Concentrations Of Ranibizumab Research Articles

Dose-Related Side Effects of Intravitreal Injections of Humanized Anti-Vascular Endothelial Growth Factor in Rats: Glial Cell Reactivity and Retinal Ganglion Cell Loss.

In a previous study, we documented that the Intravitreal injections (IVIs) of bevacizumab in rats caused a retinal inflammatory response. We now study whether the IVI of other humanized anti-VEGF: ranibizumab and aflibercept also cause an inflammatory reaction in the rat retina and if it depends on the dose administered. Finally, we study whether this reaction affects retinal ganglion cell (RGC) survival. Albino Sprague-Dawley rats received a single IVI of 5 µL of PBS or ranibizumab or aflibercept at the concentration used in clinical practice (10 µg/µL or 40 µg/µL) or at a lower concentration (0.38 µg/µL and 1.5 µg/µL) calculated to obtain within the rat eye the same concentration as in the human eye in clinical practice. Others received a single 5 µL IVI of a polyclonal goat anti-rat VEGF (0.015 µg/µL) or of vehicle (PBS). Animals were processed 7days or 1month later. Retinal whole mounts were immunolabeled for the detection of microglial, macroglial, RGCs, and intrinsically photosensitive RGCs (ipRGCs). Fluorescence and confocal microscopy were used to examine retinal changes, and RGCs and ipRGCs were quantified automatically or semiautomatically, respectively. All the injected substances including the PBS induced detectable side effects, namely, retinal microglial cell activation and retinal astrocyte hypertrophy. However, there was a greater microglial and macroglial response when the higher concentrations of ranibizumab and aflibercept were injected than when PBS, the antibody anti-rat VEGF and the lower concentrations of ranibizumab or aflibercept were injected. The higher concentration of ranibizumab and aflibercept resulted also in significant RGC death, but did not cause appreciable ipRGC death. The IVI of all the substances had some retinal inflammatory effects. The IVI of humanized anti-VEGF to rats at high doses cause important side effects: severe inflammation and RGC death, but not ipRGC death.

The role of mitochondrial genes on nuclear gene expression in neovascular age related macular degeneration: analysis of nuclear VEGF gene expression after ranibizumab treatment in cytoplasmic hybrid retinal pigment epithelial cell lines correlated with clinical evolution

PurposeThe present study tests the hypothesis that mitochondrial genes have retrograde signaling capacity that influences the expression of nuclear genes related to angiogenesis pathways. Cytoplasmic hybrid (cybrid) in vitro cell lines with patient specific mitochondria inserted into an immortalized retinal pigment epithelial cell line (ARPE-19) were used to test this hypothesis. This type of analysis can provide important information to identify the optimal regimen of anti-VEGF treatment, personalizing age-related macular degeneration (AMD) therapies.MethodsMitochondria deficient ARPE-19 cells (Rho0) were fused with AMD donor’s platelets to create individual cybrid cell lines containing mitochondria from patients with phenotypic AMD disease and nuclear DNA from the immortalized RPE cell line. The cybrids were treated with Ranibizumab (Lucentis, Genentech, San Francisco, CA), at 4 different concentrations for 24 h, and subsequently the levels of reactive oxygen species (ROS), gene expression for VEGF-A, hypoxia-inducible factor 1-alpha (HIF1-a) and manganese superoxide dismutase (SOD2) were measured. The clinical evolution of the two AMD-donors were correlated with the molecular findings found in their ‘personalized’ cybrids.ResultsCybrids from Patient-01 showed down-regulation of gene expression of VEGF-A and HIF-1a at both 1X and 4X Ranibizumab concentrations. Patient-01 AMD cybrid cultures had an increase in the ROS levels at 1X (P = 0.0317), no changes at 2X (P = 0.8350) and a decrease at 4X (P = 0.0015) and 10X (P = 0.0011) of Ranibizumab. Clinically, Patient-01 responded to anti-VEGF therapy but eventually developed geographic atrophy. Patient-02 cybrids demonstrated up-regulation of gene expression of VEGF-A and HIF-1a at Ranibizumab 1X and 4X concentrations. There was decreased ROS levels with Ranibizumab 1X (P = 0.1606), 2X (P = 0.0388), 4X (P = 0.0010) and 10X (P = < 0.0001). Clinically, Patient-02 presented with a neovascular lesion associated with a prominent production of intraretinal fluid in clinical follow-up requiring regular and repeated intravitreal injections of Ranibizumab with recurrent subretinal fluid.ConclusionsOur cybrid model has the potential to help personalize the treatment regimen with anti-VEGF drugs in patients with neovascular AMD. Further investigation is needed to better understand the role that the mitochondria play in the cellular response to anti-VEGF drugs. Future studies that focus on this model have the potential to help personalize anti-VEGF treatment.

