Enzyme inhibitor produced by cultivation of streptomyces microorganisms

A new compound, named "Griseolic acid", and its salts and can be prepared by the cultivation of Streptomyces griseoaurantiacus SANK 63479 (FERM-P 5223). Griseolic acid and its salts inhibit the activity of the enzyme cyclic adenosine monophosphate phosphodiesterase and, as a result of this, have variety of physiological activities and uses.

BACKGROUND OF THE INVENTION 
The present invention relates to a new compound, named griseolic acid, and 
salts thereof having activity as an enzyme inhibitor, and to a process for 
preparing them. 
Specifically, griseolic acid and its salts inhibit the activity of cyclic 
adenosine monophosphate (cAMP) phosphodiesterase (PDE) and can thus 
increase the level of cAMP in the cells of the patient being treated. 
It is well-known that cAMP, which is very widely distributed in animal 
tissues, functions as a second messenger for and mediates the effect of a 
large number of hormones; as a result, cAMP has a variety of very 
important physiological and biochemical roles. Additionally, it is known 
to have an effect on: division, growth and differentiation of cells; 
systole; haemapoiesis; various activities of the central nervous system; 
immune reactions; and liberation of insulin and histamine. Its 
concentration in tissues and hence its effect on these various functions 
depends upon the balance between the enzyme which synthetizes cAMP 
(adenylic acid cyclase) and the enzyme which splits cAMP (cAMP PDE). As 
inhibitor against cAMP PDE, griseolic acid and its salts would increase 
the level of cAMP in the cells and provide physiological activity of the 
type that is of value as an angiocardiokinetic agent, for example, as 
disclosed in U.S. Pat. No. 4,104,462 for the adenosine nitrates disclosed 
therein, an antiasthmatic agent, a smooth muscle relaxant, an 
antiinflammatory agent, a treatment of diabetes and a treatment of various 
types of psoriasis. 
BRIEF SUMMARY OF THE INVENTION 
It is, therefore, an object of the invention to provide, as a new 
composition of matter, a compound having an inhibitory effect against cAMP 
PDE. 
It is a further object of the invention to provide a process for producing 
such an inhibitory compound by the cultivation of an appropriate 
microorganism of the genus Streptomyces. 
The new compound of the invention is named griseolic acid and can be 
characterized by the following chemical structure and physico-chemical 
properties: 
##STR1## 
1. Colour and state: White powder. 
2. Melting point: 
Over 220.degree. C. (decomposition). 
3. Molecular weight: 
379 (by high resolution mass spectrometry). 
4. Molecular formula: 
C.sub.14 H.sub.13 N.sub.5 O.sub.8. 
5. Colour reactions: 
Positive for iodine, 2,4-dinitrophenylhydrazine and Bial's reagent. 
Negative for ferric chloride and ninhydrin. 
6. Optical rotation: 
[.alpha.].sub.D.sup.20 =+6.9.degree. [as its free acid, C=1.0, dimethyl 
sulphoxide]; 
7. Ultraviolet absorption spectrum: 
The ultraviolet absorption spectra, as measured in 0.1N hydrochloric acid 
and 0.1N aqueous sodium hydroxide, are shown in FIG. 1 of the accompanying 
drawings. 
8. Infrared absorption spectrum: 
The infrared absorption spectrum measured in a KBr pellet is as shown in 
FIG. 2 of the accompanying drawings. 
9. Nuclear magnetic resonance spectrum: 
Nuclear magnetic resonance spectrum as measured in hexadeuterated dimethyl 
sulphoxide at 60 MHz is shown is FIG. 3 of the accompanying drawings. 
The salts of griseolic acid may include, typically, alkali metal salts, for 
example, sodium, potassium and lithium salts; ammonium salt; alkaline 
earth metal salts, for example, calcium, magnesium and barium salts; and 
the like. The sodium and calcium salts are preferable. It is to be noted 
that the salts of griseolic acid are also new compounds and forming part 
of the present invention. 
DETAILED DESCRIPTION OF INVENTION 
We have found that griseolic acid and its salts can be prepared by 
cultivating a griseolic acid-producing microorganism of the genus 
Streptomyces and separating the griseolic acid or a salt thereof from the 
fermented broth. 
In particular, we have found that these compounds can be prepared by 
culturing a strain of actinomycetes hereinafter referred as "Strain No. 
SANK 63479", which has been newly isolated from soil samples taken from 
Kyoto in Japan. 
