CN1179979C - Hematopoietic arabinogalactan compositions - Google Patents

Abstract

Purified arabinogalactans isolated from Astragalus membranaceus (particularly roots), and arabinogalactan protein compositions isolated from these purified arabinogalactan compositions having a weight average molecular weight of at least 100 kilodaltons, can be formulated into liquid dosage forms for intravenous injection; after intravenous administration to a mammal, it is useful to stimulate hematopoiesis, induce proliferation or maturation of megakaryocytes, stimulate production of IL-1 β, IL-6, TNF- α, IFN- γ, GM-CSF, or G-CSF, stimulate production or activation of neutrophils, treat neutropenia, anemia, or thrombocytopenia, promote recovery of the individual following exposure to cytotoxic agents or radiation, treat cachexia, treat emesis, or withdrawal syndromes, or improve biological responsiveness, or protect liver cells from a hepatitis B patient.

Description

The arabinogalactan composition of hematopoiesis

The technical field of the invention

The present invention relates to a kind of arabogalactan.More particularly, the present invention relates to a kind of from the Radix Astragali (Astragalus membranaceus), the arabogalactan of the isolating purifying of its root particularly, and isolating weight-average molecular weight is the arabinogalactan protein composition of 100 kilodaltons (KD) at least from the arabinogalactan composition of these purifying or its intermediate.

Background technology related to the present invention

The Radix Astragali (Radix Astragali) is the root after Radix Astagali or Radix Astragali (Astragalusmembranaceus Bge.Var.mongholicus (Bge) Hsiao or A.membranaceus (Fisch) Bge. (Fabaceae)) dry.The Radix Astragali is a kind of well-known ancient Chinese medicine.Formal record is arranged in the Chinese Pharmacopoeia, and mainly be used as a kind of tonic, be used for treating ephritis and diabetes.Usually be used as decoction or be used as " tea " separately and use, or support 12 kinds of herbal medicine that hold soup (Ren-shen-yang-rong-tang) (by comprising Radix Astragali Radix Astragali) at traditional herbal medicines Xi Jialong (shi-ka-ron) (forming) and genseng and form by Asian puccoon (Lithospermum erythrorhizon) and two kinds of herbal medicine of Ligusticum wallichii (Ligusticum wallachii)) in unite use with other plant.[consult " Chinese medicine (Chinese Drugsof Plant Origin) " the 26th trifle of editing by W.Tang and G.Eisenbrand, the 191-197 page or leaf, title " Astragalusmembranaceus (Fisch) Bge. ", Springer Verlag, Berlin, 1992].

Existing human astragalus decoction, the injection of solution administration that is prepared into alcohol precipitation thing by this decoction, can improve gastric duodenal ulcer symptom and the chronic leukopenia patient's of increase white blood cell count and (consult " pharmacology of Chinese medicine and application (Pharmacology and Applications of Chinese materialMedica) " 1041-1046 page or leaf of editing by people such as H.-M.Chang and P.P.-H.But, exercise question " Radix Astragali " (Huangqi), World ScientificPublishing Co., Singapore, 1987).Known astragalus decoction, (for example molecular weight is 25 for the low molecular weight part of purifying, 000-35,000 part), decoction with the medical herbs mixture that includes the Radix Astragali, also can recover the immunity system (D.-T.Chu etc. of partial heterograft and host's opposing reaction, " herbal medicine immunotherapy " " Immunotherapy withChinese medicinal herbs. I... ", J.Clin.Lab.Immunol., 25,119-123 (1988)), and reverse is reacted D.-T.Chu etc. because of the immunosuppression of cyclic phosphoric acid amine inductive., " herbal medicine immunotherapy " " Immunotherapy with Chinese medicinal herbs.II... ", J Clin.Lab. " immunology " (Immunol.), 25,125-129 (1988)), promote " in the LAK cell by the cytotoxicity due to the rIL-2 (D.-T.Chu etc.; " separation of Radix Astragali extract " " Fractionated extract of Astragalus membranaceus "; J.Clin.Lab.Immunol.; 25; 183-187 (1988)); promote the inhibitive ability of immunity mouse immune response [K.S.Zhao etc., " Radix Astragali extract strengthens the intravital immune response of mouse " (" Enhancement of the immune response in mice by Astragalusmembranaceus extracts "), " immunopharmacology " (Immunopharmacology), 20,225-234 (1990)], stimulate monocytic reaction [Y.Sun etc., " preliminaryobservation on the effects of the Chinese medicinal herbs... ", " biological response instrumentality magazine " (J.Biol.Response Modifiers), 2,227-237 (1983)], with the marrow hemopoiesis ability that stimulates mouse [M.Rou etc., " Radix Astragali is to the influence of marrow hemopoiesis ", (The effect of Radix astragali on mouse marrow hemopoiesis), " traditional Chinese medicine " (J.Trad.Chin.Med.) 3 (3), 199-204 (1983); S.I.Miura etc., " Chinese herb drug effect " (" Effect of a traditional Chinese herbalmedicine... "), Int.J.Immunopharmacol. " immunopharmacology magazine ", 7 (11), 771-781 (1989); And Y. Ohnishi etc., " Effects of Juzen-taiho-toh (TJ-48) ... ", Exp.Hematol. " experimental hematology ", 18,18-22 (1990)].

Disclosed a kind of pharmaceutical composition in the United States Patent (USP) 4,843,067 of Liu, it contains astragalus polysaccharides (can obtain by extracting among Astragalus membranaceus Bge. or the Astragalus gummiferLabillard) and Radix Angelicae Sinensis polysaccharide is formed.This astragalus polysaccharides used water is extracted from Radix Astragali root powder or alcohol precipitation and is obtained.The United States Patent (USP) 5 of Verbiscar, 2868, delivered a kind of immunomodulatory polysaccharide in 467 from tragakanta (Astragalus tragacantha (tragacanth)) plant, be prepared from low temperatures, in order to " keep the integrity of polysaccharide structures, do not have chemistry or conformational change ".People's such as Josephson United States Patent (USP) the 5th, 336, delivered the mixture of using such as arabogalactan (separating from tamarack (Larixoccidentalis)) and this class vegetalitas polysaccharide of mannosans and the formation of some medicaments in No. 506, the energy target can cause the cell receptor of receptor-mediated pinocytosis.People's such as Adams United States Patent (USP) 5,116,969 has been delivered a kind of arabogalactan product that is suitable for the special purified of density gradient separation.People's such as Jung United States Patent (USP) 5,478,576 have delivered a kind of arabogalactan (equally from Larix occidentalis), split product and modifier thereof etc. of purifying, can be used to equally medicament is delivered to cause pinocytotic targeted cells acceptor.People's such as Lewis United States Patent (USP) 5,589,591 has disclosed a kind of endotoxic polysaccharide that do not contain, for example arabogalactan, dextran, mannosans and gum arabic; With ultrafiltration process a kind of impure polysaccharide is prepared and forms, at first, stay retentate, can remove the film of higher molecular weight part again by another kind, equally still stay the filtrate part by a kind of film that removes low molecular weight part.

But above-mentioned these reference all focus on the polysaccharide part (as arabogalactan) in the product, and may even use the technology that can get rid of arabinogalactan protein.

Arabinogalactan protein also is present in the flowering plant, and extensively is stored in the higher plant.Arabinogalactan protein (AGPs) claims the arabogalactan peptide sometimes again, is the glycoprotein that includes carbohydrate at high proportion and a small amount of protein (being usually less than 10%), though known also have an arabinogalactan protein that contains high protein.In the isolated glycoprotein that is rich in oxyproline by plant, the characteristics of arabinogalactan protein be protein content low and can with β-glucosyl group Ya Lifu (Yariv) reagent, promptly 1,3,5-three-(4-β glucosyl group furans oxygen phenylazo)-2,4,6-trihydroxybenzene bonded ability [J.H.yariv etc., Biochem.J., 85,383-388 (1962); R.L.Anderson etc., Aust.J.plant physiol., 4,143-158 (1977)].Arabinogalactan protein is the part of gum arabic (being the gluey exudate of a kind of babul (Acacia senegal)), often is taken as emulsifying agent, prevents that crystallizing agent and flavour agent from adding in the food.In plant (Henbane (Nicotiana alata), spend single leaf tobacco (Nicotianaplumbaginafolia) and European pear (Pyrus communis) in vain), isolate the method for AGP gene, United States Patent (USP) 5 in people such as Chen application, disclosure is arranged in 646,029.Going through of relevant arabinogalactan protein, please refer to " proteglycan in the vegetable cell and related compound " (Proteglycans and relatedcompounds in plant cells) of E.A.Nothnagel, Int.Rev.cytology, 174,195-291,1997).

Here the document of mentioning in disclosed and this specification sheets at this in conjunction with as a reference.

The invention summary

A first aspect of the present invention provides the arabinogalactan composition of a kind of separation from the purifying of the Radix Astragali (Astragalusmembranaceus), particularly its root.

A second aspect of the present invention provides a kind of arabinogalactan protein composition, having molecular-weight average is 100 kilodaltons at least, and it is an arabinogalactan protein composition isolating from the arabinogalactan composition of the purifying of first aspect present invention.Arabinogalactan protein composition according to purifying of the present invention, comprise protein with 5%, with the total amino acid content is that 20% of the described proteinic aminoacids content of benchmark is an oxyproline, and has sugar composition, pectinose wherein: the ratio at least 2: 1 of semi-lactosi, and comprise the pectinose of molar percentage 45% to 75%; The rhamnosyl of molar percentage 2% to 4%; The galactosonic acid of molar percentage 4% to 6%; The semi-lactosi of molar percentage 8% to 25%; Glucose with molar percentage 5% to 25%

But a third aspect of the present invention provides a kind of arabogalactan aqua of injection for intravenous, the arabinogalactan composition of purifying that comprises a kind of first aspect present invention of pharmacy effective dose, or the adjuvant solution of the arabinogalactan protein composition of second aspect present invention and a kind of injection for intravenous.

A fourth aspect of the present invention provides a kind of purifying arabinogalactan composition that uses first aspect present invention, or the arabinogalactan protein composition of second aspect present invention is treated the method (hemopoietic for example of mammalian diseases, induce megakaryocyte proliferation and maturation, stimulate IL-1 β, IL-6, TNF-α, IFN-γ, GM-CSF, or the generation of G-CSF, stimulate the generation or the activation of neutrophils, the treatment neutropenia, anaemia, or thrombocytopenia, quicken because of exposing (exposing to the open air for example to the open air because of accident or non-therapeutic, and curative exposing to the open air) in cytotoxic substance, or the recovery situation behind the radiation irradiation, the treatment emaciation, vomiting, or drug withdrawal syndrome, improve biological respinse or the protection hepatitis B patient liver cell), be included in this Mammals of receiving treatment and inject the arabinogalactan composition of purifying of the first aspect present invention of pharmacy effective dose on one's body, or the arabinogalactan protein composition of second aspect present invention, but the arabogalactan prescription of a kind of injection for intravenous of third aspect present invention particularly; Perhaps share with at least a other medicaments medicament of hemopoietic (for example can).