Immunogenicity With Ranibizumab Biosimilar SB11 (Byooviz) and Reference Product Lucentis and Association With Efficacy, Safety, and Pharmacokinetics

SB11 and reference ranibizumab (RBZ) are monoclonal anti-vascular endothelial growth factor (VEGF)-A antibodies approved for the treatment of neovascular age-related macular degeneration (nAMD) and other retinal diseases. The association of ranibizumab immunogenicity and treatment outcomes in patients with nAMD is unclear but relevant regarding concerns about immunogenicity of anti-VEGF biological products. To examine the association of immunogenicity to ranibizumab products (SB11 and RBZ) with efficacy, safety, and pharmacokinetics. This was a post hoc analysis of a randomized, double-masked, parallel-group phase 3 equivalence study with participants from 75 centers in 9 countries conducted from March 14, 2018, to December 9, 2019. Included were participants 50 years or older with nAMD and active subfoveal choroidal neovascularization lesions. Intravitreal injection of SB11 or RBZ, 0.5 mg, every 4 weeks through week 48. Serum antidrug antibodies (ADAs) were analyzed during the study period until week 52 to measure immunogenicity. Analyses were performed on immunogenicity (overall ADA positivity) with best-corrected visual acuity (BCVA) and central subfield thickness (CST). Adverse events associated with intraocular inflammation (IOI) and serum ranibizumab levels were compared between overall ADA-positive and ADA-negative participants. A total of 705 participants (mean [SD] age, 74.1 [8.5] years; 403 female individuals [57.2%]) were included in the study. The overall incidence of ADA-positivity was 32 of 657 (4.9%) at week 52. The least-squares mean (SE) differences between overall ADA-positive and ADA-negative participants up to week 52 for BCVA and CST, respectively, were 1.6 (2.2) letters (95% CI, -2.7 to 5.8; P = .46) and 3 (13) μm (95% CI, -23 to 29; P = .83). IOI-related events occurred in 1 of 32 overall ADA-positive participants (3.1%) and 4 of 620 overall ADA-negative participants (0.6%). Mean (SD) serum ranibizumab concentrations over time were slightly lower in overall ADA-positive participants compared with those of ADA-negative participants, with a maximum value of 1389.3 (875.4) pg/mL at week 16 vs 1665.4 (1124.0) pg/mL at week 36, respectively. Results of this post hoc analysis of an equivalence trial suggest that immunogenicity was not associated with efficacy and safety of SB11 and RBZ in participants with nAMD. With a low overall ADA incidence, no clear association was identified between overall ADA positivity and pharmacokinetics. These findings support the biosimilarity of SB11 and RBZ, with no safety concern identified for SB11 vs RBZ associated with immunogenicity. ClinicalTrials.gov Identifier: NCT03150589.

Pharmacokinetics of the Port Delivery System with Ranibizumab in the Ladder Phase 2 Trial for Neovascular Age-Related Macular Degeneration.

IntroductionLadder was a phase 2 trial that evaluated the Port Delivery System with ranibizumab (PDS) for neovascular age-related macular degeneration. Serum and aqueous humor samples were collected to characterize the pharmacokinetics (PK) of ranibizumab delivered through the PDS.MethodsLadder was a multicenter, randomized, active treatment-controlled, phase 2 clinical trial. Patients with neovascular age-related macular degeneration (n = 220) were randomized (3:3:3:2) to PDS 10 mg/ml, PDS 40 mg/ml, PDS 100 mg/ml, or monthly intravitreal ranibizumab 0.5 mg. Serum PK samples were collected in all arms and analyzed for ranibizumab concentration using an enzyme-linked immunosorbent assay. The main PK analyses were conducted in the PK-evaluable population (n = 68), which excluded patients who received fellow eye intravitreal treatment, supplemental ranibizumab treatment, or had previous treatment with bevacizumab in either eye within 9 months of randomization.ResultsIn the PDS 10 mg/ml arm, median serum ranibizumab concentrations were below the serum trough concentration (Ctrough; 130 pg/ml) expected with monthly intravitreal ranibizumab 0.5 mg at all time points. In the PDS 40 mg/ml and 100 mg/ml arms, median serum ranibizumab concentrations were above the Ctrough expected with monthly intravitreal ranibizumab 0.5 mg (130 pg/ml) through month 3 and month 12 after implantation, respectively, and remained above the lower limit of quantification through month 15 and month 16 after implantation, respectively.ConclusionsThese PK data indicate that the implant in the PDS 100 mg/ml arm maintained ranibizumab concentrations within the range of monthly intravitreal ranibizumab 0.5 mg injections (130–2220 pg/ml) through month 12 after implantation.Trial RegistrationClinicalTrials.gov identifier, NCT02510794.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40123-022-00532-9.

The Port Delivery System with ranibizumab: a new paradigm for long-acting retinal drug delivery

The Port Delivery System with ranibizumab (PDS) is an innovative intraocular drug delivery system designed for the continuous delivery of ranibizumab into the vitreous for 6 months and beyond. The PDS includes an ocular implant, a customized formulation of ranibizumab, and four dedicated ancillary devices for initial fill, surgical implantation, refill-exchange, and explantation, if clinically indicated. Ranibizumab is an ideal candidate for the PDS on account of its unique physicochemical stability and high solubility. Controlled release is achieved via passive diffusion through the porous release control element, which is tuned to specific drug characteristics to accomplish a therapeutic level of ranibizumab in the vitreous. To characterize drug release from the implant, release rate was measured in vitro with starting concentrations of ranibizumab 10, 40, and 100 mg/mL, with release of ranibizumab 40 and 100 mg/mL found to remain quantifiable after 6 months. Using a starting concentration of 100 mg/mL, active release rate at approximately 6 months was consistent after the initial fill and first, second, and third refills, demonstrating reproducibility between implants and between multiple refill-exchanges of the same implant. A refill-exchange performed with a single 100-µL stroke using the refill needle was shown to replace over 95% of the implant contents with fresh drug. In vitro data support the use of the PDS with fixed refill-exchange intervals of at least 6 months in clinical trials.

