DE1642699C3 - Process for the production of mycophenolic acid - Google Patents

Description

Mycophenolic acid has the formula

ΓΗ ^OH

Y "3!

HO ₂ C ■ ■ CH ₂ CH ₂ · C = CH · CH ₂ -f \ - CO _x

I ο

CH ₃ OA / -CH /

CH ₃

In GB-PS 11 57 100 it is reported that this Acid and its salts and esters are mitotic suppressors and have antitumor activity as well as immunosuppressive Have activity.

In the literature, the production of this acid is both by surface cultivation and by submerged aerobic cultivation of mycophenolic acid-producing Penicillium species, e.g. B. Penicillium brevicompactum. In general, microorganisms suitable for a person skilled in the art are the Composition of suitable nutrient media, the cultivation conditions, the method of isolation of the Mycophenolic acid from the medium and methods of making it pharmaceutically useful Commonly used with salts. The previously used process for the production of mycophenolic acid by submerse aerobic cultivation was very poor and gave a yield of only about 9 mg acid / l kililtur filtrate.

The invention was based on the object a To create a process for the production of mycophenolic acid that enables higher yields !.

It has now been made the surprising discovery that in a submerged aerobic cultivation increased yields of mycophenolic acid can be obtained by ensuring that the Nutrient solution contains a certain amount of assimilable magnesium during the entire cultivation process contains.

Accordingly, the object on which the present invention is based is achieved by a method for the production of mycophenolic acid by aerobic submerged cultivation of a mycophenolic acid-producing Penicillium representative, which is characterized in that a nutrient medium is used that at the beginning of the fermentation sources of C, N, P, S and K and small amounts of trace elements as well as a magnesium source in a concentration of 10 to per liter of the nutrient medium Contains 100 mg of magnesium, and that in the sewing medium Magnesium swell is present throughout essentially the entire process.

Suitable active Penicillium species are:

Penicillium brevicompactum, D i e r c k χ (for example, strains listed in The Commonwealth Mycological Institute, Kew, England under the designation I.M.I. 40225, 92034, 92044, 92262, 92274 and 94149),

Penicillium stoloniferum. Thorn (for example I.M.I. 39824, 91960, 92219 and 126540, the latter strain being I.C.I. No. ACC 438 is) and

Penicillium viridicatum, W e s 11 i η g (for example I.M.I 49096).

The cultivation is usually carried out at a temperature in the range of 18 to 38 ° C and preferably carried out in the range from 20 to 27 ° C.

Examples of a suitable source of magnesium are non-toxic soluble derivatives or salts of magnesium, such as. B. Magnesium sulfate and those containing magnesium Compounds found in corn extract fluids and similar materials. It's on it to point out that the entanglement of the microorganisms after magnesium the concentration of the cells which in turn is proportional to the amount of assimilable nitrogen source present in the nutrient medium is proportional. Depending on the conditions, 0.00175 to 0.00288 mg of magnesium are required so that 1 mg of cells (dry weight) can be formed that produce mycophenolic acid. If the If the nitrogen source has just been used up, then carry 0.09 to 0.26 mg of assimilable nitrogen source each according to the conditions for the formation of 1 mg cells (dry weight).

In addition to the magnesium source, the culture medium contains sources for at least at the beginning of the process essential elements, d. H. Carbon, nitrogen, phosphorus, sulfur and potassium.

The culture medium usually also contains small amounts of so-called trace elements such as z. B. iron, manganese, zinc, molybdenum and copper. The carbon source can be from a sugar, such as. B. Sucrose or glucose, a polyhydric alcohol, such as. B. glycerol or an ester thereof, a Polysaccharide, e.g. B. "Starch or a vegetable oil. The nitrogen source can be an inorganic or be an organic source. Examples of inorganic nitrogen sources are ammonium salts or nitrates, such as. B. ammonium nitrate, ammonium tar-

stepped, ammonium sulfate, ammonium phosphate, sodium nitrate, Potassium Nitrate and Calcium Nitrate Examples of organic nitrogen sources are amino acids such as Glycine, a seed meal such as cottonseed meal, corn extract, peptone, urea, yeast extracts and meat extracts. The sources of phosphorus, sulfur and potassium can for example be from Potassium dihydrogen phosphate, a sulfate or potassium chloride exist.

