Aprepitant Restores Corneal Sensitivity and Reduces Pain in DED.

Purpose: This study aims to assess the efficacy of two aprepitant formulations (X1 and X2), in a preclinical model of dry eye disease (DED) induced by benzalkonium chloride (BAK). Methods: Two aprepitant formulations were tested on 7 to 8-week-old male mice for their efficacy. In vivo corneal fluorescein staining assessed epithelial damage as the primary end point on days 0, 3, 5, 7, 9, 12, and 14 using slit-lamp microscopy. The DED model was induced with 0.2% BAK twice daily for the first week and once daily for the next week. Mice were randomly assigned to 5 treatment groups: Aprepitant X1 (n = 10) and X2 (n = 10) formulation, 2 mg/mL dexamethasone (n = 10), control vehicle X (n = 10), 0.2% hyaluronic acid (n = 10), or no treatment (n = 10). Eye wiping, phenol red, and Cochet Bonnet tests assessed ocular pain, tear fluid secretion, and nerve function. After 7 days, the mice were euthanized to quantify leukocyte infiltration and corneal nerve density. Results: Topical aprepitant X1 reduced BAK-induced corneal damage and pain compared to gel vehicle X (P = 0.007) and dexamethasone (P = 0.021). Aprepitant X1 and X2 improved corneal sensitivity versus gel vehicle X and dexamethasone (P < 0.001). Aprepitant X1 reduced leukocyte infiltration (P < 0.05) and enhanced corneal nerve density (P < 0.001). Tear fluid secretion remained statistically unchanged in both the X1 and X2 groups. Conclusions: Aprepitant formulation X1 reduced pain, improved corneal sensitivity and nerve density, ameliorated epitheliopathy, and reduced leukocyte infiltration in male mouse corneas. Translational Relevance: Aprepitant emerges as a safe, promising therapeutic prospect for the amelioration of DED's associated symptoms.

Topical fosaprepitant for the treatment of ocular pain and inflammation.

Purpose: To assess whether topical administration of fosaprepitant improves intractable chronic ocular pain and inflammation. Methods: We report three clinical cases of female patients with drug-resistant ocular pain associated with inflammatory diseases of the ocular surface. The patients were treated for 3 (case 1) and 4 (cases 2-3) weeks with fosaprepitant eyedrops (0.1 mg/mL for case 1; 10 mg/mL for case 2-3). Patients were then followed up for at least 3 weeks. We measured ocular pain with the Visual Analogue Scale (VAS), the Ocular Surface Disease Index (OSDI), and corneal sensitivity with the Cochet-Bonnet esthesiometry. Slit-lamp photography and corneal confocal imaging were used to assess ocular surface integrity/conjunctival hyperemia and corneal nerve morphology, respectively. Results: All three patients had severe ocular pain (score higher than 6/10 VAS scale). All patients reported a significant improvement in ocular pain after 1 week of treatment. We also observed reduced corneal epitheliopathy (case 1) and conjunctival hyperemia (cases 1-2). In two patients (cases 2-3) the treatment was repeated after 1 year and 9 weeks, respectively, and pain reduction was similar in magnitude to what we observed after the first administration. Conclusions: Topical administration of fosaprepitant ameliorates ocular pain and clinical symptoms in three patients with intractable ocular pain associated with inflammatory diseases of the ocular surface, without adverse effects. Importance: Fosaprepitant instillation holds promise as a treatment of chronic ocular pain, an area of unmet medical need.

Topical formulations of Aprepitant are safe and effective in relieving pain and inflammation, and drive neural regeneration.

