ITMI20112433A1 - COMPOSITIONS FOR ADIUVANT ORAL THERAPY OF ANORESSIA INCLUDING ANTIBACTERIALS, ANTIBACTERIALS, GROWTH FACTORS AND CYTOCHINES - Google Patents

Description

Description

â € œCOMPOSITIONS FOR ADJUVANT ORAL THERAPY FOR ANOREXIA INCLUDING ANTIBODIES, ANTIBACTERIALS, GROWTH FACTORS AND CYTOKINESâ €

The present invention relates to compositions for the adjuvant therapy of anorexia comprising antibodies, growth factors and cytokines, isolated from the tissues and biological fluids of mammals.

State of the art

A person has Anorexia Nervosa if they have all four of the following:

- Significant weight loss (more than 15% of the weight considered normal for age, sex and height);

- Intense fear of gaining weight even when you are underweight;

- Alteration in the way of living weight, size and body shape; - Disappearance of menstruation (absence of at least three consecutive menstrual cycles in women).

There are two forms of anorexia nervosa: restrictive anorexia, a form in which weight loss is caused by fasting and intense physical activity, and anorexia with bulimia, a form in which the person engages in behaviors that together with fasting they serve to decrease body weight (abuse of laxatives and / or diuretics, vomiting).

Anorexia and eating disorders in general are a real health emergency in western industrialized countries and, according to many authors, are constantly increasing. In reality, the various studies carried out do not agree: if some of them tend to highlight a worrying increase in cases, others underline the constant trend, without any variation. A meta-analytical study, which examined the historical evolution of the disease in the past (1995), showed that in the nineties the percentage of the affected population remained constant.

According to the data obtained from the literature, the prevalence (total number of cases in the population) of anorexia was around 0.3% in 2003, while the incidence (number of new cases in the population in a given period of time) It is 8 cases per 100,000 subjects in a year. The percentage was then updated to 0.42% in 2006 by studies conducted in Italy. Thereafter, in 2007, the prevalence would have risen slightly, settling at around 0.5% or, as another study suggests more pessimistically, would have exceeded 2%. As far as the age of onset is concerned, this is between 12 and 25 years (although several cases over 30 years have occurred in recent years), with the most critical moment between 15 and 19 years. Other studies have found incidence peaks at 14 and 18 years of age. The disease therefore affects mainly adolescents, although lately there are more and more cases in adults and also among the elderly.

Another typical characteristic of anorexia is that of being a purely female disorder: about 90% of cases, in fact, develop in the female sex. However, the problem is not just about women. Even if the studies on the male sex are minor, it has been estimated that out of the total number of patients, 5% to 10% of cases involving adolescent boys and adult males are present. According to some research, the male-female ratio is 1:10; other studies have found a smaller difference between the sexes, even reaching 1: 8. The percentage of anorexic males appears to be on the rise, but this could stem from the simple fact that more men are now seeking medical attention to treat this ailment.

The causes of this disease are still unclear.

Researches carried out on the biochemical characteristics of patients suffering from eating disorders have found that the neurotransmitters â € œserotoninâ € and â € œnoradrenalineâ € are present in smaller numbers in those suffering from anorexia, a characteristic that brings them closer to those suffering from depression. These characteristics are confirmed by studies that have shown that some antidepressants can be used successfully in the treatment of these disorders. People with anorexia often tend to have a rate of `` cortisol '' (a hormone produced by the brain in stressful situations) and `` vasopressin '' (a hormone produced by the brain in abnormal quantities in patients with `` obsessive-compulsive disorder '') higher than normal.

Other mental disorders can accompany anorexia such as "obsessive-compulsive disorder", the tendency to self-harm or "bipolar disorders".

Generally, antidepressant drugs can be effective in treating people with anorexia, but the most effective therapy has been found to be able to achieve a weight within 10% of normal. Individual-to-family psychotherapy can also help.

The starvation suffered by anorexics can cause damage to vital organs such as the heart and brain.

The respiratory rate, heart rate and blood pressure decrease and sometimes those suffering from this pathology suffer from arrhythmias or heart problems. A poor diet causes the decalcification of the bones which become brittle and prone to breakage. In severe cases, anorexics can allow themselves to die of malnutrition. Eating disorders have the highest mortality rate among personality disorders, accounting for a death rate of 6%. Fortunately, most of the side effects turned out to be reversible. From the above it is evident the need for pharmacological, dietary or food aids that can help resolve the complex picture of this pathology.

