CN109771658A - Target multi-arm conjugate - Google Patents
Target multi-arm conjugate Download PDFInfo
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- CN109771658A CN109771658A CN201711119266.8A CN201711119266A CN109771658A CN 109771658 A CN109771658 A CN 109771658A CN 201711119266 A CN201711119266 A CN 201711119266A CN 109771658 A CN109771658 A CN 109771658A
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Abstract
The invention discloses a kind of multi-arm, by water-soluble polymers decorated folacin receptor targeted drug conjugate and its salt.The drug conjugates have the following structure formula:
Description
Technical field
Present invention relates in general to multi-arm targeting conjugates.In particular it relates to the target anticancer of multi-arm PEG modification
Conjugate, more specifically, the present invention are the conjugate for connecting into targeted molecular by multi-arm PEG and anticancer drug.
Background technique
For many years, it has been proposed that for improving the stability of bioactivator and a variety of methods of delivering.With medicinal examination
The associated challenge of preparation and delivery of agent may include: water solubility, the toxicity, low biological utilisation of the difference of the medicinal reagent
Rate, unstability and rapidly degradation in vivo.Although having devised thousand and one way to improve the delivering of medicinal reagent,
It is a kind of no individual method is without its disadvantage.For example, the delivery method generallyd use aims at solution
Or at least improve one or more following problems, including the medicine such as in a kind of liposome, polymer substrate or unimolecular micelle
Composite capsule, be covalently attached on a kind of water-soluble polymer such as polyethylene glycol, the use of gene target agent, salt structure,
Etc..
WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a kind of in clinic
The drug nktr 102 of three phases, the drug are mainly used for metastatic breast cancer, are researched and developed by Nektar Therapeutics.The medicine
Object is a kind of water-soluble multibranched polymer prodrug, and to improve the load of drug, structure is as follows:
The compound is to be connect using multi-arm PEG with Irinotecan, to improve water solubility, increases drugloading rate, is made in anticancer
In the case where constant, side effect is reduced.But the drug still has the disadvantage, for example, targeting is poor, cannot act on specific
Cancer cell also will affect the performance of normal cell while killing cancer cell, and compare adverse reaction rate still
It is high.
The disadvantages of drug of traditional treatment tumour is generally existing to tumor tissues poor selectivity, and toxic side effect is big, how
Good drug delivery system is designed as research hotspot in recent years.
Folacin receptor (folate receptor, FR) is a kind of membrane glycoprotein of glycolsyl-phosphatidylinositol connection, molecule
Amount is 38~40kD, has identified three kinds of folacin receptor isomers: α-FR, β-FR and γ-FRc.FR is seldom on normal cell
Expression, there is certain expression in choroid plexus, placenta, is in low expression level in lung, thymus gland, kidney, by contrast, it is but in many
Tumor tissues are in high level expression.α-FR is in some epithelial cell line tumours, such as oophoroma, kidney, uterine cancer, carcinoma of testis, brain
The high level expressions such as tumor, colon cancer, adenocarcinoma of lung；β-FR is in other many tumours, as breast cancer, brain tumor, carcinoma of testis, incidence are swollen
The high level expressions such as tumor, grain system leukaemia；γ-FR then is difficult to detect in many tissues.Metastatic tumor is than in situ, grade malignancy
Low tumour expresses more folacin receptors.
Folic acid (folate, FA) can be specifically bound with folacin receptor.Folic acid is added in the composite makes folic acid
It is combined with the folacin receptor on tumour cell, such compound can be inhaled by cell receptor-mediated endocytosis by cell
It takes in into cell interior.A special targeted drug thus, which is provided, for the tumour cell of folate receptor-positive delivers way
Diameter.Due to this stable bond of folic acid and folacin receptor, present folic acid is widely used for delivering as various targeted drugs
Targeted system.
Summary of the invention
The invention discloses a kind of completely new multi-arm drug conjugates with targeting, shown in the conjugate such as formula (I):
To further illustrate inventive concept of the invention, above-mentioned conjugate can be expressed as formula (II):
Wherein, R is organic core, i.e., in conjugate structureRepresent the junction of atom.From
The central carbon atom of organic core is set out, and four branches are issued, and each branch is all identical.Each branch is by polymer
POLY, polyvalent linkers L, targeted molecular T, activating agent D are constituted.
Polymer P OLY is polyethylene glycol, in the present invention, specifically:
N is 113,The junction for representing atom, the oxygen for marking ampersand are former
Son is the atom connecting with organic core " R ".
Those skilled in the art should know that, in the field of polymers, n represents the degree of polymerization of the polymer, that is, polymerize
Contained number of repeat unit purpose average value on object macromolecular chain depends on the molecular weight of the polymer, for example, when n is
When 113, refer to that average value is 113.
Polyvalent linkers L are as follows:
Symbol " * " represents the tie point that polyvalent linkers L passes through cysteine and targeted molecular T, and " # " represents polyvalent linkers L
With the tie point of activating agent D, " % " represents the tie point of polyvalent linkers L and POLY.
