Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment.

The aim of this study was to investigate the influence of association state and net charge of human insulin analogues on the rate of iontophoretic transport across hairless mouse skin, and the effect of different skin pretreatments on said transport. No insulin flux was observed with anodal delivery probably because of degradation at the Ag/AgCl anode. The flux during cathodal iontophoresis through intact skin was insignificant for human hexameric insulin, and only low and variable fluxes were observed for monomeric insulins. Using stripped skin on the other hand, the fluxes of monomeric insulins with two extra negative charges were 50-100 times higher than that of hexameric human insulin. Introducing three additional charges led to a further 2-3-fold increase in flux. Wiping the skin gently with absolute alcohol prior to iontophoresis resulted in a 1000-fold increase in transdermal transport of insulin relative to that across untreated skin, i.e. to almost the same level as stripping the skin. The alcohol pretreatment reduced the electrical resistance of the skin, presumably by lipid extraction. In conclusion, monomeric insulin analogues with at least two extra negative charges can be iontophoretically delivered across hairless mouse skin, whereas insignificant flux is observed with human, hexameric insulin. Wiping the skin with absolute alcohol prior to iontophoresis gave substantially improved transdermal transport of monomeric insulins resulting in clinically relevant delivery rates for basal treatment.

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. Both insulins were infused for 4 h to achieve steady state glucose metabolism. The infusion rate ranged from 2.5-8 pmol min-1 kg-1. Parallel dose response curves were obtained with the mean glucose infusion rate from 180-240 min as the response and the logarithm of the insulin infusion rate as the dose. Standard bioassay analysis showed that the molar potency of the analogue relative to human insulin was 95.2% with a 95% confidence interval of 82.3-111.2%. To assess the clinical validity of the method a similar euglycaemic clamp study was carried out in human volunteers. The insulin infusion rates were 3 and 6 pmol min-1 kg-1, and the mean glucose infusion rate over the final 180-240 min period of the clamp was used as response. The statistical analysis showed, as in the pig clamp bioassay, no significant deviations from steady state or from the assumption of parallelism. The resulting molar potency of the analogue relative to human insulin was 85.5% with a 95% confidence interval of 49.5-128.4%. This was in agreement with the result of the pig clamp bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

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. The rate of hydrolysis at B3 is independent of the strength of the preparation, and in most cases the species of insulin, but varies with storage temperature and formulation. Total transformation at B3 is considerably reduced when insulin is in the crystalline as compared to the amorphous or soluble state, indicating that formation of the rate-limiting cyclic imide decreases when the flexibility of the tertiary structure is reduced. Neutral solutions containing phenol showed reduced deamidation probably because of a stabilizing effect of phenol on the tertiary structure (alpha-helix formation) around the deamidating residue, resulting in a reduced probability for formation of the intermediate imide. The ratio of isoAsp/Asp derivative was independent of time and temperature, suggesting a pathway involving only intermediate imide formation, without any direct side-chain hydrolysis. However, increasing formation of Asp relative to isoAsp derivative was observed with decreasing flexibility of the insulin three-dimensional structure in the formulation. In certain crystalline suspensions a cleavage of the peptide bond A8-A9 was observed. Formation of this split product is species dependent: bovine greater than porcine greater than human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.

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. With the aim to improve the absorption properties recombinant DNA technology has been utilized to design novel insulin molecules with changed physico-chemical characteristics and hence altered subcutaneous absorption kinetics. Soluble, long-acting human insulin analogues in which the isoelectric point has been increased from 5.4 to approx. 7 are absorbed very slowly, providing a more constant basal insulin delivery with lower day-to-day variation than present protracted preparations. In addition they have better storage stability. Rapid-acting human insulin analogues with largely reduced self-association are absorbed substantially faster from subcutaneous tissue than current regular insulin and thus are better suited for bolus injection. The absorption kinetics of these analogues have been able to explain the mechanism behind the dose effect on insulin absorption rate.

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. Phenol and sodium chloride exerted their stabilizing effect through independent mechanisms. Zinc ions, in concentrations that promote association of insulin into hexamers, increase the stability, whereas higher zinc content had no further influence. Protamine gave rise to additional formation of covalent protamine-insulin products which increased with increasing protamine concentration. The impact of excipients on the chemical processes seems to be dictated mainly via an influence on the three-dimensional insulin structure. The effect of the physical state of the insulin on the chemical stability was also complex, suggesting an intricate dependence of intermolecular proximity of involved functional groups. At pH values below five and above eight, insulin degrades relatively fast. At acid pH, deamidation at residue A21 and covalent insulin dimerization dominates, whereas disulfide reactions leading to covalent polymerization and formation of A- and B-chains prevailed in alkaline medium. Structure-reactivity relationship is proposed to be a main determinant for the chemical transformation of insulin.

NovoSol Basal: pharmacokinetics of a novel soluble long acting insulin analogue.

