Paclitaxel mechanism studies

After the identification of the paclitaxel mechanism of action, several additional microtubule stabilizing agents were discovered from natural sources (Chart 9). Among them, epothilones (12-15) are the most studied and characterized. Epothilones A and B (12,13) were first isolated by Hofle and coworkers [54] from a myxobacterium (Sorangium cellulosum strain 90) in 1993, while their activity in stabilizing microtubule similarly to paclitaxel was reported for the first time by Bollag and co-workers in 1995 [55], Experiments with radio-labeled paclitaxel... 

Propidium iodide vs. annexin membrane staining was performed in cell culture to assess the primary mechanism of cell death with paclitaxel targeted nanodroplets prepared with triacetin. This study showed evidence for necrosis which could be due to the toxicity of the then-used triacetin. These mechanism studies were never substantiated further. However, toxicity studies were conducted with both the triacetin and Capmul vehicles demonstrated that the toxicity of the vehicle was over three magnitudes less than paclitaxel. Thus, we are able to identify the mechanism of action. The picture below notes the evidence primarily for necrosis. 

In vitro studies have shown that paclitaxel is a potent radiation sensitizer. Survival curves using grade 3 human astrocytoma cell lines showed a sensitizer enhancement ratio of 1.8 (57). In a series of experiments using a human leukemic cell line (HL-60), a sensitizing enhancement ratio of 1.48 was noted (58). Other studies have shown that G2/M block is not the only mechanism of paclitaxel-induced radiosensitization (59). 

Our SAR study of paclitaxel analogues on their ability to activate macrophage, inducing the production of NO and TNF, has revealed stark differences in the structural requirements for cytotoxicity vs. macrophage activation. The results warrant a great deal of further study on the possible alternative or auxiliary mechanism of action for paclitaxel and taxoids, which might lead to the discovery of a new series of taxoid anticancer agents with unique mechanism of action. 

The study of the tubulin-bound conformation of paclitaxel has resulted in a number of protein-ligand models, partially or fully based on the electron diffraction structure of aP-tubulin in paclitaxel-stabilized Zn2+-induced sheets [5, 12], Obviously, the nature of the paclitaxel binding site and the paclitaxel conformation in the binding site have key implications for the design of new MSA. A deep knowledge of the bioactive conformation would also help to explain how compounds as structurally diverse as the epothilones [48], discodermolide [49], and eleutherobin [50] have very similar mechanisms of action. 

Dose-dependent clearance and distribution was then later observed in a Phase 1 study in children with solid tumors (Sonnichsen et al., 1994). In a study in adults with ovarian cancer, Gianni et al. (1995) used a 3-compartment model with saturable intercompart-mental clearance into Compartment 2 and saturable, Michaelis-Menten elimination kinetics from the central compartment to describe the kinetics after 3 hour and 24 hour infusion. Now at this point one would typically assume that the mechanism for nonlinear elimination from the central compartment is either saturable protein binding or saturable metabolism. But the story is not that simple. Sparreboom et al. (1996a) speculated that since Cremophor EL is known to form micelles in aqueous solution, even many hours after dilution below the critical micellular concentration, and can modulate P-glycoprotein efflux, that the nonlinearity in pharmacokinetics was not due to paclitaxel, but due to the vehicle, Cremophor EL. This hypothesis was later confirmed in a study in mice (Sparreboom et al., 1996b). 

In another study, radiolabeled and fluorescent lipid nanocapsules were synthesized by using a phase inversion process that followed the formation of an o/w microemulsion containing triglycerides, lecithins, and a nonionic surfactant. Results of the experiment revealed that lipid nanocapsules were rapidly accumulated within cells through active and saturating mechanisms. Nanocapsules could bypass the endo-lysosomal compartment with only 10% of the cell-internalized fraction found in isolated lysosomes. When nanocapsules were loaded with paclitaxel, smallest lipid nano capsules (LNCs) also were found to trigger the best cell death activity. ... 

Here, we describe one representative example, namely the application of direct Raman imaging to the visualization of an anticancer drug, paclitaxel, within living tumour cells (see Ling et al. (2002)). Paclitaxel is an important antimitotic agent whose mechanisms of interaction with cells are reasonably well understood thus, it can be viewed as a suitable candidate for validation of the capabilities of Raman imaging. The human breast tumour cell line MDA-435 was used in these studies. 

---