NO781035L - T METHOD OF PREPARING A PHARMACEUTICAL PREPARATION - Google Patents

Description

Method for producing a pharmaceutical preparation.

The present invention relates to a method for producing a pharmaceutical preparation comprising an extract of lytic enzymes from physarum. The invention comprises a crude extract from physarum with chitina se-, (X-1, 3 glucanase, α-1,4 glucanase, α-1,6 glucanase, (3-1,3 glucanase, |3-1,6 glucanase-, ( 3-gluco sidase, (3-galac to sidase-, |3-marmanase-, chi-thiobiase-, a-glucosidase and muramidase enzyme activity and a purified extract from physarum with a-1,3 glucanase-, a-1 ,4 glucanase-, a-1,6 glucanase-, (3-1,3 glucanase-, (3-1,6 glucanase-, P-glucosidase- , (3-galac to sida s e-, (3-mannanase - and the chitobiase enzyme activity. The invention further comprises a pharmaceutical preparation suitable for combination with a pharmaceutically acceptable carrier, where the preparation comprises a sterile, pyrogen-free, lyophilized extract of lytic enzymes from physarum. The invention further comprises preparations suitable for parenteral or topical application and further containing a conventional anti-myotic agent such as amphotericin-B, nystatin or 5-fluoro-cytosine as well as other agents specified in Chapter 12 of "Cuttings Handbook of P.harmacology," 4th edition, Ap pleton-Century-Crof ts , N.Y.,N.Y. pages 79 - 85.

As mentioned, the present invention includes a method for the production of a pharmaceutical preparation which consists in isolating cell-lytic enzymes from physarum, and then lyophilizing the enzyme extract to form a sterile, pyrogen-free preparation. The method also includes combining a product thus obtained with a suitable pharmaceutical carrier and possibly adding an antifungal agent.

The preparations according to the invention represent a supplement to conventional antifungal chemical therapy by partially breaking down fungal cell walls and thereby making fungal cells more susceptible to conventional antifungal therapy. The invention therefore relates to the treatment of mycosis, which involves alternating or simultaneous administration of physarum extract and antifungal agent.

By the term therapeutically effective amount is meant the amount of physarum extract that is effective alone or effective in connection with a conventional antifungal agent such as amphotericin-B. An antifungal agent which is ineffective alone or toxic in effective doses can be made effective in less than therapeutic doses by means of preparations according to the invention. One skilled in the art will appreciate that doses will be varied depending on the degree of infection and depending on the individual patient's response. An effective intravenous dose is typically 2 mg/kg every 12 hours i

4 days in connection with amphotericin B therapy.

An enzyme extract precipitated using 1-5 volumes of 95$> ethanol from physarium culture growth typically has chitinase-, α-1,3 glucanase-, α-1,4 glucanase-, α-1,6 glucanase-, |3-1 ,3 glucanase-, (3-1,6 glucanase-, (3-glucosidase-, (3-galactosidase), (3-mannanase-, and chitobiase) enzyme activity.

These enzymes are lyophilized for administration in sterile, pyrogen-free solutions. 2 mg/kg body weight of this preparation becomes e.g. administered in 200 ml 5$ extrose over 1-2 hours 2 times daily for 4 days in conjunction with amphotericin B therapy for pulmonary coccidiomyosis or aspergillus infection ion..

Compounds and methods according to the invention are particularly effective in the treatment of Candida, aspergillus and trichophyton infections such as those caused by Candida albicans, aspergillus fLimigatus and trichophyton mentagrophytes.

Injectable, intravenous and topical preparations are prepared as described in Remington's Pharmaceutical Science Mach Publishing Company, Easton, Pennsylvania 1965, using techniques known per se. The person skilled in the art will understand that many different dosage forms and preparations can be prepared.

Chemical abstracts 78 15787'lW (1973) describes cell wall degrading enzymes extracted from a growth of Physarum polycephalum co-cultured with yeast or bacteria. The resulting enzymes were found to break down yeast cell walls. The present invention comprises crude and purified extracts of cultures of physarum alone, pharmaceutical preparations thereof and the use of these preparations as a supplement in the therapy of mycosis.

US Patent No. 3,682,778 describes methods for extracting cell lytic enzymes from various coprinus species. M.V. Tracey, Biochem Journal 6l, 579-588 (1955) describes extracts of certain lycoperdone species and compares the chitinase activity of these with the chf<t>inase activity of coprinusex bats.

British Patent Nos. 108887 and 14-10079 describe bacterial sources of cell lytic enzymes and their in vitro activity against pathogenic fungi and Kokai et al. Chem. Abs. 79>133662 V describes (3-1,3 glucanase and chitinase as fungicide for use

on rice plants. Mirua, Tohoku Journal of Exp. Med. 59" No. k 403 (l954) suggests that invitroactivity of bacterial chitinase may indicate possible use as po tisk an ti-dermatoinyco si forged! , but this was not tested.

It has been found that lytic enzyme mixtures extracted from physarum species not only have significantly greater antifungal activity than those extracted from coprinus and lycoperdon, but the phystarum enzyme extracts also show a wider spectrum of activity which is reflected in their ability to not only attack fungi , but also bacterial cell walls. This latter property is believed to be due to the presence of muramidase in the crude enzyme extract, an enzyme not present in significant amounts in the coprinus or lycoperdone extracts.

