HK1046290A1 - Hematopoietic arabinogalactan composition - Google Patents

Abstract

Purified arabinogalactan compositions isolated from <i>Astragalus membranaceus</i>, especially from the roots of <i>Astragalus membranaceus</i> , and arabinogalactan protein compositions having a weight average molecular weight of at least 100 kiloDaltons isolated from these purified arabinogalactan compositions, are capable of reconstitution into aqueous intravenously injectable formulations; and are useful for stimulating hematopoiesis; inducing the proliferation or maturation of megakaryocytes, stimulating the production of IL-1 beta , IL-6, TNF- alpha , IFN- gamma , GM-CSF, or G-CSF, stimulating the production or action of neutrophils, treating neutropenia, anemia, or thrombocytopenia, accelerating recovery from exposure (e.g. accidental or non-therapeutic exposure, as well as therapeutic exposure) to cytotoxic agents or radiation, treating cachexia, emesis, or drug withdrawal symptoms, or modifying biological responses or protecting hepatic cells in hepatitis B, in a mammal when intravenously administered to the mammal.

Description

Hematopoietic arabinogalactan compositions

The technical field to which the invention belongs

The invention relates to arabinogalactan. More particularly, the present invention relates to a purified arabinogalactan isolated from Astragalus membranaceus (Astragalus membranaceus), particularly roots thereof, and arabinogalactan protein compositions having a weight average molecular weight of at least 100 Kilodaltons (KD) isolated from these purified arabinogalactan compositions, or intermediates thereof.

Background of the invention

Radix Astragali (Radix Astragali) is sun-dried root of Astragalus mongholicus (Astragalus mongholicus) or Astragalus membranaceus (Astragalus membranaceus) Bge. Astragalus membranaceus is a well-known ancient Chinese medicine. The Chinese pharmacopoeia is formally recorded and is mainly used as a tonic for treating nephritis and diabetes. It is usually used as a decoction or as "tea" alone, or in combination with other plants in the traditional herbal medicines of Shi-ka-ron (composed of two herbs of Lithospermum erythrorhizon and Ligusticum wallichii) and Ginseng-shang-yang-hang-tan (composed of twelve herbs including Astragalus membranaceus Radix). [ see section 26 of "Chinese medicine (Chinese drug Plant Origin)" edited by W.Tang and G.Eisenbrand, page 191-197, title "Astragalum branderaceus (Fisch) Bge.", Springer Verlag, Berlin, 1992 ].

Astragalus decoction and a solution prepared from an alcohol precipitate of the decoction have been administered by injection to improve the symptoms of gastric and duodenal ulcers and to increase the white blood cell count in patients with chronic leukopenia (see "Pharmacology and Applications of Chinese medicine" edited by H. -M. Chang and P.P. -H.But et al, pages 1041 and 1046, entitled "Astragalus" (Huang. qi), World scientific publishing Co., Singapore, 1987). It is known that decoction of Astragalus membranaceus, purified low molecular weight fractions (e.g., fractions having a molecular weight of 25,000. about.35,000), and decoction of a mixture of Astragalus membranaceus-containing herbs can also restore the immune system of local xenografts and host-resistant reactions (D. T.Chu et al, Chinese herbal Immunotherapy with Chinese medicinal herbs. I. ", J.Clin. Lab. Immunol., 25, 119. 123(1988)), and reverse the immunosuppressive reactions induced by cyclophosphamide D. T.Chu et al, Chinese herbal Immunotherapy with Chinese medicinal herbs. II.", Clin. Lab. Immunol., "Immunothricin. 129. (Immunol., 25, 125. 129(1988)), promote the cytotoxic reactions induced by IL-2 in" LAK cells (D. about.183. J., Immunol. 183. J. Immunol. 187. J. immunogical., S. J. 183. Immunol. J. 183. et al., J. Immunol. 187. J. Immunol., S. J. 183. et al., J. Immunol., S. J. 187. J. for stimulating immune system, S. immune system of mice, J. immune system, J. 2. immune system, and mouse immune system of mouse, 2. immune system, astragalus extract enhances immune response in mice ("Enhancement of The immune response in mice by Astragalus membranaceus extracts"), "Immunopharmacology" (Immunopharmacology), 20, 225-plus 234(1990) ], stimulation of monocyte response [ Y.Sun et al, "preliminaryon preservation on The effects of The Chinese medicinal products renerger.", "J.Bioresponse Modulator" (J.biol.Response modulators), 2, 227-plus 237(1983) ], and stimulation of hematopoietic capacity of mice [ M.Rou et al., "hematopoietic effect of Astragalus membranaceus on bone marrow" (The effect of Astragalus membranaceus on bone marrow), (The effect of radiation on tissue antigens), (The traditional medicine of medicine, J.199. (1983.204)), (1983); miura et al, "Chinese herbal medicine efficacy" (Effect of a traditional Chinese herbal medicine "), int.J.Immunopharmacol," J.Immunopharmacol, "J.Immunol. Pharmacol., 7(11), 771-; and Y, Ohnishi et al, "Effects of Juzen-taiho-toh (TJ-48.", exp. Hematol., "Experimental hematology," 18, 18-22 (1990)).

U.S. patent 4,843,067 to Liu discloses a pharmaceutical composition comprising Astragalus polysaccharides (obtainable by extraction from Astragalus membranaceus Bge. or Astragalus gummifer Labill) and angelicase polysaccharides. The Astragalus polysaccharides can be extracted from radix astragali root powder or ethanol precipitation with water. Verbiscar, U.S. Pat. No. 5,2868,467, discloses an immunomodulatory polysaccharide from the plant tragacanth (tragalus tragacantha) prepared at low temperature to "maintain the integrity of the polysaccharide structure without chemical or conformational changes". U.S. patent No. 5,336,506 to Josephson et al discloses that complexes formed with plant polysaccharides such as arabinogalactans (isolated from larixoccmentalis) and mannans and certain agents target cellular receptors that cause receptor-mediated endocytosis. U.S. patent 5,116,969 to Adams et al discloses a particularly refined arabinogalactan product suitable for density gradient separation. U.S. patent 5,478,576 to Jung et al discloses a purified arabinogalactan (also from Larix occidentalis), lysate, modifications thereof, and the like, which can also be used to deliver an agent to a targeted cellular receptor that causes pinocytosis. U.S. patent 5,589,591 to Lewis et al discloses an endotoxin-free polysaccharide such as arabinogalactan, dextran, mannan, and acacia; an impure polysaccharide is prepared by ultrafiltration by first passing through a membrane which removes the low molecular weight fraction, leaving a retentate, and then through another membrane which removes the higher molecular weight fraction, also leaving a filtrate fraction.

However, these references, all focus on the polysaccharide portion of the product (e.g., arabinogalactans) and may even use techniques that exclude arabinogalactan proteins.

Arabinogalactan proteins, are also present in flowering plants, and are widely present in higher plants. Arabinogalactan proteins (AGPs), sometimes also called arabinogalactan peptides, are glycoproteins with high proportions of carbohydrates and small amounts of protein (usually less than 10%), although high protein-containing arabinogalactan proteins are also known. Among hydroxyproline-rich glycoproteins isolated from plants, arabinogalactan proteins are characterized by low protein content and the ability to bind to the β -glucosylsylidene (Yariv) reagent, 1, 3, 5-tris- (4- β -glucosylfuryloxyphenylazo) -2, 4, 6-trihydroxybenzene [ j.h.yariv et al, biochem.j., 85, 383-; r.l. anderson et al, aust.j. plant physiol., 4, 143-. Arabinogalactan protein is part of gum arabic (a gum exudate from Acacia senegal), and is commonly used as an emulsifier, crystallization inhibitor, and flavor in food products. Methods for isolating the AGP gene from plants (Nicotiana alata), Nicotiana alba (Nicotiana pallibagnafia), and Pyrus communis) are disclosed in U.S. Pat. No. 5,646,029 to Chen et al. For a detailed discussion of arabinogalactan proteins, reference is made to E.A. Nothnagel, "Glucan proteins and related compounds in plant cells" (proteins and related compounds in cells), int.Rev. cytology, 174, 195- "291, 1997).

The documents disclosed herein and referred to in this specification are incorporated herein by reference.

Brief description of the invention

In a first aspect of the invention there is provided a purified arabinogalactan composition isolated from astragalus membranaceus (astraglusmbranaceae), particularly the roots thereof.

In a second aspect of the invention there is provided an arabinogalactan protein composition having an average molecular weight of at least 100 kilodaltons, isolated from the purified arabinogalactan composition of the first aspect of the invention. A purified arabinogalactan protein composition according to the invention, comprising 5% protein, 20% of the amino acid content of said protein based on the total amount of amino acids being hydroxyproline, and having a sugar composition wherein the ratio of arabinose to galactose is at least 2: 1 and comprising a molar percentage of arabinose from 45% to 75%; rhamnose in a molar percentage of 2 to 4%; galactonic acid in a molar percentage of 4% to 6%; 8 to 25 mole percent galactose; and 5 to 25 mole percent of glucose

In a third aspect of the present invention, there is provided an aqueous arabinogalactan formulation for intravenous injection comprising a pharmaceutically effective amount of the purified arabinogalactan composition of the first aspect of the present invention or the arabinogalactan protein composition of the second aspect of the present invention, and an adjuvant solution for intravenous injection.

In a fourth aspect, the invention provides a method of treating a disease in a mammal (e.g., stimulating hematopoiesis, inducing megakaryocyte proliferation and maturation, stimulating IL-1 β, IL-6, TNF- α, IFN- γ, GM-CSF, or G-CSF production, stimulating neutrophil production or activation, treating neutropenia, anemia, or thrombocytopenia, accelerating recovery from exposure (e.g., from accidental or non-therapeutic exposure, and therapeutic exposure) to cytotoxic substances, or radiation exposure, treating cachexia, emesis, or withdrawal syndromes, or improving biological response or protecting liver cells in a hepatitis B patient) using the purified arabinogalactan composition of the first aspect of the invention, or the arabinogalactan protein composition of the second aspect of the invention, comprising injecting into the mammal to be treated a pharmaceutically effective amount of the purified arabinogalactan composition of the first aspect of the invention, or the arabinogalactan protein composition of the second aspect of the invention, particularly an intravenously injectable arabinogalactan formulation of the third aspect of the invention; or in combination with at least one other agent (e.g., an agent that stimulates hematopoiesis).

