David A. Ammar

Silicone Oil Microdroplets and Protein Aggregates in Repackaged Bevacizumab and Ranibizumab: Effects of Long-term Storage and Product Mishandling

PURPOSE To quantify levels of subvisible particles and protein aggregates in repackaged bevacizumab obtained from compounding pharmacies, as well as in samples of bevacizumab and ranibizumab tested in controlled laboratory experiments. METHODS Repackaged bevacizumab was purchased from four external compounding pharmacies. For controlled laboratory studies, bevacizumab and placebo were drawn into plastic syringes and incubated at -20°C, 4°C, and room temperature (with and without exposure to light) for 12 weeks. In addition, mechanical shock occurring during shipping was mimicked with syringes containing bevacizumab. Particle counts and size distributions were quantified by particle characterization technology. Levels of monomer and soluble aggregates of bevacizumab were determined with size-exclusion high-performance liquid chromatography (SE-HPLC). RESULTS Repackaged bevacizumab from the compounding pharmacies had a wide range of particle counts (89,006 ± 56,406 to 602,062 ± 18,349/mL). Bevacizumab sampled directly from the original glass vial had particle counts of 63,839 ± 349/mL. There was up to a 10% monomer loss in the repackaged bevacizumab. Laboratory samples of repackaged bevacizumab and placebo had initial particle counts, respectively, of 283,675 ± 60,494/mL and 492,314 ± 389,361/mL. Freeze-thawing of both bevacizumab and placebo samples led to >1.2 million particles/mL. In all repackaged samples, most of the particles were due to silicone oil. SE-HPLC showed no significant differences for repackaged samples incubated in the laboratory under various conditions, compared with bevacizumab directly from vial. However, repeated freeze-thawing caused a more than 10% monomer loss. CONCLUSIONS Bevacizumab repackaged in plastic syringes could contain protein aggregates and is contaminated by silicone oil microdroplets. Freeze-thawing or other mishandling can further increase levels of particle contaminants.

Effects of benzalkonium chloride-preserved, polyquad-preserved, and sofZia-preserved topical glaucoma medications on human ocular epithelial cells

Introduction|To investigate potentially adverse effects of different topical glaucoma medications and preservatives on cultured ocular epithelial cells.Methods|Confluent cultures of human corneal (10.014 pRSV-T) and conjunctival cells (1-5c-4) were assayed with 100 μL of different glaucoma medications for 25 minutes at 37°C and 5% CO2. We also tested the preservative sofZia® (Alcon Laboratories, Fort Worth, TX, USA), as well as a range of concentrations of the preservative benzalkonium chloride (BAK; 0.001% to 0.050%). Balanced salt solution was used as the “live” control and a solution containing 70% methanol and 0.2% saponin was used as a “dead” control. The LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Carlsbad, CA, USA) was used to determine the percentage of dead and live cells via ethidium homodimer and calcein fluorescence, respectively.Results|The toxicity of the prostaglandin analogs latanoprost, tafluprost and travoprost preserved with BAK was similar to the toxicity observed in their respective BAK concentrations. The prostaglandin analog travoprost (0.004%) preserved with the oxidizing preservative sofZia had much greater corneal and conjunctival cell survival than travoprost preserved with BAK. Travoprost (0.004%) containing polyquad also performed statistically better than its BAK-preserved formulation.Conclusion|Ocular surface side effects have previously been demonstrated with chronic, long-term exposure to intraocular pressurelowering medications containing the common preservative BAK. BAK alone has significant in-vitro cytotoxicity to cultured ocular epithelial cells. Substitution of BAK with polyquad or sofZia resulted in significantly higher percentages of live conjunctival and corneal cells. Further studies are needed to understand the- clinical implications of these findings.

Sustained Elevation in Intraocular Pressure Associated With Intravitreal Bevacizumab Injections

This retrospective case series reports sustained elevation of intraocular pressure (IOP) after single or repeated intravitreal injections of bevacizumab (Avastin; Genentech, San Francisco, CA) for wet age-related macular degeneration (AMD). All six cases experienced significant and sustained elevation in IOP after single or repeated intravitreal injections of bevacizumab. Initiation or advancement of IOP-lowering therapy was required in all cases. The results support the need for further studies investigating the incidence of this potential side effect and the need for close long-term surveillance of IOP after injection of bevacizumab, particularly in patients with glaucoma or suspected glaucoma. Future in vitro and in vivo studies are needed to better understand the reasons for this observed phenomenon.