A fifth aspect of the present invention provides the preparation method, be used to prepare the Arabic galactan composition of the purifying of first aspect present invention, with the arabinogalactan protein composition of second aspect present invention, but and the arabogalactan aqua of a kind of injection for intravenous of third aspect present invention.

Accompanying drawing is briefly described

After Fig. 1 illustrates the human cancer patient and accepts chemotherapy, there is not further curer and with the arabinogalactan composition treatment of purifying of the present invention or with G-CSF treatment, the situation that its treatment back white cell mean value changes with fate.

After Fig. 2 illustrates the human cancer patient and accepts chemotherapy, there is not further curer and with the arabinogalactan composition of purifying of the present invention or with after the G-CSF treatment, total treatment back symptom value is with the situation of fate variation.

After Fig. 3 illustrates the human cancer patient and accepts chemotherapy, there is not further curer and with the arabinogalactan composition of purifying of the present invention or with after the G-CSF treatment, Karnofsky performance Index (Karnofsky performance Index) numerical value.

Detailed Description Of The Invention

Definition

" arabinogalactan protein " or " AGP " can use β-glucosyl group Ya Lifu (Yariv) reagent precipitation usually, and the protein of high glycosylation, carbohydrate accounts at least 50% of molecular weight, and main carbohydrate to form be arabinose and galactolipin, and this arabinose residue mainly is positioned at end. For arabinogalactan protein, its usually and monoclonal antibody MAC207 carry out specific reaction.

" arabinogalactan protein composition " refers to a kind of composition, at least comprise at least 70% of above-mentioned composition weight, particularly at least 80%, especially at least 90% arabinogalactan protein and relevant arabogalactan and other polysaccharide.

" arabinogalactan composition of purifying " is a kind of arabinogalactan protein (as above-mentioned) and relevant arabogalactan and composition of other polysaccharide of comprising.

" mammal " comprises human and inhuman mammal, such as pet (cat, dog etc.) and domestic animal (ox, horse, sheep, goat, pig etc.).

" disease " comprises any unsound situation of a kind of animal, comprise because of the unhealthy situation of medical treatment due to (as side effect), for example a kind of morbid state, the effect of enriching blood are the tool curative effects, particularly comprise those morbid states of " pharmacology and purposes " of the present invention part record.

" pharmaceutically useful adjuvant " is meant a kind of to the effective adjuvant of preparation pharmaceutical compositions, generally is safe, nontoxic and necessary, and comprises the adjuvant that can be used for animal doctor's medication and human medication.This class adjuvant can be solid, liquid, semisolid or aerosol composition forms and gas form.

" pharmacy effective dose " is meant a kind of dose, and with treatment during disease, this dose is enough to treat effectively this disease on being applied to the animal health.

" treatment " of disease comprises that pre-tetrandra root infects but the unlikely morbidity of animal (prophylactic treatment) that do not occur as yet being critically ill, suppress disease (slow down or suppress advancing of disease), symptom or the side effect (comprising relaxes subtracts therapy) of alleviating this disease is provided and alleviates this disease (disease is alleviated).

The arabinogalactan composition of purifying

The definition of the arabinogalactan composition of purifying of the present invention as mentioned above, it separates from the Radix Astragali (Astragalus membranaceus) (particularly root), better be from the root portion of Radix Astagali or Radix Astragali (Astragalus membranaceus Bge.Var.mongholicus (Bge) Hsiao or A.membranaceus (Fisch) Bge.) from; Preferably be grown in the Radix Astragali plant roots of inner mongolia or Shanxi Province, particularly the former; And plant root is to be selected from the Radix Astragali in 2 years of growth preferably.Have typical sugar composition (the trimethyl silicane radical derivative of analyzing the methanol extract of described composition with GLC is measured), contain about 5-15mole% (molar percentage), particularly 10% pectinose (Ara); Below 1.5%, the rhamnosyl below 1% particularly; Content reaches about 4% GalA (galactosonic acid); About Gal of 3% to 7% (semi-lactosi); With about glucose of 70% to 90% (Glc); Pectinose wherein: the ratio of semi-lactosi was not less than 1.5: 1; Particularly be not less than 1.75: 1; Especially be not less than 3: 1; Ash content is not higher than 2% (weight percent); Heavy metal content is not higher than 10ppm; Hydroxyproline content is not higher than 0.1%, particularly is not higher than 0.05%.Contain any intracellular toxin (testing [Seikagaku corporation, Tokyo, Japan]) hardly according to the intracellular toxin that the explanation of preparation merchant handbook is carried out, content is lower than the 1.0EU/ milligram, particularly be lower than the 0.8EU/ milligram, especially be lower than the 0.5EU/ milligram, preferably be lower than the 0.3EU/ milligram); Solubleness is 20 mg/ml at least in the water, and pH value of water solution is between about 4.5 to 6.5; Weight-average molecular weight is between between 20 to 60 kilodaltons, particularly between between 25 to 40 kilodaltons, especially between between 27 to 35 kilodaltons.For simplicity, all use " PAGC " to represent this material below.

The arabinogalactan composition of preparation purifying

The arabinogalactan composition of purifying is to have or this class of alkali metal-free salt (particularly potassium primary phosphate or SODIUM PHOSPHATE, MONOBASIC) is carried in the presence of the thing (co-extract) altogether, (temperature generally is not less than 80 ℃ to all hot water, particularly be not less than 90 ℃, particularly about 100 ℃), extracting root after the Radix Astragali (Astragalus membranaceus) dries under this temperature (generally is down with the section of exsiccant root with cut apart in aseptic, prune the exsiccant root, wash by rubbing with the hands with surpassing drainage, after using sterile solution flushing again such as 70% alcohol, root is thinly sliced, and it is air-dry under sterilising conditions, to call this preparation in the following text is " infuse section ") for some time, under a temperature, and optionally repeat extraction step many times, allow the stripping (generally be at about 100 ℃, extract each 3 hours three times) of can trying one's best of root arabinogalactan protein and relevant polysaccharide.All steps of carrying out behind preparation root fragment all are to use sterile equipment and reagent to operate under gnotobasis.The hot water extract is concentrated (in 60-70 ℃ of following vacuum concentration to about 1 liter/kilogram dried roots), use lower alcohols (for example alcohol, ultimate density is 70% under the room temperature) precipitation then.Clean the throw out (generally be with 95% alcohol wash three times) of these lower alcohols again with the alcohols of even lower level, and handle (generally being the 18-20% weight/volume) with the proper concn products for further that suspends in water.Then water-fast material in the suspension is removed, for example added precipitation and remove by lower aliphatic alcohols (about 35% ethanol).Then, the supernatant liquor of low fat alcohol throw out (for example 35% alcohol precipitation thing) is made further precipitation with the lower aliphatic alcohols of high density again, the alcohol of 40%-80% for example, the alcohol of 60-70% particularly will include arabinogalactan protein and be precipitated out in conjunction with the crude product of the arabinogalactan composition of polysaccharide.To precipitate soluble in waterly, and dry (spraying drying is avoided superheated) is with the crude product of isolating arabinogalactan composition again.The said composition crude product generally is a pale yellow powder, water soluble, and concentration is 100 mg/ml at least, at least 200 mg/ml particularly, because of the dry weight of drying loss is less than about 15%, and endotoxin content is lower than 0.5, particularly is lower than the 0.3EU/ milligram.

For regulate Radix Astragali coarse raw materials batch and batch between variation, in technological process, can mix, to keep the consistence of end product raw material, " infuse section " and intermediate product.

Be further purified the crude product of arabinogalactan composition with the ion-exchange chromatography chromatography.It is water-soluble, or the solution behind the resolution of precipitate is adjusted to proper concn (general about 2%), carry out ultra-filtration then, get rid of low molecular weight part, with reducing liquor capacity (for example filtering) with getting rid of the ultra-filtration system (5K MWCOUF system) of molecular weight under 5 kilodaltons.Cationic exchange tubing string (the SP sepharose cationic exchange tubing string for example of again supernatant liquor after the ultra-filtration being flowed through, 20mM acetyl sodium (NaOAc) damping fluid balance with pH 5.20), again elutriant is filled in the anionresin tubing string (for example Q sepharose anionresin tubing string, with same NaOAc damping fluid balance).Elutriant can directly be brought the arabinogalactan protein composition of preparation second aspect present invention from the anionresin tubing string, and but concentrate drying forms a kind of intermediate product that is fit to be used for preparing the arabinogalactan protein composition, or directly is used for preparing a kind of arabinogalactan composition of purifying.When the arabinogalactan composition of preparation purifying, the available suitable micro-filtration film (for example filter membrane of 0.1 μ m) that can filter bacterium carries out ultra-filtration, removes salt and reduces liquor capacity (for example using 8K MWCO UF system).(under 50-60 ℃, 20-26%), use lower aliphatic alcohols (ethanol of about 80-90%) to precipitate then the supernatant concentration after the ultra-filtration.Further washing and precipitating (for example use raw spirit, cleans three times), and drying (60-70 ℃ vacuum oven) can obtain the arabinogalactan composition of purifying.

The arabinogalactan protein composition

The definition of the arabinogalactan protein composition of purifying of the present invention is as indicated above, it separates from the Radix Astragali (Astragalus membranaceus) (particularly its root), and better be from from the root portion of Radix Astagali or Radix Astragali (Astragalus membranaceusBge.Var.mongholicus (Bge) Hsiao or A.membranaceus (Fisch) Bge.) from; And preferably be grown in the Radix Astragali (A.membranaceus) plant roots of the Inner Mongol or Chinese Shanxi Province, particularly the former; And plant root is to be selected from the Radix Astragali in 2 years of growth preferably.It has typical sugar composition, comprises about 45% to 75% (molar percentage), particularly about 50% to 70% pectinose; 2% to 4% rhamnosyl; About galactosonic acid of 4% to 6%; About 8% to 25%, 10% to 20% semi-lactosi particularly; With about 5% to 25% glucose; Pectinose wherein: the ratio of semi-lactosi was not less than 2: 1; Particularly be not less than 3: 1; Especially be not less than 4: 1; Ash content is not higher than 2% (weight percent); Heavy metal content is not higher than 10ppm; And hydroxyproline content is not higher than 0.2%, particularly is not higher than 0.3%.Contain any intracellular toxin (with above-mentioned definition) hardly; Solubleness is 20 mg/ml at least in the water, and pH value of water solution is between about 4.5 to 6.5; Molecular-weight average is not less than 100 kilodaltons, especially between between 150 to 350 kilodaltons.Contain about 95% carbohydrate (comprise can with the carbohydrate of arabinogalactan protein core saccharification) and about 5% protein.What account for total amino acid content 20% is that oxyproline is the characteristics of arabinogalactan protein.For simplicity, all use " AGPC " to represent this material below.

