Insulin Analogues Research Articles

ATOS study: effectiveness and safety of insulin glargine 300 U/mL in the real world clinical practice in insulin-naïve type 2 diabetic patients in the Russian Federation

Rationale: Basal insulin glargine 300 U/mL (Gla-300) is a second-generation basal insulin analogue that has comparable efficacy and lower variability compared to the first generation long-acting insulin analogue glargine 100 U/mL.
 Aim: To assess the effectiveness and safety of Gla-300 in insulin-nave type 2 diabetic patients in the real world practice in Russia.
 Materials and methods: ATOS (NCT03703869) was a 12-month, prospective observational international multicenter study. The study included 4422 adults ( 18 years) with uncontrolled type 2 diabetes (HbA1c 7 and 11%) with 1 oral anti-hyperglycemic drug and for whom the treating physician had decided to add Gla-300. We performed a post-hoc sub-analysis of the study participants recruited in Russia.
 Results: The Russian study group included 1493 patients receiving Gla-300. At 6 months, 25.9% of the patients achieved their predefined individualized HbA1c target and 53.3% achieved their HbA1c target at month 12. Their mean ( SD) HbA1c level decreased from 9.3 0.9% at baseline to 7.6 0.7% and 7.2 0.7 at months 6 and 12. The incidence of hypoglycemia was generally low; overall, severe hypoglycemia was reported only in 0.07% and 0.13% of the patients at 6 and 12 months. The baseline average daily dose of Gla-300 was 13.2 4.9 Units; it to 23.6 9.1 and 26.0 9.8 Units at months 6 and 12.
 Conclusion: In the real world setting, initiation of insulin Gla-300 in type 2 diabetic patients who had been out of their target glucose range with oral hypoglycemic agents is associated with improved glycemic control and low risk of hypoglycemia.

New therapeutic approaches for type 1 diabetes: Disease-modifying therapies.

It has been 100 years since the first successful clinical use of insulin, yet it remains the only treatment option for type 1 diabetes mellitus (T1DM) patients. Advances in diabetes care, such as insulin analogue therapies and new devices, including continuous glucose monitoring with continuous subcutaneous insulin infusion have improved the quality of life of patients but have no impact on the pathogenesis of the disease. They do not eliminate long-term complications and require several lifestyle sacrifices. A more ideal future therapy for T1DM, instead of supplementing the insufficient hormone production (a consequence of β-cell destruction), would also aim to stop or slow down the destructive autoimmune process. The discovery of the autoimmune nature of type 1 diabetes mellitus has presented several targets by which disease progression may be altered. The goal of disease-modifying therapies is to target autoimmune mechanisms and prevent β-cell destruction. T1DM patients with better β-cell function have better glycemic control, reduced incidence of long-term complications and hypoglycemic episodes. Unfortunately, at the time symptomatic T1DM is diagnosed, most of the insulin secreting β cells are usually lost. Therefore, to maximize the salvageable β-cell mass by disease-modifying therapies, detecting autoimmune markers in an early, optimally presymptomatic phase of T1DM is of great importance. Disease-modifying therapies, such as immuno- and regenerative therapies are expected to take a relevant place in diabetology. The aim of this article was to provide a brief insight into the pathogenesis and course of T1DM and present the current state of disease-modifying therapeutic interventions that may impact future diabetes treatment.

Pitfalls in Diagnosing Hypoglycemia Due to Exogenous Insulin: Validation and Utility of an Insulin Analog Assay

ObjectiveTo overcome the limitations of commercially available insulin immunoassays which have variable detection of analog insulin and can lead to clinically discordant results and misdiagnosis in the workup of factitious hypoglycemia. Patients and MethodsWe performed analytical validation of a liquid chromatography high resolution accurate mass (LC-HRAM) immunoassay to detect insulin analogs. We completed clinical assessment using a large cohort of human serum samples from 78 unique individuals, and subsequently used the assay in the evaluation of eight individuals with high diagnostic suspicion for factitious hypoglycemia. ResultsThe performance characteristics show that the LC-HRAM immunoassay can be applied to detect five commonly used synthetic insulin analogs (lispro, glulisine, aspart, glargine metabolite, and detemir) in human serum. Our clinical cases show that this assay could be used in the diagnosis of factitious hypoglycemia by identifying the analog insulin(s) in question. ConclusionThe LC-HRAM immunoassay reported here overcomes a gap in our diagnostic pathway for hypoglycemia. The results obtained from our studies suggest that this method is appropriate for use in clinical laboratories when factitious hypoglycemia is considered as a differential diagnosis.

