KR100209180B1 - Pharmaceutical composition containing acemannan - Google Patents

Abstract

Acemanan has been shown to be effective in treating a number of diseases in which the main mechanism of resolution or treatment requires intervention by the patient's immune system. Acemanan has a direct stimulatory effect on the immune system. Methods of treating cancer, viral diseases, respiratory and immunomodulatory diseases, inflammation, infections and invasion have been described by administering acetylated mannan derivatives such as acemannan derived from aloe. It has been found that the method can be used in tissue culture, animals and plants.

Description

[Name of invention]

Pharmaceutical Compositions Containing Acemannan

[Background of invention]

A. Field of Invention

The present invention relates to the use of biological response modifiers. More particularly, the present invention relates to the therapeutic use of polysaccharide materials which are primarily acetylated mannans or derivatives thereof, which are

1) Alleviate and / or treat viral disease symptoms in animals and plants, including humans, other mammals and birds. These polysaccharide materials, either alone or in combination with other drugs, will inhibit viral replication through direct antiviral action or their immune stimulating activity;

2) Enhances immune system response to carcinoma in humans, other mammals, animals, birds and plants. These polysaccharide materials stimulate the body's immune cells to directly change the surface of the tumor cells so that the stimulated immune cells recognize the tumor cells as not self cells;

3) alters the body's response to antigens, toxins, allergens and autoantigens present in autoimmune diseases. These polysaccharide substances make homeostasis by allowing immune regulatory cells to work more properly;

4) The final step in the removal or treatment of the disease acts as adjuvant therapy with other drugs in a wide range of disease states that require an immune response. These polysaccharide materials can be used with anti-infective, anti-tumor, anti-inflammatory and antidepressant drugs that are not toxic by them. The efficacy of the combination is better than with a single drug alone.

B. Description of General Background Information

Aloe is a member of the family Liliaceae. Aloe vera contains two main liquid sources: yellow latex (exud) and clear gel (viscous). Aloe barbadensis Anhydrous exudates of Miller leaves are referred to as aloes. Commercially available is Curacao Aloe. It consists mainly of aloe, aloe-emodin and phenol [Bruce, South African Medical Journal, 41: 984 (1967); Morrow et al., Archives of Dermatology, 116: 1064-1065 (1980); Mapp et al., Planta Medica. 18: 361-365 (1970); Rauwald, Archives Pharmazie, 315: 477-478 (1982). Many phenolic compounds, including anthraquinones and their glycosides, are known to be pharmaceutically active. Bruce, Execlsa, 5: 57-68 (1975); Suga et al., Comestics and Toiletries. 98: 105-108 (1983).

Viscous jelly from plant tissue cells is referred to as aloe vera gel. In general, no anthraquinone is present that degrades and causes the gel to decolor unless the gel is contaminated by improper processing techniques. Aloe vera gel consists of about 98.5 weight% water. At least 60% of the total solid components consist of polysaccharides from the carbohydrate source. Organic acids and inorganic compounds, in particular calcium oxalate, make up the remainder of the solid component.

Aloe vera has been a traditional medicament as a repellent, laxative, and antagonist in various cultures, and has been used in particular for leprosy, burns and allergic diseases. Cole et. al., Archives of Dermatology and Syphilology, 47: 250 (1943); Chopra et al., Glossary of Indian Medicinal Plants. Council of Scientific and Industrial Research, New Delhi (1956); Ship, Journal of the American Mdical Association. 238 (16): 1770-1772 (1977); Morton, Atlas of Medicinal Plants of Middle American Bahamas to Yucatan. Charles C. Thomas Publisher, 78-80 (1981); Diez-Martinez. La Zabila, Communicado NO. 46 Sobre Recursos Bioticos Potenciales del Pais, INIREB, Mexico (1981); Dastur, Medicial Plants of India and Pakistan: D.B. Taraporevala Sons Co., Private Ltd., Bombay 16-17 (1962).

Aloe vera has been widely characterized in the field of dermatology, especially in the treatment of radiation-induced skin diseases. Mackee, X-rays and Radium in the Treatment of Diseases of the Skin, 3rd Ed., Lea dn Febiger, Philadelphia, 319 -320 (1938); Rovatti et al., Industrial Medicine and Surgery, 28: 364-368 (1959); Zawahry et al., Quotations From Medical Journals on Aloe Research, Ed. Max B. Skuosen, Aloe Vera Research Institute, Cypress, California, 18-23 (1977); Cera et al., Journal of the American Animal Hospital Association. 18: 633-638 (1982). The substance of scientific literature that teaches degradation problems as antiviral agents, fungicides and fungicides and medical applications in gynecological diseases is extensively described in Grindley et al., Journal of Ethnopharmacology, 16: 117-151 (1986). ] Is appropriately considered.

Depending on the way the leaves are processed, viscous substances and sugars are the main components of the dehydrated gel. Sugars found are galactols, glucose, mannose, rhamnose, xylose and uronic acid. There are conflicting opinions, but the slime consists mainly of met or glucomannan. See Eberebu et al., The Chemical Charaterization of Carrisyn. in Preparation); Mandal et al., Carbohydrate Research. 86: 247-257 (1980b); Roboz et al., Journal of the American Chemical Society, 70: 3248-3249 (1948); Gowda et al., Carbohydrate Research. 72: 201-205 (1979); Segal et al., Lipydia, 31: 423 (1968).

Prior to this study, debate persisted about the identity of the active substance (s) in aloe vera. Therefore, it is important to clearly distinguish between the components present in the gel and those found in the exudate. Most gels are viscous, mainly polysaccharides, containing small amounts of various other ingredients. It has been observed that for some activities there may be synergism between the polysaccharide substrate and other components. Leung, Excelsa 8: 65-68 (1978); Henry, Cosmetics and Toiletries, 94: 42-43, 46, 48, 50 (1979). For example, some researchers report that tannins (Frtag, Pharmazie, 9: 705 (1954)) and some types of polysaccharides are effective in wound healing (Kameyama, Wound-healing compositions from Aloe arborescens extracts). . Japanese Patent # 7856995, (1979)].

However, there are many examples in the literature that point out that polysaccharides can exhibit pharmacological and physiological activity without the aid of other ingredients (see Gialdroni-Grassi, International Archieves of Allergy and Applied Immunology, 76 (Supple. 1): 119-127 (1985); Ohno et al., Chemical and Pharmaceutical Bulletin, 33 (6): 2564-2568 (1985); Leibovici et al., Chemico-Biological Interactions, 60: 191-200 (1986); Ukai et al., Chemical and Pharmaceutical Bulletin, 31: 741-744 (1983); Leibovici et al., Anticancer Research, 5: 553-558 (1985). One such example relates to the progression of atherosclerosis. Hyperlipidemia in the general population, especially in hypercholesterolemia families, is associated with coronary heart disease and death. In countries with high dietary fiber intake, atherosclerosis rarely occurs (Trowell et al., Editors, Refined Carbohydrate Foods and Disease, London, Academic Press, 207 (1975)). Pectin and guar have been reported to lower cholesterol in normal and hyperlipidemic patients (Kay et al., American Journal of Clinical Nutrition, 30: 171-175 (1977)). Locust bean gum, a polysaccharide composed of mannose and galactose, lowers plasma lipoprotein cholesterol levels in both normal and household cholesterinemia patients (Zavovor et al., American Journal of Clinical Nutrition, 38: 285-294 (1983)). Addition of guar gum to carbohydrate meals reduces the postprandial increase in glucose in both normal and diabetic patients. JJenkins et al., Lencet. 2: 779-780 (1977). See, Kuhl et al., In Dianetes Care. 6 (2): 152-154 (1983) demonstrate that guar gum can regulate blood glucose levels in pregnant insulin-dependent diabetic patients.

The antitumor activity of polysaccharides has been widely reported. Polysaccharides prepared from Lentinus cyanthiformis are known to elevate host defense against tumors (Rethy et al., Annales Immunologiae Hungaricae, 21: 285-290 (1981)). It has been reported that polysaccharides from mushroom, yeast or bacterial extracts can induce a high level of host defense activity against viral infections and tumorigenesis. Chihara, Nature, 222: 687 (1969); Shwartzman et al., Proceedings of the Society for Experimental Biology and Medicine, 29: 737-741 (1932); Suzuki et al., Journal of Pharmacobio-Dynamics, 7 (7): 492-500 (1984), also discloses polysaccharide capture (GF-1) extracted from the cultured fruiting bodies of the fungus Grifola frondosa. And antitumor activity. This fraction has been shown to inhibit activity at equivalent high levels when administered intraperitoneally (IP), intravenous (IV) and intratumoral (IT). However, oral administration (PO) is not effective. The GF-1 fraction also shows antitumor activity against Meth A fibrosarcoma and MM 46 carcinoma in solid cancer form in mice. Lentinane is a 6-branched chain similar to GF-1 -1-3-linked glucan, which is not effective against Meth A fibrosarcoma. Chihara, The antitumor polysaccharide Lentinan: an overview; Manipulation of Host Defense Mechanisms; Ed. by Aoki et al., Excerpta Medica, North Holland, 1-16 (1981); Sasaki et al., Carbohydrate Research, 47 (1): 99-104 (1976). Synthetic side chain polysaccharides have been reported to have activity on tumors. See Matsuzaki et al., Makromol. Chem., 186 (3): 449-456 (1985). See Matsuzaki et al., Makromol. Chem., 187 (2): 325-331 (1986), met ivory nut met with branched polysaccharides showing significant activity. -(1-4) -D-mannopyranose) and Synthesized from-(1-4) -linked glucomannan. Partial Acetylated Linearity Extracted from Fruiting Body of Dictyophoria indusiata Fish -(1-3) -D-met also exhibited antitumor activity. See Hara. Carbohydrate Research, 143: 111 (1982). -(1-3) -glucan type polymer Because of their higher antitumor activity than-(1-4) -glucan and hemicellulose polymers, antitumor activity appears to depend on the type of polymer backbone and its degree of polymerization. Matsuzaki et al., Makromol . Chem., 187: 325-331 (1986). Obtained from bacterial culture filtrate Carboxymethylated derivatives of-(1-3) -glucan caused significant cell loss from sarcoma 180 tumors established within 2 hours after injection. Baba, Journal of Immunopharmacology, 8 (6): 569-572 (1986) ]. The authors observed a compensatory increase in polymorphonuclear leukocytes due to injection of the material. At the same time, bestatin, a dipeptide known to have immunomodulatory and anti-tumor activity (Ishizuka, Journal of Antibiotics, 32: 642-652 (1980)), influences tumor yield and polymorphonuclear leukocyte count. (See Baba et al., Supra).

Heparin [Jolles et al., Acta Univ. Int. Cancer, 16: 682-685 (1960); Suemasu et al., Gann. 61 (2): 125-130 (1970)], sulfated laminaran and dextran (Jolles et al., British Journal of Cancer. 17: 109-115 (1963), including a number of reports of antitumor effects of sulfated polysaccharides. Yamamoto et al., In Japanese Journal of Experimental Medicine, 54: 143-151 (1984) describe the improvement of antitumor activity of fucoidan fractions by further sulfation. Sulfation products have been demonstrated to be active against L-1210 leukemia. Polysaccharides with sulfate groups are also reported to be human T cell mitotics and murine polyclonal B cell activators (Sugawara et al., Microbiological Immunology, 28 (7): 831-839 (1984)). In general, high molecular weight homo-polysaccharides with sulphate groups have these properties (Dorries, European Journal of Immunology, 4: 230-233 (1974); Sugawara et al., Cell Immunology, 74: 162-171 (1982).

It has been reported that glucans extracted from yeast Saccharomyces cerevisiae are modulators of cellular and exotropic immunity (Wooles et al., Science, 142: 1078-1080 (1963)). Polysaccharides also stimulate the proliferation of pluripotent hematopoietic stem cells, granulocyte macrophage colony-forming cells and bone marrow and erythrocyte colony forming cells in mice (Pospisil et al., Experientia, 38: 1232-1234 (1982); Burgaleta, Cancer Research, 37: 1739-1742 (1977). Maisin et al., Radiation Research, 105: 276-281 (1986) also found that intravenous administration of polysaccharides protected rat hematopoietic stem cells against X-ray exposure, resulting in mortality in such exposed mice. Is reported to be reduced.

Lackovic et al., Proceedings of the Society for Experimental Biology and Medicine, 134: 8740879 (1970), take a yeast cell wall and extract all components, leaving only the met, which is responsible for the production of α-interferon by monocytes. Found to be involved in induction. Purified mannan found to be involved in physiological reactions has a molecular weight of 5,500 to 20,000 Daltons.

Seljelid et al., Experimental Cell Research, 131 (1): 121-129 (1981), show that insoluble or gel-forming polysaccharides activate macrophages in vitro, but not corresponding soluble glycans. Was observed. Bogwald (Bogwald, Scandinavian Journal of Immunology, 20: 355-360 (1984)) fixed polysaccharides that have a stimulating effect on macrophages in vitro. This led the author to regard the spatial arrangement of polysaccharides as crucial for the effect on macrophages in vitro. Purified polysaccharides isolated from Candida albicans induce antibody responses by human peripheral blood lymphocytes in vitro (Wirz et al., Clinical Immnology and Immunopathology, 33: 199-209 (1984)). . There is a significant difference between anti- Candida antibodies in the serum of normal and Candida infected individuals (see Wirz et al., Supra).

The antiviral activity of polysaccharides bound to polysaccharides and peptides was observed. Suzuki (Suuki et al., Journal of Antibiotics, 32: 1336-1345 (1979), Suzuki et al., Supra) is a femtodonan extracted from mycelial culture of Lentinus edodes. The antiviral action of KS-2) was reported. Both oral and intraperitoneal administrations increase peak serum interferon titers, thereby protecting mice against viral infection. It is derived from dextran phosphate (DP-40) (Suzuki et al., Proceedings of the Society for Experimental Biology and Medicine, 149 (4) 1069-1075 (1975)) and 9-methylstreptimidone (9-MS) Contrary to Saito et al., Antimier, Agent Chemotherapy, 10 (1): 14-19 (1976), which induce high interferon titers in mice only when administered intravenously or intraperitoneally.

The anti-inflammatory activity of aloe vera gel has been widely reported both by oral demonstration and in authoritative scientific journals. Rubel (Rubel, Cosmetics and Toiletries, 98: 109-114 (1983)) fully discusses the possible mechanisms of anti-inflammatory action of aloe gels. Ukai et al., Journal of Pharmacobio-Dynamics. 6 (12): 983-990 (1983) note the anti-inflammatory activity of polysaccharides extracted from the fruiting bodies of some fungi. Polysaccharides have a significant inhibitory action on carrageenan-induced edema. One of the polymers, O-acetylated-D-mannan (T-2-HN), further showed a much superior inhibitory effect on burn hyperalgesia than phenylbutazone (Ukai et al., Supra).

Other researchers also found complex polysaccharides (Saeki et al., Japanese Journal of Pharmacology, 24 (1): 109-118 (1974)), glycoproteins [Arita et al., Journal of Biochemistry, 76 (4). ): 861-869 (1974) and sulfated polysaccharides (Rocha et al., Biochemical Pharmacology, 18: 1285-1295 (1969)).

The debate as to whether the polysaccharide is a glocommannan, mannan, pectin or some other composition has been resolved by a series of chemical purification steps, with a slightly modified extraction method [see Yagi et al., Planta Medica, 31]. (1): 17-20 (1977)], acetylated mannan (aloe met) from Aloe arborescens Miller Bar Natalensis. However, Ovodova (Ovodova, Khim Prior, Soedin, 11 (1): 325-331 (1975)) isolated pectin earlier as the main component of the same aloe species.

The structural and structural variant types of these immunologically active polysaccharides appear to be factors that regulate their efficacy and toxicity. Their mode of action (s) is hardly recognized; Recently, however, there is evidence that some polysaccharides induce lymphocytes and macrophages to produce a wide range of immunologically active substances. For example, 2-keto-3-deoxy-D-manno-octulonic acid (KDO) is a chemical part of lipopolysaccharide (LPS), which appears to provide a minimal signal for macrophage host defense activation [ See Lebbar et al., Eur. J. Immunol 16 (1): 87-91 (1986)]. The composition of the present invention has all the properties of these immunologically active substances; This is the most potent of all known biologically active polysaccharides, except that no toxicity is observed. Modifications of viral glycoprotein synthesis also demonstrate specific antiviral activity.

Many pharmacological studies have recently been conducted on aloe vera gel. The result is a rapid healing of radiographic burns. Rowe. J. Am. Pharm. Assoc., 29: 348-350 (1940) and accelerated healing of lesions (Lusbaugh et al., Cancer, 6: 690-698 (1953)). Thermal burns treated with aloe vera gel heal much faster than untreated burns. Ashley et al., Plast. Reconstr. Surg., 20: 383-396 (1957), Rovatto, supra, Rodriguez-Bigas et al., J. Plast. Reconstr. Surg., 81: 386-389 (1988). Gels are used for the treatment of leg ulcers (El Zawahry et al., Int. J. Dermatol., 12: 68-73 (1973)] and the promotion of postoperative healing [Payne, Thesis submitted to Faculty of Baylor University, Waco. TX, MS Degree]. Experimental evidence shows that the extract of Aloe Vera has anti-infective properties [Solar, Arch. Inst. Pasteur Madagascar, 47: 9-39 (1979)], which suggests that it improves dietary activity (Stepanova, Fiziol. Akt. Veshchestva, 9: 94-97 (1977).

