CA1107646A - Anti-microbial composition - Google Patents

Abstract

Abstract of the Disclosure The present invention encompasses an antimicrobial composition comprising an extract of lytic enzymes from Physarum. These lytic enzymes are particularly effective antimycotic agents when used in conjunction with conventional antimycotic agents. The antimicrobial composition is prepared by isolating cell lytic enzymes from Physarum cultures then lyophilizing the enzyme extract to produce a sterile, pyrogen-free composition.

Description

7~6 ANTIMICROBIAL COMPOSITION
The present invention encompasses an antimicrobial composition comprising an extract of lytic ~enzymes from Physarum. This invention encompasses a crude extract from Physarum haviny chitinase (3.2.1.14), ~-1,3 glucanase (3.2.1.59), ~-1,4 glucanase (3.2.1.1), ~-1,6 glucanase (3.2.1.11), ~-1,3 glucanase (3.2.1.6), ~-1,6 glucanase (3.2.1.75), ~-glucosidase (3.2.1.21), ~-galatosidase (3.2.1.23), ~-mannanase (3.2.1.25), chitiobiase (3.2.1.29), ~-glucosidase (3.2.1.20), and muramidase (3.2.1.17) enzyme activity and a purified extract from Physarum having ~-1,3 glucanase, ~-1,4 glucanase, ~-1,6 glucanase, ~-1,3 gluca-nase, ~-1,6 glucanase, 3-glucosidase, ~-galactosidase, ~-mannanase and chitobiase enzyme activity. The present in-vention also encompasses a pharmaceutical composition suit-able for combining with a pharmaceutically acceptable car-rier said composition comprising a sterile, pyrogen free, lyophilized extract of lytic enzymes from Physarum. The present invention also encompasses the above formulations suitable for parenteral or topical application and further containing a conventional antimycotic agent such as Ampho-tericin-B, Nystatin, or 5-fluorocytosine as well as other agents named in Chapter 12 of Cuttings Handbook of Pharma-cology, 4th edition, Appleton-Century-Crofts, N.Y., N.Y.
pages 79-85.
The present invention describes a process for pre-paring a pharmaceutical composition which comprises isolat-ing cell lytic enzymes from Physarum then lyophilizing the enzyme extract to produce a sterile, pyrogen-free composi-tion. The present invention also describes a process for preparing a pharmaceutical composition as above, combining it with a suitable pharmaceutical carrier and optionally adding an antimycotic agent.

- 2 -~The enzymes in this disclosure are identified according to the numbering scheme found in the following reference:
Florkin, M. and Stotz, E.H. "Comprehensive Biochemistry", Volume 13, 3rd Edition, Elsevier Pub. Co. New York (1973).

The compositions of the present inventions pro-v1de an adjunct to conventional antimycotic chemotherapy by partially degrading fungal cell walls and ~hereby making fungal cells more susceptible to conventional antimycotic therapy. Therefore the present invention is concerned with a method of treating mycoses which involves alternate or simultaneous administration of Physarum extract and anti-mycotic agent.
In the context of the present invention the term therapeutically effective amount refers to the amount of Physarum extract which is effective alone or effective in conjunction with a conventional antimycotic agent such as Amphotericin-B. An antimycotic agent which is ineffective alone or toxic at effective doses can be made effective at less than therapeutic doses by compositions of the present invention. Those skilled in the pharmaceutical arts will recognize that doses will be varied depending on the severity of the infection and individual patient response.
Typically an intravenous dose of 2 mglkg every 12 hours for 4 days in conjunction with Amphotericin-B therapy is an effective dose.
Typically an enzyme extract precipitated by 1-5 volumes of 95% ethanol from Physarum culture growth has Chitinase a-1,3 glucanase a-1,4 glucanase a-1,6 glucanase

3 glucanase ~-1,6 glucanase ~-glucosidase ~-galactosidase ~-mannanase Chitobiase enzyme activity.

