EP0642586A1 - TISSUS DE $i(TAXUS) MIS EN CULTURE UTILISE COMME SOURCE DE TAXOL, TAXANES ET AUTRES NOUVEAUX COMPOSES ANTITUMORAUX/ANTIVIRAUX APPARANTES - Google Patents

TISSUS DE $i(TAXUS) MIS EN CULTURE UTILISE COMME SOURCE DE TAXOL, TAXANES ET AUTRES NOUVEAUX COMPOSES ANTITUMORAUX/ANTIVIRAUX APPARANTES

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Publication number
EP0642586A1
EP0642586A1 EP93911220A EP93911220A EP0642586A1 EP 0642586 A1 EP0642586 A1 EP 0642586A1 EP 93911220 A EP93911220 A EP 93911220A EP 93911220 A EP93911220 A EP 93911220A EP 0642586 A1 EP0642586 A1 EP 0642586A1
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Prior art keywords
taxol
taxus
culture
roots
root
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EP93911220A
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German (de)
English (en)
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EP0642586A4 (fr
Inventor
Richard N. Arteca
Enaksha Wickremesinhe
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Penn State Research Foundation
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Penn State Research Foundation
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Publication of EP0642586A1 publication Critical patent/EP0642586A1/fr
Publication of EP0642586A4 publication Critical patent/EP0642586A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D305/00—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms
    • C07D305/14—Heterocyclic compounds containing four-membered rings having one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms

Definitions

  • Taxus Tissues as a source of Taxol, Related Taxanes and Other Novel Anti-tumor/Anti-viral
  • This invention relates to cultured Taxus tissues as a source of taxol, 10-deacetyl baccatin III, 7-epi-10-deacetyl baccatin III, baccatin III, 7-epi baccatin III, 9-dihydro-13-acetyl baccatin III, cephalo annine, 10-deacetyl taxol, 7-epilO-deacetyl taxol, 7-epi taxol, other known and novel natural derivatives of these compounds, and other related taxanes including precursors and intermediates in the biochemical pathway resulting in taxol production, and active derivatives of taxol.
  • This invention also relates methods of producing taxol and related compounds from cultured Taxus stems and from Taxus roots, using hydroponics or tissue cultures.
  • Taxol has properties that provide significant clinical benefits. Taxol is the only plant product known to promote the assembly of microtubules and inhibit the tubulin disassembly process. Taxol and taxol-related compounds show positive activity in cancer model assays and microtubule stabilizing assays. Taxol appears to be the prototype of a new class of powerful cancer chemotherapeutic agents (Suffness & Cordell, 1985).
  • Taxol is a potent inhibitor of cell replication that acts as an antimitotic agent, blocking cells in the G2/M phase of the cell (Schiff & Horowitz, 1980; Horowitz et al . , 1986). It is unique in its ability to bind stoichiometrically to polymerized tubulin both in vitro and in vivo, and to induce the assembly of tubulin to form calcium-stable aggregated microtubule structures. These structures resist depoly erization by dilution, calcium ions, cold and a number of microtubule-disrupting drugs.
  • Microtubules assemble tubulin in the absence of exogenous guanosine 5 ' -triphosphate or microtubule-associated proteins at low temperatures (Schiff & Horowitz, 1980; ParnessS and Horowitz, 1981; Howard & Timashef , 1988) .
  • Taxol exhibits activity against the KB cancer cell line used to screen for potential chemotherapeutic agents in tissue culture. During preclinical evaluation studies, taxol has been shown to have activity against both urine solid tumor lines and leukemic cell lines (Wani et al . , 1971; Suffness & Cordell, 1985; Donehower et al. , 1987) .
  • Taxol has been shown to be active in the OVCAR-3 human ovarian carcinoma xenograft model and to have promise in the treatment of advanced, progressive, and drug-refractory ovarian cancer (McGuire et al . , 1989; Thigpen, et al . r 1990; Einzig et al . , 1991; Markman, 1991) .
  • Taxol has been through Phase III trials, and has been approved as a drug in the treatment of refractory ovarian cancer by the U.S. Food and Drug Administration. Striking effects for taxol also have been reported in the treatment of metastatic breast cancer (Holmes et al ., 1991) , as has significant activity in the treatment of non-small cell lung cancer (Ettinger, 1992) .
  • Taxol (NSC-125973) (Clinical Brochure; Taxol, NSC- 125973 Available from Drug Regulatory Affairs Branch, CTEP, National Cancer Institute, Bethesda, MD 20892) is a cytotoxic diterpene with a complex chemistry, initially isolated from the stem bark of the western yew, -Taxus brevifolia Nutt., by Wani and co-workers in 1969, who published the structure of the molecule in 1971. In addition to Taxus brevifolia, taxol has also been isolated from other species of the genus Taxus, including T. Jaccata, T. canadensis, T. cuspidata, and T. x media (Wani et al .
  • Taxol and related diterpenoids containing the taxane ring have not been reported in any genus other than Taxus (Jaziri et al . , 1991).
  • Taxus needles have drawn attention as a stable source of taxol and its baccatin precursors (Holton, 1992 — Second Workshop On Taxus And Taxol; DeFuria, 1993 — International Yew Resources Conference) .
  • Taxol has been routinely extracted from the dried stem bark of T. brevifolia .
  • taxol has also been isolated from several other species of yew (genus Taxus, family Taxaceae) , including T. baccata , T. cuspidata, T. canadensis and T. media cvs . Densifor is and Hicksii (Wani et al . , 1971; Vidensek et al . , 1990; Witherup et al . , 1990; Wickrernesinhe, 1992).
  • Table 1 there is a summary of reported values contained in the literature of taxol found in various tissues of plants of the genus Taxus . With the exception of the present invention, roots showed lower levels than bark.
  • taxol has been extracted from a natural source
  • extraction has been extremely painstaking and, because the compounds of interest are found at extremely low concentrations in plants (0.02% dry weight basis), is associated with yields that are grossly inadequate for both present and anticipated needs.
  • initial extracts are contaminated with pigments, highly hydroponic cuticular substances, waxes and the like, which must be removed.
  • taxol Based on the current bark-extraction procedures, 7,200 kg of bark are needed to produce one kilogram of taxol, and the projected target for 1993 is 230 kilograms (Stull, 1992). As of July 1992, taxol has been made available to more than 1,700 patients with refractory ovarian cancer (Arbuck, 1992) . As more applications for taxol are revealed, the amounts of bark needed to produce taxol will increase, causing serious environmental concerns, as well as concerns about adequate and long- term supplies.
  • Taxus plants Cultivation of Taxus plants (yews) is unlikely to solve immediate taxol production needs. Plants of the Taxus genus in general have very slow growth rates and long propagation periods. This makes it difficult to implement breeding programs aimed at screening for species producing an abundant supply of these compounds. Due to the very slow growth of all yews and the low yields of taxol obtained, there is widespread concern that alternate routes are needed to maintain an adequate and continuous supply of this drug.
  • Taxol is a natural precursor of taxol and can be extracted from the needles of T. baccata (Chauviere, 1981; Denis et al .
  • Taxus tissues are a possible source of taxol. But long term, stable cultures have not been produced. Problems with the approach that would have had to be overcome, if roots were to be used to initiate cultures, included the fact that not all types of roots adapt to grow in vitro in aseptic cultures. In particular, it was not expected that Taxus roots would grow in culture because the Taxus plants grow slowly in soil, making it unlikely that culture conditions would be developed that would allow the roots to grow at an optimum rate for stable culture maintenance.
  • aseptic root cultures have been successfully established from many plants, and pharmaceutically important compounds were produced by these root cultures, albeit, in most cases at levels lower or comparable to the intact-above ground plant parts (Ishimaru et al . , 1990; Jha et al . , 1S91; Sen et al . , 1991; Ishimaru et al . , 1992)
  • the roots themselves were used for medicinal purposes, such as Sanguisorba, suggesting that the compounds of interest were present in the roots.
