Callus cell induction and the preparation of taxanes

A method for the production of taxanes such as taxol. The method includes inducing formation of callus cells by contacting an explant tissue with a liquid medium without complete submergence of the tissue in the medium. The callus cells formed are employed in a liquid suspension cell culture to produce one or more taxanes.

FIELD OF THE INVENTION 
The present invention relates to a method for the induction of callus cells 
capable of producing taxanes such as taxol from explant tissues, to the 
callus cells so produced, and to a method employing these cells in the 
suspension cell culture preparation of taxanes. 
BACKGROUND OF THE INVENTION 
Taxanes are diterpene compounds which find utility in the pharmaceutical 
field. For example, taxol, a taxane having the structure: 
##STR1## 
where Ph is phenyl, Ac is acetyl and Bz is benzoyl has been found to be an 
effective anticancer agent, particularly useful in the treatment of 
ovarian cancer. 
Taxanes such as taxol may be found in plant materials, and have been 
isolated therefrom. Such taxanes may, however, be present in plant 
materials in relatively small amounts so that, in the case of taxol, for 
example, large numbers of the slow-growing yew trees forming a source for 
the compound may be required. The art has thus continued to search for 
alternate methods for obtaining taxanes such as taxol. Particularly sought 
are efficient methods for the suspension cell culture preparation of these 
compounds. 
SUMMARY OF THE INVENTION 
The present invention provides a method for the induction of callus cells 
capable of producing at least one taxane from explant tissue, comprising 
the steps of: 
(a) contacting at least part of said explant tissue with a liquid medium 
without completely submerging said tissue in said medium; and 
(b) inducing callus cells to form. 
Induction of callus cells according to the method of the present invention 
allows direct transfer of the cells so formed to a liquid medium for the 
suspension cell culture preparation of taxanes, without need of a separate 
growth or proliferation step, thus shortening the overall development 
time. 
The present invention also provide callus cells produced by the above 
method of the present invention, and a method for the use of these cells 
in the suspension cell culture preparation of taxanes.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is described in further detail as follows. 
Definitions 
The terra "explant tissue", as used herein, denotes tissue from an original 
plant source, which tissue, for example, has not previously been contacted 
with an artificial liquid or solid medium for the formation of callus 
cells. 
The term "induction", as used herein, denotes the initial dedifferentiation 
from the aforementioned explant tissue to form callus cells. 
The term "callus cell", as used herein, denotes any cell which is 
dedifferentiated relative to the explant tissue from which it was derived. 
The term "solid medium", as used herein, denotes a medium containing 
gelling agent(s) in a quantity sufficient for solidification of the 
medium. 
The term "liquid medium", as used herein, denotes a medium containing 
gelling agent(s) in a quantity insufficient for solidification of that 
medium, or which contains no gelling agent(s) at all. 
The term "dispersed cells", as used herein, denotes those callus cells or 
callus cell clusters which, upon dedifferentiation from explant tissue, do 
not adhere to the remaining explant tissue and thus become free cells or 
cell clusters in the surrounding liquid medium. 
The term "membrane raft", as used herein, denotes a sheet-like support 
structure for the explant tissue, which structure is preferably 
sufficiently porous to allow transport of nutrients. 
The term "dedifferentiation", as used herein, denotes changes in a 
differentiated tissue, which changes are of a kind leading to the 
reversion of cell type to a common cell type. 
Explant tissue 
The explant tissue employed in the method of the present invention may be 
any plant tissue from which callus cells capable of producing a taxane may 
be induced. Exemplary sources for explant tissue include plants of the 
family Taxaceae such as plants of the genera Amentotaxus, Austrotaxus, 
Pseudotaxus, Torreya, and Taxus. Preferred as sources of explant tissue 
are plants of the genus Taxus, particularly the species T. brevifolia, T. 
baccata, T. x media (e.g. Taxus media hicksii), T. wallichiana, T. 
canadensis, T. cuspidata, T. floridiana, T. celebica and T. x 
hunnewelliana. 
Any part of the plant from which callus cells may be induced may be 
employed as the explant source such as the bark, cambium, roots, leaves or 
needles, stems, branches, twigs, wood, embryos, seeds or seedlings. 
