Mitochondrial-targeted drug dr20 and efficacy thereof in treating pulmonary arterial hypertension, pulmonary fibrosis, and lung cancer

Abstract

The present invention relates to the field of medicines, and specifically relates to a compound, a composition, and uses thereof. The provided compound can effectively inhibit diseases related to mitochondrial dysfunction and the pathological progression thereof, and promote the restoration of cellular function. Currently common mitochondrial dysfunction diseases comprise pulmonary arterial hypertension, pulmonary fibrosis, and non-small cell lung cancer.

Description

Mitochondrial-targeted drug DR20 and its efficacy in treating pulmonary hypertension, pulmonary fibrosis, and lung cancer. Technical Field

[0001] This invention relates to a mitochondrial-targeting drug for the treatment of pulmonary hypertension, pulmonary fibrosis, and lung cancer with mitochondrial dysfunction. Background Technology

[0002] Over the past three decades, significant advances in mitochondrial biology and clinical research have revealed a wide range of diseases closely linked to mitochondrial dysfunction, including cardiovascular disease, neurodegeneration, metabolic syndrome, and cancer. The development of mitochondrial-targeted therapies has become a highly promising treatment approach.

[0003] Pulmonary hypertension (PH) is a fatal disease with a low survival rate. The main pathological mechanisms include mitochondrial dysfunction in pulmonary vascular smooth muscle cells and inappropriate vascular remodeling, which further leads to right ventricular failure and patient death. Current clinical treatment focuses on symptomatic relief, inhibiting inappropriate smooth muscle proliferation, and maintaining right ventricular function to reduce mortality. However, current clinical practice has not been able to effectively inhibit disease progression and improve patient survival. To prevent the pulmonary artery vascular smooth muscle from transitioning from a contractile to a secretory state, thereby improving pulmonary artery function, preventing inappropriate remodeling, and further reducing the progression of right ventricular failure caused by pulmonary hypertension, targeting vascular smooth muscle mitochondria to further enhance their function is a promising therapeutic approach.

[0004] Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease caused by unexplained, persistent inflammation and repair of the lungs, leading to scarring (fibrosis). This disease involves dysfunction of alveolar respiratory epithelial cells, persistent inflammation, and increased activity of fibroblasts and myofibroblasts, resulting in the accumulation of large amounts of fiber in the pulmonary interstitium, compressing alveolar space, hardening of the lung parenchyma, and deterioration of lung function. Ultimately, patients die from respiratory failure. Currently, the drugs used clinically in China to treat IPF are pyrfenidone and nitedanib. These two drugs have different mechanisms of action, and while they can slow the deterioration of lung function in some patients, they cannot cure or inhibit the progression of fibrosis, and patients ultimately die from respiratory failure.

[0005] Lung cancer is the leading cause of death in China. Current treatment methods include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, lung cancer patients are often diagnosed at an advanced stage, at which point cancer cells have frequently metastasized and become highly invasive. Despite the development and application of numerous targeted therapies in recent years, improvements in lung cancer survival rates remain limited and require further advancement. Mitochondrial glycolysis has long been considered one of the main ways cancer cells generate energy in response to environmental metabolism. Furthermore, mitochondria play a crucial role in tumor cells in redox reactions, regulating calcium homeostasis, participating in transcriptional regulation, controlling cell death, and developing drug resistance. Therefore, targeting and inhibiting the function of cancer cell mitochondria is one of the most effective directions for clinical cancer treatment.

[0006] In recent years, strategies targeting mitochondrial dysfunction have emerged and demonstrated their feasibility in preclinical trials for various diseases. However, the lengthy drug development process often requires significant investment of manpower, money, and time. Addressing the remaining challenges in drug development, this invention utilizes magnolol, a traditional Chinese medicine known for its diverse biological effects and mitochondrial function inhibition, and modifies it into a novel drug with high lipophilicity that can target mitochondria: Dr20. To verify the efficacy of Dr20 in treating pulmonary hypertension, pulmonary fibrosis, and lung cancer after mitochondrial targeting, animal models and cell experiments were conducted for different diseases. Summary of the Invention

[0007] Magnoliol (MAG), an important active ingredient isolated from *Houpoea officinalis*, is a traditional Chinese medicine with few side effects and various biological benefits, including anti-inflammatory, antimicrobial, antioxidant, neuroprotective, cardiovascular protective, metabolic regulation, antitumor, antifibrotic, and mitochondrial function regulation. This invention utilizes magnoliol for synthetic modification to produce a mitochondrial-targeting derivative, "Dr20". This derivative possesses the advantages of retaining the original drug's pharmacological properties, requiring less development time, having relatively low cost, and better mitochondrial targeting ability. The structural formula of Dr20 of this invention is as follows:

[0008] The natural compound magnolol was modified, where R1 and R2 are not both hydrogen atoms. R1 or R2 may be composed of 1-12 carbon atoms (n=1-12) connected to the end of benzyltriphenylphosphonium bromide, forming the Dr20 structure. Cellular or animal experiments were then conducted using Dr20 to confirm its efficacy.

[0009] This invention confirms that Dr20 can effectively promote the function of mitochondria in pulmonary vascular smooth muscle, prevent smooth muscle from transforming into a secretory form and cell proliferation, reduce the occurrence of vascular atrophy and plexiformization, and inhibit inappropriate pathological remodeling of pulmonary vessels.

[0010] This invention confirms that Dr20 can effectively inhibit mitochondrial function of myofibroblasts, inhibit collagen proliferation and accumulation, and promote the degradation of improper extracellular matrix in the lungs, thereby further inhibiting the progression of fibrosis and preventing the deterioration of respiratory function.

