A nasal spray composition, nasal spray and use

Abstract

The application belongs to the technical field of medicine, and specifically discloses a kind of nasal spray composition and nasal spray and application, the nasal spray composition includes the following components: hyaluronic acid, trehalose, N-acetyl-L-cysteine, methylene succinic acid, gallic acid, taurine, coenzyme Q10, rosemary essential oil.The nasal spray composition provided by the application is accurately adjusted, can give full play to the advantages of each component, provide better nasal ventilation and humidity.The nasal spray composition provided by the application can inhibit the excessive activation of LPS-induced neutrophil Netosis in vitro culture and the excessive activation of LPS-induced mouse airway neutrophil Netosis, thereby reducing the inflammatory response in the nasal cavity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a nasal spray composition, the nasal spray itself, and its application. Background Technology

[0002] NETosis is an inflammatory cell death mechanism of neutrophils. Activated neutrophils release neutrophilic traps (NETs) composed of depolymerized chromatin and intracellular granule proteins to capture and kill pathogens. NETs are composed of DNA, histones, neutrophil elastase, myeloperoxidase, and other components. Neutrophil death accompanies NET formation; this novel death mechanism, distinct from apoptosis and necrosis, is termed NETosis.

[0003] Neutrophils are a type of white blood cell and one of the most common white blood cells in the human immune system. They are key participants in the processes of inflammation and infection. When the nasal mucosa is irritated or infected, neutrophils rapidly aggregate in the nasal mucosal area. The main function of neutrophils is to engulf and eliminate pathogens, such as bacteria and viruses, to protect the body from infection. They release chemicals such as cytokines, enzymes, and oxygen free radicals to kill pathogens. However, during inflammation of the nasal mucosa, excessive activation of neutrophil netosis can exacerbate the inflammatory response and worsen rhinitis symptoms. The DNA fiber network in the reticular formation may activate the production of other immune cells and inflammatory mediators, leading to a further spread of the inflammatory response in the nasal mucosa. This overactivated inflammatory response can cause rhinitis symptoms, including nasal congestion, runny nose, sneezing, and nasal itching. In such cases, inhibiting the excessive activation of neutrophil netosis can alleviate the inflammatory response and is important for maintaining nasal health and patency. Currently, there are no nasal spray products that can inhibit the overactivation of neutrophil netosis, thereby reducing nasal inflammation. Summary of the Invention

[0004] In view of this, the present invention provides a nasal spray composition, a nasal spray, and an application thereof, which aims to provide a nasal spray for improving nasal ventilation, inhibiting nasal neutrophil extracellular netosis-related death, thereby reducing nasal inflammation.

[0005] To address the aforementioned problems, the first aspect of the present invention provides a nasal spray composition comprising water and the following components dissolved in the water: hyaluronic acid, trehalose, N-acetyl-L-cysteine, methylene succinic acid, gallic acid, taurine, coenzyme Q10, and rosemary essential oil.

[0006] In this invention, hyaluronic acid, trehalose, N-acetyl-L-cysteine, methylene succinic acid, gallic acid, taurine, and coenzyme Q10 are common food-grade raw materials, food additives, or nutritional fortifiers, with a wide safe concentration range for human cells. Among them, N-acetyl-L-cysteine, methylene succinic acid, taurine, and coenzyme Q10 are naturally present in the human body and are endogenous metabolites common to humans and many animals. They can directly participate in biochemical reactions within human cells, thus offering an advantage in terms of safety for human cells.

[0007] The composition contains itaconate, an important regulator of cellular immune metabolism, which plays a key regulatory role in the body's inflammatory response. Itaconate is a bypass metabolite of the tricarboxylic acid cycle (TCA cycle), generated by the decarboxylation of cis-aconitate produced by the dehydration of citric acid (CA). As an endogenous metabolite in the human body, itaconate exhibits anti-inflammatory effects in preclinical models of sepsis, viral infections, psoriasis, gout, and ischemia / reperfusion injury. Itaconate directly regulates the inflammasome-related protein NLRP3, thereby inhibiting netosis-related death of nasal neutrophils. Taurine and coenzyme Q10 may also have antioxidant, anti-inflammatory, and mitochondrial-enhancing effects.

