Novel biological substance from a fungus and the process for producing the same

This invention relates to a biological substance extracted from the Mycelium of a Fusarium. This substance has a peptidolic structure with specific amino-acids into. This invention also relates to a process for producing the afore-said biological substance by culturing the strain of Fusarium and extracting the mycelium. The biological substance has a therapeutical use.

PRIOR ART 
The relevant prior art may be illustrated with the following references: 
Belgian patent, 847 941 
J of Antibioties, 29 (1976) 1043 
J of Antibioties, 29 (1976) 1050 
Tetrahedron, 14 (1978) 1147 
SUMMARY OF THE INVENTION 
This invention provides a biological substance obtained by extracting the 
mycelium produced during the cultures of a Fusarium equiseti. 
This biological substance contains four different components denominated A, 
B, C and D of peptidolic structure. Only the specific amino-acids vary 
from one component to another one. 
This substance is produced by culturing a strain of Fusarium equiseti under 
specific conditions, separating the mycelium and extracting it with water 
unsoluble organic solvents. The biological substance is further purified 
according to the usual methods of the biochemistry. 
This substance is of value for the immunological therapy and is used in the 
form of pharmaceutical compositions, namely those suitable for parenteral, 
permucous, per cutaneous, sublingual or rectal ways. 
PREFERRED EMBODIMENT OF THE INVENTION 
This invention relates to a novel biological substance extracted from a 
fungus. 
More precisely, this invention provides a biological substance obtained 
from the brothes of a Fusarium, a method for its production and its use in 
the form of pharmaceutical compositions. 
This invention specifically provides a biological substance having a 
peptidolic structure obtained by extraction from the mycelium of Fusarium 
equiseti. This biological substance includes four different components 
having a very close structure and giving rise to the production by 
hydrolysis in acidic medium of a single hydroxy acid, .alpha.-hydroxy 
valeric acid and of specific .alpha.-amino acids i.e. N-methyl Valine, 
N-methyl isoleucine and N-methyl allo iso leucine. 
The percentage of these four components named A, B, C, D has been 
determined after separation by high pressure liquid-chromatography in 
reverse phase, by means of an integrator. The percentage of four 
components in this biological substance is determined by measuring the 
square of each peak after high pressure liquid chromatography and the 
residual absorption in UV light at 254 .mu.m. 
The respective percentages of each component may slightly vary and in 
average range between the following figures: 
component A, 2-5% 
component B, 10-16% 
component C, 32-39% 
component D, 40-47% 
Component A after partial hydrolysis in alkaline medium provides 3 mol of 
the lactone of .alpha.-hydroxy isovaleroyl N-methyl Valine. This lactone 
results from the splitting of the ester functions and recyclizing of the 
liberated hydroxy group into a lactone. 
Component A after complete hydrolysis in acidic medium produces 3 moles of 
N-methyl Valine and 3 moles of .alpha.-hydroxy isovaleric acid. The acid 
hydrolysis produces both a split of the ester functions and of the amid 
functions. 
Component B after partial hydrolysis in alkaline medium gives raise to the 
production of 2 moles of the lactone of .alpha.-hydroxy isovaleroyl 
N-methyl Valine and a mole of the lactone of .alpha.-hydroxy isovaleroyl 
N-methyl isoleucine (or N-methyl isoalloleucine). 
Component C under similar experimental conditions provides after partial 
hydrolysis 2 moles of the lactone of .alpha.-hydroxy iso valeroyl N-methyl 
isoleucine (or N-methyl alloisoleucine) and 1 mole of the lactone of 
.alpha.-hydroxy iso valeroyl N-methyl valine. 
The complete hydrolysis in acidic medium gives rise to the production of 3 
moles of .alpha.-hydroxy isovaleric acid, 1 mole of N-methyl valine and 2 
moles of N-methyl isoleucine (or N-methyl alloisoleucine). 
Compound D provides after partial hydrolysis in alkaline medium 3 moles of 
the lactone of .alpha.-hydroxy isovaleroyl N-methyl isoleucine (or 
N-methyl alloisoleucine). 
After complete hydrolysis in acidic medium, compound D provides 3 moles of 
.alpha.-hydroxy isovaleric acid and 3 moles of N-methyl iso leucine (or 
N-methyl alloisoleucine). In summary, the complete hydrolysis of the 
depsipeptide according to the invention, provides .alpha.-hydroxy 
isovaleric acid, N-methyl valine, N-methyl iso leucine (erythro form) and 
N-methyl allo isoleucine (threo form). The N-methyl allo isoleucine has 
not yet been attributed to one specific component of the biological 
substance. 
This biological substance of peptidolic structure may be obtained by 
sumitting to an aerobic fermentation a culture of Fusarium equiseti in 
determined conditions of temperature, stirring and duration, separating at 
the end of the time of fermentation the mycelium of Fusarium equiseti from 
the broth, extracting the mycelium with a water-insoluble organic solvent, 
separating the organic phases, evaporating off, taking up the crude 
residue with a liquid hydrocarbon, submitting the organic solution to a 
counter-current extraction with a mixture of water and a lower alkanol, 
separating this alkanolic phase, concentrating it and recovering the 
biological substance which is further purified by the usual physical or 
chemical methods. 
The process of preparation according to the invention may also be defined 
by the following features, which are the presently preferred ones: 
the strain of Fusarium equiseti responsible of the production of the 
depsipeptide according to the invention is that of Fusarium equiseti 
(Corda) saccardo, deposited at the Commonwealth Mycological Institute at 
Kew (Surrey) Great Britain under the n 213 107. 
the aerobic fermentation is carried out in three stages at a pH between 5 
and 6 and for a period of time extending from 36 to 48 hours for each 
stage. 
the extraction of the mycelium of Fusarium equiseti is performed using a 
chlorinated organic solvent as for example Trichlorethylene. 
the taking up of the crude residue is performed using a saturated liquid 
hydrocarbon such as hexane or pentane. 
the purification of the peptidolic substance is performed by means of 
chromatography on silica. 
the hydrosoluble alkanol is methanol or ethanol. 
The depsipeptide according to the invention is endowed with interesting 
pharmacological properties. It possesses strong immuno-modulating 
properties evidenced by a positive response in the test of 
hypersensitivity to 4-ethoxy methylene 2-phenyloxazolone or to 
lipopolysaccharides of Escherichiacoli. Further the depsipeptide causes 
the activation of macrophages by increasing or altering the enzymatic 
activities and the total proteic content of the cells thereof. Moreover, 
the depsipeptide show an anti-exsudative and anti-oedematious activity. 
Compared to the activities of Muramyldipeptide and of the 
lipopolysaccharides from Escherichia coli, the depsipeptides from Fusarium 
equiseti appears to be at least as active as these two reference 
substances. 
