Application of halofuginone in the preparation of drugs for treating heterotopic ossification

Abstract

The present invention belongs to the field of medical technology, and in particular relates to the use of halofuginone in the preparation of a drug for treating heterotopic ossification. The present invention provides an application of halofuginone in the preparation of a drug for treating heterotopic ossification. Through imaging observation, histological section staining result observation, and vascular fluorescence staining observation, the positive control group had obvious bone hyperplasia in the Achilles tendon of mice, bone structure formation, obvious cartilage formation in the bone formation area, and obvious blood vessel formation in the Achilles tendon of mice with heterotopic ossification, while the halofuginone-treated group had no obvious bone hyperplasia, no obvious bone formation and cartilage accumulation, and no obvious blood vessel formation in the Achilles tendon of mice with heterotopic ossification. This shows that halofuginone has a good effect of improving bone formation, cartilage accumulation and blood vessel formation, that is, it shows that halofuginone has a good anti-heterotopic ossification effect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to an application of halofuginone in preparing a medicine for treating heterotopic ossification. Background Art

[0002] Heterotopic ossification, the formation of ectopic lamellar bone within soft tissue, is associated with a high disability rate, with over 20% of patients experiencing significant functional impairment due to soft tissue defects, joint contractures, and chronic pain. The incidence of heterotopic ossification can reach as high as 50% in patients with traumatic brain injury. Heterotopic ossification is recognized as a complication following trauma, burns, neurological injury, and major orthopedic surgery, and remains a clinical challenge.

[0003] Halofuginone, also known as halofuginone, is a derivative of febrifugine and a low molecular weight quinazolinone alkaloid (495 Da) isolated from the plant Dichroafebrifuga. Its chemical name is 7-bromo-6-chloro-3-[3-(3-hydroxy-2-piperidinyl)-2-oxopropyl(acetonyl)]-4(3H)-quinazolinone hydrobromide. Its molecular formula is C 16 H 17 BrClN3O3, the main pharmacological effects of halofuginone include pulmonary vasodilator, anti-fibrosis, and treatment of chronic graft-versus-host disease (cGvHD) of the skin, but there are no reports on the anti-heterotopic ossification effect of halofuginone. Summary of the Invention

[0004] The present invention aims to address one or more technical problems existing in the aforementioned prior art, and at least provide a beneficial alternative or create a condition. Specifically, the present invention provides the use of halofuginone in the preparation of a medicament for treating heterotopic ossification. This medicament can effectively reduce abnormal soft tissue bone formation, cartilage accumulation, and angiogenesis at the site of trauma, demonstrating a strong anti-heterotopic ossification effect.

[0005] A first aspect of the present invention provides a use of halofugone in the preparation of a medicament for treating heterotopic ossification.

[0006] Preferably, the active ingredient of the drug includes halofugone.

[0007] Preferably, the content of halofugone in the drug is 1.6-2.5 mg / mL; further preferably, the content of halofugone in the drug is 1.8-2.2 mg / mL; further preferably, the content of halofugone in the drug is 2 mg / mL.

[0008] Specifically, in the drug, halofuginone can be used as the sole active ingredient, or can be combined with other pharmaceutically approved substances.

[0009] Preferably, the medicament further comprises a buffer.

[0010] Preferably, the buffer comprises dimethyl sulfoxide (DMSO), polyethylene glycol (PEG300), polyoxyethylene sorbitan monooleate (Tween-80), and phosphate buffered saline (PBS).

[0011] Preferably, the mass ratio of DMSO, PEG300, Tween-80, and PBS is (1.6-3.0):(7.0-9.0):1:(8.0-10.0); further preferably, the mass ratio of DMSO, PEG300, Tween-80, and PBS is (1.8-2.8):(7.5-8.5):1:(8.5-9.5); even further preferably, the mass ratio of DMSO, PEG300, Tween-80, and PBS is 2:8:1:9.

[0012] Preferably, the drug contains pharmaceutically acceptable excipients.

[0013] Preferably, the drug is prepared into any pharmaceutically acceptable dosage form.

