The Role of Systematic Reviews in Pharmacovigilance Planning and Clinical Trials Authorisation Application: Example from the SLEEPS Trial

Background

Adequate sedation is crucial to the management of children requiring assisted ventilation on Paediatric Intensive Care Units (PICU). The evidence-base of randomised controlled trials (RCTs) in this area is small and a trial was planned to compare midazolam and clonidine, two sedatives widely used within PICUs neither of which being licensed for that use. The application to obtain a Clinical Trials Authorisation from the Medicines and Healthcare products Regulatory Agency (MHRA) required a dossier summarising the safety profiles of each drug and the pharmacovigilance plan for the trial needed to be determined by this information. A systematic review was undertaken to identify reports relating to the safety of each drug.

Methodology/Principal Findings

The Summary of Product Characteristics (SmPC) were obtained for each sedative. The MHRA were requested to provide reports relating to the use of each drug as a sedative in children under the age of 16. Medline was searched to identify RCTs, controlled clinical trials, observational studies, case reports and series. 288 abstracts were identified for midazolam and 16 for clonidine with full texts obtained for 80 and 6 articles respectively. Thirty-three studies provided data for midazolam and two for clonidine. The majority of data has come from observational studies and case reports. The MHRA provided details of 10 and 3 reports of suspected adverse drug reactions.

Conclusions/Significance

No adverse reactions were identified in addition to those specified within the SmPC for the licensed use of the drugs. Based on this information and the wide spread use of both sedatives in routine practice the pharmacovigilance plan was restricted to adverse reactions. The Clinical Trials Authorisation was granted based on the data presented in the SmPC and the pharmacovigilance plan within the clinical trial protocol restricting collection and reporting to adverse reactions.     

The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b₅ reductase (CYB5R) and cytochrome b₅ (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.     

Replacing the Orchestra? – The Discernibility of Sample Library and Live Orchestra Sounds

Recently, musical sounds from pre-recorded orchestra sample libraries (OSL) have become indispensable in music production for the stage or popular charts. Surprisingly, it is unknown whether human listeners can identify sounds as stemming from real orchestras or OSLs. Thus, an internet-based experiment was conducted to investigate whether a classic orchestral work, produced with sounds from a state-of-the-art OSL, could be reliably discerned from a live orchestra recording of the piece. It could be shown that the entire sample of listeners (N = 602) on average identified the correct sound source at 72.5%. This rate slightly exceeded Alan Turing''s well-known upper threshold of 70% for a convincing, simulated performance. However, while sound experts tended to correctly identify the sound source, participants with lower listening expertise, who resembled the majority of music consumers, only achieved 68.6%. As non-expert listeners in the experiment were virtually unable to tell the real-life and OSL sounds apart, it is assumed that OSLs will become more common in music production for economic reasons.     

Analysis of omega-3 and omega-6 fatty acid-derived lipid metabolite formation in human and mouse blood samples

Mass spectrometry techniques have enabled the identification of different lipid metabolites and mediators derived from omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFA) that are implicated in various biological processes. However, the broad-spectrum assessment of physiologically formed lipid metabolites and mediators in blood samples has not been presented so far. Here lipid mediators and metabolites of the n-6 PUFA arachidonic acid as well as the long-chain n-3 PUFA eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA) were measured in human blood samples as well as in mouse blood. There were detectable but mostly very low amounts of the assayed compounds in human native plasma samples, whereas in vitro activation of whole blood with the calcium ionophore A23187 led to highly significant increases of metabolite formation, with a predominance of the 12-lipoxygenase (12-LOX) products 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA). A23187 activation also led to significant increases in the formation of 5-LOX products including leukotriene B(4) (LTB(4)), leukotriene B(5) (LTB(5)) as well as of 15-LOX products and prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)). Levels were similar or even higher in A23187-activated mouse blood. The approach presented here thus provides a protocol for the comprehensive and concomitant assessment of the generation capacity of n-3 and n-6 PUFA-derived lipid metabolites as well as thromboxanes and prostaglandins in human and murine blood samples. Further studies will now have to evaluate lipid metabolite generation capacity in different physiological and pathophysiological contexts.     

The role of lipid peroxidation in the N-oxidation of 4-chloroaniline.

