For LAU extracted ion chromatograms of the blank
For LAU 399, extracted Amikacin disulfate chromatograms of the blank, t0, and t120 samples for the diagnostic product ion m/z 163.0216 are provided in Fig. S11, and extracted ion chromatograms of the t120 sample for m/z 163.0216 (diagnostic product ion), and m/z 308.1677 and 306.1522 (metabolites) are shown in Fig. 8. Microsomal incubation of LAU 399 yielded three metabolites M1-M3 (Table 3). M1-2 with m/z of 308.1677 were formed by aliphatic hydroxylation, whereas M3 with m/z of 306.1522 was formed through aliphatic hydroxylation followed by oxidation of the alcohol (Fig. 4). MS spectra of LAU 399 and metabolites are provided in Fig. S12.
CYP450 reaction phenotyping studies were performed with Silensomes™. The types of Silensomes™ for the studies were selected based on the prediction from the CYP450 substrates module of ACD/labs Percepta 14.0 (Fig. S1), and further validated with CYP450-specific substrates (Fig. S13). Examples of typical CYP3A4 Silensomes™ profiles with/without enzyme contribution are provided in Fig. S14, and results of the reaction phenotyping for piperine and analogs are summarized in Fig. 9. Piperine and SCT-29 were metabolized exclusively by one CYP450 isoenzyme, CYP1A2 and CYP2C9, respectively. Metabolism of LAU 397 involved CYP3A4 and CYP2C9, and LAU 399 was metabolized by CYP2C9 and CYP1A2.
Discussion In vitro metabolic stability is routinely assessed in early drug discovery to evaluate stability of leads, and to identify metabolically labile sites to guide lead optimization. We here investigated the metabolic stability of piperine and selected analogs in the presence of pooled human liver microsomes, and calculated in vitro unbound intrinsic clearance for each compound. Piperine was identified as the metabolically most stable compound, whereas the tested analogs were rapidly metabolized. Metabolites formed by microsomal incubation were analyzed. Interestingly, hydroxylation of piperine and analogs preferentially occurred on the aliphatic portion of the molecule resulting in mono-hydroxylated metabolites. The exact position of hydroxylation could not be established due to a low abundance of metabolites. However, because of the steric hindrance in proximity of the nitrogen, C1 atoms on the butyl chains are poorly accessible for enzymatic hydroxylation. CYP450 reaction phenotyping showed that piperine and SCT-29 are cleared by a single enzyme, CYP1A2 and CYP2C9, respectively, and thus are more sensitive to drug–drug interactions. CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Genetic polymorphism of CYP2C9 may lead to individual variations in drug response , . Piperine and all tested analogs exhibited extensive binding to blood constituents, which in turn resulted in a low hepatic extraction ratio calculated for all compounds. The strong protein binding can be explained by the lipophilicity of piperine (cLogP 3.27) and analogs (cLogP 4.70–5.21) . Highly lipophilic compounds tend to bind strongly to plasma proteins, and high lipophilicity also leads to higher metabolic clearance .
Conclusions Piperine analogs (SCT-29, LAU 397, and LAU 399) were rapidly metabolized and showed strong binding to blood constituents due to increased lipophilicity. The next cycle of medicinal chemistry optimizations should, therefore, focus on reducing lipophilicity, in order to decrease metabolic liabilities and extensive protein binding .
Conflict of interest
Acknowledgements This study was supported by the Swiss National Science Foundation (project 205320_126888, MH). Authors thank Orlando Fertig for technical assistance.
Introduction Corticosteroids are a class of chemicals that includes natural steroid hormones (glucocorticoids and mineralocorticoids), produced from cholesterol in the adrenal cortex of vertebrates, and their synthetic analogues. Fish interrenal glands are capable of secreting adrenocortical steroids. Cortisol has been found in the blood and tissues of several fish species, including rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta) (Sloman et al., 2001), and Mozambique tilapia (Oreochromis mossambicus); (Johnstone et al., 2013). Fish kidneys and associated adrenal glands are structured differently than other vertebrates (Perry and Capaldo, 2011). Natural corticosteroids are involved in a broad range of physiological processes (e.g., inflammation) by reducing the response to stress as well as glucose, lipid and protein metabolism. Synthetic equivalents of corticosteroids act in similar ways and can have higher corticosteroid potencird than natural variants (Bentz, 2014).