Synthesis and Evaluation of Insulin−Human Serum Albumin Conjugates

Abstract

A series of human insulin maleimido derivatives with short and long linkers was synthesized by exploiting the variations in the pK(a) values and environment of the three amino groups present in the protein. The syntheses were accomplished in organic solvent because of maleimide's instability in basic aqueous media. The derivatives thus obtained were conjugated to the free thiol on Cys34 of human serum albumin (HSA) and purified. A structure-activity relationship based on in vitro receptor binding and activation results for this series of insulin-HSA conjugates showed that the best compounds were attached at the B1 position of insulin with either short or long linkers. Two conjugates were administered subcutaneously to streptozotocin-induced diabetic rats and found to possess blood glucose normalizing activity up to 8 h post-administration. The return to diabetic plasma glucose levels was not observed within the time frame of the experiment (48 h). In comparison, the insulin-treated group's normalization activity lasted 2 h and returned to a diabetic level at 8 h. The onset of the conjugate activities were delayed by 1 h when compared to the activity of human insulin. The study results led to the identification of CJC-1575 as a potent and long lasting human insulin analogue.

Albumin-conjugated C34 Peptide HIV-1 Fusion Inhibitor

“…Preparation of Albumin Conjugates-The conjugation of maleimido-C34 and maleimido-T-20 derivatives to cysteine 34 of HSA and subsequent purification were performed as reported previously (13)(14)(15)(16)(17). Mass spectrometry of each purified sample confirmed the most abundant protein product corresponded to a 1:1 covalent complex of HSA with each maleimido derivative, and reverse phase HPLC analysis of each purified sample confirmed the removal of essentially all unbound (free) maleimido derivative.…”

“…The challenge in developing therapeutic peptides is complicated primarily by their rapid renal clearance, poor distribution, and susceptibility to peptidase degradation. Despite recent predictions that cross-linking C-peptide inhibitors to larger proteins will likely reduce their antiviral activity (11), we used albumin conjugation as a vehicle to achieve superior pharmacokinetic profiles of C34 peptide as has been performed with other classes of maleimido peptides (12)(13)(14)(15)(16)(17). Such conjugation reactions may be performed in vivo by administering the compound directly into the human patient followed by conjugation to endogenous serum albumin.…”

Preparation and Characterization of PEGylated Amylin

“…In the field of novel biotherapeutic protein design, increasing the in vivo biological activity of compounds has typically been achieved using bioconjugation strategies with large, biocompatible molecular partners, such as fatty acids, albumin, and polyethylene glycol, which directly or indirectly inhibit renal excretion (52)(53)(54)(55)(56)(57)(58). With this strategy in mind, we have designed and obtained PEGylated amylin conjugates with high reaction efficiency through the reaction of mPEG-SC and mPEG-SPA to the two amine groups of the Lys1 residue, which has been confirmed by trypsin digestion followed by MALDI-ToF-MS analysis.…”

A Covalent Inhibitor Targeting an Intermediate Conformation of the Fusogenic Subunit of the HIV-1 Envelope Complex

“…1 and 5). Although maleimido-containing compounds, A and C, are the most efficient in performing a covalent attachment to gp41, maleimido peptides are expected to bioconjugate preferentially to free thiols (such as the cysteine 34 of human serum albumin present in excess) following their administration to patients intravenously or subcutaneously (45)(46)(47)(48). This would chemically quench the reactive moiety and render the anti-fusogenic peptide less capable of forming a covalent bond to its intended target.…”