Newly designed modifier prolongs the action of short-lived peptides and proteins by allowing their binding to serum albumin.

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

Establishing the principle of reversibility in peptide/protein and small-molecule therapy.

Several important pharmacological features can be integrated into injected drugs to enhance their therapeutic efficacy following administration. Short-lived peptide/protein drugs should be converted into long-lived species in vivo to avoid multiple injections. Circulating levels of anticancer agents need to be maintained within a narrow therapeutic range for prolonged period. Water-insoluble drugs must be turned into soluble species and blood-brain barrier-impermeable agents need to be modified to cross it following peripheral administrations. The derivatization requiring for achieving those desirable pharmacological features typically result in biologically/pharmacologically inactive products, unless those derivatizations can be carried out in a reversible fashion.

Novel analogs of VIP with multiple C-terminal domains.

The effect of multiplication of the N-terminal domain of vasoactive intestinal peptide (VIP) on the binding activity of the peptide was recently evaluated. A VIP analog with multiple N-terminal domains was found to be slightly more potent as compared to [Nle(17)]VIP towards VIP receptor type 1 (VPAC1)-related cAMP production. Here, the effect of multiplication of the C-terminal domain of VIP was evaluated with the aim of possibly amplifying peptide-receptor (VPAC1) binding and activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the C-terminal domain that was obtained by either a simple linear tandem extension or by a unique branching methodology. Results show that despite significant alterations in the C-terminal domain of VIP that is considered essential to induce potent receptor binding, few peptides demonstrated only slight reduction in receptor binding and activation in comparison to [Nle(17)]VIP. Furthermore, a specific branched VIP analog with multiple C-terminal domains was equipotent to [Nle(17)]VIP in the cAMP production assay. Therefore, it is concluded that the association between the VIP ligand to the VIP receptor could be tolerable to size increases in the C-terminal region of the VIP ligand and multiplication of the C-terminal does not increase activity.

Novel extended and branched N-terminal analogs of VIP.

The effects of vasoactive intestinal peptide (VIP) are primarily mediated through VPAC1 and VPAC2, receptors that are preferentially coupled to adenylate cyclase activation. As a large majority of the potent VIP antagonists have modifications in the N-terminal domain of the peptide, the effect of multiplication of this domain on VIP was examined with the aim of possibly amplifying peptide-receptor (VPAC1) activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the N-terminal domain that was obtained by either linear tandem extension or by parallel branching. Circular dichorism (CD) analysis revealed that these extended/branched peptides maintained an alpha helical structure in organic environment, similar to VIP. A specific branched VIP analog was found to be slightly more potent towards VPAC1-related cAMP production as compared to VIP. This analog could have potential therapeutic value in several disorders, similar to VIP. Two branched N-terminal VIP sequences demonstrated superior receptor binding and activation as compared to two N-terminals in tandem. The results suggest that correct alignment of the VIP N-terminal region is important for receptor binding and activation. However, increased receptor binding was not directly associated with increased cAMP production suggesting steric dynamic interactions.

Albumin-insulin conjugate releasing insulin slowly under physiological conditions: a new concept for long-acting insulin.

The covalent linkage of peptides or protein drugs to human serum albumin (HSA) greatly prolongs their lifetime in vivo but is pharmacologically irrelevant when it irreversibly inactivates them. We retain drug bioactivity by synthesizing a heterobifunctional reagent (MAL-Fmoc-OSu, 9-hydroxymethyl-2-(amino-3-maleimidopropionate)-fluorene-N-hydroxysuccinimide) that generates HSA-Fmoc-insulin on covalent conjugation to the amino group of insulin and the Cys-34 side chain of HSA. HSA-Fmoc-insulin is water-soluble and, upon incubation in aqueous buffers reflecting normal human serum conditions, slowly, spontaneously, and homogeneously hydrolyzes to release unmodified insulin with a t 1/2 of 25 +/- 2 h. A single subcutaneous or intraperitoneal administration of HSA-Fmoc-insulin to diabetic rodents lowers circulating glucose levels for about 4 times longer than an equipotent dose of Zn2+-free insulin. Following subcutaneous administration, onset of the glucose-lowering effect is delayed 0.5-1 h and persists for 12 h. Thus, we present a prototype insulin formulation possessing three desirable parameters: high aqueous solubility, delayed action following subcutaneous administration, and prolonged therapeutic effect.