CELLULAR GROWTH REGULATOR FIELD OF THE INVENTION The present invention is generally in the field of human and veterinary medicine and is related to novel substances, which effect the growth and proliferation of cells. The present invention also relates to the uses of the substances in the prevention or therapy of diseases. The invention also relates to a method for diagnosing diseases or protecting individuals who are prone to develop a disease or who have a predisposition to develop a disease. GLOSSARY In order to outline the present description, the use will be made with several terms created.
Some of these terms and the manner in which they will be understood in the written context of the present are the following: "Cell Growth Regulator (CR)" - an agent that effects the growth or proliferation of cells. The effect of CR on growth or proliferation can be either to promote the growth or proliferation of cells or to inhibit the growth or proliferation of cells. "Endogenous RC (RCE)" - an agent secreted or discharged by a cell within the body, which effects the growth or proliferation of the same cell or other cells within the body. "RC / Cytostatic RCE". an RC / RCE that has an effect to stop or reduce considerably the growth or proliferation of the cells, this is substantially without any cellular destruction. Cytostatic RCEs typically act by preventing the cell cycle in one of the phases of the cell cycle. "RCE of Low Molecular Weight (RCE-PMB) - an RCE that has a molecular weight below about 3,000 Daltons." Muscular Factor (FM) "an RCE-PMB secreted by white blood cells or discharged by them." White Globules (FGB) "an RCE-PMB secreted by the white blood cells or discharged from them." Source Cell "- cell that secretes or discharges the RCE." Target Cell "- cell that is submitted by the RCE." Conditioned Medium (MC) "- a medium conditioned by the growth in it of origin cells.A MC thus comprises the RCE secreted by the qrigen cells." MC of Muscular Cells "and MC of White Globules" - a medium conditioned by the growth in the same of muscle cells and white blood cells, respectively. The MC of the muscle cells and the MC of the White Globules respectively comprise an FM and the FGB. The above terms will be constructed and understood with reference also to what is described below. BACKGROUND OF THE INVENTION There is a body of heavy researchers involved in the discovery, characterization, biological tests and clinical development of the RCEs known as "cytokines". All the cytokines discovered to date are protein substances that have a molecular weight that varies from several thousand Daltons to several tens of thousands of Daltons, although cytokines have different origin cells and target cells and have different mode of activities, they share a common denominator since they are all protein substances. It has been reported that a significant physical exercise inhibits the growth and progression of tumors in experimental animals (SA Hoff et al., 1962, Cancer Res. 22: 597-599, VE Baracos, 1989, Chem. J. Physiol Pharmacol., 67: 864-870). A. Szent-Gyorgy, et al., (1963, Science, 140: 1391-1392) reported that extracted from various tissues including the thymus, aorta, muscles and tendons that contain two substances, one promotes the growth of ascites tumors in mice (called by them as "Promina") and the other inhibits growth (called by them as "Retina"). The retina was described as a substance with a small molecular weight, relatively unstable when it decomposes at room temperature, in about a week. In addition, based on its mode of separation, the substance appears as lipophilic. The inhibition of ascitic tumor cells by cellular extracts of muscles has also been described by T. Namba et al.(1968, British J. of Exp. Pathol, 49: 294-301) and the inhibitory activity found in the muscle extract was dialysable through a silicon membrane. The activity was affected by heating the extract, although no effect was found on the material undergoing dialysis (dialysate). E. Watta et al., (U.S. Patent 4,708,948) discloses a high molecular weight polypeptide, which inhibits the growth of tumors obtainable also from muscle tissue. M. Djaldetti et al. (U.S. Patent 5,242,692) discloses a factor derived from muscle cells that inhibits the proliferation of tumor cells. This factor, which was separated from the cream of a muscle cell culture, was found to have an apparent molecular weight, determined by gel electrophoresis in the range of 25,000-30,000 Daltons. The present invention is based on the discovery of novel substances secreted, discharged or produced by any of the muscle cells or white blood cells that are biologically active to inhibit the proliferation of tumor cells, substantially without affecting the proliferation of normal cells without tumor. In addition, it was also found that these substances are effective in inhibiting the proliferation of stimulated immune cells. Accordingly, the present invention provides, by one of its aspects, a substantially purified cell growth regulator, which is any of: (a) an RCE-PMB, which is an agent having the following characteristics: i. produced, secreted or discharged by cells, particularly muscle cells or white blood cells, ii. it has a molecular weight of approximately less than 3,000 Daltons, iii. it is not protein, iv. is soluble in water, v. it is stable to heat, and vi. is a biological active agent that inhibits the proliferation of cells, particularly inhibits the proliferation of cells with tumors or proliferation stimulated lymphocytes; or (b) an agent that is a derivative of the low (a) agent and that is biologically active to inhibit cell proliferation. The present invention, through another of its aspects, provides the use of said substance in the prevention or therapy of diseases. According to this aspect, there is provided a method for preventing a disease or disorder comprising administering to a subject in need thereof an effective amount of said CR. According to the mode of activity of the RC to inhibit proliferation, it will typically be administered to the subject periodically over a period of time. In addition, according to this aspect, a composition comprising an amount of said RC is provided. The composition can be a pharmaceutical composition comprising a therapeutically effective amount of said CR, together with a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition can be formulated so as to be useful for the prevention of a disease or disorder or formulated in order to be useful for the therapy of a disease or disorder. The composition can be a composition without prescription, etc. Finally, the use of said RC, according to this aspect, is also provided for the preparation of such compositions. Particularly preferred is the use of said CR for the treatment or prevention of cancer, or to inhibit (eliminate or reduce) the activity of stimulated lymphocytes within the system of treatment or prevention of a variety of conditions resulting from an overactive immune system, for example, proposed treatment to combat organic rejection, treatment of autoimmune diseases, etc. According to a further aspect of the invention there is provided a method for diagnosing cancer or a cancerous state of an individual, or for the protection of individuals who are prone or have a predisposition to develop cancer, comprising determining the level of said ROSC. -PMB in a body fluid obtained from said individual, or in the cream of a cell culture obtained from said individual. A further aspect of the present invention is a process for the preparation of the RC of the invention based on a purification of active fractions of an appropriate conditioned medium. DESCRIPTION OF THE INVENTION The present invention is based on the discovery of novel RCEs, which have a low molecular weight (MBP). The term "low molecular weight" will be understood to be a molecular weight, determined by ultrafiltration, that is approximately less than 3,000 Daltons, particularly about less than 2,000 Daltons, and preferably about less than 500 Daltons. It is clear to the experienced person that these molecular weights are approximations and can not be considered as exact quantities. RCE-PMBs of the invention were found to be non-proteinaceous, ie they are not proteins, neither peptides nor any other substance having a protein or a portion of peptide that plays a role in their biological activity. (The findings of the present invention can not rule out the possibility that the RCE-PMB may exist in a form in which it is bound or complexed with a portion of protein or a peptide, that does not play any role or that has only a limited role in the activity of the RCE-PMB as a growth regulator). In accordance with the invention, the RCE-PMBs were obtained up to the present time from CM of muscle cells and MC of white blood cells. However, it is considered that the RCE-PMB according to the invention can also be obtained from other sources. The present invention is not limited to FM and FGB. But on the contrary, it is equipped with the knowledge obtained by the findings according to the invention by employing standard skills and knowledge available to it, the experienced person will have no difficulty in finding other RCE-PMBs, which fall within the scope of the present invention.
