FR2694196A1 - Compsns. active against tumour viruses - obtained by double fermentation of milk or whey using specified starter - Google Patents

Abstract

Medicinal compsns. contain protease-inhibiting peptides (I) with a molecular wt. below 30kD, one or more orotic acid salts (II), and L(+)-lactic acid (III). Pref. the compsns. are obtained by double fermentation of milk or whey with a starter comprising polysaccharides in soln., Candida pseudotropicalis, Pichia membranaefaciens, Lactococcus spp., Lactobacillus spp. and opt. Bifidobacterium, Acetobacter and Leuconostoc spp. USE - The compsns. have antiviral activity, esp. against oncogenic viruses, e.g. mammary tumour virus (MTV) in mice and Peyton sarcoma and erythromyeloblastic leukaemia viruses in chickens. In an example, the starter was prepd. by fermenting semi-skimmed milk 'RDR91A) (Bioadditive Soc.) at 30 deg.C for 20 hours and freeze drying the liq. phase. A mixt. of milk and skim-milk powder (12.5% solids) was inoculated with the starter, fermented at 30 deg.C for 6 hours and at 21 deg.C for 12 hours, pasteurised at 95 deg.C for 5 minutes, concd. by vacuum evapn. at 25 deg.C, and freeze dried. C3H/Bi mice (neonatally infected with MTV) receiving drinking water contg. 5% of the prod. showed a tumour incidence of 30% (90% for controls) and had a mean survival time of 84 days (37 days for controls).

Description

MEDICINAL COMPOSITION FOR USE AS ANTIVIRAL AGENT
The invention relates to a medicinal composition which can be used in particular as an antiviral agent; it relates in particular to a composition having an antiviral activity on oncogenic viruses.

Numerous studies have been carried out to develop compositions having antiviral effects; some authors have recommended the use of sulfated polysaccharides (dextran or curdlan type) patent EP-A-464,759; Aoki T et al, Eurodlan sulfate and
HIV-1, I. In Vitro inhibitory effects of curdlan sulfat on
HIV-1 infection, AIDS Res Hum Retrovirus, April 1991-7 (4), pages 409-415; McClure et al, Investigations into the mechanism by which sulfated polysaccharides inhib HIV HIV infection in vitro, AIDS Res Hum Retroviruses,
January 1992.8 (1), pages 19-26. These studies have revealed certain antiviral properties of these sulfated polysaccharides, but these are not currently used, probably due to too low efficacy in vivo and side effects linked to their mode of administration. .

 Other work has been carried out on uridin derivatives and has highlighted the antitumor and antiviral properties of some of these compounds.

In particular, fluorinated derivatives of uridins generally have antitumor properties (patent EP-A331.032; patent DE-A-3,828,156), while iodine derivatives of uridins generally have antiviral properties (patent EP-A-56265 ). These compounds are known to be very active but have the serious defect of being highly cytotoxic.

Recently, compounds of a peptide nature have been presented as having antiviral properties (international application WO-A-91.17760; patents
US 5,066,783 and 5,073,542). These peptides act as protease inhibitors and are very active in vitro, but have been shown to be weakly active in vivo (international application
WO-A-92.04908).

In addition, injectable formulations have been proposed combining several compounds. For example, international application WO-A-91.18629 describes a formulation in which peptides are encapsulated in a polymer matrix; international demand
WO-A-91.17660 describes a medicinal preparation containing a mixture of tetrahydrofolate (salt of folic acid) and 5-fluoro uracil; Japanese patent -A-89.252008 describes a drug combining a polygalactosamine and 5fluoro uracil; Japanese patent -A-88.078182 describes an anti-tumor composition combining derivatives of chitosan and 5-fluoro uracil; Japanese patent -A-82.142664 describes a formulation combining a polylactic copolymer and a 5-fluoro uracil derivative. These formulations are only injectable and have either a low intrinsic activity or an annoying cytotoxicity.

 The present invention proposes to provide a new medicinal composition. It relates to a composition having no cytotoxic effect and having pharmacological properties, in particular a very effective antiviral activity.

