Rapid and Sensitive Analytical Method for the Determination of Insulin in Liposomes by Reversed-Phase HPLC.

Insulin is an important anabolic hormone that regulates the metabolism of carbohydrates, lipids and proteins. In this study, a reverse-phase liquid chromatography (RP-LC) method was successfully validated and tested for the encapsulation efficiency assay of insulin and in vitro release studies. HPLC analyses were carried out using a RP C18- Luna® Phenomenex (4.6 × 250 mm, 5 ?m particle size) column maintained at room temperature, using a mobile phase constituted by a mixture of acetonitrile and 0.1% TFA aqueous solution (60:40, v/v), in an isocratic mode with a flow rate of 1.0 mL/ min, with ultraviolet detection at 214 nm and 20 ?L of injection volume. Method validation was performed according recognized guidelines for system suitability, specificity, linearity, precision, accuracy, LOD, LOQ and robustness. The method was shown to be linear in the range of 0.5-100 ?g/mL (r2 = 0.9993) selective, precise, robust, accurate with LOD and LOQ values were 0.097 ?g/mL and 0.294 ?g/mL, respectively. The developed method proved to be adequate to analyze the encapsulation efficiency and the profile of insulin release from liposomes.

Strategy for peptide quantification using LC-MS in regulated bioanalysis: case study with a glucose-responsive insulin.

AIM: Advances in technology have led to a shift for peptide quantification from traditional ligand-binding assays to LC-MS/MS-based analysis, which presents challenges, in other assay sensitivity, specificity and ruggedness, in addition to lacking of regulatory guidance, especially for the hybrid assay format. Methodology & results: This report communicates a strategy that has been employed in our laboratories for method development and assay validation, and exemplified in a case study of MK-2640, a glucose-responsive insulin, in multiple matrices. Intact MK-2640 was monitored, while immunoaffinity purification and SPE were used to support the rat/dog GLP and clinical studies, respectively. The rationale and considerations behind our approach, as well as the acceptance criteria applied to the assay validation are discussed.

A Comparison of Pharmacokinetic and Pharmacodynamic Properties Between Faster-Acting Insulin Aspart and Insulin Aspart in Elderly Subjects with Type 1 Diabetes Mellitus.

BACKGROUND: Due to population aging, an increasing number of elderly patients with diabetes use insulin. It is therefore important to investigate the characteristics of new insulins in this population. Faster-acting insulin aspart (faster aspart) is insulin aspart (IAsp) in a new formulation with faster absorption. This study investigated the pharmacological properties of faster aspart in elderly subjects with type 1 diabetes mellitus (T1DM). METHODS: In a randomised, double-blind, two-period crossover trial, 30 elderly (≥65 years) and 37 younger adults (18-35 years) with T1DM received single subcutaneous faster aspart or IAsp dosing (0.2 U/kg) and underwent an euglycaemic clamp (target 5.5 mmol/L) for up to 12 h. RESULTS: The pharmacokinetic and pharmacodynamic time profiles were left-shifted for faster aspart versus IAsp. In each age group, onset of appearance occurred approximately twice as fast (~3 min earlier) and early exposure (area under the concentration-time curve [AUC] for serum IAsp from time zero to 30 min [AUCIAsp,0-30 min]) was greater (by 86% in elderly and 67% in younger adults) for faster aspart than for IAsp. Likewise, onset of action occurred 10 min faster in the elderly and 9 min faster in younger adults, and early glucose-lowering effect (AUC for the glucose infusion rate [GIR] from time zero to 30 min [AUCGIR,0-30 min]) was greater (by 109%) for faster aspart than for IAsp in both age groups. Total exposure (AUCIAsp,0-t) and the maximum concentration (C max) for faster aspart were greater (by 30 and 28%, respectively) in elderly than in younger adults. No age group differences were seen for the total (AUCGIR,0-t) or maximum (GIRmax) glucose-lowering effect. CONCLUSION: This study demonstrated that the ultra-fast pharmacological properties of faster aspart are similar in elderly subjects and younger adults with T1DM. ClinicalTrials.gov Identifier: NCT02003677.

Moving toward the ideal insulin for insulin pumps.

Advances in insulin formulations have been important for diabetes management and achieving optimal glycemic control. Rapid-acting insulin analogs provide a faster time-action profile than regular insulin and are approved for use in pumps. However, the need remains for therapy to deliver a more physiologic insulin profile. New insulin formulations and delivery methods are in development, with the aim of accelerating insulin absorption to accomplish ultra-fast-acting insulin time-action profiles. Furthermore, the integration of continuous glucose monitoring with insulin pump therapy enables on-going adjustment of insulin delivery to optimize glycemic control throughout the day and night. These technological and pharmacological advances are likely to facilitate the development of closed-loop pump systems (i.e., artificial pancreas), and improve glycemic control and quality of life for patients with diabetes.

Old and new basal insulin formulations: understanding pharmacodynamics is still relevant in clinical practice.

