Population Pharmacokinetics of Selumetinib and Its Metabolite N-desmethyl-selumetinib in Adult Patients With Advanced Solid Tumors and Children With Low-Grade Gliomas.

Selumetinib (AZD6244, ARRY-142886), a mitogen activated protein kinases (MEK1 and 2) inhibitor, has been granted orphan drug designation for differentiated thyroid cancer. The primary aim of this analysis was to characterize the population pharmacokinetics of selumetinib and its active metabolite N-desmethyl-selumetinib in patients with cancer. Concentration-time data from adult and pediatric clinical trials were pooled to develop a population pharmacokinetic model using a sequential approach where selumetinib and N-desmethyl-selumetinib data were modeled separately. A sequential zero- and first-order absorption with lag time with a two-compartment model for selumetinib and a two-compartment model for N-desmethyl-selumetinib best described the concentration-time data. Intrapatient variability in absorption was higher than interpatient variability. The apparent drug clearance (CL/F) from the central compartment was 13.5 L/hr (RSE 4.9%). Significant covariates for CL/F were age, alanine aminotransferase, and body surface area. This study confirms that flat dosing is appropriate in adults, whereas body-surface area based dosing should be used in pediatric patients.

Recyclable chemosensor for oxalate based on bimetallic complexes of a dinucleating bis(iminopyridine) ligand.

Herein we describe bimetallic di-nickel and di-copper complexes [Ni2(L)Br4] (1) and [Cu2(L)Br4(NCMe)2] (2) (L = (1E,1'E)-N,N'-(1,4-phenylenebis(methylene))bis(1-(6-(2,4,6-triisopropylphenyl)pyridin-2-yl)methanimine)) that bind oxalate intramolecularly to form [Ni2(L)Br2(C2O4)(NCMe)] (3) and [Cu2(L)Br2(C2O4)] (4). For the di-nickel complex 1, oxalate incorporation is accompanied by a significant colour change, from red-pink (1) to deep green (3). Mass spectrometric experiments demonstrate that the compound 1 is selective for oxalate versus related mono- and di-carboxylates tested. Oxalate can be released by the addition of slight excess of calcium bromide that forms insoluble calcium oxalate and restores the original Ni2(L)Br4 species. The product of the oxalate release was crystallized as [Ni2(L)Br4]·CaBr2(THF)4 species.

Modulation of spontaneous baroreflex control of heart rate and indexes of vagal tone by passive calf muscle stretch during graded metaboreflex activation in humans.

We examined whether spontaneous baroreflex modulation of heart rate and other indexes of cardiac vagal tone could be altered by passive stretch of the human calf muscle during graded concurrent activation of the muscle metaboreflex. Ten healthy subjects performed four trials: a control trial, resting for 1.5 min (0% trial); or 1.5 min of one-legged isometric plantar flexor exercise at 30, 50, and 70% maximal voluntary contraction. The incremental increases in blood pressure (BP) caused were then partially sustained by subsequent local circulatory occlusion (CO). After 3.5 min of CO alone, sustained calf stretch and CO were applied for 3 min. Spontaneous baroreflex sensitivity (SBRS) was progressively decreased with increasing exercise intensity (P < 0.05). During CO, stretch decreased SBRS and increased BP similarly in all trials (P < 0.05). Within 15 s of stretch onset, heart rate (HR) increased by 6 +/- 1, 6 +/- 1, 8 +/- 1, and 6 +/- 2 beats/min in the 0, 30, 50, and 70% trials, respectively (P < 0.05), and root mean square of successive differences was decreased from CO-alone levels (P < 0.05). During the second and third minutes of stretch, HR fell back but remained significantly above CO levels, and common coefficient of variance of R-R interval decreased progressively with increasing prior exercise intensity (P < 0.05; 70% trial). This suggests that passive stretch of the human calf muscles decreases cardiac vagal outflow irrespective of the levels of BP increase caused by muscle metaboreflex activation and implies that central modulation of baroreceptor input, mediated by the actions of stretch-activated mechanoreceptive muscle afferent fibers, continues.

