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The current study provides a novel in-depth assessment of the extent of antipsychotic drugs transport across the blood-brain barrier (BBB) into various brain regions, as well as across the blood-spinal cord barrier (BSCB) and the blood-cerebrospinal fluid barrier (BCSFB). This is combined with an estimation of cellular barrier transport and a systematic evaluation of nonspecific brain tissue binding. The study is based on the new Combinatory Mapping Approach (CMA), here further developed for the assessment of unbound drug neuropharmacokinetics in regions of interest (ROI), referred as CMA-ROI. We show that differences exist between regions in both BBB transport and in brain tissue binding. The most dramatic spatial differences in BBB transport were found for the P-glycoprotein substrates risperidone (5.4-fold) and paliperidone (4-fold). A higher level of transporter-mediated protection was observed in the cerebellum compared with other brain regions with a more pronounced efflux for quetiapine, risperidone and paliperidone. The highest BBB penetration was documented in the frontal cortex, striatum and hippocampus (haloperidol, olanzapine), indicating potential influx mechanisms. BSCB transport was in general characterized by more efficient efflux compared with the brain regions. Regional tissue binding was significantly different for haloperidol, clozapine, risperidone and quetiapine (maximally 1.9-fold). Spatial differences in local unbound concentrations were found to significantly influence cortical 5-HT2A receptor occupancy for risperidone and olanzapine. In conclusion, the observed regional differences in BBB penetration may potentially be important factors contributing to variations in therapeutic effect and side effect profiles among antipsychotic drugs.

Objective This study addresses mothers' experiences with potty training in a Vietnamese population. Subjects and methods Forty-seven mothers were interviewed and followed from the time that their children were newborns until they were 24-months old. The interviews were analyzed using qualitative content analysis. Results According to tradition, diapers were used only rarely. The mothers used a whistling sound at certain times to remind their children to eliminate and frequently checked for signs of need. With this process, all children used the potty by the age of 9 months. At the age of 24 months the potty training was completed, and most of the children managed the whole process independent of help. Conclusion This study shows that it is possible to start potty training with good outcomes very early in life. The process described can be achieved through an ongoing communication between parent and child.

OBJECTIVES: To study outcomes of early potty training in a population of healthy children with a tradition of early potty training and to compare these findings with a group of children to whom potty training was applied later. SUBJECTS AND METHODS: Mothers and their 47 healthy children in Vietnam participated in this longitudinal study. The voiding pattern and emptying ability were followed by the 4-h voiding observation method from 3 months to 3 years of age. A comparison is made with a group of 57 Swedish children investigated in a similar manner. RESULTS: In the group of Vietnamese children, 89% were on daily potty training at the age of 6 months. At the age of 24 months, potty training was complete for 98%. In the Swedish group, just a few (5%) had started daily potty training by the age of 24 months (p

Introduction: To investigate if potty training from infancy can affect bladder dysfunction in boys with posterior urethral valves (PUV). Subjects and Methods: The voiding pattern and emptying ability were followed using the 4-hour voiding observation method in 17 Vietnamese boys with PUV aged 3 months to 4 years who had been potty-trained from infancy. This group was compared with a group of healthy Vietnamese boys. Results: In the boys with PUV, the bladder volume increased according to age, and interrupted voiding was rare. However, when comparing boys with PUV with healthy boys, a significant difference was noted with more frequent voidings and lower voided volumes in the age group 0 - 1 year (P The findings from the 4-hour voiding observation showed few signs of dysfunctional bladder in the Vietnamese boys with PUV, including residual urine, even if there were signs of dysfunction compared with the healthy Vietnamese boys. Potty training from infancy could favor early bladder rehabilitation in boys with PUV.

