Tim U. Krohne

Intraocular Pharmacokinetics of Bevacizumab After a Single Intravitreal Injection in Humans

PURPOSE To investigate intraocular concentrations and pharmacokinetics of bevacizumab after a single intravitreal injection in humans. DESIGN Prospective, noncomparative, interventional case series. METHODS We included 30 nonvitrectomized eyes of 30 patients (age range, 43 to 93 years) diagnosed with clinically significant cataract and concurrent macular edema secondary to neovascular age-related macular degeneration, diabetic retinopathy, or retinal venous occlusion in the same eye. All patients received an intravitreal injection of 1.5 mg bevacizumab. Between one and 53 days after injection, an aqueous humor sample was obtained during elective cataract surgery. Concentrations of unbound bevacizumab in these samples were quantified by enzyme-linked immunosorbent assay. RESULTS Concentration of bevacizumab in aqueous humor peaked on the first day after injection with a mean concentration (c(max)) of 33.3 microg/ml (range, 16.6 to 42.5 microg/ml) and subsequently declined in a monoexponential fashion. Nonlinear regression analysis determined an elimination half-time (t(1/2)) of 9.82 days (R(2) = 0.81). No significant differences between diagnosis subgroups were noted. CONCLUSIONS In human nonvitrectomized eyes, the aqueous half-life of 1.5 mg intravitreally injected bevacizumab is 9.82 days.

Intraocular pharmacokinetics of ranibizumab following a single intravitreal injection in humans

PURPOSE To investigate intraocular concentrations and pharmacokinetics of ranibizumab after a single intravitreal injection in humans. DESIGN Prospective, noncomparative, interventional case series. METHODS We included 18 nonvitrectomized eyes of 18 patients (age range, 61-85 years) that were diagnosed with both clinically significant cataract and macular edema secondary to either exudative age-related macular degeneration, diabetic maculopathy, or retinal vein occlusion. Each eye received a single intravitreal injection of 0.5 mg ranibizumab. An aqueous humor sample was obtained during cataract surgery between 1 and 37 days after injection. Concentrations of unbound ranibizumab in these samples were quantified by enzyme-linked immunosorbent assay. RESULTS Ranibizumab concentration in aqueous humor peaked the first day after injection (range, 36.9-66.1 μg/mL) and subsequently declined in a mono-exponential fashion. Nonlinear regression analysis determined an initial peak concentration (c(max)) of 56.1 μg/mL and an elimination half-life (t(1/2)) of 7.19 days with a coefficient of determination (R(2)) of 0.90. Correction of ranibizumab concentrations for ocular volume as calculated from axial length measurements did not alter regression analysis results significantly (t(1/2), 7.15 days; R(2), 0.89). CONCLUSIONS In human nonvitrectomized eyes, the aqueous half-life of 0.5 mg intravitreally injected ranibizumab is 7.19 days, slightly shorter than the half-life of 9.82 days previously determined for bevacizumab by comparable methods.

Astrocyte hypoxic response is essential for pathological but not developmental angiogenesis of the retina.

Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type‐specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia‐regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell‐specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia‐inducible transcription factors HIF‐1α and HIF‐2α, and the negative regulator of HIFα, von Hippel–Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte‐derived VEGF is not essential for this process. In contrast, using a model of oxygen‐induced ischemic retinopathy, we show that astrocyte‐derived VEGF is essential for hypoxia‐induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis, and ischemia‐driven, pathological neovascularization.

Effects of lipid peroxidation products on lipofuscinogenesis and autophagy in human retinal pigment epithelial cells

Several lines of evidence suggest that progressive dysfunction of the retinal pigment epithelium (RPE) is central to the pathogenesis of age-related macular degeneration (AMD). We previously demonstrated that protein modifications with lipid peroxidation products, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), induce lysosomal dysfunction in RPE cells in vitro. Here, we investigated whether phagocytosis of modified photoreceptor outer segments (POS) affects lipofuscinogenesis and autophagy, two interrelated processes directly connected to lysosomal function. Incubation of human RPE cells with HNE- and MDA-modified POS resulted in pronounced intracellular accumulation of granular material with lipofuscin-like autofluorescence. After daily treatment with modified POS for 7 days, cellular autofluorescence increased 8.2-fold as quantified by flow cytometry. In the presence of the lysosomal inhibitor ammonium chloride, unmodified POS likewise induced an 8.0-fold increase in autofluorescence. Spectral profiles of cellular autofluorescence after incubation with modified POS were unchanged compared to incubation with native POS. Autophagy activity, measured as turnover of metabolically radiolabeled endogenous proteins, was reduced by both HNE- and MDA-modified POS by 40%. Autophagy inhibition by 3-methyladenine and lysosomal inhibition by ammonium chloride induced lipofuscinogenesis even in the absence of POS. In summary, our results demonstrate that induction of lysosomal dysfunction by lipid peroxidation-derived protein modifications results in increased lipofuscinogenesis and reduced autophagy activity in RPE cells in vitro. These mechanisms may contribute to RPE cell dysfunction and degeneration in AMD.

