Use of razotide acetate in the preparation of drugs for promoting osteogenesis

Abstract

The present invention discloses the use of razotide acetate in the preparation of drugs for promoting osteogenesis. The present invention expands the intervention means of existing materials in promoting angiogenesis in the process of bone healing. At the same time, the present invention also provides the use conditions and applications of razotide acetate. The razotide acetate of the present invention can be used in the field of materials for promoting bone defect healing and promoting vascularization.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to the use of prazole acetate in the preparation of a drug for promoting osteogenesis. Background Art

[0002] Bone tissue is a highly vascularized tissue, and vascular regeneration is an important prerequisite for bone injury healing. In the early stage of vascular regeneration in bone defect healing, the oxygen tension in the local microenvironment will trigger a significant increase in pro-angiogenic factors, and a large number of new capillary beds generated by sprouting angiogenesis will be formed, which will exceed the needs of tissue regeneration for a certain period of time. Therefore, in the late stage of bone healing, some new capillaries fuse and converge to form large blood vessels, and the chaotic vascular network is pruned and stabilized to normal vascular density to maintain vascular homeostasis, so as to ensure that the tissue regeneration process, including the bone defect healing process, can proceed within a controllable range. Therefore, in the middle and late stages of bone formation, the timely stabilization and maturation of the subsequent vascular network can provide continuous power for tissue repair, thereby achieving a more ideal bone defect repair effect; and how to achieve this goal and further improve the bone healing effect is a key clinical issue worthy of exploration.

[0003] The small molecule drug larazotide acetate (AT1001) is a single-chain peptide composed of eight amino acids. As a known tight junction permeability regulator, it plays a role in regulating tight junctions (TJs) by blocking protease-activated receptor 2 (PAR2) in endothelial cells to seal leaky junctions to a normal state, thereby treating diseases caused by tight junction destruction such as aortic aneurysms. In the regulation of TJs, the activation of PAR2 promotes the activity of intracellular myosin light chain kinase and induces ZO-1 to relocate from the cell membrane to the cytoplasm through actin, thereby increasing the paracellular permeability of human umbilical vein endothelial cells (hUVECs). However, no studies have yet reported the role and significance of AT1001 in promoting osteogenesis.

[0004] Exploring the stability of the vascular network after bone defect has important clinical research significance for promoting the positive effect of bone defect. However, there are currently no reports and applications in clinical practice on regulating bone healing in the middle and late stages of tissue defect by intervening in vascular stability. Summary of the invention

[0005] In view of the limitations of traditional angiogenesis-related cytokines in regulating bone healing, the present invention provides a novel method for timely reducing the permeability of vascular walls, promoting vascular stability, and thus effectively promoting bone healing. Specifically, the present invention includes the following contents.

[0006] One aspect of the present invention provides the use of prazole acetate or a functional fragment thereof in the preparation of a drug for promoting osteogenesis.

[0007] In certain embodiments, according to the use of the present invention, wherein the osteogenesis comprises at least one of the following:

[0008] (1) Promote the stability of the vascular network;

[0009] (2) Promote bone defect healing;

[0010] (3) Reduce the permeability of blood vessel walls;

[0011] (4) increase vascular perfusion;

[0012] (5) Increase blood vessel diameter.

[0013] In certain embodiments, according to the use of the present invention, the prazole peptide acetate or a functional fragment thereof is administered systemically.

[0014] In certain embodiments, according to the use of the present invention, the razotide acetate or a functional fragment thereof is administered by intravenous injection.

[0015] In certain embodiments, according to the use of the present invention, the medicament comprises a pharmaceutically acceptable carrier.

[0016] In certain embodiments, according to the use of the present invention, the concentration of the prazole peptide acetate or its functional fragment is 0.1-50 mg / mL.

[0017] In certain embodiments, according to the use of the present invention, the dosage of the drug is 0.01-500 mg / kg.

[0018] In certain embodiments, according to the use of the present invention, the lazotide acetate or a functional fragment thereof is administered 2-4 weeks after the formation of the bone defect.

[0019] In certain embodiments, according to the use of the present invention, the drug is administered once a day for 5 days.

[0020] In certain embodiments, according to the use of the present invention, the prazole acetate or a functional fragment thereof is administered in combination with other therapeutic drugs.

