CN106139159B - Application of polyethylene glycol derivative-small molecule drug conjugate polymer micelle in inflammation targeted drug delivery system - Google Patents
Application of polyethylene glycol derivative-small molecule drug conjugate polymer micelle in inflammation targeted drug delivery system Download PDFInfo
- Publication number
- CN106139159B CN106139159B CN201610426580.XA CN201610426580A CN106139159B CN 106139159 B CN106139159 B CN 106139159B CN 201610426580 A CN201610426580 A CN 201610426580A CN 106139159 B CN106139159 B CN 106139159B
- Authority
- CN
- China
- Prior art keywords
- polyethylene glycol
- glycol derivative
- drug
- small molecule
- sensitive
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
- A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a pH-sensitive polyethylene glycol derivative-micromolecule drug conjugate shown in a formula (I), application of a polymer micelle of the conjugate as an inflammation targeted delivery system and a preparation method of the conjugate, wherein a part B is a micromolecule drug D with activity of treating inflammatory diseases, and a part A is a polyethylene glycol derivative carrier system. The polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate has good inflammation targeting property, and can obviously improve the concentration of the B part drug at a low-pH inflammation part or prolong the time of the B part drug at the low-pH inflammation part or improve the anti-inflammatory effect of the B part drug.
Description
Technical Field
The invention belongs to the field of medicines, and relates to an application of a polymer micelle of a pH-sensitive polyethylene glycol derivative-small molecule drug conjugate connected through a hydrazone bond as an inflammation targeted delivery system. .
Background
Rheumatoid Arthritis (RA) is a chronic inflammatory disease involving a symmetrical pattern of multiple joints, characterized by an inflammatory process in the synovium that ultimately leads to destruction of articular cartilage, destruction of bone surrounding the joint, and ultimately changes the function and integrity of the joint, with systemic and extraarticular inflammation occurring in many patients in addition to joint symptoms. In patients with RA, disease-related disability usually occurs relatively early, with about 35% of patients prematurely becoming incapacitated within 10 years after being diagnosed with RA. In addition, the life span of RA patients has also been found to be shorter than normal. Currently, the treatment of RA is mainly divided into drug treatment, surgical treatment and biological treatment, and the drug treatment is favored by a plurality of RA patients due to the advantages of low treatment cost, convenient administration and the like.
Glucocorticoids are capable of rapidly controlling RA conditions and relieving pain, making them important in the pharmacological treatment of RA. However, the greatest disadvantages of free Glucocorticoid (GCs) drugs include: the water solubility is poor, repeated administration is needed, the targeting is not achieved, and the toxic and side effects of the whole body are great after long-term application, so the patient compliance is poor. Therefore, in order to increase the safety and effectiveness of drugs, the research of inflammation targeted drug delivery systems is necessary.
The existing nano drug-carrying system for targeted therapy of RA comprises drug-carrying liposome, nanoparticles, polymer micelle and the like. The nano-carriers can deliver GCs drugs to inflammatory joints through passive targeting, so that the aim of treating RA is fulfilled. However, these drug delivery systems also have the following problems: (1) liposomes and common physically entrapped micelles are unstable in the blood circulation in vivo, mainly due to: first, it is phagocytosed by the reticuloendothelial system; secondly, it interacts with many components in plasma (albumin, lipoproteins, IgG, etc.); moreover, the medicine is easy to leak in advance after being diluted by blood; (2) due to the uncontrollable drug release behavior of the liposome, the nanoparticle and the ordinary physically encapsulated micelle in vivo, the full play of the curative effect is difficult to ensure. (3) The common nano drug-carrying system has low drug-carrying capacity, so that the clinical application of the nano drug-carrying system is limited.
According to the invention, the pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate is synthesized by connecting the hydrazine group at the tail end of the branched chain of the polyethylene glycol derivative with the carbonyl group of the micromolecular drug through the pH-sensitive hydrazone bond, and the polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate is prepared through a self-assembly method, so that the defects of the common nanoscale drug-carrying system are effectively avoided, and the low-pH inflammation area can be effectively targeted. Has the following advantages: (1) firstly, coupling a polyethylene glycol derivative carrier with a micromolecular drug by using a pH sensitive hydrazone bond, and then preparing the polymer micelle by a self-assembly mode, so that the defect that the drug is degraded in the systemic circulation in advance can be avoided, and the purpose of long circulation can be achieved under the protection of a polyethylene glycol derivative shell. Furthermore, on the one hand the distribution of the drug in non-inflammatory sites can be reduced and on the other hand the drug can be protected from degradation in the blood circulation. (2) The polyethylene glycol derivative carrier and the micromolecular drug are coupled through a covalent bond to prepare the polymer micelle, so that the drug molecules can still be retained in a core when the micelle is in blood circulation, and the adjustable drug release behavior is further achieved, for example, the covalent bond responding to the change of the environment can cause more specific and continuous drug release, and the accumulation at a target position is achieved. (3) By responding the characteristic that the pH of the pathological part of inflammation is lower than that of a normal tissue, the polyethylene glycol derivative carrier and the micromolecular drug are coupled by the pH sensitive hydrazone bond, and then the polymer micelle is further prepared by a self-assembly mode. (4) Compared with the traditional linear polyethylene glycol carrier, the synthesized polyethylene glycol derivative can provide more chemical modification sites, so that the drug loading capacity is greatly improved, and in addition, the drug loading capacity can be freely adjusted by changing the number of hydrazine groups of the branched-chain part of the polyethylene glycol derivative.
