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WO2001000682A1 - Composition d'arabinogalactane hematopoietique - Google Patents

Composition d'arabinogalactane hematopoietique Download PDF

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Publication number
WO2001000682A1
WO2001000682A1 PCT/US2000/018180 US0018180W WO0100682A1 WO 2001000682 A1 WO2001000682 A1 WO 2001000682A1 US 0018180 W US0018180 W US 0018180W WO 0100682 A1 WO0100682 A1 WO 0100682A1
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Prior art keywords
arabinogalactan
composition
pagc
purified
csf
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PCT/US2000/018180
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English (en)
Inventor
Jinhua An
Karen S. Leu
Edwin S. Lennox
John H. Musser
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Pharmagenesis Inc
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Pharmagenesis Inc
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Priority to AU62036/00A priority Critical patent/AU6203600A/en
Priority to HK02107822.7A priority patent/HK1046290B/zh
Priority to EP00948559A priority patent/EP1200482A1/fr
Publication of WO2001000682A1 publication Critical patent/WO2001000682A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae

Definitions

  • This invention relates to arabinogalactans.
  • this invention relates to purified arabinogalactan compositions isolated from Astragalus membranaceus, especially from the roots of Astragalus membranaceus and to arabinogalactan protein compositions having a weight average molecular weight of at least 100 kiloDaltons isolated from these purified arabinogalactan compositions or intermediates thereto.
  • Huang-qi Radix Astragali, is the dried root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao or A. membranaceus (Fisch.) Bge. (Fabaceae).
  • Huang-qi is a very old and well known drug in traditional Chinese medicine. It is officially listed in the Chinese Pharmacopoeia and used mainly as a tonic and for treatment of nephritis and diabetes.
  • Huang-qi decoctions and solutions prepared from the alcohol-precipitated decoction, have also been administered by injection, and are reported to give improvement in the symptoms of gastric and duodenal ulcers and increase the white blood cell count in chronic leukopenia [The section entitled “Huangqi”, pages 1041-1046, of “Pharmacology and Applications of Chinese Materia Medica”,
  • Huang-qi decoctions have also shown activity in restoring the immune system in local xenogeneic graft-versus-host reaction [D.-T. Chu et al., "Immunotherapy with Chinese medicinal herbs. I. . . . ", J. Clin. Lab. Immunol, 25, 119-123 (1988)], reversing cyclophosphamide-induced immune suppression [D.-T.
  • Liu US Patent No. 4,843,067, discloses a pharmaceutical composition containing polysaccharides of huang-qi (stated to be extractable from either Astragalus membranaceus Bge. ox Astragalus gummifer Labillard) and polysaccharides ofministeruei.
  • the huang-qi polysaccharides are stated to be extractable by water extraction of a powder of the roots and ethanol precipitation.
  • Verbiscar US Patent No. 5,2868,467, discloses immunomodulatory polysaccharide fractions from the plants of Astragalus tragacantha (tragacanth), prepared at low temperature to "maintain the integrity of the polysaccharide toward chemical and conformational changes".
  • Josephson et al. US Patent No. 5,336,506, discloses the use of plant polysaccharides such as arabinogalactans (isolated from larch, Larix occidentalis) and mannans to form complexes with therapeutic agents for the targeting of the therapeutic agent to a cell receptor capable of receptor-mediated endocytosis.
  • Adams et al. US Patent No. 5,116,969, discloses an ultrarefmed arabinogalactan product said to be suitable for use in density gradient separation.
  • Jung et al. US Patent No.
  • 5,478,576 discloses purified arabinogalactans (also from Larix occidentalis), degradative products, and modifications thereof, also for use in delivering therapeutic agents to cell receptors capable of receptor-mediated endocytosis.
  • Lewis US Patent No. 5,589,591, discloses endotoxin-free polysaccharides, such as arabinogalactans, dextrans, mannans, and gum arabic, prepared from impure forms of these polysaccharides by ultrafiltration through first through a low molecular weight cutoff membrane, keeping the retentate, and then through a high molecular weight cutoff membrane, keeping the filtrate.
  • Arabinogalactan proteins are also found in flowering plants, and are widely distributed in most higher plants.
  • Arabinogalactan proteins (AGPs), sometimes referred to as arabinogalactan peptides, are glycosylated proteins containing high proportions of carbohydrate and usually a low (less than 10%) protein content, although AGPs having a higher protein content are known.
  • AGPs are characterized by their generally low protein content and their general ability to bind the ⁇ -glucosyl Yariv reagent, l,3,5-tris(4- ⁇ -D-gluco- pyranosyloxyphenylazo)-2,4,6-trihydroxybenzene, [J. H.
  • AGPs are components of gum arabic, a gummy exudate from the acacia tree, Acacia Senegal, that is frequently used in food products as an emulsifier, crystallization preventer, and flavor encapsulator.
  • the isolation of plant AGP genes from Nicotiana alata, Nicotiana plumbaginafolia, and Pyrus communis is disclosed in Chen et al., US Patent No. 5,646,029. An extensive discussion of AGPs may be found in E. A. Nothnagel,
  • this invention provides a purified arabinogalactan composition isolated from Astragalus membranaceus, especially from the roots of Astragalus membranaceus.
  • this invention provides an arabinogalactan protein composition having a weight average molecular weight of at least 100 kiloDaltons isolated from the purified arabinogalactan composition of the first aspect of this invention.
  • this invention provides an aqueous intravenously injectable arabinogalactan formulation comprising a therapeutically effective amount of the purified arabinogalactan composition of the first aspect of this invention or the arabinogalactan protein composition of the second aspect of this invention, and an aqueous intravenously injectable excipient.
  • this invention provides a method of treating a disease state in a mammal capable of treatment by administration of the purified arabinogalactan composition of the first aspect of this invention or the arabinogalactan protein composition of the second aspect of this invention (such as stimulating hematopoiesis, inducing the proliferation or maturation of megakaryocytes, stimulating the production of IL-1 ⁇ , IL-6, TNF- ⁇ , IFN- ⁇ , GM-CSF, or G-CSF, stimulating the production or action of neutrophils, treating neutropenia, anemia, or thrombocytopenia, accelerating recovery from exposure (e.g.
  • this invention provides methods of preparing the purified arabinogalactan composition of the first aspect of this invention, the arabinogalactan protein composition of the second aspect of this invention, and the aqueous intravenously injectable arabinogalactan formulation of the third aspect of this invention.
  • FIG. 1 shows the mean white blood cell counts versus days after chemotherapy for human cancer patients receiving chemotherapy followed by no treatment, treatment with a purified arabinogalactan composition of this invention, or treatment with G-CSF.
  • FIG. 2 shows the total symptom scores versus days after chemotherapy for human cancer patients receiving chemotherapy followed by no treatment, treatment with a purified arabinogalactan composition of this invention, or treatment with G-CSF.
  • FIG. 3 shows the scores on the Karnofsky Performance Index for human cancer patients receiving chemotherapy followed by no treatment, treatment with a purified arabinogalactan composition of this invention, or treatment with G-CSF.
  • arabinose and galactose are a generally ⁇ -glucosyl Yariv reagent- precipitable, highly glycosylated protein in which the carbohydrate accounts for at least 50% by weight of the molecule, and in which the major carbohydrate constituents are arabinose and galactose, with the arabinosyl residues primarily in terminal positions. It generally reacts specifically with a monoclonal antibody, MAC207, for AGPs.
