0.1% RGN-259 (Thymosin ß4) Ophthalmic Solution Promotes Healing and Improves Comfort in Neurotrophic Keratopathy Patients in a Randomized, Placebo-Controlled, Double-Masked Phase III Clinical Trial.

We determined the efficacy and safety of 0.1% RGN-259 ophthalmic solution (containing the regenerative protein thymosin ß4) in promoting the healing of persistent epithelial defects in patients with Stages 2 and 3 neurotrophic keratopathy. Complete healing occurred after 4 weeks in 6 of the 10 RGN-259-treated subjects and in 1 of the 8 placebo-treated subjects (p = 0.0656), indicating a strong efficacy trend. Additional efficacy was seen in the significant healing (p = 0.0359) with no recurrent defects observed at day 43, two weeks after cessation of treatment, while the one healed placebo-treated subject at day 28 suffered a recurrence at day 43. The Mackie classification disease stage improved in the RGN-259-treated group at Days 29, 36, and 43 (p = 0.0818, 0.0625, and 0.0467, respectively). Time to complete healing also showed a trend towards efficacy (p = 0.0829, Kaplan-Meier) with 0.1% RGN-259. RGN-259-treated subjects had significant improvements at multiple time points in ocular discomfort, foreign body sensation, and dryness which were not seen in the placebo group. No significant adverse effects were observed. In summary, the use of 0.1% RGN-259 promotes rapid healing of epithelial defects in neurotrophic keratopathy, improves ocular comfort, and is safe for treating this challenging population of patients.

Adjunctive Thymosin Beta-4 Treatment Influences MΦ Effector Cell Function to Improve Disease Outcome in Pseudomonas aeruginosa-Induced Keratitis.

Our previous work has shown that topical thymosin beta 4 (Tß4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tß4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tß4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tß4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tß4 alone and adjunctive Tß4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tß4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study.

Antimicrobial Effects of Thymosin Beta-4 and Ciprofloxacin Adjunctive Therapy in Pseudomonas aeruginosa Induced Keratitis.

Prior work has indicated that thymosin beta 4 (Tß4) administered with ciprofloxacin markedly improves disease outcome for Pseudomonas aeruginosa (PA)-induced keratitis. As a result, the goal of the current study was to elucidate mechanisms by which Tß4 mitigates the corneal response; specifically, regarding its bactericidal influence and potential synergy with ciprofloxacin. An in vitro approach was carried out using minimum inhibitory concentration (MIC) assays to assess bactericidal activity against PA. In addition, antimicrobial peptide (AMP) production was evaluated at the mRNA levels using human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. The results of the MIC assays did not show direct bactericidal activity with Tß4 alone, although ciprofloxacin exhibited significant killing at concentrations far lower than clinically dosed. Tß4, however, displayed an indirect effect on bacterial killing, as shown by an upregulation of AMPs and related molecules. The cumulative data from this study indicate an indirect bactericidal role of Tß4, as well as a synergistic relationship with ciprofloxacin. Furthermore, ciprofloxacin alone was found to influence cellular functions that otherwise have yet to be reported. These results highlight a mechanism of intracellular communication for Tß4 and further strengthen its development as an adjunct therapy with antibiotics for corneal infections.

Thymosin beta 4: A potential novel adjunct treatment for bacterial keratitis.

Microbial keratitis is a rapidly progressing, visually debilitating infection of the cornea that can lead to corneal scarring, endophthalmitis, and perforation. Corneal opacification or scarring, a complication of keratitis, is among the leading causes of legal blindness worldwide, second to cataracts.Pseudomonas aeruginosaandStaphylococcus aureusare the two bacteria most commonly associated with this type of infection. Risk factors include patients who are immunocompromised, those who have undergone refractive corneal surgery, and those with prior penetrating keratoplasty, as well as extended wear contact lens users. Current treatment of microbial keratitis primarily addresses the pathogen using antibiotics. Bacterial clearance is of utmost importance yet does not guarantee good visual outcome. Clinicians are often left to rely upon the eye's innate ability to heal itself, as there are limited options beyond antibiotics and corticosteroids for treating patients with corneal infection. Beyond antibiotics, agents in use, such as lubricating ointments, artificial tears, and anti-inflammatory drops, do not fully accommodate clinical needs and have many potential harmful complications. To this end, treatments are needed that both regulate the inflammatory response and promote corneal wound healing to resolve visual disturbances and improve quality of life. Thymosin beta 4 is a small, naturally occurring 43-amino-acid protein that promotes wound healing and reduces corneal inflammation and is currently in Phase 3 human clinical trials for dry eye disease. Our previous work has shown that topical Tß4 as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages) while enhancing bacterial killing and wound healing pathway activation in an experimental model ofP. aeruginosa-induced keratitis. Adjunctive thymosin beta 4 treatment holds novel therapeutic potential to regulate and, optimally, resolve disease pathogenesis in the cornea and perhaps other infectious and immune-based inflammatory disease. We plan to establish the importance of thymosin beta 4 as a therapeutic agent in conjunction with antibiotics with high impact for immediate clinical development.

