Human Insulin Analogues Research Articles

Relationship between mortality and dosage scheme as key issue in the design of regulatory toxicological studies of different types of human insulin analogs

Relationship between mortality and dosage scheme as key issue in the design of regulatory toxicological studies of different types of human insulin analogs

Long‐term adaptation of Saccharomyces cerevisiae to the burden of recombinant insulin production

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30 g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation.

Differences in metabolic and mitogenic signalling of insulin glargine and insulin aspart B10 in rats

Aims/hypothesisIn vitro, insulin glargine (A21Gly,B31Arg,B32Arg human insulin) has an insulin receptor (IR) profile similar to that of human insulin, but a slightly higher affinity for the IGF-1 receptor (IGF1R). Insulin aspart B10 (B10Asp human insulin) (AspB10), the only insulin analogue with proven carcinogenic activity, has a greater affinity for IGF1R and IR, and a prolonged IR occupancy time. The pharmacological and signalling profile of therapeutic and suprapharmacological doses of glargine were analysed in different tissues of rats, and compared with human insulin and AspB10.MethodsMale Wistar rats were injected s.c. with human insulin or insulin analogue at doses of 1 to 200 U/kg, and the effects on blood glucose and the phosphorylation status of IR, IGF1R, Akt and extracellular signal-regulated protein kinase 1/2 in muscle, fat, liver and heart samples were investigated.ResultsGlargine, AspB10 and human insulin lowered blood glucose, with the onset of action delayed with glargine. Glargine treatment resulted in phosphorylation levels of IR and Akt that were comparable with those achieved with human insulin, although delayed in time in some tissues. AspB10 treatment resulted in at least twofold higher phosphorylation levels and significantly longer duration of IR and Akt phosphorylation in most tissues. None of the insulin treatments resulted in detectable IGF1R phosphorylation in muscle or heart tissue, whereas intravenous injection of IGF-1 increased IGF1R phosphorylation.Conclusions/interpretationThe IR signalling pattern of AspB10 in vivo is distinctly different from that of human insulin and insulin glargine, and might challenge the notion that activation of IGF1R plays a role in the observed carcinogenic effect of AspB10.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-2923-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

The challenge of improved secretory production of active pharmaceutical ingredients in Saccharomyces cerevisiae: A case study on human insulin analogs

The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. Great attention has been put on improved secretory production of active pharmaceutical ingredients, and the secretory pathway of this eukaryotic host has been the playground of diverse strain engineering studies, aiming at enhanced cellular capacities for folding and trafficking of the target proteins. However, the cellular quality assessment for secretory proteins remains mostly unpredictable, and different target proteins often do not picture similar secretion yields, underlining the dependency of efficient secretion on the physicochemical properties of the protein of interest. In this study, two human insulin analog precursors (IAPs) with minor differences in their amino acid sequences were used as model secretory proteins. No differences between cells expressing these two proteins were found in the IAP transcript levels, gene copy numbers, or intra-cellularly accumulated proteins, yet a more than sevenfold difference in their secretion yields was found. Physiological characterization of cells expressing these proteins in batch processes revealed no significant difference in their specific growth rate, but an altered overflow metabolism. Global transcriptome analysis carried out in chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated and indicated an increased degradation of the IAP with the low secretion yield. In silico protein structure modeling of the IAPs suggested a difference in conformational stability, induced by the amino acid substitution, which most likely resulted in disparity in trafficking through the secretory pathway and thus a large difference in secretion yields.

Correction of Stress Hyperglycemia in Patients with Acute Surgical Abdominal Diseases

Critically ill patients admitted to intensive care units for the clinical manifestations of stress hyperglycemia represent a group in which attempts are most frequently undertaken to employ various methods of its correction. Objective: to compare different techniques for correction of stress hyperglycemia in patients with acute surgical abdominal disease complicated by peritonitis. Materials and methods: In this single-center randomized controlled trial, stress hyperglycemia was corrected by different procedures in 60 patients with acute surgical abdominal disease complicated by peritonitis. Results: As compared with traditional procedures for correction of stress hyperglycemia, the human insulin analogue aspart (Novorapid) improves stressful glycemic control and reduces the risk of hypoglycemia. During intensive insulin therapy, the ultrashort-acting insulin group showed showed a trend for a reduction in the number of manifestations of multiple organ failure and signs of systemic inflammation and decline in the number of hospital days. The least duration of mechanical ventilation was noted in the small-volume food group.