In the prior art, CN201310756002.9 and CN2013755517.7 are respectively connected with polyethylene glycol monomethyl ether-polylactic acid, curcumin with single-ended phenol hydroxyl carbonylation and curcumin with double-ended phenol hydroxyl carbonylation by utilizing a hydrazone bond to prepare a pH-sensitive curcumin-loaded micelle (single chain) and a pH-sensitive curcumin-loaded micelle (double chain), however, the two kinds of the loaded micelles have the following problems that PLA must be added into the chemical formula ① to enhance hydrophobicity to realize the micelle, but China FDA does not approve polylactic acid (PLA) to be used for intravenous injection of pharmaceutic adjuvants, the use of the PLA can cause inestimable risks, and the drug-loaded amounts of ② two kinds of the loaded micelles are respectively not more than 15% and 5%, so that the clinical use value is to be improved.
In the prior art, CN201310229332.2 discloses a bifunctional polyethylene glycol-doxorubicin conjugate connected by a hydrazone bond and a disulfide bond and a preparation method thereof, wherein the conjugate has a drug controlled release function of pH sensitivity and reducibility in cancer cells, can self-assemble into a nano drug, and has the ability of targeting tumor tissues through an EPR effect. However, the polyethylene glycol in the conjugate is a straight chain structure, and only one small molecule drug can be connected at one end, so the maximum molecular weight of the conjugate is 3021.52, and the drug loading is 17.4%. According to research, when the molecular weight of the macromolecular drug is lower than 40000, the clearance rate of the kidney to the macromolecular drug is very high, so that the conjugate is difficult to achieve the aim of tumor targeting.
At present, no report on the targeting inflammation of the polymer micelle of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate connected by the hydrazone bond is found. According to the invention, a hydrazone bond is used for connecting the polyethylene glycol derivative and the micromolecule drug, and the polymer micelle is prepared by self-assembly, compared with the existing pH-sensitive polyethylene glycol-micromolecule drug conjugate for tumor targeting and the preparation thereof, the synthesized polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecule drug conjugate for inflammation targeting has the greatest advantage that the chemical modification sites connected with the micromolecule drug with carbonyl groups can be obviously increased by connecting hydrazine groups at the tail ends of a plurality of branched chains, so that the drug loading capacity (34-40%) of the micromolecule drug can be greatly improved.
Disclosure of Invention
The inventor discovers that the polymer micelle of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate with the structure shown in the formula (I) has an inflammation targeting effect through creative research.
The invention conception of the invention is as follows: (1) after a hydrazine group at the tail end of a branched chain of a polyethylene glycol derivative carrier is coupled with a carbonyl group of a micromolecular drug through a pH sensitive hydrazone bond, the pH sensitive polyethylene glycol derivative-micromolecular drug polymer micelle is further prepared through a self-assembly method, the defect that the drug is degraded in the systemic circulation in advance can be avoided, and the purpose of long circulation can be achieved under the protection of a polyethylene glycol derivative shell. (2) When reaching the inflammation site, the polymer micelle is passively targeted to the inflammation synovial tissue through ELVIS, and the pH sensitive hydrazone bond can be specifically broken in the inflammation synovial membrane with reduced pH, so that the small molecule drug is released, and the accumulation of the drug is increased. The polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate greatly improves the concentration of the micromolecular drug D at the low-pH inflammation part, prolongs the time of the micromolecular drug D at the low-pH inflammation part or improves the anti-inflammatory effect of the micromolecular drug D.
The invention aims to provide application of a polymer micelle of a pH-sensitive polyethylene glycol derivative-small molecule drug conjugate in preparation of a targeted drug for treating inflammation.
The prepared polymer micelle of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate can increase the targeting property of a nano-scale drug delivery system by utilizing the leakage effect of a vascular system with increased permeability of an inflammation part and the subsequent retention Effect (ELVIS) mediated by Inflammatory cells. The specific mechanism is as follows: in the chronic inflammatory environment of RA, inflammatory mediators can be persistently overexpressed, resulting in a "leaky" vasculature, similar to that of solid tumors. In addition, in RA patients, there are abundant leukocytes in the blood circulation, which increase the uptake of the conjugate, which then further infiltrate the inflammatory cells, thus promoting the accumulation of the conjugate in the inflamed tissue.
Another object of the present invention is to provide a polymer micelle of a pH-sensitive polyethylene glycol derivative-small molecule drug conjugate having a higher inflammation targeting property. The hydrazine group of the polyethylene glycol derivative and the carbonyl group of the micromolecular drug are connected through a pH sensitive hydrazone bond, the pH sensitive polyethylene glycol derivative-micromolecular drug polymer micelle is prepared through a self-assembly method, and the polymer micelle can release the micromolecular drug in a low-pH inflammatory environment, so that the drug can be actively targeted to an inflammatory part, the concentration and the side effect of the drug at other parts of the whole body are reduced, the administration dosage is reduced, and the curative effect is improved.
The invention provides a pH-sensitive polyethylene glycol derivative-small molecule drug conjugate shown in a formula (I), wherein a hydrazine group at the tail end of a branched chain of a polyethylene glycol derivative of a part A is connected with a carbonyl group of a small molecule drug of a part B through a pH-sensitive hydrazone bond, and the polyethylene glycol derivative of the part A is used as an inflammation targeting modification group of a small molecule drug D of the part B:
formula (I)
Wherein:
n = any integer from 1 to 200;
m = any integer from 1 to 100;
R1the ligand is hydrogen, hydroxyl, amino, alkoxy, sulfydryl, acetate group, acetamido, folic acid and other targeting ligands;
the alkoxy group has 1-10 carbon atoms;
connecting a hydrazine group at the tail end of a branched chain of the polyethylene glycol derivative of the part A with a carbonyl group of the micromolecular drug of the part B through a pH sensitive hydrazone bond; wherein, the carbonyl of the B part of the small molecule drug is the carbonyl of the drug or is added by modification;
the amino group is a free amino group;
x is one of methyl thioglycolate and methyl mercaptopropionate;
the drug D is preferably a chemically synthesized drug having a molecular weight of less than 1000 daltons for treating inflammatory diseases or a natural drug monomer for treating inflammatory diseases.