Intraocular pharmacokinetics after a single intravitreal injection of 1.5 mg versus 3.0 mg of bevacizumab in humans.

Purpose: To compare the concentration of unbound bevacizumab in the anterior chamber of patients with macular edema, who received a single intravitreal injection of two different dosages. Methods: This was a comparative, interventional case series. Twenty-nine nonvitrectomized eyes of 29 patients with significant lens opacities and concurrent macular edema were included in the study. All patients were treated by a single dose of intravitreal injection of bevacizumab 1.5 mg (Group A, n = 13) or 3.0 mg (Group B, n = 16). Subsequent phacoemulsification and aspiration of an aqueous humor samples were performed at various intervals (1–60 days). The axial length of the globe was measured using the IOLMaster to calculate the total volume and corresponding globe size–corrected half-time. The concentration of unbound bevacizumab was measured by enzyme-linked immunosorbent assay and pharmacokinetic parameters including half-time of elimination. Results: The mean peak concentration was observed in both groups on the first day after intravitreal bevacizumab injection. The mean concentration of unbound bevacizumab ranged in Group A from 17.5 μg/mL at baseline to 0.66 μg/mL at Day 57 and in Group B from 28.3 μg/mL at baseline to 0.00 μg/mL at 54 days. The axial lengths of all injected eyes were not significantly different between Group A (mean values, 23.026 mm, SD 1.21) and Group B (mean, 23.313 mm, SD 1.00; P = 0.31). Regression analysis determined elimination half-time values of 7.85 days (R2 = 0.75) in Group A and 11.67 days (R2 = 0.91) in Group B. Similar half-times were calculated for globe size–corrected concentrations with 8.21 days (R2 = 0.81) in Group A and 11.17 days (R2 = 0.88) in Group B. Conclusion: Single- and double-dose injections have similar pharmacokinetic characteristics in a first-order exponential decay function. The globe size–corrected concentration and half-time did not affect the calculated half-time in both groups. Doubled dosing induced a higher peak concentration at baseline, but the presumed extended biologic active concentration was no longer statistically significant after 6 weeks. The application of twice the dosage does not double the duration of its efficacy; it rather appears to extend the pharmacological duration by 1 half-time or approximately 8 days to 11 days.

Generation of Retinal Pigment Epithelial Cells from Small Molecules and OCT4 Reprogrammed Human Induced Pluripotent Stem Cells

Autologous retinal pigment epithelium (RPE) grafts derived from induced pluripotent stem cells (iPSCs) may be used to cure blinding diseases in which RPE dysfunction results in photoreceptor degeneration. Four‐, two‐, and one‐factor‐derived iPSCs (4F‐, 2F‐, and 1F‐iPSCs, respectively) were differentiated into fully functional cuboidal pigmented cells in polarized monolayers that express RPE‐specific markers. 1F‐iPSC‐RPE (1F‐iPS‐RPE) strongly resembles primary human fetal RPE (hfRPE) based on proteomic and untargeted metabolomic analyses, and using novel in vivo imaging technology coupled with electroretinography, we demonstrated that 1F‐iPS‐RPE mediate anatomical and functional rescue of photoreceptors after transplantation in an animal model of RPE‐mediated retinal degeneration. 1F‐iPS‐RPE cells were injected subretinally as a suspension and formed a monolayer dispersed between host RPE cells. Furthermore, 1F‐iPS‐RPE do not simply provide trophic support to rescue photoreceptors as previously speculated but actually phagocytose photoreceptor outer segments in vivo and maintain visual cycling. Thus, 1F‐iPS‐RPE grafts may be superior to conventional iPS‐RPE for clinical use because 1F‐iPS‐RPE closely resemble hfRPE, mediate anatomical and functional photoreceptor rescue in vivo, and are generated using a reduced number of potentially oncogenic reprogramming factors.