Pharmacotherapy in Type 1 Diabetes

Pharmacotherapy in Type 1 Diabetes

Insulin therapy and body weight: benefits of biphasic human insulin analogue NovoMix 30

Body weight (BW) excess is a characteristic problem for type 2 diabetes mellitus (T2DM) both as its pathogenetic feature and as a side effect of blood glucose lowering therapy. In the latter case reduction of glycosuria and frequent hypoglycemic events are primarily blamed for BW gain, but additional factors like direct effect of insulin on lipogenesis and influence of its supraphysiologic levels on regulation of appetite via CNS structures are also under discussion.Advances of the last years have brought new hope due to introduction of drug classes that do not affect BW. However, fraction of patients dependent on exogenous insulin shows stable trend for growth. Deterioration of beta-cell secretory capacity makes insulin an ultimately indispensable tool in the foreseeable future. As so, insulin therapy modalities with minimal impact on BW are preferable. In this regard human insulin analogues of both rapid and prolonged action have certain advantages.Current article addresses influence of pre-mixed insulin preparation NovoMix 30 (Novo Nordisk, Denmark) on BW. A summary of several studies of substantial duration (up to 3 years) suggests a neutral effect on BW in various categories of T2DM patients (including obese and elder patients).Therapy with pre-mixed preparations is an adequately safe and effective T2DM treatment modality and is advantageous for patients in whom BW gain is particularly unfavorable.

Amino acid sequence determinants in self-assembly of insulin chiral amyloid superstructures: Role of C-terminus of B-chain in association of fibrils

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 ProB28-LysB29 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 interactionsBI and BIbind by infrared spectroscopy (View interaction)HI and HIbind by atomic force microscopy (View interaction)HEWL and HEWLbind by circular dichroism (View interaction)BI and BIbind by circular dichroism (View interaction)α-LAC and α-LACbind by circular dichroism (View interaction)BI and BIbind by atomic force microscopy (View interaction)α-LAC and α-LACbind by atomic force microscopy (View interaction)HI and HIbind by circular dichroism (View interaction)PI and PIbind by circular dichroism (View interaction)PI and PIbind by atomic force microscopy (View interaction)HEWL and HEWLbind by atomic force microscopy (View interaction)

Protamine-containing insulins are strong risk factors, and human insulin analogues are possible risk factors for insulin autoantibody: case-control study

Insulin autoantibody is known to cause fluctuation of blood glucose. We examined whether medications for diabetes are risk factors for insulin autoantibody. Especially, we examined the associations between types of insulin and insulin autoantibody. We performed a case-control study. From April 2005 to March 2010, insulin autoantibody was measured 273 times in 217 patients in our hospital. Insulin autoantibody was positive (greater than 10%) 53 times in 19 patients (case), and was negative 220 times in 198 patients (control). Oral hypoglycemic agents were not risk factors for insulin autoantibody; the odds ratio was 0.0. In contrast, insulin use was a significant risk factor for insulin autoantibody; the odds ratio (95% confidence interval) was 56.3 (7.3-432.5). As for the types of insulin and insulin autoantibody, human insulins without protamine were not risk factors; the odds ratio was 0.0. For protamine-containing insulins, the odds ratio and adjusted odds ratio (adjusted by age, gender, and disease: type 1 diabetes mellitus, type 2 diabetes mellitus, and no diabetes) were 35.3 (9.6-129.5) and 29.6 (7.6- 115.4), respectively. For Aspart-containing insulins, they were 6.2 (2.2-17.9) and 3.8 (1.2- 12.0), respectively. For Glargine, they were 3.2 (0.6-16.7) and 1.3 (0.2-8.3), respectively. To decrease the problem of insulin antibody, avoiding the use of protamine-containing insulins and avoiding the use of human insulin analogues might be preferable for the patients with diabetes.

Desensitization of allergy to human insulin and its analogs by administering insulin aspart and insulin glargine

Insulin allergy is a rare clinical situation. We report a 51-year-old patient with type 2 diabetes who required multiple daily insulin injections. The patient developed allergy to human regular insulin and insulin analogs (insulin aspart and insulin glargine), which was resolved by subcutaneous insulin desensitization.