Genotyping spring viraemia of carp virus and other piscine vesiculo-like viruses using reverse hybridisation.

A simple nylon membrane-based DNA macroarray was developed to genotype spring viraemia of carp virus (SVCV) and related viruses. Twenty-six viruses were genotyped using the array, and the results were confirmed by phylogenetic analysis of a 426 bp partial glycoprotein gene sequence. The array was not only capable of discriminating between the 4 main genogroups of cyprinid vesiculo-type viruses described previously, but also accurately sub-type the SVC viruses assigned to Genogroup I. The assay offers a practical solution for diagnostic laboratories that currently lack a sequencing capability to confirm the nature of PCR products generated in suspected SVCV cases.

Cardiovascular responses to human calf muscle stretch during varying levels of muscle metaboreflex activation.

The purpose of the present study was to investigate the cardiovascular responses to muscle metaboreflex- and concurrent muscle stretch-induced mechanoreflex activation. Eight subjects (7 males, 1 female) performed 90 s of isometric calf plantarflexion at 0, 30, 50 and 70% of maximum voluntary contraction. During exercise and for 3.5 min postexercise, circulatory occlusion (PECO) was ensured by inflation of a thigh cuff. After 90 s of PECO the calf muscle was stretched for 60 s (Stretch). Heart rate (HR; assessed from ECG), blood pressure (BP; Finapres) and phase of respiratory cycle were recorded. Exercise increased diastolic BP (DBP) from rest by 1+/-0.8, 14+/-2.5, 29+/-3.9 and 35+/-3.6 mmHg, during the 0, 30, 50 and 70% conditions, respectively (ANOVA rest versus exercise, P<0.05). During PECO DBP remained elevated, by 2+/-0.4, 8+/-0.3, 12+/-0.3 and 13+/-0.9 mmHg, respectively. Stretch produced a further increase in DBP that was not different between conditions (3+/-1.4, 2+/-0.8, 3+/-1.0 and 3+/-0.9 mmHg, for the 0, 30, 50 and 70%, respectively). HR increased during exercise but returned to baseline during PECO. HR increased at Stretch onset in all conditions. No EMG was detected from the gastrocnemius and soleus during Stretch. Our data show that the cardiovascular responses to human calf Stretch are independent of the level of concurrent muscle metaboreflex activation.

Cardiovascular responses to external compression of human calf muscle vary during graded metaboreflex stimulation.

This study investigated the cardiovascular response to a standard external muscle compression during concomitant muscle metaboreflex stimulation of varying intensity in human calf muscle. Eleven healthy male subjects (mean (s.d.) age, 26 (5.6) years; height, 177 (5) cm; weight, 74.3 (6.8) kg) were seated in an isometric dynamometer with the angle of the knee at 90 deg, and the angle of the ankle at 85 deg. After a 150-s rest period, subjects were asked to either perform isometric plantar flexion at 20, 30, 40, 50, 60, 70 or 80% of previously determined maximum isometric contractile force (MVC) for 90 s, or to sit at rest for this period. A thigh cuff maintained circulatory occlusion throughout the exercise period and for 180 s post exercise. After 60 s of post-exercise circulatory occlusion (PECO), a calf cuff was inflated to 300 mmHg for 60 s followed by a further 60 s of PECO alone after which the thigh cuff was deflated. During PECO the mean arterial pressure (MAP) increase from rest was dependent upon the preceding exercise intensity (P < 0.001). Compression elicited a further significant change in MAP, and the magnitude of this change from the PECO baseline was also dependent upon the preceding exercise intensity (P < 0.01). These results are compatible with activation of a metabolically sensitised population of mechanoreceptive afferents in human muscle during external compression.

Comparison of extraction of a beta-blocker from plasma onto a molecularly imprinted polymer with liquid-liquid extraction and solid phase extraction methods.

