CN102397527B - Medicinal composition and application thereof - Google Patents
Medicinal composition and application thereof Download PDFInfo
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- CN102397527B CN102397527B CN201010285328.4A CN201010285328A CN102397527B CN 102397527 B CN102397527 B CN 102397527B CN 201010285328 A CN201010285328 A CN 201010285328A CN 102397527 B CN102397527 B CN 102397527B
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Abstract
The invention relates to a medicinal composition and application thereof. The composition contains arctigenin and interferon. The invention also provides a microemulsion preparation and an injection which contain the medicinal composition, and application of the medicinal composition to the preparation of a medicine for treating leukemia. Compared with single medicines, the medicinal composition has a synergistic effect of treating leukemia.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition and application thereof, particularly a kind of pharmaceutical composition that contains aretigenin and interferon and the application aspect treatment leukemia thereof.
Background technology
Cancer is that current serious affects human health, threatens one of principal disease of human life.Acute leukemia is a kind of common hematologic malignancy, and in each age mortality of malignant tumors of the whole nation, leukemia is occupied the first five position, in child and the crowd below 35 years old, holds pride of place.Leukemic treatment generally can be divided into inducer remission treatment and alleviate two Main Stage of rear treatment, and the latter can be further divided into again after treatment and maintain two stages for the treatment of.The object of inducer remission treatment is promptly leukaemia to be reduced as far as possible, makes the hematopoietic function recovery of bone marrow normal, reaches the standard of alleviating completely.Alleviate be completely leukaemic without hemorrhage heating and Anemia, the normal or normal of life.After alleviating, the object for the treatment of is consolidation and the intensive treatment that continues the long period by adopting, and further eliminates remaining leukaemia in body, prevents leukemia relapse, extends and alleviates and life span, strives for curing leukemia.After alleviating completely, still need to adopt positive consolidation and intensive treatment and continue quite over a long time (2-3).Becoming in the world the therapeutic scheme that human acute myeloid leukemia is conventional is the scheme that cytosine arabinoside+anthracene nucleus or Anthraquinones chemotherapeutics form, but this therapeutic scheme toxic and side effects is large, to patient, brings very large misery.
Interferon is multi-functional cytokine, a kind of glycoprotein that stimulates reticuloendothelial system, macrophage, lymphocyte and somatic cell to produce by interferon inducers viral and other kinds.This albumen has multiple biological activity, comprises antiproliferative, antiviral, immunomodulating and induction of differentiation.
Find altogether on mankind at present α, β, γ three types: alpha-interferon is produced by leukocyte, and interferon beta is produced by fibroblast, interferon gamma is that the lymphocyte in immune system produces.Three kinds of all these are all effective viral inhibitors, and have certain antitumaous effect.Alpha-interferon is clinically the cytokine of most widely used treatment tumor.It is by direct anti-tumour cell proliferative and regulate body's immunity performance antitumor action.Beta-interferon has obviously or stronger inhibited proliferation human brain cell strain and the strain of people's malignant melanoma cell, to all there being the effect of obvious inhibition tumor body propagation after nude mice by subcutaneous inoculation glioblastoma cell strain, Glioblastoma cell strain, glioblastoma multiforme cell strain, the strain of people's malignant melanoma cell.Adult's intramuscular injection or quiet administration, each (1～6) * 10
6unit/m
2, intramuscular injection after normal saline 2ml dissolves, or be dissolved in normal saline or 5% glucose injection 250～500ml, in 1 hour, drip off, one day 1 time, be used in conjunction 10 days, interval repeats for 3～5 days, and noting altogether 30 is a course for the treatment of.The biological activity of gamma interferon comprises: the expression of the MHC II class antigens such as (1) induction mononuclear cell, macrophage, dendritic cell, skin flbroblast, vascular endothelial cell, sternzellen, makes it participate in the identifying of specific immunity; (2) promote LPS stimulated in vitro mouse B cell IgG secretion 2a, reduce the generation of IgG1, IgG2b, IgG3 and IgE; The mouse B cell that inhibition is induced by IL-4 is bred, and IgG1 and IgE produce and Fc ε RII expresses, and promotes the propagation of the human B cell of SAC induction; (3) collaborative IL-2 induction LAK is active, promotes T cell IL-2R to express; (4) induction acute phase protein is synthetic, the differentiation of induction myeloid cell.
Although interferon has been used for the treatment of inflammatory, viral and proliferative disease, comprises leukemia, it has many side effect, comprise local injection reaction, influenza sample syndrome and depression etc., and these side effect increases the weight of with dosage.Exactly because many leukemic treatments need the interferon of higher dosage, the increase of dosage has increased the weight of again toxic and side effects, is forced to therapy discontinued so a lot of patient is impatient at.Therefore, need can combine with interferon to improve treatment leukemia curative effect, reduce the compound of toxic and side effects, if can further obtain, reduce dosage, minimizing administration frequency, will produce important meaning to the treatment of leukemia patient.
For example, in multiple sclerosis therapy (MS treatment), some compounds of combining use with beta-interferon by reduce potentially beta-interferon administration dosage, alleviate the side effect being produced by beta-interferon.But these conjoint therapies may not one alleviate side effect surely.For example, comprise that acetic acid lattice draw the therapeutic alliance for thunder and alpha-interferon not improve the clinical score with the mice of EAE treatment.So carry out, to find the compound combine use with interferon be very necessary to obtain enhancement curative effect, make side effect be down to minimum work simultaneously, and difficulty is also sizable.
Aretigenin (arctigenin) is Lignanoids compounds, derives from Chinese medicine Fructus Arctii.There is antibiotic, antiviral, antitumor, anti-paf receptor and calcium antagonistic activity significantly.Wang Lu etc. are at the effects anb Mechanism > > (Acta Pharmaceutica Sinica of < < arctigenin induction human leukemia cell apoptosis, the 43rd the 5th phase of volume in 2008,542-547 page) literary composition discloses arctigenin and can bring into play cytotoxicity by cell death inducing, thereby suppresses the propagation of the capable leukemia K 562 of chronic granulocyte.But do not have so far scientific evidence to show that aretigenin and interferon therapy leukemia have the effect of potentiation.