Targeted molecular T is folic acid, and activating agent D is Irinotecan, and folic acid structure is as follows:
Irinotecan structure is as follows:
The present invention be a multiarm polymers modification target anticancer conjugate, wherein it is water-soluble it is polymer-modified can
Enhance the water solubility of the conjugate, to improve drugloading rate.Targeted molecular in the present invention is folic acid, and folic acid is as targeting point
Son, active targeting increase targeting, in this way in the tumour cell of folacin receptor expressed in abundance, preferably performance antitumor efficacy
It is higher in the concentration of destination organization to allow for conjugate.
L is arbitrary jointing, and effect is first to connect targeted molecular and anticancer drug, then " will target
Molecule, anticancer drug and polymeric arms connect, so that entire conjugate forms an organic whole.Conjugate of the present invention
It is typical prodrug, by hydrolysis or enzymolysis, activating agent D is released, and is separated with parent, and physiology is played
Activity.
Conjugate of the present invention shows high loadability, can thus reduce accumulated dose to treat a kind of special disease
Disease, such as cancer etc..That is, conjugate active agent carrier of the present invention can be effectively with active agent molecule with covalently bonded
It closes, allows that further amounts of therapeutic dosage forms (namely active agent moiety) can be taken per certain coupling object amount.Present invention coupling
Object by the modification of water-soluble polymer, be substantially conjugate be also it is hydrophilic, when especially activating agent is shipwreck soluble drug,
Improve the bioavilability of conjugate.Compared to the drug not being coupled, conjugate of the present invention can show stronger effect, in people
Body or other animal in-vivo tissues are more enriched.
Conjugate prodrug unique property containing there are many in the present invention is especially an anti-cancer compounds in activating agent
In the case where object.This prodrug can inhibit the growth of tumour with greater efficiency.This small molecule that we use is one
With the small molecule of anticancer property known to kind.However, passing through as described above in conjunction with multibranched polymer, curative effect and drug generation
It thanks to dynamics compared with the small molecule (for example, anticancer compound itself), there is very big improvement.
Conjugate of the present invention, pharmaceutically acceptable salt include inorganic salts and organic salt, and typical salt includes nitrate, sulphur
Hydrochlorate, phosphate, hydrofluoride, hydrochloride, hydrobromate, hydriodate, formates, lactate, benzoate, acetate,
Trifluoroacetate, dichloroacetate, trichloroacetate, the chlorine fluoroacetate of mixing, citrate, oxalates, sulfonate, methylsulphur
Hydrochlorate, fluoroform sulphonate, heptanesulfonic acid salt etc., wherein it is preferred that trifluoroacetate and heptanesulfonic acid salt.
Typical trifluoroacetate includes one to ten dimolecular trifluoroacetate.It is preferred that each branch is respectively in connection with three points
The conjugate of sub- trifluoroacetate, the preferred conjugate are 12 molecule trifluoroacetates:
Typical heptanesulfonic acid salt includes one to ten dimolecular heptanesulfonic acid salt.It is preferred that each branch is respectively in connection with three points
The conjugate of sub- heptanesulfonic acid salt, the preferred conjugate are 12 molecule heptanesulfonic acid salt:
The applicable variety of solid tumor types of conjugate of the present invention include oophoroma, breast cancer, lung cancer, carcinoma of endometrium, brain tumor,
Mesothelial tissue cancer, kidney, gastric cancer, head and neck neoplasm, lung cancer, colorectal cancer, carcinoma of testis are particularly suitable for oophoroma, mammary gland
Cancer and lung cancer.
Specific embodiment
It present invention will be described in detail below.However, the present invention may be embodied as many different forms,
And it is not necessarily limited in embodiment described herein, and providing the purpose in these embodiments is to make disclosure
More completely and comprehensively.Agents useful for same and raw material, except providing preparation method, remaining is commercially available.4armPEG20K-
SCM is purchased from Jiankai Science and Technology Co., Ltd., Beijing, and molecular weight is about 20kDa or so.
Unless otherwise defined, the affiliated technology of meaning and claim theme that all scientific and technical terminologies have herein is led
The normally understood meaning of domain personnel is identical.
Unless otherwise indicated, term used herein has following meaning:
DMF:N, dinethylformamide
DCM: methylene chloride
Boc-Gly-OH:
DMAP:4- dimethylamino naphthyridine
DCC: dicyclohexylcarbodiimide
IPA: isopropanol
TFA: trifluoroacetic acid
TBME: t-butyl methyl ether
EA: ethyl acetate
DME: glycol dimethyl ether
HOSU:N- succinimdyl carbonate
THF: tetrahydrofuran
DIEA:N, N- diisopropylethylamine
DEPC: diethyl phosphorocyanidate
DMSO: dimethyl sulfoxide
HOBT:1- hydroxybenzotriazole
DIC:N, N- diisopropylcarbodiimide
TIS: tri isopropyl silane
PBS: phosphate buffer
EDCHCl:1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate
4armPEG20K-SCM:
The preparation of 1 compound of embodiment
The preparation of BP103a01
Under nitrogen protection, 200mL pyridine, 120g BP103a00 (1.0eq), stirring drop are added into 1000ml there-necked flask
Temperature is added portionwise 151.8g TsCl (1.0eq) to 0 DEG C, stirs 1h, is then slowly increased to room temperature, continues to stir 3-4h.Reaction
After, reaction solution is poured into the dilute hydrochloric acid solution of ice, EA is added to extract, EA layers are washed once with dilute hydrochloric acid, and saturated sodium bicarbonate is washed
It washs, saturated common salt washing, anhydrous Na2SO4Dry, evaporating solvent under reduced pressure, silica gel column chromatography obtains BP103a01 sterling 55g.
