Human Insulin Analogues Research Articles

100 Years of Insulin: A Historical Perspective.

Diabetes mellitus, a metabolic disease characterized by high blood glucose levels, has increased dramatically in recent years, prompting the need for more affordable diagnoses and treatments. This study aimed to conduct a brief historical and theoretical review on the development of insulin. Scientific and technological data have been retrieved and analyzed with a focus on the development of the active pharmaceutical ingredient insulin and insulin-based medicines. Data have been retrieved from the PubMed database available via the CAPES portal. Diabetes is one of the oldest diseases in the world. The year 2021 marked the 100th anniversary of the discovery of insulin, which transformed diabetes from a fatal disease into a chronic disease. The extraction and purification of insulin from bovine or porcine pancreases from slaughterhouses has enabled the pharmaceutical industry to produce insulin on a large scale. The introduction of insulin analogs in 1996 expanded the options. Currently, commercial insulin consists of human insulin and/or human insulin analogs. The state-of-the-art and technological development of insulin over the last 100 years has been presented in this work. The development of new pharmaceutical technologies has led to the obtainment of improved versions of insulin, as well as the emergence of different types of insulin. Alongside the innovations in the development of the active ingredient and related medicines, new formulations, methods, and routes of administration have emerged based on the pharmacodynamic, pharmacokinetic, and pharmacotechnical modulations of the drug.

B-150 Chromatographic Separation and Quantitation of Human Insulin and Lispro Analog in Serum Using Liquid Chromatography High-Resolution Mass Spectrometry

Abstract Background Factitious hypoglycemia, a deliberate attempt to induce low blood glucose levels occurring secondary to the surreptitious administration of exogenous insulin, can be difficult to diagnose due to a variety of factors including the increased use of synthetic insulin analogs in clinical practice. Traditional insulin immunoassays demonstrate variable cross-reactivity with commonly prescribed insulin analogues and their metabolites, making it difficult to measure specific analogs. The development of liquid chromatography high resolution accurate mass (LC-HRAM)-mass spectrometry (MS) assays has largely circumvented the limitations of traditional immunoassays for detection and quantitation of insulin analogs. Analytical challenges remain, however, specifically in differentiating lispro, an isomeric human insulin analog formed by the transposition of proline and lysine near the C-terminal end of the B chain, from human insulin in patient samples due to the equivalent mass-to-charge ratios of their precursor ions. Methods Immunoaffinity purification of insulin from calibrators and quality control material prepared by spiking insulin-depleted serum with pharmaceutical insulin preparations, as well as patient samples, was performed using tips coupled with anti-insulin monoclonal antibody. An automated liquid handler was programmed to perform aspiration and dispense cycles. Eluate from the purification protocol was submitted for analysis using a TLX-2 multiplex LC system paired with a Thermo Scientific Q Exactive Plus mass spectrometer. Various HPLC column chemistries were evaluated, a column passivation procedure was established, and column heater temperature was optimized to identify a combination of parameters yielding the best chromatographic separation of human insulin and lispro. Additionally, parallel reaction monitoring (PRM) targeted tandem mass spectrometry (MS/MS) analysis was utilized in combination with the full scan data to increase specificity through the monitoring of fragment ions produced from human insulin and each insulin analog. Results We found that a 1000 Å Diphenyl, 2.7 µm, 2.1 x 100 mm analytical column in a column heater maintained at a temperature of 80°C allowed for human insulin and lispro to be differentiated as two chromatographically separated extracted ion chromatographic peaks (XICs) and quantified down to 2.5 mcIU/mL with near-baseline separation. Before use, the column required passivation with a high protein buffer (0.05% bovine serum albumin in 20% acetonitrile [ACN] + 0.2% acetic acid) followed by increasingly organic buffers (20% ACN + 0.2% acetic acid and 50% ACN + 0.2% acetic acid). MS/MS was used to confirm the presence of analogs detected. Conclusions The LC-HRAM-MS method described here, coupled with optimized column chemistry, column passivation, and proper column temperature control allows for chromatographic separation of human insulin and lispro. Incorporating a PRM scan into the mass spectrometry full scan method provides confirmation of the insulin detected in the XICs. The results obtained from preliminary studies suggest this method is appropriate for use to facilitate the clinical investigation of suspected factitious hypoglycemia with the ability to differentiate isomeric compounds, such as human insulin and lispro.

