Aage Vølund

Monomeric Insulins and Their Experimental and Clinical Implications

Due to the inherent pharmacokinetic properties of available insulins, normoglycemia is rarely, if ever, achieved in insulin-dependent diabetic patients without compromising their quality of life. Subcutaneous insulin absorption is influenced by many factors, among which the associated state of insulin (hexameric) in pharmaceutical formulation may be of importance. This review describes the development of a series of human insulin analogues with reduced tendency to selfassociation that, because of more rapid absorption, are better suited to meal-related therapy. DNA technology has made it possible to prepare insulins that remain dimeric or even monomeric at high concentration by introducing one or a few amino acid substitutions into human insulin. These analogues were characterized and used for elucidating the mechanisms involved in subcutaneous absorption and were investigated in preliminary clinical studies. Their relative receptor binding and in vitro potency (free-fat cell assay), ranging from 0.05 to 600% relative to human insulin, were strongly correlated (r = 0.97). In vivo, most of the analogues exhibited ∼100% activity, explainable by a dominating receptor-mediated clearance. This was confirmed by clamp studies in which correlation between receptor binding and clearance was observed. Thus, an analogue with reduced binding and clearance gives higher circulating concentrations, counterbalancing the reduced potency at the cellular level. Absorption studies in pigs revealed a strong inverse correlation (r = 0.96) between the rate of subcutaneous absorption and the mean association state of the insulin analogues. These studies also demonstrated that monomeric insulins were absorbed three times faster than human insulin. In healthy subjects, rates of disappearance from subcutis were two to three times faster for dimeric and monomeric analogues than for human insulin. Concomitantly, a more rapid rise in plasma insulin concentration and an earlier hypoglycemic response with the analogues were observed. The monomeric insulin had no lag phase and followed a monoexponential course throughout the absorption process. In contrast, two phases in rate of absorption were identified for the dimer and three for the normal hexameric human insulin. The initial lag phase and the subsequent accelerated absorption of soluble insulin can now be explained by the associated state of native insulin in pharmaceutical formulation and its progressive dissociation into smaller units during the absorption process. In the light of these results, the effects of insulin concentration, injected volume, temperature, and massage on the absorption process are now also understood. When given to diabetic patients immediately before a standard meal, the monomeric analogue lowered postprandial glucose excursions by ∼50% when compared with human insulin given at the same time. Subsequently, it was shown that three monomeric to dimeric analogues injected separately just before a meal gave glycemic control at least comparable to that of human insulin administered 30 min earlier. Lower plasma glucose concentrations (∼50%) were observed with the analogues from 1.5 h postprandially. Thus, monomeric analogues are faster in onset of action, can be given with the meal without losing glycemic control, and have the potential to minimize late hypoglycemia. Therefore, the development of these novel insulins represents a major step in the evolution of insulin preparations to subserve meal-related insulin requirements.

The mechanism of protraction of insulin detemir, a long-acting, acylated analog of human insulin.

AbstractPurpose. 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.

Reduced Incretin Effect in Type 2 Diabetes Cause or Consequence of the Diabetic State

We aimed to investigate whether the reduced incretin effect observed in patients with type 2 diabetes is a primary event in the pathogenesis of type 2 diabetes or a consequence of the diabetic state. Eight patients with chronic pancreatitis and secondary diabetes (A1C mean [range] of 6.9% [6.2–8.0]), eight patients with chronic pancreatitis and normal glucose tolerance (NGT; 5.3 [4.9–5.7]), eight patients with type 2 diabetes (6.9 [6.2–8.0]); and eight healthy subjects (5.5 [5.1–5.8]) were studied. Blood was sampled over 4 h on 2 separate days after a 50-g oral glucose load and an isoglycemic intravenous glucose infusion, respectively. The incretin effect (100% × [β-cell secretory response to oral glucose tolerance test − intravenous β-cell secretory response]/β-cell secretory response to oral glucose tolerance test) was significantly (P < 0.05) reduced (means ± SE) in patients with chronic pancreatitis and secondary diabetes (31 ± 4%) compared with patients with chronic pancreatitis and NGT (68 ± 3) and healthy subjects (60 ± 4), respectively. In the type 2 diabetes group, the incretin effect amounted to 36 ± 6%, significantly (P < 0.05) lower than in chronic pancreatitis patients with NGT and in healthy subjects, respectively. These results suggest that the reduced incretin effect is not a primary event in the development of type 2 diabetes, but rather a consequence of the diabetic state.