In the present invention, the small molecule drug D is a hydrophobic drug, including but not limited to: anti-inflammatory drugs, antitumor drugs, and the like.
Anti-inflammatory drugs (e.g., prednisolone, dexamethasone, hydrocortisone, cortisone acetate, methylprednisolone, betamethasone, beclomethasone, leflunomide, and the like and derivatives).
Antineoplastic drugs (e.g., doxorubicin, daunorubicin, actinomycin D, epirubicin, pirarubicin, and analogs and derivatives).
Attached X includes but is not limited to: one or more of methyl thioglycolate and methyl mercaptopropionate.
The invention also aims to provide application of the polyethylene glycol derivative of the part A in the formula (I) as an inflammation targeting modification group of the small molecule drug D of the part B.
Another object of the present invention is to provide a process for the preparation of the compound of formula (I) by first successively epoxidizing Boc-aminoethanol (where Boc-amino group may also be hydroxy, amino, alkoxy, mercapto, acetate, acetamido or folic acid) as a primer with sodium hydride as an activator at 60 deg.CEthane and allyl glycidyl ether are subjected to ring opening reaction, and then are subjected to the synthesis of animo- (EO) n- (AGE) m-O-CH with methyl thioglycolate (or methyl mercaptopropionate) under the catalysis of azobisisobutyronitrile3Then the compound is connected with hydrazine hydrate to synthesize the polyethylene glycol derivative with the hydrazine group at the end. And finally, under the acidic condition of acetic acid, connecting a hydrazine modification group at the tail end of the branched chain of the polyethylene glycol derivative of the part A with an active group carbonyl of the small molecular drug D of the part B by adopting a pH sensitive hydrazone bond.
The invention provides a polymeric micelle of a pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate, which is formed by self-assembling the pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate.
The invention provides a polymer micelle formed by self-composition of a pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate shown in a formula (I):
formula I
n = any integer from 1 to 200;
m = any integer from 1 to 100;
R1the ligand is hydrogen, hydroxyl, amino, alkoxy, sulfydryl, acetate group, acetamido, folic acid and other targeting ligands;
the alkoxy group has 1-10 carbon atoms;
the amino group is a free amino group;
x is one of methyl thioglycolate and methyl mercaptopropionate;
connecting a hydrazine group at the tail end of a branched chain of the polyethylene glycol derivative of the part A with a carbonyl group of the micromolecular drug of the part B through a pH sensitive hydrazone bond; the carbonyl of the B part of the small molecule drug is provided with the drug itself or added by modification.
The invention further provides a method for preparing the polymer micelle of the inflammation targeted pH sensitive polyethylene glycol derivative-small molecule drug conjugate, which comprises the following steps:
(1) connecting an active group carbonyl on the micromolecular drug with an active group hydrazine group at the tail end of a branched chain of the polyethylene glycol derivative through a pH sensitive hydrazone bond under the action of a catalyst;
(2) the polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecule drug conjugate is prepared by self-assembly.
As a specific embodiment and a non-limiting illustrative example of the present invention, the technical route of the present invention will be described below by taking the glucocorticoid prednisolone and the anti-tumor drug daunorubicin as examples, but the overall embodiment is not limited to prednisolone and daunorubicin.
The glucocorticoid has the functions of resisting inflammation, shock, virus, immunosuppression, etc., and is used with prednisolone, dexamethasone, betamethasone, etc. and may be used clinically widely in treating inflammation. But the toxic and side effects are not negligible: firstly, the damage to the digestive system is large, and the hormonal pancreatitis, the hormonal ulcer and the small intestine perforation are often caused; secondly, disturbances in sugar, lipid, protein, water and electrolyte metabolism are caused; thirdly, its immunosuppressive action reduces cellular and humoral immunity, making the body more vulnerable to external pathogenic bacteria. One of the more unique features of the local area of an RA-inflamed joint is its lower pH than the normal area, due to the mildly acidic local environment, also called acidosis, promoted by inflammatory reactions in and around the RA-joint. Based on the characteristic of low pH of inflammatory joints, a pH sensitive hydrazone bond is adopted to couple a polyethylene glycol derivative with a small molecular drug to form a pH sensitive polyethylene glycol derivative-small molecular drug conjugate, and a polymer micelle is formed through self-assembly. The polymer micelle can release small molecular drugs in a low-pH inflammatory environment, so that the drugs can be actively targeted to inflammatory parts, the concentration and the side effect of the drugs at other parts of the whole body are reduced, the administration dosage is reduced, and the curative effect is improved. Therefore, in order to enhance the local anti-inflammatory effect of prednisolone and reduce the toxic and side effects of prednisolone, in this experiment, a pH-sensitive hydrazone bond is used to couple the polyethylene glycol derivative and prednisolone, and a polymer micelle of the pH-sensitive polyethylene glycol derivative-prednisolone conjugate is prepared in a self-assembly manner, which is very necessary for promoting the inflammation targeting of prednisolone.
