DE2022680A1 - New anti-inflammatory substances and processes for their production - Google Patents

Description

DILMOLLEIIOII DlfL-fHYI. DlMANITI DIPL-CHEAA. Dl DEUFEL DIfL-ING. FINSTEIWALD DIfL-ING. GtXMKOW

Munich, the ^l *** Lq / th - 0 1007

ΟΝΟ HUKfUCHiTICAL CO ·, LTD. 14-, Doshomachi 2-chome, Higaehi-ku, Osaka,

Japan

Nowadays, preventive substances and methods of their manufacture

Priorities: Japan from 9 May 1969
No. 35 877/69

Japan September 26 , 1969 JJr. 76 673/69

The invention relates to new, anti-inflammatory subrticles and a process for their production.

Various anti-inflammatory drugs are known. They are roughly divided into steroids, anti-discharge gowns such as cortisone, dexasiethasone, etc. and non-steroid, anti-inflammatory drugs such as salicylates, pharmaceuticals

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of the prraaolone type and indomethacin. Recently, anti-inflammatory, enzyme-based pharmaceutical compositions have become well known and have been used in practice. However, the specifics are not known dee pharmakologisehen Wirkungsmechnnismus of entzündungsverhütenden drugs, and therefore apparently the unlimited possibility for discovery of new materials with appropriate zündungsverhütend he is given effect.

On the other hand, studies on the use of metabolites of microorganisms including antibiotics for medical purposes have been made extremely widespread. In such extensive investigations it has now been found that a substance having strong entzündungsverhütender effect IR a Jernentationslöeung is present ₉ y # luiig AUB I ^ Jermentierung of dates to be explained later microorganisms · Such entzündungsverhütende substance is water soluble, resistant to Varna, acids and alkalis , and it has not yet been discovered that it exists "not only in microorganisms but also in higher plants and animals.

According to the invention, a microorganism belonging to Aerobacter cloacäe, Aerobacter aerogenes, Bacillus subtilie, Hicrococu · lysodeikticua and Pseudomonaa aeruginosa is fermented in an ordinary culture medium containing a source of carbon and nitrogen and inorganic salts, and an ent anti-ignition substance with a high molecular weight obtained from the fermentation liquor

The invention will be more \ ρψ individual described below to show this, reference is made in part to the drawing) in this:

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Fig. 1 is a diagram showing a distribution of gel filtration an anti-inflammatory substance gen £ 2 of the invention on a column with three-dimensional cross-linked polysaccharide from linear dextran lüit low Vernetaungagrsd (Sephadax G-2CC), which iia following from negated polyaccharide will be referred to reproduces! and

Fig. 2 is a diagram showing the OT absorption spectrum according to the invention Substance reproduces

At the time of the implementation of the Erfiaduaf everyone can iUkroorganiMBue which to Aerobacter cloacae, Aerobacter aerogenea, Bacillus mibtilis, Kicrococua iTeodeikticue and Pseudoaonas aerug '. icaa heard, be used. However, those T ^ r ^ «sdat, which susr species Aerobacter because these hlna i cloth d« r 'Sr $ iebl | Sk «lt of the desired, inflammatory gyarnutasdea ikw ^^ anä ^ ® &? ^ Al ^ as

They are mentioned above in the literature b "ecnrleb" ci and aisd an eich well-known, and BiQ are easily nui sfrentuchen EuIturen "rhaltiiaSi, therefore J ~ aa hiersu kain · velteran Srlünterusgsi necessary.

According to the invention, such a microorganism is distant in a culture medium. The Hadius can be derived from the usual äahrbeatandteil & n suaaamengee «tit seia ₉ the Oblicherweia when the above mentioned lükroorgenlamen Tezvesdet. Bo contains de «Hedlum sources of carbon and nitrogen and anorjanous salts. Hubes special kSn & ea usual loulllonaedlua and another sjnthetlaches medium, a. B. Papton-fiohsvucke? " or Aapaxiigi rta ^ diua, Terwaadat be ·

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The fermentation can take place in a conventional manner Ventilation must be carried out. Any of the usual stirred diving cultures.1, shuttling cultures and stationary Cultures can be applied.