 The medicinal composition according to the present invention is characterized in that it combines (a) protease inhibiting peptides, of molecular weight less than 30,000 Daltons, (b) at least one salt of orotic acid, and (c) dextrorotatory L lactic acid. Preferably, this composition also comprises (d) a polysaccharide, (e) at least one salt of folic acid, and (f) urea.

 In vivo tests, some examples of which are given below, have shown that the above-defined composition (a, b, c, optionally supplemented by d, e, f) exhibits remarkable antiviral properties, in particular with regard to oncogenic viruses. A complete absence of virus circulating in the plasma was observed in the treated animals. In addition, an immunostimulatory effect has been observed, leading to a significant increase in the survival of infected animals. These effects are due to a synergistic action of the compounds, which leads to a reestablishment of exchanges of the infected cell with the external environment (intra and extracellular and intercellular exchanges), in particular to good absorption of peptides and minerals by the cell: the infected cell is thus restored to its normal functions (respiration, metabolism, flow), while the virus can be directly attacked in particular by the inhibitory action of the peptides which have penetrated therein. The work of the inventors makes it possible, with good likelihood, to attribute the following roles to the three essential compounds (a), (b) and (c): the salt or salts of orotic acid have a transporter effect consisting in restoring exchanges, on the one hand, through the cytoplasmic membrane, on the other hand, to inside the cytoplasm, with the mitochondria, lysosomes and nucleus; in particular, the penetration of minerals and peptides is thus favored, the latter thus being able to develop in the cytoplasm their protease inhibiting action, leading to the destruction of the virus. Lactic acid L dextrorotary restores the normal acidity of the cell and allows the restoration of the respiratory function of the cell, thus contributing to the passage of the cell from the fermentative pathological state to the normal respiratory state.

 It should be emphasized that the above-mentioned effects differ fundamentally from those of known antiviral formulations; in the invention, the infected cell is restored to its normal functions, at the same time as the virus is attacked, the healthy cells not being affected, while the known formulations aim at destroying the infected cells by a cytotoxic effect simultaneously destroying many healthy cells.

 According to a preferred formulation, the medicinal composition in accordance with the invention contains the following orotic acid salts: lysine orotate, calcium orotate, magnesium orotate, iron orotate and selenium orotate in the form of selenomethionine.

The proportions of the various compounds which seem to give the best results are as follows, relative to 100 g of lactic acid L dextrorotatory
(a) between 8 and 30 g of peptide inhibitors of
protease,
(b) between 1 and 2 g of orotic acid salt,
(d) advantageously between 5 and 15 g of polysaccha
wrinkled,
(e) advantageously between 10-3 and 4.10-3g of salt
folic acid,
(f) advantageously between 1 and 3 g of urea.

The polysaccharide used is preferably a glucogalactan, consisting in particular of a glucose and galactose complex, of average molecular weight of between 5000 and 30,000 Daltons. This polysaccharide appears to have a good immunostimulatory effect on the cell; moreover, in the formulation referred to below containing vitamins, it improves their stability
In addition, the stimulation of the cell is increased, by adding to the composition the following compounds whose penetration inside the cell is favored by the salts of orotic acid
- (g) combination of the following group B vitamins: riboflavin (B2), pyridoxine (B6) and biotin (B8),
- (h) in addition, at least one of the following group B vitamins: cyanocobalamin (B12), nicotinamide (B3), pantothenate (B5), thiamine,
- (i) advantageously the following trace elements: zinc, copper.

 For example, excellent results are obtained by providing between 0.1 and 0.3 g of group B vitamins, between 2.10-2 and 8.10-2 g of zinc, and between and 3.10-3 g of copper (reported weight 100 g of lactic acid L dextrorotatory).

 The above-mentioned medicinal composition is advantageously supplemented with (j) at least one of the following excipients: lactose, milk proteins, fatty acids of lactic origin. This excipient can be provided in a proportion of between 103 and 4,103 g, based on 100 g of lactic acid L dextrorotatory.

 The medicinal composition in accordance with the invention can be prepared by mixing the compounds.