Irradiation with u.v. light of aerobic aqueous media containing both rabbit liver microsomal fraction and 4-chloroaniline results in N-oxidation of the arylamine. The reaction is severely blocked by exhaustive extraction with organic solvents of the microsomal membranes to remove lipids. Further, scavengers of OH. and O2.-impair the photochemical process. These findings suggest that the observed phenomenon may be closely associated with light-induced lipid peroxidation. Indeed, N-oxidation of 4-chloroaniline is fully preserved when either phospholipid liposomes or dispersed linoleic acid substitute for intact microsomal fraction. Co-oxidation of the amine substrate occurs during iron/ascorbate-promoted lipid peroxidation also, but H2O2 or free OH. radicals do not appear to be involved. Cumene hydroperoxide-sustained rabbit liver microsomal turnover of the amine generates N-oxy product via O2-dependent and -independent pathways; propagation of lipid peroxidation is presumed to govern the former route. Lipid hydroperoxides, either exogenously added to rabbit liver microsomal suspensions or enzymically formed from arachidonic acid in ram seminal-vesicle microsomal preparations, support N-oxidation of 4-chloroaniline. The significance, in arylamine activation, of lipid peroxidation in certain extrahepatic tissues exhibiting but low mono-oxygenase activity is discussed.     

Kausalmechanismen der Kernbewegung und -teilung während der frühen Furchung im Ei der GallmückeWachtliella persicariae L.

Zusammenfassung Im Ei vonWachtliella legen die Furchungskerne in der Zeit zwischen den Mitoseschritten große Strecken im Ooplasma zurück, was zu einer raschen Besiedlung des Eiraumes führt. Dabei findet neben der passiven Verlagerung infolge pulsierender Plasmaströmungen einaktiver Migrationsprozeß statt. Er wird von feinen Dotterpartikeln-Schwingungen begleitet, die auch in den Eiern anderer Insekten gefunden worden sind. Um diese Zitterbewegung besser analysieren zu können, sind dieEier total bzw. partiell gequetscht worden; hierdurch wird die Schichtdicke des Ooplasmas auf 30 m bzw. auf 12—4 m reduziert. Die experimentelle Verkleinerung des Krümmungsradius' der Eioberflächefördert die Neigung zur Zellwandbildung, was schon im 2-Kern-Stadium zu einerfast totalen Furchung führt. Außerdem kann derInitialbereich der Furchung (= Initialbereich der Zitterbewegung)in kernfreien Regionen liegen und darüberhinaus in einen vorderen und einen hinteren Initialbereichaufgespalten werden; Kernteilung und -wanderung werden jedoch nicht unterbunden. Die Entwicklung unbehandelter wie auch gequetschter Eier ist mit Hilfe von Zeitrafferfilmen analysiert worden, die mit dem Phasenkontrast oder dem differentiellen Interferenzkontrast-Verfahren — unter Verwendung höchstauflösender Planapochromate am umgekehrten Mikroskop — aufgenommen worden sind.Vom Initialbereich der Furchung gehen, im Rhythmus der Furchungsschritte,Wellen ungeordneter Zitterbewegung aus und durchziehen das ganze Ei. Sie bestehen aus ungeordneten Schwingungen von Dotterpartikeln, die vermutlich auf die Aktivität räumlich ungeordneter, dynamischer Elemente im Ooplasma zurückzuführen sind. Einenordnenden Einfluß aber übt die Anwesenheit vonFurchungskernen aus: Kurz bevor eine solche Welle von Zitterbewegungen bei Furchungskernen des Metaphasestadiums eintrifft, treten nämlich in deren Umgebung geordnete Schwingungen und Verlagerungen von Dotterpartikeln auf. Sie sind radial auf die Spindelpole ausgerichtet und beginnen bei demjenigen, den die Welle zuerst erfaßt. Diese radiale Zitterbewegung wird durch eingroßes Astersystem hervorgerufen, das jeweils von den Spindelpolen ausgeht und distal weit in das Ooplasma zieht. Mit dem Beginn der Verkürzungsprozesse innerhalb der Asterstrahlen tritt der Furchungskern in die Ana- und Telophase ein, und die Spindelpole werden auseinandergezogen. Auch in der darauffolgenden Migrationsphase der Tochterkerne bleibt der Spindelpol als Asterzentrum der ehemaligen Spindel erhalten und bildet den Mittelpunkt eines Migrationsasters. Seine längsten Asterstrahlen messen mindestens 80 m und inserieren distal sowohl an frei beweglichen als auch an elastisch aufgehängten oder an starren Eikomponenten.Durch wiederkehrende, kurzzeitige Insertionen und unkoordinierte, vorübergehende Verkürzung der Asterstrahlenwird der Kern, der eine starke Affinität zu seinem Asterzentrum besitzt, im Ooplasmavorwärtsgezogen, und zwar stets in Richtung auf die jeweils noch größten kernfreien Gebiete, in denen auch die räumliche Ausdehnung des Migrationsasters am größten ist. So läßt sich u.a. auch die gegenseitige Abstoßung der Energiden erklären, die zu einer gleichmäßigen Verteilung im Eiraum führt.Bei manchen Eiern ist es möglich gewesen,Furchungskerne experimentell von ihren Astersystemen zu trennen. Währendisoliert liegende Migrationsaster sich autonom, d.h. in Abwesenheit von Kernen,aktiv im Eiraum ausbreiten und vielleicht sogar teilungsfähig sind,können asterlose Furchungskerne zunächst nicht mehr wandern; sie scheinen aber in der Lage zu sein, die Bildung neuer Spindelpole und Astersysteme zu induzieren und dann Furchungsteilungen durchzuführen.Die Migrationsaster aller Furchungsenergiden gehen durch Teilung aus demjenigen Astersystem hervor, dessen Aufbau der Spermakern nach seinem Eindringen in das Ei induziert hat. Derweibliche Vorkern dagegen besitztkeinen Migrationsaster; er wird wahrscheinlichdurch permanente Insertion von Asterstrahlen des männlichen Kerns zu diesem hingezogen, und die Befruchtung ist eine Folge der Affinität zwischen (weiblichem Vor-) Kern und Migrationsaster (des männlichen Kerns).Da sich die Asterstrahlen aus den Polstrahlen des Spindelapparates ableiten lassen, ist eineBeteiligung von Mikrotubuli an ihrem Aufbau sehr wahrscheinlich; sie muß aber noch elektronenmikroskopisch geprüft werden. Mögliche funktionelle Strukturen des Migrationsasters und ihre Beziehungen zum mitotischen Apparat werden diskutiert. Migrationsaster sind wahrscheinlich nicht nur in Anpassung an die speziellen Bedingungen der Furchung in großräumigen Eisystemen entstanden, sondern sind vermutlich auch bei der Teilung zahlreicher anderer tierischer Zellen für Spindelstreckung und Auseinanderwandern der Tochterkerne von Bedeutung.

