## 4 Discussion
However, for future pharmacology and toxicity assessment, pharmacokinetic studies using male and female mice need to be performed. We previously reported higher concentrations of enavogliflozin in the kidneys, which could contribute to long-lasting UGE up to 168 h after a single oral dose by inhibiting SGLT2-mediated glucose reabsorption in the renal proximal tubular cells [7,11]. In this study, enavogliflozin showed the highest distribution in the kidneys among the tested tissues and maintained its concentration for more than 48 h (Figure 5), consistent with our previous reports [7]. We also compared dose dependency in the tissue distribution of enavogliflozin using doses of 1 mg/kg and 3 mg/kg, since drug accumulation in tissues can alter therapeutic effects and increase the possibility of side effects. Although we were not able to evaluate it at a wider range of doses, tissue distribution of enavogliflozin exhibited dose proportionality except in the kidneys between 1 and 3 mg/kg PO dose (Table 7). Enavogliflozin levels in the kidneys were decreased at the higher dose (3 mg/kg) compared with 1 mg/kg, and T\({}_{\text{max}}\) and T\({}_{\text{1/2}}\) at 3 mg/kg were significantly shorter than at 1 mg/kg. Considering that enavogliflozin is a substrate for OAT1 (organic anion transporter 1) and OAT3 [7], high enavogliflozin concentrations could cause saturation of these transporters, reducing drug influx into the kidneys at 3 mg/kg. Since these transporters are mainly expressed in the renal proximal tubular cells, enavogliflozin uptake was influenced only in the kidneys without affecting other tissues. The results suggest that enavogliflozin in the kidneys may not show a dose-tissue exposure correlation, whereas other tissues do, which provides important information for dose optimization. The rank order of enavogliflozin distribution was kidneys > stomach > small and large intestine > liver > heart and lungs > plasma > testes > spleen. However, enavogliflozin was not detected in brain samples. Compounds with MW > 400 and high polar surface area (PSA) > 80 A\({}^{2}\) and a tendency to form more than six hydrogen bonds are limited in penetrating the blood-brain barrier [34]. Enavogliflozin has an MW of 446.9, a PSA value of 99.4 A\({}^{2}\), and a hydrogen bond number of 10, and contains chloride and several hydroxy substituents (Figure 1) [8]. This may explain its limited distribution to the brain. SGLT2 is mainly expressed in the renal proximal tubules. It is also localized in pancreatic islet \(\alpha\)-cells, prostatic and pancreatic cancer cells, glomerular mesangial cells, and retinal pericytes [25,26]. Ocular SGLT2 transports glucose and sodium to pericytes, and excessive glucose and sodium in pericytes lead to pericytes swelling and loss. Therefore, SGLT2 inhibitors exert therapeutic effects by reducing glucose entry into pericytes, reducing oxidative stress, and restoring insulin signaling [25,26,27]. Based on SGLT2 expression in retinal pericytes and ocular SGLT2 function, enavogliflozin is being developed as a therapeutic for diabetic retinopathy. However, orally administered enavogliflozin did not reach the retina. Therefore, the ocular and plasma pharmacokinetics of envogliflozin in rabbits following ocular administration of envacogliflozin ophthalmic solution were evaluated. In this study, envacogliflozin administered via the ocular route distributed to the conjunctiva, cornea, and retina (the target site for retinopathy), and retinal envacogliflozin concentrations remained above its IC\({}_{50}\) value for SGLT2 for longer than 24 h [25]. Tissue distribution results also suggest that the therapeutic effects of envacogliflozin may be conferred in highly distributed tissues such as the kidneys, intestinal tract, liver, heart, and lungs. Previous research regarding the nephoprotective effects of SGLT2 inhibitors focused on the inhibition of induced AMPK/mTOR pathways as well as the HIF-1\(\alpha\) pathway and the inhibition of immune inflammation.