It can also be prepared by a natural process of double fermentation of a milk or a whey in the presence of a lactic sourdough, this process comprising the following steps
- using a lactic acid starter containing polysaccharides in solution, the yeast Candida PseudotroPicalis, the yeast Pichia membranaefaciens, bacteria of the genus Lactococcus and bacteria of the genus
Lactobacillus,
the milk or whey containing said leaven is subjected to a first fermentation at a temperature substantially between 260 ° C. and 350 ° C.,
- the product obtained is subjected to a second fermentation at a temperature substantially between 180 C and 280 C.

 The yeast and leaven bacteria act in symbiosis during the double fermentation defined above to lead to a composition combining the above-mentioned compounds; it should be noted that no addition is made between the two fermentations.

Preferably, a lactic starter is also used to carry out this double fermentation, also containing bacteria of the following genera
Bifidobacterium, acetobacter, Leuconostac.

In the case where the composition is obtained by double fermentation of a milk, the lactic sourdough used is in particular of the type referred to in patent application FR 91.02290. Such a sourdough is prepared by fermentation of a milk in the presence of a mixed lactic ferment containing a symbiosis of yeasts and bacteria, under the following conditions
the ferment used comprises a polysaccharide matrix including, on the one hand, at least one yeast of the genus Pichia species membranaefaciens, on the other hand, at least the following two bacteria: bacteria of the genus Lactococcus species lactis, bacteria of the genus
Lactobacillus fermentum species,
the milk containing the ferment is subjected to a fermentation at a substantially constant temperature of between 260 ° C. and 350 ° C. for a period of substantially between 16 hours and 25 hours.

The ferment used preferably contains all of the following microorganisms
yeast of the genus Pichia species membranaefaciens,
yeast of the genus Candida species pseudo trosicalis,
bacteria of the genus Lactococcus species lactis subspecies lactis,
bacteria of the genus Lactobacillus species fermentum,
Bifidobacterium bacteria,
Acetobacter bacteria.

 In addition, its polysaccharide matrix is preferably based on glucose and galactose.

It is possible to use a ferment previously prepared from cultures of the abovementioned microorganisms. It is also possible to use as a ferment a natural product which is a by-product of curdled milk, in particular ferment produced and distributed by the Company "Bioadditive",
Toulouse, France.

The lactic sourdough obtained is essentially characterized by the presence in the milk medium
polysaccharides in solution, with a concentration between 0.01 and 0.05 g per gram of medium,
Pichia membranaefaciens yeast, in an amount between 105 and 107 germs per gram of medium,
Lactococcus bacteria in an amount between 107 and 109 bacteria per gram of medium,
and Lactobacillus bacteria in an amount between 105 and 107 bacteria per gram of medium.

In addition to these essential germs, the leaven according to the invention may also contain the following germs
additional yeast: Candida nseudotropicalis, the amount of this yeast being between 104 and 106 germs per gram of medium,
additional bacteria: Bifidobacterium between 103 and 105 per gram, Acetobacter between 103 and 105 per gram.

The preparation of the medicinal composition in accordance with the invention by means of such a lactic leaven can be carried out under the following optimal conditions
- proportion by weight of leaven relative to milk of between 1% and 4%,
first fermentation at a temperature substantially between 260 ° C. and 350 ° C., in particular substantially equal to 300 ° C., for a period substantially between 4 and 8 hours, in particular substantially equal to 6 hours,
- second fermentation at a temperature substantially between 180 C and 240 C, in particular substantially equal to 210 C, for a period substantially between 9 and 16 hours, in particular substantially equal to 12 hours.

Furthermore, in the case where the medicinal composition in accordance with the invention is obtained by double fermentation of a whey, the lactic sourdough used is in particular of the type referred to in the patent application
FR 91.02291. Such a leaven is prepared by double fermentation of a whey in the presence of a ferment of a type analogous to that described above, under the following conditions
the whey containing the ferment is subjected to a first fermentation at a temperature substantially between 260 ° C. and 350 ° C. for a period substantially between 16 hours and 25 hours,
- The product obtained is subjected to a second fermentation at a temperature substantially between 210 C and 280 C for a period substantially between 24 and 48 hours.

 The lactic sourdough obtained contains yeasts and strains of the same nature as those already mentioned, but with different proportions.

