JPH07308197A - Production of taxane type diterpene - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the subject compound useful as a therapeutic agent for ovarian carcinoma, mastocarcinoma, lung cancer, etc., by culturing a cell or a tissue of a plant capable of producing a taxane type terpene in the presence of a specific jasmonic acid and recovering a formed product from the culture mixture. CONSTITUTION:A cell or a tissue of a plant (e.g. Taxus baccata) capable of producing taxane type terpene is cultured in the presence of a jasmonic acid of formula I {R<1> is a 1-4C alkyl, a group of formula II [R<6> is OH, an OM (M is an alkali metal, an alkaline earth metal or NH4), an NHR<7> (R<7> is a 1-4C acyl, a 1-4C alkyl or amino acid) or an OR<8> (R<8> is a 1-4C alkyl or a hydrocarbon residue; (n) is an integer of 1-7]; R<2> to R<5> are each H or R<2> and R<3>, R<3> and R<4> or R<4> and R<5> may form a double bond together; the five-membered ring may be further substituted with a hydroxyl group and may form a double bond between adjoining carbon atoms} and a formed product is recovered to give the abjective taxane type terpene (e.g. taxol).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a taxane-type diterpene containing taxol which is useful as a therapeutic agent for ovarian cancer, breast cancer, lung cancer and the like.

[0002]

2. Description of the Related Art Taxol, which is useful as a therapeutic drug for ovarian cancer, breast cancer, lung cancer, etc., is a taxane-type diterpene isolated and identified from Taxus brevifolia NUTT, which is a Taxus genus plant. Yes, with complex ester groups associated with activity. It has been reported that taxol is present in every part of the yew plant, and its content is highest in the bark.
Currently, taxol is collected from natural or cultivated plants, but yew plants are slow growing plants that take more than 10 years to grow to a height of 20 cm above the ground, and when bark is peeled off. It is difficult to easily obtain a large amount of taxol because the trees die. If taxane or a taxane-type precursor taxane-type diterpene such as taxcatin III can be synthesized by utilizing tissue culture, a large amount of taxol can be easily obtained without cutting trees, which is advantageous. Is.

[0003] For the taxol production method using the cultured cells of the plant up to now, see
Although the production of taxol by cultured cells of S. brevifolia NUTT) has been patented in the US [USP No. 5019504], the production amount of taxol is described as 1 to 3 mg / l, which is insufficient for industrial production. In addition, the productivity of taxol in cell culture is unstable, and cells with high productivity are obtained primarily by selection, but it is difficult to maintain the content by subculture (ERM Wickremesine et al., World Co
ngress on Cell and Tissue Culture (1992)].

On the other hand, as the prior art of the taxol production method, baccatin III (ba) which is a biosynthetic precursor of taxol is used.
A semi-synthesis method from ccatin III) is disclosed in US patent to Holton et al. [USP No. 5015744]. If a plant tissue culture method is used, semi-synthetic raw materials such as baccatin III can also be produced, and can also be used for taxol production by the semi-synthetic method.

[0005]

An object of the present invention is to provide a simple method for producing a taxane-type diterpene by culturing plant tissue.

[0006]

As a result of earnest research, the present inventors have conducted jasmonic acid addition to the tissue culture medium of cultured cells or cultured tissue of plants producing taxane-type diterpenes to carry out tissue culture. By accelerating the production of jasmonic acids (endogenous jasmonates) produced by the cultured cells or the cultured tissues themselves by subjecting the cultured cells or the cultured tissues to the special treatment described below, the taxane-type diterpene in the culture Finding that productivity improves,
The present invention has been completed.