Insulin injection guidelines for peri-operative and critically ill patients

Anesthesiologists and intensivists are encountering increasing number of diabetic patients in daily clinical practice. Majority of such patients may require insulin injections for control of hyperglycemia. Advancements in diabetes management have led to usage of newer insulin injections ranging from human insulin and insulin analogs to glucagon-like peptides-1 analogs. The adequacy of glycemic control and successful outcome with such therapeutic interventions depends upon the adoption of correct injection techniques and procedures. Peri-operative and critically ill diabetic patients are highly prone to develop acute complications of diabetes if appropriate therapeutic strategies are not formulated and implemented. As such, the in-depth knowledge and awareness about various injection technique guidelines is essential from the patient care and healthcare provider's perspective in the operative and critical care settings. This description is an abridged version of the Forum for Injection Techniques, India: The first Indian recommendations for best practice in insulin injection technique and their significance in peri-operative period and critically ill patients in intensive care units (ICU). These insulin injection techniques are based on evidence-based recommendations and are meant to improve the management of diabetes by the attending staff and physicians in operative and critical care arenas.

Conformational Memory Effect Reverses Chirality of Vortex-Induced Insulin Amyloid Superstructures

Formation of amyloid fibrils is often associated with intriguing far-from-equilibrium phenomena such as conformational memory effects or flow-driven self-assembly. Insulin is a model amyloidogenic polypeptide forming distinct structural variants of fibrils, which self-propagate through seeding. According to infrared absorption, fibrils from bovine insulin ([BI]) and Lys(B31)-Arg(B32) human insulin analogue ([KR]) cross-seed each other and imprint distinct structural features in daughter fibrils. In the absence of preformed [KR] amyloid seeds, bovine insulin agitated at 60 °C converts into chiral amyloid superstructures exhibiting negative extrinsic Cotton effect in bound thioflavin T. However, when agitated bovine insulin is simultaneously cross-seeded with [KR] amyloid, daughter fibrils reveal a positive extrinsic Cotton effect. Our study indicates that dramatic changes in global properties of amyloid superstructures may emerge from subtle conformational-level variations in single fibrils (e.g., alignment and twist of β-strands) that are encoded by memory effects.

Efficacy and safety of treatment with human insulin analogues in daily management of insulin naive patients with type 2 diabetes mellitus: results of multicenter 52-week observational study A1chive

Modern insulin analogues allow safe improvement of glycemic control (not accompanied with increase in body mass or rate of severe hypoglycemic events) and its efficient maintenance during long period of time. In this paper we present results of multicenter 52-week prospective observational study A1chive (Russian cohort, n=9342), designed to assess safety and efficacy of treatment with human insulin analogues (Levemir?, NovoMix? 30 and NovoRapid? (Novo Nordisk) in daily management of insulin naive patients with type 2 diabetes mellitus. Prescription of human insulin analogues resulted in clinically and statistically significant improvement of glycemic control irrespective of initial insulin regimen. Therapy by various regimens (Levemir?, NovoMix? 30 or Levemir? + NovoRapid?) under conditions of daily clinical practice leads to marked improvement in glycemic control as measured by HbA1c (-2.9% in 12 month) with no evidence for negative influence on tolerability and short-term safety of the treatment. Furthermore, decrease in HbA1c levels was associated with pronounced and stable decrease in fasting and postprandial glucose levels (-5.0 mmol/L and -5.7 mmol/L, respectively). By the end of the follow-up all regimen groups reported improvement of QoL by treatment satisfaction criterion.

Type 1 Ig-E mediated allergy to human insulin, insulin analogues and beta-lactam antibiotics

Insulin, a crucial therapeutic agent for diabetes mellitus, has been rarely associated with hypersensitivity events. We present a 69-year-old type-2 diabetic patient with urticariform lesions on the sites of subcutaneous injection of insulin. The patient denied any known allergies, except for an unspecific cutaneous reaction after intramuscular penicillin administration in childhood. Prick tests revealed positive reactions to all tested human insulins and insulin analogues. Serum IgE levels were above normal range and RAST tests were positive for human, bovine and porcine insulins, as well as beta-lactams. Type 1 IgE-mediated allergy to insulin analogues demands a prompt diagnosis and represents a significant therapeutic challenge in diabetic patients.

