Use of 6-paradol and a pharmaceutical composition for preventing and / or treating dry eye

Abstract

The application of 6-Paradol and a drug composition for preventing and / or treating dry eye belong to the technical field of pharmaceutical preparation and ophthalmology. Through the combination of β-cyclodextrin inclusion and dimethyl sulfoxide (DMSO) synergistic solubilization, and through four administration ways of eye drops, eye gel, nasal spray and eye atomization solution, the solubility and chemical stability of 6-Paradol in water phase are significantly improved, so that it can be made into clear and sterilizable eye / nasal liquid preparation. The drug breaks through the ocular surface barrier, so that the drug can effectively act on the ocular surface and secrete stably. A treatment mode of nerve reflex secretion is constructed, covering two types of dry eye of water deficiency and excessive evaporation.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of pharmaceutical formulation and ophthalmology, specifically relating to a 6-Paradol (6-gingerol) compound ophthalmic preparation and its application in the preparation of drugs for treating dry eye syndrome. It is a compound pharmaceutical preparation and its preparation method that uses β-cyclodextrin inclusion combined with dimethyl sulfoxide (DMSO) for synergistic solubilization and is administered via four routes: eye drops, ophthalmic gel, nasal spray, and ocular nebulization solution, for the treatment of dry eye syndrome, meibomian gland dysfunction, and related ocular surface inflammation. Background Technology

[0002] Dry eye disease (DED) is a multifactorial ocular surface disease caused by tear film homeostasis imbalance, with a global prevalence of approximately 5-50%. Its core pathological mechanisms involve insufficient tear secretion, excessive tear film evaporation, and a cascade of ocular surface inflammation. Currently, first-line clinical treatment primarily relies on artificial tears, supplemented by anti-inflammatory drugs such as corticosteroids and cyclosporine A. However, this approach has drawbacks including limited target action, significant adverse reactions (increased intraocular pressure, immunosuppression), and poor patient compliance.

[0003] 6-Paradol is an alkylphenol active ingredient extracted from the seeds of African chili pepper (Aframomum melegueta). It has a carbon chain length of C10 and has higher lipid solubility and better TRPV1 receptor selectivity compared to 6-gingerol, which belongs to the same ginger family. Previous studies have suggested that Paradol has anti-inflammatory, antioxidant and metabolism-promoting effects, but its application in the treatment of dry eye has not been reported. The main technical obstacles are as follows: (1) Physicochemical defects: Paradol has extremely poor water solubility (<0.02 mg / mL) and is unstable at physiological pH. It is easily oxidized and degraded, making it difficult to make a clear ophthalmic liquid preparation; (2) Delivery barrier: The corneal epithelium is tightly connected to form a strong barrier. The bioavailability of traditional eye drops is <5%, and the drug is difficult to reach the meibomian glands and the deep surface of the eye; (3) Single mechanism: Most existing dry eye preparations are "single-point administration-single-target action" and lack a multimodal approach that takes into account both local anti-inflammatory and neuroreflex regulation. Cyclodextrin inclusion technology has been used to improve the ophthalmic delivery of hydrophobic drugs. Currently marketed ophthalmic products containing cyclodextrin include 1% chloramphenicol eye drops (Clorocil) containing methylated β-CD, 0.1% diclofenac eye drops (Voltaren Ophtha) containing 2-hydroxypropyl-γ-CD, and 0.1% indomethacin eye drops (Indocollyre) containing 2-hydroxypropyl-β-CD. However, for the specific active ingredient Paradol, due to its special requirements for the size of the inclusion cavity and the polarity environment, cyclodextrin alone cannot achieve the required drug loading for treatment. Furthermore, there are no formulations supporting four routes: eye drops, nasal spray, gel, and ophthalmic atomization. These four routes address different clinical needs, such as maintaining mild to moderate dry eye, providing long-lasting overnight hydration, rapidly relieving acute attacks, and treating severe dry eye, demonstrating significant practical value.

[0004] Therefore, the problems that need to be solved are: (1) how to significantly improve the solubility and chemical stability of 6-Paradol in the aqueous phase so that it can be made into a clear, sterile ophthalmic / nasal liquid preparation; (2) how to break through the ocular surface barrier so that the drug can effectively act on the ocular surface to promote secretion and stabilize the tear film; (3) how to construct a treatment mode of neuroreflex secretion to cover both aqueous-deficient and evaporative dry eye. Summary of the Invention

[0005] To address the aforementioned problems, the present invention first provides the application of 6-Paradol, specifically its use in the preparation of medicaments for the prevention and / or treatment of dry eye syndrome.

[0006] Secondly, the present invention also provides a pharmaceutical composition for the prevention and / or treatment of dry eye syndrome, wherein the active ingredient of the pharmaceutical composition is composed of 6-Paradol, and the pharmaceutical composition further comprises a solubilizing carrier, a synergistic solubilizer, a pH adjuster, excipients and / or a formulation matrix.

[0007] Specifically, it is prepared from the following components: Active ingredient: 6-Paradol, 0.001%-1.0% (w / v); preferably 0.01%~0.1% (w / v).

[0008] Solubilizing carrier: sulfobutyl ether-β-cyclodextrin (SBE-β-CD) or hydroxypropyl-β-cyclodextrin (HP-β-CD), 5%-20% (w / v).

[0009] Synergistic cosolvent: Dimethyl sulfoxide (DMSO), less than 5% (w / v), is used to disrupt intermolecular hydrogen bonds of Paradol and assist cyclodextrin in forming inclusion complexes with high drug loading.

[0010] Formulation matrix: Thermosensitive gel material (poloxamer 407 / carbomer 980), nasal mucosal absorption enhancer or nebulization solubilizer (mannitol) are selectively added according to the route of administration.

[0011] Excipients: Glycerin (humectant, 0-2% w / v), Sodium chloride (isotonic regulator, 0.9% w / v), Benzalkonium chloride (preservative, 0.005%-0.01% w / v), EDTA-2Na (metal chelating agent, 0.025%-0.05% w / v).

[0012] Buffer solution: Use an appropriate amount of PBS buffer or triethanolamine (TEA) to adjust the pH to 7.0~7.4.

[0013] The molar ratio of 6-Paradol to the solubilizing carrier (β-cyclodextrin derivative) is 1:1.5~1:3, and the inclusion constant Ka of the resulting non-covalent inclusion complex is ≥2×10⁻⁶. 4 M⁻¹. The solubility of 6-Paradol was determined by pH 7.4 PBS at 25℃. Excess 6-Paradol was added to buffer solutions containing different concentrations of SBE-β-CD. After equilibration by shaking for 72 h, the drug concentration was determined by HPLC. The inclusion constant Ka was calculated to be 2.3 × 10^4 M⁻¹. The inclusion rate was ≥99% at a molar ratio of 1:2, which meets the drug loading requirements of the formulation.

[0014] Furthermore, the pharmaceutical composition is an eye drop, which is prepared using the following substances; 6-Paradol, 0.01%-0.1% w / v; Solubilizing carrier, 5%-20% w / v; Dimethyl sulfoxide 0.1-5% w / v; Mannitol, 0.5% w / v; Benzalkonium chloride, 0.005-0.01% w / v; EDTA-2Na, 0.025-0.05% w / v; An appropriate amount of PBS buffer.

[0015] Furthermore, the pharmaceutical composition is an ophthalmic gel, which is prepared using the following substances; 6-Paradol, 0.01-0.1% w / v; Solubilizing carrier, 5-20% w / v; Dimethyl sulfoxide <0.1% w / v; Mannitol, 1.5% w / v; Benzalkonium chloride, 0.005%-0.01% w / v; EDTA-2Na, 0.025%-0.05% w / v; Triethanolamine, in appropriate amounts.

[0016] Furthermore, the pharmaceutical composition is a nasal spray, which is prepared using the following substances; 6-Paradol, 0.01%-0.1% w / v; Solubilizing carrier, 5%-20% w / v; Dimethyl sulfoxide 0.1-5% w / v; Benzalkonium chloride, 0.005-0.01% w / v; Sodium chloride 0.9% w / v; An appropriate amount of PBS buffer.

[0017] Furthermore, the pharmaceutical composition is an ophthalmic atomizing agent, which is prepared using the following substances; 6-Paradol, 0.01%-0.1% w / v; Solubilizing carrier, 5% w / v; Dimethyl sulfoxide 0.1-5% w / v; Mannitol, 0.5% w / v; Benzalkonium chloride, 0.005-0.01% w / v; EDTA-2Na, 0.025-0.05% w / v; An appropriate amount of PBS buffer.

[0018] The present invention also provides a preparation method, the method comprising the following steps: Step 1: Preparation of Mother Solution 1: Accurately weigh 10.0 mg of 6-Paradol, add 1.0 mL of DMSO and vortex to dissolve, to obtain 10 mg / mL Mother Solution 1 (protect from light, store at 4℃, shelf life 7 days).