That is, the present invention provides a plant cell or tissue which produces a taxane-type diterpene by the general formula [I]:

[Chemical 3] [In the formula, R ¹ represents an alkyl group having 1 to 4 carbon atoms or the following formula:

Embedded image-(CH₂ )_n-CO-R⁶ {In the formula, R⁶Is a hydroxyl group, OM (where M is an alkali metal
Group atom, alkaline earth metal atom, or NH_FourThe table
You ), NHR⁷(Where R⁷Is hydrogen atom, carbon number 1
To an acyl group having 1 to 4 carbon atoms, an alkyl group having 1 to 4 carbon atoms, or an amino group.
Represents an acid residue. ), Or OR⁸(Where R⁸Is carbon
It represents an alkyl group of the numbers 1 to 4, or a carbohydrate residue. )
Where n represents an integer of 1 to 7. } Shows the group shown by
Do; R², R³, R^Four, And R^FiveAre hydrogen atoms
Yes or R²And R ³, R³And R^Four, Or R^FourWhen
R^FiveMay together represent a double bond; said 5-membered ring
May be further substituted with a hydroxyl group, and adjacent ring members
A double bond may be formed between carbon atoms. ]
Cultivated in the presence of the compound, the resulting culture yields taxane
Taxane-type diterpenes characterized by recovering the type diterpene
It is a manufacturing method of a terpene.

The present invention will be described in detail below. The taxane-type diterpene to be the subject of the production method of the present invention,
There is no particular limitation as long as it is a diterpene having a taxane skeleton, and examples thereof include taxol, baccatin III, cephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine, 10-deacetyltaxol and the like.

The taxane-type diterpene-producing plants used in the tissue culture of the present invention include, for example, common yew (Taxus baccata LINN) and yew (T. cuspidata SIE).
B.etZUCC), Carabok (T. cuspidata SIEB.et ZUCC v
ar. nana REHDER), T. brevifolia
NUTT), Canadian yew (T. canadiensis MARSH), Chinese yew (T. chinensis), T. media, etc.

The tissue culture of the plant is carried out by adding jasmonic acid and its derivative, that is, the compound represented by the general formula [I] according to the present invention, or a treatment for promoting the production of endogenous jasmonic acid. Other than the above, it can be performed by a conventionally known method.

In the compound represented by the general formula [I], the alkyl group having 1 to 4 carbon atoms represented by R ¹ , R ⁷ or R ⁸ is, for example, a methyl group, an ethyl group or an n-propyl group. , I-propyl group, n-butyl group, isobutyl group, sec-butyl group, t-butyl group can be exemplified.

When R ⁶ is OM, examples of the alkali metal atom or the alkaline earth metal atom represented by M include sodium, potassium and calcium.

In the case where R ⁶ is NHR ⁷ , R ⁷
The acyl group having 1 to 4 carbon atoms represented by may be linear or branched, and examples thereof include a formyl group, an acetyl group, a propionyl group, a butyryl group, and an acryloyl group.

In the case where R ⁶ is NHR ⁷ , R ⁷
Examples of the amino acid residue represented by are isoleucyl group, tyrosyl group, and tryptophyll group.

When R ⁶ is OR ⁸ , the carbohydrate residue represented by R ⁸ includes a glucopyranosyl group.

In the compound represented by the general formula [I], the 5-membered ring may be further substituted with a hydroxyl group,
A double bond may be formed between adjacent ring member carbon atoms.

Specific examples of the compound represented by the general formula [I] include the compounds shown below. Among them, cucurbic acid or methyl cucurbitate is particularly preferable in terms of the size for improving the productivity of the taxane-type diterpene. preferable.

[0018]

[Chemical 5]

[0019]

[Chemical 6]

[0020]

[Chemical 7]

[0021]

[Chemical 8]

The compound represented by the general formula [I] has various stereoisomers (cis trans isomer, optical isomer), and each isomer may be used alone or in the form of a mixture. You may use.

The compounds represented by the general formula [I] are
Prepared synthetically or by extraction from plants (H. Yamane et al. Agric. Biol. Chem. 44 , 2857-286.
4 (1980).

On the other hand, jasmonic acid is a plant hormone-like substance that induces various reactions involved in growth promotion, tissue maturation, and expression of disease resistance. Engineering Vol. 2, No. 4, pp. 523-531 (1990).

