NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

Purpose: To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4. Experimental Design: Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinetics, drug-target interactions, and pharmacodynamics of ixabepilone. Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepilone dose administration to 25 mg/m 2 when compared with single-agent therapy of 40 mg/m 2 . Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC 0-1 . The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral blood mononuclear cells correlated with neutropenia. Conclusions: Ixabepilone is a good CYP3A4 substrate in vitro ; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing.

3-tert-Butyl-3-N-tert-butyloxycarbonyl-4-deacetyl-3-dephenyl-3-N-debenzoyl-4-O-methoxy-paclitaxel (BMS-275183

3-tert-Butyl-3-N-tert-butyloxycarbonyl-4-deacetyl-3-dephenyl-3-N-debenzoyl-4-O-methoxycarbonyl-paclitaxel (BMS-275183)is an orally available taxane analog that has the potential to be used as an oral agent to treat cancers. The compound is similar to the two clinically intravenously administered taxanes, paclitaxel and docetaxel, in that it contains a baccatin ring linked to a side chain through an ester bond. Unlike the other taxanes, the hydrolysis of this ester bond leads to formation of a free baccatin core (M13) that was the major metabolism pathway in incubations of Paclitaxel (Taxol, Bristol-Myers Squibb Co., Wallingford, CT), initially isolated in 1967 from the bark of the Pacific yew tree, Taxus brevifolia, showed an antitumor activity for a broad range of rodent tumors (Wall and Wani, 1995). Paclitaxel has become a widely used and effective chemotherapy agent to treat patients with lung, ovarian, and breast cancer and an advanced form of Kaposi's sarcoma (Saville et al., 1995;Rose et al., 2001). Together with docetaxel, paclitaxel forms the drug category of the taxanes. Taxanes, as well as recently marketed epothilone classes (Goel et al., 2008), stop cell division by inhibition of the microtubule function through stabilizing GDP-bound tubulin in the microtubule. Paclitaxel and docetaxel are administered to patients intravenously because of their poor oral bioavailability. Oral treatment with this class of chemotherapy agents would increase convenience to patients, reduce cost of administration, and allow the use of chronic treatment regimens.BMS-275183 was synthesized as a part of a program to identify taxanes with potential for oral use in the treatment of cancers (Bröker et al., 2007). The compound is a C-4 methyl carbonate analog of paclitaxel that contains additional modifications in the side chain where the two phenyl groups of paclitaxel were replaced by t-butyl groups (Bröker et al., 2007). BMS-275183 exhibited good oral bioavailability in both rat and dog and showed antitumor activity comparable with intravenous paclitaxel (Frapolli et al., 2006).[ 14 C]BMS-275183 was metabolized to oxidative metabolites in liver microsomes of animals and humans and in bile duct-cannulated rats Kim-Kang et al., 2002). Only trace amounts of conjugated metabolites were detected in rat bile. The major in vitro metabolites were identified as M13 (hydrolysis metabolite), M20 and M20B (a carbamate metabolite resulting from hydroxylation of the oxycarbonyl t-butyl group followed by cyclization or a lactol metabolite resulting from hydroxylation of the C3Ј t-butyl groups followed by cyclization), M21 (␥-lactone metabolite), M22 (a carboxylic acid metabolite), and M23 (an oxycarbonyl t-butyl hydroxylated metabolite). In contrast to 6␣-hydroxylation of the baccatin ring of paclitaxel, the major metabolic pathways in BMS-275183 mainly occurred by oxidation and hydrolytic cleavage of the side chain.Article, publication date, and citation information can be found at

No abstract