  • arabinogalactan protein composition is a composition comprising at least 70%, particularly at least 80%, more particularly at least 90%, by weight of the composition of arabinogalactan protein (as defined above) and associated arabinogalactans and other polysaccharides.
  • a “purified arabinogalactan composition” is a composition comprising an arabinogalactan protein (as defined above) and associated arabinogalactans and other polysaccharides.
  • “Mammal” includes humans and non-human mammals, such as companion animals (cats, dogs, and the like) and farm animals (cattle, horses, sheep, goats, swine, and the like).
  • Disease includes any unhealthy condition of an animal, including an unhealthy condition resulting from medical therapy (a "side-effect"), such as disease states in which a blood tonifying effect is therapeutic, including particularly those disease states listed in the "Pharmacology and Utility” section of this application.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • a “therapeutically effective amount” means the amount that, when administered to an animal for treating a disease, is sufficient to effect treatment for that disease.
  • Treating" or “treatment” of a disease includes preventing the disease from occurring in an animal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing or arresting its development), providing relief from the symptoms or side- effects of the disease (including palliative treatment), and relieving the disease (causing regression of the disease).
  • the purified arabinogalactan composition of this invention is a "purified arabinogalactan composition" as that term is defined above that is isolated from
  • Astragalus membranaceus especially from the roots of Astragalus membranaceus; preferably from the roots of A. membranaceus Bge. var. mongholicus (Bge.) Hsiao or A. membranaceus (Fisch.) Bge.; preferably where the roots are from A. membranaceus plants grown in Inner Mongolia or Shanxi province, Peoples' Republic of China, especially the former; and preferably where the roots are from two-year-old
  • A. membranaceus plants has a typical sugar composition (determined by GLC of the trimethylsilyl derivatives of the methanolyzed composition) containing about between 5 and 15 mole %, particularly about 10%, Ara; less than about 1.5%, particularly less than about 1%, Rha; up to about 4% GalA; about between 3 and 7% Gal; and about between 70 and 90% Glc; with an Ara:Gal ratio of not less than 1.5:1, particularly not less than about 1.75:1, and typically less than about 3:1 ; an ash content of not more than about 2% by weight; a heavy metal content of not more than about 10 ppm by weight; and a hydroxyproline content of not more than about 0.1%, particularly not more than about 0.05%.
  • endotoxin i.e. having an endotoxin content, as determined by Endospecy [Seikagaku Corporation, Tokyo, Japan] assay following the manufacturer's instructions, of less than 1.0 EU/mg, particularly less than 0.8 EU/mg, more particularly less than 0.5 EU/mg, especially less than 0.3 EU/mg); soluble in water to at least 20 mg/mL, having a pH in aqueous solution between 4.5 and 6.5; and has a weight average molecular weight (Mw) between 20 and 60 kiloDaltons, particularly between 25 and 40 kiloDaltons, and more particularly between 27 and 35 kiloDaltons.
  • PGC weight average molecular weight
  • the purified arabinogalactan composition is prepared by extracting the Astragalus membranaceus (typically the sterile processed chipped or sectioned dried roots, prepared by trimming the dried roots, scrubbing with ultrafiltered (UF) water, cleaning with a disinfecting solution such as 70% ethanol, cutting into thin slices, and drying under sterile conditions), referred to as "drink chips", with hot water (typically at not less than 80 °C, particularly not less than 90 °C, especially at about 100 °C), optionally in the presence of a co-extractant such as an alkali metal salt, especially potassium or sodium dihydrogen phosphate, for a time, at a temperature, and for as many extraction cycles are necessary or desirable to cause substantial extraction of the arabinogalactan protein and associated polysaccharides from the roots (typically three times each for 3 hours at 100 °C).
  • a co-extractant such as an alkali metal salt, especially potassium or sodium dihydrogen phosphate
  • All steps following the preparation of the dried comminuted roots are typically conducted under aseptic conditions employing sterile equipment and reagents.
  • the hot water extract is concentrated (typically under vacuum at 60 - 70 °C to a concentration of about lL/Kg of "drink chips"), and then precipitated with a lower alkanol (such as ethanol, at a final ethanol concentration of about 70% at about room temperature).
  • the lower alkanol precipitate is typically washed with further lower alkanol (typically three times with 95% ethanol) and then suspended with water at a suitable concentration for further processing (typically 18 - 20% weight/volume).
  • the aqueous suspension is then treated to remove materials that are not water-soluble, such as by re-precipitating with a lower alkanol (e.g. ethanol at an ethanol concentration of about 35%).
  • a lower alkanol e.g. ethanol at an ethanol concentration of about 35%.
  • the supernatant of the lower alkanol precipitation for example 35% ethanol precipitation, is further precipitated with a higher concentration of lower alkanol, for example 40 - 80% ethanol, particularly 60-70% ethanol, to precipitate a crude arabinogalactan composition containing the arabinogalactan protein and associated polysaccharides.
  • the precipitate is re-dissolved in water and may be dried (typically by spray drying, to avoid excessive heating) to isolate the crude arabinogalactan composition.
  • the crude arabinogalactan composition will typically be a light yellow powder, soluble in water to at least about 100 mg/mL, particularly at least about 200 mg/mL, with a weight loss on drying of less than about 15% and having an endotoxin content of less than 0.5, particularly less than 0.3 EU/mg.
  • blending of the raw material, the "drink chips", and intermediates in the process may be used to achieve consistency of final product.
  • the crude arabinogalactan composition is then purified by ion-exchange chromatography. It is dissolved in water, or the re-dissolved aqueous solution of the precipitate is brought, to a suitable concentration (typically about 2%) and then ultrafiltered to further remove low molecular weight materials and to reduce the volume of the solution (such as by using a 5 kiloDalton molecular weight cutoff (5K MWCO) ultrafiltration (UF) system).
  • 5K MWCO 5 kiloDalton molecular weight cutoff
  • the retentate obtained from the ultrafiltration is then eluted through a cation exchange column (such as a SP Sepharose cation exchange column equilibrated with 20 m NaOAc buffer, pH 5.20); and the eluate loaded onto and eluted through an anion exchange column (such as a Q Sepharose anion exchange column equilibrated with the same NaOAc buffer).
  • a cation exchange column such as a SP Sepharose cation exchange column equilibrated with 20 m NaOAc buffer, pH 5.20
  • an anion exchange column such as a Q Sepharose anion exchange column equilibrated with the same NaOAc buffer.
  • the eluate from the anion exchange column may be used directly in the preparation of the arabinogalactan protein composition of the second aspect of this invention, may be concentrated and dried to form an intermediate suitable for preparation of the arabinogalactan protein composition, or may be used directly in the preparation of the purified arabinogalactan composition.
  • the purified arabinogalactan composition may be micro filtered through a suitable bacteriostatic filter (such as a 0.1 ⁇ m filter), and is ultrafiltered to desalt the solution and again reduce its volume (such as by an 8K MWCO UF system).
  • a suitable bacteriostatic filter such as a 0.1 ⁇ m filter
  • the retentate from the ultrafiltration is concentrated (such as to about 20 - 26% at 50 - 60 °C), and then precipitated with a lower alkanol (such as with ethanol at a concentration of about
  • the precipitate may be further washed (such as with anhydrous ethanol, three times), and then dried (such as in a vacuum oven at 60 - 70 °C) to give the purified arabinogalactan composition.
