Application of divalent metal lactate in preparation of product for promoting tissue growth and repair

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of divalent metal lactate in a product for promoting tissue regeneration, growth, healing and damage repair. Preferably, the divalent metal lactate comprises one or a combination of more of calcium lactate, magnesium lactate, zinc lactate and ferrous lactate. The invention provides a new treatment mode for tissue injury and repair, and prevention and treatment of tissue fibrosis and / or scar tissue generation, and has wide application prospects in the aspects of injury treatment and repair of tissues such as skin, connective tissues, tendons, fascia and the like.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and specifically relates to the application of divalent metal lactate in products for promoting tissue regeneration, growth, healing and damage repair. Background Art

[0002] Mammals have evolved to initiate a rapid response of the body to heal when injured to prevent life-threatening bleeding and infection to ensure survival. The wound healing process goes through different stages, some of which occur sequentially, while others occur simultaneously. However, all stages work in close coordination at the site of damaged tissue to regenerate tissue with normal functionality. Tissue regeneration, growth, healing and injury repair events at the site of injury involve coagulation, inflammation, tissue deposition (migration and proliferation) and final tissue reconstruction in sequence.

[0003] When tissue is injured, first blood is released from the damaged blood vessels, resulting in the formation of a fibrin fiber grid with platelets embedded therein. These fibrin fiber grids can serve as a scaffold for the recruited cells to move toward and throughout it. The activated platelets degranulate and release chemokines, including cytokines and growth factors, such as transforming growth factor-β1 (TGF-β1), resulting in the recruitment of fibroblasts and keratinocytes. A few days after the injury, fibroblasts begin to replace the damaged tissue by depositing a new collagen matrix. The thickness of the collagen fibers gradually increases and they are arranged in a row along the wound. In normal scar formation, the collagen fibers are generally arranged parallel to the epidermis. Through myofibroblasts, this newly formed granulation tissue eventually structures and contracts into a denser structure.

[0004] Scars are usually formed due to the normal progression of the wound healing response and are composed of connective tissue deposited during the healing process. Most scars show a certain degree of abnormal structure (as seen in skin scars) and the amount of abnormal connective tissue (as seen in central nervous system scars). However, if the production of normal tissue is less than optimal wound healing, excessive deposition of scarring tissue is accompanied, resulting in keloid and hypertrophic scar formation, also known as fibrosis. The formation of scars is usually harmful, which will cause the appearance of tissues such as skin, ligaments, tendons or fascia to be affected, and more seriously, it will also cause the function of these tissues to be impaired.

[0005] The purpose of treating and repairing scars clinically is to restore function, alleviate symptoms, improve appearance and prevent recurrence. Clinical methods for treating scars include: surgery, drug therapy, physical grinding, compression therapy, radiotherapy, chemical peeling, freezing, radiofrequency microneedle therapy, ion beam therapy, laser, etc. It can be seen that most of the treatment methods currently used in clinical practice are traumatic and need to be completed in the hospital, so the convenience is poor and the economic burden on patients is heavy. In terms of drug treatment, the types of drugs available are limited and the efficacy is poor. Therefore, there is a continuous and urgent need for products that are safe to use, effective, low-priced, and simple to administer to promote tissue regeneration, growth, healing, and injury repair.

[0006] The inventors have found in previous studies that during the degradation of poly-L-lactic acid (PLLA), the molecular structure of PLLA is gradually destroyed and slowly hydrolyzed into lactic acid, which can induce human fibroblasts to increase the production of collagen, resulting in an increase in collagen fibers in the dermis, producing a filling and repairing effect. On this basis, the inventors have found that PLLA and lactic acid and its related lactate compounds have beneficial effects in repairing tissues such as cartilage, connective tissue, tendons, fascia, and nerves (see CN202210028046.9). The present invention is a continuation of previous work, and the present invention finds that divalent metal lactates can not only promote tissue regeneration, growth, and healing, but also can prevent and treat tissue fibrosis and / or scar tissue production, so it has broad application prospects in the treatment and repair of damage to tissues such as skin, connective tissue, tendons, and fascia. Summary of the invention

[0007] In order to overcome the defects and deficiencies in the prior art, the present invention provides a new use of divalent metal lactate. The inventor unexpectedly discovered during the research process that divalent metal lactate can effectively regulate and promote tissue regeneration, growth, healing and damage repair, and based on this, conducted in-depth research and completed the present invention.

[0008] In order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0009] In a first aspect, the present invention provides a use of a divalent metal lactate in the preparation of a product for promoting tissue regeneration, growth, healing and / or injury repair, wherein the tissue is selected from collagen-rich tissues.

