In Vivo Ocular Pharmacokinetics and Toxicity of Siponimod in Albino Rabbits.

Siponimod is a promising agent for the inhibition of ocular neovascularization in diabetic retinopathy and age-related macular degeneration. Siponimod's development for ophthalmological application is hindered by the limited information available on the drug's solubility, stability, ocular pharmacokinetics (PK), and toxicity in vivo. In this study, we investigated the aqueous stability of siponimod under stress conditions (up to 60 °C) and its degradation behavior in solution. Additionally, siponimod's ocular PK and toxicity were investigated using intravitreal injection of two different doses (either 1300 or 6500 ng) in an albino rabbit model. Siponimod concentration was quantified in the extracted vitreous, and the PK parameters were calculated. The drug half-life after administration of the low and high doses was 2.8 and 3.9 h, respectively. The data obtained in vivo was used to test the ability of published in silico models to predict siponimod's PK accurately. Two models that correlated siponimod's molecular descriptors with its elimination from the vitreous closely predicted the half-life. Furthermore, 24 h and 7 days after intravitreal injections, the retinas showed no signs of toxicity. This study provides important information necessary for the formulation and development of siponimod for ophthalmologic applications. The short half-life of siponimod necessitates the development of a sustained drug delivery system to maintain therapeutic concentrations over an extended period, while the lack of short-term ocular toxicity observed in the retinas of siponimod-treated rabbits supports possible clinical use.

Observation of Silicone Oil Within the Vitreous and Sclera Following Intravitreal Administration of Biotherapeutics Using Insulin Syringes in Cynomolgus Monkeys.

Silicone oil droplets have been reported in the eyes of human patients following intravitreous (IVT) injections with several marketed biotherapeutic products. Intravitreous administration of a novel biotherapeutic in a 14-week cynomolgus monkey study using insulin syringes was associated with 2, non-test-article-related phenomena: "vitreous floater/clear sphere" on indirect ophthalmoscopy and intrascleral "foreign material near injection track" on histopathology. Retrospective analysis of 81 other preclinical studies of IVT administration of novel biotherapeutics found a greater frequency of clear spheres in monkey IVT studies using insulin syringes and formulations containing polysorbate. We were able to correlate microscopic findings of clear circular to oval areas in the sclera near the injection track with an energy-dispersive X-ray spectroscopy (EDS) signal for silicon at the same location in the sclera. These observations provide further evidence that silicone lubricant in insulin syringes/needles is the source of clear spheres noted in the vitreous and foreign material noted near the injection track in the sclera. Although considered inert and toxicologically insignificant, silicone deposition within the eye should form part of the risk-benefit equation in a clinical setting.

Systemic exposure following intravitreal administration of therapeutic agents: an integrated pharmacokinetic approach. 1. THR-149.

Intravitreal (IVT) injection of pharmacological agents is an established and widely used procedure for the treatment of many posterior segment of the eye diseases. IVT injections permit drugs to reach high concentrations in the retina whilst limiting systemic exposure. Beyond the risk of secondary complications such as intraocular infection, the potential of systemic adverse events cannot be neglected. Therefore, a detailed understanding of the rules governing systemic exposure following IVT drug administration remains a prerequisite for the evaluation and development of new pharmacological agents intended for eye delivery. We present here a novel mathematical model to describe and predict circulating drug levels following IVT in the rabbit eye, a species which is widely used for drug delivery, pharmacokinetic, and pharmacodynamic studies. The mathematical expression was derived from a pharmacokinetic model that assumes the existence of a compartment between the vitreous humor compartment itself and the systemic compartment. We show that the model accurately describes circulating levels of THR-149, a plasma kallikrein inhibitor in development for the treatment of diabetic macular edema. We hypothesize that the model based on the rabbit eye has broader relevance to the human eye and can be used to analyze systemic exposure of a variety of drugs delivered in the eye.

Systemic exposure following intravitreal administration of therapeutic agents: an integrated pharmacokinetic approach. 2. THR-687.