In-vitro characterization of ranibizumab release from the Port Delivery System

The Port Delivery System with ranibizumab (PDS) consists of an implant that is a permanent, indwelling drug delivery device that can be refilled through a self-sealing septum and is designed to continuously release a customized formulation of ranibizumab into the vitreous by passive diffusion through a porous titanium release control element. Target release rates of ranibizumab via the implant used in studies of the PDS in patients with neovascular age-related macular degeneration were selected based on clinical and pharmacokinetic (PK) data from previously conducted intravitreal ranibizumab injection studies. In-vitro testing was performed to verify release rates with a range of ranibizumab concentrations before the phase II Ladder (NCT02510794) and phase III Archway (NCT03677934) trials of the PDS. Implants were filled with ranibizumab and were regularly transferred to new buffer-containing tubes to represent ocular ranibizumab clearance and release kinetics. Ranibizumab concentrations were measured and release rates calculated. Release rate data were fit to an exponential model and compared with expected release kinetics of diffusion. Release profiles of the implant releasing ranibizumab at concentrations of 10 mg/mL, 40 mg/mL, and 100 mg/mL were determined in the pre–phase II in-vitro studies. At day 3.5, mean (SD) ranibizumab release rates were 1.75 (0.07), 6.42 (0.35), and 16.69 (0.67) μg/d for PDS 10 mg/mL, 40 mg/mL, and 100 mg/mL, respectively. At month 6, mean (SD) release rates were 1.68 (0.05) and 4.16 (0.05) μg/d for PDS 40 mg/mL and 100 mg/mL, respectively. Measured release rates were within 90% of theoretical release rates during the course of drug release. PDS 100 mg/mL released 73% (SD, 1.92) of drug by month 6. In the pre–phase III in-vitro studies, mean (SD) release rates with PDS 100 mg/mL were 17.97 (0.90), 4.44 (0.11), and 2.45 (0.08) μg/d at 3.5 days, 6 months, and 9 months, respectively. Cumulative release (SD) was 73% (1.92) by month 6 and 87% (1.88) by month 9. The sustained, continuous, and reproducible release from the PDS observed in the in-vitro studies was also observed in Ladder and Archway. In conclusion, in-vitro studies were a powerful tool for characterizing and verifying ranibizumab release from the PDS implant and supported clinical evaluation of the PDS. PDS 100 mg/mL, which was associated with the longest therapeutic-level delivery of ranibizumab among the concentrations tested, was selected for evaluation in the pivotal phase III Archway trial.

Ranibizumab Population Pharmacokinetics and Free VEGF Pharmacodynamics in Preterm Infants With Retinopathy of Prematurity in the RAINBOW Trial.

PurposeTo develop a population pharmacokinetic (PK) model for intravitreal ranibizumab in infants with retinopathy of prematurity (ROP) and assess plasma free vascular endothelial growth factor (VEGF) pharmacodynamics (PD).MethodsThe RAnibizumab compared with laser therapy for the treatment of INfants BOrn prematurely With retinopathy of prematurity (RAINBOW) trial enrolled 225 infants to receive a bilateral intravitreal injection of ranibizumab 0.1 mg, ranibizumab 0.2 mg, or laser in a 1:1:1 ratio and included sparse sampling of blood for population PK and PD analysis. An adult PK model using infant body weight as a fixed allometric covariate was re-estimated using the ranibizumab concentrations in the preterm population. Different variability, assumptions, and covariate relationships were explored. Model-based individual predicted concentrations of ranibizumab were plotted against observed free VEGF concentrations.ResultsElimination of ranibizumab had a median half-life of 5.6 days from the eye and 0.3 days from serum, resulting in an apparent serum half-life of 5.6 days. Time to reach maximum concentration was rapid (median: 1.3 days). Maximum concentration (median 24.3 ng/mL with ranibizumab 0.2 mg) was higher than that reported in adults. No differences in plasma free VEGF concentrations were apparent between the groups or over time. Plotted individual predicted concentrations of ranibizumab against observed free VEGF concentrations showed no relationship.ConclusionsIn preterm infants with ROP, elimination of ranibizumab from the eye was the rate-limiting step and was faster compared with adults. No reduction in plasma free VEGF was observed. The five-year clinical safety follow-up from RAINBOW is ongoing.Translational RelevanceOur population PK and VEGF PD findings suggest a favorable ocular efficacy: systemic safety profile for ranibizumab in preterm infants.

Intraocular Pharmacokinetics of 10-fold Intravitreal Ranibizumab Injection Dose in Rabbits.

