## 4 Discussion  
It is generally accepted that first-pass metabolism is a significant impediment to the bioavailability of many orally administered drugs. For these drugs, first-pass metabolism is not limited to the liver, the major site of metabolism, but other organs and tissues are involved, such as the gastrointestinal tract, blood, and vascular endothelium [37]. TU is an ester prodrug and is subject to significant first-pass metabolism [12]. Both intestinal and hepatic first-pass metabolism can markedly decrease the oral bioavailability of TU [9; 10]. Herein, we have discriminated the contribution of hepatic and intestinal tissues to the first-pass metabolism of oral TU, which represents the main obstacle for the bioavailability of TU. This can thereby support the determination of an appropriate strategy to enhance the bioavailability of TU. In the current study, it was estimated that 91% of TU can escape hepatic first-pass metabolism (\(F_{\rm H}\) = 0.915 \(\pm\) 0.009), as predicted from the hydrolysis profile in hepatic microsomal fraction in the presence of NADPH (Figure 2). To note, the hydrolysis of TU in the presence of NADPH is a summation of NADPH-independent (carboxylesterase) and NADPH-dependant (5\(\alpha\)-reductase) enzymatic activity [38]. In addition, it should be noted that esterase activity is not limited to the microsomal fraction. In fact, it has been depicted that microsomal fraction encompasses around 60% of total esterase activity in human liver homogenate, while cytosolic fraction accounts for approximately 20% of esterase activity [39]. The incorporation of cytosolic esterase activity to the observed hydrolysis profile of TU in liver microsomes can further decrease the fraction that could escape hepatic metabolism to 89%. Nevertheless, it seems that hepatic first-pass metabolism is not the main determinant for the low reported oral bioavailability of TU, since 91% was estimated to escape this metabolism. In contrast, based on the hydrolysis profile of TU in Caco-2 cell homogenate (Figure 3), it can be derived that a high percentage of the orally absorbed TU would be hydrolysed in the intestinal wall. The fraction of TU that can escape intestinal metabolism was predicted to be 11.4% (\(F_{\rm G}\) = 0.114 \(\pm\) 0.02). Accordingly, the fraction that could escape intestinal metabolism is significantly lower than fraction that could escape hepatic metabolism (\(p\) < 0.0001, Figure 4). Figure 4: Predicted fractions of testosterone undecanoate (TU) that could escape hepatic (\(F_{\rm H}\), \(n\) = 3) and intestinal (\(F_{\rm G}\), \(n\) = 4) first-pass metabolism. Values are presented as mean \(\pm\) SD. An unpaired two-tailed Student’s \(t\)-test was used to assess statistical differences between data sets. *** \(p\) < 0.0001. Taking together the predicted profiles of hepatic and intestinal metabolism of TU, it can be estimated that the oral bioavailability of TU would not exceed 10%, assuming complete absorption from the intestinal lumen. However, it is generally accepted that ester prodrugs are exposed to extensive hydrolysis in the intestinal lumen [40]. This can substantially decrease the oral bioavailability of TU below 10%, thereby delivering low levels of testosterone to the systemic circulation. Indeed, following the oral administration of TU in humans, the reported absolute bioavailability of testosterone was as low as 6.8% [1]. In that study, as well as in others [11; 12; 13], it was suggested that the fraction of TU that escaped first-pass metabolism was delivered to the systemic circulation via the intestinal lymphatic system. Through this pathway, the fraction of TU that could be metabolised during the first pass in the liver is saved. Therefore, the bioavailability is improved, as previously demonstrated for several other lipophilic ester prodrugs [31]. However, as predicted in the current study, hepatic metabolism has a minor contribution to the metabolic loss of TU compared to intestinal metabolism (Figure 4).