[Show abstract][Hide abstract] ABSTRACT: Topical bevacizumab is a potential treatment modality for corneal neovascularization, and several recent studies have demonstrated its efficacy. No previous study of the pharmacokinetics of topical bevacizumab has been performed in human eyes. The purpose of this study is to investigate the pharmacokinetics of topical administration of bevacizumab in human eyes, and also to compare the pharmacokinetics of intravitreal bevacizumab injections with previously reported data.
Twenty-two (22 eyes) were included in this study, and divided into four groups: eight patients received topical bevacizumab and aqueous samples were obtained 1 hour later during cataract extraction surgery (group 1), eight patients received topical bevacizumab and vitreous samples were obtained 1 day later during pars-plana vitrectomy (PPV) (group 2), three patients received intravitreal bevacizumab and vitreous samples were obtained during PPV (group 3). Vitreous samples from three patients who received no bevacizumab served as controls (group 4). All samples underwent enzyme-linked immunosorbent assay to detect bevacizumab.
No bevacizumab was detected in the aqueous or vitreous of any topically treated eyes. The mean vitreal half-life for intravitreally injected bevacizumab was 4.9 days in four non-vitrectomized eyes and 0.66 days in one previously vitrectomized eye.
Topically administered bevacizumab does not penetrate the cornea into the anterior chamber and vitreous cavity, indicating that topical use for treating corneal neovascularization has minimal risk of intraocular penetration and adverse events related to intraocular vascular endothelial growth factor inhibition. The half-life following intravitreal bevacizumab injection measured in this study is comparable to that of previous reports, and includes the first demonstration of a significantly reduced half-life following intravitreal injection in a previously vitrectomized eye.
Albrecht von Graæes Archiv für Ophthalmologie 10/2013; · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSE: The accuracy of predicting conversion from early-stage age-related macular degeneration (AMD) to the advanced stages of choroidal neovascularization (CNV) or geographic atrophy (GA) was evaluated to determine whether inclusion of clinically relevant genetic markers improved accuracy beyond prediction using phenotypic risk factors alone. DESIGN: Cohort study. PARTICIPANTS: White, non-Hispanic subjects participating in the Age-Related Eye Disease Study (AREDS) sponsored by the National Eye Institute consented to provide a genetic specimen. Of 2415 DNA specimens available, 940 were from disease-free subjects and 1475 were from subjects with early or intermediate AMD. METHODS: DNA specimens from study subjects were genotyped for 14 single nucleotide polymorphisms (SNPs) in genes shown previously to associate with CNV: ARMS2, CFH, C3, C2, FB, CFHR4, CFHR5, and F13B. Clinical demographics and established disease associations, including age, sex, smoking status, body mass index (BMI), AREDS treatment category, and educational level, were evaluated. Four multivariate logistic models (phenotype; genotype; phenotype + genotype; and phenotype + genotype + demographic + environmental factors) were tested using 2 end points (CNV, GA). Models were fitted using Cox proportional hazards regression to use time-to-disease onset data. MAIN OUTCOME MEASURES: Brier score (measure of accuracy) was used to identify the model with the lowest prediction error in the training set. The most accurate model was subjected to independent statistical validation, and final model performance was described using area under the receiver operator curve (AUC) or C-statistic. RESULTS: The CNV prediction models that combined genotype with phenotype with or without age and smoking revealed superior performance (C-statistic = 0.96) compared with the phenotype model based on the simplified severity scale and the presence of CNV in the nonstudy eye (C-statistic = 0.89; P<0.01). For GA, the model that combined genotype with phenotype demonstrated the highest performance (AUC = 0.94). Smoking status and ARMS2 genotype had less of an impact on the prediction of GA compared with CNV. CONCLUSIONS: Inclusion of genotype assessment improves CNV prediction beyond that achievable with phenotype alone and may improve patient management. Separate assessments should be used to predict progression to CNV and GA because genetic markers and smoking status do not equally predict both end points. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: To develop a clinical classification system for age-related macular degeneration (AMD). DESIGN: Evidence-based investigation, using a modified Delphi process. PARTICIPANTS: Twenty-six AMD experts, 1 neuro-ophthalmologist, 2 committee chairmen, and 1 methodologist. METHODS: Each committee member completed an online assessment of statements summarizing current AMD classification criteria, indicating agreement or disagreement with each statement on a 9-step scale. The group met, reviewed the survey results, discussed the important components of a clinical classification system, and defined new data analyses needed to refine a classification system. After the meeting, additional data analyses from large studies were provided to the committee to provide risk estimates related to the presence of various AMD lesions. MAIN OUTCOME MEASURES: Delphi review of the 9-item set of statements resulting from the meeting. RESULTS: Consensus was achieved in generating a basic clinical classification system based on fundus lesions assessed within 2 disc diameters of the fovea in persons older than 55 years. The committee agreed that a single term, age-related macular degeneration, should be used for the disease. Persons with no visible drusen or pigmentary abnormalities should be considered to have no signs of AMD. Persons with small drusen (<63 μm), also termed drupelets, should be considered to have normal aging changes with no clinically relevant increased risk of late AMD developing. Persons with medium drusen (≥63-<125 μm), but without pigmentary abnormalities thought to be related to AMD, should be considered to have early AMD. Persons with large drusen or with pigmentary abnormalities associated with at least medium drusen should be considered to have intermediate AMD. Persons with lesions associated with neovascular AMD or geographic atrophy should be considered to have late AMD. Five-year risks of progressing to late AMD are estimated to increase approximately 100 fold, ranging from a 0.5% 5-year risk for normal aging changes to a 50% risk for the highest intermediate AMD risk group. CONCLUSIONS: The proposed basic clinical classification scale seems to be of value in predicting the risk of late AMD. Incorporating consistent nomenclature into the practice patterns of all eye care providers may improve communication and patient care. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.