Evaluation of Insulin and Glucagon-Like Peptide-1 Analog Sales in Turkey Before the Pandemic Period; Projection for Upcoming Years: A Market Analysis Study

This study was designed to evaluate the box sale trends of insulin preparations and glucagon-like peptide-1 (Glp-1) analogs by their therapeutic categories in Turkey. Also, we aimed to project sale trends of these pharmaceuticals in next years. We analyzed the data before the covid-19 pandemic and made predictions based on these data. This study analyzed retail and hospital box sales of insulin preparations from 4 therapeutic categories (fast/short-acting, intermediate-acting, premix, long-acting) and Glp-1 analogs. Total box sales of the insulin preparations were increased by %13,30 in the period examined. While fast/short-acting and long-acting insulin box sales increased with a decreasing upward trend, intermediate-acting and premix insulin box sales decreased. Glp-1 analog sales increased rapidly in this period. Currently, newer pharmaceuticals have been entering Turkish market for diabetes treatment. Less insulin might be required if newer pharmaceuticals are more widely available in diabetes treatment. Also it will be useful to evaluate the effect of the pandemic on access to pharmaceuticals used in the treatment of chronic diseases in future studies.

LC-MS/MS based detection of circulating proinsulin derived peptides in patients with altered pancreatic beta cell function

Routine immunoassays for insulin and C-peptide have the potential to cross-react with partially processed proinsulin products, although in healthy patients these are present at such low levels that the interference is insignificant. Elevated concentrations of proinsulin and des-31,32 proinsulin arising from pathological conditions, or injected insulin analogues, however can cause significant assay interferences, complicating interpretation. Clinical diagnosis and management therefore sometimes require methods that can distinguish true insulin and C-peptide from partially processed proinsulin or injected insulin analogues. In this scenario, the high specificity of mass spectrometric analysis offers potential benefit for patient care.A high throughput targeted LC-MS/MS method was developed as a fit for purpose investigation of insulin, insulin analogues, C-peptide and proinsulin processing intermediates in plasma samples from different patient groups. Using calibration standards and bovine insulin as an internal standard, absolute concentrations of insulin and C-peptide were quantified across a nominal human plasma postprandial range and correlated strongly with immunoassay-based measurements. The ability to distinguish between insulin, insulin analogues and proinsulin intermediates in a single extraction is an improvement over existing immunological based techniques, offering the advantage of exact identification of the species being measured. The method promises to aid in the detection of circulating peptides which have previously been overlooked but may interfere with standard insulin and C-peptide immunoassays.

Single-chain insulin analogs threaded by the insulin receptor αCT domain

Insulin is a mainstay of therapy for diabetes mellitus, yet its thermal stability complicates global transportation and storage. Cold-chain transport, coupled with optimized formulation and materials, prevents to some degree nucleation of amyloid and hence inactivation of hormonal activity. These issues hence motivate the design of analogs with increased stability, with a promising approach being single-chain insulins (SCIs), whose C domains (foreshortened relative to proinsulin) resemble those of the single-chain growth factors (IGFs). We have previously demonstrated that optimized SCIs can exhibit native-like hormonal activity with enhanced thermal stability and marked resistance to fibrillation. Here, we describe the crystal structure of an ultrastable SCI (C-domain length 6; sequence EEGPRR) bound to modules of the insulin receptor (IR) ectodomain (N-terminal α-subunit domains L1-CR and C-terminal αCT peptide; “microreceptor” [μIR]). The structure of the SCI-μIR complex, stabilized by an Fv module, was determined using diffraction data to a resolution of 2.6 Å. Remarkably, the αCT peptide (IR-A isoform) “threads” through a gap between the flexible C domain and the insulin core. To explore such threading, we undertook molecular dynamics simulations to 1) compare threaded with unthreaded binding modes and 2) evaluate effects of C-domain length on these alternate modes. The simulations (employing both conventional and enhanced sampling simulations) provide evidence that very short linkers (C-domain length of −1) would limit gap opening in the SCI and so impair threading. We envisage that analogous threading occurs in the intact SCI-IR complex—rationalizing why minimal C-domain lengths block complete activity—and might be exploited to design novel receptor-isoform-specific analogs.

