Molecular engineering of safe and efficacious oral basal insulin.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.

Elucidating the Mechanism of Absorption of Fast-Acting Insulin Aspart: The Role of Niacinamide.

PURPOSE: Fast-acting insulin aspart (faster aspart) is a novel formulation of insulin aspart containing two additional excipients: niacinamide, to increase early absorption, and L-arginine, to optimize stability. The aim of this study was to evaluate the impact of niacinamide on insulin aspart absorption and to investigate the mechanism of action underlying the accelerated absorption. METHODS: The impact of niacinamide was assessed in pharmacokinetic analyses in pigs and humans, small angle X-ray scattering experiments, trans-endothelial transport assays, vascular tension measurements, and subcutaneous blood flow imaging. RESULTS: Niacinamide increased the rate of early insulin aspart absorption in pigs, and pharmacokinetic modelling revealed this effect to be most pronounced up to ~30-40 min after injection in humans. Niacinamide increased the relative monomer fraction of insulin aspart by ~35%, and the apparent permeability of insulin aspart across an endothelial cell barrier by ~27%. Niacinamide also induced a concentration-dependent vasorelaxation of porcine arteries, and increased skin perfusion in pigs. CONCLUSION: Niacinamide mediates the acceleration of initial insulin aspart absorption, and the mechanism of action appears to be multifaceted. Niacinamide increases the initial abundance of insulin aspart monomers and transport of insulin aspart after subcutaneous administration, and also mediates a transient, local vasodilatory effect.

Investigation of the Physico-Chemical Properties that Enable Co-Formulation of Basal Insulin Degludec with Fast-Acting Insulin Aspart.

PURPOSE: To study the self-association states of insulin degludec and insulin aspart alone and combined in pharmaceutical formulation and under conditions simulating the subcutaneous depot. METHODS: Formulations were made of 0.6 mM degludec at 3 and 5 Zn/6 insulin monomers, and 0.6 mM aspart (2 Zn/6 insulin monomers). Self-association was assessed using size-exclusion chromatography (SEC) monitored by UV and orthogonal reverse-phase chromatography. RESULTS: Simulating pharmaceutical formulation, degludec eluted as dihexamers, whereas aspart eluted as hexamers and monomers. Combining degludec at low zinc with aspart increased dihexamer content, indicating hybrid hexamer formation. At high zinc concentration, however, there was no evidence of this. Simulating the subcutaneous depot by removing preservative, degludec eluted as multihexamers and aspart as monomers. Aspart was incorporated into the multihexamer structures when combined with degludec at low zinc, but there was no such interaction with high-zinc degludec. SEC using progressively diluted concentrations of phenol and m-cresol showed that dissociation of aspart into monomers occurs before the formation of degludec multihexamers. CONCLUSION: Insulins degludec and aspart can be combined without forming hybrid hexamers, but this combinability is dependent on zinc and preservative concentration, and requires that degludec is fully dihexameric before addition of aspart.

Insulin analog with additional disulfide bond has increased stability and preserved activity.

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

Comparison of the pharmacokinetics of three concentrations of insulin aspart during continuous subcutaneous insulin infusion (CSII) in a pig model.

OBJECTIVES: The aim of the study was to investigate the pharmacokinetic properties of insulin aspart (IAsp) in three different concentrations given as a continuous subcutaneous insulin infusion (CSII). METHODS: A randomized cross-over study was performed in pigs, where IAsp U200, U100 or U20 was given for 8 h with the same total dose. Six pigs were included and blood was sampled during the CSII and 3 h after. KEY FINDINGS: The half-life (t(1/2) ) was 24.3 (range 17.3-41.3), 28.8 (range 19.6-54.3) and 23.6 (range 17.4-36.8) min for U200, U100 and U20, respectively. The area under the curve per dose (AUC/D) was determined to be 51.2 ± 19.5, 52.3 ± 12.5 and 51.6 ± 6.7 pm × min/kg for U200, U100 and U20, respectively. The steady state plasma concentration (C(ss) ) was 57.5 ± 27.1, 54.3 ± 10.3 and 55.1 ± 8.0 pm (mean ± SD) for U200, U100 and U20, respectively. Time to steady state (T(ss) ) was 110 ± 36, 98 ± 48 and 90 ± 27 min for U200, U100 and U20, respectively. CONCLUSIONS: In conclusion, no significant difference was found in t(1/2) , AUC/D, C(ss) or T(ss) between the three IAsp concentrations when given at a basal rate in CSII.

Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin.

PURPOSE: Basal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements. METHODS: Self-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection. RESULTS: With depletion of phenol, insulin degludec and another fatty diacid-insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T(3)R(3) conformation in presence of phenol and zinc, changing to T(6) with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly. CONCLUSIONS: Insulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.

Identification of anchor points for chemical modification of a small cysteine-rich protein by using a cysteine scan.

Chemical modifications of proteins are increasingly important in the development of protein drugs with fine-tuned properties. Regioselective modification, such as the chemoselective alkylation of an unpaired cysteine residue, is a prerequisite for obtaining homogenous protein products. The introduction of an unpaired Cys into the Cys-rich protein, insulin, was investigated by using a Cys scan. This was challenging as the introduced Cys could interfere with insulin's three existing disulfide bonds. However, eight insulin precursors were expressed in Saccharomyces cerevisiae with good yields. Although extensive post-translational modifications of the unpaired Cys were observed, the majority could be removed by selective reduction. An example Cys(7) insulin analogue was modified with a PEGylated maleimide moiety. The new variant was active in in vitro and in vivo models. Our results show that even small Cys-rich proteins can be expressed with additional unpaired Cys in meaningful yields and further chemically modified, while maintaining their biological activity.

Structural and biological properties of the Drosophila insulin-like peptide 5 show evolutionary conservation.

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ß-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ß-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.

Backbone cyclic insulin.

Backbone cyclic insulin was designed and prepared by reverse proteolysis in partial organic solvent of a single-chain precursor expressed in yeast. The precursor contains two loops to bridge the two chains of native insulin. The cyclisation method uses Achromobacter lyticus protease and should be generally applicable to proteins with C-terminal lysine and proximal N-terminal. The presence of the ring-closing bond and the native insulin disulfide patterns were documented by LC-MS peptide maps. The cyclic insulin was shown to be inert towards degradation by CPY, but was somewhat labile towards chymotrypsin. Intravenous administration of the cyclic insulin to Wistar rats showed the compounds to be equipotent to HI despite much lower insulin receptor affinity.

Evaluation of different methods for assessment of insulin sensitivity in Gottingen minipigs: introduction of a new, simpler method.

The use of animal models in diabetes research requires reliable tests for evaluation of insulin sensitivity and beta-cell function. Minipigs are being increasingly used in metabolic research, and the aim of this study was to compare different tests and indexes for evaluation of insulin sensitivity and beta-cell function in Göttingen minipigs. Hyperinsulinemic, isoglycemic clamp, intravenous (IVGTT) and oral glucose tolerance tests (OGTT), and a modified insulin tolerance test were performed in minipigs fed either low- or high-energy diet. Furthermore, the reproducibility of IVGTT-derived parameters was assessed. Previously described insulin sensitivity indexes [steady-state glucose infusion rate/glucose concentration/insulin concentration from clamp (M/G/I); oral glucose insulin sensitivity (OGIS) and ISI(comp) from OGTT; S(I) from minimal model analysis of IVGTT; and quantitative insulin sensitivity check index from fasting values] were calculated together with an insulin sensitivity index from the modified insulin tolerance test (ISI(ITT)) and a new simple index (S2) derived from the first 30 min of the IVGTT. beta-Cell function was assessed from the IVGTT and the OGTT. Reproducibility of the IVGTT-derived parameters was calculated as median intraindividual coefficient of variation (CV%).M/G/I correlated significantly only with S2 (P < 0.05, r = 0.54). S2 furthermore correlated with S(I) (P < 0.001, r = 0.81), ISI(ITT) (P < 0.001, r = 0.57), and the two indexes from OGTT, ISI(comp) (P < 0.001, r = 0.78) and OGIS (p < 0.05, r = 0.48). No correlation was found between beta-cell function indexes from OGTT and IVGTT. The median CV% of the new S2 index was 13. In conclusion, the new simple index of insulin sensitivity, S2, was revealed to be useful for evaluation of insulin sensitivity in pigs.