PurposeTo investigate intraocular pharmacokinetics of 10-fold dose of intravitreally injected ranibizumab compared with the conventional dose in an experimental model.MethodsRanibizumab 30 µL at 10 mg/mL (conventional) and 100 mg/mL (10-fold) doses was injected separately into each eye of 28 rabbits. Ranibizumab concentrations in the aqueous humor, vitreous, and retina were estimated at each time period after injection, using enzyme-linked immunosorbent assay. The pharmacokinetic properties of ranibizumab were determined using a one-compartment model in all three ocular tissues. The time–concentration profile and predictive trends were plotted to determine drug efficacy in the retina.ResultsMaximum concentrations after conventional and 10-fold dosing were observed in the retina at 1 and 4 days after injection, respectively. The half-life of ranibizumab after conventional and 10-fold dosing did not differ in the anterior chamber and vitreous, whereas the half-life was prolonged approximately twice with the 10-fold dose in the retina (36.74 h vs. 76.85 h) and serum (91.93 h vs. 179.01 h). Similarly, the estimated time for ranibizumab concentration in the retina over 27 ng/mL (minimum effective concentration of ranibizumab) was prolonged approximately twice with the 10-fold dose (1315 h [55 days] vs. 2393 h [100 days]). No adverse effects were observed in either group.ConclusionsThe retinal half-life and concentration of ranibizumab in rabbit eyes were increased approximately twice after a 10-fold dose compared with the conventional dose. This finding indicates a possibility to lengthen the injection interval to improve the efficacy of ranibizumab in human eyes.Translational RelevanceOur results highlight the potential for clinical application of a high-dose (10-fold) of anti-VEGF agents to prolong the intravitreal injection intervals, simultaneously improving the drug efficacy.

In vitro response and gene expression of human retinal Müller cells treated with different anti-VEGF drugs

In the past decade, anti-vascular endothelial growth factor (VEGF) therapy has become more popular and the standard of care for several retinal diseases including exudative age-related macular degeneration (AMD) (Brown et al., 2006; Martin et al., 2012; Rosenfeld et al., 2006; Tufail et al., 2010), diabetic macular edema (DME) (Brown et al., 2013; Do et al., 2012; Do et al., 2013; TDRCR, 2015), and macular edema (ME) due to retinal vein occlusion (RVO) (Brown et al., 2010; Brown et al., 2013; Campochiaro et al., 2010; Wu et al., 2008). Ranibizumab (Lucentis®, Genentech, San Francisco, CA) is a humanized, recombinant, monoclonal antibody fragment. Intended for intraocular use, a 0.5mg/0.05ml dose is approved by the Food and Drug Administration for exudative AMD and ME due to RVO; a 0.3mg/0.05ml dose is approved by the FDA for DME, and diabetic retinopathy (DR). Bevacizumab (Avastin®, Genentech, San Francisco, CA) is a humanized monoclonal human immunoglobulin gamma 1 (IgG1) antibody that selectively binds to circulating VEGF and inhibit its binding to cell surface receptors. A 1.25mg/0.05ml is used off-label in the United States and is commonly used worldwide for exudative AMD, DME and RVO. Aflibercept (Eylea®, Regeneron Pharmaceuticals, Tarrytown, NY) is a recombinant protein receptor decoy composed of two VEGF receptors 1 and 2 fused with the Fc region of human IgG1. Compared to the former two anti-VEGF drugs, it has a higher binding affinity for VEGF and it also binds to VEGF-B and Placental growth factor (PGF). A concentration of 2.0mg/0.05ml is approved by the FDA for wet AMD, DME, ME due to RVO, and DR in patients with DME. Ziv-aflibercept (Zaltrap®, Sanofi Aventis, Bridgewater, NJ and Regeneron Pharmaceuticals, Tarrytown, NY), also known as VEGF Trap-oncologic, contains 25 mg/ml ziv-aflibercept, however contains higher sucrose concentrations than aflibercept (Eylea), which results in higher osmolality (Bayer, 2014; Group, 2012). Ziv-aflibercept is approved by the FDA for treatment of resistant or progressing metastatic colorectal cancer. However, Mansour et al. (Mansour et al., 2015; Mansour et al., 2017a; Mansour et al., 2017b); de Oliveira Dias et al. (de Oliveira Dias et al., 2015; de Oliveira Dias et al., 2017; de Oliveira Dias et al., 2016); and Chhablani et al., (Chhablani, 2015; Chhablani et al., 2017), among other authors, have published several reports finding good outcomes and no apparent safety issues in a series of patients treated with intravitreal ziv-aflibercept. Previous studies have shown that anti-VEGF drugs in clinically significant doses do not affect cell viability in vitro in a human retinal pigment epithelium (RPE) cell line (ARPE-19) (Luthra et al., 2006; Malik et al., 2014b). Nonetheless, subtle cytotoxic changes in RPE cells, such as decrease in mitochondrial membrane potential, could be observed even at clinical doses for some anti-VEGF drugs (Malik et al., 2014b). Numerous publications have described in vitro differential responses to the different anti-VEGF drugs in various retinal cells (Deissler et al., 2012; Klettner et al., 2010; Schnichels et al., 2013). Muller cells are predominant glial cells in the retina and provide structural and metabolic support to retinal neurons. Several studies have described numerous functions of Muller cells, including regulation of cellular homeostasis and pH, modulation of neurotransmitter recycling, contribution to the blood-retinal barrier by surrounding retinal capillaries with glial processes (Limb et al., 2002; Reichenbach and Bringmann, 2013), acting as light collectors by directing light to photoreceptors (Franze et al., 2007; Reichenbach and Bringmann, 2013), and secretion and regulation of VEGF and pigment epithelium derived factor (PEDF) (Garcia and Vecino, 2003; Limb et al., 2002; Reichenbach and Bringmann, 2013). Muller cells have also been associated with retinal neuroprotection, wound healing and regeneration (Garcia and Vecino, 2003). The purpose of this study was to evaluate the in vitro response and differences in gene expression of human retinal Muller cells treated with different concentrations of ranibizumab, bevacizumab, aflibercept or ziv-aflibercept. Assays for cell viability, metabolic activity, mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) and apoptosis were used to evaluate responses to the anti-VEGF drugs. In addition, differences in the expression levels for angiogenesis-related, pro-apoptotic, inflammation and oxidative stress genes were assessed.