IDENTIFICATION OF STRAIN NO. SANK 63479 
Strain No. SANK 63479 was identified according to ISP (International 
Streptomyces Project) standards, the examination standards for the applied 
microbiological industry, Bergey's Manual (8th edition), "The 
Actinomycetes" by S. A. Waksman, and "Actinomycetes" by Krasilinikov, as 
well as other recent literature concerning the taxonomy of the 
actinomycetes. 
Strain No. SANK 63479 is found to form spiral sporephores having a chain of 
10 to 50 or even more spores. The spores have smooth surfaces and aerial 
mycelium grows on various madia. The aerial hyphae are simply branched. 
Strain No. Sank 63479 was deposited and assigned the strain designation 
NRRL 12314 on Oct. 22, 1980. 
The strain thus possesses properties similar to those of Streptomyces 
griseoaurantiacus. For the purpose of comparative tests, we used the type 
strain ATCC 19840 of Streptomyces griseoaurantiacus (Krassilnikov and Yuan 
1965) Pridham 1970. 
1. Morphological characteristics 
As shown in Table 1. 
TABLE 1 
______________________________________ 
Strain 
No. SANK 
63479 ATCC 19840 
______________________________________ 
Form of sporephores 
spiral spiral 
Surface of spores 
smooth smooth 
Branching of aerial 
simply simply 
hyphae branched branched 
Number of spores 10 to 50 or 10 to 50 or 
more more 
Special organs none none 
______________________________________ 
2. Growth on various media 
The two strains were cultivated at 28.degree. C. for 14 days on various 
agar plate media and exhibited the properties shown in Table 2. 
TABLE 2 
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Strain No. SANK 
Agar medium 
63479 ATCC 19840 
______________________________________ 
Yeast malt agar 
(ISP-2): 
Growth Very good, dull 
Good, pale 
reddish orange 
yellowish 
orange 
Aerial mycelium 
Abundant, grey 
Abundant, grey 
Reverse surface 
Dull reddish orange 
Yellowish 
orange 
Soluble pigment 
None None 
Oatmeal agar 
(ISP-3): 
Growth Very good, dull 
Very good, light 
orange reddish orange 
Aerial mycelium 
Abundant, grey 
Good, grey 
Reverse surface 
Dull red light reddish 
orange 
Soluble pigment 
None None 
Starch-inorganic 
salt agar: 
(ISP-4) 
Growth Very good, dull 
Very good, brown 
orange to pink 
Aerial mycelium 
Abundant, brownish 
Abundant, light 
white brownish white 
Reverse surface 
light brown Pink to dark 
reddish brown 
Soluble pigment 
None None 
Glycerine- 
asparagate agar: 
(ISP-5) 
Growth Very good, Good, pale brown to 
dull red pink 
Aerial mycelium 
Abundant, light 
Abundant, light 
brownish white 
brownish white 
Reverse surface 
Dull red Pale brown to pink 
Soluble pigment 
None None 
Tyrosine agar: 
(ISP-7) 
Growth Very good, dull 
Good, yellowish brown 
red 
Aerial mycelium 
Abundant, brownish 
Abundant, light 
white brownish white 
Reverse surface 
Brownish purple 
Dark brown 
Soluble pigment 
None None 
Sucrose- 
nitrate agar: 
Growth Good, dull reddish 
Moderate, light 
orange reddish orange 
Aerial mycelium 
Abundant, light 
Good, light 
brownish white 
brownish white 
Reverse surface 
Dull reddish Pale reddish 
orange orange to pale 
reddish brown 
Soluble pigment 
Pale purple Pale purple 
(slight) (slight) 
Glucose- 
asparagate agar: 
Growth Good, dull red 
Moderate, pale 
yellowish brown 
Aerial mycelium 
Abundant, grey 
Good, light 
brownish white 
Reverse surface 
Dull red Brownish white 
Soluble pigment 
Pale purple None 
(slight) 
Nutrient agar 
(Difco): 
Growth Good, pale Good, pale 
yellowish brown 
brown 
Aerial mycelium 
Scarce, brownish 
Good, light 
white brownish 
white 
Soluble pigment 
None None 
______________________________________ 
3. Physiological properties 
The physiological properties of the two strains are shown in Table 3. Three 
media were used to determine the production of melanoid pigments: 
tryptone-yeast extract broth (ISP-1), peptone extract-iron agar (ISP-6) 
and tyrosine agar (ISP-7). The results are reported as either positive (+) 
or negative (-). 
TABLE 3 
______________________________________ 
Strain No. 
