus than in healthy controls, but with high heterogeneity. Heterogeneity was lowest and reported vascular effects strongest in superficial capillary plexus assessments. Systematic review of OCTA studies revealed massive heterogeneity in the methods employed to assess retinal perfusion, supporting calls for standardisation of methodology.

Immune System, Inflammation and Autoantigens in Wet Age-Related Macular Degeneration: Pathological Significance and Therapeutic Importance.

Wet age-related macular degeneration (wAMD) is a chronic inflammation-associated neurodegenerative disease affecting the posterior part of the eye in the aging population. Aging results in the reduced functionality of cells and tissues, including the cells of the retina. Initiators of a chronic inflammatory and pathologic state in wAMD may be a result of the accumulation of inevitable metabolic injuries associated with the maintenance of tissue homeostasis from a young age to over 50. Apart from this, risk factors like smoking, genetic predisposition, and failure to repair the injuries that occur, alongside attempts to rescue the hypoxic outer retina may also contribute to the pathogenesis. Aging of the immune system (immunosenescence) and a compromised outer blood retinal barrier (BRB) result in the exposure of the privileged milieu of the retina to the systemic immune system, further increasing the severity of the disease. When immune-privileged sites like the retina are under pathological stress, certain age- and disease-related conditions may necessitate assistance from cells distant from the resident ones to help restore the functionality of the tissue. As a necessary part of tissue repair, inflammation is a major response to disease and recruits immune cells to the site of damage. We suspect that the specific reparative inflammatory responses are controlled by an autoantigen-T cell-mediated mechanism, a process that may be hindered in wAMD.

Proposed guidelines for appropriate utilization of superficial radiation therapy in management of skin cancers. Zemtsov-Cognetta criteria.

OBJECTIVE: To develop appropriate use criteria (AUC) for the treatment of basal cell and squamous cell carcinoma by superficial radiation therapy (SRT) technique. MATERIAL AND METHODS: Delphi-type discussion of the experts. RESULTS: Presented in Figure 1. CONCLUSION: These AUCs are in compliance both with the position statement of the American Academy of Dermatology (AAD) and the ASTRO Clinical Practice Guideline on this subject. It is further recommended that SRT will be only performed by either a dermatologist who is board certified in Mohs surgery (MDS) and who had adequate SRT training or by radiation oncologists. Hopefully, this publication will stimulate further discussion on this topic.

The mechanism of action of a novel neuroprotective low molecular weight dextran sulphate: New platform therapy for neurodegenerative diseases like Amyotrophic Lateral Sclerosis.

Background: Acute and chronic neurodegenerative diseases represent an immense socioeconomic burden that drives the need for new disease modifying drugs. Common pathogenic mechanisms in these diseases are evident, suggesting that a platform neuroprotective therapy may offer effective treatments. Here we present evidence for the mode of pharmacological action of a novel neuroprotective low molecular weight dextran sulphate drug called ILB®. The working hypothesis was that ILB® acts via the activation of heparin-binding growth factors (HBGF). Methods: Pre-clinical and clinical (healthy people and patients with ALS) in vitro and in vivo studies evaluated the mode of action of ILB®. In vitro binding studies, functional assays and gene expression analyses were followed by the assessment of the drug effects in an animal model of severe traumatic brain injury (sTBI) using gene expression studies followed by functional analysis. Clinical data, to assess the hypothesized mode of action, are also presented from early phase clinical trials. Results: ILB® lengthened APTT time, acted as a competitive inhibitor for HGF-Glypican-3 binding, effected pulse release of heparin-binding growth factors (HBGF) into the circulation and modulated growth factor signaling pathways. Gene expression analysis demonstrated substantial similarities in the functional dysregulation induced by sTBI and various human neurodegenerative conditions and supported a cascading effect of ILB® on growth factor activation, followed by gene expression changes with profound beneficial effect on molecular and cellular functions affected by these diseases. The transcriptional signature of ILB® relevant to cell survival, inflammation, glutamate signaling, metabolism and synaptogenesis, are consistent with the activation of neuroprotective growth factors as was the ability of ILB® to elevate circulating levels of HGF in animal models and humans. Conclusion: ILB® releases, redistributes and modulates the bioactivity of HBGF that target disease compromised nervous tissues to initiate a cascade of transcriptional, metabolic and immunological effects that control glutamate toxicity, normalize tissue bioenergetics, and resolve inflammation to improve tissue function. This unique mechanism of action mobilizes and modulates naturally occurring tissue repair mechanisms to restore cellular homeostasis and function. The identified pharmacological impact of ILB® supports the potential to treat various acute and chronic neurodegenerative disease, including sTBI and ALS.

