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

Single dose pharmacokinetics of intravenous 3,4-dihydroxyphenylacetic acid and 3-hydroxyphenylacetic acid in rats

3,4-Dihydroxyphenylacetic acid (DOPAC) and 3-hydroxyphenylacetic acid (3-HPAA) are intestinal metabolites of the dietary flavonoid quercetin. DOPAC reportedly showed anxiolytic activity after i.p. administration in rats. The fate of these metabolites after consumption, and the pharmacological properties of 3-HPAA in the body are largely unknown. The aim of the current study was to characterize pharmacokinetic properties of DOPAC and 3-HPAA after intravenous bolus application in rats. UHPLC-MS/MS methods for quantification of DOPAC and 3-HPAA levels in lithium heparin Sprague Dawley rat plasma were developed and validated according to international regulatory guidelines. Non-compartmental and compartmental analyses were performed. Pharmacokinetic profiles of DOPAC and 3-HPAA followed a two-compartment body model, with a fast distribution into peripheral tissues (half-lives of 3.27–5.26 min) and rapid elimination from the body (half-lives of 18.4–33.3 min)

Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro BBB and intestinal drug permeability studies

Sedative and anxiolytic-like properties of flavonoids such as kaempferol and quercetin, and of some of their intestinal metabolites, have been demonstrated in pharmacological studies. However, routes of administration were shown to be critical for observing in vivo activity. Therefore, the ability to cross intestinal and blood-brain barriers was assessed in cell-based models for kaempferol (KMF), and for the major intestinal metabolite of KMF, 4-hydroxyphenylacetic acid (4-HPAA). Intestinal transport studies were performed with Caco-2 cells, and blood-brain barrier transport studies with an immortalized monoculture human model and a primary triple-co-culture rat model. UHPLC–MS/MS methods for KMF and 4-HPAA in Ringer-HEPES buffer and in Hank’s balanced salt solution were validated according to industry guidelines. For all methods, calibration curves were fitted by least-squares quadratic regression with 1/X2 as weighing factor, and mean coefficients of determination (R2) were >0.99. Data obtained with all barrier models showed high intestinal and blood-brain barrier permeation of KMF, and no permeability of 4-HPAA, when compared to barrier integrity markers

GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding.

In a screening of natural products for allosteric modulators of GABAA receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABAA and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds

Andrographolide-loaded nanoparticles for brain delivery: formulation, charcterization and in vitro permeability using hCMEC/D3 cell line

Andrographolide (AG) is a major diterpenoid of the Asian medicinal plant Andrographis paniculata which has shown exciting pharmacological potential for the treatment of inflammation-related pathologies including neurodegenerative disorders. Conversely, the low bioavailability of AG still represents a limiting factor for its use. To overcome these limitations, AG was loaded into human serum albumin based nanoparticles (HSA NPs) and poly ethylcyanoacrylate nanoparticles (PECA NPs). HSA NPs were prepared by thermal (HSAT AG NPs) and chemical cross-linking (HSAC AG NPs), while PECA AG NPs were produced by emulsion-polymerization. NPs were characterized in terms of size, zeta (ζ)-potential, polydispersity, and release studies of AG. In addition, the ability of free AG and AG-loaded in PECA and HSAT NPs to cross the blood-brain barrier (BBB) was assessed using an in vitro BBB model based on human cerebral microvascular endothelial cell line (hCMEC/D3). For BBB drug permeability assays, a quantitative UPLC-MS/MS method for AG in Ringer HEPES buffer was developed and validated according to international regulatory guidelines for industry. Free AG did not permeate the BBB model, as also predicted by in silico studies. HSAT NPs improved by two-fold the permeation of AG while maintaining the integrity of the cell layer, while PECA NPs temporarily disrupted BBB integrity