Application of glutathione dry powder inhalant in preparation of medicine for treating radiation acute lung injury

Abstract

The invention belongs to the technical field of antioxidant drugs, and particularly relates to application of a glutathione dry powder inhaler in preparation of drugs for treating radiation acute lung injury, the glutathione dry powder inhaler comprises the following active ingredients by mass percentage: 50%-60% of reduced glutathione, 10%-20% of a carrier and 20%-30% of a dispersant, totaling 100%, the carrier is mannitol, and the dispersing agent is L-leucine. According to the invention, the glutathione dry powder inhalant accurately delivers high-concentration GSH to an alveolar injury area through a lung targeting delivery system, synchronously activates endogenous antioxidant enzymes, inhibits lipid peroxidation and an inflammation source, and blocks release of proinflammatory factors, so that radiation-induced oxidation-inflammation cascade reaction is directly inhibited, alveolar structure damage is relieved, and the anti-inflammatory effect is achieved. And an efficient new treatment strategy is provided for RILI.

Description

Technical Field

[0001] This invention belongs to the field of antioxidant drug technology, specifically relating to the application of glutathione dry powder inhaler in the preparation of drugs for treating acute radiation-induced lung injury. Background Technology

[0002] Radiation-induced lung injury (RILI) is a major complication of radiotherapy for thoracic tumors. Its core pathogenesis is radiation-induced oxidative stress: ionizing radiation triggers an explosive accumulation of reactive oxygen species and reactive nitrogen species in lung tissue, leading to DNA damage, lipid peroxidation, and protein carbonylation. This, in turn, activates inflammatory pathways such as NF-κB, forming an oxidative-inflammatory cascade reaction, which eventually develops into alveolar consolidation and pulmonary fibrosis.

[0003] Currently, clinical treatment for recurrent fibrosis (RILI) relies on glucocorticoids, but long-term use has side effects such as immunosuppression and limited efficacy in advanced fibrosis. Reduced glutathione, abbreviated as GSH, is an endogenous antioxidant that can repair redox imbalances by directly scavenging reactive oxygen species and nitrogenous substances and upregulating glutathione peroxidase activity. Theoretically, it can target and intervene in the core mechanism of RILI. Glutathione peroxidase, abbreviated as GSH-Px, is a key component of RILI.

[0004] However, existing GSH dosage forms are mainly oral tablets and injections. Among them, oral GSH has insufficient local concentration in the lungs after being degraded in the gastrointestinal tract and undergoing the first-pass effect in the liver, resulting in low bioavailability. Injections require high doses to maintain effective concentrations in lung tissue, which can easily cause systemic adverse reactions. They also lack targeting and cannot form effective drug accumulation in key sites of oxidative damage, such as lung epithelial / endothelial cells.

[0005] While current dry powder inhalers can achieve targeted drug delivery to the lungs, commercially available products generally suffer from low lung deposition rates (<20%) and particle aggregation, making it difficult for the active ingredients to penetrate deep into the alveolar region. Furthermore, existing inhalers often rely on complex excipient combinations, which may pose compatibility risks and cause local irritation. Summary of the Invention

[0006] To address the technical problems of existing dry powder inhalers, such as low lung deposition rates and easy particle aggregation, which hinder the penetration of active ingredients into the alveolar region, this invention provides the application of glutathione dry powder inhaler in the preparation of drugs for treating radiation-induced acute lung injury.

[0007] This invention maintains particle dispersibility and prevents glutathione aggregation through the addition of a carrier and a dispersant. Simultaneously, L-leucine is used as a dispersant, and its molecules accumulate at the gas-liquid interface of the particles, forming a molecular-level hydrophobic coating for excellent flowability. Furthermore, the "lubricating effect" of the leucine layer promotes the instantaneous deagglomeration of microspheres, ensuring a high proportion of drug penetrates deep into the alveoli. The hydrophobic properties also slow down the dissolution rate of alveolar fluid, achieving sustained release of reduced glutathione and prolonging its duration of action. The glutathione dry powder inhaler of this invention significantly improves the deposition efficiency and retention time of the drug deep in the lungs. Simultaneously, based on the core antioxidant and anti-inflammatory activities of reduced glutathione, it achieves highly effective relief of radiation-induced acute lung injury.

