DE69133298T2 - Use of acetylated mannane derivatives for the treatment of chronic respiratory diseases - Google Patents

Description

A. Field of the Invention

The invention relates to the use of agents that modify the biological response. In particular, the invention relates to therapeutic use a polysaccharide substance which is an acetylated mannan the manufacture of a medicine to reduce symptoms, associated with chronic respiratory diseases.

B. Description of the general Background information

Aloe is a member of the family of the lilies. Contains aloe vera two main sources of fluid, a yellow resin dispersion (exudate) and a clear gel (mucus, Mucilage). The dried exudate, which comes from Aloe barbadensis Miller leaves, is referred to as aloe. The trade name is Curacao Aloe. It's mostly out Composed of aloin, aloe emodin and phenols. Bruce, South African Medical Jornal, 41: 984 (1967); Morrow et al., Archives of Dermatology, 116: 1064-1065 (1980); Mapp et al., Planta Medica, 18: 361-365 (1970); Rauwald, Archives Pharmacy, 315: 477-478 (1982). A number of phenolic compounds, including the Anthraquinones and their glycosides are pharmaceutically active known. Bruce, Excelsa, 5: 57-68 (1975); Suga et al., Cosmetics and Toiletries, 98: 105-108 (1983).

The mucilaginous jelly from the parenchymal Cells in the plant are called aloe vera gel. There is in Generally no anthraquinones that break down the gel and its discoloration cause, unless the gel is caused by unsuitable manufacturing processes has been contaminated. Aloe vera gel is 98.5% (weight percent) of water. More than 60% of the total solid consists of polysaccharides, the origin of carbohydrates. The rest of the solid are organic acids and inorganic compounds, especially calcium oxalate.

Aloe vera was in many cultures a traditional medicine as an anti-worm, catarrh and stomach remedy and it was used for Leprosy, burns and used in allergic conditions. 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 Medical 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, Medicinal Plants of India and Pakistan; D. B. Taraporevala Sons & Co., Private Ltd., Bombay 16-17 (1962).

Aloe vera is in the field of Dermatology has been used extensively, particularly for treatment for skin conditions caused by radiation. Mackee, X-rays and Radium in the Treatment of Diseases of the Skin, 3rd edition, Lea and Febinger, Philadelphia, 319-320 (1938); Rovatti et al., Industrial Medicine and Surgery, 28: 364-368 (1959) Zawahry et al., Quotations From Medical Journal on Aloe Research, ed. Max B. Skousen, Aloe Vera Research Institute, Cypress, California, 18-23 (1977); Cera et al., Journal of the American Animal Hospital Association, 18: 633-638 (1982). The main body of scientific literature that the medical applications in digestive problems as a virucidal, bactericidal and fungicidal agent and documented under gynecological conditions, is extensive and by Grindley et al., [Journal of Ethnopharmacology, 16: 117-151 (1986)] have been sufficiently discussed.

Depending on how The excretions that are processed are mucus and sugar Main components of the dehydrated gel. The sugar found are galactose, glucose, mannose, rhamnose, xylose and uronic acids. Despite contradictory According to reports, the mucus consists mainly of mannan or glucomannan. Eberendu et al., The Chemical Characterization 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., Lloydia, 31: 423 (1968).

Before starting this work, the Controversy about the identity of the active substances) in aloe vera cannot be arbitrated. It is therefore important to be clear between the ingredients in the gel and distinguish those found in the exudate. The majority the gel is a mucus of mainly polysaccharidic Nature with small amounts of various other ingredients. It has been observed that there are some with some of the efficacies synergistic effects between the polysaccharide base and others Can give components. Leung, Excelsa, 8: 65-68 (1978); Henry, Cosmetics and Toiletries, 94: 42-43, 46, 48, 50 (1979). For example, some scientists report that the effective components for wound healing are tannic acid [Freytag, Pharmazie, 9: 705 (1954)] and a type of polysaccharide. Kameyama, Wound-healing compositions from Aloe arborescens extracts. Japanese Patent 7856995, (1979).

However, there are many examples in the literature that indicate that polysaccharides can show pharmacological and physiological effects without the help of other ingredients. Gialdroni-Grassi, International Archives of Allergy and Applied Immunology, 76 (Suppl. 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 development of arteriosclerosis. Hyperlipidemia is associated with coronary artery disease and death in the general population and especially in presumably hereditary hypercholesterolaemia. In countries where dietary fiber intake is high, arteriosclerosis does not appear to be known. Trowell et al., Editors, Refined Carbohydrate Foods and Disease, London, Academic Press, 207 (1975). Pectin and guar are described as lowering cholesterol in normal and hyperlipidemic patients. Kay et al., American Journal of Clinical Nutrition, 30: 171-175 (1977). Carob gum, a polysaccharide consisting of mannose and galactose, lowers plasma lipoprotein cholesterol levels in both normal people and people with suspected inherited hypercholesterolemia. Zavoral et al., American Journal of Clinical Nutrition, 38: 285-294 (1983). The addition of guar gum to carbohydrate-containing meals lowers the postprandial glucose increase in both healthy and diabetic patients. Jenkins et al., Lancet, 2: 779-780 (1977). Kuhl et al., In Diabetes Care, 6 (2); 152-154 (1983) demonstrated that guar gum showed glycemic control in pregnant insulin-dependent diabetic patients.

The antitumor effect of polysaccharides has been extensively described. From polysaccharides from Lentinus cyathiaformis is known to be the host's toughness against tumors increase. Rethy et al., Annales Immunologiae Hungaricae, 21: 285-290 (1981). There are several Reports that polysaccharides from fungal, yeast, or bacterial extracts a higher one Degree of host resistance to viral and tumorigenic susceptibility can produce. 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 described the anti-tumor activity of a polysaccharide fraction (GF-1) made from cultivated fruiting bodies of a Grifola mushroom frondosa was extracted. This faction showed when they were intraperintonal (IP), intravenously (IV) and intratumoral (IT) was administered, equivalent, high Levels of inhibitory effectiveness. Oral administration (PO) was however not effective. Fraction GF-1 also showed anti-tumor activity against the solid form of meth A fibrosarcoma and MM 46 carcinoma in mice. Lentinan, which, similar like GF-1, a 6-branched β-1-3 linked Glucan's was ineffective against meth A fibrosarcoma. Chihara, "The antitumor polysaccharide lentinan: an overview "; manipulation of host defense mechanisms; Ed. Aoki et al .; Excerpta Medica, North Holland, 1-16 (1981); Sasaki et al., Carbohydrate Research, 47 (1): 99-104 (1976). It has been described that synthetic branched polysaccharides Show efficacy against tumors. Matsuzaki et al., Makromol. Chem., 186 (3): 449-456 (1985). Matsuzaki et al., [Makromol. Chem., 187 (2): 325-331 (1986)] synthesized branched polysaccharides from stone nut mannan namely (β- (1-4) -D-mannopyranose) and β- (1-4) -bound Glucomannan, which showed essential effects. A partial acetylated linear β- (1-3) -D-mannan that from the fruiting bodies Fish extracted from Dictyophoria indusiata also showed Antitumor activity. Hara, Carbohydrate Research, 143: 111 (1982). It appears that the anti-tumor effect is of the polymer main chain type and depends on its degree of polymerization, since polymers of the β- (1-3) -glucan type a higher one Antitumor effect as β- (1-4) glucan and hemicellular Own polymers. Matsuzaki et al., Makromol. Chem., 187: 325-331 (1986). A carboxy-methylated obtained from a bacterial culture filtrate Derivative of β- (1-3) glucan caused within 2 hours after injection of the derivative cell loss in established sarcoma 180 tumors. Baba, journal of Immunopharmacology, 8 (6): 569-572 (1986). The same author observed a compensatory due to the injection of the substance Increase in polymorphonuclear leukocytes. Bestatin also influenced a dipeptide which is used for this it is known to have immunomodulating and anti-tumor effects [Ishizuka, Journal of Antibiotics, 32: 642-652 (1980)], neither the tumor yield nor the polymorphonuclear leukocyte count. Baba et al., Supra.

