A Phase 2 Trial to Test Safety and Efficacy of ST-100, a Unique Collagen Mimetic Peptide Ophthalmic Solution for Dry Eye Disease.

Objective: Dry eye disease (DED) is a worldwide source of ocular discomfort. This first-in-human phase 2 clinical study determined the efficacy of treating signs and symptoms of DED using an ophthalmic solution of synthesized mimetic of human collagen (ST-100). Design: This double-masked, randomized, study compared high (60 µg/mL) and low (22 µg/mL) dose ST-100 to vehicle utilizing the Ora, Inc. Controlled Adverse Environment (CAE) during a 28-day period. Participants: Participants included males and females ≥ 18 years of age with signs and symptoms of DED for ≥ 6 months that worsened during CAE exposure who were not taking any topical prescription therapeutic. Intervention: Participants applied ST-100 or vehicle placebo topically to both corneas (1 drop) twice daily via a blow-fill-sealed preservative-free container. Main Outcome Measures: The prespecified primary efficacy sign end point was mean change from baseline (CFB) in total corneal fluorescein staining, and the primary symptom end point was mean CFB in ocular discomfort. A secondary prespecified efficacy end point was CFB in unanesthetized Schirmer's test for tear film production. Results: Of 160 subjects in the intent-to-treat population (112 female, 48 male, median age 64), 146 completed the study. Total corneal fluorescein staining CFB improved for high-dose ST-100, with superiority over vehicle when both eyes were considered together (2-sample t test: P = 0.0394). High-dose ST-100 was superior to vehicle in Schirmer's CFB for the study eye (least squares mean difference [confidence interval] = 2.3 [0.6, 4.0], P = 0.0094). For study eyes, the proportion of Schirmer's test responders (CFB ≥ 10 mm, Schirmer's responder rate) was 12.2% for high-dose ST-100 versus 0.0% for vehicle (P = 0.0266). The CFB for ocular discomfort score improved in study eyes for high- and low-dose ST-100 (paired t test, P = 0.0133, P = 0.0151, respectively) but without superiority over vehicle (ANCOVA: P = 0.5696, P = 0.8968, respectively). ST-100 Schirmer's responders also demonstrated total elimination of worsening of corneal fluorescein stain during the stress of CAE sessions. Conclusions: ST-100 significantly improved tear production and related outcomes in DED and was well-tolerated in reducing symptoms. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Collagen in the central nervous system: contributions to neurodegeneration and promise as a therapeutic target.

The extracellular matrix is a richly bioactive composition of substrates that provides biophysical stability, facilitates intercellular signaling, and both reflects and governs the physiological status of the local microenvironment. The matrix in the central nervous system (CNS) is far from simply an inert scaffold for mechanical support, instead conducting an active role in homeostasis and providing broad capacity for adaptation and remodeling in response to stress that otherwise would challenge equilibrium between neuronal, glial, and vascular elements. A major constituent is collagen, whose characteristic triple helical structure renders mechanical and biochemical stability to enable bidirectional crosstalk between matrix and resident cells. Multiple members of the collagen superfamily are critical to neuronal maturation and circuit formation, axon guidance, and synaptogenesis in the brain. In mature tissue, collagen interacts with other fibrous proteins and glycoproteins to sustain a three-dimensional medium through which complex networks of cells can communicate. While critical for matrix scaffolding, collagen in the CNS is also highly dynamic, with multiple binding sites for partnering matrix proteins, cell-surface receptors, and other ligands. These interactions are emerging as critical mediators of CNS disease and injury, particularly regarding changes in matrix stiffness, astrocyte recruitment and reactivity, and pro-inflammatory signaling in local microenvironments. Changes in the structure and/or deposition of collagen impact cellular signaling and tissue biomechanics in the brain, which in turn can alter cellular responses including antigenicity, angiogenesis, gliosis, and recruitment of immune-related cells. These factors, each involving matrix collagen, contribute to the limited capacity for regeneration of CNS tissue. Emerging therapeutics that attempt to rebuild the matrix using peptide fragments, including collagen-enriched scaffolds and mimetics, hold great potential to promote neural repair and regeneration. Recent evidence from our group and others indicates that repairing protease-degraded collagen helices with mimetic peptides helps restore CNS tissue and promote neuronal survival in a broad spectrum of degenerative conditions. Restoration likely involves bolstering matrix stiffness to reduce the potential for astrocyte reactivity and local inflammation as well as repairing inhibitory binding sites for immune-signaling ligands. Facilitating repair rather than endogenous replacement of collagen degraded by disease or injury may represent the next frontier in developing therapies based on protection, repair, and regeneration of neurons in the central nervous system.