Synthesis of a highly thermostable insulin by phenylalanine conjugation at B29 Lysine

Globally, millions of diabetic patients require daily life-saving insulin injections. Insulin heat-lability and fibrillation pose significant challenges, especially in parts of the world without ready access to uninterrupted refrigeration. Here, we have synthesized four human insulin analogs by conjugating ε-amine of B29 lysine of insulin with acetic acid, phenylacetic acid, alanine, and phenylalanine residues. Of these, phenylalanine-conjugated insulin, termed FHI, was the most stable under high temperature (65 °C), elevated salt stress (25 mM NaCl), and varying pH levels (ranging from highly acidic pH 1.6 to physiological pH 7.4). It resists fibrillation for a significantly longer duration with sustained biological activity in in vitro, ex vivo, and in vivo and displays prolonged stability over its native counterpart. We further unravel the critical interactions, such as additional aromatic π-π interactions and hydrogen bonding in FHI, that are notably absent in native insulin. These interactions confer enhanced structural stability of FHI and offer a promising solution to the challenges associated with insulin heat sensitivity.

Pharmacological and Benefit-Risk Profile of Once-Weekly Basal Insulin Administration (Icodec): Addressing Patients' Unmet Needs and Exploring Future Applications.

Diabetes mellitus (DM) is a chronic metabolic disease affecting over 500 million people worldwide, which leads to severe complications and to millions of deaths yearly. When therapeutic goals are not reached with diet, physical activity, or non-insulin drugs, starting/adding insulin treatment is recommended by international guidelines. A novel recombinant insulin is icodec, a once-weekly insulin that successfully completed phase III trials and that has recently obtained the marketing authorization approval from the European Medicines Agency. This narrative review aims to assess icodec pharmacological and clinical features concerning evidence on benefit-risk profile, as compared to other basal insulins, addressing the potential impact on patients' unmet needs. Icodec is a full agonist, recombinant human insulin analogue characterized by an ultra-long half-life (196 h), enabling its use in once-weekly administration. Phase III randomized clinical trials involving more than 4000 diabetic patients, mostly type 2 DM, documented non-inferiority of icodec, as compared to currently available basal insulins, in terms of estimated mean reduction of glycated hemoglobin levels; a superiority of icodec, compared to control, was confirmed in insulin-naïve patients (ONWARDS 1, 3, and 5), and in patients previously treated with basal insulin (ONWARDS 2). Icodec safety profile was comparable to the currently available basal insulins. Once-weekly icodec has the potential to improve patients' adherence, thus positively influencing patients' treatment satisfaction as well as quality of life, especially in type 2 DM insulin-naïve patients. An improved adherence might positively influence glycemic target achievement, reduce overall healthcare costs and overcome some of the unmet patients' needs. Icodec has the potential to emerge as a landmark achievement in the evolution of insulin therapy, with a positive impact also for the National Health Services and the whole society.

Comparative physicochemical and structural characterisation studies establish high biosimilarity between BGL-ASP and reference insulin aspart

Biosimilar insulin analogues are increasing market access for diabetic patients globally. Scientific establishment of biosimilarity is cornerstone of this key change in the medical landscape. BGL-ASP is a biosimilar insulin aspart developed by BioGenomics Limited, India. BioGenomics has considered a stepwise approach in generating the totality of evidence required to establish similarity with reference product. Insulin aspart is a recombinant rapid-acting human insulin analogue utilised in the treatment of type-1 and type-2 diabetes mellitus. The single amino acid substitution at position B28 where proline is replaced with aspartic acid results in a decreased propensity to form hexamers, thus increasing the absorption rate on subcutaneous administration compared to native insulin. In order to establish the safety and efficacy of BGL-ASP, the critical quality attributes (CQAs) of BGL-ASP are identified based on the impact created on biological activity, pharmacokinetic/pharmacodynamic (PK/PD), immunogenicity and safety. The CQAs of insulin aspart are related to product structure, purity and functionality and are characterised using a series of state-of-the-art orthogonal analytical tools. The primary protein sequence, the secondary, tertiary and quaternary structure are found to be highly similar for BGL-ASP and reference product. The product related impurities of insulin aspart and the assay content are determined using high performance liquid chromatography (HPLC) based analysis and is similar for BGL-ASP and reference insulin aspart sourced from United States of America (US), Europe Union (EU) and India. The safety, efficacy and immunogenicity of BGL-ASP is also found to be comparable with reference product and is confirmed through the clinical trials conducted as recommended by International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH) and European Medicines Agency (EMA) guidelines. The data encompassed in this study demonstrates that reference insulin aspart and BGL-ASP are highly similar in terms of structural, physicochemical, and biological properties, thus confirming its safety and efficacy for usage as potential alternative economical medicinal treatment for diabetes mellitus.

