Nerve lesioning with direct current.

Spastic hypertonus (muscle over-activity due to exaggerated stretch reflexes) often develops in stroke and spinal cord injury (SCI) survivors and individuals who suffer from multiple sclerosis. In previous published experiments we have shown that Direct Current (DC), when used to lesion nerves, can attenuate muscle force in a gradual manner, and this attenuation can last for several months. In this paper we present initial experimental results that profile the current required to cause controlled nerve ablation.

Allele frequencies of six miniSTR loci of three ethnic populations in Singapore.

MiniSTR loci has demonstrated to be an effective approach to recover genetic information from degraded sample, due to the improved PCR efficiency of their reduced PCR product sizes. This study investigated the allele frequency of six miniSTR loci, D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045, in three Singapore populations. All loci showed a moderate degree of polymorphism with observed heterozygosity >0.6 for all three populations. The allele frequencies, forensic parameters and heterozygosity comparison with other CODIS STR in similar populations are presented.

Two new terpenoid glucosides from Aster flaccidus.

Two new terpenoid glucosides, namely 2-O-beta-D-glucopyranoside-vicodiol (1) and 10-O-beta-D-glucopyranoside-oplopanone (2), along with seven known compounds, were isolated from the aerial part of Aster flaccidus (composite), a traditional Chinese herb medicine. The structures of 1 and 2 were established by spectroscopic methods, especially 2D NMR experiments.

Formation of unusual glutamate conjugates of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogs: the role of gamma-glutamyltranspeptidase in the biotransformation of benzylamines.

The role of gamma-glutamyltranspeptidase (GGT) in transferring glutamate from endogenous glutathione (GSH) to the benzylamine moiety of a compound, such as 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423), is described. Studies were performed with structurally related analogs of DPC 423 to demonstrate that this type of reaction was common to compounds possessing a benzylamine group. Synthesizing appropriate standards and confirming by liquid chromatography (LC)/mass spectroscopy and LC/NMR made unambiguous assignments of the structures of glutamate conjugates of DPC 423. The use of stable isotope-labeled GSH for metabolism studies has not been described before. In the present study, we report the novel use of deuterated GSH in conjunction with mass spectral analysis to demonstrate the glutamate transfer to the benzylamines in the presence of GGT. To further demonstrate that the alpha protons on the benzylamines and glutamate (as part of glutathione) were unaffected during the transpeptidation, these protons were replaced with deuterium. Acivicin (AT-125), a potent and selective inhibitor of GGT, was used to abolish the formation of the glutamate conjugates of DPC 423 in vitro and in vivo. This provided further evidence of the role of GGT in forming the glutamate conjugates of benzylamines. This study demonstrated conclusively that GGT was responsible for mediating the transfer of glutamic acid from GSH to the benzylamine moiety of a series of structurally related compounds.

The species-dependent metabolism of efavirenz produces a nephrotoxic glutathione conjugate in rats.

Efavirenz, a potent nonnucleoside reverse transcriptase inhibitor widely prescribed for the treatment of HIV infection, produces renal tubular epithelial cell necrosis in rats but not in cynomolgus monkeys or humans. This species selectivity in nephrotoxicity could result from differences in the production or processing of reactive metabolites, or both. A detailed comparison of the metabolites produced by rats, monkeys, and humans revealed that rats produce a unique glutathione adduct. The mechanism of formation and role of this glutathione adduct in the renal toxicity were investigated using both chemical and biochemical probes. Efavirenz was labeled at the methine position on the cyclopropyl ring with the stable isotope deuterium, effectively reducing the formation of the cyclopropanol metabolite, an obligate precursor to the glutathione adduct. This substitution markedly reduced both the incidence and severity of nephrotoxicity as measured histologically. Further processing of this glutathione adduct was also important in producing the lesion and was demonstrated by inhibiting gamma-glutamyltranspeptidase with acivicin pretreatment (10 mg/kg, IV) prior to dosing with efavirenz. Again, both the incidence and severity of the nephrotoxicity were reduced, such that four of nine rats given acivicin were without detectable lesions. These studies provide compelling evidence that a species-specific formation of glutathione conjugate(s) from efavirenz is involved in producing nephrotoxicity in rats. Mechanisms are proposed for the formation of reactive metabolites that could be responsible for the renal toxicity observed in rats.

Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), in rats. Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne.

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.

