WO2008011884A1 - Novel immunomodulator - Google Patents

Abstract

Immunomodulator composed of seven species of inactivated whole bacteria preserved in phenol. The species are: Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Klebsiella pneumoniae and Micrococcus spp. The immunomodulator is used in chronic diseases involving immunodeficiencies or immunodepression of the cell line.

Description

 "NEW IMMUNOMODULATOR"

Field of the invention:

The present invention is related to a new immunomodulator derived from inactivated bacteria that serves to induce an immune response in diseases related to the lack of cellular immune response and specifically related to T lymphocytes.

State of the prior art:

As a first background, there have been biological products for the treatment of chronic bronchitis and its complications and treatment of bronchial asthma of bacterial origin for several decades.

However, there were some limitations, for example, there is no curative treatment (until now) of chronic bronchitis, considering that it is a degenerative disease that involves not only irreversible microscopic damage, but also anatomical, in addition, asthma currently has no bacterial origin it is a genetic disease or acquired by atopic patients mostly (because their B lymphocytes produce excess lnmunoglobulin E).

The present invention entails technical advantages in relation to products of similar constitution, for example, it is a biological product composed of the characteristic mixture of 7 pure strains of bacteria, genetically classified by the ATCC of the United States of America, unlike previous products composed of 10 strains of inactive bacteria, which, since they are not genetically classified, are nonspecific, an aspect that is overcome by this invention by having specific strains each with its code acquired from the ATCC.

The proposed invention is not limited to the treatment of chronic bronchitis, its complications and bronchial asthma of bacterial origin, but is indicated for the treatment of chronic infectious and inflammatory diseases related to cellular immunology failures.

This was intended to constitute a molecular adapter that rearranges both the genes of the Major Histocompatibility Complex (CMH) and accessory immune response genes located on the female X chromosome. It has precise indications in mild, moderate and severe immunological diseases (HIV / AIDS), which compromise the cell line.

The previous product does not specify what type of lymphocyte line it uses (TH 1 or TH2); that of this application through 10 applications, TH1 lymphocyte line is stimulated, which eliminates the possibility of allergies produced through TH2 lymphocytes.

It is known by other inventions the anti-tumor and specifically anti-cancer effect of different parts of the Klebsiella pneumoniae membrane. For example WO / 2000/054790, in this sense the present invention not only has anticancer activity, but also acts on Chronic infectious and inflammatory diseases of epithelial tissue and is composed of 6 additional bacterial strains.

Another previous antecedent we have in the vaccine based on the Gram negative bacterium Listeria monocytogenes that has in its membrane heterologous antigens induces the activation of T lymphocytes according to WO / 2004/062597; therefore they stimulate cellular immunity. The proposed invention not only comprises Gram-negative bacteria but also Gram-positive strains, which also stimulate cellular immunology and specifically T lymphocytes.

When talking about cellular immunity, it is related to the proliferation, activation and specialization of each of the cell lines; This happens because among the cytokines that are produced is the Colony Stimulating Factor (FEC) that activates the colonies of granulocytes, monocytes or both to act against localized infections of bacteria as it happens in the chronic Pyodermitis as stated in WO / 2005/097065. For its part, this invention activates the antigen presenting cells and through them the T ₁ lymphocyte line is not only effective against bacterial infectious diseases but also against viruses (HIV), intracellular parasites (Toxoplasmosis gondii), fungi, tumors and abnormal immune reactions (chronic inflammation).

According to WO / 2002/085410, both the DNA (gene), as well as the proteins and membrane residues of a mixture of bacteria of the Mycobacterium, Rhidococcus, Corynebacterium, Listeria, or E. coli species promote response immune system where the apoptosis (programmed cell death) of the malignant cells or the destruction of molecules that promote the progression of the disease with inflammation and / or chronic infections occurs. In this invention, the total inactivation of the bacteria genes used tells us that it is the membrane proteins that act as the initial stimulus and these are favorably selected by the Major Histocompatibility Complex.

