Antiproliferative and antimigratory actions of synthetic long chain n-3 monounsaturated fatty acids in breast cancer cells that overexpress cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) is overexpressed in many human cancers and converts the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid to prostaglandin E(2) (PGE(2)), which drives tumorigenesis; in contrast, n-3 PUFA inhibit tumorigenesis. We tested the hypothesis that these antitumor actions of n-3 PUFA may involve the n-3 olefinic bond. n-3 Monounsaturated fatty acids (MUFAs) of chain length C16-C22 were synthesized and evaluated in MDA-MB-468 breast cancer cells that stably overexpressed COX-2 (MDA-COX-2 cells). Longer chain (C19-C22) n-3 MUFAs inhibited proliferation, activated apoptosis, decreased PGE(2) formation, and decreased cell invasion; C16-C18 analogues were less active. Molecular modeling showed that interactions of Arg120, Tyr355, and several hydrophobic amino acid residues in the COX-2 active site with C19-C22 MUFA analogues were favored. Thus, longer-chain n-3 MUFAs may be prototypes of novel anticancer agents that decrease the formation of PGE(2) in tumor cells that contain high levels of COX-2.

Role of human CYP3A4 in the biotransformation of sorafenib to its major oxidized metabolites.

The tyrosine kinase inhibitor drug sorafenib is used in the treatment of liver and renal cancers but adverse effects may necessitate dose interruption and under-dosage may lead to therapeutic failure. Sorafenib also undergoes cytochrome P450 (CYP)-dependent biotransformation to the N-oxide and other metabolites. However, although CYPs are major determinants of efficacy and toxicity the roles of these enzymes in the formation of multiple sorafenib metabolites are unclear. In the present study CYP-mediated pathways of sorafenib oxidation in human liver were evaluated. cDNA-expressed CYP3A4 was the major catalyst in the formation of the principal N-oxide and N-hydroxymethyl metabolites of sorafenib, as well as the minor N-desmethyl metabolite. In contrast, CYP3A5 exhibited only ~5% of the activity of CYP3A4 and eleven other CYPs and three flavin-containing monooxygenases were inactive. In human hepatic microsomes metabolite formation was correlated with CYP3A4-mediated midazolam 1'-hydroxylation, but not with other CYP-specific substrate oxidations. In accord with these findings the CYP3A4 inhibitor ketoconazole selectively inhibited microsomal sorafenib oxidation pathways. From computational modeling studies atoms in the structure of sorafenib that undergo biotransformation were within ~5.4 Å of the CYP3A4 heme. Important hydrogen bonding interactions between sorafenib and amino acids Ser-119 and Glu-374 in the active center of CYP3A4 were identified. These findings indicate that sorafenib is oxidized selectively by human CYP3A4. This information could be adapted in individualized approaches to optimize sorafenib safety and efficacy in cancer patients.

Synthesis and NMR characterization of the methyl esters of eicosapentaenoic acid monoepoxides.

The activities of cytochrome P450-derived epoxide metabolites of omega-6 polyunsaturated fatty acids (PUFAs) in cellular homeostasis have generated considerable topical interest, but there is less information on the effects of omega-3 PUFA epoxides. Mass spectroscopic data on the epoxides of the omega-3 PUFA eicosapentaenoic acid (EPA) have been reported but the absence of corresponding NMR data currently hinders their biological assessment. In the present study five monoepoxy derivatives of EPA methyl ester were synthesized by treating EPA methyl ester with m-chloroperbenzoic acid. The individual regioisomers were purified by normal-phase chromatography and characterized by LC-MS/MS and a combination of NMR approaches including (1)H-, (13)C-, (1)H-(1)H-COSY, (1)H-(13)C-HSQC, and (1)H-(13)C-HMBC. The chromatographic properties for these monoepoxides were studied in normal-phase and reversephase-HPLC systems and the MS/MS fragmentation patterns using electrospray ionization were established. This paper also focuses on the NMR characterization of epoxide, olefinic and methylenic moieties and the complete assignments of the isomers.

A high-throughput assay using liquid chromatography-tandem mass spectrometry for simultaneous in vivo phenotyping of 5 major cytochrome p450 enzymes in patients.

The phenotyping cocktail is a practical approach for phenotyping of cytochrome P450 (CYP) enzymes in vivo. In this study, a liquid chromatography-tandem mass spectrometry method using a dual-extraction approach was developed and validated to quantify 5 selective substrates and their metabolites for the simultaneous phenotyping CYPs 1A2, 2C19, 2C9, 2D6, and 3A4 in patient blood samples. The assay was applied in a pilot study of 11 patients with schizophrenia. Five blood samples were collected before and at 1, 2, 4, and 6 hours after administration of a phenotyping cocktail consisting of 100 mg caffeine, 20 mg omeprazole, 25 mg losartan, 30 mg dextromethorphan, and 2 mg midazolam. The method successfully quantitated the CYP enzyme activities without serious side effects in patients. The ratios of metabolite to parent area under the concentration-time curve values were calculated over the 6-hour postdosage to reflect CYP2D6, CYP3A4, and CYP2C9 activities. The ratios of metabolite to parent plasma concentrations were calculated at 4-hour postdosage for CYP1A2 and at 4- or 6-hour postdose for CYP2C19, respectively. The plasma concentration of midazolam at 4 hours was also estimated as another phenotyping index for CYP3A4 activity. The simultaneous assay of all these analytes in a single matrix (plasma) will increase the feasibility of CYP phenotyping in patients.

