+

WO1998052585A1 - Compositions immunomodulatrices pouvant etre derives de la bile pour le traitement des troubles d'origine virale - Google Patents

Compositions immunomodulatrices pouvant etre derives de la bile pour le traitement des troubles d'origine virale Download PDF

Info

Publication number
WO1998052585A1
WO1998052585A1 PCT/CA1998/000494 CA9800494W WO9852585A1 WO 1998052585 A1 WO1998052585 A1 WO 1998052585A1 CA 9800494 W CA9800494 W CA 9800494W WO 9852585 A1 WO9852585 A1 WO 9852585A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
tnf
acid
bile
macrophages
Prior art date
Application number
PCT/CA1998/000494
Other languages
English (en)
Inventor
Paul Percheson
Original Assignee
Lorus Therapeutics Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Lorus Therapeutics Inc. filed Critical Lorus Therapeutics Inc.
Priority to AU75160/98A priority Critical patent/AU7516098A/en
Publication of WO1998052585A1 publication Critical patent/WO1998052585A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/413—Gall bladder; Bile
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]

Definitions

  • 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.
  • 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 .
  • 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.
  • 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.
  • a virus infection 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.
  • viruses 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.
  • 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.
  • RNA viruses can cause cancer and are known as tumor viruses.
  • the viral chromosome 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.
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • 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.
  • 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.
  • 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.
  • determination of the viral load might be the only manner of revealing the progress of the infection.
  • One approach is based on the antigen-specific elements of the immune system, namely antibodies and T-cells.
  • the antigen-specific elements of the immune system namely antibodies and T-cells.
  • 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.
  • 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.
  • TNF tumor necrosis factor
  • IFN interferon
  • TNF was originally termed "cachectin” because of its ability to produce the wasting syndrome cachexia. It is composed of two related proteins: mature TNF (TNF ⁇ ) and lymphotoxin (TNF ⁇ ), which are primarily produced by activated macrophages, monocytes and lymphocytes. Both TNF ⁇ and TNF ⁇ are recognized by the same cell surface receptor. TNF ⁇ 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, 72 ⁇ 3666, 1975).
  • TNF ⁇ 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 TNF ⁇ has also been described (Kriegler, et al, Cell, 53:45, 1988).
  • TNF ⁇ The expression of the gene encoding TNF ⁇ is not limited to cells of the monocyte/macrophage family. Several human non-monocytic tumor cell lines were shown to produce TNF ⁇ . 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.
  • TNF ⁇ is a regulatory cytokine with pleiotrophic biological activities. These activities include: inhibition of lipoprotein lipase synthesis, activation of polymo ⁇ honuclear leukocytes, inhibition of cell growth or stimulation of cell growth, cytotoxic action on certain transformed cell lines, antiviral activity, stimulation of bone reso ⁇ tion, 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.
  • TNF 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.
  • 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 pu ⁇ oses. 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.
  • rheumatoid arthritis 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 reso ⁇ tion disease, reperfusion injury, graft v.
  • 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.
  • cytokine production notably TNF
  • 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.
  • cytokine production notably TNF
  • TNF (U.S. Patent Nos. 5,563,143 and 5,506,340) in the treatment of AIDS.
  • TNF and IFN individually possess antiviral activity, making them potential candidates in the treatment of viral infections and tumors.
  • 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- ⁇ 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;
  • 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- ⁇ , IL- 1 ⁇ , 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 pu ⁇ oses, 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)).
  • bile has never been considered to be a source of therapeutically useful compositions with respect to neoplastic, inflammatory or infectious diseases.
  • 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 abso ⁇ tion of endotoxin from the gut.
  • 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.
  • 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-l ⁇ , IL-1 ⁇ , TNF, IL-6, IL-8, IL-4, GM-CSF or IFN- ⁇ ; e) shows no cytotoxicity to human peripheral blood mononuclear cells or lymphocytes; and f) is not an endotoxin.