Long-acting insulin analogues have been developed to mimic the physiology of basal insulin secretion more closely than human insulin formulations (Neutral Protamine Hagedorn, NPH). However, the clinical evidence in favour of analogues is still controversial. Although their major benefit as compared with NPH is a reduction in the hypoglycaemia risk, some cost/effectiveness analyses have not been favourable to analogues, largely because of their higher price. Nevertheless, these new formulations have conquered the insulin market. Human insulin represents currently no more than 20% of market share. Despite (in fact because of) the widespread use of insulin analogues it remains critical to analyse the pharmacodynamics (PD) of basal insulin formulations appropriately to interpret the results of clinical trials correctly. Importantly, these data may help physicians in tailoring insulin therapy to patients' individual needs and, additionally, when clinical evidence is not available, to optimize insulin treatment. For patients at low risk for/from hypoglycaemia, it might be acceptable and also cost-effective not to use long-acting insulin analogues as basal insulin replacement. Conversely, in patients with a higher degree of insulin deficiency and increased risk for hypoglycaemia, analogues are the best option due to their more physiological profile, as has been shown in PD and clinical studies. From this perspective optimizing basal insulin treatment, especially in type 2 diabetes patients who are less prone to hypoglycaemia, would be suitable making significant resources available for other relevant aspects of diabetes care.

Analysis of the local dynamics of human insulin and a rapid-acting insulin analog by hydrogen/deuterium exchange mass spectrometry.

Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino acids. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3-6 and B22-24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3-6, whereas they increased in B22-24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Ultra-rapid absorption of recombinant human insulin induced by zinc chelation and surface charge masking.

BACKGROUND: In order to enhance the absorption of insulin following subcutaneous injection, excipients were selected to hasten the dissociation rate of insulin hexamers and reduce their tendency to reassociate postinjection. A novel formulation of recombinant human insulin containing citrate and disodium ethylenediaminetetraacetic acid (EDTA) has been tested in clinic and has a very rapid onset of action in patients with diabetes. In order to understand the basis for the rapid insulin absorption, in vitro experiments using analytical ultracentrifugation, protein charge assessment, and light scattering have been performed with this novel human insulin formulation and compared with a commercially available insulin formulation [regular human insulin (RHI)]. METHOD: Analytical ultracentrifugation and dynamic light scattering were used to infer the relative distributions of insulin monomers, dimers, and hexamers in the formulations. Electrical resistance of the insulin solutions characterized the overall net surface charge on the insulin complexes in solution. RESULTS: The results of these experiments demonstrate that the zinc chelating (disodium EDTA) and charge-masking (citrate) excipients used in the formulation changed the properties of RHI in solution, making it dissociate more rapidly into smaller, charge-masked monomer/dimer units, which are twice as rapidly absorbed following subcutaneous injection than RHI (Tmax 60 ± 43 versus 120 ± 70 min). CONCLUSIONS: The combination of rapid dissociation of insulin hexamers upon dilution due to the zinc chelating effects of disodium EDTA followed by the inhibition of insulin monomer/dimer reassociation due to the charge-masking effects of citrate provides the basis for the ultra-rapid absorption of this novel insulin formulation.

Accelerating and improving the consistency of rapid-acting analog insulin absorption and action for both subcutaneous injection and continuous subcutaneous infusion using recombinant human hyaluronidase.

Rapid-acting insulin analogs were introduced to the market in the 1990s, and these products have improved treatment of diabetes by shortening the optimum delay time between injections and meals. Compared with regular human insulin, rapid-acting insulin formulations also reduce postprandial glycemic excursions while decreasing risk of hypoglycemia. However, the current prandial products are not fast enough for optimum convenience or control. Recombinant human hyaluronidase (rHuPH20) has been used to increase the dispersion and absorption of other injected drugs, and in the case of prandial insulin analogs, it confers both ultrafast absorption and action profiles. Animal toxicology studies have demonstrated excellent tolerability of rHuPH20, and human studies, involving over 60,000 injections of prandial insulin + rHuPH20 to date, have similarly shown excellent safety and tolerability. Studies using rapid-acting analog insulin with rHuPH20 have included clinic-based pharmacokinetic and glucodynamic euglycemic glucose clamp studies, test meal studies, and take-home treatment studies. Administration methods have included subcutaneous injection of coformulations of rapid-acting insulin + rHuPH20 as well as continuous subcutaneous infusion of coformulations or use of pretreatment of newly inserted infusion sets with rHuPH20 followed by standard continuous subcutaneous insulin infusion therapy. These studies have demonstrated acceleration of insulin absorption and action along with improvement in postprandial glycemic excursions and reduction in hypoglycemia risks. Further, rHuPH20 reduces intrasubject variability of insulin absorption and action and provides greater consistency in absorption and action profiles over wear time of an infusion set. Further studies of rHuPH20 in the take-home treatment setting are underway.