There are numerous reports on the Anti-tumor effects of sulfated polysaccharides, including 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)]. Yamamoto et al., In Japanese Journal of Experimental Medicine, 54: 143-151 (1984) described the increase in the anti-tumor activity of a fucoidan fraction through the subsequent sulfation. The sulfated product showed Efficacy against L-1210 leukemia. Polysaccharides with sulfate groups have also been used as human T cell mitogens and polyclonal B cell activators in mice. Sugawara et al., Microbological Immunology, 28 (7): 831-839 (1984). In general have homo-polysaccharides high molecular weight with sulfate groups these properties. Dorries, European Journal of Immunology, 4: 230-233 (1974); Sugawara et al., Cell Immunology, 74: 162-171 (1982).

It has been described that glucan extracted from the yeast Saccharomyces cervisiae is a modulator of cellular and humoral immunity. Wooles et al., Science, 142: 1078-1080 (1963). The polysaccharide also stimulated the proliferation of pluripotent hematopoietic mouse stem cells, granulocyte macrophages, colony-forming cells, and cell-forming myeloid cells and erythroid colonies. 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 reported that iv administration of a polysaccharide induces protection against X-rays in mouse hematopoietic stem cells, thereby reducing the mortality of the mice so irradiated.

Lackovic et al., [Poceedings of the Society for Experimental Biology and Medicine, 134: 874-879 (1970)] took cell walls of yeast and extracted all the material except the "mannans" he found that they're for induction of α-interferon production are responsible by monocytes. The "cleaned mannans", supposedly for the physiological Answer were responsible, had a molecular weight of 5 500-20 000 Dalton.

Seljelid et al., [Experimental Cell Research, 131 (1): 121-129 (1981)] have observed that insoluble or gel-forming glycans Macrophages activated in vitro, whereas the corresponding soluble glycan didn't do this. Bogwald, [Scandinavian Journal of Immunology, 20; 355-360 (1984)] immobilized glycans that stimulate in vitro Had an effect on macrophages. This led the authors to believe that the spatial Arrangement of the glycan for the in vitro effect on the macrophages was crucial. An isolated from Candida albicans, purified polysaccharide induced an antibody response in vitro human peripheral blood lymphocytes. Wirtz et al., Clinical Immunology and Immunopathology, 33: 199-209 (1984). There were significant differences between anti-Candida antibodies Sera from normal and Candida-infected individuals. Wirtz et al., supra.

The antiviral effectiveness of polysaccharides and polysaccharides bound to peptides have been observed. Suzuki et al., Journal of Antibiotics, 32: 1336-1345 (1979). Suzuki et al., supra, described an antiviral effect of peptidomannan (KS-2), extracted from Lentinus edodes' mycelium culture. Either oral and intraperitoneal administration increased the maximum serum interferon titer, the mice against viral infections protected. That was a difference Dextran phosphate (DP-40) [Suzuki et al., Proceedings of the Society for Experimental Biology and Medicine, 149 (4): 1069-1075 (1975)] and opposite 9-methylstreptimidone (9-MS) [Salto et al., Antimier. Agent & chemotherapy, 10 (1): 14-19 (1976)], which are only higher Interferon titers in mice induced when administered iv or ip.

The anti-inflammatory effectiveness of aloe vera gel has been extensively described, both through oral Statements as well as in respected scientific journals. Rubel [Cosmetics and Toiletries, 98: 109-114 (1983)] discussed extensively the possible Anti-inflammatory mechanism Effect of aloe gel. Ukai et al., (Journal of Pharmacobio-Dynamics, 6 (12): 983-990 (1983)] described the anti-inflammatory Efficacy of from fruiting bodies of various mushrooms extracted polysaccharides. The polysaccharides showed a significant inhibitory effect on carrage-induced edema. One the polymer, O-acetylated D-Mannan (T-2-HN), also showed one noticeably more inhibitory effect than phenylbutazone on scald hyperalgesia. Ukai et al., Supra.

Other researchers have that too anti-inflammatory Effects of complex polysaccharides [Saeki et al., Japanese Journal of Pharmacology; 24 (1): 109-118 (1974)], of 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 controversy over whether the polysaccharide is a glucomannan, a mannan, a pectin or something different composition has been through a number of chemical Cleaning steps solved. Yagi et al., (Planta Medica, 31 (1): 17-20 (1977)] isolated acetylated Mannan (Aloe Mannan) using a slightly modified Extraction method from Aloe arborescens Miller var. Natalensis. However, Ovoda (Khim. Prior. Soedin, 11 (1): 325-331 (1975)) isolated earlier Pectin as the main ingredient in the same aloe species.

The structure of this immunologically effective polysaccharides and the types of structural changes seem to be the factors that control their effectiveness and toxicity. Their effects) remain unclear, but the latest statements indicate indicated that several polysaccharide lymphocytes and macrophages induce an extensive range of immunologically effective To produce substances. For example, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) to be the chemical part of lipopolysaccharide (LPS) that does that Minimal signal to activate the macrophage host resistance provides [Lebbar et al., Eur. J. Immunol. 16 (1): 87-91 (1986)]. The composition the present invention has all of the properties of these biologically active substances; it is one of the most effective of all known biologically active polysaccharides, but they differ yourself in that no toxicity has been observed. It also reveals specific antivirals Effectiveness through change of viral glycoprotein synthesis.

A variety of pharmacological studies on aloe vera gel have recently been carried out. The results include faster healing of radiation burns [Rowe, J. Am. Pharm. Assoc., 29: 348-350 (1940)] and increased wound healing [Lushbaugh et al., Cancer, 6: 690-698 (1953)]. Thermal burns treated with aloe vera gel healed much faster than unhealthy delte 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)]. The gel is useful for treating leg ulcers [EI Zawahry et al., Int. J. Dermatol., 12: 68-73 (1973)] and to accelerate postoperative healing (Payne, dissertation submitted to the faculty of Baylor University, Waco, TX, MS Degree). The experimental evidence suggests that extracts from aloe vera have anti-infectious properties [Solar, Arch. Inst. Pasteur Madagascar, 47: 9–39 (1979)] and accelerate phagocytosis [Stepanova, Fizol. Veshchestva, 9: 94-97 (1977)].

The effective fraction of aloe vera gel was developed by Carrington Laboratories, Inc., Irving, Texas as a long chain polydisperse β- (1,4) -bound acetylated mannan which is penetrated by O-acetyl groups, which has a mannose monomer to acetyl group ratio of approximately 1: 0.91 own, identified. Acemannan is the trade name of the organic effective ingredient of Carrisyn, an ingredient of Carrington Laboratories, Inc. was isolated and developed. See US patent No. 4,735,935, U.S. Patent No. 4,851,224 and U.S. Patent Application Serial Number 07/229 164, and the publications cited here, their disclosure is included here by this reference.

Mannane, including Glucomannane and galactomannans have long been used by humans. For example, galactomannans in the form of plant gums are far reaching used as a binder in the control of food texture Service. In addition, some mannans have essential therapeutic properties shown (Davis and Lewis, ed. Jeanes A., Hodge J., In: American Chemical Society Symposium, Series 15th Washington DC, American Chemical Society, 1975).

Pure mannans are in higher plants relatively rare, although they are an essential structural component of some yeasts are. For example, about 45% of the Saccharomyces cell wall is made up cerevisiae from a mannan. This mannan is a water soluble Molecule, that of β- (1,6) -, β- (1,3) - and β- (1,2) -bound partially phosphorylated D-mannose residues [McMurrough et al., Biochem. J., 105: 189-203 (1967)]. Other biologically active mannans are from Candida utilis [Oka et al., Gann, 60: 287-293 (1969), Oka et al., Gann, 58: 35-42 (1968)], from Candida albicans, from Coccidioides immitis and from Rhodotorulum rubrum [Wheat et al., Infect. Immun., 41: 728-734 (1983)] been obtained. Mannans (including galactomannans and glucomannans) are relatively resistant across from attack by mannosidases, but they can be caused by exo and endo mannanases are decomposed [Emi et al., Agr. Biol. Chem., 36: 991-1001 (1972), Snaith et al., Adv. Carbohydr. Chem. Biochem., 28: 401-445 (1973), Herman, At the. 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 noted biological effects of mannans in mammals are the activation of macrophages and the stimulation of T cells. As a result, they are effective immunostimulants with an essential effectiveness against infectious diseases and against tumors [Hasenclever et al., J. Immun., 93: 763-771 (1964)].

