Isolation and identification of three potential impurities of pholcodine bulk drug substance

Three previously unreported manufacturing impurities were isolated from a pholcodine mother liquor using preparative reversed-phase HPLC. The liquor was the residue remaining after recrystallisation of a production batch of pholcodine. The impurities, which are structurally related to pholcodine, were initially detected by thin-layer chromatography (TLC). Their structures were determined after separation by preparative HPLC (Econo-Prep 5 microm C18 column, 30 cm x 21.2 mm i.d.). Structure elucidation was carried out using nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS) and ultra violet (UV) spectroscopy. The impurities were identified as alkylated derivatives of pholcodine possessing second 2-morpholinoethyl substituents at various positions.     

A physiological toxicokinetic model for exogenous and endogenous ethylene and ethylene oxide in rat, mouse, and human: formation of 2-hydroxyethyl adducts with hemoglobin and DNA

Ethylene (ET) is a gaseous olefin of considerable industrial importance. It is also ubiquitous in the environment and is produced in plants, mammals, and humans. Uptake of exogenous ET occurs via inhalation. ET is biotransformed to ethylene oxide (EO), which is also an important volatile industrial chemical. This epoxide forms hydroxyethyl adducts with macromolecules such as hemoglobin and DNA and is mutagenic in vivo and in vitro and carcinogenic in experimental animals. It is metabolically eliminated by epoxide hydrolase and glutathione S-transferase and a small fraction is exhaled unchanged. To estimate the body burden of EO in rodents and human resulting from exposures to EO and ET, we developed a physiological toxicokinetic model. It describes uptake of ET and EO following inhalation and intraperitoneal administration, endogenous production of ET, enzyme-mediated oxidation of ET to EO, bioavailability of EO, EO metabolism, and formation of 2-hydroxyethyl adducts of hemoglobin and DNA. The model includes compartments representing arterial, venous, and pulmonary blood, liver, muscle, fat, and richly perfused tissues. Partition coefficients and metabolic parameters were derived from experimental data or published values. Model simulations were compared with a series of data collected in rodents or humans. The model describes well the uptake, elimination, and endogenous production of ET in all three species. Simulations of EO concentrations in blood and exhaled air of rodents and humans exposed to EO or ET were in good agreement with measured data. Using published rate constants for the formation of 2-hydroxyethyl adducts with hemoglobin and DNA, adduct levels were predicted and compared with values reported. In humans, predicted hemoglobin adducts resulting from exposure to EO or ET are in agreement with measured values. In rodents, simulated and measured DNA adduct levels agreed generally well, but hemoglobin adducts were underpredicted by a factor of 2 to 3. Obviously, there are inconsistencies between measured DNA and hemoglobin adduct levels.     

Inhibitory effect of Trapidil on the proliferation of bovine corneal fibroblasts in vitro

· Background: In order to develop new strategies for the pharmacological modulation of posttraumatic and postsurgical wound healing of the corneal stroma, the effect of Trapidil, a competitive platelet-derived growth factor (PDGF) antagonist, on the proliferation of cultured bovine stromal fibroblasts (BSF) was investigated. · Methods: BSF, obtained from explant cultures, were seeded at a cell density of 100/mm². The effect of various concentrations of Trapidil on cell viability and cell proliferation was determined using three different culture conditions: (1) serum-free medium (WM/F12), (2) serum-containing medium (WM/F12+10% FCS), and (3) serum-free medium +50 ng/ml PDGF-BB. Trapidil was added in concentrations ranging from 100 μg/ml to 400 μg/ml. Cell numbers were determined 2 and 5 days after addition of Trapidil, using a computer-based cell-counting system. Cell viability was evaluated morphologically and by means of a repopulation assay. · Results: Addition of Trapidil (100–400 μg/ml) led to a significant, dose-dependent inhibition of both serum- and PDGF-BB-induced proliferation of BSF. In contrast, treatment of quiescent BSF, cultured in serum-free medium, did not result in a significant reduction of cell number. No cytotoxic effects were observed. · Conclusion: The results of the present study demonstrate an inhibitory effect of Trapidil on the proliferation of BSF. It can be assumed that application of Trapidil might be a useful tool in the prevention of corneal complications after trauma (e.g., scarring, astigmatism and – with respect to photorefractive procedures – formation of haze and regression of the refractive effect). Received: 16 January 1998 Revised version received: 12 March 1998 Accepted: 17 March 1998     

