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Background The discovery of insulin has changed dramatically the outcome of patients with type 1 diabetes, giving them the possibility to survive. This is of particular concern due to the fact that type 1 diabetes most frequently occurs in children who were destined to die in ketoacidosis coma. Areas of uncertainty From insulin discovery to the availability of human insulin and human insulin analogs to be used in diabetes therapy, a series of problems have arisen as the difficulty of insulin purifications, the animal insulin used by the first researches were in fact contaminated by proteins, fats, and other impurities, and the presence of side effects such as allergy, antibodies generation, and lipoatrophy. Data source literature Data strictly related to the argument have been searched in Pub Med and used. Results Starting from insulin discovery in 1921 to nowadays, significant efforts have been made by a series of researches to purify animal insulin, discover the molecular structure of human insulin, and develop methods to synthetize human insulin and then insulin analogs. Conclusions The history of insulin discovery here reported is fascinating; insulin is a hormone, a product of biotechnology, a field of research that saved and save the life of many diabetic patients.

Oral insulin delivery has revolutionized diabetes treatment, but challenges including degradation in the gastrointestinal environment and low permeation across the intestinal epithelium remain. Herein, to overcome these barriers, we developed a novel biodegradable nanocomposite microsphere embedded with metal-organic framework (MOF) nanoparticles. An iron-based MOF nanoparticle (NP) (MIL-100) was first synthesized as a carrier with an insulin loading capacity of 35%. The insulin-loaded MIL-100 nanoparticles modified with sodium dodecyl sulfate (Ins@MIL100/SDS) promoted insulin permeation across Caco-2 monolayer models in vitro. To improve resistance to the gastric acid environment, Ins@MIL100/SDS nanoparticles were embedded into a biodegradable microsphere to construct the nanocomposite delivery system (Ins@MIL100/SDS@MS). The microspheres effectively protected the MOF NPs from rapid degradation under acidic conditions and could release insulin-loaded MOF NPs in the simulated intestinal fluid. After the oral administration of Ins@MIL100/SDS@MS into BALB/c nude mice, increased intestinal absorption of the insulin was detected compared to the oral administration of free insulin or Ins@MIL100/SDS. Furthermore, significantly enhanced plasma insulin levels were obtained for over 6 h after oral administration of Ins@MIL100/SDS@MS into diabetic rats, leading to a remarkably enhanced effect in lowering blood glucose level with a relative pharmacological availability of 7.8%. Thus, the MOF-nanoparticle-incorporated microsphere may provide a new strategy for effective oral protein delivery.

BACKGROUND Chronic trophic ulcers (CTUs), especially those located over the plantar region, are a leading cause of deformity and disability in patients with leprosy. Despite the various treatment modalities available, CTUs can be chronic and refractory to treatment. The successful use of topical insulin in various types of wounds led researchers to evaluate its safety and efficacy in the treatment of plantar CTUs. METHODS Forty-two patients who had completed a multidrug treatment for leprosy were recruited and randomized into two groups. In the test group, 23 patients received 10 units (0.1 mL) of topical insulin (Actrapid) in 1 mL of normal saline twice daily over treated areas. The placebo group (n = 19) received topical normal saline only. The primary end point was the proportion of patients with complete wound closure by 12 weeks. Secondary end points included time to healing, wound area reduction, Physician Global Assessment of Efficacy scores, and Dermatology Life Quality Index scores at the end of 12 weeks. RESULTS The majority of CTUs (80%) were situated over the forefoot; the metatarsal head of the hallux was the most common site (86%). Wound healing was faster (0.61 ± 0.31 vs 0.14 ± 0.42 cm per week, P < .0001), and the number of days to complete healing was significantly shorter in the test group compared with the placebo group (31.5 ± 17.6 vs 44.3 ± 16.2 days, P = .02). The only observed adverse effect in the test group was white granular deposits over the CTU (n = 10). CONCLUSIONS Topical insulin therapy may be a safe, efficacious, cheap, and easily available treatment option in CTUs among patients with leprosy.

Measurement of insulin and its therapeutic analogs is important in diabetes, hypoglycemia, sports anti-doping and toxicology. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogs. Because of their unique sequences and masses, these analogs are readily measured and distinguished with mass spectrometric (MS) assays. Reviewed here is an insulin mass spectrometric immunoassay (MSIA) that combines micro-scale immunoaffinity capture with sensitive MS detection of insulin and its therapeutic analogs. An antibody reactive to all insulin analogs was used to affinity capture the insulin analogs. Following elution, insulins were detected with MALDI-TOF MS or LC-MS analysis. Isotopic resolution for insulin was achieved for both MS techniques, and several insulin analogs were detected at unique m/z signals. Porcine insulin, spiked in all samples, served as an internal reference standard for quantification. Linear standard curves spanning three orders of magnitude were obtained, with limits of detection of 15 pM for the MALDI-TOF MS and 1.5 pM for the LC-MS. This insulin assay was capable of detecting and quantifying not only human endogenous insulin, but also most of the therapeutic insulin analogs, which could find use in diagnosis of severe hypoglycemia and in sports anti-doping. Significance Insulin replacement therapy consists of injection of long- or fast-acting insulin analogs with slightly modified primary sequences compared to human insulin. Assays that are capable of detecting all insulin analogs are desired, not only for medical management of diabetes and severe hypoglycemia but also for sports anti-doping and toxicology.

The guinea pig is an alternate small animal model for the study of metabolism, including insulin sensitivity. However, only one study to date has reported the use of the hyperinsulinemic euglycemic clamp in anesthetized animals in this species, and the dose response has not been reported. We therefore characterized the dose-response curve for whole body glucose uptake using recombinant human insulin in the adult guinea pig. Interspecies comparisons with published data showed species differences in maximal whole body responses (guinea pig ≈ human < rat < mouse) and the insulin concentrations at which half-maximal insulin responses occurred (guinea pig > human ≈ rat > mouse). In subsequent studies, we used concomitant d-[3-3H]glucose infusion to characterize insulin sensitivities of whole body glucose uptake, utilization, production, storage, and glycolysis in young adult guinea pigs at human insulin doses that produced approximately half-maximal (7.5 mU·min−1·kg−1) and near-maximal whole body responses (30 mU·min−1·kg−1). Although human insulin infusion increased rates of glucose utilization (up to 68%) and storage and, at high concentrations, increased rates of glycolysis in females, glucose production was only partially suppressed (~23%), even at high insulin doses. Fasting glucose, metabolic clearance of insulin, and rates of glucose utilization, storage, and production during insulin stimulation were higher in female than in male guinea pigs ( P < 0.05), but insulin sensitivity of these and whole body glucose uptake did not differ between sexes. This study establishes a method for measuring partitioned glucose metabolism in chronically catheterized conscious guinea pigs, allowing studies of regulation of insulin sensitivity in this species.

