Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-03-10
2001-05-08
Lee, Howard C. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
active
06229027
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the production of paclitaxel from pacific yew, ornamental yew, or other yew species', needles, barks and roots and more particularly, the present invention relates to a process for extracting paclitaxel economically which is presently extracted by a high cost process due to an extremely high percentage of unwanted impurities carried forward in the extract during the extraction.
BACKGROUND OF THE INVENTION
In the prior art there is described an extraction method (Journal of Natural Products 53, 1249-55, 1990), which reported that paclitaxel content in the extract in the range of between 0.009% and 0.07%. After removing the hexanes soluble impurities (waxes, pigments, etc.) and after partition between water and dichloromethane, the paclitaxel concentration in the dichloromethane soluble crude extract was in the range of between 0.03% and 0.3% by weight. Paclitaxel (Taxol) is represented by the following structural formula:
is an anticancer compound. Paclitaxel exhibits a unique mechanism for preventing the growth of cancer cells by affecting the microtubules, which plays an important role in cell division and other cellular functions.
Paclitaxel is clinically effective for the treatment of retractory human ovarian and breast cancer and has exhibited promising activity against a number of other types of cancers such as liver, peritoneal, cervical, prostate, colon, and esophageal cancers.
Paclitaxel was primarily extracted from the bark of pacific yew
Taxus brevifolia.
Unfortunately, the yew grows very slowly, approximately eight inches per year and thus the bark is a limited source of paclitaxel. This lead researchers to seek alternative means for producing paclitaxel and analogues thereof which may display superior antitumor activity, such as derivatives of 9-dihydrotaxol that have enhanced water solubility. As an example 13-acetyl-9-dihydrobaccatin III, a taxane diterpene, has been found to be a useful precursor to make such analogues. This has been discussed in U.S. Pat. No. 5,440,056, issued to Klein et al., Aug. 8, 1995.
13-Acetyl-9-dihydrobaccatin III and baccatin III are represented by the following structure formulas:
This can be obtained from, for example,
Taxus canadensis
which is more widely distributed than
Taxus brevifolia.
Hence, 13-acetyl-9-dihydrobaccatin III is available more abundantly than paclitaxel.
Conventional methods for the isolation of taxanes, including paclitaxel, 13-acetyl-9-dihydrobaccatin III, and baccatin III generally involve the steps of extracting taxanes from a biomass with an alcoholic solvent and separating and purifying the individual taxane by chromatography.
Prior art methods disclose the use of various types of chromatographic technologies to separate paclitaxel and derivatives thereof. For example, U.S. Pat. No. 5,380,916, issued to Rao, Jan. 10, 1995, describes a process using C18 reverse phase liquid chromatography. Although reverse phase chromatography can be successful in separating the taxanes, this protocol employs an expensive absorbent and requires a significant time investment. Accordingly, this process is not economically and industrially viable.
U.S. Pat. No. 5,478,736, issued to Nair, Dec. 26, 1995, discloses a process using a silica gel normal phase absorption chromatography to purify taxanes. In normal phase absorption chromatography, Al
2
O
3
or silica gel is used as an absorbent which is about 100 times less expensive than the ordinary reverse phase absorbent. The problem for the normal phase silica gel absorption chromatography is that the absorbent will bind with paclitaxel which will not be eluted by solvent mixture, therefore the product yield is reduced.
U.S. Pat. No. 5,530,020, issued to Gunarvardana et al., Jun. 25, 1996, disclosed a process for isolating 13-acetyl-9-dihydrobaccatin III from
Taxus canadensis
which employs planet coil countercurrent chromatography (PCCC). Some of the disadvantages associated with this procedure are that it is complex, consumes significant amounts of solvent and can be used to purify only a small amount (milligrams) of samples.
So far, no distributive column chromatography for separating and purifying paclitaxel and related taxanes has been disclosed.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an improved method for isolating taxanes, particularly paclitaxel, 13-acetyl-9-dihydrobaccatin III, from a taxus species.
One aspect of the present invention, there is provided a method for isolating taxane from a crude extract of a taxus species comprising the step of subjecting the crude extract to dry column chromatography and distributive column chromatography.
The method of the present invention may be practiced on a small laboratory scale as well as large industry scale.
Conveniently, the taxane is obtained from extraction of the plant material with an alcoholic solvent at refluxing. Preferably, the column to be used with the method of the present invention includes a nylon tube packed with pretreated silica gel, having a micro sphere size of between 150 mesh and 250 mesh for using as dry column distribution chromatography.
A glass column packed with pretreated silica gel having a particle size of between 150 mesh and 250 mesh for using as wet distributive column chromatography.
In one embodiment of the invention, the method includes the steps of dry column distribution chromatography to obtain 13-acetyl-9-dihydrobaccatin III, normal phase distributive column chromatography to obtain paclitaxel and baccatin III.
In another embodiment of the invention, 13-acetyl-9-dihydrobaccatin III can be purified by dry column distribution chromatography with eluting solvent hexanes and ethyl acetate (4:6).
In another embodiment of the invention, paclitaxel may be further purified by normal phase distributive column chromatography. Preferably, the eluting solvent for this step is hexane:ethyl acetate (starting from 6:4 ending at 3:7). This chromatography may be repeated several times if necessary.
In accordance with another aspect of the present invention, this is provided a method for obtaining taxanes from a Taxus species, including, but not limited to
T. brevifolia, T. baccata, T. cuspidata, T canadensis, T. wallichiana, T. chinensis, T. funanesis,
T. X media ov. densiformis and T. X media ov. Hicksii.
The process comprises the steps of extracting a source material using alcoholic solvent concentrated the alcoholic extract to get a taxanes containing residue, partitioning the residue was partitioned between water and CH
2
Cl
2
, then concentrating CH
2
Cl
2
.
Solution to get a taxane rich residue, separating the taxanes into taxane containing fractions by subjecting the taxanes rich residue to dry column distribution chromatography in a dry chromatography column, and recovering any individual taxane from the column.
In another embodiment of the present invention, paclitaxel and baccatin III may further be purified by the steps of crystallizing the 13-acetyl-9-dihydrobaccatin III out of the taxane-containing solution with an alcoholic solvent to obtain 13-acetyl-9-dihydrobaccatin III, and purifying the paclitaxel-containing solution by normal phase column distribution chromatography.
The advantages of the present invention are to provide a method for the isolation of taxanes which is quick, easy to perform, cost-effective and efficient, both in small and large scale productions.
REFERENCES:
patent: 5380916 (1995-01-01), Rao
patent: 6136989 (2000-10-01), Foo et al.
Witherup, K. M. et al “Taxus spp. needles contain amounts of taxol comparable to the bark oftaxus brevifolia” J. Nat. Prod. vol. 53 No. 5 pp. 1249-1255, 1990.
(Marks & Clerk)
Lee Howard C.
Maier Leigh C.
Sharpe Paul S.
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