CN103610684B - Application of saccharides in preparing medicine for treating platelet quantity related diseases - Google Patents
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an application of saccharides in preparing a medicament for treating diseases related to platelet number. The saccharide may be one or more of a monosaccharide, a redox derivative of said monosaccharide, an oligosaccharide, a polysaccharide, a complex polysaccharide and a glycoside. The diseases related to platelet number include primary or secondary thrombocythemia and primary or secondary thrombocytopenia. Experiments show that the anti-platelet GPIb alpha N-terminal antibody can induce platelet activation and apoptosis, can cause platelets to be rapidly cleared in the liver by macrophages, and the saccharides can obviously inhibit platelet clearing caused by the anti-platelet GPIb alpha antibody and regulate and control the number of the platelets. The invention firstly provides the application of the saccharides in the preparation of the medicines for treating the diseases related to the number of the platelets, and expands the new application of the saccharides.
Description
Technical field
The present invention relates to field of biological pharmacy, be specifically related to the application of saccharide in the medicine of preparation treatment platelet counts relevant disease.
Background technology
The platelet counts that various factors causes clinically can cause relevant disease extremely, as constitutional or secondary thrombocythemia, constitutional or Secondary cases thrombocytopenia, comprises immune thrombocytopenia etc.Thrombocytosis is that a kind of agnogenic paraplasm companion platelet continues to increase is main myeloproliferative disease, its clinical characters is be more common in the adult, normal hemorrhage with spontaneous skin mucosa of more than 40 years old, recurrent exerbation, thrombosis, splenomegaly, platelet persistency showed increased can be had, etiology unknown.The Pathogenesis of Hemorrhage of this disease may strengthen relevant with dysfunction of platelet or fibrinolysis.According to American-European 5 states to the Epidemiological study of primary thrombocytosis, sickness rate is 0.59/10 ten thousand ~ 2.53/10 ten thousand, over nearly 20 years, sickness rate adds 3.2 times of (JohanssonP.Epidemiologyofthemyeloproliferativedisordersp olycytothemiaveraandessentialthrombocythemia.SemThrombHa emost, 2006,32:171-173).
Immunologic thrombocytopenic purpura (immunethrombocytopenia in thrombocytopenia, ITP) be clinical common hemorrhage, account for the 30%(Hou Ming of hemorrhage sum, Dai Kesheng, Peng Jun, chief editor, " blood platelet disorder ", the second edition, scientific and technical literature publishing house, 2012,142 pages).Research shows that ITP is a kind of autoimmune disease, this disease be owing to producing anti-megalokaryocyte or hematoblastic autoantibody in body, wherein antiplatelet antibody is primarily of the two large antibody-like composition (MichelleLeeWebster of anti-platelet membrane glycoprotein glycoprotein (GP) Ib-IX-V and GPIIb/IIIa, EbrahimSayeh, MinCrow, PingguoChen, BernhardNieswandt, JohnFreedman, andHeyuNi.RelativeefficacyofintravenousimmunoglobulinGin amelioratingthrombocytopeniainducedbyantiplateletGPIIbII IaversusGPIb α antibodies.Blood, 2006108:943-946).Two large antibody-likes are after platelet is combined in patient body, and the Fc end mainly through antibody combines with the reticuloendothelial system (reticuloendothelialsystem, RES) in human body, causes platelet to be removed by organs such as spleens.
Although in recent years, the study of incident mechanism of ITP achieves a series of progress, the megalokaryocyte quality and quantity proposing autoantibody mediation in humoral immune mechanisms is abnormal.In cellular immune mechanism, propose cytotoxic T cell directly dissolve hematoblastic theory, but the definite pathogeny about ITP is not yet clear and definite.
Due to pathogeny not yet define, therefore, there is no the method for radical cure.American I TP guideline recommendation platelet is lower than 20 ~ 30 × 10
9/ L or lower than 50 × 10
9it is hemorrhage and have hemorrhage person accepts treatment that/L merges obvious skin mucosa.Therapeutic strategy main at present suppresses the generation of antibody and hematoblastic destruction, promotes hematoblastic generation, comprises various immunosuppressant, excision spleen, short thrombocytopoiesis etc.The object for the treatment of Platelet is counted bring up to level of security, reduces case fatality rate.
