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] This invention belongs to the field of biomedical technology, specifically relating to the application of divalent metal lactates in products that promote tissue regeneration, growth, healing, and damage repair. Background Technology

[0002] Mammals have evolved a rapid response to injury, initiating a healing response to prevent life-threatening bleeding and infection, thus ensuring survival. The wound healing process involves different stages, some occurring sequentially, and others simultaneously. However, all stages work in precise coordination at the site of injury to regenerate normally functional tissue. The events of tissue regeneration, growth, healing, and damage repair at the site of injury involve, in sequence, coagulation, inflammation, tissue deposition (migration and proliferation), and ultimately, tissue reconstruction.

[0003] Upon tissue injury, blood is first released from the damaged vessel, leading to the formation of a fibrin fiber network with platelets embedded within it. This fibrin fiber network serves as a scaffold for recruited cells to move toward and throughout it. 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 injury, fibroblasts begin to replace the damaged tissue by depositing a new collagen matrix. The collagen fibers gradually increase in thickness and align along the wound. In normal scar formation, collagen fibers are generally arranged parallel to the epidermis. Through myofibroblasts, this newly formed granulation tissue eventually forms and contracts into a denser structure.

[0004] Scars typically form as a result of the normal progression of a wound healing response and consist of connective tissue deposited during the healing process. Most scars exhibit some degree of abnormal structure (as seen in skin scars) and an abnormal amount of 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 scar-forming tissue occurs, leading to keloids and hypertrophic scars, also known as fibrosis. Scar formation is generally detrimental, affecting the appearance of tissues such as skin, ligaments, tendons, or fascia, and more seriously, impairing their function.

[0005] The clinical goals of scar treatment and repair are to restore function, alleviate symptoms, improve appearance, and prevent recurrence. Clinical methods for treating scars include surgery, medication, physical abrasion, compression therapy, radiation therapy, chemical peels, cryotherapy, radiofrequency microneedling, ion beam therapy, and laser treatment. It can be seen that most of the treatment methods currently used clinically are invasive and need to be completed in a hospital, thus lacking convenience and placing a heavy financial burden on patients. Regarding medication, the selection of drugs is limited, and their efficacy is unsatisfactory. Therefore, there is a continuous and urgent clinical need for safe, effective, inexpensive products with simple administration methods to promote tissue regeneration, growth, healing, and damage repair.

[0006] In previous research, the inventors discovered that during the degradation of poly-L-lactic acid (PLLA), the molecular structure of PLLA is gradually destroyed, slowly hydrolyzing into lactic acid. Lactic acid can induce human fibroblasts to increase collagen production, leading to an increase in collagen fibers in the dermis and producing a filling and repairing effect. Building on this, the inventors further discovered that PLLA, lactic acid, and related lactate compounds have beneficial effects on repairing cartilage, connective tissue, tendons, fascia, nerves, and other tissues (see CN202210028046.9). This invention is a continuation of previous work. This invention discovers that divalent metal lactates can not only promote tissue regeneration, growth, and healing, but also prevent and treat tissue fibrosis and / or scar tissue formation. Therefore, they have broad application prospects in the treatment and repair of injuries to skin, connective tissue, tendons, fascia, and other tissues. Summary of the Invention

[0007] To overcome the defects and shortcomings of existing technologies, this invention provides a novel application for divalent metal lactates. During their research, the inventors unexpectedly discovered that divalent metal lactates can effectively regulate and promote tissue regeneration, growth, healing, and damage repair. Based on this discovery, they conducted in-depth research and completed this invention.

[0008] To achieve the objectives of this invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides the use of divalent metal lactates in the preparation of articles that promote tissue regeneration, growth, healing and / or damage repair, wherein the tissue is selected from collagen-rich tissues.

[0010] In a second aspect, the present invention provides the use of divalent metal lactates in the preparation of articles for the prevention or treatment of tissue damage, wherein the tissue is selected from collagen-rich tissues.