  • arabinogalactan protein composition of this invention is an "arabinogalactan protein composition" as that term is defined above that is isolated from Astragalus membranaceus, especially from the roots of Astragalus membranaceus; preferably from the roots of A. membranaceus Bge. var. mongholicus (Bge.) Hsiao or A. membranaceus (Fisch.) Bge.; preferably where the roots are from A. membranaceus plants grown in Inner Mongolia or Shanxi province, Peoples' Republic of China, especially the former; and preferably where the roots are from two-year-old A. membranaceus plants.
  • It has a typical sugar composition containing about between 45 and 75 mole %, particularly about between 50 and 75%, Ara; about between 2 and 4%, Rha; about between 4 and 6% GalA; about between 8 and 25%, particularly about between 10 and 20% Gal; and about between 5 and 25% Glc; with an Ara:Gal ratio of not less than 2:1, typically not less than about 3:1, and especially not less than about 4:1 ; an ash content of not more than about 2% by weight; a heavy metal content of not more than about 10 ppm by weight; and a hydroxyproline content of not less than about 0.2%, particularly not less than about 0.3%.
  • the arabinogalactan protein composition may be prepared from the purified arabinogalactan composition described above or from the eluate from the ion-exchange purification step (or a solid intermediate prepared by concentrating and drying that eluate), by ultrafiltration with a 100K MWCO UF system.
  • the eluate from the anion exchange column described in the "Preparation of the purified arabinogalactan composition" above is applied directly to the 100K MWCO UF system.
  • the retentate from this 100K ultrafiltration is then further concentrated, precipitated with a lower alkanol, optionally further washed, and dried, all in a manner similar to that described above for the preparation of the purified arabinogalactan composition, to give the arabinogalactan protein composition.
  • PBMC peripheral blood mononuclear cells
  • PAGC Since stimulation of the immune and hematopoietic system occurs via multiple cytokine interactions, the ability of PAGC to induce the production of cytokines in vitro was examined as described in Example 3.
  • PAGC triggered significant, dose-dependent release of IL-1 ⁇ , IL-6, TNF- ⁇ , IFN- ⁇ , GM-CSF and G-CSF by human PBMC after activation with PHA.
  • the three cytokines, IL-6, GM-CSF and G-CSF are known to affect the production or action of neutrophils in vitro and in vivo.
  • IL-6 by itself or in combination with other cytokines, has been reported to stimulate bone marrow megakaryocyte maturation and platelet recovery in peripheral blood in mice and nonhuman primates.
  • PAGC has been tested in a short-term, murine, ex vivo model of hematopoietic progenitor recovery as described in Example 5.
  • Fluorouracil has been widely used in mice to deplete bone marrow progenitors, colony forming units in culture (CFU-C). Because quiescent stem cells are unaffected by fluorouracil, the mice recover from this treatment along a predictable, well-documented time course [A. M. Yeager et al., "The effects of 5-fluorouracil on hematopoiesis: studies of murine megakaryocyte-CFC, granulocyte-macrophage-CFC, and peripheral blood cell levels", Exp. Hematol., 11,
  • PAGC increased, in a dose-dependent manner, the number of GM-CFC per femur on day 4 after fluorouracil ablation as can be seen from the table in Example 5.
  • the mean increase in GM-CFC per femur was 3.3-fold greater than controls (p ⁇ 0.01).
  • Animals treated with PAGC at 100 mg/Kg displayed a mean GM-CFC per femur that was 1.8-fold greater than the control, and animals treated with 50 mg/Kg PAGC displayed a mean GM-CFC similar to the control.
  • mice were sublethally irradiated and then treated subcutaneously with saline or various doses of PAGC according to the protocol described in Example 6. Irradiation dramatically decreased the number of WBC to 12% of normal on day 14 post- irradiation.
  • Treatment with PAGC increased the total WBC counts to values above those seen in the saline-treated control animals. Groups treated with 100 or 300 mg/Kg of PAGC recovered WBC counts to 80% of normal (normal range: 6000-10000 WBC/ ⁇ L blood) approximately 7-9 days ahead of the saline-treated group. In the PAGC-treated animals, normal WBC counts were reached by days 22-23, when saline-treated controls had only about 46% of normal WBC counts.
  • differential WBC counts were determined using stained peripheral blood smears. Using this information, absolute neutrophil counts and absolute lymphocyte counts were calculated. Treatment with PAGC increased the absolute neutrophil count to values above those seen in the saline- treated control animals, and also increased absolute lymphocyte counts above control values, in this model.
  • a phase II clinical trial has been completed in the Peoples' Republic of China.
  • a subpopulation of patients whose WBC counts were less than 3.0 x 10 9 WBC/L at the time of entering to the study were analyzed: of these, 168 patients were in the PAGC, 59 in the G-CSF and 23 in the no-treatment group.
  • the treatment protocol is discussed in Example 9.
  • PAGC treatment steadily increased the WBC counts of patients after chemotherapy and this increase continued to day 14.
  • administration of G-CSF showed more rapid increase of the WBC count to a maximum on day 7, this increase was not sustained and started to drop after day 7.
  • Both treatment groups achieved normal WBC earlier than the no treatment group.
  • the PAGC group achieved statistical significance when compared with the no-treatment group on day 10 and day 14, but there was no statistical difference in the WBC count between the PAGC group and the G-CSF group on day 14.
  • the above results indicate that the use of PAGC as an adjunct to chemotherapy in patients with lung, gastroenteric, and breast cancer was safe and well tolerated. Treatment with PAGC restored patients' WBC count after chemotherapy. No clinical significant adverse events were observed from the administration of PAGC.
  • BALB/c mice were sublethally irradiated on day 0 and then treated subcutaneously with saline or PAGC, as described in Example 6. Irradiation dramatically decreased the number of platelets to 7% of normal on day 10 post-irradiation. Subcutaneous PAGC treatment at 100 or 300 mg/Kg significantly enhanced platelet - 13 - recovery in the peripheral blood of the mice. PAGC induced recovery of platelet counts to 80% of normal, which ranged between 8-12 xlO 5 platelets/ ⁇ L blood, about 5-6 days earlier than the saline control group.
  • PAGC is a highly efficacious agent for enhancing platelet development and should be considered useful for the treatment of thrombocytopenia.
  • AGPC administered in the same manner in a 20-day study at doses of 100 and 250 mg/Kg promoted both red blood cell and platelet recovery with improvement over control in irradiated mice at all points post-irradiation; demonstrating the same benefit in this model as that given by PAGC.
  • PAGC accelerated the recovery of the peripheral blood platelet count in an animal model of irradiation induced bone marrow suppression as shown in the preceding paragraph, suggesting that PAGC could act by stimulating the proliferation and/or maturation of bone marrow megakaryocytes.
  • This possibility was investigated using an in vitro liquid culture system as described in Example 4. From a dose titration curve, the optimal dose of PAGC in this model was 100-200 ⁇ g/mL, with an ED 50 of 30 - 40 ⁇ g/mL. This study demonstrated that PAGC alone could promote the proliferation and/or maturation of bone marrow megakaryocytes in vitro.
  • PAGC acted synergistically with a suboptimal dose of IL-3 (50 pg/mL) to increase acetylcholinesterase (AchE) levels in a dose-dependent manner.
  • AChE acetylcholinesterase
  • PAGC may be a good candidate to remedy this situation.
  • the results from this study demonstrate that PAGC can enhance the proliferation/maturation of bone marrow megakaryocytes to a normal level in the presence of otherwise insufficient levels of endogenous cytokines.
