The Effect of PEG-Insulin and Insulin Hexamer Assembly on Stability in Solution and Dry Powders. Hexamer Assembly of PEGylated-Insulin and Insulin Studied by Multi-Angle Light Scattering to Rationally Choose the pH and Zinc Content for Analytical Methods and Formulations of Dry Powders.
SupervisorForbes, Robert T.
The University of Bradford theses are licenced under a Creative Commons Licence.
InstitutionUniversity of Bradford
DepartmentThe School of Pharmacy
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AbstractThe objective of this research is to further define the relationship between the charge state of insulin, and the self assembly properties of insulin and PEGylated insulin in solution. Polyethylene glycol (PEG) chains were covalently attached to insulin in order to evaluate their impact on insulin¿s systemic duration of action after pulmonary dosing. This thesis will focus on the assembly properties of the PEG-insulin and insulin, and also demonstrate how the charge state, which was modified by the covalent attachment of PEG, relates to different modes of behavior by anion and cation exchange chromatography. In addition, explain how modifying the assembly state extends to improving formulation properties of spray-dried insulin powders. This thesis is an investigation into the relationship of insulin¿s charge state controlled by pH and how the charge state affects the self assembly of insulin, especially when the zinc ion is removed. Ionic interaction is one of the major forces affecting insulin assembly. The theory that a change in the charge state of insulin could modulate the ionic interaction and reduce hexamer formation at alkaline conditions was investigated. Experiments were designed to measure the level of hexamer with light scattering, and the amount of hexamer was then correlated with the pH and zinc content of the solutions. The importance of the charge state of the monomer and its behavior extends to chromatography and purification modes as well. Specifically, the purification of various species of PEGylated insulin presents a challenge. By varying mobile phase pH which induces the charge to insulin, an ion exchange method demonstrated very high resolution and controllable interaction between the ion exchange media and the insulin derivatives. A highly accurate method for determining molecular weight and thus the average associated state of insulin in solution has been developed using the MALS (Multi-Angle Light Scattering). Insulin concentration, pH, and metal ion concentrations, were in pharmaceutically relevant ranges. The MALS method was developed to evaluate how the parameters above affect the self-assembly properties of insulin, and use the assembly properties to improve formulations of insulin or PEGylated insulin. To use the light scattering technique the dn/dc (change in refractive index with change in concentration) is required. During the method development, the dn/dc of insulin was measured at 690 nm, and a value of 0.185 mL/g based on theory was confirmed. A novel approach for preparing insulin powders with improved chemical stability, based on maintaining the dissociation of hexamers in solution during the spray drying process was developed. The mode presented here is to remove the zinc ions from solution, increase the pH from 6.6 to 7.8, and maintain a low concentration of insulin approximately 2 to 15 mg/mL. Each of these factors alone decreases the hexamer population in solution, but by combining all three factors, hexamers are driven to very low levels of equilibrium. The increased stability of the powders is predominately related to the decrease in covalent insulin dimer (CID). The data presented correlates a reduced hexamer population in the solution with lower levels of CID¿s in the dry powder compared to controls. The CID formation rate was reduced by 40% compared to a control.
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Improving the quality of insulin prescribing for people with diabetes being discharged from hospitalBain, A.; Silcock, Jonathan; Kavanagh, S.; Quinn, Gemma L.; Fonseca, I. (BMJ, 2019-08)Medication errors involving insulin in hospital are common, and may be particularly problematic at the point of transfer of care. Our aim was to improve the safety of insulin prescribing on discharge from hospital using a continuous improvement methodology involving cycles of iterative change. A multidisciplinary project team formulated locally tailored insulin discharge prescribing guidance. After baseline data collection, three ‘plan-do-study-act’ cycles were undertaken over a 3-week period (September/ October 2018) to introduce the guidelines and improve the quality of discharge prescriptions from one diabetes ward at the hospital. Discharge prescriptions involving insulin from the ward during Monday to Friday of each week were examined, and their adherence to the guidance measured. After the introduction of the guidelines in the form of a poster, and later a checklist, the adherence to guidelines rose from an average of 50% to 99%. Qualitative data suggested that although it took pharmacists slightly longer to clinically verify discharge prescriptions, the interventions resulted in a clear and helpful reminder to help improve discharge quality for the benefit of patient safety. This project highlights that small iterative changes made by a multidisciplinary project team can result in improvement of insulin discharge prescription quality. The sustainability and scale of the intervention may be improved by its integration into the electronic prescribing system so that all users may access and refer to the guidance when prescribing insulin for patients at the point of discharge.
