Caenorhabditis elegans as model system for rapid toxicity assessment of pharmaceutical compounds.

INTRODUCTION: The model organism Caenorhabditis elegans is widely used for genetic studies as well as a living biomonitor in ecotoxicology. In this study, we investigated whether C. elegans may represent a suitable model for rapid preliminary toxicity studies of pharmaceutical compounds. METHODS: For this purpose, we used the EGFR kinase inhibitors BIBU1361, BIBX1382, and an inactive chemical analogue BIBU1476. As a first parameter to score for toxicity, we determined lethality of the wild-type C. elegans strain N2 (Bristol) in the presence of the compounds. The transgenic C. elegans strain PC72 (lacZ, heat shock protein-16 [hsp-16] construct) was used as a report organism for toxic effects. PC72 expresses beta-Galactosidase which is induced by hsp-16 in direct response when exposed to toxic compounds. The expression of beta-Galactosidase in cells was subsequently visualized by histochemical staining with X-Gal. RESULTS: A rank order of potency with respect to lethality was established: BIBU1361>BIBX1382>>BIB1476. The induction of beta-Galactosidase was concentration-dependent for each compound and demonstrated the same order of potency as observed for lethality. Furthermore, these compounds showed the same order for lethality in rodents, the first requirement of validation. DISCUSSION: These results indicate that wild-type C. elegans and the transgenic strain PC72 are both suitable models to determine the toxicity of pharmaceutical compounds. This approach allows for an easy and fast ranking of compound toxicity, which may lead to a more rational choice for further in vivo tests.

Inhibition of epidermal growth factor receptor activity by two pyrimidopyrimidine derivatives.

Overexpression of the epidermal growth factor receptors (EGFRs) and human epidermal growth factor receptor 2 occurs frequently in human cancers and is associated with aggressive tumor behavior and poor patient prognosis. We have investigated the effects in vitro and in vivo of a new class of inhibitor molecules on the growth of several human cancer cell lines. BIBX1382 [N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine] and BIBU1361 [(3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1yl)-pyrimido[5,4-d]pyrimidin-4-yl]-amine] are two new selective EGFR kinase inhibitors that do not block the activity of other tyrosine kinases. BIBU1361 blocked epidermal growth factor-induced phosphorylation of EGFR and also prevented downstream responses such as mitogen-activated protein kinase kinase (MAPK/extracellular signal-regulated kinase kinase) and MAPK activation in cells. In accordance with these observations thymidine incorporation into EGFR-expressing KB cells was selectively and potently inhibited by BIBX1382 and BIBU1361 with half-maximally effective doses in the nanomolar range. Oral administration of these compounds inhibited the growth of established human xenografts in athymic mice, including vulval and head and neck squamous cell carcinomas. Tumor growth inhibition by BIBX1382 coincided with reduced pEGFR and Ki-67 levels in vivo, which is in accordance with the expected effect of EGFR inhibitors. Collectively, these results show that the structural class of pyrimidopyrimidines, exemplified here by BIBX1382 and BIBU1361, represents an interesting scaffold for the design of EGFR inhibitors.

The effect of R 15.7/HO, an anti-CD18 antibody, on the late airway response and airway hyperresponsiveness in an allergic rabbit model.

1. The effects of a mouse (IgG1 fraction) anti-CD 18 neutralizing antibody (R15.7) on allergen-induced late airway response (LAR), airway hyperresponsiveness (AHR) and cellular recruitment were investigated in an allergic rabbit model. 2. Litter-matched NZW rabbits immunized within 24 h of birth with Alternaria tenuis (i.p.) and subsequently exposed to the allergen (i.p.) for the first 3 months of life were challenged with inhaled allergen as adult rabbits. Lung function in terms of dynamic compliance (Cdyn; ml cmH2O-1) and total lung resistance (RL; cmH2O-1 s-1) was monitored for 6 h following the allergen challenge. On day 16, separate groups of rabbits were pretreated with either control antibody (a non-binding mouse IgG1, 1 mg kg-1, i.v.) or R15.7 (1 mg kg-1, i.v.) and 1 h later all were challenged with Alternaria tenuis and lung function monitored thereafter. Airway responsiveness to inhaled histamine was assessed by measuring RL and Cdyn 24 h before and after allergen challenge and bronchoalveolar lavage (BAL) was also performed 24 h before and after allergen challenge. 3. Pretreatment of rabbits with the control antibody had no effect on the LAR as measured by AUC (Cdyn, 0-6 h). However, the magnitude of the LAR following treatment with R15.7 was significantly reduced when compared to LAR demonstrated on 1st challenge (P < 0.001) or to that of the control group on both challenges (P < 0.01). 4. In control antibody pretreated rabbits allergen induced a significant 3.4 fold reduction in the PC50 response to inhaled histamine in terms of RL changes (P < 0.05) and a significant 2.1 fold reduction in PC35 response to inhaled histamine in terms of Cdyn changes (P < 0.05). However, in anti-CD 18 antibody pretreated rabbits there was no significant change in responsiveness to histamine 24 h following allergen, as assessed by either RL PC50 or Cdyn PC35. 5. Allergen challenge induced a significant increase in eosinophil and neutrophil numbers (P < 0.05) in rabbits pre-treated with control antibody, whereas treatment with R15.7 significantly inhibited this increase in the numbers of both cell types. 6. This study demonstrates that the neutralization of CD-18 molecules reduces allergen-induced infiltration of both eosinophils and neutrophils into the airways and abolishes the accompanying LAR and AHR. These results provide evidence to support a role for CD-18 adhesion molecules in the transmigration of inflammatory cells into airways.

