CN108309963A - A kind of stabilization hybridization nanometer suspending agent reversed for multidrug resistance - Google Patents

A kind of stabilization hybridization nanometer suspending agent reversed for multidrug resistance Download PDF

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Publication number
CN108309963A
CN108309963A CN201710849077.XA CN201710849077A CN108309963A CN 108309963 A CN108309963 A CN 108309963A CN 201710849077 A CN201710849077 A CN 201710849077A CN 108309963 A CN108309963 A CN 108309963A
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ptx
dsf
suspending agent
drug
hybridization
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尹莉芳
何伟
伊木朗
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China Pharmaceutical University
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China Pharmaceutical University
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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
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Inorganic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

The nano suspension concentrate for stablizing hybridization is to be encapsulated together by anticancer agent and multiple medicine conditioning agent drug and embedded by a certain percentage by the nanoparticle that food proteins are smeared.Food proteins are as a kind of stabilizer for stablizing hybridization nanometer suspending agent.Nano suspension concentrate transports both drugs to tumor sites, reverses the drug resistance of taxol resistance lung adenocarcinoma cell system and improves Apoptosis ratio.In addition, hybridization nanometer suspending agent is made according to anti-solvent preparation method, the use of acetone is drug solvent.This hybridization nanometer suspending agent is stable and can discharge the antitumous effect that Multidrug resistance adjusts drug inactivation P glycoprotein and reduction and raising anticancer drug.This hybridization nanometer suspending agent is the treatment that can be used for drug-resistant tumor of stable homogeneous.