[0008] This invention delivers a high concentration of glutathione dry powder inhaler to the alveolar damage area via a lung-targeted delivery system. This simultaneously activates endogenous antioxidant enzymes, inhibits lipid peroxidation and inflammatory sources, and blocks the release of pro-inflammatory factors, thereby directly suppressing the radiation-induced oxidation-inflammatory cascade reaction, reducing alveolar structural damage, and providing a highly efficient and low-toxicity new treatment strategy for RILI.

[0009] The purpose of this invention is to provide the application of glutathione dry powder inhaler in the preparation of drugs for acute lung injury caused by radiation. The active ingredient of the glutathione dry powder inhaler is prepared by the following components by mass percentage: 50% to 60% reduced glutathione, 10% to 20% carrier, 20% to 30% dispersant, totaling 100%.

[0010] It should be noted that reduced glutathione, as the core active ingredient, has thiol groups in its molecule that can directly neutralize radiation-induced reactive oxygen species and reactive nitrogen species. It also serves as an essential substrate for glutathione peroxidase, catalyzing the reduction of hydrogen peroxide and lipid peroxides, and blocking the oxidative stress chain reaction.

[0011] Preferably, the carrier is mannitol; mannitol, as a "drying skeleton", co-precipitates with reduced glutathione to form rigid microspheres, providing physical support to prevent collapse; at the same time, mannitol regulates the aerodynamic diameter of the particles to 1μm to 3μm, avoiding upper respiratory tract retention or exhalation loss; the low hygroscopicity of mannitol, with a critical relative humidity ≥85%, forms a barrier on the particle surface, significantly inhibiting the hygroscopic agglomeration of reduced glutathione, ensuring storage stability and dosage uniformity.

[0012] Preferably, the dispersant is L-leucine; leucine molecules are enriched at the gas-liquid interface of the particles through hydrophobic isobutyl side chains, forming a molecular-level hydrophobic coating. This structure can effectively shield the van der Waals forces and electrostatic interactions between particles, resulting in a powder repose angle <35° and a Karl Fischer index <15%, achieving excellent flowability; under the action of inhaled airflow, the leucine layer generates a "lubricating effect," causing the microspheres to deagglomerate instantaneously, increasing the fine particle fraction to over 50%, ensuring a high proportion of drug penetrates deep into the alveoli; at the same time, its hydrophobic properties slow down the alveolar fluid dissolution rate, achieving sustained release of reduced glutathione and prolonging the duration of action.

[0013] The preferred method for preparing glutathione dry powder inhaler is as follows: Reduced glutathione, a carrier, and a dispersant are dissolved in water to obtain a mixed solution; the mixed solution is then spray-dried to obtain a glutathione dry powder inhaler.

[0014] Preferably, the spray drying conditions are: inlet temperature of 80℃~90℃, feed rate of 3mL / min~6mL / min, and gas flow rate of 600L / h~800L / h. By controlling the spray drying conditions, the thermal decomposition of GSH is avoided, and the particle size, morphology and component distribution of glutathione dry powder inhaler are synergistically controlled to meet the requirements for deposition in the lower respiratory tract and lungs, and to achieve the retention of glutathione activity during the spray drying process.

[0015] Preferably, the solid content in the mixed solution is 0.5wt% to 2.5wt%. By controlling the solid content in the suspension, the solution viscosity and drying efficiency are balanced, and nozzle clogging or the formation of excessively large particles is avoided.

[0016] Preferably, the inlet temperature and gas flow rate are maintained for 10 to 30 minutes after spraying to completely remove residual moisture; preferably, the moisture content of the glutathione dry powder inhaler is <2%, which improves long-term storage stability by reducing the hygroscopicity of the powder.

[0017] Compared with the prior art, the present invention has the following technical effects: This invention uses reduced glutathione as the core active ingredient. The addition of a carrier and a dispersant maintains the particle dispersibility and prevents glutathione aggregation. At the same time, L-leucine is used as a dispersant. L-leucine molecules are enriched at the gas-liquid interface of the particles to form a molecular-level hydrophobic coating, achieving excellent flowability. Furthermore, the "lubricating effect" generated by the leucine layer promotes the instantaneous deagglomeration of microspheres, ensuring a high proportion of drug penetrates deep into the alveoli. The hydrophobic properties also slow down the dissolution rate of alveolar fluid, achieving sustained release of reduced glutathione and prolonging the duration of action. Glutathione dry powder inhaler delivers high concentrations of reduced glutathione precisely to the alveolar damage area via a lung-targeted delivery system. It simultaneously activates endogenous antioxidant enzymes, specifically upregulating the activity of superoxide dismutase and glutathione peroxidase in lung tissue, and reducing malondialdehyde-induced nitric oxide synthase expression. This blocks the oxidation-inflammatory cascade, significantly reducing pro-inflammatory factor levels, alleviating alveolar consolidation and inflammatory infiltration, and delaying the progression of pulmonary fibrosis. Its antioxidant efficacy is superior to tracheal administration of GSH solution and oral NAC, providing a new and highly effective targeted therapy strategy for RILI.