[0019] Step 2 Preparation of Mother Solution 2: Take 1.0 mL of Mother Solution 1, add 9.0 mL of DMSO, vortex to mix, and obtain 1 mg / mL Mother Solution 2 (protect from light and store at room temperature).

[0020] Step 3: Preparation of intermediate stock solution: Take 1.0 mL of stock solution 2 and add 9.0 mL of physiological saline to obtain 0.1 mg / mL intermediate stock solution (protect from light, store at 4℃, shelf life 3 days).

[0021] Step 4: Inclusion carrier solution: Take SBE-β-CD or HP-β-CD, dissolve it in sterile physiological saline, stir until clear, and obtain a 5%-20% w / v cyclodextrin solution.

[0022] Step 5: Preparation of final formulation: Take an appropriate amount of mother liquor according to the target concentration, add cyclodextrin solution, vortex mix, add excipients (mannitol / glycerol / sodium chloride / benzalkonium chloride / EDTA-2Na), and adjust pH to 7.0-7.4.

[0023] Step 6 Sterilization: Sterilize by positive pressure filtration through a 0.22μm PVDF filter membrane (pressure ≤0.2 bar), dispense into sterile containers, and store at 2-8℃ away from light.

[0024] The final formulation contains ≤1.0% DMSO (preferably 0.1%), which is far below the corneal / nasal mucosa irritation threshold (5%). DMSO is embedded in the cyclodextrin cavity during inclusion, resulting in extremely low levels of free DMSO residue (GC determination <0.1%), ensuring the safety of long-term use.

[0025] The beneficial effects of this invention are that it significantly improves the solubility and chemical stability of 6-Paradol in the aqueous phase, enabling it to be formulated into a clear, sterilizable ophthalmic / nasal liquid preparation; the drug can penetrate the ocular surface barrier, allowing it to effectively act on the ocular surface to promote secretion and stabilize the tear film; and it establishes a treatment mode of promoting secretion through neuroreflexion, covering both aqueous-deficient and evaporative dry eye types. Attached Figure Description

[0026] Figure 1 The results of tear secretion function measurement in each group of mice in Example 1; Figure 2 The results of tear film breakup time (BUT) measurements in each group of mice in Example 1 are shown. Figure 3 This is a graph showing the tear secretion function measurement in Example 3; Figure 4This is a graph showing the tear film breakup time measurement in Example 3. Detailed Implementation

[0027] The following explanation and description of this case will be provided in conjunction with the accompanying drawings.

[0028] Example 1: Preparation of 6-Paradol eye drops (0.01% and 0.1%) Core prescription: Active ingredient: 0.01% or 0.1% (w / v) 6-Paradol • Solubilizer: 20% (w / v) SBE-β-CD • Co-solvent: 10% (w / v) DMSO (in mother liquor), final concentration calculated to meet standards.

[0029] • Solvent: Sterile physiological saline, pH 7.0~7.4 (adjusted with PBS).

[0030] Preparation process: 1. Stock solution 1 (10 mg / mL): Accurately weigh 50 mg of 6-Paradol, add 5 mL of DMSO, vortex to dissolve, and store at room temperature protected from light.

[0031] 2. Mother liquor 2 (1 mg / mL): Take 100 μL of mother liquor 1, add 900 μL of DMSO, vortex mix, and store at room temperature away from light.

[0032] 3. Inclusion carrier: Take 2 g of SBE-β-CD, dissolve it in 10 mL of sterile physiological saline, stir until clear, and obtain a 20% SBE-β-CD solution.

[0033] 4. Preparation of final formulation: 0.1% 6-Paradol eye drops: Take 100 μL of stock solution 1, add 900 μL of 20% SBE-β-CD solution, vortex to mix, and sterilize through a 0.22 μm filter membrane;

[0034] 0.01% 6-Paradol eye drops: Take 100 μL of stock solution 2, add 900 μL of 20% SBE-β-CD solution, vortex to mix, and sterilize through a 0.22 μm filter membrane.

[0035] 5. DMSO final concentration calculation: The calculated DMSO volume fraction in the 0.1% formulation was 1.0%; the DMSO volume fraction in the 0.01% formulation was 0.1%. Although the 0.1% group was slightly higher than the conventional threshold, no obvious irritation was observed during the short-term dosing period of this experiment.

[0036] Quality inspection results Appearance: Colorless to pale yellow clear liquid, without precipitation.

[0037] pH value: 7.0±0.05.

[0038] Osmotic pressure: 298±5 mOsm / kg, isotonic with tears.

[0039] Stability: After 3 months of accelerated testing at 40℃ / RH75%, the 6-Paradol content, as determined by HPLC, remained above 98.5%, with no significant degradation.

[0040] I. Pharmacodynamic Animal Experiment Data To evaluate the therapeutic effects of 0.1% and 0.01% 6-Paradol eye drops on a mouse model of dry eye, this invention established a mouse dry eye model using a low-humidity environment combined with subcutaneous injection of scopolamine. Tear secretion (Schirmer test) and tear film breakup time (BUT) were observed after administering the eye drops. Results showed that both concentration groups significantly improved dry eye symptoms with good safety, with the 0.1% concentration group showing more significant efficacy.

[0041] 1. Grouping 160 SPF-grade C57BL / 6J female mice (8 weeks old, 20±2g) were selected and divided into 4 groups for animal experiments on each formulation. One eye of each mouse was included in the experiment. Control group 1 (Normal): Normally fed, no modeling was performed, and 3 μL of physiological saline was administered as eye drops.

[0042] Control group 2 (Vehicle): Dry eye model was created, and 3 μL of 20% SBE-β-CD solution containing the corresponding proportion of DMSO (solvent control) was administered as eye drops.

[0043] Experimental group 1 (0.01% 6-Paradol): Dry eye model was created, and 3 μL of 0.01% 6-Paradol eye drops were administered.

[0044] Experimental group 2 (0.1% 6-Paradol): Dry eye model was created, and 3 μL of 0.1% 6-Paradol eye drops were administered.

[0045] 2. Establishment of a dry eye model From D1 to D19, mice were placed in a low-humidity environment (humidity ≤40%) and were injected subcutaneously into the neck and back of the neck with 0.75 mg / mL scopolamine hydrobromide, 0.3 mL / mouse / time, twice a day (09:30 and 15:30).

[0046] 3. Dosing regimen Dosing period: D5~D19 (intervention begins on the 5th day after model initiation).

[0047] Frequency: 2 times / day, 6 hours apart.

[0048] Dosage volume: 3 μL / dose.

[0049] Route of administration: eye drops.

[0050] 4. Observation Indicators and Detection Methods Schirmer's test for tear secretion function: The phenol red cotton thread method was used. The cotton thread was fixed in the middle of the lower eyelid conjunctival sac for 30 seconds, and the wet length (mm) was measured.

[0051] Tear film breakup time (BUT) measurement: 1 μL of 0.05% sodium fluorescein saline solution was used to stain the tear film. The patient blinked three times manually, and the time (s) for the first dry spot to appear was recorded under cobalt blue light from a handheld slit lamp.

[0052] Detection time points: before modeling (D1), onset of drug administration (D5), mid-drug administration (D10), and end of drug administration (D19).

[0053] 5. Experimental Results Tear secretion function (Schirmer's test) like Figure 1 As shown, the tear secretion function of mice in each group was measured (Schirmer's Test: length of phenol red cotton thread wetted, unit: mm). After dry eye modeling (D5), the tear secretion of mice in each group decreased significantly compared with D1 (P<0.01). From D10, the tear secretion of both treatment groups began to recover. By D19, the tear secretion length of the 0.01% group and the 0.1% group was significantly higher than that of the solvent control group (P<0.01). Moreover, the improvement in the 0.1% group was better than that in the 0.01% group, showing a certain dose-dependent trend. Compared with the control group, the treatment group showed no significant difference (P<0.01).

[0054] Tear film breakup time (BUT) Figure 2 The tear film breakup time (BUT) measurements (in seconds) for each group of mice were consistent with the tear secretion results, showing a significant shortening of BUT after modeling. After drug treatment, measurements at D10 and D19 showed that the BUT in the 0.01% and 0.1% groups was significantly longer than that in the solvent control group (P<0.01). The average BUT in the 0.1% group reached 9.89 seconds on D19, close to the level of the normal group, indicating that it effectively stabilized the tear film.

[0055] II. Safety Evaluation Data Eye observation: Throughout the experiment, the mice in each drug administration group showed no redness or hair loss around their eyes; the corneas were transparent, the irises were not adhered, and no obvious congestion or edema was observed.

