EP0891189A1

EP0891189A1 - Medicament for optimising mucosal viscosity and stimulating intestinal function

Info

Publication number: EP0891189A1
Application number: EP97919487A
Authority: EP
Inventors: Michel Hooreman
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-04-05
Filing date: 1997-04-04
Publication date: 1999-01-20
Also published as: HUP9902421A3; JP4196225B2; PL188157B1; FR2747045B1; CA2251009A1; NO984632L; AU735340B2; CA2251009C; NO984632D0; CN1221347A; CZ318598A3; TR199801976T2; NO324877B1; AP9801357A0; CZ298428B6; NZ332107A; US6440413B1; AP1018A; FR2747045A1; RU2198673C2

Abstract

The invention pertains to the field of biology, and more particularly, to therapeutic chemistry, and discloses pharmaceutical compositions containing, as an active ingredient, an enzyme or an enzyme complex containing said enzyme, obtained by cultivating Streptomyces fradiae under conditions defined in French patent 2.600.340, as a mixture or in combination with a physiologically acceptable, non-toxic and inert carrier or vector. Said pharmaceutical compositions are useful for preventing or treating degenerative diseases, particularly those related to intestinal dysfunction.

Description

DRUG

OPTIMIZING THE VISCOSITY OF MUCUS AND

STIMULATING THE WORKING OF THE INTESTIN

The invention relates to the field of biology and more particularly to that of therapeutic chemistry

The subject of the invention is pharmaceutical and / or dietetic compositions, based on an enzyme or an enzymatic complex containing this enzyme, which can contribute to the prevention and / or treatment of diseases.

It relates more particularly to pharmaceutical compositions based on an enzyme or an enzymatic complex containing this enzyme stimulating the functions of the intestine, and thus being able to contribute to the prevention and / or the treatment of degenerative diseases linked to aging.

It specifically relates to pharmaceutical compositions containing as active principle one or more enzymes extracted or contained in the enzymatic complex obtained by culturing Streptomyces fradiae under the conditions defined in French patent 2,600,340 in the name of the Applicant

In the aforementioned patent, the production and use in zootechnics of a new proteolytic complex, obtained from a filamentous bacterium, Streptomyces fradiae, has been described, this complex is designated below by its current commercial name PANSTIMASE®

This filamentous bacterium can simultaneously produce at least 5 proteinases and 2 peptidases, these 7 enzymes have been described by K MORIHARA et al (Biochim Biophys Acta, 1967, 139, 382) When they are added together in the food consumed by animals, these 7 enzymes cause an excessive reduction in the viscosity of the intestinal mucus, and can then cause an ulceration of the intestinal wall These 7 enzymes therefore have negative effects in animal husbandry when used together Under the conditions indicated in the aforementioned patent, PANSTIMASE® is obtained, which consists only of 3 exocellular proteinases, with a preponderance of one of them, and excluding endocellular proteinases and peptidases. When added to food consumed by animals, PANSTIMASE® causes a moderate reduction in the viscosity of intestinal mucus, and is not likely to attack the intestinal wall.

At the same time as its relatively moderate action on the proteins of the intestinal mucus, PANSTIMASE® exerts a stronger action on the proteins of raw materials used in the composition of food, which improves their digestibility.

In addition to these direct actions in the intestine, PANSTIMASE® exerts indirect actions on the various organs, in particular on the pancreas; as already indicated in the prior patent, it has been found that it causes an increase of 50 to 80% in the concentration of enzymes or proenzymes in this organ.

Because of these favorable actions, PANSTIMASE® is now used as a nutritional supplement added to animal feed, to improve their growth or productivity. It has thus been found that it also improves the resistance of animals to infectious, bacterial or viral diseases, and that it reduces certain negative effects of aging.

These findings have suggested the possible therapeutic application, for animals as for humans, of PANSTIMASE®, or of the active ingredients of which it consists, namely, the preponderant exocellular proteinase produced by Streptomyces fradiae or the mixture of this preponderant exocellular proteinase with at least one of the other two exocellular proteinases produced by Streptomyces fradiae.

The active principles of pharmaceutical compositions require additional purification compared to the product used for the animal. Under the conditions indicated in the previous patent, an unpurified PANSTIMASE® is obtained, the titer of which is at least equal to 2,000 AU / mg.