[0010] In a second aspect, the present invention provides use of a divalent metal lactate in the preparation of a product for preventing or treating tissue damage, wherein the tissue is selected from collagen-rich tissues.

[0011] In a third aspect, the present invention provides use of a divalent metal lactate in the preparation of a product for preventing or treating tissue fibrosis and scar formation, wherein the tissue is selected from collagen-rich tissues.

[0012] As an option, in the above aspects, the tissue is selected from at least one of skin, ligament, tendon or fascia.

[0013] As an optional mode, in the above aspect, the divalent metal lactate includes one or more combinations of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate.

[0014] As an alternative, in the above aspect, the divalent metal lactate is selected from zinc lactate or a combination of zinc lactate and magnesium lactate.

[0015] Preferably, in the composition, the ratio of the zinc lactate to the magnesium lactate is 1:10-10:1 by weight.

[0016] More preferably, in the composition, the ratio of zinc lactate to magnesium lactate is 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 by weight.

[0017] Most preferably, in the composition, the ratio of the zinc lactate to the magnesium lactate is 7:1 by weight.

[0018] As an optional mode, in the above aspects, the product is selected from one or more of medicines, test kits, health products, and medical devices.

[0019] Preferably, the concentration of the divalent metal lactate in the product is 15-250 mmol / L.

[0020] More preferably, the content of the divalent metal lactate in the skin, ligament, tendon or fascia is 30-150 mmol / L.

[0021] As an optional manner, in the above aspect, the medical device includes one or more combinations of medical tape, bandage, gauze, dressing, sponge, and medical suture.

[0022] As an optional mode, in the above aspect, in the drug, the divalent metal lactate is an active ingredient, and the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0023] Preferably, in the medicament, the divalent metal lactate is the only active ingredient.

[0024] In the drug, divalent metal lactate is an active ingredient. The dosage form of the drug includes injection or external preparation.

[0025] Preferably, the external preparation comprises a combination of one or more of ointment, cream, patch, spray, solution and lotion.

[0026] Preferably, the administration of the product includes one or more of the following: intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration or application.

[0027] In addition, those skilled in the art will appreciate that the use of the preparation, as well as the dosage and volume of administration are related to the age, physical condition and disease of the subject, and can be determined by a clinician based on the circumstances.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention provides a new treatment method that is safe, effective, inexpensive, and simple to administer in terms of tissue damage repair and prevention and treatment of tissue fibrosis and / or scar tissue. In particular, the present invention has discovered the use of a divalent metal lactate comprising a combination of one or more of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate in the preparation of a product that promotes tissue regeneration, growth, healing, and / or damage repair. Therefore, the present invention has broad application prospects in the treatment and repair of damage to tissues such as skin, connective tissue, tendons, and fascia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Achilles tendon staining images of the experimental side (50mmol / L calcium lactate solution) and the control side (0.9% sodium chloride injection).

[0031] Figure 2 : Achilles tendon staining images of the experimental side (50mmol / L magnesium lactate solution) and the control side (0.9% sodium chloride injection).

[0032] Figure 3 : Achilles tendon staining images of the experimental side (50mmol / L zinc lactate solution) and the control side (0.9% sodium chloride injection). DETAILED DESCRIPTION

[0033] The present invention will be further described with reference to specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0036] Example 1: Effects of divalent metal lactates on tendon growth in experimental animals

[0037] The main experimental method steps are shown in CN202210028046.9. The details are as follows:

[0038] 1. Experimental design:

[0039] Experimental animals: SD rats weighing 200-220 g (male and female, 1:1 ratio) were selected and divided into three groups, with 10 rats in each group.

[0040] Experimental site: Tendon The right Achilles tendon of rats was selected.

[0041] The experimental groups are shown in Table 1.

[0042] Table 1: Experimental groups and treatments

[0043] Group concentration Injection volume Calcium lactate group 50mmol / L 0.1mL Magnesium lactate group 50mmol / L 0.1mL Zinc lactate group 50mmol / L 0.1mL

[0044] Experimental operation: Determine the anatomical position of the right Achilles tendon of the rat, disinfect the dorsal skin of the distal tibia with an alcohol cotton ball, and use a 26G needle and a 1mL sterile syringe to inject 0.1mL of 50mmol / L lactate solution around the Achilles tendon. The right side is set as the experimental side (injection of 0.1mL of different types of divalent metal lactate solutions), and the left side is set as the control side (injection of 0.1mL of 0.9% sodium chloride injection).

[0045] During the first 1-7 days of the experiment, the experimental solution of the corresponding group was injected at a single point on the experimental side every day, and the same volume of sodium chloride injection was injected at a single point on the control side. Three rats in each group were killed on the 3rd, 7th and 14th days after surgery, and the experimental and control sides of each rat were selected for fixation and pathological histological examination.

