IE46698B1 - Anti-microbial compositions containing extracts of lytic enzymes from physarum species - Google Patents

Abstract

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

This invention relates to anti-microbial compositions containing extracts of lytic enzymes from Physarum species.

The present invention provides a sterile, pyrogen-free, lyophilized extract of anycolytic enzymes from Physarum species alone which, in the crude form, exhibits chitinase, a-1,3, a-1,4 glucanase, a-1,6 glucanase, β-1,3 glucanase, β-1,6 giucanase, βglucosidase, β-1,4 glucanase, β-galactosidase, β-mannanase, chitobiase, α-glucosidase and muramidase activity antjl which, i in the purified form, exhibits chitinase, a-1,3, a-1^4 glucanase, a-1,6 glucanase, β-1,3 glucanase, β-1,6 glucanase, β-^1υσοΞΪ<ϊαΞβ, β-galactosidase, β-mannanase and chitobiase activity.· The preferred Physarum species is Physarum polycephalum.

Such an extract may be obtained by isolating mycqlytic enzymes from Physarum species alone and lyophilizing .the isolate.

The present invention7also provides a pharmaceutical composition which comprises such an extract and a pharmaceutically acceptable carrier or diluent. The composition may also comprise an antimycotic agent, for example amphotericin B, nystatin or 5fluorocytosine. Such a composition is generally in a form suit20 . able for parenteral, intravenous or topical administration.

These compositions may be obtained by mixing the constituents thereof.

The present invention further provides a method for the treatment of mycoses in non-human animals which ccnprises administer25 ing thereto a therapeutically effective amount (as hereinafter defined) of such an extract or such a composition. The present 6 0 3 8 pharmaceutical compositions may also comprise other agents named in Chapter 12 of Cuttings Handbook of Pharmacology, 4th edition, Appleton-Century-Crofts, N.Y., N.Y. pages 79—85.

The present compositions are useful as adjuncts to conven5 tional antimycotic chemotherapy by virtue of the ability thereof partially to degrade fungal cell walls and thereby render fungal cells more susceptible to conventional antimycotic therapy.

As used herein, the term therapeutically effective amount means that amount of Physarum species extract which is either effective alone or effective in conjunction with a conventional antimycotic agent, such as amphotericin B. An antimycotic agent which is either ineffective alone or toxic at effective doses may be made effective at less than normal therapeutic doses by combination with the present extracts. Those skilled in the pharmaceutical art will recognize that doses may vary depending on the severity of the infection and on individual patient response. Typically an intravenous dose of 2 mg/kg every 12 hours for 4 days in conjunction with amphotericin B therapy is an effective dose.

Typically an enzyme extract precipitated by from 1 to 5 volumes of 95% (v/v) ethanol from Physarum species culture growth has the enzyme activity specified above for the purified form.

The enzymes are lyophilized for administration in sterile pyrogen-free soluticns-^^i'or example, 2 mg/kg body weight of this preparation is administered in 200 ml of 5% (w/v) dextrose over from 1 to 2 hours twice daily for 4 days in conjunction with amphotericin B therapy for lung coccidiomy cases or Aspergillus infection.

The present invention is particularly effective in treating Candida, Aspergillus and Trichophyton infections, such as those caused by Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes.

Injectables, intravenous formulations and topical formula-, tions may be prepared as described in Remington's Pharmaceutical Science, Mach Publishing Company, Easton, Pennsylvania 1965 by techniques recognized in the art. Those skilled in the pharmac5 eutical art will recognize a wide variety of dosage forms and formulations.

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, but the present invention relates to crude and purified extracts of culture of Physarum species alone, pharmaceutical compositions thereof and the use thereof particularly as an adjunct in the therapy of mycoses.

U.S. Patent No. 3,682,778 describes methods for extracting cell lytic enzymes from various Coprinus species, Μ. V. Tracey, Biochem Journal 61, 579—588 (1955) discloses extracts of certain Lycoperdon species and compares the chitinase activity thereof to chitinase activity of Coprinus extracts.

