JP2006109784A - Method for producing paclitaxel - Google Patents

Abstract

【Task】
An object of the present invention is to provide a method for more efficiently producing paclitaxel by cell culture of yew plants.
[Solution]
In the production of paclitaxel by cell culture of yew plants, the productivity of paclitaxel can be significantly increased by adding and culturing an aliphatic triglyceride having an octanol / water partition coefficient (log P) of less than 4 in a culture tank.
[Selection]
None

Description

  The present invention relates to an improved method for increasing culture productivity in a method for producing paclitaxel by cell culture of yew. Paclitaxel is an important substance as an anticancer agent against ovarian cancer, lung cancer and the like.

  Paclitaxel (trade name Taxol, etc.) is a substance obtained from the bark and needles of plants of the genus Taxus, which stabilizes eukaryotic microtubules and inhibits depolymerization, thereby suppressing cell division. And has a strong antitumor activity against cancer cells such as ovarian cancer and lung cancer. Although the demand is increasing year by year, the amount of paclitaxel obtained by direct extraction from bark and needles collected from yew plants is limited due to the relationship with the amount of resources depending on the vegetation and growth rate of yew plants. It has been. For this reason, establishment of the manufacturing method of the mass and cheap paclitaxel which responds to the increasing demand is desired.

  In general, in addition to the method obtained by directly extracting from the above-mentioned yew plant, the method for producing paclitaxel is a method by complete chemical synthesis, a method for chemically modifying precursors such as baccatin, or the production of paclitaxel. A method of culturing cells is mentioned. Conventionally, commercial production of paclitaxel has been carried out mainly by a semi-synthetic method in which paclitaxel is synthesized by chemically modifying a precursor extracted from yew needles. In this case, the amount of paclitaxel produced depends on the plant resources that produce the needles, and there is a problem in that supply fluctuations occur due to the influence of weather conditions and pests.

  Recently, various studies have been made on such problems in order to produce them in large quantities and at low cost by culturing cells (callus) of yew plants (for example, see Patent Documents 1 and 2). The callus culture method has the advantage that callus can be easily derived from a small amount of plant fragments, and can be cultured stably and in large quantities because it is not affected by natural factors such as seasonal fluctuations and the occurrence of pests. . Furthermore, since the kind and amount of the target component and the mixed component contained in the product can be stabilized, the product extraction and purification process can be simplified. Thus, the culture production method using callus is a production method expected as a production method of paclitaxel.

  However, the problem of paclitaxel culture production by callus is that the paclitaxel itself released and accumulated in the culture medium inhibits the growth of callus and paclitaxel production, so the production rate of paclitaxel per unit culture medium Further, there is a problem that the productivity of paclitaxel per cell is not sufficiently increased. As a means for solving this problem, i. A method of continuously supplying and extracting a medium and recovering paclitaxel therefrom (for example, see Patent Document 3), ii. A method of improving the productivity of paclitaxel by adding an organic solvent that is not mixed with water (medium) into a culture tank and culturing while paclitaxel secreted in the medium is dissolved in the organic solvent phase. Is known (for example, see Non-Patent Documents 1 and 2).

In particular, in a method in which an organic solvent is added to a culture solution and culture is performed in a two-phase system, the paclitaxel production process can be simplified by simultaneously performing the production of paclitaxel and the paclitaxel transfer to the solvent in a culture tank. So far, it has been shown that solvents such as tricaprylin (for example, see Non-Patent Document 1), hexadecane, and dibutyl phthalate (for example, see Non-Patent Document 2) improve the productivity of paclitaxel. Conditions required for the organic solvent used in this method include that the cytotoxicity to callus is as low as possible and that the effect of improving paclitaxel productivity is large.
As a conventional example of such examination, there is an example in which the relationship between the cytotoxicity of an organic solvent to callus and the octanol / water partition coefficient (log P), which is an index of hydrophobicity of the organic solvent, is examined, and the partition coefficient is low. In order to avoid the cytotoxicity against yew callus and improve the productivity in the study on the production of paclitaxel, it is necessary to use an organic solvent having a partition coefficient of 5 or more. (For example, refer nonpatent literature 3). However, the amount of paclitaxel produced by this method using an organic solvent having a conventional octanol / water partition coefficient (log P) of 5 or more is as low as 2 to 5 times that when no organic solvent is added. Further, there is a demand for a solvent that improves productivity and a method for using the same.