Disposition of glutathione conjugates in rats by a novel glutamic acid pathway: characterization of unique peptide conjugates by liquid chromatography/mass spectrometry and liquid chromatography/NMR.

With the advent of liquid chromatography/mass spectrometry and liquid chromatography/NMR, it has become easier to characterize metabolites that were once difficult to isolate and identify. These techniques have enabled us to uncover the existence of an alternate pathway for the disposition of glutathione adducts of several structurally diverse compounds. Studies were carried out using acetaminophen as a model compound to investigate the role of the glutamic acid pathway in disposition of the glutathione adducts. Although the mercapturic acid pathway was the major route of degradation of the glutathione adducts, it was found that the conjugation of the glutathione, cysteinylglycine, and cysteine adducts of acetaminophen with the gamma-carboxylic acid of the glutamic acid was both interesting and novel. The coupling of the glutathione adduct and the products from the mercapturic acid pathway with the glutamic acid led to unusual peptide conjugates. The natures of these adducts were confirmed unequivocally by comparisons with synthetic standards. This pathway (addition of glutamic acids) led to larger peptides, in contrast to the mercapturic acid pathway, in which the glutathione adducts are broken down to smaller molecules. The enzyme responsible for the addition of glutamic acid to the different elements of the mercapturic acid pathway is currently unknown. It is postulated that the gamma-carboxylic acid is activated (perhaps by ATP) before enzymatic addition to the alpha-amino group of cysteine or glutamate takes place. The discovery of these peptide conjugates of acetaminophen represents a novel disposition of glutathione adducts of compounds. The formation of such conjugates may represent yet another pathway by which drugs could produce covalent binding via their reactive intermediates.

Identification and characterization of efavirenz metabolites by liquid chromatography/mass spectrometry and high field NMR: species differences in the metabolism of efavirenz.

Efavirenz (Sustiva, Fig. 1) is a potent and specific inhibitor of HIV-1 reverse transcriptase approved for the treatment of HIV infection. To examine the potential differences in the metabolism among species, liquid chromatography/mass spectrometry profiles of efavirenz metabolites in urine of rats, guinea pigs, hamsters, cynomolgus monkeys, and humans were obtained and compared. The metabolites of efavirenz were isolated, and structures were determined unequivocally by mass spectral and NMR analyses. Efavirenz was metabolized extensively by all the species as evidenced by the excretion of none or trace quantities of parent compound in urine. Significant species differences in the metabolism of efavirenz were observed. The major metabolite excreted in the urine of all species was the O-glucuronide conjugate (M1) of the 8-hydroxylated metabolite. Efavirenz was also metabolized by direct conjugation with glucuronic acid, forming the N-glucuronide (M2) in all five species. The sulfate conjugate of 8-OH efavirenz (M3) was found in the urine of rats and cynomolgus monkeys but not in humans. In addition to the aromatic ring-hydroxylated products, metabolites with a hydroxylated cyclopropane ring (at C14) were also isolated. GSH-related products of efavirenz were identified in rats and guinea pigs. The cysteinylglycine adduct (M10), formed from the GSH adduct (M9), was found in significant quantities in only rat and guinea pig urine and was not detected in other species. In vitro metabolism studies were conducted to show that the GSH adduct was produced from the cyclopropanol intermediate (M11) in the presence of only rat liver and kidney subcellular fractions and was not formed by similar preparations from humans or cynomolgus monkeys. These studies indicated the existence of a specific glutathione-S-transferase in rats capable of metabolizing the cyclopropanol metabolite (M11) to the GSH adduct, M9. The biotransformation pathways of efavirenz in different species were proposed based on some of the in vitro results.

Liquid chromatography/mass spectrometry and high-field nuclear magnetic resonance characterization of novel mixed diconjugates of the non-nucleoside human immunodeficiency virus-1 reverse transcriptase inhibitor, efavirenz.