Biosynthetic human insulins and analogs have replaced animal insulins and permitted structural modifications to alter the rate of absorption, duration of action, improve reproducibility of effects, and modulate relative efficacy in various target tissues. Several forms of rapidly acting insulins nearly achieve rapid pharmacokinetics and pharmacodynamics similar to first-phase insulin release. There is need for even faster-acting analogs to mimic normal physiology and improve control of postprandial glycemic excursions. Two biosynthetic insulin analogs have sufficiently long duration of action for use as once-daily basal insulins; controversy persists regarding their respective risks of hypoglycemia and relative glycemic variability.Basal-bolus therapy and insulin pump therapy, including closed-loop automated insulin delivery, require rapid-acting insulin analogs. The longer acting insulins can provide stable, reproducible basal insulin with reduced rates of hypoglycemia, particularly nocturnal hypoglycemia, greater efficacy in reducing mean glucose and glucose variability while increasing time in glucose target range. Inhalable human insulin provides very rapid action. Premixture of rapid-acting analogs with protamine has been useful for some patients with type 2 diabetes. An insulin analog with preferential efficacy at the liver has been developed and tested clinically but not marketed. Current research is aimed at developing even faster-acting insulin analogs. Long-acting basal insulins coformulated with GLP-1 receptor agonists or with a rapidly acting insulin analog have valuable clinical applications. Excipients, chaperones, local heating of the infusion site, and hyaluronidase have also been used to accelerate the absorption of insulin analogs.Biosynthetic human insulins have radically revolutionized management of both type 1 and type 2 diabetes worldwide. The ability to manipulate the structure and formulation of insulin provides for more physiologic pharmacokinetics and pharmacodynamics, enabling improved glycemic control, reduced risk of hypoglycemia, and reduced rates of long-term complications.

Examples are presented of successful data-processing, phasing and refinement strategies for non-merohedral twins, covering the range from minerals to proteins., In contrast to twinning by merohedry, the reciprocal lattices of the different domains of non-merohedral twins do not overlap exactly. This leads to three kinds of reflections: reflections with no overlap, reflections with an exact overlap and reflections with a partial overlap of a reflection from a second domain. This complicates the unit-cell determination, indexing, data integration and scaling of X-ray diffraction data. However, with hindsight it is possible to detwin the data because there are reflections that are not affected by the twinning. In this article, the successful solution and refinement of one mineral, one organometallic and two protein non-merohedral twins using a common strategy are described. The unit-cell constants and the orientation matrices were determined by the program CELL_NOW. The data were then integrated with SAINT. TWINABS was used for scaling, empirical absorption corrections and the generation of two different data files, one with detwinned data for structure solution and refinement and a second one for (usually more accurate) structure refinement against total integrated intensities. The structures were solved by experimental phasing using SHELXT for the first two structures and SHELXC/D/E for the two protein structures; all models were refined with SHELXL.

Administration of insulin for treatment of diabetes mellitus in dogs can stimulate an immune response, with a proportion of animals developing anti-insulin antibodies (AIA). For an IgG antibody response to occur, this would require B cell presentation of insulin peptides by major histocompatibility complex (MHC) class II molecules, encoded by dog leukocyte antigen (DLA) genes, in order to receive T-cell help for class switching. DLA genes are highly polymorphic in the dog population and vary from breed to breed. The aim of the present study was to evaluate AIA reactivity in diabetic dogs of different breeds and to investigate whether DLA genes influence AIA status. Indirect ELISA was used to determine serological reactivity to insulin in diabetic dogs, treated with either a porcine or bovine insulin preparation. DLA haplotypes for diabetic dogs were determined by sequence-based typing of DLA-DRB1, -DQA1 and -DQB1 loci. Significantly greater insulin reactivity was seen in treated diabetic dogs (n=942) compared with non-diabetic dogs (n=100). Relatively few newly diagnosed diabetic dogs (3/109) were found to be AIA positive, although this provides evidence that insulin autoantibodies might be involved in the pathogenesis of the disease in some cases. Of the diabetic dogs treated with a bovine insulin preparation, 52.3% (182/348) were AIA positive, compared with 12.6% (75/594) of dogs treated with a porcine insulin preparation, suggesting that bovine insulin is more immunogenic. Breeds such as dachshund, Cairn terrier, miniature schnauzer and Tibetan terrier were more likely to develop AIA, whereas cocker spaniels were less likely to develop AIA, compared with crossbreed dogs. In diabetic dogs, DLA haplotype DRB1*0015--DQA1*006--DQB1*023 was associated with being AIA positive, whereas the haplotype DLA-DRB1*006--DQA1*005--DQB1*007 showed an association with being AIA negative. These research findings suggest that DLA genes influence AIA responses in treated diabetic dogs.

Insulin injected directly into skeletal muscle diffuses rapidly through the interstitial space to cause glucose uptake, but this is blocked in insulin resistance. As glucotoxicity is associated with endothelial dysfunction, the observed hyperglycemia in diet-induced obese dogs may inhibit insulin access to muscle cells, and exacerbate insulin resistance. Here we asked whether interstitial insulin diffusion is reduced in modest hyperglycemia, similar to that induced by a high fat diet. Methods During normoglycemic (100 mg/dl) and moderately hyperglycemic (120 mg/dl) clamps in anesthetized canines, sequential doses of insulin were injected into the vastus medialis of one hindlimb; the contra-lateral limb served as a control. Plasma samples were collected and analyzed for insulin content. Lymph vessels of the hind leg were also catheterized, and lymph samples were analyzed as an indicator of interstitial insulin concentration. Results Insulin injection increased lymph insulin in normoglycemic animals, but not in hyperglycemic animals. Muscle glucose uptake was elevated in response to hyperglycemia, however the insulin-mediated glucose uptake in normoglycemic controls was not observed in hyperglycemia. Modest hyperglycemia prevented intra-muscularly injected insulin from diffusing through the interstitial space reduced insulin-mediated glucose uptake. Conclusion Hyperglycemia prevents the appearance of injected insulin in the interstitial space, thus reducing insulin action on skeletal muscle cells.

Background: Oral insulin as a preventive strategy and/or treatment of type 1 diabetes has been the target of much research. Producing oral insulins is a complex and challenging task, with numerous pitfalls, due to physiological, physical, and biochemical barriers. Our aim was to determine the impact of oral insulin on the delicate gut microbiota composition. Methods: Female nonobese diabetic mice were given oral porcine insulin 2 times a week from 5 weeks of age for 4 weeks, and then subsequently once a week for 21 weeks, or until euthanized. The mice were divided into groups on a gluten-reduced diet or a standard diet. Gut microbiota composition was analysed based on faecal samples, and the type 1 diabetes incidence of the mice was monitored. Results: We observed no influence of the oral porcine insulin on the gut microbiota composition of mice on a gluten-reduced or a standard diet at 9 weeks of age. Also, the administration of oral insulin did not influence the incidence of type 1 diabetes at 30 weeks of age. Conclusions: Oral porcine insulin does not alter the gut microbiota composition of nonobese diabetic mice on either a gluten-reduced diet or standard diet. Also, the oral porcine insulin did not influence the incidence of type 1 diabetes in the groups.