Lipid peroxidation products reduce lysosomal protease activities in human retinal pigment epithelial cells via two different mechanisms of action

In age-related macular degeneration (AMD), reduced lysosomal capacity may contribute to lipofuscinogenesis and progressive dysfunction of the retinal pigment epithelium (RPE). We previously demonstrated that lipid peroxidation-related protein modifications inhibit lysosomal degradation of photoreceptor outer segment (POS) proteins in RPE cells. Herein, we investigate the effects of lipid peroxidation products on activities of key RPE lysosomal proteases. In lysosomes isolated from primary human RPE cells, lipid peroxidation products 4-hydroxynonenal (HNE) and malondialdehyde (MDA) exerted a dose-dependent inhibitory effect on cysteine proteases cathepsin B and L, with biologically relevant concentrations of 1 muM resulting in a reduction of enzyme activities by 88-94%. This effect was confirmed in cultured RPE cells. Using mass spectrometry, covalent HNE and MDA adducts were detected in the active center region of inactivated cathepsins. POS previously modified with HNE and MDA likewise caused a dose-dependent reduction of cathepsin B and L activities in isolated lysosomes and, in addition, inhibited the aspartic protease cathepsin D. Our results indicate that lipid peroxidation products in vitro interfere with RPE lysosomal protease activities by two different mechanisms of action: (i) HNE and MDA directly inactivate lysosomal cysteine proteases by covalent binding to the active center; (ii) HNE- and MDA-mediated protein modifications convert proteolytic substrates into competitive inhibitors of lysosomal proteases. Via these mechanisms, lipid peroxidation products may induce lysosomal dysfunction and lipofuscinogenesis in the aging RPE and thus contribute to the pathogenesis of AMD.

In-depth mass spectrometric mapping of the human vitreous proteome

Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from “surrogate normal patients” with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.

New Pharmacologic Approaches to Therapy for Age-Related Macular Degeneration

As a result of a better understanding of molecular mechanisms, a variety of new Pharmacologic treatments have recently been developed for patients with age-related macular degeneration (AMD). Efficacy and tolerability have been demonstrated for drugs targeting vascular endothelial growth factor (VEGF), a key player in the pathogenesis of choroidal neovascularization. Both pegaptanib (anti-VEGF aptamer) and ranibizumab (anti-VEGF antibody fragment), applied at 4- to 6-week intervals into the vitreous, modified the natural course of the disease in phase III clinical studies. Corticosteroids with anti-angiogenic properties also represent a treatment option for wet AMD. Both intravitreal triamcinolone and anecortave acetate, administered juxtasclerally, are currently being pursued.The combination of different treatment strategies and potential synergistic effects offers new perspectives. While photodynamic therapy (PDT) combined with intravitreal triamcinolone is already frequently applied, other combinations (e.g. anti-VEGF drugs with PDT or antifibrotic agents) appear to be attractive alternatives. Pigment epithelium-derived factor represents another potential target, as well as inhibitors of matrix-metalloproteinases. With the advent of gene therapy, the use of small interfering RNA (siRNA) is also on the horizon.Prophylactic measures are still limited. The combination of vitamins C and E, β-carotene, and zinc as used in the AREDS (Age-Related Eye Disease Study) reduces risk for conversion from early- to late-stage disease in patients with high-risk features, at least to some extent. Lutein and zeaxanthin dietary supplements for improvement of macular pigment density need to be investigated in future longitudinal trials.

Pharmacokinetics and safety of intravitreally delivered etanercept

BackgroundThe anti-inflammatory drug etanercept may be an effective therapeutic agent in diabetic retinopathy. In order to further evaluate its potential, the pharmacokinetics and safety of this drug after intravitreal delivery were investigated.MethodsAfter intravitreal administration of etanercept in rabbits, clinical examination, electroretinography (ERG), visually evoked potentials (VEP) and histology were evaluated. The pharmacokinetics and distribution of etanercept were analyzed using fluorescence-coupled protein at 0, 2, 4, and 8 weeks after injection in vitreous, retina, and choroid.ResultsNo adverse effects and signs of toxicity were found. Etanercept showed peak concentrations after 4 weeks in the retina and choroid.ConclusionsIntravitreally delivered etanercept is safe and results in high concentrations in the retina and choroid over a long period of time.