[0011] This invention confirms that Dr20 can effectively inhibit the function of tumor mitochondrial metabolism and respiratory chain, and further inhibit tumor growth and lymph node invasion and metastasis.

[0012] Therefore, Dr20 has certain value and potential in the treatment of pulmonary hypertension, idiopathic pulmonary fibrosis, and end-stage or drug-resistant lung cancer.

[0013] This invention provides a compound with the following structural formula: Where R1 is H or C 1-12 -Benzyltriphenylphosphonium bromide; R2 is H or C 1-12 -Benzyltriphenylphosphonium bromide; where R1 is H, then R2 is C. 1-12 -Benzyltriphenylphosphonium bromide; and wherein when R2 is H, then R1 is C. 1-12 -Benzyltriphenylphosphonium bromide.

[0014] In a preferred embodiment, when R1 is H, R2 is C6-benzyltriphenylphosphonium bromide.

[0015] In a preferred embodiment, when R2 is H, R1 is C6-benzyltriphenylphosphonium bromide.

[0016] The present invention further provides a pharmaceutical composition comprising a therapeutically effective dose of the composition, the composition comprising: a compound of formula (I) and a pharmaceutically acceptable carrier thereof, wherein the pharmaceutically acceptable carrier is selected from approved pharmaceutical or clinical candidate pharmaceuticals suitable for treating the following diseases: cancer, cardiovascular disease, neurodegenerative disease, metabolic disease, pulmonary hypertension, pulmonary fibrosis, or lung disease.

[0017] In one embodiment, the effective dose of the composition is 2-50 mg / kg. In a preferred embodiment, the effective dose of the composition is 2.5-5 mg / kg.

[0018] In one embodiment, the composition is administered in the form of an intravenous, oral, or inhaled formulation.

[0019] The present invention further provides the use of the aforementioned compound in the preparation of a medicament for treating mitochondrial dysfunction, wherein the mitochondrial dysfunction includes pulmonary hypertension, pulmonary fibrosis, and non-small cell lung cancer.

[0020] In one embodiment, the effective dose of the drug is 2-50 mg / kg.

[0021] In one embodiment, the drug is administered via intravenous injection, oral administration, or inhalation.

[0022] In one embodiment, the compound is used in combination with one or more drugs suitable for treating mitochondrial dysfunction to enhance the therapeutic effect, wherein the drug for treating mitochondrial dysfunction includes pyruvate and coenzyme Q. 10 (Coenzyme Q 10 L-carnitine, nucleotide supplements, alpha-lipoic acid, vitamins E and C, steroids, minocycline, or NAD+ + Supplements.

[0023] In one embodiment, the compound of formula (I) is used to treat pulmonary hypertension in mitochondrial dysfunction, characterized in that an effective dose of the compound of formula (I) is administered to inhibit pathological remodeling of vascular smooth muscle cells, prevent smooth muscle from transforming into a secretory form, thereby reducing pulmonary hypertension symptoms and improving lung function.

[0024] In another embodiment, the compound of formula (I) is used to treat pulmonary fibrosis in mitochondrial dysfunction, characterized by administering an effective dose of the compound of formula (I) to inhibit collagen proliferation and extracellular matrix accumulation, maintain the structure of alveoli and respiratory membranes, thereby slowing the progression of fibrosis and the occurrence of respiratory failure.

[0025] In another embodiment, the compound of formula (I) is used to treat non-small cell lung cancer with mitochondrial dysfunction, characterized by administering an effective dose of the compound of formula (I) to inhibit mitochondrial metabolism and respiratory chain function of tumor cells, thereby further inhibiting tumor growth, promoting cancer cell death, and inhibiting lymph node metastasis.

[0026] In another embodiment, the effective dose is 2-50 mg / kg. In a preferred embodiment, the effective dose is 2.5-5 mg / kg.

[0027] In another embodiment, the compound of formula (I) is administered in a form selected from intravenous, oral, or inhaled formulations.

[0028] In another embodiment, the compound of formula (I) is administered in combination with one or more drugs suitable for treating mitochondrial dysfunction to enhance the therapeutic effect, wherein the drug for treating mitochondrial dysfunction includes, but is not limited to, pyruvate and coenzyme Q. 10 (Coenzyme Q 10 L-carnitine, nucleotide supplements, alpha-lipoic acid, vitamins E and C, steroids, minocycline, or NAD+ + Supplements.

[0029] The present invention provides the use of the aforementioned compounds and pharmaceutically acceptable salts thereof for the manufacture of remedies for treating cancer, cardiovascular diseases, neurodegenerative diseases, metabolic diseases, pulmonary hypertension, pulmonary fibrosis, or lung diseases.

[0030] In one embodiment, the use is for manufacturing a medicament to treat mitochondrial dysfunction or other related diseases or conditions. Attached Figure Description

[0031] Figure 1 shows the compound structure diagram of Dr20 and its verification by nuclear magnetic resonance (^1H-NMR spectrum). Figure 1A shows the compound structure diagram of Dr20. Figure 1B shows the NMR verification results of Dr20.

