CN106362161A - Coupling conjugate of micromolecular CD33 antibody peptide and vincristine - Google Patents
Coupling conjugate of micromolecular CD33 antibody peptide and vincristine Download PDFInfo
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- CN106362161A CN106362161A CN201610748177.9A CN201610748177A CN106362161A CN 106362161 A CN106362161 A CN 106362161A CN 201610748177 A CN201610748177 A CN 201610748177A CN 106362161 A CN106362161 A CN 106362161A
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- vincristine
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
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Abstract
The invention discloses a coupling conjugate of CD33 antibody peptide and vincristine, belonging to the field of antibody coupling medicines. The coupling conjugate is characterized that the coupling conjugate is composed of vincristine and CD33 antibody peptide, and the CD33 antibody peptide adopts a brand-new sequence; the CD33 antibody peptide is prepared by adopting a chemical synthesis method; the carbodiimide coupling is adopted between vincristine-hemisuccinate and the CD33 antibody peptide; the coupling conjugate is applied to the preparation of medicines for treating tumor. The coupling conjugate has the beneficial effects that the tumor targeting property of vincristine is promoted, the curative effects are improved, and the toxic and side effects are reduced.
Description
Technical field
The present invention is antibody coupling drug world.In particular it relates to cd33 antibody polypeptides are coupled antineoplastic agent
The conjugate of vincristine.
Background technology
Vincristine (vincristine, oncovin, vcr) is the biology extracting in apocynaceae plant Herba Catharanthi Rosei
Alkali, because antitumor action is good, its preparation is as clinical antitumor agents at present.It is clinically used for acute leukemia, especially
Virgin acute leukemia, evident in efficacy to acute lymphoblastic leukemia.Malignant lymphoma, germ cell tumor, small cell lung cancer,
Ewing sarcoma, nephroblastoma, neuroblastoma, breast carcinoma, chronic lymphocytic leukemia, digestive tract cancer, melanoma
And multiple myeloma etc..
Although vincristine is widely used in the chemotherapy regimen of cancer, because chemotherapy exists than more serious bone
Marrow suppresses, intestines and stomach reaction and lesions of liver and kidney etc..Progress is toward liposomal vincristine body direction, new for reducing Changchun now
The neurotoxicity of alkali.Liposome is a kind of targeted drug carrier, belongs to a kind of novel form of targeting drug delivery system.But, lipid
There is unstability in body dosage form, therefore, be not used for clinic all the time in itself.
Anti-tumour antibody is passed through chemical modification and is coupled with effector molecule, these conjugates existing specific recognition tumor antigen
Ability, also remain the toxicity of effector molecule killing tumor cell, be injected in tumor patient body, orientablely dense gather swollen
Tumor position, selective killing tumor cell.Therefore, inventor wants to vincristine and specific antibody coupling, from
And produce the targeting of tumor cell, improve curative effect, reduce toxic and side effects.
Cd33 is myeloid cell differentiation antigen, and molecular weight is 67kd, is mainly distributed on medullary system hemocyte, particularly in differentiation
Commitment.Patients with Acute Myeloid Leukemia more than 90% for the cd33 has expression.There is no table in hemopoietic stem cell surface
Reach, also do not express in ripe granulocyte and its hetero-organization, therefore cd33 becomes the good target spot of marrow series leukemia treatment.Mesh
The front clinical trial testing having had tumor resists the antibody of cd33 receptor.Cd33 antibody can be with targeting in tumor cell table
Face, and there is antineoplastic action in itself.The antibody commonly used at present is still many with Mus source monoclonal antibody, and molecular weight is larger, has relatively
Strong immunogenicity, is used for people for a long time and knows from experience generation immunoreation, lead to autoimmune disease.Therefore, the invention provides one
Species specificity is strong, the conjugate that the higher small molecule cd33 antibody polypeptides of purity are coupled with vincristine, promotes vincristine
Tumor-targeting, improves curative effect, reduces toxic and side effects.
Content of the invention
Goal of the invention
The present invention provides a kind of high specificity, the knot that the higher small molecule cd33 antibody polypeptides of purity are coupled with vincristine
Compound, promotes the tumor-targeting of vincristine, improves curative effect, reduces toxic and side effects.
