Bioavailability of insulin detemir and human insulin at the level of peripheral interstitial fluid in humans, assessed by open-flow microperfusion.

AIMS: To find an explanation for the lower potency of insulin detemir observed in humans compared with unmodified human insulin by investigating insulin detemir and human insulin concentrations directly at the level of peripheral insulin-sensitive tissues in humans in vivo. METHODS: Euglycaemic-hyperinsulinaemic clamp experiments were performed in healthy volunteers. Human insulin was administered i.v. at 6 pmol/kg/min and insulin detemir at 60 pmol/kg/min, achieving a comparable steady-state pharmacodynamic action. In addition, insulin detemir was doubled to 120 pmol/kg/min. Minimally invasive open-flow microperfusion (OFM) sampling methodology was combined with inulin calibration to quantify human insulin and insulin detemir in the interstitial fluid (ISF) of subcutaneous adipose and skeletal muscle tissue. RESULTS: The human insulin concentration in the ISF was ∼115 pmol/l or ∼30% of the serum concentration, whereas the insulin detemir concentration in the ISF was ∼680 pmol/l or ∼2% of the serum concentration. The molar insulin detemir interstitial concentration was five to six times higher than the human insulin interstitial concentration and metabolic clearance of insulin detemir from serum was substantially reduced compared with human insulin. CONCLUSIONS: OFM proved useful for target tissue measurements of human insulin and the analogue insulin detemir. Our tissue data confirm a highly effective retention of insulin detemir in the vascular compartment. The higher insulin detemir relative to human insulin tissue concentrations at comparable pharmacodynamics, however, indicate that the lower potency of insulin detemir in humans is attributable to a reduced effect in peripheral insulin-sensitive tissues and is consistent with the reduced in vitro receptor affinity.

Pharmacodynamics of the long-acting insulin analogues detemir and glargine following single-doses and under steady-state conditions in patients with type 1 diabetes.

AIM: The pharmacodynamic characteristics of the basal insulin analogues insulin detemir (IDet) and insulin glargine (IGlar) have been examined extensively via euglycaemic clamp studies. However, differences in clamp methodology and in the analysis of clamp data between trials have led to confusion over the duration of action of these two insulins. The aim of this study was to address these ambiguities in the literature by assessing the pharmacodynamic properties of IDet and IGlar over 30 h under single-dose and steady-state conditions using the definitions and procedures previously standardized by Heise and Pieber in 2007. METHODS: This was a single-centre, randomized, double-blind, glucose clamp trial involving 36 patients with type 1 diabetes. RESULTS: The mean duration of action of IDet was 25.9 h, compared with 19.8 h for IGlar after a single-dose (NS), and 23.3 h (IDet) versus 27.1 h (IGlar) at steady-state (p < 0.0001). IDet had a significantly higher area under the curve glucose infusion rate (AUCGIR ) than IGlar over 0-12 h after a single-dose (p = 0.0018). The steady-state AUCGIR for IDet was numerically higher than IGlar over 0-12 h (728 vs. 592 mg/kg, respectively; p = NS), but significantly lower than IGlar at 12-30 h (p = 0.0003). CONCLUSIONS: The duration of action of IDet is 23 h (range: 4.0-30.0), while that of IGlar is 27 h (range: 10.5-29.0) (95% CI: -8.1, 0.6). This suggests both insulins can be used for once-daily dosing, but individual needs must be considered.

A direct comparison of the pharmacodynamic properties of insulin detemir and neutral protamine lispro insulin in patients with type 1 diabetes.

