CN109771658B - Targeted multi-arm conjugates - Google Patents
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Abstract
The invention discloses a multi-arm folic acid receptor targeted drug conjugate modified by a water-soluble polymer and a salt thereof. The drug conjugate has the following structural formula:
Description
Technical Field
The present invention generally relates to multi-arm targeting conjugates. The invention relates to a multi-arm PEG modified targeting anticancer conjugate, in particular to a conjugate formed by connecting a targeting molecule and an anticancer drug through multi-arm PEG.
Background
Over the years, various methods have been proposed for improving the stability and delivery of bioactive agents. Challenges associated with the formulation and delivery of pharmaceutical agents may include: poor water solubility, toxicity, low bioavailability, instability, and rapid in vivo degradation of the pharmaceutical agent. Although many approaches have been devised to improve the delivery of pharmaceutical agents, none of the individual approaches have had their drawbacks. For example, drug delivery methods commonly employed aim to solve or at least ameliorate one or more of the following problems, including drug encapsulation, such as in a liposome, polymer matrix, or unimolecular micelle, covalent attachment to a water soluble polymer such as polyethylene glycol, use of gene targeting agents, salt structures, and the like.
WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a drug nktr 102 in the third clinical stage, which is mainly used for metastatic breast cancer and developed by Nektar Therapeutics. The drug is a water-soluble multi-branched polymer prodrug to improve the load of the drug, and has the following structure:
the compound is connected with irinotecan by multi-arm PEG, so as to improve water solubility, increase drug loading and reduce side effects under the condition of unchanged anticancer effect. However, the drug still has the disadvantages of poor targeting, incapability of acting on specific cancer cells, capability of killing the cancer cells and influencing the performance of normal cells, and high incidence of adverse reaction.
The traditional medicines for treating tumors generally have the defects of poor selectivity to tumor tissues, great toxic and side effects and the like, and how to design a good medicine delivery system becomes a research hotspot in recent years.
Folate Receptor (FR) is a glycosylated phosphatidylinositol-linked membrane glycoprotein with a molecular weight of 38-40 kD, and three folate receptor isomers have been identified: alpha-FR, beta-FR and gamma-FRc. FR is rarely expressed on normal cells, has some expression in choroid plexus, placenta, and is expressed at low levels in lung, thymus, and kidney, and conversely, it is expressed at high levels in many tumor tissues. alpha-FR is expressed at high levels in tumors of some epithelial cell lines, such as ovarian cancer, renal cancer, uterine cancer, testicular cancer, brain tumor, colon cancer, lung adenocarcinoma, and the like; beta-FR in many other tumors, e.g. breast cancer, brainHigh-level expression of tumor, testicular cancer, head and neck tumor, myeloid leukemia, etc.; γ -FR is difficult to detect in many tissues. Metastases express more folate receptors than in situ, less malignant tumors.
Folate (FA) specifically binds to folate receptors. The folic acid is added into the compound to combine folic acid with folic acid receptor on tumor cell, so that the compound can be absorbed by cell via endocytosis mediated by cell receptor into the cell. Thus, a specific targeted drug delivery way is provided for the folate receptor positive tumor cells. Due to this stable association of folate with the folate receptor, folate is now widely used as a targeting system for the delivery of various targeted drugs.
Disclosure of Invention
The invention discloses a brand-new multi-arm drug conjugate with targeting property, which is shown in a formula (I):
to further illustrate the inventive concept of the present invention, the above conjugate can be represented by formula (ii):
wherein R is an organic center, i.e. in the structure of the conjugate
Represents the attachment of atoms. Starting from the central carbon atom of the organic center, four branches are emitted, each branch being identical. Each branch is composed of a polymer POLY, a multivalent linker L, a targeting molecule T, and an active agent D.
The polymer POLY is polyethylene glycol, which in the present invention is specifically:
It will be understood by those skilled in the art that in the polymer art, n represents the degree of polymerization of the polymer, i.e., the average number of repeating units contained in the macromolecular chain of the polymer, depending on the molecular weight of the polymer, e.g., when n is 113, it means that the average is 113.
The multivalent linker L is:
the symbol "#" represents the point of attachment of the multivalent linker L to the targeting molecule T via a cysteine, "#" represents the point of attachment of the multivalent linker L to the active agent D, and "%" represents the point of attachment of the multivalent linker L to POLY.
The targeting molecule T is folic acid, the active agent D is irinotecan, and the folic acid has the following structure:
irinotecan has the following structure:
the invention relates to a multi-arm polymer modified targeting anticancer conjugate, wherein the water-soluble polymer modification can enhance the water solubility of the conjugate, thereby improving the drug loading rate. The targeting molecule is folic acid which is used as the targeting molecule and actively targets tumor cells with abundant expression of folic acid receptors, so that the anti-tumor effect is better exerted, and the targeting property is increased, so that the concentration of the conjugate in a target tissue is higher.