Description of the invention

According to current scientific literature, the therapeutic action of stem cells can be traced back to two mechanisms: differentiation of stem cells into resident cells and release of regenerative trophic factors by stem cells. The respective contributions of these two mechanisms remain to be clarified, even if it has been hypothesized that it is not the stem cells that transform into mature cells of the injured tissue, but that they transmit vital factors to this tissue, which can thus return to proliferate. and to differentiate, regenerating (Figure).

Stem cell therapy presents many problems related not only to costs and technical and application complications, but also to ethical and religious scruples.

Stem cell therapy is feasible only by injection or, in some cases, topically, and not by mouth. The stem cell supernatant in culture contains growth factors, cytokines, chemotactic factors, etc. believed to be responsible for the beneficial effect of stem cell therapy on tissue growth and / or repair.

The eventual use of the vital factors that can be isolated from the supernatant of the stem cells presents, however, not only the same ethical problems of the use of the stem cells themselves but also very high costs.

It was surprisingly found that the same active factors released by stem cells and therefore present in the supernatant of stem cell cultures are present in biological fluids and in some mammalian tissues. The best sources of these factors are mammalian serum, placenta, and colostrum.

The Table reports the qualitative comparison between the factors present in the supernatant of stem cell cultures and the factors isolated from these new sources which we call POOL OF MATERNAL FACTORS (P.M.F.).

Table

P.M.F. Mesenchymal NÃ € ME (pg / ml) stein ceils (pg / ml) IL 1 ra IL-1 receptor antagonist 29.05 0.0

IL-lb Interleukin - 0.09 0.0

IL-2 Interleukin-2 32.05 0.0

IL-4 Interleukin-4 0.17 21.12

IL -6 Interleukin- 6 1.26 293.01 IL-8 Interleukin - 8 0.71 359.56

IL-9 Interleu -9 3.66 0.78

IL- 10 Interleuk - 10 2.83 0.99

IL-12 Interleuk -12 1.73 0.0

IL- 15 Interleukin -15 4.33 0.5

IL- 17 Interleukin - 17 21.95 0.32 Eotaxin Eotaxin 5.01 0.0

INF-γ Gamma -interferon 5.97 11.14 MCP-1 Monocyte chemotactic factor-1 4.12 11.23

PDGF Piatelet derived growth factor 11. 3.08 TNT Tumor necrosis factor 40.00 12.30

endot helial growth 41 .00 208.04 VEGF factor

HGF Hepatocyte growth factor 52.3 100.74

F GF Fibroblast growth factor 180.15 18.21 TGF- 64.00 51.32 beta1 transforming growth factor

IGF-1 insulin-like growth factor 1.60 0.0

GM- Gramilocyte / moiiocyte-colony 183.85 0.0 CSF stimulating factor

LIF leukemia inhibitory factor 15.20 27.32 SCF stelli cell factor 10.55 4.54 SDF- 1 stromal derived factor- 1 41.75 28.8 NGF Nerve growth factor 8.33 0.0 BMP-2 Bone morphogenic protein 25.01 2.65

RNÃ € 189 ng / ml 48 pg / ml

Presence of growth factors / cytokines in P.M.F. (1 mg / ml) and in the Mesenchymal Stem Cell supernatant (1 million cells).

It is evident (table) the greater concentration of almost all the factors present in the P.M.F. compared to that present in the stem cell supernatant.

It is also evident the possibility of using even very high therapeutic concentrations of P.M.F., for example doses of 0.5 / 1 g topically or injected and doses up to 20/30 g orally, compared to the maximum therapeutic concentrations of cells used. stem cells that correspond to 1/2 million / kg and therefore to a maximum of 140 million per therapy and, therefore, to values of factors always of the order of picograms.

The object of the invention is therefore a compound (PMF) containing in very high concentration all the active factors present in the supernatant of stem cells but isolated, with simple extraction methods, from very cheap natural sources, easily available and without problems. ethical.

Furthermore P.M.F., isolated from biological fluids and mammalian tissues, also contains antibacterial factors (transferrin, lysozyme, lactoperoxidase, lactoferrin) and antibodies of the IgG and IgA classes. (P.M.F. Ab).