OBJECTIVE: To determine the courses of absorption and the interindividual and intraindividual variations in absorption of iodine-125 labelled Ultratard HM and NovoSol Basal injected subcutaneously. DESIGN: Open randomised crossover study. Each patient was tested during two study periods of five days each, during which he or she received a subcutaneous injection of either 125I-NovoSol Basal or 125I-Ultratard HM on four consecutive days. The aim was to detect a reduction in intraindividual standard deviation by a factor of two with a probability 0.95, taking 0.05 as the level of significance. This required 24 degrees of freedom and led to the choice of four courses in each of eight patients. SETTING: Referrals to the diabetes research centre in Hvidøre, Copenhagen. PATIENTS: Eight insulin dependent (type I) diabetics with low or undetectable C peptide concentrations who were receiving a multiple insulin injection regimen. One patient withdrew immediately after recruitment. INTERVENTIONS: After an overnight fast patients received 96 nmol (16 IU insulin) of either 125I-NovoSol Basal or 125I-Ultratard HM injected subcutaneously into the thigh. To ensure that the insulin entered the subcutaneous fat at the same depth, ultrasonography was performed on each patient before the first injection. A different injection site on the thigh was used each day for four days in order to facilitate monitoring of the disappearance of four different depots in each patient. MAIN OUTCOME MEASURE: Residual activity at the injection site was measured roughly every two hours throughout the day. No radioactivity measurements were performed overnight (10 pm till 8 am). The residual radioactivity after the injection on the first day (upper right thigh) was recorded for five days, that after the injection on the second day (upper left thigh) for four days, after the injection on the third day (lower right thigh) for three days, and after the last injection (lower left thigh) for two days. RESULTS: NovoSol Basal was absorbed according to first order kinetics with a mean t50% of 35.3 (SEM 1.4) hours; Ultratard HM was absorbed after a lag phase and the corresponding t50% was 25.5 (2.5) hours. The intraindividual variations in t50% were significantly smaller with NovoSol Basal than with Ultratard HM (18.4% v 44.5%; p less than 0.001). Interindividual variations, however, were not significantly different (25.2% v 36.9%; p = 0.38). The total variation in t50% was substantially smaller with NovoSol Basal than with Ultratard HM (20.3% v 42.8%). CONCLUSIONS: NovoSol Basal seems to be an appreciable advance over Ultratard HM as a soluble insulin preparation for obtaining reproducible 24 hour insulin concentrations in the blood

Soluble, prolonged-acting insulin derivatives. III. Degree of protraction, crystallizability and chemical stability of insulins substituted in positions A21, B13, B23, B27 and B30.

It was previously demonstrated that insulins to which positive charge has been added by substituting B13 glutamic acid with a glutamine residue, B27 threonine with an arginine or lysine residue, and by blocking the C-terminal carboxyl group of the B-chain by amidation, featured a prolonged absorption from the subcutis of rabbits and pigs after injection in solution at acidic pH. The phenomenon is ascribed to a low solubility combined with the readiness by which these analogs crystallize as the injectant is being neutralized in the tissue. However, acid solutions of insulin are chemically unstable as A21 asparagine both deamidates to aspartic acid and takes part in formation of covalent dimers via alpha-amino groups of other molecules. In order to circumvent the instability, substitutions were introduced in position A21, in addition to those in B13, B27 and B30, challenging the fact that A21 asparagine has been conserved in this position throughout the evolution. Biological potency was retained when glycine, serine, threonine, aspartic acid, histidine and arginine were introduced in this position, although to a varying degree. In the crystal structure of insulin a hydrogen bond bridges the alpha-nitrogen of A21 with the backbone carbonyl of B23 glycine. In order to investigate the importance of this hydrogen bond for biological activity a gene for the single-chain precursor B-chain(1-29)-Ala-Ala-Lys-A-chain(1-21) featuring an A21 proline was synthesized. However, this single-chain precursor failed to be properly produced by yeast, pointing to the formation of this hydrogen bond as an essential step in the folding process. The stability of the A21-substituted analogs in acid solutions (pH 3-4) with respect to deamidation and formation of dimers was approximately 5-10 times higher than that of human insulin in neutral solution. The rate of absorption of most insulins is decreased by increasing the Zn2+ concentration of the preparation. However, one analog with A21 glycine showed first-order absorption kinetics in pigs with a half-life of approximately 25 h, independent of the Zn2+ concentration. The day-to-day variation of the absorption of this analog was significantly lower than that of the conventional insulin suspensions, a property that might render such an insulin useful in the attempts to improve glucose control in diabetics by a more predictable delivery of basal insulin.

Soluble, prolonged-acting insulin derivatives. II. Degree of protraction and crystallizability of insulins substituted in positions A17, B8, B13, B27 and B30.

It has previously been found that insulins, to which positive charge has been added by substitutions in position B30, thus raising the isoelectric point towards pH 7, had a prolonged action when injected as slightly acidic solutions because such derivatives crystallize very readily upon neutralization. Positive charge has now been added by substituting the B13 and A17 glutamic acid residues with glutamines and B27 threonine with lysine or arginine. These substitutions were introduced by site-specific mutagenesis in a gene coding for a single-chain insulin precursor. By tryptic transpeptidation the single-chain precursors were transformed to the double-chain insulin structure, concomitantly with incorporation of residue B30. Thus insulins combining B13 glutamine, A17 glutamine and B27 lysine or arginine with B30 threonine, threonine amide or lysine amide were synthesized. The time course of blood glucose lowering effect and the absorption were studied after subcutaneous injection in rabbits and pigs. The prolonged action of B30-substituted insulins was markedly enhanced by B27 lysine or arginine substitutions and by B13 glutamine. The B27 residue is located on the surface of the hexamer, so a basic residue in this position presumably promotes the packing of hexamers at neutral pH. The B13 residues cluster in the centre of the hexamer. When the electrostatic repulsive forces from six glutamic acid residues are abolished by substitution with glutamine, a stabilization of the hexamer can be envisaged.(ABSTRACT TRUNCATED AT 250 WORDS)