Biomedical subjects

Svend Havelund

Publications and source records attributed to Svend Havelund.

8 recordsLinked to original sources

PubMed2006

Biochemical and physiological properties of a novel series of long-acting insulin analogs obtained by acylation with cholic acid derivatives.

PURPOSE: This study was conducted to assess the suitability of insulin analogs acylated by various cholic acid derivatives for use as basal insulin, and to test the most promising of these, LysB29(Nepsilon-lithocholyl-gamma-Glu) des(B30) human insulin (NN344) in pigs. METHODS: Circular dichroism spectroscopy and size-exclusion chromatography were used to explore the physicochemical properties of the analogs, and affinities for albumin and insulin receptors were determined. After subcutaneous injection in pigs, disappearance half-times were measured, and the plasma profile and glucose-lowering effect in a euglycemic clamp were assessed for NN344. RESULTS: NN344 showed glucose-lowering activity lasting more than 24 h. Glucose infusion rate was essentially constant from 5 to 19 h after injection. NN344 seemed to be a dodecamer in the presence of zinc ions and phenol. Without phenol, the apparent molecular mass was >5000 kDa. Formation of such a self-assembly at the site of s.c. injection and its subsequent slow decomposition might explain the long duration of action of NN344. A measurable affinity for albumin of the lithocholic acid ligand may also contribute to the prolonged action. CONCLUSIONS: NN344 is a candidate for a neutral soluble basal insulin that might offer people with diabetes a prolonged duration, smooth, and predictable basal insulin supplement.

Acylation↗

PubMed2006

Chemical and thermal stability of insulin: effects of zinc and ligand binding to the insulin zinc-hexamer.

PURPOSE: To study the correlation between the thermal and chemical stability of insulin formulations with various insulin hexamer ligands. MATERIALS AND METHODS: The thermal stability was investigated using differential scanning calorimetry (DSC) and near-UV circular dichroism (NUV-CD). The formation of chemical degradation products was studied with reversed-phase and size-exclusion chromatography and mass spectrometry. RESULTS: An excellent correlation between the thermal stabilization by ligand binding and the deamidation of Asn(B3) was observed. The correlation between thermal stability and the formation of covalent dimer and other insulin related products was less clear. Zinc was found to specifically increase the deamidation and covalent dimer formation rate when the insulin hexamer was not further stabilized by phenolic ligand. Thiocyanate alone had no effect on the thermal stability of the insulin zinc-hexamer but significantly improved the chemical stability at 37 degrees C. At low temperatures thiocyanate induced a conformational change in the insulin hexamer. NUV-CD thermal scans revealed that this effect decreased with temperature; when the thermal denaturation temperature was reached, the effect was eliminated. CONCLUSIONS: Thermal stability can be used to predict the rate of Asn(B3) deamidation in human insulin. Chemical degradation processes that do not rely on the structural stability of the protein do not necessarily correlate to the thermal stability.

Calorimetry, Differential Scanning↗

PubMed2006

Ligand binding and thermostability of different allosteric states of the insulin zinc-hexamer.

The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate > or = 4-hydroxy-3-nitrobenzoate >> p-aminobenzoate >> chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.

Allosteric Regulation↗

PubMed2005

Thermal dissociation and unfolding of insulin.

The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2+) ions/hexamer), only the hexamer transition is observed. The results of this study show that the thermal stability of insulin is closely linked to the association state and that the zinc hexamer remains stable at much higher temperatures than the monomer. This is in contrast to studies with chemical denaturants where it has been shown that monomer unfolding takes place at much higher denaturant concentrations than the dissociation of higher oligomers [Ahmad, A., et al. (2004) J. Biol. Chem. 279, 14999-15013].

Amino Acid Substitution↗

PubMed2005

Reversible insulin self-assembly under carbohydrate control.

Insulin with built-in pairs of boronates and polyols can produce soluble high molecular weight self-assemblies under control by carbohydrates. The illustrated principle has potential utility for general protein and peptide protraction and controlled drug release.