Transfer of single dose of intravitreal injection of ranibizumab and bevacizumab into milk of sheep.

To investigate whether single-dose intravitreal injections of bevacizumab and ranibizumab transfer into milk. This study included lactating 12 sheep and a single 3-month old suckling lamb of each sheep. Two groups consisting of 6 sheep and their lambs were constituted; the ranibizumab group and the bevacizumab group before the administration of intravitreal injections, blood and milk samples were obtained from all sheep and, following the injections, blood and milk samples of all sheep and blood samples of all lambs were collected at regular time points. Serum and milk concentrations of bevacizumab and ranibizumab were measured using an enzyme-linked immunosorbent assay (ELISA) kit. The limit of determination was 0.9 ng/mL for bevacizumab and 0.62 ng/mL for ranibizumab. At 6h after intravitreal injections, bevacizumab concentration was above the limit of determination in the blood of all sheep. At 3wk, when the study was terminated, bevacizumab concentrations were high in 4 sheep. Even though bevacizumab concentrations in milk showed fluctuations, the drug transferred into the milk of all sheep at detectable concentrations. Ranibizumab drug concentrations in the blood and milk of sheep and those in the blood of lambs were below the limit of determination by the ELISA kit. This sheep model study demonstrate that intravitreal injection of ranibizumab, which did not transfer into the milk of sheep and suckling lambs, is safer than bevacizumab during lactation period.

Topical Delivery of Anti-VEGF Drugs to the Ocular Posterior Segment Using Cell-Penetrating Peptides.

To evaluate the efficacy of anti-VEGF agents for treating choroidal neovascularization (CNV) when delivered topically using novel cell-penetrating peptides (CPPs) compared with delivery by intravitreal (ivit) injection. CPP toxicity was investigated in cell cultures. Ivit concentrations of ranibizumab and bevacizumab after topical administration were measured using ELISA. The biological efficacy of topical anti-VEGF + CPP complexes was compared with ivit anti-VEGF injections using an established model of CNV. CPPs were nontoxic in vitro. In vivo, after topical eye drop delivery, CPPs were present in the rat anterior chamber within 6 minutes. A single application of CPP + bevacizumab eye drop delivered clinically relevant concentrations of bevacizumab to the posterior chamber of the rat eye in vivo. Similarly, clinically relevant levels of CPP + ranibizumab and CPP + bevacizumab were detected in the porcine vitreous and retina ex vivo. In an established model of CNV, mice treated with either a single ivit injection of anti-VEGF, twice daily CPP + anti-VEGF eye drops or daily dexamethasone gavage for 10 days all had significantly reduced areas of CNV when compared with lasered eyes without treatment. CPPs are nontoxic to ocular cells and can be used to deliver therapeutically relevant doses of ranibizumab and bevacizumab by eye drop to the posterior segment of mouse, rat, and pig eyes. The CPP + anti-VEGF drug complexes were cleared from the retina within 24 hours, suggesting a daily eye drop dosing regimen. Daily, topically delivered anti-VEGF with CPP was as efficacious as a single ivit injection of anti-VEGF in reducing areas of CNV in vivo.

Vascular Endothelial Growth Factor (VEGF) Concentration Is Underestimated by Enzyme-Linked Immunosorbent Assay in the Presence of Anti-VEGF Drugs

Commercially available enzyme-linked immunosorbent assay (ELISA) kits are often used to monitor vascular endothelial growth factor (VEGF) levels in exudative age-related macular degeneration. To test their accuracy, this study performed measurements using the ELISA kits in the presence of anti-VEGF drugs. The concentrations of bevacizumab, pegaptanib, or ranibizumab at 28 days and aflibercept at 28 and 56 days after an injection were estimated based on previous pharmacokinetic studies. Vascular endothelial growth factor concentrations were measured with two widely used VEGF ELISA kits in the presence of anti-VEGF drugs or control mouse immunoglobulin G (IgG). The monocyte chemotactic protein-1 (MCP-1) ELISA kit was used as a non-VEGF ELISA control kit. The concentrations of aflibercept, bevacizumab, pegaptanib, and ranibizumab were estimated at 0.14 to 7.2, 4.9, 8.6, and 0.11 to 1.1 μg/mL, respectively. ELISA underestimated the VEGF concentration 2- to 100-fold lower in the presence of an anti-VEGF drug, except for pegaptanib, at all VEGF concentrations tested (7.8-1500 pg/mL). Vascular endothelial growth factor at 1000 pg/mL was measured as 92, 150, and 170 pg/mL in the presence of aflibercept (7.2 μg/mL), bevacizumab (4.9 μg/mL), and ranibizumab (1.1 μg/mL), respectively (all P < 0.0001), and the measured VEGF concentration decreased proportionately by 90% to 92% with aflibercept, 85% to 94% with bevacizumab, and 83% to 99% with ranibizumab. The control mouse IgG did not interfere with the measurement of VEGF. Ranibizumab did not affect the measurements with MCP-1 ELISA. Investigators should exercise caution when interpreting measurements of VEGF ELISA in patients being treated with an anti-VEGF drug.

