Single Intravitreal Injection Research Articles

Intravitreal Delivery of rAAV2-hSyn-hRS1 Results in Retinal Ganglion Cell-Specific Gene Expression and Retinal Improvement in the Rs1-KO Mouse.

X-linked retinoschisis (XLRS) is a monogenic recessive inherited retinal disease caused by defects in retinoschisin (RS1). It manifests clinically as retinal schisis cavities and a disproportionate reduction of b-wave amplitude compared with the a-wave amplitude. Currently there is no approved treatment. In the last decade, there has been major progress in the development of gene therapy for XLRS. Previous preclinical studies have demonstrated the treatment benefits of hRS1 gene augmentation therapy in mouse models. However, outcomes in clinical trials have been disappointing, and this might be attributed to dysfunctional assembly of RS1 complexes and/or the impaired targeted cells. In this study, the human synapsin 1 gene promoter (hSyn) was used to control the expression of hRS1 to specifically target retinal ganglion cells and our results confirmed the specific expression and functional assembly of the protein. Moreover, our results demonstrated that a single intravitreal injection of rAAV2-hSyn-hRS1 results in architectural restoration of retinal schisis cavities and improvement in vision in a mouse model of XLRS. In brief, this study not only supports the clinical development of the rAAV2-hSyn-hRS1 vector in XLRS patients but also confirms the therapeutic potential of rAAV-based gene therapy in inherited retinal diseases.

Comprehensive Ocular and Systemic Safety Evaluation of Polysialic Acid-Decorated Immune Modulating Therapeutic Nanoparticles (PolySia-NPs) to Support Entry into First-in-Human Clinical Trials.

An inflammation-resolving polysialic acid-decorated PLGA nanoparticle (PolySia-NP) has been developed to treat geographic atrophy/age-related macular degeneration and other conditions caused by macrophage and complement over-activation. While PolySia-NPs have demonstrated pre-clinical efficacy, this study evaluated its systemic and intraocular safety. PolySia-NPs were evaluated in vitro for mutagenic activity using Salmonella strains and E. coli, with and without metabolic activation; cytotoxicity was evaluated based on its interference with normal mitosis. PolySia-NPs were administered intravenously in CD-1 mice and Sprague Dawley rats and assessed for survival and toxicity. Intravitreal (IVT) administration in Dutch Belted rabbits and non-human primates was assessed for ocular or systemic toxicity. In vitro results indicate that PolySia-NPs did not induce mutagenicity or cytotoxicity. Intravenous administration did not show clastogenic activity, effects on survival, or toxicity. A single intravitreal (IVT) injection and two elevated repeat IVT doses of PolySia-NPs separated by 7 days in rabbits showed no signs of systemic or ocular toxicity. A single IVT inoculation of PolySia-NPs in non-human primates demonstrated no adverse clinical or ophthalmological effects. The demonstration of systemic and ocular safety of PolySia-NPs supports its advancement into human clinical trials as a promising therapeutic approach for systemic and retinal degenerative diseases caused by chronic immune activation.

Dose-Related Side Effects of Intravitreal Injections of Humanized Anti-Vascular Endothelial Growth Factor in Rats: Glial Cell Reactivity and Retinal Ganglion Cell Loss.

In a previous study, we documented that the Intravitreal injections (IVIs) of bevacizumab in rats caused a retinal inflammatory response. We now study whether the IVI of other humanized anti-VEGF: ranibizumab and aflibercept also cause an inflammatory reaction in the rat retina and if it depends on the dose administered. Finally, we study whether this reaction affects retinal ganglion cell (RGC) survival. Albino Sprague-Dawley rats received a single IVI of 5 µL of PBS or ranibizumab or aflibercept at the concentration used in clinical practice (10 µg/µL or 40 µg/µL) or at a lower concentration (0.38 µg/µL and 1.5 µg/µL) calculated to obtain within the rat eye the same concentration as in the human eye in clinical practice. Others received a single 5 µL IVI of a polyclonal goat anti-rat VEGF (0.015 µg/µL) or of vehicle (PBS). Animals were processed 7days or 1month later. Retinal whole mounts were immunolabeled for the detection of microglial, macroglial, RGCs, and intrinsically photosensitive RGCs (ipRGCs). Fluorescence and confocal microscopy were used to examine retinal changes, and RGCs and ipRGCs were quantified automatically or semiautomatically, respectively. All the injected substances including the PBS induced detectable side effects, namely, retinal microglial cell activation and retinal astrocyte hypertrophy. However, there was a greater microglial and macroglial response when the higher concentrations of ranibizumab and aflibercept were injected than when PBS, the antibody anti-rat VEGF and the lower concentrations of ranibizumab or aflibercept were injected. The higher concentration of ranibizumab and aflibercept resulted also in significant RGC death, but did not cause appreciable ipRGC death. The IVI of all the substances had some retinal inflammatory effects. The IVI of humanized anti-VEGF to rats at high doses cause important side effects: severe inflammation and RGC death, but not ipRGC death.