Preparation arabinogalactan protein composition

Arabinogalactan protein composition of the present invention is by 100K MWCO ultra-filtration system, and the overhead product (or with the solid-state intermediate behind the overhead product concentrate drying) of above-mentioned arabinogalactan composition or ion-exchange tubing string purification step gained is further purified and makes.The overhead product of gained in the anionresin tubing string is directly to be added in the 100K MWCO ultra-filtration system in above-mentioned " preparation arabinogalactan protein composition ".The filtrate of retaining in this 100K MWCO ultra-filtration system is further concentrated, and precipitate with rudimentary Fatty Alcohol(C12-C14 and C12-C18), perhaps further clean, drying, all similar with the arabinogalactan composition of preparation purifying, can get the arabinogalactan protein composition.

Pharmacology and purposes

The favourable activity of the arabinogalactan protein composition of the arabinogalactan composition of the purifying of first aspect present invention or second aspect present invention obtains to confirm in several tests, and following purposes is arranged.

Treatment patients undergoing chemotherapy neutropenia

1. from activatory human peripheral blood mononuclear cells (PBMC), prepare cytokine

Immunity and hemopoietic system need just can be activated by the interaction of several cytokines, have described the external evoked production of cytokines of PAGC among the embodiment 3.It is significantly that PAGC causes, the release of IL-1 β, the IL-6 that PHA activated human peripheral blood mononuclear cells is relevant with dosage, TNF-α, IFN-γ, GM-CSF and G-CSF.These three kinds of cytokines of known IL-6, GM-CSF and G-CSF can influence the generation and/or the activation of neutrophils in vitro and in vivo.IL-6 itself or with other cytokines can stimulate the megakaryocytic maturation in the marrow, can recover hematoblastic numerical value in mouse and the non-human primate's peripheral blood.These data show that PAGC can stimulate the bone marrow depression patient to produce neutrophils and recovers platelet count by producing the cytokine relevant with hemopoietic function.

2. the recovery of GM-CFC stem cell in the mouse body that Fluracil is handled

PAGC obtains measuring in the model that short-term, mouse, the intravital hemopoietic stem cell in earlier external back recover as described in example 5 above.Fluracil is widely used for removing the colony forming unit (CFU-C) that can produce in stem cell in the mouse bone marrow cells and the cell cultures.Because the stem cell that is not activated can not be subjected to the influence of Fluracil, therefore through the mouse after this processing, can be along known, expected time curve recovery [A.M.Yeager etc., " 5 FU 5 fluorouracil is to the influence of hematopoiesis: mouse megalokaryocyte-CFC, granulocyte-macrophage-CFC, the research of peripheral blood cells level " (The effects of 5-fluorouracil onhematopoiesis:studies of murine megakaryocyte-CFC, granulocyte-macrophage-CFC, and peripheral blood cell levels), " experimental hematology " (Exp.Hematol.), 11,944-952 (1983)].As can be known in dose-dependent method, handled the back the 4th day by the form among the embodiment 5 through Fluracil, along with the increase of PAGC dosage, the also and then rising of numerical quantities of GM-CFC in every Thigh bone.When using PAGC dosage and be 200 mg/kg, the GM-CFC that every mouse on average increases is 3.3 times (p＜0.01) of control group; If the PAGC with 100 mg/kg handles, the GM-CFC that every mouse on average increases is 1.8 times (p＜0.01) of control group mice; If the PAGC with 50 mg/kg handles, GM-CFC and control group difference that every mouse on average increases are little.These data show that PAGC can promote the recovery of bone marrow stem cell (GM-CFC) in the myelosuppressive mouse body of Fluracil inductive, and this is soluble at least in one aspect to be to promote due to the leukocytic increase of week of myelosuppressive mouse inside and outside because of PAGC.

3. accept the leukocytic recovery of week of sublethal dose radiating mouse inside and outside

Radiation irradiation amount with sublethal dose is shone BALB/c mouse, and then according to the method for embodiment 6 from the PAGC of subcutaneous injection normal saline solution or various dosage in the mouse body.The mouse of accepting radiation irradiation after radiation the 14th day, leukocytic numerical value can drop to 12% of normal mouse value.Than the mouse of only injecting normal saline solution, can increase leukocytic sum in the mouse body with the PAGC treatment.With white cell sum in the mouse body of the PAGC of 100 or 300 mg/kg treatment, comparable mouse of only injecting normal saline solution shifts to an earlier date the 80% (normal value: 6000-10000 white cell/microliters of blood) that returned to the normal mice number in 7-9 days.In the animal that PAGC handles, after about 22-23 days, it is normal that white blood cell count can recover; And the mouse numerical value at this moment of only injecting normal saline solution is 46% of normal value.In addition, also available peripheral blood film dyes and measures different white cell values.From these information, can calculate neutrophils and lymphocytic absolute value.In this model, to compare with the control group of the mouse of only injecting normal saline solution, the absolute value with PAGC treatment can increasing neutrophils number also can increase lymphocyte number purpose absolute value simultaneously.

4. the recovery of white count in the patients undergoing chemotherapy body

All II phase clinical experiments all are to finish in the People's Republic of China (PRC).White blood cell count is lower than 3.0 * 10 when having analyzed a small set of participation experiment in the test ⁹White cell/liter patient's data: wherein 168 patients accept the PAGC treatment, and 59 patients accept the G-CSF treatment, and 23 patients do not accept any treatment.The treatment flow process is described in detail in embodiment 9.As shown in Figure 1, PAGC treatment can stably increase accepts the intravital white blood cell count of patient after the chemotherapy, and the situation of this increase can continue until the 14th day.Though use G-CSF, can the speed that white cell is increased is very fast, reached maximum value by the 7th day, but can't continue, the intravital white blood cell count of patient promptly begins to descend after 7 days.These two treatment groups all can make white blood cell count return to normally than not treatment group is more Zao.Compare with the control group of not accepting any treatment, the white blood cell count of PAGC treatment group can produce notable difference at the 10th day and the 14th day; But PAGC group and the white blood cell count no significant difference of G-CFC group in the time of the 14th day.The above results shows uses PAGC as the treatment adjuvant to the lung cancer of accepting chemotherapy, stomach cancer and patients with mastocarcinoma, not only safety but also can be accepted by the patient.The PAGC treatment can recover the intravital white blood cell count of patients undergoing chemotherapy.And when treating with PAGC clinically, do not find any significant side effects yet.

Treatment patients undergoing chemotherapy thrombocytopenia

1. accept the recovery of all platelet cell numbers in radiating mouse inside and outside of sublethal dose

In the time of the 0th day, shine BALB/c mouse with the radiation irradiation amount of sublethal dose, and then treat according to the step of embodiment 6 PAGC with normal saline solution or various dosage.Accept the radiating mouse after radiation the 10th day, leukocytic numerical value can drop to 7% of normal mouse.The PAGC treatment mouse of subcutaneous injection 100 or 300 mg/kg can obviously be improved mouse inside and outside week platelet cell number.Early 5-6 days induced platelet cell numbers of control group that injection PAGC organizes comparable injecting normal saline return to 80% (normal value: 8-12 * 10 of normal mouse ⁵Thrombocyte/microlitre blood).The mouse of handling through PAGC, behind radiation irradiation about 24-25 days, it is normal that platelet count can recover; And the mouse of only injecting normal saline solution, numerical value at this moment only is 72% of normal value.These results show that PAGC is a kind of medicament that can effectively promote the thrombocyte growth, reduces quite effective to the treatment number of platelets.In 20 days by a definite date treatment, the mouse for the treatment of with the AGPC of 100 and 250 mg/kg can both recover red blood corpuscle and number of platelets better than the time point of control group behind all radiation irradiations with the same manner; Show that PAGC also has identical curative effect in this model system.

2. the propagation of bone marrow megakaryocyte and maturation

As previously mentioned, PAGC can add the platelet count of the interior peripheral blood of inductive bone marrow depression animal body in the quick-recovery radiation irradiation animal model, and prompting PAGC can stimulate the propagation and/or the maturation of bone marrow megakaryocyte.Can use this possibility of embodiment 4 described external liquid culture systematic studyes.By the dose titration curve as can be known, PAGC dosage best in this model is the 100-200 mcg/ml, and ED ₅₀It is the 30-40 mcg/ml.This independent use PAGC that studies show that just can promote the propagation and/or the maturation of external bone marrow megakaryocyte.In addition, PAGC also can with the IL-3 that is lower than optimal dose (50pg/ml) drug combination, increase the level of acetylcholinesterase (AchE) with the relevant mode of dosage.The AchE content that obtains in the cell culture that the IL-3 of the PAGC of 200 mcg/ml and 50pg/ml handled is suitable with the AchE content that IL-3 with optimal dose handles in the culture later.These data show that PAGC can improve the reactivity of bone marrow megakaryocyte to the IL-3 of suitable low dosage, or IL-3 can increase the reactivity of bone marrow megakaryocyte to PAGC.Because chemotherapy or radiation irradiation can destroy many cells and tissue comprises the cell that produces cytokine, the content of therefore accepting the interior endogenous cytokine of patient's body of this treatment can be lower.So low cytokine content can't the hematopoiesis support system normal function, thereby the hematopoietic cell that can't produce enough numbers is used for recovering downtrod marrow function.Therefore, PAGC may be the good medicine of this disease of treatment.Even this result of study shows that under the insufficient situation of endogenous cytokine PAGC can promote that also the propagation of bone marrow megakaryocyte and/or maturation reach normal level.As implied above, PAGC can stimulate the periphery number of platelets to recover, and this effect seemingly predisposition lead the propagation and/or ripe generation of bone marrow megakaryocyte.This data show that PAGC is effective because of the number of platelets due to the bone marrow depression reduces disease to treatment.

3. platelet cell number after the increase chemotherapy

II phase clinical experiment is as described in the embodiment 9.White blood cell count is lower than 4.0 * 10 when having analyzed a small set of participation experiment in the test ⁹White count/liter and platelet count be lower than 90 * 10 ⁹/ liter patient's data: wherein 54 patients accept the PAGC treatment.These patients' platelet count was increased at the 7th day and is higher than 100 * 10 ⁹/ liter, and continue to increase by the 14th day, shown in the tabulation of embodiment 9.These data show that PAGC can increase the intravital platelet count of these patients undergoing chemotherapies.

Improve cancer patients's quality of the life

The II clinical trial phase is described in embodiment 9 to some extent.As described in embodiment 9, all participate in the basis of patient's quality of the life of test as assessment with the numerical value that improves chemotherapy symptom and Karnofsky performance Index (Karnofsky performance Index).Record and calculate treatment during by the caused symptom of chemotherapy, comprise tired out and burnout, discomfort, perspiration, short of breath and lack appetite.As shown in Figure 2,, show that the curative effect of PAGC group is the fastest along with the carrying out of treatment, this can descend and return to positive constant (counting=0) sooner by SI than the G-CSF group find out.In addition, compared with the G-CSF group, PAGC organizes during the 10th day to the 14th day, also shows more obvious improvement the (p＜0.01) on the statistical figure.Yet the G-CSF group is compared with control group (not accepting any treatment), during whole 14 days monitoring, does not all have any obvious statistical difference.These data show that PAGC can improve the symptom of patients undergoing chemotherapy, and as if about this point, G-CSF is without any effectiveness.Karnofsky performance Index (Karnofsky performance Index) number before the analysis treatment is lower than 70 a small set of patient in the various improvement situations for the treatment of the back SI of process.Compared with G-CSF group and the control group of not accepting any treatment, the PAGC group can significantly be improved symptom, and as shown in Figure 3, statistical difference is respectively p＜0.01 and p＜0.0001.The index of patient in most of PAGC group is all than other two groups of height.

