A rapid and sensitive assay to quantify valproyl 1-O-acyl glucuronide in supernatants of sandwich-cultured rat hepatocytes using ultra-high performance liquid chromatography-tandem mass spectrometry.

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of valproyl-1-O-acyl glucuronide (VPA-G) levels in hepatocyte culture medium. Chromatographic separation was achieved using a Waters Acquity UPLC(®) BEH C18 column (1.7µm, 2.1mm×50mm) with gradient elution and a total run time of 4min. [(2)H6]-VPA-G was used as internal standard (IS). Quantification was performed in the multiple reaction monitoring (MRM) mode using the total ion current of the MRM transition pairs m/z 319.1→142.7 and m/z 319.1→175.2 for VPA-G, and m/z 325.1→149.3 and m/z 325.1→174.9 for the IS under negative electrospray ionization mode. The assay was linear over the VPA-G concentrations of 0.5-500ng/mL, with a r(2) value of 0.995±0.002 (mean±SD). The intra- and inter-day accuracy (% deviation) ranged from -10.2% to 11.1%, whereas the intra- and inter-day precision (% RSD) were ≤7.43%. The method was applied successfully to the quantification of VPA-G levels in culture supernatants of sandwich-cultured rat hepatocytes treated with valproic acid (VPA). No significant difference in the levels of VPA-G over a culture period of 6 days was observed in an experiment that investigated the effect of the age of hepatocyte culture on the extent of VPA glucuronidation. The method presented here for the direct quantification of VPA-G is an improvement of existing methods in the literature and offers a shorter run time and greater sensitivity that enables the use of small volumes of sample. To the best of our knowledge, this is the first validated UHPLC-MS/MS method applied to the quantification of VPA-G in cell culture supernatants.

Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity.

The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role for metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.

Role of oxidative metabolism in the effect of valproic acid on markers of cell viability, necrosis, and oxidative stress in sandwich-cultured rat hepatocytes.

Valproic acid (VPA) is a drug known for idiosyncratic hepatotoxicity and is associated with oxidative stress. It is metabolized extensively with at least one pathway leading to reactive metabolites. The primary aim of the present study was to determine whether oxidative metabolites of VPA generated in situ contribute to the toxicity of the parent drug in sandwich-cultured rat hepatocytes. Concentration-response experiments with VPA produced median effective concentration values (mean ± SEM) of 1.1 ± 0.4, 12.2 ± 1.4, and 12.3 ± 1.9mM in the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1; cell viability), lactate dehydrogenase (LDH; necrosis), and 2',7'-dichlorofluorescein (DCF; oxidative stress) assays, respectively. At equimolar concentrations, only the unsaturated metabolites of VPA gave responses comparable to VPA, with 2,4-diene-VPA calculated to be 3-, 6-, and 10-fold more potent than VPA in the WST-1, LDH, and DCF assays, respectively. In support of a role for reactive metabolites, 2-fluoro-2-propylpentanoic acid, which is relatively resistant to biotransformation to form a 2,4-diene metabolite, yielded little or no toxicity when compared with the nonhepatotoxic octanoic acid or the vehicle-treated control. By comparison, attenuating the in situ formation of 2-propylpent-4-enoic acid (4-ene-VPA), 3-hydroxy-2-propylpentanoic acid, 4-hydroxy-2-propylpentanoic acid, and 5-hydroxy-2-propylpentanoic acid by an inhibitor of cytochrome P450 (1-aminobenzotriazole) did not alter the effects of VPA on the WST-1, LDH, or DCF assay. Overall, VPA toxicity in sandwich-cultured rat hepatocytes is independent of the in situ formation of cytochrome P450-dependent oxidative metabolites, including 4-ene-VPA. However, the data obtained from structural analogues of VPA suggest that biotransformation does appear to play a role in VPA toxicity in rat hepatocytes.

Inhibition of experimental colorectal cancer and reduction in renal and gastrointestinal toxicities by copper-indomethacin in rats.

