CA1134764A - Producing codeine from papaver cell cultures - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The production of codeine has been achieved from modified cell cultures derived from the plants Papaver somniferum L. or Papaver setigerum DC. The cell cultures have been constrained to produce cytodifferentiated polyploid cells which are coniderably larger than the normal non-differentiated cells. In the presence of these polyploid cells the production of codeine is realized.

Description

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Field of the In~ention The morphinan alkaloid codeine is produced and recovered from certain plant cell cultures. Selected cell cultures including a novel form of cytodifferentiated cells have been found to produce codeine.

Description of the Prior Art Opium contains up to about 15% of anhydrous morphine. Codeine, which is morphine-3-methyl ether, is present in opium in up to about 2.5~ and is usually prepared from morphine by methylation. The common opium-poppy species is Papaver somniferum L. but P. setigerum DC. is also known to yield an opium product containing codeine. Codeine has antitussive and analgesic properties with little narcotic function, and is normally used as the sulfate.
Codeine is 7,8-didehydro-4,5 ~-epoxy-3-methoxy-17-methylmorphinan-6 ~ol (C18H21NO3) mol. wt. 299.36 and is a member of the morphinan group of alkaloids. Other members of this group include thebaine (ClgH2lNO3), the immediate biosynthetic precursor to codeine, and morphine (Cl7Hl9NO3) which like codeine is a medicinally-useful drug. Codeine has the structural formula:

CH30 ~
Il I

~N C}}3 H G' 1~3~764 In recent years plant cell culture techniques have been developed to the stage where callus cultures with a capacity to produce specialized plant cells can be maintainea ln vitro. Plant cells cultured in vitro retain the genetic information of the original plant, i.e. upon induction they may grow roots and shoots or produce metabolites specific for the species or variety in question. Accordingly, cell cultures derived from Papaver somniferum L. and P.
setigerum DC. can be considered a potential source for papaver-alkaloids, morphinans. The problem encountered in exploiting this potential is to provide for cyto-physiological conditions which permit the realization of the biosynthetic potential of Papa~er cell cultures. Callus tissue cultures from opium-poppy have been achieved, and recently the alkaloids present in such callus tissue have been identified. These callus tissues yielded benzo-phenanthridine-, protopine- and aporphine-type alkaloids but not morphinan-type alkaloids as were present in the original plants. See ~ Furuya et al, Phytochemistry 11, 3041-3044, 1972; ~ Ikuta et al, Phytochemistry 13, 2175-2179, 1974. In biotransformation experiments where alkaloids were added to callus tissue cultures from Papaver somniferum L., these cultures were found to lack the ability to metabolize (RS)-reticuline to thebaine, codeine and morphine but could metabo~ize added (-)-codeinone to (-)-codeine. See ~ Furuya et al, Phytochemistry 17, 891-893, 197~. Thus previous callus cultures from the cpi~l-poppy have been unable to synthesize codeine or morphine. One variety of a ~i~~erent poppy species Papaver 3C racteatum Lindl. (known to cor.tain thebaine~ has been used to derive callus tissues (from the seed heads) and these ~3~'7~

tissues have been found to yield small amounts of thebaine (0.1% thebaine based on dry wt. of seed head tissue). See United States Paten-t 4, 114,314, September 19, 1978, Shafiee et al. The prior art has not achieved the direct production of codeine from cell cultures.

Summary of the Invention We have now found, in accordance with this invention, that selected cell cultures derived from the opium-poppy P. somniferum L. or P. setigerum DC. can be adapted to yield codeine from basic nutrients. A novel cell culture containing cytodifferentiated polyploid cells able to produce codeine, has been prepared.
The invention includes a process for the production of codeine from plant cell cultures which comprises: (a) preparing an inoculum of cells from ~arieties of Papaver somniferum L. or Papaver setigerum DC. selected to form, on cell culturing in a suitable plant cell culture nutri-ent medium which becomes growth-limited, cytodifferentiated polyploid cells capable of producing codeine, said cytodiffer-entiated cells being larger than the non-differentiated cells;
(b) inoculating a suitable nutrient medium for plant cell cultures with said inoculum cells, and allowing these cells to multiply until at least one essential nutrient component becomes growth-limiting;
(c) allowing this growth-limiting culture to undergo cytodifferentiation thereby forming s~id polyploid cells;
(d) accumulating codeine in said cytodi~fer-entiated cell-containing culture; and ~e) recovering codeine ~rom the resu,lting culture.

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This invention includes the novel cells and culture achieved in process step (c).

Brief Description of Drawing In the drawing, the growth-limited and cytodifferentiated cell culture condition of cells from Papaver somniferum L. is shown. The cytodifferentiated -polyploid cells are shown at A and the non-differentiated cells at B with the latter usually as aggregates.