[0014] Preferably, the dosage form includes any one of tablets, capsules, granules, oral liquids, injections, sprays, ointments, creams, gels, and inhalants.

[0015] Preferably, the halofugone has the following structural formula:

[0016]

[0017] A second aspect of the present invention provides a drug for treating heterotopic ossification.

[0018] Specifically, the drug includes the halofugone described in the first aspect of the present invention.

[0019] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0020] The present invention provides a use of halofuginone in the preparation of a medicament for treating heterotopic ossification. Imaging observation, histological section staining observation, and vascular fluorescence staining observation showed that the positive control group had significant bone hyperplasia, bone structure formation, and cartilage formation in the bone formation area in the Achilles tendons of mice with heterotopic ossification. In contrast, the halofuginone-treated group showed no significant bone hyperplasia, bone formation, or cartilage accumulation, and no significant blood vessel formation in the Achilles tendons of mice with heterotopic ossification. This indicates that halofuginone can effectively reduce abnormal soft tissue bone formation, cartilage accumulation, and blood vessel formation at the site of injury, further demonstrating that halofuginone has a good anti-heterotopic ossification effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are CT scan images of the three groups of experimental mice treated with the drug for 10 weeks;

[0022] Figure 2 Figure 2 is a graph showing bone mass at the Achilles tendon of three groups of experimental mice treated with drugs for 10 weeks;

[0023] Figure 3 The graph shows the histological staining results of Achilles tendons in three groups of experimental mice after 10 weeks of drug intervention.

[0024] Figure 4 The results of immunofluorescence staining of Achilles tendon blood vessels in three groups of experimental mice treated with drugs for 10 weeks;

[0025] Figure 5 This is a statistical graph of the ratio of positive blood vessels to total blood vessels in the three groups of experimental mice after 10 weeks of drug intervention. DETAILED DESCRIPTION

[0026] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0027] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0028] Example 1

[0029] Study on the effect of halofuginone on heterotopic ossification

[0030] Control drug: intraperitoneal injection of solvent DMSO.

[0031] Halofuginone solution: 10 mg of halofugone was dissolved in 5 mL of buffer solution (10% DMSO, 40% PEG300, 5% Tween-80 and 45% PBS), and mixed until clear to prepare a 2 mg / mL halofugone solution.

[0032] Experimental mice: C57BL / J mice were provided by the Animal Experiment Center of Southern Medical University in Guangdong Province and housed in a sterile animal room. The cages and utensils used were strictly disinfected, and the mice had free access to food and purified water. Before the experiment, the mice were observed for clinical symptoms and their activity was checked for 2 consecutive days for future use.

[0033] Experimental methods

[0034] Experimental grouping: 70 C57BL / J mice were raised to 3 months of age, weighed one by one, and mice with poor vitality and excessive weight were eliminated. The remaining mice were randomly divided into 3 groups (Group I, Group II, and Group III), with 20 mice in each group, and the total weight of mice in each group was appropriately adjusted to be roughly equal.

[0035] Experimental treatment: Group I was the blank control group (mice did not undergo Achilles tendon transection model). Except for Group I, all other groups of mice underwent Achilles tendon transection model establishment; Group II was the positive control group, which was intraperitoneally injected with the solvent DMSO every other day, with each intraperitoneal injection of 100 μL; Group III was the halofuginone-treated group, which was intraperitoneally injected with halofuginone solution every other day, with each intraperitoneal injection of 100 μL.

[0036] The groups of experimental mice are shown in Table 1.

[0037] Table 1: Grouping of experimental mice

[0038] Group Number of mice Modeling treatment Drug treatment Group I (blank control group) 20 No modeling No Addition Group II (positive control group) 20 Achilles tendon transection DMSO Group III (halofuginone-administered group) 20 Achilles tendon transection Halofuginone solution

[0039] The mental state, food intake, and feces of the mice were observed and recorded every day. All mice were weighed individually on the 7th day after Achilles tendon transection modeling, and drug intervention was performed 7 days after modeling. Drug intervention treatment lasted for 10 weeks, and then the following experimental analysis was performed.