Summary of the invention
In order to reduce toxic and side effects, to improve interferon to leukemic curative effect, the present invention combines use by aretigenin with interferon, obtained good expection, the invention provides a kind ofly to treatment leukemia effectively pharmaceutical composition for this reason, in this pharmaceutical composition, contain active component aretigenin and interferon.The object of aretigenin and interferon administering drug combinations is to reduce the dosage of interferon, to obtain effective treatment, the side effect that alleviates interferon simultaneously.
The present invention finds that aretigenin has very strong therapeutic effect to leukemia mouse after aretigenin, interferon drug administration by injection are used for the treatment of to various types of leukemia mouse models, and wherein the weight ratio of interferon and aretigenin is (3 * 10
-5-0.1): 1, with independent drug administration by injection aretigenin, separately drug administration by injection interferon is compared and had significant difference, and there is Synergistic treatment activity.The invention provides a kind of leukemic pharmaceutical composition for the treatment of, the active component in this pharmaceutical composition is aretigenin and interferon for this reason, and the weight ratio of interferon and aretigenin is (3 * 10
-5-0.1): 1.
Above-mentioned interferon is selected from a kind of in alpha-interferon, beta-interferon and gamma interferon or between them, has the combination in any for the treatment of leukemia effect.Interferon refers to alpha-interferon, alpha-interferon analog, alpha-interferon derivant, alpha-interferon conjugates, beta-interferon, beta-interferon analog, beta-interferon derivant, beta-interferon conjugates, gamma interferon particularly.Alpha-interferon and beta-interferon gene can change and produce its beta-interferon analog by for example pointing to the oligonucleotide of sudden change, for example people's analog cysteine disappearance or cysteine displacement of recombinating.In addition, more than one amino acid whose identification or location can be suddenlyd change and be changed by targeting, and the primary amino acid sequence of protein can increase by glycosylation or by other complementarity molecule (as lipid, phosphoric acid and acetyl group).Single amino acids residue in chain can be modified by oxidation, reduction or other derivation effect.Alpha-interferon or beta-interferon protein cleavable obtain the fragment that keeps active, and whole protein or its fragment can merge with other peptides and proteins (as immunoglobulin).Alpha-interferon and beta-interferon conjugates can represent to comprise the component of the beta-interferon of the polymer coupling containing polyalkylene glycol moiety existing with non-natural.Preferred interferon comprises
alfacon-1, alpha-interferon, alpha-interferon analog, the alpha-interferon of Pegylation, the alpha-interferon of the alpha-interferon of polymerization, dimerization, the alpha-interferon closing with carrier yoke, the alpha-interferon of oral cavity inhalant form, the alpha-interferon of the alpha-interferon of injectable composition form, topical composition form,
analog,
analog,
analog,
analog,
analog, Alfacon-1 analog, beta-interferon, Avonex
tM, Betaseron
tM, Betaferon
tM, Rebif
tM, beta-interferon analog, the beta-interferon of Pegylation, the beta-interferon of the beta-interferon of polymerization, dimerization, the beta-interferon, the beta-interferon of oral cavity inhalant form, the beta-interferon of injectable composition form, the beta-interferon of topical composition form, the Avonex that close with carrier yoke
tManalog, Betaseron
tManalog, Betaferon
tManalog, Rebif
tManalog.Also can use the material that brings out alpha-interferon or beta-interferon generation or simulation alpha-interferon or beta-interferon effect.
The dosage using for every kind of interferon and professional and technical personnel in the field known and clinical before and in clinical research and business application those dosage of use similar.Owing to having strengthened these compounds and combining of aretigenin compound the curative effect of these compounds, so concentration can be lower than the general dosage using.Really, the object of aretigenin compound and interferon administering drug combinations is to reduce the dosage of interferon, to obtain effective treatment, the side effect that alleviates interferon simultaneously.
The dosage of alpha-interferon and beta-interferon is known in the art.For example, the dosage that is used for the treatment of the interferon of proliferative disease hairy cell leukemia is 100-3600 ten thousand units, once a day or twice weekly.The dosage of beta-interferon is 30 μ g-250 μ g typically.Avonex
tM, Betaseron
tM, Rebif
tMthe main distinction be the amount of given beta-interferon and the approach of administration different with frequency.Avonex
tMpreferably with 30 μ g dosage administered intramuscular each time; Betaseron
tMpreferably with the dosage subcutaneous injection administration every other day of 250 μ g; Rebif
tMpreferably with time subcutaneous injection administration on every Wendesdays of the dosage of 44 μ g.
Those of ordinary skills are by clear, and in the present composition of patient being used according to the present invention, the amount of various compositions will depend on these above-mentioned factors.The invention provides and promote or strengthen the method for interferon to leukemia therapeutic activity, described method is co-administered a certain amount of aretigenin and interferon, aretigenin and interferon simultaneously or jointly with various dose and route of administration therapeutic scheme, carry out administration, to strengthen interferon therapy leukaemic's effect (with using separately interferon and comparing).The therapeutic scheme that can adopt comprises: (1) once a day above, once a day, weekly above, once in a week, monthly once above or monthly co-administered aretigenin and interferon once, for leukemic effective treatment; (2) once a day above, once a day, weekly above, once in a week, monthly once above or monthly use aretigenin and interferon once simultaneously, for leukemic effective treatment.
In addition, in the leukemic pharmaceutical composition of above-mentioned treatment, the dosage of aretigenin intramuscular injection, subcutaneous injection or intravenous drip is 0.01-10mg/kg.