Therefore, the jasmonic acids according to the present invention are
In addition to being added from outside the culture system, the cells or culture tissues to be used can be produced by themselves. As a method of promoting the production of the endogenous jasmonic acids by the cultured cells or the cultured tissue, the culture of the microorganism or the extract thereof, the addition of the heat-treated product or the plant extract to the medium can be exemplified, and specifically, Is MJ Mueller et al., Proc. Nat
The method of adding the mold cell wall fraction described in I. Acad. Sci. USA, 90 (16), 7490-7494 (1993) can be exemplified. Furthermore, the production amount of endogenous jasmonic acid can be increased by mechanically or partially damaging the cultured cells or tissue to be used with ultraviolet rays, heat, etc. et al.,
Proc. Natl. Acad. Sci. USA, 89 (11), 4938-4941
(1989), a method of mechanically destroying a part of cells can be exemplified.

Since the compound represented by the general formula [I] is poorly soluble in water, it is usually dissolved in an organic solvent such as ethanol or methanol or a surfactant, and then added to the medium. The free form of the compound may be used as it is, or may be neutralized with an alkali to form a salt.

The compound represented by the general formula [I] needs to have a concentration of 0.01 to 1000 μM in the medium,
Among these, it is particularly preferable for the method of the present invention to adjust the concentration to the range of 0.1 to 500 μM.

[0028] The addition of jasmonic acid to plant cell cultures to induce secondary metabolites is a German patent publication [DE
4122208C1], however, in tissue culture of taxane-type diterpene-producing plants, there has been no report of tissue culture in the presence of jasmonic acids in the medium, and as a result, the amount of taxane-type diterpene produced was increased. The increase was unexpected.

The medium used in the present invention is a conventionally known medium used for tissue culture of plants, such as Murashige & Skoog (1962).
Medium of Rinsmeyer Skoog (1965) [Linsmaie
r Skoog's Medium, Woody Plant Medium
(1981) [Woody Plant Medium] medium, Gamborg B-5 medium, Mitsui M-9 medium and the like. A plant hormone may be added to these media, and if necessary, a carbon source, an inorganic component, vitamins, amino acids and the like may be added.

As the carbon source, disaccharides such as sucrose, maltose and lactose, monosaccharides such as glucose, fructose and galactose, starch, or a mixture of two or more of these sugar sources in an appropriate ratio can be used.

Examples of the inorganic component include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt. These components are, for example, Potassium nitrate, sodium nitrate, calcium nitrate,
Potassium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, It can be added as a compound such as molybdenum trioxide, potassium iodide, or cobalt chloride.

Examples of plant hormones include indole acetic acid (IAA), naphthalene acetic acid (NAA) and 2,4-
Auxins such as dichlorophenoxyacetic acid (2,4-D) and cytokinins such as kinetin, zeatin and dihydrozeatin are used.

Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B).
6), pantothenic acid, inositol, nicotinic acid, etc. are used.

As amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added.

Generally, each of the above components contains about 0.1% of inorganic components.
[mu] M to 100 mM, a carbon source of about 1 to about 30 g / l, plant hormones of about 0.01 to about 10 [mu] M, vitamins and amino acids at concentrations of about 0.1 to about 100 mg / l, respectively.

In the present invention, either a liquid medium or a solid medium containing 0.1% to 1% of agar or gellan gum can be used, but the liquid medium is usually preferable.

In the tissue culture of the present invention, the plant body of the above-mentioned plant, for example, roots, growing points, leaves, stems, seeds, pollen, anthers, sepals and the like tissue pieces or cells are used in the above-mentioned medium or other conventional medium. Cultured cells obtained by tissue culture can be used.

When these tissues or cells were tissue-cultured using a medium to which the compound represented by the general formula [I] was added according to the present invention, the taxane-type diterpene highly-producing culture tissues or Cultured cells are obtained.

The taxane-type diterpenes can be separated from the cultures such as the culture tissues, culture cells, and media obtained as described above by extraction with an organic solvent such as methanol. Further, taxon-type diterpenes can be continuously recovered by coexisting an appropriate adsorbent or an organic solvent in the medium.