The preparation of BP103a02
55g BP103a01 (1.0eq) and 160mL DMSO are added into 1000mL there-necked flask, stirs evenly, is then added
NaN323.52g (2.0eq) is heated to 50 DEG C and reacts 3 hours, is cooled to room temperature, reaction solution is poured into water, extracted with EA,
Merge organic phase, anhydrous sodium sulfate is dry, is concentrated to give BP103a02 colourless liquid 29.2g.
The preparation of BP103a03
29g BP103a02, methanol 360mL, palladium carbon 5.0g, stirring are added into 1L hydrogenation reaction cauldron, nitrogen displacement is led to
Enter hydrogen reaction 3-4h, TLC is monitored after completion of the reaction, and filtrate is concentrated to get the grease of BP103a03 by filtering reacting liquid
23.5g。
The preparation of BP103a04
23.5g compound BP103a03 (1.0eq) is added into 1L there-necked flask, 68.6g (Boc)2O (2.0eq), methanol:
The mixed solution 500ml of triethylamine (9:1), stirring are warming up to reflux, react 1h, and TLC is monitored after completion of the reaction, boil off methanol three
Ethamine is dissolved in water, and methylene chloride extracts 3 times, merges organic layer washing once, and anhydrous sodium sulfate is dry, and solvent is evaporated off, and does
It is dry, obtain the 34.8g of solid BP103a04.
The preparation of BP103a05
34.8g compound BP103a04 (1.0eq) is added into 1000mL there-necked flask, toluene and each 150ml of THF, bromine second
Sour 58.2g (3eq), stirring, is heated to 45~50 DEG C, adds sodium hydroxide 33.5g (6eq), and overnight, TLC monitoring is anti-for reaction
After answering, reaction solution is evaporated off, water and EA is added to extract, it is 3 that water phase, which adjusts pH, and water phase is extracted with dichloromethane, and merges dichloromethane
Alkane layer is concentrated to give BP103a05 oily compounds 18g after anhydrous sodium sulfate is dry.
The preparation of BP103a
18g compound BP103a05,100ml EA are added into 250mL there-necked flask, is cooled to 0 DEG C after stirring and dissolving, adds
Enter 150ml EA/HCl (3.5M), keep the temperature 0 DEG C, TLC monitors end of reaction, filtering, and filter cake washs to obtain white solid with TBME
BP103a 10.4g。
The preparation of compound 2
3.0g BP103a (1.0eq) is added to 100mL flask, 1 4.0g of compound (1.0eq), 40mlDCM, 4.0ml
DIEA (2.0eq), is stirred at room temperature, and TLC monitors end of reaction, boils off organic solvent, column chromatographs to obtain 6.4g class oily compounds 2
5.2g。
The preparation of compound 3
9.00g compound 2 (1.0eq) is added into 200mL there-necked flask, 3.96gHOSU (1.53eq), 90ml DCM,
6.60g EDCHCl (1.53eq) reacts at room temperature 2h, and TLC is monitored after completion of the reaction, with pH=6.0's after DCM dilution
The potassium dihydrogen phosphate aqueous solution of 50mmol/L washs 2 times, saturated common salt water washing, and anhydrous sodium sulfate is dry, is concentrated to give colorless oil
Shape object 5.9g compound 3.
The preparation of compound 4
2.93g compound N H is added to 200mL flask2- Lys (Boc)-OH (1.0eq), 60ml water, 2.00g NaHCO3
(2.0eq), stirring are added dropwise the solution that 5.9g compound 3 (1.0eq) is dissolved in 60ml DME, add 60ml THF, be stirred overnight,
TLC monitors end of reaction, boils off organic solvent, adjusts pH=4 with acetic acid, EA extraction, anhydrous sodium sulfate is dry, and concentration obtains
4.50g colorless oil compounds 4.
The preparation of compound 6
3.50g compound 5 (1.0eq) is added into 250mL round-bottomed flask, 52.5mlDMF is heated to 60 DEG C of dissolutions, 5-
Decompression boils off DMF after 10min, and the vacuum distillation of 300ml normal heptane is added, in triplicate, 105mlDCM, 1.08g are added after being spin-dried for
Boc-Gly-OH (1.2eq), 63mg DMAP (0.1eq) are added dropwise 1.59gDCC (1.5eq) and are dissolved in the solution of 10ml DCM, and 20 DEG C
Reaction 4 hours, TLC monitor filtering after completion of the reaction, and 120ml IPA is added when being concentrated into remaining 25% volume, boils off 75%
Solvent is added 150ml normal heptane, is stirred at room temperature 1 hour, filters, and normal heptane washs 2 times, dry faint yellow solid 4.02gization
Close object 6.