AIMS: To compare the pharmacodynamic properties of insulin detemir (detemir) and neutral protamine lispro (NPL) insulin using a euglycaemic glucose clamp. METHODS: In a double-blind, crossover study, 30 patients with C-peptide negative type 1 diabetes were randomly assigned to a single dose (0.4 U/kg) of detemir and NPL. Plasma glucose (PG) was normalized with a variable insulin infusion and then decreased stepwise, followed by a euglycaemic clamp at 5.5 mmol/l over 32 h. Duration of action was defined as time from dosing until PG exceeded 8.3 mmol/l for at least 30 min. RESULTS: Duration of action was similar for detemir [23.0 (range 2.25-32) h] and NPL [22.0 (9.5-32) h], p = 0.55. Using glucose infusion rate (GIR) parameters, detemir showed a flatter pharmacodynamic profile versus NPL: area under the curve, AUC(GIR) ((0-32)) = 1326 vs. 1841 mg/kg, p < 0.01 (detemir vs. NPL, respectively); AUC(GIR) ((0-12)) = 784 vs. 1392 mg/kg, p < 0.05; AUC(GIR) ((12-32)) = 455 vs. 274 mg/kg, p = 0.051; GIR(late) (12-32)/GIR(early) (0-12) ratio = 0.33 vs. 0.04, p < 0.001. Detemir also showed a lower and later peak of action than NPL [GIR(max) 2.0 vs. 3.2 mg/kg/min, p < 0.01; T(max) 9.1 (95% confidence interval: 3.0-14.7) vs. 7.0 h (1.8-15.2)]. CONCLUSIONS: Detemir and NPL had similar duration of action of approximately 24 h in patients with type 1 diabetes. Compared with NPL, detemir had a flatter profile with a more even distribution of metabolic effect over 24 h.

A novel automated discontinuous venous blood monitoring system for ex vivo glucose determination in humans.

Intensive insulin therapy reduces mortality and morbidity in critically ill patients but imposes great demands on medical staff who must take frequent blood samples for the determination of glucose levels. A solution to this resourcing problem would be provided by an automated blood monitoring system. The aim of the present clinical study was to evaluate such a system comprising an automatic blood sampling unit linked to a glucose biosensor. Our approach was to determine the correlation and system error of the sampling unit alone and of the combined system with respect to reference levels over 12h in humans. Two venous cannulae were inserted to connect the automatic and reference systems to the subjects. Blood samples were taken at 15 and 30 min intervals. The median Pearson coefficient of correlation between manually and automatically withdrawn blood samples was 0.982 for the sampling unit alone and 0.950 for the complete system. The biosensor had a linear range up to 20 mmoll(-1) and a 95% response time of <2 min. Clark Error Grid analysis showed that 96.93% of the data (228 data pairs) was in zone A and 3.07% in zone B. Insulin Titration Error Grid analysis suggested an acceptable treatment in 99.56% of cases. Implementation of a "Keep Vein Open" saline infusion into the automated blood sampling system reduced blood withdrawal failures through occluded catheters fourfold. In summary, automated blood sampling from a peripheral vein coupled with automatic glucose determination is a promising alternative to frequent manual blood sampling.

Insulin glulisine, insulin lispro and regular human insulin show comparable end-organ metabolic effects: an exploratory study.

AIMS: To compare the end-organ metabolic effects of insulin glulisine (glulisine), insulin lispro (lispro) and regular human insulin (RHI) in patients with type 1 diabetes mellitus. METHODS: Eighteen patients with type 1 diabetes mellitus (mean age 36.9 +/- 8.6 years, BMI 23.6 +/- 2.8 kg/m(2), haemoglobin A(1c) 7.4 +/- 0.9%) were randomized in this single-centre, double-blind, three-period cross-over, standard Latin-square, euglycaemic glucose clamp trial. Patients received sequential, primed stepwise intravenous infusions of glulisine, lispro or RHI (infusion rates were increased in a stepwise manner from an initial rate of 0.33 [180 min] to 0.66 [180 min] and 1.00 [180 min] mU/kg/min). The primary variables were the suppression of endogenous glucose production (S(EGP)) and glucose uptake (GU). RESULTS: Mean basal endogenous glucose production (EGP) was 1.88, 2.12 and 2.12 mg/kg/min for glulisine, lispro and RHI respectively. Mean (+/-s.e.) maximum absolute S(EGP) (adjusted for basal EGP) was -1.64 +/- 0.06, -1.72 +/- 0.05 and -1.56 +/- 0.05 mg/kg/min respectively. Mean (+/-s.e.) maximum absolute increase in GU (adjusted for basal GU) was 6.46 +/- 0.26, 6.23 +/- 0.24 and 6.72 +/- 0.24 mg/kg/min respectively. There were no clinically relevant differences between the three insulin treatments with respect to serum insulin, free fatty acid (FFA), glycerol or lactate levels. No serious adverse events and no episodes of severe hypoglycaemia were reported. CONCLUSIONS: This study shows that glulisine, lispro and RHI have similar effects on S(EGP), GU, FFA, glycerol and lactate levels, providing evidence for similar end-organ metabolic effects.

