Ray Bakhtiar

CYTOCHROME P450 3A4 IS THE MAJOR ENZYME INVOLVED IN THE METABOLISM OF THE SUBSTANCE P RECEPTOR ANTAGONIST APREPITANT

The contribution of human cytochrome P450 (P450) isoforms to the metabolism of aprepitant in humans was investigated using recombinant P450s and inhibition studies. In addition, aprepitant was evaluated as an inhibitor of human P450s. Metabolism of aprepitant by microsomes prepared from baculovirus-expressed human P450s was observed only when CYP1A2, CYP2C19, or CYP3A4 was present in the expression system. Incubation with CYP1A2 and CYP2C19 yielded only products of O-dealkylation, whereas CYP3A4 catalyzed both N- and O-dealkylation reactions. The metabolism of aprepitant by human liver microsomes was inhibited completely by ketoconazole or troleandomycin. No inhibition was observed with other P450 isoform-selective inhibitors. Aprepitant was evaluated also as a P450 inhibitor in human liver microsomes. No significant inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP2E1 was observed in experiments with isoform-specific substrates (IC50 > 70 μM). Aprepitant was a moderate inhibitor of CYP3A4, with Ki values of ∼10 μM for the 1′- and 4-hydroxylation of midazolam, and the N-demethylation of diltiazem, respectively. Aprepitant was a very weak inhibitor of CYP2C9 and CYP2C19, with Ki values of 108 and 66 μM for the 7-hydroxylation of warfarin and the 4′-hydroxylation of S-mephenytoin, respectively. Collectively, these results indicated that aprepitant is both a substrate and a moderate inhibitor of CYP3A4.

Immunoaffinity purification using anti-PEG antibody followed by two-dimensional liquid chromatography/tandem mass spectrometry for the quantification of a PEGylated therapeutic peptide in human plasma.

Quantification of a PEGylated peptide in human plasma using LC-MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC-MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate N(1-12)-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with <9.76% coefficient of variation (CV) for IAP, and 99.0-101.0% accuracy with <3.43% CV for PPT, over a dynamic range of 2-200 nM and 1-1000 nM, respectively. A cross comparison, performed using clinical samples, showed that the values obtained from IAP assay were about 15-30% lower than those from PPT method, which supports more specific PEG recognition provided by IAP.

Capillary electrophoresis fourier transform ion cyclotron resonance mass spectrometry with sustained off-resonance irradiation for the characterization of protein and peptide mixtures

A new approach to protein and peptide analysis that involves the coupling of on-line capillary electrophoresis-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry with a variation of sustained off-resonance irradiation is described. With this technique, multiple irradiation frequencies are broadcast simultaneously, which yields fragmentation of species at different mass-to-charge ratio values from the same waveform. In conjunction with capillary electrophoresis, this technique can provide sequence information from small amounts of proteins or peptides in complex mixtures. Initial results obtained from a mixture of gramicidin S (1141 u), bee venom melittin (2845 u), and equine apomyoglobin (16,951 u) are presented.