Concentrations of insulin glargine and its metabolites during long-term insulin therapy in type 2 diabetic patients and comparison of effects of insulin glargine, its metabolites, IGF-I, and human insulin on insulin and igf-I receptor signaling.

We investigated 1) the ability of purified glargine (GLA), metabolites 1 (M1) and 2 (M2), IGF-I, and NPH insulin to activate the insulin receptor (IR)-A and IR-B and IGF-I receptor (IGF-IR) in vitro; 2) plasma concentrations of GLA, M1, and M2 during long-term insulin therapy in type 2 diabetic patients; and 3) IR-A and IR-B activation in vitro induced by serum from patients treated with GLA or NPH insulin. A total of 104 patients (age 56.3 ± 0.8 years, BMI 31.4 ± 0.5 kg/m(2), and A1C 9.1 ± 0.1% [mean ± SE]) were randomized to GLA or NPH insulin therapy for 36 weeks. Plasma concentrations of GLA, M1, and M2 were determined by liquid chromatography-tandem mass spectrometry assay. IR-A, IR-B, and IGF-IR autophosphorylation was induced by purified hormones or serum by kinase receptor activation assays. In vitro, M1 induced comparable IR-A, IR-B, and IGF-IR autophosphorylation (activation) as NPH insulin. After 36 weeks, M1 increased from undetectable (<0.2 ng/mL) to 1.5 ng/mL (0.9-2.1), while GLA and M2 remained undetectable. GLA dose correlated with M1 (r = 0.84; P < 0.001). Serum from patients treated with GLA or NPH insulin induced similar IR-A and IR-B activation. These data suggest that M1 rather than GLA mediates GLA effects and that compared with NPH insulin, GLA does not increase IGF-IR signaling during long-term insulin therapy in type 2 diabetes.

Pharmacokinetics and pharmacodynamics of protamine zinc recombinant human insulin in healthy dogs.

Protamine zinc insulins are generally considered to be long acting, with slow absorption from subcutaneous tissue. Protamine zinc recombinant human insulin (PZIR) may be useful to treat diabetic dogs. The purpose of this study was to describe the pharmacokinetics and pharmacodynamics of PZIR in dogs. PZIR was administered subcutaneously to 10 healthy Beagles using an incomplete crossover design, at doses of 0.3 or 0.5 U/kg (each n=5), 0.8 U/kg (n=10), or 0.8 U/kg at three separate sites (n=6). Insulin and glucose concentrations were measured over 24 h. The shapes of insulin and glucose curves were variable among dogs, and the relationship between insulin dose, concentration, and glucose-lowering effect was nonlinear. For single-site 0.8 U/kg, median (range) onset of action was 3.5 h (0.5-10 h), time to glucose nadir was 14 h (5 to >24 h), and duration of action was >24 h (16 to >24 h). Mathematical model predictions of times to 50% and 90% insulin absorption, and fraction of insulin absorbed in 24 h, were not significantly different among protocols. Results confirm the tendency toward a late onset and long duration of action for PZIR in dogs. This insulin may be an alternative treatment option for diabetic dogs.

Impact of recurring intermediate insulin-induced hypoglycemia on hypothalamic paraventricular corticotropin-releasing hormone, oxytocin, vasopressin and glucokinase gene profiles: role of type II glucocorticoid receptors.

Activation of central type II glucocorticoid receptors (GR) during neutral protamine Hagedorn insulin (NPH) administration exacerbates recurring hypoglycemia. The hypothalamic paraventricular nucleus (PVN) integrates metabolic sensory input, controls autonomic and neuroendocrine motor outflow, and is characterized by abundant GR expression. The present studies investigated the hypothesis that PVN GR mediate intensification of hypoglycemia by serial NPH dosing, and that PVN glucokinase (GCK) and glucoregulatory neuropeptide genes acclimate to this treatment paradigm through GR-dependent mechanisms. Groups of adult male rats were injected subcutaneously with one or four doses of NPH, on as many days, while controls received vehicle. Bilateral administration of the selective GR antagonist, CP-472555, into the PVN prior to the first three NPH injections prevented amplification of hypoglycemia in response to the final insulin dose, while intra-PVN delivery of the GR agonist, dexamethasone, to euglycemic rats did not modify ensuing NPH-induced hypoglycemia. Quantitative real-time RT-PCR analysis of microdissected PVN tissue revealed that GCK, corticotropin-releasing hormone (CRH), oxytocin (OT), and vasopressin (VP) mRNA levels were unchanged in response to acute NPH, and baseline gene profiles measured 24 h after antecedent injections were similar to vehicle controls. In contrast, serial dosing with NPH elevated CRH and GCK, diminished OT, but did not alter VP gene transcripts. Intracerebroventricular CP-472555 delivery in conjunction with antecedent NPH dosing prevented transcriptional habituation of GCK and OT genes, but did not modify CRH or VP mRNA profiles. The present data show that activation of PVN GR during antecedent intermediate insulin-induced hypoglycemia is required for exacerbation of recurring hypoglycemia, and receptor stimulation in the absence of hypoglycemia and/or its sequelae does not intensify the effects of subsequent NPH administration. The results also provide evidence for acclimation of PVN CRH, GCK, and OT gene profiles to serial NPH dosing, and demonstrate that GR may be involved in GCK and OT transcriptional adaptation to ongoing intermediate insulin-induced hypoglycemia.

