Intelligent targeting polypeptide drug conjugate for stopping bleeding of invisible bleeding and application of intelligent targeting polypeptide drug conjugate

Abstract

The invention provides an intelligent targeting polypeptide drug conjugate for stopping invisible bleeding. The intelligent targeting polypeptide drug conjugate is formed by connecting plasmin cleavable polypeptide with von willebrand factor binding peptide and tranexamic acid. In blood circulation, the intelligent targeting polypeptide drug conjugate is specifically combined with the von Willebrand factor of a vascular rupture opening through the von Willebrand factor binding peptide so as to target a bleeding part, and under the condition that the plasmin activity is increased, the conjugate is specifically sheared by plasmin so as to release tranexamic acid, so that the tranexamic acid is released, and the target of the intelligent targeting polypeptide drug conjugate is achieved. The tranexamic acid stabilizes blood clots by inhibiting dissolution of plasmin on fibrous protein networks in the blood clots, so that the targeted intelligent hemostasis function is achieved, the effects of rapid hemostasis and stable hemostasis of the blood clots are achieved, repeated bleeding or continuous bleeding is effectively prevented, and expansion of spontaneous cerebral hemorrhage hematoma is limited.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide drug conjugates, and particularly relates to an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis. Background Art

[0002] Currently, the clinical treatment for acute spontaneous intracerebral hemorrhage (ICH) relies on hemostatic drugs. Commonly used drugs include aminocaproic acid, aminomethylbenzoic acid, and tranexamic acid (TXA). TXA can inhibit plasminogen activation and reduce fibrin degradation, and is the drug with the strongest antifibrinolytic effect. However, direct injection has insufficient efficacy or potential systemic risks, including adverse reactions such as off-target thrombosis, thromboembolism, and neuropathy. Therefore, developing a drug delivery system with the ability to target bleeding points and achieve site-specific release is crucial for improving the therapeutic effect and safety of TXA. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the deficiencies of the above-mentioned prior art, and provides an intelligent plasmin-responsive targeted polypeptide drug conjugate for invisible (occult) bleeding hemostasis, which is composed of von Willebrand factor-binding peptide (VBP), plasmin-cleavable polypeptide (KTFKC), and tranexamic acid (TXA). Among them, KTFKC is used as a linker to covalently connect VBP and TXA to form a plasmin-responsive polypeptide drug conjugate (VBP-KTFKC-TXA, RPDC). In the blood circulation, RPDC can specifically bind to von Willebrand factor (vWF) through VBP and target to the blood clot enriched at the rupture of cerebral microvessels. In the pathological environment with elevated local plasmin activity, KTFKC is specifically cleaved and releases TXA, which competitively inhibits the binding of plasminogen to fibrin, blocks the degradation of the fibrin network in the blood clot by the fibrinolytic system, thereby stabilizing the thrombus and restricting the further expansion of the intracerebral hemorrhage (ICH) hematoma.

[0004] To solve the above technical problem, the technical solution adopted by the present invention is: an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis, which is formed by a plasmin-cleavable polypeptide connecting von Willebrand factor-binding peptide and tranexamic acid.

[0005] The present invention discloses an application of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis, and the intelligent targeted polypeptide drug conjugate is used for targeted hemostasis of invisible bleeding in vivo.

[0006] Preferably, the intelligent targeted polypeptide drug conjugate is used for spontaneous intracerebral hemorrhage.

[0007] The present invention discloses the application of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis, and the intelligent targeted polypeptide drug conjugate is used for bleeding management that cannot be stopped by pressing.

[0008] Preferably, the intelligent targeted polypeptide drug conjugate is used for liver bleeding caused by surgery.

[0009] Preferably, the intelligent targeted polypeptide drug conjugate targets the bleeding point by specifically binding to von Willebrand factor at the bleeding site, releases tranexamic acid in response to plasmin at the bleeding point, and controls the site-specific release of tranexamic acid.