In the technical route, a polyethylene glycol derivative is connected with a prednisolone drug by a pH-sensitive hydrazone bond to obtain a pH-sensitive polyethylene glycol derivative-prednisolone conjugate I.
I
The preparation method of the pH-sensitive polyethylene glycol derivative-prednisolone conjugate and the polymer micelle is briefly described as follows:
firstly, the amino ethanol is protected by BOC anhydride to obtain BOC-amino ethanol, and then the BOC-amino ethanol and EO and AGE respectively carry out two-step ring-opening copolymerization reaction to obtain BOC-N- (EO) N- (AGE) m. Then BOC-N- (EO) N- (AGE) m is subjected to elimination of BOC protecting groups in a trifluoroacetic acid/dichloromethane (v/v: 5/2) solution to obtain an animo- (EO) N- (AGE) m product. Dissolving a proper amount of the product in THF, further carrying out reflux reaction with methyl thioglycolate under the catalysis of Azobisisobutyronitrile (AIBN) under the protection of nitrogen for 5 hours, and carrying out rotary evaporation to obtain a product animo- (EO) n- (AGE) m-OCH3The product is then refluxed and reacted with hydrazine hydrate in THF solution for 7 h, and the product animo- (EO) n- (AGE) m-OCH is obtained by rotary evaporation2-NH-NH2(polyethylene glycol derivatives). And then dissolving the product and prednisolone in dry DMSO, adding acetic acid and anhydrous sodium sulfate, stirring at room temperature in a dark place for reaction for 48h, filtering, separating and purifying the filtrate by a protein purifier, and finally freeze-drying to obtain the pH-sensitive polyethylene glycol derivative-prednisolone conjugate. And (3) dissolving a proper amount of the compound in double distilled water, and preparing the polymer micelle of the pH-sensitive polyethylene glycol derivative-prednisolone conjugate through self assembly.
Daunorubicin is the first generation anthracycline antitumor antibiotic, a periodic non-specific chemotherapeutic drug. It is mainly used for various acute leukemia (including granulocytic, lymphocytic and monocytic and granulo-monocytic), erythroleukemia, chronic granulocytic leukemia, malignant lymphoma, neuroblastoma, Ewing's sarcoma and nephroblastoma. The daunorubicin has wide clinical application but has limited clinical application due to serious toxic and side effects. The main adverse reactions include: 1. myocardial toxicity: if abnormal heart rhythm, urgent qi and edema of lower limbs occur, the possibility of congestive heart failure is cautioned; 2. toxicity in the blood system: leukopenia and thrombocytopenia; 3. hyperuricemia and kidney damage; 4. myelosuppression is severe; 5. can cause the central vein and lobular vein of liver to be blocked, which causes jaundice, hepatomegaly, hepatic encephalopathy and ascites. The anoxic state inside the solid tumor makes tumor cells anaerobic glycolysis to produce lactic acid, and the lack of blood vessel system inside the tumor makes the produced lactic acid not be discharged sufficiently, resulting in lower pH of local tumor environment. Based on the characteristic of low pH of a tumor part, in order to enhance the local anti-tumor effect of daunorubicin and reduce the toxic and side effects of the daunorubicin, in the experiment, a pH-sensitive hydrazone bond is adopted to couple the polyethylene glycol derivative and the daunorubicin, and the polymer micelle of the pH-sensitive polyethylene glycol derivative-daunorubicin conjugate is prepared by a self-assembly method, and can release the daunorubicin in a low-pH tumor environment, so that the daunorubicin can be actively targeted to the tumor part, the concentration and the side effects of the daunorubicin at other parts of the whole body are reduced, the administration dosage can be reduced, and the curative effect can be increased. Therefore, it is very necessary to promote tumor targeting of daunorubicin.
In the technical route, a polyethylene glycol derivative is used as a tumor targeting modification group, a medicine is daunorubicin, and the carrier and the medicine are coupled through a pH sensitive hydrazone bond to obtain a product, namely a pH sensitive polyethylene glycol derivative-daunorubicin conjugate II, wherein the structural formula of the product is as follows:
II
the preparation method of the pH-sensitive polyethylene glycol derivative-daunorubicin conjugate and the polymer micelle is briefly described as follows:
firstly, amino ethanol is protected by BOC anhydride to obtain BOC-amino ethanol, and then the BOC-amino ethanol is respectively subjected to two-step ring-opening copolymerization with EO and AGEReacting to obtain BOC-N- (EO) N- (AGE) m. Then BOC-N- (EO) N- (AGE) m is subjected to elimination of BOC protecting groups in a trifluoroacetic acid/dichloromethane (v/v: 5/2) solution to obtain an animo- (EO) N- (AGE) m product. Dissolving a proper amount of the product in THF, further carrying out reflux reaction with methyl thioglycolate under the catalysis of Azobisisobutyronitrile (AIBN) under the protection of nitrogen for 5 hours, and carrying out rotary evaporation to obtain a product animo- (EO) n- (AGE) m-OCH3The product is then refluxed and reacted with hydrazine hydrate in THF solution for 7 h, and the product animo- (EO) n- (AGE) m-OCH is obtained by rotary evaporation2-NH-NH2(polyethylene glycol derivatives). And then dissolving the product and daunorubicin in dry DMSO, adding acetic acid and anhydrous sodium sulfate, stirring at room temperature in the dark for reaction for 48h, filtering, separating and purifying the filtrate by a protein purifier, and finally freeze-drying to obtain the pH-sensitive polyethylene glycol derivative-daunorubicin conjugate. And (3) dissolving a proper amount of the compound in double distilled water, and preparing the polymer micelle of the pH-sensitive polyethylene glycol derivative-daunorubicin conjugate through self assembly.
