Metabolic activation of TM5441 in vitro and in vivo: Formation of reactive metabolites and human enzymes involved.

TM5441, a furan-containing drug, is an inhibitor of plasminogen activator inhibitor-1 (PAI-1), which can induce intrinsic apoptosis of human cancer cell lines. The aim of this study was to identify the reactive metabolites of TM5441 and to reveal the bioactivation pathways that are associated with its hepatotoxicity. The reactive metabolites were trapped by using glutathione (GSH) or N-acetyl-lysine (NAL) in rat, dog, and human liver microsomal incubation system after exposure to TM5441. Two metabolic activation pathways were disclosed. The first bioactivation pathway was dominated by Cytochrome P450 enzymes (CYP450s); TM5441 was metabolized into cis-2-butene-1,4-dial derivative dependent on NADPH, which can be trapped in the liver microsomal incubations fortified with GSH or NAL as trapping agents. Five metabolites (M1, M2, M9, M12 and M13) associated with GSH and three metabolites (M4, M7 and M14) associated with NAL were identified by liquid chromatography-high resolution mass spectrometry. The second bioactivation pathway was catalyzed by UDP-glucuronosyltransferases (UGTs); TM5441 was conjugated with glucuronide to form acyl-glucuronide (M10), which further reacted with GSH, resulting in the identification of a TM5441-S-acyl-GSH adduct (M11) in liver microsomal incubations fortified with uridine-5'-diphosphoglucuronidc acid (UDPGA) and GSH. M9, M10, M11, M12 and M13 were also detected in bile samples of rats given TM5441. Compared with rat, dog would display closer bioactivation profiles to human. The CYP450 enzyme responsible for the bioactivation of TM5441 was mainly identified as CYP3A4, using human recombinant CYP450 enzymes and specific inhibitory studies. The UGT enzymes responsible for the bioactivation of TM5441 mainly involved UGT2B7, 1A1 and 1A4. These results facilitate the understanding of the bioactivation of TM5441 and potential toxicological implications.

The stromal composition of malignant lymphoid aggregates in bone marrow: variations in architecture and phenotype in different B-cell tumours.

We present evidence that different B-cell tumours, in bone marrow, have different relationships to stroma. Marrow core biopsies from 46 patients with B-cell tumours were immunostained with antibodies for distinct stromal cells. Cases included follicular lymphoma (FL), chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL), and nodal, extranodal and splenic marginal zone lymphoma (NMZL, MALT, SMZL). In normal marrow, low-affinity nerve growth factor receptor (LNGFR) highlighted a fine network of adventitial reticular cells (ARC). The nodular aggregates of CLL/SLL, NMZL, MALT and SMZL were characterized by distortion of the ARC network and downregulation of LNGFR. In contrast, the aggregates of FL, LPL and MCL were composed of linear arrays of ARC in tight association with individual tumour cells. LNFGR+ was upregulated in ARC associated with the aggregates in FL, LPL and focally in MCL. Upregulation of CD35, vascular cell adhesion molecule (VCAM-1) and CD40 on ARC was noted exclusively in FL. Marrow lymphoid aggregates in CLL/SLL, NMZL, MALT and SMZL probably grow by displacing the pre-existing marrow stroma, while FL and LPL maintain a close association with the ARC network. In FL, expression of follicular dendritic cell-associated markers is modulated in pre-existing marrow stromal cells.