Purpose Proliferation of subconjunctival fibroblasts plays a critical role in scarring and failure of glaucoma filtering surgery. Long-term topical glaucoma medications appear to increase fibroblast proliferation. In this study, the effects of topical antiglaucoma drugs latanoprost and GLC756, a novel dopamine D2 agonist and D1 antagonist, on cultured normal human dermal fibroblasts (NHDF) were examined. Methods The NHDF cell line was incubated with latanoprost, prostaglandinF2α (PGF2α), GLC756, or 5-fluorouracil as a positive control at concentrations of 3 and 30 μM for 6, 18, and 24 hours. Fibroblast growth was measured by 5-bromodeoxyuridine (BrdU) uptake using laser scanning cytometry. Results Latanoprost and PGF2α had a biphasic response on the number of cultured NHDF positively stained with BrdU. A stimulating effect on proliferation occurred early, 6 hours after incubation, and an inhibitory effect 18 to 24 hours after incubation. GLC756, in contrast, revealed only inhibitory effects on BrdU uptake 18 to 24 hours after incubation. The pattern of GLC756 was similar to that of the positive control 5-fluorouracil. Conclusions Latanoprost seemed to have a biphasic response on the proliferation of cultured NHDF. First there was a stimulating thereafter a secondary negative modulating effect. GLC756 had a fully antiproliferative effect on the NHDF, indicating an additional potential of novel dopamine compounds for topical glaucoma medication.

The skin tolerability of the tubulin polymerisation inhibitor LAV694 was compared to that of 5% 5-fluorouracil (5-FU) and 0.5% podophyllotoxin in vitro using a human reconstructed epidermis (HRE), and in vivo using minipigs. Topical treatment of HRE for 1 or 3 days with a 0.2, 0.6 or 1% LAV694 cream or the placebo showed no signs of irritation in terms of morphology, cell viability (lactate dehydrogenase leakage) or interleukin-8 mRNA expression and release. 5-FU increased interleukin-8 production and induced morphological signs of irritation. The substances were also applied under occlusion to the back of two minipigs, twice daily, for 9 days to allow intraindividual comparison of skin effects and tolerability. Skin reactions were monitored by visual scoring, chromometry, pro-inflammatory activity, cell cycle and apoptosis by RT-PCR, laser scanning cytometry and histopathological examination of biopsies. Application of podophyllotoxin and 5-FU had to be stopped on days 4 and 8, respectively, due to severe skin lesions. LAV694 (1%) induced only moderate skin reddening after 9 days. 5-FU and podophyllotoxin, but not LAV694, increased mRNA expression of pro-inflammatory cytokines. LAV694 arrested keratinocytes in the M phase of the cell cycle and apoptosis was detected histologically in the basal layer. LAV694 increased the expression of pro-apoptotic genes in both experimental models. In conclusion, LAV694 selectively induced apoptosis, rather than necrosis, of growth-arrested keratinocytes, thus avoiding the occurrence of extensive inflammation. This resulted in an improved skin tolerability in comparison with 5-FU and podophyllotoxin.

One of the main concepts in toxicology and risk assessment is the identification of compound s with the least toxicity, gaining increased understanding of the underlying mechanisms of efficacy and toxicity so as to accelerate the early selection of compound s for development. For this purpose, “cuttingedge” technologies, such as flow cytometry (FC), laser scanning cytometry (LSC) and confocal laser scanning microscopy (CLSM), have proved to be valuable tools. FC, LSC and CLSM have been successfully applied in a wide range of areas within toxicology and research including genetics, reproduction, dermatology, pathology and target organ toxicity. The scope of this paper is to give a short overview of the usefulness of the different laser applications. Specific examples of the impact of these technologies will be presented or can be found in the references. Flow cytometry methods have been successfully applied in immunophenotyping, micronuclei scoring, polyploidy determination, apoptosis and cell cycle evaluation, cell proliferation and quantification. A three-parameter FC method for the analysis of testicular toxicity has also been established as an alternative to traditional histopathological methods. This method allows a large number of cells to be analysed in a short time and provides quantitative values to evaluate testicular damage in the rat. Laser scanning cytometry has been used in our unit for rat blood cell immunophenotyping, tumor proliferation, apoptosis and cell cycle analysis on minipig and rat skin and cardiac cells identification. The wide range of applications that can be applied with the LSC shows the enormous potential of this technology in research and development. Confocal laser scanning microscope was used in our laboratory, in collaboration with the research department, to investigate the mechanisms underlying hepatic lesions found in dogs, to detect fibrinogen influx into rat lung, to explore the mechanism of eye toxicity and to quantify dopaminergic fibers in brain sections. Integrating these technologies within discovery pathology allowed us to understand disease processes with respect to their development and subsequent consequences. It contributes to descriptive pathologic diagnostic and allows a productive interaction with research and development. These technologies offer a range of novel applications and have been shown to be useful tools in terms of specificity, sensitivity, reliability, rapidity and quantification. Expertise in cutting-edge technologies, pathology and cell and molecular biology is essential to a successful and flexible interaction across all therapeutic areas in drug discovery.