Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-05-26
2002-09-17
Solola, T. A. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
active
06452024
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an easy and quick process for the extraction and purification of Paclitaxel from natural sources. In comparison to the prior processes known in the art, this process is particularly economical because it has a limited number of steps and a reduced amount of losses during the purification.
PRIOR ART
The first work concerning Paclitaxel, also called Taxol™ or Pacitaxeline™, started in the United States in the 1960's, when the National Cancer Institute began a program for selecting plant extracts having activities against cancerous tumours, or anti-neoplasic activities.
From 1960 to 1981, more than 110,000 composites were extracted from 35,000 species of plants and were isolated and tested (Blume E. J. Natl. Cancer Inst. 1991; 83: 1054-1056).
Yew is part of the plants that were selected and tested, and the first extract from yew barks (
Taxus brevifolia
Nutt) coming from the west coast of the United States (Oregon) was obtained by Wani et al (Wani et al, J. Am. Chem. Soc., 1971; 93:2325-2327). The crude extract of these barks demonstrated a cytotoxic activity against leukaemic cells and an inhibiting action against a variety of tumours.
A couple of years later, the active compound of the extract was isolated. This active compound has been given Paclitaxel as generic name, and its molecular structure has been determined by X-ray crystallography and by 1H-NMR spectrum (Wani et al. J. Am. Chem. Soc. 1971; 93:2325-2327).
Since that time, studies on the effect of Paclitaxel towards cancerous tumours in-vitro and in-vivo have been carried out and the positive results that were obtained have lead to classify this active compound as one of the promising drugs for the treatment of ovarian cancer and breast cancer (Rowinski et al. Pharmacol. Ther. 1991; 52: 35-84). By the way, the use of Paclitaxel for the treatment of different cancers has been approved by the Food and Drug Administration since 1992.
The first variety of yew that was used to produce Paclitaxel, was
Taxus brevifolia
, but other varieties found in different regions of the earth have also been tested. They consist of
Taxus baccata, Taxus canadensis, Taxus wallichiana, Taxus yunnanensis, Taxus densiformis, Taxus hicksii, Taxus wardii, Taxus cuspidata, Taxus capitata, Taxus brownii
(Miller et al. J. Org. Chem. 1981; 46: 1469; McLaughlin et al. J. Nat. Prod. 1981; 44: 321; Kingston et al. J. Nat. Prod 1982; 45: 466; Senilh et al. J. Nat. Prod. 1984; 47: 131-137; Huang et al. J. Nat. Prod. 1986; 49: 665-669; Fett-Neto et al. Bio/Technology 1992; 10: 1572-1575).
All these species contain Paclitaxel, but in very limited amounts—about 0.0004 to 0.008%—(Kingston, Pharmacol. Ther. 1991; 52: 1-34). This low concentration of Paclitaxel makes its extraction and purification very costly because it takes time and it generally calls for repeated chromatographies.
The low concentration of Paclitaxel in all the varieties of yew produces a huge impact on the environment. To extract 1 kg of Paclitaxel from
Taxus brevifolia
barks, one needs to cut down around 3000 grown trees to get 10,000 kg of bark. The obtained quantity (1 kg) of Paclitaxel permits to treat about 500 patients but the number of patients suffering from cancer is as high as hundreds of thousands. Replanting the trees will never meet the urgent demand of Paclitaxel for human needs, because of their slow growth (Vidensek et al. J. Nat. Prod. 1990; 53: 1609-1610; Kelsey et al. J. Nat. Prod. 1992; 55: 912-917; Wheeler et al. J. Nat. Prod 1992; 55: 432-440).
Numerous processes for extracting and purifying Paclitaxel have been proposed. For example, Wani et al. (Wani et al. J. Am. Chem. Soc, 1971; 93: 2325-2327) have proposed a process for extraction of Paclitaxel from the barks of yew of the United States' west coast (
Taxus brevifolia
) comprising a treatment of the barks with alcohol followed by a few steps of purification by chromatography.