[0056] FSC score: According to the Draize eye irritation test standard, the mice were observed at 24 h, 48 h and 72 h after single and multiple administrations of 0.1% 6-Paradol eye drops. The scores of the cornea, iris and conjunctiva of the mice were all 0, indicating that there was no obvious eye irritation.

[0057] Histology: No obvious pathological changes were found in the nasal and ocular surface tissues.

[0058] 5.1 Draize eye irritation score: Vehicle control group (four dosage forms) 0 / 0 0 / 0 0 / 0 0 / 0 Non-irritating 0.01% group (gel / nasal spray) 0 / 0 0 / 0 0 / 0 0 / 0 Non-irritating 0.01% group (four dosage forms) 0 / 0 0 / 0 0 / 0 0 / 0 Non-irritating 0.1% group (eye drops / nebulizer) 0 / 0 0 / 0 1 / 0 1 / 0 Very minor (recoverable) Note: In the 0.1% nebulization group, 2 out of 10 mice developed mild conjunctival hyperemia (score 1) on day 10, which resolved spontaneously within 24 hours and did not affect subsequent drug administration. On day 19, all mice had a score of 0.

[0059] This invention establishes a mouse dry eye model and verifies the efficacy of 6-Paradol eye drops.

[0060] The proposed mechanism of action is that 6-Paradol, as an agonist of the capsaicin receptor (TRPV1), may improve dry eye symptoms by activating the trigeminal reflex arc and promoting basal lacrimal secretion. Pretreatment of dry eye model mice with the TRPV1 antagonist capsaicin (10 μM) significantly inhibited the lacrimal secretion-promoting effect of 0.1% 6-Paradol eye drops (P < 0.01), confirming that its mechanism is related to TRPV1 receptor activation.

[0061] Dose-effect: The 0.1% concentration group showed a trend of being superior to the 0.01% group in both increasing tear secretion and prolonging BUT, suggesting that the efficacy of 6-Paradol is dose-dependent within a certain range.

[0062] Formulation advantages: The SBE-β-CD inclusion technology significantly improves the water solubility of 6-Paradol, and combined with the permeation-enhancing effect of DMSO, it ensures the bioavailability of the drug.

[0063] Based on the experiment and its results, it can be seen that: Both 0.01% and 0.1% 6-Paradol eye drops significantly improved insufficient tear secretion and tear film instability in dry eye model mice.

[0064] Neither concentration of the eye drops showed significant eye irritation during the experimental period, indicating good safety.

[0065] 5.2 Nasal and respiratory tract safety test results: Nasal mucosal congestion none none none Nasal secretions none none Occasionally seen in small quantities (2 / 10, D10) sneezing frequency normal normal normal respiratory rate normal normal normal Weight change (D1→D19) +2.5±0.8 g +2.3±0.6 g +2.1±0.7 g

[0066] 5.3 Acute toxicity test Single administration: 72 h after a single administration of the highest concentration (0.1%) of the four dosage forms, no animal deaths, no significant weight loss (<5%), and Draize score ≤1.

[0067] Long-term toxicity: After 14 days of administration twice daily, blood biochemical tests (ALT, AST, BUN, Cr) were all within the normal range, and the coefficients of major organs (heart, liver, spleen, lung, kidney) showed no significant changes (P>0.05).

[0068] III. Stability Data 6.1 Accelerated stability test (40 / 75% RH) October 99.2 98.8 January 98.5 98.0 March 97.1 96.5 June 95.3 94.6 December 92.8 91.5 24 months 88.3 86.7 Conclusion: The content decrease under accelerated conditions follows first-order degradation kinetics; 0.01% group t 90 ≈36 months, 0.1% group t 90 ≈30 months. Tentative shelf life 24 months (refrigerated at 2-8℃).

[0069] 6.2 Long-term stability test (25 / 60% RH) The 0.01% group had a content of 98.5% (t) in December. 90 ≈58 months); 0.1% group had a 12-month content of 98.0% (t). 90 ≈52 months); 0.001% group had a 97.8% content (t) at 12 months. 90 (Approximately 46 months). Degradation products were identified as oxides (≤0.05%) and isomers (≤0.03%), with total impurities <0.5%.

[0070] 6.3 Drug stability before and after nebulization The content was 100% before atomization and 98.5% after atomization (loss of 1.5%), with a pH change of <0.05, confirming that the atomization process had no significant effect on the stability of Paradol.

[0071] III. Pharmacological and Toxicological Mechanisms 7.1 Mechanism of Action Neuroreflex-induced secretion: 6-Paradol, as a selective agonist of TRPV1 receptors, activates trigeminal nerve endings, promoting basal tear secretion via the trigeminal nerve-lacrimal gland reflex arc and prolonging tear film breakup time. The ophthalmic gel forms in situ at 34°C, extending its retention time on the ocular surface to 8 hours, providing targeted therapeutic effects for evaporative dry eye.

[0072] Example 2: Preparation of 6-Paradol ophthalmic gel (0.01% and 0.1%) Prescription composition (10 g per dose): Carbopol 980 30mg 30mg 0.3%w / w gel matrix 6-Paradol 1.0mg 0.1mg 0.1% / 0.01% Active ingredients Triethanolamine (TEA) ≈45 mg ≈45mg 0.45%w / w Neutralizing agent Mannitol 150mg 150mg 1.5% w / w Osmotic pressure regulation benzalkonium chloride 1.0mg 1.0mg 0.01% w / w preservative EDTA-2Na 5.0mg 5.0mg 0.05% w / w Metal chelating agents HP-β-CD 800mg 800mg 8% w / w Solubilizing carrier Polosham 407 2.0g 2.0g 20% w / w Thermosensitive materials Water for Injection Add to 10g Add to 10g margin solvent DMSO (in mother liquor) 1.0 μL 0.1μL <0.1% w / v Mother liquor for solubilization

[0073] Preparation process (swelling-neutralization method SOP, 10g per dose):

[0075] Rheological characterization data: Test instrument: TA Instruments AR2000ex rheometer, cone plate fixture (diameter 40 mm, cone angle 2°), temperature control accuracy ±0.1℃.

[0076] Power-law model fitting: 0.1% group n=0.58, K=11.2 Pa·sⁿ, R²=0.995; 0.01% group n=0.60, K=10.5 Pa·sⁿ, R²=0.994.

[0077] Temperature scanning of gel-sol transition temperature (Tgel): 0.1% group ≈ 33.5℃; 0.01% group ≈ 33.2℃. G' at 37℃<G"(tanδ> 1) It is in a free-flowing sol state. After contact with the ocular surface tear fluid, the pH rises to 7.4, and the Carbopol carboxyl groups further ionize, forming a dense gel network, achieving in-situ retention.

[0078] Thixotropic scanning recovery rate: 88.2% in the 0.1% group; 87.7% in the 0.01% group (>85%, good thixotropy √).

[0079] STF dilution triggering: After dilution, G' increased sharply from 78.6 Pa to 950 Pa, and tanδ decreased from 0.49 to 0.055, confirming the in-situ gelation property of "the more diluted, the stronger".

[0080] Brookfield verification (SC4-34 rotor, 20 rpm, 25℃): 0.1% group 4920 mPa·s; 0.01% group 4680 mPa·s, conforming to the industry standard of 3000-8000 mPa·s.

[0081] 8. Evaluation of 6-Paradol ophthalmic gel (0.01% and 0.1%) The following is an validation of a carbopol ophthalmic in-situ gel formulation with 6-Paradol as the active ingredient. This formulation utilizes the properties of Carbopol 980, which is a low-viscosity solution under acidic conditions and rapidly gels upon contact with tears (neutral pH), to prolong the drug's residence time on the ocular surface and improve bioavailability. It is suitable for the treatment of dry eye, meibomian gland dysfunction, and related ocular surface inflammation.

[0082] Quality inspection items and results Properties Colorless to pale yellow transparent gel Tongzuo Uniform and free of particles Qualified ✓ pH value 7.25 7.28 6.5–7.4 Qualified ✓ Viscosity (25℃, 20rpm) 4850 ± 320 mPa·s 4620 ± 280 mPa·s 3000–8000 Qualified ✓ osmotic pressure 298 ± 5 mOsm / kg 296 ± 4 mOsm / kg 280–320 Qualified ✓ 6-Paradol content (HPLC) 99.2% of the labeled amount 98.7% of the labeled amount 90%–110% Qualified ✓ Related substances Total impurities: 0.3% Total impurities: 0.3% ≤1.0% Qualified ✓ Aseptic examination Aseptic growth Aseptic growth Aseptic Qualified ✓ DMSO residue 0.1% w / v 0.01% w / v <0.5% Qualified ✓ 8.2 Rheological Characterization Following the rheological evaluation method for gatifloxacin ophthalmic gels, two concentrations of 6-Paradol carbomer gels were systematically rheologically characterized. The testing instrument was a TA Instruments AR2000ex rheometer with a cone-plate clamp (40 mm in diameter, 2° cone angle) and a temperature control accuracy of ±0.1℃.

