Novel peptide from cultures of Schistosoma mansoni, a process for producing it and pharmaceutical compositions containing the same

A small peptide obtained from Schistosoma mansoni has a molecular weight between 500 and 1000. It is water soluble. It is heat-stable. It inhibits the mast cell degranulation in vitro and in vivo elicited by chemical compounds or anaphylactic reactions. It prevents passive or active cutaneous anaphylactic reactions.

PRIOR ART 
The prior art may be illustrated with the following references: B. M. 
Ogilvie and V. E. Jones--Progress in Allergy 17 (1973) 93 A. Capron and 
cowork.--Path. Biol. 16 (1968) 121 J. P. Dessaint and cowork.--Europ J. 
Immunol. 7 (1977) 624 C. Mazingue--Int. archives Allergy Appl. Immuno 63 
(1980) 178 
SUMMARY OF THE INVENTION 
The peptide of small molecular weight obtained from Schistosoma mansoni 
appears dialysable, heat resistant and soluble in trichloracetic acid. It 
prevents or inhibits the mast cell degranulation in vitro and in vivo, 
induced by chemical compounds or anaphylactic reactions. 
Therefore, it inhibits the serotonin release from the mast cells induced by 
compound 48/80 or Polymyxin B or by an anaphylactic system, by mere 
incubation. 
This peptide may be formulated into pharmaceutical compositions in 
conjunction or admixture with an inert non-toxic acceptable pharmaceutical 
carrier or vehicle. It may be used for treating or preventing conditions 
correlated to allergy, anaphylaxia and migraine. 
PREFERRED EMBODIMENTS 
This invention relates to a polypeptide of low molecular weight obtained 
from the cultures of adults Schistosoma mansoni. 
This peptide shows the particular reactions of the peptides. It is not 
retained by a diafiltration membrane such as the membrane of reticulated 
dextran sold under the tradename AMICON X 100 which is devised to retain 
the polypeptide having a molecular weight higher than 1000. The 
determinations of molecular weight performed using physical methods 
(centrifugation, electrophorese), or chemical methods seem to indicate 
that the molecular weight lies between 500 and 1000 and more likely closer 
to 500 than 1000. 
It is thermostable, soluble in trichloracetic acid and in water. 
This invention also relates to a process for producing the afore-defined 
peptide, consisting in incubating for a fixed set of time adults 
schistosomes in a saline solution then separately the insoluble bodies of 
the trematods by ultra-filtration, recovering the clear filtrate, 
dialysing the latter against distilled water, separating the dialysate and 
lyophilisine it. 
The polypeptide thus obtained is a colourless powder, soluble in water and 
in saline. 
According to a more specific procedure, the process for producing the 
polypeptide of the invention, the ultra-filtration is carried out using a 
membrane sold under the tradename MILLIPORE having a mean diameter of the 
pores of about 0.22 m.mu., the dialysis is carried out using a cell fitted 
with a membrane AMICON X 100 and the first incubation lasts for 3 hours. 
The polypeptide of this invention is endowed with very useful 
pharmacological properties. It is able to inhibit "in vitro" and "in vivo" 
the mast cell degranulation elicited by chemical compounds or anaphylactic 
reactions. 
Namely serotonin release normally induced by the chemical compounds such as 
compound 48/80, Polymyxin B or by an anaphylactic system 
(ovalbumin-anti-ovalbumin) was inhibited when mast cells were previously 
incubated with the polypeptide from Schistosoma mansoni. 
Cutaneous anaphylactic reactions elicited by the compound 48/80 or 
polymyxin B and passive or active cutaneous anaphylactic reactions, were 
inhibited by the injection of the polypeptide prior the challenge. 
The anaphylactic shock evoked by ovalbumin was also inhibited in guinea 
pigs by a previous injection of the polypeptide, preferably by 
intraperitoneal way. 
These effects appear to be specific whilst the polypeptide has no action 
either on the eosinophils or the mast cell mediators. No effect of the 
polypeptide was observed on the growth of HeLa cells or of the J.111 human 
monocytic cell line. 