The preparation of the medicinal composition in accordance with the invention by means of this lactic sourdough is in this case carried out under the following optimal conditions
- proportion by weight of leaven relative to whey between 1% and 4%,
first fermentation at a temperature substantially between 260 ° C. and 350 ° C., in particular substantially equal to 300 ° C., for a period substantially between 24 and 72 hours, in particular substantially equal to 48 hours,
- Second fermentation at a temperature substantially between 210 C and 280 C lower than the first, in particular substantially equal to 250 C, for a period substantially between 48 and 96 hours, in particular substantially equal to 72 hours.

 The production methods described above (double fermentation of a milk, or double fermentation of a whey) lead to a composition comprising the various compounds (a) - (j) defined above, with relative proportions entering into the mentioned ranges.

This composition also contains microorganisms which can be preserved or, on the contrary, eliminated at the end of the second fermentation.

 Examples 1 and 2 which follow present processes for the preparation of medicinal compositions in accordance with the invention; Examples 3 and 4 describe the in vivo experiments of these compositions respectively on mice and on laying hens.

Figures 1, 2 and 3 of the accompanying drawings show diagrams obtained in Example 3
- Figure 1: diagram giving the percentage of tumor appearance in the treated mice,
- Figure 2: diagram giving the percentage of survival,
- Figure 3: diagram giving the evolution of the consumption of products.

EXAMPLE 1: Preparation of a medicinal composition by double fermentation of a milk.

Ferment used
The ferment used is the ferment distributed by the company "Bioadditive" under the reference "RDR91A". This ferment is in the form of a whitish granular mass of size between 1 and 3 centimeters.

Milk used
The milk used is semi-skimmed milk at 105 g / kg + 1 g of dry matter, 15 g / kg + 1 g of fat and 47 g / kg + 1 g of lactose. This milk was pasteurized twice at 90/950 C for a period of 30 min each pasteurization (plate pasteurizer). The milk is then cooled in a fermenter tank ("GOAVEC" type) to 300 C.

Production of lactic sourdough
The fermenter tank containing 100 liters of milk was regulated at a temperature of 300 C + 0.50. This tank being of the completely closed, sealed, steam sterilizable type at 2 bars is provided with a microbial filter system for air exchange and with aseptic seeding means.

 The inoculation using the above-mentioned ferment was carried out at the rate of 20 g of ferment per liter of milk. Stirring by stirring (40 rpm), 30 minutes at the start and 30 minutes at the end of fermentation, homogenizes the medium.

 The product is left at 300 ° C. in the fermenter tank for 20 hours. PH checks every two hours show that the pH equal at the start to 6.5 drops regularly to a final value of 4.5 after 20 hours. The product is then cooled in less than 30 min to 40 C, and the liquid fraction is drawn off from the tank aseptically. It is freeze-dried by a traditional process, using a "Serail" device (freezing at -400 ° C, vacuum and sublimation).

Leaven obtained
Before freeze-drying, the leaven thus produced was analyzed and the following results were obtained (number of germs per gram, average value)
. 6.105 Pichia membranaefaciens,
. 6.104 Candida Pseudotronicalis,
. 9.107 Lactococci,
. 25.105 Lactobacilli,
. 9.103 Bifidobacterium,
. 7.103 Acetobacter.

 After lyophilization, there is a loss of 50%.

 In addition, the leaven before lyophilization has a concentration of polysaccharides in solution of 0.03 g per gram of leaven; these polysaccharides include glucose and galactose in a relative percentage equal to 0.8 g of glucose per gram of galactose. In addition, the leaven contains 73 mg of orotic acid per liter.

Manufacture of the medicinal composition
Leaving said leaven
The milk used for this manufacture is a mixture of milk after adjustment of the fat content and skim milk powder to standardize in dry extract
- 125 g / kg + 1 g of dry matter,
- 36 g / kg + 1 g of fat,
- 50 g / kg + 1 g of lactose.

 This milk was sent to a plate exchanger to undergo preheating there before being homogenized (150-200 kg / cm2, at a temperature of 700 C) then pasteurized at 90/950 C for a period of 30 min (pasteurizer with plates). The milk is then cooled in a maturation tank ("GOAVEC" type) to 300 C.