The present invention has also carried out preferably the weight ratio of aretigenin and interferon, the screening mainly for this pharmaceutical composition at the curative effect of medication of inhibition propagation and inducing leukemia cell differentiation.Pharmaceutical composition of the present invention is selected but is not limited to the embodiment of following pharmaceutical compositions:
(1) interferon is recombinant human interferon alpha-2, and the ratio between the amount of recombinant human interferon alpha-2 and the amount of aretigenin is (0.25-500): 1, and the amount of recombinant human interferon alpha-2 represents with million international units, the amount of aretigenin represents with milligram.
(2) interferon is recombinant human interferon alpha 1 b, and the weight ratio of recombinant human interferon alpha 1 b and aretigenin is (5 * 10
-5～0.1): 1.
(3) interferon is recombinant human interferon beta 1a, and the weight ratio of recombinant human interferon beta 1a and aretigenin is (3 * 10
-5-0.1): 1.
(4) interferon is recombinant human interferon beta 1b, and the weight ratio of recombinant human interferon beta 1b and aretigenin is (5 * 10
-5-0.45): 1.
(5) interferon is recombinant human interferon gamma, and the ratio between the amount of recombinant human interferon gamma and the amount of aretigenin is 1: (0.3-1200), the amount of recombinant human interferon gamma represents with million international units, and the amount of aretigenin represents with milligram.
The present invention has investigated the compositions dialogue cell strain NB4 of interferon and aretigenin and the impact of MR2 propagation and differentiation by embodiment 26,27, experimental result shows that each experimental group compares with matched group, NB4 cell is all had to inhibited proliferation, and there is significance or utmost point significant difference.Compare with interferon-ALPHA 2a group, Arctiin tuple, high group of low group of interferon-ALPHA 2a compound recipe and compound recipe have significant difference to NB4 cell inhibitory effect, and have synergism; Compare with Interferon α1 b group, Arctiin tuple, high group of low group of Interferon α1 b compound recipe and compound recipe have significant difference to NB4 cell inhibitory effect, and have synergism; Compare with interferon beta 1a group, Arctiin tuple, high group of low group of interferon beta 1a compound recipe and compound recipe have significant difference to NB4 cell inhibitory effect, and have synergism; Compare with interferon beta 1b group, Arctiin tuple, high group of low group of interferon beta 1b compound recipe and compound recipe have significant difference to NB4 cell inhibitory effect, and have synergism; Compare with interferon gamma group, Arctiin tuple, low group of interferon gamma compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism.Each experimental group is followed successively by the power of NB4 cell inhibitory effect effect: the high group of low group of interferon compound recipe and compound recipe > interferon group > Arctiin tuple.
Each experimental group is compared with matched group, and MR2 cell is all had to inhibited proliferation, and has significance or utmost point significant difference, and inhibitory action prolongation in time strengthens gradually.Compare with interferon-ALPHA 2a group, Arctiin tuple, high group of low group of interferon-ALPHA 2a compound recipe and compound recipe have significant difference to MR2 cell inhibitory effect, and have synergism; Compare with Interferon α1 b group, Arctiin tuple, high group of low group of Interferon α1 b compound recipe and compound recipe have significant difference to MR2 cell inhibitory effect, and have synergism; Compare with interferon beta 1a group, Arctiin tuple, high group of low group of interferon beta 1a compound recipe and compound recipe have significant difference to MR2 cell inhibitory effect, and have synergism; Compare with interferon beta 1b group, Arctiin tuple, high group of low group of interferon beta 1b compound recipe and compound recipe have significant difference to MR2 cell inhibitory effect, and have synergism; Compare with interferon gamma group, Arctiin tuple, low group of interferon gamma compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Each experimental group is followed successively by the power of MR2 cell inhibitory effect effect: the high group of low group of interferon compound recipe and compound recipe > interferon group > Arctiin tuple.
No matter NBT reduction test result shows for NB4 cell or MR2 cell, compare with single medicine, the cell NBT positive rate of the high group of low group of interferon compound recipe and compound recipe has significant difference or utmost point significant difference, compare with single medicine, high group of group of low group of interferon compound recipe and compound recipe also further shows as synergism to the induction differentiation capability of NB4 cell and MR2 cell.
Based on above experimental result, the present invention also provides the application of pharmaceutical composition of the present invention in the medicine of preparation treatment inducing leukemia cell differentiation and inhibition Leukemia Cell Proliferation.Active component in described compositions is aretigenin and interferon, and the weight ratio of interferon and aretigenin is (3 * 10
-5-0.1): 1.
In application in the medicine of preparation treatment inducing leukemia cell differentiation and inhibition Leukemia Cell Proliferation, described pharmaceutical composition is selected from following pharmaceutical combination:
(1) interferon is recombinant human interferon alpha-2, and the ratio between the amount of recombinant human interferon alpha-2 and the amount of aretigenin is (0.25-500): 1, and the amount of recombinant human interferon alpha-2 represents with million international units, the amount of aretigenin represents with milligram.
(2) interferon is recombinant human interferon alpha 1 b, and the weight ratio of recombinant human interferon alpha 1 b and aretigenin is (5 * 10
-5～0.1): 1.
(3) interferon is recombinant human interferon beta 1a, and the weight ratio of recombinant human interferon beta 1a and aretigenin is (3 * 10
-5-0.1): 1.
(4) interferon is recombinant human interferon beta 1b, and the weight ratio of recombinant human interferon beta 1b and aretigenin is (5 * 10
-5-0.45): 1.
(5) interferon is recombinant human interferon gamma, and the ratio between the amount of recombinant human interferon gamma and the amount of aretigenin is 1: (0.3-1200), the amount of recombinant human interferon gamma represents with million international units, and the amount of aretigenin represents with milligram.
In addition, the discovery that inventor is surprised, the dosage of alpha-interferon, beta-interferon and gamma interferon height no matter, even in the situation that their drug dose is very low, this pharmaceutical composition to various leukemia mouse model therapeutic effect all very obviously, and be significantly higher than the interferon such as alpha-interferon, beta-interferon and gamma interferon of the high dose of independent use.
