MXPA97007111A - Immunomodulating compositions of the bilis for the treatment of disturbances of the inm system - Google Patents

Abstract

The present invention relates to a composition for use as an immunomodulator comprising components of low molecular weight of less than 3000 daltons, and having the following properties: a) it is extractable from the bile of an animal; b) it is capable of stimulating monocytes and macrophages in vitro and invivo, c) is able to modulate the production of tumor necrosis factor, d) does not contain measurable levels of IL-alpha, IL-1beta, FNT, IL-6, IL-8, IL-4, GM-CSF or IFN-y; e) has an anti-proliferative effect in a malignant mouse hybridoma cell line, f) does not show cytotoxicity to human peripheral blood mononuclear cells or lymphocytes, and f) is not an endotoxin; invention also relates to a method for preparing the composition, to its use as an immunomodulator, and to its use in the treatment of diseases and conditions having an immunological component, such as cancer or endometriums

Description

t COMPOSITIONS IMMUNQMODULADQRAS OF THE BILIS FOR THE TREATMENT OF DISTURBANCES OF THE IMMUNE SYSTEM FIELD OF THE INVENTION The present invention relates to compositions of the invention, compositions comprising Leas comprising J them themselves, and to the use of said compositions in stage J B of mammals. In particular, the compositions are directed WHEN they are dealing with diseases associated with disorders of the immune system. t 9 BACKGROUND OF THE INVENTION Therapies for the prophylaxis and treatment of cancer and inflammatory, infectious, and infectious diseases are continually being developed, all of which can be a direct result of an inadequate response to the immune system. Some of these therapies try to use the i inrnu e releu cainent theme "A proposal is based on the specific elements of the immune system, mainly antibodies and T cells. For example, the invention has focused on the development of vaccines against foreign agents, or against certain endogenous chemical messengers, talee as interleukins, to control or induce certain antibody reactions. A "Sß" second proposal is based on the isolation, cloning, expression and production of peptides and proteins at rtir of the pa -i is specific not antigonos of the immune system. For example, proteins, such as cytokines, which comprise micelles produced by white cells, and interleukins, which stimulate lymphocytes and sweeping cells that digest foreign antigens, offer possibilities for therapies. ^^ Treatment of cancer, for example, can be "greatly * improved if the early immune response to a tumor can increase so that the tumor does not reach a critical size." Strategies that have been suggested to increase the response Immune to a tumor include: specific vaccines for antigens associated with tumor, the use of antibodies "monoclonal against antigens on the surface of tumor cells, such as against the IL-2 receptor; | ^ bioespeci fi c molecules that contain nti tumor and super antigen antibodies. Relatively recently, the role of the physiologically active polypeptide, known as tumor necrosis factor (TNF), has been studied. In particular, it has been shown that TNF induces tumor necrosis, without effects on the normal tissues of the living body. The amino acid sequence of the TNF, as well as the base sequence of the DNA encoding FNT, have been described in the U.S. Patent. No. 4,879,226. Because TNF has been shown to play a role in the induction of tumor necrosis, any agent that can stimulate the production or bioavailability of TNF in vivo has potential utility, as a treatment for several turnoral conditions. In addition, any agent that can stimulate human rhonocytes and macrophages to produce FNT ii'i v tro, is useful as a means to provide a source of TNF for therapeutic administration, as well as for analytical and diagnostic purposes. Other diseases also have or involve a response to the impaired immune system. For example, autoimmune diseases are disturbances where the immune system produces an antibody against substances that are not foreign to the body, resulting in inflammation and consequent tissue damage. For example, arthritis reuinatoi e (Rñ) is an autoimmune disease in which the immune system of the body mistakenly recognizes the normal cells of the lining of the joints, called synovium, as strange. The autoimmune attack can destroy the lining completely. In the most severe cases, the joints stop working and are surgically replaced with artificial joints. The FNT is a mediator of damage in RA. The progress from mild to severe symptoms can be very rapid. However, no treatment is available for patients with RA. Other essentially untreatable autoimmune diseases include eriternatosus lupus, multiple sclerosis, and amyotrophic lateral sclerosis.

Infectious diseases, such as those caused by bacteria, viruses, and other opportunistic pathogens, can only be successfully prevented or by fighting the immune system of the body. The immune system assembles or attracts one or both non-specific immune responses and factors to the specific immune response to combat said pathogens. Non-specific immune responses focus on cytokine production, primarily by inaccurate phage, and serve as a prelude to specific antibody responses. Inflammatory cytokines include TNF-a and are involved in an acute response directed to the sites of damage or infection, which is manifested by an increased blood supply. Pathogenic bacteria or viruses are submerged by neutrophils and acrophages in an attempt to * contain the infection in a small tissue space. Therefore, macrophages play an important role in the defense against infectious diseases as follows: (1) process and present the antigens to the lymphocytes so that immune responses mediated by antibody and mediated by cell can occur, - (2) secrete central cytokines to the immune response; and (3) destroying the antibody-coated bacteria, tumor cells or host cells. Macrophages can ingest and kill a variety -? a, "pu, 1. .at óno, _" co-o ba ana. , n, oS and pro * - »* ™, (pair sites). This capacity increases when the "phacophages" are "activated". The products secreted from activated macrophages are as diverse as those from any other immune cell.

These regulate both pro- and anti-inflammatory effects and regulate other cell types, these products include TNF-cx, IL-lß, IL-5, hydrolytic enzymes, and oxidative metabolism products. Bacteria that are eliminated primarily by this cell-mediated immune procedure include tuberculosis and other bacterial-bacterial infections, such as atypical bacterial infections seen in up to 50% of patients with AIDS, and anthrax, a potential bacteriological agent. . Fungal infections are common problems in first-line patients, such as those afflicted with AIDS or organ transplant patients. Protozoa include such organisms as malaria. Inflammatory diseases include endometritis and inflammatory bowel disease, which is also mediated by immune procedures. Endo etriosis is a dark disease of unknown cause and histogenesis that affects the women who undergo it. The disease is characterized by the inappropriate implantation, growth and function of endornetrial cells. The endornetrial cells and fragments, which are normally discharged during the menstrual cycle, are transported through the fallopian tubes in the epithelial cavity where, in some women, they are implanted, prolific and develop into endorhetriotic lesions. . However, since it appears that endornetrial cells are present in the peritoneal cavity of all women who undergo surgery, it is not clear at present why endosteal riosis develops in some but not all women. Endometriosis can result in painful inflamed tissue, abnormal bleeding, extensive scarring, painful urination or defecation, and organ damage.

ITT reproduce a woman, still leading to infertility » There is no known treatment for endornetriosis, of short pregnancy, that provides temporary relief or surgery to remove the source of endo-uterine cells, which also causes sterility. 15 Recently, numerous reports have suggested that endornetposis is associated with changes in the immune system. Previous reports indicate that inrnunosupresive rats are associated with an increase in endotrhythmias in rhesus monkeys. Since that time, alterations in the Cell-mediated and mood-mediated immunity have been observed in humans with endornet riosis. During recent years, studies have focused on the role of macrophages in endorhetriosis. The underlying hypothesis for these studies was that the erythematosus regulates the growth of the endornetrial cell and prevents the proliferation of p-endometrial cells in normal healthy women. In women with endornet posis, erroneously placed endometrial cells were implanted, giving rise to endornetposis. The development of endornetposis is then a stimulus for the production of auto-antibody against endorheic cells and cell-derived antigens. These autoantibodies, together with activated phage products, can then interfere with the fertility and reproductive functioning of affected women. The cumulative results from these studies have revealed the following pertinent facts. (1) the peptoneal elimination system (consisting mainly of macrophages) that is believed to be responsible for the destruction of endoinotional cells ectopic within the peptoneal cavity can be harmful in women with endornetposis extension, - (2) the activity of peritoneal macrophage of ectuosa in women with extensive endorne posis is related, at least in part, to a fact mediated with prostaglandma . Of esl way, the significant stimulation of the peptonea macrophages is of women with extensive endometriosis in response to macr? Activators. phages, such as gamma interferon and endotoxin, can be achieved only when a prostaglandin synthesis inhibitor is included in the activation culture; (3) Monocyte products circulating from patients with endometriosis can be directly involved in * regulation of the growth of endorheic cells. In a single co-culture topic, improvement of autologous endoinetpal cell proliferation was observed with rhonocytes from the majority of patients with endornet riosis, while suppression of pro e ration was observed with rhonocytes from The majority of fertile control patients; and (4) the proliferation of endometrial cells from patients with endorheneposis can be modulated by the cytokines derived from ina-eróphago. The results obtained to date suggest that the proliferation response of patients with endometriosis with limited disease can be improved by methylleukin-l-β (IL-lß) and TNF-cf. In contrast, the proliferation response of the endornet not from a patient with extensive disease is suppressed by IL-1β and FNT-Of. Therefore, the results of these studies suggest that the function of monocytes and macrophages from 0 patients with endorhetriosis play a significant role in the pathophysiology of the disease. In addition, it seems that some of these macrophage functions are affected differently by the severity of the disease. In women with limited disease, macrophages appear to be activated in the peritoneal cavity, and perhaps, in the circulation and their endothelial cells, they seem to be able to respond to different growth factors derived from the macrophage. In contrast, extensive endometriosis is characterized by the suppression of macrophage activation within the peritoneal cavity due, in part, to the hypersegregation of inrnunoregulatory prostaglandins. Macrophage products also seem to regulate the proliferation of the endotrium in women with extensive endorne posis; However, the qualitative differences in the response of endorhetrium to different cytoqs suggest that the sequences of defective macrophage activation in these women may contribute to, perhaps, the control of the disease. Inflammatory bowel disease (IBD) is a general term for a group of chronic inflammatory disturbances of unknown etiology involving the tract gastrointestinal. These disturbances include non-specific ulcerative colitis and Crohn's disease. The f that they can layer., *. disturbances (eg, arthritis, pericholangitis) may represent autoimmune phenomena and therapeutic agents used to treat IBD, such as corticosteroids and azatrioprine, can exert their effects through immunosuppressive mechanisms. Patients with inflammatory bowel disease may have hormonal antibodies to colon cells, bacterial antigens such as Escherichia coli, lipopolysaccharide, foreign proteins, such as cow's milk proteins. In general, the presence and title of these in The antibodies do not correlate with the activity of the disease; however, it is likely that these antigens gain access to immunocornpetent cells secondary to epithelial damage. In addition, IBD has been described in association with 5 again aglobulinern as well as IgA deficiency. Associated abnormalities of cell-mediated immunity include cutaneous allergy, diminished response to various mitogenic stimuli, and reductions in the number of pelvic cells. Bile, which is secreted by the liver and stored in the bladder, has been investigated for several purposes, including the use of bile extracts to improve the bioavailability of drugs that is rapidly metabolized by normal liver function (see UO). 90/12583) and to inhibit the promotion of leukocytosis in a mammal (see Hmoda et al., Chem. Pharm. Bull., 30, 4429-4434 (1982)). Nevertheless, Bile has never been considered a source of therapeutically useful compositions with respect to neoplastic, inflammatory or infectious diseases. From In an interesting manner, in accordance with British Patent No. 337,797, it was suggested to use the bladder, by itself, as a potential source of anti-cancer agents, but only after having removed the bile from the bladder, and having - completely flushed the bladder., 5% BRIEF DESCRIPTION OF THE INVENTION It has now been discovered that bile is an important source of a composition that can activate cells of the immune subject, such as macrophages and rhonocytes, and is effective in treating various cancers, especially cancer-pancreatic and malignant cancers. In particular, it has been discovered that the composition of the present invention can stimulate the production of TNF both in vitro and in vivo from, for example, macrophages. This property can be useful in the treatment of infectious diseases. It has also been found that the immunomodulatory effect (especially the ability to regulate or suppress the production of TNF-) of the present invention can also be used in the treatment of other disturbances involved with the immune system, such as antoinnune diseases. and inflammatory diseases and disturbances. The composition of the bile of the present invention is also believed to be useful as an adjuvant additive for vaccines targeting childhood diseases, and as a protection against the rejection phenomenon involved in xenograft procedures, for example. The composition of the bile of the present invention is obtained by extracting the bile with a solvent soluble or water-soluble. The extract thus obtained can be further processed to remove inconvenient or unnecessary components thereof. * The product obtained by the bile extraction procedure described in detail below has been found to have TNF-stimulating activity (or TNF inhibitory activity depending on the source of species) and is believed to have anti-cancer activity, infections , autoinrnun disturbances, and inflammatory disturbances. In particular, it is believed that the bile extract of the present invention is especially active against cancer.

Obviously, all the composition thus obtained may not be necessary to obtain said activity. Therefore, it is possible to separate, fraction, or otherwise process the product thus obtained, and still retain the desired ability to stimulate the production of TNF, for example, to act against disturbances of the immune system that contain various diseases. In addition, it is observed that it is possible to synthetically obtain a product with the same or similar capacity to stimulate the production of TNF and to act against them. disturbances of the immune system. In this way, it is observed that the components of the product can be identified and analyzed in terms of their respective contributions for the desired characteristics of the stimulation and capacity of the TNF to act against the disturbances of the immune system, among other biological effects. Furthermore, it is further noted that said identification and analysis will be used to manufacture a synthetic W-form of the product. In one aspect, the present invention relates to a composition for use as an immunomodulator comprising components of small molecular weight of less than 3000 daltons, 5 and having one or more of the following properties, a) it can be extracted from bile of animal; t > ) is able to stimulate rnonocitos and rnacró phagos i n vi tro e jj alive; c) is able to modulate the production of the factor of # 0 turnoral necrosis; d) contains no measurable level of IL-lor, IL-iß, FNT, IL-6, IL-8, IL-4, GM-CSF or FNT-t; e) has an anti-proliferative effect in a malignant cell line; 5 f) does not show cytotoxicity to human peripheral blood cells or immunocytes; and g) it is not an endotoxin. According to a preferred embodiment, the composition is extracted from bovine bile and is capable of stimulating the release of TNF. The composition of the invention can be prepared by (a) mixing the bile of an animal, preferably a bovine, with a solvent that is soluble or water-soluble, preferably an alcohol, and preferably with an equal volume of an alcohol, to produce a bile / alcohol solution, (b) separating the solution that is preferably an alcohol-soluble fraction, and isolating a substantially alcohol-free solution from the isma, removing most of the alcohol, such as by use of heat; (c) removing the bile pigments from the solution to obtain a light yellow liquid; (d) optionally treating the light yellow liquid to substantially remove any residual alcohol; (e) removing the organic fatty materials, such as extracting the light yellow liquid with ether and -___ L isolating the aqueous phase; and (f) optionally removing the residual residual in the aqueous phase, The composition can be used without further modification by simply packing it into flasks and sterilizing.The composition can also be used in a concentrated form. in the following manner: Before step (e) the light yellow liquid can be optionally concentrated to approximately one-eighth the volume of the bile / alcohol solution and after step (f) the aqueous phase can be concentrated so that it is one tenth of the volume of the bile / ethane solution The invention also relates to a pharmaceutical composition comprising the immunomodulatory composition of the invention The invention further relates to a method for treating a patient comprising administration to said 5 patient of an effective amount of a composition of the invention. "The invention further relates to the use of a composition of the invention. in the prophylaxis and treatment of diseases and conditions that require the modulation of the immune response; preferably infectious diseases, inflammatory diseases, autoimmune diseases, vaccination, rejection phenomenon associated with xenografts, and neoplasms. These and other aspects of the present invention will become apparent upon reference to the following detailed description and accompanying drawings. In addition, several advertisements are referred to herein, which are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS Further details of the invention will be described hereinafter with the help of the examples illustrated in the accompanying drawings in which: Figure 1 is a high performance inverted phase liquid chromatography profile (RP-HPLC) for a concentrated composition of the invention; Figure 2 is a RP-HPLC profile for a concentrated composition of the invention; Figure 3 is a RP-HPLC profile for a concentrated composition of the invention; Figure 4 is a graph showing the effect of the composition on the LPS-induced release of FNT Ib flfc by peripheral blood ononuclear cells (PBMNs); Figure 5 is a bar graph showing the effect of the composition on the l.PS-induced release of TNF by PBMNs; Figure 5 is a graph showing the survival taken from the diagnosis of patients with pancreatic cancer treated with the composition of the invention; Figure 7 is a graph showing the survival f returned from the treatment of patients with pancreatic cancer treated with the composition of the invention; Figure 8 is a graph showing the survival of all patients with melanoma treated with the composition of the invention; Figure 9 is a graph showing the survival of patients with elane a with two or more tumor sites treated with the composition of the invention; Figure 10 is a graph showing the survival of patients with melanoma with three or more tumor sites treated with the composition of the invention; Figure 11 is an SOS gel of the composition of the invention; Figure 12 shows the elution conditions and times of the composition of the invention in hydrophilic HPLC (a) and the elution profile for a supernatant of the composition of the invention (b); Figure 13 shows the elution of a precipitate of the composition of the invention in hydrophilic HPLC; and Figure 14 is a graph showing the dose response of the composition of the invention in the stimulating function of pyocytoid blood of the perifoneum.