Structural characterization of insulin NPH formulations.

Insulin NPH (neutral protamine hagedorn) has for long been one of the most important therapeutic formulations for the treatment of diabetes. The protracted action profile of NPH formulations is gained from crystallizing insulin with zinc in the presence of the basic poly-arginine peptide protamine. In spite of its long history and successful use, the binding mode of the insulin-protamine complex is not known. In this study, three different systems were used to study protamine binding to insulin. In the first system, crystals of an insulin-protamine complex grown in the presence of urea and diffracting to 1.5A resolution were analyzed. In the second system, a shorter peptide consisting of 12 arginine residues was co-crystallized with insulin in order to reduce the flexibility and thereby improve the electron density of the peptide. Both systems yielded data to a significantly higher resolution than obtained previously. In addition, a third system was analyzed where crystals of insulin and protamine were grown in the absence of urea, with conditions closely resembling the pharmaceutical formulation. Data from these NPH microcrystals could for the first time be collected to 2.2A resolution at a micro focused X-ray beamline. Analysis of all three crystal forms reveal potential protamine density located close to the solvent channel leading to the centrally located zinc atoms in the insulin hexamer and support that protamine binds to insulin in a not well defined conformation.

Morning versus bedtime isophane insulin in type 2 (non-insulin dependent) diabetes mellitus.

Morning versus bedtime administration of NPH insulin was compared in 12 subjects with Type 2 diabetes and overt fasting hyperglycaemia. Subjects were studied at baseline (diet alone) and after 2 months on each of the two insulin programmes in a random crossover design, in which dosage was increased until at least one daily preprandial blood glucose was consistently in the range of 3.9 to 6.0 mmol l-1. Mean (+/- SEM) daily total insulin dosage was equivalent for the morning (0.36 +/- 0.03 units kg-1) and for the bedtime (0.37 +/- 0.03 units kg-1) insulin administration schedules. Glycaemic control was improved on both insulin regimens, but was better on bedtime than morning insulin. Fasting plasma glucose (mmol l-1) was 12.0 +/- 0.7 (baseline), 8.6 +/- 0.7 (morning), and 4.6 +/- 0.3 (bedtime), respectively. Mean 24 h plasma glucose (mmol l-1) was 13.3 +/- 1.3, 9.0 +/- 0.7, and 7.8 +/- 0.7. Glycated haemoglobin (%) was 7.65 +/- 0.35, 6.23 +/- 0.26, and 5.81 +/- 0.32. The improvement of basal glycaemia is a consequence of increased basal metabolic clearance of glucose (baseline, 47.6 +/- 3.1 ml m-2 min-1; morning 63.5 +/- 5.4, bedtime 103.5 +/- 7.1). There was no change in hepatic glucose output. It is concluded that bedtime administration of intermediate acting insulin results in increased basal insulinaemia, leading to improved basal glycaemia and consequent improved overall metabolic control, compared to morning insulin administration. Therefore, bedtime may be the preferable timing of insulin therapy for patients with Type 2 diabetes and overt fasting hyperglycaemia.

[A study of the absorption of NPH insulin administered into subcutaneous tissue: in vitro study of the mechanism of insulin release from NPH insulin crystal].