[0032] Figure 2 shows the effects of Dr20 on the morphological and weight changes of the lungs and liver in mice with pulmonary arterial hypertension (PAH). Figure 2A shows the appearance of the mouse lungs. Figure 2B shows the mouse lung images and histological analysis results obtained by hematoxylin and eosin (H&E) staining. Figures 2C to 2F show the mouse body weight (BW), lung weight (LW), lung weight to body weight ratio (LW / BW), and liver weight, respectively. In the figures, ns indicates no significant difference; compared with the control group, * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0033] Figure 3 shows the correlation between the expression level of the anti-fibrotic microRNA miR-29a-3p in the serum of patients with pulmonary hypertension and clinical cardiac function-related parameters. Figure 3A shows the expression levels of miR-29a-3p in 53 disease control patients and 95 patients with pulmonary hypertension (PAH). In addition, in 75 patients with pulmonary hypertension, the following were assessed: (Fig. 3B) mean pulmonary artery pressure (mPAP), (Fig. 3C) pulmonary vascular resistance (PVR), (Fig. 3D) right atrial (RA) size, (Fig. 3E) pulmonary capillary wedge pressure (PCWP), (Fig. 3G) left ventricle (LV) type D, (Fig. 3H) tricuspid annular systolic displacement (TASPE), (Fig. 3I) right ventricular (RV) size, and (Fig. 3J) pulmonary artery (PA) level.

[0034] Figure 4 shows that Dr20 enhances miR-29a-3p expression and regulates mitochondrial function in human pulmonary artery smooth muscle cells (HPASMCs). Figures 4A and 4B show the effects of Dr20 on mouse lung samples in a pulmonary hypertension animal model, analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization. The results show that Dr20 significantly promotes miR-29a-3p expression, which is confirmed by in situ hybridization analysis; miR-29a-3p is indicated in purple-red, and the cell nucleus is indicated in blue. Figure 4C shows the expression of mitochondrial respiratory chain proteins in mouse lung tissue samples from a pulmonary hypertension animal model using Western blot. Figure 4D simulates the pulmonary hypertension scenario in the cell model using cobalt chloride (CoCl2), and the oxygen consumption rate (OCR) of HPASMCs is measured using a Seahorse XFp analyzer. Figure 4E shows HPASMC cells transfected with either miR-29a-3p mimic or inhibitor, and the results were analyzed by RT-qPCR. Figure 4F assesses the effect of transfected miR-29a-3p mimic or inhibitor on mitochondrial function in HPASMCs, using a Seahorse XFp analyzer to measure oxygen consumption rate (OCR). In comparison to the control group, ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0035] Figure 5 shows the changes in HPASMC phenotype regulated by Dr20 in pulmonary arterial hypertension (PAH). Desmin serves as a marker of the contractile phenotype in smooth muscle cells, while osteopontin (OPN) serves as a marker of the secretory phenotype. To investigate the inhibitory effect of Dr20 on the monocrotaline (MCT)-induced transition of smooth muscle cells from the contractile to the secretory phenotype, Figures 5A and 5B show the results of real-time quantitative polymerase chain reaction (RT-qPCR). Figures 5C and 5D show the results of Western blot analysis. Figure 5E shows the results of immunostaining combined with confocal laser microscopy and Imaris 3D / 4D analysis. Compared with the control group, ns indicates no significant difference; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0036] Figure 6 shows that Dr20 inhibits collagen production and promotes fibrosis in lung tissue. Figures 6A and 6B show the mRNA expression of type I collagen (Collagen I) and type III collagen (Collagen III) analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Figures 6C and 6D show the protein expression of type I and type III collagen confirmed by Western blot. Figure 6E shows the results of immunostaining combined with confocal laser microscopy and Imaris 3D / 4D analysis of type I and type III collagen expression. Figures 6F and 6G show the mRNA expression levels of matrix metalloproteinase 19 (MMP19) and inhibitor of metalloproteinase 3 (TIMP3) analyzed by RT-qPCR, respectively. Figures 6H and 6I show the protein expression of MMP19 and TIMP3 detected by Western blot. Figure 6J shows the results of immunostaining combined with confocal laser microscopy and Imaris 3D / 4D analysis to confirm the differences between MMP19 and TIMP3. Compared with the control group, ns indicates no significant difference; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0037] Figure 7 shows that Dr20 inhibits pathological pulmonary vascular remodeling induced by monocrotaline (MCT). Figure 7A uses Masson's trichrome staining to detect the distribution of pulmonary vessels and collagen. Figure 7B uses micro-computed tomography (Micro-CT) of mouse lungs to confirm the effect of Dr20 on MCT-induced pathological changes in the lungs and blood vessels. Figure 7C shows that hematoxylin and eosin (H&E) staining reduces MCT-induced pulmonary artery smooth muscle hypertrophy, plexiform lesions, vascular occlusion, and intimal hyperplasia.

[0038] Figure 8 shows that Dr20 alleviates MCT-induced pulmonary fibrosis. To analyze the effect of Dr20 on MCT-induced pulmonary fibrosis, the following methods were used: Figure 8A was analyzed using transmission electron microscopy (TEM). Figure 8B was analyzed using second-harmonic generation (SHG). Figure 8C was measured using hydroxyproline. Figure 8D was detected using Verhoeff–Van Gieson Stain (VVG) to detect reticular fiber expression. Figures 8E and 8F were assessed using Massen trichrome staining and Picrosirius Red Staining, respectively, to evaluate type I collagen expression.

[0039] Figure 9 shows the histopathological changes and lung injury effects of Dr20 on C57BL / 6 mice after BLM exposure. Figure 9A shows the chemical structure of Dr20 and the animal experimental design. Figures 9B and 9C show the mouse body weight (BW) and the lung weight to body weight ratio (LW / BW). Figure 9D shows the external image of the mouse lungs. Figures 9E and 9F show the whole lung sections stained with hematoxylin and eosin (H&E) and analyzed according to the Ashcroft Score. *p indicates no significant difference compared to the control group; *p indicates <0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0040] Figure 10 shows that Dr20 inhibits apoptosis and promotes regeneration of type I alveolar epithelial cells (ATI). Analysis was performed using immunostaining, confocal laser microscopy, and Imaris 3D / 4D imaging. Figures 10A and 10B show that Dr20 inhibits the expression of cleaved Caspase-3 in ATI cells (with the specific protein marker: ZO-1) and type II alveolar epithelial cells (ATII) cells (with the specific protein marker: SP-C). Figures 10C and 10D show that Dr20 increases the expression of pro-differentiation factors YAP1 and TAZ in ATII cells (with the specific protein marker: SP-C), thus promoting the differentiation of ATII cells into ATI cells. Figures 10E and 10F show that Dr20 regulates alveolar ATI cell regeneration, promotes increased E-cadherin expression in ATI cells (with the specific protein marker: ZO-1), and promotes ATII cell proliferation (with the specific protein markers: SP-C and SP-D).