Different Effects of Ranibizumab and Bevacizumab on Platelet Activation Profile

Purpose: The aim of the study was to evaluate the potential influence of ranibizumab and bevacizumab on platelet activation and aggregation, which are critical processes in the pathogenesis of arterial thromboembolic events (ATEs). Methods: For the assessment of platelet function, flow cytometry and aggregometry were employed. Platelets were isolated from healthy volunteers and exposed to ranibizumab (1 mg/ml and 150 ng/ml) and bevacizumab (2.5 mg/ml and 3 μg/ml) or their solvents for 10 and 30 min prior to the addition of TRAP (25 μ<smlcap>M</smlcap>), PAR-4-AP (25 μ<smlcap>M</smlcap>) or thrombin (0.02 U/ml). The surface expression of activated GP IIb/IIIa, P-selectin (CD62P) and platelet-bound stromal cell-derived factor-1 (SDF-1) was measured on resting (nonactivated) and activated platelets by flow cytometry. The platelet aggregation capacity was examined using light transmission aggregometry. Results: The expression of surface activation markers did not differ significantly between nonstimulated and TRAP-, PAR-4-AP- or thrombin-activated platelets after incubating with ranibizumab. However, GP IIb/IIIa, CD62P and SDF-1 were significantly downregulated in PAR-4-AP- and thrombin-activated platelets after exposure to bevacizumab 2.5 mg/ml. In addition, ranibizumab- and bevacizumab-FITC were significantly increased in all activated platelets. No significant differences were observed in the aggregation of activated platelets after incubation with ranibizumab or bevacizumab. Conclusion: All ranibizumab concentrations as well as the bevacizumab concentration of 3 μg/ml had no influence on platelet activation and aggregation. Therefore, this in vitro study did not show any relationship between the exposition of activated platelets to ranibizumab or bevacizumab and the development of ATEs. However, the highest level of bevacizumab interfered with platelet activation, leading to downregulation of platelet activation markers. This observation might explain why the systemic treatment with high-dose bevacizumab could be associated with an increased risk of bleeding. Regarding the use of lower intravitreal dosages, further research should focus on the complex interactions between platelets and other cells, such as endothelial cells, which might stronger relate to a potentially increased risk of ATEs and depend on systemic vascular endothelial growth factor levels. Facing the different activation profiles, the diverse effects of the drugs on the cellular level have to be critically scrutinized for their clinical relevance.

PENETRATION OF BEVACIZUMAB AND RANIBIZUMAB THROUGH RETINAL PIGMENT EPITHELIAL LAYER IN VITRO

To determine the permeability of bevacizumab and ranibizumab through highly-polarized retinal pigment epithelial (RPE) cells in vitro. Highly-polarized RPE cells were cultured in the upper chamber of a Transwell culture system. Bevacizumab or ranibizumab was added to the upper chamber. After 3 hours, the concentrations of bevacizumab or ranibizumab were determined in the upper and lower chambers. The cytotoxicities of the two anti-vascular endothelial growth factor agents were determined histologically. The effects of inhibiting endocytosis by pharmacologic inhibitors were also evaluated. The concentration of ranibizumab was higher than that of bevacizumab in the lower chamber (P < 0.05). Vascular endothelial growth factor was found mainly in the lower chamber under normal conditions. However, the concentration of vascular endothelial growth factor in the lower chamber was significantly less when ranibizumab was added to the upper chamber than when bevacizumab was added. Histology showed no obvious changes in bevacizumab-exposed or ranibizumab-exposed RPE cells. Pretreatment with protein kinase C inhibitor staurosporine had significant negative effects on the permeability of bevacizumab and ranibizumab (P < 0.05). Ranibizumab is more permeable than bevacizumab through the highly-polarized RPE layer at clinically equivalent concentrations, and their permeability was partially protein kinase C-dependent. Ranibizumab might be more therapeutically effective than bevacizumab on choroidal neovascularization beneath the RPE layer.

Angiogenic potential of vitreous from Proliferative Diabetic Retinopathy and Eales' Disease patients.