The active fraction of aloe vera gel is a long chain polydisperse dispersed with O-acetyl groups having a mannose monomer to acetyl group ratio of about 1: 0.91. It was identified by Carrington Laboratories, Inc., Irving, Texas as a-(1,4) -bonded acetylation encounter. Acemanan is a compound isolated and developed by Carrington Laboratories, Inc. Non-patent name of a biologically active ingredient (US Pat. Nos. 4,735,935 and 4,851,224 and US Patent Application No. 07 / 229,164, which are incorporated herein by reference).

Mannans, including glucomannan and galactomannan, have long been used by humanity. For example, galactomannans in the form of plant gums are widely used as binders for the regulation of food tissues. In addition, some encounters have significant therapeutic properties [Davis and Lewis, eds. Jeanes A., Hodge J., In: American Chemical Society Symposium, Series 15. Washington, DC, American Chemical Society, 1975.

Pure encounters are relatively uncommon in higher plants, although they are a major structural component of some yeasts. For example, about 45% of Saccharomyces cerevisiae cell walls are made up of mets. This encounter is a water-soluble molecule consisting of β- (1,6)-, β- (1,3)-and β- (1,2) -linked, partially phosphorylated D-mannose residues. McMurrough et. al., Biochem. J., 105: 189-203 (1967). Other biologically active encounters are Candida utilis (Oka et al., Gann, 60: 287-293 (1969), Oka et al., Gann, 58: 35-42 (1968)). Candida albicans, Coccidioides immitis and Rhodotorulum rubrum [Wheat et al., Infect. Immun., 41: 728-734, (1983). Mannans (including galactomannans and glucomannanes) are relatively resistant to attack by mannosidase but can be degraded by exo- and endo-mannases. Emi, et. al., Agr, Biol. Chem., 36: 991-1001 (1972). Snaith, et al., Adv. Carbohydr. Chem. Biochem., 28: 401-445, (1973) Herman, Am. J. Clin. Nutr., 24: 488-498 (1971), McMaster, et al., Proc. Soc. Exp. Biol. Med., 135: 87-90 (1970), Jones et al., J. Biol. Chem., 243: 2442-2446 (1968), Eriksson et al., Acta. Chem. Scand., 22: 1924-1934 (1968). The most prominent biodegradable activity of met in mammals is the activation of macrophages and the stimulation of T cells. As a result, they have been found to be potent immunostimulants with marked activity against infectious diseases and tumors (Hasenclever et al., J. Immun., 93: 763-771 (1964)).

Saccharomyces mannan (15 mg / kg / day) improves carbon clearance in normal male ddI mice, perhaps by acting as a reticuloendothelial stimulant (Suzuki et al., Gann, 62: 553-556). 1971). This same encounter also increases the number of antibody-forming cells in the spleen (Suzuki et al., Gann, 62: 343-352 (1971)). In vitro studies with mouse peritoneal cells (macrophage and lymphocyte mixtures) indicate that some met and met-protein complexes can stimulate interferon release both in vivo and in vitro. Lackovic et al., Proc. Soc. Exp. Biol. Med., 134: 874-879 (1970). Mannan stimulates interferon release in a similar manner to endotoxins but, unlike endotoxins, causes minimal toxicity (Borecky et al., Acta Virol., 11: 264-266 (1967). Hasenclever, supra. Meetings from Candida albicans are active in this manner, but meetings from Saccharomyces cerevisiae are inactive. DeDlercq et al., Ann. NY Acad. Sci., 173: 444-461 (1970). Inconsistent or poor results have been obtained in other laboratories (DeDlercq, supra). These differences can result from minor structural or size differences in the polymers. See Suzuki et al., Jpn. J. Microbiol., 12: 19-24 (1968). The latter seems more relevant because low molecular weight meets (5.5-20 kDa) tend to be the strongest in the interferon induction assay, and Saccharomyces meets tend to be much larger than Candida meets.

20kDa galactomannan from Lipomyces starkeyi has weak interferon-induced properties. In contrast, Candida albicans mannan showed interferon activity 2 to 24 hours after intravenous administration (Borecky, supra).

DMG, a degraded mannoglucan from Microellobosporia grisea culture, stimulates the cytotoxic activity of macrophages, natural killer (NK) cells, and killer T cells, resulting in interleukin-1 (IL-1) And improves secretion of colony stimulating factor (CSF). It exhibits stronger antitumor activity than lentinane (glucans from Lentinus edodes) (Nakajima et al., Gann, 75: 260-268 (1984). Inoue et al., Carbohyd. Res., 114: 164-168 (1983). DMG stimulates macrophages to produce increased amounts of IL-1. DMG also increases 1) antibody production against sheep erythrocytes, 2) natural killer activity in the spleen as well as the spleen, and 3) cytostatic activity in the peritoneal macrophages (Nakajima et al., Gann, 75: 253-259 (1984).

In humans, the main mannose-binding protein is an acute-phase protein; These concentrations are elevated in stressed individuals. See Ezekowitz et al., J. Exp. Med., 169: 185-196 (1989). The envelope glycoproteins of human immunodeficiency virus (HIV gp 120 and gp 41) contain mannose-rich oligosaccharides that are believed to be potent ligands for mannose-binding proteins. As a result, the mannose-binding protein can inhibit HIV infection of lymphocytes and selectively bind to HIV-infected cells. Free yeast encounters can competitively prevent this protein from binding to infected cells. Thus, a factor inducing an increase in mannose-binding protein concentration may confer protection against HIV.

[Problems to Be Solved by the Invention]

Immunomodulators, viruses and cancers continue to be the main cause of both morbidity and mortality in humans, other mammals, other animals, birds and plants. The problems involved with the drugs currently in use are the development of the so-called general metrology, lack of efficacy (or both), lack of specificity and resistance by causative organisms or agents. Thus, there is a need for good nontoxic agents that are not yet therapeutically effective in treating diseases. Acemanan has been found to have a unique combination of immune regulation and antiviral properties.

[Summary of invention]

It is therefore an object of the present invention to provide a method for strengthening or stimulating an animal's immune system, including administering an amount of an acetylated met derivative that is sufficient to enhance or stimulate the immune system in the animal.

It is also an object of the present invention to include cytokines by mononuclear cells and macrophages, peripheral blood proximal cells in animals, including administering to the animal an amount of an acetylated met derivative that is sufficient to activate monocytes and macrophages. To activate, induce and / or enhance the synthesis and production of interleukins, interferons and prostaglandins).

A further object is to provide a method of stimulating macrophage phagocytosis in an animal, including administering an amount of acetylated mannan derivative sufficient to activate monocytes and macrophages.

A still further object is to provide a method of causing antiviral activity in a tissue culture, animal or plant, including administering to the tissue culture, animal or plant an amount of acetylated mandatory derivative sufficient to exhibit antiviral action. To provide.

A further object of the present invention is a person infected with a virus, comprising administering to the infected person an amount of an acetylated mannan derivative sufficient to activate monocytes and macrophages and alter viral replication in the virus infected cells. To provide a way to produce a deficiency virus.

Another object is to induce interferon synthesis, improve antibody formation, enhance T-cell activity, enhance killer cell activity, stimulate pleural activity, alter glycosylation of glycoproteins, and second messengers. (messenger) Provides a method of causing antiviral action in an animal, including altering the synthesis and activity, inhibiting viral replication or inducing a combination of acetylated mannan derivatives sufficient to induce their combined action It is.

A further object is to provide a method for producing a deficient virus in a main seed medium for vaccine production, including adding a predetermined amount of acetylated mandatory derivative sufficient to alter viral replication to the main seed culture.

A further object is to provide a method for stimulating and enhancing cytokine synthesis by immune system cells, including administering to the animal an amount of an acetylated mannan derivative sufficient to stimulate cytokine synthesis.

Another object of the present invention is that the plant or animal immune system comprises administering to the plant or animal an amount of acetylated met derivatives sufficient for the plant or animal immune system to inhibit the growth of the tumor or cancer. It is to provide a method to induce to suppress.

It is also an object of the present invention to administer a plant or animal to a plant or animal, including administering to the plant or animal an amount of an acetylated met derivative that is sufficient to cause the plant or animal's immune system to recognize the tumor or cancer as not self. It is to provide a method for the immune system of destroy or inhibit the growth of a tumor or cancer.

It is a further object of the present invention to provide a method of producing an anticancer effect in an animal suffering from a virus, chemical, radiation, cancer of genetic or other origin.

A further object is to provide an anti-tumor in said animal, comprising administering to the animal suffering from a tumor of hereditary origin an amount of acetylated mannan derivative sufficient to inhibit the expression of the first and second messengers of the oncogene. It is to provide a method of showing the effect.

Another object is to reduce tissue damage such as ulceration and / or necrosis and restore soft tissue capillary bed vascular perfusion in animals, including administering to the animal an amount of an acetylated met derivative that is sufficient to restore tissue viability. To provide a way.

Another object is to provide a method for alleviating the symptoms associated with inflammatory bowel disease in an animal, including administering to the animal an amount of an acetylated mannan derivative sufficient to reduce the symptoms associated with inflammatory bowel disease.

A further object is to provide a method for alleviating the symptoms associated with multiple sclerosis in a human, including administering to the human an amount of an acetylated mannan derivative sufficient to alleviate the symptoms associated with multiple sclerosis.

Another object is to provide a method for alleviating symptoms associated with neurochemical diseases and depression in an animal, including administering to the animal an amount of an acetylated met derivative that is sufficient to alleviate the symptoms associated with neurochemical diseases and depression. will be.

A further objective is to provide acute and chronic autoimmune diseases in animals, including administering to the animal an acetylated met derivative sufficient to induce immunosuppression and / or immunomodulation of cells and tissues involved in the autoimmune disease. It is to provide a method of treatment.

Another additional object is to provide faster healing of traumatic injuries in the animal, including the administration of an acetylated met derivative to the animal so that the animal body tissue repair mechanism and the immune system are sufficient to respond more quickly and appropriately to trauma. To provide a way.

A further object is to treat asthma, conjunctivitis by acting on the animal's respiratory system, including administering to the animal an acetylated mannan derivative sufficient to modulate the immunity of cells and tissues involved in symptoms associated with asthma, conjunctivitis, rhinitis and bronchitis To reduce the symptoms associated with rhinitis and bronchitis.

A still further object is to show a prophylactic effect in an animal such that the animal's immune system prevents infection by an infectious organism, including administering to the animal an acetylated mandatory derivative sufficient to prevent infection by the infectious organism. To provide a way.

A further object is also to provide an animal with an acetylated met derivative that is sufficient to up-regulate genes that cause the animal body and its tissues to produce cell products and allow the tissues to express and regenerate infant cell functions and properties. Including the administration, it provides a method for reactivating the enzyme system and organ system to reactivate aged tissue.

A still further object is to provide a method for immunopotentiating a vaccine by producing a cooperative effect, including adding a predetermined amount of acetylated met derivatives to the vaccine product.

Another additional object is to administer to animals an amount of an acetylated mannan derivative sufficient to activate monocytes and macrophages and to enhance specific tumor cell lysis and natural killer cell activity by cytotoxic cells and / or antibodies. It is to provide a method for treating an animal suffering from a tumor.

A still further object is to introduce acetylated mannan derivatives to (i) protect uninfected cells from viral infection, including introducing a sufficient amount of acetylated mannan derivatives into cells to alter viral glycoproteins at the cell or cell surface. A method of introducing into a cellular function of a virus-infected cell to produce a glycoprotein that disrupts or inhibits viral expression in uninfected cells and / or (ii) virus infected cells to alter the glycoprotein.

A still further object is to introduce acetylated met derivatives into the cells in an amount sufficient to impart non-infectivity to the virus, so that the virus-infected to produce a glycoprotein that prevents or inhibits viral expression in the virus-infected cells. It is to provide a method for introducing an acetylated met derivative into the cell function of the cell.

Another further aim is to acetylate an amount sufficient to (i) produce a broad spectrum of specific antibodies that provide a broader immunological response in cells prior to introduction and (ii) improve the rate of antibody production over a broad spectrum To provide a method for introducing an acetylated mannan derivative into the cellular function of a virus-infected cell to produce an altered glycoprotein that prevents or inhibits viral expression in virus infected cells, including intracellular administration of the mannan derivative. .

It is also an object of the present invention to normalize malabsorption and mucosal cell maturation syndrome in an animal, comprising administering to the animal an acetylated mannan induced cell sufficient to provide additional acetylated mannan derivatives for glycoprotein synthesis. It is to provide a method for accelerating Michaelis-menten (Km) dynamics on mannosyl transferase activity by increasing the amount of acetylated met derivatives in animals up to intracellular and extracellular metabolic pathways.

A further object is to administer into a mammalian infected cell an amount of an acetylated mannan derivative sufficient to produce the modified viral glycoprotein and express the modified viral glycoprotein on the surface of the infected cells so that they are exposed to the epidermal antibody. To express a modified viral glycoprotein antigen on the antigen surface to induce virus-infected mammalian cells to initiate antibody-dependent cellular disintegration (ADCC) by cytotoxic lymphocytes.

Another object is to reduce symptoms associated with multiple sclerosis, including administering to humans an amount of an acetylated mannan derivative sufficient to reduce scar formation and replace the scar with functional tissue in central nervous system cells. It is to provide a method of introducing an acetylated met derivative to a human.

Another object is an amount of acetyl sufficient to extinguish lesions associated with inflammatory bowel disease by increasing tissue regeneration of ulcers in lesions associated with inflammatory bowel disease and reducing autoimmune immunoglobulins in the local tissue of the lesion. To provide a method of introducing an acetylated mannan derivative into a mammal to reduce symptoms associated with inflammatory bowel disease, including administering a mad mannan derivative to the mammal.

[Brief Description of Drawings]

1 shows the synergistic antiviral effect of acemannan and AZT on the viability of HIV-infected MT-2 cells.

FIG. 2 shows the synergistic antiviral effect of acemannan and AZT quantified by percent growth rate of HIV-infected MT-2 cells.

Detailed Description of the Preferred Aspects

Carrisyn ) Is the trade name given to the purified ethyl alcohol extract of the leaf inner gel of Aloe barbadensis miller by the assignee of the present invention. The active ingredient of caricin has been designated as acemannan by the United States Adopted Name Council. At least 73% of the carcinin extract is acemannan; Carycin extract comprises about 73-90% acemannan in total. Carycin extracts are generally prepared by removing the outer sheath of leaves and then processing and removing internal flakes or viscous material in the following manner: pH adjustment, ethanol extraction, lyophilization and grinding [see: United States Patent Application No. 144,872, filed January 1988, which is part of a continuous application of United States Patent Application No. 869,261 (currently US Patent No. 4,735,935). Incorporated herein by reference]. Processing in this manner is expected to produce no toxic compounds by essentially altering any covalent bonds. These preparation steps were developed to overcome the limitations that traditional aloe product manufacturers could not standardize and stabilize polysaccharides.

Carycin is a fuzzy white amorphous powder, poorly soluble in water and dimethyl sulfoxide and insoluble in most other organic solvents. This powder essentially contains at least 73% of a polysaccharide consisting of linear β (1-4) -D-mannosyl units. Polysaccharides are long chain polymers interspersed randomly with acetyl groups bonded to the polymer via oxygen atoms. The generic name of the polymer is acemannan. It is a ratio of about 0.91 acetyl groups per monomer as measured by the acetylation or alkaline hydroxysamate method (Hestrin, Journal of Biological Chemistry, 180: 240-261 (1949)). With neutral sugar binding assay, D-galactopyranose, attached to the chain via (1-6) binding, appears to be present in about 1 ratio for every 70 sugars. The ratio of 20: 1 mannose to galactose is mainly in the galactose unit. (1-4) Glycoside bonds together. The chemical structure of acemannan is as follows:

[Term Definition]

The term virus, as used herein, includes both DNA and RNA viruses. It may be an envelope virus or a non-envelope virus. In all but one case the term envelope virus can be understood to mean a virus enclosed within a modified host cell membrane; Poxviruses produce their own envelopes. Common envelope viruses are described in Table 1.

The term tumor, as used herein, includes both malignant and non-malignant neoplasms, including tumors of viral, chemical, radiation, gene, and other origin. It may be of embryonic ectoderm origin, embryogenic mesoderm origin, or embryogenic endoderm origin. It may be from embryonic surface ectoderm, embryogenic neuroectodermal, embryonic head mesodermal embryonic paraxial mesoderm, embryogenic middle mesoderm, embryogenic side mesoderm, or embryogenic endoderm. Thus, tumors in animals include tumors of skin and soft tissues, tumors of muscles, tumors of connective and adjacent soft tissues and tumor-type lesions, tumors of bone and cartilage, tumors of lymphoid and hematopoietic tissues, tumors of the respiratory system , Tumors of the digestive tract, tumors of the liver, gallbladder and pancreas, tumors of the urinary system, tumors of the genital organs, tumors of the mammary glands, tumors of the endocrine glands, and tumors of the nervous system and the eye.

Human malignancies include acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, true polycythemia, spinal sclerosis with myelodysplasia, multiple myeloma, primary polyglobulinemia, Hodgkin's disease, non-hoho Jekyn's lymphoma, skin cancer, malignant melanoma, head and neck cancer, lung cancer, gastrointestinal cancer, breast cancer, gynecological cancer, trophoblast disease, testicular cancer, prostate cancer, kidney cancer, bladder cancer, endocrine tumor, brain tumor, retinoblastoma, neuroblastoma Wilm) tumors, osteogenic sarcoma, Ewing's sarcoma and soft tissue sarcoma.