[0041] Figure 11 shows that Dr20 promotes miR-29a-3p expression in lung tissue while inhibiting mitochondrial function in fibroblasts. Figures 11A and 11B were obtained through in situ hybridization and RT-qPCR. The results showed that Dr20 increased the expression level of miR-29a-3p in vivo. Purple-red: miR-29a-3p and U6 (RNU6-1, internal miRNA control); blue: cell nucleus. Figure 11C shows MRC5 cells transfected with miR-29a-3p mimics and inhibitors, respectively, and detected by RT-qPCR. Compared with the control group, ns indicates no significant difference; *p indicates <0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001. Figures 11D to 11F show the effects of Dr20 and miR-29a-3p on mitochondrial function in fibroblasts using a simulated pulmonary fibrosis model with TGF-β1 as a stimulant, and the analysis of their oxygen consumption rate (OCR).

[0042] Figure 12 shows that Dr20 and miR-29a-3p inhibit myofibroblast function while increasing the expression of MMP19 and TIMP3. Figures 12A and 12B show the expression of α-SMA, FN1, MMP19, and TIMP3 using RT-qPCR, immunostaining, confocal laser microscopy, and Imaris 3D / 4D analysis. Figure 12C shows that Dr20 increases the expression of cleavage-type Caspase-3 in lung fibroblasts (specific marker: α-SMA), indicating promotion of cell death, confirmed by immunostaining, confocal laser microscopy, and Imaris 3D / 4D analysis. Figures 12D and 12E show that Dr20 regulates the expression of α-SMA, FN1, MMP19, and TIMP3, detected by RT-qPCR and Western blotting. Compared with the control group, ns indicates no significant difference; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0043] Figure 13 shows that Dr20 alleviates bleomycin (BLM)-induced pulmonary fibrosis. Figure 13A is a micro-computed tomography (Micro-CT) image of the lungs of BLM-induced C57BL / 6J mice. Figure 13B shows changes in lung structure observed using H&E staining. Figure 13C confirms the expression of elastic and collagen fibers using Verhoeff-Van Gieson (VVG) staining. Figure 13D is an image of collagen fiber distribution using Massen trichrome staining. Figure 13E is an image of collagen fiber distribution using Picro-Sirius Red Stain staining. Figure 13F analyzes the distribution, aggregation, and deposition of collagen fibers in the lung interstitium using transmission electron microscopy (TEM). Figure 13G uses hydroxyproline enzyme immunoassay to analyze collagen expression. Figure 13H analyzes the expression of collagen I mRNA by RT-qPCR. Figure 13I shows the protein expression of collagen I compared to the control group using Western blotting. ns indicates no significant difference; * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.

[0044] Figure 14 shows the effect of Dr20 on inhibiting tumor growth and lymph node metastasis in non-small cell lung cancer (NSCLC). Figure 14A shows the appearance of mice in the athymic mouse xenograft model of NSCLC. Figure 14B shows the tumor appearance. Figure 14B is a statistical graph of tumor weight inhibition by Dr20. Figures 14C to 14E show the mean tumor volume, tumor weight, and tumor weight-to-body weight ratio of the mice, respectively. Compared with the control group, **** indicates p<0.0001. Figure 14F shows the image of tumor metastasis to the inguinal lymph nodes in mice. Figure 14G shows the detection of tumor cell metastasis to lymph nodes using Ki67 staining.

[0045] Figure 15 shows that Dr20 targets lung cancer cell mitochondria and promotes mitochondrial damage. Figure 15A shows the gold (Au) labeled structures of MAG and Dr20. Figure 15B uses fluorescence microscopy to analyze immunofluorescence staining to confirm the location of mitochondria. The cell membrane is stained green with wheat germ lectin (WGA), the mitochondria are stained red with mitochondrial tracking dye, and the cell nucleus is stained blue with DAPI staining. Figure 15C uses soft X-ray tomography to observe the distribution of gold atoms (Au) (indicated by blue arrows and circles) and the morphology and damage of mitochondria (indicated by red arrows and circles).

[0046] Figure 16 shows the effects of Dr20 on mitochondrial dysfunction in vitro. The following parameters were measured in A549 cells (human non-small cell lung cancer cell line): mitochondrial oxygen consumption rate (OCR), basal respiration, proton leak, maximum respiration, spare respiratory capacity, non-mitochondrial oxygen consumption, coupling efficiency (%), ATP production, and spare respiratory capacity (%). Detailed Implementation

[0047] The following is a description of the method of the present invention. Although certain specific embodiments are referenced in this description, they are for illustrative purposes only and should not be construed as limiting the invention to these specific embodiments. Rather, the spirit and scope of the invention are intended to include alternatives, modifications, and equivalents. Therefore, this specification and drawings should be regarded as illustrative rather than limiting.

[0048] This description provides numerous details of implementation to aid in a thorough understanding of the invention. However, those skilled in the art can implement the invention without these specific details. In some cases, methods, operations, and materials already known to those skilled in the art have not been described in detail to avoid obscuring the essential features of the invention.