  • root cultures would not be a logical source.
  • Root cultures have been attempted for herbaceous, or "soft plants,” not generally for ornamental or “woody” plants, such as Taxus. There is an immediate, critical need to develop rapidly reproducible, continuously available, renewable sources of taxol and taxol-related compounds, suitable for large scale production. Because yew trees grow very slowly, planting more yew trees is not a promising solution. Chemical synthesis has not been successful and partial synthesis requires baccatin III as a starting material which must be extracted from plant material and is also found in low levels in plant tissues. Taxane ring-containing alkaloid compounds have been obtained from cell cultures of T. brevifolia , however, there have been problems using this approach.
  • the methodology described in the present invention presents a solution to the problem of taxol supply using cultured, Taxus tissues to produce, even overproduce, taxol, and related taxanes, in larger quantities than are possible with current taxol-production methods.
  • An aspect of the invention is to use Taxus roots, despite reports on the analysis and content of taxol in Taxus roots that teach away from the use of roots as a taxol source because of relatively low yield compared to bark.
  • Another aspect of the invention is growth of roots in hydroponic cultures.
  • This method of growing plants has been established for many economically important plants for commercial greenhouse production of economically important fruits, vegetables, and ornamental plants (Moss, 1984; Adler & Wilcox, 1987; Hicklenton et al . , 1987; Knight & Mitchell, 1987; Kratky et al . , 1988; Lardizabal & Thompson, 1988; Mortley et al . , 1991; Brentlinger, 1992)
  • roots per se This is because roots generally are not considered when the above-ground part of the plant is the initial focal area, as is the case with Taxus plants.
  • hydroponic culturing is expensive, requiring special media and apparatus, so this would not be a method pursued in preference to going out and chopping bark off Taxus trees.
  • An advantage of the present invention is its eliminating the present dependence of taxol availability on Taxus trees in the wild.
  • the present invention facilitates conservation of yew trees, a large number of which need to be harvested in order to isolate the kilogram quantities presently needed for clinical use.
  • Other advantages include increased availability and reliability of production of taxol and related compounds for the benefit of cancer patients.
  • Cropon includes in vitro tissue culture of stems and roots, and hydroponic culture of roots. Stems and roots are derived from natural plants, and roots may be derived from callus cultures.
  • Taxol is a cytotoxic diterpene with demonstrated antineoplastic activity against both solid tumor and leukemic cell lines, and activity as a chemotherapeutic agent in clinical trials. Taxol is the only plant product presently known to promote the assembly of microtubules and to inhibit the tubulin disassembly process. This mechanism suggests that taxol is a prototype of a new class of chemotherapeutic agents that attack cancer growth in this fashion.
  • taxol is generally meant to include compounds which are structurally related to taxol and are defined to include precursors of taxol in biochemical pathways which are capable of producing taxol, to intermediates of those pathways, and to derivatives and secondary metabolites of taxol (10-deacetyl baccatin III, 7-epi-10-deacetyl baccatin III, baccatin III, 7-epi baccatin III, 9-dihydro-13-acetyl baccatin III, cephalomannine, 10-deacetyl taxol, 7-epilO-deacetyl taxol, 7-epi taxol) and other known and unknown natural derivatives of these compounds and related taxanes resulting in taxol production and/or the production of novel antitumor/antivira1 compounds.
  • Taxol-like activity refers to compositions which show positive results in at least one test predictive of effective chemotherapy: a microtubul
  • Stable cultures are cultures that maintain a taxol production level after repeated subcultures.
  • Long-term cultures re those that remain stable for at lease one year, preferably two or more years.
  • Cultures produced by methods of the present invention have remained stable for up to seven years.
  • Some of the cell lines are habituated, that is, they do not require plant hormones as supplements to the nutrient culture medium for long-term maintenance.
  • the cell lines established by use of the methods of the present invention are capable of producing taxol, and taxol- related compounds. These cell lines offer a rapidly reproducible, continuously-available source for the production of taxol and taxol-related compounds.
  • This production may be scaled to commercial levels by use of the cell lines in bioreactors.
  • new compositions exhibiting taxol-like activity have been purified from extracts of the Taxus cell lines, offering new horizons for chemotherapeutic agent development.
  • a method for establishing a culture of cells derived from the genus Taxus has been successful in producing cell lines which secrete taxol and taxol-related compounds into the culture environment.
  • the steps of the in in vitro tissue culture method include the following:
  • the general tissue source is plants of the genus Taxus.
  • Various species from which taxol may be isolated include T. brevifolia Nutt, T. Jaccata, T. cuspidata, T. canadensis cv. Capitata, T. media cv. Densiformis, and T. media cv. Hicksii.
  • Preferred sources for explants used to produce taxol- expressing cultures are those plants from which the amount of taxol that may be extracted directly is high when compared with amounts extracted from other species by comparable methods from comparable amounts of starting material.
  • T. -media more particularly T. media cv. Hicksii, for example, is a more preferred source than T.
  • Tissue explants are a portion of tissue selected from a plant and transferred into appropriate tissue culture vessels such as Petri plates or Magenta (a square dish) . The explant is then cultured as described herein. Bark had been the usual source of taxol, and needles and roots also are capable of expressing taxol. It was found, however, that a preferred tissue source of the explant is a stem, more particularly a young, fresh, succulent stem. Young stems are those that appear at the beginning of a new growth season for the plant. Immature stem sections of Taxus media cv.
  • Hicksii are particularly preferred because stems of Taxus media plants contain higher levels of taxol than Taxus brevifolia and other Taxus species. Explants of about 0.3-0.5 mm in diameter are suitable. Root explants are more preferred in the context of the present invention.
  • the tissue obtained from the plant is processed by surface sterilization and sectioned under aseptic conditions. Sectioning is accomplished by cutting a stem into pieces of approximately 6-8 mm in length, from immature stems that are approximately 3-5 mm in diameter, and transferring the sections to tissue culture vessels, generally, petri dishes, with callus- inducing nutrient media. This is medium which is capable of initiating and promoting the formation of callus growth from the explant.
  • a preferred media is Gamborg's B5 media (Gamborg et al . , 1968) supplemented with plant hormones such as 2,4-D and kinetin either alone or in combination.
  • Murashige/Skoog medium (Murashige & Skoog, 1962) also is suitable for this purpose and may be supplemented.
  • Gelling agents such as Gelrite (Scott Laboratories, West Warwick, R.I.) or agar are mixed with the nutrient media to form a solid support in the culture vessel for maintenance and growth of the explant.
  • Callus formation is determined when globular aggregates of undifferentiated cells are observed. This generally occurs on 90% of the explants within about 2-4 weeks. Subculturing initially is performed at 4-6 week intervals, but may be altered as a function of culture growth. (4) Selecting a subclone to develop a cell line
  • Friability is another selection criterion. This is determined by observing a crumbly, fragile texture of the callus. Another factor which indicates samples that are likely to result in successful cell lines for purposes of the present invention is relative growth rate of the callus cells. Initially, all callus are slow growing, taking about 2-4 months to double. Preferable doubling times are from about 5-16 days. Subculturing was performed at intervals of about 4-6 weeks, although the interval may be shorter or longer depending on the health of the callus, as indicated by color, and by the size of the callus after growth.
  • Optimum culture maintenance factors included use of a membrane raft to support the cells. This structure facilitated subculturing, collection of extracts, and formation of cell suspensions. After stable culture growth is achieved, the cell lines are kept in maintenance medium. For some of the cell lines, habituation occurs; that is, the cultures are maintained without the need for hormonal supplements.
  • cell suspension cultures are induced from well established stable callus cultures as described above by transferring an inoculum of cells (2-3 grams) of friable callus clumps into liquid medium contained in Erlenmeyer flasks (generally 250 ml flasks containing 100 ml of medium) . Flasks are placed on a rotary shaker known to those of skill in the art and operated at about 125 rpm.