Intravitreal (IVT) injection remains the preferred administration route of pharmacological agents intended for the treatment of back of the eye diseases such as diabetic macular edema (DME) and neovascular age-related macular degeneration (nvAMD). The procedure enables drugs to be delivered locally at high concentrations whilst limiting whole body exposure and associated risk of systemic adverse events. Nevertheless, intravitreally-delivered drugs do enter the general circulation and achieving an accurate understanding of systemic exposure is pivotal for the evaluation and development of drugs administered in the eye. We report here the full pharmacokinetic properties of THR-687, a pan RGD integrin antagonist currently in clinical development for the treatment of DME, in both rabbit and minipig. Pharmacokinetic characterization included description of vitreal elimination, of systemic pharmacokinetics, and of systemic exposure following IVT administration. For the latter, we present a novel pharmacokinetic model that assumes clear partition between the vitreous humor compartment itself where the drug is administered and the central systemic compartment. We also propose an analytical solution to the system of differential equations that represent the pharmacokinetic model, thereby allowing data analysis with standard nonlinear regression analysis. The model accurately describes circulating levels of THR-687 following IVT administration in relevant animal models, and we suggest that this approach is relevant to a range of drugs and analysis of subsequent systemic exposure.

[Protocol of intravitreal drug delivery. Consensus of the Expert Counsil of Retina and Optic Nerve Diseases of the All-Russian Public Organasation «Association of Ophthalmologists¼]. / Protokol vypolneniya intravitreal'nogo vvedeniya lekarstvennykh preparatov. Konsensus Ekspertnogo soveta po zabolevaniyam setchatki i zritel'nogo nerva Obshcherossiiskoi obshchestvennoi organizatsii «Assotsiatsiya vrachei-oftal'mologov¼.

Intravitreal drug administration is a procedure that has become widespread in modern ophthalmology. However, there is no global consensus on certain aspects of this manipulation, and practitioners feel the need for guidelines. In the Russian Federation, until now, such a document was not available. The expert council on diseases of the retina and optic nerve of All-Russian public organization «Association of Ophthalmologists¼, with participation of invited specialists, has studied and analyzed the existing foreign guidelines for performing intravitreal injections, as well as the regulatory framework in Russia. As a result, this Protocol was developed and approved for use in the healthcare system of the Russian Federation. The document regulates the requirements for specialists and organizations, the conditions for performing the procedure and the necessary material resources and presents an algorithm for performing intravitreal drug administration, a patient examination check-list for various conditions of the procedure, as well as parameters for evaluating and monitoring the quality of the procedure.

Intravitreal administration of known phototoxicants in the rabbit fails to produce phototoxicity: implications for phototoxicity testing of intravitreally administered small molecule therapeutics.

CONTEXT: Intravitreal (ITV) dosing has become a clinically important route of administration for the treatment of uveitis, endophthalmitis, retinal vein occlusion, diabetic macular edema and age-related macular degeneration. Despite this, there are no validated non-clinical models of phototoxicity for ITV products. OBJECTIVE: The objective of this study was to develop an ITV rabbit model of phototoxicity for use in assessing the photosafety of small molecules therapeutics. MATERIALS AND METHODS: Dutch Belted rabbits were intravitreally injected bilaterally with four known phototoxicants: 8-methoxypsoralen, lomefloxacin, doxycycline and stannsoporfrin. Triescence(®), a non-phototoxic triamcinolone acetonide steroid formulation designed for ITV administration, was used as a negative control. One eye was then irradiated with solar-simulated ultraviolet radiation for 30 min, 1 h after dosing, while the other eye was occluded, serving as a non-irradiated control. RESULTS: Despite the direct administration of known phototoxicants into the vitreous, no evidence of ocular phototoxicity was observed in any dose group. Direct (non-phototoxic) retinal toxicity was observed in the doxycycline dose group only. CONCLUSION: These data suggest that the posterior segment of the rabbit eye is protected against phototoxicity by anatomical and/or physiological mechanisms, and is not a useful model for the assessment of phototoxicity of intravitreally administered molecules.