PurposeProliferative Diabetic Retinopathy (PDR) and Eales' Disease (ED) have different aetiologies although they share certain common clinical symptoms including pre-retinal neovascularization. Since there is a need to understand if the shared end-stage angiogenic pathology of PDR and ED is driven by common stimulating factors, we have studied the cytokines contained in vitreous from both patient groups and analyzed the angiogenic potential of these samples in vitro.Material and MethodsVitreous samples from patients with PDR (n = 13) and ED (n = 5) were quantified for various cytokines using a cytokine biochip array and sandwich ELISA. An additional group of patients (n = 5) with macular hole (MH) was also studied for comparison. To determine the angiogenic potential of these vitreous samples, they were analyzed for their ability to induce tubulogenesis in human microvascular endothelial cells. Further, the effect of anti-VEGF (Ranibizumab) and anti-IL-6 antibodies were studied on vitreous-mediated vascular tube formation.ResultsElevated levels of IL-6, IL-8, MCP-1 and VEGF were observed in vitreous of both PDR and ED when compared to MH. PDR and ED vitreous induced greater levels of endothelial cell tube formation compared to controls without vitreous (P<0.05). When VEGF in vitreous was neutralized by clinically-relevant concentrations of Ranibizumab, tube length was reduced significantly in 5 of 6 PDR and 3 of 5 ED samples. Moreover, when treated with IL-6 neutralizing antibody, apparent reduction (71.4%) was observed in PDR vitreous samples.ConclusionsWe have demonstrated that vitreous specimens from PDR and ED patients share common elevations of pro-inflammatory and pro-angiogenic cytokines. This suggests that common cytokine profiles link these two conditions.

Gene profiling of human VEGF signaling pathways in human endothelial and retinal pigment epithelial cells after anti VEGF treatment.

BackgroundRanibizumab (Lucentis®) is a Fab-antibody fragment developed from Bevacizumab, a full-length anti-VEGF antibody. Both compounds are used for treating age-related macular degeneration (AMD). The influence of bevacizumab and ranibizumab on genes involved in signal transduction and cell signaling downstream of VEGF were compared in order to detect possible differences in their mode of action, which are not related to their Fab-antibody fragments.MethodsHuman umbilical vein cell lines (EA.hy926) and retinal pigment epithelial cells (ARP-19) were exposed to oxidative stress. The cells were treated with therapeutic concentrations of bevacizumab (0.25 mg/mL) and ranibizumab (125 mg/mL) for 24 hours prior to all experiments, and their effects on gene expressions were determined by RT- PCR.ResultsAfter exposure to bevacizumab, more genes in the endothelial cells were up-regulated (KDR, NFATc2) and down-regulated (Pla2g12a, Rac2, HgdC, PRKCG) compared to non-treated controls. After exposure to ranibizumab, fewer genes were up-regulated (PTGS2) and down-regulated (NOS3) compared to controls. In comparison between drugs, more genes were up-regulated (NFATc2 and KDR) and more were down-regulated (Pla2g12a, Pla2g1b, Ppp3r2, Rac2) by bevacizumab than by ranibizumab. In RPE cells, NOS3 and PGF were up-regulated and Pla2g12b was down-regulated after exposure to ranibizumab, while PIK3CG was up-regulated and FIGF was down-regulated after exposure to bevacizumab, but the differences in gene expression were minor between drugs (PIK3CGand PGF were down-regulated more by ranibizumab than by bevacizumab).ConclusionsThe different gene expressions after exposure to ranibizumab and bevacizumab in endothelial and RPE cells may indicate a somewhat different biological activity of the two compounds.

Pharmacokinetics of Ranibizumab after Intravitreal Administration in Patients with Retinal Vein Occlusion or Diabetic Macular Edema

To describe the systemic pharmacokinetics of ranibizumab after intravitreal administration in patients with retinal vein occlusion (RVO) or diabetic macular edema (DME). A population approach of nonlinear mixed-effect pharmacokinetics modeling based on serum concentrations of ranibizumab measured at various times after intravitreal administration. Patients with RVO (n = 441) and DME (n = 435) from 4 large, randomized, phase 3 clinical trials of monthly ranibizumab intravitreal administration. A 1-compartment pharmacokinetics model with first-order absorption and elimination rate constants previously developed in patients with age-related macular degeneration (AMD) was fitted separately to RVO and DME data. Population pharmacokinetic parameters and interindividual variability were estimated for each model. Baseline covariates were evaluated for potential effects on systemic pharmacokinetics. Model performance was validated using general diagnostic plots and a visual predictive check. Ranibizumab disposition was determined in RVO and DME patients and compared with that previously seen in AMD patients. The AMD pharmacokinetics model correctly predicted the measured serum ranibizumab concentration data for RVO and DME patients. Most observed data points were within the simulated 90% confidence interval, indicating that systemic ranibizumab concentrations were comparable among AMD, RVO, and DME patients. No disease-related covariates were identified by the population pharmacokinetics analysis. The systemic pharmacokinetics of ranibizumab were similar among patients with AMD, RVO, or DME. Disease-related differences and patient demographics, measured in this study, did not lead to variability in ocular elimination or in systemic exposure of ranibizumab after intravitreal administration. In all disease processes tested, ranibizumab exits the eye slowly and then is eliminated rapidly from the circulation, thus minimizing systemic exposure.

