Use of phenyllactic acid or a composition containing phenyllactic acid in the manufacture of a medicament for the relief of lung disease

Abstract

The application discloses application of phenyllactic acid or a composition containing phenyllactic acid in preparation of a drug for relieving lung diseases, wherein the lung diseases are preferably pulmonary fibrosis or acute lung injury. In an acute lung injury research model, the phenyllactic acid can effectively intervene in an inflammatory factor cascade reaction and an oxidative stress damage process, and significantly improve symptoms of acute lung injury caused by the above factors. Compared with a traditional treatment method, experiments prove that the phenyllactic acid or the composition containing the phenyllactic acid can significantly relieve symptoms of acute lung injury (ALI) of a mouse induced by lipopolysaccharide (LPS) and pulmonary fibrosis (PF) of the mouse induced by bleomycin, effectively improve pathological changes of the lung, and restore lung function.

Description

Technical Field

[0001] This invention relates to the use of DL-3-Phenyllactic acid (PA) or compositions containing DL-3-Phenyllactic acid in the preparation of drugs for relieving lung diseases, and belongs to the field of medicine and health. Background Technology

[0002] Acute lung injury (ALI) is a serious lung disease with a complex pathogenesis. It is caused by non-cardiac factors such as infection, aspiration of harmful gases, trauma, and shock, leading to a rapid deterioration of pulmonary gas exchange function. During the onset of the disease, the immune system is activated, and large amounts of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6) are released into the bloodstream. These factors chemotactically attract and activate immune cells such as neutrophils to accumulate in the lungs. The excessive inflammatory response damages the alveolar epithelium and pulmonary capillary endothelial cells, disrupting the air-blood barrier and resulting in impaired lung function. ALI causative factors are divided into direct and indirect injuries. Direct injuries include pneumonia (caused by viral, bacterial, or fungal infections), aspiration lung injury (aspiration of gastric contents, inhalation of toxic gases, etc.), and pulmonary contusion, all of which stimulate immune cells to release inflammatory factors, triggering ALI. Indirect causes of ALI include sepsis, multiple injuries, complications from massive transfusions (such as TRALI), poisoning, burns, severe pancreatitis, and pulmonary embolism. These factors, through systemic inflammatory responses, immune responses, or local ischemia and hypoxia, promote the massive release of inflammatory factors, affecting the lungs and triggering ALI. In severe cases, ALI progresses to acute respiratory distress syndrome (ARDS). ALI patients exhibit respiratory distress, rapid respiratory rate, and progressive hypoxemia. Blood gas analysis shows a decreased PaO2 / FiO2 ratio, and imaging examinations reveal bilateral pulmonary exudative lesions or consolidation. Currently, only cevelex sodium is approved for the treatment of ALI in my country, but this drug can only partially inhibit the inflammatory response and cannot comprehensively regulate the inflammatory factor network, resulting in limited therapeutic efficacy. In the ICU, the mortality rate for ALI / ARDS patients is as high as 40%-50%, highlighting the urgent need to develop new therapies that precisely regulate the inflammatory factor network and inhibit excessive inflammatory responses to reduce patient mortality and improve prognosis.