A novel high-affinity peptide antagonist to the insulin receptor.

In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.

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.

Sensory nerve desensitization by resiniferatoxin improves glucose tolerance and increases insulin secretion in Zucker Diabetic Fatty rats and is associated with reduced plasma activity of dipeptidyl peptidase IV.

Sensory nerve desensitization by capsaicin has been shown to improve the diabetic condition in Zucker Diabetic Fatty rats. However, administration of capsaicin to adult rats is associated with an increased mortality. Therefore, in this experiment, we examined the influence of resiniferatoxin, a tolerable analogue of capsaicin suitable for in vivo use, on the diabetic condition of Zucker Diabetic Fatty rats. A single subcutaneous injection of resiniferatoxin (0.01 mg/kg) to these rats was tolerable, with no mortality. When administered to early diabetic rats at 15 weeks of age, the further deterioration of glucose homeostasis was prevented by resiniferatoxin. Further, when administered to overtly diabetic rats at 19 weeks of age, resiniferatoxin markedly improved glucose tolerance at two weeks after administration and this was accompanied by an increased insulin response to oral glucose as well as a reduction in the plasma levels of dipeptidyl peptidase IV. Therefore, resiniferatoxin is a safe alternative to capsaicin for further investigations of the role of the sensory nerves in experimental diabetes.

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.

Valine pyrrolidide preserves intact glucose-dependent insulinotropic peptide and improves abnormal glucose tolerance in minipigs with reduced beta-cell mass.

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important in blood glucose regulation. However, both incretin hormones are rapidly degraded by the enzyme dipeptidyl peptidase IV (DPPIV). The concept of DPPIV inhibition as a treatment for type 2 diabetes was evaluated in a new large animal model of insulin-deficient diabetes and reduced beta-cell mass, the nicotinamide (NIA) (67 mg/kg) and streptozotocin (STZ) (125 mg/kg)-treated minipig, using the DPPIV inhibitor, valine pyrrolidide (VP) (50 mg/kg). VP did not significantly affect levels of intact GLP-1 but increased levels of intact GIP (from 4543 +/- 1880 to 9208 +/- 3267 pM x min; P <.01), thus improving glucose tolerance (area under the curve [AUC] for glucose reduced from 1904 +/- 480 to 1582 +/- 353 mM x min; P =.05). VP did not increase insulin levels during the oral glucose tolerance test (OGTT) but increased the insulinogenic index in normal animals (from 83 +/- 42 to 192 +/- 108; P <.05), but not after NIA + STZ, possibly because of less residual insulin secretory capacity in these animals. GIP seems to contribute to the antihyperglycemic effect of VP in this model; however, additional mechanisms for the effect of DPPIV inhibition cannot be excluded. The authors conclude that DPPIV inhibitors may be useful to treat type 2 diabetes, even when this is due to reduced beta-cell mass.

GLP-1 derivative liraglutide in rats with beta-cell deficiencies: influence of metabolic state on beta-cell mass dynamics.