As used herein, the term microorganism includes parasites, bacteria and other organisms and agents that cause infection. Parasites include arthropod parasites, intestinal parasites, protozoan parasites and hemaprotozoan parasites. Examples of such parasites include acne mites, duodenums and hookworms.

The term glycosylation refers to the addition of carbohydrate molecules to protein molecules. Acetylated mannan derivatives, in particular acemannan, can exert their therapeutic effect by two possible mechanisms. One is to alter glycosylation or insert acetylated met derivatives into glycoproteins, such as inhibition of glycosidase I. Another possible mechanism is to enhance the antigenicity of the virus or tumor or to enhance the host's immune adaptation. Enrichment of antigens can be achieved by presentation by macrophages; It can be achieved through uptake by T or B cells or both, altered antigen labeling or adjunctive effect. In some respects, acetylated met derivatives enhance the host's recognition of tumors or infectious agents (eg, viruses or other microorganisms) as non-magnetic.

Administration of acetylated mannan derivatives can be accomplished by topical application, oral ingestion, intraperitoneal route, intravenous route or other parenteral route of administration.

Acetylated met derivatives can be used as a single agent to be used with recipients or in combination with other known therapeutic agents that require the participation or assistance of the host immune system to achieve maximum therapeutic effect.

It has now been found that acemannan is a potent derivative of IL-1 and prostaglandin E2 (PGE2) production by human peripheral blood adjacent cells in culture. The present invention is believed to be the first practical non-toxic stimulant of IL-1 release. IL-1 is an important macrophage product reported in the literature that affects the activity and production of lymphocytes, fibroblasts, B-lymphocytes and endothelial cells. Old, Scientific American, 258 (5): 59-60, 69-75 (1988).

IL-1 induces fibroblast proliferation critical for wound healing. IL-1 also enhances (1) bone marrow activity (which may have therapeutic activity in individuals with reduced bone marrow) and (2) generally strengthens the immune system.

A series of experiments using formulated lymphocyte cultures (MLC) has shown that acemannan increases the homologous antigen response of these lymphocytes in a dose-related fashion. When acemannan is incubated with monocytes, monocyte-induced signals enhance the response of T lymphocytes to lectins. Related investigations of the acemannan effect on MLC have been shown to increase phagocytosis and activity of natural killer cells. Thus, in this in vitro test system, acemannan is non-toxic and an adjuvant.

Acemanan actively stimulates lymphocytes to secrete lymphokines and also cause HIV-infected lymphocytes to produce glycoproteins (GP-120) altered by mechanisms similar to those of glucosidase I inhibitors. Gruters et al., Nature 330: 74-77 (1987) and Pal et al., Intervirol. 30: 27-35 (1989). Acemannan performs phagocytosis and is explicitly pumped into the Golgi / glycoprotein machinery of monocytes where it is directly opposed to glycoprotein synthesis.

A. Toxicology

The toxicological effects of acemannan have been investigated in both in vivo and in vitro systems. Acemanan is not a mutant or embryogenic material in an in vitro test system. In vitro, this compound is non-toxic to H-9, MT-2 and CEM-SS lymphocyte cells. In vivo toxicology studies on acemannan include 91 days of subchronic oral toxicity studies in dogs, 180 days of chronic oral toxicity studies in rats, and 180 days of chronic oral toxicity studies in humans. In these studies, there was no observable toxic effect in dogs receiving up to 825 mg / kg of acemannan per day for 91 days. No clinical, severe pathological or toxic effects were noted in rats fed up to 38,475 ppm of acemannan in the diet for 180 days. No adverse clinical or toxic effects were observed in human patients receiving 800 mg of acemannan per day for 180 days.

In a pilot study, administration of acemannan to dogs resulted in a definite monocytic increase in blood samples taken to count complete leukocyte counts and to identify morphological differences. Within 2 hours of oral administration of high doses of acemannan, highly activated monocytes appeared in the circulation. Similar effects have been observed in humans.

Human peripheral blood mononuclear cell cultures to track the introduction or uptake of acemannan into biological systems and A study using C-labeled acemannan was performed. In these studies, The amount of detection of C-labeled acemannan was taken up or taken up by human peripheral mononuclear / macrophage cells. Peak introduction occurred at 48 hours. 5OfAt the concentration of,¹⁴C-labeled acemannan was not cytotoxic to monocytes / macrophage cells and the degraded cell mass (weight / volume: w / v) was 760 times greater than the degraded acemannan solution (w / v). These results suggest that macrophages can maintain intracellular concentrations of acemannan at very high levels but not cytotoxicity.

Pyrogenic assays are injection solutions of acemannan 1 Of Was performed in rabbits according to the pyrogen test protocol outlined in USP XXI, Biological Test [151]. Temperatures were measured more frequently than specified in the USP because of the unknown systemic effects of injected acemannan. The temperature change in the test animals did not exceed the minimum change allowed in the USP protocol: therefore, this solution met USP conditions for the absence of pyrogenic material. Injected acemannan was 0.3 in one rabbit Led to an increase in maximum body temperature. This temperature increase appeared 90 minutes after injection. Acemanan is an inducer of IL-1 secretion by macrophages and monochromatic cells in vitro. Since IL-1 is a potent pyrogen, the minimum delayed temperature increase in this lap can be explained.

Studies on the stability and tolerability of orally-administered acemannan were recorded and completed for 24 humans. Clinical trials have shown that changes outside the normal range have occurred: CO ₂ in 7 subjects, cholesterol in 3 subjects, triglycerides in 2 subjects, and phosphorus in 1 subject, 4 Hemoglobin in two subjects, basophils in two subjects, monocytes in three subjects, eosinophils in three subjects, lymphocytes in four subjects, neutrophils in two subjects, and erythrocytes and white blood cells , Few red blood cells and white blood cells were also found in urine. This change was clinically irrelevant.

Immune Profile Results, Day 1 Levels for CD-16, CD-4 (T-4), CD-8 + Leu7, CD-4 + CD-25, CD-8 + CD-16, Leu7 and TQ-1 And group differences between 7 and 7 days. The mitotic reaction was in the low range.

No fatal signs above the normal range were seen. There was no group difference in urine output. In group IV, one subject had diarrhea and had stool during the investigation. In group I, one subject had stool during the 2 to 4 days of the survey. A total of five subjects had a total of eight adverse events. All events occurred in subjects who received oral acemannane 1600-3200 mg daily for 6 days.

B. Mode of Administration

Acemanan's physical properties allow it to be formulated and incorporated into all pharmaceutical dosage forms known to those skilled in the art. The biopharmaceutical and toxicological properties of acemannan can be used in tissues and organs of bioorganisms and administered over a wide range of doses.

Acemanan is 0.001 per day Of To 1000 Of Doses of body weight can be administered to animals orally, parenterally, topically and locally.

Acemannan in combination with a suitable adjuvant can be compressed or filled into solid dosage units such as pills, tablets and gelling tablets or can be processed into capsules. These oral dosage forms are about 0.1 per kg of body weight per day To 1000 It can be administered at a dose of.

If a suitable liquid vehicle is used, acemannan may be injected as a solution, suspension or emulsion. These products are weight per day 0.001 per To 1000 It can be administered at the ratio of. Acemanan as an adjuvant for vaccines or other products ranges from 0.001 to 1000 per unit dose of adjuvant product. Can be used at the ratio of.

Topical administration of acemannan may be in the form of a processed gel, cream, lotion, solution, ointment or powder. These formulations may comprise up to 90% acemannan.

Example 1

Production of Interleukin-1 and PGE2 by Acemannan Stimulated Human Adhesion Peripheral Blood Leukocytes

A. Induction of IL-1 Production

Human mononuclear cells are isolated from heparinized whole blood by density-gradient centrifugation in Ficoll-Hypaque (Pharmacia, Sweden). After washing, cells were treated with 50 U / ml penicillin, 50 containing 25 mM Hepes. Of 2 x 10 ⁶ cells per RPM1-1640 supplemented with streptomycin and 20 mM L-glutamine Resuspend at the concentration of. Cell suspension 2 Aliquots were distributed to each well of a 6-well plate and in 5% CO ₂ -humidified atmosphere, 37 Incubate for 1 hour at. After the non-adherent cells are removed, the adherent cells are washed three times with the medium described above. Medium 2 supplemented with 5% human AB serum Is added to each well. Cultures are stimulated with different concentrations of acemannan. 20 Of Concurrent control with lipopolysaccharide (LPS) from E. coli (Sigma 0111: B4) at a final concentration of and control without background (background) are included. Cultures were allowed to stir for 37 hours as described above. Incubate in. The supernatant was harvested and centrifuged to remove cells, then dialyzed (pound once) to 500 volumes of PBS for 48 hours and then to 20 volumes of RPMI-1640 with 25 mM Hepes, antibiotics and L-glutamine as described above. Dialysis for 4 hours. Supernatant is -20 until IL-1 activity is assessed Freeze in

B. Determination of IL-1 in Supernatant

Two different processes are used to assay IL-1:

(1) thoracic cell proliferation assay and (2) ELISA assay specific for IL-1

1. Thymic cells from 5 to 8 weeks old C ₃ H / HeJ mice are used. Homogeneous cell suspension 5% FCS, 100U / Penicillin, 50 g / Prepare in minimal essential medium (MEM) supplemented with streptomycin, 2 mM L-glutamine and 5 × 10 ⁵ 2-mercaptoethanol. Cell concentration is adjusted and dispensed into 96-well plates at 1 × 10 ⁶ m cells / well. Plant Hemagglutinin (PHA) 10 Add to each well at a concentration of / well. Samples were serially diluted from 1:10 to the final dilution rate, 25 A volume is added to each well. Each dilution is tested in triplicate. Plates were placed in a humidified atmosphere with 5% CO ₂ for 72 hours, 37 Incubated for 72 hours at [ ³ H] -thymidine (0.5 Ci / well). Cells are harvested on a fiber glass filter using an automatic cell harvester and radioactivity is measured by a standard scintillation process. The results are expressed as the amount of thymidine (cpm) introduced by the pleural cells in response to the final 1:10 dilution supernatant.

2. Sandwich ELISA with two-sites for IL-1. This process has recently been described in the Journal of Immunology, 138: 4236 (1987), the description of which is specifically incorporated herein by reference. See also US Pat. No. 3,654,090 and US Pat. No. RE 31,006gh, Schuurs et al. In short, IL-1 Monoclonal Purified Antibody for IL-1-H6 (100 / Well, 10 Of 4) Overnight in vinyl black plate wells. The wells are washed with PBS / 0.5% Thimerosal and 5% anhydrous skim milk / 0.5% Thimerosal / PBS 200 at room temperature. 1 hour counter cover. After washing, sample or human recombinant IL-1 standard 50 / Well and IL-1 [1% anhydrous skim milk / 0.5% thimerosal / biotinylated IL-1 in PBS -H67 (2 μg / ml)] another monoclonal antibody against non-overlapping epitopes 50 Was added and the plate was incubated for 2 hours at room temperature. 1: 1000 dilution of streptavidin-peroxidase after washing 1000 / Well is added and plate is incubated for 1 hour. Wash wells, 100 OPD substrate solution Incubate for 30 minutes in the cow using, then 450 Absorbance at is measured.

C. Measurement of PGE ₂

PGE ₂ is assessed by radioimmunoassay in the same non-dialysis supernatant. Antibodies against PGE ₂ (ICN Biomedical, Inc., Costa Mesa. CA) are used according to the manufacturer's instructions (incorporated herein by reference).

D. Observation

Representative experiments are shown in Table 2. Acemanan is a potent derivative of IL-1 production by human adhesive peripheral blood leukocytes. 1 to 10 Of At doses of acemannan extract LPS 20, a reference inducer of IL-1 production Of Induced production of IL-1 compared to that induced by. Acemanan in the same dose range is also positive for LPS (positive control) 20 Of The production of PGE ₂ was induced to a level comparable to that induced by.

Example 2

Effect of Acemanan on In Vitro Phagocytosis

The effect of acemannan was investigated in vitro to confirm its effect on phagocytic function. CBA mice are injected intraperitoneally with 1 mg / kg of acemannan and three days later peritoneal and splenic macrophages are collected. Thioglycolate and saline are similarly tested as positive and negative controls, respectively. Macrophages are incubated with SRBC as an uptake factor in the presence and absence of anti-sheep red blood cell (SRBC) titers and phagocytologically measured as% of cells ingested SRBC. Non-specific phagocytosis was slightly increased after acemannan treatment, but phagocytosis was significantly increased in the presence of the antibody. In the presence of complement, acemannan-stimulated and antibody-mediated phagocytosis was increased to a fairly high degree. These results indicate that acemannan can increase the number of macrophages and enhance their phagocytic activity. This response may contribute to the effects of acemannan as a stimulator of wound healing and anti-infective agents.

A. Methods and Materials

Acemanan is stored in its anhydrous form at room temperature. Weigh the required amount for each experiment and microwave with 2 min exposure at 600 watts. It is then transferred to sterile plastic centrifuge tubes and microwaved for an additional minute. This material is diluted to the desired concentration in cell culture medium (RPMI-1640).

Phagocytic Cells

Mouse spleen cells are purchased from BALB / c mice purchased from Harlan Sprague-Dawley. Mice are killed by CO asphyxiation and their spleen is removed aseptically. The cells are then separated into adherent and non-adhesion populations by nylon wool column fractionation according to the method of Journal of Immunology, 71: 220, specifically incorporated herein by reference. As a result of microscopic analysis of adherent cells, macrophages (monocytes) and lymphocytes had a ratio of 4 to 1. After single-cell suspensions are obtained from monolayer destruction, both adherent single and non-adherent single cells are placed in Picol-high park and centrifuged to obtain a mixture of lymphocytes and macrophages.

[Badge generation test]

Standard embryogenic assays are outlined below. The mitotic material used in this assay is PHA-P from Burroughs Wellcome. As indicated for the individual experiments, the cultures are maintained for 72 hours in a 5% CO moisture atmosphere. Tritiated thymidine is added during the last 6 hours of the incubation period. A flat microtiter tissue culture plate is used and the cell concentration per well is 5 x 10 Dog Mouse Cells / 0.2to be. Once the cells are attached to the wells, acemannan or mitosis is added. The stimulus index (SI) is calculated from the following equation:

[Cell Staining]

Briefly, cell smears are stained with non-specific esterase stains as follows. Fixed solution 4 consisting of a mixture of 2 drops of fetal calf serum and 25 ml of 35% formaldehyde, 45 ml of acetone, 100 mg of KH ₂ PO ₄ , 10 mg of Na ₂ HPO _4, and 30 ml of water in about 2 x 10 ⁶ cells in two drops 4 Mix with drips. The slides are incubated with a mixture of 10 mg naphthyl acetate and 4.5 mg of Fast Blue dye in 1.4 ml of ethylene glycol monomethyl ether with 0.1 M trismaleate buffer pH 7.8 (light dye). The dye is allowed to react for 10 minutes and then washed in water for 20 seconds. Counter washes of 0.2 g of Giemsa dye, 12.5 ml of ethanol and 12.5 ml of glycerol are used for 30 seconds before washing again.

Induction of Peritoneal Macrophages

Peritoneal exudation macrophages are induced by intraperitoneal injection of saline thioglycolate broth (1 mg / kg) or acemannan (1 mg / kg) into female BALB / c mice. Induced cells are removed from the abdominal cavity 3 days after injection.

Macrophages are washed twice with phosphate-buffered saline (PBS), covered with 2 ml of fresh medium, and 0.1 ml of macrophage suspension is added to each tube. Cultures 37 , Humidified 5% CO ₂ -95% air incubator for 30 to 60 minutes. The cultures are washed twice with PBS and covered with 2 ml of PBS. One of each pair of cover slips is removed with a needle-nose forceps, soaked only in distilled water for 5 seconds, and then quickly placed in a petri dish. PBS is removed and the culture is covered with ice-cold glutaraldehyde. After 10 minutes, glutaraldehyde is removed and the cover slip is coated with distilled water.

The mounted coverslips are quickly irradiated using an oil immersion lens of a phase contrast microscope. Cohesion is recorded on coverslips that are not subjected to storage shock and intake is recorded on coverslips dissolved in distilled water.

[Antibody-dependent phagocytosis and antibody-independent phagocytosis

SRBC purchased from Austin Biologics Lab oratory (Austin, Texas) is washed three times in PBS (pH 7.2). BALB / c mice were injected intraperitoneally with 10 ⁶ cells, and then blood was collected 14 days after injection. Serum was collected, mixed, and then 56 for 45 minutes Heat-inactivate at. The aggregation titer was measured using a round bottom microtiter well and found to be 1024.

Antibody-independent phagocytosis is measured by culturing SRBC (0.5% v / v) and macrophages (10 ⁶ ) in RPMI-1640 with 20% fetal calf serum (FCS). Slides are prepared and stained at various time points. Macrophages with ingested red blood cells (%) are visually determined by counting 200 cells / slides and 3 slides / animals.