[0040] 1. Imaging Observation

[0041] After 10 weeks of drug intervention, the mice were killed and the lower limbs of the three groups of experimental mice were collected for CT scanning. The CT scan images are shown in the figure below. Figure 1 As shown, Figure 1 Figure (a) in the middle is the CT scan image of the blank control group. Figure 1 The middle (b) figure is the CT scan image of the positive control group. Figure 1 The middle (c) figure is a CT scan image of the halofuginone-treated group.

[0042] The bone volume (BV) of the Achilles tendon of mice was counted and the formation of heterotopic ossification in the Achilles tendon was observed. Figure 2 As shown. Among them, Figure 2 The control in the middle horizontal axis represents the blank control group, the Achilles tendon transection model represents the positive control group, and the halofuginone represents the halofuginone-treated group; the vertical axis BV (%) represents the bone formation volume in the Achilles tendon of mice, **** indicates a statistically significant difference (p<0.0001), and ns indicates no statistical difference.

[0043] Depend on Figure 1 It can be seen that obvious bone hyperplasia was observed in the mice in the positive control group. Compared with the positive control group, no obvious bone hyperplasia was found in the Achilles tendon of the mice in the halofuginone-treated group.

[0044] Depend on Figure 2 It can be seen that the bone formation volume (BV) of the Achilles tendon of mice in the halofuginose-treated group was significantly downregulated compared with the positive control group and was basically equivalent to that of the blank control group.

[0045] Depend on Figure 1 and Figure 2 It can be seen that halofuginone can improve bone hyperplasia in the Achilles tendon of mice and reduce the volume of bone formation in the Achilles tendon of mice.

[0046] 2. Observation of Histological Section Staining Results

[0047] After 10 weeks of drug intervention, the mice were killed, and the Achilles tendon specimens of the three groups of experimental mice were subjected to histological staining, including hematoxylin and eosin staining (H&E staining) and safranin O and fast green staining. The specific test methods are as follows:

[0048] (1) Sampling: After 10 weeks of drug treatment, mice were killed. The area below the knee joint to the paw of the three groups of mice was sampled and placed in a pre-prepared fixative (10% formalin fixative) to denature and coagulate the proteins of the tissues and cells to prevent autolysis of the cells or bacterial decomposition after death, thereby maintaining the original morphological structure of the cells and obtaining tissue blocks.

[0049] (2) Dehydration and transparency: Use low to high concentrations of alcohol (70%, 80%, 90%, 95%, and 100% alcohol, in that order) as a dehydrating agent for 1 hour to gradually remove the water from the tissue block. Then, dissolve the tissue block in xylene, a paraffin-based transparent agent, to make it transparent. Xylene replaces the alcohol in the tissue block to obtain a transparent tissue block.

[0050] (3) Wax embedding: Place the transparent tissue block obtained in step (2) in the melted paraffin, put it in a wax melting box to keep it warm, and embed it after the paraffin is completely immersed in the tissue block: Use a paraffin embedding box, pour in the melted paraffin, quickly clamp the tissue block that has been soaked in paraffin and put it into it, and cool it to solidify into a block;

[0051] (4) Sectioning and patching: The embedded wax block obtained in step (3) was fixed on a microtome and cut into 5 μm thick sections;

[0052] (5) Dewaxing: Before staining, use xylene to remove the paraffin from the sections, then dehydrate through high to low concentration alcohol (100%, 95%, 90%, 80%, 70%) for 20 minutes, then put into distilled water and stain;

[0053] (6) Staining: H&E and Safranin O and fast green staining are commonly used to increase the color differences of different parts of the tissue cell structure and facilitate observation;

[0054] Principle of H&E staining: Hematoxylin (H) is a basic dye that stains the cell nucleus and ribosomes in the cell into blue-purple. The structures stained by basic dyes are basophilic. Eosin (Eosin, E) is an acidic dye that stains the cytoplasm into red or light red. The structures stained by acidic dyes are eosinophilic.

[0055] Safranin O and fast green staining principle: Basophilic cartilage combines with the basic dye Safranin O to produce red, while eosinophilic bone combines with the acidic dye Fast Green to produce green or blue, which contrasts sharply with the red cartilage, thereby distinguishing cartilage tissue from bone tissue.

