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WO1993008810A1 - Utilisations de produits issus de l'aloes, tel que de l'acemannan, dans le traitement des maladies necessitant l'intervention du systeme immunitaire pour la guerison - Google Patents

Utilisations de produits issus de l'aloes, tel que de l'acemannan, dans le traitement des maladies necessitant l'intervention du systeme immunitaire pour la guerison Download PDF

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Publication number
WO1993008810A1
WO1993008810A1 PCT/US1991/008204 US9108204W WO9308810A1 WO 1993008810 A1 WO1993008810 A1 WO 1993008810A1 US 9108204 W US9108204 W US 9108204W WO 9308810 A1 WO9308810 A1 WO 9308810A1
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acemannan
tumor
acetylated mannan
virus
use according
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PCT/US1991/008204
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English (en)
Inventor
Bill H. Mcanalley
Robert H. Carpenter
Harley R. Mcdaniel
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Carrington Laboratories, Inc.
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Application filed by Carrington Laboratories, Inc. filed Critical Carrington Laboratories, Inc.
Priority to CA002122604A priority Critical patent/CA2122604C/fr
Priority to JP4501864A priority patent/JPH07500568A/ja
Priority to KR1019940701484A priority patent/KR100209180B1/ko
Priority to PCT/US1991/008204 priority patent/WO1993008810A1/fr
Priority to EP92900586A priority patent/EP0611304B1/fr
Priority to DE69131628T priority patent/DE69131628T2/de
Priority claimed from CA002122604A external-priority patent/CA2122604C/fr
Publication of WO1993008810A1 publication Critical patent/WO1993008810A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters

Definitions

  • the invention pertains to uses of biological response modifying agents. More particularly, this invention relates to the therapeutic use of a polysaccharide substance which is 1 0 predominantly an acetylated mannan or its derivatives to:
  • Aloe is a member of the lily family. Aloe vera contains two major liquid sources, a yellow latex (exudate) and the clear gel (mucilage). The dried exudate of Aloe barbadensis Miller leaves is referred to as aloe.
  • the commercial name is Curacao aloe. It is composed mainly of aloin, aloe-emodin and phenols. 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
  • Aloe vera gel The mucilaginous jelly from the parenchymal cells of the plant is referred to as Aloe vera gel.
  • Aloe vera gel There are generally no anthraquinones to decompose and cause discoloration of the gel unless the gel is contaminated by an improper processing technique.
  • Aloe vera gel is about 98.5% water by weight. More than 60% of the total solid is made up of polysaccharides of carbohydrate origin. Organic acids and inorganic compounds, especially calcium oxalate, account for the remainder of the solid.
  • Aloe vera was the traditional medicine of many cultures as an anthelmintic, cathartic and stomachic and was used inter alia for leprosy, burns and allergic conditions.
  • Aloe vera has been featured extensively in the field of dermatology, especially for treating radiation-caused skin conditions. Mackee, X-rays and Radium in the Treatment of Diseases of the Skin. 3rd Ed., Lea and Febiger, Philadelphia, 319-
  • sugars are the major components of the dehydrated gel.
  • the sugars found are galactose, glucose, mannose, rhamnose, xylose and uronic acids. Although reports conflict, the mucilage is mainly composed of mannan or glucomannan. Ebeumble et al., The Chemical Characterization of Carrisyn® (in preparation);
  • 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 proliferation of murine pluripotent hematopoietic stem cells, granulocyte macrophage colony-forming cells and cells forming myeloid and erythroid colonies.
  • Maisin et al., rRadiation Research. 105:276-281 (1986)] also reported that IV administration of a polysaccharide induced protection of murine hematopoietic stem cells against x-ray exposure, thereby decreasing the mortality of the mice so exposed.
  • composition of the present invention possesses all of the attributes of these immunologically active substances; it is among the most potent of all known biologically active polysaccharides but differs in that no toxicity has been observed. It also manifests specific antiviral activity through alteration of viral glycoprotein synthesis.
  • Aloe vera gel has been identified by Carrington Laboratories, Inc., Irving, Texas, as a long-chain polydispersed ⁇ -(l ,4)-linked acetylated mannan intersperse with O-acetyl groups having a mannose monomer-to-acetyl group ratio of approximately 1 :0.91.
  • Acemannan is the nonproprietary name of the biologically active component of Carrisyn® , a compound isolated and developed by Carrington Laboratories, Inc. See U.S. Patent No. 4,735,935, U.S. Patent No.
  • Mannans including glucomannans and galactomannans
  • galactomannans in the form of plant gums
  • binders for control of food texture.
  • some mannans have exhibited significant therapeutic properties (Davis and Lewis, eds. Jeanes A., Hodge J., In: American Chemical Society Symposium, Series 15. Washington, DC, American Chemical
  • mannans are relatively uncommon in higher plants, although they are a major structural component of some yeasts.
  • a mannan is a water soluble molecule composed of ⁇ -(l,6)-, ⁇ -(l,3)-, and ⁇ -(l,2)-linked, partially phosphorylated D-mannose residues [McMurrough et al., Biochem. J.. 105:189-203 (1967)].
  • Other biologically active mannans have been obtained from 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 glucomannans
  • mannosidases can be degraded by exo- and endo-mannanases
  • Snaith, et al., Adv. Carbohvdr. Chem. Biochem.. 28:401-445 (1973) Herman, Am. J. Clin. Nutr..
  • Saccharomyces mannan (15 mg/kg/day) enhances carbon clearance in normal male ddl mice, presumably acting as a reticuloendothelial system stimulant [Suzuki et al., Gann.
  • DMG a degraded mannoglucan from Microellobosporia grisea culture fluid, can stimulate cytotoxic activities of macrophages, natural killer (NK) cells and killer T cells, and it enhances the secretion of interleukin-1 (IL-1 ) and colony- stimulating factors (CSF). It has more potent antitumor activity than lentinan (a glucan from Lentinus edodes) [Nakajima et al.,
  • DMG stimulates macrophages to produce increased amounts of IL-1.
  • DMG enhances 1) antibody production against sheep erythrocytes, 2) natural killer activity of spleen as well as of peritoneal cells, and 3) cytostatic activity of peritoneal macrophages [Nakajima et al., Gann. 75:253-259 (1984)].
  • the major mannose-binding protein is an acute-phase protein; its levels rise in stressed individuals [Ezekowitz et al., J. Exp. Med.. 169: 185-196 (1989)].
  • the envelope glycoproteins of the human immunodeficiency virus (HIV gpl20 and gp41) contain mannose-rich oligosaccharides that appear to be potential ligands for the mannose-binding protein.
  • the mannose-binding protein can inhibit HIV infection of lymphoblasts and bind selectively to HIV- infected cells. Free yeast mannan can competitively interfere with binding of this protein to infected cells.
  • factors that induce an increase in the level of the mannose-binding protein may confer protection against HIV.
  • Virus, cancer and diseases of immune regulation continue to be major causes of both morbidity and mortality in humans, other mammals, other animals, birds, and plants.
  • Problems associated with currently used drugs are, namely, general toxicity, lack of efficacy (or both), deficiency in specificity and development of resistance by causative organisms or agents.
  • Acemannan has been shown to possess a unique combination of immunodulatory and antiviral properties.
  • cytokines such as interleukins, interferon, and prostaglandin
  • ADCC antibody-dependent cell cytolysis
  • Fig. 1 shows synergistic antiviral effects of acemannan and AZT on the viability of HIV-infected MT-2 cells.
  • Fig. 2 shows synergistic antiviral effects of acemannan and AZT as quantified by the percent increase in viability of
  • Carrisyn® is the brand name given by the assignee of the instant invention to the purified ethyl alcohol extract of the inner gel of the leaves of Aloe barbadensis Miller.
  • the active component of Carrisyn® has been designated "acemannan" by the United States Adopted Name Council.
  • Carrisyn® extract is generally produced by removing the outer sheath of the leaf, then removing and processing the inner filet or mucilage as follows: pH adjustment, ethanol extraction, freeze drying and grinding.
  • Carrisyn is a fluffy, white, amorphous powder, which is poorly soluble in water and dimethyl sulfoxide and insoluble in most other organic solvents. This powder contains not less than 73% of a polysaccharide consisting essentially of linear ⁇ (l-4)-
  • the polysaccharide is a long chain polymer interspersed randomly with acetyl groups linked to the polymer through an oxygen atom.
  • the generic name for the polymer is acemannan.
  • the degree of acetylation is approximately 0.91 acetyl groups per monomer as determined by the alkaline hydroxamate method. See Hestrin, Journal of Biolo gical Chemistry . 180:240-261 (1949).