PURPOSE: To evaluate, for the first time, the efficacy of copper-indomethacin in the inhibition of aberrant crypt foci formation using the azoxymethane-induced adenocarcinoma model, to examine cell viability in the HCT-116 colorectal cancer cell line, gastrointestinal permeability, mitochondrial oxidative damage, and renal toxicity in rat models. METHODS: Azoxymethane-induced adenocarcinoma rats were dosed with indomethacin and copper-indomethacin for 28 days and aberrant crypt foci were evaluated. HCT-116 colorectal cancer cells were exposed to indomethacin and copper-indomethacin at 0-250 microg/mL (0-698 microM for indomethacin, and 0-147 microM for copper-indomethacin), and cell viability was measured. Acute gastrointestinal toxicity was measured using gastrointestinal permeability markers, gastrointestinal ulceration and bleeding, and measurement of an acute-phase protein haptoglobin. Effects of acute and chronic administration of indomethacin and copper-indomethacin on urinary electrolyte concentrations were examined. RESULTS: Both indomethacin and copper-indomethacin resulted in a significant reduction in aberrant crypt foci in azoxymethane-treated rats. In parallel, high concentrations of indomethacin and copper-indomethacin also reduced cell viability in HCT-116 colorectal cancer cells. However, copper-indomethacin was considerably safer in all measures of gastrointestinal toxicity compared to indomethacin. In addition, indomethacin reduced urinary electrolytes at an ulcerogenic dose of 10 mg/kg acutely and chronically at 3.0 mg/kg for 28 days, whereas copper-indomethacin at equimolar doses of indomethacin affected urine electrolytes after acute dosing but not after chronic dosing for 28 days. CONCLUSIONS: Copper-indomethacin has both gastrointestinal and renal sparing properties while maintaining efficacy in experimental adenocarcinoma.

Pharmacokinetics of selected stilbenes: rhapontigenin, piceatannol and pinosylvin in rats.

The pharmacokinetics of piceatannol, pinosylvin and rhapontigenin were characterized in male Sprague-Dawley rats after single intravenous doses of 10 mg kg(-1) of each stilbene. Serial blood samples were collected via a catheter inserted into the right jugular vein and plasma samples were analysed for the selected stilbenes concentrations using reverse phase HPLC methods. After an acute intravenous dose of piceatannol, plasma AUC, urine t(1/2), CL and V(d) were 8.48+/-2.48 micro g h mL(-1), 19.88+/-5.66 h, 2.13+/-0.92 Lh(-1) kg(-1) and 10.76+/-2.88 L kg(-1)(mean+/-s.e.m.), respectively. The acute intravenous dose of pinosylvin yielded the plasma AUC, urine t(1/2), CL and V(d) values of 5.23+/-1.20 micro g h mL(-1), 13.13+/-2.05 h, 1.84+/-0.44 Lh(-1) kg(-1) and 2.29+/-0.56 L kg(-1)(mean+/-s.e.m.), respectively. Rhapontigenin intravenous dosing yielded the plasma AUC, urine t(1/2), CL and V(d) values of 8.39+/-0.10 micro g h mL(-1), 25.31+/-1.46 h, 1.18+/-0.035 Lh(-1) kg(-1) and 11.05+/-0.17 L kg(-1)(mean+/-s.e.m.), respectively. Each stilbene was extensively glucuronidated. These stilbenes were predominantly eliminated via non-urinary routes. All three stilbenes were highly distributed into tissues and were highly extracted by the liver. The detectable plasma half-lives of these xenobiotics appear to be relatively short. However, utilizing urinary concentration-time data, much longer elimination half-lives were evident. The estimates of oral bioavailability characterize these stilbenes as poorly bioavailable compounds.

Oxidative stress in children receiving valproic acid.

