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Grey-box pharmacokinetic/pharmacodynamic (PK/PD) modelling is presented as a promising way of modelling the pharmacokinetics and pharmacodynamics of the in vivo system of insulin and glucose and to estimate model and derived PK/PD parameters. The concept behind grey-box modelling consists in using a priori physiological knowledge along with information from data in the estimation of model parameters. The PK/PD properties of two types of insulin are investigated in a euglycaemic clamp study where a single bolus of insulin is injected subcutaneously. The effect of insulin on the glucose disappearance is investigated by artificially maintaining a blood glucose concentration close to the normal fasting level. The infused glucose needed to maintain the clamped blood glucose concentration can therefore be used as a measure for the glucose utilization. The PK and PD parameters are successfully estimated simultaneously thereby describing the uptake, distribution, and effect of the two different types of insulin.

The effect on postprandial blood glucose control of an immediately pre-meal injection of the rapid acting insulin analogue Aspart (IAsp) was compared with that of human insulin Actrapid injected immediately or 30 minutes before a test meal in insulin-treated type 2 diabetic patients with residual beta-cell function. In a double-blind, double dummy crossover design, patients attended three study days where the following insulin injections in combination with placebo were given in a random order: IAsp (0.15 IU/kg body weight) immediately before the meal, or insulin Actrapid (0.15 IU/kg) immediately (Act0) or 30 minutes before (Act-30) a test meal. We studied 25 insulin-requiring type 2 diabetic patients, including 14 males and 11 females, with a mean age of 59.7 years (range, 43-71), body mass index 28.3 kg/m2 (range, 21.9-35.0), HbA1c 8.5% (range, 6.8-10.0), glucagon-stimulated C-peptide 1.0 nmol/l (range, 0.3-2.5) and diabetes duration 12.5 years (range, 3.0-26.0). Twenty-two patients completed the study. A significantly improved postprandial glucose control was demonstrated with IAsp as compared to Act0, based on a significantly smaller postprandial blood glucose excursion (IAsp, 899 +/- 609 (SD) mmol/l.min versus Act0, 1102 +/- 497 mmol/l min, p < 0.01) and supported by a significantly lower maximum serum glucose concentration (Cmax) up to 360 min after dosing (IAsp, 10.8 +/- 2.2 mmol/l vs. Act0, 12.0 +/- 2.4 mmol/l, p < 0.02). No difference was demonstrated in glucose endpoints between IAsp, administered with a meal and Actrapid injected 30 minutes before the meal (AUCglucose IAsp, 899 +/- 609 mmol/l min vs. Act-30, 868 +/- 374 mmol/l min; Cmax IAsp, 10.8 +/- 2.2 mmol/l vs. Act-30, 11.1 +/- 1.8 mmol/l). No concerns about the safety of IAsp were raised. Immediate pre-meal administration of the rapid-acting insulin analogue Aspart in patients with type 2 diabetes resulted in an improved postprandial glucose control compared to Actrapid injected immediately before the meal, but showed similar control compared to Actrapid injected 30 minutes before the meal. These results indicate that the improved glucose control previously demonstrated with insulin Aspart compared to human insulin in healthy subjects and type 1 diabetic patients also applies to insulin-treated type 2 diabetic patients.

