WO1998052585A1 - Immunomodulating, bile-derivable compositions for the treatment of viral disorders - Google Patents

Abstract

The present invention relates to the use of a composition exhibiting antiviral properties, comprising small molecular weight components of less than 3000 daltons, and having the following properties: a) is extractable from bile of animals; b) is capable of stimulating monocytes and macrophages in vitro and in vivo; c) is capable of modulating tumor necrosis factor production; d) contains no measurable IL-1α, IL-1β, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-η; e) shows no cytotoxicity to human peripheral blood mononuclear cells or lymphocytes; and f) is not an endotoxin. The invention also relates to the use of the antiviral composition when used in conjunction with other drugs such as antiviral compounds or immunomodulators such as interferon.

Description

IMMUNOMODULATING , BILE-DERIVABLE COMPOSITIONS FOR THE TREATMENT OF VIRAL DISORDERS

FIELD OF THE INVENTION

The present invention relates to immunomodulating compositions for the treatment of viral infections, pharmaceutical compositions comprising the same, and the use of such compositions in the treatment of viral infections in mammals.

BACKGROUND OF THE INVENTION

Almost all life forms are susceptible to virus infections, including humans, animals, plants, and even bacteria. Viral infections cause countless diseases and are directly responsible for enormous annual losses of domestic crops and animals. Viruses are infectious agents characterized mainly by their small size and chemical simplicity. During apart of its infectious cycle in a cell, and sometimes even outside the cell, a virus consists only of nucleic acid, either DNA or RNA, though a mature virus particle normally exists in the form of nucleic acid encased in a protein shell.

Viral infections begin either by injection of viral nucleic acid into a cell, such as a bacterial cell, or by engulfment of whole virus and uncoating of the nucleic acid, as with animal and plant viruses . Once inside the cell, the viral nucleic acid competes with the genetic material of the host for control of cell processes. The viral nucleic acid interacts with the host genetic material in different ways, dependant upon the viral type. For example, some bacterial viruses induce synthesis of an enzyme that destroys the host nucleic acid, rendering the cellular synthesis under the exclusive control of the new virus directing synthesis of new virus particles. In other situations, host material is not destroyed and viral induced functions are supplemented by those of the host nucleic acid with the resultant production of new virus particles. In other situations, all or part of the viral nucleic acid appears to be physically inserted into the host nucleic acid. In some bacterial and animal tumor viruses, the viral nucleic acid may function to some extent to produce certain specific proteins, but whole new virus particles are not produced.

In some situations the virus will damage or destroy its host (cell) and in other situations it will live in somewhat of a symbiotic relationship within its host. Viral infections can therefore be determined by either the presence of the virus or by the results of its infection as manifested in a disease state.

When a virus infection is latent, persistent or slow, the infection can be inapparent and chronic in which a virus-host equilibrium is established. Slow and persistent viruses are intermittently or continuously present in the infected animal and may cause diseases usually of a chronic and degenerative nature, often associated with the central nervous system. There are many examples of natural viral infections of animals, plants, bacteria, and insects as well as humans, in which there are no obvious evidences of injury or illness in the host. At one end of the spectrum, there are examples such as subclinical forms of poliomyelitis, influenza, or yellow fever, no illness is manifested, although laboratory tests can easily show the virus to be present and multiplying in the host. At the other end of the spectrum, there are examples of viruses such as type 1 herpes simplex virus which persists in a latent infection for long periods, sometimes for life, and only adversely affects the individual when nonspecific factors such as fever, precipitate attacks of cold sores.

Both DNA and RNA viruses can cause cancer and are known as tumor viruses. In a small percentage of nonpermissive cells, which allow the virus to enter but not to replicate lytically, the viral chromosome either becomes integrated into the host cell genome, where it is replicated along with the host chromosomes, or forms a plasmid. Such nonpermissive invections sometimes result in a genetic change in the host cell, causing it to proliferate in an ill-controlled way and thus transforming it into its cancerous equivalent. RNA viruses use the enzyme reverse transcriptase to transcribe the infecting RNA chains of these viruses into complementary DNA molecules that integrate into the host cell genome.

The most familiar retrovirus, called human immunodeficiency virus (HIV) enters helper T lymphocytes by first binding to a functionally important plasma membrane protein called CD4. Not only does HIV kill the helper T cells that it infects, but it also tends to persist in a latent state in the chromosomes of an infected cell without producing virus until it is activated by an unknown rare event; this ability to hide greatly complicates any attempt to treat the infection with antiviral drugs.

The resultant acquired immune deficiency syndrome (AIDS) develops because of specific damage to helper T-lymphocytes, which are essential to the proper functioning of the cellular immune system. This component of the immune defenses is essential in controlling infections caused by fungi, viruses, protozoa and some bacteria. It is also thought to effectively control the growth of certain tumors. The case definition of AIDS was initially used to identify persons with this syndrome for s eillance purposes. Since the discovery of the human immunodeficiency virus, AIDS and AIDS-related illnesses have been reclassified by the Centers for Disease Control in Atlanta. This new classification places AIDS within the context of HIV illness as groups IV C- 1 (opportunistic infections) and group D (tumors) disease (CDC, MMWK., 35:334-339, 1986). The most frequently observed skin lesion is Kaposi's sarcoma. This is a multifocal systemic tumor that is characterized by the rapid generation of neovascular tissue. The sites most frequently involved are the skin, mucous membranes and lymph nodes. Usually the tumor presents with single or multiple pink, red or violet lesions that may take the form of macules, papules, plaques or nodules. With progressive disease, this tumor has been found in all organs.

Measurement of the viral load, or the number of copies of a viral genome per cell in a biological sample is important for a number of reasons. In some situations, the viral load may be the only manner of determining the seriousness of the infection or disease and whether the patient is responding to therapy. In the case of viruses responsible for a disease such as AIDS, where the infection is overshadowded by symptomatic complications, determination of the viral load might be the only manner of revealing the progress of the infection.

The problem of the combating virus infections has not yet been solved. One of the unsolved problems is the relatively rapid mutation of viruses which totally or partly prevents the effect of antiviral drugs or the body's own antibodies. Therapies are continuously being developed for the prophylaxis and treatment of predominant, devastating viral infections, such as HIV.

One approach is based on the antigen-specific elements of the immune system, namely antibodies and T-cells. For example, research has been aimed at developing vaccines against foreign agents, or against certain endogenous chemical messengers, such as interleukins, to control or induce certain antibody reactions. A second approach is based on the isolation, cloning, expression and production of peptides and proteins from the nonantigen-specific parts of the immune system. For example, proteins, such as cytokines, which comprise the interleukins produced by white blood cells, and interferons, which stimulate lymphocytes and scavenger cells that digest foreign antigens, offer possibilities for therapies.

Research in the field of tumor and virus biology has provided critical insights regarding substances which affect their pathology. Two substances which appear to play important roles in tumor and viral growth and replication are tumor necrosis factor (TNF) and interferon (IFN).

TNF was originally termed "cachectin" because of its ability to produce the wasting syndrome cachexia. It is composed of two related proteins: mature TNF (TNFα) and lymphotoxin (TNFβ), which are primarily produced by activated macrophages, monocytes and lymphocytes. Both TNFα and TNFβ are recognized by the same cell surface receptor. TNFα was originally discovered in the serum of animals injected sequentially with the bacterial vaccine bacillus Calmette-Guerin, and endotoxin (Carswell, et al, P.N.A.S. USA, 72ι3666, 1975). TNFα is secreted as a soluble homotrimer of 17kD protein subunits in response to endotoxin or other stimuli (Smith, et al., J.B.C., 262; 6951 , 1987). A membrane bound precursor form of TNFα has also been described (Kriegler, et al, Cell, 53:45, 1988).

The expression of the gene encoding TNFα is not limited to cells of the monocyte/macrophage family. Several human non-monocytic tumor cell lines were shown to produce TNFα. The role of the physiologically active TNF polypeptide has been studied. In particular, TNF has been shown to induce necrosis of tumors, with no effect upon the normal tissues of the living body. The amino acid sequence of TNF, as well as the base sequence of the DNA coding for TNF, have been disclosed in U.S. Patent No. 4,879,226.

The mechanism of action of TNFα appears to be derived from accumulating evidence which indicates that TNFα is a regulatory cytokine with pleiotrophic biological activities. These activities include: inhibition of lipoprotein lipase synthesis, activation of polymoφhonuclear leukocytes, inhibition of cell growth or stimulation of cell growth, cytotoxic action on certain transformed cell lines, antiviral activity, stimulation of bone resoφtion, stimulation of collagenase and prostaglandin E2 production and immunoregulatory actions including activation of T-cells, B-cells, monocytes, thymocytes and stimulation of the cell-surface expression of major histocompatibility complex class I and class II molecules.

The mechanisms by which TNF exerts its effect is not entirely known, but it has been suggested that they are mediated by the suppression of lipoprotein lipase activity. Serum TNF levels have been directly correlated with tumor burden in sarcoma bearing experimental animals, and inversely with food intake and body weight. Elevated levels of TNF have aslo been associated with HIV-infected persons with AIDS or AIDS related complex, but not in asymtomatic HIV-infected persons.

Because TNF has been shown to have a role in inducing necrosis of tumors, any agent that can stimulate the production or bioavailability of TNF in vivo has potential utility as a treatment for various tumorous conditions. Additionally, any agent that can stimulate human monocytes and macrophages to produce TNF in vitro, is useful as a means for providing a source of TNF for therapeutic administration, as well as for analytical and diagnostic puφoses. Unfortunately, treatments with high dosages of TNF alone have been associated with such side effects as hypotension, leukocytosis, fever, chills, neurotoxicity, nausea and vomiting.

TNF therapy has therefore played an important role in the field of cancer therapy, however excessive or unregulated TNF production has been implicated in exacerbating a number of disease states.

These include rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, gram negative sepsis, toxic shock syndrome, adult resipratory stress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resoφtion disease, reperfusion injury, graft v. host reaction, allograft rej ections, fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia secondary to AIDS, keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis and pyresis plus a number of autoimmune diseases such as multiple sclerosis, autoimmune diabetes and systemic lupus erythematosis.

Cytokines, specifically TNF, have been implicated in the activation of T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T-lymphocyte activation.

Therefore, extensive research has been directed towards interfering with cytokine production, notably TNF, in a HIV-infected individual. The therapeutic aim being to limit the maintenance of T-cell activation, thereby reducing the progression of HIV infectivity to previously uninfected cells, thereby resulting in a slowing or elimination of the progression of immune dysfunction caused by HIV infection. Hence there is mounting evidence supporting the use of inhibitors of cytokines, particularly

TNF, (U.S. Patent Nos. 5,563,143 and 5,506,340) in the treatment of AIDS.

Numerous clinical trials have also been carried out in patients with Kaposi's sarcoma with immune modulators such as Interferonα (J.AIDS., 1:111-118; 1988). This drug has been licensed in Canada for the treatment of Kaposi's sarcoma. Interferon has been shown to have antitumor and antiretroviral effects. Response rates to treatment with IFN are initially high (Krown, et al., Recomb. Leucocyte A IFN in Kaposi's sarcoma, N.Y. Acad. Sci., 437:431-438, 1984). However prolonged responses are not frequent, possibly because of the emergence of anti-IFN antibodies (Autavelli, et al., J.I.D. 163:882-885, 1991). Patients invariably require chemotherapy or radiotherapy to control tumor growth. Both IFN and chemotherapy have substantial toxic side effects on bone marrow resulting in the termination of therapy (Fischl, M.A., Am. J. Med., April 10, 1991).

Both TNF and IFN individually possess antiviral activity, making them potential candidates in the treatment of viral infections and tumors. However, serious side effects have been observed in the treatment with therapeutically valuable doses of TNF and IFN which have limited their clinical usefulness.

Infectious diseases, such as those caused by viruses can only succeed by avoiding or defeating the body's immune system. The immune system mounts or elicits either or both non-specific immune responses and specific immune response factors to fight such pathogens.

Non-specific immune responses are focused on cytokine production, principally by macrophages, and serve as a prelude to specific antibody responses. The inflammatory cytokines include TNF-α and mediate an acute response directed to the injury or infection sites, which is manifested by an increased blood supply. The pathogenic bacteria or viruses are engulfed by neutrophils and macrophages in an attempt to contain the infection to a small tissue space. Macrophages, therefore, play a key role in the defense against infectious diseases as follows: ( 1 ) processing and presentation of antigens to lymphocytes so that antibody-mediated and cell- mediated immune responses can occur;

(2) secretion of cytokines central to immune response; and

(3) destruction of antibody-coated bacteria, tumor cells or host cells.

Macrophages can ingest and kill a wide variety of pathogens, such as bacteria, fungi, and protozoa (parasites). This ability is augmented when the macrophages are "activated." Secreted products of activated macrophages are more diverse than those from any other immune cell. These regulate both pro- and anti-inflammatory effects and regulate other cell types. These products include TNF-α, IL- 1 β, IL-6, hydrolytic enzymes, and products of oxidative metabolism Bacteria that are eliminated primarily through this cell-mediated immune process include tuberculosis and other related mycobacterial infections, such as atypical mycobacterial infections seen in up to 50% of AIDS patients, and anthrax, a potential bacteriological warfare agent. Fungal infections are common problems in immunosuppressed patients, such as those afflicted with AIDS or organ transplant patients.

Bile, which is secreted by the liver and stored in the gall bladder, has been investigated for various puφoses, including the use of bile extracts to enhance bioavailability of drugs that are readily metabolized by normal liver function (^"see WO 90/12583) and to inhibit leucocytosis promotion in a mammal (see Shinoda et al, Chem. Pharm. Bull. 30, 4429-4434 (1982)). However, bile has never been considered to be a source of therapeutically useful compositions with respect to neoplastic, inflammatory or infectious diseases. Interestingly, in accordance with British Patent No. 337,797, it was suggested to use the gall bladder, itself, as a potential source of anti-cancer agents, but only after the bile had been removed from the gall bladder, and the gall bladder thoroughly washed.

Bile acids have been shown to inhibit endotoxin-induced release of TNF by a direct inhibitory effect on monocytes (Greve, et al, Hepatology, 10(4):454, 1989). Of the bile acids investigated, deoxycholic acid was the most effective, chenodeoxycholic acid was less effective and ur?deoxycholic acid was ineffective. Other workers (Keane, et al., Surgery, 95:439, 1964) have demonstrated that bile acids produce their effect by inactivating endotoxins. A further possible explanation for the clinical effects of bile salts in this indication is a reduction in the absoφtion of endotoxin from the gut.

SUMMARY OF THE INVENTION

It has now been discovered that bile is an important source of a composition that has antiviral activities. In particular, it has been discovered that the composition of the present invention can exert antiviral activities as demonstrated by a significant reduction in viral load of a patient infected with HIV.

The bile composition of the present invention is obtained by extraction of bile with a water-soluble or water-miscible solvent. The extract so obtained may be further processed to remove unnecessary or undesirable components therefrom.

In one aspect, the present invention relates to a composition for use as an antiviral agent comprising small molecular weight components of less than 3000 daltons, and having one or more of the following properties: a) is extractable from bile of animals; b) is capable of stimulating monocytes and macrophages in vitro and in vivo; c) is capable of modulating tumor necrosis factor production; d) contains no measurable level of IL-lα, IL-1 β, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-γ; e) shows no cytotoxicity to human peripheral blood mononuclear cells or lymphocytes; and f) is not an endotoxin.

In accordance with a preferred embodiment, the composition is extracted from the bile of bovines and is capable of stimulating the release of TNF.

The composition of the invention may be prepared by (a) mixing bile from an animal, preferably a bovine, with a solvent that is soluble or miscible with water, preferably an alcohol, and preferably with an equal volume of an alcohol, to produce a bile/alcohol solution; (b) separating the solution which preferably is an alcohol-soluble fraction, and isolating therefrom a solution substantially free of alcohol, as by removing most of the alcohol, such as by the use of heat; (c) removing bile pigments from the solution to obtain a clear, yellowish liquid; (d) optionally treating the clear, yellowish liquid to substantially remove any residual alcohol; (e) removing fatty organic materials, as by extracting the clear, yellowish liquid with ether and isolating the aqueous phase; and (f) optionally removing residual ether from the aqueous phase.

The invention also relates to a pharmaceutical composition comprising the antiviral composition of the invention.

The invention further relates to a method of treating a patient with a viral infection, comprising administering to said patient an effective amount of a composition of the invention. The invention still further relates to the use of a composition of the invention in the prophylaxis and treatment of diseases and conditions caused by viral infection. The invention also relates to the use of the antiviral composition when used in conjunction with other drugs such as antiviral compounds or immunomodulators such as interferon.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incoφorated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

Further details of the invention are described below with the help of the examples illustrated in the accompanying drawings in which: Figure 1 is a Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) profile for a concentrated composition of the invention;

Figure 2 is an 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 LPS-induced release of TNF by peripheral blood mononuclear cells (PBMNs);

Figure 5 is a bar graph showing the effect of the composition on LPS-induced release of TNF by

PBMNs;

Figure 6 is an SDS gel of the composition of the invention;

Figure 7 shows the conditions and times of elution of the composition of the invention on hydrophilic HPLC (a) and the elution profile for a supernatant of the composition of the invention (b);

Figure 8 shows the elution of a precipitate of the composition of the invention on hydrophilic HPLC; and

Figure 9 is a graph showing dose response of the composition of the invention in stimulating peripheral blood monocyte function.

DETAILED DESCRIPTION OF THE INVENTION

A new biologic response modifier called VIRULIZIN™ has been in development by IMUTEC Coφoration, a Toronto-based biopharmaceutical company, since 1986. VIRULIZIN™ is an immunomodulator which is hypothesized to exert anti-tumour activity via activation of macrophages, with subsequent enhancement of cell-mediated immune response to tumour. It is derived from bovine bile, and formulated as a sterile injectable product. Its precise mechanism of action remains unknown.

The "VIRULIZIN-2;. " (2-gamma) designation refers to drug that has been standardized for potency by the new TNF -release potency bioassay. Otherwise it is the same drug as used in previous preclinical and clinical testing, which was designated either "VIRULIZIN™" or "VIRULIZIN™-2- beta". IMUTEC Coφoration now only manufactures VIRULIZIN™-2 / .

Preclinical experimental evidence to date indicates that VIRULIZIN™-2>> activity is associated with low molecular weight fraction material derived from bovine tissue, with unique immunomodulatory properties. The cumulative results of our studies with VIRULIZIN™ have revealed following:

(1) VIRULIZIN™ does not directly stimulate lymphocytes to synthesize DNA or undergo blastogenesis and cell division. VIRULIZIN™ does not directly stimulate the development of lymphocyte-mediated cytotoxicity.