The Anson Unit (abbreviated AU) is here defined as the quantity of enzyme which, incubated for 10 min at 25 ° C. and at pH 7.5 in the presence of denatured hemoglobin, releases of this substrate the equivalent of 1 mcg of tyrosine, determined by spectrophotometric absorption at 280 nm on the filtrate which cannot be precipitated by trichloroacetic acid.

Various adjustments to the conditions indicated in the prior patent make it possible to obtain a semi-purified PANSTIMASE®, the titer of which is at least equal to 4,000 AU / mg. The conventional techniques of fractional precipitation, or of column passages, allow each of the 3 proteinases composing PANSTIMASE® to be obtained in isolation. The specific activity of the preponderant exocellular proteinase is greater than 100,000 AU / mg, it is clearly higher than that of the other 2 proteinases; its molecular weight is close to 32 kDa, and its isoelectric point is close to 7.0.

This preponderant proteinase alone explains the main physiological properties of PANSTIMASE®, due in particular to the fact that it alone exerts a characteristic action on two important proteins: cholera toxin and collagen.

Cholera toxin is the counterpart of the LT toxin produced by certain pathogenic colibacilli; it is composed of the A subunit which is aggressive, and the B subunit which is immunostimulatory. At a low concentration, comparable to that which exists in the intestine of animals receiving a food supplemented with PANSTIMASE®, the preponderant proteinase selectively destroys the A subunit without destroying the B subunit which thus retains its capacity to stimulate the immune system .

Collagen is the most abundant protein in the animal kingdom: in mammals, the different types of collagen represent about a third of total proteins. We know that the solubility of muscle collagen in hot water is high if it comes from young animals, and low if it comes from old animals.

When the collagen of old animals is treated with preponderant proteinase, its solubility in hot water is increased, which becomes comparable to that of young animals. We can conclude that this proteinase causes a certain "rejuvenation" of collagen.

Most of the results on animals correspond to tests carried out with unpurified or semi-purified PANSTIMASE®. For humans, it is possible to use either the semi-purified PANSTIMASE®, or the purified proteinase (s) of which it is composed, namely the preponderant exocellular proteinase or the mixture of this preponderant exocellular proteinase with at least one of the other two exocellular proteinases, or with one or more proteinases whose amino acid sequences have at least 60% homology with those of predominant exocellular proteinase or with those of the proteinase mixture as defined above.

The subject of the present invention is pharmaceutical and / or dietetic compositions containing, as active principle, the preponderant exocellular proteinase produced by culturing Streptomyces fradiae having a specific activity greater than 100,000 AU / mg, a molecular weight varying from 30 to 36 kDa and preferably close to 32 kDa, an isoelectric point close to 7.0, and capable of selectively destroying the cholera toxin A subunit without destroying the B subunit, in mixture or in association with an excipient or an inert vehicle , non-toxic, physiologically acceptable.

The present invention also relates to pharmaceutical and / or dietetic compositions containing, as active principle, the preponderant exocellular proteinase as defined, or the mixture of this preponderant exocellular proteinase with at least one of the other two exocellular proteinases produced by in culture of Streptomyces fradiae, or, one or more proteinases whose amino acid sequences have at least 60% homology with those of the predominant exocellular proteinase or with those of the mixture of proteinases as defined above, in mixture or in combination with an inert, non-toxic, physiologically acceptable excipient or vehicle. The homologous proteinases can be extracted from the culture of different microorganisms and in particular from Streptomyces fradiae or Streptomyces griseus.

The active ingredient is administered by oral, pulmonary or genital route in doses between 5,000 and 100,000 AU. For these purposes, the active principle is put, in a mixture or in association with conventional excipients, in one of the pharmaceutical forms suitable for these types of administration.