The preparation of compound 7
4.02g compound 6 is added into 100mL there-necked flask, 11.6mlTFA, room temperature is added dropwise after stirring and dissolving in 50ml DCM
2h is reacted, 150ml acetonitrile is added after completion of the reaction, pours into 320ml TBME solution after being evaporated under reduced pressure 120ml solvent for TLC monitoring
In, 30min, filtering are stirred, filter cake washs to obtain 7 4.00g of faint yellow solid compound with TBME.
The preparation of compound 8
3.69g compound 7,100mlDCM, 3.21g (1.05eq) compound 4,2.7ml are added into 200mL there-necked flask
DIEA (3.0eq), 1.2ml DEPC (1.5eq) react at room temperature 4h, and TLC is monitored after completion of the reaction, after DCM dilution, washing two
Secondary, saturated salt solution washed once, dry, and concentration, HPLC is lyophilized after purification obtains 8 1.85g of faint yellow solid compound.
The preparation of compound 9
1.20g compound 8 is added into 50mL round-bottomed flask, the 20%TFA/DCM of 10ml reacts at room temperature 4h, TLC monitoring
After completion of the reaction, it pours into TBME, is centrifuged, drying simultaneously purifies to obtain 9 210mg of faint yellow solid compound through HPLC preparation.
The preparation of compound 10
51mg compound 9 (4.0eq) is added into 10mL round-bottomed flask, 2ml DCM, 11ulTEA (8.0eq), 201mg
4armPEG20K-SCM (1.0eq), after room temperature reaction overnight, concentration is added in TBME, centrifugation, through HPLC preparation purifying, freeze-drying
Obtain 10 85mg of greenish yellow solid compound.
The synthesis of CDDRD-folic acid
The synthesis of peptide C DDRD-folic acid utilizes 2- using Fmoc method synthesis in solid state known to those skilled in the art
Cl-Trt Resin, Fmoc is removed using 20% piperidines/DMF, coupling reagent uses HOBT/DIC, and DMF is reaction dissolvent, instead
It should monitor using ninhydrin detection method, successively following protected amino acid is connected on resin: Fmoc-Cys (Trt)-OH,
Fmoc-Asp(OtBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-
Glu-OtBu, trifluoroacetyl group pteroic acid, after removing trifluoroacetyl group with hydrazine hydrate after DMF washing, DMF is washed, methanol washs,
It is dry after DCM washing, it is added lytic reagent (TFA: thioanisole: phenol: TIS=85:5:5:5), reaction used ice after 2 hours
TBME precipitating, centrifugation, obtain crude product, and sterling is lyophilized to obtain after purification through HPLC preparation.
500mg compound 10 (1.0eq) is added into 10mL round-bottomed flask, the PBS of 10ml Ph=7,0.01M, after dissolved clarification
For use；83mg (4.0eq) CDDRD-folic acid is separately taken, 1ml purified water is added, and water-soluble with the sodium bicarbonate of 10mmol/L
Liquid adjusts pH=7, this solution is added into the solution of compound 10 after reacting at room temperature 4 hours and is dialysed, and is concentrated, obtains compound
11, methanol dissolves crude product, and TFA is added and adjusts pH=5-6, and concentration is added in TBME, and solid is precipitated, centrifugation, dry that yellow is solid
12 367mg of body.
Compound 11 purifies (silica gel: C18,300A through reverse hplc；Mobile phase: sodium heptanesulfonate/water, acetonitrile) after, it receives
PH=4~5 are adjusted after collecting sterling, then through reverse hplc desalination (silica gel: C18,300A；Mobile phase: acetic acid/water, acetonitrile), it collects
Concentration removes organic solvent after sterling, and off-white powder compound 13 is lyophilized to obtain.
Compound 11MALDI-TOF detection molecules amount is 28887.03.
Compound 12MALDI-TOF detection molecules amount is 28920.25.
Compound 13MALDI-TOF detection molecules amount is 28951.38.
Antitumor effect experiment of the embodiment 2 to human ovarian cancer SK-OV-3 nude mouse xenograft tumor
1. experiment purpose
Tested drug compound 11 is evaluated to human ovarian cancer SK-OV-3 cell strain Nude Mouse Model tumor growth in vivo
Inhibiting effect.
2. experimental material
2.1 test sample
Irinotecan (bulk pharmaceutical chemicals) system purchase gained, nktr-102 and compound 11 are by winning auspicious biological medicine (Suzhou) stock
Part Co., Ltd provides.
Wherein, the preparation method of nktr-102 method with reference to disclosed in CN102711837A is as follows:
Compound 7 (829mg, 4.5eq) in embodiment is added in the reaction flask of 250mL, is added DCM (50mL),
Triethylamine (221mg, 9.0eq) is added 4arm-PEG20K-SCM (5.00g, 1.0eq) and is added in the reaction flask after dissolution.
After HPLC monitoring reaction is without being obviously in progress, it is evaporated under reduced pressure away about 20mL DCM, solution is poured into 300mL TBME and is stirred
Precipitating, filtering obtain 5.4g crude product, and crude product is prepared through HPLC and purified, desalination, adjust pH to 5-6 with dilute hydrochloric acid, freeze-drying obtains
2.71g pale green powder nktr-102.