ILB®, a Low Molecular Weight Dextran Sulphate, Restores Glutamate Homeostasis, Amino Acid Metabolism and Neurocognitive Functions in a Rat Model of Severe Traumatic Brain Injury.

In a previous study, we found that administration of ILB®, a new low molecular weight dextran sulphate, significantly improved mitochondrial functions and energy metabolism, as well as decreased oxidative/nitrosative stress, of brain tissue of rats exposed to severe traumatic brain injury (sTBI), induced by the closed-head weight-drop model of diffused TBI. Using aliquots of deproteinized brain tissue of the same animals of this former study, we here determined the concentrations of 24 amino acids of control rats, untreated sTBI rats (sacrificed at 2 and 7 days post-injury) and sTBI rats receiving a subcutaneous ILB® administration (at the dose levels of 1, 5 and 15 mg/kg b.w.) 30 min post-impact (sacrificed at 2 and 7 days post-injury). Additionally, in a different set of experiments, new groups of control rats, untreated sTBI rats and ILB®-treated rats (administered 30 min after sTBI at the dose levels of 1 or 5 mg/kg b.w.) were studied for their neurocognitive functions (anxiety, locomotor capacities, short- and long-term memory) at 7 days after the induction of sTBI. Compared to untreated sTBI animals, ILB® significantly decreased whole brain glutamate (normalizing the glutamate/glutamine ratio), glycine, serine and γ-aminobutyric acid. Furthermore, ILB® administration restored arginine metabolism (preventing nitrosative stress), levels of amino acids involved in methylation reactions (methionine, L-cystathionine, S-adenosylhomocysteine), and N-acetylaspartate homeostasis. The macroscopic evidences of the beneficial effects on brain metabolism induced by ILB® were the relevant improvement in neurocognitive functions of the group of animals treated with ILB® 5 mg/kg b.w., compared to the marked cognitive decline measured in untreated sTBI animals. These results demonstrate that ILB® administration 30 min after sTBI prevents glutamate excitotoxicity and normalizes levels of amino acids involved in crucial brain metabolic functions. The ameliorations of amino acid metabolism, mitochondrial functions and energy metabolism in ILB®-treated rats exposed to sTBI produced significant improvement in neurocognitive functions, reinforcing the concept that ILB® is a new effective therapeutic tool for the treatment of sTBI, worth being tested in the clinical setting.

A phase II open label clinical study of the safety, tolerability and efficacy of ILB® for Amyotrophic Lateral Sclerosis.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is an invariably lethal progressive disease, causing degeneration of neurons and muscle. No current treatment halts or reverses disease advance. This single arm, open label, clinical trial in patients with ALS investigated the safety and tolerability of a novel modified low molecular weight dextran sulphate (LMW-DS, named ILB®) previously proven safe for use in healthy volunteers and shown to exert potent neurotrophic effects in pre-clinical studies. Secondary endpoints relate to efficacy and exploratory biomarkers. METHODS: Thirteen patients with ALS were treated with 5 weekly subcutaneous injections of ILB®. Safety and efficacy outcome measures were recorded weekly during treatment and at regular intervals for a further 70 days. Functional and laboratory biomarkers were assessed before, during and after treatment. RESULTS: No deaths, serious adverse events or participant withdrawals occurred during or after ILB® treatment and no significant drug-related changes in blood safety markers were evident, demonstrating safety and tolerability of the drug in this cohort of patients with ALS. The PK of ILB® in patients with ALS was similar to that seen in healthy controls. The ILB® injection elicited a transient elevation of plasma Hepatocyte Growth Factor, a neurotrophic and myogenic growth factor. Following the ILB® injections patients reported increased vitality, decreased spasticity and increased mobility. The ALSFRS-R rating improved from 36.31 ± 6.66 to 38.77 ± 6.44 and the Norris rating also improved from 70.61 ± 13.91 to 77.85 ± 14.24 by Day 36. The improvement of functions was associated with a decrease in muscle atrophy biomarkers. These therapeutic benefits decreased 3-4 weeks after the last dosage. CONCLUSIONS: This pilot clinical study demonstrates safety and tolerability of ILB® in patients with ALS. The exploratory biomarker and functional measures must be cautiously interpreted but suggest clinical benefit and have a bearing on the mechanism of action of ILB®. The results support the drug's potential as the first disease modifying treatment for patients with ALS. TRIAL REGISTRATION: EudraCT 2017-005065-47.