The active ingredient is presented in particular in one of the usual forms in pharmacotechnology such as tablets, sugared almonds, capsules, capsules, powders, solutions or suspensions, dry aerosols, ova, vaginal tablets

Administration will be based on therapeutic need, one to three times a day

The results obtained on animals all seem to have the same origin the fundamental action of PANSTIMASE® on the intestine, this action has different aspects which will be described successively

PHARMACOLOGICAL STUDY OF PHARMACEUTICAL COMPOSITIONS ACCORDING TO THE INVENTION

PANSTIMASE ACTION IN THE BOWEL

1 - Villosities

On relatively young rats (around 2 months), light microscopic examinations show that the villi show normal development, for the control rats as for the treated rats

On relatively old rats (around 7 months), these examinations show that the villi of the control rats are atrophied, while those of the rats treated since their weaning at 4 weeks are as developed as those of the 2 month old rats. PANSTIMASE® causes a certain “rejuvenation” of the intestine, the villi of which retain their initial capacity for development and rapid renewal longer

The intestine is not only a physiologically inactive filter, it is also the largest of the endocrine glands (B LEBLANC et al Rec Med Vet 1981, 157 629 K UVNAS-MOBERG, for SCIENCE, September 1989, 24) Consisting of small cells that are rapidly renewed throughout life, the intestine is the organ richest in Nucleic Acids DNA + RNA (G DURAND et al Ann Biol Anim Bioch Biophys 1 966 6 389), it therefore has the strongest latent capacity for protein synthesis Examination of histological sections of the intestine indicates that PANSTIMASE® allows a better expression of this latent capacity, and consequently a stronger production of many physiologically active compounds digestive enzymes, hormones (acting on the pancreas, liver, brain), immunoglobms, cytokmes, neurotransmitters (for example VIP = Vasoactive Intestinal Peptide), cell growth (or survival) factors We can thus explain that PANSTIMASE®, which acts directly in the intestine, can act indirectly on the organs and physiological systems influenced by the preceding compounds, thus would be explained in particular its remarkable action on the pancreas (A ESTIVAL et al Communication to the Metabolism Days of Nancy January 25-26, 1979 - University of Nancy I)

The intestine is also the organ richest in immune cells, which circulate in its blood and lymphatic vessels, or which gather and multiply in the plates of PEYER These immune cells are a component of MALT (Mucosae Associated Lymphoid Tissue) which is subdivided into GALT located in the intestine, and BALT located in the bronchi, and which presents analogies with SALT located in the skin GALT and BALT are interdependent and act in synergy to ensure the immune defense of the mucous membranes, and to contribute to the defense of the organism as a whole

The examination of histological sections of the intestine shows that PANSTIMASE® directly stimulates GALT, it can therefore indirectly stimulate the general immune defense. This would explain the fact that PANSTIMASE® increases the resistance of animals towards Infectious diseases

2 - Microvilli

On rats, young or old, examinations by electron microscopy show that the microvilliites of the control rats are coated with hypervisquous mucus and with traces of food residues, on the contrary, those of rats treated with PANSTIMASE® appear sharper, more higher, more numerous PANSTIMASE® can therefore increase the total developed surface of microvilli, i.e. their absorption capacity

"In vitro" by treating with PANSTIMASE® intestinal fragments taken from control rats, it has been found that it is possible, without damaging the microvilli, to eliminate the hyperviscous mucus which coats them, this moderate detergent action can be carried out in a few minutes "In vivo", this cleaning of the intestinal mucosa allows the establishment of a closer contact between the infectious germs and this mucosa, which can induce the multiplication in the intestine of immune cells able to fight against these germs

3 - Secretory cells and exocrine micro-regions

Various secretory cells (producing mucus, lysozyme ...) and numerous exocrine microglands are distributed throughout the intestine into which they discharge large quantities of secretions (approximately 2,000 ml per day in humans) The flow of these secretions can be slowed down, or even blocked, by the presence of mucous micro-plugs.

Examination of histological sections of the intestine shows that PANSTIMASE® disintegrates these microbugs, restores the normal flow of these secretions, and causes a reactional increase in their production.

It is known that the secretion of saliva contains a cell growth factor Epithelial Growth Factor (E G F), similarly, the intestinal secretions can contain growth factors capable of acting on enterocytes, lymphocytes. The increase in the production of such factors, under the action of PANSTIMASE®, may explain the better development of the villi and of the GALT, observed on the histological sections of the intestine of the treated rats.

