Pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition. The pharmaceutical composition comprises a sphingosine derivative and a mitochondrial quinone derivative, wherein the molar weight ratio of the sphingosine derivative to the mitochondrial quinone derivative is (2-4): 1; according to the present invention, the sphingosine derivative and the mitochondrial quinone derivative form the pharmaceutical composition, such that the immunometabolism homeostasis of the macrophages can be improved, the immune response balance can be maintained, and the wound surface immune microenvironment can be improved by improving the mitochondrial function of the macrophages so as to promote the wound surface healing.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a pharmaceutical composition and application thereof. Background Art

[0002] Wound healing is a highly coordinated dynamic process encompassing four phases: hemostasis, inflammation, proliferation, and remodeling. The transition from inflammation to proliferation is crucial for wound healing, and the effective clearance of apoptotic and necrotic cells and pathogens by macrophages is a crucial prerequisite for this process. This prevents chronic inflammation and oxidative damage, fosters collagen deposition and angiogenesis, and promotes re-epithelialization.

[0003] Improving wound macrophage function and maintaining immune response homeostasis are important approaches to accelerate wound healing. Because the wound immune microenvironment is a prerequisite for the normal function of various cell types during the healing process, healing-promoting therapies targeting the regulation of macrophage immune responses offer significant advantages. Therefore, how to regulate macrophage immune responses has become a pressing issue. Summary of the Invention

[0004] The purpose of the present invention is to provide a pharmaceutical composition and its application to improve macrophage immune metabolic homeostasis and maintain immune response balance.

[0005] The present invention adopts the following technical solution: a pharmaceutical composition comprising a sphingosine derivative and a mitochondrial quinone derivative; wherein the molar ratio of the sphingosine derivative to the mitochondrial quinone derivative is (2-4):1.

[0006] Furthermore, the structural formula of the sphingosine derivative is Wherein, R1 is OH or H2PO4, R2 is H or a fatty acid chain, and n is a positive integer.

[0007] Furthermore, the mitochondrial quinone derivative is or its quinol form; wherein n′ is a positive integer.

[0008] Furthermore, the pharmaceutical composition is a free-state drug comprising a sphingosine derivative and a mitochondrial quinone derivative.

[0009] Furthermore, it includes a drug delivery carrier that can penetrate the cell membrane, and the drug delivery carrier is loaded with the free drug.

[0010] Furthermore, the pharmaceutical composition is in the form of an external dosage form.

[0011] Another technical solution of the present invention: an application of a pharmaceutical composition, wherein the pharmaceutical composition is used to prepare a drug for promoting wound healing.

[0012] Furthermore, the pharmaceutical composition is used to reduce the concentrations of pro-inflammatory factors TNFα and IL-6 in macrophages, and to increase the concentrations of anti-inflammatory factors IL-10, TGF-β and VEGF that promotes angiogenesis.

[0013] The beneficial effects of the present invention are as follows: the present invention comprises a pharmaceutical composition composed of sphingosine derivatives and mitochondrial quinone derivatives, which can improve the immune metabolic homeostasis of macrophages, maintain the balance of immune responses, and improve the immune microenvironment of wounds by enhancing the mitochondrial function of macrophages, thereby promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram showing the effect of the pharmaceutical composition of an embodiment of the present invention on the immune response phenotype of macrophages;

[0015] Figure 2 This is a schematic diagram showing the effect of the pharmaceutical composition of an embodiment of the present invention on the phagocytic ability of macrophages;

[0016] Figure 3 This is a schematic diagram showing the effect of the pharmaceutical composition of an embodiment of the present invention on the tube-forming behavior of vascular endothelial cells by regulating the immune response phenotype of macrophages;

[0017] Figure 4 This is a schematic diagram showing the effect of the pharmaceutical composition of an embodiment of the present invention on the expression of collagen in fibroblasts by regulating the immune response phenotype of macrophages;

[0018] Figure 5 This is a schematic diagram of the mechanism analysis of the synergistic effect of the pharmaceutical composition of the embodiment of the present invention;

[0019] Figure 6 This is a schematic diagram of the healing effect of the cream containing the pharmaceutical composition according to an embodiment of the present invention applied to a diabetic mouse wound model; DETAILED DESCRIPTION

[0020] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Sphingosine-1-phosphate (S1P) is a lipid molecule with important biological activity, exerting its effects by acting on five G protein-coupled receptors, known as S1P signaling. Research in this study has shown that enhancing S1P signaling can effectively maintain macrophage immune homeostasis. The key mechanism is to improve macrophage mitochondrial function and enhance the oxidative phosphorylation metabolic pathway.