CRISPR-Cas9-mediated deletion of carbonic anhydrase 2 in the ciliary body to treat glaucoma

The carbonic anhydrase 2 (Car2) gene encodes the primary isoenzyme responsible for aqueous humor (AH) production and plays a major role in the regulation of intraocular pressure (IOP). The CRISPR-Cas9 system, based on the ShH10 adenovirus-associated virus, can efficiently disrupt the Car2 gene in the ciliary body. With a single intravitreal injection, Car2 knockout can significantly and sustainably reduce IOP in both normal mice and glaucoma models by inhibiting AH production. Furthermore, it effectively delays and even halts glaucomatous damage induced by prolonged high IOP in a chronic ocular hypertension model, surpassing the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide. The clinical application of CRISPR-Cas9 based disruption of Car2 is an attractive therapeutic strategy that could bring additional benefits to patients with glaucoma.

Phase 1 DAVIO Trial: EYP-1901 Bioerodible, Sustained-Delivery Vorolanib Insert in Patients With Wet Age-Related Macular Degeneration

PurposeTo evaluate safety and tolerability of EYP-1901, an intravitreal insert containing vorolanib, a pan–vascular endothelial growth factor (VEGF) receptor inhibitor packaged in a bioerodible delivery technology (Durasert E™) for sustained delivery, in patients with wet age-related macular degeneration (wAMD) previously treated with anti-VEGF therapy. DesignPhase 1, multicenter, prospective, open-label, dose-escalation trial. ParticipantsPatients with wAMD and evidence of prior anti-VEGF therapy response. MethodsPatients received a single intravitreal injection of EYP-1901. Main Outcome MeasuresThe primary objective was to evaluate safety and tolerability of EYP-1901. Secondary objectives assessed biologic activity of EYP-1901 including best-corrected visual acuity (BCVA) and central subfield thickness (CST). Exploratory analyses included reduction in anti-VEGF treatment burden and supplemental injection-free rates. ResultsSeventeen patients enrolled in the 440 μg (3 patients), 1030 μg (1 patient), 2060 μg (8 patients), and 3090 μg (5 patients) dose cohorts. No dose-limiting toxicity, ocular serious adverse events (AEs), or systemic AEs related to EYP-1901 were observed. There was no evidence of ocular or systemic toxicity related to vorolanib or the delivery technology. Moderate ocular treatment-emergent AEs (TEAEs) included reduced visual acuity (2/17) and retinal exudates (3/17). One patient with reduced BCVA had 3 separate reductions of 17, 18, and 16 letters, and another had a single drop of 25 letters. One severe TEAE, neovascular AMD (i.e., worsening/progressive disease activity), was reported in 1 of 17 study eyes but deemed unrelated to treatment. Mean change from baseline (CFB) in BCVA was –3.7 letters and –5.4 letters at 6 and 12 months. Mean CFB in CST was +1.7 μm and +2.3 μm at 6 and 12 months. Reduction in treatment burden was 76% and 72% at 6 and 12 months. Of 16 study eyes, 13, 9, and 6 were injection-free up to 3, 6, and 12 months. ConclusionIn the DAVIO trial (ClinicalTrials.gov identifier, NCT04747197), EYP-1901 had a favorable safety profile and was well tolerated in previously treated eyes with wAMD. Measures of biologic activity remained relatively stable following a single EYP-1901 injection. These preliminary data support ongoing Phase 2 and planned Phase 3 trials to assess efficacy and safety.

Клинико-лабораторные факторы риска рецидива (реактивации) ретинопатии недоношенных после интравитреального введения афлиберцепта