4 - PEYER plates

PEYER plaques, which are aggregates of immune cells distributed throughout the intestine, are more numerous in treated rats. they appear from the duodenum in these rats, but only from the jejunum in the control rats, they are also more dense. their lymphocytes are smaller, tighter, and seem more active (they are more intensely stained by the reagents used in histology)

5 - Non-prolonged adhesion of infectious germs PANSTIMASE® can oppose the prolonged adhesion of infectious germs on the mucous membranes, and in particular on the intestinal mucosa.

This prolonged adhesion results from the formation of chemical bonds between adhesins fixed on these germs and their receptors fixed on the mucosa: adhesins and receptors are complex (glyco-lipo) proteins.

“In vitro” tests have shown that PANSTIMASE®, which has a certain affinity for complex proteins, can cut adhesins, or detach receptors from the mucosa where they are attached; this often results in a break in adhesion which can take place in a few tens of minutes.

In addition, ANS PANSTIMASE® can stimulate the renewal of the villi in the intestine, that is to say the elimination of old cells which are abnormal or affected by the adhesion of infectious germs; this elimination is facilitated by the intensification of the flow of intestinal secretions, the production of which is increased under the action of PANSTIMASE®.

We see that, if PANSTIMASE® can first allow a closer contact between the infectious germs and the intestinal mucosa (and induce the multiplication in the intestine of immune cells able to fight against these germs), it can also then oppose the prolonged adhesion of these germs to the mucosa and their penetration into the underlying tissue.

6 - Destruction of protein enterotoxins

At concentrations between 10 and 100 A.U./mi, and by heating to 37 ° C for a few tens of minutes, ia PANSTIMASE® destroys many protein toxins, and can thus contribute to the prevention and / or treatment of diarrhea. Among the toxins destroyed by PANSTIMASE®, we can cite the botulinum toxin, cholera toxin, LT toxin produced by pathogenic colibacilli, the Necrotizing Cytotoxic Factor also produced by these colibacilli ...

Under the same conditions, endogenous proteinases (Trypsin, Chymotrypsin, etc.) do not destroy these toxins, and therefore cannot contribute to the prevention and / or treatment of these diarrheas. In the case of the LT toxin, PANSTIMASE® destroys the A subunit which is aggressive, without destroying the B subunit which is immunostimulatory.

7 - Conclusion

PANSTIMASE® exerts, throughout the intestine, a fundamental action which stimulates the global functioning of this key organ, and consequently of the whole organism, hence the great diversity of examples of therapeutic applications

THERAPEUTIC APPLICATIONS

PHARMACEUTICAL COMPOSITIONS ACCORDING TO THE INVENTION

The fundamental action of PANSTIMASE® on the intestine makes it possible to envisage its use or the use of the active ingredients of which it is composed, namely the preponderant exocellular proteinase or the mixture of this preponderant exocellular proteinase with at least one of the two other exocellular proteinases, to help prevent or treat disease

1 - Cystic fibrosis and respiratory diseases

Cystic fibrosis is characterized by the excessive secretion of excessively viscous mucus, in the intestine as in the bronchial level All mucus have the same type of biochemical structure, this is why PANSTIMASE® can optimize the viscosity of the intestinal mucus of the bronchial mucus or genital mucus

The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the exocellular proteinase (s) of the invention, can be used for the manufacture of a drug intended to optimize the viscosity of intestinal, bronchial and / or genital mucus

The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the exocellular proteinase (s) of the invention, can be used for the manufacture of a drug intended for the treatment of cystic fibrosis and diseases related to an excess of secretion of an intestinal, bronchial and / or genital mucus, and in particular when this is too viscous

2 - Improvement of resorption and blood circulation

By its moderate detergent action on the intestinal wall, PANSTIMASE® contributes to improving absorption

This improvement in absorption can be seen by dosing in the blood of animals drugs administered orally. For example, chickens have been administered, by gastric tube, 50 mg of tetracycline without PANSTIMASE® (control batch) or in combination with 3 mg PANSTIMASE® grading 400 AU / mg (treated batch), 25 ml of blood were drawn 2 hours after this administration, and it was found that the average concentration of tetracycline in the serum is 1 35 mcg / ml for the control batch and 190 mcg / ml for the treated batch