Japanese Patent No. 2967034 Japanese Patent No. 3169968 JP 7-255495 A M. Collins-Pavao et al., Journal of Biotechnology 49, 95-100 (1996) Chuangui Wang et al., Biotechnol. Prog. 17, 89-94 (2001) Zhao-Liang Wu et al., Biochemical Engineering Journal 5, 137-142 (2000)

  An object of the present invention is to solve the above-mentioned problems in the prior art and to provide a method for more efficiently producing paclitaxel by cell culture of yew plants.

In the production of paclitaxel by callus culture of yew plants, the present inventors have conducted extensive research on the type of organic solvent added to the culture solution and the method of extracting it, and in addition to that, the octanol / water partition coefficient (log P) is After adding one or more selected from fatty acid triglycerides, particularly triacetin, tripropionine, and tributyrin, as organic solvents to the culture solution, the organic solvent phase is separated and extracted to greatly increase the productivity of paclitaxel. As a result, the present invention has been completed.
That is, the present invention provides fatty acid triglycerides having an octanol / water partition coefficient (log P) of less than 4 in a culture tank in the production of paclitaxel by cell culture of yew plants (1) to (5) It relates to a method for producing paclitaxel shown in FIG.
(1) In the production of paclitaxel by cell culture of yew plants, the cell culture is performed in a culture solution to which an aliphatic triglyceride having an octanol / water partition coefficient (log P) of less than 4 is added. Production method.
(2) The method for producing paclitaxel according to (1), wherein the fatty acid triglyceride having an octanol / water partition coefficient (logP) of less than 4 is one or more selected from triacetin, tripropionine, and tributyrin.
(3) Fatty acid triglyceride having an octanol / water partition coefficient (log P) of less than 4 is added to the culture solution in a saturated amount or more, and the cell culture is performed in a state where the triglyceride and the culture solution form two phases of an organic solvent phase and an aqueous phase. The method for producing paclitaxel according to (1) or (2).
(4) The method for producing paclitaxel according to any one of (1) to (3), wherein an aliphatic triglyceride having an octanol / water partition coefficient (log P) of less than 4 is continuously or intermittently added to the culture solution.
(5) The method for producing paclitaxel according to any one of (1) to (4), wherein an organic solvent phase containing a triglyceride having an octanol / water partition coefficient (logP) of less than 4 is fractionated continuously or intermittently.

  According to the present invention, triglyceride with a saturation concentration or higher is added to the culture medium, and the triglyceride phase containing paclitaxel can be extracted continuously or intermittently to prevent product inhibition by paclitaxel, thus promoting callus growth rate. In addition to being able to increase the production speed of paclitaxel itself, it is possible to efficiently produce a large amount of paclitaxel.

  The present invention will be described in detail below. Examples of yew plants used for the production of paclitaxel in the present invention include yew (Taxus cuspidata), yew (Taxus baccata), yew (Taxus brevifolia), Chinese yew (Taxus chinensis), tax me Illustrated.

  Known tissue culture methods can be used to obtain callus from yew plants. The type and concentration of the medium and plant hormone used to induce callus from the plant body may be appropriately selected as necessary. For example, when callus is obtained from needle cells of yew plants, auxin such as 2,4-dichlorophenoxyacetic acid or naphthaleneacetic acid, or auxin and cytokinin is added to a solid medium usually used in tissue culture. Cells are cultured using a medium to obtain callus.