 The maturation tank is an ultra clean "GOAVEC" type test tank regulated at a temperature of 300 ° C. + 0.50 C. The seeding, by means of the above-mentioned leaven, is carried out directly using a pump " MOUVEX S4 "at a rate of 2% with stirring 10 rpm during filling.

 The product is left at 300 ° C. in the maturation tank for 6 hours. PH checks are carried out every two hours to arrive at a cut-off pH of 5.2. The cutting is done by stirring at 10 rpm, with a circulation of chilled water at 0 C (+ 20 C maximum), to cool to 210 C in 30 minutes.

 The milk then undergoes a second fermentation in the same maturation tank at 210 C for 12 hours until pH 4.5.

 The liquid product obtained is pasteurized in the tank at 950 C for 5 minutes, which inactivates the microorganisms, then cooled and concentrated using a centrifugal thin layer vacuum evaporator ("EVAPOR type CEP1) at a temperature of 250 C, until its volume is halved (viscosity obtained: 4,000 centipoise) The concentrated product is then lyophilized by a traditional process ("SERAIL type CS80" device).

 A whitish powder is obtained, with a moisture content of less than 5%, soluble in an amount of 0.3 g per ml of water. This powder constitutes the medicinal composition targeted by the invention. Its administration is preferably done orally.

Galenic characterization of the composition
This characterization was carried out by the following methods - protein determination by the KJELDAHL method (on
KJELFOSS device), - assay of peptides by HPLC chromatography

(JASCO 875) followed by ultraviolet spectrography
(SECOMAM S 1000 PC), - dosage of sugars and polysaccharides by
HPLC chromatography (JASCO 875) p-Bondapak / carbohydrate column, - determination of lipids (fats) by butyrometry
and GC chromatography (HEWLETT-PACKARD 5890
series 11), - determination of minerals by ion chromatography (JASCO
875) with Wescan 315 conductimetric detector, column
SCK 01 for anions and column SCA 03 for
cations, - determination of fat-soluble vitamins by chromatography
HPLC (JASCO 875) and dosage of vitamins
water-soluble by microbiology and colorimetry, - determination of urea by the CONWAY method (using urease).

The formulation (in g, based on 100 g of lactic acid) is as follows
Milk proteins: 700
Fatty acid
lactic origin: 600
Lactose: 440
Peptides: 12.00
Orotates: 1.40
Lactic acid: 100
Glucogalactan: 10
Folates: 0.002
Urea: 2
Zinc: 0.050
Copper: 0.0018
B vitamins: 0.19
B1 thiamine: 0.01
B2 riboflavin: 0.042
B3 nicotinamide: 0.024 B5 prantothenate: 0.102
B6 pyridoxine: 0.0094
B8 biotin: 0.0004
B12 cyanocobalamin: 0.00006
Vit A: 0.0084
Vit C: 0.222
Vit E: 0.0186
Calcium: 22
Magnesium: 2
Phosphorus: 16
Potassium: 27
Sodium: 8
Iodide: 0.0008
Iron: 0.042
Manganese: 0.0006
Selenium: 0,00018
The peptides are protease inhibitor peptides with a molecular weight of the order of 10 000 to 15 000 Daltons. The orotates consist of a mixture of calcium orotate and magnesium orotate (predominant) with also iron orotate, lysine orotate and, in very small quantities, selenium orotate (selenomethionine).

 Lactic acid consists of L dextrorotatory lactic acid (more than 95%).

 Glucogalactan is a glucose and galactose complex with an average molecular weight of around 16,300 Daltons.

 Folates are tetrahydrofolates.

 The composition can be presented in the form of powders, or of compounds to be dissolved in water or to be consumed as it is orally.

EXAMPLE 2: Preparation of a medicinal composition by double fermentation of a whey
Ferment used
The ferment used is the ferment distributed by the company "Bioadditive" under the reference "RDR91B".

Whey used
The whey used is sweet whey pasteurized at 90/950 C for 30 minutes, with the following composition: 52 g / kg + 1 g of dry matter, 0.6 g / kg of fat, 44 g / kg + 1 g lactose and 0.9 g / kg protein. The pH of this whey is 5.6.

