Determination of five positive control drugs in hERG external solution (buffer) by LC-MS/MS to support in vitro hERG assay as recommended by ICH S7B.

ICH S7B recommends screening for hERG channel block using patch clamp recordings to assess a drug's proarrhythmic risk. Block of the hERG channel has been associated with clinical QTC prolongation as well as the rare, but potentially fatal ventricular tachyarrhythmia Torsade de Pointes (TdP). During recording, drug concentrations perfused to the cells can deviate from nominal concentrations due to molecule-specific properties (such as non-specific binding), thereby introducing error when assessing drug potency. To account for this potential source of error, both the original ICH S7B and the newly released ICH E14/S7B Q&As guidelines call for verifying drug solutions' concentrations. Dofetilide, cisapride, terfenadine, sotalol and E-4031 are hERG blockers commonly used as positive controls to illustrate hERG assay sensitivity. The first four compounds are also clinical drugs associated with high TdP risk; therefore, their safety margins may be useful comparators to better understand an investigational product's TdP risk. Having analytical methods to quantify these five compounds in the hERG external solution that will be used for patch clamp recordings is important from a regulatory science research perspective. However, a literature search revealed no analytical methods or stability information for these molecules in the high salt, serum-free matrix that constitutes the hERG external solution. This study was conducted to develop and validate LC-MS/MS methods to quantify these 5 molecules in hERG external solution. The bioanalytical methods for these positive controls were validated as per the FDA's bioanalytical method validation guidance along with various stabilities.

hERG block potencies for 5 positive control drugs obtained per ICH E14/S7B Q&As best practices: Impact of recording temperature and drug loss.

According to the ICH S7B guideline, drug candidates are screened for hERG block prior to first-in-human testing to predict the likelihood of delayed repolarization associated with a rare, but life-threatening, ventricular tachyarrhythmia. The new ICH E14 Q&As guideline allows hERG results to be used in later clinical development for decision-making (Q&As 5.1 and 6.1). To pursue this path, the hERG assay should be conducted following the new ICH S7B Q&A 2.1 guideline, which calls for best practice considerations of the recording temperature, voltage protocol, stimulation frequency, recording/data quality, and concentration verification. This study investigated hERG block by cisapride, dofetilide, terfenadine, sotalol, and E-4031 - positive controls commonly used to demonstrate assay sensitivity - using the manual whole cell patch clamp method and an action potential-like voltage protocol presented at 0.2 Hz. Recordings were conducted at room and near physiological temperature. Drug concentrations were measured using samples collected during real patch clamp experiments and satellite experiments. Results showed temperature effects for E-4031, terfenadine, and sotalol, but not cisapride and dofetilide. Cisapride and terfenadine showed substantial concentration losses, largely due to nonspecific binding to the perfusion apparatus. Using concentrations measured from the real and satellite experiments to assess block potencies yielded comparable results, indicating that satellite sample collection may be viable for drugs with nonspecific binding concerns only. In summary, this study provides block potencies for 5 hERG positive controls, and serves as a case study for hERG assays conducted, and results illustrated in accordance with the new ICH E14/S7B Q&As.

Characterizing the reproducibility in using a liver microphysiological system for assaying drug toxicity, metabolism, and accumulation.

