The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, L y~~~~P r o~~~-h u m a n insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.

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Advancing nanomedicines from concept to clinic requires integration of new science with traditional pharmaceutical development. The medical and commercial success of nanomedicines is greatly facilitated when those charged with developing nanomedicines are cognizant of the unique opportunities and technical challenges that these products present. These individuals must also be knowledgeable about the processes of clinical and product development, including regulatory considerations, to maximize the odds for successful product registration. This article outlines these topics with a goal to accelerate the combination of academic innovation with collaborative industrial scientists who understand pharmaceutical development and regulatory approval requirements-only together can they realize the full potential of nanomedicines for patients.

In the preceding paper [Havel, H. A., Kauffman, E. W., Plaisted, S. M., & Brems, D. N. (1986) Biochemistry (preceding paper in this issue)], an associated intermediate was shown to be highly populated during the equilibrium denaturation of bovine growth hormone. In this paper, we describe its partial characterization and propose a mechanism for association. The associated equilibrium intermediate is populated under conditions that induce partial denaturation and at protein concentrations greater than 0.2 mg/mL. The remaining nativelike helical structure present in the partially denatured species is implicated in the mechanism of association as demonstrated by similar concentration dependencies and thermal stabilities of the helix and the associated equilibrium intermediate. Furthermore, it is suggested that a putative amphiphilic helix from residues 110-127 plays a critical role in the association as demonstrated by a diminution of the associated equilibrium intermediate when mixed with the peptide fragment 96-133. A model is proposed to account for these results in which partial denaturation exposes the segment of the protein corresponding to the hydrophobic face of the putative amphiphilic helix 110-127. This metastable form is the species from which association occurs. Association is stabilized by the hydrophobic interactions resulting from intermolecular packing of the lipophilic faces of the helices. The implications of these results to protein folding studies are described.

It has been previously shown, by equilibrium denaturation, that human growth hormone (hGH) folds by a cooperative two-state process. This is in contrast to the folding pathways of other nonhuman growth hormones that contain stable monomeric and multimeric equilibrium intermediates. We have reinvestigated the equilibrium denaturation of hGH at higher protein concentrations and found smooth transitions from the native to denatured state, but the calculated free energy for unfolding, delta G, decreases with increasing protein concentration. The effect of protein concentration on the delta G of unfolding is due to the presence of folding intermediates that have a tendency to self-associate. A correlation was found between the equilibrium denaturation data and the observation of precipitation that occurs upon refolding, suggesting that the presence of self-associated folding intermediates leads to precipitation. Direct evidence for the existence of a soluble, associated intermediate was obtained by dynamic light scattering (DLS) and equilibrium analytical ultracentrifugation. Peptide fragments from the third helix of either hGH or bovine growth hormone (bGH) were capable of inhibiting the formation of this aggregated species and prevent precipitation during refolding. The data show that the folding pathway of hGH is similar to that of nonhuman growth hormones except for differences in the tendency for intermediates to self-associate. These findings are relevant to the design and interpretation of equilibrium folding experiments, and may be important to understanding mechanistic details of protein folding and aggregation in vivo.