Current medicine is mainly the glucocorticoid, monoclonal antibody etc. of Hemopoietic factor thrombopoietin, interleukin-11 and immunologic intervention.But above-mentioned various medicine has respective limitation separately, there is the potential side effect such as thrombosis, myelofibrosis, vein occlusion as ITP commonly uses medicine RhTPPO RHTPO (rhTPO); Intravenous injection immunoglobulin (IVIG) is applicable to intractable or needs emergency treatment ITP patient; side effect is light; it is unrestricted to use; but because its action time is short; expensive; limit clinical practice (BeardsleyDS, ErtemM.Plateletautoantibodiesinimmunethrombocytopenicpur pura.TransfusSci.1998; 19:237-244).Therefore a kind of medicine of effective treatment platelet counts relevant disease is found to be Lin bed Yi Han problem to be solved.
Saccharide is also called carbohydrate, the polymer of this compounds that its structure refers to polyhydroxy aldehyde, ketone, alcohol and derivant thereof and connected by glycosidic bond.Saccharide compound includes monosaccharide and oxidoreduction derivant, oligosaccharide, polysaccharide and complex polysaccharide and glucosides class.The Carbohydrate drugs of current mankind's use not lower more than 500, includes Multiple Classes of Antibiotics, nucleoside, polysaccharide, glycolipid, glycoprotein etc.Saccharide is extensively present in occurring in nature, cheap, wide material sources.But the application of Carbohydrate drugs in treatment platelet counts relevant disease not yet met report.
Summary of the invention
The object of the invention is to provide a kind of medicine effectively treating platelet counts relevant disease.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:
The application of saccharide in the medicine of preparation treatment platelet counts relevant disease.
Described saccharide in above-mentioned application can be one or more of monosaccharide, the oxidoreduction derivant of described monosaccharide, oligosaccharide, polysaccharide, complex polysaccharide and glucosides apoplexy due to endogenous wind.
All monosaccharides that described monosaccharide in above-mentioned application is is representative with N-acetyl-GLUCOSAMINE, β-N-acetyl-GLUCOSAMINE, methyl-β-D-glucoside, D-Glucose.
Platelet counts relevant disease described in above-mentioned application comprises constitutional or secondary thrombocythemia, constitutional or secondary thrombocytopenia, but is not limited to above-mentioned disease, and every disease caused by platelet counts is included.Thrombocytopenia described in above-mentioned application is representative with immune thrombocytopenia, but is not limited to immune thrombocytopenia.
Medicine described in above-mentioned application can be saccharide.
Medicine described in above-mentioned application can be the compositions comprising saccharide.
Medicine described in above-mentioned application can be prepared into various dosage form according to common mode.
Medicine described in above-mentioned application can oral or injection or topical application.
The present invention find antiplatelet GPIb α N hold antibody can induced platelet activation, apoptosis, platelet can be caused to be removed rapidly by macrophage at liver, and platelet that saccharide can significantly suppress platelet GPIbα antibody to cause is removed, regulation and control platelet counts.
The present invention also finds that antiplatelet GPIb α N holds antibody can induced platelet aggregation, and the GPIb α that antiplatelet GPIb α N holds the platelet aggregation of antibody induction to depend on antibody induction assembles, and does not rely on Fc section.
The invention has the advantages that:
(1) based on new pathogenesis, there is the feature of good effect, high specificity.
(2) the present invention has excavated saccharide in the frontier for the preparation for the treatment of platelet counts relevant disease medicine.
(3) carbohydrate source is extensive, with low cost, and potential applicability in clinical practice is wide.
Accompanying drawing explanation
Fig. 1 shows antiplatelet GPIb Alpha antibodies AN51 can induced platelet aggregation.Figure 1A. respectively to IgG and AN51 adding 2 μ g/ml in PRP, with the change of platelet aggregation instrument record platelet aggregation.Risto represents the platelet aggregation of ristomycin induction; Figure 1B. multiple monoclonal antibody is on the impact of platelet aggregation, and hematoblastic maximum agglutination rate in vertical coordinate represents in 15min, wherein only has AN51 can induced platelet aggregation significantly; After Fig. 1 C. stirs 15min respectively to IgG and AN51 adding 2 μ g/ml in PRP in gathering instrument, imaging result after object lens amplify 40 times.