SANK 63479 
ATCC 19840 
______________________________________ 
Reduction of nitrate 
+ + 
Hydrolysis of starch 
+ - 
Liquefaction of gelatin 
+ + 
Coagulation of milk 
+ + 
Peptonization of milk 
- - 
Production of melanoid 
- - 
pigments 
______________________________________ 
4. Utilization of carbon sources 
The utilization of carbon sources is as shown in Table 4. The culture 
medium employed was Pridham-Gottlieb agar (ISP-9) and evaluations were 
conducted after cultivation at 28.degree. C. for 14 days. The results are 
reported according to the following code: 
++: well utilized; 
+: utilized; 
.+-.: poorly utilized 
-: not utilized. 
TABLE 4 
______________________________________ 
Strain No. 
SANK 63479 
ATCC 19840 
______________________________________ 
D-glucose ++ ++ 
L-arabinose + ++ 
D-xylose .+-. .+-. 
D-fructose ++ + 
L-rhamnose + + + + 
i-inositol .+-. - 
galactose ++ ++ 
mannose ++ + 
sucrose - - 
D-cellobiose ++ .+-. 
lactose ++ .+-. 
maltose ++ .+-. 
raffinose - - 
D-mannitol ++ ++ 
insulin - - 
dextrin ++ ++ 
soluble starch 
++ .+-. 
salicin - - 
sodium succinate 
+ .+-. 
glycerine ++ + 
cellulose + .+-. 
control - - 
______________________________________ 
It can be seen from the results reported above that there are considerable 
points of similarity in morphological properties, physiological properties 
and carbon source utilization between Strain No. SANK 63479 and the type 
strain Streptomyces griseoaurantiacus ATCC 19840. Accordingly, Strain No. 
SANK 63479 has been identified as Streptomyces griseoaurantiacus 
(Krasilinikov and Yuan 1965) Pridham 1970. This strain was deposited on 
Oct. 9, 1979, at the Fermentation Research Institute, Agency of Industrial 
Science and Technology, Japan, from which it is available under the 
Accession No. FERM-P 5223, and on Oct. 22, 1980 at the Agricultural 
Research Service, Peoria, U.S.A., from which it is available under the 
Accession No. NRRL 12314. 
The inhibitory activity of the compounds of the invention (or of 
compositions, e.g. culture broths, containing them) may be assayed by the 
method of Pichard and Thun [Journal of Biological Chemistry, 251, 
5726-5737 (1976)], using a crude enzyme solution derived from rat brains 
as the cAMP PDE. In this method, .sup.14 C-labelled cAMP is used as the 
substrate and is reacted at 30.degree. C. for 20 minutes with 2-5 .mu.l of 
the compound or composition under test, 20 .mu.l of a snake venom and 40 
.mu.l of the crude enzyme solution in a 0.2M tris-HCl buffer (pH 8.0). The 
tris-HCl buffer is a mixture of tris(hydroxymethyl)aminomethane and 
hydrochloric acid. After completion of the reaction, the reaction mixture 
is treated with an Amberlite (Trade Mark) IRP-58 resin. From the residual 
radioactivity of the adenosine in the product, the inhibitory activity of 
the compound or composition against the cAMP PDE can be calculated as a 
percentage. 
As is well-known, the properties of actinomycetes, including Streptomyces, 
strains are not fixed and they readily undergo mutation both through 
natural causes and as the result of artificial mutation. Although the 
invention relates to the production of griseolic acid and its salts 
especially by the cultivation of the above identified Streptomyces 
griseoaurantiacus SANK 63479, it also includes within its scope the use of 
mutants of this organism and generally of any Streptomyces strain which is 
capable of producing griseolic acid and its salts. 
The cultivation of the griseolic acid-producing microorganism, in 
accordance with the process of the invention, can be performed under the 
conditions conventionally employed for the cultivation of Actinomycetes 
strains. Shaken culture in a liquid medium or a solid cultivation method 
are preferred. 
The nutrient medium used for the cultivation can be of a composition such 
as is conventionally used for the cultivation of Actinomycetes. Thus, it 
would contain an assimilable carbon source and an assimilable nitrogen 
source. Suitable assimilable carbon sources include: a concentrated 
solution of a sugar (e.g. of sucrose and/or invert sugar or of a mixture 
of sucrose with another sugar, such as glucose or corn syrup), starch, 
dextrose, mannitol, fructose, galactose or rhamnose or any combination of 
two or more thereof. The nitrogen source may be: an organic or inorganic 
compound, e.g. ammonium chloride, ammonium sulphate, urea, ammonium 
nitrate or sodium nitrate; or natural products, such as peptone, meat 
extract, yeast extract, dried yeast, live yeast, corn steep liquor, 
soybean powder, soybean flour, casamino acid or soluble vegetable 
proteins. A single such nitrogen source or a combination of any two or 
more may be employed. In addition, the nutrient medium may also contain 
inorganic salts(such as potassium chloride, calcium carbonate or 
phosphoric acid salts), optionally together with other organic or 
inorganic substances to promote the growth of the microorganism or its 
production of griseolic acid and/or salt thereof. 
