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CN-121971641-A - Hepatic stellate cell targeted conjugate, and preparation method and application thereof

CN121971641ACN 121971641 ACN121971641 ACN 121971641ACN-121971641-A

Abstract

The invention discloses a conjugate targeting hepatic stellate cells, a preparation method and application thereof, and belongs to the technical fields of biological medicine and pharmaceutical chemistry. The conjugate is RRG-TK-Ato, and the molecular formula of the conjugate is C 98 H 144 FN 27 O 14 S 2 . The conjugate is formed by covalently connecting Atorvastatin (Atorvastatin) with targeting polypeptide RRGRLKRWY through Reactive Oxygen Species (ROS) sensitive thioketal (Thioketal, TK), can specifically target aHSCs and intelligently release Atorvastatin in a hepatic fibrosis oxidative stress microenvironment, overcomes the limitation of medicines, realizes synergistic attenuation hepatic fibrosis targeting treatment, can more effectively induce activated hepatic stellate cell apoptosis, has obvious specific inhibition effect on proliferation of activated hepatic stellate cells, can more effectively inhibit migration capacity of the activated hepatic stellate cells, thereby restricting spread of fibrosis and improving liver function of hepatic fibrosis.

Inventors

  • JIN JIANBO
  • Lu Zhewen
  • MAO HAIBO

Assignees

  • 宁波大学附属阳明医院(余姚市人民医院)

Dates

Publication Date
20260505
Application Date
20260407

Claims (10)

  1. 1. A conjugate for targeting hepatic stellate cells is characterized in that the conjugate is RRG-TK-Ato, and has the structural formula: 。
  2. 2. A method for preparing a hepatic stellate cell-targeted conjugate according to claim 1, comprising the steps of: (1) Dissolving atorvastatin in anhydrous dichloromethane serving as an organic solvent, adding a condensing agent A and organic base, stirring and mixing for reaction for 30-60min, adding 2,2' - (propane-2, 2-diylbis (sulfadiyl)) diethylamine, performing condensation reaction for 10-24h at room temperature, extracting a reaction solution by using an organic phase, concentrating under reduced pressure, separating and purifying to obtain an intermediate TK-Ato; (2) The synthesis of the conjugate RRG-TK-Ato comprises the steps of dissolving an intermediate TK-Ato and polypeptide in anhydrous N, N-dimethylformamide of an organic solvent, stirring and reacting in an ice bath and nitrogen atmosphere to obtain a mixed solution, adding a condensing agent B and organic base into the mixed solution, stirring, activating and reacting for 30-60min, removing the ice bath, heating to room temperature and reacting for 4-8h, concentrating under reduced pressure, and purifying to obtain the conjugate RRG-TK-Ato.
  3. 3. The method of claim 2, wherein the condensing agent A in the step (1) is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
  4. 4. The method of claim 2, wherein the organic base in step (1) and step (2) is N, N-diisopropylethylamine.
  5. 5. The method for preparing hepatic stellate cell-targeted conjugate according to claim 2, wherein the mass ratio of atorvastatin, condensing agent A, organic base and 2,2' - (propane-2, 2-diylbis (sulfadiyl)) diethylamine in step (1) is 1-3:1-2:1:2-4.
  6. 6. The method of claim 2, wherein the polypeptide in step (2) is a polypeptide fragment RRGR LKRWY.
  7. 7. The method of claim 2, wherein the condensing agent B in the step (2) is HATU.
  8. 8. The method for preparing the hepatic stellate cell-targeted conjugate according to claim 2, wherein the mass ratio of the intermediate TK-Ato, the polypeptide, the condensing agent B and the organic base in the step (2) is 4-6:5-6:2-4:1.
  9. 9. Use of a hepatic stellate cell-targeted conjugate according to claim 1 for the preparation of a medicament for the prevention or treatment of liver fibrosis.
  10. 10. Use of a hepatic stellate cell-targeted conjugate of claim 1 for the preparation of an activated hepatic stellate cell inhibitor.

