JPH07255495A - Taxol manufacturing method - Google Patents

Abstract

(57) [Abstract] [Purpose] It is an object of the present invention to provide a production method capable of mass-producing taxanes including taxol and derivatives thereof on an industrial scale. [Constitution] When culturing cells or groups of Taxus plants, the liquid medium is intermittently or continuously replaced with a fresh medium, and the taxol-containing taxane product contained in the medium used for the culturing is produced. Is a method for producing taxol.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing taxanes containing taxol using cultured cells of Taxus plants. More specifically, the present invention, when culturing cells or groups of Taxus plants, liquid medium is intermittently or continuously exchanged with a fresh medium, the product contained in the medium used for culture The present invention relates to a method for increasing the yield of taxanes containing taxol by recovering taxol. INDUSTRIAL APPLICABILITY According to the present invention, taxanes such as taxol having antitumor activity and derivatives thereof can be produced in large amounts, and thus the pharmaceutical industry is greatly benefited.

[0002]

2. Description of the Related Art Taxol is a diterpene amide produced by plants of the genus Taxus and has antitumor activity. Its mechanism of action is unique in that it stabilizes microtubules and inhibits their depolymerization, thereby inhibiting cell division. This taxol is for ovarian cancer,
It has been reported that it has a remarkable effect on breast cancer, and is expected to have an effect on lung cancer and malignant melanoma. As described above, taxanes containing taxol are extremely useful as natural anticancer agents or raw materials for synthesizing anticancer agents.

As a raw material for taxol, it has been considered to use bark of a Taxus genus plant such as Taxus breviforia: Pacificyew. However, its taxol content is approximately 0.0 of dry weight.
It is 1%, and yew plants are extremely slow-growing plants.
Two or three adult trees aged 60 years or older are required to produce g taxol. Therefore, utilizing the bark of the Taxus plant as a raw material of taxol has many problems from the viewpoint of stable supply of raw material and protection of plant resources.

Therefore, attempts have been made to produce taxol by a chemical synthesis method, but the yield is extremely low and mass production is difficult. On the other hand, semi-synthesis of taxol from a precursor of taxol (10-deacetylbaccatin III and the like), or synthetic studies of analogues such as taxotere are underway. A plant cell culture method (tissue culture method) is required because a source of plant raw materials is required even if the semi-synthesis method is adopted.
Mass production of taxanes including taxol has been attempted.

[0005]

However, when cells of the genus Taxus are used and the cells are to be cultured by the tissue culture method, the growth rate of the cells of the taxus and the group of cells in the culture solution in culture is Since it is extremely slow and the amount of taxanes and other taxanes accumulated is small, it has not been possible to mass-produce it on an industrial scale.

The present invention has been made in view of the above circumstances, and it is possible to mass-produce taxols and their derivatives containing taxol on an industrial scale by improving the productivity of taxol by improving the liquid culture method. The purpose is to provide a possible manufacturing method.

[0007]

Means for Solving the Problems The inventors of the present invention conducted a study for a method for mass-producing taxol-containing taxanes and derivatives thereof using cells or groups of Taxus plants, and found that liquid medium It was found that the productivity of taxane-containing taxanes can be improved by intermittently or continuously exchanging a fresh medium and recovering the product contained in the medium used for culture, and completed the present invention. Let

[0008] That is, the present invention, when culturing cells or groups of Taxus plants, the liquid medium is intermittently or continuously replaced with a fresh medium, and the taxol contained in the medium used for the culturing is changed. A method for producing taxol, which comprises recovering a taxane product containing the same.

In the present invention, when the culture solution is extracted, the cells are separated by a membrane or a mesh, or the cells are immobilized on a carrier, so that the culture solution having a cell concentration lower than that in the culture system can be extracted. Alternatively, a cell settling tank or device using gravity or centrifugal force may be used.

[0010]

[Operation] In liquid culture of Taxus cells or a group of cells, the culture solution is intermittently or continuously withdrawn,
At the same time, by supplying a fresh medium and collecting the taxane products such as taxol released in the extracted medium, the production of taxane products such as taxol can be continued for a long time without destroying the cells. Can be done, resulting in high productivity.

[0011]

Examples The Taxus genus plant used in the culture of the present invention may be any plant having the ability to produce taxanes such as taxol, and examples thereof include Taxus breviforia Nutt and Taxus breviforia Nutt. cus
pidata) etc. The well-known tissue culture method can be applied to the method for inducing cultured cells (callus) suitable for liquid culture from these Taxus plants. The type and concentration of the medium and plant hormones used when deriving the cultured cells from the plant cells may be appropriately selected according to the material cells. For example, when obtaining the cultured cells from the leaves of the Taxus plant, tissue culture is performed. The cells are cultivated using a solid medium that is usually used in (1), using a auxin such as 2,4-dichlorophenoxyacetic acid or naphthaleneacetic acid, or a medium in which auxin and cytokinin are added to obtain cultured cells (callus).

The callus obtained is then cultivated in a liquid medium. In the present invention, when culturing this callus, the liquid medium is intermittently or continuously replaced with a fresh medium, and the taxane-containing taxane product contained in the medium used for the culturing is recovered. It has a feature. As the liquid medium, a liquid medium usually used in tissue culture is basically used, and a liquid medium prepared by adding about 0.1 to 5 ppm of auxin such as 2,4-dichlorophenoxyacetic acid or naphthaleneacetic acid is used.