Description

A kind of stabilization hybridization nanometer suspending agent reversed for multidrug resistance
Technical field：
The invention belongs to biomedical sectors, and repellence solid tumor drug resistance field is generated especially for conventional chemotherapy. Preparation process uses the DSF containing the PTX and β-LG without toxicological effect by downward MDR-1 genes to kill tumour, this How plus nanoparticle suspension bead dosage form be primarily applicable for generate drug resistance tumour.
Background technology：
Chemotherapy is a kind of critically important anti-cancer therapies, but monotherapy is very undesirable, and this is mainly due to treatments In side effect, minimum uptake and the ever-increasing drug resistance for chemotherapy.Combined chemotherapy can be thin with modulate tumor The signal path of born of the same parents reaches the effect that collaboration overcomes single chemotherapy drawback.However this mixing treatment also possesses many deficiencies, For example the different physicochemical properties of combination medicine, different medicines are for the different internal distribution of dynamic characteristic, drug and not Same cell membrane pass through mechanism, best release concentration cannot be reached in target position by having resulted in combination medicine.Drug The generation of drug resistance (MDR) is to influence the main barrier for the treatment of of cancer.Therefore, reversion MDR is the core of tumour cell chemistry success or failure Heart determinant.Caused by the raising of MDR is mainly overexpressed by the P- glycoprotein that MDR-1 is encoded.P- glycoprotein, a kind of film Binding transport body belongs to ATP-Binding Cassette (ABC) family, and chemotherapeutics is allowed to be flowed out from tumour cell.
The nano suspending liquid (Ns) of insoluble reactive compound is a kind of aqueous colloidal dispersion, by nano particle and stable on a small quantity Agent forms.Other Nano medications are compared, such as liposome, polymer micelle and inorganic nano transport agent, Ns possesses obviously more Good drug carrying capacity ability, this is primarily due to the drug that particle in Ns contains 100%.Further, it is also possible to reduce individual difference Property and food effect, improve therapeutic effect, reduce side effect, reduce the toxicity to normal organ.Meanwhile by β-LG in Ns The particle of package can significantly improve the half-life period in blood circulation.
Disulfiram (DSF) nineteen fifty-one is proved to be a kind of insoluble drug by FDA, can be used for treating excessive drinking, gather around simultaneously There are the potentiality of reversion MDR.DSF inhibits P- glycoprotein to adjust the ability that drug outflows by interacting with cysteine, to Restore therapeutic effect of the chemotherapy to cancer cell.
Chemotherapeutics PTX has blocked the dynamic instability of micro-pipe needed for cell division, so as to cause Apoptosis and carefully The quick division in born of the same parents' period.
Invention content：
The purpose of the present invention is prepare that a profit stablizes containing taxol and disulfiram not soluble in water in tumor targets The nanoparticle suspension dosage form to play a role.It is organic to form double medicines that PTX and DSF according to fixed 6: 1W/W ratio is dissolved in acetone Phase.β-LG are dissolved in water and heat the 30-40 minutes liquid phases for forming pH7-9 at 90-100 DEG C.It is anti-molten heavy that PTX-DSF Ns are used Prepared by shallow lake method, and organic phase is mixed into liquid phase in the ice bath less than 4-8 DEG C.PTX-DSF Ns are ultrasonically treated immediately later 15-30 minutes.
TX-DSF Ns (FITC-PTX-DSF Ns) or of fluorescein 5 (6)-isothiocyanate (FITC) labels FITC-PTX Ns are prepared using similar method, in addition to FITC, DSF and PTX are being dissolved in acetone together using previous.
PTX-DSF Ns dosage forms diameters reach current potential (- 20-30mV) in nanoscale (160-200nm) and with negative boundary.
The PTX-DSF Ns of liquid and frozen form pass through Electronic Speculum, transmission electron microscope and powder in powder form Diffraction analysis (PXRD).
The present invention has MDR adjusters and anticancer drug very high Drug loading capacity, the about PTX of 30-40% and 6- 8% DSF.
The present invention can discharge sensitivities of the DSF for reversion MDR and raising A549 cell resistance PTX drug resistances in tumor targets Degree.The A549/TAX cells of intake invention significantly improves to(for) PTX, compare PTX sensitivity A549 cells, by changing core Cysteine residues in thuja acid calmodulin binding domain CaM reduce the expression that DSF lowers P- glycoprotein, higher thin which results in absorbing Cytotoxic drugs.Two kinds of nanoparticles, FITC-PTX Ns and FITC-PTX-DSF Ns co-culture 12-24 respectively at A549/TAX A hour is used in combination fluorescein FITC to be incubated 0.5-12 hours, and concentration is fixed as 400-800ng/ml.Cell initially absorbs The speed of FITC-PTX Ns be with time correlation, then there is unexpected decaying.On the other hand, FITC-PTX- DSF Ns significantly improve fluorescence intensity over time.Cellular uptake FITC-PTX-DSF Ns are almost FITC- 2 times to 14 times of PTX Ns.By confocal detection, A549/TAX cells show the green fluorescence of cell periphery, on the contrary The processed cells of FITC-PTX-DSF Ns, show apparent intracellular green and yellow fluorescence model.