Detailed Description and Preferred Embodiments Our experience with Papa~er cultures, as with other cell strains derived from medicinal plants, shows ~hat c~tophysiological conditions favorable for metabolite production require a degree of differentiation. Generally, the higher the percentage of differentiated cells the ~reater is the amount of metabolites accumulated. As differentiation and cell division exclude each other, an extreme participation of cells in differentiation would pre~ent a culture from growing in cell number. Differentiation of cells can be induced by a decline in limiting growth factors; nitrogen, carbohydrates, growth hormones, for instance. ~ culture will arri~e at a point in time, where a limiting growth factor will be exhausted and growth by cell di~ision will be replaced by differentiation of cells.
ln growth-limited Papa~er somniferum L. cell cultures this differentiation has been found to furnish giant cells which are polyploid and which protrude from aggregates of non-differentiated smaller cells. Appearance of these cells coincides with the production of codeine in such cultures.
The giant cells are thought o b~ the site of codeine accumulation. A similarity of gian' cells with laticifers ~3~76~

(size, polyploidy, occurrence of multinucleation, pigmentation) is suggested. Initial selection of cell strains for codeine formation can, therefore, be a selection for the capability of cytodifferentiation, specifically the formation of giant cells as described.
Any cell cultures derived from explants (inocula) of Papaver somniferum L. or P. setigerum DC.
selected to exhibit ready cytodifferentiation upon induction by means of culture conditions as described can be used according to this invention.
While the biosynthetic potential of cells cultured in vitro can be tapped to provide for the synthesis and accumulation of species-specific metabolites, the rate of production and the amount of product will, at least in part, be determined by the genotype of a specific cell line.
Thus, by means of selecting for cytodifferentiation giving many giant cells, cell lines with superior qualities for metabolite production may be obtained. A feasi~le way to vary the genotype of plant material would be the induction of mutations in cells, and selection of mutant cell lines for their capacity to undergo cytodiffer-entiation to giant cells accompanied by an increased rate of codeine synthesis and accumulation.
The part of the opium-poppy plant from which the explants or initial cells are derived can vary, i.e. cells from seedling material, flower parts, seed head, leaf, stem, root, etc., may be used to start the initial cell culture. Prefera~ the cells are from seedlings, or flower parts inclu~ing microspores.

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Seedling cells (hypocot~l explants), seed head cells or anther cells are particularly preferred. These cells may be maintained on various plant cell culture nutrient media and will serve as inoculum. For codeine production, these cells will be inoculated into a suitable growth medium, and the cells allowed to multiply in the presence of light. In order to develop the cytodifferentiated polyploid cells required according to the invention, at least one growth nutrient should be allowed to become depleted, i.e. become growth-limiting. Under these limiting conditions we have found that cytodifferentiation will occur leading to the appearance of large polyploid cells. These polyploid cells are larger than the initial (non-differentiated) cells and most frequently appear attached to the surface of clumps or agglomerates of the smaller cells.
The nutrient media used for growth, cyto-differentiation tand eventual codeine production) can be the same or different from that used for the initial culture or inoculum. Any good growth medium for plant cell cultures may be used with one of the nutrients required for cell growth being proportioned so that it becomes depleted before the others, thus causing the medium to become growth-limiting. Typical suitable growth media are shown in Tables 1 and 2. Further details on such suitable media and their preparation are given in the book, "Plant Tissue Culture Methods", O.L. Gamborg and L.~. Wetter, Edito~s, 1975 (Published by National Research Council of Canada, Prairie Regional Laboratory, Sas~atoon, Sas~atchewan, Canada S7N OW9).

Composition of Mineral Salt Media for Plant Tissue and Cell Culture Macronutrients mg/Q mM mg/Q mM mg/Q mM
I
NH4NO3 165020.6 1200 15.0 - -XNO3 190018.8 1900 18.8 2500 25 CaC12 2H2 440 3.0 440 3.0 150 1.0 MgSO4 7H2O 370 1.5 370 1.5 250 1.0 KH2PO4 1701.25 340 2.5 (NH4~2SO4 ~ 134 1.0 NaH PO H2O - - - - 150 1.1 Micronutrients mg/Q ~M mg/Q ~M mg/Q ~M

XI 0.83 5.0 - - 0.75 4.5 H3BO3 6.2 100 0.6310 3.0 50 MnSO4-4H2O 22.3 100 2.2310 MnSO4 H2O - - - - 10 60 ZnSO4 4H2O 8.6 30 ZnSO4-7H2O ~ 2.0 7.0 Zn Na2EDTA - - 15 37 Na2MoO4 2H2O 0.25 1.0 0.025 0.1 0.25 1.0 CUS4-5H2 0.0250.1 0.0025 0.01 0.025 0.1 CoCl 6H O 0.0250.1 0.0025 0.01 0.025 0.1 Na2-EDTA 37.3 100 37.3100 37.3 100 FeSO 7H2O 27.8 100 27.8100 27.8 100 Sucrose 30000 40000 20000 p~ 5.7 5.8 5.5 . .