[0083] Steady-state flow curve Table 3-1 Steady-state flow curve data (shear rate 0.01–100 s⁻¹, 25℃) 0.01 8520 8140 0.085 0.081 0.05 6230 5980 0.312 0.299 0.1 4850 4620 0.485 0.462 0.5 3120 2980 1.56 1.49 1 2580 2450 2.58 2.45 5 1720 1640 8.60 8.20 10 1350 1280 13.5 12.8 50 820 790 41.0 39.5 100 610 580 61.0 58.0

[0084] Conclusion: Both concentrations of gels exhibited typical non-Newtonian fluid pseudoplastic behavior—viscosity decreased significantly with increasing shear rate (shear thinning). The 0.1% group, due to its slightly higher drug content and stronger intermolecular interactions, had a slightly higher overall viscosity than the 0.01% group, but the difference was small (<10%), indicating that the drug concentration had a limited impact on the Carbopol gel network structure within this range.

[0085] Power-law model fitting Table 3-2 Fitting results of the power-law model (τ = K·γⁿ) Flow index n 0.58 0.60 <1 is a pseudoplastic fluid✓ Consistency coefficient K (Pa·sⁿ) 11.2 10.5 Reflects gel strength Correlation coefficient R² 0.995 0.994 >0.99✓ Linear range (s⁻¹) 0.05–50 0.05–50 Fitting interval 3.3 Temperature Scan – Gel-sol Transition Temperature (Tgel) Table 3-3 Temperature scan data (heating rate 1℃ / min, fixed frequency 1Hz) 20 85.2 42.1 80.5 40.2 0.49 25 78.6 38.5 74.2 36.8 0.49 30 65.3 32.0 61.8 30.5 0.49 32 52.1 26.5 49.6 25.2 0.51 33 38.5 20.8 36.2 19.6 0.54 34 22.3 15.2 20.8 14.3 0.68 35 12.5 10.8 11.6 10.1 0.86 37 5.8 6.5 5.2 6.0 1.12 / 1.15 40 2.1 3.2 1.8 2.9 >1 Table 3-4 Determination of gel-sol transition temperature (Tgel) 0.1% group ≈33.5℃ G''>G' (tanδ=1.12) It is in a sol state at 37℃ and can flow on the surface of the eye. 0.01% group ≈33.2℃ G''>G' (tanδ=1.15) It is in a sol state at 37℃ and can flow on the surface of the eye. Conclusion: The Tgel of both concentrations of gel was around 33℃, which is lower than body temperature (37℃), indicating that the gel was in a low-viscosity sol state before administration (room temperature / body temperature), making it easy to squeeze out and spread; after being instilled into the eye and coming into contact with the tear fluid (neutral pH + body temperature), Carbopol further ionized, forming a denser gel network and achieving in-situ gelation.

[0086] 3.4 Three-stage thixotropic scanning Table 3-5 Thixotropic scanning results (low shear 0.1s⁻¹ → high shear 10s⁻¹ → recovery 0.1s⁻¹) ① Initial low shear 4850 4620 0.1 s⁻¹, 60 s Static storage viscosity ② High shear failure 1350 1280 10 s⁻¹, 60 s Viscosity during extrusion / coating ③ Low shear after recovery 4280 4050 0.1 s⁻¹, 120 s Ocular surface recovery gel Viscosity recovery rate 88.2% 87.7% ③ / ①×100% >85% ✓ Good thixotropy 3.5 STF (Artificial Tears) Dilution Triggered Rheology To simulate the scenario where the gel is diluted by tears after being instilled into the eye, the gel and artificial tears were mixed at a 1:1 volume ratio, and the rheological changes after dilution were measured.

[0087] Table 3-6 Rheological changes of STF after dilution (25℃, 1 Hz) G' (Pa) 78.6 950 74.2 880 G'' (Pa) 38.5 52.3 36.8 50.1 tanδ 0.49 0.055 0.50 0.057 Viscosity (mPa·s, 1s⁻¹) 2 580 12 500 2 450 11 800 determination G'>>G'' Strong Gel G'>>G'' Stronger Gel Tongzuo Tongzuo Conclusion: Upon dilution of STF, as the pH was further adjusted from neutral to the tear film pH (~7.4), the ionization degree of Carbopol carboxyl groups increased, and the gel modulus G' actually increased (tanδ dropped sharply from 0.49 to 0.055), forming a stronger gel network. This confirms that the formulation becomes stronger with dilution after contact with the tear film on the ocular surface, effectively prolonging the drug retention time.

[0088] 3.6 Brookfield Viscometer Verification Single-point viscosity verification was performed using a Brookfield DV2T rotational viscometer, referring to the pharmacopoeia / industry standard test method for gatifloxacin ophthalmic gel.

[0089] Table 3-7 Brookfield viscosity verification data Rotor SC4-34 SC4-34 SC4-34 Consistent rotational speed 20rpm 20rpm 20rpm Consistent temperature 25±0.5℃ 25±0.5℃ 25±0.5℃ Consistent viscosity 4920 mPa·s 4680 mPa·s 3000–8000 Qualified ✓ Torque 42.5% 40.8% 10%–90% Qualified ✓ 3-8 Summary of Rheological Characterization Steady-state flow curve Shear thinning, η(0.1s⁻¹) = 4850 mPa·s η(0.1s⁻¹) = 4620 mPa·s Power-law fitting n=0.58,K=11.2Pa·sⁿ,R²=0.995 n=0.60,K=10.5Pa·sⁿ,R²=0.994 Tgel (tanδ=1) ≈33.5℃ ≈33.2℃ Energy storage modulus G' at 37℃ 5.8 Pa (sol state, flowable) 5.2 Pa (sol state, flowable) Thixotropic recovery rate 88.2%（>85%✓） 87.7%（>85% ✓） STF diluted G' 950Pa (Strong Gel ✓) 880Pa (Strong gel ✓) Brookfield viscosity 4920 mPa·s (Qualified ✓) 4680 mPa·s (Pass)

[0090] 4. Animal Experiment Methods 4.1 Laboratory Animals and Grouping Forty female SPF-grade C57BL / 6J mice (8 weeks old, 20±2g) were randomly divided into four groups (n=10 / group), with one eye of each mouse included in the experiment.

[0091] Table 4-1 Animal Grouping Scheme Control group 1 (Normal) Normal feeding, no modeling 3μL saline eye drops Basic Comparison Control group 2 (Vehicle) Dry eye modeling 3μL gel matrix eye drops (medication-free) Solvent / Placebo Control Experimental group 1 (0.01%) Dry eye modeling 3μL 0.01% Paradol gel eye drops Low concentration experimental group Experimental group 2 (0.1%) Dry eye modeling 3μL 0.1% Paradol gel eye drops High concentration experimental group 4.2 Establishment of a dry eye model From D1 to D19, mice were placed in a low-humidity environment (humidity ≤40%) and were injected subcutaneously into the neck and back of the neck with 0.75 mg / mL scopolamine hydrobromide, 0.3 mL / mouse / time, twice a day (09:30 and 15:30).

[0092] 4.3 Dosing regimen Table 4-2 Dosing Regimen Duration of administration D5–D19 (Intervention begins on the 5th day after model initiation) frequency Twice a day, 6 hours apart Dosage volume 3μL / time / eye route of administration Eye drops (eye drops) Dosing techniques Using a 10μL micropipette, slowly drop the gel into the lower conjunctival sac, then gently close your eyes for 5 seconds. Utilization of gel properties Taking advantage of its in-situ gelling properties, it forms a low-viscosity sol when instilled into the eye and then remains as a gel after entering the eye. 4.4 Observation Indicators Tear secretion function assay (Schirmer's test): phenol red cotton thread method, D-1, D5, D10, D19.

[0093] Tear film breakup time (BUT) measurement: 0.05% sodium fluorescein staining, observation under slit lamp cobalt blue light, and detection at D-1, D5, D10, and D19.

[0094] Corneal fluorescein staining score (FSC): 0–12 points according to the NEI standard, with the D19 endpoint used to assess corneal epithelial defects.

[0095] Safety assessment: ocular observation (redness / swelling / hair loss / corneal transparency), Draize score, and histopathological examination.

[0096] 5. Experimental Results 5.1 Tear secretion function (Schirmer's test) Table 5-1 Schirmer's test tear secretion (length of phenol red cotton thread wetted, mm, mean) Control group 1 (Normal) 5.3 5.2 5.5 5.3 Control group 2 (Vehicle) 5.3 0.9 1.25 1.2 Experimental group 1 (0.01%) 5.3 1.45 3.25 3.37 Experimental group 2 (0.1%) 5.4 1.8 3.5 3.67 Compared with the Normal group, P<0.01 (modeling successful). Both drugs significantly promoted the recovery of tear secretion, with the 0.1% and 0.01% groups showing better efficacy than the control group.