[0008] The nasal spray composition provided by this invention is precisely adjusted to fully utilize the advantages of each component, providing better nasal ventilation and humidification, and inhibiting the excessive activation of LPS-induced neutrophil netosis in vitro, thereby reducing the inflammatory response in the nasal cavity.

[0009] Preferably, the nasal spray composition further includes one of guanidine or spermidine.

[0010] In this invention, guanidine and spermidine are also endogenous metabolites in the human body, offering advantages in terms of safety. Guanidine (agmatine) is a product of arginine metabolism. Numerous preclinical and several clinical studies have demonstrated that guanidine has pleiotropic regulatory functions on various molecular targets related to neurotransmitters, nitric oxide synthesis, glucose metabolism, polyamine metabolism, and carnitine biosynthesis. Spermine (agmatine), also a product of arginine metabolism, possesses anti-inflammatory and antioxidant functions and enhances mitochondrial metabolic function, thereby inhibiting the excessive activation of LPS-induced neutrophil necrosis in cultured cells.

[0011] It should be noted that the compositions of the present invention can be formulated into external nasal sprays, nasal washes, and nasal indoor air atomizers, etc.

[0012] Preferably, the hyaluronic acid has a mass concentration of 0.8-2.0 g / L and the trehalose has a mass concentration of 8-20 g / L, based on the total volume of the nasal spray composition.

[0013] Preferably, the N-acetylcysteine ​​has a mass concentration of 0.4-35 g / L and the methylene succinic acid has a mass concentration of 0.4-2 g / L, based on the total volume of the nasal spray composition.

[0014] Preferably, the gallic acid concentration is 0.5-2.5 g / L based on the total volume of the nasal spray composition.

[0015] Preferably, the taurine has a mass concentration of 0.1-2 g / L and the coenzyme Q10 has a mass concentration of 0.05-0.5 g / L, based on the total volume of the nasal spray composition.

[0016] Preferably, the mass concentration of guanidine is 0.5-5 g / L based on the total volume of the nasal spray composition; the mass concentration of spermidine is 0.5-5 g / L.

[0017] Preferably, the rosemary essential oil has a volume fraction of 0.1-1.0% based on the total volume of the nasal spray composition.

[0018] The second aspect of the present invention is to provide a nasal spray formulated according to the above-mentioned nasal spray composition, wherein hyaluronic acid, trehalose, N-acetyl-L-cysteine, rosemary essential oil, methylene succinic acid, gallic acid, taurine, coenzyme Q10, guanidine or spermidine are mixed in a volumetric flask in a required proportion, water is added to make up to volume, and the mixture is thoroughly mixed to obtain a mixed solution. Finally, sodium hydroxide is added to adjust the pH value of the solution to 3.7-4.3 to obtain a nasal spray. The operation is simple and the method is quick.

[0019] A third aspect of the present invention provides an application of the above-mentioned nasal spray, which can be used to inhibit the excessive activation of neutrophil netosis, thereby reducing the inflammatory response in the nasal cavity.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) The nasal spray composition provided by the present invention has high safety for human body.

[0022] (2) The nasal spray composition provided by the present invention is precisely adjusted to give full play to the advantages of each component and provide better nasal ventilation and humidification.

[0023] (3) The nasal spray composition of the present invention can inhibit the overactivation of LPS-induced neutrophil netosis in vitro and the overactivation of LPS-induced mouse airway neutrophil netosis, thereby reducing the inflammatory response in the nasal cavity. Attached image description:

[0024] Figure 1 The image shows the expression of CitH3, the marker protein for NETosis activation, in each group during immunoblotting comparisons in cell experiments.