Saccharomyces mannan (15 mg / kg / day) may increase carbon clearance in normal male ddl mice by acting as a stimulant of the reticuloendothelial system [Suzuki et al., Gann, 62: 553-556 (1971)]. The same mannan increased also the number of antibody-producing Spleen cells [Suzuki et al., Gann, 62: 343-352 (1971)]. In vitro studies with peritoneal mouse cells (a mixture of macrophages and lymphocytes) indicate that some mannans and mannan protein complexes the release of interferon both in vivo and in vitro can stimulate [Lackovic et al., Proc. Soc. Exp. Biol. Med., 134: 874-879 (1970)]. The mannans stimulated the release of interferon in one Way, similar to that of endotoxins unlike the endotoxins, however, they cause one low toxicity (Borecky et al., Acta Virol., 11: 264-266 (1976), Hasenclever, supra). The Mannan from Candida albicans is effective in this way, that Mannan from Saccharomyces cerevisiae is however ineffective [DeClercq et al., Ann. NY Acad. Sci., 173: 444-461 (1970)]. In other laboratories inconsistent and poor results have been obtained (DeClercq, supra). These differences can for small differences in structure or size of the polymers [Suzuki et al., Jpn. J. Microbiol., 12: 19-24 (1968)]. The latter is most likely for it responsible because of low molecular weight mannans (5.5–20 kDa) in the interferon induction assay are usually most effective, even with Saccharomyces-Mannan the tendency is higher than Candida-Mannan.

A 20 kDa galactomannan Lipomyces starkeyi had poor interferon-inducing properties. In contrast, Candida albicans induced the appearance of the Interferon activity 2–24 Hours after the intravenous Administration (Borecky, supra).

DMG, a degraded mannoglucan from Microellobosporia grisea culture fluid, can stimulate the cytotoxic effects of macrophages, natural killer cells (NK) and killer T cells and it increases the secretion of interleukin-1 (IL-1) and colony stimulating factor (CSF). It has a more potent anti-tumor activity than lentinan (a glucan 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 higher levels of IL-1. In addition, DMG 1) increases antibody production against sheep erythrocytes, 2) the natural killer activity of both spleen and peritoneal cells, and 3) the cytostatic activity of peritenoal macrophages [Nakajima et al., Gann, 75: 253-259 (1984)].

The most common mannose-binding protein in humans is an acute phase protein; his level rises when stressed To [Ezekowitz et al., J. Exp. Med., 169: 185-196 (1989)]. The envelope glycoproteins of the human immunodeficient Virus (HIV gp120 and gp41) contain high-mannose oligosaccharides that effective ligands for the mannose-binding protein seem to be. As a result, it can mannose-binding protein inhibit HIV infection of lymphoblasts and selectively bind to HIV-infected cells. Free yeast mannan can compete in binding this protein to infected Intervene cells. Therefore can the factors leading to an increase in mannose-binding protein levels induce, confer protection against HIV.

PROBLEMS TO WHICH THEY ARE THE INVENTION DOES

Virus, cancer and diseases of the Immune regulation continues to be the main cause of both morbidity and mortality in humans, other mammals, other living things, birds and plants. Problems with currently used drugs are linked to general toxicity, lack of effectiveness (or both), lack of specificity on and development of resistance to causative organisms or pests. As a result, better non-toxic, therapeutically effective agents for the treatment of these diseases is needed. Acemannan has been shown that it's a unique combination of immunomodulatory as well as having antiviral properties.

SUMMARY THE INVENTION

It is therefore a task to use of an acetylated mannan in the manufacture of a drug to reduce symptoms associated with chronic respiratory problems are to be provided.

The medicine causes one Effect on a living being's respiratory system to reduce the symptoms, those associated with asthma, conjunctivitis, runny nose and bronchitis are to alleviate. An acetylated mannane derivative must be sent to the living being administered to ensure adequate cell and immunomodulation To cause tissues for the symptoms caused by asthma, conjunctivitis, runny nose and bronchitis are responsible.

Precise description of the preferred embodiments

Carrisyn is the brand name the holder of the present invention the purified ethyl alcohol extract given from the inner gel of the layers by Aloe barbadensis Miller to have. The effective ingredient of Carrisyn is from the United States Adopted Name Council has been referred to as "Acemannan". Not less than 73% of the Carrisyn extract is acemannan; Carrisyn extract generally consists of approximately 73% –90% Acemannan. Carrisyn extract is generally due to the removal of the outer skin of the leaf, the subsequent Removal and processing of the inner fillet or slime like manufactured as follows: pH adjustment, ethanol extraction, freeze drying and grinding. See U.S. Application Serial No. 144,872 filed in January 1988, a continuation-in-part application of US application serial no. 869,261 (now U.S. Patent No. 4,735,935), the disclosure of which is by this reference is incorporated into the present invention. The Processing in this way requires essentially none covalent bonds changed and there are no toxic compounds. This Manufacturing steps have been developed to overcome the inability of traditional aloe product manufacturers to overcome the polysaccharides to standardize and stabilize.

Carrisyn is a loose, white, amorphous Powder that is sparingly soluble in water and dimethyl sulfoxide is insoluble in most other organic solvents. This powder contains not less than 73% of a polysaccharide, which is essentially from linear β- (1-4) -D-mannosyl units consists. The polysaccharide is a long chain polymer, which in random Way of acetyl groups through an oxygen atom to the polymer are bound, permeated. The generic name for the polymer is Acemannan. The one determined by the alkaline hydroxamate method Degree of acetylation is approximately 0.91 acetyl groups per monomer. See Hestrin, Journal of Biological Chemistry, 180: 240-261 (1949). The neutral sugar binding analysis indicates that D-galactopyranose, possibly through an α- (1-6) bond, in relationship from about 1 in 70 sugars to the chain. The 20: 1 ratio of Mannose to galactose also indicates that galactose units are interconnected, preferably by a β- (1-4) glycosidic Binding. The chemical structure of Acemannan can be shown as follows become:

Allgemeine Struktur von ultrareinem Acemannan

General structure of ultra pure acemannan

The administration of the acetylated Mannanderivates can by local Application, oral administration, on the ip route, on the iv route or by other related routes of administration.

The acetylated mannane derivative can the recipient not just given as a single remedy, it can also be used in conjunction can be used with other known therapeutic agents, which by their requirement to support or help the immune system of the host to achieve their maximum therapeutic effect.

It has now been found that Acemannan is an effective inducer of IL-2 and prostaglandin E2 (PGE2) production through adherent human peripheral blood cells in the culture. One is convinced that the present invention is the practically first non-toxic IL-1 release stimulator. IL-1 is an important product of macrophages, which in the literature as the activity and production of lymphocytes, fibroblasts, B-lymphocytes and endothelial cells was described influencing. See Old, Scientific American, 258 (5): 59-60, 69-75 (1988).

IL-1 induces proliferation of fibroblasts, which is fundamental to wound healing. IL-1 also increases (1) activity the bone marrow; it can be found in individuals where bone marrow activity is decreased is to be therapeutic; and (2) generally strengthens the immune system.

A series of mixed experiments Lymphocyte cultures (MLC) have shown that Acemannan is the alloantigenic Response of these lymphocytes increased in a dose-dependent manner. The Incubation of Acemannan with monocytes enabled monocyte-directed Signals to increase the T lymphocyte response to lectin. relative Studies on the effects of acemannan on MLC have an increase in phagocytosis and the activity of natural Killer cells shown. That is why Acemannan is in these in vitro systems non-toxic and it's an immune booster.

Acemannan effectively stimulates the Lymphocytes to secrete lymphokines and it also causes them Generation of modified Glycoproteins (GP-120) in HIV-infected lymphocytes by a Mechanism similar to that of the glucosidase I inhibitor. See Gruters et al., Nature, 330: 74-77 (1987) and Pal et al., Intervirol., 30: 27-35 (1989). Acemannan is phagocytosed and probably in the Golgi / glucoprotein apparatus of the monocytes are pumped where it goes directly into glycoprotein synthesis intervenes.

A. Toxicology

The toxicological effects of Acemannan have been investigated in both in vivo and in vitro systems Service. Acemannan is not mutagenic or in vitro in test systems blastogenically. In vitro, the compound was non-toxic to H-9, MT-2 and CEM-SS lymphoid cells. Toxicological in vivo studies on Acemannan conclude a 91 day subchronic oral toxicity study in dogs, a 180 day chronic oral toxicity study in rats and a 180 day chronic oral toxicity study in people. In these studies, dogs with a Period of 91 days up to 825 mg / kg acemannan per day, no toxic effects noted. In rats that have one Period of 180 days up to 38 475 ppm acemannan in their feed received, were not clinical, conspicuously pathological, or toxic Effects noticed. In human patients who were treated over a period of 180 Days received 800 mg of acemannan per day, no opposite clinical or toxic effects.

