Preclinical Safety and Efficacy Assessments for Novel Femtosecond Lasers in Corneal Refractive Surgery.

Preclinical safety requirements and test methods have been standardized over time to guide medical device developers in the path needed to manufacture safe devices and achieve regulatory approval. Today, femtosecond lasers are commonly used in cataract and refractive surgeries. Currently, an industry standard to guide developers in preclinical testing of ophthalmic lasers does not exist. Consequently, the data presented in regulatory submissions may vary between manufacturers, making the regulatory review process more ambiguous. Here, the authors present a comprehensive discussion of preclinical test methods applied to the evaluation of an ophthalmic laser. We include in vitro and ex vivo models, as well as an in vivo rabbit model subject to corneal refractive treatments, for consideration in a preclinical safety evaluation plan. Scientific rationale to support the ocular endpoints of evaluation in the rabbit model to demonstrate safety is also presented and discussed.

Preclinical Safety Evaluation of Ophthalmic Viscosurgical Devices in Rabbits and a Novel Mini-Pig Model.

INTRODUCTION: To establish the preclinical safety and equivalency of ophthalmic viscosurgical devices (OVDs) comprised of bacterially sourced sodium hyaluronate (HA) to animal sourced HA using pyrogenicity and aqueous exchange models in rabbits and a novel mini-pig model to evaluate corneal endothelial cell protection in vivo. METHODS: HEALON OVD and HEALON5 OVD containing animal-derived HA and HEALON PRO OVD and HEALON5 PRO OVD containing bacterial-derived HA were used. Two rabbit aqueous exchange studies were conducted where aqueous humor was exchanged with OVDs in six animals each to observe potential ocular inflammation, intraocular pressure (IOP) response, corneal health and pachymetry until 7 days post procedure, as well as overall assessment of the OVDs. Endothelial cell protection was evaluated in a Yucatan mini-pig cataract surgery model where HEALON PRO and HEALON5 PRO OVDs were compared to HEALON and HEALON5 OVDs, respectively. Following cataract surgery with use of OVDs in six animals per study, animals were evaluated for ocular and general health, IOP, corneal thickness, ocular inflammation, and endothelial cell protection on days 1, 3, 7 and 14 post-surgery. RESULTS: All rabbit studies demonstrated equivalence between bacterial-derived and animal-derived OVDs. Mild, post-surgical irritation, IOP increase, and corneal thickness measurements were not significantly different in HEALON PRO OVD and HEALON5 PRO OVD compared to HEALON and HEALON5 OVDs, respectively. The mini-pig model developed to investigate endothelial cell protection was successful in demonstrating equivalence between the OVDs studied. Changes in IOP mirrored actual surgical procedures, while corneal pachymetry and endothelial cell density remained constant for all OVDs used. Slit lamp observations showed expected inflammation following surgical procedures, likely due to challenges encountered during surgical procedures. CONCLUSION: Rabbit pyrogenicity and aqueous exchanged paired with a novel simulated cataract surgery mini-pig model demonstrate equivalence of OVDs regardless of HA source. Albeit with challenges, the mini-pig model was shown to be a promising tool for endothelial cell evaluation during the development of new OVDs for ophthalmic use. FUNDING: Johnson & Johnson Surgical Vision, Inc.

Correlations of In Vitro Assays for Assessing Cytotoxicity and Biocompatibility of Contact Lens Multipurpose Solutions.

OBJECTIVES: To demonstrate correlations among in vitro assays used for assessing cytotoxicity of contact lens multipurpose solution (MPS) and propose the use of multiple assays as a part of preclinical evaluation for MPS biocompatibility assessment. METHODS: The effect of four different MPS on cell cytotoxicity, metabolic activity, and membrane integrity was performed by evaluating toxicity, expression of tight junction protein zonula occludens-1, and transepithelial electrical resistance in human corneal epithelial cells and Chinese hamster fibroblast cells. RESULTS: Cytotoxicity of four MPS was assayed with five different experimental systems at various concentrations. In vitro MPS-induced cytotoxicity was dependent on assay choice, concentration of MPS used, and duration of treatment. Overall, MPS-1 and MPS-2 were comparable to MPS-4 and better than MPS-3 in maintaining corneal barrier integrity and cell viability. CONCLUSIONS: In vitro cytotoxicity testing with MPS exposure to monolayer of cells in culture could be used as a tool to understand the potential cytotoxicity profiles of MPS and possibly a predictor of clinical outcome. Furthermore, MPS effects on in vitro cytotoxicity are best demonstrated by performing multiple assays to evaluate cell viability, metabolic activity, and membrane integrity during development.

Leak-Free Clear Corneal Incisions in Human Cadaver Tissue: Femtosecond Laser-Created Multiplanar Incisions.