OBJECTIVE: To determine whether valproic acid (VPA) influences urinary levels of 15-F2t -isoprostane (15-F2t -IsoP), a marker of oxidative stress, in children. STUDY DESIGN: Morning urine samples were collected from children with epilepsy receiving VPA (n = 25), carbamazepine (n = 16), or clobazam (n = 12) for > or = 4 weeks and from age-matched control subjects (n = 39). Urinary 15-F2t -IsoP levels were determined by enzyme-linked immunosorbent assay. RESULTS: The mean (+/- standard deviation) urine 15-F2t -IsoP levels (nmol/mmol Cr) were: valproic acid (0.36 +/- 0.15); carbamazepine (0.24 +/- 0.10); clobazam (0.23 +/- 0.10); control group (0.20 +/- 0.09). Patients treated with VPA had significantly elevated 15-F2t -IsoP levels when compared with the control, carbamazepine, and clobazam groups (P < .05). Multiple linear regression analysis demonstrated that younger patient age and exposure to second-hand smoke were significant predictors of elevated urine 15-F2t -IsoP levels within the control group (r2 = 0.261, P = .05 and P = .01, respectively). Subjects not exposed to second-hand smoke receiving valproic acid therapy had a significantly elevated mean urine 15-F2t -IsoP level compared to subjects not exposed to second-hand smoke in the carbamazepine, clobazam and control groups (P < .05). CONCLUSIONS: These data demonstrate that treatment of children with VPA is associated with higher urinary levels of 15-F2t -IsoP, a marker of oxidative stress.

Distinct role of bilobalide and ginkgolide A in the modulation of rat CYP2B1 and CYP3A23 gene expression by Ginkgo biloba extract in cultured hepatocytes.

In the present study, primary cultures of rat hepatocytes were treated for 48 h with one of several extracts of Ginkgo biloba (10, 100, or 1000 microg/ml). Maximal increase in CYP2B1 and CYP3A23 mRNA levels was obtained at 100 microg/ml. This concentration of G. biloba extract also increased CYP3A2 and CYP3A18 mRNA expression in addition to CYP2B-mediated 7-benzyloxyresorufin O-dealkylation (BROD) and CYP3A-mediated testosterone 6beta-hydroxylation. In other experiments, cultured hepatocytes were treated for 48 h with bilobalide, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, kaempferol, quercetin, isorhamnetin, or a flavonol diglycoside at a concentration that represented the level present in a 100 microg/ml concentration of an extract. Only bilobalide (2.8 microg/ml) increased CYP2B1 mRNA expression, and the -fold increase (7.9 +/- 0.5; mean +/- S.E.M.) was similar to that (8.3 +/- 1.7) by the extract. By comparison, only ginkgolide A (1.1 microg/ml) increased CYP3A23 mRNA expression, but the extent (2.6 +/- 0.5-fold) was less than the 5.3 +/- 1.7-fold increase by the extract. A greater concentration (5 microg/ml) of ginkgolide A was required to elevate CYP3A2 and CYP3A18 mRNA expression. Over the range of 1 to 5 microg/ml, bilobalide increased CYP2B1 mRNA and BROD, but not CYP3A23 mRNA or testosterone 6beta-hydroxylation, whereas ginkgolide A increased CYP3A23 mRNA and testosterone 6beta-hydroxylation, but not CYP2B1 mRNA or BROD. Overall, our novel results indicate a distinct role of bilobalide and ginkgolide A in the modulation of CYP2B1 and CYP3A23 gene expression and enzyme activities by G. biloba extract in primary cultures of rat hepatocytes.

Pharmacokinetics and tissue distribution of d-alpha-tocopheryl succinate formulations following intravenous administration in the rat.

Unlike d-alpha tocopherol (T), d-alpha tocopheryl succinate (TS) has the unique ability to selectively kill tumor cells while protecting normal tissue from toxic oxidative stress. The pharmacokinetics of TS and the serum and tissue disposition of TS were studied in male Sprague-Dawley rats to delineate formulation dependent disposition between TS administered as the Tris salt (TS-T) (a liposomal formulation) or as the free acid (TS-FA) dissolved in polyethylene glycol (PEG) 400. The pharmacokinetics of TS was studied after single intravenous (i.v.) equimolar doses of 124 mg/kg TS-T and 100 mg/kg of TS-FA. Serial blood samples were collected via a catheter inserted into the right jugular vein and serum samples were analysed for TS and T levels using a reverse phase HPLC method. Terminal tissue samples were also collected at 24 and 48 h. After an acute i.v. dose of TS-T, serum AUC, t(1/2), Cl and V(d) of TS were 2601.0 +/- 351.7 microg h/ml, 9.98 +/- 1.02 h, 0.049 +/- 0. 0073 l/h/kg and 0.7 +/- 0.14 l/kg (mean +/- SD), respectively. The acute i.v. administration of TS-FA (PEG formulation) yielded results similar to those observed for TS-T, with a serum AUC, t(1/2), Cl and V(d) of 2553.3 +/- 166.4 microg h/ml, 9.83 +/- 0.86 h, 0.039 +/- 0.0027 l/h/kg and 0.56 +/- 0.09 l/kg (mean +/- SD), respectively. Distribution into tissues and a low Cl was apparent, with the highest concentrations of TS in the liver and lung, regardless of formulation. As expected, baseline endogenous concentrations of T were present in both groups, with a net increase in T levels, occurring as TS was hydrolysed to T, which slowly peaked in serum between 7-8 h post-dose. Intravenous TS administration, regardless of formulation, also resulted in significant T accumulation in all tissues examined, which was especially abundant in the liver and lung. Likewise, there was a lack of significant effect of formulation on the pharmacokinetics and tissue distribution of TS. The only significant formulation difference was a small but significant increase in serum T and liver T levels in the TS in PEG formulation group. These results indicate that TS may be especially useful for the targeted delivery of T and TS to the lung and liver for anti-oxidant and anti-cancer activity.