The increase of intracellular cyclic AMP levels suggests that modulation of 
cyclic AMP is involved on the mechanism of inhibition. The polypeptide 
from schistosoma also inhibits the mast cell-dependant eosinophil 
cytotoxicity for schistosomuls sensitized with IgG2a antibodies. 
This polypeptide is distinct of the circulating M antigen (TCA soluble, 
thermostable) previously described and purified from schistosomes. This 
factor has no inhibitory action against cutaneous allergy and very likely 
possesses a high molecular weight. 
This invention also relates to pharmaceutical compositions containing as 
active ingredient the polypeptide from schistosoma in conjunction or 
admixture with one or more non toxic, inert, pharmaceutically-acceptable 
carriers or vehicles. In a preferred way, the carrier or vehicle will be 
selected among those suitable for parenteral administration, permucous 
administration, percutaneous administration, rectal way or perlingual way 
of administration. 
Appropriate pharmaceutical compositions are, for example, injectable 
solutions or suspensions packed in ampouls, phials, multi-doses flasks, 
auto-injectable syringes, sublingual tablets, suppositories, pressurized 
solutions for permucous way of administration, solutions or suspensions in 
a polar solvent for percutaneous application. 
The usual dosology may broadly vary. It depends namely of the therapeutic 
use and the condition of the patient. Due to the very low toxicity of the 
polypeptide from schistosoma, the dosology may be very significantly 
increased when a more intensive effect is desired. 
This invention also relates to a method for preventing or treating allergic 
states or migraine caused by the "in vivo" degranulation of the 
mastocystes, which consists in administering to the humans suffering from 
said conditions, a safe but efficient amount of the afore-defined 
polypeptide from schistosoma.

The following examples are merely intended to illustrate the invention 
without restricting it in any manner. 
EXAMPLE I 
Preparation of the polypeptide from Schistosoma mansoni 
10,000 adult schistosomas recovered from 40 days infected hamsters, were 
incubated for 3 hours in 10 ml 5.permill. sodium chloride solution. 
The incubation solution was filtered on 0.22 .mu.m Millipore membrane and 
the clear filtrate was dialysed against 10 ml distilled water. The 
dialysed solution was further lyophilysed. 
The resulting powder was re-suspended in 1 ml saline for each testing. 
EXAMPLE II 
Pharmacological study of the polypeptide 
(a) Inhibition of mast cell-dependent eosinophil cytotoxicity by the 
polypeptide. 
The eosinophil cytotoxicity for antibody sensitized S. mansoni 
schistosomuls requires the presence of a threshold number of mast cells. 
The possible inhibitory role of the polypeptide was investigated in that 
system. The results obtained show that the polypeptide significantly 
inhibited (76%) the cytotoxicity by a mixed population of eosinophils and 
mast cells. A significant inhibition was also obtained when intact 
purified mast cells were added to the mast cell-depleted population (48% 
inhibition, P&lt;0.05). When the eosinophils were activated by the 
supernatant of degranulated mast cells, no inhibition was obtained in the 
presence of the polypeptide. These results indicated that the polypeptide 
does not interfere with the eosinophil counterpart but inhibits intact 
mast cells only. 
(b) Effect of washing the cells on the inhibitory activity of the 
polypeptide 
Labeled mast cells were preincubated at 37.degree. C. for 30 min. with 10 
.mu.l of the polypeptide. The mast cells were washed three times and 
resuspended in culture medium. Degranulation was then induced either by 
polymyxin B or by the ovalbumin-antivalbumin system. Results obtained show 
the persistance of the inhibitory effect of the polypeptide on mast 
degranulation after washing. 
(c) PCA reactions. 
This test performed according to the techniques of OVARY. Accordingly 
antiovalbumin serum (0.1 ml) or S. mansoni--infected rat serum was 
injected intradermally into male Wistar rats. Forty-eight hours later, PCA 
reactions were elicited by the injection into the penis dorsal vein of 4 
mg Evans blue together with 5 mg ovalbumin or 2 mg S. mansoni antigen 
respectively in 1 ml saline. The rats were killed 30 min. later and the 
areas of Evans blue diffusion on the internal face of the skin were 
measured with a planimeter. 