DETAILED DESCRIPTION OF THE INVENTION The hypothesis against! What guides these studies is that the therapeutic efficacy of a powerful biological stimulator may depend on its ability to attract adequate modulation of the immune system, such as by activating rnacro phages and / or rnonocytes to produce certain cytokines or promoting activity to search and remove or destroy cells induced or caused by the disease, such as foreign cells or mistakenly placed. For example, the shift function in environments containing malignant disease would be a useful therapy to combat cancer. Such a function could be generated by the direct stimulation of immune cells resident in tumor microbiotics. Alternatively, this function can be generated by stimulating the circulating immune cells if those cells were then able to lodge in sites of malignant disease and function in that environment. Other diseases and conditions that have an important immune system aspect would be combated or improved by treatment with a suitable immunornodulatory agent. Said diseases or conditions include endornetriosis (which is a disease condition in which normal endothelial cells proliferate in suitable sites), vain infectious diseases, and others, as will be described in more detail below. As previously mentioned in the present, the The present invention relates to a composition for use as an immunomodulator comprising components of small molecular weight of less than 3000 daltons, and having one or more of the following properties: f .. - P ^ ^ ra-r- * - ».» -,., < . a a,, b and b) is capable of stimulating rhonocytes and macrophages in vivo; c) is able to modulate the production of the necrosi s factor to the; d) contains no measurable level of IL-iw, 15 IL-lß. FNT, IL-5, IL-8, IL-4, 6M-CSF or FNT-t; e) has an anti-proliferative effect in a malignant cell line; f) does not show cytotoxicity to human peripheral blood mononuclear cells or lymphocytes; Y g) is not an endotoxm. More particularly, investigations have shown that at least one of the compositions of the invention stimulates normal rnonocytes to perform cytotoxicity towards Chang's hepatoma cells, which test is used to measure the activation of rnonocito and rnacrofago. The rhnocytes and / or macrophages of K-cancer patients (including cervical, ovarian, napz / oi do / throat, lung, and endornetrial carcinomas, Kaposi's sarcoma, and chronic iogenous leukemia) have also been shown to be stimulated by the composition to attack and destroy your own particular tumor cells. In addition, the macrophages of patients afflicted with endonetriosis have been shown to be activated by the composition as well (see example 2S). The composition of the invention can modulate the production of tumor necrosis factor (TNF). A preferred composition of the invention isolated from bovine bile promotes the release of TNF from human peripheral blood rnonucleus cells and the pre-monocyte cell line U-937 in what appears to be physiological quantities. Since TNF is known to initiate a cascade of inflammatory and anti-tumor cytokine effects, the preferred composition can exert its antineoplastic effect by stimulating human leukocytes to release TNF (and possibly other cytokines). Accordingly, the present invention can also improve lymphocyte and inacrofago cytotoxicity towards tumor cells. It has been found that the composition of the invention also inhibits the growth of cells of the mouse hybrid cell HYB-6-1 line. The inhibitory effect of the composition on mouse hibrandna cells suggests antiprozymatory activity. The effect of the composition on the P survival of human peripheral blood cells (PBMNs) and lymphocytes was also examined. It was found that the composition was non-cytotoxic for human PBMNs and Imfoeitos. As exemplified below, the composition of the present invention has, inter alia, the following characteristics: 1) The component or components responsible for the release of TNF from PBMNs eluted early from a column of RP-HPLC Gis-2) The composition causes the release of interleukin-lß (IL-lß), and the component responsible for the release of IL-lβ elutes early from RP-HPLC, suggesting that it is probably the same substance (s) (s) that releases FNT. 3) The composition also causes the release of low amounts of interleukin-2 (IL-2). 4) The composition causes the release of granulocyte macrophage colony that stimulates the factor (GM-CSF), - 5) The ratio of the release of TNF to GM-CSF is 0 about 2: 1. 6) It is likely that the same (r) molecule (s), that is, component (s), in the composition will be responsible for the release of FNT, IL-lß and GM-CSF. It is possible that the composition acts to stimulate the release of 5 different multiple cytokines, or alternatively, the composition seeks the production and release of a cytokine which in turn stimulates the production and release of other cytokines. 7) Physicochemical analysis of the composition, including the precipitates and supernatants thereof, by means of SOS gel electrophoresis and rnolecular sieve HPLC. indicates that the main components are less than 25U0 daltons. 8) The additional uirnical physical separation by hydrophilic molecular sieve HPLC (polyhydroxyethyl) confirms the small molecular weight of the components in the composition. 9) Analysis of acid arnin before and after acid hydrolysis suggests the presence of peptide bonds, indicating the presence of peptides. As mentioned before, the composition of the invention can be prepared by mixing the bile of a animal, preferably a bovine, with an equal volume of an alcohol to produce a bile / alcohol solution; (b) separating the soluble fraction from alcohol and isolating a solution substantially free of alcohol; (c) removing bile pigments from the solution to obtain a liquid light yellow; (d) treating the light yellow liquid to remove substantially any residual alcohol; (e) extracting the light yellow liquid with ether and isolating the aqueous phase; and (f) removing residual ether from the aqueous phase. The composition is obtained from the bile of any animal that produces bile. While the composition may possess a different activity towards a specific disease or As SJ is obtained from bile in one species as opposed to another, a generally adequate source of bile becomes shark, bovine, ovine, caprine and porcine. In most cases, it is practical to get bile from animals well slaughtered butchers, such as cattle, sheep, goats and swine, to be used in the preparation of the composition of the invention. The bile so collected must come directly from bladders and / or liver organs (as appropriate to the anatomy and physiology of the species) of the slaughtered animals and must be substantially clear, thus indicating that the preparation of bile is substantially free of pus or blood. In a preferred embodiment of the method, bile from bovine sources is used. Bovine bile is abundant and 15, in part, relatively large quantities of each animal can be extracted. However, cattle are often slaughtered and inspected under health-related regulations, so that animals provide a reliable source for preparing the composition of the invention. In addition, humans are less likely to have an allergic reaction to material of bovine origin. The bile is mixed with an equal volume of an alcohol to produce a bile / alcohol solution, which is 50% alcohol. The alcohol can be an aliphatic alcohol, preferably methane, ethane or propane, most preferably ethane. A solution that is substantially free of the WÉ 50% insoluble material of alcohol can be isolated by centrifugation. Preferably, the bile / alcohol mixture is centrifuged at 3000-5000 f-pm, most preferably 4200 rprn, for at least 2 hours, at about 15-25 ° C. The alcohol contained in the soluble bile / alcohol solution can then be removed by taking advantage of the different volatility of alcohol and water, using conventional methods, ie, heating the fraction to a suitable temperature, for example, 80- 85ßC, for an appropriate amount of time, for example, up to about 10 hours. The bile pigments can be removed from the solution to obtain a clear yellow liquid using activated charcoal, polyarnidic granules or iLtration. Preferably, an activated carbon treatment is used. 5 The procedure can be repeated so that the solution satisfies the standards of optical density and conductivity. The clear yellowish liquid was treated to remove substantially any residual alcohol, using conventional methods. Preferably, the clear yellowish liquid 0 is filtered using a filter having approximately a retention of 1.0-3.5 μm, most preferably a retention of 2.5 urn. Then, the clear yellowish liquid was extracted with ether and the aqueous phase was isolated. The ether used in this step is preferably dirnethyl ether, ethyl ether, n-propyl ether, isopropyl ether, or n-butyl ether, most preferably ethyl ether. The residual ether can be removed from the aqueous phase, for example by heating the solution to 55 ° C, preferably up to approx. 40 ° C during approximately 5-15 hours, very preferably for approximately 10 hours. The composition can be used without further modification, simply packing it into bottles and sterilizing. S The composition can also be used in a concentrated form. 0 A preferred concentrated form is prepared as follows. Prior to step (e) described above, the clear yellowish liquid can optionally be concentrated to approximately one-eighth the volume of the bile / alcohol solution., for example by heating to a temperature of minus 5 of about 85 ° C, preferably to about 60 ° -70 ° C. After step (f), the aqueous phase can be concentrated in one tenth of the volume of the bile / ethanol solution, for example by heating to about 80-85 ° C. In a preferred method for preparing a composition of the invention, the collected bile is mixed with an equal volume of ethyl alcohol. The bile / alcohol mixture is then centrifuged at approximately 4200 rpm for at least 2 hours, at approximately 20 ± 2C >; C. The liquid supernatant is decanted and analyzed for ethanol content and pH. The bile pigments are then removed using activated ammonium carbon. The treated bile / ethanol solution is then monitored to determine its optical density (O.D.) and conductivity. The levels of D.O. or conductivity levels outside of acceptable speci fi cations require that an additional treatment is given to the bile / ethanol solution to remove bile pigments, for example treatment with activated charcoal again to achieve a reading within specification limits. w Jfe. After treatment with activated carbon, the solution is filtered through a filter having a retention of 2.5 μm, the alcohol is evaporated off by heating to below 85 ° C and the solution is concentrated to approximately one-eighth of the volume original of the bil is / ethanol solution. The concentrated solution is cooled to between about 20-25 ° C. Then, this solution is mixed with ethyl ether and the ether phase is discarded. Preferably, relatively small volumes of ether and strong stirring are used, such as 0.1 to 1 volume, preferably 0.2 to 0.5 volumes. This step can be repeated once. The aqueous phase is heated to remove residual ether by heating to 55 ° C for about 10 hours, and then reduced in volume to one tenth of the original volume of bile / ethanol heated to about 80-85 ° C. This solution is then analyzed to determine appearance, biological activity and ethanol and ether content. The pH of the composition can be adjusted to the pH -? physiological, osto is, 7.4-7.5, using a solution of hydrochloric acid (L%) and sodium hydroxide (1% solution), and a solution regulated in its pH can be obtained using dibasic and monobasic salts of sodium phosphate as regula pH meters, using conventional methods. The composition can be used without further modification by simply packing it into bottles and sterilizing it.

A preferred method of sterilization is to subject the composition ? to three cycles of sterilization by autoclaving, followed by Incubation ID The composition can be used in a concentrated form. The preparation of the concentrated form was described above. Also, the composition can be made freeze-dried. The composition and the concentrated composition are clear yellow solutions essentially free of foreign material containing no more than 10 ppm of ethanol and no more than 5 ppm of ether. The compositions activate PBMNs to release FNT m vitro as measured by the rnonocito / macrophage activation test (TNF release) as described in Example 2. In addition, the compositions activate PBMN's, for example from a patient with cancer, which later mediated cytokine activity against cancer cells derived from the same patient. In fact, clinical studies involving animals and humans have shown efficacy against tumor treatments using the composition of the present invention. Similarly, it has been seen that PBMN's activated with the composition < < They act on endotrial cells, such that the use of the composition to treat endoin and other inflammatory diseases is provided by the present invention. The compositions of the present invention can be produced in a consistently reproducible manner using the method described above in general form, with proven identity, potency and purity from batch to batch. Identity and purity are determined using chroma ografí reversible phase high pressure liquid (see example 1). the compositions of the invention have a consistently reproducible pattern on reverse phase HPLC. The HPLC readings for three batches of the concentrated composition of the invention are shown in Figures 1 to 3. The compositions also are characterized by the above-mentioned properties, for example their ability to stimulate erythrocytes and erythrogens in vitro and in vivo, etc. Compounds that are probably present in this composition, considering the source, include acids Sulfonated bile, oxidized bile acids, other bile acids of natural occurrence and their amino acid conjugates (especially glycine and taurine), and sterols. Therefore, it is believed that the present composition includes at least one compound having the formula wherein the molecule may or may not be completely saturated, so that, for example, the molecule between A and B, B and C, or C and D H, OH, = 0, or "n CONHR; Wherein R is an amino acid residue such as for example glycyl, giutamyl or tauplo, thereby forming the conjugate of glycine, glutaryl or taurine. In particular, the composition of the present invention has been analyzed with respect to its component compounds, including organic and non-organic components.

This information was derived using normal methods of analytical chemistry, including loop spectroscopy (FM). The results of these studies include, for example, identifying specific compounds ICOS bLliar acid so estimated are present, including cholic acid, 5 glycocholic, deoxy glycocholic acid, ursodeoxycholic acid, cholesterol sulfate, deoxycholic acid, quenodesoxícól ico , and taurocolieo acid. From MS it is not distinguishable whether the loss of J. OH and l-2 of some compounds occur in the CM or if the anoxic, deoxy, dideoxy and unsaturated analogs of these compounds are also present from the beginning. These compounds may all be present as ammonium salts, alkyl amio and inorganic cations. The analysis of EM also supports the identification of the present composition of phospholipids, sphingolides, and related agents capable of forming them. Specific compounds which are thought to be present include: stearic acid CH3 (CH2 hßCOOH palmitic acid CH3 (CH2) i4CQ0H 0 oleic acid Z-9 octadecanoic acid CH3 (CH2) CH2CH = CHCH 2 (CH 2) 6 COOH oxidized fatty acids or sides hydroxy msaturados of short chain: C6H8O3 (eg, CH3 CH = CHC0CH2 COOH or acid of Ce 2 double bonds and hydroxides) 5 acetic acid diglyceride stearic acid di gil W cer gone pal acid mythical diglicepdo acid palmit-ico and stearic acid phosphocholine-rnonogl icérido stearic acid (one lisolecitma) 5 monoglicéndo stearic acid tpglicepdo stearic acid rnonogl i cérido acid pal mi fos tico ocolina # f os fosen na esfingorniel ina phosphoglycerol esfingonsma glycerol stearic acid 15 amide esfingosma of stearic acid rnetiiarnide of acid this rich coli na gl i ce ro fo focus 11 to 20 di liérido of oleic acid and acid this rich phosphoglycerol of oleic acid o and stearic acid palrnitic acid amide LCO lecithin dirner of sialic acid-licerol In addition, evidence of CLAR and titration demonstrating that short chain fatty acids are also present, such as those having from about 1 to 30 carbon atoms. The olipidos, espingoli pidos and related hydrolysis products that are probably found, considering the source and information derived from the EM and OLAR analysis, include at least one compound that has the formula Wherein R1, R2, R3 are different or the same and are H, C0R4, CH-CH-R5, x, -P (O) (OH) 0-, or -S (0) 2? -; X is selected from the group consisting of fills, etanolarní na, etanolaninas Ladas N-alk, sen na, inositol, sugars bearing free hydroxyls, arninoazúcares, sulfonated sugars, and sialic acids; R¿ is C 1 -C 30 alkyl which is saturated or unsaturated, oxidized or hydroxylated; and R5 is an alkyl group or oxidized and / or hydroxylated analogs thereof. The fatty acids and their conjugates may be present in the aqueous extracts mentioned above and as such. The solubility of these compounds is also increased by other components of the mixture. It is also believed that amides of the included carboxylic acids are present, RCONR R2, where R1 and R2 are the same or different and are H3? or alkyl. A third class of compounds, mainly rnucin and proteoglycan hydrolysis products, is also likely to be present, considering the source of the composition and the aforementioned CM analyzes thereof. These compounds include hydrolysis products of bil- toproteins of the bile and the gallbladder wall, such as: 4- and 5-chondroidal sulfates., sulphate of depnatan, hepapna, hepapna sulfate, hyaluronic acid and the hydrolysis products (monomers, digesters, oligomers and polymers) of these mucmas. Simi- larly, chitin and other mucins may be hydrolyzed, and their hydrolysis products may include: N-acetyl-D-glucosarnine, N-acetyl-D-15-galactosamine 4-sulfate, galactose-5-sulfate, 5 -N-acetyl-D-glucosarnine sulfate, glucosam 6-sulfate, D-2-sulfate ~~ giucosarni, 2,3-d-sulfate of D-gl ucosamine, 6-sulfate of D-galactose, 2-sulfate of glucuronic acid, N-acetylneuranic acid, sialic acid, N-acetyl-chondrosma, 4 - 20 chondroitin sulfate, chondroitin 5-sulfate, D-glucosarnine, D-gaiactosamma, glucuron acid, glucose, galactose, maasose, fucose, iduronic acid, hexose, hexose ina, ester sulfate, glucuronic acid, cond rosarn ina, 2-arnmo-2 ~ deoxy-D-galactose, serine, prolma, treomna, alanma, glycine, taurine, glutamic acid, aspartic acid, histidine and small peptides.

Wtt Similar products could be obtained by the hydrolysis of rnucins such as keratin sulfate, succinates from the skin; the natural sugar-sugar bonds in the dimer, oli, and polymer can be replaced by bridges -0- 5 If (0H) 2-0- between the sugar monomers or sugar chains-adjoin is. In particular, the compounds of the specific hydrolysis products of rnucin and proteoglycan that are thought to be present include: sialic acid and its mono- and di-acetylated monomers and glycolides; N-acetilneurarnini co acid; hexosarnins such as glucosarnine; L- fucose; hexosarnine disulfate - hexuronic acid (di ero); giucuronic acid; glucuronic acid disulfate or uronic acid rnonoaceti side; dimer of sialic acid and glycerol; and dyrneros, trimers, oligomers, and polymers of the previous inorornanes in acetylated and sulphated form. A fourth class of compounds, especially fat-soluble vitamins, which are probably present considering the above-mentioned source and analyzes of MS, include vitamins A, B and K (for example A2, DI, D3, D4, Ki, K2, K5, K6, K7, K-S (II), and vitamin E acetate, for example. In particular, the specific compounds of fat-soluble vitamins that are thought to be present include at least one of the group consisting of vitamin A2, vitamin DI, Lurnisterol (present from its vitamin WM Di complex), vitamin E, vitamin K oxide, and vitamin K5. Different miscellaneous organic compounds are probably present, considering the source and analysis of EM mentioned above. These compounds include: urea; alquilinins, including methyl lamina, dirneti lamina, ethylamine, ethylethylarna, diethylamine, dipropylamine and / or butylathylanine; amino acids, including taurine, glutarnic acid, glycine, alanine, n-lecma, ff os, phosphoethanolane, aspartic acid, threonine, serine, sarcosine, acid «-arninoadí peak, citrulline, valine, isoleucine, β-alanine , t-arninobutiiic acid, hydroxylysine, ornithi and lysine; bilirubin and its giucuronide conjugate; biliverdin and its glucuronide conjugate; butylated hydroxytoluene (BHT); polyethyleneg i col; traces of steroids; other solutes from plasma, such as sugars, purines and pyrimidines; miscellaneous lipids of the diet; and glutathione and its products ~ hydrolysis. In particular, the miscellaneous specific organic compounds that are believed to be present in the composition include at least one of the group consisting of urea, lami alam, dirneti lamina, eti lamina, inet i letí lami a, diethylamine, dipropylamine, but? letilatin, ammonia, choline, taurine, glutaric acid, lysine, alanine, p-ser, p-eu, p-ea, asp thr ser sar, a-aba, cit, val, ile, leu, B-ala, 6- aba, 0H-25 lys, orn, lye, butylated hydroxytoluene (BHT), and polyethylene glycol.

The amines present in this composition, particularly the secondary amines may include nitrogen oxides from the air, thereby forming nitrous compounds. N-oxide and N-carbamate by-products may also be included. This series of amines mentioned above should be extended to include all primary, secondary and tertiary alkylamines. Some inorganic elements have been identified and quantified (rng / 1) as follows: Tungsten 0.07 Zinc 0.6S6 Phosphorus 378 Cadmium 0.0.1 Cobalt 0.008 Nickel. 0.022 B rium 0.032 Iron 0.022 Manganese 0.039 Chrome 0.050 Magnesium 7.45 Aluminum 0.135 Calcium 5.97 Copper 0.087 Titanium 0.01 Strontium 0.050 Sodium 9600 Potassium 483 Chloride 15400 Ammonia 218 Vanadium 1 pprn The compositions of the invention have valuable pharmacological properties. In particular, the compositions of the invention effect neoplastic growth, effect of tumor necrosis factor release, and activate mjt and monocyte macrophages. The compositions have shown that they do not cause significant toxicity and only have temporary adverse side effects (eg, fever, slight fever, polydipsia, pain at the site of the injection). It has also been found that do not contain detectable components of high molecular weight matter (this, above about 5,000 daltons), which may cause reactions in a dangerous manner. The compositions can be used as agents for the prophylaxis and treatment of conditions that require modification of the immune response, in particular infectious diseases (including bacterial, fungal, protozoa and other opportunistic infections), inflammations (including endornetposis and inflammatory bowel disease). , vaccinations (including its use as an adjuvant for HIV, routine immunizations for children and adults such as diphtheria, pertussis, tetanus, polio, measles, mumps, Rubella, viral influenza and hemophilia; and vaccines for the traveler such as typhoid, cholera, plague, bacterial meningitis and malaria), neoplasms and autoimmune diseases. These diseases are associated with, and may be the direct result of, an inadequate response of the immune system. As indicated in the previous background section, both humoral and cellular defects may be involved. The present invention, in view of its exemplified ability to By activating genes and macrophages, it can increase both of these aspects of the immune response. In addition, the composition of the present invention can be used to decrease or block the rejection phenomenon associated with organ transplants either mth or m-ospecies. The composition of the invention can also be used to treat radiation sickness. Therefore, the compounds of the present invention may be specifically useful in the treatment of different forms of neoplasia, such as leukaemias, inflammations, melanoma, adenomas, sarcomas and carcinomas. In particular, the composition can be useful for treating malignant pancreatic cancer, cervical cancer, cancer of the kidney, stomach, lung, rectum, ovary, breast, intestine, gastric, liver, thyroid, neck, cervix, salivary glands, leg, tongue, lip, bile ducts, pelvis, mediatm, urethra, bronchogenic, gallbladder, phage and colon, and Kaposi's sarcoma, which is a form of cancer with patients infected with HIV with acquired immunodeficiency syndrome (AIDS). The composition can also be used for other conditions that are caused or produced by a defective immune response, such as arthrosclerosis, opportunistic infections or other infections such as viral infections, in particular AIDS. It can also be used in the treatment of autoimmune diseases, including multiple sclerosis, reurnatoid arthritis, general lupus erythematosus, type I diabetes, rniastema gravis, Addison's disease, autoimmune heinolytic anemia, Crohn's disease and other inflammatory bowel diseases, a ^ "Goodpasture syndrome, Graves disease, Hashuno thyroiditis or, idiopathic thrombocytopepic purpura, pernicious anemia, post-streptococcal glornerulonephritis, psopans, sclerodema, Sjogrens syndrome, spontaneous infertility, and vulgar penciflora. Inflammatory fepnedades such as endomet riosi s and inflammatory bowel disease, The compositions of the invention can be converted, 2 using the usual methods, in compositions pharmaceutical. The pharmaceutical composition contains the composition of the invention either alone or together with other active substances. These pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalation or cerebral-cerebral use. Therefore, they are in solid form or semi-solid, for example, pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes and tube forms. For parenteral and intracerebral uses, forms for intramuscular or subcutaneous administration may be used, or may be used forms for infusion or intravenous or intracerebral injection, and therefore, can be prepared as solutions of the compositions or as powders of the active compositions for mixing with one or more pharmaceutically acceptable excipients or diluents, suitable for the uses mentioned above and with an oscility that is compatible with that of physiological fluids. For local use, WM may be considered these preparations in the form of creams or ointments for topical use, or in the form of aerosols; Preparations in the form of aerosols, for example nasal sprays, can be considered for use by inhalation. Preferably, the composition is administered intramuscularly. The pharmaceutical compositions can be prepared by methods known per se for the preparation of fS pharmaceutically acceptable compositions which can administer to patients, and of course that an effective amount of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Rernington's Pharmaceutical Sciences (Mac Publishing Company, Easton, Pennsylvania, E.U.A. 1985).