[0018] The glutathione dry powder inhaler prepared in this invention directly targets the core area of ​​radiation damage, forming a high concentration of drug accumulation at the site of oxidative stress. Simultaneously, through multi-target synchronous regulation, it disrupts the core pathological cycle of RILI (radiation-induced irritation syndrome). Furthermore, local administration significantly reduces the risk of systemic exposure and avoids the immunosuppressive side effects of glucocorticoids. Attached Figure Description

[0019] Figure 1 The image shows a SEM image of the glutathione dry powder inhaler prepared in Example 1.

[0020] Figure 2 This study aimed to detect oxidative stress markers in lung tissue after different drug administrations; where A represents iNOS, B represents MDA, C represents SOD, and D represents GSH-PX.

[0021] Figure 3 Lung index after different drug administration treatments.

[0022] Figure 4HE staining of lung tissues after different drug treatments; where A1 is HE staining of lung tissue in the Nor group, A2 is a magnified view of box 1 in A1, B1 is HE staining of lung tissue in the RILI group, B2 is a magnified view of box 1 in B1, C1 is HE staining of lung tissue after treatment with GSH sol (IPA / qd), C2 is a magnified view of box 1 in C1, D1 is HE staining of lung tissue after treatment with GSH sol (IPA / q5d), D2 is a magnified view of box 1 in D1, E1 is HE staining of lung tissue after treatment with NAC sol (IPA / qd), E2 is a magnified view of box 1 in E1, F1 is HE staining of lung tissue after treatment with NAC sol (IPA / q5d), F2 is a magnified view of box 1 in F1, G1 is HE staining of lung tissue after treatment with GSH DPI (IPA / qd), G2 is a magnified view of box 1 in G1, H1 is HE staining of lung tissue after treatment with GSH DPI (IPA / qd), G2 is a magnified view of box 1 in G1, and H1 is HE staining of lung tissue after treatment with GSH DPI (IPA / qd). HE staining of lung tissue after treatment with sol (ig / qd), H2 is an enlarged view of box 1 in H1, I1 is HE staining of lung tissue after treatment with NAC sol (ig / qd), and I2 is an enlarged view of box 1 in I1.

[0023] Figure 5 The percentage of the area stained by HE in lung tissue after different drug administration treatments.

[0024] Figure 6 The values ​​represent the mRNA levels of inflammatory factors in lung tissue after different drug administrations; where A represents IL-1β, B represents CXCL-16, C represents IL-17, D represents IL-6, E represents TNF-α, F represents IL-1α, G represents IFN-γ, and H represents COX-2.

[0025] Figure 7 The levels and proportions of inflammatory cells in peripheral blood after different drug treatments are shown; where A is WBC, B is LYM percentage, C is MID percentage, D is GRAN percentage, E is LYM count, F is MID count, and G is GRAN count. Detailed Implementation

[0026] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0027] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0028] The English names for nitric oxide synthase are nitric oxide synthase (iNOS); malondialdehyde (MDA); superoxide dismutase (SOD); glutathione peroxidase (GSH-Px); neutrophils (GRAN); monocytes (MID); lymphocytes (LYM); and hematoxylin-eosin staining (HE staining).

[0029] Example 1 A glutathione dry powder inhaler.

[0030] Prescription 1 is as follows: GSH 600mg; L-leucine 300mg; mannitol 100mg; pure water 40mL.

[0031] The specific preparation method is as follows: The prescribed amounts of GSH, L-leucine, and mannitol were dissolved in 40 mL of pure water, and then filtered through a 0.45 μm filter membrane to obtain the drug solution.

[0032] The drug solution was added to a spray dryer for spray drying. The inlet temperature was set to 80℃, the spray flow rate was 1mL / min, the fan power was 100%, and the gas flow rate was 800L / h. After spraying, the gas inlet temperature and gas flow rate were maintained for 30 minutes to obtain glutathione dry powder inhaler.

[0033] Example 2 A glutathione dry powder inhaler.

