CN101810559A - Hirudin polyion micelle composition of targeted platelet - Google Patents

Abstract

The present invention relates to a hirudin polyion micelle composition targeting platelets. The composition contains hirudin, arginine-glycine-aspartic acid-polyethylene glycol grafted chitosan and polyion initiator. The preparation method of the polyion micelle composition is simple, has platelet targeting, enhances the anticoagulant and antithrombotic activity of hirudin, effectively inhibits platelet aggregation, and simultaneously significantly improves the partial thromboplastin time of plasma.

Description

Hirudin polyion micelles composition targeting platelets

technical field

The present invention relates to a polyionic micellar composition of arginine-glycine-aspartic acid-polyethylene glycol-chitosan (RGD-PEG-CS) loaded with hirudin targeting platelets for injection and its preparation method.

Background technique

Cardiovascular and cerebrovascular diseases are one of the three major fatal diseases, among which thromboembolism is the cause of many cardiovascular and cerebrovascular diseases. Due to the induction of arteriosclerosis, vascular endothelial injury, abnormal internal and external coagulation system, or abnormal hemodynamics, the components in the blood flow process form a semi-clot-like substance on the surface of the blood vessel or the intima of the heart, which is a thrombus. Aiming at various mechanisms of thrombus formation, a variety of antithrombotic drugs have come out one after another, including drugs that inhibit thrombosis (anticoagulant and antithrombotic) and platelet aggregation (antiplatelet), and drugs that dissolve thrombosis. Anticoagulant antithrombotic and antiplatelet drugs are used in the initial stage of thrombosis and in the secondary thromboprophylaxis after thrombolytic therapy. Anticoagulants such as heparin and warfarin combined with antiplatelet drugs such as aspirin are mainly used clinically before thrombosis and after thrombolysis. However, most of the above drugs have side effects such as inducing bleeding, and their clinical safety has been questioned.

Hirudin is a single-chain polypeptide with a molecular weight of 6950 Da composed of 65 amino acids, with an acidic amino acid and an isoelectric point of 3.8. Extracted from the salivary glands of leeches by Markwardt in the middle of the last century, it is the strongest thrombin inhibitor so far, with direct anticoagulant, indirect antiplatelet and indirect profibrinolytic effects. And it has won widespread attention for its high efficiency, specificity, non-antigenicity, low toxicity, and small bleeding side effects. In 1986, the successful introduction of recombinant hirudin obtained from gene fermentation engineering, with its relatively low cost and relatively high yield, marked the possibility of hirudin entering clinical application. The drug can be used for acute coronary syndrome, adjuvant therapy of streptokinase thrombolysis, deep vein thrombosis, heparin-induced thrombocytopenia, hemodialysis, disseminated intravascular coagulation, etc. However, hirudin has a short plasma half-life, lacks tissue specificity, and cannot selectively act on the lesion. Therefore, it needs to be used in large doses continuously in clinical practice, and is prone to serious complications such as bleeding, which limits its clinical application. Enhancing the specific targeting of thrombolytic agents is a key measure to reduce drug dosage, prolong drug action time in vivo, and avoid thrombus re-formation.

Arginine-glycine-aspartic acid sequence (Arg-Gly-Asp, RGD) short peptide is a cell membrane integrin receptor and activated platelet IIb/IIIa globulin (GP IIb/IIIa) or other extracellular ligands Combined recognition sites. On resting platelets, GP IIb/IIIa is inactive and cannot bind to the RGD fragment of the adhesion protein. However, once the endothelium is damaged and platelets adhere to it, inducers (such as thrombin, collagen, thrombospondin, ADP, thromboxane A2, etc.) bind to their corresponding receptors and regulate GPIIb through different intracellular signaling mechanisms The activated state of the three-dimensional configuration of /IIIa activates it, improves the adhesion ability of GP IIb/IIIa and fibrinogen, and accelerates the adhesion and aggregation of platelets. RGD tripeptide acts as a target molecule to activate platelets, brings anticoagulant and antithrombotic drugs to the vicinity of thrombus, and competitively binds to GPIIb/IIIa on activated platelets, antagonizes the combination of platelets and fibrin, and reduces the adhesion and aggregation of platelets. Therefore, the RGD peptide can serve as a homing warhead for thrombus-targeting agents. CN101176785 discloses a recombinant bifunctional hirudin oral preparation and its preparation method. This patent has carried out structural modification on hirudin, recombined the RGD gene into the hirudin gene, and obtained the anticoagulant and antiplatelet bifunctional recombination by fermentation. Hirudin. However, the production cost of the above method is extremely high, and the original chemical composition of the drug is changed. Therefore, there is still a need to develop a hirudin composition that does not change the chemical composition of the original drug, has a simple preparation process, has a long circulation time in the body, and targets platelets.