Technical scheme
Technical program of the present invention lies in providing the coupling conjugate of a kind of small molecule cd33 antibody polypeptides and vincristine,
It is characterized in that: (1) uses vincristine, succinic anhydrides and the dissolving of triethylamine 100ml acetyl triethyl, is heated to reflux 1 hour；
(2) solvent is removed in distillation, residue 5%nahco3 aqueous dissolution；(3) ether washes twice, and then carries out acid with h2so4
Change, to ph2.0；(4) use chloroform-hexane crystallization, obtain solid content；(5) 2.5g hemisuccinic acid ester is taken to be dissolved in 6.5ml thionyl chloride,
65 DEG C, heat 30 minutes, be evaporated under reduced pressure, be dried 1 hour；(6) above-mentioned product is dissolved in 15ml n, n- dimethyl-acetic acid acetyl
In amine, reaction 2 hour is stirred at room temperature；(7) 65 DEG C, after vacuum evaporation, so that crystallization is separated out with isopropanol, obtain vincristine-half
Succinate；(8) vincristine-hemisuccinic acid ester and cd33 antibody polypeptides are coupled, and obtain final product.Described cd33 antibody polypeptides adopt
Prepared by chemical synthesiss.Described vincristine-hemisuccinic acid ester is coupled with cd33 antibody polypeptides and adopts carbodiimides.Institute
State coupling conjugate, the application in preparation is for tumor.It is preferably and be used for treating in breast cancer medicines in preparation
Application.
Beneficial effect
The small molecule cd33 antibody polypeptides of the present invention and the coupling conjugate of vincristine, can be used for promoting vincristine
Tumor-targeting, improves curative effect, reduces toxic and side effects.Have the beneficial effects that (1) improves vincristine tumor tissues in vivo
Concentration；Reduce vincristine in whole blood, the distribution of the tissue such as Yi Jixin, liver, spleen, lung, kidney, brain simultaneously；(2) significance suppression is swollen
The propagation of oncocyte；(3) reduce the toxicity of vincristine；(4) the coupling conjugate of cd33 antibody polypeptides and vincristine can be with
The specific binding of tumor cell；(5) in tumor-bearing mice body, improve survival rate.
Specific embodiment
Polypeptide is by Shanghai raw work gill synthesis.
Embodiment 1
Prepare the coupling conjugate of cd33 antibody polypeptides and vincristine: with vincristine, succinic anhydrides and triethylamine
With the dissolving of 100ml acetyl triethyl, it is heated to reflux 1 hour；Solvent is removed in distillation, residue 5%nahco3 aqueous dissolution；Ether
Wash twice, be then acidified with h2so4, to ph2.0；With chloroform-hexane crystallization, obtain solid content；Take 2.5g hemisuccinic acid
Ester is dissolved in 6.5ml thionyl chloride, 65 DEG C, heats 30 minutes, is evaporated under reduced pressure, is dried 1 hour；Above-mentioned product is dissolved in 15ml n,
In n- dimethyl-acetic acid acetamide, reaction 2 hour is stirred at room temperature；65 DEG C, after vacuum evaporation, crystallization is made to separate out with isopropanol,
Obtain vincristine-hemisuccinic acid ester；Vincristine-hemisuccinic acid ester and cd33 antibody polypeptides adopt carbodiimidesization to be coupled,
Lyophilizing, obtains final product.
Embodiment 2
Internal vigor with transplanted human breast carcinoma model inspection cd33 antibody polypeptides and the coupling conjugate of vincristine.
6-8w age scid female mice, mice is randomly divided into 5 groups, every group 10.(1) blank group；(2) conjugate low dosage
Group；(3) conjugate middle dose group；(4) conjugate high dose group；(5) vincristine positive group.Set up transplanted human breast carcinoma mould
Type, the 7th day after inoculation breast cancer cell, tail vein administration administration respectively.Dosage regimen is: blank group adds same volume
Solvent, experimental group sets 3 dosage: 0.125,0.25,0.5 μm of ol/kg for conjugate, and the positive organizes the Changchun for 0.5 μm of ol/kg
New alkali, daily administration, continuous 14 days.After 21 days, observe mouse survival quantity, calculate survival rate.Result shows, conjugate can have
Effect ground protection tumor-bearing mice, can improve the survival rate of tumor-bearing mice, survival rate in 0.125,0.25,0.5 μm of ol/kg of dosage
It is respectively 57.3,75.2,89.0%.
