Variability of non-clinical behavioral CNS safety assessment: An intercompany comparison.

INTRODUCTION: Irwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a "state-of-the-art" assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests. METHODS: This assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results. RESULTS: Our survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DISCUSSION: Our survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.

Activity control of the ClpC adaptor McsB in Bacillus subtilis.

Controlled protein degradation is an important cellular reaction for the fast and efficient adaptation of bacteria to ever-changing environmental conditions. In the low-GC, Gram-positive model organism Bacillus subtilis, the AAA+ protein ClpC requires specific adaptor proteins not only for substrate recognition but also for chaperone activity. The McsB adaptor is activated particularly during heat stress, allowing the controlled degradation of the CtsR repressor by the ClpCP protease. Here we report how the McsB adaptor becomes activated by autophosphorylation on specific arginine residues during heat stress. In nonstressed cells McsB activity is inhibited by ClpC as well as YwlE.

Toxicological properties of metaflumizone.

Metaflumizone is a new insecticide developed for crop protection and urban pest control by BASF. Its mammalian toxicological profile was assessed by conducting multiple toxicity studies in the rat, mouse, and dog, covering all relevant endpoints. Metaflumizone is characterized by very low acute toxicity, is not irritating to the eye or the skin and does not possess a potential to induce skin sensitization. The substance also shows relatively low toxicity following subchronic oral or dermal exposure to mammals. In addition, metaflumizone demonstrates low toxicological potential following chronic oral exposure to rats, mice, and dogs. Overall, the lowest no observed adverse effect level (NOAEL) is 12mg/(kgday) from the 1-year chronic dog study. In a battery of in vitro and in vivo mutagenicity assays, the weight-of-the-evidence indicates a lack of potential genotoxicity for metaflumizone. Furthermore, the compound demonstrated a lack of potential oncogenicity in long-term toxicity studies in rats and mice. Results from the rat multi-generation reproductive toxicity study as well as the rat and rabbit developmental toxicity studies indicate that metaflumizone is not selectively toxic to the offspring or fetus, as compared to the parents. Also, metaflumizone is not teratogenic in the rat or rabbit. Lastly, no neurotoxicity could be detected in acute and subchronic neurotoxicity studies in rats.

Pilot study for comparison of reticulocyte-micronulei with lymphocyte-micronuclei in human biomonitoring.

Biomonitoring tries to determine the consequences for humans of exposures to environmental or pharmaceutical agents. Different end points have been employed to assess the burden of genomic damage. This is the first report comparing a recently introduced new end point, the reticulocyte-micronuclei analyzed by flow cytometry with the widely used lymphocyte-micronucleus assay, applied to two exposure scenarios leading to enhanced genomic damage. Radioiodine therapy was chosen to represent a short time exposure and hemodialysis treatment in end-stage renal failure was chosen to represent a chronic exposure. The results show that iodine radiation induced measurable genomic damage in the lymphocyte-micronucleus assay as well as in the reticulocyte-micronucleus test. Of two groups of patients under hemodialysis treatment, a reduced genomic damage was found with the lymphocyte-micronucleus test, but not with the reticulocyte-micronucleus test in the group undergoing daily hemodialysis, which removes uremic toxins more efficiently as compared to conventional hemodialysis, the treatment applied in the other group. The limited life-span of reticulocytes may make them less suitable for accumulation of chronic low level damage than lymphocytes. In conclusion, the lymphocyte-micronucleus test may be applicable to more exposure situations (including low chronic exposure), but the reticulocyte-micronucleus assay may be easier to perform in a clinical setting. The latter reflects a more rapid reduction of genomic damage after an acute exposure.

Cross-species amplification from crop soybean Glycine max provides informative microsatellite markers for the study of inbreeding wild relatives.

The development of microsatellite markers through transfer of primers from related species (cross-species amplification) remains a little-explored alternative to the de novo method in plants. In this study of 100 microsatellite loci from Glycine max, we examined two aspects of primer transfer. First, we tested if source locus properties can predict primer transfer and polymorphism in Glycine cyrtoloba and Glycine clandestina. We transferred 23 primers to G. cyrtoloba and 42 to G. clandestina, with 19 loci polymorphic within G. clandestina. However, we could not predict transfer or polymorphism from the source locus properties. Second, we evaluated the subset of 11 polymorphic loci for study in G. clandestina populations representing two local morphotypes. All loci were informative within populations (population mean He +/- SE = 0.58 +/- 0.04). We directly sequenced 28 alleles at 4 representative loci. The allelic patterns and sequencing results established that 8 of 11 loci were typical microsatellites, confirming the utility of primer transfer as an alternative to de novo development. Additionally, we found that morphotypic differentiation between populations was paralleled by changes in polymorphism level at six loci and size homoplasy at one locus. We interpret these patterns as being a product of selfing in G. clandestina. Our results demonstrate the value of allele sequence knowledge for the most effective use of microsatellites.

[Medical-surgical intuition and professional policy]. / Arztlich-chirurgische Intuition und Berufspolitik.