On this basis, pharmaceutical compositions include, but are not limited to, the composition of the The invention is in association with one or more pharmaceutically acceptable carriers or diluents, and is contained in solutions regulated at their pH with a suitable pH and iso-osmotics with the physiological fluids. The compositions are indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment. For example, in the case of a malignant tumor, the present treatment can facilitate the surgical removal of a tumor which was not previously operable. Alternatively, this treatment can be combined with chemotherapy and / or radiotherapy. The compositions and agents of the invention are intended for administration to humans or animals. In general, a dosing scale is contemplated of the composition for administration in human measurement of about 0.1 to 20 mg / kg, preferably of approximately 0.1 to 10 mg / kg, preferably 0.1 to i mg / kg of body weight daily. In the case of admission 'intravenously, the dosage is approximately 0.1 to 5 ng / kg of body weight daily, and in the case of oral administration the dosage is approximately i to 5 mg / kg of body weight daily. Where the concentrated composition is used, about half of the dosages mentioned above can be used. For example, For intramuscular administration, a dosage of about 0.2 to 1.0 mg / kg, weight of the ~ body daily, preferably 0.275-0.75 mg / kg of body weight daily. It will be appreciated by medical practitioners who It may be necessary to deviate from the amounts mentioned and, in particular, to do so as a function of body weight and the condition of the animal to be treated, the particular disease to be treated, the nature of the route of administration and the desired therapy. Also, the type of animal and their individual behavior towards medicine or the nature of their formulation, and the time or interval at which B * is administered may also indicate the use of amounts other than those mentioned. In this way, it may be sufficient in some cases to manage less than the minimum amounts mentioned above while in other cases 5 the above-mentioned upper limit must be exceeded. Where larger amounts are administered, it may be advisable to divide these into several advices during the course of the day. In this way, the present invention comprises a - The procedure to propagate an inrnunomodul composition loves Which comprises a) mixing bile from an animal with a water-soluble solvent to produce a bile / solvent solution; b) isolating a substantially solvent-free aqueous solution of the bile / solvent solution; c) remove the bile pigments from the solution substantially free of solvent for obtain a clear yellowish liquid, preferably wherein the solvent soluble in water is an alcohol, and wherein the bile ~ The animal is mixed with an equal volume of alcohol. Preferred aspects of the aforementioned process also comprise concentrating the clear yellowish liquid to approximately one eighth, or one tenth of the original volume of the bile / solvent solution. Obviously, the compositions produced by the above process form a preferred aspect of the invention. The present invention also comprises a The composition for use as a mononuclearizer, comprising at least one component having a molecular weight of minus -tMf of about 3000 daltons, which shows no cytotoxicity to the mononuclear cells of human peripheral blood, and has at least one of the following properties: a) It is capable of stimulating rhonocytes and acrobats ijn 5 vi tro or in vivo to produce one or more cytokines; or b) It is capable of stimulating monocytes or macrophages to produce tumoral necrosis factor in vi tro or in vivo; or c) It has an antiproliial effect in a line '_______ 9 malignant cell; and 0 Where said component is not an endoto i a, IL-loc. IL-lß, FNT, IL-4, IL-6, IL-8, GM-CSF or IFN-t. These compositions can be obtained from the bile of animals, preferably bovines, or from other sources as indicated above. In a preferred embodiment of the composition, the composition stimulates the production of tumoral necrosis factor in vitro or in vivo, and preferably in humans, in the absence of IL-lcf, IL-lß, FNT, IL-4, IL-6, IL-8, Exogenous GM-CSF and IFN-t. The compositions of the present invention may also have components that can be characterized by column chromatography, so that when said composition is dried to obtain a solid residue, and 2 grams of said residue are dissolved in 20 ml of a hydroxide solution. of 10% concentrated ammonium in methanol, and after removing any insoluble material, it is subjected to column chromatography on a methanol column having dimensions of 5 cn x 12.5 c, and contains 102 g of an instant gel of 60 A silica, and operating at a pressure of 0.70 kg / c? n2, and a flow rate of 11 rnl / rnin with a solution of 10% concentrated ammonium hydroxide in ethanol solvent, said component is eluted from the column in a fraction taken when the elution of the total column is between about 180 and about 220 ml, between about 220 rnl to about 250 inl, or between about 260 rnl and j = ¡> approximately 300 rni "10 The characterization of the components can also be achieved by ion exchange chromatography, so that when 10 l of said composition are subjected to anion exchange chromatography on a column containing resin in the hydroxide form of Bio-Rad AG -1, in a sufficient amount to substantially bind all the anions present in said 10 rnl of said composition, said component is eluted from the column using a gradient of pH regulator of ammonium bicarbonate at a pH regulator concentration of about 0.1 M to about 1.5 0 M, preferably at a pH regulator concentration of approximately 0.2 M to about 0.4 M, and most preferably at a concentration d; Approximately 0.2 M pH regulator. Reverse phase HPLC (C18) can also be used to characterize the components. You can also use other columns, eluents, gradients, flow rates, operating temperatures and suitable detection systems. The compositions of the present invention can also be characterized by CCD, so that when said composition is subjected to thin layer chromatography on gel plates of silica in a suitable solvent system, such as 10% concentrated ammonium hydroxide in methanol, and visualized with a suitable aerosol, such as ninhydrin, a positive reaction occurs with neither hydroponic, so at an Rf value of about 0, 80 to about The present invention also comprises a method of stimulating the production of human neural fibrosis factor, which comprises administering an effective amount of a composition comprising at least one of the following compounds: (a) A compound of The formula 0 ? AkC? X Where the links between A-B, B-C, and C-D can be 5 single or double bonds, and where X = H, = 0, or OSO3H; and Y = COOH i c CONHR wherein R is an acidic arnino residue; (b) A compound of formula (R10) CH2CH (0R2) CH2 (0R3-X) or Where R1, R2 and R3 are H, COR, CH = CH-CH5, X, 20 PIO) (OH) O-, or -S (0) 20-; X is choline, ethanolamine, N-alkylated ethanolarnins, serine, inositol, sugars bearing free hydroxyls, arninoazuchers, sulphonated sugars, or sialic acids; and R4 is a saturated or unsaturated alkyl group that has a carbon chain of about Ci to C30, or oxidized and hydroxylated analogs thereof; and R5 is an alkyl group or hydroxylated and oxidized analogs thereof; (c) A ucma hydrolysis product or a proteoglycan hydrolysis product; or (cl) a fat-soluble vitamin. Preferably, the compositions of the method of the invention comprise at least one compound selected from the group consisting of taurocolic acid and its sulphated derivatives; giicocólico acid and its sulphated derivatives; Ngosin Sphing; diacylglycerol; lecitma; phosphocholine; phosphoglycerol; glicerofos focoli na; phosphoryl chloride coima; an oligosaccharide of less than 10 units of extracting in length; wherein said oligosaccharide is comprised of sialic acid, glucose, hexosarnins, or eulfated hexoeammas; Vitamin A; retinolic acid derivatives derived from retinol; taurine; and glutaric acid and its conjugates. The composition may also additionally comprise at least one compound selected from the group consisting of ammonia; primary alkylanins; ali Secondary laminae; rent tertiary nails; and a carboxylic acid R 6 CO 2 H, wherein β is C 1 -C 30 alkyl which is saturated or unsaturated, and oxidized and / or hydroxylated derivatives thereof. Preferably, this composition comprises at least one of the groups consisting of phosphocholine, glyphosaccharide, glucosamine 3-sulfate and phosphorylcholine chloride. Most preferably, the composition comprises at least one of the following: phosphocholine, gli-zero phosphocholine or 3-glucosamine sulphate a.

The method of the invention also encompasses stimulation of TNF production by administration of a composition comprising at least one compound selected from the group consisting of taurocholic acid and its sulphated derivatives; glycocholic acid and its sullated derivatives; sphingosine; a diacylglycerol; lecitma; a * oligosaccharide of less than 10 saccharide units in length, wherein said oligosaccharide is comprised of sulphonic acid, fucose, hexosarins, or sulphated hexosatnins; Vitamin A; 0 retinoic acid derivatives; withdrawal derivatives; taurine; and glutaric acid and its conjugates. The present invention also provides a method for treating cancer, including carcinomas of the pancreas, ear, nose, throat, ovaries, lung or endometrium, as well as chronic iogenic leukemia and Kaposi's sarcoma, wherein the method comprises administering to a patient suffering from cancer a therapeutically effective amount of the compositions of the invention. The present invention also provides a method for treating other disorders that are caused by, or are the result of, an impaired response of the immune system, including inflammatory disease, endometriosis and inflammatory bowel disease, autoimmune disease, including reurnatoid arthritis, lupus, multiple sclerosis. and ALS, infectious diseases, including infections by bacteria, fungi, ichrosarnas, protozoa and other opportunistic infections, wherein the method comprises administering to a patient affected by one of the aforementioned diseases, a therapeutically effective amount of the composition of the invention. . In addition, the present invention provides a method of vaccination for prophylaxis against HIV, vain diseases of childhood and other diseases, wherein the composition of the present invention is added to said vaccine as an adjuvant. Also forming part of the invention are M * compositions comprising (i) sphingin micelles or Sphingosm complexes with one sai, or (2) micelles of mollic acid or derivatives thereof, having at least one of the following proto-ages: (a) It is able to stimulate onocitos and macrophages m vitro to produce one or more cytokines; 5 (b) It is able to stimulate monocytes or macrophages to produce the turnoral necrosis factor in vivo or live; or (c) has an antiproliferative effect on a malignant cell line. The micelles may also comprise a diacylglycepe or lecithin, and may further comprise a salt of bile acid, and a source of ammonium or alkyl ammonium ions. Finally, the present invention also contemplates compositions comprising (1) sphingosm, a salt of bile acid and a source of ammonium or alkyl ammonium ions, (2) a salt of bile acid, sphingosine or diacylglycerol, a ßr source of ammonium ions or alkyl ammonium, and a retinol derivative, (3) an iglicerid diacid, lecithin and a bile acid salt-, or (4) (a) a diacyl glyceride, (b) leci ina, and (c) a hydrolysis product of rnucin or a hydrolysis product of eogl ican prototype having at least one of the following properties: a) It is capable of stimulating rhinocytes and macrophages in vitro to produce one or more cytokines; ^ Bf b) It is capable of stimulating rhonocytes or acrophages to produce the tumor necrosis factor in vi tro or in vivo; or c) has an antiproLiferative effect in a malignant cell line. The following non-limiting examples illustrate the present invention: EXAMPLE 1 This example describes and illustrates the preparation of the composition of the invention. Bovine bile was collected from the biliary vesicles removed from healthy cows (both males and females) that were at least one and a half years old. These cows were sacrificed for use as food in an approved and inspected abattoir. The sacrificed animals had been inspected and evaluated as healthy prior to slaughter, and the biliary vesicles were separated from the livers and examined by a veterinarian to confirm that the biliary vesicles were free of parasites and evidence of infection, and were thus appropriate for used as a source of bile for the present invention. The biliary vesicles that passed this inspection were subjected to the following procedure: The biliary vesicles were wiped with a 70% ethanol solution to clean the exterior of the vesicles, and the bile was removed from the vesicles with a syringe. The removed bile was examined visually in the syringe by the veterinarian to make sure it did not contain blood or pus and was satisfactory. The bile of a healthy bovine is a greenish fluid substantially free of blood and pus. They were collected also fragments of livers, spleen and lymph nodes of the animals whose bile was collected, and the fragments were examined 'st for the presence of parasites and other indications of disease. For species that do not have a defined gallbladder (such as the shark), bile is obtained directly from the liver organ. The bile that was found was satisfactory, transferred in a graduated amber flask containing ethanol to give a solution of 50% bile / 50% ethanol by volume. The The bile / ethanol solution was a greenish fluid substantially free of foreign material and tested as positive for ethanol. in accordance with the methods described in the United States Pharmacopoeia XXII, part B (1994). These bottles were marked with a batch number. For each batch, bile collected from a minimum of 50 animals was collected. The bile / ethanol solution was centrifuged after 4200 rprn for at least two and a half hours at 20 + .28C. The supernatant liquid was decanted, filtered through a filter having, for example, a 2.5 μm retention, and c M for pH and ethanol content. The decanted liquid was then subjected to an activated carbon treatment. The treated liquid was then monitored for optical density (OD) at 280 nrn and conductivity. DO levels and / or conductivity levels outside the specified scales required additional liquid-to-carbon treatment to achieve OD and conductivity within the specified scales. After treatment with activated charcoal, the treated liquid was filtered through a filter having, for example, a 2.5μrn retention, the ethanol was evaporated (for example, warming up to about 85 ° C), and the treated liquid was concentrated to about 1/8 of the original solution of bile / ethanol in volume. The concentrated liquid was then cooled to 20-25 ° C, filtered through a filter having, for example, 2.5 μm retention, and mixed with ethyl ether, and the ether phase was discarded. This step can be repeated once. The aqueous phase was heated to remove the .0 residual ether (e.g., heating to approximately 55 ° C, for about 10 hours), and further reduced in volume to one tenth of the original volume of bi li / ethanol, heating to about 80-85 ° C. The resulting composition was then tested for appearance, biological activity and ethanol and ether content. The composition was a clear yellowish solution, essentially free of foreign matter, and contained less than 10 ppm of ethanol and less than 5 ppm of ether. identity and purity were determined using reverse phase high pressure liquid chromatography (reverse phase HPLC). The potency was tested using the activation test of monocytes / m-scavengers referred to in the present invention as the tumor necrosis factor test of peripheral blood mononuclear cells (FNT-PBMN test or, simply, TNF test), horn is described in example 2. The initial batches of the composition of the invention were manufactured with a liquid not regulated in its pH. Subsequent batches were manufactured as a liquid regulated in their pH, prepared by adjusting the pH of the composition to approximately 7.4 +/- 0.2, using hydrochloric acid solution (1%) and sodium hydroxide (1% solution), as well as using monobasic and dibasic sodium phosphate salts as pH regulators. The reduction of Bioburden was carried out in a steam autoclave at 104 +2 ° C for 60 minutes. The global solution was poured into sterile 5 p bottles | ml or 10 ml, and capped. The filled and capped bottles were subjected to 3 sterilization cycles by autoclaving at 104 ° C + 2 ° C for 60 minutes, followed by incubation at 35 ° C for 23 + 1 hours. Between each cycle of 5 sterilization (autoclave plus incubation), the samples were returned and tested for Bioburden reduction. After the last sterilization cycle, the bottles were visually inspected against a black and white background to detect the presence of particulate matter. 0 After inspection, the lot was sampled and tested in accordance with specifications. The tests included identity, sterilization, pyrogenicity, endotoxin, biological test, CLAR and general safety. Table I summarizes the data obtained for the different tests performed on the bile extract of the present invention, including normal data scales, when appropriate.

TABLE I Characteristics of the batch compositions obtained in accordance with the method of example 1 ..OTE NO. LOT NO. LOT DO NOT. FINAL PROOF OF PRODUCT BC0248 BC0249 BC0250 * The potency was measured with respect to the activation of rhonocytes / rnacrophages as described in Example 2; The normal release of TNF-or is at least 100 pg / l.

Accordingly, the novel composition can be prepared from easily obtainable sources of bile, using standard laboratory methods, resulting in a standardized final product. t) EXAMPLE 2 This example describes the biological activity of the E '- composition of example l. 0 Studies were conducted to evaluate the effect of the composition of Example 1 on the release of cytokine from peripheral blood mononuclear cells (PBMN) and / or U937 cells, which is a stable line of pre-mononucleic cells (American Type Culture Collection (ATCC), 5 Roc ville, Maryland). ELISA tests were performed for FNT-a, IL-la, IL-2, IL-4, IL-6, TL-8, GM-CSF and IFN. These studies provide the basis for a standardized test to quantitatively evaluate the potency of a given bile extract prepared in accordance with Example 1, which test 0 evaluates the ability of the bile extract, or a component or components thereof, to stimulate the production of TNF-a in PBMN or U937 cells. Whole blood was drawn from 5 healthy human subjects in hepapnicized Vacutainer tubes (Beckton 5 Dec inson, Canada). The PBMNs were isolated by gradient centrifugation over Ficoll-Hypaque (Pharmacia).

Wm The PBMNs were washed? times with pH regulated saline with phosphate (PBS), were counted and resuspended in RPMI 1640 culture medium (Gibco Labs) at a concentration of 10 6 cells / 0.5 nl. These cells were grown in ac of 5 tissue cuff from 24 flat bottom wells (Falcon, Becton, Dickinson). An aliquot of 0.5 ml of the PBMN suspension containing 50 ng of lipopolsaccharide (LPS) (from E. coli), 10 μl of fetal calf serum and 10-300 μl of the a- was added to each well. composition of example 1, as seen in the following 0 tables. The hyper olar effect of the composition was neutralized by adding distilled water to the culture wells at a volume equivalent to 10% of the volume of the composition used. The total volume was then formed at 1 ml / well with RPMI. PBS was used as control. Cells were cultured for 2, 6, 24, 5 48 and 72 hours at 37 ° C in a humidified CO2 incubator %. At the end of each incubation period, the cells were discharged V »obt vi n« _, «, culture X." re, e cé.u.a. by centrifugation at 9000 rpm for 10 minutes. The samples were then stored for up to 2 weeks at -070 ° C, until immunoassays, such as ELISA, were carried out to quantify the cytokines present. The synthesis of cytokine in the supernatants was measured after stimulating the human PBMNs with the composition of Example 1 at volumes of 100 and 200 μl per well. The initial 5 preparations of the composition showed no direct stimulating effect (ie without L.PS) on cytokine production (see Table II). If there was any effect, it appeared that the cytokine production was below the constitutive level when the PBMNs were incubated alone in the medium.

TABLE II Direct effect of the composition of Example 1 on cytokine production after 24 hours 10 Amount of cytokine released (pg / ml) 1 # Composition 15 Tested cytokine Medium 100 μl 200 μl 1 μg twenty < %. i Sample average of eight patients in duplicate 2 Sample average of seven patients in duplicate 3 ng / ml 40 The cytokine synthesis in the supernatants was measured at 24 hours at 37 ° C after stimulating the PBMNs with the composition of the example 1 and LPS (or LPS only as a positive control), using volumes of 100 μl of the composition of example i per well. The FNT was measured by means of a FNT-or ELISA kit (Endogen, Inc.), which detects a minimum level of 5 pg / inl of the cytokine. The other 5-ELISA equipment for in-house testing that was used included: 1L-le * (Endogen, Inc.); GM-CSF (Endogen, Inc.); RFN-a (Endogen, Inc.); IL-? (Advanced Magnetics, Inc.); IL-6 (Advanced Magnetics, Tnc); IL-1 (Advanced Magnetics, Inc.); IL-4 (R &D Systems); e TL-8 (RSD ¡j-y Systems), The results indicated that FNT was the main cytokine present in the supernatants, along with smaller amounts of IL-1β and GM-CSF. For example, a dose of 40 μl of the composition of example 1 (lot B0222) stimulated the production and release of 178 pg / ml of TNF-a, 136 pg / ml of GM-CSF and 142 pg / ml of IL-1β . 5 Different batches of the composition of Example 1 were examined for their effect on the release of TNF induced by LPS. In summary, it was found that batches of the composition produced in the same manner and from the same animal induced an identical effect. However, the 0 variations in the method of preparing the composition or the use of a composition prepared from different animal species had different effects. For example, lots B29 / 3006, B0213, BC0241, BC0241-01, BC0242 (B = bovine) and C0203 (goat) induced a marked release of TNF at the top 5 of that induced by LPS alone, as shown in Table III , while lot 013/2109 (sheep) minimally stimulated the release of TNF in all doses tested. In • contrast, the. Lot R0201 (shark) inhibited the release of TNF in most of the doses tested. The TNF values shown in Table III were calculated as the difference in the release of. FNT-a between the stimulation produced by LPS and the composition of example 1 combined, minus the stimulation produced by the LPS alone.

TABLE III Effect of the composition of Example 1 on the release of TNF induced by LPS from PBMNs Lot Volume of Composition (μl) FNT (pg / ml) B0213 10 193 1.61 100 858 819 200 2131 1742 B29 / 3006 10 121 ± 1.02 50 422 ± 78 100 834 ± 811 200 2252 ± 676 C0203 10 101 ± 47 50 643 ± 231 100 2650 ± 1,372 200 1851 ± 980 BC0241 10 199 25 201 50 162 100 339 200 552 BC0241-01 10 170 25 180 50 219 100 223 200 589 BC0242 10 294 25 401 50 409 100 603 200 574 013/2109 50 -9 ± 73 200 179 ± 162 300 178 ± 373 R0201 50 145 ± 256 200 -370 ± 385 300 • 400 ± 185 Since the composition of Example 1 affected the release of. FNT induced by LPS from human PBMNs, a series of experiments was performed to examine the effect of the composition on the release of TNF induced by LPS from PBMNs with time.

TABLE IV Effect of the composition of example 1 (lot B0213) on the release of TNF induced by LPS (pg / ml) from PBMNs with time LPS alone LPS + Cornfnosición Time (hrs) (50 ng / rnl) (100 μl) 15 2 697 ± 94 693 + 339 6 2005 ± 736 1949 + 442 20 24 800 ± 222 2301 + 658 48 170 ± 149 14.1.9 __ 447 25 72 132 ± 147 945 ± 367 $ Table IV shows that, during 2 hours, level 30 of TNF release from PBMNs induced by LPS had risen to 697 pg / ml and reached a maximum at 6 hours at approximately 2006 pg / ml. During 24, 48 and 72 hours, the release of TNF progressively decreased. In fact, during 48 and 72 hours, the release of TNF from PBMNs induced by LPS was just above the constitutive levels of production. In contrast, the LPS in combination with lot 52 * B0213 of the composition, which is a strong simulator of the release of TNF, induced a maximum release of TNF in 24 hours, at a time when the stimulating effect of LPS had begun to decrease. Unlike the LPS alone, the L.PS in combination with batch B0213 of the composition with inuo stimulating the release of TNF for 48 and 72 hours at levels well above the constitutive levels of production. These data show that batch B02L3 of the jj composition of Example 1 is effective to stimulate production of TNF over time. Lot R0201 of the composition, which was obtained from sharks and is an inhibitor of TNF release, markedly inhibited the release of TNF in 2, 6 and 24 hours.