  The obtained callus is then cultured in a liquid medium. The liquid medium to be used is based on the liquid medium usually used in tissue culture, and the liquid medium prepared by adding 0.1 to 5 mg / L of auxin such as 2,4-dichlorophenoxyacetic acid or naphthalene acetic acid is cultured. Used as a liquid.

  As a method of adding an aliphatic triglyceride having an octanol / water partition coefficient (log P) of less than 4 to a culture solution, a culture solution obtained by culturing in the above-mentioned medium is used as a preculture solution, and a part of the triglyceride is added. You may transplant to the liquid medium contained, or you may add a triglyceride directly to a preculture liquid. The triglyceride to be added is added so as to have a saturation concentration or higher with respect to the medium, and cultured in a state separated into two phases of the medium and the organic solvent.

Attempts have been made to improve the productivity of paclitaxel by supplying an organic solvent to the medium. In general, organic solvents having an octanol / water partition coefficient (log P) of less than 4 are highly toxic to cells. It was considered unsuitable for the production of paclitaxel because it inhibits the growth of The octanol / water partition coefficient (logP) of the organic solvent mentioned here means that a certain amount of a test substance is dissolved in 1-octanol and added to the two solvent phases of 1-octanol and water and mixed well. This is the distribution coefficient obtained by the following equation after separating into two phases and measuring the test substance concentration in each phase.
P = C _o / C _w
Co : Test substance concentration in 1-octanol layer (mol / L)
C _w : Test substance concentration in the water layer (mol / L)

Examples of the triglyceride added to the culture solution include the compounds shown in Table 1, but are not limited to these glycerides as long as they meet the object of the present invention. For comparison, distribution coefficients of substances other than triglyceride are also shown.
Table 1 Examples of organic solvent partition coefficients (octanol / water) (Note)

Compound molecular formula logP
Triglyceride
Triacetin C ₉ H ₁₄ O ₆ 0.36
Tripropionine C ₁₂ H ₂₀ O ₆ 1.8
Tribtilin C ₁₅ H ₂₆ O ₆ 3.3
Tricaproine C ₂₁ H ₃₈ O ₆ 6.3
Tricaprylin C ₂₇ H ₅₀ O ₆ 9.2
Fatty acid alcohol
Lauryl alcohol CH ₃ (CH ₂ ) ₁₁ OH 5.0
hydrocarbon
Hexane C ₆ H ₁₄ 3.6
Heptane C ₇ H ₁₆ 4.0
Nonane C ₉ H ₂₀ 5.1
Decane C ₁₀ H ₂₂ 5.6
Dodecane C ₁₂ H ₂₆ 6.6
Pentadecane C ₁₅ H ₃₂ 8.2
Hexadecane C ₁₆ H ₃₄ 8.7
Octadecane C ₁₈ H ₃₈ 9.8
Aromatic hydrocarbons
Ethylbenzene C ₈ H ₁₁ 3.1
Benzene C ₆ H ₆ 2.0
Toluene C ₇ H ₈ 2.5

(Note) Distribution coefficients are literature values or calculated values (Syracuse Research)
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  In yew cell culture, an organic solvent having an octanol / partition coefficient (log P) of less than 4, for example, an aliphatic hydrocarbon compound such as hexane was supplied to the medium as an organic solvent, and the effect was examined. Compared to the case, cell growth was inhibited, and the productivity of paclitaxel was also reduced. In contrast, nonane, decane, dodecane, hexadecane, and octadecane, which are higher aliphatic hydrocarbon compounds having a large number of carbon atoms, tend to improve the cell yield as the distribution coefficient increases, and carbonization with a distribution coefficient of 6 or more. The addition of the hydrogen compound clearly showed the effect of promoting cell yield compared to the case of no addition. However, the promotion effect when a higher aliphatic hydrocarbon having a partition coefficient of 5 or more, triacylglyceride, and fatty acid alcohol is added is within the range of conventional knowledge.