Valproic acid glucuronidation is associated with increases in 15-F2t-isoprostane in rats.

Oxidative stress has been associated with valproic acid (VPA) treatment in rats and studies are ongoing to examine the relationship between VPA biotransformation and the increase in the lipid peroxidation biomarker 15-F2t-isoprostane (15-F2t-IsoP). This study investigated the effects of modulating VPA-1-O-acyl glucuronide (VPA-G) formation on 15-F2t-IsoP levels. Adult male Sprague-Dawley rats were pretreated with phenobarbital (PB; 80 mg/kg/day for 4 days), (-)-borneol (320 mg/kg), or a combination of both before VPA treatment (500 mg/kg). Liver VPA-G levels were determined by LC/MS and plasma and liver 15-F2t-IsoP levels were measured using an EIA method. PB, an inducer of VPA glucuronidation, elevated both liver VPA-G and plasma and liver 15-F2t-IsoP levels in VPA-treated rats. (-)-Borneol, an inhibitor of glucuronidation, significantly reduced the levels of liver VPA-G and decreased plasma and liver 15-F2t-IsoP levels in both the VPA and the PB + VPA groups. (-)-Borneol and PB alone did not elevate 15-F2t-IsoP levels compared to the vehicle control groups. The fluorinated analogue of VPA, alpha-fluoro-VPA, was a poor substrate for glucuronidation and did not elevate 15-F2t-IsoP levels. In summary, the VPA-induced formation of 15-F2t-IsoP is apparently associated with VPA glucuronidation.

Valproic acid II: effects on oxidative stress, mitochondrial membrane potential, and cytotoxicity in glutathione-depleted rat hepatocytes.

Oxidative stress has been associated with valproic acid (VPA) treatment, and mitochondrial dysfunction has been implicated in the pathogenesis of VPA-idiosyncratic hepatotoxicity. The present study investigated the effect of VPA and the role of GSH on oxidative stress, mitochondrial membrane potential, and toxicity in freshly isolated rat hepatocytes. Hepatocytes were isolated from Sprague-Dawley rats, and total levels of glutathione (GSH) reduced by pretreatment with a combination of L-buthionine sulfoximine (2 mM) and diethylmaleate (0.5 mM) prior to VPA (0-1000 microg/ml) treatment. Oxidative stress was determined by measuring the levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP) and 2',7'-dichlorofluorescein (DCF). Mitochondrial membrane potential (Deltapsi(m)) was determined by using the dual-fluorescent dye JC-1, and cell viability was evaluated by the water-soluble tetrazolium salt WST-1 assay. Exposure of rat hepatocytes to VPA (0-1000 mug/ml) resulted in a time- and dose-dependent increase in 15-F(2t)-IsoP and DCF fluorescence, and these levels were further elevated in GSH-reduced hepatocytes. In control hepatocytes, VPA had no effect on cell viability; however, significant cytotoxicity was observed in the glutathione-depleted hepatocytes treated with 1000 mug/ml VPA. The Deltapsi(m) was only reduced in glutathione-reduced hepatocytes at 500 and 1000 microg/ml VPA. Our novel findings indicate that acute treatment of freshly isolated rat hepatocytes with VPA resulted in oxidative stress, which occurred in the absence of cytotoxicity, and that glutathione confers protection to hepatocytes against mitochondrial damage by VPA.

