Grapefruit juice has been found to significantly increase oral bioavailability of several drugs metabolized by cytochrome P450 3A4 (P450 3A4) through inhibiting the enzymatic activity and decreasing the content of intestinal P450 3A4. HPLC/MS/MS and HPLC/UV analyses of ethyl acetate extracts from grapefruit juice revealed the presence of several furanocoumarins of which bergamottin (BG) is the major one. BG was shown to inactivate P450 3A4 in a reconstituted system consisting of purified P450 3A4, NADPH-cytochrome P450 reductase, cytochrome b5, and phospholipids. Inactivation was time- and concentration-dependent and required metabolism of BG. The loss of catalytic activity exhibited pseudo-first-order kinetics. The values of kinactivation and KI calculated from the inactivation studies were 0.3 min-1 and 7.7 microM, respectively. While approximately 70% of the erythromycin N-demethylation activity was lost during incubation with BG in the reconstituted system, P450 3A4 retained more than 90% of the heme as determined either by UV-visible spectroscopy or by HPLC. However, approximately 50% of the apoP450 in the BG-inactivated P450 3A4 incubation mixture could not be recovered from a reverse-phase HPLC column when compared with the -NADPH control. The mechanism of the inactivation appears to involve modification of the apoP450 in the active site of the enzyme instead of heme adduct formation or heme fragmentation. These results indicate that BG, the primary furanocoumarin extracted from grapefruit juice, is a mechanism-based inactivator of P450 3A4. BG was also found to inhibit the activities of P450s 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes.

Nitrogenase catalyses the ATP-dependent reduction of N2 to NH3, and is composed of two proteins, dinitrogenase (MoFe protein or component I) and dinitrogenase reductase (Fe protein or component II). Dinitrogenase contains a unique prosthetic group (iron-molybdenum cofactor, FeMoco) comprised of Fe, Mo and S, which has been proposed as the site of N2 reduction. Biochemical and genetic studies of Nif- (nitrogen fixation) mutants of Klebsiella pneumoniae which are defective in nitrogen fixation, have shown that the nifB, nifQ, nifN, nifE and nifV genes are required for the biosynthesis of FeMo-co. Recently, a system for in vitro synthesis of FeMoco was described. The assay requires at least the nifB, nifN and nifE gene products, and a low-molecular-weight factor (V factor) produced in the presence of the nifV gene product. We have used this system to study FeMoco biosynthesis. We report here the isolation of V factor and identify it as homocitric acid ([R]2-hydroxy-1,2,4-butanetricarboxylic acid).

No abstract

Over 400 professionals representing pharmaceutical companies, CROs, and multiple regulatory agencies participated in the 6th Workshop on Recent Issues in Bioanalysis (WRIB). Like the previous sessions, this event was in the format of a practical, focused, highly interactive and informative workshop aiming for high-quality, improved regulatory compliance and scientific excellence. Numerous 'hot' topics in bioanalysis of both small and large molecules were shared and discussed, leading to consensus and recommendations among panelists and attendees representing the bioanalytical community. The major outcome of this year's workshop was the noticeable alignment of multiple bioanalytical guidance/guidelines from different regulatory agencies. This represents a concrete step forward in the global harmonization of bioanalytical activities. The present 2012 White Paper acts as a practical and useful reference document that provides key information and solutions on several topics and issues in the constantly evolving world of bioanalysis.

No abstract