(d) Active cutaneous anaphylactic reactions (ACA) 
Wistar rats were immunized by intraperitoneal injection of 10 .mu.g 
ovalbumin together with 5.times.10.sup.9 Bordetella pertussis. Thirty days 
later, ACA reactions were induced by intradermal injection of 1 .mu.g 
ovalbumin in 0.1 ml saline. Simultaneously, 4 mg Evans blue in 1 ml saline 
was administered intravenously. The rats were killed 30 min. later to 
examine the internal face of their skins as carried out for the PCA 
reactions. 
The polypeptide from Schistosoma mansoni has been tested on PCA, ACA and 
cutaneous reactions induced by compound 48/80 or Polymyxin B. Skin 
reactions elicted by the compound 48/80 or Polymyxin B were inhibited by 
prior intradermal injection of 0.1 ml of the polypeptide into the same 
site. The ACA reactions to ovalbumin were significantly inhibited by 0.1 
ml of the polypeptide but not physiological saline injected 15 min. before 
into the site of the challenge injection. The PCA reactions were markedly 
inhibited when 0.1 ml of the polypeptide was injected intradermally into 
the site of injection of the antiovalbumin or anti-S. mansoni antiserum, 
15 min. before the intravenous injection of ovalbumin of S. mansoni 
antigen respectively. No inhibition was observed when physiological saline 
was injected instead of the polypeptide. Various doses of the polypeptide 
were injected 15 min. before the antigen challenge: undiluted or diluted 
1/2 polypeptide inhibited the PCA reaction but no significant inhibition 
was obtained with higher dilutions. When the polypeptide was injected 48 
hours before the injection of the antigen, no inhibition of the PCA 
reaction was observed. However, when the polypeptide was injected 3 hours 
or 30 min. before the ovalbumin, a marked inhibition of the PCA reaction 
was obtained. The intradermal injection of the polypeptide carried out 
simultaneously with intravenous injection of the antigen did not inhibit 
the PCA reaction. 
(e) Anaphylactic shock in guinea pigs. 
Guinea pigs (Janvier Strain) were immunized by intraperitoneal injection of 
1 .mu.g ovalbumin together with 5.times.10.sup.9 Bordetella pertussis. The 
anaphylactic shock was elicited 2 months later by intraperitoneal 
injection of 1 mg ovalbumin. Thirty minutes prior to this injection, 3 ml 
saline or the polypeptide in saline were injected intraperitoneally. 
The experiments have shown that the intraperitoneal injection of 1 mg 
ovalbumin into guinea pigs sensitized to ovalbumin induced tremor, dyspnea 
and death 30 min. to 2.5 hours later. When 3 ml of the polypeptide in 
physiological saline were injected intraperitoneally before the challenge, 
death did not occur and only an urinary emission was observed. Control 
injection of 3 ml of physiological saline did not inhibit the shock. 
(f) [.sup.3 H] serotonin release assay. 
This was carried out as previously described in the litterature. Peritoneal 
cells from Wister rats were incubated with (G-.sup.3 H) 
5-hydroxytryptamine creatinine sulfate (10 Ci/mmole) at a dose of 2 
.mu.Ci/10.sup.6 /mast cells. The assay was performed in microtiter plates 
(Linbro. Flow Lab, Scotland). Mast cell degranulation was induced either 
by chemical compounds or by an antigen-antibody reaction. Fifty 
micro-liters of Polymyxin B sulfate (5 .mu.g) or of the compound 48/80 
(0.5 .mu.g) were added to 50 .mu.l of the cell suspension 
(5.times.10.sup.4 mast cells) together with 50 .mu.l of Eagle's Minimum 
Essential Medium (MEM) (Institut Pasteur Production, Paris). 