In a fifth aspect, the present invention provides a method for preparing the purified arabinogalactan composition of the first aspect of the present invention, the arabinogalactan protein composition of the second aspect of the present invention, and an intravenously injectable aqueous arabinogalactan formulation of the third aspect of the present invention.

Brief description of the drawings

Figure 1 shows the white blood cell mean values as a function of days after treatment of a human cancer patient, without further subjects, and treated with a purified arabinogalactan composition of the invention, or with G-CSF, after receiving chemotherapy.

Figure 2 shows the total symptom value after treatment as a function of days without further subjects after receiving chemotherapy, and after treatment with the purified arabinogalactan composition of the invention, or with G-CSF, in human cancer patients.

FIG. 3 shows Carnofski Performance Index (Karnofsky performance Index) values after human cancer patients received chemotherapy without further subjects, and after treatment with a purified arabinogalactan composition of the invention, or with G-CSF.

Detailed Description

Definition of

"arabinogalactan proteins" or "AGP" can usually be precipitated with beta-glucosylsilylaleff (Yariv) reagents and are highly glycosylated proteins, with carbohydrates representing approximately at least 50% of the molecular weight and the major carbohydrate components being arabinose and galactose, with the arabinose residues being predominantly terminal. For arabinogalactan proteins, it typically reacts specifically with the monoclonal antibody MAC 207.

By "arabinogalactan protein composition" is meant a composition comprising at least 70%, particularly at least 80%, especially at least 90%, by weight of the composition, of arabinogalactan proteins, and related arabinogalactans, and other polysaccharides.

A "purified arabinogalactan composition" is a composition comprising an arabinogalactan protein (as described above) and related arabinogalactans and other polysaccharides.

"mammal" includes human and non-human mammals, such as pets (cats, dogs, etc.) and livestock (cows, horses, sheep, goats, pigs, etc.).

"disease" includes any unhealthy condition of an animal, including unhealthy conditions resulting from medical treatment (e.g., side effects), such as a disease state for which the blood-enriching effect is therapeutically effective, particularly including those disease states described in the "pharmacology and use" section of this invention.

"pharmaceutically acceptable adjuvant" means an adjuvant that is effective in preparing pharmaceutical compositions, is generally safe, non-toxic, and essential, and includes both veterinary and human-useful adjuvants. Such adjuvants may be in the form of solid, liquid, semi-solid, or aerosol compositions, and in the form of gas.

By "pharmaceutically effective amount" is meant an amount of drug which, when administered to the body of an animal to treat a disease, is sufficient to effectively treat the disease.

"treating" or "treatment" of a disease includes preventing the onset of the disease in an infected, but not yet at risk animal (prophylactic treatment), inhibiting the disease (slowing or inhibiting the progression of the disease), providing relief from the symptoms or side effects of the disease (including relieving therapy), and relieving the disease (causing remission).

Purified arabinogalactan compositions

The purified arabinogalactan composition of the present invention is as defined above, isolated from Astragalus membranaceus (especially roots), preferably from the roots of Astragalus mongholicus or Astragalus membranaceus (Astragalus membranaceus Bge. var. mongholicus (Bge) Hsiao or a. membranaceus (Fisch) Bge.); preferably the roots of the astragalus plants growing in inner mongolia or shanxi province of china, especially the former; and preferably the roots are selected from Astragalus membranaceus grown for two years. Having a typical sugar composition (as determined by GLC analysis of trimethylsilyl derivatives of methanol extracts of said composition) containing about 5-15 mole% (mole percent), especially 10%, arabinose (Ara); less than 1.5%, especially less than 1% rhamnose; GalA (galactonic acid) in an amount of up to about 4%; about 3% to 7% Gal (galactose); and about 70% to 90% glucose (Glc); wherein the ratio of arabinose to galactose is not less than 1.5: 1; in particular not less than 1.75: 1; especially not less than 3: 1; ash content not higher than 2% (weight percentage); the content of heavy metal is not higher than 10 ppm; the hydroxyproline content is not higher than 0.1%, especially not higher than 0.05%. Hardly any endotoxin (endotoxin test according to the manufacturer's manual [ Seikagaku corporation, Tokyo, Japan ]), at a content of less than 1.0 EU/mg, particularly less than 0.8 EU/mg, particularly less than 0.5EU/mg, preferably less than 0.3 EU/mg); a solubility in water of at least 20 mg/ml and a pH of the aqueous solution of between about 4.5 and 6.5; the weight-average molecular weight is between 20 and 60 kilodaltons, in particular between 25 and 40 kilodaltons, especially between 27 and 35 kilodaltons. For convenience, the present material is hereinafter referred to as "PAGC".

Preparation of purified arabinogalactan composition

The purified arabinogalactan composition is prepared by extracting dried root of Astragalus membranaceus (Astragalus membranaceus) at a temperature of generally not less than 80 deg.C, particularly not less than 90 deg.C, particularly about 100 deg.C with hot water (generally not less than 80 deg.C, particularly not less than 90 deg.C, particularly about 100 deg.C) in the presence or absence of co-extract (co-extract) such as alkali metal salt (particularly potassium dihydrogen phosphate or sodium dihydrogen phosphate), slicing and cutting the dried root under sterile conditions, trimming the dried root, washing with ultra-filtered water, washing with a sterilizing solution such as 70% ethanol, slicing the root, and drying under sterile conditions, hereinafter referred to as "macerated slice"), for a period of time, at a temperature, and optionally repeating many extraction steps to allow the arabinogalactan protein and related polysaccharides in the root to be dissolved out as much as possible (generally at about 100 deg.C, three times, 3 hours each time). All steps performed after the preparation of the root fragments are performed in a sterile environment using sterile equipment and reagents. The hot water extract is concentrated (to about 1 liter/kg dry root under vacuum at 60-70 deg.C) and then precipitated with lower alcohols (e.g., ethanol, 70% final concentration at room temperature). The precipitate of these lower alcohols is washed again with lower alcohols (typically three times with 95% alcohol) and suspended in water at an appropriate concentration for further processing (typically 18-20% w/v). The water-insoluble material of the suspension is then removed, for example by adding a lower aliphatic alcohol (about 35% ethanol) to the precipitate. The supernatant of the low fatty alcohol precipitate (e.g., 35% alcohol precipitate) is then subjected to a further precipitation with a high concentration of a lower fatty alcohol, e.g., 40% to 80% alcohol, particularly 60% to 70% alcohol, to precipitate the crude arabinogalactan composition containing arabinogalactan proteins and bound polysaccharides. The precipitate is redissolved in water and dried (spray dried, avoiding excessive heating) to isolate the crude arabinogalactan composition. The crude composition is generally a pale yellow powder, soluble in water, having a concentration of at least 100 mg/ml, in particular at least 200 mg/ml, a dry weight loss on drying of less than about 15%, and an endotoxin content of less than 0.5, in particular less than 0.3 EU/mg.

To accommodate batch-to-batch variation in the astragalus membranaceus crude material, the raw material, "macerated slices," and intermediate products can be mixed during the process to maintain consistency in the final product.

The crude arabinogalactan composition is further purified by ion exchange chromatography. It is dissolved in water or the solution after dissolution of the precipitate is adjusted to a suitable concentration (typically about 2%), then ultrafiltered to remove the low molecular weight fraction and the volume of the solution is reduced (for example, filtration is carried out using an ultrafiltration system (5K MWCOUF system) which excludes molecules with a molecular weight of 5 kilodaltons). The ultrafiltered supernatant was then passed through a cation exchange column (e.g., SP Sepharose cation exchange column, equilibrated with 20mM sodium acetyl (NaOAc) buffer, pH 5.20) and the eluate was loaded into an anion exchange column (e.g., Q Sepharose anion exchange column, equilibrated with the same NaOAc buffer). The eluate from the anion exchange column may be taken directly to prepare the arabinogalactan protein composition of the second aspect of the invention and may be concentrated and dried to form an intermediate suitable for use in the preparation of the arabinogalactan protein composition or may be used directly to prepare a purified arabinogalactan composition. In preparing the purified arabinogalactan composition, ultrafiltration may be carried out using a suitable microfiltration membrane (e.g., a 0.1 μm membrane) capable of filtering bacteria, removing salts and reducing the volume of the solution (e.g., using an 8K MWCO UF system). The supernatant after ultrafiltration is concentrated (20-26% at 50-60 deg.C) and then precipitated with lower aliphatic alcohol (about 80-90% ethanol). The precipitate is further washed (e.g., three times with absolute alcohol) and dried (vacuum oven at 60-70 deg.C) to obtain a purified arabinogalactan composition.

Arabinogalactan protein composition

The purified arabinogalactan protein composition of the invention is as defined above, isolated from Astragalus membranaceus (particularly roots thereof), and preferably from the roots of Astragalus membranaceus (Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch) Bge.); and preferably roots of astragalus (a. membranaceus) plants grown in inner mongolia or shanxi province of china, in particular the former; and preferably the roots are selected from Astragalus membranaceus grown for two years. It has a typical sugar composition comprising about 45 to 75% (mole percent), especially about 50 to 70% arabinose; 2% to 4% rhamnose; about 4% to 6% galactaric acid; between about 8% and 25%, particularly between 10% and 20% galactose; and between about 5% and 25% glucose; wherein the ratio of arabinose to galactose is not less than 2: 1; especially not less than 3: 1; especially not less than 4: 1; ash content not higher than 2% (weight percentage); the content of heavy metal is not higher than 10 ppm; and the hydroxyproline content is not higher than 0.2%, especially not higher than 0.3%. Hardly any endotoxin (as defined above); a solubility in water of at least 20 mg/ml and a pH of the aqueous solution of between about 4.5 and 6.5; the average molecular weight is not less than 100 kilodaltons, in particular between 150 and 350 kilodaltons. Contains about 95% carbohydrate (including carbohydrate which can saccharify the arabinogalactan protein core), and about 5% protein. About 20% of the total amino acids are hydroxyproline, which is characteristic of arabinogalactan proteins. For convenience, the material is hereinafter referred to as "AGPC".