It was found that FM and FGB are cytostatic RCEs specific for tumors. They have a unique biological activity since they specifically inhibit the growth and proliferation of tumor cells without a perceptible effect on normal cells without tumor. In addition, it was found that both FM and FGB are for non-specific tumors (ie, they are effective for inhibiting the growth and proliferation of a variety of cells with tumor) and for non-specific species (showing activity to inhibit growth). and the proliferation of tumor cells in a variety of animal species). In other words, FM and FGB have a broad spectrum of activity to inhibit the growth and proliferation of cancer cells. Furthermore, the findings according to the invention also mean that an FM or FGB derived from an animal species, particularly in mammals, can also be used in cancer treatment of an animal of other species, particularly mammals. It will be noted that although FM and FGB were found to be cytostatic agents, it is possible that they may have some destructive effect on the cells, particularly after prolonged exposure. For example, after prolonged exposure to FM or FGB, as well as to its derivatives, cells with target tumor may eventually die, for example, as a result of apoptosis. In addition to its activity to inhibit the growth and proliferation of cells with tumor, it was found that FM and FGB are also active to inhibit the proliferation of lymphocytes as evidenced by the inhibition of the response of lymphocytes to a mitogen and a mixed reaction of lymphocytes (RML), which means that the RC of the invention can have an immunosuppressive activity. It is clear that once the RCE-PMB of a species is isolated, it is possible to find the homologue of the RCE-PMB of another species. For example, until now the FM obtained according to the invention was of rats and of human origin. There is no doubt that FM homologs of other species, particularly mammals, can also be obtained. Similarly, the FGB obtained to date according to the invention is of human origin. There is no doubt that the FGB homolog of other species, particularly mammals, can also be obtained. The present invention also comprises said homologs. The RC of the invention may also be a single molecule, a group of molecules that operate together in an additive or synergistic manner to affect the growth and proliferation of cells, or a molecular complex having such activity. Once isolated, it is possible to prepare derivatives, for example by chemical modification of the RCE-PMB, which will have a biological activity that is similar to that of the RCE-PMB. Derivatives having a biological activity similar to the RCE-PMB or the homologues of such RCE-PMB can be identified, for example, by employing the same biological tests used to characterize the RCE-PMB. For example, in the case of FM and FGB, which are active to inhibit the growth or proliferation of tumor cells, derivatives and homologs can be found by testing the synthetic derivatives or the fractionated MC of other species, as the case may be. , for the activity of inhibiting the growth or proliferation of the in vitro growth of cells with tumor or for the ability to inhibit the RLM. The person with experience will not hesitate to be able to select the appropriate biological test in each case. The derivative can be a molecule having a molecular structure similar to said RCE-PMB, in which one or more chemical groups have been replaced by another; a reduction or oxidation product of said RCE-PMB; etc. The RC of the invention can be used for a variety of therapeutic purposes, where it will be administered therapeutically in an effective amount to a subject in need thereof. A preferred therapeutic indication for which said RC can be used, is in the treatment or prevention of cancer. For treatment, CR can be administered to individuals who have a history of cancer, such as chemotherapy, radiation therapy or surgery. For prevention, said RC can be administered to cancer-free individuals or individuals prior to the diagnosis of any carcinogenic condition, particularly to high-risk individuals, who are prone or have a predisposition to develop cancer. Individuals at high risk may be such that they have a genetic predisposition to develop cancer, for example, individuals diagnosed to have a variety of known genes that are associated with cancer, individuals with a family history of cancer; individuals who are at high risk of developing cancer as a result of exposure to a variety of environmental factors, such as irradiation, exposure to carcinogens, etc .; etc. According to the biological activity of the RC, which inhibits the proliferation of the target cells, instead of destroying the target cells immediately, the RC will typically be administered periodically over a period of time. However, as indicated above, it is possible that a prolonged interruption of its growth continues, the cells with tumor eventually die, so that it will be possible to end the treatment after a certain period of time. Another preferred therapeutic indication of said RC is to inhibit the activity of the components of the immune system. Examples are the treatment of autoimmune diseases; the use within the system of transplant therapy; that is, the treatment that follows after the intended transplant to avoid the rejection of organs and tissues; etc. The RCs of the invention were tested in a variety of animal models, including animal models for primary tumors in which the tumor is induced by subcutaneous, intramuscular or intraperitoneal inoculation of cells with tumor, as well as animal models for metastasis, in the that the tumor is induced by an intravenous inoculation of the cells with tumor. It was found that CR is effective to inhibit the development of tumors both by parenteral administration and by oral administration. As is known, oral administration is physiologically much more tolerable than parenteral administration and therefore for the treatment or prevention of cancer, particularly for treatment or prevention regimens comprising the administration of RC periodically for a prolonged period of time, prefers the route of oral administration. The RC can be formulated into a pharmaceutical composition, which will comprise an effective amount of RC together with a physiologically acceptable carrier that is compatible with said RC. It was found that said RC is soluble in water and therefore, a pharmaceutically acceptable carrier can be a saline solution, for parenteral administration or it can be an edible aqueous liquid for oral administration. In addition, for oral administration, CR can be formulated in a variety of dosage forms, such as capsules, tablets, etc. In addition, the RC can also be lyophilized to be mixed with the carrier or diluent before use. The term "effective amount" as used herein will be understood as a sufficient amount to achieve a desired effect. For example, in the case of a cancer therapy, an effective amount is an amount of said CR in a given therapeutic regimen, which is sufficient to inhibit the growth and proliferation of cells with tumor, evidenced, for example, by the reduction in the proportion of mortality related to cancer. In the case of cancer prevention, an effective amount is an amount of said CR, in a regimen of preventive administration, which is sufficient to inhibit the occurrence of a primary growth of cancer. In addition to the formulation of the RC in a pharmaceutical composition, the RC of the invention can also be formulated in other types of compositions, for example composition of food additives, neutraceutical compositions, non-prescription health products, etc. Experiments carried out according to the invention have shown that the white blood cells of cancer patients secrete much lower amounts of the RCE-PMB than what the white blood cells of healthy individuals normally secrete. So when determining the level of the CER. PMB in a body fluid (for example, serum, urine, etc.), or in a cream of a cell culture, for example muscle cells or white blood cells of an individual, it is possible to diagnose cancer in individuals as well as to obtain an indication of a cancerous state of the individual. In addition, the determination of the level of the RCE-PMB in a body fluid or in such cream, can be a basis to protect the individual, who is prone or has a predisposition to develop cancer. The determination of the level of RCE-PMB can be carried out by means of a biological test which comprises testing the activity of the body fluid or cream, or the activity of an appropriate fraction thereof, to inhibit the proliferation of cells with tumor. . In addition to such a biological assay, the presence of the RCE-PMB can also be determined by a number of analytical methods, generally known but for a person with experience, including a variety of immunological assays based on the use of specific antibodies for the RCE-PMB; tests based on the use of appropriate chemicals, for example colored reagents; spectroscopic tests or assays based on the absorption of the irradiation, for example light absorption, a variety of chromatographic techniques; etc. The present invention provides, by other aspects, a process for the purification of the RC of the invention from the biological source. The process according to this aspect comprises: (a) developing cells under conditions in which the growth regulator of the cells is produced, secreted or discharged by the cells, into their surrounding environment; (b) collecting the cream from the cell culture; (c) separating between a fraction of the cream comprising the substances of a molecular weight higher than about 3,000 Daltons, and fractions of the cream comprising substances with a molecular weight below about 3,000 Daltons, and selecting the latter. The fractions selected in step (c) can be further purified by a variety of purification techniques, specifically chromatography, for example, chromatography, with a high pressure liquid (HPLC). Next, the invention will be illustrated by some experiments carried out according to the invention, with occasional reference to the attached drawings. In these experiments, in vitro activity and in vivo activity of FM and FGB are demonstrated. There is no doubt that the skilled person will appreciate that this illustration will not be constructed to limit the scope of the invention, but exemplifies the full scope of the invention as defined in the appended claims. The experienced person will have no doubt that he is capable, based on the foregoing and the following description of carrying out the invention in its full claimed scope. BRIEF DESCRIPTION OF THE DRAWINGS In the drawings: Figures 1-9 show the effect of FM and FGB contained in the filtering of an MC of muscle cells or MC of white blood cells, filtered through a membrane that has a molecular cutoff of 3,000 Daltons (such filtrate will be referred to herein as respectively an ultrafiltrate of the FM of 3,000 Daltons and an ultrafiltrate of the FGB of 3,000 Daltons.) In the Figures, the cells had a growth of 96 microwell plates and were incubated with a filtrate of FM of 3,000 Daltons, in several dilutions ("1" -not diluted, "2" -two linked dilutions, etc.) In all experiments an unconditioned medium served as the control In Figures 1-4 and 6, 8 and 9, partially in Figure 7, cell perforation was measured by incorporation of 3H-thymidine and ordering showed a count of radioactivity In Figure 5 and partially in Figure 7, proliferation was measured by cell counting and the ordering showed the number of cells. Figure 1 shows the effect of a 2000 Daltons FM ultrafiltrate obtained from a primary culture of striated muscle cells from a newborn rat, in the proliferation of two cell lines with tumor: B16, which is a cell line of murine melanoma (Figure IA); HTB-38, which is a human adenocarcinoma cell line (Figure IB). In this figure, the FM ultrafiltrate activity of 3,000 Daltons was compared with that of the crude conditioned medium ("crude MC"). Figure 2 shows the effect of a 3,000 Dalton FM ultrafiltrate derived from a primary culture of newborn rat striated muscle cells, in two types of normal cells and without tumors: fibroblasts or rat fibrocytes (Figure 2A); bone marrow cells from murine (Figure 2B). Figure 3 shows the effect of a 3,000 Daltons FM ultrafiltrate derived from a rat striated muscle cell line -L-8, in the proliferation of two cell lines with tumor: B16 (Figure 3A); MCA-105 sarcoma line induced by murine lung methylcholanthrene (Figure 3B).