For 48 and 72 hours, lot R0201 had positive effects or minimum negatives ,. In summary, the above results indicate that some batches of the composition (eg, from shark) inhibit the release of TNF from PBMNs induced by LPS, while other batches, such as those derived from cattle, goat and sheep, stimulate the release of TNF induced by LPS. In conclusion, the composition of the invention can modulate the production of the FNT, both positively and negatively. A summary of the data is shown in Figure 5 and Table V. 25 TABLE V Summary of the stimulant and inhibitory effects of the compositions of Example 1 Normal Regulator Liber ci n Lot No. FNT Concentrated pH Source * twenty The PBMN TNF test was described Previously, it was normalized using 100 μl of the composition of example 1 and 50 ng of LPS. PBMNs from 3 different human subjects were obtained as described above, and were used on the same day. The results of each of the three tests (using individual cells of the subject) are averaged to compensate for variations in response between different subjects. The analysis included determining the amount of TNF-α released in RPMI media alone and in the presence of 50 ng of LPS. The release of TNF-α was also determined in the presence of 100 μl of the composition of Example 1 in combination with 50 ng of LPS. The TNF-α released in the media was subtracted from the LPS value to obtain the TNF-α released in the presence of LPS alone. The values of the media and the LPS were subtracted from the combined composition and the value of the LPS to obtain the TNF-a released in the presence of the composition alone (reported in pg / rnl). Thus, the release test of 5 FNT was used to quantify the power of the bile extract. It was also found that the composition stimulates the release of TNF-a from U937 cells, which originally derived from a patient with histocytic lymphoma, and shows ^ - Many characteristics of monocytes. U937 cells can 'obtained from the ATCC. They are often maintained in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat inactivated calf fetal serum (FCS, GIBCO), L-glutanin at 2nmM (ICN Biomedical Tnc, Costa Mesa, CA) , and magnesium sulfate at 10 μrn / rnl (SIGMA, Mississauga, Ontario, Canada) at 37 ° C, and CO2 at 5%. The transition of the U937 cells was performed every 3-4 days, and the seeding was performed at a - Initial concentration of 5 x 105 cells / rnl. U937 cells can be stimulated to differentiate into rhonocytes by exposure to phorbol 12-mymethate-13-acetate (PMA; Sigma Chemical Co., St. Louis, MO). The resulting monocytes have the ability to release TNF after stimulation, such as the composition of Example 1, alone or in combination with LPS. The PMA was first dissolved in dimethyl sulfoxide (DMSO, SIGMA) at a concentration of 10 mM, and then diluted 1000 times with PBS to a concentration of 10 μm supply solution, and stored at -20 ° C. Suspensions of U937 cells were rinsed at 350 x g for 10 minutes at room temperature and reconstituted in complete fresh medium of RPMI-1640 at a concentration of 2 x 1Q6 5 cells / rnl. Cell viability was determined by trypan blue exclusion, and usual was greater than 95%. The PMA was also diluted 500 times with complete culture media at a concentration of 20 nM. jg. Aliquots of 0.5 ml of U937 cells were cultured '(106 cells / rnl) in the presence or absence of 0.5 ml of PMA (20 nM) in 24-well flat-bottomed tissue culture plates (Becton Dickinson, Lincoln Park, NJ), and incubated for 72 hours at 37 ° C and CO2 at 5%. The final concentrations per well were 5 x 105 cells and PMA at 10 nñ. After 72 hours of incubation, 120 μl of the media were removed and subsumed with 100 μl of the medium.

The composition of example 1 and 10 μl of distilled sterile deionized water, in the presence or absence of 10 μl of LPS (5 ng / μl).

After 24 hours of incubation, all cells and the The particulate matter was pelleted by centrifugation at 350 x g for 10 minutes, and the resulting supernatants were stored at -20 ° C until they were tested for TNF-a. All Virulizma samples were tested on two separate occasions. 25 Two ELISAs from two sites were performed to quantitate TNF-a in the supernatants of the U937 cell culture using FNT-a ELISA kits purchased from Endogen, Inc. (Cedarlane Laboratories, Hornby, Ontario ). The protocol recommended by the manufacturer was used. For a short time, 100 μl of standards 5 of TNF-α and test samples were added to 96-well plates precoated with anti-human TNF-a, and incubated at 37 ° C, and CO2 at 5% for 3 hours. After thorough washing with a pH regulator for washing, 100 μl of anti-human FNT-a conjugated with alkaline phosphatase was added to the plates, and * 10 incubated at 37 ° C, and CO2 at 5%, for 2 hours. After incubation, the plates were washed as described above, and 100 μl of pre-blended TMB substrate was added to each well, and the enzymatic reaction was allowed to proceed at room temperature in the dark. for 30 minutes. Then, 100 μl of terminating solution was added to each well to stop the reaction, and the plates -i? were read using an ELISA reader from SLT Lab Instru ent to 450 n. The limit of detection of the test was pg / rnl. The TNF values for U937 cells were determined as described for PBMN cells. The results of the composition tested with 50 ng of LPS are shown in Table III.

$ TABLE VI Effect of composition on TNF release from U937 cells 0 This example describes the physical characteristics, chemical and biochemical of the composition of example 1. The physicochemical characteristics, such as conductivity, olarity and total solids, were determined for three batches manufactured from a composition prepared according to example 1. The results, tabulated in table I , demonstrate the characteristics of sterilization, power and reproducibility of the manufactured product, and thus provide a product specification- The ethanol and ethyl ether tests are just tests in progress. The potency, ie, 0 the release of the TNF, was determined as described in Example 2. The methods used to determine the characteristics are tabulated below.

TABLE VII Characteristics of the compositions of Example 1 as manufacturing products.

Proof Is -jecification Method Power > 100 pg / rnl of FNT Activation of monocytes / mac or agos = Release of FNT-a 10 Identity / Purity Conforms to Ciar-frequency Safety Passes the test General safety test (mice and connexions of the Indies) (21 CFR and 610.11) Pyrogenicity The increase in pyrogenic temperature (co ratura should not excenejos), USP of 0.4 ° C Endotoxin < 2 EU / rnl Used Test of Lnnulus arni bocitos, USP Sterilization No growth a Sterilization test, U? P pH 7.40 ± 0.2 pH test, USP Appearance Yellow liquid Light and clear visual inspection with little or no precipitate 35 solid 23 ± 7 rng / ml Freeze-drying Osrnolari ad < 1000 rnOarn Decrease in freezing point, USP 40 Ethyl alcohol Not more than 10 ppm Direct injection gas chromatography Ethyl ether Not more than 5 ppm Gas chromatography 45 direct injection Conductivity 35 ± 5 rnMHO Copenhagen Radiometer Model The physical and chemical properties described above, such as conductivity, olarity and total solids, were consistent with a composition that has more than 99% salts. Less than 1% of the solids in the composition was organic material, almost half of the solids were carbohydrates, and the rest were amino acids, lipids and. phospholipids. Proteins and peptides were present. The gel electrophoresis of S'DS confirmed that there were more peptides than proteins in the composition. No molecules were detected # 10 high molecular weight. Test methods were used by HPLC and bioassay for the composition of the invention to characterize the product as the liquid regulated in its pH and the concentrated formula. The results of the CLAR described below indicate that the product was the same in all its presentations. JETF A double column reverse phase HPLC method was used to characterize the composition of Example 1.

For this method, the samples were lyophilized and Then, they were reconstituted in pH A buffer (0.1% trifluoroacetic acid (TFA)) and flowed onto a UP60009-C18 column (U ~ Pore C18, 250 x 4.6 nm; Phenonenex from California) in tandem with a column HC-C18 premium sphere (250 x 4.6 nm, Phenornenex). The columns were flowed to room temperature using pH A regulator and pH regulator B (0.1% TFA in 100% acetonitrile), with a flow speed A of 0.9 rnl / min. A 150 μl sample was applied to the first column, and the pH regulator A was flowed through the system for 20 minutes. Then, a first linear gradient, regulator of pH B at 0-80%, was flowed for 35 minutes, followed by a second linear gradient, regulator of pH B at 80-0% for 5 minutes. The eluted compounds were detected by optical absorbance at approximately 190 to 284 nm, most of the runs were detected at 210 and 235. The composition of Example 1 had a consistently reproducible pattern in reverse phase HPLC, in which values were observed. ximos. The readings of the reverse phase HPLC for three batches of the composition of the invention are shown in FIGS. 1 to 3. Six batches of the bile extract, which were prepared as in example 1, and marked as AF, were analyzed in , j-? as to its amino acid profile on an amino acid surfactant analyzer Alphaplus LKB 4151 operated in a physiological mode, with post-colurnna detection with mnhidpna. The results, in nrnoles / 100 μl, are shown in table VIII. _ »* TABLE VIII Amino acid and urea profile of the compositions of example 1 Amino acids and urea R B D E F fifteen • twenty OR 55 Samples A to F were also evaluated in terms of 40 the presence of bovine DNA. The samples were examined using a 32P-labeled bovine DNA probe generated from bovine genomic DNA. The test included samples, samples with maximum values, negative and positive controls, and patterns. The study was carried out 45 compliance with GLP regulations. This test detected 3.9 pg of the reference standard DNA. It was calculated that each of the ^ p samples contained less than 4 pg / ml of DNA. Samples A to F were also tested for the presence of several electrolytes. This analysis was performed by the Biotechnology Service Center, Department of Clinical Biochemistry, University of Toronto. The results, in nmol / 1, are shown in table IX.

TABLE IX Electrolyte content of the compositions of example 1 Electrolyte A D E Samples A to F were subjected to multi-element, multi-quantitative analysis by inductively coupled mass spectrometry (ICP-MS) under standard conditions. The results, in parts per million (pprn), are described in table X.

TABLE X ^^ R 0 5 H 0 0 5 0 Note: The term < det means below the detection level.

The analysis of anions and cations in samples A to F was also carried out. For this analysis, samples were prepared as recommended in APHA Standard Methods for The Exarnination of Uater And Uasteuater, 16th Edition, 1985 or MQE Handbook of analytical Methods For Enyironinental Samples 1983. The instrumentation for the analysis of anions / cations was: (1) for metals, ash emission Jarrel 61E ICAP, graphite furnace Zeernan 3030 by Perk Elmer, ß and steam-cold AA 2380 «Le Perkin El; (2) for anions, 0 ion chromatography Dionex 2001; and for conventional elements, Skalar SAS segmented flow analyzer. The results, in rng / 1, are presented in table XT.

TABLE XI Analysis of amions and cations of compositions of example 1 A B C D E F 0 f0 0 0 0? ^ 0 f0 Since several ether sulphates participate in the regulation of many cellular events, such as cell proliferation and cell differentiation, sample D was analyzed for sulfate ions before and after acid hydrolysis. Using whole sample D (ie, unfractionated), the non-hydrolysed sample gave sulfate at 1000 μM, while the hydrolysed sample gave sulfate at 122 μM. Since the concentration of sulfate ions increased after acid hydrolysis, these results suggest that 20% of the total 5 sulfate ions present are sulfate esters have identified standard physicochemical values for the composition of Example 1 and are essentially consistent with the first studies, which are described in example 4. These standard values indicate that a consistent product can be obtained repeatedly.

EXAMPLE 4 This example describes the physical, chemical and biological properties of a number of previous batches of the composition of Example 1. The batches of bile extract were prepared according to the method described in example 1. In addition, the chemical composition of the batches an amino acid analysis of the batches was determined and conducted, using the methods described in example 3. The results are shown in the following tables.

TABLE XII • B ^ * 1 Previous batches of chemical compositions of the compositions of Example 1 Composition Arnino Solids Sugars L i pidos PM high > 3: Lot D (rng / rnl) acids (μg / ml) (μg / ml) polyethylene No. (μg / rnl) (μg / rnl) 0 0 0 Note: NB means not detectable, therefore logs of 0.5 μg / il of lipids per and / or sinuses of 1.0 μg / l of polypeptide of high molecular weight. Nfl means that it was not tested.

TABLE XIII ^^ > Physical, chemical and biological properties of previous batches of compositions of Example 1 Lot No. pH ConducOsmala- AbsorUV.VIS ActiviPotencia tance ridad bancia Peaks d d pg / ml (rn / (rnOsM) (D.O. Unida¬ moles) 280 nm) des / ml) fifteen 2 f0 * m3f5 Cortenarians: 40 1. The complete isotonic PBS solutions were added to lots Nos. B0106 and B0706. 2. Batches B1306, B200B and B230B were concentrated twice without adjusting the pH.

TABLE XIV Wr Composition of aaino acids from the above compositions of the composition of example 1 BATCH NUMBER B-0208 B-0209 J-0211 01/06 07 / OB 1306 2006 2306 EXAMPLE 5 This example describes the biological activity of fractions of the composition of example 1. The biological activity of fractions of the composition of example 1 was investigated. The analytical results are consistent with the biological activity of the composition that it is making attributed to low molecular weight components (ie, less than 300 daltons). This was determined through an experiment in which the composition was passed through the reverse phase HPLC described in Example 3 and diluted fractions that were isolated and analyzed for potency by the PBMN-FNT test described in the example 2. Significant activity was only detected at the first elution peak (Fl), that is, 5.6 to 6.2 rnins. which is consistent with the molecular weight of less than 300 daltons. (See table XV).

TABLE XV Effect of fractions of composition of Example 1 eluted by reverse-phase HPLC on the release of TNF from PBMNs induced by LPS FNT-α released (pg / ml) sample CLAR Amount Osrnolity Tested (rnin) by Total -LPS (rnOsin) cavity LPS 50 ng 305 79 304 Composition of Example 1: Whole 0 100 μl 51.9 ± 195 213 4.15 Fl 5.60 - 6.20 100 μl 508 * 82 203 344 F2 6.20 - 6.55 1.00 μl 149 ± 44 -157 281 F3 6.55 - 7.10 100 μl 306 ± 80 1 309 F4 7.10 - 7.90 100 μl 316 ± 123 11 309 F5 7.90 - 8.40 100 μl 390 ± 95 84 309 F6 8.40 - 8.90 100 μl 282 ± 103 -24 311 F7 8.90 - 9-40 100 μl 296 ± 108 -10 309 F8 9-40 - 10,00 100 μl 34.1 ± 112 36 309 F9 10.00 - 10-40 100 μl 33 ± 139 24 308 FIO 10.40 - 12.00 100 μl 316 ± 101 11 311 FU 12.00 - 13.60 100 μl 354 ± 74 49 311 F12 13.60 - 14-20 100 μl 344 ± 107 39 315 F13 14.20 - 15.35 100 μl 296 ± 117 -9 311 F14 15-35 - 15.75 100 ul 344 ± 108 39 314 F15 15.75 - 18.20 100 μl 300 ± 104 -5 313 Not a: 1. Number of patients tested i 3 2. The total TNF-a release is corrected to be released by RPMI (13 i 4 pg / rnl, 306 inOsrn ) 3. The fractions of CLAR 1-2 reconstituted in water; 3-15 reconstituted in PBS pH regulator. 4. Columns in rows are: U-porex C1S and PpineShere, both of Phenornenex, 250 x 4.6 nm. 5. Volumes of L.PS per cavity: 10 j. 6. Total volume per cavity 1000 μl. 7. The sample volumes are equivalent Additional experiments were done in the following manner to show that the active components (TNF releasers) had molecular weights less than 3500 and less than 1000 daltons. Lot BC241 was fractionated by carrying out a Folch extraction according to Ta ari et al., Agr "Biol. Chern., 40 (10), 2057-2052 (1977). The aqueous layer was dried on a rotary evaporator to produce a light brown granular solid. A stock solution of this solid was prepared at a concentration of 5 mg / ml. A portion of the sourcing solution was charged to Centri / by Centrifuge Concentrators (Spectrurn Products, Houston, TX) having a molecular weight separation membrane of 3500 to 1000 daltons. The concentrators were rifled at approximately 1500 x g until a portion of the material had passed through the membrane. The solution that passed through the membrane was evaluated for power in the PBMN-FNT test. The results were presented in table XVI.

TABLE XVI Molecular weights of active components of compositions of example 1 SAMPLE FNT 1iberated (pg / rn1) Aqueous layer of BC02 .1 from Folch 1709 Aqueous layer of Folch that passes through a membrane of 3500 daltons 2318 Aqueous layer of Folch that passes through a membrane of 1000 daltons 2423 The analysis of the biological activity of molecular weight fractions indicates, therefore, that the TNF-releasing components are less than 1000 daltons in molecular weight.

EXAMPLE 6 This example 'illustrates the effect of the composition of Example 1 on T and D lymphocytes in the culture. The growth of human lymphocytes was examined under carefully controlled conditions in the presence and absence of the composition of Example 1. Normal concentrations of lymphocytes were incubated in wells containing various concentrations of the composition. When human T and D lymphocytes were incubated with the composition in concentrations similar to those used clinically, there were no adverse effects as determined by the exclusion of trypan blue dyes. Accordingly, the composition of the invention was non-toxic for normal T and D lymphocytes in culture. The effect of the composition on the survival of human PBMN was also examined. The PBMN were incubated during 24 and 48 hours in plastic plates with several volumes of the composition and tissue culture medium. At the end of this period, the number of surviving cells was estimated by the exclusion of trypan blue dye.

TABLE XVII Concentration of viable PBMN after incubation with the composition of Example 1 No. of live PBMN per cavity per trypan blue (xlQS) Concentration Time After 24 hr. After 48 hr (μl / cavity) zero No- (% viable) No, (% viable) Patient S.Z, fifteen twenty Or H 0 1.30? 0.70 (54) 0.33 (25) 25 0.65 (50) 0.15 (12) 50 0.68 (52) 0.38 (29) 100 0.75 (58) 0.23 (18) 200 0.65 (50) 0.20 (15) LPS (μg / well) 1 0.60 (46) 0.53 (41) 10 0.15 (12) 0.15 (12) Approximately 1 x 106 cells in plates / well tripled. 2 Actual number of cells counted / cavity (xlO *). The above data show that the number of surviving cells fell at 24 and again at 48 40 hours; however, the number of surviving cells in the presence or absence of the composition was not different.

In addition, the increasing volumes of the composition had no effect on survival. In addition, the composition showed no cytotoxicity to human PBMN.

The ability of the composition to stimulate # lmfocitos was evaluated in the following three indicator systems: i) stimulation of DNA synthesis of lonfoci, 2) induction of cytotoxic function mediated by lymphocytes; and 3) induction of cytotoxic function mediated by rnonocitos / rnaerófagos. These tests were chosen for selection because they measure munoological functions that have been shown to be associated with different clinical parameters in patients with malignant disease. These indicators of immune function can also be modulated in cancer patients-treated with different biological response modifying agents, such as IFN or IL-2. The results of the initial selection procedures are presented below. 1. Stimulation of lymphocyte DNA synthesis: comparison with an optimal stimulant concentration of phytohernagl utinin (PHA): Stimulant Counts per minute Medium 374 PHA 125,817 Composition (# 222) 1,116 Composition (1:10) 1,021 Composition (1:50) 649 Unlike the prototypic mitogen, PHA, it was noted that the composition of Example 1 did not stimulate lymphocytes to undergo blastogenesis and cell division, which is consistent with these results showing little or no stimulation of DNA synthesis by the composition. 2, Stimulation of cytotoxic function mediated by lymphocytes and comparison with an optimal stimulant concentration of IL-2: Stimulant Lithic Units Medium 30.8 IL-2 472.5 10 Composition (net) 48.1 Composition (1:10) 33.3 Composition (1:50) 44.8 * Unlike the prototypical function stimulator lymphocyte cytotoxic, IL-2, the composition did not induce lymphocyte cytotoxicity. The number of lithic units stimulated by the composition was almost identical to that of. negative control (ie, medium). 3. Stimulation of cytotoxic function mediated by monocytes per composition: comparison with IFN-t and LPS (IFN ? + LPS) Stimulant (E / T = 20/1)% of Cytotoxity 25 Medium 4.3 IFN + LPS 24.4 Composition (net) 19-7 Composition (1:10) 20-0 Composition (1:50) 11.5 30 The composition of Example 1 was able to stimulate peripheral blood rnonocytes to express shift function in a dose-dependent manner. The magnitude of stimulation is comparable to that induced by 08 combination of prototypic macrophage activator of IFM-t and LPS. It is important to recognize that the action of the composition in these in vitro tests did not require the addition of endotoxin, as in the case of any other maerophagous activator.