[0097] 5.2 Tear film breakup time (BUT) Table 5-2 Tear film breakup time (seconds, mean) Control group 1 (Normal) 10.83 10.97 10.97 11.03 Control group 2 (Vehicle) 10.93 3.91 3.91 3.77 Experimental group 1 (0.01%) 10.95 7.03 7.26 7.5 Experimental group 2 (0.1%) 10.96 7.64 8.23 9.89 The gel matrix itself has a moisturizing effect, but the drug group was still significantly better than the vehicle group. The 0.1% and 0.01% groups were more effective than the control group.

[0098] 5.3 Corneal fluorescein staining score (FSC) Table 5-3 D19 corneal fluorescein staining scores (NEI 0–12 points, mean) Control group 1 (Normal) 0.0±0.0 — Control group 2 (Vehicle) 0.9 — Experimental group 1 (0.01%) 0.4 P<0.01 Experimental group 2 (0.1%) 0.3 P<0.01 6. Safety Evaluation Table 6-1 Draize Ocular Irritation Score (D19 endpoint, mean) Corneal opacity (0–4) 0 0 0 0 Iris congestion (0–2) 0 0 0 Conjunctival hyperemia (0–3) 0 0.8 0.7 0.6 Conjunctival edema (0–4) 0 0.6 0.6 0.7 Total score (0–13) 0.0 1.4 1.3 1.3 All Draize total scores in all treatment groups were ≤1, falling into the "non-irritating" category (<3 points), far below the ophthalmic safety threshold, and no irritation was observed.

[0099] Advantages of Carbopol 980 in situ gel: This formulation utilizes the properties of Carbopol 980, which is a low-viscosity sol under acidic conditions and rapidly gels upon contact with tears (pH~7.4), achieving intelligent drug delivery that is "liquid when instilled and gels upon entering the eye." Rheological data confirm that Tgel≈33.5℃, lower than body temperature, ensuring good fluidity and high patient comfort before administration.

[0100] The systematic value of rheological characterization: Referring to the industry standard for gatifloxacin ophthalmic gels, this experiment established a complete rheological evaluation system (steady-state flow, power-law fitting, temperature scanning, thixotropic scanning, STF dilution triggering, and Brookfield verification). Six sets of data corroborated each other, fully demonstrating that the gel formulation process is stable and reproducible. Carbopol ionizes more fully at higher pH levels, resulting in more complete molecular chain extension and a stronger gel network. This precisely reflects the ideal "the more diluted, the stronger" characteristic of in-situ gels—its retention is enhanced after dilution by tears.

[0101] The effect of drug concentration on rheology was limited: the difference in rheological parameters between the 0.1% and 0.01% groups was <10%, indicating that within the ppm concentration range, 6-Paradol itself does not participate in gel network construction, and Carbopol dominates the rheological behavior. This facilitates subsequent concentration screening—eliminating the need to re-optimize the matrix formulation due to concentration adjustments.

[0102] The efficacy-dose effect was slightly better in the 0.1% group than in the 0.01% group in terms of Schirmer and BUT (P<0.05 or P<0.01), indicating a certain dose-dependency. Based on the safety data, 0.1% and 0.01% can be recommended as the preferred concentrations for subsequent studies.

[0103] Compared with solution formulations: Compared with the previous solution-type eye drops (DMSO final concentration 0.1%–1.0%), this gel formulation is completely free of DMSO (DMSO is only present in trace amounts of the mother liquor, final concentration 0.01%–0.1%), and has better safety; at the same time, the gel retention effect can prolong drug release, which is expected to reduce the frequency of administration.

[0104] The rheological characterization of the two concentrations (0.1% and 0.01%) of gel both met the standards for ophthalmic in situ gels: Tgel≈33℃, thixotropic recovery rate>87%, and strong gel formation after STF dilution.

[0105] Animal experiments confirmed that both concentrations of gel significantly improved dry eye symptoms, with the 0.1% group showing better efficacy. Both indicators (tear secretion and BUT) were significantly better than the control group (P<0.01).

[0106] Good safety profile: FSC score <1, Draize score <3, no irritation reactions such as corneal opacity or iris congestion.

[0107] Example 3: Preparation of 6-Paradol nasal spray (0.01% and 0.1%) Prescription composition (per 10 mL) 6-Paradol 1.0 mg 0.1 mg 0.1% / 0.01% Active ingredients SBE-β-CD 1800 mg 1800 mg 18% w / v Solubilization and inclusion DMSO (in mother liquor) 10 μL 1 μL 0.1% / 0.01% w / v Solubilizer Sodium chloride 90 mg 90 mg 0.9% w / v Isotonic adjustment benzalkonium chloride 1.0 mg 1.0 mg 0.01% w / v Antiseptic and antibacterial PBS buffer Add to 10 mL Add to 10 mL margin Solvent / pH adjustment

[0108] Preparation process: Step 1: Stock solution A (10mg / mL): Weigh 100mg Paradol + 1mL DMSO → make up to 10mL with physiological saline.

[0109] Step 2: Mother solution B (1 mg / mL): Take 1 mL of mother solution A and 9 mL of physiological saline to obtain 1 mg / mL mother solution B.

[0110] Step 3 20% SBE-β-CD solution: Weigh 2g of SBE-β-CD and add physiological saline to make up to 10mL, then stir until clear.

[0111] Step 4 0.1% Nasal Spray: Take 100 uL of stock solution A and 900 uL of 20% SBE-β-CD solution, and vortex to mix.

[0112] Step 5 0.01% Nasal Spray: Take 100 uL of stock solution B and 900 uL of 20% SBE-β-CD solution, and vortex to mix.

[0113] Step 6: Add sodium chloride to 0.9% and benzalkonium chloride to 0.01%, and adjust the pH of PBS to 7.2-7.4.

[0114] Step 7: Sterilize with a 0.22μm filter membrane, dispense into quantitative spray bottles (10mL / bottle, 50μL per spray), and store at 2-8℃.

[0115] Final DMSO concentration: 0.1% group = 0.1% w / v; 0.01% group = 0.01% w / v.

[0116] 1.3 Quality Inspection Results Appearance: All three concentrations are colorless to pale yellow clear liquids without precipitation.

[0117] pH value: 7.00.

[0118] Osmotic pressure: 298±5mOsm / kg, isotonic with tears.

[0119] Stability (accelerated test): After being placed at 40℃ / RH75% for 3 months, the 6-Paradol content, as measured by HPLC, remained above 98.5%, with no obvious degradation product peaks.

[0120] 2. Animal models and grouping Forty female SPF-grade C57BL / 6J mice (8 weeks old, 20±2g) were selected, with each mouse included in the experiment by one eye.

[0121] Dry eye model establishment: Mice were placed in a low-humidity environment (humidity ≤40%) and injected subcutaneously into the back of the neck with 0.75 mg / mL scopolamine hydrobromide, 0.3 mL / mouse / time, twice a day (09:30 and 15:30), for 19 days.

[0122] 2.1 Experimental Groups (n=10 / group): Control group 1 (Normal): Normally fed, no modeling was performed, and 3 μL of physiological saline was administered via nasal drops.

[0123] Control group 2 (Scoppoderm): Dry eye model was created, and 3 μL of 20% SBE-β-CD solution containing 0.1% DMSO was administered as nasal drops (negative control).

[0124] Experimental group 1 (0.01% 6-Paradol): Dry eye model was created, and 3 μL of 0.01% 6-Paradol solution was administered as nasal drops.

[0125] Experimental group 2 (0.1% 6-Paradol): Dry eye model was created, and 3 μL of 0.1% 6-Paradol solution was administered as nasal drops.

[0126] 2.2 Dosing regimen Dosage period: Day 5 to Day 19 (intervention begins on Day 5 after model initiation, simulating mid-stage treatment in clinical course).

[0127] Frequency: 2 times / day, 6 hours apart.

[0128] Dosage volume: 3 μL / dose.

[0129] Route of administration: Nasal drops.

[0130] 2.3 Observation Indicators and Results 2.3.1 Shirmer's test for tear secretion function Methods: Mice were restrained, and a shortened commercially available phenol red cotton thread was held with clean, toothless forceps and fixed in the middle of the conjunctival sac of the lower eyelid of the mouse for 30 seconds. The length of the moistened phenol red cotton thread was measured using vernier calipers.

[0131] Testing points: Day 5, Day 10, and Day 19 after modeling.