In pilot studies, the administration caused from acemannan to dogs complete monocytosis in blood samples, the to complete the white one Blood cell count and were used for morphological differentiation. Within Two hours after administration of high doses of Acemannan appeared strong activated monocytes in the circulation. A similar effect is in humans been observed.

A study has been conducted using human peripheral blood monocyte cell cultures and ¹⁴ C-labeled acemannan to monitor the incorporation or absorption of acemannan into a biological system. In this study, detectable levels of ¹⁴ C-labeled acemannan were absorbed or taken up by human peripheral monocyte / macrophage cells. The maximum incorporation occurred after 48 hours. At a concentration of 5 mg / ml, ¹⁴ C-labeled acemannan was not toxic to monocyte / macrophage cells and the weight / volume (w / v) of the digested cell mass was 760 times greater than the w / v of the digested Acemannan solution. These results suggest that macrophages are able to maintain the intracellular acemannan concentration at a very high level that is not cytotoxic.

A pyrogen assay was performed in accordance with the pyrogen test protocol set out in USP XXI., biological test [151], on rabbits using 1 mg / ml injectable solution Acemannan carried out. Because of the unknown systemic effects of the injected acemannan are more common Temperature measurement as specified in the USP has been carried out. The temperature changes exceeded in the test organisms not that by the USP protocol allowed minimal changes; therefore fulfilled the solution the USP requirements over the Absence of pyrogens. Injectable acemannan called in one Rabbits showed a maximum increase in body temperature of 0.3 ° C. This temperature increase occurred 90 minutes after the injection. Acemannan is an inducer of IL-1 secretion by macrophages in vitro and monocytes. Because IL-1 is an effective pyrogen, this could delay the slight, delayed rise in temperature explain in this rabbit.

Twenty-four panelists completed the study on the safety and tolerance of orally administered acemannans. Clinical laboratory results showed that the following deviations from the normal range occurred: CO ₂ in seven people; Cholesterol in three people, triglycerides in two people, phosphorus in one, hemoglobin in four, basophils in two, monocytes in three, eosinophils in three, lymphocytes in four, neutrophils in two, and one and one red and white blood cells. None of these deviations were clinically relevant.

The results of the immune profiles showed the following group differences between the values on day 1 and on Day 7: CD-16, CD-4 (T-4), CD-8 + Leu7, CD-4 + CD-25, CD-8 + CD-16, Leu7 and TQ-1. The mitogenic responses were low.

The vital signs seemed the normal ranges not to exceed. There were no group differences in urine output. A person in group IV suffered from diarrhea and diarrhea during the course of this study. One person in Group I had between Days 2 to 4 diarrhea. A total of 5 people described in total eight negative cases. All of these events occurred in people who had one Period of 6 days a day received 1600 or 3200 mg of acemannan orally.

B. Method of administration

The physical properties from Acemannan allow it to be used in all known in this field pharmaceutical dosage forms can be formulated and mixed can. The biopharmaceutical and toxicological properties of Acemannan allow it to live in tissues and organs Organisms is used and that it has a wide dosage range is administered.

Acemannan can be administered orally to a living being, parenterally, locally and locally with a daily Dose of 0.001 mg / kg to 1000 mg / kg body weight administered per day become.

Mixed with suitable aids can be pressed acemannan or in solid dosage units, such as Pills, tablets, coated tablets, can be filled, or it can too Capsules are processed. These oral dosage forms would come in administered at a dose of about 0.1 mg / kg to 1000 mg / kg body weight daily become.

With the help of suitable liquid aids can Acemannan in solutions, Suspensions or emulsions are injected. These products would come with administered at a rate of 0.001 mg / kg up to 1000 mg / kg body weight daily become. As an additional Acemannan would be part of a vaccine or other product at a rate of 0.001 up to 1000 mg per dosage unit of Additional product can be administered.

Local administration of Acemannan can be in the form of a processed gel, cream, lotion, solution, ointment or powder. These formulations can contain up to 90% acemannan.

example 1

PRODUCTION OF INTERLEUKIN-1 AND PGE2 THROUGH ADHESIVE, PERIPHERAL HUMAN BLOOD LEUKOCYTES THAT WERE STIMULATED WITH ACEMANNAN

A. Introduction of IL-1 production

Mononuclear human cells were separated from heparinized whole blood by density gradient centrifugation in Ficoll-Hypaque (Pharmacia, Sweden). After washing, the cells were resuspended at a concentration of 2 × 10 ^-6 cells / ml in RPMI-1640 with 25 mM Hepes and supplemented with 50 U / ml penicillin, 50 μg / ml streptomycin and 2 mM L-glutamine. Two ml aliquots of the cell suspensions were dispersed in each well of a six-well plate and for one hour at 37 ° C in a 5% wet atmosphere CO ₂ incubated. After removing non-adherent cells, adherent cells were washed three times with the medium described above. Two ml of medium supplemented with 5% pooled human AB serum was added to each well. Cultures were stimulated with Acemannan at various concentrations. Stimulated controls with lipopolysaccharide (LPS) from E. coli (Sigma 0111: B4) at a final concentration of 20 μg / ml and without any addition (background) were included. The cultures were incubated at 37 ° C for 24 hours as described above. The supernatants were harvested to remove cells, centrifuged and dialyzed against 500 volumes of PBS for 48 hours (it changed all at once), followed by 4 hours of dialysis against 20 volumes of RPMI-1640 with 25 mM Hepes, antibiotics and L-glutamine such as described. The supernatants were frozen at -20 ° C until IL-1 activity was assessed.

B. IL-1 determination in supernatants

• 1. Thymozyten von C₃H/HeJ-Maus, 5–8 Wochen alt, wurden verwendet. Eine homogene Zellsuspension wurde in Minimum essentiellen Medium (MEM), ergänzt mit 5% FCS, 100 U/ml Penicillin, 50 g/ml Streptomycin, 2 mM L-Glutamin und 5 × 10^–5 M 2-Mercaptoethanol, hergestellt. Die Zellkonzentration wurde eingestellt und in 96-Vertiefungsplatten bei 1 × 10⁶ Zellen/Vertiefung dispergiert. Phytohämagglutinin (PHA) wurde zu jeder Vertiefung bei einer Konzentration von 10 μg/Vertiefung gegeben. Die Proben wurden seriell verdünnt und ein Volumen von 25 μl wurde zu jeder Vertiefung, beginnend mit 1 : 10 bis zur Endverdünnung, zugegeben. Jede Verdünnung wurde in vierfacher Ausführung getestet. Die Platten wurden bei 37°C in einer feuchten Atmosphäre mit 5% CO₂ für 72 Stunden inkubiert und wurden mit [³H]-Thymidin (0,5 μCi/Vertiefung) innerhalb der letzten 16 Stunden gepulst. Die Zellen wurden auf Faserglasfiltern mit einem automatischen Zellernter geerntet und die Radioaktivität wurde durch Standardszintillationsverfahren gemessen. Die Ergebnisse werden als cpm Thymidin-Einbau durch Thymozyten in Reaktion auf die Überstände bei einer Endverdünnung von 1 : 10 wiedergegeben.
• 2. Zwei-Seiten-"Sandwich" ELISA für IL-1. Dieses Verfahren wurde kürzlich in Journal of Immunology, 138: 4236 (1987) beschrieben, dessen Offenbarung hierin speziell durch Hinweis einbezogen ist. Siehe auch US-Patent Nr. 3 654 090 und US-Patent Nr. RE 31 006 Schuurs et al. Monoclonaler, gereinigter Antikörper IL-1-H6 gegen IL-1β (100 μl/Vertiefung, 10 μg/ml) wurde somit auf Vinyl-Assayplattenvertiefungen über Nacht bei 4°C beschichtet. Die Vertiefungen wurden mit PBS/0,5% Thimerosal gewaschen und mit 200 μl 5%iger, nicht fettiger Trockenmilch/0,5%iges Thimerosal/PBS eine Stunde bei Raumtemperatur gegenbeschichtet. Nach Waschen wurden 50 μl/Vertiefung Probe oder humaner, rekombinanter IL-1-Standard und 50 μl eines weiteren monoclonalen Antikörpers gegen ein nicht überlappendes Epitop von IL-1 [biotinyliert IL-1 β-H67 (2 μg/ml) in 1%iger nicht-fetter Trockenmilch/0,5%igem Thimerosal/PBS] zugegeben und die Platten wurden 2 Stunden bei Raumtemperatur inkubier. Nach Waschen wurden 100 μl/Vertiefung einer 1 : 1000 Verdünnung von Streptavidinperoxidase zugegeben und die Platte wurde 1 Stunde inkubiert. Die Vertiefungen wurden gewaschen, 30 Minuten im Dunkeln mit 100 μl OPD-Substratlösung inkubiert und die Absorption bei 450 nm wurde gemessen.