The method of cultivation may be a liquid cultivation method, with 
reciprocal or rotatory shaking, or a solid cultivation method, a 
deep-stirring cultivation method being particularly preferred. Although 
the microorganism will grow over a wide range of temperatures, it is 
particularly preferred to effect the cultivation at a temperature of from 
20.degree. to 35.degree. C. and at a substantially neutral pH value. When 
a liquid cultivation method is employed, the cultivation is normally 
effected for a period of from 48 hours to 120 hours, during which time 
griseolic acid and/or a salt thereof is formed and accumulates in the 
culture broth. The progress of the cultivation may be monitored and the 
content of griseolic acid in the broth estimated by determining the enzyme 
inhibitory activity of the broth using the method described above. After 
completion of deep liquid cultivation, the culture broth will generally 
show an inhibitory activity of from 70 to 85%. 
Griseolic acid is an acidic, water-soluble substance and normally and 
preferably exists in the culture broth in the form of its calcium salt. It 
is, therefore, possible to employ methods of separation and purification 
of the type commonly used for the isolation of water-soluble microorganism 
metabolic products. In the case of the deep cultivation method, the 
preferred separation and purification procedure is as follows. First, the 
cells of the microorganism are separated by filtration or centrifugation 
and the resulting filter cake is washed with water. The washings and the 
filtrate or supernatant liquor from centrifugation are combined and the 
combined liquor is treated, in turn, with activated charcoal or another 
adsorbent and an ion-exchange resin. The adsorption may be conducted 
either batch wise or by continuously feeding the liquor through an 
adsorption column. In the batch method, for example, an activated charcoal 
adsorbent is preferably added in an amount of from 0.1 to 0.6% w/v, more 
preferably from 0.35 to 0.40% w/v, to the filtrate and the resulting 
mixture is stirred for a period of time of from 30 to 60 minutes. 
The activated charcoal adsorbent is then eluted with aqueous acetone or an 
aqueous lower alkanol and the eluate is concentrated by evaporation under 
reduced pressure. The residue is then further purified by means of 
ion-exchange resins, an activated charcoal column and a Sephadex column to 
give pure griseolic acid having the properties hitherto described. 
The inhibitory activity of griseolic acid against cAMP PDE in terms of its 
50% inhibitory value (I.sub.50) are shown in Table 5 against cAMP PDE from 
a variety of sources having either a high or a low Michaelis constant 
(K.sub.m). The results are reported as .mu.moles (.mu.M). 
TABLE 5 
______________________________________ 
low K.sub.m cAMP POE 
high K.sub.m cAMP POE 
______________________________________ 
cAMP 
concentration 
0.14 .mu.M 100 .mu.M 
in substrate 
cAMP POE 
source: 
rat brain 0.16 .mu.M 0.80 .mu.M 
rat heart 0.036 .mu.M 0.34 .mu.M 
rat aorta 0.031 .mu.M 0.51 .mu.M 
rat plate- 
0.041 .mu.M 8.0 .mu.M 
lets 
rat kidney 
0.12 .mu.M 0.58 .mu.M 
______________________________________ 
As can be seen from the results reported in Table 5, griseolic acid has a 
remarkably potent and specific inhibitory effect against both high and low 
Michaelis constant cAMP PDE. By comparison, Papaverine, which is known in 
the art to be an inhibitor against this enzyme, has an I.sub.50 value of 
3.5 .mu.M against a low Michaelis constant cAMP PDE (cAMP concentration 
0.14 .mu.M) derived from a rat brain. Thus, the inhibitory activity of 
griseolic acid is about 20 times stronger than that of papaverine. Indeed, 
it is believed that griseolic acid has the strongest inhibitory activity 
against this enzyme of those natural products hitherto tested. Moreover, 
basic tests on cytotoxicity using Hela S-3 strain mouse fibroblast cells 
showed no inhibition of cell growth even at concentrations of 100 
.mu.g/ml, thus indicating a probable lack of toxicity to human and other 
animals. 