(2) VIRULIZIN™ can stimulate normal peripheral blood monocytes to express cytocidal activity in a dose-dependent manner. The activity elicited by VIRULIZIN™ is equal to or greater than the activity produced in response to more conventional macrophage activators that are currently under investigation in cancer patients including: Gamma Interferon; Granulocyte- Monocyte Colony Stimulating Factor; Monocyte Colony Stimulating Factor; and Interleukin- 12.

(3) VIRULIZIN™ can stimulate both the peripheral blood monocytes and regional, tumour- associated macrophages from cancer patients to express significant cytocidal activity. This included peritoneal macrophages from women with gynaecological malignancies and alveolar macrophages from patients with lung cancer. VIRULIZIN™ has been found to stimulate macrophages from cancer patients to kill autologous and heterologous tumour cells obtained from surgical specimens of patients. Of potentially greater importance is the finding that VIRULIZIN™ can often stimulate cancer patient macrophages that are unresponsive to stimulation with conventional activators such as gamma interferon + endotoxin.

(4) The hypersecretion of prostaglandins, both by macrophages and by tumor cells from cancer patients has been shown by Dr. Braun and others to be a principal cause of the immunosuppression seen in patients with advanced malignant disease. One determinant of the biological activity of different macrophage activators in cancer patients PBMs, therefore, is the sensitivity of the activator to arachidonic acid metabolism and the secretion by the cell of prostaglandins. The development of macrophage cytocidal function in response to VIRULIZIN™ was found to be insensitive to the inhibitory effects of prostaglandins. This is considered important therapeutically because the effectiveness of many other biological activators is limited by prostaglandins.

(5) VIRULIZIN™ can stimulate cytocidal function in macrophages obtained from cancer patients (including pancreatic cancer) who are undergoing cytotoxic therapy. Of note is the fact that VIRULIZIN™ was more effective in stimulating tumouricidal function than conventional activators such as gamma interferon plus endotoxin. Thus, VIRULIZIN™ appears to have the potential to be combined with cytotoxic cancer treatment in appropriate clinical settings.

(6) VIRULIZIN™ can also stimulate cytocidal function in macrophages obtained from patients with Kaposi's sarcoma even at very late stages of the disease. Thus, the action of VIRULIZIN™ appears to be independent of the need for collaboration with other immune cell types including helper T-lymphocytes.

(7) Preliminary studies suggest that the macrophage cytocidal function that develops in response to VIRULIZIN™ may be associated with the expression of TNFα by the macrophages. However, other mechanisms for cytotoxicity may also be involved and are currently the subj ect of ongoing investigations .

(8) Demonstrates anti-tumour activity in a mouse tumour (plasmacytoma) model.

(9) Exhibits no toxicity in animals at doses up to 125 X the human clinical trial doses with no LD⁵⁰ yet reached in toxicity studies.

(10) Induces the phenomenon of apoptosis in some continuous cell lines.

The central hypothesis guiding these studies is that the therapeutic efficacy of a powerful biological stimulator can depend on its ability to elicit suitable modulation of the immune system, such as by activating macrophages and/or monocytes to produce certain cytokines or promote activity to seek and remove or destroy disease-causing viruses or cells negatively affected by such viral infections. Such function could be generated by direct stimulation of resident immune cells in viral microenvironments. Alternatively, this function could be generated by stimulation of circulating immune cells if those cells were then able to home on sites of viral infection and to function in that environment.

As hereinbefore mentioned, the present invention relates to a composition for use as an antiviral agent comprising small molecular weight components of less than 3000 daltons, and having at least one of the following properties: a) is extractable from bile of animals; b) is capable of stimulating or activating monocytes and macrophages m vitro and in vivo; c) is capable of modulating tumor necrosis factor production; d) contains no measurable level of IL-lα, IL- 1 β, TNF, IL-6, IL-8, IL-4, GN-CSF or IFN-γ; e) shows no cytotoxicity to human peripheral blood mononuclear cells or lymphocytes; and f) is not an endotoxin.

The composition of the invention can modulate tumor necrosis factor (TNF) production. A preferred composition of the invention isolated from bile from bovines, promotes the release of TNF from human peripheral blood mononuclear cells and from the pre-monocyte cell line U-937 in what appears to be physiological quantities. Because TNF is known to initiate a cascade of inflammatory and antitumor cytokine effects, the preferred composition could exert its antineoplastic effect by stimulating human leukocytes to release TNF (and possibly other cytokines).

The effect of the composition on the survival of human peripheral blood mononuclear cells (PBMNs) and lymphocytes was also examined. The composition was found to be non-cytotoxic to human PBMNs and lymphocytes.

As further exemplified below, the composition of the present invention has, among others, the following characteristics:

( 1 ) The component or components responsible for TNF -release from PBMNs eluted early from a C_lg RP-HPLC column.

(2) The composition causes the release of interleukin- 1 β (IL- 1 β), and the component responsible for the IL-lβ release elutes early from RP-HPLC, suggesting that it is likely the same substance(s) that releases TNF.

(3) The composition also causes the release of low quantities of interleukin-2 (IL-2).

(4) The composition causes the release of granulocyte macrophage colony stimulating factor (GM-CSF); (5) The ratio of TNF to GM-CSF release is about 2:1.

(6) It is likely that the same molecule(s), i.e., component(s), in the composition are responsible for releasing TNF, IL-1 β and GM-CSF. It is possible that the composition acts to stimulate the release of multiple different cytokines, or alternatively, the composition triggers the production and release of one cytokine that in turn stimulates production and release of other cytokines.

(7) Physicochemical analysis of the composition, including the precipitates and supernatants thereof, by SDS gel electrophoresis and molecular sieve HPLC indicates that the principal components are less than 2500 daltons. (8) Further physicochemical separation by hydrophilic (polyhydroxy ethyl) molecular sieve HPLC confirms the small molecular weight of the components in the composition. (9) Amino acid analysis before and after acid hydrolysis suggest the presence of peptide bonds, indicating the presence of peptides.

As hereinbefore mentioned, the composition of the invention may be prepared by (a) mixing bile from an animal, preferably a bovine, with an equal volume of an alcohol to produce a bile/alcohol solution;

(b) separating out the alcohol soluble fraction and isolating a solution substantially free of alcohol;

(c) removing bile pigments from the solution to obtain a clear, yellowish liquid; (d) treating the clear, yellowish liquid to substantially remove any residual alcohol; (e) extracting the clear, yellowish 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 toward a specific disease if obtained from the bile of one species as opposed to another, a generally suitable source of bile is that taken from sharks, bovines, ovines, caprines, and porcines. In most cases, it is practical to obtain the bile of slaughtered healthy food animals, such as bovines, ovines, caprines, and porcines, for use in the preparation of the composition of the invention. The bile thus collected should come directly from the gall bladders and/or hepatic organs (as appropriate to the species' anatomy and physiology) of the slaughtered animals and should be substantially clear, thereby indicating that the bile preparation substantially free of pus or blood.

In a preferred embodiment of the method, bile from bovine sources is utilized. Bovine bile is plentiful, because, in part, relatively large quantities can be extracted from each animal. Moreover, bovines are routinely slaughtered and inspected under health-related regulations, thus such animals provide a reliable source for preparing the composition of the invention. Furthermore, humans are less likely to have an allergic reaction to material of bovine origin.

Obviously, the entire composition so obtained may not be necessary to obtain such activity. Accordingly, it is possible to further separate, fractionate, or otherwise process the product thus obtained, and still retain the desired ability to stimulate TNF production, for example, to act against the immune system disorders that underlie various diseases. Moreover, it is envisioned that it is possible to obtain synthetically a product with the same or similar ability to stimulate TNF production and act against immune system disorders. Thus, it is envisioned that the components of the product may be identified and analyzed as to their respective contributions to the desired characteristics of TNF stimulation and ability to act against immune system disorders, among other biological effects.

Moreover, it is further envisioned that such identification and analysis will be used to manufacture a synthetic form of the product.

The composition may be used without further modification by simply packaging it in vials and sterilizing. The composition may also be used in a concentrated form. A preferred concentrated form is prepared as follows. Prior to step (e) the clear, yellowish liquid may optionally be concentrated to about one-eighth of the volume of the bile/alcohol solution and after step (f) the aqueous phase may be concentrated so that it is one-tenth of the volume of the bile/ethanol solution.

The bile is mixed with an equal volume of an alcohol to produce a bile/alcohol solution, which is 50% alcohol. The alcohol may be an aliphatic alcohol, preferably methanol, ethanol, or propanol, most preferably ethanol.

A solution that is substantially free of the 50% alcohol-insoluble material may be isolated by centrifuging. Preferably, the bile/alcohol mixture is centrifuged at 3000-5000 rpm, most preferably 4200 φm, for at least 2 hours, at about 15-25 °C. The alcohol contained in the bile/alcohol-soluble fraction then may be removed by taking advantage of the different volatility of alcohol and water, using conventional methods, i.e., heating the fraction to a suitable temperature, e.g., 80-85 °C, for a suitable amount of time, e.g., up to about 10 hours.

Bile pigments may be removed from the solution to obtain a clear, yellowish liquid by using activated charcoal, polyamidic microgranules, or filtration. Preferably, an activated charcoal treatment is utilized. The procedure may be repeated in order that the solution satisfies optical density and conductivity standards.

The clear, yellowish liquid is treated to remove substantially any residual alcohol, using conventional methods. Preferably the clear, yellowish liquid is filtered using a filter having about a 1.0-3.5 μm retention, most preferably a retention of 2.5 μm.

The clear, yellowish liquid is then extracted with ether and the aqueous phase is isolated. The ether used in this step is preferably dimethyl ether, ethyl ether, n-propyl ether, isopropyl ether, or n-butyl ether, most preferably ethyl ether.

Residual ether may be removed from the aqueous phase by, for example, heating the solution up to 55 °C, preferably up to about 40 °C for about 5-15 hours, most preferably for about 10 hours.

The composition may be used without further modification simply by packaging it in vials and sterilizing. The composition also may be used in a concentrated form. A preferred concentrated form is prepared as follows. Prior to step (e) described hereinabove, the clear, yellowish liquid optionally may be concentrated to about one eighth of the volume of the bile/alcohol solution by, for example, heating to a temperature of less than about 85 ° C, preferably, to about 60 ° -70 ° C. After step (f), the aqueous phase may be concentrated so that it is one tenth of the volume of the bile/ethanol solution by, for example, heating to about 80-85 °C.

In a preferred method to prepare 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 about 4200 φm for at least 2 1/2 hours, at about 20+2 °C. The supernatant liquid is decanted and checked for pH and ethanol content. Bile pigments are then removed using activated charcoal. The treated bile/ethanol solution is then monitored for optical density (O.D.) and conductivity. O.D. levels or conductivity levels outside acceptable specifications require that the bile/ethanol solution be given additional treatment to remove bile pigments, for example treatment again with activated carbon to achieve a reading within specification limits.

Following activated carbon treatment, the solution is filtered through a filter having a 2.5 μm retention, the alcohol is evaporated off by heating to less than 85 °C and the solution is concentrated to approximately one eighth of the original bile/ethanol solution volume. The concentrated solution is cooled to between about 20-25 ° C. This solution is then mixed with ethyl ether and the ether phase is discarded. Preferably, relatively small volumes of ether and strong agitation are used, such as 0.1 to 1 volume, preferably 0.2 to 0.5 volume. This step may be repeated once. The aqueous phase is heated to remove residual ether by heating up to 55 °C for about 10 hours, and further reduced in volume to one tenth of the original bile/ethanol volume by heating to about 80-85 °C. This solution is then tested for appearance, biological activity, and ethanol and ether content.

The pH of the composition may be adjusted to physiological pH, i.e. 7.4-7.5, using hydrochloric acid (1%) solution and sodium hydroxide (1% solution), and a buffered solution may be obtained using dibasic and monobasic sodium phosphate salts as buffers, using conventional methods.

The composition may be used without further modification by simply packaging it in vials and sterilizing. A preferred sterilization method is to subject the composition to three sterilization cycles by autoclaving followed by incubation.

The composition may be used in a concentrated form. The preparation of the concentrated form is described above. The composition may also be lyophilized.

The composition and concentrated composition are clear yellowish solutions essentially free of foreign matter, containing not more than 10 ppm ethanol and not more than 5 ppm ether. The compositions activate PBMNs to release TNF in vitro as measured by the Monocyte/Macrophage Activation Assay (TNF-Release) as described in Example 2.

The compositions of the invention can be produced in a consistently reproducible form using the method as generally described above with demonstrated identity, potency and purity from batch to batch. Identity and purity are determined using reverse-phase high pressure liquid chromatography. (See Example 1). The compositions of the invention have a consistently reproducible pattern on reverse-phase HPLC The HPLC readings for three lots of the concentrated composition of the invention are shown in Figures 1 to 3. The compositions are also characterized by the properties hereinbefore mentioned, for example their ability to stimulate monocytes and macrophages in vitro and in vivo, etc. Compounds likely to be present in the present composition, considering the source, include sulfonated bile acids, oxidized bile acids, other naturally occurring bile acids, and their amino acid (especially glycine and taurine) conjugates and sterols. Accordingly, it is believed that the present composition includes at least one compound having the formula

wherein the molecule may or may not be fully saturated, such that, for example, the bond between A and B, B and C, or C and D may be single or double bonds, and wherein X is H, OH, =O, or OSO₃H; and Y is

wherein R is an amino acid residue, such as, for example, glycyl, glutamyl, or tauryl, thereby forming the glycine, glutamyl, or taurine conjugate.

In particular, the composition of the present invention has been analyzed as to its component compounds, including organic and inorganic components. Such information was derived using standard methods of analytical chemistry, including mass spectroscopy (MS). The results of such studies include, for example, the identification of specific bile acid compounds thought to be present, including cholic acid, glycocholic acid, deoxyglycocholic acid, ursodeoxycholic acid, cholesterol sulfate, deoxycholic acid, chenodeoxycholic acid, and taurocholic acid.

From the MS it is not distinguishable if the loss of OH and H₂ of some compounds are occurring in the MS or if the deoxy, dideoxy and unsaturated analogs of such compounds are also present to begin with. These compounds may all be present as salts of ammonium, alkylammonium and inorganic cations.

The MS analysis also supports the identification in the present composition of phospholipids, sphingolipids and related agents capable of forming miscelles. Specific compounds thought to be present include: stearic acid CH₃(CH₂)₁₆COOH palmitic acid CH₃(CH₂)₁₄COOH oleic acid Z-9 octadecanoic acid CH₃(CH₂)₂CH₂CH=CHCH₂(CH₂)₆COOH oxidized or hydroxy lated/unsaturated short chain fatty acids: C₆H₈0₃

(e.g., CH₃CH=CHCOCH₂COOH or a C₆ acid with 2 double bonds and a hydroxide) acetic acid stearic acid diglyceride palmitic acid diglyceride stearic acid, palmitic acid diglyceride stearic acid-monoglyceride-phosphocholine (a lysolecithin) stearic acid monoglyceride stearic acid triglyceride palmitic acid monoglyceride phosphocholine phosphoserine phosphosphingosine sphingomyelin phosphoglycerol glycerol stearic acid-sphingosine sphingosine stearic acid amide stearic acid methylamide choline glycerophosphocholine stearic acid, oleic acid diglyceride stearic acid, oleic acid phosphoglycerol palmitic acid amide lecithin sialic acid-glycerol dimer

In addition, preliminary HPLC and titration evidence has been obtained which shows that shorter chain fatty acids are also present, such as those having from 1 to about 30 carbon atoms.

Phospholipid, sphingolipid, and related hydrolysis product compounds likely to be present considering the source and the information derived from the MS and HPLC analyses include at least one compound having the formula

— OR' CH₃(CH₂)₁₂CH=CH

I OR² I OR' or I NHR²

— OR³-X I OR³-X

where R', R², R³ are different or the same and are H, COR⁴, CH=CH-R⁵, X, -P(O)(OH)O-, or - S(O)₂O-; X is selected from the group consisting of choline, ethanol amine, N-alkylated ethanolamines, serine, inositol, sugars bearing free hydroxyls, amino-sugars, sulfonated sugars, and sialic acids; R⁴ is C_]-C₃₀ alkyl that 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 aforementioned aqueous extract as salts. The solubility of such compounds is also enhanced by other components of the mixture. Amides of the included carboxylic acids, RCONR'R², where R' and R² are the same or different and are H or alkyl, are also believed to be present.

A third class of compounds, namely, mucin and proteoglycan hydrolysis products, are also likely to be present, considering the source of the composition and the aforementioned MS analysis thereof. Such compounds include hydrolysis products of mucoproteins from bile and from the gallbladder wall, such as: chondroitin 4- and 6-sulfates, dermatan sulfate, heparin, heparin sulfate, hyaluronic acid and the hydrolysis products (monomers, dimers, oligomers and polymers) of these mucins. Chitin and other mucins may be similarly hydrolyzed, which hydrolysis products would include:

N-acetyl-D-glucosamine,N-acetyl-D-galactosamine-4-sulfate,galactose-6-sulfate, N-acetyl-D-glucosamine-6-sulfate, glucosamine-6-sulfate, D-glucosamine 2- sulfate, D-glucosamine 2,3-disulfate, D-galactose-6-sulfate, glucuronic aid 2- sulfate, N-acetylneuraminic acid, sialic acid, N-acetyl chondrosine, chondroitin 4- sulfate, chondroitin 6-sulfate, D-glucosamine, D-galactosamine, glucuronic acid, glucose, galactose, mannose, fucose, iduronic acid, hexose, hexosamine, ester sulfate, glucuronic acid, chondrosamine, 2-amino-2-deoxy-D-galactose, serine. proline, threonine, alanine glycine taurine, glutamic acid, aspartic acid, histidine. and small peptides.

Similar products would be obtained by hydrolysis of mucins such as keratin sulfates, dermatan sulfates the natural sugar-sugar linkages in the dimers, oligomers and polymers may be replaced by -O-Si(OH)₂-O- bridges between the sugar monomers or adjacent sugar chains.

In particular, specific mucin and proteoglycan hydrolysis product compounds thought to be present include: sialic acids and their mono and diacetylated and glycolylated monomers;

N-acetylneuraminic acid; hexosamines, such as glucosamine;

L-fucose; hexosamine-hexuronic acid (dimer) disulfate; glucuronic acid; glucuronic acid or iduronic acid disulfate, monoacetylated; sialic acid-glycerol (dimer); and dimers, trimers, oligomers & polymers of the above monomers in acetylated & sulfated form. A fourth class of compounds, namely fat-soluble vitamins, likely to be present considering the source and the aforementioned MS analysis, include A, D, and K vitamins (e.g., A2, D 1. D3, D4, Kl, K2, K5, K6, K7, K-S(II), and Vitamin E acetate, for example.