The interior neutropenia of cancer patients's body of chemotherapy is accepted in prevention

In the paragraph, known PAGC can treat chemotherapy inductive neutropenia effectively in front.These Notes of Key Datas PAGC may be the same effective to prevention and neutrophils minimizing, planned at present in the People's Republic of China (PRC), to carry out clinical trial.

The interior neutropenia of cancer patients's body of radiation irradiation is accepted in treatment

The pharmacological datum of front shows that also PAGC can recover radiation irradiation inductive neutropenia effectively.Begun the Chinese people at present and domesticly carried out clinical trial, in order to understand the situation of PAGC treatment non--neutropenia that Ha Jin lymphomas cancer patients causes after accepting radiation irradiation.

The cancer patients's of chemotherapy anaemia phenomenon is accepted in treatment

Radiation irradiation amount with sublethal dose (4.25Gy) is shone BALB/c mouse, and then treats according to the step of embodiment 6 PAGC with normal saline solution or various dosage.Accept the radiating mouse behind radiation irradiation the 17th day, the numerical value of RBC can acutely drop to 55% of normal mouse RBC value.The mouse of handling through PAGC can obtain quite high RBC number during at the 17th day, and the numerical value when only the mouse of injecting normal saline is this is still very low.The PAGC treatment mouse of subcutaneous injection 100 or 300 mg/kg, comparable 80% (normal value: 8.5-11 * 10 that only are increased to normal mouse with the control group 4-6 angel morning RBC number of physiological saline treatment ⁶The RBC/ microliters of blood).Mouse through PAGC handles can recover normal about RBC numeration in 20-22 days; And the mouse numerical value at this moment of only injecting normal saline solution is 65% of normal value.Therefore these results show that PAGC is a kind of red blood corpuscle development of stem cells and/or mobile medicament of quite effectively promoting, the anaemia that treatment radiotherapy or chemotherapy are caused is quite effective.In 20 days by a definite date the course of treatment, the mouse for the treatment of with the AGPC of 100 and 250 mg/kg all can recover red blood corpuscle and number of platelets when testing compared with the time point of control group behind all radiation irradiations better with the same manner; Show that PAGC also has identical curative effect in this model system.

II clinical trial phase purpose among the embodiment 9 is the leukocytic recovery situation of research patients undergoing chemotherapy, so condition is to analyze experiment according to white blood cell count.To study the clinical trial of the anaemia that causes with PAGC treatment anaemia and chemotherapy at present at PRC (People's Republic of China (PRC)) with plan.

To the independent mobile peripheral hematopoietic stem cells of the patient who accepts autologous peripheral blood stemcell transplant or with The G-CSF combined treatment

CD34 antigen is present in hemopoietic stem cell and the stem cell, comprises BFU-E (erythrocytic burst-forming unit), GM-CFC (GM-CFC) and the CFU-Mix (mixed colony forming unit) that can form cell colony.CD34 ⁺The flow cytometry analysis result of cell provides a kind of mensuration transplantation group compound (graftcomposition) best mode.As far back as 1975, just proposed to have hemopoietic stem cell (PBPC) in the human peripheral blood system.These peripheral hematopoietic stem cells only account for the sub-fraction of total cell count, most stem cell all the position marrow in.In the time of 1976, at first the someone proposed between decubation after the chemotherapy, and the hemopoietic stem cell number in the human peripheral blood system can rise.Recently, more the someone reports population of cells's stimulating factor (colony stimulatingfactor) G-CSF and GM-CSF, can directly improve the numerical value of hemopoietic stem cell.Unite during chemotherapy and use population of cells's stimulating factor than the numerical value of only treating the more effective increase hemopoietic stem cell of energy with a kind of method.At present, G-CSF is used to the mobile hemopoietic stem cell in body and heteroplastic transplantation the mankind.Transplanting hemopoietic stem cell carries out faster than traditional bone marrow transplantation.If unite use, can treat the hemopoietic stem cell number that produces by raising G-CSF by a larger margin with chemotherapy or other cytokines.Use PAGC separately and/or unite use with G-CSF, the ability of inducing hemopoietic stem cell to move is set forth to some extent at embodiment 7.Shown in the table results among the embodiment 7, PAGC can improve round-robin GM-CFC and measure about 6 times.In addition, PAGC and G-CSF unite when using, and can improve round-robin GM-CFC and measure about 83 times.39 times of the amount that this increasing amount is increased when also being higher than independent use G-CSF.The result shows that PAGC can improve the amount of round-robin GM-CFC, or with the collaborative amount that increases the GM-CFC of normal mouse body-internal-circulation of G-CSF.As shown in Table, PAGC also can increase about 9.5 times of the amount of round-robin BFU-E.4.5 times of the amount that this increasing amount is increased when being higher than independent use G-CSF.The mouse of handling with cyclic phosphoric acid amine in similar experiment is compared with the mouse of not accepting the PAGC treatment, CD34 ⁺Lin ^-The numerical value of cell can increase.PAGC can bring into play good effect aspect the increase hemopoietic stem cell number with the G-CSF synergy as the treatment preparation.This class Synergistic treatment can reduce in donor and obtains the required separating plasma metathetical number of times of hemopoietic stem cell cell, thereby reduces the expense of treatment.In addition, the Synergistic treatment of this class is for independent use G-CSF being reacted not good or not wanting to use the patient of chemotherapy also helpful.Same, AGPC is also very effective to transplanting.

Promote to be subjected to cytotoxic agent or radiation irradiation effect individuality afterwards to restore

Above result shows, when animal or patient are exposed in radiation irradiation or the cytotoxic factor modestly, PAGC is compared with more not promoting to be exposed to recovery process after radiation irradiation or the cytotoxic factor (as exposing to the open air of unexpected or non-therapeutic, also having curative exposing to the open air) with this treatment.

The treatment emaciation

The modal a kind of side effect of chemotherapy and radiation therapy causes patient's appetite to reduce exactly and loses weight.Embodiment 8 shows that PAGC can make the weight increase of the mouse after chemotherapeutic agents cyclic phosphoric acid amine and Fluracil processing.With the mouse after the processing of cyclic phosphoric acid amine, through the mouse that PAGC handled, losing weight is inhibited, and can the original body weight of faster recovery compared with independent.But these differences are also not obvious on statistics.

As above-mentioned, PAGC can obviously improve II clinical trial phase patient's quality of the life.Wherein a parameter of Ce Lianging is an appetite stimulator.Show jointly that with the mouse model result of study PAGC can help improve patient's emaciation, this is a kind of material wastage and the malnutrition that causes by such as this chronic disease of cancer or cancer therapy.

The cancer patients who accepts the inhibition bone marrow therapy with the G-CSF Synergistic treatment promotes the white blood of neutrophilia Ball recovers and reduction G-CSF consumption

But the discussion of front shows the generation of PAGC stimulating cytokine, particularly the G-CSF of human peripheral blood mononuclear cells generation; And can promote to recover, and make the cancer patients of II clinical trial phase accept after the chemotherapy that the white cell value returns to normally in the body because of GM-CFC in the radiogenic bone marrow depression animal model and periphery white blood cell count purpose.These results show that PAGC can come indirect action in hemopoietic system by generating the multiple endogenous cell factor, and these cytokines can act synergistically each other and recover the neutrophils number.Simultaneously, PAGC also can act synergistically with IL-3 and promotes the propagation/maturation of bone marrow megakaryocyte as mentioned above.

In addition, use not only safety of PAGC, and the patient can accept in the clinical trial.And for example bone pain, myalgia, headache, tired out, nausea,vomiting,diarrhea etc. of serious concurrent side effect when using G-CSF, and when using PAGC, do not find.The synergy that discussed these results and front shows that PAGC can unite with G-CSF makes the consumption that is used for reducing exogen G-CSF, and promotes to accept the recovery of neutrophils number in cancer patients's body that marrow presses down treatment.

In the treatment of high dosage cytotoxicity with after body or the transplanting of allosome hemocytoblast, unite use The G-CSF treatment recovers in order to promote neutrophils

At present treat many cancer patientss, for example breast cancer, lymphatic cancer and multiple myeloma patient with high dose chemotherapy and/or radiotherapy and supportive hemocytoblast (BPC) transplanting.Transplant BPC and can add the quick-recovery hemopoietic function.Usually can be used G-CSF after transplanting BPC, in order to adding the numerical value of quick-recovery neutrophils, prevention reduces the fever that causes because of the neutrophils due to infecting, and shortens hospital stay and reduce antibiotic dosage.Shown in embodiment 9II clinical trial phase, PAGC can recover chemotherapy cancer patients's white blood cell count; The pharmacology effectiveness that these results and leading portion literal are summed up shows to unite uses PAGC and G-CSF, very effective to the numerical value of transplanting BPC after-acceleration recovery neutrophils.

Improve the biological reactivity and the protection liver cell of hepatitis B carriers