The GPIb α that the platelet aggregation that Fig. 2 shows AN51 induction depends on antibody induction assembles, and does not rely on Fc section.Fig. 2 A.PRP respectively with mouse IgG, the IV.3 incubation 10min at normal temperatures of 10 μ g/ml, add 2 μ g/mlAN51, platelet aggregation instrument record platelet aggregation changes; The statistical result of maximum agglutination rate in Fig. 2 B. Fig. 2 A15min; Fig. 2 C. is respectively to the AN51F (ab ') adding PBS and 2 μ g/ml in PRP
2fragment, platelet aggregation instrument record platelet aggregation changes; The statistical result of maximum agglutination rate in Fig. 2 D. Fig. 2 C15min; Fig. 2 E. respectively to adding the AN51 monomer Fab fragment of 2 μ g/ml in PRP, (AN51 adds sheep anti-Mouse F (ab ') for dimer (AN51) and the tetramer
2, AN51+GAM), platelet aggregation instrument record platelet aggregation changes; The statistical result of maximum agglutination rate in Fig. 2 F. Fig. 2 E15min.
Fig. 3 shows AN51 induced platelet apoptosis.Fig. 3 is IgG, AN51 of μ g/ml and the hematoblastic ratio of apoptosis after washing platelet incubation 1-6 hour A.2; The change of Fig. 3 B.2 μ g/mlIgG, HIP1 and AN51 and washing platelet incubation 6 hours blood platelet apoptosis protein expressions.
Fig. 4 shows AN51 and platelet count in Cavia porcellus body can be caused to decline rapidly.By Cavia porcellus forearm vein respectively to different Cavia porcelluss injection normal mouse IgG, 0.05mg/kg, 0.1mg/kg and 0.2mg/kgAN51, at the appointed time put eye socket blood sampling, platelet Counting.In figure, often represents the multiple of this time point platelet count divided by the sugared thromboblast counting of injection.
Fig. 5 shows the guinea pig platelets removing that GlcNac can suppress AN51 to induce.After injecting PBS, 5mM and 10mMGlcNac by Cavia porcellus forearm vein respectively to Cavia porcellus, inject 0.2 μ g/kgAN51 by opposite side forearm vein, at the appointed time put eye socket blood sampling, platelet Counting.In figure, often represents the multiple of fixed time platelet count divided by the sugared thromboblast counting of injection.
Fig. 6 shows the guinea pig platelets removing that β-GlcNac can suppress AN51 to induce.After injecting PBS, the β-GlcNac of 5 μMs by Cavia porcellus forearm vein respectively to Cavia porcellus, inject 0.2 μ g/kgAN51 by opposite side forearm vein, 5min eye socket blood sampling before injection of antibodies and after injection of antibodies, platelet Counting.Vertical coordinate representative residue platelet count accounts for the percentage ratio of injection of antibodies platelet count.
Fig. 7 shows the guinea pig platelets removing that β-Glc can suppress AN51 to induce.To be injected the β-Glc of PBS, 25mM respectively to Cavia porcellus by Cavia porcellus forearm vein after, inject 0.2 μ g/kgAN51 by opposite side forearm vein, 5min eye socket blood sampling before injection of antibodies and after injection of antibodies, platelet Counting.Vertical coordinate representative residue platelet count accounts for the percentage ratio of injection of antibodies platelet count.
Detailed description of the invention
Saccharide described in the present invention can be prepared according to known conventional method, and (local records are outstanding, write, " Carbohydrate drugs synthesis and preparation ", the first edition, Chemical Industry Press, 2010) or patent of invention in 1991 disclosed in saccharide preparation method preparation (patent No.: 91108011, denomination of invention: the preparation method of saccharide).In addition, described saccharide also can be buied by commercial sources.
Medicine of the present invention can be only saccharide, also can be the compositions containing saccharide.