Pharmacotherapy of Diabetes Mellitus Type 2: A Review on Various Hypoglycemics

Diabetes is a cosmopolitan medical issue ramifying a large population regardless of ethnicity. Elevated glycemic condition in a patient is explained as diabetes. It can be mainly categorized into Non-Insulin Dependent Diabetes Mellitus (NIDDM) and Insulin Dependent Diabetes Mellitus (IDDM) or Type 1 or Type 2 respectively. Type 1 or IDDM needs insulin therapy exclusively while Type 2 can be controlled via various hypoglycemic agents. Conventional classes contain glitazones, biguanides, sulfonylurea’s and so on, while newly developed drug classes include gliptins or DPP-4 inhibitors, SGLT-2 receptor inhibitors and GLP-1 receptor agonists. The novelty in the anti-hyperglycemic agents have given promising results as compared to the conventional drugs. Insulin cannot be negated as far as its glycemic control is concerned. Furthermore, development of Insulin analogues like detemir, glargine, Lispro have been shown to control glucose levels in the plasma more efficiently.

Leveraging Clinical Pharmacology Data to Assess Biosimilarity and Interchangeability of Insulin Products.

There is over a hundred years of clinical experience with insulin for the treatment of diabetes. The US Food and Drug Administration (FDA) approved the first insulin biosimilar interchangeable product in 2021 for improving glycemic control in adults and pediatric patients with type 1 diabetes mellitus and in adults with type 2 diabetes mellitus. Several recombinant insulin products are available in the United States, including the recently approved biosimilar insulins. The approval of the biosimilar insulin products was based on comparative analytical characterizations and comparative pharmacokinetic (PK) and pharmacodynamic (PD) data. The primary objective of this review is to discuss the scientific considerations in the demonstration of biosimilarity of a proposed insulin biosimilar to a reference product and the role of clinical pharmacology studies in the determination of biosimilarity and interchangeability. Euglycemic clamp studies are considered a "gold standard" for insulin PK and PD characterization and have been widely used to determine the time-action profiles of rapid-acting, intermediate-acting, and long-acting insulin products. Clinical pharmacology aspects of study design, including selection of appropriate dose, study population, PK, and PD end points, are presented. Finally, the role of clinical pharmacology studies in the interchangeability assessment of insulin and the regulatory pathways used for insulin and the experience with follow-on insulins and the two recently approved biosimilar insulin products is discussed.

Effects of Different Regimens of Insulin on Body Mass Index Commonly Used in Type 2 Diabetes Mellitus

Background: Weight gain is an ongoing challenge when initiating insulin therapy in patients with Type 2 diabetes mellitus. The aim of this study is to observe and compare the effect of commonly used regimens of insulin on body mass index (BMI) among Type 2 diabetes patients. Methods: An observational study was conducted in two diabetic centers in Dhaka starting from July 2018 to June 2019. During the 12 weeks of the data collection period, a total of 100 patients were included according to selection criteria and then divided into two equal groups. Patients who were prescribed with premixed (conventional or analog) 30/70 insulin twice daily with or without oral Antidiabetic drugs were included in Group I (n = 50) and the patients who were prescribed with insulin glargine once daily with a bolus insulin (conventional or analog) three times before large meals with or without oral Antidiabetic drugs were included in Group II (n = 50). BMI was calculated from the height and weight of the patients initially as baseline data and again after 12 weeks of treatment in both groups. Fiber Bragg grating, 2 h ABF, and hemoglobin A1C (HbA1c) change were also observed among the patients of both insulin groups from diagnostic reports during the study period. Results: After 12 weeks of treatment, mean body weight (±standard deviation [SD]) was increased significantly from 59.82 ± 12.33 kg to 60.40 ± 13.38 kg in Group I (P = 0.01) and from 59.00 ± 12.36 kg to 60.33 ± 12.97 kg in Group II (P = 0.02). Mean BMI (±SD) increased significantly from 23.71 ± 4.69 kg/m2 to 24.10 ± 4.17 kg/m2 in Group I (P = 0.01) and from 24.00 ± 4.30 kg/m2 to 24.43 ± 4.59 kg/m2 in Group II (P = 0.02) during the study period. The mean BMI compared between two study groups (24.10 ± 4.17 vs. 24.43 ± 4.59) kg/m2 m+ after 12 weeks of treatment was not statistically significant (P = 0.816). About 64.0% of patients in Group I and 68.0% in Group II have shown weight gain and BMI change after 12 weeks. Mean HbA1c (±standard deviation [SD]) reduced significantly from 10.40% ± 2.17% to 7.76% ± 1.41% in Group I and from 10.41% ± 1.80% to 7.63% ± 1.37% in Group II. About 48.0% of patients in Group I and 56.0% of patients in Group II achieved glycemic target <7%. About 28.0% of patients in Group I and 20.0% of patients in Group II had hypoglycemia. Conclusion: From the current study, it can be concluded that both insulin regimens significantly cause weight gain, BMI change and also significantly effective in glycemic control. However, in comparison between the two groups, there was no significant difference. Hence, both premixed and glargine-based basal-bolus regimen can be prescribed as per patient need. This result may provide some preliminary information for further investigation