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

[0012] Alternatively, in the above aspects, the tissue is selected from at least one of skin, ligaments, tendons, or fascia.

[0013] Alternatively, in the above aspects, the divalent metal lactate includes one or more combinations of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate.

[0014] Alternatively, in the above aspects, the divalent metal lactate is selected from zinc lactate or a combination of zinc lactate and magnesium lactate.

[0015] Preferably, the ratio of zinc lactate to magnesium lactate in the composition is 1:10 to 10:1, based on parts 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, the ratio of zinc lactate to magnesium lactate in the composition is 7:1 by weight.

[0018] Alternatively, in the foregoing aspects, the article is selected from one or more of pharmaceuticals, reagent 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, ligaments, tendons or fascia is 30 to 150 mmol / L.

[0021] Alternatively, in the above aspects, the medical device includes one or more combinations of medical tape, bandage, gauze, dressing, sponge, and medical sutures.

[0022] Alternatively, in the foregoing aspects, in the medicament, the divalent metal lactate is the active ingredient, and the medicament further comprises a pharmaceutically acceptable carrier or excipient.

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

[0024] In the drug, divalent metal lactate is the active ingredient. The dosage form of the drug includes injections or topical preparations.

[0025] Preferably, the topical preparation includes one or more combinations of ointments, creams, patches, sprays, solutions, and lotions.

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

[0027] In addition, those skilled in the art will know that the manner of use of the product, as well as the dosage and volume of application, are related to the age, physical condition and disease of the subject, and can be determined by a clinician as appropriate.

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

[0029] This invention provides a safe, effective, inexpensive, and simple administration method for tissue damage repair and the prevention and treatment of tissue fibrosis and / or scar tissue formation. Specifically, this invention discovers the application of divalent metal lactates, including one or more combinations of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate, in the preparation of articles that promote tissue regeneration, growth, healing, and / or damage repair. Therefore, this invention has broad application prospects in the treatment and repair of injuries to tissues such as skin, connective tissue, tendons, and fascia. Attached Figure Description

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

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

[0032] Figure 3 Staining images of the Achilles tendon on the experimental side (50 mmol / L zinc lactate solution) and the control side (0.9% sodium chloride injection). Detailed Implementation

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

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

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

[0037] The main experimental methods and procedures are detailed in CN202210028046.9.

[0038] 1. Experimental Design:

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

[0040] Experimental site: The right Achilles tendon of a rat was selected as the tendon.

[0041] The experimental groupings are shown in Table 1 below.

[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 procedure: The anatomical location of the right Achilles tendon in rats was determined. The skin on the dorsal side of the distal tibia was disinfected with an alcohol swab. Using a 26G needle and a 1mL sterile syringe, 0.1mL of 50mmol / L lactate solution was injected around the Achilles tendon. The right side was designated as the experimental side (0.1mL of different types of divalent metal lactate solution was injected), and the left side was designated as the control side (0.1mL of 0.9% sodium chloride injection was injected).

[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 from each group were sacrificed on the 3rd, 7th and 14th days after the operation. The experimental and control sides of each rat were selected for fixation and histopathological examination.

[0046] 2. Detection method:

[0047] 1) The Achilles tendon tissue was excised for observation. After the Achilles tendon tissue was fixed, Sirius red staining was performed to observe the effect of the injection solution on the Achilles tendon tissue.

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

[0049] Average value = (3 measurements of animal #1 + 3 measurements of animal #2 + 3 measurements of animal #3) / 9

[0050] By comparing the diameters on both sides, the effect of divalent metal lactate on Achilles tendon tissue was analyzed using SPSS21 calculation results.