In the present invention, in the liquid culture of cultured cells or a group of cultured cells in which a few to several hundreds of individual cells are collected in a small mass, the medium is intermittently or continuously withdrawn, at the same time, etc. By supplying a sufficient amount of fresh medium and collecting the taxane products such as taxol released in the extracted medium, it is possible to produce taxane products such as taxol over a long period of time without destroying the cultured cells. Continue to do. As a culturing device for this culturing, a large-capacity tank-type incubator or the like can be appropriately selected and used from a small-capacity container such as a flask according to the culturing scale. The culture conditions can be the same as the culture conditions in the tissue culture of general plants, and particularly preferably, the culture temperature is about 20 to 28 ° C. and the dark conditions. Further, in this culture, it is desirable to shake, stir and mix the culture solution so as not to damage the cultured cells.

When the culture solution is extracted during this culture, it is desirable to extract the culture solution having a cell concentration lower than that in the culture system. Cultured cells may be mixed to some extent in the culture medium extracted from the culture system, and the cultured cells in the extracted culture medium may be collected by filtration or centrifugation and returned to the culture system. Further, cells in the culture medium may be crushed to extract taxanes such as taxol together with the culture medium or separately.

When the medium in the culture system is exchanged during this culturing, in order to make the cell concentration of the used medium to be withdrawn lower than that in the culturing apparatus, an opening smaller than the average diameter of the cells or cell clusters used is used. Having a mesh, for example 2 openings
The culture solution is extracted from the culture system through a nylon mesh of about 00 μm or a separation membrane having an average pore size smaller than the average diameter of cells or cell mass, for example, a precision separation membrane having a pore size of about 0.2 to 1 μm. .

In addition, in order to lower the cell concentration of the used medium to be extracted at the time of exchanging the medium, the cells may be alginate, carrageenan, agar, a photosensitive resin, a gel-like carrier such as acrylamide gel in order to lower the cell concentration. Alternatively, a method of immobilizing the same by adsorbing it on urethane foam, cellulose, dextran, porous glass, stainless steel or the like is also effective.

Further, in order to lower the cell concentration of the used medium to be withdrawn when exchanging the medium, the sedimentation tank or the sedimentation device utilizing gravity or centrifugal force is installed in order to lower the cell concentration of the used medium to be removed from the culture device. It is also effective to take out only the light liquid (liquid having a lower cell concentration).

Further, this medium exchange is performed by continuously supplying a new medium into the culture system at a constant flow rate and continuously supplying an equal amount of the culture medium (used culture medium containing taxol) from the culture system. The amount of the medium to be exchanged is not particularly limited, and any of the method of extracting the medium and the method of intermittently supplying a new medium into the culture system and extracting an equal amount of the culture solution may be used. Further, the replacement frequency in the case of intermittently exchanging the medium is not limited, and for example, the medium may be exchanged at intervals of 2 to 3 days or may be exchanged several times a day.

A usual method can be used to recover taxol from the culture solution extracted from the culture system. That is,
A target taxane product such as taxol can be extracted from the culture solution using a solvent such as ether or dichloromethane. Further, before extraction, taxol can be adsorbed and separated using an adsorbent, for example, Amberlite XAD-2 which is a nonionic polymer adsorbent bead.

(Experimental Example) For the culture, a modified Gamborg B5 medium containing 1 to 5 ppm of naphthalene acetic acid was used. 100 ml of the medium was put into a 300 ml Erlenmeyer flask, and a medium withdrawing pipe equipped with a nylon mesh having an opening of 100 to 200 μm and a supply pipe were attached thereto, and sterilized at 120 ° C. for 15 minutes. After cooling, about 1 g of wet (Taxus cuspidata) cultured cells (callus) was added by wet weight, and reciprocal shaking culture was performed at 26 ° C. The dilution rate in continuous culture was 0.09, 0.27,
It was set to 0.90. The total amount of taxol produced in the medium taken out on the 11th day of the culture was compared with the amount produced in the medium after the usual 11-day batch culture (without extracting and supplying the medium). The results are shown in Table 1. . In the quantitative analysis of taxol in the medium, the retention time was compared with that of the standard substance using HPLC.

[0021]

[Table 1]

As is clear from the results shown in Table 1, the culture medium is fed by batch culture by feeding fresh medium into the culture system and culturing while extracting the used medium and extracting taxol from the extracted medium. It was found that the production amount of taxol can be significantly increased as compared with the method of obtaining taxol.

[0023]

As described above, according to the present invention,
It is possible to provide a method for producing taxol, in which the productivity of taxol is significantly improved and taxanes such as taxol and derivatives thereof can be mass-produced on an industrial scale.

Claims (4)

[Claims] 1. A taxane containing taxol contained in a medium used for culturing, when a liquid medium is intermittently or continuously replaced with a fresh medium when culturing cells or groups of Taxus plants. A method for producing taxol, which comprises recovering a product. 2. When the liquid medium is intermittently or continuously exchanged with a fresh medium, a membrane or a mesh is used so that the cell concentration in the culture system is higher than that in the medium extracted from the culture system. The method for producing taxol according to claim 1, wherein the method is maintained. 3. When the liquid medium is intermittently or continuously exchanged with a fresh medium, the cells are immobilized by entrapping, adhering or binding to a carrier, and the cell concentration in the culture system is adjusted to that in the culture system. The method for producing taxol according to claim 1, wherein the taxol is maintained at a higher concentration than the cells in the medium extracted from the medium. 4. When the liquid medium is intermittently or continuously exchanged with a fresh medium, the cell concentration in the culture system is adjusted from within the culture system by using a sedimentation tank or a sedimentation device by gravity or centrifugal force. The method for producing taxol according to claim 1, wherein the concentration of cells in the medium to be extracted is maintained higher than that of the cells.