Preparation of arabinogalactan protein composition

The arabinogalactan protein composition of the present invention is prepared by further purifying the arabinogalactan composition or a distillate obtained in the step of purifying the arabinogalactan composition by an ion exchange column (or a solid intermediate obtained by concentrating and drying the distillate) by a 100K MWCO ultrafiltration system. The distillate from the anion exchange column in the "preparation of arabinogalactan protein composition" described above was fed directly to a 100K MWCO ultrafiltration system. The remaining filtrate from this 100K MWCO ultrafiltration system is further concentrated and precipitated with a lower aliphatic alcohol, or further washed and dried, all similar to the preparation of a purified arabinogalactan composition, to provide an arabinogalactan protein composition.

Pharmacological and use

The advantageous activity of the purified arabinogalactan composition of the first aspect of the invention or the arabinogalactan protein composition of the second aspect of the invention has been demonstrated in several tests and has the following uses.

Treating neutropenia of chemotherapy patients

1. Cytokine production from activated human Peripheral Blood Mononuclear Cells (PBMC)

The immune and hematopoietic systems need to be activated by the interaction of several cytokines, and PAGC induces cytokine production in vitro as described in example 3. PAGC priming was evident as a dose-related release of IL-1 β, IL-6, TNF- α, IFN- γ, GM-CSF and G-CSF from PHA-activated human peripheral blood mononuclear cells. Three cytokines, IL-6, GM-CSF and G-CSF, are known to affect the production and/or activation of neutrophils in vitro and in vivo. IL-6, by itself or in combination with other cytokines, stimulates megakaryocyte maturation in the bone marrow and restores the values of platelets in peripheral blood of mice and non-human primates. These data indicate that PAGC can stimulate the production of neutrophils and restore platelet counts in myelosuppressed patients by producing cytokines associated with hematopoietic function.

2. Recovery of GM-CFC stem cells in fluorouracil-treated mice

PAGC, as described in example 5, has been tested in a short-term, mouse, ex vivo in vivo model of hematopoietic stem cell recovery. Fluorouracil is widely used to remove stem cells from mouse bone marrow and colony forming units (CFU-C) that can be produced in cell culture. Mice treated with this treatment recover along a known, predictable time curve because unactivated stem cells are not affected by fluorouracil [ a.m. yeager et al, ] the effect of 5-fluorouracil on hematopoiesis: rat megakaryocyte-CFC, granulocyte-macrophage-CFC, peripheral blood cell level research (The effects of 5-fluorogenic on-hematology: students of muscle megakaryocyte-CFC, granulocyte-macrocage-CFC, and periphytol blood cells), Experimental hematology (exp. hematol.), 11, 944-952 (1983). From the table in example 5, it is seen that the amount of GM-CFC in each femur increased with increasing PAGC dose on day 4 after fluorouracil treatment in the dose-dependent method. When PAGC dose was 200 mg/kg, the average increased GM-CFC per mouse was 3.3-fold higher than the control group (p < 0.01); if 100mg/kg PAGC is used for treatment, the average increased GM-CFC of each mouse is 1.8 times that of the control group (p is less than 0.01); if 50mg/kg PAGC is used for treatment, the average increase of GM-CFC per mouse is not much different from the control group. These data indicate that PAGC can promote the reconstitution of myeloid stem cells (GM-CFC) in fluorouracil-induced myelosuppressed mice, which may be explained, at least in one aspect, by an increase in peripheral leukocytes in mice where PAGC promotes myelosuppression.

3. Recovery of peripheral leukocytes in mice receiving sub-lethal doses of radiation

BALB/c mice were irradiated with sub-lethal doses of radiation, and then injected subcutaneously with saline or various doses of PAGC according to the method of example 6. The white blood cell values of the irradiated mice decreased to 12% of the normal mouse values at 14 days after irradiation. Treatment with PAGC increased the total number of white blood cells in mice compared to mice injected with saline alone. The total number of leukocytes in mice treated with 100 or 300mg/kg of PAGC was restored to 80% of the number of normal mice (normal value: 6000-10000 leukocytes/microliter blood) 7-9 days earlier than in mice injected with physiological saline alone. In PAGC treated animals, white blood cell counts returned to normal after about 22-23 days; whereas the value of the normal saline-only mice was 46% of the normal value. In addition, peripheral blood smear staining can also be used to determine different white blood cell values. From this information, the absolute values of neutrophils and lymphocytes can be calculated. In this model, treatment with PAGC increased the absolute number of neutrophils and also the absolute number of lymphocytes, compared to the control group of mice injected with saline only.

4. Recovery of leukocyte counts in chemotherapy patients

All phase II clinical experiments are completed in the people's republic of China. In the experiment, the white blood cell count of a small group participating in the experiment is analyzed to be lower than 3.0 multiplied by 10⁹Data for white blood cells/liter patient: of these 168 patients received PAGC treatment, 59 patients received G-CSF treatment, and 23 patients received no treatment. The treatment protocol is described in detail in example 9. As shown in fig. 1, PAGC treatment steadily increased white blood cell count in patients receiving chemotherapy, and this increased condition continued until day 14. Although administration of G-CSF increases the rate of increase of leukocytes to a maximum by day 7, it does not last and the number of leukocytes in the patient begins to decrease after day 7. Both treatment groups resulted in a higher white blood cell count than the untreated groupAnd the normal state is recovered early. The white blood cell count of the PAGC treated group may be significantly different between day 10 and day 14 compared to the control group not receiving any treatment; however, there was no significant difference in the white blood cell count between the PAGC group and the G-CFC group at day 14. The above results indicate that the use of PAGC as a therapeutic adjuvant in lung, gastric, and breast cancer patients receiving chemotherapy is not only safe but also acceptable to the patient. PAGC treatment restores white blood cell counts in chemotherapy patients. And no obvious side effect is found when the PAGC is clinically used for treatment.

Treating thrombocytopenia in chemotherapy patients

1. Recovery of peripheral platelet cell count in mice receiving sublethal doses of radiation

BALB/c mice were irradiated with sub-lethal doses of radiation on day 0, and then treated with saline or various doses of PAGC according to the procedure of example 6. The number of white blood cells in the irradiated mice decreased to 7% of that in the normal mice at day 10 after irradiation. When 100 or 300mg/kg of PAGC is injected subcutaneously to treat mice, the peripheral platelet cell number in the bodies of the mice can be obviously improved. PAGC injection group induced platelet cell number recovery to 80% of normal mice 5-6 days earlier than normal saline injection control group (normal value: 8-12X 10)⁵Platelets/microliter blood). The PAGC treated mice can recover the platelet count about 24 to 25 days after the radiation irradiation; in contrast, the value of the normal saline-only mice was 72%. These results indicate that PAGC is an effective agent for promoting platelet growth and is quite effective in treating a decrease in platelet count. In the same manner, mice treated with 100 and 250mg/kg of AGPC recovered both red blood cell and platelet counts better than the control group at all time points after irradiation in a 20 day treatment period; it was shown that PAGC also had the same therapeutic effect in this model system.

2. Proliferation and maturation of bone marrow megakaryocytes

As described abovePAGC accelerates the recovery of platelet counts in peripheral blood in myelosuppressed animals induced in animal models of radiation exposure, suggesting that PAGC may stimulate proliferation and/or maturation of bone marrow megakaryocytes. This possibility can be investigated using the in vitro liquid culture system described in example 4. From the dose titration curves, the optimal PAGC dose in this model was 100-200. mu.g/ml, while ED₅₀Is 30-40 microgram/ml. This study suggests that PAGC alone may promote proliferation and/or maturation of bone marrow megakaryocytes in vitro. In addition, PAGC may also be administered in combination with a less than optimal dose of IL-3(50pg/ml) to increase acetylcholinesterase (AchE) levels in a dose-related manner. The AchE content obtained in cell cultures treated with 200. mu.g/ml PAGC and 50pg/ml IL-3 was comparable to that obtained in cultures treated with the optimal dose of IL-3. These data indicate that PAGC may increase the responsiveness of bone marrow megakaryocytes to relatively low doses of IL-3, or that IL-3 may increase the responsiveness of bone marrow megakaryocytes to PAGC. Because chemotherapy or radiation exposure can destroy many cells and tissues, including cytokine-producing cells, endogenous cytokine levels in patients receiving such treatment can be low. Such low levels of cytokines will not support the normal function of the hematopoietic system and thus will not produce a sufficient number of hematopoietic cells to restore the suppressed bone marrow function. Therefore, PAGC may be a good drug for the treatment of this disease. The results of this study indicate that PAGC promotes the proliferation and/or maturation of bone marrow megakaryocytes to normal levels even in the presence of endogenous cytokine deficiency. As shown above, PAGC stimulates the recovery of peripheral platelet numbers, and this effect appears to result from the induction of proliferation and/or maturation of bone marrow megakaryocytes. This data indicates that PAGC is effective in treating thrombocytopenia due to myelosuppression.

3. Increasing platelet cell number after chemotherapy

Phase II clinical trials are described in example 9. In the experiment, the white blood cell count of a small group participating in the experiment is analyzed to be lower than 4.0 multiplied by 10⁹White blood cell count/liter and platelet count below 90X 10⁹Per litre ofData of the patient: of these 54 patients received PAGC treatment. The platelet count of these patients increased to over 100X 10 on day 7⁹Liter, and continued to increase to day 14 as shown in the table of example 9. These data indicate that PAGC can increase platelet counts in these chemotherapy patients.

Improving the quality of life of cancer patients

The phase II clinical trial is described in example 9. As described in example 9, values for improving symptoms of chemotherapy and the carnowski performance Index (Karnofsky performance Index) were used as a basis for assessing the quality of life of all patients participating in the trial. Symptoms caused by chemotherapy during treatment, including fatigue and listlessness, discomfort, sweating, shortness of breath, and loss of appetite were recorded and calculated. As shown in fig. 2, the PAGC group showed the fastest efficacy as treatment progressed, as seen by a decline in symptom index and a faster return to normal (count ═ 0) than the G-CSF group. In addition, the PAGC group also showed statistically more significant improvement (p < 0.01) over the period from day 10 to day 14 than the G-CSF group. However, the G-CSF group did not show any significant statistical difference over the entire 14-day monitoring period compared to the control group (not receiving any treatment). These data indicate that PAGC improves the symptoms in chemotherapy patients, and in this regard, G-CSF does not appear to have any utility. A small group of patients with a pre-treatment carnofsky performance Index (Karnofsky performance Index) number below 70 was analyzed for improvement in symptom indices after various treatments. The PAGC group significantly improved symptoms compared to the G-CSF group and the control group that did not receive any treatment, as shown in FIG. 3, with statistical differences of p < 0.01 and p < 0.0001, respectively. The patients in most of the PAGC groups had higher indices than the other two groups.