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Amounts and Kinds of Vitamins, Hormones and Supplements Used with the Mineral Salt Media for Agar and Suspension Cultures Compound mg/Q mg/Q mg/~

Inositol 100 - 100 Nicotinic Acid 0.5 0.5 1.0 Pyridoxine HCl 0.5 0.5 1.0 Thiamine HCl 0.1 0.5 10.0 Glycine 2.0 2.0 NAA - 1.0 Kinetin 0.04-10 0.02 0.1

2,4-D - - 0.1-2.0 It is preferable to select and optimize the media both for initial culture and inoculum preparation, and for growth, cytodifferentiation and eventual codeine production.
We have found it beneficial to fortify the medium especially for the growth stage with organic nitrogen source materials such as casein hydrolysate, coconut milk; and with a cytokinin; each suitably within a concentration range of about 0.1 to 5 ppm. Normally, the medium will contain a carbon source, a nitrogen source, sources of Ca, Mg, P, S and K and trace elements, vitamins and phytohormones.
While 2,4-dichlorophenoxyacetic acid is the phytohormone usually added, it may be replaced with l-naphthaleneacetic acid or 3-indoleacetic acid, usually in amounts o~ about 0.1 to 2 mg/Q.
The cells are readily maintained by transferring to fresh medium or replenishing the medium at

3~13~764 intervals of about 1 to 4 weeks. These growing cells provide inoculum as required or if left in a medium that becomes growth-limiting and allowed to cytodifferentiate after growth ceases, then codeine can be accumulated and recovered. The usual temperatures for plant cell cultures will be suitable (e.g. from about 20C - 40C) for inoculum preparation, growth and codeine accumulation.
The time from inoculation to reach the growth-limiting condition is usually about 1 to 3 weeks depending on inoculum size, medium, and growth conditions.
The growth-limiting condition in these media leads to the development of cytodifferentiated polyploid cells as shown in the drawing at A. These polyploid cells are usually about 5-10 times or more larger than the non-dif~er-entiated cells, and have average diameters of at least about 100 microns. Elongated giant cells have been observed with the long dimension up to about 1000 microns or more.
The non-differentiated cells are diploid in constrast to these polyploid cytodifferentiated cells. Repeated ~icro-scopic observations have revealed that the large polyploid cells frequently cluster about clumps or aggregates of non-differentiated cells, e.g. as shown in the drawing at A
and B respectively. Since these polyploid cells have always been obser~ed when codeine production has been achieved, they are believed to coincide with and be necessary for codeine synthesis. At least about 10 percent of the total cells should be of this polyploid type for codeine accumu-lation.
The growth-limited culture is allowed to accumulate codeine ~nder light. Usually this codeine :~13476~

accumulation will take from about 15 to about 30 days before the rate of production declines~but this depends on many factors. The codeine is then recovered, e.g.
by extraction with a suitable organic solvent, followed by isolation if desired from the extract. Suitable organic solvents include ethyl acetate, chloroform, methanol, ethanol and isopropanol. Chromatographic techniques are very suitable for isolating the codeine from the extracted material as is known in the art.

Example Cell Culture Preparation Seeds of Papaver somniferum L. cv. Marianne were surface sterilized and germinated on moistened filter paper in dishes. Hypocotyls of 5 - 6 day old seedlings were explanted onto nutrient medium B5 ~as in Tables 1 and 2) which contained 1 mg/Q of 2,4-dichlorophenoxyacetic acid and which was solidified with 0.6~ agar. The medium had a pH of 5.5 prior to sterilization by autoclaving.
Within six weeks the explanted hypocotyls produced callus, neoplastic growth, of 3 to 10 mm diameter. The callus was transferred to fresh B5 medium supplemented with 1 g/~ of casein hydrolysate and 2 mg/Q kinetin (l-B5C medium). The callus material was su~cultured every four weeks using small callus pieces as inocula. After six months of subculturing, callus material was transferred to liquid l-B5C medium and from here on subcultured as 50 mQ batches in 250 mQ
Delong-flasks on gyratory shakers (150 rpm). The resulting suspension of cells and cell aggregates was transferred to fresh 3-B5C medium at wee~ly in_ervals. All of these cultures ~ere grown in continuous light of B00 - 1000 lux 1~3476'~