Embodiment 3
The in-vitro multiplication rate experiment of breast cancer cell: using mtt colorimetry.By the human breast cancer cell of logarithmic growth, with
1.0×105Add in 96 well culture plates, cultivate 24h；Experiment is divided into 5 groups, respectively blank group, positive group and low middle high dose
The cd33 antibody polypeptides that obtain of embodiment 1 and vincristine coupling conjugate；It is separately added into the dmem culture of same volume
The coupling knot of base (hyclone, containing hyclone 20%), vincristine (1mmol/ml) and cd33 antibody polypeptides and vincristine
Compound (12.5,25,50 μm of ol/ml).Every hole sets five multiple holes, cultivates 48h, and every hole adds mtt, after effect 4h, adds dmso,
Incubation 30min, mensuration absorbance a value at microplate reader 620nm, become breast cancer cell suppression ratio=(1 experimental group suction by formula
Light value/matched group light absorption value) × 100%.The positive group of vincristine and the low middle high dose experimental group of coupling conjugate of embodiment 1
Breast cancer cell suppression ratio be respectively 54.9 ± 13.6,89.1 ± 23.9,144.7 ± 37.1% (p ＜ 0.05).
Embodiment 4
Internal distribution with transplanted human breast carcinoma model inspection cd33 antibody polypeptides and the coupling conjugate of vincristine.
6-8w age scid female mice, mice is randomly divided into 3 groups, every group 6.(1) blank group；(2) conjugate group；(3)
Vincristine positive group.Set up transplanted human breast carcinoma model, the 7th day after inoculation breast cancer cell, tail vein is given respectively
Medicine is administered.Dosage regimen is: blank group adds the solvent of same volume, and experimental group is the conjugate of 2mmol/kg dosage, positive
Organize the vincristine for 2mmol/kg, daily administration, continuous 7 days.After 14 days, after sacrifice, take whole blood respectively, and
The heart, liver, spleen, lung, kidney, brain, tumor etc. are organized.Whole blood anticoagulant heparin, heparin anti-coagulating centrifugation hemocyte takes and purifies the blood
Slurry 0.5ml, adds 2.67%hclo4, centrifugation plasma protein, and 100 DEG C of supernatant heating in water bath 20 minutes is to be measured；Each tissue
After homogenate, removing protein, centrifugation, take supernatant.Detect the content of vincristine in blood plasma and the supernatant of tissue using hplc method.
Extracting method: add 5ml dichloromethane in the supernatant of blood plasma and tissue, fully mix, extract, centrifugation, take supernatant, will be organic
Solvent is flung to, then with 200 μ l flowing phased solns.Chromatographic condition: c18 chromatographic column (15cm, 5 μm of * 4.6mm), mobile phase: 0.04 phosphorus
Acid buffer-methanol (4.5:5.5, ph3.0), flow velocity: 1ml/min, Detection wavelength: 254nm, sample size 20 μ l measures.Calculate
The concentration of vincristine in mice plasma and each tissue.
Table 1 conjugate blood plasma and tissue distribution concentration in tumor model animal
* p < 0.05, * * p < 0.01 is compared with positive group.
Experimental result is shown in Table 1, compares with vincristine positive group, the coupling conjugate of cd33 antibody polypeptides and vincristine
Concentration in group in-vivo tumour tissue is significantly raised, (p ＜ 0.01), and the tissue such as the heart, liver, spleen, lung, kidney, brain, and in blood plasma
Vincristine concentration substantially reduce, especially the drug level in blood plasma will be 5.83 ± 2.04ng/ml (p ＜ 0.01), have
Significant difference.
Embodiment 5
The coupling conjugate of cd33 antibody polypeptides and vincristine toxic action.Using embodiment 1 conjugate, measure
Median lethal dose(LD 50): take 60 mices to be randomly divided into 6 groups, every group of l0 is only.Each group mice elder generation fasting 12h, lumbar injection is different respectively
Embodiment 1 conjugate of dosage, dosage be respectively 250,500,1000,2000,5000,10000mg/kg, after test medicine
Fasting 3-4 hour again.Hereafter Continuous Observation 2 weeks, have or not the phenomena of mortality, calculate median lethal dose(LD 50) ld50.And observe skin, glue
Film, hair color, eyes, breathing, circulation, autonomous and central nervous system's behavior expression.
Result: embodiment 1 conjugate is 1347.5mg/kg to median lethal dose(LD 50) ld50 of mice.