[0022] Therefore, enhancing S1P signaling can also promote wound healing by improving the immune microenvironment. However, intervention strategies that use S1P signaling as a single target are unlikely to fully leverage the advantages of immune response regulation in promoting wound healing. Therefore, exploring combination therapy strategies that can synergize with S1P signaling is of great significance. From a mechanistic perspective, mitochondrial quinones have the potential to enhance mitochondrial function in wound macrophages. Therefore, when combined with therapeutic strategies that enhance S1P signaling, they can further improve macrophage immune metabolic homeostasis, maintain immune response balance, and thus play a synergistic role in promoting wound healing.

[0023] Furthermore, S1P is chemically unstable and easily decomposes when heated, potentially destroying it during the preparation of pharmaceutical preparations for wound treatment. Furthermore, S1P is susceptible to enzymatic degradation in the wound microenvironment, posing a challenge for its application in wound healing therapy. Therefore, the present invention proposes delivering a more stable sphingosine derivative (a precursor of S1P) into cells, where it is metabolized into S1P and then released into the extracellular space via cell membrane transporters, thereby enhancing S1P signaling and overcoming the limitations of S1P's stability on its application.

[0024] The invention discloses a pharmaceutical composition comprising a sphingosine derivative and a mitochondrial quinone derivative; wherein the molar ratio of the sphingosine derivative to the mitochondrial quinone derivative is (2-4):1.

[0025] The structural formula of sphingosine derivatives is Wherein, R1 is OH or H2PO4, R2 is H or a fatty acid chain, and n is a positive integer.

[0026] Mitochondrial quinone derivatives are or its quinol form; wherein n′ is a positive integer.

[0027] The pharmaceutical composition is a free-state drug comprising a sphingosine derivative and a mitochondrial quinone derivative.

[0028] The concentration of the sphingosine derivative in the treatment system is not less than 1 μM, and the concentration of the mitochondrial quinone derivative in the treatment system is not less than 0.25 μM.

[0029] In addition, the present invention also includes a cell membrane-penetrating drug delivery vehicle loaded with the above-mentioned free drug. In other words, the pharmaceutical composition is in the form of an external dosage form, which can be one or more of a cream, gel, spray, powder, or microneedle.

[0030] The drug delivery carrier includes one or more of liposomes, polymer-based nanoparticles, lipid-based nanoparticles, and solid lipid nanoparticles. The drug delivery carrier can enable the above-mentioned compound combination to effectively penetrate the cell membrane and achieve intracellular release.

[0031] Another technical solution of the present invention is the use of a pharmaceutical composition for preparing a medicament for promoting wound healing. In the present invention, wound damage refers to skin defects and tissue damage caused by mechanical injury, burns, diabetic ulcers, radiation ulcers, etc.

[0032] Specifically, wound healing therapy involves the aforementioned drugs modulating the wound's immune microenvironment, exerting synergistic anti-inflammatory and antioxidant effects, improving pathogen and necrotic tissue clearance, promoting wound collagen deposition and angiogenesis, and thereby accelerating re-epithelialization. More specifically, the pharmaceutical composition is used to reduce the concentrations of pro-inflammatory factors TNFα and IL-6 in macrophages, while increasing the concentrations of anti-inflammatory factors IL-10 and TGF-β, as well as VEGF, which promotes angiogenesis.

[0033] Example 1:

[0034] This example shows the effect of the combination of sphingosine derivatives and mitochondrial quinone derivatives on macrophage immune response, vascular endothelial cell function and fibroblast collagen deposition.

[0035] 1. Preparation of stock solutions of the combination of sphingosine derivatives and mitochondrial quinone derivatives.

[0036] This invention uses a combination of sphingosine and mitochondrial quinone as the active ingredient. To ensure adequate cellular entry of sphingosine, liposomes are constructed to encapsulate the two compounds as a drug delivery vehicle. Specifically, soy lecithin and cholesterol are dissolved in chloroform at a mass ratio of 4:1 to create Solution A with a total lipid concentration of 20 mg / ml.

[0037] Sphingosine and mitochondrial quinone were dissolved in methanol at a mass ratio of 7:4 (molar ratio of 4:1) to prepare solution B with a total compound concentration of 8 mg / ml.