Aims: To study the effects of physiological concentrations of rat proinsulin C peptide I and II, respectively, on whole body glucose utilization in streptozotocin diabetic and healthy rats., Methods: A sequential insulin clamp procedure was used (insulin infusion rates 3.0 and 30.0 mU.kg-1.min-1) in awake animals. C-peptide infusion rates were 0.05 and 0.5 nmol.kg-1.min-1. Blood glucose was clamped at 7.7 +/- 0.3 mmol/l in the diabetic rats and at 3.9 +/- 0.1 mmol/l in the healthy rats., Results: In diabetic rats infused at lower rates of C peptide and insulin, glucose utilization increased by 79-90% (p < 0.001) compared with diabetic animals infused with saline and insulin. Increasing the rate of C-peptide infusion tenfold did not elicit a statistically significant further increase in glucose utilization. C peptide I and II exerted similar effects. The metabolic clearance rate for glucose in the diabetic animals infused with C peptide was not different from that of the healthy rats. During high-dose insulin infusion (30.0 mU.kg-1.min-1) glucose utilization increased considerably and no statistically significant C-peptide effects were observed. About 85% of the increase in glucose utilization induced by C peptide could be blocked by treatment with N-monomethyl-L-arginine., Conclusions/interpretation: Physiological concentrations of homologous C peptide stimulate whole body glucose utilization in diabetic but not in healthy rats. C peptide I and II elicit similar effects. The influence of C peptide on glucose utilization may be mediated by nitric oxide.

Recombinant DNA technology allows the production of insulin analogues with faster absorption rates from subcutaneous tissue as compared to soluble human insulin. The human insulin analogue B10Asp (mono/dimeric) is absorbed twice as fast as soluble human insulin (hexameric). A double blind, randomised crossover study with a 1-month run-in period and two 2-month treatment periods was performed in 21 male insulin-dependent diabetic (IDDM) patients aged 18-40 years in order to compare the metabolic control obtained with equimolar doses of the analogue B10Asp vs soluble human insulin (Actrapid) given as mealtime insulin and intermediate acting isophane insulin (Protaphane) at bedtime. At the end of each 2-month study period, the patients were admitted to the metabolic ward. We found significantly higher plasma insulin/analogue levels after breakfast, lunch and dinner with B10Asp as compared to Actrapid (p < 0.05). The plasma insulin/analogue levels were significantly lower before lunch and dinner with B10Asp as compared to Actrapid (p < 0.05). Also, the plasma insulin/analogue level tended to be lower at bedtime when comparing B10Asp to Actrapid. The 24-h blood glucose profiles showed identical fasting blood glucose, significantly lower blood glucose after breakfast with the analogue (p < 0.05), no differences in blood glucose after lunch and dinner but a significantly higher blood glucose at midnight using the analogue (p < 0.05). The overall 24-h mean blood glucose concentrations, the daily insulin dose, HbA1c, diet, home blood glucose monitoring and frequency of hypoglycaemia were almost identical in the two treatment periods.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of maternal hypoglycemia on early organogenesis was studied in normal and diabetic rats. Female Wistar rats were made diabetic by an intravenous injection of streptozotocin (45 mg/kg) 2-3 weeks before conception. On day 9.5 or 10.5 of embryo development, both control and diabetic dams received saline or Actrapid human insulin (400 mU/rat) intraperitoneally after 19-h starvation. The fasting plasma glucose levels in diabetic dams decreased from approximately 23 to 8 mM. Hypoglycemia as low as 3.5 mM was maintained for 60 min in insulin-treated mother rats. Pregnancy was terminated on day 11.6 of embryo development. A significant growth retardation was found in diabetic embryos as compared with normal embryos. Maternal hypoglycemia lowered the DNA content in normal but not diabetic embryos, while the teratogenic effect of maternal hypoglycemia was not pronounced in either normal or diabetic embryos. These data may suggest that maternal hypoglycemia in vivo in early pregnancy influences the embryogenesis but not teratogenesis of rat embryos.

Gastric acid outputs caused by glucose injection into the lateral hypothalamic area (LHA) were examined in insulin hypoglycemia with or without estradiol-17 beta (EST) administration in bilaterally ovariectomized (OV) female rats. The basal level of acid output was higher in OV rats without EST than in OV rats with EST. When acid response was expressed as the percentage change, glucose injection into the LHA decreased acid output in a dose-dependent fashion in OV rats, while, in OV rats with EST, glucose injection into the LHA also reduced acid output without dose dependency. It was also noted that the threshold concentration of glucose that induced an acid response was lower in OV rats without EST than in OV rats with EST. These findings suggest that glucose-sensitive neurons responsible for gastric acid secretion can be modulated by estrogen at the LHA level.