[0046] 2. Detection method:

[0047] 1) The Achilles tendon tissue to be observed was cut out, and after fixation, the Achilles tendon tissue was stained with Sirius red to observe the effect of the injection solution on the Achilles tendon tissue.

[0048] 2) Randomly select the experimental side and the control side for sectioning, measure the tissue diameter under a 100x optical microscope, and calculate the average value of the experimental side and the control side using the following formula:

[0049] Average value = (3 measurements of animal No. ① + 3 measurements of animal No. ② + 3 measurements of animal No. ③) / 9

[0050] The diameters of both sides were compared, and the effects of divalent metal lactate on Achilles tendon tissue were analyzed using SPSS 21 calculation results.

[0051] 3. Experimental results:

[0052] The experimental results are as follows Figure 1 , Figure 2 , Figure 3 As shown in Table 2 below, after the injection of 50mmol / L divalent metal lactate solution, the Achilles tendon tissue of each group of experimental animals showed significant thickening to varying degrees. It can be seen from the figure that the zinc lactate solution group showed significant thickening of the experimental side on the 3rd, 7th and 21st days, and the diameter of the Achilles tendon increased. Its effect on promoting the growth of the Achilles tendon is better than that of calcium lactate solution and magnesium lactate solution.

[0053] The values ​​of Achilles tendon tissue measured under a 100x optical microscope are shown in Table 2: Compared with the experimental side, the difference in diameter of the Achilles tendon on the experimental side of the zinc lactate treatment group on the 7th day and the 14th day was statistically significant (P<0.01), and it can be seen that the diameter of the Achilles tendon increased, showing a significant promotion effect on the growth of the Achilles tendon; Compared with the experimental side, the difference in diameter on the experimental side of the calcium lactate treatment group on the 7th day was statistically significant (P<0.01), and it can be seen that the diameter of the Achilles tendon increased, showing a promotion effect on the growth of the Achilles tendon; Compared with the experimental side, the diameter of the Achilles tendon on the experimental side of the magnesium lactate treatment group increased on the 7th and 14th days, but the promotion effect on the growth of the Achilles tendon was not so obvious.

[0054] It can be seen from these experimental results that divalent metal lactates have a significant promoting effect on animal tendon tissue regeneration, especially zinc lactate treatment shows a very significant promoting effect.

[0055] Table 2: Measurement values ​​of experimental side and control side

[0056]

[0057]

[0058] Example 2: Effects of divalent metal lactate on the treatment of ligament injury in experimental animals 1. Establishment of anterior cruciate ligament injury model and experimental grouping

[0059] New Zealand white rabbits (male and female, 1:1), 3 months old, weighing 2.5 kg ± 200 g, a total of 12 rabbits, were placed in a temperature-controlled room (22 ± 2 ° C). Before the experiment, 3% (1 mL / kg) sodium pentobarbital was injected through the rabbit ear vein. After the rabbit was anesthetized, it was fixed on the operating table, the hair of the knee joint surgery area was shaved, and a 2 cm longitudinal incision was made along the inner side of the patella. After the skin was cut, the fascia and muscle were carefully separated. After fully exposing the joint cavity, a row of 3 eye holes were punctured perpendicular to the long axis of the ligament at the middle and upper 1 / 3 of the distal femur of the anterior cruciate ligament with a 20 mL needle bevel, simulating the shear force injury of the anterior cruciate ligament in clinical practice. After the model was constructed, the surgery area was closed, sutured and pressure bandaged. After surgery, the animals were randomly divided into 4 groups, 10 in each group.

[0060] After the model was established, the control group was injected with 0.25 mL of normal saline around the damaged ligament; the experimental group was injected with 0.25 mL of the corresponding lactate aqueous solution around the damaged ligament, 0.25 mL each time, once a day, for 4 consecutive weeks.

[0061] Table 3: Experimental groups and treatments

[0062]

[0063] The limb motor function after anterior cruciate ligament injury was evaluated by comparing the vertical jump height of rabbits in different experimental groups. The results showed that the vertical jump height of the experimental group was significantly greater than that of the control group (P < 0.05). The vertical jump height of the animals in the magnesium lactate and zinc lactate combined group was greater than that of the divalent metal magnesium lactate group (P < 0.05).

[0064] 2. Tissue Sample Acquisition

[0065] The rabbits were killed by intravenous injection of an overdose of 3% sodium pentobarbital through the ear margin. The surgical area was shaved and disinfected, the skin was incised along the patella, the fascia and muscle were carefully separated, the incision was cleaned with PBS solution, and the complete anterior cruciate ligament was cut off at both ends close to the tibia and femur for total RNA extraction.