The depsipeptide according to this invention find a use in human or 
veterinary therapy, namely as a stimulant of the body defences. It is 
particularly useful for the treatment of the chronic or acute diseases of 
the respiratory tract such as chronic bronchitis and emphysema. 
For the therapeutic use, the depsipeptide is incorporated into 
pharmaceutical compositions with an inert non-toxic 
pharmaceutically-acceptable carrier or vehicle. The carriers or vehicles 
are those suitable for administration parenteral, permucous, percutaneous 
or rectal way. 
As preferred pharmaceutical compositions they may be cited the injectible 
solutions or suspensions, the sublingual tablets, the solutions or 
suspensions in a diffusible solvent to be applied on the mucosa optionally 
under pressure, the solutions in a polar solvent for the percutaneous way 
and the suppositories. 
The useful dosology may broadly vary depending of the therapeutic use, the 
way of administration, the weight or age of the patient. In the man, the 
usual dosology ranges from 0.05 and 1 mg per unit dosage and the daily 
dosage ranges from 0.2 and 5 mg. 
It may also be convenient to utilize discontinuous administrations such as 
from 2 to 4 successive administrations followed with a stay for several 
weeks and renewed again 2 to 4 times. 
The pharmaceutical compositions according to the invention may also contain 
further active ingredients having similar activities, or synergistic 
activities; they also may contain diluing agents, filling agents, binding 
agents, flavouring agents, sweetening agents, tensioactive agents, 
emulsifiers or suspending agents and propelling agents. 
The pharmaceutical compositions are prepared according to the known methods 
of pharmacotechnology.

The following examples are merely intended to illustrate the invention: 
EXAMPLE 1 
Production of the depsipeptide from Fusarium equiset. 
(A) Identication of the strain 
The strain of Fusarium equiseti which allows the production of the 
depspeptide has been identified as a Fusarium equiseti (Corda) Saccardo 
and deposited at the Commonwealth Mycological Institute at Kew under the 
No. 213,107. 
(a) fermentation brothes and mycelial growth 
For the purpose of identifying this strain it appears convenient to use the 
following methods of fermentation 
(1) fermentation medium n.degree.1 
This medium is constituted from 
______________________________________ 
saccharose 25g 
glucose 25g 
ammonium nitrate 10g 
magnesium nitrate 2,5g 
mono potassium phosphate 
5g 
water enough for 1000ml 
______________________________________ 
This medium is sterilized and the pH value is adjusted after sterilization 
to about 5. 
After inoculation this medium gives raise to the production of a vary dense 
mycelium, of a wooly appearance, and showing at its surface an orange 
colouration. At the reverse face in a Petri box thallus shows a 
yelllow-orange colouration, better known as a peach colouration turning to 
yellowish-orange in the aged cultures. 
In the cultures on gelose it, appears a yellow pigment which diffuses 
therein. 
(2) cultures on atomized corn steep (2%) agar-agar (2%) medium 
In this medium the mycelium grows in producing a thallus appearing as 
rather close-shaved, wooly with concentric typical zonings. Pigmentation 
remains slight. The back free shows in the age a dark-yellowish 
colouration. 
(3) cultures on carboxy methyl cellulose 
(Capellin+Petterson's Medium 1965) This medium is constituted with the 
following ingredients: 
______________________________________ 
carboxymethyl cellulose 
15g 
ammonium nitrate 1g 
monopotassium phoshate 
1g 
magnesium sulphate 5g 
yeast extract 1g 
______________________________________ 
Distilled water enough for 1000 ml 
This medium may be optionaly added to small pieces of dry leaves of 
Phragmites communes. 
On this medium the mycelium remains scarce, very-close-shaved, nearly 
unpercievable and colourless. 
(b) effect of the light on the sporulation of the strain 
Out the three above described medium under illumination, with white light 
during 12 hours per day only the medium containing carboxy methyl 
cellulose and further the medium in which leaves of Phragmites communes 
are added, gives rise to the formation of macroconidias. 
High amounts of macroconidias are formed in the culture media in Petri box 
enlighted with a Wood light diffuser (max. wawe lengths 360 and 365 .mu.m) 
disposed at 33 cm from the lamp, after an incubation period of 4 to 6 
days. The colouration of the media after culture under Wood light is 
slightly attenuated in comparison to that appearing in the cultures grown 
uner white light. 
(c) aspects of the Fusarium in several medias 
Fusarium equiseti N.degree. 213,107 has also been grown on usual media, 
generally utilized for the growth of various Fusarium. Experimental 
results have been thus obtained: 
medium containing 40 g/l of flour of oat 
medium containing 50 g of grated potatoes, 10 g glucose and 20 g gelose for 
1 liter water 
medium containing 2% Agar and Malt 
On these media, it appears that the conidiogenesis occurs with a very 
slight extend with production of abnormalous conidias which could induce 
the confusion between aborted macroconidias with microconidias. 
It may be of interest to note some typical aspects of the Fusarium genus, 
namely the appearance of a pigment distributed in concentric zonings. This 
coloured zones appear namely in the medium with potatoes, as well in the 
enlighted cultures as the cultures performed in the dark. 
The cultures grown on the medium with flour of oat provide mycelial thalli 
which are short-shaved, compact, heterogenous, and slightly coloured. 
Conidiogenesis did not practically occur. 
MORPHOLOGICAL DATA OF FUSARIUM EQUISETI 
Fusarium equiseti no. 213,107 shows the following particulars: 
lack of microconidias 
presence of macroconidias showing from 3 to 7 septa weared on branched 
macroconidiophors 
presence of intermediate chlamydospori, isolate or in chain. 
The whole morphological data of the macroconidias, the lack of terminal 
chlamydospori and the presence of intermediate chlamydospori allow the 
identification of this Fusarium as a Fusarium of the subgenus Gibbosum. 
Among the three species included in this genus, only the species equiseti 
corresponds to this whole of morphological data. The appearance of the 
peach colour of the cultures and the oblong shape of the basal cells in 
form of foot, confirm this identification. 
(B) Preparation of the first tank of the fermentation 
(1) culture on inclinated gelose 
The content of an ampul of lyophilizated mycelium is taken up with 2 ml 
sterile water. The thus obtained suspension is utilized for seeding 4 test 
tubes containing 10 ml of a solid medium containing: 
______________________________________ 
floaked oats 30g 
glucose 10g 
agar-agar 20g 
distilled water enough for 
1000ml 
______________________________________ 
This solution is sterilized by heating for 20 mn at 120.degree. C. The pH 
of this medium is about 6,6 before sterilization and 5,8 after 
sterilization. These slides are incubated for 6 to 8 days in a 
thermostatic oven at 28.degree. C. then let to receive the sunlight for a 
week at laboratory temperature. 