A Novel Combination Therapy Tß4/VIP Protects against Hyperglycemia-Induced Changes in Human Corneal Epithelial Cells.

Despite the prevalence of diabetic retinopathy, the majority of adult diabetic patients develop visually debilitating corneal complications, including impaired wound healing. Unfortunately, there is limited treatment for diabetes-induced corneal damage. The current project investigates a novel, peptide-based combination therapy, thymosin beta-4 and vasoactive intestinal peptide (Tß4/VIP), against high-glucose-induced damage to the corneal epithelium. Electric cell-substrate impedance sensing (ECIS) was used for real-time monitoring of barrier function and wound healing of human corneal epithelial cells maintained in either normal glucose (5 mM) or high glucose (25 mM) ± Tß4 (0.1%) and VIP (5 nM). Barrier integrity was assessed by resistance, impedance, and capacitance measurements. For the wound healing assay, cell migration was also monitored. Corneal epithelial tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were assessed to confirm our findings. Barrier integrity and wound healing were significantly impaired under high-glucose conditions. However, barrier function and cell migration significantly improved with Tß4/VIP treatment. These findings were supported by high-glucose-induced downregulation of tight junction proteins that were effectively maintained similar to normal levels when treated with Tß4/VIP. These results strongly support the premise that Tß4 and VIP work synergistically to protect corneal epithelial cells against hyperglycemia-induced damage. In addition, this work highlights the potential for significant translational impact regarding the treatment of diabetic patients and associated complications of the cornea.

Thymosin beta4 inhibits TNF-alpha-induced NF-kappaB activation, IL-8 expression, and the sensitizing effects by its partners PINCH-1 and ILK.

The mechanisms by which thymosin ß 4 (Tß(4)) regulates the inflammatory response to injury are poorly understood. Previously, we demonstrated that ectopic Tß(4) treatment inhibits injury-induced proinflammatory cytokine and chemokine production. We have also shown that Tß(4) suppresses TNF-α-mediated NF-κB activation. Herein, we present novel evidence that Tß(4) directly targets the NF-κB RelA/p65 subunit. We find that enforced expression of Tß(4) interferes with TNF-α-mediated NF-κB activation, as well as downstream IL-8 gene transcription. These activities are independent of the G-actin-binding properties of Tß(4). Tß(4) blocks RelA/p65 nuclear translocation and targeting to the cognate κB site in the proximal region of the IL-8 gene promoter. Tß(4) also inhibits the sensitizing effects of its intracellular binding partners, PINCH-1 and ILK, on NF-κB activity after TNF-α stimulation. The identification of a functional regulatory role by Tß(4) and the focal adhesion proteins PINCH-1 and ILK on NF-κB activity in this study opens a new window for scientific exploration of how Tß(4) modulates inflammation. In addition, the results of this study serve as a foundation for developing Tß(4) as a new anti-inflammatory therapy.

Whole blood, flow-chamber studies in real-time indicate a biphasic role for thymosin ß-4 in platelet adhesion.

BACKGROUND: Thymosin beta 4 (Tß(4)) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing. METHODS: In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins-fibrinogen and collagen-were immobilized onto the middle ~25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tß(4), on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers. RESULTS: The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026±0.0015s(-1), corresponding to a half-life of 26.7s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tß(4) (0.2 µM to 0.5 µM) increased both the rate constant of platelet deposition by ~1.5-fold (i.e. half-life decreased from 26.7s to 17.6s) and the total number of deposited platelets by ~3-fold. However at higher concentrations (>1 µM) the Tß(4)-potentiating effect was diminished to near control levels. Tß(4) did interact with fibrinogen with an estimated K(D) of ~126±18nM or 66±20nM under equilibrium or flow, respectively. CONCLUSION: These results suggest that Tß(4) could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tß(4) could also bind to fibrinogen and as its concentration increased would prevent platelet-fibrinogen interactions resulting in the attenuation of platelet deposition. GENERAL SIGNIFICANCE: This work suggests that Tß(4) might have a dual role in platelet function.