An optimised solid phase extraction (SPE) method developed for the extraction of a structural analogue of the beta-blocking drug propranolol from plasma utilising a molecularly imprinted polymer (MIP) has been compared with methods based on conventional liquid-liquid extraction (LLE), and SPE using C18-bonded and immobilised phenyl boronic acid (PBA). All four methods could be used for the extraction of the analyte with acceptable accuracy and precision. The MIP-based method, unlike the other methods required a protein precipitation step prior to extraction to eliminate the effects of co-extracted protein. The best performance was seen with the LLE method followed by SPE on the C18 phase. The MIP-based method represented no advantage over the comparator methods for this analyte. Indeed the performance of the MIP-based method was marginally worse as leaching of low level template impurities prevented detection of the target analyte at low concentrations (5 ngmL(-1)). This relatively poorer performance was evident as worse accuracy at low concentrations with a consequent higher limit of quantification than the conventional methods.

The effect of erythromycin on the pharmacokinetics of rosuvastatin.

RATIONALE OBJECTIVE: To examine in vivo the effect of erythromycin on the pharmacokinetics of rosuvastatin [an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase]. Erythromycin is a potent inhibitor of CYP3A4 that markedly increases circulating levels of some other HMG-CoA reductase inhibitors. METHODS: In this randomised, double-blind, two-way cross-over, placebo-controlled trial 14 healthy volunteers were given 500 mg erythromycin or placebo four times daily for 7 days. A single dose of 80 mg rosuvastatin was co-administered on day 4 of dosing. Plasma concentrations of rosuvastatin and active and total HMG-CoA reductase inhibitors were measured up to 96 h after dosing. RESULTS: Eleven volunteers had data available from both dosing periods. There was no increase in rosuvastatin plasma exposure following co-administration with erythromycin compared to placebo. In fact, following co-administration with erythromycin, rosuvastatin geometric least square mean AUC((0-t)) and C(max) were 20% and 31%, respectively, lower than with placebo. Individual treatment ratios for AUC((0-t)) ranged from 0.48 to 1.17, and for C(max) ranged from 0.33 to 2.19. Essentially all of the circulating active HMG-CoA reductase inhibitors and most (>94%) of the total inhibitors were accounted for by rosuvastatin. Erythromycin did not affect the proportion of circulating active or total inhibitors accounted for by circulating rosuvastatin. CONCLUSIONS: Erythromycin did not produce any increase in rosuvastatin plasma exposure. This indicates that CYP3A4 metabolism is not an important clearance mechanism for rosuvastatin, a result consistent with previous findings. The small decreases in rosuvastatin AUC((0-t)) and C(max) that occurred as a consequence of short-term treatment with erythromycin are unlikely to have relevance to long-term treatment with rosuvastatin.

Pharmacokinetics and pharmacodynamics of rosuvastatin in subjects with hepatic impairment.

OBJECTIVE: To assess the effect of chronic hepatic impairment on rosuvastatin disposition, pharmacodynamic activity and tolerability. METHODS: This was an open-label, non-randomised, parallel-group trial. Six subjects were enrolled in each of three hepatic-function strata: Child-Pugh class A (CP-A, mild impairment), Child-Pugh class B (CP-B, moderate impairment) and normal hepatic function; the latter two strata were age, weight, race, sex and smoking history matched. All subjects were given rosuvastatin 10 mg for 14 days. RESULTS: In subjects with CP-A, and in four of six subjects with CP-B, rosuvastatin steady-state AUC(0-24) and C(max) were similar to subjects with normal hepatic function (geometric mean values 60.7 ng h/ml and 6.02 ng/ml, respectively). Two of six subjects with CP-B who had the highest CP scores (i.e. the highest degrees of hepatic impairment) had the highest AUC(0-24) (128 ng h/ml and 242 ng h/ml) and C(max) (23.4 ng/ml and 96.7 ng/ml) values. Low-density lipoprotein cholesterol (LDL-C) was decreased in all strata, but the response was more variable in the CP-B group. Rosuvastatin was well tolerated, and the safety profile was similar in subjects with hepatic impairment and normal hepatic function. CONCLUSION: In most subjects with mild-to-moderate hepatic impairment, the steady-state pharmacokinetics of rosuvastatin were similar to subjects with normal hepatic function (more extensive hepatic impairment may increase systemic exposure to rosuvastatin), and most had LDL-C reductions similar to subjects with normal hepatic function.

