WO2010083581A1

WO2010083581A1 - Method for preparing an immunostimulant from a brucella lysate, and product

Info

Publication number: WO2010083581A1
Application number: PCT/BR2010/000019
Authority: WO
Inventors: Wânia Maria QUINTÃO COIMBRA; Breno FREITAS QUINTÃO; Genésio PACHECO DA VEIGA
Original assignee: Quintao Coimbra Wania Maria; Freitas Quintao Breno; Pacheco Da Veiga Genesio
Priority date: 2009-01-21
Filing date: 2010-01-20
Publication date: 2010-07-29
Also published as: BRPI0900215A2

Abstract

The present invention relates to a method for preparing an immunostimulant from a Brucella lysate, having the following steps: (a) cultivating colonies of at least two strains or species of Brucella spp., in defined proportions and in a suitable culture medium; (b) adding a pharmacologically acceptable solvent; (c) homogenising and inoculating the suspension with media specific to Brucella; (d) adding a pharmacologically acceptable solvent and homogenising the resulting solution; (e) adding a pharmacologically acceptable acid; (f) heating, then cooling the resulting solution; (g) adjusting the pH from 6.0 to 8.0; (h) filtering the solution; (i) adjusting the pH from 6.0 to 8.0; (j) diluting the lysate in a pharmacologically acceptable solvent and adding a preservative that is also pharmacologically acceptable; (k) adjusting the pH from 6.0 to 8.0; (l) filtering the dilution until a sterile, ready-for-use end product with a clear aspect is obtained. The invention also relates to the immunostimulating product obtained from the culture filtrate produced by this method. The immunostimulant from Brucella lysate can be indicated for the treatment of bone and joint disorders, such as arthritis, osteoarthritis and tendinitis; immune disorders, rheumatoid arthritis and related disorders, infection and inflammation in humans. In animals, moreover, the product can be indicated for bacterial and viral infections, such as parvovirus and coronavirus infection, leptospirosis, canine distemper and pneumonia; traumatic wounds and skin lesions.

Description

BRUCELLA AND PRODUCT LISADO IMMUNOSTIMULANT PREPARATION PROCESS

The present invention is a process for obtaining an immune system stimulant for veterinary and human use.

There is a great interest in the use of agents capable of producing an immunostimulant response in both humans and animals, because in this way, the organism's ability to fight an infection is increased, bringing health and vitality benefits.

Conceptually, immunostimulation has long been known in medicine. It is defined as substances that in themselves are capable of inducing the body's own defense mechanism in a specific or non-specific manner.

Certain bacteria and their extracts are known to be capable of enhancing the immune response when ingested or administered. According to some authors, Brucellas appear to have an antigen capable of producing high antibody response in patients with rheumatic arthritis.

To verify the behavior of Brucella abortus endotoxin in experimental rheumatoid arthritis, in 1991 Preza et al. (PREZA FB, AMORIM LMF, PREZA PCA. Quantification of anticollagen IgG antibodies in mice Balb / c strain, vaccinated with Brucella abortus endotoxin. Presented at the 10th Annual Meeting of the Brazilian Society of Biochemistry and Molecular Biology, p. 173, 1991) injected, At intervals of 3 days, endotoxin in four groups of rats and after 30 days all animals were inoculated with collagen C-11 to induce arthritis. After 4 weeks, those who received the vaccine had no arthritis, while those who did not get it.

Amorim, 1993 (AMORIM LMF. Study of variations in the production of antibodies against type II collagen in Balb / c mice. Master's thesis. Rio de Janeiro, UERJ, 1993) evaluated the production of anti-C-specific immunoglobulin. ll (considered to cause rheumatoid arthritis) in mice. In his research, he inoculated collagen C-11 with Freund's adjuvant, causing marked elevation of IgG immunoglobulin. It then applied Brucella extract to the inoculated animals, resulting in a marked decrease in anti-C-11 IgG production, with a high immunosuppressive effect. It is concluded, therefore, that considering the great role of anti-C- II in rheumatoid arthritis, the action of Brucella extract as a potent diminisher of the production of these antibodies in mice is beneficial.