L is any connecting joint and has the function of connecting the targeting molecule and the anticancer drug firstly and then connecting the targeting molecule, the anticancer drug and the polymer arm, so that the whole conjugate forms an organic whole. The conjugate of the invention is a typical prodrug, and the active agent D is released through hydrolysis or enzymolysis, separated from a parent body and exerts physiological activity.
The conjugates of the invention exhibit high loading capacity, which allows for a reduction in the total dose to treat a particular disease, such as cancer, etc. That is, the active agent carrier of the conjugates of the invention is capable of effectively covalently bonding to the active agent molecule, allowing a greater amount of the therapeutic agent (i.e., active agent moiety) to be administered per a given amount of conjugate. The conjugate is modified by the water-soluble polymer, is hydrophilic in nature, and particularly improves the bioavailability of the conjugate when the active agent is a water-insoluble drug. Compared with unconjugated drugs, the conjugate of the invention can show stronger effect and is more enriched in tissues in human bodies or other animal bodies.
The conjugate prodrugs of the present invention contain a number of unique properties, particularly where the active agent is an anti-cancer compound. The drug precursor can inhibit the growth of tumor with high efficiency. This small molecule we use is one known to have anti-cancer properties. However, by combining with multi-branched polymers as described above, the therapeutic efficacy and pharmacokinetics are greatly improved compared to the small molecule (e.g., the anti-cancer compound itself).
The conjugate of the present invention, pharmaceutically acceptable salts include inorganic salts and organic salts, and typical salts include nitrate, sulfate, phosphate, hydrofluoride, hydrochloride, hydrobromide, hydroiodide, formate, lactate, benzoate, acetate, trifluoroacetate, dichloroacetate, trichloroacetate, mixed chlorofluoroacetate, citrate, oxalate, sulfonate, methanesulfonate, trifluoromethanesulfonate, heptanesulfonate and the like, wherein trifluoroacetate and heptanesulfonate are preferred.
Typical trifluoroacetate salts include one to twelve molecules of trifluoroacetate salt. Preferably each branch binds to a trimolecular trifluoroacetate conjugate, and the preferred conjugate is a dodecamolecular trifluoroacetate:
typical heptane sulfonate salts include one to twelve molecules of heptane sulfonate. Preferably each branch incorporates three molecules of a heptanesulfonate conjugate, the preferred conjugate being twelve molecules of heptanesulfonate:
the solid tumor types suitable for the conjugate of the invention comprise ovarian cancer, breast cancer, lung cancer, endometrial cancer, brain tumor, mesothelial tissue cancer, kidney cancer, gastric cancer, head and neck tumor, lung cancer, colorectal cancer and testicular cancer, and are particularly suitable for ovarian cancer, breast cancer and lung cancer.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention will be described in detail below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are provided for the purpose of making the disclosure more complete and complete. The reagents and starting materials used were all commercially available except for the preparation provided. The 4armPEG20K-SCM is purchased from Kyork Tech Co., Ltd, and has a molecular weight of about 20 kDa.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs.
Unless otherwise indicated, the terms used herein have the following meanings:
DMF: n, N-dimethylformamide
DCM: methylene dichloride
Boc-Gly-OH：
DMAP: 4-dimethylaminopyridine
DCC: dicyclohexylcarbodiimide
IPA: isopropanol (I-propanol)
TFA: trifluoroacetic acid
TBME: tert-butyl methyl ether
EA: ethyl acetate
DME: ethylene glycol dimethyl ether
HOSU: n-succinimidyl carbonate
THF: tetrahydrofuran (THF)
DIEA: n, N-diisopropylethylamine
DEPC: cyanophosphoric acid diethyl ester
DMSO, DMSO: dimethyl sulfoxide
HOBT: 1-hydroxybenzotriazoles
DIC: n, N-diisopropylcarbodiimide
And (3) TIS: tri-isopropyl silane
PBS: phosphate buffer
EDC. HCl: 1-Ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride
4armPEG20K-SCM：
Preparation of the Compound of example 1
Preparation of BP103a01
Under the protection of nitrogen, 200mL of pyridine and 120g of BP103a00(1.0eq) are added into a 1000mL three-neck flask, stirred and cooled to 0 ℃, 151.8g of TsCl (1.0eq) is added in portions, stirred for 1h, then slowly warmed to room temperature, and stirred for 3-4 h. After the reaction is finished, pouring the reaction liquid intoExtracting with EA in dilute hydrochloric acid solution of ice, washing EA layer with dilute hydrochloric acid, washing with saturated sodium bicarbonate, washing with saturated salt solution, and removing anhydrous Na2SO4Drying, vacuum evaporating to remove solvent, and performing silica gel column chromatography to obtain pure product 55g of BP103a 01.