The purpose of the present study was to determine the activation of porcine insulin promoter (PIP) by three transcription factors: pancreatic and duodenal homeobox 1 (Pdx-1), v-maf musculoaponeurotic fibrosarcoma oncogene (MafA) and neurogenic differentiation 1 (NeuroD1) in non-beta islet cells cultured in vitro. In addition, the expression of the exogenous human islet amyloid polypeptide (hIAPP) gene driving by PIP in porcine kidney 15 (PK15) cells co-transfected with these transcription factors was also examined. In the present study, a series of vectors for gene overexpression were constructed, including pGL3-Pdx-1, pGL3-MafA, pGL3-NeuroD1, pGL3-PIP-LUC and pcDNA3.1-PIP-hIAPP. The dual-luciferase reporter assay showed that the PIP activity was increased in PK15 cells when overexpressing the exogenous transcription factors Pdx-1, MafA and NeuroD1. Introducing the PIP-hIAPP expression vector into PK15 cells combined with exogenous Pdx-1, MafA and NeuroD1 resulted in the efficient expression of hIAPP at the gene level, but not the protein. The current systematic porcine insulin promoter analysis provided the basic information for future utilization of porcine insulin.

Many experiments conducted in the literature have investigated the effect of microneedles (MNs) on insulin permeation across skin. There are also a number of articles that deal with the effect of MN insertion force in skin. However, there is little known on quantifying the relationship between the effect of MN insertion force and the amount of insulin permeated for given MNs. This issue is addressed in this article. MNs of 1100 µm and 1400 µm are used to conduct in vitro permeability experiments on porcine skin, using insulin. Histological images of MN treated skin are obtained from a microtome and the viscoelastic properties of the skin sample are measured using a rheometer. An in-house insertion force device is utilized that can reproducibly apply a specified force on MNs for a set period of time using compressed air. It is deduced that when porcine skin was pretreated with an applied force of 60.5 N and 69.1 N, the resultant amount of insulin permeated was approximately 3 µg and 25 µg over a 4-hour period for the MNs used. The amount of MN force applied to porcine skin was shown to be related to the amount of insulin permeated. An increase in insertion force increase the amount of insulin permeated. It was also demonstrated that using insufficient force may have reduced or prevented the amount of insulin passing through the skin, regardless of the geometry of the MNs.

The proline-rich Akt substrate of 40 kDa (PRAS40) is a component of the mammalian target of rapamycin complex 1 (mTORC1) and among the most prominent Akt substrates in skeletal muscle. Yet the cellular functions of PRAS40 are incompletely defined. This study assessed the function of PRAS40 in insulin action in primary human skeletal muscle cells (hSkMC).Insulin action was examined in hSkMC following small interfering RNA-mediated silencing of PRAS40 (also known as AKT1S1) under normal conditions and following chemokine-induced insulin resistance.PRAS40 knockdown (PRAS40-KD) in hSkMC decreased insulin-mediated phosphorylation of Akt by 50% (p0.05) as well as of the Akt substrates glycogen synthase kinase 3 (40%) and tuberous sclerosis complex 2 (32%) (both p0.05). Furthermore, insulin-stimulated glucose uptake was reduced by 20% in PRAS40-KD myotubes (p0.05). Exposing PRAS40-KD myotubes to chemokines caused no additional deterioration of insulin action. PRAS40-KD further reduced insulin-mediated phosphorylation of the mTORC1-regulated proteins p70S6 kinase (p70S6K) (47%), S6 (43%), and eukaryotic elongation 4E-binding protein 1 (100%), as well as protein levels of growth factor receptor bound protein 10 (35%) (all p0.05). The inhibition of insulin action in PRAS40-KD myotubes was associated with a reduction in IRS1 protein levels (60%) (p0.05), and was reversed by pharmacological proteasome inhibition. Accordingly, expression of the genes encoding E3-ligases F-box protein 32 (also known as atrogin-1) and muscle RING-finger protein-1 and activity of the proteasome was elevated in PRAS40-KD myotubes.Inhibition of insulin action in PRAS40-KD myotubes was found to associate with IRS1 degradation promoted by increased proteasome activity rather than hyperactivation of the p70S6K-negative-feedback loop. These findings identify PRAS40 as a modulator of insulin action.

This study evaluates the clinical efficacy and safety of NovoRapid (insulin aspart) compared to Actrapid™ (human neutral insulin) for diabetic ketoacidosis (DKA). In this retrospective study involving 40 patients, no statistically significant differences were observed between biochemical variables, infusion duration or complications in patients treated with insulin aspart or human neutral insulin. These results support the use of insulin aspart as an effective and safe alternative to human neutral insulin in DKA.

Transient neonatal diabetes mellitus is a rare disease usually associated with chromosome 6 abnormalities. Mutations of the genes encoding the potassium channel are rarely associated with these transitional forms. Herein, we report the clinical features of two siblings with a heterozygous mutation C679 G>A in the KCNJ11 gene.

The influence of insulin preparations (Actrapid and Ransulin) on the glucose and insulin blood level has been studied in patients with diabetes mellitus. It has been shown that comparable changes in the measured parameters are achieved in most patients with oral doses of Ransulin that are two to three times higher than the doses of Actrapid.

Excess lipid availability causes insulin resistance. We examined the effect of acute exercise on lipid-induced insulin resistance and TBC1 domain family member 1/4 (TBCD1/4)-related signaling in skeletal muscle. In eight healthy young male subjects, 1 h of one-legged knee-extensor exercise was followed by 7 h of saline or intralipid infusion. During the last 2 h, a hyperinsulinemic-euglycemic clamp was performed. Femoral catheterization and analysis of biopsy specimens enabled measurements of leg substrate balance and muscle signaling. Each subject underwent two experimental trials, differing only by saline or intralipid infusion. Glucose infusion rate and leg glucose uptake was decreased by intralipid. Insulin-stimulated glucose uptake was higher in the prior exercised leg in the saline and the lipid trials. In the lipid trial, prior exercise normalized insulin-stimulated glucose uptake to the level observed in the resting control leg in the saline trial. Insulin increased phosphorylation of TBC1D1/4. Whereas prior exercise enhanced TBC1D4 phosphorylation on all investigated sites compared with the rested leg, intralipid impaired TBC1D4 S341 phosphorylation compared with the control trial. Intralipid enhanced pyruvate dehydrogenase (PDH) phosphorylation and lactate release. Prior exercise led to higher PDH phosphorylation and activation of glycogen synthase compared with resting control. In conclusion, lipid-induced insulin resistance in skeletal muscle was associated with impaired TBC1D4 S341 and elevated PDH phosphorylation. The prophylactic effect of exercise on lipid-induced insulin resistance may involve augmented TBC1D4 signaling and glycogen synthase activation.

Developing combination drug delivery systems (CDDS) is a challenging but necessary task to meet the needs of complex therapy regimes for patients. As the number of multi-drug regimens being administered increases, so does the difficulty of characterizing the CDDS as a whole. We present a single-step method for quantifying three model therapeutics released from a model hydrogel scaffold using high-performance liquid chromatography (HPLC). Poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel tablets were fabricated via photoinitiated crosslinking and subsequently loaded with model active pharmaceutical ingredients (APIs), namely, porcine insulin (PI), fluorescein isothiocyanate-labeled bovine serum albumin (FBSA), prednisone (PSE), or a combination of all three. The hydrogel tablets were placed into release chambers and sampled over 21 days, and APIs were quantified using the method described herein. Six compounds were isolated and quantified in total. Release kinetics based on chemical properties of the APIs did not give systematic relationships; however, PSE was found to have improved device loading versus PI and FBSA. Rapid analysis of three model APIs released from a PEGDMA CDDS was achieved with a direct, single-injection HPLC method. Development of CDDS platforms is posited to benefit from such analytical approaches, potentially affording innovative solutions to complex disease states.