British Patent Specification Nos. 1,048,887 and 1,410,079 describe bacterial sources of cell lytic enzymes and the in vitro activity thereof against pathogenic fungi and Kokai et al. Chem. Abs. 79, 133662 V describes β-l, 3 glucanase and chitinase as a fungicide for rice blight. Mirua, Tohoku Journal of Exp. Med. 59, No. 4,403 (1954) suggests that in vitro activity of bacterial chitinase might indicate its use as a typical anti-dermatomycosis agent, but this was not tested.

It has now been found that mycolytic enzyme mixtures extrac ted from Physarum species not only have significantly greater antimycotic activity than those extracted from Coprinus and Lycoperdon species, but, in addition, the Physarum species enzyme extracts show a wider spectrum of activity deriving from ί ΰ 6 9 8 . - 5 the ability thereof'to attack not only fungal cell walls, but also bacterial cell walls. This latter property is thought to arise because of the presence of muramidase in the crude enzyme extract, an enzyme not present in significant amounts in extracts from Coprinus or Lycoperdon. (Muramidase is only present in the crude extract).

U.S. Patent No. 4,062,941 describes the use of cell lytic enzymes from Coprinus and Lycoperdon, while the present invention is concerned with medicinally useful extracts from Physarum species.

As mentioned above, the preferred source of enzyme extract for the purposes of the present invention is Physarum polycephalum. The desired enzyme extract is most conveniently obtained by growth in liquid culture, separating the supernatant liquid from the culture and then isolating the product from this liquid.

In a preferred method Physarum polycephalum is grown under aerobic conditions in shake flasks or in a stirred fermenter in a liquid medium of the following composition: Glucose 10 9 Bactopeptone 10 9 Citric Acid H^O 3.54 9 kh2po4 2.0 9 CaCl26H2O 0.9 9 MgSO4 7H2O 0.6 9 NUjEDTA 0.224 9 FeCl2 4H2O 0.06 9 ZnSO. 7H0 4 2 0.034 9 Thiamine HC1 O.O424g Biotin 0.005 9 Haemin 0.005 9 Distilled Water to 1 litre The glucose may be replaced by maltose, starch, galactose or another suitable carbohydrate.

I The medium is adjusted to pH 5, for example using 10% (w/v) HaOH.

Preferred growth conditions are as follows: pH should be maintained at from 4.5 to 6, the optimum pH being 5; the required temperature is from 25 to 29°C. 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 extra10 cellular polysaccharide (which complicates extraction) cultures are harvested after from 180 to 200 hours of growth. The crude enzyme extract is then obtained as follows: Culture supernatant is separated from the cells, for example by centrifugation (1600 g for 20 minutes), and the solid residue is discarded. The separated supernatant is cooled and residual slime is precipitated, for example by the addition of ammonium sulfate to 25% (w/v) saturation or by the addition of 1 volume of ethanol or acetone which .has been previously cooled to -2Q°C. The precipitate is separated from the liquor, for example by centrifugation at 10,000 g for 30 minutes, and the residue is discarded. The liquid is dialysed against several changes of distilled water and subsequently lyophilised. This is the crude extract which may, if desired, be further purified 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: i ύ ο 9 8 Chitinase 0.0008 u β-1,3 glucanase 0.28 u β-1,6 glucanase 0.02 u a-1,6 glucanase 0.004 u a-1,3, a-1,4 glucanase 0.031 u β-glucosidase 0.14 u Chitobiase 0.113 u β-galactosidase 0.17 u β-mannanase 0.03 u β-1,4 glucanase 0.02 u cs-glucosidase 0.06 u a-1,4 glucanase 0.03 u Muramidase 100.00 u A purified sample precipitated between 1 and 5 volumes of ιοί has the following enzyme pro: Chitinase 0.02 u β-1,3 glucanase 0.49 u β-1,6 glucanase 0.07 u a-1,6 glucanase 0.02 U a-1,3,a-1,4 glucanase 0.04 u β-glucos idase 0.35 u Chitobiase 0.25 u β-galactosidase 0.35 U β-Mannanase 0.06 u (u) 1 Unit = the amount of enzyme that will release 1μ Mole of product/1 minute/1 mg protein at 37°C.