[0132] Result: See Figure 3 As shown in the figure, the tear secretion function of mice decreased significantly after dry eye modeling. After treatment with 6-paradol solution nasal drops starting on day 5, the tear secretion function of mice on days 10 and 19 was significantly increased compared with the control group. These data were analyzed by one-way ANOVA, and the p-value was <0.001, indicating a significant difference.

[0133] 2.3.2 Tear film breakup time (BUT) measurement Methods: Mice were tethered, and 1 μL of 0.05% sodium fluorescein saline was drawn into the conjunctival sac of the mice using a micropipette. After 30 seconds of staining, the mice blinked three times artificially and observed under a handheld slit lamp with cobalt blue light. The time (seconds) for the appearance of dry spots was recorded after the mice opened their eyes after the last blink. Each eye was measured three times, and the average value was taken as the BUT for that mouse.

[0134] Testing points: Day 5, Day 10, and Day 19 after modeling.

[0135] Result: See Figure 4 As shown in the figure, the BUT of mice decreased significantly after dry eye modeling. After nasal treatment with 6-paradol solution was started on day 5, the BUT of mice on days 10 and 19 was significantly longer than that of the control group. The data of these groups were analyzed by one-way ANOVA, and the p-values ​​were all <0.0001, indicating significant differences.

[0136] 3. Safety Evaluation Eyes: Throughout the experiment, mice in each drug administration group showed no redness or hair loss around their eyes, their corneas were transparent, and their irises were not adhered.

[0137] Nasal region: The nasal mucosa of mice treated with nasal drops showed no congestion or ulceration, and the nasal septum remained intact.

[0138] FSC score: The mice were observed at 24h, 48h and 72h after a single administration of 0.05% 6-Paradol eye drops. The FSC score of the mice eyes was 0 (no irritation).

[0139] Example 4: Preparation of 6-Paradol eye atomizing solutions (0.01% and 0.1%) Prescription composition (prepare 20 mL each time) 6-Paradol 20.0 mg 2.0 mg 0.1% / 0.01% Active ingredients SBE-β-CD 1000 mg 1000 mg 5% w / v Solubilization and inclusion DMSO (in mother liquor) 200 μL 20 μL 1.0% / 0.1% w / v Solubilizer Mannitol 100 mg 100 mg 0.5% w / v Osmotic pressure regulation benzalkonium chloride 1.0 mg 1.0 mg 0.005% w / v Antiseptic and antibacterial EDTA-2Na 5.0 mg 5.0 mg 0.025% w / v Chelation stability PBS buffer Add to 20 mL Add to 20 mL margin Solvent / pH adjustment The dosage of SBE-β-CD has been reduced from 20% to 5% in gels / eye drops. Since nebulized administration does not require thickening, a low concentration of cyclodextrin can meet the solubilization requirements and reduce the burden of respiratory tract deposition.

[0140] Preparation process: Step 1: Stock solution A (10mg / mL): Weigh 100mg Paradol + 1mL DMSO → make up to 10mL with physiological saline.

[0141] Step 2: Mother solution B (1 mg / mL): Take 1 mL of mother solution A + 9 mL of physiological saline.

[0142] Step 3 5% SBE-β-CD solution: Weigh 1g of SBE-β-CD and add physiological saline to make up to 20mL, then stir until clear.

[0143] Step 4 0.1% Nebulizing Solution: Take 2 mL of stock solution A + an appropriate amount of SBE-β-CD solution → bring the volume up to 20 mL with PBS.

[0144] Step 5 0.01% Nebulizing Solution: Take 2 mL of stock solution B + an appropriate amount of SBE-β-CD solution → bring the volume up to 20 mL with PBS.

[0145] Step 6: Add mannitol, benzalkonium chloride, and EDTA-2Na. Adjust the pH to 7.2-7.4 with PBS and bring the volume to a final level.

[0146] Step 7: Sterilize by positive pressure filtration through a 0.22μm PVDF membrane (≤0.2 bar), and dispense into sterile amber bottles (5mL / bottle × 4 bottles).

[0147] Step 8: Store in the refrigerator at 2-8℃, away from light.

[0148] DMSO final concentration calculation: 0.1% group = 1.0% w / v; 0.01% group = 0.1% w / v.

[0149] Atomizer parameter verification (Yuwell 7EYJ-A01 eye atomizer): atomization rate 0.20±0.02 mL / min 0.15-0.25 mL / min Qualified √ MMAD 4.2±0.8 μm 3-5 μm Qualified √ <5μm proportion 82.3±5.1% >70% Qualified √ Residual liquid volume 0.8±0.2 mL <1.5 mL Qualified √ Atomizer cup temperature 28.5±2.0℃ <35℃ Qualified √ pH (before and after atomization) 7.25→7.22 Change < 0.3 Qualified √ Content (before and after atomization) 100%→98.5% Loss <5% Qualified √

[0150] The optimal deposition range for atomized particles is 3-5 μm. Particles that are too large (>10 μm) primarily deposit on the eyelids and eyelashes, failing to reach the cornea and conjunctiva; particles that are too small (<1 μm) enter the deep respiratory tract with respiration. Yuwell 7EYJ-A01 is specifically designed for ocular use, with a 45° nozzle angle and a 30° atomization cone angle, ensuring that the drug is primarily deposited on the ocular surface.

[0151] 2.7 Quality Inspection Results Properties Colorless to pale yellow clear liquid Colorless to pale yellow clear liquid Visual inspection All meet ✓ pH value 7.2-7.4 7.2-7.4 pH meter 7.25 ± 0.05 / 7.28 ± 0.04 osmotic pressure 290±10 mOsm / kg 290±10 mOsm / kg Osmometer 285 ± 5 / 288 ±4 6-Paradol content (HPLC) 98.5%-101.5% of the labeled amount 98.5%-101.5% of the labeled amount HPLC (C18 column) 99.2% / 98.8% Related substances Total impurities <1.0% Total impurities <1.0% HPLC 0.3% / 0.2% Aseptic examination Aseptic growth Aseptic growth Membrane filtration All sterile ✓ Atomized particle size (MMAD) 3-5μm 3-5μm Particle size analyzer 4.2μm / 4.0μm atomization rate 0.2 mL / min 0.2 mL / min graduated cylinder timing method 0.20 mL / min / 0.19 mL / min DMSO residue (GC) ≤1.0% w / v (0.1% group) / ≤0.5% w / v (0.01% group) ≤1.0% w / v (0.1% group) / ≤0.5% w / v (0.01% group) Gas chromatography 0.95% / 0.08% 3. Animal Models and Experimental Methods 3.1 Laboratory Animals and Grouping Forty female SPF-grade C57BL / 6J mice (8 weeks old, 20±2g) were randomly divided into four groups (n=10 / group), with one eye of each mouse included in the experiment.

[0152] Control group 1 (Normal) Normal feeding, no modeling, 20 mL PBS (drug-free) administered via nebulizer. no Yuwell nebulizer eye spray, 5 minutes / time, 2 times / day Control group 2 (Vehicle) Dry eye model was established, and PBS solution containing 5% SBE-β-CD + 1% DMSO was administered via nebulization. yes Yuwell nebulizer eye spray, 5 minutes / time, 2 times / day Experimental group 1 (0.01% 6-Paradol) Dry eye modeling was performed using 0.01% 6-Paradol nebulizer solution. yes Yuwell nebulizer eye spray, 5 minutes / time, 2 times / day Experimental group 2 (0.1% 6-Paradol) Dry eye modeling was performed, and 0.1% 6-Paradol nebulizer solution was administered. yes Yuwell nebulizer eye spray, 5 minutes / time, 2 times / day

[0153] 3.2 Establishment of a dry eye model From day 1 to day 19, mice were placed in a low-humidity environment (humidity ≤40%, temperature 22-25℃) and injected subcutaneously into the neck and back of the neck with 0.75 mg / mL scopolamine hydrobromide, 0.3 mL / mouse / time, twice a day (09:30 and 15:30).

[0154] 3.3 Nebulized drug delivery regimen Drug delivery device: Yuwell 7EYJ-A01 ophthalmic nebulizer Nebulizer parameters: atomization rate 0.2 mL / min, atomized particle MMAD 4.2±0.8 μm, nozzle distance from eye 5 cm, nozzle angle 45°.

[0155] Duration of administration D5-D19 (Intervention begins on the 5th day after model initiation) Simulated mid-stage treatment of clinical disease frequency Twice a day, 6 hours apart 09:30 and 15:30, synchronized with molding injection. Single atomization volume Approximately 1 mL per dose (5 min × 0.2 mL / min) Each time, take approximately 1 mL from the 5 mL dispensing bottle and add it to the nebulizer cup. Atomization time 5 min / time Ensure the liquid in the atomizing cup is almost completely depleted (residual <0.5 mL). Nozzle-eye distance 5cm Yuwell atomizer comes standard with a bracket for mounting. route of administration Atomized eye spray (open-ended, mice allowed to blink freely) Eyes are not forced to open; natural exposure is simulated. Daily dosage Approximately 2 mL / day / animal Each 20mL solution is sufficient for 10 mice for one day. Configuration frequency Prepare 20mL every 2 days. Store in the refrigerator (2-8℃), and allow to warm to room temperature before use.

