| USAN: | Paclitaxel |
| Brand Name: | Taxol (Bristol-Myers Squibb) |
| Molecular Weight: | 839.92 |
| FDA Approval: | 1992 |
| Drug Class: | Anticancer |
| Indications: | Ovarian, Breast, and Non–Small-Cell Lung Cancer; Kaposi’s Sarcoma |
| Mechanism of Action: | Microtubule-stabilizing Agent (Tubulin Polymerization Agent) |
■ 1 History
Cancer is a disease of cellular dysfunction involving a range of biological activities that promote unregulated proliferation. It is as old as the existence of animals—cancers are found even in dinosaur bones. Approximately 110 types of cancer have been characterized. In particular, breast cancer (second only to lung cancer in terms of fatality rate) strikes one in eight women and there are approximately 200,000 annual incidents in the United States alone. About 25% of women with breast carcinoma eventually will die from their disease. Genetic predisposition is largely blamed for the genesis of breast cancer, as well as colon and prostate cancers.
In terms of the genesis of cancer, the carcinogen theory emerged first. Carcinogens are agents such as chemicals, X-rays, and UV light that cause cancers. As early as 1775, British doctor Percival Pott made an astute epidemiological observation that young English boys employed as chimney sweeps were more prone to develop scrotal skin cancers than their French counterparts.1 Further scrutiny revealed that the continental sweeps bathed more frequently after work, which prompted Pott to speculate that long exposure to coal tar caused skin cancer. Pott’s theory was later confirmed experimentally by Katsusaburo Yamagiwa and Koichi Ichikawa in Japan in 1914. Nowadays, the carcinogenicity of a compound is routinely screened using the Ames test.
In 1966, Peyton Rous at the Rockefeller Institute won the Nobel Prize in Physiology or Medicine for his discovery of tumor-inducing viruses. Fifty years previously in 1909, Rous was able to artificially produce for the first time a tumor in an animal (chicken) using a tumor virus, which was later named the Rous sarcoma virus, or RSV.2 Viral infections have now been implicated—hepatic cancers are caused by hepatitis B or C viruses (HBV and HCV), cervical cancer is caused by papilloma virus, and T-cell leukemia is caused by human T-lymphotropic virus 1 (HTLV-1). In addition, the Helicobacter pylori bacterium infection is a major risk factor for stomach cancer. The past several decades saw wide acceptance of the oncogene theory,3 but that is beyond the scope of this book.
The current arsenal of treatment for breast cancer includes surgery (i.e., mastectomy or lumpectomy), radiation, chemotherapy, and hormone treatment.
An aggressive chemotherapy4 regimen typically includes cyclophosphamide (2, Cytoxan), 5-FU (3, fluorouracil), and methotrexate (4). Cytoxan (2) is a nitrogen mustard, an alkylating agent discovered in the 1940s. Alkylating agents kill both resting and multiplying cancer cells. Specifically, they work by interfering with the chemical growth processes of cancer cells, thus preventing DNA from uncoiling and thereby blocking DNA replication and cell division. Unfortunately, alkylating agents also destroy healthy cells indiscriminately. The use of such drugs has often been compared to a “carpet bombing” strategy. By the same mechanism, a common side effect to such cancer treatment is hair loss—alkylating agents also kill hair follicle cells along with active dividing cancer cells. On the other hand, 5-FU (3) and methotrexate (4), a folic acid analogue, are antimetabolites. They prevent cancer cells from metabolizing nutrients and other essential substances, thus blocking processes within the cell that lead to cell division.
The female hormone (estrogen) can fuel the growth of breast cancer cells, and pregnancy should be avoided for breast cancer patients. Tamoxifen (5, Nolvadex) was initially developed by ICI as a birth-control pill. Although it was ineffective as a contraceptive in animal models, the FDA approved tamoxifen (5) for the treatment of metastatic breast cancer in 1977, and tamoxifen is now the most frequently prescribed anticancer drug in the world. It is a partial antagonist and partial agonist of the estrogen receptor (ER). It blocks the generation of estrogen in some parts of the body while acts like estrogen in some other parts of the body. More specifically, it is a SERM, that is, a selective estrogen receptor modulator. Tamoxifen (5) is very well tolerated but recently has been shown to lead sometimes to blood clots and endometrial cancer. Since estrogen is a key trigger in two-thirds of all breast cancers, after surgery, radiation and chemotherapy, the estrogen-dependent breast cancer patient is often treated with tamoxifen. In 1998, the FDA also approved tamoxifen for prophylactic use in women at high risk of developing breast cancer (those who have ER-positive tumors)—patients taking tamoxifen are 45% less likely to get breast cancer recurrence. However, because tamoxifen acts as a stimulant of estrogen action in the uterus and thus could cause uterine cancer, it is less desirable as a breast cancer preventive. A newer SERM, raloxifene (6, Evista)5 renders a 58% reduction of breast cancer.