The roles of diabetes mellitus, insulin-like growth factor I and insulin in vascular smooth muscle proliferation in vivo were studied. Proliferation was induced by endothelial injury (balloon catheterization) of rat aorta, and was measured as 3H-thymidine incorporation into DNA. Levels of insulin-like growth factor I mRNA and insulin-like growth factor I receptor mRNA were measured with a solution hybridization assay. The increase in DNA synthesis was most pronounced 2 days after injury in both normal and diabetic rats and declined thereafter, but DNA synthesis in aortas from diabetic rats was lower throughout the time period studied. Levels of insulin-like growth factor I mRNA and the receptor mRNA were both increased in balloon catheterized aortas, and time-course studies showed an increase in receptor mRNA prior to the increase in insulin-like growth factor I mRNA. Diabetic rats were treated with equimolar concentrations of insulin (35 nmol/day) or insulin-like growth factor I (31 nmol/day) for 5 days. Insulin-like growth factor I increased DNA synthesis in injured aortas 2 days after injury without improving blood glucose, whereas the effect of insulin was associated with a decrease in blood glucose levels. In conclusion, vascular smooth muscle proliferation is impaired by diabetes and stimulated by insulin treatment. Insulin-like growth factor I infusion stimulates vascular smooth muscle proliferation without affecting blood glucose, and gene expressions of insulin-like growth factor I and its receptor are increased in proliferating vascular smooth muscle, indicating that insulin-like growth factor I and involved in vascular smooth muscle proliferation in vivo.

Recombinant DNA technology allows the production of insulin analogues with faster absorption rates from subcutaneous tissue as compared to conventional human regular insulin. We report the time-action profiles of 12 U subcutaneously injected insulin analogues (B9Asp + B27Glu or B10Asp) as evaluated against human regular insulin by means of the euglycaemic clamp technique (blood glucose 5.0 mmol/l) in healthy men. After injection of 12 U of either insulin preparation identical values were found for maximal insulin action (maximal glucose infusion rate, time to peak action), total amount of glucose infused as well as area under the curve of glucose infusion rate. Half-maximal glucose infusion rate was reached significantly earlier after injection of modified insulins (mean +/- SD 38 +/- 7 and 43 +/- 5 min) as compared to regular insulin (56 +/- 14 min, p less than 0.01). Forty-five min after injection of both insulin analogues glucose infusion rate had increased by 7.4 +/- 1.8 or 6.1 +/- 1.8 mg.kg-1.min-1, reflecting 83 +/- 27 or 67 +/- 15% of maximal regular insulin action. In conclusion, the two tested insulin analogues showed similar action profiles, but a significantly faster onset of action as compared to regular insulin.

For more than 2 years now it has been controversially debated whether awareness of hypoglycemia is reduced when type I diabetic patients are switched from porcine to human insulin. In order to address this question, we studied nine C-peptide negative diabetics (age 27.6 years, Broca index 106%, duration of diabetes 5.7 years, HbA1, 8.8%) in comparison with eight healthy volunteers (age 22.4 years, Broca index 104%). Following euglycemic monitoring overnight, a controlled hypoglycemia was induced by altering the algorithms of the Biostator. This was done in a double-blind, cross-over fashion using porcine or human insulin on 2 nonconsecutive days. There were no differences between the results obtained with respect to the time course of the study, blood glucose, amount of insulin infused, and concentration of venous free insulin achieved. Of the nine diabetics, eight were aware of hypoglycemia at a higher blood glucose level under porcine insulin. The first symptom of hypoglycemia was perceived at a mean blood glucose level of 61.1 +/- 5.4 mg/dl under porcine insulin and of 44.4 +/- 5.3 mg/dl under human insulin (P less than or equal to 0.05). Thirty symptoms were noted under porcine insulin exclusively or preferentially as opposed to only eight which were observed exclusively or preferentially under human insulin. The healthy volunteers evidenced fewer symptoms at lower blood glucose concentrations than the diabetics. The clear difference between human and porcine insulin could not unequivocally be reproduced in this group.(ABSTRACT TRUNCATED AT 250 WORDS)