Proportional dose-response relationship and lower within-patient variability of insulin detemir and NPH insulin in subjects with type 1 diabetes mellitus.

AIMS: This study was conducted to evaluate the dose ratio of insulin detemir and neutral protamine Hagedorn (NPH) insulin over a range of therapeutically relevant subcutaneous doses. METHODS: The study was a randomized, double-blind, crossover 24-h-iso-glycemic clamp trial in 12 C-peptide-negative type 1 diabetic patients. Each subject received, by an incomplete block design selection, two of three possible doses of insulin detemir (0.15, 0.3, 0.6 U/kg) and NPH insulin (0.15, 0.3, 0.6 IU/kg), respectively. A detailed assessment of endogenous glucose production (EGP) and glucose uptake was performed, by use of stable isotopic labeled glucose tracer (D-[6,6- (2)H (2)] glucose). RESULTS: Dose proportionality was observed within the tested dose range. Regarding unit dose ratio, 0.68 U insulin detemir equals 1 IU NPH insulin (95% CI [0.35; 1.30]). There was no statistically significant difference in effect on the area under the curve (AUC) of glucose infusion rate (GIR) (AUC (GIR)) and the maximal GIR (GIR (max)) values, when comparing U (insulin detemir) to IU (NPH insulin). The pharmacodynamic within-subject profile was lower with insulin detemir in regard to AUC (GIR 0-24 h), GIR (max) and duration of action ( P<0.05). There was a tendency for a greater reduction of EGP with insulin detemir than with NPH insulin in regard to the area over the curve (AOC) of EGP in 24 hours (AOC (EGP 0-24 h)) ( P=0.07) and minimal EPG (EGP (min)) ( P=0.02). CONCLUSIONS: These data show that insulin detemir is dose-proportional to NPH insulin in type 1 diabetic patients at clinically relevant doses. The data indicate that insulin detemir has a lower degree of within-subject variability.

Post-prandial administration of the insulin analogue insulin aspart in patients with Type 1 diabetes mellitus.

AIMS: In intensified insulin therapy, the recent development of short-acting insulin analogues with a very rapid onset of action forces a new discussion in terms of the optimal injection-meal interval. This study evaluated prandial glycaemia in patients with Type 1 diabetes following the subcutaneous injection of soluble human insulin (HI) and the insulin analogue insulin aspart (IAsp) at different injection-meal intervals and investigated whether administration of IAsp after the meal might provide satisfactory metabolic control. METHODS: In a randomized, double-blind, double-dummy, four-period crossover study, 20 Type 1 diabetic patients were investigated. Prandial insulin was administered 15 min before the start of the meal (HI(-15min)), immediately before the meal (HI(0min); IAsp(0min)) and 15 min after the start of the meal (IAsp(+15min)). RESULTS: Plasma glucose excursions from baseline levels during the 4 h (PGexc) were highest with HI(0min) (17.9 mmol.l(-1).h; P < 0.05 vs. other treatments) and were not statistically different for HI(-15min), IAsp(0min) and IAsp(15min) (13.6, 11.9 and 14.2 mmol.l(-1).h, respectively). Maximum concentration of plasma glucose (PGmax) was lowest with IAsp(0min) (11.2 mmol/l; P < 0.05 vs. other treatments). PGmax was comparable with HI(-15min), HI(0min) and IAsp(+15min) (13.3, 14.1 and 13.2 mmol/l, respectively). CONCLUSIONS: With regard to prandial glycaemia IAsp(+15min) is as effective as HI(-5min) and superior to HI(0min). Thus, post-prandial dosing of the insulin analogue IAsp offers an attractive and feasible therapeutic option for well-controlled patients with Type 1 diabetes mellitus.

