Pharmacokinetics and metabolism of CNS-targeted natural products

Considerable progress has been made to increase the success rate of bringing new therapeutics to the market by implementation of drug metabolism and pharmacokinetics (DMPK) screening strategies in early drug discovery. DMPK screenings help to select leads with good oral bioavailability, low clearance, optimal half-life, and a desirable metabolic profile.
In previous studies with natural products, the flavonoids kaempferol and quercetin, and the alkaloid piperine have been characterized in vivo as central nervous system (CNS) acting compounds.To gain a better understanding of anxiolytic effects reported for the flavonoids, PK studies after oral and intravenous administrations in rats were conducted. UHPLC-MS/MS methods for quantification of the compounds of interest in rat plasma were developed and validated according to the principles of regulatory guidelines for industry to support PK studies. The validated methods were successfully applied to determine the concentration levels of the analytes in rat plasma, and PK parameters were calculated with the aid of the industry standard software WinNonlin. The findings suggest that poor oral bioavailability and extensive first-pass metabolism limit plasma exposure of kaempferol. Based on the results, it is more likely that the anxiolytic effect reported for this flavonoid is rather attributed to its metabolites.The major colonic metabolites of kaempferol and quercetin are 4-hydroxyphenylacetic acid (4-HPAA), 3-hydroxyphenylacetic acid (3-HPAA), and 3,4-dihydroxyphenylacetic acid (DOPAC). Moreover, anxiolytic activity has been reported for 4-HPAA and DOPAC. Thus, we aimed to obtain PK profiles of the metabolites upon intravenous application. It has been found that the metabolites were rapidly eliminated with a half-life of 20-30 min, so that effective concentrations in the brain do not appear to be reached.
During a screening of natural products for γ-aminobutyric acid type A (GABAA) receptor activity, piperine was characterized as a positive allosteric modulator targeting a benzodiazepine-independent binding site. Due to pharmacological promiscuity of piperine, its structure was systematically modified, and a library of piperine analogs was prepared. The most potent and efficacious analogs were identified from in vitro and in vivo studies. The information on metabolically labile sites of the selected analogs was needed to guide further lead optimization process. Thus, the objective of the second part of the PhD thesis was to investigate metabolism of the selected analogs. Metabolic stability of compounds was tested in the presence of pooled human liver microsomes. Intrinsic clearance was calculated using the substrate depletion approach. Metabolites were analyzed by UHPLC-Q-TOF-MS, and with the aid of metabolite identification software Mass-MetaSite. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. CYP450 reaction phenotyping studies were carried out with Silensomes™. Microsomal stability assays revealed piperine as the metabolically most stable compound, whereas its analogs demonstrated high metabolic liability. The principal routes of oxidative metabolism were found to be aliphatic hydroxylation, and N- and O-dealkylation. It appeared that piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Moreover, extensive binding to blood constituents was observed for all compounds. Our findings showed that analogs were rapidly metabolized and demonstrated strong binding to blood constituents due to increased lipophilicity. The next cycle of medicinal chemistry optimizations should, therefore, be focused on reducing lipophilicity to lower metabolic liability and extensive binding of analogs.