This discovery result is as follows, (i) A549/TAX cell transitions express P-glycoprotein, therefore repels FITC-PTX Ns, from And in extracellular visible green fluorescence.(ii) in A549/TAX cells the DSF of FITC-PTX-DSF successfully in P- glycoprotein phases Interaction significantly suppresses FITC-PTX-DSF outflows, and gradually assembles in the cell over time.
The present invention presents the dose-dependent cytotoxicity after 24 and 48 hours, and on the whole, viability is with PTX medicines Object concentration is reduced from the raising of 0.1-100 μ g/ml.Compared with single drug, in A549 cells, PTX-DSF and PTX-DSF Ns, which is carried, has been total to more toxicity.
The present invention possesses better apoptosis-inducing ability compared with PTX-DSF, for A549/TAX persister cells, This illustrates its ability for possessing better anti-MDR associated cancers in vitro.IC50Be based on calculate free PTX, free DSF, PTX concentration obtains in PTX-DSF and PTX-DSF Ns, to determine benefit that PTX-DSF Ns compare in PTX-DSF, as a result shows With Table 1.In A549 cells, compared with cytotoxin PTX, after PTX-DSF was cultivated at 24 hours, IC is reduced50Value About 2.3 times, 2.6 times were reduced after culture at 48 hours.But PTX-DSF Ns cultivated it 24 hours and 48 hours Afterwards, IC50Value about reduces 6 times.It uses A549/TAX cells instead, similarly, uses PTX-DSF and PTX-DSF Ns cultures 24/48 2.2/2.4 times and 6/8 times are then reduced respectively after hour.This demonstrate that the present invention can play two drugs well Synergy improves about 1.75-5 times of apoptosis rate, reduces 2-7 times of IC50Value, and by reducing MDR-1 Expression, and reduce 5.8-8.9 times of PTX dosage.
The present invention can induce A549 and A549/TAX Apoptosis.Annexin-V/FITC is the results show that compared to single medicine Object, PTX-DSF and PTX-DSF Ns can significantly induce a large amount of Apoptosis.However, two kinds of dosage form inducing cell apoptosis Toatl proportion substantially similar about 77%.In contrast, PTX-DSF drug combinations can significantly improve the ratio of A549/TAX apoptosis Example, ratio apoptosis-induced PTX-DSF Ns is about that 80%, PTX-DSF is about 48%.To a certain extent, DSF independent roles Can also inducing cell system apoptosis.
The present invention can lead to the stopping of A549 and A549/TAX cell cycles, the ratio that PTX passes through reduction G0/G1 phase cells Example, and improve G2/M phase cell proportions and generate cytotoxin.In addition, cell of the DSF significant decrease A549 cells in the G0/G1 phases Ratio improves G2/M phase cell proportions.Compared with PTX, combination dosage form PTX-DSF and PTX-DSF Ns are further improved A549G2/M phase cell proportions.Joint PTX and DSF produce a large amount of cytotoxin and significantly affect A549 cell lines, especially It is the cancer cell of resistance to taxol.Therefore, this dosage form can reduce the ratio of G0/G1 phase cells, and the cell cycle is made to stop.With list One PTX is compared, hybridization nanometer suspending agent in A549 cells, significantly raise caspase-3 expression, caspase-9 expression then by PTX-DSF Ns up-regulations.Similar raising caspase-3 expression can also be by PTX-DSF and PTX-DSF Ns processing It is found in A549/TAX cells.PTX-DSF Ns up-regulation caspase-3 and caspase-9 express degree ratio PTX-DSF biggers, Illustrate that PTX-DSF Ns have the effect for promoting two kinds of drug synergisms.
The present invention has significantly lowered about 2.4 times of the expression of MDR-1.Therefore, MDR-1 is lowered in A549/TAX cells into one Step improves inhibition.
Description of the drawings：
Fig. 1：The PTX-DSF Ns that β-LG stablize
Fig. 2：Freeze-dried powder PTX-DSF Ns
Fig. 3：PTX-DSF Ns particle sizes and particle dispersion degree
Fig. 4：The boundary of PTX-DSF Ns reaches current potential
Fig. 5：The SEM of PTX-DSF Ns schemes
Fig. 6：The TEM of PTX-DSF Ns schemes
Fig. 7：PTX-DSF Ns are according to particle size and PDI in physiological saline, 7.4 and PBS pH 7.4+ of PBS pH Physical stability in 10%serum
Fig. 8：PTX-DSF Ns are according to boundary up to current potential in physiological saline, 7.4 and PBS pH 7.4+10% of PBS pH Physical stability in serum
Fig. 9：The PXRD analysis results of PTX, DSF, β-LG and PTX, DSF and β-LG, PTX-DSF Ns mixtures
Figure 10：The result of release in vitro of the DSF from predecessor and PTX-DSF Ns in pH 5.4or pH 7.4
Figure 11：The result of release in vitro of the PTX from predecessor and PTX-DSF Ns in pH 5.4or pH 7.4
Figure 12：A549/TAX cells are examined by the copolymerization coke at the place FITC-PTX-DSF Ns and FITC-PTX Ns 12 hours It surveys as a result, FITC concentration is fixed as 800ng/ml, scale is 20 μm.
Figure 13：FITC-PTX-DSF Ns and FITC-PTX Ns are absorbed by the A549/TAX cells of flow cytomery The case where.
Figure 14：Copolymerization coke picture of the A549 cells by FITC-PTX-DSF Ns processing.Scale is 20 μm
Figure 15：Copolymerization coke picture of the A549/TAX cells by FITC-PTX-DSF Ns processing.Scale is 20 μm
Figure 16：The Apoptosis of A549 and A549/TAX cells detects
Figure 17：A549 and A549/TAX cell cycle arrests.
Figure 18：The Caspase-3 of A549 cells and -9 activity.
Figure 19：The Caspase-3 of A549/TAX cells and -9 activity.
Figure 20：The RT-qPCR testing results of the MDR-1 gene expressions of A549/TAX cells.
Figure 21：The Gel electrophoresis results of MDR-1 gene expressions.