Cytokine is infectd in antagonism virus and has been played the part of considerable role.Cytokine is to be produced by the cell relevant with immunity system.For example hugely have a liking for cell; CD4+ and CD8+T lymphocyte.They at virus infection, play direct effect to individuality in the recovery as hepatitis B virus (HBV).From the animal model to human research, can know clearly that cell immune response may play some effects to solving on the HBV infection utmost point disease pathology.In acute HBV infected, the immune response of intensive polyclonal cellular was vital.CD4+T lymphocyte generation IL-2 and INF-γ indicate the release of the 1st cytokines; And induce and keep cellular immunity by IL-2 and INF-γ, this point is considerable [C.A.Biron for the antagonism virus infection, " cytokine is to reactive generation of virus infection and adjusting " (cytokines in the generation of immune response to, and regulation of, virus infection "; Cur.Opin.Immunol.), 6,530-538 (1994)].The cytokine that CD4+ and CD8+ cell discharge has also been played the part of considerable role for duplicating of downward adjusting (downregulation) HBV.If occurred defective in the acute reaction, will become chronic HBV infection.These results show the reaction of enhancing the 1st type or produce suitable cytokine in the liver part may be to treating chronic HBV infection very effective [M.J.Koziel, Sem.Liver Disease, 19 (2), 157-161 (1999)].PAGC extracts from traditional Chinese medicinal plant (Astragalus membranaceus Var.mongholicus (AM)), and this kind of plant is used to immune stimulatory and hemopoietic system already.It is widely used for treating various symptoms, and this class patient's symptom is similar to the bone marrow depression (except that neutrophils reduces, also having thrombopenia and anaemia) of chemotherapy or radiotherapy-induced.In addition, known AM stimulates the cell proliferation of mouse pancreas with external dose-dependent mode; And can improve natural killer cell (Naturalkiller, NK) activity in the pancreas cell in the animal body of having inoculated the S-180 sarcoma cell; Can also improve tool Cytotoxic T-lymphocyte (cytotoxicTlymphocytes, CTL) activity.The cytokine may command body inner virus of CTL preparation infects, and the INF-γ and the INF-α of viral narrow spectrum CTL preparation, can increase the ability that CTL removes infective virus [L.G.Guidotti etc., " transgenic mice inner cell toxic T lymphocyte suppresses the expression of hepatitis B virus with non-bacteriolyze mechanism " (Cytotoxic Tlymphocytes inhibit hepatitis B virus gene expression by a noncytolyticmechanism in transgenic mice), Proc.Natl.Acad.Sci.USA, 91,764-3768 (1994)].In addition, as mentioned above, PAGC can be after the PHA activation, and the preparation of stimulating human peripheral blood lymphocytes generates IL-1 β, IL-6, TNF-α, TNF-γ, GM-CSF and G-CSF.These studies show that PAGC can come the remote effect immunity system by the generation of the regulating cell factor.The crude extract that is obtained by AM has been used to treat chronic hepatitis patient, with reducing IgG amount too high in the body, and reduction ALT value, improve patient's immunity and liver function [Y.Liu, " oral liquid that extracts from the Radix Astragali is to the curative effect of 70 chronic hepatitis B patients " " therapeutic effect of oral solution from Astragalus in thetreatment of 70 chronic hepatitis B patients " Jiang Su Chung Yao, 15 (12), 38 (1994)].In addition, the fraction of known AM has the ability that can increase immunologic function as described above.These results show that PAGC can be used to improve and modify the biological reactivity and the protection liver cell of hepatitis B carriers.

The E vaccine adjuvant of hepatitis B patient

By the polysaccharide that Polyporus umbellatus (Polyporus umbellatus) is prepared from, the adjuvant that has been used as hepatitis B vaccine is treated chronic hepatitis B.Known for conversion hepatitis B e antigen (HBeAg) be seronegativity and eliminate hepatitis B virus DNA (HBV-DNA) have gratifying effect [S.M.Wu etc., " Polyporus polysaccharide and hepatitis B vaccine drug combination are used for the treatment of the treatment of chronic hepatitis B and find " " The therapeutic observation on the combined Polyporuspolysaccharide with hepatitis B vaccine in the treatment of chronichepatitis B " .J.Chin.Infectious Disease, 13 (3), 187-189 (1995); H.Z.Shu etc., " Polyporus polysaccharide and heavy dose of hepatitis B vaccine drug combination are used for the treatment of the treatment of 64 routine chronic hepatitis B patients and find " " The therapeutic observationon the Polyporus polysaccharide with large dose of hepatitis B vaccinein the treatment of 64 cases of chronic hepatitis B patients ", Med.J.NDFSC 6 (4), 211-212 (1996)].The known extract that is made by AM can be turned out cloudy hepatitis B e antigen (HBeAg) and anti-hepatitis B virus cAg (anti HBc), and the hepatitis B virus DNA (HBV-DNA) of removal hepatitis B patient [C.K.Liu etc., " with the clinical and experimental study of Radix Astragali extract treatment chronic hepatitis B " " Clinicaland experimental studies on effects of chronic hepatitis B treated withAstragli composita ", Chung Kuo Chung His I Chieh Ho Tsa Chin, 16 (7), 394-397 (1996); P.L.Chen etc., " use from the polysaccharide of the Radix Astragali and treat 33 routine chronic viral hepatitis B patients " " Polysaccharide from Astragalus in treating 33 casesof chronic active hepatitis B patients ", New Drugs Clin.Remedies.11 (2), 75-76 (1991)].Above-mentioned these results show that PAGC may can be used as the vaccine adjuvant of hepatitis B patient.

The withdrawal symptom that the treatment narcotic produces

The patient begins to give up when using narcotic, generally Withrawal symptom can occur.Traditional chinese medicine (TCM) viewpoint thinks that so-called Withrawal symptom is exactly gas (energy) deficiency.With regard to traditional Chinese physician's observation,, just can quicken medicine and give up process if can strengthen gas.Embodiment 9 is about one of indication of PAGC clinical experiment, can improve the quality of the life of cancer patients after the chemotherapy exactly.Quality of the life is weighed in improvement by the symptom relevant with chemotherapy; These comprise tired and tired out, uncomfortable, emit cold sweat, be short of breath and lack appetite.These symptoms all are typical case's " gas " insufficient a kind of performances, also are typical case's performances of the Withrawal symptom thought of traditional traditional Chinese medical science.Therefore, PAGC also can treat desire and give up the Withrawal symptom of using the anaesthetic patient except " gas " that can strengthen the patient.

Chemotherapy or radiotherapy cancer patients's symptoms of emesis is accepted in prevention and treatment

Chemotherapy and/or the radiotherapy side effect of normal appearance are exactly nausea and vomiting.Though chemotherapy or radiation irradiation technology are improved at present, therefore the symptom that still has quite a high proportion of patient to occur vomitting still need seek the method that can reduce this class side effect.Though the medicine listing (for example 5-hydroxytryptamine receptor antagonist, corticoid and dopamine-receptor antagonist) of existing many energy emesis,, this class medicine itself also has other side effects.The symptom relevant with these medicines be slight headache, constipation, can't fall asleep, the non-autokinetic movement and the sedation of irritated, muscle and tongue.Though the neuropharmacology about vomiting still imperfectly understands at present, but should comprising, the measure of controlling symptoms of emesis fully and taking allow the patient feel Energy and comfort as far as possible, can reduce with providing that the patient is in hospital and the treatment of bed rest time, and then medicine of promotion patient quality of the life or the like.The clinical trial of carrying out within Chinese territory shows that PAGC is useful to patients undergoing chemotherapy, the particularly available quality of making the life better.Researchist's observations and patient's reflection all support PAGC can improve the conclusion of patient's quality of the life, and show in the symptoms of emesis of PAGC after prevention and treatment chemotherapy that obvious effect is arranged.

Reduce the erythropoietin consumption of kidney dialysis patients or substituting as erythropoietin Thing

(epoetin alfa is to check and approve in 1989 to be used for treating because of chronic renal failure to accept the Anemia that the patient of dialysis treatment causes erythropoietin) to EPOGEN.It is a kind of more positive that this medicine can allow the patient cross, more great-hearted life.Before EPOGEN came out, 90% dialysis patients all suffered from anaemia, the patient was often felt tired and general weakness and influence its work capacity.Now, most dialysis patients all can be accepted EPOGEN simultaneously and treats and improve or keep red blood corpuscle level in the body.During clinical trial, the modal side effect that is caused by EPOGEN is hypertension, headache, apoplexy, nausea and thrombus; During general if these side effects, the doctor can advise that the patient reduces the EPOGEN consumption.PAGC studies show that among the embodiment 6, and it can make the mouse inside and outside week number of red blood cells of accepting the sublethal dose radiation irradiation restore.Treat the mouse of process radiation irradiation with same administering mode with the AGPC of 100 and 250 mg/kg dosage, 20 days studies show that by a definite date, all time points behind radiation irradiation are tested, and the red blood corpuscle of test group and platelet count purpose are restored situation and control group more all has improvement; Explanation has identical curative effect with PAGC in this model.In addition, shown in embodiment 7, PAGC can increase the amount of circulation BFU-E, and works in coordination with the amount that increases normal mouse body-internal-circulation BFU-E with G-CSF.In addition, normal mice after PAGC handles and the TER-119 in the peripheral blood of the mouse that cyclic phosphoric acid amine is handled ⁺Cell number significantly increases.Up to the mature erythrocyte stage, TER-119 antigen is expressed at erythrocyte surface from early stage erythroblast.TER-119 ⁺Increasing of the numerical value of cell represents that PAGC can stimulate differentiation, propagation and the maturation of the red blood corpuscle system in the marrow, and impels these cells to enter into peripheral blood.All these results show that PAGC can promote test erythrocytic propagation and maturation in the mouse body.Owing to using not only safety of PAGC in clinical trial, and never finding any pronounced side effects, so PAGC may can be used for reducing the erythropoietin consumption of kidney dialysis patients or as the surrogate of erythropoietin.

Program according to embodiment 6, treat the mouse of process radiation irradiation with same administering mode with the AGPC of 100 and 250 mg/kg dosage, 20 days studies show that by a definite date, all time points behind radiation irradiation are tested, and the red blood corpuscle of test group and platelet count purpose are restored situation and control group more all has improvement; Explanation has identical curative effect with PAGC in this model; And mean that AGPC and PAGC have same curative effect if other PAGC data are discussed in this piece research, therefore various pharmaceutical application and the index with PAGC adapts.

Pharmaceutical formulation and administering mode

In general, the arabinogalactan composition of the composition of the first aspect of the pharmacy effective dose among the present invention---purifying, or the composition of second aspect---arabinogalactan protein composition, the administration of available intravenous injection mode, can be separately or with at least a other medicaments, medicament coupling that particularly can hemopoietic.Pharmacy effective dose will with age of disease, severity of disease, administration individuality and healthy situation, and other factors different.For hemopoietic, comprise and induce megakaryocyte proliferation or maturation, stimulate to generate IL-1 β, IL-6, TNF-α, TNF-γ, IFN-γ, GM-CSF or G-CSF, stimulate generation or effect, the minimizing of treatment neutrophils, anaemia or the platelet cell reduced number disease of neutrophils; Or accelerate the patient and (for example expose to the open air in radiation, the radiation irradiation of unexpected or non-therapeutic, also comprise radiotherapy) or the following recovery afterwards of cytotoxic agent, concerning the people of the general bodily form and body weight, the pharmacy effective dose of the arabinogalactan composition of the composition purifying of first aspect present invention is approximately between 50 to 1000 mg/day, particularly between 100 to 500 mg/day, particularly about 250 mg/day.For the treatment emaciation, feel sick or drug withdrawal syndrome or improve biological reactivity or the liver cell of protection hepatitis B patient, above-mentioned dosage also is pharmacy effective dose.Because the contained Arabinogalactan-Protein content of arabinogalactan protein composition itself of the present invention is higher, estimate that the property of medicine should be stronger, therefore pharmacy effective dose decreases corresponding, for example between the pharmacy effective dose of the arabinogalactan composition of purifying 10% to 50% between.Should be able to need not under the condition of further experiment those skilled in the art,, judge the present composition that should give how many pharmacy effective doses to a specified disease according to this specification sheets and technology own.

In general, the composition of first aspect present invention---arabinogalactan composition of purifying or second aspect present invention composition---arabinogalactan protein composition will be made pharmaceutical dosage form by the administration of intravenous injection mode.Described formulation comprises the composition of first aspect present invention---arabinogalactan composition of purifying or composition of second aspect present invention---arabinogalactan protein composition and the intravenous injection assist agent solution that is fit to.The compound method that is used in intravenous proper adjuvant solution well-known to those having ordinary skill in the art can be with reference to Alfonso AR: " Remington pharmaceutical science " (Remington ' s Pharmaceutical Science), 17th ed., Mack PublishingCompany, Easton, PA, 1985.Intravenous suitable assist agent solution comprises water, normal saline solution, D/W and analogue.