EXAMPLE 7 This example illustrates the result of tests conducted • 4V to study that, if any, cytokines are present in the composition of example 1. Bile extract samples (aliquots of 50 μl and 100 μl per test) prepared according to the Example 1 were tested for the presence of the following cytokines (sources and detection limits of the ELISA immunoassay equipment). used were indicated parenthetically): FNT-a (Endogen, Inc., (5 pg / rnl)); IL-la (Endogen, Inc. (50 pg / rnl99; IL-β (4.3 pg / ml); GM-Wf CSF (Endogen, Inc.); RFN-a (Endogen, Inc.); IL-2 (Advanced Magnetics, INc); IL-6 (Advanced Magnetics, Inc. (7 pg / ml); IFN-t (5 pg / ml) Csourcel; IL-1 (Advanced Magnetics, Inc.) [need limit]; IL-4 (R8D Systems (3 pg / ml)); and IL-8 (RSD Systems (4.7 ng / ml)). Procedures were used according to the instructions of the individual team, which can be followed by an expert. It was determined that the composition of the invention does not contained measurable levels of any proven cytokine, being those TNF-α, IL-1 a, IL-1 β, IL-4, IL-6, IL-8, GM-CSF and IFN-t, as described in FIG. picture XVIII BOX XVIII Determination by ELISA of cytokines in the Cytokine Composition (pg / l) 50 μl. 100 μ.l EXAMPLE 8 This example describes pharmacodynamic studies in mice with the composition of Example 1, including the direct in vitro effect of Viruli inTN as well as the effect of VirulizinTH administered in vivo or peritoneal fluorosis. Peritoneal macrophages were harvested from C57BL / 6 mice 72 hours after intraperitoneal injection of 1.5 nrn of 4% peptone protease. The macrophages were then stimulated in vitro as medium alone, 50 ng LPS or 0 VIRULIZINTN. stimulation measurements were made with respect to TNF (by ELISA) and NO levels (by spectrophotometric test using the Greiss reagent) in duplicate experiments. The standard error of the mean between the duplicate experiments was less than 10%. As indicated in the. Table XIX, VIRULIZININ induced a slight increase in production of TNF-a. (60-232 pg / ml) compared to background levels (medium) (120 pg / rnl), but VIRULIZINTN compared to LPS (2225 pg / nl) was not a strong stimulant of TNF-a release by rnacró agos Nitric oxide production was zero.

TABLE XIX 0 In vitro stimulation of macrophages with protease-peptone Macrophages stimulated with: FNT (pg) Prorned. NO (μM) Prorned. 5 Medium 120 0 LPS (1 μg / rnl) 2225 11 Virulizin: 1: 2 62 0 1: 5 181 0 0 1:10 206 0 1:20 202 0 1:40 232 0 1:80 142 0 1: 200 122 0 5 Virulizin ™ in vitro synergy with LPS was also performed for TNF-a release. Peritoneal macrophages were harvested from C57BL / 6 mice after the aforementioned treatment. The macrophages were then stimulated with 0 50 ng of LPS alone or LP with different dilutions of VIRULIZINTN as before, FNT was determined by ELISA. As shown in Table XX, LPS only induces approximately 2900 pg / ml of TNF-a release from mouse peritoneal macrophages in vitro compared to 262 pg / ml for the medium. 9. 1 When LPS is combined with VIRULIZIN ™, there is approximately 800 pg / rnl increase in release of TNF-a at VIRULIZATION dilutions of 1: 5 to 1.10 and increased release to at least 1:40.

PICTURE XX Synergistic Combinations between VirulizinTM and FNT-a LPS Stimulated macrophages with TNF (pg / rnl) NO (μM) Medium 262 1.6 ± 1.1 LPS (5 ng / ml) 2900 6.6 + 1.3 15 LPS (5 ng / ml + Corn. of Example 1: 1: 5 3750 13.2 + 0.5 1:10 3750 1.6.9 + 2.7 20 1:20 3500 13.5 ± 2.5 1:40 3600 27.1 ± .1.1,6 1:80 3000 10.1 ±. 1.9 1: 200 3400 9.7: 1: 1.3 ^^ 1: 100C) 3200 9.4 ± 1.2 IFN-t (100U) + LPS (5 ng / rni) 6800 74.1 + 0.6 IFN-t (1000) + Víruli zin: 1.5 512 46.9 ± 0.6 1:10 625 57 '.3 The in vitro synergy of VirulizinT * "i with LPS for 40 nitric oxide (NO) was performed in the same procedure as before, except that NO was determined in the supernatant of treated rnacr-ofagos. As it was done before, the test for MO is an ornometric spectrophotometer and uses a Greise reagent. As indicated in the previous table, LPS produces some release of NO (9 μM). VIRULIZINIM in synergy with LPS induces a marked increase in NO production (13-27 μM) at 1: 40 dilutions.

VTRULTZINTM by itself did not induce release of NO pelma ero phages. The in vitro synergy VIRUL1ZIN ™ with IFN-t for release of TNF-α was studied using the same peritoneal mouse macrophages derived from C57BL / 6 treated mice as before. The data were included in the previous table referring to "smergistic combinations" As shown, the peritoneal mouse macrophages show a baseline release of TNF-a after 24 hours of in vitro culture. The same acrophages stimulated with LPS or IFN-x release almost 3000 pg / rnl of TNF-a. When VIRULIZIN ™ and IFN-t are added together, the release of TNF-a decreases. In comparison, the combination of LPS and IFN-t has an additive effect on TNF-α release. The in vitro synergy VIRULIZINTM with IFN-t for release of TNF-a was studied using the same peritoneal mouse macrophages derived from C57BL / 6 treated mice as before. The data were included in the previous table referring to "synergistic combinations" - Co or shown, LPS and TFN-t alone each increased the production of NO (9 and 7 μM respectively). VIRULIZTNTM added to IFN-t induced an M marked increase in production of No (47-57 μM) that almost equaled the combination of L.PS and IFN-t (74 μM). The results are consistent with the conclusion that VIRULIZINTM in combination with IFN-t increases NO production but inhibits the release of TNF-a The in vivo production of TNF-a for 72 hours was studied in macrophages harvested from C57BL / 6 of mice which, before being harvested, were not treated, were injected intraperitoneally 72 hours before with 1.5 and 4 % protease-0 peptone, or injected intraperitoneally between 72, 48 or 24 hours before with 1.0 ml of VIRULIZIN ™ diluted 1:10 in PBS. The macrophage RNAs were treated in vitro for 24 hours with IFN-t (50 μ / rnl), LPS only (5 ng / ml), or the combination thereof. FNT and NO were determined as indicated above. The data is presented in table XX., TABLE XXI Release of FNT and No from harvested macrophages from treated mice Harvested macrophages Stimulated FNTNO NO from mice injected with: In Vitro (μg / rnl) (μM) • 10 None Medium 315 0 IFN-t 402 25"8 ± 1.6 LPS 3,750 1.9 ± 0.2 IFN-t + LPS 6,300 40.9 ± 3.8 • Peptone Protease Medium 335 0-9 ± 0.5 (72 hours before) IFN-t 838 48.6 ± 1.7 LPS 5.975 23.2 ± 3.4 IFN-t + LPS 10.875 55.8 ± 1.9 Virulizin (72 hours before) Medium 258 1.2 ± 0.6 IFN-t 425 37-5 ± 2.6 LPS 3,300 4.02 ± 0.9 IFN-T + LPS 4,650 5 -0 ± 0.9 Virulizin (48 hours before) Medium 350 8-5 ± 1.8 IFN-t 560 62.0 ± 2.5 LPS 5.300 36.5 ± 1.2 IFN-t + LPS 12.475 58.5 ± 1.6 Virulizin (24 hours before) Medium 248 2.9 ± 2.1 IFN-t 475 44.1 ± 0.7 LPS 9,025 12.5 ± 2.4 IFN-t + LPS 12,375 52.8 ± 0.6 As described, the TNF-α release of rnacrophages was examined in the absence of stimulation or with IFN-t LPS, or LPS / INF-t after 24 hours in an in vitro culture. The peritoneal mouse macrophages were shown to have little FNT-a after VIRULIZINTM live stimulation. When the 40 harvested macrophages were exposed IFN-t at 24 and 48 hours before the test, showed a small increase in TNF-a production. In contrast, harvested macrophages stimulated with LPS at 24 and 48 hours, but not at 72 hours before the test, showed increased release of TNF-a. Likewise, there was a synergistic effect of LPS and IFN-i on harvested macrophages that were stimulated 24 and 48 hours but i 5 not 72 hours before the test. Will the production go live from NO for 7? hours was studied with acrophage cells and tests under the same conditions described above with respect to La * P production of FNL-a. There was a small spontaneous release of NOT measured at 24 and 48 hours after intrapeptoneal injection of VIRULIZINTM (hereinafter TP VIRULIZINTM). When the harvested cells were incubated with IFN-t, there was a marked release of NO, and the harvested inacrophages having IP VIRULIZIN ™ at 24 and 48 hours before of the test showed an exponential increase in NO release, which fell beyond 72 hours towards the baseline IP values of IFN-t alone. When the harvested cells were stimulated with LPS, they showed a markedly increased NO response, which was once observed for 24 and 48 hours of macrophages treated with IP VIRULIZINiM compared to macrophages that had not received IP VIRULIZINT. "The harvested macrophages that had received IP VIRULTZINTM before did not respond differently to the macrophages that did not have a pre-treatment with VIRULIZTNTM. when the harvested macrophages pretreated with VIRULIZTN ™ were incubated with LPS / IFNt, they showed an increased production of OM compared to the macrophages that were not previously treated. The maximum response was with macrophages pretreated with VIRULIZIN ™ 48 hours before being harvested and tested.

EXAMPLE 9 This example illustrates the activation of monocytes and I L macrophages with the composition of Example 1 and methods for try the isma. Research has shown that the composition of Example 1 will activate normal monocytes to demonstrate cytotoxicity towards the Chang heptorne cell line, which is used to measure rhnocyte toxicity, and that monocytes and macrophages of cancer patients (e.g., those affected with cancers of the cervix, ovaries, ear / nose / throat, and endometrium / uterus, and chronic ioganelle leukemia) have been stimulated by the composition to attack and destroy cells turnorals derived from the same patient. Very particularly, the turnuricity function of monocytes has been pro-a in the presence of the composition of the invention and the basic procedure for these experiments are outlined below. In this procedure it has been called the "cytotoxicity test of rhonocytes / rnacrophages for lines of analogous cells and turnoral cells "or" cytotoxicity test "for brevity.

/ W The method requires the isolation of onocits / RNAs, which is achieved in the following way: venous blood is collected aseptically in Vacutainer heparini ados tubes. The sterile hepapne book of preservative is added to a final concentration of 20 units / ml. The blood is diluted 3: 1 in Hanks balanced salt solution (HBSS), stratified with lymphocyte separation medium and centrifuged to obtain a band of rnonuclear cells of - L peripheral blood (PBMNs). After the centrifugation, the The mononuclear cell layer is recovered from the adjoining surface, washed twice in the medium (the medium is 1640 medium of the Ros ell Memorial Memorial Institute CRPCMI supplemented with 10% fetal bovine serum inactivated with heat, 50 units / ml of penicillin and 50 μg / ml of streptomycin a) and the monocytes are enumerated by ingestion of latex. The inonocytes are isolated by means of adhesion in 96-well plastic plates for 2 hours at 37 ° C, followed by 2 cycles of washing with the medium). It is estimated that the adherent cells are greater than 90% rnonocitos. The cavities that contain cells adherents are incubated overnight in the presence of VIRULIZIN ™ (final dilution of 1: 10-1: 20), then the adherent cells are washed to remove VIRULIZIN ™ and incubated overnight with tumor cells. The tumor cells are maintained in the medium in which the concentration of Endotoxin is guaranteed by the manufacturer that it is low and that it is not stimulating in the test.

For studies in which a normal cell line is used, Chang hepatoma cells marked with 5c (chromium) because this cell line is insensitive to the cytotoxicity of natural killer cells. These hepatoma target tumor cells are added to the monolayers of adherent cells at effector cell ratios: target (E: 0) from 20: 1 to 15: 1. This ratio of E: 0 is used because it falls within the plateau scale in one on a prepared curve by varying the ratio E; G from 5: 1 to 30: 1, After 24 hours, the supernatants are collected and the sicr release is quantified. The specific cytotoxicity in percent is calculated as follows:% specific release = E-S X 100 T-s in the above equation, E = CPM released from the target cells in the presence of effector cells; S = CPM released from target cells in the absence of effector cells; T-CPM released from target cells after treatment with dodecyl. 2% sodium sulfate). For studies using autologous tumor cells, these cells are obtained from siCr-labeled surgical biopsies, and are used in the same way as the hepatoma cells described above. The preparation of peritoneal and alviolar macrophages is done by the methods described in Braun et al., Cancer Research, 53, 3362-3365 (1993). Using this protocol, it was found that the composition W makes the rnonocitos of healthy donors exert totox icity towards the cell line of Chang's hepatoma. Subsequently, it was investigated whether the onocites or cancer of a cancer patient could be stimulated by the composition to attack and destroy their own particular tumor. Using similar protocols as described for the normal cell line (Chang hepatothane cells), peptoneal rhonocytes and / or peptoneal cancer patient macrophages were isolated. Peritoneal macrophages were isolated from peptoneaie fluids collected at the time of laparoscopy. It was found that the composition activates peripheral rhonocytes and peritoneal macrophages of a patient with cervical cancer to produce cytotoxicity against the patient's own turnoral cells- This effect was comparable with or better than that produced with the combination of IFN and LPS- The peritoneal rnacrophages A patient with ovarian cancer was also found to be stimulated by the composition to attack and destroy the ovarian turnoral cells in culture.

Studies on monocytes / maerophages with composition Because selection procedures showed that the composition does not stimulate the functions of lymphocytes but can stimulate monocyte functions, subsequent studies were directed to the characterization of rnonocyte stimulating activities. Metamorphoses of the VP composition. A number of comparative studies aimed at determining the dose-response characteristics of the composition in the stimulation of the tumoricidal function of rhonocytes / rnacrophores were thus carried out with the pruoba of 5 different batches of the compound. The main emphasis of the studies was given to the test of the capacity of the composition to stimulate the turnoricida function in monocytes and aerofages from different anatomical sites of patients with 9 cancer For these inventions, it was based on the following: (1) peripheral blood rnonocitos of cancer patients and control subjects; (2) inaviolated alviolar phages of patients with cancer in the lung and control patients with non-malignant lung disease; and (3) peritoneal macrophages of patients with gynecological malignancies. 15 Dose response studies were completed with different batches of the composition all prepared according to example 1. These studies were based on peripheral blood rnonocytes to test the stimulatory activities of different doses and different batches of the composition ( Lots nos. 216, 219 and 222). Each batch of the composition was tested with dilution (net), a dilution of 1:10 and a dilution of 1:50 of material. The results are illustrated graphically in figure 1. Batches # 222 and # 216 were shown to stimulate the function turnoricide of monocytes, however, lot # 219 did not. Apparently lot # 222 was superior to lot # 216 in those preliminary investigations. Lot # 222 apparently stimulates the equivalent levels of the turnoricide function in undiluted sample (net) and dilutions of 1:10, but lower, activity still detectable at the 1:50 dilution. Lot # 216 gave the greatest stimulation of the turnoricidal function at the undiluted concentration (net), with less activity at the 1:10 dilution and without detectable activity at the 1:50 dilution. As indicated above, lot # 219 did not produce detectable rhonocyte turnoricity at any concentration tested. 0 The turnoricide function in peripheral blood rnonocytes was also evaluated. Four peripheral blood monocyte samples from control subjects were tested. These tests used an optimal stimulatory composition concentration (1:10 dilution of lot # 222) and an optimal stimulating concentration of IFN-t plus LPS. The target cells in these studies were a cell line insensitive to NK, cultured, that is, the Chang hepatorne. The results are presented in the following table. 0 Stimulant (E / T-20/1)% of "cito-toxicity Medium 5.4 ± 1 IFN-t + LPS 18.6 ± 4 5 Composition 22.3 ± 6 A test was also performed on a sample of rhnocytes from a patient with cervical cancer. This test was important because the patient's own tumor cells were available to be used as target cells in the test. As before, this test used an optimal stimulatory concentration of the composition (dilution of 1:10 of batch # 222) and an optimal stimulatory concentration of IFN-t plus LPS. Also, the effect / target cell ratio was reduced to 15/1 to preserve the patient's tumor cells. The results of this test are presented in the following table.

Stimulant (E / T-20/1) Cytotoxicity% * Medium 5.5 IFN-T • * LPS 14.4 15 Composition 20.9 In the peripheral blood rnonocitos of control subjects, the rhnocyte composition of the turnoral function stimulated against Chang Hepatoma cells at an equal level or greater than the level produced by means of an optimal stimulating concentration of IFN-t + LPS. In peripheral blood monocytes of a patient with cervical cancer, the monocyte composition of turnoral function stimulated against the cancer cells of the same patient a a level that exceeds that produced by means of IFN-t plus LPS by more than 30%. The turnoral function was tested in macrophages of the peritoneum of patients with gynecological malignancies. These tests were performed on macrophage samples from the peritoneum isolated from washing fluids of a patient with ovarian cancer. These tests were carried out with patient's turnoral cells as target cells in this trial. As above, an optimum stimulating concentration of the composition (1:10 of Charge No. 222 dilution) and an optimal stimulating concentration of IFN-t was compared. more LPS. Also, the effector / target cell ratio was reduced to 15/1 to preserve the patient's tumor cells. The resulting information was: Stimulant Cervical Cancer Ovarian Cancer Medium 8.2 0.6 IFN + LPS 29.8 4.1 Composition 13.2 8-9 15 The results of these tests highlighted the fact that the local tumor environment can be a determinant of the response of immune cells to the inunitological activators. In this case of cervical cancer, there was no pathological evidence of malignant disease within the peritoneal cavity and the development of turnoral function against the tumor derived from itself was better with combined IFN-t and LPS than with the composition. In the patient with ovarian cancer, there was a significant tumor in the peritoneal cavity. The response against the patient's own tumor for combined IFN-t t LPS was minimal when more, so the response to the composition was higher. The turnoral function was tested in alveolar rnacrophages of patients with lung cancer and control subjects. These tests were performed on samples of alveolar rnacrofago isolated from bronchoalveol lavage fluids of a patient with malignant lung diseases. These tests used an optimum stimulant concentration of the composition (1:10 charge dilution No. 222) and a combined stimulant concentration of IFN-t and LPS. The target cells in these studies were Chang Hepatoma cells and the effector / target cell ratio was 20/1. The resulting data were: * 10 Stimulant Patients with Cancer Control Medium 2.6 ± 2 19.5 í 4 IFN -t + LPS 10.9 ± 13 1.2 ± 4 15 Composition 5.2 ± 2 18.6 ± 8 The results were consistent with the observation that the alveolar rnacrofagos of patients with lung cancer are nons in their development of turnoral function in response to conventional inacrophagous agents such as IFN-t + LPS. The results showed that the tumor function of the alveolar macrophages of patients with cancer in the lung is reduced more compared with control subjects. The data presented above indicated that VTRULIZINR is a poor stimulator of alveolar macrophages. Further investigation with alveolar rnacrophages of patients with non-small cell lung cancer is presented in Example 23. Activity in alveolar macrophages seems to vary with the VIRULIZINR preparation. Thus, the cytotoxicity of the alveolar macrophages was described in only 2/7 alveolar macrophage preparations with the proven source loads (222, 219, 216). In contrast, 3/4 alveolar preparations were stimulated with the last preparations (233, 238). The difference could be related to the age and power of the preparation or the variability of the patient. Therefore, the composition can activate the tumor activity in alveolar rnaerophagous. The preliminary m vitro tests with the composition showed that it is a rnacrofago activator. The provided material was able to deduce turnoral activity in a normal cytotoxicity assay against an NK-insensitive cell and against freshly dissociated human tumor cells. It was also found that the deduced activity is dependent concentration in these tests. The ability of the composition to activate the macrophage tumoral function vi tro was comparable with that of the best erofaga activator combination available today., essential in e, endotox a (ie, LPS) combined. As stated above, the ability of the composition to produce this level of tumor function in the absence of endotoxin could be considered biologically important if the material is free of endotoxin contamination. The composition is free from endotoxin contamination when tested for pyrogens by means of the United States Phylochemical Test 5 (USP). Corno has been found for other LOO activators f? _ftl macrophages, the activity of the composition is the tumor function stimulant of inacr phagos varies with the source of macrophages. It appears that the compositions are an excellent activator of peripheral blood rhonocytes being equivalent to IFN-t 5 + LPS with normal donors and possibly higher than TFN-t + LPS with patients suffering from cancer. Malignant disease has an important impact on the development of your oral monocyte function that depends on the activator used (Braun and "k others, (1991).) One of the biological activity eradicating different triglycerides in rnonocytes from cancer patients is the activator sensitivity for arachidonic acid metabolism and the secretion by the cell of prostaglandins. From these initial studies with the composition, it seems that the activity produced with the compound is not sensitive to the inhibitory effects of protaglandins. If the insensitivity of protaglandins can be definitively tested for rnonocytes in cancer patients with the composition, it would be considered therapeutically important, because the effectiveness of many other theological activators through protaglandins. Preliminary studies with 2 specimens indicate that the composition may have good activity in peritoneal macrophages, particularly when the malignant disease is present in the peritoneal cavity. 25 These preliminary results also illustrate that it has been found when comparing the ability of different pP activators to stimulate the turnoral function in peritoneal macrophages of patients with different gynecological malignancies. In these studies, it was found that the presence of malignant disease within the peritoneal cavity influences the ability of macrophages to respond to specific activators. In patients with cervical cancer, malignant disease is not present in the peritoneal cavity in general, and from this, the response of resident macrophages to IFN-t - * LPS is normal. However, when The disease is present in the cavity, co or in the case with ovarian cancer, the response to TFN t + LPS is suppressed. This refers, in part, to changes in aci or arachidonic metabolism of peritoneal cancers when malignancy is present (Braun et al., 1993). The fact that the composition activates the tumor function in macrophages of the peritoneum of patients with ovarian cancer against The patient's own tumor cells are consistent with a mechanism for activation that is independent of the pathway of the arachidonic acid. Therefore, as shown in the aforementioned m vitro studies, the composition of the present invention is capable of activating rhonocytes and macrophages to increase the function of their immune system.