Purpose. To study the causes of reactivation of retinopathy of prematurity (ROP) after intravitreal aflibercept injections. Material and methods. This study was carried out at V.F. Voino-Yasenetsky Scientific and Practical Center for Specialized Medical Care in Children from September 2021 to February 2023. The study included 101 premature infants (202 eyes) with retinopathy of prematurity who received intravitreal administration of aflibercept. The patients were divided into two groups: the control group included patients who received a single intravitreal injection of a VEGF inhibitor (aflibercept) with a positive effect. The main group included children who received two or more intravitreal injections of VEGF inhibitors (aflibercept) after reactivation of ROP. All patients were monitored for reactivation of retinopathy of prematurity after intravitreal injection of aflibercept. Results. The study noted a statistically significant difference in blood pH levels, stage of ROP, and APROP. In the group with reactivation ROP, APROP was noted 2 times more often (48.94% versus 27.78% in the control group). The group with more intravitreal aflibercept injections had lower blood pH levels (7.348±0.044 versus 7.372±0.030 in the control group). Conclusion. The study showed that clinical and paraclinical risk factors for the development of recurrent (reactivation) of ROP after intravitreal treatment with aflibercept are: severe eye disease (stage of ROP, aggressive posterior ROP), a change in the acid-base balance of the blood towards increased acidity. Blood acidity reflects a generalized process of impaired angiogenesis throughout the body. The more severe the patient’s initial eye condition, the higher the incidence of reactivation of ROP. Key words: recurrence, reactivation, retinopathy of prematurity, risk factor, clinical and paraclinical, aflibercept

Частота рецидивов ретинопатии недоношенных после введения ингибиторов ангиогенеза (афлиберцепта) в зависимости от массы тела ребенка

Purpose. To study the effectiveness of treatment and the causes of reactivation of retinopathy (ROP) in premature children after aflibercept injection. Material and methods. This study was conducted at the V.F. Voino-Yasenetsky Scientific and Practical Center for Specialized Medical Care in Children from September 2021 to February 2023. The study included 122 premature infants (244 eyes) with ROP who received intravitreal administration of aflibercept. Patients were divided into two groups: the main group (reactivation group) consisted of children who received two or more intravitreal injections of VEGF inhibitors (aflibercept) and the control group consisted of children who received a single intravitreal injection of VEGF inhibitor (aflibercept) with a positive effect. In addition, 3 subgroups were identified depending on birth weight: 37 children had a birth weight of less than 900 g (1st subgroup), 41 children weighed from 900 to 1200 g (2nd subgroup), and 44 children weighed more than 1200 g (3rd subgroup). All patients were monitored for reactivation of ROP after intravitreal injection of aflibercept. Results. Since 2010, we have been using angiogenesis inhibitors in the treatment of ROP. The effectiveness of one dose of aflibercept (0,5 mg/0,0125 ml) is 84,84%. Repeated injections are easily tolerated by patients and increase the effectiveness of treatment to 99,76%. The work is devoted to the analysis of reactivation of ROP after one injection of aflibercept and the study of the reasons for repeated injections. 37/244 eyes (15,16%) showed reactivation of ROP after one injection. Factors influencing the reactivation of retinopathy of prematurity after intravitreal injection of aflibercept include: gestational age, postconceptual age at the 1st injection, birth weight, weight at the 1st injection, birth height, newborn head circumference, head circumference at the 1st injection, newborn chest circumference, history of blood transfusions, body mass index (BMI) at the 1st injection, nuchal cord complications. A large dependence of the reactivation rate on the child’s weight was noted. The birth weight subgroup of less than 900 g had a recurrence rate of 28,38%, followed by the average birth weight subgroup (12,20%) and the subgroup weighing more than 1200 g (6,82%). The reactivation time in the three subgroups of patients was 9,82±5,76, 10,20±3,27 and 12,33±9,24 weeks, respectively. The time of reactivation was also determined by the child’s weight. Reactivation occurred earlier in children with lower weight: in three subgroups of patients, 9.82±5.76, 10.20±3.27 and 12.33±9.24 weeks, respectively. Conclusion. Based on our research, the frequency of reactivation of ROP depends on the weight of the premature baby. A change in weight, even by 100 grams, changes the course of ROP. The lower the birth weight is, the higher the reactivation rate of ROP becomes. Reactivation of ROP occurs 4 times more often (in every 3–4 children) in children with very low birth weight (less than 900 g). The frequency of reactivation is also determined by the weight of the child during the first injection of an angiogenesis inhibitor. Weight also determines the time of reactivation, occurring in children weighing less than 900 or 9–10 weeks after the first injection. If there are risk factors, it is advisable to: carry out simultaneous injections in both eyes, which will increase the dose of aflibercept in total, carefully monitor them in the first 2 months with examination of the fundus, or increase the dose of the drug. Effectiveness can be enhanced by ingesting synergists or impact of physiotherapy. Key words: recurrence, reactivation, retinopathy of prematurity, risk factor, birth weight, intravitreal injection, aflibercept, vascular endothelial growth factor

Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma

Mutations in myocilin (MYOC) are the leading known genetic cause of primary open-angle glaucoma, responsible for about 4% of all cases. Mutations in MYOC cause a gain-of-function phenotype in which mutant myocilin accumulates in the endoplasmic reticulum (ER) leading to ER stress and trabecular meshwork (TM) cell death. Therefore, knocking out myocilin at the genome level is an ideal strategy to permanently cure the disease. We have previously utilized CRISPR/Cas9 genome editing successfully to target MYOC using adenovirus 5 (Ad5). However, Ad5 is not a suitable vector for clinical use. Here, we sought to determine the efficacy of adeno-associated viruses (AAVs) and lentiviruses (LVs) to target the TM. First, we examined the TM tropism of single-stranded (ss) and self-complimentary (sc) AAV serotypes as well as LV expressing GFP via intravitreal (IVT) and intracameral (IC) injections. We observed that LV_GFP expression was more specific to the TM injected via the IVT route. IC injections of Trp-mutant scAAV2 showed a prominent expression of GFP in the TM. However, robust GFP expression was also observed in the ciliary body and retina. We next constructed lentiviral particles expressing Cas9 and guide RNA (gRNA) targeting MYOC (crMYOC) and transduction of TM cells stably expressing mutant myocilin with LV_crMYOC significantly reduced myocilin accumulation and its associated chronic ER stress. A single IVT injection of LV_crMYOC in Tg-MYOCY437H mice decreased myocilin accumulation in TM and reduced elevated IOP significantly. Together, our data indicates, LV_crMYOC targets MYOC gene editing in TM and rescues a mouse model of myocilin-associated glaucoma.

Wide-Field Choroidal Thickness Analysis after Half-Fluence Photodynamic Therapy Combined with Intravitreal Aflibercept Injection in Pachychoroid Neovasculopathy.

Background: Pachychoroid neovasculopathy (PNV) is a pachychoroid-spectrum disease. As blood circulation throughout the choroid may be involved in PNV pathogenesis, analysis using ultra-wide-field (UWF) fundus imaging is crucial. We evaluated choroidal thickness after half-fluence photodynamic therapy (PDT) combined with intravitreal aflibercept injection for PNV using UWF swept-source optical coherence tomography. Methods: Seventeen eyes with PNV that underwent half-fluence PDT with an adjuvant single intravitreal aflibercept injection were analyzed. To compare choroidal thicknesses in the central and peripheral choroids, we set subfields <3, <9, and 9-18 mm from the fovea. The <9 and 9-18 mm subfields were divided into four quadrants. Results: Choroidal thickness in each subfield decreased significantly after half-fluence PDT (p < 0.001); this reduction was more pronounced in the central area. We also investigated the relationship between the dominant side of the deep choroidal veins that harbor choroidal vein efflux from the macula. When choroidal thickness in the supratemporal and infratemporal 9 mm subfields were evaluated, the ratio of choroidal thickness reduction was not significantly different between the dominant and non-dominant sides. The dominant side was not associated with the extent of choroidal thickness reduction in PNV. Conclusions: Half-fluence PDT caused thinning of the entire choroid, especially in the central area, in PNV.

In-situ forming biodegradable implants for sustained Fluocinolone acetonide release to the posterior eye: In-vitro and in-vivo investigations in rabbits

Delivering medication to the posterior segment of the eye presents a significant challenge. Intravitreal injection has emerged as the preferred method for drug delivery to this area. However, current injectable non-biodegradable implants for fluocinolone acetonide (FA) require surgical removal after prolonged drug release, potentially affecting patient compliance. This study aimed to develop an in-situ forming biodegradable implant (ISFBI) optimal formulation containing PLGA504H and PLGA756S (50:50 w/w%) with the additive NMP solvent. The goal was to achieve slow and controlled release of FA over a two-month period with lower burst release, following a single intravitreal injection. Through morphology, rheology, stability and in-vitro release evaluations, the optimal formulation demonstrated low viscosity (0.12–1.25 Pa. s) and sustained release of FA at a rate of 0.36 µg/day from the third day up to two months. Furthermore, histopathology and in-vivo studies were conducted after intravitreal injection of the optimal formulation in rabbits’ eye. Pharmacokinetic analysis demonstrated mean residence time (MRT) of 20.02 ± 0.6 days, half-life (t1/2) of 18.80 ± 0.4 days, and clearance (Cl) of 0.29 ± 0.03 ml/h for FA in the vitreous humor, indicating sustained and slow absorption of FA by the targeted retinal tissue from vitrea over the two-month period and eliminating through the anterior section of the eye, as revealed by its presence in the aqueous humor. Additionally, FA exhibited no detection in the blood and no evidence of systemic side effects or damage on the retinal layer and other organs. Based on these findings, it can be concluded that in-situ forming injectable biodegradable PLGA implants can show promise as a long-acting and controlled-release system for intraocular drug delivery.