Effects of benzalkonium chloride- and polyquad-preserved combination glaucoma medications on cultured human ocular surface cells

IntroductionThe aim of this study is to investigate potential adverse effects of fixed combination glaucoma medications preserved with either benzalkonium chloride (BAK) or Polyquad® (PQ; Alcon Research Ltd., Fort Worth, TX, USA) on cultured ocular epithelial cells.MethodsConfluent cultures of human cornea and conjunctival cell lines were exposed for 25 minutes to different glaucoma medications as well as a range of concentrations of BAK (0.001%–0.050%). Balanced salt solution was used as the “live” control and a solution containing 70% methanol and 0.2% saponin was used as a “dead” control. The number of dead and live cells were determined via ethidium homodimer (Eth-1) and calcein acetoxymethyl ester (AM) fluorescence, respectively.ResultsThe toxicity of the prostaglandin analog with beta-blocker timolol fixed-combination formulations preserved with BAK was different from that observed in the respective BAK concentrations. Travoprost plus timolol fixed combination with BAK performed better than its respective BAK concentration alone, while the latanoprost plus timolol fixed combination performed worse than its respective BAK concentration. Travoprost plus timolol fixed combination preserved with PQ had greater corneal and conjunctival cell survival than either the travoprost plus timolol fixed combination preserved with BAK or the latanoprost plus timolol fixed combination.ConclusionOcular surface side effects have previously been demonstrated with chronic, long-term exposure to intraocular-pressure-lowering medications containing the common preservative BAK. BAK alone has significant in-vitro cytotoxicity to cultured ocular epithelial cells. Substitution of BAK with PQ resulted in significantly higher percentages of live conjunctival and corneal cells. Further studies are needed to understand the clinical implications of these findings.

Multiphoton Microscopy for Ophthalmic Imaging

We review multiphoton microscopy (MPM) including two-photon autofluorescence (2PAF), second harmonic generation (SHG), third harmonic generation (THG), fluorescence lifetime (FLIM), and coherent anti-Stokes Raman Scattering (CARS) with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

Simultaneous spatial and temporal focusing for tissue ablation

Simultaneous spatial and temporal focusing (SSTF) of femtosecond pulses was originally conceived as a novel method for increasing the field-of-view in multiphoton imaging applications. Multiphoton imaging with SSTF deviated from traditional nonlinear systems in that it enabled the use of low numerical aperture beams to be used to increase the field-of-view, but retain the axial sectioning of a high numerical aperture beam. In this manner efficiency gains in the imaging process were achieved without compromising axial resolution [1,2].

In vitro effects of antivascular endothelial growth factors on cultured human trabecular meshwork cells.

PurposeTo investigate the effects of a neutralizing vascular endothelial growth factor antibody and antibody fragment as well as vehicle components on primary cultures of human trabecular meshwork (TM) cells. MethodsAssays of cellular metabolism were performed using the diphenyl tetrazolium bromide assay in confluent cultures of cells. Proliferative effects were determined by measuring 5′-bromo-2′-deoxyuridine uptake in subconfluent cultures of cells. ResultsTwenty-four-hour treatment with 4 mg/mL bevacizumab reduced TM metabolism to 34.4±12.4% (mean±SD) as compared with human immunoglobulin G controls (P<0.0001). 4 mg/mL bevacizumab also reduced TM cell proliferation to 62.7±9.2% of controls (P<0.0001). No significant decrease was seen at 2 mg/mL bevacizumab, or with molar equivalents of the related anti-vascular endothelial growth factor agent ranibizumab. Exposure of TM cells to the components of bevacizumab and ranibizumab vehicle did not lead to significant antimetabolic effects. ConclusionsOur data reveal that high concentrations of bevacizumab are harmful to TM cells in vitro whereas no such effect was noted with human immunoglobulin G controls or ranibizumab. Further studies are needed to better understand the antimetabolic effects of higher concentrations of bevacizumab on intraocular cell lines and whether smaller concentrations may have a similar effect on TM cells after repeated exposures.

Preclinical Investigation of Ab Interno Trabeculectomy Using a Novel Dual-Blade Device

