Insulin detemir is a fully efficacious, low affinity agonist at the insulin receptor.

AIM: To compare the properties of insulin detemir with human insulin or insulin aspart in various in vitro and in vivo experiments, thereby highlighting the importance of performing dose-response studies when investigating insulin analogues, in this study specifically insulin detemir. METHODS: Displacement of membrane-associated insulin receptors from human and rat hepatocytes, and from Chinese Hamster Ovary cells over-expressing human insulin receptor (CHO-hIR) at varying albumin concentrations is measured. Lipogenesis in primary rat adipocytes over time and the effects in the simultaneous presence of insulin detemir and human insulin or insulin aspart are assessed. The hyperinsulinaemic euglycaemic clamp technique in rats is used to establish dose-response curves for multiple metabolic endpoints and to investigate the effects of the simultaneous presence of insulin detemir and human insulin. RESULTS: Both in vitro and in vivo, insulin detemir shows full efficacy and right-shifted parallel dose-response curves compared with human insulin. The potency estimates are different between the in vivo and in vitro conditions and among different in vitro conditions, that is the potency decreases in vitro with increasing albumin concentration. The effects of insulin detemir and human insulin are additive both in vitro and in vivo. CONCLUSIONS: Insulin detemir is fully efficacious compared with human insulin on all metabolic endpoints measured in vitro and in vivo. The fact that the potency estimates are method-dependent emphasizes the importance of establishing full dose-response relationships when characterizing insulin detemir.

Beta-cell function and islet morphology in normal, obese, and obese beta-cell mass-reduced Göttingen minipigs.

Herein, we bridge beta-cell function and morphology in minipigs. We hypothesized that different aspects of beta-cell dysfunction are present in obesity and obesity with reduced beta-cell mass by using pulsatile insulin secretion as an early marker. Measures for beta-cell function (glucose and arginine stimulation plus baseline and glucose-entrained pulsatile insulin secretion) and islet morphology were studied in long-term (19-20 mo) obese (n = 5) and obese beta-cell-reduced [nicotinamide + streptozotocin (STZ), n = 5] minipigs and normal controls, representing different stages in the development toward type 2 diabetes. Acute insulin response (AIR) to glucose and arginine were, surprisingly, normal in obese (0.3 g/kg glucose: AIR = 246 +/- 119 vs. 255 +/- 61 pM in control; 67 mg/kg arginine: AIR = 230 +/- 124 vs. 214 +/- 85 pM in control) but reduced in obese-STZ animals (0.3 g/kg glucose: AIR = 22 +/- 36, P < 0.01; arginine: AIR = 87 +/- 92 pM, P < 0.05 vs. control). Baseline pulsatile insulin secretion was reduced in obese (59 +/- 16 vs. 76 +/- 16% in control, P < 0.05) and more so in obese-STZ animals (43 +/- 13%, P < 0.01), whereas regularity during entrainment was increased in obese animals (approximate entropy: 0.85 +/- 0.14 vs. 1.13 +/- 0.13 in control, P < 0.01). Beta-cell mass (mg/kg body wt) was normal in obese and reduced in obese-STZ animals, with pancreatic fat infiltration in both groups. In conclusion, obesity and insulin resistance are not linked with a general reduction of beta-cell function, but dynamics of insulin secretion are perturbed. The data suggest a sequence in the development of beta-cell dysfunction, with the three groups representing stages in the progression from normal physiology to diabetes, and assessment of pulsatility as the single most sensitive marker of beta-cell dysfunction.

Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26.

A subset of prolyl oligopeptidases, including dipeptidyl-peptidase IV (DPP IV or CD26, EC ), specifically cleave off N-terminal dipeptides from substrates having proline or alanine in amino acid position 2. This enzyme activity has been implicated in the regulation of the biological activity of multiple hormones and chemokines, including the insulinotropic peptides glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Targeted inactivation of the CD26 gene yielded healthy mice that have normal blood glucose levels in the fasted state, but reduced glycemic excursion after a glucose challenge. Levels of glucose-stimulated circulating insulin and the intact insulinotropic form of GLP-1 are increased in CD26(-/-) mice. A pharmacological inhibitor of DPP IV enzymatic activity improved glucose tolerance in wild-type, but not in CD26(-/-), mice. This inhibitor also improved glucose tolerance in GLP-1 receptor(-/-) mice, indicating that CD26 contributes to blood glucose regulation by controlling the activity of GLP-1 as well as additional substrates. These data reveal a critical role for CD26 in physiological glucose homeostasis, and establish it as a potential target for therapy in type II diabetes.

