ABSTRACT: We have exploited a prandial insulin analog to elucidate the underlying structure and dynamics of insulin as a monomer in solution. A model was provided by insulin lispro (the active component of Humalog(®); Eli Lilly and Co.). Whereas NMR-based modeling recapitulated structural relationships of insulin crystals (T-state protomers), dynamic anomalies were revealed by amide-proton exchange kinetics in D(2)O. Surprisingly, the majority of hydrogen bonds observed in crystal structures are only transiently maintained in solution, including key T-state-specific inter-chain contacts. Long-lived hydrogen bonds (as defined by global exchange kinetics) exist only at a subset of four ?-helical sites (two per chain) flanking an internal disulfide bridge (cystine A20-B19); these sites map within the proposed folding nucleus of proinsulin. The anomalous flexibility of insulin otherwise spans its active surface and may facilitate receptor binding. Because conformational fluctuations promote the degradation of pharmaceutical formulations, we envisage that "dynamic re-engineering" of insulin may enable design of ultra-stable formulations for humanitarian use in the developing world.
Project description:Glide force, average glide force, and glide force variability of the insulin lispro 200 units/mL pen (Eli Lilly and Company, Indianapolis, IN, USA) were compared to the Humalog KwikPen 100 units/mL pen (hereafter, KwikPen; Eli Lilly and Company, Indianapolis, IN, USA). Data were collected on 2 doses, 2 injection speeds, and 2 needle types. Insulin lispro 200 units/mL pen showed significantly lower maximum glide force, average glide force, and glide force variability than the KwikPen across all combinations of dose size, dose speed, and needle type. The lower glide force observed with the insulin lispro 200 units/mL pen offers another treatment option for patients with type 1 or type 2 diabetes who require greater than 20 units of mealtime insulin daily.
Project description:Isolation and identification of unknown impurities of recombinant insulin lispro (produced at IBA) formed during accelerated stability testing of pharmaceutical solutions. For comparative purposes also commercially available formulations of recombinant human insulin (Humulin S®; Lilly), recombinant insulin lispro (Humalog®; Lilly), recombinant insulin aspart (NovoRapid® Penfill®; Novo Nordisk), recombinant insulin detemir (Levemir®; Novo Nordisk) and recombinant insulin glargine (Lantus®; Sanofi-Aventis) were analyzed.The impurities of insulin analogs were isolated by RP-HPLC and identified with peptide mass fingerprinting using MALDI-TOF/TOF mass spectrometry.The identified derivatives were N-terminally truncated insulin analog impurities of decreased molecular mass of 119, 147 and 377 Da related to the original protein. The modifications resulting in a mass decrease were detected at the N-terminus of B chains of insulin lispro, insulin aspart, human insulin, insulin glargine, insulin detemir in all tested formulations. To our knowledge it is the first time that these impurities are reported.The following derivatives formed by truncation of the B chain in insulin analogs were identified in pharmaceutical formulations: desPheB1-N-formyl-ValB2 derivative, desPheB1 derivative, pyroGluB4 derivative.
Project description:AIMS:To compare the pharmacokinetic (PK) and glucodynamic (GD) characteristics of ultra rapid lispro (URLi; Eli Lilly and Company, Indianapolis, Indiana), Fiasp® (Novo Nordisk, Bagsvaerd, Denmark), Humalog® (Eli Lilly and Company) and NovoRapid® (Novo Nordisk), in patients with type 1 diabetes (T1D). MATERIALS AND METHODS:This was a randomized, double-blind, four-period, crossover study, conducted in 68 patients with T1D. Patients received the same individualized subcutaneous dose of each study drug immediately prior to a liquid test meal. For comparison, 12 healthy subjects received the same test meal. RESULTS:URLi had a significantly faster insulin absorption compared to the other insulins tested. Early half-maximal drug concentration was reached 13?minutes after administration of URLi, which was 6 minutes faster than Fiasp, 13?minutes faster than Humalog, and 14?minutes faster than NovoRapid (all P?<0.0001). Early insulin exposure was significantly greater and late insulin exposure was reduced after URLi compared to the other insulins. URLi achieved the greatest numerical reduction in postprandial glucose (PPG) at 2 hours post-meal (7 mg/dL vs Fiasp) and was significantly different from Humalog (21?mg/dL) and Novo Rapid (29?mg/dL). Additionally, glucose excursions over the first 3 hours post-meal with URLi were comparable to those in healthy subjects. CONCLUSIONS:URLi demonstrated the fastest insulin absorption and the greatest numeric PPG-lowering effect compared to the other insulins tested. URLi more closely matched the early physiological glucose control observed in healthy subjects.