[0157] Preparation volume calculation: 10 mice per group × 2 mL / day = 20 mL / day. Preparing 20 mL each time is exactly enough for one group's daily dosage. In the experiment, there are 2 concentration groups + 1 vehicle group = 3 groups that need to be administered the drug, therefore, 3 doses × 20 mL = 60 mL need to be prepared daily.

[0158] 3.4 Observation Indicators and Detection Methods Tear secretion (Schirmer test) Phenol red cotton thread method: Fix the lower eyelid conjunctival sac for 30 seconds and measure the wet length (mm). D-1 (before molding), D5, D10, D19 Tear film breakup time (BUT) 1 μL of 0.05% sodium fluorescein was used for staining, and the time (s) of the first drying spot was recorded under cobalt blue slit lamp light. D-1, D5, D10, D19 Corneal fluorescein staining (FSC) Stain with 0.05% sodium fluorescein and score under a slit lamp from 0 to 12 (3 zones × 4 points per eye). D5, D10, D19 Atomizer deposition efficiency Weigh the periorbital patches of mice before and after nebulization and calculate the amount of drug deposited (μg). D10 Single Measurement

[0159] 4. Experimental Results 4.1 Tear secretion function (Schirmer's test) Following dry eye modeling (D5), tear secretion in all mouse groups decreased significantly compared to D-1 (P<0.01). From D10 onwards, tear secretion began to recover in both nebulized drug administration groups. By D19, the tear secretion lengths in the 0.01% and 0.1% groups were 3.9 mm and 4.1 mm, respectively, both significantly higher than those in the Vehicle control group (1.3, P<0.01). The improvement in the 0.1% group was better than that in the 0.01% group, showing a dose-dependent trend.

[0160] Normal control group 5.1 5.2 5.1 5.2 Vehicle control group 5.1 1.05 1.25 1.3 0.01% 6-Paradol group 5.2 1.15 3.8** 3.8** 0.1% 6-Paradol group 5.3 1.15 3.9** 4.1**

[0161] ** P < 0.01 compared with the Normal group; P < 0.01 compared with the Vehicle group. Data format: mean (mm).

[0162] 4.2 Tear film breakup time (BUT) Consistent with the tear secretion results, the tear film endoscopic transition time (BUT) was significantly shortened after modeling. Following nebulized drug treatment, measurements at D10 and D19 showed that the BUT in the 0.01% and 0.1% groups was significantly longer than that in the Vehicle group (P<0.01). The 0.1% group had an average BUT of 9.89 s on D19, close to the normal group level (10.96 s), indicating that nebulized drug delivery effectively stabilizes the tear film.

[0163] Normal control group 10.99 10.80 10.73 10.96 Vehicle control group 10.87 3.91 3.85 3.80 0.01% 6-Paradol group 10.97 4.01 7.26 8.23 0.1% 6-Paradol group 11.03 3.77 7.6 9.89

[0164] The experimental group was compared with the normal group (P<0.01); the experimental group was compared with the vehicle group (P<0.01). Data format: mean (s).

[0165] 4.3 Corneal fluorescein staining score (FSC) The FSC score reflects the integrity of the corneal epithelium. The Vehicle group had a score of 1.4 on day 19, indicating persistent dry eye damage. The FSC scores of both Paradol nebulization groups decreased significantly with treatment time; the 0.1% group's score dropped to 0.5 on day 19, which was better than the 0.7 in the 0.01% group (P<0.05), confirming that nebulized administration promotes corneal epithelial repair.

[0166] Normal control group 0 — Vehicle control group 1.4 — 0.01% 6-Paradol group 0.7 P<0.01 0.1% 6-Paradol group 0.5 P<0.01

[0167] The FSC score ranges from 0 to 12, with higher scores indicating more severe corneal damage.

[0168] 4.4 Atomization Deposition Efficiency To verify the actual drug delivery efficiency of nebulized drug delivery, the amount of Paradol deposited on the periocular patch was determined by HPLC on day 10: 0.01% group 200 μg (1 mL × 0.01% × 1000 mL / L after correction) 42.5±8.3 μg 21.3±4.2% 78.7% 0.1% group 2000μg (1mL×0.1%) 435.0±62.5 μg 21.8±3.1% 78.2% The periocular deposition efficiency of nebulized drug delivery is approximately 21-22%, with the remainder deposited primarily in the eyelids, facial hair, and air. Although the absolute deposition amount is limited, Paradol, as a TRPV1 agonist, can activate the trigeminal reflex arc and promote tear secretion even at extremely low concentrations, thus the nebulization route still has significant therapeutic efficacy.

[0169] 5. Safety Evaluation 5.1 Eye observation Throughout the experiment, mice in all treatment groups showed no redness or hair loss around their eyes; their corneas were clear, their irises were not adhered, and no obvious congestion or edema was observed. No irritation caused by splashing of the nebulized drug was observed in the nebulized drug treatment group.

[0170] 5.2 Draize rating Following the Draize eye irritation test criteria, corneal, iris, and conjunctival scores were observed and recorded at 1 hour, 24 hours, 48 ​​hours, and 72 hours after daily nebulized drug administration. Vehicle control group D10 / D19 0 / 0 0 / 0 0 / 0 0 / 0 Non-irritating 0.01% 6-Paradol group D10 / D19 0 / 0 0 / 0 0 / 0 0 / 0 Non-irritating 0.1% 6-Paradol group D10 / D19 0 / 0 0 / 0 11 / 0 1 / 0 Very minor (recoverable) In the 0.1% group, 2 out of 10 mice developed mild conjunctival hyperemia (score 1) on day 10, which resolved spontaneously within 24 hours and did not affect subsequent drug administration. By day 19, all mice had a score of 0.

[0171] 5.3 Observation of the nose and respiratory tract During nebulized drug delivery, some drug particles can enter the nasal cavity and respiratory tract through respiration. Nasal examinations will be performed weekly during the experiment. Nasal mucosal congestion none none none Nasal secretions none none Occasionally seen in small quantities (2 / 10 of each, D10) sneezing frequency normal normal normal respiratory rate normal normal normal Weight change (D1→D19) +2.5±0.8 g +2.3±0.6 g +2.1±0.7 g Conclusion: Nebulized administration did not produce significant irritation or toxicity to the nasal cavity and respiratory tract. A small amount of nasal discharge was occasionally observed in the 0.1% group, which resolved spontaneously within 24 hours and did not affect animal welfare.

[0172] 6. Discussion 6.1 Advantages and limitations of nebulized drug delivery Nebulized drug delivery, as a non-invasive method of ocular surface drug delivery, has the following advantages: (1) the drug is uniformly covered on the ocular surface with 3-5μm particles, without the need to force the eyes to open; (2) the nebulizer formulation is simple and does not require thickeners (such as carbomer), resulting in good patient compliance; (3) the Yuwell ocular nebulizer is specially designed for the eyes, with optimized nozzle angle and atomization cone angle, and can activate reflexive tear secretion at extremely low concentrations, so the therapeutic effect is still significant.

[0173] 6.2 Dosage effect and safety This study further validated the dose-dependent efficacy of 6-Paradol: the 0.1% concentration group was superior to the 0.01% group in all three indicators: Schirmer test, BUT, and FSC score (P<0.05). Regarding safety, nebulized administration did not cause significant eye or respiratory irritation, and the Draize scores were all 0-1 (very mild and reversible), confirming the good safety profile of this route of administration.

[0174] 7. Conclusion 0.01% and 0.1% 6-Paradol nebulizers administered via the Yuyue Eye Nebulizer significantly improved insufficient tear secretion and tear film instability in dry eye model mice.

[0175] Neither of the two concentrations of nebulized solution showed significant eye or respiratory irritation during the 14-day experimental period, with a Draize score of 0-1, indicating good safety.

[0176] The atomization deposition efficiency is approximately 21-22%, which is lower than that of ophthalmic drops. However, Paradol's potent reflex secretion-promoting effect as a TRPV1 agonist compensates for the insufficient absolute delivery amount.

[0177] Considering both efficacy and safety, 0.01% and 0.1% 6-Paradol nebulized solutions are the preferred concentrations for subsequent preclinical large animal experiments and long-term toxicity studies. The current 14-day data shows no abnormalities in blood biochemistry and organ function, meeting the requirements for this stage of preclinical research. A 6-month long-term toxicity study will be conducted next.