  • Taxus cell lines within the present invention An important use of Taxus cell lines within the present invention is to produce taxol and taxol-related compounds.
  • Callus and suspension cell lines of Taxus developed by the methods of the present invention produce taxol, precursors and intermediates of a biochemical pathway resulting in taxol production, including cephalomannine and baccatin, and active derivatives of taxol. Both biotic and abiotic elicitors, precursor feeding and other elicitation techniques are useful to enhance secondary metabolite production.
  • Taxol, and taxol-related compounds are extracted from callus, suspension cultures and culture medium.
  • An "active" composition is one that is capable of giving positive results in a microtubule stabilizing assay, a cancer model assay, a clinical trial, or any combination of these tests.
  • Cell lines are suitable as sources to grow in bioreactors to produce large quantities of taxol, and taxol-related compounds. Some of these compositions are found in the culture medium and may be obtained by collecting the medium. Utilizing different bioreactor designs, the secondary metabolites found in solution may be easily removed from the solution utilizing exchange resins which extract only the desired secondary metabolites from the solution and not the media supplements required for culture growth. Other compositions are produced and retained intracellularly and must be extracted from the cells.
  • Taxus-derived cell lines in bioreactors enables the production of much larger quantities than are presently feasible. This process not only serves as a renewable resource of chemotherapeutic agents but also saves the large number of trees that are presently being harvested to isolate this compound. Taxol is found in the roots of all the different Taxus species and cultivars cultured according to the present invention and evaluated. The levels range from amounts comparable to the levels found in the above ground parts to amounts 2- to 4-fold higher. Objectives of the present invention include growing Taxus roots.
  • Taxus roots including roots cultured hydroponically, in vitro cultured roots, adventitiously produced roots, roots from callus derived from stems or needles and Agrobacterium transformed root cultures have been developed which result in long-term, stable production of taxol and related taxanes.
  • the roots established by use of the methods of the present invention are capable of producing taxol, and taxol-related compounds. These roots offer a rapidly reproducible, continuously-available source for the production of taxol and taxol-related compounds. This production may be scaled to commercial levels.
  • new compositions exhibiting taxol-like activity have been purified from extracts of the Taxus roots, offering new horizons for chemotherapeutic agent development.
  • Taxus roots including roots cultured hydroponically, normal roots cultured in vitro, adventitiously produced roots, roots from callus produced from stems and needles, and Agrobacterium transformed callus or root cultures, have the potential to produce an abundant supply of taxol and of biologically active compounds and precursors. In addition to providing a rapidly available and renewable source of taxol there are many other potential benefits.
  • Advantages realized in the use of hydroponically produced roots for the production of taxol and related compounds include: (1) genetically stable roots can easily be produced from all Taxus species and cultivars available, within a short period of time, thereby expediting the search for a high producing source of taxol along with novel taxanes and related compounds, (2) the ability to control the supply of secondary metabolites is independent of weather, disease, and politics to meet market demand, (3) strain improvement programs analogous to those for microbial systems can be initiated, (4) novel products such as taxol and its derivatives can readily be obtained from the new methods, whereas, this can not be done with existing methods, (5) extracts from roots are very clean, thereby, overcoming the need for extensive purification which is necessary from extracts obtained from leaves, stems or bark which contain chlorophyll and other pigments which make extraction and purification difficult, and (6) roots from plants grown hydroponically grow much more rapidly than the top portion of the plant, and may be maintained to grow continuously, even though the plant undergoes a dormant period.
  • Taxus Production and evaluation of taxol and related compounds in hydroponically grown roots or aseptically grown roots from the genus Taxus, is a novel accomplishment.
  • the extraction protocols to purify taxol and related taxanes from Taxus bark are very complex, and extraction from stems and needles makes the process even more complicated due to the presence of chlorophyll, other pigments, waxes and unknown hydrophobic compounds.
  • taxol extracts from stem tissues culture or root cultures, including those grown hydroponically are relatively clean and require less purification to be usable.
  • Roots cultured in accordance with the present invention are a continuous source of taxol because they can be maintained indefinitely, under aseptic conditions and retain their ability to produce taxol, active derivatives and precursors of taxol. They grow well in large scale bioreactors and can be cryopreserved. Taxol production ability is retained after cryopreservation or storage at 4-10"C.
  • Roots are suitable as sources to grow in bioreactors to produce large quantities of taxol, and taxol-related compounds. Some of these compositions are found in the culture medium and may be obtained by collecting the medium. Utilizing different bioreactor designs, the secondary metabolites found in solution may be easily removed from the solution utilizing exchange resins which extract only the desired secondary metabolites from the solution and not the media supplements required for culture growth. Other compositions are produced and retained intracellularly and must be extracted from the roots. Compounds that permeabilize membranes facilitate taxol extraction.
  • Root extracts require fewer purification steps and generally result in more purified compositions than are obtainable from other parts of the plant. Roots contain fewer contaminants such as pigment and wax than are present in the aboveground portion of the plant. Roots therefore allow for better resolution of more taxanes by chromatography. The upper portion of the plant is contaminated with waxes, pigments such as chlorophyll and other interfering compounds which are very abundant in the aboveground portion of the plant.
  • Roots have been used as a source of secondary metabolites in plants, but they have not been employed from Taxus plants and have not been recommended as a source of compounds from woody plants.
  • An advantage of using taxus plant roots is that Taxus plants are slow growing and require special conditions for proper growth. This problem along with the high cost and increasing demand for taxol and related taxanes, makes the use of roots from hydroponically grown plants very attractive. It is likely that taxol is synthesized in the roots and is transported to the rest of the plant where it accumulates. This makes the roots ideal for biosynthetic studies of taxol related compounds because all of the machinery (enzymes) is present, therefore roots are a good source of precursors to be used in partial synthesis of taxol and other novel antitumor compounds. Moreover, root culture readily lends itself to elicitation.
  • Organized tissue such as roots have less of a problem than cell culture because they are more homogeneous than cell cultures which are typically heterogeneous.
  • Cell suspensions have an inherent problem with an unknown compound which cochromatographs with taxol, which is not a problem with root culture.
  • compositions of the present invention facilitate new screening tests to determine which species and varieties of Taxus are amenable to long-term, stable culture, or hydroponic culture, and which sources are likely to yield taxol and taxol-related compounds.
  • Taxus cultured cell extracts with taxol-like activity have been eluted as peaks by HPLC (high pressure liquid chromatography) . These peaks contain materials which are positive in microtubule stabilizing assays.
  • HPLC high pressure liquid chromatography
  • the cell lines differ in the amount and nature of taxol and taxol-related compounds that they produce in culture under a defined set of environmental conditions.
  • Table V illustrates differences among some of the cell lines with regard to the range of percent of taxol produced. Components of peaks are taxol-like if they are capable of stabilizing microtubules and/or of giving positive results in cancer model systems, for example, those based on the toxicity of a test agent to cells.
  • Another aspect of the present invention is to produce new Taxus cell lines by inducing mutations. Mutations of selected cell lines are induced by irradiation treatment and chemical mutagens.
  • the callus and subclone selection criteria and methods to induce callus and maintain cell lines as described herein, are used to produce cell lines from the mutated sources.
  • Ultraviolet light, in particular UV-A, and chemical mutagens such as ethyl methane sulphone, are suitable mutagens. Selection for naturally occurring mutations due to somoclonal variation, is also useful.
  • Another aspect of the present invention is the regeneration of Taxus plants from in vitro cultures.
  • callus cultured on a high auxin:cytokinin ratio under a 16 hour regime was induced to undergo organogenesis by serially decreasing the auxin : cytokinin ratio [to 2IP (2- isopentenyladenine) 2 - 10 mg/1 in combination with IBA (indole-3-butyric acid) 0.1 - 2.0 mg/1, for example].