  • PAGC stimulates the recovery of peripheral blood platelets and seems to do so by inducing the proliferation and/or the maturation of megakaryocytes.
  • the data suggest that PAGC may prove useful in the treatment of thrombocytopenia that occurs secondary to bone marrow suppression.
  • Example 9 A Phase II clinical trial is described in Example 9. A subpopulation of patients had WBC count less than 4.0 x 10 9 /L and platelet counts less than 90 x 10 9 /L at the time of entering the study. There were 54 such patients in the PAGC group. These patients' platelet counts increased to greater than 100 x 10 9 /L on day 7 and the counts continued to increase through day 14 as seen in the table in Example 9. These data show that PAGC increased the platelet counts of these patients after chemotherapy.
  • PAGC was shown effective for treatment of chemotherapy- induced neutropenia.
  • the data suggest also that PAGC may be useful in the prevention of neutropenia as well as in the treatment of neutropenia, and clinical trials are planned in the PRC for this indication.
  • mice were sublethally irradiated (4.25 Gy) and then treated with saline or various doses of PAGC according to the protocol described in Example 6. Irradiation dramatically decreased the number of RBC to 55% of normal on day 17 post-irradiation. PAGC treatment resulted in significantly higher RBC counts on day 17, when RBC counts of saline-treated control animals were still at their lowest point. In animals given 100 or 300 mg/Kg of PAGC, RBC counts rose to 80% of normal (8.5 - 11 x 10 6 / ⁇ L blood) approximately 4-6 days ahead of the saline-treated group.
  • PAGC-treated animals showed RBC counts in the normal range by days 20-22, whereas, on those same days, the saline-treated controls had only about 65% of normal RBC counts.
  • AGPC administered in the same manner in a 20-day study at doses of 100 and 250 mg/Kg promoted both red blood cell and platelet recovery with improvement over control in irradiated mice at all points post-irradiation; demonstrating the same benefit in this model as that given by PAGC.
  • Example 9 The phase II trial of Example 9 was targeted to study the recovery of WBC counts in patients after chemotherapy, and the inclusion criteria were based on WBC counts only.
  • An additional clinical trial has been planned in the PRC to study the use of PAGC for the treatment of anemia, targeting patients with anemia after chemotherapy.
  • the CD34 antigen is present on hematopoietic stem cells and progenitor cells, including colony-forming cells such as BFU-E (burst-forming unit - erythroid), GM-CFC (granulocyte macrophage colony forming cells), and CFU-Mix (colony forming unit - mixture) cells.
  • colony-forming cells such as BFU-E (burst-forming unit - erythroid), GM-CFC (granulocyte macrophage colony forming cells), and CFU-Mix (colony forming unit - mixture) cells.
  • BFU-E burst-forming unit - erythroid
  • GM-CFC granulocyte macrophage colony forming cells
  • CFU-Mix colony forming unit - mixture
  • Example 7 The ability of PAGC to induce PBPC mobilization when administered as a single agent and/or to synergize with G-CSF to induce PBPC mobilization when administered in combination with G-CSF was demonstrated in Example 7. As can be seen in the table in Example 7, PAGC resulted in a 6-fold mean increase in circulating GM-CFC. Additionally, PAGC synergized with G-CSF, resulting in an 83-fold mean increase in circulating GM-CFC. This increase was significantly better than the 39-fold increase observed with G-CSF alone. The results showed that PAGC increased the numbers of circulating GM-CFC and synergized with G-CSF to increase the numbers of circulating GM-CFC in normal mice.
  • PAGC also resulted in a 3 -fold mean increase in circulating BFU-E, as shown in the table, and synergized with G-CSF, resulting in a 9.5-fold mean increase in circulating BFU-E. This increase was significantly better than the 4.5-fold increase observed with G-CSF alone.
  • Therapeutic agents such as PAGC which synergize with G-CSF to increase the yields of PBPC will likely be very useful. This type of synergy may reduce costs by decreasing the number of aphereses necessary to harvest PBPC from donors. Additionally, this type of combination therapy may help in situations where recipients are not sufficiently responsive to G-CSF alone or where the use of chemotherapeutic drugs is not desirable. Similarly, AGPC should offer the same benefit.
  • PAGC will also be useful in accelerating recovery from exposure (e.g. accidental or non-therapeutic exposure, as well as therapeutic exposure) to cytotoxic agents or radiation.
  • PAGC could increase the body weight of mice treated with chemotherapeutic agent cyclophosphamide or fluorouracil.
  • the mice treated with PAGC lost less weight and regained weight faster than those treated with the chemotherapeutic agents alone, although these differences were not statistically significant.
  • PAGC has clearly demonstrated to be effective in improving patients' quality-of-life in a Phase II clinical trial.
  • One of the parameters measured was improvement in appetite. This, together with the mouse study, suggests that PAGC could help patients with cachexia, a general physical wasting and malnutrition caused by a chronic disease such as cancer or the therapies for it.
  • PAGC stimulates the production of cytokines especially G-CSF from activated human peripheral blood mononuclear cells, promotes recovery of GM-CFC and peripheral WBC counts in radiation-induced myelosuppression animal model, and restores the WBC counts of cancer patients after chemotherapy in a Phase II clinical trial.
  • cytokines especially G-CSF from activated human peripheral blood mononuclear cells
  • PAGC may act indirectly on the hematopoiesis system through production of multiple endogenous cytokines and these cytokines may act in synergy to promote neutrophil recovery.
  • PAGC is also shown to have a synergistic effect with IL-3 in promoting the proliferation/maturation of bone marrow megakaryocytes as discussed above.
  • BPC blood progenitor cell
  • PAGC can restore cancer patients' WBC counts after chemotherapy as shown in the Phase II clinical trial of Example 9; and that together with the pharmacological effects summarized in previous sections suggest that PAGC may also be useful as a combination therapy with G-CSF after BPC transplantation to accelerate neutrophil recovery.
  • Cytokines play an important role in the defense against viral infections.
  • the cytokines produced by cells involved in the immune response such as macrophages and CD4+ and CD8+T lymphocytes, play a more direct role in recovery from viral infection such as hepatitis B viral (HBV) infection.
  • HBV hepatitis B viral
  • IFN- ⁇ by CD4+ T cells which prime and maintain antigen-specific cellular immunity, is important in defense against viral infection [C. A. Biron, "Cytokines in the generation of immune response to, and resolution of, virus infection", Curr. Opin. Immunol. , 6, 530-538 (1994)].
  • the cytokines released by CD4+ and CD8+ cells also play an important role in the downregulation of HBV replication. If there is a defect in the acute response, HBV becomes chronic.
  • PAGC is prepared from the traditional Chinese medicinal plant Astragalus membranaceus var. mongholicus (AM) and this plant has been used historically to stimulate the immune and hematopoietic systems. It is widely used to treat patients with various ailments, which are similar to the symptoms of chemotherapy- or radiotherapy-induced myelosuppression (thrombocytopenia and anemia in addition to neutropenia). Additionally, AM has been reported to stimulate mouse spleen cell proliferation in a dose-dependent manner in vitro, increase the natural killer (NK) cell activity in spleen cells of animals inoculated with S-180 sarcoma tumor cells, and also increase the activity of cytotoxic T lymphocytes (CTL).