[0021] In certain embodiments, according to the use of the present invention, the drug is used for pathological bone regeneration and repair in diabetes and / or bone regeneration and repair in elderly patients. The present invention expands the intervention means of existing materials in promoting angiogenesis in the process of bone healing, and provides the use conditions and applications of razolidine acetate. The razolidine acetate of the present invention can be used in the field of materials for promoting bone defect healing and promoting vascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure shows the bone healing effect of the defect site after administration of AT1001.

[0023] Figure 2 The figure shows the imaging of the vascular network at the bone defect site after AT1001 administration.

[0024] Figure 3 Shown are the immunofluorescence results of bone tissue sections after AT1001 stimulation.

[0025] Figure 4 It was shown that in a nude mouse subcutaneous tumor-bearing model, AT1001 has a promoting effect on the integrity of the regenerated vascular barrier in vivo. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] use

[0030] In one aspect of the present invention, use of lazotide acetate or a functional fragment thereof in the preparation of a drug for promoting osteogenesis is provided. If, when provided in an effective amount, the fragment has the property of promoting osteogenesis in a mammalian individual, then the fragment derived from lazotide acetate can be considered functional. Such properties can be measured by any suitable means, including, for example, determining the amount of new bone using computed tomography or determining the morphology of the vascular network using confocal microscopy or the like.

[0031] It is understood that the prazole acetate or functional fragment of the present invention is soluble, or the prazole acetate or functional fragment may also be contained in a fusion protein.

[0032] The razolidine acetate or functional fragment of the present invention can be prepared by any technique known to those skilled in the art, including the expression of proteins, polypeptides or peptides by standard molecular biology techniques, the separation of protein compounds from natural sources, or the chemical synthesis of protein materials. In addition, various commercial preparations of razolidine acetate or functional fragments are known to those skilled in the art.

[0033] In certain embodiments, prazole peptide acetate or a functional fragment may be purified. Generally, "purified" refers to a specific polypeptide that has been separated to remove various other proteins, polypeptides or peptides, and the resulting composition substantially retains its activity, which can be assessed, for example, by a known assay for the specific or desired polypeptide activity by a person skilled in the art.

[0034] Those skilled in the art will appreciate that biological functional equivalents of razolidine acetate can be used, for example, modifications and / or changes can be made in the structure of the polynucleotide and / or protein expressing the razolidine acetate of the present invention, so as to obtain razolidine acetate or its functional fragments having similar or improved properties, and therefore such biological functional equivalents are also included in the present invention.

[0035] In the present invention, promoting osteogenesis or promoting bone healing may mean that the degree of promotion or healing is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% compared to an untreated reference group under the same conditions, measured by any standard technique.

[0036] In the present invention, promoting osteogenesis or promoting bone healing includes at least one of the following:

[0037] (1) Promote the stability of the vascular network;

[0038] (2) Promote bone defect healing;

[0039] (3) Reduce the permeability of blood vessel walls;

[0040] (4) increase vascular perfusion;

[0041] (5) Increase blood vessel diameter.

[0042] In the present invention, promoting osteogenesis or promoting bone healing also includes increasing the amount of new bone, the density of regenerated blood vessels, promoting the integrity of blood vessel walls, and the like.

[0043] In the present invention, the drug comprising prazole acetate or a functional fragment thereof can be in any suitable dosage form. In a preferred embodiment, the drug is in a dosage form suitable for systemic administration (or parenteral administration), such as an injection administered by intravenous injection.

[0044] In the present invention, the drug comprising prazole acetate or its functional fragment includes a pharmaceutically acceptable carrier, and the term "pharmaceutically acceptable" refers to a carrier, excipient or stabilizer that is non-toxic to cells or mammals exposed to the dose and concentration used or has an acceptable toxicity level determined by a person skilled in the art. A non-limiting example of a pharmaceutically acceptable carrier is water for injection or an aqueous pH buffer solution. Pharmaceutically acceptable excipients may also include one or more of the following: antioxidants, such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin; gelatin; immunoglobulins; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids; carbohydrates, such as glucose, mannose or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium and non-ionic surfactants, such as Tween and polyethylene glycol (PEG), etc.