[0034] Prescription 2 is as follows: GSH 500mg; L-leucine 300mg; mannitol 200mg; pure water 40mL.

[0035] The specific preparation method is as follows: The prescribed amounts of GSH, L-leucine, and mannitol were dissolved in 40 mL of pure water, and then filtered through a 0.45 μm filter membrane to obtain the drug solution.

[0036] The drug solution was added to a spray dryer for spray drying. The inlet temperature was set to 80℃, the spray flow rate was 1mL / min, the fan power was 100%, and the gas flow rate was 800L / h. After spraying, the gas inlet temperature and gas flow rate were maintained for 30 minutes to obtain glutathione dry powder inhaler.

[0037] Example 3 A glutathione dry powder inhaler.

[0038] Prescription 3 is as follows: GSH 600mg; L-leucine 200mg; mannitol 200mg; pure water 40mL.

[0039] The specific preparation method is as follows: The prescribed amounts of GSH, L-leucine, and mannitol were dissolved in 40 mL of pure water, and then filtered through a 0.45 μm filter membrane to obtain the drug solution.

[0040] The drug solution was added to a spray dryer for spray drying, with the inlet temperature set at 80℃, the spray flow rate at 1mL / min, the fan power at 100%, and the gas flow rate at 800L / h. After spraying, the gas inlet temperature and gas flow rate were maintained for 30 minutes to obtain glutathione dry powder inhaler.

[0041] Experimental test: 1. Scanning electron microscopy characterization.

[0042] like Figure 1 As shown, the glutathione dry powder inhaler obtained in Example 1 is a white porous microsphere. Scanning electron microscopy shows that the surface is wrinkled, which is characteristic of leucine. The bulk density is 0.10~0.25 g / cm³. 3 It can be directly loaded into HPMC capsules and used with standard inhalers.

[0043] 2. In vivo testing.

[0044] The glutathione dry powder inhaler prepared in Example 1 was selected for dry powder administration, and GSH raw material was selected to prepare a solution for administration; the drug preparation is as follows: Weigh 12.5 mg of GSH into an EP tube, add 500 μL of PBS to dissolve, and obtain a 25 mg / mL GSH solution; weigh 62.5 mg of GSH into an EP tube, add 500 μL of PBS to dissolve, and obtain a 125 mg / mL GSH solution; weigh 7.5 mg of GSH into an EP tube, add 500 μL of PBS to dissolve, and obtain a 15 mg / mL NAC solution; weigh 37.5 mg of GSH into an EP tube, add 500 μL of PBS to dissolve, and obtain a 75 mg / mL NAC solution.

[0045] Male C57BL / 6J mice, aged 6-8 weeks and weighing 20-25g, were selected and housed in the animal facility of the School of Pharmacy, Fujian Medical University, under a 12-hour / 12-hour light-dark cycle with free access to water and food. This study was approved by the Experimental Animal Ethics Committee of Fujian Medical University (No.: FJMU IACUC 2021-0465). Mice were anesthetized with 70 mg / kg of 3% sodium pentobarbital via intraperitoneal injection and fixed in a specially designed device. A single 6 MV X-ray irradiation of approximately 1.8 cm width of the mouse lungs was performed using a linear accelerator (Clinac 600C / D) at a dose rate of 500 cGy / min, resulting in a total irradiation dose of 18 Gy and an absorbed dose of 15 Gy, thus establishing a recurrent respiratory injury (RILI) model.

[0046] Based on previous research, daily and five-day dosing groups were established to investigate the immediate and sustained-release effects of GSH and NAC, as well as the stimulating effect of intratracheal administration of liquid on the lungs of mice. The specific administration methods for the RILI model mice are as follows:

[0047] Each mouse received the following treatments via intratracheal inhalation: 50 μL of 25 mg / mL GSH solution (GSH sol / qd); 50 μL of 125 mg / mL GSH solution (GSH sol / q5d); 50 μL of 15 mg / mL NAC solution (NAC sol / qd); 50 μL of 75 mg / mL NAC solution (NAC sol / q5d); intratracheal administration of 50 mg / kg of the GSH powder preparation prepared in Example 1 (GSH DPIs / qd); and oral administration of 50 μL of 25 mg / mL GSH solution (GSH DPIs / qd). The mice were divided into two groups: the NAC sol (ig / qd) group and the NAC sol (ig / qd) group. Each mouse was orally administered 50 μL of 15 mg / mL NAC solution. Mice with the RILI model received no treatment and were designated as the RILI group. Normally sham-irradiated mice served as controls and were designated as the Nor group. Administration was started on day 1 post-irradiation and continued daily orally or intratracheally for 14 days. Mice were sacrificed on day 14, and blood and lung tissue were collected for subsequent analysis.