Efavirenz (Sustiva) is a potent and specific inhibitor of the HIV-1 reverse transcriptase and is approved for the treatment of HIV infection. The metabolism of efavirenz in different species has been described previously. Efavirenz is primarily metabolized in rats to the glucuronide conjugate of 8-OH efavirenz. Electrospray ionization-liquid chromatography/mass spectrometry analyses of bile samples from rats dosed with either efavirenz or with 8-OH efavirenz revealed three polar metabolites, M9, M12, and M13, with pseudomolecular ions [M-H](-) at m/z 733, 602, and 749, respectively. The characteristic mass spectral fragmentation patterns obtained for metabolites M9 and M13 suggested that these were glutathione-sulfate diconjugates, and the presence of a glutathione moiety in metabolite M9 was confirmed by liquid chromatograpy/nuclear magnetic resonance (NMR) analysis of bile extracts. Metabolite M12 was characterized by liquid chromatography/mass spectrometry as a glucuronide-sulfate diconjugate. Unambiguous structures of M9, M12, and M13 were obtained from one-dimensional proton and carbon NMR as well as proton-proton (correlated spectroscopy, two-dimensional shift correlation), proton-carbon heteronuclear multiple quantum correlation, and long-range proton-carbon (heteronuclear multiple bond correlation) correlated two-dimensional NMR analyses of metabolites isolated from rat bile. The mass spectral and NMR analyses of M10, which was isolated from rat urine, suggested a cysteinylglycine-sulfate diconjugate. The isolation of these polar metabolites for further characterization by NMR was aided by mass spectral analyses of HPLC fractions and solid phase extraction extracts during the isolation steps. The complete characterization of these novel diconjugates demonstrates that further phase II metabolism of polar conjugates such as sulfates could take place in vivo.

Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration.

The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichlorofluorescein were excreted into and concentrated in the bile canalicular lumen of day 5 sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca(2+)-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca(2+)-free buffer. The cumulative uptake of [(3)H]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca(2+)-free buffer, due to accumulation of taurocholate in canalicular spaces. When [(3)H]taurocholate was preloaded in the day 5 sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca(2+)-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from approximately 8% at day 0 to approximately 60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.

Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration.

PURPOSE: This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. METHODS: Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. RESULTS: By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. CONCLUSIONS: A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

Discovery of bioavailable inhibitors of secretory phospholipase A2.

Substrate-mimetic inhibitors of sPLA2 with submicromolar in vitro potency were discovered by use of a novel dual substrate screening strategy. In vivo evaluation of selected inhibitors in the rat carrageenan paw edema model of inflammation, however, indicated that in vitro potency was not a good predictor of in vivo activity. Studies of the metabolic stability of early examples of these inhibitors suggested that the metabolic lability of these compounds was a major contributing factor to the observed weak in vivo activity. In an attempt to achieve improved in vivo activity, we prepared and tested compounds designed to overcome the observed metabolic instability. The design of the new compounds involved two types of changes in the inhibitor molecules. First, the C-2 ester moiety was replaced with an amide function so that direct cleavage by stomach acid and blood esterases at this site was minimized. Second, omega-oxidation of the decanamide moiety was eliminated by substitution of hydrogen with fluorine in this position. Compounds containing fluorine in the terminal positions of the alkyl chain retained sPLA2 inhibitory activity and also possessed improved in vitro metabolic stability and pharmacokinetic parameters relative to nonfluorinated inhibitors in this series. As exemplified by GW 4776, improvements in metabolic stability alone, however, were not sufficient to ensure oral activity. Thus, GW 4776 did not show oral activity in the carrageenan edema model and had only modest activity after i.v. dosing in the same model. In fact, the results for GW 9624 and GW 8219 suggested that factors in addition to potency of sPLA2 inhibition and metabolism affect the observed in vivo activity. Despite the fact that these two compounds varied only by a single oxygen-to-sulfur substitution, one was active whereas the other was not. One possible explanation for the observed variability is a compound-dependent difference in the rate of equilibration into tissue. This possibility is relevant as both the carrageenan paw edema model and the phorbol ester edema model involve a localized inflammation. No measurements were made to assess differences in the distribution of the different inhibitors between the blood and the localized site of inflammation. In summary, a series of bioavailable inhibitors of sPLA2 was prepared using an iterative approach that combined medicinal chemistry, in vitro and in vivo evaluation of biological activity, and metabolic and pharmacokinetic studies. Although some compounds in the series showed in vivo activity, the anti-inflammatory effect observed in animal models was modest and a decision was made to abandon sPLA2 as a molecular target for the development of anti-inflammatory agents.

Modulation of the tight junctions of the Caco-2 cell monolayers by H2-antagonists.