Claims (10)

  1. The taxol and disulfiram hybridization nanometer suspending agent (PTX-DSF Ns) that 1.Beta- lactoglobulins uniformly wrap up are prepared and mirror It is fixed as follows：
    (1) Beta- lactoglobulins (β-LG) a concentration of 0.5-2W/V.
    (2) a concentration of 1/0.1-10/1W/W of double medicines (taxol-disulfiram).
    (3) acetone concentration is 0.5-2%.
  2. A diameter of nanoscale of 2.PTX-DSF Ns simultaneously reaches current potential with negative boundary.
  3. 3. stability study is implemented in 10% physiological saline, 10%PBS (pH7.4), 10%PBS (pH7.4)+10%FBS.It receives Rice suspending agent can be stablized according to particle size, polydispersity and boundary up to current potential judgement in a variety of solution.
  4. 4.PTX-DSF Ns Senile Mouses are observed using electron-microscope scanning and transmission electron microscope, and form is needle-shaped knot Structure.
  5. 5. crystallinity is obtained by PXRD analyses, and is compared with PTX, DSF, β-LG and PTX-DSF Ns mixtures.
  6. 6. drugloading rate is detected by HPLC, no matter PTX-DSF Ns are owned by high load medicine for MDR conditioning agents or anticancer drug Ability.
  7. 7.PTX-DSF Ns transmit two kinds of drugs (Paclitaxel-Disulfiram) in pH5.4-7.4.
  8. 8 hours and 12 hours after cellular uptake PTX-DSF Ns are about 2-14 times of difference, phase in 8.A549/TAX cell lines Than being free of DSF in cellular uptake PTX Ns.
  9. 9.PTX-DSF Ns improve the apoptosis rate of A549/TAX cell lines, reduce minimum inhibitory concentration and anticarcinogen dosage.Drop Low amounts is related with toxicity.
  10. 10. hybridization nanometer suspending agent raises the expression of caspase-3 and caspase-9 albumen.Micro-pipe unstability is fissional Necessary condition.PTX is microtubule stabilizing agents, and therefore, the tumour cell cycle that the high speed that PTX DSF Ns can cause divides stops.
CN201710849077.XA 2017-09-14 2017-09-14 A kind of stabilization hybridization nanometer suspending agent reversed for multidrug resistance Pending CN108309963A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101217956A (en) * 2005-05-05 2008-07-09 康宾纳特克斯公司 Compositions and methods for treatment for neoplasms
CN103432072A (en) * 2013-09-11 2013-12-11 中国药科大学 In-dissolvable drug food protein stabilizing suspension for injection and preparation method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101217956A (en) * 2005-05-05 2008-07-09 康宾纳特克斯公司 Compositions and methods for treatment for neoplasms
CN103432072A (en) * 2013-09-11 2013-12-11 中国药科大学 In-dissolvable drug food protein stabilizing suspension for injection and preparation method thereof

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