Preparation of BP103a02
55g BP103a01(1.0eq) and 160mL DMSO were added to a 1000mL three-necked flask, stirred well, and then NaN was added323.52g (2.0eq), heated to 50 ℃ for reaction for 3 hours, cooled to room temperature, poured into water, extracted with EA, combined organic phases, dried over anhydrous sodium sulfate and concentrated to obtain 29.2g of BP103a02 colorless liquid.
Preparation of BP103a03
29g of BP103a02, 360mL of methanol and 5.0g of palladium carbon are added into a 1L hydrogenation reaction kettle, the mixture is stirred and replaced by nitrogen, hydrogen is introduced for reaction for 3 to 4 hours, and after the reaction is monitored by TLC, the reaction solution is filtered, and the filtrate is concentrated to obtain 23.5g of oily matter of BP103a 03.
Preparation of BP103a04
Into a 1L three-necked flask were charged 23.5g of Compound BP103a03(1.0eq), 68.6g (Boc)2O (2.0eq), methanol: and (3) stirring and heating 500ml of mixed solution of triethylamine (9: 1) to reflux, reacting for 1h, after TLC monitoring reaction is finished, evaporating methanol triethylamine, adding water to dissolve, extracting for 3 times by dichloromethane, combining organic layers, washing once, drying by anhydrous sodium sulfate, evaporating to remove a solvent, and drying to obtain 34.8g of solid BP103a 04.
Preparation of BP103a05
Adding 34.8g of compound BP103a04(1.0eq), toluene and THF (150 mL each), bromoacetic acid 58.2g (3eq) into a 1000mL three-necked flask, stirring, heating to 45-50 ℃, adding sodium hydroxide 33.5g (6eq), reacting overnight, monitoring by TLC (thin layer chromatography), evaporating to remove the reaction solution, adding water and EA for extraction, adjusting the pH of the water phase to 3, extracting the water phase with dichloromethane, combining dichloromethane layers, drying with anhydrous sodium sulfate, and concentrating to obtain the oily compound BP103a05 (18 g).
Preparation of BP103a
18g of the compound BP103a05 and 100mL of EA were added to a 250mL three-necked flask, and after dissolution by stirring, the temperature was lowered to 0 ℃ and 150mL of EA/HCl (3.5M) was added thereto, and the mixture was kept at 0 ℃ to monitor the reaction by TLC, followed by filtration, and the filter cake was washed with TBME to obtain BP103a 10.4.4 g as a white solid.
Preparation of Compound 2
A100 mL flask was charged with 3.0g of BP103a (1.0eq), 14.0 g of compound (1.0eq), 40mL of DCM, and 4.0mL of DIEA (2.0eq), stirred at room temperature, monitored by TLC for completion of the reaction, the organic solvent was distilled off, and column chromatography gave 25.2 g of 6.4g of the oily compound.
Preparation of Compound 3
In a 200mL three-necked flask, 9.00g of compound 2(1.0eq),3.96g of housu (1.53eq), 90mL of DCM, 6.60g of EDC · HCl (1.53eq) were added, the reaction was carried out at room temperature for 2 hours, and after completion of the reaction monitored by TLC, after dilution of DCM, the mixture was washed with 50mmol/L potassium dihydrogen phosphate aqueous solution having a pH of 6.0 for 2 times, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 5.9g of compound 3 as a colorless oil.
Preparation of Compound 4
Into a 200mL flask was added 2.93g of Compound NH2-Lys (Boc) -OH (1.0eq), 60ml water, 2.00g NaHCO3(2.0eq), stirred, 5.9g of compound 3(1.0eq) dissolved in 60ml of DME was added dropwise, 60ml of THF was added, stirred overnight, TLC monitored the reaction, the organic solvent was evaporated, pH was adjusted to 4 with acetic acid, EA extracted, dried over anhydrous sodium sulfate, and concentrated to give 4.50g of compound 4 as a colorless oil.
Preparation of Compound 6
Adding 3.50g of compound 5(1.0eq) and 52.5mL of DMF into a 250mL round bottom flask, heating to 60 ℃ for dissolution, distilling off the DMF under reduced pressure after 5-10min, adding 300mL of n-heptane, distilling under reduced pressure for three times, adding 105mL of DCM, 1.08g of Boc-Gly-OH (1.2eq) and 63mg of DMAP (0.1eq) after spin-drying, adding a solution of 1.59g of DCC (1.5eq) dissolved in 10mL of DCM dropwise, reacting for 4 hours at 20 ℃, filtering after TLC monitoring the reaction, adding 120mL of IPA when the reaction is finished, distilling off 75% of the solvent, adding 150mL of n-heptane, stirring for 1 hour at room temperature, filtering, washing for 2 times with n-heptane, and drying to obtain 4.02g of compound 6 as a light yellow solid.