In particular, specific fat-soluble vitamin compounds thought to be present include at least one of the group consisting of Vitamin A2, Vitamin DI, Lumisterol (present from its vitamin DI complex), Vitamin E, Vitamin Kl oxide, and Vitamin K5.

Various miscellaneous organic compounds are likely to be present, considering the source and the aforementioned MS analysis. Such compounds include: urea; alkylamines, including methylamine, dimethylamine, ethylamine, methylethylamine, diethylamine, dipropylamine, and/or butylethylamine; amino acids, including taurine, glutamic acid, glycine, alanine, n-leucine, phosphoserine, phosphoethanolamine, aspartic acid, threonine, serine, sarcosine, α-amino adipic acid, citrulline, valine, isoleucine, β-alanine, γ-amino butyric acid, hydroxylysine, ornithine, and lysine; bilirubin, and its gluconuride conjugate; biliverdin, and its gluconuride conjugate; butylatedhydroxy toluene (BHT); polyethylene glycol; traces of steroids; other plasma solutes, such as sugars, purines and pyrimidines; miscellaneous dietary lipids; and glutathione and its hydrolysis products.

In particular, specific miscellaneous organic compounds believed to be present in the composition include at least one of the group consisting of urea, methyl amine, dimethylamine, ethylamine, methylethylamine, diethylamine, dipropylamine, butylethylamine, ammonia, choline, taurine, glutamic acid, glycine, alanine, p-ser, p-eu, p-ea, asp thr ser sar, a-aba, cit, val, ile, leu, B-ala, G- aba, OH-lys, orn, lys, butylated hydroxy toluene (BHT), and polyethylene glycol.

Amines present in the present composition, particularly the secondary amines, may include nitrogen oxides from the air, thus forming nitroso compounds. N-oxides and N-carbamate byproducts may also be included. This series of amines cited above should be extended to include all primary, secondary and tertiary alkylamines.

Certain inorganic elements have been identified and quantified (mg/1) as follows:

Tungsten 0.07

Zinc 0.666

Phosphorus 378

Cadmium 0.01

Cobalt 0.008

Nickel 0.022

Barium 0.032

Iron 0.022

Manganese 0.039

Chromium 0.060

Magnesium 7.46

Aluminum 0.136

Calcium 5.97

Copper 0.087

Titanium 0.01

Strontium 0.060

Sodium 9600

Potassium 483

Chloride 15400

Ammonia 218

Vanadium 1 ppm

The compositions of the invention may be converted using customary methods into pharmaceutical compositions. The pharmaceutical composition contain the composition of the invention either alone or together with other active substances. Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant, or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, and tubelets. For parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be used, and can therefore be prepared as solutions of the compositions or as powders of the active compositions to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity that is compatible with the physiological fluids. For local use, those preparations in the form of creams or ointments for topical use or in the form of sprays may be considered; for inhalant uses, preparations in the form of sprays, for example nose sprays, may be considered. Preferably, the composition is administered intramuscularly.

The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Nack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit not exclusively, the composition of the invention in association with one or more pharmaceutically acceptable vehicles or diluents, and are contained in buffered solutions with a suitable pH and iso-osmotic 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, other antiviral compounds, including but not limited to; 3TC, interferon, ganciclovir, famciclovir, rimantadine, foscarnet sodium, zidovudine, amantadine hydrochloride, valacyclovir, ribavirin, acyclovir, may be used in combination with the composition of the present invention. The compositions and agents of the invention are intended for administration to humans or animals.

In general, a dosage range of the composition is envisaged for administration in human medicine of from about 0.01 to 20 mg/kg, preferably from about 0.1 to 10 mg/kg, most preferably 0.1 to 1 mg/kg of body weight daily may be employed. In the case of intravenous administration, the dosage is about 0.1 to 5 mg/kg of body weight daily, and in the case of oral administration the dosage is about 1 to 5 mg/kg of body weight daily. Where the concentrated composition is used, approximately half the above mentioned dosages may be used. For example, for intramuscular administration, a dosage of about 0.2 to 1.0 mg/kg of body weight daily, preferably 0.275-0.75 mg/kg of body weight daily may be used.

It will be appreciated by medical practitioners that it may be necessary to deviate from the amounts mentioned and, in particular, to do so as a function of the body weight and condition of the animal to be treated, the particular disease to be treated, the nature of the administration route and the therapy desired. In addition, the type of animal and its individual behavior towards the medicine or the nature of its formulation and the time or interval at which it is administered may also indicate use of amounts different from those mentioned. Thus it may suffice, in some cases, to manage with less than the above-mentioned minimum amounts while in other cases the upper limit mentioned must be exceeded. Where major amounts are administered, it may be advisable to divide these into several administrations over the course of the day.

Thus, the present invention comprises a process for preparing an antiviral composition comprising (a) mixing bile from an animal with a water-soluble solvent to produce a bile/solvent solution; (b) isolating an aqueous solution substantially free of solvent from the bile/solvent solution; and (c) removing bile pigments from the substantially solvent-free solution to obtain a clear, yellowish liquid, preferably where the water soluble solvent is an alcohol, and where the bile from the animal is mixed with an equal volume of the alcohol. Preferred aspects of the aforementioned process also comprise further concentrating the clear, yellowish liquid to about one-eighth, or one-tenth, the original volume of the bile/solvent solution. Obviously, compositions produced via the above process form a preferred aspect of the invention.

The present invention also comprises a composition for use as an immunomodulator, comprising at least one component having a molecular weight of less than about 3000 daltons, which shows no cytotoxicity to human peripheral blood mononuclear cells, and has at least one of the following properties:

(a) is capable of stimulating monocytes and macrophages in vitro or in vivo to produce one or more cytokines; and/or

(b) is capable of stimulating monocytes or macrophages to produce tumor necrosis factor in vitro or in vivo; and wherein said component is not an endotoxin, IL-lα, IL-lβ, TNF, IL-4, IL-6, IL-8, GM- CSF or IFN-γ. Such compositions may be obtained from the bile of animals, preferably bovines, or from other sources as noted above. In a preferred embodiment of the composition, the composition stimulates tumor necrosis factor production in vitro or in vivo, and most preferably in humans, in the absence of exogenous IL-lα, IL-lβ, TNF, IL-4, IL-6, IL-8, GM-CSF, and IFN- γ.

The compositions of the present invention also have components that can be characterized by column chromatography such that when said composition is dried to obtain a solid residue, and 2 grams of said residue are dissolved in 20 ml of a 10% concentrated ammonium hydroxide solution in methanol, and after any insoluble material is removed, is subjected to column chromatography in a methanol column having dimensions of 5 cm x 12.5 cm, and containing 102 g of 60 A flash silica gel, and operating at a pressure of 10 pounds per square inch and a flow rate of 11 ml/min with a 10% concentrated ammonium hydroxide in methanol solvent solution, said component is eluted from the column in a fraction taken when the total column elution is between about 180 and about 220 ml, between about 220 ml to about 260 ml, or between about 260 ml and about 300 ml.

Characterization of components may also be accomplished by ion-exchange chromatography, such that when 10 ml of said composition is subjected to anion-exchange chromatography in a column containing Bio-Rad AG- 1 hydroxide form resin in an amount sufficient to bind substantially all the anions present in said 10 ml of said composition, said component is eluted from the column using a step gradient of ammonium bicarbonate buffer at a buffer concentration from about 0.1 M to about 1.5 M, preferably at a buffer concentration from about 0.2 M to about 0.4 M, and most preferably at a buffer concentration of about 0.2 M.

Reversed-phase (C 18) HPLC can also be used for characterization of components. Other suitable columns, eluents, gradients, flow rates, operating temperatures and detection systems may be used.

The compositions of the present invention can also be characterized by TLC, such that when said composition is subjected to thin layer chromatography on silica gel plates in a suitable solvent system, such as 10% concentrated ammonium hydroxide in methanol, and visualized with a suitable spray, such as ninhydrin; a positive reaction with ninhydrin occurs at, for example, an R_f value from about 0.80 to about 0.90.

The present invention also comprises a method of stimulating tumor necrosis factor production in humans, comprising administering an effective amount of a composition comprising at least one of the following compounds: (a) a compound of the formula

where the bonds between A-B, B-C, and C-D may be single or double bonds, and where X = H, OH, =O, or OSO₃H; and Y=

where R is an amino acid residue; (b) a compound of the formula

(R¹O)CH₂CH(OR²)CH₂(OR³-X) or CH₃(CH₂)₁₂CH=CH

OR¹

|- NHR² OR³-X where R¹, R² and R³ are H, COR⁴, CH=CH-R⁵, X, P(O)(OH)O-, or -S(O)₂O-; X is choline, ethanolamine, N-alkylated ethanolamines, serine, inositol, sugars bearing free hydroxyls, amino-sugars, sulfonated sugars, or sialic acids; and

R⁴ is a saturated or unsaturated alkyl group having a carbon chain from about C_j to C₃₀, or oxidized and hydroxylated analogs thereof; and

R⁵ is an alkyl group or oxidized and hydroxylated analogs thereof;

(c) a mucin hydrolysis product or a proteoglycan hydrolysis product; or

(d) a fat-soluble vitamin.

Preferably, compositions of the inventive method comprise at least one compound selected from the group consisting of taurocholic acid and its sulphated derivatives; glycocholic acid and its sulphated derivatives; sphingosine; adiacyl glycerol; lecithin; phosphocholine; phosphoglycerol; glycero-phosphocholine; phosphoryl choline chloride; an oligosaccharide of less than 10 saccharide units in length, where said oligosaccharide is comprised of sialic acid, fucose, hexosamines, or sulphated hexosamines; Vitamin A; retinolic acid derivatives; retinol derivatives; taurine; and glutamic acid and its conjugates. The composition may also additionally comprise at least one compound selected from the group consisting of ammonia; primary alkyl amines; secondary alkyl amines; tertiary alkyl amines; and a carboxylic acid R⁶CO₂H, wherein R⁶ is C,-C₃₀ alkyl that is saturated or unsaturated, and oxidized and/or hydroxylized derivatives thereof. More preferably, such a composition comprises at least one of the group consisting of phosphocholine, glycero-phosphocholine, glucosamine-3 -sulfate, and phosphorylcholine chloride. Most preferably, the composition comprises at least one of the following: phosphocholine, glycero-phosphocholine, or glucosamine-3-sulfate.

The method of the invention also embraces 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 sulphated derivatives; sphingosine; a diacyl glycerol; lecithin; an oligosaccharide of less than 10 saccharide units in length, where said oligosaccharide is comprised of sialic acid, fucose, hexosamines, or sulphated hexosamines; Vitamin A; retinoic acid derivatives; retinol derivatives; taurine; and glutamic acid and its conjugates.

Also forming part of the present invention are compositions comprising ( 1 ) micelles of sphingosine or sphingosine complexed with a salt, or (2) micelles of retinolic acid or its derivatives, which have at least one of the following properties:

(a) is capable of stimulating monocytes and macrophages in vitro to produce one or more cytokines; and/or (b) is capable of stimulating monocytes or macrophages to produce tumor necrosis factor in vitro or in vivo. The micelles may also comprise a diacyl glyceride or lecithin, and may further comprise a bile acid salt, and a source of ammonium or alkyl ammonium ions.

Finally, the present invention also contemplates compositions comprising ( 1 ) sphingosine, a bile acid salt and a source of ammonium or alkyl ammonium ions, (2) a bile acid salt, sphingosine, a diacyl glycerol, a source of ammonium or alkyl ammonium ions, and a retinol derivative, (3) a diacyl glyceride, lecithin, and a bile acid salt, or (4) (a) a diacyl glyceride, (b) lecithin, and (c) a mucin hydrolysis product or a proteoglycan hydrolysis product, which has at least one of the following properties: (a) is capable of stimulating monocytes and macrophages in vitro to produce one or more cytokines; and/or

(b) is capable of stimulating monocytes or macrophages to produce tumor necrosis factor in vitro or in vivo.

The following non-limiting examples are illustrative of the present invention:

Example 1

This example describes and illustrates preparation of the composition of the invention.

Bovine bile was collected from the gall bladders removed from healthy cows (both males and females) that were at least one and one-half years old. These cows were slaughtered for food use at a licensed and inspected abattoir. The slaughtered animals had been inspected and evaluated as healthy prior to slaughter and the gall bladders were separated from the livers and examined by a veterinarian to confirm that the gall bladders were free of parasites and evidence of infection, and thus suitable for use as a source of bile for the present invention. Gall bladders that passed this inspection were subjected to the following procedure: Gall bladders were wiped with a solution of 70% ethanol to sanitize the exterior of the bladders and bile was removed from the bladders with a syringe. The bile removed was visually examined in the syringe by the veterinarian to assure that it contained no blood or pus and was otherwise satisfactory. Bile from a healthy bovine is a greenish fluid substantially free of blood and pus. Fragments of livers, spleen, and lymph nodes were also collected from the animals whose bile was collected and the fragments were examined for the presence of parasites and other indications of disease.

For species that do not have a defined gall bladder (such as shark), bile is obtained directly from the hepatic organ.

Bile found to be satisfactory was transferred into a graduated amber bottle containing ethanol to give a 50% bile/50% ethanol solution by volume. The bile/ethanol solution was a greenish fluid substantially free of foreign material and tested positive for ethanol in accordance with methods recited at United States Pharmacopeia XXII. Part B (1994). These bottles were labelled with a lot number. Bile collected from a minimum of fifty animals was collected for each lot.

The bile/ethanol solution was then centrifuged at 4200 φm for at least 2-1/2 hours at 20 ± 2° C.

The supernatant liquid was decanted, filtered through a filter having, for example, a 2.5 μm retention, and checked for pH and ethanol content. The decanted liquid was then subjected to an activated charcoal treatment. The treated liquid was then monitored for Optical Density (OD) at 280 nm and conductivity. OD levels and/or conductivity levels outside specified ranges necessitated additional treatment of the liquid with activated carbon to achieve an OD and conductivity within specified ranges.

Following activated carbon treatment, the treated liquid filtered through a filter having, for example, a 2.5 μm retention, the ethanol was evaporated off (for example, by heating up to about 85° C), and the treated liquid was concentrated to approximately one-eighth of the original bile/ethanol solution volume. The concentrated liquid was then cooled to 20-25 °C, filtered through a filter having, for example, a 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 residual ether (for example, by heating up to about 55 °C for about 10 hrs) and further reduced in volume to one-tenth of the original bile/ethanol volume by heating to around 80-85 ° C. The resultant 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 ethanol and less than 5 ppm ether.

Identity and purity were determined using reverse-phase high pressure liquid chromatography (reverse-phase HPLC). Potency is assayed using the monocyte/macrophage activation test referred to herein as the peripheral blood mononuclear cell-tumor necrosis factor assay (PBMN- TNF assay or, simply, TNF assay), as described in Example 2.

Initial batches of the composition of the invention were manufactured as a non-buffered liquid. Subsequent batches were manufactured as a buffered liquid, prepared by adjusting the pH of the composition to about 7.4 ± 0.2, using hydrochloric acid (1 %) solution and sodium hydroxide (1 % solution), as well as using dibasic and monobasic sodium phosphate salts as buffers. Bioburden reduction was conducted in a steam autoclave at 104 ±2° C for 60 mins. The bulk solution was filled into 5 ml or 10 ml sterile bottles and capped. The filled and capped bottles were subjected to three sterilization cycles by autoclaving them at 104°C ± 2°C for 60 mins followed by incubation at 35° C for 23 ± 1 hrs. Between each cycle of sterilization (autoclave plus incubation), samples were taken and tested for bioburden. Following the last cycle of sterilization, the bottles were visually inspected against a black and a white background to detect the presence of particulates.

Following inspection, the lot was sampled and tested for conformance to specifications. Tests included identity, sterility, pyrogenicity, endotoxin, bioassay, HPLC and general safety. Table I summarizes the data obtained for the various tests performed on the bile extract of the present invention, including normal ranges of data, where appropriate.

Table I: Characteristics of Batch Compositions Obtained In Accordance with Method of Example 1

FINAL PRODUCT TEST BATCH # BATCH # BATCH # BC0248 BC0249 BC0250

Potency (pg/ml)* 210 183 304

Identity/Purity Pass Pass Pass

Agrees with reference

Safety (passes test according to 81 CFR § 610.11) Pass Pass Pass FINAL PRODUCT TEST BATCH # BATCH # BATCH # BC0248 BC0249 BC0250

Pyrogenicity (temp, increase shall not exceed 0.4° C) Pass Pass Pass

Endotoxin <0.4 EU/ml <0.25 <0.25 <0.25

Sterility (no growth) Pass Pass Pass pH (7.40 ± 0.2) 7.20 7.27 7.22 Appearance - Visual (clear, light yellowish liquid with Pass Pass Pass little or no precipitate)

Appearance - OD (passes test) 1.34 1.38 1.85

Osmolarity ( < 1000) 877 854 832

Solids (23 +/- 7mg/ml) 18 15 20 Ethanol (not more than 10 ppm) Pass Pass Pass

Ethyl Ether (not more than 5 ppm) Pass Pass Pass

Conductivity (35 +/- 5 mMho) 33 35 38

* Potency was measured with respect to monocyte/macrophage activation as described in Example 2; normal TNF-K release is at least 100 pg/ml.

Accordingly, the inventive composition can be prepared from readily available sources of bile, using standard laboratory methods, resulting in a standardized final product.

Example 2

This example describes the biological activity of the composition of Example 1.

Studies were conducted to evaluate the effect of the composition of Example 1 on cytokine release from peripheral blood mononuclear cells (PBMN) and/or U937 cells which is a stable line of pre-monocyte cells (American Type Culture Collection (ATCC), Rockville, Maryland).

ELISA assays for TNF-α, IL-la, IL-2, IL-4, IL-6, IL-8, GM-CSF and IFN were conducted.

These studies provided the basis for a standardized test for quantitatively evaluating the potency of a given batch of bile extract prepared according to Example 1, which test evaluates the ability of the bile extract, or a component or components thereof, to stimulate TNF-α production in the PBMN or U937 cells.