The Physarum species extract is of low toxicity. To demonstrate this, female BALB/c mice weighing from 20 to 25 g were injected i.p. with purified extract (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. LD^0 was calculated by plotting survivors against dose and deaths against dose, the LD^0 being the intersection of the two curves.

An LD5q of 670 mg/kg was found.

In rhesus monkeys, toxicity tests established that no illeffects resulted when the animals were given a dose approximately ten times the expected normal human dose of Physarum species extract in saline. Respiration, pulse and heart rate, biochemistry and haematology remained within normal tolerances.

The present invention is further illustrated by the following Examples: EXAMPLE 1.

The effects of Physarum species extract, with or without conventional antimycotic drugs, in inhibiting growth of Candida albicans in vitro was assessed by a turbidimetric method. x 10^ cells of C. albicans were inoculated int;o bottles of broth containing various amounts of crude Physarum species extract, antimycotic drugs or mixtures of the two. Samples of these suspensions were taken (T_) and the turbidity thereof 6 measured using a spectrophotometer at 560 nm. The cultures were incubated for 24 hours at 37°C., the turbidity was then measured again (T24? and compared with that of a control culture (T24 control). The results are shown in Table 1 below. The value for T24 control was 0.61. f ) o 6 i) 8 Effects of Physarum extract and antimycotic drugs on growth of Candida albicans in φ μ •P «—I ΰ Ό Ή & *rl Is* 3* 00 o o o ό ό ό 4-1 4-i 4-1 σ» in xp Ο Ο Ο θ' Ο Ο +1 +1 +1 ιη σ -) Ο U β 05 ρμ d •μ β pH μ Φ Ο 4J 3 a Μ Κ Ο 01 Φ •μ 3 01 •Ρ ω >1 01 0 13 φ φ d •μ μη υ C ΜΗ φ φ •μ ο φ a μ Λ •μ σ\ϊ Ο . . . tP >1 3 Λ β tP O o o 3. Λ ΜΗ •Ρ Ο Λ pH a •μ +1 +1 +1 01 £ O + O o φ 3 1 m σι io Λ rH β 05 pH m O ro CP •P pH •μ 3 * 3 •μ +> O o’ o H β σ d φ μ & φ φ •μ (M m CP ζ a ε •—1 o o O 3 •μ • 01 •1"' Π3· 3 ε • O Φ CM ι< •μ μη MH ό o’ o rH Λ o &> Η C • 3. 4-1 +1 +1 4- o o ε Η 0 rH 01 ίΰ c o r—i σι n CP •μ 2 •Ρ μ Μη d OJ . ,O r-H 3 IP υ φ 0 Φ • O μ R Ρ •Ρ £ 6 O O pH φ μ d 01 C •Ρ rH φ >1 X μ o r-t CP 01 φ φ 0 Φ o o 3 Λ a 10 r4 • « O μ 0) •Ρ 0) e o o io d Φ Q φ •μ d| O tP n o +1 4-1 4- o o pH Ο 0 Φ •μ ο pH r-H ο επ CQ β •Η □ •Η μ C Q) ·μ π3 Φ ω εη β ·γ4 ο •Η Μ Φ •Ρ Ο Λ Οι 01 ε 4- + -Ρ rH Φ μ φ ω φ Λ Εη ϋ •rl 4J ϋ £ •μ •Ρ β rd φ Λ •Ρ a ω ε μ d > ΐμ 4J υ Φ μη Μη W Η Η Ο μ a a d tP c •μ C •H d •P - 10 infection, mice were treated by intraperitoneal injection with 0.2 ml saline (control); or with 0.2 ml saline containing lOug Physarum species extract and l|J.g Amphotericin B; or with 0.2 ml saline containing 10ug Physarum extract only; or with 0.2 ml saline containing Amphotericin B only. The course of infection was followed daily. The results are given in Table II below.