It has recently been proposed that the combination of skin barrier impairment using microneedles (MNs) coupled with iontophoresis (ITP) may broaden the range of drugs suitable for transdermal delivery, as well as enabling the rate of delivery to be achieved with precise electronic control. However, no reports exist on the combination of ITP with in situ drug loaded polymeric MN delivery systems. Furthermore, although a number of studies have highlighted the importance of MN design for transdermal drug delivery enhancement, to date, there has been no systematic investigation of the influence of MN geometry on the performance of polymeric MN arrays which are designed to remain in contact with the skin during the period of drug delivery. As such, for the first time, this study reports on the effect of MN heigth and MN density upon the transdermal delivery of small hydrophilic compounds (theophylline, methylene blue, and fluorescein sodium) across neonatal porcine skin in vitro, with the optimised MN array design evaluated for its potential in the electrically faciliatated delivery of peptide (bovine insulin) and protein (fluorescein isothiocyanate—labelled bovine serum albumin (FTIC-BSA)) macromolecules. The results of the in vitro drug release investigations revealed that the extent of transdermal delivery was dependent upon the design of the MN array employed, whereby an increase in MN height and an increase in MN density led to an increase in the extent of transdermal drug delivery achieved 6 h after MN application. Overall, the in vitro permeation studies revealed that the MN design containing 361 MNs/cm2 of 600 μm height resulted in the greatest extent of transdermal drug delivery. As such, this design was evaluated for its potential in the MN mediated iontophoretic transdermal delivery. Whilst the combination of MN and ITP did not further enhance the extent of small molecular weight solute delivery, the extent of peptide/protein release was significantly enhanced when ITP was used in combination of the soluble PMVE/MA MN arrays. For example, the cumulative amount of insulin permeated across neonatal porcine skin at 6 h was found to be approximately 150 μg (3.25%), 227 μg (4.85%) and 462 μg (9.87%) for ITP, MN, and MN/ITP delivery strategies, respectively. Similarly, the cumulative amount of FTIC-BSA delivered across neonatal porcine skin after a 6 h period was found to be approximately 110 μg (4.53%) for MN alone and 326 μg (13.40%) for MN in combination with anodal ITP (p

OBJECTIVE Animal studies indicate a prominent role of brain insulin signaling in the regulation of peripheral energy metabolism. We determined the effect of intranasal insulin, which directly targets the brain, on glucose metabolism and energy expenditure in humans. RESEARCH DESIGN AND METHODS In a double-blind, placebo-controlled, balanced within-subject comparison, 19 healthy normal-weight men (18–26 years old) were intranasally administered 160 IU human insulin after an overnight fast. Energy expenditure assessed via indirect calorimetry and blood concentrations of glucose, insulin, C-peptide, and free fatty acids (FFAs) were measured before and after insulin administration and the subsequent consumption of a high-calorie liquid meal of 900 kcal. RESULTS Intranasal insulin, compared with placebo, increased postprandial energy expenditure, i.e., diet-induced thermogenesis, and decreased postprandial concentrations of circulating insulin and C-peptide, whereas postprandial plasma glucose concentrations did not differ from placebo values. Intranasal insulin also induced a transient decrease in prandial serum FFA levels. CONCLUSIONS Enhancing brain insulin signaling by means of intranasal insulin administration enhances the acute thermoregulatory and glucoregulatory response to food intake, suggesting that central nervous insulin contributes to the control of whole-body energy homeostasis in humans.

In cartilage regenerative medicine, which is highly expected in the face of our aging society, insulin is the potent factor for culture media. To secure the safety of culture media, we attempted to use medical insulin formulations, and compared their effects on human articular or auricular chondrocytes between regular human insulin (R) and neutral protamine hagedorn insulin (N). In monolayer culture with the media containing either R or N, the cell growth reached approximately 15-fold-increase in 6 days, which showed no significant difference between them. These cells showed the equivalent ability to produce cartilage matrices, both in vitro and in vivo. Also, in the 3D culture of the dedifferentiated chondrocytes, either R or N increased gene expression of type II collagen at 3-4 folds in the combination with other growth factors, compared with basal medium, while insulin could similarly enhance both the redifferentiation and cartilage maturation. The in vitro half-life of each insulin in the presence of chondrocytes neither decreased within 3 days, suggesting little degradation in the culture media, unlike in the body. Although both R and N showed similar biological effects on cultured chondrocytes, we may choose the R for clinical practice because of its pure composition.

Udgivelsesdato: 2010 Some patients with type 2 diabetes continue to have high postprandial blood glucose levels on twice-daily regimens of 'low-ratio' premix insulin formulations (up to 30% rapid-acting, with 70% protracted insulin). These patients require intensified insulin therapy, which can be provided by a twice- or thrice-daily regimen of mid-ratio (50% rapid-acting and 50% protaminated intermediate-acting insulin - human or analogue) or high-ratio (70% rapid-acting and 30% protaminated insulin - analogue only) premix insulin. Alternatively, a third daily injection of low-ratio premix insulin can be added to the regimen, with the option of incorporating one or more injections of mid- or high-ratio premix as required, and as an alternative to basal-bolus therapy. How these mid- and high-ratio formulations differ from the low-ratio premix insulins is reviewed here, with the aim of identifying the role of these formulations in diabetes management. Glucose clamp studies have shown that premix analogues give serum insulin levels proportional to their percentage of rapid-acting uncomplexed insulin: the higher the proportion, the greater the maximum level reached. Other pharmacokinetic parameters were not always significantly different between the mid- and high-ratio formulations. In clinical trials, postprandial plasma glucose and glycated haemoglobin A1c (HbA(1c)) levels were significantly reduced with thrice-daily mid- /high-ratio premix analogue when compared with twice-daily low-ratio biphasic human insulin (BHI) 30/70 or once-daily insulin glargine. Moreover, glycaemic control with mid-/high-ratio premix analogue was found to be similar to that with a basal-bolus therapy. Mid- and high-ratio premix regimens are generally well tolerated. The frequency of minor hypoglycaemia was reportedly higher with mid- /high-ratio premix analogues than with BHI 30, but nocturnal hypoglycaemia was less frequent. Although there is little evidence that clinical outcomes with mid-ratio premix analogues are different from those with high-ratio, they are useful additions to the low-ratio formulations for the management of diabetes, and addressing postprandial hyperglycaemia in particular.