The role of superfast insulin analogues in diabetes mellitus

In recent decades, significant advances in sugar therapy have been associated with the introduction of human insulin analogues. Superfast human insulin analogues have become an important tool for achieving optimal glycemic control and improving the quality of life of patients. Due to its unique pharmacokinetics, this group of insulin preparations provides a faster and more predictable onset of action, which allows its profile to be as close as possible to the secretion of endogenous insulin. A number of clinical trials have confirmed the advantage of superfast insulin analogues in the control of postprandial hyperglycemia compared with insulin preparations of the previous generation.

Acetone-induced structural variant of insulin amyloid fibrils

Self-propagating polymorphism of amyloid fibrils is a distinct manifestation of non-equilibrium conditions under which protein aggregation typically occurs. Structural variants of fibrils can often be accessed through physicochemical perturbations of the de novo aggregation process. On the other hand, tiny changes in the amino acid sequence of the parent protein may also result in structurally distinguishable amyloid fibrils. Here, we show that in the presence of acetone, the low-pH fibrillization pathway of bovine insulin (BI) leads to a new type of amyloid with the infrared features (split amide I′ band with the maximum at 1623 cm−1) bearing a striking resemblance to those of the previously reported fibrils from recombinant LysB31-ArgB32 human insulin analog formed in the absence of the co-solvent. Insulin fibrils formed in the presence ([BI-ace]) and absence ([BI]) of acetone cross-seed each other and pass their infrared features to the daughter generations of fibrils. We have used dimethyl sulfoxide (DMSO) coupled to in situ infrared spectroscopy measurements to probe the stability of fibrils against chemical denaturation. While both types of fibrils eventually undergo DMSO-induced disassembly coupled to a β-sheet→coil transition, in the case of [BI-ace] amyloid, the denaturation is preceded by the fibrils transiently acquiring the [BI]-like infrared characteristics. We argue that this effect is caused by DMSO-induced dehydration of [BI-ace]. In support to this hypothesis, we show that, even in the absence of DMSO, the infrared features of [BI-ace] disappear upon drying. We discuss this very peculiar aspect of [BI-ace] fibrils in the context of recently accessed in silico models of plausible structural variants of insulin protofilaments.

Detection of recombinant Pichia pastoris producing glargine

The global prevalence of diabetes continues to rise. Diabetes management involves the administration of insulin to help regulate blood glucose levels. Currently, two main types of insulin are used for diabetes treatment: human insulin and insulin analogs. One such insulin analog is insulin glargine, categorized as a long-acting insulin. Four prominent organisms used for insulin production are Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha. Pichia pastoris was used to produce insulin glargine in this study. The pPICZαA-G plasmid containing the synthetic glargine gene was inserted into Pichia pastoris. Recombinant Pichia pastoris containing the pPICZαA-G plasmid was detected using selection media with Zeocin, Polymerase Chain Reaction (PCR), and DNA sequencing. Through the implementation of selection media containing Zeocin, PCR, and DNA sequencing techniques, it was known that the recombinant Pichia pastoris contained the synthetic glargine gene. Further research will be carried out testing the expression of glargine in recombinant Pichia pastoris.

