Application of divalent metal lactates in the preparation of products promoting tissue growth and repair

Abstract

The present invention belongs to the field of biomedicine and specifically relates to the use of divalent metal lactates in products that promote tissue regeneration, growth, healing, and injury repair. Preferably, the divalent metal lactate comprises a combination of one or more of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate. The present invention provides a new treatment for tissue injury and repair, as well as for the prevention and treatment of tissue fibrosis and / or scar tissue formation. It has broad application prospects in the treatment and repair of injuries to tissues such as skin, connective tissue, tendons, and fascia.

Description

Technical Field

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

[0002] Mammals have evolved to rapidly initiate a healing response when injured to prevent life-threatening bleeding and infection, ensuring survival. The wound healing process progresses through different stages, some of which occur sequentially, while others occur simultaneously. However, all stages work together meticulously at the site of damaged tissue to regenerate tissue with normal function. The events of tissue regeneration, growth, healing, and wound repair at the site of injury sequentially involve coagulation, inflammation, tissue deposition (migration and proliferation), and ultimately tissue reconstruction.

[0003] When tissue is damaged, blood is first 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 them. The activated platelets degranulate and release chemotactic factors, including cytokines and growth factors, such as transforming growth factor-β1 (TGF-β1), which leads to 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, collagen fibers are generally arranged parallel to the epidermis. Through myofibroblasts, this newly formed granulation tissue eventually constructs 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 may occur, leading to keloid and hypertrophic scar formation, also known as fibrosis. The formation of scars is usually harmful, as it will affect the appearance of tissues such as skin, ligaments, tendons or fascia, 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 they are less convenient and impose a heavy financial burden on patients. 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, have a definite effect, are inexpensive, and have simple administration methods to promote tissue regeneration, growth, healing, and injury repair.

[0006] The present inventors have found in previous studies that during the degradation process of poly-L-lactic acid (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 generation of collagen, resulting in an increase in collagen fibers in the dermis, and a filling and repair effect. On this basis, the present inventors have found that PLLA and lactic acid and its related lactate compounds have a beneficial effect in repairing tissues such as cartilage, connective tissue, tendon, 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, 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, tendon, fascia. Summary of the Invention

[0007] To overcome the shortcomings and deficiencies of the prior art, the present invention provides a novel use of divalent metal lactates. The inventors unexpectedly discovered during their research that divalent metal lactates can effectively regulate and promote tissue regeneration, growth, healing, and injury repair. Based on this discovery, they 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 solutions:

[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 tissue.

[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 tissue.

[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 tissue.

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

[0013] As an optional manner, 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 the zinc lactate to the 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 a drug, a kit, or a medical device.

[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 to 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 injections or external preparations.

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

[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 topical application.

[0027] In addition, those skilled in the art will appreciate that the mode of 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 modality that is safe, effective, inexpensive, and simple to administer for tissue damage repair and the prevention and treatment of tissue fibrosis and / or scar formation. Specifically, the present invention has discovered the use of divalent metal lactates comprising a combination of one or more of calcium lactate, magnesium lactate, zinc lactate, and ferrous lactate in the preparation of products that promote 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 (50 mmol / L calcium lactate solution) and the control side (0.9% sodium chloride injection).

[0031] Figure 2 : Achilles tendon staining images of the experimental side (50 mmol / 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 below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0034] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. 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 methods and 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, with a ratio of 1:1) were selected and divided into three groups, with 10 rats in each group.

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

[0041] The experimental groups 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 of rats was determined. The dorsal skin of the distal tibia was disinfected with an alcohol swab. Using a 26G needle and a 1mL sterile syringe, 0.1mL of a 50mmol / L lactate solution was injected into the area surrounding the Achilles tendon. The right side was designated as the experimental side (injection of 0.1mL of different divalent metal lactate solutions), and the left side was designated 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 into the experimental side at a single point every day, and the control side was injected with the same volume of sodium chloride injection at a single point. 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 and fixed, and then stained with Sirius red to observe the effect of the injection solution on the Achilles tendon tissue.

