In vitro evaluation of enteral tube administration of lansoprazole orally disintegrating tablets.

Lansoprazole orally disintegrating tablets (ODTs) can be administered orally or through a nasogastric (NG) tube for patients who are unable to swallow. In addition, off-label administration through gastrostomy (G) or jejunal (J) tubes has been reported. The purpose of this study was to develop in vitro methods to assess the risk of clogging during administration of two lansoprazole ODTs through enteral feeding tubes. Feeding tubes of various compositions and geometries were selected for testing. Disintegration, sedimentation, percent recovery, acid phase dissolution testing, and particle size distribution measurements were performed. The results indicated that G tubes had the greatest risk of clogging compared to NG and J tubes. In addition, larger particles and an increased amount of insoluble excipients observed in Product B resulted in more irreversible enteral tube clogging than compared to Product A. The geometry and design of the tube also had an impact on the amount of lansoprazole recovered after enteral tube administration. Lansoprazole ODTs demonstrated acid resistance stability regardless of the water used for suspension. The in vitro methods discussed in this work could be used to evaluate in vitro equivalence and to assess the risk of delivering a drug product through an enteral feeding tube.

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.

Characterization of water-soluble organic matter in urban aerosol by 1H-NMR spectroscopy.

The functional and 13C isotopic compositions of water-soluble organic carbon (WSOC) in atmospheric aerosol were determined by nuclear magnetic resonance (1H-NMR) and isotope ratio mass spectrometry (IRMS) in an urban location in the Southern Mississippi Valley. The origin of WSOC was resolved using the functional distribution of organic hydrogen, δ13C ratio, and positive matrix factorization (PMF). Three factors were retained based on NMR spectral bins loadings. Two factors (factors 1 and 3) demonstrated strong associations with the aliphatic region in the NMR spectra and levoglucosan resonances. Differences between the two factors included the abundance of the aromatic functional group for factor 1, indicating fresh emissions and, for factor 3, the presence of resonances attributed to secondary ammonium nitrate and low δ13C ratio values that are indicative of secondary organic aerosol. Factors 1 and 3 added 0.89 and 1.08 µgC m-3, respectively, with the highest contribution in the summer and fall. Factor 2 retained resonances consistent with saccharides and was attributed to pollen particles. Its contribution to WSOC varied from 0.22 µgC m-3 in winter to 1.04 µgC m-3 in spring.

Utilizing G-quadruplex formation to target 8-oxoguanine in telomeric sequences.

Utilizing G-quadruplex specific ligands that can induce/bind G-quadruplex DNA in human telomeric regions has recently become an attractive means for cancer chemotherapy because the formation of G-quadruplex structures inhibits the activity of telomerase, a reverse transcriptase mainly expressed in cancer cells. In the present work, we synthesized a type of bifunctional molecules that selectively bind to telomeric DNA via G-quadruplex formation and subsequently react with proximate OxodG in the presence of one-electron oxidant. Such molecules could be useful for telomerase inhibition. Perylene derivatives (7 and 9) containing 1,3-diamino moieties were prepared for demonstration. The binding of 7 with G-quadruplex DNA was determined using UV thermal denaturation and the corresponding binding constant was derived from UV titration. The interactions of 7 with G-quadruplex DNA containing OxodG were characterized using circular dichroism. Gel electrophoresis revealed that 7 can form more adducts with OxodG in G-quadruplex regions than that in duplex DNA.

Investigation on the Interactions of NiCR and NiCR-2H with DNA.

We report here a biophysical and biochemical approach to determine the differences in interactions of NiCR and NiCR-2H with DNA. Our goal is to determine whether such interactions are responsible for the recently observed differences in their cytotoxicity toward MCF-7 cancer cells. Viscosity measurement and fluorescence displacement titration indicated that both NiCR and NiCR-2H bind weakly to duplex DNA in the grooves. The coordination of NiCR-2H with the N-7 of 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) is stronger than that of NiCR as determined by (1)H NMR. NiCR-2H, like NiCR, can selectively oxidize guanines present in distinctive DNA structures (e.g., bulges), and notably, NiCR-2H oxidizes guanines more efficiently than NiCR. In addition, UV and (1)H NMR studies revealed that NiCR is oxidized into NiCR-2H in the presence of KHSO(5) at low molar ratios with respect to NiCR (

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.

Comparative Evaluation of U.S. Brand and Generic Intravenous Sodium Ferric Gluconate Complex in Sucrose Injection: Physicochemical Characterization.