[0010] Preferably, after the polypeptide drug conjugate is made into an injection, the polypeptide drug conjugate enters the blood circulation, and the von Willebrand factor binding peptide in the polypeptide drug conjugate can specifically bind to the von Willebrand factor exposed at the blood vessel rupture, so as to target the blood clot formed at the rupture opening of the bleeding blood vessel. Under the action of high-level plasmin locally in the blood clot, the plasmin in the conjugate can cleave the polypeptide and be degraded, releasing the antifibrinolytic drug tranexamic acid. Tranexamic acid is released by cleavage by high-level plasmin in the blood vessel, and the released tranexamic acid further inhibits the dissolution of the fibrin network in the blood clot by plasmin. Tranexamic acid further inhibits the dissolution of the fibrin network in the blood clot, stabilizes the coagulation thromboembolism, thereby exerting a targeted intelligent hemostasis function and effectively limiting the expansion of the hematoma caused by spontaneous intracerebral hemorrhage.

[0011] After the occurrence of hemorrhagic diseases, vascular endothelial cell injury exposes collagen. Free hemophilia factor in plasma binds to the collagen fibers exposed under the endothelium, enabling platelets to adhere to the collagen fibers. On the basis of platelet activation, platelet aggregation is further induced, and finally the aggregated platelets form a primary embolism, activating the coagulation cascade reaction. Among them, the formation and stabilization of fibrin are the core of hemostasis. Soluble fibrinogen at the site of vascular injury is cleaved by thrombin to form insoluble fibrin, and fibrin cross-links to form a fibrin network to stabilize the blood clot and capture more platelets and red blood cells. Normally, during the restoration of vascular wall integrity, endothelial cells secrete tissue plasminogen activator (tPA) to convert plasminogen into plasmin to dissolve the fibrin network, and at the same time plaque lysis occurs. However, continuous or repeated bleeding after spontaneous intracerebral hemorrhage usually leads to hyperfibrinolysis. Due to the upregulation of tPA activity and the overexpression of plasmin, the fibrin network is dissolved, and the hemostatic clot becomes unstable. Therefore, using antifibrinolytic drugs to stop bleeding and limit hematoma expansion is of great significance for improving the prognosis of ICH patients. Currently, tranexamic acid (TXA) is the drug with the strongest antifibrinolytic effect. TXA is a lysine analogue, which competitively occupies the lysine binding sites in plasminogen and tPA, weakening their interaction with the lysine residues exposed on the surface of fibrin. TXA inhibits the binding of plasminogen and tPA and the subsequent generation of plasmin on the surface of fibrin, protecting the fibrin clot from plasmin hydrolysis. However, the existing direct injection of TXA has insufficient efficacy or potential systemic risks, including off-target thrombosis, thromboembolism, and neuropathy and other problems. Therefore, achieving precise targeted delivery of TXA has become the key to solving the above problems. Although some platelet-mimicking nanoparticles with bleeding site targeting functions have been developed in recent years, due to the low drug loading capacity and the requirement for drug release at the bleeding site to exert the coagulation function, the application of these methods is limited to a certain extent. Based on the fact that the existing drug loading methods cannot effectively solve the technical problems faced by this application, the present invention has developed an intelligent targeted polypeptide drug conjugate. The conjugate consists of von Willebrand factor binding peptide (VBP), plasmin-cleavable polypeptide (KTFKC), and tranexamic acid (TXA). Among them, the plasmin-cleavable polypeptide (KTFKC) serves as a linker to connect the von Willebrand factor binding peptide (VBP) and tranexamic acid (TXA), forming a plasmin-responsive intelligent targeted polypeptide drug conjugate (VBP-KTFKC-TXA, RPDC).