From the studies presented, it was concluded that Brucella extract decreases only IgG immunoglobulin, which causes rheumatoid arthritis and increases IgM gamma globulin, as demonstrated by Meiselas et al., 1961 (MEISELAS LE, ZINGALE SB, LEE SL, RICHMAN S, SIEGEL M. Antibody production in rheumatic diseases.The effect of Brucella antigen (J.Clin.Invest, 40: 1872,1961), whose purpose is to protect the organism. This selective mechanism of intervening in the immune response in rheumatoid arthritis was the factor that drew attention to the research that culminated in the development of the present invention.

The present invention is a simple process for obtaining an immunostimulant product which increases the cytophageal opsonic index of blood, nontoxic, sterile, ready for use and rarely causes side effects.

Brucella lysate immunostimulant may be indicated in the treatment of osteoarticular diseases, such as arthritis, arthritis and tendonitis; immune disorders, rheumatoid arthritis and variants, infections and inflammation in humans. In animals, it may be indicated for bacterial and viral infections, such as Parvovirus, Coronavirus, Leptospirosis, Distemper and Pneumonia; wounds and skin lesions caused by trauma.

The present invention claims a process for preparing Brucella lysate immunostimulant which comprises the propagation of two or more Brucella strains or species such as: B. melitensis, B. abortus, B. suis, B. canis, B. ovis , B. neotomae, B. maris, B. cetaceae, B. pinnipediae, in certain proportions. A pharmacologically acceptable solvent is then added. The solution is homogenized and the suspension inoculated in Brucellas-specific medium. Then a pharmacologically acceptable solvent is added and homogenized. A pharmacologically acceptable acid is then added and the mixture is heated. After cooling the pH is adjusted from 6.0 to 8.0 followed by a serial filtration process. The pH is again adjusted to between 6.0 and 8.0. One or more dilutions of the product are made in a pharmacologically acceptable solvent. A pharmacologically acceptable preservative is then added and, if necessary, adjusted to pH 6.0 to 8.0. The dilution is filtered to a clear, sterile ready-to-use final product. The invention also claims the immunostimulant product from culture filtrate obtained by the process described above.

Brucella strains are picked and propagated according to techniques known to man in the art. Two or more Brucella strains or species may be used such as: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, B. neotomae, B. maris, B. cetaceae, B. pinnipediae, in certain proportions.

Mainly β species are used. melitensis, B. abortus and B. suis, preferably B. abortus and β. suis in certain proportions, from 6 to 8 parts of B. suis and 12 to 16 parts of B. abortus in relation to the total mass of bacteria, preferably 6 parts of B. suis and 12 parts of B. abortus, ie , in the ratio 1: 2 respectively.

After incubation of the test tubes for 24 to 72 hours, preferably 48 hours at a temperature of 34.5 to 36.5 ° C, preferably 35 ° C, 1 to 3 ml, preferably 2 ml, is added to each tube. of a solvent, which may be any pharmacologically acceptable colloid or crystalloid solution, preferably 0.9% saline, maintaining a concentration of 1 to 60 billion bacteria / ml, preferably 30 billion bacteria / ml, on the scale. Farland's nephelometric test in each test tube. The solution is homogenized and the suspension is inoculated from each tube into a Roux bottle containing Brucellas specific medium in the inoculation ratio of 1 test tube to each Roux bottle, always maintaining the ratio 1: 2, that is, 1 bottle of B. suis Roux for every 2 bottles of B. abortus, regardless of the amount of bottles.

After incubating the Roux bottles for 24 to 72 hours, preferably 48 hours at 34.5 to 36.5 ° C, preferably 35 ° C, 10 to 50 ml of solvent, preferably 30 ml of serum, is then added. 0.9% in each Roux bottle, once again maintaining a concentration of 1 to 60 billion bacteria / mL, preferably 30 billion bacteria / mL in each bottle.