[Show abstract][Hide abstract] ABSTRACT: The development of new drugs and drug delivery devices for the treatment of posterior eye diseases is critically dependent
on the potential for that drug to be approved by the United States Food and Drug Administration (FDA). This approval process
is predicated on the successful achievement of endpoints in large multicenter clinical trials. This chapter will discuss the
history and evolving nature of endpoints for these clinical trials. Updates on recent novel endpoints will be discussed as
well as the potential for the use of readouts from various imaging tools of the retina as FDA acceptable endpoints for clinical
[Show abstract][Hide abstract] ABSTRACT: To compare the pharmacokinetics and tissue response between intravitreal and microcannulation injections into the suprachoroidal space using bevacizumab.
Sixty-two pigs were studied. Either a pars plana intravitreal bevacizumab or a viscoelastic-enhanced microcannula suprachoroidal injection was performed with either 1.25 mg (group 1) or 3 mg (group 2). In group 1, six animals were euthanatized at 0.5, 7, 30, 60, 90, and 120 days after injection (n = 36). In group 2, six animals were euthanatized at 0.5, 7, 14, and 32 days (n = 24). Eyes were enucleated, dissected, and snap-frozen, or they were fixed for histology. Analysis of drug tissue levels was performed at two separate laboratories using masked specimens.
Both laboratories were confirmatory. Intravitreal bevacizumab pharmacokinetics demonstrated a gradual decline in tissue levels over 30 to 60 days in both groups 1 and 2. In addition, suprachoroidal bevacizumab tissue levels declined rapidly and were not measurable at or beyond 7 days. Vitreitis and granulomatous vasculitis were noted in 7 of 30 intravitreal injection eyes. Immunohistology suggested a distinctive drug distribution.
Direct intravitreal injection of bevacizumab has a more sustained pharmacologic profile than does a similar dose delivered to the suprachoroidal space. Intravitreal injections distributed more to the inner retina, whereas suprachoroidal delivery occurred primarily at the choroid, retinal pigment epithelium, and photoreceptor outer segments. Sustained release formulation of larger biological molecules should be considered to optimize suprachoroidal delivery. Inflammation from injections is granulomatous, seen only with intravitreal injections, and may result from either an altered immune response or a dose-related effect.
[Show abstract][Hide abstract] ABSTRACT: We recently demonstrated increased frequency and growth potential of late outgrowth endothelial progenitor cells (OECs) in patients with neovascular age-related macular degeneration (nvAMD). This study investigated the effects of short- and long-term in vitro inhibition of vascular endothelial growth factor (VEGF) Receptor-2 (VEGFR-2) signaling by SU5416 and other inhibitors of the VEGF signaling pathway in OECs.
OECs, from the peripheral blood of patients with nvAMD, and human umbilical vein endothelial cells were grown in the presence of SU5416, other VEGFR-2 tyrosine kinase inhibitors (TKIs), and inhibitors of phosphatidylinositol 3'-Kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) in complete angiogenic medium. Apotosis was assessed after 48 h using the fluorescein isothiocyanate Annexin V method. Cell counts were performed for 10 days, and features of senescence were analyzed using senescence-associated β-galactosidase staining, the telomeric repeat amplification protocol for telomerase activity, Southern blot analysis for mean telomere length, flow cytometric analysis for cell-cycle arrest, and western blot for p53 and p21. Control OECs, cells treated for 7 days with inhibitors, as well as naturally senescent OECs were analyzed for expression of different endothelial antigens, including VEGFR-2 and the receptor for stromal cell-derived factor 1, chemokine receptor 4 (CXCR-4). Migration in vitro to VEGF and stromal cell-derived factor 1 of OECs was assessed.