  • NK natural killer
  • the cytokines produced by CTL can mediate control of viral infection in vivo, and the production of IFN- ⁇ and TNF- ⁇ by virus-specific CTL can amplify the ability of CTL to clear viral infection [L. G. Guidotti et al., "Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice", Proc. Natl. Acad. Sci. USA, 91, 764-3768 (1994)]. Furthermore, as shown above, PAGC stimulated the production of cytokines IL-l ⁇ , IL-6, TNF- ⁇ , IFN- ⁇ , GM-CSF and G-CSF from human PBMC after activation with PHA.
  • PAGC may affect immune function indirectly through modulation of cytokine production.
  • Crude extracts prepared from AM have been used in chronic hepatitis patients to reduce the elevated IgG, to lower ALT value, and to improve patients' immune and liver functions [Y. Liu, "Therapeutic effect of oral solution from Astragalus in the treatment of 70 chronic hepatitis B patients", Jiang Su Chung Yao, 15(12), 38 (1994)].
  • fractionated AM has been shown to have immunopotentiating activity, as discussed earlier.
  • Polysaccharide prepared from Polyporus umbellatus has been used as an adjuvant to hepatitis B vaccine in treating chronic hepatitis B patients. It has been shown to have statistically significant sero-negative conversion of hepatitis B e antigen (HBeAg) and disappearance of hepatitis B viral DNA (HBV-DNA) [S. M. Wu et al., "The therapeutic observation on the combined Polyporus polysaccharide with hepatitis B vaccine in the treatment of chronic hepatitis B". J. Chin. Infectious Disease, 13(3), 187-189 (1995); H. Z.
  • Extracts prepared from AM also has been reported to have sero-negative conversion of HBeAg and anti hepatitis B core antigen (anti HBc), and elimination of HBV-DNA in hepatitis B patients [C. K. Liu et al, "Clinical and experimental studies on effects of chronic hepatitis B treated with Astragali composita", Chung Kuo Chung Hsi I Chieh Ho Tsa Chih, 16(7), 394-397 (1996); P.
  • TCM qi (energy) deficiency
  • Nausea and vomiting is a common side effect of chemotherapy and/or radiation therapy. Although there are many improvements with chemotherapy or radiation therapy, a significant number of patients still experience emesis, and efforts to reduce this side effect of treatment must continue.
  • antiemetic drugs such as serotonin receptor antagonists, corticosteroids, and dopamine receptor antagonists
  • Symptoms that have been associated with these drugs are light headache, constipation, trouble sleeping, restlessness, involuntary movements of the muscles and tongue, and sedation.
  • the goals related to the complete control of emesis include providing care that is convenient for the patient, treatment that reduces hospitalization and time in the ambulatory setting, and therapy that enhances the patient's quality-of-life.
  • the clinical trial in the PRC has demonstrated that PAGC is beneficial to patients after chemotherapy, especially in - 22 - improving patients' quality-of-life. Observations from the investigators and patients all support that PAGC can really improve the overall well-being of the patients, and this suggests that PAGC may have a role in preventing and treating emesis after chemotherapy.
  • EPOGEN epoetin alfa, erythropoietin
  • PAGC peripheral red blood cell counts in sublethally irradiated mice as shown in Example 6.
  • AGPC administered in the same manner in a 20-day study at doses of 100 and 250 mg/Kg promoted both red blood cell and platelet recovery with improvement over control in irradiated mice at all points post-irradiation; demonstrating the same benefit in this model as that given by PAGC.
  • PAGC increased the numbers of circulating BFU-E when administrated as a single agent and synergized with G-CSF to increase the numbers of circulating BFU-E in normal mice.
  • PAGC-treated mice showed a significant increase of TER-119 cells in peripheral blood of both normal and cyclophosphamide-treated mice.
  • the TER-119 antigen is expressed on erythroid cells from the early erythroblast through mature erythrocyte stages.
  • An increase in the number of TER-119 + cells indicates that PAGC could stimulate the differentiation, proliferation and maturation of the erythroid lineage in the bone marrow, and the mobilization of these cells to the peripheral blood. All these findings suggest that PAGC can promote the production and maturation of red blood cells in the mice studied. Since the use of PAGC in the clinical trials was safe and no clinically significant adverse events were reported, it is therefore suggested that PAGC may be able to reduce or even replace the use of EPOGEN for treatment of anemia in kidney dialysis patients.
  • the purified arabinogalactan composition of the first aspect of this invention or the arabinogalactan protein composition of the second aspect of this invention will be administered in therapeutically effective amounts by intravenous injection, either singly or in conjunction with of at least one other therapeutic agent, especially a therapeutic agent capable of stimulating hematopoiesis.
  • a therapeutically effective amount may vary widely depending on the disease, its severity, the age and relative health of the animal being treated, and other factors.
  • a therapeutically effective amount of the purified arabinogalactan composition of the first aspect of this invention ranges about between 50 and 1000 mg/day, particularly about between 100 and 500 mg/day, especially about 250 mg/day, for a human of average body mass.
  • the arabinogalactan protein composition of this invention has a higher content of the arabinogalactan protein itself, it is expected to be more potent, and will have a correspondingly lower therapeutically effective amount, such as about between 10% and 50% of the therapeutically effective amount of the purified arabinogalactan composition.
  • a person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine a therapeutically effective amount of the compositions of this invention for a given disease.
  • the purified arabinogalactan composition of the first aspect of this invention or the arabinogalactan protein composition of the second aspect of this invention will be administered as pharmaceutical formulations by intravenous injection.
  • the formulation will comprise the purified arabinogalactan composition of the first aspect of this invention or the arabinogalactan protein composition of the second aspect of this invention in combination with an aqueous intravenously injectable excipient.
  • Suitable aqueous intravenously injectable excipients are well known to persons of ordinary skill in the art, and they, and the methods of formulating the formulations, may be found in such standard references as Alfonso AR: Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton PA, 1985.
  • Suitable aqueous intravenously injectable excipients include water, aqueous saline solution, aqueous dextrose solution, and the like.
  • the purified arabinogalactan composition of the first aspect of this invention or the arabinogalactan protein composition of the second aspect of this invention will be administered by intravenous injection, especially by continuous intravenous infusion over a period of a few minutes to an hour or more, such as around fifteen minutes.
  • the amount of a compound of this invention in the composition may vary widely depending on the type of composition, size of a unit dosage, kind of excipients, and other factors well known to those of ordinary skill in the art.
  • the final composition may comprise from 0.001 percent by weight (%w) to 10 %w of the compound of this invention, preferably 0.01 %w to 1 %w, with the remainder being the excipient or excipients.
  • the purified arabinogalactan composition of the first aspect of this invention and the arabinogalactan protein composition of the second aspect of this invention may optionally be administered in conjunction with at least one other therapeutic agent for the - 25 - disease state being treated, especially another agent capable of stimulating hematopoiesis such as, for example, erythropoietin, thrombopoietin, granulocyte colony stimulating factor (G-CSF), IL-3, and the like.
  • another agent capable of stimulating hematopoiesis such as, for example, erythropoietin, thrombopoietin, granulocyte colony stimulating factor (G-CSF), IL-3, and the like.
  • the size exclusion chromatography method was as follows: A Shimadzu HPLC system, equipped with an SCL-10A system controller, LC-IOAD pump, DGU-4A degasser, RID-6A refractive index detector, and SPD-1 OAV UV detector, and using GS-701 and GS-620 columns (Shodex Asahipak, 7.6 x 500 mm) equilibrated with 0.2 N sodium chloride.