In spring cases, the cultivation or fermentation can be carried out at a temperature of about JO ⁰ C to 40 ° C, preferably at eWa 25 to 37 ° C;

As the microorganism grows, an inflammation-preventing substance is produced which is enriched in the culture medium. The concentration of the substance usually reaches its maximum after about 16 to 24 hours of exposure.

The enriched anti-inflammatory substance in the Culture and fermentation broths according to different Modes, which are explained below, are isolated and purified.

Working method I

During the cultivation of the microorganism HRMi, the resulting fermentation solution is subjected to centrifugal separation into supernatant liquid and cells a ·· rotary evaporator is concentrated »and el« is obtained by sterilization 2. B. at about 100 ° C for about 20 minutes. Bech On cooling, to remove unlSeUch "" Katerial the liquid s ^ trlfueiert, and ei® "ill 4ann in" in "Malj" i "3> *

Überfuhrt and Hinter linf © Mta HtuBiiez ¹ wSIwsbö 2 ■ 3 d zur i & tfernusig τοη Sti & s ^ sia® ^ with nl® & £ lfgtii Holt ·

Entrifugiert «u» everything milo3llelie liat ^ rlal fsn

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tind it is concentrated to a suitable volume in the same way as explained above. The concentrate is after the acetone fractionation or without fractionation by means of a filter, a.'B. crosslinked polysaccharide with a not quite as low degree of crosslinking (Sephadex G-100) gel-filtered. The anti-inflammatory substance appears only in the fraction at the frontal sod. The active fraction is further gel-filtered with crosslinked polysaccharide with a low degree of crosslinking (Sephadex G-2Q0). In this case, too, the fraction generally at the front has an activity, it represents a practically colorless solution. This solution is lyophilized, or it is dehydrated by suspending it with acetone. A purified powder of the anti-inflammatory substance is obtained

The anti-inflammatory substance can also be obtained from the cells. If a small amount of cells is treated, the extraction can be carried out by grinding the cells with quartz-containing glass powder in a mortar . However, treatment can give me a great propensity of cells folgeMeaArbeltswelseaaogewandt be ι

(1) Application of osaotic pressure.

In this way of working , the washed cells. suspended in a small amount of Vaeser, and the suspension is placed in a 3 HK ₂ HPO ^ -LO solution. The whole is left to stand overnight in a tree with low (temperature and then ia a · * large Vsesox * »■ · given Änge The suspension is konsentriert means" Inas Botationsverdampf ers and sentviftigier |. ₉ are shown below in order supernatant liquid, d ^ se is used as partially purified material

(2) Freezing and thawing method «

In this way of working, the Gewascnonwi tmllmt are suspended in a small amount of water.

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Buopansion is placed in its freezer below -20 ° C and left frozen longer than overnight. Daa frozen product is then placed in warm Melted water. This operation is repeated two or three times. Then the suspension centrifuged and the supernatant! liquid is used as partially purified material.

In either case, the partially purified material becomes concentrated and heated, e.g. B. to 100 ° C for 20 min to protein and nucleic acid by precipitation to remove. The heated solution is cooled and then centrifuged, and the supernatant liquid is dialyzed with water. The subsequent © cleaning is carried out in the same way as before in With regard to the solution of the KuXtur £ iIts? Sftes es-AÄ ^ tert.

Working method II

The fermentation solution is adjusted to pH 3-4 with an acid, e.g. B. 3 N HCl, and it is separated into a supernatant liquid and cells by means of centrifugation.The supernatant liquid is cooled to M- ~ £ ° C, and qb % dxd lalium chloride is added so that there is an Eo & sent ^ atiosi of 1 - J> % * preferably about 3% beeitzt® Bum. If ethanol is added "so that the AikolioienakonEefitrmtioA QQ " eats 80%, preferably about 50 % "The solution is left at 4 -" 5 ° C ® "r HacJs.t" Dojqs. the larger rope d®r übersttaeA is uad "the liquid removed by BelEintierea" of precipitation durcli centrifugation is collected · Ber Miefitrselalag is dissolved in Masser, and he is as ei h again with I in the same way the brush in © Aa ¥ esoa & © lf "v @ a IsliumcfelorM alt Xtbanol eusfalls ·

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The precipitate is dialyzed wildly with water, and then the internal liquid from dialysis is lyophilized, in order to obtain an inflammation-preventing subetans.