Two different methods were used to determine IL-1: (1) the thymocyte proliferation assay and (2) an ELISA assay specific for IL-1.

• 1. C ₃ H / HeJ mouse thymocytes 5-8 weeks old were used. A homogeneous cell suspension was prepared in minimum essential medium (MEM) supplemented with 5% FCS, 100 U / ml penicillin, 50 g / ml streptomycin, 2 mM L-glutamine and 5 × 10 ^-5 M 2-mercaptoethanol. The cell concentration was adjusted and dispersed in 96-well plates at 1 x 10 ⁶ cells / well. Phytohemagglutinin (PHA) was added to each well at a concentration of 10 µg / well. The samples were serially diluted and a volume of 25 ul was added to each well, starting at 1:10 to the final dilution. Each dilution was tested four times. The plates were incubated at 37 ° C in a humid atmosphere with 5% CO ₂ for 72 hours and were pulsed with [ ³ H] thymidine (0.5 µCi / well) within the last 16 hours. The cells were harvested on fiberglass filters with an automatic cell harvester and radioactivity was measured by standard scintillation methods. The results are reported as cpm thymidine incorporation by thymocytes in response to the supernatants at a final dilution of 1:10.
• 2. Two-sided "sandwich" ELISA for IL-1. This method was recently described in Journal of Immunology, 138: 4236 (1987), the disclosure of which is specifically incorporated herein by reference. See also U.S. Patent No. 3,654,090 and U.S. Patent No. RE 31,006 Schuurs et al. Monoclonal, purified antibody IL-1-H6 against IL-1β (100 μl / well, 10 μg / ml) was thus coated on vinyl assay plate wells at 4 ° C. overnight. The wells were washed with PBS / 0.5% thimerosal and counter-coated with 200 μl of 5% non-fatty dry milk / 0.5% thimerosal / PBS for one hour at room temperature. After washing, 50 μl / well of sample or human, recombinant IL-1 standard and 50 μl of another monoclonal antibody against a non-overlapping epitope of IL-1 [biotinylated IL-1 β-H67 (2 μg / ml) in 1% non-fat dry milk / 0.5% thimerosal / PBS] was added and the plates were incubated for 2 hours at room temperature. After washing, 100 ul / well of a 1: 1000 dilution of streptavidin peroxidase was added and the plate was incubated for 1 hour. The wells were washed, incubated for 30 minutes in the dark with 100 ul OPD substrate solution and the absorbance at 450 nm was measured.

C. Determination of PGE2

PGE ₂ was assessed using the radioimmunoassay in the same non-dialyzed supernatants. The antibody to PGE ₂ (ICN Biomedical, Inc., Costa Mesa, CA) was used according to the manufacturer's instructions, which are incorporated herein by reference.

D. Observations

Representative experiments are shown in Table 2. Acemannan is a strong inducer of IL-1 production through adherent, peripheral human blood leukocytes. At doses between 1 and 10 μg / ml, the acemannan extract induces the production of IL-1, which is comparable to that by 20 μg / ml LPS, which is the reference inducer of IL-1 production. Acemannan in the same dose range also induced the generation of PGE ₂ with levels comparable to those induced by 20 μg / ml LPS (positive control).

Table 2 INDUCTION OF PGE ₂ SYNTHESIS BY PERIPHERAL, ADHESIVE HUMAN BLOOD CELLS STIMULATED BY ACEMANNAN AND BY LIPOPOLYSACCHARID (LPS).

Example 2

EFFECT OF ACEMANNAN ON PHAGOZYTOSIS IN VITRO

The effect of Acemannan has been shown in tested in vitro to determine its effect on phagocyte function assess. CBA mice were injected ip with 1 mg / kg acemannan and peritoneally and spleen macrophages were collected 3 days later. thioglycolate and saline were similar tested as positive or negative controls. The macrophages were ingested with red blood cells from sheep (SRBC) as ingestion particles Presence and absence of anti-SRBC titers incubated and phagocytosis was measured histologically as percent cells that ingested SRBC. Although nonspecific phagocytosis is mild after acemannan treatment elevated phagocytosis was significantly increased in the presence of antibody. In the presence of Acemannan-stimulated, antibody-mediated phagocytosis was complemented increased to an even greater extent. This Results indicate that Acemannan's macrophage count increase can and their phagocytic effect increases. Such reactions can the effectiveness of Acemannan as a stimulant of wound healing and as an anti-infectious Contribute funds.

A. Procedures and materials

Acemannan was at room temperature stored in its dried form. The amount needed for each attempt was balanced and in 2-minute exposures at 600 watts of power microwaved. It was then transferred to a sterile plastic centrifuge tube and Microwave treated for 1 additional minute. The material was in cell culture medium (RPMI-1640) to the desired one Concentration diluted.

Phagocytic cells: Mouse spleen cells were obtained from BALB / c mouse obtained from Harlan Sprague-Dawley. The mice were killed by CO ₂ fumigation and their spleens were removed aseptically. The cells were then separated into adherent and non-adherent populations by nylon wool, column fractionation according to the method of Journal of Immunology, 71: 220, the disclosure of which is specifically incorporated herein by reference. Adherent cells were determined by microscopic analysis as below as macrophages (monocytes) and lymphocytes in a ratio of 4 to 1. After the single cell suspensions were obtained by interrupting the single layer, both adherent and non-adherent single cell preparations were placed on Ficoll-Hypaque and centrifuged to obtain a mixture of lymphocytes and macrophages.

Blastogenesis Assay: A standard blastogenesis assay was set up as shown below. The mitogen used in the assay was PHA-P obtained from Burroughs Wellcome. As indicated for individual experiments, the cultures were kept in a moist atmosphere with 5% CO ₂ for 72 hours. Tritiated thymidine was added to the culture during the last 6 hours. Cell concentrations per well using flat-bottom microtiter tissue culture plates were 5 x 10 ⁵ mouse cells / 0.2 ml. The cells were separated in the wells and acemannan or mitogen was added. A simulation index (SI) was calculated using the formula:

Cell staining: Cell smears were stained by nonspecific esterase staining as follows. Approximately 2 × 10 ⁶ cells in 2 drops were treated with 2 drops of fetal calf serum and 4 drops of a fixative solution consisting of a mixture of 25 ml, 35% formaldehyde, 45 ml acetone, 100 mg KHP ₂ O ₄ , 10 mg NaHP ₂ O ₄ and 30 ml of water, mixed. The slides were incubated with a mixture of 10 mg naphthyl acetate and 4.5 mg fast blue staining in 1.4 ml ethylene glycol monomethyl ether with 5 ml 0.1 M trismaleate buffer, pH 7.8 (Wright staining). The staining was allowed to react for 10 minutes, then washed in water for 20 seconds. A counterstain of 0.2 g Giemsa stain, 12.5 ml ethanol and 12.5 ml glycerin was used for 30 seconds before washing again.

Introduction of peritoneal macrophage cells: saline thioglycolate broth (1 mg / kg) or acemannan (1 mg / kg) was injected ip into female BALB / c mouse to induce peritoneal exudate macrophage cells. Induced cells were removed from the peritoneal cavity 3 days after injection. Macrophages were washed twice with phosphate buffered saline (PBS) and covered with 2 ml of fresh medium; 0.1 ml of the macrophage suspension was added to each well. The cultures were placed in a 5% CO ₂ -95% air incubator humidified at 37 ° C for 30 to 60 minutes. The cultures were washed twice with PBS and covered with 2 ml PBS. One of each pair of deck slides was removed with a needle eye clip, immersed in distilled water for 5 seconds, and immediately replaced in the culture dish. The PBS was removed and the cultures were covered with ice cold glutaraldehyde. After 10 minutes the glutaraldehyde was removed and the cover slides were covered with distilled water.