[0012] The present invention has the following advantages compared with the prior art:

[0013] In the blood circulation, the intelligent targeted polypeptide drug conjugate of the present invention can specifically recognize and bind to von Willebrand factor (vWF) around the vascular rupture site through VBP, thereby targeting to the bleeding site. In the hyperfibrinolysis environment caused by spontaneous intracerebral hemorrhage, plasmin in the conjugate can cleave the polypeptide to specifically release TXA. The released TXA competitively occupies the lysine binding sites in plasminogen and tissue-type plasminogen activator, inhibits the generation of plasmin, prevents the dissolution of the fibrin network in the blood clot, stabilizes the thrombus plug, thereby exerting a targeted intelligent hemostatic function, achieving the effects of rapid hemostasis and stable hemostatic clot, preventing repeated bleeding or continuous bleeding, and thus limiting the hematoma expansion of ICH. Therefore, the polypeptide drug conjugate of the present invention improves the therapeutic effect and safety performance of TXA.

[0014] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Brief Description of the Drawings

[0015] Figure 1 Schematic diagram of the mechanism of action of the intelligent targeted polypeptide drug conjugate (RPDC) of the present invention applied to invisible bleeding hemostasis.

[0016] Figure 2 Cumulative release curve diagram of TXA of NPDC and RPDC of the present invention under the condition of plasmin.

[0017] Figure 3 Fluorescence images of the brain and fluorescence intensity control diagrams of Free Cy5 and Cy5-labeled RPDC of the present invention at different time periods after collagenase-induced intracerebral hemorrhage in mice.

[0018] Figure 4 Immunofluorescence distribution diagram of Free Cy5 and Cy5-labeled RPDC in the brain of collagenase-induced intracerebral hemorrhage in mice of the present invention.

[0019] Figure 5 Schematic diagram of different groups treating collagenase-induced intracerebral hemorrhage in mice of the present invention.

[0020] Figure 6 Statistical chart of the bleeding volume of different groups treating collagenase-induced intracerebral hemorrhage in mice of the present invention. Compared with the Sham group: ***P < 0.001, **P < 0.01, *P < 0.05; compared with the Saline group: ### P < 0.001, ## P < 0.01, # P < 0.05; compared with the TXA group: &&& P < 0.001, && P < 0.01, & P < 0.05;

[0021] Figure 7 Schematic diagram of liver puncture bleeding treatment in different groups of the present invention;

[0022] Figure 8 Statistical chart of cumulative blood amount during liver puncture treatment in different groups of the present invention. Compared with the Saline group: ### P < 0.001, ## P < 0.01, # P < 0.05; Figure 9 Statistical chart of liver puncture bleeding time in different groups of the present invention. Compared with the Saline group: ### P < 0.001, ## P < 0.01, # P < 0.05; Detailed implementation manners

[0023] An intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis disclosed in this embodiment. The intelligent targeted polypeptide drug conjugate is composed of von Willebrand factor binding peptide (VBP), plasmin-cleavable polypeptide (KTFKC), and tranexamic acid (TXA). The plasmin-cleavable polypeptide (KTFKC) connects the von Willebrand factor binding peptide (VBP) and tranexamic acid (TXA) to form a plasmin-responsive intelligent targeted polypeptide drug conjugate (VBP-KTFKC-TXA, RPDC). In this study, a non-responsive polypeptide drug conjugate (NPDC, VBP-TXA) was designed as a control to verify the targeted intelligent hemostasis effect of RPDC through in vitro and in vivo experiments, with the expectation of providing a new technical means for improving the clinical treatment of ICH.

[0024] 1. Study on plasmin-responsive drug release

[0025] To study the drug release characteristics of RPDC in a plasmin environment, a comparative experiment was carried out with a non-responsive polypeptide drug conjugate (NPDC). RPDC and NPDC were respectively added to a solution containing u-PA and incubated at 37 °C for 15, 30, 45 min, 1, 2, 4, 6, 8, 12, 24 h. Subsequently, liquid chromatography-mass spectrometry (LC-MS) technology was used to measure the content of free TXA in the solution, and the release curve of TXA was plotted. As Figure 2 shown, it can be seen from the figure that the cumulative drug release amount of RPDC is significantly higher than that of NPDC.