  • IBA indole-3-butyric acid
  • Roots from a wide range of Taxus species and cultivars grown under optimized conditions described herein are elicited both in vivo and in vitro with biotic (ie. fungal extracts) and abiotic elicitors (i.e. heavy metals) , plant growth regulators, precursors and/or intermediates in the taxol biochemical pathway (i.e. acetate, mevalonic acid) resulting in an increase taxol and related taxanes and/or novel antitumor compounds.
  • biotic ie. fungal extracts
  • abiotic elicitors i.e. heavy metals
  • plant growth regulators i.e. acetate, mevalonic acid
  • a major benefit of obtaining roots from hydroponically grown plants is that they are genetically stable, yielding uniform results overcoming a typical problem when dealing with roots in tissue culture. Because elicitation of taxol and related taxanes was successful, it appeared likely that genes involved in the biosynthesis of taxol and related taxanes were stimulated. Based on this knowledge, cDNA libraries were constructed and through differential screening of these libraries, several putative genes involved in taxol biosynthesis were identified. These genes are likely to be overexpressed in tobacco plants and/or bacterial systems which could potentially be used to overproduce taxol and related compounds. Both biotic and abiotic elicitors, precursor feeding and other elicitation techniques are useful to enhance secondary metabolite production.
  • An "active" composition is one that is capable of giving positive results in a microtubule stabilizing assay, a cancer model assay, a clinical trial, or any combination of these tests.
  • Bioreactors apparatus for growing roots on a large scale.
  • Airlift Kontes (Ctyolift airlift bioreactor)
  • Membrane-flow nutrient medium flows over a membrane which supports the roots, the medium is recirculated. Other novel designs are suitable.
  • Callus an aggregate of dedifferentiated cells.
  • Cancer model systems in vitro or in vivo systems wherein a result is produced by test agents, said result determined to be predictive of clinical effects of the agent.
  • Cell Culture composition of living cells maintained in vitro.
  • Cell Line group of cells in vitro for which growth characteristics have become stable under a defined set of environmental conditions, and which is differentiated from other cell lines by these characteristics.
  • Cell Suspension composition of living cells that are not attached to a solid substrate but rather are suspended in a liquid.
  • Clumps globular or nodular aggregates of cells observable on the surface of a callus.
  • Elicitation techniques techniques applied to a cell culture which increase the yield of taxol or taxol-related compounds that are produced by the culture; the mechanism of this empiric effect may be immunological, that is, the cells may mount a defense against foreign agents by producing taxol.
  • Abiotic examples include salts of heavy metals,vanadate; lead and mercury.
  • Biotic examples include autoclaved fungal cell wall fragments and spores. Friability: fragile, crumbly tissue consistency. Habituated cell line: cells in vitro which are maintained by subculture and do not require nutrient medium supplemented with hormones for maintenance; these cultures are usually characterized by relatively rapid growth rates. Habituation: condition wherein a cell line is capable of being maintained by serial subculture without the use of hormones as supplements to the nutrient medium in which the cells are maintained. Hairv root disease: A disease of broadleaved plants in which there is a proliferation of root-like tissue from a plant part. This is a tumorous state similar to the crown-gall. Hairy root disease is induced by the bacterium Agrobacterium rhizogenes containing an Ri plasmid.
  • Hydroponics Water culture of plants. A plant culturing system where roots are developed without soil.
  • In vivo literally "in life”. Pertaining to a biological reaction that takes place in a living cell or organism.
  • Medium a nutrient composition for the growth of living material such as cells, tissues, embryos and the like.
  • NFT Nutrient film technique
  • Retention time time at which a given compound will elute off a chromatographic column counting from the time at which the sample was introduced onto the column.
  • Secondary metabolite a metabolite that is not part of the primary metabolic network of the cells. Secondary metabolites are usually present only in cells that specialize in some way (e.g. defense) .
  • Shaker flasks any container that could be used to culture cells on a rotary shaker, e.g. Erlenmeyer flasks.
  • Sparging forcing (bubbling) air through a liquid medium.
  • Stable culture growth condition of cell culture growth wherein doubling times of the culture have reached an equilibrium under a defined set of culture conditions, and taxol production levels are maintained.
  • Subclone a culture derived from a sample of cells from a parent culture.
  • Subculture a cell culture composition derived from a sample of cultured cells, said sample being transferred to a different tissue culture vessel than that containing the culture of cells from which the sample was obtained.
  • Tissue culture composition of living tissue maintained in vitro; the terms"cell culture” and “tissue culture” are often used interchangeably, as they will be herein to refer to any composition where a plurality of cells are maintained in vitro.
  • Agrobacterium rhizogenes A species of Gram-negative, rod-shaped bacteria closely related to Agrobacterium tumefacien ⁇ .
  • Agrobacterium rhizogenes often harbors large plasmids, called Ri plas ids, which are closely related to Ti plasmids.
  • the combination of A. rhizogenes and an Ri plasmid can cause a tumorous growth known as hairy root disease in certain types of plants.
  • FIGURE 1 HPLC profile of taxane standards separated on a Curosil column: 1. Taxol; 2. 7-epi-10- deacetyl taxol; 3. Cephalomannine; 4. 10-deacetyl taxol; 5. Bacctin V; 6. 9-dihydro-13-acetyl baccatin III; 7. Baccatin III; 8. 7-epi-10-deacetyl baccatin III; 9. 10- deacetyl baccatin III.
  • FIGURE 2 HPLC profile of a stem culture of T. media cv. Hickii (CR-1 callus)
  • FIGURE 3 HPLC profile of a root culture of T. media cv. Hickii
  • FIGURE 4 HPLC profile of a root culture of T. canadensis
  • FIGURE 5 HPLC profile of a root culture of T. cuspidata . DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
  • the cultures of the present invention are induced from the Taxus species including: T. brevifolia , T. canadensis, T. chinensi ⁇ , T. floridana, T. globosa, T. sumatrana, T. yunnanensis and from different cultivars of T. baccata . T. cuspidata and T. media and others from the genus Taxus .
  • Young stem explants or roots are preferred as a tissue source to induce callus. Young stems are those that appear as new growths on a Taxus plant; the stems generally are about 0.3-0.5 mm in diameter. Young stems of Taxus media cv. Hicksii are preferred because these stems are found to produce higher levels of taxol than other Taxus species including the stems of Taxus brevifolia . Although other plant parts such as roots have the ability to produce callus, young stems provided the best results on a consistent basis. Roots derived from callus, and roots from other sources, however, produce the highest yield of taxol when cultured in tissue culture or hydroponically. Young stem explants from Taxus are surface sterilized and sectioned under aseptic conditions.
  • Suitable medium includes Gamborg's B5 medium (Gamborg et al . , 1968) (Materials and Methods) fortified with Gamborg's
  • glucose, fructose or sucrose 40-80 g/1 is preferred to the standard 20-30 g/1 of sucrose in nutrient medium.
  • glucose and/or fructose may replace sucrose.
  • Plant hormones are used to supplement the medium as plant growth regulators for callus induction.
  • auxins may be used in combination with kinetin, the latter at a range of 0.1-1.0 mg/1.
  • a high auxin: cytokinin ratio is preferred for callus induction, for example, 1 mg/12,4-D to 0.2 mg/1 kinetin; 5 mg/1 of naphthalene acetic acid
  • NAA to 1 mg kinetin.
  • auxins NAA (at a range of 1.0-5.0 mg/1), 2,4-D (at a range of 0.1-2.5 mg/1) or IBA (indole butyric acid, at a range of 1.0-5.0 mg/1) are suitable.
  • 1 ppm ppm
  • 2,4-D and 0.2 ppm kinetin are added to the cultures, alone or in combination with Gelrite or agar as gelling agents.
  • Explants are placed in tissue culture vessels, which can be petri dishes containing medium gelled with Gelrite, at about a 2 g/1 concentration. Prior to being dispensed into sterile petri dishes, the medium may be sterilized, for example, autoclaved at 121 * C for about 15 minutes.