[0003] Pulmonary fibrosis (PF) is a highly complex and refractory respiratory disease. PF patients often experience progressive disease, and once diagnosed, their survival is extremely limited. Related clinical research data shows that the median survival for most PF patients after diagnosis is only 2-3 years. Furthermore, the high mortality rate of PF places a heavy burden on patients and their families. Due to its high mortality rate and severity, it is figuratively called a "tumor-like disease," posing a serious threat to human health. Clinically, drugs used to treat pulmonary fibrosis mainly include glucocorticoids, pirfenidone, nintedanib, and N-acetylcysteine. Glucocorticoids, such as prednisone and methylprednisolone, slow the progression of pulmonary fibrosis by inhibiting the inflammatory response, but long-term use can lead to many adverse reactions, such as osteoporosis, elevated blood sugar, increased risk of infection, and adrenal insufficiency. Pirfenidone is a drug with anti-fibrotic, anti-inflammatory, and antioxidant effects. However, some patients experience adverse reactions such as gastrointestinal discomfort (nausea, vomiting, loss of appetite), and photosensitivity during use. Furthermore, its relatively high price places a significant financial burden on patients. Nintedanib, also used to treat pulmonary fibrosis, inhibits the tyrosine kinase activity of various pro-fibrotic growth factor receptors, but it also has adverse reactions such as diarrhea, nausea, vomiting, and abnormal liver function, and is expensive. N-acetylcysteine, a mucolytic agent with antioxidant properties, is also used in the treatment of pulmonary fibrosis, but its efficacy is relatively poor when used alone, and some patients may experience adverse reactions such as nausea, vomiting, and rash. In summary, these commonly used anti-pulmonary fibrosis drugs generally suffer from numerous adverse reactions, high prices, and poor efficacy, making it difficult to meet the actual needs of clinical treatment. Developing more effective, safe, and economical treatments for pulmonary fibrosis has significant clinical and social value.

[0004] Previous research on phenyllactic acid has primarily focused on its applications in food preservation and antibacterial properties. However, there are currently no reports on the use of phenyllactic acid or compositions containing phenyllactic acid in the preparation of drugs to alleviate lung diseases. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the use of phenyllactic acid or a composition containing phenyllactic acid in the preparation of a medicine for relieving lung diseases.

[0006] The technical solution of this invention is summarized as follows:

[0007] Use of phenyllactic acid or compositions containing phenyllactic acid in the preparation of medicaments for relieving lung diseases.

[0008] Preferably, the lung disease is pulmonary fibrosis.

[0009] Preferably, the lung disease is acute lung injury.

[0010] In acute lung injury research models, phenyllactic acid effectively intervened in the inflammatory cascade and oxidative stress damage process, significantly improving the symptoms of acute lung injury caused by these factors. Compared with traditional treatments, experimental verification showed that phenyllactic acid or compositions containing phenyllactic acid can significantly alleviate the symptoms of lipopolysaccharide (LPS)-induced acute lung injury (ALI) and bleomycin-induced pulmonary fibrosis (PF) in mice, effectively improving lung pathological changes and restoring lung function. Attached Figure Description

[0011] Figure 1 HE staining of the lungs in the mouse model of acute lung injury evaluated by phenyllactic acid in Example 1;

[0012] Figure 2 The results of white blood cell count in the bronchoalveolar lavage fluid of mice in the evaluation of the mouse model of acute lung injury by phenyllactic acid in Example 1;

[0013] Figure 3 This refers to the determination of protein content in bronchoalveolar lavage fluid in mice during the evaluation of the acute lung injury mouse model using phenyllactic acid in Example 1.

[0014] Figure 4 The image shows the mRNA expression of inflammatory factors TNF-α, IL-6, and IL-1β in the mouse lung tissue during the evaluation of the mouse model of acute lung injury using phenyllactic acid in Example 1.

[0015] Figure 5 The mRNA expression of inflammatory factors COX2 and iNOS in the mouse lung tissue during the evaluation of the mouse model of acute lung injury by phenyllactic acid in Example 1;

[0016] Figure 6 The expression of lactate dehydrogenase in the mouse model of acute lung injury evaluated by phenyllactic acid in Example 1;

[0017] Figure 7 The results are CT scans from the evaluation of phenyllactic acid in a mouse model of pulmonary fibrosis in Example 2.

[0018] Figure 8 HE staining of the lungs in the mouse model of pulmonary fibrosis evaluated by phenyllactic acid in Example 2;

[0019] Figure 9 This is a MASSON staining image of the lungs in the mouse model of pulmonary fibrosis evaluated by phenyllactic acid in Example 2.