[0026] Chromatographic conditions: A ZIC-HILIC column (50 mm × 2.1 mm, 3.5 μm) was used, with 25 mM ammonium acetate and acetonitrile (3:7, v / v) as the mobile phase, and the flow rate was 0.2 mL / min. Mass spectrometry parameters: Positive ion mode electrospray ionization (ESI+) was used, the spray voltage was 3000 V, the capillary temperature was 700 °C, and the nebulizing gas pressure was 60 psi. Selected ion monitoring (SRM) was used for quantitative analysis, and the ion transition of TXA was from m / z 158.1 to 123.1.

[0027] 2. Having bleeding site targeting

[0028] To verify the targeting ability of the intelligent targeting polypeptide drug conjugate (RPDC) of the present invention to the intracerebral hemorrhage site, a collagenase-induced intracerebral hemorrhage model in ICR mice was established. After the model was completed, Free Cy5 and Cy5-labeled RPDC solutions were respectively injected into the tail vein. Fluorescent images of the mouse brain were collected through a small animal in vivo imaging system at 1, 12, and 24 h, and the fluorescence intensity was analyzed (as Figure 3 shown). Free Cy5 was used as a non-targeted drug control for comparative analysis of the specific targeting ability of RPDC to the intracerebral hemorrhage site.

[0029] 3. Study on bleeding site targeting

[0030] A collagenase-induced intracerebral hemorrhage model in ICR mice was established. Free Cy5 and Cy5-labeled RPDC solutions were respectively injected into the tail vein for 24 h. The brain tissues of mice in different groups were collected, fixed and sectioned, and then immunofluorescence staining was performed to evaluate the bleeding site targeting of RPDC (as Figure 4 shown). It can be seen from the figure that the intracerebral hemorrhage area of the mice injected with the Cy5-labeled RPDC solution showed significantly higher fluorescence intensity, and its fluorescence signal showed a strong co-localization phenomenon with the thrombus distribution, further proving the targeting distribution ability of RPDC.

[0031] 4. Evaluation of in vivo hemostatic effect

[0032] To verify the in vivo hemostatic effect of RPDC, taking the intracerebral hemorrhage model and the liver puncture injury model as examples. Theoretically, RPDC can specifically target the bleeding site through the von Willebrand factor-binding peptide and can release TXA under the stimulation of high levels of plasmin, thereby exerting a hemostatic function. While NPDC is a non-responsive polypeptide drug conjugate, which only has bleeding targeting but does not release drugs, and thus does not exert a hemostatic function.

[0033] (1) Mouse intracerebral hemorrhage experiment: Grouping was designed according to experimental requirements, including Sham, Saline, TXA, RPDC, and NPDC groups. Among them, the Sham group was the sham operation group, Saline was the model group, and TXA, RPDC, and NPDC were the treatment groups. After the establishment of the intracerebral hemorrhage model, mice were treated with intravenous administration according to different groups. After 24 hours, the brain tissues were perfused and collected and fixed with 4% paraformaldehyde. Serial mouse brain tissue sections with a thickness of 1 mm were prepared. Then, digital photography was performed on the serial sections, and the hemorrhage area was calculated using the image analysis program ImageJ software. The head side of the brain section was considered the A side, and the tail side was considered the B side. The hemorrhage areas on both sides of A and B were recorded respectively. Then, the area values on both sides of A and B were added and averaged to calculate the intracerebral hemorrhage area of the mouse, and the intracerebral hemorrhage volume of the mouse was obtained by multiplying the area by the thickness of each section.

[0034] The experimental results of the intracerebral hemorrhage volume of mice in different groups are as Figure 5 shown. Collagenase can significantly induce ICH in ICR mice. Both TXA and RPDC can reduce the hematoma volume induced by collagenase. The statistical analysis results of the intracerebral hemorrhage volume of mice in different groups are as Figure 6 shown. Compared with the TXA group, RPDC can reduce more ICH hematoma volume ( Figure 6 in which Hemorrhagic Volume represents the amount of bleeding).