[0048] 2) Randomly select the 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 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 SPSS21 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 injection of 50 mmol / L divalent metal lactate solution, the Achilles tendon tissue of each group of experimental animals showed significant thickening to varying degrees. As can be seen from the figure, the experimental side of the zinc lactate solution group showed significant thickening and increased Achilles tendon diameter on the 3rd, 7th, and 21st days. Its promoting effect on Achilles tendon growth 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 Achilles tendon diameter on the experimental side of the zinc lactate treatment group on the 7th and 14th days was statistically significant (P<0.01), and the Achilles tendon diameter increased, showing a significant promotion effect on Achilles tendon growth; 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 the Achilles tendon diameter increased, showing a promotion effect on Achilles tendon growth; compared with the experimental side, the Achilles tendon diameter of the experimental side of the magnesium lactate treatment group increased on the 7th and 14th days, but the promotion effect on Achilles tendon growth was not so obvious.

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

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

[0056]

[0057]

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

[0059] Twelve New Zealand white rabbits (male and female, 1:1 ratio), 3 months old and weighing 2.5 kg ± 200 g, were housed in a temperature-controlled room (22 ± 2°C). Before the experiment, 3% (1 mL / kg) sodium pentobarbital was injected into the rabbit's ear vein. After anesthesia, the rabbits were secured to the operating table. The hair at the knee joint surgery site was shaved, and a 2 cm longitudinal incision was made along the medial side of the patella. The skin was opened and the fascia and muscle were carefully separated. After fully exposing the joint cavity, a 20 mL needle was used to puncture a row of three holes perpendicular to the long axis of the anterior cruciate ligament in the upper and middle thirds of the distal femoral end to simulate shear force injury to the anterior cruciate ligament in clinical practice. After model establishment, the surgical area was closed, sutured, and a pressure bandage was applied. After surgery, the animals were randomly divided into four groups, with 10 animals 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 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] Limb motor function after anterior cruciate ligament injury was evaluated by comparing the vertical jump heights of rabbits in different experimental groups. The results showed that the vertical jump heights of the experimental group were significantly greater than those in the control group (P < 0.05). The vertical jump heights of the animals in the combined magnesium lactate and zinc lactate groups were greater than those in the divalent metal magnesium lactate group (P < 0.05).

[0064] 2. Tissue Sample Acquisition

[0065] Rabbits were sacrificed by intravenous injection of an overdose of 3% sodium pentobarbital at the ear margin. The surgical area was shaved, prepared, and disinfected. The skin was incised along the patella, and 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 0.5 mL of Trizol, grind thoroughly, and centrifuge to remove the supernatant. Add 0.1 mL of chloroform, centrifuge to remove the supernatant. Add 0.8 mL of isopropanol, centrifuge to remove the supernatant. Add 1 mL of 75% ethanol, centrifuge to remove the supernatant. Add 20 μL of DEPC-treated water to dissolve the RNA. Determine the concentration and purity of the total RNA solution using a nucleic acid protein analyzer.

[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 5ˋ-GAGCTTGGAACGGTACGATA-3ˋ

[0073] Downstream primer 5-CCGTACCAGAGCGAGATGAC-3

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

[0075] Downstream primer 5-CTTTGGTCTGCATTCACATTTGT-3

[0076] Caspase-3 plays a key 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 results of real-time quantitative PCR for caspase-3 are shown in Table 4. The results showed no significant changes in caspase-3 mRNA expression in the control group at various time points. Compared with the control group, caspase-3 mRNA expression in the divalent metal lactate group was significantly downregulated on days 1, 3, and 7 after modeling (P < 0.05). Furthermore, caspase-3 mRNA levels in the divalent metal lactate group gradually decreased over time within 7 days after modeling (P < 0.05). Compared with the magnesium lactate and zinc lactate groups, caspase-3 mRNA expression in the magnesium lactate + zinc lactate group was significantly downregulated on days 1, 3, and 7 (P < 0.05). These results suggest that divalent metal lactates can reduce Caspase-3 protein expression in animals following ligament injury and mitigate apoptosis caused by nerve damage. The combined use of magnesium lactate and zinc lactate demonstrated a synergistic effect in reducing Caspase-3 protein expression.