  • Neutral sugars linkage analysis indicates that attached to the chain, probably through an ⁇ (l-6) linkage, is a D-galactopyranose in the ratio of approximately one for every 70 sugars.
  • the 20:1 ratio of mannose to galactose indicates that galactose units are also linked together, primarily by a ⁇ (l-4) glycosidic bond.
  • the chemical structure of acemannan may be represented as follows:
  • virus as used herein includes both the DNA and the RNA virus. It can either be an enveloped or a non- enveloped virus.
  • enveloped virus in all cases but one is understood to mean a virus encased within a modified host cell membrane; the poxviruses produce their own envelope. Typical enveloped viruses are set forth in Table 1. TABLE 1
  • enveloped viruses as divided into family and common species or genus:
  • Orthomyxoviridae Influenza virus types A, B, and C
  • Paramyxoviridae Newcastle disease virus of fowl human parainfluenza viruses Sendai virus mumps virus paramyxoviruses measles virus rinderpest virus of cattle canine distemper virus peste-des-petits-ruminants virus of sheep and goats respiratory syncytial virus of man bovine respiratory syncytial virus pneumonia virus of mice
  • Bunyaviridae bunyavirus (Bunyamwera, Bwamba,
  • Alphaviruses aura virus Chikungunya virus eastern equine encephalitis virus getah virus mayaro virus middleburg virus mucamba virus ndumu virus O'Nyong-nyong virus pixuna virus ross river virus semliki forest virus Sindbis virus una virus
  • Retroviridae type C oncovirus group type B oncovirus group type D retrovirus group avian complex leukemia virus Rous sarcoma virus murine complex leukemia virus mouse sarcoma virus murine mammary tumor virus feline leukemia complex virus feline sarcoma complex virus woolly monkey sarcoma virus gibbon leukemia virus Mason-Pfizer virus hamster leukemia virus rat leukemia virus bovine lymphoma virus human T cell leukemia viruses: types 1 and 2 etc.
  • spumaviridae syncytial and foamy viruses of humans, monkeys, cattle, cats visna virus of sheep Maedi virus progressive pneumonia viruses of sheep *human immunodeficiency viruses: (include HTLV HI/LAV) HIV, HTLV IV,
  • virus-like agents viroids-prions kuru virus
  • Lymphadenopathy virus human immunodeficiency virus (HIV) simian T-lymphotropic virus type III (STLV-IIlAGM) human T-lymphotropic virus type IV
  • HTLV-IV HTLV III and LAV are now usually referred to as HIV
  • tumor includes both malignant and non-malignant neoplasms including tumors of viral, chemical, radiation, genetic and other origins. It can be of embryonic ectodermal origin, embryonic mesodermal origin, or embryonic endodermal origin. It can be from the embryonic surface ectoderm, the embryonic neuroectoderm, the embryonic head mesoderm, the embryonic paraxial mesoderm, the embryonic intermediate mesoderm, the embryonic lateral mesoderm, or the embryonic endoderm.
  • tumors in an animal include: tumors of the skin and soft tissues; tumors of the muscle; tumors and tumor-like lesions of joints and adjacent soft tissues; tumors of bone and cartilage; tumors of the lymphoid and hematopoietic tissues; tumors of the respiratory system; tumors of the alimentary tract; tumors of the liver, gall bladder and pancreas; tumors of the urinary system; tumors of the genital systems; tumors of the mammary gland; tumors of the endocrine glands; and tumors of the nervous system and eye.
  • Human malignant tumors include: acute lymphoid leukemia; acute myeloid leukemia; chronic myeloid leukemia; chronic lymphoid leukemia; polycythemia vera; myelosclerosis with myeloid metaplasia; multiple myeloma; primary macroglobulinemia; Hodgkin's disease; non-Hodgkin's lymphoma; skin cancer; malignant melanoma; head and neck cancer; lung cancer; gastrointestinal cancer; breast cancer; gynecologic cancer; trophoblastic disease; testicular cancer; prostate cancer; renal carcinoma; bladder cancer; endocrine tumor; brain tumor; retinoblastoma; neuroblastoma; Wilm's tumor; osteogenic sarcoma; Ewing's sarcoma; and soft-tissue sarcoma.
  • microorganism as used herein includes parasites, bacteria, and other organisms and agents causing infestation.
  • Parasites include arthropod parasites, helminth parasites, protozoal parasites, and hemaprotozoal parasites. Examples of these parasites include demodex mange, hookworm and coccidia.
  • glycosylation means the addition of carbohydrate molecules to a protein molecule.
  • An acetylated mannan derivative in particular acemannan, may exert its therapeutic effect by two possible mechanisms.
  • One is the altering of glycosylation, such as inhibition of glucosidase I or the incorporation of the acetylated mannan derivative into glycoprotein.
  • the other possible mechanism is enhancement of the antigenicity of the virus or the tumor, or the enhancement of immunocompetency of the host.
  • the enhancement of antigen can be achieved through the presentation by macrophage; reception by T or B cells or both, altered antigen presentation, or adjuvant effect.
  • acetylated mannan derivative enhances the recognition of a tumor or of an infectious agent, such as a virus or another microorganism, as "not self" by the host.
  • acetylated mannan derivative can be achieved by topical application, oral ingestion, IP route, IV route or other parenteral routes of administration.
  • acetylated mannan derivative be given to the recipient as a single agent, it can also be used in combination with other known therapeutic agents that are characterized by their requirement of the participation or aid of the host's immune system to achieve their maximal therapeutic effect.
  • Acemannan has now been discovered to be a potent inducer of IL-I and prostaglandin E2 (PGE2) production by human peripheral blood adherent cells in culture.
  • PGE2 prostaglandin E2
  • the instant invention is believed to be the first practical non-toxic stimulator of IL-1 release.
  • IL-1 is an important macrophage product reported in the literature to influence the activity and production of lymphocytes, fibroblasts, B-lymphocytes and endothelial cells. See Old,. Scientific American. 258(5):59-60, 69-75 (1988).
  • IL-1 induces fibroblast proliferation which is fundamental to wound healing.
  • IL-1 also: (1) enhances bone marrow activity; it may be therapeutic in individuals whose bone- marrow is depressed; and (2) enhances the immune system in general.
  • acemannan increases the alloantigenic response of these lymphocytes in a dose-related fashion. Incubation of acemannan with monocytes permitted monocyte- driven signals to enhance the T lymphocyte response to lectin.
  • acemannan is non-toxic and is an immunoenhancer.
  • Acemannan actively stimulates lymphocytes to secrete lymphokines and also causes HIV-infected lymphocytes to produce altered glycoproteins (GP-120) by a mechanism similar to that of glucosidase I inhibitors.
  • GP-120 glycoproteins
  • Acemannan is phagocytized and apparently pumped to the Golgi/glycoprotein apparatus of the monocyte where it interferes directly with glycoprotein synthesis.
  • acemannan The toxicological effects of acemannan have been studied in both in vivo and in vitro systems. Acemannan is not mutagenic or blastogenic in in vitro test systems. In vitro, the compound was non-toxic for H-9, MT-2 and CEM-SS lymphoid cells. In vivo toxicology studies on acemannan include a 91 -day subchronic oral toxicity study in dogs, a 180-day chronic oral toxicity study in rats and an 180-day chronic oral toxicity study in humans. In these studies, no toxic effects were noted in dogs receiving up to 825 mg/kg of acemannan per day for 91 days.
  • detectable amounts of 14 C-labeled acemannan were absorbed or ingested by human peripheral monocyte/macrophage cells. Peak incorporation occurred at 48 hours.
  • the 14 C -labeled acemannan was not cytotoxic to the monocyte/macrophage cells, and the weight/volume (w/v) digested cell mass was 760 times greater than the w/v of the digested acemannan solution.
  • macrophage is capable of maintaining intracellular concentration of acemannan at very high levels that are not cytotoxic.
  • a pyrogen assay was performed in rabbits in accordance with the pyrogen test protocol outlined in the U.S.P. XXI, Biological Test [151], using a 1 mg/ml injectable solution of acemannan. More frequent temperature measurements were taken than specified in the U.S.P. because of the unknown systemic effects of injected acemannan. Temperature changes in test animals did not exceed minimum changes allowed by the U.S.P. protocol; therefore, the solution met the U.S.P. requirements for absence of pyrogens. Acemannan injectable elicited a maximum body temperature increase of 0.3 °C in one rabbit. This temperature rise occurred 90 minutes after injection. Acemannan is an inducer of IL-1 secretion by macrophages and monocytes in vitro. Since IL-1 is a potent pyrogen, this might explain the minimal, delayed temperature rise in this rabbit.