Atherosclerosis is a chronic inflammatory disease of the endothelium that leads to its destruction, but as indicated in WO / 2006/048628, the administration of enteric bacteria such as Lactobacillus Spp, Bifidobacteria Spp.Streptococcus, and E. coli can counteract the disease. The present invention is distinguished by being a macromolecule constituted by other bacterial strains, its administration is subcutaneously (safer) and not only inflammation of the vascular endothelium but also epithelial tissue cells.

Bacterial proteins alone or fused can be injected subcutaneously and constitute a specific immune antigen, which is what WO / 2006/007366 refers to where a bacterial exotoxin protein treats, prevents or inhibits the action of an enterotoxigenic E. coli infection . The proposed invention is not really a vaccine because it does not form immunoglobulins, therefore it is not useful for disease prevention, but it is a heterologous immunomodulator that is used for the treatment of chronic infectious and inflammatory diseases including certain tumor diseases. According to WO / 2002/056909 using bacterial polysaccharide antigens, an immune response related to T cells that recover patients infected by bacteria (Streptoccocus, Meningococcus, Pneumococcus, H. influenzae) or by virus (Poliovirus, Encephalomyocarditis, Influenza) is induced. There is some similarity of strains but there are also variants and this in itself is already a difference. The present formula not only uses bacterial polysaccharides as antigens, but also the complete and intact membrane of 7 bacterial strains.

The mixture of inactivated bacteria constitutes a potent antigen that slows or stimulates the immune system becoming a potent immunomodulator as mentioned in WO / 2004/096270. The clinical indications also depend on the bacterial strains that are used. The present application is differentiated by the method of inactivation of bacteria (via phenol to

4%), because it is a fixed, constant and proportional mixture of bacteria and because it is used in all the clinical indications mentioned in the same way and technique.

Description:

There are diseases in the medical field that have the characteristic of being chronic and even irresolvable, which are related to an immunological failure that can be of a humoral (antibody) or cellular type and in many cases a combination of both. The immunological explanation of an acute inflammation is that, after trauma of any kind, the immune system must resort to mechanisms where the defense reaction begins, until it achieves full control; for example: a trauma (stroke) is manifested by the latent acute inflammatory response due to the presence of pain, heat, flushing, malfunction of the traumatized tissue, and edema due to what the immune system has to devote to solving these problems.

After the trauma, the immune system acts through the antigen presenting cells to capture the detritus and liquids that escape from the circulation towards the hit site, trying to identify the molecule or epitope characteristic of the traumatic attack. Antigen presenting cells are inflammatory cells that will initially be neutrophils and subsequently macrophages, dendritic cells and lymphocytes. The conglomeration of these cells constitutes an inflammatory focus, which must resolve the immune system and does so through the major histocompatibility complex that the antigen presenting cells possess, which pass the identity (the epitope) to the lymphocyte receptors T, until reaching the suppressor T cell, modulator or TH3, who will give the final warning of having finished the inflammatory process, which means that acute inflammation is an effective immune defense mechanism.

This means that the acute inflammatory process has a beginning and an end; on the other hand, the chronic inflammatory process as its name indicates active indefinitely for both chronic inflammation is an immune failure and an aggression.

There are characteristic diseases that occur only with chronic inflammation, but others are accompanied by an infection that can be viral, bacterial, fungal (fungal), parasitic, allergic or tumor.

At present, the use of anti-inflammatory drugs is frequent continuously and often accompanied by antibiotics, antiviral, antifungal, antiparasitic, antiallergic or antitumor agents, as the case may be, which do not always have the desired effect, due to the resistance of germs to these medications.

If a chronic infection is treated properly, with ideal drugs and despite this the disease persists, it means that the problem is due to lack of immunological response of the host, but not due to lack of medication.

The present invention overcomes these limitations based on the repetitive stimulation of the immune system until it becomes competent against germs.

The immunomodulator consists of the mixture of seven pure bacterial strains, genetically characterized, grown and harvested under known techniques, measuring the optical density in a spectrophotometer calibrated to 600 nrm, washing them in double-distilled and autoclaved water three times to remove contaminants.

