Interference of carbamazepine and carbamazepine 10,11-epoxide in the fluorescence polarization immunoassay for tricyclic antidepressants: estimation of the true tricyclic antidepressant concentration in the presence of carbamazepine using a mathematical model.
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We evaluated effects of carbamazepine and its metabolite, carbamazepine 10,11-epoxide, on the measurement of tricyclic antidepressant (TCA) concentrations in serum using the fluorescence polarization immunoassay (FPIA). We determined apparent TCA concentrations in 30 patients who were receiving carbamazepine but no TCAs. Carbamazepine concentrations ranged from 1.4 to 20.9 microg/mL (5.9-88.4 micromol/L); the observed apparent TCA concentrations ranged from 31.8 to 130.1 ng/mL (113.4-463.9 micromol/L). When aliquots of the drug-free serum pool were supplemented with known concentrations of carbamazepine or its metabolite, we observed significant apparent TCA concentrations using the FPIA; however, interference of carbamazepine was more than 3-fold more than its metabolite. When serum pools prepared from patients receiving TCA but no anticonvulsant medications were supplemented with known amounts of carbamazepine or its metabolite, we observed falsely elevated TCA concentrations. We formulated an equation to calculate the apparent TCA concentration from known carbamazepine concentrations. If carbamazepine and TCAs are present in a specimen, the true TCA concentration can be estimated by subtracting the calculated TCA concentration (due to carbamazepine) from the observed TCA concentration as measured by the TCA FPIA. This mathematical modeling is feasible because TCAs, even at very high concentrations, showed no interference with the carbamazepine FPIA. (+info)
Membrane sphingolipid-ergosterol interactions are important determinants of multidrug resistance in Candida albicans.
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In this study, we examined the importance of membrane ergosterol and sphingolipids in the drug susceptibilities of Candida albicans. We used three independent methods to test the drug susceptibilities of erg mutant cells, which were defective in ergosterol biosynthesis. While spot and filter disk assays revealed that erg2 and erg16 mutant cells of C. albicans became hypersensitive to almost all of the drugs tested (i.e., 4-nitroquinoline oxide, terbinafine, o-phenanthroline, itraconazole, and ketoconazole), determination of the MIC at which 80% of the cells were inhibited revealed more than fourfold increase in susceptibility to ketoconazole and terbinafine. Treatment of wild-type C. albicans cells with fumonisin B1 resulted in 45% inhibition of sphingolipid biosynthesis and caused cells to become hypersensitive to the above drugs. Although erg mutants displayed enhanced membrane fluidity and passive diffusion, these changes alone were not sufficient to elicit the observed hypersusceptibility phenotype of erg mutants. For example, the induction in vitro of a 12% change in the membrane fluidity of C. albicans cells by a membrane fluidizer, benzyl alcohol, did not affect the drug susceptibilities of Candida cells. Additionally, the surface localization of green fluorescent protein-tagged Cdr1p, a major drug efflux pump protein of C. albicans, revealed that any disruption in ergosterol and sphingolipid interactions also interfered with its proper surface localization and functioning. A 50% reduction in the efflux of the Cdr1p substrate, rhodamine 6G, in erg mutant cells or in cells with a reduced sphingolipid content suggested a strong correlation between these membrane lipid components and this major efflux pump protein. Taken together, the results of our study demonstrate for the first time that there is an interaction between membrane ergosterol and sphingolipids, that a reduction in the content of either of these two components results in a disruption of this interaction, and that this disruption has deleterious effects on the drug susceptibilities of C. albicans cells. (+info)
Analytical performance of the CEDIA cyclosporine PLUS whole blood immunoassay.