Project description:<b>Background and objective: </b>Ultra rapid lispro (URLi) is a novel insulin lispro formulation developed to more closely match physiological insulin secretion and improve postprandial glucose control. This study compared the insulin lispro pharmacokinetics and glucodynamics, safety and tolerability of URLi and Humalog<sup>®</sup> after a single subcutaneous dose in patients with type 2 diabetes mellitus (T2DM).<br><br><b>Methods: </b>This was a phase I, randomised, two-period, two-treatment, double-blind, crossover study in 38 patients with T2DM. At each dosing visit, patients received either 15 units of URLi or Humalog, followed by a 10 h automated euglycaemic clamp procedure. Serum insulin lispro and blood glucose were measured.<br><br><b>Results: </b>Insulin lispro appeared in the serum 5 min faster (p?<?0.0001) and exposure was 6.4-fold greater in the first 15 min (p?<?0.0001) with URLi versus Humalog. Exposure beyond 3 h postdose was 26% lower and the duration of exposure was 51 min shorter with URLi versus Humalog. Onset of insulin action was 13 min faster (p?<?0.0001) and insulin action was 4.2-fold greater within the first 30 min (p?<?0.0001) with URLi versus Humalog. Insulin action beyond 4 h postdose was 20% lower (p?=?0.0099) with URLi versus Humalog. Overall insulin lispro exposure and total glucose infused were similar for URLi and Humalog. Both treatments were well tolerated.<br><br><b>Conclusions: </b>This is the first study to investigate URLi in patients with T2DM using a euglycaemic clamp procedure. URLi demonstrated ultra-rapid pharmacokinetics and glucodynamics in patients with T2DM. CLINICALTRIALS.<br><br><b>Gov identifier: </b>NCT03305822.
Project description:Insulin lispro 200 U/mL (IL200) is a new strength formulation of insulin lispro (Humalog®, IL100), developed as an option for diabetic patients on higher daily mealtime insulin doses. This phase 1, open-label, 2-sequence, 4-period crossover, randomized, 8-hour euglycemic clamp study aimed to demonstrate the bioequivalence of IL200 and IL100 after subcutaneous administration of 20 U (U) to healthy subjects (n = 38). Pharmacokinetic (PK) and pharmacodynamic (PD) responses were similar in both formulations. All 90%CIs for the ratios of area under the concentration-versus-time curve from time zero to the time of the last measurable concentration (AUC0-tlast) and maximum observed drug concentration (Cmax), as well as the total glucose infused throughout the clamp (Gtot) and the maximum glucose infusion rate (Rmax), were contained within 0.80 and 1.25. Time of maximum observed drug concentration (tmax) was similar between formulations, with a median difference of 15 minutes and a 95%CI of the difference that included zero. Inter- and intrasubject variability estimates were similar for both formulations. Both formulations were well tolerated. IL200 was bioequivalent to IL100 after subcutaneous administration of 20-U single doses, and PD responses were comparable between formulation strengths.