Technological Developments and Quality of Life in Type 1 Diabetes Mellitus Patients: A Review of the Modern Insulin Analogues, Continuous Glucose Monitoring and Insulin Pump Therapy.

Type 1 Diabetes Mellitus (T1DM) is a chronic autoimmune disease, which is characterized by an increased prevalence worldwide, which, in fact, tends to take extensive dimensions. The recent rapid development of science and technology has significantly contributed to the improvement of the management of type 1 diabetes mellitus, both in achieving the required euglycaemic regulation and reducing the psychological burden associated with the disease, consequently improving the quality of life of the patients with type 1 diabetes mellitus. A literature review from 2010, related to the contribution of the modern insulin analogues, continuous glucose monitoring and the insulin pump, was performed using Scopus, ScienceDirect and PubMed databases. Studies included in the review support a direct and indirect association of technological innovations with the quality of life. The use of type 1 diabetes mellitus technology was negatively associated with the frequency of the hypoglycaemias and the value of the glycosylated hemoglobin, while at the same time, the development and use of the related technology were highly associated with an improvement in the quality of life. Patients' quality of life is an indicator of the management of type 1 diabetes mellitus, and it is just as important as glycaemic regulation. Through this review, it was concluded that a better quality of life of T1DM patients was associated with the improvement of glycosylated hemoglobin and hypoglycemic episodes.

Pharmacokinetic and pharmacodynamic similarity evaluation between an insulin glargine biosimilar product and Lantus® in healthy subjects: Pharmacokinetic parameters of both parent insulin glargine and M1 were used as endpoints.

Insulin glargine is a long-acting insulin analog, which plays an important role in the treatment of diabetes mellitus. Biosimilar products of insulin glargine can provide patients with additional safe, high-quality, and potentially cost-effective options for treating diabetes. This article presents a randomized, double-blind, single-dose, two-treatment, four-period, replicate crossover, euglycemic clamp study which was designed to evaluate the PK and PD similarity between the recombinant insulin glargine developed by Wanbang (test) and Lantus® (reference) in healthy volunteers. Subjects received subcutaneous administration of the insulin glargine formulation (0.4 U/kg) on two occasions for the test and reference drug, respectively, and a 20% dextrose solution was infused at variable rate to clamp the blood glucose concentrations at 0.3 mmol/L below the subjects’ fasting glucose for 24 h. Taking advantage of the improved sensitivity of the bioanalytical method applied and the solution of the matrix stability problem, the parent insulin glargine was determined in the vast majority of plasma samples using a fully validated UHPLC-MS/MS method. The PK characteristics of the parent insulin glargine were revealed for the first time: after subcutaneous injection, concentrations of the parent insulin glargine increased to a relative high level within 3 h, and then, a relatively flat concentration–time profile lasting for at least 12 h post-dose was observed. For the first time, the pharmacokinetic parameters of the parent insulin glargine were used as endpoints for similarity evaluation, which complied with the regulatory guidance better and made the similarity conclusion more powerful. The ratios of geometric means of all PK and PD endpoints were close to 100.00%. For the PK endpoints (AUC0–24h, Cmax, AUC0–12h, and AUC12–24h of the parent insulin glargine and its metabolite M1), the 90% confidence intervals of geometric mean ratios of test to reference were entirely contained within 80.00%–125.00%. For the PD endpoints [AUCGIR(0–24h), GIRmax, AUCGIR(0–12h), and AUCGIR(12–24h)], the 95% confidence intervals of geometric mean ratios of test to reference were entirely contained within 80.00%–125.00%. Based on the above mentioned results, it can be concluded that the PK and PD characteristics of the biosimilar drug developed by Wanbang are similar to those of Lantus.