Aim: Randomized, open, single-centre, two-way crossover study comparing the pharmacokinetic (PK) and pharmacodynamic (PD) properties of subcutaneous (sc) regular human insulin (Actrapid) and oral insulin in a capsule form (Capsulin). Methods: Sixteen persons (12 males) with type 2 diabetes on oral hypoglycaemic agents (OHAs) participated. Mean (s.d.) age 60.2 (5.5) years, BMI 28.3 (3.4) kg/m2, haemoglobin A1c (HbA1c) 7.4% (1.1). Two 6-h isoglycaemic glucose clamp studies were conducted 11 days apart. All subjects received in random order 12U sc Actrapid on one clamp study day and either 150U or 300U Capsulin (Cap) on the other day. Glucose infusion rates (GIRs), plasma insulin and C-peptide concentrations were determined throughout each 6-h isoglycaemic clamp. Between the clamp study days, all patients received 150U Capsulin twice daily, dropping all their standard OHAs apart from metformin. Self-monitored blood glucose (SMBG) levels were taken four times a day between the clamp study days. Results: Administration of either Actrapid or Capsulin (150 and 300U) increased GIRs reaching a maximum values at approximately 280–330 min. Overall values for maximum GIR values were higher for Actrapid than either dose of Capsulin (p < 0.05). The significantly greater systemic insulin concentrations following Actrapid were reflected in the AUC0–6 h (910 ± 270 vs. 472 ± 245 pmol h/L; 950 ± 446 vs. 433 ± 218 pmol h/L; both p < 0.05 for Actrapid vs. 150U Capsulin and 300U Capsulin respectively). No difference was observed between 150U and 300U Capsulin. During the repeat-dosing period, good safety and tolerability were observed with Capsulin, and SMBG levels remained stable. At the poststudy visit, significant falls in HbA1c, weight and triglycerides were observed. Conclusions: Administration of the oral insulin Capsulin preparation demonstrated a significant hypoglycaemic action over a period of 6 h associated with only a small increase in circulating plasma insulin concentrations.

Easy-to-use insulin devices are an important tool in the treatment of diabetes. In this study, injection force requirements and dosage scale displays were evaluated for NovoPen 3 and NovoPen 4 (both from Novo Nordisk A/S, Copenhagen, Denmark).To simulate 5 years of expected lifetime usage, 5,475 injections were performed automatically. Before and after lifetime testing, the force required to expel 60 U of insulin from NovoPen 3 and NovoPen 4 with 30-gauge or 31-gauge needles was measured. To compare dosage scale displays, digital images of multiple settings were made, and the total inked areas of digits were converted to mm(2).At baseline, the mean +/- SD injection force of NovoPen 4 was 9.14 +/- 0.87 N and 16.55 +/- 1.17 N, which was significantly lower (P0.001) than for NovoPen 3, at 18.36 +/- 1.06 N and 29.81 +/- 1.26 N, with the 30-gauge and 31-gauge needle, respectively. After simulated lifetime testing, mean +/- SD injection force was 10.93 +/- 0.77 N and 17.77 +/- 1.14 N for NovoPen 4, which was significantly lower than the injection force of 18.54 +/- 0.94 N and 31.69 +/- 1.98 N for NovoPen 3 (P0.001). The mean dosage scale digit size was 1.63 mm(2) for NovoPen 3 and 7.82 mm(2) for NovoPen 4, with a mean difference of 6.19 mm(2). The display for NovoPen 4 was 4.74 times larger (P0.001).The mean injection force required to operate NovoPen 4 was reduced up to 50% compared with NovoPen 3 (P0.001), and the mean dosage display for NovoPen 4 was over four times larger than for NovoPen 3 (P0.001). Based on these findings, patients with diabetes who have manual or visual impairment should find it easier to dose insulin with NovoPen 4.

Circulating insulin binding capacity was studied for 10 to 24 months in forty diabetic patients who were being treated with insulin for the first time. They received either neutral highly purified pork insulin, bovine or porcine desphe insulin containing formulations, semisynthetic human insulin, or "conventional" acid chromatographed bovine surfen insulin over the entire duration of the study. In only 6 of the 23 patients on pork insulin, porcine desphe insulin, or human insulin were minimal antibody concentrations (less than or equal to 2 IU/l) observed. The bovine desphe insulin containing beef insulin, was markedly more immunogenic: only 3 out of 12 patients did not produce any antibodies. All five patients treated with bovine surfen insulin reacted with a strong antibody formation, which was, on average, six times as high as that in the patients on bovine desphe insulin (approximately 90 vs 14 IU/l). According to these results, bovine desphe insulins should be considered more valuable in insulin therapy than formulations of unmodified beef or of mixed beef/pork insulins.

Aims Activity in the renin-angiotensin system (RAS) may influence the susceptibility to cardiac arrhythmia. To study the effect of basal RAS activity on cardiac repolarization during myocardial stress induced by hypoglycaemia or hypoxaemia in healthy humans. Methods and results Ten subjects with high RAS activity and 10 subjects with low RAS activity were studied on three different occasions: (i) hypoglycaemia (nadir P-glucose 2.7 +/- 0.5 mmol/L), (ii) hypoxaemia (nadir pO(2) 5.8 +/- 0.5 kPa), and (iii) normoglycaemic normoxia (control day). QT parameters were registered by Hotter monitoring. Hypoglycaemia and hypoxaemia induced QTc prolongation (P < 0.001, both stimuli). The QT/RR slope and the VR increased as a function of hypoglycaemia, but were unaffected by hypoxaemia. Low RAS activity was associated with a steeper QT/RR slope in the recovery phase after both stimuli: hypoglycaemia: P = 0.04; hypoxia: P = 0.03. RAS activity had no impact on QTc [P = 0.48 (hypoglycaemia) and P = 0.40 (hypoxaemia)] or any of the other outcome variables. Conclusion Basal RAS activity has significant impact on QT dynamics, but not the corrected QT interval, during recovery from hypoglycaemia and hypoxaemia. The impact, however, is modest and more subtle than initially expected. The clinical relevance is unclear.

This study investigated whether strict control of plasma glucose levels with mealtime dosing of a rapid-acting insulin analog improves early morning fasting plasma glucose (FPG) levels in patients with type 2 diabetes. A rapid-acting insulin analog was administered at each mealtime to 40 Japanese patients with type 2 diabetes whose existing antidiabetic medication was discontinued. Approximately one-half (52.5%) of the patients achieved a minimum early morning FPG levels achievable (nadir FPG) of

The influence of the deposition pattern and spray characteristics of nasal powder formulations on the insulin bioavailability was investigated in rabbits. The formulations were prepared by freeze drying a dispersion containing a physical mixture of drum dried waxy maize starch (DDWM)/Carbopol 974P (90/10, w/w) or a spray-dried mixture of Amioca starch/Carbopol 974P (25/75, w/w). The deposition in the nasal cavity of rabbits and in a silicone human nose model after actuation of three nasal delivery devices (Monopowder, Pfeiffer and experimental system) was compared and related to the insulin bioavailability. Posterior deposition of the powder formulation in the nasal cavity lowered the insulin bioavailability. To study the spray pattern, the shape and cross-section of the emitted powder cloud were analysed. It was concluded that the powder bulk density of the formulation influenced the spray pattern. Consequently, powders of different bulk density were prepared by changing the solid fraction of the freeze dried dispersion and by changing the freezing rate during freeze drying. After nasal delivery of these powder formulations no influence of the powder bulk density and of the spray pattern on the insulin bioavailability was observed.