Valproic acid I: time course of lipid peroxidation biomarkers, liver toxicity, and valproic acid metabolite levels in rats.

A single dose of valproic acid (VPA), which is a widely used antiepileptic drug, is associated with oxidative stress in rats, as recently demonstrated by elevated levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP). To determine whether there was a temporal relationship between VPA-associated oxidative stress and hepatotoxicity, adult male Sprague-Dawley rats were treated ip with VPA (500 mg/kg) or 0.9% saline (vehicle) once daily for 2, 4, 7, 10, or 14 days. Oxidative stress was assessed by determining plasma and liver levels of 15-F(2t)-IsoP, lipid hydroperoxides (LPO), and thiobarbituric acid reactive substances (TBARs). Plasma and liver 15-F(2t)-IsoP were elevated and reached a plateau after day 2 of VPA treatment compared to control. Liver LPO levels were not elevated until day 7 of treatment (1.8-fold versus control, p < 0.05). Liver and plasma TBARs were not increased until 14 days (2-fold vs. control, p < 0.05). Liver toxicity was evaluated based on serum levels of alpha-glutathione S-transferase (alpha-GST) and by histology. Serum alpha-GST levels were significantly elevated by day 4, which corresponded to hepatotoxicity as shown by the increasing incidence of inflammation of the liver capsule, necrosis, and steatosis throughout the study. The liver levels of beta-oxidation metabolites of VPA were decreased by day 14, while the levels of 4-ene-VPA and (E)-2,4-diene-VPA were not elevated throughout the study. Overall, these findings indicate that VPA treatment results in oxidative stress, as measured by levels of 15-F(2t)-IsoP, which precedes the onset of necrosis, steatosis, and elevated levels of serum alpha-GST.

Stereospecific high-performance liquid chromatographic analysis of hesperetin in biological matrices.

A method of analysis of hesperetin (+/--3,5,7-trihydroxy-4'-methoxyflavanone) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and tissue distribution. A simple high-performance liquid chromatographic method was developed for simultaneous determination of hesperetin enantiomers in rat serum, and rat and human urine. Serum and urine (0.1 ml) were precipitated with cold acetonitrile after addition of the internal standard, 7-methoxycoumarin. Separation was achieved on a Chiralpak AD-RH column with UV detection at 298 nm. The calibration curve was linear ranging from 0.5 to 100 microg/ml for each enantiomer. The mean extraction efficiency was >98%. Precision of the assay was <5% (CV), and was within 5% at the limit of quantitation (0.5 microg/ml). Bias of the assay was lower than 5%, and was within 5% at the limit of quantitation. The assay was applied successfully to the urinary excretion of hesperetin in rats and humans.

Lack of evidence for induction of CYP2B1, CYP3A23, and CYP1A2 gene expression by Panax ginseng and Panax quinquefolius extracts in adult rats and primary cultures of rat hepatocytes.

Treatment of rats with a single oral dose (10-30 mg/kg) of a crude Panax ginseng extract of unknown ginsenoside content has been reported to modestly increase hepatic microsomal cytochrome P450-mediated aminopyrine N-demethylation activity. In the present study, we compared the effect of P. ginseng and Panax quinquefolius extracts on rat hepatic CYP2B1, CYP3A23, and CYP1A2 gene expression. Adult male Sprague-Dawley rats (250-275 g) received, by oral gavage or i.p., P. ginseng extract [4% (w/w) total ginsenosides; 30 or 100 mg/kg/day for 1 or 4 days], P. quinquefolius extract [10% (w/w) total ginsenosides; 100 or 400 mg/kg/day for 21 consecutive days), or an equivalent volume (2 ml/kg) of the vehicle (0.9% NaCl or 0.3% carboxymethylcellulose) and were terminated 1 day after the last dose. P. ginseng and P. quinquefolius extracts did not affect body weight gain, absolute or relative liver weight, hepatic CYP2B1, CYP3A23, or CYP1A2 mRNA expression, or microsomal CYP2B-mediated 7-benzyloxyresorufin O-dealkylation (BROD) or CYP1A-mediated 7-ethoxyresorufin O-dealkylation (EROD) activity. In contrast, results from positive control experiments indicated that phenobarbital increased CYP2B1 mRNA and BROD activity, dexamethasone increased CYP3A23 mRNA, and beta-naphthoflavone increased CYP1A2 mRNA and EROD activity levels. Treatment of primary cultures of rat hepatocytes with either of the ginseng extracts (0.1-1000 microg/ml for 2 days) also did not affect CYP2B1 or CYP3A23 mRNA expression. Overall, our data indicate that P. ginseng and P. quinquefolius extracts do not increase rat hepatic CYP2B1, CYP3A23, or CYP1A2 gene expression.