Figure 4 shows the effect of an ultrafiltrate of 3,000 Daltons FM derived from L-8 cells, on two normal types of cells: bone marrow cells of the murine and primary culture of fibrocytes or rat fibroblasts. Figure 5 shows the effect of a 3,000 Daltons FM ultrafiltrate derived from a primary culture of striated muscle cells from a newborn rat in the proliferation of cells from the rat lymphoma line NB2-11C, in a cell growth based on cell count. In this assay the cells were synchronized in the GVG1 phase, the crude CM of the 3,000 Daltons ultrafiltrate EM or a control medium was added and then the development was stimulated by the addition of hGH. Figure 6 shows the effect of an FM ultrafiltrate of 3,000 Daltons derived from L-8 cells, in the proliferation of several, tumor cell lines. The result in each case is a percentage of the control in the respective experiment. Figure 7 shows the effect of an ultrafiltrate of 3,000 Daltons FM derived from human myoblasts. The proliferation of B-16 and K562 cells was determined by the incorporation of 3H-thymidine; the proliferation of NBT cells was determined by means of a cell count. Figure 8 shows the effect of a 3,000 Daltons FGB ultrafiltrate derived from human lymphocytes on the proliferation of human and murine tumor cells. Proliferation was shown as a percentage of control. Figure 9 shows the effect of an ultrafiltrate of 3,000 Daltons FM derived L-8 cells in the response of lymphocytes to PHA and in a mixed reaction of lymphocytes (RML). Figure 10 is a representation showing the exposed peritoneum of two representative mice, in which a tumor was induced by an intra-peritoneal injection of 2xl05 MCA-105 cells. After injection, the mouse on the right was treated twice daily with an intra-peritoneal injection of 0.5 ml of a fraction containing FM eluted by a preparative reverse phase C18 high pressure liquid chromatography (HPLC) column. (RP) (this fraction is referred to as "FM-SP" in the following text (see 7.62)); the mouse on the left is injected with a control RPMI medium. Figure 11 is a representation showing the exposed peritoneum of two representative mice in which a tumor has been induced by intraperitoneal injection of cells with B16 melanoma of 5xl05. The representation on the right is a mouse treated once per day with an intra-peritoneal injection of an FM ultrafiltrate of 3,000 Daltons of 1 ml (in PBS) derived from the L-8 cells; the representation on the left is of a mouse treated once a day with a PBS control medium injected intra peritoneally. Figure 12 is a representation showing the exposed peritoneum of a representative mouse injected intraperitonally with MCA-105 cells of 2.5xlOs. The mice shown in Figure 12A are from a group of mice that were treated daily (beginning on the day the tumor was inoculated) per ounce (po) per 1 ml of a 3,000 Daltons ultrafiltered FM rS r derived from the cells
L-8. The mice shown in Figure 12B are from a control group of mice treated p.o. with a control RPMI medium. Figure 13 is a representation showing the lungs separated from the mice that were injected
intravenously with 5 × 10 5 B16 melanoma cells The lungs in the upper row were from a group of control animals administered daily by p.o. with 1 ml of a control PBS solution. The lungs in the lower row of the experimental group, which consisted of
animals treated by p.o. of 1 ml of a 3,000 Daltons FM filtrate obtained from the L-8 cells. Figure 14 shows the effect of an ultrafiltrate of 3,000 Daltons of a cream of white blood cells of patients with cancer and blood of healthy individuals,
to inhibit the proliferation of three cell lines with tumor (B-16, AK and K-562). Figure 14a shows the proliferation of three cell lines, as compared to the control ones, after being exposed to 3,000 Daltons ultrafiltrate. and Figure 14b shows the disseminated results given as percentages of inhibition (inhibition that is reciprocal to proliferation- 100% proliferation that is 0% inhibition, etc. (results greater than 100% proliferation also provided "0%" markers. Figure 15 shows an elution profile of 220 nm of a solution containing FM, which was again chromatographed through an HPLC-RP column (chromatography was carried out again in the same way as The same column is used as the first chromatography.) Figures 16-21 show the activity of several fractions eluted by several HPLC columns that inhibit the proliferation of Nb2 cells, determined by the cell count, the abscissa in each of these The photos showed the tube number (with respect to the flow rate and the volume of each tube, see below in the text.) The ordering showed the relative number of cells in comparison n with a preform (growth of the cells without addition of any solution to develop in the middle of Nb2 cells, which is 100%). Figures 16 and 17 show the activity of various fractions eluted in two different runs, from a preparative RP-HPLC column (C-18) to inhibit NB2 cell proliferation. The solutions that were fed into the columns were either a 3,000 Daltons FM ultrafiltrate 5 derived from L-8 cells (in a PBS solution) or a control PBS solution. In the Figure
16, the solution containing FM - empty circles (or); the control PBS solution - full circles (•). In the Figure
*. 17, the solution containing FM full triangles (V); the control PBS solution - full circles (•). Figure 18 shows the activity of several fractions eluted from the analytical RP-HPLC column (C-18) to inhibit the proliferation of NB2 cells. The solution that was fed to this column consisted of a combination of
fractions 5 and 6 of Figure 16. The solution of FM - full circles (•); control-empty circles (or). Figure 19 shows the activity of several fractions eluted from a superdex column to inhibit the proliferation of Nb2 cells. The solution that was fed to the column was a combined solution of the elution product of Figure 17 which consisted of 250 ml of tubes 19-10, 520 ml of tubes 10-11 and 600 ml of fractions 11-12 . Figure 20 shows the activity of several r ~ • s. eluted fractions from an analytical RP-HPLC column to inhibit the proliferation of Nb2 cells. The solution fed to the column was a combination of tubes 6-12 of Figure 17. The FM solution - Full squares (D) 5 control, PBS solution - empty circles (o). Figure 21 shows the activity of several fractions eluted from a size exclusion (SE) column to inhibit the proliferation of Nb2 cells. The solution r that was fed into the column was different from the fractions 10 eluted from the preparative HPLC-RP column shown in Figure 17: Fraction "A", Tubes 6-8, empty circles (o); Fraction "B", Tubes 8-10 - full circles (•); Fraction "C", Tubes 10-12-empty triangles (V); and Fraction "D", Tubes 12-14-full triangles (V). Figure 22 shows the activity of several fractions eluted from a superdex column to inhibit the proliferation of Nb2 cells. The solutions fed into the column consisted of several fractions of the elution product of Figure 21, as follows: Figure 22A shows the results of the following solutions fed: tubes 28-30 of Fraction C - empty circles (o); tubes 22-23 of Fraction C - full circles (•); tubes 29-33 of Fraction B-empty triangles- (V); and / - Figure 22B shows the results of the following fed solutions: tubes 31-32 of the Fraction
C - full circles (•); tubes 23-26 of Fraction C - empty circles (o); tubes 29-33 of Fraction B-triangles
empty (V). Figure 23 shows the activity of the fraction profile eluted from an analytical RP-HPLC column to inhibit the proliferation of Nb2 cells. The solutions ", fed to the column consisted of active fractions 10 (eluted in acetonitrile of 18% -28%) of a preparative HPLC-RP column, Figure 24 shows the activity profile of the fractions eluted from an exclusion column. of size to inhibit the proliferation of Nb2 cells.