PURPOSE: The tight junctions in the intestinal epithelium represent highly specialized intercellular junctions. Ranitidine, an H2-antagonist, causes a tightening of the tight junctions. Hence, we have investigated the effect of ranitidine and other H2-antagonists on the function of the intestinal tight junctions. METHODS: Effect of the H2-antagonists on the tight junctions has been investigated using the transepithelial electrical resistance (TEER) and the transport of mannitol across the Caco-2 cell monolayers. RESULTS: Four different H2-antagonists caused an increase in the TEER across the Caco-2 cell monolayers, accompanied by a decrease in the permeability for mannitol. The effect was concentration-dependent and saturable. Ranitidine and famotidine, caused a decrease in their own transport rate across the Caco-2 cells. Ranitidine competitively inhibited the increase in TEER caused by famotidine, whereas compounds which represent molecular fragments of ranitidine had no effect. The relative potency of the four H2-antagonists in causing an increase in the TEER correlated inversely with the oral bioavailability of these compounds in humans. CONCLUSIONS: We hypothesize that the H2-antagonists exert their effect on the tight junctions of Caco-2 cells by modulation of interactions among proteins associated with the tight junctional complex.

CYP3A-like cytochrome P450-mediated metabolism and polarized efflux of cyclosporin A in Caco-2 cells.

Transport of cyclosporin A (CsA) across Caco-2 cells is modulated by its directional efflux, mediated by a p-glycoprotein-like pump (Augustijns et al., Biochem. Biophys. Res. Comm. 197:360-365, 1994). In addition to this unidirectional flux, oxidative metabolism of CsA by cytochrome P450 is likely to influence the absorption of this cyclic peptide across intestinal mucosa. Thus, metabolism of CsA in the in vitro Caco-2 cell culture system was investigated. Formation of several metabolites was observed during the course of CsA transport across Caco-2 cell monolayers. Results from LC/MS/MS experiments revealed that the major metabolite was 1eta-hydroxy CsA (M-17), one of the three major metabolites produced by CYP3A4 present in both the liver and small intestine in humans. Preincubation of Caco-2 cell monolayers with troleandomycin, a specific inhibitor for the microsomal CYP3A protein, reduced the formation of the metabolite M-17, suggesting that an enzyme that functionally resembles CYP3A is responsible for the formation of this metabolite. However, formation of only the M-17 metabolite suggests that the isozyme present in the Caco-2 cells is distinct from CYP3A4, which also catalyzes the formation of significant quantities of the metabolites 9gamma-hydroxy cyclosporin A (M-1) and 4N-desmethyl cyclosporin A (M-21) from CsA. Interestingly, the amount of M-17 accumulating on the apical (AP) side was much greater than that on the basolateral (BL) side during the AP --> BL transport of CsA across Caco-2 cell monolayers. This is consistent with p-glycoprotein pump-mediated efflux of the metabolite to the apical side. Furthermore, formation of the M-17 metabolite on the AP side of cell monolayers during the AP --> BL transport of CsA was much greater than that during the BL --> AP transport. This result suggests that the p-glycoprotein efflux pump causes an increase in the metabolism of CsA during the course of its AP --> BL transport by effectively slowing down the transport of CsA molecules across Caco-2 cells. Thus, Caco-2 cells serve as an excellent model to dissect the relative roles played by p-glycoprotein-mediated efflux and CYP3A-catalyzed oxidation in modulating the overall absorption of CsA and other such compounds.

High-performance liquid chromatographic purification, optimization of the assay, and properties of ribonucleoside diphosphate reductase from rabbit bone marrow.

Partial purification of ribonucleoside diphosphate reductase from rabbit bone marrow was achieved by size exclusion HPLC of the crude homogenate. This step, requiring < 15 min, led to 9- to 13-fold purification of the reductase and removal of 64% of the contaminating kinase/phosphatase activities, which in the crude extract degrade > 95% of substrate CDP when reductase is assayed. A systematic study was conducted to evaluate the influence of contaminating kinase/phosphatase activities on CDP concentration during the reductase-catalyzed reaction with either ATP or its kinase-inhibiting analog, 5'-adenylylimidodiphosphate (AMP-PNP), as the allosteric effector. Our studies demonstrated that in the presence of ATP, CDP levels fell instantly to < 24% but thereafter remained fairly constant due to recycling via CTP. In contrast, in the presence of AMP-PNP, CDP levels decreased continuously. The Km values of the reductase for CDP determined in the presence of ATP were significantly higher than those in the presence of AMP-PNP. Furthermore, we also found that the concentration of the ultimate electron donor dithiothreitol (DTT) required for optimum activity of the reductase varies significantly with the level of purity of the reductase preparation. Interestingly, DTT is an inhibitor of the reductase above the optimum concentration. This purification method and the optimized assay together with the understanding of the fate of CDP in partially purified preparations should find application in studies with reductases from other eukaryotic sources.