Body temperature falls during hypoglycaemia, perhaps as a protective mechanism. To test the hypothesis that the skin blood flow response to hypoglycaemia is specifically designed to facilitate heat loss we studied both nutritional blood flow and arteriovenous shunting of blood in skin during prolonged, controlled hypoglycaemia in man. We studied eight otherwise healthy, male, Type 1 (insulin-dependent) diabetic patients. Under Biostator control blood glucose was clamped at 8.0 (7.9-8.9), mmol/l (median and range) for 30 min, reduced to symptomatic hypoglycaemia, 1.7 (1.0-2.6) mmol/l for 20 min then raised to 4.9 (3.3-6.7) mmol/l. Interdigital skin web blood flow (laser doppler flowmeter, nutritional flow) fell during hypoglycaemia from 3.1 (2.2-3.8) to 2.4 (1.2-2.8) volts and remained depressed. In contrast, finger blood flow (venous occlusion plethysmography, arteriovenous shunt flow) started high at 54.7 (17.4-85.6), remained high at 52.7 (38.1-81.4) during hypoglycaemia but fell sharply to 25.3 (4.2-66.2) ml.min-1.100 ml-1 when symptoms were relieved. Plasma adrenaline and vasopressin both rose during hypoglycaemia from 0.4 (0.05-0.8) to 4.5 (2.3-20.2) nmol/l and from 0.5 (0.5-3.5) to 4.4 (2.0-13.9) pg/ml, respectively, and both fell sharply thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

The biological potency of semisynthetic human insulin (Actrapid HM, Novo) and purified pork insulin (Actrapid MC, Novo) was assessed in normal and diabetic subjects. The blood glucose lowering effect and the related counter-regulatory response were initially tested in six healthy subjects who received an i.v. injection of 0.15 U/kg body weight of either insulin preparation. The attained insulin levels were very similar (peak at 15 min: HM 139 +/- 7, MC 129 +/- 7 microU/ml), as well as the resulting blood glucose curves. A prolonged suppression of C-peptide values was observed after injecting both preparations. The evoked counter-regulatory response [glucagon, growth hormone (GH), cortisol and catecholamines] showed minimal differences. Prolactin secretion was almost identical after HM and MC injection. A glucose clamp study was subsequently performed in six insulin-dependent diabetic (IDD) patients. Blood glucose levels were maintained at 80 mg/dl by the artificial pancreas during a 180 min infusion of MC or HM insulin (30 mU/kg/h). The amounts of dextrose infused during the last 60 min of the study were not significantly different (121 +/- 14 vs 137 +/- 11 mg/kg/h for MC and HM, respectively). It is clear from our results that at the dose levels used in this study, the biological potency of i.v. injected HM is very similar to that of MC.