[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] Forty-two 200g Sprague-Dawley rats (6 per group) were anesthetized and shaved on the back. A 5mm deep, 3cm long incision was made on the back with a scalpel. The control group was smeared with 0.5mL of normal saline, while the wounds of the experimental groups were smeared 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, or 25mmol / L zinc lactate aqueous solution plus 25mmol / L magnesium lactate aqueous solution. Starting from the day of modeling, each animal was smeared once in the morning and evening, 0.5mL each time, for 14 days. Wound healing rates were recorded on days 3, 7, and 14 (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 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 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 combined 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 used for the experiment after 7 days of adaptive feeding with no obvious abnormalities.

[0090] Scar model establishment: The rabbits were anesthetized by intravenous injection of 30 g / L pentobarbital solution at the ear margin. Circular wounds with a diameter of 1 cm were made along the long axis of the ventral side of the rabbit ears. Two wounds were made on each ear, approximately 3.0 cm apart. The full-thickness skin was completely removed to form the wound.

[0091] Experimental Grouping and Dosing: Thirty New Zealand white rabbits were randomly divided into four groups, each consisting of six animals, for a total of 24 wounds. These groups included a control group, a 50 mmol / L zinc lactate aqueous solution group, a 50 mmol / L magnesium lactate aqueous solution group, and a 25 mmol / L zinc lactate aqueous solution plus a 25 mmol / L magnesium lactate aqueous solution group. After wound initiation, the control group received 0.5 mL of normal saline applied to the wound surface once daily, morning and evening. In the experimental groups, each animal received 0.5 mL of the corresponding lactate aqueous solution applied to each wound surface, morning and evening. Evaluation was performed on the 13th day.

[0092] Efficacy evaluation: The Vancouver Scar Scale (VSS) was used to evaluate the efficacy of the treatment. 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 software. 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 in New Zealand white rabbit ears

[0099]

[0100]

[0101] The results on day 13 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 wound scar formation. In addition, the VSS scores of the group treated with a combination of zinc lactate aqueous solution and magnesium lactate aqueous solution were significantly different from those of the zinc lactate group and the magnesium lactate group (P<0.01), indicating that the combination group unexpectedly showed a synergistic effect in preventing wound scar formation.

[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 those of the above magnesium lactate, and will not be repeated here.

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

Claims

1. Use of a divalent metal lactate in the preparation of a product for promoting skin wound healing, characterized in that: The divalent metal lactate is selected from a composition of zinc lactate and magnesium lactate, and in the composition, the ratio of the zinc lactate to the magnesium lactate is 1:1, and the product is selected from one or more of a medicine, a kit or a medical device, and the medical device is selected from one or more of a bandage, a gauze, a dressing or a sponge.

2. The use according to claim 1, wherein: The concentration of the divalent metal lactate in the product is 15-250 mmol / L.

3. The use according to claim 2, characterized in that: The content of the divalent metal lactate in the skin is 30-150 mmol / L.

4. The use according to claim 1, characterized in that: In the medicine, divalent metal lactate is an active ingredient, and the medicine further comprises a pharmaceutically acceptable carrier or excipient.

5. The use according to claim 4, characterized in that: The dosage form of the medicine is an external preparation.

6. The use according to claim 5, characterized in that: The external preparation is selected from one or more combinations of ointments, patches, sprays, solutions or lotions.

7. The use according to claim 1, characterized in that: The administration mode of the product is application.

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