[0045] In a preferred embodiment, the purpose of promoting osteogenesis or promoting bone healing is achieved by intravenously administering a therapeutically effective amount of prazole acetate or a functional fragment thereof or a medicament comprising the same to a subject in need thereof.

[0046] The term "subject" used in the present invention refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, and the like, that are about to receive a specific treatment. Typically, "subject" and "patient" are used interchangeably in the present invention and both refer to a human subject.

[0047] The term "therapeutically effective amount" used in the present invention refers to an effective dosage considered pharmaceutically, that is, the amount of active drug (lazotide acetate) is sufficient to significantly improve the condition without causing serious side effects. The dosage depends on many factors, such as the nature and severity of the disease to be treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can vary widely.

[0048] In the present invention, the concentration of the lazolidinedione acetate or its functional fragment is 0.1-50 mg / mL, preferably 0.1-40 mg / mL, also preferably 1-20 mg / mL, further preferably 1-10 mg / mL, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL.

[0049] In the present invention, the dosage of the drug is 0.01-500 mg / kg, preferably 1-100 mg / kg, and further preferably 10-100 mg / kg, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg / kg.

[0050] The present invention also provides the use of prazole acetate or a functional fragment thereof in combination with other therapeutic drugs in the preparation of a combined administration drug for promoting osteogenesis or bone healing.

[0051] In the present invention, other therapeutic agents may be any therapeutic agents used for bone healing or not used for bone healing, and are not particularly limited.

[0052] Example

[0053] 1. Experimental Methods

[0054] All animals and surgical procedures used in this example have been approved by the Institutional Animal Care and Use Committee of Peking University (approval number: LA2023209). 60 male C57BL / CJ mice (8 weeks old) and 24 male BALB / c nude mice (4 weeks old). In order to minimize potential confounding factors related to sex and estrous cycle, only male animals were used in this example.

[0055] In order to study the effect of AT1001 on vascular permeability in vivo, 1×10 7 A mixture of human umbilical vein endothelial cells (hUVECs) and 0.1 mL Matrigel (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was subcutaneously injected into nude mice (6 per group) to construct subcutaneous tumors on ice at 4°C. One day after cell inoculation, the experimental group was injected with 50 mg / kg / day AT1001 (MCE, Monmouth Junction, NJ, USA), while the control group mice were injected with IgG. Four days after inoculation, 0.1 mL of FITC-dextran (70 kDa, Invitrogen, Carlsbad, CA, USA) at a concentration of 10 mg / mL was administered by intravenous tail injection. Five minutes later, after anesthesia, the mouse skin was peeled off to expose the tumor, and vascular leakage images were observed under a two-photon confocal microscope.

[0056] To investigate the effects of AT1001 on vascular stability and bone defect healing, a skull defect model was established. Before surgery, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (100 mg / kg). The surgical area of ​​the skull of the mice was then exposed. A saline-cooled round drill was used to create a circular defect of approximately 1.8 mm in diameter on both sides of the middle suture line in the parietal region. Subsequently, the animals were divided into different experimental categories using a random number table. The test animals were initially divided into a control group and a drug intervention group. Subsequently, the drug intervention group was further stratified, and the animals were randomly assigned to one of three subgroups, each corresponding to a specific time point after surgical intervention: 1 week, 2 weeks, and 4 weeks after surgery. The sample size of each subgroup was standardized to 6 animals per group (N=6). At each designated time point, a single intervention of AT1001 (50 mg / kg / day) was administered by tail vein injection. After a course of drug treatment was administered, the vascular morphology and bone condition were evaluated.

[0057] 2. Experimental Results

[0058] 1. Role of AT1001 in vascular network stability and bone formation

[0059] To determine the effect of AT1001 on vascular network stability and bone formation, mice were stimulated with AT1001 and IgG (as a control) 1 week, 2 weeks, or 4 weeks after skull defect surgery. Figure 1 As shown, Figure 1 (A) is a Micro-CT skull three-dimensional reconstruction image after AT1001 treatment at 2 and 4 weeks after surgery and 8 weeks after sampling. The imaging results show that the amount of new bone increased significantly in the middle and late stages (2 weeks and 4 weeks). The red circle indicates the defect modeling area. (B) is a statistical chart of new bone increment (n=6) (**, P<0.01). Micro-CT results show that AT1001 administration can also effectively promote the healing of bone defects, and the later the administration time, the better the effect on bone defect repair.

