Peptide Inhibitors of Vascular Endothelial Growth Factor A : Current Situation and Perspectives

Vascular endothelial growth factors (VEGFs) are the family of extracellular signaling proteins involved in the processes of angiogenesis. VEGFA overexpression and altered regulation of VEGFA signaling pathways lead to pathological angiogenesis, which contributes to the progression of various diseases, such as age-related macular degeneration and cancer. Monoclonal antibodies and decoy receptors have been extensively used in the anti-angiogenic therapies for the neutralization of VEGFA. However, multiple side effects, solubility and aggregation issues, and the involvement of compensatory VEGFA-independent pro-angiogenic mechanisms limit the use of the existing VEGFA inhibitors. Short chemically synthesized VEGFA binding peptides are a promising alternative to these full-length proteins. In this review, we summarize anti-VEGFA peptides identified so far and discuss the molecular basis of their inhibitory activity to highlight their pharmacological potential as anti-angiogenic drugs.Peer reviewe

Rabbit as an animal model for intravitreal pharmacokinetics : Clinical predictability and quality of the published data

Corrigendum: Experimental Eye Research 169 (2018) 60 doi: 10.1016/j.exer.2018.01.006 WOS: 000430158400008Intravitreal administration is the method of choice in drug delivery to the retina and/or choroid. Rabbit is the most commonly used animal species in intravitreal pharmacokinetics, but it has been criticized as being a poor model of human eye. The critique is based on some anatomical differences, properties of the vitreous humor, and observed differences in drug concentrations in the anterior chamber after intravitreal injections. We have systematically analyzed all published information on intravitreal pharmacokinetics in the rabbit and human eye. The analysis revealed major problems in the design of the pharmacokinetic studies. In this review we provide advice for study design. Overall, the pharmacokinetic parameters (clearance, volume of distribution, half-life) in the human and rabbit eye have good correlation and comparable absolute values. Therefore, reliable rabbit-to-man translation of intravitreal pharmacokinetics should be feasible. The relevant anatomical and physiological parameters in rabbit and man show only small differences. Furthermore, the claimed discrepancy between drug concentrations in the human and rabbit aqueous humor is not supported by the data analysis. Based on the available and properly conducted pharmacokinetic studies, the differences in the vitreous structure in rabbits and human patients do not lead to significant pharmacokinetic differences. This review is the first step towards inter-species translation of intravitreal pharmacokinetics. More information is still needed to dissect the roles of drug delivery systems, disease states, age and ocular manipulation on the intravitreal pharmacokinetics in rabbit and man. Anyway, the published data and the derived pharmacokinetic parameters indicate that the rabbit is a useful animal model in intravitreal pharmacokinetics. (C) 2015 The Authors. Published by Elsevier Ltd.Peer reviewe

Quantitative pharmacokinetic analyses of anterior and posterior elimination routes of intravitreal anti-VEGF macromolecules using published human and rabbit data

The purpose of this study was to evaluate the contribution of the anterior elimination route for four anti-vascular endothelial growth factor (anti-VEGF) macromolecules (aflibercept, bevacizumab, pegaptanib and ranibizumab) after intravitreal injection using published human and rabbit data and three previously described pharmacokinetic (PK) modeling methods. A PubMed search was used to identify published studies with concentration-time data. The data were utilized only if the intravitreally injected drugs were used as plain solutions and several criteria for a well-performed PK study were fulfilled. The three methods to analyze rabbit data were (1) the equation for vitreal elimination halflife based molecular size assuming anterior elimination, (2) Maurice equation and plot for the ratio of aqueous humor (AH) to vitreal concentration assuming anterior elimination, and (3) the equation for amount of macromolecule eliminated anteriorly based on the area under the curve in AH. The first and third methods were used for human data. In the second and third methods, AH flow rate is a key model parameter, and it was varied between 2 and 3 ill/min. The methods were applied to data from 9 rabbit studies (1 for aflibercept, 5 for bevacizumab, and 3 for ranibizumab) and 5 human studies (1 for aflibercept, 3 for bevacizumab, and 1 for ranibizumab). Experimental half-lives of anti-VEGF macromolecules in both vitreous and aqueous humor were close to those calculated with the equations for vitreal elimination half-life in humans and rabbits. Rabbit data analyzed with Maurice plot indicated that the contribution of anterior elimination was usually at least 75%. In most human and rabbit studies, the calculated percentage of anterior elimination was at least 51%. Variability between studies was extensive for bevacizumab and ranibizumab. The results suggest that the anterior elimination route dominates after intravitreal injection of anti-VEGF macromolecules. However, the clinical data are sparse and variability is extensive, the latter emphasizing the need of proper experimental design.Peer reviewe