PURPOSE: To test long-term ocular toxicity and analgesic/anti-inflammatory efficacy of two novel ocular formulations of neurokinin 1 receptor (NK1R) antagonist Aprepitant. METHODS: for toxicity studies, two Aprepitant formulations (X and Y) were tested on C57BL/6 N mice. Gold standards were 0.4% Oxybuprocaine, 0.1% Diclofenac, or saline. For efficacy studies, C57BL/6 N mice underwent corneal alkali burn, and then received Aprepitant formulation X, Dexamethasone or saline. Eye-drops were applied 3 times/day for 90 days (toxicity) and 14 days (efficacy). Stromal opacity, corneal epithelial damage, nociception and sensitivity were assessed in vivo. The eye-wiping test and corneal sensitivity were assessed to evaluate analgesic efficacy and nerve function. At the end of the experiments mice were euthanized, and corneas were dissected for immunohistochemistry and RT-PCR analyses. RESULTS: In normal mice, formulation X was not toxic when topically administered for 90 days. Formulation Y was associated with increased leukocyte infiltration in the cornea (p < 0.001). X1 and X2 formulations significantly reduced corneal pain, as Diclofenac and Oxybuprocaine, but did not reduce corneal sensitivity. Formulation Y, instead, was not analgesic at any time point. In the alkali burn model, X1 and X2 formulation enhanced epithelial damage recovery, and reduced inflammation both at day 7 and 14. Moreover, formulation X showed a stronger analgesic effect when compared to the saline and Dexamethasone groups (p < 0.01). Finally, formulation X1 and X2 restored corneal sensitivity by promoting corneal nerve regeneration. CONCLUSIONS: Aprepitant X formulation is a promising candidate for the treatment of pain associated with inflammation of the ocular surface.

Vitamin D Supplementation Impacts Systemic Biomarkers of Collagen Degradation and Copper Metabolism in Patients With Keratoconus.

Purpose: To evaluate the impact of vitamin D (Vit D) supplementation on systemic biomarkers of collagen degradation, inflammation, oxidative stress, and copper metabolism in adolescent patients with keratoconus (KC). Methods: This was a prospective observational pilot study. Twenty patients (age range, 16-19 years) presenting KC and Vit D insufficiency (<30 ng/mL) were included. Vit D supplementation was prescribed by their general practitioner as per the standard of care. Patients were followed up for 12 months. At each visit, best spectacle-corrected visual acuity (BSCVA), maximal keratometry (Kmax), and thinnest corneal thickness (TCT) were evaluated. The primary outcome of the study was the proportion of patients with Kmax progression of less than 1 D throughout the 12-month follow-up time. Blood samples were collected at different time points to evaluate Vit D levels and systemic markers of collagen degradation, inflammation, oxidative stress, and copper metabolism by ELISA or RT-PCR. Results: Lower Vit D levels in the plasma were correlated with higher levels of systemic biomarkers of collagen degradation. Vit D supplementation increased the cell availability of copper. Moreover, stabilization of KC progression was found in 60% of patients (72% of eyes) after 12 months with Vit D supplementation. BSCVA, Kmax, and TCT rates remained stable during the observation period. Conclusions: Our findings support that Vit D administration could affect ocular and systemic biomarkers in KC and illuminate a possible mechanism that can be used to develop new treatment alternatives. Translational Relevance: Although KC therapy currently relies exclusively on surgical procedures, Vit D supplementation may offer a non-invasive and inexpensive alternative with minimal associated side effects.

Corneal endothelial cell reduction and increased Neurokinin-1 receptor expression in a graft-versus-host disease preclinical model.

The aim of this work was to assess corneal endothelial morphology in a well-established acute graft-versus-host disease (GVHD) murine model and to quantify the expression of neurokinin-1 receptor (NK1R) in the corneal endothelium during ocular GVHD (oGVHD). Pre-conditioning was performed in BALB/c using myeloablative total body irradiation. Subsequently, allogeneic bone marrow transplantation was performed without (BM) or with mature T cells (BM + T). Corneal transparency was monitored with in vivo biomicroscopy. After sacrifice, corneal thickness and endothelial cell number were measured, and the expression of NK1R was investigated in the corneal endothelium through immunofluorescence and quantified by immunohistochemistry. Mice presenting oGVHD showed a significant reduction in endothelial cell number compared to control animals (p < 0.0001). NK1R expression was significantly increased in oGVHD mice endothelium (p < 0.05). Corneal transparency and thickness were unchanged in all groups. Our results suggest that oGVHD affects the corneal endothelium, inducing a reduction of the cell number, and that this is associated with increased expression of the pro-inflammatory marker NK1R.

Corneal and Epidermal Nerve Quantification in Chemotherapy Induced Peripheral Neuropathy.