Prevention of neutropenia in cancer patients receiving chemotherapy

In the preceding paragraph, PAGC is known to be effective in treating chemotherapy-induced neutropenia. These data suggest that PAGC may be as effective as neutropenia prevention and clinical trials in the people's republic of china have been planned.

Treatment of neutropenia in cancer patients receiving radiation

The previous pharmacological data also indicate that PAGC is effective in restoring radiation-induced neutropenia. Clinical trials have been initiated in the Chinese and domestic setting to understand the condition of PAGC in treating neutropenia in non-Hamgold lymphoma cancer patients after exposure to radiation.

Treatment of anemia in cancer patients undergoing chemotherapy

BALB/c mice were irradiated with sub-lethal dose (4.25Gy) of radiation and then treated with saline or various doses of PAGC according to the procedure of example 6. RBC values for irradiated mice dropped dramatically to 55% of normal mouse RBC values at day 17 post irradiation. PAGC treated mice received a considerably higher RBC count on day 17, while saline-only mice were still low. Mice treated with 100 or 300mg/kg of PAGC by subcutaneous injection increased RBC numbers to 80% of normal mice (normal value: 8.5-11X 10) 4-6 days earlier than the control group treated with physiological saline alone⁶RBC/microliter blood). The RBC count of the PAGC-treated mice can be recovered to be normal after about 20-22 days; whereas the value of the normal saline-only mice was 65% of the normal value. These results indicate that PAGC is a relatively effective agent for promoting the development and/or migration of red blood cell stem cells and is therefore relatively effective in treating anemia arising from radiation or chemotherapy. In the same manner, mice treated with 100 and 250mg/kg of AGPC recovered both red blood cell and platelet counts better than the control group when tested at all time points after irradiation during a 20 day treatment period; it was shown that PAGC also had the same therapeutic effect in this model system.

The phase II clinical trial in example 9 was conducted for the purpose of studying the recovery of leukocytes of chemotherapy patients, and therefore, it was conditioned to conduct an analysis experiment based on the number of leukocytes. The clinical trials for treatment of anemia and chemotherapy-induced anemia with PAGC are currently being studied in PRC (the people's republic of china).

Moving peripheral blood stem cells alone or in combination with a patient receiving peripheral blood stem cell transplantation G-CSF binding therapy

The CD34 antigen is present in hematopoietic stem cells and stem cells, and includes BFU-E (erythrocyte burst forming unit), GM-CFC (granulocyte-macrophage colony forming unit), and CFU-Mix (mixed colony forming unit) which are capable of forming cell colonies. CD34⁺Flow cytometry analysis of cells provides a best way to determine the graft composition. The presence of hematopoietic stem cells (PBPCs) in the human peripheral blood system has been proposed as early as 1975. These peripheral blood stem cells account for only a small fraction of the total cell number, with the majority of the stem cells being located in the bone marrow. In 1976, it was first proposed that the number of hematopoietic stem cells in the human peripheral blood system would increase during the recovery period following chemotherapy. Recently, it has been reported that G-CSF and GM-CSF, which are cell colony stimulating factors (colony stimulating factors), directly increase the number of hematopoietic stem cells. The combination of a cell population stimulating factor with chemotherapy is more effective in increasing the number of hematopoietic stem cells than treatment with only one method. Currently, G-CSF is used to mobilize hematopoietic stem cells in human autologous and allogeneic transplantations. Transplantation of hematopoietic stem cells proceeds more rapidly than traditional bone marrow transplantation. The number of hematopoietic stem cells produced by G-CSF treatment can be increased even more if used in combination with chemotherapy or other cytokines. The ability of PAGC alone and/or in combination with G-CSF to induce migration of hematopoietic stem cells is illustrated in example 7. As shown by the results in the table in example 7, PAGC can increase the amount of GM-CFC circulated by about 6-fold. Furthermore, PAGC in combination with G-CSF can increase the amount of circulating GM-CFC by about 83-fold. This increase was also 39 times higher than the increase when G-CSF alone was used. The results indicate that PAGC can increase the amount of circulating GM-CFC, or synergistically increase normal mouse body with G-CSFThe amount of GM-CFC internally recycled. As shown in the table, PAGC may also increase the amount of BFU-E circulated by about 9.5 times. This increase was 4.5 times higher than the increase obtained when G-CSF alone was used. Mice treated with cyclophosphamide in similar experiments, CD34, compared to mice not receiving PAGC treatment⁺Lin^-The number of cells will increase. PAGC as a therapeutic agent can exert a good effect in increasing the number of hematopoietic stem cells by synergistic effect with G-CSF. Such co-therapy can reduce the number of apheresis required to obtain hematopoietic stem cell cells from a donor, thereby reducing the cost of the therapy. In addition, such synergistic treatment is also helpful for patients who do not respond well to G-CSF alone or do not want to use chemotherapy. Similarly, AGPC is also effective for transplantation.

Promoting recovery of individuals after exposure to cytotoxic agents or radiation

The above results indicate that when an animal or patient is discreetly exposed to radiation exposure or cytotoxic agents, PAGC promotes a more recovery process following exposure to radiation exposure or cytotoxic agents (e.g., accidental or non-therapeutic exposure, as well as therapeutic exposure) than without such treatment.

Treatment of cachexia

One of the most common side effects of chemotherapy and radiation therapy is the loss of appetite and weight in the patient. Example 8 demonstrates that PAGC can increase the body weight of mice treated with the chemotherapeutic agents cyclophosphamide and fluorouracil. Compared to mice treated with cyclophosphamide alone, the mice treated with PAGC were inhibited from losing weight and were able to recover the original weight more quickly. But these differences are not statistically significant.

As described above, PAGC significantly improved the quality of life in patients in phase II clinical trials. Wherein one parameter measured is an increase in appetite. Together with the results of the mouse model studies, it was shown that PAGC helps to improve cachexia in patients, a substance waste and malnutrition phenomenon caused by chronic diseases such as cancer or cancer treatment.

Treatment of cancer patients receiving myelosuppressive therapy with G-CSF for promoting neutrophilic leukemia Ball recovery and reduced G-CSF usage

The previous discussion shows that PAGC can stimulate cytokine production, particularly G-CSF produced by human peripheral blood mononuclear cells; and can promote the recovery of GM-CFC and peripheral leucocyte number in the bone marrow suppression animal model caused by radiation, and recover the leucocyte value in vivo of the cancer patients in the phase II clinical test to normal after receiving chemotherapy. These results indicate that PAGC may act indirectly on the hematopoietic system by producing multiple endogenous cytokines that act synergistically with each other to restore neutrophil numbers. Also, PAGC may act synergistically with IL-3 to promote proliferation/maturation of bone marrow megakaryocytes, as described above.

Furthermore, administration of PAGC is not only safe, but acceptable to patients in clinical trials. While severe and complicated side effects such as bone headache, muscle pain, headache, fatigue, nausea, vomiting, diarrhea, etc. were observed when G-CSF was used, which was not observed when PAGC was administered. These results, together with the synergistic effects discussed above, indicate that PAGC can be used in combination with G-CSF to reduce the amount of exogenous G-CSF and to promote recovery of neutrophil counts in cancer patients undergoing myelosuppression.

Combined use after high dose cytotoxic therapy and autologous or allogeneic stem cell transplantation G-CSF therapy to promote neutrophil recovery

Many cancer patients, such as breast cancer, lymphoma, and multiple myeloma patients, are currently treated with high-dose chemotherapy and/or radiation and supportive stem cell (BPC) transplantation. Transplantation of BPC accelerates the recovery of hematopoiesis. G-CSF is usually used in combination with BPC transplantation to accelerate recovery of neutrophil count, prevent fever due to neutropenia caused by infection, shorten hospital stay and reduce antibiotic usage. As shown in phase II clinical trial in example 9, PAGC can restore white blood cell counts in cancer patients undergoing chemotherapy; these results, together with the pharmacological effects summarized in the preceding paragraph, indicate that the combined use of PAGC and G-CSF is very effective in accelerating the recovery of neutrophil values after transplantation of BPC.

Improving biological reactivity of hepatitis B carrier and protecting liver cells

Cytokines play a very important role in combating viral infections. Cytokines are produced by cells associated with the immune system. Such as macrophages; CD4+ and CD8+ T lymphocytes. They play a direct role in the recovery of viral infections, such as Hepatitis B Virus (HBV), in individuals. From animal models to human studies, it is clear that cellular immune responses may play some role in addressing the pathology of the most diseased form of HBV infection. In acute HBV infection, a strong multiple cellular immune response is crucial. The production of IL-2 and INF-gamma by CD4+ T lymphocytes is indicative of the release of type 1 cytokines; and cellular immunity is induced and maintained by IL-2 and INF-gamma, which are important for combating viral infection [ C.A. Biron, production and regulation of cytokine responsiveness to viral infection (cytokines in the generation of immune response to infection, and regulation of viral infection, Cur. Opin. Immunol.), 6, 530-. Cytokines released by CD4+ and CD8+ cells also play a significant role in down-regulation (down-regulation) of HBV replication. If a defect occurs in the acute response, it becomes a chronic HBV infection. These results indicate that enhancement of type 1 responses or local production of appropriate cytokines in the liver may be very effective in treating chronic HBV infection [ M.J.Koziel, Sem.Liver Disease, 19(2), 157-161(1999) ]. PAGC is extracted from traditional Chinese medicine plants (Astragalus membranaceus var. mongholicus (AM)), which have been used for a long time to stimulate the immune and hematopoietic systems. It is widely used to treat a variety of conditions similar to chemotherapy or radiotherapy-induced myelosuppression (thrombocytopenia and anemia in addition to neutropenia). In addition, AM is known to stimulate mouse pancreatic cell proliferation in an in vitro dose-dependent manner; and can increase Natural Killer (NK) activity in pancreatic cells of animals inoculated with S-180 sarcoma cells; can also improve cytotoxic T-lymphocyte (CTL) activity. Cytokines produced by CTLs control viral infection in vivo, while viral-specific CTLs produce INF- γ and INF- α, which increase the ability of CTLs to eliminate infectious virus [ L.G. Guidotti et al. ], the use of a non-lytic mechanism for Cytotoxic T lymphocytes in transgenic mice to inhibit the expression of hepatitis B virus (Cytotoxic T lymphocytes inhibition of hepatitis B virus expression by a non-lytic mechanism), Proc.Natl.Acad.Sci.USA, 91, 764-3768(1994) ]. In addition, as described above, PAGC can stimulate the production of IL-1 β, IL-6, TNF- α, TNF- γ, GM-CSF, and G-CSF by human peripheral blood mononuclear cells after PHA activation. These studies suggest that PAGC can indirectly affect the immune system by modulating cytokine production. The crude extract from AM has been used to treat patients with chronic hepatitis by reducing the amount of IgG which is too high in the body and by reducing the ALT value, and by improving the immune and liver functions of the patients [ Y.Liu, therapeutic effect of oral liquid extracted from Astragalus membranaceus on 70 patients with chronic hepatitis B ] "therapeutic effect of organic metabolism from the sources in the treatment of the patients with 70 chronic hepatitis B" Jiang Su Chung Yao, 15(12), 38(1994) ]. In addition, fractions of AM are known to have the ability to increase immune function as described previously. These results indicate that PAGC can be used to improve and modify the biological reactivity of hepatitis b carriers and protect liver cells.