Die experimentelle Frühsommer-Encephalomyelitis (FSEM) der weißen Maus

Zusammenfassung In der vorliegenden Arbeit wurde die Topik der Hirnläsionen nach einer experimentellen subcutanen Infektion der Maus mit FSEM-Viren der Stämme VIE 415 B, V 175,Hypr undWill beschrieben. Die bevorzugten Lokalisationen der Encephalitis waren die Großhirnrinde, der Linsenkern, einige Thalamuskerne (Nucl. parataenalis, Nucl. centralis, Nucl. paracentralis, Zona incerta), die Formatio reticularis und die Trigeminus- und die Vestibulariskerngebiete. Eine weitere Praedilektionsstelle für das Auftreten encephalitischer Infiltrate waren die Grenzregionen zwischen den einzelnen Kernen. Die rostralen und die ventralen Hirnabschnitte waren bevorzugt befallen. Auch einige Fasersysteme zeigten entzündliche Veränderungen.Bei der durch das FSEM (CEE)-Virus hervorgerufenen Encephalitis handelt es sich um eine fleckförmige Encephalitis mit perivasculären Gewebsalterationen.

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Summary The present investigation deals with the topography of brain lesions in albino mice as consequence of subcutaneous infection with spring-summer-encephalitisvirus (strains VIE 415 B, V 175, HYPR and WILL). The lesions could be located preferably in the cortex, nucleus lentiformis, some nuclei of the thalamus- such as nucleus parataenalis, centralis, paracentralis, the zona incerta and the nucleus reticularis-, the formatio reticularis and the trigeminal and vestibular region. Cellular infiltration were most often found along the borders of these nuclei, respectively in their rostral or ventral parts. Similar infiltrations were found in fibertracts of adjacent regions. The experimental spring-summer-encephalomyelitis in albino mice apparently leads to spotted perivascular alterations of gray and white matter as well.

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Frau Prof. Dr.Carmen Coronini-Cronberg zum Geburtstag in Verehrung gewidmet.     

Zur Epidemiologie des Carcinoms

V. Zusammenfassung Die Unterschiede in der Krebssterblichkeit zwischen den österreichischen Bundesländern in den Zeiträumen 1939/41, 1952/54 und 1959/61 wurden analysiert. Das Bild wird durch Unterschiede im Niveau der gesundheitlichen Betreuung der Bevölkerung wesentlich beeinträchtigt; eine Klärung der epidemiologisch wichtigen Fragen nach der Existenz reeler regionaler Unterschiede und zeitlicher Entwicklungstendenzen ist dadurch sehr erschwert. Eine annähernde Abschätzung der gröbsten Verzerrungen führt zu einer wesentlichen Einengung der Differenzen, wobei bei den Frauen keine statistisch signifikante Differenzierung nachweisbar ist. Bei den Männern sprechen gewichtige Momente für eine echte Übersterblichkeit in Wien, welche auch in der Gesamtmortalität ihren Niederschlag findet. Eine Tendenz zur Angleichung der Werte zwischen Wien einerseits und den übrigen Bundesländern andererseits dürfte wahrscheinlich sein; auch diese Tendenz findet in der Entwicklung der Gesamtsterblichkeit eine gewisse Parallele.

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Summary The differences in the cancer mortality among the Austrian Federal States in the years 1939/41, 1952/54, and 1959/61 were analyzed. The variations in the level of health care of the population prejudice these differences considerably. Consequently, it becomes difficult to elucidate the important epidemiological questions regarding the existence of real regional differences and the developmental tendencies occurring with time. An estimation of the most obvious distortions leads to an approximation of the differences, whereby women stastistically manifest no significant differentiation. With men, however, important factors suggest a truly excessive mortality in Vienna which expresses itself in the entire mortality. A tendency for the values between Vienna and the remaining Federal States to equilibrate seems likely. The counterpart of this tendency is found in the development of the entire mortality.