[0048] like Figure 2As shown, the indicators representing oxidative stress damage, iNOS and MDA, significantly increased after lung injury. In the GSH DPIs (IPA / qd) group treated with the GSH dry powder formulation prepared in Example 1, the levels of MDA and iNOS in lung tissue were lower than in the RILI model group, and superior to those in the GSH solution inhalation and oral GSH solution groups. The lung homogenate SOD and GSH-Px activities in the GSH DPIs (IPA / qd) group were also increased compared to the model group, showing better effects than the solution inhalation and oral groups.

[0049] like Figure 3 As shown, the lung index in the RILI group was significantly increased, indicating significant pulmonary edema, while the lung index in the dry powder group was significantly lower than that in the RILI group; and neither inhalation nor oral administration of the solution had any beneficial effect on the lung index.

[0050] like Figure 4 As shown in (A) and (B) in the figures, the RILI model group showed impaired alveolar structure, lung tissue consolidation, and inflammatory cell infiltration compared to the Nor group. Figure 3 As shown in (G), the GSH dry powder preparation prepared in Example 1 significantly improved the pathological condition in each mouse after administration of the dry powder to each mouse, and the effect was better than that of GSH solution inhalation therapy and GSH solution oral therapy.

[0051] like Figure 5 As shown, compared with the HE lung tissue area of ​​normal sham-irradiated mice, the percentage of HE lung tissue area in the RILI model group was significantly increased; after each mouse was treated with the GSH dry powder preparation prepared in Example 1, the percentage of HE lung tissue area was significantly reduced, and the effect was better than that of mice treated with GSH solution inhalation and GSH solution oral administration.

[0052] like Figure 6 RT-qPCR results showed that, compared to the RILI model group, the dry powder group showed significantly reduced mRNA levels of IL-1β, IL-6, TNF-α, IL-1α, IL-17, COX-2, CXCL-16, and IFN-γ in lung tissue after treatment, and exhibited superior anti-inflammatory effects compared to mice treated with GSH solution inhalation and oral GSH solution. This indicates that the glutathione dry powder inhaler prepared in this embodiment of the invention comprehensively inhibits the RILI process by blocking the oxidative stress-inflammation positive feedback loop.

[0053] like Figure 7 Peripheral blood analysis showed that the GSH dry powder formulation prepared in Example 1, when administered to each mouse, restored the radiation-induced elevated GRAN and MID levels to normal and corrected the LYM reduction.

[0054] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0055] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of glutathione dry powder inhaler in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, The active ingredient of the glutathione dry powder inhaler is prepared by the following components by mass percentage: 50%–60% reduced glutathione, 10%–20% carrier, 20%–30% dispersant, totaling 100%; The carrier is mannitol, and the dispersant is L-leucine.

2. The application of the glutathione dry powder inhaler according to claim 1 in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, Drugs for treating acute lung injury caused by radiation are drugs for treating lung injury caused by radiation-induced oxidative stress.

3. The application of the glutathione dry powder inhaler according to claim 1 in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, The geometric particle size of the glutathione dry powder inhaler is 0.5 μm to 5 μm.

4. The application of the glutathione dry powder inhaler according to claim 1 in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, The preparation method of the glutathione dry powder inhaler is as follows: Reduced glutathione, carrier, and dispersant were dissolved in water to obtain a mixed solution; The mixed solution was spray-dried to obtain glutathione dry powder inhaler.

5. The application of the glutathione dry powder inhaler according to claim 3 in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, The solid content in the mixed solution is 0.5wt% to 2.5wt%.

6. The application of the glutathione dry powder inhaler according to claim 3 in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, The spray drying conditions are: inlet temperature of 80℃~90℃, feed rate of 3mL / min~6mL / min, and gas flow rate of 600L / h~800L / h.

7. The application of the glutathione dry powder inhaler according to claim 3 in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, After spray drying is complete, continue to maintain the gas inlet temperature and gas flow rate for 10 to 30 minutes to remove moisture.

8. The application of the glutathione dry powder inhaler according to claim 3 in the preparation of drugs for treating acute radiation-induced lung injury, characterized in that, The water content of glutathione dry powder inhaler is <2%.

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