A simple and green capillary electrophoresis-mass spectrometry method for therapeutic drug monitoring of colistin in clinical plasma samples

Colistin and other polymyxin antibiotics have become increasingly used in clinical settings as a result of treating multidrug-resistant infections in critically ill patients. The highly variable pharmacokinetics of colistin in these patients is accompanied by a high risk of toxicity or underdosing. An effective tool that allows rational optimization of the drug dosage regimen is therapeutic drug monitoring. Therefore, there is a need to dispose with appropriate, sensitive, and accurate analytical methods. Here, a simple, specific, and accurate on-line capillary electrophoresis – tandem mass spectrometry method was developed and applied for the first time to determine colistin in human plasma. Protein precipitation using acidified acetonitrile was the solitary procedure used to achieve sample pretreatment. A bare fused silica capillary was employed for the separation process, and the background electrolyte used was 50 mM formic acid (pH 2.54). The FDA's bioanalytical method validation guidelines were followed in the validation of the proposed method. For colistin A and colistin B, favorable performance and validation parameters were obtained (such as linearity, limit of detection, lower limit of quantitation, intra-day and inter-day precision, accuracy, and stability).The validated method was then effectively used to analyze real clinical samples taken from patients who were in critical condition. Our newly developed method is comparable with previously published liquid chromatography approaches and has the potential to be applied in the therapeutic monitoring of colistin in routine clinical laboratories. Moreover, according to the greenness assessment, the developed capillary electrophoresis – mass spectrometry method represents a very interesting green and sustainable tool in the field of bioanalysis

Implementation of Modern Therapeutic Drug Monitoring and Lipidomics Approaches in Clinical Practice: A Case Study with Colistin Treatment

Nowadays, lipidomics plays a crucial role in the investigation of novel biomarkers of various diseases. Its implementation into the field of clinical analysis led to the identification of specific lipids and/or significant changes in their plasma levels in patients suffering from cancer, Alzheimer’s disease, sepsis, and many other diseases and pathological conditions. Profiling of lipids and determination of their plasma concentrations could also be helpful in the case of drug therapy management, especially in combination with therapeutic drug monitoring (TDM). Here, for the first time, a combined approach based on the TDM of colistin, a last-resort antibiotic, and lipidomic profiling is presented in a case study of a critically ill male patient suffering from Pseudomonas aeruginosa-induced pneumonia. Implementation of innovative analytical approaches for TDM (online combination of capillary electrophoresis with tandem mass spectrometry, CZE-MS/MS) and lipidomics (liquid chromatography–tandem mass spectrometry, LC-MS/MS) was demonstrated. The CZE-MS/MS strategy confirmed the chosen colistin drug dosing regimen, leading to stable colistin concentrations in plasma samples. The determined colistin concentrations in plasma samples reached the required minimal inhibitory concentration of 1 μg/mL. The complex lipidomics approach led to monitoring 545 lipids in collected patient plasma samples during and after the therapy. Some changes in specific individual lipids were in good agreement with previous lipidomics studies dealing with sepsis. The presented case study represents a good starting point for identifying particular individual lipids that could correlate with antimicrobial and inflammation therapeutic management

Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just “dilute and shoot”), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91–15.12% and with accuracy yielding relative errors in the range of 85.47–112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines

FUNDAMANT: an interventional 72-week phase 1 follow-up study of AADvac1, an active immunotherapy against tau protein pathology in Alzheimer’s disease

