Pyridine Metabolite Research Articles

LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods.

Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier “indolome” to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us “indolome”) in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100–1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition.

Effects of orally administered antibiotics on the bioavailability of amlodipine: gut microbiota-mediated drug interaction.

Amlodipine is a representative calcium channel blocker that is frequently prescribed for the treatment of hypertension. In this study, the possibility of drug-drug interactions between amlodipine and coadministered antibiotics (ampicillin) was investigated in rats; thus, changes in the metabolic activities of gut microflora and the consequent pharmacokinetic pattern of amlodipine following ampicillin treatment were characterized. In human and rat fecalase incubation samples, amlodipine was metabolized to yield a major pyridine metabolite. The remaining amlodipine decreased and the formation of pyridine metabolite increased with incubation time, indicating the involvement of gut microbiota in the metabolism of amlodipine. Pharmacokinetic analyses showed that systemic exposure of amlodipine was significantly elevated in antibiotic-treated rats compared with controls. These results showed that antibiotic intake might increase the bioavailability of amlodipine by suppressing gut microbial metabolic activities, which could be followed by changes in therapeutic potency. Therefore, coadministration of amlodipine with antibiotics requires caution and clinical monitoring.

P450-mediated O-demethylated metabolite is responsible for rat hepatobiliary toxicity of pyridyltriazine-containing PI3K inhibitors.

The dysregulation of phosphatidylinositol 3-kinase (PI3K)-dependent pathways is implicated in several human cancers making it an attractive target for small molecule PI3K inhibitors. A series of potent pyridyltriazine-containing inhibitors of class Ia PI3Ks were synthesized and a subset of compounds was evaluated in exploratory repeat-dose rat toxicology studies. Daily oral dosing of compound 1: in Sprague Dawley rats for four consecutive days was associated with hepatobiliary toxicity that included biliary epithelial hyperplasia and hypertrophy, periductular edema, biliary stasis, and acute peribiliary inflammatory infiltrates. These histological changes were associated with clinical pathology changes that included increased serum liver enzymes, total bile acids, and bilirubin. The predominant clearance pathway of 1: was shown in vitro and in a bile-duct cannulated rat (14)C-ADME study to be P450-mediated oxidative metabolism. An O-demethylated pyridine metabolite, M3: , was identified as a candidate proximal metabolite that caused the hepatotoxicity. Co-administration of the pan-P450 inhibitor 1-aminobenzotriazole with 1: to rats significantly reduced the formation of M3: and prevented liver toxicity, whereas direct administration of M3: reproduced the toxicity. Structural changes were introduced to 1: to make the methoxypyridine ring less susceptible to P450 oxidation (compound 2: ), and addition of a methyl group to the benzylic carbon (compound 3: ) improved the pharmacokinetic profile. These changes culminated in the successful design of a clinical candidate 3: (AMG 511) that was devoid of liver toxicity in a 14-day rat toxicity study. Herein, we describe how a metabolism-based structure-activity relationship analysis allowed for the successful identification of a PI3K inhibitor devoid of off-target toxicity.

Trigonelline and related nicotinic acid metabolites: occurrence, biosynthesis, taxonomic considerations, and their roles in planta and in human health

This review describes the occurrence and biosynthesis of trigonelline (N-methylnicotinic acid) and related nicotinic acid metabolites. High concentrations of trigonelline are found in seeds of coffee, and some members of the Fabaceae, while trace amounts occur in many other species. In contrast, the occurrence of other pyridine alkaloids derived from nicotinic acid is limited. Nicotinic acid, a precursor of the secondary pyridine metabolites, is derived from pyridine nucleotides. In planta, pyridine nucleotide biosynthesis de novo is initiated from aspartic acid. The degradation of NAD and its regeneration from the catabolites is called the pyridine nucleotide cycle (PNC). Isotopic labelling and enzymatic studies indicate a seven-component PNC VII pathway is the major route. All plants examined convert exogenous nicotinic acid to trigonelline and/or nicotinic acid N-glucoside (NaG). In general, NaG formation is restricted to ferns and selected orders of angiosperms, whereas other plants produce trigonelline. The biosynthesis of other pyridine alkaloids, of which many details remain to be resolved, is discussed briefly. The potential in planta roles of trigonelline, including detoxification, nyctinasty and host selection are discussed. Coffee beverage is the major food containing trigonelline and some vegetables also contain trigonelline. The possible effects of trigonelline on health mediated via hypoglycaemic, neuroprotective, anti-cancer, estrogenic, and antibacterial activities are reviewed. Finally, potential genetic manipulation of biosynthetic pathways to create trigonelline- and vitamin B3-rich plants and agricultural uses of trigonelline-rich genetically modified crops and trees are discussed.