E vaccine adjuvant for hepatitis B patients

Polysaccharides prepared from the genus Polyporus umbellatus (Polyporus umbellatus) have been used as an adjuvant for hepatitis B vaccines for the treatment of chronic hepatitis B. It is known that it is seronegative for converting hepatitis B e antigen (HBeAg) and has satisfactory effects on eliminating hepatitis B virus DNA (HBV-DNA) [ S.M.Wu et al, "therapeutic findings of The combination of Polyporus polysaccharide and hepatitis B vaccine for The treatment of chronic hepatitis B" [ The therapeutic inhibition on The combined Polyporus polysaccharide with hepatitis B vaccine in The treatment of The chronic hepatitis B "[ J.Chin.Infectious diseases Disase, 13(3), 187-189(1995) ]; H.Z.Shu et al, "The therapeutic findings of The therapeutic administration of Polyporus polysaccharide with large size of hepatitis B vaccine of hepatitis B vaccine for The treatment of 64 patients with chronic hepatitis B," The therapeutic administration of 64 cases of chronic hepatitis B vaccines ", Med.J.NDFSC 6(4), 211-212(1996) ]. It is known that the extract prepared from AM turns hepatitis B e antigen (HBeAg) and anti-hepatitis B virus core antigen (anti HBc) negative and removes hepatitis B virus DNA (HBV-DNA) from hepatitis B patients [ C.K.Liu et al., "clinical and experimental studies on the treatment of chronic hepatitis B with extracts of Astragalus," clinical and experimental studies on chronic hepatitis B patients, Chung Kuo Chung His I Chieh Ho Tsa Chin, 16(7), 394-397 (1996); chen et al, "treatment of 33 patients with chronic hepatitis B with polysaccharides from Astragalus" polysaccharides from Astragalus in treating 33 patients with chronic hepatitis B ", New Drugs clin. Remedia.11 (2), 75-76 (1991)". These results indicate that PAGC may be useful as a vaccine adjuvant for hepatitis b patients.

Treating withdrawal symptoms caused by narcotics

Withdrawal symptoms typically occur when a patient begins to quit using narcotics. From the Traditional Chinese Medicine (TCM) viewpoint, the so-called withdrawal symptom is insufficient qi (energy). As observed by TCM, if qi can be strengthened, the withdrawal process can be accelerated. Example 9 one of the indications for PAGC clinical trials is to improve the quality of life of cancer patients after chemotherapy. Quality of life is measured by improvement in symptoms associated with chemotherapy; these include tiredness and exhaustion, discomfort, cold sweats, shortness of breath, and lack of appetite. These symptoms are both a manifestation of typical 'qi' deficiency and also a typical manifestation of withdrawal symptoms considered by traditional Chinese medicine. Thus, in addition to strengthening the "qi" of a patient, PAGC may also treat withdrawal symptoms in patients who are to be abstained from the use of narcotic drugs.

Preventing and treating emesis symptoms in cancer patients receiving chemotherapy or radiotherapy

The most common side effects of chemotherapy and/or radiation therapy are nausea and vomiting. Although current chemotherapy or radiation techniques have improved greatly, a significant proportion of patients still experience symptoms of emesis, and methods for reducing such side effects are sought. Although a number of antiemetic drugs are available on the market (e.g. 5-hydroxytryptamine receptor antagonists, corticoids and dopamine receptor antagonists), such drugs themselves have other side effects. The symptoms associated with these drugs are mild headache, constipation, failure to fall asleep, irritability, involuntary movements of muscles and tongue, and sedated state. Although the neuropharmacology associated with emesis is not currently fully understood, the measures taken to completely control the symptoms of emesis should include trying to make the patient feel convenient and comfortable, and providing medications that reduce the time the patient is hospitalized and bedridden, thereby promoting the quality of life of the patient, and the like. Clinical trials conducted in china have shown that PAGC is beneficial to chemotherapy patients, particularly in improving quality of life. Both the investigator observations and the patient's responses support the conclusion that PAGC improves the quality of life of patients and indicates that PAGC has significant efficacy in preventing and treating emesis symptoms following chemotherapy.

Reducing erythropoietin use in renal dialysis patients or as an alternative to erythropoietin Article (A)

EPOGEN (epoetin alfa, erythropoetin) was approved in 1989 for the treatment of anemia symptoms in patients undergoing dialysis treatment for chronic renal failure. The medicine can make patients live more active and more active. Before the advent of EPOGEN, 90% of dialysis patients suffered from anemia, often causing them to become tired and physically weak and affecting their ability to work. At present, most dialysis patients are treatedEPOGEN therapy is concurrently administered to increase or maintain red blood cell levels in the body. During clinical trials, the most common side effects caused by EPOGEN are hypertension, headache, stroke, nausea/vomiting, and thrombosis; typically, if these side effects occur, the physician will advise the patient to reduce the amount of EPOGEN. The PAGC study in example 6 shows that it restores the number of peripheral red blood cells in mice irradiated with sub-lethal doses of radiation. The irradiated mice were treated with 100 and 250mg/kg of AGPC in the same manner, and the 20-day study showed that the recovery of the red blood cell and platelet counts in the test group was improved compared to the control group, all at all time points after irradiation; indicating the same efficacy as PAGC in this model. Furthermore, as shown in example 7, PAGC can increase the amount of circulating BFU-E, and synergistically with G-CSF increase the amount of circulating BFU-E in normal mice. In addition, TER-119 in peripheral blood of normal mice treated with PAGC and mice treated with cyclophosphamide⁺The number of cells increased significantly. From the early erythroblast stage through to the mature erythrocyte stage, the TER-119 antigen is expressed on the surface of erythrocytes. TER-119⁺An increase in the number of cells indicates that PAGC stimulates differentiation, proliferation and maturation of the erythrocyte system in bone marrow and promotes the entry of these cells into peripheral blood. All of these results indicate that PAGC promotes the proliferation and maturation of red blood cells in the test mice. Since the use of PAGC is not only safe in clinical trials, but never found any significant side effects, PAGC may be useful in reducing erythropoietin use levels or as an alternative to erythropoietin in renal dialysis patients.

The irradiated mice were treated with 100 and 250mg/kg doses of AGPC according to the procedure of example 6 in the same manner, and the 20-day study showed that the recovery of the numbers of red blood cells and platelets in the test group compared to the control group was improved at all time points after irradiation; indicating the same efficacy as PAGC in this model; and means that AGPC has the same therapeutic effect as PAGC, if discussed in conjunction with other PAGC data in this study, and thus is compatible with various pharmaceutical applications and indications of PAGC.

Pharmaceutical dosage forms and modes of administration

In general, the pharmaceutically effective amount of the purified arabinogalactan composition of the first aspect of the present invention, or the arabinogalactan protein composition of the second aspect of the present invention, is administered intravenously, alone or in combination with at least one other agent, particularly one that stimulates hematopoiesis. The pharmaceutically effective amount will vary with the disease, the severity of the disease, the age and health of the individual to whom it is administered, and other factors. For stimulating hematopoiesis, including inducing megakaryocyte proliferation or maturation, stimulating the production of IL-1 β, IL-6, TNF- α, TNF- γ, IFN- γ, GM-CSF, or G-CSF, stimulating the production or action of neutrophils, treating neutropenia, anemia, or thrombocytopenia; or accelerating recovery of the patient after exposure to radiation (e.g., accidental or non-therapeutic radiation exposure, also including radiation therapy) or cytotoxic agents, the composition of the first aspect of the invention may be used in a pharmaceutically effective amount of about 50 to 1000 mg/day, particularly about 100 to 500 mg/day, particularly about 250 mg/day, of the purified arabinogalactan composition for humans of general size and weight. The above dosage is also a pharmaceutically effective amount for treating cachexia, nausea, or withdrawal syndrome, or for improving biological responsiveness, or for protecting liver cells in a hepatitis B patient. Since the arabinogalactan protein composition of the invention itself contains a higher content of arabinogalactan proteins, it is expected that the potency will be stronger and therefore the pharmaceutically effective amount will be correspondingly lower, e.g. between 10% and 50% of the pharmaceutically effective amount of the purified arabinogalactan composition. One skilled in the art will be able to determine without further experimentation, based on the present description and the skill in the art, how many pharmaceutically effective amounts of the compositions of the present invention should be administered for a particular disease.

Generally, the composition of the first aspect of the invention, the purified arabinogalactan composition, or the composition of the second aspect of the invention, the arabinogalactan protein composition, will be formulated into a pharmaceutical dosage form for administration by intravenous injection. The dosage form comprises the composition of the first aspect of the invention, a purified arabinogalactan composition, or the composition of the second aspect of the invention, an arabinogalactan protein composition, and a suitable intravenous adjuvant solution. Methods of formulating suitable adjuvant solutions for intravenous injection well known to those skilled in the art may be found in Alfonso AR: remington's Pharmaceutical Science, 17th ed., Mack publishing company, Easton, Pa, 1985. Suitable adjuvant solutions for intravenous injection include water, physiological saline, aqueous dextrose, and the like.