Whole blood was drawn from 5 healthy human subjects into heparinized Vacutainer tubes (Beckton Dickinson, Canada). PBMNs were isolated by gradient centrifugation on Ficoll- Hypaque (Pharmacia) . The PBMNs were washed twice with phosphate-buffered saline {PBS) , counted and resuspended in RPMI 1640 culture medium (Gibco Labs) at a concentration of 10⁶ cells/0.5 ml. These cells were cultured in 24-well, flat-bottomed tissue culture plates (Falcon, Becton, Dickinson). A 0.5 ml aliquot of the PBMN suspension was added to each well, which contained 50 ng lipopolysaccharide (LPS) (from E. coli), 10 μl fetal calf serum and 10-300 μl of the composition of Example 1, as noted in the tables below. The hyperosmolar effect of the composition was neutralized by adding distilled water to the culture wells at a volume equivalent to 10% of the volume of composition used. The total volume was then made up to 1 ml/well with RPMI. PBS was used as a control. The cells were cultured for 2, 6, 24, 48 and 72 hrs at 37° C in a humidified 5 % CO₂ incubator. At the end of each incubation period, the cells were harvested and cell-free culture fluids were obtained by centrifugation at 9000 rpm for 10 mins. The samples were then stored for up to 2 weeks at -70 °C until immunoassays, such as ELISA, were conducted to quantify the cytokines present.

Cytokine synthesis in the supernatants was measured after stimulating human PBMN with the composition of Example 1 at volumes of 100 and 200 μl per well. The initial preparations of the composition showed no direct (i.e. , no LPS) stimulatory effect on cytokine production (see Table II). If there was any effect, it appeared that cytokine production was below the constitutive level when PBMNs were incubated in medium alone.

Table II: Direct Effect of Composition of Example 1 on Cytokine Production

Composi tion LPS

Cytokine Assayi sd Medium 100 μl 200 μl i μ

IL-lα 61.6 + 12 59.6 ± 7.8 54.3 + 6.0 315 + 117

IL-lβ 199 + 184 218 + 165 188 + 174 965 + 99

TNF² 203 + 149 151 ± 117 107 + 120 1501 + 284

IL-6 928 + 776 853 + 673 829 + 543 2016 + 41

IL-8 126 + 70³ 94 ± 50³ 77 + 41³ 361 ± 165³

GM-CSF 13 + 4 13 + 7 15 + 11 54 + 20

IFN-γ 11 ± 18 9 ± 14 5 + 6 54 + 94

IL-4 < 3.0 <3.0 < 3.0 < 3.0

1 Mean of eight patient samples in duplicate

Cytokine synthesis in the supernatants was measured at 24 hrs at 37 °C after stimulating PBMNs with the composition of Example 1 and LPS (or LPS alone as positive control), using volumes of 100 μl of the composition of Example 1 per well. TNF was measured by a TNF-α ELISA kit (Endogen, Inc.), which detects a minimum level of 5 pg/ml of the cytokine. The other ELISA immunoassay kits that were used included: IL-lα (Endogen, Inc.); GM-CSF (Endogen, Inc.); RFN-α (Endogen, Inc.); IL-2 (Advanced Magnetics, Inc.); IL-6 (Advanced Magnetics, Inc.); IL-1 (Advanced Magnetics, Inc.); IL-4 (R&D Systems); and IL-8 (R&D Systems). The results indicated that TNF was the major cytokine present in the supernatants, along with smaller amounts of IL-lβ and GM-CSF. For example, a 40 μl dose of the composition of Example 1 (batch B0222) stimulated the production and release of 178 pg/ml of TNF-α, 136 pg/ml GM-CSF, and 142 pg/ml of IL-lβ.

Different batches of the composition of Example 1 were examined for their effect on LPS- induced release of TNF. In summary, it was found that batches of the composition produced in the same way and from the same animal induced an identical effect. However, changes in the method of preparation of the composition or use of a composition prepared from different animal species had different effects. For example, batches B29/3006, B0213, BC0241, BC0241-01, BC0242 (B = bovine) and C0203 (goat) induced a strong release of TNF above that induced by LPS alone, as shown in Table III, whereas batch 013/2109 (sheep) minimally stimulated TNF release at all doses tested. In contrast, batch R0201 (shark) inhibited TNF release at most doses tested. The TNF values shown in Table III were calculated as the difference in TNF-α release between the stimulation produced by LPS and the composition of Example 1 combined, less the stimulation produced by LPS alone.

Table III: Effect of Composition of Example 1 on LPS-induced Release of TNF from PBMNs Batch Composition Volume (μl) TNF (pg/ml)

B0213 10 193 + 161

100 858 ± 819

200 2131 + 1742

B29/3006 10 121 ± 102

50 422 + 78 Batch Composition Volume (μl) TNF (pg/ml)

100 834 + 811

200 2252 ± 676

C0203 10 101 + 47

50 643 + 231

100 2650 ± 1372

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

Table IV 300 178 + 373

R0201 50 145 + 256

200 -370 + 385

300 -400 + 185

Given that the composition of Example 1 affected LPS-induced release of TNF from human PBMNs, a series of experiments were conducted to examine the effect of the composition on LPS-induced release of TNF from PBMNs over time.

Table IV: Effect of Composition of Example 1 (Batch B0213) On LPS-induced Release Of TNF (pg/ml) from PBMNs Over Time LPS only LPS + Composition

Time (hrs) (50 ng/ml) (100 μl)

2 697 + 94 693 + 339

6 2006 ± 736 1949 + 442

24 800 + 222 2301 ± 658

48 170 ± 149 1419 + 447

72 132 + 147 945 + 367

Table IV shows that, by 2 hours, the level of TNF release from PBMNs induced by LPS had risen to 697 pg/ml and peaked at 6 hours at about 2006 pg/ml. At 24, 48 and 72 hours, the release of TNF progressively decreased. In fact, by 48 and 72 hrs, the TNF release from LPS- induced PBMNs was just above constitutive production levels. In contrast, LPS in combination with Batch B0213 of the composition, which is a strong stimulator of TNF release, induced peak TNF release at 24 hrs, at a time when the stimulatory effect of LPS had begun to fall. Unlike LPS alone, LPS in combination with batch B0213 of the composition continued to stimulate TNF release at 48 and 72 hrs at levels well above constitute production levels. These data show that Batch B0213 of the composition of Example 1 is effective in stimulating TNF production over time.

Batch R0201 of the composition, which was derived from sharks and is an inhibitor of TNF release, markedly inhibited TNF release at 2, 6 and 24 hrs. At 48 and 72 hrs, batch R0201 had minimal positive or negative effects.

In summary , the above results indicate that some batches of the composition (e.g. , from shark) inhibit TNF release from LPS-induced PBMNs, whereas other batches, such as those derived from bovine, goat, and sheep, stimulate LPS-induced TNF release. In conclusion, the composition of the invention can modulate TNF production, in both positive and negative manners. A summary of the data is shown in Figure 5 and in Table V.

Table V: Summary Of Stimulatory And Inhibitory Effects Of Compositions Of Example 1

Normal or Batch No. Source Concentrated Buffer TNF Release

B0213 Bovine Normal Yes Stimulate C0203 Caprine Normal Yes Stimulate

013/2109 Ovine Concentrated Yes Stimulate

R0201 Shark Normal Yes Inhibit

B29/3006 Bovine Normal Yes Stimulate

B27/2806 Bovine Normal Yes Stimulate

B15/1606 Bovine Concentrated Yes Stimulate

The PBMN-TNF assay as described above was standardized 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 used the same day. The results of each of the three assays (using individual subject cells) were averaged to compensate for variations in response between different subjects. The analysis involved determining the amount of TNF-α released in RPMI media alone and in the presence of 50 ng LPS. The TNF-α released in the presence of 100 μl of the composition of Example 1 in combination with 50 ng LPS was also determined. The TNF-α released in media was subtracted from the LPS value to obtain the TNF-α released in the presence of LPS alone. The media and LPS values were subtracted from the combined composition and LPS value to obtain the TNF-α released in the presence of the composition alone (reported in pg/ml). Accordingly, the TNF release assay served to quantify the potency of the bile extract.

The composition was also found to stimulate release of TNF-α from U937 cells, which were originally derived from a patient with histocytic lymphoma and display many characteristics of monocytes. U937 cells can be obtained from the ATCC. They are routinely maintained in RPMI- 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS, GIBCO), 2 mM L-glutamine (ICN Biomedical Inc, Costa Mesa, CA), and 10 μg/ml Gentamycin Sulfate (SIGMA, Mississauga, Ontario, Canada) at 37°C, 5% CO₂. Passage of the U937 cells was performed every 3-4 days and seeding was at an initial concentration of 5 x 10⁵ cells/ml. The U937 cells can be stimulated to differentiate to monocytes by exposure to phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO). The resulting monocytes have the capacity to release TNF upon stimulation, such as with the composition of Example 1, alone or in combination with LPS.

PMA was first dissolved in dimethyl sulfoxide (DMSO, SIGMA) at a concentration of 10 mM and then diluted 1000-fold with PBS to a stock solution concentration of 10 μM and stored at -20° C. U937 cell suspensions were centrifuged at 350 x g for 10 mins at room temperature and reconstituted in fresh complete RPMI- 1640 medium at a concentration of 2 x 10⁶ cells/ml. Cell viability was determined by trypan blue exclusion and was routinely greater than 95 % . PMA was further diluted 500-fold with complete culture media to a concentration of 20 nM.

Aliquots of 0.5 ml of U937 cells (10⁶ cell/ml) were cultured in the presence or absence of 0.5 ml of PMA (20 nM) in 24-well, flat-bottom tissue culture plates (Becton Dickinson, Lincoln Park, NJ) and incubated for 72 hrs at 37°C, 5% CO₂. The final concentrations per well were 5 x 10⁵ cells and 10 nM PMA.

After 72 hrs of incubation, 120 μl of media were removed and replaced by 100 μl of the composition of Example 1 and 10 μl of sterile deionized distilled water, in the presence or absence of 10 μl of LPS (5 ng/μl). After 24 hrs of incubation, any cells and particulate matter were pelleted by centrifugation at 350 x g for 10 min and the resulting supernatants were stored at -20 °C until they were assayed for TNF-α. All the Virulizin samples were tested on two separate occasions.

Two-site sandwich ELISAs were performed to quantify TNF-α in the U937 cell culture supernatants using TNF-α ELISA kits purchased from Endogen, Inc. (Cedarlane Laboratories, Hornby, Ontario). The protocol recommended by the manufacturer was used. Briefly, 100 μl of TNF-α standards and test samples were added to antihuman TNF-α pre-coated 96-well plates and incubated at 37° C, 5 % CO₂ for 3 hrs. After extensive washing with washing buffer, 100 μl of antihuman TNF-α conjugated to alkaline phosphatase were added to plates and incubated at 37°C, 5% CO₂ for 2 hrs. After incubation, the plates were washed as described above and 100 μl of premixed TMB substrate was added to each well and the enzymatic color reaction was allowed to develop at room temperature in the dark for 30 min. Then 100 μl of stop solution was added to each well to stop the reaction and the plates were read using an SLT Lab Instrument ELISA reader at 450 run. The detection limit of the assay was 5 pg/ml.

TNF values for U937 cells were determined as described for PBMN cells. Results of the composition tested with 50 ng LPS are presented in Table III. Table VI: Effect of Composition on TNF Release from U937 Cells

Composition Batch Number TNF (pg/ml) (100 μl)

BC0241 4900

BC0241-01 4028

BC0242 6746

BC0247 5534

BC0248 6053

BC0249 5540

BC0250 5794

E: sample 3

This example describes the physical, chemical and biochemical characteristics of the composition of Example 1.

Physicochemical characteristics, such as conductivity, osmolarity, and total solids, for three manufactured batches of a composition prepared in accordance with Example 1 were determined. The results, tabulated in Table I, demonstrate the sterility, potency, and reproducibility of the manufactured product, and thereby provide a product specification. The ethanol and ethyl ether tests are in-process tests only. Potency, i.e. , the TNF release was determined as described in Example 2. The methods used to determine the characteristics are tabulated below.

Table VII: Characteristics Of Compositions Of Example 1 As Products Of Manufacture

Test Specification Method

Potency > 100 pg/ml TNF-α Monocyte/macrophage activation: TNF-α release

Identity /Purity Agrees with reference HPLC

Safety Passes test General safety test (mice and guinea pigs) (21 C.F.R. § 610.11)

Pyrogenicity Temperature increase shall Pyrogen test (rabbits) USP not exceed 0.4 °C

Endotoxin < 2 EU/ml Limulus Amoebocyte Lysate Test USP Test Specification Method

Sterility No growth Sterility Test USP

pH 7.40 ± 0.2 pH test USP

Appearance Clear, light yellowish liquid Visual Inspection with little or no precipitate

Solids 23 ± 7 mg/ml Lyophilization

Osmolarity < 1000 mOsm Freezing point depression USP

Ethyl Alcohol Not more than 10 ppm Direct Injection Gas Chromatography

Ethyl Ether Not more than 5 ppm Direct Injection Gas Chromatography

Conductivity 35 ± 5 mMHO Copenhagen Radiometer Model

The above-described physical and chemical properties, such as conductivity, osmolarity and total solids, were consistent with a composition that is over 99% salt. Less than 1 % of the solids in the composition was organic material, around half of the solids were carbohydrates, and the rest were amino acids, lipids, and phospholipids. Proteins and peptides were present. SDS gel electrophoresis confirmed that there were more peptides than proteins in the composition. High molecular weight molecules were not detected.

HPLC and bioassay test methods for the composition of the invention were used to characterize the product as the buffered liquid and the concentrated formula. The HPLC results described below indicate that the product was the same in all of its presentations.

A tandem column reverse-phase HPLC method was used to characterize the composition of Example 1. For this method, samples were lyophilized and then reconstituted in Buffer A (0.1 % trifluoroacetic acid (TFA)) and were run on a WP60009-C 18 column (W-Pore C 18, 250 X 4.6 mm; Phenomenex of California) in tandem with a prime-sphere HC-C 18 column (250 X 4.6 mm; Phenomenex). The columns were run at ambient temperature using Buffer A and Buffer B (0.1 % TFA in 100% acetonitrile), with a flow rate of 0.9 ml/min. A 150 μl sample was applied to the first column and Buffer A was run through the system for 20 mins. Next, a first linear gradient, 0-80% Buffer B, was run over 35 mins, followed by a second linear gradient, 80-0% Buffer B, over 5 mins. Eluted compounds were detected via optical absorbance at from 190 to 284 nm, with most runs being detected at 210 and 235.

The composition of Example 1 had a consistently reproducible pattern on reverse-phase HPLC in which peaks were seen. The reverse-phase HPLC readings for three lots of the composition of the invention are shown in Figures 1-3.

Six batches of bile extract, which were prepared as in Example 1 and labeled A-F, were analyzed for their amino acid profiles on an LKB 4151 Alphaplus amino acid analyzer operated in a physiological mode, with post-column detection with ninhydrin. The results, in nmoles/100 μl, are shown in Table VIII.

Table VIII : Amino Acids And Urea Profiles Of Compositions Of Example 1

Amino Acids and Urea B C D

P-Ser 0.342 0.429 0.473 3.239 1.454 1.048

Tau 3.438 8.325 2.515 1 1.297 23.005 47.019

Urea 23.318 35.224 146.806 608.984 98.489 115.26

Asp 0.606 1.060 1.163 - - -

Thr 0.649 0.483 - 0.345 12.646 1.548

Ser 1.104 0.833 0.452 0.821 - -

Glu 2.112 8.257 8.029 13.333 36.169 43.632

Gly 5.465 15.667 6.341 12.625 38.842 82.418

Ala 2.634 4.449 3.572 6.093 32.662 23.202

Val 0.942 0.645 0.550 1.311 15.521 4.362 lie - - - - 3.089 -

Leu - - 0.186 1.079 7.300 1.197

B-Ala 0.387 0.503 0.450 1.060 1.461 2.640

Orn - - - 0.102 0.412 0.336

Samples A-F were also assessed for presence of bovine DNA. The samples were examined utilizing a ³²P-labeled bovine DNA probe generated from bovine genomic DNA. The assay included the samples, spiked samples, negative and positive controls, and standards. The study was conducted in compliance with GLP regulations. This assay detected 3.9 pg of reference standard DNA. Each of the samples was calculated to contain less than 4 pg/ml DNA. Samples A-F were also tested for the presence of various electrolytes. This analysis was provided by the Biotechnology Service Centre, Department of Clinical Biochemistry, University of Toronto. The results, in mmole/1, are shown in Table IX.

Table IX: Electrolyte Content of Compositions of Example 1

Electrolyte A B D

NA 55 68 127 359 250 309

K 0.9 0.9 2.5 10.2 3.6 4.2

Ca 0.06 0.10 0.006 0.2 0.13 0.27

Mg 0.25 0.15 0.09 0.35 0.14 0.17

Cl 50 59 118 386 207 263

P0₄ 0.06 0.03 0.05 0.27 0.18 0.24

S0₄ 2.17 1.89 2.05 1.15 7.13 11.36

Samples A-F were submitted to semi-quantitative multi-element analysis by inductively coupled mass spectrometry (ICP-MS) under standard conditions. The results, in parts per million (ppm), are described in Table X.

Table X: Elemental Analysis of Compositions of Example 1

A B C D E F

Scandium 0.620 0.820 1.030 1.030 2.020 1.900

Titanium 0.210 0.310 0.260 0.720 0.920 1.180

Vanadium 0.030 0.040 0.080 0.180 0.140 0.160

Chromium 0.030 0.040 0.060 0.080 0.170 0.190

Iron 0.300 0.380 0.510 4.310 0.690 0.760

Manganese 0.020 0.020 0.030 0.530 0.050 0.060

Nickel <det <det 0.030 0.250 0.130 0.160

Cobalt <det <det 0.001 0.013 0.003 0.005

Copper 0.700 0.940 0.840 1.520 2.140 2.470

Zinc 15.600 18.300 8.800 0.830 29.800 32.900

Gallium 0.008 0.008 0.004 0.003 0.013 0.015

Selenium 1.020 1.590 2.060 7.710 3.810 7.860

Arsenic 0.030 0.070 0.100 0.200 0.250 0.350

Strontium 0.010 0.010 0.020 0.060 0.040 0.050

Rubidium 0.090 0.110 0.190 0.320 0.410 0.490

Ruthenium <det 0.001 <det 0.001 <det 0.001

Palladium 0.002 < det 0.003 0.005 0.003 0.003

Cadmium < det <det < det 0.002 0.005 0.003

Silver <det <det 0.002 0.002 0.001 <det

Tellurium 0.003 0.003 0.050 0.090 0.080 0.070 A B C D E F

Antimony <det 0.002 0.003 0.002 0.007 0.006

Barium 0.017 0.019 0.035 0.040 0.057 0.080

Cesium 0.001 0.002 0.004 0.008 0.005 0.006

Note: The term < det means below level of detection.

