Degradation chemistry of a Vitamin D analogue (ecalcidene) investigated by HPLC-MS, HPLC-NMR and chemical derivatization

Ecalcidene (1-[(1alpha,3beta,5Z,7E,20S)-1,3-dihydroxy-24-oxo-9,10-secochola-5,7,10(19)-trien-24-yl]-piperidine) is a new 1-hydroxyvitamin D analogue. In this report, the thermal degradation, acid induced degradation and iodine induced degradation of ecalcidene were investigated using HPLC-MS, HPLC-NMR and chemical derivatization. In solution ecalcidene was thermally and reversibly transformed to a pre-Vitamin D type isomer 1 which subsequently produced the dehydrated pyrocalciferol and isopyrocalciferol type isomers 2 and 3 by cyclization and dehydration at elevated temperatures. Acidic conditions resulted in the formation of a novel C9-hydroxylated isomer 4 of ecalcidene, possibly via a tachysterol type intermediate, followed by the acid facilitated nucleophilic addition of water. In the presence of iodine, cis/trans isomerization of both ecalcidene and its pre-Vitamin D type isomer 1 occurred. The results may shed light on the stability and metabolism of ecalcidene, provide useful information for its potential pharmaceutical development, and enrich the knowledge of Vitamin D chemistry.     

Safety and feasibility of percutaneous autologous skeletal myoblast transplantation in the coil-infarcted swine myocardium

INTRODUCTION: Autologous skeletal myoblast transplantation (ASMT) for myocardial regeneration is a promising new treatment for patients with congestive heart failure secondary to myocardial infarction (MI). However, non-surgical delivery could broaden the utility of this approach. The present study was designed to evaluate the safety and feasibility of transplanting autologous skeletal myoblast (ASM) via endovascular delivery into the infarcted swine myocardium. METHODS: Seven female Yorkshire swine successfully underwent induced left ventricular MI. ASM biopsies were obtained from the hind limb of each animal and myoblasts were expanded in vitro. In a pilot experiment, ASM were labeled with iridium and short-term retention and biodistribution was determined 2 h after ASM delivery via the MyoStar needle-injection catheter inserted through the femoral artery. At 30 days post-infarction, the remaining animals were divided into three groups containing 2 animals each for percutaneous catheter delivery into the infarcted zone: group 1 control animals were injected with media only, group 2 and 3 animals were injected with approximately 300 x 10(6) and 600 x 10(6) ASM, respectively. Sixty days post-transplantation, the swine hearts were harvested. RESULTS: During the 60-day period between transplantation and harvest, no adverse events were recorded, and continuous rhythm monitoring revealed no arrhythmias. In the small sampling size, myocardial function assessments revealed a trend toward improvement in the treatment groups with respect to ejection fraction, viability, and cardiac index. However, histology of treated swine hearts identified no skeletal muscle cells. DISCUSSION: Percutaneous ASMT into an infarcted swine myocardium is feasible and safe, and may contribute to overall improved heart function.     

From the laboratory to real life: a pilot study of an expectancy challenge with "heavy drinking" young people on holiday

The Alcohol Expectancy Challenge (EC) is a promising program for changing alcohol expectancies and reducing alcohol consumption in "heavy drinking" young men in a bar-lab setting. In this study the EC was adapted for use in mixed-gender groups in a holiday setting and its feasibility tested in camping resorts in the Netherlands where a lot of binge drinking takes place (summer 2002). Male and female participants (N = 170; mean age, 18.8 years) were randomly assigned to an EC or to an assessment-only control group. One day before the intervention, alcohol expectancies were measured by a Visual Analogue Scale of arousal-sedation expectancies (VAS expectancies questionnaire). At the same time, alcohol use in everyday life and on holiday was assessed by a General Drinking Questionnaire and a 24-hour drinking diary, respectively. Twenty-four hours after the intervention, the VAS expectancies questionnaire was administered again and alcohol use over the previous 24 hours was reported in the drinking diary. Six weeks after the intervention, participants were telephoned and administered oral versions of the VAS expectancies questionnaire and General Drinking Questionnaire. Data were analyzed using mixed ANOVAs. Although the study was hampered by recruitment difficulties, the EC proved feasible in this setting, was well received by youngsters, and effects on their alcohol expectancies may have been present. No effect was found on alcohol use. In conclusion, implementation must be improved and more studies are needed to come to more definite conclusions about the value of the EC in a real-life targeted intervention.     

Dose individualization of carboplatin after a 120-hour infusion schedule: higher dose intensity but fewer toxicities