Preparation of Compound 7
Adding 4.02g of compound 6 and 50mL of DCM into a 100mL three-necked flask, stirring and dissolving, then adding 11.6mL of TFA dropwise, reacting for 2h at room temperature, monitoring by TLC, adding 150mL of acetonitrile after the reaction is finished, distilling 120mL of solvent under reduced pressure, pouring into 320mL of TBME solution, stirring for 30min, filtering, and washing a filter cake by TBME to obtain 74.00 g of a light yellow solid compound.
Preparation of Compound 8
Into a 200mL three-necked flask were added 3.69g of Compound 7, 100mL of DCM, 3.21g (1.05eq) of Compound 4, 2.7mL of DIEA (3.0eq), 1.2mL of DEPC (1.5eq), and the mixture was reacted at room temperature for 4 hours, followed by TLC monitoring, dilution of DCM, washing twice with water, washing once with saturated saline, drying, concentration, purification by HPLC, and lyophilization to obtain 81.85 g of a pale yellow solid compound.
Preparation of Compound 9
A50 mL round bottom flask was charged with 1.20g of Compound 8, 10mL of 20% TFA/DCM and allowed to react at room temperature for 4h, after completion of the TLC monitoring, poured into TBME, centrifuged, dried and preparative purified by HPLC to give 9210 mg of the compound as a pale yellow solid.
Preparation of Compound 10
To a 10mL round bottom flask was added 51mg of Compound 9(4.0eq), 2mL of DCM, 11ul of TEA (8.0eq), 201mg of 4arm PEG20K-SCM (1.0eq), reacted overnight at room temperature, concentrated, added to TBME, centrifuged, purified by HPLC, and lyophilized to give 1085 mg of a yellow-green solid compound.
Synthesis of CDDRD-folic acid
The polypeptide CDDRD-folic acid is synthesized by Fmoc method solid phase synthesis well known by the professional, 2-Cl-Trt Resin is used, 20% piperidine/DMF is used for removing Fmoc, HOBT/DIC is used as a coupling reagent, DMF is used as a reaction solvent, ninhydrin detection method is used for reaction monitoring, and the following protected amino acids are connected to Resin in sequence: Fmoc-Cys (Trt) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Glu-OtBu and trifluoroacetyl pteroic acid, washing DMF, removing trifluoroacetyl group with hydrazine hydrate, washing DMF, washing methanol, washing DCM, drying, adding cracking reagent (TFA: phenylmethyl sulfide: phenol: TIS 85: 5: 5), reacting for 2 hours, precipitating with ice TBME, centrifuging to obtain crude product, purifying by HPLC, and freeze-drying to obtain pure product.
Adding 500mg of compound 10(1.0eq) and 10mL of PBS (0.01M) with Ph 7 into a 10mL round bottom flask, and dissolving and cleaning for later use; and adding 83mg (4.0eq) of CDDRD-folic acid into 1ml of purified water, adjusting the pH value to 7 by using a 10mmol/L sodium bicarbonate aqueous solution, adding the solution into the solution of the compound 10, reacting for 4 hours at room temperature, dialyzing, concentrating to obtain a compound 11, dissolving a crude product in methanol, adding TFA to adjust the pH value to 5-6, concentrating, adding the solution into TBME, precipitating a solid, centrifuging, and drying to obtain 12367 mg of a yellow solid.
And (2) purifying the compound 11 by reverse phase HPLC (silica gel: C18, 300A; mobile phase: sodium heptanesulfonate/water and acetonitrile), collecting a pure product, adjusting the pH value to 4-5, desalting by reverse phase HPLC (silica gel: C18, 300A; mobile phase: acetic acid/water and acetonitrile), collecting the pure product, concentrating to remove the organic solvent, and freeze-drying to obtain the white-like powder compound 13.
The molecular weight of compound 11 in MALDI-TOF detection is 28887.03.
The molecular weight of compound 12 in MALDI-TOF detection is 28920.25.
The molecular weight of compound 13 in MALDI-TOF detection is 28951.38.
Example 2 antitumor Effect test on human ovarian carcinoma SK-OV-3 nude mouse xenograft tumor
1. Purpose of experiment
The inhibitory effect of the test drug compound 11 on the in vivo growth of the tumor of a nude mouse transplantation tumor model of the SK-OV-3 cell line of human ovarian cancer is evaluated.
2. Experimental Material
2.1 test article
Irinotecan (bulk drug) was purchased and both nktr-102 and compound 11 were provided by borrelid biopharmaceutical (suzhou) corporation.
The preparation method of nktr-102 refers to the method disclosed in CN102711837A, and comprises the following steps:
compound 7(829mg,4.5eq) from example was added to a 250mL reaction flask, DCM (50mL), triethylamine (221mg,9.0eq) were added, and after dissolution, 4arm-PEG20K-SCM (5.00g,1.0eq) was added to the reaction flask. After no significant progress of the reaction was monitored by HPLC, about 20mL of DCM was distilled off under reduced pressure, the solution was poured into 300mL of TBME and precipitated with stirring, filtered to give 5.4g of crude product, which was purified by HPLC preparative, desalted, adjusted to pH 5-6 with dilute hydrochloric acid, and lyophilized to give 2.71g of light green powder nktr-102.