[0056] The process of H&E staining is as follows:

[0057] ① Place the sections that have been immersed in distilled water into a hematoxylin aqueous solution for staining for 5 minutes;

[0058] ②Separate in hydrochloric acid and ethanol for 20 seconds;

[0059] ③ Rinse with running water for 1 hour and then add distilled water for 10 seconds;

[0060] ④ Dehydrate in 70% and 90% alcohol for 10 minutes each;

[0061] ⑤ Place in alcohol eosin staining solution for 3 minutes;

[0062] The process of Safranin O and fast green staining is as follows:

[0063] ① Place the sections that have been immersed in distilled water in a hematoxylin aqueous solution and stain with hydrochloric acid for 5 minutes;

[0064] ②Separation in hydrochloric acid and ethanol, 20 seconds;

[0065] ③ Rinse with running water for 1 hour and then add distilled water for 10 seconds;

[0066] ④ After staining with fast green dye for 5 minutes, quickly wash the sections with acetic acid solution for 10-15 seconds to remove residual fast green and air dry;

[0067] ⑤ Transfer to safranin dye and dye for 20 minutes, then rinse with 90% and 100% alcohol to remove excess safranin dye;

[0068] (7) Dehydration and transparency: The stained sections were dehydrated with pure alcohol and then transparentized with xylene to obtain transparent sections.

[0069] (8) Sealing: Add Canada balsam to the transparent section obtained in step (7), cover with a cover glass, and seal. After the balsam is slightly dry, a label is affixed to obtain a section specimen;

[0070] (9) Observation: Observe and analyze the H&E-stained, Safranin O-stained, and fast green-stained sections under a microscope.

[0071] The histological staining results of Achilles tendons of the three groups of experimental mice are as follows Figure 3 As shown. Among them, Figure 3 Figures (a) and (d) are the results of H&E staining and safranin green staining of the blank control group, respectively; Figure 3 Figures (b) and (e) are the results of H&E staining and safranin green staining of the positive control group, respectively; Figure 3 Figures (c) and (f) in the middle are the results of H&E staining and safranin green staining of the halofuginone-treated group, respectively.

[0072] Depend on Figure 3 It can be seen that for the positive control group, H&E staining can be observed in the Achilles tendon of mice with a clear boundary with the surrounding Achilles tendon and a large amount of bone-like tissue inside. Safranin O and fast green staining can be observed in the bone formation area with chondrocytes that bind to basophilic dyes and appear red, indicating obvious cartilage formation. However, no obvious bone structure formation and cartilage accumulation were observed in the halofuginone-treated group, indicating that halofuginone has a good effect in improving bone formation and cartilage accumulation.

[0073] 3. Vascular fluorescence staining results

[0074] Vascular immunofluorescence staining was performed on Achilles tendon specimens from the three groups of experimental mice. H-type vessels are specifically present in bone tissue. This type of vessel is mainly distinguished from other vascular types by immunofluorescence co-staining of specific cell surface markers (platelet endothelial cell adhesion molecule (CD31) and endothelial cell membrane-bound mucin (Endomucin)). The specific method is as follows:

[0075] (1) Systemic perfusion: To avoid interference of intravascular platelets or red blood cells with fluorescent staining, mice should be perfused before knee joint specimen collection.

[0076] a: After 10 weeks of drug treatment, the three groups of mice were intraperitoneally injected with 300-400 μL of anesthetic. After anesthesia, the mice were fixed in a supine position with their limbs stretched out on a foam board, which was placed on a washbasin at a 45° angle to the ground.

[0077] b: The mouse skin is separated along the xiphoid process to expose the abdominal wall. After the abdominal wall is exposed, the abdominal cavity is opened along the lower edge of the ribs to expose the liver and diaphragm. The diaphragm is opened to expose the heart and lungs. The right atrial appendage is cut with microscissors to allow the perfusion fluid to flow out of the right atrial appendage after passing through the entire systemic circulation, fully flushing the blood cells in the experimental animal.