Isolation of a rhabdovirus during outbreaks of disease in cyprinid fish species at fishery sites in England.

A virus was isolated during disease outbreaks in bream Abramis brama, tench Tinca tinca, roach Rutilis rutilis and crucian carp Carassius carassius populations at 6 fishery sites in England in 1999. Mortalities at the sites were primarily among recently introduced fish and the predominant fish species affected was bream. The bream stocked at 5 of the 6 English fishery sites were found to have originated from the River Bann, Northern Ireland. Most fish presented few consistent external signs of disease but some exhibited clinical signs similar to those of spring viraemia of carp (SVC), with extensive skin haemorrhages, ulceration on the flanks and internal signs including ascites and petechial haemorrhages. The most prominent histopathological changes were hepatocellular necrosis, interstitial nephritis and splenitis. The virus induced a cytopathic effect in tissue cultures (Epithelioma papulosum cyprini [EPC] cells) at 20 degrees C and produced moderate signals in an enzyme immunoassay (EIA) for the detection of SVC virus. The virus showed a close serological relationship to pike fry rhabdovirus in both EIA and serum neutralisation assays and to a rhabdovirus isolated during a disease outbreak in a bream population in the River Bann in 1998. A high degree of sequence similarity (> or = 99.5% nucleotide identity) was observed between the English isolates and those from the River Bann. Experimental infection of juvenile bream, tench and carp with EPC cell-grown rhabdovirus by bath and intraperitoneal injection resulted in a 40% mortality of bream in the injection group only. The virus was re-isolated from pooled kidney, liver and spleen tissue samples from moribund bream. The field observations together with the experimental results indicate that this rhabdovirus is of low virulence but may have the potential to cause significant mortality in fishes under stress.

The effect of fluconazole on the pharmacokinetics of rosuvastatin.

OBJECTIVE: To examine the effect of fluconazole, a potent inhibitor of CYP2C9 and CYP2C19, on the pharmacokinetics of rosuvastatin in healthy volunteers. Significantly increased plasma concentrations of fluvastatin have been observed following co-administration with fluconazole. METHODS: This was a randomised, double-blind, two-way crossover, placebo-controlled trial. Healthy male volunteers ( n=14) were given fluconazole 200 mg or matching placebo once daily for 11 days; rosuvastatin 80 mg was co-administered on day 8 of dosing. Plasma concentrations of rosuvastatin, N-desmethyl rosuvastatin, and active and total 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors were measured up to 96 h post-dose. RESULTS: Following co-administration with fluconazole, rosuvastatin geometric least-square mean area under the plasma concentration-time curve (AUC(0-t)) and peak plasma concentration (C(max)) were increased by 14% and 9%, respectively, compared with placebo (90% confidence intervals for the treatment ratios: 0.967 to 1.341 and 0.874 to 1.355, respectively). Individual treatment ratios for AUC(0-t) ranged from 0.59 to 2.23, and for C(max) ranged from 0.52 to 2.28. The limited data available for the N-desmethyl metabolite show that geometric mean C(max) was decreased by approximately 25% compared with placebo. Rosuvastatin accounted for essentially all of the circulating active HMG-CoA reductase inhibitors and most (>90%) of the total inhibitors. Fluconazole did not affect the proportion of circulating active or total inhibitors accounted for by circulating rosuvastatin. CONCLUSIONS: Fluconazole produced only small increases in rosuvastatin AUC(0-t) and C(max), which were not considered to be of clinical relevance. The results support previous in-vitro findings that CYP2C9 and CYP2C19 metabolism is not an important clearance mechanism for rosuvastatin.

Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection.

An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS) was developed and validated for the quantification of rosuvastatin (Crestor) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1-30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than +/-15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both -20 and -70 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined.

Determination of mevalonic acid in human urine as mevalonic acid lactone by gas chromatography-mass spectrometry.

Mevalonic acid in urine is converted to its lactone form by incubation overnight at acidic pH, extracted and analysed by GC-MS. The lower limit of quantitation of 7.5 ng/ml provides adequate sensitivity to measure changes in urinary excretion of MVA following administration of drugs which affect cholesterol synthesis. The assay is linear up to 300 ng/ml and has acceptable precision (<15%) and accuracy (<+/-20%) across the calibration range.

An unexpected selectivity of a propranolol-derived molecular imprint for tamoxifen.

During the evaluation of molecular imprinted polymers (MIPs) prepared against the drug tamoxifen a propranolol-derived MIP was used as a positive control. Surprisingly the propranolol-derived MIP showed considerable selectivity towards tamoxifen, and was indeed much more selective than the MIP prepared using tamoxifen as the imprint molecule. The consequences of this unexpected, cross reactivity for the use of MIPs in analytical chemistry is discussed.

Locomotion towards a goal alters the synchronous firing of neurons recorded simultaneously in the subiculum and nucleus accumbens of rats.

Rats were implanted with recording electrodes aimed at the subiculum and nucleus accumbens. They were subsequently placed in a cylindrical environment, where they searched for locations where they would receive rewarding medial forebrain bundle stimulation. At times a tone was sounded, indicating that the reward location was in the center of the environment. Animals quickly learned to switch from random running to goal directed locomotion when the tone was on. To quantify the synchronous firing between simultaneously recorded neurons in the subiculum and nucleus accumbens, a gravitational clustering algorithm was employed. Individual neurons were modeled as particles in N dimensional space. Every spike discharge of the neuron augmented the 'gravitational charge' on its model particle. Synchronous firing between two cells caused their corresponding particles to draw together over time, due to the concurrent appearance of gravitational charge. All pair-wise combinations of cells isolated in subiculum and nucleus accumbens were examined using this algorithm. The firing of nine out of 52 subicular-accumbens cell pairings was significantly more synchronous when the tone was on, and the rat was running towards the central goal. This was also seen for 15 out of 22 subicular-subicular cell pairings. Conversely, only two out of 51 accumbens-accumbens pairings displayed significant tone dependent changes in synchronous firing. Thus, synchronous interactions between subiculum and nucleus accumbens occur preferentially when the animal is required to locate a fixed goal in space, i.e., the functional connectivity is altered by the navigational demands of the spatial reward task.

A brief history of the formation of DNA databases in forensic science within Europe.

The introduction of DNA analysis to forensic science brought with it a number of choices for analysis, not all of which were compatible. As laboratories throughout Europe were eager to use the new technology different systems became routine in different laboratories and consequently, there was no basis for the exchange of results. A period of co-operation then started in which a nucleus of forensic scientists agreed on an uniform system. This collaboration spread to incorporate most of the established forensic science laboratories in Europe and continued through two major changes in the technology. At each step agreement was reached on which systems to use. From the beginning it was realised that DNA databases would provide the criminal justice systems with an efficient way of crime solving and consequently some local databases were created. It was not until the introduction of the amplification technology linked to the analysis of short tandem repeats that a sufficiently sensitive and robust system was available for the formation of efficient and effective DNA databases. Comprehensive legislation enacted in the UK in 1995 enabled forensic scientists to set up the first national DNA database which would hold both personal DNA profiles together with results obtained from crime scenes. Other countries quickly followed but in some the legislation has severely restricted the amount and type of data which can be retained and, therefore, effectiveness of the databases is limited. The widespread use of commercially produced multiplex kits has produced a situation in which nearly all European laboratories are using compatible systems and there is, therefore, the potential for the introduction of a pan-European DNA database. However, the exchange of results between countries is hampered by the various legislations which currently exist.