  • the sample amount loaded was 80 ⁇ g ( 40 ⁇ L of sample solution at 2 mg/mL in water), and samples were eluted at 1 mL/min. Pullulan standards with different average molecular weight were used to prepare a calibration curve; and the molecular weights were determined from the calibration curve.
  • the sugar content and composition of the PAGC and AGPC of this invention was determined by GLC analysis of the trimethylsilyl methyl glycoside derivatives.
  • the polysaccharide was first methanolyzed in methanolic HCl, followed by a trimethylsilyl (TMS) derivatization to generate volatile monosaccharide derivatives.
  • TMS trimethylsilyl
  • the derivatives were analyzed by Gas Liquid Chromatography (GLC) using a DB-1 column with a Flame Ionization Detector (FID).
  • An internal standard, myo- inositol was derivatized and analyzed together with the composition sample to quantitate the sugar content and composition.
  • Hydroxyproline content was determined by a colorimetric assay.
  • the sample was first hydrolyzed with hydrochloric acid, then treated with sodium hypobromite (a solution of bromine in sodium hydroxide), hydrochloric acid, and dimethylamino- benzaldehyde.
  • sodium hypobromite a solution of bromine in sodium hydroxide
  • hydrochloric acid a solution of bromine in sodium hydroxide
  • dimethylamino- benzaldehyde dimethylamino- benzaldehyde.
  • the optical density of the final solution was measured on a colorimeter, with the hydroxyproline content determined from a calibration curve made from hydroxyproline of various concentrations prepared in the same manner.
  • Dried Astragalus membranaceus roots 300 Kg were processed into drink chips by removing any contaminated parts, sterile washing and scrubbing with ultrafiltered water, soaking in 70% ethanol overnight, cutting into chips with a thickness of 3 - 5 mm, and sterile oven drying at 60 - 70 C. These dried "drink chips" have a loss on drying of ⁇ 15%.
  • Step B Crude arabinogalactan composition extraction
  • the pooled water extract was concentrated with a concentrator at 60 - 70 °C to 200 L under reduced pressure. Ethanol, 95%, was added to the concentrate to give a final ethanol concentration of 70%, with stirring at room temperature for fifteen minutes, to precipitate polysaccharides. The supernatant was decanted and the precipitate was washed three times with 95% ethanol.
  • the precipitate was re-suspended in UF water to a concentration of 18 - 20%, as measured by a refractometer, and 95% ethanol was added to give a final ethanol concentration of 35%.
  • the ethanol suspension was centrifuged, the precipitate was discarded, and 95% ethanol was added to the supernatant with stirring to give a final ethanol concentration of 70%.
  • the precipitate was collected, re-dissolved in UF water, and spray dried to generate the crude arabinogalactan composition.
  • the crude arabinogalactan composition was a light-yellow powder, soluble in water at 200 mg/mL, and had a loss on drying of ⁇ 15%.
  • the endotoxin content was ⁇ 0.3 EU/mg.
  • the crude arabinogalactan composition, 3.5 Kg, from Step B was dissolved in UF water to a concentration of 2% (volume of 175 L).
  • the solution was filtered through a 5K MWCO UF system to a final volume of 35 - 40 L.
  • the concentrated solution was adjusted to a 20 m NaOAc buffer concentration at pH 5.2 by the addition of 1.0 M NaOAc buffer, pH 5.2.
  • the solution was loaded onto an SP Sepharose cation exchange column (20 L volume, 30 cm bed height) and eluted with 20 mM NaOAc, collecting 2.5 - 3.0 bed volumes of the eluate.
  • the collected eluate was loaded onto a Q Sepharose anion exchange column with the same resin volume and bed height as the SP column and eluted with 20 mM NaOAc, collecting 3 - 3.5 bed volumes of the eluate.
  • the collected eluate from the Q Sepharose column was filtered through a 0.1 ⁇ m filter and then ultrafiltered with an 8K MWCO UF system.
  • the retentate was condensed to 20 - 26% by a concentrating system at 50 - 60 °C and precipitated by addition of anhydrous ethanol to a final ethanol concentration of 80 - 90%.
  • the precipitate was washed three times with anhydrous ethanol and dried in a vacuum oven at 60 - 70 °C to give the purified arabinogalactan composition.
  • the purified arabinogalactan composition was a white powder, soluble in water, saline, and 5% glucose at 20 mg/mL, and having a water content of ⁇ 6.0%>.
  • the composition contained ⁇ 2.0% of ash, ⁇ 10 ppm of heavy metals, and ⁇ 0.1 EU/mg of endotoxin.
  • An aqueous solution of the composition had a pH between 4.5 and 6.5.
  • the composition had a sugar content of ⁇ 85% (determined by the phenol-H 2 SO method with glucose as standard), and an Ara:Gal ratio of > 1.5:1 (determined by GLC of the TMS-methylglycoside derivatives of the composition).
  • the collected eluate from the Q Sepharose column of Step C of Example 1 was ultrafiltered with a 100K MWCO UF system.
  • the retentate from the ultrafiltration was further concentrated, and precipitated by addition of anhydrous ethanol to a final ethanol concentration of 80 - 90%.
  • the precipitate was washed three times with anhydrous - 28 - ethanol and dried in a vacuum oven at 60 - 70 °C to give the purified arabinogalactan composition.
  • the arabinogalactan protein composition was a white powder, soluble in water, saline, and 5% glucose at 20 mg/mL, with an aqueous solution having a pH between 4.5 and 6.5.
  • the composition contained ⁇ 0.5 EU/mg of endotoxin, and ⁇ 10 ppm of heavy metals. It had an Ara:Gal ratio of > 2:1 and contained more than 0.2% hydroxyproline.
  • the weight average molecular weight of the composition was > 100 kiloDaltons.
  • PBMC Human peripheral blood mononuclear cells
  • Human blood buffy coat samples approximately 25 mL/donor, were obtained from the Stanford University Medical Center Blood Bank. Using sterile techniques, each buffy coat samples was gently resuspended in a total volume of 100 mL with the addition of calcium- and magnesium-free Hank's balanced salt solution (HBSS, Gibco) at room temperature.
  • HBSS Hank's balanced salt solution
  • a volume of 25 mL of the cell suspension was then layered onto 15 mL of Ficoll-Paque (Pharmacia LKB Biotechnology, Inc.) in a 50 mL conical centrifuge tube, and the tube was centrifuged in a Beckman GPR tabletop centrifuge (GH-3.7 rotor) at 400 x g for 30 minutes at 15 °C. Following centrifugation, the PBMC suspension at the interface was transferred to a new 50 mL tube, resuspended in a total volume of 45 mL HBSS, and centrifuged at 354 x g for 10 minutes at 15 °C.
  • the supernatant was discarded, the cell pellets was resuspended to a total of 45 mL with HBSS, and centrifuged again at 265 x g for 10 minutes at 15 °C.
  • the cell pellet was resuspended in 10 ml of X- Vivo tissue culture medium (Bio Whittaker, MD) and counted using a hemocytometer.
  • Polystyrene tubes (Falcon # 2057, Becton Dickinson) and PBMC from 2 different donors were used in the following experiment.
  • PBMC suspensions were diluted to 4 x 10 6 /mL; 1 mL was incubated in the presence of 0.5 mL phytohemagglutinin P (PHA-P, Pharmacia 27-3707-01) at a final concentration of 3 ⁇ g/mL together with 0.5 mL of a solution of one of the compositions of this invention at various concentrations.
