Reversible lipidization of peptide and protein drugs for prolonging the plasma half-lives and passive liver targeting

Abstract

Restricted until 18 Nov. 2009. The interest in developing peptide and protein drugs has been shadowed by their short elimination half-life and impermeability through biological membranes. Pegylation is widely attempted to extend their half-lives. However, pegylation results in heterogeneous species, leading to inconsistent drug efficacy, immunogenecity and side effects. An at least-equally-efficient delivery system that produces homogenous species is highly desired to substitute pegylation. Reversible lipidization takes a unique approach by conjugating fatty acids specifically to cysteines of peptide and protein through reducible disulfide linkage. This approach produces a single form of site-specific and controlled-release conjugate.; Using this approach, we successfully lipidized octreotide (OCT) and Tyr3-octreotide (TOC). Lipidization augmented the pharmacological effect of OCT on inhibiting growth hormone release in growth hormone releasing factor (GRF)-stimulated rats. Further pharmacokinetic studies demonstrated that lipidization increased the plasma half-life and enzymatic stability of the peptide. Tissue distribution of the lipidized peptide was distinguished by marked liver retention. Within the liver, the lipidized peptide had high association with both parenchymal cells (PC) and nonparenchymal cells (NPC).; Can reversible lipidization be applied to protein drugs since this approach involves disruption of the disulfides that are generally crucial for protein structure and function? To address this question, we lipidized human interferon-alpha (IFN-alpha), a 19.2 KD protein containing two disulfide bonds (cys1-cys98; cys29-cys138) and generated a homogenous conjugate, PAL-IFN. The far-UV circular dichroism (CD) spectra of PAL-IFN was virtually overlapped with IFN, indicating that the lipid-lipid interaction in adjacent lipidized cysteinyl residues upheld native IFN structure. PAL-IFN is a controlled-release conjugation. Upon reduction of the disulfide bonds in vivo, IFN is slowly released from PAL-IFN into circulation, as supported by the evidence that a low level of serum IFN activity was sustained more than 8 hours post PAL-IFN injection, while serum IFN activity rapidly diminished to an undetectable level at 2 hours post IFN injection. Furthermore, lipidization increases the affinity of IFN with hepatocytes. The liver-targeting effect of PAL-IFN resulted in enhanced antiviral activity as indicated by induction of the 2'-5' oligoadenylate synthetase (OAS), an indicator of IFN antiviral activity, locally in the liver at a level significantly higher than that produced by IFN. PAL-IFN is therefore potentially useful for the patients with chronic hepatitis B and C who are not responsive to current IFN therapy.; In conclusion, reversible lipidization could be used as an alternative delivery system of pegylation to prolong the plasma half-lives of peptide and protein drugs. Superior to pegylation, reversible lipidization produces a homogenous conjugate with controlled-release and liver-targeting properties.