Anion and cation analysis was also conducted on samples A-F. For this analysis, the samples were prepared as recommended in APHA Standard Methods For The Examination Of Water And Wastewater. 16th Edition, 1985 or MOE Handbook Of Analytical Methods For Environmental Samples, 1983. Instrumentation for the anion/cation analysis was: (1) for metals, Jarrell Ash 61E ICAP emission, Perkin Elmer 3030 Zeeman Graphite Furnace, and Perkin Elmer 2380 Cold Vapour AA; (2) for anions, Dionex 2000i Ion Chromatograph; and for conventionals, Skalar SA5 Segmented Flow Analyzer. The results, in mg/1, are presented in Table XI.

Table XI: Anion And Cation Analysis Of Compositions Of Example 1

A B C D E F

Silver <0.007 <0.007 O.007 O.007 <0.007 <.007

Beryllium O.003 <0.003 O.003 <0.003 O.003 <.003

Cadmium O.003 <0.003 <0.003 0.004 <0.003 <.003

Bismuth <0.04 <0.04 <0.04 <0.04 <0.04 <0.04

Cobalt <0.005 0.005 O.005 0.006 <0.005 < 005

Copper 0.013 0.036 0.043 0.138 0.1 12 0.210

Manganese 0.007 0.006 0.007 0.283 0.018 0.029

Molybdenum 0.014 0.012 0.012 0.015 O.006 <.006

Nickel <0.01 0.012 <0.01 0.058 0.020 0.020

Lead O.025 <0.025 O.025 O.025 O.025 025

Strontium 0.01 0.019 0.015 0.126 0.040 0.063

Vanadium 0.009 0.008 0.004 0.011 O.003 <.003

Zinc 6.04 5.93 1.95 0.383 14.5 15.2

Tungsten 0.587 0.436 0.315 0.435 0.498 0.481

Phosphorus 10.6 11.4 3.34 676 22.8 14.1

Titanium <0.003 <0.003 0.006 0.005 O.003 0.004

Barium 0.062 0.056 0.055 0.105 0.079 0.1 17 A B C D E F

Chromium 0.025 0.036 0.028 0.107 0.102 0.124

Sodium 1250 1570 2770 13900 5350 6570

Potassium 30.2 32.8 65.4 686 125 154

Iron 0.018 0.023 0.024 0.008 0.036 0.037

Aluminum 0.240 0.238 0.052 O.025 0.790 0.361

Calcium 1.22 5.34 2.00 10.2 5.04 10.4

Magnesium 0.757 0.756 0.891 15.8 2.27 3.70

Fluoride <100 <100 <100 <100 <100 <100

Chloride 2120 1860 3110 30400 10900 9110

Sulphate 144 154 152 332 1150 1590

Phosphate-P 1.8 1.3 1.5 <det <det <det

Nitrate as N <10 <10 <10 <10 <10 <10

Nitrite as N <100 <100 <100 <100 <100 <100

Bromide <35 <35 <35 <35 <35 <35

Ammonia as N 98.0 125 130 492 425 592

As several sulfate esters participate in the regulation of many cellular events, such as cell proliferation and differentiation, Sample D was analyzed for sulfate ions before and after acid hydrolysis. Using whole sample D (i.e., imfractionated), the nonhydrolyzed sample yielded 1000 μM sulfate, whereas the hydrolyzed sample yielded 1200 μM sulfate. Since the sulfate ion concentration increased after acid hydrolysis, these results suggest that 20%> of the total sulfate ions present are sulfate esters.

Physicochemical standards have been identified for the composition of Example 1 and are essentially consistent with earlier studies, which are described in Example 4. These standards indicate that a consistent product can be repeatedly obtained.

Example 4

This example describes the physical, chemical, and biological properties of a number of earlier batches of the composition of Example 1.

Batches of bile extract were prepared in accordance with the method described in Example 1. In addition, the chemical composition of the batches was determined and an amino acid analysis of the batches was conducted, using the methods disclosed in Example 3. The results are shown in the following tables.

Table XII: Chemical Composition Earlier Batches of Compositions of Example 1

Composition High M.W.

Amino Acids > 3kD Poly-

Batch No. Solids (mg/ml) (μg/ml) Sugars (μg/ml) Lipids (μg/ml) peptide (μg/ml)

B0201 15.3 4.59 40.85 ND NA

B0202 15.7 13.16 54.95 ND NA

B0203 15.0 72.67 25.5 ND NA

B0208 7.8 4.53 30 ND ND

B0209 8.5 2.27 24 ND ND

B0211 5.6 1.47 19.2 ND ND

B0106 32.2 1.16 32.6 ND ND

B0706 32.7 1.42 26.2 ND ND

B1306 22.3 8.01 48 ND ND

B2006 21.7 9.73 38.4 ND ND

B2306 28.5 16.35 42 ND ND

B0213 31.6 21 61 ND ND

R0201/-pH 52.5 1553 216 ND ND

R0201/+pH 55.8 1530 280 ND ND

C0203 36.1 113 42 ND ND

0-13/2109 12.1 149 36 ND ND

B27/2806 17.5 28 37 ND ND

B29/3006 28.7 26 60 ND ND

B15/1606 26.8 41 45 75 ND

Note: ND means not detectable, thus less than 0.5 μg/ml lipids per and/or less than 1.0 μg/ml high molecular weight polypeptide. NA means not assayed.

Table XIII: Physical, Chemical and Biological Properties of Earlier Batches of Compositions of Example 1

Batch Conductance Osmolarity Absorbance UV, VIS Activity Potency No. pH (mMho) (mOsM) (P.P. 280 nm) Peaks (Units/ml) pg/ml

B0201 7.37 16.9 361 0.98 404 nm 10.5

B0202 7.35 17.3 298 0.777 None 6.5

B0203 7.3 17.7 360 0.67 365 nm 21.0

B0208 7.00 16.1 250 0.453 None 8.1 Batch Conductance Osmolarity Absorbance UV, VIS Activity Potency

No. pH (mMho) (mOsM) (O.D. 280 nm) Peaks (Units/ml) pg/ml

B0209 7.31 11.2 259 0.594 None 6.7

B0211 7.35 34.9 175 0.287 None 7.5

B0106 7.57 34.3 627 0.341 None 17.2

B0706 7.57 11.6 627 0.387 None 23.0

B1306 8.02 35.6 790 1.147 None 17.0

B2006 8.56 33.9 651 1.024 None 21.0

B2306 8.01 35.1 623 1.054 None 19.0

B0213 7.75 29.5 628 0.48 none 858

R0201-pH 7.95 44.5 877 1.59 271 nm 0.65 NA O.D.

R0201/- 7.60 50.0 1162 2.29 266 nm 1.6 NA +pH O.D.

C0203 7.90 34.8 657 0.96 none NA

0-13/2109 7.73 17.0 316 0.83 none NA

B27/2806 7.71 22.0 453 0.49 none NA

B29/3006 7.67 28.8 605 0.55 none NA

B15/1606 7.84 35.0 753 1.04 none NA

Comments:

1. Full isotonic PBS solids were added to batches No. B0106 and B0706 .

2. Batches B1306, B2006 and B2306 were concentrated two times without adjustinj Ϊ PH

Table XIV: Amino Acid Composition of Earlier Batches of Composition of Example 1

BATCH NUMBER

B- B-0209 B-0211 01/06 07/06 1306 2006 2306

0208

Asparagine 365 113 289

Serine 69 12 7 17 144 119 308

Glycine 22 449 274 279 417 3731 5314 10371

Histidine 192 90 68 938 1335 2114

Arginine 161 533

Threonine 19 13 30 148 142 250

Alanine 173 112 24 64 949 1002 1423

Proline 1092 74 817 639 1075

Tyrosine 15 55 57 43 39 205 135 45

Valine 121 63 31 10 15 367 335 224

Methionine 970 461 462 13 107 121 70

Cysteine 103 90 41 12 86 49 10

Isoleucine 2721 84 95 17 232 216 68

Leucine 58 9 221 242 84

Phenylalanine 57 200 16 45 80 23

Lysine 191 36 123 6 18 15

Total AA μg/ml 4.53 2.27 1.47 1.16 1.42 8.01 9.73 16.35

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 being attributed to small molecular weight components (i.e., less than 3000 daltons). This was determined through an experiment in which the composition was passed through the reverse-phase HPLC described in

Example 3 and eluted fractions who isolated and analyzed for potency by the PBMN-TNF assay described in Example 2. Significant activity was only detected in the early-eluting peak (F 1 ), i.e., 5.6 to 6.2 mins. which is consistent with a molecular weight of less than 3000 daltons (see Table XV). Table XV: Effect of Fractions of Composition of Example 1 Eluted by Reverse-Phase HPLC on TNF Release From LPS-Induced PBMNs

TNF-α Released (pg/ml)

Sample Tested HPLC Quantity per Total -LPS Osmolarity

(min) Well (mOsm)

LPS 50 ng 305 ± 79 0 304

Composition of Example 1:

Whole 0 00 μl 519 + 195 213 415

FI 5.60-6.20 100 μl 508 + 82 203 344

F2 6.20-6.55 ] 100 μl 149 + 44 -157 281

F3 6.55-7.10 1 00 μl 306 ± 80 1 309

F4 7.10-7.90 1 00 μl 316 ± 123 11 309

F5 7.90-8.40 1 00 μl 390 + 95 84 309

F6 8.40-8.90 1 00 μl 282 + 103 -24 311

F7 8.90-9.40 1 00 μl 296 ± 108 -10 309

F8 9.40-10.00 1 00 μl 341 + 112 36 309

F9 10.00-10.40 1 00 μl 33 ± 139 24 308

F10 10.40-12.00 1 00 μl 316 ± 101 11 311

Fl l 12.00-13.60 1 00 μl 354 + 74 49 311

F12 13.60-14.20 1 00 μl 344 + 107 39 315

F13 14.20-15.35 1 00 μl 296 ± 117 -9 311

F14 15.35-15.75 1 00 μl 344 + 108 39 314

F15 16.75-18.20 1 00 μl 300 + 104 -5 313

Note:

1. Number of patients tested: 3.

2. Total TNF-a Released is corrected for release by RPMI Media (13 + 4 pg/ml, 306 mOsm).

3. HPLC fractions 1-2 reconstituted in water; 3-15 reconstituted in PBS buffer. 4. Columns in tandem are: W-Porex C18 and PrimeSphere. Both from Phenomenex, 250 x 4.6 mm.

5. Volume of LPS per well: 10 μl 6. Total volume per well: 1000 μl

7. Sample volumes are equivalent.

Additional experiments were done as follows to show that the active (TNF-releasing) components had molecular weights less than 3500 and less than 1000 daltons. BatchBC0241 was fractionated by carrying out a Folch extraction according to Tamari et al.. Agr. Biol. Chem.. 40 (10). 2057- 2062 (1977). The water layer was dried on a rotovap to yield a light brown, granular solid. A stock solution of this solid was prepared at a concentration of 5 mg/ml. A portion of the stock solution was loaded into Centri/por Centrifuge Concentrators (Spectrum Products, Houston, TX) having a 3500 or 1000 dalton molecular weight cutoff membrane. The Concentrators were centrifuged at approximately 1500 x g until a portion of the material had passed through the membrane. The solution that passed through the membrane was assessed for potency in the PBMN-TNF assay. The results are presented in Table XVI.

Table XVI: Molecular Weights of Active Components of Composition of Example 1 SAMPLE TNF Released (pg/ml) Folch water layer from BC0241 1709 Folch water layer passed through 3500 dalton membrane 2318 Folch water layer passed through 1000 dalton membrane 2423

The analysis of the biological activity of molecular weight fractions indicates, accordingly, that the TNF-releasing components are less than 1000 daltons molecular weight.

Example 6

This example illustrates the effect of the composition of Example 1 on T and B lymphocytes in culture.

The growth of human lymphocytes was examined under carefully controlled conditions in the presence and absence of the composition of Example 1. Standard concentrations of lymphocytes were incubated in wells containing various concentrations of the composition. When normal T and B human lymphocytes were incubated with the composition in concentrations similar to those that are used clinically, there were no adverse effects as judged by trypan blue dye exclusion. Accordingly, the composition of the invention was non-toxic to normal T and B lymphocytes in culture.

The effect of the composition on the survival of human PBMN also was examined. PBMNs were incubated for 24 and 48 hrs in plastic microwell plates with various volumes of the composition and tissue culture medium. At the end of this period, the number of surviving cells was estimated by trypan blue dye exclusion.

Table XVII: Concentration of Viable PBMNs After Incubation with Composition of Example 1

No. of Live PBMN per Well by Trypan Blue (xlO⁶)¹

Concentration After 24 hrs After 48 hrs (μl/well) Zero time No. (% viable) No. (% viable)

Patient S.Z.

0 0.70² 0.23 (33) 0.10 (14)

25 0.43 (61) 0.15 (21)

50 0.10 (14) 0.23 (33)

100 0.15 (21) 0.18 (26)

200 0.48 (69) 0.23 (33)

LPS (μg/well)

1 0.30 (43) 0.28 (40)

10 0.25 (36) 0.13 (18)

Patient E.S.

0 1.30² 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)

¹ Approximately 1 x 10⁶ cells plated/well in triplicate.

² Actual number of cells counted/well (xlO⁶).

The above data show that the number of surviving cells fell at 24 and again at 48 hours; however, the number of surviving cells in the presence or absence of the composition was not different. Moreover, increasing volumes of the composition had no effect on survival. Thus, the composition showed no cytotoxicity to human PBMN.

The ability of the composition to stimulate lymphocytes was evaluated in the following 3 indicator systems: 1) stimulation of lymphocyte DNA synthesis; 2) induction of lymphocyte-mediated cytotoxic function; and 3) induction of monocyte/macrophage-mediated cytotoxic function. These tests were chosen for the screen because they measure immunological functions that have been shown to be associated with different clinical parameters in patients with malignant disease. These indicators of immune function also can be modulated in cancer patients treated with different biological response modifying agents, such as IFN or IL-2. The results of the initial screening procedures are presented below.

1. Stimulation of lymphocyte DNA synthesis: comparison with an optimal stimulating concentration of phytohemagglutinin (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 lymphocyte-mediated cytotoxic function and comparison with an optimal stimulating concentration of IL-2:

Lytic

Stimulant Units

Medium 30.8

IL-2 472.5

Composition (neat) 48.1

Composition (1 :10) 33.3

Composition (1 :50) 44.8

Unlike the prototypic stimulator of lymphocyte cytotoxic function, IL-2, the composition did not elicit lymphocyte cytotoxicity. The number of lytic units stimulated by the composition was virtually identical to that of the negative control (i.e., medium). 3. Stimulation of monocyte-mediated cytotoxic function by the composition: comparison with IFN-γ and LPS (IFN + LPS)

Stimulant (E T=20/l) % Cytotoxicity

Medium 4.3 IFN+LPS 24.4 Composition (neat) 19.7 Composition (1:10) 20.0 Composition (1:50) 11.5

The composition of Example 1 was capable of stimulating peripheral blood monocytes to express tumoricidal function in a dose-dependent manner. The magnitude of stimulation is comparable to that elicited by the prototypic macrophage activator combination of IFN-γ and LPS. It is important to recognize that the action of the composition in these in vitro assays did not require the addition of endotoxin, as in the case with any other macrophage activator.

Example 7

This example illustrates the results of assays conducted to survey what, if any, cytokines are present in the composition of Example 1.

Samples of a bile extract (50 μl and 100 μl aliquots per test) prepared according to Example 1 were tested for the presence of the following cytokines (sources and detection limits of the ELISA immunoassay kits used are noted parenthetically): TNF-α (Endogen, Inc. (5 pg/ml)); IL-l (Endogen, Inc. (50 pg/ml)); IL-lβ (4.3 pg/ml); GM-CSF (Endogen, Inc. ); RFN-α (Endogen,

Inc.); IL-2 (Advanced Magnetics, Inc.); IL-6 (Advanced Magnetics, Inc. (7 pg/ml)); IFN-γ (5 pg/ml) [source]; IL-1 (Advanced Magnetics, Inc.) [need limit]; IL-4 (R&D Systems (3 pg/ml)); and IL-8 (R&D Systems (4.7 ng/ml)). Procedures used were according to the individual kit's instructions, which can be easily followed by an ordinary artisan.

It was determined that the composition of the invention contained no measurable levels of any cytokine tested, those being TNF-α, IL-1 α, IL-1 β, IL-4, IL-6, IL-8, GM-CSF and IFN-γ, as described in Table XVII. Table XVIII: Elisa Determination of Cytokines In Composition

Cytokine (pg/ml) 50 μl 100 μl

TNF <5 <5

IL-lβ ~ 6.5

GM-CSF <5 -

IL-6 <7 -

IFNγ <5 -

IL-lα <50 ~

IL-4 ~ <3

IL-8 (ng/ml) <4.7

Example 8

This example describes pharmacodynamic studies in mice with the composition of Example 1, including the direct in vitro effect of Virulizin™ as well as the effect of Virulizin™ administered in vivo on murine peritoneal macrophages.

Peritoneal macrophages were harvested from C57BL/6 mice 72 hours after intraperitoneal injection of 1.5 ml of 4% protease peptone. The macrophages were then stimulated in vitro with medium alone, 50 ng LPS, or VIRULIZIN™. Measurements of the stimulation was done with respect to TNF (by ELISA) and NO (by spectrophotometric assay using the Greiss reagent) levels in duplicate experiments. Standard error of the mean between duplicate experiments was less than 10%. As noted in Table XIX, VIRULIZIN™ induced a slight increase in TNF-α production (60-

232 pg/ml) compared to background (medium) levels ( 120 pg/ml), but VIRULIZIN™ in comparison to LPS (2225 pg/ml) was not a strong stimulant of macrophage TNF-α release. Nitric oxide production was zero.

Table XIX: In Vitro Stimulation of Protease Peptone Macrophages

Macrophages Stimulated With TNF (pg) Mean NO (μM) Mean

Medium 120 0

LPS (1 μg/ml) 2225 11

Virulizin 1:2 62 0 1 :5 181 0

1 : 10 206 0

1:20 202 0

1:40 232 0

1:80 142 0

1 :200 122 0

In vitro synergy of Virulizin™ with LPS for TNF-α release was also addressed. Peritoneal macrophages were harvested from C57 L/6 mice after the same aforementioned treatment. The macrophages were then stimulated with 50 ng LPS alone or LPS with different dilutions of VIRULIZIN . As above, TNF was determined via ELISA. As noted in Table XX, LPS alone induces about 2900 pg/ml of TNF-α release from mouse peritoneal macrophages in vitro compared to 262 pg/ml for medium. When LPS is combined with VIRULIZIN , there is about an 800 pg/ml increase in TNF-alpha release at dilutions of VIRULIZIN 1 :5 and 1 :10 and enhanced release to at least 1 :40.

