Pharmacokinetics of oseltamivir phosphate and oseltamivir carboxylate in non-pregnant and pregnant rhesus monkeys.

Oseltamivir is an antiviral drug approved to treat influenza in humans. Although the dosing regimen of this drug is well established for non-pregnant patients, it is not clear if the significant physiological alterations associated with pregnancy affect the pharmacokinetics of oseltamivir and, thus, warrant different dosing regimens to assure efficacy. In this study, we investigated the suitability of rhesus macaques as an animal model for studying oseltamivir pharmacokinetics during all trimesters of pregnancy in comparison to pre-pregnant conditions. Specifically, we compared the pharmacokinetics of oseltamivir and its pharmacologically active metabolite oseltamivir carboxylate in rhesus monkeys after intravenous and nasogastric administration of 2.5 mg oseltamivir phosphate/kg body weight given prior to and during the first, second, and third trimesters of pregnancy. Pregnancy had only a modest effect upon the pharmacokinetic parameters of oseltamivir and oseltamivir carboxylate. Monkeys treated intravenously in the third trimester had a reduction in Vd and CL, compared to non-pregnant monkeys. These changes did not occur in the other two trimesters. Pregnant monkeys treated intravenously had 20-25% decrease in AUC0-∞ of oseltamivir carboxylate and a corresponding increase in Vd and CL. Pregnant monkeys treated nasogastrically with oseltamivir phosphate demonstrated a pattern that recapitulated intravenous dosing. Taken together these data indicate that rhesus monkeys are an acceptable model for studying drug-pregnancy interactions.

Use of a physiologically-based pharmacokinetic model to explore the potential disparity in nicotine disposition between adult and adolescent nonhuman primates.

The widespread use and high abuse liability of tobacco products has received considerable public health attention, in particular for youth, who are vulnerable to nicotine addiction. In this study, adult and adolescent squirrel monkeys were used to evaluate age-related metabolism and pharmacokinetics of nicotine after intravenous administration. A physiologically-based pharmacokinetic (PBPK) model was created to characterize the pharmacokinetic behaviors of nicotine and its metabolites, cotinine, trans-3'-hydroxycotinine (3'-OH cotinine), and trans-3'-hydroxycotinine glucuronide (3'-OH cotinine glucuronide) for both adult and adolescent squirrel monkeys. The PBPK nicotine model was first calibrated for adult squirrel monkeys utilizing in vitro nicotine metabolic data, plasma concentration-time profiles and cumulative urinary excretion data for nicotine and metabolites. Further model refinement was conducted when the calibrated adult model was scaled to the adolescents, because adolescents appeared to clear nicotine and cotinine more rapidly relative to adults. More specifically, the resultant model parameters representing systemic clearance of nicotine and cotinine for adolescent monkeys were approximately two- to three-fold of the adult values on a per body weight basis. The nonhuman primate PBPK model in general captured experimental observations that were used for both model calibration and evaluation, with acceptable performance metrics for precision and bias. The model also identified differences in nicotine pharmacokinetics between adolescent and adult nonhuman primates which might also be present in humans.

Developmental neurotoxicity of inorganic arsenic exposure in Sprague-Dawley rats.

High levels of inorganic arsenic (iAs) exposure are associated with severe health effects. Less clear are effects of lower exposure levels on neurodevelopment. Relative to maternal intake, there is limited lactational transfer of arsenic in humans or rodents, yet there are few rodent studies which directly exposed preweaning animals. To more clearly determine iAs developmental neurotoxicity, 28 pregnant Sprague-Dawley rats were exposed to arsenate (AsV) via drinking water (0, 23.6, 47.7, 71.0 ppm) (n = 5-7/group) from gestational day (GD) 6 through GD 22 with targeted doses of 0, 2.33, 4.67, 7.00 mg/kg/day, respectively. Offspring were dosed by gavage daily with the same mg/kg AsV dose as intended for their dam from postnatal day (PND) 1 to 21. Gestational water intake was reduced at all AsV doses, but returned to control levels on lactational day (LD) 1 when control water was returned. Gestational body weight was reduced only at the highest dose on GD 22 and lactational body weight was unaffected. Food intake was unaffected. iAs exposure did not alter offspring body weight (PNDs 1-21) or age at fur development and bilateral ear opening. Incisor eruption, however, was significantly delayed in offspring of the 4.67 and 7.00 mg/kg groups. Further, all iAs groups were significantly delayed in bilateral eye opening. Righting reflex (PNDs 3-6) was unaffected, while slant board performance (PNDs 8-11) was significantly poorer at the highest dose. Brains of culled pups (PND 1) showed dose-dependent increases of iAs. There were no significant AsV-related effects on PND 21 brain regional concentrations of dopamine, DOPAC, HVA, 5-HT or 5-HIAA. These hazard identification results will guide the study designs of developmental iAs exposure at human-relevant levels essential for risk-assessment.