Evidence for predominantly paracellular transport of thyrotropin-releasing hormone across CACO-2 cell monolayers.

Uptake, transport, and metabolism of tripeptide thyrotropin-releasing hormone were examined in the human intestinal epithelial cell line, Caco-2. A linear relationship between rate and concentration was observed for both the uptake and the transport of thyrotropin-releasing hormone across Caco-2 cell monolayers. Transport of thyrotropin-releasing hormone was not affected by the presence of dipeptide glycylsarcosine, amino acid glycine, tripeptide thyrotropin-releasing hormone free acid as well as active transport inhibitors 2,4-dinitrophenol, sodium azide, ouabain, and amiloride. There was no formation of metabolites during the course of thyrotropin-releasing hormone transport across Caco-2 cells. Incubation of Caco-2 cell homogenate with thyrotropin-releasing hormone, however, showed a time-dependent hydrolysis of thyrotropin-releasing hormone and the formation of thyrotropin-releasing hormone free acid. Increased rate of transport in the presence of EDTA indicates a paracellular passive diffusion as the major route for the transport of TRH. The hydrolytic enzyme present in Caco-2 cells appeared to have little or no access to TRH during the transcellular transport across Caco-2 cell monolayers.

Mechanism of intestinal absorption of ranitidine and ondansetron: transport across Caco-2 cell monolayers.

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability co-efficients (Papp) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. Papp values for ranitidine and ondansetron (bioavailability of 50 and approximately 100% in humans, respectively) were 1.03 +/- 0.17 x 10(-7) and 1.83 +/- 0.055 x 10(-5) cm/sec, respectively. The Papp value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.

Evidence for a polarized efflux system in CACO-2 cells capable of modulating cyclosporin A transport.

The characteristics of cyclosporin A (CsA) transport across Caco-2 monolayers were investigated. CsA (0.25-5.0 microM) was transported in a time and concentration dependent manner. The total amount of apical (AP) to basolateral (BL) transport was non-linearly related to CsA concentration from 0.25 to 1 microM and was linear from about 1 to 5 microM. Average permeability coefficient (Papp) values obtained in the AP to BL direction showed CsA concentration (0.5 and 5.0 microM) dependence, whereas those of the reverse (BL to AP) process did not. Papp values for the AP to BL direction were also markedly lower. When the P-glycoprotein pump inhibitors, chlorpromazine and progesterone, were included in the transport medium we observed a significant increase in CsA (0.5 and 5.0 microM) transport from the AP to BL direction; transport was decreased in the reverse direction. This study suggests that CsA is transported across Caco-2 cells by passive diffusion, but that a polarized efflux system (presumably a P-glycoprotein pump) located at the apical membrane can attenuate the net AP to BL transport.

Gas chromatographic-mass spectrometric analysis of diols and tetrols from reactions of polycyclic aromatic hydrocarbon epoxides with hemoglobin.

We have evaluated both electron ionization (EI) and negative-ion chemical ionization (NICI) methods for the analysis of trimethylsilyl derivatives of a series of polycyclic aromatic hydrocarbon (PAH) alcohols including styrene diol, benzo[e]pyrene diol and tetrols, cyclopenta[c,d]pyrene diols, benzo[a]pyrene-4,5-diols, chrysene tetrols, benz[a]anthracene tetrols I and II, and syn- and anti-benzo[a]pyrene tetrols. NICI is the more sensitive method for all compounds except styrene diol. Detection limits are compound-dependent and range from 1 fmol for cyclopenta[c,d]pyrene diol to 1 pmol for benzo[e]pyrene diol. The EI detection limit for styrene diol is 60 fmol. PAH alcohols related to the compounds listed above were observed following hydrolysis of hemoglobin which had been reacted with PAH epoxides in vitro. Benzo[a]pyrene tetrols and a chrysene tetrol were observed following hydrolysis of hemoglobin isolated from human smokers' blood. Hydrolysis of styrene oxide treated hemoglobin in 18O-labeled water revealed at least two mechanisms of ester hydrolysis, including the BAL 1 pathway.