  • 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.
  • 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.
  • FIG. 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
  • 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
  • Figure 9 is a graph showing dose response of the composition of the invention in stimulating peripheral blood monocyte function.
  • VIRULIZINTM A new biologic response modifier called VIRULIZINTM has been in development by IMUTEC Co ⁇ oration, a Toronto-based biopharmaceutical company, since 1986.
  • VIRULIZINTM 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.
  • 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 "VIRULIZINTM” or "VIRULIZINTM-2- beta". IMUTEC Co ⁇ oration now only manufactures VIRULIZINTM-2 / .
  • VIRULIZINTM-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 VIRULIZINTM have revealed following:
  • VIRULIZINTM does not directly stimulate lymphocytes to synthesize DNA or undergo blastogenesis and cell division. VIRULIZINTM does not directly stimulate the development of lymphocyte-mediated cytotoxicity.
  • VIRULIZINTM can stimulate normal peripheral blood monocytes to express cytocidal activity in a dose-dependent manner.
  • the activity elicited by VIRULIZINTM 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.
  • VIRULIZINTM 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. VIRULIZINTM 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 VIRULIZINTM can often stimulate cancer patient macrophages that are unresponsive to stimulation with conventional activators such as gamma interferon + endotoxin.
  • VIRULIZINTM can stimulate cytocidal function in macrophages obtained from cancer patients (including pancreatic cancer) who are undergoing cytotoxic therapy.
  • cancer patients including pancreatic cancer
  • VIRULIZINTM was more effective in stimulating tumouricidal function than conventional activators such as gamma interferon plus endotoxin.
  • VIRULIZINTM appears to have the potential to be combined with cytotoxic cancer treatment in appropriate clinical settings.
  • VIRULIZINTM 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 VIRULIZINTM appears to be independent of the need for collaboration with other immune cell types including helper T-lymphocytes.
  • 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.
  • 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.
  • 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.
  • 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-l ⁇ , IL- 1 ⁇ , TNF, IL-6, IL-8, IL-4, GN-CSF or IFN- ⁇ ; 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.
  • TNF tumor necrosis factor
  • 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 composition was found to be non-cytotoxic to human PBMNs and lymphocytes.
  • composition of the present invention has, among others, the following characteristics:
  • composition causes the release of interleukin- 1 ⁇ (IL- 1 ⁇ ), and the component responsible for the IL-l ⁇ release elutes early from RP-HPLC, suggesting that it is likely the same substance(s) that releases TNF.
  • IL- 1 ⁇ interleukin- 1 ⁇
  • component responsible for the IL-l ⁇ release elutes early from RP-HPLC, suggesting that it is likely the same substance(s) that releases TNF.
  • composition also causes the release of low quantities of interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • 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.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • 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.
  • 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.
  • Further physicochemical separation by hydrophilic (polyhydroxy ethyl) molecular sieve HPLC confirms the small molecular weight of the components in the composition.
  • Amino acid analysis before and after acid hydrolysis suggest the presence of peptide bonds, indicating the presence of peptides.
  • 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;
  • 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.
  • Bovine bile from bovine sources is utilized.
  • Bovine bile is plentiful, because, in part, relatively large quantities can be extracted from each animal.
  • bovines are routinely slaughtered and inspected under health-related regulations, thus such animals provide a reliable source for preparing the composition of the invention.
  • humans are less likely to have an allergic reaction to material of bovine origin.
  • 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.
  • 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.
  • the bile/alcohol mixture is centrifuged at 3000-5000 rpm, most preferably 4200 ⁇ m, 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.
  • activated charcoal 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.
  • the clear, yellowish liquid is filtered using a filter having about a 1.0-3.5 ⁇ m retention, most preferably a retention of 2.5 ⁇ m.
  • 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.