The absence of antibodies and antibacterials of the stem cell supernatant is another reason for the use of P.M.F. Ab in many therapeutic, topical and oral uses.

The serum shows the maximum peak of the factors in the last days before the birth, the colostrum in the first hours after the birth and no later than the 6th hour.

Already after 12 hours from the birth the factors decrease considerably, at 24 hours many of them are no longer measurable.

These factors are genetically very conserved in the various species and therefore it is possible to use on humans factors isolated from other mammal species such as cattle, horses, camels, marine mammals, etc.

The factors are controlled with specific ELISA methods for the species, even if the interspecies cross reaction is very high as the factors are phylogenetically very conserved and, therefore, they are only qualitatively measurable even with ELISA used for different species (eg: human-bovine and viceversa).

A first source of the factors of the invention is the serum of mammals which, in the last days (5-15) before giving birth, is very rich in the active factors object of the invention compared to non-pregnant mammals or in the first months of pregnancy. A serum extraction method is described below, by way of example.

1 liter of blood is taken in 4 days for a total of 4 samples in order not to harm the animal, preferably bovine or equine.

The blood is serumed at room temperature for 24 h and then centrifuged to squeeze the clot.

The serum is recovered (about 30/40% of the total volume) to which are added, as antiseptic agents, 2.5% phenoxyethanol and 1% diazolidinyl-urea. The treated serum is then fractionated with the following steps.

Ultrafiltration 300,000 From:

The serum sample (frozen at â € “20 ° C) obtained by coagulation and centrifugation from mammalian blood is thawed at room temperature and diluted with 2 volumes of demineralized water. The solution obtained is ultrafiltered on a 300'000 Da polyethersulfone Millipore Pellicon Biomax tangential flow flat membrane at a Pi of 0.5Ã · 1 bar, in a cold room at 4 ° C.

The retentate and a fraction corresponding to approximately 1:10 of the permeate are transferred to a 1000 Da regenerated cellulose Spectrum Spectrapor dialysis tube and dialysed against demineralized water.

Ultrafiltration 5'000 From:

The remaining permeate is ultrafiltered on a 5000 Da membrane. The 300'000 Da ultrafiltration permeate is concentrated on a Millipore Pellicon Biomax cross flow flat membrane in polyethersulfone of 5000 at a Pi of 0.5Ã · 1 bar, in the chamber cold to 4 ° C.

The retentate is transferred to a 1000 Da regenerated cellulose Spectrum Spectrapor dialysis tube and dialyzed against demineralised water (preservatives are also eliminated with this dialysis). The mixture is then immediately freeze-dried.

A second source of the factors of the invention is the placenta. An extraction method is described below, by way of example.

Bovine or equine placentas are preferably used.

Homogenization

The placenta (frozen at â € “20 ° C) is thawed at room temperature, cut into small pieces, washed with abundant cold saline (0.9% NaCl) (4 ° C) and homogenized by means of a Siramm cutter in a buffer of lysis composed as follows: 50 mM Tris / HCl, 25mM EDTA, 0.001% triton X-100 at pH 7.4. To the suspension obtained is added NaCl up to a concentration of 0.9%, and the preservatives (phenoxyethanol at 2.5% and diazolidinyl-urea at 1%). The suspension is stirred (magnetic stirrer) for 2 hours and kept static overnight in a cold chamber at 4 ° C.

Centrifugation

The suspension is centrifuged at 13,000 rpm with a Sorvall RC6 centrifuge and SLA 15000 rotor for 45 minutes at 4 ° C. The supernatant of the centrifugation is recovered, pre-filtered under vacuum on Dicalite and on regenerated cellulose filters of 0.45 µm and 0.22 µm.

Ultrafiltration 300'000 Da

The filtered product is ultrafiltered on a 300'000 Da polyethersulfone Millipore Pellicon Biomax tangential flow flat membrane at a Pi of 0.5 Ã · 1 bar, in a cold room at 4 ° C.

Ultrafiltration 5'000 Da

The 300'000 Da ultrafiltration permeate is concentrated on a 5'000 cross-flow Millipore Pellicon Biomax polyethersulfone flat membrane at a Pi of 0.5 Ã · 1 bar, in a cold room at 4 ° C. The retentate is transferred into a 1000 Da regenerated cellulose Spectrum Spectrapor dialysis tube and dialyzed against demineralised water (therefore also the preservatives are eliminated) and then immediately freeze-dried.