Embodiment 6
The combination rate of conjugate and breast cancer cell: using elisar method, by the human breast cancer cell of logarithmic growth, with
1.0×105Add in 96 hole elisa Plates, 4 DEG C, be coated overnight；After pbs washes plate tri- times, by the conjugate of embodiment 1, by concentration
0,12.5,25,50 μm of ol/ml of gradient are separately added in 96 orifice plates, and every hole adds quantitative anti-cd33 antibody competition to combine simultaneously,
37 DEG C, after incubation 2 hours；After pbs washes plate tri- times, add coupling horseradish peroxidase (horseradish
Peroxidase, hrp) anti-human igg antibody, 37 DEG C, be incubated 1 hour；After pbs washes plate tri- times again, add abc solution, incubation
10-20 minute, finally uses flowing water terminating reaction；Micro- sem observation, the cell that described embodiment 1 conjugate is combined with tumor tissues
In brownish black, it is positive cell, calculate the quantity of the cell that is positive using computer software.
Result: the quantity that embodiment 1 conjugate is combined with breast cancer cell, with dosage escalation, has notable compared with blank group
Sex differernce, when dosage is 50 μm of ol/ml, positive cell quantity reaches 692.0 ± 84.1 (p ＜ 0.05).
sequence listing
<110>Suzhou Pu Luoda bio tech ltd
<120>conjugate that a kind of cd33 antibody polypeptides are coupled with vincristine
<130>
<160> 1
<170> patentin version 3.3
<210> 1
<211> 48
<212> prt
<213>artificial sequence
<400> 1
gln glu thr arg ala gly val val his gly ala ile gly gly ala gly
1 5 10 15
val thr ala leu leu ala leu cys leu cys leu ile phe phe ile val
20 25 30
lys thr his arg arg lys ala ala arg thr ala val gly arg asn asp
35 40 45
Claims (6)
1. a kind of small molecule cd33 antibody polypeptides and vincristine coupling conjugate it is characterised in that: vincristine and cd33
The coupling conjugate of antibody polypeptides；The sequence of described cd33 antibody polypeptides is seq id no:1.
2. according to claim 1 be coupled conjugate preparation it is characterised in that: step includes: (1) is new with described Changchun
Alkali, succinic anhydrides and triethylamine are dissolved with 100 ml acetyl triethyls, are heated to reflux 1 hour；(2) solvent, residue are removed in distillation
With 5% nahco3 aqueous dissolution；(3) ether washes twice, and is then acidified with h2so4, to ph2.0-3.0；(4) use chlorine
Imitative-hexane crystallization, obtains solid content；(5) take 2.5g hemisuccinic acid ester to be dissolved in 6.5ml thionyl chloride, 65 DEG C, heat 30 minutes,
It is evaporated under reduced pressure, be dried 1 hour；(6) above-mentioned product is dissolved in 15ml n, in n- dimethyl-acetic acid acetamide, is stirred at room temperature anti-
Answer 2 hours；(7) 65 DEG C, after vacuum evaporation, so that crystallization is separated out with isopropanol, obtain vincristine-hemisuccinic acid ester；(8) Changchun
New alkali-hemisuccinic acid ester and described cd33 antibody polypeptides are coupled, and obtain final product.
3. according to claim 2 be coupled conjugate it is characterised in that: described cd33 antibody polypeptides adopt chemical synthesiss
Preparation.
4. according to claim 2 be coupled conjugate it is characterised in that: described vincristine-hemisuccinic acid ester and cd33
Antibody polypeptides are coupled and adopt carbodiimides.
5. according to the arbitrary described coupling conjugate of claim 1-4 it is characterised in that: in preparation in tumor
Application.
6. according to claim 5 be coupled conjugate it is characterised in that: preparation for treat in breast cancer medicines should
With.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610748177.9A CN106362161A (en) | 2016-08-29 | 2016-08-29 | Coupling conjugate of micromolecular CD33 antibody peptide and vincristine |
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| CN201610748177.9A CN106362161A (en) | 2016-08-29 | 2016-08-29 | Coupling conjugate of micromolecular CD33 antibody peptide and vincristine |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103298489A (en) * | 2010-10-29 | 2013-09-11 | 伊缪诺金公司 | Novel EGFR-binding molecules and immunoconjugates thereof |
| CN105440112A (en) * | 2015-12-07 | 2016-03-30 | 国家纳米科学中心 | Polypeptide-albumin coupling drug and preparing method and application thereof |
-
2016
- 2016-08-29 CN CN201610748177.9A patent/CN106362161A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103298489A (en) * | 2010-10-29 | 2013-09-11 | 伊缪诺金公司 | Novel EGFR-binding molecules and immunoconjugates thereof |
| CN105440112A (en) * | 2015-12-07 | 2016-03-30 | 国家纳米科学中心 | Polypeptide-albumin coupling drug and preparing method and application thereof |
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