Effects of human sulfotransferases on the cytotoxicity of 12-hydroxynevirapine.

Nevirapine, a non-nucleoside reverse transcriptase inhibitor used for the treatment of AIDS, can cause serious skin rashes and hepatotoxicity. Previous studies have indicated that the benzylic sulfate 12-sulfoxynevirapine, the formation of which is catalyzed by human sulfotransferases (SULTs), may play a causative role in these toxicities. To characterize better the role of 12-sulfoxynevirapine in nevirapine-induced cytotoxicity, the ability of 12 expressed human SULT isoforms to conjugate 12-hydroxynevirapine was assessed. Of the 12 human SULTs, no detectable 12-sulfoxynevirapine was observed with SULT1A3, SULT1C2, SULT1C3, SULT2B1, SULT4A1, or SULT6B1. As determined by the Vmax/Km ratio, SULT2A1 had the highest overall 12-hydoxynevirapine sulfonation activity; lower activities were observed with SULT1A1, SULT1A2, SULT1B1, SULT1C4, and SULT1E1. Incubation of 12-sulfoxynevirapine with glutathione and cysteine led to adduct formation; lower yields were obtained with deoxynucleosides. 12-Hydroxynevirapine was more cytotoxic than nevirapine to TK6, TK6/SULT vector, and TK6/SULT2A1 cells. With nevirapine, there was no difference in cytotoxicity among the three cell lines, whereas with 12-hydroxynevirapine, TK6/SULT2A1 cells were more resistant than TK6 and TK6/SULT vector cells. Co-incubation of 12-hydroxynevirapine with the competitive SULT2A1 substrate dehydroepiandrosterone decreased the level of 12-sulfoxynevirapine and increased the cytotoxicity in TK6/SULT2A1 cells. These data demonstrate that although 12-sulfoxynevirapine reacts with nucleophiles to form adducts, sulfonation of 12-hydroxynevirapine decreases the cytotoxicity of 12-hydroxynevirapine in TK6 cells.

A simple and highly sensitive UPLC-ESI-MS/MS method for the simultaneous quantification of nicotine, cotinine, and the tobacco-specific carcinogens N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in serum samples.

According to the World Health Organization, the consumption of tobacco products is the single largest cause of preventable deaths in the world, exceeding the total aggregated number of deaths caused by diseases such as AIDS, tuberculosis, and malaria. An important element in the evaluation of the health risks associated with the consumption of tobacco products is the assessment of the internal exposure to the tobacco constituents responsible for their addictive (e.g. nicotine) and carcinogenic (e.g. N-nitrosamines such as NNN and NNK) properties. However, the assessment of the serum levels of these compounds is often challenging from an analytical standpoint, in particular when limited sample volumes are available and low detection limits are required. Currently available analytical methods often rely on complex multi-step sample preparation procedures, which are prone to low analyte recoveries and ex-vivo contamination due to the ubiquitous nature of these compounds as background contaminants. In order to circumvent these problems, we report a facile and highly sensitive method for the simultaneous quantification of nicotine, cotinine, NNN, and NNK in serum samples. The method relies on a simple "one pot" liquid-liquid extraction procedure and isotope dilution ultra-high pressure (UPLC) hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry. The method requires only 10µL of serum and presents a limit of quantification of 0.02nmol (3000pg/mL) nicotine, 0.6pmol (100pg/mL) cotinine, 0.05pmol NNK (10pg/mL), and 0.06pmol NNN (10pg/mL), making it appropriate for pharmacokinetic evaluations.

Cytochrome P450-mediated metabolism of triclosan attenuates its cytotoxicity in hepatic cells.