In order better to express the form of this pharmaceutical composition, the present invention has prepared the microemulsion formulation of this pharmaceutical composition.Described microemulsion formulation can be prepared according to conventional preparation method, and the mean diameter of the microemulsion formulation of preparation is 15-80nm.Described ejection preparation can be prepared according to conventional formulation.The present invention has also studied the microemulsion formulation dialogue cell strain NB4 of pharmaceutical composition provided by the invention and the impact of MR2 propagation and differentiation, result of the test has obtained unexpected effect, is suppressing all to obtain good synergism aspect Leukemia Cell Proliferation and inducing leukemia cell differentiation.
In a word, pharmaceutical composition provided by the invention compared with the existing technology, has following outstanding advantage:
The first, prior art mostly adopts the successively administration successively in order of two or more cancer therapy drugs, and the present invention is prepared into a kind of pharmaceutical preparation administration simultaneously by two kinds of cancer therapy drugs, greatly facilitates patient to use, and has improved the compliance of disease treatment.
The second, to compare with single medicine or other drug combination, pharmaceutical composition provided by the invention has significant synergism aspect treatment leukemia.
The 3rd, this pharmaceutical composition has outstanding feature.From natural plants Fructus Arctii, extract the aretigenin obtaining, toxicity is low, once and be used in combination with interferon, guaranteeing under the prerequisite of stronger active anticancer, can significantly reduce the using dosage of interferon, reduce toxic and side effects, improve the drug resistance of cancer therapy drug.
The specific embodiment
By specific embodiment, further describe the present invention below, the present invention is not limited only to following examples.
Embodiment 1 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon alpha-2 250MIU
Soybean oil 5g
Polyoxyethylene-23-lauryl ether 20g
1,2-PD 6g
Preparation technology: take recipe quantity soybean oil, polyoxyethylene-23-lauryl ether, 1,2-propylene glycol, after mixing, stir, then add aretigenin, recombinant human interferon alpha-2 to dissolve, also can ultrasonic Treatment with accelerate dissolution, concentrated solution be must clarify, aretigenin and recombinant human interferon alpha-2 microemulsion concentrate are.Laser granulometry is measured its particle diameter, and mean diameter is 15nm.
Embodiment 2 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon alpha-2 5 * 10
5mIU
Hydrogenation cocos nucifera oil glyceride 5g
Lauroyl Polyethylene Glycol-32-glyceride 20g
1,2-PD 5g
PEG3350 10g
Preparation technology: take recipe quantity hydrogenation cocos nucifera oil glyceride, lauroyl Polyethylene Glycol-32-glyceride, 1; 2-propylene glycol, PEG3350; after mixing, stir; then add aretigenin, recombinant human interferon alpha-2 to dissolve; also can ultrasonic Treatment with accelerate dissolution; concentrated solution be must clarify, aretigenin and recombinant human interferon alpha-2 microemulsion concentrate are.Laser granulometry is measured its particle diameter, and mean diameter is 40nm.
Embodiment 3 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon alpha-2 3000MIU
Safflower oil 15g
Polyoxyethylene-23-lauryl ether 6g
1,2-PD 10g
Preparation technology is with embodiment 2.Laser granulometry is measured its particle diameter, and mean diameter is 36nm.
Embodiment 4 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 0.1g
Recombinant human interferon alpha 1 b 0.01g
Oleum Gossypii semen 10g
SY-Glyster MSW 750 15g
1,2-PD 7.5g
Preparation technology: take recipe quantity Oleum Gossypii semen, SY-Glyster MSW 750,1,2-propylene glycol, after mixing, stir, then add aretigenin, recombinant human interferon alpha 1 b to dissolve, also can ultrasonic Treatment with accelerate dissolution, must clarify concentrated solution, be the microemulsion concentrate of aretigenin and recombinant human interferon alpha 1 b.Laser granulometry is measured its particle diameter, and mean diameter is 76nm.
Embodiment 5 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon alpha 1 b 5 * 10
-5g
Soybean oil 20g
SY-Glyster MSW 750 25g
1,2-PD 5g
Preparation technology is with embodiment 4.Laser granulometry is measured its particle diameter, and mean diameter is 47nm.
Embodiment 6 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon alpha 1 b 0.01g
Soybean oil 32g
SY-Glyster MSW 750 16g
1,2-PD 8g
Preparation technology is with embodiment 4.Laser granulometry is measured its particle diameter, and mean diameter is 58nm.
Embodiment 7 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon beta 1a 30 μ g
Soybean oil 13g
SY-Glyster MSW 750 9g
1,2-PD 15g
Preparation technology: take recipe quantity soybean oil, SY-Glyster MSW 750,1,2-propylene glycol, after mixing, stir, then add aretigenin, recombinant human interferon beta 1a to dissolve, also can ultrasonic Treatment with accelerate dissolution, must clarify concentrated solution, be the microemulsion concentrate of aretigenin and recombinant human interferon beta 1a.Laser granulometry is measured its particle diameter, and mean diameter is 66nm.
Embodiment 8 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon beta 1a 0.01g
Soybean oil 6g
SY-Glyster MSW 750 15g
1,2-PD 15g
Preparation technology is with embodiment 7.Laser granulometry is measured its particle diameter, and mean diameter is 38nm.
Embodiment 9 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon beta 1a 0.1g
Soybean oil 20g
SY-Glyster MSW 750 15g
1,2-PD 11g
Preparation technology is with embodiment 7.Laser granulometry is measured its particle diameter, and mean diameter is 44nm.
Embodiment 10 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon beta 1b 0.45g
Soybean oil 5.5g
SY-Glyster MSW 750 7.5g
1,2-PD 5g
Preparation technology: take recipe quantity soybean oil, SY-Glyster MSW 750,1,2-propylene glycol, after mixing, stir, then add aretigenin, recombinant human interferon beta 1b to dissolve, also can ultrasonic Treatment with accelerate dissolution, must clarify concentrated solution, be the microemulsion concentrate of aretigenin and recombinant human interferon beta 1b.Laser granulometry is measured its particle diameter, and mean diameter is 56nm.