Antibody-dependent phagocytosis is measured using SRBC (0.5% in RPMI-1640 with 20% FCS) mixed with anti-SRBC serum or IgM fraction (minimum titer: 2000). The mixture is incubated at 37 ° C. for 15 minutes, then washed twice in PBS pH 7.2 and resuspended to initial volume.

Serum Fractionation

The whole serum is fractionated to remove IgM by euglobulin precipitation and dialysis against distilled water. 4 for 24 hours After dialysis at, centrifugation at 1500 x G for 20 minutes to remove the precipitate and the supernatant is analyzed by ion electrophoresis and complement-mediated dissolution. Less than 5% of the original IgM remains.

B. Results

To assess the effects of acemannan on macrophages, the first experiment uses mouse spleen cells cultured in vitro with acemannan (Table 3).

(a) Macrophages (monocytes) are measured by esterase staining. The results are expressed as mean ± standard deviation. The results are obtained from six experiments, examining 200 cells per experiment. Lymphocytes are cells that are not stained with esterase, and lymphocytes appear by wright stain.

Cultures are incubated for 72 or 96 hours and coated samples are made at the end of the experiment and stained by light and esterase methods. Relative percentages of macrophages and lymphocytes are measured. At 72 hours, the number of macrophages showed a dose-related increase from 30% when not treated with acemannan to 45% when treated with 0.2 μg of acemanan per well. Lymphocytes were concomitantly reduced because the data are expressed in% of cells. At 96 hours, macrophages (%) showed a dose-related increase in the presence of acemannan. At 96 hours, cultures with 0.2 μg of acemannan per well showed significant acidosis as indicated by the yellow phase. In addition, 96-hour cultures have lower macrophage levels (%), perhaps due to longer incubation times. Similar experiments were performed using mitotic PHA-P to correlate acemannan-induced increases in macrophage numbers to known standards. The results are shown in Table 4.

(a) Monocytes are measured by esterase staining. The results are expressed as mean ± standard deviation. The results are from six experiments. Lymphocytes are cells that are not stained by esterase, and lymphocytes were seen by Wright staining.

The percentage of macrophages did not change at 72 hours, but macrophages showed a dose-related increase after 96 hours of incubation with PHA-P. In comparison, acemannan is twice as effective as PHA-P. The percentage of macrophages increased up to 16% with acemannan compared to 7% with PHA-P (Table 3 and Table 4).

Acemanan was shown to increase the percentage of macrophages, so it was measured whether the activity of the phagocytes was also increased. Peritoneal effusion cells from CBA mice treated with saline, thioglycolate broth or acemannan were used as phagocytes and positive red blood cells were used as particles to be ingested (Table 5).

(a) This result is determined by counting 200 cells / slides with two slides per animal. Results are based on two experiments.

(b) Phagocytosis (%) represents the percentage of cells showing red blood cell intake. The results are expressed as mean ± standard deviation.

(c) As assessed at the 95% confidence level by the Student T-test, there is a significant difference from the saline control.

Over 120 minutes, the phagocytosis in cells from thioglycolate broth-treated animals increased to 89%, although the nonspecific phagocytosis increased by 3% to 53% in the saline control group. Phagocytosis in acemannan-treated animals increased to 63% at 120 minutes. Acemanan-stimulated phagocytosis was greater than that of the control group after 20 to 120 minutes; However, the difference is not statistically significant.

To determine whether the effects of acemannan on phagocytosis are antibody-dependent, similar experiments are performed using anti-SRBC (Table 6).

(a) Phagocytosis is the average percentage of cells with red blood cell intake Expressed as standard deviation.

(b) The antibody titer by aggregation was found to be 1: 1024. Pre-treatment and cell sources are described in the method.

(c) As assessed at the 95% confidence level by the Student T-test, significant differences from the saline control group are shown.

Heat serum (56 for 30 minutes Inactivate). The antibody titer used was 2 × 10 ^{3, which} was greater than the hemagglutination titer. In this experiment, macrophages are obtained from two sources: the abdominal cavity and the spleen. Again, mice are pretreated with intraperitoneal injection of 1 ml 0.9% saline, 1 mg / kg thioglycolate or 1 mg / kg acemannan. At titers of 2 × 10 ³ , the phagocytic activity of thioglycolate-induced peritoneal macrophages was two times greater than the activity of saline-induced controls (89% vs 43%), whereas acemannan-induced macrophages were control Compared to 30% more active (73% vs 43%). The difference in phagocytic activity in the acemannan-treated and saline control groups is statistically significant.

Similar results were obtained with macrophages from the mouse spleen. Phagocytic activity was lower than that of macrophages obtained from the abdominal cavity, probably due to the treatment of spleen cells. Again, at titers of 2 × 10 ³ , acemannan-induced macrophages had significantly higher phagocytic activity than saline controls at the 95% confidence level; Phagocytic activity was similar to the control at a titer of 8 × 10 ³ .

To determine the effect of complement (C ′) on antibody-mediated phagocytosis, experiments were performed in which C ′ was added to the medium (Table 7).

(a) Positive red blood cell intake after incubation for 30 minutes (%) Measure with standard deviation. Add guinea pig complement.

(b) As assessed at the 95% confidence level by the Student T-test, there is a significant difference compared to -C '.

To confirm that lysis did not occur, IgM-depleted mouse serum is used (see method). The titer used was 3000, as determined by hemagglutination and coombs techniques. Although the difference was only statistically significant for acemannan-induced peritoneal cells, cells from both the peritoneal and spleen were more active in phagocytosis than at the C 'addition without their addition.

Finally, experiments are conducted to distinguish the effects of acemannan phagocytosis and adhesion (Table 8).

(a) Incubate the cell mixture for 7 minutes.

(b) The results indicate that the phagocytes show phagocytosis or adhesion Expressed as standard deviation. This result is obtained from one experiment in which 200 cells are recorded per animal using 3 animals.

(c) As assessed at the 95% confidence level by the Student T-test, significant differences from the saline control group are shown.

In this experiment, the antibody against SRBC was used at a titer of 2,000, but the experiment stopped after 7 minutes. Acemanan-induced macrophages from both the peritoneum and the spleen were more effective at adhesion than the saline control and were less effective than the thioglycolate-induced group as already shown.

C. Discuss

These results indicate that acemannan stimulates phagocytosis either directly or indirectly. It also indicates that acemannan enhances phagocytosis by macrophages through nonspecific and specifically antibody-mediated responses. This demonstrates that acemannan has immunostimulatory properties against phagocytes.

Example 3

Effect of Acemanan on Nonspecific Tumor Lysis

This example investigates the possibility of nonspecific tumor killing induced by acemannan-stimulated phagocytes.

A. Process

[Acetane polymer]

Keeps acemannan in anhydrous form; The amount required for each experiment is weighed and microwave treated with 2 minutes exposure at 600 Watt power. The material is transferred to a sterile centrifuge tube (15 ml) and microwaved for an additional minute. This material is diluted to the desired concentration in Hanks Balanced Salt Solution (HBSS). In some experiments, the material is autoclaved and sterilized without a significant loss of activity.

[cell]

Macrophages are harvested from the abdominal cavity of female BALB / c mice purchased from Harlan / Sprague Dawley. Tier glycolate broth (25 Of ) Or acemannan (25 Of 6 days prior to harvesting, some groups of animals are injected intraperitoneally. Saline stimulated cells are used as additional controls. Harvested cells were washed three times in HBSS and 5 × 10 ⁶ cells / Dilute in RPMI-1640 at a concentration of.

[Target cell]

Target cells are purchased from the American Type Culture Collection (ATCC) (C ₃ H / HeN fibrosarcoma L929) and passaged. Labeled with ⁵¹ Cr 150 mCi mixed with 1 ml of a cell suspension containing 10 ⁷ cells in RPMI-1640. After incubating the cells for 1 hour, washed three times with RPMI-1640 and 5 x 10 ⁴ cells / Adjust the final concentration of

B. black

An aliquot of effector cells (100 cells / μl) is placed on a flat microtiter plate and ⁵¹ Cr-labeled cells are added with at least 3 replicates per experiment time point. The test plate was placed in 7% CO ₂ (formerly 5% CO ₂ ) for 20 hours, 37 Incubate in. Centrifuge the plate at 250 x G for 15 minutes to obtain supernatant (100 μl). The amount of radioactivity is assayed on a packcard gamma counter. The control consists of thymus bulbs. Cytotoxicity (CT%) is determined as follows:

C. Results

Table 9 shows the results of the initial experiment.

(a) total cpm of target cells = 42,000

Thioglycolate-stimulated macrophages incubated with Cr target cells averaged 2800 cpm. Cr was released and acemannan-labeled cells released an average of 3100 cpm of radiation. There is no statistical difference between these groups. Unstimulated macrophages were released in the range of 2800 cpm. However, in vitro, acemannan-stimulated macrophages produced 21,000 cpm. Cr was released. This indicates that (1) acemannan does not induce a long-lasting cell lysis effect and (2) its activation can occur in a relatively short time during tissue culture. The cytotoxicity rate corresponds to the cpm released from the target cell when the target cell is destroyed.

Serial experiments over time using the cytotoxicity assay are shown in Table 10.

(a) time after injection

(b) cpm of control cells = 39,000

The cytotoxic effect of acemannan begins within 6 hours of stimulation and increases to a maximum at 9 hours. The mechanism of this activation has not been investigated.

The data presented in this example indicate that acemannan may play an important role in nonspecific treatment of cancer.

[Selection of potent efficacy against horse sarcoma]

Three sarcoids of two horses are treated parenterally and intralesionally with acemannan. The goal of this trial is to observe horses for side effects and whether acemannan is an effective treatment for horse sarcoma. In horse 1, one sarcoid was completely healed while the size of the second sarcoma was not reduced. A third sinter sarcoma developed during treatment. In Equine 2, a single sarcoma was completely healed. These results suggest that acemannan may be effective in the treatment of horse sarcoma.

Two horses with three suspected lesions are purchased at a low price. Photos are taken, examined, and confirmed by sarcomas by histopathology.

[Horse 1]

1 day. Treatment is by direct injection (needle of 20-gauge) with 50 mg of acemanan diluted in 10 ml of saline (lesion 1) and 5 ml of saline (lesion 2) of each of the two lesions on the right hind limb. 25 mg of acemannan diluted in 7.5 ml saline is administered intravenously.

7 days. Disease 1 (upper lesion) is treated with 50 mg of acemannan (18 gauge needles) diluted in 10 ml of saline. Lesion 2 is treated with 25 mg of acemannan diluted in 7.5 ml saline. 50 mg in 10 ml of saline is administered intravenously.

14 days. Treatment of lesion 1 with 50 mg of acemannan in 10 ml of saline and treatment of lesion 2 with 25 mg of acemannan in 5 ml of saline. 75 mg in 25 ml saline is administered intravenously.

21 days. Disease 1 is treated with 50 mg in 10 ml of saline and lesion 2 is treated with 25 mg in 10 ml of saline. 100 mg in 25 ml saline is administered intravenously.

29 days. Treat lesion 1 as in day 21, but do not treat lesion 2 directly because of local swelling, 100 mg in 25 ml of saline is administered intravenously.

42 days. Do not treat lesion 1 directly, lesion 2 is treated with 25 mg of acemannan in 10 ml of goat. 100 mg in 50 ml of saline is administered intravenously.

57 days. Euthanize horse 1. Tissue samples are taken from the site of lesion 1 and from lesion 2 on inguinal lymph nodes and left shoulder developed during the course of treatment.

[Horse 2]

1 day. The lesion on the bottom left chest is treated with 50 mg of acemannan diluted in 30 ml of saline. 1/2 is injected subcutaneously (S / Q) and the other half is injected intralesionally.

At day 1, 16, 24, 30, 49, 56, 63, 70 and 77, 100 mg of acemannan diluted in 60-120 ml of saline at end 2 is administered intravenously. The amount of dilution varies to the amount needed to make the clear solution.

On days 105, 113 and 120, lesions are treated intra- and subcutaneously depending on the lesion with 25 mg of acemannan diluted in 5 ml of brine. An additional 75 mg is administered intravenously.

Result-end 1: 1 day. The size of lesion 1 was 2.5 cm (width) x 2.5 cm (length) x 1 cm (thickness). The resolution of this lesion is as follows:

[Horse 1-focal 1]

Daily measure

1 2.5cm x 2.5cm x 1cm

7 2.5cm x 1.75cm x 1cm

14 2.0cm x 1cm x 1cm

21 2.0cm x 1cm x Skin level fever scarring

29 2.0cm x 1cm x Flat and Dry

42 All Healed

Lesion 2 was 2cm x 2cm x 1cm per day and there was no significant change.

[Horse 1-lesion 2]

Daily measure

1 2cm x 2cm x 1cm

7 2cm x 2cm x 1cm

14 2cm x 2cm x 1cm

29 2cm x 2cm x 1cm-whole ferrous iron, still swollen and painful

42 slightly smaller but still swollen, no pain

54 same size, reduced swelling of joint to 65%

Result-Horse 2: 1 day. The size of the lesion is 5cm x 3.5cm x 2.5cm and has a diameter standard of 2.5cm. Changes until fully resolved are as follows:

[Horse 2-Lesion 1]

Daily measure

1 5cm x 3.5cm x 2.5cm

6 no change

16 no change-severe granulomatosis

24 5cm x 3cm x 2.5cm

30 shows reduced granulomatosis

49 4cm x 3cm x 2cm

56 4cm x 3cm x 2cm

63 3.8cm x 3cm x 2cm

70 3.7cm x 2.6cm x 1.8cm

77 2.7cm x 2cm x 1.3cm

105 2.5 x 2 cm x 1.3 cm

113 3.5cm x 2.25cm x 1.5cm

120 2.5cm x 2.4cm x 0.6cm

177 Lesions Completely Disappeared

After intravenous administration to these horses, there was no change in heart rate and no obvious signs of sweating, muscle spasms, or pain. A slight increase in depth of breath was noted only in horses 1. Locally, horse 1 showed moderate inflammatory vesicular inflammation in lesion 1 and rapid inflammatory vesicular inflammation in lesion 2, to a degree sufficient to avoid injection into the lesion, as planned on day 29. Because lesion 2 is more fibrous and more difficult to inject, there was more subcutaneous leakage of blood. This explains the lack of effect on lesion 2. Horse 2 did not show cellulitis.

The fact that nodular sarcoma develops during the course of treatment can induce sarcoma into intralesional treatment by intravenous administration, but it doubts whether the main effect of acemannan is a local tissue reaction rather than a systemic one.

The exact date on which horse 2 phase lesions heal is unknown because the investigator was a 60-day sick leave between 113 and 177 days. Judging from the fact that tumor size did not decrease significantly around 56 days, intravenous alone administration during the day seems to have little effect on equine type 2 sarcoma.

Example 4

[Allo-Reactive Enhancement of Human Lymphocytes by Acemanan]

This example is designed to test acemannan's ability to potentiate an immune response to allogenegens and to test whether strong potentiation is a monocyte-induced phenomenon. Acemanan does not enhance lymphocyte response to common genotype antigens in mixed lymphocyte cultures (MLC), but importantly, acemannan is a dose-response mode (2.6 x 10). To 2.6 x 10 M) increased homologous response. This effect of acemannan has been shown to correspond to the concentration of acemannan in vitro that is a specific response and achievable in vivo. A separate series of mixing experiments demonstrated that incubating acemannan with monocytes enhanced the T cell response to lectin by monocyte-induced signal. We have concluded that acemannan is an active ingredient in aloe vera plants and is an important immunopotentiator that increases lymphocyte response to homologous antigens. This suggests that the mechanism enhances mononuclear cell release of IL-1 under the protection of homologous antigens. This mechanism may partly explain the ability of acemannan to combat viral infections in experimental animals and humans.

This example was designed to directly assess the effect of acemannan as an immune enhancer (cell-cell interaction with homologous antigens in mixed lymphocyte cultures) in a model of monocyte-T-lymphocytes. This model tests the ability of acemannan to stimulate further monocyte-macrophage action in immunologically relevant models.

A. Materials and Methods

1.Notify and consent to the production of mononuclear leukocytes under the protection of an investigation approved by the Institutional Review Board of the University of Texas Southwestern Medical Center in Texas, Dallas. Obtained from peripheral blood of normal volunteers obtained. Peripheral blood is diluted 1: 3 in Hank Balanced Salt Solution (HBSS) and placed on top of the Piccol-Hyparq gradient. Cells from subjects known to have major histocompatibility hypertrophy are obtained on each irradiation day to confirm a positive mixed lymphocyte response. For certain experiments, more closely characterized lines of cells that inhibit mononuclear leukocyte infusions are isolated. Standard nylon wool separation technology separates T-lymphocytes. Nylon effluent cells comprise about 90% pure T cells. T-8 lymphocytes and monocyte-macrophages preferentially adhere to the column. A plunger is used to force the medium through the column to dislodge the coalescence. In order to concentrate monocytes (macrophages), 95% or more pure cell populations are generated using free adhesion processes.

2. Prepare 0.5% (w / v) solution in acemannan RPMI-1640 medium, 2.6 x 10 M, 2.6 x 10 M and 2.6 x 10 Dilute to a working concentration of M to test acemannan in these investigations.