[0038] Sphingosine was dissolved in methanol to prepare solution C with a compound concentration of 5 mg / ml.

[0039] Mitochondrial quinone was dissolved in methanol to prepare solution D with a compound concentration of 3 mg / ml.

[0040] Solution E contained only methanol solvent.

[0041] Solution A was mixed with solutions B, C, D, and E at a volume ratio of 2:1 to prepare solutions F, G, H, and I. The organic solvent of solutions F, G, H, and I was removed by rotary evaporation to form thin films. Liposome mother solution J with a final sphingosine concentration of 2 mM and a final mitochondrial quinone concentration of 0.5 mM, liposome mother solution K with a final sphingosine concentration of 2 mM, liposome mother solution L with a final mitochondrial quinone concentration of 0.5 mM, and liposome mother solution M without compound encapsulation were obtained by thin film hydration method, respectively.

[0042] This example also uses a combination of sphingosine-1-phosphate and mitochondrial quinone as active ingredients, with both components prepared as free-state stock solutions. Specifically, sphingosine-1-phosphate was dissolved in methanol to produce stock solution N with a final concentration of 2 mM, and mitochondrial quinone was dissolved in dimethyl sulfoxide to produce stock solution O with a final concentration of 0.5 mM.

[0043] 2. Preparation of Macrophage Conditioned Medium.

[0044] In this study, mouse bone marrow-derived macrophages were used as an in vitro model. They were stimulated with 10 ng / ml LPS and 12 ng / ml IFN-γ for 12 hours to induce polarization of macrophages into a pro-inflammatory phenotype. Fresh culture medium was replaced with fresh medium, and the aforementioned liposome stock solutions J, K, L, and M were added at a 1:1000 dilution ratio. After a further 12 hours of culture, the macrophage culture supernatant was collected, centrifuged, and frozen for use as macrophage-conditioned medium.

[0045] The group with added liposome mother solution J was set as experimental group 1, the group with added liposome mother solution K was set as experimental group 2, the group with added liposome mother solution L was set as experimental group 3, and the group with added liposome mother solution M was set as the liposome control group.

[0046] In addition, the above-mentioned mother liquors N+O, N, O and an equal amount of methanol + dimethyl sulfoxide were added respectively after dilution at a ratio of 1:1000. After continuing to culture for 12 hours, the macrophage culture supernatant was collected, centrifuged and frozen as macrophage conditioned medium. The group with added mother liquor N+O was set as experimental group 4, the group with added mother liquor N was set as experimental group 5, the group with added mother liquor O was set as experimental group 6, and the group with added equal amount of methanol + dimethyl sulfoxide was set as a free control group. It should be noted that the "+" in Examples 1 and 2 of the present invention are all plus signs.

[0047] 3. Cytokine detection in macrophage conditioned medium.

[0048] In this embodiment, the levels of cytokines TNF-α, IL-6, IL-10, TGF-β, and VEGF in the macrophage conditioned medium were detected by ELISA. Figure 1 As shown in the figure, it is a schematic diagram of the test results. It can be seen from the figure that compared with the control group, the pro-inflammatory factors TNFα and IL-6 in the macrophage conditioned medium of experimental groups 1 and 4 were significantly decreased, and the anti-inflammatory factors IL-10, TGF-β and the angiogenesis-promoting VEGF levels were significantly increased. However, the inflammatory response level of macrophages in experimental groups 2, 3, 5 and 6 was only partially improved. This shows that the combination of the two compounds can promote the polarization of macrophages toward a pro-repair phenotype, and the combined use of the two compounds can exert a synergistic effect.

[0049] exist Figure 1 The evaluation indicators included the levels of cytokines TNF-α, IL-6, IL-4, IL-13, TGF-β, and VEGF in the macrophage conditioned medium. The data shown are mean ± standard deviation (sample number 6); Figure 1 、 Figure 4 and Figure 5 In the middle, *P<0.05, **P<0.01, ***P<0.001.

[0050] 4. Detection of macrophage phagocytic ability.

