WO2013039374A2

WO2013039374A2 - Method for extracting, testing and counting dialysed leukocyte extract from shark spleen in order to obtain an enhanced transfer factor, specifically designed to be used as a treatment against the disease known as vitiligo

Info

Publication number: WO2013039374A2
Application number: PCT/MX2012/000085
Authority: WO
Inventors: Héctor Manuel ZEPEDA LÓPEZ
Original assignee: Zepeda Lopez Hector Manuel
Priority date: 2011-09-15
Filing date: 2012-09-13
Publication date: 2013-03-21
Also published as: WO2013039374A3

Abstract

The invention relates to a leukocyte extract which contains polypeptides up to 10,000 daltons from shark spleen and to the use thereof as a drug for treating the disease known as vitiligo. Said extract is a dialyzable leukocyte extract, specifically from the spleen, which is part of the lymphatic system and is the main organ in the immune system of the Selachimorpha, a superorder of the Chondrichthyes, commonly known as sharks, for obtaining enhanced transfer factor.

Description

METHOD OF EXTRACTION, CHECKING AND COUNTING OF DIALIZED EXTRACT OF LEUCOCITS OF ORIGIN BANK OF SHARK, FOR THE OBTAINING OF POTENTIALIZED TRANSFER FACTOR, SPECIFICALLY DESIGNED FOR USE AS TREATMENT AGAINST DISEASE KNOWN

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a leukocyte extract containing polypeptides equal to or less than 10,000 daltons of shark spleen origin and its use as a medicament in the treatment of the disease known as vitiligo.

It is also the object of the present invention to provide a method of extracting and processing dialysable leukocyte extract from setacimorphs to obtain a potentialized transfer factor used for the treatment of the disease known as vitiligo.

BACKGROUND OF THE INVENTION

Vitiligo is a degenerative skin disease in which melanocytes (the cells responsible for skin pigmentation) die, thus stopping producing melanin (the substance that produces skin pigmentation) in the area where it has occurred cell death

In most cases it begins between 10 and 30 years and is manifested by the appearance of white spots that result from the absence of the pigment in the skin. In principle they are usually circular areas with defined edges and with a variable extension that is usually seen more frequently in the extremities (hands and feet), areas of extension and flexion (knees and elbows), the face or the genitals. Depigmented areas may grow and spread over time to any other part of the body. Vitiligo is not contagious, either by touch or by any other type of contact, since the processes that trigger it are inherent in the person. Its consequences are mild: it increases the susceptibility to sunburn in areas without pigmentation and mainly causes aesthetic problems.

The prevalence of this condition is between 0.5 and 3% of the population. There are no differences by sex or race.

The causes of this disease have not yet been fully elucidated and the mechanisms by which this alteration is unleashed are still being studied, although it seems that in 30-40% of cases there is a family history of this pathology that would be inherited by a multifactorial polygenic model with variable expression.

There are three theories that attempt to explain the etiopathogenic mechanism:

1. The autoimmune theory: Melanocytes are destroyed by certain activated lymphocytes. It would be similar to what happens in other better known autoimmune processes (such as some thyroiditis), and is confirmed by the response of some cases to treatments with immunosuppressive drugs. 2. Neurogenic theory: A possible interaction between melanocytes and nerve cells that would release a toxic neurochemical mediator is postulated. This would be the cause of the destruction of melanocytes.

3. The theory of self-destruction: According to this proposal, melanocytes would be destroyed by toxic substances formed in the metabolic processes of melanin biosynthesis (through certain metabolic pathways active only in some subjects).