3. Mixed Lymphocyte Culture (MLC) Single Oriented MLC is prepared on microtiter flat tissue culture plates (Costar Co., Cambridge. MA). Mononuclear cells isolated by the Ficoll-Hyparque density gradient technique described above were exposed to 2000 rad for 30 minutes at a cesium source (Gammacell. Atomic Energy of Canada, Ontario, Canada) and used as stimulating cells. 1.3 x 10 of similarly isolated reaction cells and stimulants Adjust to cell / ml. To each well, 25 μl of acemannan or medium (control), 25 μl of RPMI-1640 supplemented with 10% fetal calf serum and 75 μl of each cell population are added. Cells are incubated at 37 ° C. under 5% CO: 95% air for 6 days. The cultures were kept for 4 hours Cells are harvested and counted after pulses with 25 μl of H-thymidine (1 μCi / well). To investigate the specificity of acemannan to afferent recognization and response to MLC, cells were recruited. An additional single oriented MLC is prepared with the added agent 20 minutes before pulsed with H-thymidine.

4. Mononuclear Cell-T Cell Interaction Lewis female rat spleens are chopped into sterile steel mesh into RPMI-1640 medium. Mononuclear leukocytes are collected from the interface of the Ficoll-Highpark density gradient as described above. Concentrate on a glass Petri dish and 10 monocytes adjusted to a final concentration of / ml were cultured with varying doses of acemannan or medium (control) at a total volume of 2 mlIncubate for 24 hours at. After monocytes were harvested and washed thoroughly with fresh medium, the ratio of the common genotype T lymphocytes and 10 T-cells: 1 monocytes was 37Co-culture by adding plant lectin botanical hemagglutinin (Difco, Detroit, MI) (1: 100) for 48 hours at. Cells are harvested on MASH II (Whittaker, MA Bioproducts, Walkersville, MD), placed in fluorine and counted with a scintillation counter (Beckman Laboratories, Chicago, IL). The T lymphocytes are incubated with acemannan, washed, and co-cultured with freshly prepared T lymphocytes and again with 10: 1 PHA-P to perform a control experiment.

B. Results

Homologous Antigen Responses Acemanan has no statistically significant effect on T-cell responses to autoantigens. When the agonist was added at the initiation of the MLC, the cells that were subjected to the co-type stimulus introduced the same triple hydrogenated thymidine in the presence or absence of the test agonist at the doses described. In the absence of oral acemannan, these MLCs incorporated thymidine 2616 ± 1099 cpm tri-treated at the end of the 4 hour pulse. Although the applied action of water capacity (2.6 x 10 ^-9 M from ^{3281 ± 1355, 2.6 x 10 -8} M in 3742 ± 1670 and 2.6 x 10 ^-7 M in 3828 ± 1978) is up-trend, but for, this to DNA There is no difference in the isotope incorporation ratio statistically significant.

In contrast to the absence of acemannan's effect on autoresponse in MLC, there was an effect of the agonist on alloresponse in the same immunological assay. First, acemannan does not interfere with lymphocyte capacity, so MLC recognizes and responds to type II homologous differences. This is evident when comparing homogenic cultures to homologous genotyping in the presence of the lowest concentration of drug. Secondly, there was a dose-response-related enrichment of allergic reactions by acemannan, so the culture treated with the highest dose of 2.6 x 10 ^-7 M reflects an almost 60% increase over the non-acemanan culture. . The dose response relationship is most convincingly demonstrated since the enhanced homologous genotype response appears to be pronounced for each dose of acemannan tested for acemannan non-addition conditions.

To determine whether acemannan exerts a specific effect on lymphocyte allogeneic reactions or a nonspecific effect on tritiated thymidine incorporation, lymphocyte cultures mixed for 7 days 20 minutes prior to addition of the tracer to the culture Reagent is added at the end of the water MLC. There was no effect of acemannan when applied in this way as a pulse at the end of MLC. These data support the specificity of the effects of acemannan on the enhancement of lymphatic responses in MLC.

2. Co-operation of Acemannans and Monocyte-T Cells To test the hypothesis that acemannans directly stimulate mononuclear cells that respond to homologous antigens and enhance lymphatic responses to antigens and / or mitotic substances, Purification populations are incubated for 24 hours with various doses of acemannan. The cells are thoroughly washed at the end of the culture and then co-cultured with T lymphocytes at a ratio of 10: 1 to simulate the natural ratio found in peripheral blood. Co-cultured cells are stimulated with plant hemagglutinin. Coculture with acemannan and precultured mononuclear cells significantly increased mitotic response in a dose-related fashion.

C. Discuss

This example examines the ability of acemannan to act as an immune stimulating drug of significant clinical value.

Acemanan is believed to be able to limit DNA and retroviral infections that cause significant disease in animals and humans. For example, acemannan improved feline virus non-bronchiolitis in animal models. Further evidence indicates that acemannan can be effective in vitro and in vivo against type II herpes simplex virus, measles virus and possibly HIV. This evidence indicates that immunological mechanisms can enhance monocytes as cells that contribute to the afferent recognition of phagocytes and antigens. These studies, on the one hand, directly enhance the phagocytic properties of monocytes and, on the other hand, increase the absolute number of important cells. Evidence supports the idea that acemannan enhances the manufacture of the signal substance IL-1 by activated monocytes.

The study described in this example specifically examines the mechanism by which acemannan may be an immune-enhancing reagent. Mixed lymphocyte cultures are in vitro models of how immunocompetent cells participate in the response to viruses and the response to various antigens required for recognition. In this reaction, there is an important monocyte-T-lymphocyte interaction that produces a response to homologous antigens. This is the model adopted to test the ability of acemannan to act as an immunoactivator.

Thus, acemannan is an important potentiator for homoantigen responses in MLC. There is a dose-response relationship that is enhanced at the highest dose tested at about 60% or more of the base. This not only shows statistical significance but also indicates a biologically relevant increase in response to homologous antigens, indicating that the drug can act as a means of assisting the organism's response to viral attack. This effect of acemannan is effective when the drug is added at the end of the MLC, unless the drug enhances the basic response to non-specific incorporation or tri-hydrogenated thymidine, a tracer DNA precursor, It has been shown to be specific for homogeneous stimulation.

In a second series of experiments, we test the hypothesis that monocyte-T-lymphocyte interactions may be involved, at least in part, in enhanced homologous reactions in MLC. In this series of experiments, acemannan is incubated with monocytes and then the treated and fully washed mononuclear cells are mixed with freshly prepared allogeneic T-lymphocytes that have never been exposed to acemannan. These experiments demonstrate a T-lymphocyte response enhancing and dose-response relationship to polyclonal mitotic vegetable hemagglutinin at the same level as the response previously seen in MLC (more than about 55% of the base).

The lowest dose tested in studies that were effective in MLC was ineffective in mononuclear cell experiments. It is not surprising that the limit doses are different for the two models tested and may differ for polyclonal response to mitotics and for homoantigen response in MLC. It can also be observed that a mononuclear cell experiment is a more rigorous test of the effects of acemannan on T cells that exhibit immune stimulation in the absence of drugs because they represent the cell type being treated (monocytes). Homoantigen responses can only or predominantly measure acemannan-enhanced monocyte production of IL-1, while polyclonal mitotic-enhanced responses each have a different limiting response to acemannan. May be the result of

The dose of acemannan used in these experiments is clinically relevant. The selected dose range is correctly chosen to lump the concentrations of acemannan that can be expected to be achieved in the plasma when the drug is dispensed into extracellular water and absorbed at a rate of 1/3 of the orally administered dose, and the aspect is Selection is based on previous pharmacological investigations in dogs. It has also been found that the actual concentrations obtainable in humans are in this range, further supporting the potential relevance of these investigations for clinical practice.

These experiments have shown that acemannan enhances T4 cell response to lectins by causing monocytes to release monocyte-induced signals. Acemanan did not enhance lymphocyte response to allogeneic antigens in MLC, while increasing MLC homoantigen response in a dose-related manner. This response has been found to be an acemannan-specific response at acemannan concentrations achievable in vivo.

This experimental documentation demonstrates that acemannans are immune enhancers and biological response modifiers in increasing lymphocyte responses to homologous antigens. The proposed mechanism of action involves the stimulation of monocytes to release IL-1; In the presence of acemannan, IL-1 has been shown to be released from monocyte culture. Pharmacological action on acemannan stimulation of monocytes may explain acemannan activity against viral infections in animals and humans.

Example 5

[Pharmacodynamic Basis for Correlation of In Vitro and In Vivo Efficacy of Acemanan]

To assess the pharmacodynamic aspects of acemannan, ¹⁴ C-labelled substances are injected intraperitoneally and intravenously and administered orally. Based on the results of previous pilot studies, an aqueous dose of ¹⁴ C-labeled acemannan 200 mg / 200 ml with specific activity of 17.4 cpm / μg is administered to the bitch (about 20 mg / kg). Blood, urine, and stool samples are taken at appropriate intervals of at least 48 hours. Organ and tissue samples are taken after the animal is sacrificed, and all samples are analyzed for radioactivity using a scintillation spectrometer.

Epidemiological aspects of acemannan are common as seen in most pharmacological preparations; However, its biological half life (t 1/2) is particularly long. Significant absorption occurs by administration of three routes. Maximal blood concentrations are achieved following intravenous injection followed by intraperitoneal injection and oral administration. Blood concentrations showing a maximum of 200 μg / ml immediately after intravenous injection decrease to t 1/2 of 50 to 60 hours; Plasma concentration is about twice the blood concentration. By comparison, blood levels after intraperitoneal injection peaked at 45 μg / ml at 24 hours and then decreased at a rate similar to that seen intravenously; In fact, blood levels reach a nearly 90% maximum in eight hours. By oral administration, blood levels are measurable after 3 hours and peak at 4-5 μg / ml. Based on the relatively long half-life in the blood, a therapeutic dosing interval of about 7 days is reasonable when considering the time required for three half-lives.

Radiolabeled acemannans are mainly distributed in the liver and spleen after intraperitoneal or intravenous injection. Liver, bone marrow, thymus and lymph nodes are the main sites of distribution after oral administration, and this finding is consistent with the immunological site of action on acemannan. Levels of radiolabeled compounds in tissues selected after 48 to 52 hours range from the lowest of about 1 μg / g in the brain to the highest of 85 μg / g in the spleen after intravenous injection. Interestingly, concentrations in the brain and spinal cord are higher after oral administration when compared to parenteral administration (about 3 μg / g tissue). This is because during the first pass the liver breaks down the polymer into smaller molecular weight fractions, allowing it to penetrate the blood-brain barrier.

In summary, from a clinical pharmacodynamic standpoint, the data indicate that ¹⁴ C-labeled acemannans (1) reach peak blood concentrations within 8 hours by all pathways studied, and (2) 7 days of treatment with relatively long biological half-lives. Allow for pharmacological dosing intervals, and (3) reach measurable levels in all tissue systems evaluated, including the central nervous system.

These pharmacodynamic data indicate that acemannan levels in the blood and / or tissue can multiply known levels that can achieve therapeutic anti-tumor or antiviral effects in vitro after injection or oral administration. For example, mice implanted with virus-infected Norman Murine Myxosarcoma (NMM) cells and intraperitoneally injected with 1 mg / kg acemannan within 24 hours were treated with NMM-treated control mice [Peng et al. Submitted for the study, 1990] and a survival rate of 35% after 60 days. The expected peak blood concentration at the intraperitoneal dose of 1 mg / kg is similar to 2 μg / ml (45 μg / ml x 1/20 mg / kg). Acemanan (2.6 ^10-9 M; 60,000 MW) added to T-lymphocyte culture at a concentration of 0.15 μg / ml increased the production of cytotoxic T-cells by 230% and performed the action of the resulting cytotoxic T-cells. Is increased by 138% to destroy target cells to which they are sensitized. Womble et al., Int. J. Immunopharmac. 10 (8): 967-974 (1988). Cytotoxic T-cells are believed to be produced against tumor cells such as NMM cells.

Blood concentrations of 4-5 μg / ml obtained after oral administration of acemannan were also found in vitro. This is significant because it corresponds to the concentration of acemannan that results in an optimal synergistic effect with (AZT). For example, 0.001 μg / ml AZT or 3.2 μg / ml of acemannan alone increased the viability of CEM cells infected with HTLV-III _RFII virus up to 10%. Together the protective effect of the antiviral combination exceeds 70%. Similarly, the combination of 0.1 μg / ml AZT and 1 mg / ml acemannan shows a protective effect of at least 80% [see; Kemp et al., Submitted for publication. 1990].

Thus, in conclusion, these biomedical studies have been studied in vitro and demonstrate that acemannan concentrations above known concentrations can be obtained in vivo.

Example 6

[Two Early Clinical Pilot Study Reports of Acemanan in HIV-1-infected Patients]

Prior to the discovery that human immunodeficiency virus type 1 (HIV-1) infections are a global health threat, there was limited development of antiviral drugs. Despite the recognition that more than 60% of all diseases in developing countries are caused by defined viral diseases, progress in this area is almost insignificant. Treatment in most areas consists of the application of mitigation measures designed to provide symptomatic relief and schistiness rather than disrupt viral replication. Due to the overall invalidity of palliative or symptomatic treatment, pandemic AIDS disease has begun the study of colostrum with novel antiviral compounds targeted to interfere with the replication cycle of HIV.

In the case of human immunodeficiency viruses, most attention has been directed to the synthesis and development of 2 ', 3'-dideoxynucleoside homologs, which are a family of antiviral agents that inhibit virus-encoding reverse transcriptase elements. One of these compounds, AZT, is the only drug approved for AIDS treatment. Unfortunately, numerous studies have shown that this compound is extremely toxic in vivo and its efficacy is significantly lower in vivo, although it is high in vitro (Richman et al., N. Engl. J. Med. 317: 192-197 (1987).

Two studies evaluated the response of human immunodeficiency virus type 1 (HIV-1) to acemannan and measured whether experimental values can be used to predict response to treatment. The protocol was submitted to the FDA as an example of a study of new medicine by an individual physician and included the Institutional Review Board of the Dallas-Ft. Worth Medical Center. Clinically evaluated using modified Walter Reed (MWR) clinical score measurements by treating HIV-1 antibody positive and symptomatic patients with about 400-800 mg oral acemannan daily. It was. CD4 / CD8 lymphocyte counts and HIV-1 (p24) core antigen levels showed immune competition and active viral load. In the first study, 15 original patients had an average NWR of 5.6, while 13 patients who survived 350 days after treatment had an average of 1.8. CD10 levels in 10 patients increased from 346 / mm 3 to 471 / mm 3 within 90 days and to 610 / mm 3 on 180 days. 5 of 15 patients have detectable serum core antigens; By 350 days only 3 of 13 were detectable but reduced serum antigens. Data from this first study suggests that CD4 and serum antigen levels can predict responses to acemannan. The second study in 26 patients confirms this. The aggregated group exhibits a mean group MWR value of 3.0 at start and a mean of 1.8 after 90 days. The CD4 levels of the 16 responders rose from 313 / kV to 372 / kV during this period, while the other 10 increased only from 63 / kV to 77 / kV. Fifteen of the 16 predicted to respond predominantly had elevated MWRs, increased CD4 counts and antigen reduction, indicating that the degree of immunosuppression and viral load affect the response to treatment.

Example 7

[Phase II Study with Acemanan Alone and AZT in Asymptomatic and Asymptomatic HIV Patients]

Forty-seven HIV + patients (23 asymptomatic patients, 24 ARC patients) participated in the double-blind randomized phase II study of acemannan. The protocol was approved by the relevant ethics committee at St. Pierre Hospital in Brussels. To assess safety and tolerability with or without concurrent AZT therapy, acemannan is administered for 24 weeks at a daily dose of 1000 mg (two 125 mg capsules, four times daily). Twenty-three asymptomatic patients were blindly placed to administer acemannan (11 patients. Group 1) or placebo (12aud patients, group 2). Of the 24 ARC patients who received AZT 1000 mg daily during the study, 12 patients (group 3) also received acemannan 1000 mg (2 capsules of 125 mg, 4 times daily) and 12 patients (group 4) also received Receive placebo; 33 of the 47 patients (70%) completed the 24 week study period (6, 9, 9 and 9 in four groups, respectively). The reasons for the decrease in the number of patients were: clinical development (8 patients: 3, 3, 0, 2), patient's own will (5 patients: 2, 0, 2, 1) or death (group 1, 1 patient). Suicide). No patient is dropped from the protocol due to side effects or poor resistance. Due to AZT therapy, there are statistically significant differences in drug side effects, mainly nausea cases, between groups 1 and 2 and 3 and 4. There is no difference between the acemannan groups 1 and 3 and the placebo groups 2 and 4. Hematological data is statistically compared between the four groups at the beginning of the study. There is a statistically significant difference in erythrocyte cell number and mean blood cell volume between patients with or without AZT at week 24, but not between placebo and acemannan patients. Of the four groups, there is no liver or nephrotoxicity.

Twelve patients (Group 3) treated with AZT and acemannan were treated to treatment Hugh Kartovsky score K when compared to patients treated with AZT alone (Group 4) (initial mean K = 81, 83 at end). Statistically significant (p 0.008) (initial mean K = 84, 90 at end). Two patients treated with AZT alone developed AIDS, which was not under combination therapy (one Kaposi, one esophageal candidiasis), but there were no statistically significant differences between groups 3 and 4 as to side effects. Comparison of CD4 cell counts in AZT-treated patients showed a significant increase (p = 0.01) at the end of the study among patients treated with combination therapy (group 145 / dL compared to group 4 and 252 / dL at end) Mean 263 / mm3 at 3 and 369 / mm3 at end CD4). The inventors concluded that acemannan is a very widely tolerated compound without biotoxicity and that among aRC patients, acemannan acts as an adjuvant therapy for AZT in the management of HIV infection.