This study provides a new method for quantifying the cyclotide kalata B1 in both plasma and brain homogenate. Cyclotides are ultra‐stable peptides with three disulfide bonds that are interesting from a drug development perspective as they can be used as scaffolds. In this study we describe a new validated LC‐MS/MS method with high sensitivity and specificity for kalata B1. The limit of quantification was 2 ng/mL in plasma and 5 ng/gmL in brain homogenate. The method was linear in the range 2–10,000 ng/mL for plasma and 5–2000 ng/g for brain. Liquid Chromatographic separation was performed on a HyPurity C18 column, 50 × 4.6 mm, 3 µm particle size. The method had inter‐ and intra‐day precision and accuracy levels

A previous pharmacokinetic study on dosing of colistin methanesulfonate (CMS) at 240 mg (3 million units [MU]) every 8 h indicated that colistin has a long half-life, resulting in insufficient concentrations for the first 12 to 48 h after initiation of treatment. A loading dose would therefore be beneficial. The aim of this study was to evaluate CMS and colistin pharmacokinetics following a 480-mg (6-MU) loading dose in critically ill patients and to explore the bacterial kill following the use of different dosing regimens obtained by predictions from a pharmacokinetic-pharmacodynamic model developed from an in vitro study on Pseudomonas aeruginosa . The unbound fractions of colistin A and colistin B were determined using equilibrium dialysis and considered in the predictions. Ten critically ill patients (6 males; mean age, 54 years; mean creatinine clearance, 82 ml/min) with infections caused by multidrug-resistant Gram-negative bacteria were enrolled in the study. The pharmacokinetic data collected after the first and eighth doses were analyzed simultaneously with the data from the previous study (total, 28 patients) in the NONMEM program. For CMS, a two-compartment model best described the pharmacokinetics, and the half-lives of the two phases were estimated to be 0.026 and 2.2 h, respectively. For colistin, a one-compartment model was sufficient and the estimated half-life was 18.5 h. The unbound fractions of colistin in the patients were 26 to 41% at clinical concentrations. Colistin A, but not colistin B, had a concentration-dependent binding. The predictions suggested that the time to 3-log-unit bacterial kill for a 480-mg loading dose was reduced to half of that for the dose of 240 mg.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of the opioid peptide DAMGO in rat plasma, as well as DAMGO and the microdialysis recovery calibrator [(13)C(2),(15)N]-DAMGO in microdialysis samples, is described. The microdialysis samples consisted of 15 μL Ringer solution containing 0.5% bovine serum albumin. Pretreatment of the samples involved protein precipitation with acetonitrile followed by dilution with 0.01% formic acid. The lower limits of quantification were 0.52 ng/mL and 0.24 ng/mL for DAMGO and [(13)C(2),(15)N]-DAMGO respectively and the response was linear up to 5000 fold higher concentrations. The plasma samples (50 μL) were precipitated with acetonitrile containing the isotope labeled analog [(13)C(2),(15)N]-DAMGO as internal standard. The method was linear in the range of 11-110,000 ng/mL. The separations were conducted on a HyPurity C18 column, 50×4.6 mm, 3 μm particle size, with a mobile phase consisting of acetonitrile, water and formic acid to the proportions of 17.5:82.5:0.01. Low energy collision dissociation tandem mass spectrometric (CID-MS/MS) analysis was carried out in the positive ion mode using multiple reaction monitoring (MRM) of the following mass transitions: m/z 514.2→453.2 for DAMGO and m/z 517.2→456.2 for [(13)C(2),(15)N]-DAMGO. The intra-day precision and accuracy did not exceed 5.2% and 93-104% for both compounds and sample types described. The inter-day precision an accuracy were6.8% and 95-105% respectively. The method described is simple, reproducible and suitable for the analysis of small sample volumes at low concentrations.