Visual deterioration in patients with photoreceptor loss after retinal reattachment surgery.

PURPOSE: Assess the relationship between photoreceptor degeneration and visual function after retinal reattachment surgery (RRS) in a prospective cohort. METHODS: Patients with rhegmatogenous retinal detachment (RRD) were reviewed before and 6 months after vitreoretinal surgery. Optical coherence tomographical thickness of the outer nuclear layer (ONL), outer retinal segment (ORS), retinal pigmented epithelium to ellipsoid zone (RPE-EZ) and external limiting membrane to EZ (ELM-EZ) were recorded 6 months post-operatively. These were compared to best corrected visual acuity (BCVA) and retinal sensitivity (Humphrey visual field). RESULTS: Thirteen macula-off and 8 macula-on RRD patients were included. The mean ONL thickness was higher after macula-on RRD compared to macula-off RRD (97.70 ± 3.62 µm vs. 73.10 ± 4.98 µm). In all RRD eyes, every 1 µm decrease in ONL thickness correlated with a 0.052 dB decrease and in retinal sensitivity and every 1 µm decrease in ORS thickness was associated with a 0.062 dB reduction in retinal sensitivity. ORS, ELM-EZ and RPE-EZ thickness did not correlate with BCVA post-RRS. CONCLUSION: There was greater ONL and ORS thinning following macula-off compared to macula-on RRD. Correlations between ONL and ORS thinning with decreased retinal sensitivity may be explained by RRD-induced photoreceptor death.

Determining the effect of ocular chemical injuries on topical drug delivery.

Ocular chemical injuries (OCIs) commonly cause ocular damage and visual loss and treatment uses topical therapies to facilitate healing and limit complications. However, the impact of chemical injury on corneal barrier function and treatment penetration is unknown. Therefore, the aim of this study was to determine the effect of OCI on drug penetration and absorption. Porcine corneal explants were used to assess histological damage, electrical resistance, and the trans-corneal penetration/corneal adsorption of reference compounds (sodium fluorescein and rhodamine B) and dexamethasone. Corneal explants were injured with either 1 M sulfuric acid, or 1 M sodium hydroxide. Dexamethasone penetration was measured using high-performance liquid chromatography (HPLC) and that of fluorescein and rhodamine using fluorescence. Dexamethasone corneal adsorption was measured using enzyme-linked immunoabsorbant assay (ELISA). Both acid and alkaline injuries reduced trans-corneal electrical resistance. NaOH injury increased hydrophilic fluorescein penetration (NaOH 8.59 ± 1.50E-05 cm.min-1 vs. Hanks' Balanced Salt Solution (HBSS) 1.64 ± 1.01E-06 cm.min-1) with little impact on hydrophobic rhodamine B (1 M NaOH 6.55 ± 2.45E-04 cm.min-1 vs. HBSS 4.60 ± 0.972E-04 cm.min-1) and dexamethasone penetration (1 M NaOH 3.00 ± 0.853E-04 cm.min-1 vs. HBSS 2.69 ± 0.439E-04 cm.min-1). By contrast, H2SO4 decreased trans-corneal penetration of hydrophilic fluorescein (H2SO4 1.16 ± 14.2E-07 cm.min-1) and of hydrophobic dexamethasone (H2SO4 1.88 ± 0.646E-04 cm.min-1) and rhodamine B (H2SO4 4.60 ± 1.42E-05 cm.min-1). Acid and alkaline OCI differentially disrupted the corneal epithelial barrier function. Acid injury reduced penetration of hydrophobic dexamethasone and rhodamine B as well as hydrophilic fluorescein, which may translate clinically into reduced drug penetration after OCI, while alkaline injury increased fluorescein penetration, with minimal effect on dexamethasone and rhodamine B penetration.