Production of lactic sourdough
An Erlen Meyer type container of 0.5 liters of whey with back and forth shaking (80 oscillations / minute) was regulated at a temperature of 300 ° C. + 0.50. Inoculation using the above-mentioned ferment was carried out at the rate of 50 g of ferment per liter of whey.

 The product is left at 300 ° C. in the container for 20 hours with continuous stirring. PH checks every two hours show that the pH equal to 5.6 at the start drops regularly to a final value of 4.4 after 20 hours.

 The product is then cooled in less than 30 min to 250 C, and it is left 36 hours at this temperature with continuous stirring for a new fermentation; the final pH is 4.4.

 The liquid fraction is withdrawn from the container aseptically. It is freeze-dried by a traditional process, using a "Serail" device (freezing at -40 ° C., placing under vacuum and sublimation).

Leaven obtained
Before freeze-drying, the leaven thus produced was analyzed and the following results were obtained (number of germs per gram, average value)
4.105 Pichia membranaefaciens,
6.105 Candida oseudotroPicalis,
2.108 Lactocoaues,
. 15.105 Lactobacilli, 104 104 Bifidobacterium,
7.103 Acetobacter,
. 8.103 Leuconostoc.

 After lyophilization, there is a loss of 50%.

 In addition, the leaven before lyophilization has a concentration of polysaccharides in solution of 0.03 g per gram of leaven; these polysaccharides include glucose and galactose in a relative percentage equal to 0.8 g of glucose per g of galactose. In addition, the leaven contains 71 mg of orotic acid per liter.

Preparation of the medicinal composition from the aforementioned sourdough
The sweet whey used for this preparation is the same as that used for making sourdough.

 This whey is introduced into an ultra-clean maturation tank (600 liters) of the "GOAVEC" type regulated at a temperature of 300 C + 0.50 C.

 The seeding by means of the lyophilized sourdough is carried out directly using a "MOUVEX S4" pump, at a rate of 2% by weight with stirring 10 rev / min during filling.

 The product is left at 300 ° C. in the maturation tank for 48 hours. The pH after 48 hours is 4.9.

 The whey which has undergone this first fermentation is cooled to 250 ° C. and is left at this temperature for 72 hours to undergo a second fermentation; its final pH is 4.5.

 The product is then drawn off by a positive "MOUVEX S4" type pump with slow rotation speed and regulated flow.

 The product is packaged in a 100 1 container. It is then powdered by atomization under traditional conditions.

Aalenian characterization of the composition
In addition to the microorganisms present, the formulation of the composition is as follows (in g, based on 100 g of lactic acid)
Milk proteins: 1143
Fatty acid
of lactic origin: 257
Lactose: 300
Peptides: 21.43
Orotates: 1.43
Lactic acid: 100
Glucogalactan: 7.43
Folate: 0.003 Urea: 2
Zinc: 0.043
Copper: 0.002
B vitamins: 0.15 B1 thiamine: 0.01
B2 riboflavin: 0.04
B3 nicotinamide: 0.02 B5 prantothenate: 0.08
B6 pyridoxine: 0.0073
B8 biotin: 0.0004
B12 cyanocobalamin: 0.00000
Vit A: 0.0040
Vit C: 0.08
Vit E: 0.064
Calcium: 20.71
Magnesium: 2.14
Phosphorus: 10.00
Potassium: 17.14
Sodium: 7.43
Iodide: 0,0009
Iron: 0.02
Manganese: 0.0004
Selenium: 0.0002
The same comments as above can be made regarding the nature of the compounds present. However, the majority orotate is lysine orotate and the peptide content is increased (almost doubled).

 The composition can be presented in the form of a powder which can be dissolved in water or granules for consumption as such.

EXAMPLE 3: Experiments on mice
The mice used are C3H / Bi mice neonatal infected with the oncogenic virus MTV (Mamary
Tumor Virus) which is a latent viral infection.

C3H / Bi mice are characterized by the development with high frequency (90% of female mice) of mammary tumors between the ages of 5 and 8 months and by an alteration of natural cytotoxic functions.

LOTS
C3H / Bi mice are divided into three lots of 12 mice named A, B and C.

FOOD
Food in the form of granular compound foods is distributed twice a day.

It is identical in quantity and quality for the three lots A, B, C.