Example 8

[Concentration-dependent Inhibition of HIV-1 Replication and Pathogenesis by In Vitro Acemanan]

Peripheral blood mononuclear (PBM) cells and two defined CD4 + cell lines, MT-2 and CEM-SS, are used as target cells for HIV-1 infection and treated with various concentrations of acemannan. The viability was determined by trypan blue dye-exhaust test or by vitreous cells to formazan of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] Measured by metabolic conversion. Viral replication and load are measured by hybridization of cell-associated viral RNA and cell-free RNA with HIV-1 probes prepared from the POL gene. The protection of PBM cells by acemannan treatment appears to be concentration dependent. The percentage protection is in the range of 14 to 100% for 3.2 to 100 μg / ml acemannan treated length. Protection by acemannan treatment of HIV-1 infected MT-2 cells is not only concentration-dependent but also multiplicity of infection (MOI) dependent. The protection rate of CEM-SS cells infected with MOI = 0.01 and treated with 62.5 μg / ml acemannan was greater than 85%. In addition to an increase in cell viability, a concentration-dependent decrease in coform formation is observed. The conjugates could not be detected in cultures treated with acemannan of 62.5 µg / ml or more. Concentration-dependent decrease in virus replication was also observed for treated PBM cells. PBM cells are treated with acemannan at a concentration of 62.5 μg / ml or greater to reduce 95-100% of detectable cell-associated viral RNA. Virus-infected CEM-SS cells were treated with acemannan at concentrations of 62.5 μg / ml or higher resulting in a reduction of at least 60% of cell-free virus. Acemannan treatment inhibits virus-induced cell fusion, increases infected cell viability, reduces viral load and inhibits the production and / or release of free virus. Cytotoxicity due to acemannan is not observed at any test concentration.

Example 9

Synergistic Antiviral Effect of Acemanan in Combination with AZT

The protective action of AZT and acemannan combination is measured in vitro using HIV-1-infected MT-2 cells at a MOI of 0.03. Checkerboard titration of the two drugs indicated that a synergistic protective effect occurred. Acemanans at concentrations below 125 μg / ml are most effective in this regard.

It can be clearly appreciated that some forms of combination chemotherapy are required to increase the efficacy of AZT while limiting its long-term toxic effects and bypassing further development of resistant HIV strains. For this reason and the apparent beneficial action of acemannan against clinical HIV infection when administered with AZT determines the determination of whether these two compounds exhibit synergistic inhibitory action in HIV replication in vitro.

The HTLV-IIIB strain of virus strain-HIV-1 was described in Bethesda NIH, Maryland. Obtained from Dr. Gallo. Virus stocks are prepared by propagating virus in H9 lymphocytes. Stock preparations of the virus are stored at -80 ° C. A 50% tissue culture infection dose (TCID50) / cell-free virus blend stock ml is measured by endpoint titration with MT-2 cells. Infection multiplicity (MOI) is determined by the Reed and Muench method.

Cell line-MT-2 cells are grown in RPMI-1640 supplemented with 2 mM L-glutamine and 15% (v / v) calf serum. MT-2 cells naturally express CD4 on their surface, making them excellent target cells for HIV-1 infection. In addition, they rapidly progress cell lysis at low viral replication levels.

Primary Test of Antiviral Activity—MT-2 cells are first treated with polybrene (2 mg / ml) for 30 minutes and then infected with HIV at MOI 0.03. After virus uptake, cells are pelleted and resuspended in complete medium. Infected cells are then dispensed into 96-well microtiter plates (2 × 10 ⁴ cells / 100 μl / well).

Each drug is diluted in dilution ratio of 1/2 log _{10 for} 6 consecutive times in media from 2 mg / ml stock solution. AZT is tested from the highest concentration of 10 μg / ml to a low concentration of 0.032 μg / ml. Acemanan is tested at 500 μg / ml and gradually diluted to a low concentration of 15.62 μg / ml. Similar assays are performed three times and drug cytotoxicity is measured twice at similar concentrations. Controls include uninfected, untreated cell cultures and virus-infected untreated cultures. Plates are incubated for 7 days in a humidified atmosphere of 5% CO ₂ in air. On day 7 after infection, MTT (450 μg / ml) is added to the test plate to measure cell viability. Then, 10% sodium dodecyl sulfate solution in 0.01 N HCl is added to dissolve the resulting MTT promazan. Color intensity is a function of the amount of formazan produced, which is proportional to the number of viable cells in each well. The plate is read under a wavelength of 570 nm on a Vmax plate reader (Moleculer Devices, Inc.). % Growth rate of cells is calculated by the following formula:

Where TI is optical density (OD) in treated infected cells, TO is OD in treated non-ramitis cells, OI is OD of untreated infected celllets, and OO is OD in untreated uninfected cells to be.

Antiviral Activity of Acemannan-AZT Combination—Antiviral activity of acemannan in combination with AZT to varying degrees is assessed using the micro; titer infection assay described above. For each mixture, a defined amount of test compound is dissolved in RPMI-1640 and 0.1 ml of each dilution is added to the test wells. The combination is evaluated twice and drug cytotoxicity is measured using the treated uninfected control. Each compound is also evaluated alone at non-cytotoxic concentrations. Thus AZT is tested at a concentration range of 0.32 to 10 μg / ml and acemannan is evaluated at a concentration range of 15.62 to 500 μg / ml. % Cell viability change is measured as above.

The effect of the AZT-acemannan combination on HIV-infected MT-2 cell viability is shown in FIGS. 1 and 2.

Acemanan alone shows up to 40% protection at a dose of 60 μg / ml. At higher doses the protective effect is not constant. AZT alone shows up to 60-67% protection in a dose range of 0.03 to 0.32 μg / ml. However, the cytopathic effect can be completely eliminated by 0.32 μg / ml AZT in the presence of 15.62 to 250 μg / ml acemannan.

The data here suggest that AZT and acemannan cause synergistic protective action on MT-2 cells infected with HIV-1 in vitro. These results support clinical evidence for the same effect. By synergistic effect is meant that the effect of a mixture of compounds, when acting alone, is greater than the sum of their effects. Thus, when 0.01 μg / ml AZT is mixed with 15.62 μg / ml acemannan (each of which does not show protection), it shows 32% cell growth. In fact, at unprotected acemannan concentrations of 31.25 and 15.62 μg / ml, a significant increase in the 0.1 μg / ml AZT protective effect can be observed.

When 0.32 μg / ml AZT (65% protection) is mixed with 62.5 μg / ml acemannan (40% protection), the mixture shows 100% protection. Obviously, over 100% viability cannot be detected, so in this case any synergistic effect is concealed but the effect exhibits an additional pseudo appearance.

The synergistic effect appears to indicate that AZT and acemannan interfere with viral replication at different stages of the virus cycle. As a result, hits below the lethal dose in one stage of the HIV replication cycle can compensate for hits in the other stage. The combination of the two hits together represents a lethal action for the virus. AZT is, of course, well known as a reverse transcriptase inhibitor of viruses. Acemanan, on the other hand, can interfere with glycosylation and HIV envelope processing.

Example 10

[Acemannan Used to Treat Skin Ulcers]

A 83-year-old female patient, TB, has a 25 mm diameter ulcer at the lateral edge of her left foot. Ulcers have progressed for months and have not responded to various treatment regimens.

The affected area is treated with the product of Example 3 of US Pat. No. 4,735,935 and the product of Example 7 of US Pat. No. 4,735,935 using three treatment schedules per day. The washed lesions are immersed for 15 minutes with the product of Example 2 of US Pat. No. 4,735,935. Excess product from the affected area is absorbed with dry sterile 4 × 4 gauze. The product of Example 7 of US Pat. No. 4,735,935 is then applied in an amount sufficient to coat the affected area and to prevent dehydration in the affected area when changing the dressing.

Wound healing progression is measured by differential imaging and area meter. The progression of the wound closure is shown in Table 11.

In fact, the epidermal defect site is sutured in 12 weeks, and by 14 weeks it is fully closed.

Example 11

[Acemannan used as a treatment for trigeminal neuralgia]

The fifth trigeminal neuralgia or neuralgia is characterized by severe and intolerable pain over one or more branches of the trigeminal nerve. This pain is usually temporary and can be sedated by touching some areas of the face, called so-called trigger zones.

The cause and cure of diseases involving such pain are not known. Several attempts to cure these diseases have rarely or never succeeded. Various therapies include analgesics, phenytoin, peripheral excretion of related nerve branches when leaving the skull and injection of 98% alcohol into the meniscus ganglion.

More potent treatment --- segmentation of the sensory muscles of the nerves close to the ganglion --- leaves the patient permanently numb in the area provided by the segmented nerve. Another recent treatment attempt uses carbamazepine and phenolriofendylate infusion. However, these infusions can be complicated by unpleasant numbness and severe side effects.

None of the treatments described above is desirable.

Diagnose a 43-year-old woman with trigeminal neuralgia. The infected area comprises the first and third segment of the right trigeminal nerve.

The patient may cause pain by combing or brushing the right hair. This patient failed to treat with diazepam (Valium), antihistamines, analgesics, propranolol hydrochloride (Inderal) and phenobarbital. The patient said there has been no painless day since the onset.

The proposed treatment involves drinking 1 to 2 oz per day for three months of the product of Example 2 of US Pat. No. 4,735,935. After this period, treatment is evaluated.

Patient pain is significantly reduced within two weeks of treatment. The patient said he was comfortable for several weeks. However, symptoms and pain recurred during the two weeks the patient had not consumed the product. However, after taking the medication again, the pain disappeared within a few days. Over the next few weeks, she again felt comfortable.

After drinking the product every day without interruption for more than six months, the patient said he could brush or comb the hair without causing pain. The patient's appearance improved and the patient said that he was more comfortable than before.

Example 12

[Investigational Clinical Pilot Study Using Acemanan in Inflammatory Bowel Disease]

Inflammatory bowel disease (IBD) is a collective term for Crohn's disease, ulcerative colitis and other symptoms of the gastrointestinal tract. Crohn's disease mainly occurs in the ileum and colon, while ulcerative colitis is limited to the colon. Three or more reliable hypotheses have been proposed to explain the etiology of IBD. One hypothesis is that unknown pathogens, such as slowly growing bacteria or viruses, cause the immune system to initiate a chronic inflammatory response. The second hypothesis is that the same process is caused by toxic substances such as pollutants or environmental pollutants contained in food. The third hypothesis is that the inflammatory response is an autoimmune symptom. However, the exact cause (s) of these diseases is not known.

A. Patient Selection

The study protocol was submitted to the FDA as a research immune drug by a physician and approved by the Institutional Review Board (IRB) of the Dallas-Fort Ward Medical Center. Patients are selected regardless of age, gender, ethnicity or ethnic background and are all volunteers. Each person must sign a signed informed consent form with a informed consent briefed by the physician.

Only patients with a combination of the following symptoms and IBD symptoms are allowed:

Diarrhea (intestinal exercise)

2. Blood is discharged from the stool (latent blood)

3. excessive mucus production

4. Spontaneous abdominal pain

5. Abdominal pain at palpation

6. Consecutive spasms

7. Others (weight loss, etc.)

The above-mentioned symptom is used when evaluating a clinical score of 0 to 7, with 1. indicating a single symptom as 7 showing all symptoms. A score of zero indicates no symptoms in the patient.

B. Endoscopic Evaluation

Endoscopy is used to evaluate patients before and after treatment according to the following criteria assessment:

Ulceration

Convergence

Oscillation

-On board

Splitability

2. Congestion

3. Exudation

4. Other

The same skilled endoscopy examines mucus appearance according to distribution and depth. A rating of 0 indicates that the mucous appearance of the patient is normal and a rating of 5 is the most severe.

C. Histological Assessment

Histological evaluation scores are recorded as follows: exudate, ulcerative mucus, edema, plasma cell lymphocytes, polymorphonuclear cells, eosinophils, granulomas, yin abscess, fibrosis, etc.

The clinical, endoscopic and histopathological criteria are used to grade the manifestation of IBD and to quantify the response to acemannan treatment. Physical examination with histoscope and histological sampling to limit regularly scheduled visits. Drug administration can be discontinued at any time without cause or shock during normal treatment. Acemanan is supplied by Carrington Laboratories, Inc.

D. Clinical Results

Nine IBD patients are allowed and treated with 200 mg of acemannan in capsules daily. Patients range in age from 14 to 46 years and include four women and five men. Typically, patients have abdominal pain, diarrhea or a number of bowel movement symptoms, and there is a blood or aqueous substance, or a combination of these elements, typically with increased mucus production in the bowel movement. Initial endoscopy reveals a spectrum with mucus changes from vascular bleeding with mucous brittleness to local, widespread fusion ulcer, ie, pan-colitis. Histological examination of the intestinal biopsy showed that the extent of the damage ranges from non-specific increases in chronic inflammatory cells to true ulcers with multiple polymorphonuclear cells and eosinophils. Two patients have microgranulomas and yin and abscess. All patients have been shown to be unresponsive to conventional reagents, including one or more azupidine, prednisone, 6-mercaptopurine, and flagyl. Imodium and mental stabilizers are often added to the reagents.

In all patients, the response to the acemannan drug was uniformly desirable and all scores were improved. Mean ratings before and after drug treatment were as follows:

Average clinical rating before treatment 4.56

(Average of nine patients)

Average clinical rating after treatment 0.44

(Average of nine patients)

Average Endoscopic Score Before Treatment 3.88

(Average of eight patients)

Average Endoscopy Rating After Treatment 0.00

(Average of two patients)

Average histologic grade before treatment 6.25

(Average of eight patients)

Average histologic grade after treatment N / A

(All applicants refuse biopsy)

During the study, no side effects caused by acemannan were observed. Some patients, whose symptoms of illness were quite severe, said they had virtually no pain and symptoms within two to five days. Other patients, especially those with locally divided diseases (Crohn's disease and ileitis), show the effects of acemannan slowly, less rapidly. All patients refused post-treatment biopsy, and only two patients allowed post-treatment endoscopy. The following reasons were given by the patients:

Costs are unjustified due to (1) inconvenient biopsy procedures and (2) improved symptoms.

Two patients had episodes of inhaling acemannan only when symptoms occurred by chance. Both said symptoms were alleviated 24 to 48 hours after using the drug, but weak symptoms were reproduced 4 to 6 weeks after the acemannan treatment was not continued. Subsequently, when acemannan was treated again for two to three days, the symptoms were alleviated. Acemanan provides a rapid clinical advance in the acute inflammatory state of disease

Example 13

[In vitro Effect of Acemanan on Measles Virus]

Measles virus is incubated with various concentrations of acemannan and then added to infectious cultures of VERO cells. The purpose of this experiment is to determine whether acemannan inhibits infection or inactivates measles virus treated with acemannan before introduction into infectious cell culture. Acemanan-treated viruses do not infect VERO monolayers, as evidenced by the absence of the cellular disease effect (CPE) of the virus at a threshold concentration of 2.5 mg / ml. Complete absence of CPE is obtained at 5 mg / ml acemannan in virus inoculum.

African green monkey kidney cells (VERO cells) are used as target cells. Measles virus is titrated to obtain a total spot number of 30-50 spots / ml (20 TCID units / 0.05 ml) on virus / cell monolayer. Acemanan is then introduced into the medium containing this fixed amount of virus at different concentrations.

Acemannan concentrations are in complete tissue culture medium. An attenuated Rubella virus vaccine aliquot is used for each titration. The mixture is preincubated at 30 ° C. for 1 hour 30 minutes and added to a VERO monolayer in a previously prepared tissue culture chamber.

The combined results of various concentrations of acemannan incubated for 5 days on measles virus and confluent VERO cell monolayers are provided in Table 12.

In case of repeated challenge with various concentrations of acemannan, a protective concentration is obtained at 2-4 mg / ml, which is a transition zone for measles virus infectivity inhibition. Note Table 12. Acemannan concentrations of 5 mg / ml provide constant protection against VERO cell monolayers challenged with measles virus pretreated with acemannan.

In this pilot study, the effects of acemannan against measles virus were: 1) VERO cells alone (negative control), 2) VERO cells inoculated with measles virus (positive control), and 3) measles virus pretreated with acemannan. Evaluation is made by comparing the prepared VERO cells. Spot production is significantly reduced in acemannan-pretreated virus-infected cultures (# 3) as measured by spot total analysis. Full protection of the culture from viral infection is obtained when the virus is pretreated with 5 mg / ml of acemannan.

Example 14

[Performance of Acemannan to Reverse Measles Virus Infection in VERO Cell Cultures]

VERO cells are incubated in medium containing 40 TCID / ml measles virus for various periods (0.5-6 hours), followed by addition of 5 mg / ml of acemannan. When cells are exposed to measles and viruses and incubated with acemannan, they do not protect VERO cells from infection.

VERO cells are incubated for 0.5 to 6 days with medium containing 40 TCID / ml measles virus. VERO cells are then washed with fresh medium to remove unbound virus. Subsequently, a medium containing 5 mg / ml of acemannan is added to the culture and the culture is examined after 5 days for cytopathology.

The results of this experiment are shown in Table 13.

It is noted that the infection of the acemannan preculture culture of 1 hour 30 minutes is lower. VERO cells do not appear to be clinically significantly protected in cultures pre-cultured with acemannan for longer periods of time.