Embodiment 11 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon beta 1b 5 * 10
-5g
Soybean oil 13.2g
SY-Glyster MSW 750 10.5g
1,2-PD 7.5g
Preparation technology is with embodiment 10.Laser granulometry is measured its particle diameter, and mean diameter is 48nm.
Embodiment 12 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1g
Recombinant human interferon beta 1b 0.1g
Soybean oil 10g
SY-Glyster MSW 750 8g
1,2-PD 15g
Preparation technology is with embodiment 10.Laser granulometry is measured its particle diameter, and mean diameter is 57nm.
Embodiment 13 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 0.3mg
Recombinant human interferon gamma 1MIU
Soybean oil 12.0g
SY-Glyster MSW 750 8.5g
1,2-PD 7.8g
Preparation technology: take recipe quantity soybean oil, SY-Glyster MSW 750,1,2-propylene glycol, after mixing, stir, then add aretigenin, recombinant human interferon gamma to dissolve, also can ultrasonic Treatment with accelerate dissolution, must clarify concentrated solution, be the microemulsion concentrate of aretigenin and recombinant human interferon gamma.Laser granulometry is measured its particle diameter, and mean diameter is 75nm.
Embodiment 14 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 0.1g
Recombinant human interferon gamma 1MIU
Soybean oil 12.5g
SY-Glyster MSW 750 5.0g
1,2-PD 15.0g
Preparation technology is with embodiment 13.Laser granulometry is measured its particle diameter, and mean diameter is 28nm.
Embodiment 15 pharmaceutical composition microemulsion formulation of the present invention
Aretigenin 1.2g
Recombinant human interferon gamma 1MIU
Soybean oil 6.8g
SY-Glyster MSW 750 15.5g
1,2-PD 10.5g
Preparation technology is with embodiment 13.Laser granulometry is measured its particle diameter, and mean diameter is 63nm.
Embodiment 16 drug combination injection of the present invention
Aretigenin 1g
Recombinant human interferon alpha-2 500MIU
Sodium chloride 0.85g
1,2-PD 4.5g
Preparation technology: get aretigenin and the recombinant human interferon alpha-2 of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 17 drug combination injection of the present invention
Aretigenin 1g
Recombinant human interferon alpha-2 0.25MIU
Sodium chloride 1.75g
1,2-PD 16g
Preparation technology is with embodiment 16.
Embodiment 18 drug combination injection of the present invention
Aretigenin 1g
Recombinant human interferon alpha 1 b 0.1g
Sodium chloride 2.5g
1,2-PD 4.5g
Preparation technology: get aretigenin and the recombinant human interferon alpha 1 b of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 19 drug combination injection of the present invention
Aretigenin 0.01g
Recombinant human interferon alpha 1 b 0.005g
Sodium chloride 1.85g
1,2-PD 1.8g
Preparation technology: get aretigenin and the recombinant human interferon alpha 1 b of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 20 drug combination injection of the present invention
Aretigenin 1g
Recombinant human interferon beta 1a 0.1g
Sodium chloride 1.5g
1,2-PD 6.5g
Preparation technology: get aretigenin and the recombinant human interferon beta 1a of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 21 drug combination injection of the present invention
Aretigenin 0.01g
Recombinant human interferon beta 1a 0.003g
Sodium chloride 1.95g
1,2-PD 2.75g
Preparation technology: get aretigenin and the recombinant human interferon beta 1a of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 22 drug combination injection of the present invention
Aretigenin 1g
Recombinant human interferon beta 1b 0.45g
Sodium chloride 2.5g
1,2-PD 6g
Preparation technology: get aretigenin and the recombinant human interferon beta 1b of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 23 drug combination injection of the present invention
Aretigenin 0.01g
Recombinant human interferon beta 1b 0.005g
Sodium chloride 1.85g
1,2-PD 1.8g
Preparation technology: get aretigenin and the recombinant human interferon beta 1b of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 24 drug combination injection of the present invention
Aretigenin 0.3mg
Recombinant human interferon gamma 1MIU
Sodium chloride 0.85g
1,2-PD 1.5g
Preparation technology: get aretigenin and the paclitaxel of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
Embodiment 25 drug combination injection of the present invention
Aretigenin 1.2g
Recombinant human interferon gamma 1MIU
Sodium chloride 0.85g
1,2-PD 7.5g
Preparation technology: get aretigenin and the paclitaxel of recipe quantity, add appropriate distilled water, then add 1,2-PD fully to dissolve, add sodium chloride, filter, embedding, 100 degrees Celsius of sterilizing 30min and get final product.
The impact of embodiment 26 pharmaceutical composition low dose group of the present invention dialogue cell strain NB4 and MR2 propagation and differentiation
1 materials and methods
1.1 material
NB4 cell strain and MR2 cell strain are all purchased from Shanghai Ruijin Hospital Blood Research Institute.ATRA, purchased from Sigma company, is mixed with 10 with dehydrated alcohol
-3the storage liquid of mol/L, filtration, packing ,-20 ℃ keep in Dark Place.Recombinant human interferon alpha-2, recombinant human interferon alpha 1 b, recombinant human interferon beta 1a, recombinant human interferon beta 1b, recombinant human interferon gamma are purchased from Shanghai clone Biology high technology Ltd.The anti-human PML polyclonal antibody of rabbit (I is anti-) is purchased from Chemicon company.Goat-anti rabbit FITC-IgG (II is anti-) is purchased from KPL company.
1.2 cell culture
By NB4, MR2 cell respectively with 1 * 10
6the concentration of/ml is inoculated in RPMI-1640 culture fluid, the CO that is 5% in volume fraction under 37 ℃, saturated humidity condition
2in incubator, cultivate.Getting the cell of exponential phase of growth tests.
