A tag-free collisionally induced fragmentation approach to detect drug-adducted proteins by mass spectrometry.

Rationale: The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag-free technique capable of detecting protein-small molecule adducts based solely on the collision-induced dissociation (CID) of the protein-small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.

A simple litmus test for aldehyde oxidase metabolism of heteroarenes.

The bioavailability of aromatic azaheterocyclic drugs can be affected by the activity of aldehyde oxidase (AO). Susceptibility to AO metabolism is difficult to predict computationally and can be complicated in vivo by differences between species. Here we report the use of bis(((difluoromethyl)sulfinyl)oxy)zinc (DFMS) as a source of CF2H radical for a rapid and inexpensive chemical "litmus test" for the early identification of heteroaromatic drug candidates that have a high probability of metabolism by AO.

Pharmacokinetics, metabolism, and excretion of the antidiabetic agent ertugliflozin (PF-04971729) in healthy male subjects.

The disposition of ertugliflozin (PF-04971729), an orally active selective inhibitor of the sodium-dependent glucose cotransporter 2, was studied after a single 25-mg oral dose of [(14)C]-ertugliflozin to healthy human subjects. Mass balance was achieved with approximately 91% of the administered dose recovered in urine and feces. The total administered radioactivity excreted in feces and urine was 40.

Molecular etiology of atherogenesis--in vitro induction of lipidosis in macrophages with a new LDL model.

Background: Atherosclerosis starts by lipid accumulation in the arterial intima and progresses into a chronic vascular inflammatory disease. A major atherogenic process is the formation of lipid-loaded macrophages in which a breakdown of the endolysomal pathway results in irreversible accumulation of cargo in the late endocytic compartments with a phenotype similar to several forms of lipidosis. Macrophages exposed to oxidized LDL exihibit this phenomenon in vitro and manifest an impaired degradation of internalized lipids and enhanced inflammatory stimulation.

Deuterium isotope effects on drug pharmacokinetics. I. System-dependent effects of specific deuteration with aldehyde oxidase cleared drugs.

The pharmacokinetic properties of drugs may be altered by kinetic deuterium isotope effects. With specifically deuterated model substrates and drugs metabolized by aldehyde oxidase, we demonstrate how knowledge of the enzyme's reaction mechanism, species differences in the role played by other enzymes in a drug's metabolic clearance, and differences in systemic clearance mechanisms are critically important for the pharmacokinetic application of deuterium isotope effects. Ex vivo methods to project the in vivo outcome using deuterated carbazeran and zoniporide with hepatic systems demonstrate the importance of establishing the extent to which other metabolic enzymes contribute to the metabolic clearance mechanism.

A rapid and specific derivatization procedure to identify acyl-glucuronides by mass spectrometry.

A simple procedure is described to identify acyl-glucuronides by coupled liquid chromatography/mass spectrometry after derivatization to a hydroxamic acid with hydroxylamine. The reaction specificity obviates the need for isolation of the acyl-glucuronide from an extract. Glucuronides derived from carbamic acids, and alkyl- and aromatic amines, are inert to the derivatization reaction conditions, making the hydroxamic acid derivative a fingerprint for acyl-glucuronides.

The use of 18O-exchange and base-catalyzed N-dealkylation with liquid chromatography/tandem mass spectrometry to identify carbinolamide metabolites.

Oxidation of N-alkyl-substituted amides is a common transformation observed in metabolism studies of drugs and other chemicals. Metabolism at the alpha carbon atom can produce stable carbinolamide compounds, which may be abundant enough to require complete confidence in structural assignments. In a drug discovery setting, rapid structural elucidation of test compounds is critical to inform the compound selection process.

Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections.

Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.

Pharmacokinetics, metabolism, and excretion of torcetrapib, a cholesteryl ester transfer protein inhibitor, in humans.

The pharmacokinetics, metabolism, and excretion of torcetrapib, a selective inhibitor of human cholesteryl ester transfer protein, were investigated in healthy human male volunteers after oral administration of [(14)C]torcetrapib (120-mg dose). The total mean recovery of radiolabeled dose after 21 days was 75.7%, and most of the dose (63%) was excreted in the urine.

Metabolism and disposition of a gamma-aminobutyric acid type A receptor partial agonist in humans.

The metabolism and disposition of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide (1), a potent subtype-selective partial agonist at the gamma-aminobutyric acid type A receptor complex, were elucidated in humans following a p.o. dose of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-[3-(14)C]indole-3-carboxamide monomethane-sulfonate ([(14)C]1).

Metabolic activation of the nontricyclic antidepressant trazodone to electrophilic quinone-imine and epoxide intermediates in human liver microsomes and recombinant P4503A4.

Therapy with the antidepressant trazodone has been associated with several cases of idiosyncratic hepatotoxicity. While the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of trazodone play a causative role. Studies were initiated to determine whether trazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates.

MT FdR: a ferredoxin reductase from M. tuberculosis that couples to MT CYP51.

We report the molecular cloning, expression and partial characterization of MT FdR, an FAD-associated flavoprotein, from Mycobacterium tuberculosis similar to the oxygenase-coupled NADH-dependent ferredoxin reductases (ONFR). We establish, through kinetic and spectral analysis, that MT FdR preferentially uses NADH as cofactor. Furthermore, MT FdR forms a complex with mycobacterial ferredoxin (MT Fdx) and MT CYP51, a cytochrome P450 (CYP) from M.

Bioactivation of the nontricyclic antidepressant nefazodone to a reactive quinone-imine species in human liver microsomes and recombinant cytochrome P450 3A4.

The therapeutic benefits of the antidepressant nefazodone have been hampered by several cases of acute hepatotoxicity/liver failure. Although the mechanism of hepatotoxicity remains unknown, it is possible that reactive metabolites of nefazodone play a causative role. Studies were initiated to determine whether nefazodone undergoes bioactivation in human liver microsomes to electrophilic intermediates.

In vitro metabolism studies on the isoxazole ring scission in the anti-inflammatory agent lefluonomide to its active alpha-cyanoenol metabolite A771726: mechanistic similarities with the cytochrome P450-catalyzed dehydration of aldoximes.

The 3-unsubstituted isoxazole ring in the anti-inflammatory drug leflunomide undergoes a unique N-O bond cleavage to the active alpha-cyanoenol metabolite A771726, which resides in the same oxidation state as the parent. In vitro studies were conducted to characterize drug-metabolizing enzyme(s) responsible for ring opening and to gain insight into the mechanism of ring opening. Under physiological conditions, leflunomide was converted to A771726 in rat and human plasma (rat plasma,t1/2 = 36 min; human plasma, t1/2 = 12 min) and whole blood (rat blood, t1/2 = 59 min; human blood, t1/2 = 43 min).

In vitro drug interactions of cytochrome p450: an evaluation of fluorogenic to conventional substrates.

Clinically observed drug interactions with cytochrome p450 (p450) enzymes have increased the need to assess drug interactions of new chemical entities early in the discovery process. To meet this need, fluorogenic substrates have been commercialized. However, only limited evaluations of their utility and comparisons to drug probes have been reported.