Pharmacokinetic and pharmacodynamic properties of long-acting insulin analogue NN304 in comparison to NPH insulin in humans.

NN304 is a long-acting insulin analogue that is acylated with a 14-C-fatty acid chain. Protraction of action of this novel insulin analogue is due not to slow absorption after subcutaneous administration but to reversible binding to albumin. We investigated the pharmacokinetic and pharmacodynamic properties of insulin analogue NN304 (0.3 and 0.6 U/kg) in comparison to NPH insulin (0.3 and 0.6 IU/kg) in 10 healthy volunteers performing a randomised, double-blind, cross-over, placebo-controlled glucose clamp study. During the observation period of 24 hours the areas under the insulin curve for NPH[0.3 IU/kg] vs. NPH[0.6 IU/kg] were 60 vs. 102 nmol min l(-1) (p<0.01) and for insulin analogue NN304[0.3 U/kg] vs. NN304[0.6 U/kg] 490 vs. 932 nmol min l(-1) (p <0.001), suggesting a clear dose-response relationship for both NPH insulin and NN304. The amount of disposed glucose (area under the curve of glucose infusion) differed with statistical significance between the five treatments and was highest with NPH[0.6 IU/kg] (2671 mg/kg) and lowest with placebo (265 mg/kg). However, area under the curve of glucose infusion after treatment with NN304 was only 36% (dose of 0.3 U/kg) and 24% (dose of 0.6 U/kg) of that observed with corresponding doses of NPH insulin. Moreover, increasing dosages of NN304 failed to demonstrate a significant dose-response with regard to the area under the curve of glucose infusion. This study demonstrates that the principle of protracted insulin action of NN304 by reversible binding to albumin is effective in humans albeit at a much lower rate of glucose utilisation when compared to NPH insulin. Thus, in contrast to animal studies NN304 and NPH insulin can not be considered equipotent in humans.

Measurement of interstitial albumin in human skeletal muscle and adipose tissue by open-flow microperfusion.

The absolute concentration of albumin was measured in the interstitial fluid of subcutaneous adipose tissue and skeletal muscle in six healthy volunteers by combining the method of open-flow microperfusion and the no-net-flux calibration technique. By use of open-flow microperfusion, four macroscopically perforated double lumen catheters were inserted into the tissue regions of interest and constantly perfused. Across the macroscopic perforations of the catheters interstitial fluid was partially recovered in the perfusion fluid. Catheters were perfused with five solutions, each containing different concentrations of albumin. Absolute interstitial albumin concentrations were calculated by applying linear regression analysis to perfusate vs. sampled albumin concentration (no-net-flux calibration technique). Interstitial albumin concentrations were significantly lower (P < 0.0001) in adipose tissue (7.36 g/l; r = 0.99, P < 0.0003; range: 4.3-10.7 g/l) and in skeletal muscle (13.25 g/l; r = 0.99, P < 0.0012; range: 9.7 to 15.7 g/l) compared with the serum concentration (48.9 +/- 0.7 g/l, mean +/- SE, n = 6; range: 46.4-50.4 g/l). Furthermore, interstitial albumin concentrations were significantly higher in skeletal muscle compared with adipose tissue (P < 0.01). The study indicates that open-flow microperfusion allows stable sampling of macromolecules from the interstitial space of peripheral tissue compartments. Moreover, the present data report for the first time in healthy humans in vivo the true albumin concentrations of interstitial fluid of adipose tissue and skeletal muscle.

Plasma and interstitial glucose dynamics after intravenous glucose injection: evaluation of the single-compartment glucose distribution assumption in the minimal models.