[0078] c: Use a scalp vein needle to puncture the heart upward along the apex of the heart. Use 20 mL of PBS buffer solution containing heparin to flush out the blood in the systemic circulation. When the liver turns from dark red to brown, replace the perfusion solution and use 4% paraformaldehyde to perfuse and fix the blood vessels in the body. During paraformaldehyde perfusion, the mouse's muscles will spasm throughout the body, and the mouse tail will become rigid and tilt upward due to spasm, indicating that the 4% paraformaldehyde has flowed through the entire systemic circulation of the mouse. At this time, the perfusion is completed and the next step of sampling is carried out;

[0079] (2) Sampling and decalcification: The lower femur, knee joint, and upper tibia of the knee joint specimen were cut off; at the same time, the ex vivo specimen was fixed in 4% paraformaldehyde for 24 hours, and then replaced with 10% ethylenediaminetetraacetic acid (EDTA) rapid decalcification solution after 24 hours. The specimen was decalcified on a shaking table at 4°C for 14 days until the syringe needle could easily pierce the bone cortex, and the decalcification was completed to obtain the decalcified bone tissue specimen;

[0080] (3) Dehydration and embedding: The decalcified bone tissue specimen obtained in step (2) is placed in an embedding box before ethanol dehydration, and rinsed under running water for 30 minutes to fully wash the decalcification liquid on the surface of the specimen; then immersed in gradient ethanol dehydration solution for dehydration: I 70% ethanol 1 hour → II 70% ethanol 1 hour → 80% ethanol 1 hour → I 95% ethanol 1 hour → II 95% ethanol 1 hour → I anhydrous ethanol 1 hour → II anhydrous ethanol 1 hour → III anhydrous ethanol 1 hour → I xylene 30 minutes → II xylene 30 minutes → I wax tank 1 hour → II wax tank 2 hours → tissue embedding; after embedding, the wax block is placed at -20°C overnight to fully condense the wax block, and sliced ​​every other day, with the thickness of the paraffin section being 0.4 microns;

[0081] (4) Immunofluorescence staining:

[0082] a: Dewax and rehydrate the paraffin section specimen obtained in step (3): I xylene for 15 min → II xylene for 15 min → I anhydrous ethanol for 5 min → II anhydrous ethanol for 5 min → I 95% ethanol for 5 min → II 95% ethanol for 5 min → 80% ethanol for 5 min → 70% ethanol for 5 min → distilled water for 5 min → PBS for 2 min;

[0083] b: Antigen retrieval: For subchondral bone vascular endothelial marker protein antigen retrieval, we used pH = 9.0 EDTA antigen retrieval solution for retrieval. The retrieval solution was poured into a glass trough and placed in a constant temperature water bath to heat to 70 degrees Celsius. The specimen sections were then placed in the retrieval solution for 30 minutes. After retrieval, the glass trough was removed from the water bath and placed at room temperature until the temperature of the retrieval solution and the slides returned to room temperature. The sections were then washed twice with phosphate buffered saline (PBST) and three times with phosphate buffered saline (PBS), each for 5 minutes.

[0084] c: Blocking: Wipe the water stains on the slide, circle the specimen with an immunohistochemistry pen, and block with 15% goat serum. Drop 50 μL of blocking solution on each specimen, immerse the entire specimen in the blocking solution, and block at room temperature for 1 hour;

[0085] d: Antibody incubation: Use 15% goat serum blocking solution as the antibody diluent, add two primary antibodies to the blocking solution at their respective dilution ratios (CD31 (1:200), Endomucin (1:50)). To avoid cross-reaction, the primary antibodies are selected from different species. CD31 is a rabbit polyclonal antibody, and Endomucin is a rat monoclonal antibody. After absorbing the blocking solution on the specimen, add the antibody dropwise to the specimen to immerse the entire specimen. Incubate the specimen with the primary antibody in a 4-degree Celsius cold storage overnight. The next day, take the specimen box back from the 4-degree Celsius cold storage to the staining room and return it to room temperature. The primary antibody recovery can be reused 2-3 times. Place the slide in room temperature PBS buffer and wash it 3 times, 5 minutes each time.