(2) culture in a Roux's Flask 
The aerial mycelium from a test tube is taken up with 10 ml sterile water. 
This suspension is utilized for seeding 5 Roux's Flasks containing 200 ml 
of the same medium containing gelose as in the first step. The incubation 
is carried out for 6 to 8 days in a thermostatic oven at 28.degree. C. 
then subjected to enlightening in the sun light for a week at the 
laboratory's temperature. 
(C) Preparation of the inoculates for the second tank 
(a) first stage 
From the culture in a Roux's flask, a suspension of mycelium in 100 ml 
water is prepared by scraping the whole surface of the culture. This 
suspension is used for seeding a 10-liters Flask containing 4 liters of 
the following medium: 
______________________________________ 
saccharose 25g 
glucose 25g 
ammonium nitrate 10g 
mono potassic phosphate 5g 
magnesium sulphate 2,5g 
water enough for 1000ml 
______________________________________ 
This solution is made sterile by heating it for 30 mn at 120.degree.. The 
pH values decrease from 5,45 before sterilization to 4.75 after 
sterilization. The aerobic incubation is performed at 28.degree. C. in a 
thermostatic jacket under bubbling of sterile air which insures the 
stirring for at least 48 h and better 72 hours. 
(b) second stage 
The seeding of the second tank is performed starting from the culture in 
the 10 liters flask to a tank containing 75 liters of the following 
medium: 
______________________________________ 
saccharose 40g 
celerose 5g 
ammonium nitrate 10g 
monopotassium phosphate 5g 
magnesium sulphate 2,5g 
water enough for 1000ml 
______________________________________ 
sterilization of this medium at 120.degree. for 30 mn; pH after 
sterilization, 5.6. 
The fermentation of this second stage is performed under a stream of 
sterile air of 3.5 m.sup.3 /h a stirring of 70 RPM, a temperature of 
29.degree. C.+1 for at least 36 hours. 
During the fermentation, the controls of sterility, the determination of pH 
a nicroscopic evaluations and determination of the amounts of reducing 
sugars are performed on samples sterilely taken up. 
(c) third stage 
A sample of the cultures of the second stage is used for seeding 12,001 of 
the same milieu as previously (amount of culture: 6.25%). 
______________________________________ 
aeration 70 m.sup.3 per hour 
stirring 30 RPM 
temperature 29.degree. C. + 1 
duration from 36 to 40 h. 
______________________________________ 
The broth is submitted during this period to the same determination as 
previously exposed. 
(D) Industrial Manufacture 
The 12,001 of inoculum (3rd stage) are utilized as a whole to seed an 
industrial tank containing 7 m.sup.3 of a medium having the following 
composition: 
______________________________________ 
saccharose 50g 
Celerose 5g 
ammonium nitrate 10g 
monopotassium phosphate 5g 
magnesium sulfate 2.5g 
zinc sulfate .04g 
calcium carbonate .2g 
tap water enough 1l 
______________________________________ 
This medium is sterilized by heating at 120.degree. for 30 mn. 
The pH of the medium after sterilization is about 6.5. This culture is 
carried under the following experimental conditions. 
______________________________________ 
aeration 250 m.sup.3 /h 
stirring 20 RPM 
temperature 29.degree. C. + 1 
duration about 40 hours 
______________________________________ 
The pH value is of about 3 at the 24th hour and increases steadily to reach 
the values of about 5.3, 5.5 at the end of the fermentation. After 
termination of the fermentation, the mycelium is separated from the broth 
by filtration. The mycelium is dried in a ventilated oven 120 Kg mycelium 
are obtained after drying and grinding. 
(E) Extraction of the depsipeptide 
(1) Extraction of the raw product 
100 kg of the ground dried mycelium previously obtained are taken up in 400 
l trichlorethylene. After 2 hours stirring the unsoluble material is 
separated by filtration and pressed. This mass is a new washed with 100 l 
trichlorethylene then extracted a second time with 300 l trichlorethylene 
under stirring for one hour and filtered. The insoluble matters are 
further washed with 100 l trichlorethylene. The organic solutions are 
united, concentrated under reduced pressure until a thick mass is 
obtained. This mass is separated and weighs about 10 Kg. 
It is further taken up in 90 liters hexane. The solution is filtered and 
the clear filtrate is extracted using a counter current device with 
aqueous methanol (methanol 80: water 20 vv). Once with 50 l, once with 25 
l, for the third time with 12.5 l and for the fourth time with 10 l of the 
mixture. The methanolic solutions are united and distilled off until dry 
under reduced pressure. 
(2) Purification of the raw product 
1 Kg of alumina is suspended in 5 l water and concentrated hydrochloric 
acid is slowly added until the pH value reaches 4. The stirring is kept 
for 24 h while maintaining the pH at this value. Alumina is filtered on a 
gauze of nylon, washed with water then with acetone until dry and finally 
dried in an oven at 200.degree. for a night. 
20 Kg of the methanolic dry residue are dissolved in 80 liters methylene 
chloride and the solution is filtered. The filtrate is poored on a 
chromatography column filled with 40 Kg of alumina prepared as previously 
indicated and further washed with methylene chloride. The methylenic 
solution is slowly added at a speed of about 15 liters per hour. After 
completion of the addition the column is washed at the same rate with 80 
liters of methylene chloride. 
The methylenic eluates are recovered and concentrated until thick 
consistence. This paste is taken up in 200 liters ethanol, filtered until 
clear, cooled to 4.degree. C. and slowly added to 400 liters distilled 
water. The addition of water lasts about 8 hours and the thus formed 
suspension is kept under stirring for 20 hours at 4.degree. C. The 
precipitated crystalls are separated by filtration, washed with 20 l of a 
mixture of ethanol: 1-water: 2 previously cooled at 4.degree. then with 50 
l water. The crystalls are further dried at 40.degree. C. in an oven under 
reduced pressure until constant weight. 
The depsipeptide appears as a white crystalline powder, odourless and of 
bitter taste. It is sparingly soluble in water but easily soluble in 
chloroform, methanol and 95% ethanol. 
The melting point of the anhydrous product determined on Koffler bank is 
about 121.degree. (+5); Its rotatory power is [.alpha.].sub.D.sup.20 
=-80.degree. (+5) (C=5% methanol) 
Nitrogen content: 6,2+0.3% 
The infra-red spectrum in KBr is supplied with hereafter. 
(3) Separation and identification of the four components by TLC 
A solution of 10 mg/ml of the depsipeptide in methanol is prepared and 10 
.mu.l of this solution is deposited on a plate of 20 cm.times.20 cm 
charged with a mixture of cellulose Mn 300 and cellulose Mn 300 Q and 
impregnated with a mixture of formamide-acetone (1:4) until 2 cm from the 
top. 
