Single ocular injection of a sustained-release anti-VEGF delivers 6months pharmacokinetics and efficacy in a primate laser CNV model.

A potent anti-vascular endothelial growth factor (VEGF) biologic and a compatible delivery system were co-evaluated for protection against wet age-related macular degeneration (AMD) over a 6month period following a single intravitreal (IVT) injection. The anti-VEGF molecule is dimeric, containing two different anti-VEGF domain antibodies (dAb) attached to a human IgG1 Fc region: a dual dAb. The delivery system is based on microparticles of PolyActive™ hydrogel co-polymer. The molecule was evaluated both in vitro for potency against VEGF and in ocular VEGF-driven efficacy models in vivo. The dual dAb is highly potent, showing a lower IC50 than aflibercept in VEGF receptor binding assays (RBAs) and retaining activity upon release from microparticles over 12months in vitro. Microparticles released functional dual dAb in rabbit and primate eyes over 6months at sufficient levels to protect Cynomolgus against laser-induced grade IV choroidal neovascularisation (CNV). This demonstrates proof of concept for delivery of an anti-VEGF molecule within a sustained-release system, showing protection in a pre-clinical primate model of wet AMD over 6months. Polymer breakdown and movement of microparticles in the eye may limit development of particle-based approaches for sustained release after IVT injection.

Carboxylesterases: Dual roles in lipid and pesticide metabolism.

Carboxylesterases (CES, EC 3.1.1.1) are members of a superfamily of serine hydrolases that hydrolyze ester, amide, and carbamate bonds. Several different CES genes exist in mammalian species with evidence of multiple gene duplication events occurring throughout evolutionary history. There are five CES genes reported in the Human Genome Organization database, although CES1 and CES2 are the two best characterized human genes. An emerging picture of the CES family suggests that these enzymes have dual roles in the metabolism of xenobiotic and endobiotic compounds. Pesticides, such as the pyrethroids, are important xenobiotic substrates that are metabolized by CES, whereas cholesteryl esters, triacylglycerols, and 2-arachidonoylglycerol are examples of endobiotics known to be substrates for CES. Functional studies using selective chemical inhibitors, siRNA, and gene knockout models are providing valuable insights into the physiological functions of CES, and suggest that CES may be a novel target for the treatment of diseases such as diabetes and atherosclerosis. This review will examine the known physiological functions of CES, the interactions between xenobiotics (primarily pesticides) and lipids that occur with CES enzymes, and where possible the implications that these findings may have in terms of health and disease.

Evaluation of the 'side door' in carboxylesterase-mediated catalysis and inhibition.

Abstract Structures of mammalian carboxylesterases (CEs) reveal the presence of a 'side door' that is proposed to act as an alternative pore for the trafficking of substrates and products. p-Nitrobenzyl esterase (pnb CE) from Bacillus subtilis exhibits close structural homology and a similar side-door domain as mammalian CEs. We investigated the role of a specific 'gate' residue at the side door (i.e., Leu 362) during pnb CE-catalyzed hydrolysis of model esters, pesticides, and lipids. Recombinant pnb CE proteins containing mutations at position 362 demonstrated markedly lower kcat and kcat/Km values. The mutation with the most significant impact on catalysis was the L362R mutant (kcat/Km was 22-fold lower). Moreover, the ability of the L362R mutant to be inhibited by organophosphates (OP) was also lower. Investigation into the altered catalytic proficiency using pH-activity studies indicated that the catalytic triad of the mutant enzyme was preserved. Furthermore, viscosity variation and carbamate inhibition experiments indicated that rates of substrate association and acylation/deacylation were lower. Finally, recombinant CEs were found to possess lipolytic activity toward cholesteryl oleate and 2-arachidonylglycerol. In summary, the L362R mutant CE markedly slowed the rate of ester hydrolysis and was less sensitive to OP inhibition. The apparent causes of the diminished catalysis are discussed.

Pyrethroids: cytotoxicity and induction of CYP isoforms in human hepatocytes.

Deltamethrin [(S)-alpha-cyano-3-phenoxybenzyl-cis-(1 R,3R)-3(2,2-dibromovinyl)(2,2-dimethyl-cyclopropane-carboxylate] and permethrin [3-phenoxybenzyl(1RS)-cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylate] are pyrethroid insecticides used in agriculture, public health and military deployments. Pyrethroids are known to be capable of inducing cytochrome P450 (CYP) 2B1/2B2, CYP1A1 and overall CYP content in rat liver. The objectives of this study were to evaluate the potential of deltamethrin and permethrin to cause cytotoxicity and to induce CYP isoforms in human hepatocytes. Permethrin and deltamethrin showed dose-dependent effects on adenylate kinase activity in HepG2 cells, in which 50 and 100 microM doses, respectively, induced a 3-5 fold increase in activity, and also induced adenylate kinase activity in primary human hepatocytes. An approximately 3-fold induction was noted at 200 microM deltamethrin and a 4-fold induction at 100 microM permethrin. Cytotoxicity was noted in HepG2 cells following 48-72 h exposure to 100 or 200 microM deltamethrin and permethrin, respectively. Dose-dependent induction of caspase-3/7 was initiated by 12.5 microM deltamethrin or by 3.125 microM permethrin. Actinomycin D, a positive control for induction of caspase 3/7, induced caspase-3/7, an effect completely abrogated by the specific inhibitor Z-DEVD-FMK. At 100 microM deltamethrin 2-3 fold induction of CYP1A1 and CYP2B6 mRNA was observed, while at the same time an approximately 25-fold induction of CYP3A4 was noted. Permethrin-mediated CYP induction was much less potent, 4-fold or less for CYP1A1, CYP3A4, CYP3A5, CYP2B6 and CYP2A6. It has also been shown that these pyrethroids are ligands for the pregnane X receptor (PXR).