  • 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.
  • 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.
  • the collected bile is mixed with an equal volume of ethyl alcohol.
  • the bile/alcohol mixture is then centrifuged at about 4200 ⁇ m 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.
  • the solution is filtered through a filter having a 2.5 ⁇ m 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.
  • 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.
  • compositions 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.
  • 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.
  • compositions likely to be present in the present composition 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
  • R is an amino acid residue, such as, for example, glycyl, glutamyl, or tauryl, thereby forming the glycine, glutamyl, or taurine conjugate.
  • 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).
  • MS mass spectroscopy
  • 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.
  • the MS analysis also supports the identification in the present composition of phospholipids, sphingolipids and related agents capable of forming miscelles.
  • 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
  • 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;
  • R 4 is C ] -C 30 alkyl that is saturated or unsaturated, oxidized or hydroxylated; and
  • R 5 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'R 2 , where R' and R 2 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:
  • mucin and proteoglycan hydrolysis product compounds thought to be present include: sialic acids and their mono and diacetylated and glycolylated monomers;
  • N-acetylneuraminic acid N-acetylneuraminic acid
  • hexosamines such as glucosamine
  • 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.
  • 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.
  • 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, ⁇ -amino adipic acid, citrulline, valine, isoleucine, ⁇ -alanine, ⁇ -amino butyric acid, hydroxylysine, ornithine, and lysine; bilirubin, and its gluconuride conjugate; biliverdin, and its gluconuride conjugate; butylatedhydroxy toluene (BHT); polyurea, including methylamine, dimethylamine, ethylamine, methylethy
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the composition is administered intramuscularly.
  • 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).
  • 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.
  • compositions are indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment.
  • 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.
  • 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.
  • 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.
  • the concentrated composition is used, approximately half the above mentioned dosages may be used.
  • 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.
  • the present invention comprises a process for preparing an antiviral composition
  • 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.
  • 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;
  • compositions are 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-l ⁇ , IL-l ⁇ , TNF, IL-4, IL-6, IL-8, GM- CSF or IFN- ⁇ .
  • Such compositions may be obtained from the bile of animals, preferably bovines, or from other sources as noted above.
  • the composition stimulates tumor necrosis factor production in vitro or in vivo, and most preferably in humans, in the absence of exogenous IL-l ⁇ , IL-l ⁇ , TNF, IL-4, IL-6, IL-8, GM-CSF, and IFN- ⁇ .
  • 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.
  • 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.
  • a suitable solvent system such as 10% concentrated ammonium hydroxide in methanol
  • 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
  • X is choline, ethanolamine, N-alkylated ethanolamines, serine, inositol, sugars bearing free hydroxyls, amino-sugars, sulfonated sugars, or sialic acids;
  • R 4 is a saturated or unsaturated alkyl group having a carbon chain from about C j to C 30 , or oxidized and hydroxylated analogs thereof;
  • R 5 is an alkyl group or oxidized and hydroxylated analogs thereof
  • 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 R 6 CO 2 H, wherein R 6 is C,-C 30 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
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m 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 ⁇ m 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.
  • the treated liquid filtered through a filter having, for example, a 2.5 ⁇ m 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 ⁇ m 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.
  • 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.
  • 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.
  • the inventive composition can be prepared from readily available sources of bile, using standard laboratory methods, resulting in a standardized final product.
  • This example describes the biological activity of the composition of Example 1.