Preferably, the plant organs comprising the root, stem, leaf or embryo are 
employed, particularly where the source of root tissue is from the root 
meristem (growing tip) or root cambrium (root bark), where the stem tissue 
is from bark, branches or twigs, and where the embryo tissue is from 
immature embryos or germinated mature embryos from seeds. The age or 
maturity of the plant employed as the explant source may range from that 
of immature embryos, embryos, seedlings, up to and including mature trees. 
Stem tissue is most preferred. 
Preferably, prior to use in the present invention, the explant tissue is 
sectioned into pieces of a size suitable for use therein, such as sizes 
ranging from about 1 cm to about 5 cm in length. The surface of the 
explant tissue is also preferably sterilized before use. Sterilization may 
be conducted by any appropriate method such as by the use of chlorinated 
bleach, an alcohol solution such as an ethanol/water (e.g. 70% ethanol) 
solution, or a mixture thereof. Antimicrobial agents may also be employed 
to achieve and maintain sterility. 
Support 
The explant tissue employed in the present invention is maintained in a 
position such that at least part of the tissue is in contact with the 
liquid medium, while complete submersion in the liquid medium, which is 
undesirable for callus induction, is avoided. Thus, a support may be 
employed which maintains the explant tissue in a position where a portion 
of the tissue is in contact with the surrounding atmosphere, most 
preferably air, while the remaining portion is in contact with the liquid 
medium. 
Any support so positioning the explant tissue of the present invention may 
be employed. The explant tissue may, for example, be placed on a membrane 
raft, which is preferred, or on a sponge. Preferred materials for use as a 
membrane raft include microporous polypropylene or cellulose acetate. It 
is preferred that the average diameter of the pores of the raft is smaller 
than the average diameter of the individual callus cells which are formed. 
Particularly preferred are those membrane materials which do not allow the 
transfer of callus cells across the membrane. It is also preferred that 
the level of the liquid medium be above the level of the membrane raft, 
such as where the raft is positioned below the level of the liquid medium 
so that there is liquid both above and below the level of the raft. These 
embodiments facilitate the formation of dispersed cells, and, especially, 
clusters of dispersed cells. 
Liquid Medium 
Any liquid medium allowing callus induction may be employed. Exemplary 
liquid media are aqueous media of Gamborg's B5 (Table 1 following), 
Murashige and Skoog (Table 2 following), Anderson's Rhododendron Basal 
Salts (Table 3 following), Whites (Table 4 following), as well as 
variations of these media. Exemplary variations of the aforementioned 
media include the addition of sugars such as sucrose, glucose or maltose, 
casamino acids (e.g. 0.2%), enzyme hydrolyzed casein (e.g. 0.02%), and 
glycine (e.g. 0.002%) and various auxins and cytokinins. The use of 
aqueous Gamborg's B5 medium is preferred. 