Polyethylene glycol grafted chitosan is an improved product of chitosan, which introduces hydrophilic long-chain polyethylene glycol into the amino group of chitosan, which effectively improves the water solubility and biocompatibility of chitosan , under the action of polyion initiators, they can self-assemble in aqueous solution to form polyion micelles. Polyethylene glycol-grafted chitosan polyion micelles, as a new type of drug carrier, have the characteristics of traditional polymer micelles: stable structure, small particle size and uniform distribution; good safety; simple preparation process; easy to In addition to storage, it also has its own unique advantages: a large number of free amino groups on chitosan are protonated in an acidic environment, relying on electrostatic interaction to combine with negatively charged drugs to form the hydrophobic end of micelles, and the grafted polyethylene glycol on it A dense hydration shell is formed outside the hydrophobic inner core, which can prevent micelles from being taken up by the immune system, prolong the circulation time of drugs in the body, and achieve the purpose of long circulation. In addition, the polyionic micelles drug loading system also has the advantages of high encapsulation efficiency, wide range of drug loading, the drug loading process is basically carried out in aqueous solution, avoids the use of organic solvents, and can eliminate the toxic and side effects caused by solvent residues. If RGD is linked to chitosan through polyethylene glycol, it can play the role of long circulation and targeting platelets at the same time.

Contents of the invention

On the one hand, the present invention provides a polyion micelles composition targeting platelets with hirudin entrapped in polyethylene glycol grafted chitosan.

On the other hand, the present invention provides the application of polyion micelles composition targeting platelets with polyethylene glycol grafted chitosan loaded with hirudin for injection.

According to one aspect of the present invention, it provides a hirudin-loaded polyionic micellar composition for platelet-targeted RGD-PEG-CS for injection administration, which contains hirudin, and RGD-PEG-CS, Pharmaceutical excipients such as polyion initiators.

The present invention also relates to methods of preparing platelet-targeting polyionic micellar compositions. Including: prepare RGD-PEG-CS solution with acetic acid-sodium acetate buffer solution, add hirudin solution under electromagnetic stirring, add polyion initiator aqueous solution dropwise after stirring reaction, seal and stir and filter with filter to obtain RGD loaded with hirudin - PEG-CS polyionic micelles. The pH range of the acetic acid-sodium acetate buffer is 4.0-5.4, preferably 4.6-5.2, more preferably 4.8-5.0. The weight ratio of RGD-PEG-CS to hirudin is 5:2-20:2, preferably 10:2-16:2. The weight ratio of RGD-PEG-CS to TPP is 10:1-0.5:1, preferably 4:1-3:1. Wherein the average particle size of the micelles is below 200nm, and the encapsulation efficiency is higher than 80%.

The composition of the present invention can be formulated into injection preparations according to any conventional method, such as injection solution, freeze-dried powder injection and the like.

The composition of the invention can be used to prepare medicines for treating acute coronary syndrome, adjuvant therapy of streptokinase thrombolysis, deep vein thrombosis, heparin-induced thrombocytopenia, hemodialysis, disseminated intravascular coagulation and other related diseases.

The medicines of the composition of the present invention for treating the above diseases can be administered by injection.

The composition of the invention contains hirudin as an active ingredient, and also includes polyion micelles composed of RGD-PEG-CS and TPP. After the composition of the invention is administered by injection, the anticoagulant and antithrombotic activity of the hirudin is enhanced, effectively inhibits platelet aggregation, and simultaneously significantly increases the partial thromboplastin time of plasma. Representative examples of hirudin in the composition of the present invention are natural hirudin, recombinant hirudin obtained by genetic engineering fermentation, and variants thereof. The hirudin in the composition of the present invention is at least one of them, and the content of the hirudin in the composition is 0.1-50%, preferably 0.3-40%, more preferably 0.5-20%, based on the total weight of the composition.