OBJECTIVES: To evaluate femtosecond (FS) laser-assisted leak-free clear corneal incisions (CCI) and paracentesis (P) in human eyes of deceased donors. METHODS: Multiplanar CCI and P were created using an FS laser on human eyes of deceased donors (whole globe and corneal rims). Laser settings were programmed to multiplanar for CCI and single plane for P. Corneas were imaged by optical coherence tomography (OCT) and evaluated for leak by Seidel testing at various intraocular pressure (IOP) levels, and the wound was manipulated to mimic cataract surgery. Corneal endothelium cell damage and histological architecture were evaluated by microscopy. RESULTS: The corneal incision software of the FS laser was used to create homogeneous CCI and P incisions. Morphological changes assessed by OCT and light microscopy/scanning electron microscopy showed consistent true multiplanar incisions with predefined intersecting planes. All Seidel testing was negative, indicating that FS laser-assisted incisions did not leak. Trypan blue stain of the endothelial surface showed limited cell damage from the FS laser incisions. CONCLUSIONS: The FS laser-created incisions corresponded well with the treatment plans, as evidenced by true multiplanar architecture. Incisions were sharply demarcated and demonstrated limited cell damage. No postprocedure leaking at extreme IOP or postcataract surgery-simulated conditions was noted. The FS laser may potentially reduce postoperative complications, such as infections that may be associated with CCI.

Penetrating and Intrastromal Corneal Arcuate Incisions in Rabbit and Human Cadaver Eyes: Manual Diamond Blade and Femtosecond Laser-Created Incisions.

OBJECTIVES: To compare morphologic differences between freehand diamond or femtosecond laser-assisted penetrating and intrastromal arcuate incisions. METHODS: Freehand diamond blade, corneal arcuate incisions (180° apart, 60° arc lengths) and 150 kHz femtosecond laser (80% scheimpflug pachymetry depth corneal thickness) arcuate incisions were performed in rabbits. Intrastromal arcuate incisions (100 µm above Descemet's membrane, 100 µm below epithelium) were performed in rabbit corneas (energy 1.2 µJ, spot line separation 3 × 3 µm, 90° side cut angle). Eyes were examined by slit lamp and light microscopy up to 47 days post-procedure. Freehand diamond blade penetrating incisions, and femtosecond laser penetrating and intrastromal arcuate incisions (energy 1.8 µJ, spot line separation 2 × 2 µm) were performed in cadaver eyes. Optical coherence tomography was performed immediately after surgery and the corneas were fixed for light scanning and transmission electron microscopy. RESULTS: The rabbit model showed anterior stromal inflammation with epithelial hyperplasia in penetrating blade and laser penetrating wounds. The laser intrastromal and penetrating incisions showed localized constriction of the stromal layers of the cornea near the wound. In cadaver eyes, penetrating wound morphology was similar between blade and laser whereas intrastromal wounds did not affect the cornea above or below incisions. CONCLUSION: Penetrating femtosecond laser arcuate incisions have more predictable and controlled outcomes shown by less post-operative scarring than incisions performed with a diamond blade. Intrastromal incisions do not affect uncut corneal layers as demonstrated by histopathology. The femtosecond laser has significant advantages in its ability to make intrastromal incisions which are not achievable by traditional freehand or mechanical diamond blades.

Comparison of wavefront aberrations in rabbit and human eyes.

BACKGROUND: The rabbit is one of the most common animal models used for preclinical safety evaluation of new cataract surgery and laser vision-correction technologies in ophthalmic research; however, the distributions of wavefront aberrations in rabbit eyes are unknown. The purpose of this study was to investigate the similarities and differences of wavefront aberrations between rabbit and human eyes. METHODS: Monochromatic wavefront aberrations of left and right eyes of 12 rabbits and 12 human subjects with normal vision were measured by a commercial aberrometer (WaveScan Wavefront System, Abbott Medical Optics Inc, California, USA). Comparison of wavefront aberrations in rabbit and human eyes is based on a 6.0 mm pupil. RESULTS: The rabbit eyes have an average spherical refraction of 1.51 ± 0.83 D and a cylindrical refraction of -1.03 ± 0.63 D. The average spherical refractive error of the human eyes used in this study was -2.03 ± 2.59 D with a cylindrical refraction of -1.27 ± 1.01 D. The average wavefront error root-mean-square (RMS) from higher-order aberrations is 0.34 µm in rabbits (6.0 mm pupil), which is higher compared to the wavefront error RMS value of human eyes (0.26 µm). The largest higher-order aberration in rabbit eyes is vertical coma (Z7, 0.19 ± 0.16 µm), whereas the largest higher-order aberration in human eyes is spherical aberration (Z12, 0.07 ± 0.13 µm). Wavefront error RMS, vertical coma and some higher-order aberrations are significantly correlated between the right and left rabbit eye. CONCLUSION: Compared to wavefront aberrations in the human eye measured in this study, the rabbit eye has less refractive error but larger higher-order aberrations both in wavefront error RMS and some higher-order aberration terms. Similar to human eyes, wavefront error and some higher-order aberrations are significantly correlated between the right and left rabbit eye.