In general, the composition of the first aspect of the present invention, the purified arabinogalactan composition, or the composition of the second aspect of the present invention, the arabinogalactan protein composition, will be administered by intravenous injection, particularly by intravenous drip for a period of several minutes to an hour, or more, for example, about 15 minutes, when used as a hematopoietic agent. The amount of the compound of the present invention in the composition will vary depending upon the type of composition, the size of the unit dose, the type of adjuvant and other well-known factors. In general, the composition may eventually comprise from 0.001% to 10% by weight of a compound of the invention, preferably between 0.01% and 1% by weight, the remainder being adjuvant.

The composition of the first aspect of the invention, the purified arabinogalactan composition, or the composition of the second aspect of the invention, the arabinogalactan protein composition, may optionally be combined with at least one other agent useful in the treatment of disease, particularly other agents capable of stimulating hematopoiesis, such as heme, thrombopoietin, granulocyte colony stimulating factor (G-CFS), IL-3, and the like.

Examples

The invention is further illustrated by the following non-limiting examples. All purified arabinogalactan compositions and arabinogalactan protein compositions were assayed by size exclusion chromatography (size exclusion chromatography).

Size exclusion chromatography (size exclusion chromatography) consisted of: a Shimadzu HPLC system equipped with an SCL-10A system controller, an LC-10AD pump, a DGU-4A degasser, a RID-6A refractive index detector, and an SPD-10AV UV detector, and GS-701 and GS-620 columns (Shodex Asahipak, 7.6X 500mm) equilibrated with 0.2N sodium chloride. The amount of sample loaded was 80. mu.g (40. mu.l of sample solution at a concentration of 2 mg/ml) and the rate of sample elution was 1 ml/min. Standards of different average molecular weights were used to prepare a calibration curve; the molecular weight of the sample can be read from the standard curve. Weight average molecular weight (Mw ═ Σ (a) of sample_iM_i/∑A_i) Number average molecular weight (Mn ═ Sigma A)_i/∑(A_i/M_I) And polydispersity (Mw/Mn) were determined using a standard curve prepared using Shimadzu SEC software version 2.4.

The sugar content and PAGC and AGPC compositions of the invention were determined by Gas Liquid Chromatography (GLC) analysis of trimethylsilylglycoside derivatives. In this method, the polysaccharide is first methanolated by addition to methanolic hydrochloric acid acidified methanol, followed by Trimethylsilyl (TMS) derivatization to produce volatile monosaccharide derivatives. After removal of impurities, the derivatives were analyzed by gas-liquid chromatography (GLC) equipped with a DB-1 column including a Flame Ion Detector (FID). The sugar content and composition were determined using myo-inositol as an internal standard and analyzed with samples of the composition.

The hydroxyproline content was determined by colorimetric assay. The sample was hydrolyzed with hydrochloric acid and then treated with sodium hypobromite (sodium hydroxide solution of bromine), hydrochloric acid and dimethylaminobenzaldehyde. The optical density of the final solution was measured using a colorimeter and the hydroxyproline content was read from a standard curve drawn using the same method for preparing various hydroxyproline concentration gradients.

EXAMPLE 1 preparation of arabinogalactan composition

Step A "immersion slicing" treatment

Cleaning root of 300 kg of sun-dried radix astragali (Astragalus membranaceus), sterilizing, washing, rubbing with ultra-filtered water, soaking in 70% ethanol overnight, cutting into slices with thickness of about 3-5 mm, and oven drying in a sterilizing oven at 60-70 deg.C to obtain "soaked slices". These dried "dip slices" lose less than about 15% weight due to drying.

Step B crude extraction of arabinogalactan composition

200 kg of "macerated slices" prepared in step A were extracted 3 times with UF water (deionized water ultrafiltered with a 10KMWCO UF system) at 100 ℃ for 3 hours each time. Concentrating the mixed extractive solution at 60-70 deg.C under reduced pressure to about 200L. Adding 95% ethanol to the concentrated solution to make the final concentration of ethanol reach 70%, stirring at room temperature for 15 min, and precipitating polysaccharide. The supernatant was decanted and the pellet was washed 3 times with 95% ethanol. The precipitate was suspended in UF water at a concentration of about 18-20% (as measured by refractometer) and 95% alcohol was added to make the alcohol a final concentration of 35%. The alcohol suspension was centrifuged, the precipitate was discarded, and 95% alcohol was added to the supernatant with stirring to reach an alcohol concentration of 70%. Collecting the precipitate, dissolving with UF water, and spray drying to obtain crude arabinogalactan composition.

The crude arabinogalactan composition was a light yellow powder with a water solubility of 200 mg/ml and a baked weight loss of about < 15%. The endotoxin content was about < 0.3 EU/mg.

Step C purification of arabinogalactan composition

3.5 kg of the crude arabinogalactan composition prepared in step B was dissolved in UF water to give a 2% strength solution (175 l volume). The solution was filtered with a 5K mwcoff system to a final volume of 35-40 liters. The concentrate was prepared to 20mM NaOAc buffer pH5.2 by adding 1.0M sodium acetyl (NaOAc) buffer pH 5.2. The solution was loaded onto an SP Sepharose cation resin exchange column (volume 20L, bed height 30 cm) and eluted with 20mM NaOAc, collecting 2.5-3.0 bed volumes of eluate. The collected eluate was loaded onto a Q sepharose anion exchange column (same height as the SP column volume and bed) and eluted with 20mM NaOAc, and 3 to 3.5 bed volumes of the eluate were collected. The eluate collected from the Q sepharose anion resin exchange column was filtered through a 0.1 μm filter and then ultrafiltered through an 8K MWCO UF system. Concentrating the residual liquid to 20-26% with a concentration system at 50-60 deg.C, and precipitating with anhydrous alcohol to obtain alcohol with final concentration of 80-90%. Washing the precipitate with anhydrous alcohol for 3 times, and oven drying at 60-70 deg.C to obtain purified arabinogalactan composition.

The purified arabinogalactan composition is a white powder, dissolved in water, physiological saline, and 5% dextrose in water, having a concentration of 20 mg/ml and a water content of 6.0% or less. The ash content of the composition is less than or equal to 2.0 percent, the heavy metal content is less than or equal to 10ppm, and the endotoxin content is less than or equal to 0.1 EU/mg. The pH value of the composition solution is between 4.5 and 6.5. The composition has a sugar content of less than or equal to 85% (measured by the phenol-sulfuric acid method using glucose as standard) and an Ara: Gal ratio of greater than or equal to 1.5: 1 (determined by analysis of the trimethylsilylglucoside derivative of the composition by GLC).

EXAMPLE 2 preparation of arabinogalactan protein composition

The Q sepharose anion exchange column eluate obtained in step C of example 1 was ultrafiltered with a 100K MWCO UF system. Further concentrating the residual liquid after ultrafiltration, and precipitating with anhydrous alcohol to obtain final alcohol concentration of 80-90%. Washing the precipitate with anhydrous alcohol for 3 times, and oven drying at 60-70 deg.C to obtain purified arabinogalactan protein composition.

The purified arabinogalactan protein composition is a white powder, dissolved in water, physiological saline and 5% dextrose in water at a concentration of 20 mg/ml, and has a pH of the composition solution of between 4.5 and 6.5. The composition has endotoxin content not more than 0.5EU/mg and heavy metal content not more than 10 ppm. The proportion of arabinose and galactose (Ara: Gal) is more than or equal to 2: 1, and the hydroxyproline content is more than 0.2 percent. The weight average molecular weight of the composition is more than or equal to 100 kilodaltons.

Example 3 preparation of cytokines from activated human peripheral blood mononuclear cells

Human Peripheral Blood Mononuclear Cells (PBMC) were prepared as described by Boyum [ A.Boyum, [ Isolation of monocytes and granulocytes from human blood ] ", Scan.JLAb.Invest., 97, 77-89(1968) ], [ A.Boyum, [ Isolation of monocytes and granulocytes from human blood ]" ]]. Human buffy coat (blood sediment) samples, about 25 ml/donor, were obtained from the blood bank of the medical center at the university of histodanver. Each buffy coat sample was gently resuspended in a total volume of 100 ml of calcium-free, magnesium-free Hank's balanced salt solution (HBSS, Gibco) at room temperature. 25 ml of the cell suspension was placed in a 50 ml centrifuge tube containing 15 ml of Ficoll-Paque (Pharmacia LKB biotechnology, Inc.) and centrifuged at 400 Xg for 30 minutes at 15 ℃ using a Beckman GPR bench top centrifuge (GH-3.7 rotor). After centrifugation, the interfacial peripheral blood mononuclear cell suspension was transferred to another 50 ml centrifuge tube, resuspended in HBSS to a final volume of 45 ml, and centrifuged at 354 Xg for 10 minutes at 15 ℃. The supernatant was discarded and the cell pellet resuspended in HBSS to a final volume of 45 ml and centrifuged at 265 Xg for 10 min at 15 ℃. 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 Dickson) and peripheral blood mononuclear cells from two different donors were used in the following experiments. Peripheral blood mononuclear cell suspension was diluted to 4X 10⁶To 1 ml of the cell suspension, 0.5 ml of phytohemagglutinin P (PHA-P, Pharmacia 27-3703-01) was added at a final concentration of 0.3. mu.g/ml, and 0.5 ml of any one of the various concentrations of the composition solution of the present invention was added to conduct cell culture. The total volume in each tube was 2 ml. By P aloneHA-treated cell solution served as control group. At 37 deg.C, 7% CO₂After 24 hours incubation, the tubes were centrifuged at 1600 Xg for 10 minutes at 15 ℃ using a Beckman GPR bench top centrifuge (GH-3.7 rotor), the supernatant was collected and stored at-70 ℃ before analysis. Cytokine levels, such as the human cytokines IL-1. beta., IL-6, TNF-. alpha., GM-CSF and G-CSF (R) were measured using commercially available ELISA kits according to the manufacturer's instructions&D Systems, MN) and human TNF-. gamma. (Endogen, MA). The optical density was determined by a fully automatic quantitative plotter microplate reader (Thermo max, molecular devices, CA). The results were calculated using the software provided in a fully automatic quantitative plotter microplate reader and expressed in terms of the cytokine content (pg/ml) contained in the supernatant. All results are expressed as the ratio of test group to control group (S/C), where S represents the amount of cytokine produced after stimulation of peripheral blood mononuclear cells with PHA and the test sample, and C is the amount of cytokine produced after stimulation of peripheral blood mononuclear cells with PHA alone. The following table shows that PAGC can increase the amount of cytokines produced by activated human peripheral blood mononuclear cells.