Collagen Mimetic Peptides Promote Repair of MMP-1-Damaged Collagen in the Rodent Sclera and Optic Nerve Head.

The structural and biomechanical properties of collagen-rich ocular tissues, such as the sclera, are integral to ocular function. The degradation of collagen in such tissues is associated with debilitating ophthalmic diseases such as glaucoma and myopia, which often lead to visual impairment. Collagen mimetic peptides (CMPs) have emerged as an effective treatment to repair damaged collagen in tissues of the optic projection, such as the retina and optic nerve. In this study, we used atomic force microscopy (AFM) to assess the potential of CMPs in restoring tissue stiffness in the optic nerve head (ONH), including the peripapillary sclera (PPS) and the glial lamina. Using rat ONH tissue sections, we induced collagen damage with MMP-1, followed by treatment with CMP-3 or vehicle. MMP-1 significantly reduced the Young's modulus of both the PPS and the glial lamina, indicating tissue softening. Subsequent CMP-3 treatment partially restored tissue stiffness in both the PPS and the glial lamina. Immunohistochemical analyses revealed reduced collagen fragmentation after MMP-1 digestion in CMP-3-treated tissues compared to vehicle controls. In summary, these results demonstrate the potential of CMPs to restore collagen stiffness and structure in ONH tissues following enzymatic damage. CMPs may offer a promising therapeutic avenue for preserving vision in ocular disorders involving collagen remodeling and degradation.

Collagen mimetic peptide repair of the corneal nerve bed in a mouse model of dry eye disease.

The intraepithelial sub-basal nerve plexus of the cornea is characterized by a central swirl of nerve processes that terminate between the apical cells of the epithelium. This plexus is a critical component of maintaining homeostatic function of the ocular surface. The cornea contains a high concentration of collagen, which is susceptible to damage in conditions such as neuropathic pain, neurotrophic keratitis, and dry eye disease. Here we tested whether topical application of a collagen mimetic peptide (CMP) is efficacious in repairing the corneal sub-basal nerve plexus in a mouse model of ocular surface desiccation. We induced corneal tear film reduction, epithelial damage, and nerve bed degradation through a combination of environmental and pharmaceutical (atropine) desiccation. Mice were subjected to desiccating air flow and bilateral topical application of 1% atropine solution (4× daily) for 2 weeks. During the latter half of this exposure, mice received topical vehicle [phosphate buffered saline (PBS)] or CMP [200 µm (Pro-Pro-Gly)7, 10 µl] once daily, 2 h prior to the first atropine treatment for that day. After euthanasia, cornea were labeled with antibodies against ßIII tubulin to visualize and quantify changes to the nerve bed. For mice receiving vehicle only, the two-week desiccation regimen reduced neuronal coverage of the central sub-basal plexus and epithelial terminals compared to naïve, with some corneas demonstrating complete degeneration of nerve beds. Accordingly, both sub-basal and epithelial ßIII tubulin-labeled processes demonstrated increased fragmentation, indicative of nerve disassembly. Treatment with CMP significantly reduced nerve fragmentation, expanded both sub-basal and epithelial neuronal coverage compared to vehicle controls, and improved corneal epithelium integrity, tear film production, and corneal sensitivity. Together, these results indicate that topical CMP significantly counters neurodegeneration characteristic of corneal surface desiccation. Repairing underlying collagen in conditions that damage the ocular surface could represent a novel therapeutic avenue in treating a broad spectrum of diseases or injury.