  • Immune profile results showed group differences between Day 1 to Day 7 values for the following: CD-I 6, CD-4 (T-4), CD-8+Leu7, CD-4+CD-25, CD-8+CD-16, Leu7 and TQ-1.
  • acemannan The physical properties of acemannan 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 permit it to be used in tissues and organs of living organisms and to be administered over a wide range of doses.
  • Acemannan may be administered to an animal orally, parenterally, topically and locally, in a daily dosage of 0.001 mg/kg to 1000 mg/kg body weight per day.
  • acemannan may be compressed or filled into solid dosage units such as pills, tablets and coated tablets, or it may be processed into capsules. These oral dose forms would be administered at a dosage of about 0.1 mg/kg to 1000 mg/kg of body weight per day.
  • acemannan By means of suitable liquid vehicles, acemannan can be injected in solutions, suspensions or emulsions. These products would be administered at a rate of 0.001 mg/kg to 1000 mg/kg of body weight per day. As an adjunctive component of a vaccine or other product, acemannan would be used at a rate of 0.001 to 1000 mg per unit dose of adjuvanted product.
  • Topical administration of acemannan can be in the form of a processed gel, cream, lotion, solution, ointment or powder.
  • formulations could contain up to 90% acemannan.
  • Thymocytes from C3H/HeJ mice 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 x 10" 5 M 2-mercaptoethanol.
  • the cell concentration was adjusted and dispersed into 96- well plates at 1 x 10 6 cells/well.
  • Phytohemagglutinin (PHA) was added to each well at a concentration of 10 ⁇ g/well. Samples were diluted serially and a volume of 25 ⁇ l was added to each well, starting from 1:10 to the final dilution. Every dilution was tested in quadruplicate.
  • PGE2 was evaluated with a radioimmunoassay in the same non-dialyzed supernatants.
  • the antibody to PGE2 (ICN)
  • Acemannan is a potent inducer of IL-1 production by human adherent peripheral blood leukocytes. At doses between 1 and 10 ⁇ g/ml, acemannan extract induced production of IL-1 comparable to that induced by 20 ⁇ g/ml LPS, which is the reference inducer of IL-1 production. Acemannan in the same dose range also induced the production of PGE2 at levels comparable to those induced by 20 ⁇ g/ml LPS (positive control). Table 2
  • acemannan-stimulated, antibody-mediated phagocytosis was increased to an even greater extent. These results indicate that acemannan may increase the number of macrophages and enhance their phagocytic activity. Such responses may contribute to acemannan's effectiveness as a stimulant of wound healing and as an anti-infectious agent.
  • Acemannan was stored at room temperature in its dried form. The amount needed for each experiment was weighed out and microwaved in 2-minute exposures at 600 watts of power. It was then transferred to a sterile plastic centrifuge tube and microwaved for 1 additional minute. The material was diluted in cell culture medium (RPMI-1640) to the desired concentration.
  • Phagocytic Cells Mouse spleen cells were obtained from BALB/c mice purchased from Harlan Sprague-Dawley. The mice were killed by CO 2 asphyxiation, and their spleens were removed aseptically. Subsequently, the cells were separated into adherent and non-adherent populations by nylon wool column fractionation according to the method of Journal of Immunolo gy . 71:220, the disclosure of which is hereby specifically incorporated by reference. Adherent cells were determined by microscopic analysis, as described below, to be macrophages (monocytes) and lymphocytes in a ratio of 4 to 1. After single-cell suspensions were obtained by monolayer disruption, 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 outlined below.
  • the mitogen used in the assay was PHA-P obtained from Burroughs Wellcome. As indicated for individual experiments, the cultures were maintained for 72 hours in a 5% CO2, humidified atmosphere. Tritiated thymidine was added during the last 6 hours of the culture. Cell concentrations per well, using flat bottom microtiter tissue culture plates, were 5 x 10 5 mouse cells/0.2 ml. Cells were deposited in the wells and acemannan or mitogen was added.
  • a stimulation index (SI) was calculated using the formula:
  • Sl cpm control - cpm background Cell Staining: Briefly, smears of cells were stained by non ⁇ specific esterase stain as follows. Approximately 2 x 10 6 cells in 2 drops were mixed with 2 drops of fetal calf serum and 4 drops of a fixative solution consisting of a mixture of 25 ml of 35% formaldehyde, 45 ml of acetone, 100 mg of KH2PO4, 10 mg of Na2HPO4 and 30 ml of water.
  • the slides were incubated with a mixture of 10 mg of naphthyl acetate and 4.5 mg of Fast Blue stain in 1.4 ml of ethylene glycol monomethyl ether with 5 ml of 0.1 M Trismaleate buffer, pH 7.8 (Wright's stain). The stain was allowed to react for 10 minutes, then washed in water for
  • 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 tube. Cultures were placed for 30 to 60 minutes into a 37°C, humidified 5% CO 2-95% air incubator. Cultures were washed twice with PBS and covered with 2 ml of PBS. One of each pair of coverslips was removed with needle-nosed forceps, dipped for 5 seconds only in distilled water, and promptly 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 coverslips were overlaid with distilled water.
  • PBS phosphate-buffered saline
  • SRBC Antibody-Dependent and Antibody-Independent Phagocytosis: SRBC, obtained from Austin Biologies 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 on day 14 post-injection. Serum was collected, pooled and heat inactivated at 56°C for 45 minutes. Agglutination titers were determined to be 1024 using round-bottomed microtiter wells.
  • Antibody-independent phagocytosis was determined by incubation of SRBC (0.5% v/v) with macrophages (10 6 ) in RPMI- 1640 containing 20% fetal calf serum (FCS). Slides were prepared at various intervals and stained. The percent macrophages that had ingested red cells was determined visually by counting 200 cells/slide and three slides/animal.
  • Antibody-dependent phagocytosis was determined using
  • SRBC (0.5% in RPMI-1640 with 20% FCS) mixed with anti-SRBC serum or IgM fraction (minimum titer of 2000). The mixture was incubated for 15 minutes at 37°C, then washed twice in PBS
  • Serum Fractionation Whole serum was fractionated to remove IgM by euglobulin precipitation and dialysis against distilled water. After dialysis at 4°C for 24 hours, precipitate was removed by centrifugation at 1500 x G for 20 minutes, and supernatant was analyzed by ion electrophoresis and complement-mediated lysis. Less than 5% of the original IgM remained.
  • B. Results To evaluate the effect of acemannan on macrophages, the first experiment utilized mouse spleen cells cultured in vitro with acemannan (Table 3).
  • Macrophages were determined by esterase staining. The results are expressed as mean ⁇ S.D. The results are from six experiments with 200 cells studied/experiment. "Lymphocytes” are cells that did not stain by esterase and had the appearance of lymphocytes by Wright's stain.
  • Acemannan-stimulated phagocytosis was greater than that in controls after 20-120 minutes; however, the differences were not statistically significant.
  • Phagocytosis is expressed as the mean % of cells showing erythrocyte ingestion ⁇ S.D.
  • the phagocytic activity of thioglycolate-induced peritoneal macrophages was twice as great (89% vs. 43%) as activity from the saline-induced controls, whereas acemannan- induced macrophages were more active by 30% (73% vs. 43%) compared to controls.
  • the difference between phagocytic activity in the acemannan-treated and saline control groups was statistically significant. Similar results were seen with macrophages obtained from mouse spleens. Phagocytic activity was lower than that of macrophages obtained from the peritoneal cavity, possibly due to manipulations of the spleen cells.
  • acemannan-induced macrophages were significantly higher in phagocytic activity than saline controls at the 95% confidence level; phagocytic activity was similar to control at a titer of 8 x 10 3 .
  • Phagocytosis is measured as percent uptake of sheep erythrocytes ⁇ S.D. after incubation for 30 minutes. Guinea pig complement was added.
  • IgM-depleted mouse serum was used (see Methods).
  • the titer utilized was 3000, as determined by hemagglutination and the Coombs technique. Cells from both the peritoneal cavity and spleen were more active in phagocytosis with the addition of C than without C, although the difference was statistically significant only with peritoneal cells induced by acemannan.
  • Results are reported as percent phagocytes showing phagocytosis or adherence ⁇ S.D. The results are from one experiment with 200 cells scored/animal with three animals used.
  • acemannan both directly and indirectly stimulates phagocytosis.