In one embodiment, the method of the invention is implemented in a so-called unbalanced manner Culture medium carried out, i.e. that the amount of various nutrients in the culture medium such it is chosen that at an intermediate stage of the procedure a source from a vital one Element (with the exception of magnesium), e.g. B. the nitrogen or carbon source is consumed, while the sources of the other vital elements are still there. With one of these Method, the nitrogen source is preferably consumed first. You can proceed in such a way that the Culture medium at the beginning of the process 1 to 300 g carbon source (e.g. 100 g glucose) per liter Culture medium and 0.01 to 10 g, e.g. B. 0.65 g. Nitrogen source (expressed as the weight of elemental nitrogen) per liter of culture medium If that contains The method according to the invention is carried out in an> unbalanced culture medium in which the If the nitrogen source is consumed first, then experience has shown that better yields of mycophenolic acid are obtained versus the use of a balanced culture medium (i.e., i-ines culture medium in which the Nutrients are used up at approximately the same time). When applying an unbalanced Culture medium one can also proceed in such a way that the nitrogen source is consumed while still some carbon source and of course magnesium source are present in the culture medium, whereupon the available carbon source is maintained or increased by adding more amounts of the carbon source be admitted.

After cultivation has ended, the mycophenolic acid can be extracted from the culture medium in a manner known per se Way to be separated. The improved method according to GB-PS 11 58 387 can be used for this purpose. Here, the pH of the culture broth is first adjusted to 10 to 11 before filtering, whereupon then the filtrate and washing liquids at an alkaline or acidic pH with a suitable one Solvent or solvent mixture are extracted.

The invention is illustrated in more detail by the following examples.

example 1

A culture medium was prepared which contained the following components per liter:

ZnSO ₄
MnSO ₄ · 4 H ₃ O

glucose

NH ₄ NO ₃

KH ₂ PO ₄

MgSO ₄ · _{7H 2} O

Trace element concentrate

Distilled water on

100 g
3.72 g
5g

Ig
2 ml
1 ml

The composition of the trace element concentrate was as follows:

Distilled water on

I ₁ og
0.1g
0.1g
11th

A few drops of concentrated hydrochloric acid were added to make the solution clear.

51 culture medium with spores of the organism Penicillium stoloniferum (I.C.I. strain no.

κι ACC 438, i.e. l.M.I. strain 126540) in one vessel inoculated, which at a stirring speed of 712 rpm and was maintained at 25 ° C with an air flow of half a volume / volume / minute. After 93 st were 65 ml of an 80% (w / v) glucose solution were added.

After 215 hours, the mycelium was filtered off from the culture broth. 1 1 of the filtrate was treated with concentrated hydrochloric acid adjusted to pH 2 and extracted with ethyl acetate. The first extraction was carried out with 200 ml of solvent and the second and third extraction each with 100 ml

: »Solvent. The combined ethyl acetate extracts were dried with anhydrous sodium sulfate, after which the sodium sulfate was removed by filtration became. The ethyl acetate solution was evaporated to dryness in vacuo yielding 2.5 g of a pale brown

: ". Solid, which was dissolved in 100 ml of ethyl acetate. The solution then became 5 g Activated charcoal added. The mixture was! Refluxed for 0 min and then the charcoal was turned off removed by filtration. Now 5CC ml were diluted

in hydrochloric acid (4% [V / V] solution of concentrated Hydrochloric acid in water) was slowly added, whereupon the mixture was left to stand until crystallization ended. The mixture was filtered and the crystalline residue was first diluted with

j-, hydrochloric acid and then washed with petroleum ether (bp. 40 to 60 ⁰ C). The crystals were then dried in vacuo. In this way, 190 g of crystals in the form of needles with a melting point of 139 ° C. and a content of 95.4% mycophenolic acid (were determined

4 (i in methanol solution by absorption at 304 μπι) obtain.

Example 2

In a jar with a capacity of 1136 l, 654 l became one ι ". Introduced culture medium of the following composition.

glucose

NH ₄ NO ₃

KH ₂ PO ₄

MgSO ₄ · H ₂ O

Yeast extract

Trace element concentrate

(see example 1)

12% w / v
0.52% w / v
0.5% w / v
0.056% w / v
0.1% w / v

0.2% v / v

FeSO.!
CuSO.,

7H ₂ O
5 H ₂ O

1.0g
0.15g

After inoculation with 73 l of a growing culture of Penicillium stoloniferum (ICI strain No. ACC 438; ie IMI strain No. 126540), the culture medium was incubated at 23 ° C. with stirring at 200 rpm, the hourly air supply was about 25.5 m ³ . After 99 hours, the amount of mycophenolic acid according to fluorescence spectrophotometry (see Example 3) had increased to 1114 mg per liter. Harvesting took place as usual.