TABLE 1 
______________________________________ 
Composition of Gamborg's B5 Medium 
mg/L 
______________________________________ 
Basal Salts 
Ammonium sulfate 134.000 
Boric acid 3.000 
Calcium chloride anhydrous 
113.240 
Cobalt chloride hexahydrate 
0.025 
Cupric sulfate pentahydrate 
0.025 
Disodium EDTA dihydrate 
37.300 
Ferrous sulfate heptahydrate 
27.800 
Magnesium sulfate anhydrous 
122.090 
Manganese sulfate monohydrate 
10.000 
Potassium iodide 0.750 
Potassium nitrate 2500.000 
Sodium molybdate dihydrate 
0.250 
Sodium phosphate monobasic anhydrous 
130.500 
Zinc sulfate heptahydrate 
2.000 
Vitamins 
Myo-inositol 100.0 
Thiamine HCl 10.0 
Pyridoxine HCl 1.0 
Nicotinic acid 1.0 
Sugars 
Sucrose 20,000.0 
Hormones 
2,4-Dichlorophenoxyacetic acid ("2,4-D") 
1.5 
______________________________________ 
TABLE 2 
______________________________________ 
Composition of Murashige and Skoog Medium 
mg/L 
______________________________________ 
Basal Salts 
Boric acid 6.20 
Calcium chloride anhydrous 
332.20 
Cobalt chloride hexahydrate 
0.025 
Cupric sulfate pentahydrate 
0.025 
Disodium EDTA dihydrate 
37.260 
Ferrous sulfate heptahydrate 
27.800 
Magnesium sulfate anhydrous 
180.70 
Manganese sulfate monohydrate 
16.90 
Potassium iodide 0.830 
Potassium nitrate 1900.00 
Sodium molybdate dihydrate 
0.250 
Potassium phosphate monobasic anhydrous 
170.00 
Zinc sulfate heptahydrate 
8.60 
Ammonium Nitrate 1650.00 
Vitamins 
Myo-inositol 100.0 
Thiamine HCl 10.0 
Pyridoxine HCl 1.0 
Nicotinic acid 1.0 
Sugars 
Sucrose 20,000.0 
Hormones 
2,4-Dichlorophenoxyacetic acid 
1.5 
______________________________________ 
TABLE 3 
______________________________________ 
Composition of Anderson's Rhododendron Basal Salts Medium 
mg/L 
______________________________________ 
Basal Salts 
Ammonium nitrate 400.00 
Boric acid 6.200 
Calcium chloride anhydrous 
332.20 
Cobalt chloride hexahydrate 
0.025 
Cupric sulfate pentahydrate 
0.025 
Disodium EDTA dihydrate 
74.500 
Ferrous sulfate heptahydrate 
55.70 
Magnesium sulfate anhydrous 
180.70 
Manganese sulfate monohydrate 
16.90 
Potassium iodide 0.300 
Potassium nitrate 480.00 
Sodium molybdate dihydrate 
0.250 
Sodium phosphate monobasic anhydrous 
330.60 
Zinc sulfate heptahydrate 
8.60 
Vitamins 
Myo-inositol 100.0 
Thiamine HCl 10.0 
Pyridoxine HCl 1.0 
Nicotinic acid 1.0 
Sugars 
Sucrose 20,000.0 
Hormones 
2,4-Dichlorophenoxyacetic acid 
1.5 
______________________________________ 
TABLE 4 
______________________________________ 
Composition of Whites Medium 
mg/L 
______________________________________ 
Basal Salts 
Boric acid 1.50 
Calcium nitrate tetrahydrate 
208.40 
Cupric sulfate pentahydrate 
0.010 
Ferric sulfate 2.50 
Magnesium sulfate anhydrous 
366.20 
Manganese sulfate monohydrate 
3.788 
Potassium iodide 0.750 
Potassium nitrate 80.00 
Sodium sulfate 200.00 
Sodium phosphate monobasic anhydrous 
16.50 
Zinc sulfate heptahydrate 
3.00 
Potassium chloride 65.00 
Vitamins 
Myo-inositol 100.0 
Thiamine HCl 10.0 
Pyridoxine HCl 1.0 
Nicotinic acid 1.0 
Sugars 
Sucrose 20,000.0 
Hormones 
2,4-Dichlorophenoxyacetic acid 
1.5 
______________________________________ 
Callus Induction 
Callus cells may be induced by holding the explant tissue/liquid medium 
system at suitable conditions therefor. 
The temperature employed during induction is preferably between about 
20.degree. C. and about 30.degree. C., most preferably about 22.degree. 
C.. The portion of the explant tissue not in contact with the liquid 
medium is preferably in contact with air in which the relative humidity is 
controlled, for example, by tightly sealing the system. The relative 
humidity may, for example, be near saturation such as between about 80% 
and about 100%. Diffuse, that is, ordinary room lighting, is preferred. 
The portion of the explant tissue which is in contact with the liquid 
medium is preferably from about 10% to about 25% of the total volume of 
the tissue section. Induction is preferably conducted over a period 
between about 10 days to about 30 days. Gentle agitation of the liquid 
medium in contact with the explant tissue during induction may be 
employed, although quiescent conditions are preferred. 