SU5416, other VEGFR-2 TKIs, and inhibitors of PI3K, Akt, and PKC induced apoptosis, inhibited long-term proliferation, reduced telomerase activity, and induced premature senescence and cell-cycle arrest in OECs as well as in human umbilical vein endothelial cells. Naturally senescent cells and cells rendered senescent by VEGFR-2 TKIs had reduced VEGFR-2 and CXCR-4 expression and demonstrated reduced migratory ability to VEGF.
This study demonstrates apoptosis upon short-term inhibition and inhibition of long-term survival of OECs from patients with nvAMD by SU5416, presumably via PI3K/Akt and/or PKC-mediated reduction in telomerase activity and subsequent induction of premature senescence, which is accompanied by impaired endothelial activity. Therefore, induction of premature senescence in endothelial cells may represent a potential therapeutic target in nvAMD.
[Show abstract][Hide abstract] ABSTRACT: The goal of this study was to examine elimination pathways when delivering subconjunctivally administered hydrophilic agents to the retinas of rat eyes.
The distribution of sodium fluorescein released from an episcleral implant was compared in live and postmortem eyes. Elimination of the subconjunctivally administered hydrophilic agent IgG through blood and lymphatic vessels was investigated by immunohistochemistry. Additionally, lymphatic elimination of subconjunctivally injected sodium fluorescein was quantitatively evaluated.
NaFl released from an episcleral implant was successfully delivered to the subretinal space in the postmortem eye but failed to do so in the live eye. Immunohistochemical visualization of the conjunctival tissue demonstrated dense distribution of blood and lymphatic vessels while also confirming the elimination of subconjunctivally administered IgG through these same vessels. The lymphatic elimination rate after injection of 75.6 μg of a hydrophilic agent, sodium fluorescein, into the subconjunctival space was determined to be 105 ng/min between 30 and 60 minutes.
Conjunctival blood and lymphatic vessel elimination considerably limit transscleral hydrophilic drug delivery to the retina.
[Show abstract][Hide abstract] ABSTRACT: Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation, and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically exposed to HQ, whereas Hsp25 expression is decreased. Our data suggest that phosphorylated Hsp27 might be a key mediator in AMD and HQ-induced oxidative injury to the RPE, which may provide helpful insights into the early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation relevant to the pathogenesis of AMD.
American Journal Of Pathology 09/2010; 177(3):1198-213. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We quantified episcleral drug clearance of sodium fluorescein (NaFl) in rats to examine the hypothesis that there is rapid clearance of episcleral water soluble drugs, and that this rapid clearance may limit the amount of drug that is able to reach the posterior segment from an episcleral location. 2 mm implants containing either 12 or 22 microg of NaFl were manufactured and in vitro release rates were determined. Implants were placed in the sub-Tenon's space and the amount of drug remaining in the conjunctiva/sclera/choroid complex (CSCC) at various time points was quantified following tissue solubilization and fluorescence quantification using a spectrofluorometer. Kinetics of NaFl clearance was determined in live animals, following euthanasia and in animals in which choroidal non-perfusion had been achieved with indocyanine green-enhanced 810 nm diode laser thrombosis of the choroidal vasculature. Choroidal non-perfusion in these laser-treated rats was verified with Concavalin-A staining of choroidal flatmounts. In vitro, >99% of drug was released by 25 min for the low dose implants, and by 60 min for the high dose implants. In vivo, both implant doses were >99% cleared from the episcleral tissue by 3 h. By 7 h, an average of only 0.14 +/- 0.131 ng of NaFl per mg of wet tissue weight (mean +/- SD) remained in the CSCC with the low dose implant, and 0.29 +/- 0.428 ng of NaFl per mg of wet tissue weight remained in animals with the high dose implant. By comparison, in euthanized animals at 7 h following sub-Tenon's implantation, 432.0 +/- 181.40 ng of NaFl per mg of wet tissue weight was in the episcleral tissue of animals with the low dose implant, and of 787.8 +/- 409.89 ng of NaFl per mg of wet tissue weight remained in the animals with the high dose implant. In live animals with selective thrombosis of the choroidal vasculature, the difference in the amount of drug remaining in the episcleral tissue as compared to control live animals was not significant at all time points for both implant doses. In conclusion, there is rapid clearance of episcleral NaFl delivered from a bioerodible sub-tenon's implant. The clearance mechanisms are dramatically reduced following euthanasia, suggesting that elimination is occurring via active physiologic mechanisms, rather than by passive diffusion clearance (CL(diff)) (Pfister et al., 2003). Interestingly, the choroid does not appear to play a prominent role as clearance of episcleral NaFl was not affected by elimination of choroidal blood flow. Further work is needed to delineate the pathways of episcleral drug clearance.