持続血糖モニター（CGM）による血糖変動の把握が有用であったインスリン抗体陽性の糖尿病の1例

A 76-year-old male was diagnosed to have diabetes mellitus at 57 years of age and oral hypoglycemic agents were started. Aspart, a human insulin analogue, was initiated because of a poor glycemic control in July 2009, at 74 years of age. He complained of dizziness and faintness caused by hypoglycemia in the early morning and afternoon. He was admitted to another hospital in August 2010; his plasma insulin levels were markedly elevated (11,000 μU/ml), and 125 I-insulin binding rate of anti-insulin antibodies was 89.5 %. He was transferred to this hospital in September 2010, and a Scatchard analysis of his anti-insulin antibodies showed low affinity and high capacity. The daily profiles of blood glucose were evaluated precisely by continuous glucose monitoring (CGM). CGM showed low blood glucose levels in the early morning and afternoon, when human insulin injections were given twice daily. In addition, insulin and diet therapies were adjusted to prevent hypoglycemia, and accordingly, blood glucose control was improved. Hence, CGM is therefore considered to be useful in the management of brittle diabetes cause by anti-insulin antibodies. (Less)

Cross-Seeding of Fibrils from Two Types of Insulin Induces New Amyloid Strains

The irreversibility and autocatalytic character of amyloidogenesis and the polymorphism of amyloid fibrils underlie the phenomenon of self-propagating strains, wherein the mother seed, rather than the seeding environment, determines the properties of daughter fibrils. Here we study the formation of amyloid fibrils from bovine insulin and the recombinant Lys(B31)-Arg(B32) human insulin analog. The two polypeptides are similar enough to cross-seed but, upon spontaneous aggregation, form amyloid fibrils with distinct spectral features in the infrared amide I' band region. When bovine insulin is cross-seeded with the analog amyloid (and vice versa), the shape, absorption maximum, and even fine fingerprint features of the amide I' band are passed from the mother to daughter fibrils with a high degree of fidelity. Although the differences in primary structure between bovine insulin and the Lys(B31)-Arg(B32) analog of human insulin lie outside of the polypeptide's critical amyloidogenic regions, they affect the secondary structure of fibrils, possibly the formation of intermolecular salt bridges, and the susceptibility to dissection and denaturation with dimethyl sulfoxide (DMSO). All these phenotypic features of mother fibrils are imprinted in daughter amyloid upon cross-seeding. Analysis of noncooperative DMSO-induced denaturation of daughter fibrils suggests that the self-propagating polymorphism underlying the emergence of new amyloid strains is encoded on the level of secondary structure. Our findings have been discussed in the context of polymorphism of fibrils, amyloid strains, and possible implications for mechanisms of amyloidogenesis.

Clinical application of the different cross-reactivities of anti-insulin antibodies to insulin lispro to evaluate endogenous insulin secretion

BackgroundInsulin analogs are often used to treat patients with diabetes. We evaluated the cross-reactivities of anti-insulin antibodies in two insulin immunoassay kits (Architect and ECLusys) against recombinant human insulin and insulin analogs, and measured insulin concentrations in the serum of the diabetic patients treated with only insulin lispro. MethodsTen-fold dilutions of recombinant human insulins and insulin analogs were measured using Architect and ECLusys kits. The serum samples of 4 type 2 diabetic patients at fasting, and several time points after breakfast (25kcal/kg) following subcutaneous injection of insulin lispro were measured by Architect, ECLusys and LISPro RIA kit. ResultsThe ECLusys kit could detect human insulin but not insulin analogs. The Architect kit detected human insulin and insulin analogs with similar recovery ratios. The difference in serum insulin concentrations measured by Architect and ECLusys assays reflected the concentration measured by LISPro insulin kit in the patients. The differences in the AUC between Architect and ECLusys assays were significantly correlated with the AUC for LISPro assay (p<0.01). ConclusionsBy exploiting the different cross-reactivities of anti-insulin antibodies to insulin analogs, it may be possible to measure the endogenous and exogenous insulin concentrations in diabetic patients treated with insulin analogs.