Thermosensitive collagen/fibrinogen gels loaded with decorin suppress lesion site cavitation and promote functional recovery after spinal cord injury.

The treatment of spinal cord injury (SCI) is a complex challenge in regenerative medicine, complicated by the low intrinsic capacity of CNS neurons to regenerate their axons and the heterogeneity in size, shape and extent of human injuries. For example, some contusion injuries do not compromise the dura mater and in such cases implantation of preformed scaffolds or drug delivery systems may cause further damage. Injectable in situ thermosensitive scaffolds are therefore a less invasive alternative. In this study, we report the development of a novel, flowable, thermosensitive, injectable drug delivery system comprising bovine collagen (BC) and fibrinogen (FB) that forms a solid BC/FB gel (Gel) immediately upon exposure to physiological conditions and can be used to deliver reparative drugs, such as the naturally occurring anti-inflammatory, anti-scarring agent Decorin, into adult rat spinal cord lesion sites. In dorsal column lesions of adult rats treated with the Gel + Decorin, cavitation was completely suppressed and instead lesion sites became filled with injury-responsive cells and extracellular matrix materials, including collagen and laminin. Decorin increased the intrinsic potential of dorsal root ganglion neurons (DRGN) by increasing their expression of regeneration associated genes (RAGs), enhanced local axon regeneration/sprouting, as evidenced both histologically and by improved electrophysiological, locomotor and sensory function recovery. These results suggest that this drug formulated, injectable hydrogel has the potential to be further studied and translated into the clinic.

Stability of OCT and OCTA in the Intensive Therapy Unit Setting.

To assess the stability of retinal structure and blood flow measures over time and in different clinical settings using portable optical coherence tomography angiography (OCTA) as a potential biomarker of central perfusion in critical illness, 18 oesophagectomy patients completed retinal structure and blood flow measurements by portable OCT and OCTA in the eye clinic and intensive therapy unit (ITU) across three timepoints: (1) pre-operation in a clinic setting; (2) 24-48 h post-operation during ITU admission; and (3) seven days post-operation, if the patient was still admitted. Blood flow and macular structural measures were stable between the examination settings, with no consistent variation between pre- and post-operation scans, while retinal nerve fibre layer thickness increased in the post-operative scans (+2.31 µm, p = 0.001). Foveal avascular zone (FAZ) measurements were the most stable, with an intraclass correlation coefficient of up to 0.92 for right eye FAZ area. Blood flow and structural measures were lower in left eyes than right eyes. Retinal blood flow assessed in patients before and during an ITU stay using portable OCTA showed no systematic differences between the clinical settings. The stability of retinal blood flow measures suggests the potential for portable OCTA to provide clinically useful measures in ITU patients.

ILB® Attenuates Clinical Symptoms and Serum Biomarkers of Oxidative/Nitrosative Stress and Mitochondrial Dysfunction in Patients with Amyotrophic Lateral Sclerosis.

Oxidative/nitrosative stress and mitochondrial dysfunction is a hallmark of amyotrophic lateral sclerosis (ALS), an invariably fatal progressive neurodegenerative disease. Here, as an exploratory arm of a phase II clinical trial (EudraCT Number 2017-005065-47), we used high performance liquid chromatography(HPLC) to investigate changes in the metabolic profiles of serum from ALS patients treated weekly for 4 weeks with a repeated sub-cutaneous dose of 1 mg/kg of a proprietary low molecular weight dextran sulphate, called ILB®. A significant normalization of the serum levels of several key metabolites was observed over the treatment period, including N-acetylaspartate (NAA), oxypurines, biomarkers of oxidative/nitrosative stress and antioxidants. An improved serum metabolic profile was accompanied by significant amelioration of the patients' clinical conditions, indicating a response to ILB® treatment that appears to be mediated by improvement of tissue bioenergetics, decrease of oxidative/nitrosative stress and attenuation of (neuro)inflammatory processes.