[0086] e) Incubation with secondary antibodies: Use 15% goat serum blocking solution as the antibody diluent. Add the two secondary antibodies to the blocking solution in their respective proportions. The secondary antibody preparation process should be carried out in a dark room away from light to prevent quenching of the fluorescent secondary antibody. Prepare the corresponding fluorescent secondary antibodies. For CD31, use goat anti-rabbit 549 fluorescent antibody, and for Endomucin, use goat anti-rat FITC fluorescent antibody. Incubate in the dark for 1 hour.

[0087] f: After the secondary antibody incubation, the specimens were washed three times with PBS buffer in a dark room, each time for 5 minutes. The liquid around the specimens was wiped dry, and the sections were mounted with anti-fade 4',6-diamidino-2-phenylindole (DAPI) mounting medium. The edges of the coverslips were sealed with nail polish to prevent the coverslips from slipping. h: Observed under an Olympus confocal microscope, photos were collected, and positive cells were quantitatively counted.

[0088] The immunofluorescence staining photos of blood vessels in the three groups of experimental mice are shown in Figure 4 As shown. Among them, Figure 4 Middle (a) is a photo of blood vessels immunofluorescence staining of mice in the blank control group; Figure 4 Middle (b) is a photo of blood vessels immunofluorescence staining of mice in the positive control group; Figure 4The middle (c) figure shows the immunofluorescence staining of blood vessels in mice in the halofuginose-treated group.

[0089] The statistical diagram of the ratio of positive blood vessels to total blood vessels in the three groups of experimental mice is shown in the figure below. Figure 5 As shown in the figure, the vertical axis HV / VV (%) represents the ratio of positive blood vessels to total blood vessels, **** represents a significant statistical difference (p<0.0001), and ns represents no statistical difference.

[0090] Depend on Figure 4 The results of fluorescence staining showed that for the positive control group, obvious yellow fluorescence was produced in the Achilles tendons of heterotopic ossification mice, indicating that blood vessels were formed, while for the halofuginone-treated group, no obvious blood vessels were formed in the Achilles tendons of heterotopic ossification mice.

[0091] Depend on Figure 5 It can be seen that after the use of halofuginone, the number of H-type blood vessels was significantly reduced compared with the positive control group, indicating that halofuginone has the effect of inhibiting angiogenesis, that is, it has a good effect of resisting heterotopic ossification.

[0092] Through imaging observation, histological section staining results observation, vascular fluorescence staining observation, the positive control group had obvious bone hyperplasia in the Achilles tendon of mice, the presence of bone structure formation, obvious cartilage formation in the bone formation area, and obvious blood vessel formation in the Achilles tendon of mice with heterotopic ossification, while the halofuginone-treated group did not show obvious bone hyperplasia, no obvious bone formation and cartilage accumulation, and no obvious blood vessel formation in the Achilles tendon of mice with heterotopic ossification. This shows that halofuginone has a good effect in improving bone formation, cartilage accumulation and blood vessel formation, that is, it shows that halofuginone has a good effect in preventing heterotopic ossification.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of halofuginone in the preparation of drugs for the treatment of heterotopic ossification.

2. The use according to claim 1, characterized in that The active ingredient of the drug includes halofugone.

3. The use according to claim 2, characterized in that In the medicine, the content of halofugone is 1.6-2.5 mg / mL.

4. The use according to claim 1, characterized in that The medicament also includes a buffer.

5. The use according to claim 4, characterized in that The buffer includes dimethyl sulfoxide, polyethylene glycol, polyoxyethylene sorbitan monooleate and phosphate buffer saline.

6. The use according to claim 5, characterized in that The mass ratio of the dimethyl sulfoxide, polyethylene glycol, polyoxyethylene sorbitan monooleate and phosphate buffer is (1.6-3.0): (7.0-9.0): 1: (8.0-10.0).

7. The use according to claim 1, characterized in that The drug contains pharmaceutically acceptable excipients.

8. The use according to claim 1, characterized in that The drug is prepared into any pharmaceutically acceptable dosage form.

9. The use according to claim 8, characterized in that The dosage form includes any one of tablets, capsules, granules, oral liquids, injections, sprays, ointments, creams, gels, and inhalants.

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