Comprehensive Ocular and Systemic Safety Evaluation of Polysialic Acid-Decorated Immune Modulating Therapeutic Nanoparticles (PolySia-NPs) to Support Entry into First-in-Human Clinical Trials.

An inflammation-resolving polysialic acid-decorated PLGA nanoparticle (PolySia-NP) has been developed to treat geographic atrophy/age-related macular degeneration and other conditions caused by macrophage and complement over-activation. While PolySia-NPs have demonstrated pre-clinical efficacy, this study evaluated its systemic and intraocular safety. PolySia-NPs were evaluated in vitro for mutagenic activity using Salmonella strains and E. coli, with and without metabolic activation; cytotoxicity was evaluated based on its interference with normal mitosis. PolySia-NPs were administered intravenously in CD-1 mice and Sprague Dawley rats and assessed for survival and toxicity. Intravitreal (IVT) administration in Dutch Belted rabbits and non-human primates was assessed for ocular or systemic toxicity. In vitro results indicate that PolySia-NPs did not induce mutagenicity or cytotoxicity. Intravenous administration did not show clastogenic activity, effects on survival, or toxicity. A single intravitreal (IVT) injection and two elevated repeat IVT doses of PolySia-NPs separated by 7 days in rabbits showed no signs of systemic or ocular toxicity. A single IVT inoculation of PolySia-NPs in non-human primates demonstrated no adverse clinical or ophthalmological effects. The demonstration of systemic and ocular safety of PolySia-NPs supports its advancement into human clinical trials as a promising therapeutic approach for systemic and retinal degenerative diseases caused by chronic immune activation.

Efficacy and Safety Evaluation of Mometasone Furoate in Treating Ocular Inflammation.

Mometasone furoate (MF) is a medium-potency synthetic glucocorticosteroid with anti-inflammatory, antipruritic, and vasoconstrictive properties. However, its role in the treatment of ocular inflammation has not yet been explored. This work investigated the anti-inflammatory activity of MF in ocular tissues. First, the in vivo safety of the intravitreal (IVT) injection of MF (80, 160, and 240 µg) was evaluated via clinical examination (including the assessment of intraocular pressure), electroretinography (ERG), and histopathology. Second, MF was tested in an experimental model of bacillus Calmette-Guérin (BCG)-induced uveitis in Wistar rats. Intraocular inflammation was then evaluated via a slit-lamp and fundus examination, ERG, histopathology, and the quantification of pro-inflammatory markers. Intravitreal MF showed no toxicity in all the investigated doses, with 160 µg leading to attenuated disease progression and improvement in clinical, morphological, and functional parameters. There was a significant reduction in the levels of inflammatory markers (myeloperoxidase, interleukins 6 and 1ß, CXCL-1, and tumor necrosis factor-alpha) when compared to the levels in untreated animals. Therefore, MF should be further investigated as a promising drug for the treatment of ocular inflammation.

Ocular Posterior Segment Distribution and Pharmacokinetics of Brimonidine After Intravitreal Administration in Guinea Pigs.

Purpose: Brimonidine is a highly alpha-2 adrenergic agonist, which provides a potential myopia control effect. This study aimed to examine the pharmacokinetics and concentration of brimonidine in the posterior segment tissue of eyes in guinea pigs. Methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was successfully used for brimonidine pharmacokinetics and tissue distribution research in guinea pigs following intravitreal administration (20 µg/eye). Results: Brimonidine concentrations in the retina and sclera were maintained at a high level (>60 ng/g) at 96 h postdosing. Brimonidine concentration peaked in the retina (377.86 ng/g) at 2.41 h and sclera (306.18 ng/g) at 6.98 h. The area under curve (AUC0-∞) was 27,179.99 ng h/g in the retina and 39,529.03 ng h/g in the sclera. The elimination half-life (T1/2e) was 62.43 h in the retina and 67.94 h in the sclera. Conclusions: The results indicated that brimonidine was rapidly absorbed and diffused to the retina and sclera. Meanwhile, it maintained higher posterior tissue concentrations, which can effectively activate the alpha-2 adrenergic receptor. This may provide pharmacokinetic evidence for the inhibition of myopia progression by brimonidine in animal experiments.