were fed into the column were Fractions 13-17 of the aliquot shown in Figure 23. Figure 25 showed the activity profile of several fractions eluted in a hydrophilic interaction column (CHO) to inhibit the proliferation of the 20 cells of Nb2. The solutions fed into the column were the combined Fractions 27-30 of the aliquot shown in Figure 24. Figure 26 shows a scanning device of NMR of active fractions obtained from a column; ^ ~ of size exclusion (fractions eluted between 27-32 minutes). In the following reference it will take time to discuss the activity of the active fractions of "FM" and
"FGB" obtained from MC of muscle cells and MC of white blood cells. It will be understood that it is possible that these two
CM contain more than one factor that has the activities described below. In fact, the results of the "" HPLC fractions obtained according to the invention,
some of which are shown below, can be explained in this way (although other explanations are also possible). For example, it is possible that MC of muscle cells contain more than one factor that has an inhibitory effect on tumor growth. By
For example, at this time when referring to "FM", etc., it will not be constructed to mean the MC of muscle cells containing only a single RCE-PMB, when in fact the
CM of muscle cells may contain more than one substance that can be referred to as "FM". 20 1. Medium Condition 1.1 FM The MF was obtained from conditioned media (MC) of three types of muscle cell preparations: 1.1.1 Primary muscle cultures of newborn rats, - *. The muscles of the hind legs of the newborn rats were separated and shredded in small pieces with 24-48 hours of birth. After trypsinization with a 0.25% trypsinversan solution, the cells were previously plated in the tissue culture dishes to remove fibroblasts or fibrocytes and monocytes. The cells were counted and plated in an Enriched Dulbeco-modified Eagle medium (DMEM). Five days * "later, the cultures contained the muscle cells
contracted. The medium was then discarded and the PBS or RPMI medium was added. The cells were incubated in the RPMI medium for 24 hours and the PBS for a period of 8 to 24 hours. The cream or supernatant was subsequently collected, centrifuged and stored in a refrigerator at -20 ° C, until processing
below. 1.1.2 Rat muscle cell line (L-8) Line L-8 (obtainable from the ATCC American Type Culture Collection, CRL designation 1769) is a line of muscle myoblasts from the rat skeleton of freshly
born, comprising undifferentiated myoblasts that proliferate without the addition of growth factors. The L-8 cells were seeded in culture dishes and grown in RPMI medium (this medium will hereinafter be referred to as "RPMI") containing 4% glucose. 3 days
after the division, the cream of the crop was discarded, and / -i. re-placed either with the RPMI medium or with phosphate buffered saline (PBS) followed by an additional incubation for 24 hours. The creams or the supernatant were then collected and if not processed
immediately, they were stored in the refrigerator at -20 ° C. 1.1.3 Human myoblasts Human myoblasts obtained from a biopsy were cultured in spinner culture flasks up to their confluence (see U.S. Patent 5,130,4419.
culture was removed and put back in either medium
DMEM or a PBS solution, and the cells were further incubated in the solutions for 24 hours. The supernatant or cream was then harvested and the cells were separated therefrom by centrifugation. 15 1.1 FGB FGB was obtained from a conditioned cell medium
* Mononuclear. The mononuclear cells were isolated from venous blood, as follows: 20 ml of venous blood was extracted from a human donor with a syringe treated with heparin. The
blood treated with heparin was diluted 1: 1 with the solution
PBS, was layered on 15 ml of Ficoll-Hypaque ™
(Pharmacia, Sweden) or Histopaque ™ (Sigma, in St. Louis,
E.U.A.) and centrifuged at 400 x g for 30 minutes. The interface containing the mononuclear cells is
collected and washed three times with PBS.
2xl06 / ml mononuclear cells were suspended in the PBS solution and incubated at 37 ° C in an atmosphere moistened with 5% C02, 95% air for 48 hours. The cell suspension was then centrifuged and the supernatant was collected. 2. Ultrafiltration The MC obtained from the above sources was subjected to ultrafiltration using filters having molecular separations of 500, 2,000, 3,000 and 10,000 Daltons 10 (Centricon ™, from Amicon, E.U.A.). We found the FM that is present in the ultrafiltration through the membranes that have molecular separations of 10,000, 3,000, 2,000 Daltons, as well as in the ultrafiltrate of the membrane that has 15 molecular separations of 500 Daltons, as will be shown below . The FGB was ultrafiltered through a
*** - * .. membrane that has molecular separations of 3,000 Daltons and an ultrafiltrate that contains the FGB. The ultrafiltrate of the MC of muscle cells through the filter with molecular separations of 500, 2000, etc. will be referred to herein as a "500 Daltons FM Ultrafiltrate, 2,000 Daltons FM Ultrafiltrate", etc., the ultrafiltrate of the lymphocyte MC through the filters having molecular separations of 3,000 Daltons will be referred to in the present as the "ultrafiltrate of FGB, * 3,000 Daltons." 3. In vitro inhibition of the proliferation of tumor cells by FM and FGB 3.1 Methods 5 3.1.1 Cell Lines The effect of FM and FGB in this in vitro assay It was tested in several cell lines with tumors and several cells without tumor.The cells tested were the following: / «*« -> (a) cell lines with tumor: 10 HT-29, which is a cell line of an adenocarcinoma derived from a human colon (ATCC, designation HTB-38); MCA-105, which is a murine lung sarcoma cell line induced by methyl-colanthrene; 15 B16-F1, which is a cell line of a murine melanoma, SK-28, which is a cellulose line ar of a human melanoma; K-562, which is a murine 20 leukemia cell line; DA3 cells, which are a breast carcinoma cell line; MCF-7, which is a breast carcinoma cell line; 25 Nb2-llC, which is a lymphoid cell line of a rat, which is hormone dependent (ie growth of these cells requires the addition of growth hormone) (Gertler et al., 1985 Endocrinol ., 1 ^ 6: 1636-1644); and Nb2-SP, which is a rat lymphoma cell line independent of lactongénico hormone; (b) cells without tumor: murine bone marrow cells; rat primary fibrocytes; and IM-9; which is a cell line of a human lymphocyte. 3.1.2. Assay of 3 H-thymidine incorporation The test cells (cells in which 3 H-thymidine incorporation was assayed) were seeded in a 96 well plate with an initial cell density of 1x10 * cells / microwell. Each microwell contained a mixture of RPMI and a solution tested, either in any conditioned medium (an MC of muscle cells or an MC of white blood cells in any RPMI or PBS medium), a fractionated MC, an RPMI or PBS medium (ie, unconditioned), or an RPMI or PBS medium of fractionated control. The results of each tested solution were compared with the corresponding control (for example, a fractionated solution tested in PBS was compared to a fractionated PBS, etc.). After 2 hours of incubation at 37 ° C, each microwell was - * «. subjected to pulsations with 10 μC of 3H-thymidine, then by an additional incubation of 6 hours with a radioactive marker. The amount of 3 H-thymidine uptake was measured in a liquid scintillation counter. 5 3.1.3 Assay for cellular determination Cells from the rat lymphoma Nb2-llC cell line were synchronized and cultured as described previously (Gertler et al., 1985, Endocrinol., 116: 1636- * '1644) with the exception that the cells were grown in a
medium RMPI supplemented with 5% of a fetal calf serum. The synchronization of the Nb2-llC cells in the G0 / G? Phase was carried out. and the monitoring of self-proliferation as previously described (Gertler et al., supra). The cells were briefly transformed into a supplemented medium
with horse serum and incubated overnight. Then the cells were diluted in 3xl05 cells / ml and distributed
/ ** - * 'in a plurality of wells, well of 0.5 ml in a placate of 24 microwells. Then an amount of up to 0.5 ml of the tested solution was added and self-proliferation was started by the
addition of hGH at a final concentration of 2 mg / ml. The cells were incubated at 37 ° C in an atmosphere containing 5% C02, and after incubation for 72 hours, they were counted in a Coulter-Counter counter. Each experiment was carried out in two repetitions.