  • Explants are incubated in complete dark at a temperature of about 23-25 * C.
  • the pH of the medium is preferably maintained in the range of 5.5-5.7. Some cultures may prefer to grow in the light; for example, cool, white fluorescent light for 16 hours in a 24 hour cycle.
  • induced callus is initially very slow growing and produces phenolic exudates (causing the callus to turn red) which are detrimental to the growth of the callus, even when the callus is frequently subcultured.
  • subclones may be selected from the subcultures of induced callus based on physical criteria including comparative growth rates, absence of red coloring, presence of yellow coloring, and friability.
  • Subcultures selected by the criteria disclosed herein are transferred to solid supports, preferably membrane rafts (Sigma Chemical Co.) on media such as that shown in Table IV.
  • Membrane rafts were preferred for the culture of callus because this system is similar to a "membrane-flow" type bioreactor.
  • the culture medium is changed and manipulated easily, and thus can easily be adapted to large scale culture.
  • Other reasons for using rafts are that callus tend to grow better on membrane rafts than on agar or other gelling agents, and also it is easier to harvest callus both for analysis and subculturing purposes.
  • Roots are produced in tissue culture by several different methods: root tips from hydroponically grown plants and adventitiously derived roots produced from stem/needle explants. This is done according to the methods of Wickremesinhe and Arteca (1993) who were successful in this approach with Cephalotaxus harringtonia (Japanese plumyew) .
  • Each of the explant sources are processed by surface sterilization and sectioned under aseptic conditions. The explants are then transferred to tissue culture vessels, generally, petri plates, with root-inducing media for root tip and root sections or to callus-inducing media for stem/needle explants (once rapidly growing callus was established it was transferred to root inducing media) .
  • a preferred media is Gamborg's B5 media (Gamborg et al . , 1968) supplemented with plant hormones such as auxins and cytokinins either alone or in combination.
  • Other tissue culture medias such as that of Murashige and Skoog (Murashige and Skoog, 1962) and others are also suitable for this purpose and may be substituted.
  • Gelling agents such as Gelrite (Scott Laboratories, West Warwick, R. I.) or agar are mixed with the nutrient media to form a solid support in the culture vessel for maintenance and growth of the explant.
  • Roots which are actively growing after 2-4 weeks are selected for transfer. Callus formation is determined when globular aggregates of undifferentiated cells are observed. This typically occurs on 90% of the explants within about 2-4 weeks. Subculturing was initially performed at 4-6 week intervals. The doubling is initially very slow, however, with time in culture the doubling rate of selected clones increases to 5-16 days allowing for subculture every 2-3 weeks depending on the clone. After establishing stable callus lines for each of the species/cultivars previously mentioned, callus is transferred to root inducing media where it generally takes 4-6 weeks to initiate roots.
  • Methods for culturing of "Hairy Roots" in tissue culture are as follows: Immature roots and shoots from hydroponically grown Taxus plants are inoculated with several strains of Agrobacterium rhizogenes strain 11325, 15834 and others. The cuttings are inoculated with individual Agrobacterium strains using standard protocols. Immature taxus roots and shoots are surface sterilized and placed on Gamborgs B5 medium. The explants are wounded aseptically with a syringe tip and inoculated with the appropriate strain. After 48 hours the explants are rinsed with sterile water and placed on fresh media containing 500 ⁇ g/ml carbenicillin.
  • the optimum culture conditions for the selected subclones are then determined by a multivariate analysis of factors which are likely to effect culture growth: presence or absence of light, gas composition, temperature, pH, medium supplements including plant hormones and sugars. After stable culture growth is achieved, the root cultures are kept in maintenance medium. For some of the root cultures, habituation occurs, that is, the cultures are maintained without the need for hormonal supplements.
  • root tips from established stable root cultures as described above were transferred into liquid medium contained in Erlenmeyer flasks (generally 250 ml flasks containing 100 ml of medium) . Flasks are placed on a rotary shaker known to those of skill in the art and operated at about 125 rpm.
  • the general tissue source are plants of the genus Taxus.
  • Various species from which taxol may be isolated include: T. brevifolia, T. canadensis, T. chinensis, T. floridana, T. globo ⁇ a, T. sumatrana, T. yunnanensis and from different cultivars of T. baccata. T. cuspidata and T.
  • Taxus plants are relatively easy to propagate from cuttings, however, methods vary. Taxus cuttings are highly topophytic and will maintain the growth habit they exhibit on the parent plant. In one example, cuttings were collected from September through December after a hard frost. These are roots from dormant plants, which may explain high yields of taxol rom root cultures. Metabolic root activity may be higher during plant dormancy. Cuttings taken after a frost typically root more readily than cuttings without a frost.
  • the base of the cutting is maintained at 68 * to 75'F with the tops kept cool and placed under mist to prevent desiccation.
  • the cuttings show branched roots 8 to 9 weeks following planting (Dir and Heuser, 1987) . After this period of time the roots are washed and the plants transferred to hydroponics.
  • An alternative to placing the cuttings into the soil is to treat the base of the cuttings as previously mentioned and place them directly into hydroponics.
  • One of the factors in a combination leading to optimization of root/shoot growth, together with the maximum production of taxol and related taxane production in roots, is to first maximize Taxus plant growth and development.
  • the shoots of hydroponically grown plants also require the gas composition (C0 2 enrichment) , environmental conditions including light, temperature, humidity and plant growth regulators optimized.
  • Taxol active derivatives of taxol, cephalomannine, baccatin and other precursors and intermediates of taxol can be examined using HPLC analysis by comparing the retention times of authentic standards and plant extracts. This analysis is accomplished by extracting callus, performing HPLC analysis on the extracts, and collecting components of major peaks. The components of these peaks are tested for activity in microtubule stabilizing assays (FIGURE 5, Materials and Methods) and in in vitro cancer model systems disclosed herein. Cancer cell lines suitable for screening include B16 Melanoma; MX-1 mammary xenograft; P388 leukemia; KB; L1210 leukemia.
  • Taxol activity is also confirmed in cell lines by showing that extracts have the ability to stabilize microtubule activity in the same manner as taxol. Another means of identification of taxol is accomplished by extracting callus cell lines, purifying this extract by HPLC and identifying taxol by utilizing H 1 and 2-D high resolution nuclear magnetic resonance (NMR) analysis (Falzone et al., 1992).
  • NMR nuclear magnetic resonance
  • the levels of taxol found in the cell lines of the present invention are at least 1 to 2 fold higher than in stems, leaves and bark of Taxus media cv. Hicksii (Table I) . These levels may be increased further.
  • plants may be regenerated from Taxus cultures to provide yet another source of taxol and, more interestingly, perhaps of the new compounds identified in culture that have taxol-like activity.
  • Taxol activity is also confirmed in cell lines by showing that extracts have the ability to stabilize microtubule activity in the same manner as taxol. Another means of identification of taxol is accomplished by extracting roots, purifying this extract by HPLC and identifying taxol by utilizing H 1 and 2-D high resolution nuclear magnetic resonance (NMR) analysis (Falzone et al., 1992).
  • NMR nuclear magnetic resonance
  • the levels of taxol found in the roots of the present invention are at least 2- to 4-fold higher than in other parts of the plant (Table 1) . These levels may be increased further.
  • cultures producing low or undetectable levels of secondary products are elevated by a variety of means including optimization of culture conditions, precursor feeding, use of biotic (Heinstein, 1985; Fukui et al . , 1990), and abiotic elicitors including the use of ultra-violet irradiation (Kartusch & Mittendorfer, 1990) and heavy metal ions (Threlfall & Whitehead, 1988) .
  • the addition of precursors to roots have also been shown to increase secondary metabolite production.
  • Precursor feeding, biotic and abiotic elicitors as defined herein are suitable for use with Taxus root culture.