A Sustainable Retinal Drug Co-Delivery for Boosting Therapeutic Efficacy in wAMD: Unveiling Multifaceted Evidence and Synergistic Mechanisms.

Sustainable retinal codelivery poses significant challenges technically, although it is imperative for synergistic treatment of wet age-related macular degeneration (wAMD). Here, a microemulsion-doped hydrogel (Bor/PT-M@TRG) is engineered as an intravitreal depot composing of temperature-responsive hydrogel (TRG) and borneol-decorated paeoniflorin (PF) & tetramethylpyrazine (TMP)-coloaded microemulsions (Bor/PT-M). Bor/PT-M@TRG, functioning as the "ammunition depot", resides in the vitreous and continuously releases Bor/PT-M as the therapeutic "bullet", enabling deep penetration into the retina for 21 days. A single intravitreal injection of Bor/PT-M@TRG yields substantial reductions in choroidal neovascularization (CNV, a hallmark feature of wAMD) progression and mitigates oxidative stress-induced damage in vivo. Combinational PF&TMP regulates the "reactive oxygen species/nuclear factor erythroid-2-related factor 2/heme oxygenase-1" pathway and blocks the "hypoxia inducible factor-1α/vascular endothelial growth factor" signaling in retina, synergistically cutting off the loop of CNV formation. Utilizing fluorescence resonance energy transfer and liquid chromatography-mass spectrometry techniques, they present compelling multifaceted evidence of sustainable retinal codelivery spanning formulations, ARPE-19 cells, in vivo eye balls, and ex vivo section/retina-choroid complex cell levels. Such codelivery approach is elucidated as the key driving force behind the exceptional therapeutic outcomes of Bor/PT-M@TRG. These findings highlight the significance of sustainable retinal drug codelivery and rational combination for effective treatment of wAMD.

Topical Ophthalmic Liposomes Dual-Modified with Penetratin and Hyaluronic Acid for the Noninvasive Treatment of Neovascular Age-Related Macular Degeneration.

Since intrinsic ocular barrier limits the intraocular penetration of therapeutic protein through eye drops, repeated intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents are the standard therapy for neovascular age-related macular degeneration (nAMD), which are highly invasive and may cause particular ocular complications, leading to poor patient compliance. Using Penetratin (Pen) as the ocular penetration enhancer and hyaluronic acid (HA) as the retina-targeting ligand, a dual-modified ophthalmic liposome (Penetratin hyaluronic acid-liposome/Conbercept, PenHA-Lip/Conb) eye drop was designed to non-invasively penetrate the ocular barrier and deliver anti-VEGF therapeutic agents to the targeted intraocular tissue. PenHA-Lip effectively penetrates the ocular barrier and targets the retinal pigment epithelium via corneal and non-corneal pathways. After a single topical administration of conbercept-loaded PenHA-Lip (PenHA-Lip/Conb), the intraocular concentration of conbercept peaked at 18.74 ± 1.09 ng/mL at 4 h, which is 11.55-fold higher than unmodified conbercept. In a laser-induced choroidal neovascularization (CNV) mouse model, PenHA-Lip/Conb eye drops three times daily for seven days inhibited CNV formation and progression without any significant tissue toxicity and achieved an equivalent effect to a single intravitreal conbercept injection. PenHA-Lip efficiently and safely delivered conbercept to the posterior eye segment and may be a promising noninvasive therapeutic option for nAMD.

Intravitreal injection of Noggin ameliorates retinal degeneration in a mouse model of retinitis pigmentosa

Purpose: To determine the therapeutic efficacy of intravitreal injections of Noggin in a rodent model of retinitis pigmentosa (RP). Noggin is expressed by Myo/Nog cells which are critical for eye morphogenesis and have been shown to be both neuroprotective and a source of contractile myofibroblasts in ocular diseases. Noggin is a bone morphogenic protein (BMP) inhibitor. BMPs play important roles in eye development but the strength of this signalling pathway must be carefully regulated. Furthermore, BMP levels increase in the central nervous system under stress and injury.Methods: Male and female C3H/HEJ mice (n = 30) exhibiting Pde6b gene mutation, considered a model of RP, were divided into 3 experimental groups (n = 10 each). At week 2.5 post birth, mice received either a bilateral, single intravitreal injection (1 μL) of phosphate buffer saline (PBS; Control 1), mouse serum albumin (MSA; Control 2), or human recombinant Noggin (25 ng; Treatment). At weeks 3 and 5, electroretinograms (ERGs) were performed to assess retinal function, and mice were sacrificed. Then, eyes were enucleated, fixed, frozen, cryosectioned, and immunolabelled to evaluate apoptosis (TUNEL), Myo/Nog cells (BAI1), and neuroretinal thickness.Results: Apoptosis was significantly decreased in the Noggin treated group compared to controls (PBS and MSA) at both time points. Noggin treatment also resulted in increased thickness of the neuroretina. The number of Noggin producing Myo/Nog cells was higher in the retinas of all the control groups compared to the Noggin‐injected group. ERGs showed significant improvement of the A‐ and B‐waves in Noggin injected mice when compared to control groups at 3‐ and 5‐weeks post‐birth.Conclusions: Intravitreal injection of Noggin in a RP rodent model improved retinal function and decreased apoptosis and retinal thinning. Myo/Nog cells increase in number in response to stress and injury. The decrease in the number of Myo/Nog cells in response to Noggin treatment further supports the conclusion that this BMP inhibitor reduced tissue damage. These results suggest that inhibition of BMP signalling may be a therapeutic approach to reducing retinal degeneration in RP.

