Biomedical subjects

A H Pekar

Publications and source records attributed to A H Pekar.

16 recordsLinked to original sources

PubMed1998

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.

Amino Acid Sequence↗

PubMed1996

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

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, LysB28ProB29-human 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.

Allosteric Regulation↗

PubMed1995

Effect of salts on the structure of a potent analog of growth hormone releasing hormone as determined by optical spectroscopy.

Optical spectroscopic methods (circular dichroism, analytical ultracentrifugation, and static light scattering) were employed to study the solution behavior of an N-terminal-acylated 76-residue analog of growth hormone releasing hormone (GHRH). The GHRH analog had a 30% helical configuration in aqueous acidic solution, unlike other GHRH analogs that had a random coil configuration in aqueous solutions, and self-associated. High concentrations (7 M) of urea were required to obtain monomeric peptide, but such urea concentrations unfolded the peptide. Therefore, the folding and self-association were related events. The self-association and helix content were increased by the addition of various cations (e.g., Na+, Ca2+). In 7 M urea, when these cations were added, the peptide started to refold toward its aqueous conformation in a pH dependent manner; at low pH (2.5-3.5) the peptide folded to approximately 50% of the native configuration. At neutral pH (> 6) only small changes were seen when salts were added. When CaCl2 was added to a solution containing 7 M urea at pH 2.5, the self-association of the peptide increased with the concentration of CaCl2. Therefore, the salt dependent self-association observed in aqueous solvents was present in 7 M urea. Residues 45-76 in this GHRH analog, which are not present outside the hypothalamus, are probably needed to interact with a folding chaperone in vivo and provide stability for successful membrane transport and maintenance of biological activity.

Amino Acid Sequence↗

PubMed1993

Acid stabilization of insulin.

The effect of pH on the conformational stability of insulin was studied. Surprisingly, the Gibbs free energy of unfolding increased approximately 30% by acidification. pH titration of insulin's conformational stability is described by a transition involving a single proton with an apparent pK(a) of 7.0. The acid stabilization of insulin's conformation was attributed to the protonation of histidine at position 5 on the B-chain (HB5) as determined by 1H-NMR of the histidines, selective amino acid alteration, and enthalpies of ionization. Further acidification (at least to pH 2) does not decrease the free energy of unfolding. A conformational change in the tertiary structure, as indicated by the near-UV circular dichroism spectrum, accompanies this change in stability. We propose that this acid stabilization of insulin is physiologically important in maintaining insulin stability in the acid environment of the secretory/storage granules of the beta-cell of the pancreatic islets of Langerhans.

Chemical Phenomena↗

PubMed1993

Evidence for a self-associating equilibrium intermediate during folding of human growth hormone.

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.

Chromatography, High Pressure Liquid↗

PubMed1992

Detection of an equilibrium intermediate in the folding of a monomeric insulin analog.

To determine the conformational properties of the C-terminal region of the insulin B-chain relative to the helical core of the molecule, we have investigated the fluorescence properties of an insulin analog in which amino acids B28 and B29 have been substituted with a tryptophan and proline residue respectively, ([WB28,PB29]insulin). The biological properties and far-UV circular dichroism (CD) spectrum of the molecule indicate that the conformation is similar to that of native human insulin. Guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation of the analog as monitored by CD intensity at 224 nm indicates a single cooperative transition with a midpoint of 4.9 M GdnHCl. In contrast, when the equilibrium denaturation is observed by steady-state fluorescence emission intensity at 350 nm, two distinct transitions are observed. The first transition accounts for 60% of the observed signal and has a midpoint of 1.5 M GdnHCl. The second transition roughly parallels that observed by CD measurements with an approximate midpoint of 4.5 M GdnHCl. The near-UV CD spectrum, size-exclusion, and ultracentrifugation properties of [WB28,PB29]insulin indicate that this analog does not self-associate in a concentration-dependent manner as does human insulin. Thus, the observed fluorescence changes must be due to specific conformational transitions which occur upon unfolding of the insulin monomer with the product of the first transition representing a stable folding intermediate of this molecule.