The properties of the so-obtained, anti-inflammatory sub-tones?, En oind depending on the type de · for the initial addiction of applied boctery is not so different.

Thus, the anti-inflammatory substances obtained according to the invention are tasteless, odorless, colorless, water-soluble and neutral. They are in common solvents such as methanol, ethanol, acetone, bensol, JLthar-etc. irrevocably. According to the investigation «! «Ittsl · a gel filtration, c · B« up front · t »te · poly» acch * rid with low degree of crosslinking (Sephadex 0 * 200) or eof Eic-Gel F-300 is weighted on el® molecular weight vcsa isJ5fe «r nls 200,000 geechloeeen (Fig. 1) "Ussaels of the Sjeldahl method bestiaate Ajuonialcstifflsit ^ ff about 5% The UT-Absorption pectniA (fig. 2) shows ms® an absorption with short waves, however barking maximum absorption at 280 and 260 ap, which is special for protein and nucleic acids kemuteiohaeBd «lad · sesQglieb of the color reaction is the molar reaction, which is the common color reaction to saccharides, poaitiT · bit symcydrin reaction, which is a larbreaärtion is, and also the biuretre ctioA, which is on peptide bonds, are positive. However, the xanthoprotein reaction in response to £ rotei & is negative. «The Hhodaminf coloring ale Hachveis reaction for lipids is negative for direct coloring, however it is for« ix »». Cbloroformmethsnolextrakt positive. Bio specific * WI ⁶ «5« + 45 ° (C - 1, water),

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"is about 0.22%. Furthermore, the substance cannot be dialysed and it is stable at 100 ° C for 10 minutes, it is also stable at a pH value of 4 to 10, even treatment with such enzymes like Pro tea 30 or Amylase v. ^r, the anti-inflammatory activity is not reduced.

The anti-inflammatory activity of this substance is explained in Table I, using a sample obtained using Aerobacter cloacae ale microorganism .

The anti-inflammatory activity was determined by the Martin method on thigh edema in hats. For this purpose, male Wister rats, each weighing 100 s, were used, each group consisting of five rats. A group of three rats was also used in the clinical liver search. The test sample was dissolved in a gram of physiological saline solution to a concentration of 10 Ω / lcg, and the solution obtained was applied. 0.1 ml of 0.5% carragheenin were injected subcutaneously into the rear left thigh and 0.1 ml of a physiological saline solution subcutaneously into the right rear thigh within JO min after application of the test sample . Every hour for 2-6 hours after the injection of the Carragheenine , the anti-inflammatory effect was examined by measuring the volume of the edema.

From Table I it can be seen that not only the usual application but also the administration through the duodenum is effective.

The toxicity in rats was LD ₅₀ " 750 T / kg in the case of intravenous injection, 1 mg / kg in the" case of intr "- peritoneal injection and 5" 1 mg / kg in the case of intramuscular injection.

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Table I.

i-pp 1 i kai- i ο nsweg

Do as the extent (%) * of edema inhibition

(rag / kg)

2h 3h 4h 5h

-injection-

Lntr & peritorial Cnj-ection

in trainuEkular injection

sub cu taue injection

0.0025 0.020

0.025 0.100

70

72

ApplDeatiott

54 58

53 52

75 57

42

64 56

52 32

Until 3etzt, entzündungsverhüten-drug which is so stable was bu no non-sterq ^ of a © inflammatory

Has an effect at a small amount and possesses the ability to be injected, Deher iet the invention surprising and significant'-for pharmaceutical Applications * It can also be used as anti-inflammatory Substance according to the invention with a reliable reproducibility be produced in large quantities.