RGD-PEG-CS is the main component that constitutes micelles in the composition of the present invention, is the graft product of arginine-glycine-aspartic acid-polyethylene glycol and chitosan, wherein arginine-glycine - Aspartic acid (RGD) is a short peptide with an RGD-Y structure, and Y may not be any amino acid, but may also be amino acids such as valine (V), serine (S), and phenylalanine (F). The polyethylene glycol has a molecular weight of 400-20000Da, preferably 2000Da-6000Da. The chitosan is chitosan or its derivatives, with a molecular weight of 5-2000kDa, preferably 10-1000kDa, more preferably 10-600kDa. The chitosan or its derivatives have a deacetylation degree of 70%-95%, preferably 75%-90%. The molar ratio of RGD-PEG to amino groups on chitosan is 1:4-1:30, preferably 1:10-1:20.

A polyionic initiator is also an ingredient of the compositions of the present invention, preferably sodium tripolyphosphate.

In addition, the compositions of the present invention also include optional pharmaceutically acceptable additives.

In order to achieve the purpose of the present invention, the present invention carried out the determination of the partial thromboplastin time and platelet aggregation rate of the composition of the present invention to evaluate the targeting of the composition of the present invention.

Description of drawings

The above and other objects and features of the present invention will be apparent from the following description of the present invention with reference to the accompanying drawings 1, 2 and Table 1. Accompanying drawing 1 has shown the particle size distribution of the embodiment 1 of the present invention. Accompanying drawing 2 has shown the partial thromboplastin time (APTT) of embodiment 1 of the present invention and commercially available hirudin injection.

As mentioned above, the composition of the present invention can effectively inhibit platelet aggregation, significantly increase plasma APTT, and enhance the anticoagulant and antithrombotic activity of hirudin.

The following examples are used to further describe the present invention in detail, but are absolutely not intended to limit the scope of the present invention.

specific implementation plan

Embodiment 1: the preparation of injection

Dissolve 16mg of RGD-PEG-CS in 10ml of acetic acid-sodium acetate buffer solution with pH4.9, add 10ml of hirudin solution with a concentration of 0.2mg/mL under electromagnetic stirring, stir and react for 30min, then add 5ml of hirudin solution with a concentration of 1.1mg/mL dropwise Sodium phosphate aqueous solution, sealed and stirred for 30min. Filter with a 0.45 μm filter to obtain the platelet-targeted hirudin-polyethylene glycol-grafted chitosan polyion micelles composition.

An appropriate amount of benzyl alcohol aqueous solution is added to the obtained composition, mixed evenly, filtered, potted and sterilized by radiation.

Embodiment 2: the preparation of freeze-dried powder injection

Dissolve 32mg of RGD-polyethylene glycol grafted chitosan in 10ml of acetic acid-sodium acetate buffer solution with pH 4.8, add 10ml of hirudin solution with a concentration of 0.5mg under electromagnetic stirring, and add 4ml of a solution with a concentration of 1.5mg after stirring for 45min. mg/mL sodium tripolyphosphate aqueous solution, sealed and stirred for 40min. Filter with a 0.45 μm filter to obtain the platelet-targeted hirudin-polyethylene glycol-grafted chitosan polyion micelles composition.

Add appropriate amount of glucose and mannitol into the obtained micelle composition, mix uniformly, freeze-dry, seal and sterilize by radiation.

Test Example 1: APTT Determination of Polyionic Micelles

15 SD rats were randomly divided into three groups, respectively hirudin solution group, hirudin common micelles group (PEG-CS), hirudin targeted micelles group (RGD-PEG-CS) (Example 1) . Take 1.6mL of blood from the orbital venous plexus (sodium citrate anticoagulant, whole blood:anticoagulant=8.75:1.25), absorb 300μl of anticoagulated whole blood, add 30μl of each solution of different concentration, vortex shake for 1min to mix, 4000rpm Centrifuge for 10 min, absorb 100 μl of supernatant, and freeze at -20°C until testing. APTT was measured on a 37°C water bath. Take the concentration as the abscissa, and the ratio of the measured time to the plasma APTT of the blank sample as the ordinate to obtain a dose-effect relationship diagram. The result is shown in Figure 2.