Carboplatin (CBDCA) is a widely used anticancer agent for which dose-effect and dose-toxicity relationships have been demonstrated, thus stressing the need for a controlled exposure to this drug. So far, carboplatin administration could only be individualized a priori following 2 classic methods, which are based on the evaluation of renal clearance: Calvert's and Chatelut's formulas. This study was designed to develop and evaluate the performance of an alternative CBDCA 120-hour schedule coupled to a Bayesian adaptive dosing with feedback strategy. Precision of the dosing method was assessed in 84 patients (256 courses performed during a 10-year period), by comparing CBDCA plasma concentrations observed at the end of the infusion with initial target values. A comprehensive monitoring of treatment-related toxicities also was performed. Finally, the authors compared doses actually delivered following the dose-tailoring method with the theoretical, standard, ones calculated retrospectively with Calvert's and Chatelut's formulas. No significant differences were found between experimental and theoretical concentrations. According to the target exposure chosen (3 levels), the mean doses administered to our patients were 517, 719, and 902 mg of CBDCA compared with 550, 509, and 538 or 657, 604, and 644 mg, which would have been given following Calvert or Chatelut formulas, respectively. These results showed that our Bayesian method led to the administration of up to 60% higher doses of carboplatin compared with those based only on the evaluation of renal clearance. Despite the markedly higher doses administered, no severe toxicities were reported in the patients treated following this new schedule. It is noteworthy that neither hematologic growth factors nor stem cells, usually associated with high-dose regimen, were used as support in this study. These data strongly suggest that it is possible to deliver higher dose- intensities of carboplatin, even in elderly, unselected patients, without increasing toxicities and with no growth factor support, provided that a therapeutic drug monitoring strategy with real-time tailored dosing is performed.     

Toxicogenomics in drug discovery and development: mechanistic analysis of compound/class-dependent effects using the DrugMatrix database

A range of genomics technologies are increasingly becoming integrated with existing scientific disciplines to broaden and strengthen existing capabilities and open new avenues of research in drug discovery and development. Examples of these new research fields are proteomics, pharmacogenomics, metabolomics and toxicogenomics. Here we review the application of toxicogenomics to improve the evaluation of drug safety, mechanism of action and toxicity in the drug discovery and development process.     

Autoradiographische Untersuchung mit H3-Thymidin über die Desoxyribonucleinsäure-Neubildung in den Geweben des Rattenauges

Zusammenfassung Die Neubildung von Desoxyribonucleinsäure (DNS) in den Zellkernen der verschiedenen Gewebe des Auges wurde bei der Ratte autoradiographisch nach Gabe von H³-Thymidin untersucht. Das markierte Thymidin wird ausschließlich in die DNS eingebaut und zwar bei denjenigen Kernen, welche sich im Stadium der DNS-Verdoppelung, d.h. im allgemeinen vor einer Mitose befinden.Zahlreiche H³-markierte Zellen fanden sich im Epithel der Conjunctiva bulbi, weniger im Hornhautepithel—peripher häufiger als in der Hornhautmitte—und vereinzelt in Aderhaut, Ciliarkörper und Iris. In Hornhautstroma,-endothel, Sklera und Netzhaut zeigten die Autoradiogramme keine markierten Zellkerne. Mitosen waren nur in den basalen Epithelien der Hornhaut zu sehen.Für die Hornhautepithelien betrug der H³-Index 4,0% und der Mitose-Index 0,62%. Bei einer Mitosedauer von 1 Std läßt das auf eine DNS-Verdoppelungszeit von etwa 7 Std und eine Lebensdauer der Zellen von etwa 7 Tagen schließen.Es ist ein Vorteil der Methode, daß eine mitotische Tätigkeit auch in solchen Geweben erkannt werden kann, die keine sichtbaren Mitosen zeigen.Mit 3 TextabbildungenHerrn Prof. Dr.Maurer danken wir für seine freundliche Mithilfe bei der Durchführung der Arbeit.     

ISOLATION AND CHARACTERIZATION OF THE CYANOGEN BROMIDE FRAGMENTS OF LOBSTER ARGININE KINASE (HOMARUS VULGARIS)

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10%) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to different gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position; therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

H244R VSX1 is associated with selective cone ON bipolar cell dysfunction and macular degeneration in a PPCD family

PURPOSE: To elucidate the retinal dysfunction and the molecular basis of posterior polymorphous corneal dystrophy (PPCD) associated with macular dystrophy, both inherited in a dominant manner through a three-generation family. METHODS: Ophthalmologic examinations including slit lamp examination, visual acuity tests, fundus visualization by scanning laser ophthalmoscopy, fluorescein angiography, color vision tests, electro-oculography, photopic and scotopic electroretinography (ERG) according to the International Society for Clinical Electrophysiology of Vision (ISCEV) protocols, and oscillatory potential (OP) recordings were conducted on affected family members. Corneal button from one affected patient was examined by transmission electron microscopy. All exons and intron-exon boundaries of the VSX1 and the COL8A2 genes were amplified by polymerase chain reaction and sequenced. RESULTS: The presence of endothelial cells that have epithelial-like features with multiple layers, desmosomal junctions, and microvillous projections supports the diagnosis of PPCD. Sequence analysis indicated that the H244R variant in the VSX1 segregated with corneal and macular disease phenotypes in this family. Electrophysiologic studies indicated normal scotopic ERG findings, decreased amplitude of the photopic b-wave, photopic OP2 and OP3 barely recordable with a preserved OP4 amplitude, and variably decreased 30-Hz flicker amplitude. CONCLUSIONS: The human VSX1 is required for cone ON bipolar cell function but not for rod and cone OFF bipolar cells, giving a unique example of such a selective heritable retinal defect in humans. Furthermore, the authors provide the first clinical support for a new alternative role of VSX1 in cone biology, probably similar to that proposed for its goldfish ortholog during retinal differentiation.