The invention is illustrated by the following examples.

isl 1

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2 1 one duich the pre-roasting of a breed of Aerobacter cloacae produced brood in 100 l of a culture medium inoculated, which consists of 1 # Petpton and tap water existed., »After the inoculation, the Eultiviening

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performed with ventilation at 57 ° C for 16 h. After the K elimination, the cells were removed by centrifugation. The filtrate was brought to a concentration of 3% with potassium chloride. Then Loriui ethanol was added in such an amount that the final concentration of the alcohol was 50 %. "The solution was left to stand overnight"

When observing the precipitation of the inflammatory Gubotanz as a function of the concentration of the added potassium chloride, it was found that the precipitate remained floating and did not settle on the ground until the added potassium chloride made up 2% . However, a 2% concentration is not sufficient to allow the precipitate to settle out satisfactorily. If potassium chloride is added so that the concentration of 3 - is 5%, sits down, the precipitate almost completely off, "so that the decantation could be easily performed ·

The white precipitate produced was removed by decanting «! collected} with water to remove ithanol di * iy- B.lert and then lyophilized. The yield was 64 g. The anti-inflammatory effect of this robe is shown in Table IX.

Example 2 '-

The same procedure as in Example 1 was repeated, except that one of Basse Aerobacter genes -Uses was · Ea 7.0 g of a lyophilislerten Pi'oduktös received. The anti-inflammatory effect of this sample is shown in Table II.

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Example. 3

The bio-procedure of Example 1 was repeated with the exception that a breed of Bacillue eubtilis was used - 5.8 β of a lyophilized product were obtained. The entzündungsverhüt effect of this sample eats shown in! Table II.

Example 4

The procedure of Example 1 was repeated with the exception that a Raeae of Kicrococu · lyeodetkticu · vei-turned ν, -urde. ΕεKfurden 4 _t 8 g obtain a lyophilised ·· wobbled Produkteß. The anti-inflammatory effect of diosc-r Prcbe is shown in Table II .

Di? The procedure of Example 1 was repeated with. Äusnohu.0 de-3 a sasse of PeeudoÄonae aeruginosa was venveudet. 8 "1 g of lyophilized" oil were obtained in this way. The harmful effects of this sample are shown in Table II.

Table Π

Test sample * Appllka- . Doeie AusulB (%) of the OFDM ti (/ ^) 2 h 4 h j

51 45 43 32 22nd 73 62 58 51 43

pp (%)

tionatweg (eg / ^ e) 2 h ; j> η 4h 5 h 6h

Example 1 intrmpe-

ritonale 0.01 71 65 43 33

injection

^M 2 "0.10

■ '"3 ■" ■■. ■ 0.20

"■■ 4 ■■ 0.20 57 51 53 37

"5" 0.050 52 56 51 45

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A Ras so from Aerobacter cloacae vr was in a culture medium, which from; 1 c meat extract, 1 β peptone, 0.3 g lia ^- criuiachlorid and 100 ml tap water (adjusted to a pH value of 7-0), inoculated into an ar iicnuttalilaBcLie with a capacity of 500 ml and potted for cultivation at 37 ° C for about 20 h. The resulting incubated solution was centrifuged at 10,000 rpm for 10 minutes, then it was separated into cells and a liquid was removed. The translucent supernatant was concentrated to about 1/20 part using a cationic evaporator. This concentrate was heated in boiling water on an oil bath for about 20 minutes. The generated precipitate was removed by centrifugation. The supernatant liquid was "transferred" into an oil plian tube and analyzed with a rolling vase. The dialyslert®, internal liquid was seized to remove insoluble material, it was entrapped and crosslinked! was etched, gel-filtered, the elutioa was passed through with Var-ner. The fraction was followed by means of the UY-Absorptioa - linhydrin dyeing and the colic reectioa. The anti-inflammatory substance is eluted in a fraction which is "due to the outdated polyaccharide ( Sepha & ex G-100) passed through "You eatsundMJQGßverhütende Substana was collected and lyophilized or dehydrated by adding acetone to obtain a purified product. The yield of 1 liter of the original fermentation liquid was 250 mg.