Attached deck gliders were immediately in the oil dip lens of a phase contrast microscope. Clinging to the deck glider, which was not subjected to hypotonic shock was evaluated, whereas ingestion was assessed on the deck glider, which in distilled Water was lysed.

Antibody-dependent and antibody-independent phagocytosis: SRBC, obtained from the Austin Biologics Laboratory, Austin, Texas, were washed three times in PBS (pH 7.2). BALB / c mice were given ip injections of 10 ⁶ cells and bled to death on day 14 after the injection. The serum was collected, pooled and heat inactivated at 56 ° C for 45 minutes. Agglutination titers were determined to be 1024 using round bottom microtiter wells.

Antibody-independent phagocytosis was determined by incubating SRBC (0.5% volume / volume) with macrophages (10 ⁶ ) in RPMI-1640 containing 20% fetal calf serum (FCS). The coverslips were prepared and stained at various intervals. The percentage of macrophages that had red cells ingested was determined visually by counting 200 cells / coverslip and three coverslips / animals.

Antibody-dependent phagocytosis has been reported Use SRBC (0.5% in RPMI-1640 with 20% FCS) mixed with Anti-SRBC serum or IgM fraction (minimum titer of 2000), determined. The mixture was incubated at 37 ° C for 15 minutes, then twice in PBS (pH 7.2) washed and resuspended to the original volume.

Serum fraction: Whole blood became Removal of IgM by euglobulin precipitation and dialysis against distilled Water fractionated. After dialysis at 4 ° C for 24 hours, the precipitate became by centrifugation at 1500 × G for 20 Minutes away and the supernatant was analyzed by ion electrophoresis and complement-mediated lysis. Less than 5% of the original IgM remained.

B. Results

To the effect of Acemannan on To evaluate macrophages were the ones used in the first experiment Mouse spleen cells cultivated in vitro with Acemannan (Table 3).

Table 3 PERCENT CELL TYPES BY HISTOLOGICAL EVALUATION OF MOUSE MOLT CELLS IN CULTURE

Cultures were 72 or 96 hours incubated and smeared at the end of the experiment made and with Wright stain and stained by the esterase method. The relative percentage on macrophages and lymphocytes was determined. At 72 hours there it's a dose-related increase the macrophage numbers from 30% without Acemannan to 45% with 0.2 μg Acemannan per well. Since data is expressed as a percentage of cells, there was a simultaneous decrease in lymphocytes. At 96 hours there was also a dose related increase in the percentage Macrophages in the presence of Acemannan. At 96 hours they showed Cultures at 0.2 µg Acemannan per well significant acidolysis, such as by a yellow staining expelled. Furthermore, 96 hour cultures have a lower percentage on macrophages, possibly because of the longer Cultivation time. To increase the acemannan-induced to refer to a known standard in macrophage numbers a similar attempt performed with the Mitogen PHA-P. The results are shown in Table 4.

Table 4 PERCENT CELL TYPES BY HISTOLOGICAL EVALUATION OF MOUSE MOLT CELLS IN CULTURE

Although the percentage of macrophages didn’t change after 72 hours, there was a dose-related increase on macrophages after incubation with PHA-P for 96 hours. By comparison Acemannan was twice as effective as PHA-P. The percentage of macrophages increased a maximum of 16 with Acemannan compared to 7 with PHA-P (Tables 3 and 4).

Because Acemannan apparently the percentage increased on macrophages, was decided to determine whether the effect of the phagocytes was also elevated has been. CBA mouse peritoneal exudate cells given in saline, thioglycolate broth or Acemannan were ingested with red blood cells from the sheep, such as those Particles (Table 5) used [as phagocytes].

Table 5

Within a period of 120 Minutes increased unspecific phagocytosis from 3% to 52% in saline controls, whereas percent treated phagocytosis in cells from thioglycolate broth Living things rose to 89%. Phagocytosis in Acemannan-treated Living things rose to 63% at 120 minutes. Acemannan-stimulated phagocytosis was bigger than those in controls after 20-120 minutes; however, the differences were not statistically significant.

To determine if the Acemannan effect was dependent on antibody for phagocytosis, became a similar one Test carried out with Anti-SRBC (Table 6).

Table 6

Sera were heat inactivated (56 ° C for 30 minutes) and the antibody titer used was 2 × 10 ³ , well above the hemagglutination titer. In this experiment, macrophages were obtained from two sources, the peritoneal cavity and the spleen. Again, mice were pretreated with ip injections of 1 ml 0.9% saline, 1 mg / kg thioglycolate or 1 mg / kg acemannan. At a titer of 2 × 10 ³ , the phagocytic effect of thioglycolate-induced peritoneal macrophages was twice as high (89% vs. 43%) as the effect of the saline-induced controls, whereas acemannan-induced macrophages were 30% compared to controls ( 73% versus 43%) were more active. The difference between phagocytic activity in the Acemannan-treated and saline control groups was statistically significant.

Similar results were observed with macrophages obtained from mouse spleen. The phagocytic effect was lower than that of macrophages obtained from the peritoneal cavity, possibly due to manipulations of the spleen cells. Again, with a titer of 2 × 10 ³ acemannan-induced macrophages were significantly higher in phagocytic activity than saline controls with a 95% confidence interval; Phagocytic activity was similar to the control at 8 × 10 ³ titer.

To the effect of complementary (C ') on antibody-mediated To determine phagocytosis, the experiment was performed using the Add C 'on Media undertaken (Table 7).

Table 7 COMPLEMENT-MEDIATED PHAGOCYTOSIS COMPARISON

To ensure that Lysis is not would take place became IgM deleted Mouse serum used (see methods). The titer used was 3000, as if by hemagglutination and determined the Coombs technique. Cells from the fluid of the peritoneal cavity and spleen were more active in the phagocytosis the addition of C 'as without C ', though the difference only with peritoneal cells induced by acemannan were statistically significant.

Finally an attempt was made to Differentiate the effects of acemannan phagocytosis and adherence (Table 8th).

Table 8

Antibody was detected in this experiment SRBC was used in a titer of 2000, however the attempt was made stopped after 7 minutes. Acemannan-induced macrophages from both the peritoneum as well as the spleen were more effective at adhering than the saline controls and, more specifically, less effective than the thioglycolate-induced Group.

C. discussion

The results show that Acemannan stimulates phagocytosis both directly and indirectly. The results also demonstrate that Acemannan phagocytosis by both macrophages unspecific as well as specific by antibody-mediated reactions elevated. This shows that Acemannan has immunostimulatory properties Has phagocytes.

Example 3

REINFORCEMENT OF ALLO-reactivities FROM HUMAN LYMPHOCYTE BY ACEMANNAN

This example was designed to test Acemannan's capacity to boost immune response to alloantigen and to test whether the potential boost is a monocyte-driven phenomenon. Acemannan did not enhance the lymphocyte response to syngeneic antigens in the mixed lymphocyte culture (MLC), but more importantly, it increased the alloantigen response in a dose-response manner (2.6 x 10 ^-7 -2.6 × 10 ^-9 M). This effect of Acemannan proved to be a special reaction and competed with concentrations of in vitro Acemannan that are achievable in vivo. A separate series of mixing experiments showed that acemannan incubation with monocytes allowed monocyte-driven signals to enhance the T cell response to lectin. It is concluded that acemannan is the active ingredient of the aloe vera plant and is an important immune booster in that it enhances the lymphocyte response to alloantigen. The mechanism is suggested to involve monocyte enhancement release of IL-1 under the aegid of alloantigen. This mechanism can explain in part the ability of Acemannan, viral infections in experimental animals and Pick up people.

This example was designed to directly study the effects of Acemannan as an immune enhancer to evaluate the model of monocyte T lymphocyte, with cell-cell interaction reaction reproduced on alloantigen in the mixed lymphocyte culture becomes. This model tests the capacity of Acemannan, additional Monocyte-macrophage functions in an immunologically relevant Stimulate model.

