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The relative oral bioavailability of alkylamides from two different Echinacea dosage forms (liquid and tablet) were compared in a small two-way crossover study in humans (n=3). The liquid preparation investigated contained a mixture of Echinacea purpurea root (300 mg/ml) and Echinacea angustifolia root (200 mg/ml) extracted in 60% ethanol. The tablet preparation investigated was also a mixture of E. purpurea root (675 mg/tablet) and E. angustifolia root (600 mg/tablet), but was prepared from the dried 60% ethanolic extracts of these two Echinacea species. Alkylamides were found to be rapidly absorbed and measurable in plasma from both preparations. No significant differences in the tetraene alkylamide pharmacokinetic parameters for T(1/2), AUC(t-lin) and C(max) in the two different preparations were found. T(max) increased from 20 min for the liquid to 30 min for the tablet, which is not unexpected as the tablet required time for disintegration before absorption could occur. These results suggested that there was no significant difference in the bioavailability of alkylamides from the liquid and tablet Echinacea formulations. Furthermore, the results also indicated that the absorption site and any alkylamide loss due to digestive processes were similar in both preparations.

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Echinacea is a widely used herbal remedy for the treatment of colds and other infections. However, almost nothing is known about the disposition and pharmacokinetics of any of its components, particularly the alkamides and caffeic acid conjugates which are thought to be the active phytochemicals. In this investigation, we have examined serial plasma samples from 9 healthy volunteers who ingested echinacea tablets manufactured from ethanolic liquid extracts of Echinacea angustifolia and Echinacea purpurea immediately after a standard high fat breakfast. Caffeic acid conjugates could not be identified in any plasma sample at any time after tablet ingestion. Alkamides were rapidly absorbed and were measurable in plasma 20 min after tablet ingestion and remained detectable for up to 12 h. Concentration-time curves for 2,4-diene and 2-ene alkamides were determined. The maximal concentrations for the sum of alkamides in human plasma were reached within 2.3 h post ingestion and averaged 336+/-131 ng eq/mL plasma. No obvious differences were observed in the pharmacokinetics of individual or total alkamides in 2 additional fasted subjects who took the same dose of the echinacea preparation. This single dose study provides evidence that alkamides are orally available and that their pharmacokinetics are in agreement with the one dose three times daily regimen already recommended for echinacea.

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The nonsteroidal anti-inflammatory drug naproxen is primarily metabolized in humans by acyl glucuronidation to form naproxen acyl glucuronide and by O-dealkylation to form 6-O-desmethylnaproxen (DMN). DMN contains both carboxy and phenolic groups and has been shown to form acyl glucuronide and sulfate conjugates. This project aimed to investigate whether DMN formed a phenolic glucuronide and diglucuronide(s) (with both the carboxy and phenolic groups glucuronidated). Male Sprague-Dawley rats (300-350 g) with exteriorized bile flow were dosed i.v. with DMN at 50 mg/kg. Four major DMN-related peaks were detected in bile by high-performance liquid chromatography (HPLC) analysis at 225 nm, including the known acyl glucuronide and sulfate conjugates. Selective hydrolyses using acidic and alkaline conditions and digestion with beta-glucuronidase allowed tentative identification of the two unknown peaks as the phenolic glucuronide of DMN and a novel acyl glucuronide-sulfate diconjugate of DMN (i.e., formed by sulfonation of the phenolic group and glucuronidation of the carboxy group). The identities were confirmed by liquid chromatography-tandem mass spectrometry analysis of individual HPLC fractions. Total recovery of the DMN dose was approximately 80%, with the sulfate conjugate (50%) and unchanged DMN (10%) being excreted predominantly in urine and the acyl glucuronide (10%), phenolic glucuronide (6%), and acyl glucuronide-sulfate diconjugate (4%) being excreted predominantly or exclusively in bile. No evidence for a diglucuronide metabolite of DMN was found in either bile or urine of the DMN-dosed rats.