Chemotherapy-induced neurotoxicity is an increasingly recognized clinical issue in oncology. in vivo confocal microscopy (IVCM) of corneal nerves has been successfully used to diagnose peripheral neuropathies, including diabetic neuropathy. The purpose of this study was to test if the combination of corneal nerve density and morphology assessed by IVCM is useful to monitor the neurotoxic effects of chemotherapy compared to epidermal nerve quantification. Overall, 95 adult patients with different cancer types were recruited from the oncology and hematology departments of the San Raffaele Hospital. Neurological examination, including clinical Total Neuropathy Score, and in vivo corneal confocal microscopy (IVCM), were performed before and after chemotherapy. In a group of 14 patients, skin biopsy was performed at the first and last visit. In the group of 14 patients who underwent both skin biopsy and corneal nerve imaging, clinical worsening (+69%, p = 0.0018) was paralleled by corneal nerve fiber (CNF) density reduction (-22%, p = 0.0457). Clinical Total neuropathy score significantly worsened from the first to the last visit (+62%, p < 0.0001). CNF length was not significantly reduced overall. However, CNF density/tortuosity ratio significantly decreased after therapy. Correlation analysis showed that the CNF density/tortuosity ratio was also correlated with the number of chemotherapy cycles (r = -0.04790, P = 0.0009). Our data confirm that in vivo corneal confocal microscopy is a helpful, non-invasive tool which shows promise for the diagnosis of chemotherapy-induced peripheral neuropathies. IVCM could allow a rapid, reproducible and non-invasive quantification of peripheral nerve pathology in chemotherapy-associated neuropathy.

Nutritional and Metabolic Imbalance in Keratoconus.

Keratoconus (KC) is a progressive corneal degeneration characterized by structural changes consisting of progressive thinning and steepening of the cornea. These alterations result in biomechanical weakening and, clinically, in vision loss. While the etiology of KC has been the object of study for over a century, no single agent has been found. Recent reviews suggest that KC is a multifactorial disease that is associated with a wide variety of genetic and environmental factors. While KC is typically considered a disease of the cornea, associations with systemic conditions have been well described over the years. In particular, nutritional and metabolic imbalance, such as the redox status, hormones, metabolites, and micronutrients (vitamins and metal ions), can deeply influence KC initiation and progression. In this paper, we comprehensively review the different nutritional (vitamins and minerals) and metabolic (hormones and metabolites) factors that are altered in KC, discussing their possible implication in the pathophysiology of the disease.

Immunity and pain in the eye: focus on the ocular surface.

Most ocular diseases are associated with pain. While pain has been generally considered a mere (deleterious) additional symptom, it is now emerging that it is a key modulator of innate/adaptive immunity. Because the cornea receives the highest nerve density of the entire body, it is an ideal site to demonstrate interactions between pain and the immune response. Indeed, most neuropeptides involved in pain generation are also potent regulators of innate and adaptive leukocyte physiology. On the other hand, most inflammatory cells can modulate the generation of ocular pain through release of specific mediators (cytokines, chemokines, growth factors, and lipid mediators). This review will discuss the reciprocal role(s) of ocular surface (and specifically: corneal) pain on the immune response of the eye. Finally, we will discuss the clinical implications of such reciprocal interactions in the context of highly prevalent corneal diseases.

The two-faced effects of nerves and neuropeptides in corneal diseases.

Corneal nerves are instrumental to maintain cornea integrity through regulation of key physiological functions such as tear secretion, blink reflex, and neuropeptide turnover. Corneal nerve injury/stimulation can follow many insults including mechanical/chemical trauma, infections and surgeries. Nerve disruption initiates a process named neurogenic inflammation which leads to edema, pain, and recruitment and activation of leukocytes. Interestingly, leukocyte influx in the cornea can further damage nerves by releasing inflammatory mediators-including neuropeptides. The clinical outcome of neuroinflammation can be beneficial or detrimental to corneal integrity. On one side, it ensures prompt wound healing and prevents infections. On the other, prolonged and/or deranged neuroinflammation can permanently disrupt corneal integrity and impair vision. The cornea is an ideal site to study peripheral neuroinflammation and neurogenic inflammation since it receives the highest density of sensory nerves of the entire body. We will review the corneal nerve anatomy and neurochemistry, discuss the beneficial and detrimental effects of neurogenic inflammation in corneal wound healing, inflammatory processes, and pain. We will also examine the emerging remote impact of corneal nerve disruption on the trigeminal ganglion and the brain, highlighting the key role of neuropeptide Substance P. Finally, we will discuss the clinical relevance of such neuroinflammatory network in the context of severe and highly prevalent ocular diseases, including potential treatments.