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

NAD(+) is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD(+) pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD(+) metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD(+) levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD(+) homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.

Phosphate-responsive Signaling Pathway Is a Novel Component of NAD+ Metabolism in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in various cellular biochemical reactions. To date the signaling pathways that regulate NAD(+) metabolism remain unclear due to the dynamic nature and complexity of the NAD(+) metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD(+) pool. In this study we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD(+) metabolism. We show that the phosphate-responsive signaling (PHO) pathway contributes to control NAD(+) metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a potential NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular nicotinic acid mononucleotide (NaMN) and mediates nicotinamide mononucleotide (NMN) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD(+) metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.

Simultaneous determination of nitrendipine and one of its metabolites in plasma samples by gas chromatography with electron-capture detection

A sensitive method for GC-ECD simultaneous determination of nitrendipine and its pyridine metabolite M1 in human plasma is described. Felodipine was used as the internal standard. The plasma samples were extracted with toluene. One microlitre of the extract was injected onto the capillary column (polymethylsiloxane) and measured with electron-capture detector. The developed method showed to be linear over the range 0.25–70 for nitrendipine and 0.3–61 ng/ml for its metabolite M1 with an inter-day and intra-day precision in terms of R.S.D. lower than 8% except the concentrations near lowest limit of quantification (LLOQ) (<11% R.S.D.). The LLOQ for nitrendipine was 0.25 and 0.3 ng/ml for its metabolite, respectively. The analytical recovery was 94% for nitrendipine and 89% for its pyridine metabolite M1. This GC-ECD method was developed for being used in clinical pharmacokinetic studies.

Liquid chromatography–tandem mass spectrometry in chiral study of amlodipine biotransformation in rat hepatocytes

A high proportion of drugs are chiral compounds used as racemic mixtures in a clinical practice. Very often only one of two enantiomers exhibits a desired pharmacological effect. Amlodipine, 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-1,4-dihydropyridine, is a chiral calcium channel blocker, currently used as a racemate in clinical practice. Racemic mixture is used even though it is known that R- and S-amlodipine do not have the same biological activity and only S-amlodipine possesses vasodilating properties. In this work a novel reversed phase liquid chromatography (RP-LC) separation method for amlodipine and its metabolites was developed. Based on this separation chiral aspects of amlodipine biotransformation were studied by incubation of amlodipine and its two individual enantiomers with primary culture of rat hepatocytes. Structure of the metabolites was elucidated using a liquid chromatography (LC) separation with ultraviolet (UV) and mass spectrometry (MS) detection. An LC–tandem MS (MS/MS) method was used to establish fragmentation pattern of amlodipine and its metabolites. Eight metabolites presented in the highest amount were identified and semiquantified by employing an LC separation. Basically two types of metabolites were detected, reduced type – dihydropyridine metabolites and oxidized type – pyridine metabolites. Other metabolic modification included changes of functional groups, e.g., methylester hydrolysis or acetylation of amino group. The results exhibited that R-amlodipine was stereoselectively metabolized by the respective biotransformation enzymes in rat liver hepatocytes and it is also demonstrated by greater extent of R-amlodipine conversion into metabolites where the values for R-amlodipine are for the most metabolites higher than those for metabolites of S-amlodipine.

Detection of the calcium antagonist nicardipine and its metabolites by gas chromatography–mass spectrometry

An 89-year-old male patient, hospitalized with Parkinson’s syndrome, suddenly died shortly after an intravenous drug injection. The conditions indicated that an overdose of nicardipine (1.3 mg/(ml kg)) may be given to the patient. At the autopsy, no pathological changes were noted. With a capillary gas chromatograph with mass spectrometer (GC–MS), nicardipine (4.97 μg/ml) and its pyridine metabolite (M-5, 5.0 μg/ml) were detected in the heart blood of the deceased. This result indicated that an overdose of intravenous nicardipine caused a sudden death of a patient in a poor condition.

Screening for inhibitory effects of antineoplastic agents on CYP3A4 in human liver microsomes.