The cells of the Nb2-SP and IM-19 cell lines were tested in a similar manner. 3.2 results 3.2.1. FM Figures 1 and 2 show the results with the 3,000 Dalton FM ultrafiltrate derived from a primary culture of newborn rat striated muscle cells; Figures 3 and 4 show the results with an FM ultrafiltrate of 3,000 Daltons derived from L-8 cells. In each of these figures, the numbers in the abscissa represent the reciprocal number of dilution ("1" undiluted, "2" diluted twice, etc.) and the orderings show the radioactivity (counts per minute)
-CPM); the control (left column in each case) was with an unconditioned medium processed in the same way to the MC. As can be observed in Figures 1 and 3, the FM of a primary culture of rat striated muscle cells (Figure 1) or of striated muscle cell lines L-8 (Figure 3), inhibits the proliferation of tumor cells, as long as they do not have such an inhibitory effect on normal cells (Figures 2 and 4, respectively). In addition, it can also be observed that FM filters through the membrane having molecular separations of 3,000 Daltons, evidenced by the fact that the anti-proliferative activity present in the crude MC is restricted in the ultrafiltrate. Figures 1 and 2 can also be observed, the effect of the decrease of the FM with the increase in dilution, which also indicates that this effect is caused by a specific factor in the ultrafiltrate. Figure 5 shows the activity of the crude MC derived from the L-8 line, as well as in an FM ultrafiltrate of 3,000 Daltons thereof, both in various dilutions. As can be seen, the crude MC shows an anti-proliferative activity, which is retained in the ultrafiltrate. In addition, the effect decreases with the increase in dilution that shows this again, that is, it is an activity caused by a specific factor. The effect of an FM ultrafiltrate of 3,000 Daltons (undiluted) derived from L-8 cells is also shown in Figure 6 (the results are shown as percentages of control, the control in each experiment was evaluated at 100%). As can be seen, FM is active to inhibit the proliferation of all cell lines with tumors tested. Figure 7 shows the activity of an FM ultrafiltrate of 3,000 Daltons derived from human myoblasts, to inhibit the proliferation of 3 cell lines (the results are shown as the percentage of control). The proliferation of cell lines B-16 and K562 were tested by determining the incorporation of 3H-thymidine; the proliferation of the Nb2 cell line was tested by means of a cell determination. It can be observed that FM derived from man is active to inhibit the proliferation of the tumor cell lines tested. 5 In another experiment, fractions subjected to preparative HPLC-RP chromatography, which were found to be active to inhibit the proliferation of tumor cells (Fractions 5 and 6 of Figure 15), they were combined together, evaporated at 45 ° C and then dissolved in 5 ml of water, transferred to the tubes and again dried under vacuum, and finally dissolved in 2 ml of water and sterilized. The amounts of these fractions were tested to determine the ability to inhibit the proliferation of three different cell lines: 15 Nba-llC, Nbz-SP and IM-9. The results are shown in the following Table 1 (proliferation determined by cell counting). TABLE I CELL QUANTITY (ml / ml)% INHIBITION 20 Nb2-llC 0.05 49 0.015 33 0.005 22 Nb2-SP 0.05 46 0.015 26 25 0.005 18 TABLE I (Continued) CELL AMOUNT (ml / ml)% INHIBITION
IM-9 0.05 1 0.015 1 0.005 7 As can be observed, the FM inhibits the proliferation of the hormone dependent on the cell line with tumor Nb2-llC as well as the hormone independent of the cell line with tumor Nb2-SP. Against the human lymphocyte cell line IM-9, the FM had essentially no effect. Figure 8 shows the effect of ultrafiltration of FGB of 3,000 Daltons on the proliferation of human and murine cell lines. As can be observed again in the present, FGB inhibits the proliferation of all tumor cell lines tested both those of murine origin and those of human. 4. In vitro inhibition of lymphocyte response to PHA and MLR by FM and FGB: When the lymphocytes of two individuals are cultured together, the HLA antigens from each individual will cause a cellular reaction that will cause the proliferation of lymphocytes ( proliferation is directly related to the difference between the HLA antigens of two individuals). To explore the effect of FGB and FM on this * X, reaction, mononuclear cells of a size of lxlO6 cells / ml were incubated from two donors (cells prepared as described above according to 1.2) in PBS containing 10% FCS and different dilutions of the FM ultrafiltrate or FGB of 3,000 Daltons were added to the cells. The cultures were incubated for five days at 37 ° C in a humidified atmosphere of 5% C02, 95% air. During the last 6 hours of incubation, each well was pulsed with 1 μCi H-thymidine. The cells were
harvested and the uptake of 3H-thymidine was determined in a liquid scintillation counter LKB (LKB, Piscataway, NJ, E.U.A.). The PHA (phyto hema agglutinin) is a mitogen, which binds in the superficial sugars of the cellular of
the lymphocytes, inducing the transformation of the lymphocyte into a blast form with subsequent cell proliferation. To explore the effect of FM or FGB on the reaction induced by PHA, mononuclear cells were seeded at a concentration of 106 cells / ml in plates
of 96-well microwells, each well contains 0.2 ml of RPMI supplemented with 10% of fetal calf serum (Israel Industries, Bet. Ha-Emek, Israel) and 1 μg / ml of pHA (Wellcome Laboratories, United Kingdom). In some wells 0.2 ml of RPMI consisted of half pure RPMI and half a
FM ultrafiltrate of 3,000 Daltons of the L-8 cells in the * "• * RPMI medium.The cultures were incubated for four days in a C02 incubator and at the end of the incubation period the cells were subjected to a pulse with 1 μCi of 3 [H] - thymidine, incubated for an additional 24 hours, and harvested with a Dyatech cell harvester and the radioactivity was counted with a LKB scintillation counter Figure 9 shows the effect of FM to inhibit the lymphocyte reaction in PHLA and in the MLR (results - * that were shown for a 1: 1 dilution, and were provided as control percentages) Qualitatively similar results were obtained with FGB In addition, the effect of both FM and FGB was proportional to the dilution (the effect decreases with increasing dilutions) (the results were not shown) 15 5. In vitro studies 5.1 Induction of a tumor (MCA 105) by intraperitoneal inoculation, intraperitoneal treatment with FM: 30 r C57BL6 / J atons were injected intraperitoneally (i.p.) with 2.5xl05 of MCA-105 cells. Mice were treated twice daily by intraperitoneal injections of 0.5 ml of the HPLC-RP fraction designated below as "FM-SP" (Clause 6.5.2). The mice were sacrificed on day 33, and the 25 tumor foci were evaluated.
s ** Representative results are represented in the
Figure 10 showing two animals with the open peritoneum, wherein the animal shown in Figure 10A was treated with the FM-SP fraction and the animal shown in Figure 10B was treated with the RPMI control medium. As can be seen, a very large growth of the tumor can be observed in the control animal (arrow in Figure 10B) while, only very small detectable tumor foci X ~ * were observed (one is shown by an arrow in the Figure).