Icodec insulin - revolution in diabetes type 2 insulin therapy

Introduction: Basal glucose control in diabetes type 2 is commonly maintained by a single, once-daily administration of insulin through subcutaneous injection. Insulin icodec, a novel ultralong-acting lipidated analog validates the concept of a once-weekly basal injection that is less burdensome, yet equally safe and efficacious as conventional once-daily treatment 1.Aim: The aim of the study is to introduce once-weekly injected insulin therapy and compare it with conventional basal insulin therapy in diabetes type 2 patients.Results: The results from phase II trials suggest that switching from an existing basal insulin to icodec, with or without a loading dose, provides effective glycemic control with comparable risk of hypoglycemia.In one of the studies TIR improved from baseline (mean: A 57.0%; B 55.2%; C 51.0%; IGlar U100 55.3%) through weeks 15 and 16 (estimated mean: A 76.6%; B 83.0%; C 80.9%; IGlar U100, 75.9%). No unexpected side effects were observed. It was reassuring that not a single episode of severe hypoglycemia (level 3) was reported for any treatment group throughout the trial duration and that the time spent below range (<3.9 mmol/L [<54 mg/dL]) during weeks 15 and 16 was well below the 4% target recommended by the International Consensus on Time in Range across all treatment groups 2.Conclusion: In conclusion,insulin icodec, a new acylated basal insulin analog, has been developed with optimized modifications to provide a long half-life suitable for weekly insulin dosing. In a patient population with type 2 diabetes receiving daily basal insulin therapy, switching to once-weekly icodec resulted in effective glycemic control without a transient elevation of fasting glucose levels during the switch and without increasing the risk of clinically relevant hypoglycemia compared with IGlar U100 2.Key words: Icodec, insulin, diabetes type 2

Structural models of viral insulin-like peptides and their analogs.

The insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF1R), and the insulin/IGF1 hybrid receptors (hybR) are homologous transmembrane receptors. The peptide ligands, insulin and IGF1, exhibit significant structural homology and can bind to each receptor via site-1 and site-2 residues with distinct affinities. The variants of the Iridoviridae virus family show capability in expressing single-chain insulin/IGF1 like proteins, termed viral insulin-like peptides (VILPs), which can stimulate receptors from the insulin family. The sequences of VILPs lacking the central C-domain (dcVILPs) are known, but their structures in unbound and receptor-bound states have not been resolved to date. We report all-atom structural models of three dcVILPs (dcGIV, dcSGIV, and dcLCDV1) and their complexes with the receptors (μIR, μIGF1R, and μhybR), and probed the peptide/receptor interactions in each system using all-atom molecular dynamics (MD) simulations. Based on the nonbonded interaction energies computed between each residue of peptides (insulin and dcVILPs) and the receptors, we provide details on residues establishing significant interactions. The observed site-1 insulin/μIR interactions are consistent with previous experimental studies, and a residue-level comparison of interactions of peptides (insulin and dcVILPs) with the receptors revealed that, due to sequence differences, dcVILPs also establish some interactions distinct from those between insulin and IR. We also designed insulin analogs and report enhanced interactions between some analogs and the receptors.