Callus cells may form which remain adhered to the remaining explant tissue 
and/or which slough off the remaining explant tissue to form free callus 
cells or cell clusters dispersed in the surrounding liquid medium 
("dispersed cells"). Some initial proliferation of the callus cells formed 
may occur during the present induction method. 
Use of a liquid, rather than solid, medium in the induction of callus cells 
according to the method of the present invention provides significant 
advantages. Specifically, use of a solid medium induces callus formation 
in a relatively dry environment in which the callus cells formed remain 
adhered to the explant tissue. Callus cells formed in such an environment, 
in order to ultimately grow and produce taxanes efficiently in liquid 
suspension cell culture, must be acclimated to a liquid environment. Due 
to the change in oxygen availability, osmotic differences and the like, 
callus cells induced on a solid medium, during acclimation to a liquid 
environment, undergo a decrease in growth rate and taxane production, and 
may exhibit an increase in cell-type abnormalities and lysis. Moreover, 
acclimation, particularly when achieved during a separate, subsequent 
growth or proliferation step, lengthens the overall development time from 
explant tissue to liquid suspension cell culture. 
The method of the present invention obviates the above difficulties. Callus 
cells induced in contact with a liquid medium, particularly dispersed 
callus cells so induced as described above, are more readily acclimated to 
liquid suspension cell culture conditions. Callus cells induced by the 
method of the present invention may be transferred directly to a liquid 
suspension cell culture medium without employing a separate growth or 
proliferation step. Thus, the induction method of the present invention is 
efficient in reducing the overall development time from explant tissue to 
suspension cell culture, improving productivity. 
Other advantages may also be obtained by the method of the present 
invention. For example, callus induction may be achieved for a longer time 
period on a liquid, rather than solid, medium so that a greater number of 
callus cells may be obtained. Undesirable compounds, such as phenolics, 
produced during callus induction are more readily diffused into the medium 
and away from the callus induction site when employing a liquid medium. 
Further, the callus formed by the method of the present invention has a 
healthy, green appearance and contains fewer brown callus cell areas than 
callus induced on a solid medium. 
The preferred embodiments of the present invention provide additional 
advantages. For example, the formation of dispersed cells and dispersed 
cell clusters is desirable as such cells are most readily acclimated to 
liquid suspension cell culture conditions. Thus, use of a liquid medium in 
contact with a sufficient portion of the explant tissue so as to allow and 
promote the formation of dispersed cells and cell clusters, such as where 
the level of the liquid medium is above that of the explant tissue 
supporting structure (e.g. membrane raft), is advantageous. 
Most advantageous is the formation of clusters of dispersed cells. Clusters 
of dispersed cells, when transferred to a liquid suspension cell culture 
system, most rapidly achieve the critical mass of cells required for 
maximum cell growth and taxane output. Thus, use of an explant tissue 
supporting structure which does not allow the transfer of dispersed cell 
clusters across the structure is preferred. For example, use of a membrane 
raft which has pores the average diameter of which is smaller than the 
average diameter of individual dispersed cells, retains cell clusters 
above the level of the raft and inhibits the transfer of cells across the 
membrane during which cell clusters may be broken up into individual 
cells. 
Taxane Production 
The present invention also provides a method for the production of at least 
one taxane, comprising the steps of: 
(A) inducing callus cells capable of producing at least one taxane 
according the above-described method of the present invention for callus 
cell induction; and 
(B) culturing said cells in a liquid suspension cell culture system to 
produce said taxane(s). 
It is preferred to proceed from step (A) to step (B) without use of an 
intermediate, separate growth or proliferation step. By "separate growth 
or proliferation step", as used herein, is meant transferring the cells to 
a site physically distinct from that where steps (A) and (B) are 
conducted, and growing or proliferating callus cells. 
The suspension cell culture of any taxane capable of being produced by such 
a method is contemplated within the scope of the present invention. It is 
understood herein that a single, or two or more taxanes, may be produced 
during practice of the method of the present invention. 
The culturing step (B) of the above method of the present invention may be 
carried out according to suspension cell culture methods such as those 
known to the skilled artisan. See U.S. Pat. No. 5,019,504, incorporated by 
reference. Exemplary media which may be employed include those discussed 
above with respect to induction media. Agar (e.g. 0.1%) or phytagel (e.g. 