NPH (Neutral Protamine Hagedorn) insulin has been widely used for the patients with diabetes mellitus. NPH insulin crystal (NPH-C) is an insoluble ionically bonded complex which insulin and protamine formed at neutral pH. It has been thought that NPH-C dissolve in the injected site by splitting of crystals into protamine and insulin. But the mechanism of the dissolution of NPH-C injected into subcutaneous tissue has not been fully clarified. To investigate the mechanism of the release of insulin from NPH-C, we measured the concentrations of immunoreactive insulin (IRI) and arginine, major component of protamine, recovered from NPH-C incubated with human serum. The concentrations of IRI and arginine, recovered from NPH-C incubated with human serum at 37 degrees C, time-dependently increased, but remained unchanged at 4 degrees C. And the concentrations of IRI and arginine recovered from NPH-C incubated with Krebs-Ringer phosphate buffer at 37 degrees C or 4 degrees C did not change. The release of IRI and arginine from NPH-C with human serum was enhanced by the addition of CoCl2 and was inhibited by the addition of CdCl2 or EDTA. Human serum was fractionated using starch block electrophoresis and the NPH-C dissolving activities of each fractions were investigated. NPH-C dissolved in carboxypeptidase N rich-fraction, but not in plasmin-rich fraction. Carboxypeptidase N was purified from human serum and the activity of NPH-C dissolution was investigated. The relative activity of NPH-C dissolution in the purified Carboxypeptidase N was about 250-fold higher than that in the unpurified human serum.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the pharmacokinetics and metabolic effects of human regular and NPH insulin mixtures.

The effects of three human premixes (Mixtard, Actraphane, Humulin M3), syringe mixed 30% regular and 70% NPH insulin, regular insulin alone and NPH insulin alone, on intermediary metabolism, plasma free insulin levels and action profiles were compared using the euglycemic clamp technique. Seven normal volunteers received 20 IU of each insulin subcutaneously in a randomized fashion on separate days. The first and last 60 min of the 6 h clamp were chosen as summary measures of clinical importance. Significantly elevated plasma free insulin levels were found with all treatments compared to NPH insulin alone during the first hour, although by the final hour only Mixtard produced significantly higher levels compared to NPH (19.4 +/- 1.2, 10.5 +/- 0.3 mU/l P less than 0.01, respectively). Analysis of area under the incremental insulin absorption curve demonstrated that Mixtard produced significantly increased levels compared to syringe-mixed regular: NPH (7.6 +/- 0.8), Actraphane (9.6 +/- 1.0) and Humulin M3 (9.0 +/- 0.8 mU/l all P less than 0.05). Mixtard also resulted in significantly higher glucose infusion rates compared to the other premixes. No difference in action was found between regular and pre- or syringe-mixed human insulins during the first hour of the studies. The effects on intermediary carbohydrate and lipid metabolism were similar for syringe and premixed insulins. We conclude that: (1) fixed human insulin mixtures with NPH cause no blunting of the action of the soluble component. (2) Actraphane and Humulin M3 are similar but Mixtard may have a greater effect on some aspects of insulin action. (3) In clinical practice, fixed human insulin mixtures will be as efficacious as syringe-mixed preparations but may be easier and more convenient to use.

A comparison of the pharmacokinetics of human protamine sodium insulin with human isophane insulin following subcutaneous injection in normal subjects.

The pharmacokinetics of human protamine sodium insulin 0.6 mg% were compared with those of human isophane zinc insulin using the glucose clamp technique. The insulin preparations were administered subcutaneously at a dose of 0.5 U/kg in 7 normal subjects; a placebo injection served as control. Plasma insulin and C-peptide concentrations were then measured over 24 hours. After correction of the results to allow for endogenous insulin secretion the two insulin preparations gave similar values throughout the study except that protamine sodium insulin achieved an earlier peak in plasma insulin concentration (90 minutes) compared to isophane (120 minutes). When measured by an index of the dextrose infusion rate required to maintain euglycaemia the two insulin preparations gave similar values for the first 16 hours, maximum values being obtained during the fourth hour after administration. Using this index isophane zinc insulin gave higher values during the 17th, 19th and 21st hours following administration suggesting it may have a slightly more prolonged action than protamine sodium insulin.

Absorption kinetics and action profiles of mixtures of short- and intermediate-acting insulins.