Melanin targeting for intracellular drug delivery: Quantification of bound and free drug in retinal pigment epithelial cells

Melanin binding affects drug distribution and retention in pigmented ocular tissues, thereby affecting drug response, duration of activity and toxicity. Therefore, it is a promising possibility for drug targeting and controlled release in the pigmented cells and tissues. Intracellular unbound drug concentrations determine pharmacological and toxicological actions, but analyses of unbound vs. total drug concentrations in pigmented cells are lacking. We studied intracellular binding and cellular drug uptake in pigmented retinal pigment epithelial cells and in non-pigmented ARPE-19 cells with five model drugs (chloroquine, propranolol, timolol, diclofenac, methotrexate). The unbound drug fractions in pigmented cells were 0.00016–0.73 and in non-pigmented cells 0.017–1.0. Cellular uptake (i.e. distribution ratio Kp), ranged from 1.3 to 6300 in pigmented cells and from 1.0 to 25 in non-pigmented cells. Values for intracellular bioavailability, Fic, were similar in both cells types (although larger variation in pigmented cells). In vitro melanin binding parameters were used to predict intracellular unbound drug fraction and cell uptake. Comparison of predictions with experimental data indicates that other factors (e.g. ion-trapping, lipophilicity-related binding to other cell components) also play a role. Melanin binding is a major factor that leads to cellular uptake and unbound drug fractions of a range of 3–4 orders of magnitude indicating that large reservoirs of melanin bound drug can be generated in the cells. Understanding melanin binding has important implications on retinal drug targeting, efficacy and toxicity.Peer reviewe

Intravitreal hydrogels for sustained release of therapeutic proteins

This review highlights how hydrogel formulations can improve intravitreal protein delivery to the posterior segment of the eye in order to increase therapeutic outcome and patient compliance. Several therapeutic proteins have shown excellent clinical successes for the treatment of various intraocular diseases. However, drug delivery to the posterior segment of the eye faces significant challenges due to multiple physiological barriers preventing drugs from reaching the retina, among which intravitreal protein instability and rapid clearance from the site of injection. Hence, frequent injections are required to maintain therapeutic levels. Moreover, because the world population ages, the number of patients suffering from ocular diseases, such as age-related macular degeneration (AMD) and diabetic retinopathy (DR) is increasing and causing increased health care costs. Therefore, there is a growing need for suitable delivery systems able to tackle the current limitations in retinal protein delivery, which also may reduce costs. Hydrogels have shown to be promising delivery systems capable of sustaining release of therapeutic proteins and thus extending their local presence. Here, an extensive overview of preclinically developed intravitreal hydrogels is provided with attention to the rational design of clinically useful intravitreal systems. The currently used polymers, crosslinking mechanisms, in vitro/in vivo models and advancements are discussed together with the limitations and future perspective of these biomaterials.Peer reviewe

Pullulan Based Bioconjugates for Ocular Dexamethasone Delivery

Posterior segment eye diseases are mostly related to retinal pathologies that require pharmacological treatments by invasive intravitreal injections. Reduction of frequent intravitreal administrations may be accomplished with delivery systems that provide sustained drug release. Pullulan-dexamethasone conjugates were developed to achieve prolonged intravitreal drug release. Accordingly, dexamethasone was conjugated to similar to 67 kDa pullulan through hydrazone bond, which was previously found to be slowly cleavable in the vitreous. Dynamic light scattering and transmission electron microscopy showed that the pullulan-dexamethasone containing 1:20 drug/glucose unit molar ratio (10% w/w dexamethasone) self-assembled into nanoparticles of 461 +/- 30 nm and 402 +/- 66 nm, respectively. The particles were fairly stable over 6 weeks in physiological buffer at 4, 25 and 37 degrees C, while in homogenized vitreous at 37 degrees C, the colloidal assemblies underwent size increase over time. The drug was released slowly in the vitreous and rapidly at pH 5.0 mimicking lysosomal conditions: 50% of the drug was released in about 2 weeks in the vitreous, and in 2 days at pH 5.0. In vitro studies with retinal pigment epithelial cell line (ARPE-19) showed no toxicity of the conjugates in the cells. Flow cytometry and confocal microscopy showed cellular association of the nanoparticles and intracellular endosomal localization. Overall, pullulan conjugates showed interesting features that may enable their successful use in intravitreal drug delivery.Peer reviewe