A further source of the factors of the invention is colostrum. Bovine colostrum is preferably used due to the ease of supply and the quantities available. Colostrum from Holstein (Friesian) and Guernsey cows is particularly preferred. Such cows have been proven to produce colostrum with the highest concentration of active factors. The cows are preferably in the second or third calving. The colostrum is preferably collected between the 1st and the 6th hour from the birth as, in this period, there is the greatest concentration of active ingredients. In colostrum from the sixth hour after birth the active factors decrease rapidly (after 24 hours only 15% are present).

Collected colostrum is tested for tuberculosis, cytotoxicity on cell cultures, control of mycoplasma, prions and human and bovine viruses.

The colostrum in the mammary cistern is practically sterile, but once milked, despite all precautions, due to the high concentration of growth factors, its bacterial load rises very rapidly during freezing and thawing, rather slow processes given the ™ high density of colostrum in the first few hours.

The use of γ rays allows to obtain a sterile colostrum only if radiations greater than 10 Kgy are used which however destroy most of the active factors, and on the other hand this method does not prevent the formation of pyrogens whose use is inadvisable topically and prohibited injecting.

An innovative collection chain was therefore developed to obtain a sterile hypoallergenic compound, without preservatives and without pyrogens.

To the colostrum collected in sterile tanks (vacuum sterilized at 25 Kgy) antiseptic agents are added in such quantities as to guarantee sterility and the absence of pyrogens. Phenoxyethanol at a concentration of 2.5% and diazolidinyl urea at a concentration of 1% are preferably used.

The colostrum thus treated can, for short periods (max 30 days) not be stored frozen before the extraction processes of the active factors, with evident savings in industrial costs.

Active factors can be extracted with the following steps:

Centrifugation

Bovine colostrum is diluted 1:10 with demineralized water, NaCl is added until a concentration of 0.9% is obtained.

The suspension is centrifuged continuously at 8'000 rpm at a temperature of 20-25 ° C with an Alfa Laval centrifuge, discarding the pellet corresponding to the lipid part.

300,000 Da ceramic ultrafiltration

The product obtained from centrifugation is ultrafiltered on a ceramic membrane with a cut-off of 300,000 Da at a temperature of 20-25 ° C, recovering the permeate.

Ultrafiltration on 5000 Da

The permeate of the ultrafiltration 300'000 Da is concentrated on a spiral membrane wrapped by 5'000 Da in polyethersulfone at a temperature of 20-25 ° C and dialyzed against demineralized water up to a conductivity of the retentate of about 600 µs / cm <2> (preservatives are also eliminated with this dialysis).

Lyophilization

The 5000 Da ultrafiltration retentate is vacuum filtered on 0.2 µm regenerated cellulose Millipore filters, frozen at -20 ° C and freeze-dried.

The compound, from any isolated source, was called P.M.F. Ab and contains the following factors:

Immunoglobulins of the IgG2 and IgA class (γ / mg), in a proportion equal to about 60% of the P.M.F. Ab (about 50% IgG2 and about 10% IgA), with a natural specificity against many bacteria and viruses, some of which are responsible for the infectious overlaps of anorexia.

COMPLEMENT C3 / C4: The complement consists of circulating proteins capable of interacting with biological membranes and with specific receptors located on the surface of different cell types and which have the ability to induce inflammatory reactions that help fight infections.

ANTIBACTERIAL / ANTIVIRAL FACTORS TRANSFERRIN: releases iron to red blood cells and prevents iron chelation by bacteria and viruses.

LACTOFERRIN: removes the iron they need to grow from bacteria and viruses.

LYSOZYME: has antibacterial effects, due not only to its enzymatic properties, but also to its cationic and hydrophobic properties.

LACTOPEROXIDASE: It is the most powerful enzyme contained in colostrum. It inhibits bacterial metabolism by oxidizing the sulfhydryl groups of essential proteins.

GROWTH FACTORS TGF-β1- TRASFORMIG GROWTH FACTOR: stimulates the production of class A immunoglobulins, responsible for mucosal immune defenses. It modulates cell proliferation and stimulates extracellular matrix deposition.