Many decisions in business and policy are made intuitively. According to scientific studies intuitive persons do not belong to a certain social group; they are unconventional, self-confident and self-reliant. In professional policy facts established by documentary evidence and legally guaranteed are the basis for decisions. Facts are dealing with the past. In order to consider the future and to react in situations of immediate interest intuitive abilities are just as important. (The appropriate argument at the right time at the right place). Beside the "Leitlinien" in surgical therapy the "Methodenfreiheit", which is connected to intuitive thinking, must be lasting.

Use of the recursion formula of the Gompertz function for the quantitation of PCR-amplified templates.

One common drawback of the currently used procedures to quantitate the polymerase chain reaction (PCR) is that the statistical evaluation of the experimental data depends on many, not just trivial, model assumptions. In the present study we report on an improvement in this crucial step of the quantitative PCR. The experimental design underlying the introduced method is exactly the same as in the case of the so-called PCR. However, by applying growth curve analysis based on the recursion formula of the Gompertz function the kinetics of the accumulation of the amplicon are estimated conjointly from data spanning both the and phases of the reaction. We demonstrate the method by determining the relative number of templates (a 206 bp segment spanning the exon 3 of the X-chromosomal murine Hprt-gene) contained in known orders of dilutions of DNA isolated from the spleen of the C57BL/6J-mouse. [32P]-dATP incorporation was used in duplicate experiments to quantify the amplicons as a function of amplification cycles. Our results: i) indicate that the accumulation of the PCR product as a function of PCR cycles follows a sigmoidal pattern compatible with the Gompertz growth model (P<0.0000001); ii) directly support the thesis that the kinetical pattern of accumulation of amplicons of a given DNA fragment does not depend on the number of corresponding DNA templates provided to the reaction; iii) permit a simple direct evaluation of the parallelity in the course of the accumulation of amplicons from different template numbers as a function of amplification cycles, which is a silent preposition in the evaluation of the so-called PCR; iv) allow an easy quantitation of the relative number of provided templates.

[The status of perioperative pain therapy in Germany. Results of a representative, anonymous survey of 1,000 surgical clinic. Pain Study Group]. / Situation der perioperativen Schmerztherapie in Deutschland. Ergebnisse einer repräsentativen, anonymen Umfrage von 1000 chirurgischen Kliniken. AG Schmerz.

To evaluate the status of perioperative pain management we mailed a anonymous postal survey to all 2,254 surgical departments in Germany. We received answers from 1,000 clinics (44.4%) which were representative related to their regional distribution. We asked the responsible surgeons to report their organizational structure and responsibilities for treating pain patients, the significance of the problem, their methods of measuring pain, and the usage of different analgesic drugs and methods. In 47% the surgeon and the anesthesist together had responsibility for adequate postoperative pain treatment; in 33% and 14%, respectively, it was the surgeon and anesthesist alone. Only 41% knew the interdisciplinary statement on pain therapy of the Professional Societies of German Surgeons and Anaesthesists from 1992. Although the importance of postoperative pain is globally acknowledged, only 19.1% of all departments had a written concept for pain treatment. Pain was measured in only 11% of the clinics mainly by using the visual analogue scale. Most surgeons relieve pain solely with systemic drugs. Regional analgesia was used by 18% only 51% of the surgeons decide on the choice and dosage of analgesic therapy on the ward; 33% admit that pain therapy often starts after complaints of the patient. 70% of all surgeons never participated in a congress on pain. We conclude that postoperative pain management in most German surgical departments still lacks effectiveness, adequacy, and organizational and scientific background.

Improved determination of variant erythrocytes at the glycophorin A (GPA) locus and variant frequency in patients treated with radioiodine for thyroid cancer.

Red blood cells from individuals with the blood group MN express each form of the allelic GPA protein (GPAM and GPAN) on their cell surface. Variant cells have lost one form of the protein. Their frequency is about 10(-5) in blood from unexposed persons. The BR6 assay is currently the most widely used assay to determine variant frequency (VF) by immunolabelling and flow cytometry. The precision of the BR6 assay is mainly limited by the Poisson error because only small numbers of variant cells are detected in each assay. The BR6 assay has been improved by magnetic cell separation (MACS) of variant erythrocytes prior to their determination by this assay. This new version of the assay is named 'MACS-BR6'. It allows enumeration of variant cells from 2 x 10(8) or more blood cells instead of 5 x 10(6) in the BR6 assay with a precision which is about 5 times higher than that of the BR6 assay. The MACS-BR6 assay was used to determine the VF of GPAN/0 and GPAN/N variant cells in 12 healthy adults and 11 patients treated with radioiodine for thyroid cancer 2 to 16 years before. The average red bone marrow dose was 347 mGy. In healthy adults the mean VF of GPAN/0 and GPAN/N variant cells was 16.1 x 10(6) and 5.3 x 10(-6) respectively. In patients the corresponding mean VF was 25.4 x 10(6) and 11.9 x 10(-6), respectively. The patients GPAN/0 VF was significantly higher than that of controls. In patients VF increases linearly with the dose. The linear regression parameters of VF were 16.6 x 10(-6) (intercept), 23.7 x 10(-6) GY-1 (slope) and 6.3 x 10(-6) (intercept), 12.9 x 10(-6) Gy-1 (slope) for GPAN/0 and GPAN/N variant cells, respectively.