10A) in the animal treated with FM. 5.2 Induction of a tumor (B-16 by intraperitoneal inoculation, treatment by intraperitoneal injection of FM 40 C57BL6 / J mice were injected
intraperitoneally (i.p.) with 2.5xl05 of B-16 melanoma cells. 20 mice served as control and were injected daily with 1 ml of a 3,000 Dalton FM ultrafiltrate derived from L-8 cells. Mice were sacrificed on day 15, and the area of tumor growth
in the peritoneum of the animals was tested. It can be seen in Figure 11, which represents the open peritoneum of two representative mice of each experimental group, while numerous large tumor foci can be observed in the control group animal,
Only in the treated animal there are only a few and much smaller tumor foci. 5.3 Induction of a tumor (MCA-105) by intraperitoneal inoculation; FM treatment by intraperitoneal administration and by osteopathic administration (p.o.) 30 C57BL6 / J mice were injected intraperitoneally (i.p.) with 2.5xl05 of MCA-105 cells.
mice were treated daily by osteopathic administration of 1 ml of the 3,000 Dalton FM ultrafiltrate derived from the muscle cells L-8. 10 mice were treated daily by an intraperitoneal injection of the same solution; and 10 mice served as the control group and were treated daily by p.o. with the RPMI medium. The mice were sacrificed on day 30, their peritoneum was exposed and was tested based on the extent of tumor growth therein. Two representative mice of each group are shown in Figure 12
(Figure 12A-mice treated with FM, Figure 12B-control). As can be seen in Figure 12A, there are no signs of tumor growth in the peritoneum of the animals treated with FM, while large tumor foci appear in the peritoneum of the animals in the control group. In this experiment, 90% of the mice in the control group developed tumor foci, while foci * only developed in 40% of the mice treated with FM either by i.p. or p.o. 5.4 Induction of a tumor by intramuscular inoculation (i.m.) (DA3 breast carcinoma); treatment
with FM by intraperitoneal administration and by osteopathic administration (p.o.) lxlO6 DA3 cells were injected intramuscularly into the foot of 30 BALB / C mice. 10
, and- mice served as control and were treated
daily by an intraperitoneal injection of PBS; 10 mice were treated daily by i.p. administration. 1 ml of the 3,000 Dalton FM ultrafiltrate derived from the muscle cells L-8 (prepared in PBS); and 10 mice were treated with the same solution that contains FM
by osteopathic administration (p.o.). The mice developed large tumors in the paw and after three weeks the mice were sacrificed and metastatic foci were detected in the lung and counted in the lung. In the group, 23.4 ± 6 metastatic foci were counted in comparison
with 6.2 ± 1.3 foci in the group treated by i.p. and 2.2 ± 0.6 in the group treated by p.o. 5.5 Induction of a tumor by intravenous inoculation (i.v.) (Melanoma B-16); FM treatment by osteopathic administration (p.o.) 25 30 C57BL / 6J mice were injected with 5x105 B-l6 melanoma cells. 20 mice were treated daily by p.o. injection, starting on the day of tumor inoculation with 1 ml of an FM ultrafiltrate of 3,000 Daltons derived from L-8 cells; 10 mice served as the control and were administered by p.o. only with PBS. On day 18, the mice were sacrificed and it was possible to observe in their lungs that they developed foci of black tumor. In Figure 13 you can see the lungs representative of two groups (in the upper row, the control group, in the lower row the group with FM treatment). As can be seen, while there are many black metastatic foci in the lungs of the control group only few and small ones can be observed in the experimental group. 6. Grade of FGB secreted from white blood cells of healthy individuals and cancer patients Mononuclear cells were isolated from venous blood of 23 healthy individuals (samples were obtained from blood banks) and from 33 hospitalized cancer patients. The procedure was as described in 1.2. Supernatants were collected from the 2xl06 / ml mononuclear cells, supernatants were collected and then subjected to ultrafiltration through a filter having molecular separations of 3,000 Daltons as described in 2.
-s Ultrafiltrates were tested for their ability to inhibit the growth of cancer cells from 3 cancer cell lines: murine melanoma cell line B-16, human melanoma cell line SK, and cell line
human K-562 leukemia. The results are shown in Figure 14. As can be seen in Figure 14A, while the ultrafiltrate of the mononuclear cells of healthy T-individuals showed a marked inhibition of proliferation,
which was below 50% of the cell lines tested, there was only very little inhibition of proliferation with the ultrafiltrate of cancer patients. As can also be seen in Figure 14b, there is absolutely no overlap between the two groups. 15 The results show that there is a considerably lower degree in the level of FGB secreted by the mononuclear cells of cancer patients, compared to those secreted by the lymphocytes of healthy individuals. These results thus demonstrate the importance for the diagnosis of testing the level of RCE-PMB of the invention. In addition, the power level of the FGB is also of therapeutic importance. 7. Characterization of FM 7.1 Bioassay 25 The MC of the muscle cells was fractioned, as will be detailed below, and the various fractions were tested using the cell lines mentioned above. The effect of each fraction on cell growth was determined by the uptake of 3H-thymidine or by the
cell count assay described above. 7.2 Test for the putative nature of FM protein In order to determine if FM is a substance
< ** > protein or not, the MC of muscle cells (derived from a
primary culture of rat muscle cells) was subjected to a series of treatments including sensitivity to the stability of proteolytic enzymes, during lyophilization and incubation at various temperatures. After such treatment, the MC was returned to the salt
original and the protein concentration was taken into account by dilution with the original medium or if it was diluted, the dilution factor of the protein concentration was taken into account to evaluate the results. The assay that was used was the 3H-thymidine uptake assay. 20 7.2.1. Effect of proteolytic enzymes. Trypsin and pronase, two proteolytic enzyme preparations, were tested. In order to determine the effect of trypsin, two procedures were used: 25 (i) A solution containing FM with trypsin (0.5-2 μg / ml) was incubated for 4 hours at 37 ° C, after which the Trypsin activity was stopped by the approximate addition of 2 molar access links of the soybean bean trypsin inhibitor (STI). An unconditioned medium 5 (free of MF) was used as a control; (ii) A solution containing FM with trypsin (0.5-2 μg / ml) was incubated for 1 to 4 hours at 37 ° C, or overnight at room temperature, and after
"* d incubation, the enzyme was removed on a P-10 aminobenzamidine-agarose column, an FM-free medium served as control, and trypsin-only control tests have shown that the tryptic activity is not eluted in this column according to the operating conditions of the column, bicarbonate buffer 15 In order to test the effect of pronase, the MC of muscle cells was treated by putting them in contact with the pronase immobilized on a sepharose gel Also in the present, an FM free medium was used as a control.The results are shown in the following Table II (the number
indicates the% inhibition compared to the unconditioned control medium) TABLE II Experiment Trypsin + SITa 'T ripsin + column < b) Pronaze-Sepharose Type of Cell Untreated Treated Untreated Treated Untreated Treated
HTB 38 35 30 40 40 53 75 MCA - < c > - 25 27 62 70