Usually, medicine can be made various dosage form, described medicine can be oral or inject or topical application.
In the present invention, platelet counts relevant disease comprises constitutional or secondary thrombocythemia, constitutional or secondary thrombocytopenia, but is not limited to above-mentioned disease, and every disease caused by platelet counts is included.
Inventor finds, platelet GPIbα born of the same parents outer end can be caused to flock together, make β-N-acetyl-GLUCOSAMINE that GPIb α N-end connects flock together simultaneously after the antibody of anti-platelet membrane glycoprotein GPIb-IX-V is combined with the platelet of people.β-N-acetyl-GLUCOSAMINE after gathering is to hepatic macrophages surface α
mβ
2the affinity of integrin receptor increases greatly, makes platelet be engulfed removing by hepatic macrophages.Based on above-mentioned discovery, the present inventor completes the present invention.
The present invention finds that saccharide can suppress platelet destruction, regulation and control platelet counts.Experiment confirms that the platelet that one of mechanism of saccharide regulation and control platelet counts causes for suppression antibody A N51 is removed.In a preferred embodiment, described platelet counts relevant disease is selected from ITP.Certainly, platelet counts relevant disease does not only include ITP, also comprises constitutional or secondary thrombocythemia, non-immunity thrombocytopenia etc.
Illustrate the present invention with experimental example below, but do not limit the present invention in any form.
Experiment material
Mouse anti human platelet GPIbα N holds antibody A N51, anti-human GPIX antibody SZ1 and anti-human GPIIIa antibody SZ21 to be obtained by proteinG affinity chromatography purification by mouse ascites.Mouse anti human GPIb Alpha antibodies WM23 is so kind as to give by Du little Ping.Other anti-human GPIb Alpha antibodies AK2, HIP1 and VM16d are respectively purchased from Abcam, eBioscience and ThermoScientifc company.N-acetyl-GLUCOSAMINE (N-acetyle-D-glucosamine, GlcNac), β-N-acetyl-GLUCOSAMINE (β-N-acetyle-D-glucosamine, β-GlcNac), methyl-β-D-glucoside (methyl-β-D-glucopyranoside, β-Glc), D-Glucose (D-glucose, Glc), ristomycin, buys from Sigma-Aldrich company.Mouse IgG fraction test kit is purchased from ThermoScientific company.ProteinG agarose gel is purchased from GEhealthcare.Normal mouse IgG buys from SantaCruz company.Anti-human Fc γ RIIA monoclonal antibody IV.3 is purchased from StemCell company.The antibody of JC-1, anti-Bak, Bad, Bcl-2, Bcl-xl is purchased from green skies biotechnology research institute.
Hematoblastic separation and abstersion
Get normal person fresh venous ACD with 1:7 anticoagulant.300g isolates Platelet-rich plasm (PRP), and PRP obtains platelet by 1500g is centrifugal, after CGS buffer solution 2 times, is resuspended in Tyrode ' the s buffer of improvement to 3 × 10
8/ ml, leaves standstill 1-2h at normal temperatures.For the blood 3.8% sodium citrate 1:9 anticoagulant of platelet aggregation test, 300g isolates PRP.
Platelet aggregation
The PRP getting sodium citrate anticoagulant adds normal mouse IgG, antiplatelet GPIb α many kinds of monoclonal antibodies, ristomycin or AN51F (ab ') respectively
2, detect platelet aggregation with platelet aggregation instrument (Chrono-log).In the platelet aggregation test that the monoclonal antibody IV.3 of Effect of Anti Fc γ RIIA induces AN51,10 μ g/mlIV.3 first with PRP incubation 5 minutes at normal temperatures, then add AN51 detection of aggregation.
Flow cytometry blood platelet apoptosis
Washing platelet and 2 μ g/mlAN51 difference incubation are after 1-6 hour, JC-1 labelling platelet mitochondrial membrane potential, Flow cytometry platelet mitochondrial membrane potential changes, platelet red fluorescence being converted into green fluorescence is defined as apoptosis platelet, and remembers the percentage ratio of apoptosis platelet colony.