The effects of mixing short- and intermediate-acting insulins (lente and NPH) on plasma insulin levels and action profiles, assessed by the euglycaemic clamp technique, were studied in 10 volunteers. Four protocols were used: (1) comparison between two semi-synthetic human soluble insulins in seven subjects (0.22 IU/kg); (2) assessment of insulin levels and action profiles of lente insulin in six subjects and of NPH insulin in five subjects (0.33 IU/kg); (3) comparison between mixtures of soluble with lente insulin and soluble with NPH insulin, administered immediately after mixing, in eight subjects (0.55 IU/kg, 40% short-acting); (4) same mixtures, administered 2 days after preparation, in seven subjects. No differences in insulin levels and action profiles during the first 4 h after injection were found between both short-acting insulins and the soluble + NPH insulin mixtures. After the administration of NPH insulin, plasma insulin levels rose slightly faster in comparison with lente insulin, with no significant differences between the action profiles for either insulin. Onset of action was delayed after soluble + lente insulin, both when administered immediately after mixing and to a greater extent when stored for 2 days before administration. After the latter procedure, the onset of action was markedly retarded and only slightly faster than after lente insulin alone. We conclude, therefore, that mixing soluble with NPH insulin in a ratio of 2:3 does not affect the absorption kinetics of soluble insulin, whereas the onset of action is delayed when soluble is combined in the syringe with lente insulin, even when administered immediately after mixing.

Absorption of soluble and isophane semi-synthetic human and porcine insulin in insulin-dependent diabetic subjects.

A double-blind cross-over study of the sc absorption of radiolabelled semi-synthetic human (SHI) and purified porcine (PPI) insulin was made. Absorption of both isophane (n = 10) and soluble insulin (n = 8) was studied. There was no significant difference between the disappearance from the injection site, the plasma free insulin concentrations, or blood glucose levels after sc injection of the isophane preparations. A faster disappearance of the soluble SHI (as judged from T/50 and AUC) was found (both P-values less than 0.01). However, no difference was observed between the plasma insulin concentrations at any time point (P less than 0.05). Blood glucose levels showed no statistical differences between the two soluble preparations. The data indicate minor differences between the pharmacokinetics of SHI and PPI, but these seem of no clinical importance.

Dose-dependent subcutaneous absorption of porcine, bovine and human NPH insulins.

The absorption of 125I-labelled porcine, bovine and human neutral protamine Hagedorn (NPH) insulins was compared in 40 diabetic patients after subcutaneous injections in the thighs using doses of 6 and 24 IU (40 IU/ml). Furthermore, the absorption of porcine NPH insulin was compared with bovine NPH insulin and porcine lente type insulin in the same dose (6 IU). All these intermediately acting insulins showed similar absorption rates when administered in the same dose. However, an increase in the injected dose from 6 to 24 IU resulted in a decrease of approximately 30% in the absorption rate of all NPH insulins.

Clinical factors influencing the absorption of 125I-NPH insulin in diabetic patients.

Clinical factors which might influence the absorption of subcutaneously injected 125I-NPH insulin were studied in 101 diabetics. The disappearance curve was monoexponential after a delay period of 1.5 +/- 0.8 h (mean +/- SD). Lipohypertrophy significantly prolonged insulin absorption (half life (T1/2) = 11.2 +/- 3.1 h, p = 0.0001). Low bicarbonate levels increased the absorption (T1/2 3.9 +/- 2.3 h, p less than 0.05). Lean diabetics had a faster absorption (6.2 +/- 1.9 h) than normal weight diabetics (7.5 +/- 2.0 h, p less than 0.02). Sex, age, diabetes duration and injection depth did not influence T1/2. The half life was significantly inversely correlated to the resting subcutaneous blood flow (r = 0.882, p less than 0.01). The overall interindividual coefficient of variation for insulin absorption in nonketotic diabetics was 27.4%. Also considerable intra-patient day-to-day variation was found (24.5%), and between different injection sites (30.2%). These variations emphasize the drawbacks of conventional insulin therapy in the management of insulin-requiring diabetics.

Insulin concentrations and time-action profiles of three different intermediate-acting insulin preparations in nondiabetic volunteers under glucose-controlled glucose infusion technique.