Hypothalamic CART is a new anorectic peptide regulated by leptin.

The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems. Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y. Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus. In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus. Peripheral administration of leptin to obese mice stimulates CART mRNA expression. When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y. An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

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. The several fold higher binding affinity in rabbit serum than in pig serum is reflected in a relatively more protracted effect after sc injection in rabbits than in pigs. Due to the similar binding affinities in pig serum and human serum, the pig model should provide a useful estimate of the degree of protraction of acylated insulin in humans. The results emphasize that species differences in ligand binding can be of major importance in the preclinical evaluation of highly albumin bound drugs.

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 = 0.97), suggesting that the mechanism of prolonged disappearance is binding to albumin in subcutis. Most protracted was LysB29-tetradecanoyl des-(B30) insulin. The time for 50% disappearance was 14.3 +/- 2.2 h, significantly longer than that of Neutral Protamine Hagedorn (NPH) insulin, 10.5 +/- 4.3 h (p < 0.001), and with less inter-pig variation (p < 0.001). Intravenous bolus injections of LysB29-tetradecanoyl des-(B30) human insulin showed a protracted blood glucose lowering effect compared to that of human insulin. The relative affinity of LysB29-tetradecanoyl des-(B30) insulin to the insulin receptor is 46%. In a 24-h glucose clamp study in pigs the total glucose consumptions for LysB29-tetradecanoyl des-(B30) insulin and NPH were not significantly different (p = 0.88), whereas the times when 50% of the total glucose had been infused were significantly different, 7.9 +/- 1.0 h and 6.2 +/- 1.3 h, respectively (p < 0.04). The glucose disposal curve caused by LysB29-tetradecanoyl des-(B30) insulin was more steady than that caused by NPH, without the pronounced peak at 3 h. Unlike the crystalline insulins, the soluble LysB29-tetradecanoyl des-(B30) insulin does not elicit invasion of macrophages at the site of injection. Thus, LysB29-tetradecanoyl des-(B30) insulin might be suitable for providing basal insulin in the treatment of diabetes mellitus.

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 degree of protraction of Co(3+)-insulin is similar to that of NPH insulin when evaluated in an euglycemic glucose clamp. We suggest that the long plasma half-life and a gradual in vivo activation contribute to prolong the effect of Co(3+)-insulin. The Co(3+)-insulin hexamer provides a novel principle of protraction of potential use for basal insulin delivery to the diabetic patient.

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. The binding apparently involves both non-polar and ionic interactions with the protein. The acylated insulins bind at the long-chain fatty acid binding sites, but the binding affinity is lower than that of the free fatty acids and depends to a relatively small degree on the number of carbon atoms in the fatty acid chain. Differences in affinity of the acylated insulins for albumin are reflected in the relative timing of the blood-glucose-lowering effect after subcutaneous injection into rabbits. The acylated insulins provide a breakthrough in the search for soluble, prolonged-action insulin preparations for basal delivery of the hormone to the diabetic patient. We conclude that the biochemical concept of albumin binding can be applied to protract the effect of insulin, and suggest that derivatization with albumin-binding ligands could be generally applicable to prolong the action profile of peptide drugs.

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)

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.

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. Two analogues, B28Asp and A21Gly + B27Arg + B30Thr-NH2, had nearly the same molar in vitro potency as human insulin. Another two showed increased adipocyte potency and receptor binding, B10Asp 194% and 333% and A8His + B4His + B10Glu + B27His 575% and 511%, while B9Asp + B27Glu showed 29% and 18% and the B25Asp analogue only 0.12% and 0.05% potency. Bioassays in mice or rabbits of the analogues except B25Asp showed that they had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation had the same in vivo potency as human insulin 1.00 IU = 6.00 nmol. Thus the variation in in vivo potency reflects the differences in receptor binding affinity. Relative to human insulin a low concentration is sufficient for a high affinity analogue to produce a given receptor complex formation and metabolic response. In conclusion, human insulin and analogues with markedly different in vitro potencies were equipotent in terms of hypoglycaemic effect. This is in agreement with the concept that elimination of insulin from blood and its subsequent degradation is mediated by insulin receptors.