  • results also indicate that acemannan enhances phagocytosis by macrophages, both non-specifically and specifically, through antibody-mediated reactions. This demonstrates that acemannan has immunostimulatory properties on phagocytes.
  • Acemannan Polymer Acemannan was kept in a dried form. The amount needed for each experiment was weighed and microwaved in 2-minute exposures at 600 watts of power. The material was transferred to a sterile centrifuge tube (15 ml) and microwaved for one additional minute. The material was diluted in Hanks Balanced Salt Solution (HBSS) to the concentration needed. In some experiments, material was sterilized by autoclaving, with no apparent loss in activity.
  • HBSS Hanks Balanced Salt Solution
  • Macrophages were harvested from the peritoneal cavity of BALB/c female mice obtained from Harlan/Sprague
  • Target cells were obtained from the
  • %CT percent of cytotoxicity
  • %CT cpm in test cells - cpm in control cells CT total cpm of target cells
  • 51 Cr target cells released 51 Cr at an average of 2800 cpm
  • acemannan-labeled cells released radioactivity at an average of 3100 cpm. There was no statistical difference between these groups.
  • macrophages stimulated with acemannan in vitro had a 51 Cr release of 21,000 cpm. This indicates two things: 1) acemannan does not induce a long standing cytolytic effect, and 2) its activation can occur in a relatively short time in tissue culture. The percent cytotoxicity is parallel to the cpm released from target cells when destroyed.
  • cytotoxic effect of acemannan began within 6 hours after stimulation and increased to its maximum by 9 hours. The mechanism of this activation has not been investigated.
  • Equine Sarcoid Three sarcoids on two horses were treated both parenterally and intralesionally with acemannan. The goals of this trial were to determine whether acemannan might be an effective treatment against equine sarcoid and also to observe the horses for adverse reactions. On horse 1, one sarcoid completely resolved while a second sarcoid did not decrease in size. A third nodular sarcoid developed during treatment. On horse 2, a single sarcoid completely resolved. These results suggest that acemannan may be useful in the treatment of equine sarcoid.
  • Horse 1 Day 1. Each of the two lesions on the right rear leg was treated by direct injection (20-ga. needle), with 50 mg acemannan diluted in 10 ml saline (lesion 1) and 5 ml saline (lesion 2). Twenty-five mg acemannan diluted in 7.5 ml saline was also given IV.
  • Lesion 1 (upper lesion) was treated (18 ga. needle) with 50 mg acemannan diluted in 10 ml saline.
  • Lesion 2 was treated with 25 mg diluted in 7.5 ml saline. Fifty mg in 10 ml saline was given IV.
  • Lesion 1 was treated with 50 mg in 10 ml saline, whereas lesion 2 was treated with 25 mg in 5 ml saline. Seventy-five mg in 25 ml saline was given IV.
  • Lesion 1 was treated with 50 mg in 10 ml saline, and lesion 2 was treated with 25 mg in 10 ml saline. One hundred mg in 25 ml saline was injected IV.
  • Lesion 1 was treated as on day 21, but because of local swelling lesion 2 was not treated directly. One hundred mg in 25 ml saline was given IV. Day 42. Lesion 1 was not treated directly. Lesion 2 was treated with 25 mg in 10 ml saline. One hundred mg in 50 ml saline was given IV.
  • Horse 1 was euthanized. Tissue samples were taken at the site of lesion 1 and from lesion 2, inguinal lymph nodes and a nodular lesion on his left shoulder that had developed during the course of treatment.
  • Horse 2 Day 1. The lesion on the lower left thorax was treated with 50 mg acemannan diluted in 30 ml saline. One half was injected subcutaneously (S/Q) and the other half intralesionally. On days 6, 16, 24, 30, 49, 56, 63, 70 and 77 horse 2 was given 100 mg acemannan IV diluted in 60-120 ml saline, the amount of diluent varying as required to make a clear solution.
  • the lesion was treated with 25 mg acemannan diluted in 5 ml saline, intralesionally and S/Q at the base of the lesion. An additional 75 mg was given IV.
  • Results-Horse 1 Day 1. Lesion 1 measured 2.5 cm (length horizontally) x 2.5 cm (height vertically) x 1 cm (thickness). The resolution of this lesion can be followed below:
  • Lesion 2 measured 2 cm x 2 cm x 1 cm on Day 1 and never changed significantly.
  • Results-Horse 2 Day 1. The lesion measured 5 cm x 3.5 cm x 2.5 cm with a pedunculated base of 2.5 cm. The changes until complete resolution are shown below:
  • This example was designed to test the capacity of acemannan to enhance immune response to alloantigen and to test whether the potential enhancement is a monocyte-driven phenomenon. Acemannan did not enhance lymphocyte response to syngeneic antigens in the mixed lymphocyte culture
  • acemannan is the active ingredient of the Aloe vera plant and is an important immunoenhancer in that it increased lymphocyte response to alloantigen. It is suggested that the mechanism involves enhancement of monocyte release of IL-1 under the aegis of alloantigen. This mechanism may explain in part the capacity of acemannan to abrogate viral infections in experimental animals and man.
  • MLC Mixed Lymphocyte Cultures
  • Monocyte-T Cell Interaction Lewis female rat spleens were teased through a sterile steel mesh into RPMI- 1640 medium. 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 with varying doses of acemannan or medium (control) in a total volume of 2 ml and incubated for 24 hours at 37°C.
  • the monocytes were harvested, extensively washed with fresh medium and co-cultured with syngeneic T lymphocytes at a ratio of 10 T-cells:l monocyte, with the plant lectin phytohemagglutinin (Difco, Detroit, MI) (1 :100) for 48 hours at 37°C.
  • Cells were harvested over a MASH II (Whittaker, MA Bioproducts, Walkersville, MD), placed in fluor and counted in a scintillation counter (Beckman Laboratories, Chicago, IL).
  • a control experiment was performed by incubating T lymphocytes with acemannan, followed by wash and co-culture with freshly prepared T lymphocytes, again at 10:1 along with PHA-P.
  • acemannan did not interfere with the capacity of lymphocytes to recognize and respond to class II alloantigenic differences in the MLC; this was apparent when the syngeneic cultures were compared to the allogeneic response in the presence of the lowest concentration of drug.
  • acemannan exerts a specific effect on lymphocyte alloresponse or a nonspecific effect on tritiated thymidine incorporation
  • the reagent was added at the conclusion of a 7 day mixed lymphocyte culture MLC, 20 minutes before addition of the tracer to the culture. There was no effect of acemannan when added in this manner as a pulse at the conclusion of the MLC.
  • Acemannan is believed to be capable of limiting DNA and retrovirus infections that cause significant diseases in animals and in man. For example, in an animal model, acemannan ameloriated feline viral rhinotracheitis. Additional evidence shows that acemannan in vitro and in, vivo may be effective against Herpes simplex II virus, the measles virus, and perhaps
  • HIV HIV.
  • Acemannan is therefore an important enhancer of the alloantigenic response in MLC. There is a dose-response relationship with enhancement at the highest dose tested of about 60% above basal. This represents not only a statistically significant but also a biologically relevant increase in response to alloantigen and may serve as one means by which the drug can aid the response of the organism to viral assault. This effect of acemannan was shown to be specific for the allogeneic stimulus, provided the drug did not enhance either basal response to self (syngeneic MLC) or non-specific incorporation of a tracer DNA precursor, tritiated thymidine, when drug was added at the conclusion of the MLC.
  • acemannan was incubated along with monocytes, after which the treated, extensively washed monocytes were mixed with freshly prepared, syngeneic T- lymphocytes that had not been exposed to and would not be exposed to acemannan.
  • These experiments demonstrate the enhancement of T-lymphocyte response to the polyclonal mitogen phytohemagglutinin at a magnitude equal to the response that had been seen previously in the MLC-- approximately 55% above baseline and dose-response relationship.
  • the threshold dose may be different for the two models tested, polyclonal response to mitogen and alloantigenic response in the MLC. It can also be observed that the monocyte experiment is a more stringent test of the effect of acemannan because it presents a treated cell type, the monocyte, to T cells that then see an immune stimulus in the absence of the drug. While the alloantigenic response may be due solely or in great measure to acemannan-enhanced monocyte production of IL-1 , the lesser polyclonal mitogen- enhanced response may be a consequence of an assay of immune stimulations, each with a different threshold response to acemannan.
  • acemannan used in these experiments is clinically relevant.