Ocular surface expression and in vitro activity of antimicrobial peptides.

PURPOSE: Human ocular surface epithelia express four antimicrobial peptides (APs): beta -defensin (hBD) 1-3 and LL-37. Here the expression of additional APs (hBD 4-6, HE2beta 1; histatin-1, -3; liver expressed antimicrobial peptide-1, -2; macrophage inflammatory protein (MIP)-3alpha, and thymosin (T)beta -4) was sought and activity against common ocular pathogens studied. METHODS: AP expression was determined in human corneal and conjunctival epithelial cells (HCEC, HCjEC) by RT-PCR and in corneal sections by immunostaining. Antimicrobial assays were performed to assess peptide (hBD 1-3, LL-37, MIP-3alpha, and Tbeta 4) activity against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE) in the presence of NaCl or tears. RESULTS: HCEC and HCjEC expressed MIP-3alpha and Tbeta 4. hBD 1-3, MIP-3alpha, and Tbeta 4 showed activity against PA. hBD-3 had potent activity against SA and SE, whereas hBD-2, MIP-3alpha and Tbeta 4 had moderate activity and hBD-1 had none. NaCl markedly attenuated, and tears almost completely inhibited the activity of hBD 1-2 and Tbeta 4, but not that of hBD-3. CONCLUSIONS: The ocular surface epithelia additionally express MIP-3alpha and Tbeta 4 which have moderate antimicrobial activity. The current data support a role for hBD-3 as an antimicrobial peptide in vivo, but call in to question the effectiveness of some other APs. However, further study is required to conclusively elucidate the physiological role of each AP.

Cathelicidin-deficient (Cnlp -/- ) mice show increased susceptibility to Pseudomonas aeruginosa keratitis.

PURPOSE: To examine the clinical progression and innate immune responses during Pseudomonas aeruginosa (PA) keratitis in cathelicidin-deficient (KO) mice. METHODS: PA (ATCC 19660) keratitis was induced in KO mice and wild-type (WT) littermates generated on a 129/SVJ background. Clinical score and histopathology were used to monitor the progression of infection at postinfection (PI) days 1, 3, 7, 14, and 21. Mouse corneas were harvested for viable bacteria quantitation, and myeloperoxidase (MPO) assays were performed to determine the number of infiltrating neutrophils. ELISA was used to quantitate interleukin (IL)-1beta, IL-6, macrophage inflammatory peptide (MIP)-2, keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-alpha, and vascular endothelial growth factor (VEGF) levels in the corneas. RESULTS: WT mice were resistant (cornea healed), whereas KO mice showed increased susceptibility (corneas failed to recover by 21 days or perforated) to PA infection. Clinical scores were significantly elevated in the infected corneas of KO mice versus WT mice at 7, 14, and 21 days PI. Absence of cathelicidin resulted in significantly delayed clearance of PA in the cornea and an increased number of infiltrating neutrophils at 1, 3, 7, and 14 days PI. KO mice also exhibited differential expression of protein levels for IL-1beta, IL-6, MIP-2, KC, TNF-alpha, and VEGF up to day 21 PI compared with the WT mice. CONCLUSIONS: Cathelicidin-deficient mice showed considerable susceptibility to PA keratitis. The present study demonstrates direct in vivo evidence that endogenous expression of cathelicidin provides defense against corneal PA infection indicating its importance in host innate immunity at the ocular surface.

In vitro activity of human beta-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid.

Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human beta-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

Multifunctional roles of human cathelicidin (LL-37) at the ocular surface.