Criminal DNA databases: the European situation.

In the last 5 years, a number of European countries have successfully introduced national databases holding the DNA profiles from suspected and convicted criminal offenders as well as from biological stain materials from unsolved crime cases. At present, DNA databases are fully or partially in operation in the UK, The Netherlands, Austria, Germany, Finland, Norway, Denmark, Switzerland and Sweden. Furthermore, in the other European countries, specific legislation will be enacted soon, or the introduction of such databases is being discussed to initiate a legislative process. Numerous differences exist regarding the criteria for a criminal offender to be included in the database, the storage periods and the possibility to remove database records, the possibility to keep reference samples from the offenders as long as their respective records are being held, and the role of judges in the process of entering a database record or to perform a database search. Nevertheless, harmonization has been achieved regarding the DNA information stored in national databases, and a European standard set of genetic systems has been recommended which is included either in part or completely in the DNA profiles of offenders and crime stains for all European databases. This facilitates the exchange of information from database records to allow the investigation of crime cases across national borders.

Knowledge and attitudes of family physicians about clinical practice guidelines and the care of patients with type 2 diabetes mellitus.

This study examined the attitudes of Louisiana family physicians toward clinical practice guidelines in general and specifically how attitudes and familiarity with the American Diabetes Association Clinical Recommendations (ADACR) correlated with knowledge and evidence-based "best practice" in the care of type 2 diabetes. Surveys were mailed to a random sample of 278 eligible physicians from which a 32% response rate was obtained (n = 90). Family physicians' general attitudes towards guidelines were neutral. Attitude correlated significantly with knowledge of the ADACR (P = .03) but not with "best practice". Despite low scores for knowledge, all but one of the ADACR were adhered to by more than 85% of respondents. Physician attitudes do not appear to be barriers to guideline implementation. Results may be used to focus studies of processes and outcomes in guideline implementation.

Adolescent premarital sexual activity, cohabitation, and attitudes toward marriage.

Societal trends indicate ambivalent attitudes about marriage. Specifically, there is greater acceptance of divorce and nontraditional living arrangements such as cohabitation, as well as acceptance and prevalence of premarital sex, than in the past. The authors examine adolescent attitudes toward marriage and their association with premarital sexual activity and cohabitation. Recommendations for helping adolescents understand the realities of marriage and family life are shared.

Effects of reward anticipation, reward presentation, and spatial parameters on the firing of single neurons recorded in the subiculum and nucleus accumbens of freely moving rats.

The subiculum is the major output of the hippocampal formation (involved in spatial processing). Subicular afferents innervate the nucleus accumbens, which is thought to integrate limbic reward information with motor output. Rats were chronically implanted with extra-cellular recording electrodes aimed at both structures to investigate the functional relationship between them. Animals were then trained on a spatial task in which they searched for random locations where they would receive rewarding medial forebrain bundle stimulation. At random times a cue tone was sounded, indicating that the reward location was in the center of the environment. Rats quickly learned to run to the center upon hearing the tone in order to receive a reward. Simultaneously recorded groups of up to eight subicular and accumbens neurons were found to display alterations in firing rate after rewarding medial forebrain bundle stimulation. Moreover, neurons in both subiculum and accumbens displayed alterations in firing rate prior to arrival at the center during cued runs, i.e. they anticipated predictable rewards. Subicular and accumbens firing was also correlated with spatial location. However, neurons in accumbens were more likely to respond to task events, and these responses were more varied, than those seen in subiculum. Thus, while convergence of spatial and reward information occurs at the level of single cells in both subiculum and nucleus accumbens, these structures also display functional localization.