Biosimilars and interchangeable biosimilars: facts every prescriber, payor, and patient should know. Insulins perspective

ABSTRACT Introduction For many of the 537 million adults living now with diabetes, the cost of insulin is becoming prohibitive as the insulin prices have tripled between 2002–2013. Globally, the direct annual cost of healthcare expenditure due to diabetes will soon be US$1 Trillion. Biosimilars provide access to high-quality, affordable biologic therapy that is otherwise inaccessible due to the high costs of original biologics. Areas covered A primer to the development of biosimilars shows comparable structural and analytical characterization to the original biologics (e.g. insulins), with no clinically significant or meaningful differences in efficacy and safety. ‘Interchangeability’ status, a regulatory designation by the US FDA, bestowed to some biosimilars, enables confidence in high-quality, bio-equivalent biosimilar of insulin with key global approvals. This can allow rapid uptake of biosimilars by the prescribers, formulary decision-makers, and payors. Biocon-Viatris’s biosimilar Insulin Glargine (Semglee®) is the first interchangeable biosimilar insulin approved by the US FDA. Expert opinion The ‘interchangeable’ status can prompt faster and wider uptake of insulin biosimilars and keep the insulin expenditure under control, especially for patients who otherwise practice non-adherence or rationing of life-saving insulin. Education, support, and awareness can ensure that interchangeable biosimilars gain wider acceptance.

BrazIliaN Type 1 & 2 DiabetEs Disease Registry (BINDER): longitudinal, real-world study of diabetes mellitus control in Brazil.

This study aimed at assessing the patterns of care and glycemic control of patients with diabetes (DM) in real life during a follow-up of 2 years in the public and private health sectors in Brazil. BINDER was an observational study of patients >18 years old, with type-1 (T1DM) and type-2 DM (T2DM), followed at 250 sites from 40 cities across the five regions of Brazil. The results for the 1,266 participants who were followed for 2 years are presented. Most patients were Caucasians (75%), male (56.7%) and from the private health sector (71%). Of the 1,266 patients who entered the analysis, 104 (8.2%) had T1DM and 1162 (91.8%) had T2DM. Patients followed in the private sector represented 48% of the patients with T1DM and 73% of those with T2DM. For T1DM, in addition to insulins (NPH in 24%, regular in 11%, long-acting analogues in 58%, fast-acting analogues in 53%, and others in 12%), the patients received biguanide (20%), SGLT2-I (4%), and GLP-1Ra (<1%). After 2 years, 13% of T1DM patients were using biguanide, 9% SGLT2-I, 1% GLP-1Ra, and 1% pioglitazone; the use of NPH and regular insulins decreased to 13% and 8%, respectively, while 72% were receiving long-acting insulin analogues, and 78% fast-acting insulin analogues. Treatment for T2DM consisted of biguanide (77%), sulfonylureas (33%), DPP4 inhibitors (24%), SGLT2-I (13%), GLP-1Ra (2.5%), and insulin (27%), with percentages not changing during follow-up. Regarding glucose control, mean HbA1c at baseline and after 2 years of follow-up was 8.2 (1.6)% and 7.5 (1.6)% for T1DM, and 8.4 (1.9)% and 7.2 (1.3)% for T2DM, respectively. After 2 years, HbA1c<7% was reached in 25% of T1DM and 55% of T2DM patients from private institutions and in 20.5% of T1DM and 47% of T2DM from public institutions. Most patients did not reach the HbA1c target in private or public health systems. At the 2-year follow-up, there were no significant improvements in HbA1c in either T1DM or T2DM, which suggests an important clinical inertia.

Preclinical pharmacology of RA15127343: In vitro and in vivo activity of a novel ultralong-acting basal insulin.

To report the in vitro and in vivo preclinical pharmacokinetic (PK) and pharmacodynamic (PD) properties of RA15127343, a novel ultralong-acting insulin analogue targeting once-weekly administration, in female Göttingen minipigs. In vitro binding and activation of human insulin receptor isoforms (IR-A/IR-B), glucose uptake in rat myocytes, as well as mitogenic activity of RA15127343 were evaluated. In vivo, the PK and PD activities of RA15127343 were assessed in female, normoglycaemic Göttingen minipigs. The half-life (t1/2 ) and time to maximum plasma concentration (Tmax ) of subcutaneously (SC) administered RA15127343 (10/30/45/60 nmol/kg) were estimated. In vivo blood glucose and endogenous plasma C-peptide concentrations after single SC administration (10/30/45/60 nmol/kg) or repeated dosing (15 nmol/kg) were analysed. In comparison to human insulin, RA15127343 showed lower in vitro binding affinity (19.9/6.31 μM vs. 1.10/1.14 nM) and activation (2.054 μM/669.6nM vs. 26.04/18.24 nM) of IR-A/IR-B, lower potency to activate glucose uptake (855.2 vs. 3.37 nM) and lower mitogenic activity (17.92 μM vs. 10.78 nM; proliferation in MCF7 cells). In vivo, the mean t1/2 and Tmax of RA15127343 after SC administration ranged from 48 to 59 and 30 to 39 hours, respectively. Blood glucose and plasma C-peptide concentrations were significantly lower with RA15127343 (single/repeated doses) versus vehicle. RA15127343 showed an ultra-long t1/2 with a slow onset of action. The preclinical pharmacological outcomes suggest RA15127343 could be a potential ultralong-acting insulin analogue with low risk of hypoglycaemia in humans.