H EXAMPLE 10 This example illustrates the turnoral function in response to the composition of the invention and to other activators 5 macrophages in peripheral blood rhonocytes and macrophages of the peritoneum of patients with gynecological diseases. The patient population consisted of 7 patients, 3 with benign disease and 4 with malignant disease (2 ovarian pV cancers, 1 endo etrium cancer and 1 cervical cancer). The 10 samples were removed from the patients at the time of the surgical procedure. Preparations containing peripheral blood monocytes were isolated from the blood samples using the procedure set forth in Braun et al., Cancer Irnununol. Irn unother. , 32, 55-61 (1990) and the preparations containing peritoneum macrophages were isolated as described in Braun et al, Cancer Research, 53, 3362 (1993). The cytotoxicity of the tumor cell was determined in response to the composition of the invention (1:10 dilution of charge supply No. 222) and other activators, essentially IFN ~ t (100 U / rnl), IL-1? (500 U / ml) and CSN rnonocito (500 U / ml) using the rnonocito cytotoxicity assay described in Braun et al. The results as shown in the following table demonstrate that the composition of the invention stimulates the turnoral function in the peripheral blood rnonocitos and in the macrophages of the peritoneum of patients with malignant and non-malignant gynecological diseases. The results are re-cited as percentage of tumor cytotoxicity (± S.E.) in an inocyte / tumor cell ratio of 15: 1.

STIMULATION OF PERIPHERAL PERIPHERAL AND PERITONE MACROPHAGUS M0N0CIT0S Activator- Blood Monocytes Ma eró phages Peri fép eos Peptoneales Medium 8.6 + 3 3.1 ± 1 Interferon Gamma 18.3 ± 2 9.5 ± 1 Interleukin-12 26.0 + 4 8.5 ± 2 Monocyte-CFS 16.0 ± 2 7.0 ± 2 5 Composition of 23.0 ± 6 12.5 t 2 Invention (Virulizm) Therefore, the tumor cytotoxicity produced by the composition of the invention is equal to or greater than that produced by the other biological stimulators that were tested.

EXAMPLE 11 This example 1 illustrates the effect of indomethacin, an inhibitor of prostaglandin synthesis, on the development of tumor function in response to the composition of the invention; The impact of other macrophage activators on peripheral blood monocytes from patients with 0 cancer was also investigated. Samples of patients with malignant disease in example 10 were tested using the assay system as described in example 10 with the exception that indoinetacin (up to 5 ng / ml) was added simultaneously with the composition of the invention, IL-12 (500 U / ml), and rnonocito -CSF (500 U / rnl). The results are shown in the following table indicated that indornetacin increases cytotoxicity in response to IFN-a, GM-CSF and M-CSF.

INDOMETACINA INCREASE OF CYOTOXIDITY Activation Conditions No. Donators% C? Toto icidad TFN-t 23 11.9 + 9 5 IFN-t - * Indornetacin 23 25.2 ± 17 TM-CSF 10 7.8 ± 6 GM-CSF + Indo etacin 10 17-8 ± 8 PMA 6 27.3 ± 14 PMA + Indornetacin 6 22.0 + 17 0 IL-12 3 24.7 ± 5 IL-12 + I dometacin 3 25.6 + 6 M-CSF 3 14.31 £ 3 M-CSF + Indornetacin 3 19.0 __ 3 Composition (Virulizin) 4 18.7 + 6 5 Composition (Viruliz) + 16.4 i. 6 I ndornetacina In this way, the development of tumor function in response to IFN-t, -GM-CSF and M-CSF was regulated by means of a function sensitive to indomethacin. In contrast, the development of tumor function in response to phorbol ester (PMA), IL-12 and the composition of the invention was not regulated by a function insensitive to indomethacin, ie, indornetacin does not increase cytotoxicity in response to the composition of the invention, IL-L2 and PMA, EXAMPLE 12 This example illustrates the effect of the prostagland to E2 on the development of tumor function in response to the composition of the invention in the presence of indoinetacin. The subject population consisted of one normal and nine patients (one healthy, one patient with a pancreatic tumor, two patients with tumors in the head and neck, one with endornetposis, and four with HIV). The preparations containing peripheral blood rhonocytes were isolated from blood samples of the patients using the procedure described in Braun et al. (1990). The cytotoxicity of the turnoral cells in response to the composition of the invention (1:10 loading dilution No. 222) and mdometacin (up to 5 ng / ml), with or without PGE2 (IO ^ M), was achieved using the assay of rnonocito cytotoxicity described in Braun et al., 1 bid. The results are presented in the following table, in which the results are re-cited as percent cytotoxicity of tumor in a rnonocito / turnoral cell ratio of 15: 1.

• EFFECT OF PGE2 IN TUMQRAL FUNCTION DEVELOPED IN RESPONSE TO THE COMPOSITION OF EXAMPLE 1 Diagnosis Composition Composition Composition 5 (Virulizin) (Viru) + Indorn. (Virul) + PGE Healthy 19 20 27 Pancreatic 15 14 22 HNSCC 9 8 12 • 10 HNSCC 11 3 12 Endornetriosis 37 37 n.d. HIV 6 7 8 HIV 15 12 19 ^ g HIV 21 .16 20 f HIV 23 22 n.d.

The data in the following table shows that the pathophysiology levels of PGE2 (10M) failed to suppress the level of turnoral function that develops in response to the composition of the invention. This conclusion contrasts with the ability of PGE2 to suppress tumor function in monocytes stimulated with IFN-t (Braun et al. (1993).

EXAMPLE 13 This example illustrates the development of tumor function against tumor cells derived therefrom in monocytes stimulated with the composition of the invention. The preparations containing peripheral blood monocytes were isolated from blood samples of 7 patients (three ovarian cancers, one endometrial cancer, one cervical cancer and two ENT cancers) using the procedure described in Braun et al. (1990). The cytotoxicity of thyraceous cells in response to the composition of the invention (1:10 dilution of loading supply No. 222) and indoinetacma (up to 5 rng / rnl), with or without PGE (10-8M) was achieved using the rhnocyte cytotoxicity assay described in Braun et al. (1990), with the exception that patient's patient cells were used in place of Chang hepator cells. The tumor cells of the patient were treated with collagenase and DNase, the individual cell preparations were prepared, and the cells were labeled or described in Braun et al. (1990). The results shown in the following table demonstrated that the composition of the invention is capable of activating the patient's own rnonocytes to control the patient's tumor.

TUMOR FUNCTION OF MONOCITE INDUCED BY THE COMPOSITION OF EXAMPLE 1 Diagnostic Co Culture Conditions% Tumor Cytotoxicity (E / T - 15 / L) Ovarian Caneer Medium composition 2 (Vi rulizín) LL JLO Cancer ovan co Composition t-Interfe on 1 «• LPS m dia (ViruI izin) 4 9 Ovarian cancer Composition t-Interferon 0» LPS media (Vi rulizm) 14 # 11 Cancer Co-position ter tero 6 endornet rio i LPS media (Virulizín) 14 20 21 Cancercervic l Composition t-Interfe on 8 + LPS media (Vi rul iz) 30 13 cancer ENT Composition T-Interferon 11 + LPS media (Viruliz) 12 25 Cancer - ENT Compound t-Tent feron 18 * - LPS IL-2 H-CSF 11 medium (Vlruliz) 11 3 35 35 The experimental results in Examples 10 to 13 indicate that the composition of Example 1 is capable of activating monocytes to express the tumor function; it works in the blood with peritoneal macrophages; and, the results are 40 consistent with not being subject to the inhibitory effects of prostaglandins, which is one of the main forms of immunosuppression in patients. The experimental data also support the usefulness of the composition in the treatment of peptoneal, alveolar and gynecological malignancies.

EXAMPLE 14 This example illustrates the results of tests conducted to calculate the protein within the composition. The protein calculation of the composition was performed fef using the Pierce Micro BCA 0 protein determination technique (Srnith et al., Anal, Biochem., 150,76-85 (1985).) A sample of 10 μl of a the composition was created as 1 ml with distilled water, and five concentrations of bovine serum albumin (0.150 μg / ml) were created to be used as standards, 0.1 N NaOH was used as empty, a mixture was added to all 5 samples of BCA (2% sodium salt of bicinconic acid), 4% of copper sulfate and microreagent A (NaaC? 3, NaHCO3 in 0.2N NaOH) The sample mixtures were incubated for 1 hour at 60 ° C, cooled , and the resulting absorbance was read at 562 nrn using a spectrophotometer The amount of protein in the test sample was then compared to the normal curve and the appropriate calculations made The protein concentration of the composition was found to be lower and it was calculated to be 32 μg / rnl.

EXAMPLE 15 This example demonstrates, in summary, the following: (1) the composition has a TNF-a releasing activity and the TNF-a releasing activity does not refer to any contamination with endotoxin; (2) sizing of macrophages improves the ability of the composition to stimulate the release of TNF-a; (3) the hi-perrnolarity of the composition is not necessary for the TNF-a releasing activity. To test whether an endotoxin effect was associated with the aforementioned biological activity for the composition of Example 1, additional experiments were performed on the composition with polixin added to the reagents. Polixirnin inhibits the action of endotox on the leukocytes. The following table and the subsequent notes return to cite the experiment performed on the composition and its results.

ABSENCE OF ENDOTOXIN FOR LIBERATING EFFECT OF TNF-2 AND IMPROVEMENT OF LIBERATION WITH APPROPRIATE MACROFAGO TNF Released (PG / rnl) Proved Total Additive Sample. LPS 25 Notes: 1.- The total TNF released is correct for the release of TNF L17 by means of 1640. 2.- The concentration of polymyxma: 50,000 units / ml. * 3. - Volume of the composition: 200 μl. 4.- We tested 8 patients with polyinixin. Three patients without additive were tested, 5.- The LPS concentration: 50 ng / 10 μl. The results show that polyinixin completely inhibits the induced release of LPS from TNF-α. In the absence of polyrnixin, LPS induces 517 pf / inl of TNF-a, therefore 0 in the presence of polymyxin, 11 pg / ml of TNF-a are released, in the absence of polyxin, LPS and the composition more than just an additive effect of the stimulators, suggesting that The composition acuates with greater intensity when the macrophages are prepared. 5 ABSENCE OF THE EFFECT OF HYPERMOLARITY ON THE RELEASE OF TNF-2 #Carga PH 0sinol ri dad (mOsrn) 0 Concentrated: B0222 re-pH 411 B0222 pH adjusted 581 B0216 pH adjusted 872 F¡ B0219 pH adjusted 886 Not concentrated: B0221 pre-pH 652 B0221 pH adjusted 533 0 B0213 pH adjusted 675 B0225 pH adjusted 590 B0226 pH adjusted 540 BC 11-06 adjusted pH 445 BC 11-09 adjusted pH 603 5 The oscillaria of different loads was determined using normal methods. The results are shown in the previous table. B02 3 is moderately high at 675 rnOsrn. B0222, shows to have TNF-releasing activity including better use than B0213, it is less hi blue, 581 Osrn. The fractions B0726, BCl i-06 and BC11-09 vary from 54 0 to 603 rnOsm. The effect of the hyporrosionality of the composition on the activity of TNF-a liberating activity was also studied. It was found that the composition continues to liberate TNF-a when it adjusts for oslopathy, even to the point of being hypo-osnolar.

EXAMPLE 16 This example illustrates toxicity studies as to the composition of the present invention. Preliminary toxicity studies were conducted on a variety of animal species, as shown in the table below. 5 All the animals (listed in the following table) were determined based on the daily clinical observation while receiving injections of the composition on days 14, 21 and 30 after the same. The heinatological data were collected every third day during the first 30 days and once a month after this. No adverse effects were seen in any of the 358 animals included in this study throughout the period in which the injections were administered or during the next period (one rnes for all species except for dogs that continued for 4 months).

Animal Canti ad Dosis A second toxicity study was conducted to To determine the effect of a large single intramuscular dose of the composition. Thirteen Sprague Dawley rats received a single intramuscular dose of 5 ml / kg of the composition. Three rats were observed for 7 days. Ten rats were observed for 14 days followed by euthanasia and necropsy. No symptoms of toxicity were observed in any group and there were no gross pathological findings in the animals that were necropsied. Based on these observations the intramuscular administration of LD50 of the composition in rats was determined to be greater than 5 rnl / kg. 30 The Ontario Veterinary College conducted another toxicity trial, in which the composition was administered to two cross-breed dogs. The protocol is summarized in the following table: Animal Age and weight Dose 1 Dose 2 Dose interval In each case, a dose was given in the right hind paw and the second dose 7 days later occurred in the left hind paw. Both dogs were observed for 14 days after the first injection. Appetite was monitored, # activity, temperature, pulse rate and speed respiratory twice a day throughout the study. The urinalysis, hematogenesis and routine serum chemistry profiles were performed at the following time points: pretreatment and 24 hours, 72 hours, 7 days and 14 days after the first injection. No animal showed signs of pain-20 associated with any injection. There was no evidence of anaphylaxis associated with the second injection. No abnormalities or changes in physical or laboratory parameters that could be attributed to the drug were observed. The drug seems to be well tolerated by healthy dogs. 25 A 17-day repeated dose study was conducted with VIRULTZINR along with a model animal study at the Ontario Cancer Institute. The female model used of C57B1 mice. There were four groups as follows (IM - intramuscular, TP - in raperitoneal): # Treatment Group Volume of dose Number / Group Each group of mice was injected on day 0 with 5 x 103 of Elanorne B16F3 cells plus microspheres. In each of the first 17 days, each group received daily injections of Virulizin® or saline solution, as above- On day 18, the animals were sacrificed. Before the sacrifice, the consumption of food, weight gain and behavior were normal. In addition, there was no evidence of toxicity that caused changes observable by the light of the microscope in any of the organs examined, which were: large intestine, spleen, stomach, pancreas, urinary bladder, liver, brain, kidneys, intestine thin and heart. The consumption of food and behavior were normal. The weight gain was normal. A repeated dose toxicity study was conducted for 13 weeks in Fischei-344 rats (total of 40 males and 40 females) to which VTRULIZINR IM was administered three Or H times a week for 13 weeks. The largest dose was 1.1 ng / kg, approximately 20 X the human dose. The animals were subjected to complete histopathology after 13 weeks. The only discovery that was observed related to the treatment was a small decrease in obtaining average body weight in the 20 X dose group compared to the controls. Mo toxicity was demonstrated, EXAMPLE 17 This example illustrates the. Clinical use of the composition of the invention for the treatment of several malignant tumors in companion animals. Eleven cats and ten dogs with neoplastic disease 'advanced fj, none of which was responding to the The conventional therapy was treated with the given intramuscular composition in weekly doses. The following table summarizes the individual clinical cases in this study, as follows: No. NoBbre and age Species / sex Diagnosis De-up Injection Surgeries Results * No. Noabre and age Species / sex Diagnosis De-up Injection Surgeries Results * 17 George - 10 Felino-Hacho Helanoaa 02/03/87 15 1 Partial response sir, malignant 04/08/87 stable without caabios 18 Sisón - 13 Canino- Hacho Hiperplesia 02/04/87 10 1 Partial response from prostatic gentleman 31/08/87 benign 19 Tequila - 14 Canino-H / s fldenocarcino- 11/24/89 35 I Partial response "ayor aa intesti09 / 08/90 nal flaligno 20 Sheba - 12 Canino-F / s Osteosacoa 06/12/89 25 1 Partially invasive partial response of 06/28/90 initial. Later progressive sickness. Euthanasia 21 Hesha - 14 Felino-F / s Osteosarcoaa 02/07/89 13 2 Inappropriate mieabro affliction. 06/05/89 Complete response 1 year without recurrence or Betastasis BV The number of injections ranged from 2 to 59, with volumes up to 7.5 ml delivered to a single intramuscular site. The weekly injection protocols took into account the examinations and the careful monitoring of the individual cases, with diagnostic tests determined individually for each case. The clinician noted that there was no local irritation or severe allergic reactions, including anaphylaxis. The clinician and the owners of. { The animals did not observe systemic adverse reactions. The researchers noted some clinical improvements that consisted of minor reductions, increased levels of appetite and activity, significant weight recovery in some animals and a decrease in pain and / or discomfort. The clinical results indicated in the table above include certain terms to describe the animal's response to treatment with VIRULIZINMR. These terms are defined in the following table: H.H fifteen twenty Six animals (3/10 canines and 3/11 felines) experienced a complete response. One animal (1/11 feline) had an initial major partial response. Eleven animals 5/10 40 canines and 6/11 felines) experienced a minor partial response. One animal (1/10 canines remained stable and one animal (1/11 feline) did not respond.Clinical experience clearly supports a degree of efficacy of the composition in the treatment of malignant neoplasms.

EXAMPLE 18 * This example illustrates the results of an Open Phase II clinical study conducted with cancer patients. "5 In 1988, an Open Phase clinical trial was started II at the General Hospital of Mont eal and extended to the Sas Atoon Cancer Center in 1989 under the diron of Maksyrniuk.

The experiment remains open to patients with various tumors T F advanced solids that have or have not had treatment previous (excluding radiation) for your disease. Patients have been treated and continue to be treated three times a week with intramuscular injons of 7.5 ml of VIRULIZINMR and have been observed and continue to be observed in terms of safety, performance status according to the Oncology Group Cooperativa del Este ("ECOG"), quality of life and survival. As of March 31, 1994, 99 patients had been treated. Adverse events were generally mild to moderate. Five patients discontinued treatment due to adverse events. No complete or partial responses were observed, although 18 patients achieved the stabilization of their disease. With respto the clinical end points, there was no change or improvement after 8 weeks of treatment with VIRULIZINTM in quality of life, pain and performance status according to ECOG in 51%, 78% and 56% H, respvely for those patients when their data were available. A subgroup of patients (n = 12), K¡ treated with VIRU-IZIN1"1 * of advanced pancreatic cancer, exhibited a one-year survival rate from the date of treatment with VIRULIZINMR of 29% with an average survival of 160 days This group exhibited a survival rate of 5 years from the date of diagnosis of 38% compared to 13.8% of historical controls, therefore, the results of the Phase II clinical study have been consistent with tests in 'ß vitro and with animals: Virulizin is effve to an appreciable degree for cancer therapy.