[0051] 3. Experimental Results:

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

[0053] The Achilles tendon tissue values ​​measured under a 100x optical microscope are shown in Table 2. Compared with the experimental side, the difference in Achilles tendon diameter on day 7 and day 14 in the zinc lactate treatment group was statistically significant (P<0.01), indicating an increase in Achilles tendon diameter and a significant promoting effect on Achilles tendon growth. Compared with the experimental side, the difference in diameter on day 7 in the calcium lactate treatment group was statistically significant (P<0.01), indicating an increase in Achilles tendon diameter and a promoting effect on Achilles tendon growth. Compared with the experimental side, the Achilles tendon diameter on day 7 and day 14 in the magnesium lactate treatment group was increased, but the promoting effect on Achilles tendon growth was not so obvious.

[0054] These experimental results show that divalent metal lactates have a significant promoting effect on the regeneration of animal tendon tissue, especially zinc lactate treatment, which shows a very significant promoting effect.

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

[0056]

[0057]

[0058] Example 2: The therapeutic effect of divalent metal lactates on ligament injuries in experimental animals

[0059] 1. Establishment of an anterior cruciate ligament injury model and experimental grouping

[0060] Twelve New Zealand white rabbits (male and female, 1:1 ratio), 3 months old, weighing 2.5 kg ± 200 g, were placed in a temperature-controlled chamber (22 ± 2℃). Before the experiment, 3% (1 mL / kg) sodium pentobarbital was injected via the marginal ear vein to anesthetize the rabbits. After anesthesia, the rabbits were fixed on the operating table, and the hair in the knee joint surgical area was shaved. A 2 cm longitudinal incision was made along the medial side of the patella, and the fascia and muscles were carefully separated after the skin was opened. After fully exposing the joint cavity, three eye holes were punctured perpendicularly to the long axis of the anterior cruciate ligament (ACL) at the upper 1 / 3 of the distal femur using a 20 mL needle with an oblique cut, simulating the shear force injury to the ACL in clinical practice. After the model was established, the surgical area was closed, sutured, and pressure bandaged. Post-operatively, the animals were randomly divided into four groups of ten each.

[0061] After the model was established, the control group received an injection of 0.25 mL of physiological saline around the damaged ligament; the experimental group received an injection of 0.25 mL of the corresponding lactate solution around the damaged ligament, once a day for 4 consecutive weeks.

[0062] Table 3: Experimental Grouping and Treatment

[0063]

[0064] The vertical jump height of rabbits in different experimental groups was used to evaluate limb motor function after anterior cruciate ligament injury. The results showed that the vertical jump height of the experimental groups was significantly greater than that of the control group (P < 0.05). The vertical jump height of the group treated with magnesium lactate and zinc lactate was greater than that of the group treated with divalent magnesium lactate (P < 0.05).

[0065] 2. Tissue Sample Acquisition

[0066] Rabbits were euthanized by intravenous injection of an excessive amount of 3% sodium pentobarbital in the ear. The surgical area was shaved, prepared, and disinfected. The skin was incised along the patella, and the fascia and muscles were carefully separated. The incision was cleaned with PBS solution, and the complete anterior cruciate ligament was excised from both ends close to the tibia and femur for total RNA extraction.

[0067] 3. Real-time quantitative PCR

[0068] 3.1 Extraction of total RNA

[0069] Remove the ligament, add 0.5 mL of Trizol, grind thoroughly, centrifuge and collect the supernatant. Add 0.1 mL of chloroform, centrifuge and collect the supernatant. Add 0.8 mL of isopropanol, centrifuge and discard the supernatant. Add 1 mL of 75% ethanol, centrifuge and discard the supernatant. Add 20 μL of DEPC-treated water to dissolve the RNA. Use a nucleic acid protein analyzer to determine the concentration and purity of the total RNA solution.

[0070] 3.2 Real-time quantitative PCR reaction

[0071] The expression of Caspase-3 and VEGFα in the anterior cruciate ligament tissue of each group was detected by RT-qPCR.