EGF-EPIDERMAL GROWTH FACTOR: regulates the development of the mucosa. Promotes the formation of epithelial cells.

IGF 1- INSULIN-LIKE GROWTH FACTOR: modulates cell proliferation, adhesion and migration and induces maturation of the mucous membranes.

VEGF- VASCULAR ENDOTHELIAL GROWTH FACTOR: stimulates the production of blood vessels. It has mitogenetic activity and activation of vascular permeability.

FGF- b- FIBROBLAST GROWTH FACTOR BASIC: stimulates the proliferation of cells of mesenchymal origin such as fibroblasts, endothelial cells, astrocytes, keratinocytes. It acts as a chemotactic and phytogenetic factor.

GH-GROWTH HORMONE: general growth factor of each tissue.

NGF-NERVE GROWTH FACTOR: stimulates the activity and regulation of the growth and differentiation of the sympathetic system.

SCF- STEM CELL FACTOR: pleiotropic factor that acts, in utero and in the newborn, on the development of germinal and neural cells and on hematopoiesis.

CHEMOTACTIC FACTORS EOTAXIN: binds to chemokine receptors to call up eosinophils in inflamed tissues.

IP-10-Chemochin ligand 10: induced by interferon gamma aggregates inflammatory cells.

MCP-1 Monocyte chemotactic factor-1: favors the aggregation of monocytes to inflamed tissues.

CYTOKIN IL1-ra: member of the interleukin 1 family. It modulates the immune and anti-inflammatory response.

IL-2: induces the proliferation of T lymphocytes.

IL-9: regulator of hematopoietic cells, stimulates cell proliferation and prevents apoptosis.

IL-17: Regulates the activities of NF-KB and enhances the production of nitric oxide (NO).

IL-10: has pleiotropic effects in immunoregulation and inflammation. It improves the survival of β cells and therefore the production of antibodies. Studies on knock-out mice show that this protein is essential in the immunoregulation of the intestinal tract.

INTERFERON-gamma: has known antiviral, anticancer and immunoregulatory activities. It is a powerful activator of macrophages.

TNF-Î ± -Tumor necrosis factor: stimulates the migration of neutrophils and monocytes to the site of infections.

The fraction thus obtained and containing the factors listed above is able to provide a valid therapeutic effect in all cases of anorexia. The fraction of the invention can be formulated in compositions suitable for oral administration for the treatment of anorexia. The oral dosage of P.M.F. Ab is typically between 20 and 40 g once or twice a day on an empty stomach.

THERAPEUTIC RESULTS

Twenty female patients aged between sixteen and thirty, suffering from anorexia nervosa with weight loss between 15 and 30% of that considered normal for their age and physical structure, were placed in adjuvant oral therapy with 40 g of P.M.F. Ab with daily intakes on an empty stomach.

It is interesting to note that all the patients, despite the serious feeding problems, have accepted to take the product as it has been shown to them that the product itself does not increase fat mass but only muscle mass, positively influencing vital phenomena. in the heart, lungs and brain.

After three months of adjuvant therapy, 50% had regained more than 30% of their body weight with a notable improvement in physical and intellectual abilities. Blood chemistry parameters had returned to normal.

Surprisingly, a further 25% of these patients even resumed normal nutrition with body mass restitutio ad integrum. The remaining 25% did not respond.

Claims (6)

CLAIMS 1. Compositions comprising immunoglobulins, complement, antibacterial / antiviral factors, growth factors, chemotactic factors and cytokines isolated from serum, placenta or colostrum for the therapy of anorexia. 2. Compositions according to claim 1 comprising Immunoglobulins of the class IgG2 and IgA, complement c3 / c4, Transferrin, Lactoferrin, Lysozyme, Lactoperoxidase, TGF-β1, EGF, IGF 1, VEGF, FGF- b-, GH, GHRF, NGF SCF , Eotaxin, IP-10-Chemochin ligand 10, MCP-1, IL1-ra, IL-2, IL-9, IL-17, IL-10, Interferon-gamma, TNF-Î ±. 3. Compositions according to claim 1 or 2 obtained from mammalian serum. 4. Compositions according to claim 1 or 2 obtained from placenta. 5. Compositions according to claim 1 or 2 obtained from colostrum. 6. Compositions according to one of claims 1 to 5 suitable for oral administration. Milan, 30 December 2011