2.2 reagent
McCoy's 5A culture solution, fetal calf serum (FBS), trypsase, blueness-chain is dual anti-, physiological saline.
2.3 experimental animal
Female BAl BIc/c nude mouse (number of elements: 150；Week old: 6~8 weeks) have from Beijing dimension tonneau China experimental animal technology
The purchase of limit company, raising is in Suzhou Sheng Su new drug development Co., Ltd SPF animal house, and 20~25 DEG C of temperature, relative humidity 40%
~70%, light and shade illuminates each 12 hours；Animal free water and feeding.After normal nursing about 1 week, through veterinary inspector, sign shape
The good mouse of condition can enter anthology experiment.It is identified using marking pen in animal root of the tail portion before grouping, every animal is equal after grouping
Scarce mode is cut with ear to identify.
2.4 transplanted tumor tumor strains
Human ovarian cancer SK-OV-3 derives from the Chinese Academy of Sciences's American Type Culture Collection committee cell bank (CAS, this experiment
Room liquid nitrogen cryopreservation).
3 experimental methods
3.1 cell culture
SK-OV-3 is incubated at McCoy's 5A culture medium (GIBCO, the U.S.), containing 10% fetal calf serum FBS (GIBCO, beauty
State), it is incubated at containing 5%CO237 DEG C of incubators.
The preparation of 3.2 animal models
Cell inoculation method establishes tumour nude mice by subcutaneous transplantation model: collecting the tumour cell of logarithmic growth phase, weight after counting
It is suspended from 1 × PBS, adjustment concentration of cell suspension to 5 × 107/ ml is simultaneously mixed with Matrigel 1:1.With 1ml syringe (No. 4 needles
Head) the dorsal sc inoculated tumour cell on the right side of nude mice, 5 × 106/ mouse is inoculated with 50 animals altogether.Reach in gross tumor volume
100-300mm3When, animal is grouped at random by randomized blocks.Experiment measures weekly 2 knurl footpaths after starting, calculate swollen
Knurl product, while weighing the weight of animals and recording.
Gross tumor volume (TV) calculation formula is as follows:
TV(mm3)=l × w2/2
Wherein, l indicates tumour major diameter (mm)；W indicates tumour minor axis (mm).
3.3 solvents are prepared
It weighs 0.5g D-sorbite to be fitted into 50mL centrifuge tube, 50mL water for injection, vortex oscillation is added in centrifuge tube
It is completely dissolved solid matter, the aqueous sorbitol solution (w/v) of concentration 1% is configured to, it is spare to be stored in 4 DEG C of refrigerators.
3.4 drug-delivery preparations are prepared
3..4.1 Irinotecan drug-delivery preparation is prepared
1% lactic acid of 0.15mL is added in the Irinotecan for weighing 12.0mg, and vortex oscillation makes complete drug dissolution, then divides
Not Jia Ru 2.85mL 1% aqueous sorbitol solution, vortex oscillation be uniformly mixed, 1% lactic acid, 1% D-sorbite water in solution
The ratio of solution is about 5:95 (v/v).Irinotecan free form concentration is 4.0mgmL in solution-1。
3.4.2nktr-102 drug-delivery preparation is prepared
Every time before administration, the physiological saline of 2.3mL is added in the nktr-102 of precise 101.5mg, and vortex oscillation makes medicine
Object is completely dissolved, and Irinotecan free form concentration is 4.0mgmL in solution-1。
3.4.3 11 drug-delivery preparation of compound is prepared: the physiological saline of certain volume, whirlpool is added in precise compound 11
Rotation oscillation makes complete drug dissolution, and Irinotecan free form concentration is 4.0mgmL in solution-1。
3.5 animal packets and administration
Animal packet and dosage regimen are shown in Table 1.Started to be administered for the first time in the grouping same day, terminates experiment, administration after 21 days
Volume is 10mLkg-1.1st group is solvent control group, and tail vein injection is given blank solvent, every 4 days 1 time, is administered 3 times altogether
(Q7D×3).Tail vein injection gives given the test agent Irinotecan, nktr-102, compound 11, dosage to 2-4 group respectively
It is 40mgkg-1(with the calculating of Irinotecan content).
1 animal packet of table and dosage regimen
3.6 experiments terminate
It after experiment, weighs in, measure animal euthanasia (CO after knurl footpath2).It strips tumor tissues and weighs, calculate
Knurl weight tumour inhibiting rate.
4. data record, calculation formula
The calculation formula of relative tumour volume (RTV) are as follows:
RTV=TVt/TVinitial
Wherein, TVinitialFor the gross tumor volume measured when grouping administration；TVtIt is swollen when to be measured each time during administration
Knurl product.
The calculation formula of Relative tumor proliferation rate (%T/C) are as follows:
%T/C=100% × (RTVT/RTVC)
Wherein, RTVTIndicate treatment group RTV；RTVCIndicate solvent control group RTV.
5. statistical analysis technique
Test data calculate with 2007 software of Microsoft Office Excel and ASSOCIATE STATISTICS is handled.Number
It according to unless otherwise indicated, is indicated with mean ± standard error (Mean ± SE), two comparison among groups are examined using t-.