Delayed noninfectious intraocular inflammation and retinal vasculitis after single intravitreal Brolucizumab® injection for polypoidal choroidal vasculopathy

A 52-year-old woman with extrafoveal polypoidal choroidal vasculopathy (PCV) in her right eye was treated with a single intravitreal injection of 6 mg/0.05 ml brolucizumab (BZB) and focal thermal laser therapy. The patient complained of redness and blurred vision in her right eye for the previous week at the 4-week follow-up visit. PCV-related clinical findings revealed complete regression. There was circumcorneal congestion, diffuse nongranulomatous keratic precipitates, grade 2+ anterior chamber flare and cells, grade 1+ vitreous haze, posterior vitreous cells, hyperemic disc with perivascular sheathing, and retinal vasculitis. Following treatment with topical and systemic steroids, the intraocular inflammation (IOI) worsened, and vision was reduced to 6/60 after a week of steroid therapy at a subsequent follow-up visit. A pars plana vitrectomy was performed, and vitreous cytology revealed small lymphocytes with no evidence of intraocular infection. At the 6-month follow-up visit, the patient’s vision had improved to 6/6 and the signs of IOI had resolved. This case demonstrates that even after a single intravitreal injection of BZB, an eye that had previously received no antivascular endothelial growth factor injection can develop IOI.

Idiosyncratic responses of RGCs to injury and protection

Objectives: The objectives of the study were to investigate the responses of different retinal ganglion cell (RGC) populations to diverse injuries [transient ischemia induced by acute ocular hypertension (AOH), excitotoxicity induced by intravitreal injection of 100 mM N‐methyl D‐aspartate (NMDA) or intra‐orbital optic nerve transection (IONT)] and protection with selective agonists of the tropomyosin related kinase B (TrkB) receptor, brain‐derived neurotrophic factor (BDNF) or 7,8‐Dihydroxyflavone (DHF) or with a voltage‐dependent calcium channel blocker (ITH12657).Methods: Adult Sprague–Dawley rats were used. In the first group, the left eye received a single intravitreal injection of 5 μL vehicle or 5 μg BDNF and was connected to a saline reservoir elevated above the eye to induce an acute ocular hypertension (AOH) and transient ischemia for 75 min. These rats were analysed at different survival intervals from 3 to 45 days.A second group were treated daily subcutaneously with vehicle or ITH5263 (10 mg/kg), or intraperitoneally with vehicle or DHF (5 mg/kg), starting 12 h prior to an intraocular injection of 100 mM N‐methyl‐D‐aspartate (NMDA) to induce retinal excitotoxicity. These rats were analysed at different survival intervals from 3 to 21 days.A third group received a left intra‐orbital optic nerve transection (IONT) to induce axotomy of the RGC population and were treated daily with an intraperitoneal injection of vehicle (0.9% NaCl containing 1% DMSO) or DHF (5 mg/kg diluted in vehicle). These rats were analysed at survival intervals from 3 to 60 days.The retinas were prepared as wholemounts and immunolabelled for Brn3a, melanopsin (m), Osteopontin (OPN) and the T‐box transcription factor T‐brain‐2 (Tbr2) to identify the following retinal ganglion cell populations: Brn3a+, melanopsin+, α‐like (OPN+), α‐ON sustained RGCs (OPN+Tbr2+), α‐ON transient RGCs (OPN+ Brn3a− Tbr2−) and α‐OFF like (OPN+Brn3a+). The labelled RGCs were quantified automatically (Brn3a) or dotted manually and quantified with a graphic software, and distribution of the different populations were represented on topographical maps.Results: Our studies document that:(i) AOH induces progressive loss of Brn3a+RGCs and a significant but not progressive loss of m+RGCs. Treatment with BDNF afforded significant long‐lasting protection for both RGC populations.(ii) Intravitreal injection of NMDA resulted at 7 days in the abrupt significant loss of Brn3a+RGCs, αRGC and αONs‐RGCs without further progression, whereas αOFF‐RGCs died massively. Both m+RGCs and αONtRGCs appear fully resistant to NMDA‐induced excitotoxicity. Both ITH12657 or DHF protect Brn3a+RGC, αRGCs and αONs‐RGCs, but not αOFF‐RGCs.(iii) IONT and vehicle treatment result in a characteristic rapid loss of Brn3a, melanopsin RGCs, αRGCs, αONs‐RGCs and αOFF‐RGCs. DHFs protect Brn3a + RGC, m + RGCs, αRGCs and αONs‐RGCs, but not αOFF‐RGCs.Conclusions: Thus, three different RGC populations (Brn3a+, m+ and α‐like) respond distinctly to different injuries and neuroprotective agents, further adding to the idea that each type of RGC has a unique response to injury and protection. Deciphering the molecular mechanisms of each RGC type will help understand neuronal responses to injury and its possible prevention.