The human cytochrome P450 enzyme CYP3A4 is involved in the metabolism of many anticancer drugs. Since these drugs are usually administered in a polychemotherapy regimen, the objective of this study was to examine their inhibitory potency on CYP3A4 with regard to possible mutual drug interactions. CYP3A4 activities in human liver microsomes from 2 donors were determined using the oxidation of the dihydropyridine denitronifedipine, a specific CYP3A4 substrate, at a concentration of 50 microM (= KM). Formation of the pyridine metabolite was measured using HPLC. Inhibitor concentrations used were 0.5, 5 and 50 microg/ml, except for cyclophosphamide and ifosfamide (0.5, 2.5 and 5 mg/ml) and for paclitaxel (0.05, 0.15, 0.5, 1.5 and 5 microg/ml). The following substances showed an inhibitory effect on CYP3A4 (IC50 values for the 2 microsome samples are parenthesized): cyclophosphamide (12.3/9.2 mmol/l), mafosfamide generated 4-OH-cyclophosphamide (152/163 [micromol/l), ifosfamide (3.6/2.5 mmol/l), vinblastine sulfate (20/44 micromol/l), vincristine sulfate (67/176 micromol/l), daunorubicin hydrochloride (206/200 micromol/l), doxorubicin hydrochloride (160/215 micromol/l), teniposide (64/84 micromol/l) and docetaxel (6.4/12.7 micromol/l). No inhibitory effect on CYP3A4 was observed with epirubicin, etoposide, paclitaxel, cytarabine, 5-FU, 6-mercaptopurine, methotrexate, cisplatin, carboplatin, bleomycin, busulfan, chlorambucil and mitomycin. Comparing IC50 values with plasma concentrations present during antineoplastic therapy, the agents cyclophosphamide, ifosfamide, vinblastine, teniposide and docetaxel could possibly cause clinical drug interactions by inhibition of CYP3A4. Some recently described clinical interactions with antineoplastic agents may be explained by these results.

Comparative Induction of CYP1A1 Expression by Pyridine and Its Metabolites

We compared pyridine and five of its metabolites in terms of (i) in vivo induction of CYP1A1 expression in the lung, kidney, and liver in the rat and (ii) in vitro binding to, and activation of, the aryl hydrocarbon receptor (AhR) in cytosol from rat liver or Hepa1c1c7 cells. Following a single 2.5 mmol/kg ip dose of either pyridine, 2-hydroxpyridine, 3-hydroxypyridine, 4-hydroxypyridine, N-methylpyridinium, or pyridine N-oxide, CYP1A1 activity (ethoxyresorufin O-deethylase), protein level (as determined by Western blotting), and mRNA level (as determined by Northern blotting) were induced by pyridine, N-methylpyridinium, and pyridine N-oxide in the lung, kidney, and liver. The induction by N-methylpyridinium or pyridine N-oxide was comparable to or greater than that by pyridine in some tissues. 2-Hydroxypyridine and 3-hydroxypyridine caused tissue-specific induction or repression of CYP1A1, whereas 4-hydroxypyridine had no effect on the expression of the enzyme. Pyridine and its metabolites elicited weak activation of the aryl hydrocarbon receptor in a gel retardation assay in cytosol from rat liver but not Hepa 1c1c7 cells. However, the receptor activation did not parallel the in vivo CYP1A1 induction by the pyridine compounds, none of which inhibited binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin to AhR in a competitive assay in rat liver cytosol. The findings are consistent with a role of pyridine metabolites in CYP1A1 induction by pyridine but do not clearly identify the role of aryl hydrocarbon receptor in the induction mechanism.

The identification of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.

Pharmacokinetics of the multidrug-resistance-converting drug dexniguldipine and its pyridine metabolite M-1 in the plasma, tumor, and renal tissue of tumor-bearing Wag/Rij rats.

The pharmacokinetics of oral dexniguldipine, a new multidrug-resistance-modifying agent under clinical evaluation, and its pyridine metabolite M-1 were determined in plasma, tumor, and renal tissue in Wag/Rij rats bearing a multidrug-resistant CC531 colon adenocarcinoma tumor under the renal capsule. The pharmacokinetics were studied in four experiments. After a single administration of dexniguldipine (30 mg/kg), tumors and kidneys were collected after 5 (experiment 1), 24 (experiment 2), and 48 h (experiment 3). In the fourth experiment, dexniguldipine was given once daily for 3 consecutive days at a dose of 30 mg/kg. In all experiments, plasma samples were collected at regular intervals. The concentrations of dexniguldipine and M-1 could be determined in plasma in most of the rats at up to 32 h after drug administration. The area under the curve (AUC) of dexniguldipine and M-1 varied by a factor of 2-6 in the four experiments. High tumor-tissue concentrations of dexniguldipine were observed. The concentrations were highest in the multiple-dose experiment (2014 +/- 1005 ng/g tissue). High degrees of correlation (> 0.8) were established between the concentrations of dexniguldipine measured in plasma and tumor as well as renal tissue. Overall, tumor-tissue concentrations of M-1 comprised one-third of the dexniguldipine concentrations measured.