6. result
For people's cancer heteroplastic transplantation tumor model, recommend to be used as test evaluation index using Relative tumor proliferation rate T/C (%),
Proliferation rate is lower, illustrates to inhibit tumor effect the better, is shown in Table 2.
2 compound on tumor proliferation rate T/C (%) of table
* compared with the RTV of blank solvent, Irinotecan and nktr-102 group, P < 0.05
# is compared with the %T/C of blank solvent, Irinotecan and nktr-102 group, P < 0.05
Experimental result shows that the compounds of this invention is raw to human ovarian cancer SK-OV-3 cell strain Nude Mouse Model tumour
With good inhibiting effect, and it is better than Irinotecan and nktr-102.
Antitumor effect experiment of the embodiment 3 to human breast carcinoma MDA-MB-231 nude mouse xenograft tumor
1. experiment purpose
Tested drug compound 11 is evaluated to raw in human breast carcinoma MDA-MB-231 cell strain Nude Mouse Model tumour body
Long inhibiting effect.
2. experimental material
2.1 test sample
Irinotecan (bulk pharmaceutical chemicals) system purchase gained, nktr-102 and compound 11 are by winning auspicious biological medicine (Suzhou) stock
Part Co., Ltd provides.
2.2 reagent
DMEM culture solution, fetal calf serum (FBS), trypsase, blueness-chain is dual anti-, physiological saline.
2.3 experimental animal
Female BAl BIc/c nude mouse (number of elements: 150；Week old: 6~8 weeks) have from Beijing dimension tonneau China experimental animal technology
The purchase of limit company, raising is in Suzhou Sheng Su new drug development Co., Ltd SPF animal house, and 20~25 DEG C of temperature, relative humidity 40%
~70%, light and shade illuminates each 12 hours；Animal free water and feeding.After normal nursing about 1 week, through veterinary inspector, sign shape
The good mouse of condition can enter anthology experiment.It is identified using marking pen in animal root of the tail portion before grouping, every animal is equal after grouping
Scarce mode is cut with ear to identify.
2.4 transplanted tumor tumor strains
Human breast carcinoma MDA-MB-231, from Chinese Academy of Sciences's American Type Culture Collection committee cell bank (CAS, sheet
Laboratory liquid nitrogen cryopreservation).
3 experimental methods
3.1 cell culture
MDA-MB-231 is incubated at DMEM culture medium (DMEM, the U.S.), (GIBCO, the U.S.) containing 10% fetal calf serum FBS,
It is incubated at containing 5%CO237 DEG C of incubators.
The preparation of 3.2 animal models
Cell inoculation method establishes tumour nude mice by subcutaneous transplantation model: collecting the tumour cell of logarithmic growth phase, weight after counting
It is suspended from 1 × PBS, adjustment concentration of cell suspension to 1 × 107/ml.With 1ml syringe (No. 4 syringe needles) dorsal sc on the right side of nude mice
Inoculated tumour cell, 1 × 106/ mouse is inoculated with 50 animals altogether.Reach 100-300mm in gross tumor volume3When, by animal press with
Machine district's groups method is grouped at random.Experiment measures weekly 2 knurl footpaths after starting, calculate gross tumor volume, while weighing the weight of animals
And it records.
Gross tumor volume (TV) calculation formula is as follows:
TV(mm3)=l × w2/2
Wherein, l indicates tumour major diameter (mm)；W indicates tumour minor axis (mm).
3.3 solvents are prepared
It weighs 0.5g D-sorbite to be fitted into 50mL centrifuge tube, 50mL water for injection, vortex oscillation is added in centrifuge tube
It is completely dissolved solid matter, the aqueous sorbitol solution (w/v) of concentration 1% is configured to, it is spare to be stored in 4 DEG C of refrigerators.
3.4 drug-delivery preparations are prepared
3..4.1 Irinotecan drug-delivery preparation is prepared
1% lactic acid of 0.15mL is added in the Irinotecan for weighing 12.0mg, and vortex oscillation makes complete drug dissolution, then divides
Not Jia Ru 2.85mL 1% aqueous sorbitol solution, vortex oscillation be uniformly mixed, 1% lactic acid, 1% D-sorbite water in solution
The ratio of solution is about 5:95 (v/v).Irinotecan free form concentration is 4.0mgmL in solution-1。
3.4.2nktr-102 drug-delivery preparation is prepared
Every time before administration, the physiological saline of 2.3mL is added in the nktr-102 of precise 101.5mg, and vortex oscillation makes medicine
Object is completely dissolved, and Irinotecan free form concentration is 4.0mgmL in solution-1。
3.4.3 11 drug-delivery preparation of compound is prepared: the physiological saline of certain volume, whirlpool is added in precise compound 11
Rotation oscillation makes complete drug dissolution, and Irinotecan free form concentration is 4.0mgmL in solution-1。
3.5 animal packets and administration
Animal packet and dosage regimen are shown in Table 3.Started to be administered for the first time in the grouping same day, terminates experiment, administration after 21 days
Volume is 10mLkg-1.1st group is solvent control group, and tail vein injection is given blank solvent, every 4 days 1 time, is administered 3 times altogether
(Q7D×3).Tail vein injection gives given the test agent Irinotecan, nktr-102, compound 11, dosage to 2-4 group respectively
It is 40mgkg-1(with the calculating of Irinotecan content).