Biological activities of thymosin beta4 defined by active sites in short peptide sequences.

Thymosin beta(4), a small ubiquitous protein containing 43 aa, has structure/function activity via its actin-binding domain and numerous biological affects on cells. Since it is the major actin-sequestering molecule in eukaryotic cells and is found essentially in all cells and body fluids, thymosin beta(4) has the potential for significant roles in tissue development, maintenance, repair, and pathology. Several active sites with unique functions have been identified, including the amino-terminal site containing 4 aa (Ac-SDKP) that generally blocks inflammation and reduces fibrosis. Another active site at the amino terminus contains 15 aa, including Ac-SDKP, and promotes cell survival and blocks apoptosis, while a short sequence containing LKKTETQ, the central actin-binding domain (aa 17-23) plus 1 additional amino acid (Q), promotes angiogenesis, wound healing, and cell migration. Several additional biological activities have been identified but not yet localized in the molecule, including its antimicrobial activity, the induction of various genes (including laminin-5, MMPs, TGF beta, zyxin, terminal deoxynucleotidyl transferase, and angiogenesis-related proteins), and the ability to activate ILK/PINCH/Akt, and other signaling molecules important in both apoptosis and inflammatory pathways. This review details these important physiologically and pathologically active sites and their potential therapeutic uses.

Adjunctive Thymosin Beta-4 Treatment Influences PMN Effector Cell Function during Pseudomonas aeruginosa-Induced Corneal Infection.

Previous work examining the therapeutic efficacy of adjunct thymosin beta 4 (Tß4) to ciprofloxacin for ocular infectious disease has revealed markedly reduced inflammation (inflammatory mediators and innate immune cells) with increased activation of wound healing pathways. Understanding the therapeutic mechanisms of action have further revealed a synergistic effect with ciprofloxacin to enhance bacterial killing along with a regulatory influence over macrophage effector cell function. As a natural extension of the aforementioned work, the current study uses an experimental model of P. aeruginosa-induced keratitis to examine the influence of Tß4 regarding polymorphonuclear leukocyte (PMN/neutrophil) cellular function, contributing to improved disease response. Flow cytometry was utilized to phenotypically profile infiltrating PMNs after infection. The generation of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and PMN apoptosis were investigated to assess the functional activities of PMNs in response to Tß4 therapy. In vitro work using peritoneal-derived PMNs was similarly carried out to verify and extend our in vivo findings. The results indicate that the numbers of infiltrated PMNs into infected corneas were significantly reduced with adjunctive Tß4 treatment. This was paired with the downregulated expression of proinflammatory markers on these cells, as well. Data generated from PMN functional studies suggested that the corneas of adjunctive Tß4 treated B6 mice exhibit a well-regulated production of ROS, NETs, and limited PMN apoptosis. In addition to confirming the in vivo results, the in vitro findings also demonstrated that neutrophil elastase (NE) was unnecessary for NETosis. Collectively, these data provide additional evidence that adjunctive Tß4 + ciprofloxacin treatment is a promising option for bacterial keratitis that addresses both the infectious pathogen and cellular-mediated immune response, as revealed by the current study.

Thymosin beta4 is cytoprotective in human gingival fibroblasts.

Thymosin beta4 (Tbeta(4)) is a naturally occurring, ubiquitous, non-toxic protein with documented wound-healing, anti-inflammatory, anti-apoptotic, and tissue-repair properties in skin, the ocular surface, and the heart. The ability of Tbeta(4) to demonstrate similar protective properties in cells of the oral cavity was analyzed using an in vitro model of cultured human gingival fibroblasts. Thymosin beta 4 significantly suppressed the secretion of interleukin-8 (IL-8) following stimulation with tumor necrosis factoralpha (TNF-alpha), suggesting that it may suppress the inflammatory response initiated by pro-inflammatory cytokines. By contrast, Tbeta(4) was not effective in protecting fibroblasts from challenge with lipopolysaccharide purified from Porphyromonas gingivalis or Escherichia coli. Thymosin beta 4 was able to protect gingival fibroblasts against the known cytotoxic effects of chlorhexidine digluconate, a mouthrinse containing chlorhexidine digluconate, and carbamide peroxide. Additionally, Tbeta(4) was able to protect gingival fibroblasts from the apoptosis that is induced by stimulation with TNF-alpha or by exposure to chlorhexidine. Because of its multifunctional roles in protecting cells against damage, Tbeta(4) may have significant potential for use as an oral heathcare aid with combined antimicrobial, anti-inflammatory, anti-apoptotic, and cytoprotective properties.

