Introduction Endogenous biomarkers are promising tools to assess transporter-mediated drug-drug interactions (DDI) early in humans.Methods We evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulfate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein (MRP), and assessed the in vivo biomarker sensitivity towards the strong organic anion transporters (OAT) inhibitor probenecid at 500mg every 6h to reach close to complete OAT inhibition.Results PDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and MRP4; GCDCA-S was more selective, having affinity only towards OAT3 and MRP2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to the ones of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter. ConclusionThe current findings illustrate a clear benefit of measuring PDA plasma exposure during Phase 1 studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data.Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended in order to tease out the involvement of OAT1/3 in the inhibition interaction.

The use of in vitro drug metabolism data in the understanding of in vivo pharmacokinetic, safety and toxicity data has become a large area of scientific interest. This has stemmed from a trend in the pharmaceutical industry to use in vitro data generated from human tissue as a criterion to select compounds for further investigation. As well as measuring metabolic stability in vitro using human liver microsomal preparations, the identification of possible metabolite(s) formed may play a vital role in Hit-to-Lead and Lead optimisation processes. The data-dependent scan function mode with the ion-trap instrumentation provides the ability to measure the metabolic stability and identification of possible metabolites of a compound. A gradient liquid chromatographic method with a run time of 6 min/injection was developed for this purpose. The approach of simultaneous metabolic stability measurements and rapid identification of metabolites of drugs with high (verapamil), medium (propranolol and cisapride) and low (flunarazine) metabolic stabilities using ion-trap mass spectrometry is described. The metabolites identified after 15 min incubation for verapamil, propranolol and cisapride are in good agreement with those reported as the major metabolites in human in vivo studies.

A liquid chromatography/mass spectrometry (LC/MS) method using an atmospheric pressure chemical ionisation source was used to measure the metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivative (1) in human liver microsomes. After 15 min incubation with human liver microsomes, compound 1 exhibited metabolic turnover of 44%. Data-dependent tandem mass spectrometry (MS/MS) scanning was used to generate product ion spectra from the protonated ions of the compound and its metabolites. An unusual metabolite at m/z 407 corresponding to the [M-24+H]+ ion was identified for compound 1. Interestingly, the formation of the [M-24+H]+ ion was not observed in the analogues wherein the fused thieno double bond was substituted (2) and the thieno group replaced by a fused benzo derivative (3). Compounds 2 and 3 exhibited metabolic turnovers of 24 and 30%, yielding oxidative metabolites corresponding to [M+16] and [M+32]+, respectively. Based on these facts the mechanism for [M-24]+ formation in compound 1 through an initial epoxide formation on the double bond of the fused thieno ring followed by hydrolytic ring opening and deacylation is envisaged.

A LC/MS method using atmospheric pressure chemical ionization, positive ion mode and full scan to measure the in vitro metabolic stability of cyanoalkyl functionalized compounds with the human liver microsomes was employed. Percentage metabolism examined for the five cyanoalkyl piperidines revealed the optimal chain length and positioning of these functions to produce the most metabolically stable compound. The 4-cyanomethyl piperidine derivative was the most stable compound with 15% metabolism after 15 min incubation with human liver microsomes. In general, the major metabolites formed from the cyanoalkyl piperidine derivatives were due to oxidation of the cyanoalkyl chain or the piperidine fragment, resulting in a M+16 ion. However, the 2-cyanomethyl piperidine derivative exhibited an interesting biotransformation pathway with unusual metabolite peaks corresponding to M+5, M-11 and M+21 ions. Data-dependent MS/MS scanning was used to generate daughter ion spectra from the parent compound and its metabolite peaks. Based on the fragmentation analysis, a carboxylic acid, aldehyde and oxidative metabolite of the carboxylic acid structure have been proposed for M+5, M-11 and M+21 ions, respectively.

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