[0051] The present invention uses flow cytometry to detect the phagocytic level of fluorescent latex microspheres with a diameter of 1 μm by macrophages after the above treatment. Figure 2 As shown, the horizontal axis represents fluorescence intensity and the vertical axis represents the number of macrophages. The results show that compared with the two control groups, experimental groups 1 and 4 significantly improved the phagocytic ability of macrophages, while experimental groups 2, 3, 5, and 6 only partially improved the phagocytic ability of macrophages, indicating that the pharmaceutical composition of the present invention can effectively enhance the ability of macrophages to clear pathogens and apoptotic and necrotic cells, and the combination of the two compounds can exert a synergistic effect.

[0052] 5. Vascular endothelial cell tube formation experiment.

[0053] The vascular endothelial cells HUVEC were resuspended in the above macrophage conditioned medium and the cell density was adjusted to 10 5 / ml, seeded in a 24-well plate covered with Matrigel matrix gel, and observed the tube formation of vascular endothelial cells after 9 hours.

[0054] like Figure 3 The figure below shows the effect of different treatments on the tube-forming behavior of vascular endothelial cells. The magnification of each sub-graph is the same. The results show that compared with the control group, experimental groups 1 and 4 significantly promoted the tube-forming behavior of HUVECs, while experimental groups 2, 3, 5, and 6 only partially improved the tube-forming ability. This indicates that the combination of the two compounds can promote angiogenesis during wound healing by improving the immune response phenotype of macrophages, and the combined use of the two compounds can exert a synergistic effect.

[0055] 6. Fibroblast collagen synthesis experiment.

[0056] This example uses real-time quantitative fluorescence PCR to examine the effect of macrophage-conditioned medium on fibroblast collagen expression at the transcriptional level. Col1a1 and Col1a2 are key genes encoding type I collagen and play an important role in extracellular matrix formation. During the wound healing stage, the expression levels of these two genes are significantly upregulated upon fibroblast activation.

[0057] like Figure 4 As shown in the data, compared with the control group, experimental groups 1 and 4 had a significant promoting effect on the expression of Col1a1 and Col1a2 genes, while experimental groups 2, 3, 5, and 6 could only partially enhance the collagen deposition effect, indicating that the combination of the two compounds can promote the collagen deposition process in the wound healing process by improving the macrophage immune response phenotype, and the combined use of the two compounds can exert a synergistic effect.

[0058] 7. Study on the synergistic mechanism of the combined application of the two compounds.

[0059] like Figure 5 As shown, the present invention uses transcriptome sequencing, flow cytometry, and Seahorse oxygen consumption rate measurement to explore the synergistic mechanism of the combined use of the two drugs. The combined use of the two drugs synergistically mediates the transition of macrophages from a pro-inflammatory phenotype to a pro-repair phenotype, and the key mechanism is to enhance macrophage mitochondrial function.

[0060] like Figure 5 As shown in Figure A, compared with a single compound, the combined use of the two compounds can significantly enhance the oxidative phosphorylation metabolic pathway of macrophages. Further analysis of the synergistic mechanism, such as Figure 5 B and Figure 5 As shown in Figure C, sphingosine derivatives have an outstanding performance in increasing the mitochondrial membrane potential level of macrophages, reflecting better mitochondrial activity, but have a weak effect in increasing the number of mitochondria. The insufficient number of mitochondria limits the enhancement of the oxidative phosphorylation metabolic pathway when sphingosine derivatives are used alone; while mitochondrial quinone derivatives cause a significant increase in the number of mitochondria, which complements the effect of sphingosine derivatives. Therefore, the combined use of the two can significantly improve the level of oxidative phosphorylation metabolism, which is one of the mechanisms for the synergistic effect of the two. On the other hand, Figure 5 As shown in Figure D, the combined use of the two compounds caused an increase in the overall level of mitochondrial reactive oxygen species (mtROS) in macrophages, reflecting a better scavenging ability and the mechanism by which the two compounds exerted a synergistic effect. It should be noted that the observed phenomenon is that the drug combination containing mitochondrial quinone derivatives caused an increase in the total mtROS level in macrophages, which is different from the previous report that mitochondrial quinone derivatives act as mtROS scavengers. Figure 5As shown in Figure E, the reason is that the combined use of the two compounds causes a decrease in the level of mtROS in single mitochondria, thereby alleviating mitochondrial oxidative stress damage, and the increase in the number of mitochondria is the cause of the increase in the total mtROS level in macrophages. This finding is an innovative supplement to the mechanism of action of mitochondrial quinone derivatives, not a contradiction.