High-performance liquid chromatographic analysis of a selective cyclooxygenase-1 inhibitor SC-560 in rat serum: application to pharmacokinetic studies.

A method of analysis of SC-560 (5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism. A simple high-performance liquid chromatographic method was developed for simultaneous determination of SC-560 and other products of metabolism in rat serum. Serum (0.1 ml) was precipitated with acetonitrile after addition of the internal standard, testosterone 17-propionate. Separation was achieved on a C(8) column with UV-detection at 240 nm. The calibration curve was linear ranging from 0.02 to 100 microg/ml. The mean recovery was >86.7%. Precision of the assay was <10% (R.S.D.%), and was within 15% at the limit of quantitation (20 ng/ml). Bias of the assay was lower than 15.5%. The limit of detection was 10 ng/ml for a 0.1 ml sample. The assay was applied successfully to the in vivo kinetic study of SC-560 in rats.

Determination of piceatannol in rat serum and liver microsomes: pharmacokinetics and phase I and II biotransformation.

A method of analysis of piceatannol in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of piceatannol and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL), were precipitated with acetonitrile after addition of the internal standard, 4-methylumbelliferone. Separation was achieved on a phenomenex C(18) column (250 x 4.6 mm i.d., 5 microm) equipped with a phenomenex C(18) (4 x 3.0 mm i.d., 5 microm) guardcolumn with fluorescence excitation at 320 nm and emission at 420 nm. Separation was also possible with UV detection at 310 nm. The fluorescent calibration curves were linear ranging from 0.05 to 100 microg/mL. The mean extraction efficiency was >95%. Precision of the assay was <10% (coefficient of variation), and was within 10% at the limit of quantitation (0.05 ng/mL). Bias of the assay was lower than 7%. The limit of detection was 50 ng/mL for a 0.1 mL sample. The assay was applied successfully to the in vitro kinetic study of metabolism of piceatannol in rat liver microsomes and pharmacokinetics in rats. Three metabolites of piceatannol have been identified. .

Mometasone furoate degradation and metabolism in human biological fluids and tissues.

The in vitro metabolic and non-metabolic degradation kinetics of mometasone furoate (MF) was investigated in selected human biological fluids and subcellular fractions of tissues. Qualitative and quantitative differences in transformation profiles of MF were observed among human biological media. Degradation was the major event in plasma and urine with four new degradation products identified; A: 21-chloro-17alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4-diene-3,20-dione 17-(2-furoate), B: 9alpha,21beta-dichloro-11beta,21alpha-dihydroxy-16alpha-methylpregna-1,4,17,20-tetraen-3-one 21-(2-furoate), C: 21beta-chloro-21alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4,17,20-tetraen-3-one 21-(2-furoate), and D: 21-chloro-17alpha-hydroxy-16alpha-methyl-9beta,11beta-oxidopregna-1,4-diene-3,20-dione. A, B and C were predominant and D was minor in plasma while A and C were predominant in urine. Hydrolysis of the 17-ester bond of MF was not a major event in plasma. The turnover of MF in plasma was faster than that in phosphate buffers of pH 7.4. Metabolism of MF occurred primarily and rapidly in liver, appreciably in intestine, but negligibly in in vitro lung tissue. While 6beta-hydroxylation was a major metabolic pathway for MF in microsomes of both human liver and intestine, other parallel and subsequent metabolism pathways could also be involved. If these degradation and metabolic products are also formed and active in humans in vivo, both MF and its 'active' products need to be taken into account when determining the systemic bioavailability of MF and in establishing concentration-effect relationships with this drug.

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum.

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.

