Anders R. Sørensen

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, 0.6 U kg−1, 0.8 U kg−1) in random order on five separate days. Intranasal insulin was absorbed in a dose‐dependent manner with a mean plasma insulin peak 23 ± 7 (± SE) min after administration. Mean plasma glucose nadir was seen after 44 ± 6 min, 20 min later than following intravenous injection. Furthermore, intranasal administration of insulin resulted in a faster time‐course of absorption than subcutaneous injection, with significantly reduced intersubject variation (p < 0.001). Bioavailability for the nasal formulation was 8.3% relative to an intravenous bolus injection when plasma insulin was corrected for endogenous insulin production estimated by C‐peptide. A dose‐dependent suppression of C‐peptide and stimulation of glucagon secretion occurred after intranasal administration of insulin. Nasal irritation from spraying was absent or slight.

Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding†

Conjointly, the solvent-exposed residues of the central alpha-helix of the B chain form a well-defined ridge, which is flanked and partly overlapped by the two described insulin receptor binding surfaces on either side of the insulin molecule. To evaluate the importance of this interface in insulin receptor binding, we developed a new powerful method that allows us to introduce all the naturally occurring amino acids into a given position and subsequently determine the receptor binding affinities of the resulting insulin analogues. The total amino acid scanning mutagenesis was performed at positions B9, B10, B12, B13, B16, and B17, and the vast majority of the insulin analogue precursors were expressed and secreted in amounts close to that of the wild-type (human insulin) precursor. The analogue binding data revealed that positions B12 and B16 were the two positions most affected by the amino acid substitutions. Interestingly, the receptor binding affinities of the B13 analogues were also markedly affected by the amino acid substitutions, suggesting that GluB13 indeed is a part of insulins binding surface. The B10 library screen generated analogues covering a wide range of (20-340%) of relative binding affinities, and the results indicated that a structural stabilization of the central alpha-helix and thereby a more rigid presentation of the binding epitope at the insulin receptor is important for receptor recognition. In conclusion, systematic amino acid scanning mutagenesis allowed us to confirm the importance of the B chain alpha-helix as a central recognition element serving as a linker of a continual binding surface.

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 < 0.05; 197 ± 19 pM, P < 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.

Intranasal administration of insulin to rabbits using glycofurol as an absorption promoter

The bioavailability of insulin from a nasal formulation with 5% glycofurol (GF) was studied in vivo in rabbits. A pronounced decrease in the plasma glucose level, comparable to the response for Novolin® Nasal, was observed. A nadir after 30 min was observed where the plasma glucose was 59% of the initial value. The potential for local side effect from 5% GF was found to be very low in the frog-palate model, where the mucociliary clearance rate was only reduced 9%.

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.

Engineering of Insulin Receptor Isoform-Selective Insulin Analogues

Background The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. Methodology/Principal Findings Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. Conclusions/Significance We have discovered a new class of IR isoform-selective insulin analogues with 2–4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.

Receptor-isoform-selective insulin analogues give tissue-preferential effects.

The relative expression patterns of the two IR (insulin receptor) isoforms, +/- exon 11 (IR-B/IR-A respectively), are tissue-dependent. Therefore we have developed insulin analogues with different binding affinities for the two isoforms to test whether tissue-preferential biological effects can be attained. In rats and mice, IR-B is the most prominent isoform in the liver (> 95%) and fat (> 90%), whereas in muscles IR-A is the dominant isoform (> 95%). As a consequence, the insulin analogue INS-A, which has a higher relative affinity for human IR-A, had a higher relative potency [compared with HI (human insulin)] for glycogen synthesis in rat muscle strips (26%) than for glycogen accumulation in rat hepatocytes (5%) and for lipogenesis in rat adipocytes (4%). In contrast, the INS-B analogue, which has an increased affinity for human IR-B, had higher relative potencies (compared with HI) for inducing glycogen accumulation (75%) and lipogenesis (130%) than for affecting muscle (45%). For the same blood-glucose-lowering effect upon acute intravenous dosing of mice, INS-B gave a significantly higher degree of IR phosphorylation in liver than HI. These in vitro and in vivo results indicate that insulin analogues with IR-isoform-preferential binding affinity are able to elicit tissue-selective biological responses, depending on IR-A/IR-B expression.

Systematic Evaluation of the Metabolic to Mitogenic Potency Ratio for B10-Substituted Insulin Analogues

Background Insulin analogues comprising acidic amino acid substitutions at position B10 have previously been shown to display increased mitogenic potencies compared to human insulin and the underlying molecular mechanisms have been subject to much scrutiny and debate. However, B10 is still an attractive position for amino acid substitutions given its important role in hexamer formation. The aim of this study was to investigate the relationships between the receptor binding properties as well as the metabolic and mitogenic potencies of a series of insulin analogues with different amino acid substitutions at position B10 and to identify a B10-substituted insulin analogue without an increased mitogenic to metabolic potency ratio. Methodology/Principal Findings A panel of ten singly-substituted B10 insulin analogues with different amino acid side chain characteristics were prepared and insulin receptor (both isoforms) and IGF-I receptor binding affinities using purified receptors, insulin receptor dissociation rates using BHK cells over-expressing the human insulin receptor, metabolic potencies by lipogenesis in isolated rat adipocytes, and mitogenic potencies using two different cell types predominantly expressing either the insulin or the IGF-I receptor were systematically investigated. Only analogues B10D and B10E with significantly increased insulin and IGF-I receptor affinities as well as decreased insulin receptor dissociation rates displayed enhanced mitogenic potencies in both cell types employed. For the remaining analogues with less pronounced changes in receptor affinities and insulin receptor dissociation rates, no apparent correlation between insulin receptor occupancy time and mitogenicity was observed. Conclusions/Significance Several B10-substituted insulin analogues devoid of disproportionate increases in mitogenic compared to metabolic potencies were identified. In the present study, receptor binding affinity rather than insulin receptor off-rate appears to be the major determinant of both metabolic and mitogenic potency. Our results also suggest that the increased mitogenic potency is attributable to both insulin and IGF-I receptor activation.