0.025%) may optionally further be added to such media. The temperature 
employed during suspension cell culture is preferably between about 
22.degree. and 25.degree. C.; the relative humidity employed is preferably 
between about 40 and about 60%; and the degree of agitation is preferably 
from about 30 to about 200 revolutions per minute (RPM). Inducers such as 
fungal elicitors, vanadyl sulfate, 3,4-dichlorophenoxy triethyl(amine), 
etc. may be added. Taxane production may also be conducted by employing 
cells which are encapsulated in calcium alginate beads, as well as when in 
a slurry, e.g. made by incorporation of 0.1% agar into the media. 
Recovery of the taxanes produced during culturing may be accomplished by 
methods known to the skilled artisan. For example, adsorbent beads may be 
employed to expedite recovery of taxanes such as taxol. Beads remaining in 
the culture during the production of taxanes such as taxol may also allow 
greater production by binding the taxane product(s). Additionally, 
extraction of the taxane product(s) from the cell supernatant or beads is 
readily accomplished with solvents such as ether or methylene chloride. 
Taxane Compounds 
Taxanes are diterpene compounds containing the taxane carbon skeleton: 
##STR2## 
which skeleton may contain ethylenic unsaturation in the ring system 
thereof (e.g., where the 11,12-positions are bonded through an ethylenic 
linkage). The preparation of all taxanes, whether pharmacologically active 
or inactive, is contemplated within the scope of the present invention. 
Taxanes may be produced by the callus cells of the present invention which 
are (i.e. are naturally occuring), or are not, found in the original 
explant tissue. 
Exemplary taxanes which may be produced by the cell culture method of the 
present invention include those of the following formula I: 
##STR3## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, 
R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, R.sub.13, R.sub.14, 
R.sub.15, R.sub.16 and "a" are as defined in the following Table 5. 
3 TABLE 5 
- Compound.sup.4/ R.sub.1.sup.1/ R.sub.2 R.sub.3 R.sub.4 R.sub.5 
R.sub..sub.6 R.sub.7 .sup.2/ R.sup.8 R.sub.9.sup.5/ R.sub.10 R.sub.11 
R.sub.12 R.sub.13 R.sub.14 R.sub.15 R.sub.16 a.sup.3/ 
1) taxol tax CH.sub.3 H .beta. a 
cetyloxy O .beta. 
CH.sub.3 .beta. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
2) 10-desacetyl- ceph CH.sub.3 H .beta. O 
H O .beta. 
CH.sub.3 .beta. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
cephalomannine 
3) 7-epitaxol tax CH.sub.3 H .beta. a 
cetyloxy O .beta. 
CH.sub.3 .alpha. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
4) 10-desacetyl- tax CH.sub.3 H .beta. O 
H O .beta. 
CH.sub.3 .alpha. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
7 epitaxol 
5) 7-epicephalo- ceph CH.sub.3 H .beta. a 
cetyloxy O .beta. 
CH.sub.3 .alpha. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
mannine 
6) baccatin III .alpha. O 
H CH.sub.3 H .beta. 
acetyloxy O .beta. 
CH.sub.3 .beta. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
7) 10-desacetyl- .alpha. O 
H CH.sub.3 H .beta. 
OH O .beta. 
CH.sub.3 .beta. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
baccatin III 
8) cephalomannine ceph CH.sub.3 H .beta. a 
cetyloxy O .beta. 
CH.sub.3 .beta. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
9) 10-desacetyl- tax CH.sub.3 H .beta. O 
H O .beta. 
CH.sub.3 .beta. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
taxol 
10) xylosyl taxol tax CH.sub.3 H .beta. 
acetyloxy O .beta. 
CH.sub.3 .beta. 
xylosyl H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
11) xylosyl ceph- ceph CH.sub.3 H .beta. 
acetyloxy O .beta. 
CH.sub.3 .beta. 
xylosyl H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
alomannine 
12) taxagifine O .alpha. 