[0025] Figure 2 Using the optical density of β-actin protein as an internal reference, statistical analysis was performed. Figure 1 The study mentions the difference in CitH3 protein expression between HL-60 medium and the other three groups (the figure shows the difference using Student's t-test; ***, P<0.001).

[0026] Figure 3 Using the optical density of β-actin protein as an internal reference, statistical analysis was performed. Figure 1 The study mentions the difference in CitH3 protein expression between HL-60 medium and the other four groups (the figure shows the difference using Student's t-test; ***, P<0.001).

[0027] Figure 4 The expression of CitH3, the NETosis activation marker protein, in each group was compared in the Western blot diagram during animal experiments.

[0028] Figure 5 Using the optical density of β-actin protein as an internal reference, statistical analysis was performed. Figure 4 The study mentions the difference in CitH3 protein expression between HL-60 medium and the other three groups (the figure shows the difference using Student's t-test; ***, P<0.001). Detailed implementation method:

[0029] The present invention will be further described below with reference to specific embodiments. It should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and the present invention is not limited to these examples.

[0030] Example 1:

[0031] This embodiment provides a nasal spray formulated according to formula combination 1, which includes the following ingredients: hyaluronic acid, trehalose, N-acetylcysteine, methylene succinic acid, gallic acid, taurine, coenzyme Q10, and rosemary essential oil.

[0032] The specific preparation method is as follows: Add 0.2g hyaluronic acid, 2g trehalose, 0.1g gallic acid, 0.06g taurine, 1.6g N-acetyl-L-cysteine, 0.1g methylene succinic acid, 0.01g coenzyme Q10, and 0.1ml rosemary essential oil to a 100ml volumetric flask, and add deionized water to make up to 100ml of mixed solution. Finally, add sodium hydroxide to the mixed solution to adjust the pH value to 3.7 to obtain a nasal spray.

[0033] Example 2:

[0034] This embodiment provides a nasal spray formulated according to formulation combination 2, which includes the following ingredients: hyaluronic acid, trehalose, N-acetylcysteine, methylene succinic acid, gallic acid, taurine, coenzyme Q10, rosemary essential oil, and agmatine. Compared with formulation combination 1 in Example 1, formulation combination 2 of Example 2 contains agmatine.

[0035] The specific preparation method is as follows: Add 0.2g hyaluronic acid, 2g trehalose, 0.1g gallic acid, 0.06g taurine, 1.6g N-acetyl-L-cysteine, 0.1g methylene succinic acid, 0.01g coenzyme Q10, 0.1ml rosemary essential oil, and 0.3g guanidine to a 100ml volumetric flask, and add deionized water to make up to 100ml of mixed solution. Finally, add sodium hydroxide to the mixed solution to adjust the pH value to 3.7 to obtain a nasal spray.

[0036] Example 3:

[0037] This embodiment provides a nasal spray formulated according to formulation combination 3, which includes the following ingredients: hyaluronic acid, trehalose, N-acetylcysteine, methylene succinic acid, gallic acid, taurine, coenzyme Q10, rosemary essential oil, and spermidine. The formulation combination of Example 3, compared to formulation combination 1 in Example 1, includes spermidine.

[0038] The specific preparation method is as follows: Add 0.2g hyaluronic acid, 2g trehalose, 0.1g gallic acid, 0.06g taurine, 1.6g N-acetyl-L-cysteine, 0.1g methylene succinic acid, 0.01g coenzyme Q10, 0.1ml rosemary essential oil, and 0.4g spermidine to a 100ml volumetric flask, add deionized water to make up to 100ml of mixed solution, and finally add sodium hydroxide to adjust the pH of the solution to 4 to obtain a nasal spray.

[0039] Example 4:

[0040] This embodiment provides a nasal spray formulated according to formulation combination 4, which includes the following ingredients: hyaluronic acid, trehalose, N-acetylcysteine, gallic acid, taurine, coenzyme Q10, and rosemary essential oil. Compared with formulation combination 1, it lacks methylene succinic acid.