  • Example 1 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).
  • PBMN peripheral blood mononuclear cells
  • U937 cells which is a stable line of pre-monocyte cells (American Type Culture Collection (ATCC), Rockville, Maryland).
  • ELISA assays for TNF- ⁇ , IL-la, IL-2, IL-4, IL-6, IL-8, GM-CSF and IFN were conducted.
  • a 0.5 ml aliquot of the PBMN suspension was added to each well, which contained 50 ng lipopolysaccharide (LPS) (from E. coli), 10 ⁇ l fetal calf serum and 10-300 ⁇ l of the composition of Example 1, as noted in the tables below.
  • LPS lipopolysaccharide
  • 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 % CO 2 incubator.
  • 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.
  • immunoassays such as ELISA
  • Cytokine synthesis in the supernatants was measured after stimulating human PBMN with the composition of Example 1 at volumes of 100 and 200 ⁇ l 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.
  • IL-l ⁇ Endogen, Inc.
  • GM-CSF Endogen, Inc.
  • RFN- ⁇ Endogen, Inc.
  • IL-2 Advanced Magnetics, Inc.
  • IL-6 Advanced Magnetics, Inc.
  • IL-1 Advanced Magnetics, Inc.
  • IL-4 R&D Systems
  • IL-8 R&D Systems
  • a 40 ⁇ l dose of the composition of Example 1 stimulated the production and release of 178 pg/ml of TNF- ⁇ , 136 pg/ml GM-CSF, and 142 pg/ml of IL-l ⁇ .
  • Table III Effect of Composition of Example 1 on LPS-induced Release of TNF from PBMNs Batch Composition Volume ( ⁇ l) TNF (pg/ml)
  • 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 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.
  • 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.
  • the PBMN-TNF assay as described above was standardized using 100 ⁇ l 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 were averaged to compensate for variations in response between different subjects.
  • the analysis involved determining the amount of TNF- ⁇ released in RPMI media alone and in the presence of 50 ng LPS.
  • the TNF- ⁇ released in the presence of 100 ⁇ l of the composition of Example 1 in combination with 50 ng LPS was also determined.
  • the TNF- ⁇ released in media was subtracted from the LPS value to obtain the TNF- ⁇ 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- ⁇ 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.
  • 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 ⁇ g/ml Gentamycin Sulfate (SIGMA, Mississauga, Ontario, Canada) at 37°C, 5% CO 2 .
  • FCS heat-inactivated fetal calf serum
  • FCS heat-inactivated fetal calf serum
  • ICN Biomedical Inc Costa Mesa, CA
  • 10 ⁇ g/ml Gentamycin Sulfate SIGMA, Mississauga, Ontario, Canada
  • 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).
  • PMA phorbol 12-myristate 13-acetate
  • 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 ⁇ M and stored at -20° C.
  • DMSO dimethyl sulfoxide
  • 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 10 6 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.
  • Example 1 After 72 hrs of incubation, 120 ⁇ l of media were removed and replaced by 100 ⁇ l of the composition of Example 1 and 10 ⁇ l of sterile deionized distilled water, in the presence or absence of 10 ⁇ l of LPS (5 ng/ ⁇ l). 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- ⁇ . All the Virulizin samples were tested on two separate occasions.
  • TNF- ⁇ ELISA kits purchased from Endogen, Inc. (Cedarlane Laboratories, Hornby, Ontario). The protocol recommended by the manufacturer was used. Briefly, 100 ⁇ l of TNF- ⁇ standards and test samples were added to antihuman TNF- ⁇ pre-coated 96-well plates and incubated at 37° C, 5 % CO 2 for 3 hrs. After extensive washing with washing buffer, 100 ⁇ l of antihuman TNF- ⁇ conjugated to alkaline phosphatase were added to plates and incubated at 37°C, 5% CO 2 for 2 hrs.
  • the plates were washed as described above and 100 ⁇ l 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 ⁇ l 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
  • Example 1 This example describes the physical, chemical and biochemical characteristics of the composition of Example 1.
  • 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.
  • Example 1 A tandem column reverse-phase HPLC method was used to characterize the composition of Example 1.
  • 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.
  • Buffer A and Buffer B 0.1 % TFA in 100% acetonitrile
  • 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.
  • Samples A-F were also assessed for presence of bovine DNA.
  • the samples were examined utilizing a 32 P-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.
  • ⁇ det means below level of detection.
  • Titanium ⁇ 0.003 ⁇ 0.003 0.006 0.005 O.003 0.004
  • Sample D was analyzed for sulfate ions before and after acid hydrolysis. Using whole sample D (i.e., imfractionated), the nonhydrolyzed sample yielded 1000 ⁇ M sulfate, whereas the hydrolyzed sample yielded 1200 ⁇ M sulfate. Since the sulfate ion concentration increased after acid hydrolysis, these results suggest that 20%> of the total sulfate ions present are sulfate esters.