These studies were undertaken to assess the influence of storage temperature of insulin vials on pharmacokinetics and pharmacodynamics of a mixture of lente insulin (Monotard HM) and regular insulin (Actrapid HM) injected subcutaneously. Seven subjects with Type 1 (insulin-dependent) diabetes mellitus were studied twice after overnight normalization of plasma glucose. A mixture of lente insulin (0.22 U/kg) and regular insulin (0.11 U/kg) was prepared from insulin vials kept either refrigerated (approximately 4 degrees C) or at room temperature (approximately 18 degrees C) and injected subcutaneously (abdomen). Euglycaemia was maintained for the following 16 h by glucose infusion at variable rate. With refrigerated insulin, the plasma free insulin peak was greater (53 +/- 5 versus 45 +/- 6 mU/l) and occurred earlier (2.5 +/- 0.2 versus 6 +/- 0.3 h), and the glucose infusion rate showed a greater (16.5 +/- 1.2 versus 14.5 +/- 0.9 mumol.kg-1.min-1) and earlier peak (3.2 +/- 0.2 versus 6 +/- 0.4 h) as compared to that occurring with the non-refrigerated insulin (p less than 0.05). However, 6 h after insulin injection, both plasma free insulin and glucose infusion rate were 30% lower with the mixture of refrigerated as compared to that of non-refrigerated insulin (p less than 0.05). In contrast, when NPH-insulin (Protaphane HM) was mixed with regular insulin and injected in 4 out of the 7 diabetic patients, the storage temperature of insulin vials had no effect on the pharmacokinetics and pharmacodynamics of the mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin antibodies expressed as insulin binding capacity of IgG were determined in 50 Type 1 (insulin-dependent) diabetic children who have been treated with monocomponent porcine insulin from the onset of the disease. During the follow-up period of 0.5-5.5 years (mean +/- SD: 3.2 +/- 1.6 years), 26 out of 50 patients (52%) developed detectable insulin antibodies. These patients had significantly lower maximal C-peptide responses to a standardized breakfast 9 months after onset of diabetes (mean 0.24 pmol/ml, p less than 0.001) than those without insulin antibodies (mean 0.47 pmol/ml). In addition, patients with antibodies showed both significantly higher insulin requirements at 9 months (p less than 0.05), and shorter remissions (p less than 0.01) than those without. It is concluded that even 'small' amounts of insulin antibodies may be biologically significant and have negative effects on B-cell function and metabolic balance.

Plasma cholesterol, triglycerides, HDL2-cholesterol, and HDL3-cholesterol were studied in 18 patients with Type 2 diabetes. Prior to entering the clinical trial, the study subjects were in stable control under a fixed mixture of 20% regular and 80% NPH (isophane) biosynthetic human insulin twice daily. The patients were randomized to treatment with either biosynthetic human proinsulin or biosynthetic human NPH insulin (controls) twice daily. Glucose control was kept constant in both groups throughout the study. Of the nine patients treated with proinsulin, eight exhibited a decrease of plasma triglycerides (median decrease by 0.13 mmol/l). In contrast, all nine controls showed a rise (median increase by 0.69 mmol/l) of plasma triglycerides (p less than 0.001). In keeping with the fall of plasma triglycerides, HDL2-cholesterol rose in all but one proinsulin-treated patients. Both treatment modalities reduced HDL3-cholesterol with a median decrease of 0.20 mmol/l (p less than 0.01) with proinsulin and 0.26 mmol/l with NPH insulin (p less than 0.05). We conclude that human proinsulin is able to reduce plasma triglycerides and to increase HDL2-cholesterol in the majority of patients with Type 2 diabetes and thus appears to alter favourably risk factors for coronary heart disease.

The therapeutic efficiacy of biosynthetic human insulin (BHI) is compared to that of porcine insulin into groups of 10 children each with so far untreated type I diabetes. No significant difference between the two groups could be demonstrated throughout the first months of treatment: both the initial substitution dose, and the rate of partial remission were similar. However, the regimen of the BHI treated patients had to be changed to two injections a day earlier, since BHI given subcutaneously results in a faster and shorter lasting response than porcine insulin. Thus, after 9 and 12 months of treatment a higher BHI dose than porcine insulin dose had to be given. Thirty children pretreated with regular and NPH porcine insulin, who were in the post remission period, were transferred to BHI. First, the needed dose of insulin was significantly reduced. Two to three months later, however, the need in insulin increased, so that after 9 and 12 months the original dose had to be given again. The relationship between morning (2/3) and evening (1/3) dose was constant and in the morning as well as in the evening the ratio between regular and NPH-BHI was 40%/60%. There was no significant improvement or deterioration after replacing porcine by human insulin, since both insulins have the same metabolic effects.