The inactivation process is done using 4% phenol, which will cause the inactivation of the genes but keeping the surface molecules intact, after removing the 4% phenol, the amounts in millions per milliliter of each bacterial strain are calculated , which is then mixed and resuspended in 0.20% phenol that would have the characteristic of being a preservative. The result of this solution gives a concentration per milliliter of 50 million streptococus pneumoniae; 40 million streptococus pyogenes: 500 million Stafilococus aureus; 40 million Haemophilus influenzae; 250 million Pseudomona aeruginosa; 40 million Klebsiella pneumoniae and 80 million Micrococus species. Conservative agent: max. 0.25 of phenol and finally the measurement of the optical density that should be around 0.600 nm is made.

The compound is originated in a mixture of inactivated bacteria (between Gram. Positive and Gram. Negative), which are equivalent to being "anesthetized", but integral, that when injected subcutaneously are captured by antigen presenting cells (CPA) of the skin (macrophages, Langerhans cells, dendritic cells, B lymphocytes) which trap them and remove the identity through the Major Histocompatibility Complex (CMH) to present to the T h lymphocyte receptor (helper), the same that produces cytokines (IL-2, FNT alpha and gamma INF) that activate the proliferation and specialized response of Th1 lymphocytes are TCD4 and / or TCD8 (also through cytokines) so that they specifically and definitively act against the infecting antigens. The composition is characterized in that it contains for each ml the following strains:

50 million streptococus pneumoniae; 40 million streptococus pyogenes: 500 million Stafilococus aureus; 40 million Haemophilus influenzae; 250 million Pseudomona aeruginosa; 40 million Klebsiella pneumoniae and 80 million Micrococus species. Conservative agent: max.

0.25 phenol

The product of this mixture was subjected to multiple cultures (in vivo tests) and the bacteria did not develop until 15 days, which indicates the effective inactivation of the bacteria and the null possibility of their pathogenicity.

In vivo tests to demonstrate lethal concentration were performed in mice and white rabbits. Injecting them up to 1,200 times the concentration per milliliter compared to human doses, without causing morbidity or mortality, which means that there is no lethal dose and is only an immunostimulant.

The local response at the immunomodulator inoculation site was the same as the therapeutic dose was injected, than the 1,200 times higher concentration. Injected this product subcutaneously should normally produce an erythematous area that begins its appearance the day after inoculation and lasts approximately 48 hours. This kind of answer discovered Robert Koch in 1895 calling it "infection allergy" and that is what is now known as a delayed type IV or simply cellular delayed hypersensitivity immune response.

The procedure in humans is the application of 5 first ascending doses and 5 of uniform reinforcement in 48 days as follows:

First to fifth dose: 0.1 / 0.3 / 0.5 / 0.7 / 0.9 / mi, after a week of rest it is injected 5 times 1 mi. Both initial and booster doses should be applied every four days.

The immunological explanation is the following: it is known that an antigen of more than 600,000 daltons of molecular weight (macromolecule) when injected subcutaneously takes time to dissolve and therefore the cells of the immune system have enough time to process the antigen and remove the epitopes or epitopes that represent the macromolecule; This is what antigen presenting cells do through the Major Histocompatibility Complex. This information is transferred to the T lymphocyte receptors at the level of the lymph nodes so that through Interleukin-1, Tumor Necrosis Factor-alpha and Interferon range reach TH1 lymphocytes, who upon receiving the stimulus proliferate and activate their genes to give a specific response against the infecting microorganism. In addition, this signal may reach the suppressor, modulator or TH3 T lymphocyte that is responsible for terminating the chronic inflammation after eliminating the infection (see Figure 1a and 1b).

DESCRIPTION OF DRAWINGS

The present invention can be described graphically with the purpose of demonstrating by way of examples the object of the present invention:

Fig. 1st: After trauma, inflammatory cells accumulate consisting of macrophages, T lymphocytes and B lymphocytes.