Table XX: Synergistic Combinations between Virulizin™ and IFN-α or LPS

Macrophag es Stimulated With TNF (pg/ml) NO (μM)

Medium 262 1.6 rt 1.1

LPS (5 ng/ml) 2900 8.6 ± 1.3

LPS (5 ng/ml + Composition of Example 1 :

1 :5 3750 13.2 ± 0.5

1: 10 3750 16.9 ± 2.7

1:20 3500 13.5 ± 2.5

1:40 3600 27.1 ± 11.6

1 :80 3000 10.1 ± 1.9

1 :200 3400 9.7 ± 1.3

1:1000 3200 9.4 ± 1.2

IFN-γ (100U)+LPS (5 ng/ml) 6800 74.1 ± 0.6

IFN-γ (100U)+Virulizin:

1:5 512 46.9 ±0.6

1:10 625 57.3 In vitro synergy of Virulizin™ with LPS for nitric oxide (NO) was addressed in the same procedure as above, except NO was determined in the supernatant of the treated macrophages. As above, the assay for NO is spectrophotometric and uses a Greiss reagent. As noted in the table above, LPS causes some release of NO (9 μM). VIRULIZIN in synergy with LPS induces a marked increase in NO production (13-27 μM) to dilutions of 1 :40. VIRULIZIN by itself did not induce release of NO by macrophages.

In vitro synergy of Virulizin™ with IFN-γ for TNF-α release was studied, using the same peritoneal mouse macrophages derived from C57 L/6 mice treated as above. The data are included in the table above concerning "Synergistic Combinations." As shown, peritoneal mouse macrophages exhibit a baseline release of TNF-α after 24 hours of in vitro culture. The same macrophages stimulated with either LPS or IFN-γ release almost 3000 pg/ml of TNF-α. When VIRULIZIN and IFN-γ were added together, the release of TNF-α was diminished, y comparison, the combination of LPS and IFN-γ have an additive effect on TNF-α release.

In vitro synergy of Virulizin™ with IFN-γ for NO release was studied, using the same peritoneal mouse macrophages derived from C57 L/6 mice treated as above. The data are included in the table above concerning "Synergistic Combinations." As shown, LPS and IFN-γ alone each enhanced NO production (9 and 7 μM, respectively). VIRULIZIN added to IFN-γ induced a marked increase in NO production (47-57 μM) that almost equaled the combination of LPS and IFN-γ (74 μM). The results are consistent with the conclusion that VIRULIZIN in combina- tion with IFN-γ enhances NO production but inhibits TNF-α release.

In vivo production of TNF-α over 72 hours was studied on macrophages harvested from C57 L/6 mice that, prior to harvest, were treated with nothing, in ected intraperitoneally 72 hours previously with 1.5 and 4% protease peptone, or in ected intraperitoneally 72, 48, or 24 hours previously with 1.0 ml Virulizin™ diluted 1 : 10 in P S . The macrophage monolayers were treated in vitro for 24 hours with IFN-γ (50 μ/ml), LPS only (5 ng/ml), or the combination thereof. TNF and NO were determined as recited above. The data are presented in Table XXI.

Table XXI: TNF and No Release From Macrophages Harvested From Treated Mice

Nothing Medium 315 0

IFN-γ 402 25.8 + 1.6

LPS 3,750 1.9 + 0.2 IFN_-γ_+LP_S_ 6,300 fO-^± J

Protease Peptone (72 hrs prior) Medium 335 0.9 + 0.5

IFN-γ 838 48.6 ± 1.7

LPS 5,975 23.2 ± 3.4 IFN_j;γ_+LPS 10,875 55.8_±_L9_

Virulizin (72 hrs prior) Medium 258 1.2 +0.6

IFN-γ 425 37.5 + 2.6

LPS 3,300 4.0 ± 0.9 IFN_-γ_+LP_S_ - ⁶!⁰. -?i;0_±-.9-.9-.

Virulizin (48 hrs prior) Medium 350 8.5 + 1.8

IFN-γ 560 62.0 + 2.5

LPS 5,300 36.5 + 1.2 IPN_-γ_+k^p_^s_ ϊ<.⁴A §A ±_1A.

Virulizin (24 hrs prior) Medium 248 2.9 + 2.1

IFN-γ 475 44.1 + 0.7

LPS 9,025 12.5 ± 2.4 IFN-γ + LPS 12,375 52.8 ± 0.6

As described, the release of TNF-α from macrophages was examined in the absence of a stimulus or with IFN-γ, LPS, or LPS/IFN-γ after 24 hrs in vitro culture. Mouse peritoneal macrophages were shown to release little TNF-α after in vivo stimulation with VIRULIZIN™ . When the harvested macrophages were exposed to IFN-γ at 24 and 48 hrs prior to testing, they showed a small increase in production of TNF-α. By contrast, harvested macrophages stimulated with LPS at 24 and 48 hrs, but not 72 hrs prior to testing, showed enhanced release of TNF-α. Likewise, there was a synergistic effect of LPS and IFN-γ on harvested macrophages that were stimulated 24 and 48 hrs but not 72 hrs before testing.

In vivo production of NO over 72 hrs was studied with macrophage cells and tests under the same conditions described above with respect to TNF-α production. There was a small spontaneous release of NO measured at 24 and 48 hrs after intraperitoneal injection of VIRULIZIN™ (hereinafter IP Virulizin™). When the harvested cells were incubated with IFN-γ, there was a marked release of NO, and the harvested macrophages that had IP VIRULIZIN™ at 24 and 48 hrs prior to testing showed an exponential increase in release of NO, which fell back at 72 hrs towards the baseline values of IFN-γ alone. When the harvested cells were stimulated with LPS, they showed a markedly enhanced output of NO, which was once again observed for the 24 and 48 hrs VIRULIZIN™ -treated macrophages compared to macrophages that had not received IP VIRULIZIN™. The harvested macrophages that had received IP VIRULIZIN™ 72 hrs before responded no differently than macrophages that had no VIRULIZIN™ pretreatment. Finally, when harvested macrophages pretreated with IP VIRULIZIN™ were incubated with LPS/IFN-γ , they showed enhanced production of NO compared to macrophages not so pretreated. The maximum response was with macrophages pretreated with VIRULIZIN™ 48 hrs before harvesting and testing.

Example 9

This example illustrates the results of assays conducted to estimate protein within the composition.

Protein estimation of the composition was done using the Pierce Micro BCA Protein determination technique (Smith et al. , Anal. Biochem.. 150. 76-85 (1985)). A 10 μl sample of a batch of the composition was made up to 1 ml with distilled water. Five concentrations of bovine serum albumin (0.150 μg/ml) was also made up to be used as standards. As a blank, 0.1 N NaOH was used. To all these samples was added a mixture of BCA (2% bicinchonic acid sodium salt; Pierce), 4% copper sulfate and microreagent A (NaCO₃, NaHCO₃, Na tartrate in 0.2N NaOH). The sample mixtures were incubated for 1 hr at 60°C, cooled, and the resultant absorbency read at 562 nm using a spectrophotometer. The amount of protein in the test sample was then compared to the plotted standard curve and the appropriate calculations made. The protein concentration of the composition was found to be low and estimated to be 32 ng/ml.

Example 10 This Example demonstrates, in summary, the following: (1) the composition has TNF-α releasing activity and the TNF-α releasing activity is not related to any contamination with endotoxin; (2) priming of macrophages enhances the ability of the composition to stimulate release of TNF-α; and (3) the hyperosmolarity of the composition is not responsible for TNF-α releasing activity.

To test whether an endotoxin effect was associated with the biological activity noted above for the composition of Example 1, further composition experiments were performed with polymyxin added to the reactants. Polymyxin inhibits the action of endotoxin on leukocytes. The following table and succeeding notes recite the composition experiment performed and its results.

Table XXII: Absence of Endotoxin for TNF-2 Releasing Effect and Enhancement of Release With Macrophage Priming

TNF Released (pg/ml)

Sample Tested Additive Total -LPS

LPS Polymyxin 11 + 7 0

None 517 ± 118 0

Composition (#B02- Polymyxin 1591 + 413 1581 13)

None 5256 ± 2585 4738

Notes:

1. Total TNF released is correct for TNF release by 1640 medium.

2. Polymyxin concentration: 50,000 units/ml. 3. Composition volume: 200 μl.

4. With polymyxin, 8 patients tested. With no additive, 3 patients tested.

5. LPS concentration: 50 ng/10 μl.

The results show that polymyxin completely inhibits the LPS-induced release of TNF-α. In the absence of polymyxin, LPS induces 517 pg/ml of TNF-α, whereas in the presence of polymyxin,

11 pg/ml of TNF-α is released. The composition, on the other hand, releases 1591 pg/ml ofTNF- α in the presence of polymyxin. In the absence of polymyxin, LPS and the composition show more than just an additive effect of the stimulators, suggesting that the composition acts with greater intensity when macrophages are primed. Table XXIII: Absence of Effect of Hyperosmolarity on TNF-2 Release

Osmolarity

Batch # PH (mOsm)

Concentrated:

B0222 pre-pH 41 1

B0222 pH adjusted 581

B0216 pH adjusted 872

B0219 pH adjusted 886

Osmolarity

Batch # pH (mOsm)

Nonconcentrated:

B0221 pre-pH 652

B0221 pH adjusted 533

B0213 pH adjusted 675

B0225 pH adjusted 590

B0226 pH adjusted 540

BC 11-06 pH adjusted 445

BC 11-09 pH adjusted 603

The osmolarity of different batches was determined using standard methods. The results are shown in the previous table. B0213 is moderately high at 675 mOsm. B0222, shown to have

TNF releasing activity even better than B0213, is less hyperosmolar, 581 mOsm. The fractions B0226, BCl 1-06 and BCl 1-09 range from 540 to 603 mOsm. The effect of the hyperosmolarity of the composition on TNF-α releasing activity was also studied. It was found that the composition, when adjusted for osmolarity, even to the point of being hypoosmolar, continued to release TNF-α.

Example 11

This example illustrates toxicity studies regarding the composition of the present invention. Preliminary toxicity studies were conducted on a variety of animal species, as tabulated below.

All animals (listed in the following table) were assessed on the basis of daily clinical observation while receiving the injections of the composition on days 14, 21 and 30 thereafter. Hematologic data was collected every third day for the first 30 days and once monthly thereafter. No adverse effects were noted in any of the over 358 animals included in this study throughout the period that injections were administered or during the follow-up period (one month for all species except the dogs which were followed for 4 months).

Animal Quantity Dose ite mice 100 0.2 ml i.m. at 3-day intervals 4 times le Wistar rats 100 2.0 ml i.m. at 3-day intervals 4 times

Iden hamsters 60 1.5 ml i.m. at 4-day intervals 4 times Guinea pigs 60 3.0 ml at 3-day intervals 4 times

Rabbits 15 5.0 ml i.m. at 3-day intervals 4 times

Cats 10 3.0 ml i.m. at 3-day intervals 6 times

Dogs 12 2 ml/kg i.m. given once - observed for 4 months

A second toxicity study was conducted to determine the effect of a single large 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 either group and no gross pathologic findings were observed in the animals that were necropsied. Based on these observations the LD₅₀ for intramuscular administration of the composition in rats was determined to be greater than 5 ml/kg.

Another toxicity trial was conducted by the Ontario Veterinary College, wherein the composition was administered to two mixed breed dogs. The protocol is summarized in the following table:

Animal Age and Weight Dose 1 Dose 2 Dose Interval Male Mixed Breed Adult 5.5 ml i.m. 0.6 ml i.m. 7 days

5 kg

Female Mixed Breed 6 months 12.5 ml i.m. 1.3 ml i.m. 7 days 13

In each case, one dose was given in the right rear leg and the second dose 7 days later was given in the left rear leg. Both dogs were observed for 14 days after the first injection. Appetite, activity, temperature, pulse rate, and respiratory rate were monitored twice daily throughout the study. Routine urinalyses, hematology and 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. Neither animal showed signs of pain associated with either injection. There was no evidence of anaphylaxis associated with the second injection. No abnormalities or changes in physical or laboratory parameters were observed that could be attributed to the drug. The drug appeared to be well tolerated by healthy dogs.

A 17-day repeat dose toxicity study was carried out with VIRULIZIN™ in conjunction with an animal model study at the Ontario Cancer Institute. The model used female C57B1 mice. There were 4 groups as follows (IM = intramuscular, IP = intraperitoneal):

Group # Treatment Dose Volume Number/Group

1 Saline, IM 0.05 ml 10

2 Virulizin™, IM 0.05 ml 10

3 Virulizin™, IM 0.05 ml X2 10

4 Virulizin™, IP 0.5 ml 10

Each group of mice were injected at day 0 with 5 X 10³ of B16F1 melanoma cells plus microspheres. On each of the first 17 days, each group received daily injections of Virulizin™ or saline, as above. On day 18, the animals were sacrificed.

Prior to sacrifice, food intake, weight gain, and behavior were normal. In addition, there was no evidence of toxicity causing changes observable by light microscopy in any of the organs examined, which were: large intestine, spleen, stomach, pancreas, urinary bladder, liver, brain, kidneys, small intestine, and heart. Food intake and behavior were normal. Weight gain was normal.

A 13-week repeat dose toxicity study in Fischer-344 rats (total of 40 males and 40 females) was carried out administering VIRULIZIN™ IM three times per week for 13 weeks. The largest dose was 1.1 ml/kg, about 20 X the human dose. Animals were subjected to full histopathology after 13 weeks. The only treatment related finding observed was a small decrease in mean body weight gain in the 20 X dose group as compared to controls. No toxicity was demonstrated.

Example 12

This example illustrates the isolation of active fractions.

A 300 ml sample of the composition was evaporated to dryness on a rotovap in which the temperature of the bath did not exceed 40 °C. In order to ensure that the solution remained basic during the evaporation, 5 drops of a concentrated ammonium hydroxide solution was added every half hour to the composition until the evaporation was complete. The resulting residue had a weight of 11.6g. 20 ml of a 10% concentrated ammonium hydroxide in methanol solution was then added to 2 g of the above residue. The insoluble material was filtered off and the filtrate was chromatographed through 101.93 g of 6θA flash silica gel in a column with dimensions of 5 cm x 12.5 cm. The solvent system used was 10% concentrated ammonium hydroxide in methanol solution. The column was run at a pressure of 10 p.s.i. and a flow rate of 11 ml/min. After 100 ml of solvent had passed through the column, twelve 20 ml. fractions were collected. The collection of these fractions correlated to the appearance of an off-white band that was quickly moving down the column.

Thin layer chromatography (TLC) of these fractions was run on silica gel plates in a 10% concentrated ammonium hydroxide solution in methanol and visualized with a ninhydrin spray.

Fractions having similar TLC profiles were combined, resulting in the following fraction combinations, which were dried on a rotovap:

Volume Through Column to Obtain Fraction

Fractions Yield (g)

1-4 100-180 0 5-6 180-220 0.1175

7-8 220-260 0.1969

9-10 260-300 0.0151

11-12 300-340 0.0053

Fractions 5-6, 7-8 and 9-10 had a positive reaction with ninhydrin at an R_f value of 0.81. Fractions 5-6 and 9-10 were tested in vitro for TNF stimulation (in accordance with Example 19).

The results are shown below:

Fraction Activity

5-6 50 pg/mg

9-10 1814 pg/mg

Thus, fraction 9-10 was an extremely active TNF stimulator. Samples of Fraction 5-6 were analyzed by Electron Impact Mass Spectroscopy (El MS) and Electrospray Mass Spectroscopy to identify specific compounds likely to be present in the fraction. The Electrospray MS was performed on a Perkin-Elmer Sciex API-Ill spectrometer, using 5%> acetic acid in water as the solute. In some instances, methanol was added to aid dissolution. The El MS using a direct insertion probe was performed on a VG Analytical model

ZAB-SE spectrometer using glycerol as a matrix, and using a DCI probe on a Kratos Analytical Profile Mass Spectrometer.

A review of the resultant spectra indicated that the following compounds were likely present in Fraction 5-6: phosphocholine, taurocholic acid, choline-stearic acid diglyceride, stearic acid, stearic acid diglyceride, palmitic acid-stearic acid diglyceride, and a sphingosine-oleic acid conjugate.

Example 13

This example illustrates an expanded procedure to isolate active fractions.

Example 12 was repeated on a larger scale, as follows. 10 ml of a concentrated ammonium hydroxide solution was added to 900 ml of the composition and the resulting solution evaporated to dryness on a rotovap in which the temperature of the bath did not exceed 40 °C. In order to ensure that the solution remained basic during the evaporation, 5 drops of a concentrated ammonium hydroxide solution was added every half hour to the composition until the evaporation was complete, leaving a residue.

150 ml of a 10% concentrated ammonium hydroxide in methanol solution was then added to the total residue. The solution was sonicated for 15 min. and the insoluble material was filtered off. The filtrate was chromatographed through 1695g of 6θA flash silica gel in a column with dimensions of 30cm x 12cm. The solvent system used was 10% concentrated ammonium hydroxide in methanol solution. The column was run at a pressure of 6 p.s.i. and a flow rate of 30 ml./min. The results of the column are summarized in the table below.

Volume of each tio n # fraction (ml.) Observations

1 550 clear, yellowish Volume of each

Fraction # fraction (ml.) Observations

2 450 clear, yellowish

3 400 clear, yellowish

4 150 clear, yellowish

5 100 clear, yellowish

6-7 75 clear, yellowish

8-13 50 clear, yellowish

14 50 tan colored solution begins to elute

15-35 50 tan colored solution

36-40 50 clear, yellowish

TLC was run on silica gel plates in a 10% concentration ammonium hydroxide solution and visualized with a ninhydrin spray. Fractions having similar TLC profiles were combined, resulting in the following fraction combinations, which were dried on a rotovap:

Volume Through

Column to Obtain action # Fraction Yield ( ) Comments

3 1000-1400 0.0504 white powdery solid

4-5 1400-1650 0.0855 white powdery solid

6-8 1650-1850 0.1555 white powdery solid

9-12 1850-2050 0.3014 white powdery solid

13-14 2050-2150 0.3595 white powdery solid

15-16 2150-2250 0.6914 slight brown color - solid is tacky

17-18 2250-2350 1.0284 tan color - solid is clumpy

19 2350-2400 0.3432 tan color - solid is clumpy

20-23 2400-2600 1.1531 brown color - solid is clumpy

24-30 2600-2950 0.8517 brown color - solid is clumpy

31-34 2950-3150 0.0813 brown oil All fraction combinations from 15-16 through Fraction 31-34 had a positive reaction with ninhydrin at an R_f value of 0.87, a value very similar to the R_f value for the active fractions of Example 28. Fractions 24-30 and 31-34 had an additional positive reaction with ninhydrin at an R_f value of 0.85.