Prednisolone or daunorubicin and polyethylene glycol derivatives are coupled by adopting a pH sensitive hydrazone bond, and the polymer micelle of the conjugate is prepared in a self-assembly mode.
The invention also provides a clinically acceptable preparation of the polymer micelle, such as a solution, an aerosol or a suspension, and an injection, preferably an injection.
The invention also aims to provide the polymer micelle of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate and the application of the preparation thereof in preparing drugs for treating inflammatory diseases or tumors.
The invention also aims to provide the polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecular drug conjugate and the application of the preparation thereof in preparing drugs for preventing and controlling rheumatoid arthritis, other inflammatory diseases and tumors.
① polymeric micelles of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugates can increase the stability in blood circulation under the protection of polyethylene glycol derivative shells to achieve a long circulation effect, ② polymeric micelles of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugates can be passively targeted into inflammatory synovium through ELVIS, ③ polymeric micelles of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugates can be degraded at low-pH inflammatory parts in vivo to release small molecule drugs to play a drug effect, ④ polymeric micelles of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugates have pH dependence in vivo drug release, so that the drug release behavior can be controlled, ⑤ polymer micelles of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugates have improved drug loading, simple connection methods, easy large-scale production, low production cost and good application prospects.
The invention has the advantages of
The invention also aims to provide the application of the polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecule drug conjugate in obviously improving the concentration of part B micromolecule drug D at a low-pH inflammation part or a low-pH tumor part, prolonging the time of the part B micromolecule drug D at the low-pH inflammation part or the low-pH tumor part, or improving the anti-inflammatory or anti-tumor effect of the part B micromolecule drug D.
In order to prove the inflammation targeting property of the polymer micelle of the pH sensitive polyethylene glycol derivative-micromolecule drug conjugate, in-vivo drug distribution experiments are carried out on the polymer micelle. Experiments prove that the polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecule drug conjugate has higher distribution concentration at inflammatory parts with low pH value than original drugs under the same administration mode and administration dosage.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a synthetic route for pH sensitive polyethylene glycol derivative-prednisolone conjugates.
FIG. 2 is a synthetic route for pH sensitive polyethylene glycol derivative-daunorubicin conjugates.
FIG. 3 is a graph showing the in vitro release of polymer micelles of the pH sensitive polyethylene glycol derivative-prednisolone conjugate at pH5.5, pH6.0, pH6.5, pH6.8 and pH7.4, respectively.
FIG. 4 is a graph showing the concentration distribution of the polymer micelle of the pH-sensitive polyethylene glycol derivative-prednisolone conjugate and the prednisolone bulk drug in plasma, tissues and organs after being administered in the tail vein for 1 hour.
FIG. 5 is a time-resolved mapping of the polymer micelle of pH-sensitive polyethylene glycol derivative-prednisolone conjugate and prednisolone bulk drug in inflammatory joints after tail vein administration
Detailed Description
The following examples are further illustrative of the present invention and are in no way intended to limit the scope of the invention. The present invention is further illustrated in detail below with reference to examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation method used. Also, equivalent substitutions, combinations, improvements or modifications of the invention may be made by those skilled in the art based on the description of the invention, but these are included in the scope of the invention.
Example 1
β -aminoethanol (7.37 g, 0.12 mol) was first protected with BOC anhydride (32.72 g, 0.15 mol) to give BOC-aminoethanol, then BOC-aminoethanol (0.88 g, 5 mmol) was taken and subjected to two-step ring-opening copolymerization with EO (100 mL, 1.98mol) and AGE (30.1 g, 0.26 mol), respectively, to give BOC-N- (EO) N- (AGE) m. then BOC-N- (EO) N- (AGE) m was subjected to elimination of BOC protecting group in trifluoroacetic acid/dichloromethane (v/v: 5/2) solution to give product animo- (EO) N- (AGE) m. the product animo- (EO) N- (AGE) m (6.72 g, 7.53 mmol C = C) was dissolved in THF, then reacted with methyl thioglycolate in Azobisisobutyronitrile (AIBN) under protection and reflux for 5h, after evaporation to give product animo- (EO) m3. Then all the products are refluxed and reacted with hydrazine hydrate (37.7g, 36.6 mL, 753 mmol) in THF solution for 7 h, and the product animo- (EO) n- (AGE) m-OCH is obtained by rotary evaporation2-NH-NH2(polyethylene glycol derivatives). Finally dissolving polyethylene glycol derivative (189 mg, 176 mu mol hydrazide) and prednisolone (21 mg, 58.3 mu mol) in dry DMSO, adding acetic acid and anhydrous sodium sulfate, stirring at room temperature in dark for 48h, filtering, purifying the filtrate with proteinSeparating and purifying by a chemical analyzer, and finally freeze-drying to obtain the pH-sensitive polyethylene glycol derivative-prednisolone conjugate.