Despite the significant benefit that tamoxifen has bestowed on breast cancer patients, the third-generation aromatase inhibitors are rapidly replacing tamoxifen (5) as the first-line treatment for breast cancers. Aromatase is the enzyme (in body fat, the liver, and the adrenal glands) that converts androgen to estrogen. Aromatase inhibitors may be classified into three types. Type I aromatase inhibitors bind to the aromatase enzyme irreversibly, so they are called inactivators. In some cases they are dubbed mechanism-based or “suicide” inhibitors when they are metabolized by the enzyme into reactive intermediates that bind covalently to the active site. Type I aromatase inhibitors are usually steroidal in structure as represented by Pharmacia’s exemestane (7, Aromasin), Novartis’s formestane (8, Lentaron), and Schering’s atamestane (9).6
Type II aromatase inhibitors reversibly bind to the enzyme. The first Type II aromatase inhibitor marketed for the treatment of breast cancer was aminoglutethimide (10), marketed by Ciba-Geigy since 1981. Aminoglutethimide (10) is not very selective, binding to several steroidal hydroxylases that have the CYP prosthetic group. Thankfully, continued SAR development led to the latest type III aromatase inhibitors such as anastrozole (11) and letrozole (12) with exceptional specificity for the aromatase P450 enzyme. Therefore, there are fewer selectivity-related toxicities with the drugs. While formestane (10) is a Type II aromatase inhibitor, anastrozole (11) and letrozole (12) are considered Type III aromatase inhibitors.6
Paclitaxel (1, Taxol), has had considerable success in treating ovarian and breast cancer since 1992. It was initially isolated as part of the NCI-USDA (National Cancer Institute-United States Department of Agriculture) plant-screening program. In August 1962, Arthur Barclay at USDA traveled to the Gifford Pinchot Forest in Washington State and found a little known Pacific yew tree, Taxus brevifolia. He collected samples of twigs, leaves, and fruits, labeled them B-1046 and shipped them to the NCI.7 After the cytotoxic effect of B-1046 extract was observed in 1966, Monroe E. Wall and Mansuhk C. Wani at the Research Triangle Institute took on the challenge to isolate and characterize the principal ingredient. Using bioactivity-directed fractionation, they were able to purify the crude extract so that the cytotoxic potency increased 1,000-fold. In 1967, they isolated the active principle as white crystals in a 0.014% yield from the dried bark of the Pacific yew. The molecule was later determined to have a molecular formula of C47H51NO4 and a molecular weight of 839.8 Wall christened the molecule “Taxol,” where “tax” signified the origin of the molecule from Taxus brevifolia, and “ol” indicated that the molecule contained one or more alcohol functionalities.
Because of its insolubility, scarce supply, and low potency, there was no overwhelming enthusiasm for Taxol until 1979 when Susan Howitz,9 an assistant professor at the Albert Einstein College of Medicine, discovered that Taxol had a completely novel MOA, unlike any other drug known at the time. It turns out that Taxol exerts its action by stabilizing the microtubules, resulting in inhibition of mitosis and induction of apoptosis. Taxol stops malignant tumors from growing by interfering with the microtubules that are responsible for dividing the chromosomes during cell division.
This novel mechanism rekindled interest in Taxol. The NCI took the torch from Wall, with Mathew Suffness as the champion. In 1984, the NCI amassed enough positive data to commence a phase I clinical trial of Taxol with about 30 patients. The drug was shown to be relatively safe. There was tremendous difficulty in procuring enough Taxol—it took about 20,000 lb of yew tree bark to isolate 1 kg of Taxol—but the NCI moved forward with the phase II trials in 1987. Taxol is notoriously insoluble in water but this was overcome by the addition of ethanol and Cremophor EL, a surfactant made of polyoxyethylated castor oil, which later proved to be important in reversing multidrug resistance. Although tested in ovarian cancer, renal cancer, and melanoma, Taxol initially was only found to be efficacious for ovarian cancer. Eventually, its chemotherapeutic applications were expanded to breast cancer, non–small-cell lung cancer, and Kaposi’s sarcoma.10
Securing proof of concept (POC) from the phase II trial was a triumph for the NCI, which had overseen the clinical development of Taxol from the beginning, more than 20 years previously. However, the NCI was not equipped to take on the expensive and long phase III trials involving numerous disciplines such as oncology, pharmaceutical science, pharmacokinetics and drug metabolism, statistics, drug safety science, and others. Following a competitive selection process, Bristol-Myers Squibb (BMS), the only major US pharmaceutical company to have made a bid, was awarded the molecule under the Cooperative Research & Development Agreement (CRADA) with the NCI in 1991. At the time, the commercial potential of Taxol had not fully manifested for the breast cancer indication. The NCI’s choice of BMS over the French company Rhône-Poulenc made sense because Rhône-Poulenc already had a competing—drug decetaxel (14, Taxotere) prepared from 10-deacetylbacctin (13, 10-DAB), which can be easily extracted in high yield from the leaves of the English yew tree T. baccata L. Taxotere was discovered by Frenchman Pierre Potier by making a minor modification of Taxol (replacing the benzoyl group on Taxol with a tert-butoxyl-carbonyl group).11,12 Taxotere (14), about 1.5-fold more potent than Taxol, had annual sales of $1.54 billion in 2003.