Usually, the composition of first aspect present invention---arabinogalactan composition of purifying or composition of second aspect present invention---arabinogalactan protein composition, to come administration with the intravenous injection mode when using as the hematopoiesis agent, particularly continued several minutes to one hour or intravenous drip mode for more time, for example about 15 minutes.The content of The compounds of this invention is with kind, unitary dose size, adjuvant kind and other well-known factors vary of composition in the described composition.In general, composition finally can comprise the The compounds of this invention of 0.001% to 10% (weight percent), and preferably between 0.01% to 1% (weight percent), all the other are adjuvants.

The composition of first aspect present invention---arabinogalactan composition of purifying or composition of second aspect present invention---arabinogalactan protein composition, optionally can treat the medicament drug combination of disease with at least a other, especially be other medicaments of energy hemopoietic, for example protoheme, thrombopoietin, granulocyte colony stimulating factor (G-CFS), IL-3, or the like.

Embodiment

The present invention is further described by following non-qualitative embodiment.The arabinogalactan composition of all purifying and arabinogalactan protein composition are examined and determine by size exclusion chromatography (size exclusion chromatography).

(size exclusion chromatography) is composed as follows for size exclusion chromatography: Shimadzu HPLC system, SCL-10A central controller, LC-10AD pump, DGU-4A de-aerator, RID-6A RI-detector and SPD-10AV UV detector be equipped with, and with 0.2N sodium-chlor equilibrated GS-701 and GS-620 tubing string (Shodex Asahipak, 7.6 * 500mm).The sample amount of loading is 80 μ g (concentration is the 40 μ l sample solutions of 2mg/ml), and it is 1 ml/min that sample washes out speed.Standard substance with different molecular-weight average comes the preparation standard curve; The molecular weight of sample can be read from typical curve.The weight average molecular weight of sample (Mw=∑ (A _iM _i/ ∑ A _i), number-average molecular weight (Mn=∑ A _i/ ∑ (A _i/ M _I)), and polymolecularity (Mw/Mn) is the standard curve determination of drawing with 2.4 editions Shimadzu SEC software.

Employing gas-liquid phase chromatography (GLC) analysis trimethyl silicane methylglycoside derivative is measured PAGC and the AGPC composition among sugared content and the present invention.In this method, earlier polysaccharide is joined methanolizing in the methyl alcohol of hcl acidifying, then prepare volatile monosaccharide derivatives with trimethyl silicane (TMS) derivatization method.After removing impurity, analyze derivative with the gas-liquid phase chromatography (GLC) that is equipped with DB-1 tubing string (comprising flame-type ionization sensor (FID)).Do interior mark with meso-inositol, perform an analysis with composition sample and measure sugar degree and component.

Measure hydroxyl proline content with colorimetric method.Sample is used hydrochloric acid hydrolysis earlier, and then handles with sodium hypobromite (sodium hydroxide solution of bromine), hydrochloric acid and dimethylamino benzaldehyde.Measure the optical density(OD) of final solution with colorimeter, the content of hydroxyl proline is read from using to prepare the typical curve that various hydroxyl proline concentration gradients draw with quadrat method.

The preparation of embodiment 1 arabinogalactan composition

Steps A " infuse section " is handled

With the scrubbing of 300 kilograms of drying in the sun Radixs Astragali (Astragalus membranaceus) root process, sterilizing and washing is also washed by rubbing with the hands with surpassing drainage, is immersed in then in 70% alcohol and spends the night, and is cut into the thin slice of thick about 3-5 millimeter, in 60-70 ℃ sterilizing oven, dry, make " infuse section "." infuse section " after these oven dry is because of dry loss of weight about＜15%.

The thick extraction of step B. arabinogalactan composition

With 200 kilograms, " the infuse section " for preparing in the steps A extracted 3 times each 3 hours down with UF water (deionized water that carries out ultra-filtration with 10KMWCO UF system) at 100 ℃.Under 60-70 ℃, mixed extract is evaporated to about 200 liters.The alcohol of adding 95% makes the alcohol ultimate density reach 70% in concentrated solution, stirs 15 minutes the precipitation polysaccharide under the room temperature.Decant supernatant liquor, precipitate 3 times with 95% alcohol wash.Precipitation is suspended in the UF water, and the about 18-20% of concentration (being measured by refractometer) adds 95% alcohol again, and making the alcohol ultimate density is 35%.Alcohol suspension is centrifugal, abandon the precipitation part, and in supernatant liquor, add 95% alcohol, and stirring makes ethanol concn reach 70%.The collecting precipitation part is used the UF water dissolution again, can get the crude product of arabinogalactan composition after the spraying drying.

The crude product of arabinogalactan composition is the light yellow powder of quality, and solubleness is 200 mg/ml in the water, baking loss of weight about＜15%.Endotoxin content pact＜0.3EU/ milligram.

The purifying of step C. arabinogalactan composition

With 3.5 kilograms, the crude product of the arabinogalactan composition for preparing among the step B, obtaining concentration with the UF water dissolution is 2% solution (175 liters of volumes).With 5K MWCOUF system solution being filtered to final volume is the 35-40 litre.1.0M acetyl sodium (NaOAc) damping fluid that adds pH5.2 is mixed with concentrated solution the 20mM NaOAc damping fluid of pH5.2.Solution is loaded in the SP sepharose cation exchange resin column (20 liters of volumes, bed is high 30 centimetres), and with 20mM NaOAc wash-out, collects the elutriant of 2.5-3.0 times of bed volume.The elutriant of collecting is loaded in the Q sepharose resin anion(R.A) exchange column (identical with SP column volume and bed height), and with 20mM NaOAc wash-out, the elutriant of 3-3.5 times of bed volume of collection.The elutriant of collecting from Q sepharose resin anion(R.A) exchange column with the filtration of 0.1 μ m filter after, carry out ultra-filtration with 8K MWCO UF system again.Residual liquid is concentrated into 20-26% with concentration systems under 50-60 ℃, adds raw spirit again and precipitate, the alcohol ultimate density is 80-90%.With raw spirit washing and precipitating 3 times,, can get the arabinogalactan composition of purifying then 60-70 ℃ of oven dry down.

The arabinogalactan composition of purifying is a kind of white powder, water-soluble, normal saline solution and 5% G/W, and concentration is 20 mg/ml, and water content≤6.0%.The contained ash content of composition≤2.0%, heavy metal≤10ppm, intracellular toxin≤0.1EU/mg.The pH value of composition solution is between 4.5-6.5.The sugar degree of composition≤85% (as standard substance, detecting), and Ara: Gal ratio 〉=1.5: 1 (adopting GLC to analyze described composition trimethyl silicane methylglycoside derivative measures) by the phenolsulfuric acid method with glucose.

Embodiment 2 preparation arabinogalactan protein compositions

The Q sepharose resin anion(R.A) exchange column elutriant that step C among the embodiment 1 is obtained carries out ultra-filtration with 100K MWCO UF system.Liquid residual after the ultra-filtration is further concentrated, add raw spirit again and precipitate, the alcohol ultimate density is 80-90%.With raw spirit washing and precipitating 3 times, can get the arabinogalactan protein composition of purifying then 60-70 ℃ of following oven dry.

The arabinogalactan protein composition of purifying is a kind of white powder, water-soluble, normal saline solution and 5% G/W, and concentration is 20 mg/ml, and the pH value of composition solution is between 4.5-6.5.Said composition endotoxin content≤0.5EU/mg, heavy metal content≤10ppm.Its pectinose and semi-lactosi (Ara: ratio Gal) 〉=2: 1, and and hydroxyproline content greater than 0.2%.The heavy molecular-weight average of composition is 〉=100 kilodaltons.

Embodiment 3 prepares cytokine from the activatory human peripheral blood mononuclear cells

Prepare human peripheral blood mononuclear cells (PBMC) [A.Boyum by the described method of Boyum, " separating monocytic cell and granulocyte from human blood " (" Isolation ofmononuclear cells and granulocytes from human blood... "), Scan.JLab.Invest., 97,77-89 (1968)].Human blood buffycoat (erythrocyte sedimentation rate) sample, about 25 milliliters/donor is to obtain from medical center blood bank of stanford university.With each buffycoat sample, at room temperature be suspended in again lightly 100 milliliters of cumulative volumes, no calcium-, no magnesium-the Hank balanced salt solution in (HBSS, Gibco).25 milliliters cell suspending liquid put into include 15 milliliters of Ficoll-Paque (Pharmacia LKB biotechnology, Inc.) in 50 milliliters of centrifuge tubes, with Beckman GPR desk centrifuge (GH-3.7 type rotor) 15 ℃ with 400 * g centrifugal 30 minutes.After centrifugal, the peripheral blood lymphocytes suspension between the interface is moved in another centrifuge tube of 50 milliliters, being suspended in again and making final volume among the HBSS is 45 milliliters, and centrifugal 10 minutes of 15 ℃ of speed with 354 * g.Abandon supernatant liquor, and cell precipitation is suspended in again to make final volume among the HBSS be 45 milliliters, and centrifugal 10 minutes of 15 ℃ of speed with 265 * g.Cell precipitation is suspended in again in 10 milliliters of X-Vivo tissue culture medium (TCM)s (Bio Whittaker MD), and counts with hemocytometer.In following experiment, use polystyrene tube (Falcon #2057, Becton Dickson) and peripheral blood lymphocytes from two different donors.Peripheral blood lymphocytes suspension is diluted to 4 * 10 ⁶/ milliliter, the phytohemagglutinin P (PHA-P, Pharmacia 27-3703-01) that adds 0.5 milliliter in 1 milliliter the cell suspending liquid, ultimate density is 0.3 μ g/ml, and add any in the present composition solution of 0.5 milliliter of various concentration, carry out cell cultures.The every interior cumulative volume of pipe is 2 milliliters.The cell solution of handling with PHA is organized in contrast separately.At 37 ℃, 7%CO ₂Under cultivated 24 hours, with these centrifuge tubes with Beckman GPR desk centrifuge (GH-3.7 type rotor) centrifugal 10 minutes of 15 ℃ of speed with 1600 * g, collect supernatant liquor and and before analyzing, store-70 ℃ of temperature.Measure cytokine content with the ELISA test kit that market can be purchased according to manufacturer's operation instruction, as human cell's factor IL-1 β, IL-6, TNF-α, GM-CSF and G-CSF (R﹠amp; D Systems, MN) and human TNF-γ (Endogen, MA).(MolecularDevices CA) measures optical density(OD) to microplate reader for microplate reader, Thermo max.The computed in software result who provides with microplate reader, and represent with contained cytokine content (pg/ml) in the supernatant liquor.All results use the ratio (S/C) of test group and control group to represent, wherein S represents to stimulate the cytokine amount that produces after the peripheral blood lymphocytes with PHA and specimen, and C stimulates the cytokine amount that produces after the peripheral blood lymphocytes with PHA separately.Following table shows that PAGC can improve the cytokine amount of generation by the activatory human peripheral blood mononuclear cells.