Intraocular Pharmacokinetics of Ranibizumab in Vitrectomized Versus Nonvitrectomized Eyes

To analyze the intraocular pharmacokinetic properties of intravitreally injected ranibizumab in vitrectomized and nonvitrectomized rabbit eyes. A procedure consisting of 25-gauge pars plana vitrectomy without lensectomy and posterior vitreous detachment was performed in 18 rabbit eyes, and 18 nonvitrectomized rabbit eyes served as controls. Ranibizumab (0.25 mg/0.025 mL) was intravitreally injected in all the vitrectomized and nonvitrectomized eyes. The eyes were enucleated at 1 hour or 1, 2, 5, 14, or 30 days after the intravitreal injections and frozen at -80°C. Ranibizumab concentrations in the vitreous, aqueous humor, and retina were determined using indirect enzyme-linked immunosorbent assay. Vitreous clearance of ranibizumab showed a 2-phase elimination. The vitrectomized and nonvitrectomized eyes showed comparable rates of vitreous clearance of ranibizumab. The vitreous half-life of ranibizumab for up to 14 days was 2.51 and 2.75 days in vitrectomized and nonvitrectomized eyes, respectively. Throughout the 30-day period after intravitreal injection, there were no statistically significant differences between the concentrations of ranibizumab in the vitreous, aqueous humor, and retina of vitrectomized eyes and those in nonvitrectomized eyes. Concentrations of ranibizumab in the vitreous peaked 1 hour after injection, with a mean concentration of 118.01 and 91.61 μg/mL in vitrectomized and nonvitrectomized eyes, respectively. The elimination rate constant of intravitreal ranibizumab in 1-phase analyses showed only a 9% increase in vitrectomized eyes compared to nonvitrectomized eyes. Overall intraocular pharmacokinetic properties of ranibizumab in vitrectomized eyes were similar to those in nonvitrectomized eyes. Our data do not support the use of different dosing regimens of ranibizumab in vitrectomized eyes.

Aqueous vascular endothelial growth factor and ranibizumab concentrations after monthly and bimonthly intravitreal injections of ranibizumab for age-related macular degeneration

To evaluate vascular endothelial growth factor (VEGF) and ranibizumab concentrations in eyes with age-related macular degeneration (AMD) after monthly and bimonthly intravitreal ranibizumab (IVR) injections. Aqueous humor samples were obtained from 26 eyes with AMD before and after IVR injections. Nine eyes received three monthly injections and 17 eyes received two bimonthly injections. The VEGF and ranibizumab concentrations were measured by enzyme-linked immunosorbent assay. The aqueous VEGF concentrations in the monthly injection group decreased below the lowest detectable limit in eight of nine eyes 1 month after the first injection and seven of nine eyes 1 month after the second injection (P < 0.001, mean baseline value, 94.7 pg/ml); the aqueous VEGF concentrations in the bimonthly injection group decreased below the lowest detectable limit in two of 17 eyes 2 months after the first injection (P < 0.001, mean baseline value, 152.4 pg/ml). The mean aqueous ranibizumab concentrations with monthly injections were 71.2 ng/ml 1 month after the first injection, and 96.3 ng/ml 1 month after the second injection. The mean aqueous ranibizumab concentrations in the bimonthly injection group were 2.5 ng/ml in 15 of 17 eyes, and below the lowest detectable limit in two of 17 eyes 2 months after the first injection. In this pilot study with limited follow-up, intravitreal injection of ranibizumab can suppress aqueous VEGF completely for 1 month in most cases. Its effect does not last for 2 months enough to suppress VEGF completely in most cases, although aqueous VEGF at 2 months after intravitreal injection of ranibizumab is less than that before injection in most cases.

Pharmacokinetics of Ranibizumab in Patients with Neovascular Age-Related Macular Degeneration: A Population Approach

To characterize ranibizumab pharmacokinetics in patients with AMD. A population approach of nonlinear mixed-effect pharmacokinetic modeling based on concentration-time data from 2993 serum samples from 674 AMD patients enrolled in 5 phase 1 to 3 clinical trials of single or multiple intravitreal (ITV) doses of ranibizumab (0.3-2.0 mg/eye) administered biweekly or monthly for up to 24 months. A TOTAL OF 696 CONCENTRATION-TIME RECORDS FROM 229 SUBJECTS WITH ONE OR MORE MEASURABLE TOTAL SERUM RANIBIZUMAB CONCENTRATIONS WERE ANALYZED. THE SYSTEMIC CONCENTRATION-TIME DATA FOR RANIBIZUMAB WERE BEST DESCRIBED BY A ONE-COMPARTMENT MODEL WITH FIRST-ORDER ABSORPTION INTO AND FIRST-ORDER ELIMINATION FROM THE SYSTEMIC CIRCULATION. VITREOUS ELIMINATION HALF-LIFE (T1/2) WAS CALCULATED TO BE 9 DAYS AND THE INTRINSIC SYSTEMIC ELIMINATION T1/2 WAS CALCULATED TO BE APPROXIMATELY 2 HOURS. FOLLOWING ITV ADMINISTRATION, RANIBIZUMAB EGRESSES SLOWLY INTO THE SYSTEMIC CIRCULATION, RESULTING IN AN APPARENT SERUM T1/2 OF 9 DAYS. SYSTEMIC-TO-VITREOUS EXPOSURE RATIO WAS ESTIMATED TO BE 1: 90,000. With monthly and quarterly ITV regimens, the serum concentrations of ranibizumab at steady-state for both the 0.3 and 0.5 mg/eye dose levels were estimated to be below the range needed to inhibit VEGF-A-induced endothelial cell proliferation in vitro by 50% at all times. Systemic exposure to ranibizumab after ITV injection was very low due to elimination on reaching systemic circulation from the vitreous. Population pharmacokinetic analysis of data from a representative sample of AMD patients did not identify clinically significant sources or correlates of variability in ranibizumab exposure. (ClinicalTrials.gov numbers, NCT00056836, NCT00056823.).