---

Causal mechanisms of nuclear movement and division during early cleavage stages in the egg of a gall midge,Wachtliella persicariae L.

Summary Between each mitotic cycle, the cleavage nuclei ofWachtliella move over long distances, thus populating the ooplasm within a short time. Besides being shifted passively by flowing pulses of the ooplasm, thenuclei are also migrating actively. The active movements are accompanied by such oscillations of yolk particles as are known from the eggs of other insects, too. For a closer analysis of these quivering movements the inseminated eggs werepressed, either totally or partially, reducing their smaller diameters to ooplasmic layers of 30 m or between 12 and 4 m, respectively. Along with the experimental reduction of the radius of the curvature at the egg surface, there is anincreased tendency of cell membrane formation, resulting in anearly total cleavage already at the 2-nuclei-stage. Furthermore, theinitial region of cleavage (= initial region of quivering movements)may be shifted to a site free from nuclei; the initial region even may becomesplit up into two, one near each of the egg's poles. Yet, in flattened eggs, division and migration activities of the nuclei are not prevented. Untreated as well as flattened eggs have been analysed by means of time-lapse motion pictures taken either by the phase contrast or by the differential interference contrast method, using apochromatic objectives of maximum resolution, combined with an inverted microscope.According to the rhythm of the cleavage divisions,waves of irregular quivering movements spread from the initial region(s) of cleavage throughout the whole egg space. They are composed of irregular oscillations of yolk particles, probably caused by the effect of actively shortening, dynamic elements irregularly spread within the ooplasm. The presence ofcleavage nuclei obviously exerts a kind ofregulative effect: Shortly before such a wave of quivering movements reaches a metaphasic cleavage energide, regular oscillations and approximations of yolk particles are visible in the surroundings of the nucleus. The movements in question are radially adjusted towards the spindle poles, starting at the one which is reached first by the wave of quivering movements. These radial quivering movements are caused by abig cytaster, each originating from its spindle pole and distally reaching far into the ooplasm. Synchronous with the beginning of the shortening process of the astral rays, the cleavage nucleus passes through anaphase and telophase, and the spindle poles arepulled apart. During the then following migration of each daughter nucleus, its spindle pole—the kinetocentre of the previous spindle—is preserved and becomes the centre of a migration cytaster. Its longest rays measure up to at least 80 m. Their distal ends temporarily insert either in motile, or in elastically suspended, or in rigid egg components.By the recurrent short-time insertions and irregular shortening processes of the astral rays,the nucleus, displaying a strong affinity to its own kinetocentre,is pulled foreward. This movement always occurs in the direction of the biggest ooplasmic region still free from nuclei and therefore permitting the greatest spacial extension of the migration cytasters. This could explain the so-called mutual repulsion of the energides, leading to their even dispersion all over the egg space.In some of the eggs it has been possible toseparate the cleavage nuclei from their cytaster systems experimentally. Deprived of their nuclei such migration cytasters behave autonomously, i. e. they are actively moving within the ooplasm, possibly even retaining their division power. On the other hand, thenuclei without their cytasters have lost their mobility and therefore at first remain in their sites. But they seem to be capable ofinducing new spindle poles and migration cytasters of their own and to carry out further cleavage divisions.The migration cytasters of all cleavage energides develop by division from the very cytaster whose formation had been induced by the sperm nucleus after entering the egg. On the other hand thefemale pronucleus, remainingwithout a migration cytaster and therefore lacking migration activity, ismoved towards the male nucleus, pulled by the probably permanently inserted astral rays of the latter. Thus the final act of fertilization, i.e. nuclear fusion, comes about by the affinity between the (female pro-)nucleus and the (alien) migration cytaster (of the male nucleus).Judged by their derivation from the polar rays of the spindle apparatus, the astral rays with high probability are built up oftubuli, the evidence being left to electron microscopical investigations. Functional structures related to the causal mechanism of the migration cytaster are suggested and their supposed derivation from the mitotic apparatus is discussed. The existence of migration cytasters might not only represent an adaptation to the specific conditions of cleavage within spacious eggs, but also could be essential for the stretching of the spindle and the separation of the daughter nuclei during the division process of many other animal cells.