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.

An autopsy case of fatal insulin preparation overdose

Insulin preparations, a group of drugs used to treat diabetes, are of great forensic interest because they are difficult to detect in blood while they are sometimes intentionally administered in large doses for suicide or murder. In this study, we validated a quantification method for insulin glargine and insulin lispro using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the blood sample from a case of suicidal overdose with insulin. Case: A woman in her 30s had a history of mental illness and diabetes. A day before her death, she informed her partner that she had self-administered large doses of insulin preparations and prescription drugs. Bovine insulin was used as the internal standard (IS). The blood sample (250 μL) was added to the diluent and IS solution, followed by 250 μL each of hexane and N,N-dimethylformamide, and the mixture was centrifuged (10000 g × 10 min). The supernatant was collected and 900 μL of 5% ammonium solution was added. The extract was analyzed using Nexera 30A, LCMS8060 (Shimadzu), L-column3 ODS C18 metal-free column (150 × 2.1 mm, 3 μm), ionization method: electrospray ionization+, multiple reaction monitoring conditions: insulin glargine [M + 6H]6+ 1011.5 > 1179.4, insulin lispro[M + 6H]6+ 968.9 > 217.0, bovine insulin (IS) [M + 6H]6+ 956.3 > 1120.9. The autopsy revealed no evidence of fatal injuries. Coronary artery stenosis was observed as a finding of endogenous disease, but there were no significant histopathological changes in the myocardium. Biochemical tests revealed the following: blood glucose level, 6 mg/dL; glucose in the vitreous region, 6 mg/dL; glycated hemoglobin (HbA1c), 11.2%; blood insulin level estimated using the chemiluminescent enzyme immunoassay method, 1.03 μU/mL (6.27 pmol/mL); and C-peptide concentration, 0.6 ng/mL (16.8 pmol/L). A drug screening test and simple quantification using LC-MS/MS detected many prescription drugs, but none of them were at intoxicating concentrations. The insulin analysis showed insulin glargine concentrations of 429 μU/mL in femoral blood and 1362 μU/mL in cardiac blood, and insulin lispro was detected in femoral and cardiac blood below the lower limit of quantification (<50 μU/mL). The cardiac/peripheral blood ratio, a measure of postmortem redistribution, was 3.2. Although the therapeutic ranges of insulin glargine and insulin lispro have not been defined, the femoral and cardiac blood concentrations of insulin lispro in this case was lower than the serum concentration (155 μU/mL) listed in the package insert. However, while the femoral and cardiac blood insulin glargine concentrations were considerably higher than the serum concentration (15 μU/mL) listed in the package insert. Considering that insulin is easily degraded in whole blood due to hemolysis, it is possible that insulin concentrations were even higher at the time of administration and death than those observed in autopsy specimens on the third postmortem day. However, since insulin lispro is a fast-acting analog of insulin (1–3 hours), it may have contributed to hypoglycemia but was not the direct cause of death, and hypoglycemia due to insulin glargine overdose was the most likely cause of death in this case. We quantified insulin lispro and insulin glargine in a case of suicide to determine insulin glargine's involvement in causing death. In future, it is necessary to examine postmortem degradation and redistribution of insulin and consider the appropriate concentration in postmortem samples of insulin poisoning.

