Cytostatic assay

To screen for bone marrow toxicity early in drug discovery, assays must be able to evaluate hundreds of compounds, be inexpensive, report results within two weeks (in order to impact chemistry cycle times) and be able to detect toxicity irrespective of cytotoxic or cytostatic mechanisms. Only cell line-based assays can meet all of these various criteria. 

Meegan et al. reported the synthesis of a number of [3-lactams and these were evaluated in a series of in vitro assays, which determined their antiproliferative activity in MCF-7 and MDA-MB-231 breast cancer cell lines and also their affinity for the oestrogen receptor (Fig. 6). The cytotoxicity of the [3-lactams was determined in the LDH assay to establish that the antiproliferative effects observed were due to cytostasis rather than cellular necrosis. Most of the compounds demons-treated low cytotoxicity, indicating that their action is cytostatic rather than cytotoxic. Cytotoxicity values considerably below that obtained for tamoxifen (13.4%, 10 pM) were observed with a most potent compound (3%, 10 pM in MCF-7 cell... 

This cytostatic mechanism of action of paclitaxel in SMCs differentiates its antirestenotic activities from the antineoplas-tic activities in cancer cells, Paclitaxel has also been shown to be a potent inhibitor of SMC migration and chemotaxis (3,16,47,51), As shown in Figure 6, paclitaxel inhibited the migration of SMCs in a cell-culture wound assay at very low concentrations (16). 

Recently, it was shown that various nitrogen mustard-based cytostatics, for example, melphalan and cyclophosphamide, reacted with the cysteine 34 residue of human serum albumin in an analogous way. The tripeptide assay could be applied to samples of cancer patients treated with these cytostatics (28), which holds promise for optimization of chemotherapy with these agents by intensive screening of adduct levels in patients. 

Edelstein MB, Smink T, Ruiter DJ, Visser W, van Putten LM. Improvements and limitations of the sub-renal capsule assay for determining tumour sensitivity to cytostatic drugs. Eur J Cancer Clin Oncol 1984 20 1549-56. 

Substances capable of interaction with ROS are claimed to play an important role in the prevention of cancer. Some compoimds of the unsaponifiable fraction from virgin oil (oleuropein, tyrosol, sterols and triterpenoids) were assayed to determine their cytostatic activity in McCoy cells (originated from synovial fluid from a patient suffering degenerative arthritis) [92]. Oleuropein and the sterol+triterpenoid fraction potently inhibited cell growth at 6 Xg/ml (83 and 89% respectively), a concentration recommended by the protocols of the National Cancer Institute of USA [93]. IC50 for oleuropein and sterol+terpenoid fraction was 4.4 and 0.11 ig/ml, respectively. Tyrosol exhibited lower inhibition IC50 was 10 pg/ml. 

The number of cells within each colony depends on the number of cell doublings and can be used as an estimate of the effects, if any, of the drug on the cell doubling time. A clonogenic assay can thus discriminate between cytotoxic (cell kill) and cytostatic (decreased growth rate) effects. Because a cytostatic effect may be lost upon removal of the drug a cytotoxicity assay based on colony formation is also described since this allows continuous drug exposure. 

In an early report by Janes et al. [135], the interaction between DOX-loaded PEC nanoparticles (consisting of CHT and polyanions) and human melanoma cells was investigated with reference to the polyanion type and preparation protocol. Evidence was found that DOX-loaded PEC particles consisting of DS maintained the cytostatic activity with respect to free DOX, whereas DOX precomplexed with CHT before complexation with the polyanion showed slightly decreased activity (according to the MTT assay). Furthermore, CLSM studies revealed that DOX release did not take place in the cell culture medium, but that DOX was taken up into the melanoma cells by endocytosis while stUl bound to PEC particles and finally released intracellularly. 

In rodent cells, the phenotypic lag for expression of FsTdR or BUdR resistance is much shorter (less than 2 days) than the phenotypic lag for expression of 6TG resistance. The mechanism for resistance should be similar for both selection systems. Resistance to the selective agent requires a reduction in enzyme activity per mutant cell via division, dilution, and protein degradation, so that a toxic or cytostatic level of the analogue metabolite is not produced. The difference in expression times for FgTdR and 6TG resistance is probably caused by a faster degradation rate of TK or by a difference in sensitivity to the respective cytotoxic or cytostatic metabolites. In any case, such a short phenotypic lag is an obvious technical advantage in a cell culture mutation assay. 

In the granuloma pouch assay 3 showed no mutagenic activity. Interestingly it revealed a pronoimced cytostatic effect in this test. 

Bachurin, S. O. Dubova, L. G. Kireeva, E. G. Shevtsova, E. F. Screening assay using safranin dye for determining the effect of cjdoprotectants and cytostatics on mitochondrial permeabUity and... 

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