Recent experimental evidence suggests that estimates of glucose effectiveness (S(G)) from the minimal model of unlabeled glucose disappearance (Cold-MM) are in error. The single-compartment glucose distribution assumption embedded in the model has been indicated as a possible source of error. In this study, to directly examine the single-compartment assumption, we measured plasma and interstitial glucose concentrations after intravenous glucose injection. Additionally, we compared the accuracy of the estimates of glucose effectiveness from the Cold-MM and the single-compartment tracer minimal model (Hot-MM). Paired labeled intravenous glucose tolerance tests (IVGTTs) were performed in each of six C-peptide-negative type 1 diabetic subjects. Two different insulin infusion protocols were used: an infusion at constant basal rates and an infusion at variable rates to mimic a normal insulin response. During the labeled IVGTT with basal insulin infusion, the microperfusion technique was employed to sample adipose tissue interstitial fluid. Marked differences between the plasma and interstitial dynamics of (cold) glucose were observed during the first 22 min after glucose injection. These results suggest that the requirements for a single-compartment representation of glucose kinetics are not satisfied during at least the first 22 min of an IVGTT. Data from the labeled IVGTT with normal insulin response were used to identify the minimal-model parameters. The measure of S(G) derived using the Cold-MM was 3.44-fold higher than the direct measure obtained from the labeled IVGTT with basal insulin infusion (0.0179+/-0.0027 vs. 0.0052+/-0.0010 min(-1), P<0.01). The measure of glucose effectiveness (S(G)*) derived by the Hot-MM was 1.36-fold higher than the direct measure available from the labeled IVGTT with basal insulin infusion (0.0079+/-0.0013 vs. 0.0058+/-0.0004 min(-1), P>0.26). These results suggest that the Hot-MM is more appropriate for the evaluation of glucose effectiveness than the Cold-MM.

Direct access to interstitial fluid in adipose tissue in humans by use of open-flow microperfusion.

To gain direct access to the interstitial fluid (ISF), a new technique called open-flow microperfusion has been evaluated. This method is based on a double-lumen catheter with macroscopic (0.3-0.5 mm diameter) perforations that is inserted into the subcutaneous adipose tissue and constantly perfused. Thus partial equilibration between the ISF and the perfusion fluid occurs. The glucose concentration of the ISF was determined by established (zero flow rate, no net flux, and recirculation procedures) and new (ionic reference and suction technique) calibration methods by use of open-flow microperfusion. The data show that 1) the glucose concentration in the ISF is significantly lower than the corresponding arterialized venous plasma values during basal steady-state conditions (adipose tissue 3.2 +/- 0.10 mM, plasma 5.27 +/- 0.12 mM) as well as during hyperglycemic clamp experiments (adipose tissue 7.3 +/- 0.13 mM, plasma 9.91 +/- 0.16 mM), and 2) it is possible to determine the recovery continuously by using the ion concentration of the ISF as an internal standard (ionic reference).

Lactate metabolism of subcutaneous adipose tissue studied by open flow microperfusion.

Open flow microperfusion and a novel calibration technique (ionic reference technique) were evaluated for the frequent measurement of the absolute lactate concentration in sc adipose tissue. Furthermore, the influence of the plasma insulin concentration on the lactate concentration of sc adipose tissue was investigated during hyperglycemia. Sixteen lean healthy young men participated in the studies. In the postabsorbtive state the mean sc lactate concentrations were 1.29 and 1.36 mmol/L for the ionic reference technique and the no net flux protocol, respectively (not significant, P > 0.05). The simultaneously measured arterialized plasma lactate concentration was significantly lower at 0.77 mmol/L (P < 0.05). Both the sc lactate concentration (1.8+/-0.33 mmol/L) and the plasma lactate concentration (0.96+/-0.03 mmol/L) were significantly elevated during a hyperinsulinemic euglycemic clamp experiment. During a hyperglycemic clamp experiment the sc lactate concentration reached a significantly elevated plateau (2.15+/-0.27 mmol/L) that was not influenced by the increasing plasma insulin concentration. It is concluded that 1) open flow microperfusion combined with the ionic reference technique enables frequent measurement of the sc lactate concentration; 2) sc adipose tissue is a significant source of lactate release in the postabsorbtive state as well as during hyperinsulinemic clamp conditions; and 3) insulin concentrations greater than 180 pmol/L have no further influence on adipocyte stimulation of sc adipose tissue with respect to lactate release.