The FDA approved Taxol for use in refractory ovarian cancer in December 1992, for breast cancer in 1994, and later for non–mall-cell lung cancer and Kaposi’s sarcoma. By 2000, Taxol was the best-selling cancer drug of all time, with annual sales of $1.6 billion.
The raw material for isolating Taxol became an extremely contentious issue in the late 1980s and early 1990s. Since it takes one hundred years for a Pacific yew tree to become useful in terms of Taxol content, harvesting the trees for stem bark meant destruction of the forest. To make matters worse, the forest that harbors the trees is home to the endangered spotted owl. The battle raged for many years between the environmentalists, who wanted to save the trees, and cancer patients/oncologists, who were eager to get access to the drug. It ended abruptly in early 1993 when BMS started to use a semi-synthetic route to make Taxol which did not require the Pacific yew at all. Instead, BMS extracted 10-deacetylbacctin (13, 10-DAB) from T. baccata, the English yew, a common ornamental plant. BMS then used the side-chain installation process patented by Robert A. Holton at the Florida State University to make Taxol. More than three tons of English yew needles, a renewable source, need to be collected and processed in order to produce 1 kg of 10-DAB. The switch is worthwhile because Taxol cost $600,000 per kilogram at its most expensive, and costs nearly $400,000 per kilogram even now. The worldwide market for Taxol is about 400 kg per year. Currently, Taxol is produced in large fermentation tanks using plant cells.
■ 2 Pharmacology
2.1 Mechanism of Action
Many traditional anticancer drugs, such as cyclophosphamide (2, Cytoxan), 5-FU (3, fluorouracil) and methotrexate (4), work through, interact with, and cause irreparable damage to DNA; or to inhibit specific enzymes as exemplified by tamoxifen (5, Nolvadex) and raloxifene (6, Evista).
There is a third type of anticancer drug, known as antimitotics, whose MOA is through cell mitosis. One common characteristic of most cancer cells is their rapid rate of cell division. There are five stages of cell division: prophase, metaphase, anaphase, telophase, and interphase. Other cells are also adversely affected, but since cancer cells divide much faster than noncancerous cells, they are far more susceptible to antimitotic treatment. In order to accommodate antimitotics the cytoskeleton of a cell undergoes extensive restructuring. Microtubules become the scaffolding of the cell structure, forming the mitotic spindle, which is responsible for the segregation of chromosome during cell division. As the mitosis progresses, protein microtubules in the cell pull apart the chromosomes to each pole of the cell before cell division. Meanwhile, the cell walls begin to converge and the microtubules at the center of the convergence start to disassemble. The cell splits and the microtubules disappear in a process of depolymerization.
In the 1960s, colchicine (15) was found to be the first antimitotic. It works by binding to and inhibiting a protein called tubulin, the building block of microtubules. Colchicine–tubulin complex has been isolated and characterized. Tubulins come together to assemble microtubules in a process called polymerization, which leads to the arrest of cell division. Later on, the vinca alkaloids vinblastine (16, Velban) and vinorelbine (Navelbine) were discovered also to be antimitotics whose MOA is through inhibiting the polymerization of tubulin as well. Inhibition of microtubule formation arrests cell division at the metaphase stage of the cell cycle.
In 1977, Horwitz and Schiff began to elucidate the MOA of Taxol (1). Within a month, they were convinced that Taxol has a novel mechanism of action: Behaving like an antimitotic, it blocked cell division at the metaphase. But unlike colchicine (15) and vinblastine (16, Velban), which blocked cells in the mitotic phase of the cell cycle, cells treated with Taxol reorganized their microtubules so that distinct bundles of microtubules are formed in the cells.9,13,14
In essence, Taxol hyperstabilizes the microtubule’s structure. This destroys the cell’s ability to use its cytoskeleton in a flexible manner. Specifically, Taxol binds to the β subunit of tubulin and the binding of Taxol locks these building blocks in place. The resulting Taxol–microtubule complex does not have the ability to disassemble. This adversely affects cell function because the shortening and lengthening of microtubules (termed dynamic instability) is necessary for their function as a mechanism to transport other cellular components. For example, during mitosis, microtubules position the chromosomes during their replication and subsequent separation into the two daughter-cell nuclei. In the presence of Taxol, cells can no longer divide into two daughter cells, and the tumor gradually dies. The ability of Taxol to polymerize tubulin into stable microtubules in the absence of any cofactors such as cold or CaCl2 and to induce the formation of stable microtubules in cells is the unique characteristic of Taxol.13
Fig. 4.1 shows the Taxol binding site on mammalian β-tubulin of the Taxol–tubulin complex.14 After considering data on Taxol binding to mammalian tubulin and recent modeling studies, it was hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. Mutagenesis experiment confirmed the hypothesis.
