Disposition and metabolic fate of prasugrel in mice, rats, and dogs.

The disposition and metabolism of prasugrel, a thienopyridine prodrug and a potent inhibitor of platelet aggregation in vivo, were investigated in mice, rats, and dogs. Prasugrel was rapidly absorbed and extensively metabolized. In the mouse and dog, maximum plasma concentration of radioactivity was observed in less than 1 h after an oral [14C]prasugrel dose. Most of the administered prasugrel dose was recovered in the faeces of rats and dogs (72% and 52-73%, respectively), and in mice urine (54%). Prasugrel is hydrolysed by esterases to a thiolactone, which is subsequently metabolized to thiol-containing metabolites. The main circulating thiol-containing metabolite in the three animal species is the pharmacologically active metabolite, R-138727. The thiol-containing metabolites are further metabolized by S-methylation and conjugation with cysteine.

Synthesis and biological activity of some known and putative duloxetine metabolites.

Several putative phase I duloxetine metabolites, 4-hydroxy-, 5-hydroxy-, 6-hydroxy-, 5-hydroxy-6-methoxy-, 6-hydroxy-5-methoxy-, 5,6-dihydroxy-, and 4,6-dihydroxyduloxetine were synthesized, and their phase II metabolite as glucuronide or sulfate conjugates were also synthesized. Their in vitro binding activities were compared to that of parent compound duloxetine.

Metabolism, excretion, and pharmacokinetics of duloxetine in healthy human subjects.

Duloxetine is a potent and balanced dual inhibitor of serotonin and norepinephrine reuptake being investigated for the treatment of depression and urinary incontinence. The disposition of duloxetine was studied in four healthy human subjects after a single 20.2-mg (100.6 microCi) oral dose of [14C]duloxetine in an enteric-coated tablet. The mean total recovery of radioactivity (+/- S.E.M.) after 312 h was 90.5% (+/-0.4%) with 72.0% (+/-1.1%) excreted in the urine. Duloxetine was extensively metabolized to numerous metabolites primarily excreted into the urine in the conjugated form. The major biotransformation pathways for duloxetine involved oxidation of the naphthyl ring at either the 4-, 5-, or 6-positions followed by further oxidation, methylation, and/or conjugation. The major metabolites found in plasma were glucuronide conjugates of the following: 4-hydroxy duloxetine (M6), 6-hydroxy-5-methoxy duloxetine (M10), 4, 6-dihydroxy duloxetine (M9), and a sulfate conjugate of 5-hydroxy-6-methoxy duloxetine (M7). The major metabolites found in plasma were also found in the urine, but the urine contained many additional metabolites. In addition to duloxetine, 4-hydroxy duloxetine (M14) and an unidentified polar metabolite were observed in feces. Following [14C]duloxetine administration, Cmax was reached at a median of 6 h for both duloxetine and total radioactivity. Duloxetine accounted for less than 3% of the circulating radioactivity based on mean area under the curve values. The elimination half-life of total radioactivity (120 h) was substantially longer than that of duloxetine (10.3 h).

Bacterial contamination of children's toys used in a general practitioner's surgery.

General practitioners--and other healthcare professionals are encouraged to make their premises child friendly. One way of doing this is to provide toys for children to use. We looked at the appearance and bacterial colonisation of 50 toys after a busy morning surgery in an inner city general practice. The toys appeared generally unclean and 10% were contaminated by potential pathogens. Bacteria were cultured more frequently from soft toys than from hard toys (odds ratio 8.14; 95% confidence range 0.74-107.49). Although toys may appear to be physically dirty after use, the bacteria isolated from their surfaces are generally non-pathogenic to children with normal immune function and probably no worse than other objects in the environment. However, there does exist an appreciable (1 in 10) risk of cross-infection with the use of toys in a clinic. Toys with a hard surface are preferred as these are less likely to be contaminated and are more easily disinfected.

Eradication of a resistant Pseudomonas aeruginosa strain after a cluster of infections in a hematology/oncology unit.

OBJECTIVES: This report chronicles an outbreak of a multiply resistant strain of Pseudomonas aeruginosa and the measures required to contain this outbreak. METHODS: Laboratory-based ward-liaison surveillance allowed the detection of a multiply resistant strain of P. aeruginosa infecting patients in our hematology/oncology unit. Sampling of the immediate environment was carried out. Pulsed field gel electrophoresis was used to compare the patients' organisms with those found in the environment. Extensive dismantling of the drainage system, repeated cleaning and disinfection, and a review of the departmental antibiotic policy were some of the infection control measures instigated. RESULTS: During a period of 11 months, three patients in the hematology department and two patients in the oncology department were infected with multiply resistant P. aeruginosa. There were two cases of pneumonia, one of which was fatal, and two cases of neutropenic septicaemia. Pulsed field gel electrophoresis performed on the isolates showed that the isolates from geographically separate areas could be divided into two strains that were closely related but distinct. Two genotypically identical strains were also isolated from the plumbing systems in the areas of each ward where patients had been treated. CONCLUSIONS: The potential for serious nosocomial infections with P. aeruginosa is well recognized. Eradication of the organism from the environment may require the co-ordinated efforts of clinicians, nurses, pharmacy and hospital engineers, working in collaboration with the hospital infection control team. To date, the same strains have not been isolated despite repeated surveillance over the past 18 months and therefore these measures have, in our opinion, successfully removed the potential for nosocomial infection with this resistant organism in our hospital.