VERO cells preincubated with measles virus are not significantly protected from infection by adding 5 mg / ml of acemannan after the infection phase is over.

Example 15

Project for Determination of Acemannan Efficacy in Evoking a Protective Immune Response in Normal Poultry

Nationwide, the poultry industry loses more than $ 2 billion annually on costs related to disease and management problems. Retroviruses that cause death and / or disease associated with immunosuppression, pathogens such as infectious bursitis disease virus (IBDV), cause significant economic losses to the livestock industry. IBDV specifically targets precursor B-cells in the Fabrycis mucin capsule that cause selective destruction of the humoral arm of the immune system. This results in an immunosuppressive condition similar to acquired immune deficiency syndrome (AIDS).

The poultry industry routinely vaccinates sheep for IBDV by oral administration of live virus or subcutaneous injection of inactivated virus. Although both immunization methods can effectively induce an immune response, inherent problems associated with the use of the vaccine are introduced. Live viral vaccines are more effective in inducing a protective immune response against specific strains, but the virus itself may restore virulence or may be temporarily immunosuppressed to increase the infectivity of both populations against positive secondary pathogens. have. Killed viral vaccines do not have the same problems associated with live viral vaccines, but their immune responsiveness is reduced and dose dependent. Many alternative methods for vaccination involving complex high-tech solutions are being evaluated, but direct variation in immune response by incorporation of additional components in a killed viral vaccine implies a potentially simple solution.

Based on preliminary observations, acemannan acts as an immunomodulator, and this project is designed to determine if the compound induces an immune response against an isolated infectious IBDV vaccine.

A. Animals

Chickens hatched from eggs (purchased from SPAFAS, Inc) are used for all experiments. One day after the eggs hatch, the chicks are placed in Horsefall Units.

B. Antigens

BursaVac K (oil emulsion)-used acemannan: lot # 80226-001; Resuspend at 1 or 2 mg / ml (see experimental design).

C. Design of Experiments:

Study # 1 (Group 1). For Study # 1, 25 2 week old chicks were grouped into 5 groups. Each group of chicks is vaccinated as follows:

Group 1-mock inoculated control

Inoculate subcutaneously on the back with group 2-0.5 ml oil emulsion vaccine.

Group 3-Subcutaneous inoculation with a 1: 1 mixture of 0.25 ml acemannan (0.5 mg / ml) and 0.25 ml oil emulsion vaccine (purchased from Bio-Burs K: Key Vet., Gainesville, GA) suspended in water box.

Orally inoculated with 0.5 ml microcapsules suspended in group 4-acidic water.

Oral inoculation with 0.5 ml microcapsules suspended in acidic water with group 5-0.5 mg of acemannan.

Study # 2 (Group 2). For Study # 2, 117 SPF chicks, one week old, were divided into six groups. Each group of chicks is vaccinated as follows:

Group 1-mock inoculated control

Group 2- Inoculated subcutaneously in the back with 0.5 ml (2 mg / ml) of acemannan suspended in water.

Inoculated subcutaneously with a group 3-0.5 ml oil emulsion vaccine (Buy-Bio-Burs K; Key Vet., Gainesville, GA).

Inoculate subcutaneously with a 0.25 ml oil emulsion vaccine mixed 1: 1 with 0.25 ml acemannan (1 mg / ml) suspended in group 4-water.

Group 5- Inoculated subcutaneously with a 0.25 ml oil emulsion vaccine mixed 1: 1 with 0.25 ml acemannan (2 mg / ml) suspended in water.

Group 6-0.5ml oil emulsion vaccine subcutaneously inoculated on the back and inoculated subcutaneously into the femur with 0.5ml of acemannan (2mg / ml) suspended in water.

For both studies, serum is taken from each chick at weekly intervals and serum IBDV ELISA titers are measured using a commercial AgriTech IBDV ELISA kit. FlockTech software program (source: AgriTech Inc.) is also used for potency measurements.

D. Results

The chicks were comfortable with subcutaneous or oral administration of acemannan suspended in water or oil emulsion and had no side effects.

For Study # 1 (Group 1), average ELISA titers over 6 weeks post vaccination are shown in Table 14.

Two weeks after the first vaccination, the titer for IBDV begins to increase in chicks treated with oil emulsions or oil emulsions fed with acemannan. Chicks treated with an oil emulsion vaccine fed with acemannan, the overall average titer is about 3.9 times higher than that of chicks vaccinated with the oil emulsion vaccine. Three weeks after vaccination, chicks are re-inoculated and each chick receives the same antigenic mixture as presented at the first vaccination. One week after the second vaccination, the difference in mean titer ratio is increased to about 4.1 times. Two weeks after the second vaccination, when the average titer for both groups reaches the peak, this ratio drops to about 2.1. By three weeks after the second vaccination, the mean titers for the two vaccinated groups began to decrease, but the decrease in titers for chicks pre-inoculated with oil tanning was even more severe, with a 55% reduction in titers compared to The titer reduction of chicks vaccinated with the oil emulsion with mannan was 31%. In birds treated with an oil emulsion fed with acemannan, the titer remains higher due to the prolonged immune stimulating action of acemannan.

Three weeks after the second pre-inoculation, the chicks of the oil emulsion vaccine group (# 2) and the oil emulsion vaccine group (# 3) fed acemannan were divided back into two groups (A and B). Chicks in group A are challenged with homologous live vaccine strains, and chicks in group B are challenged with virulent strains. Three days after challenge, all chicks are autopsied. There is no effect on the immune system in chicks of group A challenged with the vaccine strain. However, all chicks in group B are lesioned as evidenced by histological examination. These results are expected, but when challenged with chicks receiving only the first vaccination, the lesions of chicks only receiving the oil tanning vaccine were more predominantly observed. When chicks are preinoculated with live viral vaccines, lesions appear in the lymphatic organs of chicks that are resistant to homologous viral challenges.

For study # 2, group size and vaccination protocol are changed. As can be seen from Table 15, the results are inconsistent.

Differences between birds are initially commented two weeks after injection. There are more runts than expected and appear to be infected in some areas of the banded chick; In addition to secondary bacterial infections, there is compression necrosis that releases toxins. To avoid the latter problem, the chicks are re-banded and treated with topical antibiotics. However, the described problem will probably lead to overall immunosuppression, thus invalidating the results of this study. Thus, the experiment is terminated.

In spite of the negative factor associated with Study # 2, acemannan results in an overall immune stimulating effect of the immune system, ie, an enhanced immune response to test antigens administered at sites far from the site of acemannan administration. At first it seemed that acemannan had to be mixed with an oil emulsion vaccine, but even when the antigen and acemannan are present separately, an enhanced immune response seems to be induced. These results develop applications and alternative vaccination methodologies for these compounds.

Acemanan has secondary properties. This increases the persistence or effective expression of IBDV antigens in the body, releasing lymphokines and enhancing lymphocyte responses.

Example 16

[Acemannan Used to Treat Absorption Syndrome]

Human malabsorption syndrome can eventually lead to wasting symptoms that can lead to death. Disruption of certain particles containing complexed polysaccharides and enzyme inhibitory peptides from the diet can alleviate certain human syndromes such as sprue and celiac disease. The consequences of this dietary change alleviate the symptoms. However, important physiological problems remain with the patient; Maturation of the small intestine mucosa is inhibited due to the inhibition of glycoprotein synthesis, which is essential for cell maturation. Failure of these small intestinal interactions reduces the absorption surface and further prevents absorption of essential amino acids, fatty acids, minerals, vitamins and other important molecular substances present in the diet.

A 56-year-old man who lost more than 40 pounds of weight due to chronic gluten-sensitive sprue rapidly gained weight gradually after chronic diarrhea after oral administration of 500-800 mg / day acemannan.

Mannose is required for glycoprotein synthesis. Feeding the diet with additional mannose probably increases the rate of glycoprotein synthesis by changing the Km rate. Enzyme synthesis is facilitated by the availability of critical mannose substrates that promote ribosomal / glycoprotein synthesis by mannose-metabolase enzymes. Increased synthesis and availability of these glycoproteins matures small intestinal mucosa cells and reduces symptoms associated with sprue and celiac disease. In addition, these thermodynamic changes in glycoprotein synthesis have applicability to other categories of diseases for which current therapy is invalid.

Example 17

[Acemannan of symptomatic drug agent related to multiple sclerosis]

Multiple sclerosis (MS) is an unknown etiological neurological disease without effective treatment. Patient data and demographic analysis indicate that this disease is probably initiated by pathogens of viral origin. Central nervous system lesions, spinal fluid and serum analysis show that autoimmune components are also present. This autoimmune response breaks down myelin.

A 36-year-old patient who has had multiple sclerosis for more than six years and has been lying for four months is treated with steroids and walked in the house using a walking aid available. Wheelchairs are required to go out of the house. The attending physician advised the patient that he would return to seat preservation within six months without chemotherapy (including treatment with cytoxane).

The patient discontinued all prescribed therapies and took orally about 500 mg of acemannan daily. The patient's condition had not changed until about 10 weeks, and by about 10 weeks, the patient was more comfortable than before. After two weeks, the patient can walk longer using a walker, after a month or more using only a cane, and for several hours after using the cane or walker after the patient's seating condition (as the physician said). You will be able to shop.

Patients remain at this level for more than 10 months and continue to receive approximately 500 mg of acemannan per day without any other therapy.

In 1986, F.G., a 57-year-old female patient, had a history of weakening of the legs, gradual decline in the volume, and a number of other neuromuscular symptoms. MS was diagnosed by NMR at the Johns Hopkins Medical School. An extensive training program was also initiated as patients began receiving oral administration of 800 mg / day acemannan.

Six weeks after the start of treatment, patients were able to wander considerably longer with limited physical protection. The patient's volume grew stronger and other symptoms improved. At six months, the patient's neurologist returned spots visualized by NMR technology. The patient continued to relieve MS related symptoms.

Example 18

[Acemannan used as antiviral agent of water]

Asemannan is evaluated as an antiviral agent for the LaFrance virus, a major problem in the mushroom cultivation industry. The cultured soil used is prepared by modifying the method of Flegg et al. La France virus-infected Agaricus bisporus M8 span is added to prepare a culture soil with 3% anhydrous material. The trays on which mycelium is produced are covered with plastic and incubated at 24 ° C. for 14 days. Acemanan is added to the culture soil from which mycelium is produced at a dosage of 0.01 to 2% (calculated on anhydrous weight basis) and placed in a 11 '' x 7 '' tray with 1.0 pound anhydrous / tray. The material is mixed evenly with the culture soil by adding two components to the sampling bag and thoroughly mixing. Invert the treated cultured soil with mycelium formed and place peat moss of water moss on top 24 Incubate for 7 days at more; The material placed on top is then gently mixed, the plastics replaced and the trays incubated for an additional 10 days. Then remove the plastic and replace the tray with 18 Incubate in the environmental chamber for an additional 7 days. The mushrooms are harvested every week after three weeks.

The scoliosis is analyzed for double stranded (ds) viral RNA by homogenizing in 20 ml of STE (1.0 M, NaCl; 0.5 M Trizma base, pH 8.0 and 0.01 M EDTA), 20 ml of LiCl and 10 ml of 10% SDS. The ds RNA is extracted in phenol and the aqueous phase is passed through a Bio-Rad LC column containing Whatman CF-11. Cellulose bound ds RNA is washed and eluted with STE. Aliquots are characterized by agarose gel electrophoresis after precipitation with ethanol and resuspension in citrate solution. ds RNA patterns are visualized by ethidium bromide staining. A decrease in ds RNA is seen in the coliforms analyzed from the experimental trays containing 0.01-1.0% acemannan compared to the untreated group.

Example 19

[Acemannan Used as a Treatment for Chronic Fatigue Syndrome]

Acemanan has been shown to affect chronic viral syndromes in humans. A 41-year-old woman with a history of markedly debilitating chronic fatigue syndrome (CFS) and increased Epstein-Barr titer over two years consumed 800 mg / day of acemannan for six months, resulting in complete drowsiness. Relaxed. After three months without symptoms, the patient ceased oral administration of acemannan. By taking acemannan again, symptoms of these syndromes were alleviated quickly.

One physician sister had a longer period of chronic fatigue syndrome with increased Epstein-Barr antibodies. Many clinical evaluations and therapeutic diets were also invalid. This symptom was eliminated by a significant improvement two to three months after the initiation of acemannan therapy, in which the patient consumed 800 mg of acemannan daily.

Example 20

[Combination of Radiation, Chemotherapy and Acemanan for the Treatment of Tumors of Sub-Tissue Origin]

W.H., a 41-year-old medical center, had severe chest pain. The radiographs show the mediastinal mass of possible vascular origin and the patient is transferred to the cardiovascular center. The core extended from the lower neck to the diaphragm and grew between the lungs and measured to include the heart basal. Biopsy revealed a malignant fetal ductal tumor. Oral administration with acemannan (approximately 500 mg / day combined with radiation and chemotherapy). After six years of treatment, the patient had a lively life with normal normal chest X-rays. A review of the literature showed that 20 tumor patients died 100% 9 to 12 months after diagnosis.

Example 21

[Combination Drug Combination Chemotherapy and Acemannan Therapy of Liver Tumors]

Patient M.A. said that the main complaint was that he did not fasten the zipper on his pants and did not tie the belt around the abdomen. Computerized tomography (CAT) injections in April 1988 revealed multiple tumors that spread to the bladder in the liver. More than 20 tumor masses, usually less than 10 cm in diameter, were located in the liver. Expected lifespan was 4-6 weeks. Patients have been given oral administration of acemannan for HIV-1 infection at 800 mg / day. Several chemotherapy treatments are initiated and acemannan continues to be administered. Patients had minimal common toxicity and side effects associated with cancer chemotherapy. Three months later, CAT injections estimated a 60% reduction in tumor mass. After 6 months, 85% of the tumor mass was reduced. By 12 months, tumors are minimal, and by 24 months only the smallest scars of questionable tumor masses appear. All clinical experimental studies are normal and the patient is doing well now.

Example 22

[Acemannan Treatment of HIV-Related Skin Tumors]

Patient S.G. showed two black lesions that were already diagnosed as Kaposi's sarcoma by biopsy that could be palpable in the arm. Acemannan gels containing 5% DSMO are applied topically onto the skin mass of the arm, while similar masses of other body parts are not treated. Reexamination at weekly intervals markedly flattened and discolored the lesion. After 60 days of treatment, only the flat scar area remains. Subsequent treatment of other lesions of the same patient shows the same result.

Patient TPD exhibited palpable ankle lesions with typical Kaposi's sarcoma pigmented. Remixed 1cc of interferon (Roche) is injected subcutaneously. Size and pigmentation are enhanced in about three days. One week after the topical acemannan gel was applied to the band, there was no sign of lesion. There are no scars or changes in pigmentation.

Example 23

[Acemannan Treatment of Premalignant Skin Lesions]

W.B., a medical practitioner whose head began to peel, developed a number of sunburn keratosis on sun-exposed skin. Acemannan gel was applied to these lesions every night for two weeks, eliminating the appearance of premalignant skin, skin, and smoothness. This response also occurs in many other older patients.

Example 24

[Acemannan Treatment of Edema Associated with Cancer Surgery]

In January 1984, J.J., a 32-year-old male patient with unsuccessful surgical incisions, radial neck incisions, radiation and chemotherapy of the primary pharyngeal tumor, had unbearable pain due to the overall occlusion of the larynx and esophagus. Due to lymph node blockage, the head is about twice its normal size and edema has lost its entire facial features. Life was retained due to gastroscopy and tracheostomy. Topical acemannan gel (CDWG 0.025%) is applied every 8 hours throughout the head, neck and shoulders (TID). By about three days, the edema was markedly reduced, and by about ten days, tissue fluids became excessive. Around 14 to 16 days, the base of the neck, the angular area of the chin and the area behind the ear became undulating. Upon incision of the skin, the necrotic tissue began to drain from the site of the previous hard, impermeable nodular tumor. The patient began to spill off the white, degenerated tumor mass. Multiple transfusions for continued bleeding. Slowly, the patient talked, ate the soup, and breathed through the mouth and nose.

Example 25

[Cancer Treatment of 5-Fluorouracil and Acemanan Combination]

Patient C.M. underwent peritoneal resection with a slight degree of rectal adenocarcinoma and mesenteric lymph node metastasis. The patient refused radiation treatment and received intravenous 5-fluorouracil (5-FU) weekly and 800 mg of acemannan daily. Patients usually did not show nausea with oral ulceration, severe fatigue or vomiting associated with 5-FU treatment. After extensive investigation by isotope injection and computerized tomography (CAT), the patient showed no detectable adenocarcinoma 24 months after surgery and continued normal.

Example 26

[Multiple Drug Combination and Chemotherapy and Acemannan Cancer Treatment]

Patient H.H. showed colon adenocarcinoma, and resection increased CEA tumor nodules and increased liver nodules by CAT injection. Beginning oral administration with acemannan (800 mg / day), 5-FU (500 mg) dicarbazide (50 mg) was intravenously injected weekly and acemannan was orally administered (800 mg). Gradually the tumor size and CEA values decrease and there is no evidence of side effects or biochemical or hematological toxicity.