PAGC μg/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

Propagation/the maturation of embodiment 4 bone marrow megakaryocytes

Experimentize with the male mouse of the G3H/HeJ in 9-14 week.The liquid culture assays method of analyzing the megakaryocytic maturation situation is a kind of bone marrow megakaryocyte (peripheral blood platelet stem cell) propagation and/or sophisticated in vitro tests method studied.Introduce in detail referring to S.A.Burstein, " interleukin-13 accelerate muroid external Megakaryocytic maturation " (" Interleukin 3 promotesmaturation of murine megakaryocytes in vitro ") " hemocyte " (BloodCells) 11,469-479 (1986).At first isolate mouse normal bone marrow monocyte, at room temperature will be suspended in and include 0.5mM di-isopropyl fluorinated phosphate salt (St.Louis in matrix MO) 20 minutes, makes endogenous acetylcholinesterase (AchE) inactivation for DFP, Sigma.Clean cell, with 4 * 10 ⁶The concentration of/milliliter is suspended in 15%FCS-IMDM again, and (Gibco BRL, Gaithersburg MD), are placed in tissue culturing plastic's bottle then, utilize stroma cell and scavenger cell to remove them attached to the characteristic on the bottle.The tissue culture flasks that includes the medullary cell that suspends again is placed on 37 ℃, 5%CO ₂Under cultivated 1.5 hours.Collect those not attached to the cell on the bottle, with 1 * 10 ⁶The concentration of/milliliter be suspended in 1%Nutridoma SP (Boehringer Mannheim, Indianapolis, IN)-analyze among the IMDM.Cell is added to 96 holes, and in the U-type tissue culture ware, every hole contains 10 ⁵The including various PAGC concentration and contain or do not contain the mouse reorganization IL-3 (R﹠amp of 50pg/ml of 0.2 milliliter of individual cell and cumulative volume; D Systems, Minneapolis, tissue culture medium (TCM) MN).At 37 ℃, 5%CO ₂Under cultivated 7 days.The PAGC activity is to measure by the raising situation of measuring acetylcholine esterase active, and the raising of acetylcholine esterase active is rodent megakaryocyte proliferation or sophisticated special relatively cell sign thing.After 7 days, the centrifugal and abandoning supernatant with culture dish.In each culture hole, add solution I (0.2%Triton X-100,1mM EDTA, 0.12mM NaCl, 50mM HEPES, pH 7.5) acetylthiocholine iodide (sigma of the 6.27mM of 0.2 milliliter and 20 microlitres, St.Louis, MO) (ultimate density of acetylthiocholine iodide is 0.57mM).The tissue culture ware was cultivated 4 hours down at 37 ℃, each hole reaction mixture of taking out 10 μ l is put into (black plate) (Labsystems in the black flat board in 96 holes then, Helsinki, Finland), the tonka bean camphor benzene maleimide (coumarinphenylmaleimide) that adds 10 μ l, 0.4mM solution then, (CPM, Molecular Probes Inc., Eugene, OR) DMSO solution and 0.2 milliliter solution II (5mM sodium acetate, 1mM EDTA, 0.2%Triton X-100, pH 5.0).Swing plate makes liquid mixing even gently.With the emission filter disc of the excitation filter of 390nm and 460nm in photofluorometer, measure the fluorescence intensity that discharges (Fluoroskan II, Labsystems, Helsinki, Finland).Fluorescence intensity is directly proportional with the AchE amount that the megalokaryocyte of being cultivated produces.PAGC concentration is situated between can increase the megalokaryocyte number of being cultivated when 12.5 μ g/ml and 400 μ g/ml when being 200 μ g/ml (crest appear at concentration), and PAGC concentration is situated between when 12.5 μ g/ml and 400 μ g/ml when being 200 μ g/ml (crest appear at concentration), and the IL-3 of combined utilization 50pg/ml will increase the megalokaryocyte number of being cultivated (concentration makes the megalokaryocyte number increase twice when being 50 to 200 μ g/ml) greatly.

Embodiment 5 restores GM-CFC stem cell number in the mouse body that Fluracil is handled

At the 0th day, be that the Fluracil (Sigma) of 150 mg/kg is expelled to the big female mouse intraperitoneal of BALB/c of 8-10 week with consumption; And at 1-3 days, respectively by the PAGC of subcutaneous injection normal saline solution, various concentration or the recombinant human G-CSF of 100 μ g/ kilograms (Neupogen, Amgen).And, put to death test mice at the 4th day.Collect Thigh bone marrow, behind the counting, with 5 * 10 ⁴Individual white corpuscle is cultivated in 35 millimeters petri dish, the pancreatic cell substratum (pokeweed mitogen spleen cell conditionedmedium) of having put 1 milliliter methylcellulose gum perfect medium and ripple grass cytokinin in each culture dish as the source thing that group's stimulating factor can be provided (StemCellTechnologies, Inc).After cultivating 6-7 days under 37 ℃, surpass 50 population of cells with Nikon Diaphot microscope (30-60 doubly) counting cells number.Following table shows that PAGC can promote through fluoridizing the recovery of the mouse GM-CFC stem cell after uracil is handled.

Handle	Normal saline solution	PAGC 50mg/Kg	PAGC 100mg/Kg	PAGC 200mg/Kg	G-CSF 100μg/Kg
						GM-CFC/Thigh bone	601	608	1075	2020	1196

Embodiment 6 accepts the recovery of all blood in mouse inside and outside of sublethal dose radiation irradiation

With mean body weight is 20 grams, and the female mouse of the BALB/c in 9-14 week carries out this experiment.Tested each time preceding 5 days, and the Xin Meisu of adding 40 mg/litre in the unacidified drinking-water of mouse (Neomycin, Sigma, St.Louis, MO).The mouse of random choose control group or experimental group, every group each 6, and at the 0th day X ray with 4.25Gy (Philips Germany) carries out radiation irradiation for 250KVP, 0.35mm Cu filter.Behind the radiation irradiation, all blood white cells in mouse inside and outside and hematoblastic numerical value are all low than normal mice, and the red blood corpuscle number slightly reduces.PAGC by subcutaneous injection 300 and 100mg/Kg.Initial 5 days (from the 0th day to the 4th day), inject once every day, is 3 times weekly then, injected for 3 weeks continuously, comprising injecting the first time behind the radiation irradiation.The shared PAGC of 14 injection amounts.Control group is the normal saline solution through 0.1 milliliter of subcutaneous injection.Experimental session, weekly by twice of mouse tail vein haemospasia, and blood sample left in (Sarstedt in the pipe that has applied EDTA, Germany), and with Serono 9010+ cell counter (Serono BakerDiagnostics Inc.Allentown PA) comes computational analysis peripheral blood white cell, thrombocyte and erythrocytic numerical value and content of hemachrome.Compare with control group, 100 and the PAGC of 300mg/Kg obviously (p＜0.01) promote the leukocytic recovery of mouse of the 17th to 30 day (experiment finishes) behind the radiation irradiation; And obviously (p＜0.05 generally is p＜0.01) promotes behind the radiation irradiation recovery of the 14th to 25 day mouse platelets.Also obviously (p＜0.05 generally is p＜0.01) promotes behind the radiation irradiation that the 14th to 25 day mouse is erythrocytic extensive former.During research in 20 days, use 100 and the AGPC of 250mg/Kg with same way as, no matter in which time point test, all can improve red blood corpuscle and hematoblastic recovery in the mouse body behind the radiation irradiation, showing with PAGC has identical usefulness in this model.

Embodiment 7 separately with PAGC or with granulocyte colony stimulating factor (G-CSF) the mobile peripheral hematopoietic stem cells of coming together

Allow the BALB/c mouse in 8-10 week freely take acidified water and food.Normal mouse was handled 7 days according to following manner, once a day: normal saline solution (200 μ l, subcutaneous injection), PAGC (100mg/Kg, subcutaneous injection), G-CSF (100 μ g/Kg, or PAGC+G-CSF (G-CSF of the PAGC+100 μ g/Kg of 100mg/Kg, subcutaneous injection) subcutaneous injection).The about 200 μ L of the injection volume of every mouse.Each group has five mouse.After above-mentioned processing in seven days, in the time of the 8th day in mouse peritoneum the heparin of injection 20 units (Elkins-Sinn, Inc., Cherry Hill, NJ), then with sucking 30 minutes CO ₂Mode mouse is put to death, and collect peripheral blood with the cardiac puncture mode.With water-soluble poly-sucrose-Paque (Ficoll-Paque) (Pharmacia Biotech AP, Uppsala, Sweden) the density centrifugation method is isolated peripheral blood lymphocytes, cleans twice with phosphoric acid buffer, is suspended in the substratum and with cell counter again and calculates cell number.Will be between 2.5 * 10 ⁴To 1 * 10 ⁵The peripheral blood lymphocytes of number is cultivated in 35 millimeters petri dish, put 1 milliliter in each culture dish and contained the methylcellulose gum perfect medium (complete erythropoietin-containing methylcellulose medium) of erythropoietin and IL-3+IL-6+ STEM CELL FACTOR (the StemCell Technologies Inc. of reorganization, Vancouver, B.C.).After cultivating 7-14 days under 37 ℃, surpass 50 population of cells with Nikon Diaphot microscope (30-150 doubly) counting cells number.Counting can form grain thin huge have a liking for population of cells (GM-CFC) and erythrocytic burst-forming unit (burst-formingunit-erythroid (BFU-E)).Following table shows that PAGC can work in coordination with the GM-CFC of increase normal mice body-internal-circulation and the numerical value of BFU-E with G-CSF.

	Normal saline solution	PAGC 100mg/Kg	G-CSF 100μg/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

Handle mouse like in testing with the cyclic phosphoric acid amine of 200 mg/kg another kind of, and then handled 11 days, suspend into 2 * 10 again after the collection peripheral blood lymphocytes with the PAGC of normal saline solution or 100 or 300 mg/kg ⁷Cells/ml, with fluorescein isothiocyanic acid-anti--CD34 and PE-blood lineage mark (CD3, CD4, CD6, CD19, CD11b, GR-1, CD41 and Ter-119) (Pharmingen, San Diego, CA) dye, use fluid cytometer (FACSCalibur, Becton Dickson, San Jose again, CA) analyze, can see that PAGC can quicken CD34 ⁺Lin ^-Cell moves in the peripheral blood.