CH.sub.3 .beta. 
OH .beta. 
acetyloxy .alpha. 
acetyloxy .beta. 
CH.sub.3 .beta. 
acetyloxy H .alpha. 
cinnamoyloxy methylene .alpha. 
acetyloxy .beta. 
H H cyclo.sup.6/ .alpha. 
CH.sub.3 -- 
(CH.sub.2) 
13) 8-benzoyloxy O .alpha. 
CH.sub.3 .beta. 
OH .beta. 
acetyloxy .alpha. 
acetyloxy .beta. 
benzoyl- .beta. 
acetyloxy H .alpha. 
cinnamoyloxy methylene .alpha. 
acetyloxy .beta. 
H H cyclo .alpha. 
CH.sub.3 -- 
taxagifine oxymethyl (CH.sub.2) 
14) 9-acetyloxy- .alpha. 
acetyloxy CH.sub.3 H .beta. 
acetyloxy .alpha. 
acetyloxy .beta. 
CH.sub.3 H H .alpha. 
acetyloxy methylene H H H CH.sub.3 CH.sub.3 X 
taxusin (CH.sub.2) 
15) 9-hydroxytaxusin .alpha. 
acetyloxy CH.sub.3 H .beta. 
acetyloxy .alpha. 
OH .beta. 
CH.sub.3 H H .alpha. 
acetyloxy methylene H H H CH.sub.3 CH.sub.3 X 
(CH.sub.2) 
16) taiwanxan H CH.sub.3 H .beta. 
acetyloxy .alpha. 
acetyloxy .beta. 
CH.sub.3 H H .alpha. 
OH methylene .alpha. 
acetyloxy H 2-methylbuta- CH.sub.3 CH.sub.3 X 
noxloxy 
17) taxane Ia tax CH.sub.3 H O O .beta. 
CH.sub.3 .alpha. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
18) taxane Ib taxsub CH.sub.3 H O O .beta. 
CH.sub.3 .alpha. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
19) taxane Ic taxsub CH.sub.3 H O O .beta. 
CH.sub.3 .alpha. 
acetyloxy H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
oH H CH.sub.3 CH.sub.3 X 
20) taxane Id .alpha. 
acetyloxy CH.sub.3 H .beta. 
acetyloxy .alpha. 
acetyloxy .beta. 
CH.sub.3 .beta. 
acetyloxy H .alpha. 
OH epoxide.sup.7/ .alpha. 
acetyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
21) 7-epibaccatin III .alpha. 
OH CH.sub.3 H .beta. 
acetyloxy O .beta. 
CH.sub.3 .alpha. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
22) taxotere taxot CH.sub.3 H .beta. 
OH O .beta. 
CH.sub.3 .beta. 
OH H oxetane .alpha. 
acetyloxy .alpha. 
benzoyloxy .beta. 
OH H CH.sub.3 CH.sub.3 X 
Footnotes 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
3/"a" denotes a double bond present between the 11 and 12positions 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
Taxanes which may be produced by the cell culture method of the present 
invention may also be represented by the following formulae II or III: 
##STR14## 
where R.sub.4 is keto (.dbd.O) or acetyloxy; 
R.sub.7 is hydrogen, .alpha.-hydroxy or .beta.-hydroxy; 
R.sub.9 is acetyloxy, cinnamoyloxy or hydroxyl; 
R.sub.10 and R.sub.11 together form a methylene or epoxide group; 
R.sub.12 is hydrogen, benzoyloxy or acetyloxy; and 
R.sub.17 is hydrogen or acetyloxy. 
Preferred taxanes include taxol, baccatin III, 10-desacetylbaccatin III, 
10-desacetyl taxol, xylosyl taxol, 7-epitaxol, 7-epibaccatin III and 
10-desacetyl-7-epitaxol. Cell culture production of taxol is a 
particularly preferred embodiment of the present invention. 
Taxanes are compounds which find utility in the pharmaceutical field, such 
as in the treatment of cancer. Taxol is exemplary of the pharmacologically 
active taxanes which also include, for example, cephalomannine, the latter 
reported as a chemotherapeutic agent for the remission of leukemia in U.S. 