Objective To describe longitudinally the development of micturition patterns in children who are potty trained early. Subjects and methods Healthy children in Vietnam from newborn up to 1 year were investigated every 3 months. This included mapping of the micturition pattern through the 4-h micturition observation method. Results Forty-seven children participated in all five investigations. At the ages of newborn, 3, 6, 9 and 12 months, 70%, 82%, 91%, 99% and 100% of the mothers, respectively, were potty training their children. Mean lowest bladder volume (voided volume + residual urine) triggering a micturition was 18 ml at the age of 2 weeks compared to 33 ml at 12 months. Post-void residual urine decreased according to age, and already at 9 months was less than 7 ml (mean 0.7 ml). Conclusions The dyscoordination between the sphincter and detrusor muscle seems to have already disappeared at the age of 9 months in infants who are potty trained very early. These findings suggest that potty training can be beneficial in small children with urinary tract infections or renal scars.

An analytical method for quantitation of colistin A and colistin B in plasma and culture medium is described. After protein precipitation with acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), the supernatants were diluted with 0.03% TFA. The compounds were separated on an Ultrasphere C18 column, 4.6 mm x 250 mm, 5 microm particle size with a mobile phase consisting of 25% ACN in 0.03% TFA and detected with tandem mass spectrometry. The instrument was operating in ESI negative ion mode and the precursor-product ion pairs were m/z 1167.7-->1079.6 for colistin A and m/z 1153.7-->1065.6 for colistin B. The lower limit of quantification (LLOQ) for 100 microL plasma was 19.4 and 10.5 ng/mL for colistin A and B, respectively, with CV

Objective To investigate the relationship between incidence and nature of life events within families and age of bladder control acquisition in healthy children. Subjects and methods Thirty-five parents of 36, 6-year-old children were interviewed using the Coddington life events questionnaire. Results The children had experienced a total of 185 life events (mean 5, median 4.5, range 1–12) before the time of dryness. The most common were related to childcare/occupation of parent (51%), illness/injury/death (24%), family composition (16%) and living conditions (9%). There was a strong correlation between the number of life events and the age of dryness; the more life events and the older the child was when experiencing them, the later the child became dry. The only single event that was significantly associated with a later age of dryness was the birth of a sister or brother. In general, children had reacted positively (39%) or neutrally, but to 30% of events there was a negative reaction. There was a correlation between the reaction of the child and adaptation to the life event, and if the child had difficulty adapting to an event, that child became day dry at a later age. Conclusion Although life events are a natural part of daily life, this study supports the conclusion that a large number of events and events to which the child has difficulty adapting can be of importance for the age of acquisition of bladder control.

A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were

Sensitive and reproducible methods for the determination of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue by liquid chromatography/mass spectrometry are described. Deuterated analogs of the substances were used as internal standards. Samples in Ringer solution were analyzed by direct injection of 10 microL Ringer solution diluted by an equal volume of water. The limit of quantification was 0.5 ng/mL and the method was linear in the range of 0.5-150 ng/mL for all substances. To analyze oxycodone and oxymorphone in rat plasma, 50 microL of plasma were precipitated with acetonitrile, and the supernatant was directly injected onto the column. To analyze oxycodone, oxymorphone and noroxycodone in rat plasma, 100 microL of rat plasma were subjected to a C18 solid-phase extraction (SPE) procedure, before reconstituting in mobile phase and injection onto the column. For both methods the limit of quantification in rat plasma was 0.5 ng/mL and the methods were linear in the range of 0.5-250 ng/mL for all substances. To analyze the content of oxycodone, oxymorphone and noroxycodone in rat brain tissue, 100 microL of the brain homogenate supernatant were subjected to a C18 SPE procedure. The limit of quantification of oxycodone was 20 ng/g brain, and for oxymorphone and noroxycodone 4 ng/g brain, and the method was linear in the range of 20-1000 ng/g brain for oxycodone and 4-1000 ng/g brain for oxymorphone and noroxycodone. All methods utilized a mobile phase of 5 mM ammonium acetate in 45% acetonitrile, and a SB-CN column was used for separation. The total run time of all methods was 9 min. The intra-day precision and accuracy were