Audit of Safety Profile of Drugs Being Used to Treat Elderly Type II Diabetics: Observations from a Tertiary Care Hospital

ABSTRACT Background and Objective: The treatment of elderly people who have developed Type II diabetes is a major challenge. Therefore, this study was conducted to ascertain the drug prescription pattern and the adverse effects of drugs in a tertiary care hospital. Methodology: This was a cross-sectional, observational study. This study was carried out on 164 elderly patients with diabetes mellitus attending inpatient and outpatient of the institute. This study assesses the prescribing pattern of antidiabetic drugs and adverse drugs reaction to the drugs administered. Naranjo-Algorithm Probability of American depositary receipt (ADR) scale and WHO prescribing indicators were used for assessment. This study also identifies the drug utilization in diabetic patients associated with other co-morbidities. Results: Mean age of the patient was 72.5 in both inpatient and outpatient and male:female ratio among inpatient and outpatient 1.5:1 and 1:1.3, respectively. Metformin (biguanide) was the most commonly prescribed antidiabetic drug and was followed by glimepiride. Insulin was used in 65% of inpatient. Potential drug-drug interactions were noted in 71% of inpatient and 50.8% of outpatient. The incidence of ADR observed in this study was3.6%. Conclusion: Metformin and glimepiride were the most common drugs used in both inpatient and outpatient groups. Human regular insulin analog was commonly used in both patients group. The poor reporting in pharmacovigilance although mandated by the Indian Council of Medical Research is falling short; in preempting ADR. Effective pharmacovigilance will improve the quality of life in geriatric patients and would help the treating physicians to steer towards safe and effective healthcare.

Pharmacoepidemiological analysis of type 1 diabetes mellitus insulin therapy (According to the Moscow segment of the State Register of Diabetes Mellitus)

BACKGROUND: When developing programs for state reimbursement of the cost of medical services and medicines, when providing assistance to patients suffering from type 1 diabetes mellitus (DM1), data based on the results of pharmacoepide-miological studies (FEI) conducted in real clinical practice of treating patients are needed. The obtained results of the analysis of drug consumption allow us to study the most commonly used insulin therapy regimens and their compliance with modern clinical recommendations. Calculations of the weighted average cost of conducting hypoglycemic therapy in DM1 can be used for further pharmacoeconomical calculations, including analysis of the medical and social cost of the disease.AIM: To conduct a pharmacoepidemiological analysis of the consumption of insulin preparations according to the ATX /DDD methodology, to study the modes of insulin administration and the ratio of the weighted average cost of insulin therapy components per 1 patient with DM1 per year in the outpatient practice of treatment with the introduction of insulin in the mode of multiple injections of insulin (MII) and during continuous subcutaneous insulin infusion (CSII).MATERIALS AND METHODS: A retrospective continuous cohort analytical pharmacoepidemiological study was conducted on the basis of the SRDM database (status as of 01/01/2020), in which 17,617 patients with T1DM aged 18 years and older were registered at the time of the study.RESULTS: 90% of patients received analogues of human insulin in the MII mode using syringe pens, 8.6% used the CSII mode. In 34.5% of patients with DM1 HbA1c was <7%, the proportion of patients with HbA1c >8% was 28%. The average cost of insulin therapy per 1 person per year was 54,977 rubles. The cost of basal insulin treatment per 1 patient per year was approximately 1.5 times higher than the cost of bolus insulin during insulin therapy in the MII mode (33,960 rubles and 21,017 rubles, respectively). The weighted average cost of insulin per 1 patient per year for CSII was 31,822 rubles.CONCLUSION: The majority of patients with DM1 in the study cohort received analogues of human insulin in the basic bolus mode of MII using syringe pens, which corresponds to existing clinical recommendations. The proportion of patients achieving the goals of glycemic control is comparable to the data obtained on the basis of registers of diabetic patients in other economically developed countries