The objective of this study was to evaluate physicochemical equivalence between brand (i.e., Ferrlecit) and generic sodium ferric gluconate (SFG) in sucrose injection by conducting a series of comparative in vitro characterizations using advanced analytical techniques. The elemental iron and carbon content, thermal properties, viscosity, particle size, zeta potential, sedimentation coefficient, and molecular weight were determined. There was no noticeable difference between brand and generic SFG in sucrose injection for the above physical parameters evaluated, except for the sedimentation coefficient determined by sedimentation velocity analytical ultracentrifugation (SV-AUC) and molecular weight by asymmetric field flow fractionation-multi-angle light scattering (AFFF-MALS). In addition, brand and generic SFG complex products showed comparable molecular weight distributions when determined by gel permeation chromatography (GPC). The observed minor differences between brand and generic SFG, such as sedimentation coefficient, do not impact their biological activities in separate studies of in vitro cellular uptake and rat biodistribution. Coupled with the ongoing clinical study comparing the labile iron level in healthy volunteers, the FDA-funded post-market studies intended to illustrate comprehensive surveillance efforts ensuring safety and efficacy profiles of generic SFG complex in sucrose injection, and also to shed new light on the approval standards on generic parenteral iron colloidal products.

Simple and rapid quantification of brominated vegetable oil in commercial soft drinks by LC-MS.

We report here a simple and rapid method for the quantification of brominated vegetable oil (BVO) in soft drinks based upon liquid chromatography-electrospray ionization mass spectrometry. Unlike previously reported methods, this novel method does not require hydrolysis, extraction or derivatization steps, but rather a simple "dilute and shoot" sample preparation. The quantification is conducted by mass spectrometry in selected ion recording mode and a single point standard addition procedure. The method was validated in the range of 5-25µg/mL BVO, encompassing the legal limit of 15µg/mL established by the US FDA for fruit-flavored beverages in the US market. The method was characterized by excellent intra- and inter-assay accuracy (97.3-103.4%) and very low imprecision [0.5-3.6% (RSD)]. The direct nature of the quantification, simplicity, and excellent statistical performance of this methodology constitute clear advantages in relation to previously published methods for the analysis of BVO in soft drinks.

Human Sulfotransferases Enhance the Cytotoxicity of Tolvaptan.

Tolvaptan, a vasopressin receptor 2 antagonist used to treat hyponatremia, has recently been reported to be associated with liver injury. Sulfotransferases (SULTs) have been implicated as important detoxifying and/or activating enzymes for numerous xenobiotics, drugs, and endogenous compounds. To characterize better the role of SULTs in tolvaptan metabolism, HEK293 cells stably overexpressing 12 human SULTs were generated. Using these cell lines, the extent of tolvaptan sulfate formation was assessed by reversed-phase high-performance liquid chromatography through comparison to a synthetic standard. Of the 12 known human SULTs, no detectable sulfation of tolvaptan was observed with SULT1A1, SULT1A2, SULT1A3, SULT1C2, SULT1C4, SULT4A1, or SULT6B1. The affinity of individual SULT isozymes, as determined by Km analysis, was SULT1C3 >> SULT2A1 > SULT2B1 ∼ SULT1B1 > SULT1E1. The half inhibitory concentration of tolvaptan on cell growth in HEK293/SULT1C3 cells and HEK293/CYP3A4 & SULT1C3 cells was significantly lower than that in the corresponding HEK293/vector cells or HEK293/CYP3A4 & SULT vector cells. Moreover, exposing cells to tolvaptan in the presence of cyclosporine A, an inhibitor of the drug efflux transporters, significantly increased the intracellular levels of tolvaptan sulfate and decreased the cell viability in HEK293/SULT1C3 cells. These data indicate that sulfation increased the cytotoxicity of tolvaptan.

Direct analysis in real time (DART) mass spectrometry of nucleotides and nucleosides: elucidation of a novel fragment [C5H5O]+ and its in-source adducts.

Direct analysis in real time (DART) mass spectrometry is a recently developed innovative technology, which has shown broad applications for fast and convenient analysis of complex samples. Due to the ease of sample preparation, we have recently initiated an investigation of the feasibility of detecting nucleotides and nucleosides using the DART-AccuTOF instrument, which we will refer to as the DART mass spectrometer. Our experimental results reveal that the ions representing the intact molecules of nucleotides are not detectable in either positive-ion or negative-ion mode. Instead, all four natural nucleotides fragment in the DART ion source, and a common fragment ion, [C(5)H(5)O](+) (1), is observed, which is probably formed via multiple-elimination reactions. Interestingly, 1 can form adducts with nucleobases in different molar ratios in the DART ion source. In contrast to nucleotides, the ions representing the intact molecules of nucleosides are detected in both positive-ion and negative-ion mode using DART mass spectrometry. Surprisingly, the fragmentation pattern of nucleosides is different from that of nucleotides in the DART ion source. In the cases of nucleosides (under positive-ion conditions), the production of 1 is not observed, indicating that the phosphate group plays an important role for the multiple eliminations observed in the spectra of nucleotides. The in-source reactions described in the present work show the complexity of the conditions in the DART ion source, and we hope that our results illustrate a better understanding about DART mass spectrometry.