Liver microphysiological systems (MPSs) are promising models for predicting hepatic drug effects. Yet, after a decade since their introduction, MPSs are not routinely used in drug development due to lack of criteria for ensuring reproducibility of results. We characterized the feasibility of a liver MPS to yield reproducible outcomes of experiments assaying drug toxicity, metabolism, and intracellular accumulation. The ability of the liver MPS to reproduce hepatotoxic effects was assessed using trovafloxacin, which increased lactate dehydrogenase (LDH) release and reduced cytochrome P450 3A4 (CYP3A4) activity. These observations were made in two test sites and with different batches of Kupffer cells. Upon culturing equivalent hepatocytes in the MPS, spheroids, and sandwich cultures, differences between culture formats were detected in CYP3A4 activity and albumin production. Cells in all culture formats exhibited different sensitivities to hepatotoxicant exposure. Hepatocytes in the MPS were more functionally stable than those of other culture platforms, as CYP3A4 activity and albumin secretion remained prominent for greater than 18 days in culture, whereas functional decline occurred earlier in spheroids (12 days) and sandwich cultures (7 days). The MPS was also demonstrated to be suitable for metabolism studies, where CYP3A4 activity, troglitazone metabolites, diclofenac clearance, and intracellular accumulation of chloroquine were quantified. To ensure reproducibility between studies with the MPS, the combined use of LDH and CYP3A4 assays were implemented as quality control metrics. Overall results indicated that the liver MPS can be used reproducibly in general drug evaluation applications. Study outcomes led to general considerations and recommendations for using liver MPSs. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Microphysiological systems (MPSs) have been designed to recreate organ- or tissue-specific characteristics of extracellular microenvironments that enhance the physiological relevance of cells in culture. Liver MPSs enable long-lasting and stable culture of hepatic cells by culturing them in three-dimensions and exposing them to fluid flow. WHAT QUESTION DID THIS STUDY ADDRESS? What is the functional performance relative to other cell culture platforms and the reproducibility of a liver MPS for assessing drug development and evaluation questions, such as toxicity, metabolism, and pharmacokinetics? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? The liver MPS systematically detected the toxicity of trovafloxacin. When compared with spheroids and sandwich cultures, this system had a more stable function and different sensitivity to troglitazone, tamoxifen, and digoxin. Quantifying phase II metabolism of troglitazone and intracellular accumulation of chloroquine demonstrated the potential use of the liver MPS for studying drug metabolism and pharmacokinetics. Quality control criteria for assessing chip function were key for reliably using the liver MPS. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? Due to its functional robustness and physiological relevance (3D culture, cells expose to fluid flow and co-culture of different cell types), the liver MPS can, in a reproducible manner: (i) detect inflammatory-induced drug toxicity, as demonstrated with trovafloxacin, (ii) detect the toxicity of other drugs, such as troglitazone, tamoxifen, and digoxin, with different effects than those detected in spheroids and sandwich cultures, (iii) enable studies of hepatic function that rely on prolonged cellular activity, and (iv) detect phase II metabolites and drug accumulation to potentially support the interpretation of clinical data. The integration of MPSs in drug development will be facilitated by careful evaluation of performance and reproducibility as performed in this study.

A Method for Diagnosing Organophosphate Pesticide Exposure in Humans using Liquid Chromatography Coupled Tandem Mass Spectrometry.

Organophosphate (OP) pesticides are commonly utilized worldwide for agricultural purposes and pose a health threat through air, ground, and water contamination. Here, we present a convenient method for diagnosing exposure to OP pesticides in humans. This immunoprecipitation method relies on extraction of butyrylcholinesterase (BChE), a biomarker of OP poisoning that adducts OP compounds, from human serum using agarose beads conjugated to anti-BChE antibodies. Extracted BChE was then digested with pepsin and analyzed for unadducted and OP-adducted peptides by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). To characterize and validate this method, pooled human plasma was exposed to parathion and dichlorvos to form diethoxyphospho, aged ethoxyphospho and dimethoxyphospho adducts with BChE. Untreated plasma was also analyzed for unadducted peptides. Additionally, samples were analyzed using Ellman's assay to measure BChE functional activity. The percent inhibition of BChE was 53.5±5.76 and 95.2±0.37%, respectively, for plasma treated with parathion for 1 hour and 24 hours. The percent inhibition was 97.2±0.98 for plasma treated with dichlorvos for 1 hour. The percent inhibition was 97.9±0.41% when the plasma treated with parathion for 1 hour, parathion for 24 hour and dichlorvos for 1 hour were mixed. Individual adducts were quantified in a single chromatographic run. Untreated plasma contained 26.4±1.87 ng/mL of unadducted BChE and no adducted peptides. In contrast, the plasma sample treated with both pesticides contained no unadducted BChE, but did contain 9.46±1.10, 10.9±0.98 and 14.1±1.10 ng/mL of diethoxyphospho, aged-ethoxy, and dimethoxyphospho peptides, respectively. The ability to identify and measure BChE and BChE adducts to parathion and dichlorvos is expected to be useful for diagnosing human exposure to multiple OP pesticides.

Simultaneous UHPLC-MS/MS method of estradiol metabolites to support the evaluation of Phase-2 metabolic activity of induced pluripotent stem cell derived hepatocytes.

