Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7-300 mM) at 37ae in 0.1 M KH 2 PO 4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC 50 values were determined for each substrate at respective K M concentrations. Silibinin had little effect (IC 50 Ͼ200 mM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(π)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC 50 Ω173 mM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC 50 Ω29 mM and 46 mM) and S(ª)-warfarin 7-hydroxylation (CYP2C9; IC 50 Ω43 mM and 45 mM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (K i,c Ω2.3 mM and 2.4 mM). Inhibition was competitive for S(ª)-warfarin 7-hydroxylation (K i,c Ω18 mM and 19 mM) and mainly non-competitive for denitronifedipine oxidation (K i,n Ω9 mM and 12 mM). With therapeutic silibinin peak plasma concentrations of 0.6 mM and biliary concentrations up to 200 mM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.

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Trospium chloride, an atropine derivative used for the treatment of urge incontinence, was tested for inhibitory effects on human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using the following selective substrates: dextromethorphan (CYP2D6), denitronifedipine (CYP3A4), caffeine (CYPlA2), chlorzoxazone (CYP2E1), S-( +)-mephenytoin (CYP2C19), S-(-)-warfarin (CYP2C9) and coumarin (CYP2A6). Incubations with each substrate were carried out without a possible inhibitor and in the presence of trospium chloride at varying concentrations (37-3000 pM) at 37" in 0.1 M KH2P04 buffer containing up to 3% DMSO. Metabolite concentrations were determined by high-performance liquid chromatography (HPLC) in all cases except CYP2A6 where direct fluorescence spectroscopy was used. First, trospium chloride IC5,, values were determined for each substrate at respective KM concentrations. Trospium chloride did not show relevant inhibitory effects on the metabolism of most substrates (IC50 values considerably higher than 1 mM). The only clear inhibition was seen for the CYP2D6-dependent high-affinity 0-demethylation of dextromethorphan, where ICs0 values of 27 pM and 44 pM were observed. Therefore, additional dextromethorphan concentrations (0.L2000 pM) were tested. Trospium chloride was a competitive inhibitor of the reaction with K, values of 20 and 51 pM, respectively. Thus, trospium chloride has negligible inhibitory effects on CYP3A4, CYPIA2, CYP2E1, CYP2C19, CYP2C9 and CYP2A6 activity but is a reasonably potent inhibitor of CYP2D6 in vitro. Compared to therapeutic trospium chloride peak plasma concentrations below 50 nM, the 1000-times higher competitive inhibition constant K, however suggests that inhibition of CYP2D6 by trospium chloride is without any clinical relevance.Trospium chloride, a hydrophilic atropine derivative containing a quaternary ammonium group ( fig. l), is obtained from tropan alkaloid precursors and has mainly peripheral parasympatholytic properties. It is used therapeutically for the treatment of incontinence due to detrusor hyperreflexia (Madersbacher et al. 1995). The mechanism of action is the inhibition of muscarinic receptors of the musculus detrusor vesicue, subject to parasympathic control which is responsible for the micturition (Madaus AG, data on file).The bioavailability of orally administered trospium chloride in man is approximately 3-10 YO. The maximal plasma concentration of trospium chloride after oral medication of therapeutic doses of 20 mg is below 50 nM. Most of the absorbed fraction is found unchanged in the urine, with a small part is being metabolised by esterases to form the spiro alcohol (Schladitz-Keil et al. 1986; Madaus AG, data on file).Although there is no evidence for the participation of cytochrome P-450 enzymes in the metabolism of trospium chloride, this does not exclude that it affects the activity of these important rate-limiting drug-metabolising enzymes, giving rise to metabolic drug-drug interactions (Fuhr...

Hybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A-chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome-inactivating proteins. Anti-MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin.z 1999 Federation of European Biochemical Societies.