Thymosin beta 4 and the eye: the journey from bench to bedside.

INTRODUCTION: Thymosin beta 4 (Tß4) has important applications in ocular repair and Phase 3 clinical trials using Tß4 to treat dry eye and neurotrophic keratopathy are currently ongoing. These exciting clinical possibilities for Tß4 in the eye are the result of seminal basic scientific discoveries and contributions from so many talented investigators. Areas covered: My personal Tß4 journey began at the NIH in 1998 and propelled my career as a clinician scientist. As a tribute to the amazing individuals who have guided and supported me along with my brilliant colleagues and students who have contributed and collaborated with me over the years, this review will tell the cumulative story of how Tß4 became a major potential new therapy for corneal wound healing disorders. The journey has been marked by the thrilling exhilaration from fundamental breakthroughs in the laboratory and clinic, combined with the challenging and often harsh realities of submitting grants and obtaining funding. Expert opinion: The electrifying possibility of Tß4 as a revolutionary novel dry eye therapy is something that could have only been dreamed about just a few years ago. We believe that Tß4 eyedrops will help many patients suffering from several ocular surface related disorders.

RGN-259 (thymosin ß4) improves clinically important dry eye efficacies in comparison with prescription drugs in a dry eye model.

This study evaluated the clinical activity of RGN-259 (thymosin ß4) in comparison with cyclosporine A (CsA), diquafosol (DQS), and lifitegrast (LFA) in a murine model of dry eye. The model was NOD.B10-H2b mice in a 30-40% humidified environment together with daily scopolamine hydrobromide injections for 10 days. After desiccation stress, all drugs were evaluated after 10 treatment days. RGN-259 increased tear production similar to that in the DQS- and LFA-treated mice while CsA was inactive. RGN-259 improved corneal smoothness and decreased fluorescein staining similar to that of LFA group while CsA and DQS were inactive. Corneal epithelial detachment was reduced by RGN-259, and DQS and LFA showed similar activity but the CsA was inactive. RGN-259 increased conjunctival goblet cells and mucin production comparable to that seen with CsA, while DQS and LFA were inactive. RGN-259 reduced the over-expression of inflammatory factors comparable to that with CsA and LFA, while DQS was inactive. RGN-259 increased mucin production comparable to that observed with CsA, while DQS and LFA were inactive. In conclusion, RGN-259 promoted recovery of mucins and goblet cells, improved corneal integrity, and reduced inflammation in a dry eye mouse model and was equal to or more effective than prescription treatments.

Thymosin Beta-4 and Ciprofloxacin Adjunctive Therapy Improves Pseudomonas aeruginosa-Induced Keratitis.

With increasing multidrug resistance and contraindication for corticosteroid use, the goal of this study was to develop thymosin beta-4 (Tß4) as an adjunctive therapy to antibiotics for the treatment of bacterial keratitis that effectively promotes enhanced wound healing, host defense, and inflammation resolution. Disease outcome was assessed by clinical score, slit lamp photography, and histopathology. Cytokine profile, bacterial load, PMN infiltration, and Griess and reactive oxygen species (ROS) levels were determined. Adjunct Tß4 treatment resulted in a significant improvement compared to PBS, Tß4, and most remarkably, ciprofloxacin, correlating with changes in mediators of inflammation and wound healing. Collectively, these data provide evidence that wound healing is an essential aspect in the development of new therapies to treat corneal infection. Use of adjunctive Tß4 provides a more efficacious approach for bacterial keratitis by addressing both the infectious pathogen and deleterious host response.

Thymosin beta-4 and the eye: I can see clearly now the pain is gone.

