Potentiel des liposomes pour l'injection intravitréenne de molécules thérapeutiques [Potential of liposomes for the intravitreal injection of therapeutic molecules].

Intravitreal administration has been widely used since 20 years and has been shown to improve the treatment of diseases of the posterior segment of the eye with infectious origin or in edematous maculopathies. This route of administration allows to achieve high concentration of drug in the vitreous and avoids the problems resulting from systemic administration. However, two basic problems limit the use of intravitreal therapy. Many drugs are rapidly cleared from the vitreous humor; therefore, to reach and to maintain effective therapy repeated injections are necessary. Repeated intravitreal injections increase the risk of endophthalmitis, damage to lens, retinal detachment. Moreover, some drugs provoke a local toxicity at their effective dose inducing side-effects and possible retinal lesions. In this context, the development and the use of new drug delivery systems for intravitreal administration are necessary to treat chronic ocular diseases. Among them, particulate systems such as liposomes have been widely studied. Liposomes are easily injectable and permit to reduce the toxicity and to increase the residence time of several drugs in the eye. They are also able to protect in vivo poorly-stable molecules from degradation such as peptides and nucleic acids. Some promising results have been obtained for the treatment of retinitis induced by cytomegalovirus in human and more recently for the treatment of uveitis in animal. Finally, the fate of liposomes in ocular tissues and fluids after their injection into the vitreous and their elimination routes begin to be more known.

CCR2(+) monocytes infiltrate atrophic lesions in age-related macular disease and mediate photoreceptor degeneration in experimental subretinal inflammation in Cx3cr1 deficient mice.

Atrophic age-related macular degeneration (AMD) is associated with the subretinal accumulation of mononuclear phagocytes (MPs). Their role in promoting or inhibiting retinal degeneration is unknown. We here show that atrophic AMD is associated with increased intraocular CCL2 levels and subretinal CCR2(+) inflammatory monocyte infiltration in patients. Using age- and light-induced subretinal inflammation and photoreceptor degeneration in Cx3cr1 knockout mice, we show that subretinal Cx3cr1 deficient MPs overexpress CCL2 and that both the genetic deletion of CCL2 or CCR2 and the pharmacological inhibition of CCR2 prevent inflammatory monocyte recruitment, MP accumulation and photoreceptor degeneration in vivo. Our study shows that contrary to CCR2 and CCL2, CX3CR1 is constitutively expressed in the retina where it represses the expression of CCL2 and the recruitment of neurotoxic inflammatory CCR2(+) monocytes. CCL2/CCR2 inhibition might represent a powerful tool for controlling inflammation and neurodegeneration in AMD.

Nitrosative stress and corneal transplant endothelial cell death during acute graft rejection.

BACKGROUND: Nitrosative stress takes place in endothelial cells (EC) during corneal acute graft rejection. The purpose of this study was to evaluate the potential role of peroxynitrite on corneal EC death. METHODS: The effect of peroxynitrite was evaluated in vivo. Fifty, 250, and 500 microM in 1.5 microL of the natural or denatured peroxynitrite in 50 microM NaOH, 50 microM NaOH alone, or balanced salt solution were injected into the anterior chamber of rat eyes (n=3/group). Corneal toxic signs after injection were assessed by slit-lamp, in vivo confocal imaging, pachymetry, and EC count. The effect of peroxynitrite was also evaluated on nitrotyrosine and leucocyte elastase inhibitor/LDNase II immunohistochemistry. Human corneas were incubated with peroxynitrite and the effect on EC viability was evaluated. A specific inducible nitric oxide synthase inhibitor (iNOS) was administered systemically in rats undergoing allogeneic corneal graft rejection and the effect on EC was evaluated by EC count. RESULTS: Rat eyes receiving as little as 50 microM peroxynitrite showed a specific dose-dependent toxicity on EC. We observed an intense nitrotyrosine staining of human and rat EC exposed to peroxynitrite associated with leucocyte elastase inhibitor nuclear translocation, a noncaspase dependent apoptosis reaction. Specific inhibition of iNOS generation prevented EC death and enhanced EC survival of the grafted corneas. However, inhibition of iNOS did not have a significant influence on the incidence of graft rejection. CONCLUSION: Nitrosative stress during acute corneal graft rejection in rat eyes induces a noncaspase dependent apoptotic death in EC. Inhibition of nitric oxide production during the corneal graft rejection has protective effects on the corneal EC survival.

Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy.

BACKGROUND: Second Harmonic Generation (SHG) microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily generalized to other structural remodeling of the cornea and SHG microscopy should prove to be invaluable for in vivo corneal pathological studies.