Amino Acid Sequence↗

PubMed2005

Insulins with built-in glucose sensors for glucose responsive insulin release.

Derivatization of insulin with phenylboronic acids is described, thereby equipping insulin with novel glucose sensing ability. It is furthermore demonstrated that such insulins are useful in glucose-responsive polymer-based release systems. The preferred phenylboronic acids are sulfonamide derivatives, which, contrary to naïve boronic acids, ensure glucose binding at physiological pH, and simultaneously operate as handles for insulin derivatization at LysB29. The glucose affinities of the novel insulins were evaluated by glucose titration in a competitive assay with alizarin. The affinities were in the range 15-31 mM (K(d)), which match physiological glucose fluctuations. The dose-responsive glucose-mediated release of the novel insulins was demonstrated using glucamine-derived polyethylene glycol polyacrylamide (PEGA) as a model, and it was shown that Zn(II) hexamer formulation of the boronated insulins resulted in steeper glucose sensitivity relative to monomeric insulin formulation. Notably, two of the boronated insulins displayed enhanced insulin receptor affinity relative to native insulin (113%-122%) which is unusual for insulin LysB29 derivatives.

Amino Acid Sequence↗

PubMed2004

Crystallographic and solution studies of N-lithocholyl insulin: a new generation of prolonged-acting human insulins.

The addition of specific bulky hydrophobic groups to the insulin molecule provides it with affinity for circulating serum albumin and enables it to form soluble macromolecular complexes at the site of subcutaneous injection, thereby securing slow absorption of the insulin analogue into the blood stream and prolonging its half-life once there. N-Lithocholic acid acylated insulin [Lys(B29)-lithocholyl des-(B30) human insulin] has been crystallized and the structure determined by X-ray crystallography at 1.6 A resolution to explore the molecular basis of its assembly. The unit cell in the crystal consists of an insulin hexamer containing two zinc ions, with two m-cresol molecules bound at each dimer-dimer interface stabilizing an R(6) conformation. Six covalently bound lithocholyl groups are arranged symmetrically around the outside of the hexamer. These form specific van der Waals and hydrogen-bonding interactions at the interfaces between neighboring hexamers, possibly representing the kinds of interactions which occur in the soluble aggregates at the site of injection. Comparison with an equivalent nonderivatized native insulin hexamer shows that the addition of the lithocholyl group disrupts neither the important conformational features of the insulin molecule nor its hexamer-forming ability. Indeed, binding studies show that the affinity of N-lithocholyl insulin for the human insulin receptor is not significantly diminished.

Binding Sites↗

PubMed2004

The mechanism of protraction of insulin detemir, a long-acting, acylated analog of human insulin.

PURPOSE: Insulin detemir has been found in clinical trials to be absorbed with very low variability. A series of experiments were performed to elucidate the underlying mechanisms. METHODS: The disappearance from an injected subcutaneous depot and elimination studies in plasma were carried out in pigs. Size-exclusion chromatography was used to assess the self-association and albumin binding states of insulin detemir and analogs. RESULTS: Disappearance T50% from the injection depot was 10.2+/-1.2 h for insulin detemir and 2.0+/-0.1 h for a monomeric acylated insulin analog. Self-association of acylated insulin analogs with same albumin affinity in saline correlated with disappearance rate and addition of albumin to saline showed a combination of insulin detemir self association and albumin binding. Intravenous kinetic studies showed that the clearance and volume of distribution decreased with increasing albumin binding affinity of different acylated insulin analogs. CONCLUSIONS: The protracted action of detemir is primarily achieved through slow absorption into blood. Dihexamerization and albumin binding of hexameric and dimeric detemir prolongs residence time at the injection depot. Some further retention of detemir occurs in the circulation where albumin binding causes buffering of insulin concentration. Insulin detemir provides a novel principle of protraction, enabling increased predictability of basal insulin.

Acylation↗