[0035] (2) Mouse liver puncture model injury experiment: Grouping was designed according to experimental requirements, including Saline, TXA, RPDC, and NPDC groups. Mice were pretreated with intravenous administration according to different groups. Then, a transverse abdominal incision was made to expose the liver, and the residual peritoneal fluid on the liver surface was cleaned. A pre-weighed filter paper was placed at the predetermined bleeding site, and the liver was punctured with an 18G needle to cause liver bleeding. Subsequently, the time required for complete blood coagulation was recorded, and the weight of the filter paper was weighed to calculate the cumulative blood loss.

[0036] The results are as Figures 7-9 shown. Figure 8 、 9 The statistical analysis results show that both TXA and RPDC can not only reduce the cumulative blood loss in the liver puncture bleeding injury model ( Figure 8 in which Cumulative blood amount represents the total cumulative blood loss), but also significantly shorten the time required for hemostasis ( Figure 9 in which Hemostatic time represents the hemostasis time). The application of the intelligent targeted polypeptide drug conjugate of the present invention to the hemostasis mechanism of invisible bleeding is as Figure 1 shown. The specific meanings of each symbol are as follows:

[0037] vWF: represents von Willebrand factor;

[0038] VBP: von Willebrand factor-binding peptide represents the vWF-binding peptide;

[0039] KTFKC: represents a plasmin-cleavable peptide sequence;

[0040] VBP-KTFKC-TXA constitutes RPDC; Collagen: represents collagen;

[0041] Activated Platelet: represents activated platelets;

[0042] Clot: represents blood clots;

[0043] Circulating Platelets: represents circulating platelets;

[0044] Fibrinogen: represents fibrinogen;

[0045] Fibri: represents fibrin;

[0046] TXA: represents the tranexamic acid group;

[0047] RPDC: represents a plasmin-responsive polypeptide drug conjugate (VBP-KTFKC-TXA);

[0048] NPDC: represents a non-plasmin-responsive polypeptide drug conjugate (VBP-TXA);

[0049] Figure 1 The mechanism of action of the intelligent targeting polypeptide drug conjugate in blood circulation is elaborated in detail. Specifically, RPDC can specifically recognize and bind to vWF aggregated around the blood vessel rupture site through VBP, thereby targeting to the bleeding site. In an environment of hyperactive plasmin, the plasmin-cleavable polypeptide (KTFKC) in RPDC is specifically cleaved to release TXA, which competitively occupies the lysine binding sites in plasminogen and tissue-type plasminogen activator (tPA), inhibits the generation of plasmin, further prevents the dissolution of the fibrin network in the blood clot by plasmin, and stabilizes the thrombus plug, thus achieving the function of targeted intelligent hemostasis.

[0050] As described above, it is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change, and equivalent change made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis, characterized in that: The intelligent targeted peptide drug conjugate is composed of a plasmin-cleavable peptide connected to a von Willebrand factor-binding peptide and tranexamic acid.

2. An application of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis, characterized in that: The intelligent targeted polypeptide drug conjugate is used for targeted hemostasis treatment of invisible bleeding in vivo.

3. The use of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis according to claim 2, characterized in that: The intelligent targeted polypeptide drug conjugate is used for the treatment of spontaneous cerebral hemorrhage.

4. An application of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis, characterized in that: The smart targeted polypeptide drug conjugate is used for the management of bleeding that cannot be stopped by compression.

5. The use of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis according to claim 4, characterized in that: The intelligent targeted polypeptide drug conjugate is used for the treatment of liver hemorrhage caused by surgery.

6. The use of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis according to any one of claims 2 to 5, characterized in that: The intelligent targeted polypeptide drug conjugate specifically binds to the von Willebrand factor at the bleeding site, targets the bleeding point, releases tranexamic acid at the bleeding point in response to the action of plasmin, and realizes the site-specific release of tranexamic acid.

7. The use of an intelligent targeted polypeptide drug conjugate for invisible bleeding hemostasis according to any one of claims 2 to 5, characterized in that: The polypeptide drug conjugate is prepared in the form of an injection.

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