P-glycoprotein-associated resistance to taxol and taxotere and its reversal by dexniguldipine-HCl, dexverapamil-HCl, or cyclosporin A

A series of different human MDR (multidrug-resistant) cell lines including a HeLa-MDR1 transfectant which exhibit high overexpression of the MDR1/P-glycoprotein gene, but no enhanced expression of the MRP (multidrug resistance associated protein) gene, showed different ratios of relative resistances to the taxanes taxol and taxotere. Using these cell lines the chemosensitizing efficacies of several structurally different chemosensitizers, i.e. the dihydropyridine dexniguldipine-HC1 (B8509-035), its main pyridine metabolite M1 (B8909-008), the cyclic peptide cyclosporin A, or the phenylalkylamine dexverapamil-HCl, were examined applying a 72 h tetrazolium based colorimetric MTT-assay, or a 96 h sulforhodamine B assay. Remarkably, we observed in some instances that the modulating efficacy of a particular chemosensitizer was strongly dependent on the cell line used for experimentation. Thus, dexniguldipine-HCl efficiently modulated taxane resistances of the ovarian carcinoma MDR cell line 2780AD in the submicromolar concentration range, whereas cyclosporin A and the other chemosensitizers were rather ineffective. Dexniguldipine-HCl or cyclosporin A, however, both showed a similarly strong modulating activity on the HeLa-MDR1 transfectant in clear contrast to the effects observed using the pyridine B8909-008, or dexverapamil-HCl, respectively, at the same final concentrations. Our results point to additional, as yet unidentified factors beyond the expression levels of P-glycoprotein which could contribute to the susceptibility of MDR cells to a combined treatment using taxanes and different chemosensitizing compounds. This result appears to be important considering the clinical application of chemosensitizers for combination therapy of tumors of different origin.

Modipafant, a new PAF antagonist: pharmacokinetics and disposition in rat, dog and man.

1. The pharmacokinetics and disposition of modipafant, a dihydropyridine PAF antagonist, were studied in rat and dog following intravenous and oral administration of the drug or its radiolabelled analogue. In addition, the pharmacokinetics were studied in man following single administration of escalating oral doses of the drug. Modipafant is a lipophilic weak base with log D(octanol) 7.4 and pKa of 4.3 and 5.3 respectively. 2. Following intravenous administration of [14C]-modipafant to rat, radioactivity is rapidly distributed throughout the body, except for the brain. A significant amount of radioactivity (probably modipafant) is rapidly distributed to the alimentary tract, particularly in the stomach. This is believed to be due to 'ion trapping' of modipafant in the acidic environment of the upper GI tract. The re-circulated modipafant may be subject to reabsorption and/or faecal excretion. 3. Following intravenous administration to rats, systemic clearance is five times greater in the male than female. The magnitude of this difference is in keeping with the clearance of other dihydropyridines such as nilvadipine. In dog, the clearance values are similar for both sexes, as expected. In this latter species, the systemic clearance decreases 6-fold with increasing dose size, indicative of saturation of a pathway of metabolism. 4. Following oral administration over a dose range of 1-12 mg/kg, modipafant is incompletely (27-67%) bioavailable in rat and dog. In the male dog, systemic exposure to drug (AUC/infinity) increased non-linearly with dose. Following oral administration to man, absorption was rapid with a mean value for Tmax of 1 h, and Cmax's ranging non-linearly from 90 to 2100 ng/ml following dosing at 12.5 to 150 mg respectively. 5. The elimination of modipafant is characterized by short half-life (mean values for t1/2 range from 1 to 3 h). However, the nature of the receptor kinetics of modipafant (slow offset) means that the drug shows a long duration of action in spite of short pharmacokinetics at pharmacologically relevant doses. 6. Following oral and intravenous administration of 14C-modipafant to rat and dog, the majority of radioactivity (mean 92%) is recovered in the faeces. The excretion of modipafant in rat and dog is characterized by metabolism, mostly to pyridine metabolites, accounting for between 38 and 75% of total clearance, the rest being cleared as unchanged drug.