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Herrn Prof. Dr. A. Pischinger zum 65. Geburtstag gewidmet.

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Diese Arbeit wurde vom Österreichischen Forschungsrat unterstützt.     

Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis.

Dendritic spines receive most excitatory inputs in the vertebrate brain, but their function is still poorly understood. Using two-photon calcium imaging of CA1 pyramidal neurons in rat hippocampal slices, we investigated the mechanisms by which calcium enters into individual spines in the stratum radiatum. We find three different pathways for calcium influx: high-threshold voltage-sensitive calcium channels, NMDA receptors, and an APV-resistant influx consistent with calcium-permeable AMPA or kainate receptors. These pathways vary among different populations of spines and are engaged under different stimulation conditions, with peak calcium concentrations reaching >10 microM. Furthermore, as a result of the biophysical properties of the NMDA receptor, the calcium dynamics of spines are exquisitely sensitive to the temporal coincidence of the input and output of the neuron. Our results confirm that individual spines are chemical compartments that can perform coincidence detection. Finally, we demonstrate that functional studies and optical quantal analysis of single, identified synapses is feasible in mammalian CNS neurons in brain slices.     

Collagen type Ⅲ is an important linking molecule between generated renal tubules and an artificial polyester interstitium Reticulin as a linker between generated tubules and the interstitium

在再生医学领域,已经有很多人考虑在将来用干,祖细胞治疗急慢性肾功能衰竭.但是,要使这种治疗方案行之有效,需要了解关于患病肾脏肾小管发育的细胞生物学信息.与干/祖细胞治疗急慢性肾功能衰竭相关尚待明晰的细胞生物学问题包括:①干/祖细胞的整合.②干/祖细胞的定向分化类型.⑨新形成肾小管的空间构成.为了更好的了解这项技术的相关机制,文章应用了先进的培养技术在人工非细胞外基质材料界面条件下构建肾小管.将新生兔肾来源的干/祖细胞用多层聚酯纤维网覆盖,放置在灌注培养器中,用含1×10-7mol/L醛固酮的新鲜合成的在DMEM基础上改良的IMDM培养基诱导培养13 d.扫描电镜下,在大豆凝集素、银染和抗Ⅲ型胶原或层粘连蛋白Y 1单克隆抗体标记的全标本包埋爬片或冰冻切片上观察肾小管的生长情况.扫描电镜观察结果显示,新牛成的肾小管完全被基底膜覆盖,纤维网状板有大量的纤维与牛成的肾小管基底面和周围人工聚酯材料相互交联.抗Ⅲ型胶原染色和银染结果表明,在新牛成的肾小管的基底膜和邻近的人工聚酯材料间有大量的纤维交织,在发育成熟的肾小管中有与新生成肾小管相同排列形式的Ⅲ型胶原纤维.以上实验结果说明.Ⅲ胶原是联系新生成的肾小管基底面和人工基质聚酯纤维的分子纽带.     

In vitro susceptibility of clinical isolates of Brucella melitensis to fucidic acid

Brucella species are facultative intracellular bacteria, and therefore a limited number of antibiotics are effective against these organisms. The side effects of drug combination schemes, and the incidences of relapses and therapeutic failures, have led to investigations of new drugs to treat brucellosis. The purpose of this study was to test the in vitro susceptibility of 50 Brucella melitensis isolates to fucidic acid, which has not previously been used for the treatment of brucellosis. The minimum inhibitory concentrations (MICs) of fucidic acid to 50 B. melitensis isolates that were obtained from blood and bone marrow cultures of patients with brucellosis were studied by the broth microdilution method. The MIC₅₀ and MIC₉₀ values for the 50 B. melitensis strains susceptibility to fucidic acid were determined to be 0.5 and 2µg/ml, respectively, and the MIC range was 0.125–2.0µg/ml. Further experiments are needed to reassess the activity of fucidic acid against intracellular Brucella spp.