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Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium . Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Pseudomonas aeruginosa forms stable biofilms, providing a major barrier for multiple classes of antibiotics and severely impairing treatment of infected patients. The biofilm matrix of this Gram-negative bacterium is primarily composed of three major exopolysaccharides: alginate, Psl, and Pel. Here, we studied the antibiofilm properties of sponge-derived natural products ianthelliformisamines A-C and their combinations with clinically used antibiotics. Wild-type P. aeruginosa strain and its isogenic exopolysaccharide-deficient mutants were employed to determine the interference of the compounds with biofilm matrix components. We identified that ianthelliformisamines A and B worked synergistically with ciprofloxacin to kill planktonic and biofilm cells. Ianthelliformisamines A and B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin to 1/3 and 1/4 MICs, respectively. In contrast, ianthelliformisamine C (MIC = 53.1 µg/mL) alone exhibited bactericidal effects dose-dependently on both free-living and biofilm populations of wild-type PAO1, PAO1Δ pslA (Psl deficient), PDO300 (alginate overproducing and mimicking clinical isolates), and PDO300Δ alg8 (alginate deficient). Interestingly, the biofilm of the clinically relevant mucoid variant PDO300 was more susceptible to ianthelliformisamine C than strains with impaired polysaccharide synthesis. Ianthelliformisamines exhibited low cytotoxicity towards HEK293 cells in the resazurin viability assay. Mechanism of action studies showed that ianthelliformisamine C inhibited the efflux pump of P. aeruginosa . Metabolic stability analyses indicated that ianthelliformisamine C is stable and ianthelliformisamines A and B are rapidly degraded. Overall, these findings suggest that the ianthelliformisamine chemotype could be a promising candidate for the treatment of P. aeruginosa biofilms.

Widespread resistance in parasitic nematodes to most classes of anthelmintic drugs demands the discovery and development of novel compounds with distinct mechanisms of action to complement strategic or integrated parasite control programs. Products from nature-which assume a diverse 'chemical space'-have significant potential as a source of anthelmintic compounds. In the present study, we screened a collection of extracts ( n = 7616) derived from marine invertebrates sampled from Australian waters in a high throughput bioassay for in vitro anti-parasitic activity against the barber's pole worm ( Haemonchus contortus )-an economically important parasitic nematode of livestock animals. In this high throughput screen (HTS), we identified 58 active extracts that reduced larval motility by ≥70% (at 90 h), equating to an overall 'hit rate' of ~0.8%. Of these 58 extracts, 16 also inhibited larval development by ≥80% (at 168 h) and/or induced 'non-wild-type' (abnormal) larval phenotypes with reference to 'wild-type' (normal) larvae not exposed to extract (negative controls). Most active extracts (54 of 58) originated from sponges, three from chordates (tunicates) and one from a coral; these extracts represented 37 distinct species/taxa of 23 families. An analysis of samples by 1 H NMR fingerprinting was utilised to dereplicate hits and to prioritise a set of 29 sponge samples for future chemical investigation. Overall, these results indicate that a range of sponge species from Australian waters represents a rich source of natural compounds with nematocidal or nematostatic properties. Our plan now is to focus on in-depth chemical investigations of the sample set prioritised herein.

Chemical investigations of two specimens of the Australian crinoid Comatula rotalaria afforded five new taurine-conjugated anthraquinones, comatulins A-E ( 1 - 5 ), together with 11 known marine natural products ( 6 - 16 ). The chemical structures of all the compounds were elucidated by detailed spectroscopic and spectrometric data analysis. The first X-ray crystal structure of a crinoid-derived acyl anthraquinone, rhodocomatulin 5,7-dimethyl ether ( 8 ), is reported here. Compounds 1 , 2 , 6 - 13 , and two additional naphthopyrone derivatives, 17 and 18 , were evaluated for their ability to inhibit HIV-1 replication in vitro ; none of the compounds were active at 100 μM. Furthermore, compounds 1 , 2 , 6 - 10 , 14 , 15 , 17 , and 18 were screened for nematocidal activity against exsheathed third-stage larvae of Hemonchus contortus , a highly pathogenic parasite nematode of ruminants. Compound 17 , known as 6-methoxycomaparvin 5,8-dimethyl ether, showed an inhibitory effect on larval motility (IC 50 = 30 μM) and development (IC 50 = 31 μM) and induced the eviscerated ( Evi ) phenotype.

There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus -a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu -1, Ha -1 and Ha -2) from two marine sponges ( Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus . Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata , a known pentacyclic guanidine alkaloid, fromiamycalin ( 1 ), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC 50 ) = 26.6 ± 0.74 µM) and L4 motility (IC 50 = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC 50 ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A ( 5 ). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts., Competing Interests: The authors declare no conflicts of interest. Funding bodies played no role in the design of the study, collection, analysis or interpretation of data, or in the writing of the manuscript or in the decision to publish the results.