Fractions 4-5, 15-16 and 17-18 were tested in vitro for TNF stimulation (in accordance with

Example 19), resulting in no TNF stimulation activity. Elemental analysis of the above fractions showed them to be high in NH₄C1, which is known to inhibit TNF production.

Samples of fractions 15-16 and 24-30 were dialyzed and then analyzed by mass spectroscopy, using the methods described in Example 12. Undialyzed samples from fractions 17-18 and 24-30 were also analyzed. A review of the resultant spectra indicated that the following compounds were likely present: glycocholic acid, a trihexosamine trimer, and taurocholic acid (Fraction 15- 16); stearic acid, and a hexosamine dimer; and glycocholic acid (Fraction 24-30).

Example 14

This example illustrates the application of further methods to fractionate and analyze the active components of the inventive composition.

Having identified that TNF, IL-lβ and GM-CSF releasing activity can be precipitated, in part, by 80%o acetonitrile and that much of the releasing activity elutes early from C_]g RP-HPLC, the physicochemical properties of the precipitate fraction have been studied and compared to the whole composition and supernatant fraction of the composition.

Figure 6 shows an SDS gel electrophoresis of whole composition and precipitates and supernatants of the composition. In all three instances, the composition runs near the SDS front, indicating a low molecular weight. The smallest standard used was 14,400 daltons.

The molecular size of the composition was also examined by determining its time of elution from a molecular sieve HPLC column. The elution times of whole composition, precipitate and supernatant compared to standards. All three eluted later than insulin, which eluted at 24.5 min.

Once again, physicochemical analysis indicates a mol. wt. less than 2,400 daltons. The TNF-releasing component elutes early. Thus a column with the opposite effect was chosen, a hydrophilic column in the presence of organic solvents. The ideal eluting conditions for the polyhydroxyethyl column is 80% acetonitrile. However, as indicated in the prior Example, some of the substances in the preparation precipitated at this concentration. Consequently, the composition was analyzed at a low concentration of acetonitrile where the column functions mostly as a molecular sieve column. Figures 7 and 8 show the profile of whole supernatant and precipitate. The front sheet summarizes the elution time for the different peaks. The elution times indicate the active component of the composition has a low molecular weight.

The composition and its precipitate and supernatant were separated by ion-exchange HPLC. Both by AX300 (anion exchange) chromatography and by CMX 300 (cation exchange) chromatography, there was no significant separation of components. Hydrophobic reverse phase chromatography did not separate the peaks.

In another series of experiments, 10 ml of VIRULIZIN™ was loaded onto an anion exchange chromatography column (Bio-Rad AG-1, hydroxide form, total resin wet volume was 10 ml, equilibrated with Millipore deionized water) . The volume of resin was calculated to be sufficient for the binding of all the anions present in the extract. The unbound fraction was collected and reloaded onto the column in order to maximize the binding to the resin. The unbound fraction from this second passage was collected and saved. Any unbound material remaining on the column's void volume was removed by washing with deionized water (2 X 20 ml). Bound molecules were eluted with a step gradient of ammonium bicarbonate, 20 ml/step. Free ammonium bicarbonate was removed by lyophilization. Samples from all the fractions were tested for TNF- releasing activity in the monocyte/macrophage activation assay. TNF-releasing activity was not found in the unbound fraction (effluent), but the majority was found in the eluate eluted with 0.2 M ammonium bicarbonate. These results indicate that the active components are polar, anionic, acidic in nature.

Samples from all the fractions were analyzed for TNF stimulation activity, in accordance with the procedures of Example 2. The results are shown below: TNFα release-

Sample inducing activity-LPS

(pg/ml)

O M -496

0.1 M -156

0.2 M 1638

0.3 M -36

0.4 M 256

0.5 M -27

0.6 M -175

1.0 M -246

1.5 M -346

VIRULIZIN™ control 1961

The results from the activity assays show that TNF production stimulation was found in the 0.2 M and 0.4 M fractions.

The composition was subjected to dialysis and drying of the dialysate, as follows: 100 ml of the composition was placed inside a Spectra/Por® CE membrane tubing which had a molecular weight cut off of 100. The ends of the tubing were sealed with clips and the tubing was placed into a stirred bath of 10 L of distilled water. The dialysis was monitored daily by removing 1 ml. of solution from the dialysis tubing and adding 3-4 drops of a 1/10 N silver nitrate solution. 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. Dialysis completion occurred after 3-4 days. After dialysis was complete, the dialyzed material was dried on a rotovap to yield an average of 0.3 mg of solid per ml of original volume.

A sample of the solid material was then dissolved in HPLC grade water, and TLC was run on silica gel plates in a 10%> concentrated ammonium hydroxide solution in methanol, and visualized with a ninhydrin spray. A positive reaction with ninhydrin was obtained at an R_f value of 0.83.

A sample of the solid material was also analyzed by mass spectroscopy, using the methods described in Example 12. A review of the resultant spectra indicated that the following compounds were likely present: a sphingosine-oleic acid conjugate, diacetyl sialic acid, a fucose- hexosamine dimer, deoxyglycocholic acid, taurocholic acid, a sialic acid-fiicose dimer, and a di(fucose)hexosamine trimer.

Example 15

This example will illustrate the use of Reverse Phase - HPLC (RP-HPLC) to analyze the inventive composition.

Samples were lyophilized and then reconstituted in 0.1% trifluoroacetic acid (TFA) in water (buffer A) and subsequently run in the following columns and conditions:

Column: WP60009-C18 column (W-Pore C18, 250 X 4.6 mm, Phenomenex,

California) in row with prime-sphere HC-C18 column (250 X 4.6 mm,

Phenomenex, California)

Eluents: Buffer A:0.1%TFA in H₂O

Buffer B:0.1%TFA in acetonitrile

Gradient: 150 μl sample applied to column

Run buffer A for 20 minutes

Start linear gradient, 0-80% buffer B, run over 35 minutes

Run 80-0% buffer B over 5 minutes

Flow: 0.9 ml/minute

Temperature: Ambient

Detection: Absorbance from 290 to 284 nm, with most runs being detected at 210 and

235

Fifteen eluent fractions were collected, at the approximate times from injection noted in the following table. In addition, a TNF release essay, as described in Example 2, was performed on each fraction, with the following results: Fraction # Time (min.) TNF (pg/ml)

1 5.6-6.25 203

2 6.25-6.6 -157

3 6.6-7.1 1

4 7.1-7.9 11

5 7.9-8.4 84

6 8.4-8.9 -24

7 8.9-9.4 -10

8 9.4-10.0 36

9 10.0-10.4 24

10 10.4-12.0 11

11 12.0-13.6 49

12 13.6-14.2 39

13 14.2-15.35 -9

14 15.35-16.75 39

15 16.75-18.20 -5

Whole VIRULIZIN™ 213

Accordingly, the majority of the active components of VIRULIZIN™ eluted in Fraction 1. Activity was also found in Fractions 4-5, 8-9, 11-12, and 14.

Samples from all RP-HPLC fractions were analyzed by mass spectroscopy in accordance with

Example 12. A review of the resultant spectra for the fractions indicated that the following compounds were likely present: taurocholic acid, a sialic acid-glycerol dimer, NaCl, trimeth- ylamine, methylethylamine, and propylamine.

Example 16

This example illustrates the compounds that have been identified in the inventive composition.

The inventive composition was prepared in accordance with Example 1 and subjected to standard methods of fractionation, including (1) dialysis in 100 MWCO dialysis membrane; (2) classical organic extractions including Folch extractions, (Tamari et al., Agr. Biol. Chem..40 (10).2057- 2062 (1976)); (3) silica column chromatography; (4) ion exchange chromatography); and (5) preparative silica TLC fractionation using butanol: acetic acid: water 6:2:2 as the eluant and ninhydrin as the visualization reagent, using standard methods as disclosed in Dying Reagents for Thin Layer and Paper Chromatography. E. Merck, Darmstadt, Germany, 1971.

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-MS, CI-MS (OH-,), and FAB-MS (in glycerol or thioglycerol matrices). A VG Analytical Model ZAB-SE 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 Packard model 5890. A Kratos profile spectrometer was used to obtain EI-MS, LSIM-MS (in glycerol and NPOE matrices), and GC-MS mass spectra. The GC used in conjunction with the instrument was also a Hewlett Packard model

5890. MS-MS, electrospray using either water or water alcohol (methanol or isopropyl alcohol) mixtures as solutes, EI-MS and FAB-MS in glycerol and thioglycerol were performed on a perkin- Elmer Sciex API-Ill spectrometer. Fractions were derivatized for MS analysis as required by acetylation with acetic anhydride/pyridine or methylation with diazomethane. Conversion of molecules into sodiated species was accomplished by addition of sodium acetate to the electrospray solute. Protonation of molecules for electrospray MS was achieved using acetic acid or trifluoroacetic acid. TLCs of extracts and standards were run on silica TLC plates using butanol: acetic acid: water 6:2:2 or cited eluants as mobile phases and several reagent sprays for visualization.

Standard methods were used in connection with the aforementioned instruments, which are further recited in the following references: Rigler et al., J. Chromatography. 277, 321-327 (1983); Sundaram, et al., Clinica ChimicaActa. 34425-429 (1971); Bandurski et al., J. Biol. Chem.. 193 405-410 (1951); and Larsen et al., J. Chromatography. 226 484-487 (1981).

Typical TLC profiles on silica plates (using butanol: acetic acid: water, 6:2:2 as the eluant) are as tabulated for active lots of VIRULIZIN™:

Analysis of the inventive composition using the aforementioned instrumentation and methods revealed the following compounds contained therein: 1) BILE ACIDS: cholic acid; glycocholic acid; deoxyglycocholic acid; cholesterol sulfate; deoxycholic acid; chenodeoxycholic acid; and taurocholic acid. Note: From the MS it is not distinguishable if -OH and -H₂ are occurring in the MS or if the deoxy, dideoxy and unsaturated analogs are also present to begin with. These compounds may all be present as salts of ammonium, alkylammonium and inorganic cations. 2) PHOSPHOLIPIDS, SPHINGOLIPIDS AND RELATED (HYDROLYSIS) PRODUCTS : stearic acid CH₃(CH₂)₁₆COOH; palmitic acid CH₃(CH₂),₄COOH; oleic acid Z-9 octadecanoic acid: CH₃(CH₂)₂CH₂CH=CHCH₂(CH₂)₆COOH oxidized or hydroxylated/unsaturated short chain fatty acids, such as C₆H₈O₃ (CH₃CH=CH-COCH₂COOH or a C₆ acid with 2 double bonds and a hydroxide); acetic acid; stearic acid diglyceride; palmitic acid diglyceride; stearic acid, palmitic acid diglyceride; stearic acid monoglyceride-phosphocholine (a lysolecithin); stearic acid monoglyceride; stearic acid triglyceride; phosphocholine; phosphoserine; phosphosphingosine; sphingomyelin; lecithin; stearic acid-sphingosine; sphingosine; phosphoglycerol; glycerol; choline; glycero-phosphocholine; stearic acid, oleic acid diglyceride; stearic acid, oleic acid phosphoglycerol; stearic acid amide; stearic acid methylamide; and palmitic acid amide.

In addition, preliminary HPLC and titration evidence has been obtained which shows that shorter chain fatty acids are also present (acids range from C, to C₃₀). 3) MUCIN HYDROLYSIS PRODUCTS: sialic acids and their mono and diacetylated monomers;

N-acetylneuraminic acid; hexosamines, such as glucosamine; L-fucose; hexosamine-hexuronic acid (dimer) disulfate; glucuronic acid; glucuronic acid or iduronic acid disulfate, monoacetylated; sialic acid-glycerol (dimer); and dimers, trimers, oligomers and polymers of the above monomers in acetylated and sulfated form.

4) FAT-SOLUBLE VITAMINS: Vitamin A2; Vitamin DI; lumisterol (present from its vitamin DI complex);

Vitamin E;

Vitamin Kl oxide; and

Vitamin K5.

5) MISCELLANEOUS ORGANIC: urea; alkyl amines, including methyl amine, dimethylamine, ethylamine, methylethylamine, diethylamine, dipropylamine, butylethylamine; amino acids, including taurine, glutamic acid, glycine, alanine, n-leucine, phosphoserine, phosphoethanolamine, aspartic acid, threonine, serine, sarcosine, α-amino adipic acid, citrulline, valine, isoleucine, β-alanine, γ-amino butyric acid, hydroxy lysine, ornithine, and lysine; butylated hydroxy toluene (BHT); and polyethylene glycol. Example 17

This example illustrates the saccharide components of the invention.

The monosaccharide composition of the samples was determined before and after hydrolysis. All reagents used to analyze the monosaccharides were of analytical grade. THF (trifluoroacetic acid) obtained from Aldrich after dilution with deionized water, was used for the hydrolysis of samples.

A 50% (W/W) NaOH solution (low in carbonate) was purchased from Fisher Scientific. Sodium acetate was from Fluka-Gerantie, New York.

To release the monosaccharides, the samples were treated with 4M trifluoroacetic acid for 4 hours at 100° C. The samples were lyophilized and analyzed by high performance liquid chromatogra- phy-anion exchange using a Dionex Bio-LC System for carbohydrates with Carbopack Pal separating column (250 x 4 mm i.d.) and HPLC-AG6 guard column (50 x 4 mm i.d.) equipped with a25 ul sample loop. Detection of eluting monosaccharides was accomplished with PAD, i.e., pulsed amperometric detector. Conditions were as follows:

Before Hydrolysis

For detection of inositol, sialic acid and glucuronic acid, isocratic elution eluant (100 mM

NaOH+150 mM NaOAc mixture) was used. The eluant was protected from the atmosphere with a helium module degasser. The flow rate was 1 ml/min through the column.

Detection of monosaccharides, including fucose, galactosamine, galactose, glucose and mannose, also was accomplished via isocratic elution, eluant (15 mM NaOH) with a post column 300 mM NaOH, at a flow rate 1 ml/min.

The detector settings E1=0.05V, E2=0.60V, E3=0.60 V, tl=120ms, t2=120ms, t3=300ms; gold working electrode; silver-silver chloride reference electrode; output range 1-3 KnAmp full scale; chart speed 0.5 cm/min.

Measurements were performed of the detector for uronic acid and monosaccharides. A linear response was obtained for concentrations varying from 0.5 -2.5 ug/ml by a progressive dilution of a standard mixture.

After Hydrolysis

Monosaccharides were detected after hydrolysis of the sample after applying a gradient elution, eluant A (50 mM NaOH) and eluant B (50 mM NaOH/150 mM NaOAc mixture). The eluants were protected from the atmosphere with a helium module degasser. A Spectra-Physics (SP 4270) integrator was used to analyze the output. The standard gradient was injection in 100% eluant A, followed by a linear progression to 80% A:20% B over the next 10 minutes. This condition was maintained for 20 minutes and then the eluant returned to 100% A over 5 minutes followed by at least 10 minutes of equilibration before injection of the next sample.

The results of the monosaccharide analysis as described are presented in the following table:

As noted in the table, only the ethyl acetate extract of green bile (batch MU100 GB) was shown to include any monosaccharide prior to hydrolysis, those being sialic and glucuronic acids, in microgram per milliliter concentration. After hydrolysis, no sialic acid was detected and the glucuronic acid was present at approximately 20% the concentration. After hydrolysis, other preparatives of the inventive compositions were shown to contain sialic acid, glucuronic acid, glucosamine, and inositol.

Example 18

This example illustrates the antiviral effects of VIRULIZIN on a patient infected with an HIV virus and demonstrating advanced stage IV-D disease with Kaposi's sarcoma. This patient experienced a reduction in viral load which was associated temporarily with the administration of VIRULIZIN.

This patient was enrolled in Imutec's Corporation Inc., open, non-comparative Phase I/II trial of VIRULIZIN-2y in HIV infected patients with stage IV-D disease (Protocol CO5-107). The objective of this study was to determine the safety, toxicity and effect of VIRULIZIN when administered intramuscularly to HIV patients.

Eligible patients had: (i) documented HIV infection (by confirmatory serologic testing) that meets

CDC stage IV-D classification, advanced Kaposi's sarcoma (and lymphoma) recalcitrant to conventional therapy; (ii) ECOG performance status of < 2 or Karnofsky status of ≥ 50% and life- expectancy of at least 12 weeks; (iii) adequate bone marrow and organ function with a haemoglobin count of ≥ 85g/L, neutrophil count of ≥ 1.0 x 10⁹ / L, and a platelet count of > 75 x 10⁹ / L, Liver function tests ALT and AST < 5 times above normal values, alkaline phosphate and total bilirubin < 3 times above normal values. Prothrombin time and partial thromboplastin time should not be more that 2 seconds above normal control value. BUN should be normal (< 10 mmol/L) and serum creatinine < 150 μmol/L).

Baseline clinical evaluations were performed within two weeks prior to patient entry into trial. Evaluations included medical history, physical examination, ECOG/Karnofsky performance status, ECG and chest X-ray. Baseline clinical evaluations also involved laboratory tests including: (i) Hematology; hemoglobin, hematocrit, WBC with differential and platelet coults, prothrombin and partial thromboplastin times, (ii) Blood Chemistry; blood urea nitrogen (BUN), creatinine, uric acid, biliribin, alkaline phosphatase, lactic dehydrogenase (LDH), total protein, albumin, calcium, phosphorus, blood sugar, serum glutamic-oxaloacetic transaminase [SOGT, (AST)], serum glutamate-pyruvate transaminase [SGPT, (ALT)], (iii) Urinalysis; pH, specific gravity, albumin, glucose, protein, ketones and microscopic (WBC, RBC, casts and bacteria).

Further evaluations involved Quality of Life Assessment, symptom assessment, immune status assessment, delayed hypersensitivity skin test (modified Sokal method) tumor measurements, photography of lesions and recording of previous and concomitant medications. The patient was followed until death to generate survival analysis data.

The patient was male, 33 years of age, fulfilled the inclusion criteria for enrolment into the clinical trial, and was diagnosed as having Kaposi's sarcoma.

During the course of treatment, the patient received 15 intramuscular injections with VIRULIZIN over a 3 week period. On day 5 of this clinical study, this patient also began to receive 150mg BID of Lamivudine (3TC). In addition, starting on day 14, 800mg TID of Indinavir (Ceixivan) was also provided.

Viral Load (Plasma Branch DNA)

Baseline 70,690 copies/mL Day O

Week 2 727 copies/mL Day 14

Week 3 500 copies/mL Day 22

CD4 Counts

Baseline CD4% 2 Abs 38 Day O Week 2 CD4% 1 Abs 13 Day 14

Week 3 CD4% 2 Abs 38 Day 22

Example 19

This example illustrates the activation of monocytes and macrophages with the composition of Example 1 and methods for testing same.

