Biopharmaceutical characterization of the telomerase inhibitor BRACO19.

PURPOSE: To characterize the telomerase inhibitor and G-quadruplex stabilizing substance 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis (3-pyrrolodino-propionamido) acridine x 3HCl (BRACO19) in terms of biopharmaceutical properties such as solubility, protein binding, interaction with membrane lipids, cytotoxicity, and permeability across pulmonary epithelial cells. METHODS: Protein binding and interaction with membrane lipids were investigated by two high-performance liquid chromatography methods with immobilized human serum albumin and immobilized phosphatidylcholine, respectively. Cytotoxicity (methyl-thiazolyl-tetrazolium assay) and transport studies were performed with the bronchial cell lines 16HBE14o- and Calu-3, primary human alveolar epithelial cells, and the intestinal cell line Caco-2. Transport experiments were also done in the presence of cyclosporin A (10 microM) and tetraethylammonium chloride (5 mM) and at low temperature (4 degrees C). RESULTS: BRACO19 has good solubility of at least 2 mg/mL in water and in physiological buffers of pH 7.4 and below. Protein binding to human serum albumin was 38%. No interaction with membrane lipids could be found. Cytotoxicity in 16HBE14o-, Calu-3, and human alveolar epithelial cells was in the range of IC50 = 3.5 to 13.5 microM. Caco-2 cells were not affected at concentrations up to 50 microM. No transport of BRACO19 was detected across either cell monolayer in absorptive direction. In secretory direction, permeability was very low, with P (app) values in the range of 0.25 x 10(-7) to 0.98 x 10(-7) cm/s for all epithelial cell cultures tested. The transport was not influenced by cyclosporin A or tetraethylammonium chloride or at 4 degrees C, indicating that no efflux/influx systems or active transport are involved. CONCLUSIONS: From these results, we conclude that the very poor permeability of BRACO19 is its main biopharmaceutical limitation. Further applications will require a suitable formulation to warrant adequate delivery across cellular barriers.

Reconstructed epidermis versus human and animal skin in skin absorption studies.

European chemical policy in general and the REACH initiative in particular will increase the number of chemical substances submitted to toxicological evaluation by several orders of magnitude compared to the current status. To limit animal exposure the resulting enormous increase in testing, however, asks for validated in vitro test systems. While the OECD favours in vitro testing for cutaneous absorption using viable human and animal skin (Guideline 428) the availability of viable human skin is already limited today. We present a comparison of various in vitro techniques suitable for routine skin absorption studies including commercially available reconstructed human epidermis which may be a reliable alternative to excised human and animal skin. In order to develop a protocol for the subsequent transfer to partner laboratories the experimental set-up was analysed stepwise using the OECD reference compounds caffeine and testosterone. Franz cell type, the donor and receptor media for hydrophilic/lipophilic substances, albumin and tensid addition, and storage conditions of the excised skins were systematically varied. A protocol has been developed which now allows to proceed to the pre-validation process.

[Marker transport across biological barriers in vitro: comparison of cell culture models for the gastrointestinal barrier, the blood-brain barrier and the alveolar epithelium of the lung]. / Markertransport über biologische Barrieren in vitro: Vergleich von Zellkulturmodellen für die Dünndarmschleimhaut, die Blut-Hirn Schranke und das Alveolarepithel der Lunge.

In order to respond to the flood of new active ingredients currently being generated by combinatorial chemistry or molecular biological synthesis, selection procedures able to filter out rapidly and economically those drug candidates with the highest development potential are required. This necessitates the measurement of fundamental biopharmaceutical parameters very early in the drug development process. Any pharmaceutically active agent must be able to overcome the body's natural protective mechanisms. A broad variety of biological barriers can be simulated in the laboratory by cell monolayer models. Apart from ethical aspects, the advantage of these in vitro test systems is that permeability studies can be performed at high throughput rates under controlled and reproducible conditions. The validity of such a model is ultimately reflected in its ability to accurately predict the behaviour of an active ingredient at the corresponding in vivo barrier.

[Ultrasonography guided versus anatomically oriented puncture of the internal jugular vein for central venous catheterization]. / Ultraschallgesteuerte versus anatomisch orientierte Punktion der Vena jugularis interna zur zentralvenösen Katheterisierung. Eine randomisierte Studie.