DRINK
The drink contained in 100 ml bottles is renewed every day. It is identical in quantity and different in quality according to lots A, B, C.

 - lot A: sterile water.

 - batch B: sterile water + 5% of skimmed milk powder standardized and freeze-dried.

 - batch C: sterile water + 5% of medicinal composition prepared in Example 1 (in powder form).

EXPERIMENTATION
It begins from the 4th month of life of C3H / Bi mice. The mice have permanent and free access to baby bottles.

RESULTS
We notice by touching three times a week the appearance of breast tumors, and we note the consumption of drinks every day. Blood samples are taken every week. Mortality is noted progressively.

 The diagram in FIG. 1 gives the percentage of appearance of tumors by batch.

 The control curve A (drink: sterile water) conforms to the known genetics of the mice used (90% of the mice present a mammary tumor at the 8th month).

 Curve B shows a lag of approximately 1 month from this appearance (drink: milk), the rate of appearance remaining identical at the end of the 9th month.

 Curve C obtained with the composition according to the invention shows, on the one hand, a time shift, on the other hand and above all a very significant reduction in the rate of appearance of tumors (30% of appearance to be compared 90% for the other two lots).

 This renewed test gives substantially identical results.

 The diagram in FIG. 2 gives the percentage of survival for the three lots. For lots A and B, 10% survival is observed at the end of the 10th month (with a slight shift in favor of lot B), while for lot C (composition according to the invention), the survival rate is 90% at the end of the 10th month (a single dead mouse).

The average difference in days of survival between the moment of appearance of a tumor and the death of the mouse is as follows
- lot A: 37 days,
- lot B: 27 days,
- lot C: 84 days
These results demonstrate a remarkable antiviral and antitumor efficacy of the composition.

 The diagram in Figure 3 shows the evolution of consumption for the three lots. We note that consumption is higher for lot C and stabilizes at the end of the 8th month, while this consumption drops continuously for lots A and B.

 The blood samples were the subject of research on the tumor response and on the cellular response. Identical tumor and cellular responses were observed for lots A and B demonstrating the existence of circulating viruses, while for lot C, the absence of circulating viruses was observed. In addition, for the dead mouse of batch C, a hystopathological analysis showed that its tumor was necrotic, containing inactive viruses.

EXAMPLE 4 Treatment of tumors induced by viral oncogene (sarcoma and avian leukemia) in a
Laying hens
The farm is a French farm with 10,550 laying hens 20 weeks old when treatment begins. Historically, in this farm, we could observe until the date of the tests, a spontaneous appearance of viral tumors, sarcoma type
Peyton and leukosis (erythro-myeloblastic leukemia) which cause around 90% of natural mortality.

The hens are divided into two lots
A. Control lot of 5300 hens,
B. Treated batch of 5250 hens.

 The basic food (cereals ...) is identical for the two lots.

 The control batch A receives a dose of sweet whey powder mixed with the basic food, while the treated batch B receives the same dose of powder but consisting of the medicinal composition prepared in Example 2.

 The dose is provided as follows - from the 22nd to the 37th week: 0.5 g / kg of basic food, - from the 28th to the 62nd week: 1 g / kg of food.

 The research focuses on the number of laying hens died per tumor induced after histopathological analysis of the carcasses.

From the 22nd to the 37th week the number of dead hens
- in lot A: 55 of which 50 per induced tumor,
- in lot B: 35, including 20 per induced tumor.

From the 28th to the 62nd week
- in lot A: 165 of which 151 per induced tumor,
- in lot B: 86 including 44 by induced tumor.

The results show that the treatment with the medicinal composition makes it possible to reduce the total mortality of the hens by 55% and to decrease the rate of tumors induced by a viral oncogene
- 91% of the dead hens in lot A presented induced tumors,
- 53% of the dead hens in lot B presented induced tumors.