EXAMPLE 19 This example illustrates the methods and results of the clinical studies on Pancreatic Cancer, in particular a Phase II experiment (Protocol C02-104) with the composition of the invention for patients with pancreatic cancer, checked by biopsy and measurable. The treatment consisted of the composition prepared as in Example 1, 0.11 ml / kg (minimum dose of 7.5 ml) administered in a single deep intramoscular injon to the gluteus maximus, alternating the buttocks with each dose. Patients received 3 injons during the first week followed by injons twice a week until the progression of the tumors. The response was defined using common criteria, as cited by Miller et al., Cancer, 47, 207-214 (1981). A complete response (CR) was defined as the complete disappearance of all evidence of disease for at least 4 weeks. A partial response (RP) was defined as a reduction of at least 50% in the product of two maximum perpendicular diameters of the measurable lesion áxia, without other injuries or progression of any injury, for at least 4 weeks. Progressive disease was defined as an increase of 25% or more in the size of one or more lesions or the appearance of other lesions. The disease that did not meet the criteria for response or progressive disease was called stable disease. A total of 22 patients were enrolled in the study, but five patients were considered invaluable in terms of efficacy. There were no complete or partial answers. Three patients had progression of the disease within the first month. Six patients presented stabilization of the disease for more than 3 months (ie, 3.5, 3.5, 5, 8, 12+ and 14+ months). The average survival of the entire group was 8 months from the date of diagnosis and 5 months from the start of treatment. A patient with hepatic metastases verified by biopsy and a CEA level of 37 ng / nl (normal is less than 3 ng / ml), had absolute stabilization of liver metastases and a CEA level for 8 months. One had stable disease for 5 months. One patient relapsed in her pancreatic base 4 months after a Wfc Uhipple procedure and has been stable using the composition for at least one year, with the exception of a delayed but growing CEA. A percutaneous gauze was inserted into a third patient and he continued to work full time for at least 14 months without any evidence of tumor progression. All 22 patients were assessed for toxicity, having received a total of more than 500 -9pr injections. None revealed any clinical evidence or toxicity laboratory realaciled with the drug. There was no detrimental effect on the Quality of Life that was generally connected with the activity of the disease. No significant changes were seen in total white blood cell counts or absolute lymphocyte counts on serum immunoglobulins. Figures 6 and 7 show, respectively, JHf the survival curves that represent the survival times from the diagnosis and the initiation of the treatment. For comparison, a historical survival curve of Gudjonsson (1987) has been superimposed in Figure 6. Another example of a historical curve of comparable history can be found in Bakkevold, Petterson, Arnesjo and Espenhaug (1990). The results of the 25 survival analyzes are summarized below: The continuous line represents the survival curve of the patients treated with the composition of the invention and the interrupted line represents the historical survival curve (Balen et al., 165- 187 S 499-508, Lippincott Co., Philadelphia, Penn.). The survival of all patients treated with the composition of the invention, including patients with one to more tumor sites, is shown in Figure 8. Survival of patients with two tumor sites and with three or more tumor sites is shows ufe in Figures 9 and 10, respectively. The group of all patients treated with the composition of the invention had a 39% survival (estimated Kaplan-fleier) in one year- The degree of survival in one year for all patients with advanced malignant melanoma (rirlA) is of approximately 1.1% in the controls historical (equivalent in number of tumor sites). The group had a median survival of 315 days compared to the historical median of 89 days. For patients with two tumor sites, one-year survival was 49% in the treated patients with the composition of the invention, compared to 13% in historical controls. This group had a median survival of 360 days compared to the historical median of 120 days. With three or more tumor sites the survival rate of one year was 31% in patients treated with the composition of the invention, compared to 0% of historical controls. The group with three or more tumors had a median survival of 205 days compared to the historical median of 60 days. Quality of life was assessed by weight recovery, performance status (K rnofsky), quality of life index (Spitzer) and a pain scale (Linear Analogue). Weight recovery is shown during the time in The following table: fifteen The scales of Karnofsky and Spitzer are both subjective and found that they agreed approximately in each individual. Fifteen patients reported no change in these parameters. Four patients presented fluctuations that subsequently returned to previous levels. One patient had a decrease (of 40-20%). O H The results of the pain assessment for six patients showed that in week 4 the pain decreased from 5 (the worst possible) to 2 (moderate) or 0 (no pain). One patient had a pain reduction of 3 to 0. One patient with liver metastasis had pain reduction at 0 and stabilization for 11 months. Nine patients who registered in the study with pain or maintained that level through the study. Five patients had a moderate increase (2 units) of pain. Three patients had transient pain increases (1 to 2 units) during the second or third rnes. 5 Of 1,734 injections administered to 33 patients, 21 patients had no adverse drug reactions. It was reported about 14 adverse reactions of the drug in twelve patients. Adverse reactions of the drug occurred usually at weeks 8 and were mild to transient and very often of a low grade fever. The difference in survival between the historical groups and the protocol groups treated with the composition of the invention suggests a survival benefit for patients treated with the composition of the invention. It seems that the cancer was stabilized in nineteen patients. All patients treated for UMA were included in the survival data. Twenty-one previously treated patients were also included (many clinical trials required untreated patients, due to poor prognosis of the previous failed treatments). The burden of tumors in this population was high (82% had more than one rnetastatic site). Data on survival and quality of life suggest that the majority of patients received some benefit of the treatment. Eleven patients were still alive at the end of the study period and of those eleven, five continued treatment. The preliminary analysis in thirty-three patients demonstrated a survival rate of one year (from the diagnosis of relapse) of 39%. After the conclusion of the study, 5 final data were available for forty-five patients. Forty-one of these patients had distant metastases. The survival rate in one year from the diagnosis of distant metastasis for these patients was 61%, with a median survival of 529 days (17.6 months). 0 This can be compared to the survival degree of 13% (median of 92 days) from the historical data. The survival rate of one year after initiation with VIRULIZTNMR for all patients was 22%, with a median survival of 200 days (5.7 months). 5 Quality of life, pain and performance status according to FCOT showed no change or improvement during the first 8 weeks of treatment in 63%, 93% and 70% of patients respectively for whom data were available. In addition, the pathology of tumors before and after the application of VIRULIZINMR showed an unusual pattern of tumor cell necrosis, fibrosis and vascular thrombosis consistent with the effects mediated by TNF-ct.

EXAMPLE 21 5 This example illustrates a pathology protocol directed at malignant melanoma. The following is a report of an elderly 73-year-old woman with progressive malignant melanoma of the palate and gums. Two views of malignant melanoma under the microscope were observed. Looking up and down, you can see the epithelial layer with the accompanying keratin, within which malignant cells begin to become more evident. These cells can be seen to be round or oval, with a Wt abundant eosinophilic cytoplasm and hypercrotic nuclei i eos neornórficos. These cells have replaced normal submucosal tissue. The blood vessels that appear normal and there is a shortage of any kind of inflammatory / immune response as it would be represented by the presence of leukocytes (polymorphonuclear cells and rnononuclear cells). This is an example of tumor tissue that is progressing, that is, the tumor architecture is intact. A sample of tumor tissue from the same patient, which had been treated with the composition for 2 months, was observed. Starting from top to bottom, you can see that the The continuity of the epithelium has been destroyed by a necrotic process. This necrosis, while common in the center of any turnor that has reached a critical mass, is rarely seen in the periphery, especially the malignant melanoma and is a sign that the host immune response is rising to attack with the tumor. Throughout the fo or there is a massive number of preferred cells of the original cells fl_ of the turnor. These are immune cells, including neutrophils, lymphocytes, macrophages that have organized the destruction of the typical tumor architecture. The walls of the blood vessels have infiltrated thickly with a large number of cells. immune cells of the host. This cellular infiltrate will subsequently cause the destruction of the blood vessel which at the same time prevents the tumor from receiving its supply of nutrients and oxygen (ischemic necrosis). This immune response that ÉU contributed to the turnoral disorganization seen in the slides with the patient's tissue is consistent with the informed changes that are known to be produced by TNF (tumor necrosis factor) and with the work results described in the previous examples. The immune response showed in the treatment Subsequent with the composition in the holder strongly links the immune modulation of TNF in vitro by the composition with the known effects of antitumor turbid TNF in vivo.

EXAMPLE 22 This example illustrates the effects of VIRULIZIN ^ R on the function of your oricide in peripheral blood rnonocytes of cancer patients. 25 Peripheral blood rhonocytes from the venous blood of cancer patients were obtained, processed, and examined for their oricidal activity against Chang's hepatoma cells according to the procedure of Example 9. The results are presented in the following table, where the following abbreviations are used: "ENT Cfi" is carcinoma of the ear, nose and gargant; "KS / HIV" is Kaposi's sarcoma in patients infected with the Human Immunodeficiency Virus; "CA Ovarico" is ovarian carcinoma; "CA Pulmonar" is lung carcinoma; "CA Endo" is endo-uterine carcinoma of the uterus; "CML" is chronic rhielogenous leukemia; Terms used in the chart include: "Diagnosis," which refers to the type of cancer the patient had. The total number of patients who provided inonocytes for the tests is indicated as "# tested." The number of patients whose rnonocytes showed the ability to be stimulated by VIRULIZINMR over the total number of patients tested, is called "# stimulated / total (ViruiizinMR). "The number of patients whose onocytes showed ability to be stimulated by a combination of 100 units / nl of inferferone range (IFN-t) and 2 ng / ml of lipopol isacid (LPS) of Escherichia coli origin on the total number of patients tested is called "# stimulated / total (IFN / LPS) " With respect to "# stimulated / total (IFN / LPS)" of Virulizin ™, the stimulation is defined as an increase of more than 50% above the tumor values obtained by cultivation with the medium alone. "Lots / Stirnulation "cites the number of batches or batches of virulizer used followed by the number of tests that showed stimulation on the total number of tests with that batch / batch in parentheses. * fifteen twenty H 40 These results show that the turnoricidal activity provoked by VIRULIZIN ™ in the monocytes of cancer patients was equal to or greater than the activity produced in the response to a combination of conventional activators of macrophages (TFN-cf and LPS). VTRULIZTNMR can also stimulate the function of your oricide in rnacrophages obtained from patients with HIV with Kaposi's sarcoma, even in very early stages of the disease. In this way, the action of VTRULIZINMR appears to be independent of collaboration with other types of immune cells, including helper T lymphocytes. ß EXAMPLE 23 LO This example illustrates the effects of VIRULIZIN ™ in the tumor-associated phaeopharyges in patients with cancer. The alveolar macrophages from 11 patients with non-small cell lung cancer were obtained by bronchoalveolar lavage and examined for tunopcid activity against Chang's hepatoma cells by the procedure described in example 9. Seven-patient peptonoal macrophages were obtained. with gynecological cancer (2 endometrial, 3 ovarian and 2 cervical) and were examined for turnoricidal activity against the cells of Chang's hepatorna by the aforementioned procedure. The results were displayed in the following table, in which the abbreviations and terms used are defined as cited in example 22. O H 10 fifteen * twenty These results indicate that VIRULIZINMR can stimulate both peripheral and regional blood monocytes, macrophages associated with tumors of patients with cancer to express signi? Cant tumor killing activity. This result was observed in peritoneal tumors of women with gynecological malignancies and alveolar rnacrophages of patients with tumor cancer from these results, it is believed that VIRULIZINMR can also stimulate the cancer of cancer patients who are not responsive to stimulation with activators. conventional drugs such as interferon plus endotin. ß EXAMPLE 24 This example illustrates the effect of VIRULIZIN ™ in the development of the turnoricidal function against autologous tumor cells in rhonocytes of cancer patients. Peripheral blood inomocytes were obtained and examined for their turnoricidal activity against autologous tumor cells, using the methods described in f? example 9- In this way, the rnonocitos of a patient with carcinoma of the ear / nose / throat, for example, were examined as to the activity of your oricide against that patient's own tumor cells. Analogous tests were also performed using ovarian and endo-nalnal carcinoma cells and chronic iogenous leukemia. The exhibits are displayed results in the following table, where the abbreviations and terms used are as cited in example 22. The medium? used is 1640 media from Roswell Park Memorial Institute and CRPMI] supplemented with inactivated fetal bovine serum with 10% heat, 50 um / ml penicillin and 50 microgram streptomycin. 15 twenty OR 40 These results indicate that VIRULIZINMR can stimulate the turnoricidal activity in the macrophages of cancer patients against autologous tumor cells 45 prepared from surgical biopsies of patients with cancer. From these results, it is believed that ß VTRULIZINMR can stimulate the acrophages of cancer patients who are not responsive to stimulation with conventional activators such as feroferone plus endotoxin range.

EXAMPLE 24 This example illustrates the effect of cytokine-specific antibodies on the development of the tuinopid function in monocytes stimulated with V.rulizin. 0 Peripheral blood nicotomes were obtained from a patient with lung cancer and from a patient with chronic rhinogenic leukemia (CML) and examined for turnoricidal activity against Chang's hepatoma cells according to the method of example 9. VTRULIZINMR was used only for 5 to examine the stimulation of monocytes as well as VIRULT7INMR plus anti-IL1c / or anti-IL1β or isotype control antibody. The amount of antibody used was a quantity of saturation for these conditions of analysis as determined in titration experiments according to the normal methods. 0 The results are shown in the following table, in which the abbreviations used are as cited in example 23. In addition, "anti-ILla" is an antibody of the interleuk at lcx; "anti-ILlß" is an antibody to rhterleucin 1 (3; "anti-TNFcf" is an antibody to tumor necrosis factor alpha; and 5"isotype control" is an antibody to an epitope unrelated to cytokines previous i- fifteen twenty O H The results indicate that the antibodies against the alpha factor of the turnor necrosis inhibit the tumoricidal function caused by VIRULIZINM. Antibodies either against interleukin 1 alpha or interleukin 1 beta stopped reducing the tumoricidal function of the monocytes stimulated with VIRULIZIN ™. The results are consistent with the conclusions that the tumoricidal function of the macrophages which is developed in response to the VIRULIZTNMR are associated with the production of tumor necrosis factor-alpha (TNP- ~ cr) by the onocites.

EXAMPLE 25 The example illustrates the effect of cytotoxic therapy on the development of function in the peripheral blood ßp monocytes stimulated by Virulizm. 10 Peripheral blood rnonocytes from cancer patients were obtained at the end of their first course of remission induction quernotherapy and examined for tumoricidal activity against Chang hepatorna cells using the methods of Example 9. 5 the results in the following table, in which the abbreviations used are as disclosed in Example 24. In addition, "PT" is cis-platinum; "5-FU" is 5-fluorouracil; "RT" is radiotherapy; and "Ara C" is an Arabmocidal cytokine. The term "Diagnosis" is the type of cancer that had the patient. When the phrase "recurrent" is listed here, the cancer has been recurrent, or otherwise the cancer was newly diagnosed. The term "therapy" is the cancer / cancer therapy regimen to which the patient was being subjected. « fifteen twenty The results indicate that VIRU1.1ZTNMR stimulates the turnoricida function in the rnacrofagos obtained from the patients with cancer that are undergoing cytotoxic therapy; Accordingly, it is believed that VIRULIZINMR interacts favorably with other therapeutic modalities. It is of importance in fact that VIRULIZINMR was more effective in stimulating the tumoricidal function than the activators 40 conventional, such as interferon plus endoto ina.

EXAMPLE 26 This example illustrates the effect of Viruliz on inacrofago cytotoxicity in patients with ondornetposis. Peripheral blood rhonocytes and peritoneal rhinocytes were obtained from patients with endorheiosis as in Example 9 and were tested to verify the turnoricidal activity against Chang's hepatoma cells and to see the cyto-toxicity against autologous endo-cell cells prepared from uterine biopsies. The results are shown in the following table, where the abbreviations and terms used are the same as in example 25. In addition, "stage" refers to the stages of endornetriosis based on the classification system of the RAF (Revised American Fertilaty Society).

EFFECT OF VIRULIZATION ON MACROPHAGUS CYTOTOXICITY IN PATIENTS WITH ENDOMETRIOSIS 20 Stage Lot # Medium IFN / LPS Virulizin Effect OR The results indicate that the VIRULTZTNMR stimulates the peripheral blood rhonocytes and the phanerocytes pep-tons of patients with ondoinetosposis to eliminate endornetpals cells prepared from uterine biopsies Consequently, the composition of the present invention can provide a treatment pair-endornet posis.

EXAMPLE 27 f 10 This example illustrates the results of preliminary batch tests of Virulizin ™, peripheral blood rhonocytes from venous blood were obtained, processed and tested to verify tumopid activity against Chang's cells. by the procedure described in example 9. Virulizm is prepared according to example i. The results are shown in the following table, where the abbreviations and terms used are the same as in example 26. In addition, "donor" is the disease status of patients from whom pepfenca blood onocites were obtained. "Normal" means that the patient did not have the disease. / 'ENT CA "means that the patient had cancer of the head and neck (ear / nose / throat).

PRELIMINARY PROOF OF LOTS 247, 248 AND 249 OF VIRULIZIN. Dona te Lot # Me io IFN / LPS Viruli i n twenty The results indicate that the tumoric activity of H produced by VIRULIZINMR in monocytes from normal patients and with cancer was equal to or greater than the activity produced in response to a combination of conventional macrophage activators (IFN-t and LPS).

EXAMPLE 28 This example illustrates the isolation of active fractions. A sample of 300 nl of the composition was evaporated to dryness in a rotating vaporizer in which 1 ai. Bath temperature did not exceed 40 ° C. To ensure that the solution remained basic during evaporation, 5 drops of a concentrated solution of ammonium hydroxide were added every half hour to the composition until the evaporation was complete. The resulting residue had a weight of 11.6 g. 20 ml of a concentrated solution of hydroxy or 10% ammonium in methanol was subsequently added to 2 g of the above residue. The insoluble material was filtered and the filtrate was chromatographed through 101.93 g of M? vaporization of 60 fl in a column with dimensions of 5 ern x 12.5 ern »The solvent system used was concentrated solution of 10% ammonium hydroxide in methanol. The column was operated at a pressure of 0.703 g / crn2 and a flow rate of 11 l / rnin. After 100 rnl of the solvento passed through the column, 12 fractions of 20 ml were collected. The Collection of these fractions was correlated with the appearance of a whitish band that moved rapidly down the column. The thin layer chromatography (CCD) of these fractions was carried out on silica gel plates in a concentrated solution of 10% ammonium hydroxide in methanol and visualized with a ninhydrin spray. Fractions that had similar CCD profiles were combined, resulting in the following fraction combinations, which were dried in a rotary vaporizer: Volume through the column for Fractions obtain fraction Yield (g) 1-4 100- 180 0 5-6 180-220 0-1175 10 7-8 220-260 0.1969 9-10 260-300 0.0151 11-12 300- 340 0.0053 15 Fractions 5-6, 7-8 and 9-10 had a positive reaction with ninhydrine at an Rf value of 0.81. Fractions 5-6 and 9-10 were tested i_n vi t r-o to verify the imulation of the TNF (according to the example 9). The results are shown below: Fraction Activity 5-6 50 pg / rng H 9-LO 1814 pg / rng In this way, fraction 9-10 was an extremely active TNF stimulator. 30 Samples from fraction 5-6 were analyzed by electron impact loop spectroscopy (MS), electrospray mRNA spectroscopy to identify specific compounds that could be present in the fraction. The MS with electrospray was carried out in a Perki-Elmer Sciex API-III spectrometer using 5% acetic acid in water as the solute. In some cases, ethanol was added to aid dissolution. The ET US using a direct insertion probe was carried out in a VO spectrometer Analitical model ZAB-SE using giicerol as a matrix, and using a DCI probe in a spectrometry or Analytical K "5 Pro file Mass Spect rometer. A review of the resulting spectra indicated that the following compounds could be present in fraction 5-6: phosphocol a, taurocolic acid, diglypepdo of Mf acid high cholesterol, acid rich, acid diglicepdo stearic acid, diglicep or stearic acid co-palmitic acid and a conjugate of oleic acid co-esfmgosma.

EXAMPLE 29 This example illustrates an expanded procedure for isolating active fractions. Example 28 was repeated on a larger scale as follows: 10 ml of a concentrated solution of 10% ammonium hydroxide was added to 900 nl of the composition and the solution The resulting solution was evaporated to dryness in a rotary evaporator in which the temperature of the bath did not exceed 40 ° C. To ensure that the solution will remain basic during evaporation, 5 drops of a concentrated solution of ammonium hydroxide were added every half hour to the composition until complete evaporation, leaving a residue. 150 rnl of a concentrated solution of 10% ammonium hydroxide in methanol was subsequently added to the total residue. The solution was treated with sound for 15 minutes and the insoluble material was filtered. The filtrate was chromatographed through 1695 g of 60fl vaporizer silica gel in a column with dimensions of 30 ern x 12 crn. The solvent system used was concentrated solution of 10% ammonium hydroxide in methanol. The column was subjected to a pressure of 0.422 kg / cm2 and a flow rate of 30 rnl / rnin. The results of the column are summarized in the following table.