The effect of epithelium removal on leukotriene E4-induced histamine hyperresponsiveness in guinea-pig tracheal smooth muscle.

1. Removal of the epithelium resulted in a threefold increase in guinea-pig tracheal sensitivity to histamine without increasing the maximal response. 2. Preincubation of epithelially-denuded guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased the subsequent maximal response of the tissues to histamine. The sensitivity of the tissues to histamine was unaffected by LTE4 pretreatment. 3. Pretreatment of the epithelially-denuded tissues with the LTE4-analogue, 20-COOH LTE4, did not affect the maximal response to histamine. 4. LTE4 pretreatment increased the maximal response of the epithelially-denuded tissues to substance P (SP) but did not affect the maximal response to carbachol, KCl nor to the beta-adrenoceptor agonist, isoprenaline. 5. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM) and atropine (1 microM). 6. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 7. It is proposed that LTE4 induces an increased maximal response of epithelially-denuded guinea-pig airway smooth muscle to both histamine and substance P via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. Further, it is suggested that the increased maximal response of the epithelially-intact tissues to both histamine and substance P, after LTE4 pretreatment, may be suppressed by an epithelially-derived factor.

The mechanism of LTE4-induced histamine hyperresponsiveness in guinea-pig tracheal and human bronchial smooth muscle, in vitro.

1. Preincubation of guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased its subsequent responsiveness to histamine. 2. LTE4 pretreatment of guinea-pig tracheal strips did not affect the subsequent responsiveness to either the contractile agents, carbachol and KCl, or to the relaxant beta-adrenoceptor agonist, isoprenaline. 3. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM), atropine (1 microM) and tetrodotoxin (1 microM). 4. U46619, a stable thromboxane A2-analogue, at a non-contractile concentration of 4 nM, increased tracheal smooth muscle sensitivity to histamine. 5. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 6. Preincubation of human bronchial spirals with LTE4 in vitro increased its subsequent responsiveness to histamine. 7. LTE4-induced histamine hyperresponsiveness of human bronchus was inhibited by GR32191 (3 microM) and atropine (1 microM). 8. It is proposed that LTE4 induces guinea-pig airway smooth muscle hyperresponsiveness to histamine via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. In addition, it is suggested that LTE4 may induce histamine hyperresponsiveness of human bronchus in vitro by a similar mechanism as to that seen in guinea-pig central airway smooth muscle.

The contractile activities of lipoxin A4 and lipoxin B4 for guinea-pig airway tissues.

1. The isometric contractile activities of lipoxin A4 (LxA4) and lipoxin B4 (LxB4) were evaluated on guinea-pig lung tissue over the concentration range, 10(-8) to 10(-5) M. 2. LxA4 contracted guinea-pig lung parenchymal strips; the concentration eliciting 50% maximum histamine response was 3 x 10(-6) M. LxA4 did not contract tracheal spirals. 3. The LxA4 dose-response curve was parallel to that of leukotriene D4 (LTD4) with LxA4 being approximately 10,000 fold less potent than LTD4. 4. The time course of the contraction elicited by LxA4 was similar to that of LTD4 and it was slow in onset and did not plateau for 20 min. 5. Pre-incubation of parenchymal strips with leukotriene receptor antagonists at a concentration of 1 x 10(-6) M to 3 x 10(-5) M FPL 55712 or 3 x 10(-5) M L 649923 inhibited LxA4 activity. 6. Pre-incubation of tissues with 1 x 10(-5) M L 651392, a 5-lipoxygenase inhibitor, or 1 x 10(-5) M indomethacin, a cyclo-oxygenase inhibitor, did not affect the contractile activity of LxA4. 7. LxB4 did not constrict parenchymal strips or tracheal spirals.