  • the dose range selected was chosen precisely to bracket that concentration of acemannan that could be expected to be achieved in plasma if the drug distributes in extracellular water and is absorbed at the rate of a third of the orally-administered dose, figures that were based on previous pharmacologic studies in dogs. The actual concentrations achievable in man have also been shown to be in this range, further supporting the potential relevance of these studies for clinical practice.
  • Acemannan was shown by these experiments to cause monocytes to release monocyte-driven signals to enhance T4 cell response to lectin. While acemannan did not enhance lymphocyte response to syngeneic antigens in MLC, it did increase MLC alloantigenic response in a dose-related manner. This response was shown to be an acemannan-specific response at acemannan concentrations achievable in vivo.
  • acemannan is an immunoenhancer and biological response modifier in that it increases lymphocyte response to alloantigen.
  • a proposed mechanism of action involves stimulation of monocytes to release IL-1; in the presence of acemannan, IL-1 has been shown to be released from monocyte cultures.
  • the pharmacologic action of acemannan stimulation of monocytes may explain acemannan activity against viral infection in animals and man.
  • Blood levels which were immediately maximal at 200 ⁇ g/ml after IV injection, declined with a t ⁇ /2 of 50-60 hours; plasma levels were approximately twice those of blood.
  • blood levels peaked at 45 ⁇ g/ml at 24 hours and then declined at a rate similar to that seen with IV; in fact, blood levels were nearly 90% maximal after only 8 hours.
  • oral administration blood levels were measurable after 3 hours and peaked at 4-5 ⁇ g/ml. Based on the relatively long half-life in blood, a therapeutic dosing interval of approximately 7 days would be justified, considering the time required for three half- lives.
  • Radiolabeled acemannan distributed mainly in liver and spleen following IP or IV injection.
  • Liver, marrow, thymus, and lymph nodes were primary sites of distribution after oral dosing, a finding consistent with the immunologic sites of action for acemannan.
  • Levels of radiolabeled compound in tissues sampled after 48-52 hours ranged from a low of approximately 1 ⁇ g/g brain to a high of 85 ⁇ g/g spleen after IV injection.
  • levels in brain and spinal cord were higher (approximately 3 ⁇ g/g tissue) after oral, compared to parenteral, administration. This could be the result of the liver's partial breakdown of the polymer into smaller molecular weight fractions during the first pass, thus rendering it capable of penetrating the blood-brain barrier.
  • acemannan levels in blood and/or tissue can duplicate those levels known after injection or oral administration to produce therapeutic antitumor or antiviral effects in vitro.
  • mice implanted with virally-infected Norman Murine Myxosarcoma (NMM) cells and injected IP within 24 hours with 1 mg/kg of acemannan showed 35% survival after 60 days compared to 0% survival in NMM-treated control mice (Peng et al., submitted for publication, 1990).
  • Expected peak blood levels at an IP dose of 1 mg/kg would be on the order of 2 ⁇ g/ml (45 ⁇ g/ml x 1/20 mg/kg).
  • Blood levels of 4-5 ⁇ g/ml obtained after oral administration of acemannan are also significant, since they correspond to the concentration of acemannan that gives optimal synergism with Zidovudine® (AZT) in vitro.
  • AZT Zidovudine®
  • alone 0.001 ⁇ g/ml AZT or 3.2 ⁇ g/ml acemannan increased the viability of CEM cells infected with HTLV-IIIRFII virus by no more than 10%. Together the protective effect of the antiviral combination exceeded 70%.
  • a combination of 0.1 ⁇ g/ml of AZT and 1 mg/ml acemannan resulted in a protective effect exceeding 80% (Kemp et al. submitted for publicationl990).
  • HIV-1 human immunodeficiency virus type 1
  • HIV-1 human immunodeficiency virus type 1
  • acemannan Two studies assessed the response of human immunodeficiency virus type 1 (HIV-1) infection to acemannan and determined whether laboratory values could be used to predict response to treatment.
  • the protocol was submitted to the FDA, as an individual physician investigational new drug exemption and approved by the Institutional Review Board of the Dallas-Ft. Worth Medical Center.
  • Subjects who were HIV-1 antibody positive and symptomatic were treated with approximately 400-800 mg oral acemannan daily and evaluated clinically using modified Walter Reed (MWR) clinical scoring.
  • MWR Walter Reed
  • the 15 original subjects had an average MWR of 5.6, but after 350 days of therapy the surviving 13 had an average of 1.8.
  • CD4 levels in ten subjects increased from 346/mm 3 to 471/mm 3 within 90 days and to 610/mm 3 at 180 days.
  • Five of the 15 patients had detectable serum core antigen; by 350 days only 3 of 13 had detectable, but reduced, serum antigen.
  • Data from this first study suggested that values for CD4 and serum antigen levels could predict the response to acemannan.
  • the aggregate group had an average MWR of 3.0 at the start, and 90 days later their average was 1.8.
  • the CD4 levels of 16 "responders" rose from 313/mm 3 to 372/mm 3 during this period, but in 10 others went from 63/mm 3 to only 77/mm 3 .
  • Example 7 A PHASE II STUDY OF ACEMANNAN ALONE AND WITH AZT AMONG SYMPTOMATIC
  • acemannan was administered during 24 weeks at a daily dose of 1000 mg (2 capsules of 125 mg, 4 times daily).
  • the 23 asymptomatic patients were blindly allocated to receive either acemannan (11 patients, group 1) or placebo (12 patients, group 2).
  • acemannan 11 patients, group 1
  • placebo 12 patients, group 2.
  • PBM Peripheral blood mononuclear
  • MT-2 and CEM-SS two defined CD4+ cell lines
  • Viabilities were determined either by the trypan blue dye-exclusion test or by metabolic conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] to formazan by viable cells.
  • Virus replication and load were measured by hybridization of cell- associated viral RNA and cell -free RNA with an HIV-1 probe prepared from the P OL gene. Protection of PBM cells by acemannan treatment was shown to be concentration- dependent.
  • Percent protection ranged from 14-100% for cells treated with 3.2-100 ⁇ g/ml of acemannan. Protection by acemannan treatment of HIV- 1 -infected MT-2 cells was not only concentration-dependent but also multiplicity of infection- (MOI) dependent. Protection of CEM-SS cells infected at an
  • MOI 0.01 and treated with 62.5 ⁇ g/ml of acemannan exceeded 85%. In addition to an increase in cell viability, a concentration-dependent reduction in syncytium formation was observed. Syncytia could not be detected in cultures treated with > . 62.5 ⁇ g/ml of acemannan. A concentration -dependent reduction in virus replication was also observed for treated PBM cells. Treatment of PBM cells with concentrations of acemannan >62.5 ⁇ g/ml resulted in a 95-100% reduction in detectable cell- associated viral RNA. Treatment of virus-infected CEM-SS cells with acemannan concentrations > 62.5 ⁇ g/ml caused >60% reduction in cell-free virus. Acemannan treatment inhibits virus-induced cell fusion, increases infected cell viability, reduces virus load and suppresses production and/or release of free virus. Cytotoxicity due to acemannan was not observed at any test concentration.
  • Virus Strains The HTLV-IIIB strain of HIV-1 was obtained from Dr. R. Gallo, NIH, Bethesda, Maryland. Viral stocks were prepared by propagating the virus in H9 lymphoid cells. A stock preparation of the virus was stored at -80°C. The 50% tissue culture infective dose (TCID50)/ml of cell-free virus pool stock was determined by end-point titration using MT-2 cells. Multiplicity of infection (MOI) was determined by the method of Reed and Muench.
  • MT-2 cells were propagated in RPMI-1640 supplemented with 2 mM L-glutamine and 15% (v/v) fetal bovine serum. MT-2 cells naturally express CD4 on their surface and are thus good target cells for HIV-1 infection. In addition, they rapidly undergo cytolysis at low levels of virus replication. Primary testing of antiviral activity - MT-2 cells were first treated with polybrene (2 mg/ml) for 30 min and then infected with HIV at a MOI of 0.03. After virus absorption, the cells were pelleted and resuspended in complete medium. The infected cells were then dispensed (2 x 10 4 cells/100 ⁇ l/well) into 96-well microtiter plates.
  • Each drug was diluted in medium from a stock solution of 2 mg/ml in six serial half logi o dilutions.
  • AZT was tested from the highest concentration of 10 ⁇ g/ml to a low concentration of 0.032 ⁇ g/ml.
  • Acemannan was tested at 500 ⁇ g/ml and diluted down to a low concentration of 15.62 ⁇ g/ml.