Comparison of Insulin Analogs and Human Insulins: A Narrative Review

Abstract Introduction Since insulin analogs have pharmacological properties that are similar to the normal physiological action of insulin, it has been suggested that they provide better glucose control and less rates of hypoglycemia compared to human insulins. Methods We performed a narrative, nonsystematic review of the literature including clinical trials, systematic reviews, meta-analyses, and professional guidelines related to the comparison of human insulins and insulin analogs in terms of glucose control, safety profile, and cost. Results Long-acting basal insulins result in mild improvement in glucose control and less rates of nocturnal hypoglycemic compared to neutral protamine Hagedorn insulin, mainly among patients with type 1 diabetes. Rapid-acting insulin analogs provide better glucose control and lower rates of hypoglycemia compared to regular insulin among patients with type 1 diabetes, whereas no advantage has been shown for insulin analogs among patients with type 2 diabetes for glucose control or hypoglycemia. Premixed insulin analogs provided no advantage in glucose control and inconsistent benefit in lowering the rates of hypoglycemia compared to human premixed insulins among patients with type 2 diabetes. The cost of insulin analogs is significantly higher than human insulins, and favorable cost-effectiveness has only been demonstrated for rapid-acting insulin analogs in type 1 diabetes. Conclusion Currently available evidence supports the use of rapid-acting insulin analogs and possibly long-acting basal insulin over human insulins for patients with type 1 diabetes. For patients with type 2 diabetes, the use of long-acting insulin analogs may be recommended for selected patients who are at an increased risk of significant hypoglycemia, while no clear benefits of meal insulin analogs over human insulins have been observed.

Insulin: Know what your immunoassay detects. Evaluation of two new immunoassays

BackgroundInsulin is essential for glycemic regulation but diseases can cause a default or an excess of insulin secretion leading to dysregulated glycemia. Hence, measurement of insulinemia is useful to investigate hypoglycemia, determine the pathogenesis of diabetes and evaluate β-cell function. Thus, diabetic patients need supplementation with recombinant human insulin and/or insulin analogues. Analogues have primary sequences different from native human insulin and may not be detected by some immunoassays. The objective of our study was to evaluate new insulin immunoassays by determining their ability to detect different types of human insulin or analogues. MethodsThis study compared the reactivity of two new insulin immunoassays with five well-established immunoassays on ten commercial insulins. We also measured insulin in blood samples from diabetic or pancreas transplant patients with known treatment. ResultsContrary to recombinant human insulin, there were differences in the specificity to insulin analogues. We distinguished three immunoassay categories: those recognizing all types of insulin such as the non-specific BI-INS-IRMA®, Architect® and Access® immunoassays; those recognizing human insulin only (Cobas®); and those recognizing human insulin and analogues in variable proportions (Liaison XL®, iFlash® and Maglumi®). ConclusionAn accurate biological interpretation of insulinemia relies on knowledge of the specificity of the immunoassay used.

Optimisation of induction conditions for a bacterial strain producing proinsulin aspart

Diabetes poses a serious threat to the health of people around the world. Therefore, in 2021, the World Health Organisation launched the Global Diabetes Compact, an initiative aimed at improving the management and prevention of diabetes. The rapid growth in the number of diabetic patients has increased the need for insulin. Rapid-acting human insulin analogues, including insulin aspart, improve the efficacy of insulin therapy. Methods for insulin aspart production include its biosynthesis in the proinsulin form in Escherichia coli. However, the yield of the recombinant protein largely depends on the optimisation of the production process.The aim of the study was to optimise the induction conditions for an E. coli strain expressing recombinant proinsulin aspart through applying the Design of Experiment (DoE) approach to enhance bacterial cell productivity.Materials and methods. The study focused on a strain of E. coli producing proinsulin aspart. The authors planned the experiment using MODDE software and the reduced face-centred central composite design (CCF) enabling the assessment of factor interactions and the creation of design spaces. The authors carried out fermentations of the producing strain in a 5 L Biostat® B bioreactor and measured proinsulin aspart concentrations by capillary gel electrophoresis. The results were analysed using GraphPad Prism 6.Results. Using the DoE approach, the authors optimised the conditions for the growth of the producer strain and the biosynthesis of proinsulin aspart. Based on data from response surface plots for wet biomass concentration, specific productivity, and volumetric productivity, as well as plotted models, the authors established design spaces for the induction of proinsulin aspart expression in E. coli. The plotted models demonstrated high predictive power and high reproducibility of the results. The authors successfully validated the induction process for the synthesis of proinsulin aspart in a bioreactor under optimised conditions. The volumetric productivity of the strain producing proinsulin aspart increased from 3.06±0.16 g/L (conventional conditions) to 4.93±0.80 g/L (optimised conditions).Conclusions. The authors achieved a 60% increase in the volumetric yield of proinsulin aspart. The study results may be used to intensify the industrial production of insulin aspart.

