AIR3-CT94-1979 Selection, Cultivation and Harvesting of Yew: Silviculture and Biotechnical Production as Alternatives in the Development of Taxanes with Anticancer Activity

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AIR Cluster V - Speciality Chemicals : Biotechnology : Pharmaceuticals/Cosmetics

	Proposal No:	AIR3-CT94-1979
	Date Prepared:	September 1999
	Source:	Final technical report

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Final technical report

Introduction

Taxol^® (= Paclitaxel) was first isolated from the stem bark of the Pacific yew, Taxus brevifolia. It can now be prepared from 10-deacetylbaccatin III (10-DAB), extracted from the needles of various Taxus species. One active analog, Taxotere^® (= docetaxel), is also prepared by semisynthesis from 10-DAB. Both molecules are used in the treatment of several cancers.

In spite of recent, sophisticated, chemically but not economically beautiful total syntheses, extraction of selected plants and semisynthesis from abundant natural precursors still remain methods of choice to produce Taxol^® and Taxotere^®. Biotechnological production can become a further or alternative route.

Objectives

The project aimed at:

• collecting and selecting species, varieties and cultivars of yew whose leaves (renewable material) would contain as large as possible amounts of Taxolo and known or potential precursors (I O-DA.B, taxine B, isotaxine B)
• establishing tissue and cell cultures and optimizing them : a biotechnological production of Taxolo, precursors or/and analogs would provide a further route for the taxane production
• determining the best conditions allowing the development of conunercial yew plantations and the mechanization of their harvesting, preserving the native strands of trees
• conceiving new semisynthetic pathways towards Taxolo analogs from precursors extracted from leaves and/or tissue and cell cultures

Activities

By the end of the contract, 221 samples of needles of various Taxus had been analysed for their contents in Taxol, 10-DAB, total alkaloids, taxines B and for their activity in tubulin test. Samples have been collected from various species, varieties, cultivars of Taxus coming from different geographical origins.

Efficient methods for the analyses have been developed that are now available for large-scale quantitative screening. The best samples, selected at the end of the second year of the contract, have been used to begin new experiments in silviculture and in tissue and cell cultures establishment. Once this selection had been made, the best conditions were determined and optimized with different Taxus samples.

Many experiments have been undertaken to evaluate rooting capacity, possible micropropagation of plantlets and production of hairy roots from very numerous samples. These experiments were begun as soon as the contract started. The best conditions for Taxus cell cultures have been established and optimized, mainly for Taxus chinensis during the first 2 years, while during the third year they have been applied to the selected trees but the results have not yet been finalised. Requirements for nutritients and light as well as effects of fertilizers or growth retardants have been studied. Open field plantations have been established to evaluate the factors able to influence growth, biomass and taxoid yields.

The same strain was used for biosynthetical investigations. The preparation of taxane derivatives needed to obtain haptens and develop immunoassays was difficult and took a long time: this delayed the establisment of immuno- and radioimmunoassays.

The development of a microanalytical method, consisting of high pressure liquid chromatography (HPLC) combined with nuclear magnetic resonance (NMR) was used to analyse crude extracts of yew, cell cultures and hairy roots.

Semisynthesis of Taxol^® analogs from natural precursors easily extracted from the leaves of Taxus sp. led to several new molecules, prepared either from 10-DAB or taxines B. Several compounds were found to be almost as active as Taxol^® in the tubulin test and in terms of cytotoxicity.

Discussion

It was found that three years was too short a period to conclude such a project. However, the results already obtained have provided better knowledge of the variations of the contents of significant constituents of Taxus, within species, cultivars, geographical origin and seasonal variations. Results were obtained enabling comparisons between the content of several metabolites for each sample. The methods, adapted for the analysis of a significant number of samples, can now be easily used.

On the basis of this analysis several trees have been selected for propagation, with cuttings inserted into soil for rooting or used to establish cell cultures.

Experiments showed the need to develop culture of selected Taxus, on a large scale to enable mechanized harvest of needles as source of Taxol^®, Taxotere^® and precursors of other active analogs and preserve the wild trees. Some results were positive, but this work also revealed various difficulties.

Taxus is a slow-rooting and a slow-growing tree. Several hundred cuttings were established in greenhouses, those that root over several months, will be used to develop cultures in open field. Studies carried out to establish plant and cell cultures provided interesting results, but again indicated difficulties. Optimal experimental conditions and good elicitors have been determined, which could be applied to yield high-taxane producing cell cultures from any other Taxus species.

Biosynthetic investigations were successfull and resulted in informatio that will be useful in the future biotechnological production of taxoids. Analogs of Taxol^® and Taxotere^® were prepared by semisynthesis from 10- DAB and taxines B. Several molecules are as active as Taxol^®: the structurally closest will probably not be developed. Activity of the others needs to be explored in more depth in order to establish whether their effects would justify further development.

Other prepared molecules, although inactive, have provided new data concerning structure- activity relationships. A lot of work in this area has already been carried out worldwide before and during the contract and will probably continue due to the importance of these relationships. Hence, semisynthesis of other new molecules continues.

It could not be expected that silviculture of Taxus, biotechnological production of Taxol^® or precursors and the semisynthesis of the next generation of analogs could be achieved in three years, in spite of studies, all begun before the beginning of the contract. However, although interesting results have been obtained that increase knowledge in this area, it is too early to take commercially advantage of the results obtained to date.

The participants are, at presentt, the main beneficiaries, since they can use the results to achieve further objectives. The improved knowledge of Taxus physiology will assist in development of silviculture and biotechnological production of Taxus metabolites that can become precursors in semisynthesis of active analogs of Taxol^® or Taxotere^®.