Triclosan is a widely used broad-spectrum anti-bacterial agent. The objectives of this study were to identify which cytochrome P450 (CYP) isoforms metabolize triclosan and to examine the effects of CYP-mediated metabolism on triclosan-induced cytotoxicity. A panel of HepG2-derived cell lines was established, each of which overexpressed a single CYP isoform, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1. The extent of triclosan metabolism by each CYP was assessed by reversed-phase high-performance liquid chromatography with online radiochemical detection. Seven isoforms were capable of metabolizing triclosan, with the order of activity being CYP1A2 > CYP2B6 > CYP2C19 > CYP2D6 ≈ CYP1B1 > CYP2C18 ≈ CYP1A1. The remaining 11 isoforms (CYP2A6, CYP2A7, CYP2A13, CYP2C8, CYP2C9, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1) had little or no activity toward triclosan. Three metabolites were detected: 2,4-dichlorophenol, 4-chlorocatechol, and 5'-hydroxytriclosan. Consistent with the in vitro screening data, triclosan was extensively metabolized in HepG2 cells overexpressing CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP2C18, and these cells were much more resistant to triclosan-induced cytotoxicity compared to vector cells, suggesting that CYP-mediated metabolism of triclosan attenuated its cytotoxicity. In addition, 2,4-dichlorophenol and 4-chlorocatechol were less toxic than triclosan to HepG2/vector cells. Conjugation of triclosan, catalyzed by human glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also occurred in HepG2/CYP-overexpressing cells and primary human hepatocytes, with a greater extent of conjugation being associated with higher cell viability. Co-administration of triclosan with UGT or SULT inhibitors led to greater cytotoxicity in HepG2 cells and primary human hepatocytes, indicating that glucuronidation and sulfonation of triclosan are detoxification pathways. Among the 18 CYP-overexpressing cell lines, an inverse correlation was observed between cell viability and the level of triclosan in the culture medium. In conclusion, human CYP isoforms that metabolize triclosan were identified, and the metabolism of triclosan by CYPs, UGTs, and SULTs decreased its cytotoxicity in hepatic cells.

Timing and route of exposure affects crystal formation in melamine and cyanuric exposed male and female rats: gavage vs. feeding.

Effects of the dosing matrix and timing on the onset of renal crystal formation were evaluated in male and non-pregnant female rats (Fisher 344) exposed to both melamine (MEL) and cyanuric acid (CYA) for 28 days. Rats were fed ground feed containing 60 ppm MEL and 60 ppm CYA, (5 mg/kg bw/day equivalent), or exposed via oral gavage to carboxymethylcellulose containing 5 mg/kg bw MEL followed by 5 mg/kg bw CYA either consecutively (<1 min apart) or delayed 45 min after MEL. Staggered gavage exposure to MEL/CYA caused extensive renal crystal formation as compared to when the two compounds were administered consecutively or in feed. Treatment related effects included reduced weight gain, feed consumption, and testicular weight and increased kidney weight, water consumption and urine output. Animals from the staggered MEL/CYA gavage exposure group became ill and were removed after 9 days of exposure. Approximately 1 week after the initiation of exposure microscopic urinalysis revealed MEL/CYA crystals in both groups of gavaged animals but not in the MEL/CYA feed treatment groups. Urinary crystals were smaller (10 µm) in animals consecutively gavaged. In contrast the urinary crystals were larger (20-40 µm) and frequently clumped in the animals in the staggered gavage group.

Cyclofructan 6 based stationary phases for hydrophilic interaction liquid chromatography.

New stationary phases for hydrophilic interaction liquid chromatography (HILIC) were synthesized by covalently attaching native cyclofructan 6 (CF6) to silica gel. The chromatographic characteristics of the new stationary phases were evaluated and compared to three different types of commercial HILIC columns. The CF6 columns produced considerably different retention and selectivity patterns for various classes of polar analytes, including nucleic acid compounds, xanthines, ß-blockers, salicylic acid and its derivatives, and maltooligosaccharides. Univariate optimization approaches were examined including organic modifier (acetonitrile) contents and buffer pH and salt concentration. The thermodynamic characteristic of the CF6 stationary phase was investigated by considering the column temperature effect on retention and utilizing van't Hoff plots. CF6 based stationary phases appear to have exceptionally broad applicability for HILIC mode separations.