Experimental and mathematical approaches for drug delivery for the treatment of wet age-related macular degeneration.

Several chronic eye diseases affect the posterior segment of the eye. Among them age-related macular degeneration can cause vision loss if left untreated and is one of the leading causes of visual impairment in the world. Most treatments are based on intravitreally injected therapeutics that inhibit the action of vascular endothelial growth factor. However, due to the need for monthly injections, this method is associated with poor patient compliance. To address this problem, numerous drug delivery systems (DDSs) have been developed. This review covers a selection of particulate systems, non-stimuli responsive hydrogels, implants, and composite systems that have been developed in the last few decades. Depending on the type of DDS, polymer material, and preparation method, different mechanical properties and drug release profiles can be achieved. Furthermore, DDS development can be optimized by implementing mathematical modeling of both drug release and pharmacokinetic aspects. Several existing mathematical models for diffusion-controlled, swelling-controlled, and erosion-controlled drug delivery from polymeric systems are summarized. Compartmental and physiologically based models for ocular drug transport and pharmacokinetics that have studied drug concentration profiles after intravitreal delivery or release from a DDS are also reviewed. The coupling of drug release models with ocular pharmacokinetic models can lead to obtaining much more efficient DDSs for the treatment of age-related macular degeneration and other diseases of the posterior segment of the eye.

Design, optimization, and in vitro characterization of idebenone-loaded PLGA microspheres for LHON treatment.

Biodegradable poly(lactic-co-glycolic acid) microspheres (PLGA MSs) are attractive delivery systems for site-specific maintained release of therapeutic active substances into the intravitreal chamber. The design, development, and characterization of idebenone-loaded PLGA microspheres by means of an oil-in-water emulsion/solvent evaporation method enabled the obtention of appropriate production yield, encapsulation efficiency and loading values. MSs revealed spherical shape, with a size range of 10-25 µm and a smooth and non-porous surface. Fourier-transform infrared spectroscopy (FTIR) spectra demonstrated no chemical interactions between idebenone and polymers. Solid-state nuclear magnetic resonance (NMR), X-ray diffractometry, differential scanning calorimetry (DSC) and thermogravimetry (TGA) analyses indicated that microencapsulation led to drug amorphization. In vitro release profiles were fitted to a biexponential kinetic profile. Idebenone-loaded PLGA MSs showed no cytotoxic effects in an organotypic tissue model. Results suggest that PLGA MSs could be an alternative intraocular system for long-term idebenone administration, showing potential therapeutic advantages as a new therapeutic approach to the Leber's Hereditary Optic Neuropathy (LHON) treatment by intravitreal administration.

Formulation and Evaluation of Polymer-Based Nanoparticles for Intravitreal Gene-Delivery Applications.

The advent of the first-ever retinal gene therapy product, involving subretinal administration of a virus-based gene delivery platform, has garnered hope that this state-of-the-art therapeutic modality may benefit a broad spectrum of patients with diverse retinal disorders. On the other hand, clinical studies have revealed limitations of the applied delivery strategy that may restrict its universal use. To this end, intravitreal administration of synthetic gene-delivery platforms, such as polymer-based nanoparticles (PNPs), has emerged as an attractive alternative to the current mainstay. To achieve success, however, it is imperative that synthetic platforms overcome key biological barriers in human eyes encountered following intravitreal administration, including the vitreous gel and inner limiting membrane (ILM). Here, we introduce a series of experiments, from the fabrication of PNPs to a comprehensive evaluation in relevant experimental models, to determine whether PNPs overcome these barriers and efficiently deliver therapeutic gene payloads to retinal cells. We conclude the article by discussing a few important considerations for successful implementation of the strategy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation and characterization of PNPs Basic Protocol 2: Evaluation of in vitro transfection efficacy Basic Protocol 3: Evaluation of PNP diffusion in vitreous gel Basic Protocol 4: Ex vivo assessment of PNP penetration within vitreoretinal explant culture Basic Protocol 5: Assessment of in vivo transgene expression mediated by intravitreally administered PNPs.

