Effects of non-covalent self-association on the subcutaneous absorption of a therapeutic peptide.

Purpose: To utilize an acylated peptide as a model system to investigate the relationships among solution peptide conformation, non-covalent self-association, subcutaneous absorption and bioavailability under pharmaceutically relevant solution formulation conditions.

Methods: CD spectroscopy, FTIR spectroscopy, equilibrium sedimentation, dynamic light scattering, and size exclusion chromatography were employed to characterize the effects of octanoylation on conformation and self-association of the 31 amino acid peptide derivative des-amino-histidine(7) arginine(26) human glucagon-like peptide (7-37)-OH (IP(7)R(26)GLP-1). Hyperglycemic clamp studies were performed to compare the bioavailability, pharmacokinetics, and pharmacodynamics of solution formulations of oct-IP(7)R(26)GLP-1 administered subcutaneously to normal dogs.

Results: Octanoylation of IP(7)R(26)GLP-1 was shown to confer the propensity for a major solvent-induced conformational transition with an accompanying solvent- and temperature-dependent self-association behavior. Formulations were characterized that give rise to remarkably different pharmacodynamics and pharmacokinetics that correlate with distinct peptide conformational and self-association states. These states correspond to: (i) a minimally associated alpha-helical form (apparent molecular weight = 14 kDa), (ii) a highly associated, predominantly beta-sheet form (effective molecular diameter 20 nm), and (iii) an unusually large, micelle-like soluble beta-sheet aggregate (effective molecular diameter 50 nm).

Conclusions: Bioavailability and pharmacokinetics of a self-associating peptide can be influenced by aggregate size and the ease of disruption of the non-covalent intermolecular interactions at the subcutaneous site. Hydrophobic aggregation mediated by seemingly innocuous solution formulation conditions can have a dramatic effect on the subcutaneous bioavailability and pharmacokinetics of a therapeutic peptide and in the extreme, can totally preclude its absorption. A size exclusion chromatographic method is identified that distinguishes subcutaneously bioavailable aggregated oct-IP(7)R(26)GLP-1 from non-bioavailable aggregated oct-IP(7)R(26)GLP-1.