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The anti-inflammatory activity of this substance is

shown in table III.

Table III

intravenous injection

M dd

intraperitoneal injection

intramuscular injection

subcutaneous injection

a «wi 4v« 4 »i« «« «« * «■ Boßi's extent (%) of edema inhibition

Application route (mg / kg) 2h 3h 4h 5 & 6h

0.3

87

88 89

74 V.

0.1 79 74 72 68 56 0.03 42 36 35 10 0 0.3 83 86 88 79 72 1.0 56 57 58 62 65 1.0 21 28 25th 56 56

In the case of intravenous application to rattan, about 50 was 77kg and LDg _n was 7 »5 ng / kg»

Example 7

14.5 g (hate weight) of the cells obtained from 1 l of the permentation liquid from Example 6 were washed with water and suspended in 150 ml of water the freezing container was inserted and the same operation was repeated.Ditee solution was centrifuged at 20,000 rpm for 20 min and then the supernatant liquid was treated were received »

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5 β (hate weight) of the same cells as in Example 7 were washed with water and suspended in 100 ml of a 3 M KJSPO ₁ ^ solution. The suspension was left to stand in a low-temperature room and it was gradually poured into 20 l of water while bubbling Remove the cells below. The protruding surface was dialyzed with Vaaeer. Then, in the same way as in Example 1, the separation and purification were carried out, 130 mg of an inflammation-preventing substance being obtained.

Example, ft

Aerobacter cloacae was placed in a culture medium containing 0.5 g of KH, PO, 0.2 g of MgO, TE, and 100 ml of pipe wafers (adjusted to a pH of 7, 0), inoculated into a rubble eleven la see with a capacity of 500 ml, and shaken during cultivation at 37 ° C for about 20 h. You lermentierungsflüsaigkeit was treated in the same Veiee as in Example 1, 550 vobei an ent "ündungBV8rhüt" ADCN Substens mg of 1 X warden received the ureprüngliche & 7ermentierung9flilesigkeit ·

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Claims (1)

2Ö22B80 Claims Geoclur.ackloee * odorless, colorless, water-soluble and neutral anti-inflammatory substance with one Molecular weight of more than 200,000 obtained from a distant liquid resulting from breeding from a su Aerobacter cloacae, Aerobacter aerogenee, Bacillua subtiliB, Micrccocue lyaodeikticua or Pceudcmonae aeruginoea belonging to Raeee atammt. Process for the nucleus division of an inflammation-preventing gubutance, according to claim 1, characterized in that a microorganism, the aerobacter cloacae _f aerogenoo, Bacillua subtilis, Micrococue ο α-. ¹ IV. Ti cn D oiler rseudoinonas aeruginoaa heard, searched in ITähi'nediuia et. vird, which contains an EoiilenuelTe, a Stichc'oifquelle and inorganic Seize ", and an inflammation ever hat end sub starts with a high Holekrilargevicht according to the Penaen preserved» is won. J) ο ancestors according to objection 2, thereby identified. ο t, c.a3 the inflammation preventive substance from aL, Filtral; the fermentation liquid by the addition of ltlianol in the presence of potassium chloride is also confused. 'f. The method of claim 3 t characterized g "k"&& engraving et n that the potassium chloride in the Filtrmt a Kenge of 1 - 5%, and then sugeaetat Ithex »! is added in such an amount that the Alkono! concentration is ^ O - 60 % and - 15 - 009847/1811 BATH ORIGINAL ? O226IO Method according to claim 2, characterized in that g t; k e η η E e i c hn β t that a piltrate of a Fencenticrunge solution concentrated and then removed from the subtle dialjosed with low hell weight and then for gaining an anti-inflammatory effect. Dubetan " containing fraction subjected to gel filtration will. 009847/1611 - 16 - BATH ORIGINAL