TABLE II Treatment Survival (5 mics/group) days None 3 χ-lOug Physarum extract 12.5+1.6 25.2+2.3 3 x lug Amphotericin B 14.4+1.5 3 x (lone Physarum extract + lug Amphotericin B) All alive at day 50 These results demonstrate clearly that the Physarum species extract alone is effective to an appreciable extent in controlling the infection and shows better results than those obtained with Amphotericin B alone. The results obtained using the extract together with the antimycotic drug demonstrates a strongly synergistic action.

EXAMPLE 3.

Treatment of superficial fungal infection in guinea pigs with Physarum extract or 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 the inoculation, the animals were checked visually for signs of infection and samples of hair and skin taken and cultured, (these were T controls taken to confirm o infection). Treatment then commenced. All treatments were applied in Carbopol (Registered Trade Mark) buffered gel at a rate of 0.5 g/animal/day according to the following scheme. Each group contained 5 animals : 1GG0S - 11 Group 1: Control-treated with placebo gel only 2: Gel + 0.1% (w/w) Physarum species extract 3: Gel + 0.5% (w/w) Physarum species extract 4: Gel + 0.5% (w/w) Nystatin : 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 T^ samples). The results are given in Table III.

TABLE III Group 1 2 3 4 5 T Samples (% animals infected) 100 100 100 100 100 T^ Samples ( % animal's infected) 100 60 40 60 20 Number of colonies from samples 212 91 70 96 28 These results show a marked reduction in numbers of animals infected and numbers of fungal colonies developing as a result of all treatments. However, Group 3 (0.5% (w/w) Physarum) and group 5 (0.1% (w/w) Physarum + 0.05 Nystatin) gave the best results once again showing the synergism between the Physarum species extract and Nystatin.

EXAMPLE 4.

Measurement of antibacterial activity of Physarum species extract as a function of reduction of turbidity of suspensions of cells of Micrococcus lysodeikticus.

The potential antibacterial activity of Physarum species enzyme extract resulting from the muramidase activity of the crude extract was measured as the reduction in the optical density (at 570 nm) of a suspension of lyophilized cells of Micrococcus lysodeikticus. Enzyme extract from Physarum species (2 mg/ml) was compared with an enzyme extract from Coprinus (2 mg/ml) species and with a series of standard lysozyme solutions (0—16p.g/ml). The optical density (OD) was read at Tq and at T2Q (after 20 minutes incubation at 37°C). The results are as follows.

Lysozyme Standards hg/ml p-g/ml [ig/ml μg/ml hg/ml T control o Coprinus T20 T0 Physarum T T OD at 570 nm 0.112 0.220 0.360 0.408 0.417 0.476 0.456 0.465 0.120 0.470 o These results show that Coprinus species extract has little or no muramidase activity, while the Physarum extract at 2 mg/ml shows a muramidase activity equivalent to approximately 15 (ig/ml of lysozyme, i.e. pure muramidase.

EXAMPLE 5 (1) 4 groups of BALB/c mice were infected by intravenous Q injection of 0.2 ml .of suspension containing 5 x 10 spores of Aspergillus fumigatus and treated in the 2 days following infection by intraperitonea1 injection. ,6898 - 13 (a) 0.2 ml saline 24 hrs and 48 hrs. after infection (control) (b) 0.2 ml saline containing 200 μ-g Physarum species extract precipitated between 1 and 5 volumes of 95% (v/v) ethanol at 24 hours and 0.2 ml saline at 48 hours. (c) 0.2 ml saline at 24 hours and 1 μ-g amphotericin B in 0.2 ml saline at 48 hours. (d) 200 u.g Physarum extract in 0.2 ml saline at 24 hours. 1 μ-g amphotericin B in 0.2 ml saline at 48 hours.