Collagen Mimetic Peptides Promote Adherence and Migration of ARPE-19 Cells While Reducing Inflammatory and Oxidative Stress.

Epithelial cells of multiple types produce and interact with the extracellular matrix to maintain structural integrity and promote healthy function within diverse endogenous tissues. Collagen is a critical component of the matrix, and challenges to collagen's stability in aging, disease, and injury influence survival of adherent epithelial cells. The retinal pigment epithelium (RPE) is important for maintaining proper function of the light-sensitive photoreceptors in the neural retina, in part through synergy with the collagen-rich Bruch's membrane that promotes RPE adherence. Degradation of Bruch's is associated with RPE degeneration, which is implicated early in age-related macular degeneration, a leading cause of irreversible vision loss worldwide. Collagen mimetic peptides (CMPs) effectively repair damage to collagen helices, which are present in all collagens. Our previous work indicates that in doing so, CMPs promote survival and integrity of affected cells and tissues in models of ocular injury and disease, including wounding of corneal epithelial cells. Here, we show that CMPs increase adherence and migration of the ARPE-19 line of human RPE cells challenged by digestion of their collagen substrate. Application of CMPs also reduced both ARPE-19 secretion of pro-inflammatory cytokines (interleukins 6 and 8) and production of reactive oxygen species. Taken together, these results suggest that repairing collagen damaged by aging or other pathogenic processes in the posterior eye could improve RPE adherence and survival and, in doing so, reduce the inflammatory and oxidative stress that perpetuates the cycle of destruction at the root of age-related diseases of the outer retina.

Intraocular Delivery of a Collagen Mimetic Peptide Repairs Retinal Ganglion Cell Axons in Chronic and Acute Injury Models.

Vision loss through the degeneration of retinal ganglion cell (RGC) axons occurs in both chronic and acute conditions that target the optic nerve. These include glaucoma, in which sensitivity to intraocular pressure (IOP) causes early RGC axonal dysfunction, and optic nerve trauma, which causes rapid axon degeneration from the site of injury. In each case, degeneration is irreversible, necessitating new therapeutics that protect, repair, and regenerate RGC axons. Recently, we demonstrated the reparative capacity of using collagen mimetic peptides (CMPs) to heal fragmented collagen in the neuronal extracellular milieu. This was an important step in the development of neuronal-based therapies since neurodegeneration involves matrix metalloproteinase (MMP)-mediated remodeling of the collagen-rich environment in which neurons and their axons exist. We found that intraocular delivery of a CMP comprising single-strand fractions of triple helix human type I collagen prevented early RGC axon dysfunction in an inducible glaucoma model. Additionally, CMPs also promoted neurite outgrowth from dorsal root ganglia, challenged in vitro by partial digestion of collagen. Here, we compared the ability of a CMP sequence to protect RGC axons in both inducible glaucoma and optic nerve crush. A three-week +40% elevation in IOP caused a 67% degradation in anterograde transport to the superior colliculus, the primary retinal projection target in rodents. We found that a single intravitreal injection of CMP during the period of IOP elevation significantly reduced this degradation. The same CMP delivered shortly after optic nerve crush promoted significant axonal recovery during the two-week period following injury. Together, these findings support a novel protective and reparative role for the use of CMPs in both chronic and acute conditions affecting the survival of RGC axons in the optic projection to the brain.

Corneal collagen as a potential therapeutic target in dry eye disease.

