Effect of fatty acids and selected drugs on the albumin binding of a long-acting, acylated insulin analogue.

NN304 (LysB29-tetradecanoyl, des(B30)-insulin) is a new soluble, long-acting insulin analogue that is tightly bound to human serum albumin differentiating it from human insulin. In the present study, we investigate the effect of fatty acids and selected drugs on the binding of NN304 to human serum albumin in vitro. Binding of the first fatty acid equivalent to albumin does not affect the binding of NN304.

Peptomers: a versatile approach for the preparation of diverse combinatorial peptidomimetic bead libraries.

This report describes a versatile approach in the generation of peptidomimetic bead libraries. The method is based on the preparation of peptide-peptoid hybrids using the portioning-mixing procedure, which gives diverse peptidomimetic bead libraries composed of peptides, peptoids and peptide-peptoid hybrids. We term these peptomers, from peptide-peptoid hybrid polymers.

Solution structure of an engineered insulin monomer at neutral pH.

Insulin circulates in the bloodstream and binds to its specific cell-surface receptor as a 5808 Da monomeric species. However, studies of the monomer structure and dynamics in solution are severely limited by insulin self-association into dimers and higher oligomers. In the present work we use site-directed mutagenesis of the dimer- and hexamer-forming surfaces to yield the first insulin species amenable for structure determination at neutral pH by nuclear magnetic resonance (NMR) spectroscopy.

Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs.

We have synthesized insulins acylated by fatty acids in the epsilon-amino group of LysB29. Soluble preparations can be made in the usual concentration of 600 nmol/ml (100 IU/ml) at neutral pH. The time for 50% disappearance after subcutaneous injection of the corresponding TyrA14(125I)-labelled insulins in pigs correlated with the affinity for binding to albumin (r = 0.

Albumin binding and time action of acylated insulins in various species.

Insulins acylated with fatty acids at the epsilon-amino group of LysB29 constitute a new class of insulin analogs, which are prolonged-acting due to albumin binding. In the present study it is shown that the affinity of fatty acid acylated insulins for albumin varies considerably (> 50-fold) among species. The relative affinities of acylated insulin for albumin in human, pig, and rabbit serum are about 1:1:5:35.

Albumin binding of insulins acylated with fatty acids: characterization of the ligand-protein interaction and correlation between binding affinity and timing of the insulin effect in vivo.

Albumin is a multifunctional transport protein that binds a wide variety of endogenous substances and drugs. Insulins with affinity for albumin were engineered by acylation of the epsilon-amino group of LysB29 with saturated fatty acids containing 10-16 carbon atoms. The association constants for binding of the fatty acid acylated insulins to human albumin are in the order of 10(4)-10(5) M-1.

Action profile of cobalt(III)-insulin. A novel principle of protraction of potential use for basal insulin delivery.

The Co(3+)-insulin hexamer is an extraordinary stable insulin hexamer that has no affinity for the insulin receptor per se but is converted into active insulin in vivo. In the present study, we evaluated the action profile of Co(3+)-insulin after subcutaneous injection into nondiabetic pigs and showed that the Co(3+)-hexamer does not dissociate before absorption. After absorption, Co(3+)-insulin is accumulated in the bloodstream because the complex is distributed and eliminated more slowly than human insulin.

The cobalt(III)-insulin hexamer is a prolonged-acting insulin prodrug.

The insulin hexamer has two high-affinity metal ion binding sites, each involving three HisB10 residues, one from each dimer. Insulin hexamers containing Co2+ at both these sites were oxidized to form a stable Co(3+)-insulin complex. It is shown that the Co(3+)-coordinated insulin monomers are released extremely slowly in aqueous solution at pH 8.

The high resolution solution structure of the insulin monomer determined by NMR.

Studies of naturally occurring and chemically modified insulins indicate that relatively few of the 51 amino acid residues may be assigned specific roles in insulin-receptor interactions. Most of the insulin X-ray structural information is derived from aggregated species (notably hexamers). Because insulin exerts its physiological effect as a 5808 Dalton monomeric species, it is necessary to consider whether crystal-packing forces have modified the structure from that required for biological action.

High-resolution structure of an engineered biologically potent insulin monomer, B16 Tyr-->His, as determined by nuclear magnetic resonance spectroscopy.

Site-directed mutagenesis is used in conjunction with 1H nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy in order to find an insulin species amenable for structure determination in aqueous solution by NMR spectroscopy. A successful candidate in this respect, i.e.

Engineering stability of the insulin monomer fold with application to structure-activity relationships.

To evaluate the possible relationship between biological activity and structural stability in selected regions of the insulin molecule, we have analyzed the guanidine hydrochloride induced reversible unfolding of a series of mutant insulins using a combination of near- and far-UV circular dichroism (CD). The unfolding curves are reasonably described on the basis of a two-state denaturation scheme; however, the observation of subtle differences between near- and far-UV CD detected unfolding indicates that intermediates may be present. Three regions of the insulin molecule are analyzed in detail with respect to their contribution to folding stability, i.

Ligand-induced conformation change in folate-binding protein.

C.d. and fluorescence spectroscopy have been used to investigate the effect of ligand binding on the structure and stability of folate-binding protein (FBP) from cow's whey.