Experimental Eye Research 04/2010; 90(4):501-6. · 3.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose. Pars plana vitrectomy (PPV) has been reported to reduce macular thickness and improve visual acuity in patients with diabetic macular edema (ME). The hypothesis for the study was that after PPV, clearance is accelerated and VEGF concentrations are reduced. To test this hypothesis, hVEGF(165) injections were performed in rabbit eyes, with and without PPV, and vitreous VEGF levels were measured as a function of time. Methods. The PPV group rabbits had a bilateral 25-gauge PPV, and in the no-PPV group, rabbits had intact vitreous. Intravitreal injections of hVEGF(165) were performed, and the animals were euthanatized at time points up to 7 days. The vitreous was isolated and an enzyme-linked immunosorbent assay was used to measure the VEGF levels. Pharmacokinetic parameters were determined in a noncompartmental analysis approach. Results. Mean vitreous VEGF levels decreased more rapidly in eyes subjected to PPV than in no-PPV eyes. The vitreous VEGF half-life (t([)(1/2)(])) in PPV eyes was 10 times shorter than that in normal eyes. In addition, mean clearance and mean area under the curve (AUC) increased and decreased, respectively, in eyes that underwent PPV. Conclusions. VEGF clearance is increased after PPV. Reducing VEGF concentrations in the vitreous post-PPV may partially explain the improvement in macular thickness in some patients with ME. Unexpectedly, the half-life of VEGF in the vitreous, even in no-PPV eyes, was <3 hours, whereas compounds of similar molecular weight typically have longer vitreous half-lives. The back of the eye may be uniquely adapted with rapid-clearance mechanisms to regulate vitreous VEGF levels. Further study is suggested.
[Show abstract][Hide abstract] ABSTRACT: Choroidal neovascularization (CNV) is the major cause of severe vision loss in patients with age-related macular degeneration (AMD). Present drug delivery may be limited by poor delivery to the choroid where CNV originates. The goal of this study was to develop a drug delivery system to deliver an integrin-antagonist peptide to the sub-retinal space. We developed polylactic acid/polylactic acid-polyethylene oxide nanoparticles (PLA/PLA-PEO) encapsulating the water-soluble integrin-antagonist peptide, C16Y (C16Y-NP). The PLA/PLA-PEO nanoparticles were 302+/-85.1 nm in size and demonstrated a two-week sustained release, in vitro, of encapsulated C16Y. Injected nanoparticles did not demonstrate retinal toxicity as determined by histopathology. C16Y peptide solution or C16Y-NP was injected 5 or 9 days post laser photocoagulation. A single intravitreal injection of C16Y peptide and C16Y-NP solution at both 5 days and 9 days post laser photocoagulation statistically inhibited CNV (p<0.05). However, for the day 5 injections the area of choroidal neovascularization on day 12 was smaller for C16Y-NP than for C16Y peptide solution (p<0.05) because of the short vitreous half-life of C16Y peptide solution. These results demonstrate the importance of sustained release delivery for the treatment of choroidal neovascularization associated with age-related macular degeneration. The intravitreally administered PLA/PLA-PEO containing coumarin was found to penetrate the retina and localize to the RPE. These results suggest that nanoparticles of biodegradable polymers may be a potential useful delivery system for intravitreal injection of drugs in the treatment of AMD.
Journal of Controlled Release 11/2009; 142(2):286-93. · 7.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate potential safety risks associated with nonspecific inhibition of vascular endothelial growth factor (VEGF).
A perspective, reviewing the current literature.
Herein, we discuss the systemic safety of VEGF-targeted therapies, address safety issues for VEGF-targeted therapies in neovascular age-related macular degeneration, and propose the consideration of methods for identifying low rate systemic safety signals from patients treated with these agents.
Several prospective, randomized clinical trials have demonstrated that intravitreal anti-VEGF therapies generally are well tolerated. However, within these trials, there is some circumstantial evidence that links systemic VEGF inhibition to systemic adverse events, particularly systemic thromboembolic events. Because all of the intravitreal anti-VEGF agents have been associated with detectable levels in the systemic circulation, there is a scientific rationale for the occurrence of potential systemic adverse events. However, if safety issues are present, they occur at very low rates and may go undetected in controlled clinical trials of premarketed drugs.
We propose that highly sensitive methodologies be put into place for identifying low rate safety signals, including postmarketing clinical trials, chart reviews, electronic medical records, and various national and international registries and databases, to evaluate the systemic safety of antiangiogenic agents in ocular diseases such as neovascular age-related macular degeneration.
American Journal of Ophthalmology 09/2009; 148(5):647-56. · 4.02 Impact Factor