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1. An isolated perfused rat liver (IPRL) preparation was used to investigate separately the disposition of the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP), its reactive acyl glucuronide metabolite (NAG) and a mixture of NAG rearrangement isomers (isoNAG), each at 30 microg NAP equivalents ml perfusate (n = 4 each group). 2. Following administration to the IPRL, NAP was eliminated slowly in a log-linear manner with an apparent elimination half-life (t 1/2) of 13.4 +/- 4.4h. No metabolites were detected in perfusate, while NAG was the only metablolite present in bile in measurable amounts (3.9 +/- 0.8% of the dose). Following their administration to the IPRL, both NAG and isoNAG were rapidly hydrolysed (t 1/2 in perfusate = 57 +/- 3 and 75 +/- 14 min respectively). NAG also rearranged to isoNAG in the perfusate. Both NAG and isoNAG were excreted intact in bile (24.6 and 14.8% of the NAG and isoNAG doses, respectively). 3. Covalent NAP-protein adducts in the liver increased as the dose changed from NAP to NAG to isoNAG (0.20 to 0.34 to 0.48% of the doses, respectively). Similarly, formation of covalent NAP-protein adducts in perfusate were greater in isoNAG-dosed perfusions. The comparative results suggest that isoNAG is a better substrate for adduct formation with liver proteins than NAG.

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The plasma profiles of valproate (VPA), its beta-oxidation metabolites E-2-en-VPA and 3-oxo-VPA and its terminal desaturation metabolite 4-en-VPA, have been measured in a patient receiving NaVPA 1000 mg twice per day from early in the course of serious hepatotoxicity and for 2 weeks after the drug was stopped. Concurrent profiles of liver, renal and haematological function parameters were available. Relative to concurrent plasma VPA concentrations, E-2-en-VPA concentrations were not different to those of the VPA-treated epileptic population at any stage of the illness, whereas 3-oxo-VPA concentrations relative to concurrent VPA concentrations were abnormally high early in the toxicity, abnormally low at its peak (3-5 days later), and comfortably within normal limits for the treated epileptic population late in the recovery phase (9-13 days from the onset). When measurable, plasma 4-en-VPA concentrations were not elevated. The elimination half-life of VPA during the recovery phase was 100 h, which is some 6-12 times greater than values reported for this parameter in normal patients. These data clearly define, in this patient, a link between idiosyncratic VPA-associated hepatotoxicity at its onset and peak and the later stages of VPA beta-oxidation. Whether the beta-oxidation abnormalities are causative or a consequence of an as yet undefined defect is unknown. In this patient, 4-en-VPA was unlikely to have been involved in the pathogenesis of the toxicity.

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The effects of co-administration of the antiepileptic agent valproic acid (VPA) and the non-steroidal anti-inflammatory drug naproxen (NAP) on their relative dispositions (particularly with respect to glucuronidation) were investigated in human volunteers. Seven healthy males received each drug alone and then in combination (orally twice daily for seven days, 500 mg sodium VPA, 500 mg NAP). On day 7 of each dosing phase, serial plasma and 24 h urine samples were collected for analysis. Co-administration of NAP resulted in significant increases (about 20%, p<0.05) in the apparent plasma clearance of total VPA and in the unbound fraction of VPA in plasma, with the apparent plasma clearance of unbound VPA being unchanged. There were associated increases in the formation clearances to urinary VPA-glucuronide and 3-oxo-VPA, though these were relatively greater for the glucuronidation pathway (and remained significant when formation clearances were calculated using the unbound fraction of drug in plasma). The data thus point to a shift towards glucuronidation as a result of the NAP-induced increase in the unbound fraction of VPA in plasma. By contrast, VPA co-administration caused a decrease (of about 10%, p<0.05) in the apparent plasma clearance of total NAP. Taken in hand with in vitro results showing a VPA-induced displacement (of about 40%) of NAP from plasma protein binding sites, the data strongly support a role for diminished glucuronidation of NAP and its desmethyl metabolite in the presence of co-administered VPA.