1H NMR (400 MHz, DMSO-d6): 0.90 (t, 3H), 1.54 (m, 2H), 1.779 (m, 2H),2.635 (t, 2H), 3.235 (s, 2H), 3.37 (t, 2H), 3.644 (s, 3H)，3.676（m, 1H），5.426(s, 1H)，5.923 (s, 1H)，6.176 (dd, 1H)，7.323 (dd, 1H), 9.113（m, 1H）。
Example 2
β -aminoethanol (7.37 g, 0.12 mol) was first protected with BOC anhydride (32.72 g, 0.15 mol) to give BOC-aminoethanol, then BOC-aminoethanol (0.88 g, 5 mmol) was taken and subjected to two-step ring-opening copolymerization with EO (100 mL, 1.98mol) and AGE (30.1 g, 0.26 mol), respectively, to give BOC-N- (EO) N- (AGE) m. then BOC-N- (EO) N- (AGE) m was subjected to elimination of BOC protecting group in trifluoroacetic acid/dichloromethane (v/v: 5/2) solution to give product animo- (EO) N- (AGE) m. the product animo- (EO) N- (AGE) m (6.72 g, 7.53 mmol C = C) was dissolved in THF, then reacted with methyl thioglycolate in Azobisisobutyronitrile (AIBN) under protection and reflux for 5h, after evaporation to give product animo- (EO) m3. Then all the products are refluxed and reacted with hydrazine hydrate (37.7g, 36.6 mL, 753 mmol) in THF solution for 7 h, and the product animo- (EO) n- (AGE) m-OCH is obtained by rotary evaporation2-NH-NH2(polyethylene glycol derivatives). And finally, dissolving the polyethylene glycol derivative (189 mg, 176 mu mol of hydrazide) and the daunorubicin (31 mg, 58.3 mu mol) in dry DMSO, adding acetic acid and anhydrous sodium sulfate, stirring at room temperature in the dark for reaction for 48h, filtering, separating and purifying the filtrate by a protein purifier, and finally freeze-drying to obtain the pH-sensitive polyethylene glycol derivative-daunorubicin conjugate.
Example 3
Respectively and precisely weighing 30 mg of the two pH-sensitive polyethylene glycol derivative-micromolecule drug conjugates, dissolving the two pH-sensitive polyethylene glycol derivative-micromolecule drug conjugates in 7.5 ml of double distilled water, and then stirring the two pH-sensitive polyethylene glycol derivative-micromolecule drug conjugates for 0.5 hour at 25 ℃ along the same direction to obtain the polymer micelle of the pH-sensitive polyethylene glycol derivative-micromolecule drug conjugate of 4 mg/ml.
Example 4
First, 5 parts of 1 mL of a pH-sensitive polymeric micelle of a polyethylene glycol derivative-prednisolone conjugate at 4 mg/mL were placed in a dialysis bag (MW 1000). Then, the above 5 dialysis bags filled with the drug were immersed in a round-bottomed flask containing equal amounts of acetate buffer solutions of pH5.5, pH6.0, pH6.5, pH6.8 and pH7.4, respectively, and then incubated in a shaker at 37 ℃ and a speed of 100 rpm per minute. At the specified time points, 300. mu.L of release medium was injected into the HPLC assay and simultaneously supplemented with an equal amount of buffer in a round bottom flask, and all samples were assayed for release rate over 60 hours (n = 3).
Test example 1
Establishment of RA CIA model of mouse
DBA/1 mouse, weighing 20 + -2 g, male, on day 0, taking equal volume of bovine type II collagen and Freund's complete adjuvant, preparing into emulsion (the emulsion is dropped into ice water to form drops which are not dispersed and are regarded as the emulsification end point), and injecting 100 μ l each mouse subcutaneously. Then, a second boost is carried out on day 21, equal volume of bovine type II collagen and incomplete Freund's adjuvant is taken to prepare emulsion (the emulsion is dripped into ice water to form a non-dispersed drop which is regarded as an emulsification end point), each mouse is injected with 50 mul subcutaneously, and the diseased mouse is selected on day 50 to carry out an in vivo distribution experiment.
In vivo distribution experiment in mice
The affected DBA/1 mice were randomly divided into two large groups of 30 mice each. The first large group was a free prednisolone control group, and 6 individuals were divided into 5 groups, and administered by tail vein injection at 15.0 mg/kg (prednisolone/body weight); the second group is a polymer micelle experimental group of pH-sensitive polyethylene glycol derivative-prednisolone conjugate, 6 are divided into 5 groups, and 15mg/kg (calculated according to the dose equivalent to 15mg/kg of prednisolone) is administered by tail vein injection. The above two groups are taken at 0.5, 1, 2, 4 and 6 h after administration, the eye sockets are killed after blood is taken, the whole blood is centrifuged at 4500 rpm/min for 5min, and the plasma is taken and frozen at-40 ℃ for standby. The heart, liver, spleen, lung and kidney tissues were immediately separated, homogenized by adding twice the amount of physiological saline, and then frozen at-40 ℃ for use. Then cutting off joint tissues, adding twice of physiological saline water respectively after cutting, homogenizing, centrifuging, taking supernatant, and freezing at-40 ℃ for later use. After thawing the plasma viscera homogenate and the joint tissue homogenate, respectively processing and measuring according to a sample pretreatment method and a sample measuring method. The results of the in vivo distribution experiments are shown in FIG. 4.
Taking 0.3 ml of mouse plasma and 0.3 ml of homogenate, placing in a 5 ml EP tube, adding 10 μ l of 6N HCl, vortexing for 5min, and adding 6N NH with equal amount4OH, vortex for 5 min. Adding 300 mul acetonitrile solution, carrying out vortex extraction for 5min, 12000 r/min, centrifuging for 10 min, precisely absorbing 50 mul supernatant, injecting sample, and recording chromatogram and peak area of the drug. HPLC detection conditions: a chromatographic column: dikma Diamond column, ODS-C185 μm, 150 × 4.6 mm, gradient elution, mobile phase 0.05 mol/L trisodium citrate buffer (ph 4.1 adjusted with phosphoric acid) from 70% to 30%, detection wavelength: 254 nm, column temperature: 35 ℃, flow rate: 1 ml/min, detection time: 7.2 min, sample size: 50 μ l.