ILB® resolves inflammatory scarring and promotes functional tissue repair.

Fibrotic disease is a major cause of mortality worldwide, with fibrosis arising from prolonged inflammation and aberrant extracellular matrix dynamics. Compromised cellular and tissue repair processes following injury, infection, metabolic dysfunction, autoimmune conditions and vascular diseases leave tissues susceptible to unresolved inflammation, fibrogenesis, loss of function and scarring. There has been limited clinical success with therapies for inflammatory and fibrotic diseases such that there remains a large unmet therapeutic need to restore normal tissue homoeostasis without detrimental side effects. We investigated the effects of a newly formulated low molecular weight dextran sulfate (LMW-DS), termed ILB®, to resolve inflammation and activate matrix remodelling in rodent and human disease models. We demonstrated modulation of the expression of multiple pro-inflammatory cytokines and chemokines in vitro together with scar resolution and improved matrix remodelling in vivo. Of particular relevance, we demonstrated that ILB® acts, in part, by downregulating transforming growth factor (TGF)ß signalling genes and by altering gene expression relating to extracellular matrix dynamics, leading to tissue remodelling, reduced fibrosis and functional tissue regeneration. These observations indicate the potential of ILB® to alleviate fibrotic diseases.

Spectroscopic detection of traumatic brain injury severity and biochemistry from the retina.

Traumatic brain injury (TBI) is a major burden on healthcare services worldwide, where scientific and clinical innovation is needed to provide better understanding of biochemical damage to improve both pre-hospital assessment and intensive care monitoring. Here, we present an unconventional concept of using Raman spectroscopy to measure the biochemical response to the retina in an ex-vivo murine model of TBI. Through comparison to spectra from the brain and retina following injury, we elicit subtle spectral changes through the use of multivariate analysis, linked to a decrease in cardiolipin and indicating metabolic disruption. The ability to classify injury severity via spectra of the retina is demonstrated for severe TBI (82.0 %), moderate TBI (75.1 %) and sham groups (69.4 %). By showing that optical spectroscopy can be used to explore the eye as the window to the brain, we lay the groundwork for further exploitation of Raman spectroscopy for indirect, non-invasive assessment of brain chemistry.

Low Molecular Weight Dextran Sulfate (ILB®) Administration Restores Brain Energy Metabolism Following Severe Traumatic Brain Injury in the Rat.

Traumatic brain injury (TBI) is the leading cause of death and disability in people less than 40 years of age in Western countries. Currently, there are no satisfying pharmacological treatments for TBI patients. In this study, we subjected rats to severe TBI (sTBI), testing the effects of a single subcutaneous administration, 30 min post-impact, of a new low molecular weight dextran sulfate, named ILB®, at three different dose levels (1, 5, and 15 mg/kg body weight). A group of control sham-operated animals and one of untreated sTBI rats were used for comparison (each group n = 12). On day 2 or 7 post-sTBI animals were sacrificed and the simultaneous HPLC analysis of energy metabolites, N-acetylaspartate (NAA), oxidized and reduced nicotinic coenzymes, water-soluble antioxidants, and biomarkers of oxidative/nitrosative stress was carried out on deproteinized cerebral homogenates. Compared to untreated sTBI rats, ILB® improved energy metabolism by increasing ATP, ATP/ adenosine diphosphate ratio (ATP/ADP ratio), and triphosphate nucleosides, dose-dependently increased NAA concentrations, protected nicotinic coenzyme levels and their oxidized over reduced ratios, prevented depletion of ascorbate and reduced glutathione (GSH), and decreased oxidative (malondialdehyde formation) and nitrosative stress (nitrite + nitrate production). Although needing further experiments, these data provide the first evidence that a single post-injury injection of a new low molecular weight dextran sulfate (ILB®) has beneficial effects on sTBI metabolic damages. Due to the absence of adverse effects in humans, ILB® represents a promising therapeutic agent for the treatment of sTBI patients.

Inflammatory and Fibrogenic Factors in Proliferative Vitreoretinopathy Development.