(a) Previous procedure (i) of Trypsin (b) Previous procedure (ii) of Trypsin (c) "-" = not tested The above shows that treatment with trypsin by both procedures as well as with pronase, does not had a significant effect on the inhibitory activity of FM tumor growth. 7.2.2. Lyophilization The MC of the muscle cells was lyophilized without a previous dialysis and the lyophilisate was then dissolved again in water at its original volume. After this treatment, the loss of inhibitory activity to FM tumor growth was not appreciable, indicating that FM is stable to lyophilization. 7.2.3. Heat Treatment An MC of muscle cells (derived from a previous culture of rat muscle cells) was treated for several periods of time at temperatures ranging from 4 ° 00 ° C, after these treatments the samples were tested with both cells MCA and HTB. The results are shown in the following Table III (the numbers indicate the change (in%) to inhibit the power of the FM after the heat treatment):
TABLE III Temperature 4 22 40 60 100 ° C / Time 1 min - +13 5 min - +26
1 hour - - +16 +29 +6 22 hours +61 +42 - "-" = not tested under such conditions In other groups of experiments, an ultrafiltrate of 3,000 Daltons of the conditioned medium was treated by heating to boiling, without no loss of activity of this low molecular weight fraction to inhibit the proliferation of tumor cells. The above results show that there was no decrease in the inhibitory potency under all the tested temperatures including boiling at 100 ° C. 7.2.4 Summary No decrease in the power of FM was observed to inhibit the growth of tumor cells under the conditions tested, which clearly indicates that FM is not a protein. Against this, the results showed an increase to inhibit the activity after some treatments, which can be explained by the fact that the medium containing FM contains a protein factor, which exerts an opposite effect to FM and which is destroyed in the treatment . 7.3 Size of the FM The MC that contains FM has been fractionated by ultrafiltration in Amicon membranes with molecular separations of 10, 2 and 0.5 kD (the retentive element is filtered twice in each case with at least one additional volume of PBS). In the cases of membranes 10 and 2 kD, essentially all the inhibitory activity (above 90%) was found in the first two filtrates, and with the 0.5 kD membrane, it was found in approximately 80% of the activity in the filtered and some activity was retained
(20%) in the second retentive element. The inhibitory activity was assayed in each case in both the HTB 38 cells and the MCA cells. The dialysis of the MC containing FM through the membranes with the molecular separations of 12 and 3 kD, showed that the active component escaped through the membranes. The above results show that the FM has a molecular weight in the order of 500 Daltons or less than this value. 7.4 Characterization of the HPLC-RP chromatography of FM (derived from a primary culture of rat muscle cells) ~ < - ± The filtrates of the 10 kD membranes were subjected to reverse phase chromatography on a column (4x250 mm)
(RP) C-18. The filtrate was brought to the original concentration by dilution in an acid solution
trifluoroacetic at 0.15 (TFA) before it is applied to the column, which was developed with a gradient of 5 to 355 acetonitrile (these components were previously tested to determine that they do not effect the inhibitory activity of the
^ "FM) Activity that was tested with HTB-38 cells
subjected to elution in 15% acetonitrile in 0.1% TFA. The activation fraction was again subjected to chromatography on the same column to this acetonitrile gradient and the partially purified MF was subjected to dilution in the same position as before (time
approximate retention was 21 minutes). A third run of reverse phase chromatography (RP) was carried out on this last fraction under the same conditions as the first two runs. Only a maximum inhibitory point was found, and coincided with the position of a maximum
absorbance of 220 nm in the elution profile that can be observed in Figure 15. 7.5 Large-scale purification of FM (derived from a primary culture of rat muscle cells) 7.5.1 Experimental Protocol 25 The MC of muscle cells of The rat was subjected to ultrafiltration in Amicon 10 kD membranes. The 10 kD filter was subjected to chromatography on a reverse phase (RP) C-18 column (47x300 mm) fixed to a preparative HPLC column. Fractions were tested in a cell proliferation assay using HTB 38 cells, and the active fraction was re-chromatographed on a second reverse phase (RP) C18 column (analytical chromatography: 4x250 mm) and the active fraction was I identify as before. * - \ 7.5.2 Activity of purified FM fractions
The material obtained in the last step of the
Section 7.5.1 was called "FM-P" considering that the material obtained after the first reverse phase column RP was called "FM-SP" (this fraction was also tested in Section 5.1). Table IV shows the activities of
crude condition medium (MC), fraction FM-P and FM-SP as well as the retentive element (R) of ultrafiltration
"" 'i' * '' (the activity is expressed in μ / ml: lμ is defined as the amount of the material that causes 50% of the inhibition in the proliferation assay) TABLE IV MC Cell Line "FM-P "" FM-SP "" R "HTB-38 < a > 18.0 8.4 2. 16 - HTB-38 < a > 30. 0 10 .2 MCA - 7.4 8. Four. Five . 0 25 (a) = Experiments were performed with HTB-38 cells separately in two different locations "-" = not tested 7.6 Characterization by FMLC chromatography of FM derived from cell line L-8 In Sections 7.6.1 - 7.6.5 Some representative results of HPLC chromatography are demonstrated. An exemplary method for FM purification is provided in Section 7.6.1. 7.6.1. Reverse Phase HPLC (RP) 160 ml of a 3,000 Daltons FM ultrafiltrate in PBS (obtained after 8 hours of incubation in the muscle cells with PBS) was subjected to chromatography through a RP-HPLC column (C-18). ). The elution fluid had an HPLC grade water gradient prepared in a B-pure ™ device (Barnstead, Dubuque, Iowa) and an HPLC grade acetonitrile (G.T. Baker, E.U.A.). The gradient of the elution fluid was between 0% for acetonitrile and 60% acetonitrile during a period of 30 minutes: The flow rate was 100 ml / min. Two fractions were collected per minute (Each consisted of 200 ml). 20 ml of each fraction obtained by HPLC were evaporated to dry in a centrifugal concentration apparatus, and the dried fractions were then suspended in 100 μl of water, then evaporated again until it was dried, then dissolved in 2 ml of PBS, and the fractions were tested for their inhibitory activity of proliferation in the cell count assay, by applying 0.2 ml of each fraction in a microwell containing the tester cells. Figures 16 and 17 show the activity of several fractions in two different runs. The results of both cases show the presence of a specific inhibition, which was eluted in Fractions 5 and 6 of Figure 16 and Fractions 8-12 of Figure 17. Fractions 5 and 6 of Figure 16 were combined, evaporated at 45 ° C, dissolved in 5 ml of water, transferred into tubes and again dried under vacuum, dissolved in 2 ml of water and sterilized. 1 ml of these concentrated fractions were subjected to chromatography on an analytical RP-HPLC column (C-18) at a gradient of the operating fluids between 100% for water and 60% for acetonitrile within a period of 20 minutes, the flow rate was 1 ml / min. One fraction was collected per minute (each of 1 ml), then dried, and dissolved in 0.4 ml of RPMI containing 5% HS and sterilized. 0.15 ml was added in each well containing cell (the activity was determined by the cell count assay). For control, the PBS solutions were fractionated by HPLC and tested for their activity in a similar manner.