3 animal packet of table and dosage regimen
3.6 experiments terminate
It after experiment, weighs in, measure animal euthanasia (CO after knurl footpath2).It strips tumor tissues and weighs, calculate
Knurl weight tumour inhibiting rate.
4. data record, calculation formula
The calculation formula of relative tumour volume (RTV) are as follows:
RTV=TVt/TVinitial
Wherein, TVinitialFor the gross tumor volume measured when grouping administration；TVtIt is swollen when to be measured each time during administration
Knurl product.
The calculation formula of Relative tumor proliferation rate (%T/C) are as follows:
%T/C=100% × (RTVT/RTVC)
Wherein, RTVTIndicate treatment group RTV；RTVCIndicate solvent control group RTV.
5. statistical analysis technique
Test data calculate with 2007 software of Microsoft Office Excel and ASSOCIATE STATISTICS is handled.Number
It according to unless otherwise indicated, is indicated with mean ± standard error (Mean ± SE), two comparison among groups are examined using t-.
6. result
For people's cancer heteroplastic transplantation tumor model, recommend to be used as test evaluation index using Relative tumor proliferation rate T/C (%),
Proliferation rate is lower, illustrates to inhibit tumor effect the better, is shown in Table 4.
4 compound on tumor proliferation rate T/C (%) of table
* compared with the RTV of blank solvent, Irinotecan and nktr-102 group, P < 0.05
# is compared with the %T/C of blank solvent, Irinotecan and nktr-102 group, P < 0.05
Experimental result shows that the compounds of this invention is to human breast carcinoma MDA-MB-231 cell strain Nude Mouse Model tumour
Growth has good inhibiting effect, and is better than Irinotecan and nktr-102.
Antitumor effect experiment of the embodiment 4 to human lung cancer SPC-A-1 nude mouse xenograft tumor
1. experiment purpose
Evaluate suppression of the tested drug compound 11 to Lung cancer SPC-A-1 cells strain Nude Mouse Model tumor growth in vivo
Production is used.
2. experimental material
2.1 test sample
Irinotecan (bulk pharmaceutical chemicals) system purchase gained, nktr-102 and compound 11 are by winning auspicious biological medicine (Suzhou) stock
Part Co., Ltd provides.
2.2 reagent
RPMI-1640 culture solution, fetal calf serum (FBS), trypsase, blueness-chain is dual anti-, physiological saline.
2.3 experimental animal
Female BAl BIc/c nude mouse (number of elements: 150；Week old: 6~8 weeks) have from Beijing dimension tonneau China experimental animal technology
The purchase of limit company, raising is in Suzhou Sheng Su new drug development Co., Ltd SPF animal house, and 20~25 DEG C of temperature, relative humidity 40%
~70%, light and shade illuminates each 12 hours；Animal free water and feeding.After normal nursing about 1 week, through veterinary inspector, sign shape
The good mouse of condition can enter anthology experiment.It is identified using marking pen in animal root of the tail portion before grouping, every animal is equal after grouping
Scarce mode is cut with ear to identify.
2.4 transplanted tumor tumor strains
Human lung cancer SPC-A-1 derives from Chinese Academy of Sciences's American Type Culture Collection committee cell bank (CAS, this laboratory
Liquid nitrogen cryopreservation).
3 experimental methods
3.1 cell culture
SPC-A-1 is incubated at RPMI-1640 culture solution, and (GIBCO, the U.S.) containing 10% fetal calf serum FBS is incubated at and contains
5%CO237 DEG C of incubators.
The preparation of 3.2 animal models
Cell inoculation method establishes tumour nude mice by subcutaneous transplantation model: collecting the tumour cell of logarithmic growth phase, weight after counting
It is suspended from 1 × PBS, adjustment concentration of cell suspension to 2 × 107/ml.With 1ml syringe (No. 4 syringe needles) dorsal sc on the right side of nude mice
Inoculated tumour cell, 2 × 106/ mouse is inoculated with 50 animals altogether.Reach 100-300mm in gross tumor volume3When, by animal press with
Machine district's groups method is grouped at random.Experiment measures weekly 2 knurl footpaths after starting, calculate gross tumor volume, while weighing the weight of animals
And it records.
Gross tumor volume (TV) calculation formula is as follows:
TV(mm3)=l × w2/2
Wherein, l indicates tumour major diameter (mm)；W indicates tumour minor axis (mm).
3.3 solvents are prepared
It weighs 0.5g D-sorbite to be fitted into 50mL centrifuge tube, 50mL water for injection, vortex oscillation is added in centrifuge tube
It is completely dissolved solid matter, the aqueous sorbitol solution (w/v) of concentration 1% is configured to, it is spare to be stored in 4 DEG C of refrigerators.
