A randomized phase 2 clinical trial of phentolamine mesylate eye drops in patients with severe night vision disturbances.

PURPOSE: Dim light vision disturbances (DLD) comprise a wide range of symptoms affecting the quality of vision at low illumination including glare, halos, and starbursts. This exploratory study investigated 1.0% phentolamine mesylate ophthalmic solution (PMOS) as a treatment to improve vision and image quality for patients with DLD. METHODS: In this placebo-controlled, randomized, double-masked clinical trial, 24 adult patients with severe DLD were randomized in a 2:1 ratio to receive either one dose of PMOS or placebo. Subjects were eligible if they reported experiencing severe night vision difficulty that was not eliminated by distance spectacle correction and scored ≥0.3 log units below the normal range of contrast sensitivity assessed under mesopic conditions with glare at ≥2 spatial frequencies. Key efficacy outcomes were change from baseline in pupil diameter, contrast sensitivity, and visual acuity. Safety measures including intraocular pressure, conjunctival hyperemia, and systemic effects were also assessed. RESULTS: Eight subjects were randomized to placebo (63% female; mean age 47 years) and 16 were randomized to PMOS (75% female; mean age 42 years). Mean (SD) pupil diameter of PMOS-treated subjects decreased significantly - 1.3 mm (0 to - 2.8 mm) with p < 0.0001. Mean contrast sensitivity with glare in PMOS-treated subjects improved significantly post-treatment at spatial frequencies 3, 6, 12, and 18 cycles per degree (p ≤ 0.03). PMOS also demonstrated improvements in the numbers of letters read for mesopic and photopic, high- and low-contrast visual acuity (LCVA). Importantly, a statistically greater proportion of PMOS-treated eyes registered mesopic LCVA 5 letter (69% vs. 31%, p = 0.029) and 10 letter (34% vs. 6%, p = 0.04) improvement, with a trend at 15 letters (19% vs. 0%, p = 0.16). PMOS was well tolerated with the only reported side effect being a mild increase in conjunctival hyperemia. CONCLUSION: PMOS was well tolerated and effectively reduced pupil size with improvements in contrast sensitivity and visual acuity in adults with severe DLD. Future Phase 3 studies should be conducted to further evaluate its potential to treat DLD. TRIAL REGISTRATION: The trial registration number is NCT04004507 (02/07/2019). Retrospectively registered.

Phentolamine Eye Drops Reverse Pharmacologically Induced Mydriasis in a Randomized Phase 2b Trial.

SIGNIFICANCE: After a dilated eye examination, many patients experience symptoms of prolonged light sensitivity, blurred vision, and cycloplegia associated with pharmacological mydriasis. Phentolamine mesylate ophthalmic solution (PMOS) may expedite the reversal of mydriasis in patients, potentially facilitating return to functional vision and reducing barriers to obtaining dilated eye examinations. PURPOSE: The protracted reversal time after pharmacologically induced pupil dilation impairs vision. We tested the hypothesis that PMOS rapidly reduces pupil diameter in this acute indication. METHODS: In this double-masked placebo-controlled, randomized, two-arm crossover phase 2b trial, we evaluated the effects of one drop of 1% PMOS applied bilaterally in subjects who had their pupils dilated by one of two common mydriatic agents: 2.5% phenylephrine or 1% tropicamide. End points included change in pupil diameter, percent of subjects returning to baseline pupil diameter, and accommodative function at multiple time points. RESULTS: Thirty-one subjects completed the study (15 dilated with phenylephrine and 16 with tropicamide). Change in pupil diameter from baseline at 2 hours after maximal dilation with 1% PMOS was -1.69 mm and was significantly greater in magnitude compared with placebo for every time point beyond 30 minutes (P < .05). At 2 hours, a greater percentage of study eyes given 1% PMOS returned to baseline pupil diameter compared with placebo (29 vs. 13%, P = .03), which was this also seen at 4 hours (P < .001). More subjects treated with PMOS in the tropicamide subgroup had at least one eye returning to baseline accommodative amplitude at 2 hours (63 vs. 38%, P = .01). There were no severe adverse events, with only mild to moderate conjunctival hyperemia that resolved in most patients by 6 hours. CONCLUSIONS: Phentolamine mesylate ophthalmic solution at 1% reversed medically induced pupil dilation more rapidly than placebo treatment regardless of which mydriatic was used (adrenergic agonists and cholinergic blockers) with a tolerable safety profile.

