Transplacental passage of hyperforin, hypericin, and valerenic acid.

Safe medications for mild mental diseases in pregnancy are needed. Phytomedicines from St. John's wort and valerian are valid candidates, but safety data in pregnancy are lacking. The transplacental transport of hyperforin and hypericin (from St. John's wort), and valerenic acid (from valerian) was evaluated using the ex vivo cotyledon perfusion model (4 h perfusions, term placentae) and, in part, the in vitro Transwell assay with BeWo b30 cells. Antipyrine was used for comparison in both models. U(H)PLC-MS/MS bioanalytical methods were developed to quantify the compounds. Perfusion data obtained with term placentae showed that only minor amounts of hyperforin passed into the fetal circuit, while hypericin did not cross the placental barrier and valerenic acid equilibrated between the maternal and fetal compartments. None of the investigated compounds affected metabolic, functional, and histopathological parameters of the placenta during the perfusion experiments. Data from the Transwell model suggested that valerenic acid does not cross the placental cell layer. Taken together, our data suggest that throughout the pregnancy the potential fetal exposure to hypericin and hyperforin - but not to valerenic acid - is likely to be minimal.

Phytochemical Comparison of Medicinal Cannabis Extracts and Study of Their CYP-Mediated Interactions with Coumarinic Oral Anticoagulants.

Introduction: Treatment with cannabis extracts for a variety of diseases has gained popularity. However, differences in herb-drug interaction potential of extracts from different plant sources are poorly understood. In this study, we provide a characterization of cannabis extracts prepared from four cannabis chemotypes and an in vitro assessment of their Cytochrome P450 (CYP)-mediated herb-drug interaction profiles. Methods: Plant extracts were either commercially obtained or prepared using ethanol as solvent, followed by overnight decarboxylation in a reflux condenser system. The extracts were characterized for their cannabinoid content using NMR and HPLC-PDA-ELSD-ESIMS. CYP inhibition studies with the cannabis extracts and pure cannabinoids (tetrahydrocannabinol [THC] and cannabidiol [CBD]) were performed using pooled, mixed gender human liver microsomes. Tolbutamide and testosterone were used as specific substrates to assess the inhibitory potential of the extracts on CYP2C9 and CYP3A4, and the coumarinic oral anticoagulants warfarin, phenprocoumon, and acenocoumarol were studied as model compounds since in vivo herb-drug interactions have previously been reported for this compound class. Results: In accordance with the plant chemotypes, two extracts were rich in THC and CBD (at different proportions); one extract contained mostly CBD and the other mostly cannabigerol (CBG). Residual amounts of the corresponding acids were found in all extracts. The extracts with a single major cannabinoid (CBD or CBG) inhibited CYP2C9- and CYP3A4-mediated metabolism stronger than the extracts containing both major cannabinoids (THC and CBD). The inhibition of CYP3A4 and CYP2C9 by the extract containing mostly CBD was comparable to their inhibition by pure CBD. In contrast, the inhibitory potency of extracts containing both THC and CBD did not correspond to the combined inhibitory potency of pure THC and CBD. Although being structural analogs, the three coumarin derivatives displayed major differences in their herb-drug interaction profiles with the cannabis extracts and the pure cannabinoids. Conclusion: Despite the fact that cannabinoids are the major components in ethanolic, decarboxylated cannabis extracts, it is difficult to foresee their herb-drug interaction profiles. Our in vitro data and the literature-based evidence on in vivo interactions indicate that cannabis extracts should be used cautiously when co-administered with drugs exhibiting a narrow therapeutic window, such as coumarinic anticoagulants, regardless of the cannabis chemotype used for extract preparation.

Placental Passage of Protopine in an Ex Vivo Human Perfusion System.

The placental passage of protopine was investigated with a human ex vivo placental perfusion model. The model was first validated with diazepam and citalopram, 2 compounds known to cross the placental barrier, and antipyrine as a positive control. All compounds were quantified by partially validated U(H)PLC-MS/MS bioanalytical methods. Protopine was transferred from the maternal to the fetal circuit, with a steady-state reached after 90 min. The study compound did not affect placental viability or functionality, as glucose consumption, lactate production, and beta-human chorionic gonadotropin, and leptin release remained constant. Histopathological evaluation of all placental specimens showed unremarkable, age-appropriate parenchymal maturation with no pathologic findings.

Placental Passage of Humulone and Protopine in an Ex Vivo Human Perfusion System.