3.4 drug-delivery preparations are prepared
3..4.1 Irinotecan drug-delivery preparation is prepared
1% lactic acid of 0.15mL is added in the Irinotecan for weighing 12.0mg, and vortex oscillation makes complete drug dissolution, then divides
Not Jia Ru 2.85mL 1% aqueous sorbitol solution, vortex oscillation be uniformly mixed, 1% lactic acid, 1% D-sorbite water in solution
The ratio of solution is about 5:95 (v/v).Irinotecan free form concentration is 4.0mgmL in solution-1。
3.4.2nktr-102 drug-delivery preparation is prepared
Every time before administration, the physiological saline of 2.3mL is added in the nktr-102 of precise 101.5mg, and vortex oscillation makes medicine
Object is completely dissolved, and Irinotecan free form concentration is 4.0mgmL in solution-1。
3.4.3 11 drug-delivery preparation of compound is prepared: the physiological saline of certain volume, whirlpool is added in precise compound 11
Rotation oscillation makes complete drug dissolution, and Irinotecan free form concentration is 4.0mgmL in solution-1。
3.5 animal packets and administration
Animal packet and dosage regimen are shown in Table 5.Started to be administered for the first time in the grouping same day, terminates experiment, administration after 21 days
Volume is 10mLkg-1.1st group is solvent control group, and tail vein injection is given blank solvent, every 4 days 1 time, is administered 3 times altogether
(Q7D×3).Tail vein injection gives given the test agent Irinotecan, nktr-102, compound 11, dosage to 2-4 group respectively
It is 40mgkg-1(with the calculating of Irinotecan content).
5 animal packet of table and dosage regimen
3.6 experiments terminate
It after experiment, weighs in, measure animal euthanasia (CO after knurl footpath2).It strips tumor tissues and weighs, calculate
Knurl weight tumour inhibiting rate.
4. data record, calculation formula
The calculation formula of relative tumour volume (RTV) are as follows:
RTV=TVt/TVinitial
Wherein, TVinitialFor the gross tumor volume measured when grouping administration；TVtIt is swollen when to be measured each time during administration
Knurl product.
The calculation formula of Relative tumor proliferation rate (%T/C) are as follows:
%T/C=100% × (RTVT/RTVC)
Wherein, RTVTIndicate treatment group RTV；RTVCIndicate solvent control group RTV.
5. statistical analysis technique
Test data calculate with 2007 software of Microsoft Office Excel and ASSOCIATE STATISTICS is handled.Number
It according to unless otherwise indicated, is indicated with mean ± standard error (Mean ± SE), two comparison among groups are examined using t-.
6. result
For people's cancer heteroplastic transplantation tumor model, recommend to be used as test evaluation index using Relative tumor proliferation rate T/C (%),
Proliferation rate is lower, illustrates to inhibit tumor effect the better, is shown in Table 6.
6 compound on tumor proliferation rate T/C (%) of table
* compared with the RTV of blank solvent, Irinotecan and nktr-102 group, P < 0.05
# is compared with the %T/C of blank solvent, Irinotecan and nktr-102 group, P < 0.05
Experimental result shows that the compounds of this invention is to Lung cancer SPC-A-1 cells strain Nude Mouse Model tumour growth
There is good inhibiting effect, and is better than Irinotecan and nktr-102.
It is practical to play antitumaous effect herein it should be noted that since conjugate of the present invention enters in human body or animal body
Be free state conjugate, with conjugate of the present invention specifically at any salt without direct relation, therefore free coupling can be used
The pharmacological datum of object proves the antitumaous effect of conjugate and its salt.
Claims (9)
1. having the following structure the highly branched chain drug conjugates or its pharmaceutically acceptable salt of formula:
2. conjugate pharmaceutically acceptable salt as described in claim 1 includes nitrate, sulfate, phosphate, hydrofluoric acid
Salt, hydrochloride, hydrobromate, hydriodate, formates, lactate, benzoate, acetate, trifluoroacetate, dichloroacetic acid
Salt, trichloroacetate, the chlorine fluoroacetate of mixing, citrate, oxalates, sulfonate, mesylate, fluoroform sulphonate,
Heptanesulfonic acid salt.
3. salt as claimed in claim 2 is ten two molecule trifluoroacetates of each branch respectively in connection with three molecule salt:
With each branch respectively in connection with 12 molecule heptanesulfonic acid salt of three molecule salt:
4. conjugate as described in claim 1 the preparation method comprises the following steps:
5. highly branched chain drug conjugates a method according to any one of claims 1-3 answering in the preparation of medicament for cancer treatment
With.
6. cancer as claimed in claim 5 includes including oophoroma, breast cancer, lung cancer, carcinoma of endometrium, brain tumor, mesothelium group
Knit cancer, kidney, gastric cancer, head and neck neoplasm, lung cancer, colorectal cancer, carcinoma of testis.
7. a kind of pharmaceutically acceptable composition, it includes any highly branched chain drug conjugates of claim 1-3, with
And pharmaceutically acceptable excipient.
8. the application of composition as claimed in claim 7 in the preparation of medicament for cancer treatment.
9. cancer as claimed in claim 8 includes including oophoroma, breast cancer, lung cancer, carcinoma of endometrium, brain tumor, mesothelium group
Knit cancer, kidney, gastric cancer, head and neck neoplasm, lung cancer, colorectal cancer, carcinoma of testis.
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