Intentional overdose of glargine insulin: Determination of the parent compound in postmortem blood by LC‐HRMS

Insulin glargine is a long-acting insulin analog that is converted after enzymatic cleavage of the arginine pair of the β-chain into its main metabolite M1 (21A -Gly-insulin), which is responsible for the hypoglycemic activity. In all the overdose cases described in the literature, only M1 concentrations have been reported, whereas insulin glargine was always absent or below the limit of quantitation. In this study, we present a case of suicide of a young nurse by injection of insulin glargine in which the parent molecule was found at a toxic concentration in blood. The determination and the discrimination of insulin glargine from human insulin and other synthetic analogs in the blood specimen were performed by liquid chromatography coupled to high-resolution mass spectrometry (Waters XEVO G2-XS QToF) and extraction after precipitation in the presence of bovine insulin (internal standard), with a mixture of acetonitrile/methanol +1% formic acid followed by purification on solid phase extraction cartridges C18. Glargine insulin tested highly positive in the blood with a concentration of 1.06 mg/L. Due to the difficulty in obtaining a M1 pure standard, the metabolite could not be dosed. This unique presence of the parent molecule, reported for the first time, can be explained by inter-individual variability in the rate of conversion to metabolite. Intravenous injection versus subcutaneous injection can also explain the presence of insulin glargine. Finally, the dose injected may have been so high that saturation of the proteolytic enzymes responsible for conversion to M1 should have occurred.

Regulatory Verification by Health Canada of Content in Recombinant Human Insulin, Human Insulin Analog, and Porcine Insulin Drug Products in the Canadian Market Using Validated Pharmacopoeial Methods Over Nonvalidated Approaches.

For diabetes mellitus treatment plans, the consistency and quality of insulin drug products are crucial for patient well-being. Because biologic drugs, such as insulin, are complex heterogeneous products, the methods for drug product evaluation should be carefully validated for use. As such, these criteria are rigorously evaluated and monitored by national authorities. Consequently, reports that describe significantly lower insulin content than their label claims are a concern. This issue was raised by a past publication analyzing insulin drug products available in Canada, and, as a result, consumers and major patient organizations have requested clarification. To address these concerns, this study independently analyzed insulin drug products purchased from local Canadian pharmacies-including human insulin, insulin analogs, and porcine insulin-by compendial and noncompendial reversed-phase high-performance liquid chromatography (RP-HPLC) methods. We demonstrated the importance of using methods fit for purpose when assessing insulin quality. In a preliminary screen, the expected insulin peak was seen in all products except two insulin analogs-insulin detemir and insulin degludec. Further investigation showed that this was not caused by low insulin content but insufficient solvent conditions, which demonstrated the necessity for methods to be adequately validated for product-specific use. When drug products were appropriately assessed for content using the validated type-specific compendial RP-HPLC methods for insulin quantitation, values agreed with the label claim content. Because insulin drug products are used daily by over a million Canadians, it is important that researchers and journals present data using methods fit for purpose and that readers evaluate such reports critically.

Influence of Preservatives on Insulin in Postmortem Blood: Application to a Case of Insulin Aspart Suicide.

Insulin aspart (NovoRapid®, NovoMix®, Novolog® and Fiasp®) is a fast-acting analog of human insulin, indicated in the treatment of type I and II diabetes. It is administered before meals to mimic the physiological insulin secretion that follows a rise in blood glucose. Its misuse for the purposes of suicide and murder and in the context of factitious order has often been described. In forensic medicine, the identification of insulin in biological samples has always been complex. In this paper, we present a case of suicide of a 64-year-old man who died after the injection of insulin aspart. He was suffering from terminal lung cancer and left a letter explaining the reasons for his suicide. Four empty NovoRapid® pens were found near the body. Body examination was unremarkable, and the femoral blood was collected in two dry Vacutainer™ tubes (red cap) and two sodium fluoride (NaF) tubes (gray cap). A liquid chromatography coupled to high-resolution mass spectrometry method was used to identify and discriminate insulin aspart from human insulin after immunopurification in the blood samples and in the pens. Blood specimens tested positive for insulin aspart with the concentrations of 36 and 37 ng/mL in dry tubes and 58 and 71 ng/mL in tubes containing NaF when tested ∼3 weeks after the collection of the specimens. The contents of the pens also matched with insulin aspart. The stability of insulin in blood is a critical point in the interpretation of the concentrations due to their rapid decrease caused by the activity of proteases in blood. During a degradation study implemented to compare three preservatives and dry tubes, suitable insulin aspart stability was observed with disodium salt of ethylenediaminetetracetic acid and NaF. Given that NaF is standard in forensic toxicology for measuring blood alcohol concentrations, the authors suggest its use for blood collection when insulin intoxication is suspected.

