Preclinical toxicity profile of oral bilastine.

As part of the bilastine development program, and as mandated by regulatory authorities, several studies were performed with oral bilastine in different animal species to evaluate its toxicity profile. Toxicokinetic analyses conducted in tandem to evaluate systemic exposure, gender differences, and dose proportionality in the different animal species indicated that animals were systemically exposed to bilastine during treatment. Repeated-dose toxicity studies in beagle dogs (52 weeks) and in rats and mice (13 weeks) showed that bilastine at doses up to 2,000 mg/kg/day was not associated with any mortality, ocular effects, or nodules/masses. Likewise, no bilastine-associated neoplastic lesions were observed in rats and mice after 104 weeks of treatment with bilastine at doses up to 2,000 mg/kg/day. In general, bilastine-related clinical signs, body-weight changes, food consumption, clinical chemistry, haematology, and macro- and microscopic findings were of low order and reversible, with effects present only at the highest doses administered. Bilastine (up to 1,000 mg/kg/day) was well tolerated in pregnant/lactating rats and in their offspring and subsequent generations. With respect to effects on embryofoetal development in rabbits, bilastine at 400 mg/kg/day (the highest dose evaluated) was assessed to be the no observed adverse effects level. Overall, bilastine demonstrated a favorable toxicity profile in all animal models investigated and at higher doses than the corresponding recommended daily human dosage.

Human tumor cell lines express low levels of oncomodulin.

Different human carcinoma cell lines were screened for the presence of Ca2(+)-binding oncomodulin. A specific polyclonal antibody was raised against a synthetic peptide (amino acids 99-108) of oncomodulin coupled to hemocyanin. Extracts of tumor cell lines (several human, one rat) were analyzed for the presence of oncomodulin by immunoblotting. A strong immunoreaction of oncomodulin was obtained in chemically transformed rat fibroblasts (T14c) in contrast to all human tumor cell lines investigated, where no immunoreaction was obtained. These results suggest that oncomodulin cannot be used in diagnostics of human tumors.

A chemically transformed rat fibroblast cell line expresses high levels of oncomodulin.

Chemically (by N-methyl-N'-nitro-N-nitrosoguanidine) treated rat fibroblasts (T14c) exhibited growth characteristics and a morphology typical for transformed cells and markedly different from untreated, parental cells. In contrast to untransformed cells, T14c fibroblasts produced significant levels of oncomodulin mRNA as analyzed on Northern blots even when compared to rat Morris hepatomas, the richest source of oncomodulin known so far. The levels of transcripts for both calmodulin and oncomodulin in T14c cells were higher in log phase growth as compared to confluent stages. The T14c model system may be useful in the elucidation of mechanisms involved in the regulation of oncomodulin synthesis.

Expression of the tumor-specific and calcium-binding protein oncomodulin during chemical transformation of rat fibroblasts.

An in vivo-in vitro approach to studying neoplastic development in carcinogen-exposed rat fibroblasts was evaluated. In the model described, oncomodulin (Mr 12,000; pI 3.9), a tumor-associated and Ca2+-binding protein, was used as a specific marker of malignant transformation. A rapidly proliferating granulation tissue was exposed in vivo or in vitro to potent carcinogens like N-methyl-N'-nitro-N-nitrosoguanidine and procarbazine. As an endpoint of transformation anchorage independent (AI) colony formation in the soft agar assay was chosen. Exposure to various doses of N-methyl-N'-nitro-N-nitrosoguanidine in vivo or in vitro, or to procarbazine in vivo, led to induction of AI, transformed cells. Exposure of the cells to various doses of procarbazine in vitro produced neither formation of AI cells in the agar nor expression of oncomodulin in extracts of the exposed cell population. Almost all of the chemically induced AI cell lines tested have been found to be tumorigenic in athymic mice. In contrast, a very low rate (zero to two colonies per 10(6) cells tested) of spontaneous AI populations derived from untreated cells. None of these control AI colonies yielded tumors. In our transformation assay the appearance of neoplastic phenotypes seems very rapid, probably due to the increased cell division at the time of carcinogen-exposure. Expression of oncomodulin was found in extracts of transformed cells harvested from agar colonies, derived from carcinogen-exposed granulation tissue, but not from normal, untreated fibroblasts, as shown by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatographic analysis, as well as 45Ca2+-transblot electrophoresis. The presence of oncomodulin in extracts of transformed cells correlates well with the chemically induced colony formation in the soft agar assay. Oncomodulin might be a suitable neoplastic marker to study chemical carcinogenesis.

Carbonic anhydrase activity in primary sensory neurons. I. Requirements for the cytochemical localization in the dorsal root ganglion of chicken and mouse by light and electron microscopy.