r - "- ** - The activity of the various fractions subjected to elution is shown in Figure 18. These results indicate the presence of a specific inhibition in Fraction 15. 7.6.2 HPLC with a Superdex 5 column The active fractions of Figure 17 were divided into two combined fractions, a first fraction named "FRl" was obtained from the combination of 1200 ml of tubes 6.-8, 550 ml of tubes 8-9, 300 ml of tubes 9-10 * and 30 ml of tubes 10-11, a second fraction was obtained
named "FR2" of 250 ml of tubes 9-10, 520 ml of tubes 9-11, and 600 ml of tubes 11-12. Each one of these
Fractions FR1 and FR2 were dried and dissolved in 10 ml of distilled water. Since an insoluble precipitate formed after centrifugation, these fractions were extracted
in addition to 10 ml (in the case of FRl) and 5 ml (in the case of
FR2) of PBS. Even after the second extraction, a considerable precipitate remained. The results showed that the majority of the activity was present in FRl; FR2 only had one
activity between 15-20% of the activity of FRl. The extract of
PBS of the insoluble preparation produced only 4% of the activity compared to FRl. 2 ml of FR2 was dried and dissolved in 0.4 ml of water. 0.2 ml were applied to the superdex column
balanced with PBS. The column was developed at a rate of ml / min. with PBS as the operating fluid and fractions of 1 ml were collected. The collection started 0.3 minutes after starting the separation. The fractions subjected to elution were sterilized and 0.2 ml was added in each well. The inhibition of the various fractions eluted in the superdex column are shown in Figure 19. Specific inhibition can be observed in Fractions 18-22. Fractions 18-22 were extracted and re-chromatographed on the same column under the same conditions. The results of re-chromatographing indicate a possibility that there is a mixture of two or more active agents (FM) in these fractions. A similar elution profile was also obtained with the FR2, although the results seem to indicate that the FR1 was approximately 10 times more concentrated in its FM content compared to the FR2. 7.6.3 Analytical HPLC-RP Chromatography after HPLC chromatography with Superdex column FR1 and FR2 fractions were combined and 1 ml aliquots were applied to an analytical RP-HPLC chromatography column (C-18). 1 ml fractions were collected, dried and dissolved in 0.4 ml of RPMI containing 5% HS and 0.5 ml of this solution was added to each well containing the cells. The results are shown in Figure 20. As can be seen, there is a maximum defined value of activity in tube 16. In addition, there was also an inhibitory activity in tube 4. These results indicate that there may be more than one inhibitory agent of the proliferation of tumors in the MC of muscle cells. 7.6.4. Size exclusion HPLC chromatography (SE) Fractions 6.8 ("A"), 8-10 ("B"), 10-12 ("C") and 12-14 ("D") of HPLC-RP chromatography were chromatographed separately on a column containing particles of a silicon gel coated with polyhydroxyethyl aspartamide (PolyHYDROXYETHYL-Am, manufactured by Poly-LC). The operating fluid was an isocratic solution (ie, without gradient) which was an aqueous solution of 50 μM formic acid. Fractions of 0.5 ml were collected, dried and dissolved in 0.8 ml of PBS. Subsequently, 0.15 ml of each fraction was added to each well containing the cells. The results of the chromatography can be seen in Figure 21. The results show that there was no activity in Fractions 6-8, except perhaps a weak activity in tube 32. In Fraction B a clear and broad maximum value of activity 29-37. In Fractions C there are two maximum values of the activity clearly, one in tubes 21-23 and the other in tubes 28 - "-" - 31. Finally in Fraction D there is a single maximum value of activity in tubes 21-22. 7.6.5. HPLC chromatography with Superdex column following size exclusion HPLC chromatography (SE) 5 The active fractions eluted from the size exclusion column (SE) were combined and dried in a high vacuum concentration centrifuge (Hetovac ™ VR-1), manufactured by Heto, Denmark), is dissolved in 0.4 ml and then 0.2 samples are separated in superdex 10 columns equilibrated with PBS. 1 ml fractions were collected and 0.2 ml was added to each well containing cells. As can be seen in Figure 22A, tubes 29-33 subjected to HPLC-SE of Fraction B showed an inhibitory activity in tubes 21-24 eluted in column superdex. The profile of this activity may indicate the possibility that there is more than one active agent, or that the active agent appears in several ways. Figure 22B shows a maximum specific value eluted between 17 to 21 minutes. 20 7.6.6. HPLC Chromatography by Interaction
Hydrophilic: Fractions 27-32 of the aliquot of HPLC size exclusion chromatography shown in the following Figure 25, were combined together, dried
and dissolved in 0.2 ml of water. Each two 0.085 ml quantities were injected into an HPLC column by hydrophilic interaction (CHO) (PolyGLYCOPLEX ™, manufactured by Poly-LC). The column was then developed with a solution consisting of 70% acetonitrile in water at an operating rate of 1 ml / min. 1 ml fractions were collected, dried and dissolved in 0.6 ml of PBS, and then 0.1 ml fractions were added to each well of a 96 microtiter plate and tested in the saline count assay (Section 3.1.3). The activity profile of the solution eluted from this column can be seen in Figure 23. It can be seen that the highly specific inhibition in aliquot by CHO was found in Fractions 2-8, although a maximum inhibitory value can also be observed secondary in Fraction 10. A weak activity can also be observed at a wide maximum value in Fractions 21-30. 8. Purification of FM 8.1 Procedure A method was developed for FM purification consisting of the following steps: (a) Preparation of the conditioned medium: The conditioned medium was prepared from the L-8 cell in PBS as described in 1.1. 2. (b) Ultrafiltration: The conditioned medium was subjected to ultrafiltration "*" - through a membrane with a molecular separation of 3,000 Daltons, as described in 2. (c) Preparative HPLC-RP Chromatography: The ultrafiltrate of 3, 000 Daltons then submitted
to chromatography on a preparative RP-HPLC C-18 column. The first column is typically washed with water grade
HPLC for 20 minutes, then 200 ml of the 3,000 Daltons ultrafiltrate is loaded onto the column and washed with
^ Water continues for an additional 10 minutes. The
The column was then developed with acetonitrile: the water gradient between 0% acetonitrile at 600% acetonitrile over a period of 30 minutes. The speed of the operating fluid is approximately 100 ml / min. The active fractions eluted in the resulting fractions of the column
approximately between 8-14 minutes. The active fractions are then combined together and the combined fraction is then concentrated in a rotary evaporator (Rotovap ™, from Buchi, Switzerland) followed by a concentration centrifuge. ^ 0 (d) HPLC-RP Analytical Chromatography: The combined concentrated and dried fraction is then dissolved in 5 ml of water, dried again under vacuum and then dissolved in 2 ml of water. 1 ml of this concentrated fraction is then subjected to chromatography on a column
C-18, analytical HPLC-RP with an operating fluid that is a gradient of acetonitrile: water, between 0% acetonitrile to 60% acetonitrile over a period of 20 minutes with a flow rate of 1 ml / min. The column is loaded and then washed with water for 5 minutes. After this washing, the column develops with a gradient of acetonitrile. The active fraction was eluted into fractions resulting from the column between 12.19 minutes. The active fractions are then concentrated and dried in a concentrated centrifuge. (e) Size Exclusion Chromatography: The dried fractions are then mixed with formic acid and 200 ml samples of this solution are loaded onto the size exclusion column described in 7.6.4, the development fluid and the operating conditions. also as described herein. The active fractions were eluted after 27 to 32 minutes. It will be noted that different columns even those with specifications similar to those mentioned above, and slight variations in the elution condition, can produce different elution profiles and therefore the active fractions can be eluted after different retention times to the reported previously. Nevertheless, it will be possible for the experienced person to test each fraction for an inhibitory activity as described above, without undue difficulty in locating and isolating the purified fractions containing the RC of the invention. 8.2 Purification Results Figure 24 shows the profile of the inhibitory activity subjected to elution by an analytical RP-HPLC column and the activity of the fractions subjected to elution in an HPLC size exclusion chromatography (Figure 25), the procedure of purification is as described above in Section 8.1 9. NMR-Nature of possible oligosaccharide in the FM The active fractions of a size exclusion column such as that shown in Figure 25, were dried and then dissolved in methanol (( there were 3 consecutive drying cycles and then they were dissolved in methanol.) The methanol extract was then evaporated to dryness, and the preparation was redissolved in 0.5 ml of methanol with Deutero.The remainder of the methanol extraction was dissolved in 0.5 ml of water with Deutero. The NMR spectrum can be seen in the Figure
26. As can be observed, the scanning or NMR scan of the water fraction showed a maximum value, probably negligible, considering that the NMR exploration of the methanol extract showed several maximum X - * values in a field of 3-4. ppm. These maximum values are characteristic of protons of the C-OH groups, typical of oligosaccharides. 10. MC ultrafiltration activity of 3,000 Daltons white blood cells and their fractions A 3,000 Daltons ultrafiltrate obtained from a white blood cell MC was tested for its activity and compared to the 3,000 Daltons ultrafiltrate of MC of L-8 cells . f ^ "The results are shown in the following Table V: 10 TABLE V Tested Medium Quantity (ml) Inhibition% Ultrafiltrate of 3,000 0.4 43 Daltons in an MC of L-8 0.3 29 0.2 11 15 0.1 0 Ultrafiltrate of 3,000 0.4 32 Daltons of an MC of gl0.3 21 white bubbles 0.2 17 0.1 6 20 200 ml of the 3,000 Daltons ultrafiltrate of an MC white cell was separated on a preparative HPLC-RP column as described above (see Section 7.6.6 .) Fractions of 200 ml were collected, aliquots of 10 (out of 200 ml) were dried and
dissolved in 0.6 ml. Subsequently 0.1 ml was applied in f - »- each well. For the comparison of the 3,000 Daltons ultrafiltrate of an MC of L-8 cells, it was fractionated in the same way. The results are shown in Table VI: TABLE VI
FRACTION N °? INHIBITION / * N 2 4 22 10 3 5 25 4 30 47 5 28 58 6 21 45 7 2 33 15 ll Ultrafiltrate of 3,000 Daltons of an MC of white blood cells, 2 > Ultrafiltration of 3,000 Daltons of an MC of L-8 cells As can be seen, both aliquots show a maximum inhibitory value in Fractions 4-6. twenty