Difficulties in interpreting a therapeutic blood concentration of insulin lispro in postmortem blood: A case report

Illustrate the complexity of interpreting a postmortem therapeutic blood concentration of insulin in a probable case of suicide using insulin lispro. The identification and quantification of insulin and its analogues continues to be a challenge in the field of forensic toxicology. Its use is not always limited to the treatment of diabetes but is often used illicitly. Numerous cases of suicides and suicide attempts have been described in the literature and nevertheless, this number still seems to be underestimated due to both the inability of laboratories to identify and discriminate between synthetic analogues of human insulin and due to the chemical instability of insulin especially in postmortem samples. Our case concerns a 63-year-old man who was found dead by his brother at home. Three empty Humalog® insulin pens and a letter mentioning his suicide intention were found next to his bed. At the request of the prosecutor, an external examination of the corpse was performed and revealed only an asphyxial syndrome not specific to a cause of death. The presence of a blood glucose monitoring device on the right arm was also observed, suggesting that the subject was diabetic. External examination was unable to determine the exact cause of death. For this reason, the pathologist was oriented on a toxic death. In order to proceed to further toxicological investigations, peripheral blood, urine and hair samples were collected. It is important to underline that, due to the long period elapsed between the external examination and the pathologist's conclusion (enabling the initiation of the toxicological analysis) and due to administrative delays, insulin testing was performed only 9 months after the samples collection. Blood and urine samples were stored at +4 °C until analysis. The identification and quantification of insulin lispro was performed in the postmortem blood after precipitation in the presence of bovine insulin (internal standard), by a mixture of acetonitrile/methanol (ratio 1:1) followed by a step of ultrafiltration using Amicon® centrifugal filters (3 kDa) and a step of immunopurification using Iso-Insulin ELISA coated wells. The analysis was performed with a UHPLC- Q-TOF (XEVO G2-XS). Detection of insulin lispro was performed using the precursor molecular ion m/z 1162.543 (charge state 5 +). Since human insulin and insulin lispro share the exact same molecular mass (5807 Da), discrimination cannot be made using the multi-charged molecular ion. The distinction is based on fragment m/z 217 which is present only in insulin lispro spectrum. Toxicological tests performed on the blood sample did not reveal any xenobiotic of toxic nature. Insulin lispro was detected in the femoral blood sample at 1,1 ng/mL, whereas human insulin was not detected. Following the administration of a therapeutic dose of 0.2 U/kg of insulin Humalog ®, a maximum concentration of 1.66 ± 0.42 ng/mL is achieved. Consequently, the concentration found in our case was therapeutic. Nevertheless, given the unstable nature of insulin, especially in postmortem blood, it is not possible to exclude that death was caused by an insulin overdose. The long period between collection and analysis (9 months), the hemolyzed state of the blood, the storage of the sample in unsuitable tubes and the fact that we were able to detect even small concentrations of insulin lispro suggest that the concentration at the time of death was much higher. The pathologist concluded that the cause of death was probably an overdose of insulin lispro. This case highlights that the interpretation of a postmortem insulin concentration requires a complex approach involving the assessment of many factors such as the subject's medical history and the time, mode and state of preservation of the sample.

Insulin: evolution of insulin formulations and their application in clinical practice over 100years.