Fluroxypyr- and Triclopyr-Specific Enzyme-Linked Immunosorbent Assays: Development and Quantitation in Soil and Water

Enzyme-linked immunosorbent assays (ELISAs) were developed to quantitate fluroxypyr [[(4-amino-3,5-dichloro-6-fluoro-2-pyridinyl)oxy]acetic acid] and triclopyr [[(3,5,6-trichloro-2-pyridinyl)oxy]acetic acid] in soil and water. The linear working range of the fluroxypyr ELISA was from 0.1 to 10 ng·mL-1 with a limit of detection (LOD) of 0.1 ng·mL-1 and an IC50 value of 1.6 ng·mL-1. The linear working range of the triclopyr ELISA was from 0.1 to 5 ng·mL-1 with a LOD of 0.1 ng·mL-1 and an IC50 value of 1.7 ng·mL-1. Cross-reactivity to selected pyridine metabolites and agrochemicals was determined. Significant cross-reactivity (within the linear working range of the ELISA) using the fluroxypyr ELISA was found only to the metabolite 4-amino-3,5-dichloro-6-fluoro-2-methoxypyridine. Significant cross-reactivity using the triclopyr ELISA was found only to the auxinic herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The ELISAs accurately estimated fluroxypyr and triclopyr in water at concentrations as low as 0.1 ng·mL-1. Analysis of two different soil types with different textures (clay loam and sandy clay loam) required cleanup procedures using filtration and solid phase extraction to accurately estimate fluroxypyr and triclopyr concentrations. Keywords: ELISA; residue; pyridine; herbicide

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection.

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(-)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.

Capillary gas chromatographic determination with nitrogen—phosphorus detection of the calcium antagonist nicardipine and its pyridine metabolite M-5 in plasma

A capillary gas chromatographic method with nitrogen—phosphorus detection for the simultaneous determination of nicardipine and its pyridine metabolite M-5 was developed. The method involves extraction of the plasma with hexane—methylene chloride (1:1, v/v), followed by evaporation of the organic phase. The extract is injected into a fused-silica capillary column coated with cross-linked 5% phenyl—methylsilicone. A temperature gradient (85–285°C) is applied and the two products and the internal standard can be separated within 22 min. The limit of detection is 0.5 ng/ml for both products. The method is suitable for pharmacokinetic studies in humans.

Influence of Single and Repeated Doses of Oxcarbazepine on the Pharmacokinetic Profile of Felodipine

Eight healthy male volunteers (age 25-41 years) entered an open-label, within-subject study. They were treated for 13 consecutive days with felodipine (FEL) extended-release tablets, 10 mg daily. On day 6, oxcarbazepine (OXC) 600 mg was given in the morning, and from day 7 to 13, the daily dose was increased to 450 mg b.i.d. Blood samples for measurement of FEL and its pyridine metabolite (determined by gas-chromatography) were drawn just before dosing and at 2, 4, 6, 8, 10, 12, and 24 h after dosing on days 5, 6, and 13. Steady-state pharmacokinetic parameters of FEL and its pyridine metabolite were not influenced by the single dose of OXC. Repeated coadministration of OXC significantly reduced the area under the concentration-time curve (AUC0-24) of FEL by 28% and the FEL maximum plasma concentration (Cmax) by 34%. This reduction in FEL bioavailability is much smaller than that observed after co-administration of carbamazepine (CBZ) (i.e., 94%).

Reversal of multidrug resistance in friend leukemia cells by dexniguldipine-HCl

Dexniguldipine-HCl (DNIG)--a prospective clinical modulator of p170-glycoprotein (pgp170)-mediated multidrug resistance (MDR)--was evaluated in a drug-accumulation assay in MDR murine leukemia cell strain F4-6RADR expressing pgp170. The compound elevated low accumulation of either doxorubicin (DOX), daunorubicin (DNR), or mitoxantrone (MITO) in resistant F4-6RADR cells to the very levels observed in drug-sensitive F4-6 precursor cells. In parallel with the increase in DNR content (F4-6RADR, solvent: 303 +/- 27 pmol/mg protein; DNIG (3.3 mumol/l): 1,067 +/- 174 pmol/mg protein; F4-6P, solvent: 948 +/- 110 pmol/mg protein; n = 8-9, SEM), the amount of DNR tightly bound to the acid precipitate pellet obtained from F4-6RADR (i.e., protein, DNA, RNA) increased 3.9-times to the levels observed in sensitive F4-6 cells. The main pyridine metabolite of DNIG displayed similar activity. Concentration-response analysis revealed that DNIG and R,S-verapamil (VER) induced 100% reversal of the DNR accumulation shortage associated with the MDR phenotype but DNIG was 8 times more potent than VER (50% inhibitory concentration (IC50), 0.73 vs 5.4 mumol/l). In keeping with the accumulation assay, DNIG was about 10 times more potent than VER in sensitizing F4-6RADR cells to the cytostatic and cytotoxic effects of DNR in proliferation assays. In conclusion, DNIG is a potent in vitro modulator, improving (a) the accumulation of anthracycline-like cytostatics, (b) drug access to cellular binding sites, and (c) the cytostatic action of DNR in F4-6RADR leukemia cells of the MDR phenotype.