AIM: Our goal was to investigate whether artemisinin autoinduction is caused by an increase in cytochrome P450 (CYP) 2B6 or CYP2C9 activities, we evaluated the effects of multiple-dose artemisinin administration on S-mephenytoin N-demethylation in healthy subjects. METHODS: Fourteen subjects, 6 poor metabolizers of CYP2C19 and 8 extensive metabolizers, received a single oral dose of 200 mg racemic mephenytoin (CYP2B6 in vivo marker) before (day -28) and during multiple-dose artemisinin administration (250 mg/d orally for 9 days and 500 mg on the tenth day). A 500-mg single dose of artemisinin was administered on day -28. The CYP2C9 in vivo marker tolbutamide was administered on day -28 and on days 7, 12, and 17 to monitor the minor involvement of CYP2C9 in S-mephenytoin N-demethylation. RESULTS: Artemisinin oral clearance increased 5.3-fold (P

Using a liquid chromatography-tandem mass spectrometry method, the serum and cerebrospinal fluid (CSF) concentrations of colistin were determined in patients aged 1 months to 14 years receiving intravenous colistimethate sodium (60,000 to 225,000 IU/kg of body weight/day). Only in one of five courses studied (a 14-year-old receiving 225,000 IU/kg/day) did serum concentrations exceed the 2 μg/ml CLSI/EUCAST breakpoint defining susceptibility to colistin for Pseudomonas and Acinetobacter. CSF colistin concentrations were

Purpose: We describe the development of voiding patterns and bladder control in healthy children during the first 3 years of life. Materials and Methods: We determined voiding patterns, bladder capacity and post-void residual urine volume per 4 hours individually and noninvasively every 3 months in 36 female and 23 male healthy infants using the 4-hour voiding observation. Results: Voiding frequency decreased slowly from 5 to 2 voiding episodes per 4 hours from ages 3 months to 3 years. We noted interrupted voiding in 33% of subjects at age 3 months but this condition was rare after age 2 years. Voiding during sleep occurred mainly during the first 7 months of life and did not continue after age 18 months. Bladder capacity increased from a median of 52 to 67, 68 and 123 ml. during years 1 to 3, respectively. As measured by post-void residual urine volume, bladder emptying was unchanged during years 1 and 2 but it decreased during year 3 (median 6 versus 0 and mean 4 versus 3 ml. per 4 hours). Conclusions: During the first 3 years of life the number of voiding episodes, including interrupted voiding, post-void residual urine and voiding during sleep, decreased while bladder capacity increased.

Aims The purpose of this human intestinal perfusion study was to investigate the effect of ketoconazole on the jejunal permeability and first-pass metabolism of (R)- and (S)-verapamil in humans. Methods A regional single-pass perfusion of the jejunum was performed using a Loc-I-Gut® perfusion tube in six healthy volunteers. Each perfusion lasted for 200 min and was divided into two periods of 100 min each. The inlet concentration of (R/S)-verapamil was 120 mg l−1 in both periods, and ketoconazole was added at 40 mg l−1 in period 2. (R/S)-verapamil was also administered as a short intravenous infusion of 5 mg, over a period of 10 min. The appearance ratios of the CYP3A formed metabolites (R)- and (S)-norverapamil were also estimated in the outlet jejunal perfusate. Results The effective jejunal permeability (Peff) of both (R)- and (S)-verapamil was unaffected by the addition of ketoconazole in period 2 suggesting that ketoconazole had no effect on the P-glycoprotein mediated efflux. However, the appearance ratio of both (R)- and (S)-norverapamil in the outlet jejunal perfusate decreased in the presence of ketoconazole. The rate of absorption into plasma of (R)- and (S)-verapamil increased despite the low dose of ketoconazole added, indicating an inhibition of the gut wall metabolism of (R/S)-verapamil by ketoconazole. Conclusions Ketoconazole did not affect the jejunal Peff of (R/S)-verapamil, but it did increase the overall transport into the systemic circulation (bioavailability), probably by inhibition of the gut wall metabolism of verapamil. This might be due to ketoconazole being less potent as an inhibitor of P-glycoprotein than of CYP3A4 in vivo in humans.