This improvement in absorption can be analyzed more precisely by studying the fixation of drugs in different organs. For example, trout were given, for 2 days, a medicated feed containing 4g / kg of Oxytetracycline not supplemented with PANSTIMASE® ( control group), or supplemented with 300 mg / kg of PANSTIMASE grading 400 AU / mg (treated group) These fish were then sacrificed and the antibiotic was assayed in the liver, muscles and kidneys It was found that PANSTIMASE ® provides a significant increase in tissue antibiotic concentration, which varies greatly depending on the organ considered + 100% in the liver, ie 5.75 mcg / g for the treated batch vs 2.5 mcg / g for the batch witness

+ 400% in the muscles, ie 1.75 mcg / g for the treated batch vs 0.3 mcg / g for the control batch + 1000% in the rems, i.e. 5.75 mcg / g for the treated batch vs 0.5 mcg / g for the control batch

It appears that PANSTIMASE® causes a better absorption of Oxytetracycline, and a better distribution of this antibiotic throughout the body, which seems to imply an improvement of the blood circulation

The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the protein (s) exocellular proteinases of the invention, can be used for the manufacture of a medicament intended for the treatment of malabsorption of medicaments or nutrients, as well as for the treatment of disorders of the blood circulation, in particular, this medicament is intended for the treatment of undernutrition found in the elderly or in patients who have received drugs whose side effects affect the proper functioning of the intestine

3 - Digestive ulcers and enterotoxemia

Digestive ulcers originate from the adhesion to the stomach wall of a bacterium (Helicobacter pyloπ), and some enterotoxemias originate from the adhesion to the intestinal wall of various infectious germs

PANSTIMASE® opposes the prolonged adhesion of infectious germs on the mucous membranes and destroys many enterotoxins, it can thus help stimulate the renewal of the intestine and can prevent and / or treat digestive ulcers and enterotoxemias

In fact, in industrial farming, it has been found that PANSTIMASE® is capable of reducing the number of pigs suffering from digestive ulcers, as well as the mortality from enterotoxemia

The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the exocellular proteinase (s) of the invention, can be used for the manufacture of a medicine intended to prevent and / or treat the prolonged adhesion of infectious germs on the mucous membranes, to facilitate the regeneration of atrophied or destroyed villi of the intestinal mucosa, as well as to prevent and / or treat digestive ulcers and enterotoxemias

4 - Diabetes, liver failure and intestinal transit disorders

In the previous patent, it was already indicated that PANSTIMASE® stimulates the exocrine function of the pancreas, we have indeed observed a significant increase in the concentration in the pancreas of all enzymes or proenzymes dosed + 50% for amylase, + 70% for trypsinogen and chymotrypsinogen, + 80% for lipase (A. ESTIVAL et al. Communication at the Nancy Metabolism Days 25-26 January 1979 - University of Nancy I).

Examination of histological sections of the pancreas then showed that the islets of LANGERHANS are more numerous and have a better appearance in the treated rats. These reviews suggest that PANSTIMASE® first stimulates the endocrine function of the pancreas, resulting in a general improvement in protein anabolism, hence the subsequent stimulation of its exocrine function.

This stimulation which is exerted on the pancreas, is also exerted on the liver, which results in an improvement of the intestinal transit. In intensive farming, constipation is often observed in attached pregnant sows; it has been found that supplementing their diet with PANSTIMASE® makes it possible to avoid these harmful disturbances of intestinal transit.

The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the exocellular proteinase (s) of the invention, can be used for the manufacture of a medicine intended to prevent and / or treat diabetes, liver failure and intestinal transit disorders.

5 - Infectious diseases, immune dysfunctions, and vaccination

It has already been indicated that PANSTIMASE® acts favorably on GALT, and consequently on the whole immune system. This results in improvements in 3 different areas.

A - Infectious diseases

Some observations in intensive farming have shown that PANSTIMASE® often improves the health status of animals, which was first attributed to the fact that it facilitates the absorption of antibiotics which are added in most industrial feeds. But it was then found that this improvement was particularly marked in the case of infections by different viruses, while these are generally insensitive to the action antibiotics These findings relate to influenza and AUJESKY disease in pigs, GUMBORO disease in chickens and ecthyma in kids

In the latter case, the viral infection observed during a test at an experimental station was the subject of precise observations, covering a total of 120 kids, the percentage of animals not affected was 34% for the control group, and 70% for the treated group, the percentage of animals very affected was 28% for the control group, while no animal was very affected in the treated group

This antiviral action can be explained by the fact that PANSTIMASE® increases the number and density of PEYER plaques, which are aggregates of immune cells distributed throughout the intestine.

