Stepwise candidate drug screening for myopia control by using zebrafish, mouse, and Golden Syrian Hamster myopia models.

BACKGROUND: We developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia. METHODS: A morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-ß2 (TGF-ß2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice. FINDINGS: In the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes. INTERPRETATION: Stepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials. FUNDING: This research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare.

Pharmacokinetic and Safety Evaluation of a Transscleral Sustained Unoprostone Release Device in Monkey Eyes.

Purpose: We evaluate the ocular tissue distribution and retinal toxicity of unoprostone (UNO) during 12 months, after transscleral sustained-UNO administration using a drug delivery device in monkey eyes. Methods: The device consisted of a reservoir, controlled-release cover, and a drug formulation of photopolymerized polyethylene glycol dimethacrylate. Six mg UNO was loaded into the device (length, 17 mm; width, 4.4 mm; height, 1 mm). The concentrations of M1, a primary metabolite of UNO, in the retina, choroid, vitreous, lens, aqueous humor, iris, ciliary body, and plasma were determined by liquid chromatography-tandem mass spectrometry at 3, 6, and 12 months after implantation. Retinal toxicity was evaluated by electroretinography (ERG), optical coherence tomography (OCT), and IOP at preimplantation, and at 6, 9, and 12 months after implantation. Focal ERGs were performed at 9 and 12 months after implantation. Results: M1 was detected in the choroid and retina with maximum peaks of 243.2 and 8.41 ng/g at 6 months, respectively. M1 in the ciliary body and iris was detected with maximum peaks of 7.66 and 10.4 ng/g at 6 and 12 months, respectively. Less than 1 ng/mL or ng/g of M1 was detected in the aqueous humor, vitreous, and lens. No changes were observed in retinal function as assessed by ERG, IOP, or macula thickness and retinal histology by OCT examinations during the 12-month period. No differences in focal ERG amplitudes, especially in the macula, were observed. Conclusions: The device provided intraocular sustained delivery of UNO for 12 months without producing severe retinal toxicity.

Contractile markers distinguish structures of the mouse aqueous drainage tract.

PURPOSE: Structures of the aqueous humor drainage tract are contractile, although the tract is not entirely composed of muscle. We characterized the mouse aqueous drainage tract by immunolabeling contractile markers and determined whether profiling these markers within the tract distinguished its key structures of the trabecular meshwork (TM) and ciliary muscle (CM). METHODS: Enucleated eyes from pigmented C57BL/6 (n=8 mice) and albino BALB/c (n=6 mice) mice were processed for cryo- and formalin-fixed paraffin-embedded sectioning. Immunofluorescence labeling was performed for the following: (a) filamentous actin (using fluorescence-conjugated phalloidin), representing a global contractile marker; (b) α-smooth muscle actin (α-SMA), caldesmon, and calponin, representing classic smooth muscle epitopes; and (c) nonmuscle myosin heavy chain, representing a nonmuscle contractile protein. Tissue labeling was identified by confocal microscopy and analyzed quantitatively. Hematoxylin and eosin staining provided structural orientation. RESULTS: A small portion of the TM faced the anterior chamber; the rest extended posteriorly alongside Schlemm's canal (SC) within the inner sclera. Within the drainage tract, filamentous actin labeling was positive in TM and CM. α-SMA and caldesmon labeling was seen primarily along the CM, which extended from the anterior chamber angle to its posterior termination beyond the SC near the retina. Low intensity, patchy α-SMA and caldesmon labeling was seen in the TM. Myosin heavy chain immunoreactivity was primarily found in the TM and calponin was primarily observed in the CM. C57BL/6 and BALB/c comparison showed that pigment obscured fluorescence in the ciliary body. CONCLUSIONS: Our strategy of profiling contractile markers distinguished mouse aqueous drainage tract structures that were otherwise indistinguishable by hematoxylin and eosin staining. The mouse TM was seen as an intervening structure between SC, a part of the conventional drainage tract, and CM, a part of the unconventional drainage tract. Our findings provide important insights into the structural and functional organization of the mouse aqueous drainage tract and a basis for exploring the role of contractility in modulating aqueous outflow.

