Comparison of Water Displacement and WinRHIZO Software for Plant Root Parameter Assessment.

Water-displacement and WinRHIZO root-scanning methods were compared for efficacy of root damage assessment. Results from both methods were similar and a highly significant relationship was found between the two methods in trial one (r2 = 0.9968, P < 0.0001) and trial two (r2 = 0.9988, P < 0.0001). Both protocols provide consistent root volume measurements; however, water displacement is preferred as an economical method if a quick evaluation of a large amount of roots is essential. For a more detailed root morphological and architectural analysis, WinRHIZO root scanning provides additional information about several root parameters that cannot be measured by simple water displacement.

Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial, yeast and human lymphoblastoid cells.

1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters K(m) or S(50), V(max) and intrinsic clearance (CL(int)) or maximum clearance (CL(max)) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The K(m) or S(50) for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the V(max) for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CL(int) or CL(max). In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.

Multisite kinetic models for CYP3A4: simultaneous activation and inhibition of diazepam and testosterone metabolism.

Some substrates of cytochrome P450 (CYP) 3A4, the most abundant CYP in the human liver responsible for the metabolism of many structurally diverse therapeutic agents, do not obey classical Michaelis-Menten kinetics and demonstrate homotropic and/or heterotropic cooperativity. The unusual kinetics and differential effects observed between substrates of this enzyme confound the prediction of drug clearance and drug-drug interactions from in vitro data. We have investigated the hypothesis that CYP3A4 may bind multiple molecules simultaneously using diazepam (DZ) and testosterone (TS). Both substrates showed sigmoidal kinetics in B-lymphoblastoid microsomes containing a recombinant human CYP3A4 and reductase. When analyzed in combination, TS activated the formation of 3-hydroxydiazepam (3HDZ) and N-desmethyldiazepam (NDZ) (maximal activation 374 and 205%, respectively). For 3HDZ, V(max) values remained constant with increasing TS, whereas the S(50) and Hill values decreased, tending to make the data less sigmoidal. Similar trends were observed for the NDZ pathway. DZ inhibited the formation 6beta-hydroxytestosterone (maximal inhibition, 45% of control), causing a decrease in V(max) but no significant change to the S(50) and Hill values, suggesting that DZ may inhibit via a separate effector site. Multisite rate equation models have been derived to explore the analysis of such complex kinetic data and to allow accurate determination of the kinetic parameters for activation and inhibition. The data and models presented are consistent with proposals that CYP3A4 can bind and metabolize multiple substrate molecules simultaneously; they also provide a generic solution for the interpretation of the complex kinetic data derived from CYP3A4 substrates.

In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model.

Strategies for the prediction of in vivo drug clearance from in vitro drug metabolite kinetic data are well established for the rat. In this animal species, metabolism rate-substrate concentration relationships can commonly be described by the classic hyperbola consistent with the Michaelis-Menten model and simple scaling of the parameter intrinsic clearance (CL(int) - the ratio of V(max) to K(m)) is particularly valuable. The in vitro scaling of kinetic data from human tissue is more complex, particularly as many substrates for cytochrome P450 (CYP) 3A4, the dominant human CYP, show nonhyperbolic metabolism rate-substrate concentration curves. This review critically examines these types of data, which require the adoption of an enzyme model with multiple sites showing cooperative binding for the drug substrate, and considers the constraints this kinetic behavior places on the prediction of in vivo pharmacokinetic characteristics, such as metabolic stability and inhibitory drug interaction potential. The cases of autoactivation and autoinhibition are discussed; the former results in an initial lag in the rate-substrate concentration profile to generate a sigmoidal curve whereas the latter is characterized by a convex curve as V(max) is not maintained at high substrate concentrations. When positive cooperativity occurs, we suggest the use of CL(max), the maximal clearance resulting from autoactivation, as a substitute for CL(int). The impact of heteroactivation on this approach is also of importance. In the case of negative cooperativity, care in using the V(max)/K(m) approach to CL(int) determination must be taken. Examples of substrates displaying each type of kinetic behavior are discussed for various recombinant CYP enzymes, and possible artifactual sources of atypical rate-concentration curves are outlined. Finally, the consequences of ignoring atypical Michaelis-Menten kinetic relationships are examined, and the inconsistencies reported for both different substrates and sources of recombinant CYP3A noted.

CYP3A4 drug interactions: correlation of 10 in vitro probe substrates.

AIMS: Many substrates of cytochrome P450 (CYP) 3A4 are used for in vitro investigations of drug metabolism and potential drug-drug interactions. The aim of the present study was to determine the relationship between 10 commonly used CYP3A4 probes using modifiers with a range of inhibitory potency. METHODS: The effects of 34 compounds on CYP3A4-mediated metabolism were investigated in a recombinant CYP3A4 expression system. Inhibition of erythromycin, dextromethorphan and diazepam N-demethylation, testosterone 6beta-hydroxylation, midazolam 1-hydroxylation, triazolam 4-hydroxylation, nifedipine oxidation, cyclosporin oxidation, terfenadine C-hydroxylation and N-dealkylation and benzyloxyresorufin O-dealkylation was evaluated at the apparent Km or S50 (for substrates showing sigmoidicity) value for each substrate and at an inhibitor concentration of 30 microM. RESULTS: While all CYP3A4 probe substrates demonstrate some degree of similarity, examination of the coefficients of determination, together with difference and cluster analysis highlighted that seven substrates can be categorized into two distinct substrate groups. Erythromycin, cyclosporin and testosterone form the most closely related group and dextromethorphan, diazepam, midazolam and triazolam form a second group. Terfenadine can be equally well placed in either group, while nifedipine shows a distinctly different relationship. Benzyloxyresorufin shows the weakest correlation with all the other CYP3A4 probes. Modifiers that caused negligible inhibition or potent inhibition are generally comparable in all assays, however, the greatest variability is apparent with compounds causing, on average, intermediate inhibition. Modifiers of this type may cause substantial inhibition, no effect or even activation depending on the substrate employed. CONCLUSIONS: It is recommended that multiple CYP3A4 probes, representing each substrate group, are used for the in vitro assessment of CYP3A4-mediated drug interactions.