The V3 loop of the human immunodeficiency virus (HIV) surface protein, gp 120, constitutes a principal neutralizing determinant. HIV strains lacking a naturally conserved N-linked oligosaccharide (at position 306) within the V3 loop are highly sensitive to neutralization. We subjected molecular clones of HIV(LAI) lacking this 306N-glycan to in vitro immune selection with MAbs directed against the V3 loop. In all, ten clones were characterized, and all proved resistant to V3-directed neutralization. Sequencing of the V3 loop revealed that six of the clones had become resistant at least partly by reacquisition of the 306N-glycan. Only two of the clones possessed mutations within the binding site of the antibody itself, while the two remaining clones did not display changes within the V3 loop itself. Thus, HIV strains lacking the 306N-glycan primarily develop resistance to V3-directed neutralization through acquisition of the specific oligosaccharide. This demonstrates that protein glycosylation can be a primary modifier of virus antigenicity of possible importance for the interaction of HIV with the host immune response.

A conserved N-glycan present within the V3 loop of gp120 modulates the sensitivity to neutralization by antibodies directed to the V3 loop. A glycan-deficient mutant of HIV LAI , designated HIV A308 , displayed a 100-fold increase in sensitivity to neutralization by anti-V3 MAb NEA-9205 compared to wild-type HIV LAI . This difference in sensitivity was not caused by an alteration of the antibody binding site itself, as NEA-9205 had equal affinity for both wild-type and mutant monomeric gp120. In contrast, virion-associated wild-type gp120 was immunoprecipitated less efficiently with NEA-9205 than virion-associated mutant gp120. This difference was completely abrogated, if immunoprecipitation were carried out in the presence of detergent. Furthermore, treatment of virion preparations with detergent exposed the C-terminal D7324 epitope, which is inaccessible on virion-associated gp120 but readily accessible on monomeric, soluble gp120. Finally, both wild-type and mutant monomeric, soluble gp120 were precipitated equally efficiently by NEA-9205 in the absence of detergent. Thus, the NEA-9205 epitope was readily accessible on monomeric gp120 regardless of the presence of the 306 N -glycan, and inaccessibility of the NEA-9205 epitope imparted by the 306 N -glycan was observed only on the intact envelope oligomer.

It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation of such threonine or serine residues could decrease the sensitivity to infectivity inhibition by Tn or sialosyl-Tn specific antibodies. All potentially O-glycosylated threonine and serine residues in the V3 loop of cloned HIV-1 BRU were mutagenized to alanine thus abrogating any O-glycosylation at these sites. Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O-linked oligosaccharides Tn or sialosyl-Tn. Deletion of the N-linked oligosaccharide at N306 increased the neutralization sensitivity to antibodies specific for the tip of the loop, which indicates that N-linked glycosylation modulates the accessibility to this immunodominant epitope. However, none of the mutants with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp 120.

Site-directed mutagenesis was used to study the biological significance of three N-linked glycans (linked to Asn406, Asn448, and Asn463), situated in the CD4-binding region of gp120. Mutagenesis was carried out in a phage M13 system, and the mutated env genes were inserted into recombinant vaccinia virus (r-vaccinia virus). To evaluate if the level of expression affected the biological phenotype of mutant gp120, we expressed the envelope glycoproteins using either a weak (7.5 K) or a strong (11 K) promoter of vaccinia virus. The expression of mutated env proteins was analyzed after infecting CD4-expressing HeLa cells with the r-vaccinia virus, by monitoring the ability of the infected cells to generate CD4-dependent syncytia. Env gene products lacking all three glycans as well as env gene products lacking different permutations of one or two glycans were analyzed. All mutated gp120 species had the expected electrophoretical mobility as anticipated from elimination of one, two, and three N-linked glycans, respectively. Moreover, all mutant env gene products demonstrated the same capacity to induce formation of syncytia, irrespective of using the weak or strong promoter for expression. These data indicate that the three N-linked glycans studied are dispensable for HIV env gene products to function in CD4-binding and the subsequent fusion step.

Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.

Diphenhydramine (DPHM) and oxycodone are weak bases that are able to form cations. Both drugs show active uptake at the blood-brain barrier (BBB). There is thus a possibility for a pharmacokinetic interaction between them by competition for the same uptake transport system. The experiments of the present study were designed to study the transport of DPHM across the BBB and its interaction with oxycodone in vitro and in vivo. In vitro ,t he interaction between the drugs was studied using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13 cells). The in vivo relevance of the in vitro findings was studied in rats using brain and blood microdialysis. DPHM was actively transported across the BBB in vitro (TR-BBB13 cells). Oxycodone competitively inhibited DPHM uptake with a Ki value of 106 :M. DPHM also competitively inhibited oxycodone uptake with a Ki value of 34.7 :M. In rats, DPHM showed fivefold higher unbound concentration in brain interstitial fluid (ISF) than in blood, confirming a net active uptake. There was no significant interaction between DPHM and oxycodone in vivo. This accords with the results of the in vitro experiments because the unbound plasma concentrations that could be attained in vivo, without causing adverse effects, were far below the Ki values. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3912-3923, 2011

The aim was to develop a model to describe the population pharmacokinetics of nevirapine in South African human immunodeficiency virus (HIV)-infected patients who were taking nevirapine-based antiretroviral therapy concomitantly or in the absence of rifampicin-based tuberculosis therapy.Patients were divided into two groups: (1) patients receiving nevirapine-containing antiretroviral regimen (200 mg twice daily) and continuation phase rifampicin-containing tuberculosis therapy (n = 27) in whom blood samples were obtained before and not less than 14 days after they completed tuberculosis therapy; (2) patients without tuberculosis who were receiving a nevirapine-containing antiretroviral regimen for at least 3 weeks (n = 26). The population pharmacokinetics of nevirapine was described using nonlinear mixed effects modelling with NONMEM software. Based on the developed model, plasma concentration profiles after 300, 400 and 500 mg of nevirapine twice daily were simulated.Concomitant administration of rifampicin increased nevirapine oral clearance (CL/F) by 37.4% and reduced the absorption rate constant (k(a)) by almost sixfold. Rifampicin reduced the nevirapine average minimum concentration by 39%. Simulated doses of 300 mg twice daily elevated nevirapine concentrations above subtherapeutic levels in most patients, with minimum exposure above the recommended maximum concentration. The area under the concentration-time curve of 12-hydroxynevirapine was not different in the presence of rifampicin. 2-, 3- and 8-Hydroxynevirapine were not detectable (LLOQ = 0.025 mg/L).The developed model adequately describes nevirapine population pharmacokinetics in a South African population when taken with/and in the absence of rifampicin treatment. The simulations suggest that an increased dose of 300 mg twice daily would achieve adequate nevirapine concentrations in most patients during rifampicin-containing treatment for tuberculosis.