The course of infection was followed daily.

Group (a) (b) (c) (d) Mean Survival (days) 13.4+1.14 12.6+1.82 19.4+1.52 16.6+2.07 14.6+2.41 .0+2.24 All alive at 41 days and sacrificed for autopsy.

A strong synergistic effect is demonstrated between the Physarum species extract and amphotericin B.

At autopsy no 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 experiment to Example 5. Six groups of 5 mice were infected intravenously with 5 x 10θ spores of A. fumigatus and treated at 24 and 48 hours after infection by intraperitonea1 injection with Physarum species extract and with amphotericin B. (a) 0.2 ml saline 24 and 48 hours after infection. (b) 0.2 ml saline at 24 hours; 1 μg of amphotericin B in 0.2 ml saline at 48 hours. (c) 100 μ-g Physarum extract precipitated between 1 and 5 volumes of 95% (v/v) ethanol in 0.2 ml saline at 24 ’-6698 - 14 hours; 0.2 ml saline at 48 hours. (d) 10 ng Physarum extract in 0.2 ml saline at 24 hours; 0.2 ml saline at 48 hours. (e) 100 ng Physarum extract in 0.2 ml saline at 24 hours; 1 ng amphotericin B in 0.2 ml saline at 48 hours; (f) 10 ng Physarum extract in 0.2 ml saline at 24 hours; 1 ng of amphotericin B in 0.2 ml saline at 48 hours.

Mean Survival (days) Group (a) 14.0+2.0 (b) 16.Qt2.55 (c) 16.2+3.19 (d) 14.4+2.07 j (e) All alive at day 50 15 (f) 42.0+2.74 Again, synergism between Physarum species extract and amphotericin B was demonstrated.

Claims (15)

1. A sterile, pyrogen-free, lyophilized extract of mycolytic enzymes from Physarum species alone which, in the crude form, exhibits chitinase, a-1,3, a-1,4 glucanase, a-1,6 glucanase, β-1,3 glucanase, β-1,6 glucanase, β-glucosidase, β-1,4 glucanase, βgalactosidase, β-mannanase, chitobiase, α-glucosidase and muramidase activity and which in the purified form, exhibits chitinase, a-1,3, a-1,4 glucanase, a-1,6 glucanase, β-1,3 glucanase, β-1,6 glucanase, β-glucosidase, β-galactosidase, β-mannanase and chitobiase activity.

2. An extract as claimed in claim 1 from Physarum poiycephalum.

3. An extract as claimed in claim 1 substantially as herein described.

4. A process for the preparation of an extract as claimed in claim 1 which comprises isolating mycolytic enzymes from Physarum species alone and lyophilizing the isolate.

5. A process as claimed in claim 4 substantially as herein described.

6. An extract as claimed in claim 1 when prepared by a process as claimed in claim 4 or claim 5.

7. A pharmaceutical composition which comprises an extract as claimed in any of claims 1 to 3 or 6 and a pharmaceutically acceptable carrier or diluent.

8. A composition as claimed in claim 7 comprising an antimycotic agent.

9. A composition as claimed in claim 8 comprising an antimycotic agent which is amphotericin B, nystatin or 5-fluorocytosine.

10. A composition as claimed in claim 7 substantially as herein described.

11. A process for the preparation of a composition as claimed in claim 7 substantially as herein described.

12. A composition as claimed in claim 7 when prepared by a process as claimed in claim 11. 5

13. A composition as claimed in any of claims 7 to 10 or 12 in a form suitable for parenteral, intravenous or topical adminis tration.

14. A method for the treatment of mycoses in non-human animals which comprises administering thereto a therapeutically 10 effective amount (as hereinbefore defined) of an extract as claimed in any of claims 1 to 3 or 6 or a composition as claimed in any of claims 7 to 10, 12 or 13.

15. A method as claimed in claim 14 substantially as herein described.