B - Immune dysfunctions

Other observations on piglets in intensive breeding showed that PANSTIMASE® reduced the duration of arthritis which are inflammatory reactions following injuries to the joints affecting animals reared on metal gratings. After healing of the wounds, these inflammatory reactions can gradually fade under the action of so-called suppressive immune cells, which are numerous in the intestine, and which can migrate to areas of inflammation It is possible that PANSTIMASE® stimulates the multiplication of these cells in the intestine, and that '' facilitates their migration, which would explain the fact that it reduces the duration of arthritis by about 50%

C - Vaccinations

The improvements in health observed in intensive farming can be explained by a better response to vaccinations in animals receiving a feed supplemented with PANSTIMASE® Preliminary laboratory tests on mice have effectively confirmed that PANSTIMASE® can increase the effectiveness of vaccinations, especially when they are given orally The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the exocellular proteinase (s) of the invention, can be used for the manufacture of a medicine intended to prevent and / or treat infectious diseases and immune dysfunctions, as well as to increase the effectiveness of vaccinations.

6 - Hormonal insufficiencies and degenerative diseases linked to aging

PANSTIMASE® has been found to reduce the weaning-fertilizing interval (which tends to increase in sows), and to stimulate sexual drive, (which tends to decline in older pigs in older breeding pigs). boars). It thus appears that PANSTIMASE® makes it possible to remedy hormonal insufficiencies, and probably with other negative effects due to aging.

PANSTIMASE® has been shown to cause "rejuvenation" in older breeding rats and a reduction in the negative effects of aging in older breeding sows and chickens.

A. The first projection of the rats is normally affected around the age of 70 days; when performed around 140 days, the reproductive performance is poor for the control batch and is better for the treated batch (receiving the same food supplemented with 300 mg / kg of PANSTIMASE® grading 400 AU / mg).

It is found that the control rats protruding at 140 d give poor results but that the treated rats also protruding at 140 d give results which approximate the normal results given by the control rats protruding at 70 d, and even become identical in the case of the perinatal mortality rate. We can therefore say that PANSTIMASE® caused a “rejuvenation” of 140-day-old rats.

_B. Breeding sows are culled as soon as they appear in "poor condition", for example when the farmer feels that they can no longer stand well, or lie down flexibly (instead of dropping heavily in may crush newborn piglets). In a farm with 80 sows, before using PANSTIMASE®, the sows are culled after 4.4 breeding cycles on average; six months after this use, it appeared that sows could be culled after 5.8 breeding cycles on average. We can therefore consider that the

PANSTIMASE® has made it possible to extend the productive life of sows by around 30%, without unacceptable alteration of their “good condition” and their performance.

As they age, laying hens tend to produce eggs with a more fragile shell, so the percentage of cracked eggs can become greater than 10

% at the end of laying. PANSTIMASE® has made it possible to reduce this percentage by half, which implies stimulation of the phosphocalcic metabolism.

The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the exocellular proteinase (s) of the invention, can be used for the manufacture of a medicine intended to prevent and / or treat hormonal insufficiencies and degenerative diseases linked to aging, for example arthritis, osteoarthritis, osteoporosis.

PRELIMINARY EVALUATION OF THE TOLERANCE OF PHARMACEUTICAL COMPOSITIONS ACCORDING TO THE INVENTION

Toxicological tests have shown that PANSTIMASE® has no toxicity on piglets when administered for 1 month at a dose 20 times higher than the currently recommended use dose which is 40,000 AU. per kg of food, nor any toxicity on the rat when it is administered during 3 months at a dose 200 times higher than this dose of employment.

Consequently, a preliminary tolerance test in humans has been able to be initiated at a dose which is relatively high compared to the dose currently planned; we have in fact used capsules containing 750,000 AU of purified PANSTIMASE® (ie 50 mg of a batch drawing 15,000 AU / mg) which have been administered at the rate of 2 to 3 capsules per day for 2 or 3 weeks to patients.