  • the total volume per tube was 2 mL. Another aliquot of cells treated with PHA alone served as control.
  • Optical density was determined using a microplate reader (Thermo max, Molecular Devices, CA). Results were calculated using the software provided with the microplate reader and expressed as pg/mL of cytokine produced in the supematants. All results were expressed as a ratio of sample to control (S/C) where S is the amount of cytokine produced in PBMC stimulated with PHA plus test sample and C is the amount of cytokine produced in PBMC stimulated with PHA alone. The following table shows that PAGC increased cytokine production by activated human PBMC.
  • the liquid culture assay for megakaryocyte maturation is an in vitro assay for studying the proliferation and/or maturation of bone marrow megakaryocytes (the progenitors of peripheral blood platelets).
  • a detailed protocol is given in S. A. Burstein, "Interleukin 3 promotes maturation of murine megakaryocytes in vitro ", Blood Cells, 11, 469-479 (1986).
  • Normal mouse bone marrow mononuclear cells were isolated and suspended in medium containing 0.5 mM diisopropylflourophosphate (DFP, Sigma, St.
  • acetylcholinesterase AchE
  • the cells were washed, resuspended, and placed in a plastic tissue culture flask at 4 x 10 6 /mL in 15% FCS-IMDM (Gibco BRL, Gaithersburg, MD) to remove stromal cells and macrophages by their adherence to the flask.
  • FCS-IMDM Gibco BRL, Gaithersburg, MD
  • the flask containing resuspended bone marrow cells was incubated at 37 °C, 5% CO 2 for 1.5 hours.
  • Non-adherent cells were collected and resuspended at 1 x 10 /mL in 1% Nutridoma SP (Boehringer Mannheim, Indianapolis, LN)-IMDM for assay.
  • the cells were added to 96-well,
  • Solution I (0.2% Triton X-100, 1 mMEDTA, 0.12 mMNaCl, 50 m HEPES, pH 7.5), 0.2 mL, and 20 ⁇ L 6.27 mM acetylthiocholine iodide (Sigma, St. Louis, MO) were added to each well (the final concentration of acetylthiocholine iodide is 0.57 mM).
  • the emitted fluorescence was measured in a fluorimeter (Fluoroskan II, Labsystems, Helsinki, Finland) with an excitation filter of 390 nm and an emission filter of 460 nm.
  • the readings from the fluorimeter are directly proportional to the production of AChE from megakaryocytes in the culture.
  • PAGC at concentrations between 12.5 ⁇ g/mL and
  • mice Female BALB/c mice, 8-10 weeks old, were treated with 150 mg/Kg fluorouracil (Sigma) intraperitoneally on day 0, dosed subcutaneously with saline, various concentrations of PAGC or 100 ⁇ g/Kg recombinant human G-CSF (Neupogen, Amgen) on days 1 -3, and sacrificed on day 4. Femoral bone marrow was harvested, counted, and 5 x 10 4 leukocytes were plated in 35 mm Petri dishes in triplicate 1 mL cultures in complete methylcellulose medium with pokeweed mitogen spleen cell conditioned medium as a source of colony stimulating factors (StemCell Technologies Inc).
  • Colonies of greater than 50 cells were scored using a Nikon Diaphot microscope (30-60 ⁇ ) after 6-7 days of incubation at 37°C, with the following table showing that PAGC augmented GM-CFC progenitor recovery in fluorouracil-treated mice.
  • mice Female BALB/c mice with an average body weight of 20 grams, 9-14 weeks old, were used for the study. Five days before each experiment, Neomycin (Sigma, St. Louis, MO), 40 mg/L, was added to non-acidified drinking water. Mice were randomly assigned to control or treated group, 6 mice per group, and were irradiated were irradiated with 4.25 Gy of X-rays (250 KVP, 0.35 mm Cu filter, Philips, Germany) on day 0. Following this dose of irradiation, the peripheral blood leukocyte and platelet counts are significantly lower than that of normal mice, and the erythrocyte count is moderately lower. PAGC was given by subcutaneous injection at 300 and 100 mg/Kg.
  • Treatments were given each for the first 5 days (from day 0 to day 4), and then 3 times a week for the next 3 weeks, with the first dose given 4-5 hours after irradiation. A total of 14 doses of PAGC were administered.
  • the control group was given 0.1 mL saline subcutaneously. Mice were bled through the tail veins twice/week during the experiment; blood samples were collected into EDTA-coated tubes (Sarstedt, Germany); and peripheral blood white blood cells, platelets, red blood cells, and hemoglobin were analyzed in a Serono 9010+ cell counter (Serono Baker Diagnostics Inc., Allentown, PA).
  • PAGC at doses of 100 and 300 mg/Kg promoted white blood cell recovery in irradiated mice with statistically significant improvement (p ⁇ 0.01) over control from days 17 through 30 (end of the trial) post-irradiation; promoted platelet recovery in irradiated mice with statistically significant improvement (p ⁇ 0.05, generally p ⁇ 0.01) over control from days 14 through 25 post-irradiation; and promoted red blood cell recovery in irradiated mice with statistically significant improvement (p ⁇ 0.05, generally p ⁇ 0.01) over control from days 14 through 25 post-irradiation.
  • mice Female BALB/c mice, 8-10 weeks old, were given acidified water and food ad libitum. Normal mice were treated for seven days, once per day, as follows: saline (200 ⁇ L, subcutaneously), PAGC(100 mg/Kg, subcutaneously), G-CSF (100 ⁇ g/Kg, subcutaneously), or PAGC+G-CSF (100 mg/Kg PAGC plus 100 ⁇ g/Kg G-CSF, subcutaneously). Injection volumes were approximately 200 ⁇ L per mouse. Each group consisted of 5 animals each.
  • mice were treated intraperitoneally with 20 units of heparin (Elkins-Sinn, Inc., Cherry Hill, NJ), sacrificed by CO 2 inhalation 30 minutes post-heparin treatment, and peripheral blood was collected by cardiac puncture.
  • Peripheral blood mononuclear cells were isolated by density centrifugation using Ficoll-Paque (Pharmacia Biotech AP, Uppsala, Sweden), washed twice in phosphate buffered saline, resuspended in medium and counted using a hemocytometer.
  • peripheral blood mononuclear cells were plated in 35 mm Petri dishes in triplicate 1 mL cultures in complete erythropoietin-containing methylcellulose medium with recombinant IL-3 + IL-6 + stem cell factor (StemCell Technologies Inc., Vancouver, B.C.). Colonies of greater than 50 cells were scored using a Nikon Diaphot microscope (30-150 ⁇ ) after 7-14 days of incubation at 37°C. Both granulocyte macrophage colony forming cells
  • GM-CFC burst forming units - erythroid
  • BFU-E burst forming units - erythroid
  • mice were treated intravenously with 200 mg/Kg cyclophosphamide and then dosed with saline or PAGC at 100 or 300 mg/Kg for eleven days, the PBMC collected and suspended at 2 x 10 7 cells/mL, stained with fluorescein isothiocyanate-anti-CD34 and PE-lineage markers (CD3, CD4, CD6, CD 19, CD1 lb, GR-1, CD41, and Ter-119) (Pharmingen, San Diego, CA) and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA), it was seen that PAGC increased the mobilization of CD34 + Lin " cells into the peripheral blood.