After development the plates are dried at 100.degree. C. for 1 hour, and 
sprayed with a 0.5% chloroformic solution of iodine. The plates show 4 
different yellow strains. The two most important have a Rf respectively of 
0.55 and 0.45, one less important has a Rf of about 0.35 and the slighest 
one has a Rf of about 0.3. 
(4) Separation of the components by HPLC 
The percentages of the four components after HPLC have been determined with 
an integrator by reference to the square of the 4 peaks and assuming that 
the absorption in UV light at 254 m.mu. is the same for each component. 
The HPLC in reverse phase has been carried out with a 30 cm long column 
filled with Bonsapak CL 8 ml in a Waters apparatus. The solvent of elution 
is a mixture of methanol 8V-water .alpha.v. The output of the eluents is 
of about 1.5 ml/mn and 10 .mu.l of a 10 mg/ml solution of the depsipeptide 
in methanol is injected. The peaks are detected by measure of the 
absorbance at 254 m.mu., and the squares thereof is measured with an 
integrator ICAP 5. 
The results obtained correspond to the average of the values obtained with 
several samples of different productions of the depsipeptide. 
______________________________________ 
Compound A in average 
3.9 
Compound B in average 
15.3 
Compound C in average 
38.1 
Compound D in average 
42.7% 
______________________________________ 
The following graph shows the separation of the four components by HPLC 
EXAMPLE II 
Pharmaceutical compositions containing as active ingredient the 
depsipeptide from Fusarium equiseti. 
(a) pressurized solution in Isopropyl Myristate 
______________________________________ 
Depsipeptide .050g 
Oil of Neroli .050g 
Eucalyptol .050g 
Saccharine .00125g 
Isopropyl Myristate enough for 
2.5ml 
Flugene 12 as propellent 
7.5ml 
______________________________________ 
(b) aqueous suspension 
______________________________________ 
Depsipeptide .050g 
polyoxyethylenesorbitane mono oleate 
sold under the trade name "Tween 80" 
.010g 
Sodium chloride .180g 
Oil of Neroli .050g 
Eucalyptol .050g 
Sodium saccharinate .010g 
Water enough for 20ml 
______________________________________ 
The two pharmaceutical compositions are packed in a flask fitted with a 
pump which delivers each time 0.080 g of solution or suspension i.e. 200 
.mu.g of active ingredient by delivered dosis. 
(c) pressurized solution in Isopropyl Myristate 
______________________________________ 
Depsipeptide .015g 
Flavouring agent .0075g 
Isopropyl Myristate enough for 
.75ml 
Freon 12 2.25ml 
______________________________________ 
This solution is packed in a container fitted with a valvedelivering 25 
.mu.l each time. The amount of depsipeptide delivered at each 
pulverization is of about 125 .mu.g. 
(d) pressurized solution in Isopropyl Myristate 
______________________________________ 
Depsipeptide .030g 
Flavouring agent .015g 
Isopropyl Myristate .75ml 
Freon 12 2.25ml 
______________________________________ 
This solution is packed in a container fitted with a valve delivering 250 
.mu.g of depsipeptide each pulverization. This container is intended for 
120 pulverizations. 
(e) Solution in oil 
______________________________________ 
Depsipeptide .05g 
Saccharine .0015g 
Flavouring agent .05g 
Isopropyl Myristate enough for 
6ml 
Propelling agent .05g 2ml 
______________________________________ 
The container is fitted with a valve delivering 30 .mu.l each time. 
(f) aqueous suspension 
______________________________________ 
Depsipeptide .050g 
polyoxyethylenesorbitane mono oleate 
(sold under the trade name "Tween 80") 
120mg 
Potassium chloride 3mg 
Disodium phosphate 17.25mg 
Dipotassium phosphate 3mg 
Calcium chloride 1.5mg 
Magnesium chloride 1.5mg 
Sodium Mercurithiolate .0375mg 
Water enough for 15ml 
Propellent enough 
______________________________________ 
This suspension is packed in a container fitted with a pump delivering 75 
.mu.l at each pulverization. 
Each pulverization then provides 250 .mu.g of active ingredient. 
(g) injectible aqueous suspension 
______________________________________ 
Depsipeptide 100mg 
Cremophor EL 100mg 
Sodium chloride 800mg 
Sterile water enough for 
100ml 
______________________________________ 
This suspension is packed in ampuls of 1 ml each containing 1 mg active 
ingredient per unit dosage. 
EXAMPLE III 
Pharmacological study of the depsipeptide of this invention 
(A) Study of the stimulation of the humoral immunity in the mice after 
sensibilization with red blood cells of the sheep 
This test is performed on batches of 10 mice of swiss CD strain weighing 
about 20 g. Each mouse receives a threshold immunizing dosis of 10 
millions of red blood cells by intravenous way. 
The mice are treated at various periods of time before immunization either 
by intravenous or intraperitoneous way. The depsipeptide from Fusarium 
equiseti is dissolved in propylene glycol (84 mg/ml) then this 
concentrated solution is diluted with a saline solution. The mice are 
injected with 10 or 50 .mu.g of depsipeptide. 
A lot of controls receives only the red blood cells and the solvent. 
A lot of mice receives a suspension of a culture of Corynebacterium simplex 
in a saline solution as a reference product. 
The bloods of the mice are taken at the established days through 
retroorbital ponction. The sera are stored at -80.degree. C. For each 
batch the sera are tested individually as well as a pool of sera. 
DETERMINATION OF THE CONTENT IN ANTIBODIES 
(a) determination of the hemagglutinine titer 
0.05 ml of a suspension of red blood cells of sheep in the Mayer buffer, 
titrating 7.107 cells/ml is added to 0.025 ml of serum diluted in a saline 
s solution. The plates are placed for two hours at room temperature in a 
closed vessel then for a night at +4.degree.. The hemagglutinations are 
evidenced by reading through a magnifier lens. The hemagglutinizing titer 
is calculated from the highest dilution of the serum providing a clear 
hemagglutination. It is expressed using the exponent at the log 2 of this 
dilution (for example dilution 1/64 titer=6) 
The clear hemagglutinations are only shown from titer=4, the lowest values 
being usually not clearly concluding. 
(b) determination of the hemolytic titer 
0.025 ml of a suspension of red blood cells of sheep in the Mayer's buffer 
(10.sup.8 /ml) are added to 0.025 ml of guinea pig's complement previously 
diluted in the Mayer's buffer (1/200) and to 0.025 ml serum diluted with 
saline solution (1/2, 1/4, 1/8 . . . ) 
The plates are placed in an oven at 37.degree. for 1 hour then at 
+4.degree. C. for 2 hours. The determination is effected by means of a 
glass. 
The hemolytic titer is appreciated as the highest dilution of the serum 
causing the complete lysis of the red blood cells of sheep. It is 
expressed in the same fashion as the hemagglutining titer. 
