1-O-hexadecyl-SN-glycero-3-phosphocholine for use in the treatment of skin scarring

Abstract

The application discloses application of 1-O-hexadecyl-SN-glyceryl-3-choline phosphate in preparation of a medicine for treating skin scars and belongs to the technical field of medicines. The application finds that 1-O-hexadecyl-SN-glyceryl-3-choline phosphate can inhibit excessive proliferation of fibroblasts and excessive deposition of collagen in the process of scar formation, promote normal remodeling of scar tissues, thereby reducing scar proliferation and improving the appearance of scars; the 1-O-hexadecyl-SN-glyceryl-3-choline phosphate can be used alone or in combination with other active ingredients to prepare a medicine for treating skin scars such as hypertrophic scars, keloids and atrophic scars, and provides a new, safe and effective medicine selection for the treatment of skin scars.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of 1,1-O-hexadecyl-SN-glycero-3-choline phosphate in the preparation of drugs for treating skin scars. Background Technology

[0002] Skin scars are the result of abnormal healing after skin tissue injury, characterized by excessive proliferation of fibroblasts, excessive deposition of extracellular matrix mainly composed of collagen, and pathological tissue remodeling. Scars not only affect appearance, but in severe cases can also lead to functional impairment, itching, and pain, making clinical treatment urgently needed.

[0003] Studies have shown that lysophosphatidyllipids play a crucial role in the abnormal repair process following tissue injury. Lysophosphatidic acid (LPA) can mediate the recruitment of fibroblasts to the injury site and promote vascular leakage through its G protein-coupled receptor LPA1, thereby driving the development of fibrosis; inhibiting the LPA-LPA1 signaling pathway is considered a promising anti-fibrotic strategy. Lysophosphatidylcholine (LPC) is an important class of lysophosphatidyllipids in vivo, possessing various biological activities such as anti-inflammation. However, there are currently no records of using lysophosphatidylcholine compounds in the preparation of drugs for treating skin scars.

[0004] 1-O-hexadecyl-SN-glycero-3-choline phosphate is a monoalkyl glycerophosphate choline metabolite with CAS number 52691-62-0 and molecular formula C1. 24 H 52 NO6P, with a molecular weight of 481.65 g / mol, has been reported in the literature to be associated with synaptic dysfunction in Alzheimer's disease (Ryan SD et al., Amyloid-beta42 signals tauhyperphosphorylation and compromises neuronal viability by disrupting alkylacylglycerophosphocholine metabolism. Proc Natl Acad Sci US A. 2009,106(49):20936-41.), but its application in scars has not yet been reported. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings and defects of the prior art, the present invention provides a new use for 1-O-hexadecyl-SN-glycero-3-choline phosphate, for the preparation of a drug for treating skin scars.

[0006] Technical solution: The application of 1-O-hexadecyl-SN-glycero-3-choline phosphate of the present invention in the preparation of drugs for treating skin scars.

[0007] The skin scars mentioned herein are hypertrophic scars, keloids, atrophic scars, or flat scars.

[0008] The drug is a topical preparation, which may be an ointment, cream, gel, lotion, spray, patch, or transdermal drug delivery preparation.

[0009] The drug contains 0.05 wt% to 1 wt% 1-O-hexadecyl-SN-glycero-3-choline phosphate.

[0010] The drug is an injectable preparation, which is a subcutaneous or intra-scar injection preparation.

[0011] The drug contains 0.01wt% to 0.25wt% 1-O-hexadecyl-SN-glycero-3-choline phosphate.

[0012] The drug also contains a second active ingredient, which is at least one of glucocorticoids, 5-fluorouracil, or retinoic acid drugs.

[0013] The drug also contains a pharmaceutically acceptable carrier, which includes at least one of a thickener, emulsifier, penetration enhancer, humectant, preservative, and pH adjuster.

[0014] The mechanism of action of this invention is as follows: 1-O-hexadecyl-SN-glycero-3-choline phosphate inhibits the expression level of PKM2 protein, thereby inhibiting the excessive proliferation of skin fibroblasts and excessive collagen deposition, thus exerting the effect of inhibiting scar hyperplasia.