  • Example 1 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 1 describes the physical, chemical, and biological properties of a number of earlier batches of the composition of Example 1.
  • ND means not detectable, thus less than 0.5 ⁇ g/ml lipids per and/or less than 1.0 ⁇ g/ml high molecular weight polypeptide. NA means not assayed.
  • 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
  • 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.
  • 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 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.
  • composition (#222) 1,116 Composition (1:10) 1,021 Composition (1 :50) 649
  • 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.
  • 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).
  • Stimulation of monocyte-mediated cytotoxic function by the composition comparison with IFN- ⁇ and LPS (IFN + LPS)
  • Example 1 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- ⁇ 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 1 illustrates the results of assays conducted to survey what, if any, cytokines are present in the composition of Example 1.
  • a bile extract 50 ⁇ l and 100 ⁇ l 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- ⁇ (Endogen, Inc. (5 pg/ml)); IL-l (Endogen, Inc. (50 pg/ml)); IL-l ⁇ (4.3 pg/ml); GM-CSF (Endogen, Inc. ); RFN- ⁇ (Endogen,
  • IL-2 Advanced Magnetics, Inc.
  • IL-6 Advanced Magnetics, Inc. (7 pg/ml)
  • IFN- ⁇ 5 pg/ml [source]
  • IL-1 Advanced Magnetics, Inc.
  • IL-4 R&D Systems (3 pg/ml)
  • IL-8 R&D Systems (4.7 ng/ml)
  • composition of the invention contained no measurable levels of any cytokine tested, those being TNF- ⁇ , IL-1 ⁇ , IL-1 ⁇ , IL-4, IL-6, IL-8, GM-CSF and IFN- ⁇ , as described in Table XVII.
  • Table XVIII Elisa Determination of Cytokines In Composition
  • This example describes pharmacodynamic studies in mice with the composition of Example 1, including the direct in vitro effect of VirulizinTM as well as the effect of VirulizinTM 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 VIRULIZINTM. 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, VIRULIZINTM induced a slight increase in TNF- ⁇ production (60-
  • VIRULIZINTM in comparison to LPS (2225 pg/ml) was not a strong stimulant of macrophage TNF- ⁇ release. Nitric oxide production was zero.
  • TNF- ⁇ In vivo production of TNF- ⁇ 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 VirulizinTM diluted 1 : 10 in P S .
  • the macrophage monolayers were treated in vitro for 24 hours with IFN- ⁇ (50 ⁇ /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.
  • This example illustrates the results of assays conducted to estimate protein within the composition.
  • 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- ⁇ releasing activity and the TNF- ⁇ 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- ⁇ ; and (3) the hyperosmolarity of the composition is not responsible for TNF- ⁇ releasing activity.
  • Total TNF released is correct for TNF release by 1640 medium.
  • LPS concentration 50 ng/10 ⁇ l.
  • 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- ⁇ 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- ⁇ .
  • 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
  • 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 LD 50 for intramuscular administration of the composition in rats was determined to be greater than 5 ml/kg.
  • mice were injected at day 0 with 5 X 10 3 of B16F1 melanoma cells plus microspheres. On each of the first 17 days, each group received daily injections of VirulizinTM or saline, as above. On day 18, the animals were sacrificed.
  • a 13-week repeat dose toxicity study in Fischer-344 rats (total of 40 males and 40 females) was carried out administering VIRULIZINTM 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.
  • 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.
  • 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 6 ⁇ A 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.
  • TLC Thin layer chromatography
  • 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).
  • 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
  • Fraction 5-6 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.
  • 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.
  • Example 19 resulting in no TNF stimulation activity. Elemental analysis of the above fractions showed them to be high in NH 4 C1, which is known to inhibit TNF production.
  • This example illustrates the application of further methods to fractionate and analyze the active components of the inventive composition.
  • TNF, IL-l ⁇ 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.
  • 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.
  • VIRULIZINTM VIRULIZINTM 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.
  • 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.
  • 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 also analyzed by mass spectroscopy, using the methods described in Example 12.
  • 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.
  • 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.