The results in Figure 2 show that, compared with the commercially available hirudin injection, Example 1 can significantly increase the APTT of plasma. Compared with ordinary PEG-CS micelles, Example 1 has a stronger effect of prolonging APTT, indicating that RGD plays a targeting role, guiding micelles to platelets activated and aggregated by exogenous coagulation, and binding to them, while To achieve anti-platelet and enhance the anticoagulation and antithrombotic activity of hirudin.

Test Example 2: Determination of Polyionic Micellar Platelet Aggregation Rate

Blood was collected from the ear veins of New Zealand big-eared rabbits, anticoagulated with sodium citrate 1:9, and equal concentrations of hirudin solution, hirudin common micelles (PEG-CS), and hirudin targeted micelles (RGD -PEG-CS) (embodiment 1), the maximum platelet aggregation rate was determined by nephelometric method after mixing for 5 minutes. The results are shown in Table 1.

The results in Table 1 show that the maximum platelet aggregation rate of Example 1 is lower than that of common micelles and commercially available injections at the same concentration, indicating that Example 1 can effectively inhibit platelet aggregation and enhance the anticoagulant and antithrombotic activity of hirudin.

Table 1: Determination results of maximum platelet aggregation rate

Concentration Commercially available injections Ordinary micelles Example 1 10μg/mL 16.6% 13.2% 5.90% 2.0μg/mL 35.9% 37.4% 23.3% 0.50μg/mL 69.4% 68.7% 53.8%

Although the present invention has been described using the specific embodiments above, it should be recognized that those skilled in the art can also make various improvements and changes, and they should also be within the scope of the present invention as defined in the claims Inside.

Claims (15)

1. A composition containing hirudin, arginine-glycine-aspartic acid-polyethylene glycol grafted chitosan (RGD-PEG-CS) and a polyion initiator. 2. The composition as claimed in claim 1, wherein the weight ratio of RGD-PEG-CS to hirudin is 5:2-20:2, preferably 10:2-16:2. 3. The composition as claimed in claim 1, wherein the hirudin is selected from natural hirudin, recombinant hirudin obtained by genetic engineering fermentation, and variants thereof. 4. composition as claimed in claim 1, wherein arginine-glycine-aspartic acid (RGD) is the short peptide with RGD-Y structure, and Y can not be any amino acid, also can be valine (V), Serine (S), Phenylalanine (F). 5. The composition as claimed in claim 1, wherein the polyethylene glycol has a molecular weight of 400-20000Da, preferably 2000-6000Da. 6. The composition according to claim 1, wherein the chitosan is chitosan or a derivative thereof, having a deacetylation degree of 70%-95%, preferably 75%-90%. 7. The composition according to claim 1, wherein the chitosan is chitosan or a derivative thereof, and the molecular weight is 5-2000kDa, preferably 10-1000kDa. 8. The composition according to claim 1, wherein the molar ratio of polyethylene glycol to amino groups on the chitosan is 1:4-1:30, preferably 1:10-1:20. 9. The composition of any one of claims 1-8, wherein the polyion initiator is sodium tripolyphosphate. 10. The preparation method of the composition as described in any one of claim 1-9, comprises preparing RGD-PEG-CS solution with acetic acid-sodium acetate buffer solution, drips into hirudin solution under stirring, drips polysaccharide after stirring reaction Aqueous solution of ionic initiator, stirred and filtered. 11. The method according to claim 10, wherein the pH range of the acetic acid-sodium acetate buffer is 4.0-5.4, preferably 4.6-5.2, more preferably 4.8-5.0. 12. The method according to claim 10, wherein the weight ratio of RGD-PEG-CS to TPP is 10:1-0.5:1, preferably 4:1-3:1. 13. A pharmaceutical composition, which comprises the composition according to any one of claims 1-9 and pharmaceutically acceptable excipients. 14. The composition according to any one of claims 1-9 is used for the preparation of treatment of acute coronary syndrome, adjuvant therapy of streptokinase thrombolysis, deep vein thrombosis, heparin-induced thrombocytopenia, hemodialysis, diffuse Use of drugs for related diseases such as intravascular coagulation. 15. The use according to claim 14, characterized in that the drug is administered by injection.

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