[0015] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention discovers that 1-O-hexadecyl-SN-glycero-3-choline phosphate can be used to prepare drugs for treating skin scars. It can inhibit excessive proliferation of fibroblasts and excessive collagen deposition during scar formation, promote the normalization and remodeling of scar tissue, thereby reducing scar hyperplasia and improving scar appearance. The above effects are closely related to its inhibition of PKM2 protein expression levels. This compound has good safety and convenient administration methods. It can be used alone or in combination with other active ingredients to prepare drugs for treating hypertrophic scars, keloids, atrophic scars, and other skin scars, providing a new, safe, and effective drug option for the treatment of skin scars. Attached Figure Description

[0016] Figure 1The effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on the viability (OD value) of human hypertrophic scar dermal fibroblasts in Example 1 is shown in the figure. Figure 2 This is a flow cytometry diagram showing the effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on apoptosis of scar dermal fibroblasts in Example 2; wherein, Figure 2 In the diagram, A represents a two-dimensional dot plot of Annexin V-FITC / PI dual-stain flow cytometry. Figure 2 B in the graph represents the quantitative analysis of apoptosis. Figure 3 This is a graph showing the inhibition of collagen COL1A1 and COL1A2 protein expression in scar dermal fibroblasts and the major marker ACTA2 protein expression in myofibroblasts by 1-O-hexadecyl-SN-glycero-3-choline phosphate in Example 3. Figure 4 This is a diagram showing the inhibition of PKM2 protein expression in scar dermal fibroblasts by 1-O-hexadecyl-SN-glycero-3-choline phosphate in Example 4. Figure 5 This is a diagram illustrating the inhibition of rabbit ear scar hyperplasia by 1-O-hexadecyl-SN-glycero-3-choline phosphate in Example 5; wherein, Figure 5 In the diagram, A represents the injection flowchart; Figure 5 B in the image represents a gross observation of the scar. Figure 5 In the figure, C represents the collagen content obtained from HE staining and Masson staining. Figure 5 In the figure, D represents the quantitative analysis of the percentage of fibrotic area; Figure 5 E in the figure represents a quantitative analysis diagram of scar thickness; Figure 6 This is an immunohistochemical image showing the inhibition of PKM2 protein expression in rabbit ear scar tissue by 1-O-hexadecyl-SN-glycero-3-choline phosphate in Example 6. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the embodiments. It should be understood that the following embodiments are only for illustrative purposes and are not intended to limit the scope of protection of the present invention. Materials, reagents and instruments not specifically described in the present invention are all products that are conventionally available in the art. All equivalent substitutions or improvements made based on the substantive content of the present invention should fall within the scope of protection claimed by the present invention. The 1-O-hexadecyl-SN-glycero-3-choline phosphate used in each experimental example can be prepared according to conventional methods in the art, or can be purchased directly from commercial sources (Shanghai Yuanye Biotechnology Co., Ltd., Y38370, purity greater than 99%).

[0018] Example 1:

[0019] This example was used to investigate the effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on the proliferative activity of human hypertrophic scar dermal fibroblasts.

[0020] Primary dermal fibroblasts for hypertrophic scars were obtained from patients with hypertrophic scars treated surgically (n≥3). All tissue samples were taken during the proliferative phase and had not received other treatments. Immediately after extraction, the tissues were placed on ice. Under aseptic conditions, subcutaneous adipose tissue was removed as much as possible. After washing three times with PBS to remove residual blood and fat, the tissue was cut into soybean-sized pieces and placed epidermal-side down in six-well plates. High-glucose DMEM containing 5 mg / mL dispersin II (Sigma) was added, and the cells were digested overnight at 4°C. The next day, the epidermis was removed, and the dermis was minced and placed in 50 mL centrifuge tubes. The cells were digested with 2 mg / mL collagenase I (Sigma) in a 37°C water bath for 2 h, shaking every 30 min. After digestion, the cells were filtered through a 100 μm cell sieve, centrifuged at 1200 rpm for 4 min at room temperature, and the supernatant was discarded to collect the cells. Cells were cultured in fibroblast-specific medium FM (ScienCell), with the serum concentration increased to 10% for primary culture. They were cultured at 37℃ in a 5% CO2 incubator, and adherence was observed after 72 h. The medium was changed after adherence, and cells were passaged when the cell density reached 95% or higher. This study used proliferative scar dermal fibroblasts from passages 3 to 6. The cells were divided into a DMSO solvent control group and treatment groups with 10, 25, 50, and 100 µM compounds. Each group was treated with the corresponding concentration of 1-O-hexadecyl-SN-glycero-3-choline phosphate for 24 h. Cell viability (OD value) was measured using the CCK-8 assay. The results are shown below. Figure 1 As shown.