  • Parallel assays were performed in triplicate, and drug cytotoxicity was measured at parallel concentrations in duplicate. Controls included uninfected, untreated cell cultures and virus-infected, untreated cultures. Plates were incubated for 7 days in a humidified atmosphere of 5% CO2 in air. On day
  • cell viability was measured by the addition of MTT (450 ⁇ g/ml) to the test plates.
  • a solution of 10% sodium dodecyl sulfate in 0.01N HC1 was then added to dissolve the
  • MTT formazan that was produced.
  • the color intensity is a function of the amount of formazan produced which, in turn, is proportional to the number of viable cells in each well. Plates were read at a wavelength of 570 nm on a Vmax plate reader (Molecular Devices, Inc.). The percent change in cell viability was calculated using the following formula:
  • TI is the optical density (OD) in treated, infected cells
  • TO is the OD in treated, uninfected cells
  • Ol is the OD in untreated, infected cells
  • OO is the OD in untreated, uninfected cells.
  • Antiviral activity of acemannan in combination with AZT at various concentrations was evaluated using the microtiter infection assay described above. For each mixture, defined amounts of the test compounds were dissolved in RPMI-1640, and 0.1 ml of each dilution was added to test wells. Combinations were evaluated in duplicate, and treated uninfected controls were used to determine drug cytotoxicity. Each compound was also evaluated alone at non-cytotoxic concentrations. Thus AZT was tested at concentrations ranging from 0.32 to 10 ⁇ g/ml and acemannan was evaluated at concentrations ranging from 15.62 to 500 ⁇ g/ml. The percent change in cell viability was determined as described above.
  • HIV replication cycle may complement a hit at another stage.
  • ACEMANNAN USED IN TREATING CUTANEOUS ULCERS An 83-year-old female patient, TB, developed an ulcer, 25 mm in diameter, on the lateral margin of her left foot. The ulcer had been present for several months and had failed to respond to several treatment regimens.
  • the wound was treated with the product of Example 3 of U.S. Pat. 4,735,935 and the product of Example 7 of U.S. Pat. 4,735,935 using a three-times-daily treatment schedule.
  • the clean wound was soaked for 15 minutes with the product of Example 2 of U.S. Pat. 4,735,935.
  • Excessive product was absorbed from the wound with a dry, sterile 4 x 4 gauze.
  • the product of Example 7 of U.S. Pat. 4,735,935 was then applied in a quantity sufficient to cover the wound and to prevent wound dehydration between dressing changes.
  • the progression of wound healing was measured by interval photographs and planimetry of the wound defect. The progression of wound closure is shown in Table 11.
  • Tic douloureux or neuralgia of the fifth cranial nerve, is characterized by attacks of severe, unbearable pain over one or more branches of the trigeminal nerve.
  • the pain usually is transient, and attacks may be precipitated by touching some area of the face—the so-called trigger zone.
  • Another recent treatment attempt uses carbamazepine and phenoliophendylate injections. However, these injections can be complicated by unpleasant numbness and serious side effects.
  • the patient could trigger the pain by brushing or combing her hair on the right side. She had been treated unsuccessfully with diazepam (Valium), antihistamines, analgesics, propranolol hydrochloride (Inderal) and phenobarbital. The patient said she had not had a pain-free day since the onset of the disease.
  • the proposed therapy involved drinking 1 to 2 oz. of the product of Example 2 U.S. Pat. 4,735,935 daily for 3 months. After that period, the therapy was evaluated.
  • IBD Inflammatory bowel disease
  • Diarrhea (number of bowel movements)
  • Endoscopy was utilized to score patients pre- and post- therapy according to the following criteria:
  • Example 13 THE IN VITRO EFFECTS OF ACEMANNAN ON MEASLES VIRUS Measles virus was incubated with various concentrations of acemannan and then added to susceptible cultures of VERO cells. The purpose of this experiment was to determine whether acemannan would inhibit infection or inactivate measles virus treated with acemannan prior to introduction into a susceptible cell culture. Acemannan-treated virus did not infect the VERO monolayer as evidenced by the absence of cytopathic effects (CPE) of the virus at a threshold concentration of 2.5 mg/ml. Complete absence of CPE was achieved at 5 mg/ml of acemannan in the virus inoculum.
  • CPE cytopathic effects
  • African Green Monkey kidney cells were used as the target cells. Measles virus was titrated to obtain a plaque count of 30-50 plaques/ml (20 TCID units/0.05 ml) on the virus/cell monolayer. Acemannan at different concentrations was then introduced into media containing this fixed amount of virus.
  • the concentrations of acemannan were made in complete tissue culture medium. An aliquot of rubella attenuated virus vaccine was used for each titration. The mixtures were pre- incubated at 30°C for one-half hour and added to previously prepared VERO monolayer in tissue culture chambers.
  • VERO cells were incubated with medium containing 40 TCID/ml of measles virus for various periods of time (0.5 to 6 hours) prior to the addition of 5 mg/ml of acemannan. Incubation with acemannan after cells were exposed to the measles virus did not protect the VERO cells from infection.
  • VERO cells were incubated for 0.5 to 6 hours with medium containing 40 TCID/ml of measles virus. The VERO cells were then washed with fresh medium to remove any unbound virus. Medium containing 5 mg/ml acemannan was then added to the cultures, and the cultures were examined for cytopathology after five days.
  • IMMUNE RESPONSE IN COMMERCIAL POULTRY Nationally, losses from disease and management related problems cost the poultry industry in excess of $2 billion annually. Infectious agents such as infectious bursal disease virus (IBDV), a retrovirus that induces mortality and/or morbidity associated with immunosuppression, cause severe economic losses to the poultry, industry. IBDV specifically targets precursor B -cells in the bursa of Fabricius leading to selective destruction of the humoral arm of the immune system. This causes an immunosuppressed state akin to Acquired
  • IBDV by oral administration of live virus or by subcutaneous injection of inactivated virus. Although both methods of vaccination may effectively elicit an immune response, inherent problems associated with the use of vaccines are introduced.
  • Live virus vaccines are more effective in the elicitation of a protective immune response to a specific strain, but the virus itself may revert to virulence, or replication of the vaccine strain may cause transient immunosuppression leading to increased susceptibility of the flock to secondary pathogens.
  • Killed virus vaccines do not have the same problems as those associated with live virus vaccines, but immune responsiveness is diminished and is dose-dependent. Numerous alternatives to vaccination that involve complicated high-tech solutions are being evaluated, but directed modulation of the immune response by inclusion of an additional component in a killed- virus vaccine represents a potentially simple solution.
  • Acemannan acts as an immunomodulator, and this project was designed to determine whether this compound stimulates the immune response to a killed infectious IBDV vaccine.
  • Study #1 (Group 1). For Study #1, 25 2-week-old chicks were divided into five groups. The chicks in each group were vaccinated as follows:
  • Group 5 inoculated orally with 0.5 ml of microcapsules suspended in acidic water with 0.5 mg of acemannan Study #2 (Group 2).
  • Group 2 117 1 -week-old SPF chicks were divided into six groups. The chicks in each group were vaccinated as follows:
  • chicks from the oil emulsion vaccine group (#2) and the oil emulsion vaccine supplemented with acemannan group (#3) were redivided into two groups (A and B).
  • Group A chicks were challenged with the homologous live vaccine strain, and Group B chicks were challenged with a virulent field strain.
  • acemannan caused an overall stimulatory effect of the immune system, i.e., as an enhanced immune response to test antigens administered at sites remote from the site of acemannan administration.
  • the initial impression was that acemannan had to be mixed with the oil emulsion vaccine, it appears that an enhanced immune response was elicited when the antigen and acemannan were presented separately as well. This result allows for exploration of alternative vaccination methodologies and applications for this compound.
  • Acemannan has adjuvant properties. It increases the persistence or effective presentation of IBDV antigen within the body, possibly leading to release of lymphokines and an enhanced lymphocyte response.
  • Specific human syndromes such as sprue and celiac disease can be ameliorated if certain grains containing complex polysaccharides and enzyme-inhibiting peptides are withdrawn from the diet. The result of this diet change is to reduce the symptoms.
  • a major physiological problem remains for the patient; maturation of small bowel intestinal mucosa is arrested due to inhibition in synthesis of glycoproteins essential for cell maturation. This failure of small bowel interaction reduces absorption surface and further results in failure to absorb essential amino acids, fatty acids, minerals, vitamins and other critical molecular substances present in the diet.