Amino Acid Sequence↗

PubMed1992

Altering the association properties of insulin by amino acid replacement.

The importance of ProB28 and LysB29 on the self-association of insulin was established by systematically truncating the C terminus of the B chain. The relationship between structure and association was further explored by making numerous amino acid replacements at B28 and B29. Association was studied by circular dichroism, size-exclusion chromatography and ultracentrifugation. Our results show that the location of a prolyl residue at B28 is critical for high-affinity self-association. Removal of ProB28 in a series of C-terminal truncated insulins, or amino acid replacement of ProB28, greatly reduced association. The largest disruption to association was achieved by replacing LysB29 with Pro and varying the amino acid at B28. Several of the analogs were predominantly monomers in solutions up to 3 mg/ml. These amino acid substitutions decreased association by primarily disrupting the formation of dimers. Such amino acid substitutions also substantially reduced the Zn-induced insulin hexamer formation. The formation of monomeric insulins through amino acid replacements was accompanied by conformational changes that may be the cause for decreased association. It is demonstrated that self-association of insulin can be drastically altered by substitution of one or two key amino acids.

Amino Acid Sequence↗

PubMed1990

Hypoglycemic potency and metabolic clearance rate of intravenously administered human proinsulin and metabolites.

Since circulating proinsulin has been suggested to be important in the pathogenesis of noninsulin-dependent diabetes, and biosynthetic human proinsulin (HPI) may have a therapeutic role in patients with diabetes mellitus, the biological activity of proinsulin metabolites is of potential significance. Moreover, recent studies have suggested that the majority of circulating proinsulin immunoreactivity consists of metabolites. We, therefore, compared the blood glucose-lowering ability and MCR of the two proinsulin metabolites des-(31,32)HPI and des-(64,65)HPI with intact HPI in seven anesthetized dogs after an overnight fast. Intravenous bolus injections of 12.5 micrograms HPI/kg BW and equimolar amounts of des-(31,32)HPI and des-(64,65)HPI were given on three separate occasions. In addition to blood glucose, des-(31,33)HPI, des-(64,65)HPI, and HPI were measured using an insulin RIA and peptide-specific standard curves. Kinetic parameters were derived by fitting two exponentials to the respective decay curves. The MCR of HPI (3.3 +/- 0.1 ml/kg.min) was significantly lower (P less than 0.05) than that of des-(64,65)HPI (6.4 +/- 0.6 ml/kg.min), but was not significantly different from that of des-(31,32)HPI (3.8 +/- 0.4 ml/kg.min). The MCR of biosynthetic insulin (17.2 +/- 1.8 ml/kg.min), as measured in three of the dogs, was higher than that of HPI or the two metabolites. The blood glucose-lowering ability (defined as nadir glucose/fasting glucose, expressed as a percentage) of des-(64,65)HPI (49.3 +/- 5.0%) was significantly greater (P less than 0.05) than that of intact HPI (87 +/- 2.2%), and the glucose-lowering ability of des-(31,32)HPI (75.2 +/- 3.8%) was intermediate. In conclusion, HPI metabolites are more biologically active than intact HPI. The extent of in vivo conversion of proinsulin to metabolites may enhance the biological activity of proinsulin and, thus, have physiological, pathophysiological, and therapeutic significance.

Animals↗

PubMed1984

Isolation and characterization of a genetic variant of bovine proinsulin.

A genetic variant of bovine proinsulin has been isolated using preparative reverse-phase HPLC. The new proinsulin (bovine proinsulin II) differs from the known proinsulin (bovine proinsulin I) by a single amino acid residue at position C-48 in the connecting peptide. The amino acid replacement is a leucine substitution for proline. The two proinsulins were found in a ratio of approximately 9:1, proinsulin I: proinsulin II. No chemical or biological differences were observed for the two proinsulins other than their different elution times on reverse-phase HPLC.

Amino Acid Sequence↗

PubMed1974