Neurotoxicity is a principal concern in nonclinical drug development. However, standardized and universally accepted fluid biomarkers for evaluating neurotoxicity are lacking. Increasing clinical evidence supports the potential use of neurofilament light chain (NfL) as a biomarker of several neurodegenerative diseases; therefore, we investigated changes in the cerebrospinal fluid (CSF) and serum levels of NfL in SD rats treated with central nervous system (CNS) toxicants [trimethyltin (TMT, 10 mg/kg p.o., single dose), kainic acid (KA, 12 mg/kg s.c., single dose), MK-801 (1 mg/kg s.c., single dose)], and a peripheral nervous system (PNS) toxicant (pyridoxine, 1200 mg/kg/day for 3 days). Animals were euthanized 1 (Day 2), 3 (Day 4), or 7 days after administration (Day 8). Increased serum NfL was observed in TMT- and KA-treated animals, which indicated neuronal cell death in the brain on Day 2, 4, and/or 8. MK-801-treated animals exhibited no changes in the serum and CSF levels of NfL and no histopathological changes in the brain at any time point. Pyridoxine induced chromatolysis of the dorsal root ganglion on Day 2 and degeneration of peripheral nerve fiber on Day 4; additionally, serum NfL was increased. A strong correlation was observed between the serum and CSF levels of NfL and brain lesions caused by TMT and KA, indicating that NfL could be a useful biomarker for detecting CNS toxicity. Additionally, PNS changes were correlated with serum NfL levels. Therefore, serum NfL could serve as a useful peripheral biomarker for detecting both CNS and PNS toxicity in rats.
We developed a highly sensitive and specific high-performance liquid chromatography with tandem mass spectrometry method with an atmospheric pressure chemical ionization interface to determine 24S-hydroxycholesterol, a major metabolite of cholesterol formed by cytochrome P450 family 46A1, in human plasma without any derivatization step. Phosphate buffered saline including 1% Tween 80 was used as the surrogate matrix for preparation of calibration curves and quality control samples. The saponification process to convert esterified 24S-hydroxycholesterol to free sterols was optimized, followed by liquid-liquid extraction using hexane. Chromatographic separation of 24S-hydroxycholesterol from other isobaric endogenous oxysterols was successfully achieved with gradient mobile phase comprised of 0.1% propionic acid and acetonitrile using L-column2 ODS (2 μm, 2.1 mm id × 150 mm). This assay was capable of determining 24S-hydroxycholesterol in human plasma (200 μL) ranging from 1 to 100 ng/mL with acceptable intra- and inter-day precision and accuracy. The potential risk of in vitro formation of 24S-hydroxycholesterol by oxidation from endogenous cholesterol in human plasma was found to be negligible. The stability of 24S-hydroxycholesterol in relevant solvents and human plasma was confirmed. This method was successfully applied to quantify the plasma concentrations of 24S-hydroxycholesterol in male and female volunteers.
Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5–250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.
The pharmacokinetics of TAK-802, a potential agent of amelioration for voiding dysfunction, were investigated in rats, dogs and monkeys after a single oral administration of 14C-labeled TAK-802. The values of the bioavailability for the compound ranged from 10.2 to 19.9% and the extent of absorption of 14C were higher than the values for the bioavailability in the tested animals. TAK-802 and its related compounds distributed widely in the tissues including the target organ, the urinary bladder, in rats. TAK-802 was highly bound to the plasma proteins and the protein-binding % was independent of the spiked concentrations in all the species tested. Meanwhile, the erythrocyte distribution decreased significantly with the increase in the TAK-802 concentrations. After oral dosing, the dosed 14C was predominantly excreted into the feces of the test animals and the hepato-biliary route mainly contributed to the excretion of 14C in rats. The major components of 14C in the plasma and excreta were unidentified polar metabolites in the test animals. These results indicated that TAK-802 was extensively metabolized by first-pass metabolism during the absorption process and its related metabolites were excreted predominantly into the feces via the bile in the test animals. The concentration-dependent erythrocyte distribution was characterized as the most influential property on the pharmacokinetics of TAK-802. For the clinical safety in the development of TAK-802, the effect of the concentration-dependent erythrocyte distribution on the pharmacokinetics of TAK-802 in animals and humans should be addressed.
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