Description

Hepatic stellate cell targeted conjugate, and preparation method and application thereof Technical Field The invention relates to the technical field of biological medicine and pharmaceutical chemistry, in particular to an Atorvastatin conjugate of targeted activated hepatic stellate cells, a preparation method and application thereof, and more particularly relates to a conjugate RRGRLKRWY-TK-Atorvastatin (RRG-TK-Ato) formed by covalently connecting Atorvastatin (Atorvastatin) with targeted peptide RRGRLKRWY through Reactive Oxygen Species (ROS) sensitive thioketal (Thioketal, TK) linker, a preparation method of the conjugate and application of the conjugate in preparation of medicines for preventing or treating hepatic fibrosis. Background Liver fibrosis is a common pathological link in the progression of various chronic liver diseases to cirrhosis, liver cancer, and is mainly characterized by an imbalance in the excessive deposition and degradation of extracellular matrix (Extracellular Matrix, ECM). Activation of hepatic stellate cells (HEPATIC STELLATE CELLS, HSCS) is a central event in the development of hepatic fibrosis. In normal liver, HSCs are in a static state, when the liver is damaged, the HSCs are activated and converted into myofibroblasts, alpha-smooth muscle actin (alpha-SMA) is highly expressed, ECM components such as collagen are secreted in a large amount, and the activity of Matrix Metalloproteinases (MMPs) is inhibited, so that ECM synthesis is far greater than degradation, and finally fibrous scars are formed. Therefore, induction of apoptosis of activated hepatic stellate cells (ACTIVATED HEPATIC STELLATE CELLS, AHSCS), inhibition of activation thereof and ECM secretion have become key strategies for anti-hepatic fibrosis drug development. Statin drugs are lipid-regulating drugs widely used in clinic, and reduce cholesterol by inhibiting hydroxymethylglutaryl-CoA (HMG-CoA) reductase. In recent years, a great deal of researches show that statin drugs have multiple effects and show potential anti-hepatic fibrosis effect. It was found that Atorvastatin (Ato) can exert an anti-fibrosis effect by acting on aHSCs by a mechanism comprising down-regulating the expression of anti-apoptotic protein Bcl-2 in aHSCs, up-regulating the expression of pro-apoptotic protein Bax, thereby inducing the collapse of mitochondrial membrane potential and starting the apoptosis program of aHSCs, and simultaneously, atorvastatin can directly reduce the generation of ECM components such as type I and type III collagens in aHSCs, and through reducing the expression of matrix metalloproteinase tissue inhibitor (TIMP-1 and TIMP-2), inhibit MMPs and promote the degradation of deposited ECM. The findings provide theoretical basis for anti-fibrosis application of statin drugs. However, atorvastatin itself is not selective for aHSCs. It is widely distributed in liver, and not only acts on aHSCs, but also affects other cells such as liver cells and liver sinus endothelial cells. The nonselective distribution causes two main problems, namely that the effective treatment concentration of the medicine can not be achieved in aHSCs, the anti-fibrosis curative effect is limited, and the risk of side effects such as drug liver injury, muscle toxicity and the like can be increased when the medicine is used at high dose or for a long time. Therefore, how to accurately deliver atorvastatin to aHSCs to realize targeted therapy is a key to improving the curative effect and reducing toxic and side effects. As the surface markers of aHSCs were studied intensively, a variety of membrane proteins were found that were specifically highly expressed during activation, with Platelet-derived growth factor receptor β (Platelet-Derived Growth Factor Receptor β, PDGFR- β) being one of the recognized, most significant up-regulation receptors. PDGFR-beta is expressed on aHSCs surface in large quantity, mediates proliferation and migration of cells, and is an ideal drug delivery target. Research reports that through screening and design, polypeptides with high affinity and specificity to PDGFR-beta, such as a polypeptide with a sequence RRGRLKRWY, can simulate the combination of natural ligand and PDGFR-beta, thereby realizing active targeting to aHSCs. In addition, hepatic fibrosis microenvironment is characterized by elevated levels of oxidative stress, and activated inflammatory cells and damaged hepatocytes release large amounts of reactive oxygen species (Reactive Oxygen Species, ROS), such as hydrogen peroxide (H 2O2). By utilizing the pathological characteristics, the design of the connector which can be selectively broken under the ROS environment is an effective means for realizing the intelligent release of the medicine at the target site. The thioketal (Thioketal, TK) linker is a class of chemical groups that are highly sensitive to ROS, can undergo oxidative cleavage under conditions of high concentration ROS, and is relatively stabl