There are many reasons to believe that in the near future, the treatment of patients with Type 2 diabetes will be characterised by an increased use of insulin therapy. To ensure that insulin regimens are acceptable to patients, and implemented by physicians, they should be as simple and efficient as possible. Simplicity is synonymous with the regimen of once-daily basal insulin glargine given at any time of the day (at the same time each day). With such a strategy, the dose is adjusted by titrating to target fasting blood glucose values of 5.0 - 7.2 mmol/L (90 - 130 mg/dL). When these targets can no longer be achieved with reasonable doses of long-acting insulin, a rapid-acting insulin analogue should be added at meal times. A step-by-step strategy can be used; it is recommended that initially, a single daily prandial bolus of a rapid-acting insulin analogue is administered before the meal that leads to the highest post-meal blood glucose excursions. Further boluses can be added at other meal times as necessary, i.e, when post-meal blood glucose values remain above 10.0 mmol/L (180 mg/dL) and 7.8 mmol/L (140 mg/dL) at mid-morning and 2h-post-lunch or post-dinner times, respectively. This stepwise strategy may eventually lead to a standard basal-bolus regimen with 3 pre-meal injections of rapid-acting insulin analogues, a potentially small trade-off for achieving fairly-well controlled diabetes.

Porous spherical calcium carbonate (PS-CaCO(3)), in contrast to regular calcium carbonate (CaCO(3)), which has a cuboidal particle shape, has a characteristic spherical particle shape with a large number of porous, sliver crystals. The effect of PS-CaCO(3) as a drug carrier on intranasal insulin absorption was investigated in cynomolgus monkeys and healthy human volunteers. Each insulin formulation (powder) containing PS-CaCO(3) or regular CaCO(3) was administered intranasally. Serum insulin and glucose levels after administration were evaluated. The insulin absorption after intranasal administration with each CaCO(3) was found to be much more rapid than that after subcutaneous administration. The serum insulin level after intranasal insulin delivery (16 U per monkey) with PS-CaCO(3) showed a higher C(max) (403.5 microU/mL) and shorter T(max) (0.167 h) when compared with regular CaCO(3). The serum glucose level reduction rate after intranasal delivery using PS-CaCO(3) was faster than that of regular CaCO(3), reflecting the difference in absorption rates. Following repeated intranasal administrations for 4 weeks in monkeys, no toxicity was observed even with a maximum insulin dose level of 25 U. Furthermore, the intranasal insulin absorption rate with PS-CaCO(3) in healthy humans was also observed to be considerably faster than that with regular CaCO(3). Effects of PS-CaCO(3) on a more effective absorption behavior of insulin were considered to be the result of a greater affinity between the nasal mucosa layer and PS-CaCO(3), which is closely related to its structural characteristics. Thus, intranasal insulin delivery using PS-CaCO(3) is thought to be a safe and highly available system enabling more effective insulin absorption behavior with the appearance of endogenous postprandial insulin secretion in healthy humans. We believe that our intranasal insulin delivery system enabling a rapid and short-acting pharmacological effect against postprandial hyperglycemia will be more beneficial than pulmonary insulin delivery systems in the treatment of diabetes.

The altered pharmacokinetics of new rapid-acting insulin analogs make them very effective in controlling blood glucose peaks after meals. However, simple replacement of regular human insulin by rapid-acting analogs may not be sufficiently effective for overall daily glycemic control. Both basal and bolus insulins need to be addressed with overall changes in insulin regimens for long-term reductions of glycated hemoglobin (HbA(1c)) levels. Clinical studies in patients with type 1 diabetes have shown that better control of postprandial peaks together with optimal use of basal insulin, in multiple injection regimens or with continuous subcutaneous insulin infusion, resulted in improved HbA(1c) levels of 0.3-0.4% in comparison with regular human insulin. In patients with type 2 diabetes the combination therapy of insulin lispro with an oral agent improved metabolic control in the range of 1.5-2.5%. Such decreases in HbA(1c) should lead to a reduction in risk of complications with both type 1 and type 2 diabetes. The better overall metabolic control with rapid-acting insulin analogs was not accompanied by any increased risk for hypoglycemia.

For a long time, scientists believed that macromolecules can only be introduced through the skin with a hypodermic needle or some other harsh treatment that locally damages the skin barrier. It is now clear that macromolecules can be administered epicutaneously, so that insulin, for example, can exhibit therapeutic effects in patients with type 1 diabetes mellitus. When carriers are employed for the purpose, the drugs must be associated with specifically designed vehicles in the form of highly deformable aggregates and applied on the skin non-occlusively. Using such optimised carriers, so-called Transfersomes, ensures reproducible and efficient transcutaneous carrier and drug transport. Insulin-loaded Transfersomes, for example, can deliver the drug through the non-compromised skin barrier with a reproducible drug effect that resembles closely that of an ultralente insulin injected under the skin; the pharmacokinetic and pharmacodynamic properties of the injected and transdermal insulin are also comparable. The efficacy of transcutaneously delivered insulin in Transfersomes is not affected by the previous therapy, similar results having been measured in patients normally receiving intensified insulin therapy or a continuous subcutaneous infusion of insulin solution. Systemic normoglycaemia that lasts at least 16 hours has been achieved using a single non-invasive, epicutaneous administration of insulin in Transfersomes. Experience with other drugs suggests that the biodistribution of injected and transcutaneously delivered drugs can be very similar. This notwithstanding, Transfersomes can be designed and applied so as to mediate site-specific drug delivery into peripheral musculoskeletal tissues or into the skin, as may be desired.

As a result of a number of factors, the treatment of insulin-dependent diabetes has moved away from using insulin of beef or pork origin to using recombinant (biosynthetic) insulin preparations. However, some people with type 1 diabetes can manage their diabetes better using animal-sourced insulin. Despite dwindling options and decreased production, animal-sourced insulin (and pork insulin in particular) is still available on the Canadian market. This communication describes the actions taken by Health Canada with respect to the availability of animal insulin.Communication courte -- Le rôle de l’insuline d’origine animale dans le traitement du diabète de type 1 et sa disponibilitéEn raison d’un certain nombre de facteurs, on traite désormais davantage le diabète insulinodépendant au moyen de préparations d’insuline recombinante (biosynthétique) qu’au moyen d’insuline d’origine bovine et porcine. Cependant, certaines personnes atteintes du diabète de type 1 parviennent à mieux prendre leur diabète en charge à l’aide d’insuline d’origine animale. Malgré la disponibilité de plus en plus réduite de l’insuline d’origine animale et le déclin de sa production, on peut encore s’en procurer sur le marché canadien, en particulier l’insuline porcine. Cette communication décrit les mesures prises par Santé Canada relativement à la disponibilité de l’insuline d’origine animale.