  In contrast to this, triacetin, which is a triglyceride having a partition coefficient of 0.36, showed a significantly higher cell yield promoting effect than when the above-described higher aliphatic hydrocarbon compound was added. In addition, triacylglycetripropionin and tributyrin, which have a higher partition coefficient than triacetin, also improve cell proliferation compared to tricaproin and tricaprylin. It was higher than lauryl alcohol.

  Organic compounds with a low partition coefficient are relatively soluble in water. However, in the case of triglycerides, cytotoxicity to plant cultured cells is not observed even if triglycerides coexist in the culture solution. There is no problem in the method of recovering paclitaxel dissolved in the two-phase separated triglyceride present as the organic solvent phase while continuously or intermittently extracting the organic solvent phase.

  The amount of triglyceride added to the culture solution is not particularly limited, but it is desirable to add the triglyceride so that the culture solution is saturated or more and the culture solution and triglyceride form two phases. In addition, after paclitaxel is secreted from the callus into the culture solution, and after paclitaxel is diverted from the culture solution to the organic solvent phase, the process of separating the organic solvent phase containing paclitaxel is performed efficiently, and paclitaxel In order to increase the productivity, the amount of triglyceride added and the amount of organic solvent phase extracted should be adjusted continuously or intermittently so that the volume ratio of the culture liquid phase to the organic solvent phase in the culture tank is in a specific range. It is more desirable from the viewpoint of production efficiency to culture while adjusting.

The organic solvent phase extracted from the culture tank is separated and recovered by using an extraction method using an organic solvent that utilizes the difference in solubility between paclitaxel and impurities, or a column separation method using a molecular sieve, porous resin, ion exchange resin, etc. can do.

EXAMPLES Hereinafter, although this invention is demonstrated more concretely with an Example and a comparative example, this invention is not limited by these examples. In addition, the amount of paclitaxel in the callus cells, the medium, and the organic solvent phase was measured using an HPLC method using a reverse phase column.
Example 1
Kasul cells are induced from needles of Taxus baccata, modified Gamborg B5 agar medium (potassium nitrate concentration is reduced to 10 mM, ammonium sulfate is not added, sucrose 20 g / L, casamino acid 3 g / L, naphthalene acetic acid 2 mg Was added to a 100 ml Erlenmeyer flask, and cultured under rotary shaking at 25 ° C. for 10 to 14 days in order to increase the growth of callus cells.
The callus cells thus prepared were aseptically isolated and collected, and planted in a 35 ml L-shaped test tube charged with a modified Gamborg B5 agar medium and triacetin. Incubator was cultured for 10 days in the dark at 25 ° C. and 55 r / min. The triacetin added to the medium was aseptically filtered through a 0.22 μm membrane filter.
After completion of the culture, the whole culture broth was extracted using an aspirator, and then callus cells and other liquid phases were separated using a membrane filter, and subjected to measurement of callus live weight and paclitaxel amount.
The medium phase recovered as the other liquid phase and the organic solvent phase consisting of added triacetin were each collected with a glass pipette. Next, the organic solvent phase was mixed with a solvent of cyclohexane: acetone = 77: 23 (volume ratio), and then the lower aqueous phase was separated. Furthermore, the upper layer of cyclohexane was washed and extracted with the same volume of water, and both aqueous phases were combined. Dichloromethane was added thereto and mixed, and the dichloromethane phase was separated. This dichloromethane phase was dried with a centrifugal vacuum dryer and redissolved in methanol to prepare a sample for HPLC analysis.
On the other hand, the aqueous phase after removing the organic solvent phase was mixed with dichloromethane, and then the dichloromethane phase was separated, dried in a centrifugal vacuum dryer, and redissolved in methanol to prepare a sample for HPLC analysis. The paclitaxel production amount is the total production amount in the medium, and is the sum of the paclitaxel production amounts of the aqueous phase and the organic solvent phase. In the callus used in this study, most of the produced paclitaxel was present in the medium, so that paclitaxel in the callus was negligible.
As a result, the raw weight of the callus cells was 41.6 g / L, the paclitaxel concentration in the medium was 4.4 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of triacetin was 160 μg / L.