[0072] Primer design

[0073] Caspase-3 upstream primer 5`-GAGCTTGGAACGGTACGATA-3`

[0074] Downstream primer 5`-CCGTACCAGAGCGAGATGAC-3`

[0075] VEGFα upstream primer 5`-GGAGTACCCTGATGAGATCGA-3`

[0076] Downstream primer 5'-CTTTGGTCTGCATTCACATTTGT-3'

[0077] Caspase-3 plays a crucial role in apoptosis and is a potential drug target. Studies have shown that caspase-3 can severely inhibit DNA replication, transcription, and damage repair, ultimately leading to irreversible apoptosis. Therefore, reducing caspase-3 activation can effectively inhibit apoptosis. The real-time quantitative PCR results of caspase-3 are shown in Table 4. The results showed that the expression of caspase-3 mRNA in the control group did not change significantly at any time point. Compared with the control group, the expression level of caspase-3 mRNA in the divalent metal lactate group was significantly downregulated on days 1, 3, and 7 after modeling (P<0.05). Simultaneously, within 7 days after modeling, the caspase-3 mRNA level in the divalent metal lactate group gradually decreased over time (P<0.05). Compared with the magnesium lactate and zinc lactate groups, the expression level of caspase-3 mRNA in the magnesium lactate + zinc lactate group was significantly downregulated on days 1, 3, and 7 (P<0.05). The results indicate that divalent metal lactates can reduce Caspase-3 protein expression after ligament tissue injury in animals, thereby reducing apoptosis induced by nerve damage. The combination of magnesium lactate and zinc lactate showed a synergistic effect in reducing Caspase-3 protein expression.

[0078] Table 4: Comparison of Caspase-3 mRNA expression at different time points after modeling and postoperatively

[0079]

[0080] 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; Day 3 compared with Day 1 of the same group, e P<0.05; compared with day 3 of the same group, day 7, f P<0.05.

[0081] Example 3: Effects of divalent metal lactates on wound healing in experimental animals

[0082] Forty-two SD rats (n=6 per group) weighing approximately 200g were used. After anesthesia, the hair on their backs was shaved, and a 5mm deep, 3cm long incision was made on each back with a scalpel. The control group received 0.5mL of physiological saline, while the experimental groups received 25mmol / L zinc lactate solution, 50mmol / L zinc lactate solution, 25mmol / L magnesium lactate solution, 50mmol / L magnesium lactate solution, or a combination of 25mmol / L zinc lactate solution and 25mmol / L magnesium lactate solution, respectively. Starting from the day of model establishment, each animal received 0.5mL of the solution twice daily, morning and evening, for 14 days. The wound healing rate was recorded on days 3, 7, and 14 (see Table 5).

[0083] Table 5: Skin wound healing rate of rats in different time points

[0084]

[0085]

[0086] The experimental results in Table 5 show that, compared with the control group, the experimental animals treated 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, and 25 mmol / L zinc lactate aqueous solution + 25 mmol / L magnesium lactate aqueous solution had significantly higher wound healing rates at each time point.

[0087] The experimental results on days 3 and 7 in the table show that although magnesium lactate is not as effective as zinc lactate in promoting skin wound healing, the combination of zinc lactate and magnesium lactate unexpectedly showed a synergistic effect. The wound healing rate of the experimental animals in the combination group was better than that of the groups using zinc lactate and magnesium lactate alone on days 3 and 7, respectively.

[0088] Example 4: The role of divalent metal lactates in preventing scar formation on wounds

[0089] 1. Experimental Methods:

[0090] Thirty New Zealand white rabbits, both male and female, weighing 1.8-2.2 kg, were used in the experiment. After 7 days of acclimatization feeding, none of them showed any obvious abnormalities.

[0091] Establishment of the scar model: Anesthesia was performed by injecting a 30g / L pentobarbital solution into the ear vein. A circular wound with a diameter of 1cm was made along the long axis on the ventral side of the rabbit ear. Two wounds were made on each ear, with an interval of about 3.0cm. The full thickness of the skin was completely removed to form the wound.