The results in fig. 3 show that the polymer micelle of the pH sensitive polyethylene glycol derivative-prednisolone conjugate releases prednisolone much faster in a more acidic environment at pH5.5 and pH6.0 than in a more neutral environment at pH6.5, pH6.8 and pH 7.4. At pH5.5 and pH6.0, the drug release rate reached 100% within 60 hours, and even approached 80% within 10 hours. The drug release rate was less than 70% within 60 hours at pH6.5, whereas the drug release rate was less than 40% within 60 hours at more neutral pH6.8 and pH7.4, both of which indicate that the in vitro release of the polymeric micelles of the pH sensitive polyethylene glycol derivative-prednisolone conjugate is pH dependent.
The results in fig. 4 show that after the tail vein administration of the polymer micelle of the pH-sensitive polyethylene glycol derivative-prednisolone conjugate, the drug concentration in the joint is much higher than that of the prednisolone bulk drug, which is 3.36 times higher than that in the case of 1 hour.
The results in fig. 5 show that the drug concentrations in the joints at different time points (0.5 h, 1 h, 2 h, 4 h, 6 h) after the tail vein administration of the polymer micelle of the pH-sensitive polyethylene glycol derivative-prednisolone conjugate are much higher than those of the prednisolone bulk drug, which are 2.58 times, 3.36 times, 4.89 times, 7.41 times and 40.8 times of those of the prednisolone bulk drug.
In conclusion, the polyethylene glycol derivative and the prednisolone are chemically connected to form the pH-sensitive polyethylene glycol derivative-prednisolone conjugate, and the polymer micelle is prepared by self-assembly, so that the drug can be specifically delivered to the inflammatory joint with low pH, the concentration and the anti-inflammatory effect of the drug at the inflammatory joint with low pH are improved, the administration dosage can be reduced, and the toxic and side effects are reduced.
Claims (8)
1. A pH-sensitive polyethylene glycol derivative-small molecule drug conjugate of formula I, characterized in that:
formula I
n = any integer from 1 to 200;
m = any integer from 1 to 100;
R1hydrogen, hydroxyl, amino, alkoxy, mercapto, acetate group and acetamido;
the alkoxy group has 1-10 carbon atoms;
the amino group is a free amino group;
x is one of methyl thioglycolate and methyl mercaptopropionate;
b, the hydrophobic small molecule medicine D is an anti-inflammatory medicine containing carbonyl and is selected from prednisolone, dexamethasone, hydrocortisone, cortisone, methylprednisolone, betamethasone, beclomethasone and leflunomide;
the preparation method comprises the steps of firstly, using Boc-aminoethanol, Boc-hydroxyethanol, Boc-alkoxyethanol, Boc-mercaptoethanol, Boc-acetate-based ethanol or Boc-acetaminoethanol as a primer, using sodium hydride as an activating agent, sequentially carrying out ring opening reaction of ethylene oxide and allyl glycidyl ether, then reacting with methyl thioglycolate or methyl mercaptopropionate, connecting the compound with hydrazine hydrate to synthesize a polyethylene glycol derivative with a hydrazine group at the tail end, finally connecting the hydrazine group at the tail end of a branched chain of the polyethylene glycol derivative of the part A with the carbonyl of the hydrophobic micromolecule drug D of the part B by adopting a pH sensitive hydrazone bond, and connecting the carbonyl of the hydrophobic micromolecule drug D of the part B with the drug self.
2. A polymer micelle of a pH-sensitive polyethylene glycol derivative-small molecule drug conjugate, which is characterized in that the polyethylene glycol derivative-small molecule drug conjugate of claim 1 is self-assembled to form the polymer micelle.
3. The use of the polymer micelle of the pH sensitive polyethylene glycol derivative-small molecule drug conjugate of claim 2 in the preparation of a medicament for significantly increasing the concentration of part B small molecule drug D at the inflammatory site of low pH or prolonging the time at the inflammatory site of low pH or increasing the anti-inflammatory effect thereof.
4. A pharmaceutical preparation, characterized in that the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate micelle of claim 2 is made into a pharmaceutically acceptable preparation; administration is by injection.
5. Use of the polymer micelle of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate of claim 1 or the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate of claim 2 in the preparation of a medicament for treating an inflammatory disease.
6. Use according to claim 5, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, chronic glomerulonephritis and pneumonia.
7. A method for preparing the conjugate of formula I according to claim 1, characterized in that the hydrazine group at the end of the polyethylene glycol derivative branch of moiety a is linked to the carbonyl group of the small molecule drug of moiety B via a pH sensitive hydrazone bond.
