Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2001-02-23
2003-01-07
Drodge, Joseph W. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S198200, C210S511000, C210S656000, C424S770000, C549S510000, C549S511000
Reexamination Certificate
active
06503396
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and an apparatus for extracting and purifying taxol and derivatives thereof from source materials containing the same. In particular, the methods and apparatus can be used in separating taxol and its derivatives from needles and other parts of yew trees.
BACKGROUND OF THE INVENTION
Numerous compounds that exhibit therapeutic activity in the treatment of diseases have been isolated and identified in organic solvent extracts from plant material, microorganisms and marine organisms. Some of the compounds that have been demonstrated to have a particularly good physiological activity have been used as chemotherapeutic or anti-HIV agents. However, investigation into the therapeutic utility of these compounds has been hindered by the extreme abundance of natural sources to be screened and the inefficiencies of conventional extraction techniques.
Examples of such useful compounds include plant alkaloid derived from
Vinca rosea
and its semisynthetics vinblastine and vincristine, and taxol (NSC 125973), a diterpene alkaloid plant product derived from the pacific yew,
Taxus brevifolia.
Taxol
Taxol is one of a class of drugs inhibiting mitosis of eukaryotic organisms. It promotes polymerization of tubulin and stabilizes the structure of intracellular microtubules to produce an anti-cancer effect (see, Schiff, P. B. et al.,
Nature
277, 665-667 (1979)).
In the 1960's, taxol was discovered as a result of a plant screening program organized by the US National Cancer Institute (NCI). After cytotoxic activity was first found in a crude extract from the bark of the Pacific yew tree,
Taxus brevifolia
, the active compound was isolated in 1966. It was named taxol in 1967, and its molecular structure was characterized by Wani et al. in 1971 (Wani, M. C. et al.,
J. Am. Chem. Soc.,
93: 2325, 1971). Horwitzi et al. (1979) elucidated its active mechanism (Horwitz, F. B., et al.,
Nature,
1979, 277, 665 and Eric K. Rowinsky et al.,
J. National Can. Inst.,
82, 11247 (1990)). According to NCI's reports, the response rate to taxol of ovarian cancer, breast cancer or lung cancer patients, all who have exhibited no response to conventional anti-cancer drugs, were 30%, 50% and 20%, respectively (David, G., et al.,
J. Nat. Prod.,
53, 1 (1990)).
Marketing approval of taxol as an anti-cancer drug started from 1992, when the US Food and Drug Agency (FDA) and the HPB (Canada) approved the use of taxol for the treatment of ovarian cancer. In 1994, the use of taxol for the treatment of breast cancer and Kaposi's sarcoma was approved by the FDA. Subsequently, the FDA approved taxol for treating non-small-cell lung cancer (NSCLC) in 1997 and expanded the use of taxol to early breast cancer in 1999. In addition, taxol has been reported in studies to exhibit excellent effects on other types of cancer (Spencer, C. M. and Faulds, D.,
Drug.
48, 794-847 (1994)).
Conventional Preparation of Taxol
The conventional purification method is performed in four major steps. First, the acetone:hexane mixture from the extraction process is chromatographed on Florisil columns in a 70/30 hexane:acetone mixture to separate the taxol containing fractions. The taxol fractions are then concentrated to dryness. This step may be repeated as many as nine times. Second, taxol concentrates are crystallized from a methanol:water mixture and then recrystallized from an acetone:hexane mixture yielding 85 to 95% pure taxol. Third, the taxol is chromatographed on silica gel packed with either 2.5% isopropanol or 2.5% n-butanol in methylene chloride to yield approximately 98% pure taxol. Fourth, the taxol is dissolved in acetone, the solution filtered, and taxol recrystallized from an acetone:hexane mixture. This organic phase extraction and chromatographic purification process yields 99% pure taxol, which is about 0.014% of the milled bark (
J. Liquid Chromatography,
12(11): 2117-2132 (1989); WO 92/07842).
The production of taxol by this technique is encumbered by the following: (i) time consuming extraction and purification procedures; (ii) long residence times in a harsh environment; and (iii) low overall yields. Also, the bark of
T. brevifolia
is usually obtained from mature trees (100 to 200 years old). The bark is thus in limited supply.
Many studies have been conducted to overcome such problems and to provide alternate routes for obtaining taxol. Some of the alternate routes include total chemical synthesis and cell culturing synthesis. Several total chemical synthesis methods were reported in 1994. However, these methods were difficult to commercialize due to their complicacy. This problem originated from the fact that taxol has several asymmetric carbon atoms and a complex structure. The production of taxol through cell culturing has also undergone some difficulties in commercializing because of the slow rate of cell growth and the easy browning of cells (U.S. Pat. No. 5,019,504, issued May 28, 1991).
Yew trees can be classified into the following genera: Amentotaxus, Austrotaxus, Cephalotaxus, Pseudotaxus, Taxus, Torreya, etc. Among them, Taxus genus includes, for examples,
T. brevifolia, T. baccata, T. media, T. wallichiana, T. Canadensis, T. cuspidate
, etc, which are generally considered to be suitable sources for extraction of taxol and its derivatives.
Yew trees, such as
T. cuspidate
, which have a high taxol content in their needles, have been planted plentifully with regard to landscape and are easy to cultivate in farm fields. However, the trees have high levels of wax and non-polar components, which makes the separation of taxol therefrom difficult. The total amount of taxol in organic solvent extracts from the bark was 25%, but the content of impurities was very high in the range of 35-42%. In order to easily separate taxol from
T. cuspidate
, one must first develop selective removal techniques of materials such as waxes and chlorophyll.
The term “impurities” herein refers to all components of extracts from plants with the exception of taxol and its derivatives.
The use of supercritical fluid in the extraction of taxol has been proposed for improved extraction. The term “supercritical fluid” refers to a fluid that is above its critical pressure and above its critical temperature. A supercritical fluid has both the gaseous property of being able to penetrate anything and the liquid property of being able to dissolve materials into their components. In addition, it offers the advantage of being able to change density to a great extent in a continuous manner and by adjustment of the system pressure and temperature. This advantage is connected to simple control of its dissolving capability. As such, use of supercritical fluids is offered as a substitute for organic solvents in the fields of food industry and medical supplies.
Furthermore, supercritical fluids do not extract chlorophyll (M. T. Tena et. al.
Analytical Chemistry, Vol.
69, No. 3, 521 (1997)). Particularly, supercritical carbon dioxide is advantageous in extracting desired ingredients from plants without chlorophyll extraction.
KR patent application Nos.1994-7829, 1994-36099, 1995-703845, 1996-19486 and 1996-0055302 describe methods for extracting taxol from plants with supercritical carbon dioxide.
In particular, KR patent application No.1994-7829 discloses a method and an apparatus for continuously separating and purifying taxol with high purity wherein supercritical fluid extraction is accomplished in a countercurrent manner. Cosolvents are also employed in the invention.
KR patent application No.1994-36099 discloses the use of n-hexane in removing non-polar substances prior to supercritical carbon dioxide extraction. Namely, it suggests a combining method of organic solvent extraction with supercritical carbon dioxide extraction.
KR patent application No.1995-703845 is a method for extracting taxol from the needles of ornamental yew comprising the steps: (a) dewaxing the needles by subjecting said needles to supercritical fluid, (b) subjecting said dewaxed needles to
Choi Young Hae
Han Joo Hee
Kim Jin Woong
Noh Min Jeong
Yoo Ki Pung
Drodge Joseph W.
Fish & Richardson P.C. P.A.
Hanwha Chemical Corporation
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