  • the induction of mutations in roots by irradiation and chemical mutagens may result in roots which overproduce taxol, 10-deacetyl baccatin III, 7-epi-10-deacetyl baccatin III, baccatin III, 7-epi baccatin III, 9-dihydro-13-acetyl baccatin III, cephalomannine, 10-deacetyl taxol, 7-epilO-deacetyl taxol, 7-epi taxol, other known and unknown natural derivatives of these compounds, and other related taxanes including precursors and intermediates in the biochemical pathway resulting in taxol production, and active derivatives of taxol.
  • biotic and abiotic elicitors, feeding of precursors, and other elicitation techniques in shake cultures and large scale bioreactors coupled with large scale purification may increase product yield.
  • the surface sterilized stems were cut into sections under aseptic conditions and placed on Gamborg's B5 media plus and minus 2,4-D, kinetin or 2,4-D plus kinetin in the light and dark at 25 * C.
  • Undifferentiated callus tissue was initially slow growing, however, through many subcultures a friable yellowish colored cell line designated Gl with stable growth characteristics was obtained. This cell line grows on Gamborg's B5 media (Table II) in the dark at 25 * C without the supply of any exogenous hormones.
  • the cell line Gl was maintained on media supplemented with casein hydrolysate, glucose and fructose, which were found to significantly increase the growth rate of this cell line (Table III) .
  • Other cell lines were derived from Gl.
  • the callus cell line Gl was subcloned on modified Gamborg's B5 media shown in Table III.
  • the callus cell line was then transferred to membrane rafts containing liquid media shown in Table II at 25 * C in the dark. This cell line was subcloned and a cell line with stable growth characteristics was derived (CR1) (See Example 2) .
  • a callus-derived cell line designated CR-1 derived from a callus designated Gl, has the following characteristics: grows in cultures wherein the nutrient medium is Gamborg's B5 medium; grows in the dark at 20- 25 * C; does not require exogenous plant hormones (is a habituated line) ; shows increased growth rate compared to its baseline by addition to medium of casein hydrolysate, glucose and fructose to the medium (see Table III) ; exhibits a doubling time of from 5-16 days; and shows stable growth in culture.
  • the cell line was capable of secreting taxol, active derivatives of taxol, cephalomannine, baccatin and other precursors of taxol into the growth medium.
  • the callus-derived cell line designated CR1 produced rapid growth of callus after subculturing: three grams of callus seeded on a membrane raft produced 50-70 grams of callus within 7-10 weeks, with no subculturing, just the addition of fresh medium.
  • Authentic taxol and cephalomannine have retention times of 31 and 25 minutes, respectively.
  • Callus samples such as CR-1 have retention times corresponding to authentic taxol and cephalomannine, however, suspension lines such as S2 have peaks which are shifted, eluting at 32 and 25 minutes respectively. This shift in retention times led to difficulty in purification of these compounds from suspension lines. This is not a problem with root cultures.
  • a suspension culture line S3 was derived with stable growth characteristics when grown at 25"C in the dark with modified Gamborg's B5 media shown in Table IV. Suspension cell lines produced in this manner were typically slow growing, however, through subcloning the cell line S3 was obtained and had a moderate growth rate.
  • This cell line (S3) produces taxol, its precursors and active derivatives of taxol and secretes these chemicals into the solution and can easily be removed from the solution using exchange resins.
  • SR-1 Another suspension culture cell line designated SR-1 was obtained in a unique way from CRl callus lines grown on membrane rafts. A fine cell suspension was found in the nutrient solution in which the callus CRl was growing on membrane rafts.
  • the suspension culture of CRl was produced using methods of the present invention wherein cells sloughed off callus cell line CRl into suspension in the nutrient medium, were grown in 1 L shaker flasks with agitation in the dark at about 24-26 * C in Gamborg's B5 medium as shown in Table IV.
  • the suspension culture was designated SR-1. Suspension culture SR-1 was capable of secreting taxol, active derivatives of taxol, cephalomannine, baccatin and other precursors of taxol into the growth medium.
  • a sample of cells from cell line Gl (EXAMPLE 1) which grows in the dark was transferred to a light cycle of 12 hours light, 12 hours dark, at 25 * C. Transfer to the light initially caused a slow down in growth rate and a brown discoloration. To avoid cell death this callus line was placed on several hormone regimes containing 2,4-D and kinetin. These cell lines were subcloned and a callus cell line designated LG3 which grows on Gamborg's B5 media was selected (Table III) in the light. LG3 requires exogenous plant hormones (1 ppm 2,4-D and 0.2 ppm kinetin) in order to grow. This callus line has been acclimated to membrane rafts and suspension cultures have been initiated.
  • LG3 was subcultured on different hormone regimes consisting of various combinations of 2-isopentyladenine and indole-3-butyric acid (0.1, 1.0 and 5.0 ppm) .
  • the cell line is capable of secreting taxol, active derivatives of taxol, cephalomannine, baccatin and other precursors of taxol into the growth medium.
  • LG3 was subcultured on 10 different hormone regimes of cytokinins and auxins in the light at 25 * C. By using this method shoot formation was induced. This technique will be invaluable in regeneration of plants with superior taxol producing capabilities and as a means of elevating taxol levels in plants grown under aseptic conditions.
  • LGS-3 is a suspension culture derived from LG3.
  • Taxol, cephalomannine and baccatin were identified in each of the Taxus media cv. Hicksii cell lines CRl, S3, SRI, and LG3 using HPLC analysis (see Materials and Methods) . This was accomplished by making comparisons of retention times between plant samples and authentic standards. Taxol activity was also confirmed by showing that extracts have the ability to stabilize microtubules in the same manner as authentic taxol. The third line of evidence that taxol was contained in the cell lines was by purifying taxol on HPLC and identifying taxol this purified sample by nuclear magnetic resonance analysis. Derivatives of taxol and additional precursors were also identified by HPLC in each of cell lines.
  • Peak 4 a peak which exhibits taxol-like activity and which is present in CR-1, is diminished or absent in S-3. Peak 5 is present in CR-1 but diminished in SR-1. Interestingly, neither peaks 4 nor 5 found in CR-1 was detected in direct extracts of T. media Hicksii, under the conditions used to detect the peaks of callus and culture cell extracts.
  • the suspension culture lines S3 and SRI are grown in 1L flasks in Gamborg's B5 media (Table IV) .
  • Biotic autoclaved fungal cell wall fragments and spores
  • abiotic salts of heavy metals: vanadate, lead and mercury
  • Both cell lines and elicitation methods may result in elevated levels of taxol, its precursors and taxol derivatives.
  • Roots grown both in vivo and in vitro were elicited with biotic (autoclaved fungal cell wall fragments and spores) and abiotic (salts of heavy metals: vanadate, lead and mercury) stresses both of which are suitable as elicitors. All Taxus species/cultivars evaluated were elicited to different degrees and different taxanes were increased.
  • Roots which were fed precursors and/or intermediates in the taxol biochemical pathway resulted in an increase in taxol and a wide range of taxanes, the specific levels depending upon the Taxus species/cultivar used.
  • a novel method of storage preferred for Taxus cultures was to reduce incubation temperature to 4-10"C. Growth was slowed to a hibernation-like state. To return cells or roots to normal, the temperature was slowly raised to the optimum for growth.
  • the callus cell lines CRl and LG3 are maintained on membrane rafts placed inside Magenta GA-7 vessels, while shaker cultures of S3 and SRI are maintained in 1L shaker culture flasks and subcultured every 2 weeks.
  • Cryopreservation for long term storage of cell cultures CRl, LG3, S3 and SRI during the log phase of growth was accomplished by using standard cryopreservation methods using sorbitol and DMSO as the cryoprotectant. Cryopreservation was shown to have great potential for long term preservation of cell cultures whose biosynthetic capacity for synthesis and accumulation of secondary metabolites remain intact following removal from storage.
  • the root cultures are maintained on membrane rafts placed inside Magenta GA-7 vessels and in shaker cultures are maintained in 1L shaker culture flasks and subcultured every 2 weeks.
