SummaryWe have carried out a cell-based screen aimed at discovering small molecules that activate p53 and have the potential to decrease tumor growth. Here, we describe one of our hit compounds, tenovin-1, along with a more water-soluble analog, tenovin-6. Via a yeast genetic screen, biochemical assays, and target validation studies in mammalian cells, we show that tenovins act through inhibition of the protein-deacetylating activities of SirT1 and SirT2, two important members of the sirtuin family. Tenovins are active on mammalian cells at one-digit micromolar concentrations and decrease tumor growth in vivo as single agents. This underscores the utility of these compounds as biological tools for the study of sirtuin function as well as their potential therapeutic interest.
Cyclophosphamide (CPA) is an anticancer prodrug that is dependent on cytochrome P450 (CYP) metabolism for its therapeutic effectiveness. In spite of the use of CPA in the clinic for over 50 years, little is known about the relationship between its toxicokinetics and therapeutic response. We have employed a powerful new model, the Hepatic Cytochrome P450 Reductase Null (HRN) mouse, which has almost no hepatic cytochrome P450 activity, to study the toxicokinetics of CPA and to establish in vivo the role of hepatic P450 metabolism in its pharmacokinetics. In HRN mice the in vitro metabolism and intrinsic clearance of CPA was over 6-fold lower than in wild-type animals. This change in CPA metabolism was also reflected in vivo, with a profound difference in the pharmacokinetics of both CPA and its metabolites. At a CPA dose of 100 mg/kg, the C max , plasma area under the curve (AUC) and half-life were increased by 2.6-, 6.2-, and 3.2-fold, respectively, in the HRN mice. Similar changes were also observed at a dose of 300 mg/kg. These data confirm that hepatic metabolism is the major route of CPA elimination and disposition. The primary metabolites of CPA, 4-hydroxycyclophosphamide (4-OH-CPA) and 3-dechloroethylcyclophosphamide, were still formed, but at altered rates in the HRN mice. At 100 mg/kg the t 1/2 for 4-OH-CPA was increased 1.8-fold, the C max reduced 1.7-fold, and the AUC remained unchanged. This latter finding shows that P450-mediated oxidative metabolism is essential for the clearance of this compound. Toxicokinetic analysis of CPA-induced myelosuppression and granulocytopenia showed that at high doses (z z100 mg/kg) there was no difference in myelotoxicity between the wild-type and HRN mice. However, at lower doses (V V70 mg/kg) a significant difference was observed, with little toxicity seen in HRN mice but at least a 45% reduction in the bone marrow granulocyte population in wild-type mice. Meta-analysis of the toxicity experiments showed the myelotoxicity of CPA was found to be closely correlated with the C max of 4-OH-CPA (r 2 = 0.80, P = 0.002). As the therapeutic effectiveness of CPA has been linked to the AUC for 4-OH-CPA, the finding that 4-OH-CPA C max may determine its level of myelotoxicity indicates that the therapeutic index could be altered by changing the method of CPA administration.
1. This study reports on the pathways of metabolism and enzyme kinetics of the Eucalyptus terpene, 1,8-cineole, by liver microsomes from the brushtail possum (Trichosurus vulpecula) and koala (Phascolarctos cinereus) (animals that normally include this terpene in their diet), rat and human. 2. The rank order of the ability to metabolize 1,8-cineole with respect to overall 1,8-cineole intrinsic clearance (CL'int = Vmax/Kmax in microl mg protein(-1) min(-1)) was koala (188) > possum (181)>> rat (28) > human (12). This order supports the hypothesis that adaptation to a Eucalyptus diet involves enhanced metabolism of terpenes. 3. The metabolism of 1,8-cineole was also studied in the liver from brushtail possum pretreated with a mixture of terpenes, which have previously been shown to induce cytochrome P450 enzymes. Rats were pretreated with the same mixture of terpenes or phenobarbitone. 4. Terpene pretreatment more than doubled the CL'int of 1,8-cineole by brushtail possum liver microsomes (from 180 to 394 microl mg protein(-1) min(-1)) and increased rat CL'int by nearly 10-fold (from 28 to 259 microl mg protein(-1) min(-1)), but still less than the induced possum value. However, phenobarbitone had the greatest inducing effect, increasing the rat CL'int to 1,825 microl mg protein(-1) min(-1). 5. A regioselective preference of oxidation was evident between adapted and non-adapted species. In rat and human oxidation was preferred at the aliphatic ring carbons over methyl substituents. In possum, many of the available carbons were utilized, however metabolism at methyl substituents was preferred. In the koala, oxidation occurred primarily at the methyl substituents.
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