Characterization of ADME properties of [¹⁴C]asunaprevir (BMS-650032) in humans

<div><p></p><p>1. Asunaprevir (ASV, BMS-650032), a highly selective and potent NS3 protease inhibitor, is currently under development for the treatment of chronic hepatic C virus infection. This study describes <i>in vivo</i> biotransformation in humans and the identification of metabolic enzymes of ASV.</p><p>2. Following a single oral dose of [¹⁴C]ASV to humans, the majority of radioactivity (>73% of the dose) was excreted in feces with <1% of the dose recovered in urine. Drug-related radioactivity readily appeared in circulation and the plasma radioactivity was mainly attributed to ASV. A few minor metabolites were observed in human plasma and are not expected to contribute to the pharmacological activity because of low levels. The area under the curve (AUC) values of each circulating metabolite in humans were well below their levels in animals used in the long-term toxicological studies. In bile and feces, intact ASV was a prominent radioactive peak suggesting that both metabolism and direct excretion played important roles in ASV clearance.</p><p>3. The primary metabolic pathways of ASV were hydroxylation, sulfonamide hydrolysis and the loss of isoquinoline. <i>In vitro</i> studies with human cDNA expressed CYP enzymes and with human liver microsomes (HLM) in the presence of selective chemical inhibitors demonstrated that ASV was primarily catalyzed by CYP3A4 and CYP3A5.</p></div

Quantitative Analysis of Polyethylene Glycol (PEG) and PEGylated Proteins in Animal Tissues by LC-MS/MS Coupled with In-Source CID

The covalent conjugation of polyethylene glycol (PEG, typical MW > 10k) to therapeutic peptides and proteins is a well-established approach to improve their pharmacokinetic properties and diminish the potential for immunogenicity. Even though PEG is generally considered biologically inert and safe in animals and humans, the slow clearance of large PEGs raises concerns about potential adverse effects resulting from PEG accumulation in tissues following chronic administration, particularly in the central nervous system. The key information relevant to the issue is the disposition and fate of the PEG moiety after repeated dosing with PEGylated proteins. Here, we report a novel quantitative method utilizing LC-MS/MS coupled with in-source CID that is highly selective and sensitive to PEG-related materials. Both ^40KPEG and a tool PEGylated protein (ATI-1072) underwent dissociation in the ionization source of mass spectrometer to generate a series of PEG-specific ions, which were subjected to further dissociation through conventional CID. To demonstrate the potential application of the method to assess PEG biodistribution following PEGylated protein administration, a single dose study of ATI-1072 was conducted in rats. Plasma and various tissues were collected, and the concentrations of both ^40KPEG and ATI-1072 were determined using the LC-MS/MS method. The presence of ^40kPEG in plasma and tissue homogenates suggests the degradation of PEGylated proteins after dose administration to rats, given that free PEG was absent in the dosing solution. The method enables further studies for a thorough characterization of disposition and fate of PEGylated proteins