Taxol

Taxol^®: discovery and elucidation

Both of Arthur Barclay’s samples of Taxus brevifolia were sent to the Wisconsin Alumni Research Foundation for extraction. One of the samples, PR-4960, the bark, showed cytotoxicity against KB. Though the in vivo tests on L1210 leukemia yielded mixed results, further KB tests confirmed anticancer activity. Accordingly, the crude plant extract was designated NSC670549 to conform to CCNSC protocols. And Jonathan Hartwell of the Center instructed his counterpart at the USDA, Robert Perdue, to take steps to collect larger quantities of the bark of T. brevifolia for further evaluation.

On September 8, 1964 Barclay returned to the spot where he had originally collected PR-4960 and bagged thirty pounds of bark from T. brevifolia, which was shipped to Monroe Wall at the Research Triangle Institute after other laboratories turned it down. These laboratories were reluctant because plants that exhibited high cytotoxicity turned out to be poor drug candidates since they are toxic to everything, killing healthy cells as well as cancerous ones. But Wall was interested because of his experience with Camptotheca acuminata, which was highly cytotoxic but from which his group had isolated a compound that showed great promise.¹

When it received Barclay’s shipment of bark, RTI began a bioassay-directed fractionation to isolate the pure compound in T. brevifolia responsible for its anticancer activity. In September 1966 the researchers isolated from one of the fractions a crystalline substance they designated K172 (after NCI practice in which pure compounds were identified with a “K” prefix). Since it was common practice for a laboratory to name compounds extracted from material assigned to it, Wall decided to name the substance “taxol.” He came up with the name for two reasons. First, while the RTI scientists did not yet know the compound’s complete structure, they were sure it contained some hydroxyl groups, signifying it was an alcohol. And second, it was common to name a molecule after the genus of the plant from which it came. Accordingly, the molecule became known as Taxol® – “tax” for Taxus and “ol” for alcohol. Besides, the name “had a nice ring to it.”²

Initially, Mansukh Wani reported some success in characterizing the isolated compound. But by early 1967 Wani had to admit failure in his attempts to characterize the active compound, unable even to determine its molecular weight. This initial work on the Pacific yew coincided with the isolation of camptothecin, which was “looking very hot” and on which NCI was ready to begin clinical trials.³ Moreover, RTI had many other plant extracts to evaluate.

At that point Wall told Wani “to forget about” Taxus brevifolia. But Wani persisted. He refused to give up on the yew completely, continuing to work on it “on a low priority basis.” Even this proved difficult because, as Wall wrote thirty years later, “the basic problem was that NCI had at that time (1966-1973) a rather rigid protocol which had to be met in order to obtain larger quantities of a natural product, and for carrying out expensive antitumor assays, toxicology assays, etc.”⁴

The need for more and more bark was compounded by the fact that the yield of Taxol® from collected bark was very low. This slowed the process to determine the molecule’s structure. To definitively learn the structure of Taxol®, Wall and Wani used mass spectrometry, X-ray crystallography, and NMR spectroscopy, a technique then in its infancy. These various tools produced the molecular structure with a formula of C₄₇H₅₁NO₁₄. Wall and Wani also discovered that Taxol® was two molecules in one – a large molecule connected to a small tail or side chain. In 1971 Wall, Wani, and co-workers published the results of their work on Taxol® in the Journal of the American Chemical Society as a communication to the editor in which they stressed the antileukemic and antitumor activity of the newly isolated and elucidated molecule.⁵ Interestingly, the structure of Taxol® is so complex that Wall and Wani almost went to press with the side chain of the molecule placed in the wrong position.⁶

Wall and Wani at this point passed Taxol® to the NCI for further study of its anticancer properties in the hope that advanced animal studies and toxicological tests would eventually lead to human clinical trials. But NCI administrators showed great reluctance to pursue Taxol® for a number of reasons. Isolation and extraction of Taxol® from T. brevifolia was arduous and the tree, not all that prevalent, produced only small amounts of the compound. Each tree yielded only about two kilograms of bark and it took in the early days of testing about 12 kilograms of dried bark to obtain one-half gram of Taxol®. Moreover, bark is a finite resource; a tree stripped of its bark dies.

Because of the supply problem Taxol® literally stayed on the shelf at RTI for the next few years. But then several things happened to bring renewed interest in Taxol®. Matthew Suffness joined NCI, conducting a review of the natural products program and encouraging tests that showed Taxol® very active against B16 melanoma, a newly adopted tumor model. Almost at the same time, NCI introduced other new tumor models, called xenografts, in which human tumors are planted in what are called nude mice. These specially bred mice are called nude because they are hairless. More importantly, these mice lacked a functioning immune system, which meant their bodies would not reject transplanted human tumors. In November 1978 Taxol® showed the ability to cause considerable regression in a mammary tumor xenograft. And finally, interest in Taxol® increased greatly in 1979 when Susan Horwitz demonstrated that Taxol’s® mechanism of action was unique.

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¹ Interview with Dr. Mansukh Wani conducted by Judah Ginsberg on January 13, 2003.

² Research Triangle Institute, Progress Report No. 21, June 26, 1967; Wall, Monroe and Mansukh Wani, “Camptothecin and Taxol: Discovery to Clinic,” Cancer Research 55, 1995, p. 757.

³ Inteview with Dr.Wani.

⁴ Monroe Wall to Jordan Goodman, October 21, 1996.

⁵ Wani, M. C., H. L. Taylor, Monroe Wall, P. Coggon, A. T. McPhail, 1971, “Plant Antitumor Agents. VI. The Isolation and Structure of Taxol, a Novel Antileukemic and Antitumor Agent from Taxus brevifolia,” Journal of the American Chemical Society, 93: 2325-2327.

⁶ Interview with Dr. Wani.

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