  • Mannose is required for glycoprotein synthesis. Providing additional mannose in a diet predictably shifts the velocity of K m , increasing the rate of glycoprotein synthesis. Enzyme synthesis is promoted by the availability of the critical mannose substrate that fosters ribosomal/glycoprotein synthesis by mannose-metabolizing enzymes. This increase in glycoprotein synthesis and availability results in small intestine mucosal cell maturation and reduction in symptoms associated with sprue and celiac disease. In addition, this thermodynamic shift in glycoprotein synthesis has applications to other categories of disease for which no effective existing therapy exists.
  • MS Multiple sclerosis
  • PLANT Acemannan was evaluated as an antiviral agent against the LaFrance virus, a major problem in the mushroom farming industry.
  • the compost used was prepared by a modification of the method of Flegg et al.
  • LaFrance virus-infected Agaricus bisporus M8 span was added to prepared compost at 3% of dry matter.
  • the spawned trays were covered with plastic and incubated for 14 days at 24° C.
  • Acemannan was added to spawned compost in a range of doses from 0.01% to 2%
  • Sporophores were analyzed for double stranded (ds) viral RNA by homogenization in 20 ml STE (1.0 M NaCl; 0.5 M Trizma base, pH 8.0; and 0.01 M EDTA), 20 ml LiCl, and 10 ml 10% SDS.
  • the dsRNA was extracted in phenol and the aqueous phase was passed through a Bio-Rad LC column containing Whatman CF- 11. The cellulose-bound dsRNA was washed and then eluted with STE. After precipitation with ethanol and resuspension in citrate solution, an aliquot was characterized by agarose gel electrophoresis. The dsRNA patterns were visualized by ethidium bromide staining. A reduction in dsRNA was shown in the sporophore analyzed from experimental trays containing
  • Example 19 ACEMANNAN USED AS A TREATMENT FOR CHRONIC FATIGUE SYNDROME Acemannan has been shown to affect chronic viral syndromes in humans.
  • a 41 -year-old female with a 2 year history of markedly debilitating "chronic fatigue syndrome" (CFS) and elevated Epstein-Barr viral titers reported that taking 800 mg/day of acemannan orally for 6 months resulted in complete relief of lethargy. After three excellent months without symptoms, the patient discontinued oral acemannan and there was a slow return of tiredness with fatigue. Resumption of acemannan rapidly alleviated the symptoms of the syndrome.
  • a physician's sister had a prolonged period of chronic fatigue syndrome with elevated Epstein-Barr antibodies. Multiple clinical evaluation and therapeutic regimens had no effect. The patient started consuming 800 mg acemannan daily and reported a marked improvement followed by elimination of symptoms after 2-3 months of acemannan therapy.
  • Patient M.A. presented with the chief complaint of inability to zip up his pants or get his belt around his abdomen.
  • CAT scans in April 1988 revealed multiple tumors in the liver which extended to the urinary bladder.
  • the liver was largely replaced by over 20 tumor masses up to 10 cm in diameter.
  • a life expectancy of 4 to 6 weeks was given.
  • the patient had been on 800 mg/day oral acemannan for HIV-1 infection.
  • a multiple chemotherapeutic treatment was initiated and the acemannan was continued.
  • the patient had minimal side-effects and toxicity usually associated with cancer chemotherapy.
  • Example 22 ACEMANNAN TREATMENT OF SKIN TUMORS ASSOCIATED WITH HIV Patient S.G. presented with two black lesions, palpable on his arm that had been previously diagnosed by biopsy as
  • Kaposi's sarcoma Acemannan gel with 5% DSMO was topically applied to the skin masses on the arm while similar masses on other parts of the body were not treated. Reexamination at weekly intervals revealed noticeable flattening and depigmentation of the lesions. Sixty days after therapy was begun, only flat scarred areas remained. Subsequent treatment of other lesions on the same patient showed the same results.
  • Patient T.P.D. presented with a palpable ankle lesion with pigmentation of classic Kaposi's sarcoma. The lesion was subcutaneously injected with 1 cc recombinant alpha interferon (Roche). The size and pigmentation were improved by 3 days. Topical acemannan gel on a bandage was applied and, by the end of 1 week, no evidence of a lesion remained. There was no scarring or alteration of pigmentation.
  • Subject W.B. a balding physician, had numerous solar keratoses over sun-exposed skin. Two weeks of nightly applications of acemannan gel to these lesions resulted in removal of the scales, crusts and skin irregularities that were pre-malignant in appearance. This response has been noted in many other mature patients.
  • Patient CM. had an abdomino-peritoneal resection of low rectal adenocarcinoma with high mesenteric lymph node metastasis.
  • the patient refused radiation therapy but elected to accept weekly 5-fluorouracil (5-FU) IV infusions and 800 mg oral acemannan/day.
  • the patient did not suffer the oral ulcers, severe fatigue or nausea with vomiting usually associated with 5-FU treatment.
  • CAT computerized tomography
  • Patient H.H. had adenocarcinoma of the colon with a resection followed by discovery of rising CEA tumor nodules and CAT scan evidence of a liver nodule.
  • Treatment with oral acemannan (800 mg/day) was begun, along with weekly IV injections of 5-FU (500 mg) dicarbazide (50 mg), and acemannan orally (800 mg).
  • Progressive reduction in tumor size and CEA values occurred with no evidence of side effects or biochemical or hematological toxicity.
  • Patient J.R. a 72-year-old male, presented post-surgery and post-radiation with metastatic adenocarcinoma of the prostate with rising acid phosphatase (PAP) and prostate specific antigen (PSA).
  • PAP acid phosphatase
  • PSA prostate specific antigen
  • the patient was started on oral acemannan (800 mg/day) 5-FU (500 mg) and dicarbazide (50 mg). The rise in tumor markers plateaued, reaching a high of 15 units for PAP and 186 U. for PSA.
  • the anti-hormonal agent Eulexin was added to the regimen.
  • the PAP dropped within 60 days to 3.0 and the PSA to 15. This response was accomplished with no toxicity or side effects at any stage. This patient continues to be monitored while the total regimen is continued.
  • Example 28 ACEMANNAN TREATMENT OF VENOMOUS ANIMAL BITES
  • Acemannan had been shown to alter the body's response to antigens, toxins, allergens and "self" antigens.
  • Two cases of acemannan gel were sent to Swangi City in Southern China.
  • the Red Cross received the product to be used for burns, bed sores, stasis ulcers, and diabetic skin ulcerations.
  • acemannan gel had been a useful treatment of the above conditions; additionally, he reported that it was the best treatment they had ever used for water snake bite, a common occurrence in the manually-worked rice paddies.
  • Acemannan-treated cultures of fibroblasts obtained from a 60-year-old man revealed a change in the morphology of these aging cells. This change appeared to evidence a reversal in the aging process in these human cells in vitro. Longer-term fibroblast cultures treated with acemannan (1 mg/ml) in the culture medium resulted in expression of biochemical and morphological characteristics of neonatal cells.
  • Acemannan was used to ameliorate the inflammatory effect of plant allergens.
  • Subject H.R.M. with a known family history of seasonal hayfever, experienced annual episodes of itching, burning, congestion and watering of mucosal membranes. Starting in 1988, it was found that 800 mg/day oral acemannan for 5 days virtually eliminated hayfever symptoms including sinus headaches produced by the swollen nasal mucosa. In 1989, it was found that acemannan gel applied topically to the mucosa of the eyes and nasal passages at bedtime and every 8 hours thereafter resulted in a similar effect and benefit to H.R.M.
  • Example 33 ACEMANNAN TREATMENT OF ALLERGIES RESULTING FROM HYPERSENSITIVITY TO CHEMICALS
  • Acemannan was used to ameliorate the inflammatory effect of chemical allergens.
  • Subject T.R. a professional painter, was on the verge of quitting his profession due to wheezing and bronchitis induced by vapors from his paints and solvents. After taking 800 mg/day oral acemannan for five days, his symptoms were relieved. The patient continues to consume
  • Histopathological staining of biopsies disclosed cytomegalovirus organisms in the epithelial cells.
  • Example 37 ACEMANNAN TREATMENT OF SEQUELA TO A RHEUMATIC FEVER EPISODE
  • S.M. had the acute onset of arthritis, tendonitis, joint edema, leukocytes, and elevated sedimentation rate following a sore throat caused by an acute episode of post-streptococcal rheumatic fever.
  • the ASO titer was markedly elevated.
  • Her sisters and mother had histories of severe, multiple bouts of acute rheumatic fever with incapacitation lasting for up to a year.
  • RA acute rheumatoid arthritis
  • a psychologist gave 800 mg of oral acemannan per day to patients who had failed to respond to psychotherapy and antidepressant drugs taken for severe depression and anxiety.