US patent no. h 062 9^1 describes the use of cell-lytic enzymes from coprinus and lycoperdone, while the present invention, on the other hand, concerns medically useful extracts from physarum.

The preferred source for enzyme extract is Physarum polycephalum. The desired enzyme extract is most conveniently obtained by growth in liquid culture, separation of the supernatant liquid from the culture and isolation of the product from this liquid.

In a preferred method, physarum polycephalum is grown under aerobic conditions in shaking bottles or in a fermentation vessel with a stirrer in a liquid medium with the following composition:

The glucose can be replaced by maltose, starch, galactose or another suitable carbohydrate.

The medium is adjusted to pH 5 with 10% NaOH. Preferred growth conditions are the following: the pH value should be kept in the range 4.5-6, with the optimum pH value being 5, the required temperature is in the range 25 to 29°C and it is desirable to have continuously very high oxygenation.

Enzyme release increases with cell number up to late exponential phase and continues to increase after this, but. with reduced speed. For optimal enzyme production and minimal extracellular polysaccharide (which complicates extraction), cultures are harvested after approximately 180-200 hours of growth. The crude ensynek extract is obtained as follows: The culture supernatant is separated from the cells, e.g. e.g. by centrifugation (l600 x g for 20 minutes). The solid residue is discarded. The separated supernatant liquid is cooled and remaining mucilaginous matter is precipitated, e.g. by adding ammonium sulfate to 25°C melting or by adding a part by volume of ethanol or acetone which has previously been cooled to -20°C. The precipitate is separated from the liquid, e.g. by centrifugation at 10,000 x g for 30 minutes and the remainder discarded. The liquid is dialysed against several exchanges of distilled water and lyophilized. Thus, a crude extract is obtained which, if desired, can be further purified using conventional methods such as membrane filtration, gel filtration or affinity chromatography. '

A typical crude extract from physarum polycefaluin has the following enzyme activity profile:

A purified sample precipitated between 1 and 5 parts by volume of 95% ethanol has the following enzyme profile:

(u) 1 unit - the amount of enzyme that will release 1 u mol product/l minute/l mg protein at 37°C.

The physarum extract has low toxicity. BALB/c female mice weighing 20-25 g were injected i.p. with purified extract (purified by precipitation with h volumes of ethanol) in doses of from 0 to 800 mg/kg in saline solution. The mice were observed for 7 days and deaths recorded as they occurred. LD_,_-

50 value was calculated by plotting the number of survivors against dose and by plotting the number of deaths against dose, the LD^ value being the crossing point for the curves. An LD^ value of 670 mg/kg was found.

In rhesus monkeys, toxicity tests showed no adverse effects when the animals were given a dose of approximately 10 times the expected human dose of physarium extract in saline. Respiration, pulse and heart rate, biochemistry and hematology remained within the normal tolerance limits.

The following examples further illustrate the invention.

Example 1.

The effects of physarum extract with or without conventional antifungal drugs in inhibiting the growth of Candida.albicans in vitro were determined using a turbidimetric method. 1 x 10 cells of C. albicans were inoculated into 5 µl bottles with broth containing various amounts of crude fusarium extract, antifungal drugs or mixtures of these substances. Samples of these suspensions were taken (T ) and their turbidity measured in a spectrophotometer at 560 nm. The cultures were incubated for 2k hours at 37°C and the turbidity measured again (T^^) and compared to that of a control culture (T^-control). The results are shown in Table I. The value for the control sample was 0.6l.

These results show a clear synergism between the physarum extract and the antibiotic drugs.

Example 2.

Effect of physarum extract on Aspergillus fumigatus infection in mice. BALB/c mice were injected via the lateral tail vein with 0.2 ml of a suspension containing approximately 5

x 10^ spores of A. fumigatus. On the third day after infection, the mice were treated by intraperitoneal injection with 0.2 ml of saline (control animals), or with 0.2 ml of saline containing 10 µg of physarum extract and 1 µg of Amphotericin-B; or with 0.2 ml of saline. containing only 10 ug of physarum; or with 0.2 ml of saline containing only Amphotericin-B. The course of the infection was followed daily,

and the results obtained are indicated in table II below.

These results clearly show that physaurn extract alone is significantly effective in treating the infection, achieving better results than with Amphotericin alone. The results obtained by using the extract together with the antifungal drug are shown here in a strong synergistic effect.

Example 3-

Treatment of superficial fungal infection in guinea pigs with physarum extract with or without nystatin.

Shaved dorsal areas of 25 guinea pigs were inoculated with 5 mm~ 2 of a culture of Trichophyton mentagrolytes, a common skin pathogen. k days after inoculation the animals were visually checked for signs of infection and samples of hair and skin were taken and cultured (these were T controls taken to confirm infection). Then the treatment began. All treatment material was applied with "carbopol" buffered gel in an amount of 0.5 g/animal/day according to the scheme below. Each group consisted of 5 animals:

Group 1: control - treated only with placebo gel

2: Gel + 0.1% (w/w) physarum extract 3: Gel + 0.5% (w/w) physarum extract h: 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. 5 samples were taken from each animal and the number of separated colonies on each culture plate was counted, the number of colonies being roughly equivalent to the amount of viable fungus on the animals (these were T samples). The results are given in table III.