RESULTS 
(1) Study on the threshold dosis 
Several dosis of red blood cells of sheep have been tested in order to 
determine the minimal dosis which causes an humoral answer in the swiss 
mice and provokes a supraliminal answer. The maximal answer obtained with 
10.sup.8 red blood cells/mouse is magnified by the immunostimulant agents. 
Moreover 10.sup.6 red blood cells do not provoke any sensible humoral 
answer. In contrast thereof 10.sup.7 red blood cells/mouse give a homoral 
answer the cinetic of which is of value for the testing of the 
immunostimulant agents. 
The threshold dosis may vary broadly without any scientific explanation. 
(2) Study of the kinetics in the controls and in reference animals 
Several batches of controls which receive only 10.sup.7 red blood 
cells/mouse have been studied from a point of view of kinetics and have 
shown that the hemagglutinizing antibodies appear from the fourth day 
after immunization for certain batches and from the seventh day for other 
batches. The hemagglutining titers are usual lower than 4. It appears also 
that the hemolytic titers of the sera of the controls are practically 
equal to zero until the seventh day. 
Cultures of corynebacterium parvum have been used as a reference substance 
and show they stimulate the immunological answer being injected 3 to 6 
days before the injection of red blood cells. Antibodies appear in 
immunostimulated animals on the fourth day. 14 days after the significance 
of the immunological answer is statistically of greater value. 
(3) Study of the depsipeptide from Fusarium equiseti 
The depsipeptide from Fusarium equiseti has been administered to mice 
during 3 days from the 6th to the third day before injection of red blood 
cells. It provokes an increase in the content of hemolytic and 
hemagglutining antibodies together with an induction in the appearance of 
hemolytic antibodies from the fourth day after injection of red blood 
cells. 
Doses from 10 .mu.g to 50 .mu.g/day/mouse cause the same result. The 
immunostimulation caused by the depsipeptide is of the same order of that 
caused with 500 .mu.g/day of cultures of corynebacterium parvum (Institute 
Pasteur of Paris). The same results have been obtained with the 
depsipeptide with doses which are 10 to 50 times lower than those of 
corynebacterium parvum. 
(B) Lymphoblastic transformation Test (TTL) 
The determination of the lymphoblasts by administration of the depsipeptide 
from Fusarium equiseti has been carried out using the method described by 
R. P. Danicle and S. K. Hollan Proceed Nat. Acad Sci (New York) 73, (1976) 
3599. This test is performed at the optimal concentration of the mitogenic 
agent, the product to be tested remaining in contact with the cells during 
the whole set of time. Under this experimental conditions the, 
incorporation of the biological precursor is quite reproductible and 
maximal. The biological effect produced by the product is evidenced by 
inhibition of the incorporation of the tagged inhibitor. 
The determination of the optimal doses of the mitogenic agent is based on 
the curves Effect/Doses using the beginning of the plateau. 
The reference substances are Phytohemagglutinin (optimal dosis 1 .mu.g/ml) 
and lipopolysaccharides of Escherichia coli (optimal dosis 5 .mu.g/ml) 
From the results hereafter exposed, it appears that in contrast to the 
reference substances the depsipeptide from Fusarium equiseti is not a 
mitogenic agent at the tested doses and curbes the incorporation of 
tritiated Thymidine usually induced by the mitogenic agents. 
(C) Study of the effects of the depsipeptide from Fusarium equiseti on the 
immunological response 
Depending on the administered doses the tested substance show a stimulant 
or depressant effect either on the humoral or cellular immunological 
answer in the mice. 
The humoral answer has been evidenced after administration of 
lipopolysaccharide from Escherichia coli (Vujanovic 1973-Le Bouteiller 
1974) which increase the amount of blood cells of medullar aspect. 
The cellular response has been evidenced after administration of 
ethoxymethylene oxazolone according to the methods described by Turk 
(1967) and Anderson (1972). 
The characteristics of the cells from an immunological and ultrastructural 
point of view have been determined according to the method of Jeannesson 
(1975) 
The number of white blood cells producing the antibodies is quantitatively 
determined using the test of hemolysis zones in a semi solid medium 
containing the target red blood cells. This test utilizes the zones of 
direct hemolysis according to the method of Jerne and Nordin (1963) which 
detect the cells producing Ig M 
In view of the auto-radiographie study, the suspensions of cells are 
incubated at 37.degree. C. with tritiated thymidine at a dosis of 20 
.mu.Ci/ml for 30 minutes before being treated for the determination of the 
Ig M of surface. 
The cells are fixed with glutaraldehyde then stained with diaminobenzidine 
according to the method of Graham and Karnovsky (1966), then treated with 
osmic acid and inbedded into EPON. 
RESULTS 
Immunological response to ethoxymethylene oxazolone 
(a) number of cells 
On the 5th day after administration of the depsipeptide from Fusarium equi 
seti the increase of the number of cells is usually higher than 50 
percent, namely after intraperitoneal administration in the mice. On the 
9th day the increase is slightly less significant and thereafter 
discontinues. The number of cells having incorporated some tritiated 
thymidine is of the same order by controls and tested animals, i.e. of 
about 4-6%. 
(b) number of cells able to recognize the antigenic substances 
On the 5th day the percentage of cells recognizing the antigenic substance 
is very significantly increased (257 percent) and this increase results 
both from an increase of the amounts of cells in the ganglions and an 
increase of the percentage of the cells specifically activated for a 
determined number of cells (10.sup.6 cells). This increase progressively 
disappears and cannot be evidenced after the 9th day. 
(c) number of cells forming zones of hemolysis 
The very significant increase of cells forming zones of hemolysis appears 
on the 5th day and is caused by an increase in the number of the cells. On 
the 9th day this increase namely results from the number of cells forming 
hemolysis zones for a determined number of cells (10.sup.6 cells). 
(d) effect on the content in antibodies 
On the 5th day the content in antibodies remain constant from the 
beginning. On the 9th day the percentages of hemagglutining antibodies and 
of hemolyzing antibodies is broadly increased namely that of hemolyzing 
antibodies. 
(e) study on the number of cells having Ig on their surfaces 
The cells having Immunoglobulines (Ig) at their surface have been counted 
on their slides from the initial cellular suspension in the controls and 
in the treated mice. The percentage is of about 35 percent. 
From a microscopic inspection it appears that the nature of the cells 
causing hemolysis and the cells forming rosettes are the same in the 
controls and in the treated mice. For the rosette forming cells they are 
lymphokystes with Ig on their surface and white T cells the proportion of 
which reaches 40 to 50 percent on the 9th day. For the hemolysis zones 
forming cells they are namely plasmocyts. 