Choroidal neovascularization as first sign of Best's vitelliform dystrophy in a child

Aims/Purpose: To report a case of choroidal neovascularization secondary to Best's vitelliform dystrophy in a child managed by intravitreal aflibercept.Methods: Interventional, case report.Results: A 9‐year‐old boy reported decreased visual acuity (VA) with metamorphopsia in his right eye (RE) for the previous month. VA was RE 20/100 and left eye (LE) 20/20. Funduscopy revealed right‐eye macular oedema with deep macular haemorrhage associated to a bilateral orange‐yellowish deposit. Optical coherence tomography (OCT) showed sub‐ and intraretinal fluid in the foveal area along with a hyperreflective subretinal deposit and a localized retinal pigment epithelium (RPE) elevation. Blue autofluorescence (BAF) imaging disclosed a central hyper autofluorescence area as well as a larger defect corresponding to the haemorrhage and the subretinal deposit. The LE was positive for a central deposit located under the RPE with hyper autofluorescence at BAF. Familiar history was unknown for ocular conditions and the clinical history was irrelevant for fever, inflammation, adenopathy, articular, neurological or respiratory conditions. The condition was diagnosed as choroidal neovascularization (CNV) secondary to Best's vitelliform dystrophy and a single intravitreal injection of aflibercept was performed under general anaesthesia. Genetic study revealed a likely pathogenic mutation c.874G&gt;C (p.Glu292Gln) in the BEST1 gene (missense change, not previously published) affecting a functional dominion. The subretinal bleeding and fluid resolved after one single injection of aflibercept and VA improved to 20/20. No recurrences have been observed during the subsequent five years in either eye.Conclusions: We report a case of CNV as first manifestation of a previously not reported mutation of Best's vitelliform dystrophy in a 9‐year‐old boy that was successfully treated with one single intravitreal injection of aflibercept.

Intravitreal injection of methotrexate in persistent diabetic macular edema: a 6-month study.

Diabetic macular edema (DME) affects approximately 10% of patients with diabetes mellitus. This condition can cause blurred or distorted vision, which significantly affects the quality of life of these patients. We evaluated the therapeutic effects of intravitreal methotrexate (MTX) injections on persistent DME. This prospective interventional case series included patients with confirmed persistent DME that was unresponsive to previous standard treatments. The patients underwent comprehensive eye examinations and macular imaging with optical coherence tomography (OCT). A single intravitreal MTX injection (400 µg MTX in 0.16 mL solution) was administered, followed by patient assessments at 1, 3, and 6 months after injection. Best-corrected distance visual acuity (BCDVA), intraocular pressure (IOP), macular thickness (MT), and central subfield thickness (CST) were measured at baseline and post-injection to evaluate treatment efficacy. We included 33 eyes of 30 patients with a mean (standard deviation [SD], range) age of 62.7 (8.3, 44 to 77) years, of whom 17 (56.7%) were men and 13 (43.3%) were women. All participants had type 2 diabetes mellitus, with a mean (SD, range) duration of 17.0 (6.8, 10 to 31) years. Most participants (n = 27 eyes, 81.8%) had non-proliferative diabetic retinopathy, and six eyes (18.2%) had regressed proliferative diabetic retinopathy. Four eyes (12.1%) had undergone prior macular laser photocoagulation. The mean (SD) number of prior intravitreal bevacizumab injections was 3.4 (0.8), and 29 eyes (87.8%) had received one intravitreal triamcinolone injection. During the study period, a statistically significant difference was observed in CST (P < 0.05); however, no statistically significant differences were observed in BCDVA, MT, or IOP (P > 0.05). Pairwise comparison revealed a significant decrease in CST at 6 months post-injection compared to the baseline value (P < 0.05). During the investigation period, no side effects of MTX, such as macular edema, retinal tears, vitreous hemorrhage, endophthalmitis, or vision loss, were observed. A single intravitreal MTX injection significantly reduced CST in patients with persistent DME, without relevant safety concerns. However, no significant improvement in functional outcomes was observed. Therefore, there is no strong evidence to recommend its use as a treatment for pDME. Further studies, preferably randomized clinical trials with long-term follow-ups, are warranted to assess the long-term efficacy, safety, and potential benefits of intravitreal MTX for the treatment of persistent DME.