The cells were incubated 15 min. at 37.degree. C., then centrifuged at 
1,000.times.g for 10 min. Fifty microliters of the supernatant were 
transferred to counting vials. Treatment of cells with 50 .mu.l of 
digestin (Merck, Darmstadt, R.F.A.) was used to measure the amount of 
serotonin releasable by mast cells. Ten milliliters of butyl-PPD (7 g/l) 
in toluene-triton (1/1) scintillation fluid were added to the counting 
vials (Block, Paris). Radioactivity was measured in a liquid scintillation 
counter (Nuclear Chicago, Ill). 
For the antigen-anaphylactic antibody reaction, 50 .mu.l of the undiluted 
anti-ovalbumin serum was added to 50 .mu.l of the cell suspension. Passive 
sensitization of mast cells was achieved by incubation for 15 min. at 
37.degree. C. 
The activity of the polypeptide has been tested on rat mast cells in vitro 
sensitized with 50 .mu.l of undiluted antiovalbumin antiserum and 
incubated further with 50 .mu.l ovalbumin. The preincubation of mast cells 
with 10 to 30 .mu.l of the polypeptide 30 min. before the sensitization, 
significantly inhibited .sup.3 H serotonin release by ovalbumin. No 
significant release was induced by the polypeptide added to unsensitized 
mast cells. The activity of the polypeptide was also tested on mast cell 
degranulation induced by the compound 48/80 and Polymyxin B. The serotonin 
release normally induced by these chemical compounds was inhibited by 
preincubation of the mast cells with 10 or 20 .mu.l of the polypeptide. 
The activity of the polypeptide was also assayed after heating the 
fraction 1 hour at 100.degree. C. The mast cells were incubated 30 min. 
respectively with 10 .mu.l of unheated polypeptide before degranulation 
induced by ovalbumin-antiovalbumin. Similar inhibitions were observed with 
the untreated and heated compound (46 and 48% respectively). Therefore, it 
may be assumed that it is perfectly thermostable. 
(g) Effect of the polypeptide on cAMP levels in mast cells. 
As mast cell degranulation is associated with the decrease of the levels of 
intracellular cyclic AMP, the activity of the polypeptide on intracellular 
AMP levels was investigated. 
Mast cells were purified by centrifugation of rat peritoneal cells through 
38% bovine serumalbumin. Purified mast cells (1.times.10.sup.5) were 
incubated 5 min. with 20 .mu.l of the polypeptide. The cells were lysed by 
50 .mu.l 0.5 N sodium hydroxyde, neutralized with 50 .mu.l 0.5 N 
perchloric acid and then boiled for 5 min. at 100.degree. C. AMP was 
measured using the Becton-Dickinson radioimmunoassay kit. The results are 
expressed in picomoles per million cells (pmoles/10.sup.6 cells). 
From four different experiments, it was observed that AMP levels in 
unstimulated mast cells was 4.74.+-.1.75 pmoles/10.sup.6 cells and was 
458%.+-.124 increased after 5 min. incubation with the polypeptide. No 
measurable AMP was found in the polypeptide alone. 
(h) Effect of the polypeptide on Hela and human monocytic cells. 
The polypeptide has been shown to inhibit the proliferation of normal 
lymphocytes in vitro induced by mitogens or in mixed lymphocyte culture as 
well as the proliferation of 14 day-S. mansoni infected rat lymphocytes 
induced by S. mansoni antigen. Although the identity of the factors that 
inhibit lymphocyte proliferation and mast cell degranulation remains to be 
demonstrated, it was important to investigate the activity of the 
polypeptide from S. mansoni on other cells from various origins. The 
effect of the polypeptide (10 .mu.l) was tested on 5.times.10.sup.5 Hela 
cells or 2.5.times.10.sup.5 (J.111) human monocytic cells. The count of 
viable cells 3 to 72 hours after the addition of the polypeptide showed 
only a slight and transcient effect on cell viability and proliferation. 
The technique used was the following: 
Hela cells (ATCC CCL 2.2) and human monocytic cell lines J.111 (ATCC CCL 
24) were used as target cells. Cells were cultured in Petri dishes 
(Nunclon, Denmark) with Eagle's MEM supplemented with 15% heat-inactivited 
foetal calf serum together with 10 .mu.l of the polypeptide. Viable cells 
were counted using the trypan blue exclusion technique.