2.2 reagents
McCoy's 5A culture medium, Fetal Bovine Serum (FBS), trypsin, cyan-chain double antibody and normal saline.
2.3 Experimental animals
Female BALB/c nude mice (the number is 150; the week age is 6-8 weeks) are purchased from Beijing Wintolite laboratory animal technology, Inc., and are bred in an SPF animal house of St.Su.Sco, Suzhou, the temperature is 20-25 ℃, the relative humidity is 40-70%, and the illumination is carried out for 12 hours respectively; animals had free access to water and food. After about 1 week of normal feeding, mice with good signs can be selected for this experiment by veterinary examination. Marking the tail and root of the animals by using a marker pen before grouping, and marking each animal after grouping by using an ear clipping mode.
2.4 transplantable tumor strains
Human ovarian cancer SK-OV-3, from China academy of sciences type culture Collection cell Bank (CAS, this laboratory frozen with liquid nitrogen).
3 Experimental methods
3.1 cell culture
SK-OV-3 was cultured in McCoy's 5A medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) in 5% CO237 ℃ incubator.
3.2 animal model preparation
Establishing a tumor nude mouse subcutaneous transplantation model by a cell inoculation method: collecting tumor cells in logarithmic growth phase, counting, re-suspending in 1 × PBS, and adjusting cell suspension concentration to 5 × 107Perml and mixed well with Matrigel 1: 1. The right dorsal part of the nude mice was inoculated subcutaneously with 5X 10 tumor cells using a 1ml syringe (No. 4 needle)6Mice, a total of 50 animals were inoculated. The tumor volume reaches 100-300mm3At this time, animals were randomly grouped by the random block method. Tumor size was measured 2 times per week after the start of the experiment, tumor volume was calculated, and animal body weight was weighed and recorded.
Tumor Volume (TV) calculation formula is as follows:
TV(mm3)＝l×w2/2
wherein l represents the tumor major axis (mm); w represents the tumor minor diameter (mm).
3.3 preparation of solvent
0.5g of sorbitol was weighed into a 50mL centrifuge tube, 50mL of water for injection was added to the centrifuge tube, and the solid matter was dissolved completely by vortex oscillation to prepare a 1% sorbitol aqueous solution (w/v) and stored in a refrigerator at 4 ℃ for future use.
3.4 preparation of drug delivery preparation
3, 4.1 irinotecan administration preparation
Weighing 12.0mg irinotecan, adding 0.15mL of 1% lactic acid, vortex vibrating to dissolve completely, and adding 2.85mL of 1% lactic acid solution% sorbitol aqueous solution, vortex and mix well, the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution is about 5:95 (v/v). The concentration of irinotecan in the free form in the solution was 4.0 mg/mL-1。
3.4.2nktr-102 administration preparation
Before each administration, 101.5mg of nktr-102 was accurately weighed, 2.3mL of physiological saline was added, and the solution was vortexed to completely dissolve the drug, and the concentration of irinotecan in the solution was 4.0 mg. multidot.mL in the free form-1。
3.4.3 formulation of compound 11 for administration: accurately weighing compound 11, adding a certain volume of normal saline, and vortexing to completely dissolve the medicine, wherein the free form concentration of irinotecan in the solution is 4.0 mg/mL-1。
3.5 animal grouping and administration
Animal groups and dosing regimens are shown in table 1. The first administration was started on the day of grouping, and the experiment was ended after 21 days, and the administration volumes were all 10 mL/kg-1. Group 1 was a solvent control group, and the solvent was administered to the tail vein by injection 1 time every 4 days for 3 times (Q7D × 3). In groups 2 to 4, test samples of irinotecan, nktr-102 and Compound 11 were administered by tail vein injection at a dose of 40 mg/kg-1(calculated as irinotecan content).
TABLE 1 animal grouping and dosing regimens
3.6 end of experiment
After the experiment was completed, the animals were weighed, and the animals were euthanized after tumor size measurement (CO)2). And stripping and weighing tumor tissues, and calculating the tumor weight inhibition rate.
4. Data recording, calculation formula
The formula for the Relative Tumor Volume (RTV) is:
RTV＝TVt/TVinitial
wherein, TVinitialTumor volume measured when administered in groups; TV (television)tThe tumor volume at each measurement during dosing.
The relative tumor proliferation rate (% T/C) was calculated by the following formula:
％T/C＝100％×(RTVT/RTVC)
wherein, RTVTRepresenting treatment group RTV; RTVCRepresenting the solvent control RTV.