Phentolamine Mesylate Ophthalmic Solution Provides Lasting Pupil Modulation and Improves Near Visual Acuity in Presbyopic Glaucoma Patients in a Randomized Phase 2b Clinical Trial.

PURPOSE: Phentolamine mesylate ophthalmic solution (PMOS), applied to the eye topically, was shown previously to have beneficial effects in patients with dim light vision disturbances (DLD), including decreased pupil diameter (PD), improved best-corrected distance visual acuity (BCDVA), as well as lower intraocular pressure (IOP). The ORION-1 trial evaluated the long-term safety and efficacy of PMOS in a glaucomatous, presbyopic population. PATIENTS AND METHODS: In this randomized, double-masked, multi-center, placebo-controlled, multiple-dose Phase 2b trial, 39 patients with elevated IOP were randomized to receive one evening dose of study medication or placebo for 14 days. The primary outcome measure was mean change in diurnal IOP, and the key secondary outcome measures included changes in PD, distance-corrected near visual acuity (DCNVA), and conjunctival hyperemia. RESULTS: Use of 1% PMOS did not lead to a statistically significant decrease in diurnal IOP compared to placebo (P = 0.89) but trended toward a greater decrease in patients with lower IOP baselines. PMOS produced a statistically significant mean 20% PD reduction under both photopic and mesopic conditions that was sustained for 36 hours post-dosing. A statistically significant number of patients with PMOS compared to placebo demonstrated ≥1 line of improvement in photopic DCNVA at day 8 (P = 0.0018), day 15 (P = 0.0072), and day 16 (P = 0.0163), with a trend for 2- and 3-line improvements at all time points. There was no statistical difference in conjunctival hyperemia compared to placebo. CONCLUSION: Although mean IOP was not lowered significantly, daily evening dosing of 1% PMOS was found to be well tolerated with no daytime conjunctival redness and demonstrated improvement in DCNVA with sustained PD reduction in a glaucomatous and presbyopic population. Smaller pupil size can have beneficial effects in improving symptoms of presbyopia and DLD, which will be the focus of further studies.

MBNL Sequestration by Toxic RNAs and RNA Misprocessing in the Myotonic Dystrophy Brain.

For some neurological disorders, disease is primarily RNA mediated due to expression of non-coding microsatellite expansion RNAs (RNA(exp)). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain, resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound-knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNA(exp) in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases.

Loss of MBNL leads to disruption of developmentally regulated alternative polyadenylation in RNA-mediated disease.

Inhibition of muscleblind-like (MBNL) activity due to sequestration by microsatellite expansion RNAs is a major pathogenic event in the RNA-mediated disease myotonic dystrophy (DM). Although MBNL1 and MBNL2 bind to nascent transcripts to regulate alternative splicing during muscle and brain development, another major binding site for the MBNL protein family is the 3' untranslated region of target RNAs. Here, we report that depletion of Mbnl proteins in mouse embryo fibroblasts leads to misregulation of thousands of alternative polyadenylation events. HITS-CLIP and minigene reporter analyses indicate that these polyadenylation switches are a direct consequence of MBNL binding to target RNAs. Misregulated alternative polyadenylation also occurs in skeletal muscle in a mouse polyCUG model and human DM, resulting in the persistence of neonatal polyadenylation patterns. These findings reveal an additional developmental function for MBNL proteins and demonstrate that DM is characterized by misregulation of pre-mRNA processing at multiple levels.

Compound loss of muscleblind-like function in myotonic dystrophy.

Myotonic dystrophy (DM) is a multi-systemic disease that impacts cardiac and skeletal muscle as well as the central nervous system (CNS). DM is unusual because it is an RNA-mediated disorder due to the expression of toxic microsatellite expansion RNAs that alter the activities of RNA processing factors, including the muscleblind-like (MBNL) proteins. While these mutant RNAs inhibit MBNL1 splicing activity in heart and skeletal muscles, Mbnl1 knockout mice fail to recapitulate the full-range of DM symptoms in these tissues. Here, we generate mouse Mbnl compound knockouts to test the hypothesis that Mbnl2 functionally compensates for Mbnl1 loss. Although Mbnl1(-/-) ; Mbnl2(-/-) double knockouts (DKOs) are embryonic lethal, Mbnl1(-/-) ; Mbnl2(+/-) mice are viable but develop cardinal features of DM muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in Mbnl1(-/-) knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the hypothesis that compound loss of MBNL function is a critical event in DM pathogenesis and provide novel mouse models to investigate additional pathways disrupted in this RNA-mediated disease.

Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

Muscleblind-like 2-mediated alternative splicing in the developing brain and dysregulation in myotonic dystrophy.

The RNA-mediated disease model for myotonic dystrophy (DM) proposes that microsatellite C(C)TG expansions express toxic RNAs that disrupt splicing regulation by altering MBNL1 and CELF1 activities. While this model explains DM manifestations in muscle, less is known about the effects of C(C)UG expression on the brain. Here, we report that Mbnl2 knockout mice develop several DM-associated central nervous system (CNS) features including abnormal REM sleep propensity and deficits in spatial memory. Mbnl2 is prominently expressed in the hippocampus and Mbnl2 knockouts show a decrease in NMDA receptor (NMDAR) synaptic transmission and impaired hippocampal synaptic plasticity. While Mbnl2 loss did not significantly alter target transcript levels in the hippocampus, misregulated splicing of hundreds of exons was detected using splicing microarrays, RNA-seq, and HITS-CLIP. Importantly, the majority of the Mbnl2-regulated exons examined were similarly misregulated in DM. We propose that major pathological features of the DM brain result from disruption of the MBNL2-mediated developmental splicing program.

Developments in RNA splicing and disease.

Pre-mRNA processing, including 5'-end capping, splicing, editing, and polyadenylation, consists of a series of orchestrated and primarily cotranscriptional steps that ensure both the high fidelity and extreme diversity characteristic of eukaryotic gene expression. Alternative splicing and editing allow relatively small genomes to encode vast proteomic arrays while alternative 3'-end formation enables variations in mRNA localization, translation, and stability. Of course, this mechanistic complexity comes at a high price. Mutations in the myriad of RNA sequence elements that regulate mRNA biogenesis, as well as the trans-acting factors that act upon these sequences, underlie a number of human diseases. In this review, we focus on one of these key RNA processing steps, splicing, to highlight recent studies that describe both conventional and novel pathogenic mechanisms that underlie muscle and neurological diseases.

Partners in crime: bidirectional transcription in unstable microsatellite disease.

Nearly two decades have passed since the discovery that the expansion of microsatellite trinucleotide repeats is responsible for a prominent class of neurological disorders, including Huntington disease and fragile X syndrome. These hereditary diseases are characterized by genetic anticipation or the intergenerational increase in disease severity accompanied by a decrease in age-of-onset. The revelation that the variable expansion of simple sequence repeats accounted for anticipation spawned a number of pathogenesis models and a flurry of studies designed to reveal the molecular events affected by these expansions. This work led to our current understanding that expansions in protein-coding regions result in extended homopolymeric amino acid tracts, often polyglutamine or polyQ, and deleterious protein gain-of-function effects. In contrast, expansions in noncoding regions cause RNA-mediated toxicity. However, the realization that the transcriptome is considerably more complex than previously imagined, as well as the emerging regulatory importance of antisense RNAs, has blurred this distinction. In this review, we summarize evidence for bidirectional transcription of microsatellite disease genes and discuss recent suggestions that some repeat expansions produce variable levels of both toxic RNAs and proteins that influence cell viability, disease penetrance and pathological severity.

Pathogenic RNAs in microsatellite expansion disease.

The expansion of unstable microsatellites is the cause of a number of inherited neuromuscular and neurological disorders. While these expanded repeats can be located in either the coding or non-coding regions of genes, toxic RNA transcripts have been primarily implicated in the pathogenesis of non-coding expansion diseases. In this review, we briefly summarize studies which support this RNA-mediated toxicity model for several neurologic disorders and highlight how pathogenic RNAs might negatively impact nervous system functions. However, it is important to note that the distinction between coding versus non-coding regions has become muddled by recent observations that the transcribed portion of the genome or transcriptome is considerably larger than previously appreciated. Thus, we also explore the possibility that a combination of protein and RNA gain-of-function events underlie some microsatellite expansion diseases.