PURPOSE: The goals of this study were to examine the expression of the antimicrobial peptide LL-37 in the corneal epithelium during wound healing and to investigate whether LL-37 stimulates human corneal epithelial cell (HCEC) migration, proliferation, and cytokine production. METHODS: Expression of LL-37 was determined by RT-PCR and immunostaining in tissue sections and HCECs scraped from corneas before (original) and after (regrown) re-epithelialization. The antimicrobial activity of LL-37 against Pseudomonas aeruginosa (PA) was determined in the presence of NaCl and tears. Blind-well chamber assays were performed to study the effect of LL-37 on migration. Proliferation was determined using calcein-AM, and cytotoxicity was evaluated by MTT assay. ELISA was performed to assess the ability of LL-37 to stimulate HCEC cytokine secretion. RESULTS: LL-37 peptide was present throughout the corneal epithelium (n=4). All original corneal epithelial samples expressed a low level of LL-37 (n=10). Regrown epithelial samples collected 24 (n=3 of 5) or 48 (n=4 of 5) hours after wounding showed upregulated expression of LL-37. LL-37 killed PA in the presence of NaCl (EC50=10.3+/-2.5 microg/mL) and retained its activity in tears (n=3). LL-37 induced HCEC migration (n=5) and secretion of IL-8, IL-6, IL-1beta, and TNF-alpha (2- to 23-fold, n=4-7). Inhibitor studies indicated that LL-37's effects are mediated through multiple pathways involving a G protein-coupled receptor (formyl peptide receptor-like 1 in migration) and the epidermal growth factor receptor (n=2 to 5). LL-37 did not stimulate HCEC proliferation (n=3) and high concentrations (>10 microg/mL) were cytotoxic (n=3). CONCLUSIONS: LL-37 expression is upregulated in regenerating human corneal epithelium, has antibacterial activity against ocular pathogens under physiologically relevant conditions, and stimulates HCEC migration and cytokine production. These findings suggest that LL-37 acts as a multifunctional mediator that helps protect the cornea from infection and modulates wound healing.

Human cathelicidin (LL-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity.

PURPOSE: This study determined whether LL-37 (cathelicidin) is expressed by conjunctival and corneal epithelia as part of ocular host defense. The antimicrobial activity of LL-37 was also assessed in vitro against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), herpes simplex virus type 1 (HSV-1), and adenovirus (Ad). METHODS: Expression of LL-37/hCAP 18 mRNA and LL-37 protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting, respectively, in scraped human corneal epithelium and primary cultured human corneal and conjunctival epithelial cells. The EC50 values for three strains of PA and one each of SA and SE were determined for LL-37. LL-37 antiviral inhibition of HSV-1 and adenovirus was assessed by direct inactivation assays. Toxicity of LL-37 to A549 cells was evaluated by a MTT assay. RESULTS: LL-37/hCAP18 mRNA and LL-37 peptide were expressed by human corneal and conjunctival epithelial cells. Antibacterial activity for LL-37 was demonstrated (EC50 values for the three PA strains were 2.8 +/- 1.3, 1.9 +/- 0.3, and 3.6 +/- 2.1; for SA: 1.6 +/- 1.5; for SE: 1.3 +/- 1.9 microg/ml). LL-37 produced a significant reduction (p < 0.001 ANOVA) in HSV-1 and Ad19 viral titers with distinctly different time-kill curves (p < 0.001). LL-37 (up to 111 microM) produced no toxicity in A549 cells. CONCLUSIONS: Corneal and conjunctival epithelia express LL-37 as part of mucosal innate immunity to protect against bacterial and viral ocular infections.

Effect of preservative-free artificial tears on the antimicrobial activity of human beta-defensin-2 and cathelicidin LL-37 in vitro.

PURPOSE: Human beta-defensin-2 (hBD-2) and cathelicidin LL-37 are salt-sensitive cationic antimicrobial peptides expressed by ocular surface epithelia. The goal of this study was to investigate the effect of preservative-free artificial tears on hBD-2 and LL-37 antimicrobial activity against Pseudomonas aeruginosa. METHODS: P. aeruginosa was incubated with hBD-2 or LL-37 in the absence or presence (70% vol/vol) of different preservative-free artificial tears--Visine Tears (300 mOsm/kg), Tears Naturale Free (261 mOsm/kg), TheraTears (185 mOsm/kg), and Refresh Plus (325 mOsm/kg)--for 2 hours at 37 degrees C. In some experiments, P. aeruginosa was incubated with hBD-2 or LL-37 and Visine Tears or Tears Naturale Free with or without carboxymethylcellulose (0.5% vol/vol final concentration). Plates were inoculated with samples of each reaction mixture and then incubated for 24 hours at 37 degrees C. RESULTS: Visine Tears and Tears Naturale Free had little or no effect on the antimicrobial activity of 100 microg/mL hBD-2 or LL-37. In the presence of Refresh Plus and TheraTears, the activity of 100 microg/mL hBD-2 or LL-37 was reduced by 90% to 100%. Carboxymethylcellulose, at a concentration comparable to that present in Refresh Plus, reduced the effectiveness of hBD-2 or LL-37 by 40% to 90% in the presence of Tears Naturale Free and Visine Tears. CONCLUSION: Human beta-defensin-2 and cathelicidin LL-37 inhibit the growth of P. aeruginosa in vitro, but this activity is markedly reduced in the presence of Refresh Plus and TheraTears. These results suggest that carboxymethylcellulose-containing artificial tears may reduce the activity of the endogenously produced antimicrobial peptides.