Reduced Sample Number for Calculation of Insulin Sensitivity and Glucose Effectiveness From the Minimal Model: Suitability for Use in Population Studies

The FSIGT has been extensively applied to the minimal model of glucose kinetics to obtain noninvasive measures of SI. The protocol has been modified by the addition of a bolus tolbutamide or insulin injection 20 min after glucose. Although the modified protocol has improved the S, estimate, the method still requires a relatively large number of samples (n = 30). To reduce the total number of samples, we choose a sample schedule that minimizes the variance of the parameter estimates and the error in reconstructing the plasma insulin profile. With data from 10 subjects (BMI 30 ± 7 kg/m2; SI 0.9–10.2 × 10−4 min−1 · μU−1 · ml−1), a schedule consisting of 12 samples (0, 2, 4, 8,19, 22, 30, 40, 50, 70, 90, and 180 min) was obtained. Estimates of SI obtained from the reduced sampling schedule were then compared with those obtained with the full sampling schedule. In all 10 individuals, the SI estimates were almost identical. A second, much larger data base consisting of 118 modified FSIGTs performed in 87 subjects (67 men, 20 women; BMI from 19.6 to 40 kg/m2 for men and 26.7 to 52.5 for women; SI, from 0.35 to 14.1 × 10−4 min−1 · μU−1 · ml−1) was then used to independently assess the efficacy of the reduced sampling protocol. For this data base, the correlation between SI, which was calculated from the full versus the reduced sampling schedule, was 0.95. The mean relative deviation was –1.5% (not significantly different from zero), and the SD of the relative deviation was 20.2%. Relative deviation was defined as the percentage of difference between SI calculated from the full sample protocol and SI calculated from the reduced sample protocol. Thus, the reduced sampling schedule provides an unbiased estimate of a populations SI, and an individual estimate is generally within 20% of that obtained with the full sampling schedule. A similar analysis of SG showed that this parameter was equally well determined from the reduced compared with the full sample schedule.

Insulin analogs with improved pharmacokinetic profiles

The aim of insulin replacement therapy is to normalize blood glucose in order to reduce the complications of diabetes. The pharmacokinetics of the traditional insulin preparations, however, do not match the profiles of physiological insulin secretion. The introduction of the rDNA technology 20 years ago opened new ways to create insulin analogs with altered properties. Fast-acting analogs are based on the idea that an insulin with less tendency to self-association than human insulin would be more readily absorbed into the systemic circulation. Protracted-acting analogs have been created to mimic the slow, steady rate of insulin secretion in the fasting state. The present paper provides a historical review of the efforts to change the physicochemical and pharmacological properties of insulin in order to improve insulin therapy. The available clinical studies of the new insulins are surveyed and show, together with modeling results, that new strategies for optimal basal-bolus treatment are required for utilization of the new fast-acting analogs.

Decrease in nickel sensitization in a Danish schoolgirl population with ears pierced after implementation of a nickel‐exposure regulation

Summary Background To reduce the skin nickel exposure of the population the Danish Ministry of Environment issued a regulation that was implemented in 1992, and the European Union countries have recently adopted an expanded regulation.

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 (α-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 > porcine > human insulin. The hydrolytic cleavage of the peptide backbone takes place only in preparations containing rhombohedral crystals in addition to free zinc ions.

Subcutaneous Insulin Absorption Explained by Insulin's Physicochemical Properties: Evidence From Absorption Studies of Soluble Human Insulin and Insulin Analogues in Humans

Objective To study the influence of molecular aggregation on rates of subcutaneous insulin absorption and to attempt to elucidate the mechanism of absorption of conventional soluble human insulin in humans. Research Design and Methods Seven healthy male volunteers aged 22-43 yr and not receiving any drugs comprised the study. This study consisted of a single-blind randomized comparison of equimolar dosages of 125I-labeled forms of soluble hexameric 2 Zn2+ human insulin and human insulin analogues with differing association states at pharmaceutical concentrations (AspB10, dimeric; AspB28, mixture of monomers and dimers; AspB9, GluB27, monomeric). After an overnight fast and a basal period of 1 h, 0.6 nmol/kg of either 125I-labeled human soluble insulin (Actrapid HM U-100) or 125I-labeled analogue was injected subcutaneously on 4 separate days 1 wk apart. Absorption was assessed by measurement of residual radioactivity at the injection site by external γ-counting. Results The mean ± SE initial fractional disappearance rates for the four preparations were 20.7 ± 1.9 (hexameric soluble human insulin), 44.4 ± 2.5 (dimeric analogue AspB10), 50.6 ± 3.9 (analogue AspB28), and 67.4 ± 7.4%/h (monomeric analogue AspB9, GluB27). Absorption of the dimeric analogue was significantly faster than that of hexameric human insulin (P < 0.001); absorption of monomeric insulin analogue AspB9, GluB27 was significantly faster than that of dimeric analogue AspB10 (P < 0.01). There was an inverse linear correlation between association state and the initial fractional disappearance rates (r = −0.98, P < 0.02). Analysis of the disappearance data on a log linear scale showed that only the monomeric analogue had a monoexponential course throughout. Two phases in the rates of absorption were identified for the dimer and three for hexameric human insulin. The fractional disappearance rates (%/h) calculated by log linear regression analysis were monomer 73.3 ± 6.8; dimer 44.4 ± 2.5 from 0 to 2 h and 68.9 ± 3.5 from 2.5 h onward; and hexameric insulin 20.7 ± 1.9 from 0 to 2 h, 45.6 ± 5.0 from 2.5 to 5 h, and 70.6 ± 6.3 from 5 h onward. Conclusions Association state is a major determinant of rates of absorption of insulin and insulin analogues. The lag phase and the subsequent increasing rate of subcutaneous soluble insulin absorption can be explained by the associated state of native insulin in pharmaceutical formulation and its progressive dissociation into smaller units during the absorption process.