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Cyclosporine A (CsA) is a potent immunosuppressive agent used in solid organ and bone marrow transplantation. Because of the narrow therapeutic range and variable pharmacokinetics, blood levels of CsA are routinely monitored. The performance of the CEDIA CsA PLUS whole blood immunoassay was evaluated on the Olympus AU400 trade mark, and results were compared to those obtained by high-performance liquid chromatography (HPLC), enzyme-multiplied immunoassay technique (EMIT), and fluorescence polarization immunoassay (FPIA). A total of 592 whole blood samples from patients receiving CsA were tested by each of the assays. CEDIA was linear from 25 to 2000 micro g/L. Total imprecision ranged from 2.7% to 8.7% at CsA values between 48 and 1502 micro g/L. Recovery of added CsA was within 10% of assigned values and was unaffected by bilirubin and lipemia. Metabolite cross-reactivity at 500 micro g/L was 8.1% for AM1, 21.7% for AM4n, and 32.5% for AM9. Regression analysis revealed the following: HPLC = 0.93. CEDIA - 21.2 (r = 0.975), EMIT = 1.08. CEDIA - 25.2 (r = 0.982), and FPIA = 1.14. CEDIA + 13.4 (r = 0.984). CEDIA has acceptable analytical performance for routine CsA monitoring. Advantages are the absence of an extraction step and an extended measuring range. The disadvantage is the high metabolite cross-reactivity; however, results were similar to EMIT (+info)
A combined HPLC-immunoenzymatic comprehensive screening for suspected drug poisoning in the emergency department.
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OBJECTIVE: To review the results of a comprehensive drug screening as first line diagnostic tool in patients attending an emergency department for suspected drug poisoning. METHODS: A comprehensive drug screening was carried out in plasma or urine, or both, of 310 patients combining an HPLC multidrug profiling system and a fluorescence polarisation immunoassay. RESULTS: In 64.2% of cases the screening confirmed the diagnosis of drug poisoning, in 13.9% suspected drugs were measurable at non-toxic concentrations, and in 21.9% no drugs were found. The suspected drugs were fully confirmed in a minority of cases, (symptomatic patients: 28.2% compared with asymptomatic: 16.5%). Symptomatic patients were less likely to have at least one suspected drug (29.6% compared with 57.7%; p<0.001), and more likely to have at least one unsuspected drug found at analysis (17.4% compared with 3.1%; p = 0.005). In 5% of patients, asymptomatic when first observed, one or more unsuspected drugs were found. In 6 of 29 patients, with suspected poisoning of an unspecified drug, the screening identified the specific drug and excluded acute intoxication in the remaining cases. CONCLUSION: A rapid comprehensive drug screening adds to the diagnosis of patients with suspected drug poisoning, identifying unsuspected drugs in symptomatic patients and excluding drugs in asymptomatic subjects. (+info)
Identification of intermediates in the bile acid synthetic pathway as ligands for the farnesoid X receptor.
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Bile acid synthesis from cholesterol is tightly regulated via a feedback mechanism mediated by the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids. Synthesis via the classic pathway is initiated by a series of cholesterol ring modifications and followed by the side chain cleavage. Several intermediates accumulate or are excreted as end products of the pathway in diseases involving defective bile acid biosynthesis. In this study, we investigated the ability of these intermediates to activate human FXR. In a cell-based reporter assay and coactivator recruitment assays in vitro, early intermediates possessing an intact cholesterol side chain were inactive, whereas 26- or 25-hydroxylated bile alcohols and C27 bile acids were highly efficacious ligands for FXR at a level comparable to that of the most potent physiological ligand, chenodeoxycholic acid. Treatment of HepG2 cells with these precursors repressed the rate-limiting cholesterol 7alpha-hydroxylase mRNA level and induced the small heterodimer partner and the bile salt export pump mRNA, indicating the ability to regulate bile acid synthesis and excretion. Because 26-hydroxylated bile alcohols and C27 bile acids are known to be evolutionary precursors of bile acids in mammals, our findings suggest that human FXR may have retained affinity to these precursors during evolution. (+info)
Delivery of gentamicin to the rabbit eye by drug-loaded hydrogel iontophoresis.