Epidemiology of Corneal Neovascularization and Its Impact on Visual Acuity and Sensitivity: A 14-Year Retrospective Study.

Purpose: To quantify the severity and location of corneal neovascularization (cNV) and its impact on the visual acuity and corneal sensitivity in a cohort of the patients referred to a specialist cornea clinic and also to describe the etiology of cNV in the cohort. Methods: We retrospectively evaluated the charts of 13,493 subjects referred to the San Raffaele Cornea Unit between January 2004 and December 2018 to search for cNV diagnosis. The corneal neovascularization severity was measured in the quadrants (range: 1-4) and location was defined as superficial, deep, or both. Best spectacle corrected visual acuity (BSCVA) was measured in logMar. We used the multiple regression analysis to identify the independent predictors of logMAR, after adjusting for age, gender, keratoconus, herpes keratitis, penetrating keratoplasty, trauma, and cataract surgery. Results: Corneal neovascularization was diagnosed in 10.4% of the patients analyzed. The most prevalent etiology of cNV in our population was non-infectious corneal dystrophies/degenerations followed by herpes simplex virus infection. cNV affected OD, OS, or both eyes in 35.6, 40.2, and 24.2 of cases, respectively. Mean BSCVA (SD) was 0.59 (0.76), 0.74 (0.94), and 1.24 (1.08) in cNV one, two, and three or four of the quadrant groups. Superficial, deep, or mixed cNV occurred in 1,029, 348, and 205 eyes. Severe cNV (three or four of the quadrants) was a significant predictor of low visual acuity (p < 0.001) and reduced corneal sensitivity (p < 0.05). cNV location and its severity were associated (p < 0.05). In addition, corneal anesthesia was associated with lower BSCVA (p < 0.001). Conclusion: Severe and deep cNV are associated with the reduced visual acuity and corneal sensitivity. Our data strongly support the relevance of appropriate follow-up as cNV is a major risk factor for graft rejection.

Topical neurokinin-1 receptor antagonist Fosaprepitant ameliorates ocular graft-versus-host disease in a preclinical mouse model.

PURPOSE: to assess the effect of topical administration of the Neurokin-1 receptor (NK1R) antagonist Fosaprepitant in a pre-clinical model of ocular Graft-versus-Host disease (GVHD). METHODS: BALB/c mice were pre-conditioned by myeloablative total body irradiation and subjected to allogeneic bone marrow transplantation and mature T cell infusion (BM + T). BM-transplanted mice (BM) were used as controls. Ocular GVHD was specifically assessed by quantifying corneal epithelial damage, tear secretion, blepharitis and phimosis, 3 times/week for 28 days post-transplantation. A group of BM + T mice received Fosaprepitant 10 mg/mL, 6 times/day, topically, from day 7-29 after transplantation. After sacrifice, the expression of NK1R, CD45, CD3, and CXCL10 was quantified in the cornea, conjunctiva, and lacrimal gland by immunohistochemistry. RESULTS: BM + T mice developed corneal epithelial damage (day 0-29, p < 0.001), blepharitis (day 0-29, p < 0.001), and phimosis (day 0-29, p < 0.01), and experienced decreased tear secretion (day 21, p < 0.01) compared to controls. NK1R was found upregulated in corneal epithelium (p < 0.01) and lacrimal gland (p < 0.01) of BM + T mice. Fosaprepitant administration significantly reduced corneal epithelial damage (p < 0.05), CD45+ (p < 0.05) and CD3+ (p < 0.01) immune cell infiltration in the cornea and conjunctiva (p < 0.001 and p < 0.001, respectively). In addition, Fosaprepitant reduced the expression of CXCL10 in the cornea (p < 0.05) and in the lacrimal gland (p < 0.05). CONCLUSIONS: Our results suggest that NK1R represents a novel druggable pathway for the therapy of ocular GVHD.

