´╗┐Similarly, unbound average hepatic concentrations of 0

´╗┐Similarly, unbound average hepatic concentrations of 0.33?M and 0.36?M, respectively, were calculated for the two individuals receiving 100?mg AZD1981. For the N\deacetylated metabolite, plasma protein binding assessed was 99.04% (five pooled donors) and, with a B/P value of 0.7, an average unbound concentration in systemic blood was calculated to be 5?nM and 8?nM, respectively, for the two subjects receiving the 400?mg dose; the free hepatic concentrations were therefore calculated to be 64? nM and 100?nM, respectively, using a Kp,uu value of 12 (Equation (7)). 100?mg (= 13) HhAntag and 400?mg (= 11) AZD1981 administration, Capn1 respectively. CYP inhibition and hepatocyte uptake data were used to explain the interaction. Conclusions N\deacetylated AZD1981 can be added to the small list of drug metabolites reported as sole contributors to clinical drugCdrug interactions, with weak time\dependent inhibition exacerbated by efficient hepatic uptake being the cause. data showing active hepatic uptake of the parent and metabolite, coupled with the greater reversible cytochrome P450 (CYP) 2C9 inhibitory potency of the metabolite and weak time\dependent CYP2C9 inhibition, raised concerns of a possible S\warfarin interaction. A clinical study was performed and the observed DDI with S\warfarin was rationalized from the combination of and clinical data for the metabolite. This study highlights the need for vigilance in predicting likely drugCdrug interactions and shows that detailed analyses are required early in clinical drug development when considering possible interactions involving drug metabolites. Tables of Links human hepatocyte metabolite identification work and subsequent investigations into the possibility of a clinically relevant metabolite\dependent DDI risk. Finally, a clinical AZD1981Cwarfarin interaction study is described and all the data are discussed with reference to the available regulatory guidance documents. Methods Drugs and chemicals Unless otherwise specified, all reagents were purchased from Sigma\Aldrich (St HhAntag Louis, MO, USA). Human CYP2C9/CYP reductase coexpressed in Escherichia coli (Cypex, Dundee, UK) were used to assess the reversible inhibition potential of evaluated compounds. A 7\hydroxy warfarin (7\OH warfarin, Toronto Research Chemicals, North York, ON, Canada) standard curve was used for metabolite quantification. Human liver microsomes for the time\dependent inhibition study were obtained from the BD Gentest 150 donor UltraPool (Lot no. 38?289, Franklin Lakes, NJ, USA) at a concentration of 20?mg mlC1 protein, and NADPH for these incubations was purchased from Roche Diagnostics GmbH (Mannheim, Germany). For hepatic uptake experiments, Liverpool? 10\donor mixed gender (five female/five male) pooled cryopreserved human hepatocytes (lot IRK, BioreclamationIVT, Baltimore, MD, USA) were used. Hepatocyte incubations were performed in Williams medium E containing L\glutamine (2?mM) buffered to pH?7.4 with 4\(2\hydroxyethyl)\1\piperazineethanesulfonic acid (HEPES) (25?mM), and for the separation of cells from incubation media after incubation with AZD1981 or N\deacetylated AZD1981, microtubes (Beckman 0.5?ml, Fisher Scientific GTF AB, V?stra Fr?lunda, Sweden) were used. AstraZeneca rigorously ensures that human biological samples are handled in a responsible and ethical manner, requiring donor informed consent designed to protect the rights and expectations of donors and families. No human biological sample can be acquired unless it is from an approved source which has appropriate ethical approval procedures in place. Both BioreclamationIVT and BD Gentest are approved suppliers for AstraZeneca and, as such, retain the appropriate ethical approval documentation. A phase I study to assess the metabolism, excretion and pharmacokinetics of [14C]\AZD1981 in healthy male volunteers (Study D9830C00006) Four healthy male subjects received a single oral dose of [14C]\AZD1981 (250?mg, 33?kBq, 150?ml) administered as an oral solution (150?ml) through a drinking straw. The container was then rinsed with water (90?ml) and the subject was asked to drink the rinsing through the same drinking straw. Blood, urine and faeces samples (for analysis of total radioactivity, metabolite profiling and identification) were collected predose at specified times up to 11?days after dosing. Uptake of AZD1981 and AZD1981 metabolites into isolated human hepatocytes HhAntag The method and the data modelling were performed as described previously 22. Microtubes containing caesium chloride (15?l, 4?g 100?mlC1) and mineral oil/silicon oil mixture (140?l, with a specific gravity of 1 1.015?g mlC1) were prepared and centrifuged.

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