8. Use of the polymer micelle of the pH-sensitive polyethylene glycol derivative-small molecule drug conjugate according to claim 2 in the preparation of a medicament as an inflammation targeted delivery system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610426580.XA CN106139159B (en) | 2016-08-30 | 2016-08-30 | Application of polyethylene glycol derivative-small molecule drug conjugate polymer micelle in inflammation targeted drug delivery system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610426580.XA CN106139159B (en) | 2016-08-30 | 2016-08-30 | Application of polyethylene glycol derivative-small molecule drug conjugate polymer micelle in inflammation targeted drug delivery system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106139159A CN106139159A (en) | 2016-11-23 |
| CN106139159B true CN106139159B (en) | 2020-03-27 |
Family
ID=57353798
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610426580.XA Active CN106139159B (en) | 2016-08-30 | 2016-08-30 | Application of polyethylene glycol derivative-small molecule drug conjugate polymer micelle in inflammation targeted drug delivery system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106139159B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111635508B (en) * | 2020-06-04 | 2022-09-06 | 南方医科大学南方医院 | Polyethylene glycol-aliphatic saturated polyester block copolymer with amino-containing side chain and application thereof |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102475891A (en) * | 2010-11-23 | 2012-05-30 | 中国科学院化学研究所 | PH-responsive polyethylene glycol-anticarcinogen conjugate, and synthetic method and application thereof |
| CN102276826B (en) * | 2011-06-03 | 2013-01-02 | 中国科学院长春应用化学研究所 | Antineoplastic prodrugs and preparation method thereof |
| CN102343099B (en) * | 2011-09-30 | 2012-11-28 | 中国人民解放军第四军医大学 | Preparation method and application of folacin mediated tumor targeting adriamycin prodrugs |
| CN103357022B (en) * | 2013-06-08 | 2014-11-05 | 西安交通大学 | Bifunctional polyethylene glycol and adriamycin conjugate and preparation method thereof |
| CN103736101A (en) * | 2013-12-31 | 2014-04-23 | 天津大学 | Preparation method of pH value-sensitive curcumin-loading micelle (single chain) precursor |
| CN103961715A (en) * | 2014-04-25 | 2014-08-06 | 浙江大学 | Self-assembled adriamycin nano medicine precursor and preparation method and application thereof |
| CN104857525B (en) * | 2015-05-27 | 2017-12-15 | 湘潭大学 | It is a kind of using polyethylene glycol b poly epsilon caprolactones lactone as pH response type anti-tumor predrugs of carrier and preparation method thereof |
| CN105418909B (en) * | 2015-11-20 | 2017-11-24 | 中国人民解放军第四军医大学 | Polyethyleneglycol modified water-soluble couroupitine A polymer derivant and its preparation method and application |
-
2016
- 2016-08-30 CN CN201610426580.XA patent/CN106139159B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106139159A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zheng et al. | Improving breast cancer therapy using doxorubicin loaded solid lipid nanoparticles: Synthesis of a novel arginine-glycine-aspartic tripeptide conjugated, pH sensitive lipid and evaluation of the nanomedicine in vitro and in vivo | |
| Zhang et al. | Construction of a tumor microenvironment pH-responsive cleavable PEGylated hyaluronic acid nano-drug delivery system for colorectal cancer treatment | |
| Niu et al. | A novel chitosan-based nanomedicine for multi-drug resistant breast cancer therapy | |
| Guo et al. | Functional alginate nanoparticles for efficient intracellular release of doxorubicin and hepatoma carcinoma cell targeting therapy | |
| Ren et al. | Development of transferrin functionalized poly (ethylene glycol)/poly (lactic acid) amphiphilic block copolymeric micelles as a potential delivery system targeting brain glioma | |
| Cao et al. | Polymeric prodrugs conjugated with reduction-sensitive dextran–camptothecin and pH-responsive dextran–doxorubicin: an effective combinatorial drug delivery platform for cancer therapy | |
| CN105727307B (en) | Lipoic acid modified nano-polypeptide carrier and preparation method and application thereof | |
| Ren et al. | Carrier-free supramolecular hydrogel composed of dual drugs for conquering drug resistance | |
| KR101444274B1 (en) | Block copolymer for drug complex and pharmaceutical composition | |
| CN105727309A (en) | Preparation and application of dual-sensitivity amphiphilic polysaccharide-doxorubicin conjugate and pharmaceutical composition thereof | |
| CN109875964B (en) | Preparation and application of adriamycin carrier-free nano-drug | |
| JP2014518862A (en) | Polymer nanoparticles for drug delivery | |
| Lu et al. | Shell cross-linked and hepatocyte-targeting nanoparticles containing doxorubicin via acid-cleavable linkage | |
| Song et al. | Erythrocyte-biomimetic nanosystems to improve antitumor effects of paclitaxel on epithelial cancers | |
| CN101480377B (en) | Method for preparing tumor enzyme-targeted nano medicine-carrying polymer microsphere | |
| CN109999197B (en) | Tumor-targeted nano-composite, preparation method and application thereof in precise sonodynamic-mediated tumor treatment | |
| CN103656650B (en) | A kind of brain tumor twin-stage targeted nano delivery system of pH sensitivity and preparation method and application | |
| CN105999299A (en) | Small-molecular micelle drug-loaded nano-system, as well as preparation method and application thereof | |
| WO2022199408A1 (en) | Preparation method for and application of novel injection abiraterone derivative | |
| WO2021190495A1 (en) | Pegylated heparin nano-micelle loaded with carboxylic acid anti-tumor drug and preparation method therefor | |
| CN106139159B (en) | Application of polyethylene glycol derivative-small molecule drug conjugate polymer micelle in inflammation targeted drug delivery system | |
| Xu et al. | The design and synthesis of redox-responsive oridonin polymeric prodrug micelle formulation for effective gastric cancer therapy | |
| CN109771660A (en) | It is a kind of to respond the preparation of pectin-adriamycin/tripterine nanometer particle with pH | |
| KR101429668B1 (en) | Nanoparticles comprising amphiphilic low molecular weight hyaluronic acid complex and a process for the preparation thereof | |
| CN113827567A (en) | Application of small-molecule-drug-carrying polymer vesicle in preparation of drugs for treating acute lymphatic leukemia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |