Quantification and pharmacokinetic study of tumor-targeting agent MHI148-clorgyline amide in mouse plasma using liquid chromatography-electrospray ionization tandem mass spectrometry

Abstract

A high-performance liquid chromatography-electrospray ionization tandem mass spectrometric (HPLC-ESI-MS/MS) method was developed for the quantification of MHI148-clorgyline amide (NMI-amide), a novel tumor-targeting monoamine oxidase A inhibitor, in mouse plasma. The method was validated in terms of sensitivity, precision, accuracy, recovery and stability and then applied to a pharmacokinetic study of NMI-amide in mice following intravenous administration. NMI-amide together with the internal standard (IS), MHI-148, was extracted by protein precipitation using acetonitrile. Multiple reaction monitoring was used for quantification of NMI-amide by detecting m/z transition of 491.2–361.9, and 685.3–258.2 for NMI-amide and the IS, respectively. The lower limit of quantification (LLOQ) of the HPLC–MS/MS method for NMI-amide was 0.005 μg/mL and the linear calibration curve was acquired with R2 > 0.99 in the concentration range of 0.005–2 μg/mL. The intra- and inter-day precisions of the assay were assessed by percentage of the coefficient of variations, which was within 9.8% at LLOQ and 14.0% for other quality control samples, whereas the mean accuracy ranged from 86.8% to 113.2%. The samples were stable under storage and experimental conditions. This method was successfully applied to a pharmacokinetic study in mice following intravenous administration of 5 mg/kg NMI-amide.

Hows and Whys of Tumor-Seeking Dyes

“…Careful LC-MS analyses must be used to ascertain half-lives for disappearance of 1-Cl reflecting displacement of the meso-Cl by nucleophiles (like albumin); these show the in vivo blood half-life is 36.6 min for a derivative of 1-Cl. 35 In collaboration with Prof. H. S. Choi (Harvard Medical School), we studied in vivo tumor accumulation of 1-Cl, 1-Ph, 5-Cl, and 5-Ph, and persistence of each dye for up to 48 h. This dye series is complementary insofar as two (1-Cl {initially} and 1-Ph) form noncovalent albumin adducts, a different pair forms covalent albumin adducts (1-Cl and 5-Cl, slowly and quickly respectively), and 5-Ph does not bind albumin in any way. An orthotopic model was used to avoid complications to the pharmacokinetics data that might arise in a model involving immune compromised mice, with subcutaneous triple negative breast tumors (E0771).…”

“…However, albumin adducts cannot be detected in such experiments. Careful LC-MS analyses must be used to ascertain half-lives for disappearance of 1 -Cl reflecting displacement of the meso -Cl by nucleophiles (like albumin); these show the in vivo blood half-life is 36.6 min for a derivative of 1 -Cl …”

“…Overall, these data are consistent with the following interpretation of the mechanism of accumulation and persistence of tumor seeking dyes. After administration via iv injection and entry into the bloodstream, the dye is instantly converted into a noncovalent albumin adduct, which transforms into a covalent one over a period that varies with structure, but is about 36 min for 1 -Cl (see above) . The albumin adducts will accumulate in tumors via the EPR effect (originally observed for accumulation of labeled-albumin in tumors, − albumin is an ellipsoidal molecules of ∼3.8 nm diameter, and 14 nm length) and intracellularly via uptake through the various receptors that import albumin and are overexpressed in cancer cells. , In cell culture experiments monitored by confocal microscopy, we observed that fluorescein (FITC)-labeled albumin permeated into cancer cells and colocalized with the near-IR fluorescence of 1 -Cl and 34 Cys- blocked FITC-albumin did the same.…”