[0049] The following examples are non-limiting and only represent various aspects and features of the present invention.

[0050] Example 1: Preparation of Dr20

[0051] The preparation process of Dr20 of the present invention includes the following steps:

[0052] To effectively enhance the effects of the original natural compound, magnolol (MAG), on mitochondria, this embodiment modifies the structure of natural MAG by introducing triphenylphosphonium (TPP) into its molecular structure. + The modification introduces a positive charge and enhances the lipophilicity of the compound, allowing it to rapidly penetrate cell and mitochondrial membranes and eventually accumulate in the mitochondrial matrix, forming the mitochondrial-directing compound Dr20, as shown in Figure 1A.

[0053] In the structure of Dr20, R1 or R2 can be linked to benzyl-triphenylphosphonium bromide via an alkyl chain containing 1 to 12 carbon atoms (n = 1–12).

[0054] The synthesis of Dr20 is simple, stable, quick, and cost-effective. Compared with the original natural product, the modified Dr20 has several advantages, including better mitochondrial targeting, higher cell penetration efficiency, and more stable chemical properties.

[0055] Further, using nuclear magnetic resonance (NMR)... 1 As shown in Figure 1B, the NMR spectrum of Dr20 exhibits representative proton signals, including aromatic hydrogen atoms (δ 6.8–8.0 ppm), allyl hydrogen atoms (δ 5.0–6.0 ppm), and aliphatic hydrogen atoms (δ 1.0–4.0 ppm). These signals are consistent with the predicted chemical structure, confirming that the magnolol structural unit has been successfully conjugated with the triphenylphosphine cation via an alkoxy linkage. Furthermore, mass spectrometry analysis also confirms the correct molecular weight of Dr20.

[0056] Example 2: Establishment of MCT-induced pulmonary hypertension model and preliminary evaluation of Dr20 treatment

[0057] C57BL / 6J mice were injected intratracheally with monocrotaline (MCT) (60 mg / kg / time) to induce pulmonary hypertension. Twenty-four hours later, the mice were randomly assigned to receive Dr20 treatment: a low-dose group (intraperitoneal injection, 2.5 mg / kg, three times a week) and a high-dose group (IP, 5 mg / kg, three times a week). Treatment lasted for 28 days, and body weight changes were recorded.

[0058] Subsequent embodiments will conduct imaging and tissue analyses on the MCT-induced mouse model to explore the effect of Dr20 on MCT-induced pulmonary hypertension and further investigate its detailed mechanism.

[0059] Example 3: Effects of Dr20 on changes in lung appearance and weight in mice after MCT exposure

[0060] Lungs were harvested from mice after sacrifice for observation: Dr20 significantly reduced lung swelling and ecchymosis caused by MCT (Fig. 2A) without affecting liver morphology or histological structure (Fig. 2B). Furthermore, Dr20 effectively prevented MCT-induced weight loss in mice (Fig. 2C), while inhibiting the increase in lung weight (Fig. 2D) and the lung weight-to-body weight ratio (LW / BW ratio) (Fig. 2E). Dr20 had no significant effect on liver weight changes, further confirming that Dr20 is primarily metabolized by the liver but does not cause morphological changes in the liver (Fig. 2F).

[0061] Example 4: Relationship between serum miR-29a-3p expression level and clinical cardiac function-related parameters

[0062] This embodiment analyzes the relationship between serum miR-29a-3p expression levels and mean pulmonary artery pressure (mPAP), pulmonary vascular resistance (PVR), right atrial (RA) size, pulmonary capillary wedge pressure (PCWP), type D left ventricle (LV), tricuspid annular systolic displacement (TAPSE), right ventricular systolic velocity (RV`S), and pulmonary artery (PA) in healthy controls (NR) and patients with pulmonary hypertension (PAH), and Spearman correlation analysis is used. The results showed that the expression level of miR-29a-3p in the serum of PAH patients was significantly lower than that in the normal control group (p<0.001, Figure 3C). Further analysis showed that the expression level of serum miR-29a-3p in PAH patients was negatively correlated with mPAP (p=0.0033, Figure 3D), PVR (p=0.0096, Figure 3E), RA size (p=0.0037, Figure 3F), and PA (p=0.0165, Figure 3K), while it was positively correlated with PCWP (p=0.0017, Figure 3G), TAPSE (p=0.0004, Figure 3I), and RV'S. Meanwhile, PAH patients with low serum miR-29a-3p expression were more likely to develop type D left ventricle (p=0.0189, Figure 3H). Therefore, the results of this embodiment show that plasma miR-29a-3p level can serve as a potential indicator for predicting cardiac function and prognosis in PAH patients.

[0063] Example 5: Dr20 regulates miR-29a-3p expression and enhances mitochondrial dysfunction caused by pulmonary hypertension.

[0064] As shown in Figures 4A and 4B, Dr20 significantly promotes miR-29a-3p expression, preventing the MCT-induced decrease in miR-29a-3p expression. Simultaneously, as shown in Figure 4C, Dr20 effectively prevents the MCT-induced decrease in mitochondrial respiratory chain protein expression.

[0065] To investigate the effects of Dr20 and miR-29a-3p on mitochondrial function in pulmonary vascular smooth muscle cells (HPASMCs), this study analyzed oxygen consumption rate (OCR). The results, shown in Figure 4D, indicate that Dr20 significantly enhances mitochondrial function in HPASMCs. Furthermore, as shown in Figures 4E and 4F, increasing miR-29a-3p expression effectively mitigated the decrease in mitochondrial function in HPASMCs treated with cobalt chloride (COCl2, mimicking the in vitro PAH state); conversely, inhibiting miR-29a-3p expression exacerbated the COCl2-induced decrease in mitochondrial function.