Neutral protamine Hagedorn (NPH) insulin is one of the most commonly used insulins in insulin pens. NPH in pen cartridges is in a two-phase solution with either a solvent or a short-acting insulin, and needs adequate mixing for complete resuspension. We assessed whether NPH insulin is accurately resuspended by patients and the association of suspension errors with diabetes control.109 patients (39 with type 1 diabetes) who had received conventional diabetic education had the NPH content of their cartridges measured by an optical system; a control cartridge was designated as 100%. A questionnaire was used to assess clinical details and insulin suspension habits. After the information about residual insulin error was known, all 109 patients were instructed to resuspend their insulin by rolling and tipping the pen 20 times. 52 patients were randomly selected to have cartridges re-analysed 3 months or 6 months later and to complete another questionnaire.Only 10 (9%) of 109 patients tipped and rolled their pen more than ten times. NPH insulin content ranged from 5% to 214% and varied by more than 20% in 71 (65%) of 109 cartridges. There was no relation between inadequate suspension and the frequency of hypoglycaemic episodes (r=0.2, p=0.08). For all patients, there was a correlation between the absolute error of NPH suspension and cycles of rolling and tipping the pen (r=-0.23, p0.05). After education on resuspending the pen's contents, data were available from 44 of 52 patients; suspension errors decreased in 35 (80%), were unchanged in three (7%), and increased in six (13%). The 35 patients with improved NPH insulin suspension had fewer mean hypoglycaemic episodes per month compared with the previous period (0.4 [SD 0.1] vs 1.0 [0.3], p0.05). Mean HbA1c values in patients with improved suspension quality did not differ from baseline (8.4% [0.3] vs 8.9% [0.4], p=0.07). Mixing of NPH insulin by a mechanical device showed that at least 20 cycles were necessary before complete resuspension was obtained.Inadequate NPH insulin suspension is common. We recommended that patients tip pens that contain NPH insulin at least 20 times, since inadequate mixing may impair diabetes control.

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. [Diabetologia (1999) 42: 958–964]

Hypoglycemia has well-described effects on glucose metabolism, whereas the possible effects on hepatic amino nitrogen conversion in relation to muscle amino nitrogen flux are more uncertain.We studied six healthy young male subjects three times, i.e. for 6 h in the basal state, during a 6-h euglycemic hyperinsulinemic (1.5 mU/kg/min) clamp and during a 6-h hypoglycemic (plasma glucose below 2.8 mmol/l) clamp. Alanine (2 mmol/kg body weight/h) was infused for 3 h to describe the relationship between blood amino nitrogen concentrations and hepatic ureagenesis estimated from urea urine excretion and accumulation in body water. The slope of this relationship is denoted functional hepatic nitrogen clearance (FHNC) and quantifies substrate-independent alterations in hepatic amino nitrogen degradation. In parallel, amino nitrogen balances across muscles were estimated by the forearm flux method.Euglycemia decreased circulating glucagon values (100+/-25 ng/l vs. 160+/-30 ng/l), whereas hypoglycemia doubled glucagon (350+/-45 ng/l, p0.05). Hepatic nitrogen clearance (FHNC) decreased during hyperinsulinemic euglycemia (19.5+/-3.4 l/h vs. 30.6+/-5.7 l/h, p0.01), whereas forearm net uptake of amino nitrogen increased (130+/-40 nmol/100 ml x min vs. control: -10+/-4 nmol/100 ml x min). During hypoglycemia there was a 3-fold increase in hepatic nitrogen clearance up to 83.0+/-16.8 l/h (p0.01) and increased release of amino nitrogen from the forearm (-100+/-30 nmol/100 ml x min, p0.01).Hypoglycemia in man induces a marked increase in hepatic amino- to urea-N clearance. This catabolic response to hypoglycemia in the liver may be of primary importance for muscle amino acid release. Our data are compatible with the notion that liver and muscle together are responsible for catabolism during hypoglycemia, and that glucagon may be the primary mediator via its effect on liver metabolism.

OBJECTIVE Since 1985, we have used indwelling catheters (Insuflon, Maersk Medical, Lynge, Denmark; Chronimed, Minnetonka, MN) to lessen pain when injecting insulin. However, some patients experience a rise in blood glucose after using indwelling catheters for a few days. We therefore studied the absorption of 125I-labeled insulin when using indwelling catheters. RESEARCH DESIGN AND METHODS Five men and five women participated (age 18–25 years, C-peptide negative, HbA1c 9.0 ± 1.0% [mean ± SD, DCA-2000 method], diabetes duration 5–21 [median 9.5] years). After thyroid blockage with potassium iodide, we injected 5IU of 125I-labeled short-acting insulin subcutaneously in the abdomen (“ordinary injection”) and 5 IU on the contralateral side through an indwelling catheter (“catheter injection”). The injection/insertion area was free of lipohyper- and lipohypotrophies. Disappearance rate was measured for 180 min with a gamma camera. The patients injected all premeal injections of short-acting insulin through the same indwelling catheter in the following 4 days. The investigation procedure was repeated day 3 and 5. RESULTS We found no statistically or clinically (95% CI) significant difference in residual activity of 125I-insulin after 60 min or in time for 50% of the injected depot to disappear (T-50%) among catheter injections on day 1, 3, and 5; ordinary injections on days 1, 3, and 5; or catheter and ordinary injections on days 1, 3, and 5, respectively. HbA1c correlated both to T-50% (r = 0.73, P = 0.016) and residual activity of 125I-insulin after 60 min (r = 0.69, P = 0.028), indicating that patients with a slower absorption will have a less ideal metabolic control when using premeal bolus injections. CONCLUSIONS We conclude that using indwelling subcutaneous catheters for insulin injections for up to 4 days does not affect the absorption of short-acting insulin.

The authors present a case of severe electrolyte and acid-base disturbances after administration of sorbitol. The rate of sorbitol infusion was in excess of the stated maximum safe infusion rate in parenteral nutrition. Sorbitol was used for treating raised intracranial pressure. The mechanisms of sorbitol metabolism leading to hyperlactacemia, lactic acidosis, hyperuricemia, depletion of intracellular ATP and ADP and depletion of inorganic phosphates are described.

This article concerns the development and co-validation of a porcine insulin (pINS) certified reference material (CRM) produced by the National Institute of Metrology, People’s Republic of China. Each CRM unit contained about 15 mg of purified solid pINS. The moisture content, amount of ignition residue, molecular mass, and purity of the pINS were measured. Both high-performance liquid chromatography–isotope dilution mass spectrometry and a purity deduction method were used to determine the mass fraction of the pINS. Fifteen units were selected to study the between-bottle homogeneity, and no inhomogeneity was observed. A stability study concluded that the CRM was stable for at least 12 months at -20 °C. The certified value of the CRM was (0.892 ± 0.036) g/g. A co-validation of the CRM was performed among Chinese, Japanese, and Korean laboratories under the framework of the Asian Collaboration on Reference Materials. The co-validation results agreed well with the certified value of the CRM. Consequently, the pINS CRM may be used as a calibration material or as a validation standard for pharmaceutical purposes to improve the quality of pharmaceutical products.