[0066] 3. Real-time Quantitative PCR

[0067] 3.1 Extraction of total RNA

[0068] Remove the ligament, add 0.5mL Trizol, grind thoroughly, centrifuge and take the supernatant. Add 0.1mL chloroform, centrifuge and take the supernatant. Add 0.8mL isopropanol, centrifuge and discard the supernatant. Add 1mL 75% ethanol, centrifuge and discard the supernatant. Add 20μL DEPC-treated water to dissolve RNA. Use a nucleic acid protein detector to measure the concentration and purity of the total RNA solution.

[0069] 3.2 Real-time quantitative PCR reaction

[0070] The expressions of Caspase-3 and VEGFα in anterior cruciate ligament tissues of each group were detected by RT-qPCR.

[0071] Primer design

[0072] Caspase-3 upstream primer 5`-GAGCTTGGAACGGTACGATA-3`

[0073] Downstream primer 5`-CCGTACCAGAGCGAGATGAC-3`

[0074] VEGFα upstream primer 5`-GGAGTACCCTGATGAGATCGA-3`

[0075] Downstream primer 5`-CTTTGGTCTGCATTCACATTTGT-3`

[0076] Caspase-3 plays a key role in the process of cell apoptosis and is also a potential drug target. Studies have shown that Caspase-3 can severely inhibit DNA replication, transcription and damage repair functions, ultimately leading to irreversible apoptotic effects in cells. Therefore, reducing the activation of Caspase-3 can effectively inhibit the process of cell apoptosis. The results of real-time quantitative PCR of Caspase-3 are shown in Table 4. The results showed that there was no significant change in the expression of Caspase-3 mRNA in the control group at each time period. Compared with the control group, the expression of Caspase-3 mRNA in the divalent metal lactate group was significantly downregulated on the first, third and seventh days after modeling (P<0.05). At the same time, within 7 days after modeling, the level of Caspase-3 mRNA in the divalent metal lactate group gradually decreased over time (P<0.05). Compared with the magnesium lactate and zinc lactate groups, the expression of Caspase-3 mRNA in the magnesium lactate + zinc lactate group was significantly downregulated on the first, third and seventh days (P<0.05). The above results show that divalent metal lactate can reduce the expression of Caspase-3 protein after animal ligament tissue injury and reduce the occurrence of cell apoptosis caused by nerve injury. The combination of magnesium lactate and zinc lactate showed a synergistic effect in reducing the expression of Caspase-3 protein.

[0077] Table 4: Comparison of Caspase-3 mRNA expression at different time points after modeling and at different time points after surgery

[0078]

[0079] Note: Compared with the control group, a P<0.05; compared with the magnesium lactate group, b P<0.05; compared with the zinc lactate group, dP<0.05; compared with the first day of the same group on the third day, e P<0.05; compared with the 3rd day in the same group on the 7th day, f P<0.05.

[0080] Example 3: Effects of divalent metal lactates on wound healing in experimental animals

[0081] 42 SD rats weighing about 200g (6 in each group) were selected. After anesthesia, the backs were shaved and a 5 mm deep and 3 cm long incision was made on the backs with a scalpel. The control group was smeared with 0.5 mL of normal saline, and the wounds of each experimental group were smeared with 25 mmol / L zinc lactate aqueous solution, 50 mmol / L zinc lactate aqueous solution, 25 mmol / L magnesium lactate aqueous solution, 50 mmol / L magnesium lactate aqueous solution or 25 mmol / L zinc lactate aqueous solution + 25 mmol / L magnesium lactate aqueous solution. Starting from the day of modeling, each animal was smeared once in the morning and evening every day, 0.5 mL each time, for 14 days, and the wound healing rate was recorded on the 3rd, 7th and 14th days (see Table 5).

[0082] Table 5: Skin wound healing rate of rats in each group at different time points

[0083]

[0084]

[0085] The experimental results in Table 5 show that the wound healing rates of the experimental animals treated with 25mmol / L zinc lactate aqueous solution, 50mmol / L zinc lactate aqueous solution, 25mmol / L magnesium lactate aqueous solution, 50mmol / L magnesium lactate aqueous solution, and 25mmol / L zinc lactate aqueous solution + 25mmol / L magnesium lactate aqueous solution were significantly improved at each time point compared with the control group animals.