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OBJECTIVE: The effects of coadministration of the non-steroidal anti-inflammatory drug diflunisal (DF) on glucuronidation and beta-oxidation of the antiepileptic agent valproic acid (VPA), and of VPA on DF glucuronidation, were studied in human volunteers. METHODS: Seven healthy male volunteers received sodium valproate (NaVPA, 200 mg) orally twice daily for 7 days, after which all drug intake ceased for 1 month. The volunteers then took DF (250 mg) orally twice daily for 7 days. Both drugs were then taken (at the same doses as previously) twice daily for 7 days. On day 7 of each dosing phase, serial blood samples and all urine passed over the 12-h inter-dosing interval were collected. VPA, DF and selected metabolites were analysed using validated methods. Statistical comparisons of pharmacokinetic parameters were made using paired Student's t-tests. RESULTS: Mean plasma concentrations of total VPA were lower and apparent plasma clearances significantly higher during DF coadministration. This was associated with a significant 20% increase in the unbound fraction of VPA (from 6.6+/-1.3% to 7.9+/-1.8%). The apparent clearance of unbound VPA was not different. There was no evidence of any significant effect of DF coadministration on VPA metabolism: urinary recoveries of and formation clearances to urinary VPA-glucuronide, E-2-en-VPA, 3-oxo-VPA and 4-en-VPA were not significantly altered. However, there was a highly significant 35% increase in the area under the plasma concentration-time curve from 0-12 h (AUC0-12h) of 3-oxo-VPA and its renal clearance was lower, though not significantly so. VPA coadministration had no effect on DF pharmacokinetics or formation clearances of DF to its acyl glucuronide (DAG), phenolic glucuronide (DPG) or sulfate (DS) conjugates. However, plasma AUC0-12h values of the glucuronides were significantly lower and their renal clearances higher (though significantly so only in the case of DPG) during VPA coadministration. CONCLUSIONS: Steady-state coadministration of VPA and DF leads to a significant displacement of VPA from plasma protein binding sites. There was no evidence of competition for glucuronidation capacity or other metabolic interactions. Rather, the interactions detected appeared to be renal in nature, with renal clearance of 3-oxo-VPA being reduced by DF coadministration, and renal clearance of DPG and perhaps DAG being increased by VPA coadministration.

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OBJECTIVE: Grapefruit juice increases the oral bioavailability of several drugs metabolized by cytochrome P450 3A4. This study investigated the influence of grapefruit juice on the pharmacokinetics of oral cisapride, a substrate of CYP3A4. METHODS: Fourteen healthy volunteers received in random order 10 mg cisapride (Prepulsid) with 250 mL water or grapefruit juice after an overnight fast. Blood samples were taken for 25 hours and urine was collected for 36 hours after dosing. Plasma concentrations of cisapride and urinary norcisapride were measured by HPLC. The influence of grapefruit juice on pharmacokinetic parameters (mean +/- SD) was assessed with the Wilcoxon matched pairs test for 13 subjects (1 subject did not fast as instructed). RESULTS: Grapefruit juice increased cisapride maximum measured plasma concentration (Cmax; water, 65+/-398 ng/mL; grapefruit juice, 87+/-40 ng/mL; P = .009) and area under the plasma concentration-time curve from 0 to 25 hours [AUC(0-25); water, 418+/-280 h x ng/mL; grapefruit juice, 580+/-289 h x ng/mL; P = .005] and prolonged the time to reach Cmax (water, 1.26+/-0.36 hours; grapefruit juice, 1.72+/-0.55 hours; P = .02). Half-life was not affected. Urinary norcisapride recovery was similar and thus the partial apparent metabolic clearance to norcisapride was lower (P = .046) after grapefruit juice (89.5+/-41.2 mL/min) than after water (121.5+/-54.7 mL/min). There was considerable interindividual variation in the grapefruit juice effect [range of AUC(0-25) grapefruit juice/water ratio, 0.90 to 2.65). CONCLUSIONS: Grapefruit juice increases the oral bioavailability of cisapride, with large interindividual variation in the change in Cmax and AUC. Because cisapride has a wide therapeutic index, the interaction may not be of major clinical significance for efficacy, but further studies are necessary at steady state to rule out the possibility of side effects in susceptible individuals.

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A simple high-performance liquid chromatography assay using fluorescence detection for the major metabolite of the gastric prokinetic drug cisapride, norcisapride, is presented. Analysis is performed using an Alltech Platinum EPS C8 column with a mobile phase made up of methanol and 0.02M sodium dihygrogen phosphate (45:55, v/v) containing triethylamine (1 g/L). Complete resolution is achieved among norcisapride, the internal standard (metoclopramide), and endogenous urinary components. The assay is linear over the range 50-2000 ng/mL with a mean recovery of 71.2% across the analytical range following solvent extraction with toluene-isoamyl alcohol (95:5, v/v). Intraday coefficients of variation (precision) determined at 200 and 1000 ng/mL are 6.0 and 9.8%, respectively, and interday coefficients of variation are 8.8 and 6.6%, respectively. Intra- and interassay accuracy (as mean relative error) determined at the same concentrations is within 10% in all cases. An analysis of urine samples from a healthy volunteer following the administration of a single 10-mg oral dose of cisapride is shown.