[0020] Figure 10 This is a statistical analysis of the lung coefficient in the evaluation of the pulmonary fibrosis mouse model using phenyllactic acid in Example 2;

[0021] Figure 11 The figure shows the changes in body weight in the mouse model of pulmonary fibrosis evaluated by phenyllactic acid in Example 2.

[0022] Figure 12 This refers to the determination of hydroxyproline content in the lungs during the evaluation of the pulmonary fibrosis mouse model using phenyllactic acid in Example 2.

[0023] Figure 13 This shows the mRNA expression of pulmonary fibrosis factors α-SMA, Fibronectin, E-cadherin, and Collage I in the evaluation of the pulmonary fibrosis mouse model by phenyllactic acid in Example 2. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The CAS number of DL-3-Phenyllactic acid involved in each embodiment is 828-01-3. Purity: 99.70%, structural formula is shown in Formula I.

[0026]

[0027] Example 1

[0028] Protective effect of phenyllactic acid on lipopolysaccharide (LPS)-induced acute lung injury in mice.

[0029] I. Grouping and Treatment of Laboratory Animals

[0030] Male C57BL / 6J mice aged 6-8 weeks were randomly divided into two groups: a model group (LPS modeling) and a phenyllactic acid group (LPS + phenyllactic acid), with 6 mice in each group. Mice underwent a 3-day acclimatization period. The phenyllactic acid group received daily intragastric administration of a phenyllactic acid-saline solution (50 mg / kg, based on the mass of phenyllactic acid); the model group received the same volume of saline via intragastric administration daily. Gavage administration was performed once daily for 5 days. Intervention began 5 days before modeling in all groups and continued until the end of the experiment.

[0031] II. Construction of Acute Lung Injury Model

[0032] On day 5, LPS was injected intratracheally to establish a mouse model of acute lung injury. The specific procedure was as follows:

[0033] Mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg / kg). After anesthesia took effect, the mice were fixed in a supine position, and a sterilized endotracheal dosing device was used to draw up an aqueous solution containing LPS-physiological saline (10 mg / kg, by mass of LPS), which was then inserted into the trachea and injected. After injection, the mice were quickly upright and rotated to promote even distribution of the drug in the lungs. After the mice naturally awoke, they were placed in cages for rearing. On the second day, the mice exhibited symptoms such as dull fur, piloerection, lethargy, and curling up, indicating successful establishment of the model.

[0034] III. Sample Collection and Testing

[0035] Gross observation of the lungs and collection of bronchoalveolar lavage fluid (BALF): After the drug intervention, the mice were weighed, and anesthetized again by intraperitoneal injection of tribromoethanol (300 mg / kg). After fixing the mice in a supine position, the thoracic cavity was opened to expose the trachea. A 22G indwelling arterial and venous catheter was inserted 1 cm into the trachea, and 1 ml of physiological saline was injected into the lungs. The fluid was then collected and repeated twice to obtain BALF. Subsequently, the incision was extended, and the trachea was cut near the thyroid cartilage. The lungs were completely removed, and the gross morphology, elasticity, and presence of congestion of the lung tissue were observed.

[0036] Pathological and histological examination: After the mice in each group were sacrificed, a portion of lung tissue was excised from the right lobe of the lung, fixed with formaldehyde, embedded in paraffin, and prepared into 4μm sections. The sections were then stained with hematoxylin and eosin (HE) according to routine methods, and the degree of inflammation of the lung tissue was observed under a microscope. Figure 1 The results showed that the lung tissue structure of the model group mice was damaged, with extensive inflammatory cell infiltration, alveolar capillary congestion and dilation, and wider interstitial and alveolar septa. Compared with the model group, the pathological changes in the phenyllactic acid group were relatively mild. Inflammatory cell infiltration in the lung tissue was less pronounced, and the interstitial and alveolar septa were narrower, indicating a less severe degree of tissue damage. BALF cell counting and protein content determination: 1 ml of BALF solution was used to detect the white blood cell (WBC) count. Figure 2 The results showed that the number of white blood cells in the lungs was lower in the phenyllactic acid group. The remaining BALF solution was taken, allowed to stand at room temperature for 5 min, centrifuged at 3500 rpm for 10 min, the supernatant was discarded, and the solution was resuspended in 100 μl of PBS. Protein content was determined using the BCA protein quantification method. Figure 3 The results showed that the content of phenyllactic acid histone was significantly low.