Due to the widespread occurrence and spread of anthelmintic resistance, there is a need to develop new drugs against resistant parasitic nematodes of livestock animals. The Nobel Prize-winning discovery and development of the anti-parasitic drugs avermectin and artemisinin has renewed the interest in exploring natural products as anthelmintics. In the present study, we screened 7500 plant extracts for in vitro-activity against the barber's pole worm, Haemonchus contortus, a highly significant pathogen of ruminants. The anthelmintic extracts from two plants, Cryptocarya novoguineensis and Piper methysticum, were fractionated by high-performance liquid chromatography (HPLC). Subsequently, compounds were purified from fractions with significant biological activity. Four α-pyrones, namely goniothalamin (GNT), dihydrokavain (DHK), desmethoxyyangonin (DMY) and yangonin (YGN), were purified from fractions from the two plants, GNT from C. novoguineensis, and DHK, DMY and YGN (= kavalactones) from P. methysticum. The three kavalactones induced a lethal, eviscerated (Evi) phenotype in treated exsheathed third-stage larvae (xL3s), and DMY and YGN had moderate potencies (IC 50 values of 31.7 ± 0.23 μM and 23.7 ± 2.05 μM, respectively) at inhibiting the development of xL3s to fourth-stage larvae (L4s). Although GNT had limited potency (IC 50 of 200-300 μM) at inhibiting L4 development, it was the only compound that reduced L4 motility (IC 50 of 6.25-12.50 μM). The compounds purified from each plant affected H. contortus in an irreversible manner. These findings suggest that structure-activity relationship studies of α-pyrones should be pursued to assess their potential as anthelmintics., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A sensitive high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) assay has been developed for the quantitative analysis of vardenafil in human plasma. Vardenafil and the internal standard, alprazolam, were extracted from 0.2 mL aliquots of alkalinized plasma by a single solvent extraction into hexane : dichloromethane. Reversed-phase chromatographic separation was affected by gradient elution with mobile phases consisting of 10 mM ammonium formate pH 7.0 (solvent A) and methanol (100%, solvent B), delivered at a flow rate of 0.4 mL/min. The analytes were detected by using an electrospray ion source on a 4000 QTrap triple quadrupole mass spectrometer operating in positive ionization mode. The mass transitions were m/z 489.3 --> 312.2 for vardenafil and m/z 309.2 --> 281.0 for alprazolam. The assay was linear over the concentration range of 0.2-100 ng/mL, with correlation coefficients > or = 0.995. The intra- and inter-day precision was less than 5.4% in terms of relative standard deviation and the accuracy was within 12.7% in terms of relative error. The lower limit of quantitation was set at 0.2 ng/mL. The high sensitivity and acceptable performance of the assay allowed its application to the analysis of plasma samples obtained following the oral administration of vardenafil to healthy male volunteers in a pharmacokinetic study.

The two fixed-dose combinations of dihydroartemisinin and piperaquine (Artekin and Arterakine) were found to be bioinequivalent in healthy Vietnamese subjects. However, because the peak plasma concentrations and areas under the concentration-time curves of dihydroartemisinin and piperaquine were only marginally different between the two formulations, similar therapeutic efficacies are expected in the treatment of malaria infections.

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.

Lamotrigine concentrations were measured simultaneously (as far as was feasible) in stimulated and unstimulated saliva samples, and in plasma, from seven adult volunteers over a 32 h period following a single 50 mg dose of the drug, and in 20 children and adolescents during the course of routine antiepileptic therapy. In individuals there was a close correlation between the measurements at least 2 h after ingestion of the drug. Concentrations in stimulated and unstimulated saliva were similar; the stimulation produced little change in the saliva secretion rate. The saliva-to-plasma concentration ratio increased linearly by 0.78% for each 1 mg/L plasma lamotrigine concentration, with a mean value of 48.8% at a plasma lamotrigine concentration of 10 mg/L. With appropriate precautions as to the timing of saliva collections, and a single plasma lamotrigine concentration measurement to calibrate the salivary values in the individual, salivary lamotrigine concentration measurement appears to be a practicable approach to therapeutic drug monitoring. This has significant implications for the elucidation of the pharmacokinetics of lamotrigine in the paediatric population.

Purpose: PI-88 is a mixture of highly sulfated oligosaccharides that inhibits heparanase, an extracellular matrix endoglycosidase, and the binding of angiogenic growth factors to heparan sulfate. This agent showed potent inhibition of placental blood vessel angiogenesis as well as growth inhibition in multiple xenograft models, thus forming the basis for this study., Experimental Design: This study evaluated the toxicity and pharmacokinetics of PI-88 (80-315 mg) when administered s.c. daily for 4 consecutive days bimonthly (part 1) or weekly (part 2)., Results: Forty-two patients [median age, 53 years (range, 19-78 years); median performance status, 1] with a range of advanced solid tumors received a total of 232 courses. The maximum tolerated dose was 250 mg/d. Dose-limiting toxicity consisted of thrombocytopenia and pulmonary embolism. Other toxicity was generally mild and included prolongation of the activated partial thromboplastin time and injection site echymosis. The pharmacokinetics were linear with dose. Intrapatient variability was low and interpatient variability was moderate. Both AUC and C(max) correlated with the percent increase in activated partial thromboplastin time, showing that this pharmacodynamic end point can be used as a surrogate for drug exposure. No association between PI-88 administration and vascular endothelial growth factor or basic fibroblast growth factor levels was observed. One patient with melanoma had a partial response, which was maintained for >50 months, and 9 patients had stable disease for >or=6 months., Conclusion: The recommended dose of PI-88 administered for 4 consecutive days bimonthly or weekly is 250 mg/d. PI-88 was generally well tolerated. Evidence of efficacy in melanoma supports further evaluation of PI-88 in phase II trials.