11~37G46 These enzymes are lyophilized for admin~stration in sterile pyrogen free solutions. For example, 2 mg/kg of body weight of this preparation is administered in 200 ml of 5~ dextrose over 1-2 hours twice daily for 4 days in conjunction with Amphotericin B therapy for lung coccidiomy-cosis or aspergillus infection.
Compounds and methods of the present invention are particularly effective in treating Candida, Aspergillus and Trichophyton infections such as those caused by Candida albicans, Aspergillus fumigatus and Trichophyton mentagro-phytes~
Injectables, intravenous, and ~opical formulations are prepared as described in Re~ington's Pharmaceut;cal Science Mach Publishing Company, Easton, Pennsylvania 1965 by art recognized techniques. Those skilled in the pharma-ceutical arts will recognize a wide variety of dosage forms and formulations n Chemical abstracts 78 157874W (1973) describes cell wall degrading enzymes extracted from a growth of Physarum polycephalum cultivated together with yeast or bacteria.
The resulting enzymes were shown to degrade yeast cell walls.
The present invention involves crude and purified extracts of cultures of Physarum alone, pharmaceutical compositions thereof and the use of these compositions as an adjunct in therapy of mycoses.
U.S. Patent 3,682,778 describes methods for ex-tracting cell lyt-c enzymes from various Coprinus species.
M.V. Tracey, Biochem Journal 61, 579~588 (lg55) discloses extracts of certain Lycoperdon species and compares the chitinase activity of these to chitinase activity of Coprinus extracts.

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British,Patent specification Nos. 1,048,887 and 1,410,079 describe bacterial sources of cell lytic enzymes and their in vitro activlty against pathogenic fungi and Kokai _t al. Chem. Abs. 79, 133662 Y describes ~ 3 glu-canase and chitinase as fungicide for rice blight. Mirua,Tohoku Journal of Exp. Med. 59, No 4,403 (1954) suggests that in vitro activity of bacterial chltinase might indicate its use as a pot~cal anti-dermatomycosis agent, but this was not tested.
It has been found that lytic enzyme mixtures extracted from Physarum species not only have significantly greater antimycotic activity than those extracted from Coprinus and Lycoperdon but in addition the Physarum enzyme extracts show a wider spectrum of activity deriving from their ability to attack not only fungal but also bacterial cell walls. This latter property is thought to arise be-cause of the presence of muramidase in the crude enzyme extract, an enzyme not present in significant amounts in extracts from Coprinus or Lycoperdon.
U.S. Patent 4,062,941 describes the use of cell lytic enzymes from Coprinus and Lycoperdon whereas the pre-sent invention is concerned with medicinally useful extracts from Physarum.
The preferred source of enzyme extract is Physarum polycephalum. The desired enzyme extract is most conveniently obtained by growth in liquid culture9 separating the super-natant liquid from the culture and isolating the product from this liquid.
In a preferred method Physarum polyce~halum is grown under aerobio conditions in shake flasks or in a stirred fermenter in a liquid medium of the following compo-~ 7sition:
Glucose 10 9 Bactopeptone 10 9 Citric Acid H20 3.54 9 K~2P4 2.0 9 CaC126H2 0.9 g Mg S04 7H20 0.6 9 Na2EDTA 0.224 g FeC124H2 0.06 g ZnS04 7H20 0 034 9 Thiamine HC1 0.0424 9 Biotin 0.005 9 Haemin 0.005 g Distilled Water to 1 litre 1~ The glucose can be replaced by maltose, starch, galactose, or other suitable carbohydrate.
The medium is adjusted to pH 5 with 10% NaOH.
Preferred growth conditions are as follows:
pH should be maintained at from 4.5 to 6, the optimum pH
bein~ 5; the re~uîred temperature is from 25 to 29C. and continuous very high oxygenation is desirable.
Enzyme release increases with cell numbers up to late exponential phase and continues to rise thereafter, but at a reduced rate. For optimal enzyme production and minimal extracellular polysaccharide ~which complicates extraction) cultures are harvested after approximately 180-200 hours growth. The crude enzyme extract is obtained as follows:
Cul ture supernatant is separated from the cel 1 s, for example by centrifuga~ion (1600 xg for 20 minutes).