[0021] The results showed that, compared with the DMSO control group, cell viability was not significantly inhibited in the 10 µM and 25 µM treatment groups, but slightly increased in the 25 µM group; cell viability was significantly decreased in the 50 µM treatment group. P <0.0001), cell viability was almost completely inhibited in the 100 µM treatment group. The above results suggest that the inhibitory effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on the proliferation of scar fibroblasts is concentration-dependent, and the inhibition is significant at high concentrations (50-100 µM).

[0022] Example 2:

[0023] This example was used to investigate the effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on apoptosis of scar dermal fibroblasts.

[0024] Cell treatment was the same as in Example 1, with a DMSO control group and treatment groups at 10, 25, 50, and 100 µM. Cells were collected after treatment, and apoptosis was detected using Annexin V-FITC / PI double staining combined with flow cytometry. Early apoptosis was identified as Annexin V single-positive cells, and late apoptosis as Annexin V / PI double-positive cells. The results are as follows: Figure 2 As shown.

[0025] The results showed that, compared with the control group, the proportion of apoptotic cells in the compound-treated groups increased in a concentration-dependent manner. Specifically, the 100 µM treatment group showed a significant increase in both early and late apoptotic cell proportions, with the increase in late apoptotic cell proportion being particularly pronounced. P <0.01). The above results suggest that 1-O-hexadecyl-SN-glycero-3-choline phosphate can inhibit scar hyperplasia by inducing fibroblast apoptosis.

[0026] Example 3:

[0027] This example was used to investigate the effects of 1-O-hexadecyl-SN-glycero-3-choline phosphate on the expression of type I collagen and myofibroblast marker proteins in scar fibroblasts.

[0028] Cell treatment was the same as in Example 1. Cells from the DMSO control group and the 100 µM treatment group were used. Total protein was extracted, and the expression levels of COL1A1 (180 kDa), COL1A2 (110 kDa), and ACTA2 (α-SMA, 42 kDa) proteins were detected by Western blot. β-actin (42 kDa) was used as an internal control, and its grayscale value was quantitatively analyzed. The results are as follows. Figure 3 As shown.

[0029] The results showed that, compared with the control group, the protein expression levels of COL1A1, COL1A2, and ACTA2 were significantly downregulated in the 100 µM treatment group (COL1A1, COL1A2, ACTA2, ...). P <0.05; ACTA2, P <0.01). The above results suggest that 1-O-hexadecyl-SN-glycero-3-choline phosphate can inhibit collagen synthesis and the differentiation of fibroblasts into myofibroblasts.

[0030] Example 4:

[0031] This embodiment was used to investigate the effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on PKM2 protein expression in scar fibroblasts in order to verify its mechanism of action.

[0032] Cell treatment was the same as in Experiment 1. Cells from the DMSO control group and the 100 µM treatment group were used. Total protein was extracted, and the expression level of PKM2 (58 kDa) protein was detected by Western blot. β-actin (42 kDa) was used as an internal control, and its grayscale value was quantitatively analyzed. The results are as follows: Figure 4 As shown.

[0033] The results showed that, compared with the control group, the expression level of PKM2 protein was significantly downregulated in the 100 µM treatment group ( P <0.05). The above results suggest that the inhibitory effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on scar hyperplasia is related to its downregulation of PKM2 protein expression levels.

[0034] Example 5:

[0035] This embodiment was used to investigate the therapeutic effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on a rabbit ear scar hyperplasia animal model.

[0036] A rabbit ear scar hyperplasia model was established using male New Zealand white rabbits (approximately 2.5 kg, 4 months old). Circular full-thickness skin wounds were created on the ventral side of the rabbit ears, with 6 wounds per ear (modeling on Day 0). After successful modeling, the animals were randomly divided into a model control group and a 1-O-hexadecyl-SN-glycero-3-choline phosphate treatment group, with 4 animals in each group. From Day 14, intra-scar injections were administered weekly for a total of 4 weeks (Days 14, 21, 28, and 35). The treatment group received 100 µL of 100 µM 1-O-hexadecyl-SN-glycero-3-choline phosphate at each wound site. The model control group received an equal volume of solvent as a control. Animals were sacrificed and tissue samples were collected on Day 42. The experimental procedure is as follows: Figure 5 As shown in A in the diagram.