IMMUNOLOGICAL RESPONSE AFTER PRETREATMENT WITH LIPOPOLYSACCHARIDES 
The injection of the depsipeptide from Fusarium equiseti induces a slight 
increase in the number of cells on the 9th day. 
The effect on cells recognizing the antigenic substance is none and even 
negative. The decrease of the cells able to recognize the antigenic 
substance is more significative on and after the 9th day. 
No effect has been evidenced on the number of cells forming hemolysis 
zones. 
A slight decrease in the content of hemagglutining antibodies appears 
whilst an important increase in the content of hemolyzing antibodies is 
shown on the 5 th day. 
CONCLUSION 
The immunological effects of the depsipeptide from Fusarium equiseti may be 
summarized in the following fashion. 
Immunological response to ethoxymethylene oxazolone is increased. The 
increase of the cells in or around the ganglions is significant and 
maximal on the 5th day (50 to 80%). The number of cells recognizing the 
antigenic substance is very frequently increased either due to the 
increase of the number of cells or due to the increase in this specific 
activity. The increase in the hemolysis zones forming cells reflects only 
the increase in the number of cells in the ganglions. 
The immunological response after pretreatment with lipopolysaccharides is 
decreased. The number of cells recognizing the immunogenic agent remains 
constant or is decreased. The content in antibodies is decreased or remain 
unaltered. 
(D) Effect of the depsipeptide on the immunological response. 
(1) Potentializing action on the humoral immunity. 
This test has been performed on batches of 10 mice (C 57 B6 male mice 
strain) which are injected each with 300 .mu.g of purified bovine serum 
albumine (fraction V of Cohn), purchased from the Armour Pharmaceutical 
Company, in the posterior plantar paws. 
At the first day 20 mice received each 0.2 ml of a mixture containing the 
solution of BSA in a phosphate buffer and 10 .mu.g of the depsipeptide 
from Fusarium equiseti (2 vol) and Freund's uncomplete adjuvant (3 vol). 
A batch of 10 mice is used as controls. It receives only the same mixture 
but the depsipeptide and the Freund's adjuvant. 
Anoter batch receives only the injection of BSA together with the Freund's 
adjuvant. 
At the day 2I the half of the mice in each bath is sacrifized to determine 
the content of anti BSA-antibodies by passive hemagglutination. The second 
half of the mice receives a reniewed injection of the mixture at half 
dosage (0.1 ml instead of 0.2 ml). 
At the day 35 the remaining mice are sacrifized and the content of specific 
anti BSA antibodies is determined using the same method. 
A very significant increase in the content of specific antibodies is 
evidenced on the 35th day in the treated mice. 
(2) Effects on the cellular immunological response. 
(a) mitogenic potency of the depsipeptide 
500.000 splenic lymphokytes from CBA strain mice suspended in 0.1 ml RPMI 
are incubated at 37.degree. C. for 3 days in a stream of carbonic 
anhydride with various concentrations of the depsipeptide from Fusarium 
equiseti. 
In the same fashion the white cells are also incubated with 
phytohemagglutinine at a concentration of 1/200, Concanavaline at a 
concentration of 10 .mu.g/ml as reference substances. 
4 hours before the termination of this incubation period, 1 .mu.Ci 
tritiated thymidine is added to each culture. Afer completion of the 
incubation the cells are filtered and twice washed with saline solution. 
The radio activity on the filter is counted with a .beta.-ray counter. The 
incorporation of Thymidine is calcuated as the ratio between the amount of 
incorporated thymidine in the presence of the mitogenic substance and the 
amount of incorporated thymidine in the lymphokytes without any mitogenic 
substance. 
RESULTS 
The following results have been obtained and are tabulated hereinafter: 
The results show that the depsipeptide curbs the nucleic metabolism at very 
low dosages through a very strong effect on the immunosuppressive cells or 
a modification of the metabolism or cellular permeability. 
The depsipeptide is not a mitogenic substance as such. In contrast thereof, 
it may have a stimulant effect on the lymphokytes in the presence of the 
usual mitogenic substances. 
______________________________________ 
Amount of Ratio of 
active substance 
incorporation 
Statystical value 
______________________________________ 
0,1 pg/ml = 0,41 (.+-. 0,1) 
p = 0,0047 
1 pg/ml = 0,62 (.+-. 0,07) 
p = 0,044 
5 pg/ml = 0,84 (.+-. 0,06) 
Not significative 
10 pg/ml = 0,90 (.+-. 0,06) 
Not significative 
110 pg/ml = 0,32 (.+-. 0,12) 
p = 0,0078 
1 ng/ml = 0,40 (.+-. 0,10) 
p = 0,0082 
10 ng/ml = 0,56 (.+-. 0,08) 
Not significative 
100 ng/ml = 0,62 (.+-. 0,07) 
Not significative p = 0,054 
1 .mu.g/ml = 
0,36 (.+-. 0,1) 
p = 0,0044 
10 .mu.g/ml = 
0,63 (.+-. 0,07) 
p = 0,028 
100 .mu.g/ml = 
0,80 (.+-. 0,06) 
Not significative 
PHA 1/200 = 51,15 (.+-. 0,006) 
p = 0,0028 
ConA 10 .mu.g = 
50,30 (.+-. 0,006) 
p = 0,0025 
LPS 100 .mu.g = 
14,81 (.+-. 0,012) 
p = 0,0028 
______________________________________ 
(b) stimulation of the activities of the macrophages 
The peritoneal macrophages from unstimulated rats (Fisher strain) are 
prelevated through peritoneal washing and isolation on plastic rack. They 
are cultivated for a night with dosages of the depsipeptide ranging from 1 
picogramm to 1 nanogramm/ml. 
The content in proteins, in lysosomal .beta.-glucoronidase, and in 
cytoplasmic leucine-amino peptidase are determined on the cells previously 
lysed with 0.05% Triton X-100. 
In the same manner, incubation has also been performed with a solution of 
lipopolysaccharide from Escherichia coli and Muramyldipeptide as reference 
substances. 
The depsipeptide induces a significant increase of the hydrolases in the 
macrophages at a dose ranging from 1 pg to 10 ng/ml. The proteic content 
is also increased with doses ranging from 10 to 100 pg/ml. As higher doses 
and namely from 100 ng/ml the depsipeptide appears to be toxic for the 
cells as evidenced by the strong decrease in the proteic content and the 
increase in the content of intracellular macrophagic enzymatic activities. 
The same test has also been performed in vitro. The rats received 6 days 
before the peritoneal washings an intrapeitoneal injection of depsipeptide 
(1 to 100 .mu.g in 1 ml saline solution). 
Lipopolysaccharides from Escherichia coli Muramyl dipeptide and water 
soluble adjuvant are injected to other batches of rats as reference 
substances. The peritoneal macrophages recovered after washings are 
incubated for 2 hours in order to delete the non-adhering cells and 
further for 4 hours. The cells are then lysed and the enzymatic and 
proteic contents determined as above given. 