Embodiment 8 PAGC can recover to fluoridize the mouse body weight that uracil or cyclic phosphoric acid amine were handled

Carry out this experiment with the female mouse of the BALB/c in 8-10 week.10 mouse are divided into 5 groups at random: be respectively normal control group, cyclic phosphoric acid amine (CY)-treatment group, fluoridize uracil (FU)-treatment group, CY+PAGC-treatment group or FU+PAGC-treatment group.For normal control group, by the peritoneal injection normal saline solution, by the 1st day to the 12nd day, be then in the time of the 0th day by the subcutaneous injection normal saline solution.To cyclic phosphoric acid amine (CY)-treatment group or CY+PAGC-treatment group, in the time of the 0th day,,, be PAGC by subcutaneous injection normal saline solution or 200 mg/kg then by the 1st day to the 12nd day by the cyclic phosphoric acid amine of peritoneal injection 200 mg/kg.To Fluracil (FU)-treatment group or FU+PAGC-treatment group, be in the time of the 0th day by the FU of peritoneal injection 150 mg/kg, then by the 1st day to 12 days, be PAGC by subcutaneous injection normal saline solution or 200 mg/kg.Mouse claimed one time body weight in the time of the 0th day, every other day claim one time body weight then again, up to the 12nd day.The result shows that it is minimum to lose weight through the mouse that CY+PAGC-handles and FU+PAGC-handled, and than the faster recovery body weight of handling with CY or FU separately of mouse.

The PAGC human clinical trial that embodiment 9 carries out in China

The PAGE human clinical trial who carries out in China carries out according to the GCP standard of the regulation of Ministry of Health of the People's Republic of China.

First phase clinical trial is administered to 32 normal volunteers with PAGC with the dilution of normal physiological salt solution and with intravenous mode, injects continuously 7 days.Do not find any serious clinical side effects after using three multiple doses of clinical dosage (250 mg/day).

II clinical trial phase assessment PAGC is to suppressing the leukopenia (＜4.0 * 10 that lung cancer, stomach cancer or patients with mastocarcinoma cause after accepting chemotherapy ⁹The usefulness of white cell/L=.Amount to six medical centers, 487 patients have participated in current clinical trial, wherein 328 patients are in PAGC treatment group, 84 patients are in G-CSF treatment group, and 75 patients are at control group.Whenever at whole 14 days chemotherapeutic period, if patient's white cell value is hanged down 4 * 10 ⁹/ L is about to this patient and includes in one of wantonly three groups.To first group of patient, be dissolved in 250 milligrams of PAGC in 500 milliliters of normal saline solutions after, intravenous injection was once a day injected 7 days in patient's body continuously.During using PAGC, the patient is observed, and after drug withdrawal, continue to observe 7 days.The patient of G-CSF treatment group, by the G-CSF of subcutaneous injection 75 μ g once injected continuously 5 days every day, during the medication patient observed, and continue to observe 9 days after drug withdrawal.At control group, after chemotherapy, do not give the medicine that other can strengthen hemopoietic system, and observed the patient 14 days.Treat white cell, RBC and the thrombocyte numerical value of observing all 3 groups of patients in back 14 days.By patient and chemotherapy related symptoms and its Karnofsky performance Index, evaluate the usefulness that it improves patient's quality of the life simultaneously.The symptom relevant with chemotherapy comprises tired out and burnout, discomfort, perspiration, short of breath and lack appetite, and evaluated by qualified traditional Chinese physician.The mark of each class is as follows: 0 minute-do not have symptom, 1 minute-symptom was slight, and 2 minutes-medium symptom, 3 minutes-serious symptom.Therefore, the most serious example sign is 15 minutes.Karnofsky performance Index is the evaluation method that The World Health Organization (WHO) is used for assessing the general health state.After Fig. 1 showed chemotherapy, the white cell number was with the fate change curve; After Fig. 2 chemotherapy, Zong the symptom mark is with the fate change curve; Fig. 3 is mark (PAGC and the GCSF comparison: U=18.36, p＜0.01 of Karnofsky performance Index; PAGC and control group compare: U=15.62, p＜0.0001=; Following table is listed the variation situation of average platelet number and fate after the chemotherapy; All results show with 250 milligrams of PAGC treatments 7 days, really can promote white cell and platelet cell number to recover, improve the symptom that chemotherapy causes, and improve the Karnofsky performance Index of patients undergoing chemotherapy, generally speaking on statistics, have clear improvement than control group; And and had better statistical effect in 5 days than G-CSF treatment usually with 75 μ g.

Thrombocyte, 10 9/ L, PAGC-treatment group patient (n=54)
					The 0th day	The 4th day	The 7th day	The 10th day	The 14th day
65±21	87±61 *	118±94 **	149±107 **	178±111 **
					*p＜0.05， **p＜0.001

Though the present invention describes by certain specific embodiments, but for a person skilled in the art, availablely carry out various changes easily according to the present invention, and do not break away from purpose of the present invention and spirit, all such modifications all comprise within the scope of the appended claims.

Claims (34)

1. the arabinogalactan protein composition of a purifying, described arabinogalactan protein composition is isolating from the Radix Astragali, the weight-average molecular weight of described arabinogalactan protein composition is 100 kilodaltons at least, and comprise 5% protein, with the total amino acid content is that 20% of the described proteinic aminoacids content of benchmark is an oxyproline, and has sugar composition, pectinose wherein: the ratio at least 2: 1 of semi-lactosi, and comprise the pectinose of molar percentage 45% to 75%; The rhamnosyl of molar percentage 2% to 4%; The galactosonic acid of molar percentage 4% to 6%; The semi-lactosi of molar percentage 8% to 25%; Glucose with molar percentage 5% to 25%.

2. the arabinogalactan protein composition of purifying according to claim 1, the wherein said Radix Astragali is meant Radix Astagali or Radix Astragali.

3. the arabinogalactan protein composition of purifying according to claim 1 and 2 separates from the Radix Astragali that originates in inner mongolia or Shanxi Province.

4. the arabinogalactan protein composition of purifying according to claim 1, the wherein said Radix Astragali are the Radixs Astragali in 2 years of growth.

5. the arabinogalactan protein composition of purifying according to claim 1, wherein the ratio of pectinose/semi-lactosi is 3: 1 at least.

6. the arabinogalactan protein composition of purifying according to claim 1, its contained intracellular toxin amount is lower than 0.5EU/mg.

One kind can intravenous arabinogalactan protein aqua, comprising:

(a) the arabinogalactan protein composition according to the described purifying of any one claim in the claim 1,2,4,5 or 6 of pharmacy effective dose; And

(b) a kind of can intravenous liquid adjuvant.

8. claim 1,2,4, the arabinogalactan protein composition of the described purifying of any one claim is used for the treatment of in preparation that application in the medicine of mammalian diseases state or claim 7 are described can intravenous arabinogalactan protein aqua to be used for the treatment of application in the medicine of mammalian diseases state in preparation in 5 or 6, described Mammals is to the treatment sensitivity of stimulating immune system and hemopoietic system, the use of described medicine comprise the intravenous injection pharmacy effective dose according to claim 1,2,4, in 5 or 6 the arabinogalactan protein composition of the described purifying of any one claim or according to claim 7 can intravenous arabinogalactan protein aqua.

9. application according to claim 8; wherein said treatment mammalian diseases state is a hemopoietic; induce Megakaryocytic propagation or maturation; stimulate and generate IL-1 β; IL-6; TNF-α; IFN-γ; GM-CSF; or G-CSF; stimulate neutrophils to generate; or activate; the treatment neutropenia; anaemia; or thrombocytopenia; promote to be subjected to cytotoxic agent; or the individuality after the radiation irradiation effect restores; the treatment emaciation; vomiting; or drug withdrawal syndrome, improve biological reactivity; or the liver cell of protection hepatitis B patient.

10. application according to claim 9, wherein said treatment mammalian diseases state is a hemopoietic, induce Megakaryocytic propagation or maturation, stimulate and generate IL-1 β, IL-6, TNF-α, IFN-γ, GM-CSF or G-CSF, stimulate neutrophils to generate or activation, or treatment neutropenia, blood or thrombocytopenia.

11. application according to claim 8, wherein said Mammals are human.

12. according to claim 10 or 11 described application, wherein said Mammals suffers from bone marrow depression.

13. application according to claim 12, wherein said bone marrow depression result from the treatment of cancer chemotherapy or radiation irradiation.

14. further comprising, application according to claim 8, wherein said treatment mammalian diseases state use at least a other treatment medicament.

15. application according to claim 14, wherein said at least a other treatment medicament are a kind of medicaments of energy hemopoietic.

16. application according to claim 15, wherein said at least a other treatment medicament are selected from protoheme and generate element, thrombopoietin or granulocyte colony stimulating factor, or IL-3.

17. an arabinogalactan protein method for compositions for preparing the described purifying of claim 1 comprises:

(a) a kind of liquid extract that contains the arabinogalactan protein composition that from the Radix Astragali, extracts;

(b) lower aliphatic alcohols of interpolation q.s in the described liquid extract in step (a) comes out the arabinogalactan protein composition precipitates, and separates described sedimentary arabinogalactan protein composition;

(c) the described sedimentary arabinogalactan protein composition of step (b) is water-soluble, form the solution that contains the arabinogalactan protein composition;

(d) the described solution that contains the arabinogalactan protein composition that obtains from step (c) with the ion exchange chromatography purifying;

(e) the described solution that contains the arabinogalactan protein composition of treatment step (d) is got rid of the material that any molecular weight is lower than 100 kilodaltons; And

(f) in the treated solution that contains the arabinogalactan protein composition of step (e), isolate the arabinogalactan protein composition of purifying.

18. method according to claim 17 also comprises:

(g) solution concentration of the arabinogalactan composition that step (f) is obtained precipitates with raw spirit again; And

(h) throw out that obtains of washing step (g) obtains the arabinogalactan protein composition.

19. application according to claim 9, wherein said Mammals are human.

20. application according to claim 10, wherein said Mammals are human.

21. application according to claim 19, wherein said Mammals suffers from bone marrow depression.

22. application according to claim 20, wherein said Mammals suffers from bone marrow depression.

23. further comprising, application according to claim 9, wherein said treatment mammalian diseases state use at least a other treatment medicament.

24. further comprising, application according to claim 10, wherein said treatment mammalian diseases state use at least a other treatment medicament.

25. further comprising, application according to claim 11, wherein said treatment mammalian diseases state use at least a other treatment medicament.

26. further comprising, application according to claim 12, wherein said treatment mammalian diseases state use at least a other treatment medicament.

27. further comprising, application according to claim 13, wherein said treatment mammalian diseases state use at least a other treatment medicament.

28. application according to claim 23, wherein said at least a other treatment medicament are a kind of medicaments of energy hemopoietic.

29. application according to claim 24, wherein said at least a other treatment medicament are a kind of medicaments of energy hemopoietic.

30. application according to claim 25, wherein said at least a other treatment medicament are a kind of medicaments of energy hemopoietic.

31. application according to claim 26, wherein said at least a other treatment medicament are a kind of medicaments of energy hemopoietic.

32. application according to claim 27, wherein said at least a other treatment medicament are a kind of medicaments of energy hemopoietic.

33. the arabinogalactan protein composition of purifying according to claim 1, wherein said composition are from obtaining from the astragalus root part.

34. the arabinogalactan protein composition of purifying according to claim 1 and 2 separates from the Radix Astragali that originates in the inner mongolia.

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