Comparison of HPLC enantioseparation of substituted binaphthyls on CD-, polysaccharide- and synthetic polymer-based chiral stationary phases.

Retention and enantioseparation behavior of ten 2,2'-disubstituted or 2,3,2'-trisubstituted 1,1'-binaphthyls and 8,3'-disubstituted 1,2'-binaphthyls, which are used as catalysts in asymmetric synthesis, was investigated on eight chiral stationary phases (CSPs) based on beta-CD, polysaccharides (tris(3,5-dimethylphenylcarbamate) cellulose or amylose CSPs) and new synthetic polymers (trans-1,2-diamino-cyclohexane, trans-1,2-diphenylethylenediamine and trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid CSPs). Normal-, reversed-phase and polar-organic separation modes were employed. The effect of the mobile phase composition was examined. The enantiomeric separation of binaphthyl derivatives, which possess quite similar structures, was possible in different enantioselective environments. The substituents and their positions on the binaphthyl skeleton affect their properties and, as a consequence, the separation system suitable for their enantioseparation. In general, the presence of ionizable groups on the binaphthyl skeleton, substitution with non-identical groups and a chiral axis in the 1,2' position had the greatest impact on the enantiomeric discrimination. The 8,3'-disubstituted 1,2'-binaphthyl derivatives were the most easily separated compounds in several separation systems. From all the chiral stationary phases tested, cellulose-based columns were shown to be the most convenient for enantioseparation of the studied analytes. However, the polymeric CSPs with their complementary behavior provided good enantioselective environments for some derivatives that could be hardly separated in any other chromatographic system.

Analysis for estrogens as environmental pollutants--a review.

The approaches to the analysis for estrogen compounds as environmental pollutants are critically reviewed and evaluated on the basis of significant, recent original publications. The importance of sample pretreatment and analyte preconcentration techniques is pointed out, with an emphasis on SPE and on the use of highly selective interactions such as molecular recognition. The hyphenated systems of high-performance gas or liquid chromatography and mass spectrometric techniques are discussed as the basic methods of determination of estrogens in environmental samples. Immunochemical procedures are shown to be useful in semiquantitative screening of estrogen pollutants (e.g. ELISA kits). Classical HPLC and GC with common UV/Vis, fluorescence and electrochemical detection are useful in routine checking on higher pollutant concentrations.

Cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phases as effective tools for enantioselective HPLC separation of structurally different disubstituted binaphthyls.

Cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phases (CSPs) were used for a study of the HPLC retention and enantioseparation behavior of 2,2'-disubstituted or 3,2,2'-trisubstituted 1,1'-binaphthyls and 8,3'-disubstituted 1,2'-binaphthyls. The effects of the mobile phase composition in normal- (NP) and reversed-phase (RP) separation modes were investigated. The NP mobile phases contained n-hexane and propane-2-ol at various volume ratios, the RP ones were obtained by mixing acetonitrile with water or a 20 mM phosphate buffer of pH 6.0 or 3.0. The RP separation mode has been found more suitable for enantioresolution of most of the analytes. The best enantioseparation of 2,2'-diacetyl-1,1'-binaphthyl, 2-hydroxy-2'-(phenylamino)-1,1'-binaphthyl-3-carboxylic acid and 2-amino-2'-hydroxy-1,1'-binaphthyl-3-carboxylic acid was obtained in the mobile phase of ACN/20 mM phosphate buffer, pH 3.0, 40/60 (v/v), whereas N-(2'-hydroxy-1,1'-binaphthyl-2-yl)acetamide, N-(3'-methoxy-1,2'-binaphthyl-8-yl)acetamide, and N-(3'-hydroxy-1,2'-binaphthyl-8-yl)acetamide yielded better results in ACN/water at the same v/v ratio. The analyte-CSP interaction mechanism was found to be temperature independent but the enantioresolution improved at an elevated temperature. The mechanism of the enantioselective discrimination is discussed on the basis of the thermodynamic parameters obtained. Semi-preparative separation conditions have been proposed for 2-amino-2'-hydroxy-1,1'-binaphthyl-3-carboxylic acid, N-(3'-methoxy-1,2'-binaphthyl-8-yl)acetamide, and N-(3'-hydroxy-1,2'-binaphthyl-8-yl)acetamide.