ABSTRACT:A systematic analysis of the heteroactivation of CYP3A-mediated carbamazepine 10,11-epoxidation has been investigated in three different in vitro systems, namely recombinant CYP3A4 and CYP3A5, human liver microsomes (HLMs) and cryopreserved human hepatocytes. The effect of 10 endogenous steroids and flavonoids was studied over a range of substrate and effector concentrations. A novel heteroactivation model was used to obtain the parameters EC 200 (concentration of effector required to produce 200% control) and heteroactivation ratio (the ratio of maximum observed reaction velocity to control). The EC 200 values obtained in HLMs and human hepatocytes were corrected for nonspecific binding. Heteroactivation of CYP3A5 has been demonstrated with mean heteroactivation ratios in CYP3A5, HLMs and hepatocytes on average 2-fold greater than in recombinant CYP3A4 for most of the effectors investigated. In recombinant CYP3A4, heteroactivation was greatest at substrate concentrations below K m . Heteroactivation increased with effector concentration in a nonlinear manner and differed between effectors (mean heteroactivation ratios varied up to 12-fold). A greater extent of heteroactivation was observed in HLMs than in human hepatocytes for steroid effectors, but the opposite was true for flavonoid effectors. The observed heteroactivation of CYP3A in intact cells supports an in vivo relevance. From the in vitro heteroactivation data, a significant increase in clearance in vivo was predicted for substrates with a high dependence on CYP3A4 to the overall elimination, indicating that heteroactivation of CYP3A may be a potential source of interindividual variability.Cytochromes P450 (P450s) 3A are the most abundant P450 enzymes in the human liver and small intestine (Paine et al., 2006). In vitro assays of CYP3A-mediated metabolism are used routinely in the drug discovery effort to quantitatively predict in vivo pharmacokinetic parameters. However, these estimates may be confounded by atypical (non-Michaelis-Menten) kinetics such as autoactivation (homotropic cooperativity, evident as a sigmoidal kinetic profile), heteroactivation (heterotropic cooperativity, the activation of a substrate's metabolism by a separate effector compound), or substrate inhibition (inhibition of a substrate's own metabolism) (Houston and Galetin, 2005). A number of studies have addressed the issue of atypical kinetics (Tang and Stearns, 2001;Davydov et al., 2007). Heteroactivation is displayed by a number of CYP3A substrates in vitro, including the antiepileptic, carbamazepine (Nakamura et al., 2002;Egnell et al., 2003a). Heteroactivator-enzyme interaction has been rationalized either by the simultaneous binding of substrate and effector molecules (Shou et al., 1994) or assuming the existence of a separate effector-binding site (Houston and Galetin, 2005).Evidence exists that heteroactivation of CYP3A may occur in vivo. Coadministration of quinidine resulted in an acute increase in diclofenac clearance in monkeys, an effect also observed in m...

The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIβ. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIβ was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIβ inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.

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Treatment of etiolated pea seedlings with a short exposure to red light caused ;i stimulation of growth (size and dry wt production) and carotenoid synthesis during the following 48 hr compared with seedlings kept entirely in darkness. The effect is nullified by a following dose of far red light and thus the phenomenon is probably phytochrome-controlled.Similar treatment with red light one hour before continuous illumination with white light tended to reduce the lag period for chlorophyll synthesis. Again a following dose-of far red light reversed this response.COHEN AND GOODWIN(*) showed that if 4-day-old etiolated maize seedlings were exposed for a short period (5-10 min) to red light then 24 hr later the carotenoid levels and, to a lesser extent, dry weight of the coleoptiles were considered greater than the levels in control seedlings which had been kept in complete darkness for 5 days. After a further 24 hr the carotenoid and dry weight of the coleoptiles had increased equally in the seedlings exposed to red light so that the concentration of carotenoid per unit dry wt was no greater than in the controls, although the absolute amounts were still much greater. As the red light effect was nullified by a following exposure to far red light and far red light alone was without action it was concluded that the effect was phytochrome-mediated. Virgin(") demonstrated that pre-illumination of wheat seedlings with red light eliminated the induction period of chlorophyll synthesis when the seedlings were continuously illuminated with white light. Far-red light showed some but not complete ability to reverse the red light effect. Mitrakod8) and Klein and Price(') made similar observations on the coleoptiles of Leyidunz sutivum and found that far red treatment reversed the red light effect by 30 per cent. Margulies(*), on the other hand, found that far red light stimulated chlorophyll synthesis in beans.The present investigation was undertaken primarily to determine whether "the carotenoid effect" also occured in peas. At the same time because of the varying reports of the effect of red light on chlorophyll synthesis this aspect of the problem was also investigated. MATERIALS and METHODSSeeds. "Pilot" peas (Carters Ltd., Raylies Park, Middlesex) were used throughout the investigation. The dried seeds were shaken with a commercial preparation (Seed Saver, I.C.I., Ltd.) and then planted 3 in. deep in trays of vermiculite and watered. The water: 243

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