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A sensitive, robust gas chromatographic-mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean+/-SD): Cmax, 32.73+/-9.21 ng/ml; AUC0-infinity, 1627+/-1372 ng/ml h; Tmax, 3.08 h (median); ke, 0.022+/-0.007 h(-1); elimination half-life, 37.69+/-21.70 h.

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An enhanced, sensitive GC-MS assay is presented for the highly specific angiotensin-converting enzyme (ACE) inhibitor, captopril. This method improves previously published assays by using solid NEM as stabilizer in the collection tubes, a rapid extraction technique with dichloromethane and back-extraction into base, a commercially available internal standard (thiosalicylic acid) and a capillary GC column. Captopril and the internal standard are measured as their bis-pentafluorobenzyl derivatives. The assay was linear from 10 to 5000 ng/ml with a mean recovery following solvent extraction at 50, 200 and 1000 ng/ml of 77%. At mean values of 45.9, 187 and 980 ng/ml inter-assay precision and accuracy were 4.0, 2.9 and 3.5% and 8.2, 6.5 and 3.1%, respectively. Analysis of captopril concentrations in plasma samples from 20 volunteers following oral administration of 100 mg of captopril provided the following pharmacokinetic data (mean+/-S.D.): Cmax, 1470+/-467 ng/ml; AUC(0-infinity), 1736+/-481 ng/ml.h; Tmax, 0.73 h; k(e), 0.468+/-0.122 h(-1); elimination half life, 1.58/-0.41 h.

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We describe a method for multiple indicator dilution studies in the isolated perfused human placental lobule developed to investigate the relationships between changes in pressure and flow and solute clearance. A peripheral lobule of a human placenta is perfused with a tissue culture-based medium and the perfusate oxygen tension, arterial and venous pressures, pH and perfusion temperature continuously monitored by a computerized system. Flow rates are readily changed. Bolus injections of vascular, extracellular and water space markers, and study compounds can be made into either maternal or fetal circulations, and precisely timed outflow fractions can be collected with computer-controlled fraction collectors, allowing simultaneous determination of concentration-time profiles of each marker.

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Propylthiouracil (PTU) is widely believed to cross the placenta less freely than methimazole (MMI) and is therefore regarded as the preferred drug for treatment of hyperthyroidism in pregnancy. Clinical studies comparing the two drugs show, however, no differences in maternal or fetal thyroid function. We investigated transfer from the maternal to the fetal circuit in the isolated perfused term human placental lobule of low and high doses of PTU (4 micrograms/mL and 40 micrograms/mL) and MMI (1.5 micrograms/mL and 15 micrograms/mL) in protein-free perfusate and low doses of both drugs with addition of 40 g/L of bovine albumin. Both drugs readily crossed the placenta, reaching equilibrium in all experiments in about 2 h. Drug concentrations in the two circuits fitted a two compartmental model. Transfer kinetics for the two drugs were similar, nonsaturable, and unaffected by addition of albumin. Clearances (mL.min-1.g-1, means +/- SD) of PTU from maternal to fetal circuits were: 0.229 +/- 0.110, 0.216 +/- 0.065, and 0.170 +/- 0.032; and for transfer of MMI: 0.165 +/- 0.025, 0.232 +/- 0.153, and 0.174 +/- 0.009 (for low doses without, low doses with, and high doses without albumin, respectively). Clearances of PTU from fetal to maternal circuits were: 0.147 +/- 0.072, 0.109 +/- 0.014, and 0.116 +/- 0.028; and for transfer of MMI: 0.095 +/- 0.029, 0.122 +/- 0.088, and 0.12 +/- 0.005 (in the same experiments). There was no significant difference between drugs or drug doses and no effect of addition of albumin. We conclude that PTU and MMI have similar placental transfer kinetics.