Equivalent in vivo biological activity of insulin analogues and human insulin despite different in vitro potencies.

In vivo biological potency of two human insulin analogues, AspB9,GluB27 insulin and AspB10 insulin with low and high affinity to the insulin receptor, respectively, was assessed by intravenous infusion of equimolar amounts in pigs, with the euglycemic clamp technique. Human insulin and the low- and high-affinity analogues showed equivalent glucose utilization rates in the steady state (mean +/- SE 14.7 +/- 1.4, 12.7 +/- 1.5, and 12.2 +/- 1.2 mg.kg-1.min-1, respectively; n = 7). The corresponding plasma insulin levels, however, were markedly different (329 +/- 25 and 856 +/- 46 pM, P less than 0.05; 197 +/- 19 pM, P less than 0.05). There was an inverse relationship between the insulin levels and the in vitro activities measured by binding to human hepatoma cells (HepG2; 100, 20, and 308%) or by incorporation of glucose into lipids in mouse free fat cells (100, 31, and 207%). The total amount of glucose infused during and after insulin infusion was equal for the three insulins, whereas glucose utilization as a function of time was somewhat different. By describing the individual plasma concentration courses with an open two-compartment model with elimination from the receptor compartment, the time courses for binding and elimination of the three insulins in the receptor compartment were estimated. The effect seems closely linked to the elimination of insulin from the receptors rather than to the amount of insulin bound to the receptors. In conclusion, the total effect of equimolar amounts of human insulin and the two insulin analogues on glucose utilization is equal regardless of the different receptor affinities of the insulins.

Monomeric insulins obtained by protein engineering and their medical implications.

The use of insulin as an injected therapeutic agent for the treatment of diabetes has been one of the outstanding successes of modern medicine. The therapy has, however, had its associated problems, not least because injection of insulin does not lead to normal diurnal concentrations of insulin in the blood. This is especially true at meal times when absorption from subcutaneous tissue is too slow to mimic the normal rapid increments of insulin in the blood. In the neutral solutions used for therapy, insulin is mostly assembled as zinc-containing hexamers and this self-association, which under normal physiological circumstances functions to facilitate proinsulin transport, conversion and intracellular storage, may limit the rate of absorption. We now report that it is possible, by single amino-acid substitutions, to make insulins which are essentially monomeric at pharmaceutical concentrations (0.6 mM) and which have largely preserved their biological activity. These monomeric insulins are absorbed two to three times faster after subcutaneous injection than the present rapid-acting insulins. They are therefore capable of giving diabetic patients a more physiological plasma insulin profile at the time of meal consumption.

Soluble, prolonged-acting insulin derivatives. I. Degree of protraction and crystallizability of insulins substituted in the termini of the B-chain.

Hydrophilic insulins, more positively charged than human insulin at neutral pH, have been prepared by substitution with basic amino acids at the termini of the B-chain and by blocking the C-terminal carboxyl group of the B-chain. The isoelectric pH of the insulin is thereby moved from 5.4 towards physiological levels. Slightly acid solutions of derivatives, in which charge has been added in the C-terminus of the B-chain, have a prolonged action in vivo, in particular if the carboxyl group is blocked. It is found that the prolonged-acting hydrophilic insulins crystallize instantly when the pH is adjusted to 7. The prolonged action is ascribed to this readiness to crystallization combined with a low solubility, which may be further decreased by increased concentration of zinc ions. Hydrophobic insulins have a prolonged action independent of the site of substitution even if the derivative is soluble at physiological pH. Some derivatives were prepared from porcine insulin by tryptic transpeptidation. N-terminal B-chain substituted insulins were prepared by alkylation of a biosynthetic single-chain insulin precursor, followed by tryptic transpeptidation rendering the double chain insulin derivative. The observed blood glucose lowering in the rabbits implies that neither N- nor C-terminal B-chain substitution results in substantial deterioration of biological potency. An index for the degree of protraction based on the blood glucose data is used to compare the insulins.

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)

Pharmacokinetics of theophylline and 3-methylxanthine in guinea pigs. I. Single dose administration.