The placental passage of humulone and protopine was investigated with a human ex vivo placental perfusion model. The model was first validated with diazepam and citalopram, 2 compounds known to cross the placental barrier, and antipyrine as a positive control. All compounds were quantified by partially validated U(H)PLC-MS/MS bioanalytical methods. Only a small portion of humulone initially present in the maternal circuit reached the fetal circuit. The humulone concentration in the maternal circuit rapidly decreased, likely due to metabolization in the placenta. Protopine was transferred from the maternal to the fetal circuit, with a steady-state reached after 90 min. None of the study compounds affected placental viability or functionality, as glucose consumption, lactate production, beta-human chorionic gonadotropin, and leptin release remained constant. Histopathological evaluation of all placental specimens showed unremarkable, age-appropriate parenchymal maturation with no pathologic findings.

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).

Design, Synthesis, and Characterization of a Paclitaxel Formulation Activated by Extracellular MMP9.

The concept of triggered drug release offers a possibility to overcome the toxic side effects of chemotherapeutics in cancer treatment by reducing systemic exposure to the active drug. In the present work, the concept foresees the use of the extracellular enzyme MMP9 as an enzymatic trigger for drug release in the proximity of tumor cells. METHODS: A paclitaxel-hemisuccinate-peptide conjugate as a building block for self-assembling nanoparticles was synthesized using standard conjugation approaches. The building block was purified via preparative HPLC and analyzed by LC-MS. Nanoparticles were formed using the nanoprecipitation method and characterized. For selection of a suitable in vitro model system, common bioanalytical methods were used to determine mRNA expression, enzyme amount, and activity of MMP9. RESULTS: The MMP9-labile prodrug was synthesized and characterized. Nanoparticles were formed out of MMP9-labile conjugate-building blocks. The nanoparticle's diameter averaged at around 120 nm and presented a spherical shape. LN-18 cells, a glioblastoma multiforme derived cell line, were chosen as an in vitro model based on findings in cancer tissue and cell line characterization. The prodrug showed cytotoxicity in LN-18 cells, which was reduced by addition of an MMP9 inhibitor. CONCLUSION: taken together, we confirmed increased MMP9 in several cancer tissues (cervical, esophageal, lung, and brain) compared to healthy tissue and showed the effectiveness of MMP9-labile prodrug in in vitro tests.

Thyroid Hormones Are Transport Substrates and Transcriptional Regulators of Organic Anion Transporting Polypeptide 2B1.

Levothyroxine replacement therapy forms the cornerstone of hypothyroidism management. Variability in levothyroxine oral absorption may contribute to the well-recognized large interpatient differences in required dose. Moreover, levothyroxine-drug pharmacokinetic interactions are thought to be caused by altered oral bioavailability. Interestingly, little is known regarding the mechanisms contributing to levothyroxine absorption in the gastrointestinal tract. Here, we aimed to determine whether the intestinal drug uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1) may be involved in facilitating intestinal absorption of thyroid hormones. We also explored whether thyroid hormones regulate OATP2B1 gene expression. In cultured Madin-Darby Canine Kidney II/OATP2B1 cells and in OATP2B1-transfected Caco-2 cells, thyroid hormones were found to inhibit OATP2B1-mediated uptake of estrone-3-sulfate. Competitive counter-flow experiments evaluating the influence on the cellular accumulation of estrone-3-sulfate in the steady state indicated that thyroid hormones were substrates of OATP2B1. Additional evidence that thyroid hormones were OATP2B1 substrates was provided by OATP2B1-dependent stimulation of thyroid hormone receptor activation in cell-based reporter assays. Bidirectional transport studies in intestinal Caco-2 cells showed net absorptive flux of thyroid hormones, which was attenuated by the presence of the OATP2B1 inhibitor, atorvastatin. In intestinal Caco-2 and LS180 cells, but not in liver Huh-7 or HepG2 cells, OATP2B1 expression was induced by treatment with thyroid hormones. Reporter gene assays revealed thyroid hormone receptor α-mediated transactivation of the SLCO2B1 1b and the SLCO2B1 1e promoters. We conclude that thyroid hormones are substrates and transcriptional regulators of OATP2B1. These insights provide a potential mechanistic basis for oral levothyroxine dose variability and drug interactions.

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, characterisation 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.

Pharmacokinetics of dietary kaempferol and its metabolite 4-hydroxyphenylacetic acid in rats.