Formulation dependent pharmacokinetics, bioavailability and renal toxicity of a selective cyclooxygenase-1 inhibitor SC-560 in the rat.

PURPOSE: To delineate formulation dependent pharmacokinetics and bioavailability of SC-560, a relatively new cycloooxygenase-1 (COX-1) specific inhibitor, in the rat and examine its influence on the renal tubular enzyme, N-acetyl-beta-D-glucosaminidase (NAG), and urinary electrolytes. METHODS: The pharmacokinetics of SC-560 was studied in Sprague-Dawley rats (n = 5 per group) after a single intravenous (i.v.) and oral dose (10 mg/kg) in polyethylene glycol (PEG) 600 and a single oral dose (10 mg/kg) in 1% methylcellulose (MC). Serial blood samples were collected via a catheter inserted in the right jugular vein and serum samples were analysed for SC-560 using reverse phase HPLC. After oral administration of SC-560 in PEG, urine was also collected for 24 h and analysed for urinary sodium, chloride, and potassium as well as NAG. RESULTS: After an iv dose (10 mg/kg) of SC-560, serum AUC, t(1/2), CL and Vd were 9704 +/- 4038 ng h/mL, 5.4 +/- 0.8 h, 1.15 +/- 0.46 L/h/kg and 9.1 +/- 4.6 L/kg (mean +/- SD, n = 5), respectively. Oral administration of 10 mg/kg SC-560-PEG and MC (n=5 rats) yielded serum AUC, C max, t (max )and t (1/2) of 1203.4 +/- 130.3 and 523 +/- 208 ng h/mL, 218.5 +/- 86.9 and 119.8 +/- 15.5 ng/mL, 1.00 +/- 1.8 and 2.0+/- 0 h, 3.7 +/- 1.6 and 2.7 +/- 1.7 h (mean +/- SD, n = 5), respectively. A single oral dose 10 mg/kg of SC-560 in PEG resulted in an increase in NAG excretion in urine and a reduction in 0-24 h urinary sodium, potassium, and chloride excretion. CONCLUSIONS: SC-560 extensively distributes into rat tissues, and has a CL approaching hepatic plasma flow. The drug displays low <15% and formulation dependent bioavailability after oral administration and demonstrates kidney toxicity.

Stereospecific high-performance liquid chromatographic analysis of flurbiprofen: application to pharmacokinetic studies.

A method of analysis of flurbiprofen (+/- 2-(2-fluoro-4-biphenyl)-propionic acid) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and tissue distribution. A simple high-performance liquid chromatographic method was developed for simultaneous determination of flurbiprofen enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane-isopropanol (95:5, v/v) after addition of the internal standard (IS), S-naproxen and acidification with H(2)SO(4). Separation was achieved on a Chiralpak AD-RH column with UV detection at 247 nm. The calibration curve was linear ranging from 0.05 to 50 microg/ml for each enantiomer. The mean extraction efficiency was >95%. Precision of the assay was <11% (CV), and was within 12.6% at the limit of quantitation (LOQ) (0.05 microg/ml). Bias of the assay was lower than 13.1%, and was within 12.8% at the LOQ. The assay was applied successfully to the in vivo kinetic study of flurbiprofen in rats.

Degradation kinetics of mometasone furoate in aqueous systems.

Mometasone furoate (MF) is a synthetic glucocorticoid. There is little information available on the stability of MF and no degradation products have been unequivocally identified. Thus, the primary objective of this study was to characterize the degradation of MF, qualitatively and quantitatively. Stability of MF decreased with increasing pH (>4) and decreasing ionic strength in aqueous media. The chemical stability of MF in aqueous systems was significantly dependent on pH. MF appeared to be stable at pH < 4 but degraded to four products at higher pH. The degradation of MF in aqueous solutions follows pseudo-first-order kinetics and involved a series of parallel and consecutive reactions. The turnover of MF and its products appears to be catalyzed by the hydroxide ion. The pH dependence of these reactions should be considered, when formulating or extemporaneously compounding MF formulations. An optimal pH of stability was below pH 4. The changes in pH, however, do not appear to be the only factor of importance, since an increase in ionic strength and buffer concentration displayed a stabilizing effect on this glucocorticoid in the buffers tested. Trace metal ions are unlikely to be involved in degradation of MF in aqueous solution.