1.3MMT method detects cell proliferation situation
By NB4 and MR2 cell respectively with 5 * 10
4the concentration of/ml is inoculated in 96 well culture plates, and every hole inoculum concentration is 100 μ l.Experimental group adds respectively normal saline, aretigenin, recombinant human interferon alpha-2, recombinant human interferon alpha 1 b, recombinant human interferon beta 1a, recombinant human interferon beta 1b, recombinant human interferon gamma, recombinant human interferon alpha-2+aretigenin, recombinant human interferon alpha 1 b+aretigenin, recombinant human interferon beta 1a+ aretigenin, recombinant human interferon beta 1b+ aretigenin, recombinant human interferon gamma+aretigenin.The final concentration of effective active composition is respectively:
Matched group: RPMI-1640 culture fluid,
Arctiin tuple: the final concentration of aretigenin is 0.01mg/ml,
Interferon-ALPHA 2a group: the final concentration of recombinant human interferon alpha-2 is 2.5 * 10
-3mIU/ml,
Interferon α1 b group: the final concentration 5 * 10 of recombinant human interferon alpha 1 b
-7mg/ml,
Interferon beta 1a group: the final concentration 3 * 10 of recombinant human interferon beta 1a
-7mg/ml,
Interferon beta 1b group: the final concentration 5 * 10 of recombinant human interferon beta 1b
-7mg/ml,
Interferon gamma group: the final concentration 0.033MIU/ml of recombinant human interferon gamma,
Low group of interferon-ALPHA 2a compound recipe: the final concentration of recombinant human interferon alpha-2+aretigenin is 2.5 * 10
-3the recombinant human interferon alpha-2 of MIU/ml and the aretigenin of 0.01mg/ml,
Low group of Interferon α1 b compound recipe: the final concentration of recombinant human interferon alpha 1 b+aretigenin is 5 * 10
-7the recombinant human interferon alpha 1 b of mg/ml and the aretigenin of 0.01mg/ml,
Low group of interferon beta 1a compound recipe: the final concentration of recombinant human interferon beta 1a+ aretigenin is 3 * 10
-7the recombinant human interferon beta 1a of mg/ml and the aretigenin of 0.01mg/ml,
Low group of interferon beta 1b compound recipe: the final concentration of recombinant human interferon beta 1b+ aretigenin is 5 * 10
-7the recombinant human interferon beta 1b of mg/ml and the aretigenin of 0.01mg/ml,
Low group of interferon gamma compound recipe: the recombinant human interferon gamma that the final concentration of recombinant human interferon gamma+aretigenin is 0.033MIU/ml and the aretigenin of 0.01mg/ml.
The total measurement (volume) in every hole is 200 μ l.Matched group adds 100 μ lRPMI-1640 culture fluid.After the 1st, 3,5,8 days, by MIT method, detect cell absorbance (A) value, 3 multiple holes are established in every kind of processing, repeat 3 times, get its meansigma methods.(note: within every 3 days, to cell, change culture fluid one time. matched group RPMI-1640 culture fluid.The corresponding RPMI-1640 culture fluid that contains aretigenin, interferon or aretigenin+interferon for experimental group.)
Inhibitory rate of cell growth=[(matched group average A value-experimental group average A value)/matched group average A value] * 100%.Inhibitory rate of cell growth represents the inhibited proliferation to leukaemia, and the higher expression of inhibitory rate of cell growth is stronger to leukaemia's inhibited proliferation.
The detection of 1.4 cell differentiations
By NB4 and MR2 cell respectively with 5 * 10
4the concentration of/ml is inoculated in 25ml culture bottle, and every bottle of inoculum concentration is 5ml.Experimental group adds respectively the active component of above-mentioned concentration, and matched group adds normal saline.Collecting cell after 3 days, adopts nitro nitroblue tetrazolium reduction experiment to detect the differentiation of cell.The centrifugal supernatant that goes, adds 0.1% NBT reactant liquor 0.5ml (NBT containing the 0.1% and TPA of 100ng/ml), cultivates the centrifugal supernatant that goes of 1h. for 37 ℃, precipitation smear, air-dry, Giemsa-Wright dyeing, under oil mirror, amplify 1000 times of observations, count 200 cells, calculate NBT positive rate.NBT positive rate=(NBT positive cell number/total cellular score) * 100%.NBT positive rate represents the induction differentiation capability to NB4 cell and MR2 cell, and NBT positive rate is higher, represents stronger to the induction differentiation capability of NB4 cell and MR2 cell.Repeat to get its meansigma methods 3 times.
1.5PML Protein Detection
Cell culture processes is the same.Collecting cell after 3 days, the centrifugal supernatant that goes, is 1 * 10 with PBS dilution
6the cell suspension of/ml, gets 0.1ml system and drips sheet, carries out immediately indirect immunofluorescence experiment.4% paraformaldehyde is 20min fixedly, adds 0.1%TritonX-100 effect 10min.Add 1: 200 and exempt from anti-human anti-PML, 37 ℃ of water-bath 1h.Add 1: 10 goat-anti rabbit FITC-IgG, 37 ℃ of water-bath 1h.Add buffering glycerol, coverslip mounting.Fluorescence microscopy Microscopic observation, (excitation wavelength 488nm) takes pictures.After fixing and antibody incubation, all with PBS and BSA-PBS, soak respectively rinsing 2 times.
1.6 statistical analysis
Calculate respectively inhibitory rate of cell growth and the NBT positive rate of each experimental group, and respectively each experimental group inhibitory rate of cell growth and NBT positive rate are carried out to χ
2check.If χ
2> χ
2 0.05.1=3.84, with p < 0.05, there is significant difference.
2, experimental result
Each experimental group is compared with matched group, and NB4 cell is all had to inhibited proliferation, and has significance or utmost point significant difference, and inhibitory action prolongation in time strengthens gradually.In the time of the 5th day, the growth of NB4 cell is obviously suppressed, in the time of the 8th day each experimental group to the growth inhibition ratio of NB4 cell in Table 1.Compare with interferon-ALPHA 2a group, Arctiin tuple, low group of interferon-ALPHA 2a compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Compare with Interferon α1 b group, Arctiin tuple, low group of Interferon α1 b compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Compare with interferon beta 1a group, Arctiin tuple, low group of interferon beta 1a compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Compare with interferon beta 1b group, Arctiin tuple, low group of interferon beta 1b compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Compare with interferon gamma group, Arctiin tuple, low group of interferon gamma compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Each experimental group is followed successively by the power of NB4 cell inhibitory effect effect: low group of > interferon group > Arctiin tuple of interferon compound recipe.