5. Statistical analysis method
Experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software. Data are expressed as Mean ± standard error (Mean ± SE) and the two comparisons are performed using t-test, unless otherwise specified.
6. Results
For the human cancer xenograft tumor model, the relative tumor proliferation rate T/C (%) is recommended as the test evaluation index, and the lower the proliferation rate, the better the tumor inhibition effect, see table 2.
TABLE 2 Compound vs. tumor proliferation rate T/C (%)
P < 0.05 compared to RTV of the group of blank solvent, irinotecan and nktr-102
# P < 0.05 compared to% T/C for the blank solvent, irinotecan, and nktr-102 group
Experimental results show that the compound has good inhibition effect on tumor growth of a nude mouse transplantation tumor model of human ovarian cancer SK-OV-3 cell strains, and is superior to irinotecan and nktr-102.
Example 3 antitumor Effect test on human Breast cancer MDA-MB-231 nude mouse xenograft tumor
1. Purpose of experiment
The inhibitory effect of the test drug compound 11 on the in vivo growth of tumors in a nude mouse transplanted tumor model of human breast cancer MDA-MB-231 cell line was evaluated.
2. Experimental Material
2.1 test article
Irinotecan (bulk drug) was purchased and both nktr-102 and compound 11 were provided by borrelid biopharmaceutical (suzhou) corporation.
2.2 reagents
DMEM medium, Fetal Bovine Serum (FBS), trypsin, cyan-chain diabody, physiological saline.
2.3 Experimental animals
Female BALB/c nude mice (the number is 150; the week age is 6-8 weeks) are purchased from Beijing Wintolite laboratory animal technology, Inc., and are bred in an SPF animal house of St.Su.Sco, Suzhou, the temperature is 20-25 ℃, the relative humidity is 40-70%, and the illumination is carried out for 12 hours respectively; animals had free access to water and food. After about 1 week of normal feeding, mice with good signs can be selected for this experiment by veterinary examination. Marking the tail and root of the animals by using a marker pen before grouping, and marking each animal after grouping by using an ear clipping mode.
2.4 transplantable tumor strains
Human breast cancer MDA-MB-231 was obtained from the cell bank of the Committee for culture Collection of the Chinese academy of sciences (CAS, the laboratory is frozen in liquid nitrogen).
3 Experimental methods
3.1 cell culture
MDA-MB-231 was cultured in DMEM medium (DMEM, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and in 5% CO237 ℃ incubator.
3.2 animal model preparation
Establishing a tumor nude mouse subcutaneous transplantation model by a cell inoculation method: collecting tumor cells in logarithmic growth phase, counting, re-suspending in 1 × PBS, and adjusting cell suspension concentration to 1 × 107And/ml. The right dorsal part of the nude mice was inoculated subcutaneously with 1X 10 tumor cells using a 1ml syringe (No. 4 needle)6Mice, a total of 50 animals were inoculated. The tumor volume reaches 100-300mm3At this time, animals were randomly grouped by the random block method. Tumor size was calculated by measuring tumor size 2 times per week after the start of the experiment, and animals were weighed at the same timeBody weight and recording.
Tumor Volume (TV) calculation formula is as follows:
TV(mm3)＝l×w2/2
wherein l represents the tumor major axis (mm); w represents the tumor minor diameter (mm).
3.3 preparation of solvent
0.5g of sorbitol was weighed into a 50mL centrifuge tube, 50mL of water for injection was added to the centrifuge tube, and the solid matter was dissolved completely by vortex oscillation to prepare a 1% sorbitol aqueous solution (w/v) and stored in a refrigerator at 4 ℃ for future use.
3.4 preparation of drug delivery preparation
3, 4.1 irinotecan administration preparation
12.0mg of irinotecan was weighed, 0.15mL of 1% lactic acid was added, the mixture was vortexed to completely dissolve the drug, 2.85mL of 1% sorbitol aqueous solution was added, and the mixture was vortexed and mixed uniformly, whereby the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The concentration of irinotecan in the free form in the solution was 4.0 mg/mL-1。
3.4.2nktr-102 administration preparation
Before each administration, 101.5mg of nktr-102 was accurately weighed, 2.3mL of physiological saline was added, and the solution was vortexed to completely dissolve the drug, and the concentration of irinotecan in the solution was 4.0 mg. multidot.mL in the free form-1。
3.4.3 formulation of compound 11 for administration: accurately weighing compound 11, adding a certain volume of normal saline, and vortexing to completely dissolve the medicine, wherein the free form concentration of irinotecan in the solution is 4.0 mg/mL-1。
3.5 animal grouping and administration
Animal groups and dosing regimens are shown in table 3. The first administration was started on the day of grouping, and the experiment was ended after 21 days, and the administration volumes were all 10 mL/kg-1. Group 1 was a solvent control group, and the solvent was administered to the tail vein by injection 1 time every 4 days for 3 times (Q7D × 3). In groups 2 to 4, test samples of irinotecan, nktr-102 and Compound 11 were administered by tail vein injection at a dose of 40 mg/kg-1(in irinotecan contentCalculation).