The solid residue is discarded. The separated supernatant is cooled and residual slime precipitated for example by the addition of ammonium sulfate to 25% saturation or by the addition of 1 volume of ethanol or acetone previously cooled to -20C. The precipitate is separated from the liquor, for example by centrifugation at 10,000 xg for 30 minutes and the residue discarded. The liquid is dialysed against several changes of distilled water and lyophilized. This is the crude extract which can, if desired, be further puri-fied by conventional methods such as membrane filtration,gel filtration or affinity chromatography.
A typical crude extract from Physarum polycephalum has the following profile of enzyme activity:

Chitinase 0.0008 u ~-1,3 glucanase 0.28 u ~-1,6 glucanase 0.02 u ~-1,6 glucanase 0.004 u ~-1,3, ~-1,4 glucanase ~3.2.1.61) 0.031 u ~-glucosidase 0.14 u Chitobiase 0.113 u ~-galactosidase 0.17 u ~-mannanase 0.03 u ~-1,4 glucanase (3.2.1.4)0.02 u ~-glucosidase 0.06 u Muramidase 100.00 u A purified sample precipitated between 1 and 5 volumes of 95% ethanol has the following enzyme profile:

Chitinase 0.0~ u ~-1,3 glucanase 0.49 u ~-1,6 glucanase 0.07 u ~-1,6 glucanase 0.02 u ~-1,3, ~-1,4 glucanase0.04 u ~-glucosidase 0.35 u Chitobiase 0.25 u ~-galactosidase 0.35 u ~-mannanase 0.06 u (u)l Unit - the amount of enzyme that will release 1 ~ Mole of product/l minute/l mg protein at 37C.
The Physarum extract is of low toxicity. Female BALB/c mice weighing from 20-25g were injected i.p. with ' ~ 6~
purified extract (purif~ed by preciPitation with 4 vols. of ethanol) at doses of from 0 to 800 mg/kg in saline. Mice were observed for 7 days and deaths recorded as they occurred.
LD50 was calculated by plotting survivors against dose and deaths against dose, the LD50 being the intersection of the curves. An LD50 of 670 mg/kg was observed.
In rhesus monkeys toxicity tests established that no ill-effects were shown when the animals were given a dose approximately ten times the expected human dose of Physarum extract in saline. Respiration, pulse and heart ratel biochemistry and haematology remained within normal tolerances.
The invention is further illustrated by the following examples:

The effects of Physarum extract, with or without conventional antimycotic drugs in inhibiting grow~h of Candida albicans in vitro was assessed by a turbidimetric method.
1 x 105 cells of C. albicans were inoculated into bottles of broth containing various amounts of crude Physarum extract, antimycotic drugs or mixtures of the two.
Samples of these suspensions were tak~n (To) and their turbidity measured ln a spectrophotometer at 560 nm. The 25 cultures were incubated for 24 hours at 37C. and the tur-bidity measured again (T24) and compared with that of a control cul ture (T24 control). The results are shown in Table I. The value for T24 control was 0.61.

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~ ~ ~0 ~0 ~0 ~1 Q ~3 o o o o o o ~ ~ ~ o n ~
~ O O o O O

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~ O ~ -i O O o ~
o n ~ o o o o~
~ O o o ~1 'o ~ ~1 +1 +~ + o o o ~ ~ ~ o~ ,,~
.~ O o o .
~ ~1 ~ ~
~ O O o o +l +l + o o ~0 ~I~D O

S~ ~
m o ~ ~ o .,, ." X ,~ .,,

4~ ~ o a~ ~ + o +
o ~ ,~ ~ .,, S-l h ~ ~ ~1 ~1 a) o ~ ~ a) ~ o a) ~ ~ ~ u~ ~ u~
4~ ~ P~ ~ ~ ~ ~ ~ ~ ~.q 4~ O ~ ~ .C O , Z ~ t~ ~ ~ P~ Z

~7'f ~,~

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These results show a clear synergism between the Physarum extract and the antimycotic drugs.

Effect of Physarum extract on Aspergillus fumi-~atus infection in mice. ~ALB/c mice were injected via the lateral tail vein with 0.2 ml of a suspension containing approximately 5 x 106 spores of A. fumi~atus. On the three days following infection, mice were treated by intraperi-toneal injection with 0.2 ml saline (control); or with 0.2 ml saline containing lO~g Physarum extract and 1~9 Amphotericin B; or with 0.2 ml saline containing lO~g Physarum only; or with 0.2 ml saline containing Amphotericin B only. The course of infection was followed da~ly. The results are given in Table II.