Then homogenize and remove from 10 to 50mL, preferably

30 mL of the bacterial broth from each Roux bottle, by combining the volumes of each bottle into a single container, maintaining a concentration of 1 to 60 billion bacteria / mL, preferably 30 billion bacteria / mL, on the scale. Farland's nephelometric analysis and also the 1: 2 ratio between B. suis and B. abortus, respectively. Subsequently, 0.3 to 0.5%, preferably 0.4% of an organic and / or inorganic acid, preferably concentrated hydrochloric acid - which has the function of destroying bacteria, while maintaining the antigenic characteristics of Brucellas and neutralizing them, are added thereafter. the possible production of ammonia. Heat for 1 to 3 hours, preferably 2 hours. After cooling, the pH is adjusted to between 6.0 and 8.0, preferably 7.4. Serial filtrations are then performed, the last membrane filtration being 0.22 microns. The pH is adjusted from 6.0 to 8.0, preferably 7.4. One or more dilutions of the product are then made in colloid or crystalloid solution, preferably in 0.9% physiological solution, in varying concentrations between 1: 25,000 to 1: 1, preferably in the following concentrations: 1: 5000, 1 : 1000, 1: 500, 1: 50, 1: 20 and 1: 5 and add from 1 to 200 ppm, preferably 100 ppm of a preservative solution, preferably thimerosal in each dilute. The pH is adjusted if necessary to from 6.0 to 8.0, preferably 7.2 to 7.4. Again filter through 0.1 to 0.8 micron pre-filter membranes, preferably 0.2 to 0.6 microns until a clear, sterile, final product is obtained and after the necessary adjustments, It may be used by any route of administration: oral, topical, anal, sublingual, ocular, nasal, auricular, inhalational, intradermal, subcutaneous, intra-muscular, intravenous and intra-tecal.

Importantly, the concentrations of bacteria, the amounts of solvents, acids and preservatives used are always directly proportional. Therefore, if a larger amount of lysate is made, the amounts of these components will also increase proportionally.

Examples follow to further illustrate the invention, but they are not intended to restrict the invention described herein.

Example 1 (Preparation of immunostimulant):

Brucella strains are picked and propagated according to techniques known to man in the art. 6 parts of B. suis and 12 parts of B. abortus are used, in 1: 2 ratio respectively. Eighteen test tubes are made, being 6 B. suis and 12 B. abortus tubes, that is, in the ratio 1: 2 respectively. After incubation for 48 hours at 35 ° C in a bacteriological oven, 2 ml of 0.9% saline is added to each tube and the concentration of 30 billion bacteria / ml is maintained in each test tube. . The suspension is homogenized and the suspension is removed from each tube by inoculating it into 18 Roux bottles containing Brucellas specific medium. After incubation Roux bottles for 48 hours at 35 ° C, 30 ml of 0.9% physiological solution is added to each bottle, maintaining a concentration of 30 billion bacteria / ml in each bottle, totaling 6 bottles of Roux with B. suis and 12 bottles of B. abortus. Then, 30 ml of the bacterial broth is homogenized and removed from each Roux bottle by combining the volumes of each into a single container containing a final volume of 540 ml, ie 180 ml of broth. B. suis + 360 mL of B. abortus broth, always maintaining a concentration of 30 billion bacteria / mL. Then 2.3 mL of concentrated hydrochloric acid is added and heated for 2 hours. After cooling, the pH is adjusted to 7.4. Serial filtrations are then performed, the last membrane filtration being 0.22 microns. The filtrate is then diluted in 0.9% saline at 1: 5000, 1: 1000, 1: 500, 1: 50, 1: 20 and 1: 5 concentrations and 100 ppm of thimerosal solution in each of the diluted. The pH is adjusted, if necessary, to from 7,2 to 7,4. Again filter through 0.2 to 0.6 micron membranes until a clear, sterile, ready-to-use liquid is obtained.

Example 2 (Human Area):

1750 patients with various types of rheumatic diseases underwent Brucella lysate immunostimulant. Of these, 400 patients met the classic diagnosis criteria for rheumatoid arthritis, according to the American Council of Rheumatology classification, of which 377 agreed to participate in the study and all patients included in this study met the criteria for rheumatoid arthritis. active disease and were seropositive for rheumatoid arthritis.

The immunostimulant was prepared from Brucella abortus and Brucella suis and was separated into 6 dilutions: 1/5000, 1/1000, 1/500, 1/50, 1/20 and 1/5.

Brucella lysate immunostimulant was administered intradermally, 0.1 ml 4/4 days, increasing 0.1 ml every 5 applications until reaching 0.4 ml in each vial 5 times, then applying , as follows: 0.1 mL 5 times; 0.2 mL 5 times; 0.3 mL 5 times; 0.4 mL 5 times in all vials of the different dilutions.

Four to six months after initiation of immunotherapy, attempts to decrease anti-inflammatory drugs, corticosteroids and slow-acting drugs (gold salts, anti-malarials and immunosuppressants) began.

RESULTS Of the 377 patients who underwent immunotherapy, 322 were female (85%) and 55 male (15%). The age ranged from 21 to 80 years.

