The Development of Capillary and Microchip Electrophoresis Methods for the Analysis of Pharmaceuticals in Developing Countries

Maintaining a consistent supply of pharmaceuticals to developing countries could save millions of lives per year. One of the major roadblocks in this effort is an abundance of substandard and counterfeit drugs in the supply chain. To prevent these products from being distributed, improved methodology and instrumentation for drug screening is needed. In these low-resource areas the availability of proper funding and laboratory space is limited. To replace the expensive liquid chromatography and mass spectrometry instrumentation traditionally used for quality control, capillary electrophoresis (CE) can be used. CE has a low startup cost and, because of the small sample and reagent volume requirements, the cost per test is kept to a minimum. Additionally, any methodology developed for CE can be transferred to the miniaturized platform of microchip electrophoresis (ME). ME further reduces the cost-per-test and provides the potential for a fully portable analytical device that can be used for on-site analysis. This method is particularly useful for screening pharmaceuticals throughout the many distribution lines, store houses, and clinics across a large country. In this dissertation, CE was used to develop methodology for the analysis of the peptide drug oxytocin (OT). OT is needed in developing countries to prevent death from post-partum hemorrhage. A major concern regarding the supply chain of peptide-based drugs is the degradation of these products when then are shipped and stored incorrectly. Several degradation products are produced when OT is subjected to heat-stress conditions. Deamidation, in particular, produces small molecular changes that have significant effects on the biological activity of the peptide. Initially, methodology for the separation and detection of desamino degradation products of OT was developed for CE-UV. OT contains three potential sites of deamidation, which leads to seven distinct desamino products. The separation was achieved following an optimization of the background electrolyte to include a pseudostationary phase and an organic modifier to increase selectivity and resolve the eight structurally similar peptides. To improve the functionality of the CE assay for OT integrity screening, the next step of was to further optimize the method for a separation of all of the known degradation products formed under heat-stressed OT formulations. However, current literature on the degradation of OT does not yet addressed the degradation of Pitocin pharmaceutical formulations that are prepared in water containing a small amount of acetic acid, to adjust the pH between 3-5, and 0.5% chlorobutanol as a preservative. To investigate the effect that CB and CB-like products have on the degradation of OT, LC-UV-MS was used to monitor the formation of degradation products as a function of time. The preliminary data shows that CB, and the structurally similar trichloroethanol (TCE), significantly stabilize OT from the heat-stress degradation. The addition of either of these trichloro species substantially reduces the rate of degradation observed over a 48 h period at 70 °C. Further work is needed on this project to determine the cause of this protective effect by performing structural studies of OT in the presence of CB or TCE. Finally, initial steps were taken towards the development of a low-cost portable ME device. A significant percentage of small molecule counterfeit drugs are formulated with the wrong amount of active ingredient. Current portable analytical methods available to monitor the pharmaceutical supply chain are limited in their quantitative abilities. By coupling ME to conductivity detection, it is possible to perform quantitative analysis of multiple species simultaneously. Initial experiments to evaluate system performance were performed using two first-line anti-tuberculosis drugs, ethambutol and isoniazid

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)