TABLE II

Treatment SurviYal (5 mice/group)days . . _ .
None 12.5 ~ 1.6 3 x 10~9 Physarum extract 25.2 + 2.3 3 x 1~9 Amphotericin B 14.4 1 1.5 20 3 x (10~ Physarum extract 1~9 Amphotericin B) all alive at day 50 These results show clearly that the Physarum extract alone is effective to an appreciab~e extent in controllin~
the infection, showing better results than are obtained with Amphotericin alone. The results obtained using the extract together with the antimycotic drug demonstrates a strongly synergistic action.

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Treatment of superficial fungal infection in guinea pigs with Physarum extract with or without Nystatin.
The shaved backs of 25 guinea pigs were inoculated with 5 mm squares of a culture of Trichophyton mentagrophytes, a common skin pathogen. Four days after inoculation, the animals were checked visually for signs of infection and samples of hair and skin taken and cultured, (These were To controls taken to confirm infection). Treatment then commenced. All treatments were applied in "*Carbopol" ~
buffered gel at a rate of 0.5 g/animal/day according to ~-the following scheme. Each group contained 5 animals:

Group 1: Control - treated with placebo gel only 2: Gel + 0.1% ~w/w) Physarum extract 3: Gel ~ 0.5% (w/w) Physarum extract 4: Gel ~ 0.5% (w/w) Nystatin

5: Gel ~ Physarum 0.1% ~ Nystatin 0.05%

Treatment was continued for 5 days and further ; samples of skin and hair were taken and cultured. Five samples were taken from each animal and the number of discrete colonies on each culture plate was counted, the number of colonies being approximately equivalent to the amount of viable fungus remaining on the animals. (These were T5 samples). The results are given in Ta~le III.

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r~6 T_E I I I

Group 1 2 3 4 5 T Samples 100 100 100 100 100 (% animals infected) T5 Samples 100 60 40 60 20 (% animals infected) Number of colonies 212 91 70 96 28 from samples These results show a marked reduction in numbers of animals infected and numbers of fungal colonies de~eloping as a result of all treatments. However, Group 3 (0.5~
(w/w)Physarum) and Group 5 (0.1% Physarum ~ 0.05 Nystatin) gave the best results again showing the synergism between the extract and Nystatin.

Measurement of antibacterial activity of Physarum extract as a function of reduction of turbidity of suspen-sions of cells of Micrococcus lysodeikticus.
The potential antibacterial activity of Physarum enzyme extract resulting from the muramidase activity of the extract was measured as the reduction in the optical density (at 570 nm) of a suspension of Iyophilized cells of Micro-coccus lysodeikticus. Enzyme extract from Physarum (2 mg/ml) was compared with an enzyme extract from Coprinus (2 mg/ml) species and with a series of standard lysozyme solutions (0-76~g/ml). The optical density (OD) was read at To and at T20 (after 20 minutes incubation at 37C). The results are as follows:

~ 6~
L~sozyme-Standards OD at 570 nm 16~g/ml 0.112 8~g/ml 0.220 4~g/ml 0.360 2~g/ml 0.408 l~g/ml 0.417 To control 0.476 Coprinus T20 0 . 456 10~ To 0.465 Physarum T20 0.120 To 0.470 These results show that Coprinus extract has little or no muramidase activity whereas the Physarum ex-tract at 2 mg/ml shows a muramidase activity equivalent to approximately.l5~g/ml of lysozyme, i.eO pure muram-dase.

1~ 4 groups of BAL8/c mice were infected by intravenous iniection of 0.2 ml of suspension containing 5 x 10~ spores of Aspergillus fumigatus and treated in the days followi-ng infection by intraperitoneal injection.
(a) 0.2 ml saline 24 hrs, and 48 hrs. after infection (control) (b) 0.2 ml saline containing 20011~ Physarum extract precipi-tated between 1 and 5 volumes of 95~ ethanol at 24 hours and 0.2 ml saline at 48 hours.
(c) 0.2 ml saline at 24 hours and 1~9 Amphotericin B in 0.2 ml salin~ at 48 hours.