Each experimental group is compared with matched group, and MR2 cell is all had to inhibited proliferation, and has significance or utmost point significant difference, and inhibitory action prolongation in time strengthens gradually.In the time of the 5th day, the growth of MR2 cell is obviously suppressed, in the time of the 8th day each experimental group to the growth inhibition ratio of MR2 cell in Table 1.Compare with interferon-ALPHA 2a group, Arctiin tuple, low group of interferon-ALPHA 2a compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Compare with Interferon α1 b group, Arctiin tuple, low group of Interferon α1 b compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Compare with interferon beta 1a group, Arctiin tuple, low group of interferon beta 1a compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Compare with interferon beta 1b group, Arctiin tuple, low group of interferon beta 1b compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Compare with interferon gamma group, Arctiin tuple, low group of interferon gamma compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Each experimental group is followed successively by the power of MR2 cell inhibitory effect effect: low group of > interferon group > Arctiin tuple of interferon compound recipe.
NBT positive rate represents the induction differentiation capability to NB4 cell and MR2 cell, and NBT positive rate is higher, represents stronger to the induction differentiation capability of NB4 cell and MR2 cell.No matter NBT reduction test result shows for NB4 cell or MR2 cell, compare with single medicine, the cell NBT positive rate that interferon compound recipe is low group has significant difference or utmost point significant difference, compare with single medicine, low group of interferon compound recipe also further shows as synergism to the induction differentiation capability of NB4 cell and MR2 cell.Specifically in Table 2.
The impact on NB4 and MR2 cell proliferation of table 1 interferon and aretigenin compositions
△compare p < 0.05 with matched group;
△ △compare p < 0.01 with matched group;
*compare p < 0.05 with Arctiin tuple;
*compare p < 0.01 with Arctiin tuple;
#compare p < 0.05 with interferon-ALPHA 2a group;
$compare p < 0.05 with Interferon α1 b group;
aMP.AMp.Ampcompare p < 0.05 with interferon beta 1a group;
■compare p < 0.05 with interferon beta 1b group;
▲compare p < 0.05 with interferon gamma group.
The impact on the NBT positive rate of NB4 and MR2 cell of table 2 interferon and aretigenin compositions
△compare p < 0.05 with matched group;
△ △compare p < 0.01 with matched group;
*compare p < 0.05 with Arctiin tuple;
*compare p < 0.01 with Arctiin tuple;
#compare p < 0.05 with interferon-ALPHA 2a group;
$compare p < 0.05 with Interferon α1 b group;
aMP.AMp.Ampcompare p < 0.05 with interferon beta 1a group;
■compare p < 0.05 with interferon beta 1b group;
▲compare p < 0.05 with interferon gamma group.
The impact of embodiment 27 pharmaceutical composition high dose group of the present invention dialogue cell strain NB4 and MR2 propagation and differentiation
1, experimental procedure
With embodiment 26.Just the final concentration of effective active composition is adjusted into respectively:
Matched group: RPMI-1640 culture fluid,
Arctiin tuple: the final concentration of aretigenin is 0.01mg/ml,
Interferon-ALPHA 2a group: the final concentration of recombinant human interferon alpha-2 is 5MIU/ml,
Interferon α1 b group: the final concentration 0.001mg/ml of recombinant human interferon alpha 1 b,
Interferon beta 1a group: the final concentration 0.001mg/ml of recombinant human interferon beta 1a,
Interferon beta 1b group: the final concentration 0.0045mg/ml of recombinant human interferon beta 1b,
High group of interferon-ALPHA 2a compound recipe: the recombinant human interferon alpha-2 that the final concentration of recombinant human interferon alpha-2+aretigenin is 5MIU/ml and the aretigenin of 0.01mg/ml,
High group of Interferon α1 b compound recipe: the recombinant human interferon alpha 1 b that the final concentration of recombinant human interferon alpha 1 b+aretigenin is 0.001mg/ml and the aretigenin of 0.01mg/ml,
High group of interferon beta 1a compound recipe: the recombinant human interferon beta 1a that the final concentration of recombinant human interferon beta 1a+ aretigenin is 0.001mg/ml and the aretigenin of 0.01mg/ml,
High group of interferon beta 1b compound recipe: the recombinant human interferon beta 1b that the final concentration of recombinant human interferon beta 1b+ aretigenin is 0.0045mg/ml and the aretigenin of 0.01mg/ml,
2, experimental result:
Each experimental group is compared with matched group, and NB4 cell is all had to inhibited proliferation, and has significance or utmost point significant difference, and inhibitory action prolongation in time strengthens gradually.In the time of the 5th day, the growth of NB4 cell is obviously suppressed, in the time of the 8th day each experimental group to the growth inhibition ratio of NB4 cell in Table 3.Compare with interferon-ALPHA 2a group, Arctiin tuple, high group of interferon-ALPHA 2a compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Compare with Interferon α1 b group, Arctiin tuple, high group of Interferon α1 b compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Compare with interferon beta 1a group, Arctiin tuple, high group of interferon beta 1a compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Compare with interferon beta 1b group, Arctiin tuple, high group of interferon beta 1b compound recipe has significant difference to NB4 cell inhibitory effect, and has synergism; Each experimental group is followed successively by the power of NB4 cell inhibitory effect effect: the high group of interferon compound recipe > interferon group > Arctiin tuple.