The cornea epithelium responds to injury by synthesizing several cytokines, growth factors, and tissue remodeling molecules. Proinflammatory cytokines have been implicated in the inflammation that follows corneal epithelial injury and cytokine-mediated processes play a significant role in corneal epithelial wound healing. Poorly regulated corneal inflammatory reactions that occur after injury can retard healing. In turn, persistent corneal epithelial defects and inflammation may lead to ocular morbidity and permanent visual loss. Therefore, treatments with agents that enhance corneal reepithelialization and regulate the inflammatory response without the deleterious side effects of currently used agents, such as corticosteroids, would result in improved clinical outcome and would represent a major advance in the field. Evidence is mounting to support the idea that thymosin beta-4 (Tbeta-4) has multiple, seemingly diverse, cellular functions. In the cornea, as in other tissues, Tbeta-4 promotes cell migration and wound healing, has anti-inflammatory properties, and suppresses apoptosis. Prior studies from our laboratory have demonstrated the potent wound healing and anti-inflammatory effects of Tbeta-4 in numerous models of corneal injury. Recently, we demonstrated that Tbeta-4 suppresses the activation of the transcription factor, nuclear factor-kappa b (NF-kappaB) in TNF-alpha-stimulated cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NF-kappaB, thus both are known mediators of the inflammatory process. These results have important clinical implications for the potential role of Tbeta-4 as a corneal anti-inflammatory and wound-healing agent.

Ocular dirofilariasis: Ophthalmic implication of climate change on vector-borne parasites.

PURPOSE: To describe a geographically rare case of ophthalmic dirofilariasis. OBSERVATIONS: An 81-year-old male of good socioeconomic status living in the state of Michigan in the United States, presented to the eye clinic with a painful red left eye. He had not traveled outside of the state of Michigan in over three years. He was found to have a 7 cm long subconjunctival roundworm, which was ultimately extracted. CONCLUSIONS AND IMPORTANCE: With increasing global temperatures, ocular dirofilariasis is being introduced in more northern climates and should be included in the differential diagnosis in areas previously isolated from these vector-borne parasites.

Thymosin-beta4 modulates corneal matrix metalloproteinase levels and polymorphonuclear cell infiltration after alkali injury.

PURPOSE: Corneal alkali injury is highly caustic, and present clinical therapies are limited. The purpose of this study was to investigate the ability of thymosin-beta4 (Taubeta4) to promote healing in an alkali injury model and the mechanisms involved in that process. METHODS: Corneas of BALB/c mice were injured with NaOH, irrigated copiously with PBS, and treated topically with either Tbeta4 or PBS twice daily. At various time points after injury (PI), corneas from the Tbeta4- versus the PBS-treated group were examined for polymorphonuclear leukocyte (PMN) infiltration, chemokine, and matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) expression. RESULTS: Tbeta4-treated corneas demonstrated improved corneal clarity at day 7 PI. Whereas Tbeta4 decreased corneal MMP-2 and -9 and MT6-MMP levels after alkali injury, no change in TIMP-1 and -2 expression was detected. Tbeta4 treatment also decreased corneal KC (CXCL1) and macrophage inflammatory protein (MIP)-2 chemokine expression and PMN infiltration. Immunohistochemistry studies demonstrated MMP-9 expression at the leading edge of the epithelial wound, in the the limbus (containing stem cells), and in stromal PMNs. CONCLUSIONS: Tbeta4 treatment decreases corneal inflammation and modulates the MMP/TIMP balance and thereby promotes corneal wound repair and clarity after alkali injury. These results suggest that Tbeta4 may be useful clinically to treat severe inflammation-mediated corneal injuries.

Proteinase and growth factor alterations revealed by gene microarray analysis of human diabetic corneas.

PURPOSE: To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I. METHODS: RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agilent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10. RESULTS: More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin alpha4 chain, and thymosin beta(4) genes were downregulated. The data were corroborated by QPCR and immunohistochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin alpha(3)beta(1), whereas in normal corneas MMP-10 decreased laminin-10 and integrin alpha(3)beta(1) expression. CONCLUSIONS: Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.

Primary Mechanisms of Thymosin ß4 Repair Activity in Dry Eye Disorders and Other Tissue Injuries.

