Note: Descriptions are shown in the official language in which they were submitted.
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.
--3--
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.
~L~r~,'76~
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:
EXAMPLE 1
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.
~6~
~ ~ '
~ ~ ~ o o o o
o ~ o
.~ O 000 ~D
~ ~ ~0 ~0 ~0 ~1
Q ~3 o o o o o o
~ ~ ~ o n ~
~ O O o O O
O -~ ~0 ~o o
o O o
o +l +l +l + o o
H t~ ~ O r~) o r~ t~
~ 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
~,~
~ 7 6~
These results show a clear synergism between the
Physarum extract and the antimycotic drugs.
EXAMPLE 2
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.
-1 O-
~57~
EXAMPLE 3
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.
--11--
* Trade Mark
, .
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.
EXAMPLE 4
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:
-12-
~ 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.
EXAMPLE 5
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.
EXAMPLE 6
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.
-14-
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.
-15-