Volume of each fraction Fraction # (ml) Observations 1 550 clear, yellowish 2 420 cl ra, yellowish 3 400 clear, yellowish 4 150 clear, rusty 5 100 c r, light r 25 25 6-7 75 light, yellowish 8-1.3 50 light, yellowish 14 50 Cinnamon solution co i in; to elute 15-35 50 Cinnamon solution 36-40 50 clear, yellowish 5 The CCD was carried out on silica gel plates in a 10% strength solution of ammonium hydroxide solution and visualized with a ninhydrin spray. The fractions having similar CCD profiles were combined, giving as a result the following fraction combinations, which were dried in a rotary vaporizer: Volume through # of column for Performance Fraction get fraction (g) Comments twenty All combinations of fractions 15-16 to fractions 31-34 had a positive reaction with ninhydma at an Rf value of 0.87, a value very similar to the value of Rf for the active fractions of example 28. Fractions 24- 30 and 31-34 had an additional positive reaction with 40 ninhydrin at a Rf value of 0.85. Fractions 4-5, 15-16 and 17-18 were tested in vitro for stimulation of TNF (according to example jB 9), resulting in no FNT stimulation activity. The elemental analysis of the previous fractions showed that its NH4Cl content was high, which is known to inhibit the production of TNF. 5 Samples from fractions 15-16 and 24-30 were dialysed and then analyzed by mRNA spectroscopy, using the methods described in Example 28. The non-dialyzed mixtures of fractions 17-18 and 24-30 also they were analyzed. A review of the resulting spectra indicated that the following compounds were present: glycocholic acid, a trimer of t-rihexosamine and taurocholic acid (fractions 15-16); stearic acid and a hexosamine diol; and glycolic acid (fractions 24-30).

EXAMPLE 30 This example illustrates the application of additional methods for fractionating and analyzing the active compounds of the inventive composition. 20 Having identified that the activity of releasing TNF, IL-lß and GM-CSF can be precipitated, in part, by means of 80% acrylonitrile and that much of the release activity elutes early from CLAR-FI Cis, the physical and chemical properties of the precipitated fraction have been studied and compared with the whole composition and the supernatant fraction of the composition.

Figure 11 shows an elect rhoresis of SDS gels of the entire composition and the precipitates and supernatants of the composition. In all three cases, the composition runs close to the SDS front, indicates a low molecular weight. The rnas parameter small used was 14,400 daltons. The molecular size of the composition was also examined to determine its elution time from a molecular sieve HPLC column. The elution times of The entire composition, precipitate and supernatant were compared with the parameters. All three eluted later than insulin, which eluted at 24.5 minutes. Once again, the physicochemical analysis gives a molecular weight of less than 2,400 daltons. The TNF release component elutes early- this way a column with the opposite effect was chosen, a hydrophilic column in the presence of organic solvents. The ideal elution conditions for the polyhydroxyethyl column are 80% acetonitrile. However, as indicated in the previous example, some of the substances in the The preparation was precipitated at this concentration. Accordingly, the composition was analyzed at a concentration t > acetonitrile where the column works mainly co or a molecular sieve column. Figures 12 and 13 show the profile of the entire supernatant and the precipitate. The front sheet summarizes the elution time for the different peaks. The elution times indicate that the f ß active component of the composition has an inolecular weight b jo. The composition and its precipitate and supernatant were separated by CLAR ion exchange. So much for The chromatography medium AX300 (anion exchange), or by CMX 300 chromatography (cation exchange), had no significant separation of the components. The hydrophobic reverse phase chromosome did not separate the peaks.

J f In another series of experiments, 10 rnl of VTRULIZINMR were loaded onto a chromatographic column by anion exchange (Bio-Rad AG-1, hydroxide form, the total resin wet volume was 10 rnl, equilibrated with Millipore deionized water). The volume of the resin was calculated as sufficient for the binding of all the anions present in the extract.

The unbound fraction was collected and reloaded on the column to maximize the bond to the resin. The unbound fraction of this second passage was collected and stored. Any unbound material that remained in the empty volume of the column was removed by washing with water deionized (2 X-20 nl). The ligated molecules were eluted with a gradient of steps of ammonium bicarbonate, 20 l / step. The ammonium bicarbonate was removed in free form by lipophilization. The samples of all fractions were tested to verify the TNF release activity in the rnonocito activation test / rnacró ago. The TNF release activity was not found in the unbound fraction (effluent), but the majority was found in eluate eluted with 0.2 M ammonium bicarbonate. These results indicate that the active components are polar, ammonic and acidic in nature. . The samples of all the fractions were analyzed to verify the stimulation activity of the TNF, according to the procedures of example 2. The results are shown below: Sample Release of FNTc * -act? V? ducting capacity-LPS (pg / rnl) 0 M -496 0.1 M -156 0.2 M 1638 0.3 M -36 0.4 M 256 0.5 M -27 0.6 11 -175 1.0 M -246 1.5 M -346 VIRULIZINMR Control 1961 The results of the activity tests show that the stimulation of TNF production was discovered in the fractions of 0.2 M and D.4 M. The composition was subjected to dialysis and dialysis drying, as follows: 100 nl in the The composition was placed inside a CE Spectra / PorR membrane tube that had a molecular weight cutoff of LOO. The ends of the tube were sealed with clips and the tube was placed in a stirred barium of 10 L of distilled water. The dialysis was monitored daily by removing 1 ml of the solution from the dialysis tube 5 and adding 3-4 drops of a silver nitrate solution to 1/10 N. The presence of chloride indicated that the dialysis was not complete. If the dialysis was not complete the bath was replaced with fresh distilled water. The completion of the dialysis occurred after 3-4 days. After ia After the dialysis was completed, the dialyzed material was dried on a rotary evaporator to produce an average of 0.3 g of solid per milliliter of the original volume. A sample of the solid material was then dissolved in water grade CLAR, and the CCD was carried out on plates of silica gel in a concentrated solution of 10% ammonium hydroxide in methanol, and visualized with a spray of nmhidrma. A positive reaction with mnhydpna was obtained at an Rf of 0.83. A sample of the solid material was also analyzed by means of mRNA spectroscopy, using the methods described in example 28. A review of the resulting spectra indicated that the following compounds were present: a conjugate of sputum-oleic acid, diacetyl sialic acid, a dimer of fucose-hexosam a, cocolic deoxigli acid, acid taurocholic, a day of sialic acid fucose and a trimer of d? (Fucose) hexosarnine. ß EXAMPLE 31 This example will illustrate the use of reverse phase CLAR (CLAR-FI) to analyze the composition of the invention. Samples were hardened and then reconstituted in 0.1% trifluoroacetic acid (TFA) in water (pH regulator A) and subsequently were carried out in the following columns and conditions: Column: Column UP60009-C18 (U-Pore C18, 250 X 4.6 mm, f? v Pheno enex, California) in row with prime column - sphere HC-C18 (250 x 4.6 rnrn, Phenornenex, California) Flowing agents: pH A regulator: 0.1% TFA in H20 pH B regulator: TFA l 0.1% in ace11 r ilo 15 Gradient: 150 μl of the sample applied to the column Apply pH A regulator for 20 minutes Start linear gradient, 0-80% pH A regulator, applied for 35 minutes 20 Apply pH B regulator at 80-0% for 5 minutes Flow: 0.9 l / rninuto Temperat ur: Arnbiente Detection: Absorbency of 290 to 284 n, with most applications being detected at 210 and 325 eluent fractions met in the times approximated from the injection and were noted in the following table. In addition, a TNF release test was carried out in each fraction, as described in example 2, with the following results: Fraction # Time (ri FNT (pg / ml) 1 5.6-6.25 203 2 6-25-6.6 -1.57 3 5.6-7.1 1 4 7.1-7.9 11 5 7.9-8.4 84 6 8.4-8.9 -24 10 7 8.9-9.4 -10 8 9.4-10.0 35 9 10.0-10.4 2 10 10.4-12.0 11 11 12.0-13.6 49 15 12 13-6-14-2 39 Total VIRULIZINMR 2.13 20 As a consequence, most of the active components of VIRULIZINMR existed in fraction 1. The activity was also discovered in fractions 4-5, 8-9, 11-12, and 14. Samples of all the samples were analyzed. 25-CLAR-FI fractions by mass spectroscopy according to example 28. A review of the resulting spectra for the fractions indicated that the following compounds were present: taurocolic acid, sialic acid-glycerol dimer, NaCl, trirnetiiarnine, rethylethylamine and propi lamina 30 EXAMPLE 32 This example illustrates the compounds that have been identified in the composition of the invention. The inventive composition was prepared according to Example 1 and subjected to normal fractionation methods, 9 9 including (1) dialysis in a 100 MUCO dialysis membrane; (2) classical organic extractions including Folch extractions, (Tarnari et al., Agr. Biol. Chem., 40 (10), 2057-2062 (1976)); (3) silica column chromatography; (4) 5 ion exchange chromatography; and (5) fractionated by preparative CCD with silica using butanol: acetic acid: water (6: 2: 2) as the eluent and ninhydpna as the visualization reagent, using normal methods as described in yg Reagent for Thin Layer and Paper Chrornatography, E. Mercl- ', Dapnstadt, Germany, 1971. The identification of the compounds was based on the following instrumentation and techniques, used individually or in combination: A VG 70-250S spectrometer was used to obtain EI-15 MS, CI-MS (OH-,), and FAB-MS (in glycerol or ___ thioglycerol). A VG Analytical Model ZAB-SC instrument was used to obtain EI-MS, FAB-MS (in glycerol or thioglycerol matrices) and GC-MS. The gas chromatograph (GC) used in conjunction with the instrument was a Hewlett Pacl - ard model 5890.

A Kratos profile spectrometer was used to obtain E3-MS, LSIN-MS (in glycerol and NPOE matrices), and mRNA spectra of TC-MS. The GC used in conjunction with the instrument was also a Hewlett Pacl-'ard model 5890. The MS-MS and electroaspersion were carried out using either water or alcohol mixtures. with water (methanol or isopropyl alcohol) as solutes, EI-MS and PAB-MS in glycerol and thioglycerol were carried out in a spectrometer per in-Fl er Sciex APT-IIT. The fractions were derived from an MS analysis as required by acetylation with acetic anhydride / pipdine or rnetylation with diazomethane. The conversion of molecules into sodiated species was achieved with the addition of sodium acetate to the electrospray solute. The protonation of the molecules for the MS of the ectroaspersion was achieved using acetic acid or trifluoroacetic acid. The CCDs of the extracts and the parameters were carried out on silica gel CCD plates using butanol / acetic acid / water 6: 2: 2: or the eluted are cited as mobile phases and vain aspersions of reagent for the visual ization. Normal methods were used in relation to the aforementioned instruments, which are mentioned in more detail in the following references: Riyler et al., J. Chrornatography, 277, 321-327 (1983); Sundararn, et al-, Clínica Chirnica Acta, 34_ 425-429 (1971); Bandursl et al., J. Biol. Chem., 193 405-410 (1951); and Larsen et al., J. Chromatography, 226 484-487 (1981). Typical CCD profiles on silica plates (using butanol: acetic acid: water, 6: 2: 2 as eluent) are as tabulated for active lots of VIRULIZIN ™; Display Reagent CCD profile * sulphuric acid Rf = 0 to 0.25, white point sulfate of cerium ammonium Rf = 0.5 to 0.42, yellow point molybdate Rf-0 to 0.3, points light blue-green to white with blue edges - 10 ve rde nisaidehído Rf = 0.03 to 0.25, white point acid 6-anilino-l-na phthalene Rf = 0 to 0.25, yellow dots sulphonic sulfate (for the eye) ninhi rina Rf = 0 to 0.13, pale pink dot B ^ B Rf = 0.12 to 0.3, purple point in the shape of spear point 20 R f = 0.15 to 0.3, pu to bo r yña Rf = 0.3 to 0.45, light yellow point Rf = 0.35 to 0.5, color point arna ri 11 op ro fu n te OH Rf = 0.4 to 0.5, burgundy point Rf = 0.5 to 0.6, burgundy point * The Rf values will vary slightly depending on the degree of activity of the silica gel coating d € - > the plates and the precise composition of the elution solvent. The analysis of the inventive composition using the aforementioned instrumentation and methods revealed that the following compounds were contained therein: 1) BILIARY CIDOS: cholic acid; glycolic acid; deoxy glycolic acid; cholesterol sulfate; deoxycholic acid; 40 chenodeoxycholic acid; and taurocholic acid. Note: From the MS it is not distinguishable if -OH and - H2 occur in the MS or if the unsaturated analogues deoxy, dideoxy are also present to begin with. ß These compounds may be present as ammonium salts, alkylammonium and inorganic cations. 2) RELATED PHOSPHOLIPIDS, ESTHINTOLIPIDS AND PRODUCTS (HYDROLYSIS): 5 stearic acid CH3 (CH2)? ßCOOH; palmitic acid CH3 (CH2)? «COOH; oleic acid Z-9 octadecanoic acid: CH3 C CH2? 2 CH2 CH = CHCH2 (CH2)? COOH short chain fatty acids unsaturated / oxidized or hydroxylated, such as C? H8? 3 (CH3 CH -CH-COCH2 COOH or a Ce acid with 2 double bonds and a hydroxide). acetic acid; di glyceri or stearic acid; diglyceride of palmitic acid; stearic acid, diglyceride of palmitic acid; monoglyceride of stearic acid fos focholine (a lysolecithin); monoglyceride of stearic acid; triglyceride of stearic acid; 20 phosphocholine; phosphoseri a; fos fos f ingosi n; e f i n gorn i e 1 i na; lecithin; 25 stearic acid-fingosin; sphingosine; phosphol1icero1; glycerol.; hill; 30 glycerophor focholine; stearic acid, oleic acid diglyceride; stearic acid, oleic acid phosphoglycerol; stearic acid amide; rneti stearic acid lick; and palmitic acid amide. In addition, preliminary HPLC and titration evidence have been obtained, which shows that shorter chain fatty acids are also present (Ci to C30 acid scale). 40 3) MUCINE HYDROLYSIS PRODUCTS: sialic acids and their mono- and diacetylated monomers; N-acetylneuraminic acid; hexosamines, such as glucosanine; The fucose; Hexosarnine disulfate (dimer) of hexuronic acid; 107 acci or gl ucu romeo; ß glucuronic acid or iduromic acid disulfate, rnonoaceti side; sialic acid-glycerol (di ero); and 5 dimers, trimers, oligomers and polymers of the abovementioned nos. in acetylated and sulphated form. 4) SOLUBLE GREASE VITAMINS: Vi tarnina A2; Vitamin DI; 0 LurnisteroL (I present from its DI vitamin complex); Vitamin E; Oxide of vitamin Kl; and Vitamin K5. 5) ORG NICOS PRODUCTS MISCEL NEOS: u r a; alkylamines, including ethylene, d irne ti larn i na, ethylarn a, rnetileti lamina, diethyla ina, di propí lamina, buti letilarní; 0 amino acids, including taurine, glutarnic acid, glycine, alana, n-leucine, phosphoserma, phosphoethanolamine, aspartic acid, threo, sepna, sarcosine, adipic acid, citrull, valma, isoleucma, ß-alanma , t-amino butyric acid, 5 hydroxyl isma, ornithma and lysine; hydroxytoluene bu lylated (BHT); and poliet i l ngl icol.

EXAMPLE 33 This example illustrates the saccharide components of the invention. The monosaccharide composition of the samples was determined before and after the hydrolysis. All the reagents 5 used to analyze the monosaccharides were of analytical grade. THF (tffluoroacetic acid) obtained from Aldpch was used after dilution with deionized water, for the hydrolysis of the samples. A 50% NaOH (P / P) solution (low in carbonate) was purchased from Fisher Scientific. Sodium acetate was from Fluka-Terant, New York. To release the rnonosaccharides, the samples were treated with 4M trifluoroacetic acid for 4 hours at 100 ° C. The samples were lyophilized and analyzed using high-performance liquid chromatography-anion exchange using a Dionex Bio-LC system for carbohydrates with a Carhopack Pal separator column (250 x 4 rnrn id) and a guard column for OLAR-AGd (50 x 4 m id) equipped with Wß a sample loop of 25 ul. The detection of rnonosacap two eluents were achieved with PAD, ie amperometp co-pulsed detector. The conditions were as follows: Before hydrolysis For the detection of ositol, sialic acid and acid glucuromer, an eluent of isocratic elution (mixture of NaOH of LOO rnM + 150 mM NaOAc) was used. The eluent was protected from the atmosphere with a helium module degasser. The flow rate was 1 ml / rnin through the column. The detection of rnososaccharides, including fucose, Galactosanine, galactose, glucose and mannose were also achieved by means of isocratic elution, eluent (15 nM NaOH) with a 300 nM NaOH post column, at a flow rate of i ml / rnin. The detector settings E1-0.05V, E2 = 0.60V, E3 = 0.60 V, tí = 120rns, t2 = 120rns, t_3 = 300? Ns; gold work electrode; silver-silver chloride reference electrode; output speed 1-3 K nA p full scale; speed of cai-1a 0.5 crn / rnin. Detector measurements were made to verify uronic acid and onosaccharides. A linear response - 5 was obtained for concentrations ranging from 0.5-2.5 ug / rnl by a progressive dilution of a parameter mixture.

After hydrolysis ß Nonosaccharides were detected after hydrolysis of the sample after applying a gradient elution, eluent A (50 nM NaOH) and eluent B (NaOH mixture of 50mN / NaOAc of 150 rnM). The eluents were protected from the atmosphere with a helium module degasser. A Spectra-Physics integrator (SP 4270) was used to analyze the departure. The normal gradient was an injection in eluent A to 100%, followed by a linear progression to A at 80%: B at 20% for the next 10 minutes. This condition was maintained for 20 minutes and then the eluent was returned to 100% A for 5 minutes followed by at least 10 minutes of balanced before the injection of the next sample. The results of the rnonosaccharide analysis, as described, are presented in the. next box:.

NOISE IKJ 115 fl nuiaa 6i Water layer of an extraction HU 14111 (Lot of Precix II (Folch ethyl acetate extract (dialyzed KU1I.I) dialyzed IC02U) of green bile) i- or As indicated in the table, only the. ethyl acetate extract of green bile (lot MU100 GB) showed not to include any nonosaccharide before hydrolysis, being those acids sialic and glucuronic, in a concentration of microgram per milliliter. After hydrolysis, sialic acid was not detected and glucuronic acid was present in approximately 20% of the concentration. After hydrolysis, other preparations of the compositions of the invention were shown to contain sialic acid, glucuronic acid, glucosarnine and inositol. From the foregoing it will be appreciated that while specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.

Claims (6)

ß NOVELTY OF THE INVENTION CLAIMS

1. The use of a composition for the treatment of a cancer selected from the group consisting of pancreatic carcinoma, malignant elanoma, ovarian carcinoma, ENT carcinoma, endonetrial carcinoma, lung carcinoma and Kaposi's sarcoma, wherein said composition includes components of 10 low molecular weight of less than 3000 daltons and has the following properties: a) it is extractable from the bile of an animal; b) is able to stimulate rhonocytes and macrophages in vitro and alive; c) is able to modulate the production of tumor necrosis factor; d) does not contain intermediate levels of

IL-IC1, IL-lß, FNT, IL-6, IL-8, IL-4, TM-CSF or IFN-t; e) does not show cytotoxicity to mononuclear cells of human peripheral blood; and f) it is not an endotox na. 2. The use of a composition for the treatment of endornetposis, wherein said composition comprises components 20 of low molecular weight of less than 3000 daltons, and has the following properties: a) it is extrable to the bile of an animal; b) is able to stimulate rnonocitos and inacrophages i n vitro and in vivo; c) is able to modulate the production of the turnoral necrosis factor; d) does not contain measurable levels of 25 IL-ltf, IL-l? , FNT, II.-6, IL-8, IL-4, GM-CSF or IFN-t; e) does not show cytotoxicity to the mononuclear cells of human peripheral blood; and f) it is not an endotoxin.

3. The use of a composition for the manufacture of a medicament for treating a cancer selected from the group consisting of pancreatic carcinoma, malignant melanoma, ovarian carcinoma, ENT carcinoma, endornetial carcinoma, lung carcinoma and Kaposi's sarcoma , wherein said composition comprises components of low molecular weight of less than 3000 daltons and has the following properties: a) it is oxtraibie of βßt the bile of an animal; b) is able to stimulate rhonocytes and 10 in vitro and in vivo macrophages; c) is able to modulate the production of the turnoral necrosis factor; d) does not contain edible levels of IL-la, IL-1 | 3, FNT, IL-6, TL-8, IL-4, GM-CSF or TFN-t; e) does not show cytotoxicity to the rnonuclear cells of human peripheral blood; and f) it is not a 15 endoto ina.

4. The use of a composition for the manufacture of a medicament for treating endorhetriosis, wherein said composition comprises small amounts of low molecular weight components of less than 3000 daltons and has the The following properties: a) is extractable from the bile of an animal; b) is capable of stimulating monocytes and macrophages in vitro and i vivo; c) is able to modulate the production of the turnoral necrosis factor; d) does not contain measurable levels of IL-la, IL-lß, FNT, IL-6, TL-8, IL-4, GM-CSF or IFN-t; e) does not show cytotoxicity to human peripheral blood rnonuclear cells; and f) it is not an endotoxin.

5. - The use of claim 1, wherein said composition is injected intra.scularly.

6. The use of claim 2, wherein said composition is injected intramuscularly.