Fig. 1.b: The cellular detritus or microorganisms are captured by the Antigen Presenting Cell (CPA) and processed by means of the Major Histocompatibility Complex and presented to the Th lymphocyte (LTh), which produces lnterleukin-2 (IL- 2), Tumor Necrosis Factor-α (FTN-α) and Interferon-γ (INF-Y) so that Th 1 or CD4 Lymphocytes proliferate and specialize, which will send stimuli to Th3 Lymphocyte (LTh3), suppressor or moderator that through lnterleukin-10 (IL-10) and lnterleukin-12 (IL-12) will send the signal of completion of the inflammatory process by blocking IL-2, FNT-α and INF-γ

Fig. 2: After injecting into the skin a dose of inactivated bacteria, these are captured, processed and identified by the Major Histocompatibility Complex of the Antigen Presenting Cell (CPA), which will identify the epitopes and pass the signal through cytokines to the helper or helper (LTh), which will express the lnterleukin-2 cytokines (IL-2), Tumor Necrosis Factor - α (FNT-α) and Interferon

Y (INF-Y).

IL-2 induces the proliferation and specialization of ThL lymphocytes In the case of the female sex, IL-2 also activates anti-HIV immune response genes that are expressed through the production of anti-HIV protein that upon entering Ia circulation will reach the shock organs to control HIV virulence; therefore, the TCD4 lymphocyte index rises with a tendency to normality and HIV virulence is canceled.

Claims

REINVINDICATIONS 1. A procedure for the elaboration of an immunomodulator, characterized by the peculiar combination of the following elements: (i) Seven pure strains between gram positive and gram negative in proportional concentrations, genetically characterized each with its code and inactivated, which will constitute a stimulus to the Immune System through the TH 1 (LTH 1) lymphocyte line becoming an immunomodulator . (¡I) The seven strains comprise the following concentrations per 1 ml: 50 million streptococus pneumoniae; 40 million streptococus pyogenes; 500 million Stafilococus aureus; 40 million Haemophilus influenzae; 250 million Pseudomona aeruginosa; 40 million Klebsiella pneumoniae and 80 million Micrococus species (iii) üπ maximum conservative agent of phenol at 0.20% that prevents the development or reactivation of said bacteria. 2. A procedure according to reinvindication 1, characterized by the treatment and inactivation of bacterial strains using 4% phenol and maintaining the inactivation at a maximum phenol concentration of 0.20%. 3. A subcutaneous or intramuscular dosage form of a microbiological product (Immunomodulator), according to claims 1, 2 and 3, characterized in that it comprises the peculiar mixture of the following elements: (i) Seven pure strains between gram positive and gram negative in proportional concentrations, genetically characterized each with its code and inactivated, which will constitute a stimulus to the Immune System to through the TH 1 (LTH 1) lymphocyte line becoming an immunomodulator. (ii) The seven strains comprise the following concentrations per 1 ml: 50 million streptococus pneumoniae; 40 million streptococus pyogenes; 500 million Stafilococus aureus; 40 million Haemophilus influenzae; 250 million Pseudomona aeruginosa; 40 million Klebsiella pneumoniae and 80 million Micrococus species. (iii) A 0.20% maximum phenol preservative agent that prevents the development or reactivation of said bacteria. 4. An immunomodulator, according to the preceding claims, characterized in that it acts through the Major Histocompatibility Complex (CMH) which has the function of recognizing the molecules that belong to the organism to defend them and reject those that do not belong to it. 5. An immunomodulator, according to the preceding claims, which acts through the line of TH 1 lymphocytes, characterized by not having the ability to form antibodies, so that it does not produce allergic reaction, typical of the TH2 lymphocyte line. 6. A method of treatment, according to the preceding claims, characterized in that it is applied for diseases that occur with immunosuppression or immunodeficiencies belonging to the cell line. 7. A method, according to the preceding claims characterized in that it comprises the application of 5 ascending doses starting with 0.1 / 0.3 / 0.5 / 0.7 / 0.9 mi and 5 booster doses of one mi with an interval of four days between them. 8. A method that serves to control rebel allergic states, because activating the TM ₁ line balances or corrects the Th2.