[0086] From the experimental results on the 3rd and 7th days in the table, it can be seen that although the effect of magnesium lactate in promoting skin wound healing is not as good as that of zinc lactate, when zinc lactate and magnesium lactate are used together, they unexpectedly show a synergistic effect. The wound healing rates of the experimental animals in the combination group on the 3rd and 7th days were better than those of the zinc lactate and magnesium lactate single-use groups, respectively.

[0087] Example 4: Effect of divalent metal lactate on preventing scar formation on wound surface

[0088] 1. Experimental methods:

[0089] Thirty New Zealand white rabbits, both male and female, weighing 1.8-2.2 kg, were selected for the experiment. After 7 days of adaptive feeding, there was no obvious abnormality.

[0090] Scar model establishment: 30 g / L pentobarbital solution was injected into the ear vein for anesthesia, and a circular wound with a diameter of 1 cm was made along the long axis of the ventral side of the rabbit ear, two wounds in each ear, about 3.0 cm apart, and the whole layer of skin was completely removed to form the wound.

[0091] Experimental grouping and administration: 30 New Zealand white rabbits were randomly divided into 4 groups, with 6 animals in each group, with a total of 24 wounds. They were control group, 50mmol / L zinc lactate aqueous solution group, 50mmol / L magnesium lactate aqueous solution group, and 25mmol / L zinc lactate aqueous solution + 25mmol / L magnesium lactate aqueous solution group. After the wound was established, the control group applied normal saline to the wound once in the morning and evening, 0.5mL each time; each animal in the experimental group applied the corresponding lactate aqueous solution to each wound once in the morning and evening, 0.5mL each time, and the evaluation was performed on the 13th day.

[0092] Efficacy evaluation: The Vancouver Scar Scale (VSS) was used to evaluate the efficacy. The higher the score, the more severe the scar.

[0093] Table 6: Vancouver Scar Scale Scoring Criteria

[0094]

[0095] Statistical analysis: The data were analyzed using SPSS 21. The experimental results are expressed as x±SD.

[0096] 2. Experimental results:

[0097] The experimental results are shown in Table 7.

[0098] Table 7: Comparison of VSS values ​​of local scars on ears of New Zealand white rabbits

[0099]

[0100]

[0101] The experimental results on the 13th day showed that compared with the control group, the VSS scores of the rabbits in each experimental group decreased to varying degrees, indicating that each experimental group had a certain effect on preventing scar formation on the wound surface. In addition, the VSS scores of the zinc lactate aqueous solution and magnesium lactate aqueous solution combined group were significantly different from those of the zinc lactate group and magnesium lactate group (P<0.01), indicating that the combined group unexpectedly showed a synergistic effect in preventing scar formation on the wound surface.

[0102] In addition, in the above application, the results of other divalent metal lactates including calcium lactate, zinc lactate, ferrous lactate and various combinations are similar to the above magnesium lactate results, which will not be repeated here.

[0103] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of divalent metal lactate in the preparation of products for promoting tissue regeneration, growth, healing and / or injury repair, characterized in that: The tissue is selected from collagen-rich tissues.

2. Use of divalent metal lactate in the preparation of a product for preventing or treating tissue damage, characterized in that: The tissue is selected from collagen-rich tissues.

3. Use of divalent metal lactate in the preparation of products for preventing or treating tissue fibrosis and scar formation, characterized in that: The tissue is selected from tissues rich in collagen.

4. The use according to any one of claims 1 to 3, characterized in that: The tissue is selected from at least one of skin, ligament, tendon or fascia.

5. The use according to any one of claims 1 to 3, characterized in that: The divalent metal lactate includes one or more combinations of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate.

6. The use according to claim 5, characterized in that: The divalent metal lactate is selected from zinc lactate or a combination of zinc lactate and magnesium lactate. Preferably, in the composition, the ratio of zinc lactate to magnesium lactate is 1:10-10:1 by weight.

7. The use according to any one of claims 1 to 3, characterized in that: The product is selected from one or more of drugs, kits, health products, and medical devices. Preferably, the concentration of the divalent metal lactate in the product is 5-250 mmol / L, and more preferably, the content of the divalent metal lactate in the skin, ligaments, tendons or fascia is 30-150 mmol / L.

8. The use according to claim 7, characterized in that: The medical device includes one or more combinations of medical adhesive tape, bandage, gauze, dressing, sponge, and medical suture.

9. The use according to claim 7, characterized in that: In the medicine, divalent metal lactate is an active ingredient, and the medicine further comprises a pharmaceutically acceptable carrier or excipient.

10. The use according to claim 7, characterized in that: The dosage form of the drug includes injection or external preparation. Preferably, the external preparation includes one or more combinations of ointments, creams, patches, sprays, solutions, lotions. Preferably, the administration methods of the product include one or more of the following: intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration or application.

Images