High-throughput, low-cost quantification of 11 therapeutic antibodies using caprylic acid precipitation and LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring of treatment with therapeutic antibodies is hampered by the application of a wide range of different methods in the quantification of serum levels. LC-MS based methods could significantly improve comparability of results from different laboratories, but such methods are often considered complicated and costly. We developed a method for LC-MS/MS based quantification of 11 therapeutic antibodies concomitantly measured in a single run, with emphasis on simplicity in sample preparation and low cost. RESULTS: After a single-step sample purification using caprylic acid precipitation to remove interfering proteins, the sample underwent proteolysis followed by LC-MS/MS analysis. Infliximab is used as internal standard for sample preparation while isotope-labeled signature peptides identified for each analyte are internal standards for the LC-MS/MS normalization. The method was validated according to recognized guidelines, and pipetting steps can be performed by automated liquid handling systems. The total precision of the method ranged between 2.7 and 7.3 % (5.1 % average) across the quantification range of 4-256 µg/ml for all 11 drugs, with an average accuracy of 96.3 %. Matrix effects were xamined in 55 individual patient samples instead of the recommended 6, and 147 individual patient samples were screened for interfering compounds. SIGNIFICANCE AND NOVELTY: Our method for simultaneous quantification of 11 t-mAb in human serum allows an unprecedented integration of robustness, speed and reduced complexity, which could pave the way for uniform use in research projects and clinical settings alike. In addition, the first LC-MS protocol for signature peptide-based quantification of durvalumab is described. This high throughput method can be readily adapted to a drug panel of choice.

Nurses as adverse drug reaction reporting advocates.

Adverse drug reactions (ADRs) is a challenge in modern healthcare, particularly given the increasing complexity of drug therapy, an ageing population, rising multimorbidity, and a high patient turnover. The core activity of detecting potential ADRs over the last half century has been spontaneous reporting systems. A recent Norwegian regulation commits healthcare professionals other than physicians and dentists to report serious ADRs. In this discussion paper, we share our preliminary experience with a training programme using nurses as ADR advocates to stimulate ADR reporting among the clinical staff in a hospital department.

A Patient With Sepsis-Induced Multiorgan Failure and Increasing Serum Methadone Concentration: A Case Study.

ABSTRACT: The authors describe a patient with substance use disorder admitted to the hospital with septic shock and multiorgan failure, in whom the serum concentration of methadone kept increasing despite discontinuation of the drug. Therapeutic drug monitoring was performed to monitor the methadone serum concentration during treatment of the underlying diseases.

Perception of prescribing factors and purchase statistics of non-steroidal anti-inflammatory drugs in an orthopedic clinic.

OBJECTIVES: Nonsteroidal anti-inflammatory drugs (NSAIDS) are associated with concern of adverse drug reactions (ADRS) including gastrointestinal, cardiovascular, renal, and musculoskeletal. Non-selective and selective NSAIDS are proposed to differ with regard to their potential to cause ADRS. The aim of this pilot study was to compare perception of prescribing factors and purchase statistics of NSAIDS among physicians in a Norwegian orthopedic clinic. RESULTS: Forty-five (55%) of 82 invited physicians from the orthopedic clinic participated anonymous in a survey in February 2017. Effect and ADRS were rated as the most important factors for prescribing of NSAIDS. The participants were equally concerned about specific ADRS for prescription of non-selective and selective NSAIDS irrespective of type of ADR. They were generally more concerned about cardiovascular, gastrointestinal and renal ADRS than musculoskeletal. Purchase statistics from 2015 and 2016 showed that celecoxib, a selective NSAID, dominated in the orthopedic clinic. The discrepancy between perception of prescribing factors and purchase statistics of NSAIDS was possibly explained by a high degree of conformity to clinic guidelines. Our preliminary results indicate that perception of prescribing factors of NSAIDS among orthopedics should be surveyed in multicenter or multinational studies.

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines.

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.