A. Materials and methods

• 1. Cell preparations. Mononuclear leukocytes were from the peripheral blood of normal, informed and to this approved human subjects under the aegide of a study that by the Institutional Review Board of the University of Texas Southwestern Medical Center in Dallas received. peripheral Blood was diluted 1: 3 in Hanks Balanced Saline (HBSS) and layered on top of a Ficoll-Hypaque gradient. cell from subjects known to differ in major histocompatibility were obtained after each day of investigation to get a positive mix Secure lymphocyte response. For special experiments more carefully characterized pedigrees of cells that the mononuclear Inhibit leukocyte pole, isolated. T-lymphocytes were made using the standard nylon wool separation technique isolated. The cells converged in nylon contained approximately 90% pure T cells. T-8 lymphocytes and monocyte macrophages adhere predominantly on the pillar. The attachment population was forced back of media through the pillar removed with a valve piston. To on monocytes (macrophages) To enrich, the glass attachment process was used to create a Population larger than Is 95% pure.
• 2. Acemannan. Acemannan was tested in these studies to prepare a 0.5% (weight / volume) solution in RPMI-1640 medium with further dilution to the following working concentrations: 2.6 × 10 -7 M, 2.6 x 10 -8th M and 2.6 x 10 -9 M manufacture.
• 3. Mixed Lymphocyte Cultures (MLC). Unidirectional MLC were set up in flat-bottom tissue culture microtiter plates (Costar Co., Cambridge, MA). Mononuclear cells isolated by the Ficoll-Hypaque density gradient technique discussed above served as stimulator cells after exposure to 2000 rads for 30 minutes in a cesium source (Gammacell, Atomic Energy of Canada, Ontario, Canada). The responder cells, which were similarly isolated, and stimulators were set at 1.3 × 10 ⁶ cells / ml. The following was added to each well: 25 ul Acemannan or media (control), 25 ul RPMI-1640 supplemented with 10% fetal bovine serum and 75 ul from each cell population. The cells were incubated at 37 ° C in 5% CO ₂ : 95% air for 6 days. The cultures were pulsed with 25 µl of ³ H-thymidine (1 µCi / well) for 4 hours, after which the cells were harvested and counted. To test specificity for acemannan for attachment, detection and response to MLC, additional unidirectional MLC to the agent added just 20 minutes before the cells were pulsed with ³ N-thymidine were adjusted.
• 4. Monozote T cell interaction. Spleens from female Lewis rats were disrupted in RPMI 1640 medium through a sterile steel sieve. Mononuclear leukocytes were collected from the interface of a Ficoll-Hypaque density gradient as described above. Monocytes, obtained by enrichment on glass petri dishes and adjusted to a final concentration of 10 ⁶ / ml, were incubated by varying doses of Acemannan or medium (control) in a total volume of 2 ml and incubated at 37 ° C. for 24 hours. The monocytes were harvested, washed extensively with fresh medium and co-cultured with syngeneic T lymphocytes at a ratio of 10 T cells: 1 monocytes with the plant lectin phytohemagglutinin (Difco, Detroit, MI) (1: 100) for 48 hours at 37 ° C. The cells were harvested over a MASH II (Whittaker, MA Bioproducts, Walkersville, MD), placed in fluorine and counted in a scintillation counter (Beckman Laboratories, Chicago, IL). A control experiment was carried out by incubating T lymphocytes with Acemannan, followed by washing and co-culture with freshly prepared T lymphocytes again at 10: 1 together with PHA-P.

B. Results

• 1. Alloantigenic response. Acemannan had no statistically significant effects on the response of T cells to autoantigens. When the agent was added at the start of MLC, the cells took up syngeneic stimulation incorporated by tritiated thymidine in the presence or absence of test reagent at the doses described. In the absence of oral acemannan, this MLC built 2616 ± 1099 cpm of tritiated thymidine at the end of a 4 hour pulse. Although there was an upward trend in the dose of added agent (3281 ± 1355 at 2.6 × 10 ^-9 M, 3743 ± 1670 at 2.6 × 10 ^-8 M and 3828 ± 1978 at 2.6 × 10 ^-7 M), none of these rates of isotope incorporation in DNA was different from a statistically significant degree.

In contrast to the absence of Acemannan's effect on auto-response in the MLC, the agent's effect on alloreaction was in the same immunological assay. First, Acemannan did not interact with lymphocyte capacity to detect and respond to class II alloantigenic differences in the MLC; this was evident when the syngeneic cultures were compared to the allogeneic response in the presence of the lowest concentration of drug. Second, there was a dose response-related increase in alloreaction by acemannan so that the culture treated with the highest dose of 2.6 x 10 ^-7 M reflects an almost 60% increase over the non-acemannan culture. The dose-response relationship is most convincingly demonstrated as the enhanced allogeneic response, which is significant for each dose of acemannan tested for no acemannan condition.

• 2. Acemannan und Monozoten-T-Zellkooperation. Um die Hypothese zu testen, dass Acemannan direkt dem Alloantigen entsprechende Monozyten von Bereitstellen von dem/den Signalen) stimuliert, um die lymphoide Reaktion auf Antigen und/oder Mitogen zu verstärken, wurden gereinigte Populationen von Monozyten 24 Stunden mit verschiedenen Dosen an Acemannan inkubiert. Am Schluss der Inkubation wurden die Zellen ausgedehnt gewaschen und dann mit T-Lymphozyten bei einem Verhältnis von 10 : 1 zum Simulieren des natürlichen Verhältnisses, das in peripherem Blut gefunden wird, co-kultiviert. Co-kultivierte Zellen wurden mit Phytohämagglutinin stimuliert. Die Co-Kulturen mit Monozyten, die vorher mit Acemannan inkubiert wurden, hatten eine wesentlich erhöhte mitogene Reaktion in einer dosisverwandten Weise.

To assess whether acemannan has a specific effect on lymphocyte alloreaction or an unspecific effect on tritiated thymidine incorporation, the reagent was added to the culture at the end of a mixed 7 day MLC lymphocyte culture 20 minutes before the addition of the vehicle. There was no effect of Acemannan when added as a pulse at the confluence of the MLC. These data support the specificity of the acemannan effect on the enhancement of lymphoid response in the MLC.

• 2. Acemannan and monocote T cell cooperation. To test the hypothesis that Acemannan directly stimulates mono-cells corresponding to the alloantigen (by providing the signal (s)) to enhance the lymphoid response to antigen and / or mitogen, purified populations of monocytes were incubated for 24 hours with different doses of Acemannan. At the end of the incubation, the cells were washed extensively and then co-cultured with T-lymphocytes at a ratio of 10: 1 to simulate the natural ratio found in peripheral blood. Co-cultivated cells were stimulated with phytohaemagglutinin. The co-cultures with monocytes previously incubated with Acemannan had a significantly increased mitogenic response in a dose related manner.

C. Discussion

This example has the assets of Acemannan as an immunostimulating drug, with important clinical To act episode illustrated.

Acemannan is believed to it is able to limit DNA and retrovirus infections, which are essential Cause diseases in living beings and in humans. For example, alleviates Acemannan in an animal model in cats with viral rhinotracheitis. Further evidence shows that Acemannan is in vitro and in vivo against Herpes simplex 11 virus, measles virus and possibly HIV may be effective can. The evidence indicates that the immunological mechanism increase of the monocyte as both a phagocyte cell and a cell which contributes to the afferent recognition of the antigen. studies have direct reinforcement of phagocytic properties of the monocyte on the one hand and one increase the absolute numbers of the important cell, on the other hand. mounting evidence supports that Concept that Acemannan elaboration by the activated monocyte the signal substance IL-1 amplified.

Studies described in this example have been specifically researched into the mechanism by which Acemannan an immuno-reinforcing Reagent can be directed. Mixed lymphocyte cultures are in vitro models of the type in which immunocompetent cells respond on antigen of the type necessary for detection and response participate in virus. There are important monocyte-lymphocyte interactions in this reaction, that produce a reaction to alloantigen. It was this model that for testing capacity des Acemannans selected to act as an immunoactivator.

Acemannan is therefore an important one amplifier the alloantigenic reaction in MLC. There is a dose-response relationship with increase at the highest Dose tested at approximately 60% above basal. This gives not just statistical significance again, but also one biologically relevant increase in Response to alloantigen and can serve as a means by which the drug can support the organism's response to viral attack. This effect of Acemannan turned out to be specific for the allogeneic Stimulus, provided that the drug is not either the basal reaction itself (syngeneic MLC) or non-specific incorporation a tracer DNA precursor, tritiated thymidine, enhanced when Drugs were added to the confluence on MLC.