Aminophylline in the doses 6.31 . 10(-5) mol kg-1 and 2.37 . 10(-4) mol kg-1 (corresponding to 13.8 and 52.0 mg kg-1 respectively) and 3-methylxanthine 2.37 . 10(-4) mol kg-1 (39.4 mg kg-1) was administered to guinea pigs by intravenous bolus injection, n = 7, 10, and 6, respectively. The shape of semilogarithmic plots of all measured plasma theophylline concentrations versus time was compatible with the use of an open 2-compartment pharmacokinetic model assuming first-order distribution and elimination processes, but in the comparison of the mean values of the pharmacokinetic parameters obtained after the administration of the low and the high theophylline dose, statistical analysis by Student's t-test showed beta and V1 to be significantly altered (low dose: beta = 0.00338 min.-1, V1 = 392 ml kg-a; high dose: beta = 0.00198 min.-1, V1 = 528 ml kg-1; P less than 0.05). Following the administration of 3-methylxanthine, the observed plasma concentration time course of this pharmacologically active theophylline metabolite could be adequately described be means of the 2-compartment open model. The administered 3-methylxanthine was eliminated unchanged with a first-order rate constant ten times larger than the total elimination rate constant of theophylline itself, the latter being observed after the administration of the equimolar dose of aminophylline (kel 3MX = 0.029 min.-1, kel theophylline = 0.00293 min.-1). Clearance was calculated to 14.4 ml kg-1 min.-1 for 3-methylxanthine and 1.50 ml kg-1 min.-1 for theophylline. When aminophylline 2.37 . 10(-4) mol kg-1 had been administered, 3-methylxanthine was renally eliminated at a constant rate for the first hours after the injection, but it did not only constitute a few per cent of the theophylline-derived urine products.

Pharmacokinetics of terbutaline and theophylline in guinea pigs when administered simultaneously.

Simultaneous administration of terbutaline and theophylline to guinea pigs did not cause significant alterations of the pharmacokinetic properties of any of the drugs. Terbutaline sulphate 24 microgram kg-1 and aminophylline 52 mg kg-1 (7.42 . 10(-8) and 2.37 . 10(-4) mol kg-1 respectively) were given by a bolus intravenous injection, producing plasma concentrations in the range 0-15 ng terbutaline sulphate per ml (0-46 nanomol 1(-1)) and 10-85 microgram theophylline per ml (50-429 mumol 1(-1)). Pharmacokinetic analyses of time courses of plasma concentrations of intact drugs and investigations of tissue distribution 1 hour after the administration were performed. The results showed a weak, statistically insignificant trend of the peripheral compartment of the 2-compartment model to sequester a larger fraction of the drugs when these were administered simultaneously.

Pharmacokinetics of theophylline and 3-methylxanthine in guinea pigs. II. Multiple dose administration.

Following intravenous bolus injection of theophylline 2.37 . 10(-) mol kg-1 to guinea pigs (administered as aminophylline 52.0 mg kg-1), the elimination of the dose was more rapid in a group of 8 guinea pigs that had received theophylline 2.02 . 10(-4) mol kg-1 (aminophylline 44.3 mg kg-1 (aminophylline 44.3 mg kg-1) intraperitoneally twice daily for 12 days prior to the experiment than in a group of 10 non-pretreated guinea pigs of the same age. The difference was statistically significant in Student's t-test (P less than 0.02). The mean values in the group of pretreated animals were: kel 0.00457 min.-1, beta 0.00296 min.-1, clearance 2.04 ml kg-1 min.-1, Vd beta 693 ml kg-1. By way of comparison, the values obtained in non-pretreated guinea pigs were: kel 0.00293 min.-1 beta 0.00198 min.-1, clearance 1.50 ml kg-1 min.-1, Vd beta 757 ml kg-1. This result suggests enzyme induction to occur. The pharmacologically active theophylline metabolite 3-methylxanthine did not accumulate in the plasma during the long-term theophylline administration. The general plasma concentration level was 0-1.8 . 10(-8) mol ml-1 (0-3 microgram ml-1). In 5 per cent of the samples were detected concentrations in the range 1.8 . 10(-8) mol ml-1 (3-12 microgram ml-1), but the time of occurrence was sporadic.