Three-dimensional quantitative structure activity relationship analyses of substrates for CYP2B6.

To begin to build an understanding of the interactions of CYP2B6 with substrates, two different 3-dimensional quantitative structure activity relationship (3D-QSAR) models were constructed using 16 substrates of B-lymphoblastoid expressed CYP2B6. A pharmacophore model was built using the program Catalyst, which was compared with a partial least-squares (PLS) model using molecular surface-weighted holistic invariant molecular (MS-WHIM) descriptors. The Catalyst model yielded a 3-dimensional model of the common structural features of CYP2B6 substrates, whereas PLS MS-WHIM generated a model based on statistical analyses of molecular descriptors for size and shape of the substrate. The pharmacophore model obtained with Catalyst consisted of three hydrophobes and one hydrogen bond acceptor region. The cross-validated PLS MS-WHIM model gave a good q2 value of 0.607. Size, positive electrostatic potential, hydrogen bonding acceptor capacity, and hydrophobicity were found to be the most relevant descriptors for the model. These models were then used to predict the Km (apparent) values of a test set of structurally diverse substrates for CYP2B6 not included in the model building, specifically lidocaine, amitriptyline, bupropion, arteether, and verapamil. Overall, both 3D-QSAR methods yielded satisfactory Km (apparent) value predictions for the majority of the molecules in the test set. However, PLS MS-WHIM was unable to reliably predict the Km (apparent) value for verapamil, whereas Catalyst did not predict the Km (apparent) value for lidocaine. In both of these cases the residual of the Km (apparent) value was greater than one log unit. The strengths and limitations of both of these 3D-QSAR approaches are discussed.

Characterization of olanzapine (LY170053) in human liver slices by liquid chromatography/tandem mass spectrometry.

Olanzapine metabolism was investigated using incubation of olanzapine with human liver slices. The intent of the investigation was to identify olanzapine metabolites and determine if the human liver slice incubations could potentially produce quantities of the olanzapine glucuronides for future studies. Along with known Phase 1 olanzapine metabolites, N-desmethyl-, 2-hydroxymethyl-, and 4'-N-oxide-, a new hydroxylated species was detected. Detection of Phase 2 metabolites included known N-10-glucuronides, a quaternary glucuronide and a novel glucuronide conjugate. This investigation showed the feasibility of using human liver slices to produce sufficient quantities of olanzapine glucuronides for further studies.

Development of an instrument to measure cardiac illness dependency.

OBJECTIVE: Dependency is frequently mentioned in the literature as a response of patients with cardiac disease. The purpose of this study was to develop and test a measure of dependency occurring in response to a cardiac illness. Illness dependency is defined as the need for emotional protection and social support after a significant change in health. DESIGN: Instrument development study. SAMPLE: Convenience sample of 311 patients with cardiac disease. RESULTS: The final version of the instrument has 25 items, each of which is measured on a 5-point Likert-type scale. Content validity was demonstrated using a panel of experts. Internal consistency of the total scale was 0.90; subscale alpha coefficients ranged from 0.64 to 0.81. Exploratory factor analysis supported a four factor solution: Attention, Reassurance, Concern, and Assistance, which accounted for 57.4% of the variance in scores. Discriminant validity was demonstrated by a low correlation with neuroticism. Social desirability of responses was minimal. CONCLUSION: Internal consistency reliability, content validity, and discriminant validity of the Illness Dependency Scale have initial support. This instrument is ready for use in research in which the investigator wishes to measure dependency associated with cardiac illness.

Application of packed capillary liquid chromatography/mass spectrometry with electrospray ionization to the study of the human biotransformation of the anti-emetic drug dolasetron.

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

Determination of the muscarinic agent [(3-(3-1-butylthio)-1,2,5-thiadiazol-4-yl)-1-azabicyclo-2.2.2-oct ane], in rat, rabbit, and monkey plasma, using high-performance liquid chromatography in conjunction with tandem mass spectrometry.

A method for determining a selective muscarinic agent, LY297802 (compound I), [(3-(3-1-butylthio)-1,2,5-thiadiazol-4-yl)-1-azabicyclo-2.2.2-octa ne], indicated in the treatment of pain, in rat, rabbit, and monkey plasma is described. The analytes, including an internal standard, were extracted from plasma at basic pH with hexane. The organic fraction was evaporated to dryness and the residue reconstituted with mobile phase. The analytes were detected utilizing HPLC in conjunction with electrospray (ES) tandem mass spectrometry (MS-MS). The limit of quantitation was 0.25 ng/ml, and the response was linear to at least 100 ng/ml.