Miller et al. (1981) have extracted Paclitaxel from the
Taxus wallichiana
Zucc by a the following process:
1) extraction from the plant and concentration of the extract;
2) fat removal by separation of water and hexane;
3) extraction with chloroform and concentration;
4) first purification by chromatography in a first silica column;
5) second purification by chromatography in a second silica column;
6) a first countercurrent distribution;
7) a second countercurrent distribution;
8) preparative HPLC chromatography.
Senilh et al. (1984) have isolated Paclitaxel (or Taxol™ A: 0.0165%), Cephalomannine (or Taxol™ B: 0.0064%) and other compounds from barks of the
Taxus baccata
by the following process:
1) extraction with alcohol and concentration;
2) separation of water and dichloromethane;
3) filtration chromatography;
4) chromatography in a silica column;
5) alumina chromatography;
6) chromatography in a medium pressure silica column;
7) HPLC chromatography.
For other analog, two or three other column chromatography treatments followed by a preparative HPLC chromatography are necessary.
Another process used by Polysciences Inc. comprises the following steps:
1) dried ground barks are treated with methanol or ethanol and the obtained extract is concentrated to remove the alcohol,
2) the concentrate is then treated with dichloromethane and the obtained solvent extract is concentrated to yield a powder,
3) the powder is dissolved with a mixture of acetone and ligroin (1:1) and filtered to remove insoluble matter,
4) the organic phase which contains Paclitaxel is concentrated, dissolved in 30% of ligroin, and applied to a column of Florisil®,
5) the Paclitaxel fraction from the column is purified by double crystallization,
6) the so obtained crystalline Paclitaxel is subjected to chromatography on a silica column. The Paclitaxel is separated from the other taxanes (related analogs, cephalomannine, etc.) in this step,
7) the purified Paclitaxel obtained from the previous step is crystallized twice, and
8) unseparated mixtures and mother liquors are recycled through the silica column to obtain additional amounts of pure Paclitaxel.
Of course, there are other processes for purifying Paclitaxel from natural sources, as is explained in the following references:
Kingston et al., who disclose new taxanes obtained from
Taxus brevifolia
(J.
Nat. Prod. 1982; 45: 466-470); and
Witherup et al., who disclose a method for the separation of Paclitaxel and related compounds from
Taxus brevifolia
(J. Liq. Chrom. 1989; 12: 2117-2132).
U.S. Pat. No. 5,279,949 issued to Nair in 1994 discloses the use of tissue from ornamental yew (Taxus×media
Hicksii
) for Paclitaxel purification. In this patent:
1) fresh needles are extracted with 70% alcohol,
2) the extract is decolorized with charcoal and filtered,
3) the filtered extract is concentrated to remove most of the organic solvent,
4) the aqueous concentrate is centrifuged to separate the solids which contain Paclitaxel,
5) the solids are then subjected to a first normal phase silica chromatography,
6) the obtained crude Paclitaxel fraction is subjected to a second, low pressure silica chromatography; and
7) final purification is carried out in a reverse phase column.
U.S. Pat. No. 5,654,448 issued to Pandey et al., discloses a process for extraction and purification of Paclitaxel from barks of
Taxus brevifolia
. In this process:
1) the barks are treated thrice with methanol, each extraction being performed over a period of 5 days, the so-obtained extract being then concentrated;
2) the methanol concentrate is separated to obtain methylene chloride and water and the methylene chloride extract is evaporated to dryness, the solid residue containing the Paclitaxel amounting to about 1.8-2.2% w/w;
3) the solid residue is dissolved in acetone and mixed with one volume of hexane to remove polar impurities, and the mixture is evaporated to ⅓ of its volume;
4) the acetone/hexane residue is added drop-wise to hexane (1.5 L-3.0 L of residue for 10-15 L of hexane) to yield a precipitate which is filtered and dried under high vacuu
Bui-Khac Trung
Dupuis Nicolas
Chaichem Pharmaceuticals International
Solola T. A.
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