RESULTS 
At a dosis of 1 .mu.g the depsipeptide from Fusarium equiseti induces a 
very high increase of the lysomal enzymes (.beta.-glucuronidase+35%) and 
of the cytoplasmic enzymes (leucine amino peptidase+35%). The content in 
proteins is also increased (+24%). At this dosis the water soluble 
adjuvant induces an increase of the hydrolases of 39% and the 
lipopolysaccharides an increase of 42% of the leucine aminopeptidase and 
35% of the proteins. These activities are thus of about the same level. 
In contrast thereof at a dosis of 100 .mu.g the depsipeptide induces an 
increase of 32% of the protein content and of 50% of the cytoplasmic 
enzymes. These activities are far higher than those of the other 
immunostimulating agents. 
At a dosis of 1 ng the stimulation is weaker. 
protein content, +11% 
.beta.-glucouronidase, +23% 
leucine amino peptidase, +16% 
The following tables summarized the various results obtained with several 
doses of the depsipeptide in comparison with the reference substances. 
TABLE I 
__________________________________________________________________________ 
1 .mu.g 
P 100 .mu.g 
P 
__________________________________________________________________________ 
Depsipeptide 
Proteins + 24% 
= 0,0125 
+ 32% 0,005 
.beta.-Glucuronidase 
+ 35% 0,025 
+ 37% 0,025 
L.A.P. + 35,5% 
0,025 
+ 59% 0,0025 
(leucine amino- 
peptidase) 
L.P.S. Proteins + 35% 0,01 
+ 14% 0,025 
.beta.-Glucuronidase 
- 14% 0,025 
- 3% N.S. 
L.A.P. + 42% 0,001 
+ 2% N.S. 
M.D.P. Proteins + 7,5% N.S. 
+ 2% N.S. 
.beta.-Glucuronidase 
+ 27% 0,01 
+ 5% N.S. 
L.A.P. + 24% 0,025 
+ 5% N.S. 
W.S.A. Proteins + 3,6% N.S. 
+ 1% N.S. 
(Water soluble 
.beta.-Glucuronidase 
+ 39% 0,001 
+ 46% 
= 0,001 
adjuvant) 
L.A.P. - 10,5% 
= 0,025 
- 7% N.S. 
__________________________________________________________________________ 
Amounts of cellular enzymes and proteins in the macrophages after 16 hours 
incubation. 
TABLE II 
__________________________________________________________________________ 
Lipopoly- 
Muramyl- 
Depsipeptide 
saccharide 
dipeptide 
__________________________________________________________________________ 
Saline 
Proteins 45.7 .+-. 0,1 
46.0 .+-. 1,1 
46,0 .+-. 1,1 
.beta.-glucuronidase 
29,5 .+-. 0,1 
29,1 .+-. 2,2 
29,1 .+-. 2,2 
1 pg/ml 
Proteins 44,9 .+-. 4,2 NS 
55,1 .+-. 2,2.sup.b 
46,1 .+-. 2,2 NS 
.beta.-glucuronidase 
35,8 .+-. 1,9.sup.c 
39,2 .+-. 1,5.sup.b 
36,5 .+-. 0,1.sup.b 
10 pg/ml 
Proteins 53,3 .+-. 4,3.sup.b 
47,7 .+-. 2,2 NS 
44,8 .+-. 0,7 NS 
.beta.-glucuronidase 
35,4 .+-. 4,8.sup.c 
37,1 .+-. 5,8.sup.a 
36,3 .+-. 6,4 NS 
100 pg/ml 
Proteins 50,1 .+-. 4,2.sup.b 
52,2 .+-. 6,3 NS 
44,6 .+-. 1,9 NS 
.beta.-glucuronidase 
33,0 .+-. 2,4.sup.c 
38,7 .+-. 9,9 NS 
41,0 .+-. 2,9.sup.b 
1 ng Proteins 48,2 .+-. 4,3 NS 
45,0 .+-. 0,4 NS 
44,0 .+-. 1,8 NS 
.beta.-glucuronidase 
34,2 .+-. 3,7.sup.c 
21,0 .+-. 0,5.sup.a 
34,0 .+-. 5,3 NS 
10 ng/ml 
Proteins 45,9 .+-. 1,4 NS 
47,1 .+-. 0,4 NS 
43,4 .+-. 1,8 NS 
.beta.-glucuronidase 
32,8 .+-. 5,6.sup.b 
25,2 .+-. 6,6 NS 
39,6 .+-. 6,1.sup.a 
100 ng/ml 
Proteins 44,3 .+-. 4,0 NS 
52,3 .+-. 0,4.sup.b 
40,5 .+-. 0,7.sup.b 
.beta.-glucuronidase 
29,8 .+-. 8,8 NS 
34,9 .+-. 3,4.sup.a 
23,9 .+-. 6,2 NS 
1 ng/ml 
Proteins 44,2 .+-. 5,9 NS 
46,5 .+-. 1,3 NS 
45,1 .+-. 0,9 NS 
.beta.-glucuronidase 
27,1 .+-. 0,3.sup.c 
26,0 .+-. 2,1 NS 
30,6 .+-. 3,8 NS 
10 ng/ml 
Proteins 41,9 .+-. 7,0.sup.b 
43,7 .+-. 2,5 NS 
48,1 .+-. 1,8 NS 
.beta.-glucuronidase 
25,2 .+-. 9,3.sup.b 
23,4 .+-. 1,7.sup.c 
32,2 .+-. 3,0 NS 
100 ng/ml 
Proteins 36,6 .+-. 5,6.sup.c 
41,2 .+-. 3,8.sup.b 
55,3 .+-. 3,1.sup.b 
.beta.-glucuronidase 
7,5 .+-. 3,5.sup.c 
11,6 .+-. 2,4.sup.c 
39,0 .+-. 1,6.sup.c 
__________________________________________________________________________ 
The content in proteins is expressed in g/10.sup.6 cells and that in 
.beta.-glucuronidase in mMol of substrate hydrolysed by 10.sup.6 cells per 
hour. 
Degrees of statistical significance 
______________________________________ 
a = p 0;05 
b = p 0,025 
c = p 0,001 
NS = without signification 
______________________________________ 
(3) Effect on the release of chromium induced in the larvae of schistosoma 
mansoni after treatment by an immunostimulant agent. 
The depsipeptide from Fusarium equiseti has been tested to determine its 
action in vivo on the cytotoxicity induced in the intraperitoneal 
macrophages of the rat against the larvae of schistosoma mansoni. 
The macrophages are stimulated either in vitro or in vivo and are contacted 
with a suspension of larvae of schistosoma mansoni previously tagged with 
51 chromium. 