SCOPE: Kaempferol is a major flavonoid in the human diet and in medicinal plants. The compound exerts anxiolytic activity when administered orally in mice, while no behavioural changes were observed upon intraperitoneal administration, or upon oral administration in gut sterilized animals. 4-Hydroxyphenylacetic acid (4-HPAA), which possesses anxiolytic effects when administered intraperitoneally, is a major intestinal metabolite of kaempferol. Pharmacokinetic properties of the compounds are currently not clear. METHODS AND RESULTS: UHPLC-MS/MS methods were validated to support pharmacokinetic studies of kaempferol and 4-HPAA in rats. Non-compartmental and compartmental analyses were performed. After intravenous administration, kaempferol followed a one-compartment model, with a rapid clearance (4.40-6.44l/h/kg) and an extremely short half-life of 2.93-3.79min. After oral gavage it was not possible to obtain full plasma concentration-time profiles of kaempferol. Pharmacokinetics of 4-HPAA was characterized by a two-compartment model, consisting of a quick distribution phase (half-life 3.04-6.20min) followed by a fast elimination phase (half-life 19.3-21.1min). CONCLUSION: Plasma exposure of kaempferol is limited by poor oral bioavailability and extensive metabolism. Both compounds are rapidly eliminated, so that effective concentrations at the site of action do not appear to be reached. At present, it is not clear how the anxiolytic-like effects reported for the compounds can be explained.

Caco-2 Permeability Studies and In Vitro hERG Liability Assessment of Tryptanthrin and Indolinone.

Tryptanthrin and (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolinone (indolinone) were recently isolated from Isatis tinctoria as potent anti-inflammatory and antiallergic alkaloids, and shown to inhibit COX-2, 5-LOX catalyzed leukotriene synthesis, and mast cell degranulation at low µM to nM concentrations. To assess their suitability for oral administration, we screened the compounds in an in vitro intestinal permeability assay using human colonic adenocarcinoma cells. For exact quantification of the compounds, validated UPLC-MS/MS methods were used. Tryptanthrin displayed high permeability (apparent permeability coefficient > 32.0 × 10(-6) cm/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and unchanged apparent permeability coefficient values in the presence of the P-glycoprotein inhibitor verapamil (50 µM) indicated that tryptanthrin was not involved in P-glycoprotein interactions. For indolinone, a low recovery was found in the human colon adenocarcinoma cell assay. High-resolution mass spectrometry pointed to extensive phase II metabolism of indolinone (sulfation and glucuronidation). Possible cardiotoxic liability of the compounds was assessed in vitro by measurement of an inhibitory effect on human ether-a-go-go-related gene tail currents in stably transfected HEK 293 cells using the patch clamp technique. Low human ether-a-go-go-related gene inhibition was found for tryptanthrin (IC50 > 10 µM) and indolinone (IC50 of 24.96 µM). The analysis of compounds using various in silico methods confirmed favorable pharmacokinetic properties, as well as a slight inhibition of the human ether-a-go-go-related gene potassium channel at micromolar concentrations.

Development and full validation of an UPLC-MS/MS method for the quantification of the plant-derived alkaloid indirubin in rat plasma.

An UPLC-MS/MS method for the quantification of indirubin in lithium heparinized rat plasma was developed and validated according to current international guidelines. Indirubin was extracted from rat plasma by using Waters Ostro™ pass-through sample preparation plates. The method was validated with a LLOQ of 5.00ng/mL and an ULOQ of 500ng/mL. The calibration curve was fitted by least-square quadratic regression, and a weighting factor of 1/X was applied. Recoveries of indirubin and I.S. were consistent and ≥75.5%. Stability studies demonstrated that indirubin was stable in lithium heparinized rat plasma for at least 3 freeze/thaw cycles, for 3h at RT, for 96h in the autosampler at 10°C, and for 84days when stored below -65°C. Preliminary pharmacokinetic (PK) data were obtained from Sprague Dawley rats after intravenous administration of indirubin (2mg/kg b.w.) and blood sampling up to 12h after injection. PK parameters were determined by non-compartmental analysis. Indirubin had a half-life (t1/2) of 35min, and a relatively high clearance (CL) of 2.71L/h/kg.

Validation of UHPLC-MS/MS methods for the determination of kaempferol and its metabolite 4-hydroxyphenyl acetic acid, and application to in vitro blood-brain barrier 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/X(2) as weighing factor, and mean coefficients of determination (R(2)) 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.

Pharmacokinetics and In Vitro Blood-Brain Barrier Screening of the Plant-Derived Alkaloid Tryptanthrin.