A Hypothalamic-Controlled Neural Reflex Promotes Corneal Inflammation.

Purpose: To test whether an acute corneal injury activates a proinflammatory reflex, involving corneal sensory nerves expressing substance P (SP), the hypothalamus, and the sympathetic nervous system. Methods: C57BL6/N (wild-type [WT]) and SP-depleted B6.Cg-Tac1tm1Bbm/J (TAC1-KO) mice underwent bilateral corneal alkali burn. One group of WT mice received oxybuprocaine before alkali burn. One hour later, hypothalamic neuronal activity was assessed in vivo by magnetic resonance imaging and ex vivo by cFOS staining. Some animals were followed up for 14 days to evaluate corneal transparency and inflammation. Tyrosine hydroxylase (TH), neurokinin 1 receptor (NK1R), and neuronal nitric oxide synthase (nNOS) expression was assessed in brain sections. Sympathetic neuron activation was evaluated in the superior cervical ganglion (SCG). CD45+ leukocytes were quantified in whole-mounted corneas. Noradrenaline (NA) was evaluated in the cornea and bone marrow. Results: Alkali burn acutely induced neuronal activation in the trigeminal ganglion, paraventricular hypothalamus, and lateral hypothalamic area (PVH and LHA), which was significantly lower in TAC1-KO mice (P < 0.05). Oxybuprocaine application similarly reduced neuronal activation (P < 0.05). TAC1-KO mice showed a reduced number of cFOS+/NK1R+/TH+ presympathetic neurons (P < 0.05) paralleled by higher nNOS expression (P < 0.05) in both PVH and LHA. A decrease in activated sympathetic neurons in the SCG and NA levels in both cornea/bone marrow and reduced corneal leukocyte infiltration (P < 0.05) in TAC1-KO mice were found. Finally, 14 days after injury, TAC1-KO mice showed reduced corneal opacity and inflammation (P < 0.05). Conclusions: Our findings suggest that stimulation of corneal sensory nerves containing SP activates presympathetic neurons located in the PVH and LHA, leading to sympathetic activation, peripheral release of NA, and corneal inflammation.

Modulating Ocular Surface Pain Through Neurokinin-1 Receptor Blockade.

Purpose: The purpose of this study was to test the role of substance P (SP) and its receptor neurokinin 1 (NK1R) on ocular surface pain. Methods: Eight-week-old C57BL6/N (wild type [WT]) and B6.Cg-Tac1tm1Bbm/J (TAC1-KO) male mice were used. 5 M NaCl was topically applied on the cornea, followed by topical fosaprepitant 2, 10, and 50 mg/mL; 4 mg/mL oxybuprocaine chloride, or 0.1% diclofenac. Th eye wiping test was used to quantify ocular surface pain. SP content was quantified in the tear fluid and trigeminal ganglia (TG), and TAC1 mRNA was assessed in the cornea. Corneas were immunostained for ß3-tubulin and NK1R, or CD45, to quantify leukocyte infiltration. Results: TAC1-KO mice displayed a significant reduction of ocular pain (P < 0.001). Similarly, a single dose of 10 or 50 mg/mL fosaprepitant applied topically to WT mice reduced ocular pain as compared to vehicle (P < 0.001). Fosaprepitant 2 mg/mL, instead, induced corneal analgesia only when it was administered for 10 days, 6 times/day (P < 0.05). Diclofenac or oxybuprocaine reduced corneal nociception when compared to vehicle or fosaprepitant (P < 0.05). Fosaprepitant or oxybuprocaine groups showed lower SP content in tear secretions and TG (P < 0.05), and reduction in TAC1 mRNA (P < 0.05), and leukocyte infiltration (P < 0.05) in the cornea. Colocalization of NK1R and ß3-tubulin was detected in mouse corneas. Conclusions: Topical administration of the NK1R antagonist fosaprepitant effectively reduces ocular surface nociception by decreasing SP release in the tear fluid and TG, and corneal leukocyte infiltration. Fosaprepitant repurposing shows promise for the treatment of ocular pain.