A second series of experiments tested the hypothesis of monocyte-T-lymphocyte interactions at least in part for the increased alloreaction in which MLC may be responsible. In this series In experiments, Acemannan was incubated with monocytes, after which the treated extensively washed monocytes were mixed with freshly prepared syngeneic T lymphocytes that were not or would not be exposed to Acemannan. These experiments demonstrate the enhancement of the T lymphocyte response to the polyclonal mitogen phytohemagglutinin to an extent equal to the response previously seen in the MLC - approximately 55% above baseline and dose response relationship.

The lowest dose in the Study that was effective in the MLC had no effect in the monocyte experiment. It was not surprising that the threshold dose difficult for the two models tested, polyclonal response to mitogen and can be alloantigenic reaction in the MLC. It can also be observed be that the monocyte trial is a stricter test of the effect of Acemannan because it reflects a treated cell type the monocyte on T cells, which is then an immune stimulus in the absence of the drug sees. Although the alloantigenic response is only or in the large the Acemannan-reinforced Monocyte production of IL-1 measures the less polyclonal Mitogen-reinforced Response may be a result of an assay of immune stimulations, where each represents a different threshold response to Acemannan.

The dose of used in the experiments Acemannan is clinically relevant. The selected dose range became accurate selected to determine the concentration of acemannan that is in the plasma could be expected to achieve to narrow it down if the drug is extracellular in water distributed and at the rate of a third of the orally administered Dose absorbed. The numbers were based on pharmacological previously given Studies in dogs. The actual Concentrations that are achievable in humans have also been considered shown in this area, further supporting the potential relevance to them Clinical practice studies.

Acemannan proved through this Try as an occasion for Monocytes Monocyte-driven signals emit the T4 cell response intensify on lectin. While Acemannan non-lymphocyte response to syngesic antigens in MLC enhanced, increased it MLC alloantigenic response in a dose relevant manner. This Reaction turned out to be an acemannan-specific reaction achievable acemannan concentrations in vivo.

This experimental documentation shows that Acemannan is an immuno-reinforcing and is a biologically reactive modifier in that that it enhances the lymphocyte response to alloantigen. On proposed mechanism of action includes stimulation of Monocytes for the release of IL-1; in the presence of Acemannan, IL-1 was found to be released from monocyte cultures. The pharmacological effects of acemann stimulation on monocytes may have the acemannan effect against viral infection in living things and illustrate people.

Example 4

Pharmacokinetic base for the correlation of in vitro and in vivo efficacy of Acemannan

To calculate the pharmacokinetic behavior of Acemannan, ¹⁴ C-labeled material was given by ip and iv injections and by PO administration. Based on the results of the previous pilot work, an aqueous dose of 200 mg ¹⁴ C-labeled acemannan / 200 ml with a specific activity of 17.4 cpm / μg (approximately 20 mg / kg) was administered to female dogs. Blood, urine and faecal samples were taken in certain sections over a period of 48 hours or more. After the killing, organ and tissue samples were taken and all samples were analyzed for radioactivity using scintillation spectrometry.

Acemannan's kinetic behavior was similar to that observed with most pharmacological agents; however, its biological half-life (t _1/2 ) was extremely long. There was substantial absorption on all three routes of administration. Maximum blood levels were reached after the iv injection, followed by ip and then by po. Blood levels that were a maximum of 200 μg / ml immediately after the iv injection decreased with a t _1/2 over 50-60 hours; the plasma levels were approximately twice those of the blood. In the comparison, the blood levels after the ip injection reached their maximum after 24 hours at 45 μg / ml and then decreased at the same rate as that observed with iv; in fact, blood levels were close to 90% after only 8 hours. When administered orally, the blood levels were measurable after 3 hours and reached their maximum at 4-5 μg / ml. Based on the relatively long half-life in blood, a therapeutic dose interval of approximately 7 days would be justified considering the time period required for the three half-lives.

After ip and iv injection, radiolabeled acemannan was mainly distributed in the liver and spleen. The liver, marrow, thymus, and lymph nodes were the main distribution sites after oral dosing, a finding consistent with the immunological sites of acemannan's efficacy. Radioactive levels in tissues collected after 48-52 hours were sufficient the iv injection from a low value of approximately 1 μg / g brain to a high value of 85 μg / g spleen. Interestingly, levels in the brain and spinal cord were higher (approximately 3 μg / g tissue) after oral administration compared to parenteral administration. This may be the result of the partial degradation of the polymer into low molecular weight fractions in the liver during the first passage, which enables the blood-brain barrier to be penetrated.

In summary, the data with regard to clinical pharmacokinetic considerations indicate that ¹⁴ C-labeled acemannan (1) achieves maximum blood levels within 8 hours or less in all routes studied, (2) has a relatively long biological half-life, the therapeutic dosing interval of several days and (3) measurable levels in all tissues examined, including the central nervous system.

These pharmacokinetic data indicate that Acemannan can duplicate known levels in the blood and / or tissue after injection or oral administration, thereby achieving therapeutic or antiviral effects in vitro. For example, mice implanted with virally infected Norman mouse myxosarcoma cells (NMM) and injected with 1 mg / kg Acemannan IP within 24 hours showed a survival rate of 35% after 60 days compared to a survival rate of 0% in NMM-treated control mice (Peng et al., submitted for publication, 1990). The expected blood level maxima at an ip dose of 1 mg / kg would be in the range of 2 μg / ml (45 μg / ml × 1/20 mg / kg). Acemannan, which was added to T lymphocyte cultures at a concentration of only 0.15 μg / ml ( ^2.6-9 M; 60,000 MW), increased the number of cytotoxic T cells by 230% and the functional capacity of the cytotoxic T cells generated by 138%, thereby destroying the target cells to which they have been sensitized [Womble et al., Int. J. Immunopharmac., 10 (8): 967-974 (1988)]. Cytotoxic T cells are believed to be generated against tumor cells such as NMM cells.

Even after oral administration Blood levels of 4-5 μg / ml obtained from Acemannan were essential as they correspond to the concentration of Acemannan, which in vitro with Zidovudine (AZT) results in optimal synergism. For example increased 0.001 μg / ml alone AZT or 3.2 μg / ml Acemannan's vitality of the CEM cells infected with the HTLV-III virus no more than 10%. The protective effect of the antiviral combination exceeded together 70%. Similar led a combination of 0.1 μg / ml AZT and 1 mg / ml acemannan for a protective effect that exceeded 80% (Kemp et al., Filed for publication, 1990).

In summary, this shows pharmacokinetic So study that acemannan concentrations are the are at least as high as those known from in vitro work are reachable in vivo.

Example 5

ACEMANNAN TREATMENT OF ALLERGIES DUE TO SENSITIVITY RESULT ON CHEMICALS

Acemannan was used to relieve the inflammatory Effect of chemical allergens used. Subject T. R., a professional painter, was there because of wheezing and Bronchitis caused by vapors from its paints and solvents was initiated to give up his job. After oral ingestion of 800 mg / day acemannan for five days his symptoms were relieved. The patient continues consumption of 800 mg / day of oral acemannan before work every day and is able to continue painting.

Example 6

Acemannan treatment of Symptoms associated with asthma

A 76-year-old man, T. T., with a 72 year old History of asthma took 800 mg of acemannan daily for an unrelated one Status. After one year of therapy, T. T. told the person in charge Doctor that he used his aerosolized bronchodilator for 1 year hadn't used and a significant amount was still in unity. His wife confirmed that he's taken 2 units a month on average for 15 years and had used a handheld atomizer for 50 years he had chronic asthma. The patient continued to take 800 mg acemannan daily away and no longer had wheezing or chronic bronchitis.

Example 7

ACEMANNAN TREATMENT OF SYMPTOMS RELATED TO CYSTIC FIBROSIS

A college-age woman with a 6-month history of cystic fibrosis syndrome reported one abrupt return of energy within 2 weeks of the introduction of oral Acemannan therapy at a dose of 800 mg / day.

It is therefore assumed that the Operation and administration of the present invention from previous description becomes clear. While the shown (s) and described method (s) and techniques characterized as preferred , it can be seen that various changes and modifications herein without departing from the scope of the invention as in the following claims defined, executed can be.

Claims (2)

Use of an acetylated mannan in the Manufacture of a medication to reduce symptoms that are associated with chronic respiratory diseases. Use according to claim 1, wherein the acetylated Mannan Acemannan is.