*3.9. Ocular Irritation*

The eye irritation potential of the inducing agen<sup>t</sup> was classified into four grades; practically non-irritating, score 0–3; slightly irritating, score 4–8; moderately irritating, score 9–12; and severely irritating (or corrosive), score 13–16 [20]. The eye irritation score was calculated by dividing the total score for all rabbits by the total number of rabbits tested. The observed eye irritation score in the control was 0.33 and for MM3 was 0.66, which signifies excellent ocular tolerance. Moreover, no ocular damage or abnormal clinical signs pertaining to the cornea, iris, or conjunctivae were visible. In addition, no signs of redness, watering of the eye or swelling were noticed for both MM3 and control. Overall the results of this study revealed that MM3 is safe for ocular application.

### *3.10. Pharmacokinetics in the Aqueous Humor*

The concentration of the drug permeated into the aqueous humor after administration to the rabbit eyes was quantified to evaluate the ocular bioavailability of moxifloxacin from MM3 as well as the control. Di fferent pharmacokinetic properties were analyzed by using a non-compartmental method [33]. The determined pharmacokinetic parameters are summarized in Table 5. Figure 6 compares the mean moxifloxacin concentration in the aqueous humor following topical installation of MM3 and control in rabbits. It is evident from Figure 6 that the kinetic profiles are distinct for MM3 and control. Indeed, moxifloxacin absorption was rapid and available in the aqueous humor after 30 min in MM3 (113.98 ± 51.45 ng/mL) and control (209.44 ± 64.53 ng/mL), however, the amount of drug was di fferent. At 1 h, the drug level in the aqueous humor increased to 305.99 ± 94.95 ng/mL and 454.19 ± 126.91 ng/mL in MM3 and control, respectively. The drug absorption in MM3 was further prolonged and the drug level in the aqueous humor continued to rise to attain the peak drug concentration ( *C*max; 555.73 ± 133.34 ng/mL) and the time corresponding to peak concentration ( *T*max) was 2 h (which is 1 h in control). Comparing the drug absorption with the ex vivo permeation data, one can easily corroborate that the delay in absorption of moxifloxacin into the aqueous humor in MM3 is probably due to its slow permeation rate in the first hour observed in Figure 5. Followed by the rapid absorption, the drug level declined in both groups. At 4 h, the drug level in aqueous humor was considerably higher in MM3 (*p* < 0.0001) as compared to control. At 8 h, the drug level in aqueous humor for MM3 was 35.90 ± 13.01 ng/mL while no moxifloxacin was detected in the control. The mean value of area under the aqueous humor concentration versus time curve (*AUC*0–8 h) for MM3 (Table 5) was ~2 fold higher (*p* < 0.001), relative to the control, suggesting significant improvement in ocular bioavailability by MM3 in comparison to the control. The possible reason for the greater observed *AUC* could be due to the longer retention of vesicles (MM3) in the ocular surface which in turn prolongs the contact time of medicament with the eye and thereby improve corneal penetration, when compared to conventional eye drops, as described in the literature [34].


**Table 5.** Mean pharmacokinetic parameters of moxifloxacin in aqueous humor following topical installation of nanoemulsion (MM3) and control in rabbits.

\* Significant difference (*p* < 0.001) observed in moxifloxacin level in nanoemulsion (MM3) group compared to control. Area under the aqueous humor concentration versus time curve (*AUC*0–8 h).

**Figure 6.** Comparison of mean moxifloxacin concentration in the aqueous humor following topical installation of optimized nanoemulsion (MM3) and control (commercial eye drops) in rabbits. The data represents average ± SD of six trials.