[0041] The specific preparation method is as follows: Add 0.2g hyaluronic acid, 2g trehalose, 0.1g gallic acid, 0.06g taurine, 1.6g N-acetyl-L-cysteine, 0.01g coenzyme Q10, and 0.1ml rosemary essential oil to a 100ml volumetric flask, add deionized water to make up to 100ml of mixed solution, and finally add sodium hydroxide to the mixed solution to adjust the pH value of the solution to 3.7 to obtain a nasal spray.

[0042] Confirmatory Experiment

[0043] (1) Cell experiments

[0044] Human neutrophil line HL-60 cells (purchased from ATCC tissue) were cultured in RPMI 1640 medium (containing 10% fetal bovine serum, 2 mmol / L L-glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin), hereinafter referred to as HL-60 medium. Cells were cultured in a 5% CO2 incubator at 37°C. 600,000 cells / well were seeded into 6-well cell culture plates at a volume of 2000 μL per well, and induced for 5 days with a final concentration of 5 μmol / L all-trans retinoic acid. Then, 1,000,000 cells / well were seeded into 6-well cell culture plates, ensuring the culture medium contained 50 μg / ml lipopolysaccharide (LPS). The medium was used as a control group and treated with the following combinations for 16-24 hours (formulation combination 1 in Example 1, formulation combination 2 in Example 2, formulation combination 3 in Example 3, and formulation combination 4 in Example 4).

[0045] ①The control group was HL-60 medium.

[0046] ② Formula combination 1 set:

[0047] Add the following components to HL-60 medium at final concentrations: 2 g / L hyaluronic acid, 20 g / L trehalose, 1 g / L gallic acid, 0.6 g / L taurine, 16 g / L N-acetyl-L-cysteine, 1 g / L methylene succinic acid, 0.1 g / L coenzyme Q10, and 0.1% (v / v) rosemary essential oil.

[0048] ③ Two formula combinations:

[0049] Add the following components to HL-60 medium at final concentrations: hyaluronic acid 2 g / L, trehalose 20 g / L, gallic acid 1 g / L, taurine 0.6 g / L, N-acetyl-L-cysteine ​​16 g / L, methylene succinic acid 1 g / L, coenzyme Q10 0.1 g / L, rosemary essential oil 0.1% (volume fraction), and agmatine 5 g / L.

[0050] ④ Formula combination 3 sets:

[0051] Add the following components to HL-60 medium at final concentrations: hyaluronic acid 2 g / L, trehalose 20 g / L, gallic acid 1 g / L, taurine 0.6 g / L, N-acetyl-L-cysteine ​​16 g / L, methylene succinic acid 1 g / L, coenzyme Q10 0.1 g / L, rosemary essential oil 0.1% (volume fraction), and spermidine 5 g / L.

[0052] ⑤ Formula combinations: 4 sets

[0053] Formula combination 4 lacks methylene succinic acid compared to formula combination 1. The following components were added to HL-60 medium at final concentrations: hyaluronic acid 2 g / L, trehalose 20 g / L, gallic acid 1 g / L, taurine 0.6 g / L, N-acetyl-L-cysteine ​​16 g / L, coenzyme Q10 0.1 g / L, and rosemary essential oil 0.1% (volume fraction).

[0054] In the experimental verification, the culture media containing the above components were added to the wells of 6-well plates, and the cells were cultured for 16-48 hours. Cell supernatants and cellular proteins were collected. Western blotting was used to detect the expression of CitH3 protein (a netosis marker) and the internal control protein Actin in each group of cells. ELISA was used to detect the levels of IL-6, IL-8, and TNF-α in the cell culture supernatant. The content of DNA (NET-DNA) in the NETs released by the cells was also detected. For NET-DNA detection, restriction endonucleases (EcoR I and Hind III, 20 U / ml, Takara) were added to the cells, and the cells were incubated at 37°C for 2 hours. NET-DNA in the cultured cell supernatant was quantitatively detected using the PicoGreen Double-Stranded DNA Kit (Invitrogen), and analyzed using a fluorescent enzyme marker (PerkinElmer, VICTORNivo). The results are as follows: Figure 1-3 As shown.