Purpose: Proliferative vitreoretinopathy (PVR) occurs in 5%-10% of rhegmatogenous retinal detachment cases and is the principle cause for failure of retinal reattachment surgery. Although there are a number of surgical adjunctive agents available for preventing the development of PVR, all have limited efficacy. Discovering predictive molecular biomarkers to determine the probability of PVR development after retinal reattachment surgery will allow better patient stratification for more targeted drug evaluations. Methods: Narrative literature review. Results: We provide a summary of the inflammatory and fibrogenic factors found in ocular fluid samples during the development of retinal detachment and PVR and discuss their possible use as molecular PVR predictive biomarkers. Conclusions: Studies monitoring the levels of the above factors have found that few if any have predictive biomarker value, suggesting that widening the phenotype of potential factors and a combinatorial approach are required to determine predictive biomarkers for PVR. Translational Relevance: The identification of relevant biomarkers relies on an understanding of disease signaling pathways derived from basic science research. We discuss the extent to which those molecules identified as biomarkers and predictors of PVR relate to disease pathogenesis and could function as useful disease predictors. (http://www.umin.ac.jp/ctr/ number, UMIN000005604).

Rapid assessment of ocular drug delivery in a novel ex vivo corneal model.

Drug delivery by topical application has higher patient acceptance and lower morbidity than intraocular injection, but many ophthalmic treatments are unable to enter the eye or reach the posterior segment after topical application. The first stage towards posterior segment delivery after topical application is ocular surface penetration and existing models are in vivo or use large quantities of tissue. We therefore developed a novel ex vivo model using discs of porcine and human cornea and sclera (5 mm diameter) to assess penetration of a candidate neuroprotective siRNA. siRNA against caspase 2 or control solutions of known penetrance were applied to the corneal epithelial surface and trans-corneal penetration and corneal adsorbance measured at fixed time points. To demonstrate that leakage did not occur, we applied dextran blue, which should not penetrate the intact cornea and did not do so in our model. Fluorescein penetration (0.09%) was less than rhodamine B (6.98%) at 60 min. siCASP2 penetration was 0.01% by 60 min. When the applied siCASP2 was washed off after 2 min, (representing lacrimal drainage) 0.071% penetrated porcine cornea by 60 min and 0.0002% penetrated human cornea and 0.001% penetrated human sclera. Our ex vivo model rapidly and cost-effectively assesses transcorneal penetration of candidate topical therapies, allowing rates of trans-corneal penetration for potential therapies such as siRNA to be evaluated with small quantities of human or animal tissue.

Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema.

Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.

Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Transection is Driven by Cellular Intravitreal Sciatic Nerve Grafts.

Neurotrophic factors (NTF) secreted by Schwann cells in a sciatic nerve (SN) graft promote retinal ganglion cell (RGC) axon regeneration after either transplantation into the vitreous body of the eye or anastomosis to the distal stump of a transected optic nerve. In this study, we investigated the neuroprotective and growth stimulatory properties of SN grafts in which Schwann cells had been killed (acellular SN grafts, ASN) or remained intact (cellular SN grafts, CSN). We report that both intravitreal (ivit) implanted and optic nerve anastomosed CSN promote RGC survival and when simultaneously placed in both sites, they exert additive RGC neuroprotection. CSN and ASN were rich in myelin-associated glycoprotein (MAG) and axon growth-inhibitory ligand common to both the central nervous system (CNS) and peripheral nervous system (PNS) myelin. The penetration of the few RGC axons regenerating into an ASN at an optic nerve transection (ONT) site is limited into the proximal perilesion area, but is increased >2-fold after ivit CSN implantation and increased 5-fold into a CSN optic nerve graft after ivit CSN implantation, potentiated by growth disinhibition through the regulated intramembranous proteolysis (RIP) of p75NTR (the signalling trans-membrane moiety of the nogo-66 trimeric receptor that binds MAG and associated suppression of RhoGTP). Mϋller cells/astrocytes become reactive after all treatments and maximally after simultaneous ivit and optic nerve CSN/ASN grafting. We conclude that simultaneous ivit CSN plus optic nerve CSN support promotes significant RGC survival and axon regeneration into CSN optic nerve grafts, despite being rich in axon growth inhibitory molecules. RGC axon regeneration is probably facilitated through RIP of p75NTR, which blinds axons to myelin-derived axon growth-inhibitory ligands present in optic nerve grafts.