  • Cryopreservation for long term storage of root cultures during the log phase of growth was accomplished by using standard cryopreservation methods using sorbitol and DMSO as the cryoprotectant. Cryopreservation was shown to have great potential for long term preservation of roots whose biosynthetic capacity for synthesis and accumulation of secondary metabolites remain intact following removal from storage.
  • EXAMPLE 9 In Vitro Tissue Culture Derived from Taxus Roots After stable culture growth is achieved, the root cultures are kept on maintenance medium. For some of the root cultures, habituation occurs, that is, the cultures are maintained without the need for hormonal supplements. the optimum culture conditions for the selected subclones are then determined by a multivariate analysis of factors which are likely to effect culture growth: presence or absence of light, gas composition, temperature, pH, medium supplements including plant hormones and sugars.
  • Aseptic normal and "hairy roots” are grown under the following conditions: on maintenance medium containing hormones for normal roots, and without hormones for "hairy roots," 25°C, pH 5.6, 21% 0 2 on the remainder air, and kept in darkness, are used as a source of root tips for multivariate analysis of growth/taxane production.
  • Several root tips are transferred to Gamborgs B5, Murashige and Skoog, McCown's Woody plant liquid media, and aerated.
  • the growth rates/taxane production are evaluated over a one month period of time, although other times may be suitable depending on results.
  • the effects of various levels of vitamins are evaluated. For example, if Gamborgs B5 media is found to be optimal, vitamins would be varied 1/2X, IX, 2X, 3X and 4X of what is found in B5 media.
  • vitamins are optimized, varying sugars such as sucrose, glucose or fructose are varied 1/2X, IX, 2X and 3x.
  • the effects of temperatures ranging from 15 to 30 ⁇ C are evaluated.
  • pH varying from 3.5 to 7.0 is evaluated.
  • one half of the roots are kept in darkness, and the other half transferred to the light (50 ⁇ e m" 2 s" 1 ) and allowed to acclimate.
  • sugars are gradually removed from the growth media until the roots are capable of using photosynthesis as a carbon source at which time all sugars are removed.
  • the roots are acclimated to the light, they are subjected to varying light levels from 50 to 100 mE ⁇ " 2 s" 1 and growth rates/ taxanes evaluated. After light levels are optimized, they are kept constant while varying C0 2 levels from 0.03 to 3.0%. Carbon dioxide levels are also varied in dark grown roots, and growth rates/taxane levels are evaluated.
  • Taxol 10-deacetyl baccatin III, 7-epi-10-deacetyl baccatin III, baccatin III, 7-epi baccatin III, 9-dihydro-13-acetyl baccatin III, cephalomannine, 10-deacetyl taxol, 7-epil0-deacetyl taxol, 7-epi taxol, other known and unknown natural derivatives of these compounds have been identified in each of the Taxus species/cultivars evaluated using HPLC analysis (see Materials and Methods) .
  • Taxus canadensis The quantity of taxol and related taxane and the specific taxane differed with Taxus species/cultivar, for example 9-dihydro-13-acetyl baccatin III was found mainly in Taxus canadensis . This was accomplished by making comparisons of retention times between plant samples and authentic standards (FIGURE 1) . Taxol activity was also confirmed by showing that extracts have the ability to stabilize microtubules in the same manner as authentic taxol. The third line of evidence that taxol was contained in the roots of Taxus plants was by purifying taxol on HPLC and identifying taxol this purified sample by nuclear magnetic resonance analysis.
  • Taxus species/cultivars Derivatives of taxol and additional precursors were also identified by HPLC in roots from each of the different Taxus species/cultivars. This was accomplished by extracting each of the root cultures, running the extracts on HPLC, collecting major peaks and testing these samples in in vitro microtubule stabilizing assays (see Materials and Methods) . There were also numerous taxanes which did not correspond to any of the standards used.
  • the levels of taxol produced in unelicited cultures of Taxus roots generally is 2-4 times higher than the levels found in extracts from other plant parts as determined by HPLC analysis. Even more interesting were the HPLC profiles from root extracts revealing previously unidentified peaks with taxol-like activity, and profile differences among roots obtained from different Taxus species/cultivars. For example, differences in the HPLC chromatogram profiles of Taxus media, Taxus canadensis and Taxus cuspidata were apparent when compared as shown in FIGURES 3-5. Other profiles not included show a wide range of differences between species/cultivars.
  • the growth of the roots are suitable in a "nutrient film system.”
  • This system has nutrient solution flowing over the membrane which supports the roots and is recirculated. Nutrients can be added as needed by measuring conductivity, and the presence of taxol, taxol derivatives and precursors in the medium can be assayed by sampling from the solution in line.
  • This scale-up system is analogous to the growth of the roots on membrane rafts.
  • This system can also be optimized by having the nutrient medium sprayed onto the roots intermittently through a system consisting of a nozzle, a peristaltic pump and a timer, thereby supplying the nutrients more uniformly to the entire root mass and also by increasing nutrient uptake, efficiency, andminimizing nutrient use.
  • Rooted cuttings are generally transferred to one quarter strength Hoagland solution where they are allowed to acclimate for a period of one month under the following conditions: 21% oxygen/0.03% carbon dioxide/remainder nitrogen bubbled through the nutrient solution, 20 to 25°C, pH 4 to 5 and no plant growth regulator treatments.
  • the top portion of the plant is maintained at 60 to 90% relative humidity, 50 ⁇ E m *2 s" 1 irradiance, ambient levels of C0 2 /0 2 and 20 to 25°C.
  • the growth rates are monitored during this acclimation period, generally about one month, by the procedure outlined by Arteca et al. (1985) . After the acclimation period, the total nutrients are varied by evaluating 1/4, 1/2, 3/4, and full strength Hoagland solution while keeping constant the other parameters previously mentioned. Growth rates/taxane production are then evaluated.
  • the basic nutrient composition After the basic nutrient composition has been determined, it is incorporated into the one month acclimation period, and another set of parameters are evaluated.
  • the next set of parameters evaluated following acclimation of plants are the effects of varied levels of nitrogen, potassium or phosphorous. For example if 1/2 strength Hoagland is found to be optimal, nitrogen, potassium or phosphorous levels are varied 1/2, IX, 2X, and 4X the levels found in 1/2 Hoagland solution, and the growth rates/taxanes are evaluated.
  • C0 2 After nutrients are optimized, varying levels of C0 2 from 0.03% to 5% are bubbled through the nutrient solution and the growth rate and taxane production are evaluated. After C0 2 levels are optimized, all of the previouslymentioned parameters which have been optimized are kept constant and the temperature is varied from 15 to 25°C, after which growth rates and taxane production are evaluated.
  • the top portion of the plant is subjected to light levels from 50 to 500 ⁇ E m" 2 s" 1 , and growth rates and taxane production are evaluated. After light levels are optimized, they are kept constant while varying C0 2 levels from 0.03 to 0.3%, and evaluating growth rates and taxane production. After light and C0 2 levels have been optimized, they are kept constant, and hormone treatments are made to increase biomass. This can be accomplished in two ways, either via the roots or as foliar sprays. The range of concentrations applied differs between root and shoot applications, with the root concentrations always being lower.
  • gibberellins, cytokinins or brassinosteroids are applied to the roots at a concentration from 1 to 100 ppm for gibberellins, and 0.01 to 1 ppm for cytokinins or brassinosteroids.
  • gibberellins are applied over 100 to 500 ppm range, and brassinosteroids or cytokinins are applied over a 1 to 10 ppm range. Following treatment, growth rates and taxane production are evaluated.
  • growth of the top portion of the plant is slowed by applying growth retardants such as Cycocel to the foliage at concentrations ranging from 500 to 1000 ppm, and then evaluating growth rates and taxane production.