  • 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
  • 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.
  • BILE ACIDS cholic acid; glycocholic acid; deoxyglycocholic acid; cholesterol sulfate; deoxycholic acid; chenodeoxycholic acid; and taurocholic acid.
  • N-acetylneuraminic acid 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.
  • 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 mono
  • FAT-SOLUBLE VITAMINS Vitamin A2; Vitamin DI; lumisterol (present from its vitamin DI complex);
  • 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, ⁇ -amino adipic acid, citrulline, valine, isoleucine, ⁇ -alanine, ⁇ -amino butyric acid, hydroxy lysine, ornithine, and lysine; butylated hydroxy toluene (BHT); and polyethylene glycol.
  • BHT butylated hydroxy toluene
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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;
  • 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 ⁇ mol/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
  • 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.
  • 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.
  • This example illustrates the activation of monocytes and macrophages with the composition of Example 1 and methods for testing same.
  • 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.
  • cancer patients e.g., those afflicted with cancers of the cervix, ovaries, ear/nose/throat, and endometrium/uterus, and chronic myelogenous leukemia
  • 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).
  • HBSS Hanks balanced salt solution
  • PBMNs peripheral blood mononuclear cells
  • 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 ⁇ g/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 VIRULIZINTM (1: 10-1:200 final dilution). Then, adherent cells are washed to remove VIRULIZINTM 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.
  • medium is Roswell Park Memorial Institute [RPMI] 1640 media supplemente
  • 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 51 Cr release is quantitated. The percent specific cytotoxicity is calculated as:
  • 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).
  • these cells are obtained from surgical biopsies, labelled with 51 C, and used in the same way as the hepatoma cells described above.
  • 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.
  • peripheral blood monocytes from cancer patients and control subjects 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.
  • 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- ⁇ 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.
  • 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- ⁇ + LPS.
  • the composition stimulated tumoricidal function against the patient's own tumor cells at a level which exceeded that elicited by IFN- ⁇ 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- ⁇ plus LPS were compared. Also, the effector/target cell ratio was reduced to 15/1 to conserve patient tumor cells. The resulting data were:
  • alveolar macrophage cytotoxicity was elicited in only 2/7 alveolar macrophage preparations with the origin batches tested (222, 219, 216).
  • 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- ⁇ and endotoxin (i.e., LPS) combined.
  • 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.
  • USP United States Pharmacoepeia
  • 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- ⁇ + LPS with normal donors and possibly superior to IFN- ⁇ + LPS with cancer patient donors.
  • 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.
  • the composition of the present invention is able to activate monocytes and macrophages to increase their immune system function.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Physiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nutrition Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne l'utilisation d'une composition présentant des propriétés antivirales, constituée de composants de petit poids moléculaire inférieur à 3000 daltons, et possédant les propriétés suivantes: a) on peut l'extraire de la bile des animaux; b) elle est capable de stimuler les monocytes et les macrophages in vitro et in vivo; c) elle est capable de moduler la production du facteur de nécrose tumorale; d) elle ne contient pas d'IL-1α, d'IL-1β, de TNF, d'IL-6, d'IL-8, d'IL-4, de GM-CSF ou d'IFN-η mesurable; e) elle ne présente aucune toxicité pour les cellules mononucléées ou les lymphocytes du sang périphérique chez l'homme; et f) ce n'est pas une endotoxine. L'invention concerne également l'utilisation de ladite composition antivirale en association avec d'autres médicaments, tels que des composés antiviraux ou des immunomodulateurs comme l'interféron.
PCT/CA1998/000494 1997-05-23 1998-05-22 Compositions immunomodulatrices pouvant etre derives de la bile pour le traitement des troubles d'origine virale WO1998052585A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU75160/98A AU7516098A (en) 1997-05-23 1998-05-22 Immunomodulating, bile-derivable compositions for the treatment of viral disorders