  • Feline leukemia is a retrovirus (class oncovirus) infection in which cats present with diverse clinical signs. Infection of the lymphoreticular system is predominant with the majority of animals dying within 3 years. Current treatment for this disease is symptomatic; no cure exists.
  • Example 44 ACEMANNAN AND ANTIFUNGAL DRUG TREATMENT OF FUNGAL INFECTIONS ASSOCIATED WITH HIV
  • Three HIV-1 patients' records reveal a similar pattern in that these patients developed hairy leukoplakia and/or monilial plaques and/or ulcers of the oral cavity. Their conditions were usually extensive and painful. The use of Ketaconazole had improved the condition of some patients, but others were unresponsive.
  • the patients reported elimination of these mucocutaneous lesions. Taking acemannan and Ketaconazole for 3 to 5 days cleared the outbreak for weeks to months. Continued acemannan administration eliminated or reduced outbreaks of the mucocutaneous infections.
  • PCP Pneumocystis carinii pneumonia
  • CUTANEOUS FUNGAL INFECTIONS A 40-year-old physician applied acemannan gel to itching, cracked,and burning lesions between and above the bases of his toes. In less than a week, the doctor reported that the response and healing proved acemannan to be the most effective medication he had used in over 20 years of periodic treatment of his chronic athlete's foot. Other patients have reported similar improvement. Remarkably, some patients who were taking acemannan for other conditions reported improvement of athlete's foot lesions.
  • Acemannan has been shown to be effective in treating a number of conditions where the principal mechanism of resolution or cure requires intervention by the patient's immune system. Acemannan has direct stimulatory effects on the immune system. In addition, acemannan directly interacts with virus or other infectious organisms, infected cells, and tumor cells to produce changes in their immunologically sensitive surface composition to alter the appearance of these agents and cause them to be recognized by the body's immune system and then destroyed.

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Abstract

On a découvert que l'acemannan est efficace dans le traitement de nombreuses pathologies dans lesquelles le mécanisme principal de résolution ou de guérison nécessite l'intervention du système immunitaire du patient. Cette invention concerne des procédés de traitement du cancer, des maladies virales, des maladies respiratoires et du système de régulation immun, des inflammations, des infections et des infestations, dans lesquels on administre un dérivé de mannan acétylé, tel que de l'acemannan dérivé de l'aloès. Ce procédé est utile pour effectuer des cultures de tissus, et chez les animaux et les plantes.
PCT/US1991/008204 1991-11-05 1991-11-05 Utilisations de produits issus de l'aloes, tel que de l'acemannan, dans le traitement des maladies necessitant l'intervention du systeme immunitaire pour la guerison WO1993008810A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA002122604A CA2122604C (fr) 1991-11-05 1991-11-05 Utilisation de produits derives de l'aloes tels que l'acemannane dans le traitement de maladies dont la guerison necessite une intervention du systeme immunitaire
JP4501864A JPH07500568A (ja) 1991-11-05 1991-11-05 アロエ生成物の利用
KR1019940701484A KR100209180B1 (ko) 1991-11-05 1991-11-05 아세만난을 포함하는 약제학적 조성물
PCT/US1991/008204 WO1993008810A1 (fr) 1991-11-05 1991-11-05 Utilisations de produits issus de l'aloes, tel que de l'acemannan, dans le traitement des maladies necessitant l'intervention du systeme immunitaire pour la guerison
EP92900586A EP0611304B1 (fr) 1991-11-05 1991-11-05 Utilisation de mannane acetyle (acemannan) dans la regulation des taux de cholesterol dans le serum et pour l'elimination de plaques des vaisseaux sanguins
DE69131628T DE69131628T2 (de) 1991-11-05 1991-11-05 Verwendung von acetyliertem mannan (acemannan) zur regulierung von blutcholesterin-spiegeln und entfernung von plaques in blutgefässen

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CA002122604A CA2122604C (fr) 1991-11-05 1991-11-05 Utilisation de produits derives de l'aloes tels que l'acemannane dans le traitement de maladies dont la guerison necessite une intervention du systeme immunitaire
PCT/US1991/008204 WO1993008810A1 (fr) 1991-11-05 1991-11-05 Utilisations de produits issus de l'aloes, tel que de l'acemannan, dans le traitement des maladies necessitant l'intervention du systeme immunitaire pour la guerison

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Cited By (10)

* Cited by examiner, † Cited by third party
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EP0626008A1 (fr) * 1992-01-17 1994-11-30 American Cyanamid Company Vaccin contenant de l'acemannane comme adjuvant
WO1995017898A1 (fr) * 1993-12-30 1995-07-06 Novadex Pharmaceuticals Limited Procede de prevention ou de reduction du risque d'infection par des germes pathogenes bacteriens a l'aide de dextranes conjugues
US6258796B1 (en) 1996-11-20 2001-07-10 The University Of Montana Water soluble lipidated arabinogalactan
WO2002076474A1 (fr) * 2001-03-27 2002-10-03 Pro-Pharmaceuticals, Inc. Co-administration d'un polysaccharide avec un agent chimiotherapeutique permettant le traitement du cancer
US7893252B2 (en) 2003-09-08 2011-02-22 Pro-Pharmaceuticals, Inc. Selectively depolymerized galactomannan polysaccharide
WO2011146635A1 (fr) * 2010-05-21 2011-11-24 North Texas Medical Associates Protocole de traitement d'une tumeur maligne
US10543247B2 (en) 2007-05-11 2020-01-28 Woodcliff Skincare Solutions, Inc. Aloe preparation for skin enhancement
EP3773612A4 (fr) * 2018-03-28 2022-01-12 Herbalife International of America, Inc. Acétylation de polysaccharides
CN117064972A (zh) * 2023-06-27 2023-11-17 海南九面通科技有限公司 芦荟果肉粉或其活性成分在制备治疗抑郁症药物中的应用
CN118141799A (zh) * 2024-05-11 2024-06-07 中国人民解放军军事科学院军事医学研究院 芦荟素联合干扰素在制备抑制乙肝病毒复制的药物中的应用

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Mol. Biother., vol. 3, no. 1, March 1991, M.A. SHEETS et al.: "Studies of the effect of acemannan on retrovirus infections: clinical stabilization of feline leukemia virus-infected cats", pages 41-45, see abstract; pages 41-42,44-55 *
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0626008A1 (fr) * 1992-01-17 1994-11-30 American Cyanamid Company Vaccin contenant de l'acemannane comme adjuvant
EP0626008A4 (en) * 1992-01-17 1995-11-15 Solvay Animal Health Inc Vaccine containing acemannan as an adjuvant.
WO1995017898A1 (fr) * 1993-12-30 1995-07-06 Novadex Pharmaceuticals Limited Procede de prevention ou de reduction du risque d'infection par des germes pathogenes bacteriens a l'aide de dextranes conjugues
US5514665A (en) * 1993-12-30 1996-05-07 University Of British Columbia Method of preventing or reducing the risk of infection by bacterial pathogens utilizing simple and conjugated dextrans
US6258796B1 (en) 1996-11-20 2001-07-10 The University Of Montana Water soluble lipidated arabinogalactan
US6303584B1 (en) 1996-11-20 2001-10-16 The University Of Montana Water soluble lipidated arabinogalactan
WO2002076474A1 (fr) * 2001-03-27 2002-10-03 Pro-Pharmaceuticals, Inc. Co-administration d'un polysaccharide avec un agent chimiotherapeutique permettant le traitement du cancer
US7893252B2 (en) 2003-09-08 2011-02-22 Pro-Pharmaceuticals, Inc. Selectively depolymerized galactomannan polysaccharide
US10543247B2 (en) 2007-05-11 2020-01-28 Woodcliff Skincare Solutions, Inc. Aloe preparation for skin enhancement
WO2011146635A1 (fr) * 2010-05-21 2011-11-24 North Texas Medical Associates Protocole de traitement d'une tumeur maligne
EP3773612A4 (fr) * 2018-03-28 2022-01-12 Herbalife International of America, Inc. Acétylation de polysaccharides
US11547719B2 (en) 2018-03-28 2023-01-10 Herbalife International Of America, Inc. Acetylation of aloe polysaccharides
CN117064972A (zh) * 2023-06-27 2023-11-17 海南九面通科技有限公司 芦荟果肉粉或其活性成分在制备治疗抑郁症药物中的应用
CN118141799A (zh) * 2024-05-11 2024-06-07 中国人民解放军军事科学院军事医学研究院 芦荟素联合干扰素在制备抑制乙肝病毒复制的药物中的应用

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