These results show a marked reduction in the number of infected animals and the number of fungal colonies that develop as a result of all treatments. However, group 3 (0.5 (wt/wt) physarum) and group 5 (0.1% physarum + 0.05% nystatin) gave the best results, which also shows synergism between the extract and nystatin.

Ek se nip el k .

Measurement of the antibacterial activity of physarumex tracts as a function of reduction in turbidity in suspensions of cells of Micrococcus lysodeic ticus.

The potential antibacterial activity of the physarum en syn extract resulting from the muramidase activity of the extract was measured as the decrease in optical density (at 570 nm) of a suspension of lyophilized cells of Micrococcus lyso-deicticus.

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 (0.16 µg/ml). The optical density (OD) was read at T^ and at T (after 20 minutes of incubation at 37°C). The following results were obtained:

These results show that Coprinus extract has little or no muramidase activity, while on the other hand the Fysarum extract at 2 mg/ml shows a muramidase activity equivalent to approximately 15 ug/ml lysozyme, i.e. pure muramidase.

River see ni pel 5.

l) Four groups of BALB/c mice were infected by intravenous injection of 0.2 ml of a suspension containing 5

x 10^ spores of Aspergillus fumigates and treated in the two days after the infection by intraperitoneal injection. a) 0.2 ml saline top solution for 24 hours and 48 hours after infection (control) b) 0.2 ml saline solution containing 200 ug of Physarum extract precipitated between 1 and 5 parts by volume 95% ethanol at 2k hours and

0.2 ml of saline solution at <4>8 hours.

c) 0.2 ml saline solution at 24 hours' and 1 µg Amphotericin B in 0.2 ml saline solution at 48 hours. d) 200 ug of Physarum extract in 0.2 ml saline solution at 24 hours. 1 ug Amphotericin B in 0.2 ml saline at 48 hours.

The course of the infection was followed daily.

Average survival (days).

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

At autopsy, no abnormalities and no fungal activity were found. Some material from kidney tissue could have been fungal tissue, but no positive cultures were obtained.

Example 6.

2) Similar experiment as stated in example 5. Six groups of 5 mice were infected intravenously with 5 x 10^ spores of A. fumigatus and treated 24 and 48 hours after the infection by intraperitoneal injection with Fysarum extract and with Amphotericin-B. a) 0.2 ml saline solution 24 and 48 hours after infection. b) 0.2 ml saline at 24 hours; 1 mg Amphotericin-B in 0.2 ml saline solution at 48 hours. c) 100 ug of Physarum extract precipitated between 1 and 5 parts by volume of 95% ethanol in 0.2 ml of sal top solution at 24 hours; 0.2 ml

saline solution at 4 8 hours.

d) 10 ug Physarum extract in 0.2 ml saline solution at 24 hours; 0.2 nil saline solution at 4 8 hours. e) 100 ug of Physarum extract in 0.2 ml saline solution at

24 hours; 1 ug Amphotericin-B in 0.2 ml saline at 48

hours.

f) 10 ug Physarum extract in 0.2 ml saline solution at 24 hours; 1 ug Amphotericin-B in 0.2 ml saline at 48 hours.

Here too, synergism between Fysarum extract and Amphotericin-B was demonstrated.

Claims (8)

1. Process for the production of a pharmaceutical preparation, characterized by isolating cell-lytic enzymes from Fysarum, after which the enzyme extract is lyophilized to form a sterile, pyrogen-free preparation. 2. Method according to claim 1, characterized in that the aforementioned lyophilized Fysarum enzyme extract is combined with a suitable pharmaceutical carrier. 3. Method according to claim 2, characterized in that one combines lyophilized Physarumensym- extracts with a suitable pharmaceutical carrier and an antifungal agent. 4. Method according to claim 3, characterized in that the antifungal agent is Amphotericin-B, Nystatin or 5-fluorocytosine, 5. Method according to claim 1, for the production of a purified extract of cell-lytic enzymes from physarum, characterized in that these enzymes have α-1,3 glucanase, α-1,k glucanase, α-1,6 glucanase, (3 -1,3 glucanase, |3-1,6 glucanase, (3-gluco sidase , (3-galac to sidase , (3-mannana s e , chitobiase, chitinase activity. 6. Method for treating fungal infections in animals, characterized in that an effective amount of mycolytic enzymes extracted from Fysarum is administered to an animal in need of anti-fungal treatment. 7. Procedure for the treatment of fungal infections in animals, characterized in that one to an animal which in need of antifungal treatment administer an effective amount of mycolytic enzyme extracts from Fysarum in conjunction with an antifungal agent. 8. Method according to claim 7, for the treatment of fungal infections in animals, characterized in that one to an animal in need of anti-fungal treatment administers an effective amount of mycolytic enzymes extracted from Fysarum in conjunction with Amphotericin-B or Nystatin.