[0037] Gene expression detection: 20 mg of mouse lung tissue was minced, lysed with zirconium oxide beads and 1 ml of Trizol solution, and then ground in a low-temperature homogenizer for 10 min. After homogenization, 0.2 mL of chloroform was added, and the mixture was vigorously shaken for 15 s and incubated at room temperature for 5 min. The mixture was centrifuged at 12,000 rpm for 10 min at 4 °C, and the aqueous phase was transferred to a new centrifuge tube. The mixture was gently mixed with an equal volume of pre-chilled isopropanol, incubated at 30 °C for 10 min, and then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was discarded to obtain the RNA precipitate. The RNA precipitate was washed with 1 mL of pre-chilled 75% ethanol solution, centrifuged at 7500 rpm for 5 min at 4 °C, the ethanol solution was removed, and the RNA precipitate was air-dried for 10 min. 20 μL of RNase-free water was added, and the mixture was repeatedly pipetted 5 times to obtain the RNA solution, which was stored at -80 °C. After determining the concentration and purity of the RNA solution, cDNA was synthesized using a reverse transcription kit as a template for quantitative PCR amplification. The PCR amplification conditions were: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ annealing for 30 s, for a total of 40 cycles. Using β-actin as an internal control, the expression of TNFα, IL-1β, IL6, COX2, and INOS mRNA in mouse lung tissue was calculated using the 2-ΔΔCt method. Figure 4 , Figure 5 It can be seen that, compared with the model group, the expression levels of inflammatory factor mRNA in the phenyllactic acid group were relatively low.

[0038] Enzyme activity assay:

[0039] Weigh 20 mg of mouse lung tissue, mince it, add steel balls and 200 μL of PBS, and grind it in a low-temperature homogenizer for 10 min. After centrifugation, collect the supernatant and determine the lactate dehydrogenase content of each group of mouse lung tissue according to the lactate dehydrogenase kit method. Figure 6 It can be seen that, compared with the model group, the lactate dehydrogenase content in the phenyllactic acid group was relatively low.

[0040] Example 2

[0041] Protective effect of phenyllactic acid on bleomycin-induced mouse pulmonary fibrosis model

[0042] I. Grouping and Treatment of Laboratory Animals

[0043] Male C57BL / 6J mice aged 6-8 weeks were selected and, after 3 days of acclimatization, were randomly divided into two groups: a model group (bleomycin-induced model) and a phenyllactic acid group (phenyllactic acid administered after model), with 6 mice in each group.

[0044] II. Construction of a Pulmonary Fibrosis Model

[0045] On day 1, a mouse pulmonary fibrosis model was established using bleomycin intratracheal injection. Specifically, mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg / kg). After anesthesia took effect, the mice were fixed in a supine position, and 0.05 mL of bleomycin-containing saline solution (3 U / kg) was drawn from the trachea using a sterilized intratracheal dosing device and injected into the trachea. After injection, the mice were quickly upright and rotated to ensure even distribution of the drug in the lungs. After the mice naturally recovered, they were caged. The model was considered successfully established when the mice exhibited dull fur, piloerection, lethargy, slow or decreased weight gain, and curling up on day 7.

[0046] III. Drug Intervention and Detection

[0047] Drug intervention: Starting from day 2, the phenyllactic acid group was given 20 mg / kg of drinking water daily, while the model group was given physiological saline daily at the same volume, until the end of the experiment, which lasted for 28 days.

