Use of designed peptide linkers and recombinant hemoglobin mutants for drug delivery: in vitro release of an angiotensin II analog and kinetic modeling of delivery.

We have designed and tested specific peptide linkers for the glutathione-mediated reductive release of the angiotensin II analog N-acetyl-CGDKVYIHPF attached to recombinant human hemoglobin mutants by a disulfide bond. Inclusion of negatively charged residues decreased the rate of release by as much as 5-fold for the N-terminal linker DCD when compared to that of the control linker CG. Two different surface cysteine mutants in the second domain of the alpha,alpha-chain of recombinant human hemoglobin, D75C and K16C, were examined for their effect on the release of peptide by reduced glutathione. The reaction of the D75C-peptide conjugate with glutathione for release of the peptide is slow, with a second-order rate constant of 2.1 M-1 S-1, allowing the possibility of long term delivery. The rate of peptide release from the K16C vs the D75C mutant was decreased 15-fold. Thus, different peptide release rates can be obtained by changing both peptide linker residues and the surface location of peptide attachment. Kinetic modeling of this release using either measured or literature values for different parameters suggests boundary conditions for application to the in vivo release of peptidomimetics, small molecules, or other drugs bound to the hemoglobin surface.