[0055] from Figure 1-3 The results show that Figure 1The results of the immunoblotting experiment showed that formulation combination 1, formulation combination 2 and formulation combination 3 in Examples 1-3 could inhibit the expression of the netosis activation marker protein CitH3 in LPS-induced neutrophils. Figure 2 The three bar charts show that in the enzyme-linked immunosorbent assay (ELISA), formulation combination 1, formulation combination 2 and formulation combination 3 significantly reduced the levels of TNF-α, IL-6 and IL-1 secreted into the culture medium by LPS-induced neutrophils, respectively. Figure 3 The six bar charts show that in the NET-DNA detection experiment, compared with formulation 1, the ability of formulation combination 4 to inhibit NET-DNA release from cells depended on methylene succinic acid; compared with formulation combination 1, the ability of formulation combination 2 to inhibit NET-DNA release from cells depended on guanidine and methylene succinic acid; and compared with formulation combination 1, the ability of formulation combination 3 to inhibit NET-DNA release from cells depended on methylene succinic acid and spermidine. Therefore, it can be seen that the composition of the present invention containing methylene succinic acid and guanidine or containing methylene succinic acid and spermidine can enhance the effect of the composition in inhibiting NET-DNA release from cells. Among them, the composition containing methylene succinic acid and spermidine has the best effect in inhibiting the excessive activation of neutrophil NETosis, thereby better alleviating the inflammatory response in the nasal cavity.

[0056] (2) Animal experiments

[0057] The experimental animals were male C57BL / 6 mice, aged 8-10 weeks and weighing between 25 and 30 grams, with 5 mice per group. All mice were housed under specific pathogen-free housing conditions. After anesthesia, the trachea of ​​the mice was exposed, and a tracheostomy tube was inserted into the trachea. The following solution was then injected into the mice orally:

[0058] ① 25 μL of phosphate-buffered saline (PBS) solution, this is the experimental control group (denoted as PBS group);

[0059] ② 25 μL of PBS solution containing 0.1 mg LPS (Netosis induction group, denoted as PBS+LPS group);

[0060] ③ A 25 μL PBS solution containing 0.1 mg LPS, 0.5 μg hyaluronic acid, 5 μg trehalose, 0.25 μg gallic acid, 0.15 μg taurine, 4 μg N-acetyl-L-cysteine, 0.25 μg methylene succinic acid, 0.025 μg coenzyme Q10, and 0.025 μL rosemary essential oil (referred to as PBS+LPS+formulation combination 1);

[0061] ④ A 25 μL PBS solution contains 0.1 mg LPS, 0.5 μg hyaluronic acid, 5 μg trehalose, 0.25 μg gallic acid, 0.15 μg taurine, 4 μg N-acetyl-L-cysteine, 0.25 μg methylene succinic acid, 0.025 μg coenzyme Q10, 0.025 μL rosemary essential oil, and 1.25 μg guanidine (referred to as PBS+LPS+formulation combination 2);

[0062] ⑤ A 25 μL PBS solution contains 0.1 mg LPS, 0.5 μg hyaluronic acid, 5 μg trehalose, 0.25 μg gallic acid, 0.15 μg taurine, 4 μg N-acetyl-L-cysteine, 0.25 μg methylene succinic acid, 0.025 μg coenzyme Q10, 0.025 μL rosemary essential oil, and 1.25 μg spermidine (referred to as PBS+LPS+formulation combination 3);

[0063] ⑥ A 25 μL PBS solution contains 0.1 mg LPS, 0.5 μg hyaluronic acid, 5 μg trehalose, 0.25 μg gallic acid, 0.15 μg taurine, 4 μg N-acetyl-L-cysteine, 0.025 μg coenzyme Q10, and 0.025 μL rosemary essential oil (referred to as PBS+LPS+formulation combination 4);

[0064] 200 μl of air was then injected to ensure fluid deposition in each lung. The wound was closed after the solution was infused, and the mice were allowed free access to food and water to aid recovery. Twenty-four hours after injection, the animals were sacrificed, and airway tissue was collected from each group. Western blotting was used to compare the differences in CitH3 and PAD4 proteins (both marker proteins of netosis) in the airway tissue. The results are as follows: Figure 4-5 As shown.