Most patients had a diagnosis of rheumatoid arthritis more than 5 years old, with 37% of them over 10 years old (TABLE 1 ANNEX).

At the time of initiation of immunotherapy, 100% of patients had joint pain and the vast majority complained of: morning stiffness (84%), increased joint volume (84%), symmetrical joint pain with heat and edema (82%) , subcutaneous nodules (53%) plus general symptoms such as fatigue (75%), decreased appetite (42%) and fever (37%) despite the use of non-hormonal anti-inflammatory drugs, corticosteroids and slow acting drugs (TABLE). 2 ANNEX).

25% of patients had 1 to 2 joints, 33% of 3 to 4, 18% of 5 to 7, and 24% of all joints had been affected by the disease (TABLE 3 ANNEX). Pain, assessed by the patient himself without physician interference, was severe or very severe in 71.5% of the study population (TABLE 4 ANNEX).

Immediately before the beginning of immunotherapy, 41% of patients had difficulty moving their hands and wrist, 29% had difficulty walking, 28% had arm and foot movements, and 23% could not flex their knees (TABLE 5 ANNEX).

In the months leading up to immunotherapy until its initiation, 100% of patients were receiving non-hormonal anti-inflammatory drugs, 94% of non-hormonal anti-inflammatory drugs and corticosteroids, and 51% of non-hormonal anti-inflammatory drugs, corticosteroids and slow-acting drugs. Almost half of the study group had already undergone infiltration of one or more joints (TABLE 6 ANNEX).

Treatment with anti-inflammatory drugs, corticosteroids and slow acting drugs was causing side effects in 84% of patients (TABLE 7 ANNEX), with 57% of them presenting with gastritis, 18% weight gain, 10% digestive bleeding, 7% gastrointestinal ulcer. duodenal and 7% rash (TABLE 8 ANNEX).

Self-assessment of clinical improvement with conventional treatment showed the poor performance of the therapy so far employed: only 15.5% reported excellent or good results, 47% fair and 37.5 poor or very poor (TABLE 9 ANNEX). Regarding the use of Brucella lysate immunostimulant, 28% of patients received 1 to 3 vials, 46% from 4 to 6 and 26% more than 6 (TABLE 10 ANNEX). Thus, 2/3 of the patients referred in the present study received a maximum of 6 immunostimulant vials.

Side effects of Lysate Immunostimulant Immunotherapy

Brucella were observed in only 12% of the patients, the most frequent being: increased joint pain (14/275) and increased inflammatory signs in some joint (7/275). Other, much rarer effects were: fatigue, dizziness, rash, nausea, tachycardia, cramps, drowsiness, headache, and local reaction (Tables 11 and 12 ANNEXES). Eighty-five percent of these side effects disappeared by decreasing the immunostimulant dose or continuing treatment.

During the course of immunotherapy, 80% of the patients had a marked improvement or total regression of joint pain, inflammatory signs, functional impotence and general symptoms. Subcutaneous nodules disappearance in half of the treated patients (TABLE 13 ANNEX). Congestive, ie reversible deformities also disappeared in 40% of cases.

Self-assessment of clinical improvement without physician interference showed the good performance of immunotherapy in the patients studied: 79.5% reported excellent and good results, 16% fair and 4.5% poor or very poor (TABLE 14 ANNEX).

Importantly, after the immunological approach, it was possible to suspend corticosteroids and anti-inflammatory drugs in 40% of patients. In 54.5% it was possible to reduce such drugs and in 4.5% the need for such drugs remained unchanged (TABLE 15 ANNEX).

Thus, it is concluded that Brucella lysate immunostimulant is safe and very effective, since with minimal side effects it was possible to obtain in 80% of the treated patients, great improvement or total regression of joint pain, inflammatory signs. , functional impotence, decreased use of strong medications, and general symptoms.

Example 3 (Veterinary Area):

Female French Bulldog, 11 months old with various injuries caused by another dog. Eight doses of the immunostimulant were applied at 1/5 dilution. each dose of 1 to 2 mL / kg of the animal every other day. There was total recovery of the animal.

Example 4 (Veterinary Area):

Kennel with numerous cases of virus confirmed by laboratory examination. The animals had diarrhea and airway involvement. For 2 weeks, treatment was performed with 1/5 inhalations of 4 mL of the immunostimulant every 8 h, in addition to subcutaneous applications at 1/5 dilution of 1 to 2 mL / kg of the animal. full recovery.

