Prediction of adverse events by in vivo gene expression profiling exemplified for phytopharmaceuticals containing salicylates and the antidepressant imipramine.

BACKGROUND AND OBJECTIVE: Gene expression profiles of Sprague-Dawley (SD) rats treated with a standardized willow bark extract (WB), its salicin rich ethanol fraction (EtOH-FR) or the tricyclic antidepressant imipramine were evaluated for their potential to induce adverse events. Treatments had shown antidepressant-like effects. METHODS: Gene expression profiles (Agilent Whole Genome Array, n=4/group) obtained from the peripheral blood of male SD rats treated with WB (STW 33-I), EtOH-FR (30 mg/kg bw) or imipramine (20 mg/kg bw) were analysed comparatively by the Ingenuity Systems Programme, which allows to conduct model calculations of thresholds for theoretical potential adverse events (AE). RESULTS: The number of genes regulated by the three treatments were 1673 (WB), 117 (EtOH-FR) and 1733 (imipramine). The three treatments related to 47 disease clusters. The WB extract reached the threshold for a potential AE in one disease cluster (cardiac hypertrophy), whereas the EtOH-FR exceeded the threshold in 5 disease clusters (cardiac arteriopathy and stenosis, glomerular injury, pulmonary hypertension, alkaline phosphatase levels ⇑). Imipramine treatment hit 13 disease clusters: tachycardia, palpitation, myocardial infarction, arrhythmias, heart block, precipitation of congestive heart failure; urinary retention, altered liver functions. Those correspond to known potential adverse events. Glomerular injury and altered liver functions are part of the side effect profile of salicylic acid derivatives in agreement with the findings for the salicin rich EtOH-FR. CONCLUSION: There is no linear relationship between the number of constituents of a drug (preparation) and the number of different targets hit in a biological system on the gene expression level. Therefore, the number of genetic targets in a biological system does not necessarily increase with the complexity of the treatment corresponding to the non-linear behaviour of biological systems. Regarding gene expression levels AE of single treatments are not necessarily additive in combination treatments. The applied method appears to be an interesting screening tool for the prediction of potential AE. The phenomena that imipramine crossed the potential threshold for AEs several times whereas the WB extract did reach the threshold level only once, however not backed by clinical data for this AE, deserves to be further investigated. It questions the commonly assumed principle that substances with low number or without AE will have a poor efficacy.

Interfacial regulation of acid ceramidase activity. Stimulation of ceramide degradation by lysosomal lipids and sphingolipid activator proteins.

The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis(monoacylglycero)phosphate, phosphatidylinositol, and dolichol phosphate were incorporated into substrate-bearing liposomes. Higher ceramide degradation rates were observed in vesicles with increased membrane curvature. Dilution assays indicated that acid ceramidase remained bound to the liposomal surface during catalysis. Not only SAP-D, but also SAP-C and SAP-A, were found to be stimulators of ceramide hydrolysis in the presence of anionic phospholipids. This finding was confirmed by cell culture studies, in which SAP-A, -C, and -D reduced the amount of ceramide storage observed in fibroblasts of a patient suffering from prosaposin deficiency. Strong protein-lipid interactions were observed for both SAP-D and acid ceramidase in surface plasmon resonance experiments. Maximum binding of SAP-D and acid ceramidase to lipid bilayers occurred at pH 4.0. Our results demonstrate that anionic, lysosomal lipids are required for efficient hydrolysis of ceramide by acid ceramidase.

Cell type specific localization of sphingomyelin biosynthesis.

We have studied the incorporation of [(14)C]serine and of [(3)H]sphingosine into sphingomyelin in the presence or absence of brefeldin A (BFA) in three different cell types. Administration of BFA (1 microgram/ml) to fibroblasts for 24 h increased the incorporation of label into sphingomyelin 1.5-3 fold compared with untreated controls. In contrast, BFA strongly decreased sphingomyelin biosynthesis (4-5 fold) in cerebellar neurons as well as in neuroblastoma cells. The effect of BFA on glycosphingolipid formation, however, was similar in all three cell types studied: an increased labeling of the precursor glycolipids GlcCer, LacCer, GM3 and GD3 was paralleled by a decreased formation of complex gangliosides, GM1, GD1a, GT1b and GQ1b. Our data therefore suggest that in neuronal cells sphingomyelin synthesis, like the formation of complex gangliosides, is localized primarily distal to the BFA block, in a post-Golgi compartment, most probably the trans-Golgi network, whereas in fibroblasts sphingomyelin biosynthesis is mainly localized prior to the BFA block, in the Golgi apparatus, as has been shown for LacCer, GlcCer, GM3 and GD3 synthases.

Bcl-2 antagonizes apoptotic cell death induced by two new ceramide analogues.

Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N-thioacetylsphingosine and FS-5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations > or = 14 microM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly-(ADP-ribose) polymerase (PARP). Additional experiments in bcl-2-transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl-2. Furthermore, neither N-thio-acetylsphingosine nor FS-5 induced PARP cleavage in bcl-2-transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.