Dry eye disease (DED) is a major cause of ocular discomfort, inflammation and dysfunction worldwide. Tear film instability in DED both causes and is exacerbated by disruption of the corneal epithelium. This tandem leads to a cycle of inflammation at the corneal surface involving immune cell dysregulation and increased chemokines and cytokines, which activate mitogen-activated protein kinases in the epithelium and elevates matrix metalloproteinases (MMPs). We review evidence suggesting that corneal collagen might be highly susceptible in DED to MMP-induced disruption, digestion, and thinning. We also summarize that collagen is far from inert and contains binding sites that serve as ligands for multiple inflammatory and immune regulators. Fragmented collagen not only challenges these receptor-ligand binding relationships, but also can promote recruitment and motility of pro-inflammatory immune cells. Current physician-directed therapies for DED focus on reducing inflammation, but do not directly ameliorate the underlying corneal damage that could exacerbate surface inflammation. We argue that an important gap in practice is lack of a direct therapeutic reparative for damaged corneal collagen, which is slow to heal, and likely amplifies sight-threatening inflammation. Healing fragmented collagen in the cornea may represent a more effective means to interrupt the "vicious cycle" of inflammation in DED and other conditions that damages, sometimes irreversibly, the ocular surface.

Restoring the Extracellular Matrix: A Neuroprotective Role for Collagen Mimetic Peptides in Experimental Glaucoma.

Optic neuropathies are a major cause of visual disabilities worldwide, causing irreversible vision loss through the degeneration of retinal ganglion cell (RGC) axons, which comprise the optic nerve. Chief among these is glaucoma, in which sensitivity to intraocular pressure (IOP) leads to RGC axon dysfunction followed by outright degeneration of the optic projection. Current treatments focus entirely on lowering IOP through topical hypotensive drugs, surgery to facilitate aqueous fluid outflow, or both. Despite this investment in time and resources, many patients continue to lose vision, underscoring the need for new therapeutics that target neurodegeneration directly. One element of progression in glaucoma involves matrix metalloproteinase (MMP) remodeling of the collagen-rich extracellular milieu of RGC axons as they exit the retina through the optic nerve head. Thus, we investigated the ability of collagen mimetic peptides (CMPs) representing various single strand fractions of triple helix human type I collagen to protect RGC axons in an inducible model of glaucoma. First, using dorsal root ganglia maintained in vitro on human type I collagen, we found that multiple CMPs significantly promote neurite outgrowth (+35%) compared to vehicle following MMP-induced fragmentation of the α1(I) and α2(I) chains. We then applied CMP to adult mouse eyes in vivo following microbead occlusion to elevate IOP and determined its influence on anterograde axon transport to the superior colliculus, the primary RGC projection target in rodents. In glaucoma models, sensitivity to IOP causes early degradation in axon function, including anterograde transport from retina to central brain targets. We found that CMP treatment rescued anterograde transport following a 3-week +50% elevation in IOP. These results suggest that CMPs generally may represent a novel therapeutic to supplement existing treatments or as a neuroprotective option for patients who do not respond to IOP-lowering regimens.

Collagen Mimetic Peptides Promote Corneal Epithelial Cell Regeneration.

The cornea of the eye is at risk for injury through constant exposure to the extraocular environment. A highly collagenous structure, the cornea contains several different types distributed across multiple layers. The anterior-most layer contains non-keratinized epithelial cells that serve as a barrier to environmental, microbial, and other insults. Renewal and migration of basal epithelial cells from the limbus involve critical interactions between secreted basement membranes, composed primarily of type IV collagen, and underlying Bowman's and stromal layers, which contain primarily type I collagen. This process is challenged in many diseases and conditions that insult the ocular surface and damage underlying collagen. We investigated the capacity of a collagen mimetic peptide (CMP), representing a fraction of a single strand of the damaged triple helix human type I collagen, to promote epithelial healing following an acute corneal wound. In vitro, the collagen mimetic peptide promoted the realignment of collagen damaged by enzymic digestion. In an in vivo mouse model, topical application of a CMP-containing formulation following a 360° lamellar keratectomy targeting the corneal epithelial layer accelerated wound closure during a 24 h period, compared to vehicle. We found that the CMP increased adherence of the basal epithelium to the underlying substrate and enhanced density of epithelial cells, while reducing variability in the regenerating layer. These results suggest that CMPs may represent a novel therapeutic to heal corneal tissue by repairing underlying collagen in conditions that damage the ocular surface.