SDS-PAGE and WesternBlot
Washing platelet and normal mouse IgG, control antibodies HIP1 and AN51 respectively at 37 DEG C incubation add 6 × SDS sample-loading buffer on ice after cracking 30min through cell pyrolysis liquid after 6 hours.Albumen carries out WesternBlot after being needed on nitrocellulose filter after being separated by SDS-PAGE.Primary antibodie is respectively the antibody of anti-Bak, Bad, Bcl-2, Bcl-xl, and two resist the goat anti-rabbit antibody for HRP labelling, and ECL luminescence method develops the color.
Zoopery
By Cavia porcellus forearm vein injection 0.05mg/kg, 0.1mg/kg and 0.2mg/kgAN51 or 0.2mg/kg normal mouse IgG, at the appointed time put and taken a blood sample by eye socket, with 3.8% sodium citrate anticoagulant.Anticoagulation whole bliid platelet analyzer platelet Counting.Remove in the experiment of impact at research sugar on platelet, first pass through the N-acetyl-GLUCOSAMINE of Cavia porcellus forearm vein injection variable concentrations, β-N-acetyl-GLUCOSAMINE, methyl-β-D-glucoside or D-Glucose, after 5 minutes, again by opposite side forearm vein injection 0.2mg/kgAN51, the same blood sampling platelet Counting.
Experimental result
1, antiplatelet GPIb α N holds antibody A N51 to cause platelet aggregation
The epi-position of monoclonal antibody AN51 is positioned at human blood platelets GPIb α N and holds 1-35 amino acid residue place, as shown in accompanying drawing 1A and B, can assemble (aggregation rate is 10%-20%) by induced platelet with AN51 and PRP incubation.The antibody of other epi-positions of antiplatelet GPIb α is as AK2(35-59), HIP1(59-81), VM16d(201-168), WM23(macroglycopeptide) or the antibody of other albumen anti-as SZ1(GPIX), SZ21(GPIIIa) (accompanying drawing 1B) can not be there is by induced platelet aggregation significantly.Micro-imaging result shows that AN51 induced platelet forms little aggregation (accompanying drawing 1C).
2, the GPIb α that the platelet aggregation that AN51 induces depends on antibody induction assembles, and does not rely on Fc section
The Fc section of antiplatelet antibody is by occurring to activate and assemble with Fc γ RIIA receptors bind induced platelet.In order to study AN51 whether by with Fc γ RIIA zygotic induction platelet aggregation, have detected the antibody I of antagonism Fc γ RIIA V.3 on the impact of the platelet aggregation of AN51 induction.Accompanying drawing 2A shows the gathering that IV.3 does not suppress AN51 and induces.In addition, the AN51F (ab ') of Fc section is not comprised
2fragment also can induced platelet aggregation (accompanying drawing 2B), further illustrates AN51 and causes platelet aggregation not rely on Fc section.
Platelet GPIbα multimerization can induced platelet aggregation, whether being assembled thus activated blood platelet by induction GPIb α to inquire into AN51, have detected AN51 monomer Fab fragment, AN51IgG(dimer) and the anti-coupling AN51(tetramer of sheep anti-Mouse (GAM) two) impact on platelet aggregation.As shown in accompanying drawing 2C, monomer (Fab) can not induced platelet aggregation; Compared with dimer (AN51), the tetramer (AN51+GAM) can induce larger gathering that (10%vs20%) occurs.
Therefore, the GPIb α that the platelet aggregation that AN51 induces depends on antibody induction assembles, and does not rely on Fc section.
3, AN51 can induced platelet apoptosis
One of mitochondrial membrane potential depolarization index that can be used as blood platelet apoptosis.Washing platelet with respectively with mouse IgG, AN51 incubation 1-6 hour at 37 DEG C of 2 μ g/ml, detect apoptosis by the depolarization of detection line mitochondrial membrane potential.Fig. 3 A showed incubation after 4 hours, and the ratio of the apoptotic cell that AN51 causes has significant difference compared with IgG, and in time-dependent trend.Have detected mouse IgG, control antibodies HIP1 and the AN51 impact on blood platelet apoptosis protein expression in addition.2 μ g/ml antibody and washing platelet incubation cracking platelet after 6 hours, WesternBlot result (accompanying drawing 3B) shows, compare with control antibodies HIP1 with IgG, AN51 can cause the up-regulated of pro apoptotic protein Bad and Bak, presses down apoptotic proteins Bcl-2 and Bcl-xl down-regulated expression.Therefore, AN51 can inducing mitochondrial approach rely on blood platelet apoptosis.