A preferred example of the tissue culture of the present invention is the following method.

First, plant pieces of plants belonging to the genus Taxus, for example, plant pieces collected from roots, growing points, leaves, stems, seeds, etc. are sterilized and then placed on Woody Plant Medium solid medium solidified with gellan gum. Place it on the floor and at 10-35 ° C for 14-
After about 60 days, a part of the tissue piece is turned into callus. When the callus thus obtained is subcultured, the growth rate gradually increases and a stable callus is obtained. The term "stabilized callus" as used herein refers to one in which a part of the callus does not differentiate into shoots or roots during culture and retains the state of the callus and has a uniform cell growth rate.

The stabilized callus is transferred to a liquid medium suitable for growth, for example, a liquid medium of Woody Plant Medium and allowed to grow. The growth rate is further increased in the liquid medium. In the present invention, the stabilized callus or cells constituting the callus is cultured in a solid medium or a liquid medium containing the compound represented by the general formula [I].
The stabilized callus or the cells constituting the callus are divided into a plurality of layers according to the difference in specific gravity, and the cells contained in at least one layer are cultured in a medium containing the compound represented by the general formula [I]. May be.

As a method for separating cells by specific gravity, generally, a density gradient is prepared by using a medium for centrifugation,
A method in which cells are layered and then centrifuged is known.
Ficoll and Percoll (both Pharma
cia LKB Biotechnology), sucrose, cesium chloride and the like are used. In Example 5, the density gradient was created using Ficoll, but there is no particular limitation as long as it does not damage the cells.

There is no particular limitation on the number of layers forming the density gradient. The specific gravity difference between the layers is not particularly limited, and the specific gravity differences may be the same or different. Therefore, the definition of the density gradient includes the case where the gradient changes continuously (the number of layers forming the density gradient is infinite, and the difference in specific gravity between layers is close to 0).

By thus forming a density gradient and superposing the cells on one another and centrifuging, the cells can be divided into a plurality of layers according to the difference in specific gravity. The specific gravity of the formed layer is usually 1.00 to 1.20 g / ml, preferably 1.03 to 1.11 g / ml. As the layer to be cultured, at least one layer may be selected, or all layers may be selected and cultured.

When a plurality of layers are selected and cultured, these plurality of layers can be individually cultured, but two or more layers of the selected plurality of layers are mixed and cultured. You can also

Cultured cells having a high taxane-type diterpene-producing ability are usually obtained by culturing cells contained in a layer having a specific gravity of 1.07 or less, but this may vary depending on the cells to be cultivated and the culturing conditions. It is not limited to the range. Further, it is recognized that the cells in the layer having a high specific gravity tend to have a higher taxane-type diterpene content simply by fractionating the cells by the difference in the specific gravity. Therefore, in order to more reliably obtain the taxane-type diterpene high-producing cultured cells, after culturing the cells of all the fractionated layers for a certain period of time, the taxane-type diterpene concentration contained in the cells of each layer was measured, and Among these, it is desirable to select a layer containing highly taxane-type diterpene-producing cells.

In the present invention, a centrifuge medium having a specific gravity of 1.07 g / ml is prepared,
The cultured cells can also be divided into a plurality of layers according to the difference in specific gravity by performing centrifugation according to the above method.

The compound represented by the general formula [I] is most effectively added to the cultured cells during the growth phase or stationary phase. Among them, the compound represented by the general formula [I] is most effective at the time of transition to the growth phase or stationary phase. ] It is preferable for the method of this invention to add the compound shown by these. For example, when cells are transplanted every 21 days, the 7th to 16th days are represented by the general formula [I].
This is an appropriate period for the addition of the compound represented by. As a method of addition, a predetermined amount may be added at once, or may be added in a plurality of times and sequentially added.

The culture temperature of the tissue culture in the present invention is preferably about 10 to about 35 ° C., particularly about 23 to 28 ° C., because the growth rate is high. The culture period is 14 ~
42 days is preferred.