The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes are affected by artemisinin and to what degree the artemisinin derivatives differ with respect to their respective induction and inhibition capacity. Seventy-five healthy adults were randomized to receive therapeutic oral doses of artemisinin, dihydroartemisinin, arteether, artemether or artesunate for 5 days (days 1-5). A six-drug cocktail consisting of caffeine, coumarin, mephenytoin, metoprolol, chlorzoxazone and midazolam was administered orally on days -6, 1, 5 and 10 to assess the activities of CYP1A2, CYP2A6, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Four-hour plasma concentrations of parent drugs and corresponding metabolites and 7-hydroxycoumarin urine concentrations were quantified by liquid chromatography-tandem mass spectrometry. The 1-hydroxymidazolam/midazolam 4-h plasma concentration ratio (CYP3A) was increased on day 5 by artemisinin [2.66-fold (98.75% CI: 2.10-3.36)], artemether [1.54 (1.14-2.09)] and dihydroartemisinin [1.25 (1.06-1.47)] compared with day -6. The S-4'-hydroxymephenytoin/S-mephenytoin ratio (CYP2C19) was increased on day 5 by artemisinin [1.69 (1.47-1.94)] and arteether [1.33 (1.15-1.55)] compared with day -6. The paraxanthine/caffeine ratio (CYP1A2) was decreased on day 1 after administration of artemisinin [0.27 (0.18-0.39)], arteether [0.70 (0.55-0.89)] and dihydroartemisinin [0.73 (0.59-0.90)] compared with day -6. The alpha-hydroxymetoprolol/metoprolol ratio (CYP2D6) was lower on day 1 compared with day -6 in the artemisinin [0.82 (0.70-0.96)] and dihydroartemisinin [0.83 (0.71-0.96)] groups, respectively. In the artemisinin-treated subjects this decrease was followed by a 1.34-fold (1.14-1.58) increase from day 1 to day 5. These results show that intake of artemisinin antimalarials affect the activities of several principal human drug metabolizing CYP450 enzymes. Even though not significant in all treatment groups, changes in the individual metrics were of the same direction for all the artemisinin drugs, suggesting a class effect that needs to be considered in the development of new artemisinin derivatives and combination treatments of malaria.

The aim of this study was to investigate whether, the vaccine Priorix causes less immediate injection pain than MMR-II in vaccination of infants aged 18-24 months who were offered vaccine against measles, mumps and rubella. The infants were randomised into one of the two vaccine groups (Priorix/MMR-II in a double-blind study. One observer assessed pain, using CHEOPS (Children's Hospital Eastern Ontario Pain scale), and the parent used the Visual Analogue Pain scale (VAS). Pain assessment was made in 295 infants. Mean age was 19.08 months. Mean VAS value was 2.3/5.2 for Priorix and MMR-II, respectively (p

A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.

Purpose: We describe the voiding pattern and acquisition of bladder control in healthy children up to age 6 years. Materials and Methods: We determined age for daytime and nighttime dryness, voiding patterns, voiding volumes and post-void residual volume per 4 hours individually and noninvasively every 3 months up to age 3 years and every 6 months up to age 6 years in 36 female and 23 male patients using 4-hour voiding observation and uroflowmetry/ultrasound. Results: Median age for attaining daytime and nighttime dryness was 3.5 and 4 years, respectively. No significant difference was found between girls and boys. All but 1 child attained daytime dryness an average of 10 months before attaining nighttime dryness. Bladder sensation was reported in 31%, 79% and 100% of patients at ages 2, 3 and 4 years, respectively. Median bladder capacity was 67 ml, 123 ml and 140 ml at years 1, 3 and 6, respectively. Median post-void residual volume was 5.5 ml, 0 ml and 2 ml at ages 1, 3 and 6 years, respectively. Conclusions: Today bladder control is acquired at a later stage despite earlier awareness of bladder function. The occurrence of bladder sensation from age 1.5 years motivates an earlier start with toilet training. Infants with small post-void residual volume at age 6 months or large bladder capacity will probably attain daytime dryness earlier than those with large post-void residual volume at age 6 months or small bladder capacity.