[0060] To further investigate the potential role of AT1001 in enhancing bone defect healing by stabilizing blood vessels, Micro-VCC was used to visualize the final outcome of vascular regeneration. Figure 2 As shown, Figure 2(A) is a Micro-VCC imaging 3D reconstruction of vascular regeneration in the defect area and a vascular network identification diagram. The dotted area represents the original edge of the bone defect. The histograms of (B and C) represent the density and diameter of regenerated blood vessels in the defect area. The control group was compared with the experimental group (AT1001 treatment group) at the same time (n=6) (*, P<0.05; **, P<0.01). The results showed that AT1001 significantly increased the vascular diameter and blood flow velocity at the 8th week of IGFBP7 treatment and enhanced the stability of blood vessels in the defect area, especially in the final healing stage of 8 weeks. This further confirmed that AT1001 regulates bone defect healing by enhancing vascular stability.

[0061] 2. The role of AT1001 in vascular wall integrity and regulation of vascular stability

[0062] A skull defect model was constructed and AT1001 protein solution was injected into the tail vein 2 weeks and 4 weeks after surgery. Skull samples were collected 8 weeks after treatment, and the localization of CD31 and ZO-1 proteins in each group was detected by immunofluorescence. Figure 3 As shown in the figure, AT1001 significantly promoted the enhanced expression of ZO-1 protein co-localized with CD31, indicating that AT1001 treatment effectively strengthened the tight junction of cells in the vascular wall within the bone defect site, promoted the integrity of the vascular wall, and regulated vascular stability. Blue fluorescence is DAPI, green fluorescence is CD31, and red fluorescence is ZO-1.

[0063] 3. In vivo angiogenesis experiment

[0064] The nude mouse subcutaneous cell tumor model was used to simulate the angiogenesis process in vivo. Matrigel matrix gel containing human umbilical vein endothelial cells was injected into the nude mouse subcutaneously to form local cell clusters, and AT1001 protein solution was injected through the tail vein to stimulate the systemic blood vessels of the nude mice. Four days after tumor inoculation, (70kDA) FITC-dextran was injected into the tail vein and the lumen permeability of the blood vessels in the cell tumor was observed in vivo under a two-photon confocal microscope. The results are as follows Figure 4 As shown, (A) is the sham control group (sham) and the AT1001 stimulation experimental group. Scale bar: 100 μm; (B) is the relative quantification of the fluorescence of the extravascular leakage area and the average value of the total fluorescence of the image (n=5) (***, P<0.001). The results showed that the walls of the regenerated blood vessels in the subcutaneous tumors of the AT1001-treated group were more complete and smooth, while the walls of the blood vessels in the control group had discontinuous shadows, and FITC-dextran infiltrated the blood vessel walls, diffused more around the blood vessels, and had obvious penetration, showing the incompleteness of the vessel walls. The above shows that AT1001 has the effect of promoting the integrity of the vascular wall barrier and reducing vascular permeability.

[0065] In summary, the results of the present invention clearly show that AT1001 effectively strengthens endothelial tight junctions, especially in the neovascular network, and promotes bone defect healing by upregulating ZO-1 protein expression. Therefore, this confirms the feasibility of a therapeutic intervention strategy based on promoting bone repair using AT1001 in the defect area.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of prazole acetate in the preparation of drugs for promoting osteogenesis.

2. The use according to claim 1, characterized in that The osteogenesis includes at least one of the following situations: (1) Promote the stability of the vascular network; (2) Promote bone defect healing; (3) Reduce the permeability of blood vessel walls.

3. The use according to claim 1, characterized in that The prazole acetate is administered to a subject in need thereof by intravenous injection.

4. The use according to claim 1, characterized in that The concentration of the prazole acetate is 0.1-50 mg / mL.

5. The use according to claim 1, characterized in that: The medicament includes a pharmaceutically acceptable carrier.

6. The use according to claim 1, characterized in that: The razotide acetate is administered 2-4 weeks after bone defect formation.

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