Comparative example I.

Two cultivation samples were carried out (on a 5-liter basis). carried out under the same conditions with the following culture media.

Container A

conditions

Air flow (l / min)
Temperature CQ
Stirring speed (rpm)

Culture medium

glucose
NH ₄ NO ₃
KH ₂ PO ₄

Trace element concentrate (see example 1)
MgSO ₄ · _{7H 2} O

2.5
23
712

12% w / v 0.19% w / v 0.01% w / v 0.2% v / v

2.5 23 712

12% w / v 0.19% w / v 0.5% w / v 0.2% v / v

0.1% w / v 0.002% w / v The containers were filled with spores of Penicillium stoloniferum (I.CI. Strain No. ACC 438; i.e. I.M.I. Strain No. 126540), after which samples were taken at intervals. Each sample was with a 10 N potassium hydroxide solution brought to a pH value of 10, stirred and filtered. The filtrate was then brought to a pH of 2 with citric acid and then extracted with benzene Benzene solution was back extracted with a 0.02N sodium hydroxide solution. The concentration of mycophenolic acid (present as the sodium salt) in the aqueous solution was fluorescence spectral photometry measured (measurement of the fluorescence emission at 428 μm with an activation wavelength of 338 μm).

The results obtained were as follows:

Old container A

Dry magnesium weight

(st) (mg / g) (m _c -nil)

Mycophenolic acid

(mg / 1) container B

Dry weight

(mg / g)

Magnesium MylOphenol-

(mg / ml)

(mg / 1)

168
191

10.1
9.5

0.12 0.12

125 195 6.0

zero
zero

55 <50

These results demonstrate the benefits of using a source of magnesium.

Although the culture medium contained different levels of KH2PO4, the results are meaningful, namely, because there is no indication that the production stimulated by mycophenolic acid due to a lack of phosphorus. This was demonstrated by the comparative example 2 confirmed.

Comparative example 2

In order to confirm the essential role of magnesium, experiments were carried out in shaking flasks with carried out with the following culture medium:

Culture medium

glucose

Ammonium tartrate
KH ₂ PO ₄

Trace element concentrate
(see example 1)

10% w / v 0.427% w / v 0.5% w / v

0.2% v / v

Temperature 25 ° C

Shaker speed 200 rpm

Shaker amplitude 50.8 mm

The concentration of the product in each bottle was measured as in Comparative Example 1 with the difference is that the measurement sensitivity is adapted to the lower concentrations to be expected became. The results were as follows:

Various amounts of Magnesium, calcium, strontium or barium salts, as indicated below, are added. After sterilization three replicates were made from each sample with 'spores of Penicillium stoloniferum (I.CI.-Strain No. ACC 438; i.e. I.M.I.-Strain No. 126540), after which cultivation was carried out under the following conditions:

Salt added

Mycophenolic acid after 164 st

(mg / 1)

4-, comparative tests
MgSO ₄ · _{7H 2} O

Ca (NO,) ₂ -4 H ₂ O
Ba (NO ₁ ):
Si (NOj) ₂ -4 H ₂ O

(no

Salt addition)

0.001%

0.010%

0.050%

0.006%

0.030%

0.002%

0.010%

0.003%

0.016%

42

48

211

226

28

42

21

21

14th

14th

The results show
Magnesium.

the essential role of the

Claims (4)

Patent claims: 1. Process for the production of mycophenolic acid by aerobic submerged cultivation of a Mycophenolic acid-producing Penicillium representative -. ters, characterized in that one a nutrient medium is used that contains sources of C, N, P, S and K and at the beginning of the fermentation small amounts of trace elements as well as an additional magnesia ι ο per liter of the nutrient medium umquelle in a concentration of 10 to 100 mg Magnesium keeps, and that in the nutrient medium Magnesium swell is present throughout essentially the entire process 2. The method according to claim 1, characterized in that r> draws that the amounts of the nutrient medium components in the nutrient medium at the beginning of the fermentation so be chosen so that the N source is consumed at an intermediate stage of the process, while the sources of the other vital ones: “Elements are still there. 3. The method according to claim 2, characterized in that the nutrient medium at the beginning of the Fermentation 1 to 300 g of C source per liter of nutrient medium and 0.01 to 10 g of N source, as weight r> of elemental N, per liter of nutrient medium 4. The method according to claim 2, characterized in that the N source is consumed while there is still some C source in the nutrient medium m, whereupon the available C amount is maintained or increased by adding further amounts of the carbon source to the nutrient medium became.