Example 5 (Veterinary Area):

Maltese dog, 2 years old, with severe weight loss and numerous wounds, hair loss and extreme weakness. The picture was reversed with subcutaneous applications of the immunostimulant, at 1/5 dilution, and 3 doses of 1 to 2 mL / kg were applied at 10-day intervals. There was a 15% increase in weight, hair growth and wound healing.

Example 6 (Veterinary Area):

Akita dog, 2 years old, with purulent wet dermatitis. The treatment consisted of subcutaneous application, at 1/5 dilution, of a dose of 1 to 2 mLJkg of the animal every 8 days for 1 month. Most of the wounds dried and the hair grew back.

Example 7 (Veterinary Area):

Whippet dog, female, 3 years old, with piercing-leg injury. Treated with the subcutaneous application of 1 mL of the immunostimulant at 1/5 dilution, the second dose being administered 4 days later, however, 0.5 mL subcutaneously. The injury was completely healed.

Table 1: Time interval between diagnosis and initiation of immunotherapy in 377 rheumatoid arthritis patients.

Table 2: General and specific symptoms in 377 rheumatoid arthritis patients at the beginning of immunotherapy.

Table 3: Number of joints affected by rheumatoid arthritis in 320 patients at the beginning of immunotherapy.

Not answered: 57 patients (15%) Table 4: Intensity of joint pain in 377 patients with rheumatoid arthritis at the beginning of immunotherapy.

PAIN INTENSITY NUMBER%

PATIENTS

Weak j 23 6.0

Average j 85 22.5

Forte j 150 40.0

Very strong J 119 31.5

Table 5: Functional impotence in 377 rheumatoid arthritis patients at the beginning of immunotherapy

FUNCTIONAL EMPOWERMENT j NUMBER OF%

PATIENTS

Hand - fist j 155 41

Ambulation | 109 29

Arms - feet | 104 28

Knee flexion j 86 23

Bending down j 22 6

In wheelchair or 21 6 crutches J

Shoulders j 20 5

Load weight j 20 5

Go down or climb stairs | 19 5

Getting up j 16 4

Turn your neck j 12 3

Write | 10 3 Table 6: Therapy in use immediately prior to initiation Brucella lysate immunostimulant immunotherapy in 377 rheumatoid arthritis patients.

NSAID: Non-hormonal Anti-Inflammatory

Slow acting drugs: gold salts, antimalarials and immunosuppressants

Table 7: Side effects of non-hormonal anti-inflammatory drug, corticosteroid, and slow-acting therapy in 283 rheumatoid arthritis patients.

! SIDE EFFECTS NUMBER OF PATIENTS | %

j Gifts 238 | 84 j Missing 45 j 16

They did not answer: 94 pacien! es (25%)

Table 8: Side effects of conventional therapy in 279 rheumatoid arthritis patients.

Not answered: 98 patients (26%) Table 9: Self-assessment of 330 rheumatoid arthritis patients undergoing classic or usual therapy.

No answers: 47 patients (12%)

Table 10: Number of vials of Brucella lysate immunostimulant administered to 346 rheumatoid arthritis patients.

No answers: 31 patients (8%)

Table 11: Side Effects of Brucella Lysate Immunostimulant in 275 Rheumatoid Arthritis Patients

Not answered: 102 patients (27%) Table 12: Side effects of Brucella lysate immunostimulant in 32 patients with rheumatoid arthritis out of 275 patients who answered this question.

Table 13: Results of Brucella Lysate Immunostimulant Treatment in 377 Rheumatoid Arthritis Patients

SYMPTOM NUMBER RETURN% TOTAL PATIENTS

377 Joint pain 294 78

349 Difficulty in 282 81

movements

313 General Symptoms 251 80

292 Increase from 230 79

volume / edema

242 Heat 181 75

192 Subcutaneous nodules 106 55 Table 14: Self-assessment of 365 rheumatoid arthritis patients who received Brucella lysate immunostimulant

No answers: 12 patients (3%)

Table 15: Need for anti-inflammatory drugs and corticosteroids after the use of Brucella lysate immunostimulant in 333 patients with rheumatoid arthritis.