The first preparation of insulin extracted from a pancreas and made suitable for use in humans after purification was achieved 100 years ago in Toronto, an epoch-making achievement, which has ultimately provided a life-giving treatment for millions of people worldwide. The earliest animal-derived formulations were short-acting and contained many impurities that caused adverse reactions, thereby limiting their therapeutic potential. However, since then, insulin production and purification improved with enhanced technologies, along with a full understanding of the insulin molecule structure. The availability of radio-immunoassays contributed to the unravelling of the physiology of glucose homeostasis, ultimately leading to the adoption of rational models of insulin replacement. The introduction of recombinant DNA technologies has since resulted in the era of both rapid- and long-acting human insulin analogues administered via the subcutaneous route which better mimic the physiology of insulin secretion, leading to the modern basal-bolus regimen. These advances, in combination with improved education and technologies for glucose monitoring, enable people with diabetes to better meet individual glycaemic goals with a lower risk of hypoglycaemia. While the prevalence of diabetes continues to rise globally, it is important to recognise the scientific endeavour that has led to insulin remaining the cornerstone of diabetes management, on the centenary of its first successful use in humans.

The role of pre-pregnancy care for women with pregestational diabetes mellitus for improving maternal and perinatal outcomes

The occurrence of diabetes mellitus in women before pregnancy (pregestational diabetes mellitus can have various adverse consequences for the mother, fetus, child, and pregnancy. Pregnancy planning in women with pregestational diabetes mellitus significantly improves the outcomes of pregnancy and childbirth. The introduction of diabetes self-control skills, the use of human insulin and insulin analogues, the use of constant subcutaneous insulin infusion and family planning allows for optimal glycemic control in early pregnancy and reduces the incidence of fetal congenital anomalies and perinatal mortality. Lifestyle optimization, weight loss, taking adequate doses of folic acid, discontinuation of teratogenic medications, and quitting smoking are essential components of preconception care. Of significant clinical value is the training of women planning pregnancy at the Diabetes school, training in self-control and insulin therapy skills, consultation with an endocrinologist, an obstetrician-gynecologist, an ophthalmologist, a nephrologist, and a nutritionist.
 The aim of this study was to present current data on pre-pregnancy care in patients with pregestational diabetes mellitus and to show its possibilities in reducing the frequency of obstetric and perinatal complications. We analyzed 74 domestic and foreign literature sources using the following databases: PubMed, PubMed Central, Google Scholar, and UpToDate.

The clinical-economic characteristic of current basis-bolus insulin therapy schemes in diabetes mellitus type 1 in adults

Basis-bolus insulin therapy is a cornerstone of Diabetes Mellitus type 1 (DM1T) control. Basal insulin analogs — glargine 300 U/ml (iGla 300), glargine 100 U/ml (iGla 100), detemir (iDet) degludec (iDeg), — as well as prandial insulins — glulisine (iGlu), aspart (iAsp) and lispro — are used widely during last 10–15 years. Aim. Evaluation of a comparative economic efficacy of the different basis-bolus schemes of insulin therapy in DM1T in adults. Materials and methods. Analysis has been performed for the following schemes: iGla 300 + iGlu, iGla 100 + iGlu, iDet+iAsp, iDeg+iAsp from Govt position based on modelling of the efficacy for 5 years. Data regarding probability of complications based on glycated hemoglobin (HbA1c) reduction for human insulin treatment and insulin analogs were taken into modelling. Direct medical costs were calculated for insulins, complications, hypoglycemic including severe events. Sensitivity analysis has been performed for validation of the received results. Results: Insulin analogs have economic advantages in compare with human insulins for DM1T control for 6.5 years. They could reduce expenditures in 1.89 times. iGla 300 + iGlu and iDeg+iGlu reduced HbA1c more effective among analogs and hypo events were more rare also (35.0 episodes/patient/year), including severe (0.57 and 0.70 episodes/patient/ year accordingly) vs iGla 100 + iGlu and iDet+iAsp (37.8 and 39.9 episodes/patient/year and 1.10 and 1.21 episodes/patient/ year for severe accordingly). Calculated direct medical costs were less for iGla 300 + iGlu, after that were following iGla 100 + iGlu, iDet+iAsp and last (highest) were expenditures for iDeg+iAsp. Conclusion. Created model prognoses complications of DM1T depending on schemes of insulin therapy and calculates of direct costs. iGla 300 + iGlu has economic advantages vs iGla100 + iGlu, iDet+iAsp and iDeg+iAsp in DM1T control during 5 years horizon.