The BIGTT Test A novel test for simultaneous measurement of pancreatic β-cell function, insulin sensitivity, and glucose tolerance

OBJECTIVE—Insulin resistance and impaired β-cell function are key elements in the pathogenesis of type 2 diabetes. We aimed to develop valid algorithms for estimation of the insulin sensitivity index (SI) and acute insulin response (AIR) derived from simple and cheap physiological measurements that could be used in large-scale metabolic, genetic, and epidemiological studies. RESEARCH DESIGN AND METHODS—For our purpose, data from an oral glucose tolerance test (OGTT) (18 samples during 240 min) and a tolbutamide-modified intravenous glucose tolerance test (IVGTT) (33 samples during 180 min) from 258 individuals with fasting plasma glucose <7 mmol/l and 2-h plasma glucose <7.8 mmol/l were used for model development and internal validation. Data from an additional 28 individuals were used for external validation. Bergman’s minimal model was used to calculate SI, and the trapezoidal method was used to calculate AIR0–8 min. Multiple linear regression was applied to derive predictive equations of log(SI) and log(AIR0–8 min) using data on sex, BMI, plasma glucose, and serum insulin levels obtained during the OGTT. RESULTS—We demonstrate that it is possible to obtain estimates of SI (BIGTT-SI) and AIR (BIGTT-AIR) that are highly correlated to IVGTT-derived values of SI (R2 = 0.77) and AIR (R2 = 0.54). In the two validation datasets we obtained similar results. CONCLUSIONS—Data from OGTTs can provide accurate measures of insulin sensitivity and β-cell function, which can be used in large scale metabolic, genetic, and epidemiological studies.

Calculated Pattern of Intraportal Insulin Appearance Without Independent Assessment of C-Peptide Kinetics

Prehepatic (β-cell insulin release can be calculated with C-peptide measurements, but this requires independent determination of kinetics of C-peptide disappearance from plasma. We introduce an approach by which a prehepatic insulin release pattern is calculated from plasma insulin and C-peptide, without separate C-peptide kihetic analysis. Human insulin and C-peptide were infused intraportally into conscious dogs (n = 11) at equimolar rates; endogenous insulin and C-peptide release were suppressed with somatostatin (0.8 μg · kg−1 · min−1). Insulin and C-peptide were infused at basal and equimolar rates (range 19–72 pmol/min in dogs), and the infusions were slowly increased, in stepwise fashion, to a maximum at 60 min (range 152—613 pmol/min) and subsequently renormalized at either 85 (n = 6) or 195 (n = 5) min. Plasma insulin and C-peptide measurements were described simultaneously by a composite model of insulin and C-peptide plasma kinetics, with the molar intraportal appearance rate due to the infusion R(t) as an unknown input for both insulin and C-peptide catabolism. The model assumes one-compartment disappearance kinetics for both peptides. Fitting the model to the measured insulin and C-peptide data, we were able to compute the insulin-appearance pattern accurately for every experiment; calculated and actual secretion rates were highly correlated (r = .93−.97) and had very similar temporal patterns. Also calculated were the fractional disappearance rates for human insulin (t1/2 = 6.9 min) and C-peptide (t1/2 = 14 min) in the dog, as well as the C-peptide distribution volume (12.3 ±0.5% body wt). Islet cell insulin secretion can be determined in vivo from insulin and C-peptide measurement without separate analysis of C-peptide kinetics. This method will be applicable for quantification of β-cell secretion for diagnostic and experimental purposes.