Associations of Circulating Calcium and 25-Hydroxyvitamin D With Glucose Metabolism in Pregnancy: A Cross-Sectional Study in European and South Asian WomenWhitelaw, D.C.; Scally, Andy J.; Tuffnell, D.J.; Davies, T.J.; Fraser, W.D.; Bhopal, R.S.; Wright, J.; Lawlor, D.A. (2014-03)Background: Vitamin D deficiency is thought to impair insulin action and glucose metabolism; however, previous studies have not examined ethnic differences or the influence of calcium and parathyroid hormone. We investigated this in a cohort of predominantly white European and south Asian women during pregnancy. Methods: In this cross-sectional study from an urban population in northern England (53.8°N), 1467 women were recruited when undergoing glucose tolerance testing (75 g oral glucose tolerance test) at 26 weeks' gestation. Results: Gestational diabetes mellitus (GDM) was diagnosed in 137 women (9.3%). Median 25-hydroxyvitamin D concentration for the study population was 9.3 ng/mL (interquartile range 5.2, 16.9) and was higher in European [15.2 ng/mL (10.7, 23.5)] than in south Asian women [5.9 ng/mL (3.9, 9.4), P < .001]. After appropriate adjustment for confounders, 25-hydroxyvitamin D showed a weak inverse association with fasting plasma glucose (FPG; mean difference 1.0% per 1 SD; the ratio of geometric means (RGM) 0.99, 95% confidence interval (CI) 0.98, 1.00), and PTH was weakly associated with FPG (RGM 1.01, 95% CI 1.00, 1.02), but neither was associated with fasting insulin, postchallenge glucose, or GDM. Serum calcium (albumin adjusted) was strongly associated with fasting insulin (RGM 1.06; 95% CI 1.03, 1.08), postchallenge glucose (RGM 1.03, 95% CI 1.01, 1.04), and GDM (odds ratio 1.33, 95% CI 1.06, 1.66) but not with FPG. Associations were similar in European and south Asian women. Conclusions: These findings do not indicate any important association between vitamin D status and glucose tolerance in pregnancy. Relationships between circulating calcium and glucose metabolism warrant further investigation.
A high-fat-diet-induced cognitive deficit in rats that is not prevented by improving insulin sensitivity with metforminMcNeilly, A.D.; Williamson, Ritchie; Balfour, D.J.; Stewart, C.A.; Sutherland, C. (2012)AIMS/HYPOTHESIS: We previously demonstrated that animals fed a high-fat (HF) diet for 10 weeks developed insulin resistance and behavioural inflexibility. We hypothesised that intervention with metformin would diminish the HF-feeding-evoked cognitive deficit by improving insulin sensitivity. METHODS: Rats were trained in an operant-based matching and non-matching to position task (MTP/NMTP). Animals received an HF (45% of kJ as lard; n = 24), standard chow (SC; n = 16), HF + metformin (144 mg/kg in diet; n = 20) or SC + metformin (144 mg/kg in diet; n = 16) diet for 10 weeks before retesting. Body weight and plasma glucose, insulin and leptin were measured. Protein lysates from various brain areas were analysed for alterations in intracellular signalling or production of synaptic proteins. RESULTS: HF-fed animals developed insulin resistance and an impairment in switching task contingency from matching to non-matching paradigm. Metformin attenuated the insulin resistance and weight gain associated with HF feeding, but had no effect on performance in either MTP or NMTP tasks. No major alteration in proteins associated with insulin signalling or synaptic function was detected in response to HF diet in the hypothalamus, hippocampus, striatum or cortex. CONCLUSIONS/INTERPRETATION: Metformin prevented the metabolic but not cognitive alterations associated with HF feeding. The HF diet protocol did not change basal insulin signalling in the brain, suggesting that the brain did not develop insulin resistance. These findings indicate that HF diet has deleterious effects on neuronal function over and above those related to insulin resistance and suggest that weight loss may not be sufficient to reverse some damaging effects of poor diet.