Investigations have shown that the composition of Example 1 will activate normal monocytes to demonstrate cytotoxicity towards the Chang hepatoma cell line, which is used to measure monocyte toxicity, and that the monocytes and macrophages from cancer patients (e.g., those afflicted with cancers of the cervix, ovaries, ear/nose/throat, and endometrium/uterus, and chronic myelogenous leukemia) have been stimulated by the composition to attack and destroy tumor cells derived from the same patient.

More particularly, the monocyte tumor icidal function has been tested in the presence of the composition of the invention and the basic procedure for these experiments is outlined below. This procedure has been named the "Monocyte/Macrophage Cytotoxicity Assay to Cell Lines and Autologous Tumor Cells," or "Cytotoxicity Assay" for short.

The method requires isolation of monocytes/macrophages, which is accomplished as follows: Venous blood is collected aseptically in heparinized Vacutainer tubes. Sterile preservative-free heparin is added to a final concentration of 20 units/ml. The blood is diluted 3: 1 in Hanks balanced salt solution (HBSS), layered onto lymphocyte separation medium and centrifuged to obtain a band of peripheral blood mononuclear cells (PBMNs). After centrifugation, the mononuclear cell layer is recovered from the interface, washed twice in medium (medium is Roswell Park Memorial Institute [RPMI] 1640 media supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin) and monocytes are enumerated by latex ingestion. Monocytes are isolated by adherence in 96-well plastic plates (for 2 hours at 37° C, followed by two cycles of washing with medium). Adherent cells are estimated to be greater than 90% monocytes. Wells containing adherent cells are incubated overnight in the presence of VIRULIZIN™ (1: 10-1:200 final dilution). Then, adherent cells are washed to remove VIRULIZIN™ and incubated overnight with tumor cells. The tumor cells are maintained in medium in which endotoxin concentration is guaranteed by the manufacturer to be low and is non-stimulatory in the assay.

For studies using a standard cell line, ⁵¹Cr (chromium) labelled Chang hepatoma cells are used because this cell line is insensitive to natural killer cell cytotoxicity. These hepatoma target tumor cells are added to adherent cell monolayers at effector: target (E:T) cell ratios of 20: 1 to 15: 1. This E:T ratio is used because it falls well into the plateau range on a curve prepared by varying the E:T ratio from 5: 1 to 30: 1. After 24 hours, supernatants are collected and ⁵¹Cr release is quantitated. The percent specific cytotoxicity is calculated as:

E - S z. specific release - x 100

T - s

In the equation above, E = CPM released from 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 2% sodium dodecyl sulfate).

For studies using autologous tumor cells, these cells are obtained from surgical biopsies, labelled with ⁵¹C, and used in the same way as the hepatoma cells described above.

Preparation of peritoneal and alveolar macrophages is done by the methods described in Braun et al., Cancer Research. 53, 3362-3365 (1993).

Using this protocol, the composition was found to cause monocytes from healthy donors to exert cytotoxicity toward the Chang hepatoma cell line. Subsequently, whether monocytes and macrophages from a cancer patient could be stimulated by the composition to attack and destroy their own particular tumor was investigated. Using similar protocols as described for the standard cell line (Chang hepatoma cells), monocytes and/or peritoneal macrophages from cancer patients were isolated. Peritoneal macrophages were isolated from peritoneal fluids collected at the time of laparoscopy. The composition was found to activate peripheral monocytes and peritoneal macrophages from a patient with cervical cancer to produce cytotoxicity against the patient' s own tumor cells. This effect was comparable to or better than that produced by the combination of IFN and LPS. Peritoneal macrophages from a patient with ovarian cancer were also found to be stimulated by the composition to attack and destroy the ovarian tumor cells in culture.

Monocyte/Macrophage Studies with the Composition

Because the screening procedures demonstrated that the composition does not stimulate lymphocyte functions but can stimulate monocyte functions, subsequent studies were aimed at further characterization of the monocyte/macrophage stimulatory activities of the composition. A number of comparative studies aimed at determining the dose response characteristics of the composition in stimulating monocyte/macrophage tumoricidal function were performed as well as testing different batches of the compound. The main emphasis of the studies was to test the capacity of the composition to simulate tumoricidal function in monocytes and macrophages from different anatomical sites of cancer patients. For these investigations, the following were relied upon: (1) peripheral blood monocytes from cancer patients and control subjects; (2) alveolar macrophages from lung cancer patients and control patients with non-malignant lung diseases; and (3) peritoneal macrophages from patients with gynecological malignancies.

Dose response studies with different batches of the composition, all prepared in accordance with Example 1, were completed. These studies relied on peripheral blood monocytes to test the stimulatory activities of different doses and different batches of the composition (Batch nos. 216, 219 and 222). Each batch of the composition was tested without dilution (neat), a 1 : 10 dilution and a 1:50 dilution of material. The results are depicted graphically in Figure 14.

Batch #222 and #216 were shown to stimulate monocyte tumoricidal function, however, Batch #219 did not. It appeared that #222 was superior to #216 in these preliminary investigations. Batch #222 appeared to stimulate equivalent levels of tumoricidal function at the undiluted (neat) and 1: 10 dilutions, but lesser, still detectable activity at the 1:50 dilution. Batch #216 gave the greatest stimulation of tumoricidal function at the undiluted (neat) concentration, with less activity at the 1:10 dilution and no detectable activity at the 1:50 dilution. As stated above, Batch #219 did not elicit detectable monocyte tumoricidal function at any concentration tested. Tumoricidal function in peripheral blood monocytes was also evaluated. Tests were performed on 4 peripheral blood monocyte samples from control subjects. These tests utilized an optimal stimulating concentration of the composition (1: 10 dilution of batch #222) and an optimal stimulating concentration of IFN-γ plus LPS. The target cells in these studies were a cultured, NK-insensitive cell line, namely the Chang Hepatoma. Results are presented in the following table.

Stimulant (E/T-20/1) % Cytotoxicity

Medium 5.4 ± 1 IFN-γ + LPS 18.6 ± 4 Composition 22.3 ± 6

A test was also performed on 1 monocyte sample 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 assay. As before, this test utilized an optimal stimulating concentration of the composition (1:10 dilution of Batch #222) and an optimal stimulating concentration of IFN-γ plus LPS. Also, the effector/target cell ratio was reduced to 15/1 to conserve patient tumor cells. Results of this test are presented in the following table.

Stimulant (E/T-20/1) % Cytotoxicity

Medium 5.5 IFN-γ + LPS 14.4 Composition 20.9

In the peripheral blood monocytes from control subjects, the composition stimulated monocyte tumoricidal function against the Chang Hepatoma cells at a level equal to or greater than the level elicited by an optimal stimulating concentration of IFN-γ + LPS. In the peripheral blood monocytes from a patient with cervical cancer, the composition stimulated tumoricidal function against the patient's own tumor cells at a level which exceeded that elicited by IFN-γ plus LPS by greater than 30%.

Tumoricidal function in peritoneal macrophages from patients with gynecological malignancies was tested. These tests were performed on peritoneal macrophage samples isolated from lavage fluids of 1 patient with cervical cancer and 1 patient with ovarian cancer. These tests^{^}were performed with the patient's own tumor cells as target cells in the assay. As before, an optimal stimulating concentration of the composition (1 :10 dilution of Batch #222) and an optimal stimulating concentration of IFN-γ plus LPS were compared. Also, the effector/target cell ratio was reduced to 15/1 to conserve patient tumor cells. The resulting data were:

Stimulant Cervical Cancer Ovarian Cancer Medium 8.2 0.6 IFN + LPS 29.8 4.1 Composition 13.2 8.9

These test results highlighted the fact that the local tumor environment may be a determinant of the response of immune cells to immunological activators. In this case of cervical cancer, there was no pathological evidence of malignant disease within the peritoneal cavity and the development of tumoricidal function against the autologous tumor was better with IFN-γ and LPS combined 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 to IFN-γ and LPS combined was minimal at best, whereas the response to the composition was greater.

Tumoricidal function in alveolar macrophages from lung cancer patients and control subj ects was tested. These tests were performed on alveolar macrophage samples isolated from bronchoalveolar lavage fluids of a patient with non-small cell lung cancer and three (3) patients with non-malignant diseases of the lung. These tests utilized an optimal stimulating concentration of the composition (1 : 10 dilution of batch #222) and an optimal stimulating concentration of IFN- γ and LPS combined. The target cells in these studies were the Chang Hepatoma cells and the effector/target cell ratio was 20/1. The resulting data were:

Stimulant Cancer Patients Control Medium 2.6 ± 2 19.5 ± 4 IFN-γ+LPS 10.9 ± 13 1.2 ± 5 Composition 5.2 ± 2 18.6 --- 8

The results were consistent with the observation that alveolar macrophages from lung cancer pa- tients are impaired in their development of tumoricidal function in response to conventional macrophage activators such as IFN-γ + LPS. The results showed that the tumoricidal function of alveolar macrophages from lung cancer patients is greatly reduced compared to control subjects. The data presented earlier indicated VIRULIZIN™ to be a poor stimulator of alveolar macrophages. Further investigation with alveolar macrophages from non-small cell lung cancer patients is presented in Example 23. The activity in alveolar macrophages appears to vary with the VIRULIZIN™ preparation. Thus, alveolar macrophage cytotoxicity was elicited in only 2/7 alveolar macrophage preparations with the origin batches tested (222, 219, 216). In contrast, 3/4 alveolar preparations were stimulated with the later preparations (233, 238). The difference could be related to age and potency of the preparation or patient variability. Accordingly, the composition can activate tumoricidal activity in alveolar macrophages.

The preliminary in vitro tests with the composition demonstrate that it is a macrophage activator. The material provided was able to elicit tumoricidal activity in a standard cytotoxicity assay against both an NK insensitive cell line and against freshly dissociated human tumor cells. The activity elicited was also found to be concentration-dependent in these tests. The capacity of the composition to active macrophage tumoricidal function in vitro was comparable to that of the best macrophage activating combination presently available, namely, IFN-γ and endotoxin (i.e., LPS) combined. As stated above, the capacity of the composition to elicit this level of tumoricidal function in the absence of endotoxin would be considered important biologically if the material is free of endotoxin contamination. The composition is free of endotoxin contamination when tested for pyrogens by the United States Pharmacoepeia (USP) rabbit pyrogen test.

As has been found for other macrophage activators, the activity of the composition in stimulating macrophage tumoricidal function varies with the source of the macrophages. It appears that the composition is an excellent activator of peripheral blood monocytes being equivalent to IFN-γ + LPS with normal donors and possibly superior to IFN-γ + LPS with cancer patient donors.

Malignant disease has a significant impact on the development of monocyte tumoricidal function depending on the activator used (Braun et al., (1991 )). One determinant of the biological activity of different macrophage activators in cancer patients monocytes is the sensitivity of the activator to arachidonic acid metabolism and the secretion by the cell of prostaglandins. From these initial studies with the composition, it appears that activity elicited with the compound is not sensitive to the inhibitory effects of prostaglandins. If prostaglandin insensitivity can be proven definitively for cancer patient monocytes stimulated with the composition, this would be considered important therapeutically because the effectiveness of many other biological activators is limited by prostaglandins. Preliminary studies with 2 specimens indicate that the composition may have good activity in peritoneal macrophages, particularly when malignant disease is present in the peritoneal cavity.

These preliminary results also illustrate what has been found when comparing the capacity of different activators to stimulate tumoricidal function in peritoneal macrophages of patients with different gynecological malignancies. In those studies, it was found that the presence of malignant disease within the peritoneal cavity influences the responsiveness of the peritoneal macrophages to specific activators. In patients with cervical cancer, malignant disease is not present in the peritoneal cavity in general, and thus, the response of the resident macrophages to IFN-γ + LPS is normal. When disease is present in the cavity, however, as in the case with ovarian cancer, the response to lFN-γ + LPS is suppressed. This is related, in part, to changes in the arachidonic acid metabolism of the peritoneal macrophages when malignant disease is present (Braun et al., 1993). The fact that the composition activates tumoricidal function in peritoneal macrophages from ovarian cancer patients against the patient's own tumor cells is consistent with a mechanism for activation that is independent of the arachidonic acid metabolic pathway.

Accordingly, as shown in the aforestated in vitro studies, the composition of the present invention is able to activate monocytes and macrophages to increase their immune system function.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An antiviral cocktail comprising:

(i) a composition comprising small molecular weight components of less than 3000 daltons, and having the following properties: a) is extractable from bile of animals; b) is capable of stimulating monocytes and macrophages in vitro; c) is capable of modulating tumor necrosis factor production; d) contains no measurable level of IL-la, IL-lb, TNF, IL-6, IL-8, IL-4, GM- CSF or IFN-gamma; e) shows no cytotoxicity to human peripheral blood mononuclear cells; f) is not an endotoxin; and

(ii) an effective amount of one or more antiviral agent(s).

2. The antiviral cocktail of claim 1, as characterized wherein the composition or antiviral agent(s) have a specific activity greater than that of the composition or antiviral agent(s) alone.

3. The antiviral cocktail of claim 1, wherein said antiviral agent(s) are selected from the group comprising 3TC, interferon, ganciclovir, famciclovir, rimantadine, foscarnet sodium, zidovudine, amantadine hydrochloride, valacyclovir, ribavirin and acyclovir.

4. A method for diminishing the viral load in a host infected with a virus, comprising the administration of the antiviral cocktail of claim 1.

5. An antiviral pharmaceutical composition comprising, as an active ingredient, an effective antiviral amount of the cocktail of claim 1 and a non-toxic pharmaceutically acceptable carrier of diluent.

6. The antiviral pharmaceutical composition of claim 5, formulated into a sterile solution, a lyophilate, pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, and tubelets.

7. The use of the antiviral pharmaceutical composition of claim 5, to diminish the viral load in a host infected with a virus, comprising the administration of said pharmaceutical composition by means of oral, topical, rectal, parenteral, local, inhalant, or intracerebral delivery.

8. The use according to claim 7, wherein said parenteral delivery is achieved via intramuscular injection.

9. The use according to claim 7, wherein said virus is a retrovirus.

10. The use according to claim 9, wherein said retrovirus is human immunodeficiency virus.

11. The use according to claim 7, wherein said host is human.

12. A method for diminishing the viral load in a host infected with a virus, comprising the administration of an antiviral effective amount of a composition comprising small molecular weight components of less than 3000 daltons, and having the following properties: a) is extractable from bile of animals; b) is capable of stimulating monocytes and macrophages in vitro; c) is capable of modulating tumor necrosis factor production; d) contains no measurable level of IL-la, IL-lb, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-gamma; e) shows no cytotoxicity to human peripheral blood mononuclear cells; and f) is not an endotoxin.

13. The method of claim 12, wherein said antiviral effective amount of the composition additionally comprises a non-toxic pharmaceutically acceptable carrier of diluent.

14. The method of claim 12, wherein said composition is formulated into a sterile solution, a lyophilate, pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, and tubelets

15. The method of claim 12, said administration is achieved by means of oral, topical, rectal, parenteral, local, inhalant, or intracerebral delivery.

16. The method of claim 12, wherein said parenteral administration is achieved via intramuscular injection.

17. The method of claim 12, wherein said virus is a retrovirus.

18. The method of claim 17, wherein said retrovirus is human immunodeficiency virus.

19. The method of claim 12, wherein said host is human.

20. The method of claim 12, wherein the viral load is diminished by stimulating peripheral blood monocytes and/or tumor associated macrophages to express cytocidal activity in a manner that is insensitive to the inhibitory effects of prostaglandins.

21. The method of claim 12, wherein the viral load is diminished by eliciting suitable modulation of the immune system in a patient in need of such modulation by activating macrophages and/or monocytes to produce cytokines or promote activity to seek and remove or destroy disease-causing viruses or cells negatively affected by such viral infections.

22. The method of claim 12, wherein the viral load is diminished by stimulating the release of TNF, Il-lβ and GM-CSF.

23. A use of a composition to diminish the viral load in a host infected with a virus, comprising administering an effective amount of said composition, extracted from bile, comprising at least one of the following compounds: (a) a compound of the formula

where the bonds between A-B, B-C, and C-D may be single or double bonds, and where x=H, OH, =O, or OSO₃H; and Y=

where R is an amino acid residue; (b) a compound of the formula

(R¹0) CH₂CH (OR² ) CH₂ (OR³ -X) or

where R\ R² and R³ are H, COR⁴, CH=CH-R\ X, P(O)(OH)O-, or -S(O)₂O- X is choline, ethanolamine, N-alkylated ethanolamines, serine, inositol, sugars bearing free hydroxyls, amino-sugars, sulfonated sugars, or sialic acids; and

R⁴ is a saturated or unsaturated alkyl group having a carbon chain from about C, to C₃₀, or oxidized and hydroxylated analogs thereof; and

R⁵ is an alkyl group or oxidized and hydroxylated analogs thereof;

(c) a mucin hydrolysis product or a proteoglycan hydrolysis product; or

(d) a fat-soluble vitamin, with the proviso that retinol and retinol derivatives are not included.

24. The use according to claim 23, wherein said composition comprises at least one compound selected from the group consisting of taurocholic acid and its sulphated derivatives; glycocholic acid and its sulphated derivatives; sphingosine; a diacyl glycerol; phosphocholine; glucosamine-3-sulfate; glycero-phosphocholine; phosphoryl choline chloride; lecithin; an oligosaccharide of less than 10 saccharide units in length, where said oligosaccharide is comprised of sialic acid, fucose, hexosamines, or sulphated hexosamines; taurine; and glutamic acid and its conjugates.

25. The use according to claim 23, wherein said composition additionally comprises at least one compound selected from the group consisting of ammonia; primary alkyl amines; secondary alkyl amines; tertiary alkyl amines; and a carboxylic acid R⁶CO₂H, wherein R° is -C_JQ alkyl, saturated or unsaturated, and oxidized or hydroxylized derivatives thereof.

26. The use according to claim 23, wherein said composition comprises at least one compound selected from the group consisting of taurocholic acid and its sulphated derivatives; glycocholic acid and its sulphated derivatives; sphingosine; a diacyl glycerol; phosphocholine; glucosamine-3-sulfate; glycero-phosphocholine; phosphoryl choline chloride; lecithin; an oligosaccharide of less than 10 saccharide units in length, where said oligosaccharide is comprised of sialic acid, fucose, hexosamines, or sulphated hexosamines; Vitamin A; retinolic acid derivatives; taurine; and glutamic acid and its conjugates.

27. The use according to claim 23, wherein said virus is a retrovirus.

28. The use according to claim 27, wherein said retrovirus is human immunodeficiency virus.

29. The use according to claim 23, wherein said host is a human.