[0061] Moreover, if Figure 5 As shown in Figure F, the transcriptome sequencing results demonstrated that the combined use of the two compounds could enhance the promoting effect on the expression of genes related to mitochondrial generation and further inhibit mitochondrial autophagy, which clarified the synergistic promoting effect of the combined use of the two compounds on improving the immune metabolic homeostasis of macrophages from the gene expression level.

[0062] In summary, the above mechanisms jointly enhance mitochondrial function and metabolic homeostasis in macrophages, thereby playing a synergistic role in improving the immune response pattern of macrophages.

[0063] In addition, according to the known metabolic pathway of sphingosine, it can be determined that other sphingosine derivatives of the present invention can be metabolized into sphingosine-1-phosphate after entering cells, and have an equivalent effect to the sphingosine in this embodiment.

[0064] Example 2:

[0065] In this example, the effect of a combination of a sphingosine derivative and a mitochondrial quinone derivative in promoting diabetic wound healing was verified.

[0066] 1. Establish a diabetic mouse wound model.

[0067] The present invention uses adult male db / db mice weighing 45-50g to construct a diabetic mouse wound model. The experimental animals are kept in an SPF-grade animal room with free food and water, and a 12-hour light / dark cycle. After isoflurane inhalation anesthesia, the back hair is removed with a small electric shaver, the back skin is disinfected with iodine tincture, and deiodinated with 75% alcohol. Use a 6mm round skin punch to press vertically to the middle of the mouse's back, and rotate to remove the full-thickness skin tissue under tension-free conditions to avoid damaging the deep muscle tissue. Each mouse needs to be kept in a single cage after surgery to prevent licking and biting the wound.

[0068] 2. Preparation and application of a cream containing a combination of sphingosine derivatives and mitochondrial quinone derivatives.

[0069] Mother liquor J and mother liquor N+O from Example 1 were diluted 100-fold with sterile deionized water. An equal volume of sterile deionized water was used as a control. These dilutions were slowly added in portions to the O / W cream base at a volume ratio of 1:9. Stir continuously during incorporation and maintain the temperature at 37°C. After mixing thoroughly, the cream was slowly cooled to room temperature and used. The final concentrations of the sphingosine derivative and mitochondrial quinone derivative in the cream were 2 μM and 0.5 μM, respectively.

[0070] About 100 μl of cream was applied to the wound surface of the diabetic mice once a day, and photos were taken on days 0, 7, and 14 after surgery. Figure 6 As shown, compared with the control group, the cream containing liposomes encapsulating sphingosine and mitochondrial quinone and the cream containing free 1-phosphate sphingosine and mitochondrial quinone can significantly promote wound healing, indicating that the combination of the two compounds is effective in promoting wound healing.

[0071] In summary, the pharmaceutical compositions of the present invention can synergistically promote wound healing, improving the wound's immune microenvironment, alleviating chronic inflammation and oxidative damage, and promoting angiogenesis and collagen deposition. These two compounds possess advantages such as stable chemical properties, readily available raw materials, and low cost, demonstrating promising development prospects and application value.

Claims

1. A pharmaceutical composition, characterized in that The invention comprises a sphingosine derivative and a mitochondrial quinone derivative; wherein the molar ratio of the sphingosine derivative to the mitochondrial quinone derivative is (2-4):

1.

2. A pharmaceutical composition according to claim 1, characterized in that The structural formula of the sphingosine derivative is Wherein, R1 is OH or H2PO4, R2 is H or a fatty acid chain, and n is a positive integer.

3. A pharmaceutical composition according to claim 2, characterized in that The mitochondrial quinone derivative is or its quinol form; wherein n′ is a positive integer.

4. A pharmaceutical composition according to claim 2 or 3, characterized in that The pharmaceutical composition is a free-state drug comprising a sphingosine derivative and a mitochondrial quinone derivative.

5. A pharmaceutical composition according to claim 4, characterized in that The invention comprises a drug delivery carrier capable of penetrating cell membranes, wherein the drug delivery carrier is loaded with the free-state drug.

6. A pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of an external dosage form.

7. A use of a pharmaceutical composition, characterized in that: The pharmaceutical composition of claims 1-6 is used to prepare a medicament for promoting wound healing.

8. The use of the pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition is used to reduce the concentrations of pro-inflammatory factors TNFα and IL-6 in macrophages, and to increase the concentrations of anti-inflammatory factors IL-10 and TGF-β and VEGF that promotes angiogenesis.

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