CHEMTECH
June 1998
CHEMTECH 1998, 28(6), 31-36.
Copyright © 1998 by the American Chemical Society.


ENABLING SCIENCE

New weapons in the fight against cancer

Some tumor cells are resistant to chemotherapy. An understanding of the mechanism of this resistance has led to the development of taxane drugs that can sensitize the tumor to chemotherapy with few systemic side effects.

Iwao Ojima
Pierre-Yves Bounaud
Ralph J. Bernacki

C ancer is a major health concern; an estimated 6 million new cases are diagnosed each year worldwide. Cancer is second only to heart disease as a cause of death in the United States. The threat posed by cancer was officially addressed early in 1971 with the enactment of the National Cancer Act, which increased research funding and was instrumental in the development of several national research centers. Considerable progress has been made since then to understand the disease and to control it, both with prevention and therapy (1, 2).

Almost half of the new cases of cancer are diagnosed early enough to be treated with surgery or radiotherapy. In the remaining cases, however, the cancer has metastasized and must be treated with chemotherapy. Chemotherapy can effectively cure several cancers, such as leukemia, lymphomas, and sarcomas, and can improve the long-term survival for patients with breast cancer, the most common malignancy in women. The drugs mostly used in cancer treatment are vinca alkaloids (vinblastine), anthracyclines (doxorubicin), epipodophyllotoxins (etoposide), cisplatin, and paclitaxel (generic name for Taxol, a drug licensed to Brystol-Myers Squibb).

Multidrug resistance
Although nearly 50 antineoplastic drugs are in use, only a few are effective in the treatment of each specific tumor type because of intrinsic or primary drug resistance. The initial and subsequent chemotherapy allow a tumor to develop acquired or secondary resistances because malignant cells that survive the drug are, of course, resistant to that drug. Ultimately, by a variety of mechanisms, a multidrug-resistant tumor evolves. At this point, additional chemotherapy only induces toxic effects in the patient without reducing the tumor. For example, paclitaxel is strongly cytotoxic against drug-sensitive breast cancer cells (IC50 = 2.5nM; IC50 is the concentration of a compound necessary to inhibit the cell growth by 50%), but it exhibits only very weak activity against the drug-resistant breast cancer cells (IC50 = 860 nM).

TO SIDEBAR: Paclitaxel and docetaxel

The broad-spectrum resistance to structurally and mechanistically diverse anticancer agents constitutes the multidrug resistance (MDR) phenotype. Tumor cells carrying this phenotype are characterized by the overexpression of an energy-dependent drug transport protein, P-glycoprotein (Pgp) (Figure 1). The overexpression results in a decreased accumulation of the drug within the cancer cell because the cell can efficiently pump out the hydrophobic anticancer drug molecules (9, 10).

Figure 1 thumbnail Figure 1.

Located across the cell membrane, Pgp detects and expels the drug as it enters the plasma membrane much like a hydrophobic "vacuum cleaner" (Figure 2, left). Often, cancer cells develop a natural defense mechanism against cytotoxic agents, and any effective chemotherapy must find a way to disable or circumvent that defense. Fortunately, specific drugs are able to inhibit the MDR mechanism in cancer cells, clearing the path for effective delivery of the anticancer agent. By binding preferentially to Pgp, these inhibitors block the efflux channel, allowing the anticancer agent to slip into the cancer cell unnoticed and then kill the cell (Figure 2, right). These inhibitors are called MDR reversal agents.

Figure 2 thumbnail Figure 2.

Since Tsuruo and co-workers discovered that verapamil (a calcium channel blocker) was able to affect the MDR mechanism (11), many other reversal agents have been found, such as calmodulin antagonists (trifluoperazine), antiarrhythmics (amiodarone), antihypertensive agents (reserpine), antipsychotics (phenothiazines), and immunosuppressants (cyclosporine A, FK-506) (12-16). However, undesirable side effects limit their use in clinical trials. New, potent noncytotoxic reversal agents with a minimum of undesirable side effects are in demand.

Taxanes as new MDR reversal agents
Our work on the medicinal chemistry of paclitaxel and its congeners ("taxoids") has focused on the development of strategies to combat cellular MDR. One approach has been to develop the "second-generation" taxoid anticancer agents - paclitaxel congeners that would not be recognized by Pgp and yet would still display potent anticancer activity. We have discovered and developed such second-generation taxoids that possess activity two orders of magnitude better than paclitaxel against MDR breast cancer cell lines (17-19). These taxoids are synthesized by coupling ß-lactams as N-acylisoserine precursors with properly protected 10-deacetylbaccatin III (DAB), 14ß-hydroxy-10-deacetylbaccatin III (14-OH-DAB), and their derivatives (Figure 3).

Figure 3 thumbnail Figure 3.