Subpopulations of primary sensory neurons in mammalian dorsal root ganglion (DRG) exhibit carbonic anhydrase (CA) activity. To identify these subpopulations in DRG cells of mouse and chicken, the reliability of the cytochemical localization of the enzyme requires that several conditions be fulfilled: Preservation of the enzyme activity in glutaraldehyde-containing fixative; accessibility of the cytoenzymatic reaction throughout 20-micron thick Vibratome sections; retention of the reaction product in situ during OsO4 post-fixation; specificity of the cytoenzymatic reaction for CA activity as corroborated by the immunocytochemical detection with antibodies anti-CA II in mouse DRG; strict correlation between the CA activity and the cytological characteristics in a given subclass of neurons. On the basis of these criteria, it is concluded that the CA activity may be used as a cell marker to identify cytologically defined neuronal subpopulations and their axons in mouse DRG. In chicken DRG, CA activity is not consistently expressed in a given subclass of ganglion cells and their axons. Hence, it is assumed that the expression of CA activity by DRG cells in chicken is modulated by functional or environmental conditions.

Neuronal subpopulations in the dorsal root ganglion of the mouse as characterized by combination of ultrastructural and cytochemical features.

The neuronal population of dorsal root ganglia in mouse consists of various classes of ganglion cells which may be divided in turn into subclasses by using several criteria. In class A cells, membrane-bound organelles are distributed ubiquitously throughout the large perikarya. Subclass A alpha (12%), which is characterized by large clumps of Nissl substance separated by narrow strands of neuroplasm, lacks detectable carbonic anhydrase activity. Subclass A beta (16%) displays small clusters of Nissl substance isolated by broad channels of neuroplasm and a moderate activity of carbonic anhydrase. Subclass A gamma (8%) shows the most intense carbonic anhydrase activity and a lack of uptake for [3H]L-glutamine. In class B cells (63%), the small perikarya display a zonal distribution of the organelles. Subclass B alpha contains parallel cisternae of rough endoplasmic reticulum and acid phosphatase isoenzymes present in long and curved Golgi saccules. Subclass B beta displays straight Golgi saccules rich in acid phosphatase isoenzymes and a high affinity uptake for glutamine. Subclass B gamma is characterized by the absence of acid phosphatase isoenzymes and by the presence of substance P-immunoreactivity. Class C cells (1%) have the smallest size and the highest affinity uptake for glutamine. Thus subtypes of primary sensory ganglion cells may be identified by the concomitant use of multiple criteria.

The retina-lamina projection in the visual system of the bee, Apis mellifera.

Single Golgi impregnated visual cells and their axons were treated from the retina to the first synaptic layer (lamina) in serial electron microscopic sections. This analysis of the retina-lamina projection was undertaken in the upper dorso-median eye region which is known to be involved in the perception of polarized light. For identification of individual visual cells and their fibres a numbering system was used which relates the number of each of the nine visual cells within one retinula to the transverse axis of the rhabdom (TRA) (Fig. 1). Because of the twist of the retinula along its course to the basement membrane (Fig. 6), individual visual cells change their position relative to any eye-constant co-ordinate system. Each axon bundle originating from one 9-celled retinula performs a 180 degrees-rotation before entering the lamina (Fig. 2). The direction of rotation (clockwise or counter-clockwise), which may differ even between adjacent bundles, is related to the two mirror-image types of rhabdoms in the corresponding retinulae and is opposite to the direction of rhabdom twist. Thus, even in small groups of the in total 5500 ommatidia in the eye of the bee, two types of retinulae exist which can be characterized by the geometry of the rhabdoms as well as by the direction of rotation of the retinulae and the axon bundles (Fig. 1). Visual cell numbers 1, 2, and 9, the microvilli of which are oriented in the direction of TRA, form three long visual fibres terminating in the second synaptic layer (medulla). In cross sections of laminar pseudocartridges they appear as the smallest fibre profiles arranged in a symmetrical line of the pseudocartridge bundle (=the transverse axis of the pseudocartridge; TPA) (Fig. 4). The remaining six fibres (cell numbers 3-8) only project to the lamina (short visual fibres; svf's). Two of them (cell numbers 5 and 6), which are the largest cells in the proximal retinula and have their microvilli perpendicularly arranged to TRA (Fig. 1), give rise to the two thickest axons of the underlaying pseudocartridge. In cross sections, t he connecting line of these two axons is orthogonally oriented to TPA (Fig. 5). A model was developed, in which all long visual fibres originate from ultraviolet receptors and in which the polarization sensitivity of the basal ninth cell is enhanced by the twist of the rhabdom. Finally, this model is discussed in light of behavioral experiments revealing the ultraviolet receptors as the only cells involved in the detection of polarized light.