Brain delivery of proteins via their fatty acid and block copolymer modifications

Abstract

It is well known that hydrophobic small molecules penetrate cell membranes better than hydrophilic molecules. Amphiphilic molecules that dissolve both in lipid and aqueous phases are best suited for membrane transport. Transport of biomacromolecules across physiological barriers, e.g. the blood-brain barrier, is greatly complicated by the unique structure and function of such barriers. Two decades ago we adopted a simple philosophy that to increase protein delivery to the brain one needs to modify this protein with hydrophobic moieties. With this general idea we began modifying proteins (antibodies, enzymes, hormones, etc.) with either hydrophobic fatty acid residues or amphiphilic block copolymer moieties, such as poy(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (pluronics or poloxamers) and more recently, poly(2-oxasolines). This simple approach has resulted in impressive successes in CNS drug delivery. We present a retrospective overview of these works initiated in the Soviet Union in 1980s, and then continued in the United States and other countries. Notably some of the early findings were later corroborated by brain pharmacokinetic data. Industrial development of several drug candidates employing these strategies has followed. Overall modification by hydrophobic fatty acids residues or amphiphilic block copolymers represents a promising and relatively safe strategy to deliver proteins to the brain.

Figure 1

Methods of protein/peptide modification by fatty acid. Chemical acylation can be achieved in either aqueous (A and B) or organic (C and D) solution. Reaction in aqueous solution in general better preserves protein activity than in organic solvent. However, fatty acids do not solubilize well in aqueous solution and the obtained products often show low yield with high heterogeneity (A). Increasing fatty acid solubility by adding detergent to the aqueous solution can result in a relatively higher yield and more homogeneous product (B). Reacting protein/peptide with fatty acid directly in organic solvent is not recommended because of protein inactivation and solubility issues (C). However, reacting protein with fatty acid using a hydrated reverse micelle system in organic solution proceeds efficiently and in most cases allows recovering more-or-less uniformly modified protein retaining relatively high activity (D). A site-specific fatty acylation can be achieved via enzymatic catalysis. Endogenous ghrelin has shown to be octanoylated specifically at the serine 3 position, a post-translation step that is catalyzed by ghrelin O-acylatransferase (GOAT) (E). Since organic solvent has less impact on peptide activity, fatty acylation of short sequence peptides can be done during solid phase synthesis cycle, allowing for a clean product with high yield (F).

Figure 2

Hypothetical mechanisms of the blood to brain transport of Pluronic conjugated proteins. (A) In a protein modified with a single Pluronic molecule the block copolymer hydrophobic chains (PPO) bind to lipid rafts resulting in protein transport across the BBB. (B) The protein modified with a single Pluronic molecule is capable of binding with the BBB transporter as well as lipid rafts in the BBB. Both mechanisms lead to the transport across the BBB albeit the protein transporter makes the greater contribution (likely case of leptin-P85 with single Pluronic chain attached). (C) In a protein modified with multiple Pluronic molecules the PPO chains bind to lipid rafts resulting in protein transport across the BBB. (D) The protein modified with a multiple Pluronic molecules binds with lipid rafts, which serve as major route for the conjugate CNS entry. The binding of this protein with its transporter is either inhibited or does not result in efficient transport across the BBB (likely case of leptin-P85 with multiple Pluronic chains attached).