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Placental deiodination of T4 to rT3 has been proposed as the factor controlling materno-fetal transmission of T4. We investigated T4 transfer in the isolated perfused human placental lobule with and without addition of the deiodinase inhibitor, iopanoic acid. T4 (150 nmol/L) in protein-free medium was added to the maternal circuit. Without iopanoic acid, the appearance of T4 in the fetal circuit was very low, with fetal T4 levels reaching only 4.1 +/- 0.84 pmol/L at 6 h. Levels of rT3 rose progressively in both circuits, reaching 28.8 +/- 5.5 nmol/L in the maternal and 12.4 +/- 3.2 nmol/L in the fetal circuit by 6 h. No T3 could be measured in either circuit. Addition of 0.5 nmol/L iopanoic acid to maternal perfusate, however, resulted in significant reduction in the appearance of rT3 [maternal levels, 0.58 +/- 0.06 nmol/L (2% of control values); fetal levels, 0.33 +/- 0.03 nmol/L (2.7% of control values)] and a major (approximately 2700-fold) increase in T4 appearance in the fetal circuit, with fetal T4 levels reaching 10.1 +/- 3.4 nmol/L at 6 h. These results support the hypothesis that placental inner ring (type III) deiodination is a major factor controlling placental transmission of maternal T4.

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A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 micrograms ml-1) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma were all 100 +/- 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8 - 56.8 micrograms ml-1, for DOH in the concentration range 0.5 - 30.0 micrograms ml-1 and for MOH in the range 1.4-25.1 micrograms ml-1, in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C.V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (fu) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb.

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Isolated limb perfusion with melphalan is a long-standing treatment for melanoma but the clinical conditions have not been subjected to a systematic evaluation. In order to establish optimal conditions for perfusion, three human melanoma cell lines were cultured with melphalan in vitro under conditions comparable to in vivo therapy. The most important findings were that: (a) 41.5 degrees C was synergistic for melphalan killing of three human melanoma cell lines; (b) prolonging the treatment time beyond 1 h had little additional toxicity; and (c) varying the initial pH of the culture medium had no effect. After 1 h of treatment, cells accumulated more melphalan at 41.5 degrees C than at 37 degrees C, relative to the extracellular concentration. A cell line (MM418) derived from a primary tumour was the most resistant of the three lines; pigmented or non-pigmented sublines were equally resistant. The A2058 line showed the lowest level of synergism with hyperthermia, and displayed a marked plateau at 10% of controls in the dose-response for survival, yet no melphalan-resistant subpopulation could be isolated. The implications of this work are that (a) enhanced cellular uptake of melphalan may account for hyperthermic synergism of melphalan; (b) varying conditions other than treatment time will be necessary to deal with the variation in resistance between tumours; and (c) repeated cycles of treatment may be needed for phenotypes such as A2058 where melphalan resistance appears to be based on an epigenetic mechanism.

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Prednisolone is metabolized in the perfused human placental lobule to prednisone, 20 alpha-dihydroprednisone, 20 beta-dihydroprednisone and 20 beta-dihydroprednisolone. The pathway of metabolite formation was defined in perfusions of placental lobules using prednisone and 20 beta-dihydroprednisone separately as substrates and with prednisolone co-perfused with glycyrrhetinic acid, a potent inhibitor of the 11-oxidase component of the 11 beta-hydroxysteroid dehydrogenase enzyme system. The pattern of metabolites identified from 6 h samples indicated a reversible formation of prednisone from prednisolone, the production of the 20 alpha- and 20 beta-dihydro metabolites of prednisone from prednisone, the formation of 20 beta-dihydroprednisolone from 20 beta-dihydroprednisone only and no direct formation of 20 beta-dihydroprednisolone from prednisolone. Kinetic analysis at two substrate concentrations confirmed that the formation of three of the four steroid metabolites followed first order kinetics. In perfusions with an initial prednisolone concentration of 1 microgram/ml (n = 4) or 100 ng/ml (n = 3), the rate constants obtained were (mean +/- SD, maternal compartment, h-1): prednisone, 1.97 +/- 0.49 and 2.25 +/- 0.15, P > 0.1; 20 alpha-dihydroprednisone, 0.0006 +/- 0.0004 and 0.0017 +/- 0.0006, P < 0.1; 20 beta-dihydroprednisone, 0.15 +/- 0.022 and 0.15 +/- 0.0077, P > 0.1. In contrast, the rate constant for formation of 20 beta-dihydroprednisolone at an initial prednisolone concentration of 100 ng/ml (0.083 +/- 0.0095 h-1) was significantly (P < 0.01) greater than the corresponding rate constant at the higher initial prednisolone concentration (0.039 +/- 0.015 h-1). A significant increase (P < 0.05) was observed for the formation of 20 beta-dihydroprednisolone at the end of 6 h perfusions at the lower initial substrate concentration (11.2 +/- 1.9%) compared with the 1 microgram/ml concentration (6.0 +/- 2.5%).

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