[0037] After drug administration, gross observation of the scars was performed, and scar tissue was collected for HE staining and Masson staining to observe histological changes and collagen deposition. Simultaneously, image analysis software was used to quantitatively analyze the percentage of fibrosis area and scar thickness. The results showed that, compared with the model control group, scar hyperplasia was significantly inhibited and the scars tended to be flatter in the drug-treated group. Figure 5 In B), collagen deposition is reduced ( Figure 5 In the treatment group (C), the percentage of fibrosis area was significantly reduced (0.63% in the model control group and 0.45% in the treatment group). P <0.01, Figure 5 (D) The scar thickness was significantly reduced (42 µm in the model control group and 28 µm in the treatment group). P <0.01, Figure 5The results suggest that 1-O-hexadecyl-SN-glycero-3-choline phosphate has a significant therapeutic effect on hypertrophic skin scars.

[0038] Example 6:

[0039] This embodiment was used to investigate the effect of 1-O-hexadecyl-SN-glycero-3-choline phosphate on the expression of PKM2 protein in rabbit ear scar tissue, and to further verify its in vivo mechanism of action.

[0040] Scar tissues from each group obtained in Example 5 were used to detect the expression of PKM2 protein using immunohistochemistry, and the percentage of PKM2-positive area was semi-quantitatively analyzed. The results are as follows: Figure 6 As shown. The results indicated that, compared with the model control group, the PKM2-positive staining in the scar tissue of the drug-treated group was significantly lighter and the percentage of positive area was significantly reduced (as shown). P <0.01). The above results suggest that 1-O-hexadecyl-SN-glycero-3-choline phosphate also exerts its inhibitory effect on scar hyperplasia in vivo by downregulating PKM2 protein expression, consistent with the in vitro results.

[0041] Example 7:

[0042] This embodiment provides an ointment containing 1-O-hexadecyl-SN-glycero-3-choline phosphate and its preparation method.

[0043] This embodiment is an illustrative example illustrating the preparation method of an ointment containing 1-O-hexadecyl-SN-glycero-3-choline phosphate. Those skilled in the art can adjust the formulation according to actual needs. Exemplarily, 0.05 to 1 part (recommended 0.1 part, based on in vitro and animal effective concentration 100 µM ≈ 0.0048 wt% and combined with topical transdermal absorption efficiency, the final formulation shall prevail) and an appropriate amount of pharmaceutically acceptable excipients are weighed, including but not limited to at least one of petrolatum, lanolin, liquid paraffin, glycerin, polyethylene glycol, thickeners, emulsifiers, penetration enhancers, humectants, preservatives, and pH adjusters. The preparation method can be carried out according to conventional processes in the art, such as heating and emulsifying the aqueous and oil phases separately, then cooling and grinding until homogeneous. The resulting ointment contains 0.05 wt% to 1 wt% 1-O-hexadecyl-SN-glycero-3-choline phosphate, preferably 0.1 wt%.

[0044] This ointment is for external skin use, applied to the scar area 1-2 times daily, with the frequency of use adjusted according to the condition. The resulting ointment can be evaluated for quality, stability, and irritation using conventional methods in the art; specific evaluation results are subject to actual testing. Those skilled in the art can also formulate 1-O-hexadecyl-SN-glycero-3-choline phosphate into creams, gels, lotions, sprays, patches, transdermal drug delivery formulations, or subcutaneous / intra-scar injection formulations, or use them in combination with other active ingredients, using conventional methods in the art.

[0045] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the present invention shall fall within the protection scope of the present invention.

Claims

1. 1-O-hexadecyl-SN-glycero-3-choline phosphate in the preparation of drugs for treating skin scars.

2. The application according to claim 1, characterized in that: The skin scars mentioned are hypertrophic scars, keloids, atrophic scars, or flat scars.

3. The application according to claim 1, characterized in that: The drug is a topical preparation, which may be an ointment, cream, gel, lotion, spray, patch, or transdermal drug delivery preparation.

4. The application according to claim 1, characterized in that: The drug is an injectable preparation, which is a preparation for subcutaneous or intra-scar injection.

5. The application according to claim 3 or 4, characterized in that: The drug also contains a second active ingredient, which is at least one of glucocorticoids, 5-fluorouracil, or retinoic acid drugs.

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