The indolo[2,1-b]quinazoline alkaloid tryptanthrin was previously identified as a potent anti-inflammatory compound with a unique pharmacological profile. It is a potent inhibitor of cyclooxygenase-2, 5-lipooxygenase-catalyzed leukotriene synthesis, and nitric oxide production catalyzed by the inducible nitric oxide synthase. To characterize the pharmacokinetic properties of tryptanthrin, we performed a pilot in vivo study in male Sprague-Dawley rats (2 mg/kg bw i. v.). Moreover, the ability of tryptanthrin to cross the blood-brain barrier was evaluated in three in vitro human and animal blood-brain barrier models. Bioanalytical UPLC-MS/MS methods used were validated according to current international guidelines. A half-life of 40.63 ± 6.66 min and a clearance of 1.00 ± 0.36 L/h/kg were found in the in vivo pharmacokinetic study. In vitro data obtained with the two primary animal blood-brain barrier models showed a good correlation with an immortalized human monoculture blood-brain barrier model (hBMEC cell line), and were indicative of a high blood-brain barrier permeation potential of tryptanthrin. These findings were corroborated by the in silico prediction of blood-brain barrier penetration. P-glycoprotein interaction of tryptanthrin was assessed by calculation of the efflux ratio in bidirectional permeability assays. An efflux ratio below 2 indicated that tryptanthrin is not subjected to active efflux.

In vitro blood-brain barrier permeability predictions for GABAA receptor modulating piperine analogs.

The alkaloid piperine from black pepper (Piper nigrum L.) and several synthetic piperine analogs were recently identified as positive allosteric modulators of γ-aminobutyric acid type A (GABAA) receptors. In order to reach their target sites of action, these compounds need to enter the brain by crossing the blood-brain barrier (BBB). We here evaluated piperine and five selected analogs (SCT-66, SCT-64, SCT-29, LAU397, and LAU399) regarding their BBB permeability. Data were obtained in three in vitro BBB models, namely a recently established human model with immortalized hBMEC cells, a human brain-like endothelial cells (BLEC) model, and a primary animal (bovine endothelial/rat astrocytes co-culture) model. For each compound, quantitative UHPLC-MS/MS methods in the range of 5.00-500ng/mL in the corresponding matrix were developed, and permeability coefficients in the three BBB models were determined. In vitro predictions from the two human BBB models were in good agreement, while permeability data from the animal model differed to some extent, possibly due to protein binding of the screened compounds. In all three BBB models, piperine and SCT-64 displayed the highest BBB permeation potential. This was corroborated by data from in silico prediction. For the other piperine analogs (SCT-66, SCT-29, LAU397, and LAU399), BBB permeability was low to moderate in the two human BBB models, and moderate to high in the animal BBB model. Efflux ratios (ER) calculated from bidirectional permeability experiments indicated that the compounds were likely not substrates of active efflux transporters.

Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model.

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.

Quantification of Bufadienolides in Bryophyllum pinnatum Leaves and Manufactured Products by UHPLC-ESIMS/MS.

A quantitative assay for determination of the main bufadienolides bersaldegenin-1-acetate (1), bersaldegenin-3-acetate (2), bryophyllin A (3), and bersaldegenin-1,3,5-orthoacetate (4) in Bryophyllum pinnatum leaves and manufactured products was developed and validated. The assay involved extraction by pressurised liquid extraction, followed by quantification by ultrahigh performance liquid chromatography-tandem mass spectroscopy. The ultrahigh performance liquid chromatography-tandem mass spectroscopy method was applied to various batches of leaves harvested on several dates from plants grown at two locations (Brazil and Germany). In addition, press juices prepared from plants cultivated in Germany and Brazil were analysed. The total bufadienolide content ranged from 16.28 to 40.50 mg/100 g dry weight in leaves from plants grown in Brazil. The total content of these four bufadienolides was significantly lower in plants cultivated in Germany (3.78-12.49 mg/100 g dry weight, resp.). The total amounts of bufadienolides were 0.091-0.163 mg/100 mL and 0.89-1.16 mg/100 mL in press juices obtained from plants cultivated in Germany and Brazil, respectively. When analysing single leaves from individual plants, the content of bufadienolides was markedly higher in young leaves. For comparative purposes, the content of these bufadienolides was also determined in Bryophyllum daigremontianum and Bryophyllum tubiflorum. Bersaldegenin-1,3,5-orthoacetate (4) was predominant in the leaves of B. daigremontianum and in the stems of B. tubiflorum, while the leaves of B. tubiflorum contained very low amounts of 1-4.