ANTI-INFLAMMATORY DOSE AND]% NUMBER

CORTICOSTEROIDS | PATIENTS

Diminished j 182 54.5

Suspended j 133 40.0

Equal | 15 4.5

Augmented J 3 1.0

Not answered: 44 patients (12%)

Claims

Brucella lysate immunostimulant preparation process, comprising the following steps:

The. propagation of colonies of at least two strains or species of Brucella spp, in certain proportions and in appropriate culture medium;

B. addition of pharmacologically acceptable solvent;

ç. homogenization and inoculation of the suspension in Brucellas specific culture medium;

d. addition of pharmacologically acceptable solvent and homogenization of the obtained solution;

and. pharmacologically acceptable acid addition;

f. heating of the obtained solution, followed by its cooling;

g. pH adjustment from 6.0 to 8.0;

H. solution filtration;

I. pH adjustment to between 6.0 to 8.0;

j. diluting the lysate in a pharmacologically acceptable solvent and adding a pharmacologically acceptable preservative;

k. pH adjustment from 6.0 to 8.0;

I. dilution filtration to a clear, sterile, ready-to-use end product.

Brucella lysate immunostimulant preparation process according to claim 1, characterized in that the Brucella melitensis, Brucella abortus, Brucella canis, Brucella ovis, Brucella maris, Brucella cetaceae, Brucella pinnipediae strains are employed.

Brucella lysate immunostimulant preparation process according to claims 1 and 2, characterized in that it mainly employs the Brucella melitensis, Brucella abortus and Brucella suis strains, preferably the Brucella abortus and Brucella suis strains. Brucella lysate immunostimulant preparation process according to claim 3, characterized in that the propagation of the colonies can be carried out using from 6 to 8 parts of B. suis and from 12 to 16 parts of B. abortus in relation to the total mass. of bacteria; preferably 6 parts B. suis and 12 parts B. abortus, that is, in the 1: 2 ratio, respectively.

Brucella lysate immunostimulant preparation process according to claim 1, characterized in that the solvent employed is any pharmacologically acceptable colloid or crystalloid solution, preferably 0.9% physiological solution.

Brucella lysate immunostimulant preparation process according to claim 1 or 5, characterized in that the amount of solvent used in step "b" is 1 to 3 ml, preferably 2 ml for each test tube used.

Brucella lysate immunostimulant preparation process according to claim 1 or 5, characterized in that the amount of solvent used in step "d" is from 10 to 50 ml, preferably 30 ml for each Roux bottle used.

Brucella lysate immunostimulant preparation process according to claim 1, characterized in that the concentration of bacteria in the lysate is from 01 to 60 billion bacteria / mL, preferably 30 billion bacteria / mL, on the Mc Farland nephelometric scale.

Brucella lysate immunostimulant preparation process according to claim 1, characterized in that the acid employed is any pharmacologically acceptable organic and / or inorganic acid, preferably concentrated hydrochloric acid.

Brucella lysate immunostimulant preparation process according to claim 9, characterized in that the amount of concentrated hydrochloric acid employed is between 0.3 and 0.5%, preferably 0.4%.

Brucella lysate immunostimulant preparation process according to claim 1, characterized in that the final dilution pH is 6.0 to 8.0, preferably 7.4.

Brucella lysate immunostimulant preparation process according to claim 1, characterized in that the preservative employed is any pharmacologically acceptable preservative solution, preferably thimerosal.

Brucella lysate immunostimulant preparation process according to claim 12, characterized in that the amount of preservative solution employed is from 1 to 200 ppm, preferably 100 ppm.

Brucelia lysate immunostimulant preparation process according to claim 1, characterized in that the final product is in dilutions ranging from: 1: 25,000 to 1: 1.

Brucelia lysate immunostimulant preparation process according to claim 14, characterized in that the dilutions are preferably: 1: 5000, 1: 1000, 1: 500, 1: 50, 1: 20 and 1: 5.

Brucelia lysate immunostimulating product characterized in that it is prepared according to claims 1 to 15.

Brucelia lysate immunostimulating product according to Claim 16, characterized in that it can be used after any necessary administration via any route of administration: oral, topical, anal, sublingual, ocular, nasal, atrial, inhalation, intradermal, subcutaneous. -cutaneous., intra-muscular, intravenous and intra-tecal.

Brucelia's lysate immunostimulating product according to claim 16, characterized in that it can be employed in the treatment of osteoarticular, immunological, infections and inflammation in humans.

Brucelia's immunostimulating lysate product according to claim 16, characterized in that it can be used in the treatment of bacterial, viral, wound and skin injury caused by animal trauma.