[The study of opportunity of aqueous humor filtration increase after nondestructive laser exposure of sclera in the site of pars plana projection (experimental study)].

Increase of scleral water permeability due to formation of porous structure after exposure of pulsed periodic radiation of erbium-glass optical fiber laser with wave length 1,56 pm was demonstrated in experimental study of cadaver human eyes in vitro and eyes of experimental animals (rabbits) in vivo. Simultaneous complex laser exposure of pars plana and ciliary processes results in summation of morphological changes that provide decrease of aqueous humor secretion, uveal drainage and extension of suprachoroid space. A base for new noninvasive technology of nondestructive laser exposure in glaucoma treatment is established.

Knockdown of zebrafish lumican gene (zlum) causes scleral thinning and increased size of scleral coats.

The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans approximately 4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5'-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5'-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.

Effects of novel ethacrynic acid derivatives on human trabecular meshwork cell shape, actin cytoskeletal organization, and transcellular fluid flow.

To determine efficacy and therapeutic index in the context of ocular hypotensive activity of the new ethacrynic acid (ECA) derivatives of the series (SA8,248 and SA8,389), 9,000 series (SA9,000, SA9,622 and SA9,995) and ticrynafen, we undertook a comparative evaluation of the dose-dependent effects of these compounds on human trabecular meshwork (HTM) cell shape, actin cytoskeletal organization, focal adhesions and transcellular fluid flow. Responses were either scored using an arbitrary scale of 1-5 or quantified. Compounds of the 9000 series (SA9,995>SA9,000>SA9,622) were found to be 14- to 20-fold more potent than ECA, ticrynafen or analogs from the 8,000 series (SA8,389>SA8,248) in terms of ability to induce cell shape alterations in HTM cells. Similarly, compounds of the 9,000 series (SA9,995>SA9,622>SA9,000) were found to be much stronger (2 to 20 fold) than ECA, ticrynafen or analogs of the 8000 series in terms of affecting decreases in actin stress fiber content in HTM cells. Analogs of the 9000 series (SA9,622>SA9,995>SA9,000) were also observed to be 8 to 10 fold more potent than ECA (SA8,389>ECA>SA8,248>ticrynafen) at eliciting decreases in cellular focal adhesions. Interestingly, analogs of the 9000 series (SA9,000>SA9,622>SA9,995) and SA8,248 demonstrated a huge increase (by many folds) in transcellular fluid flow of HTM cell monolayers as compared to ECA and ticrynafen. Collectively, these analyses revealed that the structural modification of ECA improves its ocular hypotensive efficacy, indicating that the SA9,000 series compounds might be promising novel ocular hypotensive drugs.

Preclinical evaluation of a novel episcleral cyclosporine implant for ocular graft-versus-host disease.

PURPOSE: To develop a local drug delivery system that provides therapeutic cyclosporine levels to treat lacrimal gland graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. METHODS: Episcleral cyclosporine implants were manufactured with a silicone-based matrix design, and in vitro release rates were determined. Preclinical evaluation included toxicology (clinical examination, serial electroretinography, and histopathology) in normal rabbits and dogs, pharmacokinetics in normal rabbits, and pharmacodynamics in a canine model of aqueous tear deficiency and keratoconjunctivitis sicca. RESULTS: The cyclosporine implants showed sustained release of drug over time with in vitro assays. Histopathology showed normal ocular tissues in both dogs and rabbits 6 months after implantation. The cyclosporine implant produced lacrimal gland drug levels 1 to 2 log units higher than those reported with a variety of topical cyclosporine formulations and oral administration. The cyclosporine implant was effective in a canine model of keratoconjunctivitis sicca, with all animals able to discontinue topical cyclosporine and maintain normal Schirmer scores over a 6-month follow-up. CONCLUSIONS: This preclinical evaluation showed that the episcleral cyclosporine implant was safe, delivered potentially therapeutic cyclosporine levels to the lacrimal gland, and showed efficacy in a clinically relevant model of keratoconjunctivitis sicca. The episcleral cyclosporine implant shows promise in reducing the morbidity associated with lacrimal gland graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. In addition, continuous release of cyclosporine in the subconjunctival space with the episcleral implant was an effective means of delivering drug to the ocular surface and may have potential in treating other ocular inflammatory diseases.