[0066] Example 6: Dr20 regulates the phenotype changes of pulmonary vascular smooth muscle caused by pulmonary hypertension

[0067] Normal pulmonary vascular smooth muscle exhibits a contractile phenotype, which is crucial for regulating the balance between proliferation and apoptosis and maintaining the normal integrity of pulmonary vascular structure and function. However, in PAH (pulmonary angina), pulmonary vascular smooth muscle transforms into a secretory phenotype. This change leads to excessive proliferation of pulmonary vascular smooth muscle while inhibiting apoptosis, ultimately resulting in increased thickness of the vascular media and pathological vascular remodeling (including reduced vessel number, decreased and narrowed vascular branches, endothelial proliferation, vascular plexiformation, vascular occlusion, and increased vascular rigidity).

[0068] To determine whether Dr20 can inhibit MCT-induced conversion of pulmonary vascular smooth muscle to a secretory phenotype, the expression of the contractile marker desmin and the secretory-associated osteopontin (OPN) was examined. In Figure 5, Dr20 inhibited MCT-induced OPN mRNA and protein expression but preserved desmin expression. These results indicate that Dr20 can inhibit the MCT-induced conversion of pulmonary vascular smooth muscle from a contractile to a secretory phenotype in vivo.

[0069] Example 7: Dr20 inhibits collagen production in pulmonary blood vessels and promotes fibrosis degradation

[0070] MCT induces the production and accumulation of type I and type III collagen in pulmonary vessels and surrounding lung tissue. As shown in Figures 6A to 6E, Dr20 significantly reduces the production and deposition of type I and type III collagen induced by MCT. MMP19 acts as a digestive enzyme for collagen components, while TIMP3 has the ability to degrade the extracellular matrix (ECM).

[0071] To clarify the degradation effect of Dr20 on pre-existing collagen and matrix, the expression of MMP19 and TIMP3 was detected in this embodiment. The results are shown in Figures 6F to 6J, indicating that Dr20 significantly increased the expression levels of MMP19 and TIMP3.

[0072] Example 8: Dr20 inhibits MCT-induced pulmonary vascular remodeling

[0073] To evaluate the effectiveness of Dr20 in reducing MCT-induced pathological vascular remodeling, this study examined pathological changes in pulmonary vascular distribution and structure. The results, shown in Figures 7A and 7B, demonstrate that Dr20 significantly prevented the reduction and atrophy of pulmonary vessels caused by MCT. Furthermore, as shown in Figure 7C, Dr20 effectively reduced and prevented MCT-induced pulmonary vascular hypertrophy, plexiform vessel morphology, vascular occlusion, and intimal hyperplasia.

[0074] Example 9: Dr20 alleviates MCT-induced secondary pulmonary fibrosis

[0075] In Figure 8, the anti-fibrotic effect of Dr20 was confirmed by analysis using transmission electron microscopy (TEM) in Figure 8A, SHG microscopy (SHG) in Figure 8B, hydroxyproline detection in Figure 8C, VVG staining in Figure 8D, Massen's trichrome staining in Figure 8E, and Picro-Sirius Red Staining in Figure 8F. Specifically, Dr20 effectively reduced the accumulation of collagen and elastic fibers in pulmonary vessels, peribronchiolar regions, and alveolar interstitial areas induced by MCT. These results demonstrate that Dr20 inhibits the formation and deposition of pulmonary collagen and elastic fibers, thereby alleviating PAH-derived pulmonary fibrosis.

[0076] Example 10: Overall assessment of Dr20 in C57BL / 6 mice after BLM exposure

[0077] As shown in Figure 9A, after the initial intratracheal injection of BLM (2 mg / mL), mice were randomly divided into two groups: those treated with Dr20 and those not treated. The results showed that Dr20 effectively prevented weight loss caused by BLM (Figure 9B), reduced the increase in the lung weight / body weight (LW / BW ratio) caused by BLM (representing pulmonary parenchymal sclerosis) (Figure 9C), and improved the appearance of lung swelling and ecchymosis (Figure 9D). Furthermore, analysis of the Ashcroft Score showed that Dr20 significantly improved the occurrence of BLM-induced lung injury (Figures 9E and 9F).

[0078] Example 11: Dr20 inhibits alveolar cell apoptosis and promotes regeneration

[0079] To assess BLM-induced apoptosis in type I (ATI) and type II alveolar epithelial cells (ATII) due to pulmonary fibrosis progression, immunostaining, confocal laser microscopy, and Imaris 3D / 4D analysis were used to analyze the expression of cleaved-caspase-3 in ATI (specific marker ZO-1) and ATII (specific marker SP-C). The results, shown in Figures 10A and 10B, indicate that Dr20 significantly reduced BLM-induced cleaved-caspase-3 expression in both ATI and ATII cells, thus decreasing apoptosis. Furthermore, as shown in Figures 10C and 10D, compared to other groups, Dr20 promoted ATII cell proliferation, primarily by upregulating the expression of YAP1 and TAZ.

[0080] As shown in Figures 10E and 10F, Dr20 was further found to significantly promote E-cadherin expression in ATI cells, indicating intact intercellular structures and tight connections. Furthermore, Dr20 effectively increased the expression of YAP1 and TAZ, thereby promoting ATII cell proliferation and the expression of surfactant-related proteins SP-C and SP-D. These results demonstrate that Dr20 can effectively promote the proliferation and differentiation of ATII cells into ATI cells, repair damaged type I alveolar epithelial cells in one step, and promote lung function.

[0081] Example 12: Dr2 regulates miR-29a-3p expression and inhibits mitochondrial function in myofibroblasts

[0082] As shown in Figures 11A and 11B, after BLM treatment, Dr20 effectively avoided the decrease in miR-29a-3p expression caused by BLM.