Design of insulin analogues for meal-related therapy.

The human insulin in replacement therapy has a hexameric structure. Hexamerization of the insulin molecule facilitates biosynthesis and beta-cell storage of insulin, but is unnecessary for biologic activity and appears to contribute to delayed absorption of exogenous insulin from the subcutis. Insulin analogues with reduced self-association that are produced through recombinant DNA techniques have been shown to have in vivo activity comparable to that of human insulin and absorption kinetics characterized by higher and more constant rates of disappearance from the subcutaneous injection site.

Insulin structure and stability.

Insulin is composed of 51 amino acids in two peptide chains (A and B) linked by two disulfide bonds. The three-dimensional structure of the insulin molecule (insulin monomer), essentially the same in solution and in solid phase, exists in two main conformations. These differ in the extent of helix in the B chain which is governed by the presence of phenol or its derivatives.

Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during storage of pharmaceutical preparations.

Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4-45 degrees C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures greater than or equal to 25 degrees C parallel or consecutive formation of covalent oligo- and polymers can also be observed.

Chemical stability of insulin. 1. Hydrolytic degradation during storage of pharmaceutical preparations.

Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Insulin deteriorates rapidly in acid solutions due to extensive deamidation at residue AsnA21. In neutral formulations deamidation takes place at residue AsnB3 at a substantially reduced rate under formation of a mixture of isoAsp and Asp derivatives.

Application of the euglycaemic clamp technique to bioassay of insulin analogues.

The euglycaemic clamp method may offer a precise and clinically valid approach to assess the in vivo potency of new insulin analogues or derivatives relative to a human insulin standard. The proposed protocol was designed to overcome problems due to differences in pharmacokinetics between the test and standard preparations. An analogue of human insulin, GlyA21+ArgB27+ThrB30-NH2, which is absorbed very slowly after subcutaneous injection, and human insulin were compared in intravenous clamp experiments in pigs.

Intranasal administration of insulin with phospholipid as absorption enhancer: pharmacokinetics in normal subjects.

The pharmacokinetics of intranasal insulin containing a medium-chain phospholipid (didecanoyl-L-alpha-phosphatidylcholine) as absorption enhancer, was studied in normal volunteers by measuring plasma glucose, insulin, C-peptide, and glucagon. Eleven fasting subjects received 4 U insulin intravenously, 6 U subcutaneously, or three doses intranasally (approximately 0.3 U kg-1, 0.

The reactivity of anti-porcine gastric inhibitory polypeptide (GIP) rabbit serum R65 with synthetic human and porcine GIP.

Porcine gastric inhibitory peptide (GIP) has been used extensively as a standard and as [125I]-GIP in radio-immunoassays (RIAs) to determine immunoreactive GIP (IR GIP) in human plasma even though porcine and human GIP have slightly different amino acid sequences. A rabbit anti-porcine GIP serum (R65) which has been widely used in the RIA for IR GIP reacts with a part of the GIP molecule which is identical in human and porcine GIP. In order to validate the use of R65 the IR GIP contents of synthetic human and porcine GIP were measured, using natural porcine GIP as standard, and found to be 40% and 35% on a weight basis respectively.

Insulin analogues with improved absorption characteristics.

The insulin preparations available today are not ideal for therapy as s.c. injection does not provide a physiological insulin profile.

Chemical stability of insulin. 3. Influence of excipients, formulation, and pH.

The influence of auxiliary substances and pH on the chemical transformations of insulin in pharmaceutical formulation, including various hydrolytic and intermolecular cross-linking reactions, was studied. Bacteriostatic agents had a profound stabilizing effect--phenol > m-cresol > methylparaben--on deamidation as well as on insulin intermolecular cross-linking reactions. Of the isotonicity substances, NaCl generally had a stabilizing effect whereas glycerol and glucose led to increased chemical deterioration.

Chemical stability of insulin. 5. Isolation, characterization and identification of insulin transformation products.

During storage of insulin formulated for therapy, minor amounts of various degradation and covalent di- and polymerization products are formed [1-3]. The main chemical transformation products were isolated from aged preparations and characterized chemically and biologically. The most prominent products formed in neutral medium were identified as a mixture of deamidation products hydrolyzed at residue B3, namely isoAsp B3 and Asp B3 derivatives.

Chemical stability of insulin. 4. Mechanisms and kinetics of chemical transformations in pharmaceutical formulation.

Insulin decomposes by a multitude of chemical reactions [1-3]. It deamidates at two different residues by entirely different mechanisms. In acid, deamidation at AsnA21 is intramolecularly catalyzed by the protonated C-terminal, whereas above pH 6 an intermediate imide formation at residue AsnB3 leads to isoAsp and Asp derivatives.

In vitro and in vivo potency of insulin analogues designed for clinical use.

Analogues of human insulin designed to have improved absorption properties after subcutaneous injection have been prepared by recombinant DNA technology. Five rapidly absorbed analogues, being predominantly in mono- or di-meric states in the pharmaceutical preparation, and a hexameric analogue with very low solubility at neutral pH and slow absorption, were studied. Receptor binding assays with HEP-G2 cells showed overall agreement with mouse free adipocyte assays.