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA002206047A CA2206047A1 (fr) 1997-05-23 1997-05-23 Compositions immunomodulantes pour le traitement de troubles attribuables aux virus
CA2,206,047 1997-05-23

Publications (1)

Publication Number Publication Date
WO1998052585A1 true WO1998052585A1 (fr) 1998-11-26

Family

ID=4160728

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1998/000494 WO1998052585A1 (fr) 1997-05-23 1998-05-22 Compositions immunomodulatrices pouvant etre derives de la bile pour le traitement des troubles d'origine virale

Country Status (3)

Country Link
AU (1) AU7516098A (fr)
CA (1) CA2206047A1 (fr)
WO (1) WO1998052585A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001045727A3 (fr) * 1999-12-20 2002-06-06 New Pharma Res Sweden Ab Compositions veterinaires
US20050009757A1 (en) * 2001-11-21 2005-01-13 Maurizio Fraziano Immunoregulator compounds
US8394418B2 (en) 2000-11-08 2013-03-12 Erin Mills Biotech Inc. Combination preparation of a biological response modifier and an anticancer agent and uses thereof
CN115414370A (zh) * 2022-10-14 2022-12-02 华中农业大学 牛磺胆酸钠在制备治疗或预防流感病毒感染的药物中的应用
US12186329B2 (en) 2018-08-23 2025-01-07 President And Fellows Of Harvard College Compositions and methods related to cholic acid 7-sulfate as a treatment for diabetes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007089A1 (fr) * 1993-09-09 1995-03-16 Imutec Corporation Compositions immunomodulatrices tirees de la bile
CN1108556A (zh) * 1994-03-28 1995-09-20 李秀彩 一种眼药水的配制方法
CN1114566A (zh) * 1994-07-06 1996-01-10 云南滇东北制药总厂 预防艾滋病及性病的喷雾剂及制法
WO1996028175A1 (fr) * 1995-03-16 1996-09-19 Imutec Pharma Inc. Compositions immunomodulatrices d'origine biliaire destinees au traitement de troubles du systeme immunitaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995007089A1 (fr) * 1993-09-09 1995-03-16 Imutec Corporation Compositions immunomodulatrices tirees de la bile
CN1108556A (zh) * 1994-03-28 1995-09-20 李秀彩 一种眼药水的配制方法
CN1114566A (zh) * 1994-07-06 1996-01-10 云南滇东北制药总厂 预防艾滋病及性病的喷雾剂及制法
WO1996028175A1 (fr) * 1995-03-16 1996-09-19 Imutec Pharma Inc. Compositions immunomodulatrices d'origine biliaire destinees au traitement de troubles du systeme immunitaire