~ 7 ~ ~ ~
(d) 200~9 Physarum extract in 0.2 ml saline at 24 hours.
1 ~9 Amphoter;cin B in 0.2 ml saline at 48 hours.
The course of infection was ~ollowed daily.

Mean Survival (days) 5Group (a) 13.4 + 1.14 12.6 ~ 1.82 (b) 19.4 + 1.52 14.6 + 2.41 (c) 16.6 + 2.07 15.Q + 2.24 (d) All alive at 41 days and sacrificed for autopsy.

A strong synergistic effect is demonstrated be-tween the Physarum extract and Amphotericin B.
At autopsy n~ abnormalities were found and no fungal activity was found. Some material from kidney tissue might have been fungal tissue, but no positive cultures were obtained.

2) Similar e~periment to Example 5. Six groups of 5 mice were infected intravenously with 5 x lo6 spores of A. fumigatus and treated 24 and 48 hours after ~nfection by intraperitoneal injection with Physarum extract and with Amphotericin-B.
(aj 0.2 ml saline 24 and 48 hours after infection.
(b3 0~2 ml saline at 24 hours; 1 ~g of Amphotericin-B
in 0.2 ml saline at 48 hours.
(c) 100 ~g Physarum extract precipitated between 1 and 5 volumes of 95% ethanol in O.Z ml saline at 24 hours;
0.2 ml saline at 48 hours.
(d) 10 ~9 Physarum extract in OJ2 ml saline at 24 hours;
0.2 ml saline at 48 hours.

7 ~ ~ ~
(e) 100 ~9 Physarum extract ~n 0.2 ml saline at 24 hours;
1 ~9 Amphotericin B in 0.2 ml saline at 48 hours;
(f) 10 ~9 Physarum extract in 0.2 ml saline at 24 hours, 1 ~9 of Amphotericin B in 0.2 ml saline at 48 hours.

Mean Survival (days) Group (a) 14.0 + 2.0 (b) 16.0 + 2.~5 (c) 16.2 + 3.19 (d) 14.4 + 2.07 10(e) All alive at day 50 (f) 42.0 + 2.74 -~

Again, synergism between Physarum extract and Amphotericin B was demonstrated.

Claims (11)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a medicinal product which comprises isolating a cell lytic enzyme extract from a Physarum species and then lyophilizing said enzyme extract to produce a sterile, pyrogen free medicinal product.

2. The process of claim 1 wherein the Physarum species is Physarum polycephalum.

3. The process of claim 1 wherein the Physarum species is grown in a liquid culture medium and the supernatant culture liquid is thereafter separated from the culture medium and the enzyme extract is then isolated from said supernatant liquid.

4. The process of claim 3 wherein the supernatant liquid is subjected to a centrifugation and/or precipitation technique to remove unwanted impurities and the residual liquid is then dialysed to provide the desired enzyme extract.

5. The process as in claim 1 wherein the enzyme extract so obtained is further purified by dissolving it in a suitable solvent and adding ethanol thereto to provide a purified enzyme extract which is then lyophilized.

6. The process as in claim 1 wherein the product so obtained has chitinase .alpha.-1,3 glucanase .alpha.-1,4 glucanase .alpha.-1,6 glucanase .beta.-1,3 glucanase .beta.-1,6 glucanase .beta.-glucosidase .beta.-galactosidase .beta.-mannanase .beta.-1,4 glucanase .alpha.-glucosidase chitobiase and muramidase enzyme activity.

7. The process of claim 5 wherein the product so obtained has chitinase .alpha.-1,3 glucanase .alpha.-1,4-glucanase .alpha.-1,6 glucanase .beta.-1,3 glucanase .beta.-1,6 glucanase .beta.-glucosidase .beta.-galactosidase .beta.-mannanase and chitobiase enzyme activity.

8. A medicinal product, whenever prepared according to the process of claim 1, 2 or 3.

9. A medicinal product, whenever prepared according to the process of claim 4 or 5.

10. A medicinal product, whenever prepared by the process of claim 6.

11. A medicinal product, whenever prepared by the process of claim 7.