[0092] Experimental grouping and drug administration: Thirty New Zealand white rabbits were randomly divided into four groups of six animals each, resulting in a total of 24 wounds. These groups were: control group, 50 mmol / L zinc lactate aqueous solution group, 50 mmol / L magnesium lactate aqueous solution group, and 25 mmol / L zinc lactate aqueous solution + 25 mmol / L magnesium lactate aqueous solution group. After wound establishment, the control group received 0.5 mL of physiological saline twice daily (morning and evening) for each wound. In the experimental groups, each animal received 0.5 mL of the corresponding lactate aqueous solution twice daily (morning and evening) for each wound. Evaluation was conducted on day 13.

[0093] Therapeutic effect evaluation: The therapeutic effect was evaluated using the Vancouver Scar Scale (VSS) as shown in Table 6. The higher the score, the more severe the scar.

[0094] Table 6: Vancouver Scar Scale Scoring Criteria

[0095]

[0096]

[0097] Statistical analysis: Data were analyzed using SPSS 21 software. Results are expressed as mean ± SD.

[0098] 2. Experimental Results:

[0099] The experimental results are shown in Table 7.

[0100] Table 7: Comparison of VSS values ​​of local ear scars in New Zealand White rabbits

[0101]

[0102] The results of the experiment on day 13 showed that, compared with the control group, the VSS scores of rabbits in each experimental group decreased to varying degrees, indicating that each experimental group had a certain effect on preventing wound scar formation. Furthermore, the VSS scores of the combined zinc lactate and magnesium lactate solution group were significantly different from those of the zinc lactate and magnesium lactate groups (P<0.01), indicating that the combined group unexpectedly showed a synergistic effect in preventing wound scar formation.

[0103] In addition, in the above applications, the results of other divalent metal lactates, including calcium lactate, zinc lactate, ferrous lactate, and various combinations, are similar to those of magnesium lactate, and will not be repeated here.

[0104] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of divalent metal lactate salts in the manufacture of an article to promote tissue regeneration, growth, healing and / or repair of injury, characterized in that: The divalent metal lactate is selected from a combination of zinc lactate and magnesium lactate, and the tissue is at least one selected from ligament, tendon or fascia.

2. Use of a divalent metal lactate salt for the preparation of a preparation for the prevention or treatment of tissue damage, characterized in that: The divalent metal lactate is selected from a combination of zinc lactate and magnesium lactate, and the tissue is at least one selected from ligament, tendon or fascia.

3. Use of a divalent metal lactate salt for the preparation of a preparation for the prevention or treatment of tissue fibrosis and scarring, characterized in that: The divalent metal lactate is selected from a combination of zinc lactate and magnesium lactate, and the tissue is at least one selected from ligament, tendon or fascia.

4. Use according to any one of claims 1 to 3, characterized in that: In the composition, the ratio of the amount of zinc lactate to magnesium lactate is 1:10-10:1 by weight, preferably, the ratio of the amount of zinc lactate to magnesium lactate is 1:

1.

5. Use according to any one of claims 1 to 3, characterized in that: The product is selected from one or more of a drug, a kit or a medical device.

6. The use according to claim 5, wherein the concentration of the divalent metal lactate in the product is 5-250 mmol / L, preferably, the content of the divalent metal lactate in skin, ligament, tendon or fascia is 30-150 mmol / L.

7. Use according to claim 6, characterized in that: The medical device comprises a combination of one or more of medical adhesive tape, bandage, gauze, plaster, sponge, medical suture.

8. Use according to claim 6, characterized in that: In the drug, the divalent metal lactate is an active ingredient, and the drug further comprises a pharmaceutically acceptable carrier or excipient.

9. Use according to claim 6, characterized in that: The dosage form of the drug comprises injection or external preparation, preferably, the external preparation comprises a combination of one or more of ointment, cream, patch, spray, solution, lotion.

10. Use according to claim 9, characterized in that: The administration mode of the product comprises one or more of intravenous injection, in situ injection, intramuscular injection, subcutaneous injection, oral administration or smearing.

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