Pharmacokinetic Simulations of Intravitreal Biologicals : Aspects of Drug Delivery to the Posterior and Anterior Segments

Biologicals are important ocular drugs that are be delivered using monthly and bimonthly intravitreal injections to treat retinal diseases, such as age-related macular degeneration. Long acting delivery systems are needed for prolongation of their dosing interval. Intravitreal biologicals are eliminated from the eye via the aqueous humor outflow. Thus, the anterior and posterior segments are exposed to the drug. We utilized a kinetic simulation model to estimate protein drug concentrations in the vitreous and aqueous humor after bolus injection and controlled release administration to the vitreous. The simulations predicted accurately the experimental levels of 5 biologicals in the vitreous and aqueous humor. The good match between the simulations and experimental data demonstrated almost complete anterior segment bioavailability, and major dose sparing with ocular controlled release systems. Overall, the model is a useful tool in the design of intraocular delivery of biologicals.Biologicals are important ocular drugs that are be delivered using monthly and bimonthly intravitreal injections to treat retinal diseases, such as age-related macular degeneration. Long acting delivery systems are needed for prolongation of their dosing interval. Intravitreal biologicals are eliminated from the eye via the aqueous humor outflow. Thus, the anterior and posterior segments are exposed to the drug. We utilized a kinetic simulation model to estimate protein drug concentrations in the vitreous and aqueous humor after bolus injection and controlled release administration to the vitreous. The simulations predicted accurately the experimental levels of 5 biologicals in the vitreous and aqueous humor. The good match between the simulations and experimental data demonstrated almost complete anterior segment bioavailability, and major dose sparing with ocular controlled release systems. Overall, the model is a useful tool in the design of intraocular delivery of biologicals.Peer reviewe

Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation

The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein’s stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89–93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His6 fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.Peer reviewe

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.Peer reviewe

Sulfobutyl Ether b-Cyclodextrin (SBE-b-CD) in Eyedrops Improves the Tolerability of a Topically Applied Pilocarpine Prodrug in Rabbits

This is the publisher's version, also available electronically from http://dx.doi.org/10.1089/jop.1995.11.95The effects of a novel, modified β-cyclodextrin (SBE4-β-CD; a variably substituted sulfobutyl ether with an average degree of substitution of four) on eye irritation and miotic response of an ophthalmically applied pilocarpine prodrug, O,O'-dipropionyl-(1,4-xylylene) bispilocarpate, in albino rabbits were studied. Compared to the commercial pilocarpine eyedrop solution (163 mM, equivalent to 3.4% pilocarpine), 12 - 24 mM pilocarpine prodrug solutions (equivalent to 0.5 - 1.0% pilocarpine, respectively) decreased peak miotic intensity (Imax) and increased the time to reach peak (tmax), but did not significantly affect values for the area under the miosis versus time curves (AUC), i.e. 12 - 24 mM pilocarpine prodrug appeared to be equivalent to 163 mM pilocarpine. Ocularly applied 12 - 24 mM pilocarpine prodrug solutions, however, were more irritating than a commercial pilocarpine eyedrop solution. Coadministered SBE4-β-CD significantly decreased the eye irritation of the pilocarpine prodrug solutions. Coadministered SBE4-β-CD did not affect the miotic response of prodrug solution when the molar ratio of SBE4-β-CD to prodrug was low. However, increasing the molar ratio of SBE4-β-CD to prodrug decreased the Imax and AUC values. The results show that eye irritation of the pilocarpine prodrug is prevented by levels of SBE4-β-CD that do not affect the apparent ocular absorption of the prodrug