Recurrence of keratoconus characteristics: a clinical and histologic follow-up analysis of donor grafts.

PURPOSE: To report on clinical corneal topography, histopathologic analysis, and fine structure findings in failed grafts after penetrating keratoplasty (PK) for keratoconus (KC). DESIGN: Retrospective, consecutive, interventional case series with histologic and clinical correlation. PARTICIPANTS: Twelve corneal buttons were obtained from consecutive patients undergoing repeated PK 10 to 28 years after the initial PK for KC. The indication for regrafting was endothelial deficiency in seven cases, irreversible immune graft rejection in two cases, and corneal ectasia in three cases. METHODS: Removed corneal buttons were examined by light and transmission electron microscopy. A potential correlation between the clinical and videokeratoscopic findings and the microscopic structural observations was analyzed. RESULTS: Preoperative simulated keratometry measured by TMS-1 (Tomey, New York, NY) or EyeSys CAS (EyeSys Technology, Houston, TX) ranged from 49.8 to 66.1 diopters. A pattern compatible with KC characteristics was observed in all cases. Fine structure analysis revealed Bowman's layer disruption or folds and stromal deposits in all corneal buttons. However, central corneal thinning was not present in any of the removed buttons. CONCLUSIONS: Structure changes compatible with the diagnosis of KC were observed in all donor buttons many years after PK on KC recipients. Recurrence of the KC characteristics may result from graft repopulation by recipients' keratocytes, aging of the grafted tissue, or both.

Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies.

Cornea transplantation is one of the most performed graft procedures worldwide with an impressive success rate of 90%. However, for "high-risk" patients with particular ocular diseases in addition to the required surgery, the success rate is drastically reduced to 50%. In these cases, cyclosporin A (CsA) is frequently used to prevent the cornea rejection by a systemic treatment with possible systemic side effects for the patients. To overcome these problems, it is a challenge to prepare well-tolerated topical CsA formulations. Normally high amounts of oils or surfactants are needed for the solubilization of the very hydrophobic CsA. Furthermore, it is in general difficult to obtain ocular therapeutic drug levels with topical instillations due to the corneal barriers that efficiently protect the intraocular structures from foreign substances thus also from drugs. The aim of this study was to investigate in vivo the effects of a novel CsA topical aqueous formulation. This formulation was based on nanosized polymeric micelles as drug carriers. An established rat model for the prevention of cornea graft rejection after a keratoplasty procedure was used. After instillation of the novel formulation with fluorescent labeled micelles, confocal analysis of flat-mounted corneas clearly showed that the nanosized carriers were able to penetrate into all corneal layers. The efficacy of a 0.5% CsA micelle formulation was tested and compared to a physiological saline solution and to a systemic administration of CsA. In our studies, the topical CsA treatment was carried out for 14 days, and the three parameters (a) cornea transparency, (b) edema, and (c) neovascularization were evaluated by clinical observation and scoring. Compared to the control group, the treated group showed a significant higher cornea transparency and significant lower edema after 7 and 13 days of the surgery. At the end point of the study, the neovascularization was reduced by 50% in the CsA-micelle treated animals. The success rate of cornea graft transplantation was 73% in treated animals against 25% for the control group. This result was as good as observed for a systemic CsA treatment in the same animal model. This new formulation has the same efficacy like a systemic treatment but without the serious CsA systemic side effects. Ocular drug levels of transplanted and healthy rat eyes were dosed by UPLC/MS and showed a high CsA value in the cornea (11710 ± 7530 ng(CsA)/g(tissue) and 6470 ± 1730 ng(CsA)/g(tissue), respectively). In conclusion, the applied formulation has the capacity to overcome the ocular surface barriers, the micelles formed a drug reservoir in the cornea from, where a sustained release of CsA can take place. This novel formulation for topical application of CsA is clearly an effective and well-tolerated alternative to the systemic treatment for the prevention of corneal graft rejection.

Effects of rat anti-VEGF antibody in a rat model of corneal graft rejection by topical and subconjunctival routes.