PDB74 Effective Health Care Budget Planning for Innovative Drugs in Diabetes Treatment in Turkey; From the Perspective of the Social Security Institution (SSI)

Diabetes therapies constitute one of the highest budgets allocated by the Social Security Institution (SSI) each year in Turkey (within top 15). However, a standard budget allocation plan for reimbursement of innovative drugs in diabetes does not exist in Turkey. This study aims to offer a perspective for effective health care budget planning for innovative drugs in diabetes for the SSI in order to achieve patient access. Total budget spent by SSI for diabetes treatment is analyzed for the last three years. All treatment groups are defined for % unit and Turkish Lira (TL) growth and those values are investigated relative to the total diabetes budget. The current budget allocation structure for innovative drugs is defined and a new methodology is offered. The highest budget in diabetes is allocated for human insulin analogues with a total spending of 949,278,748 TL (45.7%), followed by glitazones 388,374,291 TL (18.7%) and sulphonylurea 244,190,634 TL (11.8%). However, the total budget allocated for innovative drugs as DPP-4 inhibitors and GLP-1 agonists is around 4.2%. The following steps for forward planning of budgets are proposed: Analysis of each treatment option within the same group with respect to effectiveness; defining reimbursement conditions (price-volume) for each option based on effectiveness; analysis of each treatment group with respect to effectiveness; defining budget allocated for each treatment group based on effectiveness; biannual review of the budget realizations. Re-evaluation of current therapies in terms of budget contributions and effectiveness may identify savings in SSI budget that could be freed up for new treatment options becoming available. A stepwise approach including the reevaluation of existing treatments in diabetes would be necessary in Turkey.

National trends in anti-diabetic treatment in Taiwan, 2000–2009

The impact of the introduction of newer anti-diabetic agents on the treatment pattern in the booming diabetic population remains unclear. We examined the patterns and temporal trends of anti-diabetic drug use in Taiwan, with particular emphasis on combination therapy. We searched the Taiwan National Health Insurance Database during 2000-2009 to identify outpatient prescriptions of anti-diabetic drugs, including human insulins and insulin analogues, sulfonylureas, glinides, metformin, thiazolidinediones, alpha-glucosidase inhibitors, and dipeptidyl peptidase-4 inhibitors. Glucose-lowering treatments were classified according to pattern (oral agents only, insulins only, and oral agents and insulins combined) and a number of different classes of anti-diabetic drugs. Insulin therapy and combination therapy with two oral anti-diabetic drugs (OAD) were further classified according to individual drug combination patterns. Although metformin remained the mainstay of anti-diabetic treatment, patients receiving combination therapy of oral glucose-lowering agents, either with or without insulin, significantly increased, from approximately 40% in 2000 to 60% in 2009, particularly in relation to the newer agents, including glinides, alpha-glucosidase inhibitors, and long-acting insulin analogues. Use of sulfonylureas and thiazolidinediones decreased substantially. For insulin therapy, the most commonly prescribed drugs were premix insulin analogues and basal insulin analogues, accounting for one-third of total insulin prescriptions in 2009. We found an increasing complexity of anti-diabetic therapy during the past decade in Taiwan. Further studies are needed to evaluate whether this treatment pattern will lead to improved clinical outcomes in terms of cost-effectiveness.

Insulin gene therapy from design to beta cell generation

Despite the fact that insulin injection can protect diabetic patients from developing diabetes-related complications, recent meta-analyses indicate that rapid and long-acting insulin analogues only provide a limited benefit compared with conventional insulin regarding glycemic control. As insulin deficiency is the main sequel of type-1 diabetes (T1D), transfer of the insulin gene-by-gene therapy is becoming an attractive treatment modality even though T1D is not caused by a single genetic defect. In contrast to human insulin and insulin analogues, insulin gene therapy targets to supplement patients not only with insulin but also with C-peptide. So far, insulin gene therapy has had limited success because of delayed and/or transient gene expression. Sustained insulin gene expression is now feasible using current gene-therapy vectors providing patients with basal insulin coverage, but management of postprandial hyperglycaemia is still difficult to accomplish because of the inability to properly control insulin secretion. Enteroendocrine cells of the gastrointestinal track (K cells and L cells) may be ideal targets for insulin gene therapy, but cell-targeting difficulties have limited practical implementation of insulin gene therapy for diabetes treatment. Therefore, recent gene transfer technologies developed to generate authentic beta cells through transdifferentiation are also highlighted in this review.