Neuroprotective Effect of siRNA Entrapped in Hyaluronic Acid-Coated Lipoplexes by Intravitreal Administration.

Since the possibility of silencing specific genes linked to retinal degeneration has become a reality with the use of small interfering RNAs (siRNAs), this technology has been widely studied to promote the treatment of several ocular diseases. Despite recent advances, the clinical success of gene silencing in the retina is significantly reduced by inherent anatomical and physiological ocular barriers, and new strategies are required to achieve intraocular therapeutic effectiveness. In this study, we developed lipoplexes, prepared with sodium alginate as an adjuvant and strategically coated with hyaluronic acid (HA-LIP), and investigated the potential neuroprotective effect of these systems in a retinal light damage model. Successful functionalization of the lipoplexes with hyaluronic acid was indicated in the dynamic light scattering and transmission electron microscopy results. Moreover, these HA-LIP nanoparticles were able to protect and deliver siRNA molecules targeting caspase-3 into the retina. After retinal degeneration induced by high light exposure, in vitro and in vivo quantitative reverse transcription-PCR (RT-qPCR) assays demonstrated significant inhibition of caspase-3 expression by HA-LIP. Furthermore, these systems were shown to be safe, as no evidence of retinal toxicity was observed by electroretinography, clinical evaluation or histology.

Design, development, and characterization of an idebenone-loaded poly-ε-caprolactone intravitreal implant as a new therapeutic approach for LHON treatment.

Leber's Hereditary Optic Neuropathy (LHON) is a hereditary mitochondrial neurodegenerative disease of unclear etiology and lack of available therapeutic alternatives. The main goal of the current pilot study was based on the evaluation of the feasibility and characteristics of prolonged and controlled idebenone release from a PCL intravitreal implant. The design, development, and characterization of idebenone-loaded PCL implants prepared by an homogenization/extrusion/solvent evaporation method allowed the obtention of high PY, EE and LC values. In vitro characterization was completed by the assessment of mechanical and instrumental properties. The in vitro release of idebenone from the PCL implants was assessed and the implant erosion was monitored by the mass loss and surface morphology changes. DSC was used to estimate stability and interaction among implant's components. The present work demonstrated the controlled and prolonged idebenone delivery from the PCL implants in an in vitro model. A consistent preclinical base was established, supporting the idea of idebenone-loaded PCL implants as a new strategy of long-term sustained intraocular delivery for the LHON treatment.

Host cell proteins induce inflammation and immunogenicity as adjuvants in an integrated analysis of in vivo and in vitro assay systems.

INTRODUCTION: Host cell proteins (HCPs) are contaminated proteins remaining after purification of biopharmaceuticals. Recent reports revealed clinical implications of HCPs in anti-drug antibody (ADA) development in patients without any inflammatory effects. Therefore, we evaluated the inflammatory effects and immunogenicity of HCPs in an in vivo study by intravitreal administration to rabbits and an in vitro THP-1 cells assay. METHODS: Escherichia coli-derived HCPs at 200 ng/eye with or without ranibizumab at 0.25 mg/eye were administrated intravitreally to rabbits. For in vitro examination, differentiated THP-1 cells were stimulated with HCPs at 0.17 to 10.88 µg/mL with or without ranibizumab at 0.2 mg/mL. RESULTS: Co-administration of HCPs with ranibizumab, but not HCPs alone, induced ocular inflammation. Presence of ADA (anti-ranibizumab) was detected in the vitreous fluid of rabbits in which HCPs and ranibizumab were co-administered. HCPs increased cytokine release and upregulated cell surface markers involved in the antigen presentation in the THP-1 cell assay, which was enhanced by co-stimulation with ranibizumab. DISCUSSION: These finding suggests that HCPs may induce inflammation and immunogenicity as an adjuvant. Furthermore, integrated analyses by an in vivo rabbit model and in vitro assay system using THP-1 cells would be useful to evaluate the immunological risk of HCPs.