Griseolic acid or its salt of this invention can be applied for the 
treatment of various diseases as explained hereinabove. For instance, 
griseolic acid may be used as an antiasthmatic agent, a smooth muscle 
relaxant especially for chronic bronchitis and also as an 
angiocardiokinetic agent for various types of coronarisms caused by 
arteriosclerosis or thrombus. The compound of this invention may be 
administered by any conventional means, for example (e.g. by subcutaneous, 
intravenous or intramuscular injection or topical application) or orally 
(e.g. in the form of tablets, capsules, powders or granules). The daily 
dose for adults will, of course, vary depending upon the age, body weight 
and condition of the patient, as well as upon the route and times of 
administration. However, the compounds of this invention are in general 
administered in an amount of 1 to 100 mg/day via oral route or in an 
amount of 0.1 to 10 mg/day via parenteral route in several divided doses.

The invention is further illustrated by the following non-limiting 
Examples. 
EXAMPLE 1 
30 liters of medium having pH of 7.0 before sterilization and the following 
composition (percentages are w/v) were prepared: 
______________________________________ 
Glucose 5% 
Soybean powder 1% 
Yeast extract 0.1% 
Polypeptone 0.4% 
Meat extract 0.4% 
Sodium chloride 0.25% 
Calcium carbonate 0.5%. 
______________________________________ 
15 liters of this medium were charged into each of two 30 liter jar 
fermenters, which were then sterilized under pressure at 121.degree. C. 
for 30 minutes. After cooling, 150 ml of a culture broth of strain SANK 
63479 (which had previously been incubated in the medium described above 
by means of a rotatory shaking cultivator at 28.degree. C. for 72 hours) 
were innoculated into each fermenter. Cultivation was then carried out at 
28.degree. C. for 72 hours under aeration at the rate of 10 liters/minute 
and with agitation at 200 rpm. After completion of the cultivation, 2 
.mu.l of the culture filtrate was found to exhibit an inhibitory activity 
of 76%. 
The combined filtrates (pH 6.5) from the two jar fermenters, a total amount 
of 28 liters, were passed through Diaion HP-20 (a trade mark for an ion 
exchange resin produced by Mitsubishi Chemical Co., Limited) and were then 
adsorbed on carbon and washed with water, after it was eluted with a 60:40 
by volume mixture of acetone and water. After evaporating off the acetone 
from the eluate, the aqueous solution was concentrated and then subjected 
to lyophilization to give 120 mg of a crude powder. 
This product was dissolved in a samll abount of distilled water and 
adsorbed on Dowex 1.times.4 Cl.sup.- form (a trade mark for an ion 
exchanger produced by Dow Chemical Co.) to convert the calcium salt to 
free griseolic acid. The desired acid was then eluted using various 
concentrations of aqueous sodium chloride and column chromatography was 
thereafter repeated using a Sephadex LH-20 resin produced by Pharmacia Co. 
The active fractions thus obtained were adjusted to a pH value of 3.0 by 
the addition of 0.1N hydrochloric acid and column chromatography was again 
repeated using Sephadex LH-20. Finally, the product was subjected to 
preparative thin layer chromatography using silica gel 60 F.sub.254 
(available from Merck & Co.) with a plate thickness of 0.25 mm developed 
with a 4:1:2 by volume mixture of butanol, acetic acid and water; Rf value 
0.08. There were obtained 31 mg of the desired griseolic acid. 
Alternatively, in the procedure described above, an excess of a saturated 
aqueous solution of calcium hydroxide can be added to the pH-adjusted 
active fractions and the resulting mixture cooled to 4.degree.-5.degree. 
C. to separate a precipitate. This precipitate was recovered by 
filtration, giving 42 mg of the desired calcium salt of griseolic acid. 
The appearance, melting point, colour reactions and ultraviolet adsorption 
spectrum are the same as those reported above for the free acid, but this 
product was confirmed to be the calcium salt by X-ray fluorescence 
spectrometry, which showed an energy position exactly corresponding to 
calcium. 
EXAMPLE 2 
The procedure described in Example 1 was repeated, except that 600 liter 
tanks, each containing 300 liters of the medium described in Example 1, 
were employed. After cultivating the microorganism for 48 hours, 5 .mu.l 
of the culture broth were found to exhibit an inhibitory activity of 76%. 
The desired product was separated and purified from 280 liters of the 
culture broth, following the procedure described in Example 1, to give 531 
mg of a product showing a single spot on thin layer chromatography using 
the same plate and developing solvent as in Example 1. The product was 
then dissolved in 0.1N hydrochloric acid to complete conversion to the 
free acid and was then subjected to repeated procedures to convert it to 
the calcium salt. There were finally obtained 400 mg of a pure product 
having the same properties as that obtained in Example 1.