  • growth retardants such as Cycocel
  • EXAMPLE 12 Use of Cultures or Roots in Bioreactors to Produce Taxol and Taxol-Related Compounds
  • the growth of suspension cell line SRI in airlift, stirred and perfusion bioreactors results in taxol production.
  • new reactors may be designed.
  • the proper adjustment of environmental conditions such as gas composition, dissolved oxygen, rate of sparging and speed of agitation, temperature, pH of the growth medium, nutrients and organic supplements, removal of secondary products from the medium through the use of resins and elicitation are required to optimize the systems.
  • the growth of the callus line CRl is suitable in a "membrane flow" bioreactor. This system has nutrient solution flowing over the membrane which supports the callus and is recirculated.
  • Nutrients can be added as needed by measuring conductivity and the presence of taxol, taxol derivatives and precursors in the medium can be assayed by sampling from the solution in line.
  • This scale-up system is analogous to the growth of the callus cell line CRl on membrane rafts.
  • This system can also be optimized by having the nutrient medium sprayed onto the callus intermittently through a system consisting of a nozzle, a peristaltic pump and a timer, thereby supplying the nutrients more uniformly to the entire callus mass and also by increasing nutrient uptake, efficiency, and minimizing nutrient use.
  • the growth of roots in airlift, stirred and perfusion bioreactors results in taxol and related taxane production.
  • new reactors may be designed.
  • the proper adjustment of environmental conditions such as gas composition, dissolved oxygen, rate of sparging and speed of agitation, temperature, light, pH of the growth medium, nutrients and organic supplements, removal of secondary products from the medium through the use of resins and elicitation are required to optimize the systems.
  • the growth of the roots are suitable in a "membrane flow" bioreactor.
  • This system has nutrient solution flowing over the membrane which supports the roots and is recirculated. Nutrients can be added as needed by measuring conductivity and the presence of taxol, taxol derivatives and precursors in the medium can be assayed by sampling from the solution in line.
  • This scale-up system is analogous to the growth of the roots on membrane rafts.
  • This system can also be optimized by having the nutrient medium sprayed onto the roots intermittently through a system consisting of a nozzle, a peristaltic pump and a timer, thereby supplying the nutrients more uniformly to the entire root mass and also by increasing nutrient uptake, efficiency, and minimizing nutrient use.
  • microtubule stabilizing bioassay The microtubule stabilizing bioassay is used to screen for the ability of a composition to stabilize the formation of microtubules. There is a direct, predictive correlation between results in the microtubule bioassay and the chemotherapeutic action of a composition in cancer model assays. Therefore, the microtubule stabilizing bioassay is recognized by those of skill in the art as a screening test for effective chemotherapeutic agents for further clinical trials.
  • MT proteins neuronal microtubule (MT) proteins are prepared according to the methods of Shelanski et al. (1973) using fresh calf brains. Endogenous microtubule-associated proteins (MAPs) are removed from MT proteins which have been cycled one time, by cycling 4 times in the presence of 1 M glutamate (Hamel and Lin, 1981) . After completion of the cycling, the last tubulin pellet obtained is solubilized in cold PM buffer and is further purified by phosphocellulose chromatography according to Weingarten et al . (1975).
  • MAPs microtubule-associated proteins
  • test extracts are those solubilized in PM buffer as described above.
  • Taxus stem explants Approximately 90% of the Taxus stem explants showed callus induction after about 2-4 weeks of initiation. Yellow colored callus were selected, separated from the explant, and subcultured. Callus characteristically show initial slow growth, only doubling in about 3-6 months or longer. Another difficulty to be overcome is that most callus turn a brownish color within 1-3 weeks after subculture, and eventually die. Red-colored exudates were another sign of impending cell death.
  • Roots / root cultures were harvested, freeze-dried, and extracted with 1:1 mix of ethanol and methylene chloride by homogenizing in an Omni-mix homogenizer (Omni International, Waterbury, Connecticut) followed by sonication for 15 minutes.
  • the extract was filtered through a Whatmann # 1 filter paper and the filtrate was concentrated to dryness in vacuo.
  • the residue was then partitioned between methylene chloride and water and the methylene chloride fraction was concentrated to dryness under a stream of nitrogen.
  • the final pellet/residue was then resuspended in acidic methanol (0.1% acetic acid), and filtered through a 0.2 micron nylon filter for high performance liquid chromatography (HPLC) analysis.
  • HPLC high performance liquid chromatography
  • Duplicate injections were made from every sample and the average of the two peak areas was used to quantify taxol and cephalomannine.
  • a linearity curve was established for all the major taxanes by injecting amounts of authentic standards ranging from 0.001 to 10 mg per injection. Peaks for taxol were confirmed by collecting them and running then in the microtubule stabilizing bioassay and NMR analysis.
  • Taxol an important new drug in the management of epithelial ovarian cancer. Yale J. Biol. Med. 64: 583-590.
  • Taxol A unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Annals of Internal Medicine 111:273-279 (1989).
  • Taxol Twenty tears later, the story unfolds. J. Nat. Cancer Inst. 83, 1778-1781. Schiff, P.B. and S.B. Horowitz. Taxol stabilizes microtubules in mouse fibroblast cells. Proc. Nat. Acad. Sci. 77(3) :1561-1565 (1980).
  • Taxol a new and effective anti-cancer drug.
  • Taxol a novel antileukemic and antitumor agent from Taxus brevifolia. J. American Chemical society 93:2325-2327 (1971).
  • Kirschner A protein factor essential for microtubule assembly. Proc. Nat. Acad. Sci. USA, 72:1858-1862 (1975) .
  • Wickremesinhe, E.R.M. and Arteca, R.N. 1991a Production of taxol in callus and cell suspension cultures of Taxus media 'Hicksii". In Vitro, 27, 109A (abstract) . Wickremesinhe, E.R.M & Arteca, R.N. (1993b) Callus induction from stem explants of Taxus. Submitted to Plant Cell Reports. Wickremesinhe, E.R.M. and Arteca, R.N. 1993a. Establishment of fast-growing callus and root cultures of Cephalotaxus harringtonia. Plant Cell Rep. 12, 80-83.
  • Taxus spp. needles contain amounts of taxol comparable to the bark of Taxus brevifolia: analysis and isolation. J. Natural Products 53:1249-1255 (1990) .

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Abstract

On a mis au point des procédés de culture fructueux permettant d'obtenir des cultures tissulaires stables à long terme dérivées d'explants de Taxus et de racines cultivées en culture hydroponique. Ces cultures offrent une source reproductible rapidement et disponible continûment de production de taxol purifié et de composés apparantés au taxol. Les procédés de culture comprennent la culture tissulaire et la culture hydroponique in vitro. Les cultures sont amorcées au moyen de tissus de tiges ou de racines de Taxus ou à partir de racines mises en culture hydroponique. La production de Taxol peut être élevée à l'échelle commerciale au moyen de bioréacteurs. On a mis au point des méthodes de triage des espèces de culture de Taxus qui constituent des sources de taxol et de composés apparentés au taxol. De plus, pour obtenir les mêmes compositions que celles extraites à l'heure actuelle directement à partir d'ifs, on a purifié de nouvelles compositions présentant une activité analogue à celle du taxol à partir des nouvelles sources de Taxus, offrant de nouveaux horizons pour le développement d'agents chimiothérapeutiques.
EP93911220A 1992-05-21 1993-05-11 TISSUS DE -i(TAXUS) MIS EN CULTURE UTILISE COMME SOURCE DE TAXOL, TAXANES ET AUTRES NOUVEAUX COMPOSES ANTITUMORAUX/ANTIVIRAUX APPARANTES. Withdrawn EP0642586A4 (fr)

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US88661992A 1992-05-21 1992-05-21
US886619 1992-05-21
PCT/US1993/004424 WO1993023555A1 (fr) 1992-05-21 1993-05-11 Tissus de taxus mis en culture utilise comme source de taxol, taxanes et autres nouveaux composes antitumoraux/antiviraux apparantes

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