Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer-preventive activity. Although a recent study suggested that methylated polyphenols are less cancer-protective, the molecular basis is unknown. We previously reported that ester bond-containing GTPs, for example (-)-epigallocatechin-3-gallate [(-)-EGCG] or (-)-epicatechin-3-gallate [(-)-ECG], potently and specifically inhibit the proteasomal chymotrypsin-like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome-inhibitory abilities. To test this hypothesis, methylated (-)-EGCG and (-)-ECG analogs that can be found in vivo were synthesized and studied for their structure-activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (-)-EGCG or (-)-ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate-protected (-)-EGCG molecule, Pro-EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro-EGCG (1) to (-)-EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate-protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate-protected, monomethylated (-)-EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate-protected, trimethylated (-)-EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome-inhibitory activity, contributing to decreased cancer-preventive effects of tea consumption.

Purpose Veliparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, undergoes renal excretion and liver metabolism. This study quantitatively assessed the interactions of veliparib with metabolizing enzyme (CYP2D6) and transporter (OCT2) in disease settings (renal impairment). Experimental Design Veliparib in vitro metabolism was examined in human liver microsomes and recombinant enzymes carrying wild-type CYP2D6 or functional defect variants (CYP2D6*10 and *4). Plasma pharmacokinetics were evaluated in 27 patients with cancer. A parent–metabolite joint population model was developed to characterize veliparib and metabolite (M8) pharmacokinetics and to identify patient factors influencing veliparib disposition. A physiologically based pharmacokinetic model integrated with a mechanistic kidney module was developed to quantitatively predict the individual and combined effects of renal function, CYP2D6 phenotype, and OCT2 activity on veliparib pharmacokinetics. Results In vitro intrinsic clearance of CYP2D6.1 and CYP2D6.10 for veliparib metabolism were 0.055 and 0.017 μL/min/pmol CYP, respectively. Population mean values for veliparib oral clearance and M8 clearance were 13.3 and 8.6 L/h, respectively. Creatinine clearance was identified as the significant covariate on veliparib oral clearance. Moderate renal impairment, CYP2D6 poor metabolizer, and co-administration of OCT2 inhibitor (cimetidine) increased veliparib steady-state exposure by 80%, 20%, and 30%, respectively. These factors collectively led to >2-fold increase in veliparib exposure. Conclusions Renal function (creatinine clearance) is a significant predictor for veliparib exposure in patients with cancer. Although a single factor (i.e., renal impairment, CYP2D6 deficiency, and reduced OCT2 activity) shows a moderate impact, they collectively could result in a significant and potentially clinically relevant increase in veliparib exposure.

Purpose: BMS-247550 is a semisynthetic derivative of epothilone B with mechanism of action analogous to paclitaxel. It has shown impressive antitumor activity in preclinical studies including in taxane-resistant models. We conducted a phase I trial, based on accelerated titration ''2B'' design, of BMS-247550 given as a 1-hour infusion every 3 weeks. Experimental Design: Seventeen patients (M:F, 10:7; median age, 54 years; performance status, 0-2) were treated on the trial. Forty-five cycles (1-9 cycles) of BMS-247550 were given at dosages ranging from 7.4 to 56 mg/m 2 . All patients received prophylaxis for hypersensitivity reactions, related to Cremophor-EL, with steroids and histamine antagonists. Results: First-course dose-limiting toxicity (DLT) was observed in two of three patients at 56 mg/m 2 (neutropenic sepsis, prolonged grade 4 neutropenia) and in one of six patients at 40 mg/m 2 . Nonhematologic grade 3 to 4 toxicities observed were emesis and fatigue and they occurred only at 56 mg/m 2 . Grade 1to 2 peripheral neuropathy was also observed. Other grade 1 to 2 toxicities were myalgias, arthralgias, rash, hand/foot syndrome, and mucositis. AUC and C max seemed proportional to the dose and the DLT. Development of neutropenia with BMS-247550 is related to the duration of drug exposure above a threshold. Conclusions:The maximum tolerated dose (MTD) of BMS-247550 is 40 mg/m 2 given every 3 weeks. Neutropenia is the DLT. The accelerated titration ''2B''design may help in determining MTD with fewer patients enrolled and more being treated closer to the MTD. However, the accelerated titration design did not seem to shorten the study duration.Among the many new cytotoxic agents introduced over the last two decades, the taxanes have shown significant antitumor activity in many tumor types. Despite the impressive clinical benefits observed, the overall antitumor activity of taxanes still is limited, both due to the lack of activity in certain tumor types and development of resistance. The development of drugs that can improve upon the activity and circumvent resistance of taxanes is ongoing.Epothilones A and B belong to a new class of nontaxane tubulin polymerization agents obtained by fermentation of the myxobacteria Sorangium cellulosum (1). These agents show impressive in vitro antitumor activity including activity in taxane-resistant cell lines (2). Despite this impressive in vitro activity, studies with these agents in in vivo models have revealed only modest activity. This has been attributed to their metabolic instability, unfavorable pharmacokinetics, and narrow therapeutic window (3).BMS-247550 is a semisynthetic derivative of epothilone B developed to overcome the metabolic instability and the narrow therapeutic window of the natural product. This analogue is metabolically more stable partly due to the substitution of the lactone with a lactam, which reduces the product's metabolism by carboxylestrase. The mechanism of cytotoxicity of BMS-247550 is related to the stabilization of the microtubul...