[0083] To clarify the effect of Dr20-induced miR-29a-3p expression on mitochondrial function in myofibroblasts, myofibroblasts were transfected with miR-29a-3p mimics and inhibitors for 48 hours, respectively, and the expression level of miR-29a-3p was detected, as shown in Figure 11C. To further investigate the effects of Dr20 and miR-29a-3p on mitochondrial function in myofibroblasts, the oxygen consumption rate (OCR) of the cells was analyzed, as shown in Figure 11D. The results, as shown in Figure 11E, indicate that Dr20 significantly inhibited mitochondrial function in myofibroblasts.

[0084] Furthermore, overexpression of miR-29a-3p can effectively attenuate TGF-β1 (in vitro simulated pulmonary fibrosis conditions)-induced mitochondrial function in myofibroblasts, while inhibition of miR-29a-3p enhances TGF-β1-induced mitochondrial function in fibroblasts, as shown in Figure 11F.

[0085] Example 13: Dr20 reduces the activation of myofibroblasts and increases the expression of extracellular matrix degradation-related genes.

[0086] To clarify the role of miR-29a-3p in inhibiting myofibroblast activation and promoting extracellular matrix (ECM) degradation, RT-qPCR, immunostaining, confocal laser microscopy, and Imaris 3D / 4D analysis were used to assess the expression levels of α-SMA, FN1, MMP19, and TIMP3. As shown in Figures 12A and 12B, increasing miR-29a-3p expression significantly reduced the expression levels of pro-fibrosis genes α-SMA and FN1, while simultaneously increasing the expression of extracellular matrix degradation-related genes MMP19 and TIMP3.

[0087] To further verify the effects of Dr20 on inhibiting myofibroblast proliferation and activation and promoting ECM degradation, the expression of α-SMA, FN1, MMP19, and TIMP3 was analyzed. As shown in Figures 12C to 12E, Dr20 significantly inhibited the expression of α-SMA and FN1, and increased the expression of MMP19 and TIMP3.

[0088] Example 14: Dr20 reduces bleomycin (BLM)-induced pulmonary fibrosis

[0089] C57BL / 6J mice were injected intratracheally with BLM (5 mg / kg / time) to induce pulmonary fibrosis. After 24 hours, the mice were divided into groups and treated with Dr20 (IP, 2.5 mg / kg, 3 times a week) for 28 days.

[0090] The anti-fibrotic effect of Dr20 in a BLM-induced pulmonary fibrosis model was analyzed using mini-CT scanning, as shown in Figure 13A. Histological analysis, as shown by H&E staining in Figure 13B and Verhoeff-Van Gieson (VVG) staining in Figure 13C, demonstrated that Dr20 effectively improved the formation of elastic and collagen fibers, lung injury, and tissue structure destruction induced by BLM.

[0091] To clarify the production and accumulation of collagen fibers in the lungs, collagen deposition analysis was performed using the Massen trichrome staining method (Figure 13D) and the Picro-Sirius Red Stain (Figure 13E). The results were then confirmed using transmission electron microscopy (TEM), as shown in Figure 13F.

[0092] The above analyses all confirm that Dr20 has a significant anti-fibrotic effect. Specifically, Dr20 can effectively reduce BLM-induced collagen deposition around the bronchioles and in the alveolar interstitium.

[0093] Further molecular mechanism analysis, using the hydroxyproline enzyme immunoassay in Figure 13G, RT-qPCR in Figure 13H, and protein immunoblotting analysis in Figure 13I, revealed that Dr20 can inhibit BLM-induced hydroxyproline production and collagen expression.

[0094] The results above indicate that Dr20 alleviates BLM-induced pulmonary fibrosis on multiple levels by inhibiting collagen production and accumulation.

[0095] Example 15: Dr20 inhibits tumor growth and lymph node metastasis in non-small cell lung cancer (NSCLC)

[0096] 3x10 via subcutaneous injection 6 A number of small cell lung cancer cell lines: H520 cells were subcutaneously introduced into athymic mice to establish a xenograft model. Mice were subsequently injected with Dr20 (IP, 2.5 mg / kg, 3 times a week). At the end of the experiment on day 28, the tumors were removed, photographed, weighed, and the results were recorded and analyzed. The photographs are shown in Figures 14A and 14B.

[0097] As shown in Figures 14C to 14E, the analysis of tumor changes in mice showed that the tumor volume, weight, and tumor weight to body weight ratio in the Dr20 treatment group were significantly lower than those in the control group (P<0.0001), indicating that Dr20 can effectively inhibit lung cancer tumor growth.

[0098] Because advanced lung cancer is highly aggressive, and to detect whether there is tumor metastasis and invasion in the inguinal lymph nodes on the same side of the tumor, this embodiment further observes whether there is tumor cell metastasis and invasion by photographing the inguinal lymph nodes at the time of animal sacrifice and staining them with Ki67. As shown in Figures 14F and 14G, Dr20 can significantly reduce the ability of lung cancer cells to metastasize and invade lymph nodes.

[0099] Example 16: Dr20 targets mitochondria and induces mitochondrial damage

[0100] To evaluate the effect of Dr20 on mitochondria, in this embodiment, MAG and Dr20 were labeled with gold atoms to track their delivery efficiency and distribution in cancer cells, as shown in Figure 15A.

[0101] To verify the mitochondrial targeting of Dr20, immunostaining was used to select cells and locate mitochondria. Soft X-ray tomography was then used to observe and reconstruct the images to analyze the distribution of gold atoms and further assess its impact on mitochondrial damage. As shown in Figures 15B and 15C, compared to unmodified MAG, Dr20 was delivered more efficiently and significantly to the outer mitochondrial membrane of cancer cells, further accumulating between the inner and outer mitochondrial membranes and promoting severe mitochondrial damage and vacuole formation.