Recently there have been studies that have shown that vitiligo is caused by the increase in the amount of adrenaline in the bloodstream that causes an overload in the functioning of the spleen, liver, kidney and pancreas, due to the excess of toxins (free radicals) that They are not able to eliminate. These toxins are accumulated in the liver and in all those organs responsible for their elimination, and this leads to melanocytes remaining undifferentiated in the basal layer as a result of the lack of blood flow, leading to melanocytes to lose their functions and remain intact in the basal area as undifferentiated cells (that is, without the function of creating melanin, which is the substance that gives color to the skin) and, added to this, the inability that keratinocytes acquire to retain the little melanin produced by melanocytes. As part of the accumulation of these residues in the liver, the symptoms that are usually present in these patients, such as headaches, abdominal pain, reluctance, depression, and itching in areas of lesions, hearing loss (all may be present) as only one of them). PROBLEM TO SOLVE:

At present there is no effective treatment against the destruction of melanocytes that occurs in vitiligo. Repigmentation has been attempted through the use of steroids or immunomodulators (topical and systemic) with poor results. Psoralens are also used, with quite limited efficacy, in combination with ultraviolet light sessions.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide an effective medical treatment that activates the production of melanocytes to normalize melanin levels in the body for the treatment of vitiligo.

It is also the object of the present invention, to provide a method for the processing of a dialyzed extract of leukocytes of shark spleen origin, for obtaining a potentialized transfer factor, specifically designed for use as a treatment against the disease known as vitiligo. In recent studies, no document was found to support the use of the potentialized transfer factor for the treatment of the disease known as vitiligo. The means for obtaining a dialysable leukocyte extract containing polypeptides less than or equal to 10,000 Daltons whose source or origin is from shark cells, tissues or organs, more specifically shark spleen, of the present invention, is considered novel. , since when analyzing the state of the art, methods were found for the extraction of leukocytes, white cells or transfer factor, by means of leukocyte packages from healthy (human) donors; reptile eggs, amphibians, fish and birds; in addition to colostrum (milk produced by mammals) and crocodile spleen. But no document was found that mentions the possibility of extracting leukocyte cells from selachimorphs, which are a superorder of condrict (cartilaginous fish) commonly known as sharks.

Likewise, no source was found in the state of the art to provide a transfer factor concentration of about 10 ¹² leukocytes x mm ³ . Since in the state of the art, the transfer factor concentrations of the extraction means described above, ranges from 10 ⁴ to 10 ⁸ leukocytes x mm ³ . BRIEF DESCRIPTION OF THE OBJECT OF STUDY

The potentialized transfer factor of the present invention is specifically obtained for the treatment of the symptomatology of the disease known as vitiligo, since it has the ability to reactivate the cells, specifically the individual's immune system, which gives the body the ability to reactivate the cells responsible for skin pigmentation, that is to say melanocytes, and therefore balance the production of melanin in the skin through such activation mobile.

In studies conducted on patients with vitiligo disease, it was found that when consuming the potentialized transfer factor, about 80% resulted in significant improvements, first stopping the progress of skin depigmentation in the first months, therefore, begin to regenerate depigmented parts around month 6.

The Potentialized Transfer Factor specifically designed for the medical treatment of the disease known as vitiligo of the present invention, helps neurotransmitters restore chemical signals that travel from the brain to the bone marrow, so that, as is known in the state of the art of the transfer factor, this is coupled to the chemical signals of the neui'otransmors so as to potentiate the signal, which manages to establish the correct chemical signaling to the melanocytes and thus "order" that they begin to produce melanin. In this way, the melanocytes are forced to restore the correct levels of melanin production in order to achieve pigmentation of the depigmented areas by the low amount of melanin and therefore treat a disease known as vitiligo. This means that by continuous administration of the transfer factor of the present invention, the typical symptomatology of the disease known as vitiligo can be eliminated, since by helping the chemical signals travel from the brain to the bone marrow, the white blood cell production, which are the raw material for the production of melanocytes and these in turn are responsible for producing melanin, which is the latter deficiency found in the skin of patients with the disease known as vitiligo. It is well known for an expert in the state of the art, that the transfer factor has as its main function to help the production and proper restoration of white cells and their excitation, in order to achieve the optimal functioning of the immune system. Likewise, it is therefore understandable that the potentialized transfer fac.ci of the present invention, helps the treatment of the known disease with the name of vitiligo, since it gives the possibility of alleviating the characteristic symptomatology thereof, by means of cell excitation immune system of the individual and correct operation of ios neurotransmitters, which are responsible chemical signals of various body functions, including prod ^j ction ae lancos globules c leukocytes which in turn result among other things in melanocytes and in turn melanin.