Each experimental group is compared with matched group, and MR2 cell is all had to inhibited proliferation, and has significance or utmost point significant difference, and inhibitory action prolongation in time strengthens gradually.In the time of the 5th day, the growth of MR2 cell is obviously suppressed, in the time of the 8th day each experimental group to the growth inhibition ratio of MR2 cell in Table 3.Compare with interferon-ALPHA 2a group, Arctiin tuple, high group of interferon-ALPHA 2a compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Compare with Interferon α1 b group, Arctiin tuple, high group of Interferon α1 b compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Compare with interferon beta 1a group, Arctiin tuple, high group of interferon beta 1a compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Compare with interferon beta 1b group, Arctiin tuple, high group of interferon beta 1b compound recipe has significant difference to MR2 cell inhibitory effect, and has synergism; Each experimental group is followed successively by the power of MR2 cell inhibitory effect effect: the high group of interferon compound recipe > interferon group > Arctiin tuple.
NBT positive rate represents the induction differentiation capability to NB4 cell and MR2 cell, and NBT positive rate is higher, represents stronger to the induction differentiation capability of NB4 cell and MR2 cell.No matter NBT reduction test result shows for NB4 cell or MR2 cell, compare with single medicine, the cell NBT positive rate of the high group of interferon compound recipe has significant difference or utmost point significant difference, compare with single medicine, high group of interferon compound recipe also further shows as synergism to the induction differentiation capability of NB4 cell and MR2 cell.Specifically in Table 4.
The impact on NB4 and MR2 cell proliferation of table 3 interferon and aretigenin compositions
△compare p < 0.05 with matched group;
△ △compare p < 0.01 with matched group;
*compare p < 0.05 with Arctiin tuple;
*compare p < 0.01 with Arctiin tuple;
#compare p < 0.05 with interferon-ALPHA 2a group;
$compare p < 0.05 with Interferon α1 b group;
aMP.AMp.Ampcompare p < 0.05 with interferon beta 1a group;
■compare p < 0.05 with interferon beta 1b group.
The impact on the NBT positive rate of NB4 and MR2 cell of table 4 interferon and aretigenin compositions
△compare p < 0.05 with matched group;
△ △compare p < 0.01 with matched group;
*compare p < 0.05 with Arctiin tuple;
*compare p < 0.01 with Arctiin tuple;
#compare p < 0.05 with interferon-ALPHA 2a group;
$compare p < 0.05 with Interferon α1 b group;
aMP.AMp.Ampcompare p < 0.05 with interferon beta 1a group;
■compare p < 0.05 with interferon beta 1b group.
Claims (1)
1. a pharmaceutical composition, is characterized in that its active component is comprised of interferon and aretigenin, and wherein said interferon is at least selected from a kind of in alpha-interferon, beta-interferon and gamma interferon.
2. pharmaceutical composition as claimed in claim 1, is characterized in that described alpha-interferon is recombinant human interferon alpha-2 or recombinant human interferon alpha 1 b.
3. pharmaceutical composition as claimed in claim 2, it is characterized in that the described amount of recombinant human interferon alpha-2 and the ratio between the amount of aretigenin are for (0.25-500): 1, the amount of recombinant human interferon alpha-2 represents with million international units, and the amount of aretigenin represents with milligram.
4. pharmaceutical composition as claimed in claim 2, is characterized in that described recombinant human interferon alpha 1 b and the weight ratio of aretigenin are (5 * 10
-5-0.1): 1.
5. pharmaceutical composition as claimed in claim 1, is characterized in that described beta-interferon is recombinant human interferon beta 1a or recombinant human interferon beta 1b.
6. pharmaceutical composition as claimed in claim 5, is characterized in that described recombinant human interferon beta 1a and the weight ratio of aretigenin are (3 * 10
-5-0.1): 1.
7. pharmaceutical composition as claimed in claim 5, is characterized in that described recombinant human interferon beta 1b and the weight ratio of aretigenin are (5 * 10
-5-0.45): 1.
8. pharmaceutical composition as claimed in claim 1, is characterized in that described gamma interferon is recombinant human interferon gamma.
9. pharmaceutical composition as claimed in claim 8, it is characterized in that the described amount of recombinant human interferon gamma and the ratio between the amount of aretigenin are 1: (0.3-1200), the amount of recombinant human interferon gamma represents with million international units, and the amount of aretigenin represents with milligram.
10. the application of pharmaceutical composition as claimed in claim 1 in the medicine of preparation inhibition Leukemia Cell Proliferation and inducing leukemia cell differentiation.
11. application as described in claim 10, it is characterized in that described interferon is recombinant human interferon alpha-2, ratio between the amount of recombinant human interferon alpha-2 and the amount of aretigenin is (0.25-500): 1, the amount of recombinant human interferon alpha-2 represents with million international units, and the amount of aretigenin represents with milligram.
12. application as claimed in claim 10, is characterized in that described interferon is recombinant human interferon alpha 1 b, and the weight ratio of recombinant human interferon alpha 1 b and aretigenin is (5 * 10
-5-0.1): 1.
13. application as claimed in claim 10, is characterized in that described interferon is recombinant human interferon beta 1a, and the weight ratio of recombinant human interferon beta 1a and aretigenin is (3 * 10
-5-0.1): 1.
14. application as claimed in claim 10, is characterized in that described interferon is recombinant human interferon beta 1b, and the weight ratio of recombinant human interferon beta 1b and aretigenin is (5 * 10
-5-0.45): 1.
15. application as claimed in claim 10, it is characterized in that described interferon is recombinant human interferon gamma, ratio between the amount of recombinant human interferon gamma and the amount of aretigenin is 1: (0.3-1200), the amount of recombinant human interferon gamma represents with million international units, and the amount of aretigenin represents with milligram.
16. pharmaceutical compositions as described in as arbitrary in claim 1-10, is characterized in that it is microemulsion formulation or injection.
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| CN101049484A (en) * | 2007-05-14 | 2007-10-10 | 郝来勤 | Strong effective Chinese traditional medicine for treating lung cancer and other various cancers |
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| CN101049484A (en) * | 2007-05-14 | 2007-10-10 | 郝来勤 | Strong effective Chinese traditional medicine for treating lung cancer and other various cancers |
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