Determination of xanomeline and active metabolite, N-desmethylxanomeline, in human plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

We have developed a method for the determination of xanomeline and its pharmacologically active N-desmethyl metabolite. The validated method uses hexane to extract xanomeline and its N-desmethyl metabolite from basified plasma. The hexane extract is dried, reconstituted, and analyzed using a liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometry system. The method was developed to support phase II clinical trials and has proven to be extremely sensitive, fast, and rugged. The method has a limit of quantitation of 75 and 200 pg/ml plasma for xanomeline and the N-desmethyl metabolite, respectively. Sample analysis times were less than 3 min from one injection to the next.

Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection.

A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (% C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma.

Determination of xanomeline in human plasma by ion-spray tandem mass spectrometry.

Xanomeline is a muscarinic receptor agonist currently in phase II clinical trials for the treatment of Alzheimer's disease. A fast, sensitive and specific assay has been developed to determine xanomeline plasma concentrations using ion-spray tandem mass spectrometry. Xanomeline and a structural analog, LY282122, were extracted from basifed plasma into hexane. The dried hexane extracts were reconstituted and injected onto a 10 x 1 mm C18 reversed-phase column. A mobile phase of 33 mM ammonium acetate and 0.33% acetic acid in 30/70 (v/v) water-acetonitrile was pumped through the column at 50 microliters min-1. The mobile phase eluant was introduced directly into the ion-spray interface. The mass spectrometer was operated in the positive ion mode for specific detection of the product ions of xanomeline and the internal standard. The method has a linear range of 0.075-5.0 ng xanomeline per milliliter of plasma. Sample run times were 2.5 min from one injection to the next.

Determination of dolasetron and its reduced metabolite in human plasma by GC-MS and LC.

Both a GC-MS and an LC method have been developed for the simultaneous quantitation of dolasetron and reduced dolasetron in human plasma. The GC-MS method has been utilized in preliminary human pharmacokinetic studies of dolasetron mesylate. Selected ion monitoring was used in these initial studies to obtain the sensitivity and specificity required for quantitation. The GC-MS method has been used in the range of 1-120 ng ml-1 for dolasetron and 1-240 ng ml-1 for reduced dolasetron in plasma. The limit of quantitation for both compounds by GC-MS was 1 ng ml-1. Recently, an LC method has been utilized for quantitation of both compounds on a routine basis. This method utilizes essentially the same sample preparation procedure as the GC-MS method. The LC method has been used in the range of 5-200 ng ml-1 in plasma for dolasetron and reduced dolasetron. In addition, the relationship between the LC and GC-MS methods has been assessed using data obtained from human male volunteers following intravenous administration of 3.0 mg kg-1 of dolasetron mesylate monohydrate.

Differences in the low-energy collision-activated dissociation of carboxylate anions from structurally similar prostaglandins: E2, F 2α, D 2, and DHKF 2α.

Some intriguing discoveries were made concerning the collision-activated dissociation behavior of the derivatized carboxylate anions of PGE2 and PGF2α. The carboxylate anion [MPFB](-) formed from electron-capture negative chemical ionization of the pentafluorobenzyl ester-trimethylsilyl derivative of PGF2α showed little fragmentation under typical collision gas pressures and energies (<2.0 mtorr N2 and <20 eV). In contrast, the daughter spectra of the carboxylate anion of the methoxime-pentafluorobenzyl ester-trimethylsilyl derivative of PGE2 produced many intense fragments under the same conditions.

Effects of dobutamine in patients with acute myocardial infarction.

This study was designed to evaluate the effects of dobutamine, a new cardioselective beta adrenergic agonist, on cardiac performance and myocardial injury in patients with evolving myocardial infarction. Results in 16 patients given dobutamine (1 to 40 microng/kg per min for 24 hours) were compared with those in two groups of control patients: one of 16 patients matched for predicted infarct size, and the other of 16 patients matched for early ventricular dysrhythmia, analyzed by computer. Infarct size was predicted from plasma creatine kinase (CK) values during the first 7 hours after the initial elevation, before infusion of dobutamine. Overall observed infarct size was estimated from hourly CK values for 48 hours (including those before and after administration of dobutamine). In all patients technetium-99m (stannous) pyrophosphate scans were positive for myocardial infarction. Dobutamine increased cardiac output (assessed by thermodilution) from 4.9 +/- 0.37 (mean +/- standard error) to 6.0 +/- 0.38 liters/min (P less than 0.05) and decreased pulmonary arterial occlusive pressure from 21.5 +/- 2.7 to 16.7 +/- 1.6 mmHg (P less than 0.01) without significantly altering heart rate or systemic arterial blood pressure. The ratio of observed to predicted infarct size, the frequency of independently detected reinfarction or extension of infarction and the frequency of premature ventricular complexes were similar in control and treated patients. Thus administration of dobutamine in doses sufficient to improve ventricular performance after myocardial infarction does not exacerbate myocardial injury or ventricular dysrhythmia.