PAGCμg/ml	ELISA(S/C)
							IL-1β	IL-6	TNF-α	IFN-γ	GM-CSF	G-CSF
	25	9.1	11.8	4.9	3.1	6.5							49.9
10							4.3	5.9	2.3	1.2	2.1	16.5

Example 4 proliferation/maturation of bone marrow megakaryocytes

Experiments were performed with 9-14 weeks of G3H/HeJ males. A liquid culture assay for analyzing the maturation of megakaryocytes is an in vitro assay that can study the proliferation and/or maturation of bone marrow megakaryocytes (peripheral platelet stem cells). For details, see S.A. Burstein, "maturation of murine megakaryocytes in vitro accelerated by Interleukin 3 (" Interleukin 3 protein depletion of muscle megakaryocytes in vitro ")" blood cells (Bloodcells)11, 469-479 (1986). Mouse normal bone marrow mononuclear cells were first isolated and suspended in a medium containing 0.5mM diisopropylfluorophosphate (DFP, Sigma, St. Louis, Mo.) for 20 minutes at room temperature to inactivate endogenous acetylcholinesterase (AchE). Washing the cells at 4X 10⁶The suspension was resuspended in 15% FCS-IMDM (Gibco BRL, Gaithersburg, Md.) at a concentration of one ml and then allowed to settleIn tissue culture plastic bottles, stromal cells and macrophages are removed by taking advantage of their property of adhering to the bottle. Placing the tissue culture flask containing the resuspended bone marrow cells at 37 deg.C and 5% CO₂The cells were cultured for 1.5 hours. Those cells not attached to the flask were collected at 1X 10⁶The concentration per ml was analyzed IN 1% Nutridoma SP (Boehringer Mannheim, Indianapolis, IN) -IMDM. Cells were loaded into 96-well, U-shaped tissue culture dishes containing 10 cells per well⁵Individual cells and a total volume of 0.2 ml containing various PAGC concentrations and with or without 50pg/ml of mouse recombinant IL-3 (R)&D Systems, Minneapolis, MN). At 37 deg.C, 5% CO₂The cells were cultured for 7 days. PAGC activity was measured by measuring the increase in acetylcholinesterase activity, a relatively specific cellular marker of rodent megakaryocyte proliferation or maturation. After 7 days, the plates were centrifuged and the supernatant discarded. To each well was added 0.2 ml of solution I (0.2% Triton X-100, 1mM EDTA, 0.12mM NaCl, 50mM HEPES, pH 7.5) and 20. mu.l of 6.27mM thioacetyl choline iodide (sigma, St. Louis, Mo.) (the final concentration of thioacetyl choline iodide was 0.57 mM). Tissue culture dishes were incubated at 37 ℃ for 4 hours, then 10. mu.l of the reaction mixture was removed from each well and placed in a 96-well black plate (black plate) (Labsystems, Helsinki, Finland), followed by 10. mu.l of 0.4mM Coumarin Phenylmaleimide (CPM), Molecular Probes Inc., Eugene, OR) in DMSO, and 0.2 ml of solution II (5mM sodium acetate, 1mM EDTA, 0.2% Triton X-100, pH 5.0). Gently shake the plate to mix the liquids well. The intensity of the released fluorescence was measured in a fluorimeter with a 390nm light-sensitive filter and a 460nm emission filter (Fluoroskan II, Labsystems, Helsinki, Finland). The fluorescence intensity is proportional to the amount of AchE produced by the cultured megakaryocytes. PAGC concentrations of 12.5. mu.g/ml and 400. mu.g/ml (peaks appear at concentrations of 200. mu.g/ml) increase the number of megakaryocytes cultured, and a PAGC concentration of 12.5. mu.g/ml and 400. mu.g/ml (peaks appear at concentrations of 200. mu.g/ml) greatly increase the amount of IL-3 in combination with 50pg/mlThe number of megakaryocytes cultured was added (the concentration was 50 to 200. mu.g/ml, which increased the number of megakaryocytes by two-fold).

Example 5 restoration of GM-CFC Stem cell numbers in Fluorouracil-treated mice

On day 0, fluorouracil (Sigma) in an amount of 150 mg/kg was injected intraperitoneally into BALB/c females 8-10 weeks old; and on days 1-3, saline, PAGC at various concentrations, or recombinant human G-CSF (Neupogen, Amgen) at 100. mu.g/kg were injected subcutaneously, respectively. And on day 4, the test mice were sacrificed. Collecting bone marrow of femur, counting, and mixing 5 × 10⁴The individual leukocytes were cultured in 35mm petri dishes, each of which was placed with 1 ml of methylcellulose complete medium and a prokineticin-containing pancreatic cell culture medium as a source for providing colony stimulating factors (stemcell technologies, Inc). After 6-7 days of culture at 37 ℃, cell populations with cell numbers exceeding 50 were counted using a Nikon Diapthot microscope (30-60 fold). The following table shows that PAGC promotes the recovery of mouse GM-CFC stem cells treated with fluorinated uracil.

Treatment of	Physiological saline solution	PAGC50mg/Kg	PAGC100mg/Kg	PAGC200mg/Kg	G-CSF100μg/Kg
						GM-CFC/femur	601	608	1075	2020	1196

Example 6 reconstitution of peripheral blood in mice irradiated with sublethal doses of radiation

This experiment was performed with BALB/c females with an average body weight of 20 g for 9-14 weeks. Neomycin (Neomycin, Sigma, st. louis, MO) was added at 40 mg/l to the mouse's unacidified drinking water 5 days prior to each experiment. Mice from control or experimental groups were randomly selected, 6 each, and irradiated with 4.25Gy of X-ray radiation (250KVP, 0.35mm Cu filter, Philips, Germany) on day 0. After irradiation, the mice had lower numbers of peripheral blood leukocytes and platelets than normal mice and slightly reduced numbers of erythrocytes. 300 and 100mg/Kg of PAGC were injected subcutaneously. Injections were given once daily for the first 5 days (from day 0 to day 4), followed by 3 times weekly for 3 consecutive weeks, including the first injection after irradiation. A 14 needle dose of PAGC was used. The control group was injected subcutaneously with 0.1 ml of physiological saline. During the experiment, blood was drawn twice weekly from the tail vein of mice, and blood samples were deposited in EDTA-coated tubes (Sarstedt, Germany) and analyzed for peripheral blood leukocyte, platelet, and erythrocyte values, as well as for hemoglobin content, were calculated using a Serono 9010+ cell counter (Serono baker diagnostics inc. Compared with the control group, 100 and 300mg/Kg PAGC can obviously (p is less than 0.01) promote the recovery of the white blood cells of the mice 17 to 30 days (the end of the experiment) after the irradiation; and significantly (p < 0.05, typically p < 0.01) promotes platelet recovery in mice from day 14 to day 25 after irradiation. It is also evident (p < 0.05, typically p < 0.01) that it promotes recovery of mouse red blood cells from day 14 to day 25 after irradiation. Administration of 100 and 250mg/Kg of AGPC in the same manner during the 20 day study, regardless of the time point tested, improved the recovery of red blood cells and platelets in the irradiated mice, indicating the same potency as PAGC in this model.

Example 7 migration of peripheral blood Stem cells with PAGC alone or in combination with granulocyte colony stimulating factor (G-CSF)

BALB/c mice, 8-10 weeks, were given free access to acidified water and food. Normal mice were treated for 7 days, once a day, in the following manner: physiological saline (200. mu.l, subcutaneous injection), PAGC (100mg/Kg, subcutaneous injection), G-CSF (100. mu.g/Kg, subcutaneous injection), or PAGC + G-CSF (100mg/Kg PAGC + 100. mu.g/Kg G-CSF, subcutaneous injection). The injection volume per mouse was about 200. mu.L. Each group had five mice. Seven days after the above treatment, mice were injected intraperitoneally with 20 units of heparin (Elkins-Sinn, inc., Cherry Hill, NJ) on day 8, followed by CO inhalation for 30 minutes₂Mice were sacrificed and peripheral blood was collected by cardiac puncture. Peripheral blood mononuclear cells were separated by density centrifugation using water-soluble Ficoll-Paque (Pharmacia Biotech AP, Uppsala, Sweden), washed twice with phosphate buffer, resuspended in culture medium and counted in a cell counter. Will be between 2.5 × 10⁴To 1X 10⁵A number of peripheral blood mononuclear cells were cultured in 35mm petri dishes containing 1 ml of complete methylcellulose medium containing erythropoietin (complete erythropoietin-containing methylcellulose) and recombinant IL-3+ IL-6+ stem cell factor (StemCell Technologies Inc., Vancouver, B.C.) per dish. After 7-14 days of culture at 37 ℃, cell populations with cell numbers exceeding 50 were counted using a Nikon Diaphot microscope (30-150 fold). The number of colonies forming granulocyte macrophage colony (GM-CFC) and erythroid burst-forming units (BFU-E) were counted. The following table shows that PAGC can synergistically increase the values of GM-CFC and BFU-E circulating in normal mice with G-CSF.

	Physiological saline solution	PAGC100mg/Kg	G-CSF100μg/Kg	PAGC，100mg/Kg+G-CSF，100μg/Kg
					GM-CFC，100/ml	0.8	4.6	30	64
BFU-E，1 00/ml	1.0	3.1	4.5	9.5

In another similar experiment, mice were treated with 200 mg/kg cyclophosphamide and then with physiological saline or 100 or 300mg/kg PAGC for 11 days, peripheral blood mononuclear cells were collected and resuspended to 2X 10⁷Cells/ml, stained with fluorescein isothiocyanate-anti-CD 34 and PE-lineage markers (CD3, CD4, CD6, CD19, CD11b, GR-1, CD41, and Ter-119) (Pharmingen, San Diego, Calif.) and analyzed by fluid cytometry (FACSCalibur, Becton Dickson, San Jose, Calif.), it was seen that PAGC accelerates CD34⁺Lin^-The cells move into the peripheral blood.