4, AN51 can cause platelet in Cavia porcellus body to be eliminated rapidly
The antibody of anti-GPIb α has removes hematoblastic effect in Mice Body.AN51 specificly can be combined with Cavia porcellus GPIb α and causes GPIb α to assemble.Whether platelet in Cavia porcellus body can be removed for inquiring into AN51, by to Cavia porcellus forearm vein injection 0.05mg/kg, 0.1mg/kg and 0.2mg/kg AN51, before injection of antibodies, after injection of antibodies 5min, 20min, 1hr, 3hr respectively eye socket get blood platelet Counting.Accompanying drawing 4 shows, AN51 can remove platelet after 5 minutes, and in dose-dependant trend.Guinea pig platelets returns to normal level in injection of antibodies after 3 hours gradually.The F of AN51 (ab ')
2fragment also can cause guinea pig platelets to reduce rapidly.Injection normal mouse IgG is on the not impact of guinea pig platelets counting.
5, the platelet that N-acetyl-GLUCOSAMINE significantly suppresses antibody A N51 to cause is removed
Before injecting AN51, injected the GlcNac of 5mM and 10mM respectively by Cavia porcellus forearm vein after, then inject the AN51 of 0.2mg/kg to opposite side forearm vein, at the appointed time put the rear platelet Counting of eye socket blood sampling respectively.The platelet that the GlcNac that accompanying drawing 5 shows 10mM can significantly suppress AN51 to induce is removed.
6, the platelet that β-N-acetyl-GLUCOSAMINE significantly suppresses antibody A N51 to cause is removed
Injected the β-GlcNac of 10 μMs respectively by Cavia porcellus forearm vein before injecting AN51 after, then inject the AN51 of 0.2mg/kg to opposite side forearm vein, at the appointed time put the rear platelet Counting of eye socket blood sampling respectively.Accompanying drawing 6 shows the injection of antibodies platelet that after 5 minutes, the β-GlcNac of 10 μMs can significantly suppress AN51 to induce and removes.
7, the platelet that methyl-β-D-glucoside significantly suppresses antibody A N51 to cause is removed
Injected the β-Glc of 25mM respectively by Cavia porcellus forearm vein after, then inject the AN51 of 0.2mg/kg to opposite side forearm vein, at the appointed time put the rear platelet Counting of eye socket blood sampling respectively.The platelet that the β-Glc that accompanying drawing 7 shows injection of antibodies 25mM after 5 minutes can significantly suppress AN51 to induce is removed.
8, the platelet that D-Glucose significantly suppresses antibody A N51 to cause is removed
Inject the Glc of 100mM respectively by Cavia porcellus forearm vein before injecting AN51 after, then inject the AN51 of 0.2mg/kg to opposite side forearm vein, at the appointed time put the rear platelet Counting of eye socket blood sampling respectively.The platelet that the Glc of injection of antibodies 100mM after 5 minutes can significantly suppress AN51 to induce is removed.
Claims (6)
- The application in preparation treatment platelet counts relevant disease medicine of 1.N-acetyl-GLUCOSAMINE, β-N-acetyl-GLUCOSAMINE, methyl-β-D-glucoside.
- 2. application according to claim 1, is characterized in that: described platelet counts relevant disease comprises constitutional or secondary thrombocythemia, constitutional or secondary thrombocytopenia.
- 3. application according to claim 2, is characterized in that: described platelet counts relevant disease is immune thrombocytopenia.
- 4. application according to claim 1 and 2, is characterized in that: described medicine is saccharide.
- 5. the application according to claim 1 or 3, is characterized in that: described medicine is the compositions comprising saccharide.
- 6. application according to claim 5, is characterized in that: described medicine is oral or injects or topical application.
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