Pat. No. 4,206,221. The present invention contemplates preparation of such 
pharmacologically active taxanes, as well as preparation of slightly 
active or inactive taxanes, or those having a less desired activity, which 
may be used as intermediates to prepare other, pharmacologically active 
taxanes. The method of the present invention may thus facilitate 
preparation of pharmacologically active taxanes by providing an efficient 
means for obtaining the taxane starting material through cell culture. 
The methods of the present invention are further described by the following 
Examples. These Examples are illustrative only, and are in no way intended 
to limit the scope of the instant claims. 
EXAMPLE 1 
Induction of Callus Cells and Preparation of Taxol 
Explants were cut from Taxus media hicksii plant stems after sterilizing in 
70% alcohol and 25% bleach. Each explant, approximately 2-3 cm in length, 
was placed on a microporous polypropylene membrane raft which was floating 
on a medium consisting of Gamborg's B5 Basal salts and vitamins (see 
previous Table 1), sucrose (2%), 2,4-D (1.5 mg/L) and casamino acids (2 
g/L). The medium and raft were contained in a 4.times.4 inch polycarbonate 
container fitted with a tight polypropylene lid so as to maintain high 
humidity. 
Prior to placing the explant on the raft, the vessel and medium were 
autoclaved under standard conditions for 15 minutes. After placing the 
explant on the raft, 5-10 ml of the above medium were placed on top of the 
explant. The raft vessel was incubated at 22.degree. C. for 3-4 weeks 
until a callus had been generated. After incubation, the explant and 
callus were removed and the loose cells remaining on the raft were 
pipetted off and added to 25 ml of the above Gamborg's medium to which had 
been added 0.1 g/L agar in a 125 ml flask. The loose cells from several 
rafts may be combined into one flask if cell numbers appear to be low. The 
inoculated flask was incubated at 22.degree. C. on a 50 RPM shaker with 
45% humidity and diffuse light. 
After 3 weeks, flasks were harvested and analyzed for taxol. Suspension 
flasks were found to contain 0.01 to 0.02 mg/L taxol or expressed on a dry 
cell weight basis: 0.0002 to 0.0004% taxol based on dry cell weight. The 
presence of other taxanes including cephalomannine and baccatin III was 
also detected. (Based on two runs as follows: 
______________________________________ 
Taxol Obtained 
Dry Cell weight 
Run (.mu.g/ml) (mg/ml) 
______________________________________ 
1 0.0207 5.5 
2 0.0131 5.7 ) 
______________________________________ 
EXAMPLE 2 
Variation in Medium 
Explants were cut from Taxus media hicksii plant stems after sterilizing in 
70% alcohol and 25% bleach. Each explant, approximately 2-3 cm in length, 
was placed on a microporous polypropylene membrane raft which was floating 
on a medium consisting of Anderson's Rhododendron basal salts (see 
previous Table 3) and Gamborg's vitamins (see previous Table 1), sucrose 
(2%), 2,4-D (1.5 mg/L) and casamino acids (2 g/L). The medium and raft 
were contained in a 4.times.4 inch polycarbonate container fitted with a 
tight polypropylene lid so as to maintain high humidity. 
Prior to placing the explant on the raft, the vessel and medium were 
autoclaved under standard conditions for 15 minutes. After placing the 
explant on the raft, 5-10 ml of the above medium were placed on top of the 
explant. The raft vessel was incubated at 22.degree. C. for 5-10 weeks 
until a callus had been generated. After incubation, the explant and 
callus were removed and the loose cells remaining on the raft were 
pipetted off and added to 25 ml of the above Anderson's medium to which 
had been added 0.1 g/L agar in a 125 ml flask. (Loose cells from several 
rafts may be combined as described above in Example 1.) The inoculated 
flask was incubated at 22.degree. C. on a 50 RPM shaker with 45% humidity 
and diffuse light. 
After 3 weeks, flasks were harvested and analyzed for taxol. Suspension 
flasks were found to contain 0.0194 mg/L taxol or expressed on a dry cell 
weight basis: 0.0062% taxol based on dry cell weight.