Seven type I insulin-dependent diabetic patients on continuous ambulatory peritoneal dialysis treatment were selected for this study. Each patient participated in three different 6-hour 'single-dwell' studies on 3 consecutive days. A mean dose of 33 +/- 1.3 U Insulin Actrapid Human was given intraperitoneally each day. The procedures for intraperitoneal insulin administration were: (1) with 1,000 ml Ringer lactate; (2) with 1,000 ml 3.86% glucose-containing dialysate, and (3) into an empty peritoneal cavity. The calculation of the intraperitoneal volume was done with a single injection indicator dilution technique in which 100 kBq radioiodinated serum albumin (RISA) was added into the fluid prior to instillation. Free insulin and glucose were analyzed at 16 time intervals in blood and in dialysate during each dwell. After drainage the peritoneal cavity was rinsed with 1,000 ml Ringer lactate followed by two consecutive 5-hour exchanges with 2,000 ml glucose-containing dialysate. Recovery of insulin and RISA was measured in rinsing fluid and in sampled dialysate during the 6-hour dwell. The kinetic calculations made for insulin were disappearance rate (mU/min) from the peritoneal cavity, and appearance rate in circulating blood. After drainage and rinsing, 66.0 +/- 10 and 71.8 +/- 9.8% of the insulin instilled had disappeared after 6 h from the glucose fluid and from the Ringer solution respectively and did not differ significantly. However, the estimated disappearance rate from the peritoneal cavity was significantly higher in Ringer than in glucose from the time interval 120 to 360 min. A high and peak-shaped insulin concentration in the plasma was found following insulin injection into an empty peritoneal cavity, and was significantly higher than when insulin was dissolved in a 1,000-ml fluid volume. However, a higher blood concentration was also found when Ringer was instilled than when a hyperosmolal glucose solution was instilled. A high first-pass elimination in the liver is suggested. In conclusion, fluid volume and also the osmolality of the solution in the peritoneal cavity decreases the transport rate, but not the bioavailability of insulin given intraperitoneally. Both a high peak shape and a continuous insulin appearance in blood can be achieved. It is suggested that there is a high first-pass elimination of insulin during absorption from the peritoneal cavity. However, the values are uncertain and extended investigations must be done.

Background. The tumor-bearing state is associated with increased circulating glucagon levels that may play an etiologic role in cancer cachexia. The secretion of glucagon can be inhibited with long-term somatostatin analogs, and, in combination with insulin, should maximally reverse the low insulin/glucagon ratio seen in cancer cachexia. The goal of this study is to examine the effect of somatostatin (octreotide) and insulin in a model of cancer cachexia and to determine whether inhibition of glucagon secretion will reverse some of the abnormalities in carbohydrate metabolism to selectively benefit host versus tumor metabolism Methods. Sixty-seven female Lewis rats were subcutaneously inoculated with 1 × 106 metastasizing mammary adenocarcinoma tumor cells. On day 30 the animals were randomized into four groups to receive (1) tumor-bearing control (saline injections); (2) octreotide, 150 μg/kg intraperitoneally twice a day; (3) neutral protamine Hagedorn insulin, 5 units/kg subcutaneously twice a day; or (4) both insulin and octreotide injections. A fifth group of non-tumor-bearing controls was included. The animals received treatment for 5 days and were then killed. Results. The tumor-bearing state was found to be associated with an increase in glucagon levels and a significant decrease in the insulin/glucagon ratio. The combination of somatostatin + insulin resulted in a 23-fold increase in the insulin/glucagon ratio without causing significant host morbidity from hypoglycemia. This increased insulin/glucagon ratio was associated with increased carcass weight, increased muscle weight, increased muscle protein, increased liver cellular protein, increased liver microsomal P-450 content, and decreased tumor protein content compared with the tumor-bearing controls. These results were not seen with insulin or somatostatin alone. Hepatic lactate dehydrogenase, glucose-6-phosphatase, and fructose-1,6-diphosphatase activities were increased as a result of combination hormone treatment. Conclusions. Combination hormone treatment with somatostatin and insulin results in a marked increase in the insulin/glucagon ratio and a selective nutritional benefit to the host. The inhibition of tumor-associated hyperglucagonemia should be considered in the treatment of cancer cachexia

OBJECTIVE To study the absorption rate of rapid-acting insulin from subcutaneous injection sites in nonobese and obese non-insulin-dependent diabetes mellitus (NIDDM) patients. RESEARCH DESIGN AND METHODS Ten nonobese and 10 obese NIDDM patients (body mass indexes 24.1 ± 0.4 and 31.4 ± 0.8 kg/m2, respectively) received four subcutaneous injections of 125I-labeled rapid-acting insulin (Actrapid Human, 5 U): three in the abdominal wall above, lateral to, and below the umbilicus; and one in the thigh. The depth of the subcutaneous fat layer was measured using ultrasound techniques. The residual radioactivity was monitored externally for 270 min. RESULTS The disappearance half-life of 125I-insulin was between 4 and 6 h from all injection sites, with the exception of the upper abdominal area in the nonobese subjects, where it measured ∼ 3 h. The residual radioactivity did not differ between nonobese and obese patients measured from any of the sites. In the nonobese group, the most rapid absorption of 125I-insulin was found from the upper abdominal area and the slowest from the thigh. In the obese group, the absorption rates did not differ between sites. No correlation was found between the depth of the fat layer and the residual radioactivity when measured at any site. CONCLUSIONS Our results indicate that the absorption of rapid-acting insulin is markedly slow in both obese and nonobese NIDDM patients compared with IDDM patients and healthy subjects studied previously. In the nonobese group, the most rapid absorption of 125I-insulin is obtained after injection into the upper abdominal area. Inter- and intraregional differences are small in the obese patients. Consequently the choice of injection site is of little importance in this group.

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

Formation of chiral amyloid superstructures is a newly recognised phenomenon observed upon agitation-assisted fibrillation of bovine insulin. Here, by surveying several amyloidogenic precursors we examine whether formation of such entities is unique to bovine insulin. Our results indicate that only bovine, human, and porcine insulins are capable of chiral superstructural self-assembly. A tiny covalent perturbation consisting in reversal of Pro B28 -Lys B29 residues in a human insulin analog is sufficient to prevent this process. Our study suggests that insulin’s dimer-forming interface – specifically the B-chain’s C-terminal fragment – may acquire the new role of a molecular velcro upon lateral alignment of individual fibrils into superstructures. Structured summary of protein interactions BI and BI bind by infrared spectroscopy ( View interaction ) HI and HI bind by atomic force microscopy ( View interaction ) HEWL and HEWL bind by circular dichroism ( View interaction ) BI and BI bind by circular dichroism ( View interaction ) α-LAC and α-LAC bind by circular dichroism ( View interaction ) BI and BI bind by atomic force microscopy ( View interaction ) α-LAC and α-LAC bind by atomic force microscopy ( View interaction ) HI and HI bind by circular dichroism ( View interaction ) PI and PI bind by circular dichroism ( View interaction ) PI and PI bind by atomic force microscopy ( View interaction ) HEWL and HEWL bind by atomic force microscopy ( View interaction )

The development of insulin therapy has not been stopped since the manufacturing of human insulin, because better mimic of physiological insulin response made it necessary to modify the human insulin molecule in order to create rapidly absorbing insulin analogues and 24-hour acting basal insulin analogues. Clinical observations indicate that the complete switch from human basal-bolus therapy to insulin analogues means not only “unit-for-unit” switch but it represents a transfer to an insulin therapy with different basal/bolus ratio as a result of different pharmacokinetic and pharmacodynamic properties of insulin and the level of insulin resistance of the patient. With reference to a case-history, the author presents his experience on a switch from human insulin to insulin analogue. Furthermore, the author summarizes data obtained from a few cases reported in international literature which draw the attention to the fact that the basal/bolus ratio should be adjusted individually, which may be the key for the success in the therapy in these cases. Orv. Hetil., 2012, 153, 1589–1593.