Comparative Example 1
The same procedure as in Example 1 was performed except that triacetin was not added. As a result, the raw weight of callus was 34.0 g / L, and the paclitaxel concentration in the medium was 14.2 μg / L.

Comparative Example 2
The same procedure as in Example 1 was performed except that hexane was added instead of triacetin. As a result, the raw weight of callus cells was 31.6 g / L, the paclitaxel concentration in the medium was 12.5 μg / L, and the amount of paclitaxel in the organic solvent phase consisting of hexane was below the detection limit.

Comparative Example 3
The same procedure as in Example 1 was performed except that lauryl alcohol was added instead of triacetin. As a result, the raw weight of callus cells was 40.5 g / L, the paclitaxel concentration in the medium was 7.7 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of lauryl alcohol was 32.4 μg / L.

Comparative Example 4
The same procedure as in Example 1 was conducted except that nonane was added instead of triacetin. As a result, the raw weight of the callus cells was 32.4 g / L, the paclitaxel concentration in the medium was 10.3 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of nonane was 10.7 μg / L.

Comparative Example 5
The same procedure as in Example 1 was performed except that decane was added instead of triacetin. As a result, the raw weight of the callus cells was 33.9 g / L, the paclitaxel concentration in the medium was 3.9 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of decane was 51.2 μg / L.

Comparative Example 6
The same procedure as in Example 1 was performed except that dodecane was added instead of triacetin. As a result, the raw weight of the callus cells was 37.3 g / L, the paclitaxel concentration in the medium was 8.6 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of dodecane was 18.4 μg / L.

Comparative Example 7
The same procedure as in Example 1 was performed except that hexadecane was added instead of triacetin. As a result, the raw weight of the callus cells was 40.5 g / L, the paclitaxel concentration in the medium was 2.4 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of hexadecane was 11.5 μg / L.

Comparative Example 8
The same procedure as in Example 1 was performed except that tricaprylin was added instead of triacetin. As a result, the raw weight of callus cells was 39.1 g / L, the paclitaxel concentration in the medium was 6.3 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of tricaprylin was 27.0 μg / L.

Comparative Example 9
The same procedure as in Example 1 was performed except that octadecane was added instead of triacetin. As a result, the raw weight of callus cells was 39.9 g / L, the paclitaxel concentration in the medium was 7.3 μg / L, and the paclitaxel concentration in the organic solvent phase consisting of octadecane was 26.4 μg / L.

Claims (5)

  A method for producing paclitaxel, characterized in that in the production of paclitaxel by cell culture of yew plants, the cell culture is carried out in a culture solution to which an aliphatic triglyceride having an octanol / water partition coefficient (log P) of less than 4 is added.   The method for producing paclitaxel according to claim 1, wherein the fatty acid triglyceride having an octanol / water partition coefficient (logP) of less than 4 is at least one selected from triacetin, tripropionine, and tributyrin.   A fatty acid triglyceride having an octanol / water partition coefficient (log P) of less than 4 is added to the culture solution in a saturated amount or more, and cell culture is performed in a state where the triglyceride and the culture solution form two phases of an organic solvent phase and an aqueous phase. The manufacturing method of the paclitaxel of Claim 1 or 2.   The method for producing paclitaxel according to any one of claims 1 to 3, wherein an aliphatic triglyceride having an octanol / water partition coefficient (log P) of less than 4 is continuously or intermittently added to the culture solution.   The method for producing paclitaxel according to any one of claims 1 to 4, wherein an organic solvent phase containing a triglyceride having an octanol / water partition coefficient (log P) of less than 4 is fractionated continuously or intermittently.