AIM: Ultrasonography guided puncture (UGP) of the internal jugular vein (IJV) carried out by an expert is considered to be superior to the anatomically orientated puncture (AOP). Whether routine use of the UGP lowered the number of unsuccessful punctures and complications was unknown. Both puncture techniques were compared in a random study. METHOD: Between 10/93 and 3/94, 77 patients who needed central venous catheterisation for thorax or cardiac surgery were included in the study. 84 (42 vs 42) punctures were performed by seven anesthetists, all of whom had a great deal of experience with AOP, using either one or the other technique. The UGP was demonstrated and assisted once by an expert. A conventional ultrasound unit with a 7.5 MHz transducer was used. The number of unsuccessful first punctures, overall rate of unsuccessful punctures, morbidity and puncture time were compared. RESULTS: The unsuccessful first punctures using UGP occurred significantly less frequently (7 vs 19, p < 0.005). Fewer unsuccessful punctures were evidenced (9 vs 73, p < 0.001). One arterial puncture occurred with each of the puncture techniques. Five hematomas were observed in patients where AOP was used. Four of these patients were adipous and at least six unsuccessful punctures had already been carried out. The punctures lasted comparatively the same length of time (UGP: 59 s vs AOP: 60 s, p > 0.05). CONCLUSION: UGP is superior to AOP of the IJV also in routine use. It should be used more frequently in elective situations.

Transport of proteolytic enzymes across Caco-2 cell monolayers.

PURPOSE: To investigate the mechanisms by which proteolytic enzymes, such as trypsin, chymotrypsin, papain, and bromelain, are able to cross the intestinal mucosal barrier after oral administration to man. METHODS: Filter-grown Caco-2 cell monolayers were incubated with proteolytic enzymes and then the transepithelial electrical resistance (TEER) and the transport of the paracellular marker fluorescein were monitored. The effects of the enzymes on the cells were investigated by light microscopy and by biochemical assays. Transport of intact proteases across the cells was verified by monitoring the proteolytic activity and MALDI-TOF mass spectroscopic identification of undegraded trypsin. RESULTS: Depending on time, concentration, and side of exposure to Caco-2 cell monolayers, all proteases decreased the TEER and increased the transport of fluorescein. Some morphological and metabolic changes were observed. The effects were reversible, but until 24 hours after removal of the proteases. Under the conditions of this in-vitro model, approximately 10% of the apically applied dose reached the basolateral compartment as biologically active, non-degraded molecules. CONCLUSIONS: Proteolytic enzymes were found to exert considerable effects on the barrier function of Caco-2 monolayers, facilitating the transport of normally non-absorbable compounds. This suggests the also reported, but so far unexplained, systemic absorption of proteolytic enzymes after oral administration in vivo may occur by self-enhanced paracellular transport.

[Undesirable drug reactions in simultaneous administration of high-dosage phenobarbital and beta-lactam antibiotics]. / Unerwünschte Arzneimittelreaktionen bei gleichzeitiger Gabe von hochdosiertem Phenobarbital und Betalaktam-Antibiotika.

The present paper reports on a striking increase in undesirable drug induced reactions during simultaneous administration of high-dosage phenobarbital and beta-lactam antibiotics to children in intensive care. In a 30-month study period, reactions which had to be classified as drug-induced were seen in 24 out of a total of 49 children (mainly exanthematous skin reactions, in some cases severe). The complications were mainly observed in patients receiving a combination of cefotaxime and phenobarbital. Etiologically, a toxic reaction to the combination of hepatically metabolized beta-lactam antibiotics with an enzyme-inducing drug must be considered in addition to the allergenic potential of the individual components.

Phagocytic activity of blood cells in diabetic risk probands and newly diagnosed type 1 diabetics.

The phagocytic activity of granulocytes and mononuclear blood cells was compared in probands at risk for insulin-dependent Type 1 diabetes mellitus and in newly diagnosed diabetics before and during short-term insulin treatment. Healthy persons without family history of Type 1 diabetes were used as controls. Furthermore, the relationship between phagocytic activity and the proportion on monocytes in the granulocyte- and mononuclear blood cell fractions was estimated. The phagocytic activity of the mononuclear cells from the risk subjects was reduced. This observation suggests that defective phagocytosis might be important in the pathogenesis of Type 1 diabetes. But the phagocytic activity of mononuclear cells from newly diagnosed diabetics was not severely impaired and was fully normal under insulin treatment. We found no differences in the phagocytic activity of the granulocytes between patients and healthy probands. The proportion of monocytes in the mononuclear cell fraction was significantly enhanced in newly diagnosed diabetics and remained high throughout the 6-month period of insulin therapy. We assume that the increased monocyte level and the phagocytic activity of mononuclear cells in diabetics are not related to each other. But the increased monocyte level could also be interpreted as a compensatory reaction against the impaired phagocytic activity observed in the risk probands.

[Serum levels and urine excretion of L-carnitine in patients with normal and impaired kidney function]. / Serumspiegel und Urinausscheidung von L-Carnitin bei Patienten mit normaler und eingeschränkter Nierenfunktion.

The influence of age, sex, and renal function on serum levels and urinary excretion of free carnitine was studied in 187 subjects. Sixty-one subjects with normal renal function (creatinine clearance greater than 100 ml/min) showed a serum carnitine level of 72.2 +/- 23.2 mumol/l. The carnitine values of males (76.8 +/- 23.3 mumol/l, n = 39) were higher (p less than 0.05) than those of females (64.0 +/- 21.0 mumol/l, n = 22). Carnitine levels did not correlate with age. Values in patients with normal renal function did not differ from serum carnitine levels in healthy controls (74.7 +/- 17.5 mumol/l, n = 49). The mean urinary carnitine excretion per day was 163.5 mumol (range 63.7-419.6 mumol) in patients with intact renal function. Extreme impairment of glomerular filtration rate (creatinine clearance less than 20 ml/min) resulted in higher carnitine concentrations in serum (108.9 +/- 39.4 mumol/l, n = 18, p less than 0.05), lower carnitine elimination per day (78.5 mumol, range 14.5 - 424.3 mumol, n = 18, p less than 0.05) and a decreased carnitine clearance (0.8 ml/min, range 0.2 - 3.8 ml/min). These data together with earlier results obtained in dialysis patients suggest that carnitine metabolism in renal failure is altered by reduction of both endogenous carnitine biosynthesis and renal carnitine clearance.

[Stress on the eyeball caused by mechanical vibration. Studies of patient transport and experimental models]. / Belastung des Augapfels durch mechanische Schwingungen. Untersuchungen beim Krankentransport und im Modellversuch.

The acceleration forces which occur in the eye and in the head region in transport of patients were investigated. It was to be established whether the finding often deteriorates appreciably in perforating eye injuries between the accident site and the ophthalmological operating theater. The investigation showed that smaller acceleration forces become active in the eye in the sitting position than in the lying position in patient transport. In transport in the hospital area, e.g., in the patient lift and on a stretcher, the maximum acceleration amplitudes which occur are greater than those occurring in the ambulance.--In the second part of the study, the region of the eyeball most sensitive to oscillation was determined experimentally in cadaver eyes in order to permit an evaluation of the acceleration parameters relevant in patient transport. It was shown that the eye is largely insensitive to the accelerations occurring in patient transport. It was shown that the eye is largely insensitive to the accelerations occurring in patient transport. Furthermore, it was shown that a vitreous prolapse cannot occur for this reason in an experimentally induced sclera injury.--Therefore, deteriorations in the condition of the bulb occurring in patient transport are due to movements of the eyelid and eyeball.

Gas chromatographic determination of some biogenic amines as their pentafluorobenzoyl derivatives in the picogram range and its applicability to biological materials.

The catecholamines dopamine and noradrenaline were converted into the stable pentafluorobenzoyl (PFB) derivatives for their specific and quantitative gas chromatographic (GC) assay. This allowed their detection in the picogram range using an electron-capture detector. Acylation was performed with pentafluorbenzoyl chloride in the presence of pyridine and with acetonitrile as solvent. The structures of the PFB-catecholamines were confirmed by GC-mass spectrometry. A good separation was obtained on 5% OV-17 at 265 degrees C. The high adsorption activity of the PFB-catecholamines could be overcome by optimizing the reaction conditions and applying special GC precautions. Linearity of the method was demonstrated for 50- 500 ng of the catecholamines with detection at the picogram level. The application of the method to biological materials is demonstrated.

[Functional significance of the columnar structure of the stratum corneum and its occurrence on free autologous skin grafts]. / Uber die funktionelle Bedeutung der Columnärstruktur des Stratum corneum und deren Vorhandensein auf freien autologen Vollhauttransplantaten

The morphological presentation of the stratum corneum and simultaneously measurements of the alkali resistance and alkali neutralization time of the skin have been carried out in 26 guinea pigs. Whereas we could not demonstrate any association between the alkali neutralization time and the structure of the stratum corneum, a significantly better alkali resistance of the skin could be found when the columnar structure of the stratum corneum was most pronounced. The structure of the stratum corneum on full thickness skin autografts was also compared to that of normal skin in 26 animals. The columnar structure can be just as well pronounced on grafts as on normal skin. Significant differences between graft and normal skin could not be demonstrated but random samples showed that a disturbance in the columnar structure occurred more often in the grafts.