Influence of charge, hydrophobicity, and hydrodynamic radius on vitreous pharmacokinetics of large molecules

Abstract

Purpose : Development of long acting large molecule therapeutics for human posterior segment ocular disease is hampered by an inadequate understanding of the pharmacokinetic drivers following intravitreal injection. Previous work has shown that charge and hydrophobicity can influence systemic clearance rates, and that hydrodynamic radius is linearly correlated with vitreal half-life over the range of 3 – 7 nm. A better understanding of the molecular attributes contributing to vitreal elimination half-life could enable the design of improved therapeutics.

Methods : The contribution of electrostatic charge, hydrophobicity, and hydrodynamic radius to vitreal clearance was assessed for soluble large molecules including antibodies and antibody fragments (Fabs) following intravitreal injection in rabbits and nonhuman primates. A systematic study of molecule charge was accomplished by determining vitreal half-life in rabbit of variants of ranibizumab whereby charge was altered through introduction of amino acid changes in two of the hypervariable regions of the light chain. Charge was assessed by measuring isoelectric point (pI). Hydrophobicity was determined experimentally via analytical hydrophobic interaction chromatography, and hydrophobicity of the variable domain unit (Fv) was also calculated from the amino acid sequence. Hydrodynamic radius was determined using light scattering.

Results : No significant correlation was observed between vitreal half-life and pI or hydrophobicity. Equivalent rabbit vitreal pharmacokinetics were observed for ranibizumab and its charge variants with pIs in the range of 6.8-10.2. A strong correlation was observed between rabbit vitreal half-life and hydrodynamic radius between 2 – 45 nm (p<0.0001).

Conclusions : These results indicate that diffusive properties of soluble large molecules, as quantified by the hydrodynamic radius, make a key contribution to vitreal elimination whereas minor or negligible contributions arise from differences in charge or hydrophobicity.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.