Sensitive enantioselective liquid chromatographic assays using tandem mass spectrometric detection were developed and validated for the determination of S-cetirizine (S-CZE) and R-cetirizine (R-CZE) in guinea pig plasma, brain tissue, and microdialysis samples. Enantioselective separation was achieved on an alpha1-acid glycoprotein column within 14 min for all methods. A cetirizine analog, ucb 20028, was used as internal standard. Cetirizine and the internal standard were detected by multiple reaction monitoring using transitions m/z 389.1 --> 200.9 and 396.1 --> 276.1, respectively. The samples were prepared using protein precipitation with acetonitrile. For guinea pig plasma, the assay was linear over the range 0.25-5000 ng/mL for both S-CZE and R-CZE, with a lower limit of quantification (LLOQ) of 0.25 ng/mL. For the brain tissue and microdialysis samples, the assays were linear over the range 2.5-250 ng/g and 0.25-50 ng/mL, respectively, and the LLOQ values were 2.5 ng/g and 0.25 ng/mL, respectively. The intra- and inter-day precision values were < or =7.1% and < or =12.6%, respectively, and the intra- and inter-day accuracy varied by less than +/-8.0% and +/-6.0% of the nominal value, respectively, for both enantiomers in all the matrices investigated.

Sensitive and reproducible methods for the determination of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue by liquid chromatography/mass spectrometry are described. Deuterated analogs of the substances were used as internal standards. Samples in Ringer solution were analyzed by direct injection of 10 microL Ringer solution diluted by an equal volume of water. The limit of quantification was 0.5 ng/mL and the method was linear in the range of 0.5-150 ng/mL for all substances. To analyze oxycodone and oxymorphone in rat plasma, 50 microL of plasma were precipitated with acetonitrile, and the supernatant was directly injected onto the column. To analyze oxycodone, oxymorphone and noroxycodone in rat plasma, 100 microL of rat plasma were subjected to a C18 solid-phase extraction (SPE) procedure, before reconstituting in mobile phase and injection onto the column. For both methods the limit of quantification in rat plasma was 0.5 ng/mL and the methods were linear in the range of 0.5-250 ng/mL for all substances. To analyze the content of oxycodone, oxymorphone and noroxycodone in rat brain tissue, 100 microL of the brain homogenate supernatant were subjected to a C18 SPE procedure. The limit of quantification of oxycodone was 20 ng/g brain, and for oxymorphone and noroxycodone 4 ng/g brain, and the method was linear in the range of 20-1000 ng/g brain for oxycodone and 4-1000 ng/g brain for oxymorphone and noroxycodone. All methods utilized a mobile phase of 5 mM ammonium acetate in 45% acetonitrile, and a SB-CN column was used for separation. The total run time of all methods was 9 min. The intra-day precision and accuracy were11.3% and+/-14.9%, respectively, and the inter-day precision and accuracy were14.9% and+/-6.5%, respectively, for all the concentrations and matrices described.

Cefuroxime is a second-generation cephalosporin used against different kinds of bacterial infections. To be able to optimize the dosing it is necessary to characterize the pharmacokinetics of cefuroxime which requires a selective and sensitive analytical method for cefuroxime in plasma or serum. A new rapid liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) method, using cefotaxime as internal standard, was developed for analysis of cefuroxime in human serum. The work-up procedure consisted of protein precipitation with acetonitrile/cefotaxime, and after centrifugation the supernatant was dissolved in mobile phase. The sample was injected on a SB-CN column and the detection was performed using tandem mass spectrometry (MS/MS). The limit of quantification was determined to 0.025 microg/mL. The method was linear in the range 0.025-50 microg/mL with a coefficient of correlation >0.999. The limit of quantification and intra-day variability were found to be the same for plasma samples, which indicates that the method is valid for serum as well as plasma samples.

A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C(2) column in tandem with a chiral alpha(1)-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation

Purpose The aim of this study was to describe parents’ experience of how their children achieved dryness. Material and Methods Twenty-two parents of 21 healthy children were interviewed about the process. The transcribed interviews were analyzed using the qualitative content analysis, according to Krippendorf. Results From the interview text eight subcategories were identified about the parents´experiences of how their children became dry: Making a desicion and Having time, Creating a positive athmospere and Doing activities, Cognitive skills and Personal traits of the children, Unspoken rules and Comparing to fitting in. Conclusions How children became dry, as reported mainly by their mothers, seems to be influenced by knowing the time had come, implementing new daily routines, the child's willingness, and a desire to be like others.