A Meta-Analysis of Cardiovascular Events Associated with Intravitreal Anti-VEGF Treatment in Patients with Retinal Vein Occlusion.

Purpose: Retinal vein occlusion is associated with an increased risk of cardiovascular diseases. Anti-vascular endothelial growth factor has been widely used as a treatment option. However, the systemic safety of intravitreal anti-vascular endothelial growth factor for retinal vein occlusion patients is still unclear.Materials and Methods: A meta-analysis was conducted to investigate all randomized controlled trials published up to February 2019 of retinal vein occlusion patients who received intravitreal anti-vascular endothelial growth factor vs. control treatments. Fixed effect models were used and results were reported as odds ratios and 95% confidence intervals.Results: Eight trials that evaluated 2320 patients were retrieved. Anti-vascular endothelial growth factor did not significantly increase the risks of cardiovascular events (odds ratio,1.54; 95% confidence interval, 0.66-3.57), hypertension (odds ratio, 0.92; 95% confidence interval, 0.63-1.33), or heart rate disorders (odds ratio,1.53; 95% confidence interval, 0.37-6.28) when compared with control treatment. Subgroup analyses did not show a significant increase of cardiovascular events in aflibercept (odds ratio,1.96; 95% confidence interval, 0.44-8.81) vs. ranibizumab trials (odds ratio, 1.47; 95% confidence interval, 0.54-4.02); 0.5 mg ranibizumab trials (odds ratio, 1.73; 95% confidence interval, 0.61-4.96) vs. 0.3 mg ranibizumab trials (odds ratio, 0.70; 95% confidence interval, 0.14-3.59); nor branch retinal vein occlusion (odds ratio, 1.32; 95% confidence interval, 0.40-4.33) vs. central retinal vein occlusion trials (odds ratio, 1.93; 95% confidence interval, 0.59-6.29).Conclusions: Intravitreal administration of anti-vascular endothelial growth factor did not significantly increase the risks of cardiovascular events, hypertension or heart rate disorders in retinal vein occlusion patients.

Drug Delivery to the Posterior Segment of the Eye: Biopharmaceutic and Pharmacokinetic Considerations.

The treatment of the posterior-segment ocular diseases, such as age-related eye diseases (AMD) or diabetic retinopathy (DR), present a challenge for ophthalmologists due to the complex anatomy and physiology of the eye. This specialized organ is composed of various static and dynamic barriers that restrict drug delivery into the target site of action. Despite numerous efforts, effective intraocular drug delivery remains unresolved and, therefore, it is highly desirable to improve the current treatments of diseases affecting the posterior cavity. This review article gives an overview of pharmacokinetic and biopharmaceutics aspects for the most commonly-used ocular administration routes (intravitreal, topical, systemic, and periocular), including information of the absorption, distribution, and elimination, as well as the benefits and limitations of each one. This article also encompasses different conventional and novel drug delivery systems designed and developed to improve drug pharmacokinetics intended for the posterior ocular segment treatment.

Trans-Ocular Electric Current In Vivo Enhances AAV-Mediated Retinal Transduction in Large Animal Eye After Intravitreal Vector Administration.