Twenty-four patients (12 men - 12 women) were selected, aged 44 years with an average age ranging from 25 to 70 years; 17 of these patients suffered from dyspepsia in the broadest sense of the word without any organic lesion being able to be revealed by the paraclinical investigations (laboratory examinations, radiologies, in some cases endoscopy; 7 of these patients were carriers organic lesions: 3 pancreatic, 2 gastrectomy (one for ulcer, the other for neoplasm), 2 colectomy (one for ulcerative ulcerative colitis, the other for neoplasm).

On all of these 24 patients, the evolution of the symptomatology and the weight were studied, when an underweight, with or without undernutrition, existed at the start of the treatment. Several subjects have reported an improvement in the regulation of intestinal transit in the event of constipation or, on the contrary, diarrhea, as well as weight gain in the event of undernutrition.

A preliminary trial for cystic fibrosis was also initiated in an 18-year-old patient who received 2 capsules each containing 10,000 AU a day for 3 weeks. PANSTIMASE®; there has been an improvement in symptoms, intestinal level (less abundant and less sticky faeces) and bronchial level (easier and less frequent sputum).

In conclusion, we can say that PANSTIMASE® has been well tolerated, and that it seems particularly effective in the case of undernutrition.

The exocellular proteinase (s) according to the invention, or the proteinase (s) whose amino acid sequences have at least 60% homology with those of the exocellular proteinase (s) of the invention, can be used for the manufacture of a dietary composition for the food of the fragile or malnourished subjects.

Claims

1. Pharmaceutical and / or dietetic compositions containing, as active principle, the preponderant exocellular proteinase produced by culturing Streptomyces fradiae having a specific activity greater than 100,000 AU / mg, a molecular weight varying from 30 to 36 KDa, an isoelectric point close to 7.0 and capable of selectively destroying the A subunit of cholera toxin without destroying the B subunit, in mixture or in association with an inert, non-toxic, physiologically acceptable excipient or vehicle.

2. Pharmaceutical and / or dietetic compositions containing, as active principle, the preponderant exocellular proteinase according to claim 1, or the mixture of this preponderant exocellular proteinase with at least one of the two other exocellular proteinases produced by Streptomyces fradiae, or, a or proteinases whose amino acid sequences have at least 60% homology with those of the predominant exocellular proteinase or with those of the proteinase mixture as defined above, in mixture or in association with an inert excipient or vehicle, not toxic, physiologically acceptable.

3. Pharmaceutical and / or dietetic compositions according to one of claims 1 or 2, in which the active principle is administered by oral, pulmonary and / or genital route at doses of between 5,000 and 100,000 AU.

4. Use of the proteinase (s) according to one of claims 1 or 2, for the manufacture of a medicament intended to optimize the viscosity of the intestinal, bronchial and / or genital mucus.

5. Use of the proteinase (s) according to one of claims 1 or 2, for the manufacture of a medicament intended for the treatment of diseases related to an excess of secretion of intestinal, bronchial and / or genital mucus, and especially when it is too viscous.

6. Use of the proteinase (s) according to one of claims 1 or 2, for the manufacture of a medicament intended for the treatment of malabsorption of medicaments or nutrients, as well as for the treatment of disorders of the blood circulation.

7. Use of the proteinase (s) according to one of claims 1 or 2, for the manufacture of a medicament intended to prevent and / or treat the prolonged adhesion of infectious germs on the mucous membranes, to facilitate the regeneration of the villi atrophied or destroyed of the intestinal mucosa, as well as to prevent and / or treat digestive ulcers and enterotoxemias.

8. Use of the proteinase or proteins according to one of claims 1 or 2, for the manufacture of a medicament intended to prevent and / or treat diabetes, hepatic insufficiencies and intestinal transit disorders.

9. Use of the proteinase (s) according to one of claims 1 or 2, for the manufacture of a medicament intended to prevent and / or treat infectious diseases, immune dysfunctions, as well as to increase the effectiveness of vaccinations.

10. Use of the proteinase or proteins according to one of claims 1 or 2, for the manufacture of a medicament intended for preventing and / or treating hormonal insufficiencies and degenerative diseases linked to aging.

11. Use of the proteinase (s) according to one of claims 1 or 2, for the manufacture of a dietetic composition for the feeding of fragile or malnourished subjects.