Simultaneous, quantitative determination of intracellular nucleoside triphosphates and other polar metabolites using liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS) represents a bioanalytic challenge because of charged, highly hydrophilic analytes presented at a large concentration range in a complex matrix. In this study, an ion pair LC-MS/MS method using triethylamine (TEA) – hexafluoroisopropanol (HFIP) ion-pair mobile phase was optimized and validated for simultaneous and unambiguous determination of 8 nucleoside triphosphates (including ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) in cellular samples. Compared to the the less volatile ion-pair reagent, triethylammonium acetate (100 mM, pH 7.0), the combination of HFIP (100 mM) and TEA (8.6 mM) increased the MS signal intensity by about 50-fold, while retaining comparable chromatographic resolution. The isotope-labeled internal standard method was used for the quantitation. Lower limits of quantitation were determined at 0.5 nM for CTP, UTP, dATP, dCTP, and dTTP, at 1 nM for ATP, and at 5 nM for GTP and dGTP. The intra- and inter-day precision and accuracy were within the generally accepted criteria for bioanalytical method validation (< 15%). While the present method was validated for the quantitation of intracellular nucleoside triphosphates, it had a broad application potential for quantitative profiling of nucleoside mono- and bi-phosphates as well as other polar, ionic metabolic intermediates (including carbohydrate derivatives, carboxylic acid derivatives, co-acyl A derivatives, fatty acyls, and others) in biological samples.

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.25 ml of plasma with 10μl of 1M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C18 column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1-2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration-time profile of ABT-888 after its oral administration in cancer patients.

The development and validation of a LC-MS/MS method is often performed using pooled human plasma, which may fail to account for variations in interindividual matrices. Since calibrator standards and quality control samples are routinely prepared in pooled human plasma, variations in the extraction recovery and/or matrix effect between pooled plasma and individual patient plasma can cause erroneous measurements. Using both pooled human plasma as well as individual healthy donor and cancer patient plasma samples, we evaluated the analytical performance of two classes of internal standards (i.e., non-isotope-labeled and isotope-labeled) in the quantitative LC-MS/MS analysis of lapatinib. After exhaustive extraction with organic solvent, the recovery of lapatinib, a highly plasma protein-bound drug, varied up to 2.4-fold (range, 29 – 70%) in 6 different donors of plasma and varied up to 3.5-fold (range, 16 – 56%) in the pretreatment plasma samples from 6 cancer patients. No apparent matrix effects were observed for lapatinib in both pooled and individual donor or patient plasma samples. The calibration curve range was 5 – 5000 ng/ml of lapatinib in plasma. Both the non-isotope-labeled (zileuton) and isotope-labeled (lapatinib-d3) internal standard methods showed acceptable specificity, accuracy (within 100 ± 10%), and precision (< 11%) in the determination of lapatinib in pooled human plasma. Nevertheless, only the isotope-labeled internal standard could correct for the interindividual variability in the recovery of lapatinib from patient plasma samples. As inter- and intra-patient matrix variability is commonly presented in the clinical setting, this study provides an example underscoring the importance of using a stable isotope-labeled internal standard in quantitative LC-MS/MS analysis for therapeutic drug monitoring or pharmacokinetic evaluation.

Traditional PK sampling designs were inadequate to describe XK469 disposition. XK469 and related structures work through a unique mechanism of action. A further understanding of the specific mechanism of these compounds might uncover a unique avenue for future drug development.

A 41-year study of the orchids of the Catoctin Mountains, Frederick County, Maryland reveals that 19 of 21 species have experienced precipitous declines. Four of these species are currently considered Threatened or Endangered by the State of Maryland and another two are considered Rare. Annual census data at 167 sites from throughout the Catoctin Mountains on protected and unprotected lands (private and public) show a loss of three species from the study area, a decline of[90 % (ranging from 99 to 91 %) in seven species, and a decline of \90 % (ranging from 51 to 87 %) for nine species. Each species was analyzed using Ordinary Least Squares Analysis to show trends and document corresponding R 2 and p values. We tested the hypothesis that this decline is due to intensified herbivory by white-tailed deer. The overall orchid census data is significantly inversely-correlated (R = -0.93) to the white-tailed deer harvest data of Frederick County (a surrogate for population size), which includes the entirety of the study area. Platanthera ciliaris showed a huge expansion at a single site explicitly managed for this species otherwise this orchid showed a decline similar to the other species. Proper management is critical for the continuation of the orchid species in this study, be it control of the white-tailed deer herd or combating woody plant succession in the case of P. ciliaris.