4-Hydroxynonenal modifies the effects of serum growth factors on the expression of the c-fos proto-oncogene and the proliferation of HeLa carcinoma cells.

In this study, the effect of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-poly-unsaturated fatty acids, on the expression of the c-fos proto-oncogene and growth factor-induced proliferation of HeLa carcinoma cells in vitro was investigated. The Fos protein forms the heterodimer AP-1 with the Jun protein and regulates the cell cycle by inducing cyclin D1. Agents that are able to induce c-fos include serum, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), all of which were used in this study. The proliferation rate was determined by cell counting (viable and dead cells according to trypan blue exclusion) and the BrdU assay. The c-fos mRNA level was monitored by the reverse transcriptase/polymerase chain reaction. In the absence of HNE, serum-deprived cells responded to serum stimulation with a more than 10-fold increase of the c-fos mRNA level as well as with an increased rate of DNA synthesis and cell multiplication. Both EGF and PDGF (applied in combination with insulin) were able to substitute for FCS and induced rapid growth of the tumor cells preincubated in serum-deprived medium. In the absence of growth factors a negative correlation between the HNE concentration (range: 1-250 microM) and the c-fos mRNA level was observed. We suppose that HNE interferes in this case with the basal activity of the c-fos promoter. EGF, when applied after the HNE treatment, induced rapid growth of the tumor cells preincubated in serum-free medium, if HNE was used in a physiological concentration (1 microM). No difference was observed compared to the HNE-free control. c-fos mRNA level was nearly unchanged. In contrast, a cytotoxic concentration of the aldehyde (100 microM) caused a complete inhibition of proliferation, although a twofold increase of the c-fos mRNA level immediately after the aldehyde treatment was observed. A similar effect of HNE in cytotoxic concentration on c-fos expression was observed when cells were grown in presence of PDGF instead of EGF. Hence, in both cases HNE possibly interferes with the signal transduction pathway, which is initiated by external growth factors. The increased c-fos expression might be part of an abortive attempt to overcome the stressful condition raised by a cytotoxic concentration of HNE.

Validation of home blood glucose meters with respect to clinical and analytical approaches.

OBJECTIVE: To evaluate the clinical and analytical accuracy of home blood glucose meters. RESEARCH DESIGN AND METHODS: Six blood glucose meters--Reflolux S (Boehringer Mannheim, Mannheim, Germany), One Touch II (LifeScan, Milpitas, CA), Glucocard Memory (Menarini, Florence, Italy), Precision QID (Medisense, Cambridge, U.K.), HaemoCue (HaemoCue, Angelholm, Sweden), and Accutrend alpha (Boehringer Mannheim, Mannheim, Germany)--were compared with a reference method (Beckman Glucose Analyzer II) under controlled conditions (glucose clamp technique). Validation of the blood glucose meters was accomplished by clinically oriented approaches (error grid analysis), statistical approaches (variance components analysis), and by the criteria of the American Diabetes Association (ADA), which recommend a target variability of < 5%. RESULTS: A total of 1,794 blood glucose monitor readings and 299 reference values ranging from 2.2 to 18.2 mmol/l were analyzed (705 readings < 3.89 mmol/l, 839 readings between 3.89 and 9.99 mmol/l, and 250 readings > 9.99 mmol/l). According to error grid analysis, only Reflolux S and Glucocard M had 100% of estimations within the clinically acceptable zones A and B. Assessment of analytical accuracy revealed substantial differences between the glucose meters after separation of the data into defined glycemic ranges. None of the devices met the ADA criteria. CONCLUSIONS: To evaluate accuracy of blood glucose meters, error grid analysis, as well as statistical models, are helpful means and should be performed together. Analytical performance of currently available home blood glucose meters differs substantially within defined glycemic ranges.