[0048] Lung CT scan: On day 21, lung CT scans were performed on mice. Mice were first anesthetized with tribromoethanol (300 mg / kg) via intraperitoneal injection to ensure complete anesthesia and absence of pain response. The anesthetized mice were placed on the CT scanner table, and their position was adjusted so that the lungs were within the scanning range before the scan was performed to acquire lung images. Figure 7 It can be seen that the lung CT scans of the model group showed reticular shadows and honeycomb-like changes, while the lung CT scans of the phenyllactic acid group showed no obvious shadows, indicating that phenyllactic acid can significantly inhibit bleomycin-induced pulmonary fibrosis.

[0049] Pathological and histological examination: Part of lung tissue was excised from the right lobe of the lung, fixed in formaldehyde, embedded in paraffin, and prepared into 4μm sections. The sections were then stained with routine HE and Masson staining, and the degree of inflammation and collagen fiber proliferation in the lung tissue was observed under a microscope. Figure 8 HE staining results showed that after bleomycin modeling, the lesions in the model group were unevenly distributed, the lung tissue structure at the lesion site was destroyed, the alveolar cavities were irregularly collapsed and fused, the alveolar septa were significantly wider, and inflammatory cell infiltration and fibroblast proliferation were observed. The alveolar structural changes, inflammatory cell infiltration, collagen deposition, and vasodilation in the lung tissue of mice in the phenyllactic acid group were less severe than those in the model group. Figure 9 Masson staining results showed that the model group sections had obvious patches of blue fibrosis, while the phenyllactic acid group had a lighter degree of fibrosis than the model group.

[0050] Lung coefficient and hydroxyproline content determination: The lungs were rinsed with physiological saline, blotted dry with filter paper, and weighed using an analytical balance. The lung coefficient was calculated (lung coefficient = lung wet weight (mg) / mouse body weight at sacrifice (g)). Figure 10 , Figure 11It was found that the body weight of the phenyllactic acid group was larger than that of the model group. Since hydroxyproline is one of the main amino acids constituting collagen, and collagen is a characteristic protein of fibrotic tissue, measuring the hydroxyproline content in lung tissue can reflect the degree of lung fibrosis. 20 mg of lung tissue homogenate was taken, and the hydroxyproline content in the lung tissue of each group of mice was measured according to the kit method. Figure 12 The results showed that, compared with the model group, the hydroxyproline content in the lung tissue of mice in the phenyllactic acid group was significantly lower, indicating a lower degree of fibrosis.

[0051] Gene expression detection: 20 mg of mouse lung tissue was weighed, minced, and lysed with zirconia beads and 1 ml of Trizol solution. The tissue was then ground in a low-temperature grinder for 10 min. Subsequent procedures were the same as the RNA extraction steps for gene expression detection in Example 1. After determining the RNA concentration and purity, cDNA was synthesized using a reverse transcription kit as a template for quantitative PCR amplification. The PCR amplification conditions were: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, and 60℃ annealing for 30 s, for a total of 40 cycles. β-actin was used as an internal control, and the expression of α-SMA, Fibronectin, E-cadherin, and Collagen I in mouse lung tissue was calculated using the 2-ΔΔCt method. Figure 13 The results showed that, compared with the model group, the expression of fibrin-related genes was significantly lower in the phenyllactic acid group.

[0052] Example 3

[0053] Experiments have shown that compositions of phenyllactic acid with pharmaceutically acceptable excipients provide similar relief for pulmonary fibrosis and acute lung injury as phenyllactic acid alone. It can be formulated as an inhaled aerosol, oral solution, tablet, capsule, granule, or injection to meet diverse patient needs.

Claims

1. The use of phenyllactic acid or a composition containing phenyllactic acid in the preparation of a medicament for relieving lung disease, wherein the lung disease is pulmonary fibrosis.

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