[0102] To verify that Dr20 promotes tumor mitochondrial dysfunction, this embodiment further elucidates the inhibitory effect of Dr20 on mitochondrial energy production and metabolism using a Seahorse XFp analyzer. As shown in Figure 16, Dr20 significantly inhibited mitochondrial oxygen consumption rate (OCR), basal respiration, proton leakage, maximal respiration, reserve respiratory volume, non-mitochondrial oxygen consumption, coupling efficiency (%), ATP production, and reserve respiratory volume (%). These results indicate that Dr20 can effectively inhibit mitochondrial energy production and metabolism.

[0103] While the invention has been described and illustrated in sufficient detail to enable those skilled in the art to make and use it, various alternatives, modifications and improvements should be apparent without departing from the spirit and scope of the invention.

[0104] Those skilled in the art will readily understand that the present invention is highly suitable for achieving the above-described objects and obtaining the aforementioned objects and advantages, as well as those inherent therein. The processes and methods described above for producing them represent preferred embodiments, are exemplary, and do not limit the scope of the invention. Modifications and other uses will be apparent to those skilled in the art. These modifications are contained within the spirit of the invention and are defined by the scope of the claims.

Claims

1. A compound with the following structural formula: Where R1 is H or C 1-12 -Benzyltriphenylphosphonium bromide; R2 is H or C 1-12 -Benzyltriphenylphosphonium bromide; Where R1 is H, then R2 is C. 1-12 -Benzyltriphenylphosphonium bromide; and Where R2 is H, then R1 is C. 1-12 -Benzyltriphenylphosphonium bromide.

2. The compound according to claim 1, wherein when R1 is H, then R2 is C6-benzyltriphenylphosphonium bromide.

3. The compound according to claim 1, wherein when R2 is H, then R1 is C6-benzyltriphenylphosphonium bromide.

4. A pharmaceutical composition comprising a therapeutically effective dose of the composition, the composition comprising the compound of claim 1 and a medically acceptable carrier, wherein the medically acceptable carrier is selected from approved pharmaceutical or clinical candidate pharmaceutical products suitable for treating the following conditions: cancer, cardiovascular disease, neurodegenerative disease, metabolic disease, pulmonary hypertension, pulmonary fibrosis, or lung disease.

5. The pharmaceutical composition according to claim 4, wherein the effective dose is 2-50 mg / kg.

6. The pharmaceutical composition of claim 4, wherein the composition is administered in the form of an intravenous, oral, or inhaled formulation.

7. Use of the compound of claim 1 in the preparation of a medicament for treating mitochondrial dysfunction, wherein the mitochondrial dysfunction includes pulmonary hypertension, pulmonary fibrosis, and non-small cell lung cancer.

8. The use according to claim 7, wherein the effective dose of the drug is 2-50 mg / kg.

9. The use according to claim 7, wherein the drug is administered via intravenous injection, oral administration, or inhalation.

10. The use according to claim 7, wherein the compound is used in combination with one or more drugs suitable for treating mitochondrial dysfunction to enhance the therapeutic effect, wherein the drug for treating mitochondrial dysfunction comprises pyruvate, coenzyme Q. 10 (Coenzyme Q 10 L-carnitine, nucleotide supplements, alpha-lipoic acid, vitamins E and C, steroids, minocycline, or NAD+ + Supplements.

11. A method of treating an individual with mitochondrial dysfunction, comprising administering to the individual with mitochondrial dysfunction an effective dose of the compound of claim 1 to inhibit pathological progression associated with mitochondrial dysfunction and promote the recovery of cellular function, wherein the mitochondrial dysfunction includes pulmonary hypertension, pulmonary fibrosis, and non-small cell lung cancer.

12. The method according to claim 11, wherein the compound of claim 1 is used to treat pulmonary hypertension in mitochondrial dysfunction, characterized in that... Administering an effective dose of the compound of claim 1 inhibits pathological remodeling of vascular smooth muscle cells, prevents smooth muscle from transforming into a secretory form, thereby reducing symptoms of pulmonary hypertension and improving lung function.

13. The method according to claim 11, wherein the compound of claim 1 is used to treat pulmonary fibrosis in mitochondrial dysfunction, characterized in that... The compound of claim 1 is administered in an effective dose to inhibit collagen proliferation and extracellular matrix accumulation, maintain the structure of alveoli and respiratory membranes, thereby slowing the progression of fibrosis and preventing respiratory failure.

14. The method according to claim 11, wherein the compound of claim 1 is used to treat non-small cell lung cancer with mitochondrial dysfunction, characterized in that... The compound of claim 1, when administered in an effective dose, inhibits tumor mitochondrial metabolism and respiratory chain function, thereby further inhibiting tumor growth, promoting cancer cell death, and inhibiting lymph node metastasis, thus exhibiting antitumor effects.

15. The method according to claim 11, wherein the effective dose is 2-50 mg / kg.

16. The method of claim 11, wherein the compound of claim 1 is administered in a manner selected from intravenous, oral, or inhaled formulations.

17. The method of claim 11, wherein the compound of claim 1 is administered in combination with one or more drugs suitable for treating mitochondrial dysfunction to enhance the therapeutic effect, wherein the drug for treating mitochondrial dysfunction comprises pyruvate, coenzyme Q. 10 (Coenzyme Q 10 L-carnitine, nucleotide supplements, alpha-lipoic acid, vitamins E and C, steroids, minocycline, or NAD+ + Supplements.

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