The phosphosulfomannan 1 (PI-88) is a mixture of highly sulfated oligosaccharides that is currently undergoing clinical evaluation in cancer patients. As well as its anticancer properties, 1 displays a number of other interesting biological activities. A series of analogues of 1 were synthesized with a single carbon (pentasaccharide) backbone to facilitate structural characterization and interpretation of biological results. In a fashion similar to 1, all compounds were able to inhibit heparanase and to bind tightly to the proangiogenic growth factors FGF-1, FGF-2, and VEGF. The compounds also inhibited the infection of cells and cell-to-cell spread of herpes simplex virus (HSV-1). Preliminary pharmacokinetic data indicated that the compounds displayed different pharmacokinetic behavior compared with 1. Of particular note was the n-octyl derivative, which was cleared 3 times less rapidly than 1 and may provide increased systemic exposure.

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.

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.

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.

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.

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.

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., (Copyright 2000 John Wiley & Sons, Ltd.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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%).

The isolated perfused lobule of human placenta was used as an in-vitro model to study the effect of intravenous immunoglobulin (IVGG) on the placental transfer of a human platelet-specific antibody (anti-P1A1). Normal human IgG was shown to transfer from the maternal to the fetal circulation of the placental model after a lag period of 2-3 h. IVGG also transferred across the placenta but only after a longer lag period (3-4 h) than normal human IgG at the same concentration, which suggests that IVGG may contain a factor that inhibits the transfer of its own component IgG. The sensitive Western immunoblotting technique was used to demonstrate progressive transfer of anti-P1A1 antibody to the fetal circulation after a 2-3 h lag period. When IVGG and anti-P1A1 antibody were added simultaneously to the maternal circulation, the transfer of platelet-specific antibody was strongly inhibited by IVGG. The inhibitory effect of IVGG on anti-P1A1 antibody transfer was consistent for three different batches of the same IVGG product (Sandoglobulin). These studies provide the first scientific data to support the use of IVGG to inhibit antiplatelet antibody transfer as part of the antenatal management of neonatal alloimmune thrombocytopenia.

Previous studies of the metabolism of 11 beta-hydroxy corticosteroids by placental tissue have indicated that the only product is the C11-oxidized metabolite. In the present study we have re-examined the metabolism of prednisolone in the isolated, perfused, dual recirculating human placental lobule, using a perfusate based on tissue culture medium 199. Four metabolites were identified in both the maternal and fetal compartments in 6 h perfusions by comparison of relative retention times measured by HPLC and capillary gas chromatography (GC) and of mass spectra recorded by capillary gas chromatography-mass spectrometry (GC-MS) with those of authentic reference standards. The steroids were derivatized as the MO-TMS ethers for mass spectral measurements. Analysis of samples from five perfusion experiments resulted in the following percentage conversions after 6 h perfusion (mean +/- SD, maternal and fetal perfusate, respectively): prednisone (49.1 +/- 7.8, 49.1 +/- 6.6), 20 alpha-dihydroprednisone (0.84 +/- 0.29, 0.81 +/- 0.35), 20 beta-dihydroprednisone (39.1 +/- 6.7, 39.2 +/- 5.9), 20 beta-dihydroprednisolone (6.8 +/- 2.7, 6.3 +/- 1.6) and unmetabolized prednisolone (4.1 +/- 1.8, 4.6 +/- 2.1). No evidence was found for metabolites formed by 6 beta-hydroxylation or cleavage of the C17-C20 bond.

A reversed-phase liquid chromatographic assay was developed to quantitate prednisolone, prednisone and the 20 alpha-dihydro and 20 beta-dihydro reduced metabolites of both parent compounds in tissue culture media from in vitro perfusions of the human placental lobule. Steroids were extracted from perfusate, using reversed-phase cartridges, with average recoveries of 95.2% or greater. The internal standard for the analyses was 6 alpha-methylprednisolone. In this assay cortisol coelutes with prednisolone, however, no other significant interferences were found. Assay of each steroid was linear in the range 0-1 microgram/ml. Intra-assay coefficients of variation were measured at 10 and 750 ng/ml with ranges of 3.4% (20 alpha-dihydroprednisone) to 8.8% (20 beta-dihydroprednisolone) and 4.1% (20 beta-dihydroprednisone) to 8.8% (prednisone). The corresponding inter-assay coefficients of variation were 3.3% (20 alpha-dihydroprednisone) to 9.1% (20 beta-dihydroprednisolone) and 1.9% (prednisolone) to 3.5% (prednisone). The analyses utilized two C18 columns which were linked together and maintained at 40 degrees C.