In 1990, a 66-year-old male patient, V.G., showed bone and liver metastases after pulmonary resection to sedimentary cancer of bronchioletic origin. Alkaline and total liver enzymes are three times more than the upper limit of normal. One week of treatment with 500 mg 5-FU and 50 mg dicarbazide intravenously and 800 mg of acemannan daily orally daily reduces alkaline phosphatase and hepatic enzymes to half of pretreatment levels with overall patient improvement .

Example 27

Treatment of Acemannan Cancer with Male Hormone Therapy and Multiple Drug Combination Chemotherapy

A 72-year-old male patient, J.R., showed an increase in sun phosphatase (PAP) and prostate specific antigen (PSA) after prostate metastatic adenocarcinoma surgery and after radiation therapy. Patients were orally administered acemannan (800 mg / day), 5-FU (500 mg) and dicarbazide (50 mg). Tumor marker growth is constant, reaching a peak of 15 units in PAP and 186U in PSA. Anti-hormonal agent eulexin is added to the diet. PAP decreases to 3.0 and PSA to 15 within 60 days. This reaction occurs at no stage without toxicity or side effects. The patient constantly monitors the entire diet.

Example 28

[Acemannan Treatment of Toxin-releasing Animal Occlusion]

Acemanan has been shown to alter the body's response to antigens, toxins, allergens and autoantigens. Two acemannan gels were sent to Swangi Province in southern China. The product was received for use in burns, bedsores, congestive ulcers and diabetic skin ulceration in a red cross apparatus. About a year later, the Rep. Delegation stated in a letter that Acemannan gel was a useful treatment for the disease, and it also recorded the best therapeutic effect than anything that has been used for bite snake bites that commonly occur in manual work paddy fields. Doing. Snake occlusion is often infectious and does not respond to antibiotics, so necrosis resulting from deep ischemia in soft tissues causes significant loss of skin and muscle tissue. The wound is dressed closed with an acemannan gel to remove non-control infections and assist in capillary circulation to surrounding tissues. This treatment preserves hands and toes, muscles, nerves and soft tissues.

Example 29

[Return to Neoplasia and Function of Acemannan-treated Cultures of Fibroblasts]

Acemannan-treated cultures of fibroblasts obtained from 60 year old males show changes in the morphology of these senescent cells. Such changes appear to demonstrate the reversal of the aging process in these human cells in vitro. Long-term fibroblast cultures treated with acemannan (1 mg / ml) in the culture medium showed biochemical and morphological properties of neoplastic cells.

In 1989, a physician at Duke University examined facial skin biopsies from surgical specimens submitted to the pathology department. Biopsies are obtained from Mohl's surgery performed for skin cancer treatment. Post-operative acemannan gel was applied. The specimen destroys damaged collagen fibers, rapidly regenerates immature collagen fibers by expanded fibroblasts, and exhibits a rare necrobiosis pattern. The result is a rapid remodeling of the structure in these aging skin samples.

In 1990, the same medical practitioner examined the thymus of dogs receiving acemannan. These thymus are 2 to 6 times larger than the thymus of control dogs. The thymus of treated dogs is microscopic cell hyperplasia, which demonstrates hyperplasia and activation of thymic cells. Increased numbers of T-lymphocytes and nursing-cells indicate a clear expansion in T-lymphocyte clones. Induction of thymic activity and fibroblast activity in tissues of acemannan-treated animals results in the relapse of senile tissue.

Example 30

[Acemannan Effect on Cholesterol Levels in Animals and Humans]

Sucats receiving 855 mg / kg / day of acemannan were found to have a statistically significant decrease in serum cholesterol levels during the 91-day toxicity study. Similar results were observed in normal adult male volunteers receiving acemannan at a dose of 400-3200 mg daily. An important effect of the observed acemannan therapy is the statistically significant decrease in serum cholesterol levels to normal levels in these subjects. The mean cholesterol concentration in the first 24 study subjects was 189 mg / dl; At the end of the study the concentration is 174 mg / dl. Statistical analysis using the following [CRUNCH software version of Wilcoxen Sogned Ranks Tests] shows a 98% probability that cholesterol lowering is not due to an opportunity change. Since cholesterol levels in 7 of 24 patients decrease to more than 20 mg / dl after 6 days of treatment, this effect is not due to a simple dietary improvement in the subject's controlled tolerance during the study.

Example 31

[Acemannan Treatment of Injuries from Plants]

Two rival hunters, who supply veterinary acemannan, carried it to Africa. After several diarrhea, the hunters decided to consume acemannan themselves. Acemanan was taken at a dosage level of 800 mg / day. Hunters record less diarrhea than other hunters. In addition, due to brier bushes, hunters have suffered numerous injuries to their arms and legs. The hunters who consumed acemannan report that their Beige Harquin wounds have actually healed overnight. Redness or inflammation does not appear. All the abrasions were fully healed until they returned to the United States. Other members of the Hunting Team suffered severe infections from cuts and abrasions that required their medical practitioner's visit for systemic and local antibiotic therapy. Two hunters who consumed acemannan showed no infection and no scars.

Example 32

[Acemannan Treatment of Allergies from Hypersensitivity to Plants]

Acemanan is used to reduce the inflammatory effects of plant allergens. Known household history of seasonal high fever, H.R.M., experiences pruritis, plasticity, congestion and mucosal wetting every year. Beginning in 1988, it was found that 800 mg / day of acemannan was orally administered for 5 days to substantially eliminate symptoms of hyperthermia including the headache caused by the swollen nasal mucosa. In 1989, it was found that acemannan gels applied topically to the ocular and nasal mucosa upon sleep and every 8 hours thereafter resulted in similar effects and benefits as in H.R.M.

Similar results are shown by topical administration of acemannan to ivy poison lesions in humans and animals. The severity and healing time of the lesion is significantly reduced.

Example 33

[Acemannan Treatment of Allergies Caused by Hypersensitivity to Chemicals]

Acemanan is used to reduce the inflammatory effects of chemical allergens. A professional painter, T.R., was about to stop his work due to wheezing and bronchitis induced by vapors from paint and solvents. After oral administration of 800 mg / day acemannan for 5 days, his symptoms were alleviated. The painter could continue to work by orally administering 800 mg of acemannan daily before working.

Example 34

[Acemannan Treatment of Asthma-Related Symptoms]

T.T., a 76-year-old male with asthma for 72 years, took 800 mg of acemannan daily for an unrelated disorder. After one year of treatment, T.T. told the attending physician that he had not used his aerosol bronchodilator for more than a year and that a significant amount remained in units. His wife stated that he took an average of 2 units per month for 15 years and used a hand nebulizer for 50 years before for chronic asthma. The patient continued to take 800 mg of acemannan daily and no longer suffered from wheezing or chronic bronchitis.

Example 35

[Acemannan Treatment of Symptoms Associated with Cystic Fibrosis]

A six-month college student with cystic fibrosis symptoms reported a sudden recovery of energy within two weeks of starting oral acemannan therapy at a dose of 800 mg / day.

Example 36

[Acemannan Treatment of Cytomegalovirus Infection as a Result of HIV Infection]

The 27-year-old HIV-1 positive male M.M. suffers from substernal pain for two months, which is not alleviated by drug administration. Esophageal examination describes the erosion of the distal esophageal mucosa. Histopathological staining of biopsies describes cytomegalovirus organisms in endothelial cells. As a lozenge, 1000 mg of acemannan was orally administered for 3 days to eliminate all symptoms.

Example 37

[Acemannan Treatment of Secondary Syndrome for Rheumatic Fever Case]

A 25-year-old female S.M. shows acute onset of arthritis, tendonitis, joint edema, lymphocytes and elevated sedimentation rate after sore throat caused by acute streptococcal rheumatic fever. ASO titers are significantly elevated. Her sister and mother have a history of severe complex seizures of acute rheumatic fever, with incapacitation lasting up to one year.

The patient is orally administered 800 mg of acemannan daily. Sedimentation rate and leukocyte count are normal by 6 weeks of treatment and all clinical symptoms disappear. Patients recover manual labor workability, requiring a high degree of dexterity after 8 weeks of treatment.

Example 38

[Acemannan Used to Treat Autoimmune Diseases]

A 21-year-old woman (E.M.) with a history of pancreatopenia, more than two years of progressive anemia, leukopenia, and thrombocytopenia began treatment in June 1988. She has failed to respond to treatment provided at eight major medical centers in the United States. Bone marrow transplantation served as the next step of treatment. When tested for the first time, the patient exhibits sciatica, punctate bleeding, fatigue, low hemoglobin (6.1 g%) and low platelets (20,000 to 25,000 / dl) and a total leukocyte count of 1500 cells / dl. Antibodies to her own white blood cells have been reported by specialized clinical pathology laboratories. The patient receives oral administration of acemannan 800 mg for 60 days. A slight increase in research figures is noted and her fatigue is reduced to a minimum. Blood patients received low doses of prednisone and horse antithymocyte globulin. The former has already been shown to be ineffective. By October 1988, the patient's hemoglobin was above 10 g%, the leukocyte count was 3,500 cells / dl and the platelet was 60,000. By mid-1998, 800 mg of acemannan daily was recovered by oral administration for 6 months. In January 1990, the patient's hemoglobin was reported to be 12.7 g%, platelet 120,000 and leukocyte count 5,200 cells / dl. The patient currently does not administer any medication and is a graduate professional choreographer.

Example 39

[Acemannan Treatment of Computational Lupus Erythematosus]

Patients with pre-shingled lupus erythematosus were treated by oral administration of 800 mg / day acemannan by New York medical staff. The medical staff reported that after eight weeks of treatment with acemannan, all symptoms disappeared and laboratory values increased significantly. Ongoing acemannan treatment has been shown to control the symptoms associated with these patient diseases.

Example 40

[Acemannan Treatment of Acute Rheumatoid Arthritis]

The staff reported elevated RA latex levels and sedimentation rates in a group of patients with acute rheumatoid arthritis (RA). Six to eight weeks after oral administration of 800 mg / day acemannan, the patient's symptoms were significantly reduced and showed a significantly elevated clinical trial.

Example 41

[Acemannan Treatment of Chronic Rheumatoid Arthritis]

B.L.H., a 65-year-old woman with a 20-year history of chronic RA, deformed joints, subcutaneous nodules, and nodules, suffers from chronic pain. When the patient was examined in the fall of 1988, she was performing a number of treatments, including a gold shot with little benefit. Oral administration of acemannan (800 mg / day) was recommended. Six months were reported to be ineffective. In mid-1989, two or more weekly administrations of acemannan were reported to cause diarrhea in patients, but arthritis symptoms improved. At this time, 800 mg of acemannan was allowed 3 times a week and RA symptoms were remarkably improved. In January 1990, the patient reported that she had experienced up to six months of her last 15 years. The pain associated with the required effort and her daily routine was significantly reduced.

Example 42

[Acemannan and Antidepressant Therapy in Depression and Anxiety]

The physician orally administered 800 mg of acemannan per day to patients who failed to respond to antidepressant drugs and psychotherapy taken for severe depression and anxiety. He reported that by the end of the week the patient's emotions were more stable and his first significant improvement in attitude during long-term follow-up.

Example 43

[Acemannan Treatment of Cat Leukemia]

Feline leukemia (FeLV) is a retrovirus (oncovirus family) infection in which cats display various clinical signs. Infections of the lymphatic system predominate and most animals die within three years. Current treatment of such diseases is symptomatic; There is no cure.

Forty-five cats with end-stage FeLV disease were treated with intraperitoneal injection of acemannan six times a week for six more weeks. The cat is monitored weekly during the treatment period. In addition, laboratory data is obtained at the beginning, middle and end of the study.

67% of the treated animals improved during the study. The average survival time of animals that did not respond to acemannan was less than 28 days.

Example 44

[Acemannan Antifungal Drug Treatment of HIV-associated Fungal Infections]

Three HIV-1 patient reports describe similar modalities in which these patients develop hair vitiligo and / or monolithic marks and / or oral ulcers. Their condition is usually widespread and painful. The use of ketaconazole improved the condition in some patients while the others were unresponsive. Within one week of the addition of 800 mg / day acemannan oral administration of these therapies, the patient was reported to have removed these mucous skin lesions. Acemannan and ketaconazole are taken for 3-5 days to eliminate outbreaks for weeks to months. Ongoing acemannan administration eliminates or reduces the incidence of mucous skin infections.

Example 45

[Acemannan and Antibiotic Drug Treatment of HIV-associated Pneumocystis carinii Infections]

HIV-1 study patients orally administered 800 mg / day of acemannan are admitted to a friendly hospital in Arkansas for X-ray diagnostic-compatibility and objectively received pneumocystis carini pneumonia (PCP). V.A. In hospital trials, HIV-1 patients with PCP take more than two weeks to respond to therapeutic measurements if they respond. The patient responded to a week of aerosol pentamidine and continued with acemannan therapy. Symptoms disappeared from him.

Example 46

[Acemannan and Antibiotic Treatment of HIV-associated Cryptosporidiosis Infections]

Patients with HIV-1 suffering from chronic diarrhea and weight loss have been shown to have common acidic spores in diagnosing cryptosporidiosis. 800 mg / day oral acemannan and Q.I.D. A combination of 250 mg of ansamycin (rifbutin) was administered daily for two weeks to ameliorate diarrhea and cause weight gain in the patient.

Example 47

[Acemannan and Anti-TB Drug Treatment of HIV-Resistant Human Tuberculosis]

HIV-1 positive patient S.G. undergoes weight loss and experiences a low grade fever of unknown etiology. Post-peritoneal lymphadenopathy and biopsy findings of lumps have emerged as a culture of human tuberculosis. Patients received 800 mg / day of acemannan daily. The anti-tuberculosis treatment institution is a combination of 300 mg / day isoniazid, 600 mg / day rifampin and 1000 mg / day ethambutol, indicating that the patient is febrile within 24 hours and gains weight within 10 days.

Example 48

[Acemannan and Anti-TB Drug Treatment of Resistant Avian Tuberculosis Infection in HIV-Related Persons]

HIV-1 patients suffer from persistent diarrhea and eventually appear as smears and cultures of mycobacterium avian intercellular tuberculosis (MAI). No formulation is known to be effective; However, ansamycin was supplied by C.D.C. Q.I.D. Diarrhea was terminated within 1 week by mixing anzamycin 250 mg and 800 mg / day oral acemannan. Patients who lose weight at a rate of 3 to 4 pounds a week begin to gain weight within two weeks. Fatigue and general weakness disappear. Another HIV-1 patient C.C. showing biopsy and culture-proven MAI shows similar rapid degradation and negative culture tests after oral acemannan administration.

Example 49

[Acemannan and Multidrug Chemotherapy Including Cisplatin Cancer Treatment of Breast Cancer]

J.F., 38, was 5 years after mastectomy with vascular cell carcinoma of the breast. The patient appears to be pregnant due to ascites of peritoneal metastasis. Evidence of CAT injections in the liver and complex bone areas of soluble tumors is verifiable. Patients were assessed to survive four to six months longer when chemotherapy was extended. Patients start with an oral dose of acemannan at 800 mg / day and initiate the recommended combination cytotoxic chemotherapy (adriamycin, cyclophosphamide, mitomycin-C and 5-FU) prepared by her local oncologist. It is said to have been done. She received cisplatin weekly intravenously in combination with adriamycin and other agents. Subsequent examination showed re-ossification of bone metastasis, ascites removal, accelerated abdominal mass removal, liver mass disappearance and weight gain.

Example 50

[Acemannan Treatment of Fungal Infections of the Skin]

A 40-year-old doctor applied acemannan gel to pruritic, cracked plastic lesions between and on the base of his toes. Within a week, doctors report the reactions and healings that acemannan has proven to be the most effective medication for 20 years since he has been treating his chronic athlete's foot. Other patients reported similar improvements. Surprisingly, some patients who took acemannan for other diseases have also reported improvements in athlete's foot.

Example 51

[Acemannan Treatment of Carcinoma of Marine Animals]

A 40-year-old woman, who was scuba diving, injured her knee in a coral reef. The lesion covers about 9 cm 2 of the skin surface. The normal clinical course of this injury in humans is the delayed healing of skin lesions for 14 hours after about 8 hours of intense inflammation. When acemannan gel is applied to the wound, it heals completely and scars are scarcely visible.

[summary]

Acemanan has been shown to be effective in the treatment of many diseases in which the main mechanism of disease resolution or treatment requires intervention by the patient's immune system. Acemannans have a direct stimulating effect on the immune system. In addition, acemannans interact with viruses or other infectious organisms, infected cells and tumor cells to cause changes in their immunological sensitization surface composition to alter the appearance of these substances. Alter and cause them to be destroyed after being recognized by the body's immune system.

Accordingly, the operation and administration of the present invention is considered to be apparent from the foregoing description. Although the methods and techniques illustrated and described have been characterized as being preferred, it will be apparent that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the following claims.

Claims (5)

A pharmaceutical composition for treating chronic respiratory disease, comprising acemannan and a pharmaceutically acceptable adjuvant. A pharmaceutical composition for delaying the aging process of cells, comprising acemannan and a pharmaceutically acceptable adjuvant. A pharmaceutical composition for treating diabetes, comprising acemannan and a pharmaceutically acceptable adjuvant. A pharmaceutical composition for controlling blood cholesterol concentration, comprising acemannan and a pharmaceutically acceptable adjuvant. A pharmaceutical composition for removing scars formed in a blood vessel, comprising acemannan and a pharmaceutically acceptable adjuvant.