When a liquid medium is used in the culture of the present invention, the cultured cells are separated from the medium by a method such as decantation or filtration after the completion of the culture, and the taxanes produced and accumulated from the separated cultured cells and / or the culture medium. The type diterpenes can be separated by a method such as extraction with an organic solvent.

[0052]

EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples.

[Example 1] 2% antiformin solution or 70% ethanol solution was previously added to Woody Plant Medium solid medium (gellan gum 0.25% by weight) to which naphthalene acetic acid was added at a concentration of 10 ^-5 M. A part of the stem of Taxus baccata LINN, which had been sterilized by the above method, was placed and cultured at 25 ° C. in the dark to obtain Taxus callus. Next, 1g of this callus (fresh weight),
Woody plant with the above components added at the same concentration
Transfer to medium Erlenmeyer flask containing 20 ml of liquid medium,
Spin culture on a rotary shaker (amplitude 25 mm, 120 rp
m) and planted every 21 days to accelerate the growth rate of the callus.

1 g of the cultured cells (fresh weight) thus obtained was added to Woody
The culture medium was transferred to an Erlenmeyer flask containing 20 ml of liquid medium of Plant Medium and cultured at 25 ° C. for 14 days with shaking. On the 14th day of culture, methyl ester of cucurbic acid as jasmonic acid [in the above formula (I), R ¹ is CH ₂ COOCH ₃ , R ² and R ³ are H, and R ⁴ and R ⁵ are combined. Double bond forming compound) with a final concentration of 0.01 ~ 1000μ
M was added thereto, and the cells were further cultured for 7 days.

After the completion of the culture, the Taxus vulgaris cultured cells were collected by filtration, freeze-dried, and the dry weight thereof was measured to determine the growth weight of the cultured cells per liter of the liquid medium. The taxane-type diterpene was extracted from the obtained dry callus with methanol and the like, and the taxane-type diterpene yield was measured by comparative quantification with standard products taxol, cephalomannine and baccatin III using high performance liquid chromatography. The results are shown in Table 1.

Comparative Example 1 The procedure of Example 1 was repeated except that the methyl ester of cucurbic acid was not added. The results are shown in Table 1.

[Example 2] In Example 1, the methyl ester of cucurbic acid was added every 4 days from the 7th day of culture for a total of 4 times.
Sequential addition (final concentration 25 μM per time, total 100 μM
The same operation as in the example was carried out except that M) was performed. The results are shown in Table 1.

Example 3 In Example 1, 100 μM of the methyl ester of cucurbic acid was added on the first day of culture,
The same operation was performed as in the example except that the culture was continued for 20 days. The results are shown in Table 1.

Example 4 In Example 1, 100 μM of the methyl ester of cucurbic acid was added on the 7th day of culture,
The same operation as in the example was carried out except that the culture was continued for 14 days. The results are shown in Table 1.

[0060]

[Table 1]

[Example 5] The cells having an increased growth rate obtained by the method of Example 1 were first separated by a stainless mesh into cell clumps having a size of 250 to 840 µm. Next, using Ficoll, a medium having a density of 1.07 (g / ml) was prepared, and the cells were overlaid and centrifuged at 700 rpm for 6 minutes. The cells were separated into two layers according to the difference in specific gravity. 1.
The cells contained in the layer of 07 g / ml or less were fractionated and washed with 2% sucrose solution at least 3 times to wash away Ficoll. After washing, 1 g of cells (fresh weight) was transferred to an Erlenmeyer flask containing 20 ml of Woody Plant Medium liquid medium, and cultured by shaking at 25 ° C. for 14 days. On the 14th day of culturing, methyl ester of cucurbic acid was added so that the final concentration thereof would be 250 μM, and the cells were further cultured for 7 days. After the culture was completed, the same operation as in Example 1 was performed. The results are shown in Table 2. The productivity of taxane-type diterpenes could be significantly improved by combining the selection of cells with a specific gravity and the addition of cucurbic acid methyl ester.