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PURPOSE: To assess the corneal iontophoretic delivery of gentamicin by drug-loaded hydrogel probe, and to determine the resultant ocular disposition and elimination of the drug from the cornea and anterior chamber. METHODS: Corneal iontophoresis of gentamicin sulfate was studied in healthy white rabbits by using drug-loaded disposable hydroxyethyl methacrylate (HEMA) hydrogel disk probes and a portable mini-ion device designed in the authors' laboratory. The iontophoretic treatment was performed with a current intensity of 1 mA for 60 seconds only. Three control groups were used: mock iontophoresis (no current) for 60 seconds, topical eye drops of fortified gentamicin (1.4%) every 5 minutes for 1 hour, and subconjunctival injection of 0.25 mL of 40 mg/mL gentamicin solution. The animals in the iontophoretic experimental groups were killed at predetermined time points. The gentamicin concentrations in the cornea and aqueous humor were assayed with a fluorescence polarization immunoassay. Analysis of the gentamicin eye pharmacokinetics was performed with a modeling approach. RESULTS: Peak gentamicin concentrations in the cornea (363.1 +/- 127.3 microg/g) and in the aqueous humor (29.4 +/- 17.4 microg/mL) were reached at 0 and 2 hours after the iontophoretic treatment, respectively. The peak gentamicin concentrations after a single iontophoresis treatment were 12 to 15 times higher than those obtained after gentamicin injection or after topical eye drop instillation, and much higher than in mock iontophoresis. The concentration versus time profile of gentamicin in the cornea and the anterior chamber after iontophoresis was appropriately described by applying a two-compartment pharmacokinetic model. CONCLUSIONS: A short iontophoretic treatment using gentamicin-loaded hydrogels has potential clinical value in increasing drug penetration to the anterior segments of the eye and maintaining therapeutic drug levels in the cornea for more than 8 hours. (+info)
Pharmacokinetics of lidocaine delivered from a transmucosal patch in children.
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The DentiPatch lidocaine transoral delivery system (Noven Pharmaceuticals) is indicated for mild topical anesthesia of mucosal membranes in the mouth. The DentiPatch is a mucoadhesive patch containing 46.1 mg of lidocaine (20% concentration). Current studies in adults report that DentiPatch application produces very low plasma concentrations of lidocaine. However, it is not known what plasma levels are obtained when the same dosage is used in children. The purpose of this study was to determine whether the plasma lidocaine concentrations generated by the DentiPatch are within a safe range for children. The sample in this study was 11 children aged 2-7 years requiring general anesthesia for comprehensive dental care. A lidocaine DentiPatch was placed on the buccal mucosa above the maxillary incisors for 5 minutes. Blood samples were drawn before placing the DentiPatch and at various time intervals after removing it. Blood samples were analyzed by fluorescence polarization immunoassay to determine the plasma concentrations of lidocaine and its major metabolite, monoethylglycinexylidide. The lidocaine and monoethylglycinexylidide absorbed from the DentiPatch did not reach toxic plasma levels in children. However, plasma concentrations were much higher than in adults and were high enough to require inclusion in the calculation of total lidocaine administered to a pediatric patient. (+info)
Detection of benzodiazepine intake in therapeutic doses by immunoanalysis of urine: two techniques evaluated and modified for improved performance.
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We evaluated the EMIT (enzyme-multiplied immuno technique) and FPIA (fluorescence polarization immunoassay) urine screening systems for detection of benzodiazepine intake. Healthy male volunteers were given single oral therapeutic doses of alprazolam (2 mg), chlordiazepoxide (25 mg), flunitrazepam (1 mg), lorazepam (3.75 mg), nitrazepam (5 mg), and triazolam (0.25 mg), after which urine was collected for the next 32 h. The EMIT method failed to detect the intake of flunitrazepam, lorazepam, and nitrazepam. FPIA did not detect the intake of chlordiazepoxide, flunitrazepam, lorazepam, nitrazepam, and triazolam. Modification of the EMIT method to include enzymatic hydrolysis did not significantly alter the results obtained with this method. A modification of the FPIA method to include enzymatic hydrolysis and a lower cutoff value improved the results considerably, so that we reliably detected all studied substances but flunitrazepam. We conclude that (a) both EMIT and FPIA techniques, when used as intended by the manufacturers, are unreliable for the detection of intake of therapeutic doses of these benzodiazepines, and (b) the described modification of the FPIA should provide a much improved tool for detection of benzodiazepine intake. (+info)