Generic dealkylation: a tool for increasing the hit-rate of metabolite rationalization, and automatic customization of mass defect filters.

The use of exact mass liquid chromatography/mass spectrometry (LC/MS) for drug metabolism studies has increased significantly in recent years. Firstly, exact mass measurements facilitate identification of standard biotransformations through the use of narrow window extracted ion chromatograms, which are typically highly selective relative to signals from matrix or dosing components. Secondly, novel metabolites can be characterized via elemental formula calculations and high-resolution product ion spectra. Furthermore, biological background ions can be removed by the use of mass defect filters (MDFs) which filter out ions based on the decimal component of their m/z value. Here, we describe an approach which we term 'generic dealkylation' that in association with other data interpretation tools adds significant value to the assignment process. Generic dealkylation uses a simple strategy to identify those bonds which have the potential to be cleaved by metabolism. In combination with standard phase 1 and phase 2 biotransformations, this allows creation of a chemically intelligent MDF which balances the need to remove matrix background with the requirement of avoiding filtering true metabolites. Secondly, generic dealkylation increases the hit-rate at which non-trivial (i.e. not covered by simple phase 1 oxidations or direct phase 2 conjugations) metabolites can be directly rationalized. The value of the generic dealkylation approach is illustrated by its application to determination of in vitro metabolic routes for two commercial drugs, nefazodone and indinavir.

IsoScore: automated localization of biotransformations by mass spectrometry using product ion scoring of virtual regioisomers.

We describe a novel approach for the automated localization of biotransformations, which we term IsoScore. Accurate mass measurement spectra of a parent drug and its metabolites are acquired. All virtual regioisomers of a given biotransformation are generated in silico by iterating over all plausible sites of oxidation around the parent drug. Each is then fragmented virtually using an exhaustive approach supplemented with chemical intelligence. Each fragment is scored based on the likelihood that it can be formed from the precursor structure. The fragment library of each virtual isomer is then compared with the experimentally observed ions. The likelihood that a regioisomer explains the observed fragmentation data is contained in its cumulated score. We include additional weightings, which take into account the level of similarity between the mass spectra of the metabolite and the parent compound.This concept was tested on a variety of metabolites from different chemical platforms formed via single biotransformations. For a very large proportion of the metabolites, IsoScore correctly located the biotransformation to the expected position. All ions above a defined threshold in the spectrum are used to contribute to the score with no predisposition to ignore minor ions or to weight conclusions based on readily interpretable fragments. The approach is found to be most successful when differential scoring is observed between related ions in the parent and the metabolite. Further improvements in the scoring function will result in increased differentiation between likely and unlikely structures, even when the parent and the metabolite spectra show little similarity.

Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR140333 and NKP-608.

Extensive screening of compound libraries was undertaken to identify compounds with high affinity for the rat NK(1) receptor based on inhibition of [(125)I]-substance P binding. RP67580, SR140333, NKP-608 and GR205171 were selected as compounds of interest, with cloned rat NK(1) receptor binding K(i) values of 0.15-1.9 nM. Despite their high binding affinity, NKP-608 and GR205171 exhibited only a moderate functional antagonism of substance P-induced inositol-1-phosphate accumulation and acidification rate at 1 microM using cloned or native rat NK(1) receptors in vitro. The ability of the compounds to penetrate the CNS was determined by inhibition of NK(1) agonist-induced behaviours in gerbils and rats. GR205171 and NKP-608 potently inhibited GR73632-induced foot drumming in gerbils (ID(50) 0.04 and 0.2 mg/kg i.v., respectively). In contrast, RP67580 and SR140333 were poorly brain penetrant in gerbils (no inhibition at 10 mg/kg i.v.) and were not examined further in vivo. In rats, only high doses of GR205171 (10 or 30 mg/kg s.c.) inhibited NK(1) agonist-induced sniffing and hypertension, whilst NKP-608 (1 or 10 mg/kg i.p.) was without effect. GR205171 (3-30 mg/kg s.c.) caused only partial inhibition of separation-induced vocalisations in rat pups, a response that is known to be NK(1) receptor mediated in other species. These observations demonstrate the shortcomings of currently available NK(1) receptor antagonists for rat psychopharmacology assays.