  • fluorescein isothiocyanate-anti-CD34 and PE-lineage markers CD3, CD4, CD6, CD 19, CD1 lb, GR-1, CD41, and Ter-119
  • flow cytometry FACSCalibur, Becton Dickinson, San Jose, CA
  • Example 8 Recovery of body weight by administration of PAGC in fluorouracil- or cyclophosphamide-treated mice
  • mice Female BALB/c mice, 8-10 weeks old, were used in this study. Ten mice each were randomized to one of the 5 groups, normal control, cyclophosphamide (CY)- treated, fluorouracil (FU)-treated, CY+PAGC-treated or FU+PAGC-treated.
  • CY cyclophosphamide
  • FU fluorouracil
  • CY+PAGC-treated or FU+PAGC-treated mice were injected intraperitoneally with saline on day 0 and subcutaneously with saline from day 1 through day 12.
  • the CY-treated or CY+PAGC- treated group were given 200 mg/Kg cyclophosphamide intraperitoneally on day 0, and then treated subcutaneously with saline or 200 mg/Kg PAGC from day 1 through day 12.
  • mice were given 150 mg/Kg fluorouracil intraperitoneally on day 0, and then treated subcutaneously with saline or 200 mg/Kg PAGC from day 1 through day 12. The mice were weighed on day 0 and then every other day through day 12. The results show that mice treated with CY+PAGC and FU+PAGC lose less weight and regain weight faster than those treated with CY or FU alone.
  • Example 9 Human clinical trials with PAGC in the Peoples' Republic of China
  • PAGC diluted in normal saline was administered intravenously to 32 normal volunteers by continuous infusion for seven days. There were no clinically significant adverse reactions associated with the administration of PAGC at up to three times the intended clinical dosage (250 mg/day).
  • a Phase II trial was conducted to evaluate the efficacy of PAGC in alleviating chemotherapy-induced leukopenia ( ⁇ 4.0 x 10 9 WBC/L) in patients with lung, gastroenteric, or breast cancer.
  • Six centers were involved in this study and had 487 patients entered into the study: of these, 328 patients were in the PAGC group, 84 in the G-CSF group, and 75 in the no-treatment group.
  • a patient's WBC count was less than 4.0 x 10 9 /L anytime during the fourteen days after receiving chemotherapy, the patient was assigned to one of the three groups.
  • 250 mg PAGC dissolved in 500 mL of normal saline was administered intravenously once daily for seven days.
  • the Karnofsky Performance Index is a generic health status measurement according to WHO.
  • FIG. 1 shows the mean white blood cell counts versus days after chemotherapy;
  • FIG. 2 shows the total symptom scores versus days after chemotherapy;

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Abstract

Cette invention se rapporte à des compositions d'arabinogalactane purifiées, isolées à partir de l'Astragalus membranaceus, en particulier à partir des racines de l'Astragalus membranaceus, et à des compositions de protéines d'arabinogalactane ayant un poids moléculaire moyen en poids d'au moins 100 kilodaltons isolées à partir de ces compositions d'arabinogalactane purifiées, ces compositions étant capables de se reconstituer en formulations aqueuses injectables par voie intraveineuse. Chez un mammifère, lorsqu'elles lui sont administrées par voie intraveineuse, ces compositions servent à stimuler l'hématopoïèse, à induire la prolifération ou la maturation des mégacariocytes, à stimuler la production de IL-1β, IL-6, TNF-α, IFN-η, GM-CSF ou G-CSF, à stimuler la production ou l'action des neutrophiles, à traiter la neutropénie, l'anémie ou la thrombocytopénie, à accélérer la guérison après une exposition (aussi bien une exposition accidentelle ou non thérapeutique qu'une exposition thérapeutique) à des agents cytotoxiques ou à un rayonnement cytotoxique, à traiter la cachexie, les vomissements ou les symptômes de sevrage de drogue, ou à modifier les réponses biologiques ou à protéger les cellules hépatiques dans l'hépatite B.
PCT/US2000/018180 1999-06-30 2000-06-30 Composition d'arabinogalactane hematopoietique Ceased WO2001000682A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002607A3 (fr) * 2000-06-29 2003-01-23 Pharmagenesis Inc Composition de proteine arabinogalactane modifiee par un acide
RU2208440C2 (ru) * 2001-07-20 2003-07-20 Иркутский институт химии им. А.Е. Фаворского СО РАН Средство, обладающее противоанемической и иммуномодуляторной активностью
WO2006025068A1 (fr) 2004-09-01 2006-03-09 Lupin Limited Préparation à base d'arabinogalactane-protéine (agp) de pureté élevée
US8137710B2 (en) 2008-12-15 2012-03-20 EcoPharm Corporation Treating idiopathic thrombocytopenic purpura with comprising extracts of Astragalus membranaceus
CN104147254A (zh) * 2014-08-21 2014-11-19 陈勇 一种治疗化疗后白细胞减少症的中药制剂
EP3191110A4 (fr) * 2014-08-18 2018-04-18 Pharmagenesis, Inc. Composition de polygalacturonan rhamnogalacturonane (pgrg1)

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CN1854158B (zh) * 2005-04-25 2011-09-07 蒋来高 运用超临界流体萃取技术提取黄芪多糖的工艺
CN109432242A (zh) * 2018-11-07 2019-03-08 郑毅男 一种落叶松阿拉伯半乳聚糖制备方法及其在医疗方面的应用
CN110551230B (zh) * 2019-09-21 2022-02-15 天津赛诺制药有限公司 一种黄芪多糖的制备方法

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Cited By (11)

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WO2002002607A3 (fr) * 2000-06-29 2003-01-23 Pharmagenesis Inc Composition de proteine arabinogalactane modifiee par un acide
JP2004502703A (ja) * 2000-06-29 2004-01-29 ファーマジェネシス，　インコーポレイテッド 酸修飾アラビノガラクタンタンパク質組成物
US6991817B2 (en) 2000-06-29 2006-01-31 Pharmagenesis, Inc. Acid-modified arabinogalactan protein composition
RU2208440C2 (ru) * 2001-07-20 2003-07-20 Иркутский институт химии им. А.Е. Фаворского СО РАН Средство, обладающее противоанемической и иммуномодуляторной активностью
WO2006025068A1 (fr) 2004-09-01 2006-03-09 Lupin Limited Préparation à base d'arabinogalactane-protéine (agp) de pureté élevée
US7601368B2 (en) 2004-09-01 2009-10-13 Lupin Limited Purified Arabinogalactan-Protein (AGP) composition useful in the treatment psoriasis and other disorders
US8137710B2 (en) 2008-12-15 2012-03-20 EcoPharm Corporation Treating idiopathic thrombocytopenic purpura with comprising extracts of Astragalus membranaceus
EP2376096A4 (fr) * 2008-12-15 2013-10-02 Ecopharm Llc Traitement de purpura thrombocytopénique idiopathique par des compositions comprenant des extraits d'astragalus membranaceus
US8728543B2 (en) 2008-12-15 2014-05-20 EcoPharm Corporation Methods of treating idiopathic thrombocytopenic purpura with compositions comprising extracts of Astragalus membranaceus
EP3191110A4 (fr) * 2014-08-18 2018-04-18 Pharmagenesis, Inc. Composition de polygalacturonan rhamnogalacturonane (pgrg1)
CN104147254A (zh) * 2014-08-21 2014-11-19 陈勇 一种治疗化疗后白细胞减少症的中药制剂

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EP1200482A1 (fr) 2002-05-02
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