Phase 1: Animal Preparation and DEX-Induced OHT Model -- Days 1-14
C57BL/6 mice (8-12 weeks, n=10 per group) housed in standard conditions. Baseline IOP measurements using rebound tonometer (TonoLab) under light isoflurane anesthesia. DEX-induced OHT model: topical dexamethasone acetate 0.1% drops (5 μL) applied twice daily to right eyes for 14 days. Left eyes receive vehicle (saline) as controls. Daily IOP monitoring at consistent times (10-11 AM) to track development of ocular hypertension.
...Phase 1: Animal Preparation and DEX-Induced OHT Model -- Days 1-14
C57BL/6 mice (8-12 weeks, n=10 per group) housed in standard conditions. Baseline IOP measurements using rebound tonometer (TonoLab) under light isoflurane anesthesia. DEX-induced OHT model: topical dexamethasone acetate 0.1% drops (5 μL) applied twice daily to right eyes for 14 days. Left eyes receive vehicle (saline) as controls. Daily IOP monitoring at consistent times (10-11 AM) to track development of ocular hypertension.
Phase 2: ML-402 Treatment Preparation -- Days 15-16
ML-402 (TREK-1 agonist) dissolved in sterile saline with 0.1% DMSO. Prepare fresh solutions daily. Treatment concentrations: Vehicle control (saline + 0.1% DMSO), ML-402 0.1%, ML-402 0.3%. Osmolality verified (290-320 mOsm) and pH adjusted to 7.0-7.4. Sterility confirmed by plating on agar plates.
Phase 3: Acute IOP Response to ML-402 -- Days 17-18
After 14 days of DEX treatment and confirmed IOP elevation (>15 mmHg), administer single topical ML-402 dose (5 μL) to hypertensive eyes. IOP measurements at: baseline (pre-drop), 30 minutes, 1h, 2h, 4h, 6h, and 8h post-treatment. Use multiple measurements per timepoint (n=5 readings averaged) with coefficient of variation <10%.
Phase 4: Chronic ML-402 Treatment Protocol -- Days 19-25
Chronic treatment phase: ML-402 drops administered twice daily for 7 days while continuing DEX treatment. IOP measured daily at consistent times. Monitor for signs of ocular irritation or systemic effects. Include washout period assessment (days 26-28) to evaluate duration of effects.
Phase 5: Dose-Response and Mechanism Validation -- Days 26-30
Conduct dose-response study with ML-402 concentrations: 0.03%, 0.1%, 0.3%, 1.0%. Include TREK-1 antagonist control (spadin 0.1% + ML-402 0.3%) to confirm mechanism. Measure IOP response magnitude and duration for each concentration. Calculate ED50 for IOP reduction.
Phase 6: Statistical Analysis and Safety Assessment -- Days 31-35
Primary endpoint: percent IOP reduction from elevated baseline. Statistical analysis using repeated measures ANOVA with post-hoc Tukey tests. Safety assessment: ophthalmoscopy for corneal damage, anterior chamber inflammation, lens changes. Histological examination of anterior segment tissues. Power analysis validation for observed effect sizes.