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ANON.: "Virulizin Trial for Kaposi's Sarcoma", AIDS PATIENT CARE AND STANDARDS, vol. 11, no. 1, February 1997 (1997-02-01), pages 37 - 38, XP002079172 *
BABA ET AL: "Selective Activity of Several Cholic Acid Derivatives Against HIV", JOURNL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES, vol. 2, 1989, pages 264 - 271, XP002046179 *
DATABASE WPI Week 9733, Derwent World Patents Index; AN 97-351773, XP002079178 *
DATABASE WPI Week 9740, Derwent World Patents Index; AN 97-425702, XP002079177 *
LLOYD ET AL: "Cholic Acid Derivatives Against HIV Replication", JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES, vol. 3, no. 290, 1990, pages 290 *
LLOYD ET AL: "Effect of Bile Salts and of Fusidic Acid on HIV-1 Infection of Cultured Cells", LANCET, no. 1, 25 June 1988 (1988-06-25), pages 1418 - 1421, XP002079174 *
MCPHEE ET AL: "Bile Pigments as HIV-1 Protease Inhibitors and their Effects on HIV-1 Viral Maturation and Infectivity In Vitro", BIOCHEMICAL JOURNAL, vol. 320, no. 2, 1996, pages 681 - 686, XP002079176 *
MEYER ET AL: "In Vitro Evaluation of Phosphocholine and Quaternary Ammonium Containing Lipids as Novel Anti-HIV Agents", JOURNAL OF MEDICINAL CHEMISTRY, vol. 34, no. 4, 1991, pages 1377 - 1383, XP002079173 *
THIRLWELL ET AL: "In Vitro Evidence for the Effects of Virulizin on Cytokine Production", CLINICAL AND INVESTIGATIVE MEDICINE, vol. 17, no. s4, 1994, pages b88, XP002079175 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001045727A3 (fr) * 1999-12-20 2002-06-06 New Pharma Res Sweden Ab Compositions veterinaires
US8394418B2 (en) 2000-11-08 2013-03-12 Erin Mills Biotech Inc. Combination preparation of a biological response modifier and an anticancer agent and uses thereof
US20050009757A1 (en) * 2001-11-21 2005-01-13 Maurizio Fraziano Immunoregulator compounds
US8377910B2 (en) * 2001-11-21 2013-02-19 Universita Degli Studi Di Roma “Tor Vergata” Immunoregulator compounds
US9078907B2 (en) 2001-11-21 2015-07-14 Universita Degli Studi Di Roma “Tor Vergata” Immunoregulator compounds
US12186329B2 (en) 2018-08-23 2025-01-07 President And Fellows Of Harvard College Compositions and methods related to cholic acid 7-sulfate as a treatment for diabetes
CN115414370A (zh) * 2022-10-14 2022-12-02 华中农业大学 牛磺胆酸钠在制备治疗或预防流感病毒感染的药物中的应用

Also Published As

Publication number Publication date
CA2206047A1 (fr) 1998-11-23
AU7516098A (en) 1998-12-11

Similar Documents

Publication Publication Date Title
US6596319B2 (en) Immunomodulating compositions for treatment of immune system disorders
US6280774B1 (en) Immunomodulating compositions from bile
Parages et al. Acidic polysaccharides of Arthrospira (Spirulina) platensis induce the synthesis of TNF-α in RAW macrophages
US20080275120A1 (en) Leucine rich composition
US20080279957A1 (en) Immunomodulating compositions from bile
WO1998052585A1 (fr) Compositions immunomodulatrices pouvant etre derives de la bile pour le traitement des troubles d'origine virale
US6551623B1 (en) Immunomodulating compositions from bile
RU2205010C2 (ru) Природные противоопухолевые или противовирусные вещества и их применение
JP2004339220A (ja) 免疫を増強するための医薬組成物、およびポリア抽出物
EP1085909B1 (fr) Complexe d'une alpha-fetoproteine (AFP) et d'un dérivé d'acide polyunsaturée pour le traitement des deficiences immunitaires
JP3519749B2 (ja) Ｉｆｎ産生増強用薬剤
MXPA97007111A (en) Immunomodulating compositions of the bilis for the treatment of disturbances of the inm system
JP3879938B2 (ja) 抗炎症剤
US20060110405A1 (en) Plasma or serum fraction for treatment and prevention of viral infections and related conditions
US20090311236A1 (en) Therapeutic Peptide Compositions And Methods Of Making And Using Same
AU732816B2 (en) Immunomodulation compositions from bile
US20220378870A1 (en) A composition for boosting the immune system

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998549739

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载