PURPOSE To evaluate the effects of a novel ab interno trabeculectomy device on human trabecular meshwork (TM). DESIGN Laboratory evaluation. METHODS The TM from human cadaveric corneal rim tissue was incised using 3 instruments: (1) novel dual-blade device; (2) microvitreoretinal (MVR) blade; and (3) Trabectome. Tissue samples underwent histologic processing and comparative analyses. Subsequently, human eye perfusion studies were performed to evaluate intraocular pressure (IOP)-lowering effects of each device. Main outcome measures were degree of TM removal by histology and IOP in a perfusion model. RESULTS The MVR blade exhibited minimal removal of TM and obvious injury to the adjacent sclera. The Trabectome removed a large portion of the central TM, but leaflets of residual tissue remained and thermal injury was noted in all samples. The dual-blade device achieved a more complete removal of TM without injury to surrounding tissues. All devices resulted in statistically significant lowering of IOP during perfusion model studies. MVR blade treatment across 170.0 ± 14.1 degrees of TM resulted in a decrease of IOP from 18.5 ± 1.9 mm Hg to 12.8 ± 2.2 mm Hg (P < .01). Trabectome treatment across 117.5 ± 12.6 degrees resulted in a decrease of IOP from 18.8 ± 1.7 mm Hg to 11.3 ± 1.0 mm Hg (P < .01). Dual-blade device treatment across 157.5 ± 26.3 degrees resulted in a decrease of IOP from 18.3 ± 3.0 mm Hg to 11.0 ± 2.2 mm Hg (P < .01). CONCLUSIONS The novel dual-blade device demonstrated a more complete removal of TM without residual TM leaflets or damage to surrounding tissues and significantly reduced IOP in a human eye perfusion model.

Effects of glaucoma medications and preservatives on cultured human trabecular meshwork and non-pigmented ciliary epithelial cell lines.

Aims We investigated the potential cytotoxicity of various topical ophthalmic glaucoma formulations containing different preservatives in cultured human trabecular meshwork (TM) and non-pigmented ciliary epithelial (NPCE) cell lines. Methods We tested 0.004% travoprost preserved with either 0.015% benzalkonium chloride (BAK), sofZia or 0.001% Polyquad (PQ); and 0.005% latanoprost preserved with 0.020% BAK. We also tested a range of BAK concentrations in balanced salt solution (BSS). TM cells were treated for 10 min at 37°C with solutions diluted 1:10 to mimic the reduced penetration of topical preparations to the anterior chamber. Viability was determined by the uptake of the fluorescent vital dye calcein-AM (n=6). Results BAK solutions (diluted 1:10) demonstrated a dose-dependent reduction in cell viability in both cell types (TM and NPCE). With a 1:10 dilution of 0.020% BAK, there were significantly more living NPCE cells (89±6%) than TM cells (57±6%; p<0.001). In TM cells, travoprost + BAK had statistically fewer live cells (83±5%) than both travoprost + sofZia (97±5%) and travoprost + PQ (97±6%; p<0.05). Compared with BSS-treated NPCE cells, travoprost had statistically fewer live cells (p<0.05) when preserved with BAK (85±16%), sofZia (91±6%) or PQ (94±2%). Conclusions These results demonstrate that substitution of BAK from topical ophthalmic drugs results in greater viability of cultured TM cells, the cells involved in the conventional outflow pathway. Cultured NPCE, responsible for aqueous inflow, appear more resilient to BAK.

In Vitro Toxicity of Topical Ocular Prostaglandin Analogs and Preservatives on Corneal Epithelial Cells

PURPOSE To determine the effect of 4 formulations of commercially available prostaglandin analogs (PGAs) on human corneal epithelial cells in vitro. METHODS The test solutions (PGAs) examined were tafluprost 0.005% with 0.010% benzalkonium chloride (BAK), travoprost 0.004% with 0.015% BAK, travoprost 0.004% with sofZia, and latanoprost 0.005% with 0.020% BAK. Also tested independently were the 4 respective BAK or sofZia concentrations related to each PGA. Balanced salt solution (BSS) was used as the live control, and a fixative solution containing 70% methanol and 0.2% saponin was used as the dead control. Immortalized human corneal epithelial cells were exposed to test or control solution for 25 min at 37 degrees C and 5% CO(2). A live/dead assay was used to measure the toxicity of the PGAs. RESULTS The percentage of live cells in the PGA groups ranged from 2% to 72% of the BSS group (live control). The PGA with the highest relative live cell percentage, at 72% of the live control, was travoprost with sofZia. The next highest PGA, exhibiting 14% live cells, was the formulation of travoprost containing BAK. The other 2 PGAs, tafluprost and latanoprost, had few surviving cells, with 3% and 2% live cells, respectively. The BAK concentrations exhibited 4%, 3%, and 3% for the 0.01%, 0.015%, and 0.02% concentrations, respectively. The stand-alone sofZia cell survival was 68% of the live control. CONCLUSIONS All 4 PGA formulations tested demonstrated significantly more toxicity in human corneal epithelial cells than the live control, but there were significant differences among the PGAs. Travoprost with sofZia exhibited the least toxicity, followed by travoprost with BAK, and then tafluprost and latanoprost. The stand-alone preservative systems were also tested and showed similar survival percentages to each respective PGA. The true clinical implications of these findings require further investigation.