Isolated from the leaves of the European yew, Taxus baccata (20), DAB is an important natural product for the commercial production of paclitaxel and docetaxel. We were pleasantly surprised to discover that DAB, noncytotoxic itself, can serve as an important scaffold for a new series of MDR reversal agents with high potency (21). 14-OH-DAB, isolated from the Himalayan yew, T. wallichiana Zucc., as well as the European yew, is another source of the essential scaffold for our taxane-based MDR reversal agents (TRAs). It should be noted that Kobayashi and co-workers (22, 23) have isolated noncytotoxic natural taxanes from the Japanese yew, T. cuspidata, that display a reversal activity equivalent to that of verapamil.

DAB

We have systematically modified DAB and 14-OH-DAB in the search for even better TRAs. These compounds bear several hydroxyl groups that can be modified with hydrophobic ester side chain(s). Previous structure-activity relationship (SAR) studies of structurally different classes of MDR reversal agents pointed out the importance of a hydrophobic, conjugated, planar ring (9, 10). Accordingly, benzophenone- and naphthalene-containing carboxylic acids were chosen as hydrophobic modifiers.Figure 4 illustrates the synthesis of a highly promising lead compound, SB-RA-31012.



Figure 4 thumbnail Figure 4.

MDR reversal activity of these new taxanes was evaluated by testing the cytotoxicity of paclitaxel with a TRA against drug-resistant human breast cancer cell lines MCF7-R and MDA-435/LCC6-MDR. In our initial SAR study, we investigated the positional requirements for the hydrophobic ester side chains on their MDR reversal activity. The SAR study clearly shows that the modification at the C-2 or C-13 position gave poor results, whereas that of the C-7 and/or C-10 position(s) exhibits excellent MDR reversal activity (Figure 5).

Figure 5 thumbnail Figure 5.

As can be seen in Figure 6, paclitaxel recovers 95-99.8% of its efficacy against the resistant human breast cancer cells when TRAs are coadministered at 1.0 µM. Two of these TRAs, SB-RA-131012 and SB-RA-31012, exhibit high MDR reversal activity even at 0.1 µM. The TRAs possess much higher activity than verapamil and slightly better activity than cyclosporine A; however, cyclosporine A is rather cytotoxic (IC50 = 3 µM). A promising reversal agent PSC833 (24), a potent analogue of cyclosporine A, also exhibits appreciable cytotoxicity (IC50 = 10 µM). Therapeutic index, defined as the ratio of MDR activity to cytotoxicity, is an important factor for further studies of these agents. The TRAs are noncytotoxic even at 30\tmM, which is the highest concentration tested so far because of the solubility limit in the current TRA formulations. Thus, it is clear that the TRAs have excellent therapeutic indexes. It is worth mentioning that DAB and the hydrophobic modifiers do not show any MDR reversal activity at all by themselves, and therefore the MDR reversal activity is unique to these taxane structures.

Figure 6 thumbnail Figure 6.

To prove our hypothetical mechanism of action for these TRAs, we investigated the effects of SB-RA-31012 on the paclitaxel uptake by the drug-resistant cancer cells MDA-435/LCC6-MDR in the presence and absence the taxane (Figure 7). As a control experiment, we looked at the effects of the taxane on the paclitaxel concentration in the parent MDA-435/LCC6-WT cells that are drug sensitive. As can be seen in Figure 7, only a small percentage of paclitaxel accumulates in the drug-resistant cancer cells (MDA-435/LCC6-MDR) in the absence of the TRA. However, the addition of SB-RA-31012 (1 µM) dramatically increases the accumulation of paclitaxel in the drug-resistant cells, comparable with that of the drug-sensitive cancer cells (MDA-435/LCC6-WT). No appreciable effects of the TRA were observed on the accumulation of paclitaxel in the drug-sensitive cells, as expected. These results clearly indicate that the Pgp efflux mechanism is blocked by the TRA, allowing the anticancer drug to penetrate the drug-resistant cancer cells and exert its chemotherapeutic effect.

Figure 7 thumbnail Figure 7.

Paclitaxel is not the only anticancer drug that can be used with the TRAs. The sensitization of MCF7-R cells by a TRA was observed with another commonly used anthracycline-class anticancer agent, doxorubicin. SB-RA-31012 exhibited 96\% MDR reversal activity when coadministered with doxorubicin at 1 µM (21).

Conclusions
We have discovered that the taxanes derived from DAB and 14-OH-DAB bearing hydrophobic ester side chains at the C-7 and/or C-10 positions are potent MDR reversal agents against drug-resistant breast cancer cells when coadministered with anticancer drugs such as paclitaxel and doxorubicin. These TRAs exhibit MDR reversal activity >95% at 1 µM, and two TRAs have 92-94% reversal activity even at 0.1 µM when coadministered with paclitaxel against MDA-435/LCC6-MDR breast cancer cells. The negligible cytotoxicity of these TRAs makes them attractive candidates for clinical trials.

Additional studies on these TRAs for their MDR reversal activity in animal models and for pharmacological properties are under way. The SAR study for discovery of more potent TRAs through systematic structural modifications is also continuing.

Acknowledgements

References
ACS Pubs Chem Center