The biological effects, hypoglycaemic symptoms, endocrine counterregulatory responses and glucose recovery following the injection of purified porcine and human insulin preparations were compared in a number of controlled clinical investigations and prospective clinical trials. In these studies involving healthy volunteers, Type 1 (insulin-dependent) diabetic patients on continuous subcutaneous insulin infusion or intensified conventional insulin therapy and insulin treated Type 2 (non-insulin-dependent) diabetic patients, no differences with regard to biological effects, counterregulatory responses, hypoglycaemic awareness or the long-term incidence of severe hypoglycaemia between porcine and human insulin preparations were identified. These data fail to confirm any specific risk of severe hypoglycaemia attributable to the use of human insulin preparations in the treatment of patients with diabetes mellitus.

The polymerization and precipitation of highly purified insulins which causes major problems in portable infusion systems does not occur with sulphated insulin. To compare the biological behaviour of sulphated insulin with that of a neutral highly purified monocomponent insulin, insulin receptor studies were performed on human and rat adipocytes and rat hepatocytes. Sulphated insulin displayed a lower affinity for binding to both human and rat adipocytes compared with neutral insulin, approximately four times the concentration being required to achieve half-maximal displacement of monoiodoinsulin (p less than 0.05 and 0.01, respectively). A 20-fold higher concentration of sulphated insulin was required for half-maximal displacement from rat hepatocytes (p less than 0.025). However, sulphated insulin bound to liver membranes with an affinity more closely resembling that for adipocytes rather than hepatocytes. Differences in the intracellular processing of the negatively charged insulin could account for the observed lower affinity of binding to hepatocytes.

The aim of the present study was to evaluate various functional tests for the differentiation of hyperinsulinaemic hypoglycaemia. The pathophysiological and histological findings in six infants, aged 2-7 months, with persistent hyperinsulinaemic hypoglycaemia are described. Islet cell adenoma was found in four infants and pancreatic nesidioblastosis in two others. Circulating levels of blood glucose (BG), immunoreactive insulin and C-peptide immunoreactivity were measured under basal conditions and during both stimulation and suppression. The diagnosis of hyperinsulinaemia was made by estimation of the BG/serum insulin ratio, which was the most important diagnostic criterion of hyperinsulinism. Control subjects of comparable age showed a ratio of 8.3 +/- 4.4 (range 4.1-13.3), whereas the six patients had values between 0.3 and 5.1. At least four determinations with ratios lower than 2.6 were necessary for confirming the diagnosis. Preoperatively we performed oral glucose tolerance, diazoxide infusion, somatostatin infusion and C-peptide suppression tests. It is suggested that the various function tests, especially the suppression tests, do not differentiate hyperinsulinism caused by an adenoma from that caused by diffuse pancreatic nesidioblastosis.

The plasma glucose, C-peptide and insulin responses to subcutaneously administered highly purified porcine, 'semi-synthetic' and 'biosynthetic' human isophane (NPH) insulin and diluting medium as control in normal male subjects were evaluated. Porcine and semi-synthetic human NPH insulins were administered at two dose levels of 0.15 and 0.30 U/kg body weight and biosynthetic human NPH at 0.15 U/kg body weight only. At the low dose level the three insulin preparations resulted in a similar maximal hypoglycaemic effect within 3-5 h after administration. However, over the remainder of the 11 h post-injection period, the plasma glucose level was lower after semi-synthetic human insulin. In contrast, at the 0.30 U/kg dose level, there was no difference in the early or late hypoglycaemic response between porcine and semi-synthetic human NPH insulins of equivalent pharmaceutical formulation. The clinical relevance of these findings needs further evaluation. The data suggest that for the 'intermediate-acting' NPH insulin preparations, both the species of insulin, nature and quantity of the retarding protein and their subsequent interaction may determine their time-action characteristics.