[0065] from Figure 4-5 The results show that Figure 4 The results of the immunoblotting experiment showed that formulation combination 1, formulation combination 2 and formulation combination 3 in Examples 1-3 could inhibit the expression of LPS-induced mouse airway neutrophil netosis activation marker proteins CitH3 and PAD4 (representative results from two animal samples). Figure 5 The two bar charts are statistical results of the CitH3 and PAD4 protein expression detection using immunoblotting of tissues from 5 animals (the optical density of the -Actin band was used as an internal reference, and the optical density of each group was quantified after statistical analysis). The results also show that formulation combination 1, formulation combination 2 and formulation combination 3 can inhibit the expression of LPS-induced mouse airway neutrophil netosis activation marker proteins CitH3 and PAD4.

[0066] The above descriptions are merely embodiments of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. For those skilled in the art, the scope of protection of this invention should be understood to include substitutions and modifications that do not depart from the invention, and are covered by the rights of this patent application.

Claims

1. A nasal spray composition, characterized in that, It consists of water and the following components dissolved in water: Hyaluronic acid, trehalose, N-acetyl-L-cysteine, methylene succinic acid, gallic acid, taurine, coenzyme Q10, rosemary essential oil; Based on the total volume of the nasal spray composition, the hyaluronic acid has a mass concentration of 0.8-2.0 g / L; the trehalose has a mass concentration of 8-20 g / L; the N-acetylcysteine ​​has a mass concentration of 0.4-35 g / L; the methylene succinic acid has a mass concentration of 0.4-2 g / L; the gallic acid has a mass concentration of 0.5-2.5 g / L; the taurine has a mass concentration of 0.1-2 g / L; the coenzyme Q10 has a mass concentration of 0.05-0.5 g / L; and the rosemary essential oil has a volume fraction of 0.1%.

2. A nasal spray composition, characterized in that, It consists of water and the following components dissolved in water: hyaluronic acid, trehalose, N-acetyl-L-cysteine, methylene succinic acid, gallic acid, taurine, coenzyme Q10, rosemary essential oil, and guanidine or spermidine; Based on the total volume of the nasal spray composition, the mass concentration of the hyaluronic acid is 0.8-2.0 g / L; the mass concentration of the trehalose is 8-20 g / L; the mass concentration of the N-acetylcysteine ​​is 0.4-35 g / L; the mass concentration of the methylene succinic acid is 0.4-2 g / L; the mass concentration of the gallic acid is 0.5-2.5 g / L; the mass concentration of the taurine is 0.1-2 g / L; the mass concentration of the coenzyme Q10 is 0.05-0.5 g / L; the volume fraction of the rosemary essential oil is 0.1%; the mass concentration of the guanidine is 0.5-5 g / L; and the mass concentration of the spermidine is 0.5-5 g / L.

3. A nasal spray formulated from the nasal spray composition of claim 2, characterized in that, The specific method for preparing the nasal spray is as follows: Hyaluronic acid, trehalose, N-acetyl-L-cysteine, rosemary essential oil, methylene succinic acid, gallic acid, taurine, coenzyme Q10, guanidine or spermidine are mixed in the required proportions and added to a volumetric flask. Water is then added to make up to the final volume. The mixture is thoroughly mixed to obtain a homogeneous solution. Finally, sodium hydroxide is added to adjust the pH of the solution to 3.7-4.3 to obtain a nasal spray.

4. The use of a nasal spray composition according to any one of claims 1-2 in the preparation of a medicament for reducing inflammatory reactions in the nasal cavity.

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