Several groups of methods of enhanced bioproduction and extraction of taxanes have been invented. Taxanes (see figure) have exhibited potential as drugs for treatment of cancer and congenital polycystic kidney disease. One taxane — paclitaxel (more widely known under the trade name Taxol™) — has been approved by the Food and Drug Administration for treatment of several types of cancer. Currently, paclitaxel and other taxanes are produced by extraction and by semisynthesis from precursor taxanes isolated from Taxus sp. The present inventive methods are intended to enable the production of taxanes and related products in quantities sufficient for testing them as potential anticancer drugs, without destroying Taxus trees. In addition, the methods afford versatile capabilities for tailoring the chemical structures of taxanes and related products to maximize their beneficial effects.
One group of methods exploits the discovery that haploid and haploid derived cultures of cells of any plant of the genus Taxus can produce taxanes. More specifically, female gametoyphytic tissues taken from immature seeds can be cultured to produce significant amounts of taxanes. The method of acquiring the tissues for culturing includes sterilization of seed surfaces and careful dissection to keep diploid cells (in which taxane production may in some cases be inhibited by dominant and quantitative gene interactive effects) out of the cultures. The cultures are grown under aseptic conditions that are monitored and controlled. One essential component of control is the use of culture media that do not contain nitrates. The growth process can include aging until the culture reaches a condition regarded as optimum (e.g., a condition that favors steady-state production of taxanes). Once a desired cell culture has been established, it can easily be scaled up for growth in a bioreactor to produce taxanes in larger quantities.
Taxanes can be recovered from cell cultures by one or more of methods or combinations of methods, some of which were previously established, others of which are parts of the present invention. Established methods include the use of absorbent beads, the use of adsorbent particulate material released by cultured cells, and the use of solvents to extract taxanes from the aforementioned beads and particles. Methods that are parts of the present invention include enzymatic treatments, hydrolysis under suitably controlled acid or alkaline conditions, and controlled use of radiation or heat. In the cases of taxanes that are chemically bound in cells or cell fragments, such extraction may give rise to chemical alterations that could be exploited to enhance the beneficial properties of the final taxane products. In addition, alkaloids related to taxanes can be produced by the aforementioned methods.
Another group of methods, which amounts to a generalization of the aforementioned group of methods, is based on the observation that taxanes can be obtained from parts of a tree other than bark and seeds and from conifers other than those of the genus Taxus. These methods include (1) antibody- based screening methods to identify conifer tissues that contain taxanes and (2) taxane-recovery methods include extraction by use of solvents, enzymatic treatments, and hydrolysis.
A third group of methods, embodying a further generalization, is based on the concept of obtaining taxanes from any plant capable of producing them. This group includes antibody based screening methods to identify taxane-producing plants, methods to isolate taxane-producing cells from cultures, methods of stimulating taxane production through control of physical and chemical conditions in cultures, and methods of extraction. Among the methods of isolating taxane-producing cells from a culture, the one that is preferred involves the use of paramagnetic beads to which are bound molecules of an antibody that recognizes another antibody that, in turn, binds taxanes that are present in or on cell walls. The cells thus isolated can then be cultured under controlled conditions for enhanced production of taxanes.
This work was done by Don J. Durzan and Frank F. Ventimiglia of the University of California for Johnson Space Center. In accordance with Public Law 96-517, the contractor has elected to retain title to this invention. Inquiries concerning rights for its commercial use should be addressed to
University of California
1111 Franklin Street, 5th Floor
Oakland, CA 94607-5200
Refer to MSC-23442/151/152, volume and number of this NASA Tech Briefs issue, and the page number.