**2. Materials and Methods**

The study utilized an HDM model of asthma in mice to compare the impact of 7 days of treatment with OmeGo compared to either fevipiprant or cod liver oil on lung and serum inflammatory markers and lung fibrosis.

The study was conducted according to GLP guidelines and in accordance with the laws and regulations of India, where the studies were performed. The study was approved by the Institutional Animal Ethics Committee (proposal number 214429) before the start of the study. The health status of the animals was assessed by a veterinarian, and all were noted to be in good health. The animals were acclimated to the laboratory conditions, and randomisation to the five treatment groups occurred the day before the trial commenced. The five groups were: no treatment (negative control), 0.5 mL of cod liver oil (vehicle control), 18 mg or 32 mg of OmeGo (test item), or 2 mg fevipiprant (positive control), respectively.

As per the experimental procedure shown in Figure 1, the study involved 50 healthy young adult female mice of 10 animals per group.

**Figure 1.** Experimental design. The mice were randomised into five groups of 10 animals per group. On day 1, animals were anaesthetised and sensitised intranasally with 1 μg HDM protein in 40 μL phosphate buffered saline (PBS). Following this, the mice were challenged daily with 10 μg HDM protein intranasally from day 7 to day 11. Oral interventions of PBS, cod liver oil, OmeGo low or high dose, or fevipiprant were given on days 7–14. BAL fluid was collected on day 15.

On day 1, the mice were anaesthetized using ketamine (100 mg/kg) and xylazine (10 mg/kg) given via intraperitoneal injection. HDM sensitisation was achieved with the intranasal application of 1 μg HDM protein in 40 μL phosphate buffered saline (PBS). Subsequently, daily intranasal HDM challenge was performed from day 7 to day 11 using 10 μg HDM protein in 40 μL PBS.

Between days 7 and 14, the mice received either cod liver oil, OmeGo, or fevipiprant treatment, all given orally. The fifth group of mice received no treatment (negative control). On day 15, the mice were anaesthetised and their trachea exposed to enable bronchoalveolar lavage (BAL) fluid collection using 3 mL of PBS containing 1 mM EDTA. The spleen was also removed from each animal, frozen and subsequently assessed for the extent of eosinophilia.

After collection, the BAL fluid was centrifuged (400× *g* at 4 ◦C for 7 min). The resulting cell pellet was collected, stored at −20 ◦C and subsequently analysed for total leucocyte cell count and differential cell count to provide eosinophil, neutrophil, lymphocyte and alveolar macrophage levels. A hemocytometer was used to measure total cell counts in the cell pellets and spleen tissue. For total leukocyte counts, the resuspended cell pellets in 50 μL phosphate buffered saline (PBS) were measured using a hemocytometer [15].

Serum HDM-specific IgE was assessed using the antigen-capture ELISA method [16]. Total lung collagen content was assessed using the calorimetric Quickzyme Total Assay Kit. Collagen content was normalized to the weight of each lung to be able to compare the total collagen value across groups.

Further details are contained in Appendix A. All analyses were performed in duplicate.

The animals were observed in the morning and evening to check for morbidity and mortality and were weighed during randomisation and on day 1, day 7 and day 15 of the study.

Necropsy at end of treatment was according to the guidelines of the CPSCEA committee.

The number of animals selected was guided by our previous HDM work, in which OmeGo was dosed intraperitoneally (IP) in five animals per group [12] and showed a significant 42% reduction in serum eosinophil count. To be conservative, in case of greater inter-animal responses with oral OmeGo compared to IP dosing, we chose to double the size to 10 animals per group in the present study. All raw data from this study were analysed using "Sigma Plot" v14 statistical software, and this was used to calculate the mean and standard deviations. All continuous data were checked for their homogeneity using the Shapiro–Wilk test. Once homogeneity was confirmed, the data were analysed using ANOVA, and data showing significance in their variances were subjected to unpaired *t*-test. *p* values ≤ 0.05 were deemed statistically significant.