Purpose: Electric micro-current has been shown to enhance penetration and transduction of adeno-associated viral (AAV) vectors in mouse retina after intravitreal administration. We termed this: "electric-current vector mobility (ECVM)." The present study considered whether ECVM could augment retinal transduction efficiency of intravitreal AAV8-CMV-EGFP in normal rabbit and nonhuman primate (NHP) macaque. Potential mechanisms underlying enhanced retinal transduction by ECVM were also studied. Methods: We applied an electric micro-current across the intact eye of normal rabbit and monkey in vivo for a brief period immediately after intravitreal injection of AAV8-CMV-EGFP. Retinal GFP expression was evaluated by fundus imaging in vivo. Retinal immunohistochemistry was performed to assess the distribution of retinal cells transduced by the AAV8-EGFP. Basic fibroblast growth factor (bFGF) was analyzed by quantitative RT-polymerase chain reaction (PCR). Müller glial reactivity and inner limiting membrane (ILM) were examined by the glial fibrillary acidic protein (GFAP) and vimentin staining in mouse retina, respectively. Results: ECVM significantly increased the efficiency of AAV reaching and transducing the rabbit retina following intravitreal injection, with gene expression in inner nuclear layer, ganglion cells, and Müller cells. Similar trend of improvement was observed in the ECVM-treated monkey eye. The electric micro-current upregulated bFGF expression in Müller cells and vimentin showed ILM structural changes in mouse retina. Conclusions: ECVM promotes the transduction efficiency of AAV8-CMV-GFP in normal rabbit and monkey retinas following intravitreal injection. Translational Relevance: This work has potential translational relevance to human ocular gene therapy by increasing retinal expression of therapeutic vectors given by intravitreal administration.

Polysaccharides in Ocular Drug Delivery.

Polysaccharides, such as cellulose, hyaluronic acid, alginic acid, and chitosan, as well as polysaccharide derivatives, have been successfully used to augment drug delivery in the treatment of ocular pathologies. The properties of polysaccharides can be extensively modified to optimize ocular drug formulations and to obtain biocompatible and biodegradable drugs with improved bioavailability and tailored pharmacological effects. This review discusses the available polysaccharide choices for overcoming the difficulties associated with ocular drug delivery, and it explores the reasons for the dependence between the physicochemical properties of polysaccharide-based drug carriers and their efficiency in different formulations and applications. Polysaccharides will continue to be of great interest to researchers endeavoring to develop ophthalmic drugs with improved effectiveness and safety.

A Proximity Extension Assay (PEA)-based method for quantification of bevacizumab.

INTRODUCTION: Proximity Extension Assay (PEA) is a direct one-step protein quantification method using a pair of DNA oligonucleotides linked to antibodies against the target molecule. It requires polyclonal or two monoclonal antibodies (mAbs) that bind to target epitopes close enough to form a DNA duplex which is quantified by real-time PCR. Bevacizumab, an anti-cancer drug, is a mAb against vascular endothelial growth factor with common cardiovascular adverse effects. It is widely used off-label to treat neovascular eye disorders by intravitreal application of small doses. Even then, certain amount reaches systemic circulation which is considered relevant regarding safety. We aimed to set-up a PEA-based assay for bevacizumab in human plasma and to preliminary evaluate it in patients treated intravitreally. METHODS: We tested (PEA, quantitative PCR) several combinations of commercial mAbs and a Fab fragment against bevacizumab. The best combination was used to quantify bevacizumab in three patients donating plasma before and 24 h after the first intravitreal injection. RESULTS: A combination of a mAb and a Fab fragment (HCA184 and HCA182, Bio-Rad Laboratories, Inc.) performed best: standard curve R2 0.98, linear dynamic range 1-1000 pM, lower limit of quantification 1 pM (149 pg/mL) and a satisfactory precision (coefficient of variation 12%). All pre-dose patient concentrations were zero, while post-dose concentrations were 10.94, 13.73 and 55.49 ng/mL, in line with previous reports. DISCUSSION: This is the first set-up of a PEA-based assay for quantification of bevacizumab in human plasma. Its good performance and high sensitivity support further evaluation for potential uses particularly when the expected concentrations are low.