The Transfer Factor Fo.er.cia; raised for the treatment of the disease known as vitiligo of the present invention, consists of a potency of 10 ¹² leukocytes x mm ³ (understood as potency to the quantity of leukocytes and quality of the smooth, round and innocuous cells)), quantity necessary for leukocyte excitation and optimization of chemical signals for the production of white blood cells , which in turn produce melanocytes and the latter melanin. It should be noted that with a concentration of transfer factor lower than that established in the present invention, the treatment of the symptomatology of the disease known as Vitiligo would not be possible, since it would not have sufficient power to achieve the production of chemical signals acting on leukocytes that in turn induce the production of melanueiíos and the latter melanin.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the type of cytosines induced from the inoculation of the eucocyte extract in Balb-c mice.

DETAILED DESCRIPTION OF THE INVENTION

Method of extraction and processing of leukocyte extract specifically designed or ^r s treating the condition known as vitiligo

The process of the present invention for obtaining a dialysable extract of leucine that has polypeptides less than or equal to 10,000 Daltons whose source or origin is from cells, tissues or organs of semipermeable membrane. Then, the leukocyte extract, after the separation and rupture of components has been carried out, is placed in a semipermeable dialysis bag, as for example, in a cellulose membrane with pores, and the bag is sealed. The sealed dialysis bag is placed in a container with a different solution, or pure water. The leukocyte extract, being small enough to pass through the pores, tends to move in or out of the dialysis bag in the direction of the lowest concentration. Larger molecules (often proteins, DNA, or polysaccharides) that have dimensions significantly larger than the pore diameter are retained within the dialysis bag. In this way, leukocyte extracts less than or equal to 10,000 Daltons are separated.

Filtration and sterilization.- After dialysis, the leukocyte extract is filtered through a pore size membrane between 2 and 4 micrometers. Also, the solution is sterilized again.

Formulation.- A lyophilization process is carried out to remove the water from the leukocyte extract through the generation of a vacuum, likewise in this step the aggregation of a vehicle is carried out, such as milk, water, gel or artificial flavoring, to give a presentation and pleasant taste to the product.

Physicochemical Evaluation At this point the physical-chemical processes such as density, PH, color, smell and taste are evaluated. Fits mention that if no vehicle was added to the product, the transfer factor obtained will be colorless, colorless and insaboro.

Evaluation of biological activity. The cytosines required for the treatment of the disease known as vitiligo are induced, this from the dilution of the leukocyte extract until reaching the title of the cytosines required for the production of interleukins that activate the melanocytes and therefore, level the production of melanin in the subject, thus achieving pigmentation of depigmented areas due to the lack or lack of melanin. The leukocyte extract with mtcroarray membranes is also analyzed to determine the type of cytokine found in the leukocyte extract.

The result of the above process is a potentialized transfer factor specifically designed for the treatment of the disease known as vitiligo by administering the appropriate cytosine (s) for the activation of melanocytes that will produce melanin and thus repigment areas depigmented by the lack of melanin The transfer factor of the present invention is in powder form, which gives it the virtue of being easily transported and stored, does not require refrigeration and a transfer factor power of 10 ¹² leukocytes x mm ^{3 is obtained} , which It is highly superior to any known transfer factor, understood as leukocyte potency concentration per mm ³ and cell quality (smooth, round and innocuous). Figure 1 illustrates the type of cytosines induced from inoculation of the leukocyte extract in Balb-c mice. Where groups of 8 mice are used, which will be inoculated with the equivalent amount to the weight-unit ratio of transfer factor (0.005 unit of transfer factor).