---

---

Herrn Prof. Dr. G. Krause danke ich sehr für wertvolle Anregungen bei der kritischen Durchsicht dieser Veröffentlichung. Mein besonderer Dank gilt meiner Frau für ihre Hilfe bei der Ausarbeitung des Manuskripts. Die Untersuchung wurde durch Sachbeihilfen der Deutschen Forschungsgemeinschaft unterstützt.     

Mapping and genomic characterization of the gene encoding diacylglycerol kinase γ (DAGK3): assessment of its role in dominant optic atrophy (OPA1)

The family of diacylglycerol kinases (DAGKs) is known to play an important role in signal transduction linked to phospholipid turnover. In the fruitfly Drosophila melanogaster, a human DAGK ortholog, DGK2, was shown to underlie the phenotype of the visual mutant retinal degeneration A (rdgA). Previously, the gene encoding a novel member of the human DAGK family, termed DAGK3, was cloned and demonstrated to be abundantly expressed in the human retina. Based on these findings we reasoned that DAGK3 might be an excellent candidate gene for a human eye disease. In the present study, we report the genomic organization of the human DAGK3 gene, which spans over 30 kb of genomic DNA interrupted by 23 introns. In addition, we have mapped the gene locus by fluorescence in situ hybridization to 3q27–28, overlapping the chromosomal region known to contain the gene underlying dominant optic atrophy (OPA1), the most common form of hereditary atrophy of the optic nerve. Mutational analysis of the entire coding region of DAGK3 in 19 unrelated German OPA1 patients has not revealed any disease-causing mutations, therefore excluding DAGK3 as a major cause underlying OPA1. Received: 24 August 1998 / Accepted: 13 October 1998     

Reproductive biology of the epiphytic bromeliad Werauhia gladioliflora in a premontane tropical forest

The floral phenology, fruit and seed production, and self-compatibility of Werauhia gladioliflora, an epiphytic bromeliad with a wide distribution, were studied in a premontane forest in the Monteverde area in Costa Rica. The species presents the pollination syndrome of chiropterophily, and it is visited by the small bats Hylonycteris underwoodi and Glossophaga commissarisi (Glossophaginae). The population flowering period extended from October to early December (end of rainy season) and seed dispersal occurred from February to April (dry season). Most plants opened a single flower per night, either every day or at one-day intervals during the flowering period. In natural conditions, the average fruit set amounted to almost half of the potential output, but individual fecundity (number of seeds) remained high. Seed number per fruit and germination capacity after artificial selfing and out-crossing treatments did not differ from natural pollination conditions. Werauhia gladioliflora exhibited high levels of autonomous self-pollination and self-compatibility at the individual and population level, characters associated with the epiphytic habitat. These reproductive traits are also associated with early colonizer species, yet life history traits, such as seed dispersal, seedling establishment success, and growth, are likely to have a major role in determining the presence of this species in the successional vegetation patches scattered over the studied premontane area.