Comparative Example 2 The procedure of Example 5 was repeated except that the methyl ester of cucurbic acid was not added. The results are shown in Table 2.

[0063]

[Table 2]

Example 6 250 μM of methyl ester of cucurbic acid was added to the cultured cells of Taxus communis (day 14 of culture) obtained by the same method as in Example 1 and cultured for 7 days. After the completion of the culture, the same operation as in the example was performed. The results are shown in Table 3.

Comparative Example 3 The procedure of Example 6 was repeated except that the methyl ester of cucurbic acid was not added. The results are shown in Table 3.

[Embodiment 7] me
The procedure was the same as in the example except that the dia culture cells were used. The results are shown in Table 3.

Comparative Example 4 The procedure of Example 7 was repeated except that the methyl ester of cucurbic acid was not added. The results are shown in Table 3.

[0068]

[Table 3]

[0069]

According to the present invention, a large amount of taxane-type diterpenes can be easily obtained by culturing the tissues of taxane-type diterpene-producing plants in the presence of jasmonic acids.

Claims (9)

[Claims] 1. A plant producing a taxane-type diterpene
The cells or tissues can be expressed by the general formula [I]:[In the formula, R¹Is an alkyl group having 1 to 4 carbon atoms or the following formula:₂ )_n-CO-R⁶ {In the formula, R⁶Is a hydroxyl group, OM (where M is an alkali metal
Group atom, alkaline earth metal atom, or NH_FourThe table
You ), NHR⁷(Where R⁷Is hydrogen atom, carbon number 1
To an acyl group having 1 to 4 carbon atoms, an alkyl group having 1 to 4 carbon atoms, or an amino group.
Represents an acid residue. ), Or OR⁸(Where R⁸Is carbon
It represents an alkyl group of the numbers 1 to 4, or a carbohydrate residue. )
Where n represents an integer of 1 to 7. } Shows the group shown by
Do; R², R³, R^Four, And R^FiveAre hydrogen atoms
Yes or R²And R ³, R³And R^Four, Or R^FourWhen
R^FiveMay together represent a double bond; said 5-membered ring
May be further substituted with a hydroxyl group, and adjacent ring members
A double bond may be formed between carbon atoms. ]
Cultivated in the presence of the compound, the resulting culture yields taxane
Taxane-type diterpenes characterized by recovering the type diterpene
Method for manufacturing terpenes. 2. The taxane-type diterpene is taxol,
Baccatin III, cephalomannine, 10-deacetylbaccatin III, 10-deacetylcephalomannine and / or 1
The method for producing a taxane-type diterpene according to claim 1, which is 0-deacetyltaxol. 3. The compound represented by the general formula [I] is
The method for producing a taxane-type diterpene according to claim 1, which is cucurbic acid or methyl cucurbitate. 4. The method for producing a taxane-type diterpene according to claim 1, wherein the taxane-type diterpene-producing plant is a Taxus genus plant. 5. The yew plant is a taxus (Taxus).
baccata LINN), yew (T. cuspidata SIEB.et ZUC
C), Karaboku (T. cuspidata SIEB.et ZUCC var. Nan
a REHDER), T. brevifolia NUT
The method for producing a taxane-type diterpene according to claim 4, which is selected from T), Canadian yew (T. canadiensis MARSH), Chinese yew (T. chinensis), and T. media. 6. The method for producing a taxane-type diterpene according to claim 1, wherein the concentration of the compound represented by the general formula [I] in the tissue culture medium is 0.01 to 1000 μM. 7. A compound represented by the general formula [I]
The method for producing a taxane-type diterpene according to claim 1, which is added during the growth phase or stationary phase of the cultured cells. 8. A compound represented by the general formula [I]
The method for producing a taxane-type diterpene according to claim 1, wherein the taxane-type diterpene is added to the culture medium in a plurality of times. 9. The taxane-type diterpene according to claim 1, wherein the cells of the plant producing the taxane-type diterpene are divided into a plurality of layers according to the difference in specific gravity, and the cells contained in at least one layer are cultured. Production method.