at 25-28C. For the production of biomass and cytodiffer-entia~ion of the culture leading to formation of codeine, inoculum was collected from these suspension cultures (on sterile filters) and about 2 g of cell mass was incubated with about 300 mQ of l-B5C medium in 1 Q vessels.
The 50 mQ batch cultures which were trans-ferred at weekly intervals, consisted mainly of small cell aggregates up to about 1 mm in diameter. These aggregates occasionally included less than 5 percent of cells of large size (average diameter > lO0 microns) having one or several polyploid nuclei (these cells were not growth-limited and were unable to produce codeine). These aggregates varied in pigmentation from dark brown to white.
When these cultures (or the 300 mQ production cultures) were allowed to become completely growth-limited, cytodiffer-entiation leading to the appearance of giant cells in increasing numbers occurred. From about 10% to about 50%
or more of the cells can be of this giant type.
A typical cell arrangement from one of the 300 mQ growth-limited, differentiated cultures is shown in the drawing wherein A are the giant cells and B the noxmal cells. This drawing is based on several photo-micrographs.

Isolation of Codeine After incubation and accumulation of codeine in l-B5C medium for 3 weeks, the medium (300 mQ) was extracted with ethyl acetate (2 x 50 m~) while the cells were treated with boiling metharol (150 mQ). Both the methanol and ethyl acetate extracts were combined and 3~ evaporated to sive a yello~ish light brown material which was redissol~ed in an ethyl acetate/1 ~ HCl (l:l) mixture (2 x S0 mQ). The two layers were collected separately.

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The ethyl acetate solution was washed with 1 N HCl (10 mQ) and the washings collected and combined with the acidic layer obtained previously. The acidic solution was treated with sodium bicarbonate to pH 7.5 - 8.0 and filtered.
After filtration the filtrate was extracted with ethyl acetate (2 x 25 mQ) and these extracts were combined with the ethyl acetate extract obtained previously. Evaporation yielded a yellowish brown material (98 mg) which was purified by PLC on silica gel plates (0.25 mm) using EtOAc:MeOH:NH40H (85:10:5) for development and CHC13:MeOH (8:2) as the eluent. This afforded codeine of reasonable purity (1.2 mg) having the same Rf (0.22) as authentic codeine when cochromatographed on TLC as above (M.P. 146-148C uncorrected, lit. 154-155). Yield was about 0.15~ based on the dry cell wt. (722.2 mg). Mass spectrometer data also served to identify the codeine product. The mass spectrum displayed a molecular ion at m/e 299, corresponding to C18H21N03, the molecular formula for codeine and was identical with that of the authentic codeine.

Claims (13)

CLAIMS:

1. A process for the production of codeine from plant cell cultures which comprises:
(a) preparing an inoculum of cells from a selected variety of Papaver somniferum L. or Papaver setigerum DC.
selected to form, on cell culturing in a suitable plant cell culture nutrient medium which becomes growth-limited, cyto-differentiated polyploid cells capable of producing codeine substantially free of other alkaloids, said cytodifferentiated cells being larger than the non-differentiated cells;
(b) inoculating a suitable nutrient medium for plant cell cultures with said inoculum cells, and allowing these cells to multiply until at least one essential nutrient component becomes growth-limited;
(c) allowing this growth-limited culture to undergo cytodifferentiation thereby forming said polyploid cells;
(d) accumulating codeine substantially free of other alkaloids in said cytodifferentiated cell-containing culture under growth-limited conditions; and (e) recovering codeine from the resulting culture.

2. The process of claim 1 wherein the variety is a selected Papaver somniferum L. one.

3. The process of claim 2 wherein the selected variety is of the type cv. Marianne.

4. The process of claim 1, 2 or 3 wherein the nutrient medium is of the type MS, ER or B5 as described in Tables 1 and 2 herein.

CLAIMS CONT:

5. The process of claims 1, 2 or 3 wherein the medium contains added organic nitrogen source nutrient for the multiplication stage.

6. The process of claims 1, 2 or 3 wherein the nitrogen-source nutrient material becomes growth-limiting.

7. The process of claims 1, 2 or 3 wherein the large cytodifferentiated cells are at least about 10% of the total cell population.

8. The process of claims 1, 2 or 3 wherein the cytodifferentiated cells have an average diameter of at least about 100 microns.

9. The process of claims 1, 2 or 3 wherein the inoculum is derived from seedling tissue.

10. The process of claims 1, 2 or 3 wherein the inoculum is derived from flower parts or pollen.

11. A plant cell culture from a selected variety of Papaver somniferum L. or Papaver setigerum DC. wherein the culture is growth-limited by depletion of growth nutrient and at least 10% of the cells cytodifferentiated to form large polyploid cells which accumulate codeine substantially free of other alkaloids.

12. The plant cell culture of claim 10 wherein from about 10% to about 50% or more of the total cells are large polyploid cells.

13. The plant cell culture of claim 11 or 12 wherein the selected variety is a Papaver somniferum one of the type cv. Marianne.