(1) Liraglutide is a long-acting GLP-1 derivative, designed for once daily administration in type II diabetic patients. To investigate the effects of liraglutide on glycemic control and beta-cell mass in rat models of beta-cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in 60% pancreatectomized rats. (2) When liraglutide was dosed s.c. at 150 microg kg-1 b.i.d. for 6 weeks in ZDF rats 6-8 weeks of age at study start, diabetes development was markedly attenuated. Blood glucose was approximately 12 mm lower compared to vehicle (P<0.0002), and plasma insulin was 2-3-fold higher during a normal 24-h feeding period (P<0.001). Judged by pair feeding, approximately 53% of the antihyperglycemic effect observed on 24-h glucose profiles was mediated by a reduction in food intake, which persisted throughout the study and averaged 16% (P<0.02). (3) Histological analyses revealed that beta-cell mass and proliferation were significantly lower in prediabetic animals still normoglycemic after 2 weeks treatment compared to vehicle-treated animals that had begun to develop diabetes. When the treatment period was 6 weeks, the liraglutide-treated animals were no longer completely normoglycemic and the beta-cell mass was significantly increased compared to overtly diabetic vehicle-treated animals, while beta-cell proliferation was unaffected. (4) In the experiments with 60% pancreatectomized rats, 8 days treatment with liraglutide resulted in a significantly lower glucose excursion in response to oral glucose compared to vehicle treatment. Again, part of the antihyperglycemic effect was due to reduced food intake. No effect of liraglutide on beta-cell mass was observed in these virtually normoglycemic animals. (5) In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of beta-cell deficiencies, and the in vivo effect of liraglutide on beta-cell mass may in part depend on the metabolic state of the animals.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks.

Insulin is thought to elicit its effects by crosslinking the two extracellular alpha-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

NN2211: a long-acting glucagon-like peptide-1 derivative with anti-diabetic effects in glucose-intolerant pigs.

Glucagon-like peptide-1 (GLP-1) is an effective anti-diabetic agent, but its metabolic instability makes it therapeutically unsuitable. This study investigated the pharmacodynamics of a long-acting GLP-1 derivative (NN2211: (Arg(34)Lys(26)-(N- epsilon -(gamma-Glu(N-alpha-hexadecanoyl)))-GLP-1(7-37)), after acute and chronic treatment in hyperglycaemic minipigs. During hyperglycaemic glucose clamps, NN2211 (2 micrograms kg(-1) i.v.) treated pigs required more (P < 0.005) glucose than control animals (5.8 +/- 2.1 vs. 2.9 +/- 1.8 mg kg(-1) min(-1)). Insulin excursions were higher (P < 0.01) after NN2211 (15,367 +/- 5,438 vs. 9,014 +/- 2,952 pmol l(-1) min), and glucagon levels were suppressed (P < 0.05). Once-daily injections of NN2211 (3.3 micrograms kg(-1) s.c.) reduced the glucose excursion during an oral glucose tolerance test, to 59 +/- 15% of pre-treatment values by 4 weeks (P < 0.05), without measurable changes in insulin responses. Fructosamine concentrations were unaltered by vehicle, but decreased (from 366 +/- 187 to 302 +/- 114 micromol l(-1), P = 0.14) after 4 weeks of NN2211. Gastric emptying was reduced (P < 0.05) by NN2211. NN2211 acutely increases glucose utilization during a hyperglycaemic glucose clamp and chronic treatment results in better daily metabolic control. Therefore, NN2211, a GLP-1 derivative that can be administered once daily, holds promise as a new anti-diabetic drug with a minimal risk of hypoglycaemia.

Chamber-dependent expression of brain natriuretic peptide and its mRNA in normal and diabetic pig heart.

Brain natriuretic peptide (BNP) is produced in cardiac myocytes, and increased secretion is closely associated with cardiac dysfunction. However, several fundamental aspects of BNP expression in the myocardium have not yet been resolved. In the present study, we report the presence of a precursor BNP mRNA transcript and a mature BNP mRNA transcript in normal porcine hearts. In normal pigs, the amount of precursor BNP mRNA was similar in atrial and ventricular myocardium, whereas the mature BNP transcript was 10- to 50-fold more abundant in atrial than in ventricular myocardium. Quantitation of proBNP in normal porcine hearts by radioimmunoassay disclosed abundant proBNP in the atria, whereas proBNP was undetectable in the ventricles. Laser confocal microscopy revealed proBNP in secretory granules of atrial but not in the ventricular myocardium of normal pigs. Mild streptozotocin-induced diabetes doubled the expression of BNP mRNA in porcine atrial myocardium (P=0.03), but was without effect on BNP mRNA in the ventricular myocardium. The data suggest that BNP mRNA processing and proBNP storage differ between the atrial and ventricular myocardium. The results also imply that diabetes increases cardiac BNP expression in a chamber-dependent manner.