Project description:INTRODUCTION:Identification of subgroups of patients that may benefit most from certain treatment is important because individual treatment response varies due to multiple contributing factors. The present study used the subgroup identification based on the differential effect search (SIDES) algorithm to identify subgroups with different treatment responses to insulin intensification therapies. METHODS:This was a post hoc analysis of a 24-week, multicenter, open-label, randomized, parallel study comparing prandial premixed therapy (PPT) to basal-bolus therapy (BBT). Patients with type 2 diabetes mellitus were randomized to PPT (insulin lispro mix 50/50 thrice daily with meals) or BBT (glargine at bedtime plus mealtime insulin lispro) insulin intensification therapies. The SIDES algorithm was used to identify the subgroups from at-goal patients [glycated hemoglobin (HbA1c) <7.0% (53.0 mmol/mol) at the end of 24 weeks; n = 182] who could have benefitted from insulin intensification therapies. RESULTS:Baseline characteristics of overall at-goal patients were comparable between PPT and BBT groups. The SIDES algorithm identified patients with race other than Caucasian (i.e., African-American, Asian, and Hispanic) and baseline fasting blood glucose (FBG) <8.89 mmol/L as a subgroup that could respond better to PPT relative to BBT than the overall at-goal patient population. In this identified subgroup population, the HbA1c mean (standard deviation) changes from baseline to endpoint in PPT and BBT groups were -2.27 (0.88)% versus -2.05 (0.75)%; p = 0.40, respectively; while in the overall at-goal patients, the HbA1c changes were -2.17 (0.79)% versus -2.34 (1.00)%; p = 0.19, respectively. CONCLUSIONS:The preliminary results showed that the subgroup of patients with race other than Caucasian and FBG <8.89 mmol/L may respond better to premixed intensification therapy. This result provides some preliminary information for further investigation in prospective studies. FUNDING:Eli Lilly and Company. CLINICAL TRIAL REGISTRATION:Clinicaltrials.gov ID number: NCT00110370.
Project description:We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-?-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS.
Project description:INTRODUCTION:Few prospective clinical trials have investigated the role of fasting blood glucose (FBG) and/or postprandial glucose (PPG) in assessing overall glycemic control by using different insulin regimens. In the present post hoc analysis, we assessed the contribution of FBG and/or PPG in overall glycemic control in Chinese patients under insulin treatment. METHODS:CLASSIFY is a phase IV, randomized, open-label, 26-week, parallel-arm, treat-to-target, multinational, controlled study in patients with type 2 diabetes mellitus to compare the efficacy and safety of insulin lispro mix 25 (LM25) and insulin lispro mix 50 (LM50) as starter insulins. Insulin was titrated with an aim to target pre-meal blood glucose (BG) levels at >?3.9 and ??6.1 mmol/L before breakfast and dinner. The primary outcome assessed was the change in HbA1c from baseline. RESULTS:Chinese patients contributed 38.7% (N?=?156) of the total population. The majority of patients were male (52.6%). The mean (SD) body mass index was 24.54 (3.04) kg/m2 and mean (SD) HbA1c was 8.54 (1.10) % at baseline. At week 26, LM50 showed a significantly greater reduction from baseline in HbA1c (- 2.03% vs - 1.55%, P?<?0.001), average daily BG (- 3.21 vs - 2.34 mmol/L, P?<?0.001), average post-meal BG (- 3.58 vs - 2.39 mmol/L, P?<?0.001), and average prandial BG excursion (- 1.01 vs - 0.22 mmol/L, P?=?0.006) than the LM25 group. The reductions in average pre-meal BG (- 2.59 vs - 2.28 mmol/L, P?=?0.137) were not significantly different between the groups. The proportion of patients achieving HbA1c targets (<?7% or ??6.5%) without nocturnal hypoglycemia or weight gain was greater (P?<?0.05) with LM50 compared with LM25. CONCLUSION:LM50 achieved better overall glycemic control than LM25 as a starter insulin in Chinese patients, which may be due to greater improvement in PPG levels. TRIAL REGISTRATION:Clinicaltrials.gov identification number: NCT01773473. FUNDING:Eli Lilly and Company, Shanghai, China.
Project description:<h4>Purpose</h4>Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions.<h4>Methods</h4>Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism.<h4>Results</h4>Glulisine forms compact hexamers in formulation even in the absence of Zn<sup>2+</sup>. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn<sup>2+</sup> and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs.<h4>Conclusion</h4>Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
Project description:In order to provide comprehensive information on the differences in bioactivity between human insulin and insulin analogues, published in vitro comparisons of human insulin and the rapid acting analogues insulin lispro (Humalog®), insulin aspart ( NovoRapid®), insulin glulisine (Apidra®), and the slow acting analogues insulin glargine (Lantus®), and insulin detemir (Levemir®) were gathered from the past 20 years (except for receptor binding studies). A total of 50 reports were retrieved, with great heterogeneity among study methodology. However, various differences in bioactivity compared to human insulin were obvious (e.g. differences in effects on metabolism, mitogenesis, apoptosis, intracellular signalling, thrombocyte function, protein degradation). Whether or not these differences have clinical bearings (and among which patient populations) remains to be determined.