Development of uniform fenofibrate-loaded biodegradable microparticle by membrane emulsification

Fenofibrate has shown therapeutic effects on diabetic retinopathy. However, fenofibrate can be rapidly cleared from the eye after a single intravitreal injection. Here, we aim to develop fenofibrate loaded PLGA microparticles (Feno-MP) with high drug loading and sustained in vitro release up to 6 months suitable for intravitreal injection. First, orthogonal array experimental design was applied for formulation optimization. The selected formulation parameters were used to formulate Feno-MP using homogenization method and direct membrane emulsification method. Both methods generated Feno-MP with high drug loading and sustained in vitro drug release more than 140 days. Unlike the polydisperse Feno-MP prepared using homogenization method, membrane emulsification method generated Feno-MP with uniform size distribution. By controlling the membrane pore size, 1.5 µm, 8 µm and 16 µm Feno-MP were formulated and we found that larger Feno-MP demonstrated higher drug loading, more sustained drug release in vitro with less burst drug release than the smaller Feno-MP. In conclusion, we developed Feno-MP with high drug loading and sustained release profile, and elucidated that changing the particle size could have notable impacts on drug loading and release kinetics. Formulating Feno-MP with uniform size distribution by membrane emulsification method would benefit the batch-to-batch repeatability.

Safety and tolerability of intravitreal umedaptanib pegol (anti-FGF2) for neovascular age-related macular degeneration (nAMD): a phase 1, open-label study

ObjectiveTo evaluate the efficacy and safety of a single-dose intravitreal umedaptanib pegol (anti-FGF2, investigational new drug) for the treatment of neovascular age-related macular degeneration (nAMD).MethodsNine participants who had a diagnosis of refractory nAMD were enrolled and received a single intravitreal injection of umedaptanib pegol at increasing doses of 0.2, 1.0 or 2.0 mg in the study eye.ResultsAll three doses of umedaptanib pegol evaluated in the study were safe and well tolerated. No severe adverse event (AE) was observed in the study. There was an improvement in retinal fluid measured by central subfield thickness (CST) in most subjects. Remarkably, all three subjects who received 2.0 mg/eye showed improvement of more than 150 μm.ConclusionsIntravitreal umedaptanib pegol was safe, well tolerated, and demonstrated an indication of bioactivity in participants that have persistent subretinal fluid refractory to the treatment with anti-VEGFs.

Long-Acting Microparticle Formulation of Griseofulvin for Ocular Neovascularization Therapy.

Neovascular age-related macular degeneration (nAMD) is a leading cause of vision loss in older adults. nAMD is treated with biologics targeting vascular endothelial growth factor; however, many patients do not respond to the current therapy. Here, a small molecule drug, griseofulvin (GRF), is used due to its inhibitory effect on ferrochelatase, an enzyme important for choroidal neovascularization (CNV).For local and sustained delivery to the eyes, GRF is encapsulated in microparticles based on poly(lactide-co-glycolide) (PLGA), a biodegradable polymer with a track record in long-acting formulations. The GRF-loaded PLGA microparticles (GRF MPs) are designed for intravitreal application, considering constraints in size, drug loading content, and drug release kinetics. Magnesium hydroxide is co-encapsulated to enable sustained GRF release over >30 days in phosphate-buffered saline with Tween 80. Incubated in cell culture medium over 30 days, the GRF MPs and the released drug show antiangiogenic effects in retinal endothelial cells. A single intravitreal injection of MPs containing 0.18µg GRF releases the drug over 6 weeks in vivo to inhibit the progression of laser-induced CNV in mice with no abnormality in the fundus and retina. Intravitreally administered GRF MPs prove effective in preventing CNV, providing proof-of-concept toward a novel, cost-effective nAMD therapy.