Can postmortem metabolomics be used as a tool in the identification of deaths attributed to hypoglycaemia

To establish a metabolomic profile that could be used as a screening tool for possible deaths attributed to hypoglycaemia. Postmortem metabolomics can be used to assist death investigations by characterising new biomarker profiles differentiating causes of death. Hypoglycaemia-related deaths, particularly insulin intoxications, are difficult to identify without support from antemortem blood samples. The biological analysis of exogenous insulins in postmortem cases is troublesome, as immunoassays are not precise when handling haemolysed blood samples from postmortem cases. However, in recent years advances have been made in identification of insulin analogues, particularly in vitreous fluid. Nevertheless, due to such limitations it is probable that a proportion of hypoglycaemia-related deaths remain unknown. The aim of this investigation is to build a postmortem metabolomics model to be used as a tool to identify deaths related to hypoglycaemia. Data from ultra-high-performance liquid chromatography-quadrupole time-of-flight (UHPLC-QTOF) mass spectrometry was obtained from postmortem cases including; 19 insulin intoxication cases (hypoglycaemia group), 19 diabetic coma cases (hyperglycaemia group), and 38 hanging cases (control group). Additionally, a random test group of postmortem cases ( n = 726) were used for test screening for possible unidentified hypoglycaemic deaths. Mass spectrometry data was processed in XCMS using R. Orthogonal-partial least squares discriminant analysis (OPLS-DA), in SIMCA, was used to build models comparing metabolomic features between hypoglycaemia and control groups, and hypoglycaemia and hyperglycaemia groups. These two models were combined into a shared-unique-structures (SUS) plot, and the discriminating features specific for hypoglycaemia group were selected for identification and used to build the screening model. Features were identified using the HMDB and METLIN databases. XCMS data analyses resulted in the identification of 1775 metabolomic features. The OPLS-DA model for hypoglycaemia vs control resulted in complete group separation with R2 = 0.90 and Q2 = 0.62, and the model for hypoglycaemia vs hyperglycaemia resulted in complete group separation with R2 = 0.93 and Q2 = 0.86. The SUS plot of these two models resulted in 83 features that were considered to discriminate the hypoglycaemia group. An OPLS-DA model using only those 83 discriminating features was built, resulting in complete group separation of hypoglycaemia, hyperglycaemia, and control groups (R2 = 0.73, Q2 = 0.56). Group classification prediction was performed for the random test group on this model, which resulted in 46 (6.0%), 17 (2.2%), and 332 (43.5%) of cases being classified as belonging to hypoglycaemia, hyperglycaemia, and control groups, respectively. Cases classified as belonging to no group were 363 (47.6%), falling below the threshold of group classification in the model, and the remaining four cases were classified as belonging to two of the three groups in various combinations. The reported causes of death for those classified as hypoglycaemia showed a varied list of causes, including cardiovascular complications, substance overdose/poisoning, asphyxia, amongst others. However, six cases have an undetectable cause of death reported. The difficulties in the identification of hypoglycaemia-related deaths and the toxicological analysis of exogenous insulin remains. However, based on the results here we can see that postmortem metabolomics is able to discriminant deaths attributed to hypoglycaemia, hyperglycaemia, and non-glycaemic deaths (controls). Additionally, the model for postmortem case screening classified a discrete group of the random test group (only 6%) as hypoglycaemia, and whilst the individual cases still require review, this low number of classifications is a good indication that a viable screening model could be developed. Postmortem metabolomics is a useful tool in the screening of possible hypoglycaemia-related deaths. Further studies are required to expand on the potential of postmortem metabolomics as a useful tool in the investigation of hypoglycaemia-related deaths, and the future application of identifying potential insulin intoxications.

Non-Covalent Albumin Ligands in FDA-Approved Therapeutic Peptides and Proteins.

An increasing number of drugs that consist of a therapeutic peptide or protein linked to an albumin-binding structure are being approved. In this perspective, the pharmacokinetic data of currently marketed drugs of this type will be presented. Acylation with fatty acids or fatty α,ω-dicarboxylic acids has been used successfully to prepare long-acting analogs of insulin, GLP-1, and other peptides but not of larger proteins. With a tetrazole-sulfonylamide fatty acid bioisostere, it has now been possible to prepare a long-acting analog of human growth hormone (191 amino acids), which is suitable for once-weekly administration.