The goal of this study was to develop and validate a high-throughput UHPLC-MS/MS method for simultaneous quantitation of three estradiol metabolites namely estradiol 3-ß-D-glucuronide (E3G), estradiol 17-ß-D-glucuronide (E17G) and estradiol 3-sulfate (E3S) in cell culture medium to support the characterization of metabolic function of induced pluripotent stem cell (iPSC) derived hepatocytes. To achieve this goal, a simple protein precipitation method was used for sample cleanup. All the metabolites were separated chromatographically using a C-18 column where 10 mM ammonium acetate and acetonitrile was used in gradient flow for 4 min. The analytes were quantitated by triple quadrupole mass spectrometer with the use of isotopically labeled internal standard (IS). This method was validated as per the U.S Food and Drug Administration's Bioanalytical Method Validation, Guidance for Industry. Linearity for E3G and E17G was in the range of 2-1500 ng/mL whereas for E3S it was 0.3-500 ng/mL. Inter-day and intra-day accuracy and precision of this method were in the acceptable limits. In addition, multiple stability tests (freeze thaw, autosampler, water bath (37 °C), bench top and long term) were performed for all the metabolites in cell culture medium. All the metabolites were stable up to 3 freeze thaw cycles at -20 °C and - 80 °C, 48 h in autosampler, 24 h at 37 °C, 48 h at room temperature and 173 days at -20 °C. Extraction recoveries for the metabolites were reproducible and were in the range of 94-108%. This method was used to quantitate estradiol metabolites generated by iPSC hepatocytes in-vitro studies.

Multiplexed ELISA screening assay for nine paralytic shellfish toxins in human plasma.

Paralytic shellfish poisoning is a lethal syndrome that can develop in humans who consume shellfish contaminated with paralytic shellfish toxins. These toxins have a short half-life in the human body, so a rapid diagnostic assessment of the poisoning is necessary. In this paper, we have developed and validated a rapid ELISA screening assay using anti-saxitoxin antibodies to screen nine toxins: saxitoxin; decarbamoyl saxitoxin; gonyautoxin 2,3; decarbamoyl GTX 2,3; neosaxitoxin; and gonyautoxin 1,4, in human plasma with lower limits of detection of 0.02, 0.08, 0.12, 1.2, 5.0, and 25 ng mL-1, respectively. Intra-day and inter-day precision experiments showed good reproducibility with a percent coefficient of variation less than 15%. The assay was 100% accurate in determining the presence or absence of these toxins in human plasma specimens. Blank specimens were assessed as negative for toxin content indicating that the method has excellent analyte specificity. This rapid screening assay can be used to quickly diagnose exposure to paralytic shellfish toxins, though an additional confirmatory method will be necessary to identify and quantitate the specific toxin in an exposure.

Evaluation of triazole-chelated lanthanides as chemically stabile bioimaging agents.

Europium (Eu), dysprosium (Dy), samarium (Sm), and terbium (Tb) complexes were prepared using the neutral tridentate chelator 2,6-bis(1-benzyl-1,2,3-triazol-4-yl)pyridine and one equivalent of each lanthanide salt. The physicochemical, aerodynamic, and in vitro cellular properties of each lanthanide metal complex were studied to determine their viability as cell surface fluorescent probes. Each compound was characterized by electrospray ionization mass spectroscopy (ESI-MS), ultraperformance liquid chromatography (UPLC), differential scanning calorimetry (DSC), and thermogravimetic analysis (TGA). Upon excitation at 320 nm each complex displayed characteristic lanthanide-based fluorescence emission in the visible wavelength range with stokes shifts greater than 200 nm. Each complex was found to be chemically stable when exposed to pH range of 1-11 for 72 h and resistant to photobleaching. To simulate pulmonary administration of these fluorophores, the aerodynamic properties of the Eu and Tb complexes were determined using a next generation impactor (NGI). This measurement confirmed that the complexes retain their fluorescence emission properties after nebulization. Cellular cytotoxicity was determined on A-549 lung cancer cell line using methylthiazol tetrazolium (MTT) cytotoxicity assay at 24, 48, and 72 h postexposure to the complexes. The complexes showed a dose and time-dependent effect on the percent viability of the cells.