Example 8 PAGC can restore body weight in fluorinated uracil or cyclophosphamide treated mice

This experiment was performed with BALB/c females at 8-10 weeks. 10 mice were randomly divided into 5 groups: normal control, cyclic amine phosphate (CY) -treated, Fluorinated Uracil (FU) -treated, CY + PAGC-treated, or FU + PAGC-treated groups, respectively. For the normal control group, the normal saline was injected intraperitoneally on day 0, and then subcutaneously from day 1 to day 12. For the cyclic amine phosphate (CY) -treated group or CY + PAGC-treated group, 200 mg/kg of cyclic amine phosphate was injected intraperitoneally on day 0, and then from day 1 to day 12, either saline or 200 mg/kg of PAGC was injected subcutaneously. For the Fluorouracil (FU) -treated group or FU + PAGC-treated group, 150 mg/kg of FU was injected intraperitoneally on day 0, and then from day 1 to 12, either physiological saline or 200 mg/kg of PAGC was injected subcutaneously. Mice were weighed once on day 0 and then again every other day until day 12. The results show that CY + PAGC-treated and FU + PAGC-treated mice lost minimal weight and recovered weight more rapidly than CY or FU treated mice alone.

Example 9 PAGC human clinical trials in China

The clinical test of PAGE human body in China was performed according to the GCP standard stipulated by the Ministry of health of the people's republic of China.

In the first clinical trial, PAGC was diluted with normal saline and administered intravenously to 32 normal volunteers for 7 consecutive days. Three times the clinical dose (250 mg/day) was administered without any serious clinical side effects.

Phase II clinical trials to evaluate PAGC inhibition of leukopenia (< 4.0X 10) following chemotherapy in lung, gastro-intestinal, or breast cancer patients⁹White blood cell/L ═ potency. A total of six medical centers, 487 patients, were enrolled in this clinical trial, with 328 patients in the PAGC treatment group, 84 patients in the G-CSF treatment group, and 75 patients in the control group. Throughout the 14 day chemotherapy period, the patient had a low white blood cell count of 4X 10 at any time⁹L, i.e. the patient is enrolled in one of any three groups. For the first group of patients, 250mg of PAGC was dissolved in 500 ml of physiological saline, and the solution was intravenously injected into the patients once a day for 7 days. The patient is administered a PAGCAnd observing for 7 days after stopping the drug. The patients in the G-CSF treatment group were subcutaneously injected with 75. mu.g of G-CSF once a day for 5 consecutive days, and observed for 9 days during the administration period and after the withdrawal period. In the control group, no other hematopoietic-boosting drug was administered after chemotherapy, and the patients were observed for 14 days. White blood cell, RBC, and platelet values were observed in all 3 groups of patients within 14 days after treatment. The efficacy of the patient in improving the quality of life of the patient is assessed by the patient's symptoms associated with chemotherapy and by the patient's carnowski performance index. Symptoms associated with chemotherapy include fatigue and listlessness, malaise, sweating, shortness of breath, and anorexia, and are assessed by qualified physicians in traditional Chinese medicine. The score for each class is as follows: score 0-no symptoms, score 1-mild symptoms, score 2-moderate symptoms, and score 3-severe symptoms. Therefore, the most serious example is indicated by 15 points. The carnivsky performance index is an evaluation method used by the World Health Organization (WHO) to assess general health status. FIG. 1 shows the number of white blood cells as a function of days after chemotherapy; FIG. 2 is a plot of total symptom score versus days after chemotherapy; figure 3 is a score for the carnivosyl performance index (PAGC: U18.36, p < 0.01 for GCSF; PAGC: U15.62, p < 0.0001 for control; the table below lists mean platelet number versus days after chemotherapy; all results show that treatment with 250mg PAGC for 7 days does promote recovery of leukocyte and platelet number, improves symptoms caused by chemotherapy, and improves the carnivosyl performance index for patients with chemotherapy, overall statistically significantly better than the control and generally statistically better than treatment with 75 μ G of G-CSF for 5 days.

Platelets, 109Patients in the treatment group (n ═ 54)
					Day 0	Day 4	Day 7	Day 10	Day 14
65±21	87±61*	118±94**	149±107**	178±111**
					*p＜0.05，**p＜0.001

While the invention has been described with respect to specific embodiments, it will be apparent to those skilled in the art that various changes in light of the disclosure can be made without departing from the spirit and scope of the disclosure, and all such modifications are intended to be included within the scope of the appended claims.

Claims (34)

1. A purified arabinogalactan protein composition isolated from astragalus membranaceus, said arabinogalactan protein composition having a weight average molecular weight of at least 100 kilodaltons, and comprising 5% protein, wherein 20% of the amino acid content of said protein, based on total amino acids, is hydroxyproline, and having a sugar composition wherein the ratio of arabinose to galactose is at least 2: 1, and comprising a molar percentage of arabinose between 45% and 75%; rhamnose in a molar percentage of 2 to 4%; galactonic acid in a molar percentage of 4% to 6%; 8 to 25 mole percent galactose; and 5 to 25 mole percent glucose.

2. The purified arabinogalactan protein composition of claim 1, wherein the astragalus membranaceus is astragalus mongholicus, or astragalus membranaceus.

3. The purified arabinogalactan protein composition of claim 1 or 2, isolated from Astragalus membranaceus (Fisch.) bge. var. mongholicus (R) Hsiao, inner Mongolia, or Shanxi province, China.

4. The purified arabinogalactan protein composition of claim 1, wherein the astragalus membranaceus is a two year old astragalus membranaceus.

5. The purified arabinogalactan protein composition of claim 1, wherein the arabinose/galactose ratio is at least 3: 1.

6. The purified arabinogalactan protein composition of claim 1 comprising less than 0.5EU/mg endotoxin.

7. An intravenously injectable aqueous arabinogalactan protein formulation comprising:

(a) a pharmaceutically effective dose of the purified arabinogalactan protein composition of any one of claims 1, 2, 4, 5, or 6; and

(b) an intravenous liquid adjuvant.

8. Use of the purified arabinogalactan protein composition of any one of claims 1, 2, 4, 5, or 6 or the aqueous intravenously injectable arabinogalactan protein formulation of claim 7 in the manufacture of a medicament for the treatment of a disease state in a mammal susceptible to treatment by stimulation of the immune and hematopoietic systems, said use comprising intravenously injecting a pharmaceutically effective amount of the purified arabinogalactan protein composition of any one of claims 1, 2, 4, 5, or 6 or the aqueous intravenously injectable arabinogalactan protein formulation of claim 7.

9. The use according to claim 8, wherein the treatment of a mammalian disease state is stimulation of hematopoiesis, induction of megakaryocyte proliferation or maturation, stimulation of IL-1 β, IL-6, TNF- α, IFN- γ, GM-CSF, or G-CSF production, stimulation of neutrophil production, or activation, treatment of neutropenia, anemia, or thrombocytopenia, promotion of individual recovery following exposure to cytotoxic agents, or radiation exposure, treatment of cachexia, emesis, or withdrawal syndromes, improvement of biological responsiveness, or protection of liver cells in a hepatitis b patient.

10. The use according to claim 9, wherein the treatment of a mammalian disease state is the stimulation of hematopoiesis, the induction of megakaryocyte proliferation, or maturation, the stimulation of IL-1 β, IL-6, TNF- α, IFN- γ, GM-CSF, or G-CSF production, the stimulation of neutrophil production, or activation, or the treatment of neutropenia, blood, or thrombocytopenia.

11. The use of claim 8, wherein the mammal is a human.

12. The use of claim 10 or 11, wherein the mammal suffers from myelosuppression.

13. The use of claim 12, wherein the myelosuppression results from cancer chemotherapy, or radiation exposure therapy.

14. The use of claim 8, wherein the treatment of a mammalian disease state further comprises administration of at least one additional therapeutic agent.

15. The use of claim 14, wherein the at least one other therapeutic agent is an agent that stimulates hematopoiesis.

16. The use according to claim 15, wherein the at least one other therapeutic agent is selected from the group consisting of hemopoietin, thrombopoietin, or granulocyte colony stimulating factor, or IL-3.

17. A method of making the purified arabinogalactan protein composition of claim 1, comprising:

(a) a liquid extract containing arabinogalactan protein composition extracted from astragalus;

(b) adding a sufficient amount of a lower aliphatic alcohol to the liquid extract of step (a) to precipitate an arabinogalactan protein composition and isolating the precipitated arabinogalactan protein composition;

(c) dissolving the precipitated arabinogalactan protein composition of step (b) in water to form a solution comprising the arabinogalactan protein composition;

(d) purifying the arabinogalactan protein composition-containing solution from step (c) by ion exchange chromatography;

(e) treating the arabinogalactan protein composition-containing solution of step (d) to remove any materials having a molecular weight below 100 kilodaltons; and

(f) isolating a purified arabinogalactan protein composition from the treated arabinogalactan protein composition-containing solution of step (e).

18. The method of claim 17, further comprising:

(g) concentrating the solution of arabinogalactan composition obtained in step (f), and precipitating with anhydrous alcohol; and

(h) washing the precipitate obtained in step (g) to obtain an arabinogalactan protein composition.

19. The use of claim 9, wherein the mammal is a human.

20. The use of claim 10, wherein the mammal is a human.

21. The use of claim 19, wherein the mammal has myelosuppression.

22. The use of claim 20, wherein the mammal has myelosuppression.

23. The use of claim 9, wherein the treatment of a mammalian disease state further comprises administration of at least one additional therapeutic agent.

24. The use of claim 10, wherein the treatment of a mammalian disease state further comprises administration of at least one additional therapeutic agent.

25. The use of claim 11, wherein the treatment of a mammalian disease state further comprises administration of at least one additional therapeutic agent.

26. The use of claim 12, wherein the treatment of a mammalian disease state further comprises administration of at least one additional therapeutic agent.

27. The use of claim 13, wherein the treatment of a disease state in a mammal further comprises the administration of at least one additional therapeutic agent.

28. The use of claim 23, wherein the at least one other therapeutic agent is an agent that stimulates hematopoiesis.

29. The use of claim 24, wherein the at least one other therapeutic agent is an agent that stimulates hematopoiesis.

30. The use of claim 25, wherein the at least one other therapeutic agent is an agent that stimulates hematopoiesis.

31. The use of claim 26, wherein the at least one other therapeutic agent is an agent that stimulates hematopoiesis.

32. The use of claim 27, wherein the at least one other therapeutic agent is an agent that stimulates hematopoiesis.

33. The purified arabinogalactan protein composition of claim 1, wherein the composition is isolated from the roots of astragalus membranaceus.

34. The purified arabinogalactan protein composition of claim 1 or 2, isolated from astragalus membranaceus, produced in inner mongolia, china.