TABLE 3 animal grouping and dosing regimens
3.6 end of experiment
After the experiment was completed, the animals were weighed, and the animals were euthanized after tumor size measurement (CO)2). And stripping and weighing tumor tissues, and calculating the tumor weight inhibition rate.
4. Data recording, calculation formula
The formula for the Relative Tumor Volume (RTV) is:
RTV＝TVt/TVinitial
wherein, TVinitialTumor volume measured when administered in groups; TV (television)tThe tumor volume at each measurement during dosing.
The relative tumor proliferation rate (% T/C) was calculated by the following formula:
％T/C＝100％×(RTVT/RTVC)
wherein, RTVTRepresenting treatment group RTV; RTVCRepresenting the solvent control RTV.
5. Statistical analysis method
Experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software. Data are expressed as Mean ± standard error (Mean ± SE) and the two comparisons are performed using t-test, unless otherwise specified.
6. Results
For the human cancer xenograft tumor model, the relative tumor proliferation rate T/C (%) is recommended as the test evaluation index, and the lower the proliferation rate, the better the tumor inhibition effect, see table 4.
TABLE 4 tumor proliferation rate T/C (%)
P < 0.05 compared to RTV of the group of blank solvent, irinotecan and nktr-102
# P < 0.05 compared to% T/C for the blank solvent, irinotecan, and nktr-102 group
Experimental results show that the compound has good inhibition effect on tumor growth of a nude mouse transplanted tumor model of human breast cancer MDA-MB-231 cell line, and is superior to irinotecan and nktr-102.
Example 4 antitumor Effect test on human Lung cancer SPC-A-1 nude mouse xenograft tumor
1. Purpose of experiment
The inhibitory effect of test compound 11 on the in vivo growth of tumors in a nude mouse transplanted tumor model of human lung cancer SPC-A-1 cell line was evaluated.
2. Experimental Material
2.1 test article
Irinotecan (bulk drug) was purchased and both nktr-102 and compound 11 were provided by borrelid biopharmaceutical (suzhou) corporation.
2.2 reagents
RPMI-1640 culture medium, Fetal Bovine Serum (FBS), trypsin, cyan-chain double antibody and normal saline.
2.3 Experimental animals
Female BALB/c nude mice (the number is 150; the week age is 6-8 weeks) are purchased from Beijing Wintolite laboratory animal technology, Inc., and are bred in an SPF animal house of St.Su.Sco, Suzhou, the temperature is 20-25 ℃, the relative humidity is 40-70%, and the illumination is carried out for 12 hours respectively; animals had free access to water and food. After about 1 week of normal feeding, mice with good signs can be selected for this experiment by veterinary examination. Marking the tail and root of the animals by using a marker pen before grouping, and marking each animal after grouping by using an ear clipping mode.
2.4 transplantable tumor strains
Human lung cancer SPC-A-1, derived from the cell bank of the culture Collection of the typical culture Collection of the academy of sciences of China (CAS, the laboratory is frozen in liquid nitrogen).
3 Experimental methods
3.1 cell culture
SPC-A-1 was cultured in RPMI-1640 medium containing 10% fetal bovine serum FBS (GIBCO, USA) and in 5% CO237 ℃ incubator. .
3.2 animal model preparation
Establishing a tumor nude mouse subcutaneous transplantation model by a cell inoculation method: collecting tumor cells in logarithmic growth phase, counting, re-suspending in 1 × PBS, and adjusting cell suspension concentration to 2 × 107And/ml. Nude mice were inoculated with tumor cells subcutaneously on the right dorsal side with a 1ml syringe (No. 4 needle), 2X 106Mice, a total of 50 animals were inoculated. The tumor volume reaches 100-300mm3At this time, animals were randomly grouped by the random block method. Tumor size was measured 2 times per week after the start of the experiment, tumor volume was calculated, and animal body weight was weighed and recorded.
Tumor Volume (TV) calculation formula is as follows:
TV(mm3)＝l×w2/2
wherein l represents the tumor major axis (mm); w represents the tumor minor diameter (mm).
3.3 preparation of solvent
0.5g of sorbitol was weighed into a 50mL centrifuge tube, 50mL of water for injection was added to the centrifuge tube, and the solid matter was dissolved completely by vortex oscillation to prepare a 1% sorbitol aqueous solution (w/v) and stored in a refrigerator at 4 ℃ for future use.
