Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
1999-04-28
2002-06-11
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S244000, C435S420000, C435S431000, C549S510000, C549S511000
Reexamination Certificate
active
06403343
ABSTRACT:
TECHNICAL FIELD
This invention relates to a method of producing a taxane-type diterpene including taxol which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like, and a method of obtaining cultured cells which produce the taxane-type diterpene at a high rate.
BACKGROUND ART
Taxol, which is useful as a therapeutic agent for ovarian cancer, mammary cancer, lung cancer and the like, is a taxane-type diterpene identified after being isolated from
Taxus brevifolia
NUTT, which is a plant belonging to genus Taxus, family Taxaceae and has a complex ester group which is related to its activity. Taxol can be found in all the parts of the plant body of
Taxus brevifolia
NUTT, but the bark has been reported to exceed all others in its content of the taxol. At present, taxol is collected from a natural or a cultivated plant body, however, the plant belonging to genus Taxus grows slowly, and it takes more than 10 years to grow to a height of 20 cm above the ground. Also the tree dies after its bark is taken off, thus it has been difficult to easily obtain a large amount of taxol. It would be advantageous if a taxane-type diterpene such as taxol and baccatin III which is a precursor of taxol, can be produced by the use of tissue culture, since a large amount of taxol can be easily obtained without cutting down the trees.
As a conventional method of producing taxol by utilizing cultured plant cells, a US patent was issued on a production method utilizing cultured cells of
Taxus brevifolia
NUTT (U.S. Pat. No. 5,019,504), however, the yield of taxol production described therein is 1-3 mg/l, and that is insufficient for the industrial production. Also, the production of taxol by the cell culture is unstable and even when a primary cell of high productivity can be obtained by selection, it is difficult to keep its content by subculturing [E. R. M. Wickremesine et al., World Congress on Cell and Tissue Culture (1992)].
On the other hand, as a prior art in the taxol production, a semisynthetic method from baccatin III, which is a precursor in biosynthesis of taxol, is disclosed in the specification of U.S. Pat. No. 5,015,744 issued to Holton et al. By the use of the plant tissue culture, a raw material for the semisynthetic process such -as baccatin III can be produced, thus the plant tissue culture can be also utilized for taxol production by the above-mentioned semisynthetic process.
DISCLOSURE OF INVENTION
The first object of the present invention is to provide a simple method of producing a taxane-type diterpene by plant tissue culture.
The second object of the present invention is to provide a method of obtaining cultured cells which produce a taxane-type diterpene at a high rate.
The first invention of the present application is a method of producing a taxane-type diterpene wherein a tissue or a cell of a plant which produces a taxane-type diterpene is cultured in the presence of at least one substance selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents, then the taxane-type diterpene is recovered from the resulting cultures.
The second invention of the present application is a method of producing a taxane-type diterpene wherein a tissue or a cell of a plant which produces a taxane-type diterpene is cultured by controlling the oxygen concentration in a gas phase in a culture vessel to less than the oxygen concentration in the atmosphere, from the initial stage of the culture, or by controlling the dissolved oxygen concentration in a fluid medium which is in contact with the tissue or the cell to less than the saturated dissolved oxygen concentration at that temperature, from the initial stage of the culture, then the taxane-type diterpene is recovered from the resulting cultures.
The third invention of the present application is a method of obtaining cultured cells which produce a taxane-type diterpene at a high rate, wherein cells of a plant which produces a taxane-type diterpene are fractionated into a plurality of layers according to the difference in their specific gravities, and cells contained in at least one layer are cultured, then such cultured cells that produce the taxane-type diterpene at a high rate are selected from among those cultured cells.
The present invention will be described in further detail.
The taxane-type diterpene, which is an object for the present invention, is not particularly limited to any diterpene as far as it has a taxane skeleton, and the illustrative examples include taxol, 7-epitaxol, baccatin III, 7-epibaccatin III, cephalomannine, 7-epicephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine, 10-deacetyltaxol, taxagifine, an analogue thereof, taxane 1a, an analogue thereof, xylosyl cephalomannine, xylosyl taxol and the like.
Examples of the plant to be used in the present invention which produces the taxane-type diterpene are those belonging to genus Taxus, such as Taxus baccata LINN, Taxus cuspidata SIEB. et ZUCC, Taxus cuspidata SIEB. et ZUCC var. nana REHDER, Taxus brevifolia NUTT, Taxus canadiensis MARSH, Taxus chinensis, and Taxus media.
According to the first invention of the present application, culture of the above-mentioned plant can be carried out by the previously known method except that the tissue or the cell of the plant which produces the taxane-type diterpene is cultured in the presence of at least one substance selected from the group consisting of jasmonic acids, compounds containing a heavy metal, complex ions containing a heavy metal, heavy metal ions, amines and antiethylene agents.
Examples of jasmonic acids, which are objects for the first invention of the present application, include a compound represented by the general formula (I):
[wherein, R
1a
, R
1b
, R
1
c, R
1d
, R
1e
and R
1f
respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;
R
2
, R
3
, R
4
, R
5
and R
6a
respectively represent hydrogen atom or alkyl group having 1 to 6 carbon atoms;
a side chain consisting of C
1
—C
2
—C
3
—C
4
—C
5
—C
6
may contain one or more double bonds;
R
6b
represents hydroxyl group or —O—carbohydrate residue;
R
7
represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH
4
), NHR
8
(wherein R
8
represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR
9
(wherein R
9
is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms;
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms],
a compound represented by the general formula (II):
[wherein, R
1a
, R
1b
, R
1c
, R
1d
, R
1e
and R
1f
respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;
R
2
, R
3
, R
4
, R
5
and R
6
respectively represent hydrogen atom or alkyl group having 1 to 6 carbon atoms;
a side chain consisting of C
1
—C
2
—C
3
—C
4
—C
5
—C
6
may contain one or more double bonds;
R
7
represents hydroxyl group, OM (wherein M is alkali metal atom, alkaline earth metal atom or NH
4
), NHR
8
(wherein R
8
represents hydrogen atom, acyl group having 1 to 6 carbon atoms, alkyl group having 1 to 6 carbon atoms or amino acid residue), OR
9
(wherein R
9
is alkyl group having 1 to 6 carbon atoms or carbohydrate residue), or alkyl group having 1 to 6 carbon atoms;
n is an integer of 1-7;
and in the above-mentioned five-membered ring, a double bond may be formed between the neighboring member carbon atoms],
and a compound represented by the general formula (III):
[wherein, R
1a
, R
1b
, R
1c
, R
1d
, R
1e
and R
1f
respectively represent hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or alkoxy group having 1 to 6 carbon atoms;
R
Hara Yasuhiro
Higashi Yosuke
Matsubara Kouichi
Ohnishi Naoto
Suga Chuzo
Marx Irene
Mitsui Chemicals Inc.
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