Claims (20)

 1 / - Medicinal composition, combining (a) protease inhibitor peptides, of molecular weight less than 30,000 Daltons, (b) at least one salt of orotic acid, and (c) lactic acid L dextrorotatory.  2 / - Medicinal composition according to claim 1, comprising (b) the following orotic acid salts: lysine orotate, calcium orotate, magnesium orotate, iron orotate and selenium orotate in the form of selenomethionine.  3 / - Medicinal composition according to one of claims 1 or 2, comprising, based on 100 g of lactic acid L dextrorotatory  (a) between 8 and 30 g of peptides,  (b) between 1 and 2 g of orotic acid salt.  4 / - Medicinal composition according to one of claims 1, 2 or 3, combining (a) protease inhibitor peptides, of molecular weight less than 30,000 Daltons, (b) at least one salt of orotic acid, (c ) lactic acid L dextrorotatory, (d) a polysaccharide, (e) at least one salt of folic acid, and (f) urea.  5 / - Medicinal composition according to claim 4, in which (d) the polysaccharide is a glucogalactan.  6 / - Medicinal composition according to claim 5, in which (d) the glucogalactan consists of a glucose and galactose complex, of average molecular weight of between 5,000 and 30,000 Daltons.  7 / - Medicinal composition according to one of claims 4, 5 or 6, comprising, based on 100 g of lactic acid L dextrorotatory  (a) between 8 and 30 g of peptides,  (b) between 1 and 2 g of orotic acid salt,  (d) between 5 and 15 g of polysaccharide,  (e) between 10-3 and 4.10-3 g of folic acid salt,  (f) between 1 and 3 g of urea.  8 / - Medicinal composition according to one of claims 1 to 7, further comprising (g) the following group B vitamins: riboflavin (B2), pyridoxine (B6) and biotin (B8).  9 / - Medicinal composition according to claim 8, also comprising (h) at least one of the following group B vitamins: cyanocobalamin (B12), nicotinamide (B3), pantothenate (B5), thiamine (B1).  10 / - Medicinal composition according to one of claims 8 or 9, comprising, based on 100 g of lactic acid L dextrorotatory, between 0.1 and 0.3 g of vitamins of group B.  11 / - Medicinal composition according to one of claims 1 to 10, comprising (i) the following trace elements: zinc, copper.  12 / - Medicinal composition according to claim 11, comprising, based on 100 g of lactic acid L dextrorotatory  . between 2.10-2 and 8.10-2 g of zinc,  . between 10-3 and 3.10-3 g of copper.  13 / - Medicinal composition according to one of claims 1 to 12, comprising (j) at least one of the following excipients: lactose, milk proteins, fatty acids of lactic origin.  14 / - Medicinal composition according to claim 13, comprising between 103 and 4,103 g of excipients compared to 100 g of lactic acid L dextrorotatory.  15 / - Composition according to one of claims 1 to 14, for use as an antiviral agent, having in particular an antiviral activity on oncogenic viruses.  16 / - Medicinal composition according to one of the preceding claims, obtained by a process of double fermentation of a milk or a whey in the presence of a lactic acid starter, comprising the following steps  - using a lactic acid starter containing polysaccharides in solution, the yeast Candida pseudotroPicalis, the yeast Pichia membranaefaciens, bacteria of the genus Lactococcus and bacteria of the genus Lactobacillus,  the milk or whey containing said leaven is subjected to a first fermentation at a temperature substantially between 260 ° C. and 350 ° C.,  - the product obtained is subjected to a second fermentation at a temperature substantially between 180 C and 280 C.  17 / - Medicinal composition according to Claim 16, obtained by double fermentation in the presence of a lactic acid starter also containing bacteria of the following genera: Bifidobacterium, Acetobacter, Leuconostoc.  18 / - Medicinal composition according to one of claims 16 or 17, obtained by double fermentation of a milk, the first fermentation carried out between 260 C and 350 C having a duration substantially between 4 and 8 hours, the second fermentation carried out between 180 C and 240 C having a duration substantially between 9 and 16 hours.  19 / - Medicinal composition according to one of claims 16 or 17, obtained by double fermentation of a whey, the first fermentation carried out between 260 C and 350 C having a duration substantially between 24 and 72 hours, the second fermentation carried out between 210 C and 280 C having a duration substantially between 48 and 96 hours.  20 / - Medicinal composition according to one of claims 16, 17, 18 or 19, obtained by double fermentation, the microorganisms being eliminated at the end of the second fermentation.