High-performance liquid chromatography assays for desmethoxyyangonin, methysticin, kavain and their microsomal metabolites.

Three novel, simple and reproducible high-performance liquid chromatography quantitative assays with UV detection were developed and validated for three major kavalactones--desmethoxyyangonin, methysticin and kavain--in rat liver microsomes using diazepam as an internal standard; liquid-liquid extraction was used for sample preparation and analysis was performed on a Shimadzu 10A high-performance liquid chromatography system. The analysis was carried out in reversed-phase mode with a Luna C(18) column (150 x 2.00 mm, 3 microm) at 40 degrees C. The limit of quantitation was 0.1 microg/mL using 0.25 mL of microsomal solution. The assays were linear over the range 0.1-10 microg/mL for desmethoxyyangonin, methysticin and kavain. Quality control samples exhibited good accuracy and precision with relative standard deviations lower than 15% and recoveries between 85 and 105%. The assays exhibited satisfactory performance with high sensitivity for quantifying desmethoxyyangonin, methysticin and kavain in rat liver microsomes and were successfully used to determine the three kavalactones and their microsomal metabolites.

A liquid chromatography/electrospray ionization mass spectrometry (LC-MS/MS) assay for the determination of irinotecan (CPT-11) and its two major metabolites in human liver microsomal incubations and human plasma samples.

A sensitive, rapid LC-MS/MS assay has been developed and validated for the simultaneous quantification of CPT-11 and its two principal metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38), and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxy-camptothecin (APC) in human liver microsomal fractions and plasma. The method was linear over the ranges of 1.56-100 ng/mL, 3.13-150 ng/mL, and 0.78-100 ng/mL for CPT-11, SN-38, and APC, respectively. The total run time was 7.0 min. This assay offers advantages in terms of expediency, recovery of analytes, and suitability for the analysis of CPT-11 and its metabolites in various biological fluids.

Monoepoxy octadecadienoates and monoepoxy octadecatrienoates 2: mass spectral characterization.

Methyl esters of C18 polyunsaturated fatty acids, including gamma-linolenic acid, alpha-linolenic acid and stearidonic acid, were epoxidised using m-chloroperbenzoic acid. Nine monoepoxides were obtained by normal-phase HPLC, identified using LC-MS and NMR, and characterized by NMR spectroscopy and mass spectrometry. This study is focused on structural characterization using LC-MS and LC/APCI/MS/MS. The elution profiles of these monoepoxides in RP-HPLC are determined as 12,13->9,10->6,7-epoxy, 9,10->15,16->12,13-epoxy and 15,16->12,13->9,10-epoxy derivatives of gamma-linolenic, alpha-linolenic and stearidonic acid methyl esters, respectively. The major diagnostic fragmentations in MS/MS identified are postulated to be induced by cleavages of the epoxide ring and alpha-bond cleavage to the epoxy group from [M+H]+ and/or [M+H-MeOH]+.

Plasma protein distribution and its impact on pharmacokinetics of liposomal amphotericin B in paediatric patients with malignant diseases.

OBJECTIVE: This study investigates the association of liposomal amphotericin B (L-AmB) with plasma proteins and its impact on the pharmacokinetics of L-AmB in paediatric patients with malignant diseases. METHODS: Paediatric oncology patients (n = 39) who received multiple-doses of L-AmB were recruited into this study. The association of the drug with plasma lipoprotein was investigated using single vertical spin density gradient ultracentrifugation and quantitated with a validated HPLC assay. The unbound amphotericin B (AmB) in the plasma was separated by ultrafiltration and determined with a validated LC/MS/MS assay. RESULTS: The ex vivo lipoprotein distribution of L-AmB found that 68.3 +/- 11.8% of the drug was associated with the high density lipoprotein (HDL) fraction, which demonstrated a significant inverse correlation with posterior Bayesian estimates of L-AmB clearance (r = -0.690, p < 0.01). The average of unbound fraction of AmB in plasma of patients administered with L-AmB was 0.005, but its relationship with L-AmB clearance did not reach a statistical significance. CONCLUSION: L-AmB displays different lipoprotein distribution profile from that of the conventional AmB formulation, with L-AmB preferentially associated with HDL in plasma. The inverse correlation of L-AmB clearance to its HDL distribution contributes to the difference in the pharmacokinetic profile of L-AmB.

Phytoestrogens and indicators of breast cancer prognosis.

Breast cancer incidence is lower and survival is longer in Asian women residing in Japan, China, or the Philippines than Caucasian women residing in the United States. Phytoestrogen intake has been examined as a possible reason for the disparity in breast cancer incidence and survival. This study examined the association between phytoestrogen intake prior to diagnosis of breast cancer and indicators of breast cancer prognosis (tumor size, estrogen and progesterone receptor status, histological grade, lymphovascular invasion, nodal spread, and stage) in 128 women, aged 40-79 yr, newly diagnosed with invasive breast cancer. After controlling for significant confounding factors, higher intakes of phytoestrogens were associated with favorable indicators of breast cancer. In women with higher intakes of phytoestrogens, there was a 32% reduction in the odds of being diagnosed with any stage of cancer other than stage 1 (95% confidence interval, CI = 0.49-0.93; P = 0.02), a 38% reduction in odds of being diagnosed with positive lymphovascular invasion (95% CI = 0.40-0.95; P = 0.03), and a 66% increase in the odds of being diagnosed with a positive progesterone receptor (95% CI = 1.06-2.58; P = 0.03). We conclude that phytoestrogen intake prior to diagnosis may improve prognosis of breast cancer.