Dry eye disorders are becoming more common due to many causes, including an aging population, increased pollution, and postrefractive surgery. Current treatments include artificial tears; gels; lubricants; tear duct plugs; and anti-inflammatory agents such as steroids, doxycycline, and cyclosporine. For more severe forms of the disease, serum tears and scleral contact lenses are employed. Despite these therapies, successful resolution of the problem is limited because none of these treatments fully addresses the underlying causes of dry eye to promote ocular surface repair. Thymosin ß4 (Tß4), a small, naturally occurring protein, promotes complete and faster corneal healing than saline alone or prescription agents (doxycycline and cyclosporine) in various animal models of eye injury. In human trials, it improves both the signs and symptoms of moderate to severe dry eye with effects lasting beyond the treatment period. This review will cover the multiple activities of Tß4 on cell migration, inflammation, apoptosis, cytoprotection, and gene expression with a focus on mechanisms of cell migration, including laminin-332 synthesis and degradation, that account for this paradigm-shifting potential new treatment for dry eye disorders. We will also speculate on additional mechanisms that might promote eye repair based on data from other tissue injury models. Such studies provide the rationale for use of Tß4 in other types of eye disorders beyond dry eye. Finally, we will identify the gaps in our knowledge and propose future research avenues.

Thymosin beta 4 ophthalmic solution for dry eye: a randomized, placebo-controlled, Phase II clinical trial conducted using the controlled adverse environment (CAE™) model.

BACKGROUND: The purpose of this study was to evaluate the safety and efficacy of thymosin beta 4 ophthalmic solution (RGN-259; Tß4) in subjects with moderate to severe dry eye using the CAE™ model. METHODS: This single-center, prospective, double-masked, placebo-controlled Phase II study randomized 72 qualifying subjects 1:1 to receive either 0.1% Tß4 or placebo treatment for a total of 28 days. The study consisted of six visits over a 32-day period, including a screening visit (day -1), controlled adverse environment challenge (CAE) visits (day 1, day 28), and follow-up visits (days 14, 29, and 30). The primary efficacy endpoints were ocular discomfort scores and inferior corneal staining measured at visit 5 on day 29. Secondary endpoints included central and superior corneal staining, conjunctival staining, conjunctival redness, tear-film break-up time, and daily symptom scores recorded over the course of the study. Safety measures included visual acuity, slit-lamp evaluation, conjunctival redness, tear film break-up time, intraocular pressure, dilated funduscopy, and corneal sensitivity. RESULTS: Neither of the primary endpoints, ie, ocular discomfort or inferior corneal staining, showed a significant difference between treatment and control groups at visit 5. Despite this, significant differences between treatment groups were observed for a number of secondary endpoints. The discomfort scores in the CAE on day 28 were reduced by 27% in 0.1% Tß4-treated subjects compared with the placebo group (P=0.0244). Subjects in the 0.1% Tß4 treatment group also showed statistically significant improvements in central and superior corneal staining compared with staining scores in the control group (P=0.0075 and P=0.0210). No adverse events were observed. CONCLUSION: This study confirms the efficacy of 0.1% Tß4 as a topical treatment for relief of signs and symptoms of dry eye. Significant improvements in both signs and symptoms of dry eye were observed, and the treatment exhibited a large safety window, with no adverse events reported by any subjects enrolled in the study.

Thymosin ß4 significantly improves signs and symptoms of severe dry eye in a phase 2 randomized trial.

PURPOSE: Standard therapies for severe dry eye are limited and fail to resolve the problem. The purpose of this study was to evaluate the safety and efficacy of Thymosin ß4 eye drops (RGN-259) as a novel therapy for severe dry eye disease (including that associated with graft vs. host disease). METHODS: A small, multicenter, randomized, double-masked, placebo-controlled 56-day phase 2 clinical trial including a 28-day follow-up at 2 US sites. Nine patients with severe dry eye were treated with either RGN-259 (0.1%) or vehicle control 6 times daily over a period of 28 days. Dry eye sign and symptom assessments, such as ocular discomfort (using the OSDI questionnaire) and corneal fluorescein staining (using the NEI workshop grading system), were evaluated at various time points. RESULTS: Statistically significant differences in both symptom and sign assessments, were seen at various time points throughout the study. Of particular note at day 56, the RGN-259-treated group (12 eyes) had 35.1% reduction of ocular discomfort compared with vehicle control (6 eyes) (P = 0.0141), and 59.1% reduction of total corneal fluorescein staining compared with vehicle control (P = 0.0108). Other improvements seen in the RGN-259-treated patients included tear film breakup time and increased tear volume production. CONCLUSIONS: In this small trial, RGN-259 eye drops were safe and well tolerated and met key efficacy objectives with statistically significant symptom and sign improvements, compared with vehicle control, at various time intervals, including 28-days posttreatment. CLINICAL TRIAL REGISTRATION--URL: http://www.clinicaltrials.gov. Unique identifier: NCT01393132.