Abstract Background Neurofibrillary pathology composed of tau protein is closely correlated with severity and phenotype of cognitive impairment in patients with Alzheimer’s disease and non-Alzheimer’s tauopathies. Targeting pathological tau proteins via immunotherapy is a promising strategy for disease-modifying treatment of Alzheimer’s disease. Previously, we reported a 24-week phase 1 trial on the active vaccine AADvac1 against pathological tau protein; here, we present the results of a further 72 weeks of follow-up on those patients. Methods We did a phase 1, 72-week, open-label study of AADvac1 in patients with mild to moderate Alzheimer’s disease who had completed the preceding phase 1 study. Patients who were previously treated with six doses of AADvac1 at monthly intervals received two booster doses at 24-week intervals. Patients who were previously treated with only three doses received another three doses at monthly intervals, and subsequently two boosters at 24-week intervals. The primary objective was the assessment of long-term safety of AADvac1 treatment. Secondary objectives included assessment of antibody titres, antibody isotype profile, capacity of the antibodies to bind to AD tau and AADvac1, development of titres of AADvac1-induced antibodies over time, and effect of booster doses; cognitive assessment via 11-item Alzheimer’s Disease Assessment Scale cognitive assessment (ADAS-Cog), Category Fluency Test and Controlled Oral Word Association Test; assessment of brain atrophy via magnetic resonance imaging (MRI) volumetry; and assessment of lymphocyte populations via flow cytometry. Results The study was conducted between 18 March 2014 and 10 August 2016. Twenty-six patients who completed the previous study were enrolled. Five patients withdrew because of adverse events. One patient was withdrawn owing to noncompliance. The most common adverse events were injection site reactions (reported in 13 [50%] of vaccinated patients). No cases of meningoencephalitis or vasogenic oedema were observed. New micro-haemorrhages were observed only in one ApoE4 homozygote. All responders retained an immunoglobulin G (IgG) antibody response against the tau peptide component of AADvac1 over 6 months without administration, with titres regressing to a median 15.8% of titres attained after the initial six-dose vaccination regimen. Booster doses restored previous IgG levels. Hippocampal atrophy rate was lower in patients with high IgG levels; a similar relationship was observed in cognitive assessment. Conclusions AADvac1 displayed a benign safety profile. The evolution of IgG titres over vaccination-free periods warrants a more frequent booster dose regimen. The tendency towards slower atrophy in MRI evaluation and less of a decline in cognitive assessment in patients with high titres is encouraging. Further trials are required to expand the safety database and to establish proof of clinical efficacy of AADvac1. Trial registration The studies are registered with the EU Clinical Trials Register and ClinicalTrials.gov: the preceding first-in-human study under EudraCT 2012-003916-29 and NCT01850238 (registered on 9 May 2013) and the follow-up study under EudraCT 2013-004499-36 and NCT02031198 (registered 9 Jan 2014), respectively

ADAMANT: a placebo-controlled randomized phase 2 study of AADvac1, an active immunotherapy against pathological tau in Alzheimer’s disease

Alzheimer’s disease (AD) pathology is partly characterized by accumulation of aberrant forms of tau protein. Here we report the results of ADAMANT, a 24-month double-blinded, parallel-arm, randomized phase 2 multicenter placebo-controlled trial of AADvac1, an active peptide vaccine designed to target pathological tau in AD (EudraCT 2015-000630-30). Eleven doses of AADvac1 were administered to patients with mild AD dementia at 40 μg per dose over the course of the trial. The primary objective was to evaluate the safety and tolerability of long-term AADvac1 treatment. The secondary objectives were to evaluate immunogenicity and efficacy of AADvac1 treatment in slowing cognitive and functional decline. A total of 196 patients were randomized 3:2 between AADvac1 and placebo. AADvac1 was safe and well tolerated (AADvac1 n = 117, placebo n = 79; serious adverse events observed in 17.1% of AADvac1-treated individuals and 24.1% of placebo-treated individuals; adverse events observed in 84.6% of AADvac1-treated individuals and 81.0% of placebo-treated individuals). The vaccine induced high levels of IgG antibodies. No significant effects were found in cognitive and functional tests on the whole study sample (Clinical Dementia Rating-Sum of the Boxes scale adjusted mean point difference −0.360 (95% CI −1.306, 0.589)), custom cognitive battery adjusted mean z-score difference of 0.0008 (95% CI −0.169, 0.172). We also present results from exploratory and post hoc analyses looking at relevant biomarkers and clinical outcomes in specific subgroups. Our results show that AADvac1 is safe and immunogenic, but larger stratified studies are needed to better evaluate its potential clinical efficacy and impact on disease biomarkers