This study describes the pharmacokinetics of three intermediate-acting insulin preparations, NPH porcine insulin, NPH human insulin (recombinant DNA), and "Depot-A" insulin, a mixture of 20% regular and 80% NPH human insulin from Eli Lilly and Company. Metabolic healthy normal weight volunteers were selected for the study. After overnight fasting, each test person received 0.4 U of each insulin per kg body weight injected subcutaneously in the triceps area of the arm. To prevent severe hypoglycemia, the test persons were connected to a "GCIIS Biostator" with blood glucose clamp at the 60 mg/dl level. Peripheral blood was sampled at regular intervals for glucose, insulin, and C-peptide determination. More elevated insulin levels were measured after application of both NPH human insulin and "Depot-A" insulin than after NPH porcine insulin. A more rapid decrease in the blood glucose concentration was observed after injection of both human insulin preparations than after porcine insulin. The dextrose output of the "GCIIS Biostator" was more pronounced in both human insulins than after the porcine preparation. After the injection of NPH human and NPH porcine insulin, significant differences were calculated between the concentrations of these two insulins in the blood, from the 2nd to the 10th hour (P less than 0.05-P less than 0.005) and between the dextrose output of the "GCIIS Biostator" from the 3rd to the 8.5th hour (P less than 0.05). The fall of the C-peptide concentration to the lower detection limit of the assay reflects suppression of the endogenous B-cell secretion and confirms the measure of peripheral insulin concentrations as a result of the exogenously applied insulin. Although all investigations were performed under identical experimental conditions and equal dosages of each insulin were injected, higher insulin concentrations and a stronger biologic effect, shown by larger amount of dextrose delivered, were observed in both human insulins than in porcine insulin. Why this phenomenon occurs is as yet unclear. The clamp technique used with the "GCIIS Biostator" enables establishment of the biologic profile of any insulin, and thus represents a valuable tool in comparative studies.

Absorption of isophane (NPH) insulin and its clinical implications.

Absorption of 125I-NPH insulin (125I-isophane insulin) (40 IU/ml) was studied in eight diabetics given 50% and 150% of their normal daily dose of insulin. Insulin absorption correlated with plasma insulin (r = 0.97, p less than 0.001) and blood glucose (r = -0.87, p less than 0.01) concentrations. Absorption was slower at higher doses, so that trebling the insulin dose only doubled the amount absorbed over the first 24 hours. The plasma elimination half time (t12) of insulin was about five minutes. Thus, the disappearance of radiolabelled insulin is a reliable and quantitative index of insulin absorption; subcutaneous degradation, if present, is minimal and constant. Changes in dise of intermediate-acting insulin further increases the large variation in insulin absorption. This implies that minor adjustments of intermediate insulin dosage are probably futile.

Kinetics of subcutaneous NPH insulin in diabetics.

Kinetics of subcutaneous NPH insulin were studied in newly diagnosed insulin-dependent diabetics. Using a biotelemetric technique with small Geiger-Müller detectors applied to the skin surface, the disappearance of 125I-NPH insulin fron subcutis was monoexponential with a mean half-life of 6.6 +/- 3.3 (SD) hr. A model is presented to compare the disappearance rates of subcutaneous 125I-NPH insulin and the calculated plasma appearance insulin curve derived from the actual plasma insulin concentration measurements, assuming a one-compartment model and first-order kinetics. Areas under the absorption- and appearance-time curves calculated from external measurements and from plasma insulin concentrations were identical. There was a strong correlation between plasma concentrations and absorbed amounts of labeled insulin (r = 0.8782, P less than 0.001), as there was between the blood glucose-lowering effects and percent absorbed insulin per hour (r = 0.7659, P less than 0.001). Our results indicate that the disappearance rate of iodine-labeled insulin was a relevant biological expression of insulin absorption from subcutis and a reliable noninvasive method of quantitative determination of insulin concentration in blood.

Biotelemetric detection of the disappearance of subcutaneously injected 125I-NPH insulin.

A biotelemetric method with Geiger-Müller (GM) detectors fixed to the skin surface was used for continuous registration of the disappearance rate of subcutaneously injected 125I-NPH insulin. Methodological problems concerning counting geometry were investigated by comparing the disappearance of radioactivity, measured the GM- and NaI-detectors, respectively, and by scanning of the radioactive source. The size and position of the subcutaneous depot was unchanged throughout the study. Movement artifacts could be avoided. The coefficient of variation for distribution of ratios between count rates for GM- and NaI-detectors was 3.0% +/- 1.1 (SD) (range 0.9-4.0%) over periods of 24 h. It is concluded that the biotelemetry technique proved to be a clinically useful procedure for insulin absorption studies.