Whey is extracted from each mouse, 50 microliters of serum are used, exposed against the microarray membranes that contain the cytosine receptor antibodies and the wells that develop color will be induced cytosines. The serum is diluted with a regulatory solution in multiples of 2 initially and then diluted in multiples of 100. The baseline dilution of time 0 and the dilution that preserves the color development in the microarrays prior to dilution where it is no longer present is eliminated. Color development is the title of the leukocyte extract.

Claims

1. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose source is a specific area of the spleen that is part of the lymphatic system and is the center of selacimorph immune system activity, commonly known like sharks; which is rich in B lymphocytes that produce chemical signals that interact with leukocytes produce interleukins that activate melanocytes. To obtain the potentialized transfer factor specifically designed for the treatment of the disease known as vitiligo.

2. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose specific source is an area of the spleen, rich in B lymphocytes, which is part of the lymphatic system and is the center of activity of the immune system of the selacimorphs, which are the superorder of the condrict, commonly known as sharks, to obtain a potentialized transfer factor specifically designed for the treatment of the disease known as vitiligo, which according to claim 1, is characterized in that its method of extraction, checking and counting consisting of the following steps: a) Sterilization.- Involves that any instrument used for the extraction of the leukocyte extract is sterile.

b) Spleen Extraction.- This step consists in surgically extracting from the shark spleen, a specific area rich in B lymphocytes (which has the particularity that when administered it produces chemical signals, which when interacting with the leukocytes of the subject, occur the interleukins that activate the melanocytes for the production of melanin), in order to extract the leukocyte extract.

c) Counting and qualification.- The number of leukocytes per field in the Neubauer grid is counted through the Neubauer Chamber and microscope, to know the power of the transfer factor to be obtained, that is, the number of leukocytes per cubic millimeter . Likewise, it is necessary to assess the quality of said cells, that there is no anisocytosis, that is, by microscopy it is observed that the cells are round and smooth.

d) Breaking or separation of components.- Leukocyte cells must be separated from proteins, lipids, carbohydrates and toxins. e) Dialysis.- Dialysis is the process of separating the molecules in a solution by the difference in their diffusion rates through a semipermeable membrane. Then, the leukocyte extract, after the separation and breakdown of components has been performed, is placed in a semipermeable dialysis bag, such as a cellulose membrane with pores, and the bag is sealed. The sealed dialysis bag is placed in a container with a different solution, or pure water. The leukocyte extract, being small enough to pass through the pores, tends to move in or out of the dialysis bag in the direction of the lowest concentration. Larger molecules (often proteins, DNA, or polysaccharides) that have dimensions significantly larger than the pore diameter are retained within the dialysis bag. In this way, the leucocyte extracts less than or equal to 10,000 Daltons are separated.

f) Filtration and sterilization.- After dialysis the leukocyte extract is filtered through a pore size membrane between 2 and 4 micrometers. Also, the solution is sterilized again.

g) Formulation.- A lyophilization process is carried out to remove the water from the leukocyte extract through the generation of a vacuum, likewise in this step the aggregation of a vehicle is carried out, such as milk, water, gel or flavoring artificial, to give a presentation and pleasant taste to the product.

h) Physicochemical Evaluation. At this point the physical-chemical processes such as density, PH, color, smell and taste are evaluated. It should be mentioned that if no vehicle was added to the product, the transfer factor obtained will be colorless, colorless and insaboro.

i) Evaluation of biological activity. The cytosines required for the treatment of the disease known as vitiligo are induced, this from the dilution of the leukocyte extract until it reaches to the title of the cytosines required for the production of interleukins that activate the melanocytes and therefore, level the production of melanin in the subject, thus achieving pigmentation of the depigmented areas due to the lack or lack of melanin. The leukocyte extract with microarray membranes is also analyzed to determine the type of cytokine found in the leukocyte extract.

3. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose specific source is an area of the spleen, rich in B lymphocytes, which is part of the lymphatic system and is the center of activity of the immune system of the selacimorphs, which are the superorder of the condrict, commonly known as sharks, for obtaining transfer factor, which according to claim 1, is characterized in that the product obtained is a potentialized transfer factor, specifically designed to the treatment of the disease known as vitiligo, as it consists of induced cytosines suitable for the activation of melanocytes that will produce melanin to repigment areas depigmented by melanin deficiency.

4. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose specific source is an area of the spleen, rich in B lymphocytes, which is part of the lymphatic system and is the center of activity of the immune system of selacimorphs, which are the superorder of the condrict, commonly known as sharks, for obtaining transfer factor, which according to claim 1, is characterized in that the product obtained is a potentialized transfer factor, specifically designed for the treatment of the disease known as vitiligo, in powder form, which can be easily transported and stored, and does not require refrigeration.

5. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose specific source is an area of the spleen, rich in B lymphocytes, which is part of the lymphatic system and is the center of activity of the immune system of the selacimorphs, which are the superorder of the condrict, commonly known as sharks, to obtain a potentialized transfer factor specifically designed for the treatment of the disease known as vitiligo, which according to claim 1, is characterized in that it it obtains a transfer factor power of 10 ¹² leukocytes x mm ³ , meaning leukocyte concentration per mm ³ and the quality of the cells (smooth, round and knockouts).

6. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose specific source is an area of the spleen, rich in B lymphocytes, which is part of the lymphatic system and is the center of activity of the immune system of selacimorphs, which are the superorder of the condrict, commonly known as sharks, to obtain potentialized transfer factor specifically designed for the treatment of the disease known as vitiligo, which according to claim 1, characterized in that the method of checking the potency of the leukocyte extract from the inoculation of the leukocyte extract in Balb-c mice, performed in the step of checking the biological activity, consists of: they use groups of 8 mice, which will be used at each time of the kinetics, will be inoculated with the amount equivalent to the weight-unit ratio of transfer factor (0.005 unit of transfer factor), the mice are maintained at certain times , the time 0 being the basal level of induced cytosines of the mice, which will be eliminated with the int In order to know the type, title and permanence of the induced cytosines. Whey is extracted from each mouse in its time according to the kinetics and 50 microliters of serum are used, exposed against the microarray membranes that contain the cytosine receptor antibodies and the wells that develop color will be induced cytosines. The serum is diluted with a regulatory solution in multiples of 2 initially and then diluted in multiples of 100. The baseline dilution of time 0 and the dilution that preserves the color development in the microarrays prior to dilution where it is no longer present is eliminated. Color development is the title of the leukocyte extract. The group of mice that retain the maximum titer with the longest induction time will be the residence time of the cytosine induction.

7. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose specific source is an area of the spleen, rich in B lymphocytes, which is part of the lymphatic system and is the center of activity of the immune system of the selacimorphs, which are the superorder of the condrict, commonly known as sharks, for obtaining a potentialized transfer factor specifically designed for the treatment of the disease known as vitiligo, which according to claim 6, is characterized in that a Higher title and induction time found, higher transfer factor power.

8. Dialyzable leukocyte extract from leukocyte cells containing polypeptides equal to or less than 10,000 daltons, whose specific source is an area of the spleen, rich in B lymphocytes, which is part of the lymphatic system and is the center of system activity immune system of selacimorphs, which are the superorder of the condrict, commonly known as sharks, to obtain a potentialized transfer factor specifically designed for the treatment of the disease known as vitiligo, which according to any of the preceding claims, is It characterizes because it promotes cell excitation and optimization of chemical signals, to regulate the production of melanocytes and in turn melanin, within the body of the individual who consumed it.