Cytotoxicity in vivo 
This contact is intended to demonstrate an optional cytotoxic effect 
measured by the amounts of chromium released in the culture medium. This 
measure reflects the significance of cellular lesions on the surface of 
the schistosomules. The radio activity released in the medium is expressed 
as a percentage of the total radio-activity present in the schistosomules 
at the beginning of the experiments. 
The obtained results are summarized in the table III 
______________________________________ 
Substances 1 ng 1 g 100 g 
______________________________________ 
Depsipeptide 
+0,5% (NS) +13% (NS) +10% (NS) 
Lipopolysaccharides +24% +50% 
(p 0.025) (p 0.005) 
Muramyldipeptide +6% (NS) +10% (NS) 
WSA +23% +30% 
(p 0.025) (p 0.025) 
______________________________________ 
The despsipeptide causes a release of 51 Cr but it is not statistically 
significant; it is also for Muramyldipeptide the same. 
The lipopolysaccharides and the WSA make the macrophages significantly more 
toxic for the larvae as are the macrophages of untreated animals. 
The depsipeptide is practically devoid of any effect on the macrophages in 
vivo against the schistosomules. 
CYTOTOXICITY IN VITRO 
After incubation for 24 hours of normal macrophages it is not any increase 
of the cytotoxicity of the macrophages against the tagged schistosomules. 
The doses of depsipeptide which stimulate the macrophages induce only a 
slight decrease of the release of the tagged tracer. 
The table IV explained the obtained results 
______________________________________ 
Release of 51 Cr 
Saline 100 pg/ml 1 ng/ml 10 ng/ml 
100 ng/ml 
______________________________________ 
24.03% 19.67% 18.21% 21.71% 23.46 
(.+-. 2.4) 
.+-. 3.1 .+-. 3.9 .+-. 3.5 
.+-. 2.7 
______________________________________ 
(4) Action of the depsipeptide on the survival time of the mice after 
injection of leukemic cells L 1210 
Due to the fact that the depsipeptide from Fusarium equiseti acts as an 
immunostimulant in vivo and acts namely as activating the macrophages (as 
does BCG), it appears of value to study comparatively these two 
substances. 
As a model they are utilized batches of mice inoculated with leukemic cells 
L 1210 and previously treated with Cyclophosphamide. The injection of the 
depsipeptide or of BCG allows the determination of their action on the 
residual action disease. 
METHOD 
This test has been performed in batches of mice (strain BDF) of 7-8 weeks 
old. The animals are treated according to the method described by Mathe 
and Cowork "Immunotherapie active des cancers" (ESP Paris 1976) 
At the day 0 all the animals receive intravenously a suspension of 1000 
leukemic living cells L 1210. 
At the day 1 the animals are divided in 4 lots of 10 animals of the same 
weight. The controls receive only the saline solution by intraperitoneal 
way. The three other batches receive intraperitoneously an injection of 
cyclophosphamide at the suboptimal dosis of 80 mg/Kg. The batches which 
receive the cyclophosphamide are further treated in the following manner: 
one batche receives intravenously 0.1 ml of saline solution at the day +6. 
This batch is merely intended to determine the time of survival of the 
mice previously treated by the cyclophosphamide. 
one batch receives intravenously an injection of 1 mg/mouse of BCG 
(Institut Pasteur) in 0.1 ml saline solution. 
They are intended to show the effects of the active immunotherapy after 
administration of cyclophosphamide, BCG appearing as an active agent in 
these experimental conditions. 
another batch receives intraperitoneously 50 .mu.g Depsispeptide/mouse in 
0.5 ml saline solution. They are intended to determine the survival time 
of the mice treated by the depsipeptide from Fusarium equiseti. 
For each batch the survey of the mice is maintained for 40 days. The role 
of each product on the survival time is estimated and the statistical 
significance is determined using the test "t" of student by comparing the 
results of each batch to those of the controls and if necessary with the 
results obtained with the treated batches. 
RESULTS 
The results obtained in this test are gathered in table V. Cyclophosphamide 
at suboptimal dosis extends in a statiscally significative manner, the 
survival time of the animals. The mixture cyclophosphamide-BCG merely 
improves the survival time in a non statistically significative manner. 
The mixture cyclophosphamide-depsipeptide provokes a statistically 
significative improvement of the survival time in the thus treated 
animals. However no significative difference can be seen between the 
results from the two batches treated with the mixture of therapeutic 
agents (cyclophosphamide+BCG and cyclophosphamide+depsipeptide) 
This test shows that the depsipeptide from Fusarium equiseti increases the 
survival time of the mice previously injected with 1000 L1210 living 
leukemic cells and treated with cyclophosphamide. The results are of the 
same order than those obtained with BCG the number of animals used in this 
experiment is to small to establish a statistically significative 
differentiation. 
Moreover it has been determined in the mice that a single injection of the 
depsipeptide from Fusarium equiseti (50 to 250 .mu.g) induces the 
appearance of Hassal's corpuscules in the thymus and the appearance of the 
germinative center in the spleen. 
DETERMINATION OF THE ACUTE TOXICITY 
The average lethal dosis of the depsipeptide has been determined on batches 
of 10 heteroxenic mice (strain CD) or on batches of 10 rats (strain 
Wistar). All the animals receive the depsipeptide either in aqueous 
suspension or in a solution into Isopropyl Myristate. 
This compound is administered orally, intraperitoneously or intravenously 
at increasing dosages. The animals are kept under survey for 8 days, and 
the deathes when they are any, are numbered. The average lethal dosis is 
graphically determined using the method of Lichtfield and Wilcoxon. 
The thus obtained results are hereinafter summarized: 
(a) aqueous suspension 
______________________________________ 
in the mice orally 
LD.sub.50 = 5000 mg/Kg 
intraperitoneously 
LD.sub.50 = 67 mg/Kg 
intravenously LD.sub.50 = 17 mg/Kg 
in the rats orally 
LD.sub.50 = 5000 mg/Kg 
Intraperitoneously 
LD.sub.50 = 67 mg/Kg 
intravenously LD.sub.50 = 11 mg/Kg 
______________________________________ 
(b) solutions in Isopropyl Myristate 
______________________________________ 
in the mice intraperitoneously 
male mice LD.sub.50 = 169 mg/kg 
female mice LD.sub.50 = 182 mg/Kg 
in the mice intravenously 
male mice LD.sub.50 = 41 mg/Kg 
female mice LD.sub.50 = 41 mg/Kg 
______________________________________ 
The phenomena of toxicity after intraperitoneal or intravenous injection 
are namely due to the physical state of the suspended particules in the 
aqueous medium. 
When dissolved in isopropyl Myristate the toxicity of the depsipeptide is 
largely decreased.