Three years ago, it was unclear whether the Taiwan yew could be used as a source of the cancer-inhibiting drug taxol. Today, Union Chemical Laboratories' work to develop biotechnology processes to mass-produce taxol is at the leading edge of international research, and is not only bringing hope for cancer sufferers, but also a greater chance of survival for the endangered yew itself.

Since the 1980s, when US researchers confirmed that taxol (derived from the Pacific yew, Taxus brevifolia, which belongs to the same genus as the Taiwan yew, Taxus mairei) is effective in treating ovarian and breast cancers, clinical trials have been conducted or are planned to test the drug against more than 60 types of tumor. As more and more suitable applications are discovered, the demand for taxol is expected only to rise. Furthermore, it is over 25 years since taxol's structure was first published, and it was never patented, so there are no licensing problems. Thus many countries have set their sights on the market for taxol and are rushing to develop means of producing it in bulk.

Although semi-synthetic versions of taxol have already reached the market in Germany and the USA, there is still a need to harvest large quantities of Pacific yew bark. This means that the crisis of survival for the Pacific yew has not passed. Furthermore, since taxol cannot as yet be reliably manufactured in large quantities, the supply of injectable taxol falls far short of demand, and that its price is very high. Therefore developing the means to mass-produce taxol is of great importance not only for cancer patients, but also for the survival of Taxus species.

Cell cloning

Taiwan is fortunate in having a native Taxus species, so that the raw materials for experimentation are easily available. Furthermore, unlike the Pacific yew in which the highest concentrations of taxol are found in the bark, the Taiwan yew contains large amounts of taxol in its foliage. Thus harvesting reasonable quantities does not endanger the trees' survival. This represents a great opportunity, and three years ago Taiwan's Union Chemical Laboratories (UCL) began R&D work on taxol, and the Taiwan Forestry Research Institute (TFRI), building on its knowledge of Taiwan's forest resources, also began culturing Taiwan yew to extract the drug.

How can taxol be produced in large amounts? Li Cheng-yu, a researcher at UCL, explains that it is possible to exploit the plants' own biological resources, by using tissue culture. "By tissue culture we mean promoting the growth of those parts of the plant which we need, under laboratory conditions. For instance, if we need roots we grow roots, or if we need cells we grow cells. The idea is to enhance the part which people need, and produce it in large quantities. With the Taiwan yew, for example, we had to determine which parts of the yew tissue contain the highest concentrations of taxol, and then continuously 'clone' those parts in order to extract the taxol from them."

With the assistance of the Taiwan Forestry Bureau (TFB), UCL first extracted taxol from yew foliage and measured its concentration. They found that although the concentration of taxol varied at different seasons and in different parts of the foliage, high concentrations were not difficult to find. In the laboratory, Li Cheng-yu discovered that callus cells in leaf cuttings contain taxol, and he began to culture these cells, in the hope of producing them consistently and in large quantities as a means to produce taxol.

If taxol can be produced by tissue culture, it takes little more than a small quantity of yew foliage to provide an inexhaustible source of the drug. Thus in a small space and with minimum manpower one can replace plantation growing, which requires large areas of land and is very slow. This is why tissue culture is currently the main thrust of research in laboratories in countries such as Japan and Germany.

A "small" job requiring great skill

This production process sounds simple enough, for as Li Lian-tzu, director of UCL's Pharmaceutical Chemicals Research Program explains, performing tissue culture of yew cells simply means cloning the cells in large quantities. But the first obstacle which has to be overcome is that the cells produced must contain the substances required and not unwanted substances, and the second is to raise yew cells with a high taxol content. Thirdly, the cells raised have to be suitable for the chemical extraction process.

To stimulate the yew cells to keep growing continuously, the researchers have to prepare them the best "menu"-in other words, they must determine the most appropriate growth medium. The "wrong" nutrients would give the cells the wrong signals, and they might die or mutate, bringing all the previous work to nothing.

Preparing the cells' "food" is a seemingly small task, but one which requires enormous skill. "It depends on practical manipulation and control by the researchers in the laboratory. You can't just rely on theory," says Chang Shu-hua, an associate research scientist at the TFRI's silviculture division. The nitrogen, phosphorus, potassium, calcium and magnesium which are essential to plant growth come in hundreds of different forms. With so many "recipes" available, researchers trying to prepare the most "nutritious" menu, to encourage the yew cells to eat their fill and to grow quickly, must rely on experience, patience and constant experimentation. Not only is every laboratory searching for the best growth medium, but each lab keeps its formulations secret, for they are the stuff by which the researchers earn their living.

In biotechnology, tissue culture is seen as having as great a future as the electronics industry. As well as using tissue culture to try to produce taxol, the TFRI is also using similar methods to improve the germination rate of yew seeds. Chang Shu-hua explains that by removing embryos from yew seeds and serving them up a different "menu"-a growth medium which promotes germination-and by eliminating all the factors that can lead to disease during the germination process, the institute is now able to germinate Taiwan yew seeds within ten days, with a germination rate of 100%. In view of the potential this technique has for assisting the artificial propagation of yew trees, the TFRI has already applied for an ROC patent on it.

The final step

Today, both UCL and the TFRI have formulated growth media with which they can grow yew cells containing high concentrations of taxol, and both are gradually expanding their scale of production. UCL is already culturing cells in five to seven liter reaction vessels, producing up to 50 milligrams of taxol per liter, and is currently working to enlarge the scale of the process. Li Lian-tzu says that if UCL's process were scaled up to a one-tonne reactor, the cells cultured would contain 50 grams of taxol per batch, equal to the amount contained in 70 Pacific yew trees. The concentrations achieved by the laboratory are among the highest currently seen in the literature. "People around the world know that tissue culture is the way to go, but the experience Taiwan has gained since we started raising orchids by tissue culture back in the 1980s has enabled us to take the lead," Li says.

But before production can be scaled up and taxol directly extracted from the cultured cells, to finally reach the stage of commercial mass-production, the problem of eliminating unwanted substances and maintaining purity has to be overcome. Today, international competition in the field has reached a crucial juncture.

For Taiwan to produce taxol on a commercial scale from cultured yew cells "will probably take another three to five years," says UCL program director Li Lian-tzu. To exploit Taiwan's lead and enable the program to be transferred to the private sector as soon as possible, researchers from two private pharmaceutical companies are already participating in the ITRI's research.

Although there is still some way to go before manufacture of the drug in injectable form and development of the market, production of taxol in Taiwan using biotech-nology is not far away.

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Researchers at Union Chemical Laboratories hope to culture large quantities of yew callus cells in order to extract tumor-inhibiting taxol from them. The picture on this page is a micrograph of a Taiwan yew leaf cutting with callus cells. (courtesy of Union Chemical Laboratories)

(facing page) Different culture media are added to different culture vessels in the hope of finding ones in which the cells will grow in abundance. The laboratories have scaled up their tissue-culture production process to using larger tanks.

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These three ITRI researchers (from left to right: Li Cheng-yu, Li Lian-tzu and Yang Yu-lin) are optimistic that it will be possible to produce taxol in bulk, but there are many obstacles still to be overcome in the development process.

These three ITRI researchers （from left to right: Li Cheng-yu, Li Lian-t zu and Yang Yu-lin） are optimistic that it will be possible to produce taxol in bulk, but there are many obstacles still to be overcome in the development process.