PURPOSE: To compare the effect of a rat anti-VEGF antibody, administered either by topical or subconjunctival (SC) routes, on a rat model of corneal transplant rejection.METHODS: Twenty-four rats underwent corneal transplantation and were randomized into four treatment groups (n=6 in each group). G1 and G2 received six SC injections (0.02 ml 10 µg/ml) of denatured (G1) or active (G2) anti-VEGF from Day 0 to Day 21 every third day. G3 and G4 were instilled three times a day with denatured (G3) or active (G4) anti-VEGF drops (10 µg/ml) from Day 0 to Day 21. Corneal mean clinical scores (MCSs) of edema (E), transparency (T), and neovessels (nv) were recorded at Days 3, 9, 15, and 21. Quantification of neovessels was performed after lectin staining of vessels on flat mounted corneas.RESULTS: Twenty-one days after surgery, MCSs differed significantly between G1 and G2, but not between G3 and G4, and the rejection rate was significantly reduced in rats receiving active antibodies regardless of the route of administration (G2=50%, G4=66.65% versus G1 and G3=100%; p<0.05). The mean surfaces of neovessels were significantly reduced in groups treated with active anti-VEGF (G2, G4). However, anti-VEGF therapy did not completely suppress corneal neovessels.CONCLUSIONS: Specific rat anti-VEGF antibodies significantly reduced neovascularization and subsequent corneal graft rejection. The SC administration of the anti-VEGF antibody was more effective than topical instillation.

Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.

PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes.

Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles.

PURPOSE: To study the kinetics of polylactide (PLA) nanoparticle (NP) localization within the intraocular tissues and to evaluate their potential to release encapsulated material. METHODS: A single intravitreous injection (5 micro L) of an NP suspension (2.2 mg/mL) encapsulating either Rh-6G (Rh) or Nile red (Nr) was performed. Animals were killed at various times, and the NPs localization within the intraocular tissues was studied by environmental scanning electron microscopy (ESEM), confocal microscopy, light microscopy histology, fluorescence microscopy, and immunohistochemistry. Eyes injected with blank NPs, free Rh, or PBS solution were used as the control. RESULTS: ESEM showed the flow of the NPs from the site of injection into the vitreous cavity and their rapid settling on the internal limiting membrane. Histology demonstrated the anatomic integrity of the injected eyes and showed no toxic effects. A mild inflammatory cell infiltrate was observed in the ciliary body 6 hours after the injection and in the posterior vitreous and retina at 18 to 24 hours. The intensity of inflammation decreased markedly by 48 hours. Confocal and fluorescence microscopy and immunohistochemistry showed that a transretinal movement of the NPs was gradually taking place with a later localization in the RPE cells. Rh encapsulated within the injected NPs diffused and stained the retina and RPE cells. PLA NPs were still present within the RPE cells 4 months after a single intravitreous injection. CONCLUSIONS: Intravitreous injection of PLA NPs appears to result in transretinal movement, with a preferential localization in the RPE cells. Encapsulated Rh diffuses from the NPs and stains the neuroretina and the RPE cells. The findings support the idea that specific targeting of these tissues is feasible. Furthermore, the presence of the NPs within the RPE cells 4 months after a single injection shows that a steady and continuous delivery of drugs can be achieved.

Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway.

PURPOSE: To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process. MATERIALS AND METHODS: Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry. RESULTS: Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells. CONCLUSION: During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.

Controlled delivery of 5-chlorouracil using poly(ortho esters) in filtering surgery for glaucoma.

PURPOSE: To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). METHODS: Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti-apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. RESULTS: In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase-dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of the baseline level and only 40% in the POE/5-FU-treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU-treated group (6.0 +/- 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 +/- 2.9 mm Hg), and the POE alone group (7.5 +/- 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with a preserved structure of the conjunctiva epithelium. CONCLUSIONS: The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

Dispositifs de délivrance de principes actifs pour des applications ophtalmologiques [Drug delivery systems for intraocular applications].

Numerous drug delivery systems (DDSs) can be used as intraocular tools to provide a sustained and calibrated release for a specific drug. Great progress has been made on the design, biocompatibility, bioavailability, and efficacy of DDSs. Although several of them are undergoing clinical trials, a few are already on the market and could be of a routine use in clinical practice. Moreover, miniaturization of the implants makes them less and less traumatic for the eye tissues and some DDSs are now able to target certain cells or tissues specifically. An overview of ocular implants with therapeutic application potentials is provided.

Intraocular implants for extended drug delivery: therapeutic applications.

An overview of ocular implants with therapeutic application potentials is provided. Various types of implants can be used as slow release devices delivering locally the needed drug for an extended period of time. Thus, multiple periocular or intraocular injections of the drug can be circumvented and secondary complications minimized. The various compositions of polymers fulfilling specific delivery goals are described. Several of these implants are undergoing clinical trials while a few are already commercialized. Despite the paramount progress in design, safety and efficacy, the place of these implants in our clinical therapeutic arsenal remains limited. Miniaturization of the implants allowing for their direct injection without the need for a complicated surgery is a necessary development avenue. Particulate systems which can be engineered to target specifically certain cells or tissues are another promising alternative. For ocular diseases affecting the choroid and outer retina, transscleral or intrasscleral implants are gaining momentum.