[0178] Specific advantages of different dosage forms Eye drops (0.01% / 0.1%) SBE-β-CD+DMSO synergistic solubilization increases corneal bioavailability by 3 times. Daily maintenance of mild to moderate dry eye Ophthalmic gel (0.01% / 0.1%) In-situ gelation at 34℃, retention for 8 hours, and zero-order kinetics of sustained release over 24 hours. Nighttime long-lasting moisturizing / excessive evaporation Nasal spray (0.01% / 0.1%) An increased tear secretion trend can be observed 5 minutes after administration. Rapid relief during acute attack Atomizing spray (0.01% / 0.1%) MMAD 3-5μm, ocular atomization to promote secretion, and stabilize the tear film. Severe dry eye / Sjögren's syndrome 7.3 Pharmacokinetic Data (Mice) Eye drops: Corneal bioavailability is 3 times higher than that of ordinary formulations (Cmax=285ng / g vs 95 ng / g), and ocular surface retention time t1 / 2=4.2h.

[0179] Ophthalmic gel: The ocular surface retention time t1 / 2 = 8.5 h, and the corneal drug concentration can still be detected within 24 hours (>10 ng / g).

[0180] Nasal spray: Peak plasma concentration (Cmax = 125 ng / mL) is reached 5 minutes after administration. It is mainly distributed in the nasal mucosa and lacrimal glands, with no significant systemic accumulation (t1 / 2 = 1.8 h).

[0181] Nebulized inhalation solution: lung absorption rate 35%, Cmax = 450 ng / mL (15 min after administration), can penetrate the blood-tear barrier to act on the deep ocular surface (ocular tissue / plasma ratio = 0.35).

[0182] Comparative Example 1: HP-β-CD alone without DMSO The method described in the example is to replace DMSO with an equal amount of ethanol, with the rest remaining the same.

[0183] Results: Paradol still showed minor crystal precipitation in a 20% HP-β-CD aqueous solution, with the content decreasing to 92% after 48 hours; while the content in the embodiment of this invention remained >99% under the same conditions, demonstrating the necessity of DMSO for synergistic solubilization. Solubility comparison: Cyclodextrin alone increased solubility by approximately 800 times; the present invention (DMSO + cyclodextrin) increased solubility by approximately 1500 times. The water solubility of 6-Paradol active pharmaceutical ingredient was <0.02 mg / mL, with a solubility of 16 mg / mL (800-fold increase) when using 20% ​​HP-β-CD alone, and 30 mg / mL (1500-fold increase) when combined with 1% DMSO.

[0184] Comparative Example 2: DMSO alone without cyclodextrin Replace SBE-β-CD with an equal volume of physiological saline, and use only 1% DMSO to aid dissolution.

[0185] Results: Paradol's solubility in water only increased to 0.5 mg / mL, which is insufficient to achieve the 1 mg / mL concentration required for a 0.1% formulation, and it also showed significant corneal irritation (Draize score of 3), confirming the necessity of cyclodextrin inclusion.

[0186] Comparative Example 3: Ordinary eye drops (non-cyclodextrin, non-DMSO) Paradol was directly suspended in physiological saline (0.01%) without any solubilization treatment.

[0187] Results: The drug existed in particulate form (DLS particle size > 5 μm), corneal bioavailability < 1%, and Schirmer test showed no significant improvement (P > 0.05), confirming the necessity of the solubilization system.

[0188] Comparative Example 4: Gel containing only cyclodextrin and no DMSO Carbopol gel contains 20% HP-β-CD but no DMSO.

[0189] Results: Paradol was not completely dissolved, and visible particles were present in the gel. The content uniformity RSD was 8.5% (>5% is unacceptable). After 3 months of accelerated processing, the content decreased to 90%. The DMSO-containing gel of this invention had an RSD of 1.2% and a content of 97.1% after 3 months.

Claims

The application of 1,6-Paradol is characterized by, The use of 6-Paradol in the preparation of drugs for the prevention and / or treatment of dry eye syndrome.

2. A pharmaceutical composition for the prevention and / or treatment of dry eye syndrome, characterized in that, The active ingredient of the pharmaceutical composition is 6-Paradol, and the proportion of 6-Paradol in the drug is 0.001%-1.0% w / v.

3. The pharmaceutical composition for the prevention and / or treatment of dry eye as described in claim 2, characterized in that, The pharmaceutical composition further includes a solubilizing carrier, wherein 6-Paradol accounts for 0.01% to 0.1% of the drug by weight, and the molar ratio of 6-Paradol to the solubilizing carrier is 1:1.5 to 1:3; the solubilizing carrier is sulfobutyl ether-β-cyclodextrin or hydroxypropyl-β-cyclodextrin.

4. The pharmaceutical composition for the prevention and / or treatment of dry eye as described in claim 3, characterized in that, The pharmaceutical composition further includes a co-solvent, a pH adjuster, excipients and / or a formulation matrix, wherein the co-solvent accounts for less than 5% of the drug volume, and the pH adjuster is used to adjust the pH of the drug to 7.0-7.

4.

5. The pharmaceutical composition for the prevention and / or treatment of dry eye as described in claim 4, characterized in that, The excipients include humectants, isotonic regulators, preservatives, or metal chelating agents; the humectant is glycerin, the isotonic regulator is sodium chloride, the preservative is benzalkonium chloride, the metal chelating agent is EDTA-2Na, and the synergistic solubilizer is dimethyl sulfoxide.

6. The pharmaceutical composition for the prevention and / or treatment of dry eye as described in claim 5, characterized in that, The pharmaceutical composition is an eye drop, which is prepared from the following substances; 6-Paradol, 0.01%-0.1% w / v; Solubilizing carrier, 5%-20% w / v; Dimethyl sulfoxide 0.1-5% w / v; Mannitol, 0.5% w / v; Benzalkonium chloride, 0.005-0.01% w / v; EDTA-2Na, 0.025-0.05% w / v; An appropriate amount of PBS buffer.

7. The pharmaceutical composition for the prevention and / or treatment of dry eye as described in claim 5, characterized in that, The pharmaceutical composition is an ophthalmic gel, which is prepared from the following substances; 6-Paradol, 0.01-0.1% w / v; Solubilizing carrier, 5-20% w / v; Dimethyl sulfoxide <0.1% w / v; Mannitol, 1.5% w / v; Benzalkonium chloride, 0.005%-0.01% w / v; EDTA-2Na, 0.025%-0.05% w / v; Triethanolamine, in appropriate amounts.

8. The pharmaceutical composition for the prevention and / or treatment of dry eye as described in claim 5, characterized in that, The pharmaceutical composition is a nasal spray, which is prepared from the following substances; 6-Paradol, 0.01%-0.1% w / v; Solubilizing carrier, 5%-20% w / v; Dimethyl sulfoxide 0.1-5% w / v; Benzalkonium chloride, 0.005-0.01% w / v; Sodium chloride 0.9% w / v; An appropriate amount of PBS buffer.

9. The pharmaceutical composition for the prevention and / or treatment of dry eye as described in claim 5, characterized in that, The pharmaceutical composition is an ophthalmic atomizing agent, which is prepared from the following substances; 6-Paradol, 0.01%-0.1% w / v; Solubilizing carrier, 5% w / v; Dimethyl sulfoxide 0.1-5% w / v; Mannitol, 0.5% w / v; Benzalkonium chloride, 0.005-0.01% w / v; EDTA-2Na, 0.025-0.05% w / v; An appropriate amount of PBS buffer.

10. A method for preparing a pharmaceutical composition for the prevention and / or treatment of dry eye as described in any one of claims 2-9, characterized in that, The preparation method includes the following steps: Step 1: Preparation of Mother Solution 1: Weigh 6-Paradol, add DMSO, vortex to dissolve, and obtain 10 mg / mL Mother Solution 1. Store in the dark at 4°C. Step 2 Preparation of Mother Solution 2: Take Mother Solution 1, add DMSO, vortex mix to obtain 1 mg / mL Mother Solution 2, store in the dark at room temperature; Step 3: Preparation of intermediate mother liquor: Take 1.0 ml of mother liquor 2, add 9 ml of physiological saline, vortex mix well to obtain 0.1 mg / mL intermediate mother liquor, store in the dark at 4℃; Step 4: Inclusion carrier solution: Take SBE-β-CD or HP-β-CD, dissolve it in sterile physiological saline, stir until clear, and obtain a 5-20% w / v cyclodextrin solution. Step 5: Preparation of the final formulation: Take an appropriate amount of the mother liquor according to the target concentration, add the cyclodextrin solution, vortex to mix, add excipients, and adjust the pH to 7.0-7.4; Step 6 Sterilization: Sterilize by positive pressure filtration through a PVDF filter membrane, and store at 2-8℃ away from light.

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