Effects of mitomycin C on the expression of proliferating cell nuclear antigen after filtering surgery in rabbits.

BACKGROUND: The aim of the study was to evaluate the effects of mitomycin C on the cell replication activity in wound healing following experimental filtration surgery. METHODS: Trabeculectomy with or without application of mitomycin C was performed on albino rabbit eyes. At 1, 4, 7, 14, and 28 days after surgery, the expression of proliferating cell nuclear antigen, a marker of cell proliferation, in the filtering site was examined immunohistochemically using a streptavidin-biotin complex method. RESULTS: In control eyes that underwent trabeculectomy but did not receive mitomycin C, the number of immunoreactive cells increased 4-7 days after operation and decreased markedly at around 14 days. The filtering site was obstructed histologically at 4-7 days after operation. In the mitomycin C-treated eyes, immunoreactive cells appeared 4 days after surgery but disappeared by 7 days at the surgical site. The number of immunoreactive cells in the treated eyes was much lower than that in control eyes. CONCLUSION: The cell replication activity was markedly inhibited by administration of mitomycin C. The filtering site remained open for 28 days after surgery, whereas it was completely obstructed within 7 days in control eyes. Immunocytochemistry for proliferating cell nuclear antigen, as used in this study, is a simple and reliable method for detection of cell replication activity.

A serum-free clonal growth assay for limbal, peripheral, and central corneal epithelium.

The stem cells and transient amplifying cells of the corneal epithelium are thought to be localized in the limbal and corneal basal epithelium, respectively. To study the differential regulation of proliferation of these progenitor cells, a defined, serum-free, clonal growth assay was developed for central (CC) and peripheral (PC) corneal and limbal (L) epithelial cells. After incubation in Dispase II (1.2 U/ml; 1 hr for PC and CC and 3 hr for L) and subsequent brief trypsin-ethylenediaminetetraacetic acid digestion, 18 or 180 single cells/cm2 were seeded in MCDB 151 medium supplemented with insulin, transferrin, selenium, hydrocortisone, epidermal growth factor (EGF), phosphoethanolamine, ethanolamine, and calcium. During the first week of culture, the cells gradually developed an increasing number of colonies, and the mean colony-forming efficiency on day 6 for L was 4.2 +/- 2.4%, significantly lower than 11.4 +/- 5.9% for PC and 12.8 +/- 7.6% for CC (P less than 0.003). Colony morphology was identical in L, PC, and CC with small, elongated cells more cohesive in the center but more migratory in the periphery. There were no differences in immunofluorescent staining with monoclonal antibody AE-5, indicating the corneal derivation of all colonies. Cultures could be passaged on day 14 and grown for more than 3 weeks with increasing desquamation. Addition of a mixture of trace metals to yield MCDB 153 did not enhance growth; increased selenium concentrations were inhibitory. Elimination of EGF from the supplement abolished most of the clonal growth. The lower rate of L proliferation might be explained by the absence of serum and stromal mitogens. This culture system seems preferentially to support transient amplifying cells and allows investigation of the differentiation of isolated corneal stem cells to transient amplifying cells or the proliferation and differentiation of transient amplifying cells by various factors without the interaction of undefined serum components or paracrine influences from other cells.

Dissecting ocular tissue for intraocular drug studies.

This report describes a convenient reproducible ocular dissection technique which has important applications for ocular antimicrobial penetration studies. Different ocular tissues can be sectioned while frozen and then plated directly on culture medium containing the test organism; after the zones of bacterial inhibition are measured at 18 hours following incubation, the tissue specimens are weighed providing more reliable evidence regarding drug concentrations. In such a fashion, a drug can be administered topically, subconjunctivally, or systematically, and assayed from the cornea to the optic nerve at various time intervals. Analysis of antibiotic in the vitreous body, which has important application in the therapy of endophthalmitis, can be routinely performed in the experimental model.