3.4 preparation of drug delivery preparation
3, 4.1 irinotecan administration preparation
12.0mg of irinotecan was weighed, 0.15mL of 1% lactic acid was added, the mixture was vortexed to completely dissolve the drug, 2.85mL of 1% sorbitol aqueous solution was added, and the mixture was vortexed and mixed uniformly, whereby the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The concentration of irinotecan in the free form in the solution was 4.0 mg/mL-1。
3.4.2nktr-102 administration preparation
Before each administration, 101.5mg of nktr-102 was accurately weighed, 2.3mL of physiological saline was added, and the solution was vortexed to completely dissolve the drug, and the concentration of irinotecan in the solution was 4.0 mg. multidot.mL in the free form-1。
3.4.3 formulation of compound 11 for administration: accurately weighing compound 11, adding a certain volume of normal saline, and vortexing to completely dissolve the medicine, wherein the free form concentration of irinotecan in the solution is 4.0 mg/mL-1。
3.5 animal grouping and administration
Animal groups and dosing regimens are shown in table 5. The first administration was started on the day of grouping, and the experiment was ended after 21 days, and the administration volumes were all 10 mL/kg-1. Group 1 was a solvent control group, and the solvent was administered to the tail vein by injection 1 time every 4 days for 3 times (Q7D × 3). In groups 2 to 4, test samples of irinotecan, nktr-102 and Compound 11 were administered by tail vein injection at a dose of 40 mg/kg-1(calculated as irinotecan content).
TABLE 5 animal grouping and dosing regimens
3.6 end of experiment
After the experiment was completed, the animals were weighed, and the animals were euthanized after tumor size measurement (CO)2). And stripping and weighing tumor tissues, and calculating the tumor weight inhibition rate.
4. Data recording, calculation formula
The formula for the Relative Tumor Volume (RTV) is:
RTV＝TVt/TVinitial
wherein, TVinitialTumor volume measured when administered in groups; TV (television)tThe tumor volume at each measurement during dosing.
The relative tumor proliferation rate (% T/C) was calculated by the following formula:
％T/C＝100％×(RTVT/RTVC)
wherein, RTVTRepresenting treatment group RTV; RTVCRepresenting the solvent control RTV.
5. Statistical analysis method
Experimental data were calculated and statistically processed using Microsoft Office Excel 2007 software. Data are expressed as Mean ± standard error (Mean ± SE) and the two comparisons are performed using t-test, unless otherwise specified.
6. Results
For the human cancer xenograft tumor model, the relative tumor proliferation rate T/C (%) is recommended as the test evaluation index, and the lower the proliferation rate, the better the tumor inhibition effect, see table 6.
TABLE 6 tumor proliferation rate T/C (%)
P < 0.05 compared to RTV of the group of blank solvent, irinotecan and nktr-102
# P < 0.05 compared to% T/C for the blank solvent, irinotecan, and nktr-102 group
Experimental results show that the compound has good inhibition effect on tumor growth of a human lung cancer SPC-A-1 cell strain nude mouse transplantation tumor model, and is superior to irinotecan and nktr-102.
It should be noted here that since the conjugate of the present invention enters the human or animal body, it is the free conjugate, which has no direct relation to the salt of the conjugate of the present invention, and it is actually anticancer, the pharmacological data of the free conjugate can be used to demonstrate the anticancer effect of the conjugate and its salt.
Claims (9)
1. A multi-branched drug conjugate having the structural formula:
2. the multi-branched drug conjugate according to claim 1, wherein the pharmaceutically acceptable salt comprises nitrate, sulfate, phosphate, hydrofluoride, hydrochloride, hydrobromide, hydroiodide, formate, lactate, benzoate, acetate, trifluoroacetate, dichloroacetate, trichloroacetate, mixed chlorofluoroacetate, citrate, oxalate, sulfonate, methanesulfonate, trifluoromethanesulfonate, heptanesulfonate.
3. The multi-branched drug conjugate according to claim 2, wherein the pharmaceutically acceptable salt is a dodecamolecular trifluoroacetate salt that binds three molecules per branch:
and dodecyl heptane sulfonate with each branch combined with a trimolecular salt:
4. the process for preparing the multi-branched drug conjugate of claim 1, comprising:
5. use of the multi-branched drug conjugate according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of cancer.
6. The use of claim 5, wherein the cancer comprises ovarian cancer, breast cancer, lung cancer, endometrial cancer, brain tumors, mesothelial tissue cancer, renal cancer, gastric cancer, head and neck tumors, colorectal cancer, testicular cancer.
7. A pharmaceutically acceptable composition comprising the multi-branched drug conjugate of any one of claims 1 to 3, and a pharmaceutically acceptable excipient.
8. Use of a composition according to claim 7 in the manufacture of a medicament for the treatment of cancer.
9. The use of claim 8, wherein the cancer comprises ovarian cancer, breast cancer, lung cancer, endometrial cancer, brain tumors, mesothelial tissue cancer, kidney cancer, gastric cancer, head and neck tumors, colorectal cancer, testicular cancer.
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